小麦TaLEA_4-1D基因的克隆及功能验证  

Cloning and Functional Analysis of TaLEA_4-1D Gene in Wheat

作　　者：刘金轲 刘玉秀[1] 郭娇娇 张治惠 耿强 赵常乐 杨群慧[1] 武军[1] LIU Jinke;LIU Yuxiu;GUO Jiaojiao;ZHANG Zhihui;GENG Qiang;ZHAO Changle;YANG Qunhui;WU Jun(College of Agronomy,Northwest A&F University,Yangling,Shaanxi 712100,China)

机构地区：[1]西北农林科技大学农学院,陕西杨凌712100

出　　处：《麦类作物学报》2024年第8期957-965,共9页Journal of Triticeae Crops

基　　金：中国-哈萨克斯坦抗旱节水优质小麦种质资源的互换、评价与利用研究项目(2020KWZ-011);优质旱地小麦新品种选育及普冰322的示范推广项目(TGZX2021-9)。

摘　　要：干旱胁迫是影响小麦生长和产量的主要因素之一。胚胎发育晚期丰富蛋白(LEA)能够响应多种非生物胁迫,在提高植物抗逆性方面发挥重要功能。为了探索LEA蛋白在小麦抵御干旱胁迫中的作用,本研究通过对小麦耐旱材料H611干旱胁迫前后的RNA-seq数据进行分析,筛选出10个TaLEA蛋白基因;克隆了TaLEA_4-1D基因,并对其进行生物信息学分析、表达分析和亚细胞定位,同时构建TaLEA_4-1D过表达拟南芥株系进行功能验证。结果表明,TaLEA_4-1D编码214个氨基酸,含有一个LEA_4保守结构域,蛋白分子量为21.97 kD,等电点为8.89,是亲水的稳定蛋白;二级结构和三级结构预测表明,此蛋白主要由α-螺旋(85.98%)和无规则卷曲(11.21%)组成。顺式作用元件分析发现,TaLEA_4-1D含有多个与植物激素诱导、生长发育和逆境胁迫响应相关的顺式调控元件。系统进化树分析显示,TaLEA_4-1D基因与节节麦、大麦、二穗短柄草、水稻等的LEA亲缘关系较近;亚细胞定位显示该蛋白位于细胞核和细胞质中。干旱胁迫下,过表达TaLEA_4-1D转基因拟南芥根长显著增加,其对干旱胁迫的耐受性增强,过表达植株表型优于野生型。Drought stress is one of the main factors affecting wheat growth and yield.Late embryonic developmental abundant(LEA)protein can respond to various abiotic stresses and play an important function in improving plant stress resistance.In order to explore the role of LEA proteins in wheat resistance to drought,10 TaLEA genes were identified from the transcriptome sequencing data of wheat material H611 before and after drought stress.TaLEA_4-1D was cloned and subjected to bioinformatics analysis,expression pattern analysis and subcellular localization.The TaLEA_4-1D overexpressed Arabidopsis thaliana lines were constructed for preliminary functional characterization.The results showed that TaLEA_4-1D encodes 214 amino acids and contains an LEA_4 conserved domain.The molecular weight is 21.97 kD,the isoelectric point is 8.89,and it is a hydrophilic stable protein.Secondary and tertiary structure predictions showed that TaLEA_4-1D protein is mainly composed ofα-helices(85.98%)and irregular coils(11.21%).Analysis of cis-acting elements showed that TaLEA_4-1D contained several cis-regulatory elements related to plant hormone induction,growth and development,and stress response.Phylogenetic tree analysis showed that TaLEA_4-1D is closely related to the LEAs of Aegilops tauschii,Hordeum vulgare,Brachypodium distachyon,and Oryza sativa.Subcellular localization revealed that the protein is located in the nucleus and cytoplasm.Under drought stress,the root length of TaLEA_4-1D overexpressed transgenic Arabidopsis thaliana increased significantly,with enhanced tolerance to drought stress,and the phenotype of overexpressed Arabidopsis thaliana was superior to that of wild type.

关 键 词：小麦 TaLEA_4-1D基因 基因表达 转基因 抗旱性 

分 类 号：S512.1[农业科学—作物学] S330