小麦分子伴侣TaTCP22基因克隆及特性分析

发布时间：2018-11-18 09:25

【摘要】：非生物胁迫可引起蛋白的功能紊乱,维持蛋白的功能构象和阻止非天然蛋白的聚合,对于细胞存活很关键。伴侣素可以帮助蛋白质在胁迫条件下复性,在植物抵抗非生物胁迫中扮演着非常重要的角色。为分析伴侣素TaTCP22的分子特性,解析伴侣素的胁迫响应机制,以小麦的c DNA为模板扩增得到TaTCP22,利用生物信息学方法分析小麦TaTCP22的分子特性,使用MEGA5对小麦TaTCP22蛋白序列及其同源序列进行多序列比对分析并构建同源物种间的系统进化树;利用GSDS和PHYRE2在线工具分别对小麦TaTCP22基因结构和三级结构进行分析;从Phytozome数据库中获取小麦TaTCP22上游2 000 bp序列作为启动子,用PLACE数据库对小麦TaTCP22启动子的顺式作用元件进行分析;利用实时荧光定量PCR检测TaTCP22在不同组织及在脱落酸(abscisic acid,简称ABA)、聚乙二醇(polyethylene glycol,简称PEG)、水杨酸(salicylicacid,简称SA)、氯化钠(Na Cl)、低温胁迫处理下的表达量;利用In-Fusion技术构建TaTCP22-16318GFP载体,采用PEG介导法将外源基因转到小麦原生质体中,在激光共聚焦显微镜下观察基因定位情况。小麦TaTCP22全长1 643 bp,基因编码区包含UTR区和13个内含子以及13个外显子;启动子元件分析表明,TaTCP22包含多种逆境胁迫应答元件;亚细胞定位结果表明,TaTCP22蛋白主要定位在细胞核、细胞膜和细胞质中;实时荧光定量PCR结果显示,TaTCP22在不同组织中的表达水平不同;TaTCP22对于多种非生物胁迫均有不同程度的响应,说明TaTCP22可能参与了多种胁迫应答途径。
[Abstract]:Abiotic stress can cause protein dysfunction. Maintaining the functional conformation of proteins and preventing the polymerization of non-natural proteins are crucial for cell survival. Chaperone can help protein renaturation under stress and play a very important role in plant resistance to abiotic stress. In order to analyze the molecular characteristics of chaperidin TaTCP22 and analyze the stress response mechanism of chaperidin, TaTCP22, was amplified by using c DNA of wheat as template to analyze the molecular characteristics of wheat TaTCP22 by bioinformatics. The sequence of wheat TaTCP22 protein and its homologous sequence were analyzed by MEGA5 and the phylogenetic tree of the homologous species was constructed. The TaTCP22 gene structure and tertiary structure of wheat were analyzed by GSDS and PHYRE2, and the upstream 2 000 bp sequence of wheat TaTCP22 was obtained from Phytozome database as promoter, and the cis-acting elements of wheat TaTCP22 promoter were analyzed by PLACE database. Real time fluorescence quantitative PCR was used to detect the expression of TaTCP22 in different tissues and under the low temperature stress of salicylicacid, (salicylicacid, for short SA), sodium chloride (Na Cl),) under the condition of PEG), (polyethylene glycol, (PEG), (polyethylene glycol,) and abscisic acid (abscisic acid,). The TaTCP22-16318GFP vector was constructed by In-Fusion, and the exogenous gene was transferred into wheat protoplast by PEG. The location of the gene was observed under confocal laser microscope. The encoding region of 1 643 bp, gene of wheat TaTCP22 contains UTR region, 13 introns and 13 exons, and the promoter element analysis shows that TaTCP22 contains many stress response elements. The results of subcellular localization showed that TaTCP22 protein was mainly located in nucleus, cell membrane and cytoplasm, and the expression level of TaTCP22 was different in different tissues by real-time quantitative PCR. TaTCP22 had different responses to various abiotic stresses, indicating that TaTCP22 might be involved in multiple stress response pathways.
【作者单位】：西北农林科技大学生命科学学院;西北农林科技大学农学院/旱区作物逆境生物学国家重点实验室;
【基金】：国家转基因生物新品种培育重大专项(编号:2014ZX0800203B、2016ZX08002002-010) 陕西省科技攻关项目(编号:2014K02-04-05) 陕西省科技统筹创新计划(编号:2014KTZB02-01-01)
【分类号】：Q943.2;S512.1

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