脊尾白虾热休克蛋白HSP70基因的克隆及其表达分析

Cloning and expression of heat shock protein 70(HSP70) of Exopalaemon carinicauda

  • 摘要: 克隆了脊尾白虾热休克蛋白基因全长cDNA,并进行了序列分析。该基因由2 250 bp的碱基组成,开放阅读框长1 959 bp,编码由652个氨基酸组成的蛋白,基因两翼分别存在83 bp(5′端)和208 bp(3′端)的非翻译区,将该基因命名为Ec-HSP70。与其它物种HSP70s氨基酸序列进行同源性比较发现,与甲壳动物的同源性都在90%以上,表明该蛋白属于热休克蛋白HSP70家族。聚类分析表明,脊尾白虾热休克蛋白氨基酸序列与中国明对虾和凡纳滨对虾紧密聚为一支,之后聚类顺序依次为斑节对虾、日本囊对虾、刀额新对虾等。通过荧光定量RT-PCR对该基因在肝胰腺、肌肉的表达分析表明,温度、pH和氨氮胁迫都可以引起该基因的高表达,而且肝胰腺中的高表达时间相对肌肉中出现的较早。试验结果表明,温度、pH和氨氮胁迫对脊尾白虾HSP70基因表达有一定的诱导效果,但胁迫的时间过长则抑制其表达,肝胰腺对胁迫比肌肉较敏感。

     

    Abstract: In this study,full-length cDNA sequence of heat shock protein 70(HSP70) was isolated from hepatopancreas of Exopalaemon carinicauda,which was named Ec-HSP70.The full-length cDNA sequence of heat shock protein 70 is 2 250 bp,which contains a 83 bp 5′-UTR(untranslated region),208 bp 3′-UTR(untranslated region),and 1 959 bp open reading frame(ORF) that encodes 652 amino acid residues.There are three HSP70 family signatures between 8-15,199-206 and 334-348 amino acids residues of Ec--HSP70,and these sequences are highly conserved in Fenneropenaeus chinensis,Litopenaeus vannamei,Penaeus monodon,Marsupenaeus japonicus,Metapenaeus ensis.The phylogenetic analysis based on amino acid sequence shows that Ec-HSP70 has the highest similarity(99.8%) with HSP70 of L.vannamei and F.chinensis, and has above 90% similarities with other crustacean species,indicating that this protein is one of heat shock protein HSP70 family members.The expression of the gene in hepatopancreas and muscle was analyzed by quantitative Real-time PCR,the result shows that the expression of HSP70 gene was higher in hepatopancreas and muscle after stimulation with the tempreture,pH and NH4Cl.The higher expression time of Ec-HSP70 cDNA in hepatopancreas was earlier than in muscle.HSP70 mRNA expressions were inhibited with longer stress time.Hepatopancreas were more sensitive than muscle to the tempreture,pH and NH4Cl stresses.

     

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