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作物学报 ›› 2020, Vol. 46 ›› Issue (7): 1025-1032.doi: 10.3724/SP.J.1006.2020.94152

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

大豆GmNRT1.2a和GmNRT1.2b基因的克隆及功能探究

李国纪**,朱林**,曹金山,王幼宁()   

  1. 华中农业大学植物科学技术学院, 湖北武汉 430070
  • 收稿日期:2019-10-09 接受日期:2020-03-24 出版日期:2020-07-12 网络出版日期:2020-04-27
  • 通讯作者: 王幼宁
  • 作者简介:李国纪, E-mail: lgj2389868525@163.com|朱林, E-mail: onelin@163.com
    ** 同等贡献
  • 基金资助:
    国家科技重大专项(2018ZX0800919B);国家自然科学基金项目(31872873)

Cloning and functional analysis of GmNRT1.2a and GmNRT1.2b in soybean

LI Guo-Ji**,ZHU Lin**,CAO Jin-Shan,WANG You-Ning()   

  1. College of Plant Science and Technology, Huazhong Agricultural University, Wuhan 430070, Hubei, China
  • Received:2019-10-09 Accepted:2020-03-24 Published:2020-07-12 Published online:2020-04-27
  • Contact: You-Ning WANG
  • About author:** Contributed equally to this work
  • Supported by:
    National Transgenic Major Project of China(2018ZX0800919B);National Natural Science Foundation of China(31872873)

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摘要：

拟南芥中硝酸盐的吸收、转运和分配是通过硝酸盐转运蛋白(nitrate transporter, NRT)实现的。尽管之前的生物信息学分析推测大豆GmNRT1.2s可能参与共生固氮过程, 但尚未开展相应的功能研究。本研究通过对其表达模式分析发现, GmNRT1.2a和GmNRT1.2b分别在根和叶中高表达, 且受硝酸盐诱导, 在接种根瘤菌与结瘤因子(nod factors, NFs)后表达量明显升高。功能研究结果显示, 过表达GmNRT1.2a或GmNRT1.2b后大豆根瘤数目显著增加。本研究为深入探究GmNRT1.2a和GmNRT1.2b调控大豆共生固氮过程的分子机制提供了一定的数据支持。

关键词: 大豆, 根瘤, 表达分析, 结瘤, 共生固氮

Abstract:

Nitrate transporters (NRTs) have been found to be involved in nitrate uptake, transport and allocation in Arabidopsis. The role of GmNRT1.2s in soybean (Glycine max) symbiotic nitrogen fixation process has been speculated by bioinformatics analysis, however, its biological function has not been explored yet. In this study, we mainly focused on analyzing the expression pattern and biological function of GmNRT1.2a and GmNRT1.2b in soybean. The relative expression levels of GmNRT1.2a and GmNRT1.2b were higher in leaves, which induced by nitrate and up-regulated with increasing nitrate concentration. GmNRT1.2a and GmNRT1.2b expressions were also induced by rhizobial inoculation and nod factor (NF) treatment. Overexpression of GmNRT1.2a or GmNRT1.2b caused dramatic increment of nodule number. The results provide some data for further investigating the molecular mechanism of GmNRT1.2a and GmNRT1.2b in regulating the symbiotic nitrogen fixation process of soybean.

Key words: soybean, nodule, expression analysis, nodulation, symbiotic nitrogen fixation

图1

GmNRT1.2a和GmNRT1.2b过表达载体构建 A: PCR扩增后得到的GmNRT1.2a和GmNRT1.2b目的基因片段; B: pEGAD过表达载体图谱及酶切位点。"

图2

GmNRT1.2a和GmNRT1.2b在大豆不同发育时期的组织表达模式分析 标以不同小写字母的大豆不同组织中对应基因的表达水平在P < 0.05时差异显著。DAI: 接种后天数。"

图3

GmNRT1.2a和GmNRT1.2b在不同浓度下的硝酸盐下的表达模式 大豆在0N (0 mmol L-1)、LN (0.25 mmol L-1)和HN (15.75 mmol L-1) 3种硝酸盐浓度梯度下生长到15 d, 取大豆根部组织样品。用荧光定量PCR检测GmNRT1.2a (A)和GmNRT1.2b (B)的表达, GmELF1b为内参基因。图中标以不同字母的基因表达水平在P < 0.05时差异显著。"

图4

GmNRT1.2a和GmNRT1.2b响应接种根瘤菌和结瘤因子表达模式分析 标以不同小写字母的基因表达水平在根瘤菌和结瘤因子处理的大豆植株与对照间在P < 0.05水平上差异显著。"

图5

在低氮培养条件下过表达GmNRT1.2a或GmNRT1.2b增加结瘤数目 A, B: 在接种根瘤菌28 d后, 利用荧光定量PCR检测EV、35S::GmNRT1.2a和35S::GmNRT1.2b单条毛状根表达, 以GmELF1b基因作为内参基因。标以不同小写字母的柱值在不同转基因大豆植株间在P < 0.05时的差异显著。C: EV、35S::GmNRT1.2a和35S::GmNRT1.2b的单条根的根瘤表型, Bar = 5 mm。D: 统计EV、35S::GmNRT1.2a和35S::GmNRT1.2b的根瘤数目。*和***分别表示转基因株系35S::GmNRT1.2a和35S::GmNRT1.2b的根瘤数目与对照在P < 0.05和P < 0.001水平差异显著。"

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