MeJA响应的人参PgJAZ1基因克隆、表达与功能分析  

Cloning,Expression and Functional Analysis of MeJA Responsive PgJAZ1 Gene from Panax ginseng

作　　者：张儒[1,2] 谭时泉 张变玲 王媛媛[1] 曹颖 胡颖婕 ZHANG Ru;TAN Shiquan;ZHANG Bianling;WANG Yuanyuan;CAO Ying;HU Yingjie(College of Materials and Chemical Engineering,Hunan Institute of Engineering,Xiangtan 411104,China;College of Life Sciences,Hunan Normal University,Changsha 410081,China)

机构地区：[1]湖南工程学院材料与化工学院,湘潭411104 [2]湖南师范大学生命科学学院,长沙410081

出　　处：《中国细胞生物学学报》2021年第10期1964-1975,共12页Chinese Journal of Cell Biology

基　　金：国家自然科学基金(批准号:81874332);湖南省自然科学杰出青年基金(批准号:2020JJ2012);湖南省教育厅优秀青年基金(批准号:18B387);中国博士后科学基金特别资助和面上资助项目(批准号:2017T100601,2016M590746)资助的课题。

摘　　要：茉莉酸类(jasmonates,JAs)是调控人参皂苷等次生代谢产物生物合成的重要激素,JAZ(jasmonate ZIM-domain)蛋白是茉莉酸信号途径中的重要调控因子。为了探究人参皂苷生物合成与JAZ基因及茉莉酸信号调控之间的关系,从新鲜4年生人参根中提取RNA,以其反转录合成的cDNA为模板,根据茉莉酸甲酯(methyl jasmonate,MeJA)诱导后的人参发根转录组测序结果筛选候选基因并设计引物,利用PCR扩增人参JAZ基因的cDNA全长,从基因组文库中克隆JAZ基因的启动子序列,对其进行生物信息学分析,采用荧光定量PCR(qRT-PCR)分析人参不同组织和发根中JAZ基因表达水平,并测定MeJA诱导的人参发根中皂苷、内源性JA和MeJA的含量。克隆得到人参JAZ基因,将其命名为PgJAZ1,其序列完整开放阅读框(ORF)长度为702 bp,编码233个氨基酸。氨基酸序列分析显示,该蛋白包含TIFY和CCT2(Jas结构域)两个保守的功能结构域。系统进化分析显示,PgJAZ1与黄花蒿AaJAZ1亲缘关系最近。qRT-PCR结果显示,PgJAZ1在人参的根、叶、花及种子等部位中都有表达,在花中表达水平最高,而在不定根中表达水平最低。用MeJA和SA(salicylic acid)进行处理后发现,PgJAZ1受MeJA的诱导和SA的抑制,且在MeJA诱导12 h内达到最高表达水平。启动子分析显示,PgJAZ1基因启动子中包含MeJA、SA和激素响应元件,以及MYB、MYC和WRKY转录因子结合位点;MeJA诱导后的人参皂苷、内源性JA和MeJA含量与PgJAZ1表达水平高度一致。结果提示,PgJAZ1可能参与MeJA介导的人参皂苷生物合成。JAs(jasmonates)are important phytohormones involved in regulating the biosynthesis of ginsenosides and other secondary metabolites.JAZ(jasmonate ZIM-domain)protein is a key regulator in jasmonate signaling pathway.In order to explore the relationships among ginsenoside biosynthesis,JAZ gene and jasmonic acid signal regulation in Panax ginseng,the RNA from fresh 4-year roots of P.ginseng was extracted and the synthesized cDNA was used as template.The candidate genes were screened and primers were designed based on the transcriptomic sequencing results of ginseng hairy roots induced by MeJA(methyl jasmonate).The full ORF(open reading frame)of JAZ gene was cloned by RT-PCR(reverse transcription polymerase chain reaction).The promoter sequence of JAZ gene was cloned from genomic library and analyzed by bioinformatics.The expression pattern of JAZ gene in different tissues and hairy roots of P.ginseng were analyzed by qRT-PCR,and the contents of ginsenoside,endogenous JA and MeJA were determined in MeJA-induced hairy roots of P.ginseng.The full ORF of JAZ has 702 bp and encoded 233 amino acids,and it was designated as PgJAZ1.Sequence analysis showed that PgJAZ1 protein contained two conserved domains TIFY and CCT2(Jas domain).The phylogenetic analysis showed that PgJAZ1 was closely related to AaJAZ1 of Artemisia annua.qRT-PCR results showed that PgJAZ1 was expressed in roots,leaves,flowers,seeds and hairy roots of P.ginseng.The expression level of PgJAZ1 was the highest in flowers and the lowest in adventitious roots.In addition,PgJAZ1 was induced by MeJA and inhibited by SA(salicylic acid),and its expression level reached the highest after treatment by MeJA for 12 h.The PgJAZ1 promoter contained multiple putative cis-acting elements involved in MeJA,SA and hormone response,as well as binding sites of transcription factor such as MYB,MYC and WRKY.Ginsenosides content,endogenous jasmonates(JA and MeJA)induced by MeJA were highly consistent with PgJAZ1 expression.These results suggest that PgJAZ1 is involved in MeJA-med

关 键 词：人参 PgJAZ1基因 功能分析 茉莉酸类 人参皂苷 

分 类 号：S567.51[农业科学—中草药栽培]