CN101363025B

CN101363025B - Histone demethylase gene OsJMJ706 of rice, encoding protein thereof and applications

Info

Publication number: CN101363025B
Application number: CN2008100487676A
Authority: CN
Inventors: 周道绣; 孙前文
Original assignee: Huazhong Agricultural University
Current assignee: Huazhong Agricultural University
Priority date: 2008-08-12
Filing date: 2008-08-12
Publication date: 2010-11-03
Anticipated expiration: 2028-08-12
Also published as: CN101363025A

Abstract

The invention belongs to the technical field of plant gene engineering, and more particularly relates to a paddy rice histone demethyl methylase gene, coded protein and the application thereof. The invention provides a new paddy rice histone demethyl methylase gene OsJMJ706, due to inactivation, heteroplasia of rice flowerthe, heteroplasia or deficit of internal and external glumes, as well as lessening of seeds can be caused on the gene, and by being detected by the method of western blot, the gene is proved to have enzyme activity for removing histone H3K9me3 and H3K9me2 locus methylate. The invention provides nucleic acid sequence and protein sequence of the gene as well as the application thereof.

Description

A kind of paddy rice histone demethylase gene OsJMJ706 and proteins encoded and application

Technical field

The present invention relates to the plant gene engineering technology field, be specifically related to a kind of paddy rice histone demethylase gene and proteins encoded and application.The invention provides a kind of new paddy rice histone demethylase gene OsJMJ706; this gene inactivation can cause the paddy rice flower development unusual; unusual or the disappearance of inside and outside glume development; seed diminishes, and detects with western blot method and finds that it has the histone H 3 K9me3 of removal and the methylated enzymic activity in H3K9me2 site.The nucleotide sequence that the invention provides this gene with and protein sequence and its application.

Background technology

The histone modification involved enzyme plays significant role in the growth and development of plants and animals process, for example common hybrid vigour, kernel dominance, vernalization, Paramutation, transgene silencing, transposon activation, genomic imprinting, dna methylation, rna editing and x chromosome inactivation etc. all find to have this zymoid to participate in.This fermentoid comprises histon deacetylase (HDAC) (Histone deacetylases; HDAC); ZNFN3A1 (Histone methyltransferases; HMT) and recently histone demethylase (the Histone demethylase that just finds; HDM); it is comparatively clear that the function of these modifying enzymeses is studied in animal and yeast isotype biology, but in plant, particularly study in the unifacial leaf model plant paddy rice at present less.Some achievements that obtained confirm that these histone modification enzymes play an important role to growth and development of plants.

Histone demethylase (Histone demethylase, HDM) be the class I histone modification gene (Klose that finds recent years, R.J etc., JmjC-domain-containing proteins and histone demethylation.Nat.Rev.Genet. (2006), 7,715-727.), the methyl of the histone afterbody that can remove is methylated modifies is with the functions reversed of ZNFN3A1 (HMT).New discovery JmjC family member (containing common Jumonji C (JmjC) structural domain family gene) has the function of histone demethylase in the mankind in the recent period, and locus specificity (Klose is arranged, R.J etc., Regulation of histone methylation by demethylimination anddemethylation.Nature reviews, (2007), 8,307-318).

Find 20 members that have an appointment of paddy rice (Oryza sativa) JmjC family by information biology, Arabidopis thaliana has 21 JmjC family members.The wherein previous flowering time (Noh that studies confirm that AtRef6 Gene Handling Arabidopis thaliana, B etc., Divergent roles of a pair ofhomologous jumonji/zinc-finger-class transcription factor proteins in the regulation ofArabidopsis flowering time.Plant Cell, (2004), 16,2601-2613.), but do not confirm that this gene has the active of histone demethylase or active relevant with this kind of enzyme.Saze etc. are by the screening mutant, the gene IBM1 sudden change inactivation that discovery contains the jmjC structural domain can cause the BONSAI gene DNA increase (Saze that methylates, H etc., Control of genic DNA methylation by a jmjCdomain-containing protein in Arabidopsis thaliana.Science, (2008), New York, N.Y 319,462-465.), prove the dna methylation that contains jmjC domain gene IBM1 may command gene.Yet up to the present, do not see have bibliographical information to contain the function of jmjC domain gene in paddy rice.

By search and checking (detailed process is referring to embodiment) to the applicant's mutant database; OsJMJ6 has two equipotential mutant; difference called after jmj6-1 and jmj6-2; they insert the different positions of same intron; the field phenotype is observed and is found that two equipotential mutant flower developments are unusual; in; coetonium shell heteroplasia or disappearance; seed diminishes; through PCR detect for be divided into from; RT-PCR detects and finds that the OsJMJ6 gene does not have expression in the mutant; the histone modification level is compared with wild-type very big variation has been taken place in the western blot analysis discovery mutant; histone H 3 K9me2 and the H3K9me3 decorating site accumulation that methylates in the mutant, and the H3K9me1 level is on the contrary.Existing result shows that OsJMJ6 is a histone H 3 K9 position demethylase, and relevant with the paddy rice flower development.

Substantially, illustrate at present the molecule mechanism of dicotyledons development of floral organs genetic regulation, but monocotyledons development of floral organs regulation and control are still known little about it.As the unifacial leaf model plant, the flower of paddy rice is also representative in the monocotyledons floral organ.To the further investigation of OsJMJ706 mutant, will set forth the molecule mechanism of paddy rice flower development regulation and control from new angle.

By functional study to paddy rice histone demethylase gene OsJMJ706, for verifying in relation that itself and plant flowers grow and the gene transcription regulation mechanism process important meaning is arranged, also be expected in addition provide fundamental basis for crop genetic and breeding.

Summary of the invention

The objective of the invention is to clone a new paddy rice histone demethylase gene OsJMJ706 and a proteins encoded thereof.

The present invention is achieved in that

Applicant clone obtains paddy rice histone demethylase gene OsJMJ706, the having as one of described nucleotide sequence of sequence table of this gene:

Dna sequence dna shown in the SEQ ID NO:1 in the sequence table also comprises the gene order that has 50% homology with the dna sequence dna shown in the SEQ ID NO:1 at least; Be also included within the mutant allele or the derivative that add, replace, insert or delete one or more Nucleotide and produce, also contain the gene order that has identical function and can reach the object of the invention.

Protein shown in the SEQ ID NO:2 among the present invention belongs to what paddy rice histone demethylase JMJ706, wherein carries out one or several amino acid whose replacement, insertion or the disappearance functional analogue that amino acid obtained.Therefore, the present invention also comprises the functional analogue that has the homology that (comprises 60%) more than 60% with the aminoacid sequence shown in the SEQ ID NO:3.

More detailed technical scheme is as described in " embodiment ".

Description of drawings

Sequence table SEQ ID NO:1. is the genomic dna sequence of the present invention's OsJMJ706 gene of cloning, and the sequence total length is 2577bp altogether.

Sequence table SEQ ID NO:2. is the encoded protein matter sequence of OsJMJ706 gene.

Fig. 1. be the photo of the present invention's OsJMJ706 gene mutation body of cloning.Among the figure:

A. being wild-type ZH11 above, is two equipotential mutant jmj6-1 and jmj6-2 below;

B. compare mutant (two on the right) disappearance flower glume with wild-type (left side);

C. compare mutant (two on the right) disappearance glumelle with wild-type (left side);

D. compare sophisticated mutant seed (two on the right) disappearance glumelle, seed evagination and littler than wild-type with wild-type (left side);

Fig. 2. be the structure of the present invention's OsJMJ706 gene of cloning and show that T-DNA inserts the site.

Initiation codon among the figure (ATG) and termination codon (TGA) mark, and black box is represented the exon district of OsJMJ706 gene, and the lines between the square frame are represented intron, and T-DNA is inserted in the 1st intron.F1, R1, F2 and R2 represent to be used for to verify isolating primer altogether respectively.F3 and R3 are the primer of checking rna transcription level.

Fig. 3. be T1 for being divided into of mutant plant from detected result, primer as shown in Figure 2, NTLB5 is a T-DNA right-hand member primer.Among the figure: plant mutant body jmj6-1 positive test symbol A.18, ZH11 is contrast.Wherein 1,3,11,12,14,16 are the insertion mutant that isozygotys, and show the phenotype described in Fig. 1;

B.20 plant mutant body jmj6-2 positive test symbol, ZH11 is contrast.Wherein 8,12,15,17,18,20 are the insertion mutant that isozygotys, and show the phenotype described in Fig. 1;

Fig. 4. be RT-PCR detect wild-type ZH11 and isozygoty (/-) and heterozygosis (+/-) mutant in the mRNA of OsJMJ706 transcribe situation, primer is F3 and R3 that Fig. 2 indicated, actin in contrast.

Fig. 5. detect the activity of OsJMJ706 histone demethylase in the body.Extract the histone of wild-type ZH11 and mutant jmj6-2 plant, carry out western blot with 11 kinds of antibody shown in the left side and detect, the bottom is that the coomassie brilliant blue staining histone contrasts as applied sample amount.

Embodiment

The present invention is that the technology of utilizing T-DNA to insert mutant is carried out the functional analysis of gene.Insert the insertion mutant of seeking OsJMJ706 in the mutant database (http://rmd.ncpgr.cn/) at paddy rice T-DNA, find two equipotential T-DNA to insert mutant, they all are inserted in first intron of gene.These two equipotential mutant strain systems are carried out the screening of normal field condition; obtained stable paddy rice flower development equipotential mutant jmj6-1 and the jmj6-2 (seeing shown in Figure 2) of phenotype; this mutant shows as the paddy rice flower development when planting under normal field condition unusual; unusual or the disappearance of inside and outside glume development, the proterties that seed diminishes.Utilize and to have separated the flanking sequence that obtains and in genome, design primer, verified that this T-DNA sequence and mutant phenotype are isolating altogether, thereby the inactivation that can determine this gene has caused the appearance of this mutant phenotype.

Embodiment 1: the mutant plant is divided into from detecting and expression analysis

1, rice material

The original wild-type material of the paddy rice that present embodiment adopted (Oryza sativa ssp.) is a rice varieties " in the spend 11 " japonica rice variety (ZH11) that China openly applies, and also is that paddy rice T-DNA inserts the acceptor material that mutant database (http://rmd.ncpgr.cn/) uses.For the T-DNA that searches OsJMJ706 inserts mutant, does the applicant insert mutant database (http://rmd.ncpgr.cn/) web analytics instrument (http://rmd.ncpgr.cn/blast.cgi with OsJMJ706 gene order (seeing that cloned genes sequence of the present invention is shown in SEQ IDNO:1) at paddy rice T-DNA? nickname=) carry out BLAST and analyze (Zhang J etc., a rice mutant databasefor functional analysis of the rice genome.Nucl.Acids Res., 2006,34:D745-748), find two equipotential mutant, original number is 03Z11FO83 and 05Z11DO89, for simplicity, the applicant renames and is jmj6-1 (03Z11FO83) and jmj6-2 (05Z11DO89).

2, T-DNA inserts being divided into from detection of flanking sequence and mutant phenotype

For the heredity of studying T-DNA separates, plantation has T1 that T-DNA inserts two equipotential mutant family paddy rice jmj6-1 18 strains of mark and jmj6-2 20 strains for the plant (see figure 3) and analyze the genotype that its T-DNA inserts respectively.The genomic dna that will extract from the paddy rice young tender leaf of 38 strain T1 for (see figure 3) altogether is as gene type assay, to determine isozygotying or heterozygosity of they.The oryza sativa genomic dna method for extracting is conventional method for extracting, with reference to (Huang Peitang etc. translate, Science Press, 2002 for " molecular cloning experiment guide ", J. Sa nurse Brooker and D.W. Russell work).The PCR reaction conditions: 94 ℃ 1 minute, 57 ℃ 1 minute, 72 ℃ 1.5 minutes, carry out 35 circulations altogether.Primer and NTLB5 (indicating as Fig. 2) that following numbering is respectively F1, R1, F2, R2 are used for the PCR reaction.

Primers F 1:

5’--TTACCTTAGACCGGGCCAC--3’

Primer R1:

5’--GGCGGATTCAGATTGAGGAT--3’

Primers F 2:

5’--TGACTTGGTTGGGACTTGCC--3’

Primer R2:

5’--TGACTTGGTTGGGACTTGCC--3’

Primer NTLB5 (T-DNA edge Auele Specific Primer):

5’--AATCCAGATCCCCCGAATTA--3’

Primers F 1, F2 are the forward primers that derives from the OsJMJ706 gene intron shown in the synoptic diagram of Fig. 2, and primer R1, R2 are the reverse primers that derives from gene intron.Primer NTLB5 derives from T-DNA edges of regions Auele Specific Primer.Primer NTLB5 obtains the PCR fragment of about 0.5Kb respectively with R1, R2 combination amplification, having only the pulsating T1 of this amplification is homozygote for plant, and this is because can not amplification of DNA fragments (the about 14kb of T-DNA insertion fragment) because fragment is too big by primers F 1 and R1 or F2 and R2 combination amplification.But wild-type plant can utilize primers F 1 and R1 or F2 and R2 to make up the PCR fragment that obtains about single 0.4kb, and this is owing to there is not T-DNA to insert in genome, and the T1 plant of heterozygosis can obtain the pcr amplification thing of 0.5kb and 0.4kb respectively.

Experimental result as shown in Figure 4.For jmj6-1,1,3,11,12,14,16 are the insertion mutant that isozygotys among the figure, and 2,4,5,6,7,8,10,11,13,17,18 is heterozygote.9,15 is wild-type.For jmj6-2,8,12,15,17,18,20 are the insertion mutant that isozygotys, 2,4,5,6,7,9,11,14,16, be heterozygote.1,3,10,13,19 is wild-type.These results show that T-DNA is separated to the next generation altogether.The filial generation plantation in T1 generation is obtained T2 generation, analyzes the back and find that the homozygous mutation body no longer separates, the phenotype unanimity, heterozygote 3: 1 phenotype occurs and separates, PCR detect the insertion that shows T-DNA and mutant phenotype be divided into from.Prove that thus these two equipotential mutant are because T-DNA inserts the unit point recessive mutation that causes.

3, to the phenotype analytical of two equipotential mutant of OsJMJ706

Two equipotential mutant are compared with wild-type, find that the mutant flower development is unusual, and inside and outside glume development is unusual, Ying in some disappearance, and seed diminishes, but do not influence seed germination, should be that endosperm development is affected.

4, expression analysis detects mutant mRNA and transcribes situation

In order to detect the expression amount of target gene in equipotential mutant Jmj6-1 and the jmj6-2 plant, applicant's usefulness RT-PCR method (referring to: " molecular cloning experiment guide ", J. Sa nurse Brooker and D.W. Russell are outstanding, Huang Peitang etc. translate, Science Press, 2002) wild-type ZH11 and mutant jmj6-1 and jmj6-2 plant have been carried out expression analysis.Test used total RNA from the blades of above-mentioned three rice material sword-like leave phases, RNA extracting reagent is the Trizol extraction agent box (the concrete operations step is seen the test kit specification sheets) available from Invitrogen company; The step of synthetic cDNA first chain of reverse transcription is as follows among the RT-PCR: 1. join mixed solution 1: total RNA 2 μ g, DNAse I 2u, 10x DNAse I buffer 1 μ l, add DEPC (diethylpyrocarbonate, the strongly inhibited agent of RNA enzyme) treating water (0.01%DEPC) is to 10 μ l, behind the mixing mixed solution 1 is placed 20 minutes to remove DNA at 37 ℃, 2. place 70 ℃ of water-bath temperature to bathe 10 minutes mixed solution 1 after 20 minutes to remove DNAse I activity, placed then 5 minutes on ice, 3. the oligo (dT) that in mixed solution 1, adds 1 μ l, 500 μ g/ml, 4. refrigerative mixed solution 1 is placed immediately 70 ℃ of water-bath temperature to bathe 10 minutes, thoroughly to make the RNA sex change, placed then 5 minutes on ice, 5. join mixed solution 2: get 1. liquid mixture prepared 110 μ l, 5x first strand buffer 4 μ l, 0.1M DTT (mercaptoethanol) 2 μ l, 10mM dNTP mixture 1.5 μ l, DEPC treating water 0.5 μ l, ThermoScript II 2 μ l, behind the mixing mixed solution 2 placed in 42 ℃ of water-baths temperature to bathe 1.5 hours, 6. place 90 ℃ to do bath 3 minutes mixed solution 2 after reacting end, 7.-20 final product is reacted in a ℃ preservation, and the reagent of using in the reaction is all available from Invitrogen company; The system that RT-PCR uses is 20 μ l, specifically joining method is: the cDNA first chain template 1 μ l, 10xPCR buffer 2 μ l, 10mM dNTP 1.6 μ l, 2.5mMMg2+1.5 μ l, each 0.4 μ l of left and right primer, TAQ enzyme 0.2 μ l adds water to 20 μ l (used PCR buffer, dNTP, Mg2+, rTAQ enzyme etc. are all available from TAKARA company).The PCR reaction conditions is as follows: 1. 94 ℃ 2 minutes, 2. 94 ℃ 1 minute, 3. 58 ℃ 1 minute, 4. 72 ℃ 1 minute, 5. from 2.-4. circulating 30 times, 6. 72 ℃ 7 minutes, 7. 4 ℃ of preservations.The PCR product is electrophoresis detection on 1.2% sepharose.The primer (indicating as Fig. 2) of the OsJMJ706 gene that RT-PCR uses is:

Primers F 3:

5’--GTGGTTGAGCAGGGCAGTTC--3’

Primer R3:

5’--TTGATGGAACAAGCGTATGAAAG--3’

The expression analysis result as shown in Figure 4, the result is presented at the transcript that can detect OsJMJ706 in wild-type and heterozygous mutant body (+/-) plant, and in two equipotential mutant that isozygoty (/-), detect less than its transcript, show that the unusual mutant phenotype of flower development is caused by the insertion sudden change inactivation of OsJMJ706 gene.

Embodiment 2: histone modification situation analysis in the mutant body

Because the OsJMJ706 gene contains distinctive jmjC of Jumonji family and jmjN structural domain, and existing its human homology's gene of report JMJD2 family gene has the demethylation enzymic activity in histone H 3 K9 and H3K36 site, whether has the activity of demethylase for detecting the OsJMJ706 gene, the applicant utilizes western blot method (with reference to " molecular cloning experiment guide ", J. Sa nurse Brooker and D.W. Russell are outstanding, Huang Peitang etc. translate, Science Press, 2002) histone modification situation in wild-type ZH11 and the mutant jmj6-2 body is detected.Concrete steps are as follows:

1, histone method for extracting

Get the blade 4-5 sheet of the wild-type ZH11 about 40 days and mutant jmj6-2 after planting respectively, put into after the liquid nitrogen grinding 3 times of volumes the histone extraction buffer (10mM Tris-HCl, pH 7.5; 2mM EDTA; 0.25M HCl; 5mM 2-mercaptoethanol; 0.2mMPMSF) in abundant mixing, through 12000g, 4 ℃ after centrifugal 10 minutes, draw supernatant in another new centrifuge tube, by volume adding trichoroacetic acid(TCA) to final concentration is 25%, 17000g, 4 ℃ centrifugal 30 minutes, abandon supernatant, precipitation is with washing with acetone three times, be dissolved in after drying sample-loading buffer (62.5mMTris-HCl, pH 6.8; 2%SDS; 25%glycerol; 0.01%Bromophenol Blue; Among 10% β-mercaptoethanol), be that 15% SDS-PAGE electrophoresis can detect extraction rate was acquired through concentration, electrophoresis adopts the Mini-PROTEAN3 electrophoresis chamber system of Bio-Rad company, operates according to its specification sheets.

2, utilize the antibody of different histone modifications that wild ZH11 and jmj6-2 are carried out western blot analysis

Carry out the western detection with extracting good histone, western blot changes the Mini Trans-Blot Cell transfer groove system that film uses Bio-Rad company, according to its specification sheets, histone is gone on the Hybond-P pvdf membrane of Amersham company, with PBS damping fluid (the NaCl 137mmol/L that contains 2% (mass volume ratio) bovin serum albumin (BSA) for preparing, KCl2.7mmol/L, Na ₂HPO ₄4.3mmol/L, KH ₂PO ₄1.4mmol/L pH 7.5) membrane closure more than two hours, is added different antibody incubated at room about two hours with volume ratio then at 1: 10000.Used antibody is: anti-H3K9ace (07-352), H3K9me2 (07-441), H3K9me3 (07-442), H3K27me2 (07-322), H3K36me2 (07-274), H3K4me 1 (07-436) and H3K4me3 (07-473) antibody that Upstate company buys, and anti-H3K9me1 (ab9045), H3K36me1 (ab9048), H3K36me3 (ab9050) and H3 (ab1791) antibody of the purchase of Abcam company (in the bracket is article No.).Outwell one anti-after, wash film three times with the PBS damping fluid, each 15 minutes, the goat-anti rabbit two anti-(available from SouthernBiotech company) that adds the HRP mark that is dilution in 1: 10000 by volume again, incubated at room 1-2 hour, wash film three times with PBS then, each 15 minutes, use SuperSingnal Pico (available from Pierce company) test kit by specification working method to carry out the X-ray film developing, through scanner scanning X-ray sheet post analysis result.

The result as shown in Figure 5, from coomassie brilliant blue staining with carry out the result of western with H3 antibody, wild-type ZH11 and mutant jmj6-2 protein applied sample amount are basic identical, in this case, with respect to wild-type, obvious variation does not take place in the histone modification level in following site in mutant jmj6-2: H3K9ace, H3K27me2, H3K36me2, H3K4me1, H3K4me3, H3K36me1 and H3K36me3, and H3K9me2 and H3K9me3 modification level obviously raise, H3K9me1 modification level decreases, illustrate that OsJMJ706 has the activity of H3K9me2 and H3K9me3 site demethylase, because the inactivation of this gene in the mutant, cause the accumulation that methylates of mutant histone H 3 K9me2 and H3K9me3 site, thereby when detecting, show as, and the H3K9me1 level is on the contrary than wild-type height with western blot.The activity of histone H 3 K9 position demethylase in the body of the proof OsJMJ706 that this result is strong.

Sequence table

＜110〉Hua Zhong Agriculture University

＜120〉a kind of paddy rice histone demethylase gene OsJMJ706 and proteins encoded and application

<130>

<141>2008-08-08

<160>2

<170>PatentIn?version?3.1

<210>1

<211>2577

<212>DNA

＜213〉paddy rice (Oryza sativa)

<220>

<221>gene

<222>(1)..(2577)

<223>

<220>

<221>CDS

<222>(1)..(2577)

<223>

<400>1

atg?caa?cag?gtg?gag?ggc?agg?aac?tgt?ctt?cct?gcg?gag?gtc?agg?att 48

Met?Gln?Gln?Val?Glu?Gly?Arg?Asn?Cys?Leu?Pro?Ala?Glu?Val?Arg?Ile

1 5 10 15

ggc?ctc?gag?acg?ctc?aag?agg?cgc?cgg?ctt?gag?agg?atg?cgt?ttg?act 96

Gly?Leu?Glu?Thr?Leu?Lys?Arg?Arg?Arg?Leu?Glu?Arg?Met?Arg?Leu?Thr

20 25 30

gct?cag?aac?aat?gcc?ggc?gac?ggt?cct?ccg?gtg?ccc?gca?agg?agc?ggt 144

Ala?Gln?Asn?Asn?Ala?Gly?Asp?Gly?Pro?Pro?Val?Pro?Ala?Arg?Ser?Gly

35 40 45

ggg?gat?gcg?cta?agg?act?ccc?gca?aac?tgc?ggg?gtc?agg?ttg?cat?gct 192

Gly?Asp?Ala?Leu?Arg?Thr?Pro?Ala?Asn?Cys?Gly?Val?Arg?Leu?His?Ala

50 55 60

aac?aat?ggc?aca?gct?cta?cct?agc?aga?acc?acc?cag?aac?aag?gac?cct 240

Asn?Asn?Gly?Thr?Ala?Leu?Pro?Ser?Arg?Thr?Thr?Gln?Asn?Lys?Asp?Pro

65 70 75 80

ttt?gca?aag?cgc?agg?gtg?gac?aag?ttt?gat?atg?tct?agc?cta?gaa?tgg 288

Phe?Ala?Lys?Arg?Arg?Val?Asp?Lys?Phe?Asp?Met?Ser?Ser?Leu?Glu?Trp

85 90 95

att?gac?aag?atc?gaa?gaa?tgc?cct?gtg?tac?tat?cct?acc?aag?gag?gag 336

Ile?Asp?Lys?Ile?Glu?Glu?Cys?Pro?Val?Tyr?Tyr?Pro?Thr?Lys?Glu?Glu

100 105 110

ttc?gag?gat?ccc?att?ggt?tat?ata?cag?aag?att?gca?cct?gtg?gct?tcg 384

Phe?Glu?Asp?Pro?Ile?Gly?Tyr?Ile?Gln?Lys?Ile?Ala?Pro?Val?Ala?Ser

115 120 125

aaa?tac?gga?att?tgc?aaa?atc?gta?tct?cca?gta?agc?gct?tct?gtt?cct 432

Lys?Tyr?Gly?Ile?Cys?Lys?Ile?Val?Ser?Pro?Val?Ser?Ala?Ser?Val?Pro

130 135 140

gct?ggt?gtc?gtg?ttg?atg?aag?gaa?cag?cct?ggt?ttc?aag?ttc?atg?acc 480

Ala?Gly?Val?Val?Leu?Met?Lys?Glu?Gln?Pro?Gly?Phe?Lys?Phe?Met?Thr

145 150 155 160

agg?gtt?cag?ccg?ctt?cgc?ctc?gcc?aaa?tgg?gct?gaa?gat?gac?acg?gtc 528

Arg?Val?Gln?Pro?Leu?Arg?Leu?Ala?Lys?Trp?Ala?Glu?Asp?Asp?Thr?Val

165 170 175

act?ttc?ttc?atg?agc?gaa?aga?aag?tac?act?ttc?cgg?gat?tat?gag?aaa 576

Thr?Phe?Phe?Met?Ser?Glu?Arg?Lys?Tyr?Thr?Phe?Arg?Asp?Tyr?Glu?Lys

180 185 190

atg?gcc?aac?aag?gtg?ttc?gcc?aag?aaa?tac?tca?agt?gct?agt?tgt?ctc 624

Met?Ala?Asn?Lys?Val?Phe?Ala?Lys?Lys?Tyr?Ser?Ser?Ala?Ser?Cys?Leu

195 200 205

cca?gct?aag?tac?gtg?gag?gag?gaa?ttc?tgg?cgc?gaa?att?gct?ttt?ggt 672

Pro?Ala?Lys?Tyr?Val?Glu?Glu?Glu?Phe?Trp?Arg?Glu?Ile?Ala?Phe?Gly

210 215 220

aaa?atg?gat?ttt?gtt?gaa?tat?gcc?tgt?gat?gtt?gat?ggt?agt?gct?ttc 720

Lys?Met?Asp?Phe?Val?Glu?Tyr?Ala?Cys?Asp?Val?Asp?Gly?Ser?Ala?Phe

225 230 235 240

tcc?tct?tct?cct?cat?gat?caa?ctt?ggg?aaa?agc?aac?tgg?aac?ttg?aag 768

Ser?Ser?Ser?Pro?His?Asp?Gln?Leu?Gly?Lys?Ser?Asn?Trp?Asn?Leu?Lys

245 250 255

aat?ttt?tca?cgg?ctt?tcc?aat?tct?gtg?ctt?aga?ctt?ctg?cag?aca?cca 816

Asn?Phe?Ser?Arg?Leu?Ser?Asn?Ser?Val?Leu?Arg?Leu?Leu?Gln?Thr?Pro

260 265 270

att?cca?gga?gta?aca?gat?cca?atg?ctt?tat?atc?ggg?atg?ctc?ttc?agc 864

Ile?Pro?Gly?Val?Thr?Asp?Pro?Met?Leu?Tyr?Ile?Gly?Met?Leu?Phe?Ser

275 280 285

atg?ttt?gct?tgg?cat?gtg?gaa?gat?cat?tat?ttg?tac?agc?atc?aat?tac 912

Met?Phe?Ala?Trp?His?Val?Glu?Asp?His?Tyr?Leu?Tyr?Ser?Ile?Asn?Tyr

290 295 300

cat?cat?tgt?ggg?gca?ttt?aag?aca?tgg?tat?ggc?ata?ccg?ggt?gat?gct 960

His?His?Cys?Gly?Ala?Phe?Lys?Thr?Trp?Tyr?Gly?Ile?Pro?Gly?Asp?Ala

305 310 315 320

gct?cct?ggg?ttt?gaa?aag?gtg?gct?agc?cag?ttt?gta?tac?aac?aag?gat 1008

Ala?Pro?Gly?Phe?Glu?Lys?Val?Ala?Ser?Gln?Phe?Val?Tyr?Asn?Lys?Asp

325 330 335

att?ttg?gtt?ggt?gaa?gga?gag?gat?gca?gca?ttt?gat?gtt?ctc?ttg?ggg 1056

Ile?Leu?Val?Gly?Glu?Gly?Glu?Asp?Ala?Ala?Phe?Asp?Val?Leu?Leu?Gly

340 345 350

aag?aca?aca?atg?ttc?ccc?cca?aat?gtc?ttg?tta?gac?cac?aac?gtt?cct 1104

Lys?Thr?Thr?Met?Phe?Pro?Pro?Asn?Val?Leu?Leu?Asp?His?Asn?Val?Pro

355 360 365

gtt?tat?aaa?gct?gtg?caa?aaa?cct?ggg?gag?ttt?gtc?att?act?ttc?cct 1152

Val?Tyr?Lys?Ala?Val?Gln?Lys?Pro?Gly?Glu?Phe?Val?Ile?Thr?Phe?Pro

370 375 380

cgt?tcc?tac?cac?gcg?ggt?ttc?agc?cac?ggc?ttc?aat?tgt?ggc?gag?gct 1200

Arg?Ser?Tyr?His?Ala?Gly?Phe?Ser?His?Gly?Phe?Asn?Cys?Gly?Glu?Ala

385 390 395 400

gtc?aac?ttt?gct?atc?agt?gac?tgg?ttt?cct?ctg?ggt?tct?gtg?gcc?agc 1248

Val?Asn?Phe?Ala?Ile?Ser?Asp?Trp?Phe?Pro?Leu?Gly?Ser?Val?Ala?Ser

405 410 415

aga?cgc?tac?gcg?ctt?ctg?aac?aga?aca?ccc?ttg?ctt?gca?cac?gag?gag 1296

Arg?Arg?Tyr?Ala?Leu?Leu?Asn?Arg?Thr?Pro?Leu?Leu?Ala?His?Glu?Glu

420 425 430

tta?ctt?tgc?cgt?tct?gca?gtg?ctt?ctg?tcc?cac?aaa?ctg?tta?aac?agc 1344

Leu?Leu?Cys?Arg?Ser?Ala?Val?Leu?Leu?Ser?His?Lys?Leu?Leu?Asn?Ser

435 440 445

gac?cca?aaa?tcc?ctc?aat?aaa?tct?gag?cat?cca?cat?tca?cag?cgt?tgt 1392

Asp?Pro?Lys?Ser?Leu?Asn?Lys?Ser?Glu?His?Pro?His?Ser?Gln?Arg?Cys

450 455 460

ttg?aag?tct?tgc?ttt?gtg?cag?ttg?atg?cga?ttc?cag?aga?aac?aca?cgt 1440

Leu?Lys?Ser?Cys?Phe?Val?Gln?Leu?Met?Arg?Phe?Gln?Arg?Asn?Thr?Arg

465 470 475 480

ggc?cta?ctt?gct?aaa?atg?ggc?tct?cag?ata?cat?tat?aag?cca?aaa?aca 1488

Gly?Leu?Leu?Ala?Lys?Met?Gly?Ser?Gln?Ile?His?Tyr?Lys?Pro?Lys?Thr

485 490 495

tac?ccg?aat?ctc?tca?tgt?agc?atg?tgt?cgg?cgt?gat?tgc?tac?att?aca 1536

Tyr?Pro?Asn?Leu?Ser?Cys?Ser?Met?Cys?Arg?Arg?Asp?Cys?Tyr?Ile?Thr

500 505 510

cat?gtg?ttg?tgt?gga?tgc?aac?ttt?gac?cca?gtc?tgt?ctt?cat?cac?gaa 1584

His?Val?Leu?Cys?Gly?Cys?Asn?Phe?Asp?Pro?Val?Cys?Leu?His?His?Glu

515 520 525

caa?gaa?ctc?cgg?agc?tgc?cct?tgt?aaa?tct?aac?cag?gtt?gtc?tac?gtt 1632

Gln?Glu?Leu?Arg?Ser?Cys?Pro?Cys?Lys?Ser?Asn?Gln?Val?Val?Tyr?Val

530 535 540

agg?gag?gac?ata?cag?gag?cta?gaa?gct?cta?tca?aga?aaa?ttt?gag?aag 1680

Arg?Glu?Asp?Ile?Gln?Glu?Leu?Glu?Ala?Leu?Ser?Arg?Lys?Phe?Glu?Lys

545 550 555 560

gat?att?tgc?ttg?gat?aag?gaa?ata?agt?ggt?ttt?gac?tca?tac?aag?cag 1728

Asp?Ile?Cys?Leu?Asp?Lys?Glu?Ile?Ser?Gly?Phe?Asp?Ser?Tyr?Lys?Gln

565 570 575

gcc?gaa?aag?aat?gag?cca?ttt?ttt?gag?ata?act?cgg?aac?ctc?agg?aac 1776

Ala?Glu?Lys?Asn?Glu?Pro?Phe?Phe?Glu?Ile?Thr?Arg?Asn?Leu?Arg?Asn

580 585 590

act?gaa?gta?aat?ttg?ata?gag?gat?gcc?ttc?tca?gga?gca?act?gct?gct 1824

Thr?Glu?Val?Asn?Leu?Ile?Glu?Asp?Ala?Phe?Ser?Gly?Ala?Thr?Ala?Ala

595 600 605

gat?gct?gca?aag?agt?tct?cct?gca?acg?tca?aca?ctg?aca?tct?ttt?gca 1872

Asp?Ala?Ala?Lys?Ser?Ser?Pro?Ala?Thr?Ser?Thr?Leu?Thr?Ser?Phe?Ala

610 615 620

caa?cat?gat?gtg?cct?gtt?ctt?gct?gaa?gca?att?gtc?tgt?gct?aat?caa 1920

Gln?His?Asp?Val?Pro?Val?Leu?Ala?Glu?Ala?Ile?Val?Cys?Ala?Asn?Gln

625 630 635 640

gcc?gac?caa?tta?tac?tcc?acc?acc?gag?caa?acc?atc?agc?tca?cct?tta 1968

Ala?Asp?Gln?Leu?Tyr?Ser?Thr?Thr?Glu?Gln?Thr?Ile?Ser?Ser?Pro?Leu

645 650 655

gtc?aaa?gga?act?gat?gct?gtg?ggt?gca?aat?tca?tcc?agc?atg?gct?gat 2016

Val?Lys?Gly?Thr?Asp?Ala?Val?Gly?Ala?Asn?Ser?Ser?Ser?Met?Ala?Asp

660 665 670

gct?aat?aac?gga?act?ggt?tct?tgt?aat?gct?tca?gct?gtg?gaa?tac?agt 2064

Ala?Asn?Asn?Gly?Thr?Gly?Ser?Cys?Asn?Ala?Ser?Ala?Val?Glu?Tyr?Ser

675 680 685

gga?aat?tca?gat?tct?gaa?tct?gaa?ata?ttt?cga?gtc?aag?cgc?agg?tct 2112

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

690 695 700

ggc?gta?tca?gta?aag?cct?gca?tct?gat?gcc?aag?aca?tca?aac?ttg?tct 2160

Gly?Val?Ser?Val?Lys?Pro?Ala?Ser?Asp?Ala?Lys?Thr?Ser?Asn?Leu?Ser

705 710 715 720

gat?caa?cag?gtt?ctc?agg?cgg?ttg?aag?aag?gtg?cgc?cct?gaa?ata?caa 2208

Asp?Gln?Gln?Val?Leu?Arg?Arg?Leu?Lys?Lys?Val?Arg?Pro?Glu?Ile?Gln

725 730 735

cag?cac?aat?aag?cga?cca?gaa?gac?tat?ggt?cac?tgt?tca?gtt?ccc?tca 2256

Gln?His?Asn?Lys?Arg?Pro?Glu?Asp?Tyr?Gly?His?Cys?Ser?Val?Pro?Ser

740 745 750

ggt?cgt?atg?agt?atg?aag?aat?ttg?aat?tca?tcc?tcc?tca?tgt?ggt?gaa 2304

Gly?Arg?Met?Ser?Met?Lys?Asn?Leu?Asn?Ser?Ser?Ser?Ser?Cys?Gly?Glu

755 760 765

gaa?cac?tgg?agg?atg?aag?cgg?cgg?cag?ttg?gag?act?cag?cag?gat?gag 2352

Glu?His?Trp?Arg?Met?Lys?Arg?Arg?Gln?Leu?Glu?Thr?Gln?Gln?Asp?Glu

770 775 780

agc?agt?tat?tct?gca?aag?cag?aag?tcg?tac?tcg?tat?cca?tcc?acc?agc 2400

Ser?Ser?Tyr?Ser?Ala?Lys?Gln?Lys?Ser?Tyr?Ser?Tyr?Pro?Ser?Thr?Ser

785 790 795 800

tat?tct?ttc?cga?gga?gag?ttt?gtg?gaa?atg?agt?aga?gat?gct?gct?gca 2448

Tyr?Ser?Phe?Arg?Gly?Glu?Phe?Val?Glu?Met?Ser?Arg?Asp?Ala?Ala?Ala

805 810 815

gaa?gtc?cga?cca?aag?cga?ctg?aaa?atc?cgg?cta?cct?tct?tct?agc?acg 2496

Glu?Val?Arg?Pro?Lys?Arg?Leu?Lys?Ile?Arg?Leu?Pro?Ser?Ser?Ser?Thr

820 825 830

aac?aga?gtg?gtt?gag?cag?ggc?agt?tca?ggg?caa?aga?ttt?aca?agg?gat 2544

Asn?Arg?Val?Val?Glu?Gln?Gly?Ser?Ser?Gly?Gln?Arg?Phe?Thr?Arg?Asp

835 840 845

gac?aag?tcg?ctt?ggt?tgt?tgg?cct?gca?att?tag 2577

Asp?Lys?Ser?Leu?Gly?Cys?Trp?Pro?Ala?Ile

850 855

<210>2

<211>858

<212>PRT