Acta Agriculturae Boreali-Sinica ›› 2022, Vol. 37 ›› Issue (2): 49-55. doi: 10.7668/hbnxb.20192609

Special Issue: Biotechnology

• Crop Genetics & Breeding·Germplasm Resources·Biotechnology • Previous Articles     Next Articles

Cloning and Expression Analysis of 5-Phosphomevalonate Kinase PfPMK Gene in Perilla frutescens L.

LI Hui1, WEN Chunxiu2, LIU Lingdi2, WEN Saiqun2, TANG Yinghong1, JIANG Tao2   

  1. 1.Department of Life and Environmental Sciences,Hunan University of Arts and Science,Changde 415000,China
    2.Institute of Cash Crops,Hebei Academy of Agriculture and Forestry Sciences,Shijiazhuang 050051,China
  • Received:2021-12-16 Published:2022-04-28

紫苏甲羟戊酸-5-磷酸激酶基因PfPMK的克隆与表达分析

李辉1, 温春秀2, 刘灵娣2, 温赛群2, 唐映红1, 姜涛2   

  1. 1.湖南文理学院 生命与环境科学学院,湖南 常德 415000
    2.河北省农林科学院 经济作物研究所,河北 石家庄 050051
  • 作者简介:
    作者简介:李 辉(1980—),男,河北定州人,副教授,博士,主要从事作物遗传育种研究。
  • 基金资助:
    省部共建秦药特色资源研究开发国家重点实验室(培育)开放课题(QY202102)

Abstract:

In order to investigate the function of 5-phosphomevalonate kinase PMK gene in biosynthesis pathway of terpenoids in Perilla frutescens L.,we analyzed the transcriptome data of Perilla frutescens and mined the reference sequence of PMK.The PMK gene was cloned from Perilla frutescens by gene cloning technique,and was analysed using bioinformatics and Real-time PCR(qRT-PCR).The results showed that the ORF of PfPMK gene was 1 524 bp,encoding 507 amino acids.Bioinformatics analysis showed that the molecular weight of PfPMK was 54.73 ku and the isoelectric point was 5.20,which was a hydrophilic protein.The amino acids of PfPMK were higher homologous with SmPMK,SsPMK,SbPMK and PvPMK,which indicated PfPMK protein was highly conserved in the evolutionary process.The phylogenetic analysis showed that PfPMK was closely related to PMK of Salvia miltiorrhiza and Salvia splendens.WoLF-PSORT predicted that PfPMK protein might be located in the plasma membrane or endoplasmic reticulum.Real-time fluorescence quantitative PCR results showed that PfPMK gene was expressed in roots,stems and leaves of Perilla frutescens,and the expression level in roots was higher than that in leaves and stems.The fluorescence quantitative PCR analysis showed that the expression level of PfPMK gene was higher in the middle and late September.The full length of PfPMK gene was first cloned from Perilla frutescens.Bioinformatics analysis showed that PfPMK gene belonged to 5-phosphomevalonate kinase gene and participates in the biosynthesis of terpenoids in Perilla frutescens.

Key words: Perilla frutescens L., PfPMK, Terpenoids, Bioinformatics, Functional analysis

摘要:

为了研究甲羟戊酸-5-磷酸激酶(PMK)在紫苏萜类物质生物合成代谢通路中的重要作用,对紫苏转录组数据进行分析,挖掘出紫苏PMK基因参考序列,采用基因克隆技术从紫苏中克隆了PMK基因,利用生物信息学和实时荧光定量PCR(qRT-PCR)的方法对紫苏PMK基因进行了分析。结果表明,紫苏PMK基因开放阅读框(ORF)全长为1 524 bp,编码507个氨基酸。生物信息学分析显示,PfPMK的分子质量为54.73 ku,等电点为5.20,为亲水蛋白;紫苏PfPMK氨基酸与SmPMK、SsPMK、SbPMK和PvPMK同源性较高,说明紫苏PfPMK蛋白在进化过程中保守性较强。亲缘关系分析显示,PfPMK与丹参和一串红的PMK亲缘关系很近,在线软件WoLF-PSORT预测PfPMK蛋白可能定位于质膜或内质网膜上。qRT-PCR结果表明,PfPMK基因在紫苏根、茎、叶中均有表达,在根中的表达量高于在叶和茎中的表达量;紫苏不同生长发育时期的qRT-PCR分析显示,PfPMK基因在9月中下旬表达量较高。首次从紫苏中克隆出PfPMK基因,生物信息学分析该基因属于甲羟戊酸-5-磷酸激酶基因,参与了紫苏萜类物质的生物合成。

关键词: 紫苏, PfPMK, 萜类, 生物信息学, 功能分析