Crops ›› 2016, Vol. 32 ›› Issue (5): 25-30.doi: 10.16035/j.issn.1001-7283.2016.05.005

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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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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

Table 1

Primers used for the study"

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

Fig.1

Electrophoresis of total RNA"

Fig.2

PCR products of IrHMGS gene cDNA"

Fig.3

The three-dimensional model of IrHMGS"

Fig.4

Multiple alignment of IrHMGS with other HMGS"

Fig.5

Phylogenetic tree analysis of protein encoded by HMGS genes"

Fig.6

Real-time PCR amplification curves （left） and melting curves （right） of IrHMGS and GAPDH gene in different tissues of I.rubescens"

Fig.7

Expression pattern of IrHMGS in different tissues of I.rubescens Different letters indicate significant difference at 0.05 level"

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