WO2001092489A2 - Palmitate desaturase gene - Google Patents

Abstract

Desaturase enzymes, and especially animal Δ9-desaturases, and the use of such enzymes to alter fatty acid saturation, especially fatty acid saturation in oilseeds, are disclosed. In particular, the FAT-5 palmitate desaturase enzyme is disclosed. Also disclosed are nucleic acid sequences encoding animal Δ9-desaturase enzymes, including such sequences that encode FAT-5.

Description

PALMITATE DESATURASE GENE

FIELD The disclosure relates to animal desaturase enzymes and methods of using such enzymes to alter the saturation of fatty acids.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support from the United States Department of Agriculture, grant number USDA-NRICGP97-35301-4426, and from the National Science

Foundation, postdoctoral fellowship number DBI-9804125. The government has certain rights in this invention.

BACKGROUND Fatty acids are fundamental components of living systems. They make up the major component of cytoplasmic membranes, common to plants, animals, and protists alike.

The ^saturation of fatty acids in glycerolipids is essential for the proper function of biological membranes. At physiological temperatures, polar glycerolipids that contain only saturated fatty acids cannot form the liquid-crystalline bilayer that is the fundamental structure of biological membranes (Stubbs and Smith, Biochim. Biophys. Acta, 779:89-137, 1984). The introduction of an appropriate number of unsaturated bonds into the fatty acids of membrane glycerolipids decreases the temperature for the transition from the solid to the liquid phase and provides membranes with the necessary fluidity (Russel, Trends Biochem. Sci., 9:108-112, 1984; and Hazel, Annu. Rev. Physiol., 57: 19-42, 1995). Fluidity of the membrane is important for maintaining the barrier properties of the lipid bilayer and for the activation and function of certain membrane-bound enzymes (Houslay and Gordon, Curr. Top. Membr. Transp., 18:179-231, 1984; and Thompson, J. Bioenerg. Biomembr., 21:43-60, 1989). Many poikilothermic organisms respond to a decrease in temperature by desaturating the fatty acids of their membrane lipids (Cossins, Biochim. Biophys. Acta, 470:395-411, 1977; and Lee and Cossins, Biochim. Biophys. Acta, 1026:195-203, 1990). This homeoviscous adaptation (Sinensky, Proc. Natl. Acad. Sci. USA, 71 :522-525, 1974; and McElhaney,

Biomembranes, 12:249-276, 1984) improves the organisms' ability to maintain membrane fluidity over a broader temperature range and is believed to be an important component of cellular acclimation to temperature changes in poikilothermic organisms (Tiku, Science, 271:815-818, 1996). In addition to their role in adaptation to low temperatures, membranes with unsaturated fatty acids also contribute to an organism's ability to adapt to other environmental stresses. For example, membrane lipid composition and membrane fluidity affects yeast tolerance to ethanol, with higher unsaturation correlating with higher ethanol tolerance (Alexandre et al., FEMS Microbiol. Lett., 124:17-22, 1994; Sajbidor and Grego, FEMS Microbiol. Lett, 93:13-16, 1992; Beavan et al, J. hid. Microbiol, 128:1445-1447, 1982; and Del Castillo Agudo, Appl. Microbiol. Biotechnol, 37:647-651, 1992). However, the correlation is not exact (Swan and Watson, Can. J. Microbiol, 43:70-77, 1997; Guerzoni et al., Can. J. Microbiol, 43:569-476, 1997; and Swan and Watson, Can. J. Microbiol, 45:472-479, 1999). It is likely that membrane fluidity is not the only factor to ethanol-stress resistance, since the synthesis of heat-shock proteins (Li, J. Cell PhysioL, 115:116-122, 1983) and the synthesis of the disaccharide trehalose (Odumeru et al., J. Ind. Microbiol, 11:113-119, 1993) are bom induced upon exposure of yeast to ethanol. There are many indications that ethanol and oxidative stress are connected to changes in membrane fluidity in mammals, particularly in fetal tissue (Henderson et al, Front. Biosci., 4:D541-D550, 1999), reproductive tissue (Zalata et al, Int. J. Androl, 21:154-162, 1998), and in human liver (French, Clin. Biochem., 22:41-49, 1989).

The ability of cells to modulate the degree of unsaturation in their membranes is mainly determined by the action of fatty acid desaturases (Kates et al., Biomembranes, 12:379-395, 1984; Murata and Wada, Biochem. J., 308:1-8, 1995; and Tocher et al, Prog. Lipid Res., 37: 73-117, 1998). Desaturase enzymes introduce unsaturated bonds at specific positions in their fatty acyl chain substrates. One classification of fatty acid desaturases is based on the moiety to which the hydrocarbon chains are acylated. Desaturases recognize substrates that are bound either to acyl carrier protein, to coenzyme A, or to lipid molecules (Murata and Wada, Biochem. J., 308:1-8, 1995; and Shanklin and Cahoon, Annu. Rev. Plant PhysioL Plant Mol. BioL, 49:611-641, 1998). Since desaturation reactions require one molecule of oxygen and two electrons for each reaction, desaturases also can be differentiated by the electron carrier that they require. While ferredoxin is the electron donor in the desaturation reactions catalyzed by acyl-ACP desaturases, by acyl-lipid desaturases of cyanobacteria, and by acyl-lipid desaturases in the plastids of plants (McKeon and Stumpf, J Biol. Chem., 257:12141-12147, 1982; and Wada et al, J. BacterioL, 175:544-547, 1993), the acyl lipid and acyl-CoA desaturases found in the endoplasmic reticulum of all eukaryotes and many bacteria use cytochrome b₅ as a donor (Jaworski, in The Biochemistry of Plants (Stumpf et al, Eds.), Academic Press, Orlando, FL, Vol. 9:159-174, 1987; Macartney et al, in Temperature Adaptation of Biological Membranes (Cossins, Ed.), Portland Press, London, pp. 129-139, 1994; and Jaworski and Stumpf, Arch. Biochem. Biophys., 162:158-165, 1974). Desaturase enzymes also show considerable selectivity both for the chain length of the substrate and for the location of existing double bonds in the fatty acyl chain (Shanklin and Cahoon, Annu. Rev. Plant PhysioL Plant Mol. Biol, 49:611-641, 1998).

Purification and activity of fatty acid desaturases have been limited by their requirement for membrane association. One of the most fruitful approaches to examining desaturase activity has been mutational analysis. Isolation of cyanobacteria and Arabidopsis thaliana mutants with altered fatty acid compositions has permitted the isolation of genes encoding most of the transmembrane desaturases present in these organisms (Browse et al, Science, 227: 763-765, 1985; and Browse and Somerville, in. Arabidopsis (Meyerowitz and Somerville, Eds.), Cold Spring Harbor Laboratory Press, Plainview, NY, pp. 881-912, 1994). Sequence analysis of these desaturases has facilitated the cloning of a number of other desaturase genes from plants (Tocher et al, Prog. Lipid Res., 37: 73- 117, 1998; and Sayanova et al, Proc. Natl. Acad. Sci. USA, 94:4211-4216, 1997), bacteria (Aguilar et al, J. Bacteriol, 180:2194-2200, 1998), protists (Nakashima et al, Biochem. J., 317(Pt 1): 29-34, 1996), nematodes (Spychalla et al, Proc. Natl. Acad. Sci. USA, 94:1142-1147, 1997; Watts and Browse, Arch. Biochem. Biophys., 362:175-182, 1999; and Napier et al, Biochem. J., 330:611-614, 1998), and mammals (Cho et al, J. Biol. Chem., 274:471-477, 1999; and Aki et al, Biochem. Biophys. Res. Commun., 255:575-579, 1999).

Palmitoleic (16:1) and oleic (18:1) acids are major constituents of membrane phospholipids and triacylglycerol stores in animals, plants, and fungi. These organisms regulate the ratio of mono- unsaturated fatty acids to saturated fatty acids in cellular membranes to maintain proper fluidity

(Ntambi, / LipidRes., 40:1549-1558, 1999; Tika et al, Science, 271:815-818, 1996). In humans, the alteration of this ratio has been implicated in various disease states such as cancer, diabetes, obesity, immune disorders, as well as neurological, vascular and heart diseases (Ntambi, J. LipidRes., 40:1549-1558, 1999). Eukaryotic organisms synthesize mono-unsaturated fatty acids from saturated fatty acid precursors by the introduction of a double bond between carbons 9 and 10 of a saturated acyl chain. This conversion is catalyzed by fatty acyl desaturases that use molecular oxygen and reducing equivalents obtained from NAD(P)H via a short electron transport chain consisting of NAD(P)H, cytochrome b₅-reductase, and cytochrome b₅ (Tocher et al, Prog. Lipid Res., 37:73-117, 1998). The vast majority of desaturases are endoplasmic reticulum (ER) membrane-bound di-iron-oxo proteins characterized by three conserved histidine-rich motifs. Hydrophobic domains are predicted to span the membrane twice to lock the protein into the bilayer and coordinate two iron molecules at the active site on the cytosolic face of the ER (Stukey et al, J. Biol. Chem., 265:20144-20149, 1990; Shanklin et al, Biochemistry, 33(43): 12787-12794, 1994). Another type of desaturase class is the soluble acyl-acyl carrier protein (ACP) desaturases found in the stroma of higher plant plastids. While these are also di-iron-oxo proteins, they are structurally unrelated to the ER desaturases (Lindquist et α/., £ B<9./, 15:4081-4092, 1996).

Stearoyl-CoA desaturase (SCD) is one of the most studied and best-understood membrane- bound desaturases. One yeast gene, three mouse genes, two rat genes, and a single human SCD gene have been cloned and characterized (Stukey et al, J. Biol. Chem., 264:16537-16544, 1989; Ntambi et al, J. Biol. Chem., 263:17291-17300, 1988; Mihara, J. Biochem., 108:1022-1029, 1990; Parimoo et al, J. Investig. Dermatol. Symp. Proc, 4:320-322, 1999; Kaestener et al, J. Biol. Chem., 264:14755- 14761, 1989; Zhang et al, Biochem. J., 340:255-264, 1999). The Δ⁹ fatty acyl desaturase gene ^' (OLE1) from the yeast Saccharomyces cerevisiae was isolated and characterized by complementation of the ole-1 mutant which requires unsaturated fatty acids for growth (Stukey et al, J. Biol. Chem., 264:16537-16544, 1989). This mutant is incapable of growth on unsupplemented media because it is unable to synthesize mono-unsaturated fatty acids. The yeast Olelp is 36% identical to the rat SCD1 protein within a 257 AA internal region of the open reading frame. However, the Ν-terminal region shows no significant homologies and the yeast gene extends 113 amino acids beyond the C-terminal of the rat gene. The C-terminal region of Olelp has been identified as a cytochrome b₅ domain (Mitchell and Martin, J. Biol. Chem., 270:29766-29772, 1998). Heterologous expression of rat SCDl rescues the fatty acid auxotrophy of the olel mutant, even though the rat gene carries no cytochrome b₅ domain (Stukey et al, J. Biol. Chem., 265:20144-20149, 1990). This rescue depends on the presence of the S. cerevisiae microsomal cytochrome b₅ gene (YSCYb5), indicating that the rat desaturase enzyme requires this electron donor and interacts successfully with yeast components of the desaturase system (Mitchell and Martin, J. Biol. Chem., 270:29766-29772, 1998).

As the name implies, the SCDs use stearic acid (18:0) as a substrate. The other major substrate for these enzymes is palmitic acid (16:0). Expression of SCDs in the yeast olel mutant revealed the substrate preference of these Δ⁹-desaturases. Olelp readily desaturates both 16:0 and 18:0, converting 65% of 16:0 to 16:1 and 86% of 18:0 to 18:1 (Stukey et al., J. Biol. Chem., 264:16537-16544, 1989). The rat SCD gene expressed in olel was capable of desaturating 46% of 16:0 and 88% of 18:0. Recently characterized Δ⁹-desaturases from cabbage looper moth and the oleaginous fungus Mortierella alpina expressed in olel showed an even greater preference for 18:0, producing 6-13 times more 18:1 than 16:1 (Liu et al, Insect Biochem. Mol. Biol, 29:435-443, 1999; andWongwathanarat et α/., Mι^"crofø^'otøgy, 145:2939-2946, 1999).

Both the yeast and mammalian Δ⁹-desaturases are inducible enzymes. Transcription of the yeast OLE1 gene is highly regulated in response to extracellular fatty acids. Transcription is activated by saturated fatty acids and repressed by unsaturated fatty acids provided in the growth medium (Bossie and Martin, J. Bacteriology, 171:6409-13, 1998). The mammalian desaturase activity is also regulated by nutritional manipulation, as well as by hormonal and developmental factors (Ntambi, Prog. LipidRes., 34:139-150, 1995). The mouse SCDl and SCD2 genes show great differences in their expression patterns in various tissues, at different stages of development, and in their response to dietary influences. SCDl mRNA is constitutively expressed in adipose tissue and is induced in the liver in response to high carbohydrate diet, while SCD2 transcript is induced in the brain during embryonic development, is down regulated during the development of mouse lymphocytes, and is not expressed in liver under any conditions (Kaestener et al, J. Biol. Chem., 264: 14755-14761, 1989). It is clear that distinct control mechanisms must exist for each of the genes. A major health concern with respect to dietary fats is the saturated fatty acid content of different types of fats and oils. The most abundant saturated fatty acid in most oilseeds is 16:0 (palmitic acid). One strategy to reduce the amount of saturated fatty acids in an oil-producing plant is to engineer the plant in such a way that it desaturates the palmitic acid to palmitoleic (cis Δ⁹ 16:1). Thus, acquisition of genes encoding Δ⁹-desaturases, and particularly a Δ⁹-desaturase that is specifically a palmitate desaturase, that can be expressed in plants would represent important advances in efforts to alter and control saturation of fatty acids. SUMMARY

The disclosure provides, inter alia, isolated fat-5,fat-6, and/αt-7 cDNAs from Caenorhabditis elegans that are shown to affect fatty acid saturation when transformed into host cells, and the respective FAT-5, FAT-6, and FAT-7 proteins encoded by these nucleic acids. The FAT-5 Δ⁹-desaturase provides a surprisingly different desaturation activity when compared to known Δ⁹-desaturases, in that it substantially preferentially desaturates 16-carbon fatty acids. Thus, FAT-5 is a specific palmitate desaturase.

The novel animal Δ⁹-desaturase enzymes may be cloned and expressed in the cells of various organisms, including plants, to produce unsaturated (e.g., monounsaturated) fatty acids. Expression of such unsaturated fatty acids enhances the nutritional qualities of such organisms. For instance, oil-seed plants may be engineered to incorporate a Δ⁹-desaturase. Such oil-seed plants would produce seed-oil rich in polyunsaturated fatty acids. Such fatty acids could be incorporated usefully into infant formula, foods of all kinds, dietary supplements, and nutriceutical and pharmaceutical formulations. The disclosure also provides proteins differing from these proteins by one or more conservative amino acid substitutions. Proteins that exhibit "substantial similarity" (defined below) with these Δ⁹-desaturase proteins are also provided.

The disclosure provides isolated nucleic acids that encode the above-mentioned proteins, recombinant nucleic acids that include such nucleic acids and cells, plants, and other organisms containing such recombinant nucleic acids. Appropriate plants include oil palm, sunflower, safflower, rapeseed, Canola, soy, peanut, cotton, corn, rice, Arabidopsis, mustard, wheat, barley, potato, tomato, yam, apple, and pear plants.

The novel Δ⁹-desaturase proteins can be used to produce unsaturated fatty acids, such as 16:1 and 18:1 fatty acids, as well as polyunsaturated fatty acids that include a Δ⁹-unsaturation. Embodiments also include portions of nucleic acids encoding the novel Δ⁹-desaturase enzymes, portions of nucleic acids that encode polypeptides substantially similar to these novel enzymes, and portions of nucleic acids that encode polypeptides that differ from the inventive proteins by one or more conservative amino acid substitutions. Such portions of nucleic acids may be used, for instance, as primers and probes for research and diagnostic purposes. Research applications for such probes and primers include the identification and cloning of related Δ⁹-desaturases in other organisms including both eukaryotes and prokaryotes.

The disclosure also includes methods that utilize the Δ⁹-desaturase enzymes. An example of this embodiment is a yeast or plant cell that carries a nucleic acid coding for a Δ⁹-desaturase (e.g., a fat-5 nucleic acid) and that, by virtue of this desaturase, displays altered production of unsaturated fatty acids.

The foregoing and other features and advantages will become more apparent from the following detailed description of several embodiments, which proceeds with reference to the accompanying sequence listing and figures. SEQUENCE LISTING

The nucleic acid and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and the three-letter code for amino acids. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood to be included by any reference to the displayed strand.

SEQ ID NOs: 1 and 2 are oligonucleotide primers that can be used to amplify the fat-5 encoding sequence. SEQ ID NO: 3 is an encoding sequence of fat-5, with introns.

SEQ DD NO: 4 is the cDNA sequence of fat-5, and the corresponding deduced amino acid sequence of FAT-5 (W06D12.3).

SEQ ID NO: 5 is the deduced amino acid sequence of FAT-5 (also shown in Figure 1). SEQ ID NOs: 6 and 7 are oligonucleotide primers that can be used to amplify fhe fat-6 encoding sequence.

SEQ ID NO: 8 is an encoding sequence of fat-6, with introns.

SEQ ID NO: 9 is the cDNA sequence of fat-6, and the corresponding deduced amino acid sequence of FAT-6 (VZK822L.1).

SEQ ID NO: 10 is the deduced amino acid sequence of FAT-6 (also shown in Figure 1). SEQ ID NOs: 11 and 12 are oligonucleotide primers that can be used to amplify the/αr-7 encoding sequence.

SEQ ID NO: 13 is an encoding sequence of fat-7, with introns.

SEQ ID NO: 14 is the cDNA sequence of fat-7, and the corresponding deduced amino acid sequence of FAT-7 (F10D2.9). SEQ ID NO: 15 is the deduced amino acid sequence of FAT-7 (also shown in Figure 1).

BRIEF DESCRIPTION OF THE FIGURES Figure 1.

Comparison of the three C. elegans Δ⁹-desaturase predicted open reading frames with portions of the rat SCDl and the yeast Olelp open reading frames. Trans-membrane domains as predicted by TMHMM, available from the Center for Biological Sequence Analysis (CBSA),

Technical University of Denmark, are shown by bars drawn above the sequence, and the conserved histidine residues are denoted with asterisks.

Figure 2. Δ⁹-desaturation of fatty acids by transgenic olel yeast expressing the C. elegans Δ⁹- desaturases. Graph values represent the mean conversion (%) of 2-5 trials (+ SD), except for

18:lΔ^π(*røκ.s), which was measured in only one trial. DETAILED DESCRIPTION

I. Explanation of Terms

The following explanations and methods are provided to better explain the present disclosure and to guide those of ordinary skill in the art. Unless otherwise noted, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art. Definitions of common terms in molecular biology may also be found in Rieger et al, Glossary of Genetics: Classical and Molecular, 5th edition, Springer- Verlag: New York, 1991; and Lewin, Genes VII, Oxford University Press: New York, 1999. The nomenclature for DNA bases as set forth at 37 C.F.R. § 1.822 is used. The standard one- and three-letter nomenclature for amino acid residues is used.

Amplification (of a nucleic acid molecule (e.g., a DNA or RNA molecule): Use of a technique that increases the number of copies of a nucleic acid molecule in a specimen. An example of amplification is the polymerase chain reaction (PCR), in which a biological sample collected from a subject is contacted with a pair of oligonucleotide primers, under conditions that allow for the hybridization of the primers to nucleic acid template in the sample. The primers are extended under suitable conditions, dissociated from the template, and then re-annealed, extended, and dissociated to amplify the number of copies of the nucleic acid. The product of amplification may be characterized by electrophoresis, restriction endonuclease cleavage patterns, oligonucleotide hybridization or ligation, and or nucleic acid sequencing using standard techniques. Other examples of amplification include strand-displacement amplification, as disclosed in U.S. Patent No. 5,744,311; transcription- free isothermal amplification, as disclosed in U.S. Patent No. 6,033,881; repair chain reaction amplification, as disclosed in WO 90/01069; ligase chain reaction amplification, as disclosed in EP- A-320 308; gap-filling ligase chain reaction amplification, as disclosed in U.S. Patent No. 5,427,930; and NASBA™ RNA transcription-free amplification, as disclosed in U.S. Patent No. 6,025,134. cDNA (complementary DNA): A "cDNA" is a piece of DNA lacking internal, non-coding segments (introns) and regulatory sequences that determine transcription. cDNA can be synthesized in the laboratory by reverse transcription from messenger RNA extracted from cells. Desaturase: A desaturase is an enzyme that promotes the formation of carbon-carbon double bonds in a hydrocarbon molecule.

Desaturase activity may be demonstrated by assays in which a preparation containing a putative desaturase enzyme is incubated with a suitable substrate fatty acid and analyzed for conversion of the substrate to a predicted fatty acid product. Alternatively, a DNA sequence proposed to encode a desaturase protein may be incorporated into a suitable vector construct and thereby expressed in cells of a type that do not normally have an ability to desaturate a particular fatty acid substrate. Activity of the desaturase enzyme encoded by the DNA sequence then can be demonstrated by supplying a suitable form of substrate fatty acid to cells transformed with a vector containing the desaturase-encoding DNA sequence and to suitable control cells (for example, transformed with the empty vector alone). In such an experiment, detection of the predicted fatty acid product in cells containing the desaturase-encoding DNA sequence and not in control cells establishes the desaturase activity. Examples of this type of assay have been described in, for example, Lee et al, Science, 280:915-918, 1998; Napier et al, Biochem. J., 330:611-614, 1998; and Michaelson et al, J. Biol. Chem., 273:19055-19059, 1998, incorporated herein by reference.

Δ⁹-desaturase activity may be assayed by these techniques using, for example, 18:0 or 16:0 fatty acids as substrate and detecting 18:1Δ⁹ or 16:1Δ⁹ (respectively) as the product, as described herein. Other potential substrates for use in Δ^I2-activity assays include (but are not limited to) 20:0 (yielding 20: 1Δ⁹ as the product). DNA construct: The term "DNA construct" is intended to indicate any nucleic acid molecule of cDNA, genomic DNA, synthetic DNA, or RNA origin. The term "construct" is intended to indicate a nucleic acid segment that may be single- or double-stranded, and that may be based on a complete or partial naturally occurring nucleotide sequence encoding one or more of the desaturase genes. It is understood that such nucleotide sequences include intentionally manipulated nucleotide sequences, e.g., subjected to site-directed mutagenesis, and sequences that are degenerate as a result of the genetic code. All degenerate nucleotide sequences are included within the scope of the disclosure so long as the desaturase encoded by the nucleotide sequence maintains desaturase activity as described below.

Homologs: "Homologs" are two nucleotide sequences that share a common ancestral sequence and diverged when a species carrying that ancestral sequence split into two species.

Isolated: An "isolated" biological component (such as a nucleic acid or protein or organelle) is a component that has been substantially separated or purified away from other biological components in the cell of the organism in which the component naturally occurs, i.e., other chromosomal and extra-chromosomal DNA, RNA, proteins, and organelles. Nucleic acids and proteins that have been "isolated" include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids and proteins prepared by recombinant expression in a host cell, as well as chemically synthesized nucleic acids.

Mammal: This term includes both humans and non-human mammals. Similarly, the term "patient" includes both humans and veterinary subjects. Oil: An oil is a fatty acid or mixture of fatty acids, generally liquid at about 25° C. A food oil or food oil composition is any edible oil used in the production, preparation or manufacture of food products.

Operably linked: A first nucleic acid sequence is "operably linked" with a second nucleic acid sequence whenever the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein-coding regions, in the same reading frame. ORF (open reading frame): An "ORF" is a series of nucleotide triplets (codons) coding for amino acids without any termination codons. These sequences are usually translatable into respective polypeptides.

Orthologs: An "ortholog" is a gene that encodes a protein exhibiting a function that is similar to a gene derived from a different species.

Palmitate desaturase: A desaturase that displays a strong or exclusive preference for desaturating 16-carbon fatty acids. FAT-5, disclosed herein, is a prototypical palmitate desaturase.

Plant part: Any plant structure, whether or not currently connected to the plant, including, but not limited to seeds, germplasm, roots, leaves, stems, meristem, sex organ tissues (e.g., anthers, carpels, pistils, pollen, ovaries and flowers) and tissue culture of any plant tissue.

Primers: Short nucleic acids, preferably DNA oligonucleotides 10 nucleotides or more in length, that are annealable to a complementary target DNA strand by nucleic acid hybridization to form a hybrid between the primer and the target DNA strand, then extendable along the target DNA strand by a DNA polymerase enzyme. Primer pairs can be used for amplification of a nucleic acid sequence, e.g., by the polymerase chain reaction (PCR) or other nucleic-acid amplification methods known in the art.

Probes and primers as used in the present disclosure typically comprise at least 15 contiguous nucleotides. In order to enhance specificity, longer probes and primers may also be employed, such as probes and primers that comprise at least 20, 30, 40, 50, 60, 70, 80, 90, 100, or 150 consecutive nucleotides of the disclosed nucleic acid sequences.

Alternatively, such probes and primers may comprise at least 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, or 150 consecutive nucleotides that share a defined level of sequence identity with one of the disclosed sequences, for instance, at least a 50%, 60%, 70%, 80%, 90%, or 95% sequence identity.

Alternatively, such probes and primers may be nucleotide molecules that hybridize under specific conditions and remain hybridized under specific wash conditions such as those provided below. These conditions can be used to identifying variants of the desaturases. Nucleic acid molecules that are derived from the desaturase cDNA and gene sequences include molecules that hybridize under various conditions to the disclosed desaturase nucleic acid molecules, or fragments thereof. Generally, hybridization conditions are classified into categories, for example very high stringency, high stringency, and low stringency. The conditions for probes that are about 600 base pairs or more in length are provided below in three corresponding categories.

Very High Stringency (detects sequences that share 90% sequence identity)

High Stringency (detects sequences that share 80% sequence identity or greater)

Low Stringency (detects sequences that share greater than 50% sequence identity)

Methods for preparing and using probes and primers are described in the references, for example, Sambrook et al, 1989; Ausubel et al. (ed.) Current Protocols in Molecular Biology, John Wiley & Sons, New York (with periodic updates), 1998; and Innis et al, PCR Protocols, A Guide to Methods and Applications, Academic Press, Inc., San Diego, California. 1990. PCR primer pairs can be derived from a known sequence, for example, by using computer programs intended for that purpose such as Primer™ (Version 0.5, 1991, Whitehead Institute for Biomedical Research, Cambridge, MA).

Probe: An isolated nucleic acid attached to a detectable label or reporter molecule. Typical labels include radioactive isotopes, ligands, chemiluminescent agents, and enzymes.

Purified: The term "purified" does not require absolute purity; rather, it is intended as a relative term. Thus, for example, a purified enzyme or nucleic acid preparation is one in which the subject protein or nucleotide, respectively, is at a higher concentration than the protein or nucleotide would be in its natural environment within an organism. For example, a preparation of an enzyme can be considered as purified if the enzyme content in the preparation represents at least 50% of the total protein content of the preparation. Recombinant: A "recombinant" nucleic acid is one having a sequence that is not naturally occurring in the organism in which it is expressed, or has a sequence made by an artificial combination of two otherwise-separated, shorter sequences. This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques. "Recombinant" is also used to describe nucleic acid molecules that have been artificially manipulated, but contain the same control sequences and coding regions that are found in the organism from which the gene was isolated.

Sequence identity: The similarity between two nucleic acid sequences or between two amino acid sequences is expressed in terms of the level of sequence identity shared between the sequences. Sequence identity is typically expressed in terms of percentage identity; the higher the percentage, the more similar the two sequences.

Methods for aligning sequences for comparison are well known in the art. Various programs and alignment algorithms are described in the following: Smith & Waterman, Adv. Appl. Math., 2:482, 1981; Needleman & Wunsch, J. Mol. Biol, 48:443, 1970; Pearson & Lipman, Proc. Natl. Acad. Sci. USA, 85:2444, 1988; Higgins & Sharp, Gene, 73:237-244, 1988; Higgins & Sharp, CABIOS, 5:151-153, 1989; Coτpet et al., Nuc. Acids Res., 16:10881-10890, 1988; Huang, et al, Co. App. Bioscl, 8:155-165, 1992; and Pearson et al., Meth. Mol. Bio., 24:307-331, 1994. Altschul et al. (J. Mol. Biol, 215:403-410, 1990) presents a detailed consideration of sequence alignment methods and homology calculations.

The National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST™, Altschul et al, J. Mol. Biol. 215:403-410, 1990) is available from several sources, including the NCBI (Bethesda, MD), for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn, and tblastx. A description of how to determine sequence identity using this program is available at the web site. As used herein, sequence identity is commonly determined with the BLAST™ software set to default parameters. For instance, blastn (version 2.0) software may be used to determine sequence identity between two nucleic acid sequences using default parameters (expect = 10, matrix = BLOSUM62, filter = DUST (Hancock and Armstrong, Comput. Appl. Biosci. 10:67-70, 1994), gap existence cost = 11, per residue gap cost = 1, and lambda ratio = 0.85). For comparison of two polypeptides, blastp (version 2.0) software may be used with default parameters (expect 10, filter = SEG (Wootton and Federhen, Computers in Chemistry 17:149-163, 1993), matrix = BLOSUM62, gap existence cost = 11, per residue gap cost = 1, lambda = 0.85).

When aligning short peptides (fewer than about 30 amino acids), the alignment should be performed using the Blast 2 sequences function, employing the PAM30 matrix set to default parameters: open gap 9, extension gap 1 penalties.

An alternative alignment tool is the ALIGN™ Global Optimal Alignment tool (version 3.2) available from Biology Workbench, San Diego Supercomputer Center, University of California San Diego. This tool may be used with settings set to default parameters to align two known sequences. References for this tool include Meyers and Miller, CABIOS, 4: 11-17, 1989. A first nucleic acid is "substantially similar" to a second nucleic acid if, when optimally aligned (with appropriate nucleotide deletions or gap insertions) with the other nucleic acid (or its complementary strand), there is nucleotide-sequence identity in at least about, for example, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% of the nucleotide bases, depending on the embodiment. Sequence similarity can be determined by comparing the nucleotide sequences of two nucleic acids using the BLAST™ sequence-analysis software (blastn) available from The National Center for Biotechnology Information. Such comparisons may be made using the software set to default settings (expect = 10, filter = default, descriptions = 500 pairwise, alignments = 500, alignment view = standard, gap existence cost = 11, per residue existence = 1, per residue gap cost = 0.85).

Similarly, a first polypeptide is substantially similar to a second polypeptide if they show sequence identity of at least about 75%-90% or greater when optimally aligned and compared using BLAST software (blastp) using default settings.

Specific binding agent: An agent that binds substantially only to a defined target. Thus a FAT-5 protein-specific binding agent binds substantially only the FAT-5 protein. As used herein, the term "FAT-5 protein specific binding agent" includes anti-FAT-5 protein antibodies and other agents (such as soluble receptors) that bind substantially only to the FAT-5 protein.

Anti-FAT-5 protein antibodies may be produced using standard procedures described in a number of texts, including Harlow and Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1988. The determination that a particular agent binds substantially only to the FAT-5 protein may readily be made by using or adapting routine procedures. One suitable in vitro assay makes use of the Western blotting procedure (described in many standard texts, including Harlow and Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1988. Western blotting may be used to determine that a given FAT-5 protein binding agent, such as an anti-FAT-5 protein monoclonal antibody, binds substantially only to the FAT-5 protein.

Shorter fragments of antibodies also can serve as specific binding agents. For instance, FAbs, Fvs, and single-chain Fvs (SCFvs) that bind to FAT-5 would be FAT-5-specific binding agents. These antibody fragments are defined as follows: (1) Fab, the fragment that contains a monovalent antigen-binding fragment of an antibody molecule produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain; (2) Fab', the fragment of an antibody molecule obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain wherein two Fab' fragments are obtained per antibody molecule; (3) (Fab') , the fragment of the antibody obtained by treating whole antibody with the enzyme pepsin without subsequent reduction; (4) F(ab')₂, a dimer of two Fab' fragments held together by two disulfide bonds; (5) Fv, a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains; and (6) single-chain antibody ("SCA"), a genetically engineered molecule containing the variable region of the light chain, the variable region of the heavy chain, linked by a suitable polypeptide linker as a genetically fused single-chain molecule. Methods for making these fragments are routine and known in the art.

Transformed: A "transformed" cell is a cell into which a nucleic acid molecule has been introduced by molecular biology techniques. As used herein, the term "transformation" encompasses all techniques by which a nucleic acid molecule might be introduced into such a cell, including transfection with a viral vector, transformation with a plasmid vector, and introduction of naked DNA by electroporation, lipofection, and particle gun acceleration.

Vector: A "vector" is a nucleic acid molecule as introduced into a host cell, thereby producing a transformed host cell. A vector may include nucleic acid 'sequences, such as an origin of replication, that permit the vector to replicate in a host cell. A vector may also include one or more screenable markers, selectable markers, or reporter genes and other genetic elements known in the art. TJ. Identification of Δ⁹-desaturases from C. elegans

Three C. elegans open reading frames have now been identified in the recently completed C. elegans genomic sequence that display significant similarity to the rat and yeast Δ⁹-desaturases. Using the yeast strain L8-14C, in which the Δ⁹-desaturase gene olel is disrupted, it was found that all three of the C. elegans SCD-like genes rescue the unsaturated fatty acid auxotrophy of this strain when expressed on an episomal plasmid under control of a constitutive promoter. Mono-unsaturated fatty acids with double bonds at C9 are produced by all three strains. Two of the C. elegans Δ⁹- desaturase genes (fat-6 and fat-7) display a substrate preferences similar to that of the rat Δ⁹- desaturase expressed in the olel mutant. Surprisingly, however, the third gene (fat-5) readily desaturates 16:0 and other medium- chain fatty acids, yet converts stearic acid (18:0) to oleic acid (18:1) at a very low frequency, less than 3%. Thus one gene,/ t-5, does not encode a stearoyl CoA desaturase, but rather encodes a palmitate desaturase. This is the first description of a palmitoyl-CoA specific desaturase with very limited activity on stearic acid. The results reported here demonstrate the importance of functional tests of enzyme activity and substrate specificity.

At present the specific roles of each of the three C. elegans Δ⁹-desaturase genes are not known. Not intending to be bound by one theory, we hypothesize that the palmitoyl-CoA desaturase FAT-5 plays a special role in the synthesis of cis-vaccenic acid in the triacylglycerol stores or membrane phospholipids of the nematode. Unlike mammals, C. elegans posses a Δ¹²-desaturase that catalyzes the first step in the conversion of mono-unsaturated fatty acids to polyunsaturated fatty acids (Peyou-Ndi et al., Arch. Biochem. Biophys., 376:399-408, 2000). Since oleic acid (18: 1Δ⁹) is the main precursor for the C. elegans Δ¹²-desaturase, it may be important to accumulate another mono-unsaturated fatty acid that is not a substrate for this enzyme in order to preserve the optimal ratio of saturated mono-unsaturated/ polyunsaturated fatty acids in membranes. The mouse and yeast Δ⁹-desaturase genes are highly regulated at the level of transcription. The mouse genes SCDl and SCD2 are expressed in different tissues and are regulated differently in response to dietary influences and during adipocyte differentiation (Kim and Ntambi, Biochem. Biophys. Res. Comm., 266:1-4, 1999). Regulatory elements required for the repression of SCDl and SCD2 transcription by polyunsaturated fatty acids and sterols have been identified (Waters et al, Biochim. Biophys. Acta, 1349:33-42, 1999; Tabo et al, J. Biol. Chem., 274:20603-20610, 1999). The 60-base-pair polyunsaturated fatty acid (PUFA)-response element is thought to be necessary for regulation by sterol regulatory-element binding proteins (SREBPs) as well as by a PUFA-binding protein (Ntambi, J. LipidRes., 40:1549-1558, 1999). However, the two mouse SCD genes are regulated differently by the antidiabetic thiazolidinediones during pre-adipocyte differentiation (Kim and Ntambi, Biochem. Biophys. Res. Comm., 266:1-4, 1999). SCDl transcription is repressed in response to these drugs, while no detectable change in transcription of SCD2 occurs. Interestingly, comparison of the fatty acid composition of the treated and untreated pre-adipocytes reveals that the thiazolidinediones selectively decrease the desaturation of 16:0 to 16:1, but do not affect the conversion of 18:0 to 18:1, suggesting an important role for the mouse SCDl in Hie desaturation of palmitic acid (16:0). Transcription of the yeast OLE1 gene is activated by stearic acid (18:0) and repressed by unsaturated fatty acids (Bossie and Martin, J. Bacteriology, 171 :6409-13, 1998). Promoter elements required for this activation and repression by fatty acids have been identified (Choi et al, J. Biol. Chem., 271:3581-3589, 1996) but do not show similarity to the mouse PUFA response element. At this time, it has not been possible to identify sequences similar to either the mouse PUFA response element or the yeast fatty acid-regulated region in Has fat-5, fat-6, or fat-7 promoters. It is likely, however, that these genes are regulated by dietary influences and perhaps in a stage-specific or tissue-specific manner.

Materials and Methods

Identification, amplification, and cloning of C. elegans Δ⁹ -desaturase genes.

The Sanger Center C. elegans wormpep database, available on-line at the Sanger website, was searched using BLAST (Altschul et al, J. Mol. Biol, 215:403-410, 1990) with the polypeptide sequence of the rat stearoyl-CoA desaturase (SWISSPROT accession number P07308) (Thiede et al, J. Biol. Chem., 261:13230-13235, 1986). Three open reading frames were identified: W06D12.3, VZK8221.1, and F10D2.9. The predicted coding sequence of each of these genes was amplified using either RT-PCR on total RNA from mixed stage C. elegans, or by PCR amplification from a C. elegans mixed-stage lambda phage Uni-ZAP cDNA library (Stratagene, La Jolla, CA). Total RNA was prepared with TRIZOL reagent (Life Technologies) following the manufacturer's protocol.

Reverse transcription-PCR was performed with the "One-Step" kit (Life Technologies). For all three genes, the upstream primer used to amplify the cDNA sequences added an EcoKl restriction site and the downstream primer added an Xhόl restriction site. After amplification the PCR products were digested with JECORI and Xhόl and cloned directly into the episomal yeast expression vector pMK195 (Overvoorde et al., Plant Cell, 8:271-280, 1996) restricted with the same enzymes. ρMK195 encodes uracil prototrophy and contains a multiple cloning site for directional cloning of cDNAs to be expressed under the control of the constitutive alcohol dehydrogenase promoter.

For amplification of W0D12.3 the upstream primer sequence was: TCTCGGAATTCATGACTCAAATCAAAGTAGATGCG (SEQ ID NO: 1); and the downstream primer sequence was:

CCCGGGCTCGAGTTATCCCAATTTGTGGAGC (SEQ ID NO: 2).

For amplification of VZK8221.1 the upstream primer sequence was: TCTCGGAATTCAAACAGACAGTAAAATGACGG (SEQ ID NO: 6); and the downstream primer sequence was:

CCCGGGCTCGAGCCCGAATGATTCAAAACAGTAC (SEQ ID NO: 7). For amplification of F10D2.9 the upstream primer sequence was: TCTCGAATTCAAACGGTAAAATCACGG (SEQ ID NO: 11); and the downstream primer sequence was:

TCGACCTCGAGTGTGGACAACCAACGCGT (SEQ ID NO: 12).

Engineered restriction sites are underlined in the above sequences. Plasmids were amplified in the E. coli strain DH10B. Transmembrane domains were predicted by TMHMM (version 1.0), available from the Center for Biological Sequence Analysis, Technical University of Denmark.

Functional assay.

The Saccharomyces cerevisiae strain L8-14C (provided by Chuck Martin) contains a disruption of the yeast Δ⁹-desaturase gene OLE1 and requires unsaturated fatty acids for growth (Stukey et al, J. Biol. Chem., 264:16537-16544, 1989). The cells were grown in YPD medium (2% Bacto-peptone, 1% yeast extract, 2% glucose) containing 0.5 mM oleic acid and 0.5 mM palmitoleic acid (NuChek Prep) as well as 1% tergitol, type NP-40 (Sigma) to solubilize the unsaturated fatty acids. The S.c. EasyComp transformation kit (Invitrogen, Carlsbad, CA) was used to transform constructs into strain L8-14C. The transformed cells were plated onto complete minimal medium containing 0.5 mM oleic acid, 0.5 mM palmitoleic acid, and 1% tergitol, but lacking uracil. To test for genetic complementation of the olel phenotype, several URA⁺ transformant colonies from each transformation were patched onto complete YPD plates lacking supplemental unsaturated fatty acids. After incubation at 30° C for 48 hours, all of the colonies that were URA⁺ also grew on unsupplemented yeast media, indicating that expression of the cloned C. elegans desaturases rescued the olel unsaturated fatty acid auxotrophy. The genes encoding the predicted open reading frames were assigned the following names: W06D12.3: fat-5;

YZKS,221.1: fat-6; saά F10D2.9: fat-7.

Fatty acid analysis. Yeast were grown in liquid YPD medium lacking supplemental unsaturated fatty acids for one to two days, and total fatty acids were extracted from pellets composed of 1-2 mL of culture. To prepare fatty acid methyl esters (FAMEs), the pellets were washed one time with water and were resuspended in 1 L of 2.5% sulfuric acid in methanol and heated to 80° C for one hour. When the yeast were grown in the presence of exogenous fatty acids (0.2 mM, 1% tergitol), the yeast pellets were washed one time in 1% tergitol and two times in water before being suspended in 2.5% sulfuric acid in methanol. The resulting FAMEs were extracted in hexane. Fatty acid 4,4-dimethyloxazoline (DMOX) derivatives were prepared from the FAMEs by evaporating the hexane phase, resuspending the residue in 0.5 mL of 2-amino-2-methylpropanol, and heating overnight at 180° C (Fay and Richli, J. Chromato., 541:89-98, 1991). After cooling, the DMOX derivatives were dissolved in 4 mL of dichloromethane and washed twice with 1.5 mL of distilled water. The dichloromethane solution was evaporated under a stream of nitrogen and the residue was dissolved in hexane for injection. Analyses of FAMEs and DMOX derivatives by gas chromatography and mass spectroscopy were conducted using a Hewlett Packard 6890-series GC-MS equipped with a 30 m X 0.25 μm SP-2380 column operating at an ionization voltage of 70 eV with a scan range of 50-550 Daltons. Fatty acids were identified by comparison with retention times and mass spectra of FAME standards (NuChek Prep). Relative percentages of the fatty acids were calculated from peak areas. The mass spectra (m/z, rel. int.) of the DMOX derivatives of the fatty acids were compared to published values (Spitzer, Prog. Lipid Res., 35:387-408, 1997; and Chitwood et al., Lipids, 30:567-573, 1995).

RESULTS

Identification and cloning ofC elegans A⁹ -desaturases.

Three high-scoring open reading frames were identified during a search of the C. elegans genomic DNA database with the rat SCDl protein sequences. Two of the open reading frames

VZK8221.1 (fat-6) and F10D2.9 (fat-7) displayed 49% and 48% identity with the rat SCDl sequence respectively and the third reading frame, W06D12.3 (fat-5) displayed somewhat lower identity, (43%). The structure of all three C. elegans open reading frames and the rat SCDl were similar (Fig.

!)• The open reading frames display a high degree of similarity to the rat gene, especially in the areas near each of the histidine-rich regions that are predicted to be essential for coordinating the iron moieties at the active site in all members of the membrane desaturase superfamily. They also contain four stretches of hydrophobic residues which are predicted to span the membrane twice (amino acids 47-100 and 195-231) (Fig. 1). The genes display a somewhat lower amino acid identity with the yeast Δ⁹-desaturase Olelp. This protein contains an extra 60 amino acids on the N-teiminal and an extra 113 C-terminal amino acid responsible for encoding a cytochrome b⁵ domain. A cytochrome b⁵ domain is not present on the mammalian SCD proteins, nor is it encoded by any of the three C. elegans SCD-like genes.

C. elegans can synthesize a range of fatty acids using only saturated and mono-unsaturated fatty acids obtained from is. coli in its diet, or axenically grown, with no dietary fatty acids (Chitwood et al, Lipids, 30:567-573, 1995). Four other desaturase genes in C. elegans have been isolated and characterized by expression in yeast or arabidopsis. These include the Δ¹²-desarurase (Peyou-Ndi et al, Arch. Biochem. Biophys., 376:399-408, 2000), the Δ³-desaturase (Spychalla et al, Proc. Natl. Acad. Sci. USA, 94:1142-1147, 1997), the Δ⁵-desaturase (Watts and Browse, Arch. Biochem. Biophys., 362:175-182, 1999; and Michaelson et α/., £7iiS Let, 439:215-218, 1998), and the Δ⁶- desaturase (Napier et al, Biochem. J., 330:611-614, 1998). Each desaturase appears to be present in only one copy. It is not clear why there are three Δ⁹-like desaturases in C. elegans. The most abundant fatty acid in C. elegans phospholipids and triacylglycerols is 18: 1Δ¹¹, czs-vaccenic acid (Tanaka et al, Lipids, 31 :1173-1178, 1996). One possibility for producing this rø-vaccenic acid is via a D 11-specific 18:0 desaturase. Such an enzyme has been described in cabbage looper moth (Trichoplusia ni), but this desaturase is expressed only in the adult pheromone gland where it is necessary for producing pheromone precursors (Knipple et al, Proc. Natl. Acad. Sci. USA, 95:15287- 15292, 1998). Currently, it is impossible to predict if one of the C. elegans SCD-like genes encodes a Δ¹ '-desaturase by sequence comparisons alone. The amino acid identities of the C. elegans ORFS compared to the cabbage looper moth Δ⁹-desaturase is not significantly different to the identities as compared to the moth Δ¹ '-desaturase. Alternatively, cz^'s-vaccenic acid might be produced by elongation of 16:1, as in bacteria and yeast (Southwell-Kelly and Lynen, Biochim. Biophys. Acta, 337:22-28, 1974). In order to determine if all three ORFs function as Δ⁹-desaturases or Δ^u- desaturases, we expressed them in a yeast strain in which the Δ⁹-desaturase gene olel has been disrupted.

Functional characterization. The yeast strain L8-14C carries the disruption allele olelA::LEU2 and has limited and finite growth potential (4-5 divisions) unless the growth medium is supplemented with unsaturated fatty acids (Stukey et al, J. Biol. Chem., 264:16537-16544, 1989). The L8-14C strain was transformed with an episomal plasmid containing each of the three C. elegans Δ⁹-desaturase-like ORFs under control of the constitutive alcohol dehydrogenase promoter. Unlike L8-14C, all three of the transformed strains were able to grow on rich medium (YPD) as well as complete minimal medium (- uracil) that were not supplemented with unsaturated fatty acids. Expression of any of the three C. elegans Δ⁹-like desaturase genes therefore conferred upon the yeast the ability to produce unsaturated fatty acids.

In order to determine the identity of unsaturated fatty acids produced by each of the C. elegans enzymes, the fatty acid compositions of the transgenic yeast strains were analyzed. The fatty acid composition of two of the strains, L8-14C//αt-fJ (VZK8221.1) and L8-14C//αt-7 (F10D2.9), were similar (Table 1). Both strains produced 16:1 and 18:1, although the ectopically expressed desaturases preferably desaturated the 18-carbon substrate stearic acid. Yeast expressing αt-ό^' and fat-7 converted 78% and 94% of the stearic acid (18:0) to oleic acid (18:1), and 7% and 23% of the 16:0 to 16:1.

This preference is also apparent in the rat ΔSCD1 (Stukey et al, J. Biol. Chem., 265:20144- 20149, 1990), the cabbage looper moth Δ⁹-desaturase (Liu et al, Insect Biochem. Mol. Biol, 29:435- 443, 1999), and two recently characterized Mortierella alpina Δ⁹-desaturases (Wongwathanarat et al, Microbiology, 145:2939-2946, 1999) when expressed in L8-14C. The cabbage looper moth Δ⁹- desaturase under control of the olel promoter produced six times more 18:1 than 16:1, while the rat gene converted 88% of 18:0 to 18:1 and 47% of 16:0 to 16:1 when expressed in the same strain. The olel gene expressed on an episomal plasmid in the L8-14C strain displayed a higher activity on 16:0, converting 65% of 16:0 to 16:1 and 87% of 18:0 to 18:1 (Stukey et al, J. Biol. Chem., 265:20144- 20149, 1990).

Table 1. Fatty acid composition of L8-14C (olel) yeast transformed with the C. elegans Δ⁹- desaturases FAT-5, FAT-6, and FAT-7 as compared to the rat SCDl and yeast Olelp Δ⁹-desaturases expressed in the same strain.

Fatty Acid Composition¹ (%)

% conversion % conversion

Enzymes 16:0 16:1 18:0 18:1 of 16:0 of 18:0 18:1/16:1 expressed ratio

FAT-5 28.8 + 3.1 42.2 + 3.3 9.7 ± .83 .28 ± .08 59.5 ± 1.6 2.7 ± .6 .007 ± .002

FAT-6 56.2 ± 2.3 4.1 ± .37 6.9 ± .30 23. 8 ± 1.3 6.8 ± .7 77.6 ± 1.2 5.8 ± .39

FAT-7 32.2 ± 9.2 8.7 ± 1.9 3.2 ± .60 48.7 ± 10.8 22.5 ± 8.5 93.6 ± 2.0 5.7 + 1.2

Rat SCDl² 32.0± 1.5 28.1 ± .95 4.7 ± .08 33.9 ± 2.3 46.8 ± .3 87.9 ± .5 1.21 ± .12

Olelp² 21.9 41.7 4.7 29.8 65 87 0.71

'FAT-5, FAT-6, and FAT-7 values represent the relative fatty acid compositions as a percentage of total fatty acid content determined by GC peak area for five independent trials (±SD). Data from Stukey et al, J. Biol. Chem., 26₅:20144-20149, 1990.

To confirm that the desaturations occurred at the Δ -position, DMOX derivatives of yeast fatty acids were analyzed by GC-mass spectrometry. DMOX derivatives have mass spectra that were more easily interpreted than the spectra of methyl esters and permitted unambiguous determination of double-bond locations in polyunsaturated fatty acids (Rezanka, Phytochemistry, 33:1441-1444, 1993; and Spitzer, Prog. LipidRes., 35:387-408, 1997). The mass spectra of the 18:1 molecule produced by olel yeast expressing either fat-6 or fat-7 revealed that the molecule produced is indeed the Δ⁹ isomer, with the spectral peaks, separated by 12 atomic man units, (a.m.u.) occurring at 196 and 208. The third C. elegans Δ⁹-desaturase-like gene, W06D12.3,^άt-5, displayed a novel fatty acid composition in strain L4-14C// t-5. This gene product showed a high activity on 16:0, converting an average of 60% (+/- 1.6%, n=5) of palmitic acid (16:0) to palmitoleic acid (16:1) (Table 1). Analysis of DMOX derivatives confirmed that the double bond of the 16:1 molecule is present at the Δ⁹ position, as revealed by the spectral peaks, separated by 12-a.m.u., at 196 and 208. Only a very small amount of 18: 1 Δ⁹ could be detected in these yeast cells, less than 0.3% of the total yeast fatty acids, which represents less than 3% conversion of stearic acid to oleic acid. A second 18:1 peak was also present at about three times the abundance of the 18:1Δ⁹ peak, and spectral analysis of the DMOX derivative of this fatty acid revealed the 12-a.m.u.-separated peaks at 224 and 236, which represents a double bond at the Δ^u position. This fatty acid, 18: 1Δ^U, is produced naturally in yeast by the elongation of 16: 1Δ⁹. The relative amount of 18-C fatty acids compared to 16-C fatty acids is greatly reduced in the L8-14C/ αt-5 strain, as compared to L8-14C expressing other Δ⁹-desaturases. Whereas 16-C to 18-C fatty acids are generally present in a 3:2 ratio in most strains, in L8-14C/fat-5 they are present in a 6: 1 ratio (Table 1). It appears that the yeast elongation activity is regulated by the amount of 18:0 present in the cells. When the 18:0 is not converted to 18:1, the yeast maintains this amount of 18:0 at 5-10% of the total fatty acids, even though, in the absence of 18:1, the total amount of 18-C fatty is much lower than in wild type. Strain L8-14C supplemented only with 16:1 displays a similar fatty acid composition (Stukey et al, J. Biol. Chem., 264:16537-16544, 1989). A number of medium-chain and long-chain saturated and unsaturated fatty acids were also tested in order to further investigate the substrate specificities of the C. elegans Δ⁹-desaturases (Fig. 2). Since saturated fatty acids 14:0, 15:0, and 17:0 are present in small amounts in S. cerevisiae, we examined whether these fatty acids are desaturated in the transgenic strains carrying the C. elegans Δ⁹-desaturases. In addition, the yeast were propagated in the presence of several other fatty acids by adding 18:1 Δ¹¹ (trans), 18:1 Δ^π (cis) and 20:0 fatty acids to the growth medium in the presence of 1% tergitol.

FAT-5 was capable of desaturating the medium-chain fatty acids 14:0 and 15:0, but showed very little activity on fatty acids longer than 16 carbons. In contrast, FAT-6 and FAT-7 did not desaturate 14:0 or 15:0, but did show considerable activity on 17:0 and 18:1 Δⁿ (trans). Desaturation of 18:1 Δ^u (trans) resulted in the formation of 18:2 Δ⁹ (cis) Δ^π (trans), conjugated linoleic acid (CLA). None of the C. elegans desaturases showed any activity on 18:1 Δ^u (cis) or 20:0.

A Δ⁹-desaturase specific for 16:0 has been isolated from the cat's claw vine Doxantha unguis-cati (Cahoon et al., Plant Physiology, 117:593-598, 1998).

FAT-6 and FAT-7 are capable of desaturating trara-vaccenic acid. This activity has also been observed in rat liver microsomes and in mice (Santora et al, JNutr., 130:208-215, 1999; and Malifouz et al, Biochim. Biophys. Acta, 618:1-12, 1980). Since the trans double bond does not introduce a kink in the hydrocarbon chain in the manner of a cis double bond, presumably the linear frøras-vaccenic acid can fit into the substrate-binding region of SCDs due to the shape similarity to stearic acid (18:0). Desaturation of 18:1 Δ^u (trans) results in the formation of 18:2 Δ⁹ (cis), Δ¹¹ (trans), conjugated linoleic acid (CLA). This fatty acid occurs naturally in food, and was first isolated and identified from grilled ground beef extracts that exhibited anticarcinogenic activity (Ha et al, Carcinogenesis, 8:1881-1887, 1987). The effect of CLA on body fat content, lean body mass, and glucose intolerance suggests that CLA plays a role in lipid and carbohydrate metabolism. Indeed, some isomers of CLA have been shown to modify membrane fatty acid composition by decreasing the activity of stearoyl-CoA desaturase in mice (Lee et al, Biochem. Biophys. Res. Comm., 248:817-821, 1998).

III. EXAMPLES

Example 1 : Δ⁹-Desaturase Proteins and Nucleic acid Sequences

As described above, the disclosure provides, inter alia, desaturases and desaturase-specific nucleic acid sequences. With the provision herein of these desaturase sequences, the amplification methods such as polymerase chain reaction (PCR) may now be utilized as preferred methods for identifying and producing nucleic acid sequences encoding the desaturases. For example, PCR amplification of the desaturase sequences may be accomplished either by direct PCR from a plant cDNA library or by Reverse-Transcription PCR (RT-PCR) using RNA extracted from plant cells as a template. Desaturase sequences may be amplified from plant genomic libraries, or plant genomic DNA. Methods and conditions for both direct PCR and RT-PCR are known in the art and are described in Innis et al, PCR Protocols: A Guide to Methods and Applications, Academic Press: San Diego, 1990. Other examples of amplification include strand-displacement amplification, as disclosed in U.S. Patent No. 5,744,311; transcription-free isothermal amplification, as disclosed in U.S. Patent No. 6,033,881; repair chain reaction amplification, as disclosed in WO 90/01069; ligase chain reaction amplification, as disclosed in EP -A-320 308; gap-filling ligase chain reaction amplification, as disclosed in U.S. Patent No. 5,427,930; and NASBA™ RNA transcription-free amplification, as disclosed in U.S. Patent No. 6,025,134.

The selection of amplification primers is made according to the portions of the cDNA (or gene) that are to be amplified. Primers may be chosen to amplify small segments of the cDNA, the open reading frame, the entire cDNA molecule, or the entire gene sequence. Variations in amplification conditions may be required to accommodate primers of differing lengths; such considerations are well known in the art and are discussed in Innis et al, PCR Protocols: A Guide to Methods and Applications, Academic Press: San Diego, 1990; Sambrook et al. (ed.), Molecular Cloning: A Laboratory Manual 2nd ed., vol. 1-3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989; and Ausubel et al. (ed.) Current Protocols in Molecular Biology, John Wiley & Sons, New York (with periodic updates), 1998. By way of example, the cDNA molecules corresponding to additional desaturases may be amplified using primers directed towards the 5'- and 3'- ends of the prototypical C. elegans fat-5, fat-6, and fat-7 sequences. Example primers for such reactions are shown in SEQ JD NOs: 1, 2, 6, 7, 11 and 12, above. These primers are illustrative only; it will be appreciated that many different primers may be derived from the provided nucleic acid sequences. Re-sequencing of amplification products obtained by any amplification procedure is recommended to facilitate confirmation of the amplified sequence and to provide information on natural variation between desaturase sequences. Oligonucleotides derived from the desaturase sequence may be used in such sequencing methods.

Oligonucleotides that are derived from the desaturase sequences are encompassed within the scope of the present disclosure. In some embodiments, such oligonucleotide primers comprise a sequence of at least 10-20 consecutive nucleotides of the desaturase sequences. To enhance amplification specificity, oligonucleotide primers comprising at least 15, 20, 25, 30, 35, 40, 45, or 50 consecutive nucleotides of these sequences may also be used.

A. Desaturases in Other Species

Orthologs of sequences encoding FAT-5, FAT-6, and FAT-7 are present in a number of other species (e.g., of animals, plants, or microbes) that are able to produce Δ⁹ unsaturated fatty acids. With the provision herein of fke fat-5, fat-6, and fat-1 nucleic acid sequences, the cloning by standard methods of cDNAs and genes that encode Δ⁹-desaturase orthologs in these other species is now enabled. In particular, other palmitate desaturases related to the FAT-5 protein can be isolated. As described above, orthologs of the disclosed Δ⁹-desaturase genes have Δ⁹-desaturase biological activity (or more particularly, palmitate-specifϊc activity in the case of a FAT-5 ortholog) and are typically characterized by possession of at least 60% sequence identity, as counted over the full length alignment with the amino acid sequence of the disclosed Δ⁹-desaturase sequences using the NCBI Blast 2.0 (gapped blastp set to default parameters). Proteins with even greater similarity to the reference sequences will show increasing percentage identities when assessed by this method, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 90%, or at least 95% sequence identity. Both conventional hybridization and amplification (e.g., PCR amplification) procedures may be utilized to clone sequences encoding desaturase orthologs. Common to both of these techniques is the hybridization of probes or primers that are derived from the Δ⁹-desaturase nucleic acid sequences. Furthermore, the hybridization may occur in the context of Northern blots, Southern blots, or PCR. Direct PCR amplification may be performed on cDNA or genomic libraries prepared from any of various plant species, or RT-PCR may be performed using mRNA extracted from plant cells using standard methods. PCR primers will comprise at least 10 consecutive nucleotides of the Δ⁹- desaturase sequences. One of skill in the art will appreciate that sequence differences between the Δ⁹-desaturase nucleic acid sequence and the target nucleic acid to be amplified may result in lower amplification efficiencies. To compensate for this, longer PCR primers or lower annealing temperatures may be used during the amplification cycle. Where lower annealing temperatures are used, sequential rounds of amplification using nested primer pairs may be necessary to enhance specificity.

For conventional hybridization techniques the hybridization probe is preferably conjugated with a detectable label such as a radioactive label, and the probe is preferably at least 10 nucleotides in length. As is well known in the art, increasing the length of hybridization probes tends to give enhanced specificity. The labeled probe derived from the Δ⁹-desaturase nucleic acid sequence may be hybridized to a plant cDNA or genomic library and the hybridization signal detected using methods known in the art. The hybridizing colony or plaque (depending on the type of library used) is then purified and the cloned sequence contained in that colony or plaque is isolated and characterized.

Orthologs of the C. elegans Δ⁹-desaturase alternatively may be obtained by immunoscreening of an expression library. With the provision herein of the disclosed C. elegans Δ⁹- desaturase nucleic acid sequences, the enzymes may be expressed and purified in a heterologous expression system (e.g., E. coli) and used to raise antibodies (monoclonal or polyclonal) specific for Δ -desaturases. Antibodies may also be raised against synthetic peptides derived from the desaturase amino acid sequence presented herein. Methods of raising antibodies are well known in the art and are described generally in Harlow and Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. Such antibodies can then be used to screen an expression cDNA library produced from a plant. This screening will identify the desaturase ortholog. The selected cDNAs can be confirmed by sequencing and enzyme activity assays.

B. Palmitate Δ⁹-Desaturase Variants

With the identification of the C. elegans palmitate-specific desaturase gene fat-5, variants of sequences encoding the FAT-5 protein can now be created.

Variant desaturases include proteins that differ in amino acid sequence from the desaturase sequences disclosed, but that retain desaturase biological activity. Such proteins may be produced by manipulating the nucleotide sequence encoding the desaturase using standard procedures such as site- directed mutagenesis or the polymerase chain reaction. The simplest modifications involve the substitution of one or more amino acids for amino acids having similar biochemical properties, for instance one, two, three, or four substitutions. In other embodiments, a variant protein will contain up to about 5 substitutions, about 10 substitutions, about 15 substitutions, or more. So-called "conservative substitutions" are likely to have minimal impact on the activity of the resultant protein. Table 2 shows amino acids that may be substituted for an original amino acid in a protein, and that are regarded as conservative substitutions.

Table 2

More substantial changes in enzymatic function or other features may be obtained by selecting substitutions that are less conservative than those in Table 2, i.e., selecting residues that differ more significantly in their effect on maintaining: (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation; (b) the charge or hydrophobicity of the molecule at the target site; or (c) the bulk of the side chain. The substitutions that, in general, are expected to produce the greatest changes in protein properties will be those in which: (a) a hydrophilic residue, e.g., seryl or fhreonyl, is substituted for (or by) a hydrophobic residue, e.g., leucyl, isoleucyl, phenylalanyl, valyl, or alanyl; (b) a cysteine or proline is substituted for (or by) any other residue; (c) a residue having an electropositive side chain, e.g., lysyl, arginyl, or histadyl, is substituted for (or by) an electronegative residue, e.g., glutamyl or aspartyl; or (d) a residue having a bulky side chain, e.g., phenylalanine, is substituted for (or by) one not having a side chain, e.g., glycine. The effects of these amino acid substitutions or deletions or additions may be assessed for desaturase derivatives by analyzing the ability of the derivative proteins to catalyze the desaturation of, for instance, 16:0 to 16:1Δ⁹ or (especially in the case of FAT-6 or FAT-7 variants) 18:0 to l8:lΔ⁹.

Variant desaturase cDNA or genes may be produced by standard DNA mutagenesis techniques, for example, M13-primer mutagenesis. Details of these techniques are provided in Sambrook et al. (ed), Molecular Cloning: A Laboratory Manual 2nd ed., vol. 1-3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989, Ch. 15. By the use of such techniques, variants may be created that differ in minor ways from the desaturase cDNA or gene sequences, yet that still encode a protein having desaturase biological activity. DNA molecules and nucleotide sequences that are derivatives of those specifically disclosed herein and that differ from those disclosed by the deletion, addition, or substitution of nucleotides while still encoding a protein having desaturase biological activity are comprehended. In their simplest form, such variants may differ from the disclosed sequences by alteration of the coding region to fit the codon-usage bias of the particular organism into which the molecule is to be introduced.

Alternatively, the coding region may be altered by taking advantage of the degeneracy of the genetic code to alter the coding sequence in such a way that, even though the nucleotide sequence is substantially altered, it nevertheless encodes a protein having an amino acid sequence that is identical or substantially similar to the disclosed desaturase amino acid sequences. For example, the eighth amino acid residue of Hie fat-5 cDNA (SEQ ID NO: 4) is alanine. This is encoded in the open reading frame (ORF) by the nucleotide codon triplet GCG. Because of the degeneracy of the genetic code, three other nucleotide codon triplets — GCA, GCC, and GCT — also code for alanine. Thus, the nucleotide sequence of the ORF can be changed at this position to any of these three codons without affecting the amino acid composition of the encoded protein or the characteristics of the protein. Based upon the degeneracy of the genetic code, variant DNA molecules may be derived from the cDNA and gene sequences disclosed herein using standard DNA mutagenesis techniques as described above, or by synthesis of DNA sequences. Thus, this disclosure also encompasses nucleic acid sequences that encode the desaturase protein but that vary from the disclosed nucleic acid sequences by virtue of the degeneracy of the genetic code.

Variants of the desaturase also may be defined in terms of their sequence identity with the desaturase amino acid and nucleic acid sequences described supra. As described above, Δ⁹- desaturases have Δ⁹-desaturase biological activity and share at least 60%) sequence identity with the disclosed Δ⁹-desaturase sequences. In some embodiments, variants will share at least 70% sequence identity, at least 75%, at least 80%, at least 85%, at least 90%, or 95% or more (such as 96%, 97%, 98%, or even 99% sequence identity). Nucleic acid sequences that encode such proteins may be determined readily by applying the genetic code to the amino acid sequence of the desaturase, and such nucleic acid molecules may be produced readily by assembling oligonucleotides corresponding to portions of the sequence.

As previously mentioned, another method of identifying variants of the desaturase is nucleic acid hybridization. Nucleic acid molecules that are derived from the desaturase cDNA and gene sequences include molecules that hybridize under various conditions to the disclosed C. elegans Δ⁹- desaturase nucleic acid molecules, or fragments thereof. Generally, hybridization conditions are classified into categories, for example very high stringency, high stringency, and low stringency. The conditions for probes that are about 600 base pairs or more in length are provided above. The sequences encoding the desaturase identified through hybridization may be incorporated into transformation vectors and introduced into host cells to produce the respective desaturase.

Example 2 Production of Recombinant Δ⁹-Desaturase(s) in Heterologous Expression Systems

Various yeast strains and yeast-derived vectors are commonly used for the expression of heterologous proteins. For instance, Pichia pastoris expression systems, obtained from Invitrogen (Carlsbad, California), may be used. Such systems include suitable P. pastoris strains, vectors, reagents, transformants, sequencing primers, and media. Available strains include KM71H (a prototrophic strain), SMD1168H (a prototrophic strain), and SMD1168 (&pep4 mutant strain) (Invitrogen Product Catalogue, 1998, Invitrogen, Carlsbad CA).

Non-yeast eukaryotic vectors may be used with equal facility for expression of proteins encoded by modified nucleotides according to the disclosure. Mammalian vector/host cell systems containing genetic and cellular control elements capable of carrying out transcription, translation, and post-translational modification are well known in the art. Examples of such systems are the well- known baculovirus system, the ecdysone-inducible expression system that uses regulatory elements from Drosophila melanogaster to allow control of gene expression, and the sindbis viral-expression system that allows high-level expression in a variety of mammalian cell lines, all of which are available from Invitrogen (Carlsbad, CA).

The cloned expression vector encoding at least one Δ⁹-desaturase may be transformed into any of various cell types for expression of the cloned nucleotide. Many different types of cells may be used to express modified nucleic acid molecules. Examples include cells of yeasts, fungi, insects, mammals, and plants, including transformed and non-transformed cells. For instance, common mammalian cells that could be used include HeLa cells, SW-527 cells (ATCC deposit #7940), WISH cells (ATCC deposit #CCL-25), Daudi cells (ATCC deposit #CCL-213), Mandin-Darby bovine kidney cells (ATCC deposit #CCL-22) and Chinese hamster ovary (CHO) cells (ATCC deposit #CRL-2092). Common yeast cells include Pichia pastoris (ATCC deposit #201178) and Saccharomyces cerevisiae (ATCC deposit #46024). Insect cells include cells from Drosophila melanogaster (ATCC deposit #CRL-10191), the cotton bollworm (ATCC deposit #CRL-9281), and Trichoplusia ni egg cell homoflagellates. Fish cells that may be used include those from rainbow trout (ATCC deposit #CLL-55), salmon (ATCC deposit #CRL-1681), and zebrafish (ATCC deposit #CRL-2147). Amphibian cells that may be used include those of the bullfrog, Rana catesbelana (ATCC deposit #CLL-41). Reptile cells that may be used include those from Russell's viper (ATCC deposit #CCL-140). Plant cells that could be used include Chlamydomonas cells (ATCC deposit #30485), Arabidopsis cells (ATCC deposit #54069) and tomato plant cells (ATCC deposit #54003). Many of these cell types are commonly used and are available from the ATCC as well as from commercial suppliers such as Pharmacia (Uppsala, Sweden), and Invitrogen (Carlsbad, California). Expressed protein may be accumulated within a cell or may be secreted from the cell. Such expressed protein may then be collected and purified. This protein may then be characterized for activity and stability and may be used to practice any of the various methods according to the disclosure.

Example 3 Introduction of Δ -Desaturase into Plants

Using the methods described herein, Δ⁹-desaturases can be cloned and expressed in plants to produce plants with altered (e.g., enhanced) amounts of monounsaturated fatty acids. Such plants provide an inexpensive and convenient source of these important fatty acids in a readily harvestable and edible form.

For instance, the Δ⁹-desaturases could be cloned into a common food crop, such as com, wheat, potato, tomato, yams, apples, pears, or into oil-seed plants such as sunflower, rapeseed, soy, or peanut plants. The resulting plant would express the appropriate enzyme that would catalyze the formation of monounsaturated fatty acids. In the case of an oil-seed plant, the seed oil would be a rich source of Δ⁹-desaturated monounsaturated or polyunsaturated fatty acids.

Standard techniques may be used to express an identified cDNA in transgenic plants in order to modify a particular plant characteristic. The basic approach is to clone the cDNA into a transformation vector such that the cDNA is operably linked to control sequences (e.g., a promoter) directing expression of the cDNA in plant cells. The transformation vector is then introduced into plant cells by any of various techniques (e.g., electroporation, particle bombardment, etc.) and progeny plants containing the introduced cDNA are selected. Preferably all or part of the transformation vector stably integrates into the genome of the plant cell. That part of the transformation vector that integrates into the plant cell and that contains the introduced cDNA and associated sequences for controlling expression (the introduced "transgene") may be referred to as the "recombinant expression cassette".

Selection of progeny plants containing the introduced transgene may be made based upon the detection of an altered phenotype. Such a phenotype may result directly from the cDNA cloned into the transformation vector or may be manifested as enhanced resistance to a chemical agent (such as an antibiotic) as a result of the inclusion of a dominant selectable marker gene incorporated into the transformation vector.

Successful examples of the modification of plant characteristics by transformation with cloned cDNA sequences are replete in the technical and scientific literature. Selected examples, which serve to illustrate the knowledge in this field of technology, include:

U.S. Patent No. 6,051,755 ("Modification of plant lipids and seed oils utilizing yeast SLC genes); U.S. Patent No. 6,051,756 ("Particle bombardment transformation of Brassica"); U.S. Patent No. 5,510,471 ("Chimeric Gene for the Transformation of Plants"); U.S. Patent No. 5,750,386 ("Pathogen-Resistant Transgenic Plants");

U.S. Patent No. 5,597,945 ("Plants Genetically Enhanced for Disease Resistance");

U.S. Patent No. 5,589,615 ("Process for the Production of Transgenic Plants with Increased

Nutritional Value Via the Expression of Modified 2S Storage Albumins"); U.S. Patent No. 5,750,871 ("Transformation and Foreign Gene Expression in Brassica Species"); and

U.S. Patent No. 5,569,831 ("Transgenic Tomato Plants with Altered Polygalacturonase Isoforms").

These examples include descriptions of transformation vector selection, transformation techniques, and the construction of constructs designed to over-express the introduced cDNA. In light of the foregoing and the provision herein of the desaturase amino acid sequences and nucleic acid sequences, it is thus apparent that one of ordinary skill in the art will be able to introduce the cDNAs, or homologous or derivative forms of these molecules, into plants in order to produce plants having enhanced desaturase activity. Furthermore, the expression of one or more desaturases in plants may give rise to plants having altered and/or increased desaturated fatty acid production.

A. Vector Construction, Choice of Promoters

A number of recombinant vectors suitable for stable transfection of plant cells or for the establishment of transgenic plants have been described including those described in Weissbach and Weissbach, Methods for Plant Molecular Biology, Academic Press, 1989; and Gelvin et al, Plant and Molecular Biology Manual, Kluwer Academic Publishers, 1990. Typically, plant-transformation vectors include one or more cloned plant genes (or cDNAs) under the transcriptional control of 5'- and 3 '-regulatory sequences and a dominant selectable marker. Such plant transformation vectors typically also contain a promoter regulatory region (e.g., a regulatory region controlling inducible or constitutive, environmentally or developmentally regulated, or cell- or tissue-specific expression), a transcription-initiation start site, a ribosome-binding site, an RNA-processing signal, a transcription- termination site, and/or a polyadenylation signal. Examples of constitutive plant promoters that may be useful for expressing the cDNA include: the cauliflower mosaic virus (CaMV) 35S promoter, which confers constitutive, high-level expression in most plant tissues (see, e.g. , Odel et al. , Nature 313:810, 1985; Dekeyser et al. , Plant Cell, 2:591, 1990; Terada and Shimamoto, Mol. Gen. Genet, 220:389, 1990; and Benfey and Chua, Science, 250:959-966, 1990); the nopaline synthase promoter (An et al, Plant PhysioL, 88:547, 1988); and the octopine synthase promoter (Fromm et al, Plant Cell, 1 :977, 1989).

Any of a variety of plant-gene promoters that are regulated in response to environmental, hormonal, chemical, and/or developmental signals also can be used for expression of the cDNA in plant cells, including promoters regulated by: (a) heat (Callis et al, Plant PhysioL, 88:965, 1988; Ainley, et al, Plant Mol. Biol, 22:13-23, 1993; and Gilmartin et al, Plant Cell, 4:839-949, 1992); (b) light (e.g., the pea rbcS-3A promoter, Kuhlemeier et al, Plant Cell 1 :471, 1989, and the maize rbcS promoter, Schaffner and Sheen, Plant Cell, 3:997, 1991); (c) hormones, such as abscisic acid (Marcotte et al, Plant Cell 1,:969, 1989); (d) wounding (e.g., wunl, Siebertz et al, Plant Cell, 1:961, 1989); and (e) chemicals such as methyl jasmonate or salicylic acid (Gatz et al, Ann. Rev. Plant PhysioL Plant Mol. Biol, 48:9-108, 1997).

Alternatively, tissue-specific (root, leaf, flower, and seed, for example) promoters (Carpenter et al, Plant Cell, 4:557-571, 1992; Denis et al, Plant PhysioL, 101:1295-1304, 1993; Opperman et al, Science, 263:221-223, 1993; Stockhause et al, Plant Cell, 9:479-489, 1997; Roshal et al, Embo. J., 6: 1155, 1987; Schernthaner et al, Embo J., 1: 1249, 1988; and Bustos et al, Plant Cell, 1:839, 1989) can be fused to the coding sequence to obtain a particular expression in respective organs. Where enhancement of production of desaturated fatty acid is desired in a seed (e.g., an oilseed) of a plant, the use of a seed-specific promoter is beneficial. For example, the napin promoter is an appropriate seed-storage protein promoter from Brassica that allows expression specific to developing seeds. The β-conglycinin promoters also can drive the expression of recombinant nucleic acids, thereby allowing the Δ⁹-desaturases to be expressed only in specific tissues, for example, seed tissues.

Alternatively, the native desaturase gene promoters may be utilized. With the provision herein of the desaturase nucleic acid sequences, one of ordinary skill in the art will appreciate that standard molecular biology techniques can be used to determine the corresponding promoter sequences. It will also be appreciated that less than the entire promoter sequence may be used in order to obtain effective promoter activity. The determination of whether a particular region of this sequence confers effective promoter activity may readily be ascertained by operably linking the selected sequence region to a desaturase cDNA (in conjunction with suitable 3 '-regulatory region, such as the NOS 3 '-regulatory region as discussed below) and determining whether the desaturase is expressed.

Plant-transformation vectors also may include RNA-processing signals, for example, introns, that may be positioned upstream or downstream of the ORF sequence in the transgene. In addition, the expression vectors also may include additional regulatory sequences from the 3'- untranslated region of plant genes, e.g., a 3'-terminator region to increase mRNA stability of the mRNA, such as the PI-II terminator region of potato or the octopine or nopaline synthase (NOS) 3'- terminator regions. The native desaturase gene 3 '-regulatory sequence also may be employed.

Finally, as noted above, plant-transformation vectors also may include dominant selectable marker genes to allow for the ready selection of transformants. Such genes include those encoding antibiotic-resistance genes (e.g., resistance to hygromycin, kanamycin, bleomycin, G418, streptomycin or spectinomycin) and herbicide-resistance genes (e.g., phosplnnothricin acetyltransacylase) .

B. Arrangement of Δ⁹-Desaturase Sequence in a Vector The particular arrangement of the desaturase sequence in the transformation vector is selected according to the type of expression of the sequence that is desired.

Where enhanced desaturase activity is desired, the desaturase ORF may be operably linked to a constitutive high-level promoter such as the CaMV 35S promoter. As noted above, enhanced desaturase activity also may be achieved by introducing into a plant a transformation vector containing a variant form of the desaturase cDNA or gene, for example a form that varies from the exact nucleotide sequence of the desaturase ORF, but that encodes a protein that retains desaturase biological activity.

C. Transformation and Regeneration Techniques

Transformation and regeneration of both monocotyledonous and dicotyledenous plant cells are now routine, and the practitioner can determine the appropriate transformation technique. The choice of method varies with the type of plant to be transformed; those skilled in the art will recognize the suitability of particular methods for given plant types. Suitable methods may include, but are not limited to: electroporation of plant protoplasts; liposome-mediated transformation; polyethylene glycol (PEG)-mediated transformation; transformation using viruses; micro-injection of plant cells; micro-projectile bombardment of plant cells; vacuum infiltration; and Agrobacterium tumefaciens (AT)-mediated transformation. Typical procedures for fransfoirming and regenerating plants are described in the patent documents listed at the beginning of this section.

By way of example only, transformation of Arabidopsis is achieved using, for example, Agrobacterium-mediated vacuum-infiltration process (Katavic et αl., Mol. Gen. Genet, 245:363-70, 1994) or by the floral dip modification of this process (Clough and Bent, Plant J., 16:735-43, 1998).

D. Selection of Transformed Plants

Following transformation and regeneration of plants with the transformation vector, transformed plants can be selected using a dominant selectable marker incorporated into the transformation vector. Typically, such a marker confers antibiotic resistance on the seedlings of transformed plants, and selection of transformants can be accomplished by exposing the seedlings to appropriate concentrations of the antibiotic. After transformed plants are selected and grown to maturity, they can be assayed using the methods described herein to assess production levels of Δ⁹-desaturase protein and the level of Δ⁹- desaturase activity.

Example 4: Creation of Δ⁹-Desaturase-Speciflc Binding Agents

Antibodies to one or more Δ⁹-desaturase enzymes, and fragments thereof, may be useful for purification of the enzymes, as well as for other purposes. The provision of the desaturase sequences allows for the production of specific antibody-based binding agents to these enzymes.

Monoclonal or polyclonal antibodies may be produced to the desaturases, portions of the desaturases, or variants, orthologs or homologs thereof. Optimally, antibodies raised against epitopes on these antigens will specifically detect one enzyme. That is, antibodies raised against the C. elegans FAT-5 Δ⁹-desaturase would recognize and bind the C. elegans FAT-5 Δ⁹-desaturase, and would not substantially recognize or bind to other proteins. The determination that an antibody specifically binds to an antigen is made by any one of a number of standard immunoassay methods; for instance, Western blotting, Sambrook et al. (ed.), Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1-3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.

To determine that a given antibody preparation (such as a preparation produced in a mouse against FAT-5) specifically detects the desaturase by Western blotting, total cellular protein is extracted from cells and electrophoresed on an SDS-polyacrylamide gel. The proteins are then transferred to a membrane (for example, nitrocellulose) by Western blotting, and the antibody preparation is incubated with the membrane. After washing the membrane to remove non- specifically bound antibodies, the presence of specifically bound antibodies is detected by the use of an anti-mouse antibody conjugated to an enzyme such as alkaline phosphatase; application of 5- bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium results in the production of a densely blue-colored compound by immuno-localized alkaline phosphatase.

Antibodies that specifically detect a Δ⁹-desaturase will, by this technique, be shown to bind substantially only the desaturase band (having a position on the gel determined by the molecular weight of the desaturase). Non-specific binding of the antibody to other proteins may occur and may be detectable as a weaker signal on the Western blot (which can be quantified by automated radiography). The non-specific nature of this binding will be recognized by one of ordinary skill in the art by the weak signal obtained on the Western blot relative to the strong primary signal arising from the specific anti-desaturase binding.

Antibodies that specifically bind to desaturases belong to a class of molecules that are referred to herein as "specific binding agents." Specific binding agents that are capable of specifically binding to the desaturase may include polyclonal antibodies, monoclonal antibodies and fragments of monoclonal antibodies such as Fab, F(ab')₂, and Fv fragments, as well as any other agent capable of specifically binding to one or more epitopes on the proteins. Substantially pure Δ⁹-desaturase suitable for use as an immunogen can be isolated from transfected cells, transformed cells, or from wild-type cells. Concentration of protein in the final preparation is adjusted, for example, by concentration on an Amicon (Millipore, Bedford, MA) filter device, to the level of a few micrograms per milliliter. Alternatively, peptide fragments of a desaturase may be utilized as i munogens. Such fragments may be chemically synthesized using standard methods, or may be obtained by cleavage of the whole desaturase enzyme followed by purification of the desired peptide fragments. Peptides as short as three or four amino acids in length are immunogenic when presented to an immune system in the context of a major histocompatibility complex (MHC) molecule, such as MHC class I or MHC class II. Accordingly, peptides comprising at least 3 and preferably at least 4, 5, 6 or more consecutive amino acids of the disclosed desaturase amino acid sequences may be employed as immunogens for producing antibodies.

Because naturally occurring epitopes on proteins frequently comprise amino acid residues that are not adjacently arranged in the peptide when the peptide sequence is viewed as a linear molecule, it may be advantageous to utilize longer peptide fragments from the desaturase amino acid sequences for producing antibodies. Thus, for example, peptides that comprise at least 10, 15, 20, 25, or 30 consecutive amino acid residues of the amino acid sequence may be employed. Monoclonal or polyclonal antibodies to the intact desaturase, or peptide fragments thereof may be prepared as described below.

A. Monoclonal Antibody Production by Hybridoma Fusion

Monoclonal antibodies to any of various epitopes of the desaturase enzymes that are identified and isolated as described herein can be prepared from murine hybridomas according to the classic method of Kohler & Milstern (Nature, 256:495, 1975) or a derivative method thereof. Briefly, a mouse is repetitively inoculated with a few micrograms of the selected protein over a period of a few weeks. The mouse is then sacrificed, and the antibody-producing cells of the spleen isolated. The spleen cells are fused by means of polyethylene glycol with mouse myeloma cells, and the excess unfused cells destroyed by growth of the system on selective media comprising aminopterin (HAT media). The successfully fused cells are diluted and aliquots of the dilution placed in wells of a microtiter plate where growth of the culture is continued. Antibody-producing clones are identified by detection of antibody in the supernatant fluid of the wells by immunoassay procedures, such as ELISA, as originally described by Engvall (Enzymol, 70:419, 1980) or a derivative method thereof. Selected positive clones can be expanded and their monoclonal antibody product harvested for use. Detailed procedures for monoclonal antibody production are described in Harlow & Lane (Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1988). B. Polyclonal Antibody Production by Immunization

Polyclonal antiserum containing antibodies to heterogeneous epitopes of a single protein can be prepared by immunizing suitable animals with the expressed protein, which can be unmodified or modified, to enhance immunogenicity. Effective polyclonal antibody production is affected by many factors related both to the antigen and the host species. For example, small molecules tend to be less immunogenic than other molecules and may require the use of carriers and an adjuvant. Also, host animals vary in response to site of inoculations and dose, with either inadequate or excessive doses of antigen resulting in low-titer antisera. Small doses (ng level) of antigen administered at multiple intraderrnal sites appear to be most reliable. An effective immunization protocol for rabbits can be found in Vaitukaitis et al., J. Clin. Endocrinol. Metab., 33:988-991, 1971.

Booster injections can be given at regular intervals, and antiserum harvested when the antibody titer thereof, as determined semi-quantitatively, for example, by double immunodiffusion in agar against known concentrations of the antigen, begins to fall. See, for example, Ouchterlony et al, Handbook of Experimental Immunology, Wier, D. (ed.), Chapter 19, Blackwell, 1973. A plateau concentration of antibody is usually in the range of 0.1 to 0.2 mg/mL of serum (about 12 μM). Affinity of the antisera for the antigen is determined by preparing competitive binding curves using conventional methods.

C. Antibodies Raised by Injection of cDNA Antibodies may be raised against the desaturases by subcutaneous injection of a DNA vector that expresses the enzymes in laboratory animals, such as mice. Delivery of the recombinant vector into the animals may be achieved using a hand-held form of the Biolistic system (Sanford et al, Particulate Sci. Technol., 5:27-37, 1987, as described by Tang etal., Nature (London), 356:153-154, 1992). Expression vectors suitable for this purpose may include those that express the cDNA of the enzyme under the transcriptional control of either the human β-actin promoter or the cytomegalovirus (CMV) promoter. Methods of administering naked DNA to animals in a manner resulting in expression of the DNA in the body of the animal are well known and are described, for example, in U.S. Patent Nos. 5,620,896 ("DNA Vaccines Against Rotavirus Infections"); 5,643,578 ("Immunization by Inoculation of DNA Transcription Unit"); and 5,593,972 ("Genetic Immunization"), and references cited therein.

D. Antibody Fragments

Antibody fragments may be used in place of whole antibodies and may be readily expressed in prokaryotic host cells. Methods of making and using immunologically effective portions of monoclonal antibodies, also referred to as "antibody fragments," are well known and include those described in Better & Horowitz, Methods EnzymoL, 178:476-496, 1989; Glockshuber et al.

Biochemistry, 29:1362-1367, 1990; and U.S. Patents No. 5,648,237 ("Expression of Functional Antibody Fragments"); No. 4,946,778 ("Single Polypeptide Chain Binding Molecules"); and No. 5,455,030 ("Immunotherapy Using Single Chain Polypeptide Binding Molecules"), and references cited therein.

Example 5: Δ⁹-Desaturase Production in vivo

The creation of recombinant vectors and transgenic organisms expressing the vectors are important for controlling the production of desaturases. These vectors can be used to decrease desaturase production, or to increase desaturase production. A decrease in desaturase production will likely result from the inclusion of an antisense sequence or a catalytic nucleic acid sequence that targets the desaturase-encoding nucleic acid sequence. Conversely, increased production of desaturase can be achieved by including at least one additional desaturase-encoding sequence in the vector. These vectors can then be introduced into a host cell, thereby altering desaturase production. In the case of increased production, the resulting desaturase may be used in in vitro systems, as well as in vivo for increased production of Δ⁹-desaturated fatty acids.

Increased production of Δ⁹-desaturated fatty acids in vivo can be accomplished by transforming a host cell, such as one derived from a plant, specifically an oilseed plant, with a vector containing at least one nucleic acid sequences encoding at least one Δ⁹-desaturase. Furthermore, the heterologous or homologous desaturase sequences can be placed under the control of a constitutive promoter, or an inducible promoter. This will lead to the increased production of Δ⁹-desaturase, thus altering production of desaturated fatty acids, especially altering the Δ⁹-desaturation in such molecules.

Example 6: Expression of Δ⁹-Fatty Acid Desaturase in a Plant to Produce Increased

Desaturation of Fatty Acids in Plant Seeds Plant-transformation constructs

Plant-transformation vectors can be constructed, by standard DNA-cloning techniques, to introduce the fat-5, fat-6, and 'or fat-7 cDNA into plants so that the selected desaturase protein(s) are expressed during seed development.

First, a Δ⁹-desaturase cDNA (e.g., Has fat-5 cDNA, SEQ ID NO: 4) can be engineered so as to be under the control of (functionally linked to) a plant promoter chosen for its activity during

Arabidopsis seed development. For example, the promoter for phaseolin (van der Geest and Hall, Plant Mol. Biol, 32:579-588, 1996) or the promoter for napin (Stalberg et al, Plant Mol. Biol,

23:671-683, 1993) could be used. Promoters cloned specifically for this purpose also could be used.

Appropriate promoters include those found on the genomic BAC clone T24A18 (LOCUS

ATT24A18. 45980 bp Arabidopsis thaliana DNA chromosome 4, ESSA project, ACCESSION NO:

AL035680 NED g4490701, 1999) of the Arabidopsis genome, which regulate seed storage proteins of Arabidopsis, and promoters that express other genes specifically in seeds (Parcy et al, Plant Cell,

6:1567-1582, 1994).

The seed-specific promoter-desaturase construct(s) can then be transferred to one or more standard plant-transformation T-DNA vectors, such as or similar to pART27 (Gleave, Plant Mol.

Biol, 20:1203-1207, 1992), or its derivative pBART (which is identical to pART27 except that the plant selection marker is bar rather than kanamycin; Mick Graham, personal communication), pGPTV (Becker et al, Plant Mol. Biol, 20:1195-1197, 1992), or pJIT119 (Gueτinez et aL, Plant

Mol. Biol, 15:127-136, 1990). Plant-transformation procedures

Constructs produced as described can be used to transform Arabidopsis thaliana by the standard Agrobacterium-mediated vacuum-infiltration process (Katavic et αl, Mol. Gen. Genet, 245:363-370, 1994) or by the floral-dip modification of that process (Clough and Bent, Plant J., 16:735-743, 1998). After transformation, seeds can be harvested from the plants when the plants mature. Transgenic progeny can be identified by selection using the appropriate antibiotic or herbicide. Plants that survive the transgenic selection can be grown to maturity and their seed harvested. Analysis of transgenic plants The seed of plants transformed by the construct containing the Δ⁹-desaturase can be analyzed by preparation of fatty acid methyl esters, followed by gas chromatography to determine their fatty acid composition. Plants expressing the Δ⁹-desaturase will desaturate the 18:0 fatty acid that occurs naturally in the Arabidopsis seed to 18:1 (Δ⁹), or, in the case of the palmitate desaturase FAT-5, will desaturate the 16:0 fatty acid to 16:1 (Δ⁹). At maturity, seed harvested from these transformed plants can be analyzed by gas chromatography. The seeds of the Δ⁹-desaturase- expressing plants will contain increased levels of Δ⁹-unsaturated fatty acids as described herein.

Example 7: Production of Fatty Acids and Oils

With the provision herein of transgenic plants with altered expression of one or more Δ⁹- desaturase(s), the production of oil from these plants is now enabled. Such oil will have a modified fatty acid content compared to oil extracted from a plant of the same species, which is not transgenic for a Δ⁹-desaturase-encoding molecule. In some embodiments, fatty acids (and the oils containing such fatty acids) produced from plants that are transgenic for one or more Δ⁹-desaturases, for instance a plant that expresses a higher than wild-type level of such a desaturase, will be more highly desaturated than fatty acids produced from a corresponding wildtype plant of the same species.

Oil may be extracted from seeds or other plant parts by crushing, for instance by use of a pestle and mortar or a commercial crushing machine. Alternately, oil may also be extracted by the use of a hydrophobic solvent such as hexane. In this method, seed (or other plant parts) may be ground in a tube containing hexane, the solution evaporated to dryness, and ethyl ether and potassium hydroxide solution in methanol added to release fatty acid methyl esters. Samples from this solution may then be analyzed by gas chromatography using a commercial gas chromatograph machine.

Having illustrated and described the principles of the invention in multiple embodiments and examples, it should be apparent to those skilled in the art that the invention can be modified in arrangement and detail without departing from such principles. We claim all modifications coming within the spirit and scope of the following claims.

Claims

We claim:

1. A isolated desaturase protein, comprising an amino acid sequence selected from the group consisting of:

(a) an amino acid sequence as shown in SEQ ID NO: 5, SEQ ID NO: 10, or SEQ ID NO: 15;

(b) an amino acid sequence that differs from that specified in (a) by one or more conservative amino acid substitutions;

(c) an amino acid sequence having at least 60% sequence identity to the sequences specified in (a) or (b); and

(d) fragments of (a), (b), or (c), wherein the isolated protein has Δ⁹-desaturase activity. 2. The isolated desaturase protein of claim 1 , wherein the Δ⁹-desaturase activity is palmitate desaturase activity.

3. An isolated nucleic acid molecule, encoding a protein according to claim 1.

4. The isolated nucleic acid molecule of claim 3, comprising a sequence as shown in SEQ ID NO: 4, SEQ ED NO: 9, or SEQ ID NO: 14. 5. A recombinant nucleic acid molecule, comprising a control sequence operably linked to the nucleic acid molecule of claim 3.

6. A cell, transformed with the recombinant nucleic acid molecule of claim 5.

7. The cell of claim 6, wherein the cell is a plant cell.

8. A transgenic organism, comprising a recombinant nucleic acid molecule according to claim 5, wherein the transgenic organism is selected from the group consisting of plants, bacteria, fungi, and animals.

9. The transgenic organism of claim 8, wherein the organism is a plant.

10. The transgenic plant of claim 9, wherein the plant is selected from the group consisting of oil palm, sunflower, safflower, rapeseed, Canola, soy, peanut, cotton, com, rice, Arabidopsis, mustard, wheat, barley, potato, tomato, yam, apple, and pear plants.

11. A seed of the transgenic plant of claim 9.

12. A part of the transgenic plant of claim 9.

13. An oil extracted from the transgenic plant of claim 9.

14. A food oil composition comprising the oil of claim 13. 15. An isolated nucleic acid molecule that:

(a) hybridizes under high-stringency conditions with a nucleic acid probe, the probe comprising a sequence as shown in SEQ ID NO: 4, SEQ ID NO: 9, or SEQ ID NO: 14, or a fragment thereof; and (b) encodes a protein having desaturase activity.

16. A desaturase protein encoded by the nucleic acid molecule of claim 15.

17. The desaturase protein of claim 16, wherein the desaturase protein is a Δ⁹- desaturase. 18. A recombinant nucleic acid molecule, comprising a promoter sequence operably linked to the nucleic acid molecule of claim 15.

19. A cell transformed with the recombinant nucleic acid molecule of claim 18.

20. The cell of claim 19, wherein the cell is a plant cell.

21. A transgenic organism, comprising the transformed cell of claim 19, wherein the transgenic organism is selected from the group consisting of plants, bacteria, insects, fungi, and mammals.

22. The transgenic organism of claim 21 , wherein the organism is a plant.

23. The transgenic plant of claim 21 , wherein the plant is selected from the group consisting of oil palm, sunflower, safflower, rapeseed, Canola, soy, peanut, cotton, com, rice, Arabidopsis, mustard, wheat, barley, potato, tomato, yam, apple, and pear plants.

24. A seed of the transgenic plant of claim 22.

25. A part of the transgenic plant of claim 22.

26. An oil extracted from the transgenic plant of claim 22.

27. A food oil composition comprising the oil of claim 26. 28. A method for forming a double bond between two carbons in a fatty acid substrate, comprising: contacting the fatty acid substrate with the isolated desaturase protein of claim 1 under conditions that allow the desaturase protein to form a double-bond between two carbons in the fatty acid substrate. 29. The method of claim 28, wherein the fatty acid substrate is palmitoyl-CoA.

30. The method of claim 28, wherein the desaturase protein is FAT-5, or a functional fragment or variant thereof.

31. The method of claim 28, wherein the desaturase protein is expressed in a transgenic organism and the double-bond formation occurs in vivo. 32. The method of claim 31, wherein the desaturase is expressed in an organism selected from the group consisting of eukaryotes and prokaryotes.

34. The method of claim 28, wherein the desaturase is expressed in vitro, and the double-bond formation occurs in vitro.

35. The isolated desaturase protein of claim 1, which comprises the sequence as shown in SEQ ED NO: 5.

36. The isolated desaturase protein of claim 1, which comprises the sequence as shown in SEQ ID NO: 10.

37. The isolated desaturase protein of claim 1, which comprises the sequence as shown in SEQ ID NO: 15.

38. The isolated nucleic acid molecule of claim 15, which comprises the sequence as shown in SEQ ID NO: 4. 39. The isolated nucleic acid molecule of claim 15, which comprises the sequence as shown in SEQ ID NO: 10.

40. The isolated nucleic acid molecule of claim 15, which comprises the sequence as shown in SEQ ED NO: 14.