草木樨状黄芪变异系中甲硫氨酸代谢相关cDNA的分子克隆及其在大肠杆菌中的表达

Molecular cloning of methionine-metaboly correlated cDNA in Astragalus melilotoides Pall. variant line and its expression in E.coli

利用mRNA差异显示技术 differentialdisplay 从草木樨状黄芪耐甲硫氨酸变异系中分离到一差异cDNA片段,命名为tm03 360bp .mRNA杂交结果显示,该片段只在变异苗中表达,初步确定其与甲硫氨酸代谢相关.测序后在Genebank搜寻,未发现有同源序列.进一步分析表明,该cDNA克隆可编码完整的蛋白.在该基因片段两端分别合成含Hind 和BamH 酶切位点的引物,用PCR扩增后亚克隆到pRSETA表达载体,转化宿主菌JM109 DE3 ,经IPTG诱导表达了N′端含6个组氨酸的蛋白质,分子量约为14kD,为进一步研究该蛋白的结构和生物学功能奠定基础.

Differential cDNA fragment which was named tm03 (360 bp) was isolated from Astragalus melilotoides Pall. methionine-tolerant variant line by mRNA differential display analysis.After detected by mRNA dot hybridization,tm03 expressed only in the variant line,and tm03 was primarily confirmed as the methionine-metaboly correlated cDNA.tm03 was cloned and sequenced.After searching in GenBank,there were no similar sequences reported.Further research observed that the cDNA can code a complete protein.To construct high level expression plasmid,tm03 was ligated with the expression vector pRSET A,then transformed into JM109 (DE3) E.coli strain,and induced with IPTG.SDS-PAGE confirmed that protein of 6His-recombinant tm03 was expressed efficiently in inclusion bodies in E.coli,and the molecular weight was about 14 kD.It would be helpful to further study the structure of tm03 protein and its biological function.