# Patent

## Title
Gene, protein, protein complex and method for improving aroma production in a plant

## Abstract

The invention relates to a nucleic acid molecule that improves aroma production in a plant, and a cell and a transgenic plant comprising the nucleic acid molecule. A protein and a protein complex for catalyzing the synthesis of a monoterpene and a precursor thereof and uses for improving the production of aroma in a plant are also provided.

## Background
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention relates to a gene for improving aroma production in a plant, and more particularly to a gene, protein, protein complex and method for improving aroma production in a plant.
2. Description of the Related Art
Flowers are unique organs that advertise their attractiveness in form, color and fragrance for insects, birds and mammals to assure pollination. Orchidaceae is one of the largest monocotyledon families, containing more than 25,000 species. In orchids, large quantities of pollen in masses are spread by animals (bees, moths, flies and birds), and the floral scents serve as attractants for species-specific pollinators. These pollinators play an important role in orchid floral diversification advantageous to the evolution of an obviously successful family. However, the biochemistry of fragrance production and the mechanisms regulating its emission in orchids remains sketchy.
To date, no simple, efficient, and reliable culture methods for scented orchids have been developed. Although the sympatric speciation of orchids is linked to differences in their floral odors, the large genome size, long life cycle and regeneration time and inefficient transformation system render the orchid scent biology difficult to explore. Furthermore, several scent and scentless species are cross-incompatible, which leads to the difficulty of producing scented offspring via traditional breeding. In some successful cases of cross-breeding, the offspring have diluted scent or even totally expel the scent-producing ability. Thus, to increase crop quality, the molecular breeding of scent species by introducing key enzymes regulating scent production to species/cultivars already with good characters is of interest.
Terpenoids belong to a large family of plant secondary metabolites, and their corresponding alcohols possess useful properties such as fragrance, flavor, insecticidal properties and characteristics that make them useful as pharmaceutical agents. In addition, primary metabolites like abscisic acid, carotenoids, chlorophyll, gibberellins, quinine electron carriers and steroids are also terpene-derived (van Schie et al., 2007, Plant J, 52, 752-762). Monoterpens formed from geranyl diphosphate (GDP, C10, is synthesized from dimethylallyl diphosphate (DMADP, C5) and isopentenyl diphosphate (IDP, C5) by GDP synthase (GDPS). IDP and DMADP are used by prenyltransferases to catalyze the synthesis of the general terpene backbones. GDPS is a member of the short-chain trans-prenyltransferase family, and it also includes farnesyl diphosphate synthase (FDPS) and geranylgeranyl diphosphate synthase (GGDPS), which synthesize farnesyl diphosphate (FDP, C15) and geranylgeranyl diphosphate (GGDP, C20), respectively (Ogura and Koyama, 1998, Chem. Rev., 98, 1263-1276; Reiling et al., 2004, Biotechnol Bioeng, 87, 200-212). These enzymes provide the acyclic branch-point intermediates for isoprenoid biosynthesis and control the flux into various terpenoid products. However, regulation and sharing of precursor pools are only starting to be explored.
The distribution of GDPS appears to be limited within nature. It has been described in the plants Mentha piperita (Burke et al., 1999, Proc Natl Acad Sci USA, 96, 13062-13067), Arabidopsis thaliana (Bouvier et al., 2000, Plant J, 24, 241-252), Abies grandis (Burke and Croteau, 2002, Arch Biochem Biophys, 405, 130-136), Antirrhinum majus, Clarkia breweri (Tholl et al., 2004, Plant Cell, 16, 977-992), Lycopersicon esculentum (van Schie et al., 2007, Plant J, 52, 752-762) and Phalaenopsis bellina (Hsiao et al., 2008, Plant J, 55, 719-733). GDPSs are either homomeric or heteromeric; the A. grandis, Arabidopsis and tomato GDPS, which contain the Asp-rich motifs and presume function as homodimers (Bohlmann et al., 2000, Arch Biochem Biophys, 375, 261-269; Burke and Croteau, 2002, Arch Biochem Biophys, 405, 130-136; van Schie et al., 2007, Plant J, 52, 752-762). The M. piperita, A. majus, and C. breweri GDPSs comprise heterodimers of a small (Mentha_SSU, Antirrhinum_SSU and Clarkia_SSU) and a large subunit (Mentha_LSU and Antirrhinum_LSU), which share only 22-38% identity with homomeric angiosperm GDPSs or GGDPSs (Burke et al., 2004, Arch Biochem Biophys, 422, 52-60; Tholl et al., 2004, Plant Cell, 16, 977-992). Notably, the small subunit from these plants lacks the Asp-rich motifs and is inactive per se (Tholl et al., 2004, Plant Cell, 16, 977-992). It appears to control the length of the chain synthesized by the catalytic large subunit, because interaction of the small subunit with GGDPS results in the conversion of functional GGDPS into GDPS (Tholl et al., 2004, Plant Cell, 16, 977-992). Meanwhile, the GDPS large subunit shares high amino acid sequence identity with GGDPS from plants (50%-75%), but the Mentha_LSU and Antirrhinum_LSU per se forms an active GGDPS enzyme that produces GGDP (Tholl et al., 2004, Plant Cell, 16, 977-992). Thus, the frequency of occurrence of the GDPS large subunit in plants has remained an open question.
Previously, floral scents in P. bellina (Orchidaceae, monocot) are demonstrated to be rich in the monoterpenes, geraniol and linalool and their derivatives (Hsiao et al., 2006, BMC Plant Biol, 6, 14). Identification a dual-function GDPS that lacks the Asp-rich motifs normally required for scent production but contains instead a glutamate-rich (Glu-rich) motif and is able to form a homodimer. Recent researches showed that GDPS small subunit from M. piperita, A. majus, and C. breweri is capable of modifying the chain length specificity of its catalytic partner and can bind to a variety of bona fide GDPS_LSU and GGPPS enzymes (Burke and Croteau, 2002, J Biol Chem, 277, 3141-3149; Tholl et al., 2004, Plant Cell, 16, 977-992). None of the previous studies of heterodimer GDPS have been performed in plant of monocots. It is curious that whether or not the heterodimer GDPS exists in orchid flower.
SUMMARY OF THE INVENTION
The present invention provides the isolation and heterologous expression of a PbGDPS large subunit, whose sequence was similar to plant GGDPS. The mode of action either as a homodimer or as a heterodimer with PbGDPS small subunit in P. bellina flower is also illustrated.
One subject of the invention is to provide an isolated nucleic acid molecule, which nucleic acid molecule is selected form the group consisting of:

    
    
        (a) a nucleic acid molecule, PbGDPS_LSU, comprising a nucleotide sequence of SEQ ID NO: 1;
        (b) a nucleic acid molecule encoding a polypeptide comprising an amino acid sequence of SEQ ID NO: 2;
        (c) a nucleic acid molecule encoding a polypeptide comprising an amino acid sequence of SEQ ID NO: 2 with one or more of the amino acids have been modified by a deletion, an insertion and/or a substitution, wherein the polypeptide has a geranyl diphosphate synthase activity and/or farnesyl diphosphate synthase activity;
        (d) a nucleic acid molecule encoding a polypeptide comprising an amino acid sequence with at least 85% similarity to SEQ ID NO: 2, wherein the polypeptide has a geranyl diphosphate synthase activity and/or farnesyl diphosphate synthase activity;
        (e) a nucleic acid molecule complementary to any one of the nucleic acid molecules as defined in (a), (b), (c) and (d); and
        (f) a nucleic acid molecule hybridizes under stringent hybridization conditions to any one of the nucleic acid molecules as defined in (a), (b), (c), (d) and (e).
    
    


Another subject of the invention is to provide a cell comprising the isolated nucleic acid molecule mentioned above.
Still another subject of the invention is to provide a transgenic plant comprising the nucleic acid molecule mentioned above.
Still another subject of the invention is to provide a protein, which protein comprises a polypeptide selected from the group consisting of:

    
    
        (a) a polypeptide, PbGDPS_LSU, comprising an amino acid sequence of SEQ ID NO: 2;
        (b) a polypeptide comprising an amino acid sequence of SEQ ID NO: 2 with one or more of the amino acids have been modified by a deletion, an insertion and/or a substitution, wherein the polypeptide has a geranyl diphosphate synthase activity and/or farnesyl diphosphate synthase activity; and
        (c) a polypeptide comprising an amino acid sequence with at least 85% similarity to SEQ ID NO: 2, wherein the polypeptide has a geranyl diphosphate synthase activity and/or farnesyl diphosphate synthase activity.
    
    


Still another subject of the invention is to provide a method for improving the production of aroma in a plant, which comprises increasing the expression of the protein mentioned above.
Still another subject of the invention is to provide a protein complex comprising the protein mentioned and a functional geranyl diphosphate synthase small subunit, wherein the functional geranyl diphosphate synthase small subunit is selected from the group consisting of:

    
    
        (a) a polypeptide, PbGDPS_SSU, comprising an amino acid sequence of SEQ ID NO: 4;
        (b) a polypeptide comprising an amino acid sequence of SEQ ID NO: 4 with one or more of the amino acids have been modified by a deletion, an insertion and/or a substitution, wherein the polypeptide has a geranyl diphosphate synthase activity and/or farnesyl diphosphate synthase and/or farnesyl diphosphate synthase activity; and
        (c) a polypeptide comprising an amino acid sequence with at least 40% similarity to SEQ ID NO: 4, wherein the polypeptide has a geranyl diphosphate synthase activity and/or farnesyl diphosphate synthase and/or farnesyl diphosphate synthase activity.
    
    


Still another subject of the invention is to provide a method for improving aroma production in a plant, which comprises increasing the expression of the protein complex mentioned above.



BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1A, 1B and 1C illustrate alignment of amino acid sequences of GDPS-a family and GGDPS in M. piperita GDPS large subunit (GDPS_Mentha_LSU, SEQ ID NO: 14), AF182828; A. majus GDPS large subunit (GDPS_Antirrhinum_LSU, SEQ ID NO: 13), AY534687; Abies grandis GDPS (SEQ ID NOs: 11 and 12), AF513111, AF513112, AF513112; Arabidopsis GGDPS1 (SEQ ID NO: 20), NP—195399; Capsicum annuum GGDPS (SEQ ID NO: 26), X80267; A. grandis GGDPS (SEQ ID NO: 18), AF425235; Oryza sativa GGDPS (SEQ ID NO: 19), CM000132.1, Croton sublyratus GGDPS (SEQ ID NO: 23), AB034249.1, Gentiana lutea GGDPS (SEQ ID NO: 27), AB028667.1; V. vinifera GGDPS (SEQ ID NO: 25), AM438997.2; Scoparia dulcis GGDPS (SEQ ID NO: 22), AB034250.1; Helianthus annuus GGDPS (SEQ ID NO: 28), AF020041.1; Sinapis Alba GGDPS (SEQ ID NO: 21), 2J1P_A. The conserved aspartate-rich motifs (DDXXD) are shown in box. Dashes indicate gaps inserted for optimal alignment.
FIG. 2 illustrates enzyme activity of PbGDPS_LSU. (A) Expression of recombinant PbGDPS_LSU. Partial purified His-tagged PbGDPS_LSU from E. coli (lane 1), and western blot with anti-His tag antibody (lane 2). Molecular mass markers are indicated on the left margin. (B) Quantification of the products derived by enzymatic hydrolysis of prenyl diphosphate products shown on radioactive thin-layer chromatography (TLC) analysis. Prenyltransferase activity of purified recombinant PbGDPS_LSU protein (100 μg) was using various prenyldiphosphates with different chain lengths as substrates. Following incubation of boiled protein and PbGDPS_LSU recombinant protein with the [4-14C]IPP, DMADP, GDP, and FDP, the reaction products were dephosphorylated. The experiment was repeated for three times independently.
FIG. 3 illustrates expression analysis of PbGDPS_LSU transcripts in flower from day 5 pre-anthesis (D-5), flowering day (Dd) to day 14 post-anthesis (D+14; flower senescence) (lanes 1 ̃6) and different tissues (lanes 1 ̃8). (A) RT-PCR analysis of PbGDPS_LSU with gene-specific primers was performed on RNA isolated from day 5 pre-anthesis to postanthesis of flower. Pactin9 gene was used as an internal control. (B) Spatial expression of PbGDPS_LSU with gene-specific primers was analyzed on RNA isolated from root, shoot, leaf, pedicle, sepal, petal, lip and column. Pactin9 gene was used as an internal control.
FIG. 4A illustrates interaction between PbGDPS proteins (LSU and SSU) in yeast as detected by two-hybrid system. Left panel: yeast strains AH109 co-transformed with both binding and activation vectors containing PbGDPS large subunit and small subunit combinations represented by numbers were streaked on the Minimal Synthetic Dropout (SD) medium without 1 leucine, tryptophan and histidine (a). Right panel: yeast strains relative to those in the left panel were streaked on the SD medium without adenine, histidine, leucine and tryptophan. (b). 1, pGBKT7-PbGDPS_SSU+pGADT7—PbGDPS_SSU; 2, pGBKT7-PbGDPS_LSU+pGADT7—PbGDPS_LSU; 3, pGBKT7-PbGDPS_SSU+pGADT7—PbGDPS_LSU; 4, pGBKT7-PbGDPS_LSU+pGADT7—PbGDPS_SSU; 5, pGADT7-T+pGBKT7-53 (positive control); 6, pGADT7-PbGDPS_SSU+pGBKT7 (negative control). FIG. 4B illustrates the yeast two-hybrid assay showing the relative interaction strength between PbGDPS_LSU and PbGDPS_SSU proteins. Activation of HIS3 and lacZ was indicated by growth on −HIS media with 20 mM 3AT.
FIG. 5 illustrates enzyme activity of PbGDPS_LSU/PbGDPS_SSU. (A) Expression of recombinant PbGDPS_SSU, PbGDPS_LSU and PbGDPS_LSU/PbGDPS_SSU. Partial purified His-tagged PbGDPS_SSU, PbGDPS_LSU and PbGDPS_LSU/PbGDPS_SSU from E. coli (lanes 1 ̃3), and western blot with anti-His tag antibody (lanes 4 ̃6). The black circles indicate the position of the recombinant protein. Molecular mass markers are indicated on the left margin. (B) Prenyltransferase activity of purified recombinant PbGDPS_SSU, PbGDPS_LSU and PbGDPS_LSU/PbGDPS_SSU protein (100 μg) with Mg2+ (10 mM). Boiled protein was included as a negative control in the same assay. Each point column represents the average of values from three independent experiments. Vertical bars indicate standard errors. (C) Quantification of the products derived by enzymatic hydrolysis of prenyl diphosphate products shown on radioactive TLC analysis. The purified recombinant PbGDPS_SSU, PbGDPS_LSU and PbGDPS_LSU/PbGDPS_SSU was using various prenyldiphosphates with different chain lengths as substrates as well as [4-14C]IPP, DMADP, GDP, and FDP. The experiment was repeated for three times independently.



DETAILED DESCRIPTION OF THE INVENTION
One subject of the invention is to provide an isolated nucleic acid molecule, which nucleic acid molecule is selected form the group consisting of:

    
    
        (a) a nucleic acid molecule, PbGDPS_LSU, comprising a nucleotide sequence of SEQ ID NO: 1;
        (b) a nucleic acid molecule encoding a polypeptide comprising an amino acid sequence of SEQ ID NO: 2;
        (c) a nucleic acid molecule encoding a polypeptide comprising an amino acid sequence of SEQ ID NO: 2 with one or more of the amino acids have been modified by a deletion, an insertion and/or a substitution, wherein the polypeptide has a geranyl diphosphate synthase activity and/or farnesyl diphosphate synthase activity;
        (d) a nucleic acid molecule encoding a polypeptide comprising an amino acid sequence with at least 85% similarity to SEQ ID NO: 2, wherein the polypeptide has a geranyl diphosphate synthase activity and/or farnesyl diphosphate synthase activity;
        (e) a nucleic acid molecule complementary to any one of the nucleic acid molecules as defined in (a), (b), (c) and (d); and
        (f) a nucleic acid molecule hybridizes under stringent hybridization conditions to any one of the nucleic acid molecules as defined in (a), (b), (c), (d) and (e).
    
    


As utilized in accordance with the present disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings:
The term “isolated nucleic acid molecule” as used herein refers to a nucleic acid molecule that (1) is not associated with all or a portion of a nucleic acid molecule in which the isolated nucleic acid molecule is found in nature, (2) is linked to a nucleic acid molecule to which it is not linked in nature, or (3) does not occur in nature as part of a larger sequence. Preferably, the isolated nucleic acid molecule is a polynucleotide. Examples of the isolated nucleic acid molecule are genomic DNA, mRNA, cDNA, or synthetic origin or some combination thereof, which by virtue of its origin the isolated nucleic acid molecule. The isolated nucleic acid molecule according to the invention preferably comprises a guide sequence, a coding region, an exon or an intron. An additional nucleic acid that does not affect the function of the isolated nucleic acid molecule is preferably contained. For example, several numbers of nucleic acids are contained in the 5′ and 3′ untranscribed regions.
The term “polynucleotide” as referred to herein means single-stranded or double-stranded nucleic acid polymers of at least 10 bases in length. In certain embodiments, the nucleotides comprising the polynucleotide can be ribonucleotides or deoxyribonucleotides or a modified form of either type of nucleotide. Said modifications include base modifications such as bromuridine, ribose modifications such as arabinoside and 2′,3′-dideoxyribose and internucleotide linkage modifications such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate and phosphoroamidate. The term “polynucleotide” specifically includes single and double stranded forms of DNA.
In one preferred embodiment of the invention, the nucleic acid molecule is isolated from P. bellina. The complete cDNA contains 1446 nucleotides and the truncated cDNA contains 1071 nucleotides and encodes a predicted protein of 356 amino acids. The terminus of this protein has characteristic features of a plastid-targeting peptide. It is found that there was only one PbGDPS_LSU detected in P. bellina flower. The PbGDPS_LSU showed 35%-45% similarity to GDPS small subunit and 70%-80% similarity to GGDPS. It contained the two conserved aspartate-rich motifs in all members of the prenyltransferase family which are important for substrate binding.
The term “a geranyl diphosphate synthase activity” as referred to herein means ability of condensing dimethylallyl diphosphate and isopentenyl diphosphate (IPP) to geranyl diphosphate. The method for assaying the geranyl diphosphate synthase activity is as described in Burke et al., 2004, Arch Biochem Biophys, 422, 52-60 and Tholl et al., 2004, Plant Cell, 16, 977-992.
The term “a farnesyl diphosphate synthase activity” as referred to herein means ability of transforming geranyl diphosphate and isopentenyl diphosphate to farnesyl diphosphate. The method for assaying the farnesyl diphosphate synthase activity is as described in Burke et al., 2004, Arch Biochem Biophys, 422, 52-60 and Tholl et al., 2004, Plant Cell, 16, 977-992.
In one preferred embodiment of the invention, PbGDPS_LSU protein possessed prenyltransferase activity. The purified protein is assayed using [14C]-IPP IPP and DMADP as cosubstrates and MgCl2 as cofactors, and PbGDPS_LSU yields GDP (C10) as the major product but also produces the minor product of FDP (C15). However, no GGDP (C20) is detected with IDP and DMADP as co-substrates. To test the chain-length selectivity of PbGDPS_LSU, either GDP or FDP is added as a substrate together with [14C]-IDP. PbGDPS_LSU accepts GDP as the allylic cosubstrate for the production of FDP but can not utilize FDP as the allylic cosubstrate to form GGDP. These results confirmed that PbGDPS possesses prenyltransferase activity with the substrates IDP, DMADP, and GDP. PbGDPS_LSU is able to catalyze the formation of both GDP and FDP. PbGDPS_LSU cDNA is proven to encode for GGDPS protein that behaves bifunctionally and synthesizes GDP as a major product and FDP as a minor product.
In one preferred embodiment of the invention, the gene encoding PbGDPS_LSU is constitutively expressed in all developmental and flowering stages. Spatially, PbGDPS_LSU was strongly expressed in pedicle, petal and to a less extent in root, leaf, sepal and lip but no detectable signal in shoot and column.
As used herein, a polypeptide comprising an amino acid sequence with at least 85% similarity to a reference polypeptide (such as SEQ ID NO: 2) refers to a polypeptide that differ from the reference polypeptide by substitution, deletion or insertion. For example, one or more of an amino acid residue is substituted with another amino acid residue with similar properties (based on size, polarity, hydrophobicity, and the like). The amino acids may be generally categorized into three main classes: hydrophilic amino acids, hydrophobic amino acids and cysteine-like amino acids, depending primarily on the characteristics of the amino acid side chain. These main classes may be further divided into subclasses. Hydrophilic amino acids include amino acids having acidic, basic or polar side chains and hydrophobic amino acids include amino acids having aromatic or apolar side chains. Apolar amino acids may be further subdivided to include, among others, aliphatic amino acids. The definitions of the classes of amino acids as used herein are as follows:
“Hydrophobic Amino Acid” refers to an amino acid having a side chain that is uncharged at physiological pH and that is repelled by aqueous solution. Examples of genetically encoded hydrophobic amino acids include Ile, Leu and Val. Examples of non-genetically encoded hydrophobic amino acids include t-BuA.
“Aromatic Amino Acid” refers to a hydrophobic amino acid having a side chain containing at least one ring having a conjugated π-electron system (aromatic group). The aromatic group may be further substituted with groups such as alkyl, alkenyl, alkynyl, hydroxyl, sulfanyl, nitro and amino groups, as well as others. Examples of genetically encoded aromatic amino acids include Phe, Tyr and Trp. Commonly encountered non-genetically encoded aromatic amino acids include phenylglycine, 2-naphthylalanine, β-2-thienylalanine, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, 4-chloro-phenylalanine, 2-fluorophenylalanine, 3-fluorophenylalanine and 4-fluorophenylalanine.
“Apolar Amino Acid” refers to a hydrophobic amino acid having a side chain that is generally uncharged at physiological pH and that is not polar. Examples of genetically encoded apolar amino acids include Gly, Pro and Met. Examples of non-encoded apolar amino acids include Cha.
“Aliphatic Amino Acid” refers to an apolar amino acid having a saturated or unsaturated straight chain, branched or cyclic hydrocarbon side chain. Examples of genetically encoded aliphatic amino acids include Ala, Leu, Val and Ile. Examples of non-encoded aliphatic amino acids include Nle.
“Hydrophilic Amino Acid” refers to an amino acid having a side chain that is attracted by aqueous solution. Examples of genetically encoded hydrophilic amino acids include Ser and Lys. Examples of non-encoded hydrophilic amino acids include Cit and hCys.
“Acidic Amino Acid” refers to a hydrophilic amino acid having a side chain pK value of less than 7. Acidic amino acids typically have negatively charged side chains at physiological pH due to loss of a hydrogen ion. Examples of genetically encoded acidic amino acids include Asp and Glu.
“Basic Amino Acid” refers to a hydrophilic amino acid having a side chain pK value of greater than 7. Basic amino acids typically have positively charged side chains at physiological pH due to association with hydronium ion. Examples of genetically encoded basic amino acids include Arg, Lys and His. Examples of non-genetically encoded basic amino acids include the non-cyclic amino acids ornithine, 2,3-diaminopropionic acid, 2,4-diaminobutyric acid and homoarginine.
“Polar Amino Acid” refers to a hydrophilic amino acid having a side chain that is uncharged at physiological pH, but which has a bond in which the pair of electrons shared in common by two atoms is held more closely by one of the atoms. Examples of genetically encoded polar amino acids include Asx and Glx. Examples of non-genetically encoded polar amino acids include citrulline, N-acetyl lysine and methionine sulfoxide.
“Cysteine-Like Amino Acid” refers to an amino acid having a side chain capable of forming a covalent linkage with a side chain of another amino acid residue, such as a disulfide linkage. Typically, cysteine-like amino acids generally have a side chain containing at least one thiol (SH) group. Examples of genetically encoded cysteine-like amino acids include Cys. Examples of non-genetically encoded cysteine-like amino acids include homocysteine and penicillamine.
Furthermore, one or more codons encoding a cysteine residue affect a disulfide bond of a specific polypeptide, and thus a cysteine residue is deleted, and the residue can be substituted by another amino acid residue.
As compared with the case in which an amino acid residue is conservatively substituted on the basis of the aforementioned description, when an amino acid residue is arbitrarily substituted, characteristics of the resultant protein are slightly changed.
The site of the aforementioned amino acid sequence at which amino acids are deleted, substituted, or added is arbitrary, so long as a protein containing the resultant modified amino acid sequence exhibits a geranyl diphosphate synthase activity and/or farnesyl diphosphate synthase activity. Similarly, the number of amino acids which are deleted, substituted, or added is arbitrary, so long as a protein composed of the resultant modified amino acid sequence exhibits a geranyl diphosphate synthase activity and/or farnesyl diphosphate synthase activity.
In one preferred embodiment of the invention, the nucleic acid molecule of (d) encodes a polypeptide comprising an amino acid sequence with at least 90%, 95%, 95% or 99% similarity to SEQ ID NO: 2, wherein the polypeptide has a geranyl diphosphate synthase activity and/or farnesyl diphosphate synthase activity. In one more preferred embodiment of the invention, the nucleic acid molecule of (d) encodes a polypeptide comprising an amino acid sequence with at least 99% similarity to SEQ ID NO: 2, wherein the polypeptide has a geranyl diphosphate synthase activity and/or farnesyl diphosphate synthase activity. In one most preferred embodiment, the isolated nucleic acid is PbGDPS_LSU comprising a nucleotide sequence of SEQ ID NO: 1.
Modification (variation) of the aforementioned amino acid sequence may naturally occur through, for example, mutation or modification after translation. A naturally occurring gene (e.g., the PbGDPS_LSU gene of the present invention) may be modified artificially. The present invention encompasses all the modified genes having the aforementioned characteristics, regardless of causes or means for such modification and variation. The gene of the present invention encompasses alleles of the gene encoding a protein composed of the amino acid sequence represented by SEQ ID NO: 1.
Examples of the aforementioned artificial means include genetic engineering methods such as site-specific mutagenesis; chemical synthesis methods such as a phosphate triester method and a phosphate amidite method; and combinations of the above methods. More specifically, synthesis of DNA may be carried out through chemical synthesis by means of a phosphoramidite method or a triester method. Alternatively, synthesis of DNA may be carried out by use of a commercially available automatic oligonucleotide synthesis apparatus. Double-stranded DNA fragments may be produced from single-stranded products which are chemically synthesized by annealing synthesized complementary strands under appropriate conditions or by adding complementary strands by use of an appropriate primer sequence and DNA polymerase.
Specific embodiments of the gene of the present invention include a gene having a nucleotide sequence represented by SEQ ID NO: 1. The nucleotide sequence (coding region) shows an example of the combination of codons corresponding to individual amino acid residues of the amino acid sequence represented by SEQ ID NO: 2. The gene of the present invention is not limited to the gene having such a specific nucleotide sequence; the gene of the present invention may have a nucleotide sequence which is selected from the combinations of arbitrary codons corresponding to individual amino acid residues. Selection of codons may be carried out by means of a customary method. For example, selection of codons may be carried out in consideration of the frequency of use of codons of the host.
The gene of the present invention can be easily produced or obtained by means of a customary genetic engineering method on the basis of sequence information in relation to specific examples of the gene disclosed herein.
The term “complementary” is used herein to refer to the sequences of polynucleotides which is capable of forming Watson & Crick base pairing with another specified polynucleotide throughout the entirety of the complementary region. For the purpose of the present invention, a first polynucleotide is deemed to be complementary to a second polynucleotide when each base in the first polynucleotide is paired with its complementary base. Complementary bases are, generally, A and T (or A and U), or C and G. “Complement” is used herein as a synonym from “complementary polynucleotide”, “complementary nucleic acid” and “complementary nucleotide sequence”. These terms are applied to pairs of polynucleotides based solely upon their sequences and not any particular set of conditions under which the two polynucleotides would actually bind. Unless otherwise stated, all complementary polynucleotides are fully complementary on the whole length of the considered polynucleotide.
According to the invention, the nucleic acid molecule can be a nucleic acid molecule hybridizes under stringent hybridization conditions to any one of the nucleic acid molecules as defined in (a), (b), (c), (d) and (e). The stringent conditions are not particularly limited, so long as the DNA fragment can be used as a primer or a probe. For example, the hybridization can be carried out under the condition as described above; i.e., in 0.2×SSC containing 0.1% SDS at 60° C., or in 0.1×SSC containing 0.1% SDS at 60° C.
In one preferred embodiment of the invention, the nucleic acid molecule is contained in a vector. The vector is used for storing or producing the nucleic acid molecule, or introducing the nucleic acid molecule into a plant or a plant cell. Preferably, the vector is a shuttle vector. As used herein, the term “shuttle vector” refers to a vector, which can be manipulated and selected in both a plant and a convenient cloning host, such as a prokaryote. Such a shuttle vector may include a kanamycin resistance gene for selection in plant cells and an actinomycin resistance gene for selection in a bacterial host. Besides, the shuttle vector contains an origin of replication appropriate for the prokaryotic host used, and preferably at least one unique restriction site or a polylinker containing unique restriction sites to facilitate the construction.
In another aspect, the nucleic acid molecule according to the invention is preferably driven by a promoter. More preferably, the promoter has an ability to drive expression of a nucleic acid within at least one portion of the reproductive tissues in the recipient plant, such as the cauliflower mosaic virus 35S protein promoter, the α-1 and β-1 tubulin promoter, and the histone promoters. In one embodiment of the invention, the promoter is an inducible promoter comprising but not limited to heat-shock protein promoters and light-inducible promoters including the three chlorophyll a/b light harvesting protein promoters. The methods of vector construction are well known to those skilled in the art.
Another subject of the invention is to provide a cell comprising the isolated nucleic acid molecule mentioned above.
In one preferred embodiment of the invention, the cell is a prokaryotic cell, an eukaryotic cell, a plant cell, a monocot cell, an orchid cell, a Phalaenopsis spp. cell, and a cell derived from a protocorn-like body. The term “protocorn-like body” used herein refers to a tissue, which has a potential to differentiate and is an ability for strong and rapid proliferation ability. Preferably, the nucleic acid molecule is introduced to the cell with transformation. As used herein, the term “transformation” refers to a process for changing the genetic material of a cell through introducing a nucleic acid molecule. Persons skilled in this art can conduct the transformation according to the disclosure of the invention and normal knowledge in molecular biology. For example, the vector may be introduced into a bacterial by heat shock process, or the vector is introduced into a plant cell by a gene gun.
Still another subject of the invention is to provide a transgenic plant comprising the nucleic acid molecule mentioned above. Preferably, the transgenic plant is an orchid; more preferably, the transgenic plant is a Phalaenopsis spp.
According to the invention, the plants to be transformed with the genes include orchid and orchid cells, preferably Phalaenopsis spp., which may be the wild type and an artificial mutant that produced by such as chemical modification, X-ray activated random mutagenesis or recombinant techniques.
Preferably, the transgenic orchid comprising at least one cell transformed with the isolated nucleic acid molecule, which may be transformed by conventional methods known to persons skilled in the art.
In an embodiment of the invention, a transgenic plant can be obtained by regenerating a transformed plant cell with the genes of the invention that are capable of modifying the phenotype of the plant, wherein the cells of the transgenic plant all have the same genetic material. In another embodiment of the invention, a mosaic plant can be obtained by transforming some of cells in a plant, such as reproductive cells or tissues, with the genes of the invention, wherein only the transformed cells express the modified phenotype as compared to the parent plant.
In one preferred embodiment of the invention, a method for producing a transgenic orchid comprising the steps of:

    
    
        (a) introducing the nucleic acid molecule according to the invention into an orchid cell to obtain an orchid transformed cell; and
        (b) regenerating the orchid transformed cell to obtain the transgenic orchid plant.
    
    


In one embodiment of the invention, a transgenic orchid plant may be produced through a protocorn-like body in vegetative planting or aspetic seed germination. After separating the cells in a protocorn-like body, each can regenerate a new protocorn-like body and then a new plant. In step (a), the nucleic acid molecule is introduced into a protocorn-like body, and preferably through a gene gun. At this moment, the nucleic acid molecule is introduced into some cells in the protocorn-like body to form transformed cells, and some cells are not introduced with the molecule. The transformed cells can be selected with the marker of the vector. In step (b), the transformed cells are regenerated to transgenic plants. As used herein, the term “regeneration” refers to a growth process of a plant from a plant cell, a group of plant cells or a part of a plant. The method of regeneration is well known to persons skilled in this field. A transgenic orchid produced thereby is also provided in the invention.
Still another subject of the invention is to provide a protein, which protein comprises a polypeptide selected from the group consisting of:

    
    
        (a) a polypeptide, PbGDPS_LSU, comprising an amino acid sequence of SEQ ID NO: 2;
        (b) a polypeptide comprising an amino acid sequence of SEQ ID NO: 2 with one or more of the amino acids have been modified by a deletion, an insertion and/or a substitution, wherein the polypeptide has a geranyl diphosphate synthase activity; and
        (c) a polypeptide comprising an amino acid sequence with at least 85% similarity to SEQ ID NO: 2, wherein the polypeptide has a geranyl diphosphate synthase activity.
    
    


In one preferred embodiment of the invention, the protein has an amino acid sequence of SEQ ID NO: 2.
When a desired gene encoding the protein of the present invention is designed, the nucleotide sequence of the PbGDPS_LSU gene represented by SEQ ID NO: 1 is preferably utilized. If desired, the gene may be designed by appropriately selecting and modifying codons corresponding to the amino acid residues of the protein.
The protein of the present invention may be produced on the basis of the amino acid sequence represented by SEQ ID NO: 2 by means of a general chemical synthesis method. Examples of the synthesis method include a peptide synthesis method employing a usual liquid phase method or a solid phase method.
Specific examples of the peptide synthesis method include a stepwise elongation method in which amino acids are sequentially bound with one another on the basis of amino acid sequence information, to thereby elongate the chain of amino acids; and a fragment condensation method in which fragments composed of several amino acids are synthesized in advance, and the fragments are bound with one another through coupling reaction. The protein of the present invention may be synthesized by means of either of the methods.
In such peptide synthesis, condensation may be carried out by means of a customary method. Examples of the condensation method include an azide method, a mixed acid anhydride method, a DCC method, an active ester method, an oxidation-reduction method, a DPPA (diphenylphosphorylazide) method, a DCC+additive (1-hydroxybenzotriazole, N-hydroxysuccinamide, N-hydroxy-5-norbornane-2,3-dicarboxylmide) method, and a Woodward method.
A solvent employed in such a method can be appropriately selected from widely used solvents used for peptide condensation reaction. Examples of the solvent include dimethylformamide (DMF), dimethylsulfoxide (DMSO), hexaphosphoroamide, dioxane, tetrahydrofuran (THF), ethyl acetate, and solvent mixtures thereof.
In the course of the aforementioned peptide synthesis, a carboxyl group of amino acid or peptide, which group is not involved in the reaction, may be protected typically through esterification, to thereby form esters such as lower alkyl esters; e.g., a methyl ester, an ethyl ester, a tert-butyl ester, and aralkyl esters; e.g., a benzyl ester, a p-methoxybenzyl ester, and a p-nitrobenzyl ester.
An amino acid having a functional group in its side chain; e.g., a hydroxyl group in a tyrosine residue, may be protected with a group such as an acetyl group, a benzyl group, a benzyloxycarbonyl group, or a tert-butyl group. However, the protection may optionally be performed. In addition, for example, a guanidino group in an arginine residue may be protected by an appropriate protective group such as a nitro group, a tosyl group, a p-methoxybenzenesulfonyl group, a methylene-2-sulfonyl group, a benzyloxycarbonyl group, an isobornyloxycarbonyl group, or an adamantyloxycarbonyl group.
Deprotection of these protective groups included in the aforementioned amino acids, peptides, and proteins of the present invention—final products—may be carried out through a routine method; e.g., catalytic reduction or use of a reagent such as liquid ammonia/sodium, hydrogen fluoride, hydrogen bromide, hydrogen chloride, trifluoroacetic acid, acetic acid, formic acid, or methanesulfonic acid.
The thus-obtained protein of the present invention may appropriately be purified through a variety of methods as described above. For example, there may be employed methods generally employed in the field of peptide chemistry; e.g., use of ion-exchange resin, partition chromatography, gel chromatography, and counter current distribution.
The protein of the present invention can be suitably used as an immunogen for producing its specific antibody. By use of the immunogen, a desired antiserum (polyclonal antibody) and monoclonal antibody can be produced.
Still another subject of the invention is to provide a method for improving the production of aroma in a plant, which comprises increasing the expression of the protein mentioned above.
In one embodiment of the invention, the expression of the proteins can be changed by increasing the ploid of the nucleic acid molecule encoding the proteins in at least one cell of the plant. In a preferred embodiment of the invention, a gene gun is used to introduce the nucleic acid molecule into the cell for changing the expression of the protein.
Preferably, the aroma comprises a monoterpene and a precursor thereof. More preferably, the precursor comprises geranyl diphosphate and farnesyl diphosphate.
Still another subject of the invention is to provide a protein complex comprising the protein mentioned and a functional geranyl diphosphate synthase small subunit, wherein the functional geranyl diphosphate synthase small subunit is selected from the group consisting of:

    
    
        (a) a polypeptide, PbGDPS_SSU, comprising an amino acid sequence of SEQ ID NO: 4;
        (b) a polypeptide comprising an amino acid sequence of SEQ ID NO: 4 with one or more of the amino acids have been modified by a deletion, an insertion and/or a substitution, wherein the polypeptide has a geranyl diphosphate synthase activity and/or farnesyl diphosphate synthase; and
        (c) a polypeptide comprising an amino acid sequence with at least 40% similarity to SEQ ID NO: 4, wherein the polypeptide has a geranyl diphosphate synthase activity and/or farnesyl diphosphate synthase.
    
    


Preferably, a functional geranyl diphosphate synthase small subunit comprises an amino acid sequence of SEQ ID NO: 4.
The manner of construction of the polypeptide of (b) and (c) is similar to the aforementioned description.
In one preferred embodiment of the invention, the polypeptide of (c) comprises an amino acid sequence with at least 50%, 60%, 70%, 80%, 90%, 95%, 95% or 99% similarity to SEQ ID NO: 4, wherein the polypeptide has a geranyl diphosphate synthase activity and/or farnesyl diphosphate synthase activity. In one more preferred embodiment of the invention, the polypeptide of (c) comprises an amino acid sequence with at least 90%, 95%, 95% or 99% similarity to SEQ ID NO: 4, wherein the polypeptide has a geranyl diphosphate synthase activity and/or farnesyl diphosphate synthase activity.
In one preferred embodiment of the invention, the protein complex comprises a heterdimer consisting of PbGDPS_LSU and PbGDPS_SSU, wherein PbGDPS_LSU comprises an amino acid sequence of SEQ ID NO: 2 and PbGDPS_SSU comprises an amino acid sequence of SEQ ID NO: 4.
PbGDPS_LSU and PbGDPS_SSU are able to form a heterodimer. Interestingly, PbGDPS_SSU is able to form homodimer, but the homodimeric interaction was not detected for the PbGDPS_LSU. The formation of heterodimer between PbGDPS_LSU and PbGDPS_SSU is the strongest, followed by the PbGDPS_SSU homodimer, and the weakest one is PbGDPS_LSU homodimer. Preferably, the PbGDPS_LSU and PbGDPS_SSU form heterotetramers to function as an active enzyme. To our knowledge, the protein interaction behavior between PbGDPS_LSU and PbGDPS_SSU is not the same as their homologs in eudicots.
Still another subject of the invention is to provide a method for improving the production of aroma in a plant, which comprises increasing the expression of the protein complex mentioned above.
In one preferred embodiment of the invention, combination of PbGDPS_LSU and PbGDPS_SSU has produced the highest GDP and FDP than either PbGDPS_LSU or PbGDPS_SSU per se.
The following Examples are given for the purpose of illustration only and are not intended to limit the scope of the present invention.
EXAMPLE
Materials and Methods
Plant Materials
P. bellina flowers were grown under standard greenhouse conditions as described previously (Hsiao et al., 2006, BMC Plant Biol, 6, 14).
5′ and 3′ Amplification of PbGDPS Large Subunit (PbGDPS_LSU) cDNA Ends
The full-length PbGDPS_LSU cDNA was obtained by extending the 5′ and 3′ ends of the cDNA using the SMART RACE cDNA amplification kit (Clontech; Palo Alto, Calif.). The cDNA containing the 5′ and 3′ end for PbGDPS_LSU clones were obtained by PCR amplification with use of a 5′ or 3′ universal primer and 3′ or 5′ gene-specific nested primer, respectively (Clontech). The PbGDPS_LSU gene specific primers: 5′-s CGAGGAGGGAGTATCGCATCGCTTCGT-3′ (SEQ ID NO: 5) was used to amplify the 5′ end fragments and 5′-CACACCATGTCTCTCATCCACGACGATC-3′ (SEQ ID NO: 6) was used to amplify the 3′ end fragments. The PCR products were cloned into the pGEM-T Easy vector (Promega; Madison, Wis.) and both strands from 10 positive clones were sequenced.
Sequences Analysis
Sequence data used in this study were downloaded from the EMBL/GenBank data libraries under the following accession numbers: Antirrhinum majus GDPS small subunit (GDPS_Antirrhinum_SSU), AY534686; Clarkia breweri GDPS small subunit (GDPS_Clarkia_SSU), AY534745; Mentha piperita GDPS small subunit (GDPS_Mentha_SSU), AF182827; M. piperita GDPS large subunit (GDPS_Mentha_LSU), AF182828; A. majus GDPS large subunit (GDPS_Antirrhinum_LSU), AY534687; Lycopersicon esculentum GDPS, DQ2869302; Quercus robur GDPS, CAC20852; Vitis vinifera GDPS, Am457037; Arabidopsis GDPS, Y17376; Abies grandis GDPS, AF513111, AF513112, AF513112; Citrus sinensis GDPS, AJ243739; Arabidopsis GGDPS1, NP—195399; Capsicum annuum GGDPS, X80267; A. grandis GGDPS, AF425235; Oryza sativa GGDPS, CM000132.1; Croton sublyratus GGDPS, AB034249.1; Gentiana lutea GGDPS, AB028667.1; V. vinifera GGDPS, AM438997.2; Scoparia dulcis GGDPS, AB034250.1; Helianthus annuus GGDPS, AF020041.1; Sinapis Alba GGDPS, 2J1P_A; Lupinus albus FDPS, U15777; Artemisia annua FDPS, U36376; Arabidopsis FDPS1, NM—124151; C. annuum FDPS, X84695; O. sativa FDPS, NM—192229; and Zea mays FDPS, L39789.
Expression of His-Tagged PbGDPS_LSU and PbGDPS_LSU/PbGDPS_SSU Recombinant Protein
A truncated version of PbGDPS_LSU, in which the N-terminal plastid targeting sequence was deleted, was obtained using the forward primer 5′-CACCATGCCCTCTGTTGCCACAACCGAGT-3′ (SEQ ID NO: 7) which introduced a starting Met in place of Pro-26, and the reverse primer 5′-ATTCTGCCTGTAAGCGATGTAATTCGCA-3′ (SEQ ID NO: 8) for subcloning into the pBAD/D-TOPO directional expression vector (Invitrogen, Carlsbad, Calif.). To express N-terminally His-tagged PbGDPS_LSU recombinant protein, E. coli BL21 (DE3) pLysS cells were transformed with the resulting recombinant plasmid. The empty pBAD202/D-TOPO vector was also transformed into BL21 (DE3) pLysS cells to serve as a negative control. Single positive bacterial colonies were inoculated into 30-50 ml and grown overnight at 37° C. and transfer the inoculated culture into a large volume of medium until an OD600 of 0.5 was reached. Cultures were then induced by the addition of L-arabinose (Sigma) to a final concentration of 0.1% and allowed to grow for an additional 5 h at 17° C. for the induction of protein expression. For coexpression, BL21 (DE3) pLysS E. coli cells were cotransformed with two plasmids PbGDPS_LSU and PbGDPS_SSU (Hsiao et al., 2008, Plant J, 55, 719-733) in a single transformation event. Single positive bacterial colonies were used to inoculate Luriabertani medium with 100 μg/ml of ampicillin (pET15b, Hsiao et al., 2008, Plant J, 55, 719-733) and 50 μg/ml of kanamycin (pBAD202), which was induced by the addition of 0.1% L-arabinose and 0.05 mM isopropyl-1-thio-β-D-galactopyranoside.
RT-PCR
Total RNA was extracted from the developing flowers of P. bellina, from flower buds to day 14 post-anthesis (senescence). cDNA was synthesized using DNase-treated total RNA, an oligo dT primer and SuperScriptlll reverse transcriptase (Invitrogen, Carlsbad, Calif.) according to the manufacturer's protocol. The optimal gene-specific primers used to amplify the PbGDPS_LSU sequence for the RT-PCR were 5′-GGCTAGCAACAAGACAACCT-3′ (forward) (SEQ ID NO: 9) and 5′-GAAATTTGAAGCAGCCTAGC-3′ (reverse) (SEQ ID NO: 10). PCR with the actin gene-specific primers of Phalaenopsis (Hsiao et al., 2008, Plant J, 55, 719-733) was performed. To ensure that an equal amount of RNA was used for all samples and that RT reactions were equally effective.
Yeast Two-Hybrid Analysis
Yeast two-hybrid analysis was performed using the MATCHMAKER yeast two-hybrid system (Clontech, Palo Alto, Calif.). PCR was used to generate a sequence that was flanked by EcoR1 sites and that encoded the same truncated version of PbGDPS_LSU as used for the recombinant His-tagged protein. This PCR product was cloned into the binding domain vector pGBKT7 and the activation domain vector pGADT7. Introduction of the inserts in the correct reading frame was confirmed by sequencing. The resulting constructs were transformed into yeast strain AH109 by the lithium acetate method. The transformants that received both the DNA binding domain plasmid and the activation domain plasmid were selected on medium lacking adenine, histidine, leucine and tryptophan according to the instructions of the manufacturer of the yeast two-hybrid system.
For quantification the interaction strength between GDPS proteins, the colony-lift filter assays were used for β-galactosidase activity. The colony-lift filter assay was performed according to the manufacturer's instructions (Clontech). For the analysis of β-galactosidase activity, yeast strain Y187 was used (Clontech).
Results
Molecular Cloning and Sequence Analysis of PbGDPS_LSU
The partial cDNA of P. bellina GDPS large subunit (PbGDPS_LSU) was isolated from a P. bellina floral cDNA library (Hsiao et al., 2006, BMC Plant Biol, 6, 14). A full-length cDNA was obtained by 5′ and 3′ rapid amplification of cDNA ends (RACE). The complete cDNA contains 1446 nucleotides and the truncated cDNA contains 1071 nucleotides and encodes a predicted protein of 356 amino acids (FIG. 1). The terminus of this protein has characteristic features of a plastid-targeting peptide. Several plant species contain multiple GGDPS and GGDPS-related enzymes (Burke and Croteau, 2002, Arch Biochem Biophys, 405, 130-136). To confirm the PbGDPS_LSU existed in floral of P. bellina, the presence of other PbGDPS_LSU and GGDPS in various developmental stages as well as flower on flowering day (D-day) and the day 5 post-anthesis (D+5) by using PCR-based cloning methods is searched. The search results revealed that there was only one PbGDPS_LSU detected in P. bellina flower. The PbGDPS_LSU showed 35%-45% similarity to GDPS small subunit (M. piperita, A. majus, C. breweri and P. bellina) and 70%-80% similarity to GGDPS (Table 1; FIG. 1). It contained the two conserved aspartate-rich motifs (FIG. 1, blue box) in all members of the prenyltransferase family which are important for substrate binding (Wang and Ohnuma, 2000, Biochim Biophys Acta, 1529, 33-48). According to this homology, the PbGDPS_LSU may function as GGDPS.







TABLE 1





Percentage of amino acid similarity at full length sequence


of short chain prenyltransferase from different plants



































1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17





 1
GDPS_Phal_SSU

48
48
50
48
28
24
26
25
24
22
25
25
25
39
36
36


 2
GDPS_Antirrhinum_SSU


73
68
61
25
26
30
27
26
25
34
33
31
45
44
44


 3
GDPS_Mentha_SSU



67
58
24
25
29
26
26
23
31
33
32
44
44
42


 4
GDPS_Vitis




61
28
25
30
26
27
25
31
31
28
46
46
43


 5
GDPS_Clarkia_SSU





24
23
24
22
25
25
30
31
30
44
44
42


 6
FDPS_Arabidopsis






91
86
87
85
26
30
31
31
31
31
33


 7
FDPS_Lupinus







91
87
86
27
29
30
30
33
30
33


 8
FDPS_Artemisia








85
85
27
33
33
34
34
30
34


 9
FDPS_Capsicum









83
26
29
31
30
34
32
34


10
FDPS_Oryza










27
29
31
29
32
30
32


11
FDPS_Zea











29
30
29
33
31
33


12
GDPS_Arabidopsis












91
82
36
38
38


13
GDPS_Citrus













95
42
42
41


14
GDPS_Tomato














36
36
36


15
GGDPS_Abies















80
77


16
GDPS2_Abies
















80


17
GDPS3_Abies



















18
GGDPS_Oryza



















19
GGDPS1_Arabidopsis



















20
GGDPS_Sinapisalba



















21
GGDPS_Arabidopsis



















22
GDPS_Antirrhinum_LSU



















23
GGDPS_Sooparia



















24
GDPS_Mentha_LSU



















25
GGDPS_Croton



















26
GGDPS_Medicago



















27
GGDPS_Vitis



















28
GGDPS_Capsicum



















29
GGDPS_Gentiana



















30
GGDPS_Helianthus



















31
GGDPS_Mustard



















32
GDPS_Pbellina_LSU



















33
GDPS_Pequestris_LSU































18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33






 1
GDPS_Phal_SSU
37
40
40
39
41
39
37
38
36
40
36
36
39
41
37
37



 2
GDPS_Antirrhinum_SSU
43
49
48
48
49
46
44
46
45
47
46
46
46
51
44
45



 3
GDPS_Mentha_SSU
42
48
47
46
45
47
46
46
45
48
44
43
45
49
43
44



 4
GDPS_Vitis
43
48
47
48
48
47
46
48
46
48
46
45
46
52
42
43



 5
GDPS_Clarkia_SSU
46
48
48
48
46
46
44
47
46
48
48
42
45
52
45
45



 6
FDPS_Arabidopsis
31
30
30
30
32
32
31
32
32
34
29
29
32
32
31
32



 7
FDPS_Lupinus
30
31
31
32
33
30
31
31
32
32
31
29
32
30
30
31



 8
FDPS_Artemisia
32
32
33
34
33
32
33
33
32
34
33
30
33
32
32
32



 9
FDPS_Capsicum
33
32
32
32
33
31
32
32
33
33
32
30
33
31
32
33



10
FDPS_Oryza
32
32
32
33
33
32
32
33
33
33
31
31
32
32
32
32



11
FDPS_Zea
32
33
34
33
34
33
33
33
33
34
30
32
33
33
32
33



12
GDPS_Arabidopsis
42
41
41
37
41
40
39
40
40
44
40
39
40
46
42
42



13
GDPS_Citrus
43
47
46
43
44
44
44
43
44
45
44
42
44
46
44
44



14
GDPS_Tomato
39
41
41
36
40
41
38
38
40
43
39
38
38
44
41
41



15
GGDPS_Abies
65
70
70
67
72
73
67
72
72
80
71
65
72
79
71
72



16
GDPS2_Abies
65
71
73
64
71
72
66
71
69
81
69
62
71
79
71
72



17
GDPS3_Abies
64
70
70
64
68
69
65
69
68
78
68
60
69
78
69
70



18
GGDPS_Oryza

70
72
66
70
71
69
73
70
78
70
65
69
79
72
72



19
GGDPS1_Arabidopsis


88
74
78
82
78
80
80
91
74
70
76
93
76
77



20
GGDPS_Sinapisalba



76
78
81
77
79
80
90
74
71
76
95
76
77



21
GGDPS_Arabidopsis




70
73
69
72
72
82
69
66
72
83
71
71



22
GDPS_Antirrhinum_LSU





83
77
79
78
88
76
70
77
86
74
74



23
GGDPS_Sooparia






81
83
81
88
77
72
76
88
74
75



24
GDPS_Mentha_LSU







79
76
88
72
67
74
85
73
73



25
GGDPS_Croton








83
90
75
74
75
87
74
75



26
GGDPS_Medicago









91
75
72
76
88
74
75



27
GGDPS_Vitis










85
80
86
91
81
82



28
GGDPS_Capsicum











70
78
85
75
75



29
GGDPS_Gentiana












71
83
70
70



30
GGDPS_Helianthus













85
73
73



31
GGDPS_Mustard














84
84



32
GDPS_Pbellina_LSU















98



33
GDPS_Pequestris_LSU









Functional Characterization of Recombinant PbGDPS_LSU
In previous studies of GDPS and GGDPS, optimal expression of soluble recombinant enzyme was only obtained after deleting the N-terminal plastid targeting sequence (Burke and Croteau, 2002, Arch Biochem Biophys, 405, 130-136; Burke and Croteau, 2002, J Biol Chem, 277, 3141-3149). Recombinant PbGDPS showed the same characteristics. When full-length PbGDPS was expressed in E. coli, very little soluble protein was obtained, and the majority of the expressed protein formed was located in inclusion bodies. To overcome this problem, a truncated version of the PbGDPS_LSU open reading frame was cloned, which lacked the N-terminal signal sequence and its calculated molecular weight was 50 kD (FIG. 2A). To assay whether the recombinant truncated PbGDPS_LSU protein possessed prenyltransferase activity, the purified protein was assayed using [14C]-IPP and DMADP as cosubstrates and MgCl2 as cofactors. The resulting products were further hydrolyzed to the corresponding alcohols and then analyzed by using thin-layer chromatography and scintillation counting of the radioactive areas (TLC; FIG. 2B). Recombinant truncated PbGDPS_LSU yielded GDP (C10) as the major product but also produced the minor product of FDP (C15) (FIG. 2B). However, no GGDP (C20) was detected by radioactive TLC with IDP and DMADP as co-substrates. To test the chain-length selectivity of PbGDPS_LSU, either GDP or FDP was added as a substrate together with [14C]-IDP. PbGDPS_LSU could accept GDP as the allylic cosubstrate for the production of FDP but could not utilize FDP as the allylic cosubstrate to form GGDP (FIG. 2B). These results confirmed that PbGDPS possesses prenyltransferase activity with the substrates IDP, DMADP, and GDP. In the negative control experiments, the boiled protein (FIG. 2B) and the empty vector (data not shown) did not produce any products. Thus, this PbGDPS_LSU was able to catalyze the formation of both GDP and FDP. is PbGDPS_LSU cDNA is proven to encode for GGDPS protein that behaves bifunctionally and synthesizes GDP as a major product and FDP as a minor product.
Analysis of the Spatial and Temporal Expression of PbGDPS_LSU
To understand when and where PbGDPS_LSU was active, we examined both the temporal and spatial expression of the gene by use of the 3′ specific primer. Result showed the gene encoding PbGDPS_LSU was constitutively expressed in all developmental and flowering stages (FIG. 3A, lanes 1 ̃6). Spatially, PbGDPS_LSU was strongly expressed in pedicle, petal (lane 4 ̃6) and to a less extent in root, leaf, sepal and lip (lanes 1, 3, 5 and 7) but no detectable signal in shoot and column (FIG. 3B, lane 2 and lane 8). It revealed that the expression pattern of PbGDPS_LSU did not correlate well with the monoterpene emission.
Interaction of PbGDPS_LSU and PbGDPS_SSU
For understanding how PbGDPS_LSU and PbGDPS_SSU function in orchids, the interaction between these LSU and SSU of PbGDPS by using an Y2H system is analyzed, the standard way to identify in vivo protein-protein interactions. The PbGDPS_LSU and PbGDPS_SSU open reading frames were cloned into the DNA-binding-domain vector pGBKT7 and activation-domain vector pGADT7, respectively. Combination of vector containing PbGDPS_LSU or PbGDPS_SSU cDNA was then introduced into yeast, and the interactions between these proteins were assessed by growing colonies on medium lacking adenine and histidine (SD-Trp-Leu-His-Ade). As expected, PbGDPS_LSU and PbGDPS_SSU formed heterodimer as similar to those of eudicots. Interestingly, the PbGDPS_SSU was able to form homodimer, but the homodimeric interaction was not detected for the PbGDPS_LSU (FIG. 4A). The relative protein-protein interaction strength was assessed by using colony-lift filter assay. Results showed that the formation of heterodimer between PbGDPS_LSU and PbGDPS_SSU was the strongest, followed by the PbGDPS_SSU homodimer, and the weakest one was PbGDPS_LSU homodimer (FIG. 4B). These results suggested that the PbGDPS_LSU and PbGDPS_SSU might form heterotetramers to function as an active enzyme. To our knowledge, the protein interaction behavior between PbGDPS_LSU and PbGDPS_SSU was not the same as their homologs in eudicots.
Functional Characterization of Recombinant PbGDPS Large Subunit with PbGDPS Small Subunit
Previous studies of PbGDPS_LSU and PbGDPS_SSU indicated that the protein function was to produce GDP and FDP, respectively. Coexpression of the PbGDPS_LSU and PbGDPS_SSU (FIG. 5A) revealed higher prenyltransferase activity than either PbGDPS_LSU or PbGDPS_SSU alone (FIG. 5B). The resulting products were further hydrolyzed to the corresponding alcohols and then analyzed by thin-layer chromatography and scintillation counting of the radioactive areas (TLC; FIG. 5C). Results showed combination of PbGDPS_LSU and PbGDPS_SSU has produced the highest GDP and FDP than either PbGDPS_LSU or PbGDPS_SSU per se (FIG. 5C).
While embodiments of the present invention have been illustrated and described, various modifications and improvements can be made by persons skilled in the art. The present invention is not limited to the particular forms as illustrated, and that all the modifications not departing from the spirit and scope of the present invention are within the scope as defined in the appended claims.

## Claims

1. An isolated nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1.

2. A vector comprising the isolated nucleic acid molecule according to claim 1.

3. The vector according to claim 2, which is a shuttle vector that is capable of expressing the nucleic acid molecule in a plant.

4. The vector according to claim 2, which comprises an inducible promoter.

5. A cell transformed by the vector according to claim 2.

6. The cell according to claim 5, which is selected from the group consisting of a prokaryotic cell, an eukaryotic cell, a plant cell, a monocot cell, an orchid cell, a Phalaenopsis spp. cell, and a cell derived from a protocorn-like body.

7. A transgenic plant comprising the nucleic acid molecule according to claim 1.

8. The transgenic plant according to claim 7, which is an orchid.

9. The transgenic plant according to claim 8, which is a Phalaenopsis spp.