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作物杂志,2016, 第5期: 25–30 doi: 10.16035/j.issn.1001-7283.2016.05.005

• 遗传育种·种质资源·生物技术 • 上一篇    下一篇

冬凌草HMGS基因的克隆与表达分析

朱畇昊1,2,爼梦航1,苏秀红1,2,董诚明1,2,陈随清1,2   

  1. 1 河南中医学院药学院,450046,河南郑州
    2 呼吸疾病诊疗与新药研发河南省协同创新中心,450046,河南郑州
  • 收稿日期:2016-06-30 修回日期:2016-08-23 出版日期:2016-10-15 发布日期:2018-08-26
  • 通讯作者: 董诚明
  • 作者简介:朱畇昊,讲师,研究方向为药用植物分子生物学
  • 基金资助:
    国家自然科学基金(81173486);河南中医学院创新人才项目(2011XCXRC02);河南省高等学校青年骨干教师计划项目(2011GGJS-089)

Molecular Cloning and Expression Analysis of a Gene Encoding 3-Hydroxy-3-Methylglutaryl-CoA Synthase from Isodon rubescen

Zhu Yunhao1,2,Zu Menghang1,Su Xiuhong1,2,Dong Chengming1,2,Chen Suiqing1,2   

  1. 1 School of Pharmacy,Henan University of Traditional Chinese Medicine,Zhengzhou 450046,Henan,China
    2 Collaborative Innovation Center for Respiratory Disease Diagnosis and Treatment & Chinese Medicine Development of Henan Province,Henan University of Traditional Chinese Medicine,Zhengzhou 450046,Henan,China;
  • Received:2016-06-30 Revised:2016-08-23 Online:2016-10-15 Published:2018-08-26
  • Contact: Chengming Dong

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摘要：

羟甲基戊二酰辅酶A合酶（3-Hydroxy-3-methylglutaryl-coenzyme A synthase,HMGS）是甲羟戊酸途径（MVA）中的第一个催化酶。根据冬凌草转录组数据库中HMGS基因序列设计特异引物,采用RT-PCR技术克隆冬凌草IrHMGS基因cDNA全长,并对其序列进行生物信息学分析,通过荧光定量PCR的方法分析其组织表达特性。IrHMGS基因cDNA全长1 382bp,编码460个氨基酸,序列分析结果表明该基因编码的氨基酸序列含有羟甲基戊二酰辅酶A合成酶N末端、C末端等保守结构域。荧光定量PCR表明,IrHMGS在冬凌草组培苗叶和根中的表达量显著高于在组培苗花、茎和愈伤组织中的表达量。本研究为后续深入研究IrHMGS在冬凌草二萜类物质合成途径中的功能奠定了基础。

关键词: 冬凌草, HMGS基因, 生物信息学分析, 荧光定量PCR

Abstract:

3-hydroxy-3-methylglutaryl coenzyme A synthase (HMGS) catalyzes the ?rst step in the mevalonate biosynthesis pathway in plants. The specific primers were designed according to the transcript sequence of HMGS from the Isodon rubescens transcriptome database. RT-PCR trail was used to clone the full-length cDNA. IrHMGS expression profiles in different tissues were analyzed by quantitative real-time PCR. The full-length cDNA of IrHMGS was found to contain an open reading frame of 1 382bp encoding a polypeptide of 460 amino acid residues. Multiple sequence alignment of IrHGMS with some homologous HMGSs from other plants revealed the IrHMGS had several high conservation domains, such as Hydroxymethylglutaryl-coenzyme A synthase N-terminal, C-terminal domain. Expression pro?le analysis revealed that the IrHGMS expression level in leaves and roots was higher than that in flowers, stems and calluses of I. rubescens. The characterization and expression provides useful information for further studying this gene and its function in the diterpene biosynthetic pathway in I. rubescens.

Key words: Isodon rubescens, 3-hydroxy-3-methylglutaryl coenzyme A synthase, Bioinformatic analysis, qPCR

表1

所用引物序列及用途"

序号
No.		 引物名称
Primer name		 序列(5'-3')
Sequence(5'-3')		 用途
Application
1 qHMGS-F GGGCAGAGGGTCATACTGTT 荧光定量
（qPCR）
2 qHMGS-R CAGAAACGATGTTGGACAGG
3 GAPDH-F AAACGCCTAACTTCGCATCT 荧光定量内参(qPCR reference genes)
4 GAPDH-R CCCGACTGTCCCTGTAATCA

图1

总RNA电泳"

图2

IrHMGS基因序列扩增产物"

图3

IrHMGS三级结构预测"

图4

IrHMGS蛋白的多序列比对"

图5

HMGS基因的系统进化树"

图6

冬凌草不同组织中IrHMGS和GAPDH基因的扩增曲线（左）和熔解曲线（右）"

图7

IrHMGS在冬凌草不同组织中的表达 不同字母表示在 0.05水平上差异显著"

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