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

发布时间：2018-04-20 21:30

本文选题：金钗石斛 + -羟基--甲基戊二酰辅酶A合酶　；参考：《中草药》2017年12期

【摘要】：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。
[Abstract]:Objective to clone the 3-hydroxy-3-methylglutaryl-CoA synthase (3-hydroxy-3-methylglutaryl-CoA synthase-HMGS) gene from Dendrobium nobile of Dendrobium nobile and analyze its bioinformatics and expression. Methods the full length of DnHMGS gene cDNA was obtained by reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA terminal of RT-PCRN, and the physicochemical properties and domain of the encoded protein were analyzed by bioinformatics. The amino acid multi-sequence alignment and phylogenetic tree construction were analyzed by DNASTARGA-MEGA software, and the expression patterns of gene tissues were detected by real-time quantitative PCR qRT-PCRR technique. Results the total length of DnHMGS gene was 1 816 bp(GenBank registration number KX789180, encoding a polypeptide composed of 474 amino acids with relative molecular weight of 52 458.47. The isoelectric point 5.98.DnHMGS protein had the typical domain and active center of plant HMGS enzyme, and was similar to pineapple and rice. The expression of DnHMGS gene in maize and other monocotyledonous plants was higher than that in the roots and stems of DnHMGS. The expression of DnHMGS transcripts was the highest in the leaves of Dendrobium nobile before inoculation with DnHMGS, which was more than 2 times of that in the roots and stems of Dendrobium nobile. But the expression of DnHMGS gene changed to stem and leaf root after inoculation. Conclusion the full-length HMGS gene was cloned from Dendrobium nobile for the first time. The molecular identification of the gene is intended to further reveal the role of the gene in the pathway of synthesis and metabolism of terpenoids and the effect of mycorrhizal fungi on alkaloid biosynthesis of Dendrobium nobile. The control mechanism has laid the foundation.
【作者单位】：中国医学科学院北京协和医学院药用植物研究所;
【基金】：国家自然科学基金资助项目(31170314,81473331)
【分类号】：S567.239

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