WO1996004369A1

WO1996004369A1 - Polypeptide having cold-resistant pyruvate phosphate dikinase activity, dna coding for the polypeptide, recombinant vector containing the dna, and transformed plant

Info

Publication number: WO1996004369A1
Application number: PCT/JP1995/001040
Authority: WO
Inventors: Shozo Ohta; Satoru Usami; Nigel James Burnell
Original assignee: Japan Tobacco Inc.
Priority date: 1994-07-29
Filing date: 1995-05-30
Publication date: 1996-02-15
Also published as: RO118451B1; UA28003C2; CN1135770A; RU2136748C1; CN1174093C; BR9506291A; MX9601212A

Abstract

A novel polypeptide having a cold-resistant pyruvate phosphate dikinase activity as a means for imparting cold resistance to plants; a cloned DNA coding for the polypeptide; and a recombinant vector containing the DNA. The polypeptide has an amino acid sequence, in any of the following dikinase (1) and polypeptide (2), wherein at least one amino acid residue present in the region with a length of one-sixth of the total length from the C-terminus of the amino acid sequence has been replaced by another amino acid residue and also has a cold-resistant pyruvate phosphate dikinase activity: (1) a pyruvate phosphate dikinase having an amino acid sequence represented by any of the SEQ ID Nos. 1 to 4 in the Sequence Listing, and (2) a polypeptide having an amino acid sequence which is at least 50 % homologous with the amino acid sequence of the dikinase (1) and also having a pyruvate phosphate dikinase activity.

Description

Specification

Polypeptide having cold-tolerant pyruvate phosphate dikinase activity, DNA encoding the same, and recombinant vectors and transformed plants containing the DNA

Technical field

The present invention relates to a polypeptide having a novel cold-tolerant pyruvate phosphate dikinase (hereinafter sometimes referred to as “PPDK”) activity as a means for imparting cold-tolerance to plants, and a clone encoding the same. The present invention relates to a DNA and a recombinant vector containing the DNA. Furthermore, the present invention relates to a plant transformed with the DNA of the present invention.

Background art

Plants strong light and high temperature, but photosynthetic capacity is higher in conditions and low co _2, except those adapted for part of the low temperature condition, the generally low amount of photosynthesis is greatly reduced. PPDK (EC 2.7.9.1, which catalyzes the reaction of ATP, pyruvate and orthophosphate to generate AMP, phosphophenolpyruvate and pyrophosphate) is one of the key enzymes in the pathway but its activity in leaf tissue It is not enough compared to the rate of photosynthesis, and is one of the enzymes that regulates carbon fixation in photosynthesis. It was pointed out at the same time that PPDK was a cold-sensitive enzyme. In the case of corn PPDK, there is an inflection point of the enzyme activity at 11.7 ° C, which is consistent with the critical temperature for corn growth. From these facts, it is thought that P PDK is one of the factors that lowers the photosynthetic rate of C ₄ plants at low temperatures.By improving the low-temperature sensitivity of P PDK, P PDK lowers the growth limit temperature of C ₄ plants, maize. May be able to reduce it. Asteraceae plant Flaveria browniij (Flaveria browniij reticulata) / C _A is classified as intermediate type, and its PDK is known to be hardly inactivated even at 0 ° C low temperature treatment (Burnell JN: A comparative study of The cold-sensitivity of pyruvate, Pi dikinase in Flaveria species. Plant Cell Physiol. 31, 295-297 (1990)).

If the gene encoding the low temperature-resistant PPDK of Flaverii-Brownii is cloned and used to transform plants, it is expected that the plants can be given low-temperature resistance. Disclosure of the invention

Accordingly, an object of the present invention is to provide a novel cold-tolerant polypeptide having PPDK activity, a cloned DNA encoding the same, and a recombinant vector containing the DNA, as a means for imparting cold-resistance to plants. The purpose is to provide a doctor. Still another object of the present invention is to provide a plant transformed with the DNA of the present invention.

As a result of intensive studies, the present inventors have succeeded in cloning the complete P PDK gene of Flavelia bronyii and determining its nucleotide sequence and the amino acid sequence encoded thereby. Among them, the inventors succeeded in identifying a region imparting low-temperature resistance, and completed the present invention.

That is, the present invention relates to any one of the following (1) and (2), wherein at least one or more amino acid residues in the range from the C-terminus of the amino acid sequence to one sixth of the entire length are replaced with other amino acid residues. Provided is a polypeptide having a substituted amino acid sequence and having a low-temperature-resistant pyruvate phosphate dikinase activity.

(1) A pyruvate phosphate dikinase having an amino acid sequence represented by any one of SEQ ID NOs: 1 to 4 in the sequence listing.

(2) A polypeptide having an amino acid sequence at least 50% homologous to the amino acid sequence shown in (1), and having a pyruvate phosphate dikinase activity.

The present invention also provides a cloned DNA encoding a polypeptide having the cold-resistant PPDK activity of the present invention. Further, the present invention provides a recombinant vector comprising the DNA of the present invention and capable of expressing a polypeptide having a cold-resistant PPDK in a host. Further, the present invention provides a plant transformed with the DNA of the present invention.

According to the present invention, a PDKK gene having low temperature resistance was cloned and its base sequence was determined. In addition, the region conferring low-temperature tolerance in the gene was determined. Therefore, by transforming a plant having a cold-sensitive PPDK with the gene of the present invention, the cold-sensitive PPDK can be changed to cold-tolerant. Furthermore, the low-temperature-sensitive PPDK can be changed to low-temperature-resistance by incorporating the low-temperature-resistant region into the homologous portion of the low-temperature-sensitive PPDK. As a result, Since then, it is possible to grow the plant even in cold regions where the plant could not be grown. Therefore, the present invention is expected to greatly contribute to agriculture.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic diagram showing a method for constructing an expression vector containing one example of the PPDK gene of the present invention.

FIG. 2 is a diagram showing the time course of the enzyme activity when P.sup.PPDK of Flavelaria 'Brownii, Flavelaria' Paidentis and maize expressed in E. coli was maintained at 0 ° C.

FIG. 3 is a schematic diagram showing a method for constructing a chimeric gene of corn PPDK and Flavelonia brawnii PPDK.

BEST MODE FOR CARRYING OUT THE INVENTION

According to the present invention, the P. plavens of Flavelonia brawnii, which has low temperature resistance, was cloned, and its nucleotide sequence and the predicted amino acid sequence encoded thereby were determined. The nucleotide sequence and the amino acid sequence are shown in SEQ ID NO: 5 in the sequence listing. As described in detail in the Examples below, this sequence is obtained by extracting total RNA from green leaves of Flavelaria 'Brownii, preparing a cDNA library based on a conventional method, and obtaining Flaveleria bidentis (SEQ ID NO: 1) Plaque hybridization using a region with high homology to the nucleotide sequence (SEQ ID NO: 2) of the maize PPDK gene (SEQ ID NO: 2) to select and clone a positive clone and determine the nucleotide sequence by the dideoxy method Determined by The second sequence has high homology to the PPDK gene of the genus Flavelaria 'Bidentis, has relatively high homology to the PPDK of maize, and is further purified directly from the leaves of Flavelaria' Since it completely matches the N-terminal sequence, C-terminal sequence and internal sequence of the PPDK, it is clear that the gene is a PPDK gene of Flavier-Brownii. The P PDK gene sequence of Flavelia's bidentis was determined by plaque hybridization using maize cDNA as a probe, selecting and cloning positive clones, and determining the dideoxy method. The amino acid sequence shown in SEQ ID NO: 5 is novel and differs by 40 amino acid residues from the amino acid sequence of PPDK of the genus Flavelia pydentis. I have. In addition, about 180 amino acid residues differ from the amino acid sequence of maize PPDK. The amino acid sequence of Flaveria brawnii PP DK shown in SEQ ID NO: 5 thus has high homology especially to the amino acid sequence of Flaveria 'Bidentis PPDK' of the same genus, but Flaveria 'Bidentis PP DK is cold sensitive. In contrast, the Flavelaria 'Brownii PPDK is cold-tolerant and slight amino acid sequence differences result in important trait differences. The present invention provides a cloned PPDK gene encoding the amino acid sequence shown in SEQ ID NO: 5. As described above, this amino acid sequence is novel and has a remarkable effect of having low temperature resistance. The gene of the present invention is not limited to having the nucleotide sequence shown in SEQ ID NO: 5, but may have any nucleotide sequence as long as it encodes this amino acid sequence. .

The inventors of the present application also attempted to identify a region involved in imparting low-temperature resistance in the Flavella brawnii PPDK gene represented by SEQ ID NO: 5. In other words, as described in detail in the Examples below, the PPDK gene of Flavelaria bronchii was divided into three parts with restriction enzymes so that the size would be approximately equal, and the divided regions corresponded to those of the maize PPDK gene. The quinola PPDK gene was formed in exchange for the region, and it was examined whether the PPDK encoded by the chimeric gene has low temperature resistance. As a result, it was confirmed that there was a region imparting low-temperature resistance in the last one-third of the region of Flaveria 'Brownii. Furthermore, the last one-third of the region was roughly equally divided into two by restriction enzymes, and it was confirmed in a similar manner which of the two regions had a region imparting low-temperature resistance. As a result, the amino acid sequence shown in SEQ ID NO: 5 was downstream of the XhoI site of the Flavelaria 'Brownii PPDK gene, ie, the amino acid sequence shown in SEQ ID NO: 5; It has been confirmed that the amino acid sequence up to (hereinafter, this sequence may be referred to as a “cold-resistance conferring sequence”) has a function of conferring low-temperature resistance. That is, it was found that the region involved in low-temperature resistance of PPDK was present in a range of 1/6 of the entire length from the C-terminus. On the other hand, SEQ ID NO: 1 in the sequence listing contains the nucleotide sequence and the deduced amino acid sequence of the gene encoding the PPDK of Flavelaria dentis. SEQ ID NO: 2 shows the nucleotide sequence and predicted amino acid sequence of a gene encoding corn PPDK (Journal of Biochemistry 263, 11080-11083 (1988)). SEQ ID NO: 3 shows that the pacteria Pacteroides symbiosus

The nucleotide sequence and the deduced amino acid sequence of the gene encoding PDK of (Bacteroides symbiosus) are shown (Biochemistry 29, 10757-10765 (1990)). SEQ ID NO: 4 shows the nucleotide sequence and deduced amino acid sequence of the gene encoding PPDK of Entamoeba histolytica, which is a pacteria

(Molecular and Biochemical Parasitology 62, 3-156 (1993)). As described above, the region conferring low-temperature resistance to the PPDK was found by the present invention to be within 1Z6 of the full length from the C-terminus, and thus the amino acid sequences represented by SEQ ID NOS: 1 to 4 were used. By substituting at least one or more amino acid residues within one-sixth of the total length from the C-terminus with other amino acid residues, low-temperature-resistant PPDK can be obtained. Here, “low temperature resistance” means that when the enzyme is left at a temperature of 0 ° C. for 20 minutes, its activity is 60% or more of that before leaving. As described above, in the amino acid sequence shown in SEQ ID NO: 5, the region from arginine at position 832 to valine at position 955 defines low-temperature tolerance. By replacing the corresponding portion of the PDK with the amino acid sequence from arginine 832 to valine 955 of the amino acid sequence shown in SEQ ID NO: 5, it is possible to convert the cold-sensitive PPDK to cold-resistant. it can. This finding is very important and can be used to confer low temperature resistance to the desired PPDK. In addition, the method for imparting low-temperature resistance to PPDK is limited to the method for producing a quinola gene by exchanging the low-temperature-resistance-imparting sequence in Flavier-Brownii PPDK with the corresponding part of low-temperature-sensitive PPDK, as in the following example. Alternatively, the corresponding part of the plant's own cold-sensitive PPDK can be modified by site-directed mutagenesis to have the same sequence as the cold-resistance-conferring sequence of Flavelia'Brownii PPDK. It is. Therefore, any cloned DNA encoding the polypeptide having the above-mentioned low-temperature tolerance imparting sequence and having PPDK activity is included in the scope of the present invention. In particular, proline was substituted for position 869 of the amino acid sequence shown in SEQ ID NO: 1. PPDK and PPDK in which 885th and 952nd are substituted with leucine and parin, respectively, have low temperature resistance.

The PPDK that imparts low-temperature resistance is not limited to those shown in Sequence Listings 1 to 4, but may be any as long as it has a homology of 50% or more to each. Preferably, it should be homologous to the nucleotide sequence of Pflavin 'Brownie's PDK, and should be at least 48.5%, more preferably at least 90%.

In general, when the amino acid sequence of a peptide having a physiological activity is slightly changed, that is, one or more amino acids in the amino acid sequence are substituted or deleted, or one or more amino acids are substituted. It is a well-known fact that even when added, the physiological activity of the peptide may be maintained. Therefore, a polypeptide having the amino acid sequence represented by SEQ ID NO: 5 with such a modification and having low-temperature-resistant PDK activity is also included in the scope of the present invention. That is, in the amino acid sequence represented by SEQ ID NO: 5, one or more amino acids are activated! ], Polypeptides that have been deleted or substituted and have cold-tolerant PPDK activity are also included within the scope of the present invention. Similarly, a DNA encoding a polypeptide having one or more nucleotides added, deleted or substituted in the base sequence represented by SEQ ID NO: 5 and having low-temperature-resistant PPDK activity is also included in the present invention. Included in the range.

DNA that results in the addition, deletion or substitution of amino acids can be performed by, for example, well-known techniques of site-directed mutagenesis (eg, Nucleic Acid Research, Vol. 10, No. 20, p6487-6500, 1982). As used herein, the term "one or more amino acids" refers to a number of amino acids that can be added, deleted or substituted by site-directed mutagenesis.

Site-directed mutagenesis can be performed, for example, as follows using a synthetic oligonucleotide primer complementary to the single-stranded phage DNA to be mutated, except for the specific mismatch that is the desired mutation. That is, a strand complementary to a phage is synthesized using the above-mentioned synthetic oligonucleotide as a primer, and a phage-carrying host bacterium is transformed with the obtained double-stranded DNA. Plate the transformed bacterial culture on agar and form plaques from single cells containing the phage. You. Then, theoretically, 50% of the new colonies contain the phage with the mutation as a single chain, and the remaining 50% have the original sequence. The resulting plaque is hybridized with the DNA completely having the above-mentioned desired mutation at a temperature at which the DNA does not hybridize with the DNA having the original mutation, but does not hybridize with the DNA having the original mutation. Allows hybridization to occur with the synthetic probe that has been treated with the enzyme. Next, a plaque forming a hybrid with the probe is picked up, cultured, and the DNA is recovered.

One or more amino acid substitutions, deletions or insertions in the amino acid sequence of the enzyme that do not cause loss of the enzyme activity may be performed by treating the gene with a mutagen in addition to the site-directed mutagenesis described above. Alternatively, the gene may be selectively cleaved, and then the selected nucleotides may be removed, added, or substituted, and then ligated.

A low-temperature-tolerant plant can be produced by transforming a plant with the Pflank'Brownii PDK gene or a DNA having the PDK activity and containing the sequence for imparting low-temperature tolerance. At this time, preferable examples of the transformed plant include corn, sugarcane, millet, hee and sorghum, but are not limited thereto.

A method for transforming a plant has already been established, and a method using agrobacterium can be preferably employed. Transformation methods of plants using Agrobacterium medfaciens are well known in the art, and thus, dicotyledonous plants (for example, Japanese Patent Application Laid-Open No. Hei 4-330324) and monocotyledonous plants ( 10 94 00977). Alternatively, it can be introduced into a plant protoplast by a conventional method such as electroporation, or it can be transformed by attaching DNA to tungsten particles or the like and driving it into a plant embryo. is there. Specific methods for these transformations are described in the Examples below.

Example

Hereinafter, the present invention will be described more specifically based on examples. However, the present invention is not limited to the following examples.

1. Cloning and Nucleotide Sequence Determination of Flavella brawnii PPDK Gene (1) Preparation of cDNA Library and Cloning of Full-length cDNA (i) Preparation of a cDNA library

Total RNA was isolated from the leaves of F. brownii (60s) by the guanidine hydrochloride / phenol method. This method yielded 26.5 mg of RNA after lithium precipitation. Next, using a column packed with Oligo dT cellulose type 7 (Pharmacia), 118.9 polyA ( ⁺ ) RNA was obtained from 13.2 mg of RNA according to a conventional method. For the preparation of the cDNA library, the packaging reagents attached to the TimeSaver cDNA Synthesis Kit (Pharmacia), Lambda ZAP11 vector (Stratagene) and cDNA cloning system AgtlO (Amersham) were used. Using the EcoRI Notl linker, a cDNA library was prepared in which the DNA fragment was inserted into the EcoRI site of the Lambda ZAPII vector. The size of the prepared cDNA live tally was 4150,000 pfu. XU-Blue was used as a host cell.

(ii) Preparation of probe

Primer 5 'near the processing site: GACGGCTAAAAAGAGGGT (designed based on high homology between cDNA of Flavoria and Bidentis PPDK and cDNA of maize PPDK) and R primer—R: TATCGAGAAACCTTCTATAC (Flaveria' Bidentis PCR II vector (commercially available from Invitrogen), which has a fragment amplified from L brownii RNA by reverse transcription PCR using a part of the PPDK sequence (complementary strand), and amplifies the fragment by PCR using the same primer. After electrophoresis, DNA was recovered from the gel by SUPREC-01 (Takara Shuzo). In this process, a 428 bp DNA fragment starting from 24 bp downstream of the N-terminus of the mature protein can be obtained. This fragment was labeled with ³² P using a Multiprime DNA labeling system (Amersham) to prepare a probe.

(iii) Cloning of full-length Flaveria 'Brownii cDNA

Using the above DNA fragment as a probe, the cDNA library was screened by a plaque hybridization method. Hybond N + (Amersham) was used as a hybridization filter, and hybridization conditions were 65 in 6xSSC, 5x Denhardt's solution, 0.1DSSDS, 100 g ml denatured salmon testis DNA. C overnight. The washing conditions were 2xSSC, 0.1¾SDS for 5 minutes at room temperature, 2xSSC, 0.1¾SDS for 90 minutes at room temperature, and lxSSC, 0.1¾SDS for 68 minutes at 68 ° C. This resulted in 28 independent positive PT / JP95 / 01040

9

I got a rank. Of these, 11 plaques with strong signals were selected for second screening. The secondary screening was performed in the same manner as the primary screening described above, except that the second cleaning time was 60 minutes. As a result, independent positive plaques derived from a single phage were obtained from six clones. In order to determine the size of the inserted DNA fragment, PCR was performed using the above-mentioned R primer, M13PrimerM4 (GTTTTCCCAGTCACGAC, Takara Shuzo) and M13PrimerRV (CAGGAAACAGCTATGAC, Takara Shuzo) as primers, and a phage type II. As a result, two clones were full length. Next, in vivo excision was performed, and the introduced DNA fragment was subcloned into the plasmid vector pBruescript! ISK (-) (Stratagene). The subcloned recombinant plasmids were named p411 and p631.

From the above PCR, it was found that the library prepared as described above was a library suitable for cDNA screening including a sufficiently long insert. Therefore, it is advantageous to apply the present method to isolation of mRNA from Flavelonia broni, and to obtain a large amount of mRNA at once by treating a large amount of RNA as in this case.

In addition, since the cDNA library contains many inserts that are sufficiently long as described above, the full-length can be obtained by preparing a probe using a primer near the processing site of the target protein as described above. It was possible to easily screen cDNA.

(2) Determination of the entire nucleotide sequence of cDNA and comparison of the expected amino acid sequence

Deletion mutants were prepared to determine the entire base sequence of the inserted cDNA fragment of P631. ₀ Deletion mutants were prepared using the Deletion Kit for Kilo-Sequence.

(Takara Shuzo). However, the reaction of exonuclease II was stopped by transferring the enzyme to Mung Bean Nuclease Buffer which had been kept at 65 ° C in advance. To determine the nucleotide sequence, use a plasmid purified using the Qiagen Plasmid Mini Kit (Diagen), Taq Dye Deoxy Terminator Cycle Sequencing Kit (ABI) and Applied Biosystems

This was performed using a 373A DNA Sequencer (ABI). The nucleotide sequence was determined for both strands except a part. The amino acid sequence was determined based on the determined base sequence. Determined The nucleotide sequence and amino acid sequence obtained are shown in SEQ ID NO: 5 in the sequence listing.

Although we attempted to create a delivery clone to determine the nucleotide sequence of the isolated cDNA, the excision reaction using exonuclease III simply changed the reaction solution to Mangbean nuclease buffer. It was important to pre-warm the Mang Bean nuclease buffer to 65 ° C, as it did not stop. In addition, since the base sequence of about 600 to 900 bp counted from the joint with the vector cannot be determined only by cutting the inserted gene from the upstream side, cutting from both directions was necessary.

On the other hand, PPDK was directly purified from Flaveria 'Brownii, and the amino acid sequences of its N-terminal region, C-terminal region and internal region were determined. Purification of PPDK was performed as follows. Grind the green leaves of Flavelia brawnii using 3 volumes of Extraction Buffer → Centrifuge the supernatant after 30% saturation with ammonium sulfate to remove the protein that precipitates-and recover the protein with 70% saturated ammonium sulfate, and use Sephadex G25 (Pharmacia). → pass through a DEAE-Sepharose column (Pharmacia)-elute proteins adsorbed on the column with a 50-400 mM KC1 concentration gradient-collect active fractions and 70% saturation Concentrate with ammonium sulfate and desalting with Sephadex G25. Apply to a hydroxyapatite column. → Raise the concentration of phosphate in the phosphate buffer from 10 mM to 40 mM to elute the adsorbed protein. → Collect active fractions and collect 70% saturation. Concentrate with ammonium sulfate, desalting with Sephadex G25. Thereafter, SDS-PAGE electrophoresis was performed to cut off the band of PPDK, and the protein was recovered from the gel by electroelution. Approximately 5-1 Onmol of P PDK sample purified by such a procedure was obtained. The purified PPDK thus obtained showed one band in SDS-PAGE.

Next, the N-terminal sequence, C-terminal sequence and internal sequence of the obtained purified PPDK were determined. That is, the N-terminal amino acid sequence was obtained by transferring a protein onto a PVDF membrane and using a gas-phase amino acid sequencer. The C-terminal sequence was estimated from the relationship between the amino acid composition released from digestion of purified PPM with carboxypeptidase Y and the digestion time. The internal amino acid sequence was determined by revealing the amino acid sequence from the N-terminal side of the peptide generated when the protein was digested with protease. The detailed method is as follows. First, according to the method for determining the amino acid sequence at the N-terminus. 0

11

After purifying PPDK to a certain extent from the leaves of Flaveria 'Brownii, and performing normal SDS-PAGE electrophoresis, stain the gel with Coomassie Brilliant Blue R250 and cut out the PPDK band. Equilibrate the cut gel in an equilibration buffer (Tris-HCl pH 6.8 125πιπ, EDTA lmM, 0.1¾ SDS), insert it into the well, and perform the second SDS-PAGE electrophoresis. At this time, together with the equilibrated gel, an overlay solution (Tris-HCl pH 6.8 125 mM, EDTA lmM, 0.1 SDS, 0.01 BPB, 20 glycerol) and an enzyme solution (Tris-HCl pH 6.8 125 mM,

EDTA lmM, 0.1¾ SDS, 0.01¾ BPB, 10¾ glycerol, lysyl endopeptidase 1 ~ 5 / g or V8 protease 0.01 ~ 0.1) Digest (power off for 45 minutes). Thereafter, electrophoresis was restarted, transferred to a PVDF membrane according to the method for determining the N-terminal amino acid sequence, and the amino acid sequence was determined from the N-terminal of the digested fragment using a gas-phase amino acid sequencer.

The N-terminal sequence, C-terminal sequence and internal sequence determined as described above are shown below. N-terminal sequence: Asn Pro Val Ser Pro Pro Val (72 to 78)

C-terminal sequence: Leu-Ala Ala *-Val Val (948 to 955)

Internal sequence (l): Lys Leu Tyr Gly Glu Phe Leu Val Asn Ala Gin Gly-Asp Val Val Ala (349-365)

Internal sequence (2): Gin Leu Leu Ala Pro Pro Ala Met Ser Asn Ala Leu One Thr

(59 2 ~ 6 0 5)

Internal sequence (3): Leu Thr Ala Asp Thr Gly Met Ser Lys Asp Glu lie Tyr Ser Arg He Glu (72 1 to 7 38)

Internal sequence (4): Ala-one Ser Phe Gly Thr Asn Asp Leu Cys Gin Met Val Phe Gly-Ser (844-862)

In the above amino acid sequence, "*" indicates glutamine, which cannot be analyzed by the analytical instrument used, and "one" indicates that the analysis result is unclear. The numbers in parentheses indicate the amino acid numbers of the corresponding portions of the amino acid sequence shown in SEQ ID NO: 5. Although the internal sequences (2) and (4) are partially different from the sequence of the corresponding portion of SEQ ID NO: 5, it is considered that this is due to the amino acid sequencer error. As is well known, amino acid sequencer errors occur quite frequently, Errors in the DNA sequencer rarely occur.

The amino acid sequence shown in SEQ ID NO: 5 and the partial amino acid sequence determined directly from the above purified PPDK are in good agreement, and the amino acid sequence shown in SEQ ID NO: 5 is the amino acid sequence of PPDK. The amino acid sequence was confirmed. When the amino acid sequence shown in SEQ ID NO: 5 was compared with the amino acid sequences of known Flavier bidentis PPDK and maize PPDK, 40 amino acids (Flavier pidetis) and 180 in the mature protein portion were obtained. About one (corn) amino acids were different. From the above results, among the amino acid sequences shown in SEQ ID NO: 5, the 1st to 71st amino acid sequences are not present in the mature protein, and are transit peptides necessary for passage through the membrane. And is understood to be processed after passing through the membrane. Table 1 below shows the differences between the amino acid residues in the mature proteins of Flavelaria brawnii and Flavelaria bidentis.

Table 1 Bronodentis Different amino acid numbers 5 Phe 6 Ser

And amino acids 1 0 Pro 1 1 leu

1 5 Asn 16 Arg

2 8 Thr 2 9 Asn

4 0 Pro 4 1 Ser

4 1 Ala 4 2 Ser

4 8 Arg 4 7 shi eu

4 9 Arg 4 8 Thr

5 0 Lys 4 9 Pro

5 2 Ser 5 0 Ala

5 7 lie 5 5 Pro

6 1 Thr 5 9 Ser

6 2 Gly 6 0 Ser

ει

tOI0 / S6df / XDd 69CtO / 96 OAV 736 Arg 734 then ys

739 ys 737 Asn

111 Asn 775 Thr

803 Gly 801 Ser

818 leu 81 6 Val

838 Asp 836 Glu

84 1 Ala 839 Gly

845 Glu 843 Asp

871 Pro 869 Gin

875 Ser 873 Ala

887 Leu 885 lie

954 Val 952 lie

2. Production of Flaveria brawnii PPDK in Escherichia coli and measurement of low-temperature tolerance In order to confirm that a low-temperature-resistant enzyme is actually produced from Flavelia brawnii PPDK DNA isolated as described above, E. coli was used. Was expressed as follows.

The removal of the transit peptide and the introduction of a restriction enzyme site for joining the expression vector and the reading frame together were performed as follows. p631Sac (plasmid lacking p631 after Sacl) obtained by cutting p631 with Sacl and recyclizing was type III, and a primer containing an EcoRV site based on the sequence near the processing site 4 : PCR was performed using a combination of GATATCAATCCGGTGTCTCCTCC and a primer M13 RV (Takara Shuzo) complementary to the vector sequence, and the amplified fragment was subcloned into pCR II. A fragment containing the N-terminal part was excised from pCR II using EcoRV and Sacl restriction enzymes, cut with the Sacl-Hind III fragment of p631 (including the rest of PPDK cDNA) and Ncol, and blunt-ended with Klenow enzyme. After that, a three-fragment ligation reaction was performed with pKK233-2 digested with Hindi 11 (FIG. 1). This plasmid was transformed into E. coli MV1184 and used for expression experiments. Dilute 1 ml of the preculture into 9 ml of fresh LB medium (containing 50 mg of ampicillin), shake at 37 ° C for 3 hours, and add 1 PTG to 5 mM. After further culturing for 3 hours, the cells were recovered by centrifugation. The cells were suspended in 0.5 ml of extraction buffer (50 mM Hepes-KOH pH7.5, 10 mM MgSO ImM EDTA, 5 mM DTT), lysozyme was added to about 0.5 mg ral, treated on ice for 5 minutes, and then sonicated. Enzymes were extracted by treating with a crushing device (Cosmo Bio UCD-130T) at intervals of 30 seconds for 5 minutes while cooling on ice. After centrifugation in a microcentrifuge for 10 minutes, the supernatant was passed through a Sephadex G25 column equilibrated with a column buffer (50 mM Hepes-KOH pH 7.0, 10 mM MgCl, 2 mM EDTA, 10 mM DTT) to remove low molecular weight substances. The mixture was allowed to stand at 25 ° C for 30 minutes or longer to associate with the tetramer and used for activity measurement.

PPDK produced in Escherichia coli from PPPDK cDNAs of Flaveria bronyii, Flaveria bidentis and maize (Harvest queen) showed almost the same migration on SDS-PAGE as the plant-derived enzyme. The expected molecular weights of the mature enzymes from the cDNAs are all the same, and the apparent molecular weights on SDS-PAGE are quite different. This was due to the difference in the amino acid composition contained in each polypeptide, and was not due to post-translational modifications such as protein truncation processing and glycosylation. The cold tolerance of various PPDKs produced in E. coli was consistent with the corresponding plant enzymes. In other words, the low-temperature tolerance of Flaveria'Brownii PPDK does not require cofactors or post-translational processing unique to this plant.If this cDNA is introduced into corn and expressed, low-temperature-resistant PPDK is produced. Expected. FIG. 2 shows the relationship between the elapsed time since the enzyme was placed at a temperature of 0 ° C. and the relative activity of PPDK.

In the above process, in order to produce an active PPDK in E. coli, a cDNA was prepared by removing the transit peptide and incorporated into the expression vector. It was important to match the position exactly to the N-terminal position of the plant-derived enzyme.

(F. brownii E. coli production (primers used)

M1TAKKRVFTF- 1 2

MIPVSPPVTTTKKRVFTF- + 3

MINPVSPPVTTTKKRVFTF- ++ 4

NPVSPPVTTTKKRVFTF '(leaf-derived enzyme) (F. bidentis)

MITAKKRVFTF- ++

MIPVSPPVTTAKKRVFTF- TA KRVFTF- (leaf-derived enzyme)

(corn)

MATKKRVFTF- ++

TTKKRVFTF- (leaf-derived enzyme)

That is, when the expression experiment of Flavelia'brownie cDNA was carried out, expression of corn and Flavellii-Pydentis PPDK was already successful, and both were successfully expressed with reference to the cleavage site of corn. Thus, the expression vector was prepared by cutting at the same position using Primer 2 also for Flaveler brownie cDNA and expression was attempted, but no PPDK could be produced at all. Therefore, based on the N-terminal sequence of the leaf-derived enzyme, an expression vector containing 7 residues more at the N-terminus was prepared using primer 3, and expression was attempted. As a result, production of PDKK was confirmed. However, even at this stage, the expression level was still low, and the amount of enzyme sufficient to measure low-temperature resistance was not obtained. Therefore, an expression vector extended one residue further to the N-terminal side was prepared using Primer 14, and an expression experiment was performed. As a result, a large amount of PPDK was produced, and its low-temperature resistance was confirmed. The nucleotide sequences of Primer 2 and Primer 3 were as follows.

Primer 2: CGGTGTCTCCTCCGGATATCACGGCTAAAAAGAG

Primer 3: TTGATATCCCGGTTGTCTCCTCCGGTA

3. Determination of cold-tolerant region in P. flaveria P. brunii PDK gene (1) Chimera of Flaveria 'Brownii and Flavelia' bidentis

It was recombined using restriction enzymes according to a conventional method. The EcoRI-Hindi 11 fragment of pKK-brownii is equivalent to pKK-bidentis (plasmid obtained by incorporating cDNA of Flavelaria bidentis into pKK-223-2 by the same method as pKK-brownii). ΡΚΚ_011 was exchanged for the corresponding fragment, and conversely, the EcoRI-HindiΠ fragment of pKK-bidentis was exchanged for the corresponding fragment of pKK brownii to produce pKK-100. Similarly, the Ndel-Hindlll fragments were exchanged for each other to produce pKK-001 and pKK-110. Xho-HindIII cleavage of pKK-110 The pieces were exchanged for the corresponding fragments of pKK-bidentis to replace pKK-1101 with XKK of pKK-bidentis! Fragments! PKK-1110 was generated in exchange for the corresponding fragment of ^^^. In order to further recombine the Xhol-Hindll fragments with each other, the fragments were linked by PCR (linking PCR method). Primer link-F: GCAGAGATGATGTTGGCAAG and link-R: CTTGCCAACATCATCTCTGC which were complementary to each other were prepared in the place where the base sequence is the same in the Xhol-Hindi II fragment of bidentis and brownii. The first PCR was carried out using a combination of link-FRV and M4 link-R using the Xii-HindiII fragment of brownii or bidentis subcloned in Bluescript SK (-) for type I. The obtained fragments (total of 4 types) were purified by gel excision, and the first half of brownii and the latter half of bidentis, or the first half of bidentis and the latter half of brownii were mixed to form type II, and the second PCR was performed using Primer M4 RV. Was done. The amplified binding fragment was digested with Xhol and HindII, and replaced with the corresponding part of pKK-bidentis to produce pKK-1 inkO1 and pKK-1 ink10. Another set of chimeric genes was created using the PstI site between the recombination site of linking PCR and Hindi11. The Xhol-Pstl fragment of pKK-linklO and the Pstl-HindiII fragment of ρΠ-bidentis were incorporated into the Xhol-Hindi 11 site of pKK-bidentis (3-fragment ligation reaction) to give pKK-linklOl. Similarly, the Xhol-Pstl fragment of pKK-bidentis and the Pstl-Hindi 11 fragment of pKK-brownii were similarly incorporated into the Xhol-HindiII site of pKK_bidentis to obtain pKK-linkl10.

The 40 amino acid substitutions found in the mature protein portion of the PPDK of Flavelaria'Brownii and Flaverian bidentis are relatively higher in the N- and C-termini of the enzyme and less near the central active center. First, the cDNA is divided into three parts: the front, middle, and rear, using the EcoRI and Ndel sites, which are common to both genes, and these are interchanged to produce a chimeric gene. It was examined whether it was involved in resistance. As a result, low temperature tolerance was obtained when the latter half of Flaveria'Brownii cDNA was present, and low temperature sensitivity was obtained when the latter half of Flaveria'Bidentis cDNA was present. Next, the latter 13 regions were divided into two parts by the restriction enzyme Xhol, and introduced into the corresponding parts of pKK-bidentis, respectively, and the low-temperature resistance was measured. As a result, the portion after the Xhol site (most 16 region from C-terminal) was necessary and sufficient for low-temperature tolerance. Next, the second half of the 16 region (Xho Hindi II fragment, including 7 amino acid substitutions) A chimeric gene was prepared by the linking PCR method and introduced into pKK-bidentis, and the low-temperature resistance was measured. As a result, the chimeric enzyme pKK-linkO having the last region containing four substitutions was cold-resistant, and the chimeric enzyme pKK-linkOl having the first half region containing three substitutions was inactivated at low temperature. Therefore, the latter half region was recombined with the restriction enzyme Pstl, and a chimeric gene having two amino acid substitutions was prepared and the low-temperature resistance was measured. It was estimated that there were two or more locations.

(2) Chimera of maize and Flavelia brawnii

Using a primer PPDK-F: CTCACTGTTCGAAGAGAAGC and an mNdel containing an Ndel site: CATATGCTCTGTCCGGCATAATC (complementary strand side), PCR was performed using maize PPM cDNA as type III, and the obtained fragment was subcloned into pCR11. This fragment was excised from pCR II with Sacl and Ndel, and the Sacl-Smal fragment of pKK-PPM (vector fragment) and brownii PPDK cDNA were cut with Hindin, blunt-ended with Klenow enzyme, and cut with Ndel. A three-fragment ligation reaction was performed with the obtained fragments (FIG. 3) to obtain pKK-mz bro (Nde). In addition, PCR was carried out using the maize PPDK cDNA as a type I using mXhoI: CTCGAGGGATCTCAATCATTG (complementary strand side) containing the primers PPDK-F and Xhol, and the obtained fragment was subcloned into pCR II, followed by Sacl and Xhol. It was excised from pCRII and ligated with pKK-mz bro (Nde) © Sac I-Xhol fragment (vector fragment) to obtain pKK-mz bro (Xho).

Both the maize PPDK and brownii PPDK C-terminal 13 (Nde Hindi 11 fragment) or 16 (Xho Hindi II fragment) chimeric enzymes exhibited the same level of low-temperature tolerance as brownii. As described above, since the corn PPDK having a considerably different amino acid sequence could be made low-temperature-tolerant, it is considered that by introducing the latter 13 regions or 16 regions, PPDK derived from various plants can be made to be low-temperature-tolerant. The corn F._ brownii chimeric PPDK prepared this time can use the transit peptide of corn PPDK as it is, so the transit portion is also a transformant derived from Flavelaria 'Brownii' PPDK and the chloroplast of cold-resistant PPDK If there is a problem in transporting to the cell, it may be resolved by introducing this chimeric gene instead.

(3) Point mutation clone For the Xhol-Hindll I fragment of pKK-brownii, brownii-bidentis amino acid substitutions were made one by one. In addition, for the Xhol-HindiII fragment of pKK-bidentis, amino acid substitution from bidentis type to brownii type was made for each residue. Mutagenesis was performed by subcloning the Xhoto HindIII fragment of brownii and F. bidentis PPM cDNA into pBluescript I1SK (-), respectively, and using the Megalabel kit (Takara Shuzo) and Mutan-K kit (Takara Shuzo) based on the Kunkel method. Was. The sequences of the primers used for mutagenesis are as shown in Table 2. After confirming the mutated nucleotide sequence with a DNA sequencer, these fragments were inserted into the Xhol-HindiII site of pKK-bidentis.

Table 2 Primers used for generating point mutation clones

Mutation from F. brownii type to F. bidentis type

836DE 5 'GCAATCTCTTCAGCAATC

839AG 5 'GCTTCTTTTCCAATCTCATC

843ED 5 'CGAAAAGAAATCGGCTTC

869PQ 5 'GAAAGATAAATCTGCAAAAACTTG

873SA 5'GCCTTGAGCAAGATAAATC

885L1 5'TTCTGGTCAATAACCTCAATG

952V I 5'GCTTAAACAATGACTTGTGC

Mutation from F. bidentis to F. brownii

836ED 5 'CCAATCTCATCAGCTATTAAAG

839GA 5 'GCTTCTTTTGCAATCTCTTC

843DE 5'CGAAAAGAACTCAGCTTC

869QP 5 'CAAGATAAATCGGCAAAAACTTG

873AS 5 'GAATGCCTTGAGAAAGATAAATC

885IL 5 'CTTTCTGGTCAAGAACCTCAAATG

9521V 5'GCTTAAACAACGACTTGTGC Differences in the Xhol-Hindlll region of pKK-1110 All enzymes in which amino acids were replaced one by one with a bidentis type showed low temperature resistance, and it was thought that there were multiple mutations that confer low temperature resistance (one Changing the residue alone does not impair resistance). Therefore, conversely, the different amino acids in the Xhol-HindiII region of pKK-bidentis were replaced one by one with brownii type, and it was examined whether or not the enzyme became a low-temperature resistant enzyme. That is, the enzyme activity after treatment at 0 ° C for 20 minutes was examined. As a result, the 869Gln-Pro mutation acquired low-temperature tolerance (the activity after the low-temperature treatment was 60-70%), and the 885Ile → Leu and 95211e-Val mutations It is difficult to inactivate at low temperature, and considering the results of the chimeric enzyme pKK-linkllO in (1) above, it is presumed that low temperature resistance is obtained when these two mutations coexist. From the above results, it was concluded that three residues of 869Pro, 885Leu, and 952Val were involved in cold tolerance. Among these residues involved in low-temperature tolerance of brownii, 869Pro and 885Leu are also brownii-type in corn PPM. Therefore, it is considered that these residues alone are not brownii-type, but do not necessarily acquire low-temperature resistance, and are considered to be the first amino acid sequences of brownii or bidentis to confer complete resistance. When making a significantly different type of PPDK resistant to low temperatures, it is preferable not to introduce a point mutation but to produce a quinula gene introduced in a region unit as performed for corn PPM.

4. Transformation of maize with Flaveria brawnii P PDK gene

Gordon- Kamm f. J. et al. (The Plant Cell 2: 603-618, 1990), or

According to the method of Koziel MG et al. (Bio Technology 11: 194-200, 1993), pK-brownii is coated on fine particles of tungsten or gold and implanted into immature maize embryos or suspension cultured cells . After selecting transformed cells from the transfected cells, the resulting transformed callus is cultured by a conventional method, and the plant is regenerated. Transformation methods are not limited to the particle gun method, but include the electroporation method (Rhodes CA et al., Science 240: 204-207, 1988) and the PEG method (Armstrong C. L et al., Plant Cell Reports 9: 335- 339, 1990), tissue-electroporation method (D'Halluin K. et al., The Plant Cell 4: 1495-1505, 1992), or agro-pakterium method (Hiei Y. and Komari T. WO 9400977) And so on. Seeds are obtained from the obtained plant body, germinated, and PPDK is separated from the leaves of the obtained plant to examine its low-temperature tolerance. The effect of temperature on the rate of photosynthesis is examined for transformed and untransformed plants. Characteristics of corn plants that exhibit higher photosynthetic rates at low temperatures grown over generations and determined by measuring photosynthetic rates at different temperatures and by measuring the cold tolerance of PPDK isolated from the plant Ensure conversion stability.

5. Flaveria 'Brownii P FDKV Gene by PDKK Gene Transformation

An intermediate vector containing the full-length cDNA shown in SEQ ID NO: 5 and containing the reporter gene is introduced into the disarmed Ti plasmid of Agrobacterium umme faciliens ₀ This is Draper J et al eds. It can be performed by the method described in Plant Genetic Transfomation and Gene Expression-a laboratory manual, Blackwell Scientific Publications (ISBN 0-632-02172-1).

On the other hand, the leaf tissue or callus of Flaveria bidentis is infected with the Agrobacterium mme fascient. This can be performed by co-culturing the tissue or callus with Agrobacterium ctemefasciens. Infected cells are selected based on drug resistance. Plants are regenerated from the selected calli in a conventional manner. Seeds are obtained from the obtained plant body, germinated, and PPDK is isolated from the leaves of the obtained plant to examine its low-temperature tolerance. The effect of temperature on the rate of photosynthesis is examined for transformed and untransformed plants. By growing F. verdentis plants that exhibit higher photosynthetic rates at low temperatures for generations and measuring photosynthetic rates at different temperatures and by measuring the cold tolerance of PPDK isolated from the plant. Ensure the stability of the transformation being examined.

ςπ on soi οοτ λχο an ^J3 S ^J3 S 1 ³ W ^η ΐ3 Biv na usy BTV ^ TO sAq λχο λχο naq naq m 133 IXV 30V V3I OIV 3V0 130 IID IVV V30 V03 VVV V90 V09 Oil Oil

Q6 06 58 new 9S sA jaw dsy SJV usy Αχο η ο jss §JV ^ ΐθ sAq Λχο sqd J¾

331 VVV 31V DVO 90V 3VV 330 WO 丄 3V VOV V30 VVV 100 III I3V LL

08 i O

ΤΒΛ SJV SAI sXq Βχν 丄 4 丄 T¾ 〇 ■ ¾ OJJ J9S T ^ A OJd usy ngq Λ

862 丄丄 3 OOV OVV VVV 133 93V 03V 019 033 133 131 0X0 933 IVV 113 丄丄 3

59 09 59

BTV v OJd OJJ am na A13 J3S J3S Sjy naq OJJ Sjy J3S ΐ

092 V30 V03 ODD 333 I3V VII 309 I0V 33V 393 Oil 3V3 V33 VOV DOV 丄丄 3

09 ^

SJV BTV OJd jqi naq SAQ 8jy l ^l s ^ sqj s -iss uig 3jy STH stH usy

202 VOV 939 033 I3V VII 101 303 313 III 931 031 VV3 XD3 3V3 3V3 OVV

58 OS 52 02 naq gjy Sjy naq dsy Α ^ usy 8jy Sjy UTO sAq ^ e ^ S-ty ^ TV OJ ^ nsq

^ 9ΐ Oil V33 303 313 IV3 XD9 OVV V03 003 VV3 OVV 010 VOV 039 033 VII

9ΐ Οΐ 9

SAQ J3S nig Sjy ^ TV S s q naq nsq n Αχ ^ η ο 8g 9g naq 95

90ΐ 391 ODI 103 330 VDI OVV DID 丄丄 3 9IV 100 WO 113 131 Oil 001 ΐ

J3S law; sw

85 13V 9IV OIV 00VV0VV9I 39V3V3XIIV VOLOLV 丄 VOL 30IIVID9I3 1I33I31V03 圆

9 1 6 2: $ ϊ © 「ιίΜ

ΐ: Decoration 2S [Halla ¾]

OtOIO / S6df / 13d 69 € tO / 96 OAV sq ηχο ΛΙΟ sAq εχν ηχ ^ _Λ Λ 丄 usy sAq 丄 sX ηχο eight naq 8 OVV WO 300 OVV V30 WO 913 IVl 9X0 DVV OVV 3VI VVV 0V3 110 113

992

dsy sAq risq dsy BTV eiv 丄 dsy J¾ dsy na S TH ΘΤ Ι ^ 1 sAq

928 IVO VVV 113 IVO 130 133 I3V DID IVO 33V 3V9 3X3 IV3 XXV 300 VVV

582 082

n BIV sAq 9w ujo ηχ ^ naq sA ηχο dsy aqd na Jas S TH OJd 9Π

8 丄 WO 130 VVV OIV OVO 0V9 VII OVV VV9 3V3 III VII V3I IVO 933 DIV

m m m

'ίΐθ 1SW ΐ ^ Λ ΐ¾Λ ^US V ^ ΐθ 3qd 19 ^ dsy nsq 9qd Sjy v dsy OS 109 OIV V 丄 3 110 OVV 303 III 31V IVO 313 丄丄丄 00V VOV IVl 031 OVD

o \ z OOZ

_τΛ 丄 BTV 3Md SJV BTV ^ ΐθ S s Χχ9 na λχο BIV ISA ΐ ^Β Λ ⁿ I3

289 IV 丄 3dD 111 003 V30 V90 IOV VVV 300 133 VIO 109 130 V19 310

56 ΐ 06ΐ 981 081 dsy usy naq Αχ {) nsq usy naq eight jtq 丄 dsy aaj¾ OJJ IS¾ JSS

^ 9 XV9 IVV 113 930 id IVV Oil VIO 13V DV9 91V OIV 103 130 OIV 丄 :) 丄

9Ζΐ ΟΑΐ 99ΐ

9ΐ Ι Βχν εχν Α ο j as 3jy ΤΒΛ JSS Π9 Ί η 31 nsq OJJ sA js§ OJJ dsy

985 VIV 330 139 100 031 103 310 001 IID 313 310 133 VVV 131 9D3 3V9

09ΐ 55Ϊ 051

13 Π3ΐ J9S BIV -I3S 13K n ^ o sAq uif) cl clsy naq Αχο ηχο J9S

889 130 DID 131 VDO 131 DIV OVO VVV OVD 310 IVl IV9 Vll 309 WO VOL

OH 581

9i i "TO dsy dJi nsq Αχο OJJ OJJ ng J9S sAq Αχο usy UIQ UJQ JA 丄

06 LLV OVO IVO 001 Oil 100 V33 133 VI3 30V OVV VOO IVV VV3 VV3 丄 V 丄

OS! 52ΐ 03ΐ

"TO ηι;) s P] V ηχο jx ass an J¾ naq

OJJ OJJ JBA S nsq

WO OVO IDl VDO WO I3V V3I LLV I3V 313 009 133 133 113 V3I VI3 S

Ot-OlO / S6df / 10d 69h 0/96 ΟΛ \ ί ^ ςει ovv 313 oiv νιο m iiv νον οιο νοΰ ιοο ννν ιοΰ ιον ιοο ννν ς ο so

Λΐ9 jqx SJV SAQ υχο naq aa ^ 丄 na 8jy usy ηχο Λ-^ ΜΙ ^ Md

90SI 300 VOV V03 301 VV3 Oil OIV 001 113 OOV 3VV WO VV3 110 V3V :) 丄丄

00 969 06 £

ηΐ3 an dsy m dsy sA αΑχ STH Αχο naq an S Q usy

852Ϊ VV3 丄丄 V IVO OIV OIV IVO VVV DVI 3V3 VOO OVO VII 01V OVO 301 VV

S £ 088 518

πΐθ ΐΒΛ nsq ηχ ^ sA _iA 丄 ¾τν dsy OJJ n SAQ J¾ jaw 丄 q 丄 χο

0121 9V0 010 113 OVO VVV DVl VOO IVO 1DD OiV DDI I3V OVO OiV 33V ODD

OAS 098

nai dsy ηχ ^ OJJ jqi 3jy ^ Τ Ι ^ TD ^ TV ί ^ Λ ΐ dsy ηχο χ ^ uxa εχ

Ζ9 Π Oil IVO VV3 V3D VOV V9V DIV OOO 130 113 110 IVO OVO VOO OVD 130

99S 058 91 ^ 8 0 ^ 8 usv 9Ϊ Ι naq aqj Αχο ιΑχ nsq sAq s ^ J¾ J9S OJJ usy

H U IVV 31V VII ill OVO OOO DVl VI3 OVV OVV 9V9 109 33V 30V V33 OVV

9εε οεε .ε gJV 3Md nsq AIO J¾ jas J¾ usy Αχ aw usy ο aqd 9901 OOV I3V 311 113 119 103 13V VOO VOL I3V OVV VOO 3IV OVV 303 丄丄丄

028 5TS 0ΐ8

ΤΒΛ J9W J3S UTO 3ΐ [usy ΐ BTV JMI ιο sAq το Jt [丄 an UTO

8Ϊ 0Ι 310 DIV 30V VV3 IIV 3VV 113 V30 I3V 930 OVV VII V90 I3V VIV OVD

G08 Οθε G62 usy 3 1 jag 3JV 丄 sAq usy Βχν 3 ュ V OJJ jas dsy d 丄 _Τ3§ dsy siy

0Z6 VV IIV IDV VOV 3VI OVV IVV 333 OOV V33 33V 3V0 391 IDX IVO III

062 082

ΐ ^ Λ BTV usv ΐ ^ Λ ^ TV Π3ΐ nio na UT3 sAq sA OJJ dsy J¾ OJJ 3

ZZQ 119 130 IVV 319 V30 VII OVO VI3 OVD VVV OVV V33 IVO VOV 333 丄丄丄

OLZ 59Z 09Z tOl0 / S6df / XDd 69 € tO / 96 OAV Lys Arg Thr Gly Lys Gly Ala Val Arg lie Ala Val Asp Met Val Asn

420 425 430 435

GAA GGG CTA ATT GAT ACT AGA ACA GCA ATT AAG AGG GTT GAG ACT CAA 1402

Glu Gly Leu He Asp Thr Arg Thr Ala He Lys Arg Val Glu Thr Gin

440 445 450

CAT CTA GAT CAG CTT CTT CAT CCA CAG TTT GAG GAT CCG TCT GCT TAC 1450

His Leu Asp Gin Leu Leu His Pro Gin Phe Glu Asp Pro Ser Ala Tyr

455 460 465

AAA AGC CAT GTG GTA GCA ACC GGT TTG CCA GCA TCC CCC GGG GCA GCT 1498

Lys Ser His Val Val Ala Thr Gly Leu Pro Ala Ser Pro Gly Ala Ala

470 475 480

GTG GGA CAG GTT TGT TTT AGT GCA GAG GAT GCA GAA ACA TGG CAT GCA 1546

Val Gly Gin Val Cys Phe Ser Ala Glu Asp Ala Glu Thr Trp His Ala

485 490 495

CAA GGA AAG AGT GCT ATC TTG GTA AGG ACC GAA ACA AGC CCA GAA GAT 1594

Gin Gly Lys Ser Ala lie Leu Val Arg Thr Glu Thr Ser Pro Glu Asp

500 505 510 515

GTT GGT GGT ATG CAT GCA GCA GCT GGA ATC TTA ACC GCT AGA GGA GGC 1642

Val Gly Gly Met His Ala Ala Ala Gly lie Leu Thr Ala Arg Gly Gly

520 525 530

ATG ACA TCA CAT GCA GCG GTG GTG GCT CGC GGA TGG GGC AAA TGT TGT 1690

Met Thr Ser His Ala Ala Val Val Ala Arg Gly Trp Gly Lys Cys Cys

535 540 545

GTT TCC GGT TGT GCT GAT ATT CGT GTG AAC GAT GAT ATG AAG ATT TTT 1738

Val Ser Gly Cys Ala Asp lie Arg Val Asn Asp Asp Met Lys lie Phe

550 555 560

ACG ATT GGC GAC CGT GTG ATT AAA GAA GGC GAC TGG CTT TCT CTT AAT 1786

Thr l ie Gly Asp Arg Val lie Lys Glu Gly Asp Trp Leu Ser Leu Asn

565 570 575 PC / / 1 40

27

GGT ACA ACT GGT GAA GTC ATA TTG GGT AAA CAG CTA CTG GCT CCA CCT 1834

Gly Thr Thr Gly Glu Val lie Leu Gly Lys Gin Leu Leu Ala Pro Pro

580 585 590 595

GCA ATG AGC AAT GAC TTA GAA ATA TTC ATG TCA TGG GCT GAT CAA GCA 1882

Ala Met Ser Asn Asp Leu Glu lie Phe Met Ser Trp Ala Asp Gin Ala

600 605 610

AGG CGT CTC AAG GTT ATG GCA AAT GCA GAC ACA CCT AAT GAT GCA TTA 1930

Arg Arg Leu Lys Val Met Ala Asn Ala Asp Thr Pro Asn Asp Ala Leu

615 620 625

ACA GCC AGA AAC AAT GGT GCA CAA GGG ATC GGG CTC TGT AGA ACT GAA 1978

Thr Ala Arg Asn Asn Gly Ala Gin Gly lie Gly Leu Cys Arg Thr Glu

630 635 640

CAT ATG TTT TTC GCT TCT GAT GAG AGG ATC AAA GCT GTA AGA AAG ATG 2026

His Met Phe Phe Ala Ser Asp Glu Arg lie Lys Ala Val Arg Lys Met

645 650 655

ATC ATG GCG GTC ACT CCA GAA CAA AGA AAA GTG GCT CTA GAT CTC TTA 2074 l ie Met Ala Val Thr Pro Glu Gin Arg Lys Val Ala Leu Asp Leu Leu

660 665 670 675

CTC CCA TAC CAA AGA TCC GAT TTT GAG GGC ATT TTC CGA GCA ATG GAT 2122

Leu Pro Tyr Gin Arg Ser Asp Phe Glu Gly lie Phe Arg Ala Met Asp

680 685 690

GGA CTT CCT GTA ACT ATC CGC CTT CTA GAC CCT CCA CTT CAT GAG TTT 2170

Gly Leu Pro Val Thr lie Arg leu Leu Asp Pro Pro leu His Glu Phe

695 700 705

TTA CCC GAA GGT GAT CTA GAA CAC ATA GTG AAC GAA CTT GCA GTC GAC 2218

Leu Pro Glu Gly Asp Leu Glu His Lie Val Asn Glu Leu Ala Val Asp

710 715 720

ACA GGC ATG AGT GCA GAT GAA ATC TAT TCA AAA ATC GAA AAT CTA TCT 2266

Thr Gly Met Ser Ala Asp Glu lie Tyr Ser Lys lie Glu Asn Leu Ser 725 730 735

GAA GTG AAC CCT ATG CTT GGT TTC CGT GGT TGC AGA TTA GGG ATT TCA 2314 Glu Val Asn Pro Met Leu Gly Phe Arg Gly Cys Arg Leu Gly He Ser 740 745 750 755

TAC CCC GAG CTA ACA GAA ATG CAA GTT CGT GCG ATC TTT CAA GCT GCA 2362 Tyr Pro Glu Leu Thr Glu Met Gin Val Arg Ala He Phe Gin Ala Ala

760 765 770

GTG TCT ATG ACC AAT CAG GGG GTG ACT GTA ATA CCA GAG ATC ATG GTT 2410 Val Ser Met Thr Asn Gin Gly Val Thr Val lie Pro Glu lie Met Val

775 780 785

CCG TTA GTG GGG ACA CCT CAG GAA TTA CGT CAT CAA ATC ACT GTA ATT 2458 Pro Leu Val Gly Thr Pro Gin Glu Leu Arg His Gin He Ser Val l ie

790 795 800

CGT GGA GTA GCT GCA AAT GTG TTT GCT GAA ATG GGG GTG ACA TTG GAA 2506 Arg Gly Val Ala Ala Asn Val Phe Ala Glu Met Gly Val Thr Leu Glu

805 810 815

TAT AAA GTG GGA ACG ATG ATT GAG ATT CCT CGA GCT GCT TTA ATA GCT 2554 Tyr Lys Val Gly Thr Met lie Glu lie Pro Arg Ala Ala Leu lie Ala 820 825 830 830 835

GAA GAG ATT GGA AAA GAA GCT GAT TTC TTT TCG TTT GGA ACC AAT GAT 2602 Glu Glu lie Gly Lys Glu Ala Asp Phe Phe Ser Phe Gly Thr Asn Asp

840 845 850

CTG ACC CAG ATG ACA TTT GGG TAC AGC AGA GAT GAT GTT GGC AAG TTT 2650 Leu Thr Gin Met Thr Phe Gly Tyr Ser Arg Asp Asp Val Gly Lys Phe

855 860 865

TTG CAG ATT TAT CTT GCT CAA GGC ATT CTG CAG CAT GAT CCA TTT GAG 2698 Leu Gin lie Tyr Leu Ala Gin Gly lie Leu Gin His Asp Pro Phe Glu

870 875 880

GTT ATT GAC CAG AAA GGG GTG GGT CAG TTG ATT AAG ATG GCT ACG GAG 2746 Val He Asp Gin Lys Gly Val Gly Gin Leu lye Lys Met Ala Thr Glu

885 890 895

AAA GGT CGT GCA GCA AAT CCT AAC TTA AAG GTT GGG ATA TGT GGG GAG 2794 Lys Gly Arg Ala Ala Asn Pro Asn Leu Lys Val Gly He Cys Gly Glu 900 905 910 915

CAT GGT GGG GAG CCT TCT TCT GTT GCA TTT TTT GAT GGA GTT GGA CTA 2842 His Gly Gly Glu Pro Ser Ser Val Ala Phe Phe Asp Gly Val Gly Leu

920 925 930

GAT TAT GTG TCG TGC TCT CCA TTT AGG GTT CCT ATC GCA AGG TTG GCC 2890 Asp Tyr Val Ser Cys Ser Pro Phe Arg Val Pro lie Ala Arg Leu Ala

935 940 945

GCT GCA CAA GTC ATT GTT TAAGCTT 2915 Ala Ala Gin Val lie Val

950 SEQ ID NO: 2

Array length: 2 8 8 0

Sequence type: nucleic acid

Array

CGGCGCAGTA GGGGATCGGA AGG ATG GCG GCA TCG GTT TCC AGG GCC ATC TGC 53

Met Ala Ala Ser Val Ser Arg Ala lie Cys 1 5 10

GTA CAG AAG CCG GGC TCA AAA TGC ACC AGG GAC AGG GAA GCG ACC TCC 101 Val Gin Lys Pro Gly Ser Lys Cys Thr Arg Asp Arg Glu Ala Thr Ser

15 20 25

TTC GCC CGC CGA TCG GTC GCA GCG CCG AGG CCC CCG CAC GCC AAA GCC 149 Phe Ala Arg Arg Ser Val Ala Ala Pro Arg Pro Pro His Ala Lys Ala

30 35 40

CGC CGG CGT CAT CCG CTC CGA CTC CGG CGC GGG ACG GGG CCA CAT TGC 197 002 ¾6ΐ 06ΐ

3JV ^η ΐ3 ^ TD s sAq eiv BTV ^η . Ί ^ ΤΟ ^ TV ^ TV ΐ¾ "TO ^ds V usv naq

629 333 0V9 900 39V 3VV 339 030 DIO ODD 330 030 010 WO 3V3 OVV 313 m 081 5il

^ ΐθ ^Η3 Ί usv naq Λ J¾ dsy Αχο OJJ J3S Ι ^ Λ ^ TV ^ χν

185 093 013 DVV 313 910 33V 3V3 OIV OIV 300 333 OIV DDI 9ID 330 330

Oil G9I 091 591 χθ J9S 3jy ΪΒΛ J3S nsi nai OJJ 3JV UTO o ^ d DS V ^ ΐθ Interview 8 £ 300 DDI 303 DID 331 3X3 013 DID DD3 303 0V3 033 IV9 D90 013 33V

09ΐ OH

BTV ΛΤΟ 13W Yu ^ L nig cLt 丄 UTO χο dsy A an \ BTV

98 ^ 330 309 OIV 3V1 0V9 OVO 019 901 OVO 013 3D0 3V0 310 DIV 3V0 330

dJX nsq Αιο εχν OJJ na Βχν s Αχο εχν dsy 丄 uio u ^ j) SAQ ^ 301 313 000 933 333 313 313 339 DDI 900 330 3V9 3V3 DVI 0V3 0V3 391

02ΐ 9Π 0Π

^ TV ^η ΐ3 JSS ΐ ^ Λ J ^ Md ^ TO QJd ojj iB J3S naq Αχο 3Π -i3S

686 9D0 OVO 03V 931 913 03V DII 000 V30 933 019 031 OID 903 3IV DOV

50ΐ 00ΐ 96

BTV l ^ K "TO BTV n9i usy ^e IV ^ TO sAq Αχ ^ 10 ^η9 Ί ^na l ⁿ lD ^s 1 jaw in 9D9 OIV OVO 330 OID DVV 930 300 OVV DOO DOO 013 OID WO OVV OXV

06 98 08 Qi

: I usy ^ ΐθ nio J9S sA Αχο sAq Λχο 3qj STH sqj ΙΒΛ S ^ V sAq

S62 33V OVV OVV 303 OVO 30V OVV 300 9VV 300 311 DVD Oil 010 99V OVV

OZ S9 09 sq jqi Jqi UTO 3Π. · ¾ v ^ TV dsy T ^ A ΐ ^ Λ ^ TV V ^Η3 Ί OJd J3S

VVV 33V 03V 0V3 VIV 033 030 330 DV9 IID 319 333 OOV 913 033 931

55 05 ^

SAQ ST ^ OJJ ij) _r丄^{TJ) 3jy S ^ V n9 S-} ^ V ηθ 1 OJJ S TJ {gjy

OS

P0l0IS6dTILDd 69 W / 96 OAV TTC GCC TAC GAC TCC TTC CGC CGC TTC CTC GAC ATG TTC GGC AAC GTC 677 Phe Ala Tyr Asp Ser Phe Arg Arg Phe Leu Asp Met Phe Gly Asn Val

205 210 215

GTC ATG GAC ATC CCC CGC TCA CTG TTC GAA GAG AAG CTT GAG CAC ATG 725 Val Met Asp lie Pro Arg Ser Leu Phe Glu Glu Lys Leu Glu His Met

220 225 230

AAG GAA TCC AAG GGG CTG AAG AAC GAC ACC GAC CTC ACG GCC TCT GAC 773 Lys Glu Ser Lys Gly Leu Lys Asn Asp Thr Asp Leu Thr Ala Ser Asp 235 240 245 250

CTC AAA GAG CTC GTG GGT CAG TAC AAG GAG GTC TAC CTC TCA GCC AAG 821 Leu Lys Glu Leu Val Gly Gin Tyr Lys Glu Val Tyr Leu Ser Ala Lys

255 260 265

GGA GAG CCA TTC CCC TCA GAC CCC AAG AAG CAG CTG GAG CTA GCA GTG 869 Gly Glu Pro Phe Pro Ser Asp Pro Lys Lys Gin Leu Glu Leu Ala Val

270 275 280

CTG GCT GTG TTC AAC TCG TGG GAG AGC CCC AGG GCC AAG AAG TAC AGG 917 Leu Ala Val Phe Asn Ser Trp Glu Ser Pro Arg Ala Lys Lys Tyr Arg

285 290 295

AGC ATC AAC CAG ATC ACT GGC CTC AGG GGC ACC GCC GTG AAC GTG CAG 965 Ser lie Asn Gin lie Thr Gly Leu Arg Gly Thr Ala Val Asn Val Gin

300 305 310

TGC ATG GTG TTC GGC AAC ATG GGG AAC ACT TCT GGC ACC GGC GTG CTC 1013 Cys Met Val Phe Gly Asn Met Gly Asn Thr Ser Gly Thr Gly Val Leu

315 320 325 330

TTC ACC AGG AAC CCC AAC ACC GGA GAG AAG AAG CTG TAT GGC GAG TTC 1061 Phe Thr Arg Asn Pro Asn Thr Gly Glu Lys Lys Leu Tyr Gly Glu Plu

335 340 345

CTG GTG AAC GCT CAG GGT GAG GAT GTG GTT GCC GGA ATA AGA ACC CCA 1109 Leu Val Asn Ala Gin Gly Glu Asp Val Val Ala Gly lie Arg Thr Pro 350 355 360

GAG GAC CTT GAC GCC ATG AAG AAC CTC ATG CCA CAG GCC TAC GAC GAG 1157

Glu Asp Leu Asp Ala Met Lys Asn Leu Met Pro Gin Ala Tyr Asp Glu

365 370 375

CTT GTT GAG AAC TGC AAC ATC CTG GAG AGC CAC TAC AAG GAA ATG CAG 1205

Leu Val Glu Asn Cys Asn lie Leu Glu Ser His Tyr Lys Glu Met Gin

380 385 390

GAT ATC GAG TTC ACT GTC CAG GAA AAC AGG CTG TGG ATG TTG CAG TGC 1253

Asp lie Glu Phe Thr Val Gin Glu Asn Arg Leu Trp Met Leu Gin Cys 395 400 405 410

AGG ACA GGG AAA CGT ACG GGC AAA ACT GCC GTG AAG ATC GCC GTG GAC 1301

Arg Thr Gly Lys Arg Thr Gly Lys Ser Ala Val Lys lie Ala Val Asp

415 420 425

ATG GTT AAC GAG GGC CTT GTT GAG CCC CGC TCA GCG ATC AAG ATG GTA 1349

Met Val Asn Glu Gly Leu Val Glu Pro Arg Ser Ala lie Lys Met Val

430 435 440

GAG CCA GGC CAC CTG GAC CAG CTT CTT CAT CCT CAG TTT GAG AAC CCG 1397

Glu Pro Gly His Leu Asp Gin Leu Leu His Pro Gin Phe Glu Asn Pro

445 450 455

TCG GCG TAC AAG GAT CAA GTC ATT GCC ACT GGT CTG CCA GCC TCA CCT 1445

Ser Ala Tyr Lys Asp Gin Val lie Ala Thr Gly Leu Pro Ala Ser Pro

460 465 470

GGG GCT GCT GTG GGC CAG GTT GTG TTC ACT GCT GAA GAT GCT GAA GCA 1493

Gly Ala Ala Val Gly Gin Val Val Phe Thr Ala Glu Asp Ala Glu Ala 475 480 485 490

TGG CAT TCC CAA GGG AAA GCT GCT ATT CTG GTA AGG GCG GAG ACC AGC 1541

Trp His Ser Gin Gly Lys Ala Ala lie Leu Val Arg Ala Glu Thr Ser

495 500 505

CCT GAG GAC GTT GGT GGC ATG CAC GCT GCT GTG GGG ATT CTT ACA GAG 1589 599 099 599 n9i

is «" TO 3

1202 313 030 0V3 9V3 09V DID OVO 113 33V 333 130 OIV IIV OIV OVO 00V 0¾9 9 5S9 Λ BIV sAq an 3JV ητθ dsy ss BTV sqd 3Md i ^ W SIH jqi §jy 8A6I 310 130 OVVIIV 30V 130V 3 丄丄 OIV 3V3 OVO V3V D03

089 529 0Z9 s i½i Αχο sn H "TO ^ TV ^ TD usy usy SJV ^ iv -iqi ns dsy

9Ζ6Ϊ 301 VLL VOO IIV VOO VV3 V30 000 IVV 3VV V03 333 I3V Oil V39 IVO

919 019 ¾09 dsy OJJ: rii 丄 dsy Βχν usy εχν naq

dsy dsy

LL \ IVO 133 33V XVO 330 3VV 133 013 310 OVV 313 9VV VOV 119 IVO 丄 V3

009 569 069

ΐ¾ dJi BTV 1SW 3Md JMI 3 naq dsy Α ο jag naq Βχγ oaj

s

6Z8I 010 001 330 OIV 311 XDV VOO 013 IVO 130 I3V 113 033 VDD V33 DDI

589 085

n9i ojd uio sAi Ai3 ns d \ \ ΙΒΛ "TO ^ IO ^J9 S ^ 13 usy nsq ias

18 I 113 033 0V3 OVV 903 lid DIV 010 9V3 100 13V 031 933 IVV 913 031 0 595 099 995

"31 dJ 丄 ηχο Ai9 3jy na τ ^ Λ SJH S ^ ΐθ 9T I ^ Mi ΐ ^ Λ" 91 sAq εεπ 013 oox ovo 109 wo αοα οια oio ivo DOV VOO 3 丄 V OOV OIO OIO OVV

055 5 ^ 9 0 ^ 5

"TO BTV dsy usy T ^ A v 3ΐ Ι ^ IO s AJO JSS i¾A s sAq

5891 DVO 030 XVO 3VV VI9 303 XIV 390 VDX 391 VOO 031 313 331 331 VVV

525 089 929

^ ΐθ 丄 djx gjy BTV ΐ ^ Λ ΐ ^ Λ ^ TV ^ TV SIH J3S Jqi ISW ^ το ^ ΐθ ^3J V Ζ £ 9ΐ 900 031 ODX 1D3 V30 310 910 130 130 3V3 DDI 13V OIV 300 103 09V

029 919 015

ΠΤΟ -iqi nai an BTV BTV SIR 3K ^ TD ^ IO ΐ ^ Λ dsy ηχο OJJ εε

PQl0JS6dF / lDd 69 £ 96 O GAC CGT CTC TTG ACG TAT CAG AGG TCT GAC TTC GAA GGC ATT TTC CGT 2069 Asp Arg Leu Leu Thr Tyr Gin Arg Ser Asp Phe Glu Gly lie Phe Arg

670 675 680

GCT ATG GAT GGA CTC CCG GTG ACC ATC CGA CTC CTG GAC CAT CCT TCT 2117 Ala Met Asp Gly Leu Pro Val Thr lie Arg Leu Leu Asp His Pro Ser

685 690 695

TAC GAG TTC CTT CCA GAA GGG AAC ATC GAG GAC ATT GTA AGT GAA TTA 2165 Tyr Glu Phe Leu Pro Glu Gly Asn lie Glu Asp lie Val Ser Glu Leu

700 705 710

TGT GCT GAG ACG GGA GCC AAC CAG GAG GAT GCC CTC GCG CGA ATT GAA 2213 Cys Ala Glu Thr Gly Ala Asn Gin Glu Asp Ala Leu Ala Arg lie Glu 715 720 725 730

AAG CTT TCA GAA GTA AAC CCG ATG CTT GGC TTC CGT GGG TGC AGG CTT 2261 Lys Leu Ser Glu Val Asn Pro Met Leu Gly Phe Arg Gly Cys Arg Leu

735 740 745

GGT ATA TCG TAC CCT GAA TTG ACA GAG ATG CAA GCC CGG GCC ATT TTT 2309 Gly He Ser Tyr Pro Glu Leu Thr Glu Met Gin Ala Arg Ala lie Phe

750 755 760

GAA GCT GCT ATA GCA ATG ACC AAC CAG GGT GTT CAA GTG TTC CCA GAG 2357 Glu Ala Ala lie Ala Met Thr Asn Gin Gly Val Gin Val Phe Pro Glu

765 770 775

ATA ATG GTT CCT CTT GTT GGA ACA CCA CAG GAA CTG GGG CAT CAA GTG 2405 l ie Met Val Pro Leu Val Gly Thr Pro Gin Glu Leu Gly His Gin Val

780 785 790

ACT CTT ATC CGC CAA GTT GCT GAG AAA GTG TTC GCC AAT GTG GGC AAG 2453 Thr Leu lie Arg Gin Val Ala Glu Lys Val Phe Ala Asn Val Gly Lys 795 800 805 810

ACT ATC GGG TAC AAA GTT GGA ACA ATG ATT GAG ATC CCC AGG GCA GCT 2501 Thr He Gly Tyr Lys Val Gly Thr Met lie Glu lie Pro Arg Ala Ala 815 820 825

CTG GTG GCT GAT GAG ATA GCG GAG CAG GCT GAA TTC TTC TCC TTC GGA 2549 Leu Val Ala Asp Glu lie Ala Glu Gin Ala Glu Phe Phe Ser Phe Gly

830 835 840

ACG AAC GAC CTG ACG CAG ATG ACC TTT GGG TAC AGC AGG GAT GAT GTG 2597 Thr Asn Asp Leu Thr Gin Met Thr Phe Gly Tyr Ser Arg Asp Asp Val

845 850 855

GGA AAG TTC ATT CCC GTT CAT CTT GCT CAG GGC ATC CTC CAA CAT GAC 2645 Gly Lys Phe lie Pro Val His Leu Ala Gin Gly lie Leu Gin His Asp

860 865 870

CCC TTC GAG GTC CTG GAC CAG AGG GGA GTG GGC GAG CTG GTG AAG TTT 2693 Pro Phe Glu Val Leu Asp Gin Arg Gly Val Gly Glu Leu Val Lys Phe 875 880 885 890

GCT ACA GAG AGG GGC CGC AAA GCT AGG CCT AAC TTG AAG GTG GGC ATT 2741 Ala Thr Glu Arg Gly Arg Lys Ala Arg Pro Asn Leu Lys Val Gly He

895 900 905

TGT GGA GAA CAC GGT GGA GAG CCT TCG TCT GTG GCC TTC TTC GCG AAG 2789 Cys Gly Glu His Gly Gly Glu Pro Ser Ser Val Ala Phe Phe Ala Lys

910 915 920

GCT GGG CTG GAT TTC GTT TCT TGC TCC CCT TTC AGG GTT CCG ATT GCT 2837 Ala Gly Leu Asp Phe Val Ser Cys Ser Pro Phe Arg Val Pro He Ala

925 930 935

AGG CTA GCT GCA GCT CAG GTG CTT GTC TGAGGCTGCC TCCTCG 2880 Arg Leu Ala Ala Ala Gin Val Leu Val

940 945 SEQ ID NO: 3

Array length: 2 6 1 0

Sequence type: nucleic acid Array

GAATTCTCAA TCCTTTGCTC ATCGCAGCAT ATCAATGTTA ACACATAAAC TTTAGGAGGA 60 AGAAAACTT ATG GCA AAA TGG GTT TAT AAG TTC GAA GAA GGC AAT GCA TCT 11 1

Met Ala Lys Trp Val Tyr Lys Phe Glu Glu Gly Asn Ala Ser 1 5 10

ATG AGA AAC CTT CTT GGA GGC AAA GGC TGC AAC CTT GCA GAG ATG ACC 159

Met Arg Asn Leu Leu Gly Gly Lys Gly Cys Asn Leu Ala Glu Met Thr 15 20 25 30

ATC TTA GGA ATG CCG ATT CCA CAG GGC TTT ACT GTA ACA ACA GAA GCT 207 l ie Leu Gly Met Pro lie Pro Gin Gly Phe Thr Val Thr Thr Glu Ala

35 40 45

TGT ACA GAG TAC TAC AAC AGT GGA AAA CAG ATC ACA CAG GAA ATT CAG 255

Cys Thr Glu Tyr Tyr Asn Ser Gly Lys Gin lie Thr Gin Glu lie Gin

50 55 60

GAT CAG ATT TTC GAA GCT ATC ACA TGG TTA GAG GAA CTG AAC GGC AAG 303

Asp Gin lie Phe Glu Ala lie Thr Trp Leu Glu Glu Leu Asn Gly Lys

65 70 75

AAG TTC GGC GAC ACT GAA GAT CCG TTA TTA GTA TCT GTA CGT TCC GCG 351

Lys Phe Gly Asp Thr Glu Asp Pro Leu Leu Val Ser Val Arg Ser Ala

80 85 90

GCC CGC GCA TCC ATG CCG GGT ATG ATG GAT ACC ATC CTG AAC CTT GGT 399

Ala Arg Ala Ser Met Pro Gly Met Met Asp Thr lie leu Asn Leu Gly 95 100 105 110

TTA AAC GAC GTT GCA GTA GAG GGC TTT GCA AAG AAA ACG GGA AAT CCA 447

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

115 120 125

AGA TTT GCA TAT GAT TCT TAC AGA AGA TTT ATC CAG ATG TAT TCC GAC 495

Arg Phe Ala Tyr Asp Ser Tyr Arg Arg Phe lie Gin Met Tyr Ser Asp

130 135 140 GTA GTT ATG GAA GTT CCG AAG TCC CAT TTC GAG AAA ATC ATC GAT GCG 543

Val Val Met Glu Val Pro Lys Ser His Phe Glu Lys lie lie Asp Ala

145 150 155

ATG AAA GAA GAA AAG GGC GTT CAC TTC GAT ACA GAC CTG ACT GCC GAT 591

Met Lys Glu Glu Lys Gly Val His Phe Asp Thr Asp Leu Thr Ala Asp

160 165 170

GAT TTA AAA GAG CTG GCT GAG AAG TTC AAA GCT GTT TAC AAA GAG GCT 639

Asp Leu Lys Glu Leu Ala Glu Lys Phe Lys Ala Val Tyr Lys Glu Ala

175 180 185 190

ATG AAC GGC GAA GAG TTC CCA CAG GAG CCG AAG GAT CAG TTA ATG GGC 687

Met Asn Gly Glu Glu Phe Pro Gin Glu Pro Lys Asp Gin Leu Met Gly

195 200 205

GCT GTT AAA GCA GTT TTC CGT TCC TGG GAC AAC CCT CGT GCA ATC GTA 735

Ala Val Lys Ala Val Phe Arg Ser Trp Asp Asn Pro Arg Ala lie Val

210 215 220

TAC CGC CGT ATG AAC GAT ATC CCT GGA GAC TGG GGT ACT GCA GTT AAC 783

Tyr Arg Arg Met Asn Asp He Pro Gly Asp Trp Gly Thr Ala Val Asn

225 230 235

GTT CAG ACC ATG GTA TTT GGT AAC AAG GGC GAG ACC AGC GGT ACA GGC 831

Val Gin Thr Met Val Phe Gly Asn Lys Gly Glu Thr Ser Gly Thr Gly

240 245 250

GTT GCC TTC ACA CGT AAC CCA TCC ACA GGT GAA AAA GGC ATC TAC GGT 879

Val Ala Phe Thr Arg Asn Pro Ser Thr Gly Glu Lys Gly lie Tyr Gly

255 260 265 270

GAG TAC CTG ATC AAT GCA CAG GGC GAG GAC GTA GTT GCA GGT GTC CGC 927

Glu Tyr Leu lie Asn Ala Gin Gly Glu Asp Val Val Ala Gly Val Arg

275 280 285

ACA CCA CAG CCT ATC ACC CAG TTA GAG AAC GAT ATG CCT GAC TGC TAC 975

Thr Pro Gin Pro】 le Thr Gin Leu Glu Asn Asp Met Pro Asp Cys Tyr 290 295 300

AAG CAG TTC ATG GAT CTG GCC ATG AAG CTG GAG AAA CAT TTC CGT GAC 1023

Lys Gin Phe Met Asp Leu Ala Met Lys Leu Glu Lys His Phe Arg Asp

305 310 315

ATG CAG GAT ATG GAG TTC ACA ATC GAG GAA GGT AAA TTA TAC TTC TTA 1071

Met Gin Asp Met Glu Phe Thr lie Glu Glu Gly Lys Leu Tyr Phe Leu

320 325 330

CAG ACA CGT AAC GGC AAG AGA ACA GCT CCG GCT GCT CTT CAG ATT GCC 1119

Gin Thr Arg Asn Gly Lys Arg Thr Ala Pro Ala Ala Leu Gin lie Ala

335 340 345 350

TGC GAT TTA GTA GAC GAA GGC ATG ATC ACA GAG GAA GAG GCT GTT GTA 1167

Cys Asp Leu Val Asp Glu Gly Met lie Thr Glu Glu Glu Alu Val Val

355 360 365

AGA ATC GAA GCA AAA TCT CTT GAT CAG TTA CTT CAC CCG ACC TTC AAC 1215

Arg lie Glu Ala Lys Ser Leu Asp Gin Leu Leu His Pro Thr Phe Asn

370 375 380

CCG GCT GCT TTA AAG GCC GGC GAA GTA ATC GGT TCC GCT CTT CCG GCA 1263

Pro Ala Ala Leu Lys Ala Gly Glu Glu Val lie Gly Ser Ala Leu Pro Ala

385 390 395

TCT CCT GGC GCA GCA GCA GGT AAA GTA TAC TTC ACC GCT GAT GAG GCT 1311

Ser Pro Gly Ala Ala Ala Gly Lys Val Tyr Phe Thr Ala Asp Glu Ala

400 405 410

AAG GCT GCC CAC GAG AAG GGT GAG AGA GTT ATC CTT GTT CGT CTT GAG 1359

Lys Ala Ala His Glu Lys Gly Glu Arg Val lie Leu Val Arg Leu Glu

415 420 425 430

ACA TCT CCG GAA GAT ATC GAA GGT ATG CAT GCA GCC GAA GGT ATC CTG 1407

Thr Ser Pro Glu Asp He Glu Gly Met His Ala Ala Glu Gly lie Leu

435 440 445

ACA GTG CGC GGC GGT ATG ACA AGC CAT GCA GCC GTA GTT GCA CGT GGT 1455 Thr Val Arg Gly Gly Met Thr Ser His Ala Ala Val Val Ala Arg Gly

450 455 460

ATG GGA ACA TGC TGC GTA TCC GGA TGC GGT GAG ATC AAG ATC AAC GAA 1503 Met Gly Thr Cys Cys Val Ser Gly Cys Gly Glu lie Lys lie Asn Glu

465 470 475

GAA GCT AAG ACA TTC GAA CTT GGC GGA CAC ACA TTT GCA GAG GGA GAT 1551 Glu Ala Lys Thr Phe Glu Leu Gly Gly His Thr Phe Ala Glu Gly Asp

480 485 490

TAC ATC TCC TTA GAT GGT TCC ACA GGT AAG ATT TAC AAG GGC GAC ATC 1599 Tyr l ie Ser Leu Asp Gly Ser Thr Gly Lys lie Tyr Lys Gly Asp lie 495 500 505 510

GAG ACT CAG GAA CGT TCC GTA AGC GGA AGC TTC GAG CGT ATC ATG GTA 1647 Glu Thr Gin Glu Arg Ser Val Ser Gly Ser Phe Glu Arg He Met Val

515 520 525

TGG GCT GAC AAG TTC AGA ACA TTA AAG GTT CGT ACA AAT GCC GAC ACA 1695 Trp Ala Asp Lys Phe Arg Thr Leu Lys Val Arg Thr Asn Ala Asp Thr

530 535 540

CCG GAA GAT ACA CTC AAT GCC GTT AAA CTG GGT GCA GAG GGC ATC GGT 1743 Pro Glu Asp Thr Leu Asn Ala Val Lys Leu Gly Ala Glu Gly lie Gly

545 550 555

CTT TGC CGT ACA GAG CAT ATG TTC TTC GAG GCT GAC AGA ATC ATG AAG 1791 Leu Cys Arg Thr Glu His Met Phe Phe Glu Ala Asp Arg He Met Lys

560 565 570

ATC AGA AAG ATC ATC CTT TCC GAT TCA GTG GAA GCA AGA GAA GAG GCT 1839 lie Arg Lys Met lie Leu Ser Asp Ser Val Glu Ala Arg Glu Glu Ala

575 580 585 590

CTG AAC GAA TTA ATC CCG TTC CAG AAG GGC GAT TTC AAG GCT ATG TAC 1887 Leu Asn Glu Leu lie Pro Phe Gin Lys Gly Asp Phe Lys Ala Met Tyr

595 600 605 AAA GCT CTG GAA GGC AGG CCA ATG ACG GTT CGC TAC CTG GAT CCG CCG 1935

Lys Ala Leu Glu Gly Arg Pro Met Thr Val Arg Tyr Leu Asp Pro Pro

610 615 620

CTG CAT GAG TTC GTT CCT CAT ACA GAA GAG GAG CAG GCT GAA CTG GCT 1983

Leu His Glu Phe Val Pro His Thr Glu Glu Glu Gin Ala Glu Leu Ala

625 630 635

AAG AAC ATG GGC CTT ACT TTA GCA GAA GTA AAA GCA AAA GTT GAC GAA 2031

Lys Asn Met Gly Leu Thr Leu Ala Glu Val Lys Ala Lys Val Asp Glu

640 645 650

TTA CAC GAG TTC AAC CCA ATG ATG GGC CAT CGT GGC TGC CGT CTT GCA 2079

Leu His Glu Phe Asn Pro Met Met Gly His Arg Gly Cys Arg Leu Ala

655 660 665 670

GTT ACC TAT CCG GAA ATT GCA AAG ATG CAG ACA AGA GCC GTT ATG GAA 2127

Val Thr Tyr Pro Glu lie Ala Lys Met Gin Thr Arg Ala Val Met Glu

675 680 685

GCT GCT ATC GAA GTG AAG GAA GAG ACA GGA ATC GAT ATT GTT CCT GAG 2175

Ala Ala lie Glu Val Lys Glu Glu Thr Gly lie Asp lie Val Pro Glu

690 695 700

ATC ATG ATT CCG TTA GTT GGC GAG AAG AAA GAG CTT AAG TTC GTT AAG 2223 lie Met lie Pro Leu Val Gly Glu Lys Lys Glu Leu Lys Phe Val Lys

705 710 715

GAC GTA GTT GTG GAA GTA GCT GAG CAG GTT AAG AAA GAG AAA GGT TCC 2271

Asp Val Val Val Glu Val Ala Glu Gin Val Lys Lys Glu Lys Gly Ser

720 725 730

GAT ATG CAG TAC CAC ATC GGT ACC ATG ATC GAA ATT CCT CGT GCA GCT 2319

Asp Met Gin Tyr His lie Gly Thr Met lie Glu lie Pro Arg Ala Ala

735 740 745 750

CTC ACA GCA GAT GCC ATC GCT GAG GAA GCA GAG TTC TTC TCC TTC GGT 2367

Leu Thr Ala Asp Ala lie Ala Glu Glu Ala Glu Phe Phe Ser Phe Gly 25 ΐ ΙΙΟ V33 ID VOO IIV VVV VOV 01V VDV 301 113 V90 100 VOO OVV V00

51 01 5 χ na na sAq sAq usy Jqi dsy;) dsy ηχο 9qd BTV ^ 丄 ΐ ^ Λ

V09 113 313 VVV OVV 3VV 33V VOO IVO 100 IVO WO 3IX 130 IVl VXO

Ϊ

3JV "TO 19W

99 V9V VV3 OIV V1VVXV3 V3I1V31I3V OVlllVVOVV X3IIIVV00V VlllVXOVVO mm ■■ m

Z Z L Z ■

0192 0

OJd rial nsi ai l ηχ ^ Αχο S TH ηχο Αχ s

6092 VI0VVV3V33 3 Jii) V;) V 丄 933 113 1X3 31V OVO 330 3V3 OVO 390 DDI

0S8 928 028 918

3T I ^ TO SA3 SAi naq A ^ g OJJ V ^ 丄 "TO ^ ΐί) sAq sAq i BTV

6592 DIV 393 301 OVV 113 309 033 IDD V3V 3V3 ID3 300 VVV OVV 110 VD9

018 908 008

H nig ΙΒΛ n9i UTO ^ ld ^ TO "TO dsy na gjy ^ TV ³ 4d QJd

3IV DV3 VIO V 丄丄 OVO 300 XX3 300 V3V 9V3 3V9 113 VOV V30 Dll VDD

96Z 06Z, 98Z

dsy S "TO JA 丄 3T I sAi BTV sX JAX 丄 d ^ dsy ns sq ^ sAq Λχο

IVO 331 OVO IVl 丄丄 V VVV V30 VVV IVl 3VI 331 IVO 013 311 OVV 309

08A 0 / i

BTV dsv dsy V S ^ Md ^ TO ^ Md -iqi law "ID J¾" 91 dsy usy -ΐ¾

330 3V0 103 331 311 300 Oil VOV 01V OVD VOV VII DVD 3VV VDV 99L 09L 1

I P

»OIO / S6df / X3d 69h 0/96 OAV Gly Lys Gly Ala Gly Leu Cys Thr Met Thr Lys lie Gly Leu Pro Val

20 25 30 35

CCA CAA GGA TTT GTT ATT ACA ACT GAA ATG TGT AAA CAA TTC ATT GCT 200

Pro Gin Gly Phe Val lie Thr Thr Glu Met Cys Lys Gin Phe lie Ala

40 45 50

AAT GGA AAC AAA ATG CCA GAA GGA TTA ATG GAA GAA GTT AAA AAA GAA 248 Asn Gly Asn Lys Met Pro Glu Gly Leu Met Glu Glu Val Lys Lys Glu

55 60 65

TAT CAA TTA GTT GAA AAG AAA TCA GGA AAA GTC TTT GGA GGA GAA GAA 296 Tyr Gin Leu Val Glu Lys Lys Ser Gly Lys Val Phe Gly Gly Glu Glu

70 75 80

AAT CCA CTT CTT GTT TCA GTC AGA TCA GGA GCT GCT ATG TCT ATG CCA 344 Asn Pro Leu Leu Val Ser Val Arg Ser Gly Ala Ala Met Ser Met Pro

85 90 95

GGT ATG ATG GAT ACT ATT CTT AAT CTT GGA CTT AAT GAT AAA ACT GTT 392 Gly Met Met Asp Thr lie Leu Asn Leu Gly Leu Asn Asp Lys Thr Val 100 105 110 115

GTT GCT CTT GCT AAA TTA ACC AAC AAT GAA AGA TTT GCA TAT GAT TCA 440 Val Ala Leu Ala Lys Leu Thr Asn Asn Glu Arg Phe Ala Tyr Asp Ser

120 125 130

TAC AGA AGA TTT GTT TCC CTC TTC GGA AAG ATT GCT CTT AAT GCT TGT 488 Tyr Arg Arg Phe Val Ser Leu Phe Gly Lys lie Ala Leu Asn Ala Cys

135 140 145

GAT GAA GTT TAT GAT AAG ACT CTT GAA AAC AAA AAA GTT GAA AAG GGA 536 Asp Glu Val Tyr Asp Lys Thr Leu Glu Asn lys Lys Val Glu Lys Gly

150 155 160

GTT AAA TTA GAT ACT GAA TTA GAT GCT AAT GAT ATG AAA GAA CTT GCA 584 Val Lys Leu Asp Thr Glu Leu Asp Ala Asn Asp Met Lys Glu Leu Ala

165 170 175 CAA GTC TTC ATT AAA AAG ACT GAA GAA TTC ACT AAA CAA CCA TTC CCA 632

Gin Val Phe He Lys Lys Thr Glu Glu Plu Thr Lys Gin Pro Phe Pro

180 185 190 195

GTT GAT CCA TAT GCT CAA TTA GAA TTT GCC ATT TGT GCT GTA TTC AGA 680

Val Asp Pro Tyr Ala Gin Leu Glu Phe Ala lie Cys Ala Val Phe Arg

200 205 210

TCA TGG ATG GGA AAG AGA GCT GTT GAT TAC AGA AGA GAA TTC AAG ATT 728

Ser Trp Met Gly Lys Arg Ala Val Asp Tyr Arg Arg Glu Phe Lys lie

215 220 225

ACT CCA GAA CAA GCT GAT GGA ACT GCT GTT TCA GTT GTT TCT ATG GTT 776

Thr Pro Glu Gin Ala Asp Gly Thr Ala Val Ser Val Val Ser Met Val

230 235 240

TAT GGT AAT ATG GGT AAT GAT TCA GCT ACT GGT GTT TGT TTC ACT AGA 824

Tyr Gly Asn Met Gly Asn Asp Ser Ala Thr Gly Val Cys Phe Thr Arg

245 250 255

GAT CCA GGA ACA GGA GAA AAT ATG TTC TTC GGA GAA TAT CTT AAG AAT 872

Asp Pro Gly Thr Gly Glu Asn Met Phe Phe Gly Glu Tyr Leu Lys Asn

260 265 270 275

GCA CAA GGA GAA GAT GTT GTT GCT GGT ATT AGA ACA CCA CAA ATT ATT 920

Ala Gin Gly Glu Asp Val Val Ala Gly lie Arg Thr Pro Gin lie lie

280 285 290

TCA AAG ATG GCA GAA GAT CGA GAT CTT CCA GGT TGC TAT GAA CAA CTT 968

Ser Lys Met Ala Glu Asp Arg Asp Leu Pro Gly Cys Tyr Glu Gin Leu

295 300 305

CTT GAT ATT AGA AAG AAA TTA GAA GGA TAT TTC CAT GAA GTA CAA GAC 1016

Leu Asp lie Arg Lys Lys Leu Glu Gly Tyr Phe His Glu Val Gin Asp

310 315 320

TTT GAA TTC ACT ATT GAA AGA AAG AAA CTT TAC ATG CTC CAA ACT AGA 1064

Phe Glu Phe Thr lie Glu Arg Lys Lys Leu Tyr Met Leu Gin Thr. Arg 325 330 335

AAT GGA AAG ATG AAT GCA ACT GCT ACT GTC AGA ACA GGA GTT GAT ATG 1112 Asn Gly Lys Met Asn Ala Thr Ala Thr Val Arg Thr Gly Val Asp Met 340 345 350 355

GTT GAA GAA GGA CTT ATT ACA AAA GAA CAA GCC ATT ATG AGA ATT GCA 1160 Val Glu Glu Gly Leu lie Thr Lys Glu Gin Ala lie Met Arg lie Ala

360 365 370

CCA CAA TCA GTT GAT CAA TTA CTT CAT AAG AAT ATG CCA GCT AAT TAT 1208 Pro Gin Ser Val Asp Gin Leu Leu His Lys Asn Met Pro Ala Asn Tyr

375 380 385

GCA GAA GCT CCA TTA GTT AAA GGA CTT CCA GCA TCA CCA GGA GCT GCT 1256 Ala Glu Ala Pro Leu Val Lys Gly Leu Pro Ala Ser Pro Gly Ala Ala

390 395 400

ACA GGA GCT GTT GTT TTT GAT GCC GAT GAT GCA GTT GAA CAA GCT AAA 1304 Thr Gly Ala Val Val Phe Asp Ala Asp Asp Ala Val Glu Gin Ala Lys

405 410 415

GGA AAG AAA GTT CTT CTT CTT AGA GAA GAA ACT AAA CCA GAA GAT ATT 1352 Gly Lys lys Val Leu Leu Leu Arg Glu Glu Thr Lys Pro Glu Asp lie 420 425 430 435

CAT GGA TTC TTT GTT GCT GAA GGT ATT TTA ACC TGC AGA GGA GGA AAA 1400 His Gly Phe Phe Val Ala Glu Gly He Leu Thr Cys Arg Gly Gly Lys

440 445 450

ACA TCA CAC GCA GCT GTC GTT GCT AGA GGT ATG GGT AAA CCA TGT GTT 1448 Thr Ser His Ala Ala Val Val Ala Arg Gly Met Gly Lys Pro Cys Val

455 460 465

TCA GGA GCT GAA GGA ATT AAA GTT GAT GTT GCT AAG AAA ATT GCT AAG 1496 Ser Gly Ala Glu Gly lie Lys Val Asp Val Ala Lys Lys lie Ala Lys

470 475 480

ATT GGA AGC CTT GAA GTT CAT GAA GGA GAT ATT TTA ACT ATT GAT GGA 1544 He Gly Ser Leu Glu Val His Glu Gly Asp lie Leu Thr lie Asp Gly

485 490 495

TCA ACT GGA TGT GTC TAT AAG GGA GAA GTT CCA TTA GAA GAA CCA CAA 1592 Ser Thr Gly Cys Val Tyr Lys Gly Glu Val Pro Leu Glu Glu Pro Gin 500 505 510 515

GTT GGA TCA GGA TAT TTC GGA ACC ATC TTA AAA TGG GCC AAT GAA ATT 1640 Val Gly Ser Gly Tyr Phe Gly Thr lie Leu Lys Trp Ala Asn Glu lie

520 525 530

AAA AAG ATT GGA GTT TTT GCT GCT GGA GAT CTT CCA TCA GCT GCT AAG 1688 Lys Lys lie Gly Val Phe Ala Ala Gly Asp Leu Pro Ser Ala Ala Lys

535 540 545

AAA GCC CTT GAA TTT GGA GCT GAA GGT ATT GGA CTT TGC AGA ACT GAA 1736 Lys Ala Leu Glu Phe Gly Ala Glu Gly lie Gly Leu Cys Arg Thr Glu

550 555 560

CGT ATG TTC AAT GCA GTT GAA AGA CTT CCA ATT GTT GTC AAG ATG ATT 1784 Arg Met Phe Asn Ala Val Glu Arg Leu Pro He Val Val Lys Met l ie

565 570 575

CTT TCA AAT ACC CTT GAA GAA AGA AAG AAA TAT CTT AAT GAA CTT ATG 1832 Leu Ser Asn Thr Leu Glu Glu Arg Lys Lys Tyr Leu Asn Glu Leu Met 580 585 585 590 595

CCA CTT CAA AAA CAA GAT TTC ATT GGA TTA TTG AAG ACT ATG AAT GGA 1880 Pro Leu Gin Lys Gin Asp Phe He Gly Leu Leu Lys Thr Met Asn Gly

600 605 610

CTT CCA GTC ACT GTC AGA CTT CTT GAT CCA CCA TTA CAT GAA TTC CTC 1928 Leu Pro Val Thr Val Arg Leu Leu Asp Pro Pro Leu His Glu Phe Leu

615 620 625

CCA ACT CTT GAA GAG TTA ATG AGA GAA ATC TTT GAA ATG AAA CTT TCA 1976 Pro Thr Leu Glu Glu Leu Met Arg Glu lie Phe Glu Met Lys Leu Ser

630 635 640 GGT AAG ACT GAA GGA CTT GCA GAA AAA GAA GTT GTT CTT AAG AAA GTT 2024

Gly Lys Thr Glu Gly Leu Ala Glu Lys Glu Val Val Leu Lys Lys Val

645 650 655

AAA GAA CTT ATG GAA GTT AAT CCA ATG ATT GGA CAC AGA GGA ATT AGA 2072

Lys Glu Leu Met Glu Val Asn Pro Met lie Gly His Arg Gly lie Arg 660 665 670 675

CTT GGA ACT ACT AAT CCA GAA ATT TAT GAA ATG CAA ATT AGA GCA TTC 2120

Leu Gly Thr Thr Asn Pro Glu lie Tyr Glu Met Gin He Arg Ala Phe

680 685 690

TTA GAA GCT ACT CGT GAA GTT ATT AAG GAA GGA ATT AAC GAT CAT CGA 2168

Leu Glu Ala Thr Arg Glu Val lie Lys Glu Gly lie Asn Asp His Arg

695 700 705

GAA ATT ATG ATT CCA AAT GTT ACA GAA GTT AAT GAA CTT ATT AAC TTA 2216

Glu lie Met lie Pro Asn Val Thr Glu Val Asn Glu Leu lie Asn Leu

710 715 720

AGA AAG AAT GTT CTT GAA CCA GTT CAT GAA GAA GTT GAA AAG AAA TAT 2264

Arg Lys Asn Val Leu Glu Pro Val His Glu Glu Val Glu Lys Lys Tyr

725 730 735

GGT ATT AAA GTA CCA TTC TCG TAT GGT ACT ATG GTT GAA TGT GTT AGA 2312

Gly lye Lys Val Pro Phe Ser Tyr Gly Thr Met Val Glu Cys Val Arg 740 745 750 755

GCA GCA TTA ACA GCT GAT AAG ATT GCT ACA GAA GCT TCA TTC TTC TCA 2360

Ala Ala Leu Thr Ala Asp Lys lie Ala Thr Glu Ala Ser Phe Phe Ser

760 765 770

TTC GGA ACT AAT GAT CTT ACA CAA GGA ACA TTC TCA TAC TCA CGT GAA 2408

Phe Gly Thr Asn Asp Leu Thr Gin Gly Thr Phe Ser Tyr Ser Arg Glu

775 780 785

GAT TCA GAA AAC AAA TTC ATT CCA AAA TAT GTT GAA CTT AAG ATT CTT 2456

Asp Ser Glu Asn Lys Phe lie Pro Lys Tyr Val Glu Leu Lys lie Leu 790 795 800

CCA GCT AAT CCA TTT GAA ATT CTT GAT AGA CCA GGT GTT GGA GAA GTT 2504 Pro Ala Asn Pro Phe Glu He Leu Asp Arg Pro Gly Val Gly Glu Val

805 810 815

ATG AGA ATT GCT GTT ACT AAA GGA AGA CAA ACA AGA CCA GAA TTA CTT 2552 Met Arg lie Ala Val Thr Lys Gly Arg Gin Thr Arg Pro Glu Leu Leu 820 825 830 830 835

GTT GGT ATT TGT GGA GAA CAC GGA GGA GAA CCA TCA TCA ATT GAA TGG 2600 Val Gly lie Cys Gly Glu His Gly Gly Glu Pro Ser Ser lie Glu Trp

840 845 850

TGC CAC ATG ATT GGA TTG AAC TAT GTT TCA TGT TCT TCA TAC AGA ATT 2648 Cys His Met lie Gly Leu Asn Tyr Val Ser Cys Ser Ser Tyr Arg l ie

855 860 865

CCA GTT GCT AGA ATT GCT GCT GCT CAA GCC CAA ATT AGA CAT CCA AGA 2696 Pro Val Ala Arg lie Ala Ala Ala Gin Ala Gin lie Arg His Pro Arg

870 875 880

GAA AAT TAAATTAACT TTTTTGGTTT 2722 Glu Asn

885 SEQ ID NO: 5

Array length: 3 1 8 0

Sequence type: nucleic acid

Array

CTGAAATTCC CGTAATCTAT CATCATTTAC ACCACAAATC GATTCACATC CTCACCGAAT 60 AGAAATCAAA TATCATTTAC TCCATCTCAC GATCTCCTTT TGCTATTGCT GATACCTCAA 120 TTTCGCAGGT GAAGGCGGAC G ATG AGT TCG TTG TTT GTT GAA GGT ATG CCT 171

Met Ser Ser Leu Phe Val Glu Gly Met Pro 1 5 10 CTG AAG TCA GCC AAT GAG TCG TGC TTA CCG GCG AGC GTG AAG CAA CGG 219 Leu Lys Ser Ala Asn Glu Ser Cys Leu Pro Ala Ser Val Lys Gin Arg

15 20 25

CGA ACC GGT GAT CTC AGG CGA TTG AAC CAC CAC CGT CAA CCG GCG TTT 267 Arg Thr Gly Asp Leu Arg Arg Leu Asn His His Arg Gin Pro Ala Phe

30 35 40

GTC CGG GGG ATT TGC CGT CGG AAG TTG AGT GGA GTT AGC AGA ATA GAG 315 Val Arg Gly lie Cys Arg Arg Lys Leu Ser Gly Val Ser Arg lie Glu

45 50 55

TTG CGC ACC GGT GGT TTA ACT CTG CCA CGA GCG GTG CTT AAT CCG GTG 363 eu Arg Thr Gly Gly Leu Thr Leu Pro Arg Ala Val Leu Asn Pro Val

60 65 70

TCT CCT CCG GTA ACG ACG ACT AAA AAG AGG GTT TTC ACT TTT GGT AAA 411 Ser Pro Pro Val Thr Thr Thr Lys Lys Arg Val Phe Thr Phe Gly Lys 75 80 85 90

GGA AAC AGT GAA GGC AAC AAG GAC ATG AAA TCC TTG TTG GGA GGA AAA 459 Gly Asn Ser Glu Gly Asn Lys Asp Met Lys Ser Leu Leu Gly Gly Lys

95 100 105

GGT GCA AAT CTT GCA GAG ATG GCA AGC ATT GGC CTA TCA GTT CCT CCT 507 Gly Ala Asn Leu Ala Glu Met Ala Ser He Gly Leu Ser Val Pro Pro

110 115 120

GGG CTC ACT ATT TCA ACT GAA GCA TGT GAG GAA TAT CAA CAA AAT GGA 555 Gly Leu Thr lie Ser Thr Glu Ala Cys Glu Glu Tyr Gin Gin Asn Gly

125 130 135

AAA AAA CTG CCT CCA GGT TTA TGG GAT GAG ATT CTG GAA GGC TTA CAG 603 Lys Lys Leu Pro Pro Gly Leu Trp Asp Glu lie Leu Glu Gly Leu Gin

140 145 150

TAT GTC CAG AAA GAG ATG TCT GCA TCT CTC GGT GAC CCG TCT AAA GCT 651 Tyr Val Gin Lys Glu Met Ser Ala Ser leu Gly Asp Pro Ser Lys Ala 155 160 165 170

CTC CTC CTT TCC GTC CGT TCG GGT GCT GCC ATA TCG ATG CCT GGT ATG 699

Leu Leu Leu Ser Val Arg Ser Gly Ala Ala lie Ser Met Pro Gly Met

175 180 185

ATG GAC ACT GTA TTG AAT CTC GGG CTT AAT GAT GAG GTC GTA GAT GGT 747 Met Asp Thr Val Leu Asn Leu Gly Leu Asn Asp Glu Val Val Asp Gly

190 195 200

CTA GCT GCC AAA AGT GGA GCT CGC TTT GCC TAT GAC TCG TAT AGG AGG 795 Leu Ala Ala Lys Ser Cly Ala Arg Phe Ala Tyr Asp Ser Tyr Arg Arg

205 210 215

TTT CTA GAT ATG TTT GGC AAC GTT GTA ATG GGT ATC CCA CAT TCG TTA 843 Phe Leu Asp Met Phe Gly Asn Val Val Met Gly He Pro His Ser Leu

220 225 230

TTT GAT GAA AAG TTA GAG CAG ATG AAA GCT GAA AAA GGG ATT CAT CTC 891 Phe Asp Glu Lys Leu Glu Gin Met Lys Ala Glu Lys Gly lie His Leu

235 240 245 250

GAC ACT GAT CTC ACT GCT GCT GAT CTT AAA GAT CTT GCT GAG CAA TAC 939 Asp Thr Asp Leu Thr Ala Ala Asp Leu Lys Asp Leu Ala Glu Gin Tyr

255 260 265

AAG AAC GTG TAT GTG GAA GCA AAG GGC GAA AAG TTT CCC ACA GAT CCA 987 Lys Asn Val Tyr Val Glu Ala Lys Gly Glu Lys Phe Pro Thr Asp Pro

270 275 280

AAG AAA CAG CTA GAG TTA GCA GTG AAT GCG GTT TTT GAT TCT TGG GAC 1035 Lys Lys Gin Leu Glu Leu Ala Val Asn Ala Val Phe Asp Ser Trp Asp

285 290 295

AGC CCA AGG GCC AAT AAG TAC AGG AGT ATT AAC CAG ATA ACT GGG TTA 1083 Ser Pro Arg Ala Asn Lys Tyr Arg Ser lie Asn Gin lie Thr Gly Leu

300 305 310

AAG GGG ACC GCG GTT AAC ATT CAA TGC ATG GTG TTT GGC AAC ATG GGG 1 131 0 9 ^ 0 ΐΒΛ ΐ¾ STH J3S ^s Ί ^J eiv J3S ojj usy aqj UTQ OJJ STH ngq

995Ϊ V19 013 1V3 39V VVV 3V 丄 130 131 933 IVV OVO III 0V3 V33 1V3 丄丄 :)

S OQi '5 ^

ri9i uio dsy na STH UTO jqx ΤΘ 3JV sA 9χ [Βχν 丄 §JV 丄 ςΐ9ΐ no ova m via iva m IDV ovo no 33v ovv 丄丄 v voo vov vov 丄 :) v

^ o

dsy 3TI "31 io ητο usv ΐ ^ Λ 13W dsy ΐ ^ Λ ^ TV si I v A BTV ^ I tm m IIV vio ooo m ovv oio oiv vxo νοΰ 丄丄 v vov i ooo ΐΰο

S 0 9ΐ ^

s q χ9 J¾ 3jy ^ ΐθ-^ I S- ^ V SAQ ng xajy djj, nsq Sjy usv

61 ^ 1 VVV 000 13V 103 VVV 000 VOV V9D 301 VV3 013 3IV 001 113 VOV 3VV

0 GO ^ 00 ^ 968

"T9 uio ΙΒΛ 丄 sqj ΘΠ dsy 13« 13 «dsy sAq λχ SIH 3iV ηχο

U81 WO VV3 110 V3V 311 WO UV IV9 91V OXV IVO VVV 3V1 3V3 VOV 9V3

068 589 08C nai 3TI ΐ ^ Λ SAQ usy r j) naq ητο 3JV JAX BTV "TO 0Jd 18« sAo Z \ VII IIV 010 101 DVV 3V0 010 XI3 OVO VOV 3VI V30 VV9 IOD OIV 301

0A6 598

J "TO 13W -iqi ΐ ^ Λ nai dsy ηχο OJJ jqi 3jy 3U χο BTV ΐ ^ Λ T¾

¾Α2ΐ I3V DVO 9IV 33V 010 Oil IVO VV3 V33 V3V V9V 31V 090 133 110 丄丄;)

098 998 09S

dsy "TO ^ ΐθ" TO BTV usv T¾ naq a¾j ηχ ^ J 丄 nsq s q sAq n ^ g

IVO 9V3 V33 0V3 130 XVV 310 VII 111 9V0 000 IVI 013 OVV DVV OVO m 589

^ ΐθ Jqi J3S ojj usy V 丄 3qd ^ I ^ TQ Jqi ATO JSS -rm usy

6ΙΠ 100 13V 30V V33 DVV 30V IDV Oil 113 110 100 33V VOO V3I IDV DVV οεε ε 9ΐε

Λΐ3 jaw usv ΛΙΟ 9Md ΐΒ., \ 13N SAO "TO 3Π usy ΐΒΛ BTV 'ίΐθ sAq

OS

ΟίΌΪ 0 / S6df / IDd 69h 0/96 OAV P / 95/01

51

GCA ACC GGT TTG CCA GCA TCC CCT GGG GCA GCC GTG GGG CAG GTT GTG 161 1 Ala Thr Gly Leu Pro Ala Ser Pro Gly Ala Ala Val Gly Gin Val Val 475 480 485 490

TTC AGC GCA GAG GAT GCT GAA ACA TGG CAT GCA CAA GGA AAG AGT GCT 1659 Phe Ser Ala Glu Asp Ala Glu Thr Trp His Ala Gin Gly Lys Ser Ala

495 500 505

ATC TTG GTA AGG ACT GAA ACA AGC CCA GAA GAT GTT GGT GGT ATG CAT 1707 l ie Leu Val Arg Thr Glu Thr Ser Pro Glu Asp Val Gly Gly Met His

510 515 520

GCA GCA GCT GGA ATC TTA ACC GCT AGA GGA GGA ATG ACA TCA CAT GCA 1755 Ala Ala Ala Gly lie Leu Thr Ala Arg Gly Gly Met Thr Ser His Ala

525 530 535

GCA GTG GTG GCT CGC GGA TGG GGC AAA TGT TGT GTT TCT GGT TGT GCT 1803 Ala Val Val Ala Arg Gly Trp Gly Lys Cys Cys Val Ser Gly Cys Ala

540 545 550

GAT ATT CGT GTG AAC GAT GAT ATG AAG GTT TTT ACG ATA GGT GAC CGT 1851 Asp lie Arg Val Asn Asp Asp Met Lys Val Phe Thr lie Gly Asp Arg

555 560 565 570

GTG ATT AAA GAA GGT GAC TGG CTT TCA CTT AAT GGT TCA ACT GGC GAA 1899 Val lie Lys Glu Gly Asp Trp Leu Ser Leu Asn Gly Ser Thr Gly Glu

575 580 585

GTC ATA TTG GGT AAA CAG CTA CTG GCT CCA CCT GCA ATG AGC AAT GAT 1947 Val lie Leu Gly Lys Gin Leu Leu Ala Pro Pro Ala Met Ser Asn Asp

590 595 600

TTA GAA ACA TTC ATG TCA TGG GCT GAT CAA GCA AGG CGT CTC AAG GTT 1995 Leu Glu Thr Phe Met Ser Trp Ala Asp Gin Ala Arg Arg leu Lys Val

605 610 615

ATG GCA AAT GCA GAC ACA CCT AAT GAT GCA TTA ACA GCC AGA AAC AAT 2043 Met Ala Asn Ala Asp Thr Pro Asn Asp Ala Leu Thr Ala Arg Asn Asn ΖζΖ VOV VOO DID VII 033 110 91V OIV OVO VO VIV VI9 I3V 010 ΟΰΟ OVD

QLL ¾9A

usv usv 13W A BTV BTV "TO sqd 9T I BTV SJV ΐ ^ Λ WJ) 13 ^ ηχ ^

S IVV OVV OIV 131 010 VOO 130 VV3 III 31V 030 103 110 WD OIV WO

09A Q9I 051 jqi ns OJJ JA 丄 jas ST I I Q nsq 3jy SAQ Αχο 8jy sqd ^ ΐθ ίζη V3V VID DOO v 丄 nv ooo VII vra αοι ιοΰ ioo on IOO no

5 OH ςει

13W OJJ usv ΐ ^ Λ ^η ΐΰ J3S ^η9 Ί sA ηχ ^ 3 \ \ 3ay ^J3 S ⁹ ΐ Ι ^η ΐΟ dsy

6 εΖ OXV 133 OVV 10 m OOi VIJ. VVV VV9 3IV V3V V3X IV丄31V WO丄V3 QL 921 QZL 5 U sq jas 19W χ JiLL dsv ^ TV Interview ^{n \ usv A ^ ll S TH} ηχο nsq mZ VVV 33V OIV 3D0 VOV IV9 933 V3V 113 WO XVV 010 VIV 3VD VV3 VID

ou goi oo dsy ηχ9 OJJ nsq sqj η ^ st}} nsq OJJ OJJ dsy naq ng-j gjy sn 8822 XVO 100 WO 330 VID III OVO 1V3 1X3 V33 13D 3V3 VID 113 393 3XV

G69 069 989

I i} 丄 TE ojj nsi dsy 19W BTV 3 v v 311 ^ TO ^η ΐ3 ^ Md dsy J3S

VOV VIO 133 丄丄 3 VOO XVO 9IV V30 V93 311 11V 330 9V0 111 IV9 331

089 9i9 0iL9

SJV uff) j ojj naq nsq na dsy naq Β ν ^ TV sAq Sjy ^U 1 ⁿ T9 OJJ i813 VOV m 3VI V33 313 Vll 3X3 3V3 VXD 130 009 VVV V9V WD WO V33

599 099 959

• "U ΪΕΛ eiv WW 3Π 19N sAq 3JV ΐ ^ Λ ^ TV sAq an 3jy ηχο dsy J3S

13V 310 030 9IV 3IV OIV OVV VOV VI3 139 VVV 3IV 93V OVO OVO 丄 :) 丄 099 mm 9S9

BTV aqd 3Md 13W S TH nio J¾ 3 V V SAQ nsq λχ an Αχθ "TO ^E IV ^ ID

1602 133 311 III OXV XV3 WO I3V VOV 101 313 V09 3 丄 V 330 VV3 VOO 130

0S9 529 0Z9

S3

OtOlO / S6df / X3d 69 € »Ό / 96 OAV Gin Gly Val Thr Val lie Pro Glu lie Met Val Pro Leu Val Gly Thr

780 785 790

CCT CAG GAA TTA CGG CAT CAA ATC GGC GTA ATT CGT GGT GTA GCT GCA 2571 Pro Gin Glu Leu Arg His Gin lie Gly Val He Arg Gly Val Ala Ala 795 800 805 810

AAT GTT TTT GCT GAA ATG GGG CTG ACG TTG GAG TAT AAA GTG GGA ACG 2619 Asn Val Phe Ala Glu Met Gly Leu Thr Leu Glu Tyr Lys Val Gly Thr

815 820 825

ATG ATT GAG ATT CCT CGA GCT GCT TTG ATT GCT GAT GAG ATT GCA AAA 2667 Met lie Glu He Pro Arg Ala Ala Leu lie Ala Asp Glu lie Ala Lys

830 835 840

GAA GCC GAG TTC TTT TCG TTT GGA ACC AAT GAT TTG ACC CAG ATG ACA 2715 Glu Ala Glu Phe Phe Ser Phe Gly Thr Asn Asp Leu Thr Gin Met Thr

845 850 855

TTT GGG TAC AGC AGA GAT GAT GTT GGC AAG TTT TTG CCG ATT TAT CTT 2763 Phe Gly Tyr Ser Arg Asp Asp Val Gly Lys Phe Leu Pro lie Tyr Leu

860 865 870

TCT CAA GGC ATT CTG CAG CAT GAT CCA TTT GAG GTT CTT GAC CAG AAA 2811 Ser Gin Gly lie Leu Gin His Asp Pro Phe Glu Val Leu Asp Gin Lys

875 880 885 890

GGG GTG GCT CAA TTG ATC AAG ATG GCC ACG GAG AAA GGT CGT GCA GCC 2859 Gly Val Gly Gin Leu lie Lys Met Ala Thr Glu Lys Gly Arg Ala Ala

895 900 905

AAT CCT AAC TTA AAG GTT GGG ATA TGT GGG GAG CAT GGT GGA GAA CCT 2907 Asn Pro Asn Leu Lys Val Gly lie Cys Gly Glu His Gly Gly Glu Pro

910 915 920

TCT TCT GTT GCA TTT TTT GAC GGA GTT GGA CTA GAT TAT GTG TCG TGC 2955 Ser Ser Val Ala Phe Phe Asp Gly Val Gly Leu Asp Tyr Val Ser Cys

925 930 935 TCT CCA TTC AGG GTT CCT ATC GCA AGG TTG GCC GCT GCA CAA GTC GTT 3003 Ser Pro Phe Arg Val Pro lie Ala Arg Leu Ala Ala Ala Gin Val Val

940 945 950

GTT TAAGCTTTGA AAGGAGGATG GCTTATTTGC TTCATGTTTT CCGCCATTGT 3056 Val

955

ATATTATTTT GGTTTCATCC TTATTGTAAT GGTGAAAATG AACGATGTTT AAACAAAACA 3116 ACCCATTATA TTTTGGTTTG GTATGCAATA ATCTACTTTT CAAACAAAAA AAAAAAAAAA3176 AAAA 3180 SEQ ID NO: 6

Array length: 1 8

Sequence type: nucleic acid

Array

GACGGCTAAA AAGAGGGT 18 SEQ ID NO: 7

Array length: 20

Sequence type: nucleic acid

Array

TATCGAGAAA CCTTCTATAC 20 SEQ ID NO: 8

Array length: 1 7

Sequence type: nucleic acid

Array

GTTTTCCCAG TCACGAC 17 SEQ ID NO: 9 Array length: 1 7

Sequence type: nucleic acid

Array

CAGGAAACAG CTATGAC 17 SEQ ID NO: 10

Array length: 2 3

Sequence type: nucleic acid

Array

GATATCAATC CGGTGTCTCC TCC 23 SEQ ID NO: 1 1

Array length: 3 4

Sequence type: nucleic acid

Array

CGGTGTCTCC TCCGGATATC ACGGCTAAAA AGAG 34 SEQ ID NO: 1 2

Array length: 2 7

Sequence type: nucleic acid

Array

TTGATATCCC GGTTGTCTCC TCCGGTA 27 SEQ ID NO: 1 3

Array length: 20

Sequence type: nucleic acid

Array

GCAGAGATGA TGTTGGCAAG 20 SEQ ID NO: 1 4

Array length: 20

Sequence type: nucleic acid

Array

CTTGCCAACA TCATCTCTGC 20 SEQ ID NO: 15

Array length: 20

Sequence type: nucleic acid

Array

CTCACTGTTC GAAGAGAAGC 20 SEQ ID NO: 16

Array length: 2 3

Sequence type: nucleic acid

Array

CATATGCTCT GTCCGGCATA ATC 23 SEQ ID NO: 17

Array length: 2 1

Sequence type: nucleic acid

Array

CTCGAGGGAT CTCAATCATT G 21 SEQ ID NO: 18

Array length: 1 8

Sequence type: nucleic acid

Array

GCAATCTCTT CAGCAATC 18 SEQ ID NO: 1 9

Array length: 20

Sequence type: nucleic acid

Array

GCTTCTTTTC CAATCTCATC 20 SEQ ID NO: 20

Array length: 1 8

Sequence type: nucleic acid

Array

CGAAAAGAAA TCGGCTTC 18 SEQ ID NO: 21

Array length: 2 4

Sequence type: nucleic acid

Array

GAAAGATAAA TCTGCAAAAA CTTG 24 SEQ ID NO: 2 2

Array length: 1 9

Sequence type: nucleic acid

Array

GCCTTGAGCA AGATAAATC 19 SEQ ID NO: 23

Array length: 2 1

Sequence type: nucleic acid

Array TTCTGGTCAA TAACCTCAAT G 21 SEQ ID NO: 24

Array length: 20

Sequence type: nucleic acid

Array

GCTTAAACAA TGACTTGTGC 20 SEQ ID NO: 25

Array length: 2 2

Sequence type: nucleic acid

Array

CCAATCTCAT CAGCTATTAA AG 22 SEQ ID NO: 26

Array length: 20

Sequence type: nucleic acid

Array

GCTTCTTTTG CAATCTCTTC 20 SEQ ID NO: 2 7

Array length: 1 8

Sequence type: nucleic acid

Array

CGAAAAGAAC TCAGCTTC 18 SEQ ID NO: 28

Array length: 2 3

Sequence type: nucleic acid Array

CAAGATAAAT CGGCAAAAAC TTG 23 SEQ ID NO: 2 9

Array length: 2 3

Sequence type: nucleic acid

Array

GAATGCCTTG AGAAAGATAA ATC 23 SEQ ID NO: 30

Array length: 2 4

Sequence type: nucleic acid

Array

CTTTCTGGTC AAGAACCTCA AATG 24 SEQ ID NO: 31

Array length: 20

Sequence type: nucleic acid

Array

GCTTAAACAA CGACTTGTGC 20

Claims

The scope of the claims

1. In any of (1) and (2) below, at least one amino acid residue in the range of 1/6 of the full length from the C-terminal of the amino acid sequence has been replaced with another amino acid residue. A polypeptide having an amino acid sequence and low-temperature-resistant pyruvate phosphate dikinase activity.

(2) A polypeptide having an amino acid sequence 50% or more homologous to the amino acid sequence shown in (1), and having pyruvate phosphate dikinase activity.

2. An amino acid sequence represented by SEQ ID NO: 5 in the sequence listing, or a polypeptide having one or more amino acids added, deleted or substituted in the amino acid sequence, and having a low-temperature-resistant pyruvate phosphoric acid dikinase activity.

3. The polypeptide of claim 2, which has the amino acid sequence shown in SEQ ID NO: 5 in the sequence listing

4. One or more amino acids are added, deleted or substituted in the 832 to 955th portion or the partial amino acid sequence of the amino acid sequence shown in SEQ ID NO: 5 in the sequence listing, and the pyruvate phosphate is used. A polypeptide comprising a sequence capable of imparting low-temperature resistance to a polypeptide having dikinase activity, and having low-temperature-resistant pyruvate phosphate dikinase activity.

5. The polypeptide according to claim 4, which comprises a portion of the amino acid sequence represented by SEQ ID NO: 5 in the sequence listing at position 832 to 955, and has a low-temperature-resistant pyruvate phosphate dikinase activity.

6. One or more amino acids are added, deleted or substituted in the 832 to 955th part or the partial amino acid sequence of the amino acid sequence shown in SEQ ID NO: 5 in the sequence listing, and pyruvate is used. 2. The polypeptide according to claim 1, wherein the homologous portion of the amino acid sequence of pyruvate phosphate dikinase is substituted with a sequence capable of imparting low temperature resistance to phosphate dikinase.

7. The amino acid sequence shown in SEQ ID NO: 5 in the amino acid sequence of SEQ ID NO: 5 at position 8332 to 9555, wherein the homologous portion of the amino acid sequence of pyruvate phosphate dikinase has been substituted. Polypeptide.

8. The polypeptide according to claim 1, wherein the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing is substituted with proline at position 869.

9. The polypeptide according to claim 1, wherein positions 885 and 952 of the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing are substituted with leucine and valine, respectively.

10. A cloned DNA encoding the polypeptide of any one of claims 1 to 9.

1 1. The nucleotide sequence represented by SEQ ID NO: 5 in the sequence listing, or a polypeptide having one or more nucleotides added, deleted or substituted in the nucleotide sequence and having a low-temperature-resistant pyruvate kinase activity 10. The DNA of claim 10, wherein the code is a code.

1 2. A recombinant vector comprising the DNA of claim 10 and capable of expressing a polypeptide having a low-temperature-resistant pyruvate phosphate dikinase activity in a host.

13. A plant transformed with the DNA of claim 10.