高羊茅FaGST1基因克隆、亚细胞定位及表达分析  

Cloning,subcellular localization and expression analysis of FaGST1 in tall fescue

作　　者：陈锡[1,2,3] 赵德刚[1,2,3] 陈莹 吴佳海[1] 袁暘暘 王小利[1,2,3] CHEN Xi;ZHAO De-gang;CHEN Ying;WU Jia-hai;YUAN Yang-yang;WANG Xiao-li(Guizhou Institute of Prataculture,Guizhou Academy of Agricultural Sciences,Guiyang 550006,China;The National-Local Joint Engineering Research Center of Karst Region Plant Resources Utilization&Breeding of Guizhou Province,Guizhou University,Guiyang 550025,China;The Key Laboratory of Plant Resources Conservation and Germplasm Innovation in Mountainous Region,Ministry of Education,Guiyang 550025,China)

机构地区：[1]贵州省农业科学院贵州省草业研究所,贵阳550006 [2]贵州大学喀斯特山区植物资源利用与育种国家地方联合工程研究中心,贵阳550025 [3]山地植物资源保护与种质创新教育部重点实验室,贵阳550025

出　　处：《南方农业学报》2020年第7期1614-1624,共11页Journal of Southern Agriculture

基　　金：贵州省科技平台及人才团队专项(黔科合平台人才〔2018〕5634);贵州省科技计划项目(黔科合基础〔2019〕1302);教育部重点实验室开放基金项目(MOELP-201702);贵州省农科院青年基金项目(黔农科院青年基金项目〔2018〕80)。

摘　　要：【目的】克隆高羊茅谷胱甘肽-S-转移酶(GST)基因FaGST1,并进行亚细胞定位及表达分析,为深入研究该基因的抗逆分子调控提供理论依据。【方法】采用RT-PCR和RACE从高羊茅黔草1号中克隆FaGST1基因,对其进行生物信息学分析,构建植物融合表达载体pCAMBIA1300-FaGST1-GFP,通过农杆菌介导转入烟草叶片表皮细胞,观察FaGST1基因亚细胞定位情况,并利用实时荧光定量PCR检测高盐、高温、干旱及低氮胁迫处理下高羊茅叶片中该基因的表达情况。【结果】克隆获得FaGST1基因cDNA全长序列为1003 bp,开放阅读框(ORF)为681 bp,编码227个氨基酸残基,其编码蛋白的分子量约25.60 kD,理论等电点(pI)为5.58,亲水指数平均值-0.342,不稳定系数32.96,为稳定的亲水蛋白,定位于细胞核。FaGST1蛋白二级结构中,α-螺旋占50.44%,β-转角占5.75%,无规则卷曲占30.54%,延伸链占13.27%。FaGST1蛋白的保守结构区域为含有G位点的N末端结构域和含有H位点的C末端结构域。FaGST1蛋白与黑麦草GST蛋白(AMY26594.1)的氨基酸序列相似性最高,为93%,其次是海滨雀稗GST蛋白(AMN87043.1),为91%,与玉米(ACG39365.1)和小米(KQK86785.1)GST蛋白氨基酸序列相似性均在80%以上,与系统发育进化树分析结果基本相符,即FaGST1蛋白与黑麦草、海滨雀稗、玉米和小米等禾本科植物GST蛋白亲缘关系较近。高羊茅FaGST1基因在干旱、高温、高盐胁迫和低氮胁迫处理下均出现抑制表达。【结论】FaGST1基因负向调控高羊茅逆境胁迫响应。【Objective】Glutathione-S-transferase(GSTs)gene FaGST1 form tall fescue was cloned,subcellular localization and expression analysis was analyzed in order to provide reference for molecular regulation of stress resistance study of the gene.【Method】FaGST1 gene was cloned by RT-PCR and RACE from tall fescue Qiancaoyihao,the structure and function of the protein were analyzed by bioinformatic software,and plant fusion expression vector pCAMBIA1300-FaGST1-GFP was constructed,which injected to tobacco epidermal cells through agrobacterium-mediated method,the subcellular localization of FaGST1 gene was observed,and FaGST1 gene expression level in tall fescue leaves under salt stress,heat stress,drought stress and low nitrogen stress was analyzed by qRT-PCR.【Result】The sequence analysis results showed that the full-length cDNA(1003 bp)was obtained with a 681 bp open reading frame(ORF),which encoded a small molecular protein containing 227 amino acids,the relative molecular weight of FaGST1 protein was about 25.60 kDa and its theoretical isoelectric point(pI)was 5.58.The grand average of hydropathicity was-0.342 and the instability index was 32.96,which was a stable hydrophilic protein.Subcellular localization results showed that FaGST1 was located on the cell uncleus.In the secondary structure of FaGST1 protein,including 50.44%of alpha helix,5.75%of beta turn,30.53%of random coil and 13.27%of extended strand were observed.FaGST1 protein conserved domain contained the GST-specific N-terminal domain(G site)and the C-terminal domain(H site).FaGST1 protein had the highest amino acid sequence similarity with GST(AMY26594.1)of Lolium perenne,at 93%,and followed the amino acid sequence similarity with GST(AMN87043.1)of Paspalum vaginatum,at 91%.Its amino acid sequence similarities with GST(AMY26594.1)of Zea mays(ACG39365.1)and Setaria italica(KQK86785.1)were over 80%,the results were basically consistent with phylogenetic evolution tree analysis.FaGST1 had close genetic relationship with the GST protein of Lolium per

关 键 词：高羊茅 谷胱甘肽-S-转移酶(GST) 基因克隆 亚细胞定位 生物信息学分析 非生物胁迫 表达分析 

分 类 号：S543.9[农业科学—作物学]