# Patent

## Title
Flavonoid 3′,5′ hydroxylase gene sequences and uses therefor

## Abstract

The present invention relates generally to a genetic sequence encoding a polypeptide having flavonoid 3′,5′-hydroxylase (F3′5′H) activity and to the use of the genetic sequence and/or its corresponding polypeptide thereof inter alia to manipulate color in flowers or parts thereof or in other plant tissue. More particularly, the F3′5′H has the ability to modulate dihydrokaempferol (DHK) metabolism as well as the metabolism of other substrates such as dihydroquercetin (DHQ), naringenin and eriodictyol. Even more particularly, the present invention provides a genetic sequence encoding a polypeptide having F3′5′H activity when expressed in rose or gerbera or botanically related plants. The instant invention further relates to antisense and sense molecules or RNAi-inducing molecules corresponding to all or part of the subject genetic sequence or a transcript thereof. The present invention further relates to promoters which operate efficiently in plants such as rose, gerbera or botanically related plants.

## Background
This is a national stage application filed under 35 U.S.C. 371 from PCT/AU03/01111, filed Aug. 29, 2003, which claims priority to AUSTRALIA 2002951088 filed Aug. 30, 2002, and AUSTRALIA 2002952835 filed Sep. 16, 2002.
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to a genetic sequence encoding a polypeptide having flavonoid 3′,5′-hydroxylase (F3′5′H) activity and to the use of the genetic sequence and/or its corresponding polypeptide thereof inter alia to manipulate color in flowers or parts thereof or in other plant tissue. More particularly, the F3′5′H has the ability to modulate dihydrokaempferol (DHK) metabolism as well as the metabolism of other substrates such as dihydroquercetin (DHQ), naringenin and eriodictyol. Even more particularly, the present invention provides a genetic sequence encoding a polypeptide having F3′5′H activity when expressed in rose or gerbera or botanically related plants. The instant invention further relates to antisense and sense molecules or RNAi-inducing molecules corresponding to all or part of the subject genetic sequence or a transcript thereof as well as to genetically modified plants as well as cut flowers, parts and reproductive tissue from such plants. The present invention further relates to promoters which operate efficiently in plants such as rose, gerbera or botanically related plants.
2. Description of Prior Art
Reference to any prior art in this specification is not, and should not be taken as an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in any country.
Bibliographic details of references provided in the subject specification are listed at the end of the specification.
The flower or ornamental plant industry strives to develop new and different varieties of flowers and/or plants. An effective way to create such novel varieties is through the manipulation of flower color. Classical breeding techniques have been used with some success to produce a wide range of colors for almost all of the commercial varieties of flowers and/or plants available today. This approach has been limited, however, by the constraints of a particular species' gene pool and for this reason it is rare for a single species to have the full spectrum of colored varieties. For example, the development of novel colored varieties of plants or plant parts such is flowers, foliage and stems would offer a significant opportunity in both the cut flower and ornamental markets. In the flower or ornamental plant industry, the development of novel colored varieties of major flowering species such as rose, chrysanthemum, tulip, lily, carnation, gerbera, orchid, lisianthus, begonia, torenia, geranium, petunia, meremubergia, pelargonium, iris, impatiens and cyclamen would be of great interest. A more specific example would be the development of a blue rose or gerbera for the cut flower market.
In addition, the development of novel colored varieties of plant parts such as vegetables, fruits and seeds would offer significant opportunities in agriculture. For example, novel colored seeds would be useful as proprietary tags for plants. Furthermore modifications to flavonoids common to berries or fruits including grapes and apples and their juices including wine have the potential to impart altered style characteristics of value to such fruit and byproduct industries.
Flower color is predominantly due to three types of pigment: flavonoids, carotenoids and betalains. Of the three, the flavonoids are the most common and contribute a range of colors from yellow to red to blue. The flavonoid molecules that make the major contribution to flower color are the anthocyanins, which are glycosylated derivatives of cyanidin and its methylated derivative peonidin, delphinidin or delphinidin-based molecules and its methylated derivatives petunidin and malvidin and pelargonidin. Anthocyanins are localised in the vacuole of the epidermal cells of petals or the vacuole of the sub epidermal cells of leaves.
The flavonoid pigments are secondary metabolites of the phenylpropanoid pathway. The biosynthetic pathway for the flavonoid pigments (flavonoid pathway) is well established, (Holton and Cornish, Plant Cell 7: 1071-1083, 1995; Mol et al., Trends Plant Sci. 3: 212-217, 1998; Winkel-Shirley, Plant Physiol. 126: 485-493, 2001a; and Winkel-Shirley, Plant Physiol, 127: 1399-1404, 2001b) and is shown in FIGS. 1A and B. Three reactions and enzymes are involved in the conversion of phenylalanine to p-coumaroyl-CoA, one of the first key substrates in the flavonoid pathway. The enzymes are phenylalanine ammonia-lyase (PAL), cinnamate 4-hydroxylase (C4H) and 4-coumarate: CoA ligase (4CL). The first committed step in the pathway involves the condensation of three molecules of malonyl-CoA provided by the action of acetyl CoA carboxylase (ACC) on acetyl CoA and CO2) with one molecule of p-coumaroyl-CoA. This reaction is catalysed by the enzyme chalcone synthase (CHS). The product of this reaction, 2′,4,4,6′, tetrahydroxy-chalcone, is normally rapidly isomerized by the enzyme chalcone flavanone isomerase (CHI) to produce naringenin. Naringenin is subsequently hydroxylated at the 3 position of the central ring by flavanone 3-hydroxylase (F3H) to produce dihydrokaempferol (DHK).
The pattern of hydroxylation of the B-ring of dihydrokaempferol (DHK) plays a key role in determining petal color. The B-ring can be hydroxylated at either the 3′, or both the 3′ and 5′ positions, to produce dihydroquercetin (DHQ) or dihydromyricetin (DHM), respectively. Two key enzymes involved in this part of the pathway are flavonoid 3′-hydroxylase and flavonoid 3,5′-hydroxylase, both of the cytochrome P450 class of enzymes. Cytochrome P450 enzymes are widespread in nature and genes have been isolated and sequenced from vertebrates, insects, yeasts, fungi, bacteria and plants.
Flavonoid 3′-hydroxylase (F3H) is a key enzyme in the flavonoid pathway leading to the cyanidin-based pigments which, in many plant species (for example Rosa spp., Dianthus spp., Petunia spp., begonia, cyclamen, impatiens, morning glory and chrysanthemum), contribute to red and pink flower color.
Flavonoid 3′,5′-hydroxylase (F3′5′H) is a key enzyme in the flavonoid pathway leading to the delphinidin-based pigments which, in many plant species (for example, Petunia spp., Viola spp., Lisianthus spp., Gentiana spp., Sollya spp., Salvia spp., Clitoria spp., Kennedia spp., Campanula spp., Lavandula spp., Verbena spp., Torenia spp., Delphinium spp., Solanum spp., Cineraria spp., Vitis spp., Babiana stricta, Pinus spp., Picea spp., Larix spp., Phaseolus spp., Vaccinium spp., Cyclamen spp., Iris spp., Pelargonium spp., Liparieae, Geranium spp., Pisum spp., Lathyrus spp., Catharanthus spp., Malvia spp., Mucuna spp., Vicia spp., Saintpaulia spp., Lagerstroemia spp., bouchina sp., Plumbago spp., Hypocalyptus spp., Rhododedron spp., Linum spp., Macroptiltium spp., Hibiscus spp., Hydrangea spp., Cymbidium spp., Millettia spp., Hedysarum spp., Lespedeza spp., Asparagus spp. Antigonon spp., Pisum spp., Freesia sap. Brunella spp., Clarkia spp., etc.), contribute to purple and blue flower color. Many plant species such as roses, gerberas, chrysanthemums and carnations, do not produce delphinidin-based pigments because they lack a F3′5′H activity.
The next step in the pathway, leading to the production of the colored anthocyanins from the dihydroflavonols (DHK, DHQ, DHM), involves dihydroflavonol-4-reductase (DFR) leading to the production of the leucoanthocyanidins. The leucoanthocyanidins are subsequently converted to the anthocyanidins, pelargonidin, cyanidin and delphinidin or delphinidin-based molecules. These flavonoid molecules are unstable under normal physiological conditions and glycosylation at the 3-position, through the action of glycosyltransferases, stabilizes the anthocyanidin molecule thus allowing accumulation of the anthocyanins. In general, the glycosyltransferases transfer the sugar moieties from UDP sugars to the flavonoid molecules and show high specificities for the position of glycosylation and relatively low specificities for the acceptor substrates (Seitz and Hinderer, Anthocyanins. In: Cell Culture and Somatic Cell Genetics of Plants. Constabel, F. and Vasil, I. K. (eds.), Academic Press, New York, USA, 5: 49-76, 1988). Anthorcyanins can occur as 3-monosides, 3-biosides and 3-triosides as well as 3,5-diglycosides and 3,7-diglycosides associated with the sugars glucose, galactose, rhamnose, arabinose and xylose (Strack and Wray, In; The Flavonoids—Advances in Research since 1986. Harborne, J. B. (ed), Chapman and Hall, London, UK, 1-22, 1993).
Glycosyltransferases involved in the stabilisation of the anthocyanidin molecule include UDP glucose: flavonoid 3-glucosyltransferase (3GT), which transfers a glucose moiety from UDP glucose to the 3-O-position of the anthocyanidin molecule to produce anthocyanidin 3-O-glucoside.
In petunia and pansy (amongst others), anthocyanidin 3-O-glucoside are generally glycosylated by another glycosyltransferase, UDP rhamnose: anthocyanidin 3-glucoside rhamnosyltransferase (3RT), which adds a rhamnose group to the 3-O-bound glucose of the anthocyanin molecule to produce the anthocyanidin 3-rutinosides, and once acylated, can be further modified by UDP: glucose anthocyanin 5 glucosyltansferase (5GT). However, in roses (amongst others), the anthocyanidin 3-O-glucosides are generally glycosylated by another glycosyltrasferase, UDP: glucose anthocyanin 5 glucosyltransferase (5GT) to produce anthocyanidin 3,5 diglucosides.
Many anthocyanidin glycosides exist in the form of acylated derivatives. The acyl groups that modify the anthocyanidin glycosides can be divided into two major classes based upon their structure. The aliphatic acyl groups include malonic acid or succinic acid and the aromatic class include the hydroxy cinnamic acids such as p-coumaric acid, caffeic acid and ferulic acid and the benzoic acids such as p-hydroxybenzoic acid.
Methylation at the 3′ and 5′ positions of the B-ring of anthocyanidin glycosides can also occur. Methylation of cyanidin-based pigments leads to the production of peonidin. Methylation of the 3′ position of delphinidin-based pigments results in the production of petunidin, whilst methylation of the 3′ and 5′ positions results in malvidin production. Methylation of malvidin can also occur at the 5-O and 7-O positions to produce capensinin (5-O-methyl malvidin) and 5,7-di-O-methyl malvidin.
In addition to the above modifications, pH of the vacuole or compartment where pigments are localised and copigmentation with other flavonoids such as flavonols and flavones can affect petal color. Flavonols and flavones can also be aromatically acylated (Brouillard and Dangles, In: The Flavonoids—Advances in Research since 1986. Harborne, J. B. (ed), Chapman and Hall, London, UK, 1-22, 1993).
The ability to control F3′5′H activity, or other enzymes involved in the flavonoid pathway, in flowering plants would provide a means of manipulating the color of plant parts such as petals, fruit, leaves, sepals, seeds etc. Different colored versions of a single cultivar could thereby be generated and in some instances a single species would be able to produce a broader spectrum of colors.
Two nucleotide sequences (referred to herein as SEQ ID NO:1 and SEQ ID NO:3) encoding petunia F3′5′Hs have been cloned (see International Patent Application No. PCT/AU92/00334 and Holton et al, Nature, 366: 276-279, 1993a). These sequences were efficient in modulating 3′,5′ hydroxylation of flavonoids in petunia (see International Patent Application No. PCT/AU92/00334 incorporated herein by reference and Holton et al., 1993a, supra), tobacco (see International Patent Application No. PCT/AU92/00334 incorporated herein by reference) and carnations (see International Patent Application No. PCT/AU96/00296 incorporated herein by reference). Surprisingly, however, inclusion of these sequences in standard expression cassettes, did not lead to the production of intact or full-length transcripts as detectable by RNA or Northern blot analysis and consequently 3′,5′-hydroxylated flavonoids were not produced in roses. There is a need, therefore, to identify further genetic sequences encoding F3′5′Hs which efficiently accumulate and are then able to modulate 3′,5′ hydroxylation of flavonoids such as anthocyanins in roses and other key commercial plant species.
SUMMARY OF THE INVENTION
Throughout this specification, unless the context requires otherwise, the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.
Nucleotide and ado acid sequences are referred to by a sequence identifier number (SEQ ID NO:). The SEQ ID NOs: correspond numerically to the sequence identifiers <400>1 (SEQ ID NO:1), <400>2 (SEQ ID NO:2), etc.
Genetic sequences encoding a F3′5′H have been identified and cloned from a number of plant species. The F3′5′H genetic sequences when expressed in rose petal tissue results in detectable level of delphinidin or delphinidin-based molecules as determined by a chromatographic technique such as thin layer chromatography (TLC) or high performance liquid chromatography (HPLC). Alternatively, or in addition, expression of the genetic sequences in rose petal tissue results in a sufficient level and length of transcript which is capable of being translated to F3′5′H. This is conveniently measured as delphinidin or delphinidin-based molecules, detectable using a chromatographic technique such as TLC or HPLC. The genetic sequences of the present invention permit the modulation of expression of genes encoding this enzyme by, for example, de novo expression, over-expression, suppression, antisense inhibition, ribozyme activin, RNAi-induction or methylation-induction. The ability to control F3′5′H synthesis in plants and more specifically in roses or gerberas permits modulation of the composition of individual anthocyanins as well as alteration of relative levels of flavonols and anthocyanins, thereby enabling the manipulation of color of tissues and/or organs of plants such as petals, leaves, seeds, sepals, fruits etc.
Accordingly, one aspect of the present invention provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence encoding a flavonoid 3′,5′ hydroxylase (F3′5′H) or a polypeptide having F3′5′H activity wherein expression of said nucleic acid molecule in a rose petal tissue results in detectable levels of delphinidin or delphinidin-based molecules as measured by a chromatographic technique.
Another aspect of the present invention is directed to an isolated nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence encoding a F3′5′H or a polypeptide having F3′5′H activity wherein expression of said nucleic acid molecule in a rose petal tissue results in a sufficient level and length of transcript which is translated to said F3′5′H as determined by detectable levels of delphinidin or delphinidin-based molecules as measured by a chromatographic technique.
The isolated nucleic acid molecule of the present invention, therefore, encodes a F3′5′H which is capable of more efficient conversion of DHK to DHM in roses than is the F3′5′H encoded by the nucleotide sequence set forth in SEQ ID NO:1 and SEQ ID NO:3 as measured by delphinidin production in rose petals.
Efficiency as used herein relates to the capability of the F3′5′H enzyme to convert its substrate DHK or DHQ into DHM in a rose cell (or any cell of a commercially important plant such as gerber). This conversion provides the plant with a substrate (DHM) for other enzymes of the flavonoid pathway which are present in the paint to further modify the substrate. This modification may include for example, glycosylation, acylation, rhamnosylation and/or methylation, to produce various anthocyanins which contribute to the production of a range of colors. The modulation of 3′,5′-hydroxylated anthocyanins in rose is thereby enabled. Efficiency is conveniently assessed by one or more parameters selected from: extent of F3′5′H transcription, as determined by the amount of intact F3′5′H mRNA produced (as detected by Northern blot analysis); extent of translation of the F3′5′H mRNA, as determined by the amount of translation product produced; extent of F3′5′H enzyme activity as determined by the production of anthocyanin derivatives of DHQ or DHM including delphinidin or delphinidin-based pigments (as detected by TLC or HPLC); the extent of effect on flower color.
It has also been surprisingly determined that certain combinations of promoter and F3′5′H gene sequences that were functional in carnation and petunia were not functional in rose. Surprisingly, only a particular subset of promoter and F3′5′H gene sequence combinations resulted in 3′,5′-hydroxylated flavonoids in rose flowers. These included F3′5′H sequences isolated from Viola spp., Salvia spp. Lavandula spp. and Sollya spp. Furthermore, the Viola F3′5′H (or pansy F3′5′H) sequences were found to result in the highest accumulation of 3′,5′-hydroxylated flavonoids in rose. The novel promoter and F3′5′H gene sequence combinations can be employed inter alia to modulate the color or flavour or other characteristics of plants or plant parts such as but not limited to flowers, fruits, nuts, roots, stems, leaves or seeds. Thus, the present invention represents a new approach to developing plant varieties having altered color characteristics. Other uses include, for example, the production of novel extracts of F3′5′H transformed plants wherein the extract has use, for example, as a flavouring or food additive or health product or beverage or juice or coloring. Beverages may include but are not limited to wines, spirits, teas, coffee, milk and dairy products.
In a preferred embodiment, therefore, the present invention provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to a sequence encoding pansy F3′5′R, salvia F3′5′H, lavender F3′5′H, kennedia F3′5′H or sollya F3′5′H or a functional derivative of the enzyme.
The nucleotide sequences encoding the pansy F3′5′H (SEQ ID NOs:9 and 11), salvia F3′5′H (SEQ ID NOs:13 and 15), sollya F3′5′H (SEQ ID NO:17), lavender F3′5′H (SEQ ID NO:31) and kennedia F3′5′H (SEQ ID NO:26) are defined by sequence identifiers indicated in parentheses. A summary of the sequence identifiers is shown in Table 1.
Accordingly, another aspect of the present invention provides a nucleic acid molecule comprising a nucleotide sequence or complementary nucleotide sequence substantially as set forth in SEQ ID NO:9 (pansy) or SEQ ID NO:11 (pansy) or SEQ ID NO:13 (salvia) or SEQ ID NO:15 (salvia) or SEQ ID NO:17 (sollya) or SEQ ID NO:31 (lavender) or SEQ ID NO:26 (kennedia) or having at least about 50% similarity thereto or capable of hybridizing to the sequence set forth in SEQ ID NO:9 or SEQ ID NO:11 or SEQ ID NO:13 or SEQ ID NO:15 or SEQ ID NO:17 or SEQ ID NO:31 or SEQ ID NO:26 under low stringency conditions.
The amino acid sequences of the preferred F3′5′H enzymes are set forth in SEQ ID NO:10 (pansy) or SEQ ID NO:12 (pansy) or SEQ ID NO:14 (salvia) or SEQ ID NO:16 (salvia) or SEQ ID NO:18 (sollya) or SEQ ID NO:32 (lavender) or SEQ ID NO:27 (kennedia).
A further aspect of the present invention provides a method for producing a transgenic flowering plant capable of synthesizing a F3′5′H said method comprising stably transforming a cell of a suitable plant with a nucleic acid sequence which comprises a sequence of nucleotides encoding said F3′5′H under conditions permitting the eventual expression of said nucleic acid sequence, regenerating a transgenic plant from the cell and growing said transgenic plant, for a time and under conditions sufficient to permit the expression of the nucleic acid sequence. The expression of the nucleic acid sequence generally results in a transcription of sufficient level and length to encode a F3′5′H. This is conveniently determined by detectable levels of delphinidin or delphinidin-based molecules as measured by chromatographic techniques such as TLC or HPLC. The transgenic plant may thereby produce a non-indigenous F3′5′H at elevated levels relative to the amount expressed in a comparable non-transgenic plant. This generally results in a visually detectable color change in the plant or plant part or preferably in the inflorescence or flowers of said plant.
Another aspect of the present invention contemplates a method for producing a transgenic plant with reduced F3′5′H activity, said method comprising stably transforming a cell of a suitable plant with a nucleic acid molecule which comprises a sequence of nucleotides encoding or complementary to a sequence encoding a F3′5′ activity, regenerating a transgenic plant from the cell and where necessary growing said transgenic plant under conditions sufficient to permit the expression of the nucleic acid.
Yet another aspect of the present invention contemplates a method for producing a genetically modified plant with reduced F3′5′H activity, said method comprising altering the F3′5′H gene through modification of the indigenous sequences via homologous recombination from an appropriately altered F3′5′H gene or derivative or part thereof introduced into the plant cell, and regenerating the genetically modified plant from the cell.
Still another aspect of the present invention contemplates a method for producing a transgenic flowering plant exhibiting altered floral or inflorescence properties, said method comprising stably transforming a cell of a suitable plant with a nucleic acid sequence of the present invention, regenerating a transgenic plant from the cell and growing said transgenic plant for a time and under conditions sufficient to permit the expression of the nucleic acid sequence.
Still a further aspect of the present invention contemplates a method for producing a transgenic flowering plant exhibiting altered floral or inflorescence properties, said method comprising alteration of the F3′5′H gene through modification of nucleotide sequences via homologous recombination from an appropriately altered F3′5′H gene or derivative or part thereof introduced into the plant cell, and regenerating the genetically modified plant from the cell.
Even yet another aspect of the present invention extends to a method for producing a transgenic plant capable of expressing a recombinant gene encoding a F3′5′H or part thereof or which carries a nucleic acid sequence which is substantially complementary to all or a part of a mRNA molecule encoding said F3′5′H, said method comprising stably transforming a cell of a suitable plant within the isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to a sequence encoding, a F3′5′H where necessary under conditions permitting the eventual expression of said isolated nucleic acid molecule, and regenerating a transgenic plant from the cell.
Even still another aspect of the present invention extends to all transgenic plants or parts of transgenic plants or progeny of the transgenic plants containing all or part of the nucleic acid sequences of the present invention, or antisense forms thereof and/or any homologs or related forms thereof and, in particular, those transgenic plants which exhibit altered floral or inflorescence properties.
Even still another aspect of the present invention extends to all transgenic plants or parts of transgenic plants or progeny of the transgenic plants containing all or part of the nucleic acid sequences of the present invention, or antisense forms thereof and/or any homologs or related forms thereof and, in particular, those transgenic plants which exhibit altered aerial parts of the plant such as fruit, berries, sepal, bract, petiole, peduncle, ovaries, anthers or stern properties.
Another aspect of the present invention contemplates the use of the extracts from transgenic plants or plant parts transgenic plants or progeny of the transgenic plants containing all or part of the nucleic acid sequences of the present invention and, in particular, the extracts from those transgenic plants when used as a flavouring or food additive or health product or beverage or juice or coloring.
A further aspect of the present invention is directed to recombinant forms of F3′5′H.
Another aspect of the present invention contemplates the use of the genetic sequences described herein in the manufacture of a genetic construct capable of expressing a F3′5′H or down-regulating an indigenous F3′5′H enzyme in a plant.
Yet another aspect of the present invention is directed to a prokaryotic or eukaryotic organism carrying a genetic sequence encoding a F3′5′H extrachromasomally in plasmid form.
Still another aspect of the present invention extends to a recombinant polypeptide comprising a sequence of amino acids substantially as set forth in SEQ ID NO:10 or SEQ ID NO:12 or SEQ ID NO:14 or SEQ ID NO:16 or SEQ ID NO:18 or SEQ ID NO:32 or SEQ ID NO:27 or an amino acid sequence having at least about 50% similarity to SEQ ID NO:10 or SEQ ID NO:12 or SEQ ID NO:14 or SEQ ID NO:16 or SEQ ID NO:18 or SEQ ID NO:32 or SEQ ID NO:27 or a derivative of said polypeptide.
The present invention further provides promoters which operate efficiently in plants such as rose and gerbera or botanically related plants. Such promoters include a rose CHS promoter, chrysanthemum CHS promoter and a CaMV 35S promoter.
A summary of sequence identifiers used throughout the subject specification is provided in Table 1:







TABLE 1







Summary of sequence identifiers











SEQ ID


TYPE



NO:
NAME
SPECIES
OF SEQ
DESCRIPTION














1
petHf1.nt
Petunia hybrida
nucleotide
F3′5′H cDNA


2
petHf1.aa
Petunia hybrida
amino acid
translation of F3′5′H cDNA


3
petHf2.nt
Petunia hybrida
nucleotide
F3′5′H cDNA


4
petHf2.aa
Petunia hybrida
amino acid
translation of F3′5′H cDNA


5
RoseCHS promoter
Rosa hybrida
nucleotide
promoter fragment


6
D8 oligo#2
Petunia hybrida
nucleotide
oligonucleotide


7
D8 oligo #4
Petunia hybrida
nucleotide
oligonucleotide


8
chrysanCHSATG
chrysanthemum
nucleotide
oligonucleotide


9
BP#18.nt
Viola spp.
nucleotide
F3′5′H cDNA


10
BP#18.aa
Viola spp.
amino acid
translation of F3′5′H cDNA


11
BP#40.nt
Viola spp.
nucleotide
F3′5′H cDNA


12
BP#40.aa
Viola spp.
amino acid
translation of F3′5′H cDNA


13
Sal#2.nt
Salvia spp.
nucleotide
F3′5′H cDNA


14
Sal#2.aa
Salvia spp.
amino acid
translation of F3′5′H cDNA


15
Sal#47.nt
Salvia spp.
nucleotide
F3′5′H cDNA


16
Sal#47.aa
Salvia spp.
amino acid
translation of F3′5′H cDNA


17
Soll#5.nt
Sollya spp.
nucleotide
F3′5′H cDNA


18
Soll#5.aa
Sollya spp.
amino acid
translation of F3′5′H cDNA


19
FLS-Nco
Petunia hybrida
nucleotide
oligonucleotide


20
BpeaHF2.nt
Clitoria ternatea
nucleotide
F3′5′H cDNA


21
BpeaHF2.aa
Clitoria ternatea
amino acid
translation of F3′5′H cDNA


22
Gen#48.nt
Gentiana
nucleotide
F3′5′H cDNA




triflora


23
Gen#48.aa
Gentiana
amino acid
translation of F3′5′H cDNA




triflora


24
PetD8 5′
Petunia hybrida
nucleotide
oligonucleotide


25
Bpea primer
Clitoria ternatea
nucleotide
oligonucleotide


26
Kenn#31.nt
Kennedia spp.
nucleotide
F3′5′H cDNA


27
Kenn#31.aa
Kennedia spp.
amino acid
translation of F3′5′H cDNA


28
chrysCHS.nt
chrysanthemum
nucleotide
CHS cDNA


29
chrysCHS.aa
chrysanthemum
amino acid
translation of CHS cDNA


30
chrysCHS promoter
chrysanthemum
nucleotide
promoter fragment


31
LBG.nt
Lavandula nil
nucleotide
F3′5′H cDNA


32
LBG.aa
Lavandula nil
amino acid
translation of F3′5′H cDNA












BRIEF DESCRIPTION OF THE FIGURES
FIGS. 1A and 1B are schematic representations of the biosynthesis pathway for the flavonoid pigments. FIG. 1A illustrates the general production of the anthocyanidin 3-glucosides that occur in most plants that produce anthocyanins. FIG. 1B represents further modifications of anthocyanins that occur in petunia. Enzymes involved in the pathway have been indicated as follows: PAL=Phenylalanine ammonia-lyase; C4H=Cinnamate 4-hydroxylase; 4CL=4-coumarate: CoA ligase; CHS=Chalcone synthase; CHI=Chalcone flavanone isomerase; F3H=Flavanone 3-hydroxylase; DFR=Dihydroflavonol-4-reductase; ANS=Anthocyanidin synthase, 3GT=UDP-glucose: flavonoid 3-O-glucosyltransferase; 3RT=UDP rhamnose: anthocyanidin 3-glucoside rhamnosyltransferase, AR-AT=Anthocyanidin-rutinoside acyltransferase, 5GT=Anthocyanin 5-glucosyltransferase; 3′OMT=Anthocyanin 3′ O-methyltransferase, 3′5′ OMT=Anthocyanin 3′,5′ O-methyltransferase. Other abbreviations include: DHK=dihydrokaempferol, DHQ=dihydroquercetin, DHM=dihydromyricetin,







TABLE 2







Descriptions of the abbreviations used in FIGS. 2 to 52








ABBREVIATION
DESCRIPTION





Amp
ampicillin resistance gene which confers resistance to the antibiotic



ampicillin


ColE1ori
plasmid origin of replication


f1 ori (+)
f1 filamentous phage origin of replication


GcntR
gentamycin resistance gene which confers resistance to the antibiotic



gentamycin


LB
left border of the T-DNA


nptIII
the neomycin phosphotransferase III gene which confers resistance to the



antibiotic kanamycin


ori pRi
plasmid origin of replication


ori 322
plasmid origin of replication


pACYC ori
modified replicon from pACYC184 from E. coli


pVS1
a broad host range origin of replication from a plasmid from Pseuodomonas



aeruginosa


rev
approximate location of the M13 reverse primer site used in sequence



analysis


RB
right border of the T-DNA


TetR
tetracycline resistance gene which confers resistance to the antibiotic



tetracycline


−20
approximate location of the M13 −20 primer site used in sequence analysis


RK2
broad host range Gram-negative plasmid RK2 origin









FIG. 2 is a diagrammatic representation of the plasmid pCGP602, pCGP601 and pCGP176 containing petunia F3′5′H petHf1 cDNA clones from P. hybrida cv. OGB. The petunia F3′5′H petHf1 fragment was used in the preparation of constructs containing the petunia F3′5′H cDNA clone, 32P-labelled fragments of the 1.6 kb BspHI/FspI fragment were used to probe petal cDNA libraries. Selected restriction endonuclease sites are marked. Refer to Table 2 and Table 4 for a description of the abbreviations.
FIG. 3 is a diagrammatic representation of the plasmid pCGP175 containing the petunia F3′5′H petHf2 cDNA clone from P. hybrida cv. OGB. Selected restriction endonuclease sites are marked. Refer to Table 2 and Table 4 for a description of the abbreviations.
FIG. 4 is a diagrammatic representation of the plasmid pCGP1303 containing a subclone of the petunia F3′5′H petHf1 cDNA clone from pCGP601. The construction of pCGP1303 is described in Example 4. Selected restriction endonuclease sites are marked. Refer to Table 2 and Table 4 for a description of the abbreviations.
FIG. 5 is a diagrammatic representation of the binary plasmid pCGP1452. The AmCHS 5′: petHf1: petD8 3′ gene from pCGP485 was cloned into the binary vector pWTT2132 (DNAP) in a tandem orientation with the chimaeric SuRB gene. The construction of pCGP1452 is described in Example 4. Selected restriction endonuclease sites are marked. Refer to Table 2 and Table 4 for a description of the abbreviations.
FIG. 6 is a diagrammatic representation of the binary plasmid pWTT2132 (DNAP) containing the 35S 5′: SuRB selectable marker gene and a multi-cloning site. A description of pWTT2132 is given in Example 4. Selected restriction endonuclease sites are marked. Refer to Table 2 and Table 4 for a description of the abbreviations.
FIG. 7 is a diagrammatic representation of the plasmid pCGP725. The AmCHS 5′: petHf1: petD8 3′ gene from pCGP485 was cloned into pBluescript II (KS (+) vector. The construction of pCGP725 is described in Example 4. Selected restriction endonuclease sites are marked. Refer to Table 2 and Table 4 for a description of the abbreviations.
FIG. 8 is a diagrammatic representation of the binary plasmid pCGP1453. The Mac: petHf1: mas 3′ gene from pCGP628 was cloned into the binary vector pWTT2132 (DNAP) in a tandem orientation with the chimaeric SuRB gene. The construction of pCGP1453 is described in Example 4. Selected restriction endonuclease sites are marked. Refer to Table 2 and Table 4 for a description of the abbreviations.
FIG. 9 is a diagrammatic representation of the binary plasmid pCGP1457. The petD8 5′: petHf1: petD8 3′ gene from pCGP1107 was cloned into the binary vector pWTT2132 (DNAP) in a tandem orientation with the chimaeric SuRB gene. The construction of pCGP1457 is described in Example 4. Selected restriction endonuclease sites are marked. Refer to Table 2 and Table 4 for a description of the abbreviations.
FIG. 10 is a diagrammatic representation of the binary plasmid pCGP1461. The shortpetFLS 5′: petHf1: petFLS 3′ gene from pCGP497 was cloned into the binary vector pWTT2132 (DNAP) in a tandem orientation with the chimeric SuRB gene. The construction of pCGP1461 is described in Example 4. Selected restriction endonuclease sites are marked. Refer to Table 2 and Table 4 for a description of the abbreviations.
FIG. 11 is a diagrammatic representation of the binary plasmid pCGP1616. The petRT 5′: petHf1: nos 3′ gene from pCGP846 was cloned into the binary vector pWTT2132 (DNAP) in a tandem orientation with the chimaeric SuRB gene. The construction of pCGP1616 is described in Example 4. Selected restriction endonuclease sites are marked. Refer to Table 2 and Table 4 for a description of the abbreviations.
FIG. 12 is a diagrammatic representation of the binary plasmid pCGP1623. The mas/35S: petHf1: ocs 3′ gene from pCGP1619 was cloned into the binary vector pWTT2132 (DNAP) in a tandem orientation with the chimaeric SuRB gene. The construction of pCGP1623 is described in Example 4. Selected restriction endonuclease sites are marked. Refer to Table 2 and Table 4 for a description of the abbreviations.
FIG. 13 is a diagrammatic representation of the binary plasmid pCGP1638. The CaMV 35S: petHf1: nos 3′ gene from pCGP1636 was cloned into the binary vector pWTT2132 (DNAP) in a tandem orientation with the chimaeric SuRB gene. The construction of pCGP1636 is described in Example 4. Selected restriction endonuclease sites are marked. Refer to Table 2 and Table 4 for a description of the abbreviations.
FIG. 14 is a diagrammatic representation of the binary plasmid pCGP1860. The RoseCHS 5′: petHf1: nos 3′ gene from pCGP200 was cloned into the binary vector pWTT2132 (DNAP) in a tandem orientation with the chimaeric SuRB gene. The construction of pCGP1860 is described in Example 4. Selected restriction endonuclease sites are marked. Refer to Table 2 and Table 4 for a description of the abbreviations.
FIG. 15 is a diagrammatic representation of the binary plasmid pCGP2123. The CaMV35S: petHf2: ocs 3′ gene from pCGP2109 was cloned into the binary vector pCGP1988 in a tandem orientation with the chimaeric SuRB gene. The construction of pCGP2123 is described in Example 4. Selected restriction endonuclease sites are marked. Refer to Table 2 and Table 4 for a description of the abbreviations.
FIG. 16 is a diagrammatic representation of the binary plasmid pCGP1988. The multi-cloning site of the binary vector pWTT1132 (DNAP) was replaced with the multi-cloning site from pNEB193 (New England Biolabs). The construction of pCGP1988 is described in Example 4. Selected restriction endonuclease sites are marked. Refer to Table 2 and Table 4 for a description of the abbreviations.
FIG. 17 is a diagrammatic representation of the plasmid pCGP2105. The 35S 5′: ocs 3′ expression cassette with multiple restriction endonuclease sites between the promoter and terminator fragments is in a pBluescript SK (+) vector backbone. The construction of pCGP2105 is described in Example 4. Selected restriction endonuclease sites are marked. Refer to Table 2 and Table 4 for a description of the abbreviations.
FIG. 18 is a diagrammatic representation of the binary plasmid pCGP1307. The petD8 5′: GUS: petD8 3′ gene from pCGP1106 was cloned into the binary vector pCGP1548 in a tandem orientation to the chimaeric nptII selectable marker gene. The construction of pCGP1307 is described in Example 6. Selected restriction endonuclease sites are marked. Refer to Table 2 and Table 4 for a description of the abbreviations.
FIG. 19 is a diagrammatic representation of the binary plasmid pCGP1506. The longpetFLS 5′: GUS: petFLS 3′ gene from pCGP496 was cloned into the binary vector pBIN19 in a tandem orientation to the chimaeric nptII selectable marker gene. The construction of pCGP1506 is described in Example 6. Refer to Table 2 and Table 4 for a description of the abbreviations.
FIG. 20 is a diagrammatic representation of the binary plasmid pCGP1626. The ChyrsCHS 5′: GUS: nos 3′ gene from pCGP1622 was cloned into the binary vector pWTT2132 in a tandem orientation with the chimaeric SuRB gene. The construction of pCGP1626 is described in Example 6. Selected restriction endonuclease sites are marked. Refer to Table 2 and Table 4 for a description of the abbreviations.
FIG. 21 is a diagrammatic representation of the binary plasmid pCGP1641. The petRT 5′: GUS: petRT 3′ gene from pCGP1628 was cloned into the binary vector pWTT2132 in a tandem orientation with the chimaeric SuRB gene. The construction of pCGP1641 is described in Example 6. Selected restriction endonuclease sites are marked. Refer to Table 2 and Table 4 for a description of the abbreviations.
FIG. 22 is a diagrammatic representation of the binary plasmid pCGP1861. The RoseCHS 5′: GUS: nos 3′ gene from pCGP197 was cloned into the binary vector pWTT2132 in a tandem orientation with the chimaeric SuRB gene. The construction of pCGP1861 is described in Example 6. Refer to Table 2 and Table 4 for a description of the abbreviations.
FIG. 23 is a diagrammatic representation of the binary plasmid pCGP1953. The AmCHS 5′: GUS: petD8 3′ gene from pCGP1952 was cloned into the binary vector pWTT2132 in a tandem orientation with the chimaeric SuRB gene. The construction of pCGP1953 is described in Example 6. Refer, to Table 2 and Table 4 for a description of the abbreviations.
FIG. 24 is a diagrammatic representation of the binary plasmid pWTT2084 (DNAP) containing a 35S 5′: GUS: ocs 3′ gene in a convergent orientation to the chimaeric SuRB selectable marker gene. A description of pWTT2084 is given in Example 6. Refer to Table 2 and Table 4 for a description of the abbreviations.
FIG. 25 is a diagrammatic representation of the plasmid pCGP1959 containing the F3′5′H BP#18 cDNA clone from Viola spp. cv Black Pansy in a pBluescript SK II (+) backbone. A description of pCGP1959 is given in Example 7. Selected restriction endonuclease sites are marked. Refer to Table 2 and Table 4 for a description of the abbreviations.
FIG. 26 is a diagrammatic representation of the plasmid pCGP1961 containing the F3′5′H BP#40 cDNA clone from Viola spp. cv Black Pansy in a pBluescript SK II (+) backbone. A description of pCGP1961 is given in Example 7. Selected restriction endonuclease sites are marked. Refer to Table 2 and Table 4 for a description of the abbreviations.
FIG. 27 is a diagrammatic representation of the binary plasmid pCGP1972. The AmCHS 5′: BP#18: petD8 3′ gene from pCGP1970 was cloned into the binary vector pWTT2132 (DNAP) in a tandem orientation with the chimaeric SuRB gene. The construction of pCGP1972 is described in Example 7. Selected restriction endonuclease sites are marked. Refer to Table 2 and Table 4 for a description of the abbreviations.
FIG. 28 is a diagrammatic representation of the binary plasmid pCGP1973. The AmCHS 5′: BP#40: petD8 3′ gene from pCGP1971 was cloned into the binary vector pWTT2132 (DNAP) in a tandem orientation with the chimaeric SuRB gene. The construction of pCGP1973 is described in Example 7. Selected restriction endonuclease sites are marked. Refer to Table 2 and Table 4 for a description of the abbreviations.
FIG. 29 is a diagrammatic representation of the binary plasmid pCGP1967. The CaMV 35S: BP#18:ocs 3′ gene from pCGP1965 was cloned into the binary vector pWTT2132 (DNAP) in a tandem orientation with the chimaeric SuRB gene. The construction of pCGP1967 is described in Example 7. Selected restriction endonuclease sites are marked. Refer to Table 2 and Table 4 for a description of the abbreviations.
FIG. 30 is a diagrammatic representation of the binary plasmid pCGP1969. The CaMV 35S: BP#40:ocs 3′ gene from pCGP1966 was cloned into the binary vector pWTT2132 (DNAP) in a tandem orientation with the chimaeric SuRB gene. The construction of pCGP1969 is described in Example 7. Selected restriction endonuclease sites are marked. Refer to Table 2 and Table 4 for a description of the abbreviations.
FIG. 31 is a diagrammatic representation of the plasmid pCGP1995 containing the F3′5′H Sal#2 cDNA clone from Salvia spp, in a pBluescript SK II (+) backbone. A description of pCGP1995 is given in Example 7. Selected restriction endonuclease sites are marked. Refer to Table 2 and Table 4 for a description of the abbreviations.
FIG. 32 is a diagrammatic representation of the plasmid pCGP1999 containing the F3′5′H Sal#47 cDNA clone from Salvia spp in a pBluescript SK II (+) backbone. A description of pCGP1999 is given in Example 7. Selected restriction endonuclease sites are marked. Refer to Table 2 and Table 4 for a description of the abbreviations.
FIG. 33 is a diagrammatic representation of the binary plasmid pCGP2121. The AmCHS 5′: Sal#2: petD8 3′ gene from pCGP2116 was cloned into the binary vector pCGP1988 in a tandem orientation with the chimaeric SuRB gene. The construction of pCGP2121 is described in Example 7. Selected restriction endonuclease sites are marked. Refer to Table 2 and Table 4 for a description of the abbreviations.
FIG. 34 is a diagrammatic representation of the binary plasmid pCGP2122. The AmCHS 5′: Sal#47: petD8 3′ gene from pCGP2117 was cloned into the binary vector pCGP1988 in a tandem orientation with the chimaeric SuRB gene. The construction of pCGP2122 is described in Example 7. Selected restriction endonuclease sites are marked. Refer to Table 2 and Table 4 for a description of the abbreviations.
FIG. 35 is a diagrammatic representation of the binary plasmid pCGP2120. The CaMV 35S:Sal#2:ocs 3′ gene from pCGP2112 was cloned into the binary vector pCGP1988 in a tandem orientation with the chimaeric SuRB gene. The construction of pCGP2120 is described in Example 7. Selected restriction endonuclease sites are marked. Refer to Table 2 and Table 4 for a description of the abbreviations.
FIG. 36 is a diagrammatic representation of the binary plasmid pCGP2119. The CaMV 35S:Sal#47:ocs 3′ gene from pCGP2111 was cloned into the binary vector pCGP1988 in a tandem orientation with the chimaeric SuRB gene. The construction of pCGP2119 is described in Example 7. Selected restriction endonuclease sites are marked. Refer to Table 2 and Table 4 for a description of the abbreviations.
FIG. 37 is a diagrammatic representation of the plasmid pCGP2110 containing the F3′5′H Soll#5 cDNA clone from Sollya spp. in a pBluescript SK II (+) backbone. A description of pCGP2110 is given in Example 7. Selected restriction endonuclease sites are marked. Refer to Table 2 and Table 4 for a description of the abbreviations.
FIG. 38 is a diagrammatic representation of the binary plasmid pCGP2130. The AmCHS 5′: Soll#5: petD8 3′ gene from pCGP2128 was cloned into the binary vector pCGP1988 in a tandem orientation with the chimaeric SuRB gene. The construction of pCGP2130 is described in Example 7. Refer to Table 2 and Table 4 for a description of the abbreviations.
FIG. 39 is a diagrammatic representation of the binary plasmid pCGP2131. The CaMV 35S: Soll#5:ocs 3′ gene from pCGP2129 was cloned into the binary vector pCGP1988 in a tandem orientation with the chimaeric SuRB gene. The construction of pCGP2131 is described in Example 7. Refer to Table 2 and Table 4 for a description of the abbreviations.
FIG. 40 is a diagrammatic representation of the plasmid pCGP2231 containing the F3′5′H Kenn#31 cDNA clone from Kennedia spp. in a pBluescript SK II (+) backbone. A description of pCGP2231 is given in Example 7. Selected restriction endonuclease sites are marked. Refer to Table 2 and Table 4 for a description of the abbreviations.
FIG. 41 is a diagrammatic representation of the binary plasmid pCGP2256. The AmCHS 5′: Kenn#31: petD8 3′ gene from pCGP2242 was cloned into the binary vector pCGP1988 in a tandem orientation with the chimaeric SuRB gene. The construction of pCGP2256 is described in Example 7. Refer to Table 2 and Table 4 for a description of the abbreviations.
FIG. 42 is a diagrammatic representation of the binary plasmid pCGP2252. The CaMV 35S: Kenn#31:ocs 3′ gene from pCGP2236 was cloned into the binary vector pCGP1988 in a tandem orientation with the chimaeric SuRB gene. The construction of pCGP2252 is described in Example 7. Refer to Table 2 and Table 4 for a description of the abbreviations.
FIG. 43 is a diagrammatic representation of the plasmid pBHF2F containing the full-length F3′5′H BpeaHF2 cDNA clone tom Clitoria ternatea in a pBluescript SK II (+) backbone. A description of pBHF2F is given in Example 7. Selected restriction endonuclease sites are marked. Refer to Table 2 and Table 4 for a description of the abbreviations.
FIG. 44 is a diagrammatic representation of the binary plasmid pCGP2135. The AmCHS 5′: BpeaHF2: petD8 3′ gene from pCGP2133 was cloned into the binary vector pCGP1988 in a tandem orientation with the chimaeric SuRB gene. The construction of pCGP2135 is described in Example 7. Selected restriction endonuclease sites are marked. Refer to Table 2 and Table 4 for a description of the abbreviations.
FIG. 45 is a diagrammatic representation of the binary plasmid pBEBF5. The eCaMV 35S: BpeaHF2: nos 3′ gene was constructed by replacing the GUS fragment from pBE2113-GUSs with the Clitoria F3′5′H peaHF2 cDNA clone from pBHF2F. The construction of pBEBF5 is described in Example 7. Refer to Table 2 and Table 4 for a description of the abbreviations.
FIG. 46 is a diagrammatic representation of the binary plasmid pCGP2134. The CaMV 35S: BpeaHF2: ocs 3′ gene from pCGP2132 was cloned into the binary vector pCGP1988 in a tandem orientation with the chimaeric SuRB gene. The construction of pCGP2134 is described in Example 7. Refer to Table 2 and Table 4 for a description of the abbreviations.
FIG. 47 is a diagrammatic representation of the plasmid pG48 containing the F3′5′H Gen#48 cDNA clone from Gentiana triflora in a pBluescript SK II (+) backbone. A description of pG48 is given in Example 7. Selected restriction endonuclease sites are marked. Refer to Table 2 and Table 4 for a description of the abbreviations.
FIG. 48 is a diagrammatic representation of the binary plasmid pCGP1498. The AmCHS 5′: Gen#48: petD8 3′ gene from pCGP1496 was cloned into the binary vector pCGP1988 in a tandem orientation with the chimaeric SuRB gene. The construction of pCGP1498 is described in Example 7. Selected restriction endonuclease sites are marked. Refer to Table 2 and Table 4 for a description of the abbreviations.
FIG. 49 is a diagrammatic representation of the binary plasmid pBEGHF48. The eCaMV 35S: Gen#48: nos 3′ gene was constructed by replacing the GUS fragment from a pBE2113-GUSs with the Gentiana F3′5′H Gen#48 cDNA clone from pG48. The construction of pBEGHF48 is described in Example 7. Selected restriction endonuclease sites are marked. Refer to Table 2 and Table 4 for a description of the abbreviations.
FIG. 50 is a diagrammatic representation of the binary plasmid pCGP1982. The CaMV 35S:Gen#48:ocs 3′ gone from pCGP1981 was cloned into the binary vector pWTT2132 (DNAP) in a tandem orientation with the chimaeric SuRB gene. The construction of pCGP1982 is described in Example 7. Refer to Table 2 and Table 4 for a description of the abbreviations.
FIG. 51 is a diagrammatic representation of the plasmid pLFH8 containing the F3′5′H LBG cDNA clone from Lavandula nil in a pBluescript SK II (+) backbone. A description of pLFH8 is given in Example 7. Selected restriction endonuclease sites are marked. Refer to Table 2 and Table 4 for a description of the abbreviations.
FIG. 52 is a diagrammatic representation of the binary plasmid pBELF8. The eCaMV 35S: LBG: nos 3′ gene was constructed by replacing the GUS fragment from pBE2113-GUSs with the Lavandula F3′5′H LBG cDNA clone from pLHF8. The construction of pBELF8 is described in Example 7. Refer to Table 2 and Table 4 for a description of the abbreviations.



DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
In accordance with the present invention, genetic sequences encoding polypeptides having F3′5′H activity have been identified, cloned and assessed. The recombinant genetic sequences of the present invention permit the modulation of expression of genes encoding this enzyme by, for example, de novo expression, over-expression, sense suppression, antisense inhibition, ribozyme, minizyme and DNAzyme activity, RNAi-induction or methylation-induction or other transcriptional or post-transcriptional silencing activities. RNAi-induction includes genetic molecules such as hairpin, short double stranded DNA or RNA, and partially double stranded DNAs or RNAs with one or two single stranded nucleotide over hangs. The ability to control F3′5′H synthesis in plants permits modulation of the composition of individual anthocyanins as well as alteration of relative levels of flavonols and anthocyanins, thereby enabling the manipulation of petal color. Moreover, the present invention extends to plants and reproductive or vegetative parts thereof including flowers, fruits, seeds, vegetables, leaves, stems and the like. The present invention farther extends to ornamental transgenic or genetically modified plants. The term “transgenic” also includes progeny plants and plants from subsequent genetics and/or crosses thereof from the primary transgenic plants.
Accordingly, one aspect of the present invention provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence encoding a F3′5′H or a polypeptide having F3′5′H activity wherein expression of said nucleic acid molecule in a rose petal tissue results in detectable levels of delphinidin or delphinidin-based molecules as measured by a chromatographic technique.
Another aspect of the present invention is directed to an isolated nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence encoding a F3′5′H or a polypeptide having F3′5′H activity wherein expression of said nucleic acid molecule in a rose petal tissue results in a sufficient level and length of transcript which is translated to said F3′5′H as determined by detectable levels of delphinidin or delphinidin-based molecules as measured by a chromatographic technique.
A further aspect of the present invention is directed to an isolated nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence encoding a F3′5′H or a polypeptide having F3′5′H activity wherein expression of said nucleic acid molecule in a rose petal tissue results a full-length transcript which is detectable by Northern blot analysis of total RNA isolated from rose petals.
The present invention is described and exemplified herein by reference to the identification, cloning and manipulation of genetic sequences encoding a F3′5′H which acts on DHK as well as DHQ. Preferably, the F3′5′H enzyme is a pansy; salvia, sollya lavender or kennedia F3′5′H. The F3′5′H enzyme may also be considered to include a polypeptide or protein having a F3′5′H activity or F3′5′H-like activity. The latter encompasses derivatives having altered F3′5′H activities.
A preferred aspect of the present invention, therefore, is directed to an isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to a sequence encoding a F3′5′H or a functional mutant, derivative, part, fragment, homolog or analog thereof wherein the nucleic acid molecule is characterized by the following:
(i) the F3′5′H transcript in rose petal tissue is of sufficient level and size to encode a F3′5′H resulting in detectable delphinidin or delphinidin-based molecules in the rose petal tissue as measured by a chromatographic procedure (eg. TLC or HPLC);
(ii) the F3′5′H transcript in rose petal tissue is full-length and detected by Northern blot analysis of total RNA isolated from rose petal tissue
(iii) the F3′5′H in rose petal tissue results in detectable delphinidin or delphinidin-based molecules as measured by a chromatographic procedure (eg. TLC or HOPLC); and/or
(iv) the F3′5′H results in a visual color change in rose petal tissue.
The term delphinidin-based pigments includes the anthocyanidin, delphinidin or any derivatives thereof including but not limited to glycosylated, acylated, methylated or other modified forms. Methylated forms of delphinidin include but are not limited to the anthocyanidin petunidin (methylated at the 3′-position), malvidin (methylated at the 3′ and 5′ position), 5-O-methyl malvidin (methylated at the 5,7,3′ and 5′ positions), 5,7-O dimethyl malvidin (methylated at the 5,7,3′ and 5′ positions). The methylated anthocyanidins can also be modified by glycosylation and acylation. The term anthocyanins defines glycosylated forms of the respective anthocyanidins.
By the term “nucleic acid molecule” is meant a genetic sequence in a non-naturally occurring condition. Generally, this means isolated away from its natural state or synthesized or derived in a non-naturally-occurring environment. More specifically, it includes nucleic acid molecules formed or maintained in vitro, including genomic DNA fragments recombinant or synthetic molecules and nucleic acids in combination with heterologous nucleic acids. It also extends to the genomic DNA or cDNA or part thereof encoding F3′5′H or a part thereof in reverse orientation relative to its own or another promoter. It further extends to naturally occurring sequences following at least a partial purification relative to other nucleic acid sequences.
The term “genetic sequences” is used herein in its most general sense and encompasses any contiguous series of nucleotide bases specifying directly, or via a complementary series of bases, a sequence of amino acids in a F3′5′H enzyme. Such a sequence of amino acids may constitute a full-length F3′5′H such as is set forth in SEQ ID NO: 10 (pansy) or SEQ ID NO:12 (pansy) or SEQ ID NO:14 (salvia) or SEQ ID NO:16 (salvia) or SEQ ID NO:18 (sollya) or SEQ ID NO:32 (lavender) or SEQ ID NO:27 (kennedia) or an active truncated form thereof or may correspond to a particular region such as an N-terminal, C-terminal or internal portion of the enzyme. A genetic sequence may also be referred to as a sequence of nucleotides or a nucleotide sequence and includes a recombinant fusion of two or more sequences.
In accordance with the above aspects of the present invention there is provided a nucleic acid molecule comprising a nucleotide sequence or complementary nucleotide sequence substantially as set forth in SEQ ID NO:9 (pansy) or SEQ ID NO:11 (pansy) or SEQ ID NO:13 (salvia) or SEQ ID NO:15 (salvia) or SEQ ID NO:17 (sollya) or SEQ ID NO:31 (lavender) or SEQ ID NO:26 (kennedia) or having at least about 50% similarity thereto or capable of hybridizing to the sequence set forth in SEQ ID NO:9 or SEQ ID NO:11 or SEQ ID NO:13 or SEQ ID NO:15 or SEQ ID NO:17 or SEQ ID NO:31 or SEQ ID NO:26 under low stringency conditions.
Table 1 provides a summary of the sequence identifiers.
Alternative percentage similarities and identities (at the nucleotide or amino acid level) encompassed by the present invention include at least about 60% or at least about 65% or at least about 70% or at least about 75% or at least about 80% or at least about 85% or at least about 90% or above, such as about 95% or about 96% or about 97% or about 98% or about 99%, such as at least about 60%, 61%, 62%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%.
In a particularly preferred embodiment, there is provided an isolated nucleic acid molecule comprising a nucleotide sequence or complementary nucleotide sequence substantially as set forth in SEQ ID NO:9 (pansy) or SEQ ID NO:11 (pansy) or SEQ ID NO:13 (salvia) or SEQ ID NO:15 (salvia) or SEQ ID NO:17 (sollya) or SEQ ID NO:31 (lavender) or SEQ ID NO:26 (kennedia) or having at least about 50% similarity thereto or capable of hybridizing to the sequence set forth in SEQ ID NO:1 (petunia) or SEQ ID NO:3 (petunia) or complementary strands of either under low stringency conditions, wherein said nucleotide sequence encodes a polypeptide having a F3′5′H activity.
For the purposes of determining the level of stringency to define nucleic acid molecules capable of hybridizing to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:9 or SEQ ID NO:11 or SEQ ID NO:13 or SEQ ID NO:15 or SEQ ID NO:17 or SEQ ID NO:31 or SEQ ID NO:26 reference herein to a low stringency includes and encompasses from at least about 0% to at least about 15%, v/v formamide and from at least about 1M to at least about 2 M salt for hybridization, and at least about 1 M to at least about 2 M salt for washing conditions. Generally, low stringency is from about 25-30° C. to about 42° C. The temperature may be altered and higher temperatures used to replace the inclusion of formamide and/or to give alternative stringency conditions. Alternative stringency conditions may be applied where necessary, such as medium stringency, which includes and encompasses from at least about 16% v/v to at least about 30% v/v formamide and from at least about 0.5 M to at least about 0.9 M salt for hybridization, and at least about 0.5 M to at least about 0.9 M salt for washing conditions, or high stringency, which includes and encompasses from at least about 31% v/v to at least about 50% v/v formamide and from at least about 0.01 M to at least about 0.15 M salt for hybridization, and at least about 0.01 M to at least about 0.15 M salt for washing conditions. In general, washing is carried out Tm=69.3+041 (G+C) % (Marmur and Doty, J. Mol. Biol. 5: 109, 1962). However, the Tm of a duplex DNA decreases by 1° C. with every increase of 1% in the number of mismatch base pairs (Bonner and Laskey, Eur. J. Biochem. 46: 83, 1974). Formamide is optional in these hybridization conditions. Accordingly, particularly preferred levels of stringency are defined as follows: low stringency is 6×SSC buffer, 1.0% w/v SDS at 25-42° C.; a moderate stringency is 2×SSC buffer, 1.0% w/v SDS at a temperature in the range 20° C. to 65° C.; high stringency is 0.1×SSC buffer, 0.1% w/v SDS at a temperature of at least 65° C.
Another aspect of the present invention provides a nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence encoding an amino acid sequence substantially as set forth in SEQ ID NO:10 or SEQ ID NO:12 or SEQ ID NO:14 or SEQ ID NO:16 or SEQ ID NO:18 or SEQ ID NO:32 or SEQ ID NO:27 or an amino acid sequence having at least about 50% similarity thereto.
The term similarity as used herein includes exact identity between compared sequences at the nucleotide or amino acid level. Where there is non-identity at the nucleotide level, similarity includes differences between sequences which result in different amino acids that are nevertheless related to each other at the structural, functional, biochemical and/or conformational levels. Where there is non-identity at the amino acid level, similarity includes amino acids that are nevertheless related to each other at the structural, functional, biochemical and/or conformational levels. In a particularly preferred embodiment, nucleotide and sequence comparisons are made at the level of identity rather than similarity.
Terms used to describe sequence relationships between two or more polynucleotides or polypeptides include “reference sequence”, “comparison window”, “sequence similarity”, “sequence identity”, “percentage of sequence similarity”, “percentage of sequence identity”, “substantially similar” and “substantial identity”. A “reference sequence” is at least 12 but frequently 15 to 18 and often at least 25 or above, such as 30 monomer units, inclusive of nucleotides and amino acid residues, in length. Because two polynucleotides may each comprise (1) a sequence (i.e. only a portion of the complete polynucleotide sequence) that is similar between the two polynucleotides, and (2) a sequence that is divergent between the two polynucleotides, sequence comparisons between two (or more) polynucleotides are typically performed by comparing sequences of the two polynucleotides over a “comparison window” to identify and compare local regions of sequence similarity. A “comparison window” refers to a conceptual segment of typically 12 contiguous residues that is compared to a reference sequence. The comparison window may comprise additions or deletions (i.e. gaps) of about 20% or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Optimal alignment of sequences for aligning a comparison window may be conducted by computerized implementations of algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, Wis., USA) or by inspection and the best alignment (i.e. resulting in the highest percentage homology over the comparison window) generated by any of the various methods selected. Reference also may be made to the BLAST family of programs as, for example, disclosed by Altschul et al. (Nucl. Acids Res. 25: 3389-3402, 1997). A detailed discussion of sequence analysis can be found in Unit 19.3 of Ausubel et al. (“Current Protocols in Molecular Biology” John Wiley & Sons Inc, 1994-1998, Chapter 15, 1998).
The terms “sequence similarity” and “sequence identity” as used herein refers to the extent that sequences are identical or fictionally or structurally similar on a nucleotide-by-nucleotide basis or an amino acid-by-amino acid basis over a window of comparison. Thus, a “percentage of sequence identity”, for example, is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g. A, T, C, G, I) or the identical amino acid residue (e.g. Ala, Pro, Ser, Thr, Gly, Val, Lou, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. For the purposes of the present invention, “sequence identity” will be understood to mean the “match percentage” calculated by the DNASIS computer program (Version 2.5 for windows; available from Hitachi Software engineering Co., Ltd, South San Francisco, Calif., USA) using standard defaults as used in the reference manual accompanying the software. Similar comments apply in relation to sequence similarity.
The nucleic acid molecules of the present invention may be flier characterized by having, or previously having, prior to derivatization on overall lower AT content (or higher GC content) compared to a nucleic acid molecule which encodes a F3′5′H but which does not result in detectable intact transcript in rose petal tissue or, when expressed, does not result in detectable delphinidin or delphinidin-based molecules, as measured by a chromatographic procedure such as TLC or HPLC. Furthermore, the % of A's or T's in the third position of a codon is also lower than other F4′5′H enzymes. Reference herein to a chromatographic procedure includes a related procedure. By “related” means a technically related procedure or a procedure which provides a similar result. Examples of related procedures include other forms of chromatography (eg. gas chromatography).
In addition, nucleotide sequences which do not express well in rose tissue may be modified such as in reducing overall % AT or at least reduce the levels of % AT in the third position of a codon. Such time expression in rose tissue is elevated.
The nucleic acid sequences contemplated herein also encompass oligonucleotides useful as genetic probes for amplification reactions or as antisense or sense molecules capable of regulating expression of the corresponding gene in a plant. Sense molecules include hairpin constructs, short double stranded DNAs and RNAs and partially double stranded DNAs and RNAs which one or more single stranded nucleotide over hangs. An antisense molecule as used herein may also encompass a genetic construct comprising the structural genomic or cDNA gene or part thereof in reverse orientation relative to its or another promoter. It may also encompass a homologous genetic sequence. An antisense or sense molecule may also be directed to terminal or internal portions of the gene encoding a polypeptide having a F3′5′H activity or to combinations of the above such that the expression of the gene is reduced or eliminated.
With respect to this aspect of the invention, there is provided an oligonucleotide of 5-50 nucleotides such as 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55 having substantial similarity to a part or region of a molecule with a nucleotide sequence set forth in SEQ ID NO:9 or SEQ ID NO:11 or SEQ ID NO:13 or SEQ ID NO:15 or SEQ ID NO:17 or SEQ ID NO:31 or SEQ ID NO:26. By substantial similarity or complementarity in this context is meant a hybridizable similarity under low, alternatively and preferably medium and alternatively and most preferably high stringency conditions specific for oligonucleotide hybridization (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratories, Cold Spring Harbor, N.Y., USA, 1989). Such an oligonucleotide is useful, for example, in screening for F3′5′H genetic sequences from various sources or for monitoring an introduced genetic sequence in a transgenic plant. The preferred oligonucleotide is directed to a conserved F3′5′H genetic sequence or a sequence conserved within a plant genus, plant species and/or plant variety.
In one aspect of the present invention, the oligonucleotide corresponds to the 5′ or the 3′ end of the F3′5′H genetic sequences. For convenience, the 5′ end is considered herein to define a region substantially between the start codon of the structural gene to a centre portion of the gene, and the 3′ end is considered herein to define a region substantially between the centre portion of the gene and the terminating codon of the structural gene. It, is clear, therefore, that oligonucleotides or probes may hybridize to the 5′ end or the 3′ end or to a region common to both the 5′ and the 3′ ends, the present invention extends to all such probes.
In one embodiment, the nucleic acid sequence encoding a F3′5′H or various functional derivatives thereof is used to reduce the level of an endogenous a F3′5′H (e.g. via co-suppression or antisense-mediated suppression) or other post-transcriptional gene silencing (PTGS) processes including RNAi or alternatively the nucleic acid sequence encoding this enzyme or various derivatives or parts thereof is used in the sense or antisense orientation to reduce the level of a F3′5′H. The use of sense strands, double or partially single stranded such as constructs with hairpin loops is particularly useful in inducing a PTGS response. In a further alternative, ribozymes, minizymes or DNAzymes could be used to inactivate target nucleic acid sequences.
Still a further embodiment encompasses post-transcriptional inhibition to reduce translation into polypeptide material. Still yet another embodiment involves specifically inducing or removing methylation.
Reference herein to the altering of a F3′5′H activity relates to an elevation or reduction in activity of up to 30% or more preferably of 30-50%, or even more preferably 50-75% or still more preferably 75% or greater above or below the normal endogenous or existing levels of activity. Such elevation or reduction may be referred to as modulation of a F3′5′H enzyme activity. Generally, modulation is at the level of, transcription or translation of F3′5′H genetic sequences.
The nucleic acids of the present invention may be a ribonucleic acid or deoxyribonucleic acids, single or double stranded and linear or covalently closed circular molecules. Preferably, the nucleic acid molecule is cDNA. The present invention also extends to other nucleic acid molecules which hybridize under low, preferably under medium and most preferably under high stringency conditions with the nucleic acid molecules of the present invention and in particular to the sequence of nucleotides set forth in SEQ ID NO:9 or SEQ ID NO:11 or SEQ ID NO:13 or SEQ ID NO:15 or SEQ ID NO:17 or SEQ ID NO:31 or SEQ ID NO:26 or a part or region thereof. In its most preferred embodiment, the present invention extends to a nucleic acid molecule having a nucleotide sequence set forth in SEQ ID NO:9 or SEQ ID NO:11 or SEQ ID NO:13 or SEQ ID NO:15 or SEQ ID NO:17 or SEQ ID NO:31 or SEQ ID NO:26 or to a molecule having at least 40%, more preferably at least 45%, even more preferably at least 55%, still more preferably at least 65%-70%, and yet even more preferably greater than 85% similarity at the level of nucleotide or amino acid sequence to at least one or more regions of the sequence set forth in SEQ ID NO:9 or SEQ ID NO:11 or SEQ ID NO:13 or SEQ ID NO:15 or SEQ ID NO:17 or SEQ ID NO:31 or SEQ ID NO:26 and wherein the nucleic acid encodes or is complementary to a sequence which encodes an enzyme having a F3′5′H activity. It should be noted, however, that nucleotide or amino acid sequences may have similarities below the above given percentages and yet still encode a F3′5′H activity and such molecules may still be considered in the scope of the present invention where they have regions of sequence conservation. The present invention further extends to nucleic acid molecules in the form of oligonucleotide primers or probes capable of hybridizing to a portion of the nucleic acid molecules contemplated above, and in particular those set forth in SEQ ID NO:9 or SEQ ID NO:11 or SEQ ID NO:13 or SEQ ID NO:15 or SEQ ID NO:17 or SEQ ID NO:31 or SEQ ID NO:26, under low, preferably under medium and most preferably under high stringency-conditions. Preferably the portion corresponds to the 5′ or the 3′ end of the gene. For convenience the 5′ end is considered herein to define a region substantially between the start codon of the structural genetic sequence to a centre portion of the gene, and the 3′ end is considered herein to define a region substantially between the centre portion of the gene and the terminating codon of the structural genetic sequence. It is clear, therefore, that oligonucleotides or probes nay hybridize to the 5′ end or the 3′ end or to a region common to both the 5′ and the 3′ ends. The present invention extends to all such probes.
The term gone is used in its broadest sense and includes cDNA corresponding to the exons of a gene. Accordingly, reference herein to a gene is to be taken to include:
(i) a classical genomic gene consisting of transcriptional and/or translational regulatory sequences and/or a coding region and/or non-translated sequences (i.e. introns, 5′- and 3′-untranslated sequences); or
(ii) mRNA or cDNA corresponding to the coding regions (i.e. exons) and 5′- and 3′-untranslated sequences of the gene.
The term gene is also used to describe synthetic or fusion molecules encoding all or part of an expression product. In particular embodiments, the term nucleic acid molecule and gene may be used interchangeably.
The nucleic acid or its complementary form may encode the full-length enzyme or a part or derivative thereof. By “derivative” is meant any single or multiple amino acid substitutions, deletions, and/or additions relative to the naturally occurring enzyme and which retains a F3′5′H activity. In this regard, the nucleic acid includes the naturally occurring nucleotide sequence encoding a F3′5′H or may contain single or multiple nucleotide substitutions, deletions and/or additions to said naturally occurring sequence. The nucleic acid of the present invention or its complementary form may also encode a “part” of the F3′5′H, whether active or inactive, and such a nucleic acid molecule may be useful as an oligonucleotide probe, primer for polymerase chain reactions or in various mutagenic techniques, or for the generation of antisense molecules.
Reference herein to a “part” of a nucleic acid molecule, nucleotide sequence or amino acid sequence, preferably relates to a molecule which contains at least about 10 contiguous nucleotides or five contiguous amino acids, as appropriate.
Amino acid insertional derivatives of the F3′5′H of the present invention include amino and/or carboxyl terminal fusions as well as intra-sequence insertions of single or multiple amino acids. Insertional amino acid sequence variants are those in which one or more amino acid residues are introduced into a predetermined site in the protein although random insertion is also possible with suitable screening of the resulting product. Deletional variants are characterized by the removal of one or more amino acids from the sequence. Substitutional amino acid variants are those in which at least one residue in the sequence has been removed and a different residue inserted in its place. Typical substitutions are those made in accordance with Table 3.







TABLE 3







Suitable residues for amino acid substitutions








Original residue
Exemplary substitutions





Ala
Ser


Arg
Lys


Asn
Gln; His


Asp
Glu


Cys
Ser


Gln
Asn; Glu


Glu
Asp


Gly
Pro


His
Asn; Gln


Ile
Leu; Val


Leu
Ile; Val


Lys
Arg; Gln; Glu


Met
Leu; Ile; Val


Phe
Met; Leu; Tyr


Ser
Thr


Thr
Ser


Trp
Tyr


Tyr
Trp; Phe


Val
Ile; Leu; Met









Where the F3′5′H is derivatized by amino acid substitution, the amino acids are generally replaced by other amino acids having like properties, such as hydrophobicity, hydrophilicity, electronegativity, bulky side chains and the like. Amino acid substitutions are typically of single residues. Amino acid insertions will usually be in the order of about 1-10 amino acid residues and deletions will range from about 1-20 residues. Preferably, deletions or insertions are made in adjacent pairs, i.e. a deletion of two residues or insertion of two residues.
The amino acid variants referred to above may readily be made using peptide synthetic techniques well known in the art, such as solid phase peptide synthesis (Merrifield, J. Am. Chem. Soc. 85: 2149, 1964) and the like, or by recombinant DNA manipulations. Techniques for making substitution mutations at predetermined sites in DNA having known or partially known sequence are well known and include, for example, M13 mutagenesis. The manipulation of DNA sequence to produce variant proteins which manifest as substitutional, insertional or deletional variants are conveniently described, for example, in Sambrook et al. (1989, supra).
Other examples of recombinant or synthetic mutants and derivatives of the F3′5′H enzyme of the present invention include single or multiple substitutions, deletions and/or additions of any molecule associated with the enzyme such as carbohydrates, lipids and/or proteins or polypeptides.
The terms “analogs” and “derivatives” also extend to any functional chemical equivalent of a F3′5′H and also to any amino acid derivative described above. For convenience, reference to F3′5′H herein includes reference to any functional mutant, derivative, part, fragment, homolog or analog thereof.
The present invention is exemplified using nucleic acid sequences derived from pansy, salvia, sollya or lavender or kennedia since this represents die most convenient and preferred source of material to date. However, one skilled in the art will immediately appreciate that similar sequences can be isolated from any number of sources such as other plants or certain microorganisms. All such nucleic acid sequences encoding directly or indirectly a F3′5′H are encompassed by the present invention regardless of their source. Examples of other suitable sources of genes encoding F3′5′H include, but are not limited to Vitis spp., Babiana stricta, Pinus spp., Picea spp., Larix spp., Phaseolus spp., Vaccinium spp., Cyclamen spp., Iris spp., Pelargonium spp., Lipadeae, Geranium spp., Pisum spp., Lathyrus spp., Clitoria spp., Catharanthus spp., Malva spp., Mucana spp., Vicia spp., Saintpaulia spp., Lagerstroemia spp., bouchina spp., Plumbago spp., Hypocalyptus spp., Rhododendron spp., Linum spp., Macroptilium spp., Hibiscus spp., Hydrangea spp., Cymbidium spp., Millettia spp., Hedysarum spp., Lespedeza spp., Asparagus spp. Antigonon spp., Freesia spp., Brunella spp., Clarkia spp., etc.
In accordance with the present invention, a nucleic acid sequence encoding a F3′5′H may be introduced into and expressed in a transgenic plant in either orientation thereby providing a means either to convert suitable substrates, if synthesized in the plant cell; ultimately into DHM, or alternatively to inhibit such conversion of metabolites by reducing or eliminating endogenous or existing F3′5′H activity. The production of these 3′,5′-hydroxylated substrates will subsequently be converted to delphinidin-based pigments that will modify petal color and may contribute to the production of a bluer color. Expression of the nucleic acid sequence in the plant may be constitutive, inducible or developmental and may also be tissue-specific. The word “expression” is used in its broadest sense to include production of RNA or of both RNA and protein. It also extends to partial expression of a nucleic acid molecule.
According to this aspect of the present invention, there is provided a method for producing a transgenic flowering plant capable of synthesizing a F3′5′H, said method comprising stably transforming a cell of a suitable plant with a nucleic acid sequence which comprises a sequence of nucleotides encoding said F3′5′H under conditions permitting the eventual expression of said nucleic acid sequence, regenerating a transgenic plant from the cell and growing said transgenic plant for a time and under conditions sufficient to permit the expression of the nucleic acid sequence. The transgenic plant may thereby produce non-indigenous F3′5′H at elevated levels relative to the amount expressed in a comparable non-transgenic plant.
Another aspect of the present invention contemplates a method for producing a transgenic plant with reduced indigenous or existing flavonoid 3′,5′-hydroxylase activity, said method comprising stably transforming a cell of a suitable plant with a nucleic acid molecule which comprises a sequence of nucleotides encoding or complementary to a sequence encoding a F3′5′H activity, regenerating a transgenic plant from the cell and where necessary growing said transgenic plant under conditions sufficient to permit the expression of the nucleic acid.
Yet another aspect of the present invention contemplates a method for producing a genetically modified plant with reduced indigenous or existing F3′5′H activity, said method comprising altering the F3′5′H gene through modification of the indigenous sequences via homologous recombination from an appropriately altered F3′5′H gene or derivative or part thereof introduced into the plant cell, and regenerating the genetically modified plant from the cell.
As used herein an “indigenous” enzyme is one, which is native to or naturally expressed in a particular cell. A “non-indigenous” enzyme is an enzyme not native to the cell but expressed through the introduction of genetic material into a plant cell, for example, through a transgene. An “endogenous” enzyme is an enzyme produced by a cell but which may or may not be indigenous to that cell.
In a preferred embodiment, the present invention contemplates a method for producing a transgenic flowering plant exhibiting altered floral or inflorescence properties, said method comprising stably transforming a cell of a suitable plant with a nucleic acid sequence of the present invention, regenerating a transgenic plant from the cell and growing said transgenic plant for a time and under conditions sufficient to permit the expression of the nucleic acid sequence.
Alternatively, said method may comprise stably transforming a cell of a suitable plant with a nucleic acid sequence of the present invention or its complementary sequence, regenerating a transgenic plant from the cell and growing said transgenic plant or a time and under conditions sufficient to alter the level of activity of the indigenous or existing F3′5′H. Preferably the altered level would be less than the indigenous or existing level of F3′5′H activity in a comparable non-transgenic plant. Without wishing to limit the present invention, one theory of mode of action is that reduction of the indigenous F3′5′H activity requires the expression of the introduced nucleic acid sequence or its complementary sequence. However, expression of the introduced genetic sequence or its complement may not be required to achieve the desired effect: namely, a flowering plant exhibiting altered floral or inflorescence properties.
In a related embodiment, the present invention contemplates a method for producing a flowering plant exhibiting altered floral or inflorescence properties, said method comprising alteration of the flavonoid 3′,5′-hydroxylase gene through modification of the indigenous sequences via homologous recombination from an appropriately altered F3′5′H gene or derivative or part thereof introduced into the plant cell, and regenerating the genetically modified plant from the cell.
Preferably, the altered floral or inflorescence includes the production of different shades of blue or purple or red flowers or other colors, depending on the genotype and physiological conditions of the recipient plant.
Accordingly, the present invention extends to a method for producing a transgenic plant capable of expressing a recombinant gene encoding a F3′5′H or part thereof or which caries a nucleic acid sequence which is substantially complementary to all or a part of a mRNA molecule encoding the F3′5′H, said method comprising stably transforming a cell of a suitable plant with the isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to a sequence encoding, a F3′5′H, where necessary under conditions permitting the eventual expression of said isolated nucleic acid molecule, and regenerating a transgenic plant from the cell. By suitable plant is meant a plant capable of producing DHK and possessing the appropriate physiological properties required for the development of the color desired.
One skilled in the art will immediately recognise the variations applicable to the methods of the present invention, such as increasing or decreasing the expression of the enzyme naturally present in a target plant leading to differing shades of colors such as different shades of blue, purple or red.
The present invention, therefore, extends to all transgenic plants or parts or cells therefrom of transgenic plants or progeny of the transgenic plants containing all or part of the nucleic acid sequences of the present invention, or antisense forms thereof and/or any homologs or related forms thereof and, in particular, those transgenic plants which exhibit altered floral or inflorescence properties. The transgenic plants may contain an introduced nucleic acid molecule comprising a nucleotide sequence encoding or complementary to a sequence encoding a F3′5′H. Generally, the nucleic acid would be stably introduced into the plant genome, although the present invention also extends to the introduction of a F3′5′H nucleotide sequence within an autonomously-replicating nucleic acid sequence such as a DNA or RNA virus capable of replicating within the plant cell. The invention also extends to seeds from such transgenic plants. Such seeds, especially if colored, are useful as proprietary tags for plants. Any and all methods for introducing genetic material into plant cells including but not limited to Agrobacterium-mediated transformation, biolistic particle bombardment etc. are encompassed by the present invention.
Another aspect of the present invention contemplates the use of the extracts from transgenic plants or plant parts or cells therefrom of transgenic plants or progeny of the transgenic plants containing all or part of the nucleic acid sequences of the present invention and, in particular, the extracts from those transgenic plants when used as a flavouring or food additive or health product or beverage or juice or coloring.
Plant parts contemplated by the present invention includes, but is not limited to flowers, fruits, vegetables, nuts, roots, stems, leaves or seeds.
The extracts of the present invention may be derived from the plants or plant part or cells therefrom in a number of different ways including but not limited to chemical extraction or heat extraction or filtration or squeezing or pulverization.
The plant, plant part or cells therefrom or extract can be utilized in any number of different ways such as for the production of a flavouring (e.g. a food essence), a food additive (e.g. a stabilizer, a colorant) a health product (e.g. an antioxidant, a tablet) a beverage (e.g. wine, spirit, tea) or a juice (e.g. fruit juice) or coloring (e.g. food coloring, fabric coloring, dye, paint, tint).
A further aspect of the present invention is directed to recombinant forms of F3′5′H. The recombinant forms of the enzyme will provide a source of material for research, for example, more active enzymes and may be useful in developing in vitro systems for production of colored compounds.
Still a further aspect of the present invention contemplates the use of the genetic sequences described herein in the manufacture of a genetic construct capable of expressing a F3′5′H or down-regulating an indigenous F3′5′H enzyme in a plant.
The term genetic construct has been used interchangeably throughout the specification and alims with the terms “fusion molecule”, “recombinant molecule”, “recombinant nucleotide sequence”. A genetic construct may include a single nucleic acid molecule comprising a nucleotide sequence encoding a single protein or may contain multiple open reading frames encoding 2 or more proteins. It may also contain a promoter operably linked to 1 or more of the open reading frames.
Another aspect of the present invention is directed to a prokaryotic or eukaryotic organism carrying a genetic sequence encoding a F3′5′H extrachromasomally in plasmid form.
The present invention further extends to a recombinant polypeptide comprising a sequence of amino acids substantially as set forth in SEQ ID NO:10 or SEQ ID NO:12 or SEQ ID NO:14 or SEQ ID NO:16 or SEQ ID NO:18 or SEQ ID NO:32 or SEQ ID NO:27 or an amino acid sequence having at least about 50% similarity to SEQ ID NO:10 or SEQ ID NO:12 or SEQ ID NO: 14 or SEQ ID NO:16 or SEQ ID NO:18 or SEQ ID NO:32 or SEQ ID NO:27 or a derivative of said polypeptide.
A “recombinant polypeptide” means a polypeptide encoded by a nucleotide sequence introduced into a cell directly or indirectly by human intervention or into a parent or other relative or precursor of the cell. A recombinant polypeptide may also be made using cell-free, in vitro transcription systems. The term “recombinant polypeptide” includes an isolated polypeptide or when present in a cell or cell preparation. It may also be in a plant or parts of a plant regenerated from a cell which produces said polypeptide.
A “polypeptide” includes a peptide or protein and is encompassed by the term “enzyme”.
The recombinant polypeptide may also be a fusion molecule comprising two or more heterologous amino acid sequences.
The present invention is further described by the following non-limiting Examples.
Example 1
General Methods
In general, the methods followed were as described in Sambrook et al. (1989, supra) or Sambrook and Russell, Molecular Cloning: A Laboratory Manual 3rd edition, Cold Spring Harbor Laboratories, Cold Spring Harbor, N.Y., USA, 2001 or Plant Molecular Biology Manual (2nd edition), Gelvin and Schilperoot (ads), Kluwer Academic Publisher, The Netherlands, 1994 or Plant Molecular Biology Labfax, Croy (ed), Bios scientific Publishers, Oxford, UK, 1993.
The cloning vectors pBluescript and PCR script were obtained from Stratagene, USA pCR7 2.1 was obtained from Invitrogen, USA.
E. coli Transformation
The Escherichia coli strains used were:
DH5α
supE44, Δ (lacZYA-ArgF)U169, (ø80lacZΔM15), hsdR17(rk−, mk+).
recA1, endA1, gyrA96, thi-1, relA1, deoR (Hanahan, J. Mol. Biol. 166: 557, 1983)
XL1-Blue
supE44, hsdR17(rk−, mk+), recA1, endA1, gyrA96, thi-1, relA1,
lac−, [F′proAB, lac1q, lacZΔM15, Tn10(tetR)] (Bullock et al., Biotechniques 5: 376, 1987).
BL21-CodonPlus-RIL Strain
ompT hsdS(Rb− mB−) dcm+ Tetr gal endA Hte [argU ileY leuW Camr]
M15 E. coli is derived from E. coli K12 and has the phenotype NalS, StrS, RifS, Thi−, Ara+, Gal+, Mtl−, F−, RecA+, Uvr+, Lon+.
Transformation of the E. coli strains was performed according to the method of Inoue et al., (Gene 96: 23-28, 1990).
Agrobacterium tumefaciens Strains and Transformations
The disarmed Agrobacterium tumefaciens strain used was AGL0 (Lazo et at. Bio/technology 9: 963-967, 1991).
Plasmid DNA was introduced into the Agrobacterium tumefaciens strain AGL0 by adding 5 μg of plasmid DNA to 100 μL of competent AGL0 cells prepared by inoculating a 50 mL LB culture (Sambrook et al., 1989, supra) and incubation for 16 hours with shaking at 28° C. The cells were then pelleted and resuspended in 0.5 mL of 85% (v/v) 100 mM CaCl2/15% (v/v) glycerol. The DNA-Agrobacterium mixture was frozen by incubation in liquid N2 for 2 minutes and then allowed to thaw by incubation at 37° C. for 5 minutes. The DNA/bacterial mix was then placed on ice for a further 10 minutes. The cells were then mixed with 1 mL of LB (Sambrook et al., 1989 supra) media and incubated with long for 16 hours at 28° C. Cells of A. tumefaciens carrying the plasmid were selected on LB agar plates containing appropriate antibiotics such as 50 μg/mL tetracycline or 100 μg/mL gentamycin. The confirmation of the plasmid in A. tumefaciens was done by restriction endonuclease mapping of DNA isolated from the antibiotic-resistant transformants.
DNA Ligations
DNA ligations were carried out using the Amersham Ligation Kit or Promega Ligation Kit according to procedures recommended by the manufacturer.
Isolation and Purification of DNA Fragments
Fragments were generally isolated on a 1% (w/v) agarose gel and purified using the QIAEX II Gel Extraction kit (Qiagen) or Bresaclean Kit (Bresatec, Australia) following procedures recommended by the manufacturer.
Repair of Overhanging Ends after Restriction Endonuclease Digestion
Overhanging 5′ ends were repaired using DNA polymerase (Klenow fragment) according to standard protocols (Sambrook et al., 1989 supra). Overhanging 3′ ends were repaired using T4 DNA polymerase according to standard protocols (Sambrook et al., 1989 supra).
Removal of Phosphoryl Groups from Nucleic Acids
Shrimp alkaline phosphatase (SAP) (USB) was typically used to remove phosphoryl groups from cloning vectors to prevent re-circularization according to the manufacturer's recommendations.
Polymerase Chain Reaction (PCR)
Unless otherwise specified, PCR conditions using plasmid DNA as template included using 2 ng of plasmid DNA, 100 ng of each primer, 2 μL 10 mM dNTP mix, 5 μL 10×Taq DNA polymerase buffer, 0.5 μL Taq DNA Polymerase in a total volume of 50 μL. Cycling conditions comprised an initial denaturation step of 5 minutes at 94° C., followed by 35 cycles of 94° C. for 20 sec, 50° C. for 30 sec and 72° C. for 1 minute with a final treatment at 72° C. for 10 minutes before storage at 4° C.
PCRs were performed in a Perkin Elmer GeneAmp PCR System 9600.
32P-Labelling of DNA Probes
DNA fragments (50 to 100 ng) were radioactively labelled with 50 μCi of [α-32P]-dCTP using a Gigaprime kit (Geneworks). Unincorporated [α-32P]-dCTP was removed by chromatography on Sephadex G-50 (Fine) columns or Microbiospin P-30 Tris chromatography columns (BioRad).
Plasmid Isolation
Single colonies were analyzed for inserts by inoculating LB broth (Sambrook et al., 1989, supra) with appropriate antibiotic selection (e.g. 100 μg/mL ampicillin or 10 to 50 μL/mL tetracycline etc.) and incubating the liquid culture at 37° C. (for E. coli) or 29° C. (for A. tumefaciens) for  ̃16 hours with shaking. Plasmid DNA was purified using the alkali-lysis procedure (Sambrook et al., 1989, supra) or using The WizardPlus SV minipreps DNA purification system (Promega) or Qiagen Plasmid Mini Kit (Qiagen). Once the presence of an insert had been determined, larger amounts of plasmid DNA were prepared from 50 mL overnight cultures using the alkali-lysis procedure (Sambrook et al., 1989, supra) or QIAfilter Plasmid Midi kit (Qiagen) and following conditions recommended by the manufacturer.
DNA Sequence Analysis
DNA sequencing was performed using the PRISM (trademark) Ready Reaction Dye Primer Cycle Sequencing Kits from Applied Biosystems. The protocols supplied by the manufacturer were followed. The cycle sequencing reactions were performed using a Perkin Elmer PCR machine (GeneAmp PCR System 9600). Sequencing runs were generally performed by the Australian Genome Research Facility at The Walter and Eliza Hall Institute of Medical Research (Melbourne, Australia) or in-house on an automated 373A DNA sequencer (Applied Biosystems).
Sequences were analysed using a MacVectorTM application (version 6.5.3) (Oxford Molecular Ltd., Oxford, England).
Homology searches against Genbank, SWISS-PROT and EMBL databases were performed using the FASTA and TFASTA programs (Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85(8): 2444-2448, 1988) or BLAST programs (Altschul et al., J. Mol. Biol. 275(3): 403-410, 1990). Percentage sequence similarities were obtained using LALIGN program (Huang and Miller, Adv. Appl. Math. 12: 373-381, 1991) or ClustalW program (Thompson et al., Nucleic Acids Research 22: 4673-4680, 1994) within the MacVectorTM application (Oxford Molecular Ltd., England) using default settings.
Multiple sequence alignments were produced using ClustalW (Thompson et al., 1994, supra) using default settings.
Example 2
Plant Transformations
Petunia hybrida Transformations (Sw63×Skr4)
As described in Holton et al. (1993a supra) by any other method well known in the art.
Rosa hybrida Transformations
As described in U.S. patent application Ser. No. 542,841 (PCT/US91/04412) or Robinson and Firoozabady (Scientia Horticulturae, 55: 83-99, 1993). Rout et al. (Scientia Horticulturae, 81: 201-238, 1999) or Marchant et al. (Molecular Breeding 4: 187-194, 1998) or by any other method well known in the art.
Cuttings of Rosa hybrida were generally obtained from Van Wyk and Son Flower Supply, Victoria.
Dianthus caryophyllus Transformations
International Patent Application No. PCT/US92/02612 (carnation transformation). As described in International Patent Application No. PCT/AU96/00296 (Violet carnation), Lu et al. (Bio/Technology 9: 864-868, 1991), Robinson and Firoozabady (1993, supra) or by any other method well known in the art.
Cuttings of Dianthus caryophyllus cv. Kortina Chanel or Monte Lisa were obtained from Van Wyk and Son Flower Supply, Victoria.
Example 3
Transgenic Analysis
Color Coding
The Royal Horticultural Society's Color Chart (Kew, UK) was used to provide a description of observed color. They provide an alternative means by which to describe the color phenotypes observed. The designated numbers, however, should be taken only as a guide to the perceived colors and should not be regarded as limiting the possible colors which may be obtained.
Chromatographic Analysis
Thin Layer Chromatography (TLC) and High Performance Liquid Chromatography (HPLC) analysis was performed generally as described in Brugliera et al. (Plant J. 5, 81-92, 1994).
Extraction of Anthocyanidins
Prior to HPLC analysis, the anthocyanin and flavonol molecules present in petal and stamen extracts were acid hydrolysed to remove glycosyl moieties from the anthocyanidin or flavonol core. Anthocyanidin and flavonol standards were used to help identify the compounds present in the floral extracts.
Anthocyanidins in the reaction mixture were analysed by HPLC via gradient elution using gradient conditions of 50% B to 60% B over 10 minutes, then 60% B for 10 minutes and finally 60% B to 100% B over 5 minutes where solvent A consisted of TFA: H2O (5:995) and solvent B consisted of acetonitrile: TFA: H2O (500:5:495). An Asahi Pac ODP-50 cartridge column (250 mm×4.6 mm ID) was used for the reversed phase chromatographic separations. The flow rate was 1 mL/min and the temperature was 40° C. The detection of the anthocyanidin compounds was carried out using a Shimadzu SPD-M6A three dimensional detector at 400-650 nm.
The anthocyanidin peaks were identified by reference to known standards, viz delphinidin or delphinidin-based molecules, petunidin, malvidin, cyanidin and peonidin.
States of Flower Development
Petunia 
Petunia hybrida cv. Skr4×Sw63 flowers were harvested at developmental stages defined as follows:
Stage 1: Unpigmented, closed bud.
Stage 2: Pigmented, closed bud.
Stage 3: Pigmented bud with emerging corolla
Stage 4: Pigmented, opened flower with anther intact (pre-dehiscence)
Stage 5: Fully opened flower with all anthers dehisced.
For TLC or HPLC analysis, petals were collected from stage 4 flowers at the stage of maximum pigment accumulation.
For Northern blot analysis, petals were collected from stages 2 to 3 flowers at the stage of maximal expression of flavonoid pathway genes.
Carnation.
Dianthus caryophyllus flowers were harvested at developmental stages defined as follows:
Stage 1: Closed bud, petals not visible.
Stage 2: Flower buds opening: tips of petals visible.
Stage 3: Tips of nearly all petals exposed. “Paint-brush stage”.
Stage 4: Outer petals at 45° angle to stem.
Stage 5: Flower fully open.
For TLC or HPLC analysis, petals were collected from stage 4 flowers at the stage of maximum pigment accumulation.
For Northern blot analysis, petals were collected from stage 3 flowers at the stage of maximal expression of flavonoid pathway genes.
Rose
Stages of Rosa hybrida flower development were defined as follows:
Stage 1: Unpigmented, tightly closed bud.
Stage 2: Pigmented, tightly closed bud
Stage 3: Pigmented, closed bud; sepals just beginning to open.
Stage 4: Flower bud beginning to open; petals heavily pigmented; sepals have separated.
Stage 5: Sepals completely unfolded; some curling. Petals are heavily pigmented and unfolding.
For TLC or HPLC analysis, petals were collected from stage 4 flowers at the stage of maximum pigment accumulation.
For Northern blot analysis, petals were collected from stage 3 to 4 flowers at the stage of maximal expression of flavonoid pathway genes (Tanaka et al., Plant Cell Physiol., 36(6): 1023-1031, 1995).
Anthocyanin/Flavonol Measurements by Spectrophotometric Measurements
Approximately 200 mg of fresh petal tissue was added to 2 mL of methanol/1% (v/v) HCl and incubated for  ̃16 hours at 4° C. A 1 in 20 dilution (e.g. 50 μL made to 1000 μL) was then made and the absorbance at 350 nm and 530 nm was recorded.
The approximate flavonols and anthocyanin amounts (nmoles/gram) were then calculated according to the following formulae:
Anthocyanin Content




  
    
      
        
          
            (
            
              
                
                  A
                  530
                
                /
                34
              
              ⁢
              
                ,
              
              ⁢
              000
            
            )
          
          ×
          volume
          ⁢
          
            
          
          ⁢
          of
        
      
    
    
      
        
          extraction
          ⁢
          
            
          
          ⁢
          buffer
          ⁢
          
            
          
          ⁢
          
            (
            mL
            )
          
          ×
          dilution
          ⁢
          
            
          
          ⁢
          factor
          ×
          
            10
            6
          
        
      
    
  
  
    mass
    ⁢
    
      
    
    ⁢
    of
    ⁢
    
      
    
    ⁢
    petal
    ⁢
    
      
    
    ⁢
    tissue
    ⁢
    
      
    
    ⁢
    
      (
      grams
      )
    
  




Flavonol Content





  
    
      
        
          
            (
            
              
                
                  A
                  350
                
                /
                14
              
              ⁢
              
                ,
              
              ⁢
              300
            
            )
          
          ×
          volume
          ⁢
          
            
          
          ⁢
          of
        
      
    
    
      
        
          extraction
          ⁢
          
            
          
          ⁢
          buffer
          ⁢
          
            
          
          ⁢
          
            (
            mL
            )
          
          ×
          dilution
          ⁢
          
            
          
          ⁢
          factor
          ×
          
            10
            6
          
        
      
    
  
  
    mass
    ⁢
    
      
    
    ⁢
    of
    ⁢
    
      
    
    ⁢
    petal
    ⁢
    
      
    
    ⁢
    tissue
    ⁢
    
      
    
    ⁢
    
      (
      grams
      )
    
  




Northern/RNA Blot Analyst

Transcription of a transferred gene was monitored by isolating RNA and estimating the quantity and size of the expected transcript. Northern blot analysis was used to monitor the steady-state level of particular transcripts in petals. A transcript was determined to be intact or full-length based on the estimated size expected from the gene used. In general when cDNAs were used as coding sequences the size of the transcript expected would be the size of the cDNA plus any 5′ untranslated component of the fused promoter fragment plus any 3′ untranslated sequence from the fused terminator fragment. In some cases where a cDNA region contained a putative polyadenylation site and the terminator region contained a putative polyadenylation site, 2 transcripts would be detected. One would be of a size consistent with polyadenylation occurring just downstream from the polyadenylation site within the cDNA sequence. The second transcript would be larger and consistent with the transcript being polyadenylated after the polyadenylation site within the terminator fragment.
Total RNA was isolated from petals or leaves using a Plant RNAeasy kit (QIAGEN) following procedures recommended by the manufacturer. For rose samples 1% (w/v) PVP was added to the extraction buffer.
RNA samples (5 μg) were electrophoresed through 2.2 M formaldehyde/1.2% w/v agarose gels using running buffer containing 40 mM morpholinopropanesulphonic acid (pH 7.0), 5 mM sodium acetate, 0.1 mM EDTA (H 8.0). The RNA was stained with ethidium bromide and visualised under UV-light. The ribosomal RNA was generally used as a guide in confirming that the RNA had not been degraded by intra- or extra-cellular ribonucleases. The RNA was transferred to Hybond-N membrane filters (Amersham) and treated as described by the manufacturer.
Control samples were included on RNA gels as a measure of the integrity of the radiolabelled probe and as guides to expected transcript sizes. Controls for petHf1 and petHf2 genes included RNA isolated from petunia OGB petals (stages 3 to 4) or from flowers of transgenic carnations shown previously to accumulate petHf1 transcripts. Controls for other F3′5′H genes generally included RNA isolated from petals of the same species from which the F3′5′H sequence had been isolated.
RNA blots were probed with 32P-labelled fragments. Prehybridization (1 hour at 42° C.) and hybridization (16 hours at 42° C.) of the membrane filters were cared out in 50% v/v formamide, 1 M NaCl, 1% w/v SDS, 10% w/v dextran sulphate. The membrane filters were generally washed in 2×SSC, 1% w/v SDS at 65° C. for between 1 to 2 hours and then 0.2×SSC, 1% w/v SDS at 65° C. for between 0.5 to 1 hour. Membrane filters were generally exposed to Kodak XAR film with an intensifying screen at −70° C. for 16 to 72 hours.
Example 4
Introduction of Chimeric Petunia F3′5′H Genes into Rose
As described in the introduction, the pattern of hydroxylation of the B-ring of the anthocyanidin molecule plays a key role in determining petal color. The production of the dihydroflavonol DHM, leads to the production of the purple/blue delphinidin-based pigments in plants such as petunia. The absence of the F3′5′H activity has been correlated with the absence of blue flowers in many plant species such as Rosa, Gerbera, Antirrhinum, Dianthus and Dendranthema. 
Based on success in producing delphinidin-based pigments in a mutant petunia line (Holton et al., 1993a, supra and International Patent Application No. PCT/AU92/00334), in tobacco flowers (International Patent Application No. PCT/AU92/00334) and in carnation flowers (International Patent Application No. PCT/AU96/00296), similar chimeric petunia F3′5′H genes were also introduced into roses in order to produce novel delphinidin-based pigments and modify flower color.
Preparation of Chimeric Petunia F3′5′H Gene Constructs
A summary of promoter, terminator and coding fragments used in the preparation of constructs and the respective abbreviations is listed in Table 4.







TABLE 4







Abbreviations used in construct preparations








ABBREVIATION
DESCRIPTION





AmCHS 5′
1.2 kb promoter fragment from the Antirrhinum majus chalcone



synthase (CHS) gene (Sommer and Saedler, Mol Gen. Gent., 202:



429-434, 1986)


CaMV 35S
~0.2 kb incorporating BglII fragment containing the promoter



region from the Cauliflower Mosaic Virus 35S (CaMV 35S) gene -



(Franck et at., Cell 21: 285-294, 1980, Guilley et al., Cell, 30:



763-773, 1982)


35S 5′
promoter fragment from CaMV 35S gene (Franck et al., 1980,



supra) with an ~60 bp 5′ untranslated leader sequence from the



petunia chlorophyll a/b binding protein gene (Cab 22 gene)



(Harpster et al., MGG, 212: 182-190, 1988)


chrysCHS 5′
promoter region from a CHS gene from chrysanthemum (SEQ ID



NO: 30)


eCaMV 35S
enhanced CaMV 35S promoter as described in Mitsuhara et al.,



Plant Cell Physiol. 37: 49-59, 1996


GUS
β-glucuronidase (GUS) coding sequence (Jefferson, et al., EMBO



J. 6: 3901-3907, 1987)


Mac
Hybrid promoter consisting of the promoter from the mannopine



synthase (mas) gene and a CaMV 35S enhancer region (Comai et



al., Plant Mol. Biol 15: 373-381, 1990)


mas/35S
Hybrid promoter consisting of a promoter region from CaMV 35S



gene with enhancer elements from a promoter fragment of



mannopine synthase (mas) gene of Agrobacterium tumefaciens



(Janssen and Gardner, Plant Molecular Biology. 14: 61-72, 1989)


mas 5′
Promoter region from the mas of A. tumefaciens


mas 3′
Terminator region from the mas gene of A. tumefaciens


nos 5′
Promoter region from the nopaline synthase (nos) gene of A. tumefaciens



(Depicker et al., J Mol. and Appl. Genetics 1: 561-573,



1982)


nos 3′
Terminator region from the nos gene of A. tumefaciens (Depicker



et al., 1982, supra)


nptII
Kanamycin-resistance gene (encodes neomycin



phosphotransferase which deactivates aminoglycoside antibiotics



such as kanamycin, neomycin and G418)


ocs 3′
~1.6 kb terminator fragment from octopine synthase gene of A. tumefaciens



(described in Janssen and Gardner, 1989, supra)


petD8 5′
~3.2 kb promoter region from a phospholipid transfer protein gene



(D8) of Petunia hybrida (Holton, Isolation and characterization of



petal specific genes from Petunia hybrida. PhD thesis, University



of Melbourne, Australia, 1992) (SEQ ID NO: 24)


petD8 3′
~0.7 kb terminator region from a phospholipid transfer protein



gene (D8) of Petunia hybrida cv. OGB (Holton, 1992, supra)


long petFLS 5′
~4.0 kb fragment containing the promoter region from a flavonol



synthase (FLS) gene of P. hybrida


short petFLS 5′
~2.2 kb fragment containing the promoter region from FLS gene of



P. hybrida


petFLS 3′
~0.95 kb fragment containing the terminator region from FLS gene



of P. hybrida


petHf1
Petunia F3′5′H Hf1 cDNA clone (Holton et al., 1993a, supra)



(SEQ ID NO: 1)


petHf2
Petunia F3′5′H Hf2 cDNA clone (Holton et al., 1993a, supra)



(SEQ ID NO: 3)


petRT 5′
Promoter region of an anthocyanidin-3-glucoside



rhamnosyltransferase (3RT) gene from P. hybrida (Brugliera,



Characterization of floral specific genes isolated from Petunia



hybrida. RMIT, Australia. PhD thesis, 1994)


petRT 3′
Terminator region of a 3RT gene from P. hybrida (Brugliera,



1994, supra)


RoseCHS 5′
~2.8 kb fragment containing the promoter region from a CHS gene



of Rosa hybrida (SEQ ID: 5)


SuRB
Chlorsulfuron-resistance gene (encodes Acetolactate Synthase)



with its own terminator from Nicotiana tabacum (Lee et al.,



EMBO J. 7: 1241-1248, 1988)









In order to produce delphinidin or delphinidin-based molecules in rose petals, a number of binary vector constructs were prepared utilising the petunia F3′5′H cDNA fragments and various promoter and terminator fragments. The chimeric petunia F3′5′H genes had proved successful in carnation and petunia leading to detectable intact F3′5′H transcripts (as detected by Northern blot analysis) and to the production of delphinidin or delphinidin-based molecules pigments. Table 5 summarises the list of binary vector constructs containing petunia F3′5′H cDNA fragments.







TABLE 5







Summary of chimaeric petunia F3′5′H gene expression cassettes


contained in binary vector constructs used in the transformation of roses


(see Table 4 for an explanation of abbreviations).











SELECTABLE


PLASMID
F3′5′H GENE
MARKER GENE





pCGP1452
AmCHS 5′:petHf1:petD8 3′
35S 5′:SuRB


pCGP1453
Mac:petHf1:mas 3′
35S 5′:SuRB


pCGP1457
petD8 5′:petHf1:petD8 3′
35S 5′:SuRB


pCGP1461
short petFLS 5′:petHf1:petFLS 3′
35S 5′:SuRB


pCGP1616
petRT 5′:petHf1:nos 3′
35S 5′:SuRB


pCGP1638
CaMV 35S:petHf1:ocs 3′
35S 5′:SuRB


pCGP1623
mas 35S:petHf1:ocs 3′
35S 5′:SuRB


pCGP1860
Rose CHS 5′:petHf1:nos 3′
35S 5′:SuRB


pCGP2123
CaMV 35S:petHf2:ocs 3′
35S 5′:SuRB









The Isolation of Petunia F3′5′H cDNA Clones (petHf1 and petHf2)

The isolation and characterisation of cDNA clones of petunia F3′5′H (petHf1 and petHf2 contained in pCGP602 (FIG. 2) and pCGP175 (FIG. 3) respectively) (SEQ ID NO:1 and SEQ ID NO:3, respectively) have been described in International Patent Application No. PCT/AU92/00334 and Holton et al. (1993a, supra).
The plasmid pCGP601 (FIG. 2), pCGP602 (FIG. 2), pCGP176 (FIG. 2) contain homologs of the petunia petHf1 F3′5′H cDNA clone. The plasmid pCGP601 contains a petunia F3′5′H petHf1 homolog that includes 52 bp of 5′ untranslated sequence. The plasmid pCGP602 contains a petunia F3′5′H petHf1 homolog that includes 125 bp of 5′ untranslated sequence (SEQ ID NO:1). The plasmid pCGP176 (described in Holton et al., 1993a supra) contains a petunia F3′5′H petHf1 homolog that includes 27 bp of 5′ untranslated sequence and a further  ̃127 bp of 3′ untranslated sequence over the petunia F3′5′H petHf1 cDNA clone in pCGP602.
Construction of pCGP1303 (petHf1 in pUC19 Backbone).
The petunia F3′5′H cDNA clone contained in the plasmid pCGP601 (described above) (FIG. 2) included 52 bp of 5′ untranslated sequence and 141 bp of 3′ untranslated sequence including 16 bp of the poly A tail. The plasmid pCGP601 (FIG. 2) was firstly linearized by digestion with the restriction endonuclease BspHI. The ends were repaired and the petunia F3′5′H petHf1 cDNA clone was released upon digestion with the restriction endonuclease FspI. The BspHI recognition sequence encompasses the putative translation initiating codon and the FspI recognition sequence commences 2 bp downstream from the stop codon. The 1.6 kb fragment containing the petunia F3′5′H petHf1 cDNA clone was purified and ligated with repaired EcoRI ends of pUC19 (New England Biolabs). Correct insertion of the fragment was established by restriction endonuclease analysis of DNA isolated from ampicillin-resistant transformants. The resulting plasmid was designated as pCGP1303 (FIG. 4).
Construction of pCGP27 (Short petHf1 in pBluescript Backbone)
The plasmid pCGP176 (Holton et al., 1993a, supra) (FIG. 2) was digested with the restriction endonuclease SpeI and EcoRI. The ends were then repaired and allowed to religate. The resulting plasmid was designated as pCGP627 and contained the identical cDNA clone as in pCGP176 except that the restriction endonuclease sites PstI, BamHI and SmaI were removed from the multi-cloning site of the pBluescript vector at the 5′ end of the cDNA clone.
The Binary Vector pCGP1452 (AmCHS 5′: petHf1: petD8 3′).
The plasmid pCGP1452 (FIG. 5) contains a chimeric petunia F3′5′H (petHf1) gene under the control of a promoter fragment from the Antirrhinum majus chalcone synthase gene (CHS) (Sommer and Saedler, 1986, supra) with a terminator fragment from the petunia phospholipid transfer protein (PLTP) gene (petD8 3′) (Holton, 1992, supra). The chimeric petunia F3′5′H cassette is in a tandem orientation with respect to the 35S 5′: SuRB gene of the binary vector, pWTT2132 (DNA Plant Technologies, USA=DNAP) (FIG. 6).
Intermediates in the Preparation of the Binary pCGP1452
The Binary Vector pWTT2132
The binary vector plasmid pWTT2132 (DNAP) (FIG. 6) contains a chimeric gene comprised of a 35S 5′ promoter sequence (Franck et al., 1980, supra), ligated with the coding region and terminator sequence for acetolactate synthase (ALS) gene from the SuRB locus of tobacco (Lee et al., 1988, supra). An  ̃60 bp 5′ untranslated leader sequence from the petunia chlorophyll a/b binding protein gene (Cab 22 gene) (Harpster et al., MGG, 212: 182-190, 1988) is included between the 35S 5′ promoter fragment and the SuRB sequence.
Construction of pCGP725 (AmCHS 5′: petHf1: petD8 3′ in pBluescript)
A chimeric petunia F3′5′H gene under the control Antirrhinum majus CHS (AmCHS 5′) promoter with a petunia PLTP terminator (petD8 3′) was constructed by cloning the 1.6 kb BclI/FspI petunia F3′5′H (petHf1) fragment from pCGP602 (Holton et al., 1993a, supra) (FIG. 2) between a 1.2 kb Antirrhinum majus CHS gene fragment 5′ to the site of translation initiation (Sommer and Saedler, 1986, supra) and a 0.7 kb SmaI/XhoI PLTP fragment (petD8 3′) from pCGP13ΔBam (Holton, 1992, supra), 3′ to the deduced stop codon. The resulting plasmid in a pBluescript II KS (Stratagene, USA) backbone vector was designated pCGP725 (FIG. 7).
Construction of pCGP485 and pCGP1452 (AmCHS 5′: petHf1: petD8 3′ Binary Vectors)
The chimeric F3′5′H gene from pCGP725 (FIG. 7) was cloned into the binary vector pCGN1547 containing an nptII selectable marker gene cassette (McBride and Summerfelt Plant Molecular Biology 14: 269-276, 1990) to create pCGP485. A 3.5 kb fragment containing the AmCHS 5′; petHf1: petD8 3′ cassette was released upon digestion of pCGP485 with the restriction endonuclease PstI. The overhanging ends were repaired and the purified 3.5 kb fragment was ligated with SmaI ends of the binary vector, pWTT2132 (DNAP). Correct insertion of the fragment in a tandem orientation with respect to the 35S 5′: SuRB selectable marker gene cassette was established by restriction endonuclease analysis of plasmid DNA isolated from tetracycline-resistant transformants. The plasmid was designated as pCGP1452 (FIG. 5).
Plant Transformation with pCGP1452
The T-DNA contained in the binary vector plasmid pCGP1452 (FIG. 5) was introduced into rose via Agrobacterium-mediated transformation.
The Binary Vector pCGP1453 (Mac: petHf1: mas 3′)
The plasmid pCGP1453 (FIG. 8) contains a chimeric petunia F3′5′H (petHf1) gene under the control of a Mac promoter (Comai et al., 1990, supra) with a terminator fragment from the mannopine synthase gene of Agrobacterium (mas 3′). The chimeric petunia F3′5′H cassette is in a tandem orientation with respect to the 35S 5′: SuRB selectable marker gene cassette of the binary vector, pWTT2132 (DNAP) (FIG. 6).
A 3.9 kb fragment containing the Mac: petHf1: mas 3′ gene was released from the plasmid pCGP628 (described in International Patent Application No. PCT/AU94/00265) upon digestion with the restriction endonuclease PstI. The overhanging ends were repaired and the purified fragment was ligated with SmaI ends of pWTT2132 (DNAP). Correct insertion of the Mac: petHf1: mas 3′ gene in a tandem orientation with respect to the 35S 5′: SuRB selectable marker gene cassette was established by restriction endonuclease analysis of plasmid DNA isolated from tetracycline-resistant transformants. The plasmid was designated as pCGP1453 (FIG. 8).
Plant Transformation with pCGP1453
The T-DNA contained in the binary vector plasmid pCGP1453 (FIG. 8) was introduced into rose via Agrobacterium-mediated transformation.
The Binary Vector pCGP1457 (petD8 5′: petHf1: pet D8 3′)
The plasmid pCGP1457 (FIG. 9) contains a chimeric petunia F3′5′H (petHf1) gene under the control of a promoter fragment from the petunia PLTP gene (petD8 5′) with a terminator fragment from the petunia PLTP gene (petD8 3′). The chimeric petunia F3′5′H cassette is in a tandem orientation with respect to the 35S 5′: SuRB gene of the binary vector, pWTT2132 (DNAP) (FIG. 6).
Intermediates in the Preparation of the Binary Vector pCGP1457
Isolation of Petunia D8 Genomic Clone
Preparation of P. hybrida cv. OGB (Old Glory Blue) Genomic Library in λ2001
A genomic DNA library was constructed from Petunia hybrida cv. OGB DNA in the vector λ2001 (Karn et al., Gene 32: 217-224, 1984) using a Sau3A partial digestion of the genomic DNA as described in Holton, 1992 (supra). Screening of the OGB genomic library for the petunia D8 gene was as described in Holton, 1992. supra.
Isolation of D8 Genomic Clone OGB2.6
PCR was performed in order to find a non-mutant genomic clone representing D8. Oligo #2 (5′ to 3′ GTTCTCGAGGAAAGATAATACAAT) (SEQ ID NO:6) and Oligo #4 (5′ to 3′ CAAGATCGTAGGACTGCATG) (SEQ ID NO:7) were used to amplify D8 gene fragments, across the intron region, using 4 μL of phage suspension from the clones isolated from the primary screening of the OGB genomic library. The reactions were carried out in a total volume of 50 μl containing 1× Amplification buffer (Cetus), 0.2 mM dNTP mix, <1 μg of template DNA, 50 pmoles of each primer and 0.25 μL of Taq polymerase (5 units/μL-Cetus). The reaction mixtures were overlaid with 30 μL of mineral oil and temperature cycled using a Gene Machine (Innovonics). The reactions were cycled 30 times using the following conditions: 94° C. for 1 minute, 55° C. for 50 seconds, 72° C. for 2 minutes. One quarter of each PCR reaction was run on an agarose gel using TAE running buffer.
Three clones, λOGB-2.4, λOGB-2.5, and λOGB-2.6, gave fragments of approximately 1 kb whereas the mutant clone, λOGB-3.2 (described in Holton, 1992, supra), had produced a product of 1.25 kb. The λOGB-2.6 clone was chosen for further analysis.
pCGP382
The genomic clone, λOGB-2.6, contained a single 3.9 kb XbaI fragment that hybridized with the D8 cDNA. This XbaI fragment was isolated and purified and ligated with the XbaI ends of pBluescriptII KS—(Stratagene, USA). Restriction mapping of this clone revealed an internal PstI site 350 bp from the 3′ end. However, the “mutant” genomic clone in pCGP13, had an internal PstI near the putative initiating “ATG” of the coding region (approximately 1.5 kb from its 3′ end). The difference in the position of the PstI site in both clones suggested that the λOGB-2.6 XbaI fragment did not contain the whole genomic sequence of D8. A Southern blot was performed on PstI digested λOGB-2.6 DNA, and a fragment of 2.7 kb was found to hybridize with the D8 cDNA. Restriction endonuclease mapping confirmed that this fragment contained the 3′ coding region and flanking sequences.
In order to obtain a fragment containing the whole D8 genomic sequence, a number of cloning steps were undertaken. The λOGB-2.6 PstI fragment of 2.7 kb was purified and ligated with PstI ends of pBluescriptII KS—(Stratagene, USA). The resultant clone was digested with XbaI to remove the 350 bp PstI/XbaI fragment. This fragment was replaced by the 3.9 kb XbaI fragment from λOGB-2.6 to produce the plasmid pCGP382.
A 3.2 kb fragment containing the promoter region from the D8 2.6 gene in pCGP382 was released upon digestion with the restriction endonucleases HindIII and NcoI. The fragment was purified and ligated with the 4.8 kb NcoI/HindIII fragment of pJB1 (Bodean, Molecular and genetic regulation of Bronze-2 and other maize anthocyanin genes. Dissertation, Stanford University, USA, 1994) to produce pCGP1101 containing a petD85′: GUS: nos 3′ cassette.
A 1.6 kb petunia F3′5′H petHf1 fragment was released from the plasmid pCGP602 (Holton et al., 1993a, supra) (SEQ ID NO:1) (FIG. 2) upon digestion with the restriction endonucleases BspHI and BamHI. The fragment was purified and ligated with the 6.2 kb NcoI/BamHI fragment of pCGP1101 to produce pCGP1102 containing a petD8 5′: petHf1: nos 3′ expression cassette.
A 0.75 kb BamHI petD8 3′ fragment (Holton, 1992, supra) was purified from the plasmid pCGP13ΔBamHI and ligated with BamHI/BglII ends of pCGP1102 to produce the plasmid pCGP1107 containing a petD8 5′: petHf1: petD8 3′ expression cassette.
The plasmid pCGP1107 was linearised upon digestion with the restriction endonuclease XbaI. The overhanging ends were repaired and then the 5.3 kb fragment containing the petD8 5′: petHf1: petD8 3′ expression cassette was released upon digestion with the restriction endonuclease PstI. The fragment was purified and ligated with SmaI/PstI ends of the binary vector pWTT2132 (DNAP) (FIG. 6). Correct insertion of the petD8 5′: petHf1: petD8 3′ gene in a tandem orientation with respect to the 35S 5′: SuRB selectable marker gene cassette was established by restriction endonuclease analysis of plasmid DNA isolated from tetracycline-resistant transformants. The plasmid was designated as pCGP147 (FIG. 9).
Plant Transformation with pCGP1457
The T-DNA contained in the binary vector plasmid pCGP1457 (FIG. 9) was introduced into rose via Agrobacterium-mediated transformation.
The Binary Vector pCGP1461 (Short petFLS 5′: petHf1: pet FLS 3′)
The plasmid pCGP1461 (FIG. 10) contains a chimeric pea F3′5′H (petHf1) gene under the control of a promoter fragment from the petunia flavonol synthase (FLS) gene (short petFLS 5′) with a terminator fragment from the petunia FLS gene (petFLS 3′). The chimeric petunia F3′5′H gene is in a tandem orientation with respect to the 35S 5′: SuRB gene of the binary vector, pWTT2132 (FIG. 6).
Intermediates in the Preparation of the Binary Vector pCGP1461
Isolation of Petunia FLS Gene
Preparation of P. hybrida cv. Th7 Genomic Library
A P. hybrida cv. Th7 genomic library was prepared according to Sambrook et al. (1989, supra) using a Sau3A partial digestion of the genomic DNA. The partially digested DNA was cloned into EMBL-3 lambda vector (Stratagene, USA).
The Th7 genomic DNA library was screened with 32P-labelled fragments of a petunia FLS cDNA clone (Holton et al., Plant J. 4: 1003-1010, 1993b) using high stringency conditions.
Two genomic clones (FLS2 and FLS3) were chosen for further analysis and found to contain sequences upstream of the putative initiating methionine of the petunia FLS coding region with FLS2 containing a longer promoter region than FLS3.
pCGP486
A 6 kb fragment was released upon digestion of the genomic clone FLS2 with the restriction endonuclease XhoI. The fragment containing the short petunia FLS gene was purified and ligated with XhoI ends of pBluescript SK (Stratagene, USA). Correct insertion of the fragment was established by restriction endonuclease analysis of DNA isolated from ampicillin-resistant transformants. The resulting plasmid was designated as pCGP486.
pCGP487
A 9 kb fragment was released upon digestion of the genomic clone FLS3 with the restriction endonuclease XhoI. The fragment containing the petunia FLS gene was purified and ligated with XhoI ends of pBluescript SK (Stratagene, USA). Correct insertion of the fragment was established by restriction endonuclease analysis of DNA isolated from ampicillin-resistant transformants. The resulting plasmid was designated as pCGP487.
pCGP717
A 2.2 kb petunia FLS promoter fragment upstream from the putative translational initiation site was released from the plasmid pCGP487 upon digestion with the restriction endonucleases XhoI and PstI. The fragment generated was purified and ligated with XhoI/PstI ends of pBluescript II KS+ (Stratagene, USA). Correct insertion of the fragment was established by restriction endonuclease analysis of DNA isolated from ampicillin-resistant transformants. The resulting plasmid was designated as pCGP717.
pCGP716
A 0.95 kb petunia FLS terminator fragment downstream from the putative translational stop site was released from the plasmid pCGP487 upon digestion with the restriction endonucleases HindIII and SacI. The fragment generated was purified and ligated with HindIII/SacI ends of pBluescript II KS+ (Stratagene, USA). Correct insertion of the fragment was established by restriction endonuclease analysis of DNA isolated from ampicillin-resistant transformants. The resulting plasmid was designated as pCGP716.
Construction of pCGP493 (Short petFLS 5′: petFLS 3′ Expression Cassette)
A 1.8 kb fragment containing the short petunia FLS promoter fragment was amplified by PCR using the plasmid pCGP717 as template and the T3 primer (Stratagene, USA) and an FLS-Nco primer (5′ AAA ATC GAT ACC ATG GTC TTT TTT TCT TTG TCT ATA C 3′) (SEQ ID. NO:19). The PCR product was digested with the restriction endonucleases XhoI and ClaI and the purified fragment was ligated with XhoI/ClaI ends of pCGP716. Correct insertion of the fragment was established by restriction endonuclease analysis of DNA isolated from ampicillin-resistant transformants. The resulting plasmid was designated as pCGP493.
Construction of pCGP497 (Short petFLS 5′: petHf1: petFLS3′ Expression Cassette)
The petunia F3′5′H (petHf1) cDNA clone was released from the plasmid pCGP627 (described above) upon digestion with the restriction endonucleases BspHI and FspI. The BspHI recognition sequence encompasses the putative translation initiating codon and the FspI recognition sequence commences 2 bp downstream from the stop codon. The petunia F3′5′H petHf1 fragment generated was purified and ligated with ClaI (repaired ends)/NcoI ends of the plasmid pCGP493. Correct insertion of the fragment was established by restriction endonuclease analysis of DNA isolated from ampicillin-resistant transformants. The resulting plasmid was designated as pCGP497.
Construction of pCGP1461 (Short petFLS 5′: petHf1: petFLS3′ Binary Vector)
The plasmid pCGP497 was linearised upon digestion with the restriction endonuclease SacI. The overhanging ends were repaired and a 4.35 kb fragment containing the short petFLS 5′: petHf1: petFLS3′ gene expression cassette was released upon digestion with the restriction endonuclease KpnI. The fragment generated was purified and ligated with PstI (ends repaired)/KpnI ends of the binary vector pWTT2132 (DNAP) (FIG. 6). Correct insertion of the fragment in a tandem orientation with respect to the 35S 5′: SuRB selectable marker gene cassette of pWTT2132 was established by restriction endonuclease analysis of plasmid DNA isolated from tetracycline-resistant transformants. The resulting plasmid was designated as pCGP1461 (FIG. 10).
Plant Transformation with pCGP1461
The T-DNA contained in the binary vector plasmid pCGP1461 (FIG. 10) was introduced into rose via Agrobacterium-mediated transformation.
The Binary Vector pCGP1616 (petRT 5′: petHf1: nos 3′)
The plasmid pCGP1616 (FIG. 11) contains a chimeric petunia F3′5′H (petHf1) gene under the control of a promoter fragment from the P. hybrida 3RT gene (petRT 5′) (Brugliera, 1994, supra) with a terminator fragment from the nopaline synthase gene (nos 3′) of Agrobacterium (Depicker, et al., 1982, supra). The chimeric petunia F3′5′H cassette is in a tandem orientation with respect to the 35S 5′: SuRB gene of the binary vector, pWTT2132 (DNAP) (FIG. 6).
Intermediates in the Preparation of the Binary Vector pCGP1616
Isolation of Petunia 3RT Gene
P. hybrida cv. Th7 Genomic DNA Library Construction in EMBL3
A Petunia hybrida cv. Th7 genomic library was prepared according to Sambrook et al. 1989, supra using a Sau3A partial digestion of the genomic DNA. The partially digested DNA was cloned into EMBL-3 lambda vector (Stratagene, USA). Screening of the Th7 genomic library for the petunia 3RT gene was as described in Brugliera, 1994, supra.
A 3 kb fragment containing the petRT 5′: petHf1: nos 3′ cassette was released from the plasmid pCGP846 (described in Brugliera, 1994, supra) upon digestion with the restriction endonucleases PstI and BamHI. The purified fragment was ligated with PstI/BamHI ends of pWTT2132 (DNAP) (FIG. 6). Correct insertion of the fragment in a tandem orientation with respect to the 35S 5′: SuRB selectable marker gene cassette was established by restriction endonuclease analysis of plasmid DNA isolated from tetracycline-resistant transformants. The plasmid was designated as pCGP1616 (FIG. 11).
Plant Transformation with pCGP1616
The T-DNA contained in the binary vector plasmid pCGP1616 (FIG. 11) was introduced into rose via Agrobacterium-mediated transformation.
The Binary Vector pCGP1623 (mas/35S: petHf1: ocs 3′)
The plasmid pCGP1623 (FIG. 12) contains a chimeric petunia F3′5′H (petHf1) gene under the control of the expression cassette contained in pKIWI101 (Janssen and Gardner, 1989, supra) consisting of a promoter fragment from the cauliflower mosaic virus 35S gene (35S 5′) with an enhancing sequence from the promoter of the mannopine synthase gene (mas) of Agrobacterium and a terminator fragment from the octopine synthase gene of Agrobacterium (ocs 3′). The chimeric petunia F3′5′H cassette is in a tandem orientation with respect to the 35S 5′: SuRB gene of the binary vector, pWTT2132 (DNAP) (FIG. 6).
Intermediates in the Preparation of the Binary Vector pCGP1623
The  ̃1.6 kb fragment of the petunia F3′5′H petHf1 cDNA clone contained in the plasmid pCGP1303 (FIG. 4) was released upon digestion with the restriction endonucleases BspHI and SmaI. The petunia F3′5′H petHf1 fragment was purified and ligated with a  ̃5.9 kb NcoI/EcoRI (repaired ends) fragment of pKIWI101 (Janssen and Gardner, 1989, supra) to produce the plasmid pCGP1619.
A partial digest of the plasmid pCGP1619 with the restriction endonuclease XhoI released a 4.9 kb fragment containing the mas/35S: petHf1: ocs 3′ expression cassette. The fragment was purified and ligated with SalI ends of pWTT2132 (DNAP) (FIG. 6), Correct insertion of the fragment in a tandem orientation with respect to the 35S 5′: SuRB selectable marker gene cassette was established by restriction endonuclease analysis of plasmid DNA isolated from tetracycline-resistant transformants. The plasmid was designated as pCGP1623 (FIG. 12).
Plant Transformation with pCGP1623
The T-DNA contained in the binary vector plasmid pCGP1623 (FIG. 12) was introduced into rose via Agrobacterium-mediated transformation.
The Binary Vector pCGP1638 (35S 5′: petHf1: ocs 3′)
The plasmid pCGP1638 (FIG. 13) contains a chimeric petunia F3′5′H (petHf1) gene under the control of a CaMV 35S promoter (35S 5′) with an octopine synthase terminator (ocs 3′). A  ̃60 bp 5′ untranslated leader sequence from the petunia chlorophyll a/b binding protein gene (Cab 22 gene) (Harpster et al., 1988, supra) is included between the CaMV 35S promoter fragment and the petunia F3′5′H petHf1 cDNA clone. The chimeric petunia F3′5′H cassette is in a tandem orientation with respect to the 35S 5′: SuRB selectable marker gene cassette of the binary vector, pWTT2132 (FIG. 6).
Intermediates in the Preparation of the Binary Vector pCGP1638
Construction of pCGP1273
The plasmid pCGP1273 was constructed by subcloning a  ̃3 kb HindIII/HpaI fragment containing 35S 5′: GUS: ocs 3′ gene from the binary vector pJJ3499 (Jones et al., Transgenic Research, 1: 285-297, 1992) with the HindIII/SmaI ends of the plasmid pBluescript KS II (+) (Stratagene, USA).
Construction of pCGP1634
A  ̃3 kb HindIII/BamHI fragment containing the 35S 5′: GUS: ocs 3′ gene from pCGP1273 was then isolated and ligated with the HindIII/BamHI ends of the cloning vector pUC19 (New England Biolabs) to, create the plasmid pCGP1634.
Construction of pCGP1636
The GUS fragment from the plasmid pCGP1634 was removed by digesting pCGP1634 with the restriction endonucleases NcoI and XbaI and purifying the  ̃3.7 kb fragment containing the 35S 5′ promoter fragment, the ocs 3′ terminator fragment and the pUC19 vector backbone.
The petunia F3′5′H petHf1 cDNA clone was released from pCGP1303 (FIG. 4) upon digestion with the restriction endonucleases BspHI and XbaI. The resulting  ̃1.6 kb fragment was purified and ligated with the  ̃3.7 kb NcoI/XbaI fragment from pCGP1634. Correct insertion of the petunia F3′5′H petHf1 fragment was established by restriction endonuclease analysis of plasmid DNA isolated from ampicillin-resistant transformants. The resulting plasmid containing a 35S5′: petHf1: ocs 3′ gene was designated pCGP1636.
Construction of pCGP1638
The 35S 5′: petHf1; ocs 3′ gene from the plasmid pCGP1636 was released upon digestion of pCGP1636 with the restriction endonucleases PstI and EcoRI. The ends were repaired and the  ̃2.6 kb fragment was purified and ligated with the Sam ends of the binary vector, pWTT2132 (DNAP). Correct insertion of the 35S 5′: petHf1: ocs 3′ gene in a tandem orientation with respect to the 35S 5′: SuRB selectable marker gene cassette was established by restriction endonuclease analysis of plasmid DNA isolated from tetracycline-resistant transformants. The plasmid was designated as pCGP1638 (FIG. 13).
Plant Transformation with pCGP1638
The T-DNA contained in the binary vector plasmid pCGP1638 (FIG. 13) was introduced into rose via Agrobacterium-mediated transformation.
The Binary Vector pCGP1860 (RoseCHS 5′: petHf1: nos 3′)
The plasmid pCGP1860 (FIG. 14) contains a chimeric petunia F3′5′H (petHf1) gene under the control of a promoter fragment from the chalcone synthase gene of Rosa hybrida (RoseCHS 5′) with a terminator fragment from the nopaline synthase gene of Agrobacterium (nos 3′). The chimeric petunia F3′5′H cassette is in a tandem orientation with respect to the 35S 5′: SuRB selectable marker gene cassette of the binary vector, pWTT2132 (DNAP) (FIG. 6).
Intermediates in the Preparation of the Binary Vector pCGP1860
Isolation of Rose CHS Promoter
A rose genomic DNA library was prepared from genomic DNA isolated from young leaves of Rosa hybrida cv. Kardinal.
The Kardinal genomic DNA library was screened with 32P-labelled fragment of rose CHS cDNA clone contained in the plasmid pCGP634. The rose CHS cDNA clone was isolated by screening of a petal cDNA library prepared from RNA isolated from petals of Rosa hybrida cv Kardinal (Tanaka et al., 1995, supra) using a petunia CHS cDNA fragment as probe (clone 1F11 contained in pCGP701, described in Brugliera et al., 1994, supra). Conditions are as described in Tanaka et al., 1995 (supra).
A rose genomic clone (roseCHS20λ) was chosen for further analysis and found to contain  ̃6.4 kb of sequence upstream of the putative initiating methionine of the rose CHS coding region.
An  ̃6.4 kb fragment upstream from the translational initiation site was cloned into pBluescript KS (−) (Stratagene) and the plaid was designated as pCGP1114.
The plasmid pCGP1114 was digested with the restriction endonucleases HindIII and EcoRV to release a 2.7-3.0 kb fragment which was purified and ligated with the HindIII/SmaI ends of pUC19 (Now England Biolabs). Correct insertion of the rose CHS promoter fragment was established by restriction endonuclease analysis of DNA isolated from ampicillin-resistant transformants. The resulting plasmid was designated as pCGP1116. The DNA sequence of the rose CHS promoter fragment was determined using pCGP1116 as template (SEQ ID NO:5).
Construction of pCGP197 (RoseCHS 5′: GUS: nos 3′ in a pUC18 Backbone)
An  ̃3.0 kb fragment containing the rose chalcone synthase promoter (RoseCHS 5′) was released from the plasmid pCGP1116 upon digestion with the restriction endonucleases HindIII and Asp718. The fragment was purified and ligated with a HindII/Asp718 fragment from pJ18 (Bodeau, 1994, supra) containing the vector backbone, β-glucoronidase (GUS) and nos 3′ fragments. Correct insertion of the rose CHS promoter fragment upstream of the GUS coding sequence was established by restriction endonuclease analysis of DNA isolated from ampicillin-resistant transformants. The resulting plasmid was designated as pCGP197.
Construction of pCGP200 (RoseCHS 5′: petHf1: nos 3′ in pUC18 Backbone)
A 1.8 kb fragment containing the petunia F3′5′H (petHf1) fragment was released from the plasmid pCGP1303 (described above) (FIG. 4) upon digestion with the restriction endonucleases BspHI and SacI. The petunia F3′5′H petHf1 fragment was purified and ligated with NcoI/SacI ends of pCGP197. Correct insertion of the petunia F3′5′H petHf1 fragment between the rose CHS promoter and nos 3′ fragments was established by restriction endonuclease analysis of DNA isolated from ampicillin-resistant transformants. The resulting plasmid was designated as pCGP200.
Construction of pCGP1860 (RoseCHS 5′: petHf1: nos 3′ in a Binary Vector)
An  ̃4.9 kb fragment containing the RoseCHS 5′: petHf1: nos 3′ cassette was released from the plasmid pCGP200 upon digestion with the restriction endonuclease BglII. The fragment was purified and ligated with BamHI ends of the binary vector, pWTT2132 (DNAP) (FIG. 6). Correct insertion of the fragment in a tandem orientation with respect to the 35S 5′: SuRB selectable marker gene cassette of pWTT2132 was established by restriction endonuclease analysis of plasmid DNA isolated from tetracycline-resistant transformants. The resulting plasmid was designated as pCGP1860 (FIG. 13).
Plant Transformation with pCGP1860
The T-DNA contained in the binary vector plasmid pCGP1860 (FIG. 14) was introduced into rose via Agrobacterium-mediated transformation.
The Binary Vector pCGP2123 (CaMV35S: petHf2: ocs 3′)
The plasmid pCGP2123 (FIG. 15) contains a chimeric petunia F3′5′H (petHf2) gene under the control of a CaMV35S promoter with a terminator fragment from the octopine synthase gene of Agrobacterium (ocs 3′). The chimeric petunia F3′5′H cassette is in a tandem orientation with respect to the 35S 5′: SuRB selectable marker gene cassette of the binary vector, pCGP1988 (FIG. 16).
Intermediates in the Preparation of the Binary Vector pCGP2123
Construction of pCGP1988 (a Derivative of the Binary Vector, pWTT2132)
The binary vector pCGP1988 (FIG. 16) is based on binary vector pWTT2132 (DNAP) (FIG. 6) but contains the multi-cloning site from pNEB193 (New England Biolabs). The plasmid pNEB193 was firstly linearized by digestion with the restriction endonuclease EcoRI. The overhang ends were repaired and the multi-cloning fragment was released upon digestion with the restriction endonuclease PstI. The fragment was purified and ligated with SalI (ends repaired)/PstI ends of the binary vector pWTT12132 (DNAP). Correct insertion of the multi-cloning fragment into pWTT2132 was established by restriction endonuclease analysis of plasmid DNA isolated from tetracycline-resistant transformants. The resulting plasmid was designated as pCGP1988 (FIG. 16).
Construction of pCGP2000 (CaMV 35S Promoter Fragment in pBluescript)
The plasmid pCGP2000 was an intermediate plasmid containing a cauliflower mosaic virus (CaMV) 35S promoter fragment in a pBluescript SK (Stratagene, USA) backbone. The CaMV 35S promoter fragment from pKIWI101 (Janssen and Gardner, 1989, supra) was released upon digestion with the restriction endonucleases XbaI and PstI. The  ̃0.35 kb fragment generated was purified and ligated with XbaI/PstI ends of the vector pBluescript SK. Correct insertion of the fragment was established by restriction endonuclease analysis of plasmid DNA isolated from ampicillin-resistant transformants. The plasmid was designated as pCGP2000.
Construction of pCGP2105 (CaMV 35S 5′ and ocs 3′ Fragments in pBluescript)
The plasmid pCGP2105 (FIG. 17) contained a CaMV 35S promoter fragment along with a terminator fragment from the octopine synthase gene of Agrobacterium (ocs 3′) both from pKIWI101 (Janssen and Gardner, 1989, supra).
The ocs 3′ fragment from pKIWI101 (Janssen and Gardner, 1989, supra) was isolated by firstly digesting the plasmid pKIWI101 with the restriction endonuclease EcoRI, followed by repair of the overhanging ends, and finally by digestion with the restriction endonuclease XhoI to release a 1.6 kb fragment. This fragment was then ligated with HincII/XhoI ends of pCGP2000. Correct insertion of the fragment was established by restriction endonuclease analysis of plasmid DNA isolated from ampicillin-resistant transformants. The plasmid was designated pCGP2105 (FIG. 17).
Construction of pCGP2109 (CaMV 35S: petHf2: ocs 3′Gene in pBluescript)
The plasmid pCGP2109 contained the CaMV 35S: petHf2: ocs 3′ expression gene cassette in a pBluescript backbone.
The 1.8 kb petunia F3′5′H petHf2 cDNA clone was released from pCGP175 (Holton et al., 1993a, supra) upon digestion with the restriction endonucleases XbaI and SspI. The overhanging ends were repaired and the purified fragment was ligated with PstI (ends repaired)/EcoRV ends of pCGP2105 (described above) (FIG. 17). Correct insertion of the fragment was established by restriction endonuclease analysis of plasmid DNA isolated from ampicillin-resistant transformants. The plasmid was designated pCGP2109.
Construction of pCGP2123 (CaMV 35S: petHf2: ocs 3′ Binary Vector)
The CaMV 35S: petHf2: ocs 3′ cassette was released from pCGP2109 upon digestion with the restriction endonucleases Asp718 and XbaI. The overhanging ends were repaired and the resultant  ̃3.7 kb fragment containing the CaMV 35S: petHf2: ocs 3′ gene was purified and ligated with repaired ends of Asp718 of the binary vector, pCGP1988 (FIG. 16). Correct insertion of the CaMV 35S: petHf2: ocs 3′ gene in a tandem orientation with respect to the 35S5′: SuRB selectable marker gene cassette was established by restriction endonuclease analysis of plasmid DNA isolated from tetracycline-resistant transformants. The plasmid was designated as pCGP2123 (FIG. 15).
Plant Transformation will pCGP2123
The T-DNA contained in the binary vector plasmid pCGP2123 (FIG. 15) was introduced into rose via Agrobacterium-mediated transformation.
Example 5
Analysis of Transgenic Roses
The transgenic roses produced in the experiments described in Example 4 were grown to flowering. Flowers were collected and the colors of the petals were coded using the Royal Horticultural Society Colour Charts (RHSCC). The anthocyanins were extracted and the anthocyanidins (specifically the presence of delphinidin or delphinidin-based molecules) analysed by TLC and/or HPLC analysis. Total RNA was also isolated from petal tissue and Northern blot analysis was used to detect transcripts of petunia F3′5′H transgenes, endogenous rose CHS gene and SuRB transgene. The results of the transgenic analysis are summarised in Table 6.
Although over 250 transgenic Kardinal roses were produced (Table 6) none produced flowers with a change in color. TLC and/or HPLC analysis failed to detect accumulation of delphinidin or delphinidin-based molecules pigments confirming the absence of efficient F3′5′H activity. Subsequent Northern analysis on total RNA isolated from petal tissue of these transgenic roses revealed either no detectable intact petunia F3′5′H (petHf1 or petHf2) transcripts, or in some cases (see footnotes), degraded transcripts. Hybridization of the same membranes with the selectable marker gene (SuRB) or with an endogenous rose CHS cDNA probe revealed discrete hybridizing transcripts indicating that the total RNA isolated was not degraded. The detection of the SuRB transgene transcripts confirmed that the roses were transgenic.







TABLE 6







Results of transgenic analysis of rose petals transformed with the T-DNA


from various petunia F3′5′H (petHf1 or petHf2) gene expression cassettes.











PLASMID
F3′5′H GENE
EVENTS
DEL
RNA














pCGP1452
AmCHS 5′:petHf1:petD8 3′
34
0/28
0/341


pCGP1453
Mac:petHf1:mas 3′
16
0/14
0/132


pCGP1457
petD8 5′:petHf1:petD8 3′
11
0/11
0/11


pCGP1461
short petFLS 5′:petHf1:petFLS 3′
11
0/11
0/11


pCGP1616
petRT 5′:petHf1:nos 3′
4
0/4 
0/4


pCGP1623
mas/35S:petHf1:ocs 3′
27
0/20
0/123


pCGP1638
CaMV 35S:petHf1:ocs 3′
22
0/14
0/14


pCGP1860
RoseCHS 5′:petHf1:nos 3′
15
0/13
0/13


pCGP2123
CaMV 35S:petHf2:ocs 3′
40
0/26
0/10





EVENTS = number of independent transgenic events produced


DEL = number of transgenic events in which delphinidin or delphinidin-based molecules was detected (by TLC or HPLC) in petals over the total number of events analyzed


RNA = number of transgenic events in which intact F3′5′H (petHf1 or petHf2) transcripts were detected by Northern blot analysis in total RNA isolated from rose petals over the total number of events analyzed


1= Degraded transcripts were detected in 5 of the 34 analyzed


2= Degraded transcripts were detected in 8 of the 13 analyzed


3= Degraded transcripts were detected in 8 of the 12 analyzed






The fact that no intact petunia F3′5′H (petHf1 or petHf2) transcripts were ever detected in transgenic rose petals transformed with the T-DNAs described (Table 6) suggested a number of possibilities:
1. that the RNA isolated was degraded. This was not the case as the RNA had been stained by ethidium bromide and visualised under UV-light. The intact visible ribosomal RNA bands were used as an indicator of the quality of the RNA isolated. Furthermore the detection of full-length transcripts of the endogenous rose CHS and SuRB transgenes confirmed that the RNA preparation was not degraded.
2. that there was no initiation of transcription of the chimeric F3′5′H genes evaluated. This was a possibility with some of the expression cassettes analysed, as no F3′5′H transcripts were detected by Northern analysis. However all of the petunia F3′5′H expression cassettes had proven to be functional (ie. result in an intact transcript and result in the production of delphinidin-based pigments) in other plants such as carnation and petunia.
3. that the petunia F3′5′H petHf1 and petHf2 mRNAs were unstable in roses. This was also a possibility as degraded petunia F3′5′H transcripts were detected by Northern analysis in total RNA isolated from petals of some events. However the petunia petHf1 and petHf2 mRNAs had boon proven to be stable in other plants such as carnation and petunia. Such instability could be due to aberrant translation leading to mRNA turnover, some feature of the sequence inherently unstable in rose cells, some other factor or factors.
There was a need therefore to find suitable promoter fragments that would efficiently drive expression of genes in rose petals and find suitable F3′5′H sequences that would result in intact transcripts accumulating in rose petals leading to functional F3′5′H activity and to the production of delphinidin-based pigments.
Example 6
Evaluation of Promoters in Roses
Development of GUS Gene Expression Cassettes.
The evaluation of the promoter and terminator fragments was performed using the (GUS reporter gene. Therefore, a number of promoters were linked to the β-glucuronidase reporter gene (GUS) (Jefferson et al., 1987, supra) and introduced into roses in an attempt to identify expression cassettes that lead to effective initiation of transcription in rose flowers.
A summary of the promoters and terminator agents evaluated is given in Table 7.







TABLE 7







List of chimaeric GUS gene expression cassettes evaluated in roses












SELECTABLE




GUS EXPRESSION
MARKER
BACKBONE


PLASMID
CASSETTE
GENE
VECTOR





pCGP1307
petD8 5′:GUS:petD8 3′
mas
pCGN1548




5′:nptII:mas 3′


pCGP1506
long
nos 5′:nptII:nos
pBIN19



petFLS 5′:GUS:petFLS 3′
3′


pCGP1626
chrysCHS 5′:GUS:petRT
35S 5′:SuRB
pWTT2132



3′


pCGP1641
petRT 5′:GUS:petRT
35S 5′:SuRB
pWTT2132



3′


pCGP1861
RoseCHS 5′:GUS:nos 3′
35S 5′:SuRB
pWTT2132


pCGP1953
AmCHS 5′:GUS:petD8 3′
35S 5′:SuRB
pWTT2132


pWTT2084
35S 5′:GUS:ocs 3′
35S 5′:SuRB
pWTT2132









The Binary Vector pCGP1307 (petD8 5′: GUS: petD8 3′)

The plasmid pCGP1307 (FIG. 18) contains a chimeric GUS gene under the control of a promoter and terminator fragment from the petunia PLTP gene (petD8 5′ and petD8 3′, respectively). The chimeric GUS reporter gene cassette is in a tandem orientation with respect to the mas 5′: nptII: mas 3′ selectable marker gene cassette of the binary vector pCGN1548 (McBride and Summerfelt, 1990, supra).
Intermediates in the Preparation of the Binary Vector pCGP1307
The nos 3′ fragment from pCGP1101 (see Example 4) was replaced with the 0.75 kb petD8 3′ fragment (Holton, 1992, supra) to produce the plasmid pCGP1106 containing a petD8 5′: GUS: petD8 3′ expression cassette.
The 5.3 kb fragment containing the petD8 5′: GUS: petD8 3′ expression cassette was released from the plasmid pCGP1106 upon digestion with the restriction endonucleases HindIII and PstI. The fragment was purified and ligated with HindIII/PstI ends of the binary vector, pCGN1548 (McBride and Summerfelt, 1990, supra). Correct insertion of the fragment was established by restriction endonuclease analysis of DNA isolated from gentamycin-resistant transformants. The resulting plasmid was designated as pCGP1307 (FIG. 18).
Plant Transformation with pCGP1307
The T-DNA contained in the binary vector plasmid pCGP1307 (FIG. 18) was introduced into rose via Agrobacterium-mediated transformation.
The Binary Vector pCGP1506 (Long petFLS 5′: GUS: petFLS 3′)
The plasmid pCGP1506 (FIG. 19) contains a chimeric GUS gene under the control of promoter and terminator fragments from the petunia flavonol synthase gene (petFLS 5′ and petFLS 3′, respectively). The chimeric GUS reporter gene cassette is in a tandem orientation with respect to the nos 5′: nptII: nos 3′ selectable marker gene cassette of the binary vector pBIN19 (Bevan, Nucleic Acids Res 12: 8711-8721, 1984).
Intermediates in the Preparation of the Binary Vector pCGP1506
A 4 kb long petunia FLS promoter fragment upstream from the putative translational initiation site was released from the plasmid pCGP486 (described in Example 4) upon digestion with the restriction endonucleases XhoI and PstI. The fragment generated was purified and ligated with XhoI/PstI ends of pBluescript II KS+ (Stratagene, USA). Correct insertion of the fragment was established by restriction endonuclease analysis of DNA isolated from ampicillin-resistant transformants. The resulting plasmid was designated as pCGP715.
Construction of pCGP494 (Long petFLS 5′: petFLS3′ Expression Cassette)
A 4.0 kb fragment containing the long petunia FLS promoter fragment was amplified by PCR using the plasmid pCGP715 as template and the T3 primer (Stratagene, USA) and an FLS-Nco primer (5′ AAA ATC GAT ACC ATG GTC TTT TTT TCT TTG TCT ATA C 3′) (SEQ ID NO:19). The PCR product was digested with the restriction endonucleases XhoI and ClaI and the purified fragment was ligated with XhoI/ClaI ends of pCGP716 (described in Example 4). Correct insertion of the fragment was established by restriction endonuclease analysis of DNA isolated from ampicillin-resistant transformants. The resulting plasmid was designated as pCGP494.
Construction of pCGP496 (Long petFLS 5′: GUS: petFLS3′ Expression Cassette)
The GUS coding sequence from the plasmid pjB1 (Bodeau, 1994, supra) was released upon digestion with the restriction endonucleases NcoI and SmaI. The GUS fragment generated was purified and ligated with ClaI (repaired ends)/NcoI ends of the plasmid pCGP494. Correct insertion of the fragment was established by restriction endonuclease analysis of DNA isolated from ampicillin-resistant transformants. The resulting plasmid was designated as pCGP496.
Construction of pCGP1506 (Long petFLS 5′: GUS: petFLS3′ Binary Vector)
The plasmid pCGP496 was firstly linearised upon digestion with the restriction endonuclease XhoI. The overhanging ends were partially repaired (using only dTTP and dCTP in the reparation reaction) and a 6.7 kb fragment containing the long petFLS 5′: GUS: petFLS3′ gene expression cassette was released upon digestion with the restriction endonuclease SacI. The fragment generated was purified and ligated with BamHI (partially repaired ends using dGTP and dATP in the reparation reaction)/SacI ends of the binary vector pBIN19. Correct insertion of the fragment in a tandem orientation with respect to the nos 5′: nptII: nos 3′ selectable marker gene cassette was established by restriction endonuclease analysis of plasmid DNA isolated from kanamycin-resistant transformants. The resulting plasmid was designated as pCGP1506 (FIG. 19).
Plant Transformation with pCGP1506
The T-DNA contained in the binary vector plasmid pCGP1506 (FIG. 19) was introduced into rose via Agrobacterium-mediated transformation.
The Binary Vector pCGP1626 (chrysCHS 5′: GUS: petRT 3′)
The plasmid pCGP1626 (FIG. 20) contains a chimeric GUS gene under the control of promoter fragment from the chalcone synthase gene of chrysanthemum (chrysCHS 5′) and a terminator fragment from the 3RT gene of petunia (petRT 3′) (Brugliera, 1994, supra). The chimeric GUS reporter gene cassette is in a tandem orientation with respect to the 35S 5′: SuRB selectable marker gene cassette of the binary vector pWTT2132 (DNAP) (FIG. 6).
Intermediates in the Preparation of the Binary Vector pCGP1626
Isolation of Chrysanthemum CHS Promoter
A chrysanthemum genomic DNA library was prepared from genomic DNA isolated from young leaf material of the chrysanthemum cv Hero.
The chrysanthemum genomic DNA library was screened with 32P-labelled fragments of a chrysanthemum CHS cDNA clone (SEQ ID NO:28) (contained in the plasmid pCGP856) using high stringency conditions. The plasmid pCGP856 contains a 1.5 kb cDNA clone of CHS isolated from a petal cDNA library prepared from RNA isolated from the chrysanthemum cv. Dark Pink Pom Pom.
A genomic clone (CHS5) was chosen for further analysis and found to contain  ̃3 kb of sequence upstream of the putative initiating methionine of the chrysanthemum CHS coding region.
A 4 kb fragment was released upon digestion of the genomic clone CHS5 with the restriction endonuclease HindIII. The fragment containing the chrysanthemum CHS promoter was purified and ligated with HindIII ends of pBluescript SK (Stratagene, USA). Correct insertion of the fragment was established by restriction endonuclease analysis of DNA isolated from ampicillin-resistant transformants. The resulting plasmid was designated as pCGP1316.
A 2.6 kb chrysanthemum CHS promoter fragment upstream from the putative translational initiation site was amplified by PCR using pCGP1316 as template and primers “chrysanCHSATG” (5′-GTTAAGGAAGCCATGGGTGT-3′) (SEQ ID NO:8) and the M13 reverse primer (Stratagene, USA). Primer “chrysanCHSATG” incorporated an NcoI restriction endonuclease recognition sequence at the putative translation initiation point for ease of cloning. The PCR fragment was purified and ligated with EcoRV (dT-tailed) ends of pBluescript KS (Holton and Graham, Nuc. Acids Res. 19: 1156, 1990). Correct insertion of the fragment was established by restriction endonuclease analysis of DNA isolated from ampicillin-resistant transformants. The resulting plasmid was designated as pCGP1620. The nucleotide sequence of the chrysanthemum CHS promoter fragment contained in pCGP1620 is represented as SEQ ID NO:30.
Construction of pCGP1622 (chrysCHS 5′: GUS: nos 3′ pUC Backbone)
A  ̃2.5 kb fragment containing the chrysanthemum CHS promoter was released from the plasmid pCGP1620 upon digestion with the restriction endonucleases NcoI and PstI. The fragment was purified and ligated with a 4.8 kb NcoI/PstI fragment of pjB1 (Bodeau, 1994, supra) containing the backbone vector with the GUS and nos 3′ fragments. Correct insertion of the fragment was established by restriction endonuclease analysis of DNA isolated from ampicillin-resistant transformants. The resulting plasmid was designated as pCGP1622.
Construction of pCGP1626 (chrysCHS 5′: GUS: nos 3′ in Binary Vector)
A  ̃4.6 kb fragment containing the chrysCHS 5′: GUS: nos 3′ cassette was released from the plasmid pCGP1622 upon digestion with the restriction endonucleases PstI and BglII. The fragment was purified and ligated with PstI/BamHI ends of the binary vector pWTT2132 (DNAP) (FIG. 6). Correct insertion of the cassette in a tandem orientation with respect to the 35S 5′: SuRB selectable marker gene cassette was established by restriction endonuclease analysis of DNA isolated from tetracycline-resistant transformants. The resulting plasmid was designated as pCGP1626 (FIG. 20).
Plant Transformation with pCGP1626
The T-DNA contained in the binary vector plasmid pCGP1626 (FIG. 20) was introduced into rose via Agrobacterium-mediated transformation.
The Binary Vector pCGP1641 (petRT 5′: GUS: petRT 3′)
The plasmid pCGP1641 (FIG. 21) contains a chimeric GUS gene under the control of a petunia 3RT promoter (petRT 5′) covering 1.1 kb upstream from the putative 3RT translation initiation codon with a petunia 3RT terminator (petRT 3′) covering 2.5 kb downstream from the 3RT stop codon. The chimeric GUS cassette is in a tandem orientation with respect to the 35S 5′: SuRB selectable marker gene cassette of the binary vector, pWTT2132 (DNAP) (FIG. 6).
Intermediates in the Preparation of the Binary Vector pCGP1641
Isolation of Petunia 3RT Gene
The isolation of the petunia 3RT gene corresponding to the Rt locus of P. hybrida has been described in Brugliera, 1994, supra.
Construction of pCGP1625 (CaMV 35S: GUS: petRT 3′ Cassette)
The intermediate plasmid pCGP1625 contains a CaMV 35S: GUS: petRT 3′ cassette in a pUC backbone. The 2.5 kb fragment containing a petRT terminator sequences was released from the plasmid pCGP1610 (described in Brugliera 1994, supra) upon digestion with the restriction endonucleases BamHI and SacI. The fragment was purified and ligated with the BglII/SacI 4.9 kb fragment of pJB1 (Bodeau, 1994, supra) containing the vector backbone and the CaMV 35S promoter and GUS fragments. Correct insertion of the petunia 3RT terminator fragment downstream of the GUS fragment was established by restriction endonuclease analysis of DNA isolated from ampicillin-resistant transformants. The resulting plasmid was designated as pCGP1625.
Construction of pCGP1628 (petRT 5′: GUS: petRT 3′ Cassette)
A 1.1 kb petRT promoter fragment was released from the plasmid pCGP1611 (described in Brugliera, 1994, supra) upon digestion with the restriction endonucleases NcoI and PstI. The purified fragment was ligated with NcoI/PstI ends of the 7 kb fragment of pCGP1625 containing the vector backbone and the GUS and petRT 3′ fragments. Correct insertion of the petRT promoter fragment upstream of the GUS fragment was established by restriction endonuclease analysis of DNA isolated from ampicillin-resistant transformants. The resulting plasmid was designated as pCGP1628.
Construction of pCGP1641, (petRT5′: GUS: petRT 3′Binary Vector)
A 5.4 kb fragment containing the petRT 5′: GUS: petRT 3′ cassette was released from pCGP1628 upon digestion with the restriction endonuclease PstI. The fragment was purified and ligated with PstI ends of the binary vector pWTT2132 (DNAP) (FIG. 6). Correct insertion of the fragment in a tandem orientation with respect to the 35S 5′: SuRB selectable marker gene cassette was established by restriction endonuclease analysis of plasmid DNA isolated from tetracycline-resistant transformants. The resulting plasmid was designated as pCGP1641 (FIG. 21).
Plant Transformation with pCGP1641
The T-DNA contained in the binary vector plasmid pCGP1641 (FIG. 21) was introduced into rose via Agrobacterium-mediated transformation.
The Binary Vector pCGP1861 (RoseCHS 5′: GUS: nos 3′)
The plasmid pCGP1861 (FIG. 22) contains a chimeric GUS gene under the control of a promoter fragment from the CHS gene of R. hybrida (RoseCHS 5′) with a terminator fragment from the nos gene of Agrobacterium (nos 3′). The chimeric GUS reporter gene cassette is in a tandem orientation with respect to the 35S 5′: SuRB selectable marker gene cassette of the binary vector, pWTT2132 (FIG. 6).
An  ̃5 kb fragment containing the RoseCHS 5′: GUS: nos 3′ cassette was released from pCGP197 (described in Example 4) upon digestion with the restriction endonuclease BglII. The fragment was purified and ligated with BamHI ends of the binary vector, pWTT2132 (DNAP). Correct insertion of the fragment in a tandem orientation with respect to the 35S 5′: SuRB selectable marker gene cassette was established by restriction endonuclease analysis of plasmid DNA isolated from tetracycline-resistant transformants. The resulting plasmid was designated as pCGP1861 (FIG. 22).
Plant Transformation with pCGP1861
The T-DNA contained in the binary vector plasmid pCGP1861 (FIG. 22) was introduced into rose via Agrobacterium-medicated transformation.
The Binary Vector pCGP1953 (AmCHS 5′: GUS: petD8 3′)
The plasmid pCGP1953 (FIG. 23) contains a chimeric GUS gene under the control of a promoter fragment from the CHS gene of Antirrhinum majus (AmCHS 5′) with a petunia PLTP terminator (petD8 3′). The chimeric GUS reporter gene cassette is in a tandem orientation with respect to the 35S 5′: SuRB selectable marker gene cassette of the binary vector, pWTT2132 (DNAP) (FIG. 6).
Intermediates in the Preparation of the Binary Vector pCGP1953
The plasmid pJB1 (Bodeau, 1994, supra) was linearised with the restriction endonuclease NcoI. The overhanging ends were repaired and the 1.8 kb GUS fragment was released upon digestion with BamHI. The GUS fragment was purified and was ligated with the 5 kb XbaI (ends repaired)/BamHI fragment of pCGP726 containing the pBluescript backbone vector and the AmCHS 5′ and petD8 3′ fragments (described in Example 4). Correct insertion of the GUS fragment between the AmCHS 5′ and petD8 3′ fragments was established by restriction endonuclease analysis of plasmid DNA isolated from ampicillin-resistant transformants. The plasmid was designated as pCGP1952.
A 3.8 kb fragment containing the AmCHS 5′: GUS: petD8 3′ expression cassette was released from the plasmid pCGP1952 upon digestion with the restriction endonucleases EagI and PstI. The overhanging ends were repaired and the purified fragment was ligated with the repaired ends of an Asp718 digested pWTT2132 binary vector (FIG. 6). Correct insertion of the fragment in a tandem orientation with respect to the 35S 5′: SuRB selectable marker gene cassette was established by restriction endonuclease analysis of plasmid DNA isolated from tetracycline-resistant transformants. The plasmid was designated as pCGP1953 (FIG. 23).
Plant Transformation with pCGP1953
The T-DNA contained in the binary vector plasmid pCGP1953 (FIG. 23) was introduced into rose via Agrobacterium-mediated transformation.
The Binary Vector pWTT2084 (35S 5′: GUS: ocs 3′)
The plasmid pWTT2084 (DNAP) (FIG. 24) contains a chimeric GUS gene under the control of a CaMV 35S promoter (35S 5′) with an octopine synthase terminator (ocs 3′). An  ̃60 bp 5′ untranslated leader sequence from the petunia chlorophyll a/b binding protein gene (Cab 22 gene) (Harpster et al., 1988, supra) is included between the CaMV 35S promoter fragment and the GUS clone. The chimeric GUS cassette is in a tandem orientation with respect to the 35S 5′: SuRB selectable marker gene cassette of the binary vector, pWTT2084.
Plant Transformation with pWTT2084
The T-DNA contained in the binary vector plasmid pWTT2084 (FIG. 24) was introduced into rose via Agrobacterium-mediated transformation.
Transgenic Analysis of Roses Transformed with GUS Expression Cassettes
Northern blot analysis was performed on total RNA isolated from petals of developmental stages 3 to 4 of transgenic Kardinal roses transformed with the T-DNA of various GUS expression cassettes. There was either no accumulating transcript or an intact full-length transcript of the expected size of  ̃1.8 kb as detected by Northern blot hybridisation. The relative levels of GUS transcripts accumulating in the rose petals were recorded (see Table 8).







TABLE 8







Summary of Northern analysis on transgenic Kardinal rose


flowers (open bud stage) containing GUS constructs.












SELECTABLE
GUS




MARKER
TRANSCRIPT


PLASMID
GUS REPORTERGENE
GENE
LEVELS





pCGP1307
petD8 5′:GUS:petD8 3′
mas
—




5′:nptII:mas 3′


pCGP1506
petFLS 5′:GUS:petFLS 3′
nos
—




5′:nptII:nos 3′


pCGP1626
chrysCHS 5′:GUS:petRT
35S 5′:SuRB
++ to +++



3′


pCGP1641
petRT
35S 5′:SuRB
—



5′:GUS:petRT 3′


pCGP1861
RoseCHS 5′:GUS:nos 3′
35S 5′:SuRB
++++


pCGP1953
AmCHS 5′:GUS:petD8 3′
35S 5′:SuRS
—


pWTT2084
35S 5′:GUS:ocs 3′
35S 5′:SuRB
+++++





—= no transcripts detected


+ to +++++ = relative levels (low to high) of full-length GUS transcript detected by Northern blot analysis






Based on the above results (Table 8), the CaMV 35S (35S 5′) and rose CHS (RoseCHS 5′) promoters appear to drive relatively high levels of transcription in rose petals. The chrysanthemum CHS promoter (chrysCHS 5′) appears to also lead to high transcript levels but not as high as those obtained using CaMV 35S or rose CRS promoters. Surprisingly, antirrhinum (snapdragon) CHS (AmCHS 5′), petunia 3RT (petRT 5′), petunia FLS (petFLS 5′) and petunia PLTP-(petD8 5′) promoters did not appear to function in rose petals as no GUS transcripts were detected with expression cassettes incorporating these promoters. However, these same promoters fused to petHf1 and/or -GUS genes had previously been proven to function well in carnation and petunia leading to relatively high full-length transcript levels and for petHf1 genes, the production of delphinidin or delphinidin-based molecules pigments. The result obtained with the antirrhinum CHS promoter (AmCHS 5′) fused with the GUS gene was more surprising as promoter regions from homologous genes from two other species (rose and chrysanthemum) appeared to function relatively well in roses. The antirrhinum CHS promoter had also been successfully used in conjunction with petunia F3′5′H (petHf1) to produce the novel violet-colored carnations Florigene Moondust (see International Patent Application No. PCT/AU96/00296).
The evaluation of promoter and terminator fragments fused with the GUS gene also provided further evidence to suggest that the petunia F3′5′H petHf1 and petHf2 sequences were unstable in roses as constructs containing the petunia F3′5′H sequences ligated to the CaMV 35S, -rose CHS and chrysanthemum CHS promoters (which do function in rose) did not result in intact petunia F3′5′H petHf1 or petHf2 transcripts in roses (see Table 6).
Example 7
Isolation of F3′5′H Sequences from Species Other than Petunia
Since the petunia F3′5′H sequences had already been proven to function in various plants such as carnation, petunia and tobacco and ultimately resulted in the production of delphinidin-based pigments, it was reasonable to assume that these sequences would also prove functional in roses. There was an assumption that the enzyme activity may vary depending on the background of the species, indeed between cultivars of a given species, that the petunia F3′5′H was introduced into. However, there was no expectation that full-length recombinant petunia F3′5′H mRNA would not accumulate. Analysis of the petunia F3′5′H nucleotide sequences (petHf1 and petHf2) did not reveal any sequences which might lead to instability and subsequent degradation (Johnson et al., In A look beyond transcription, ASPP, USA, Bailey-Serres and Gallie, eds, 1998), intron: exon splice junctions (Brendel et al., In A look beyond transcription, ASPP, USA. Bailey-Serres and Gallie, eds, 1998), or any autocatalytic or degradation trigger sequences reported in the scientific literature to date (In A look beyond transcription, ASPP, USA, Bailey-Serres and Gallie, eds, 1998). The surprising result suggested that there were factors specific to rose that resulted in petunia F3′5′H sequences being unstable.
Since it was not obvious why the petunia F3′5′H sequences were unstable in roses but stable in carnation, petunia or tobacco, a number of F3′5′H sequences were isolated across a range of families in an attempt to determine whether any F3′5′H sequence would be stable in rose and then identify any F3′5′H sequences that would lead to the synthesis of stable F3′5′H transcripts and F3′5′H activity and ultimately the production of delphinidin-based pigments in roses leading to a change in flower color.
Construction of Petal cDNA Libraries
Petal cDNA libraries were prepared from RNA isolated from petals from bud to opened flower stages from various species of plants described in Table 9, Rosa hybrida is classified in the family Rosaciae, Order Rosales, Subclass Rosidae and so species that produced delphinidin-based pigments and so contained a functional F3′5′H and belonged to the Subclass Rosidae were selected. Petunia hybrida is classified in the Family Solanaceae, Order Solanales, Subclass Asteridae and so species from the Subclass Asteridae that produced delphinidin-based pigments were also selected.







TABLE 9







List of flowers from which total RNA was isolated for the preparation of


petal cDNA libraries. Information obtained from National Center for


Biotechnology Information (NCBI) website under Taxonomy browser


(TaxBrowser) as of August 2003.











FLOWER
SPECIES
FAMILY
ORDER
SUBCLASS





gentian
Gentiana spp.
Gentianaceae
Gentianales
Asteridae


lavender
Lavandula spp.
Lamiaceae
Lamiales
Asteridae


salvia
Salvia spp.
Lamiaceae
Lamiales
Asteridae


sollya
Sollya spp.
Pittosporaceae
Apiales
Asteridae


kennedia
Kennedia spp.
Fabaceae
Fabales
Rosidae


butterfly
Clitoria
Fabaceae
Fabales
Rosidae


pea
ternatea


pansy
Viola spp.
Violaceae
Malpighiales
Rosidae









Unless otherwise described, total RNA was isolated from the petal tissue of purple/blue flowers using the method of Turpen and Griffith (BioTechniques 4: 11-15, 1986). Poly(A)+ RNA was selected from the total RNA, using oligotex-dTTM (Qiagen) or by three cycles of oligo-dT cellulose chromatography (Aviv and Leder, Proc. Natl. Acad. Sci. USA 69: 1408, 1972).
In general a λZAPII/Gigapack II Cloning kit (Stratagene, USA) (Short et al., Nucl. Acids Res. 16: 7583-7600, 1988) was used to construct directional petal cDNA libraries in λZAPII using around 5 μg of poly(A)+ RNA isolated from petal as template. The total number of recombinants obtained was generally in the order of 1×105 to 1×106.
After transfecting XL1-Blue MRF′ cells, the packaged cDNA mixtures were plated at around 50,000 pfu per 15 cm diameter plate. The plates were incubated at 37° C. for 8 hours, and the phage were eluted in 100 mM NaCl, 8 mM MgSO4, 50 mM Tris-HCl pH 8.0, 0.01% (w/v) gelatin (Phage Storage Buffer (PSB)) (Sambrook et al., 1989, supra). Chloroform was added and the phages stored at 4° C. as amplified libraries.
In general around 100,000 pfu of the amplified libraries were plated onto NZY plates (Sambrook et al., 1989, supra) at a density of around 10,000 pfu per 15 cm plate after transfecting XL1-Blue MRF′ cells, and incubated at 37° C. for 8 hours. After incubation at 4° C. overnight, duplicate lifts were taken onto Colony/Plaque ScreenTM filters (DuPont) and treated as recommended by the manufacturer.
Plasmid Isolation
Helper phage R408 (Stratagene, USA) was used to excise pBluescript phagemids containing cDNA inserts from amplified λZAPII or λZAP cDNA libraries using methods described by the manufacturer.
Screening of Petal cDNA Libraries
Prior to hybridization, duplicate plaque lifts were washed in prewashing solution (50 mM Tris-HCl pH7.5, 1 M NaCl, 1 mM EDTA, 0.1% (w/v) sarcosine) at 65° C. for 30 minutes; followed by washing in 0.4 M sodium hydroxide at 65° C. for, 30 minutes; then washed in a solution of 0.2 M Tris-HCl pH 8.0, 0.1×SSC, 0.1% (w/v) SDS at 65° C. for 30 minutes and finally rinsed in 2×SSC, 1.0% (w/v) SDS.
The membrane lifts from the petal cDNA libraries were hybridized with 32P-labelled fragments of a 1.6 kb BspHI/FspI fragment from pCGP602 (FIG. 2) (SEQ ID NO: 1) containing the petunia F3′5′H petHf1 cDNA clone (Holton et al., 1993a, supra).
Hybridization conditions included a prehybridization step in 10% v/v formamide, 1 M NaCl, 10% w/v dextran sulphate, 1% w/v SDS at 42° C. for at least 1 hour. The 32P-labelled fragments (each at 1×106 cpm/mL) were then added to the hybridization solution and hybridization was continued at 42° C. for a further 16 hours. The filters were then washed in 2×SSC, 1% w/v SDS at 42° C. for 2×1 hour and exposed to Kodak XAR film with an intensifying screen at −70° C. for 16 hours.
Strongly hybridizing plaques were picked into PSB (Sambrook at al., 1989, supra) and rescreened to isolate purified plaques, using the plating and hybridization conditions as described for the initial screening of the cDNA library. The plasmids contained in the λZAPII or λZAP bacteriophage vectors were rescued and sequence data was generated from the 3′ and 5′ ends of the cDNA inserts. New F3′5′H cDNA clones were identified based on sequence similarity to the petunia F3′5′H petHf1 cDNA clone.
The cDNA clones isolated were given plasmid designation numbers as described in Table 10.







TABLE 10







Plasmid numbers and SEQ ID NO. of F3′5′H cDNA


clones isolated from various species













PLASMID
FIG.



SPECIES
CLONE
NUMBER
NUMBER
SEQ ID NO.














Viola spp.
BP#18
pCGP1959
25
9


Viola spp.
BP#40
pCGP1961
26
11


Salvia spp.
Sal#2
pCGP1995
31
13


Salvia spp.
Sal#47
pCGP1999
32
15


Sollya spp.
Soll#5
pCGP2110
37
17


Kennedia spp.
Kenn#31
pCGP2231
40
26


Clitoria ternatea
BpeaHF2
pBHF2F4
43
20


Gentiana triflora
Gen#48
pG48
47
22


Lavandula nil
LBG
pLHF8
51
31









Viola (Pansy) F3′5′H Constructs

Isolation of F3′5′H cDNA Clones from Petals of Viola spp. (Pansy)

Total RNA and poly (A)+ RNA was isolated from petals of young buds of Viola spp. cultivar black pansy as described above. A petal cDNA library was constructed using λZAPII/Gigapack II Cloning kit (Stratagene, USA) and screened as described above. Two full-length pansy F3′5′H cDNA clones (BP#18 (SEQ ID NO:9) in pCGP1959 (FIG. 25) and BP#40 (SEQ ID NO:11) in pCGP1961 (FIG. 26)) were identified by sequence similarity to the petunia F3′5′H petHf1 cDNA clone (SEQ D NO:1). The BP#18 and BP#40 shared 82% identity at the nucleotide level. Comparison of the nucleotide sequence of pansy F3′5′H clones (BP#18 and BP#40) with that of the petunia P3′5′H revealed around 60% identity to the petunia F3′5′H petHf1 clone and 62% identity to the petunia F3′5′H petHf2 clone.
The Binary Vectors, pCGP1972 and pCGP1973 (AmCHS 5′: BP#18 or BP#40: petD8 3′)
The plasmids pCGP1972 (FIG. 27) and pCGP1973 (FIG. 28) contain the pansy F3′5′H cDNA clone (BP#18 and BP#40, respectively) between an A. majus (snapdragon) CHS promoter fragment (Am CHS 5′) and a petunia PLTP terminator fragment (petD8 3′). The chimeric F3′5′H genes are in tandem with respect to the 35S 5′: SuRB selectable marker gene cassette of the binary vector, pWTT2132 (DNAP) (FIG. 6).
The petunia F3′5′H (petHf1) cDNA clone in pCGP725 (described in Example 4) (FIG. 7) was removed by initially digesting pCGP725 with the restriction endonuclease BamHI. The ends were repaired and the linearised plasmid was further digested with the restriction endonuclease XbaI. The  ̃4.9 kb fragment containing the vector with the AmCHS 5′ and petD8 3′ fragments was purified and ligated with the  ̃1.6 kb KpnI (ends repaired)/XbaI fragment containing the pansy F3′5′H BP#18 or BP#40 cDNA clone from pCGP1959 or pCGP1961, respectively to produce pCGP1970 and pCGP1971, respectively. The AmCHS 5′: pansy F3′5′H: petD8 3′ cassette was then isolated from pCGP1970 or pCGP1971 by firstly digesting with the restriction endonuclease NotI. The ends of the linearised plasmid were repaired and then the chimeric F3′5′H genes were released upon digestion with the restriction endonuclease EcoRV. The purified fragments were then ligated with Asp718 (repaired ends) of the binary vector pWTT2132 (DNAP). Correct insertion of the fragment was established by restriction endonuclease analysis of plasmid DNA isolated from tetracycline-resistant transformants. The resulting plasmids were designated pCGP1972 (FIG. 27) and pCGP1973 (FIG. 28), respectively.
Carnation and Petunia Transformation with pCGP1972 and 1973
The T-DNAs contained in the binary vector plasmids pCGP1972 (FIG. 27) and pCGP1973 (FIG. 28) were introduced separately into Dianthus caryoplhyllus cultivars Kortina Chanel and Monte Lisa and Petunia hybrid cv. Skr4×Sw63 via Agrobacterium-mediated transformation.
The Binary Vectors, pCGP1967 and pCGP1969 (CaMV 35S: pansy F3′5′H: ocs 3′)
The binary vectors pCGP1967 (FIG. 29) and pCGP1969 (FIG. 30) contain chimeric CaMV 35S: pansy F3′5′H: ocs 3′ genes in tandem with respect to the 35S 5′: SuRB selectable marker gene cassette of the binary vector, pWTT2132 (DNAP) (FIG. 6).
Intermediates in the Preparation of the Binary Vectors pCGP1967 and pCGP1969
The plasmids pCGP1959 (FIG. 25) and pCGP1961 (FIG. 26) were firstly linearized upon digestion with the restriction endonuclease KpnI. The overhanging KpnI ends were repaired and the pansy F3′5′H cDNA clones, BP#18 and BP#40, were released upon digestion with the restriction endonuclease PstI. The  ̃1.6 kb fragments generated were ligated with an  ̃5.9 kb EcoRI (repaired ends)/PstI fragment of pKIWI101 (Janssen and Gardner, 1989, supra). Correct insertion of each fragment was established by restriction endonuclease analysis of plasmid DNA isolated from ampicillin-resistant transformants. The resulting plasmids were designated pCGP1965 and pCGP1966, respectively.
The plasmids pCGP1965 and pCGP1966 were firstly partially digested with the restriction endonuclease XhoI. The resulting fragments were further digested with the restriction endonuclease XbaI. The overhanging ends were repaired and the 3.6 kb fragments containing the CaMV 35S: pansy F3′5′H: ocs 3′ chimeric genes were isolated and ligated with Asp718 repaired ends of pWTT2132 (FIG. 6). Correct insertion of each fragment was established by restriction endonuclease analysis of plasmid DNA isolated from tetracycline-resistant transformants. The resulting plasmids were designated pCGP1967 (FIG. 29) and pCGP1969 (FIG. 30), respectively.
Rose Transformation with pCGP1967 and pCGP1969
The T-DNAs contained in the binary vector plasmids pCGP1967 (FIG. 29) and pCGP1969 (FIG. 31) were introduced separately into Rosa hybrida cv. Kardinal and Soft Promise via Agrobacterium-mediated transformation. The T-DNA contained in the binary vector plasmids pCGP1969 (FIG. 31) was also introduced into Rosa hybrida cu. Pamela and Medeo via Agrobacterium-mediated transformation,
Salvia F3′5′H Constructs
Isolation of a F3′5′H cDNA Clone from Petals of Salvia spp.
Total RNA and poly (A)+ RNA was isolated from young petal buds of Salvia spp. (bought from a nursery) as described above. A petal cDNA library was constructed using λZAPII/Gigapack II Clog kit (Stratagene, USA). Two full-length salvia F3′5′H cDNA clones (Sal#2 (SEQ ID NO:13) in pCGP1995 (FIG. 31) and Sal#47 (SEQ ID NO:15) in pCGP1999 (FIG. 32)) were identified by sequence similarity with the petunia F3′5′H petHf1 cDNA clone. The Sal#2 and Sal#47 shared 95% identity at the nucleotide level Comparison of the nucleotide sequence of salvia F3′5′H clones (Sal#2 and Sal#47) with that of the petunia F3′5′H revealed around 57% identity to the petunia F3′5′H petHf1 clone (SEQ ID NO:1) and 58% identity to the petunia F3′5′H petHf2 clone (SEQ ID NO:3).
The Binary Vectors, pCGP2121 and pCGP2122
(AmCHS 5′: Salvia F3′5′H #2 or #47: petD8 3′)
The plasmids pCGP2121 (FIG. 33) and pCGP2122 (FIG. 34) contain the salvia F3′5′H cDNA clones (Sal#2 and Sal#47, respectively) between a snapdragon CHS promoter fragment (AmCHS 5′) and a petunia PLTP terminator fragment (petD8 3′) in tandem with the 35S 5′: SuRB selectable marker gene cassette of the binary vector pWTT2132 (DNAP) (FIG. 6).
The petunia F3′5′H (petHf1) cDNA clone in pCGP725 (described in Example 4) (FIG. 7) was removed by initially digesting pCGP725 with the restriction endonuclease BamHI. The ends were repaired and the linearised plasmid was further digested with the restriction endonuclease XbaI. The  ̃4.9 kb fragment containing the vector with the AmCHS 5′ and petD8 3′ fragments was purified and ligated with the  ̃1.6 kb XhoI/BamHI (ends repaired) fragment from pCGP 1995 (FIG. 31) containing the salvia F3′4′H #2 or XhoI/EcoRI (ends repaired) fragment from pCGP1999 (FIG. 32) containing the salvia F3′5′H #47, respectively to produce pCGP2116 and pCGP2117, respectively.
The AmCHS 5′: salvia F3′5′H: petD8 3′ cassette was isolated from pCGP2116 or pCGP2117 by firstly digesting with the restriction endonuclease NotI. The ends of the linearized plasmid were repaired and then the chimeric F3′5′H gene cassettes were released upon digestion with the restriction endonuclease EcoRV. The  ̃3.6 kb purified fragments were then ligated with Asp718 repaired ends of the binary vector pCGP1988 (FIG. 16) (described in Example 4). Correct insertion of each fragment was established by restriction endonuclease analysis of plasmid DNA isolated from tetracycline-resistant transformants. The resulting plasmids were designated pCGP2121 (FIG. 33) and pCGP2122 (FIG. 34), respectively.
Carnation and Petunia Transformation with pCGP2121 and pCGP2122
The T-DNAs contained in the binary vector plasmids pCGP2121 (FIG. 33) and pCGP2122 (FIG. 34) were introduced separately into Dianthus caryoplhyllus cultivars Kortina Chanel and Monte Lisa and Petunia hybrida cv. Skr4×Sw63 via Agrobacterium-mediated transformation.
The Binary Vectors, pCGP2120 and pCGP2119 (CaMV 35S: Salvia F3′5′H: ocs 3′)
The binary vectors pCGP2120 (FIG. 35) and pCGP2119 (FIG. 36) contain chimeric CaMV 35S: salvia F3′5′H: ocs 3′ gene cassettes in tandem with the 35S 5′. SuRB selectable marker gene cassette of the binary vector pCGP1988 (FIG. 16).
Intermediates in the Preparation of the Binary Vectors pCGP2120 and pCGP2119
The plasmids pCGP1995 (FIG. 31) and pCGP1999 (FIG. 32) were firstly linearized upon digestion with the restriction endonuclease XhoI. The overhanging XhoI ends were repaired and then the salvia F3′5′H cDNA clones Sal#2 or Sal#47 were released upon digestion with the restriction endonuclease EcoRI. In the case of pCGP1995 a partial digest with EcoRI was undertaken. The  ̃1.7 kb fragments were ligated with the ClaI (repaired ends)/EcoRI ends of pCGP2105 (FIG. 17). Correct insertion of each fragment was established by restriction endonuclease analysis of plasmid DNA isolated from ampicillin-resistant transformants. The resulting plasmids were designated pCGP2112 and pCGP2111, respectively.
The plasmids pCGP2112 and pCGP2111 were digested with the restriction endonucleases XhoI and XbaI. The resulting overhanging ends were repaired and  ̃3.6 kb fragments containing the CaMV 35S: salvia F3′5′H: ocs 3′ chimeric genes were isolated and ligated with Asp718 repaired ends of the binary vector, pCGP1988 (described in Example 4). Correct insertion of each fragment was established by restriction endonuclease analysis of plasmid DNA isolated from tetracycline-resistant transformants. The resulting plasmids were designated pCGP2120 (FIG. 35) and pCGP2119 (FIG. 36), respectively,
Rose Transformation with pCGP2120 and pCGP2119
The T-DNAs contained in the binary vector plasmids pCGP2120 (FIG. 35) and pCGP2119 (FIG. 36) were introduced separately into Rosa hybrida cv. Kardinal via Agrobacterium-mediated transformation.
Sollya F3′5′H Constructs
Isolation of a F3′5′H cDNA Clone from Petals of Sollya spp.
Total RNA and poly (A)+ RNA was isolated from young petal buds of Sollya spp. (bought from a nursery) as described above. A petal cDNA library was constructed using λZAPII/Gigapack II Cloning kit (Stratagene, USA). One full-length Sollya F3′5′H cDNA clone (Soll#S5 (SEQ ID NO:17) in pCGP2110 (FIG. 37)) was identified by sequence similarity to the petunia F3′5′H petHf1 cDNA clone. Comparison of the nucleotide sequence of the sollya F3′5′H clone with that of the petunia F3′5′H revealed around 48% identity to the petunia F3′5′H petHf1 clone (SEQ ID NO:1) and 52% identity to the petunia F3′5′H petHf2 clone (SEQ ID NO:3).
The Binary Vector pCGP2130 (AmCHS 5′: Sollya F3′5′H: petD8 3′)
The plasmid pCGP2130 (FIG. 38) contains the sollya F3′5′H Soll#5 cDNA clone between a snapdragon CHS promoter fragment (AmCHS 5′) and a petunia PLTP terminator fragment (petD8 3′) in a tandem orientation with respect to the 35S 5′: SuRB selectable marker gene cassette of the binary vector pCGP1988 (FIG. 16).
The petunia F3′5′H (petHf1) cDNA clone in pCGP725 (described in Example 4) (FIG. 7) was removed by initially digesting pCGP725 with the restriction endonucleases XbaI and BamHI. The ends were repaired the  ̃4.9 kb fragment containing the vector with the AmCHS 5′ and petD8 3′ fragments was purified and ligated with the repaired ends of the  ̃1.6 kb Asp718/PstI fragment from pCGP2110 containing the sollya F3′5′H cDNA clone to produce pCGP2128. Correct insertion of the sollya F3′5′H fragment in tandem with the AmCHS 5′ and petD8 3′ fragments was confirmed by restriction endonuclease mapping.
The AmCHS 5′: sollya F3′5′H: petD8 3′ gene cassette was then isolated from pCGP2128 by firstly digesting with the restriction endonuclease NotI. The ends of the linearized plasmid were repaired and then the chimeric F3′5′H gene was released upon digestion with the restriction endonuclease EcoRV. The  ̃3.5 kb purified fragment was then ligated with Asp718 (repaired ends) of the binary vector pCGP 1988 (described in Example 4) (FIG. 16). Correct insertion of the fragment was established by restriction endonuclease analysis of plasmid DNA isolated from tetracycline-resistant transformants. The resulting plasmid was designated pCGP2130 (FIG. 38).
Carnation and Petunia Transformation with pCGP2130
The T-DTA contained in the binary vector plasmid pCGP2130 (FIG. 38) was introduced into Dianthus caryoplhyllus cultivars Kortina Chanel and Monte Lisa and Petunia hybrida cv. Skr4×Sw63 via Agrobacterium-mediated transformation.
The Binary Vector pCGP2131 (CaMV35S: sollya F3′5′H: ocs 3′)
The binary vector pCGP2131 (FIG. 39) contains a chimeric CaMV 35S: sollya F3′5′H: ocs 3′ gene in tandem with the 35S 5′: SuRB selectable marker gene cassette of the binary vector pCGP1988 (FIG. 16).
Intermediates in the Preparation of the Binary Vector pCGP2131
The plasmid pCGP2110 was firstly linearized upon digestion with the restriction endonuclease Asp718. The overhanging ends were repaired and then the sollya F3′5′H cDNA clone was released upon digestion with the restriction endonuclease PstI. The  ̃1.7 kb fragment was ligated with the EcoRV/PstI ends of pCGP2105 (FIG. 17). Correct insertion of the fragment was established by restriction endonuclease analysis of plasmid DNA isolated from ampicillin-resistant transformants. The resulting plasmid was designated pCGP2129.
A 3.6 kb fragment containing the CaMV 35S: sollya F3′5′H: ocs 3′ chimeric gene was released upon digestion with the restriction endonucleases Asp718 and XbaI The overhanging ends were repaired and the purified fragment was ligated with of Asp718 repaired ends of the binary vector, pCGP1988 (FIG. 16). Correct insertion of the fragment was established by restriction endonuclease analysis of plasmid DNA isolated from tetracycline-resistant transformants. The resulting plasmid was designated pCGP2131 (FIG. 39).
Rose Transformation with pCGP2131
The T-DNA contained in the binary vector plasmid pCGP2131 (FIG. 39) was introduced into Rosa hybrida cv. Kardinal via Agrobacterium-mediated transformation.
Kennedia F3′5′H Constructs
Isolation of a F3′5′H cDNA Clone from Petals of Kennedia spp.
Total RNA and poly (A)+ RNA was isolated from young petal buds of Kennedia spp. (bought from a nursery) as described above. A petal cDNA library was constructed using λZAPII/Gigapack II Cloning kit (Stratagene, USA). One full-length kennedia F3′5′H cDNA clone (Kenn#31 in pCGP2231 (FIG. 40)) (SEQ ID NO:26) was identified by sequence similarity to the petunia F3′5′H petHf1 cDNA clone. Comparison of the nucleotide sequence of the kennedia F3′5′H clone with that of the petunia F3′5′H revealed around 64% identity to the petunia F3′5′H petHf1 clone (SEQ ID NO:1) and 60% identity to the petunia F3′5′H petHf2 clone (SEQ ID NO:3).
The Binary Vector pCGP2256 (AmCHS 5′: kennedia F3′5′H: petD8 3′)
The plasmid pCGP2256 (FIG. 41) contains the kennedia F3′5′H (Kenn#31) cDNA clone between a snapdragon CHS promoter fragment (AmCHS 5′) and a petunia PLTP terminator fragment (petD8 3′) in tandem with the 35S5′. SuRB selectable marker gene cassette of the binary vector pCGP1988 (FIG. 16).
The petunia F3′5′H (petHf1) cDNA clone in pCGP725 (described in Example 4) (FIG. 7) was removed by initially digesting pCGP725 with the restriction endonucleases XbaI and BamHI. The ends were repaired the  ̃4.9 kb fragment containing the vector with the AmCHS 5′ and petD8 3′ fragments was purified and ligated with the repaired ends of the  ̃1.8 kb XhoI/BamHI fragment from pCGP2231 containing the kennedia F3′5′H cDNA clone to produce pCGP2242. Correct insertion of the kennedia F3′5′H fragment in tandem with the AmCHS 5′ and petD8 3′ fragments was confirmed by restriction endonuclease mapping.
The AmCHS 5′: kennedia F3′5′H: petD8 3′ cassette was then isolated from pCGP2242 by digesting with the restriction endonucleases NotI and EcoRI. The ends were repaired and the  ̃3.7 kb purified fragment was then ligated with Asp718 repaired ends of the binary vector pCGP1988 (described in Example 4) (FIG. 16). Correct insertion of the fragment was established by restriction endonuclease analysis of plasmid DNA isolated from tetracycline-resistant transformants. The resulting plasmid was designated pCGP2256 (FIG. 41).
Petunia Transformation with pCGP2256
The T-DNA contained in the binary vector plasmid pCGP2256 (FIG. 41) was introduced into Petunia hybrida cv. Skr4×Sw63 via Agrobacterium-mediated transformation.
The Binary Vector pCGP2252 (CaMV 35S: kennedia F3′5′H: ocs 3′)
The binary vector pCGP2252 (FIG. 42) contains a chimeric CaMV 35S: kennedia F3′5′H: ocs 3′ gene in tandem with the 35S 5′. SuRB selectable marker cassette of the binary vector pCGP1988 (FIG. 16).
Intermediates in the Preparation of the Binary Vector pCGP2252
The plasmid pCGP2231 was firstly linearized upon digestion with the restriction endonuclease XhoI. The overhanging ends were repaired and then the kennedia F3′5′H cDNA clone was released upon digestion with the restriction endonuclease PstI. The  ̃1.7 kb fragment was ligated with the ClaI (repaired ends)/PstI ends of pCGP2105 (FIG. 17). Correct insertion of the fragment was established by restriction endonuclease analysis of plasmid DNA isolated from ampicillin-resistant transformants. The resulting plasmid was designated pCGP2236.
A 3.6 kb fragment containing the CaMV 35S: kennedia F3′5′H: ocs 3′ chimeric gene cassette was released from the plasmid pCGP2236 upon digestion with the restriction endonucleases XhoI and NotI. The overhanging ends were repaired and the purified fragment was ligated with Asp718 repaired ends of the binary vector, pCGP1988 (FIG. 16). Correct insertion of the fragment was established by restriction endonuclease analysis of plasmid DNA isolated from tetracycline-resistant transformants. The resulting plasmid was designated pCGP2252 (FIG. 42).
Rose Transformation with pCGP2252
The T-DNA contained in the binary vector plasmid pCGP2252 (FIG. 42) was introduced into Rosa hybrida cv. Kardinal via Agrobacterium-mediated transformation.
Butterfly Pea F3′5′H Constructs
Isolation of a F3′5′H cDNA Clone from Petals of Clitoria ternatea (Butterfly Pea)
Construction of Butterfly Pea Petal cDNA Library
A blue variety of Clitoria ternatea (butterfly pea the seeds were kindly provided by Osaka Botanical Garden) was grown in a field in Osaka. Total RNA was isolated from fresh and pigmented petals at a pre-anthesis stage as described above. PolyA+ RNA was prepared using Oligotex (Takara) according to the manufacturer's recommendation. A petal cDNA library of butterfly pea was constructed from the polyA+ RNA using a directional λZAP-cDNA synthesis kit (Stratagene, USA) following the manufacturers protocols.
Screen of Butterfly Pea cDNA Library for a F3′5′H cDNA Clone
The butterfly pea petal cDNA library was screened with DIG-labelled petunia F3′5′H petHf1 cDNA clone as described previously (Tanaka et al., Plant Cell Physiol. 37: 711-716, 1996). Two cDNA clones that showed high sequence similarity to the petunia F3′5′H petHf1 were identified. The plasmid containing the longest cDNA clone was designated pBHF2 and the cDNA clone was sequenced. Alignment between the deduced amino acid sequences of the butterfly pea F3′5′H clone and the petunia F3′5′H petHf1 clone (SEQ ID NO:2) revealed that the butterfly pea F3′5′H cDNA (contained in pBHF2) did not represent a full-length cDNA and lacked first 2 bases of the putative initiation codon. These two bases along with a BamHI restriction endonuclease recognition site were added to the cDNA clone using PCR and a synthetic primer, 5′-GGGATCCAACAATGTTCCTTCTAAGAGAAAT-3′ [SEQ ID NO:25] as described previously (Yonekura-Sakakibara et al., Plant Cell Physiol. 41: 495-502, 2000). The resultant fragment was digested with the restriction endonucleases BamHI and PstI and the subsequent DNA fragment of about 200 bp was recovered. The DNA fragment was ligated with a 3.3 kb fragment of BamHI/EcoRI digested pBHF2 to yield pBHF2F (FIG. 43). The DNA sequence was confirmed to exclude errors made during PCR (SEQ ID NO:20).
Comparison of the nucleotide sequence of butterfly pea F3′5′H clone (SEQ ID NO:20) with that of the petunia F3′5′H revealed around 59% identity to the petunia F3′5′H petHf1 clone (SEQ ID NO:1) and 62% identity to the petunia F3′5′ petHf2 clone (SEQ ID NO:3).
The Binary Vector pCGP2135 (AmCHS 5′: Butterfly Pea F3′5′H: petD8 3′)
The plasmid pCGP2135 (FIG. 44) contains the butterfly pea F3′5′H cDNA clone between a snapdragon CHS promoter fragment (AmCHS 5′) and a petunia PLTP terminator fragment (petD8 3′) in tandem with the 35S 5′: SuRB selectable marker gene cassette of the binary vector pCGP1988 (FIG. 16).
The petunia F3′5′H (petHf1) cDNA clone in pCGP725 (described in Example 4) (FIG. 7) was removed by initially digesting pCGP725 with the restriction endonucleases XbaI and BamHI. The ends were repaired the  ̃4.9 kb fragment containing the vector with the AmCHS 5′ and petD8 3′ fragments was purified and ligated with the repaired ends of the  ̃1.6 kb XhoI/BamHI fragment from pBHF2F (FIG. 43) containing the butterfly pea F3′5′H cDNA clone to produce pCGP2133. Correct insertion of the butterfly pea F3′5′H fragment in tandem with the AmCHS 5′ and petD8 3′ fragments was confirmed by restriction endonuclease mapping.
The AmCHS 5′: butterfly pea F3′5′H: petD8 3′ cassette was then isolated from pCGP2133 by firstly digesting with the restriction endonuclease NotI. The ends of the linearised plasmid were repaired and then the chimeric F3′5′H gene was released upon digestion with the restriction endonuclease EcoRV. The  ̃3.6 kb purified fragment was then ligated with Asp718 repaired ends of the binary vector pCGP1988 (described in Example 4) (FIG. 16). Correct insertion of the fragment was established by restriction endonuclease analysis of plasmid DNA isolated from tetracycline-resistant transformants. The resulting plasmid was designated pCGP2135 (FIG. 44).
Carnation and Petunia Transformation with pCGP2135
The T-DNA contained in the binary vector plasmid pCGP2135 FIG. 44) was introduced into Dianthus caryoplhyllus cultivars Kortina Chanel and Monte Lisa and Petunia hybrida cv. Skr4×Sw63 via Agrobacterium-mediated transformation.
The Binary Vector pBEBF5 (eCaMV 35S: Butterfly pea F3′5′H: nos 3′)
The binary vector, pBE2113-GUS contains a GUS coding region between an enhanced CaMV 35S promoter and nos terminator in a pBI121 binary vector (Mitsuhara et al., 1996, supra). The plasmid pBE2113-GUS was digested with the restriction endonuclease SacI. The overhang ends were repaired and then ligated with a SalI linker to yield pBE2113-GUSs. The 1.8 kb BamHI-XhoI fragment from pBHF2F was ligated with BamHI-SalI digested pBE2113-GUSs to create pBEBF5 (FIG. 45).
Rose Transformation with pBEBF5
The T-DNA contained in the binary vector plasmid pBEBF5 (FIG. 45) was introduced into Rosa hybrida cultivar Lavande via Agrobacterium-mediated transformation.
The Binary Vector pCGP2134 (CaMV 35S: Butterfly pea F3′5′H: ocs 3′)
The binary vector pCGP2134 (FIG. 46) contains a chimeric CaMV 35S: butterfly pea F3′5′H: ocs 3′ gene cassette in a tandem orientation with the 35S 5′: SuRB selectable marker gene cassette of the binary vector pCGP1988 (FIG. 16).
Intermediates in the Preparation of the Binary Vector pCGP2134
The butterfly pea F3′5′H cDNA clone was released upon digestion of the plasmid pBHF2F (FIG. 43) with the restriction endonucleases XhoI and BamHI. The overhanging ends were repaired and the  ̃1.7 kb fragment was ligated with the PstI (repaired ends)/EcoRV ends of pCGP2105 (described in Example 4) (FIG. 17). Correct insertion of the fragment was established by restriction endonuclease analysis of plasmid DNA isolated from ampicillin-resistant transformants. The resulting plasmid was designated pCGP2132.
An  ̃3.6 kb fragment containing the CaMV 35S: butterfly pea F3′5′H: ocs 3′ chimeric gene cassette was released upon digestion with the restriction endonucleases XhoI and XbaI. The overhanging ends were repaired and the purified fragment was ligated with Asp718 repaired ends of the binary vector, pCGP1988 (described in Example 4) (FIG. 16). Correct insertion of the fragment was established by restriction endonuclease analysis of plasmid DNA isolated from tetracycline-resistant transformants. The resulting plasmid was designated pCGP2134 (FIG. 46).
Rose Transformation with pCGP2134
The T-DNA contained in the binary vector plasmid pCGP2134 (FIG. 46) was introduced into Rosa hybrida cv. Kardinal via Agrobacterium-mediated transformation.
Gentia F3′5′H Contructs
Isolation of a F3′5′H cDNA Clone from Petals of Gentiana triflora (Gentian).
Construction and Screening of a Gentian Petal cDNA Library
The isolation of a gentian cDNA encoding F3′5′H has been described previously (Tanaka et al., 1996, supra) and is contained within the plasmid pG48 (FIG. 47). Comparison of the nucleotide sequence of the gentia F3′5′H clone (Gen#48) (SEQ ID NO:22) contained in the plasmid pG48 (FIG. 47) with that of the petunia F3′5′H revealed around 61% identity to the petunia F3′5′H petHf1 clone (SEQ ID NO:1) and 64% identity to the petunia F3′5′H petHf2 clone (SEQ ID NO:3).
The Binary Vector pCGP1498 (AmCHS 5′: Gentia F3′5′H: petD8 3′)
The plasmid pCGP1498 (FIG. 48) contains the gentia F3′5′H (Gen#48) cDNA clone between a snapdragon CHS promoter fragment (AmCHS 5′) and a petunia PLTP terminator fragment (petD8 3′) in tandem with the 35S 5′: SuRB selectable marker gene cassette of the binary vector pWTT2132 (FIG. 6).
The petunia F3′5′H (petHf1) cDNA clone in pCGP725 (described in Example 4) FIG. 7) was removed by initially digesting pCGP725 with the restriction endonucleases XbaI and BamHI. The ends were repaired the  ̃4.9 kb fragment containing the vector with the AmCHS 5′ and petD8 3′ fragments was purified and ligated with the repaired ends of the  ̃1.7 kb XhoI/BamHI fragment from pG48 (FIG. 47) containing the gentia F3′5′H cDNA clone to produce pCGP1496. Correct insertion of the gentia F3′5′H fragment in tandem with the AmCHS 5′ and petD8 3′ fragments was confirmed by restriction endonuclease mapping.
The AmCHS 5′: gentia F3′5′H: petD8 3′ cassette was then isolated from pCGP1496 by firstly digesting with the restriction endonuclease NotI. The overhanging ends of the linearised plasmid were repaired and then the chimeric F3′5′H gene was released upon digestion with the restriction endonuclease EcoRV. The  ̃3.6 kb purified fragment was then ligated with Asp718 repaired ends of the binary vector pWTT2132 (FIG. 6). Correct insertion of the fragment was established by restriction endonuclease analysis of plasmid DNA isolated from tetracycline-resistant transformants. The resulting plasmid was designated pCGP1498 (FIG. 48).
Carnation and Petunia Transformation with pCGP498
The T-DNA contained in the binary vector plasmid pCGP1498 (FIG. 48) was introduced into Dianthus caryoplhyllus cultivars Kortina Chanel and Monte Lisa and Petunia hybrida cv. Skr4×Sw63 via Agrobacterium-mediated transformation.
The Binary Vector pBEGHF48 (eCaMV 35S: Gentia F3′5′H: nos 3′)
The gentia F3′5′H cDNA clone was released by digestion of the plasmid pG48 with the restriction endonucleases BamHI and XhoI. The resulting  ̃1.7 kb DNA fragment was isolated and ligated with BamHI/SalI digested pBE22113-GUSs (Mitsuhara et al., 1996, supra) to create pBEGHF48 FIG. 49).
Rose Transformation with pBEGHF48
The T-DNA contained in the binary vector plasmid pBEGHF48 (FIG. 49) was introduced into Rosa hybrida cv. Lavande via Agrobacterium-mediated transformation.
The Binary Vector pCGP1982 (CaMV 35S: Gentia F3′5′H: ocs 3′)
The binary vector pCGP1982 (FIG. 50) contains a chimeric CaMV 35S: gentia F3′5′H: ocs 3′ gene cassette in tandem with the 35S 5′: SuRB selectable marker gene cassette of the binary vector pWTT2132 (FIG. 6).
Intermediates in the Preparation of the Binary Vector pCGP1982
The plasmid pG48 (FIG. 47) was linearised upon digestion with the restriction endonuclease Asp718. The overhanging ends were repaired and then the gentia F3′5′H cDNA clone (Gen#48) was released upon digestion with the restriction endonuclease BamHI. The  ̃1.7 kb fragment was ligated with the 5.95 kb EcoRI (repaired ends)/BamHI fragment of pKIWI101 (Janssen and Gardner, 1989, supra). Correct insertion of the fragment was established by restriction endonuclease analysis of plasmid DNA isolated from ampicillin-resistant transformants. The resulting plasmid was designated pCGP1981.
An  ̃3.6 kb fragment containing the CaMV 35S: gentia F3′5′H: ocs 3′ chimeric gene cassette was released upon digestion of the plasmid pCGP1981 with the restriction endonucleases XhoI and XbaI The overhanging ends were repaired and the purified fragment was ligated with repaired ends of Asp718 digested binary vector, pWTT2132. Correct insertion of the fragment was established by restriction endonuclease analysis of plasmid DNA isolated from tetracycline-resistant transformants. The resulting plasmid was designated pCGP1982 (FIG. 50).
Rose Transformation with pCGP1982
The T-DNA contained in the binary vector plasmid pCGP1982 (FIG. 50) was introduced into Rosa hybrida cv. Kardinal via Agrobacterium-mediated transformation.
Lavender F3′5′H Constructs
Isolation of a F3′5′H cDNA Clone from Petals of Lavandula nil (Lavender)
Construction of Lavender Petal cDNA Library
Cut flowers of a violet variety of Lavandula nil were purchased from a florist. Total RNA was isolated from fresh and pigmented petals as described above. PolyA+ RNA was prepared using Oligotex (Takara) according to the manufacture's recommendations. A petal cDNA library of lavender was constructed from the polyA+ RNA using a directional λZAP-cDNA synthesis kit (Stratagene, USA) following the manufacturer's protocols.
Screening of Lavender cDNA Library for a F3′5′H cDNA Clone
The lavender petal cDNA library was screened with DIG labelled petunia F3′5′H petHf1 cDNA clone as described previously (Tanaka et al. 1996, supra). One cDNA clone (LBG) that showed high similarity to petunia F3′5′H petHf1 was identified and the plasmid was designated pLHF8 (FIG. 51). The nucleotide sequence of the lavender F3′5′H (LBG) cDNA clone was designated as SEQ ID NO: 31.
Comparison of the nucleotide sequence of lavender F3′5′H clone with that of the petunia F3′5′H cDNA clones revealed around 59% identity to the petunia F3′5′H petHf1 clone (SEQ ID NO:1) and 60% identity to the petunia petHf2 clone (SEQ ID NO:3).
The Binary Vector pBELF8 (eCaMV 35S: Lavender F3′5′H: nos 3′)
The plasmid of pLHF8 (FIG. 51) was digested with the restriction endonucleases BamHI and XhoI to release a DNA fragment of approximately 1.8 kb. The  ̃1.8 kb purified fragment from pLHF8 was then ligated with the BamHI-SalI digested ends of the plasmid pBE2113-GUSs (described above) to create pBELF8 (FIG. 52).
Rose Transformation with pBELF8
The T-DNA contained in the binary vector plasmid pBELF8 (FIG. 52) was introduced into Rosa hybrida cultivar Lavande via Agrobacterium-mediated transformation.
Example8
Analysis of Transgenic Carnation, Petunia and Rose
The transgenic plants produced in the experiments described in Example 7 were grown to flowering. Flowers were collected and the colors of the petals were coded using the Royal Horticultural Society Colour Charts (RHSCC). The anthocyanins were extracted and the anthocyanidins analysed by spectrophotometric, TLC and/or HPLC analysis. Total RNA was also isolated from petal tissue of the appropriate stages of flower development and Northern blot analysis was used to detect transcripts of F3′5′H transgenes. The results of the transgenic analysis are summarised in Tables 11, 12 and 13.
Carnation
The F3′5′H genes described in Example 7 were evaluated for their ability to lead to the production of delphinidin-based pigments in carnation petals. Two carnation cultivars, Kortina Chanel (KC) and Monte Lisa (ML), were used in the transformation experiments. The carnation cultivar Kortina Chanel produces pink colored flowers that normally accumulate cyanidin-based anthocyanins. This cultivar therefore contains a carnation F3′H and DFR activity that an introduced F3′5′H would need to compete with for substrate. The carnation cultivar Monte Lisa produces brick red colored flowers that normally accumulate pelargonidin-based anthocyanins. This cultivar is thought to lack fully functional F3′H activity and contain a DFR that is capable of acting on DHK and thus an introduced F3′5′H would only be required to compete with the endogenous DFR for substrate.







TABLE 11







Results of transgenic analysis of petals from carnations transformed


with T-DNAs containing F3′5′H gene expression cassettes


(AmCHS 5′:F3′5′H:petD8 3′).





















Highest %
Av. %



F3′5′H
pCGP
cv.
#tg
TLC+
HPLC+
del
del
Northern+


















Salvia#2
2121
KC
22
 2/16
3/4
12.5%  
 7%
nd




ML
21
17/18
9/9
76%
57%
14/15


Salvia#47
2122
KC
23
 6/12
8/8
29%
12%
nd




ML
25
21/22
17/17
88%
56%
12/14


Sollya
2130
KC
30
22/27
17/17
35%
11%
nd




ML
23
14/15
14/14
76%
49%
13/14


Butterfly pea
2135
KC
22
 0/16
0/1
nd
nd
nd




ML
24
19/20
13/13
23%
10%
14/14


Gentian
1498
KC
22
 0/14
nd
nd
nd
7/8




ML
2
2/2
1/1
nd
nd
1/2


pansy BP#18
1972
KC
26
18/20
12/12
14%
9%
19/19




ML
21
15/16
8/8
80%
66%
14/16


pansy BP#40
1973
KC
26
11/15
7/8
18%
8%
13/17




ML
33
19/22
20/20
72%
52%
12/15


petunia
1452
KC
104
41/64
nd
3.5% 
1.3% 
15/17


petHf1

ML
48
39/41
26/26
75%
30%
12/13


petunia
1524
ML
27
18/19
17/17
81%
41%
12/14


petHf2





F3′5′H = F3′5′H sequence contained on the T-DNA


pCGP = plasmid pCGP number of the binary vector used in the transformation experiment


cv. = cultivar


KC = carnation cultivar Kortina Chanel (cyanidin line)


ML = carnation cultivar Monte Lisa (pelargonidin line)


#tg = total number of transgenics produced


TLC+ = number of individual events in which delphinidin or delphinidin-based molecules was detected in petals (as determined by TLC) over the total number of individual events analyzed


HPLC+ = number of individual events in which delphinidin or delphinidin-based molecules was detected in petals (as determined by HPLC) over the total number of individual events analyzed


Highest % del = Highest % delphinidin or delphinidin-based molecules detected in the petals for the population of transgenic event


Av % del = average % delphinidin or delphinidin-based molecules detected in the petals for the population of transgenic events


Northern = number of individual events in which the specific intact F3′5H transcripts were detected by Northern blot analysis in total RNA isolated from petals over the total number of events analyzed


nd = not done






The results suggest that all of the F3′5′H sequences evaluated (petunia petHf1, petunia petHf2, Salvia Sal#2, Salvia Sal#47, Sollya Sol#5, Butterfly pea BpeaHF2, pansy BP#18, pansy BP#40 and Gentian Gen#48) were stable in carnation and resulted in the production of novel delphinidin-based pigments in carnation flowers. Intact transcripts of each F3′5′H were detected by Northern blot analysis in total RNA isolated from petals of the transgenic carnations.
Petunia 
The F3′5′H genes described in Example 7 were evaluated for their ability to lead to the production of delphinidin-based pigments in petunia petals. The P. hybrida F1 hybrid Skr4×SW63 which is homozygous recessive for Hf1 and Hf2, was used in the transformation experiments. Although Skr4×SW63 is homozygous recessive for Hf1 and Hf2, these mutations do not completely block production of the endogenous F3′5′H (see U.S. Pat. No. 5,349,125) and low levels of malvidin are produced to give the petal limb a pale lilac color. Malvidin is the methylated derivative of the 3′5′-hydroxylated pigment, delphinidin or delphinidin-based molecules (FIGS. 1A and 1B). Spectrophotometric analysis was used as a measure of total anthocyanins accumulating in petals from the transgenic petunia flowers. The increased level of anthocyanins and/or the color change detected was used as a guide to the efficacy of the F3′5′H gene under evaluation.







TABLE 12







Results of transgenic analysis of petals from P. hybrida cv Skr4 × SW63 plants


transformed with T-DNAs containing F3′5′H gene expression cassettes


(AmCHS 5′:F3′5′H:petD8 3′).

























Best


F3′5′H
pCGP
#tg
TLC+
Col
↑ A/c
Best
Av.
Northern+
color





control
na
na
na
na
na
144-250

0
75C


Gentian#48
1498
22
3/5
18/20
nd


6/6
72B/78A


Butterfly pea
2135
24
18/20
22/24
23/24
4427
2397
nd
74A/78A


Kennedia
2256
24
22/24
22/24
22/24
4212
2592
nd
74A/78A


Salvia#2
2121
24
21/24
21/24
21/24
2471
1730
nd
78A


Salvia#47
2122
19
17/19
16/19
16/19
2634
1755
nd
78A/80A


Sollya#5
2130
22
14/16
13/16
13/16
3446
1565
nd
78A


pansy BP#18
1972
22
nd
20/22
nd
nd
nd
9/9
74A/B


pansy BP#40
1973
19
8/8
18/19
18/20
2583
1556
nd
74/78A


petunia
 484
16
nd
 9/16
 8/15
2683
1250
nd
74A/B


petHf1


petunia
1524
20
nd
18/20
8/8
4578
2357
8/8
74A/B


petHf2





F3′5′H = F3′5′H sequence contained on the T-DNA


pCGP = plasmid pCGP number of the binary vector used in the transformation experiment


#tg = total number of transgenics produced


TLC+ = number of individual events in which malvidin was detected in the flowers (at a level above the Skr4 × Sw63 background) (as determined by TLC) over the total number of individual events analyzed


Col = number of individual events that produced flowers with an altered flower color compared to the control over the total number examined


↑ A/c = number of individual events that had an increased level of anthocyanins in petals as measured by spectrophotometric analysis of crude extracts over the number of individual events analyzed (in μmoles/g)


Best = highest anthocyanin amount as measured by spectrophotometric analysis of crude extracts from a flower of an individual event (in μmoles/g)


Av = the average amount of anthocyanin detected as measured by spectrophotometric analysis of crude extracts from a flower in the population of transgenic flowers analysed (in μmoles/g)


Northern = number of individual events in which the specific intact F3′5H transcripts were detected by Northern blot analysis in total RNA isolated from petals over the total number of events analyzed


Best color = most dramatic color change recorded for the transgenic population


nd = not done


na = not applicable






Introduction of the F3′5′H gene expression cassettes into Skr4×SW63 led to a dramatic flower color change from pale lilac to purple with a dramatic increase in the production of malvidin in the petals.
The results suggest that all of the F3′5′H sequences tested (petunia petHf1, petunia, petHf2, Salvia Sal#2, Salvia Sal#47, Sollya Sol#5, Butterfly pea BpeaHF2, pansy BP#18, pansy BP#40, Gentian Gen#48, Kennedia Kenn#31) were stable in petunia petals and resulted in the complementation of the Hf1 or Hf2 mutation in the Skr4×SW63 petunia line leading to dramatically increased levels of malvidin cumulation with a concomitant color change
Rose
The F3′5′H genes described in Example 7 were evaluated for their ability to lead to the production of delphinidin-based pigments in rose petals. A selection of three rose cultivars, Kardinal (Kard), Soft Promise (SP) or Lavande (Lav) were used in transformation experiments. The rose cultivar Kardinal produces red colored flowers that normally accumulate cyanidin-based anthocyanins. This cultivar therefore contains rose F3′H and DFR activities that the introduced F3′5′H would need to compete with for substrate. The rose cultivar Lavande produces light pink colored flows that normally accumulate cyanidin-based anthocyanins. This cultivar therefore contains functional rose F3′H and DFR activities that the introduced F3′5′H would need to compete with for substrate. The rose cultivar Soft Promise produces apricot colored flowers that normally accumulate pelargonidin. This cultivar is thought to lack a fully functional rose F3′5′H activity and contain a DFR that is capable of acting on DHK and thus the introduced F3′5′H would only be required to compete with the endogenous rose DFR for substrate.







TABLE 13







Results of transgenic analysis of petals from roses transformed with T-DNAs


containing F3′5′H gene expression cassettes


(CaMV 35S:F3′5′H:ocs 3′).





















Highest %
Av. %



F3′5′H
plasmid
Cult
#tg
TLC+
HPLC+
del
del
Northern+





Salvia2
pCGP2120
Kard
30
18/20
21/21
12%
 5%
18/18


Salvia47
pCGP2119
Kard
22
11/16
9/9
7.1% 
 2%
12/15


Sollya
pCGP2131
Kard
27
 0/23
2/2
 1%
0.5% 
6/6


Butterfly
pCGP2134
Kard
29
 0/15
nd
na
na
0/9


pea
pBEBF5
Lav
25
nd
0/25
0%
 0%
nd


Gentian
PCGP1482
Kard
27
 0/23
nd
na
na
 0/23



pBEGHF48
Lav
23
nd
 0/23
 0%
 0%
 0/23


pansy
PCGP1967
Kard
56
30/33
33/34
58%
12%
21/21


BP18

SP
36
21/24
18/18
65%
35%
16/21


pansy
pCGP1969
Kard
22
15/15
15/15
24%
 9%
16/16


BP40

SP
37
17/17
16/17
80%
54%
11/13


Petunia
pCGP1638
Kard
22
 0/21
nd
na
na
 0/16


petHf1
pCGP1392
Lav
34
nd
 0/34
 0%
 0%
nd


Petunia
PCGP2123
Kard
41
 0/26
nd
na
na
 0/10


petHf2


Lavender
pBELF8
Lav
28
nd
 4/28
 4%
3.5% 
nd





F3′5′H = the F3′5′H sequence contained on the T-DNA


plasmid = the plasmid number of the binary vector used in the transformation experiment


Cult = Rosa hybrida cultivar


Kard = Kardinal


SP = Soft Promise


Lav = Lavande


#tg = # of independent transgenic events produced


TLC+ = number of individual events that accumulated detectable delphinidin or delphinidin-based molecules (as determined by TLC) in the petals over the number of individual events analyzed


HPLC+ = number of individual events that accumulated detectable delphinidin or delphinidin-based molecules (as determined by HPLC) in the petals over the number of individual events analyzed


Northern = number of individual events in which the specific intact F3′5H transcripts were detected by Northern blot analysis in total RNA isolated from petals over the total number of events analyzed


nd = not done






The results suggest surprisingly that not all of the F3′5′H sequences assessed (petunia petHf1, petunia petHf2, Salvia Sal#2, Salvia Sal#47, Sollya Sol#5, Butterfly pea BpeaHF2, pansy BP#18, pansy BP#40, Gentian Gen#48, Kennedia Kenn#31 and Lavender LBG) were functional in rose. In fact transcripts of the introduced F3′5′H sequences isolated from Clitoria ternatea (butterfly pea), Gentiana triflora, (gentian) and Petunia hybrida (petunia) failed to accumulate in rose petals. Only full-length F3′5′H transcripts from pansy, salvia, kennedia, sollya and lavender accumulated in rose petals. However although Kennedia F3′5′H transcripts did accumulate in rose petals, there was either no accumulation of the enzyme or the enzyme produced was either not functional or was unable to compete with the endogenous rose F3′H and DFR enzymes to allow for the production of delphinidin or delphinidin-based molecules pigments. Of the F3′5′H sequences evaluated, only the F3′5′H sequences derived from cDNA clones from Salvia spp. (Sal#2 and Sal#47), Viola spp. (BP#18 and BP#40), Sollya spp. (Soll#5) and Lavandula nil (LBG) resulted in the production of delphinidin or delphinidin-based molecules based pigments in rose petals. Based on the relative percentages of delphinidin or delphinidin-based molecules produced in rose petals, the F3′5′H sequences from pansy (BP#18 and BP#40) were revealed to be the most effective of those assessed at producing delphinidin or delphinidin-based molecules in rose petals.
Introduction of Viola spp. F3′5′H Sequence into Rosa hybrida cv. Medeo and Pamela
As described in the introduction, copigmentation with other flavonoids, further modification of the anthocyanidin molecule and the pH of the vacuole impact on the color produced by anthocyanins. Therefore, selection of rose cultivars with relatively high levels of flavonols and relatively high vacuolar pH would result in bluer flower colors upon production of delphinidin or delphinidin-based molecules pigments.
The rose cultivar Medeo generally produces cream-colored to pale apricot flowers (RHSCC 158C to 159A). HPLC analysis of the anthocyanidins and flavonols accumulating in Medeo rose petals revealed that the petals accumulate high levels of flavonols (2.32 mg/g kaempferol, 0.03 mg/g quercetin) and very low levels of anthocyanins (0.004 mg/g cyanidin, 0.004 mg/g pelargonidin). The estimated vacuolar pH of Medeo petals is around 4.6.
The rose cultivar Pamela produces white to very pale pink colored flowers. It similarly accumulates low levels of anthocyanin and relatively high levels of flavonols.
The T-DNA contained in the construct pCGP1969 (FIG. 30) incorporating the pansy F3′5′H clone, BP#40, was also introduced into the rose cultivars Medeo and Pamela resulting in the production of over 90% delphinidin or delphinidin-based molecules in these roses and leading to a dramatic color change and novel colored flowers. The most dramatic color change in transgenic Medeo flowers was to a purple/violet color of RHSCC 70b, 70c, 80c, 186b. The most dramatic color change in transgenic Pamela flowers was to a purple/violet color of RHSCC 71c, 60c, 71a, 80b.
In conclusion, two unexpected findings were revealed when gene sequences that had been proven to lead to functionality in petunia and carnation were introduced into roses.
First, the petunia F3′5′H petHf1 (and petHf2) sequences that had resulted in novel color production in carnation and also proven to lead to synthesis of a functional enzyme in petunia did not lead to full-length (or intact) transcript accumulation (as detectable by Northern blot analysis) in rose petals. In fact, there was either no accumulation of full-length or intact transcript or the transcripts that were detected were degraded and were seen as low MW (or fast migrating) smears on RNA blots indicating the presence of low MW heterologous hybridizing RNA. Therefore in order to find a F3′5′H sequence that would accumulate in rose and load to a functional enzyme, a number of F3′5′H sequences were isolated. Again it was not obvious which sequence would lead to an active enzyme in rose petals. All of the F3′5′H sequences isolated were tested for functionality in carnation and/or petunia and all led to accumulation of intact transcripts and production of a functional F3′5′H activity. However only F3′5′H sequences from pansy (BP#18 and BP#40), salvia (Sal#2 and Sal#47), sollya (Soll#5), kennedia (Kenn#31) and lavender (LBG) resulted in accumulation of intact full-length transcripts and only those from pansy (BP#18 and BP#40), salvia (Sal#2 and Sal#47), sollya (Soll#5) and lavender (LBG) resulted in production of a functional enzyme in rose as measured by the synthesis of delphinidin or delphinidin-based molecules.
Secondly that it was not obvious which promoters would be effective in rose. Promoter cassettes that had been tested and proven to be functional in carnation and petunia flowers did not lead to accumulation of detectable transcripts in rose petals. Of the promoters tested in rose, only CaMV 35S, RoseCHS 5′, ChrysCHS 5′, mas 5′ and nos 5′ promoters led to intact and detectable GUS or nptII or SuRB transcript accumulation in rose petals.
Table 14 shows a summary of the results obtained when assessing F3′5′H sequences from various species in petunia, carnation and rose.







TABLE 14







Summary of effectiveness of the F3′5′H sequences in petunia,


carnation and rose











Petunia
Carnation
Rose













F3′5′H
Mal
RNA
Del
RNA
Del
RNA





Kennedia (Kenn#31)
+
nd
nd
nd
−
+


Gentian (Gen#48)
+
+
+
+
−
−


Salvia (Sal#2)
+
nd
+
+
+
+


Salvia (Sal#47)
+
nd
+
+
+
+


Sollya (Sol#5)
+
nd
+
+
+
+


Butterfly pea
+
nd
+
+
−
−


(BpeaHF2)


Pansy (BP#18)
+
+
+
+
+
+


Pansy (BP#40)
+
nd
+
+
+
+


Petunia (petHf1)
+
+
+
+
−
−


Petunia (petHf2)
+
+
+
+
−
−


Lavender (LBG)
nd
nd
nd
nd
nd
+





nd = not done


Mal = malvidin detected in petals as analysed by TLC


Del = delphinidin or delphinidin-based molecules detected in petals as analysed by TLC or HPLC


+ = yes


− = no






Example 9
Use of Pansy F3′5′H Sequences in Species other than Rose
Gerbera
From the examples above, it was clear that the pansy F3′5′H sequences, BP18 and BP#40, resulted in functional F3′5′H activity and lead to the production of high levels of delphinidin or delphinidin-based molecules in roses and carnations.
The T-DNA from binary construct pCGP1969 (described in Example 8) (FIG. 30) containing the chimeric CaMV 35S: pansy BP#40 F3′5′H: ocs 3′ gene expression cassette was introduced into the gerbera cultivar Boogie via Agrobacterium-mediated transformation, to test the functionality of the pansy F3′5′H sequence in gerbera.
Of six events produced to date, one (#23407) has produced flowers with a dramatic color change (RHSCC 70c) compared to the control flower color (RHSCC 38a, 38c).
The color change of the petals of the transgenic gerbera has been correlated with the presence of delphinidin or delphinidin-based molecules as detected by TLC
Other Species
In order to produce delphinidin or delphinidin-based molecules pigments in plants that do not normally produce delphinidin-based pigments and does not contain a flavonoid 3′5′-hydroxylase constructs containing a F3′5′H gene (such as but not limited to a chimaeric Viola spp. and/or Salvia spp. and/or Sollya spp. and/or Lavandula spp. and/or Kennedia spp. F3′5′H gene) are introduced into a species that does not normally produce delphinidin-based pigments. Such plants may include but are not limited to carnation, chrysanthemum, gerbera, orchids, Euphorbia, Begonia and apple.
Example 10
Characteristics of F3′5′H Sequences Evaluated in Petunia, Carnation and Rose
Gene regulation in eukaryotes is, in simple terms, facilitated by a number of factors which interact with a range of sequences proximal and distal to a nucleotide sequence coding for a given polypeptide. Engineering expression cassettes for introduction into plants for the generation of one or more traits is based on an understanding of gene regulation in eukaryotes in general and, in selected cases, plants in particular. The essential elements include a series of transcriptional regulation sequences typically, but not exclusively, located upstream or 5′ to the point of transcription initiation. Such elements are typically described as enhancers and promoters, the latter being proximal to the point of transcription initiation. Immediately downstream from, or 3′ to, the initiation of transcription point is a variable region of transcribed DNA which is denoted as the 5′ untranslated region (5′utr) which plays a role in transcript stability and translational efficiency. Such sequences, when engineered into expression cassettes, are frequently chimeric and may be derived from sequences naturally occurring adjacent to the coding sequence and/or adjacent to a given promoter sequence. The coding sequence (sometimes disrupted by introns) lies 3′ to the 5′utr followed by a 3′utr important to transcript (mRNA) stability and translational efficiency. Sequences 3′ to the end of the coding region and 3′ to the 3′utr itself are denoted as terminator sequences. All these elements make up an expression cassette. In making direct comparisons between promoters or other elements it is important to maintain uniformity in the remaining elements of an expression cassette. Hence, when comparing the efficacy of various F3′5′H sequences it was possible to confine the sequences leading to instability and the subsequent autodegradation of engineered mRNA and resultant absence of tri-hydroxylated products (delphinidin or delphinidin-based molecules derivatives) to the region coding for the F3′5′H and not to other elements in the expression cassette such as 5′utr and/or 3′utr sequences for example.
In an attempt to identify motifs or similarities between the F3′5′H sequences that resulted in full-length transcripts being detected in total RNA isolated from rose flowers, and ultimately delphinidin or delphinidin-based molecules production, comparisons across a range of parameters were performed. These included sequence identities at nucleic acid and amino acid levels, sequence alignments, taxonomic classifications, % of A or T nucleotides present in the sequence, % of codons with an A or T in the third position etc.
Taxonomic Classification
The taxonomy of each species from which the F3′5′H sequences were isolated was examined (Table 15). There appeared to be no obvious link between the subclass classification and whether the F3′5′H sequence resulted in an intact transcript and subsequent delphinidin or delphinidin-based molecules production in roses.







TABLE 15







Taxonomic classifications of the species that F3′5′H sequences


were isolated from and whether the use of the sequences resulted in


intact transcript in rose petals that were detectable by RNA blot analysis.


















Intact
Delphinidin


Flower
Species
Family
Order
Subclass
transcript
in rose petals





gentian
Gentiana
Gentianaceae
Gentianales
Asteridae
NO
NO



triflora


lavender
Lavandula nil
Lamiaceae
Lamiales
Asteridae
YES
YES


salvia
Salvia spp.
Lamiaceae
Lamiales
Asteridae
YES
YES


sollya
Sollya spp.
Pittosporaceae
Apiales
Asteridae
YES
YES


petunia
Petunia hybrida
Solanaceae
Solanales
Asteridae
NO
NO


kennedia
Kennedia spp.
Fabaceae
Fabalcs
Rosidae
YES
NO


butterfly
Clitoria
Fabaceae
Fabalcs
Rosidae
NO
NO


pea
ternatea


pansy
Viola spp.
Violaceae
Malpighiales
Rosidae
YES
YES


rose
Rosa hybrida
Rosaciae
Rosales
Rosidae
na
na





Intact transcript = fall-length F3′5′H mRNA detected by Northern blot analysis in total RNA isolated from petals from transgenic roses






Comparison of F3′5′H Nucleotide Sequences

The nucleotide sequence identities between each of the F3′5′H sequences evaluated were determined using the ClustalW program (Thompson et al., 1994, supra) within the MacVectorTM version 6.5.3 application program (Oxford Molecular Ltd., England) (Table 16). There were no obvious differences between the F3′5′H sequences that resulted in the detection of intact full-length transcripts in RNA isolated from rose petals and those that didn't.







TABLE 16







Percentage of nucleic acid sequence identity between the nucleotide sequences


of the F3′5′H isolated from various species. F3′5′H sequences that resulted


in intact transcripts being detected in RNA isolated from rose petals are


underlined and in italics.



















BP18
BP40
Lav
Sal47
Sal2
Soll
Kenn
Bpea
Gent
PetHf1
PetHf2






















BP18
100
82
60
61
62
51
60
62
62
59
62


BP40

100
60
57
58
50
59
62
58
60
62


Lav


100
68
68
48
57
57
58
59
60


Sal47



100
95
48
56
57
59
57
58


Sal2




100
49
57
58
60
57
59


Soll





100
48
50
50
48
51


Kenn






100
70
56
64
60


Bpea







100
59
59
62


Gent








100
61
64


PetHf1









100
84


PetHf2










100









Comparison of F3′5′H Translated Nucleotide Sequences

The translated nucleotide sequence identities and similarities between each of the F3′5′H sequences evaluated were also determined using the ClustalW program (Thompson et al., 1994, supra) within the MacVectorTM version 6.5.3 application program (Oxford Molecular Ltd., England) (Table 17). There were no obvious differences between the F3′5′H sequences that resulted in the detection of intact full-length transcripts in RNA isolated from rose petals and those that didn't.







TABLE 17







Percentage of the amino acid sequence identity and similarity (in brackets)


between F3′5′H sequences isolated from various species. F3′5′H sequences


that resulted in intact transcripts being detected in RNA isolated from rose


petals are underlined and in italics.



















BP18
BP40
Lav
Sal47
Sal2
Soll
Kenn
Bpea
Gent
PetHf1
PetHf2






















BP18
100
91 (94)
65 (77)
65 (76)
65 (76)
44 (63)
69 (83)
64 (75)
69 (80)
74 (85)
74 (85)


BP40

100
67 (89)
66 (77)
66 (77)
46 (64)
69 (82)
64 (75)
68 (79)
74 (85)
75 (86)


Lav


100
75 (86)
75 (86)
45 (63)
63 (79)
59 (74)
66 (80)
68 (82)
69 (83)


Sal47



100
 98
45 (65)
64 (78)
60 (72)
64 (76)
68 (79)
69 (81)


Sal2




100
45 (65)
64 (78)
60 (72)
63 (75)
68 (79)
69 (81)


Soll





100
46 (66)
41 (61)
44 (62)
46 (67)
46 (66)


Kenn






100
72 (80)
65 (75)
71 (83)
72 (83)


Bpea







100
69 (81)
65 (75)
65 (74)


Gent








100
73 (82)
73 (82)


PetHf1









100
93 (95)


PetHf2










100









Percentage of Nucleotides A or T in the F3′5′H DNA Sequences

There is some evidence to suggest that the choice of codons influences the rate of translation and mRNA degradation. Certain codons are used less frequently than others are and this may be related to the abundance of isoaccepting tRNAs. Transfer RNAs corresponding to rare codons are less abundant in E. coli and yeast than tRNAs corresponding to preferred codons (van Hoof and Green, Plant Molecular Biology, 35: 383-387, 1997). Examples of altering codon usage and making a gene more “plant-like” are the bacterial B.t. toxin gene (reviewed in Diehn et al., Genet Engin, 18: 83-99, 1996) and the jellyfish gfp gene (Haseloff et al., Proc. Natl. Acad. Sci USA, 94: 2122-2127, 1997). However as commented in van Hoof and Green, (1997) (supra), the effect of eliminating the rare codons in the B.t. genes increased the GC content, thereby eliminating AU-rich sequences that may be responsible for improper recognition of introns and polyadenylation sites as well as removing instability determinants. Alteration of codon usage in the jellyfish gfp gene also resulted in removal of a cryptic intron (Haseloff et al., 1996, supra). Studies examining the effect of codon usage and instability elements have generally been limited to differences between genes isolated from species in different kingdoms ie. bacterial versus yeast versus animal versus plant. Within the plant kingdom, differences have been observed between the dicotyledons and the monocotyledons. Studies on transgenic plants have suggested that promoter fragments used to drive gene expression in dicotyledonous plants are not as effective when used in monocotyledonous plants (see Galun and Breiman, Transgenic Plants, Imperial College Press, London, England, 1997). Differences in the methylation and ultimate expression of a DFR transgene in Petunia hybrida (dicot) were detected when a maize (monocot) DFR cDNA was compared with a gerbera (dicot) DFR cDNA (Elomaa et al., Molecular, and General Genetics, 248: 649-656, 1995). The conclusion was that the gerbera DFR cDNA had a higher AT content (lower CC content) and was more, “compatible” with the genomic organization of petunia preventing it being recognised as a foreign gene and hence silenced by methylation. (Rose along with carnation and petunia are dicotyledons and the F3′5′H genes tested were all isolated from dicotyledonous plants.) These points serve to illustrate that degradation and stability mechanisms are not understood in detail and differences appear between plants and other kingdoms and within the plant kingdom.
The content of A and T was examined in the F3′5′H cDNAs evaluated along with that of four flavonoid pathway genes (F3′H, DFR, CHS, FLS) that had been isolated from rose (Table 18). The third position of each codon (within the open reading frame) was also examined and the percentage of codons with an A or a T in the third position was calculated (able 18).







TABLE 18







Summary of the percentage amount of A or T dinucleotides in the F3′5′H


sequences isolated and whether the F3′5′H resulted in full-length


transcripts being detected in rose petals by Northern blot analysis.













% A or T in




F3′5′H seq
% AT
3rd
RNA
Delphinidin





Viola BP#18
50
40
YES
YES


Viola BP#40
51
35
YES
YES


Salvia#2
48
33
YES
YES


Salvia#47
48
34
YES
YES


Sollya#5
54
54
YES
YES


LavenderLBG
50
37
YES*
YES


Kennedia#31
54
47
YES
NO


petunia petHf1
61
66
NO
NO


petunia petHf2
59
65
NO
NO


Gentian#48
57
57
NO
NO


Butterfly
57
53
NO
NO


pea#HF2


rose F3′H
47
34
**
na


rose CHS
52
42
**
na


rose DFR
53
46
**
na


rose FLS
56
43
**
na





% AT = % of nucleotides that are A or T in the nucleic acid sequence


% A or T in 3rd = the percentage of codons that have an A or T in the third position


RNA = whether a full-length mRNA transcript was detected by Northern blot analysis in total RNA isolated from rose petals


Del = whether any delphinidin or delphinidin-based molecules was detected by TLC or HPLC in rose petals


YES* = although Northern blot analysis of transgenic roses transformed with the lavender F3′5′H expression cassettes was not performed, it can be assumed that full-length transcript was produced since delphinidin or delphinidin-based molecules was detected in the rose petals.


rose F3′H (described in International Patent Application No. PCT/AU97/00124)


rose DFR (Tanaka et al., 1995, supra)


rose FLS (GenBank accession number AB038247)


rose CHS (GenBank accession number AB038246)






The AT content of the four rose sequences (above) encoding flavonoid pathway enzymes had an AT content of between 47 and 56%. In general the AT content of the F3′5′H sequences that resulted in intact transcripts in rose petals was between 48 and 54%. However the F3′5′H sequences that did not result in intact transcripts accumulating in rose petals generally had a higher AT content of between 57 and 61%. Hence the AT content of the introduced F3′5′H genes into rose may be a factor in whether an intact transcript accumulates in rose petals and so leads to production of F3′5′H and delphinidin or delphinidin-based molecules.
The nucleotide base at the third position of each codon of the four rose sequences encoding flavonoid pathway enzymes was generally an A or a T in 34 to 46% of the codons. In general F3′5′H sequences that resulted in intact transcripts in rose petals contained an A or a T in the third position of each codon in 33 to 54% of the codons. Howevvr the F3′5′H sequences that did not result in intact transcripts accumulating in rose petals generally contained an A or a T in the third position of each codon in 53 to 66% of the codons. So the percentage of codons with an A or a T in the third position of the introduced F3′5′H genes into rose may also be a factor in whether an intact transcript is accumulates in rose petals and so leads to production of F3′5′H and delphinidin or delphinidin-based molecules.
It may be that by altering the overall content of the nucleotides A and/or T in any F3′5′H DNA sequence that does not result in an intact transcript in rose such as but nor limited to the Petunia hybrida petHf1, Petunia hybrida petHf2, Clitora ternatea (butterfly pea) BpeaHF2 or Gentiana triflora (gentian) Gen#48, to a level more consistent with that found in rose genes, intact transcripts will accumulate and result in the efficient translation of F3′5′H transcripts and so to delphinidin or delphinidin-based molecules accumulation in rose petals. One way of altering the AT content of the DNA sequence without altering the amino acid sequence is to target the degeneracy of the third position of each codon.
Those skilled in the at will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said stops or features.
BIBLIOGRAPHY


    A look beyond transcription, ASPP, USA, Bailey-Serres and Gallie, eds. 1998
    Altschul et al., J. Mol. Biol. 215(3): 403-410, 1990.
    Altschul et al., Nucl. Acids Res. 25: 3389-3402, 1997.
    Ausubel et al., “Current Protocols in Molecular Biology” John Wiley & Sons Inc, 1994-1998, Chapter 15.
    Aviv and Leder, Proc. Natl. Acad. Sci. USA 69: 1408, 1972.
    Bevan, Nucleic Acids Res 12: 8711-8721, 1984.
    Bonner and Laskey, Eur. J. Biochem. 46: 83, 1974.
    Bodeau, Molecular and genetic regulation of Bronze-2 and other maize anthocyanin genes. Dissertation, Stanford University, USA, 1994
    Brendel et al., In A look beyond transcription, ASPP, USA, Bailey-Serres and Gallie, eds, 1998.
    Brouillard and Dangles, In: The Flavonoids—Advance in Research since 1986. Harborne, J. B. (ed), Chapman and Hall, London, UK, 1-22, 1993.
    Brugliera et al., Plant J. 5: 81-92, 1994.
    Brugliera, Characterization of floral specific genes isolated from Petunia hybrida. RMIT, Australia. PhD thesis, 1994.
    Bullock et al., Biotechniques 5: 376, 1987.
    Comai et al., Plant Mol. Biol. 15: 373-381, 1990.
    Depicker et al., J Mol. and Appl. Genetics I: 561-573, 1982.
    Diehn et al., Genet Engin, 18: 83-99, 1996
    Elomaa et al., Molecular and General Genetics, 248: 649-656, 1995
    Franck et al., Cell 21: 285-294, 1980.
    Galun and Breiman, Transgenic Plants, Imperial College Press, London, England, 1997
    Guilley et al., Cell 30: 763-773. 1982,
    Hanahan, J. Mol. Biol. 166: 557, 1983.
    Harpster et al., MGG 212: 182-190, 1988.
    Haseloff et al., Proc. Natl. Acad. Sci USA, 94: 2122-2127, 1997
    Holton and Cornish, Plant Cell 7: 1071-1083, 1995.
    Holton and Graham, Nuc. Acids Res. 19: 1156, 1990.
    Holton et al., Nature 366: 276279, 1993a.
    Holton et al., Plant J. 4: 1003-1010, 1993b
    Holton, Isolation and characterization of petal specific genes from Petunia hybrida. PhD thesis, University of Melbourne, Australia, 1992
    Huang and Miller, Adv. Appl. Math. 12: 373-381, 1991.
    Inoue et al., Gene 96: 23-28, 1990.
    Janssen and Gardner, Plant Molecular Biology, 14: 61-72, 1989
    Jefferson, et al., EMBO J. 6: 3901-3907, 1987.
    Johnson et al., In A look beyond transcription, ASPP, USA, Bailey-Serres and Gallie, eds, 1998.
    Karn et al., Gene 32: 217-224, 1984.
    Lazo et al., Bio/Technology 9: 963-967, 1991.
    Lee et al., EMBO J. 7: 1241-1248, 1988.
    Lu et al., Bio/Technology 9: 864-868, 1991.
    Merchant et al., Molecular Breeding 4: 187-194, 1998.
    Marmur and Doty, J. Mol. Biol. 5: 109, 1962.
    McBride and Summerfelt, Plant Molecular Biology 14: 269-276, 1990.
    Merrfield, J. Am. Chem. Soc. 85: 2149, 1964.
    Mitsuhara et al., Plant Cell Physiol. 37: 49-59, 1996.
    Mol et al., Trends Plant Sci. 3: 212-217, 1998.
    Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85(8): 2444-2448, 1988.
    Plant Molecular Biology Labfax, Croy (ed), Bios scientific Publishers, Oxford, UK, 1993.
    Plant Molecular Biology Manual (2nd edition), Gelvin and Schilperoot (eds), Kluwer Academic Publisher, The Netherlands, 1994.
    Robinson and Firoozabady, Scientia Horticulturae 55: 93-99, 1993.
    Rout et al., Scientia Horticulturae 81: 201-238, 1999.
    Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor, N.Y., USA, 1989.
    Sambrook and Russell, Molecular Cloning: A Laboratory Manual 3rd edition, Cold Spring Harbor Laboratories, Cold Spring Harbor, N.Y., USA, 2001
    Seitz and Hinderer, Anthocyanins. In: Cell Culture and Somatic Cell Genetics of Plants. Constabel, F. and Vasil, I. K. (eds.), Academic Press, New York, USA, 5: 49-76, 1988.
    Short et al., Nucl. Acids Res. 16: 7583-7600, 1988.
    Sommer and Saedler, Mol Gen. Gent., 202: 429-434, 1986.
    Strack and Wray, In: The Flavonoids—Advances in Research since 1986. Harborne, J. B. (ed), Chapman and Hall, London, UK, 1-22, 1993.
    Tanaka et al., Plant Cell Physiol. 36: 1023-1031, 1995.
    Tanaka et al., Plant Cell Physiol. 37: 711-716, 1996.
    Thompson et al., Nucleic Acids Research 22: 4673-4680, 1994.
    Turpen and Griffith, Bio Techniques 4: 11-15, 1986.
    van Hoof and Green, Plant Molecular Biology, 35: 383-387, 1997
    Winkel-Shirley, Plant Physiol. 126: 485-493, 2001a.
    Winkel-Shirley, Plant Physiol. 127: 1399-1404, 2001b.
    Yonekura-Sakakibara et al., Plant Cell Physiol. 41: 495-502, 2000.

## Claims

1. An isolated nucleic acid molecule comprising a sequence of nucleotides encoding a flavonoid 3′,5′ hydroxylase (F3′5′H), said nucleotide sequence selected from the group consisting of:
(i) a nucleotide sequence encoding the amino acid sequence as set forth in SEQ ID NO: 12;
(ii) a nucleotide sequence encoding an amino acid sequence having at least 95% identity to the amino acid sequence as set forth in SEQ ID NO: 12;
(iii) the nucleotide sequence as set forth in SEQ ID NO: 11; and
(iv) a nucleotide sequence capable of hybridizing to the complementary sequence of the nucleotide sequence as set forth in SEQ ID NO: 11 under high stringency conditions which comprise hybridization at 42° C. in 50% v/v formamide, 1M NaCl, 1% w/v SDS, and 10% w/v dextran sulphate, and washings at 65° C. first in 2×SSC, 1% w/v SDS and subsequently in 0.2×SSC, 1% w/v SDS.

2. The isolated nucleic acid molecule of claim 1 operably linked to a promoter.

3. The isolated nucleic acid molecule of claim 2 wherein the promoter is the CaMV 35S promoter.

4. A genetic construct comprising the nucleic acid molecule of any one of claims 1 to 3.

5. A genetically modified plant or progeny thereof comprising the nucleic acid molecule of claim 1 which is heterologous to said plant and said progeny thereof, wherein expression of said nucleic acid molecule results in an altered flower color in said plant and said progeny thereof.

6. The genetically modified plant of claim 5 wherein said nucleic acid molecule is operably linked to the CaMV 35S promoter.

7. The genetically modified plant according to claim 5 or claim 6, wherein the plant is a rose or a progeny thereof, and wherein said rose progeny comprises said nucleic acid molecule.