镍胁迫下安氏伪镖水蚤SSH文库的构建及铁蛋白cDNA的克隆与差异表达

为探讨镍胁迫下桡足类的分子响应机制,以安氏伪镖水蚤为研究对象,利用抑制性消减杂交(suppression subtractive hybridization,SSH)技术,构建了镍胁迫下安氏伪镖水蚤SSHcDNA文库,并随机挑取生长菌落140个克隆子,进行菌液PCR鉴定,计算文库重组率为98.6％,文库容量为1.12×106 cfu.将重组子测序,经BLAST一致性搜索比对分析发现,有一重组片段含有铁蛋白保守结构域,该片段大小为859 bp,连续编码170个氨基酸残基,Gen-Bank登录号为JK312601.半定量PCR证实,镍胁迫24 h后,安氏伪镖水蚤中铁蛋白表达显著上调.本文库的成功构建及铁蛋白cDNA片段的获得为进一步研究镍胁迫下桡足类的分子响应机制奠定了基础.

Construction of suppression subtractive hybridization(SSH) library of copepod Pseudodiaptomous annandalei and its ferritin cDNA cloning and differential expression under nickel stress

To study the molecular response mechanisms of copepod to nickel stress,a suppression subtractive hybridization(SSH) cDNA library of Pseudodiaptomous annandalei under nickel stress was constructed by using SSH technique,and a total of 140 clones were randomly picked from the growing colonies and identified by PCR.The recombinant rate of the library was 98.6%,and the volume of the library was 1.12×106 cfu.After the recombinant plasmids were sequenced,a partial cDNA fragment of ferritin was recognized based on BLAST searches in NCBI,with a size of 859 bp and continuously encoding 170 amino acid residues.The semi-quantitative PCR results showed that the ferritin cDNA under 24 h nickel stress was distinctly up-regulated.The successful construction of the SSH library and the obtaining of ferritin cDNA fragment would supply basis for the further study of the molecular response mechanisms of copepod to nickel stress.