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| 小麦基因TaLEA_4-1D的克隆及功能验证 |
| Cloning and Functional Analysis of TaLEA 4-1D Gene in Wheat |
| 投稿时间：2023-12-10 修订日期：2024-04-01 |
| DOI： |
| 中文关键词: 小麦 TaLEA_4-1D基因 基因表达 转基因 抗旱性 |
| 英文关键词:Wheat TaLEA 4-1D gene Expression analysis Transgenic Arabidopsis thaliana Drought resistance |
| 基金项目: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(2020KWZ-011)；优质旱地小麦新品种选育及普冰322的示范推广(TGZX2021-9) |
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| 中文摘要: |
| 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,氨基酸分子量为21.97kDa,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、大麦和二穗短柄草和水稻的LEA亲缘关系较近；亚细胞定位显示该基因位于细胞核和细胞质中。在干旱胁迫下,过表达TaLEA_4-1D转基因拟南芥植株表型优于野生型,显著提高了根长,增强其对干旱胁迫的耐受性。本研究结果为LEA蛋白的功能分析提供参考,并为育种工作提供有力的理论基础和优异基因资源。 |
| 英文摘要: |
| Drought stress is one of the main factors affecting wheat growth and yield. Late embryonic developmental abundant (LEA) protein can respond to a variety of abiotic stresses and plays an important role in improving plant stress resistance. In order to explore the role of LEA protein in wheat resistance to drought, 10 TaLEA protein genes were identified from the RNA-seq data of wheat material H611 before and after drought stress. TaLEA_4-1D gene was cloned, and bioinformatic analysis, expression pattern analysis and subcellular localization were carried out. The TaLEA_4-1D overexpressed Arabidopsis thaliana was constructed for preliminary functional verification. The results showed that TaLEA_4-1D encoded 214 amino acids and contained a LEA_4 conserved domain. The molecular weight is 21.97kDa, 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 of α-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 gene was closely related to the LEAs of Aegilops tauschii, Hordeum vulgare, Brachypodium distachyon and Oryza sativa. Subcellular localization revealed that the gene was located in the nucleus and cytoplasm. Under drought stress, the phenotype of Arabidopsis thaliana plants overexpressing TaLEA_4-1D was superior to that of the wild type, significantly increasing the root length. TaLEA 4-1D gene enhanced Arabidopsis thaliana tolerance to drought stress. The results of this study provide a reference for the functional analysis of LEA protein, and provide a strong theoretical basis and excellent gene resources for breeding. |
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