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作物学报 ›› 2017, Vol. 43 ›› Issue (09): 1319-1327.doi: 10.3724/SP.J.1006.2017.01319

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

碱胁迫相关基因GsWRKY15的克隆及其转基因苜蓿的耐碱性分析

朱娉慧**,陈冉冉**,于洋,宋雪薇,李慧卿,杜建英,李强,丁晓东,朱延明*   

  1. 东北农业大学农业生物功能基因重点实验室, 黑龙江哈尔滨 150030
  • 收稿日期:2016-12-03 修回日期:2017-05-10 出版日期:2017-09-12 网络出版日期:2017-05-22
  • 通讯作者: 朱娉慧, E-mail: zhupinghui@outlook.com, Tel: 15604601520
  • 基金资助:

    本研究由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(2011TD005)和东北农业大学学科团队建设项目(团队1)资助。

Cloning of Gene GsWRKY15 Related to Alkaline Stress and Alkaline Tolerance of Transgenic Plants

ZHU Ping-Hui**,CHEN Ran-Ran**,YU Yang,SONG Xue-Wei,LI Hui-Qing,DU Jian-Ying,LI Qiang,DING Xiao-Dong,ZHU Yan-Ming*   

  1. Key Laboratory of Agricultural Biological Functional Genes, Northeast Agricultural University, Harbin 150030, China
  • Received:2016-12-03 Revised:2017-05-10 Published:2017-09-12 Published online:2017-05-22
  • Contact: 朱娉慧, E-mail: zhupinghui@outlook.com, Tel: 15604601520
  • Supported by:

    This study was supported by National Natural Science Foundation of China (31171578),Heilongjiang Provincial Higher School Science and Technology Innovation Team Building Program (2011TD005), and the Northeast Agricultural University Discipline Team Construction Project (Group 1).

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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 NaHCO3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。

关键词: 野生大豆, GsWRKY15, 肇东苜蓿, 农杆菌, 耐碱性

Abstract:

WRKY proteins are members of a transcription factor family with Zinc-finger structure in higher plant, which participate in various responses to multiple stresses.In this study,we constructed a gene expression profile under alkaline treatment using RNA-seq data, from which we cloned a gene GsWRKY15.We analyzedtheexpression pattern of GsWRKY15 in roost of Glycine soja under alkaline stress, and found that this gene was up-regulated by alkaline stress,wirh the highest expression at one hour after alkaline treatment. We analyzed theexpression pattern of GsWRKY15 in different tissues of Glycine soja, and found that this gene could express in all tissues, with the highest expression level in flowers.GsWRKY15 was transformed into Zhaodong alfalfa by Agrobacteriumtumefaciens-mediated infection of alfalfa cotyledonary nodes, and 39 resistant plants were obtained. The results of PCR, Southern blot and RT-PCR showed that GsWRKY15 was integrated into the genome of Zhaodong alfalfa and expressed in transgenic plants. Alkaline tolerance analysis showed that the growth of transgenic alfalfa after two weeks of treatment with 150 mmol L–1 NaHCO3 was better than those of non-transgenic alfalfa. MDA content and relative membrane permeability were significantly higher while chlorophyll content was significantly lower in non-transgenic alfalfa than in transgenic alfalfa. And by analyzing t some stress response marker genes , we found that he expression levels of H+-Ppase, NADP-ME, KIN1, RD29A were higher in transgenic alfalfa than in non-transgenic alfalfa.Taken together we suggest that the expression of GsWRKY15 gene can enhance the alkaline-resistant ability of alfalfa.

Key words: Glycine soja, GsWRKY15, Zhaodong alfalfa, Agrobacterium tumefaciens, Alkaline tolerance

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