碱胁迫相关基因GsWRKY15的克隆及其转基因苜蓿的耐碱性分析

发布时间：2018-02-09 10:42

  本文关键词： 野生大豆 GsWRKY 肇东苜蓿 农杆菌 耐碱性　出处：《作物学报》2017年09期 　论文类型：期刊论文

【摘要】：WRKY蛋白属于锌指型转录调控因子,能够参与植物多种逆境响应。本研究利用前期野生大豆盐碱胁迫RNA-seq测序数据,从构建的碱胁迫基因调控网络中筛选并克隆到GsWRKY15基因。分析GsWRKY15在碱胁迫下野生大豆根中的表达模式,发现该基因受碱胁迫诱导显著上调表达,且在胁迫后1 h表达量最高。分析GsWRKY15基因在野生大豆各组织中的表达特异性,发现该基因在各组织中均有表达,花中表达量最高。采用根癌农杆菌侵染苜蓿子叶节方法,将GsWRKY15转化肇东苜蓿,获得39株抗性植株。通过PCR、Southern blot和RT-PCR方法分析抗性植株,获得了超量表达GsWRKY15基因的转基因株系并对其进行了耐碱性分析。在150 mmol L 1 Na HCO3处理2周后转基因苜蓿生长状态良好,而非转基因苜蓿出现萎蔫、变黄,甚至死亡;非转基因苜蓿的相对质膜透性和丙二醛含量显著高于转基因苜蓿,而叶绿素含量显著低于转基因苜蓿;同时分析碱胁迫下转基因植株中胁迫相关基因的表达模式,发现H+-Ppase、NADP-ME、KIN1、RD29A基因的表达量高于非转基因苜蓿。结果表明GsWRKY15基因的超量表达能够显著增强苜蓿的耐碱能力。
[Abstract]:WRKY protein is a zinc-finger transcription regulator and can participate in many stress responses of plants. In this study, RNA-seq sequencing data of wild soybean saline-alkali stress were used. The GsWRKY15 gene was screened and cloned from the constructed alkali stress gene regulatory network. The expression pattern of GsWRKY15 in the root of wild soybean under alkali stress was analyzed. It was found that the gene was significantly up-regulated by alkali stress. After 1 hour of stress, the expression of GsWRKY15 gene was the highest in all tissues of wild soybean. The results showed that the GsWRKY15 gene was expressed in all tissues and the highest in flower. Agrobacterium tumefaciens was used to infect cotyledons of alfalfa by Agrobacterium tumefaciens (Agrobacterium tumefaciens). 39 resistant plants were obtained by transforming GsWRKY15 into alfalfa, and the resistant plants were analyzed by Southern blot and RT-PCR. Transgenic lines with overexpression of GsWRKY15 gene were obtained and their alkaline tolerance was analyzed. After treatment with 150 mmol L ~ (-1) Na HCO3 for 2 weeks, transgenic alfalfa grew well, while non-transgenic alfalfa showed wilting, yellowing and even death. The relative plasma membrane permeability and malondialdehyde content of non-transgenic alfalfa were significantly higher than that of transgenic alfalfa, while chlorophyll content was significantly lower than that of transgenic alfalfa. It was found that the expression of RD29A gene of NADP-MEKIN1 was higher than that of non-transgenic alfalfa. The results showed that the overexpression of GsWRKY15 gene could significantly enhance the alkali-tolerant ability of alfalfa.
【作者单位】： 东北农业大学农业生物功能基因重点实验室;
【基金】：国家自然科学基金项目(31171578) 黑龙江省高校科技创新团队建设计划项目(2011TD005) 东北农业大学学科团队建设项目(团队1)资助~~
【分类号】：Q943.2;S541.9
，

本文编号：1497743