大菱鲆组成型热休克蛋白70的全长cDNA克隆及表达分析

根据感染哈维氏弧菌(Vibrio harveyi)的大菱鲆(Scophthalmus maximus)的差减cDNA文库中hsp70EST序列设计引物,用RACE方法首次克隆到长2188bp的大菱鲆HSC70(Heat-shock cognate protein 70)全长cDNA序列,包括1956bp的开放阅读框及5′和3′非翻译区。在预测的651个氨基酸序列中发现了Dnak特征性基序,胞质HSP70特征基序以及四肽简并重复序列。与其他真核生物HSP70家族成员进行同源性比较,发现大菱鲆HSC70与牙鲆(Paralichthys olivaceus)HSC71、虹鳟(Oncorhynchus mykiss)HSC71、人(Homo sapiens)HSC70、家鼠(Mus musculus)HSC70、烟草天蛾(Manduca sexta)HSC70的氨基酸相似性分别是97%,95%,94%,93%,86%,表现出较高的保守性。表达分析显示,hsc70mRNA在大菱鲆正常肝脏、鳃、肠、脾脏、头肾、肾等组织中以不同的水平存在,呈组成型表达;被哈维氏弧菌感染后,大菱鲆肝脏和脾脏组织hsc70mRNA表达水平分别在24h和12h出现上调(2.5-fold和1.6-fold);注射生理盐水组与未注射组之间差异不显著。  

CLONING AND EXPRESSION ANALYSIS OF A HSC70 GENE FROM TURBOT

Disruption of normal cellular processes can cause the rapid and increased synthesis of a group of proteins belonging to the heat shock proteins (HSPs). Of all the HSPs, HSP70 has been widely studied as a biomarker of stress, and the major inducing factor for HSP70 up-regulation is the occurrence of damaged cellular protein. HSC70 (heat-shock cognate protein 70) is a constitutively expressed member of the 70 kD class of HSP70, which plays key roles in the cell as molecular chaperone and involves in a number of cellular processes. HSC70 has been shown to be involved in protein folding in the cytoplasm, protein import into the endoplasmic reticulum, mitochondria, chloroplasts, or trafficking of the receptors and coated vesicles and so on. In previous study, suppression subtractive hybridization (SSH) was used to investigate the response of turbot (Scophthalmus maximus) to Vibrio harveyi, using a cDNA library constructed from kidney and spleen of artificially infected turbot, and several immune-related genes were identified, including a hsp70 gene. In the present study, the complete cDNA sequence of turbot HSCT0 was obtained using the method of RACE. The full length hsc70 cDNA of 2188 bp contained a 138 bp 5'-untranslated region (5'-UTR), a 1956 bp open reading frame (ORF) encoding 651 amino acids, and a 94 bp Y-untranslated region (3'-UTR). The specific mo-tif of Dnak (DLGTT-S-V, 10-18 aa), EEVD (648-651 aa) and GGMP repeated tetra-peptide (615-630 aa) were found in the deduced amino acid sequence of turbot HSC70. Compared with the members of HSC70 from other organ-isms, turbot HSC70 had 97%, 95%, 94%, 93% and 86% identity with HSC70 from flounder (Paralichthys olivaceus), rainbow trout (Oncorhynchus mykiss), human (Homo sapiens), mouse (Mus musculus) and tobacco (Manduca sexta) respectively. The overall topology of the phylogenetic tree showed that the turbot HSC70 and HSCT0s from other fish formed one cluster. Quantitative real-time PCR demonstrated that hse70 mRNA expressed constitutively in all of the test tissues, and the lowest expression level of turbot hsc70 mRNA was detected in muscle, the highest expression level was in liver by 45.4-fold. After the injection of physiological saline, the hsc70 mRNA expression levels in liver and spleen had no significant changes compared to those of non-injected turbot (P>0.05). This suggested that the injection itself had no influence on the expression of hsc70. After turbot were challenged with V. harveyi, the expression levels of hsc70 mRNA were up-regulated in liver (2.5-fold) and spleen (l.6-fold) at 24h and 12h, respectively (P<0.05). The re-sults provided a preliminary foundation for the further functional research of turbot HSC70.