CN1928084A

CN1928084A - 斑节对虾热休克蛋白90的基因的核苷酸及氨基酸序列

Info

Publication number: CN1928084A
Application number: CN 200610037558
Authority: CN
Inventors: 江世贵; 邱丽华; 周发林; 张汉华
Original assignee: South China Sea Fisheries Research Institute Chinese Academy Fishery Sciences
Current assignee: South China Sea Fisheries Research Institute Chinese Academy Fishery Sciences
Priority date: 2006-09-07
Filing date: 2006-09-07
Publication date: 2007-03-14

Abstract

本发明从近源物种肌动蛋白的基因的CDS序列的保守区设计的兼并引物，从斑节对虾的性腺中提取总RNA，并用PCR法扩增，克隆到斑节对虾热休克蛋白90基因的表达序列。斑节对虾热休克蛋白90基因的分离，对于研究软体动物体内的热休克蛋白90对环境中的污染因素的应激反应，从而探讨能否将该分子作为一种生物学指标具有重要的理论意义和实践意义。

Description

斑节对虾热休克蛋白90的基因的核苷酸及氨基酸序列

技术领域

本发明涉及分子生物学中基因克隆领域，尤其涉及斑节对虾热休克蛋白90的基因的核苷酸及氨基酸序列。

背景技术

热休克蛋白Hsp90是一类特异的分子伴侣，在进化中具有高度保守性。早期认为它可协助变性蛋白从高温或其它应激环境中复性，并可促进已产生突变的蛋白质降解。近年来的研究显示Hsp90具有广泛的生理功能，它参与调节多种蛋白质在非应激条件下的生物活性，其中包括调节DNA的复制、基因转录、亚细胞结构的蛋白质转移、细胞信号转导、免疫应答、以及生长、发育、凋亡等。Hsp90还与Ras信号途径中许多信号分子的折叠与组装密切相关，主要是Hsp90的结合与解离，介导了这些分子在非活性形式与活性形式间的转化或在一定条件下，从Hsp90等与之形成的复合物中释放，才能转位至胞膜，行使激酶的活性功能。Hsp90还具有使突变蛋白质具备类似野生型构象活性的作用。因此，研究这类进化上非常保守的蛋白的基因表达调控机制和相关的信号转导是非常重要的。

发明内容

本发明的目的是通过以近源物种热休克蛋白的基因的CDS序列的保守区设计的兼并引物，并用PCR法扩增，结合RACE技术克隆到斑节对虾热休克蛋白90基因的全表达序列。

上述目的是通过以下技术方案来实现的：

解剖斑节对虾取出性腺，提取总RNA，使之与反转录引物(oligo-dT接头引物)混合，进行反转录，合成第一链cDNA。

从GenBank中下载近源物种(如人、牛、斑马鱼、爪蛙等)热休克蛋白90同源基因CDS序列，利用Clustal W软件进行多序列比对，确定保守区，根据保守区序列设计兼并引物，扩增片段大小约为680bp。引物设计后采用β-乙睛亚磷酰胺化学法进行DNA合成得到以下引物序列：

X90F：5’-ATgATTggACAgTTYggTgT-3’

X90R：5’-TACAgYTTgATITTgTTCTT-3’。

以合成的第一链cDNA作为模板，用兼并引物进行PCR扩增，所扩增的PCR产物用1.2％的琼脂糖凝胶电泳进行检测，从凝胶中纯化回收目的产物。然后将纯化的PCR产物克隆到pMD-18T载体中，转化大肠杆菌JM-109感受态细胞，挑取阳性克隆，提取质粒DNA。经简并引物PCR检测后，将具有***片段的质粒DNA用M13通用引物进行双向测序。

本发明另外再根据已得到的第一链cDNA片段序列设计特异性引物，利用cDNA末端快速扩增技术(Rapid Amplification of cDNA ends，RACE)对目的基因的3′和5′末端进行PCR扩增。

在3′端的快速扩增中，利用半巢式(semi-nested)PCR方法，首先由基因引物和接头引物进行PCR反应，扩增片段大小约为1500bp。所述的基因引物和接头引物序列为：

基因引物：5’TCGGGGACTTCGCATACATC 3’

接头引物：5’GGCCACGCGACTAGTAC 3’。

3′端的快速扩增后，进行5′端的快速扩增，利用末端转移酶和dCTP在cDNA末端加上poly(C)尾后，以加尾后的cDNA作为模板，利用外侧特异性引物和OligodG进行第一次PCR扩增，所得PCR产物再利用内侧引物和OligodG进行第二次PCR扩增。所述引物序列为：

oligo-dG：5’GGGGGGGGGGGGGGGH 3’

基因外侧引物：5’GGCCACGCGACTAGTAC 3’

基因内侧引物：5’GCCGACAAGGTGACCGTAGT 3’。

经末端快速扩增技术进行PCR所得到的PCR产物在1.2％的琼脂糖凝胶电泳上进行分离检测后，将PCR产物纯化后，克隆到pMD-18T载体中，转化大肠杆菌JM-109感受态细胞，挑取阳性克隆，用M13引物对质粒DNA进行测序，测序所得序列再与兼并引物扩增所得的序列利用Clustal W软件进行拼接得到目的基因。

斑节对虾热休克蛋白90基因的分离，对于研究软体动物体内的热休克蛋白90对环境中的污染因素的应激反应，从而探讨能否将该分子作为一种生物学指标具有重要的理论意义和实践意义。而且进来发现该基因对性腺发育具有促进的作用，因此可以通过对基因的克隆获得纯化的蛋白，来进一步检测该基因是否影响斑节对虾的性腺发育。

具体实施方式

下面通过以下具体实施方式对本发明作进一步阐述，但是本发明的内容完全不局限于此。

1.总RNA的提取

取鲜活健康斑节对虾(体重约300g)在实验室内暂养2d后(水温约24℃，气泵充气)，解剖虾体取出性腺约100mg，放入1mL Trizol(Gibco，Japan)中进行匀浆，按照试剂盒使用说明提取总RNA。

2.cDNA第一链的合成

取斑节对虾总RNA 5μg与反转录引物(oligo-dT接头引物)1μL(10pmol/L)混合，70℃加热5min后，立即放置冰上，然后加入5×buffer，2.5mmol/L dNTP混合液，Ribonuclease Inhibitor，M-MLV反转录酶，反应体系为25μL。反应过程为42℃60min，70℃15min，最后放入-80℃保存备用。

3.兼并引物设计依据，引物合成的方法

从GenBank中下载近源物种(如人、牛、斑马鱼、爪蛙等)Hsp90同源基因CDS序列，利用Clustal W软件进行多序列比对，确定保守区，根据保守区序列设计兼并引物，扩增片段大小约为680bp。引物设计后采用β-乙睛亚磷酰胺化学法进行DNA合成。

引物序列：

X90F：5’-ATgATTggACAgTTYggTgT-3’

X90R：5’-TACAgYTTgATITTgTTCTT-3’。

4.斑节对虾热休克蛋白90基因cDNA全序列的克隆

以近源物种Hsp90基因CDS序列的保守区设计的兼并引物，扩增片段大小约为680bp。

4.1第一链cDNA用兼并引物进行PCR扩增

以上述合成的第一链cDNA作为模板，用兼并引物进行PCR扩增，反应体系为：10x PCR反应缓冲液5μL，25mmol/L MgCl₂ 3μL，2.5mmol/L dNTP 2μL，10nmol/L引物dTF和dTR各2μL，Taq酶1.25U，用PCR水将反应体系补充至50μL。反应条件为：1个循环，94℃变性5min；35个循环：94℃变性45s，55℃退火45s，72℃延伸45s；1个循环，72℃延伸10min；4℃保温。所扩增的PCR产物用1.2％的琼脂糖凝胶电泳进行检测，从凝胶中纯化回收目的产物。然后将纯化的PCR产物克隆到pMD-18T载体中，转化大肠杆菌JM-109感受态细胞，挑取阳性克隆，提取质粒DNA。经兼并引物PCR检测后，将具有***片段的质粒DNA用M13通用引物进行双向测序。

4.2第一链cDNA的快速扩增技术进行PCR扩增

根据已得到的cDNA片段序列设计特异性引物。利用cDNA末端快速扩增技术(Rapid Amplification of cDNA ends，RACE)对目的基因的3′和5′末端进行PCR扩增。

在3′RACE中，由基因引物和接头引物进行PCR反应，扩增片段大小约为1500bp。引物序列为：

接头引物：5’GGCCACGCGACTAGTAC 3’

基因引物：5’GCCGACAAGGTGACCGTAGT 3’

反应体系同兼并引物扩增体系。

反应条件为：1个循环，94℃变性5min；35个循环：94℃变性45s，59℃退火30s，72℃延伸1min；1个循环，72℃延伸10min；4℃保温。

在5′RACE中，利用末端转移酶和dCTP在cDNA末端加上poly(C)尾后，以加尾后的cDNA作为模板，利用外侧特异性引物和OligodG进行第一次PCR扩增，所得PCR产物取1μL作为模板，再利用内侧引物和OligodG进行第二次PCR扩增。引物序列为：

oligo-dG：5’GGGGGGGGGGGGGGGH 3’

外侧基因引物：5’TGTTCTTCTGCTTGCGGTTC 3’

内侧基因引物：5’TCCTCCCAGTCGTTGGTCAG 3’

反应体系反应体系同兼并引物扩增体系。

反应条件：

第一次PCR扩增为：1个循环，94℃变性5min；35个循环：94℃变性45s，60℃退火30s，72℃延伸1min；1个循环，72℃延伸10min；4℃保温。

第二次PCR扩增为：1个循环，94℃变性5min；35个循环：94℃变性45s，58℃退火30s，72℃延伸1min；1个循环，72℃延伸10min；4℃保温。

所得到的PCR产物在1.2％的琼脂糖凝胶电泳上进行分离检测后，将PCR产物纯化后，克隆到pMD-18T载体中，转化大肠杆菌JM-109感受态细胞，挑取阳性克隆，用M13引物对质粒DNA进行测序，测序所得序列再与兼并引物扩增所得的序列利用Clustal W软件进行拼接即得目的基因。

5.斑节对虾细胞周期蛋白B基因的测定

所得到的PCR产物在1.2％的琼脂糖凝胶电泳上进行分离检测后，将PCR产物纯化后，克隆到pMD-18T载体中，转化大肠杆菌JM-109感受态细胞，挑取阳性克隆，用3730测序仪对质粒DNA进行测序。测序结果用BLAST软件( http://www.ncbi.nim.nih.gov/)进行同源性测定，来确定为斑节对虾热休克蛋白90基因同源序列。比对结果为：

Score E

Sequences producing significant alignments： (Bits) Value

gi|70997669|gb|AAZ17403.1|90 kDa heat shock protein[Bemisia ta 939 0.0

gi|54873686|gb|AAV41061.1|Hsp90beta[Xenopus laevis]＞gi|506... 937 0.0

gi|70997651|gb|AAZ17402.1|90 kDa heat shock protein[Bemisia ta 934 0.0

gi|4835864|gb|AAD30275.1|heat shock protein hsp90 beta[salmo s 922 0.0

gi|42556386|gb|AAS19788.1|hsp-90[Chiromantes haematocheir] 653 0.0

根据Iron1995年的报道，只要两个序列间用BLAST软件比对后E-Value值小于0.005，则两个序列之间具有绝对的同源性，即证明序列为同一种基因序列。从对比结果看斑节对虾目的基因序列BLAST比对后，与其他物种的热休克蛋白90基因的E值绝对小于0.005，所以证明所克隆的目的基因是热休克蛋白90基因的同源序列。

序列表

<110>中国水产科学研究院南海水产研究所

<120>斑节对虾热休克蛋白90的基因的核苷酸及氨基酸序列

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<210>1

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<212>RNA

<213>斑节对虾(Penaeus monodon)

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<221>3’UTP

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<221>5’UTP

<222>(1)...(63)

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<221>CDS

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<221>PolyA site

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caacacattccaaagccaacaactttttgttcctttgtcggtcaaagcttcacacattcc 60

aaaatg cct gag gag gtc acc acc ggg gag gag gag gtc ttc gcg ttc cag 111

Met Phe Glu Glu Val Thr Thr Gly Glu Glu Glu Val Phe Ala Phe Gln 16

1 7 13

gcg gag atc gcg cag ctg atg tcc ctg atc atc aac acc ttc tac agc 159

Ala Glu Ile Ala Gln Leu Met Ser Leu Ile Ile Asn Thr Phe Tyr Ser 32

17 23 29

aac aag gag atc ttc ctg cga gag ctg atc tcg aac tcg tcc gac gcc 207

Asn Lys Glu Ile Phe Leu Arg Glu Leu Ile Ser Asn Ser Ser Asp Ala 48

33 39 45

ctc gac aag atc cgc tac gag tcg ctg acg gac ccg tcc aag ctg gag 255

Leu Asp Lys Ile Arg Tyr Glu Ser Leu Thr Asp Pro Ser Lys Leu Glu 64

49 55 61

agc ggc aag gac ctg ttc atc aag ctg gtg ccc aac aag gac gac cgc 303

Ser Gly Lys Asp Leu Phe Ile Lys Leu Val Pro Asn Lys Asp Asp Arg 80

65 71 77

acg ctc acc atc atc gac agt ggc atc ggc atg acc aag gcc gac ctg 351

Thr Leu Thr Ile Ile Asp Ser Gly Ile Gly Met Thr Lys Ala Asp Leu 96

81 87 93

gtg aac aac ctg ggc acc atc gcc aag tcg ggc aca aag gcc ttc atg 399

Val Asn Asn Leu Gly Thr Ile Ala Lys Ser Gly Thr Lys Ala Phe Met 112

97 103 109

gag gcg ctg cag gcg ggc gcc gac atc tcg atg atc ggc cag ttc ggc 447

Glu Ala Leu Gln Ala Gly Ala Asp Ile Ser Met Ile Gly Gln Phe Gly 128

113 119 125

gtg ggc ttc tac tcc gcg tac ctg gtg gcc gac aag gtg acc gta gtg 495

Val Gly Phe Tyr Ser Ala Tyr Leu Val Ala Asp Lys Val Thr Val Val 144

129 135 141

tcg agg aac aac gac gac gaa cag tac atc tgg gag tcg tcg gcg ggc 543

Ser Arg Asn Asn Asp Asp Glu Gln Tyr Ile Trp Glu Ser Ser Ala Gly 160

145 151 157

ggg tcg ttc acg gtg cgc cac gac acc ggt gaa ccc atc ggc cgt ggt 591

Gly Ser Phe Thr Val Arg His Asp Thr Gly Glu Pro Ile Gly Arg Gly 176

161 167 173

aca aag atc acc ctc cac ctg aag gag gac cag aca gag tac ctc gag 639

Thr Lys Ile Thr Leu His Leu Lys Glu Asp Gln Thr Glu Tyr Leu Glu 192

177 183 189

gag cgt cgc gtg aag gag atc gtg aag aag cac tcg caa ttc att ggc 687

Glu Arg Arg Val Lys Glu Ile Val Lys Lys His Ser Gln Phe Ile Gly 208

193 199 205

tat ccc atc aag ctc ctc gtc gag aag gag agg gac aag gaa gtg tct 735

Tyr Pro Ile Lys Leu Leu Val Glu Lys Glu Arg Asp Lys Glu Val Ser 224

209 215 221

gac gat gag gaa gag gag aaa gaa gag aag gaa gag gaa gca gag gag 783

Asp Asp Glu Glu Glu Glu Lys Glu Glu Lys Glu Glu Glu Ala Glu Glu 240

225 231 237

gac aag ccc aaa atc gaa gat gta ggc gag gac gaa gaa gcc gac aaa 831

Asp Lys Pro Lys Ile Glu Asp Val Gly Glu Asp Glu Glu Ala Asp Lys 256

241 247 253

gag aag ggc gaa gac aag aag aaa aag aag acg gtg aag gag aag tac 879

Glu Lys Gly Glu Asp Lys Lys Lys Lys Lys Thr Val Lys Glu Lys Tyr 272

257 263 269

acg gag gac gaa gag ctg aac aag acg aag ccc ctg tgg acg cgc aac 927

Thr Glu Asp Glu Glu Leu Asn Lys Thr Lys Pro Leu Trp Thr Arg Asn 288

273 279 285

ccc gac gac atc tcg aag gag gag tac ggc gag ttc tac aag tcg ctg 975

Pro Asp Asp Ile Ser Lys Glu Glu Tyr Gly Glu Phe Tyr Lys Ser Leu 304

289 295 301

acc aac gac tgg gag gac cac ctg gcc gtg aag cac ttc agc gtg gag 1023

Thr Asn Asp Trp Glu Asp His Leu Ala Val Lys His Phe Ser Val Glu 320

305 311 317

ggc cag ctt gag ttc cgc gcc ctc ctg ttc ctg ccg cgc cgc gcc ccc 1071

Gly Gln Leu Glu Phe Arg Ala Leu Leu Phe Leu Pro Arg Arg Ala Pro 336

321 327 333

ttc gac ctg ttc gag aac cgc aag cag aag aac aag atc aag ctg tac 1119

Phe Asp Leu Phe Glu Asn Arg Lys Gln Lys Asn Lys Ile Lys Leu Tyr 352

337 343 349

gtg cgt cgc gtg ttc atc atg gag aac tgc gag gaa ctg atc ccc gag 1167

Val Arg Arg Val Phe Ile Met Glu Asn Cys Glu Glu Leu Ile Pro Glu 368

353 359 365

tac ctg aac ttc atc aac ggt gtc gtc gac tcc gag gat ctg cct ctc 1215

Tyr Leu Asn Phe Ile Asn Gly Val Val Asp Ser Glu Asp Leu Pro Leu 384

369 375 381

aac atc tct cgt gag atg ctg caa cag aac aag atc ctg aaa gtt atc 1263

Asn Ile Ser Arg Glu Met Leu Gln Gln Asn Lys Ile Leu Lys Val Ile 400

385 391 397

agg aag aat ctc gtc aag aag acc ctc gaa ctt ttt gaa gaa att gtt 1311

Arg Lys Asn Leu Val Lys Lys Thr Leu Glu Leu Phe Glu Glu Ile Val 416

401 407 413

gac gac aag gaa agc tac aag aag ttc tac gaa aac ttc tcc aag aac 1359

Asp Asp Lys Glu Ser Tyr Lys Lys Phe Tyr Glu Asn Phe Ser Lys Asn 432

417 423 429

ctc aaa ctc gga atc cac gag gat tcc acc aac cgc aag aag ctt gcc 1407

Leu Lys Leu Gly Ile His Glu Asp Ser Thr Asn Arg Lys Lys Leu Ala 448

433 439 445

gaa ttc ctg agg tac cac act tct gcc tct ggc gac gaa atg tcc tcc 1455

Glu Phe Leu Arg Tyr His Thr Ser Ala Ser Gly Asp Glu Met Ser Ser 464

449 455 461

ctc aag gag tac gtg tcc cgc atg aag gag aac cag aag cac atc tac 1503

Leu Lys Glu Tyr Val Ser Arg Met Lys Glu Asn Gln Lys His Ile Tyr 480

465 471 477

ttc atc act ggc gag act cgc gaa cag gtg cag aac tct gcc ttc gtg 1551

Phe Ile Thr Gly Glu Thr Arg Glu Gln Val Gln Asn Ser Ala Phe Val 496

481 487 493

gag agg gtg aag aag cgc ggc ttc gag gtc atc tac atg acc gaa ccc 1599

Glu Arg Val Lys Lys Arg Gly Phe Glu Val Ile Tyr Met Thr Glu Pro 512

497 503 509

atc gac gaa tac tgc gtt cag cag ctg aag gaa tac gac ggg aag cag 1647

Ile Asp Glu Tyr Cys Val Gln Gln Leu Lys Glu Tyr Asp Gly Lys Gln 528

513 519 525

ctt gtc tcg gtg acg aag gaa ggc ctt gaa ctc ccc gag gac gag gag 1696

Leu Val Ser Val Thr Lys Glu Gly Leu Glu Leu Pro Glu Asp Glu Glu 544

529 535 541

gag aaa aag aag ttc gag gaa cag aag acc aag ttc gag aac ctg tgc 1743

Glu Lys Lys Lys Phe Glu Glu Gln Lys Thr Lys Phe Glu Asn Leu Cys 560

545 551 557

aag gta atg aag gac att ttg gac aag cgc gtt gag aag gtg gtg gtg 1791

Lys Val Met Lys Asp Ile Leu Asp Lys Arg Val Glu Lys Val Val Val 576

561 567 573

agc aac cgg ctg gtg acc tct ccg tgc tgc atc gtg acc tcc cag tac 1839

Ser Asn Arg Leu Val Thr Ser Pro Cys Cys Ile Val Thr Ser Gln Tyr 592

577 583 589

ggc tgg acc gcc aac atg gag cgc atc atg aag gct cag gcg ctg agg 1887

Gly Trp Thr Ala Asn Met Glu Arg Ile Met Lys Ala Gln Ala Leu Arg 608

593 599 605

gac acc tcg acc atg ggc tac atg gcc gcc aag aag cac ctt gag atc 1935

Asp Thr Ser Thr Met Gly Tyr Met Ala Ala Lys Lys His Leu Glu Ile 624

609 615 621

aac ccc gac cac agc atc atc gaa acc ctg aga caa aag gcg gat gcc 1983

Asn Pro Asp His Ser Ile Ile Glu Thr Leu Arg Gln Lys Ala Asp Ala 640

625 631 637

gac aag aac gac aag tct gtg aag gat ctg gtg atg ctg ctg ttc gag 2031

Asp Lys Asn Asp Lys Ser Val Lys Asp Leu Val Met Leu Leu Phe Glu 656

641 647 653

agc tcc ctt ctg tcg tct ggc ttc agc ctg gag gac cca ggt gtc cac 2079

Ser Ser Leu Leu Ser Ser Gly Phe Ser Leu Glu Asp Pro Gly Val His 672

657 663 669

gcc agc cgc atc tac aga atg atc aag ctt ggc ctg ggt att gac gag 2127

Ala Ser Arg Ile Tyr Arg Met Ile Lys Leu Gly Leu Gly Ile Asp Glu 688

673 679 685

gag gac gcc ccg atg gag gag gcc gag acc ttg gag gag gat atg ccc 2175

Glu Asp Ala Pro Met Glu Glu Ala Glu Thr Leu Glu Glu Asp Met Pro 704

689 695 701

ccc ctc gaa ggt gat gac gag gac gcc tct cgc atg gaa gaa gtc gat 2223

Pro Leu Glu Gly Asp Asp Glu Asp Ala Ser Arg Met Glu Glu Val Asp 720

705 711 717

taa 2226

atattcgtcacaacttaaaatattcacccattatataccaaagctaatcattgtcattca 2286

ttcgggaaccaaaatacttctctaatcttggtatattttggcttctctggctttcatcat 2346

tccgatcacgcccaacattccataagatttaaacaagcattagttttagttatagacaaa 2406

gatatattctgttataaggatttattctttcgtttatgtaaataatttgtaacaactttg 2466

ttacaataaaactcgagaccatttaaaaaaaaaaaaaaaaaaaaaaaa 2514

<210>2

<211>720

<212>PRT

<213>斑节对虾(Penaeus monodon)

<400>2

Met Phe Glu Glu Val Thr Thr Gly Glu Glu Glu Val Phe Ala Phe Gln 16

1 7 13

Ala Glu Ile Ala Gin Leu Met Ser Leu Ile Ile Asn Thr Phe Tyr Ser 32

17 23 29

Asn Lys Glu Ile Phe Leu Arg Glu Leu Ile Ser Ash Ser Ser Asp Ala 48

33 39 45

Leu Asp Lys Ile Arg Tyr Glu Ser Leu Thr Asp Pro Ser Lys Leu Glu 64

49 55 61

Ser Gly Lys Asp Leu Phe Ile Lys Leu Val Pro Asn Lys Asp Asp Arg 80

65 71 77

Thr Leu Thr Ile Ile Asp Ser Gly Ile Gly Met Thr Lys Ala Asp Leu 96

81 87 93

Val Asn Asn Leu Gly Thr Ile Ala Lys Ser Gly Thr Lys Ala Phe Met 112

97 103 109

Glu Ala Leu Gln Ala Gly Ala Asp Ile Ser Met Ile Gly Gln Phe Gly 128

113 119 125

Val Gly Phe Tyr Ser Ala Tyr Leu Val Ala Asp Lys Val Thr Val Val 144

129 135 141

Ser Arg Asn Asn Asp Asp Glu Gln Tyr Ile Trp Glu Ser Ser Ala Gly 160

145 151 157

Gly Ser Phe Thr Val Arg His Asp Thr Gly Glu Pro Ile Gly Arg Gly 176

161 167 173

Thr Lys Ile Thr Leu His Leu Lys Glu Asp Gln Thr Glu Tyr Leu Glu 192

177 183 189

Glu Arg Arg Val Lys Glu Ile Val Lys Lys His Ser Gln Phe Ile Gly 208

193 199 205

Tyr Pro Ile Lys Leu Leu Val Glu Lys Glu Arg Asp Lys Glu Val Ser 224

209 215 221

Asp Asp Glu Glu Glu Glu Lys Glu Glu Lys Glu Glu Glu Ala Glu Glu 240

225 231 237

Asp Lys Pro Lys Ile Glu Asp Val Gly Glu Asp Glu Glu Ala Asp Lys 256

241 247 253

Glu Lys Gly Glu Asp Lys Lys Lys Lys Lys Thr Val Lys Glu Lys Tyr 272

257 263 269

Thr Glu Asp Glu Glu Leu Asn Lys Thr Lys Pro Leu Trp Thr Arg Asn 288

273 279 285

Pro Asp Asp Ile Ser Lys Glu Glu Tyr Gly Glu Phe Tyr Lys Ser Leu 304

289 295 301

Thr Asn Asp Trp Glu Asp His Leu Ala Val Lys His Phe Ser Val Glu 320

305 311 317

Gly Gln Leu Glu Phe Arg Ala Leu Leu Phe Leu Pro Arg Arg Ala Pro 336

321 327 333

Phe Asp Leu Phe Glu Asn Arg Lys Gln Lys Asn Lys Ile Lys Leu Tyr 352

337 343 349

Val Arg Arg Val Phe Ile Met Glu Asn Cys Glu Glu Leu Ile Pro Glu 368

353 359 365

Tyr Leu Asn Phe Ile Asn Gly Val Val Asp Ser Glu Asp Leu Pro Leu 384

369 375 381

Asn Ile Ser Arg Glu Met Leu Gln Gln Asn Lys Ile Leu Lys Val Ile 400

385 391 397

Arg Lys Asn Leu Val Lys Lys Thr Leu Glu Leu Phe Glu Glu Ile Val 416

401 407 413

Asp Asp Lys Glu Ser Tyr Lys Lys Phe Tyr Glu Asn Phe Ser Lys Asn 432

417 423 429

Leu Lys Leu Gly Ile His Glu Asp Ser Thr Asn Arg Lys Lys Leu Ala 448

433 439 445

Glu Phe Leu Arg Tyr His Thr Ser Ala Ser Gly Asp Glu Met Ser Ser 464

449 455 461

Leu Lys Glu Tyr Val Ser Arg Met Lys Glu Asn Gln Lys His Ile Tyr 480

465 471 477

Phe Ile Thr Gly Glu Thr Arg Glu Gln Val Gln Asn Ser Ala Phe Val 496

481 487 493

Glu Arg Val Lys Lys Arg Gly Phe Glu Val Ile Tyr Met Thr Glu Pro 512

497 503 509

Ile Asp Glu Tyr Cys Val Gln Gln Leu Lys Glu Tyr Asp Gly Lys Gln 528

513 519 525

Leu Val Ser Val Thr Lys Glu Gly Leu Glu Leu Pro Glu Asp Glu Glu 544

529 535 541

Glu Lys Lys Lys Phe Glu Glu Gln Lys Thr Lys Phe Glu Asn Leu Cys 560

545 551 557

Lys Val Met Lys Asp Ile Leu Asp Lys Arg Val Glu Lys Val Val Val 576

561 567 573

Ser Asn Arg Leu Val Thr Ser Pro Cys Cys Ile Val Thr Ser Gln Tyr 592

577 583 589

Gly Trp Thr Ala Asn Met Glu Arg Ile Met Lys Ala Gln Ala Leu Arg 608

593 599 605

Asp Thr Ser Thr Met Gly Tyr Met Ala Ala Lys Lys His Leu Glu Ile 624

609 615 621

Asn Pro Asp His Ser Ile Ile Glu Thr Leu Arg Gln Lys Ala Asp Ala 640

625 631 637

Asp Lys Asn Asp Lys Ser Val Lys Asp Leu Val Met Leu Leu Phe Glu 656

641 647 653

Ser Ser Leu Leu Ser Ser Gly Phe Ser Leu Glu Asp Pro Gly Val His 672

657 663 669

Ala Ser Arg Ile Tyr Arg Met Ile Lys Leu Gly Leu Gly Ile Asp Glu 688

673 679 685

Glu Asp Ala Pro Met Glu Glu Ala Glu Thr Leu Glu Glu Asp Met Pro 704

689 695 701

Pro Leu Glu Gly Asp Asp Glu Asp Ala Ser Arg Met Glu Glu Val Asp 720

705 711 717

Claims

1、一种斑节对虾热休克蛋白90基因，其特征在于具有以下RNA核苷酸及相应的氨基酸序列：

caacacattccaaagccaacaactttttgttcctttgtcggtcaaagcttcacacattcc 60

aaaatg cct gag gag gtc acc acc ggg gag gag gag gtc ttc gcg ttc cag 111

Met Phe Glu Glu Val Thr Thr Gly Glu Glu Glu Val Phe Ala Phe Gln 16

1 7 13

gcg gag atc gcg cag ctg atg tcc ctg atc atc aac acc ttc tac agc 159

Ala Glu Ile Ala Gln Leu Met Ser Leu Ile Ile Asn Thr Phe Tyr Ser 32

17 23 29

aac aag gag atc ttc ctg cga gag ctg atc tcg aac tcg tcc gac gcc 207

Asn Lys Glu Ile Phe Leu Arg Glu Leu Ile Ser Asn Ser Ser Asp Ala 48

33 39 45

ctc gac aag atc cgc tac gag tcg ctg acg gac ccg tcc aag ctg gag 255

Leu Asp Lys Ile Arg Tyr Glu Ser Leu Thr Asp Pro Ser Lys Leu Glu 64

49 55 61

agc ggc aag gac ctg ttc atc aag ctg gtg ccc aac aag gac gac cgc 303

Ser Gly Lys Asp Leu Phe Ile Lys Leu Val Pro Asn Lys Asp Asp Arg 80

65 71 77

acg ctc acc atc atc gac agt ggc atc ggc atg acc aag gcc gac ctg 351

Thr Leu Thr Ile Ile Asp Ser Gly Ile Gly Met Thr Lys Ala Asp Leu 96

81 87 93

gtg aac aac ctg ggc acc atc gcc aag tcg ggc aca aag gcc ttc atg 399

Val Asn Asn Leu Gly Thr Ile Ala Lys Ser Gly Thr Lys Ala Phe Met 112

97 103 109

gag gcg ctg cag gcg ggc gcc gac atc tcg atg atc ggc cag ttc ggc 447

Glu Ala Leu Gln Ala Gly Ala Asp Ile Ser Met Ile Gly Gln Phe Gly 128

113 119 125

gtg ggc ttc tac tcc gcg tac ctg gtg gcc gac aag gtg acc gta gtg 495

Val Gly Phe Tyr Ser Ala Tyr Leu Val Ala Asp Lys Val Thr Val Val 144

129 135 141

tcg agg aac aac gac gac gaa cag tac atc tgg gag tcg tcg gcg ggc 543

Ser Arg Asn Asn Asp Asp Glu Gln Tyr Ile Trp Glu Ser Ser Ala Gly 160

145 151 157

ggg tcg ttc acg gtg cgc cac gac acc ggt gaa ccc atc ggc cgt ggt 591

Gly Ser Phe Thr Val Arg His Asp Thr Gly Glu Pro Ile Gly Arg Gly 176

161 167 173

aca aag atc acc ctc cac ctg aag gag gac cag aca gag tac ctc gag 639

Thr Lys Ile Thr Leu His Leu Lys Glu Asp Gln Thr Glu Tyr Leu Glu 192

177 183 189

gag cgt cgc gtg aag gag atc gtg aag aag cac tcg caa ttc att ggc 687

Glu Arg Arg Val Lys Glu Ile Val Lys Lys His Ser Gln Phe Ile Gly 208

193 199 205

tat ccc atc aag ctc ctc gtc gag aag gag agg gac aag gaa gtg tct 735

Tyr Pro Ile Lys Leu Leu Val Glu Lys Glu Arg Asp Lys Glu Val Ser 224

209 215 221

gac gat gag gaa gag gag aaa gaa gag aag gaa gag gaa gca gag gag 783

Asp Asp Glu Glu Glu Glu Lys Glu Glu Lys Glu Glu Glu Ala Glu Glu 240

225 231 237

gac aag ccc aaa atc gaa gat gta ggc gag gac gaa gaa gcc gac aaa 831

Asp Lys Pro Lys Ile Glu Asp Val Gly Glu Asp Glu Glu Ala Asp Lys 256

241 247 253

gag aag ggc gaa gac aag aag aaa aag aag acg gtg aag gag aag tac 879

Glu Lys Gly Glu Asp Lys Lys Lys Lys Lys Thr Val Lys Glu Lys Tyr 272

257 263 269

acg gag gac gaa gag ctg aac aag acg aag ccc ctg tgg acg cgc aac 927

Thr Glu Asp Glu Glu Leu Asn Lys Thr Lys Pro Leu Trp Thr Arg Asn 288

273 279 285

ccc gac gac atc tcg aag gag gag tac ggc gag ttc tac aag tcg ctg 975

Pro Asp Asp Ile Ser Lys Glu Glu Tyr Gly Glu Phe Tyr Lys Ser Leu 304

289 295 301

acc aac gac tgg gag gac cac ctg gcc gtg aag cac ttc agc gtg gag 1023

Thr Asn Asp Trp Glu Asp His Leu Ala Val Lys His Phe Ser Val Glu 320

305 311 317

ggc cag ctt gag ttc cgc gcc ctc ctg ttc ctg ccg cgc cgc gcc ccc 1071

Gly Gln Leu Glu Phe Arg Ala Leu Leu Phe Leu Pro Arg Arg Ala Pro 336

321 327 333

ttc gac ctg ttc gag aac cgc aag cag aag aac aag atc aag ctg tac 1119

Phe Asp Leu Phe Glu Asn Arg Lys Gln Lys Asn Lys Ile Lys Leu Tyr 352

337 343 349

gtg cgt cgc gtg ttc atc atg gag aac tgc gag gaa ctg atc ccc gag 1167

Val Arg Arg Val Phe Ile Met Glu Asn Cys Glu Glu Leu Ile Pro Glu 368

353 359 365

tac ctg aac ttc atc aac ggt gtc gtc gac tcc gag gat ctg cct ctc 1215

Tyr Leu Asn Phe Ile Asn Gly Val Val Asp Ser Glu Asp Leu Pro Leu 384

369 375 381

aac atc tct cgt gag atg ctg caa cag aac aag atc ctg aaa gtt atc 1263

Asn Ile Ser Arg Glu Met Leu Gln Gln Asn Lys Ile Leu Lys Val Ile 400

385 391 397

agg aag aat ctc gtc aag aag acc ctc gaa ctt ttt gaa gaa att gtt 1311

Arg Lys Asn Leu Val Lys Lys Thr Leu Glu Leu Phe Glu Glu Ile Val 416

401 407 413

gac gac aag gaa agc tac aag aag ttc tac gaa aac ttc tcc aag aac 1359

Asp Asp Lys Glu Ser Tyr Lys Lys Phe Tyr Glu Asn Phe Ser Lys Asn 432

417 423 429

ctc aaa ctc gga atc cac gag gat tcc acc aac cgc aag aag ctt gcc 1407

Leu Lys Leu Gly Ile His Glu Asp Ser Thr Asn Arg Lys Lys Leu Ala 448

433 439 445

gaa ttc ctg agg tac cac act tct gcc tct ggc gac gaa atg tcc tcc 1455

Glu Phe Leu Arg Tyr His Thr Ser Ala Ser Gly Asp Glu Met Ser Ser 464

449 455 461

ctc aag gag tac gtg tcc cgc atg aag gag aac cag aag cac atc tac 1503

Leu Lys Glu Tyr Val Ser Arg Met Lys Glu Asn Gln Lys His Ile Tyr 480

465 471 477

ttc atc act ggc gag act cgc gaa cag gtg cag aac tct gcc ttc gtg 1551

Phe Ile Thr Gly Glu Thr Arg Glu Gln Val Gln Asn Ser Ala Phe Val 496

481 487 493

gag agg gtg aag aag cgc ggc ttc gag gtc atc tac atg acc gaa ccc 1599

Glu Arg Val Lys Lys Arg Gly Phe Glu Val Ile Tyr Met Thr Glu Pro 512

497 503 509

atc gac gaa tac tgc gtt cag cag ctg aag gaa tac gac ggg aag cag 1647

Ile Asp Glu Tyr Cys Val Gln Gln Leu Lys Glu Tyr Asp Gly Lys Gln 528

513 519 525

ctt gtc tcg gtg acg aag gaa ggc ctt gaa ctc ccc gag gac gag gag 1696

Leu Val Ser Val Thr Lys Glu Gly Leu Glu Leu Pro Glu Asp Glu Glu 544

529 535 541

gag aaa aag aag ttc gag gaa cag aag acc aag ttc gag aac ctg tgc 1743

Glu Lys Lys Lys Phe Glu Glu Gln Lys Thr Lys Phe Glu Asn Leu Cys 560

545 551 557

aag gta atg aag gac att ttg gac aag cgc gtt gag aag gtg gtg gtg 1791

Lys Val Met Lys Asp Ile Leu Asp Lys Arg Val Glu Lys Val Val Val 576

561 567 573

agc aac cgg ctg gtg acc tct ccg tgc tgc atc gtg acc tcc cag tac 1839

Ser Asn Arg Leu Val Thr Ser Pro Cys Cys Ile Val Thr Ser Gln Tyr 592

577 583 589

ggc tgg acc gcc aac atg gag cgc atc atg aag gct cag gcg ctg agg 1887

Gly Trp Thr Ala Asn Met Glu Arg Ile Met Lys Ala Gln Ala Leu Arg 608

593 599 605

gac acc tcg acc atg ggc tac atg gcc gcc aag aag cac ctt gag atc 1935

Asp Thr Ser Thr Met Gly Tyr Met Ala Ala Lys Lys His Leu Glu Ile 624

609 615 621

aac ccc gac cac agc atc atc gaa acc ctg aga caa aag gcg gat gcc 1983

Asn Pro Asp His Ser Ile Ile Glu Thr Leu Arg Gln Lys Ala Asp Ala 640

625 631 637

gac aag aac gac aag tct gtg aag gat ctg gtg atg ctg ctg ttc gag 2031

Asp Lys Asn Asp Lys Ser Val Lys Asp Leu Val Met Leu Leu Phe Glu 656

641 647 653

agc tcc ctt ctg tcg tct ggc ttc agc ctg gag gac cca ggt gtc cac 2079

Ser Ser Leu Leu Ser Ser Gly Phe Ser Leu Glu Asp Pro Gly Val His 672

657 663 669

gcc agc cgc atc tac aga atg atc aag ctt ggc ctg ggt att gac gag 2127

Ala Ser Arg Ile Tyr Arg Met Ile Lys Leu Gly Leu Gly Ile Asp Glu 688

673 679 685

gag gac gcc ccg atg gag gag gcc gag acc ttg gag gag gat atg ccc 2175

Glu Asp Ala Pro Met Glu Glu Ala Glu Thr Leu Glu Glu Asp Met Pro 704

689 695 701

ccc ctc gaa ggt gat gac gag gac gcc tct cgc atg gaa gaa gtc gat 2223

Pro Leu Glu Gly Asp Asp Glu Asp Ala Ser Arg Met Glu Glu Val Asp 720

705 711 717

taa 2226

atattcgtcacaacttaaaatattcacccattatataccaaagctaatcattgtcattca 2286

ttcgggaaccaaaatacttctctaatcttggtatattttggcttctctggctttcatcat 2346

tccgatcacgcccaacattccataagatttaaacaagcattagttttagttatagacaaa 2406

gatatattctgttataaggatttattctttcgtttatgtaaataatttgtaacaactttg 2466

ttacaataaaactcgagaccatttaaaaaaaaaaaaaaaaaaaaaaaa 2514