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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 3-hydroxy-3-methylglutaryl-CoA synthase (HMGS) gene DnHMGS from Dendrobium nobile and perform bioinformatics and expression analysis. Methods The full-length cDNA of DnHMGS was obtained by reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). The bioinformatics analysis was used to analyze the physicochemical properties and domains of the protein. DNASTAR, MEGA software was used to perform multiple amino acid sequence alignment and phylogenetic tree analysis respectively. Genomic expression patterns were detected by real-time quantitative PCR (qRT-PCR). Results The full-length DnHMGS gene was 1 816 bp (GenBank accession number KX789180) encoding a polypeptide of 474 amino acids with a relative molecular mass of 52 458.47 and an isoelectric point of 5.98. DnHMGS protein has the typical domain and active center of plant HMGS enzyme, and is closely related to monocotyledonous plants such as pineapple, rice and corn. The DnHMGS gene has the specificity of tissue expression. Before inoculation, the expression of DnHMGS transcripts in Dendrobium nobile Lindl was the highest, which was more than twice of that in roots and stems. However, DnHMGS gene expression changed into stem> leaf> root after inoculation. Conclusions The full-length cDNA of HMGS gene was cloned from Dendrobium nobile for the first time. The molecular identification of this gene is further revealing the role of this gene in the biosynthesis pathway of dendrobium nobile, and the regulation of mycorrhizal fungi on the biosynthesis of dendrobium Mechanism laid the foundation.