Herbicides test method

This application claims priority PCT/EP98/03261, filed Jun. 2, 1998 which in turn claims priority to British patent applications 97 111 63.7 and 97 134 77.9 filed May 31, 1997 and Jun. 27, 1997, respectively.

Not applicable.

The invention relates to plant enzymatic activity, and aspects thereof, involved in the biosynthesis of Coenzyme A. The invention particularly relates to the plant enzyme known either as pantoate-β-alanine ligase (EC 6.3.2.1), pantoate activating enzyme or pantothenate synthetase (PS). PS catalyses the synthesis of pantothenate.

PS is an essential enzyme in the in planta biosynthesis of the vitamin and Coenzyme A precursor pantothenate. It is known to catalyse the following reaction:

ATP +( R )-pantoate+β-alanine→ AMP +pyrophosphate+( R )-pantothenate.

PS genes have previously been isolated from Escherichia coil (GenBank accession number P31663), Bacillus subtilis (GenBank accession number P52998), and the cyanobacterium Synechocystis (GenBank accession number U44896). DNA sequences from Saccharomyces cerevisiae (GenBank accession number P40459) and Schizosaccharomyces pombe (GenBank accession number Q09673) having unknown functions have been proposed to code for PS enzymes based on DNA and deduced amino acid sequence similarities. To date, however, no gene has been reported which codes for the PS enzyme in any plant species. It is therefore an object of the invention to identify, isolate and sequence a gene coding for the PS enzyme present in plants.

A number of assays have been reported for measuring PS activity. One assay developed by Maas (1950a and 1950b) uses a microbiological assay of pantothenate based on the ability to promote growth of an E. coli pantothenate auxotroph (M99-1, panC). The assay developed by Pfleiderer et al (1960) measures the AMP liberated in the PS reaction. In this assay, myokinase catalyses the production of 2 moles of ADP for each mole of AMP released in pantothenate synthesis using ATP supplied in the assay mixture. Pyruvate kinase then generates 2 moles of pyruvate and ATP for 2 moles of phosphoenolpyruvate and ADP. Finally, lactate dehydrogenase reduces 2 moles of pyruvate to yield 2 moles of lactate concomitant with stoichiometric oxidation of NADH to NAD, which can be monitored spectrophotometrically by following the absorbance at 340 nm. A third assay, developed by Miyatake et al (1979), employs an assay mix containing 14 C-β-alanine and unlabelled pantoate. In this assay any 14 C-pantothenate formed is separated from unreacted 14 C-β-alanine by cation exchange chromatography and subsequently quantified by liquid scintillation counting. These assays, however, are not suitable for use with high throughput biochemical screening and cannot be used for the large scale biochemical screening of compounds necessary to discover useful inhibitors of PS.

We have developed an invention which addresses the above-mentioned drawbacks associated with the prior art. Our invention covers a number of related aspects which encompass the same inventive concept.

According to a first aspect of the invention there is provided an isolated DNA molecule encoding a protein from a plant, which protein has PS activity. In preferred embodiments, the DNA is isolated from Lotus japonicus or Oryza sativa.

To support our invention we herein disclose the cDNA sequence from Lotus japonicus . In addition, we have shown that a previously unassigned expressed sequence tag of Oryza sativa (GenBank accession number D25017) is part of a cDNA coding sequence for a PS enzyme in Oryza sativa and disclose, as part of this invention, the full cDNA sequence of the PS gene from Oryza sativa . Furthermore, we have confirmed by sequence similarity, functional complementation of an Escherichia coli mutant devoid of PS enzyme activity, and by enzyme assays that the DNA sequence from Saccharomyces cerevisiae (GenBank accession number P40459) putatively ascribed as coding for a PS enzyme does code for the PS enzyme of S. cerevisiae . A cDNA sequence coding for a PS enzyme in L. japonicus is provided in FIG. 2 (SEQ. ID NO:1). A cDNA sequence coding for a PS enzyme in O. sativa is provided in FIG. 4 (SEQ ID NO:2). A DNA sequence coding for a PS enzyme in S. cerevisiae is provided in FIG. 8 (SEQ ID NO:3). As a result of our invention it is now possible to obtain the DNA coding sequence for the PS enzyme(s) from any plant source using methods available to those skilled in the art.

A further preferred embodiment of this aspect of our invention is an isolated DNA molecule encoding a protein from L. japonicus having PS activity wherein said protein comprises the amino acid sequence set forth in FIG. 2 (SEQ ID NO:4). A still further embodiment is an isolated DNA molecule encoding a protein from O. sativa having PS activity wherein said protein comprises the amino acid sequence in FIG. 4 (SEQ ID NO:5).

In addition, we have extended our invention to include a further aspect so as to provide a non-naturally occurring chimeric gene comprising a promoter operably linked to a DNA molecule encoding a protein from a plant having PS activity. Preferably, the protein is isolated from a dicotyledonous or a monocotyledonous plant, such as L. japonicus or O. sativa . Preferably the amino acid sequence is selected from the group set forth in FIG. 2 ( L. japonicus ) and FIG. 4 ( O. sativa ).

We have developed our invention into another aspect which provides a recombinant vector comprising a chimeric gene, wherein the vector is capable of being stably transformed into a host cell. Also comprised in this aspect is the host cell stably transformed with the vector wherein the host cell is preferably a cell selected from the group consisting of a bacterial cell, a yeast cell, and an insect cell and is further capable of expressing the DNA molecule according to the invention.

In a still further aspect we have applied our invention to the recombinant production of the PS enzyme. In particular, the invention provides a method of producing a protein having PS activity in a host organism by firstly inserting a DNA sequence encoding a protein having PS activity into an expression cassette designed for the chosen host; inserting the resultant molecule, containing the individual elements linked in proper reading frame, into a vector capable of being transformed into the host cell; growing the thus transformed host cell in a suitable culture medium; and isolating the protein product either from the transformed cell or the culture medium, or both, and purifying it.

In addition, we have developed our invention to provide methods for assaying a protein having pantothenate synthetase activity comprising; incubating pantothenate synthetase in a suitable reaction mixture in which pantothenate synthetase is capable of catalysing the conversion of pantoate, □-alanine and ATP to pantothenate, AMP and pyrophosphate; determining the amount of pyrophosphate formed by a calorimetric technique based on the assay for pyrophosphate developed by Chang et al. (1983); or converting the pyrophosphate formed by the catalytic activity of the pantothenate synthetase into inorganic phosphate by the catalytic activity of an inorganic pyrophosphatase, preferably yeast inorganic pyrophosphatase; and determining the amount of inorganic phosphate generated by the catalytic activity of said inorganic pyrophosphatase by calorimetric techniques, preferably by techniques based either on the assay for inorganic phosphate developed by Lanzetta et al. (1979) or on the assay for inorganic phosphate developed by Chifflet et al. (1988).

The production of PS, for example by using the recombinant methodology described hereinabove, has enabled us to develop methods of using purified PS to screen for novel inhibitors of PS activity which may be used as herbicides to control undesirable vegetation in fields where crops are grown, particularly agronomically important crops such as maize and other cereal crops such as wheat, oats, rye, sorghum, rice, barley, millet, turf and forage grasses, and the like, as well as cotton sugar cane, sugar beet, oilseed rape, and soybeans.

Referring to FIG. 1, there is shown a partial restriction map (FIG. 1 A), subcloning (FIG. 1 B), and nucleotide sequencing (FIG. 1C) of pLC, a L. japonicus cDNA for pantotheate synthetase. FIG. 1 A: The L. japonicus pantotheate synthetase cDNA (pLC) was isolated by functional complementation of E. coli AT1371 (panC). FIG. 1B represents the sub-clones needed for the DNA sequencing strategy which is summarized in FIG. 1 C.

Referring to FIG. 2, there is shown the nucleotide sequence of the L. japonicus cDNA for pantothenate synthetase and its predicted amino acid sequence. FIGS. 2A and 2B show the DNA sequence of the 1.33 kb EcoRI to XhoI insert of pLC (SEQ ID NO: 1) (described in FIG. 1 ). The open reading frame codes for a polypeptide of 308 amino acids with a predicted molecular mass of 34.2 kDa (SEQ ID NO: 4) and with 61% similarity to PS from E. coli . The indicated translation start site is putative and the stop codon (TAA) is translated as “*”. This ORF on pLC is in frame with lacZ which accounts for expression of functional enzyme in E. coli and hence the observed complementation effect

Referring to FIG. 3, there is shown a partial restriction map (FIG. 3 A), subcloning (FIG. 3 B), and nucleotide sequencing (FIG. 3C) of the rice pantothenate synthetase cDNA. The original cDNA for rice gene, pRC1, was subcloned in order to obtain its complete nucleotide sequence. The arrows indicate the position, direction, and length of individual sequencing runs. The open arrow indicates the EST sequence (GenBank accession no. D25017).

Referring to FIGS. 4A and 4B, there is shown the nucleotide sequence of the rice cDNA for pantothenate synthetase and its predicted amino acid sequence. The figure shows the DNA sequence of the 1.26 kb SalI to NotI insert of pRC1 (FIG. 3) (SEQ ID NO: 2). The ORF encodes a polypeptide of 313 residues with a predicted molecular mass of 33.9 kD (SEQ ID NO: 5). The indicated translation start site is putative, and the stop codon is translated as “*”.

Referring to FIG. 5, there is depicted a method for generating the lacZ-pantothenate synthetase fusion clone for the expression of rice pantothenate synthetase in E. coli . The orientation of the cDNA in pRC1 was changed to yield pRC2 where the open reading frame is under transcriptional control of the lacZ promoter. The lacZ-PS fusion was generated by deleting four base pairs from pRC2, and the resulting plasmid, pRC, was sequenced to confirm the deletion (data not shown).

Referring to FIGS. 6A and 6B, the alignment of pantothenate synthetase protein sequences is shown. The PS protein sequences predicted from known ( L. japonicus (SEQ ID NO: 4), O. sativa (SEQ ID NO: 5), E. coli (SEQ ID NO: 6), B. subtilis (SEQ ID NO: 7), Synechocystis sp. (SEQ ID NO: 8)) or putative ( S. cerevisiae (SEQ ID NO: 9), Schizos. pombe (SEQ ID NO: 10)) genes were aligned using CLUSTAL W(1.5) within the GCG software package. Fully conserved residues are marked “*”, functionally conserved ones are marked “.”. lotus: L. japonicus , rice: O. sativa , coli: E. coli (GenBank P31663), subt: B. subtilis (GenBank P52998), syne: Synechocystis sp. (GenBank U44896), yeast: S. cerevisiae (GenBank P40459), pombe: Schizos. pombe (GenBank Q09673).

Referring to FIG. 7, the subcloning of yeast pantothenate synthetase for expression in E. coli is depicted. FIG. 7A shows the γ bacteriophage clone 1PM4950 that was obtained from the Sanger Centre, Hinxton Hall, Cambridge, UK, where the yeast PS sequence had been generated. FIGS. 7B and 7C show yeast panC, subcloned in two steps to yield plasmid clone pYC1, where the gene is placed under transcriptional control of the lac promoter. The ORF position is indicated by arrows. A T3-primed sequencing reaction using pYC1 as template confirmed the identity of the EcoRV-HindIII insert of the plasmid.

Referring to FIG. 8, there is shown the nucleotide sequence of the S. cerevisiae genomic DNA fragment for pantothenate synthetase and its predicted amino acid sequence. FIGS. 8A and 8B show the nucleotide sequence of the 1.5 kb EcoRV to HindIII genomic DNA fragment of S. cerevisiae (SEQ ID NO: 3) that forms the insert of pYC1. The predicted amino acid sequence of yeast PS appears below the open reading frame (SEQ ID NO: 9). A Shine-Dalgarno-like sequence upstream of the translation initiation codon that may fortuitously serve as a RBS in E. coli is underlined. §-EcoRV; ¶ HindIII.

Referring to FIG 9 , there is shown the inverse PCR product of L. japonicus genomic regions flanking panC. FIG. 9A shows a schematic representation of the iPCR product cloned into pCRII. The EcoRI.EcoRI insert was sequenced using T7 and M13 reverse primers. Both sequence runs were performed in duplicate and spanned ca. 600 bases each. FIGS. 9B and 9C show the nucleotide sequence of the cloned iPCR product (SEQ ID NO: 11) and panC ORF (SEQ ID NO: 12). The indicated matches with panC cDNA mean identical sequences. Positions corresponding to the first base (5′-¶) or the last base (3′- §) of the panC cDNA are marked. Within the 5′ flanking genomic sequence, there is a stop-codon in frame with the panC ORF.

Referring to FIGS. 10A and 10B, the expression cassette PCR of L. japonicus pantothenate synthetase, including the 2 primers (SEQ ID NOs: 13 and 14), is shown.

Referring to FIG. 11, there is shown the anion exchange chromatographs of recombinant L. japonicus pantothenate synthetase is shown. A sample of ammonium sulphate precipitated and dialyzed PS was subjected to anion exchange chromatography on a MonoQ HR10/10 column as described in Example 7. FIG. 11A shows the PS activity profile. Fractions 29 through 32 were pooled to give sample PS-I, and fractions 57 through 60 were pooled to give sample PS-II. FIG. 11B shows the protein elution profile followed by continuous measurement of A 280 and potassium chloride gradient employed.

Referring to FIG. 12, the results of a high-throughput assay for recombinant L. japonicus pantothenate synthetase is depicted graphically. FIG. 12A shows the effect of enzyme concentration on the reaction rate. Specific amounts of MonoQ purified PS-I were assayed as described in Example 9, method 2a. The activity-response is proportional in a range from 1 to 4 μg PS per assay. FIG. 12B shows the time course of inorganic phosphate formation. 1.2 mg (), 2.4 mg (▪), and 3.0 mg (▴) of MonoQ purified PS-I were assayed as described in Example 9, method 2a. The activity-response is proportional in a range from 0 to 20 minutes of incubation.

In particular, the present invention relates to a method for assaying a chemical entity for the ability to inhibit the activity of a PS enzyme from a plant by:

a) combining said PS enzyme in a suitable reaction mixture in which

i) said PS enzyme is capable of catalysing the conversion of pantoate, β-alanine and ATP to pantothenate, AMP and pyrophosphate;

ii) the pyrophosphate liberated in the PS reaction is determined by a colorimetric method, preferably by a method based on the assay for pyrophosphate developed by Chang et al. (1983);

iii) or the pyrophosphate liberated in the PS reaction is further converted to inorganic phosphate by the catalytic activity of an inorganic pyrophosphatase, preferably yeast inorganic pyrophosphatase; and

iv) the inorganic phosphate generated by the catalytic activity of inorganic pyrophosphatase is determined by a calorimetric method, preferably by a method based on the assay for inorganic phosphate developed by Lanzetta et al. (1979) or by a method based on the assay for inorganic phosphate developed by Chifflet et al. (1983);

b) combining said chemical and said PS enzyme together in a second reaction mixture under the same conditions as in said first reaction mixture; and

c) measuring the amount of pyrophosphate or inorganic phosphate produced in said first and said—second reaction mixture;

wherein said chemical is capable of inhibiting the activity of said PS enzyme if the amount of pyrophosphate or inorganic phosphate measured in said second reaction mixture is significantly less than the amount of pyrophosphate or inorganic phosphate measured in said first reaction mixture.

The assay principle invented here is not limited to measuring PS activity but can be employed to measure any enzyme whose catalytic activity involves the formation of a substrate-nucleotidyl reaction intermediate by transfer of the nucleotidyl moiety from the corresponding nucleoside triphosphate to a suitable substrate, thereby generating inorganic pyrophosphate as one reaction product. Such enzymes include, but are not limited to, all aminoacyl-tRNA synthetases, asparagine synthetase, acetate thiokinase, dephosphocoenzyme-A-pyrophosphorylase and all enzymes catalysing the formation of nucleotide-diphosphate-sugars. The assays are preferably carried out on a microtiter scale and are preferably employed for the high-throughput biochemical screening of inhibitors of the enzymes.

The present invention is further directed to probes capable of specifically hybridising to a plant PS gene, cDNA or mRNA, wherein the probe comprises a contiguous portion of the coding sequence for a PS enzyme from a plant at least 10 nucleotides in length.

A further aspect the invention provides a method of producing a DNA molecule comprising a DNA portion encoding a protein having PS activity by,

a) preparing a nucleotide probe capable of specifically hybridising to a plant PS gene, cDNA or mRNA, wherein the probe comprises a contiguous portion of the coding sequence for a PS enzyme from a plant at least 10 nucleotides in length;

b) probing for other PS coding sequences in populations of cloned genomic DNA fragments or cDNA fragments from a chosen organism using the nucleotide probe prepared according to step a); and

c) isolating a DNA molecule comprising a DNA portion encoding a protein having PS activity.

DNA encoding the PS enzyme may be isolated from any desired plant species according to the invention. One method taught for isolating a plant PS coding sequence is represented by Example 1. In this method cDNA clones encoding a PS enzyme are identified from a library of cDNA clones derived from the plant of interest based on their ability to supply PS enzymatic activity to a mutant host organism deficient in this activity. Suitable host organisms for use in this method are those which can be used to screen cDNA expression libraries and for which mutants deficient in PS activity are either available or can be routinely generated. Such host organisms include, but are not limited to, E. coli panC (strain AT1371).

Alternatively, plant PS coding sequences may be isolated according to well known techniques based on their sequence homology to the Lotus japonicus PS coding sequence set forth in FIG. 2 or to the O. sativa PS coding sequence set forth in FIG. 4 . In these techniques all or part of the known PS coding sequence is used as a probe which selectively hybridises to other PS coding sequences present in populations of cloned genomic DNA fragments or cDNA fragments (i.e. genomic or cDNA libraries) from a chosen organism. Such state of the art techniques include hybridisation screening of plated DNA libraries and amplification by PCR using oligonucleotide primers corresponding to sequences conserved among known PS amino acid sequences.

For recombinant production of the PS enzyme in a host organism, the plant PS coding sequence may be inserted into an expression cassette designed for the chosen host and introduced into the host where it is recombinantly produced. The choice of specific regulatory sequences such as promoter, signal sequence, 5′ and 3′ untranslated sequences, and enhancer appropriate for the chosen host is within the level of skill of those skilled in the art. The resultant molecule, containing the individual elements linked in proper reading frame, may be inserted into a vector capable of being transformed into the host cell. Suitable expression vectors and methods for recombinant production of proteins are well known for host organisms such as E. coli , yeast and insect cells. Specific examples include plasmids such as pBLUESCRIPT, pFLAG, pTrcHis, and baculovirus expression vectors, for example those derived-from the genome of Autographica californica nuclear polyhedrus virus.

Recombinantly produced plant PS can be isolated and purified using a variety of standard techniques. The actual techniques which may be used will vary depending upon the host organism used, whether the PS enzyme is designed for secretion, and other such factors familiar to those skilled in the art.

Recombinantly produced plant PS is useful for a variety of purposes. For example, it may be used in an in vitro assay to screen known herbicidal chemicals whose target has not been identified to determine if they inhibit PS. Such an in vitro assay may also be used as a more general screen to identify chemicals which inhibit PS activity and which are therefore herbicide candidates. Alternatively, recombinantly produced plant PS may be used to elucidate the complex structure of this enzyme. Such information regarding the structure of the PS enzyme may be used, for example, in the rational design of new inhibitory herbicides.

Typically, the inhibitory effect on PS is determined by a significant reduction, a reduction that is greater than the margin of error inherent in the measurement technique, of pantothenate synthesis in the in vitro assay. Such a determination may be made simply by comparing the amount of pantothenate synthesised in the in vitro assay in the presence and absence of the candidate inhibitor.

The disclosures in British patent applications 97 111 63.7 and 97 134 77.9 from which this application claims priority, and in the abstract accompanying this application are incorporated herein by reference.

The invention will be further described by reference to the following detailed examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless otherwise specified.

A number of standard techniques have been used during development of our invention. These includes: cloning of plant genes by functional complementation (for example Senecoff and Meagher, 1993); the use of inverse PCR to recover fragments of genes not present in conventional libraries (Ocham et al, 1989) and the use of DNA sequence databases to discover PS genes cloned from other species which had unknown function at the time of their submission.

Bradford, M. M. (1976) Anal. Biochem. 72, 248-254.

Chang, G. G., Pan, F. Yeh, C., and Huang, T. M. (1983) Anal. Biochem. 130, 171.

Chifflet, S., Torriglia, A., Chiesa, R., and Tolosa, S. (1988) Anal. Biochem. 168, 1.

Cronan, J. E., Littel, K. J. and Jackowski, S. (1982) J. Bacteriol. 149, 916-922.

Cronan, J. E. (1980) J. Bacteriol. 141, 1291-1297.

Dellaporta, S. L., Wood, J., and Hicks, J. B. (1983) Plant Mol. Biol. Rep. 1, 19-21.

Eadie, G. S. (1942) J. Biol. Chem. 146, 85.

Hebsgaard, S. M., Korning, P. G., Tolstrup, N., Engelbrecht, J., Rouze, P., and Brunak, S. (1996) Nucl. Acids Res. 24, 3439-3452.

Hofstee, B. H. J. (1959) Nature 184, 1296.

Lanzetta, P. A., Alvarez, L. J., Reinach, P. S. and Candia, O. A. (1979) Anal. Biochem. 100, 95-97.

Laemmli, U. K. (1970) Nature 227, 680-685.

Lineweaver, H., and Burk, D. (1934) J. Am. Chem. Soc. 56, 658.

Maas, W. K. (1952a) J. Biol Chem. 198, 23-32.

Maas, W. K. (1952b) J. Bact. 63, 227-232.

MacFerrin, K. D., Terranova, M. P., Schreiber, S. L., and Verdine, G. L. (1990) Proc. Natl. Acad. Sci. USA 87,1937-1941.

Miyatake, K., Nakano, Y., and Kitaoka, S. (1979) Methods Enzymol. 62, 215-219.

Ochman, H, Ajioka, J. W., Garza, D., and Hartl, D. L. (1989) Inverse PCR in PCR Technology , pp.105-112, Erlich, H. A. ed., Stockton Press, New York.

Pfleiderer, G., Kreiling, A., and Wieland, T. (1960) Biochem. Z. 333, 302-307.

Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.

Senecoff, J. F. and Meagher, R. B. (1993). Plant Physiol. 102: 387-399.

21

1

1338

Lotus japonicus

1

gaattcggca cgagctccaa tggcaccaat ggtgatatct gataaggacg agatgcggaa 60

atggtcaagg tccatgcgat cccaaggcaa gctcatcgcc ctcgttccca ccatgggctt 120

ccttcacgaa ggccaccttt ctctcgtcag agacgctcac aaccacgctg acctcgtcgc 180

cgtctcaatc tatgtcaacc ctggccagtt ttccccgacc gaggaccttt ccgcataccc 240

ttctgatttt caaggtgatc tccaaaaact catgtctgtt cctggtggtg ttgatgttgt 300

tttccacccc cacaatttgt atgattacgg tggtgatggc ggtgatgctg tggcggagtg 360

tggtggtgat ggggtggtgt cttgtgttga taggaggagt ggttttgggc atgaaacttg 420

ggttagagct gagaagctgg agaaacccct ttgtgggaag agtaggcctg ttttctttag 480

aggggttgcc accattgtta ccaagttgtt taatattgtg gagcctgatg ttgctgtgtt 540

tgggaagaag gactatcagc aatggaaaat tattcagaga atggttcgag atcttgattt 600

ttccataaaa gtgataggtt ctgaagtaat acgtgagaaa gatggcctag caatgagttc 660

ccgtaatgtg tacctatcac ctgaagagag ggaaaaggca gtatctataa ataaatcatt 720

gtttagagct aaatcggcag cagaagatgg acagatacat tgtgagaaat tgataaactt 780

ggtcgtgcaa agtatcaccg aagctggtgg aaggattgat tatgctgaga ttgttgatca 840

aaataatttg gagaaagtgg aatggatcaa gggtcctgtt gtcttctgtg tttctgcatg 900

gtttgggaaa gccaggctta tagacaacat agaaatcaac ttgtaaatgg aagtaagatt 960

gatctaacct tgtgaataat ctcagacatg gaccatatga ttagtagttc tggcatttca 1020

tggggtatag acttcattct acaagccatg atatgactac ttgtagatgt attttactac 1080

ctcatgaaat tctaggagct gcttctattt gttggtgatg gtataatatt ttgcagagcc 1140

accactccag aggaaaacaa aattagagaa atcttgctta tgtatcaaag tgccccaggt 1200

ttactcatta atctagataa atctgagctt tctttaggct gatgtacgcc tagagataga 1260

caaacataat tctggtgctg gataaaatta acgcattgga ttcccatttg aaataaaaaa 1320

aaaaaaaaaa aactcgag 1338

2

1264

Oryza sativa

2

gtcgacccac gcgtccggtt tctccctgtc cacttctgtc cgattcctcc tcacctctta 60

tcgattggac gaccatggcg gcgccgcgcg agccggaggt gatccgcgac aaggcggcga 120

tgcgcgcatg gtcgcgccgc cgtcgcgccg agggcaagac cgtcgcggtc gtacccacca 180

tgggctacct ccaccaaggc cacctctccc tcatctccgc cgccgccgcc gccgcctccg 240

ctgatcccgt cgccatcgtc gtcaccatct acgtcaaccc cagccagttc gcgccctcag 300

aggacctcgc cacctaccct tccgacttcg ccggtgacct ccgcaagctc gcctccaccg 360

gcgtcgtgga tgccgtcttc aacccccctg acctctacgt ccgtggcgcc ggtcgccgcg 420

gggccggctc cggaggcgcg atctcctgcc tggaggaggc ggccggggat gggcacgaga 480

cgtgggttcg ggtggagcga ttggagaagg gattgtgcgg ggccagccgt cccgtgttct 540

tccgaggcgt ggccaccata gtctccaagc tgtttaacat catcgagccg gatgttcctg 600

tgttcgggaa gaaggattat cagcagtggc gcgtcatctt gccgtattgg tcgggacttg 660

attttggcat agagataatg ggatcaagaa attgtgcgag aactgatggt cttgccatga 720

actcccggaa tgtgcaccta tcacgcgagg aagggaaaaa ggcattatcc atcagtagat 780

cactggttga tgctagaact ggcgccttga agggaaacac tgattccaaa caaatcaaaa 840

acaaaatagt acagacacta actgaaactg gcggtcaggt tgactatgtt gagatcgtgg 900

agcaagaaag tttggtccct gtagaacaga tcgacggccc tgtggtcatt tgcgttgcgg 960

cgtggtttgg aaaggtcagg ctgatcgata atatcgaaat cgatacacga tcctgaggtt 1020

ttggggggat tcacttgctg tctgctgtga ccttggcatt gcgtttgaaa taccttttgt 1080

ttcgcgtgat gattcgcgtc atgttgtacg ctgtaacaat cacagagaga aaatatgcag 1140

gagtacactg actgaaggca aatttataag tacaaactgt agaggcctga tgctgtaaca 1200

ggggaaatca tgcttgttga ttacagattc cgctgaaaaa aaaaaaaaaa aaaagggcgg 1260

ccgc 1264

3

1500

Saccharomyces cerevisiae

3

gatatccggg taaatgttac tttaacgagt ttttttcttt tgtcatattt tccaacaaga 60

gattaatgaa catttctcga tgtagcgtac atatttatgt gtacaagaag tggtgtgtgt 120

tgatactgca ctgttttata caagttttta tactgcatat ccatatagaa ttgatgaaaa 180

tcttccatac tgtcgaagaa gttgttcaat ggagaacaca ggagctgagg gaaactagat 240

ttagagaaac tattgggttc gttcccacaa tgggttgcct gcattcgggt cacgctagtt 300

tgatctcgca gtctgtgaag gaaaacacct atactgtggt cagtatattt gtaaatccct 360

cccagtttgc gccaacggaa gatctagata actatcctcg aactttgcca gacgacatca 420

aattgcttga gtcgttgaag gtggatgttc tatttgctcc taatgcacac gtgatgtatc 480

cacagggaat tccgctcgac atagaagagc agaaaggccc ttttgttagt gttcttggat 540

tgagtgaaaa attagagggg aagacgagac ctaacttctt taggggcgtg gcaactgtcg 600

tgactaaact attcaatatc gttatggcgg atgtggctta ttttgggcag aaggacattc 660

aacagttcat tgttttacag tgtatggtgg acgaactgtt tgttaataca aggctacaaa 720

tgatgcctat tgtaagaaac aataatggac tggctctgag tagtagaaac aaatatcttt 780

gtccagagtc tttaaagatc tctgaaaacc tttaccgcgg gctgaaagct gcggaaaatg 840

ctattaggag actagcacca gggggacgtc tctccagatc agaaatcatc gatactgtga 900

ctcaaatatg ggcaccctac gttgattccc acgatttcaa aatcgactat gtttccttag 960

cagattttaa gactcttgat gaactctccg atgttgaaaa caccagcgaa cagcagccaa 1020

tagtcattag ttgtgctgta tacgtgactg accgcgaaaa acccgatacg gtcgtcagac 1080

taatagataa catcgttatt taaactaggt gattgggcct tcccgtgtct gtgttgcagt 1140

atataccact cttatacagt atgcacgata ttcttttaaa ccaacaacgg gatgatagat 1200

ttcacgcttg atgacttttt ttttagacgg ctgaagggac gacatcccca tcgctcaaaa 1260

cacaaatatg gaaaggacaa atcgtctttc acagtttgca tagtaaaagc aaagtttata 1320

ctacttcagc aaagttgaag ttgtttggca cttgtttcgt gctttctcaa atatcttaga 1380

tcaccgtctg tctagagcat atatctattg tttgacgcac cccttttaca aaaaaaaaaa 1440

aaagaaacag atctattaag taataaaaaa gttatttagg aaataaggtg cagtaagctt 1500

4

308

Lotus japonicus

4

Met Ala Pro Met Val Ile Ser Asp Lys Asp Glu Met Arg Lys Trp Ser

1 5 10 15

Arg Ser Met Arg Ser Gln Gly Lys Leu Ile Ala Leu Val Pro Thr Met

20 25 30

Gly Phe Leu His Glu Gly His Leu Ser Leu Val Arg Asp Ala His Asn

35 40 45

His Ala Asp Leu Val Ala Val Ser Ile Tyr Val Asn Pro Gly Gln Phe

50 55 60

Ser Pro Thr Glu Asp Leu Ser Ala Tyr Pro Ser Asp Phe Gln Gly Asp

65 70 75 80

Leu Gln Lys Leu Met Ser Val Pro Gly Gly Val Asp Val Val Phe His

85 90 95

Pro His Asn Leu Tyr Asp Tyr Gly Gly Asp Gly Gly Asp Ala Val Ala

100 105 110

Glu Cys Gly Gly Asp Gly Val Val Ser Cys Val Asp Arg Arg Ser Gly

115 120 125

Phe Gly His Glu Thr Trp Val Arg Ala Glu Lys Leu Glu Lys Pro Leu

130 135 140

Cys Gly Lys Ser Arg Pro Val Phe Phe Arg Gly Val Ala Thr Ile Val

145 150 155 160

Thr Lys Leu Phe Asn Ile Val Glu Pro Asp Val Ala Val Phe Gly Lys

165 170 175

Lys Asp Tyr Gln Gln Trp Lys Ile Ile Gln Arg Met Val Arg Asp Leu

180 185 190

Asp Phe Ser Ile Lys Val Ile Gly Ser Glu Val Ile Arg Glu Lys Asp

195 200 205

Gly Leu Ala Met Ser Ser Arg Asn Val Tyr Leu Ser Pro Glu Glu Arg

210 215 220

Glu Lys Ala Val Ser Ile Asn Lys Ser Leu Phe Arg Ala Lys Ser Ala

225 230 235 240

Ala Glu Asp Gly Gln Ile His Cys Glu Lys Leu Ile Asn Leu Val Val

245 250 255

Gln Ser Ile Thr Glu Ala Gly Gly Arg Ile Asp Tyr Ala Glu Ile Val

260 265 270

Asp Gln Asn Asn Leu Glu Lys Val Glu Trp Ile Lys Gly Pro Val Val

275 280 285

Phe Cys Val Ser Ala Trp Phe Gly Lys Ala Arg Leu Ile Asp Asn Ile

290 295 300

Glu Ile Asn Leu

305

5

313

Oryza sativa

5

Met Ala Ala Pro Arg Glu Pro Glu Val Ile Arg Asp Lys Ala Ala Met

1 5 10 15

Arg Ala Trp Ser Arg Arg Arg Arg Ala Glu Gly Lys Thr Val Ala Val

20 25 30

Val Pro Thr Met Gly Tyr Leu His Gln Gly His Leu Ser Leu Ile Ser

35 40 45

Ala Ala Ala Ala Ala Ala Ser Ala Asp Pro Val Ala Ile Val Val Thr

50 55 60

Ile Tyr Val Asn Pro Ser Gln Phe Ala Pro Ser Glu Asp Leu Ala Thr

65 70 75 80

Tyr Pro Ser Asp Phe Ala Gly Asp Leu Arg Lys Leu Ala Ser Thr Gly

85 90 95

Val Val Asp Ala Val Phe Asn Pro Pro Asp Leu Tyr Val Arg Gly Ala

100 105 110

Gly Arg Arg Gly Ala Gly Ser Gly Gly Ala Ile Ser Cys Leu Glu Glu

115 120 125

Ala Ala Gly Asp Gly His Glu Thr Trp Val Arg Val Glu Arg Leu Glu

130 135 140

Lys Gly Leu Cys Gly Ala Ser Arg Pro Val Phe Phe Arg Gly Val Ala

145 150 155 160

Thr Ile Val Ser Lys Leu Phe Asn Ile Ile Glu Pro Asp Val Pro Val

165 170 175

Phe Gly Lys Lys Asp Tyr Gln Gln Trp Arg Val Ile Leu Pro Tyr Trp

180 185 190

Ser Gly Leu Asp Phe Gly Ile Glu Ile Met Gly Ser Arg Asn Cys Ala

195 200 205

Arg Thr Asp Gly Leu Ala Met Asn Ser Arg Asn Val His Leu Ser Arg

210 215 220

Glu Glu Gly Lys Lys Ala Leu Ser Ile Ser Arg Ser Leu Val Asp Ala

225 230 235 240

Arg Thr Gly Ala Leu Lys Gly Asn Thr Asp Ser Lys Gln Ile Lys Asn

245 250 255

Lys Ile Val Gln Thr Leu Thr Glu Thr Gly Gly Gln Val Asp Tyr Val

260 265 270

Glu Ile Val Glu Gln Glu Ser Leu Val Pro Val Glu Gln Ile Asp Gly

275 280 285

Pro Val Val Ile Cys Val Ala Ala Trp Phe Gly Lys Val Arg Leu Ile

290 295 300

Asp Asn Ile Glu Ile Asp Thr Arg Ser

305 310

6

283

Escherichia coli

6

Val Leu Ile Ile Glu Thr Leu Pro Leu Leu Arg Gln Gln Ile Arg Arg

1 5 10 15

Leu Arg Met Glu Gly Lys Arg Val Ala Leu Val Pro Thr Met Gly Asn

20 25 30

Leu His Asp Gly His Met Lys Leu Val Asp Glu Ala Lys Ala Arg Ala

35 40 45

Asp Val Val Val Val Ser Ile Phe Val Asn Pro Met Gln Phe Asp Arg

50 55 60

Pro Glu Asp Leu Ala Arg Tyr Pro Arg Thr Leu Gln Glu Asp Cys Glu

65 70 75 80

Lys Leu Asn Lys Arg Lys Val Asp Leu Val Phe Ala Pro Ser Val Lys

85 90 95

Glu Ile Tyr Pro Asn Gly Thr Glu Thr His Thr Tyr Val Asp Val Pro

100 105 110

Gly Leu Ser Thr Met Leu Glu Gly Ala Ser Arg Pro Gly His Phe Arg

115 120 125

Gly Val Ser Thr Ile Val Ser Lys Leu Phe Asn Leu Val Gln Pro Asp

130 135 140

Ile Ala Cys Phe Gly Glu Lys Asp Phe Gln Gln Leu Ala Leu Ile Arg

145 150 155 160

Lys Met Val Ala Asp Met Gly Phe Asp Ile Glu Ile Val Gly Val Pro

165 170 175

Ile Met Arg Ala Lys Asp Gly Leu Ala Leu Ser Ser Arg Asn Gly Tyr

180 185 190

Leu Thr Ala Glu Gln Arg Lys Ile Ala Pro Gly Leu Tyr Lys Val Leu

195 200 205

Ser Ser Ile Ala Asp Lys Leu Gln Ala Gly Glu Arg Asp Leu Asp Glu

210 215 220

Ile Ile Thr Ile Ala Gly Gln Glu Leu Asn Glu Lys Gly Phe Arg Ala

225 230 235 240

Asp Asp Ile Gln Ile Arg Asp Ala Asp Thr Leu Leu Glu Val Ser Glu

245 250 255

Thr Ser Lys Arg Ala Val Ile Leu Val Ala Ala Trp Leu Gly Asp Ala

260 265 270

Arg Leu Ile Asp Asn Lys Met Val Glu Leu Ala

275 280

7

286

Bacillus subtilis

7

Met Arg Gln Ile Thr Asp Ile Ser Gln Leu Lys Glu Ala Ile Lys Gln

1 5 10 15

Tyr His Ser Glu Gly Lys Ser Ile Gly Phe Val Pro Thr Met Gly Phe

20 25 30

Leu His Glu Gly His Leu Thr Leu Ala Asp Lys Ala Arg Gln Glu Asn

35 40 45

Asp Ala Val Ile Met Ser Ile Phe Val Asn Pro Ala Gln Phe Gly Pro

50 55 60

Asn Glu Asp Phe Glu Ala Tyr Pro Arg Asp Ile Glu Arg Asp Ala Ala

65 70 75 80

Leu Ala Glu Asn Ala Gly Val Asp Ile Leu Phe Thr Pro Asp Ala His

85 90 95

Asp Met Tyr Pro Gly Glu Lys Asn Val Thr Ile His Val Glu Arg Arg

100 105 110

Thr Asp Val Leu Cys Gly Arg Ser Arg Glu Gly His Phe Asp Gly Val

115 120 125

Ala Ile Val Leu Thr Lys Leu Phe Asn Leu Val Lys Pro Thr Arg Ala

130 135 140

Tyr Phe Gly Leu Lys Asp Ala Gln Gln Val Ala Val Val Asp Gly Leu

145 150 155 160

Ile Ser Asp Phe Phe Met Asp Ile Glu Leu Val Pro Val Asp Thr Val

165 170 175

Arg Glu Glu Asp Gly Leu Ala Lys Ser Ser Arg Asn Val Tyr Leu Thr

180 185 190

Ala Glu Glu Arg Lys Glu Ala Pro Lys Leu Tyr Arg Ala Leu Gln Thr

195 200 205

Ser Ala Glu Leu Val Gln Ala Gly Glu Arg Asp Pro Glu Ala Val Ile

210 215 220

Lys Ala Ala Lys Asp Ile Ile Glu Thr Thr Ser Gly Thr Ile Asp Tyr

225 230 235 240

Val Glu Leu Tyr Ser Tyr Pro Glu Leu Glu Pro Val Asn Glu Ile Ala

245 250 255

Gly Lys Met Ile Leu Ala Val Ala Val Ala Phe Ser Lys Ala Arg Leu

260 265 270

Ile Asp Asn Ile Ile Ile Asp Ile Arg Glu Met Glu Arg Ile

275 280 285

8

275

Synechocystis sp

8

Val Gln Val Phe Arg Thr Ile Ala Gly Leu Gln Thr Tyr Leu Arg Gln

1 5 10 15

Ala Gly Arg Gly Lys Thr Val Gly Leu Val Pro Thr Met Gly Ser Leu

20 25 30

His Ala Gly His Gly Ser Leu Leu Lys Arg Ala Val Ala Glu Met Asp

35 40 45

Leu Val Val Leu Ser Ile Phe Val Asn Pro Leu Gln Phe Gly Pro Gly

50 55 60

Glu Asp Leu Glu Lys Tyr Pro Arg Asp Phe Asp Gly Asp Arg Gln Trp

65 70 75 80

Ala Glu Ser Leu Gly Val Ala Val Ile Phe Ala Pro Thr Val Thr Asp

85 90 95

Leu Gly Ile Asp Ala Lys Gly Asp Gln Thr Thr Val Leu Pro Pro Pro

100 105 110

Ala Met Thr Glu Val Leu Cys Gly Ala His Arg Pro Gly His Phe Gln

115 120 125

Gly Val Ala Thr Ile Val Thr Lys Leu Phe Thr Ile Val Cys Pro Asp

130 135 140

Val Ala Tyr Phe Gly Ala Lys Asp Ala Gln Gln Leu Ala Ile Ile Arg

145 150 155 160

Arg Leu Val Gln Asp Leu Asn Leu Thr Val Thr Ile Arg Ser Cys Ala

165 170 175

Thr Val Arg Glu Glu Ser Gly Leu Ala Met Ser Ser Arg Asn Gln Tyr

180 185 190

Leu Ser Pro Ile Glu Lys Glu Gln Ala Thr Val Leu Tyr Arg Ser Leu

195 200 205

Gln Ala Ala Pro Thr Ala Ile Ser Ser Arg Arg Ser Pro Ser Phe Cys

210 215 220

Phe Val Asp Arg His Pro Gly Arg Phe Gly Arg Gly Thr Val Leu Ser

225 230 235 240

Arg Cys Asn Ile Cys Asn Trp Trp Lys Leu Thr Pro Cys Gln Pro Ile

245 250 255

Thr Trp Asn Ile Thr Gly Pro Lys Ser Cys Phe Asn Gly Asp Arg Arg

260 265 270

Leu Cys Gly

275

9

345

Saccharomyces cerevisiae

9

Met Asn Ile Ser Arg Cys Ser Val His Ile Tyr Val Tyr Lys Lys Trp

1 5 10 15

Cys Val Leu Ile Leu His Cys Phe Ile Gln Val Phe Ile Leu His Ile

20 25 30

His Ile Glu Leu Met Lys Ile Phe His Thr Val Glu Glu Val Val Gln

35 40 45

Trp Arg Thr Gln Glu Leu Arg Glu Thr Arg Phe Arg Glu Thr Ile Gly

50 55 60

Phe Val Pro Thr Met Gly Cys Leu His Ser Gly His Ala Ser Leu Ile

65 70 75 80

Ser Gln Ser Val Lys Glu Asn Thr Tyr Thr Val Val Ser Ile Phe Val

85 90 95

Asn Pro Ser Gln Phe Ala Pro Thr Glu Asp Leu Asp Asn Tyr Pro Arg

100 105 110

Thr Leu Pro Asp Asp Ile Lys Leu Leu Glu Ser Leu Lys Val Asp Val

115 120 125

Leu Phe Ala Pro Asn Ala His Val Met Tyr Pro Gln Gly Ile Pro Leu

130 135 140

Asp Ile Glu Glu Gln Lys Gly Pro Phe Val Ser Val Leu Gly Leu Ser

145 150 155 160

Glu Lys Leu Glu Gly Lys Thr Arg Pro Asn Phe Phe Arg Gly Val Ala

165 170 175

Thr Val Val Thr Lys Leu Phe Asn Ile Val Met Ala Asp Val Ala Tyr

180 185 190

Phe Gly Gln Lys Asp Ile Gln Gln Phe Ile Val Leu Gln Cys Met Val

195 200 205

Asp Glu Leu Phe Val Asn Thr Arg Leu Gln Met Met Pro Ile Val Arg

210 215 220

Asn Asn Asn Gly Leu Ala Leu Ser Ser Arg Asn Lys Tyr Leu Cys Pro

225 230 235 240

Glu Ser Leu Lys Ile Ser Glu Asn Leu Tyr Arg Gly Leu Lys Ala Ala

245 250 255

Glu Asn Ala Ile Arg Arg Leu Ala Pro Gly Gly Arg Leu Ser Arg Ser

260 265 270

Glu Ile Ile Asp Thr Val Thr Gln Ile Trp Ala Pro Tyr Val Asp Ser

275 280 285

His Asp Phe Lys Ile Asp Tyr Val Ser Leu Ala Asp Phe Lys Thr Leu

290 295 300

Asp Glu Leu Ser Asp Val Glu Asn Thr Ser Glu Gln Gln Pro Ile Val

305 310 315 320

Ile Ser Cys Ala Val Tyr Val Thr Asp Arg Glu Lys Pro Asp Thr Val

325 330 335

Val Arg Leu Ile Asp Asn Ile Val Ile

340 345

10

283

Schizosaccharomyces pombe

10

Met Gln Val Leu Lys Glu Lys Leu Leu Ile His Gln Gln Val Asp Asn

1 5 10 15

Trp Arg Lys Asp Gly Asn Arg Ile Ala Phe Val Pro Thr Met Gly Asn

20 25 30

Leu His Glu Gly His Phe Ser Leu Val Arg Glu Ala Lys Arg His Ala

35 40 45

Glu Lys Val Val Val Ser Ile Phe Val Asn Pro Met Gln Phe Asn Asn

50 55 60

Pro Gln Asp Leu Leu Leu Tyr Pro Arg Thr Met Asp Gln Asp Cys Ser

65 70 75 80

Gln Leu Gln Asn Leu Gly Val Asp Leu Val Tyr Ala Pro Thr Val Glu

85 90 95

Glu Leu Tyr Pro Glu Gly Ser Gln Asp Ile Thr Phe Val Asp Val Pro

100 105 110

Lys Leu Ser Thr Met Leu Glu Gly Ala Ser Arg Pro Gly His Phe Arg

115 120 125

Gly Val Thr Thr Val Val Ser Lys Leu Phe His Ile Val Asn Pro Asp

130 135 140

Val Ala Cys Phe Gly Glu Lys Asp Phe Gln Gln Val Ala Ile Ile Lys

145 150 155 160

Lys Met Val Arg Asp Leu Asn Phe Phe Ile Glu Ile Ile Gln Val Pro

165 170 175

Ile Val Arg Ala Asp Asp Gly Leu Ala Leu Ser Ser Arg Asn Gly Tyr

180 185 190

Leu Thr Ser Glu Glu Arg Lys Ile Ala Pro Asn Leu Tyr Lys Ile Leu

195 200 205

Lys Lys Leu Ala Gln Glu Leu Ser Asn Gly Asn Gly Asp Leu Glu Lys

210 215 220

Leu Ile Ala Glu Thr Asn Thr Glu Leu Ser Arg Cys Arg Phe Ile Pro

225 230 235 240

Asp Gln Leu Glu Ile Cys Asp Ser Thr Thr Leu Glu Pro Phe Thr Ala

245 250 255

Gly Thr Lys Asn Val Val Ile Leu Ala Ala Ala Trp Leu Gly Lys Ala

260 265 270

Arg Leu Ile Asp Asn Ile Gln Thr Thr Ile Asn

275 280

11

788

Lotus japonicus

11

gaattcggct tcatcaagct tatgtatcaa agtgccccag gtttactcat taatctagat 60

aaatctgagc tttctttagg ctgatgtacg cctagagata gacaaacata attctggtgc 120

tggataaaat taacgcattg gattcccatt tgaaatatct tggtctccct attttatagg 180

aggatccaac ggcagagaac ctaggttctc atgacccctc ttgagggcca tcctagcttc 240

gggatctcat ataaaaggtc ccttacaaca aggggccaat gtaacttctc ttagctcaat 300

tactttgata acttagactt ttcatcctgc tgaaccagaa ttgacttagg catcaaagtg 360

gtttgcacga ccccctcccg ggcttacctg tacgttgcag actacctctc tccagtcgtt 420

cgctcgagct ccaacctccc tcacccccgt tccccttccc tcctacccct tcgtgggtag 480

tcttggtcga atcttcccgg atcaatatca tatatgatat catcatcatt tcaatagaat 540

gaagcgccac ctatctattt gcttcatcaa aagccttctt ttgcaagagt tcccatttgt 600

tcttatcacc ttcacgttca actagcttta cactttttcg acattcccaa taacaacacc 660

agaaccctcc tccaatggca ccaatggtga tatctgataa ggacgagatg cggaaatggt 720

caaggtccat gcgatcccaa ggcaagctca tcgccctcgt tcccaccatg gatcccgaag 780

ccgaattc 788

12

33

Lotus Japonicus

12

Met Ala Pro Met Val Val Ile Ser Asp Lys Asp Glu Met Arg Lys Trp

1 5 10 15

Ser Arg Ser Met Arg Ser Gln Gly Lys Leu Ile Ala Leu Val Pro Thr

20 25 30

Met

13

24

Lotus japonicus

13

atggcaccaa tggtgatatc tgat 24

14

21

Lotus japonicus

14

ttgtatcttt agttgaacat t 21

15

31

Lotus japonicus

15

cgggatccat ggtgggaacg agggcgatga g 31

16

30

Lotus japonicus

16

catcaagctt atgtatcaaa gtgccccagg 30

17

49

Lotus japonicus

17

cgcgctctag aaggaggaat ttaaaatggc accaatggtg atatctgat 49

18

32

Lotus japonicus

18

gcgcgctcga gttacaagtt gatttctatg tt 32

19

17

Lotus japonicus

19

Met Ala Pro Met Val Ile Ser Asp Lys Asp Glu Met Arg Lys Trp Ser

1 5 10 15

Arg

20

15

Lotus japonicus

20

Pro Met Val Ile Ser Asp Lys Asp Glu Met Arg Lys Trp Ser Arg

1 5 10 15

21

16

Lotus japonicus

21

Ala Pro Met Val Ile Ser Asp Lys Asp Glu Met Arg Lys Trp Ser Arg

1 5 10 15