Patent Publication Number: US-2004049806-A1

Title: Nucleic acid encoding a plant very long chain fatty acid biosynthetic enzyme

Description:
[0001] This application claims priority from U.S. Provisional Patent Application No. 60/206,789, which was filed May 24, 2000. 
    
    
     
       FIELD OF THE INVENTION  
       [0002] This invention relates to the isolation of a genomic DNA sequence encoding a condensing enzyme involved in very long chain fatty acid production in plants and its uses.  
       BACKGROUND  
       [0003] Living organisms synthesize a vast array of different fatty acids which are incorporated into complex lipids. These complex lipids represent both major structural component membranes, and are a major storage product in both plants and animals. Very long chain fatty acids (VLCFAs, chain length C20 or longer) are synthesized in the epidermal cells where they are either directly incorporated into waxes, or serve as precursors for other aliphatic hydrocarbons found in waxes, including alkanes, primary and secondary alcohols, ketones aldehydes and acyl-esters. VLCFAs also accumulate in the seed oil of some plant species, where they are incorporated into triacylglycerols (TAGs), as in the Brassicaceae, or into wax esters, as in jojoba. These seed VLCFAs include the agronomically important erucic acid (C22:1), used in the production of lubricants, nylon, cosmetics, pharmaceuticals and plasticizers.  
       [0004] VLCFAs are synthesized by a microsomal fatty acid elongation (FAE) system which involves four enzymatic reactions: (1) condensation of malonyl-CoA with a long chain acyl-CoA, (2) reduction to -hydroxyacyl-CoA, (3) dehydration to an enoyl-CoA and (4) reduction of the enoyl-CoA, resulting in the elongated acyl-CoA by two carbons. The condensing enzyme catalyzing reaction (1) is the key activity of the FAE system. It is the rate-limiting enzyme of the VLCFA biosynthetic pathway, which controls the amount of VLCFAs produced. In addition, the condensing enzyme determines the ultimate VLCFA acyl chain length, and thus their use.  
       SUMMARY OF THE INVENTION  
       [0005] The present invention consists of a DNA sequence encoding a condensing enzyme involved in VLCFA biosynthesis. Such a DNA fragment is desirable for use in genetic engineering projects aimed at increasing the chain length of fatty acids in seed oils. In addition, expression of this sequence in the epidermis can be used for altering the composition and accumulation of cuticular and epicuticular waxes. 
     
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
     [0006]FIG. 1 shows DNA sequence of the LfKCS3 genomic clone. The deduced amino acid sequence in shown below the nucleotide sequence of corresponding exons. Intron sequences are shown in bold and italics.  
     [0007]FIG. 2 shows sequence similarity among the Brassicaceae condensing enzymes along their entire length (FIG. 2).  
    
    
     DETAILED DESCRIPTION  
     [0008] The present invention provides an isolated genonic DNA sequence encoding a condensing enzyme involved in very long chain fatty acid production in plants. Because condensing enzymes are pivotal enzymes in the synthesis of very long chain fatty acids (VLCFA), controlling levels of accumulation of VLCFAs and their acyl chain length (Millar and Kunst, 1997), are useful for biotechnology. For instance, the accumulation of VLCFAs in tobacco seed expressing FAE1 from Arabidopsis (Millar and Kunst, 1997) indicates that VLCFAs can be produced in plant species that currently do not synthesize VLCFAs. The availability of LfKCS3 condensing enzyme may be especially useful, because it is capable of efficiently elongating hydroxy fatty acids. Thus, the expression of the LfKCS3 condensing enzyme in seeds should allow the production of crop plants capable of synthesizing hydroxylated VLCFAs in seed oil for industrial applications.  
     [0009] The methods employed in the isolation of the nucleic acid sequence of the present invention and the uses thereof are discussed in the following non-limiting examples:  
     [0010] Screening of the Genomic DNA Library:  
     [0011] A  Lesquerella fendleri  genomic DNA library was obtained from Dr. Chris Somerville of the Carnegie Institution of Washington, Stanford, Calif. The genomic library was plated on  E. coli  LE392 (Promega) and about 150,000 clones were screened using Arabidopsis FAE1 as a probe. The probe was prepared by PCR using pGEM7-FAE1 (Millar and Kunst, 1997) as a template with FAE1 upstream primer, 5′-CCGAGCTCAAAGAGGATACATAC-3′ and FAE1 downstream primer, 5′-GATACTCGAGAACGTTGGCACTCAGATAC-3′. PCR was performed in a 10 μl reaction containing 10 ng of the template, 2 mM MgCl 2 , 1.1 μM of each primer, 100 μM of (dCTP+dGTP+dTTP) mix, 50 μCi of [α-32P]dATP, 1×PCR buffer and 2.5 units of Taq DNA polymerase (Life Technologies). Amplification conditions were: 2 min of initial denaturation at 94° C., 30 cycles of 94° C. for 15 sec, 55° C. for 30 sec, 72° C. for 1 min and 40 sec, followed by a final extension at 72° C. for 7 min. The amplified hot probe was purified by QIAqiuck PCR Purification Kit (Qiagen) and denatured by boiling before adding to the hybridization solution. Hybridization took place overnight at 65° C. in a solution containing 6×SSC (1×SSC=0.15 M NaCl, 0.015 M Na-citrate, pH 7.0), 20 mM NaH 2 PO 4 . 0.4% SDS, 5×Denhardt&#39;s solution [0.1% (w/v) of Ficoll (Type 400, Pharmacia), 0.1% (w/v) of polyvinylpyrrolidone, and 0.1% (w/v) of bovine serum albumin (Fraction V, Sigma)], and 50 μg/ml sonicated, denatured salmon sperm DNA (Sigma) and washing was performed three times for 20 min each in 2×SSC, 0.5% (w/v) SDS at 65° C.  
     [0012] Construction of Plasmids (Refer to Table 1):  
     [0013] From tertiary screening, nine positive clones were purified from the  Lesquerella fendleri  genomic library. The phage DNA from those nine clones was extracted and purified using QIAGEN Lambda Mini Kit (Qiagen) according to the manufacturer&#39;s protocol. One of them was digested with EcoRI and a 4.3 kb fragment was subcloned into the pGEM-7Zf(+) vector (Promega) cut with EcoRI, resulting in the vector pMHS15. The whole insert was sequenced with ABI automatic 373 DNA sequencer using fluorescent dye terminators. The upstream region of the genomic DNA was amplified using the high fidelity Pfu polymerase (Stratagene) with a forward primer 5′-CGCAAGCTTGAATTCGGAAATGGGCCAAGT3′ and a reverse primer 5′-CGCGTCGACTGTTTTGAGTTTGTGTCGGG-3′. The amplified 573 bp promoter was inserted upstream of the GUS gene in pBI101 (Clontech) cut with HindIII and SalI, resulting in the vector pLfKCS3-GUS. The fragment containing the promoter and the coding sequence was removed from pMHS15 by digestion with EcoRI and HpaI and the insert fragment was ligated to pRD400 cut with EcoRI and SmaI, resulting in the vector pLfKCS3. As a comparison, another construct was made with LFAH12 promoter (Broun et al., 1998) and the coding sequence, which was named pLFAH12-LfKCS3.  
               TABLE 1                          Plasmids                         Name   Promoter   Coding sequence(s)                             pMHS15   4313 bp EcoRI fragment in pGEM-7Zf(+)                         pLfKCS3-GUS   LfKCS3 (573 bp)   β-glucuronidase       pLfKCS3   LfKCS3 (573 bp)   LfKCS3 ORF (2062 bp)       P1FAH12-LfKCS3   LFAH12 (2200 bp)   LfKCS3 ORF (2062 bp)                  
 
     [0014] All the plasmids shown in Table 1, pLfKCS3-GUS, pLfKCS3, and pLFAH12-LfKCS3, were introduced into  Agrobacterium tumefaciens  strain GV3101 (pMP90; Koncz and Schell, 1986) by heat-shock and selected for resistance to kanamycin (50 μg/mL). They were then used to tansform Arabidopsis (Columbia wild type) and/or fad2/fae1 double mutant by floral dip method (Clough and Bent, 1998). The fad2/fae1 double mutant is characterized by a very high level (&gt;80%) of oleic acid (18:1) in its seed oil due to deficiency in the activities of both cytoplasmic oleate Δ12 desaturase and the condensing enzyme, FAE1. Screening for transformed seed was done on 50 μg/mL kanamycin as described previously (Katavic et al., 1994).  
     [0015] Analysis of Fatty Acid Composition:  
     [0016] To determine the fatty acid composition of the tissues, fatty acid methyl esters were prepared by refluxing the samples in 2 ml of 1N methanolic-HCl for 90 min at 80° C. After cooling 2 ml of 0.9% NaCl solution and 200 μl of hexane were added and the mixture was vortexed vigorously. The fatty acid methyl esters in the hexane layer were analyzed by gas chromatography.  
     [0017] GUS Assay:  
     [0018] GUS assay was performed by immersing tissues in GUS histochemical staining solution (Jefferson, 1989) for 4 to 7 hours at 37° C. The assay solution was composed of 50 mM sodium phosphate, pH 7.0, 0.5 mM potassium ferricyanide, 0.5 mM potassium ferrocyanide, 10 mM EDTA, 0.05%(w/v) triton X-100, and 0.35 mg/ml 5-bromo-4chloro-3-indolyl-β-D-glucuronide (X-Gluc). Following staining the blue-stained samples were fixed in 70% ethanol.  
     [0019] Isolation and Characteristics of the LfKCS3 Gene:  
     [0020] A genomic clone of a putative condensing enzyme was isolated using the Arabidopsis FAE1 (James et al., 1995) to probe filters of a genomic library of  Lesquerella fendleri . The EcoRI fragment subcloned into the plasmid pMHS15 was fully sequenced and a 4313 bp consensus sequence was assembled from individual sequence fragments using GCG program (Edelman et al., 1994). The sequence included 573 bp of 5′ flaking region, a 2062 bp coding region, and an 1678 bp 3′ flanking sequence (FIG. 1). A sequence comparison between the 4313 bp genomic DNA and the Arabidopsis cDNA made using the BCM Search Launcher: Multiple Sequence Alignments (Smith et al., 1996) revealed two introns in the  L. fendleri  genomic DNA. Then the deduced amino acid sequence was obtained after removing the two introns by aid of a translation tool, “Translate” (http://www.expasy.ch/tools/dna.html). The amino acid sequence indicates that the gene, designated LfKCS3, encodes a polypeptide of 496 amino acids, and an estimated molecular mass of the LfKCS3 protein is 55.3 kD. Amino acid sequence comparisons, using the BCM Search Launcher: Pairwise Sequence Alignments (Smith et al., 1996), show that LfKCS3 shares high sequence identity, ranging from 49.9% to 80.2%, with VLCFA condensing enzymes, including Arabidopsis FAE1 (James et al., 1995),  Brassica napus  KCS (Roscoe et al., 1996: GenBank accession number U50771), CUT1 (Miller et al.; 1999), and jojoba KCS (Lassner et al., 1996). Multiple sequence alignments reveal remarkable similarity among the Brassicaceae condensing enzymes along their entire length (FIG. 2).  
     [0021] Expression Studies of LfKCS3 in Plants:  
     [0022] In order to determine the LfKCS3 expression pattern a 573 bp upstream fragment from the LfKCS3 genomic clone was translationally fused to the uidA reporter gene encoding β-glucuronidase (GUS), and introduced into Arabidopsis by floral dip method (Clough and Bent, 1998). LfKCS3 expression pattern was determined for more than 30 independent primary transgenic plants using GUS histochemical assays on leaves, stems, inflorescences, roots, and siliques at different stages of development. GUS staining was observed exclusively in the embryos. No GUS expression was detected in other tissues. Thus, the Arabidopsis LfKCS3 promoter is regulated in a tissue specific manner.  
     References  
     [0023] Clough, S. J. and Bent, A. F. (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of  Arabdiopsis thaliana . Plant J. 16, 735-743.  
     [0024] Broun, P., Boddupanli, S., and Somerville, C. (1998) A bifunctional oleate 12-hydroxylase:desaturase from  Lesquerella fendleri . Plant J. 13, 201-210  
     [0025] Doyle, J. J., and Doyle, J. L. (1990) Isolation of plant DNA from fresh tissue. Focus 12, 13-15  
     [0026] Edelman, L, Olson, D., and Devereux, J. (1994) Wisconsin Sequence Analysis Package, version 8.0, Genetic Computer Group, Inc., University Research Park, 575 Science Drive, Madison, Wis., 53711, USA  
     [0027] James, D. W., Lim, E., Keller, J., Plooy, I., Ralston, E., and Dooner, H. K. (1995) Directed tagging of the Arabidopsis FATTY ACID ELONGATION1 (FAE1) gene with the maize transposon activator. Plant Cell 7, 309-319  
     [0028] Jefferson, R. A. (1987) Assaying chimeric genes in plants: The GUS gene fusion system. Plant Mol. Bol. Rep. 5, 387-405  
     [0029] Katavic, V., Haughn, G. W., Reed, D., Martin, M., and Kunst, L. (1994) In planta transformation of  Arabidopsis thaliana  Mol.Gen.Genet 245, 363-370.  
     [0030] Koetsier, P. A., Schorr, J., and Doerfler, W. (1993) A rapid optimized protocol for downward alkaline Southern blotting of DNA. BioTechniques 15, 260-262  
     [0031] Koncz, C. and Schell, J. (1986) The promoter of T L -DNA gene 5 controls the tissue-specific expression of chimaeric genes carried by a novel type of Agrobacterium binary vector. Mol. Gen. Genet 204, 383-396.  
     [0032] Lassner, M. W., Lardizabal, K., and Metz, J. G. (1996) A jojoba β-ketoacyl-CoA synthase cDNA complements the canola fatty acid elongation mutation in transgenic plants. Plant Cell 8, 281-292  
     [0033] Millar, A. A., and Kunst, L. (1997) Very-long-chain fatty acid biosynthesis is controlled through the expression and specificity of the condensing enzyme. Plant J. 12, 121-131  
     [0034] Millar, A. A., Clemens, S., Zachgo, S., Giblin, E. M., Taylor, D. C., and Kunst, L. (1999) CUT1, an Arabidopsis gene required for cuticular wax biosynthesis and pollen fertility, encodes a very-long-chain fatty acid condensing enzyme. Plant Cell 11, 825-838  
     [0035] Smith, R. F., Wiese, B. A., Wojzynski, M. K., Davison, D. B., Worley, K. C. (1996) Search-Launcher—An integrated interface to molecular biology database search and analysis services available on the world wide web. Genome Res. 6, 454-462  
     [0036] van de Loo, F. J., Broun, P., Tumer, S., and Somerville, C. (1995) An oleate 12-hydroxylase from  Ricinus communis  L. is a fatty acyl desaturase homolog. Proc. Natl. Acad. Sci. USA 92,6743-6747  
    
     
       
         1 
         
           
             8  
           
           
             1  
             4313  
             DNA  
             Lesquerella fendleri  
             
               CDS  
               (574..1056, 1439..1859, 2049..2632)  
             
           
            1 

gaattcggaa atgggccaag tgaaatggaa atagagcttc aatccattta gtcccactca     60 

aaatggtgct cgaattatat ttagttacgt tcgaatcaga caaccaagta tttggttaat    120 

aaaaaccact cgcaacaaag gaaaaacacc aagcgcgtgc gtccaacatc cgacggaagg    180 

ggggtaatgt ggtccgaaaa ccttacaaaa atctgacgtc atctaccccc gaaaacgttg    240 

aatcgtcaac gggggtagtt ttcgaattat ctttttttta ggggcagttt tattaatttg    300 

ctctagaaat tttatgattt taattaaaaa aagaaaaaga atatttgtat atttattttt    360 

tatactcttt ttttgtccaa ctatttctct tattttggca actttaacta gactagtaac    420 

ttatgtcaat gtgtatggat gcatgagagt gagtatacac atgtctaaat gcatgcctta    480 

tgaaagcaac gcaccacaaa acgaagaccc ctttacaaat acatctcatc ccttagtacc    540 

ctcttactac tgtcccgaca caaactcaaa aca atg aca tct cta aac ata aag     594 
                                     Met Thr Ser Leu Asn Ile Lys 
                                     1               5 

ctc ctt tac cat tac atc cta acc aac ctt ttc aac ctc tgt ctc ttc      642 
Leu Leu Tyr His Tyr Ile Leu Thr Asn Leu Phe Asn Leu Cys Leu Phe 
        10                  15                  20 

cct tta aca gca ttt ctc gcc gga aaa gct tct cac tta acc aaa tcc      690 
Pro Leu Thr Ala Phe Leu Ala Gly Lys Ala Ser His Leu Thr Lys Ser 
    25                  30                  35 

gat ctc ctc atg ttc tta tct cat ctc caa gac aat ctt ata act gtc      738 
Asp Leu Leu Met Phe Leu Ser His Leu Gln Asp Asn Leu Ile Thr Val 
40                  45                  50                  55 

att gta ctc ttt act ttc act atc ttc tgt ttg gtt ctt tac att gta      786 
Ile Val Leu Phe Thr Phe Thr Ile Phe Cys Leu Val Leu Tyr Ile Val 
                60                  65                  70 

acc aaa cct aaa cag att tat ctt gtg gat tac tct tgt tac ctt cca      834 
Thr Lys Pro Lys Gln Ile Tyr Leu Val Asp Tyr Ser Cys Tyr Leu Pro 
            75                  80                  85 

cca gat cat ctt aaa gtt agt ata tca agt gtc atg gat att ttc tat      882 
Pro Asp His Leu Lys Val Ser Ile Ser Ser Val Met Asp Ile Phe Tyr 
        90                  95                  100 

gag ttg aga aaa gtt gat cct ttg tgt gag gtg ggt tgt gat gat tct      930 
Glu Leu Arg Lys Val Asp Pro Leu Cys Glu Val Gly Cys Asp Asp Ser 
    105                 110                 115 

tct ctt gag ttt atg agg aag gtt tta gaa cgt tca ggt tta ggt gat      978 
Ser Leu Glu Phe Met Arg Lys Val Leu Glu Arg Ser Gly Leu Gly Asp 
120                 125                 130                 135 

gag act tat gtt cca ctt gga ctc cat caa gtg cca cct caa aag act     1026 
Glu Thr Tyr Val Pro Leu Gly Leu His Gln Val Pro Pro Gln Lys Thr 
                140                 145                 150 

ttt gca gcg ata aag gac gag aca gaa cag gtaactagag ccggttcaat       1076 
Phe Ala Ala Ile Lys Asp Glu Thr Glu Gln 
            155                 160 

gtttttgcat gctctataga atttttataa taatcaatat aaagaaaatg acatattaat   1136 

tgataaaatt gttataaaat aaagttttga tccccttaaa aatctagaag cttataaatt   1196 

ttgaaaattt aggtcgaaaa atgtttaata tttataattt tttaaaaaaa attagatgta   1256 

aaatgtcgta aaaattgtga tactatagat gaatcgtcta ctttttgaac atgtcctggg   1316 

gtgatagaag cggaccaagc ctatgcttgt ggttgttaga ttgtttattc tttttattta   1376 

gtttgtatcc tttatttttt ctgtcgcttg tggtcttaaa atgtatagga ccggctctgc   1436 

ag gta atc aaa ggt gca ctt gag aat cta ttc gag aac aca aaa gta      1483 
   Val Ile Lys Gly Ala Leu Glu Asn Leu Phe Glu Asn Thr Lys Val 
               165                 170                 175 

aac cct aga gag att ggt ata ctt gtg ata aac tca agc atg ttt aat     1531 
Asn Pro Arg Glu Ile Gly Ile Leu Val Ile Asn Ser Ser Met Phe Asn 
            180                 185                 190 

cca aca cct tct cta tca gcc atg gtt att aat act ttc aaa ctc cga     1579 
Pro Thr Pro Ser Leu Ser Ala Met Val Ile Asn Thr Phe Lys Leu Arg 
        195                 200                 205 

agt aac atc aaa agc ttt aat ctt gga gga atg ggt tgt agt gct ggt     1627 
Ser Asn Ile Lys Ser Phe Asn Leu Gly Gly Met Gly Cys Ser Ala Gly 
    210                 215                 220 

gta atc gcc att gat cta gct aaa gac ttg ttg cat gtt cat aaa aac     1675 
Val Ile Ala Ile Asp Leu Ala Lys Asp Leu Leu His Val His Lys Asn 
225                 230                 235                 240 

act tat gct ctt gta ata agc act gag aac atc acc ata acc gct tat     1723 
Thr Tyr Ala Leu Val Ile Ser Thr Glu Asn Ile Thr Ile Thr Ala Tyr 
                245                 250                 255 

gct ggc gaa aat cga tcc atg aat gtt agt aat tgc ttg ttc cga ata     1771 
Ala Gly Glu Asn Arg Ser Met Asn Val Ser Asn Cys Leu Phe Arg Ile 
            260                 265                 270 

ggc ggg gcc gcg att ttg ctc tct aat aag cca aga gat aag aga agg     1819 
Gly Gly Ala Ala Ile Leu Leu Ser Asn Lys Pro Arg Asp Lys Arg Arg 
        275                 280                 285 

tct aag tac aag cta gct cac act gtt cga aca cat aca g               1859 
Ser Lys Tyr Lys Leu Ala His Thr Val Arg Thr His Thr 
    290                 295                 300 

gtattgaatc tagatgatta cattgcgaag aaaatgtttt attcataagc ataaggtgcc   1919 

aaataggata caattttgct atcatataat atcgtcatca tatcagaatt atatcgaata   1979 

tgatatctta taacatatga ttttctatat gatgtgattc tctaaatctt ctagtatgtt   2039 

ctacaacag  ga gct gat gac atg tct tat aga tgt gtg caa caa gaa       2086 
          Gly Ala Asp Asp Met Ser Tyr Arg Cys Val Gln Gln Glu 
                      305                 310 

gaa gat gag atg ggt aaa gta gga gtt cgt ctc tca aag gac ata act     2134 
Glu Asp Glu Met Gly Lys Val Gly Val Arg Leu Ser Lys Asp Ile Thr 
315                 320                 325                 330 

act gtc gcg ggc aca gca gtt aag aag aac ata tca aca tta ggt cca     2182 
Thr Val Ala Gly Thr Ala Val Lys Lys Asn Ile Ser Thr Leu Gly Pro 
                335                 340                 345 

ctg att ctt cct tta agc gaa aag ctt ctt tat ttc gtt tcc ttc atc     2230 
Leu Ile Leu Pro Leu Ser Glu Lys Leu Leu Tyr Phe Val Ser Phe Ile 
            350                 355                 360 

gcg aag aaa ctt ttg aag gag aag atc aag aac tat tac gtc ccg gat     2278 
Ala Lys Lys Leu Leu Lys Glu Lys Ile Lys Asn Tyr Tyr Val Pro Asp 
        365                 370                 375 

ctt aag cta gct atc aat cat ttt tgt atc cat gct ggt gga aga ggt     2326 
Leu Lys Leu Ala Ile Asn His Phe Cys Ile His Ala Gly Gly Arg Gly 
    380                 385                 390 

gtg tta gat gtg ttg gaa aag aac tta agg cta tca cca att gat gta     2374 
Val Leu Asp Val Leu Glu Lys Asn Leu Arg Leu Ser Pro Ile Asp Val 
395                 400                 405                 410 

gaa gca tct aga tcg act tta cat aga ttt ggg aat act tct tcg agt     2422 
Glu Ala Ser Arg Ser Thr Leu His Arg Phe Gly Asn Thr Ser Ser Ser 
                415                 420                 425 

tcg att tgg tat gag ttg gct tat att gaa gct aaa gga agg atg aag     2470 
Ser Ile Trp Tyr Glu Leu Ala Tyr Ile Glu Ala Lys Gly Arg Met Lys 
            430                 435                 440 

aaa ggt gat aaa gct tgg caa att gct tta ggg tca ggg ttt aag tgt     2518 
Lys Gly Asp Lys Ala Trp Gln Ile Ala Leu Gly Ser Gly Phe Lys Cys 
        445                 450                 455 

aat agt gct gtt tgg gtt gct tta cgt aat gtt aaa gct tcg gct aat     2566 
Asn Ser Ala Val Trp Val Ala Leu Arg Asn Val Lys Ala Ser Ala Asn 
    460                 465                 470 

agt cct tgg gaa cat tgt att gat agg tat cct gtt cct gat act tgt     2614 
Ser Pro Trp Glu His Cys Ile Asp Arg Tyr Pro Val Pro Asp Thr Cys 
475                 480                 485                 490 

gta gaa aat ggt aag tct tagttaatgt atcttagtgt caacgtttct            2662 
Val Glu Asn Gly Lys Ser 
                495 

tttttttttt tagttttttt tttgatgttg aagatgttct cttttcgtta taaataagaa   2722 

ataaaagttt ttttaggagg actgccattt aagtttacca atcttgtcta tactgccatt   2782 

tagcttcagc gtgaacatta atactacgga cggatatgtt ataggcttgt cattgtccgc   2842 

gaaaaaagat caatgtactc ctgtgtttac agatatttgc gattgaacct gggtaaacag   2902 

tccgttttga ttgttatgga caatcatgta ctacacgcgt tatccgtatt gttcacagaa   2962 

gatcaaaaca aaccatttta tccatattaa atcgtaaata tctgtaaaca caaagatacg   3022 

cggtctattt cgcagaaagt agcacgcttg atcgaatccg tatatggaaa ttaaaaaacg   3082 

tctttgaaaa tatcaatttc ataatttcaa taaagagaag ttaacttaac cggtagtttc   3142 

aagaaatttc caatagaaat taaccggcag tcatcatttt tattgggcct tctgtggact   3202 

atagaatcag gccctttatt gtaacttgat atagccttta gaatcaggca ggccgtttat   3262 

tcataaatcg aaaatcttgg gataattaag aatcaacaac ttggaatcgg gaccagtcgc   3322 

agcaaattga tgtcgtaacg tgtcacttgg catgttttag ttaggaccat attcgaaaaa   3382 

gtaaatgggt catgttgcca taaaacaaaa aatgctgatt ccaaacgtaa attgctaaac   3442 

tcgatgtaat aaaggttaga ttttacgaac ctcaaacccc cattttttta ctttgtttat   3502 

ctccaatccc acggtgtaag aacaacaaga acatgatgct ttaaataata gaagacaaac   3562 

tcataaaatt gaaataaata gtcatcatac tagcagatta agatccgaat tttacaacca   3622 

aaaacaaata aatgcctttt tttttttttt ttaattcgga tcttcttatt catattctta   3682 

ttaataattt ttattaaaag atactaagaa atcgtgcaat tacgtttcta cgatgataaa   3742 

tcaaacccat cgtcgcagta gtaaaaccca aagttattga tcgcagctga aaagagagtt   3802 

actctgtgat gctgcctcag ttgcaagata cggcatgttg tgaaaggaac gatcaagaca   3862 

agacacaaca ttgttgatct cgtcttcgtc ttcatcgtca tcgcttttgg tatgcatcaa   3922 

ttcatctgct cggtgcttta gagtctcaaa cattagctca ggctcatgct gcattgctcg   3982 

aagaacatca gcagaggact gaccaggcat ggctcgggta actgcaaagc tatgtggtcc   4042 

atcgctctgc gagacacgga caatgcttgg cccaccaaac aaattatgga ctccaccgag   4102 

cgcctcctca taagctcctc caagaaacat tcccagaaag tatcttcctc cgcagttgtc   4162 

aatctcgtgc aatggcatgg tggattctcc gcctatgaac ttgttgatct ttccatcgct   4222 

atcgcaagtt aaatcagata agatcccacg agttcccggc ctttgatcga gcttatggat   4282 

aggaactata ggaaataact gatccgaatt c                                  4313 

 
           
             2  
             496  
             PRT  
             Lesquerella fendleri  
           
            2 

Met Thr Ser Leu Asn Ile Lys Leu Leu Tyr His Tyr Ile Leu Thr Asn 
1               5                   10                  15 

Leu Phe Asn Leu Cys Leu Phe Pro Leu Thr Ala Phe Leu Ala Gly Lys 
            20                  25                  30 

Ala Ser His Leu Thr Lys Ser Asp Leu Leu Met Phe Leu Ser His Leu 
        35                  40                  45 

Gln Asp Asn Leu Ile Thr Val Ile Val Leu Phe Thr Phe Thr Ile Phe 
    50                  55                  60 

Cys Leu Val Leu Tyr Ile Val Thr Lys Pro Lys Gln Ile Tyr Leu Val 
65                  70                  75                  80 

Asp Tyr Ser Cys Tyr Leu Pro Pro Asp His Leu Lys Val Ser Ile Ser 
                85                  90                  95 

Ser Val Met Asp Ile Phe Tyr Glu Leu Arg Lys Val Asp Pro Leu Cys 
            100                 105                 110 

Glu Val Gly Cys Asp Asp Ser Ser Leu Glu Phe Met Arg Lys Val Leu 
        115                 120                 125 

Glu Arg Ser Gly Leu Gly Asp Glu Thr Tyr Val Pro Leu Gly Leu His 
    130                 135                 140 

Gln Val Pro Pro Gln Lys Thr Phe Ala Ala Ile Lys Asp Glu Thr Glu 
145                 150                 155                 160 

Gln Val Ile Lys Gly Ala Leu Glu Asn Leu Phe Glu Asn Thr Lys Val 
                165                 170                 175 

Asn Pro Arg Glu Ile Gly Ile Leu Val Ile Asn Ser Ser Met Phe Asn 
            180                 185                 190 

Pro Thr Pro Ser Leu Ser Ala Met Val Ile Asn Thr Phe Lys Leu Arg 
        195                 200                 205 

Ser Asn Ile Lys Ser Phe Asn Leu Gly Gly Met Gly Cys Ser Ala Gly 
    210                 215                 220 

Val Ile Ala Ile Asp Leu Ala Lys Asp Leu Leu His Val His Lys Asn 
225                 230                 235                 240 

Thr Tyr Ala Leu Val Ile Ser Thr Glu Asn Ile Thr Ile Thr Ala Tyr 
                245                 250                 255 

Ala Gly Glu Asn Arg Ser Met Asn Val Ser Asn Cys Leu Phe Arg Ile 
            260                 265                 270 

Gly Gly Ala Ala Ile Leu Leu Ser Asn Lys Pro Arg Asp Lys Arg Arg 
        275                 280                 285 

Ser Lys Tyr Lys Leu Ala His Thr Val Arg Thr His Thr Gly Ala Asp 
    290                 295                 300 

Asp Met Ser Tyr Arg Cys Val Gln Gln Glu Glu Asp Glu Met Gly Lys 
305                 310                 315                 320 

Val Gly Val Arg Leu Ser Lys Asp Ile Thr Thr Val Ala Gly Thr Ala 
                325                 330                 335 

Val Lys Lys Asn Ile Ser Thr Leu Gly Pro Leu Ile Leu Pro Leu Ser 
            340                 345                 350 

Glu Lys Leu Leu Tyr Phe Val Ser Phe Ile Ala Lys Lys Leu Leu Lys 
        355                 360                 365 

Glu Lys Ile Lys Asn Tyr Tyr Val Pro Asp Leu Lys Leu Ala Ile Asn 
    370                 375                 380 

His Phe Cys Ile His Ala Gly Gly Arg Gly Val Leu Asp Val Leu Glu 
385                 390                 395                 400 

Lys Asn Leu Arg Leu Ser Pro Ile Asp Val Glu Ala Ser Arg Ser Thr 
                405                 410                 415 

Leu His Arg Phe Gly Asn Thr Ser Ser Ser Ser Ile Trp Tyr Glu Leu 
            420                 425                 430 

Ala Tyr Ile Glu Ala Lys Gly Arg Met Lys Lys Gly Asp Lys Ala Trp 
        435                 440                 445 

Gln Ile Ala Leu Gly Ser Gly Phe Lys Cys Asn Ser Ala Val Trp Val 
    450                 455                 460 

Ala Leu Arg Asn Val Lys Ala Ser Ala Asn Ser Pro Trp Glu His Cys 
465                 470                 475                 480 

Ile Asp Arg Tyr Pro Val Pro Asp Thr Cys Val Glu Asn Gly Lys Ser 
                485                 490                 495 

 
           
             3  
             23  
             DNA  
             Artificial Sequence  
             
               FAE1 Primer  
             
           
            3 

ccgagctcaa agaggataca tac                                             23 

 
           
             4  
             29  
             DNA  
             Artificial Sequence  
             
               FAE1 Primer  
             
           
            4 

gatactcgag aacgttggca ctcagatac                                       29 

 
           
             5  
             29  
             DNA  
             Artificial Sequence  
             
               PCR Primer  
             
           
            5 

cgcaagcttg aattcggaaa tgggccaag                                       29 

 
           
             6  
             29  
             DNA  
             Artificial Sequence  
             
               PCR Primer  
             
           
            6 

cgcgtcgact gttttgagtt tgtgtcggg                                       29 

 
           
             7  
             506  
             PRT  
             Arabidopsis thaliana  
           
            7 

Met Thr Ser Val Asn Val Lys Leu Leu Tyr Arg Tyr Val Leu Thr Asn 
1               5                   10                  15 

Phe Phe Asn Leu Cys Leu Phe Pro Leu Thr Ala Phe Leu Ala Gly Lys 
            20                  25                  30 

Ala Ser Arg Leu Thr Ile Asn Asp Leu His Asn Phe Leu Ser Tyr Leu 
        35                  40                  45 

Gln His Asn Leu Ile Thr Val Thr Leu Leu Phe Ala Phe Thr Val Phe 
    50                  55                  60 

Gly Leu Val Leu Tyr Ile Val Thr Arg Pro Asn Pro Val Tyr Leu Val 
65                  70                  75                  80 

Asp Tyr Ser Cys Tyr Leu Pro Pro Pro His Leu Lys Val Ser Val Ser 
                85                  90                  95 

Lys Val Met Asp Ile Phe Tyr Gln Ile Arg Lys Ala Asp Thr Ser Ser 
            100                 105                 110 

Arg Asn Val Ala Cys Asp Asp Pro Ser Ser Leu Asp Phe Leu Arg Lys 
        115                 120                 125 

Ile Gln Glu Arg Ser Gly Leu Gly Asp Glu Thr Tyr Ser Pro Glu Gly 
    130                 135                 140 

Leu Ile His Val Pro Pro Arg Lys Thr Phe Ala Ala Ser Arg Glu Glu 
145                 150                 155                 160 

Thr Glu Lys Val Ile Ile Gly Ala Leu Glu Asn Leu Phe Glu Asn Thr 
                165                 170                 175 

Lys Val Asn Pro Arg Glu Ile Gly Ile Leu Val Val Asn Ser Ser Met 
            180                 185                 190 

Phe Asn Pro Thr Pro Ser Leu Ser Ala Met Val Val Asn Thr Phe Lys 
        195                 200                 205 

Leu Arg Ser Asn Ile Lys Ser Phe Asn Leu Gly Gly Met Gly Cys Ser 
    210                 215                 220 

Ala Gly Val Ile Ala Ile Asp Leu Ala Lys Asp Leu Leu His Val His 
225                 230                 235                 240 

Lys Asn Thr Tyr Ala Leu Val Val Ser Thr Glu Asn Ile Thr Gln Gly 
                245                 250                 255 

Ile Tyr Ala Gly Glu Asn Arg Ser Met Met Val Ser Asn Cys Leu Phe 
            260                 265                 270 

Arg Val Gly Gly Ala Ala Ile Leu Leu Ser Asn Lys Ser Gly Asp Arg 
        275                 280                 285 

Arg Arg Ser Lys Tyr Lys Leu Val His Thr Val Arg Thr His Thr Gly 
    290                 295                 300 

Ala Asp Asp Lys Ser Phe Arg Cys Val Gln Gln Glu Asp Asp Glu Ser 
305                 310                 315                 320 

Gly Lys Ile Gly Val Cys Leu Ser Lys Asp Ile Thr Asn Val Ala Gly 
                325                 330                 335 

Thr Thr Leu Thr Lys Asn Ile Ala Thr Leu Gly Pro Leu Ile Leu Pro 
            340                 345                 350 

Leu Ser Glu Lys Phe Leu Phe Phe Ala Thr Phe Val Ala Lys Lys Leu 
        355                 360                 365 

Leu Lys Asp Lys Ile Lys His Tyr Tyr Val Pro Asp Phe Lys Leu Ala 
    370                 375                 380 

Val Asp His Phe Cys Ile His Ala Gly Gly Arg Ala Val Ile Asp Glu 
385                 390                 395                 400 

Leu Glu Lys Asn Leu Gly Leu Ser Pro Ile Asp Val Glu Ala Ser Arg 
                405                 410                 415 

Ser Thr Leu His Arg Phe Gly Asn Thr Ser Ser Ser Ser Ile Trp Tyr 
            420                 425                 430 

Glu Leu Ala Tyr Ile Glu Ala Lys Gly Arg Met Lys Lys Gly Asn Lys 
        435                 440                 445 

Ala Trp Gln Ile Ala Leu Gly Ser Gly Phe Lys Cys Asn Ser Ala Val 
    450                 455                 460 

Trp Val Ala Leu Arg Asn Val Lys Ala Ser Ala Asn Ser Pro Trp Gln 
465                 470                 475                 480 

His Cys Ile Asp Arg Tyr Pro Val Lys Ile Asp Ser Asp Leu Ser Lys 
                485                 490                 495 

Ser Lys Thr His Val Gln Asn Gly Arg Ser 
            500                 505 

 
           
             8  
             505  
             PRT  
             Brassica napus  
           
            8 

Met Thr Ser Ile Asn Val Lys Leu Leu Tyr His Tyr Val Ile Thr Asn 
1               5                   10                  15 

Leu Phe Asn Leu Cys Phe Phe Pro Leu Thr Ala Ile Val Ala Gly Lys 
            20                  25                  30 

Ala Tyr Leu Thr Ile Asp Asp Leu His His Leu Tyr Tyr Ser Tyr Leu 
        35                  40                  45 

Gln His Asn Leu Ile Thr Ile Ala Pro Leu Leu Ala Phe Thr Val Phe 
    50                  55                  60 

Gly Ser Val Leu Tyr Ile Ala Thr Arg Pro Lys Pro Val Tyr Leu Val 
65                  70                  75                  80 

Glu Tyr Ser Cys Tyr Leu Pro Pro Thr His Cys Arg Ser Ser Ile Ser 
                85                  90                  95 

Lys Val Met Asp Ile Phe Phe Gln Val Arg Lys Ala Asp Pro Ser Arg 
            100                 105                 110 

Asn Gly Thr Cys Asp Asp Ser Ser Trp Leu Asp Phe Leu Arg Lys Ile 
        115                 120                 125 

Gln Glu Arg Ser Gly Leu Gly Asp Glu Thr His Gly Pro Glu Gly Leu 
    130                 135                 140 

Leu Gln Val Pro Pro Arg Lys Thr Phe Ala Arg Ala Arg Glu Glu Thr 
145                 150                 155                 160 

Glu Gln Val Ile Ile Gly Ala Leu Glu Asn Leu Phe Lys Asn Thr Asn 
                165                 170                 175 

Val Asn Pro Lys Asp Ile Gly Ile Leu Val Val Asn Ser Ser Met Phe 
            180                 185                 190 

Asn Pro Thr Pro Ser Leu Ser Ala Met Val Val Asn Thr Phe Lys Leu 
        195                 200                 205 

Arg Ser Asn Val Arg Ser Phe Asn Leu Gly Gly Met Gly Cys Ser Ala 
    210                 215                 220 

Gly Val Ile Ala Ile Asp Leu Ala Lys Asp Leu Leu His Val His Lys 
225                 230                 235                 240 

Asn Thr Tyr Ala Leu Val Val Ser Thr Glu Asn Ile Thr Tyr Asn Ile 
                245                 250                 255 

Tyr Ala Gly Asp Asn Arg Ser Met Met Val Ser Asn Cys Leu Phe Arg 
            260                 265                 270 

Val Gly Gly Ala Ala Ile Leu Leu Ser Asn Lys Pro Arg Asp Arg Arg 
        275                 280                 285 

Arg Ser Lys Tyr Glu Leu Val His Thr Val Arg Thr His Thr Gly Ala 
    290                 295                 300 

Asp Asp Lys Ser Phe Arg Cys Val Gln Gln Gly Asp Asp Glu Asn Gly 
305                 310                 315                 320 

Gln Thr Gly Val Ser Leu Ser Lys Asp Ile Thr Asp Val Ala Gly Arg 
                325                 330                 335 

Thr Val Lys Lys Asn Ile Ala Thr Leu Gly Pro Leu Ile Leu Pro Leu 
            340                 345                 350 

Ser Glu Lys Leu Leu Phe Phe Val Thr Phe Met Gly Lys Lys Leu Phe 
        355                 360                 365 

Lys Asp Glu Ile Lys His Tyr Tyr Val Pro Asp Phe Lys Leu Ala Ile 
    370                 375                 380 

Asp His Phe Cys Ile His Ala Gly Gly Lys Ala Val Ile Asp Val Leu 
385                 390                 395                 400 

Glu Lys Asn Leu Gly Leu Ala Pro Ile Asp Val Glu Ala Ser Arg Ser 
                405                 410                 415 

Thr Leu His Arg Phe Gly Asn Thr Ser Ser Ser Ser Ile Trp Tyr Glu 
            420                 425                 430 

Leu Ala Tyr Ile Glu Pro Lys Gly Arg Met Lys Lys Gly Asn Lys Val 
        435                 440                 445 

Trp Gln Ile Ala Leu Gly Ser Gly Phe Lys Cys Asn Ser Ala Val Trp 
    450                 455                 460 

Val Ala Leu Asn Asn Val Lys Ala Ser Thr Asn Ser Pro Trp Glu His 
465                 470                 475                 480 

Cys Ile Asp Arg Tyr Pro Val Lys Ile Asp Ser Asp Ser Gly Lys Ser 
                485                 490                 495 

Glu Thr Arg Val Pro Asn Gly Arg Ser 
            500                 505