小麦YSK_(2)型脱水素基因WDHN2的克隆及表达分析  

Cloning and Expression Analysis of YSK_(2)Type Dehydrin Gene WDHN2in Wheat(Triticum aestivum)

作　　者：张红梅[1] 龚明贵[1] 伍家发[1] 张敏[1] 刘帅 薛豪彬 曹智豪 潘永康 ZHANG Hong-Mei;GONG Ming-Gui;WU Jia-Fa;ZHANG Min;LIU Shuai;XUE Hao-Bin;CAO Zhi-Hao;PAN Yong-Kang(College of Food and Bioengineering,Henan University of Science and Technology,Luoyang 471000,China)

机构地区：[1]河南科技大学食品与生物工程学院,洛阳471000

出　　处：《农业生物技术学报》2022年第8期1443-1453,共11页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金(31870093);河南科技大学青年科学基金(2015QN036)。

摘　　要：非生物逆境胁迫严重影响小麦(Triticum aestivum)的生长发育和产量,脱水素(dehydrin,DHN)是一类非生物逆境胁迫诱导表达蛋白,在保护细胞免受水分亏缺和温度变化的伤害过程中发挥重要作用。为研究脱水素在植物响应非生物逆境胁迫过程中的作用,本研究利用同源克隆技术,从小麦中克隆了1个YSK_(2)型脱水素基因,命名为WDHN2(GenBank No.OK094572)。该基因的开放阅读框为501 bp,编码1个含有166个氨基酸残基的蛋白;含有4个保守区域,分别为1个Y片段、1个S片段和2个K片段,与节节麦(Aegilops tauschii)脱水素(GenBank No.XP_020188302.1)亲缘关系最近。生物信息学分析表明,WDHN2蛋白属于高度亲水性蛋白,高级结构以无规则卷曲为主;亚细胞定位预测表明,WDHN2蛋白定位于细胞核;另外,S片段为主要的磷酸化部位。qPCR分析表明,WDHN2基因受干旱、高盐、低温和脱落酸(abscisic acid,ABA)诱导表达。构建原核融合表达载体pET28a-WDHN2并转化大肠杆菌(Escherichia coli)感受态细胞BL21(DE3),用异丙基硫代半乳糖苷(isopropylβ-D-1-thiogalactopyranoside,IPTG)诱导表达,获得25 kD的WDHN2蛋白和重组大肠杆菌。重组菌的抗性实验表明,WDHN2蛋白可以增强大肠杆菌对渗透胁迫、高盐、低温和高温胁迫的抗性。上述研究结果提示WDHN2可能参与小麦响应逆境胁迫的信号途径。本研究丰富了小麦响应非生物胁迫分子机制的研究内容,为深入探讨WDHN2基因的功能提供基础资料。Abiotic stress seriously affects the growth and yield of wheat(Triticum aestivum). Dehydrin(DHN) is a kind of protein induced by abiotic stress, and plays an important role in protecting cells from damage caused by water deficiency and temperature changes. In order to study the role of dehydrin in plants responding to abiotic stress, this study isolated a YSK_(2)type dehydrin gene WDHN2(GenBank No. OK094572)from wheat(Triticum aestivum) in homology-based cloning technology. WDHN2 gene had an open reading frame with the length of 501 bp, and encoded a protein containing 166 amino acids, which contained 1segment of Y, 1 segment of S, and 2 segments of K. Phylogenetic analysis revealed that WDHN2 had the closest genetic relationship with Aegilops tauschii DHN(XP_020188302.1). The bioinformatics analysis showed that WDHN2 protein was a highly-hydrophilic protein, and its higher structure was mainly composed of random coil. Subcellular localization predicted that WDHN2 protein localized in the nucleus. In addition, S segment was the main phosphorylated site. The qPCR analysis indicated that the expression of WDHN2 gene was induced by drought, salinity, low temperature and abscisic acid(ABA). The prokaryotic fusion expression vector pET28a-WDHN2 was constructed and transformed into Escherichia coli BL21(DE3). Then isopropyl β-D-1-thiogalactopyranoside(IPTG) was used for inducible expression, the 25 kD WDHN2 fusion protein and recombinant E. coli were obtained. Resistance analysis of recombinant bacteria showed that WDHN2 protein improved the tolerance to osmotic stress, high salinity, low temperature and high temperature stresses in Escherichia coli. The above results revealed that WDHN2 might be involved in the signal transduction pathway of wheat in response to abiotic stresses. The present study could enrich the research content of the molecular mechanism of abiotic stress responses in wheat, and provide basic material for further study on the function of WDHN2 gene.

关 键 词：小麦 脱水素(DHN) 生物信息学分析 qPCR 原核表达 

分 类 号：S512.1[农业科学—作物学]