刺参耐寒基因筛选cDNA文库的构建与分析  

Construction and analysis of subtracted cDNA library of sea cucumber Apostichopus japonicus under low temperature stress

作　　者：李成泽[1] 王晓燕[1] 丁君[1] 常亚青[1] 

机构地区：[1]大连海洋大学农业部北方海水增养殖重点实验室,辽宁大连116023

出　　处：《大连海洋大学学报》2013年第6期522-527,共6页Journal of Dalian Ocean University

基　　金：国家自然科学基金资助项目(31072230);国家海洋局海洋公益项目(201105016-3);辽宁省重大科技专项(2011203003);国家"863"计划重大项目(2012AA10A412)

摘　　要：利用抑制性消减杂交(Suppression subtractive hybridization,SSH)技术,对刺参Apostichopus japonicus耐低温性状相关的基因进行研究。通过对刺参进行低温骤降试验,利用SSH技术构建刺参低温骤降双向消减cDNA文库,共获得6 800个阳性克隆,从文库中随机挑取1 000个阳性克隆测序,共获得680条有效EST序列。利用Blastx软件将测序结果与GenBank数据库进行比对,结果显示:72个克隆与已知基因高度同源,其中有6个基因与应激和免疫反应相关,存在于正向文库的有4个,分别为铁蛋白、溶菌酶、类似紫海胆冷诱导β-RNA结合蛋白和huadl3021索转铁蛋白,存在于反向文库的有2个,分别为热休克同源蛋白70和热休克蛋白gp96;有3个基因与营养代谢和运输相关,且都存在于反向文库,分别为主要卵黄蛋白1、主要卵黄蛋白2和胰淀粉酶。研究表明,刺参低温骤降文库的成功构建对阐述其耐低温分子机制有非常重要的意义,为刺参育种和生物学研究提供了新的资料。Suppression subtractive hybridization( SSH) was used to analyze the differences in expression of genes related to low temperature resistance in sea cucumber Apostichopus japonicus,induced by low temperature,in order to understand the molecular response. A total of 6 800 clones was obtained in the forward and reverse library of the sea cucumber exposed to the sharp decrease in temperature. Among the clones,1 000 clones was selected and analyzed,and 680 distinct EST sequences were obtained,with 72 clones sharing similarity to the sequences in Genbank databases by Blaster. There were 6 genes related to the stress and immune response,including 4 of ferritin, lysozyme,similar to cold inducible RNA-binding protein beta,and isolate huadl3021 melanotransferrin mRNA in the forward library and the other 2 of heat shock cognate 70,heat shock protein gp96 in the reverse library. There were 3 genes related to nutrition in the reverse library,including major yolk protein 1,major yolk protein 2 and pancreatic amylase. The findings suggest that cold stress have significant effects on gene expression,and provide the basis for further study of molecular regulation mechanisms underlying the response to cold in sea cucumber.

关 键 词：刺参 耐寒基因 抑制消减杂交 

分 类 号：S917.4[农业科学—水产科学]