| PATENT ASSIGNEE'S COUNTRY | Canada |
| UPDATE | 08.01 |
| PATENT NUMBER | This data is not available for free |
| PATENT GRANT DATE | 14.08.01 |
| PATENT TITLE |
Nucleic acids encoding a plant enzyme involved in very long chain fatty acid synthesis |
| PATENT ABSTRACT |
Nucleic acid molecules encoding an enzyme involved in very long chain fatty acid (VLCFA) elongation in plants are disclosed. The invention includes a cDNA, genomic clone and encoded protein, as well as plants having modified VLCFA composition, such as modified epicuticular waxes, and methods of making such plants. |
| PATENT INVENTORS | This data is not available for free |
| PATENT ASSIGNEE | This data is not available for free |
| PATENT FILE DATE | 10.04.98 |
| PATENT REFERENCES CITED |
Benfey et al., Science 250:959-966, Nov. 1990.* Newman et al., "AC T22193," EMBL Database, Jun. 27, 1994. Newman et al., "AC T76616," EMBL Database, Mar. 25, 1995. Sohal & Jenkins, "Epidermal-specific gene expression in Brassica and Arabidopsis," Plant Physiology Supplement, vol. 111, No. 2, p. 6, Jun. 2, 1996. Evenson & Post-Beittenmiller, "Fatty acid-elongation activity in rapidly expanding leek epidermis," Plant Physiology, vol. 109, pp. 707-716, 1995. Millar & Kunst, "Very-long-chain fatty acid biosynthesis is controlled through the expression and specificity of the condensing enzyme" The Plant Journal 12(1):121-131, 1997. Newman et al., "Genes Galore: A Summary of Methods for Accessing Results from Large-Scale Partial Sequencing of Anonymous Arabidopsis cDNA Clones" Plant Physiol., 106:1241-1255, 1994. Katavic et al., "In planta transformation of Arabidopsis thaliana " Mol Gen Genet, 245:363-370, 1994. Lemieux, "Molecular genetics of epicuticular wax biosynthesis" Trends in Plant Science, 1(9):312-318, 1996. Kunst et al., "Fatty acid elongation in developing seeds of Arabidopsis thaliana" Plant Physiol. Biochem., 30:(4) 425-434, 1992. Lassner et al., "A Jojoba .beta.-Ketoacyl-CoA Synthase cDNA Complements the Canola Fatty Acid Elongation Mutation in Transgenic Plants" The Plant Cell, 8:281-292, 1996. James et al., "Directed Tagging of the Arabidopsis Fatty Acid Elongation 1 (FAE1) Gene with the Maize Transposon Activator," The Plant Cell, 7:309-319, 1995. James & Dooner, "Isolation of EMS-induced mutants in Arabidopsis altered in seed fatty acid composition" Theor Appl Genet, 80:241-245, 1990. Lemieux et al., "Mutants of Arabidopsis with alterations in seed lipid fatty acid composition" Theor Appl Genet, 80:234-240, 1990 |
| PATENT PARENT CASE TEXT | This data is not available for free |
| PATENT CLAIMS |
We claim: 1. A recombinant nucleic acid molecule comprising a promoter sequence operably linked to a nucleic acid sequence, wherein the promoter sequence comprises a transcriptional regulatory region capable of mediating gene expression in epidermal cells of Arabidopsis wherein the transcriptional regulatory region hybridezes under stringent conditions to: Seq. I.D. No. 12 or the complement of Seq. I.D. No. 12. 2. A recombinant nucleic acid molecule according to claim 1 wherein the promoter sequence comprises at least 50 consecutive nucleotides of the sequence shown in Seq. I.D. No. 12 or the complement of Seq. I.D. No. 12. 3. The recombinant nucleic acid molecule according to claim 1, wherein the promoter sequence is at least 70% identical to the sequence set forth in Seq. I.D. No. 12. 4. A recombinant nucleic acid molecule according to claim 1, wherein the promoter sequence is at least 80% identical with the sequence set forth in Seq. I.D. No. 12 or the complement of Seq. I.D. No. 12. 5. A recombinant vector comprising a nucleic acid molecule according to claim 1. 6. A transgenic plant comprising a heterologous nucleic acid sequence, wherein the heterologous nucleic acid sequence comprises the recombinant nucleic acid molecule of claim 1. 7. The recombinant nucleic acid molecule according to claim 1, wherein the nucleic acid sequence encodes a protein having very long chain fatty acid elongase activity. 8. A method of producing a transgenic plant comprising introducing into the plant the recombinant nucleic acid molecule of claim 1. 9. A plant produced by sexual or asexual propagation of the transgenic plant produced according to the method of claim 8, or by propagation of progeny of the transgenic plant, wherein the plant comprises the recombinant nucleic acid molecule. 10. A method of isolating a nucleic acid molecule having promoter activity, comprising hybridizing under stringent conditions a nucleic acid preparation with a probe comprising Seq. I.D. No. 12 or the complement of Seq. I.D. No. 12. 11. A plant cell comprising a heterologous nucleic acid sequence, wherein the heterologous nucleic acid sequence comprises the recombinant nucleic acid molecule of claim 1. 12. A recombinant nucleic acid molecule comprising a promoter sequence operably linked to a nucleic acid sequence, wherein the promoter sequence comprises a transcriptional regulatory region capable of mediating gene expression in epidermal cells of Arabidopsis wherein the transcriptional regulatory region is obtainable from a plant VLCFA condensing enzyme gene comprising an open reading frame that hybridizes under stringent conditions to Seq. I.D. No. 3 or to the complement of Seq. I.D. No. 3. |
| PATENT DESCRIPTION |
TECHNICAL FIELD This invention relates to DNA molecules cloned from plants and methods of using such DNA molecules to produce transgenic plants with altered fatty acid composition. BACKGROUND Epicuticular waxes form the outermost layer of the aerial portion of the plant and are thus the first line of interaction between the plant and its environment. The physical properties of this wax layer protect the plant from numerous environmental stresses. For example, the hydrophobic nature of wax prevents dehydration (nonstomatal water loss) and aids in shedding rainwater. The reflective nature of wax protects the plant against UV radiation (Reicosky and Hanover, 1978). Waxes are also known to protect against acid rain (Percy and Baker, 1990) and, because they are a good solvent for organic pollutants, they are able to impede the uptake of aqueous foliar sprays (Schreiber and Schonherr, 1992). Furthermore, surface waxes protect plants from bacterial and fungal (Jenks et al., 1994) pathogens ad play a role in plant-insect interactions (Eigenbrode and Espelie, 1995). Recently it has been shown that some of the compounds found in epicuticular waxes are also present in the tryphine layer of pollen grains (Preuss et al., 1993). Without these compounds the tryphine layer erodes, resulting in pollen that is unable to function causing male sterility. Epicuticular waxes are composed of long chain, hydrophobic compounds all derived from saturated very long chain fatty acids (VLCFAs), that are synthesized within and then secreted from the epidermis. VLCFAs are defined as those fatty acids whose chain length is 20 or more carbons long. The lengths will vary from plant to plant, but typically, the wax VLCFAs are approximately 26-34 carbon long. These VLCFAs are synthesized by a microsomal fatty acid elongation (FAE) system by sequential additions of C2 moieties from malonyl-coenzyme A (CoA) to pre-existing fatty acids derived from the de novo fatty acid synthesis (FAS) pathway of the plastid. Analogous to de novo FAS it is thought that each cycle of FAE involves four enzymatic reactions; (1) condensation of malonyl-CoA with a log chain acyl-CoA, (2) reduction to .beta.-hydroxyacyl-CoA, (3) dehydration to an enoyl-CoA and (4) reduction of the enoyl-CoA, resulting in the elongated acyl-CoA (Fehling and Mukherjee, 1991). Together these four activities are termed the elongase (von Wettstein-Knowles, 1982). VLCFAs in the epidermis are then converted to the other wax components through a number of pathways consisting of multienzyme complexes. For example VLCFAs are converted to aldehydes by fatty acyl-CoA reductase (Kolattukudy, 1971). These aldehydes can either be reduced by aldehyde reductase to produce primary alcohols (Kolattukudy, 1971), or decarbonylated by an aldehyde decarbonylase to produce odd chained alkanes (Cheesbrough and Kolattukudy, 1984). Alkanes can then undergo oxidation to form firstly secondary alcohols and then ketones (for review see Post-Beittenmiller, 1996). Very little is known at the molecular level about the components that are involved in the biosynthesis of wax specific compounds and their secretion onto the plant surface. Genetic studies have shown that there are a large number of genes involved in these processes (for example, 22 loci have been reported in Arabidopsis, 84 in barley). However only a few of these genes have been isolated so far and the biochemical role of their gene products remains unknown (Lemieux, 1996). In addition to being made in the epidermal cells, VLCFAs also accumulate in the seed oil of some plant species. To date, developing seeds have been the primary focus of research into VLCFA biosynthesis. In seeds VLCFAs are incorporated into triacylglyerols (TAGs), as in the Brassicaceae, or into wax esters, as in Jojoba. The seed VLCFAs include the agronomically important erucic acid (C22:1), with oils containing this fatty acid used in the manufacture of lubricants, nylon, cosmetics, pharmaceuticals and plasticisers (Battey et al., 1989); Johnston and Fritz, 1989). Conversely, VLCFAs have detrimental nutritional effects and are therefore undesirable in edible oils. This has led to the breeding of Canola rapeseed varieties that are almost devoid of VLCFAs (Stefansson et al., 1961). The seeds of Arabidopsis contain approximately 28% [w/wt of total fatty acids (FA)] of VLCFAs, eicosenoic acid (20:1) being the predominant VLCFA (21% of wt/wt of total FA). To identify the gene products that are involved in the synthesis of seed VLCFAs and establish the VLCFA biosynthetic pathway, several groups performed mutational analysis and screened for seed that had reduced VLCFA content. Each group independently identified the FATTY ACID ELONGATION1 gene (FAE1; James and Dooner, 1990; Kunst et al., 1992; Lemieux et al., 1990). A mutation at this locus resulted in reduced VLCFA levels (<1% wt/wt of total FA) in the seed. Several other mutations that were non-allelic to FAE1 were also isolated. However, these mutations had a less pronounced effect in that VLCFAs still constituted 6.7% (wt/wt of total FA) of the seed fatty acid (Katavic et al., 1995; Kunst et al., 1992). Thus, despite the fact that four enzymatic activities are required for each elongation step, the FAE1 gene was the only one found by mutant analysis that resulted in almost complete loss of VLCFA synthesis in the seed. The Arabidopsis FAEI gene was subsequently cloned (James et al., 1995; WO 96/13582), and showed homology to three condensing enzymes: chalcone synthase, stilbene synthase and .beta.-ketoacyl-[acyl carrier protein] synthase III (17 amino acids were identical to a 50 amino acid region of a consensus sequence for condensing enzymes). Based on this homology it was proposed that FAE1 encodes a .beta.-ketoacyl-coenzyme A synthase (KCS), the condensing enzyme which catalyzes the first reaction of the microsomal fatty acid elongation system (James et al., 1995). As determined by Northern analysis, the FAE1 gene is expressed in seeds of Arabidopsis, but is absent from leaves (James et al., 1995). This result is consistent with the fact that the faeI mutation affects only the fatty acid composition of the developing seed, having no pleiotropic effects on fatty acid composition of the vegetative, or floral parts of the plant. Thus, FAE1 is regarded as a seed-specific condensing enzyme. Recently a cDNA from Jojoba seeds involved in the syntheses of VLCFAs has been isolated (Lassner et al., 1996; WO 95/15387). The protein encoded by this cDNA showed high homology to FAE1 (52% amino acid identity), and biochemical analysis demonstrated that it has a KCS activity. Using Jojoba KCS cDNA, Lassner et al. (1996) were able to complement the mutation in a Canola variety of Brassica napus, restoring a low erucic acid rapeseed line to a line that contained higher levels of VLCFAs. This suggests that in Canola, the mutation is in the structural gene encoding KCS, or a gene affecting KCS activity. Thus, both in Arabidopsis and Brassica napus, the mutations that result in the abolition of VLCFA synthesis seem to affect the condensing enzyme. If four enzyme activities are necessary for an elongation step, and FAE1 and Jojoba-KCS only encode the KCS activity, one might expect to find other complementation groups that result in very low levels of VLCFAs synthesis. Because these complementation groups were not found in mutation screenings, Millar and Kunst (1997) have hypothesized that these three activities are not seed specific, but ubiquitously present throughout the plant and shared with other FAE systems involved in VLCFA formation including wax biosynthesis. To test this FAE1 was ecotopically expressed in yeast and in tissues of Arabidopsis and tobacco, where significant quantities of VLCFAs are not found. Expression of FAE1 alone in these cells resulted in the biosynthesis and accumulation of VLCFAs. This demonstrated that the condensing enzyme is the pivotal control point of the elongase, controlling not only the amounts of VLCFAs produced, but also their chain lengths. In contrast, it appears that the other three enzyme activities of the elongase are found ubiquitously throughout the plant, are not rate limiting and play no role in the control of VLCFA synthesis. The ability of yeast containing FAE1 to synthesize VLCFAs suggests that the expression, and the acyl chain length specificity of the condensing enzyme, along with the apparent broad specificities of the other three FAE activities, may be universal eukaryotic mechanism for regulating the amounts and acyl chain length of VLCFAs synthesized in any given cell (Millar and Kunst, 1997). Thus, considering the central role of the condensing enzyme for VLCFA synthesis, the isolation of genes encoding condensing enzymes involved in the production of wax specific VLCFAs would facilitate the modification of wax composition through genetic engineering. Furthermore, since the majority of wax components are derived from VLCFAs, the availability of such genes would offer the potential to modify the wax load itself. This offers the potential to modify the susceptibility of plants to environmental stresses such as ultraviolet light, heat and drought, as well as the ability of plants to withstand insects and pathogens. The present invention is directed towards nucleic acids that encode condensing enzymes for VLCFA synthesis. SUMMARY OF THE INVENTION The present invention provides nucleic acids (cDNAs and genomic clones) that encode a key enzyme in the synthesis of VLCFAs in plant epidermal cells. The activity of this enzyme is referred to as very long chain fatty acid elongase; the activity is required for synthesis of VLCFAs of greater than 24 carbons in length. It is shown that co-suppression of the CUT1 gene in plants can disrupt VLCFA synthesis which results in plants having none of the protective wax usually found on stem surfaces. In addition, it is shown that such plants are conditionally male sterile: when grown under normal humidity, the plants are male sterile, but fertility can be restored by growth in an elevated humidity environment. The invention thus provides the CUT1 cDNA and gene nucleotide sequences ("CUT1 nucleic acids") and the amino acid sequence of the CUT1 protein. In one embodiment, the CUT1 nucleic acids disclosed are from Arabidopsis thaliana. The open reading frame of the Arabidopsis CUT1 cDNA molecule encodes an enzyme of 497 amino acids which catalyzes the addition of 2C units to preexisting C24 or longer fatty acids. Also encompassed within the scope of this invention are transformation vectors that include at least a portion of the CUT1 nucleic acid molecules. Such vectors may be transformed into plants to produce transgenic plants with modified VLCFA compositions (relative to non-transgenic plants of the same species). Depending on the particular sequences incorporated into the vector, transformation with the CUT1 cDNA, gene or derivatives thereof can be used to modify agronomically important traits, including the presence, composition and thickness of epicuticular wax layers on leaves and stems, seed coat fatty acids, seed oil composition and male sterility. Typically, such vectors include regulatory sequences, such as promoters, operably linked to the CUT1 open reading frame or a derivative of the CUT1 nucleic acids. For example, VLCFA synthesis may be altered by introducing into a plant a transformation vector that includes a sense or antisense version of the CUT1 cDNA. Transgenic plants having modified VLCFA compositions and which are transformed with such recombinant transformation vectors are also provided by this invention. In one aspect of the invention, transformation with sense or antisense versions of the CUT1 nucleic acids may be used to produce plants having modified epicuticular wax layers on the aerial parts of the plants, such as the leaves and stems. A modified epicuticular wax layer may be modified in physical respects, such as thickness of the wax layer, or in composition. Because these layers play a role in the ability of plants to resist environmental stresses, such as drought and ultraviolet light, as well as insects and pathogens, transformation with vectors including forms of the CUT1 nucleic acids may be used to produce plants with particular agronomic advantages. Producing plants with modified epicuticular wax composition may be achieved by introducing into the plants a vector in which the CUT1 nucleic acid (or a derivative thereof) is operably linked to a promoter that directs expression of the open reading frame in the epidermal cells. The CaMV 35S promoter and the endogenous CUT1 gene promoter are examples of regulatory sequences that may be suitable for this purpose. Agronomically important traits in addition to wax composition may also be modified using the CUT1 nucleic acids of the present invention. For example, the fatty acid composition of the seed coat and the fatty acid composition of seed oil may be modified by transforming plants with the CUT1 cDNA or derivatives thereof. Preferably, where it is desired to modify aspects of seed VLCFA composition, the introduced CUT1 nucleic acid sequence will be operably linked to a promoter known to direct expression in seed tissues. Seed-specific promoters include the napin promoter of Brassica napus (Lee et al., 1991). In addition, transformation with the CUT1 nucleic acids or derivatives thereof may be used to disrupt VLCFA synthesis in pollen, resulting in conditionally male sterile plants. Such plants are useful in plant breeding programs. While the invention provides CUT1-encoding nucleic acids from Arabidopsis, it additionally encompasses homologs, orthologs and variants and derivatives of these sequences, as well as homologs, orthologs and variants of the CUT1 polypeptide sequence. Thus, in one aspect of the invention, nucleic acid molecules that comprise specified regions of these sequences are provided. Exemplary of such nucleic acid molecules are oligonucleotides that are useful as probes or primers to detect and amplify CUT1-encoding nucleic acids from other plant species. Such oligonucleotides are useful as hybridization probes or PCR primers, and typically comprise at least 15 consecutive bases of the disclosed CUT1 nucleic acid sequences. In other embodiments, such oligonucleotides comprise longer regions of the disclosed CUT1 sequences, such as at least 20, 25 or 30 consecutive nucleotides. In another aspect, the invention provides compositions and methods for isolating nucleic acid sequences that encode enzymes having CUT1 activity from other plant species. Typically, such methods involve hybridizing probes or primers derived from the disclosed Arabidopsis sequences to nucleic acids obtained or derived from such other plant species. Homologous and orthologous sequences to Arabidopsis CUT1 nucleic acid and CUT1 amino acid sequences share key functional and structural characteristics with the disclosed Arabidopsis sequences. Functionally, such sequences encode (or comprise) a polypeptide that catalyzes the very long chain fatty acid elongation as described above. Structurally, such sequences share a specified structural relationship with the disclosed sequences. By way of example, in certain embodiments, homologous amino acid sequences have at least 70% sequence identity with the Arabidopsis CUT1 amino acid sequence. In other embodiments, homologous nucleic acid sequences hybridize under stringent conditions to the disclosed Arabidopsis CUT1 nucleic acid sequences. Another aspect of the invention relates to the purified CUT1 enzyme itself. Having provided nucleic acid molecules that encode this enzyme, the invention also facilitates the expression of CUT1 enzyme in heterologous systems, including E. coli, yeast and baculovirus expression systems. Thus, the invention permits the large scale production of the enzyme for agricultural and other applications. In another aspect of the invention the promoter sequence of the CUT1 gene is disclosed. This promoter sequence confers epidermis-specific expression, and may be used to express a variety of nucleic acids in an epidermis-specific manner. |
| PATENT EXAMPLES | This data is not available for free |
| PATENT PHOTOCOPY | Available on request |
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