Abstract:
The present invention relates to a novel plant enzyme called delta 12 fatty acid acetylenase. This enzyme is responsible for the conversion of fatty acids to acetylenic acids and the invention relates to production of such acids. The invention also relates to use of cDNA encoding acetylenase, preferably  Crepis alpina  delta 12 acetylenase, for transforming organisms such as oil accumulating organisms selected from the group consisting of oil crops, oleogeneous yeasts and moulds. Furthermore, the invention relates to organisms such as oil accumulating organisms transformed with acetylenase cDNA, and to oils and other acetylenic compounds from said organisms.

Description:
This is a CIP of parent, co-pending application PCT/SE97/00247, filed Feb. 14, 1997, the entire contents of which are hereby incorporated by reference. 
    
    
     TECHNICAL FIELD 
     The present invention relates to a novel plant enzyme. More specifically, the present invention relates to a method for producing acetylenic compounds in particular acetylenic fatty acids, to a cDNA encoding a plant fatty acid acetylenase, to the use of the said cDNA for transforming oil accumulating organisms for the purpose of producing acetylenic fatty acids, and to such oil accumulating organisms per se as well as oils therefrom. 
     BACKGROUND OF THE INVENTION 
     There is considerable interest, world-wide, in producing chemical feedstocks such as fatty acids for industrial use from renewable plant resources rather than from non-renewable petrochemicals. This concept has broad appeal for both manufacturers and consumers on the basis of resource conservation and in addition provides significant opportunities to develop new industrial crops for agriculture. 
     There is an enormous diversity of unusual fatty acids in oils from wild plant species which have been well characterized (see e.g. Badami &amp; Patil, 1981). Many of these acids are of potential industrial use. This has lead to an interest in domesticating relevant plant species to enable the agricultural production of particular fatty acids. However the development of genetic engineering combined with a greater understanding of the biosynthesis of unusual fatty acids make it now possible to transfer genes coding for key enzymes, involved in the synthesis of a particular fatty acid from a wild species, to a choosen domesticated oilseed crop. In this way specific fatty acids can be produced in high purity and quantities at moderate costs. 
     One class of fatty acids of particular interest are the acetylenic fatty acids; consisting of an acyl chain having two adjacent carbon atoms linked by an acetylenic or triple bond. Because of their high reactivities they may be ideally suited for the production of coatings, plastics and lubricants. By transferring the genes responsible for the production of a specific acetylenic acid from a wild species to commercial oilseeds, or any other oil accumulating organism that can be easily multiplied, it should be possible to develop a renewable primary source of this oil containing acetylenic fatty acids for industrial uses. 
     PRIOR ART 
     The formation of acetylenic bonds in fatty acids in mosses occurs via the subtraction of hydrogens from a double bond (Kohn et al., 1994) 
     Crepis species have seed oils with high contents of acetylenic acids (Badami &amp; Patil, 1981; Hirsinger, 1991). 
     SUMMARY OF THE INVENTION 
     The present invention provides a new method of producing acetylenic fatty acids from transgenic oil accumulating organisms. 
     The inventors have characterized an enzyme (acetylenase) that is responsible for the production of 9-octadecen-12-ynoic acid (crepenynic acid) from 9,12-octadecadienoic acid (linoleic acid) in membrane fractions from developing  Crepis alpina  seeds. The characterization of the acetylenase from  Crepis alpina  revealed that the acetylenase had very similar biochemical properties to the non-heme containing monooxygenases oleate delta 12 and linoleate delta 15 (omega 3) desaturases. Based on the premise that the biochemical similarities observed between the acetylenase and the enzymes producing linoleic and linolenic acid (delta 12 and delta 15 desaturases) would also be associated with similarity in the primary sequence of these proteins a full length cDNA (pCrep1) (SEQ ID NO:1), encoding a putative acetylenase (SEQ ID NO:2), was isolated from  Crepis alpina.    
     Initially, two types of cDNA fragments, obtained by using PCR and primers designed by aligning protein sequences of delta 12 desaturases, were characterised from  C. alpina.  DNA sequence analysis revealed that one was highly homologous to all the other plant endoplasmic reticular (ER) delta 12 desaturases and the castor bean hydroxylase. The other cDNA fragment characterised had a sequence that was homologous to the ER delta 12 desaturase sequences of plants but was divergent not only in a number of non-conserved amino residues but also in a number of amino acid residues that were highly conserved in all delta 12 ER desaturases. Using northern blot analysis the gene encoding this cDNA (pCrep1) was observed to be highly expressed only in a seed specific manner when compared to expression in leaf tissue. Taken together these findings, and a consideration of the unique biochemical nature of an cell in a oilseed, provided strong evidence that the isolated cDNA (pCrep1) from  C. alpina  encode an enzyme responsible for converting linoleic acid into crepenynic acid. 
     Finally, conclusive evidence that the cDNA, pCrep1, from  C. alpina  encoded a plant acetylenase enzyme was obtained by the expression of this gene in yeast. The expression of this gene together with the addition of linoleic acid when culturing these yeast resulted in the production of a delta 12 acetylenic acid, 9-octadecen-12-ynoic acid (crepenynic acid), as confirmed by mass spectrometric analysis of extracted yeast fatty acids. 
     Therefore, in a first aspect, the present invention relates to a method of producing acetylenic compounds, characterized in that a double bond is converted to an acetylenic bond by an acetylenase. 
     In a preferred embodiment of the method, the acetylenic fatty acids are produced by conversion of unsaturated fatty acids to acetylenic fatty acids by a fatty acid acetylenase. 
     In a second aspect, the invention relates to cDNA coding for acetylenase of the mixed function monoxygenase type containing three conserved histidine motifs (His Xaa (3 or 4) His and His Xaa (2 or 3) His His, and His Xaa (2 or 3) His His) according to FIGS. 8A-8D of the accompanying drawings. 
     In a further embodiment the invention relates to a cDNA encoding fatty acid acetylenase, such as  Crepis alpina  delta 12 acetylenase comprising the sequence according to FIG. 3 of the accompanying drawings or any nucleotide sequences essentially homologous therewith. 
     A third aspect of the invention concerns use of the above described cDNA for transforming organisms. The organisms may be acetylenic compound accumulating organisms or oil accumulating organisms, respectively. 
     In a fourth aspect, the invention relates to organisms transformed with a acetylenase CDNA as described above. The organisms are acetylenic compound or oil accumulating, examples of the latter being oil crops, oleaginous yeasts and moulds. 
     In a fifth aspect, the invention concerns acetylenic componds accumulated in organisms described above. 
     In a sixth aspect, the invention concerns oils from oil accumulating organisms described above. 
     In a preferred embodiment, the present invention relates to transforming oil accumulating organisms with the said isolated cDNA from  Crepis alpina  seed cDNA library for the purpose of producing acetylenic fatty acids acids and in particular 9-octadecen-12-ynoic acid (crepenynic acid). 
     DETAILED DESCRIPTION OF THE INVENTION 
       C. alpina  seed oil is rich in crepenynic acid [0-octadecen12-ynoic acid (Hirsinger, 1989)]. The inventors have studied the biosynthesis of crepenynic acid in  C. alpina  seeds. The feeding of exogenous 1- 14 C-labelled free fatty acids to intact developing cotyledons of  C. alpina  seeds demonstrated that linoleate is a precursor to crepenynic acid. This is contradictory to previous published results for the biosynthesis of crepenynic acid in  Crepis rubra  (Haigh &amp; James, 1967). Although the reaction of acetylenic acid formation in mosses has been shown to be a desaturation process (Kohn et al. 1994), such desaturation processes can be carried out by a variety of different unrelated types of plant enzymes, such as phytoene desaturases (Wieland et al. 1994) or non-heme containing proteins, the latter a class of enzymes of which some show very little amino acid sequence homologies except for three conserved histidine motifs (Shanklin et al. 1994). It has been suggested that the biosynthesis of acetylenic fatty acids occur by a sequence of intermediates catalyzed by separate enzymatic reactions. For example, acetylenic bonds were thought to be formed as a side pathway of saturated fatty acid synthesis (Diedrich &amp; Henschel, 1991); or via an epoxygenation of a double bond with subsequent conversion to a diol which in its turn is dehydrated in two steps in order to form an acetylenic bond (Van de Loo et al. 1993). Given these conflicting alternatives the nature of an acetylenase enzyme and its mechanism of action was not known at all nor obvious at the time of the present priority patent application SE 9601236-4. 
     The enzyme, according to this invention, responsible for the synthesis of crepenynic acid (called the delta 12 acetylenase), was shown by the inventors to remain only active in membrane (microsomal) fractions prepared from developing seeds of  Crepis alpina,  provided that the homogenization buffer contain NADH or NADPH, catalase and free coenzyme A. The characterisation of the microsomal acetylenase and its comparison with the delta 12 desaturase (responsible for the desaturation of oleate to linoleate) revealed that these enzymes had very similar properties. Both enzymes required O 2  and NADH or NADPH; where both coreductants worked equally well with both enzymes. Cyanide (CN—) and antibodies against cauliflower cytochrome b 5  inhibited both these enzymes whereas carbonmonoxide had no significant effect on either enzyme activity. These data suggested that both enzymes were biochemically similar. The oleate delta 12 hydroxylase from castor bean was also shown to have similar biochemical properties to the delta 12 desaturase despite catalyzing a different reaction (Bafor et al., 1991, Smith et al, 1992). The castor bean delta 12 hydroxylase gene was later shown to have significant sequence homology to the ER delta 12 desaturase genes (FAD 2 genes) (Van de Loo et al., 1995). Because the delta 12 acetylenase, like the delta-12 desaturase (FAD2), catalyzes a dehydrogenation between carbons 12 and 13 of an acyl chain, and like the delta 15 desaturase (FAD3) utilized linoleic acid as substrate the inventors considered the possibility that the acetylenase gene should have some sequence homology to the FAD2 and/or the FAD3 genes. 
     The invention will now be described more closely below in relation to the accompanying drawings and an Experimental Part. 
    
    
     THE DRAWINGS SHOW 
     FIG.  1 . Restriction map of pCrep1 
     FIG.  2 . Restriction map of pVT-Crep1 
     FIG.  3 . Superimposed single ion chromatograms of ions 333, 365, 367 from FADEA prepared from total fatty acids extracted from yeast strain YN94-1 transformed with pVT-Crep1. The letters denotes peaks representing the following diethylamide derivatives of fatty acids: A, eicosanoic acid; B, eicosaenoic acid; C, 9-octadecen-12-ynoic acid. 
     FIG.  4 . Superimposed single ion chromatograms of ions 333, 365, 367 from FADEA prepared from total fatty acids extracted from yeast strain YN94-1 transformed with empty vector (pVT100U; control). The letters denotes peaks representing the following diethylamide derivatives of fatty acids: A, eicosanoic acid; B, eicosaenoic acid. 
     FIG. 5. A total ion chromatogramme of FADEA prepared from fatty acids enriched in the putative 9-octadecen-12 ynoic acid originating from lipid extracts of YN94-1 transformed with pVT-Crep1. The letters denotes peaks representing the following diethylamide derivatives of fatty acids: A, hecadecanoic acid; B, octadecaonoic acid; C, octadeca-9,12-dienoic acid; D. 9-octadecen-12-ynoic acid. 
     FIG.  6 . Mass spectrum of compound corresponding to peak D in FIG. 5. 
     FIG.  7 . Gas liquid analysis of  A. thaliana  seed fatty acids. Methyl esters of seeds from a plant transformed with (A) an empty vector and (B) the  Crepis alpina  acetylenase (Crep1) were analyzed and their fatty acid profile determined. In (A) the indicated peaks correspond to methyl esters (Me) derivates of hexadecanoic acid, 1; octadecanoic acid, 2; 9-octadecenoic acid, 3; 9,12-octadecadienoic acid, 4; 9,12,15-octdecatrienoic acid, 5; 11-eicosaenoic acid, 6 and 13-docosaenoic acid, 7. In (B) the peak corresponding to the methyl ester derivative of crepenynic acid is indicated and corresponds to 25% of total fatty acid methyl ester peak areas. 
     FIGS. 8A-8D show an alignment of amino acid sequences from delta 12 ER and plastid desaturases, delta 15 desaturases and from the castor bean hydroxylase. Also included in this alignment is the protein sequence derived from pCrep1 (crepis). Underlined are three histidine motifs that are conserved in non-heme containing monoxygenases. Sequences given in this alignment together with their accession numbers are: 
     bnom6des.seq, delta 12 desaturase from  Brassica napus  (L29214) (SEQ ID NO:3); 
     gmom6des.seq, delta 12 desaturase from  Glycine max  (L29215) (SEQ ID NO:4); 
     atom3des.seq, delta 15 desaturase from  Arabidopsis thaliana  (L22961) (SEQ ID NO:5); 
     bnom3des.seq, delta 15 from  Brassica napus  (L22963) (SEQ ID NO:6); 
     rcom3des.seq, delta 15 desaturase from  Ricinus communis  (L25897) (SEQ ID NO:7); 
     siom3des.seq, delta 15 desaturase from oriental sesame (U25817) (SEQ ID NO:8); 
     ldd15des.seq, delta 15 desaturase from  Limnanthes douglasil  (U17063) (SEQ ID NO:9); 
     gsom3des, delta 15 desaturase from  Glycine max  (L22965) (SEQ ID NO:10); 
     atom3bdes.seq, delta 15 desaturase from  Arabidopsis thaliana  (D17579) (SEQ ID NO:11); 
     bnom31des.seq, delta 15 from  Brassica napus  (L22962) (SEQ ID NO:12); 
     gsom3bdes.seq, delta 15 desaturase from  Glycine max  (L22964) (SEQ ID NO:13); 
     atd12des.seq, delta 12 desaturase from  Arabidopsis thaliana  (L26296) (SEQ ID NO:14); 
     gmom6bdes.seq delta 12 desaturase from  Glycine max  (L43921) (SEQ ID NO:15); 
     scom12des.seq, delta 12 desaturase from  S. commersonli  (X92847) (SEQ ID NO:16); 
     gmom6ades.seq, delta 12 desaturase from  Glycine max  (L43920) (SEQ ID NO:17); 
     rchyd.seq, oleate 12-hydroxylase from  Ricinus communis  (U22378) (SEQ ID NO:18); 
     crepis,  Crepis alpina  acetylenase from this document (SEQ ID NO:2). 
    
    
     EXPERIMENTAL PART 
     Cloning of Putative Acetylenase Gene 
     An alignment of amino acid sequences (SEQ ID Nos:3-18) from different species showed that the membrane bound fatty acid desaturases could be grouped according to the homology of their putative mature protein into three distinct groups (plastid delta 12 desaturases, ER delta 12 desaturases and delta 15 desaturases; see FIGS.  8 A- 8 D). The castor bean hydroxylase (Van de Loo et al 1995) shared a high homology with the ER delta 12 desaturases to the degree that it was not easily distinguishable from these sequences. Furthermore, the sequences from all three classes of enzymes showed some degree of sequence homology with each other. 
     Based on this alignment oligonucleotide primers were designed and synthesised for these three groups of sequences and for a consensus of all of these sequences. The sequence of these primers are given below. 
     (i) consensus primers (primers designed to a consensus of all three groups of membrane-bound desaturases and the castor bean fatty acid hydroxylase): sense is GSN CAY GAN TGY GSN CAY (SEQ ID NO:19) antisense is RAN ADR TGR TGN RBN AYR TG (SEQ ID NO:20). 
     (ii) plastid delta 12 desaturase primers: sense is TGG MGN TTY AAR CAY GAY MG (SEQ ID NO:21) antisense is GTN SWC ATC CAR AAR TGR TA (SEQ ID NO:22). 
     (iii) ER delta 12 desaturase primers including the castor bean fatty acid hydroxylase: sense is CAY GAR TGY GGN CAY CAY GC (SEQ ID NO:23) antisense is CCN CKN ARC CAR TCC CAY TC (SEQ ID NO:24). 
     (iv) delta 15 desaturase primers: sense is ACN CAY CAY CARAAY CAY GG (SEQ ID NO:25) antisense is CAY TGY TTN CCN CKR TAC CA (SEQ ID NO:26). 
     Poly A+ RNA was isolated from developing seeds (100 mg) of  C. alpina  using a QuickPrep Micro mRNA purifcation kit from Pharmacia Biotech. All of the poly A+ RNA from this purification was precipitated and used to synthesise first strand cDNA which was primed with both oligo dT and random hexamers and synthesised with Superscript II reverse transcriptase from Gibco BRL. The polymerase chain reaction (PCR) was then used, with the described primers and this cDNA, to amplify products with the following cycling conditions: 1 cycle of 94° C. for 2 min, 30 cycles of (94° C., 30 sec; 50° C., 30 sec; 72° C., 30 sec) and finally one cycle of 72° C. for 5 min. 
     Products were obtained for all the primers used; particularly noticeable was that the primers against the ER delta 12 desaturases gave significantly more product than from the other primers used. The sizes of the PCR products from the delta 12 and delta 15 primers corresponded to the sizes anticipated. 
     The PCR products obtained by amplification with the ER delta 12 primers and delta 15 primers were made blunt ended with T4 and klenow polymerases and cloned into the EcoRV site of the plasmid vector Bluescript. DNA sequencing of a number of the clones revealed that at least three distinct sequences had been amplified when using these two sets of primers: (i) a highly conserved delta 15 desaturase sequence (ii), a highly conserved ER delta 12 sequence and (iii) a sequence (D12V) having homology to the ER delta 12 sequences but showing distinct differences even in some amino acid residues that were highly conserved amongst all the other desaturase sequences. 
     The analysis of fatty acids from  C. alpina  had indicated that the crepenynic acid was probably present only in seeds. Northern blot analysis at high stringency indicated that the mRNA from the D12V sequence described above was expressed highly in seeds but not in leaves which is consistent with the observation that crepenynic acid was only observed in seeds. 
     A cDNA library was made from developing seeds from  C. alpina  using a Uni-ZAP XR cloning kit for cDNA from Stratagene and screened with the random labelled D12V sequence. From this screening it was estimated that cDNAs encoding the D12V sequences were highly abundant; further emphasing the high level of expression of this gene. After the isolation of single hybridising Lambda plaques, pBluescript phagemid was excised using the ExAssist/SOLR system from Stratagene, Phagemids obtained by this were subsequently used to produce double stranded DNA plasmid. From these colonies a full length clone (pCREP1, see FIG. 1) was isolated by using DNA sequencing and restriction mapping of isolated plasmid. The insert from pCrep1, a 1.5 kb insert contained in the vector pBluescript SK, was sequenced and from this an open reading frame deduced coding for a 375 aa long protein (SEQ ID NO:2). The analysis of this protein sequence revealed approximately 60% identity and 80% similarity with other plant delta 12 desaturase proteins and had noticeable differences I homology, where, certain residues that were conserved amongst all other desaturases were not in this sequence FIGS. 8A-8D. Three histidine motifs were present which have been shown to be conserved in a number of non-heme containing monoxygenases catalyzing hydroxylation and desaturation reactions (Shanklin et al. 1994). 
     Expression of the pCrep cDNA and Detection of Crepenynic Acid in Transgenic Yeast 
     The pCrep1 open reading frame was released from pCrep1 on a SmaI/XhoI restriction fragment and the 1.5 kb Crep1 open reading frame recovered by gelpurification (Langridge et al., 1980). pVT100-U DNA (Vernet et al., 1987) was digested using PvuII and XhoI. 50 ng PvuII/XhoI-linearized pVT100 was ligated with 100 ng 1.5 kb SmaI/XhoI fragment corresponding to the Crep1 open reading frame using T4 DNA ligase (NBL Genen Science Ltd., UK). Part of the ligation mixture was used to transform competent  E. coli  DHa cells. One clone (pVT-Crep1), which contained the expected 1.5 kb insert, was chosen and the contruct checked by digestion with EcoRI, or HindIII+XbaI. Both digests gave the expected products (approx. 5.3, 2.3 and 0.8 kb for the EcoRI digest, and release of the 1.5 kb open reading frame with the HindIII+XbaI digest). pVT-Crep1 DNA (see FIG.  2 ), or empty vector pVT100U, was used to transform  Saccharomyces cerevisiae  strains YN94-1 and C13-ABYS86, using the PLATE method of Elble (1992). Overnight yeast transformants were spread on SCD minus uracil agar and single colonies were streaked onto fresh selective (minus uracil) plates. 
     The YN94-1 and C13-ABYS86 strains of yeast transformed with pVT-Crep1 DNA and with empty vector (pVT100U; control) were cultivated in shaking cultures at 28° C. for five hours in selective media (without uracil; 400 ml) whereafter 40 ml of cultivation media containing linoleic acid dispersed in Tween 40® was added to the culture to give a final concentration of 0.03% linoleic acid and 1% Tween 40® (w/w). After cultivation for an additional 78 h at 28° C. the cells were pelleted by centrifugation and washed by dispersion in 20 ml of 0.1M Tris-HCl buffer pH. 7.8 containing 1% Tween 40® and repelleted by centrifugation. The cells were further washed by resuspension in 20 ml of 0.1M Tris-HCl buffer pH. 7.8 and pelleted again. The cells were thereafter extracted in a mixture of chloroform/methanol/0.15M acetic acid (1:2:0.8 by vol.) in a Braun MSK glass bead cell homogenizer (B. Braun Biotech International, Melsungen, Germany) at 4000 r.p.m. for 20 s. The yeast lipids were extracted from the mixture into a chloroform phase by adding chloroform and 0.15M acetic acid to yield final proportions of 1:1:0.9 (by vol.) of chloroform, methanol and 0.15 M acetic acid. After centrifugation of the mixture the lipid containing chloroform phase was removed and evaporated to dryness under a stream of nitrogen. 
     The lipohilic residue were methylated with methanolic HCl (4% w/w) at 85° C. for 45 min wherafter the fatty acid methyl esters were extracted into n-hexane. Gas liquid (GC) chromatogrammes of the methyl esters separated on a glass column (2.5 m×3 mm i.d.) containing 3% SP-2300 on Supelcoport 100/120 mesh (Supelco, Bellefonte, P. USA) revealed a peak with the same retention time as authentic 9-octadecen-12 ynoic acid metyl ester constituting up to 0.3% of total peak areas in samples prepared from yeast transformed with pVT-Crep1 but not in samples prepared from yeast transformed with empty vector (pVT100U; control). 
     Since acetylenic fatty acid methyl esters can be partially separated from other fatty acid methylesters on silica gel thin layer chromatography, the methylesters prepared from YN94-1 transformed with pVT-Crep1 were separated on silica gel 60 thin layer chromatography plates (Merck, Darmstadt, Germany) by developing the plate in hexane/diethyl ether/acetic acid (85:15:1 by vol.). An area located just below the main methyl ester area was removed from the plate and the lipids were eluted with methanol/chloroform (2:1) and analyzed by gas liquid chromatography. The fraction were shown to consist of fatty acid methylesters where the peak with the same retention time as 9-octadecen-12 ynoic acid metyl ester made up 12.5% of the total peak area. 
     The methyl ester fraction enriched in the putative 9-octadecen-12 ynoic acid methyl ester as well as total fatty acid methyl esters prepared from YN94-1 transformed with pVT-Crep1 and YN94-1 transformed with empty vector (pVT100U; control) were hydrolyzed in 2.5M KOH in aqueous methanol (15% methanol, by vol.) at 90° C. for 1 h. The free fatty acids were extracted into hexane after acidicifiction with HCl and the hexane phase was evaporated to dryness under a stream of nitrogen. 
     Fatty acid diethylamides (FADEA) were prepared from the free fatty acids according to Nilsson and Liljenberg (1991). The FADEA were either injected directly on a gas liquid chromatography coupled to mass spectrometer (GC-MS) or subjected to further purification by silica gel thin layer chromatography by developing the plate in heptane/diethyleter/acteic acid (50:50:1, by vol.). 
     The FADEA were analyzed on a Hewlett-Packard 5890 II gas chromatograph equipped with a DB225 (0.25 mm i.d.×30 m, J&amp;W, Folsom, USA) in series with a Rtx 2330 (Restek Corp., PA, USA) fused silica capillary column, coupled with a Hewlett-Packard 5989A mass spectrometer working in electron impact mode at 70 eV. Injection technique was cold splitless at 100° C. and then the temperature was raised as quickly as possible to 240° C. Oven temperature was 100° C. for 7 min, then 20° C. per min to 190° C. and then 1° C. per min to a final temp. of 225° C. where it was kept for another 20 min. The double bond positions were determined according to Nilsson and Liljenberg (1991). 
     Single ion chromatogrammes of masses corresponding to the molecular ion of FADEA prepared from total fatty acids from YN94-1 transformed with pVT-Crep1 and from YN94-1 transformed with empty vector (pVT100U; control) are shown in FIG.  5  and FIG. 6, respectively. Chromatogram of FADEA from YN94-1 transformed with pVT-Crep1 showed a peak of mass 333 (corresponding to the molecular weight of 9-octadecen-12 ynoic acid diethylamide) which was absent in the chromatogram of FADEA from YN94-1 transformed with empty vector (pVT100U; control). The peak had a retention time of 57.3 min and was located between peaks corresponding to eicosanoic and eicosenoic FADEA derivatives. 
     A total ion chromatogramme of FADEA prepared from fatty acids enriched in the putative 9-octadecen-12 ynoic acid by thin layer chromatography (as described above) originating from lipid extracts of YN94-1 transformed with pVT-Crep1 is shown in FIG.  5 . Mass spectrum (FIG. 6) of the putative 9-octadecen-12 ynoic acid diethylamide derivative (peak D in FIG. 5) showed a gap in mass of 26 amu instead of regular 28 between carbon 7 and 9 indicating a double bond at position 9. Further more there was a gap of 24 amu instead of regular 28 between carbon atom 10 and 12 indicating acetylenic bond at position 12. The peak D produced a mass spectrum identical to that of authentic 9-octadecen-12 ynoic acid diethylamide prepared from oils from  Crepis alpina  seeds. Thus the peak D in the chromatogram in FIG. 5 was unambigously identified as 9-octadecen-12 ynoic acid diethylamide derivative. Since the compound was absent in yeast strains not transformed with the Crep1 cDNA it is clear that the Crep1 cDNA codes for a delta-12 fatty acid acetylenase. 
     Production of Acetylenic Fatty Acids in Seed Oils of Transgenic Plants 
     The Crep1 gene was expressed in Arabidopsis under control of the seed-specific napin promoter. Total fatty acids of bulk seed samples from individual T 0  transgenic plants consisted up to 25% crepenynic acid, with no other acetylenic fatty acids being detected while plants transformed with empty vector had no acetylenic acids in their seeds (FIG.  7 ). 
     Experimental 
     A binary vector for the acetylenase consisted of the Crep1 cDNA placed downstream of the −309 fragment from the napin promoter (Stalberg et al., 1993) in the vector pGPTV-KAN (Becker et al., 1992).  A. thaliana  columbia (C-24) was transformed with  Agrobacterium tumefaciens  (Valvekens, 1988). Methyl esters of seed fatty acids from T 0  transgenic plants were prepared by heating 10-30 whole seeds from individual plants at 85° C. for 90 min in 1 ml of 0.1 M sodium methoxide. Methyl esters were extracted with hexane and analysed by gas liquid chromatography through a 50 m×0.32 mm CP-Wax58-CB fused silica column (Chrompack). 
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             1128 
             DNA 
             Crepis alpina 
             
               CDS 
               (1)..(1125) 
             
           
            1
atg ggt ggc ggt ggc cgt ggt cgg act tcg caa aaa ccc ctc atg gaa       48
Met Gly Gly Gly Gly Arg Gly Arg Thr Ser Gln Lys Pro Leu Met Glu
  1               5                  10                  15
cgt gtc tca gtt gat cca ccc ttc acc gtg agt gat ctc aag caa gca       96
Arg Val Ser Val Asp Pro Pro Phe Thr Val Ser Asp Leu Lys Gln Ala
             20                  25                  30
atc cct ccc cat tgc ttc aag cga tct gta atc cgt tcc tct tac tac      144
Ile Pro Pro His Cys Phe Lys Arg Ser Val Ile Arg Ser Ser Tyr Tyr
         35                  40                  45
ata gtc cac gat gct att atc gcc tac atc ttc tac ttc ctt gcc gac      192
Ile Val His Asp Ala Ile Ile Ala Tyr Ile Phe Tyr Phe Leu Ala Asp
     50                  55                  60
aaa tac att ccg att ctc cct gcc cct cta gcc tac ctc gct tgg ccc      240
Lys Tyr Ile Pro Ile Leu Pro Ala Pro Leu Ala Tyr Leu Ala Trp Pro
 65                  70                  75                  80
ctt tac tgg ttc tgt caa gct agc atc ctc acc ggc tta tgg gtc atc      288
Leu Tyr Trp Phe Cys Gln Ala Ser Ile Leu Thr Gly Leu Trp Val Ile
                 85                  90                  95
ggt cac gaa tgc ggt cac cat gcc ttc agc gac tac cag tgg gtt gac      336
Gly His Glu Cys Gly His His Ala Phe Ser Asp Tyr Gln Trp Val Asp
            100                 105                 110
gac act gtg ggc ttc atc ctc cac tcg ttt ctc atg acc ccg tat ttc      384
Asp Thr Val Gly Phe Ile Leu His Ser Phe Leu Met Thr Pro Tyr Phe
        115                 120                 125
tcc tgg aaa tac agc cac cgg aac cac cat gcc aac aca aat tcg ctt      432
Ser Trp Lys Tyr Ser His Arg Asn His His Ala Asn Thr Asn Ser Leu
    130                 135                 140
gac aac gat gaa gtt tac atc ccc aaa agc aag gcc aaa gtc gcg ctt      480
Asp Asn Asp Glu Val Tyr Ile Pro Lys Ser Lys Ala Lys Val Ala Leu
145                 150                 155                 160
tac tat aaa gtt ctc aac cac cca cct ggc cga ctg ttg att atg ttc      528
Tyr Tyr Lys Val Leu Asn His Pro Pro Gly Arg Leu Leu Ile Met Phe
                165                 170                 175
atc acc ttc acc cta ggc ttc cct cta tac ctc ttt acc aat att tcc      576
Ile Thr Phe Thr Leu Gly Phe Pro Leu Tyr Leu Phe Thr Asn Ile Ser
            180                 185                 190
ggc aag aag tat gaa agg ttt gcc aac cat ttc gac ccc atg agt ccg      624
Gly Lys Lys Tyr Glu Arg Phe Ala Asn His Phe Asp Pro Met Ser Pro
        195                 200                 205
att ttc aaa gag cgt gag cgg ttt cag gtc ttg cta tcg gat ctt ggc      672
Ile Phe Lys Glu Arg Glu Arg Phe Gln Val Leu Leu Ser Asp Leu Gly
    210                 215                 220
ctt ctt gct gtg ctt tac gga gtt aaa ctt gcg gta gca gcg aaa ggc      720
Leu Leu Ala Val Leu Tyr Gly Val Lys Leu Ala Val Ala Ala Lys Gly
225                 230                 235                 240
gcc gcc tgg gtg acg tgc att tac gga att cca gtt tta ggc gtg ttt      768
Ala Ala Trp Val Thr Cys Ile Tyr Gly Ile Pro Val Leu Gly Val Phe
                245                 250                 255
atc ttt ttc gat atc atc acc tac ttg cac cac acc cat ctg tcg ttg      816
Ile Phe Phe Asp Ile Ile Thr Tyr Leu His His Thr His Leu Ser Leu
            260                 265                 270
cct cat tat gat tca tct gaa tgg aac tgg ctc aga ggg gct ttg tca      864
Pro His Tyr Asp Ser Ser Glu Trp Asn Trp Leu Arg Gly Ala Leu Ser
        275                 280                 285
aca atc gat agg gac ttt ggg ttc ctg aat agt gtg ctc cat gat gtt      912
Thr Ile Asp Arg Asp Phe Gly Phe Leu Asn Ser Val Leu His Asp Val
    290                 295                 300
aca cac act cac gtt atg cat cat ctg ttt tca tac att cca cac tat      960
Thr His Thr His Val Met His His Leu Phe Ser Tyr Ile Pro His Tyr
305                 310                 315                 320
cat gcg aag gag gca agg gat gca atc aac aca gtc ttg ggc gac ttt     1008
His Ala Lys Glu Ala Arg Asp Ala Ile Asn Thr Val Leu Gly Asp Phe
                325                 330                 335
tat aag atc gat agg act cca att ctg aaa gca atg tgg aga gag gcc     1056
Tyr Lys Ile Asp Arg Thr Pro Ile Leu Lys Ala Met Trp Arg Glu Ala
            340                 345                 350
aag gaa tgc atc ttc atc gag cct gaa aaa ggt agg gag tcc aag ggt     1104
Lys Glu Cys Ile Phe Ile Glu Pro Glu Lys Gly Arg Glu Ser Lys Gly
        355                 360                 365
gta tat tgg tac aat aaa ttc tga                                     1128
Val Tyr Trp Tyr Asn Lys Phe
    370                 375 
           
             2 
             375 
             PRT 
             Crepis alpina 
           
            2
Met Gly Gly Gly Gly Arg Gly Arg Thr Ser Gln Lys Pro Leu Met Glu
  1               5                  10                  15
Arg Val Ser Val Asp Pro Pro Phe Thr Val Ser Asp Leu Lys Gln Ala
             20                  25                  30
Ile Pro Pro His Cys Phe Lys Arg Ser Val Ile Arg Ser Ser Tyr Tyr
         35                  40                  45
Ile Val His Asp Ala Ile Ile Ala Tyr Ile Phe Tyr Phe Leu Ala Asp
     50                  55                  60
Lys Tyr Ile Pro Ile Leu Pro Ala Pro Leu Ala Tyr Leu Ala Trp Pro
 65                  70                  75                  80
Leu Tyr Trp Phe Cys Gln Ala Ser Ile Leu Thr Gly Leu Trp Val Ile
                 85                  90                  95
Gly His Glu Cys Gly His His Ala Phe Ser Asp Tyr Gln Trp Val Asp
            100                 105                 110
Asp Thr Val Gly Phe Ile Leu His Ser Phe Leu Met Thr Pro Tyr Phe
        115                 120                 125
Ser Trp Lys Tyr Ser His Arg Asn His His Ala Asn Thr Asn Ser Leu
    130                 135                 140
Asp Asn Asp Glu Val Tyr Ile Pro Lys Ser Lys Ala Lys Val Ala Leu
145                 150                 155                 160
Tyr Tyr Lys Val Leu Asn His Pro Pro Gly Arg Leu Leu Ile Met Phe
                165                 170                 175
Ile Thr Phe Thr Leu Gly Phe Pro Leu Tyr Leu Phe Thr Asn Ile Ser
            180                 185                 190
Gly Lys Lys Tyr Glu Arg Phe Ala Asn His Phe Asp Pro Met Ser Pro
        195                 200                 205
Ile Phe Lys Glu Arg Glu Arg Phe Gln Val Leu Leu Ser Asp Leu Gly
    210                 215                 220
Leu Leu Ala Val Leu Tyr Gly Val Lys Leu Ala Val Ala Ala Lys Gly
225                 230                 235                 240
Ala Ala Trp Val Thr Cys Ile Tyr Gly Ile Pro Val Leu Gly Val Phe
                245                 250                 255
Ile Phe Phe Asp Ile Ile Thr Tyr Leu His His Thr His Leu Ser Leu
            260                 265                 270
Pro His Tyr Asp Ser Ser Glu Trp Asn Trp Leu Arg Gly Ala Leu Ser
        275                 280                 285
Thr Ile Asp Arg Asp Phe Gly Phe Leu Asn Ser Val Leu His Asp Val
    290                 295                 300
Thr His Thr His Val Met His His Leu Phe Ser Tyr Ile Pro His Tyr
305                 310                 315                 320
His Ala Lys Glu Ala Arg Asp Ala Ile Asn Thr Val Leu Gly Asp Phe
                325                 330                 335
Tyr Lys Ile Asp Arg Thr Pro Ile Leu Lys Ala Met Trp Arg Glu Ala
            340                 345                 350
Lys Glu Cys Ile Phe Ile Glu Pro Glu Lys Gly Arg Glu Ser Lys Gly
        355                 360                 365
Val Tyr Trp Tyr Asn Lys Phe
    370                 375 
           
             3 
             443 
             PRT 
             Brassica napus 
           
            3
Met Ala Ser Arg Ile Ala Asp Ser Leu Phe Ala Phe Thr Gly Pro Gln
  1               5                  10                  15
Gln Cys Leu Pro Arg Ala Pro Lys Leu Ala Ser Ala Arg Leu Ser Pro
             20                  25                  30
Gly Val Tyr Ala Val Arg Pro Ile Asp Leu Leu Leu Lys Gly Thr Arg
         35                  40                  45
Arg Thr Phe Leu Val Pro Ala Lys Lys Arg Ile Gly Cys Ile Lys Ala
     50                  55                  60
Val Phe Val Pro Val Ala Pro Pro Ser Ala Asp Asn Ala Glu Asp Arg
 65                  70                  75                  80
Glu Gln Leu Ala Glu Ser Tyr Gly Phe Lys Gln Ile Gly Gln Asp Leu
                 85                  90                  95
Pro Asp Asn Val Thr Leu Lys Asp Ile Met Asp Thr Leu Pro Lys Glu
            100                 105                 110
Val Phe Glu Ile Asp Asp Val Lys Ala Trp Lys Ser Val Leu Ile Ser
        115                 120                 125
Val Thr Ser Tyr Ala Leu Gly Leu Phe Met Ile Ala Lys Ala Pro Trp
    130                 135                 140
Tyr Leu Leu Pro Leu Ala Trp Ala Trp Thr Gly Thr Ala Val Thr Gly
145                 150                 155                 160
Phe Phe Val Ile Gly His Asp Cys Ala His Lys Ser Phe Ser Lys Asn
                165                 170                 175
Lys Leu Val Glu Asp Ile Val Gly Thr Leu Ala Phe Leu Pro Leu Val
            180                 185                 190
Tyr Pro Tyr Glu Pro Trp Arg Phe Lys His Asp Arg His His Ala Lys
        195                 200                 205
Thr Asn Met Leu Val His Asp Thr Ala Trp Gln Pro Val Pro Pro Glu
    210                 215                 220
Glu Phe Asp Ser Ser Pro Val Leu Arg Lys Ala Ile Ile Phe Gly Tyr
225                 230                 235                 240
Gly Pro Ile Arg Pro Trp Leu Ser Ile Ala His Trp Val Asn Trp His
                245                 250                 255
Phe Asn Leu Arg Lys Phe Arg Pro Ser Glu Val Asn Arg Val Lys Ile
            260                 265                 270
Ser Leu Ala Cys Val Phe Ala Phe Met Ala Val Gly Trp Pro Leu Ile
        275                 280                 285
Ile Tyr Lys Val Gly Val Leu Gly Trp Val Lys Phe Trp Leu Met Pro
    290                 295                 300
Trp Leu Gly Tyr His Phe Trp Met Ser Thr Phe Thr Met Val His His
305                 310                 315                 320
Thr Ala Pro His Ile Pro Phe Lys Pro Ala Asp Glu Trp Asn Ala Ala
                325                 330                 335
Gln Ala Gln Leu Asn Gly Thr Val His Cys Asp Tyr Pro Ser Trp Ile
            340                 345                 350
Glu Ile Leu Cys His Asp Ile Asn Val His Ile Pro His His Ile Ser
        355                 360                 365
Pro Arg Ile Pro Ser Tyr Asn Leu Arg Ala Ala His Gln Ser Ile Gln
    370                 375                 380
Glu Asn Trp Gly Lys Tyr Thr Asn Leu Ala Thr Trp Asn Trp Arg Leu
385                 390                 395                 400
Met Lys Thr Ile Met Thr Val Cys His Val Tyr Asp Lys Glu Glu Asn
                405                 410                 415
Tyr Ile Pro Phe Asp Arg Leu Ala Pro Glu Glu Ser Gln Pro Ile Thr
            420                 425                 430
Phe Leu Lys Lys Ala Met Pro Asp Tyr Ala Ala
        435                 440 
           
             4 
             424 
             PRT 
             Glycine max 
           
            4
Met Ala Cys Thr Leu Ala Asp Ser Leu Leu Leu Phe Lys Gly Ser Tyr
  1               5                  10                  15
Gln Lys Pro Val Leu Arg Arg Asp Ile Ala Ala Arg Tyr Ser Pro Gly
             20                  25                  30
Ile Phe Ser Leu Asn Ser Asn Gly Leu Ile Gln Lys Arg Phe Arg Arg
         35                  40                  45
Gln Arg Asn Phe Val Thr Arg Asn Lys Val Thr Val Ile His Ala Val
     50                  55                  60
Ala Ile Pro Val Gln Pro Ala Pro Val Glu Ser Ala Glu Tyr Arg Lys
 65                  70                  75                  80
Gln Leu Ala Glu Asp Tyr Gly Phe Arg Gln Val Gly Glu Pro Leu Ser
                 85                  90                  95
Asp Asp Val Thr Leu Lys Asp Val Ile Asn Pro Leu Pro Lys Glu Val
            100                 105                 110
Phe Glu Ile Asp Asp Val Lys Ala Trp Lys Ser Val Leu Ile Ser Val
        115                 120                 125
Thr Ser Tyr Ala Leu Gly Leu Phe Met Ile Ser Lys Ala Pro Trp Tyr
    130                 135                 140
Leu Leu Pro Leu Ala Trp Val Trp Thr Gly Thr Ala Ile Thr Gly Phe
145                 150                 155                 160
Phe Val Ile Gly His Asp Cys Ala His Arg Ser Phe Ser Ser Asn Lys
                165                 170                 175
Leu Val Glu Asp Ile Val Gly Thr Leu Ala Phe Met Pro Leu Ile Tyr
            180                 185                 190
Pro Tyr Glu Pro Trp Arg Phe Lys His Asp Arg His His Ala Lys Thr
        195                 200                 205
Asn Met Leu Arg Glu Asp Thr Ala Trp His Pro Val Trp Lys Asp Glu
    210                 215                 220
Phe Glu Ser Thr Pro Leu Leu Arg Lys Ala Ile Ile Tyr Gly Tyr Gly
225                 230                 235                 240
Pro Phe Arg Cys Trp Met Ser Ile Ala His Trp Leu Met Trp His Phe
                245                 250                 255
Asp Leu Lys Lys Phe Arg Pro Ser Glu Val Pro Arg Val Lys Ile Ser
            260                 265                 270
Leu Ala Cys Val Phe Ala Phe Ile Ala Ile Gly Trp Pro Leu Ile Ile
        275                 280                 285
Tyr Lys Thr Gly Ile Met Gly Trp Ile Lys Phe Trp Leu Met Pro Trp
    290                 295                 300
Leu Gly Tyr His Phe Trp Met Ser Thr Phe Thr Met Val His His Thr
305                 310                 315                 320
Ala Pro Tyr Ile Pro Phe Lys Tyr Ser Glu Glu Trp Asn Arg Ala Gln
                325                 330                 335
Ala Gln Leu Asn Gly Thr Val His Cys Asp Tyr Pro Lys Trp Ile Glu
            340                 345                 350
Ile Leu Cys His Asp Ile Asn Val His Ile Pro His His Ile Ser Pro
        355                 360                 365
Arg Ile Pro Ser Tyr Asn Leu Arg Ala Ala His Lys Ser Leu Gln Glu
    370                 375                 380
Asn Trp Gly Gln Tyr Leu Asn Glu Ala Ser Trp Asn Trp Arg Leu Met
385                 390                 395                 400
Lys Thr Ile Met Thr Val Cys Gln Val Tyr Asp Lys Glu Lys Ser Leu
                405                 410                 415
Cys Cys Leu Arg Arg Thr Cys Pro
            420 
           
             5 
             446 
             PRT 
             Arabidopsis thaliana 
           
            5
Met Ala Asn Leu Val Leu Ser Glu Cys Gly Ile Arg Pro Leu Pro Arg
  1               5                  10                  15
Ile Tyr Thr Thr Pro Arg Ser Asn Phe Leu Ser Asn Asn Asn Lys Phe
             20                  25                  30
Arg Pro Ser Leu Ser Ser Ser Ser Tyr Lys Thr Ser Ser Ser Pro Leu
         35                  40                  45
Ser Phe Gly Leu Asn Ser Arg Asp Gly Phe Thr Arg Asn Trp Ala Leu
     50                  55                  60
Asn Val Ser Thr Pro Leu Thr Thr Pro Ile Phe Glu Glu Ser Pro Leu
 65                  70                  75                  80
Glu Glu Asp Asn Lys Gln Arg Phe Asp Pro Gly Ala Pro Pro Pro Phe
                 85                  90                  95
Asn Leu Ala Asp Ile Arg Ala Ala Ile Pro Lys His Cys Trp Val Lys
            100                 105                 110
Asn Pro Trp Lys Ser Leu Ser Tyr Val Val Arg Asp Val Ala Ile Val
        115                 120                 125
Phe Ala Leu Ala Ala Gly Ala Ala Tyr Leu Asn Asn Trp Ile Val Trp
    130                 135                 140
Pro Leu Tyr Trp Leu Ala Gln Gly Thr Met Phe Trp Ala Leu Phe Val
145                 150                 155                 160
Leu Gly His Asp Cys Gly His Gly Ser Phe Ser Asn Asp Pro Lys Leu
                165                 170                 175
Asn Ser Val Val Gly His Leu Leu His Ser Ser Ile Leu Val Pro Tyr
            180                 185                 190
His Gly Trp Arg Ile Ser His Arg Thr His His Gln Asn His Gly His
        195                 200                 205
Val Glu Asn Asp Glu Ser Trp His Pro Met Ser Glu Lys Ile Tyr Asn
    210                 215                 220
Thr Leu Asp Lys Pro Thr Arg Phe Phe Arg Phe Thr Leu Pro Leu Val
225                 230                 235                 240
Met Leu Ala Tyr Pro Phe Tyr Leu Trp Ala Arg Ser Pro Gly Lys Lys
                245                 250                 255
Gly Ser His Tyr His Pro Asp Ser Asp Leu Phe Leu Pro Lys Glu Arg
            260                 265                 270
Lys Asp Val Leu Thr Ser Thr Ala Cys Trp Thr Ala Met Ala Ala Leu
        275                 280                 285
Leu Val Cys Leu Asn Phe Thr Ile Gly Pro Ile Gln Met Leu Lys Leu
    290                 295                 300
Tyr Gly Ile Pro Tyr Trp Ile Asn Val Met Trp Leu Asp Phe Val Thr
305                 310                 315                 320
Tyr Leu His His His Gly His Glu Asp Lys Leu Pro Trp Tyr Arg Gly
                325                 330                 335
Lys Glu Trp Ser Tyr Leu Arg Gly Gly Leu Thr Thr Leu Asp Arg Asp
            340                 345                 350
Tyr Gly Leu Ile Asn Asn Ile His His Asp Ile Gly Thr His Val Ile
        355                 360                 365
His His Leu Phe Pro Gln Ile Pro His Tyr His Leu Val Glu Ala Thr
    370                 375                 380
Glu Ala Ala Lys Pro Val Leu Gly Lys Tyr Tyr Arg Glu Pro Asp Lys
385                 390                 395                 400
Ser Gly Pro Leu Pro Leu His Leu Leu Glu Ile Leu Ala Lys Ser Ile
                405                 410                 415
Lys Glu Asp His Tyr Val Ser Asp Glu Gly Glu Val Val Tyr Tyr Lys
            420                 425                 430
Ala Asp Pro Asn Leu Tyr Gly Glu Val Lys Val Arg Ala Asp
        435                 440                 445 
           
             6 
             329 
             PRT 
             Brassica napus 
           
            6
Met Ser Tyr Val Val Arg Glu Leu Ala Ile Val Phe Ala Leu Ala Ala
  1               5                  10                  15
Gly Ala Ala Tyr Leu Asn Asn Trp Leu Val Trp Pro Leu Tyr Trp Ile
             20                  25                  30
Ala Gln Gly Thr Met Phe Trp Ala Leu Phe Val Leu Gly His Asp Cys
         35                  40                  45
Gly His Gly Ser Phe Ser Asn Asp Pro Arg Leu Asn Ser Val Val Gly
     50                  55                  60
His Leu Leu His Ser Ser Ile Leu Val Pro Tyr His Gly Trp Arg Ile
 65                  70                  75                  80
Ser His Arg Thr His His Gln Asn His Gly His Val Glu Asn Asp Glu
                 85                  90                  95
Ser Trp His Pro Met Ser Glu Lys Ile Tyr Lys Ser Leu Asp Lys Pro
            100                 105                 110
Thr Arg Phe Phe Arg Phe Thr Leu Pro Leu Val Met Leu Ala Tyr Pro
        115                 120                 125
Phe Tyr Leu Trp Ala Arg Ser Pro Gly Lys Lys Gly Ser His Tyr His
    130                 135                 140
Pro Asp Ser Asp Leu Phe Leu Pro Lys Glu Arg Asn Asp Val Leu Thr
145                 150                 155                 160
Ser Thr Ala Cys Trp Thr Ala Met Ala Val Leu Leu Val Cys Leu Asn
                165                 170                 175
Phe Val Met Gly Pro Met Gln Met Leu Lys Leu Tyr Val Ile Pro Tyr
            180                 185                 190
Trp Ile Asn Val Met Trp Leu Asp Phe Val Thr Tyr Leu His His His
        195                 200                 205
Gly His Glu Asp Lys Leu Pro Trp Tyr Arg Gly Lys Glu Trp Ser Tyr
    210                 215                 220
Leu Arg Gly Gly Leu Thr Thr Leu Asp Arg Asp Tyr Gly Leu Ile Asn
225                 230                 235                 240
Asn Ile His His Asp Ile Gly Thr His Val Ile His His Leu Phe Pro
                245                 250                 255
Gln Ile Pro His Tyr His Leu Val Glu Ala Thr Glu Ala Ala Lys Pro
            260                 265                 270
Val Leu Gly Lys Tyr Tyr Arg Glu Pro Asp Lys Ser Gly Pro Leu Pro
        275                 280                 285
Leu His Leu Leu Gly Ile Leu Ala Lys Ser Ile Lys Glu Asp His Phe
    290                 295                 300
Val Ser Asp Glu Gly Asp Val Val Tyr Tyr Glu Ala Asp Pro Asn Leu
305                 310                 315                 320
Tyr Gly Glu Ile Lys Val Thr Ala Glu
                325 
           
             7 
             460 
             PRT 
             Ricinus communis 
           
            7
Met Ala Ala Gly Trp Val Leu Ser Glu Cys Gly Leu Arg Pro Leu Pro
  1               5                  10                  15
Arg Ile Tyr Ser Arg Pro Arg Ile Gly Phe Thr Ser Lys Thr Thr Asn
             20                  25                  30
Leu Leu Lys Leu Arg Glu Leu Pro Asp Ser Lys Ser Tyr Asn Leu Cys
         35                  40                  45
Ser Ser Phe Lys Val Ser Ser Trp Ser Asn Ser Lys Gln Ser Asn Trp
     50                  55                  60
Ala Leu Asn Val Ala Val Pro Val Asn Val Ser Thr Val Ser Gly Glu
 65                  70                  75                  80
Asp Asp Arg Glu Arg Glu Glu Phe Asn Gly Ile Val Asn Val Asp Glu
                 85                  90                  95
Gly Lys Gly Glu Phe Phe Asp Ala Gly Ala Pro Pro Pro Phe Thr Leu
            100                 105                 110
Ala Asp Ile Arg Ala Ala Ile Pro Lys His Cys Trp Val Lys Asn Pro
        115                 120                 125
Trp Arg Ser Met Ser Tyr Val Leu Arg Asp Val Val Val Val Phe Gly
    130                 135                 140
Leu Ala Ala Val Ala Ala Tyr Phe Asn Asn Trp Val Ala Trp Pro Leu
145                 150                 155                 160
Tyr Trp Phe Cys Gln Gly Thr Met Phe Trp Ala Leu Phe Val Leu Gly
                165                 170                 175
His Asp Cys Gly His Gly Ser Phe Ser Asn Asn Pro Lys Leu Asn Ser
            180                 185                 190
Val Val Gly His Leu Leu His Ser Ser Ile Leu Val Pro Tyr His Gly
        195                 200                 205
Trp Arg Ile Ser His Arg Thr His His Gln Asn His Gly His Val Glu
    210                 215                 220
Asn Asp Glu Ser Trp His Pro Leu Ser Glu Lys Ile Phe Lys Ser Leu
225                 230                 235                 240
Asp Asn Val Thr Lys Thr Leu Arg Phe Ser Leu Pro Phe Pro Met Leu
                245                 250                 255
Ala Tyr Pro Phe Tyr Leu Trp Ser Arg Ser Pro Gly Lys Lys Gly Ser
            260                 265                 270
His Phe His Pro Asp Ser Gly Leu Phe Val Pro Lys Glu Arg Lys Asp
        275                 280                 285
Ile Ile Thr Ser Thr Ala Cys Trp Thr Ala Met Ala Ala Leu Leu Val
    290                 295                 300
Tyr Leu Asn Phe Ser Met Gly Pro Val Gln Met Leu Lys Leu Tyr Gly
305                 310                 315                 320
Ile Pro Tyr Trp Ile Phe Val Met Trp Leu Asp Phe Val Thr Tyr Leu
                325                 330                 335
His His His Gly His Glu Asp Lys Leu Pro Trp Tyr Arg Gly Lys Ala
            340                 345                 350
Trp Ser Tyr Leu Arg Gly Gly Leu Thr Thr Leu Asp Arg Asp Tyr Gly
        355                 360                 365
Trp Ile Asn Asn Ile His His Asp Ile Gly Thr His Val Ile His His
    370                 375                 380
Leu Phe Pro Gln Ile Pro His Tyr His Leu Val Glu Ala Thr Glu Ala
385                 390                 395                 400
Ala Lys Pro Val Met Gly Lys Tyr Tyr Arg Glu Pro Lys Lys Ser Gly
                405                 410                 415
Pro Leu Pro Leu His Leu Leu Gly Ser Leu Val Arg Ser Met Lys Glu
            420                 425                 430
Asp His Tyr Val Ser Asp Thr Gly Asp Val Val Tyr Tyr Gln Lys Asp
        435                 440                 445
Pro Lys Leu Ser Gly Ile Gly Gly Glu Lys Thr Glu
    450                 455                 460 
           
             8 
             447 
             PRT 
             oriental sesame 
           
            8
Met Ala Ser Trp Val Leu Ser Glu Cys Gly Leu Arg Pro Leu Pro Arg
  1               5                  10                  15
Val Tyr Pro Lys Pro Arg Thr Gly His Pro Leu Leu Asn Ser Asn Pro
             20                  25                  30
Thr Lys Leu Arg Phe Ser Arg Thr Asp Leu Gly Asn Gly Ser Ser Phe
         35                  40                  45
Cys Leu Ser Ser Gly Ile Leu Arg Glu Lys Asn Trp Ala Leu Arg Val
     50                  55                  60
Ser Ala Pro Leu Arg Val Leu Gln Val Glu Glu Glu Glu Glu Asn Lys
 65                  70                  75                  80
Glu Gly Glu Arg Val Ile Asn Gly Gly Glu Glu Phe Asp Pro Gly Ala
                 85                  90                  95
Pro Pro Pro Phe Lys Leu Ser Asp Ile Arg Glu Ala Ile Pro Lys His
            100                 105                 110
Cys Trp Val Lys Asp Pro Trp Arg Ser Met Gly Tyr Val Val Arg Asp
        115                 120                 125
Val Ala Val Val Phe Gly Leu Ala Ala Val Ala Ala Tyr Phe Asn Asn
    130                 135                 140
Trp Val Val Trp Pro Leu Tyr Trp Phe Ala Gln Ser Thr Met Phe Trp
145                 150                 155                 160
Ala Leu Phe Val Leu Gly His Asp Cys Gly His Gly Ser Phe Ser Asn
                165                 170                 175
Asp Pro Lys Leu Asn Ser Val Val Gly His Ile Leu His Ser Ser Ile
            180                 185                 190
Leu Val Pro Tyr His Gly Trp Arg Ile Ser His Arg Thr His His Gln
        195                 200                 205
Asn His Gly His Val Glu Asn Asp Glu Ser Trp His Pro Leu Ser Glu
    210                 215                 220
Lys Ile Tyr Lys Asn Leu Asp Thr Ala Thr Lys Lys Leu Arg Phe Thr
225                 230                 235                 240
Leu Pro Phe Pro Leu Leu Ala Tyr Pro Ile Tyr Leu Trp Ser Arg Ser
                245                 250                 255
Pro Gly Lys Gln Gly Ser His Phe His Pro Asp Ser Asp Leu Phe Val
            260                 265                 270
Pro Asn Glu Lys Lys Asp Val Ile Thr Ser Thr Val Cys Trp Thr Ala
        275                 280                 285
Met Leu Ala Leu Leu Val Gly Leu Ser Phe Val Ile Gly Pro Val Gln
    290                 295                 300
Leu Leu Lys Leu Tyr Gly Ile Pro Tyr Leu Gly Asn Val Met Trp Leu
305                 310                 315                 320
Asp Leu Val Thr Tyr Leu His His His Gly His Glu Asp Lys Leu Pro
                325                 330                 335
Trp Tyr Arg Gly Lys Glu Trp Ser Tyr Leu Arg Gly Gly Leu Thr Thr
            340                 345                 350
Leu Asp Arg Asp Tyr Gly Trp Ile Asn Asn Ile His His Asp Ile Gly
        355                 360                 365
Thr His Val Ile His His Leu Phe Pro Gln Ile Pro His Tyr His Leu
    370                 375                 380
Ile Glu Ala Thr Glu Ala Ala Lys Pro Val Leu Gly Lys Tyr Tyr Arg
385                 390                 395                 400
Glu Pro Lys Lys Ser Ala Pro Leu Pro Phe His Leu Leu Gly Asp Leu
                405                 410                 415
Thr Arg Ser Leu Lys Arg Asp His Tyr Val Ser Asp Val Gly Asp Val
            420                 425                 430
Val Tyr Tyr Gln Thr Asp Pro Gln Leu Thr Gly Ala Glu Lys Ser
        435                 440                 445 
           
             9 
             436 
             PRT 
             Limnanthes douglasii 
           
            9
Met Ala Ser Trp Val Leu Ser Gln Tyr Ala Leu Asn Pro Leu Pro His
  1               5                  10                  15
Ile Phe Arg Thr Pro Arg Thr Ser Ile Thr Ser His Lys Leu Thr Val
             20                  25                  30
Ser His Thr Asn Asn Arg Ala Thr Pro Asp Leu Thr Lys Leu Ser Leu
         35                  40                  45
Ile Lys Phe Arg Glu Arg Lys Leu Gly Leu Arg Val Ser Ala Pro Phe
     50                  55                  60
Gln Ile Ala Ser Thr Thr Pro Glu Glu Glu Asp Glu Val Ala Glu Phe
 65                  70                  75                  80
Asp Pro Gly Ser Pro Pro Pro Phe Lys Leu Ala Asp Ile Arg Ala Ala
                 85                  90                  95
Ile Pro Lys His Cys Trp Val Lys Asn Gln Trp Arg Ser Met Ser Tyr
            100                 105                 110
Val Val Arg Asp Val Val Ile Val Leu Gly Leu Ala Ala Ala Ala Val
        115                 120                 125
Ala Ala Asn Ser Trp Ala Val Trp Pro Leu Tyr Trp Val Ala Gln Gly
    130                 135                 140
Thr Met Phe Trp Ala Leu Phe Val Leu Gly His Asp Cys Gly His Gly
145                 150                 155                 160
Ser Phe Ser Asn Asn His Lys Leu Asn Ser Val Val Gly His Leu Leu
                165                 170                 175
His Ser Ser Ile Leu Val Pro Tyr His Gly Trp Arg Ile Arg His Arg
            180                 185                 190
Thr His His Gln Asn His Gly His Val Glu Asn Asp Glu Ser Trp His
        195                 200                 205
Pro Met Ser Glu Lys Leu Phe Arg Ser Leu Asp Lys Ile Ala Leu Thr
    210                 215                 220
Phe Arg Phe Lys Ala Pro Phe Pro Met Leu Ala Tyr Pro Phe Tyr Leu
225                 230                 235                 240
Trp Glu Arg Ser Pro Gly Lys Thr Gly Ser His Tyr His Pro Asp Ser
                245                 250                 255
Asp Leu Phe Val Pro Ser Glu Lys Lys Asp Val Ile Thr Ser Thr Ile
            260                 265                 270
Cys Trp Thr Thr Met Val Gly Leu Leu Ile Gly Leu Ser Phe Val Met
        275                 280                 285
Gly Pro Ile Gln Ile Leu Lys Leu Tyr Val Val Pro Tyr Trp Ile Phe
    290                 295                 300
Val Met Trp Leu Asp Phe Val Thr Tyr Leu Asp His His Gly His Glu
305                 310                 315                 320
Asp Lys Leu Pro Trp Tyr Arg Gly Glu Glu Trp Ser Tyr Leu Arg Gly
                325                 330                 335
Gly Leu Thr Thr Leu Asp Arg Asp Tyr Gly Leu Ile Asn Asn Ile His
            340                 345                 350
His Asp Ile Gly Thr His Val Ile His His Leu Phe Pro Gln Ile Pro
        355                 360                 365
His Tyr His Leu Val Glu Ala Thr Gln Ala Ala Lys Pro Ile Phe Gly
    370                 375                 380
Lys Tyr Tyr Lys Glu Pro Ala Lys Ser Lys Pro Leu Pro Phe His Leu
385                 390                 395                 400
Ile Asp Val Leu Leu Lys Ser Leu Lys Arg Asp His Phe Val Pro Asp
                405                 410                 415
Thr Gly Asp Ile Val Tyr Tyr Gln Ser Asp Pro Gln Ile Ser Gly Ser
            420                 425                 430
Leu Lys Pro Glu
        435 
           
             10 
             453 
             PRT 
             Glycine max 
           
            10
Met Ala Thr Trp Tyr His Gln Lys Cys Gly Leu Lys Pro Leu Ala Pro
  1               5                  10                  15
Val Ile Pro Arg Pro Arg Thr Gly Ala Ala Leu Ser Ser Thr Ser Arg
             20                  25                  30
Val Glu Phe Leu Asp Thr Asn Lys Val Val Ala Gly Pro Lys Phe Gln
         35                  40                  45
Pro Leu Arg Cys Asn Leu Arg Glu Arg Asn Trp Gly Leu Lys Val Ser
     50                  55                  60
Ala Pro Leu Arg Val Ala Ser Ile Glu Glu Glu Gln Lys Ser Val Asp
 65                  70                  75                  80
Leu Thr Asn Gly Thr Asn Gly Val Glu His Glu Lys Leu Pro Glu Phe
                 85                  90                  95
Asp Pro Gly Ala Pro Pro Pro Phe Asn Leu Ala Asp Ile Arg Ala Ala
            100                 105                 110
Ile Pro Lys His Cys Trp Val Lys Asp Pro Trp Arg Ser Met Ser Tyr
        115                 120                 125
Val Val Arg Asp Val Ile Ala Val Phe Gly Leu Ala Ala Ala Ala Ala
    130                 135                 140
Tyr Leu Asn Asn Trp Leu Val Trp Pro Leu Tyr Trp Ala Ala Gln Gly
145                 150                 155                 160
Thr Met Phe Trp Ala Leu Phe Val Leu Gly His Asp Cys Gly His Gly
                165                 170                 175
Ser Phe Ser Asn Asn Ser Lys Leu Asn Ser Val Val Gly His Leu Leu
            180                 185                 190
His Ser Ser Ile Leu Val Pro Tyr His Gly Trp Arg Ile Ser His Arg
        195                 200                 205
Thr His His Gln His His Gly His Ala Glu Asn Asp Glu Ser Trp His
    210                 215                 220
Pro Leu Pro Glu Lys Leu Phe Arg Ser Leu Asp Thr Val Thr Arg Met
225                 230                 235                 240
Leu Arg Phe Thr Ala Pro Phe Pro Leu Leu Ala Phe Pro Val Tyr Leu
                245                 250                 255
Phe Ser Arg Ser Pro Gly Lys Thr Gly Ser His Phe Asp Pro Ser Ser
            260                 265                 270
Asp Leu Phe Val Pro Asn Glu Arg Lys Asp Val Ile Thr Ser Thr Ala
        275                 280                 285
Cys Trp Ala Ala Met Leu Gly Leu Leu Val Gly Leu Gly Phe Val Met
    290                 295                 300
Gly Pro Ile Gln Leu Leu Lys Leu Tyr Gly Val Pro Tyr Val Ile Phe
305                 310                 315                 320
Val Met Trp Leu Asp Leu Val Thr Tyr Leu His His His Gly His Glu
                325                 330                 335
Asp Lys Leu Pro Trp Tyr Arg Gly Lys Glu Trp Ser Tyr Leu Arg Gly
            340                 345                 350
Gly Leu Thr Thr Leu Asp Arg Asp Tyr Gly Trp Ile Asn Asn Ile His
        355                 360                 365
His Asp Ile Gly Thr His Val Ile His His Leu Phe Pro Gln Ile Pro
    370                 375                 380
His Tyr His Leu Val Glu Ala Thr Glu Ala Ala Lys Pro Val Phe Gly
385                 390                 395                 400
Lys Tyr Tyr Arg Glu Pro Lys Lys Ser Ala Ala Pro Leu Pro Phe His
                405                 410                 415
Leu Ile Gly Glu Ile Ile Arg Ser Phe Lys Thr Asp His Phe Val Ser
            420                 425                 430
Asp Thr Gly Asp Val Val Tyr Tyr Gln Thr Asp Ser Lys Ile Asn Gly
        435                 440                 445
Ser Ser Lys Leu Glu
    450 
           
             11 
             386 
             PRT 
             Arabidopsis thaliana 
           
            11
Met Val Val Ala Met Asp Gln Arg Thr Asn Val Asn Gly Asp Pro Gly
  1               5                  10                  15
Ala Gly Asp Arg Lys Lys Glu Glu Arg Phe Asp Pro Ser Ala Gln Pro
             20                  25                  30
Pro Phe Lys Ile Gly Asp Ile Arg Ala Ala Ile Pro Lys His Cys Trp
         35                  40                  45
Val Lys Ser Pro Leu Arg Ser Met Ser Tyr Val Val Arg Asp Ile Ile
     50                  55                  60
Ala Val Ala Ala Leu Ala Ile Ala Ala Val Tyr Val Asp Ser Trp Phe
 65                  70                  75                  80
Leu Trp Pro Leu Tyr Trp Ala Ala Gln Gly Thr Leu Phe Trp Ala Ile
                 85                  90                  95
Phe Val Leu Gly His Asp Cys Gly His Gly Ser Phe Ser Asp Ile Pro
            100                 105                 110
Leu Leu Asn Ser Val Val Gly His Ile Leu His Ser Phe Ile Leu Val
        115                 120                 125
Pro Tyr His Gly Trp Arg Ile Ser His Arg Thr His His Gln Asn His
    130                 135                 140
Gly His Val Glu Asn Asp Glu Ser Trp Val Pro Leu Pro Glu Arg Val
145                 150                 155                 160
Tyr Lys Lys Leu Pro His Ser Thr Arg Met Leu Arg Tyr Thr Val Pro
                165                 170                 175
Leu Pro Met Leu Ala Tyr Pro Leu Tyr Leu Cys Tyr Arg Ser Pro Gly
            180                 185                 190
Lys Glu Gly Ser His Phe Asn Pro Tyr Ser Ser Leu Phe Ala Pro Ser
        195                 200                 205
Glu Arg Lys Leu Ile Ala Thr Ser Thr Thr Cys Trp Ser Ile Met Phe
    210                 215                 220
Val Ser Leu Ile Ala Leu Ser Phe Val Phe Gly Pro Leu Ala Val Leu
225                 230                 235                 240
Lys Val Tyr Gly Val Pro Tyr Ile Ile Phe Val Met Trp Leu Asp Ala
                245                 250                 255
Val Thr Tyr Leu His His His Gly His Asp Glu Lys Leu Pro Trp Tyr
            260                 265                 270
Arg Gly Lys Glu Trp Ser Tyr Leu Arg Gly Gly Leu Thr Thr Ile Asp
        275                 280                 285
Arg Asp Tyr Gly Ile Phe Asn Asn Ile His His Asp Ile Gly Thr His
    290                 295                 300
Val Ile His His Leu Phe Pro Gln Ile Pro His Tyr His Leu Val Asp
305                 310                 315                 320
Ala Thr Lys Ala Ala Lys His Val Leu Gly Arg Tyr Tyr Arg Glu Pro
                325                 330                 335
Lys Thr Ser Gly Ala Ile Pro Ile His Leu Val Glu Ser Leu Val Ala
            340                 345                 350
Ser Ile Lys Lys Asp His Tyr Val Ser Asp Thr Gly Asp Ile Val Phe
        355                 360                 365
Tyr Glu Thr Asp Pro Asp Leu Tyr Val Tyr Ala Ser Asp Lys Ser Lys
    370                 375                 380
Ile Asn
385 
           
             12 
             377 
             PRT 
             Brassica napus 
           
            12
Met Val Val Ala Met Asp Gln Arg Ser Asn Ala Asn Gly Asp Glu Arg
  1               5                  10                  15
Phe Asp Pro Ser Ala Gln Pro Pro Phe Lys Ile Gly Asp Ile Arg Ala
             20                  25                  30
Ala Ile Pro Lys His Cys Trp Val Lys Ser Pro Leu Arg Ser Met Ser
         35                  40                  45
Tyr Val Ala Arg Asp Ile Phe Ala Val Val Ala Leu Ala Val Ala Ala
     50                  55                  60
Val Tyr Phe Asp Ser Trp Phe Phe Trp Pro Leu Tyr Trp Ala Ala Gln
 65                  70                  75                  80
Gly Thr Leu Phe Trp Ala Ile Phe Val Leu Gly His Asp Cys Gly His
                 85                  90                  95
Gly Ser Phe Ser Asp Ile Pro Leu Leu Asn Thr Ala Val Gly His Ile
            100                 105                 110
Leu His Ser Phe Ile Leu Val Pro Tyr His Gly Trp Arg Ile Ser His
        115                 120                 125
Arg Thr His His Gln Asn His Gly His Val Glu Asn Asp Glu Ser Trp
    130                 135                 140
Val Pro Leu Pro Glu Lys Leu Tyr Lys Asn Leu Ser His Ser Thr Arg
145                 150                 155                 160
Met Leu Arg Tyr Thr Val Pro Leu Pro Met Leu Ala Tyr Pro Leu Tyr
                165                 170                 175
Leu Trp Tyr Arg Ser Pro Gly Lys Glu Gly Ser His Tyr Asn Pro Tyr
            180                 185                 190
Ser Ser Leu Phe Ala Pro Ser Glu Arg Lys Leu Ile Ala Thr Ser Thr
        195                 200                 205
Thr Cys Trp Ser Ile Met Leu Ala Thr Leu Val Tyr Leu Ser Phe Leu
    210                 215                 220
Val Gly Pro Val Thr Val Leu Lys Val Tyr Gly Val Pro Tyr Ile Ile
225                 230                 235                 240
Phe Val Met Trp Leu Asp Ala Val Thr Tyr Leu His His His Gly His
                245                 250                 255
Asp Asp Lys Leu Pro Trp Tyr Arg Gly Lys Glu Trp Ser Tyr Leu Arg
            260                 265                 270
Gly Gly Leu Thr Thr Ile Asp Arg Asp Tyr Gly Ile Phe Asn Asn Ile
        275                 280                 285
His His Asp Ile Gly Thr His Val Ile His His Leu Phe Pro Gln Ile
    290                 295                 300
Pro His Tyr His Leu Val Asp Ala Thr Lys Ser Ala Lys His Val Leu
305                 310                 315                 320
Gly Arg Tyr Tyr Arg Glu Pro Lys Thr Ser Gly Ala Ile Pro Ile His
                325                 330                 335
Leu Val Glu Ser Leu Val Ala Ser Ile Lys Lys Asp His Tyr Val Ser
            340                 345                 350
Asp Thr Gly Asp Ile Val Phe Tyr Glu Thr Asp Pro Asp Leu Tyr Val
        355                 360                 365
Tyr Ala Ser Asp Lys Ser Lys Ile Asn
    370                 375 
           
             13 
             380 
             PRT 
             Glycine max 
           
            13
Met Val Lys Asp Thr Lys Pro Leu Ala Tyr Ala Ala Asn Asn Gly Tyr
  1               5                  10                  15
Gln Gln Lys Gly Ser Ser Phe Asp Phe Asp Pro Ser Ala Pro Pro Pro
             20                  25                  30
Phe Lys Ile Ala Glu Ile Arg Ala Ser Ile Pro Lys His Cys Trp Val
         35                  40                  45
Lys Asn Pro Trp Arg Ser Leu Ser Tyr Val Leu Arg Asp Val Leu Val
     50                  55                  60
Ile Ala Ala Leu Val Ala Ala Ala Ile His Phe Asp Asn Trp Leu Leu
 65                  70                  75                  80
Trp Leu Ile Tyr Cys Pro Ile Gln Gly Thr Met Phe Trp Ala Leu Phe
                 85                  90                  95
Val Leu Gly His Asp Cys Gly His Gly Ser Phe Ser Asp Ser Pro Leu
            100                 105                 110
Leu Asn Ser Leu Val Gly His Ile Leu His Ser Ser Ile Leu Val Pro
        115                 120                 125
Tyr His Gly Trp Arg Ile Ser His Arg Thr His His Gln Asn His Gly
    130                 135                 140
His Ile Glu Lys Asp Glu Ser Trp Val Pro Leu Thr Glu Lys Ile Tyr
145                 150                 155                 160
Lys Asn Leu Asp Ser Met Thr Arg Leu Ile Arg Phe Thr Val Pro Phe
                165                 170                 175
Pro Leu Phe Val Tyr Pro Ile Tyr Leu Phe Ser Arg Ser Pro Gly Lys
            180                 185                 190
Glu Gly Ser His Phe Asn Pro Tyr Ser Asn Leu Phe Pro Pro Ser Glu
        195                 200                 205
Arg Lys Gly Ile Ala Ile Ser Thr Leu Cys Trp Ala Thr Met Phe Ser
    210                 215                 220
Leu Leu Ile Tyr Leu Ser Phe Ile Thr Ser Pro Leu Leu Val Leu Lys
225                 230                 235                 240
Leu Tyr Gly Ile Pro Tyr Trp Ile Phe Val Met Trp Leu Asp Phe Val
                245                 250                 255
Thr Tyr Leu His His His Gly His His Gln Lys Leu Pro Trp Tyr Arg
            260                 265                 270
Gly Lys Glu Trp Ser Tyr Leu Arg Gly Gly Leu Thr Thr Val Asp Arg
        275                 280                 285
Asp Tyr Gly Trp Ile Tyr Asn Ile His His Asp Ile Gly Thr His Val
    290                 295                 300
Ile His His Leu Phe Pro Gln Ile Pro His Tyr His Leu Val Glu Ala
305                 310                 315                 320
Thr Gln Ala Ala Lys Pro Val Leu Gly Asp Tyr Tyr Arg Glu Pro Glu
                325                 330                 335
Arg Ser Ala Pro Leu Pro Phe His Leu Ile Lys Tyr Leu Ile Gln Ser
            340                 345                 350
Met Arg Gln Asp His Phe Val Ser Asp Thr Gly Asp Val Val Tyr Tyr
        355                 360                 365
Gln Thr Asp Ser Leu Leu Leu His Ser Gln Arg Asp
    370                 375                 380 
           
             14 
             383 
             PRT 
             Arabidopsis thaliana 
           
            14
Met Gly Ala Gly Gly Arg Met Pro Val Pro Thr Ser Ser Lys Lys Ser
  1               5                  10                  15
Glu Thr Asp Thr Thr Lys Arg Val Pro Cys Glu Lys Pro Pro Phe Ser
             20                  25                  30
Val Gly Asp Leu Lys Lys Ala Ile Pro Pro His Cys Phe Lys Arg Ser
         35                  40                  45
Ile Pro Arg Ser Phe Ser Tyr Leu Ile Ser Asp Ile Ile Ile Ala Ser
     50                  55                  60
Cys Phe Tyr Tyr Val Ala Thr Asn Tyr Phe Ser Leu Leu Pro Gln Pro
 65                  70                  75                  80
Leu Ser Tyr Leu Ala Trp Pro Leu Tyr Trp Ala Cys Gln Gly Cys Val
                 85                  90                  95
Leu Thr Gly Ile Trp Val Ile Ala His Glu Cys Gly His His Ala Phe
            100                 105                 110
Ser Asp Tyr Gln Trp Leu Asp Asp Thr Val Gly Leu Ile Phe His Ser
        115                 120                 125
Phe Leu Leu Val Pro Tyr Phe Ser Trp Lys Tyr Ser His Arg Arg His
    130                 135                 140
His Ser Asn Thr Gly Ser Leu Glu Arg Asp Glu Val Phe Val Pro Lys
145                 150                 155                 160
Gln Lys Ser Ala Ile Lys Trp Tyr Gly Lys Tyr Leu Asn Asn Pro Leu
                165                 170                 175
Gly Arg Ile Met Met Leu Thr Val Gln Phe Val Leu Gly Trp Pro Leu
            180                 185                 190
Tyr Leu Ala Phe Asn Val Ser Gly Arg Pro Tyr Asp Gly Phe Ala Cys
        195                 200                 205
His Phe Phe Pro Asn Ala Pro Ile Tyr Asn Asp Arg Glu Arg Leu Gln
    210                 215                 220
Ile Tyr Leu Ser Asp Ala Gly Ile Leu Ala Val Cys Phe Gly Leu Tyr
225                 230                 235                 240
Arg Tyr Ala Ala Ala Gln Gly Met Ala Ser Met Ile Cys Leu Tyr Gly
                245                 250                 255
Val Pro Leu Leu Ile Val Asn Ala Phe Leu Val Leu Ile Thr Tyr Leu
            260                 265                 270
Gln His Thr His Pro Ser Leu Pro His Tyr Asp Ser Ser Glu Trp Asp
        275                 280                 285
Trp Leu Arg Gly Ala Leu Ala Thr Val Asp Arg Asp Tyr Gly Ile Leu
    290                 295                 300
Asn Lys Val Phe His Asn Ile Thr Asp Thr His Val Ala His His Leu
305                 310                 315                 320
Phe Ser Thr Met Pro His Tyr Asn Ala Met Glu Ala Thr Lys Ala Ile
                325                 330                 335
Lys Pro Ile Leu Gly Asp Tyr Tyr Gln Phe Asp Gly Thr Pro Trp Tyr
            340                 345                 350
Val Ala Met Tyr Arg Glu Ala Lys Glu Cys Ile Tyr Val Glu Pro Asp
        355                 360                 365
Arg Glu Gly Asp Lys Lys Gly Val Tyr Trp Tyr Asn Asn Lys Leu
    370                 375                 380 
           
             15 
             383 
             PRT 
             Glycine max 
           
            15
Met Gly Ala Gly Gly Arg Thr Asp Val Pro Pro Ala Asn Arg Lys Ser
  1               5                  10                  15
Glu Val Asp Pro Leu Lys Arg Val Pro Phe Glu Lys Pro Gln Phe Ser
             20                  25                  30
Leu Ser Gln Ile Lys Lys Ala Ile Pro Pro His Cys Phe Gln Arg Ser
         35                  40                  45
Val Leu Arg Ser Phe Ser Tyr Val Val Tyr Asp Leu Thr Ile Ala Phe
     50                  55                  60
Cys Leu Tyr Tyr Val Ala Thr His Tyr Phe His Leu Leu Pro Gly Pro
 65                  70                  75                  80
Leu Ser Phe Arg Gly Met Ala Ile Tyr Trp Ala Val Gln Gly Cys Ile
                 85                  90                  95
Leu Thr Gly Val Trp Val Ile Ala His Glu Cys Gly His His Ala Phe
            100                 105                 110
Ser Asp Tyr Gln Leu Leu Asp Asp Ile Val Gly Leu Ile Leu His Ser
        115                 120                 125
Ala Leu Leu Val Pro Tyr Phe Ser Trp Lys Tyr Ser His Arg Arg His
    130                 135                 140
His Ser Asn Thr Gly Ser Leu Glu Arg Asp Glu Val Phe Val Pro Lys
145                 150                 155                 160
Gln Lys Ser Cys Ile Lys Trp Tyr Ser Lys Tyr Leu Asn Asn Pro Pro
                165                 170                 175
Gly Arg Val Leu Thr Leu Ala Val Thr Leu Thr Leu Gly Trp Pro Leu
            180                 185                 190
Tyr Leu Ala Leu Asn Val Ser Gly Arg Pro Tyr Asp Arg Phe Ala Cys
        195                 200                 205
His Tyr Asp Pro Tyr Gly Pro Ile Tyr Ser Asp Arg Glu Arg Leu Gln
    210                 215                 220
Ile Tyr Ile Ser Asp Ala Gly Val Leu Ala Val Val Tyr Gly Leu Phe
225                 230                 235                 240
Arg Leu Ala Met Ala Lys Gly Leu Ala Trp Val Val Cys Val Tyr Gly
                245                 250                 255
Val Pro Leu Leu Val Val Asn Gly Phe Leu Val Leu Ile Thr Phe Leu
            260                 265                 270
Gln His Thr His Pro Ala Leu Pro His Tyr Thr Ser Ser Glu Trp Asp
        275                 280                 285
Trp Leu Arg Gly Ala Leu Ala Thr Val Asp Arg Asp Tyr Gly Ile Leu
    290                 295                 300
Asn Lys Val Phe His Asn Ile Thr Asp Thr His Val Ala His His Leu
305                 310                 315                 320
Phe Ser Thr Met Pro His Tyr His Ala Met Glu Ala Thr Lys Ala Ile
                325                 330                 335
Lys Pro Ile Leu Gly Glu Tyr Tyr Arg Phe Asp Glu Thr Pro Phe Val
            340                 345                 350
Lys Ala Met Trp Arg Glu Ala Arg Glu Cys Ile Tyr Val Glu Pro Asp
        355                 360                 365
Gln Ser Thr Glu Ser Lys Gly Val Phe Trp Tyr Asn Asn Lys Leu
    370                 375                 380 
           
             16 
             383 
             PRT 
             Glycine max 
           
            16
Met Gly Ala Gly Gly Arg Met Ser Ala Pro Asn Gly Glu Thr Glu Val
  1               5                  10                  15
Lys Arg Asn Pro Leu Gln Lys Val Pro Thr Ser Lys Pro Pro Phe Thr
             20                  25                  30
Val Gly Asp Ile Lys Lys Ala Ile Pro Pro His Cys Phe Gln Arg Ser
         35                  40                  45
Leu Ile Arg Ser Phe Ser Tyr Val Val Tyr Asp Leu Ile Leu Val Ser
     50                  55                  60
Ile Met Tyr Tyr Val Ala Asn Thr Tyr Phe His Leu Leu Pro Ser Pro
 65                  70                  75                  80
Tyr Cys Tyr Ile Ala Trp Pro Ile Tyr Trp Ile Cys Gln Gly Cys Val
                 85                  90                  95
Cys Thr Gly Ile Trp Val Asn Ala His Glu Cys Gly His His Ala Phe
            100                 105                 110
Ser Asp Tyr Gln Trp Val Asp Asp Thr Val Gly Leu Ile Leu His Ser
        115                 120                 125
Ala Leu Leu Val Pro Tyr Phe Ser Trp Lys Tyr Ser His Arg Arg His
    130                 135                 140
His Ser Asn Thr Gly Ser Leu Glu Arg Asp Glu Val Phe Val Pro Lys
145                 150                 155                 160
Pro Lys Ser Gln Leu Gly Trp Tyr Ser Lys Tyr Leu Asn Asn Pro Pro
                165                 170                 175
Gly Arg Val Leu Ser Leu Thr Ile Thr Leu Thr Leu Gly Trp Pro Leu
            180                 185                 190
Tyr Leu Ala Phe Asn Val Ser Gly Arg Pro Tyr Asp Arg Phe Ala Cys
        195                 200                 205
His Tyr Asp Pro Tyr Gly Pro Ile Tyr Asn Asn Arg Glu Arg Leu Gln
    210                 215                 220
Ile Phe Ile Ser Asp Ala Gly Val Leu Gly Val Cys Tyr Leu Leu Tyr
225                 230                 235                 240
Arg Ile Ala Leu Val Lys Gly Leu Ala Trp Leu Val Cys Val Tyr Gly
                245                 250                 255
Val Pro Leu Leu Val Val Asn Gly Phe Leu Val Leu Ile Thr Tyr Leu
            260                 265                 270
Gln His Thr His Pro Ser Leu Pro His Tyr Asp Ser Thr Glu Trp Asp
        275                 280                 285
Trp Leu Arg Gly Ala Leu Ala Thr Cys Asp Arg Asp Tyr Gly Val Leu
    290                 295                 300
Asn Lys Val Phe His Asn Ile Thr Asp Thr His Val Val His His Leu
305                 310                 315                 320
Phe Ser Thr Met Pro His Tyr Asn Ala Met Glu Ala Thr Lys Ala Val
                325                 330                 335
Lys Pro Leu Leu Gly Asp Tyr Tyr Gln Phe Asp Gly Thr Pro Ile Tyr
            340                 345                 350
Lys Glu Met Trp Arg Glu Ala Lys Glu Cys Leu Tyr Val Glu Lys Asp
        355                 360                 365
Glu Ser Ser Gln Gly Lys Gly Val Phe Trp Tyr Lys Asn Lys Leu
    370                 375                 380 
           
             17 
             387 
             PRT 
             S. commersonii 
           
            17
Met Gly Leu Ala Lys Glu Thr Thr Met Gly Gly Arg Gly Arg Val Ala
  1               5                  10                  15
Lys Val Glu Val Gln Gly Lys Lys Pro Leu Ser Arg Val Pro Asn Thr
             20                  25                  30
Lys Pro Pro Phe Thr Val Gly Gln Leu Lys Lys Ala Ile Pro Pro His
         35                  40                  45
Cys Phe Gln Arg Ser Leu Leu Thr Ser Phe Ser Tyr Val Val Tyr Asp
     50                  55                  60
Leu Ser Phe Ala Phe Ile Phe Tyr Ile Ala Thr Thr Tyr Phe His Leu
 65                  70                  75                  80
Leu Pro Gln Pro Phe Ser Leu Ile Ala Trp Pro Ile Tyr Trp Val Leu
                 85                  90                  95
Gln Gly Cys Leu Leu Thr Gly Val Trp Val Ile Ala His Glu Cys Gly
            100                 105                 110
His His Ala Phe Ser Lys Tyr Gln Trp Val Asp Asp Val Val Gly Leu
        115                 120                 125
Thr Leu His Ser Thr Leu Leu Val Pro Tyr Phe Ser Trp Lys Ile Ser
    130                 135                 140
His Arg Arg His His Ser Asn Thr Gly Ser Leu Asp Arg Asp Glu Val
145                 150                 155                 160
Phe Val Pro Lys Pro Lys Ser Lys Val Ala Trp Phe Ser Lys Tyr Leu
                165                 170                 175
Asn Asn Pro Leu Gly Arg Ala Val Ser Leu Leu Val Thr Leu Thr Ile
            180                 185                 190
Gly Trp Pro Met Tyr Leu Ala Phe Asn Val Ser Gly Arg Pro Tyr Asp
        195                 200                 205
Ser Phe Ala Ser His Tyr His Pro Tyr Ala Pro Ile Tyr Ser Asn Arg
    210                 215                 220
Glu Arg Leu Leu Ile Tyr Val Ser Asp Val Ala Leu Phe Ser Val Thr
225                 230                 235                 240
Tyr Ser Leu Tyr Arg Val Ala Thr Leu Lys Gly Leu Val Trp Leu Leu
                245                 250                 255
Cys Val Tyr Gly Val Pro Leu Leu Ile Val Asn Gly Phe Leu Val Thr
            260                 265                 270
Ile Thr Tyr Leu Gln His Thr His Phe Ala Leu Pro His Tyr Asp Ser
        275                 280                 285
Ser Glu Trp Asp Trp Leu Lys Gly Ala Leu Ala Thr Met Asp Arg Asp
    290                 295                 300
Tyr Gly Ile Leu Asn Lys Val Phe His His Ile Thr Asp Thr His Val
305                 310                 315                 320
Ala His His Leu Phe Ser Thr Met Pro His Tyr His Ala Met Glu Ala
                325                 330                 335
Thr Asn Ala Ile Lys Pro Ile Leu Gly Glu Tyr Tyr Gln Phe Asp Asp
            340                 345                 350
Thr Pro Phe Tyr Lys Ala Leu Trp Arg Glu Ala Arg Glu Cys Leu Tyr
        355                 360                 365
Val Glu Pro Asp Glu Gly Thr Ser Glu Lys Gly Val Tyr Trp Tyr Arg
    370                 375                 380
Asn Lys Tyr
385 
           
             18 
             387 
             PRT 
             Ricinus communis 
           
            18
Met Gly Gly Gly Gly Arg Met Ser Thr Val Ile Thr Ser Asn Asn Ser
  1               5                  10                  15
Glu Lys Lys Gly Gly Ser Ser His Leu Lys Arg Ala Pro His Thr Lys
             20                  25                  30
Pro Pro Phe Thr Leu Gly Asp Leu Lys Arg Ala Ile Pro Pro His Cys
         35                  40                  45
Phe Glu Arg Ser Phe Val Arg Ser Phe Ser Tyr Val Ala Tyr Asp Val
     50                  55                  60
Cys Leu Ser Phe Leu Phe Tyr Ser Ile Ala Thr Asn Phe Phe Pro Tyr
 65                  70                  75                  80
Ile Ser Ser Pro Leu Ser Tyr Val Ala Trp Leu Val Tyr Trp Leu Phe
                 85                  90                  95
Gln Gly Cys Ile Leu Thr Gly Leu Trp Val Ile Gly His Glu Cys Gly
            100                 105                 110
His His Ala Phe Ser Glu Tyr Gln Leu Ala Asp Asp Ile Val Gly Leu
        115                 120                 125
Ile Val His Ser Ala Leu Leu Val Pro Tyr Phe Ser Trp Lys Tyr Ser
    130                 135                 140
His Arg Arg His His Ser Asn Ile Gly Ser Leu Glu Arg Asp Glu Val
145                 150                 155                 160
Phe Val Pro Lys Ser Lys Ser Lys Ile Ser Trp Tyr Ser Lys Tyr Ser
                165                 170                 175
Asn Asn Pro Pro Gly Arg Val Leu Thr Leu Ala Ala Thr Leu Leu Leu
            180                 185                 190
Gly Trp Pro Leu Tyr Leu Ala Phe Asn Val Ser Gly Arg Pro Tyr Asp
        195                 200                 205
Arg Phe Ala Cys His Tyr Asp Pro Tyr Gly Pro Ile Phe Ser Glu Arg
    210                 215                 220
Glu Arg Leu Gln Ile Tyr Ile Ala Asp Leu Gly Ile Phe Ala Thr Thr
225                 230                 235                 240
Phe Val Leu Tyr Gln Ala Thr Met Ala Lys Gly Leu Ala Trp Val Met
                245                 250                 255
Arg Ile Tyr Gly Val Pro Leu Leu Ile Val Asn Cys Phe Leu Val Met
            260                 265                 270
Ile Thr Tyr Leu Gln His Thr His Pro Ala Ile Pro Arg Tyr Gly Ser
        275                 280                 285
Ser Glu Trp Asp Trp Leu Arg Gly Ala Met Val Thr Val Asp Arg Asp
    290                 295                 300
Tyr Gly Val Leu Asn Lys Val Phe His Asn Ile Ala Asp Thr His Val
305                 310                 315                 320
Ala His His Leu Phe Ala Thr Val Pro His Tyr His Ala Met Glu Ala
                325                 330                 335
Thr Lys Ala Ile Lys Pro Ile Met Gly Glu Tyr Tyr Arg Tyr Asp Gly
            340                 345                 350
Thr Pro Phe Tyr Lys Ala Leu Trp Arg Glu Ala Lys Glu Cys Leu Phe
        355                 360                 365
Val Glu Pro Asp Glu Gly Ala Pro Thr Gln Gly Val Phe Trp Tyr Arg
    370                 375                 380
Asn Lys Tyr
385 
           
             19 
             18 
             DNA 
             Unknown 
             
               Description of Unknown Organism unknown 
             
           
            19
gsncaygant gygsncay                                                   18 
           
             20 
             20 
             DNA 
             Unknown 
             
               Description of Unknown Organism unknown 
             
           
            20
ranadrtgrt gnrbnayrtg                                                 20 
           
             21 
             20 
             DNA 
             Unknown 
             
               Description of Unknown Organism unknown 
             
           
            21
tggmgnttya arcaygaymg                                                 20 
           
             22 
             20 
             DNA 
             Unknown 
             
               Description of Unknown Organism unknown 
             
           
            22
gtnswcatcc araartgrta                                                 20 
           
             23 
             20 
             DNA 
             Unknown 
             
               Description of Unknown Organism unknown 
             
           
            23
caygartgyg gncaycaygc                                                 20 
           
             24 
             20 
             DNA 
             Unknown 
             
               Description of Unknown Organism unknown 
             
           
            24
ccncknarcc artcccaytc                                                 20 
           
             25 
             20 
             DNA 
             Unknown 
             
               Description of Unknown Organism unknown 
             
           
            25
acncaycayc araaycaygg                                                 20 
           
             26 
             20 
             DNA 
             Unknown 
             
               Description of Unknown Organism unknown 
             
           
            26
caytgyttnc cnckrtacca                                                 20