棉花GhEPSPS基因的原核表达载体的构建及诱导表达

目的在于构建棉花GhEPSPS基因的原核表达载体,并在宿主大肠杆菌中成功诱导表达。利用RT-PCR技术从陆地棉苏棉18中克隆获得GhEPSPS基因的开放阅读框序列,连接到p EASY-Blunt平端载体,构建获得重组载体p EASY-Blunt-GhEPSPS;以该重组载体为模板,PCR获得两端含有Eco RⅠ和XhoⅠ酶切位点的GhEPSPS基因CDS序列,通过这2个酶切位点插入到p ET32a载体,构建获得原核表达重组载体p ET32a-GhEPSPS,转化到宿主菌株BL21(DE3)中,经IPTG诱导后,SDS-PAGE检测目的基因的表达情况。研究结果表明,成功克隆获得了棉花GhEPSPS基因的CDS序列,并成功构建了该基因的原核表达载体,目的基因能够在宿主菌中被诱导大量表达。该研究结果将为深入研究棉花EPSPS酶的结构、功能以及酶学特性提供重要的研究基础。

Construction of a Prokaryotic Expression Vector and Induced Expression of Cotton GhEPSPS gene

The purpose of this paper was to construct a prokaryotic expression vector of cotton GhEPSPS gene, which could be induced expression in host bacteria E.coli. RT-PCR was used to obtain GhEPSPS gene open reading frame sequence from upland cotton cultivar Sumian 18. This sequence was cloned to pEASY-Blunt vector and got a pEASY-Blunt-GhEPSPS recombinant vector. A CDS sequence of GhEPSPS gene which contain EcoRⅠand XhoⅠ restriction enzyme sites in its 5' and 3' terminal was achieved by PCR in the recombinant plasmid as template. The CDS sequence through this two restriction enzyme sites digested and inserted to pET32a vector, a prokaryotic expression vector of pET32a-GhEPSPS was constructed. This recombinant expression vector was transformed to host bacteria E.coli BL21(DE3) and SDS-PAGE assay was used to test the expression of the target gene after IPTG induction. The research achievement showed that the CDS sequence of GhEPSPS gene was successfully cloned and the prokaryotic expression vector was completed constructed. SDS-PAGE result indicated that our aimed gene was abundantly induced expression by IPTG in host bacteria. The study results would provide an important foundation on the structure, function and enzyme activity trait research of cotton EPSPS.