中国农业科学 ›› 2020, Vol. 53 ›› Issue (1): 18-28.doi: 10.3864/j.issn.0578-1752.2020.01.002

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

异源表达芥菜BjMATE增强紫花苜蓿耐酸铝胁迫的机理

李小冬1,2,尚以顺1(),李世歌1,2,陈光吉1,2,裴成江1,2,孙方1,熊先勤1   

  1. 1 贵州省农业科学院草业研究所,贵阳 550006
    2 贵州鼎芯农牧科技有限公司,贵阳 550006
  • 收稿日期:2019-06-03 接受日期:2019-09-10 出版日期:2020-01-01 发布日期:2020-01-19
  • 通讯作者: 尚以顺
  • 作者简介:李小冬,E-mail:lixiaodongzl@163.com
  • 基金资助:
    国家自然科学基金(31960345);贵州省科学技术基金(黔科合J字[2015]2080号);贵州省科研机构服务企业行动计划(黔科合服企[2018]4001号);贵州省农业科学院院专项基金(黔农科院院专项[2013]003号);贵州省科研机构创新能力建设专项资金项目(黔科合服企[2019]4010号)

The Mechanism of Ectopic Expression of Brassica juncea Multidrug and Toxic Compound Extrusion (BjMATE) to Enhance the Resistance to Acid and Aluminum Stress in Alfalfa

XiaoDong LI1,2,YiShun SHANG1(),ShiGe LI1,2,GuangJi CHEN1,2,ChengJiang PEI1,2,Fang SUN1,XianQin XIONG1   

  1. 1 Guizhou Institute of Prataculture, Guizhou Academy of Agricultural Science, Guiyang 550006
    2 Guizhou Dingxin Agriculture and Animal Husbandry Technology Co. Ltd, Guiyang 550006
  • Received:2019-06-03 Accepted:2019-09-10 Online:2020-01-01 Published:2020-01-19
  • Contact: YiShun SHANG

摘要:

【目的】酸铝胁迫是南方农业生产面临的主要环境胁迫之一。研究乡土植物对酸铝胁迫的适应机理,发掘并利用优良基因培育抗酸耐铝作物新品种,从而为紫花苜蓿分子遗传改良奠定基础。【方法】贵州省草业研究所紫花苜蓿遗传改良组前期已从贵州乡土资源平坝苦油菜中克隆获得BjMATE,并将其构建过量表达载体。采用组织培养和花器官浸泡法分别对紫花苜蓿和拟南芥进行遗传转化,根据NPT抗性基因和特异基因设计引物对转化材料进行分子鉴定,并采用qRT-PCR进行表达量的鉴定。利用水培法平行比较转基因和对照紫花苜蓿在酸、铝和酸铝组合胁迫条件下的发芽率、幼苗生长状况,以及在长时间弱酸铝胁迫条件下的幼苗地上和地下部分的生长变化。采用酶标仪检测酸、铝以及酸铝组合胁迫条件下转基因和对照紫花苜蓿根系中POD、SOD、CAT等抗氧化酶的活性和MDA含量的变化,同时在BjMATE过表达的拟南芥中采用qRT-PCR的方法分析酸铝代谢途径的关键基因AtMATEAtPIN2AtALS3AtALMT1AtSTOP1在胁迫条件下的表达变化,并对其调控关系进行讨论。【结果】过量表达BjMATE的拟南芥和紫花苜蓿的阳性率分别为100%与66.7%,候选紫花苜蓿和拟南芥转基因株系中BjMATE的表达量分别比对照上调63.02和76.87倍。过量表达BjMATE紫花苜蓿株系OEMs-5和对照中苜1号紫花苜蓿的发芽率在正常、酸胁迫、铝胁迫和酸铝组合胁迫条件下都没有显著差别,但是其发芽势在胁迫条件下显著优于对照中苜1号。在长时间弱胁迫条件下,OEMs-5的株高与对照没有显著区别,但在铝离子胁迫和酸铝组合胁迫条件下,其的生物产量和根系伸长显著优于对照中苜1号。抗氧化酶活性和MDA含量在OEMs-5与中苜1号中的变化不尽相同。POD和SOD的活性在胁迫处理后的2个材料中都显著上升,但只有SOD的活性在2材料间差异显著。CAT的活性在处理间和材料间都没有显著的差异。MDA的含量略有下降,在铝离子和酸铝组合胁迫条件下的2个材料中差异达显著水平。qRT-PCR显示,在OEAt-1植株中,AtMATEAtPIN2AtALS3AtALMT1AtSTOP1受铝和酸铝组合胁迫诱导,但只有AtSTOP1OEAt-1与野生型之间差异显著。【结论】BjMATE能够正向调节紫花苜蓿在种子萌发和幼苗生长阶段对酸铝胁迫的耐受性。活性氧清除系统尤其是SOD以及酸铝代谢途径关键基因AtSTOP1可能参与BjMATE介导的酸铝胁迫调节。

关键词: 酸铝胁迫, MATE, 紫花苜蓿, 基因表达调控

Abstract:

【Objective】 Acid and aluminum stress is one of the major environmental stresses in agricultural production in the south of China. Studying the mechanism of native plants adaptation to acid and aluminum stress, and identifying and making use of the excellent gene resources would benefit to breed new varieties of acid and aluminum resistant crops, that will play a fundamental role in molecular breeding of alfalfa. 【Method】 In previous study, full length of BjMATE was cloned from Pingba bitter rape, a native Brassica juncea resource in Guizhou province, and the overexpression vector was constructed by the alfalfa genetic improvement group of Guizhou Institute of Prataculture. The transgenic plants of alfalfa and Arabidopsis thaliana were obtained by tissue culture and flower dipping methods, respectively. Molecular examination was carried out with NPT resistance gene primers and gene-specific primers; and the expression of BjMATE in transgenic plants was examined by qRT-PCR. The germination rate and seedling growth of transgenic and control alfalfa lines were analyzed by hydroponic method under acid, aluminum and acid aluminum combination stress conditions. The alteration of the seedling and root growth was also analyzed between transgenic and Zhongmu 1# alfalfa under a long term but weaker acid-aluminum stress condition. Antioxidant enzyme activity, including POD, SOD, CAT, and MDA concentration alterations between OEMs-5 and Medicago sativa Zhongmu 1# were analyzed with microplate reader under acid, aluminum and acid-aluminum combined stress conditions. At the same time, expression changes of AtMATE, AtPIN2, AtALS3, AtALMT1, and AtSTOP1 which were key genes involving in acid aluminum regulation pathways, were analyzed in the model plant Arabidopsis thaliana by qRT-PCR, and potential regulation network were discussed. 【Result】 The positive rates of transgenic Arabidopsis and alfalfa were 100% and 66.7%, respectively. Compared with the control lines, the expression levels of the candidate transgenic alfalfa and Arabidopsis lines were up-regulated by 63.02 and 76.87 times, respectively. There was no significant difference in seed germination rate between OEMs-5 and Zhongmu 1# under normal, acid stress, aluminum ion stress and acid-aluminum combination stress conditions. While the germination potential was significantly different among the treatments, as well as the materials, OEMs-5 was significantly better than Zhongmu 1#. After a long-term weak stress treatment, the plant height of OEMs-5 was not significantly different from that of Zhongmu 1#; but the biomass and root length of OEMs-5 was significantly better than that of Zhongmu 1# under aluminum stress and acid-aluminum combined stress conditions. Antioxidant enzyme activity and MDA concentration alterations between OEMs-5 and Zhongmu 1# were different. POD and SOD activity increased in both OEMs-5 and Zhongmu 1# after stress treatment, however, significant difference was only detected in SOD assay. No significant difference in CAT activity was detected between the two materials, and the treatments. The content of MDA decreased slightly, significant differences were detected between OEMs-5 and Zhongmu 1# under aluminum stress and acid-aluminum combined stress conditions. Expressions of AtMATE, AtPIN2, AtALS3, AtALMT1 and AtSTOP1 were up-regulated under aluminum stress and acid-aluminum combined stress in both OEMs-5 and Zhongmu 1#, however, no significant difference were detected between them except AtSTOP1.【Conclusion】BjMATE can positively regulate alfalfa tolerance to acid-aluminum stress during seed germination and seedling growth stages. Reactive oxygen species (ROS) scavenger system, especially the activity of SOD, and AtSTOP1 gene which is a key gene involving in acid-aluminum stress regulation pathway, may participate in BjMATE mediated acid-aluminum stress regulation.

Key words: acid aluminum stress, MATE, alfalfa, gene expression regulation