褶纹冠蚌热休克蛋白HSP70基因的克隆及表达研究  

Cloning and expression analysis of HSP70 gene from Cristaria plicata

作　　者：谢彦海[1,2] 胡宝庆[1] 文春根[1] 

机构地区：[1]南昌大学生物科学系,江西南昌330031 [2]南昌大学实验动物科学部,江西南昌330031

出　　处：《南昌大学学报（理科版）》2011年第5期457-463,共7页Journal of Nanchang University(Natural Science)

基　　金：国家自然基金资助项目(30960296);江西省科技攻关基金资助项目(2004)

摘　　要：运用兼并引物结合RT-PCR方法从褶纹冠蚌(Cristaria plicata)血细胞中克隆出热休克蛋白70(HSP70)的cDNA序列。用半定量PCR方法研究了HSP70mRNA在褶纹冠蚌不同组织的表达情况。结果表明:HSP70cDNA全长为2 664bp,5’非编码区364个核苷酸,3’非编码区383个核苷酸,开放阅读框为1 917bp,编码638个氨基酸组成的蛋白质,该蛋白理论等电点为5.61,分子量为70.3kD。HSP70基因不含内含子。氨基酸序列分析发现,推导的氨基酸序列含有HSP70家族的3个标签序列(I10DLGTTYS17,V198FDLGGGTFDVSIL211和V336VLVGGSTRIPKV QK350)。氨基酸序列同源性比较显示HSP70与紫贻贝(Mytihcs galloprovincialis)和美洲牡蛎(Crassostrea virginica)氨基酸序列同源性分别为79.47%和77%。半定量PCR分析显示在正常褶纹冠蚌血液、外套膜、鳃、闭壳肌和肝胰腺5种组织中均能检测到HSP70的基因表达。经过热休克或病原菌刺激后,蚌各组织中HSP70mRNA的表达显著增加,其中热休克处理后,蚌的鳃和血液中HSP70mRNA表达量增加最为显著;而嗜水气单胞菌刺激后蚌的肌肉和鳃组织中HSP70mRNA表达量增加最明显。The sequences of HSP70 cDNA were cloned from the haemocytes of freshwater mussel Cristaria plicata using degenerate primers to integrate the reverse transcription-polymerase chain reaction（RT-PCR）.The Semi-quantitative PCR method was applied to further study the HSP70 mRNA expression in various tissues of C.plicata.The results revealed that the HSP70 cDNA clone consisted of 2664bp（GenBank accession number：ADM64336）with a 5＇-untranslated region（UTR） of 364 bp and a 3＇-UTR of 383 bp.The open reading frame of Hsp70 was 1917 bp and intronless,.The ORF encoded a putative protein of 638 amino acids with a predicted molecular mass of 70.3 kDa.The theoretical isoelectric point of the protein is 5.61.There were three HSP70 family signatures,i.e.,IDLGTTYS（residues 10-17）,VFDLGGGTFDVSIL（198-211）,VVLVGGSTRIPKVQK（336-350）.The deduced amino acid sequence of HSP70 shared high identities with the HSP70s of Mytilus galloprovincialis and Crassostrea virginica,with 79.47% and 77% autoploidy respectively.The expression of HSP70 mRNA was all detectable in blood,mantle,gill,muscles and hepatopancreas in common situation.Challenge of Cristaria plicata with heat shock（35 ℃） or the pathogenic bacteria Aeromonas hydrophila resulted in a dramatic increase in the expression of HSP70 mRNA level in these tissues.The expression of HSP70 in hemocytes and gills by the heat shock treatment increased in the most significant,while in gills and muscles,the most significant rise of the expression of HSP70 is by injection A.hydrophila.

关 键 词：褶纹冠蚌、热休克 HSP70、表达 嗜水气单胞菌 

分 类 号：Q959.215[生物学—动物学]