滇龙胆3-羟基-3-甲基戊二酰辅酶A还原酶基因克隆、表达及分析

Gene Cloning,Prokaryotic Expression and Bioinformatics Analysis of 3-Hydroxy-3-methylglutaryl CoA Reductase(HMGR) from Gentiana rigescens

目的：从药用植物滇龙胆中克隆得到3-羟基-3-甲基戊二酰辅酶A还原酶（3-hydroxy-3-methylglutaryl CoA reductase,HMGR）基因的c DNA全长,构建原核表达载体使其在大肠埃希菌中表达,对其进行定量表达并进行生物信息学分析。方法：利用PCR扩增克隆得到滇龙胆HMGR基因c DNA全长。构建p EASY-E1-HMGR表达载体,IPTG诱导表达。利用生物信息学方法分析克隆到的c DNA序列并预测结构和功能。采用实时荧光定量聚合酶链式反应（Real-time PCR）分析滇龙胆根、茎、叶在4个不同发育时期的HMGR表达情况。结果：克隆出两组HMGR基因,其中一组HMGR c DNA全长1 747 bp,开放阅读框长1 697 bp,编码565个氨基酸,定名为GRHMGR-1;另一组HMGR c DNA全长1 731 bp,开放阅读框长1 572 bp,编码523个氨基酸,定名为GRHMGR-2。SDS-PAGE显示重组质粒成功表达目的蛋白。Blastp发现,GRHMGR-1,GRHMGR-2与同属植物黄龙胆、秦艽的亲缘关系最为相近。结构分析显示都含有2个HMG-CoA结合位点,2个NADPH结合位点,预测都定位于内质网上。HMGR在滇龙胆根、茎、叶这4个不同发育期均有表达,表达量最高的是叶。结论：首次从滇龙胆中克隆到可能编码HMGR酶的基因,可为深入探讨滇龙胆龙胆苦苷生物合成奠定基础。

Objective： To clone the full-length c DNA of 3-hydroxy-3-methylglutaryl CoA reductase（HMGR） gene from Gentiana rigescens,construct prokaryotic expression vector for expression in Escherichia coli,quantify the expressions and conduct bioinformatics analysis. Method： Polymerase chain reaction（PCR）technique was used for cloning the c DNA sequence of HMGR from G. rigescens. The p EASY-E1-HMGR expression vector was constructed to prokaryotic expression and the expression was induced by isopropyl β-D-thiogalactoside（IPTG）. The obtained c DNA sequences were analyzed and their structure and function were forecasted by bioinformatics method. The expression levels of HMGR gene of roots,stems and leaves in different growth stages were analyzed by using Real-time quantitative PCR（Real-time PCR） in G. rigescens. Result： Two groups of HMGR gene were cloned,one group of HMGR c DNA full-length was 1 747 bp,open reading frame（ORF） of 1697 bp,encoding 565 amino acids,named as GRHMGR-1; another group of HMGR c DNA full-length was 1 731 bp,ORF of 1 572 bp, encoding 523 amino acids, and named as GRHMGR-2. SDS-PAGE showed that the recombinant plasmid could successfully express the target proteins. Blastp found that GRHMGR-1 and GRHMGR-2 were closely related to the same genus plants G. lutea and G. macrophylla. The structure analysis results showed that both of them contained two HMG-CoA binding sites and two NADPH binding sites,implying that it may be localized on endoplasmic reticulum. RT-PCR showed that HMGR gene was expressed in roots,stems and leaves at four different growth stages,and the highest expression level was found in leaves. Conclusion： This was the first time to obtain cloned HMGR genes from G. rigescens. The results can provide a foundation for exploring the mechanism of gentiopicroside biosynthesis in G. rigescens.