滇龙胆1-脱氧-D-木酮糖-5-磷酸还原异构酶基因GrDXR的克隆、序列分析与原核表达

Cloning,sequence analysis,and prokaryotic expression of 1-deoxy-D-xylulose-5-phosphate reductoisomerase GrDXR gene in Gentiana rigescens

目的从滇龙胆Gentiana rigescens幼叶中克隆萜类合成关键酶1-脱氧-D-木酮糖-5-磷酸还原异构酶基因GrDXR,进行序列分析和原核表达。方法根据滇龙胆转录组中GrDXR基因序列,设计引物,通过RT-PCR扩增得到GrDXR开放阅读框(ORF)序列,并进行TA克隆、测序和序列分析;构建原核表达载体pGEX-4T-1-GrDXR,转入大肠杆菌Rosetta(DE3)中,在IPTG诱导下进行表达。结果 GrDXR ORF全长1 425 bp,编码474个氨基酸。序列分析表明,GrDXR基因是DXR家族成员;蛋白质序列系统发育分析表明,GrDXR与萝芙木RvDXR、橡胶树HbDXR和长春花CrDXR亲缘关系较近。构建pGEX-4T-1-GrDXR重组质粒,获得稳定的原核表达体系。SDS-PAGE结果表明所表达蛋白与预期蛋白大小一致。结论克隆了GrDXR基因,建立其稳定的原核表达体系,为进一步纯化GrDXR蛋白,研究其结构和功能奠定基础。

Objective To clone the 1-deoxy-D-xylulose 5-phosphate reductoisomemse gene GrDXR which is the key enzyme involving in the terpenoid biosynthesis from young leaves of Gentiana rigescens, and to pelform its sequence analysis and prokaryotic expression. Methods According to the GrDXR gene sequence of G. rigescens tmaascriptome, a pair of primers were designed, and the ORF of cDNA sequence was obtained by RT-PCR. Then TA cloning, sequencing, and sequence analysis were performed. Prokaryotic expression vector pGEX-4T- 1-GrDXR was constructed and transformed into Escherichia coli Rosetta （DE3） for expression underthe induction of IPTG Results The ORF of GrDXR had a length of 1 425 bp coding for 474 amino acids. Sequence analysis showed that GrDXR was the member of DXR family. Results of phylogenic analysis showed that GrDXR was close to RvDXR, HbDXR, and CrDXR. The pGEX-4T-1-GrDXR recombinant plasmid was constructed and the stable prokaryotic expression system was obtained. The SDS-PAGE results displayed that the expressed proteins were consistent with the anticipated size. Conclusion The GrDXR gene is successfully cloned, and the stable prokaryotic expression system is established. This study will provide a foundation for further purification, structural and functional research of GrDXR protein.