高山离子芥磷脂酶Dζ基因及启动子的克隆与表达分析  

Cloning and Expression Analysis of Phospholipase DζGene and Its Promoter from Chorispora bungeana

作　　者：刘博 杨宁[1] 李辉 周亚萍 刘锐锐 高润 Liu Bo;Yang Ning;Li Hui;Zhou Yaping;Liu Ruirui;Gao Run(College of Life Science,Northwest Normal University,Lanzhou,730070)

机构地区：[1]西北师范大学生命科学学院,兰州730070

出　　处：《分子植物育种》2021年第24期8141-8150,共10页Molecular Plant Breeding

基　　金：国家自然科学基金项目(31660116,31960061)资助。

摘　　要：磷脂酶D(phospholipose D,PLD)作为一类磷脂水解酶,在植物生长发育和各种胁迫的信号转导过程中起着重要作用。本研究以高山冰缘植物高山离子芥作为材料,用RT-RCR技术和RACE技术克隆得到一种新的高山离子芥PLD基因,并命名为CbPLDζ,该基因全长3147 bp,开放阅读框(ORF)为2197 bp,生物信息学分析显示,该蛋白是一种弱亲水性蛋白,编码989个氨基酸,二级结构中α-螺旋和无规则卷曲占主导地位。系统进化分析表明,高山离子芥CbPLDζ基因和拟南芥AtPLDζ基因亲缘关系最为密切。采用染色体步移技术(Genome walking technique)扩增CbPLDζ基因5’端上游调控序列,获得520 bp启动子序列,Plant Care分析表明,该序列含有大量基本顺式作用元件TATAbox、CAATbox,还存在温度、干旱、水杨酸(SA)、赤霉素(GA)、生长素等响应元件及多个光响应顺式调控元件。应用RT-qPCR技术研究CbPLDζ基因在不同组织的相对表达量,结果表明,CbPLDζ基因在根、茎、叶中均有表达,其中茎中表达最高,根中表达最低。CbPLDζ基因在不同胁迫下相对表达量的变化说明CbPLDζ基因积极的参与了高山离子芥在生长发育过程中对盐、甘露醇以及外源ABA等非生物胁迫的调控。本实验为进一步研究和完善高山离子芥PLD基因家族的功能提供数据基础。Phospholipase D(PLD),as a type of phospholipid hydrolase,plays an important role in the signal transduction process of plant growth and development and various stresses.In this study,the Alpine ice plant Chorispora bungeana was used as plant material,and a new Chorispora bungeana PLD gene,CbPLDζ,CbPLDζgene with a total length of 3147 bp was cloned by RT-RCR technology and RACE technology.The open reading frame(ORF)is 2197 bp.Bioinformatics analysis was performed.The protein encodes 989 amino acids.In the secondary structure,α-helix and random coils are the dominant secondary structure.Phylogenetic analysis showed that the CbPLDζgene is most closely related to the Arabidopsis AtPLDζgene.Genome walking technique was used to amplify the 5’up-stream regulatory sequence of the CbPLDζgene to obtain a 520 bp promoter sequence.Plant Care analysis showed that the sequence contained a large number of basic cis-acting elements including TATAbox and CAATbox,as well as temperature and drought,salicylic acid(SA),gibberellin(GA),auxin and other response elements and multiple light-responsive cis-regulatory elements.Real-time fluorescence quantitative PCR(RT-qPCR)technology was used to study the relative expression of the CbPLDζgene in different tissues.The CbPLDζgene was expressed in roots,stems,and leaves,in which the stems have the highest expression and the roots have the lowest expression.The change of relative expression of CbPLDζunder different stress conditions proves that CbPLDζis actively involved in the regulation of several abiotic stresses such as salt,mannitol and exogenous ABA during the growth and development in Chorispora bungeana.This experiment provides a data basis for further research and improvement of the function of the PLD gene family of Chorispora bungeana.

关 键 词：高山离子芥(Chorispora bungeana) 磷脂酶Dζ 克隆 生物信息学分析 表达分析 

分 类 号：Q943.2[生物学—植物学]