# Patent

## Title
Nucleic acid sequences and methods of use for the production of plants with modified polyunsaturated fatty acid levels

## Abstract

By this invention, novel nucleic acid sequences are provided, wherein said nucleic acid sequence is a genomic sequence of a plant desaturase encoding sequence. Also provided in the present invention are the promoter and intron sequences of the desaturase genomic sequences. Furthermore, recombinant DNA constructs employing the polynucleotide sequences are provided. The instant invention also provides methods for the modification of fatty acid compositions in host plant cells.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a divisional of U.S. patent application Ser. No. 09/638,508 filed Aug. 11, 2000 now abandoned (herein incorporated by reference), which claims priority to U.S. Provisional Application Ser. No. 60/151,224 filed Aug. 26, 1999 and U.S. Provisional Application Ser. No. 60/172,128 filed Dec. 17, 1999.


INCORPORATION OF SEQUENCE LISTING
A paper copy of the Sequence Listing and a computer readable form of the sequence listing on diskette, containing the file named 16518-077 PUFA SeqList.txt, which is 24,069 bytes in size (measured in MS-DOS), and which was created on May 15, 2002 are herein incorporated by reference.
1. Technical Field
The present invention is directed to nucleic acid sequences and constructs, and methods related thereto.
2. Background
Plant oils are used in a variety of applications. Novel vegetable oils compositions and/or improved means to obtain oils compositions, from biosynthetic or natural plant sources, are needed. Depending upon the intended oil use, various different fatty acid compositions are desired.
One means postulated to obtain such oils and/or modified fatty acid compositions is through the genetic engineering of plants. However, it is necessary to identify the appropriate nucleic acid sequences which are capable of producing the desired phenotypic result, regulatory regions capable of directing the correct application of such sequences, and the like.
Higher plants appear to synthesize fatty acids via a common metabolic pathway (fatty acid synthetase pathway). In developing seeds, where fatty acids are attached to glycerol backbones, forming triglycerides, for storage as a source of energy for further germination, the FAS pathway is located in the proplastids. The first committed step is the formation of acetyl-ACP (acyl carrier protein) from acetyl-CoA and ACP catalyzed by the enzyme, acetyl-CoA:ACP transacylase (ATA). Elongation of acetyl-ACP to 16- and 18-carbon fatty acids involves the cyclical action of the following sequence of reactions: condensation with a two-carbon unit from malonyl-ACP to form a β-ketoacyl-ACP (β-ketoacyl-ACP synthase), reduction of the keto-function to an alcohol (β-ketoacyl-ACP reductase), dehydration to form an enoyl-ACP (β-hydroxyacyl-ACP dehydrase), and finally reduction of the enoyl-ACP to form the elongated saturated acyl-ACP (enoyl-ACP reductase). β-ketoacyl-ACP synthase I, catalyzes elongation up to palmitoyl-ACP (C16:0), whereas β-ketoacyl-ACP synthase II catalyzes the final elongation to stearoyl-ACP (C18:0). Common plant unsaturated fatty acids, such as oleic, linoleic and a-linolenic acids found in storage triglycerides, originate from the desaturation of stearoyl-ACP to form oleoyl-ACP (C18:1) in a reaction catalyzed by a soluble plastid Δ-9 desaturase (also often referred to as “stearoyl-ACP desaturase”). Molecular oxygen is required for desaturation in which reduced ferredoxin serves as an electron co-donor. Additional desaturation is effected sequentially by the actions of membrane bound Δ-12 desaturase and Δ-15 desaturase. These “desaturases” thus create mono- or polyunsaturated fatty acids respectively.
Obtaining nucleic acid sequences capable of producing a phenotypic result in FAS, desaturation and/or incorporation of fatty acids into a glycerol backbone to produce an oil is subject to various obstacles including but not limited to the identification of metabolic factors of interest, choice and characterization of an enzyme source with useful kinetic properties, purification of the protein of interest to a level which will allow for its amino acid sequencing, utilizing amino acid sequence data to obtain a nucleic acid sequence capable of use as a probe to retrieve the desired DNA sequence, and the preparation of constructs, transformation and analysis of the resulting plants.
Thus, additional nucleic acid targets and methods for modifying fatty acid compositions are needed. In particular, constructs and methods to produce a variety of ranges of different fatty acid compositions are needed.
SUMMARY OF THE INVENTION
The present invention is generally directed to genomic desaturase polynucleotides, and in particular to genomic desaturase polynucleotides which encode enzymes that catalyze the insertion of a double bond into a fatty acyl moiety at the twelfth (Δ12 desaturase or fad2) or fifteenth (Δ15 desaturase or fad3) carbon position in a fatty acyl chain as counted from the carboxyl terminus. Further, the present invention provides isolated non-coding regions of such genomic polynucleotide sequences, particularly including the introns, and promoter regions. Specific oligonucleotides are provided which include partial or complete sequences which are derived from Δ12 and Δ15 desaturase promoter and intron sequences. Although the sequences disclosed herein are obtained from soybean plants, it is contemplated that additional sequences can be derived from intron and promoter regions of desaturase genomic polynucleotide sequences which are homologous or have identity to the soybean desaturase sequences. Such additional desaturase sequences can be obtained using standard methods described below from a variety of plant sources, in particular oilseed crops.
It is also an aspect of the present invention to provide recombinant DNA constructs which can be used for the modification of the fatty acid composition in a plant and in particular, to modify the transcription or transcription and translation (expression) of desaturase genes or proteins, such as Δ12 and Δ15 desaturase. The invention is particularly directed to DNA constructs which include sequences which are derived from the intron or promoter regions of a genomic clone wherein said sequences are in a sense or antisense orientation in a DNA construct. These DNA constructs are then used to transform or transfect host cells to produce plants with modified levels of fatty acids, particularly modified levels of oleic, linoleic and linolenic acid. It is particularly contemplated to provide constructs and methods for down regulating Δ12 and Δ15 desaturase gene expression, so as to increase the levels of oleic acid and to decrease the levels of linoleic acid and linolenic acid. It is particularly contemplated to alter the fatty acid composition in seed tissue of oilseed crops.
The modified plant cells, plants, seeds and oils obtained by the expression of the Δ12 and Δ15 desaturase polynucleotides are also considered part of the invention. Further, it is contemplated to produce oil compositions with specific relative levels of each fatty acid. One preferred embodiment comprises at least about 80–85% oleic acid, no more than about 1–2% linoleic acid, and no more than about 1–3% linolenic acid; and a second preferred embodiment comprising at least about 50–75% oleic acid, at least about 10–30% linoleic acid, and no more than about 3% linolenic acid.


DETAILED DESCRIPTION OF THE INVENTION
The present invention is directed to genomic desaturase sequences, particularly the isolated non-coding sequences from genomic fatty acid desaturase nucleic acid sequences from host cell sources. A desaturase sequence of this invention includes any nucleic acid genomic sequence, including all non-coding regions, encoding amino acids from a source, such as a protein, polypeptide or peptide, obtainable from a cell source, which is capable of catalyzing the insertion of a double bond into a fatty acyl moiety in a plant host cell, i.e., in vivo, or in a plant cell-like environment, i.e. in vitro. As will be described in more detail below, specific genomic polynucleotide sequences encoding enzymes which add double bonds at the twelfth (Δ12 desaturase) and fifteenth (Δ15 desaturase) carbon positions in a fatty acyl chain as counted from the carboxyl terminus are provided. In addition, provided herein are specific non-coding regions of such genomic sequences.
The term “non-coding” refers to sequences of polynucleotides that do not encode part or all of an expressed protein. Non-coding sequences include but are not limited to introns, promoter regions, and 5′ untranslated regions.
The term “intron” as used herein refers to the normal sense of the term as meaning a segment of polynucleotides, usually DNA, that does not encode part or all of an expressed protein.
The term “exon” as used herein refers to the normal sense of the term as meaning a segment of polynucleotides, usually DNA, that encodes part or all of an expressed protein.
Thus, the term “intron” refers to gene regions that are transcribed into RNA molecules, but which are spliced out of the RNA before the RNA is translated into a protein. As contrasted to the term “exon” which refers to gene regions that are transcribed into RNA and subsequently translated into proteins.
As set forth in detail in the sequence listing and the examples, genomic Δ12 desaturase and Δ15 desaturase sequences and intron and promoter regions obtained from such sequences are provided herein. In particular, two Δ12 desaturase genomic clones were identified and are set forth in SEQ ID NOs:1 and 23. A single Δ15 desaturase genomic clone was identified and is set forth in SEQ ID NO:3. A single intron region was obtained from each of the Δ12 desaturase genomic clones with the sequences provided in SEQ ID NOs:2 and 24, respectively. The promoter region from each of the Δ12 desaturase genomic clones are respectively included in SEQ ID NO:1 (base pairs 1–1094) and SEQ ID NO:23 (base pairs 1–1704). The Δ15 desaturase included seven introns in the coding region (set forth as SEQ ID NOs:4, 5, 6, 7, 8, 25 and 26). In addition, preliminary results suggest that there is an additional intron within the 5′ untranslated region.
Although the sequences described herein are obtained from soybean, it is contemplated that intron and promoter regions can be obtained from desaturase genomic polynucleotide sequences which are homologous or have identity to the soybean desaturase sequences. In particular, sequences can be obtained from other plant sources and particularly from oilseed crops. Such genomic sequences can be obtained using standard methods, certain of which are described below.
The sequences of the present invention can be used to modify the fatty acid composition in a plant (see Example 3 and Table I). In particular, it is shown that sense and antisense suppression can be used to obtain broad ranges in the levels of oleic, linoleic and linolenic acid. In particular, it is shown that levels of oleic acid can range from about 26 to 80%, levels of linoleic acid can range from about 2.97 to 49.92% and levels of linolenic acid can range from about 3.38 to 8.81%. However, these are merely representative of the broad range that be can achieved. Moreover, it is contemplated that combinations of the sequences could be used to achieve additional fatty acid compositions. Certain compositions are preferred based on the intended use of the oil.
One preferred composition includes at least about 50–75% oleic acid, at least about 10–30% linoleic acid and no more than about 3% linolenic acid. A particularly preferred embodiment includes at least about 60–70% oleic acid, at least about 15–20% linoleic acid and no more than about 3% linolenic acid.
Although the examples set forth herein utilize sense or antisense suppression to downregulate the gene of interest, it is contemplated that other means of modifying gene expression can be used. In particular, it is contemplated that gene expression can be down regulated using DNA binding proteins which can be designed to specifically bind to the non-coding regions identified herein or that ribozymes can be designed to cleave such non-coding regions. In addition, as described below, other methods of downregulation of gene expression which are well known in the art are contemplated and can be used with the sequences of the present invention.
Isolated Polynucleotides, Proteins, and Polypeptides
A first aspect of the present invention relates to isolated desaturase polynucleotides. The polynucleotide sequences of the present invention include isolated polynucleotides that are obtainable from genomic nucleic acid sequences.
The invention provides a polynucleotide sequence identical over its entire length to each sequence as set forth in the Sequence Listing. The polynucleotide includes non-coding sequences, including for example, but not limited to, non-coding 5′ and 3′ sequences, such as the transcribed, untranslated sequences, termination signals, ribosome binding sites, sequences that stabilize mRNA, introns, polyadenylation signals, and additional coding sequence that encodes additional amino acids. For example, a marker sequence can be included to facilitate the purification of the fused polypeptide. Polynucleotides of the present invention also include polynucleotides comprising a structural gene and the naturally associated sequences that control gene expression.
The invention also includes polynucleotides of the formula:

X−(R1)n−(R2)−(R3)n−Y

wherein, at the 5′ end, X is hydrogen, and at the 3′ end, Y is hydrogen or a metal, R1 and R3 are any nucleic acid residue, n is an integer between 1 and 3000, preferably between 1 and 1000 and R2 is a nucleic acid sequence of the invention, particularly a nucleic acid sequence selected from the group set forth in the Sequence Listing and preferably SEQ ID NOs: 1–8, and 23–29. In the formula, R2 is oriented so that its 5′ end residue is at the left, bound to R1, and its 3′ end residue is at the right, bound to R3. Any stretch of nucleic acid residues denoted by either R group, where R is greater than 1, may be either a heteropolymer or a homopolymer, preferably a heteropolymer.

Further preferred embodiments of the invention that are at least 50%, 60%, or 70% identical over their entire length to a polynucleotide of the invention, and polynucleotides that are complementary to such polynucleotides. More preferable are polynucleotides that comprise a region that is at least 80% identical over its entire length to a polynucleotide of the invention and polynucleotides that are complementary thereto. In this regard, polynucleotides at least 90% identical over their entire length are particularly preferred, those at least 95% identical are especially preferred. Further, those with at least 97% identity are highly preferred and those with at least 98% and 99% identity are particularly highly preferred, with those at least 99% being the most highly preferred.
Preferred embodiments are polynucleotides that are obtained from genomic polynucleotide sequences and set forth in the Sequence Listing.
The invention further relates to polynucleotides that hybridize to the above-described sequences. In particular, the invention relates to polynucleotides that hybridize under stringent conditions to the above-described polynucleotides. As used herein, the terms “stringent conditions” and “stringent hybridization conditions” mean that hybridization will generally occur if there is at least 95% and preferably at least 97% identity between the sequences. An example of stringent hybridization conditions is overnight incubation at 42° C. in a solution comprising 50% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5× Denhardt's solution, 10% dextran sulfate, and 20 micrograms/milliliter denatured, sheared salmon sperm DNA, followed by washing the hybridization support in 0.1×SSC at approximately 65° C. Other hybridization and wash conditions are well known and are exemplified in Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, cold Spring Harbor, N.Y. (1989), particularly Chapter 11.
The invention also provides a polynucleotide consisting essentially of a polynucleotide sequence obtainable by screening an appropriate library containing the complete gene for a polynucleotide sequence set forth in the Sequence Listing under stringent hybridization conditions with a probe having the sequence of said polynucleotide sequence or a fragment thereof; and isolating said polynucleotide sequence. Fragments useful for obtaining such a polynucleotide include, for example, probes and primers as described herein.
As discussed herein regarding polynucleotide assays of the invention, for example, polynucleotides of the invention can be used as a hybridization probe for RNA, cDNA, or genomic DNA to isolate full length cDNAs or genomic clones encoding a polypeptide and to isolate cDNA or genomic clones of other genes that have a high sequence similarity to a polynucleotide set forth in the Sequence Listing. Such probes will generally comprise at least 15 bases. Preferably such probes will have at least 30 bases and can have at least 50 bases. Particularly preferred probes will have between 30 bases and 50 bases, inclusive.
The region of each gene that comprises or is comprised by a polynucleotide sequence set forth in the Sequence Listing may be isolated by screening using a DNA sequence provided in the Sequence Listing to synthesize an oligonucleotide probe. A labeled oligonucleotide having a sequence complementary to that of a polynucleotide of the invention is then used to screen a library of cDNA, genomic DNA or mRNA to identify members of the library which hybridize to the probe. For example, synthetic oligonucleotides are prepared which correspond to the desaturase promoter and intron sequences. In particular, screening of cDNA libraries in phage vectors is useful in such methods due to lower levels of background hybridization.
Typically, a desaturase sequence obtainable from the use of nucleic acid probes will show 60–70% sequence identity between the target desaturase sequence and the encoding sequence used as a probe. However, lengthy sequences with as little as 50–60% sequence identity may also be obtained. The nucleic acid probes may be a lengthy fragment of the nucleic acid sequence, or may also be a shorter, oligonucleotide probe. When longer nucleic acid fragments are employed as probes (greater than about 100 bp), one may screen at lower stringencies in order to obtain sequences from the target sample which have 20–50% deviation (i.e., 50–80% sequence homology) from the sequences used as probe. Oligonucleotide probes can be considerably shorter than the entire nucleic acid sequence encoding an desaturase enzyme, but should be at least about 10, preferably at least about 15, and more preferably at least about 20 nucleotides. A higher degree of sequence identity is desired when shorter regions are used as opposed to longer regions. It may thus be desirable to identify regions of highly conserved amino acid sequence to design oligonucleotide probes for detecting and recovering other related desaturase genes. Shorter probes are often particularly useful for polymerase chain reactions (PCR), especially when highly conserved sequences can be identified. (See, Gould, et al., PNAS USA (1989) 86:1934–1938.).
“Identity”, as is well understood in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, “identity” also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as determined by the match between strings of such sequences. “Identity” can be readily calculated by known methods including, but not limited to, those described in Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York (1988); Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M. and Griffin, H. G., eds., Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press (1987); Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., Stockton Press, New York (1991); and Carillo, H., and Lipman, D., SIAM J Applied Math, 48:1073 (1988). Methods to determine identity are designed to give the largest match between the sequences tested. Moreover, methods to determine identity are codified in publicly available programs. Computer programs which can be used to determine identity between two sequences include, but are not limited to, GCG (Devereux, J., et al., Nucleic Acids Research 12(1):387 (1984); suite of five BLAST programs, three designed for nucleotide sequences queries (BLASTN, BLASTX, and TBLASTX) and two designed for protein sequence queries (BLASTP and TBLASTN) (Coulson, Trends in Biotechnology, 12: 76–80 (1994); Birren, et al., Genome Analysis, 1: 543–559 (1997)). The BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBI NLM NIH, Bethesda, Md. 20894; Altschul, S., et al., J. Mol. Biol., 215:403–410 (1990)). The well known Smith Waterman algorithm can also be used to determine identity.
Parameters for polypeptide sequence comparison typically include the following:
Algorithm: Needleman and Wunsch, J. Mol. Biol. 48:443–453 (1970)
Comparison matrix: BLOSSUM62 from Hentikoff and Hentikoff, Proc. Natl. Acad. Sci USA 89:10915–10919 (1992)
Gap Penalty: 12
Gap Length Penalty: 4
A program which can be used with these parameters is publicly available as the “gap” program from Genetics Computer Group, Madison Wis. The above parameters along with no penalty for end gap are the default parameters for peptide comparisons.
Parameters for polynucleotide sequence comparison include the following:
Algorithm: Needleman and Wunsch, J. Mol. Biol. 48:443–453 (1970)
Comparison matrix: matches=+10; mismatches=0
Gap Penalty: 50
Gap Length Penalty: 3
A program which can be used with these parameters is publicly available as the “gap” program from Genetics Computer Group, Madison Wisconsin. The above parameters are the default parameters for nucleic acid comparisons.
For immunological screening, antibodies to the protein can be prepared by injecting rabbits or mice with the purified protein or portion thereof, such methods of preparing antibodies being well known to those in the art. Either monoclonal or polyclonal antibodies can be produced, although typically polyclonal antibodies are more useful for gene isolation. Western analysis may be conducted to determine that a related protein is present in a crude extract of the desired plant species, as determined by cross-reaction with the antibodies to the encoded proteins. When cross-reactivity is observed, genes encoding the related proteins are isolated by screening expression libraries representing the desired plant species. Expression libraries can be constructed in a variety of commercially available vectors, including lambda gt11, as described in Sambrook, et al. (Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).
Plant Constructs and Methods of Use
Of particular interest is the use of the polynucleotide sequences in recombinant DNA constructs to direct the transcription of the desaturase genomic sequences of the present invention in a host plant cell. The expression constructs generally comprise a promoter functional in a host plant cell operably linked to a nucleic acid sequence of the present invention and a transcriptional termination region functional in a host plant cell.
Those skilled in the art will recognize that there are a number of promoters which are functional in plant cells, and have been described in the literature. Chloroplast and plastid specific promoters, chloroplast or plastid functional promoters, and chloroplast or plastid operable promoters are also envisioned.
One set of promoters are constitutive promoters such as the CaMV35S or FMV35S promoters that yield high levels of expression in most plant organs. Enhanced or duplicated versions of the CaMV35S and FMV35S promoters are useful in the practice of this invention (Odell, et al. (1985) Nature 313:810–812; Rogers, U.S. Pat. No. 5,378,619). In addition, it may also be preferred to bring about expression of the sequences of the present invention in specific tissues of the plant, such as leaf, stem, root, tuber, seed, fruit, etc., and the promoter chosen should have the desired tissue and developmental specificity.
Of particular interest is the expression of the nucleic acid sequences of the present invention from transcription initiation regions which are preferentially expressed in a plant seed tissue. Examples of such seed preferential transcription initiation sequences include those sequences derived from sequences encoding plant storage protein genes or from genes involved in fatty acid biosynthesis in oilseeds. Examples of such promoters include the 5′ regulatory regions from such genes as napin (Kridl et al., Seed Sci. Res. 1:209:219 (1991)), phaseolin, zein, soybean trypsin inhibitor, ACP, stearoyl-ACP desaturase, soybean α′ subunit of β-conglycinin (soy 7s, (Chen et al, Proc. Natl. Acad. Sci., 83:8560–8564 (1986))) and oleosin.
It may be advantageous to direct the localization of proteins conferring desaturase to a particular subcellular compartment, for example, to the mitochondrion, endoplasmic reticulum, vacuoles, chloroplast or other plastidic compartment. For example, where the genes of interest of the present invention will be targeted to plastids, such as chloroplasts, for expression, the constructs will also employ the use of sequences to direct the gene to the plastid. Such sequences are referred to herein as chloroplast transit peptides (CTP) or plastid transit peptides (PTP). In this manner, where the gene of interest is not directly inserted into the plastid, the expression construct will additionally contain a gene encoding a transit peptide to direct the gene of interest to the plastid. The chloroplast transit peptides may be derived from the gene of interest, or may be derived from a heterologous sequence having a CTP. Such transit peptides are known in the art. See, for example, Von Heijne et al. (1991) Plant Mol. Biol. Rep. 9:104–126; Clark et al. (1989) J. Biol. Chem. 264:17544–17550; della-Cioppa et al. (1987) Plant Physiol. 84:965–968; Romer et al. (1993) Biochem. Biophys. Res Commun. 196:1414–1421; and, Shah et al. (1986) Science 233:478–481.
Depending upon the intended use, the constructs may contain the entire genomic nucleic acid sequence or a particular non-coding region of such a sequence or a portion of such sequences. For example, where antisense inhibition of a given desaturase protein is desired, the entire sequence is not required. Furthermore, where desaturase sequences used in constructs are intended for use as probes, it may be advantageous to prepare constructs containing only a particular portion of a desaturase sequence, for example a sequence which encodes a highly conserved desaturase region.
The skilled artisan will recognize that there are various methods for the inhibition of expression of endogenous sequences in a host cell. Such methods include, but are not limited to, antisense suppression (Smith, et al. (1988) Nature 334:724–726), co-suppression (Napoli, et al. (1989) Plant Cell 2:279–289), ribozymes (PCT Publication WO 97/10328), combinations of sense and antisense (Waterhouse, et al. (1998) Proc. Natl. Acad. Sci. USA 95:13959–13964), promoter silencing (Park, et al. (1996) Plant J. 9(2):183–194), DNA binding proteins (Beerli, et al. (1997) Proc. Natl. Acad. Sci. USA, 95:14628–14633; and Liu, et al. (1998) Proc. Natl. Acad. Sci. USA, 94:5525–5530). Methods for the suppression of endogenous sequences in a host cell typically employ the transcription or transcription and translation of at least a portion of the sequence to be suppressed. Such sequences may be homologous to coding as well as non-coding regions of the endogenous sequence.
Regulatory transcript termination regions may be provided in plant expression constructs of this invention as well. Transcript termination regions may be provided by the DNA sequence encoding the desaturase or a convenient transcription termination region derived from a different gene source, for example, the transcript termination region which is naturally associated with the transcript initiation region. The skilled artisan will recognize that any convenient transcript termination region which is capable of terminating transcription in a plant cell may be employed in the constructs of the present invention.
Alternatively, constructs may be prepared to direct the expression of the desaturase sequences directly from the host plant cell plastid. Such constructs and methods are known in the art and are generally described, for example, in Svab, et al. (1990) Proc. Natl. Acad. Sci. USA 87:8526–8530 and Svab and Maliga (1993) Proc. Natl. Acad. Sci. USA 90:913–917 and in U.S. Pat. No. 5,693,507.
A plant cell, tissue, organ, or plant into which the recombinant DNA constructs containing the expression constructs have been introduced is considered transformed, transfected, or transgenic. A transgenic or transformed cell or plant also includes progeny of the cell or plant and progeny produced from a breeding program employing such a transgenic plant as a parent in a cross and exhibiting an altered phenotype resulting from the presence of a desaturase nucleic acid sequence.
Plant expression or transcription constructs having a desaturase polynucleotide of the present invention as the DNA sequence of interest for increased or decreased expression thereof may be employed with a wide variety of plant life, particularly, plant life involved in the production of vegetable oils for edible and industrial uses. Most especially preferred are temperate oilseed crops. Plants of interest include, but are not limited to, rapeseed (Canola and High Erucic Acid varieties), sunflower, safflower, cotton, soybean, peanut, coconut and oil palms, and corn. Depending on the method for introducing the recombinant constructs into the host cell, other DNA sequences may be required. Importantly, this invention is applicable to dicotyledons and monocotyledons species alike and will be readily applicable to new and/or improved transformation and regulation techniques.
Of particular interest, is the use of plant desaturase promoter and/or intron constructs in plants to produce plants or plant parts, including, but not limited to leaves, stems, roots, reproductive, and seed, with a modified fatty acid composition. Of particular interest in the desaturase promoter and/or intron constructs is the use of the promoter and/or intron sequences of the Δ-12 and Δ-15 desaturase genomic sequences in sense or antisense orientations for the modification of fatty acid compositions in host cells.
The polynucleotides of the present invention can be used in the preparation of constructs for use in a variety of host cells. Host for use in the present invention include, but are not limited to plant cells, bacterial cells, fungal cells (including yeast), insect cells, and mammalian cells.
For example, to confirm the activity and specificity of the proteins encoded by the identified nucleic acid sequences as desaturase enzymes, in vitro assays can be performed in insect cell cultures using baculovirus expression systems. Such baculovirus expression systems are known in the art and are described by Lee, et al. U.S. Pat. No. 5,348,886, the entirety of which is herein incorporated by reference.
The method of transformation in obtaining such transgenic plants is not critical to the instant invention, and various methods of plant transformation are currently available. Furthermore, as newer methods become available to transform crops, they may also be directly applied hereunder. For example, many plant species naturally susceptible to Agrobacterium infection may be successfully transformed via tripartite or binary vector methods of Agrobacterium mediated transformation. In many instances, it will be desirable to have the construct bordered on one or both sides by T-DNA, particularly having the left and right borders, more particularly the right border. This is particularly useful when the construct uses A. tumefaciens or A. rhizogenes as a mode for transformation, although the T-DNA borders may find use with other modes of transformation. In addition, techniques of microinjection, DNA particle bombardment, and electroporation have been developed which allow for the transformation of various monocot and dicot plant species.
Normally, included with the DNA construct will be a structural gene having the necessary regulatory regions for expression in a host and providing for selection of transformant cells. The gene may provide for resistance to a cytotoxic agent, e.g. antibiotic, heavy metal, toxin, etc., complementation providing prototrophy to an auxotrophic host, viral immunity or the like. Depending upon the host species the expression construct or components thereof are introduced, one or more markers may be employed, where different conditions for selection are used for the different hosts.
Where Agrobacterium is used for plant cell transformation, a vector may be used which may be introduced into the Agrobacterium host for homologous recombination with T-DNA or the Ti- or Ri-plasmid present in the Agrobacterium host. The Ti- or Ri-plasmid containing the T-DNA for recombination may be armed (capable of causing gall formation) or disarmed (incapable of causing gall formation), the latter being permissible, so long as the vir genes are present in the transformed Agrobacterium host. The armed plasmid can give a mixture of normal plant cells and gall.
In some instances where Agrobacterium is used as the vehicle for transforming host plant cells, the expression or transcription construct bordered by the T-DNA border region(s) will be inserted into a broad host range vector capable of replication in E. coli and Agrobacterium, there being broad host range vectors described in the literature. Commonly used is pRK2 or derivatives thereof. See, for example, Ditta, et al., (Proc. Nat. Acad. Sci., U.S.A. (1980) 77:7347–7351) and EPA 0 120 515, which are incorporated herein by reference. Alternatively, one may insert the sequences to be expressed in plant cells into a vector containing separate replication sequences, one of which stabilizes the vector in E. coli, and the other in Agrobacterium. See, for example, McBride and Summerfelt (Plant Mol. Biol. (1990) 14:269–276), wherein the pRiHRI (Jouanin, et al., Mol. Gen. Genet. (1985) 201:370–374) origin of replication is utilized and provides for added stability of the plant expression vectors in host Agrobacterium cells.
Included with the expression construct and the T-DNA will be one or more markers, which allow for selection of transformed Agrobacterium and transformed plant cells. A number of markers have been developed for use with plant cells, such as resistance to chloramphenicol, kanamycin, the aminoglycoside G418, hygromycin, or the like. The particular marker employed is not essential to this invention, one or another marker being preferred depending on the particular host and the manner of construction.
For transformation of plant cells using Agrobacterium, explants may be combined and incubated with the transformed Agrobacterium for sufficient time for transformation, the bacteria killed, and the plant cells cultured in an appropriate selective medium. Once callus forms, shoot formation can be encouraged by employing the appropriate plant hormones in accordance with known methods and the shoots transferred to rooting medium for regeneration of plants. The plants may then be grown to seed and the seed used to establish repetitive generations and for isolation of vegetable oils.
For the alteration of unsaturated fatty acid production in a host cell, a second expression construct can be used in accordance with the present invention. For example, the desaturase expression construct can be introduced into a host cell in conjunction with a second expression construct having a nucleotide sequence for a protein involved in fatty acid biosynthesis.
There are several possible ways to obtain the plant cells of this invention which contain multiple expression constructs. Any means for producing a plant comprising a construct having a DNA sequence encoding the expression construct of the present invention, and at least one other construct having another DNA sequence encoding an enzyme are encompassed by the present invention. For example, the expression construct can be used to transform a plant at the same time as the second construct either by inclusion of both expression constructs in a single transformation vector or by using separate vectors, each of which express desired genes. The second construct can be introduced into a plant which has already been transformed with the desaturase expression construct, or alternatively, transformed plants, one expressing the desaturase construct and one expressing the second construct, can be crossed to bring the constructs together in the same plant.
The invention now being generally described, it will be more readily understood by reference to the following examples which are included for purposes of illustration only and are not intended to limit the present invention.
EXAMPLES
Example 1
Cloning of Desaturase Genomic Sequences
1A. Soybean Δ-12 Desaturase (fad2-1)
The soybean fad 2-1A sequence was identified by screening a soybean genomic library using a soybean fad2-1 cDNA probe. Three putative soy fad 2-1 clones were identified and plaque purified. Two of the three soy fad 2-1 clones were ligated into pBluescript II KS+ (Stratagene) and sequenced. Both clones (14-1 and 11-12) were the same and matched the soy fad 2-1 cDNA exactly. The sequence of the entire fad2-1A clone is provided in SEQ ID NO: 1.
Prior to obtaining this full length clone, a portion of the fad2-1A genomic clone was PCR amplified using PCR primers designed from the 5′ untranslated sequence (Primer 12506, 5′-ATACAA GCCACTAGGCAT-3′, SEQ ID NO:9) and within the cDNA (Primer 11698: 5′-GATTGGCCATGCAATGAGGGAAAAGG-3′, SEQ ID NO:10. The resulting PCR product, which contained the fad2-1A intron, was cloned into the vector pCR 2.1 (Invitrogen) and sequenced. The soy fad 2-1A partial genomic clone(SEQ ID NO:27) and its intron region (SEQ ID NO:2) were identified by comparison to the soybean cDNA sequence using the Pustell comparison program in Macvector. The intron sequence begins after the ATG start codon, and is 420 bases long.
A second fad2-1 gene family member was also identified and cloned, and is referred to herein as fad2-1B. The soy fad 2-1B partial genomic clone (SEQ ID NO:23) (contains the promoter (base pairs 1–1704); 5′UTR (base pairs 1705–1782); intron#1 (base pairs 1786–2190); and a portion of the fad2-1B coding region (base pairs 1783–1785 and 2191–2463)) and its intron region (SEQ ID NO:24) were identified by comparison to the soybean cDNA sequence using the Pustell comparison program in Macvector. The intron sequence begins after the ATG start codon and is 405 bases long.
1B. Soybean Δ15 Desaturase (fad3)
The partial soybean fad 3 genomic sequence was PCR amplified from soybean DNA using primers 10632, 5′-CUACUACUACUACTCGAGACAAAGCCTTTAGCCTATG-3′ (SEQ ID NO:11), and 10633: 5′-CAUCAUCAUCAUGGATCCCATGTC TCTCTATGCAAG-3′ (SEQ ID NO:12). The Expand Long Template PCR system (Boehringer Mannheim) was used according to the manufacturers directions. The resulting PCR products were cloned into the vector pCR 2.1 (Invitrogen) and sequenced. The soy fad 3 partial genomic clone sequence and the intron regions were confirmed by comparisons to the soybean fad 3 CDNA sequence using the Pustell program in Macvector. From the identified partial genomic soybean fad3 sequence (SEQ ID NO:3), seven introns were identified (SEQ ID NO:4 (intron #1), SEQ ID NO:5 (intron #2), SEQ ID NO:6 (intron #3A), SEQ ID NO:7 (intron #4), SEQ ID NO:8 (intron #5), SEQ ID NO:25 (intron #3B) and SEQ ID NO:26 (intron #3C)). Intron #1 is 192 base pairs long and is located between positions 294 and 485, intron #2 is 348 base pairs long and is located between positions 576 and 923, intron #3A is 142 base pairs long and is located between positions 991 and 1132, intron #3B is 98 base pairs long and is located between positions 1225 and 1322, intron #3C is 115 base pairs long and is located between positions 1509 and 1623, intron #4 is 1231 base pairs long and is located between positions 1705 and 2935, and intron #5 is 626 base pairs long and is located between positions 3074 and 3699.
Example 2
Expression Constructs
The soybean fad2-1A intron sequence was amplified via PCR using the fad2-1A partial genomic clone (SEQ ID NO:27) as a template and primers 12701 (5′-ACGAATTCCTCGAGGTAAA TTAAATTGTGCCTGC-3′ (SEQ ID NO:13)) and 12702 (5′-GCGAGATCTATCG ATCTGTGTCAAAGTATAAAC-3′ (SEQ ID NO:14)). The resulting amplification products were cloned into the vector pCR 2.1 (Invitrogen) and sequenced. The soyfad2-1A intron was then cloned into the expression cassette, pCGN3892, in sense and antisense orientations. The vector pCGN3892 contains the soybean 7S promoter and a pea RBCS 3′. Both gene fusions were then separately ligated into pCGN9372, a vector that contains the CP4 gene regulated by the FMV promoter. The resulting expression constructs (PCGN5469 sense and pCGN5471 antisense) were used for transformation of soybean using biolistic methods described below.
The soybean fad2-1B intron sequence was amplified via PCR using the fad2-1B partial genomic clone (SEQ ID NO:23) as a template and primers 13883 (5′-GCGATCGATGTATGATGCTAAATTAAATTGTGCCTG-3′ (SEQ ID NO:30)) and 13876 (5′-GCGGAATTCCTGTGTCAAAGTATAAAGAAG-3′ (SEQ ID NO:31)). The resulting amplification products were cloned into the vector pCR 2.1 (Invitrogen) and sequenced. The soyfad2-1B intron was fused to the 3′ end of the soy fad 2-1A intron in plasmids pCGN5468 (contains the soybean 7S promoter fused to the soy fad2-1A intron (sense) and a pea RBCS 3′) or pCGN5470 (contains the soybean 7S promoter fused to the soy fad2-1A intron (antisense) and a pea RBCS 3′) in sense or antisense orientation respectively. The resulting intron combo fusions were then ligated separately into pCGN9372, a vector that contains the CP4 gene regulated by the FMV promoter. The resulting expression constructs (pCGN5485, fad2-1A&B intron sense and pCGN5486, fad2-1A&B intron antisense) were used for transformation of soybean using biolistic methods described below.
Four of the seven introns identified from the soybean fad 3 genomic clone were PCR amplified using the soy fad 3 partial genomic clone as template and primers as follows: Intron #1, primers 12568: GATCGATGCCCGGGGTAATAATTTTTGTGT (SEQ ID NO:15) and 12569: CACGCCTCGAGTGTTCAATTCAATCAATG (SEQ ID NO:16); Intron #2, primers 12514: 5′-CACTCGAGTTAGTTCATACTGGCT (SEQ ID NO:17) and 12515: 5′-CGCATCGATTGCAAAATCCATCAAA (SEQ ID NO:18); Intron #4, primers 10926: 5′-CUACUACUACUACTCGAGCGTAAATAGTGGGTGAACAC (SEQ ID NO:19) and 10927: 5′-CAUCAUCAUCAUCTCGAGGAATTCGTCCATTTTAGTACACC (SEQ ID NO:20); Intron #5, primers 10928: 5′-CUACUACUACUACTCGAGGCGCGT ACATTTTATTGCTTA (SEQ ID NO:21) and 10929: 5′-CAUCAUCAUCAUCT CGAGGAATTCTGCAGTGAATCCAAATG (SEQ ID NO:22). The resulting PCR products for each intron were cloned into the vector pCR 2.1 (Invitrogen) and sequenced. Introns #1, #2, #4 and #5 were all ligated separately into the, pCGN3892, in sense or antisense orientations. pCGN3892 contains the soybean 7S promoter and a pea RBCS 3′. These fusions were ligated into pCGN9372, a vector that contains the CP4 gene regulated by the FMV promoter for transformation into soybean. The resulting expression constructs (pCGN5455, fad3 intron#4 intron sense; pCGN5459, fad3 intron#4 intron antisense; pCGN5456, fad3 intron#5 intron sense; pCGN5460, fad3 intron#5 intron antisense; pCGN5466, fad3 intron#2 intron antisense; pCGN5473, fad3 intron#1 intron antisense;) were used for transformation of soybean using biolistic methods described below.
The soy fad3 Intron #3C and #4 were also PCR amplified from a second fad3 gene family member, herein referred to as fad3-1B. The soy fad3-1B introns #3C and #4 were PCR amplified from soybean DNA using the following primers, 5′ CATGCTTTCTGTGCTTCTC 3′ (SEQ ID NO:32) and, 5′ GTTGATCCAACCATAGTCG 3′ (SEQ ID NO:33). The PCR products were cloned into the vector pCR 2.1 (Invitrogen) and sequenced. The sequences for the soy fad3-1B introns #3C and #4 are provided in SEQ ID NOs:28 and 29.
Example 3
Plant Transformation and Analysis
Linear DNA fragments containing the expression constructs for sense and antisense expression of the Δ12 and Δ15 desaturase introns were stably introduced into soybean (Asgrow variety A4922) by the method of McCabe, et.al. (1988) Bio/Technology 6:923–926. Transformed soybean plants were identified by selection on media containing glyphosate.
Fatty acid compositions were analyzed from seed of soybean lines transformed with the intron expression constructs using gas chromatography. T2 pooled seed and T2 single seed oil compositions demonstrate that the mono and polyunsaturated fatty acid compositions were altered in the oil of seeds from transgenic soybean lines as compared to that of the seed from non-transformed soybean. Table I provides a summary of results which were obtained using the described constructs. These data clearly show that sense and antisense expression of the non-coding regions of the desaturase gene results in the modification of the fatty acid compositions. The data also shows that introns can be used to obtain a variety of lines with varying fatty acid compositions. Selections can be made from such lines depending on the desired relative fatty acid composition. In addition, since each of the introns is able to modify the levels of each fatty acid to varying extents, it is contemplated that combinations of introns can be used depending on the desired compositions.









TABLE I









orien-













tation
event
Oleic
Linoleic
Linolenic
















Fad 2







wildtype

5469-5 null T2 pool
18.15%
55.59%
7.97%


(control)

10 seed average
13.89%
55.89%
9.067%




5469-27 null T2 pool
19.15%
54.62%
9.32%




A4922
15.75%
56.1%
8.75%




5471-13 null T2 pool
17.02%
56.49%
9.08%




10 seed average
13.86%
56.14%
9.49%




A4922
14.95%
55.95%
9.07%


full length
sense
5462-133 T2 pool
84%
2.17%
1.55%


cDNA

best 5462-133 T2 seed
84%
0.59%
1.76%


(control)


intron 1
sense
5469-6 T2 pool
29.93%
46.53%




5469-8 T2 pool
36.5%
42.11%
5.98%




best 5469-6 T2 seed
44.41%
29.34%
6.68%




best 5469-8 T2 seed
41.26%
33.16%
5.74%




5469-14 T2 pool
61.06%
16.42%
7.75%




5469-20 T2 pool
48.89%
31.61%
4.89%




5469-22 T2 pool
80%
2.97%
4.78%




best 5469-14 T2 seed
62.21%
11.97%
8.81%




5485-3 T2 pool
63.54%
14.09%
7.32%




5485-53 T2 pool
47.58%
27.64%
7.81%



anti-
5471-8 T2 pool
31.05%
43.62%
7.07%



sense
5471-2 T2 pool
27.98%
48.88%
6.83%




5471-26 T2 pool
32.66%
44.54%
6.76%




best 5471-8 T2 seed
57.4%
23.37%
5.73%




best 5471-2 T2 seed
28.08%
46.14%
6.52%




best 5471-26 T2 seed
43.3%
34.15%
5.6%




5486-33 T2 pool
32.37%
43.66%
6.87%




5486-12 T2 pool
27.32%
46.97%
6.4%




5486-40 T2 pool
26.79%
48.72%
6.55%


Fad 3


wildtype

5473-7 null T2 pool
15.65%
56.74%
9.55%


(control)

A4922 T2 pool
19.84%
56.79%
7.48%


full length
sense
5464-50 T2 pool
18.06%
62.03%
2.75%


cDNA

best 5464-50 T2 seed
17.08%
62.44%
1.72%


(control)


intron 1
anti-
5473-8 T2 pool
33.47%
45.97%
5.54%



sense
5473-1 T2 pool
33.34%
42.67%
7.59%


intron 2
anti-
5466-20 T2 pool
28.43%
48.83%
6.37%



sense
5466-16 T2 pool
27.61%
49.92%
5.96%


intron 4
sense
5455-19 T2 pool
40.35%
39.97%
4.61%




5455-10 T2 pool
35.14%
43.59%
5.53%




5455-57 T2 pool
38.04%
42.44%
5.24%




5455-76 T2 pool
37.24%
42.42%
5.37%




5455-107 T2 pool
36.44%
42.72%
5.62%




best 5455-57 T2 seed
45.36%
35.55%
4.92%




best 5455-76 T2 seed
35.3%
43.54%
5.53%




best 5455-107 T2 seed
45.56%
34.85%
5.12%



anti-
5459-2 T2 pool
34.5%
43.87%
5.59%



sense
5459-6 T2 pool
33.78%
44.12%
5.62%




5459-20 T2 pool
28.26%
49.48%
5.5%




best 5459-2 T2 seed
61.45%
23.45%
3.38%




best 5459-6 T2 seed
53.51%
29.68%
3.53%




best 5459-20 T2 seed
30%
50.55%
4.15%


intron 5
sense
5456-38 T2 pool
28.23%
49.59%
6.74%




5456-62 T2 pool
28.94%
48.66%
6.25%




best 5456-62 T2 seed
29.5%
43.69%
5.4%



anti-
5460-9 T2 pool
29.78%
48.57%
5.54%



sense
5460-21 T2 pool
28.37%
49.79%
5.54%




best 5460-21 T2 seed
35.18%
40.52%
5.33%









All publications and patent applications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.
Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.

## Claims

1. An isolated polynucleotide comprising SEQ ID NO: 24.

2. An isolated polynucleotide comprising at least 50 consecutive nucleotides of SEQ ID NO: 24.

3. An isolated polynucleotide comprising at least 30 consecutive nucleotides of SEQ ID NO: 24.

4. An isolated polynucleotide having at least 90% identity to SEQ ID NO: 24.

5. The isolated polynucleotide of claim 4 having at least 95% identity to SEQ ID NO: 24.

6. The isolated polynucleotide of claim 4 having at least 97% identity to SEQ ID NO: 24.

7. The isolated polynucleotide of claim 4 having at least 98% identity to SEQ ID NO: 24.

8. The isolated polynucleotide of claim 4 having at least 99% identity to SEQ ID NO: 24.