牙鲆SRF基因的克隆及真核表达载体的构建  

Cloning of SRF from Paralichthys olivaceus and eukaryotic expression vector construction

作　　者：苏艳芳[1] 付元帅[1] 施志仪[1] 张红梅[1] 高丽娜[1] 

机构地区：[1]上海海洋大学,农业部淡水水产种质资源重点实验室,上海201306

出　　处：《海洋渔业》2015年第4期331-340,共10页Marine Fisheries

基　　金：国家自然科学基金项目(31172392);上海海洋大学博士科研启动基金(A-0209-13-0105387)

摘　　要：采用RACE-PCR技术,从牙鲆(Paralichthys olivaceus)组织中克隆了血清应答因子(SRF)基因全长序列,该序列全长2 477 bp,开放阅读框1 503 bp,编码500个氨基酸。通过氨基酸同源序列比对,牙鲆SRF与其它物种的同源性较高,在氨基酸序列的N端具有NLS结构域和高度保守的MADS结构域。用PCR方法扩增SRF基因的编码区片段,克隆到p EGFP-N1载体中,构建真核表达载体p EGFP-N1-SRF,将重组质粒转染牙鲆胚胎细胞,在荧光显微镜下观察转染细胞有绿色荧光蛋白表达。荧光定量PCR和Western blot实验进一步证实,SRF在转染细胞中高表达。说明真核表达载体p EGFP-N1-SRF构建成功,为进一步研究SRF在牙鲆变态发育中的作用奠定了实验基础。Serum response factor （SRF） is a MADS-box transcription factor that regulates the expression of genes involved in development, metabolism, cell proliferation and differentiation. In the present study, the full-length cDNA of SRF was cloned from Paralichthys olivaceus by RACE-PCR, the cDNA sequence was 2 477 bp, including the coding region of 1 503 bp, a 574 bp 5＇untranslated region （UTR） and a 400bp 3＇ UTR. The deduced 501 amino acid sequence of the SRF protein contained MADS-box and NLS domain at the N-terminus, similar to other organisms, and it is also highly phylogenetically conserved, indicating that SRF may play an important role in the development of P. olivaceus. To further study the function of SRF in flounder, the coding region of SRF gene obtained by PCR was inserted into eukaryotic expression vector of pEGFP-N1, and the recombinant vector of pEGFP-N1-SRF was transfected into flounder embryonic cell （FEC）. The transfected cells expressed green fluorescent protein observed under fluorescence microscope, real-time quantitative PCR and western blot were futher confirmed that the vector pEGFP-N1-SRF can up- regulate expression of SRF. The eukaryotic expression vector of pEGFP-N1-SRF has been constructed successfully, which laid floundation for the analysis on the function of SRF gene in the development of P.olivaceus.

关 键 词：SRF 牙鲆 克隆 真核表达 

分 类 号：Q782[生物学—分子生物学]