金钗石斛中3-羟基-3-甲基戊二酰辅酶A合酶基因的克隆及特征分析

Molecular characterization of 3-hydroxy-3-methylglutaryl-CoA synthase gene from Dendrobium nobile

目的克隆金钗石斛Dendrobium nobile中3-羟基-3-甲基戊二酰辅酶A合酶(3-hydroxy-3-methylglutaryl-CoA synthase,HMGS)基因DnHMGS,并进行生物信息学和表达分析。方法采用反转录聚合酶链式反应(RT-PCR)、cDNA末端快速扩增(RACE)技术获得DnHMGS基因cDNA全长;生物信息学分析编码蛋白的理化特性、结构域等特征;用DNASTAR、MEGA软件分别进行氨基酸多序列比对和进化树构建分析;借助实时荧光定量PCR(qRT-PCR)技术检测基因组织表达模式。结果 DnHMGS基因全长为1 816 bp(GenBank注册号KX789180),编码一条由474个氨基酸组成的多肽,相对分子质量为52 458.47,等电点5.98。DnHMGS蛋白具有植物HMGS酶的典型结构域和活性中心,与凤梨、稻、玉米等单子叶植物亲缘关系较近。DnHMGS基因具有组织表达特异性,接菌前,DnHMGS转录本在金钗石斛叶中的表达量最高,为根、茎中的2倍以上。但接菌后DnHMGS基因表达情况转变为茎>叶>根。结论首次从金钗石斛中克隆得到HMGS基因的全长cDNA,该基因的分子鉴定为进一步揭示该基因在金钗石斛萜类物质合成代谢途径中的作用及菌根真菌影响石斛碱生物合成的调控机制奠定了基础。

Objective To clone and characterize a 3-hydroxy-3-methylglutaryl-CoA synthase（HMGS） gene DnHMGS in Dendrobium nobile. Methods RT-PCR and RACE technologies were used for gene cloning. Characteristics including the physicochemical properties and conserved domain of the deduced DnHMGS protein were determined by a series of bioinformatics tools. The analyses of multiple alignment and phylogenetic tree were performed by DNASTAR and MEGA softwares, respectively. qRT-PCR was employed to examine the tissue specific expression pattern of DnHMGS. Results The full length cDNA of DnHMGS was 1 816 bp（GenBank accession No. KX789180） and encoded a 474-amino-acid protein with a molecular weight of 52 458.47 and an isoelectric point（p I） of 5.98. The deduced DnHMGS protein, like other HMGS proteins, constituted typical domain and active site. Multiple sequence alignment and phylogenetic analyses demonstrated that DnHMGS was closely related to Ananas comosus, rice, and maize monocots. Proteins analysis revealed that DnHMGS was expressed in the three included organs. The transcripts were the most abundant in the leaves with more than twice that in the roots and stems. However, the expression of DnHMGS changed to stems leaves roots when D. nobile infected by Mycena sp. Conclusion The full length cDNA of DnHMGS is indentified from D. nobile for the first time. Molecular characterization of DnHMGS will be useful for further functional elucidation of the gene involving in isoprenoid biosynthesis pathway in D. nobile, and help us understand the molecular mechanism of Mycena sp. which better encourages the biosynthesis of dendrobine.