滇龙胆8-羟香叶醇氧化还原酶基因的克隆与表达分析

Cloning and expression analysis of 8-hydroxygeraniol oxidoreductase gene in Gentiana rigescens

关键词:  滇龙胆, 8-羟香叶醇氧化还原酶, 克隆, 生物信息学, 基因表达

Key words:  Gentiana rigescens, 8-hydroxygeraniol oxidoreductase, cloning, bioinformatics, gene expression

摘要:

滇龙胆主要药效成分为龙胆苦苷,而8-羟香叶醇氧化还原酶基因Gr8HGO是龙胆苦苷生物合成途径的结构基因。为了研究Gr8HGO基因的功能,该文克隆了滇龙胆Gr8HGO基因,并进行表达分析。结果表明:(1)共克隆到5个Gr8HGO基因,其GenBank登录号分别为KP722029.1(Gr8HGO-1)、KP722030.1(Gr8HGO-2)、KP722031.1(Gr8HGO-3)、KP722032.1(Gr8HGO-4)、KP723852.1(Gr8HGO-5)。(2)Gr8HGO-1基因全长1 062 bp,编码353个氨基酸,其他4个基因全长1 131 bp,编码376个氨基酸; 理化性质分析结果表明5个蛋白单体相对分子质量约40 kD,理论等电点在5.47～5.95之间,均为疏水稳定蛋白。(3)信号序列分析结果表明5个蛋白均不含信号肽、跨膜螺旋和叶绿体转运肽; 亚细胞定位分析结果表明5个蛋白可能定位于细胞质; 结构域预测结果表明除Gr8HGO-1蛋白仅包含乙醇脱氢酶N端结构域(IPR013154)和C端结构域(IPR013149)外,其他4个蛋白还包含聚酮合酶、烯酰还原酶结构域(IPR020843)。(4)系统发育分析结果表明这些Gr8HGO蛋白与长春花Cr8HGO蛋白亲缘关系最近。(5)qPCR结果表明Gr8HGO基因主要在叶中表达,在根和茎中表达量很低。该研究为后续龙胆苦苷生物合成途径的解析奠定基础。

Abstract:

Gentiopicroside is the main active ingredient in Gentiana rigescens, while 8-hydroxygeraniol oxidoreductase gene Gr8HGO is a structural gene involved in gentiopicroside biosynthesis. In order to study the function of Gr8HGO gene, the Gr8HGO gene in G. rigescens was cloned and its expression analysis was conducted in this study. The results were as follows:(1)Five Gr8HGO genes were cloned and their GenBANK accession numbers were KP722029.1(Gr8HGO-1), KP722030.1(Gr8HGO-2), KP722031.1(Gr8HGO-3), KP722032.1(Gr8HGO-4)and KP723852.1(Gr8HGO-5), separately.(2)The length of Gr8HGO-1 gene was 1 062 bp encoding 353 amino acids, while the other four genes were 1 131 bp encoding 376 amino acids; The results from physicochemical analysis showed that the relative molecular weight of these five Gr8HGO proteins were approximately 40 kD, and their theoretical pI ranged from 5.47 to 5.95, which were all hydrophobic stable proteins.(3)Signal sequence analysis showed that five proteins did not contain signal peptides, transmembrane helixes and chloroplast transit peptides; Subcellular localization analysis indicated that these five proteins might be localized in cytoplasm; Domain prediction results showed that beside the Gr8HGO-1 contained only alcohol dehydrogenase N-terminal(IPR013154)and C-terminal(IPR013149)conserved domains, the other four also contained polyketide synthase, enoylreductase domain(IPR020843).(4)Phylogenic analysis showed that these five Gr8HGO proteins had the closest relationship with Cr8HGO in Catharanthus roseus.(5)The results of qPCR suggested that Gr8HGO gene was mainly expressed in leaves, but very low in roots and stems. This study will lay a foundation for further analysis of the biosynthesis pathway of gentiopicroside.