金钗石斛HMGR基因克隆及菌根真菌诱导表达分析

李清,李标,郭顺星

中国药学杂志 ›› 2017, Vol. 52 ›› Issue (22) : 1976-1982.

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中国药学杂志 ›› 2017, Vol. 52 ›› Issue (22) : 1976-1982. DOI: 10.11669/cpj.2017.22.004
论著

金钗石斛HMGR基因克隆及菌根真菌诱导表达分析

  • 李清,李标*,郭顺星*
作者信息 +

Cloning and Expression Analysis of HMGR Gene in Dendrobium nobile in Response to Mycorrhizal Fungal Inoculation

  • LI Qing, LI Biao*, GUO Shun-xing*
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文章历史 +

摘要

目的 克隆金钗石斛(Dendrobium nobile Lindl.)3-羟基-3-甲基戊二酸单酰辅酶A还原酶(3-hydroxy-3-methylglutaryl-CoA reductase,HMGR)基因,并进行生物信息学及接菌前后差异表达分析。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DnHMGR2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;借助实时定量PCR技术检测基因接菌前后表达模式。结果 从金钗石斛中分离得到一个HMGR基因,命名为DnHMGR2 (GenBank注册号KX825920)。DnHMGR2基因cDNA全长2 095 bp,编码一条由562个氨基酸组成的多肽,相对分子质量为59.68×103,等电点6.18。DnHMGR2蛋白具有植物HMGR酶的典型结构域和结合基序。DnHMGR2与多种植物HMGR基因高度同源,与铁皮石斛的亲缘关系最近。DnHMGR2基因具有组织表达特异性,在金钗石斛叶、茎中的表达量较高,为根中的2.59和1.60倍;但接菌后各器官的表达量表现为茎>叶>根。结论 首次克隆得到金钗石斛中HMGR基因的全长cDNA,为进一步解析该基因在金钗石斛萜类物质合成代谢途径中的分子调控作用,及菌根真菌对石斛碱生物合成调控机制奠定了基础。

Abstract

OBJECTIVE To clone and characterize a 3-hydroxy-3-methylglutaryl-CoA reductase(HMGR) gene DnHMGR2 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 DnHMGR2 protein were determined using a series of bioinformatics tools. The analyses of multiple alignment and phylogenetic tree were performed using DNASTAR and MEGA softwares, respectively. qRT-PCR was employed to examine the specific expression pattern of DnHMGR2 before and after inoculation. RESULTS The full length cDNA of DnHMGR2 was 2 095 bp (GenBank accession No. KX825920) and encoded a 562-amino-acid protein with a molecular weight of 59.68×103 and an isoelectric point (pI) of 6.18. The deduced DnHMGR2 protein, like other HMGR proteins, constituted typical domain and active site. Multiple sequence alignment and phylogenetic analyses demonstrated that DnHMGR2 had highly similar identity to a number of HMGR genes from various plants and was closely related to Dendrobium candidum. Real time quantitative PCR (qRT-PCR) analysis revealed that DnHMGR2 was expressed in the three included organs. The transcripts were abundant in the leaves and stems with more than 2.59 and 1.60 fold over that in the roots, respectively. However, the expression levels of DnHMGR2 changed to the sequence of stems > leaves > roots when D. nobile was infected by Mycena sp.. CONCLUSION The full length cDNA of DnHMGR2 from D. nobile is indentified. Molecular characterization of DnHMGR2 will be useful for further functional elucidation of this gene involving in isoprenoid biosynthesis pathway in D. nobile, and help us understand the molecular mechanism of Mycena sp., which encourages the biosynthesis of dendrobine.

关键词

金钗石斛 / 3-羟基-3-甲基戊二酸单酰辅酶A还原酶 / 基因克隆 / 实时荧光定量PCR

Key words

Dendrobium nobile Lindl. / 3-hydroxy-3-methylglutaryl-CoA reductase / gene cloning / real time quantitative PCR

引用本文

导出引用
李清,李标,郭顺星. 金钗石斛HMGR基因克隆及菌根真菌诱导表达分析[J]. 中国药学杂志, 2017, 52(22): 1976-1982 https://doi.org/10.11669/cpj.2017.22.004
LI Qing, LI Biao, GUO Shun-xing. Cloning and Expression Analysis of HMGR Gene in Dendrobium nobile in Response to Mycorrhizal Fungal Inoculation[J]. Chinese Pharmaceutical Journal, 2017, 52(22): 1976-1982 https://doi.org/10.11669/cpj.2017.22.004
中图分类号: R284   

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国家自然科学基金资助项目(31170314,81473331)
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