| 摘要: |
| 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cDNA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ClustW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qRT-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(P<0.05)。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。 |
| 关键词: 蓝点马鲛 Wap65 基因克隆 表达 功能研究 |
| DOI：10.11693/hyhz20150700206 |
| 分类号: |
| 基金项目:宁波市重大(重点)科技攻关计划项目,2012C10035号。 |
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| MOLECULAR CLONING AND FUNCTIONAL ANALYSIS OF TWO DIFFERENT Wap65 GENES FROM SCOMBEROMORUS NIPHONIUS |
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WANG Dan-Ni1, ZHENG Chun-Jing2, JING Ting-Jia1, LIU Jun1
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1.China Jiliang University, Hangzhou 310018, China;2.Ningbo Institute of Marine and Fishery, Ningbo 315010, China
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| Abstract: |
| 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. |
| Key words: Scomberomorus niphonius Wap65 gene cloning expression function research |
