栀子(Gardenia jasminoides)GGPPS基因小亚基的克隆及表达分析  

Cloning and Expression Analysis of Gardenia jasminoides GGPPS Small Subunit Gene

作　　者：陈建荣 毛凯权 陈果 刘芷翾 邓文月 刘芳 Chen Jianrong;Mao Kaiquan;Chen Guo;Liu Zhixuan;Deng WenyueLiu Fang(College of Biological and Environmental Engineering,Changsha University,Changsha,410022)

机构地区：[1]长沙学院生物与环境工程学院,长沙410022

出　　处：《分子植物育种》2020年第10期3199-3206,共8页Molecular Plant Breeding

基　　金：湖南省自然科学基金项目(2017JJ2294);长沙市科技计划项目(K1705043);湖南省科技创新计划(2018NK2041)共同资助。

摘　　要：香叶基焦磷酸(geranyl pyrophosphate,GPP)是单萜的前体物质,而单萜是栀子内重要的活性成分。香叶基香叶基焦磷酸合酶(GGPPS)在萜类化合物、类胡萝卜素类化合物生物合成中处于关键位置,GGPPS主要分为香叶基香叶基焦磷酸合成酶大亚基(GGPPSlsu)和香叶基香叶基焦磷酸合成酶小亚基(GGPPSssu)。为了研究栀子GGPPS基因在栀子萜类生物合成中的作用,本研究成功从栀子品种‘林海一号’中提取RNA反转录合成cDNA,根据转录组测序拼接结果设计引物扩增得到栀子GGPPSssu的cDNA序列,分别命名为GjGGPPSssu1和GjGGPPSssu2,ORF区分别为1053 bp和915 bp。通过构建基因进化树,栀子GGPPSssu基因和其他植物的GGPPSssu基因聚为一类,属于香叶基香叶基焦磷酸合成酶小亚基基因。通过蛋白质序列比对表明GjGGPPSssu1有一个CxxxC(x为碱性氨基酸)的保守基元和一个FARM的保守基元,没有SARM保守基元,与SSUⅡ类基因相似;GjGGPPSssu2有两个CxxxC的保守基元,没有FARM保守基元和SARM保守基元,与SSUⅠ类基因相似。荧光定量PCR试验结果表明,GjGGPPSssu1在T4和T5时期有较高的表达水平,GjGGPPSssu2在栀子各生长时期的根部和果实中有较高水平的表达。推测GjGGPPSssu1基因与栀子黄色素合成相关,GjGGPPSssu2基因与单萜合成相关。本研究初步解析了栀子GGPPS基因的功能,为提高栀子药用品质提供了理论依据。Geranyl pyrophosphate(GPP)is a precursor of monoterpenes,and monoterpenes are important active ingredients in gardenias.Geranylgeranyl pyrophosphate synthase(GGPPS)is in a key position in the biosynthesis of terpenoids and carotenoids.GGPPS is mainly divided into geranylgeranyl pyrophosphate synthase large subunit(GGPPSlsu)and geranylgeranyl pyrophosphate synthase small subunit(GGPPSssu).To study the role of the GGPPS gene in the biosynthesis of Gardenia jasminoides In this study,total RNA was successfully extracted from Gardenia jasminoides cultivar'Linhai 1'and was reversely transcribed into cDNA.According to the results of transcriptome sequencing and splicing,primers were designed to amplify the cDNA region sequence of gardenia GGPPSssu,named GjGGPPSssu1 and GjGGPPSssu2.The ORF regions were 1053 bp and 915 bp,respectively.By constructing gene evolutionary tree,it is known that gardenia GGPPSssu gene and other plants GGPPSssu genes were clustered into one group,belonging to the small subunit gene of geranyl geranyl pyrophosphate synthase.Protein sequences alignment showed that GjGGPPSssu1 has a conserved CxxxC(x is an alkaline amino acid)and a conserved FARM unit,and no SARM conserved unit.It is similar to SSU classⅡgene.GjGGPPSssu2 have two conserved CxxxC,no FARM and SARM conserved units,similar to SSU classⅠgene.Fluorescence quantitative PCR results showed that GjGGPPSssu1 has a high expression level at T4 and T5 stages,leaf and fruit with spatio-temporal specificity,while GjGGPPSssu2 was expressed in Gardenia jasminoides have a high level in root and fruit.It is speculated that GjGGPPSssu1 gene is related to Gardenia jasminoides yellow pigment synthesis and GjGGPPSssu2 gene is related to Gardenia jasminoides monoterpene synthesis.The function of GGPPS gene in Gardenia jasminoides was preliminarily analyzed,which provided a theoretical basis for improving the quality of Gardenia jasminoides.

关 键 词：栀子(Gardenia jasminoides) 基因克隆 香叶基香叶基焦磷酸合酶小亚基 荧光定量 

分 类 号：S567.19[农业科学—中草药栽培]