蓝点马鲛(Scomberomorus niphonius)两种Wap65基因克隆及蛋白功能的研究

采用同源克隆及Race技术获取了蓝点马鲛Wap65-1和Wap65-2基因的c DNA全长序列,长度分别为1659 bp和1725 bp,各自编码426和436个氨基酸。通过Clust W同源性及进化树分析表明:蓝点马鲛Wap65-1与Wap65-2基因的同源性为60%,处于不同的分支上。进一步对该鱼的幼体进行热诱导,通过q RT-PCR分析表明:两种基因在高温诱导下均有上调,而Wap65-2基因上调呈现显著性差异(P0.05)。在此基础上,构建两种基因冷休克表达系统,成功实现了两种蛋白的可溶性表达,而且发现Wap65-2对高温胁迫下E.coli BL21(DE3)的存活具有较强的保护作用。本研究为蓝点马鲛耐热品种的培育奠定了理论基础。

MOLECULAR CLONING AND FUNCTIONAL ANALYSIS OF TWO DIFFERENT Wap65 GENES FROM SCOMBEROMORUS NIPHONIUS

Full-length cDNA sequences of Wap65-1 and Wap65-2 were cloned first time from the Scomberomorus niphonius by homology cloning and RACE techniques. The Wap65-1 and Wap65-2 sequences are 1659bp and 1725bp, encoding 426 and 436 amino acids respectively. Sequences comparison and phylogenetic analysis showed that the homology of Wap65-1 and Wap65-2 is 60%, which located in different branches. Furthermore, the fish larva were induced by high temperature, the real-time quantitative PCR results showed that the expression of two different genes are up-regulated under the high temperature induced, and the expression of Wap65-2 gene show significant difference(P<0.05). On this basis, we succeeded getting the soluble expression of two proteins by contracting two kinds of Cold-Shock expression system, and found Wap65-2 had better strong effect on survival protection under high temperature stress. The study laid a theoretical foundation for breeding heat resistant varieties of S. niphonius.