滇龙胆GrHMA基因的克隆和原核表达

Cloning and Prokaryotic Expression of GrHMA Gene in Gentiana rigescens

HMA蛋白(heavy metal transporting ATPase)是一种在植物中广泛存在的多功能蛋白。根据滇龙胆转录组GrHMA基因序列,设计特异性引物,通过RT-PCR扩增GrHMA基因序列,并进行TA克隆、测序及序列分析;构建原核表达载体pGEX-4T-1-GrHMA,转入E.coli Rosetta(DE3)中,并在37℃、1.0 mmol/L IPTG下成功诱导表达。序列分析表明,GrHMA基因是HMA超家族的成员;GrHMA氨基酸序列系统发育分析表明,GrHMA与TcHMA处于同一进化枝。SDS-PAGE结果表明所表达蛋白与预期蛋白大小一致。这些结果为GrHMA蛋白的进一步纯化及结构和功能的研究奠定基础。

HMA （heavy metal transporting ATPase） is a kind of ubiquitous and muhifunctional protein in plant. Specific primers were designed according to the GrHMA gene sequence of Gentiana rigescens transcriptome and the GrHMA gene sequence was amplified by RT-PCR from young leaves of G. rigescens. Also the TA cloning, sequencing and sequence analysis were performed. Prokaryotic expression vector pGEX-4T- 1-GrHMA was constructed and transformed into E. coli Rosetta （DE3）, and successfully expressed under 37 ℃ inducing by 1 mmol/L of IPTG. Sequence analysis showed that GrHMA gene belonged to the HMA super family. Results of phylogenic analysis with its amino acids showed that GrHMA was at the same evolutionary branch with TcHMA. The SDS-PAGE results displayed that the expressed protein was consistent with the anticipated size. These results may provide a foundation for further purification, structural and functional researches of GrHMA protein.