虾夷扇贝热休克蛋白60基因克隆及表达特性研究

doi:10.13560/j.cnki.biotech.bull.1985.2015.10.025

生物技术通报 ›› 2015, Vol. 31 ›› Issue (10): 157-164.doi: 10.13560/j.cnki.biotech.bull.1985.2015.10.025

虾夷扇贝热休克蛋白60基因克隆及表达特性研究

孔繁东¹,史井运¹,鲍相渤²,高祥刚²,赫崇波²,刘卫东²

1.辽宁省大连工业大学食品学院, 大连 116034;
2.辽宁省海洋水产科学研究院辽宁省海洋水产分子生物学重点实验室, 大连 116023

收稿日期:2015-01-14 出版日期:2015-10-28 发布日期:2015-10-28
作者简介:孔繁东, 男, 教授, 研究方向：蛋白质资源开发利用; E-mail：kfd456@163.com
基金资助:
辽宁省农业攻关计划项目（2014203005）, 水产种质资源平台项目（2012DKA30470_012）

Gene Cloning and Expression Characterization of Gene for a Heat Shock Protein 60 from Japanese Scallop Mizuhopecten yessoensis

Kong Fandong¹, Shi Jingyun¹, Bao Xiangbo², Gao Xianggang², He Chongbo², Liu Weidong²

1. School of Food Science and Technology, Liaoning Dalian Polytechnic University, Dalian 116034; 2. Liaoning Key Lab of Marine Fishery Molecular Biology, Liaoning Ocean and Fisheries Science Research Institute, Dalian 116023

Received:2015-01-14 Published:2015-10-28 Online:2015-10-28

摘要/Abstract

摘要： 旨在探讨虾夷扇贝高温应激的分子机理并为解决该种夏季死亡问题打下基础。基于HSP60的转录组序列设计特异引物, 采用RACE技术成功克隆获得虾夷扇贝热休克蛋白60（MyHSP60）cDNA全序列。其cDNA序列全长3 368 bp, 包括68 bp的5' UTR, 1 569 bp的3' UTR和1 731 bp的开放阅读框, 共编码576个氨基酸。利用基因步移技术扩增MyHSP60基因启动子区域序列并测序, 获得MyHSP60基因启动子区域序列1 236 bp, 该区域包含2个TATA盒, 4个CCAAT盒, 3个热激元件。基于氨基酸序列构建的系统进化树与传统物种进化规律基本吻合。通过实时PCR检测发现HSP60在虾夷扇贝各组织中均有表达, 升温后肾脏中该基因的转录量升高极显著, 而肝胰腺、外套膜、血淋巴和闭壳肌中的转录量升高显著（P<0.05）, 说明该基因在热应激过程中发挥一定作用。

关键词: 虾夷扇贝, 热休克蛋白60, 克隆, 启动子序列, 表达特性

Abstract: In order to investigate the molecular mechanism of high temperature stress in Mizuhopecten yessoensis, as well to find effective ways to solve the problem of summer mortality. The HSP60（heat shock protein 60）as a member of the highly conserved heat shock protein family, not only can help other protein fold correctly and restore the natural conformation in the condition of stress, but also can be used as a danger signal to the natural immune system. Specific primer was designed based on transcriptome sequence of HSP60, and the full sequence of cDNA of heat shock protein 60（MyHSP60）from Japanese scallop Mizuhopecten yessoensis was cloned by RACE. The cDNA sequence was 3 368 bp in length, including 68 bp 5' UTR, 1 569 bp 3' UTR and 1 731 bp open reading frame encoding 576 amino acids. Using gene walking the promoter region of MyHSP60 gene sequences was amplified and sequenced, the sequences of promoter region of MyHSP60 gene was 1 236 bp, which contained 2 TATA boxes, 4 CCAAT boxes, and 3 heat shock elements. The phylogenetic tree based on amino acid sequence matched with the traditional species trees based on basic anastomosis. The Real-time PCR result showed that HSP60 in all tissues of M. yessoensis was expressed, the expression in the tissues after heating increased extremely significantly in kidney, and significantly in hepatopancreas, mantle, hemolymph and muscle（P<0.05）, indicating that the gene plays a certain role in the process of heat stress.

Key words: Mizuhopecten yessoensis, heat shock protein 60, cloning, promoter sequence, expression characterization

孔繁东,史井运,鲍相渤,高祥刚,赫崇波,刘卫东. 虾夷扇贝热休克蛋白60基因克隆及表达特性研究[J]. 生物技术通报, 2015, 31(10): 157-164.

Kong Fandong, Shi Jingyun, Bao Xiangbo, Gao Xianggang, He Chongbo, Liu Weidong. Gene Cloning and Expression Characterization of Gene for a Heat Shock Protein 60 from Japanese Scallop Mizuhopecten yessoensis[J]. Biotechnology Bulletin, 2015, 31(10): 157-164.

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虾夷扇贝热休克蛋白60基因克隆及表达特性研究

Gene Cloning and Expression Characterization of Gene for a Heat Shock Protein 60 from Japanese Scallop Mizuhopecten yessoensis

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