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作物学报 ›› 2018, Vol. 44 ›› Issue (02): 185-196.doi: 10.3724/SP.J.1006.2018.00185

• • 上一篇    下一篇

R2R3-MYB转录因子GmMYB184调节大豆异黄酮合成

朱莹1,**, 褚姗姗2,**, 张培培1, 程浩1, 喻德跃1, 王娇1,*   

  1. 1南京农业大学大豆研究所 / 作物遗传育种与种质创新国家重点实验室 / 江苏省现代作物生产协同创新中心 / 国家大豆改良中心, 江苏南京210095
    2河南农业大学农学院 / 河南省粮食作物协同创新中心, 河南郑州 450002
  • 收稿日期:2017-05-15 接受日期:2017-09-10 出版日期:2018-02-12 网络出版日期:2017-10-27
  • 通讯作者: 朱莹,褚姗姗,王娇
  • 作者简介:

    第一作者联系方式: 朱莹, E-mail: 2014101144@njau.edu.cn; 褚姗姗, E-mail: chushan3@163.com; ** 同等贡献(Contributed equally to this work)

  • 基金资助:
    本研究由国家重点研发计划(2016YFD0100304, 2016YFD0100504),国家自然科学基金项目(31301342, 31370034)和中国博士后科学基金(2017M612400)资助

An R2R3-MYB Transcription Factor GmMYB184 Regulates Soybean Isoflavone Synthesis

Ying ZHU1,**, Shan-Shan CHU2,**, Pei-Pei ZHANG1, Hao CHENG1, De-Yue YU1, Jiao WANG1,*   

  1. 1 Soybean Research Institute / National Key Laboratory of Crop Genetics and Germplasm Enhancement / Jiangsu Collaborative Innovation Center for Modern Crop Production / National Center for Soybean Improvement, Nanjing Agricultural University, Nanjing 210095, Jiangsu, China
    2 Collaborative Innovation Center of Henan Grain Crops / Department of Agronomy, Henan Agricultural University, Zhengzhou 450002, Henan, China
  • Received:2017-05-15 Accepted:2017-09-10 Published:2018-02-12 Published online:2017-10-27
  • Contact: Ying ZHU,Shan-Shan CHU,Jiao WANG
  • Supported by:
    This study was supported by National Key R&D Program for Crop Breeding (2016YFD0100304, 2016YFD0100504), the National Natural Science Foundation of China (31301342, 31370034), and China Postdoctoral Science Foundation (2017M612400).

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摘要：

异黄酮是一类主要含在豆科植物中的次生代谢物, 在植物防御体系中发挥重要作用, 并与人类健康密切相关。大豆异黄酮含量受多基因和复杂代谢网络控制, 调控代谢途径上的结构基因不能显著改变大豆异黄酮含量, 与异黄酮代谢途径相关的转录因子的鉴定和应用可能会有效解决这个问题。本研究克隆了一个与大豆异黄酮合成相关的R2R3类型MYB转录因子GmMYB184, 并进行了初步的功能验证。亚细胞定位研究结果表明GmMYB184转录因子定位于细胞核。组织表达分析结果表明该转录因子基因与IFS2 (异黄酮合酶2编码基因)的表达模式相同。同时, GmMYB184和IFS2的表达模式与异黄酮的积累模式相似。谷胱甘肽诱导表达分析表明该转录因子基因与IFS2共同被诱导, 说明这两个基因可能参与同一或相似的生物过程。采用双荧光素酶报告系统分析其对异黄酮合成途径关键基因的转录激活活性影响, 发现GmMYB184能够促进IFS2和CHS8启动子表达活性分别提高5倍和7倍。最后, 通过发根农杆菌介导的遗传转化系统, 找到该转录因子在异黄酮合成调控中的直接作用证据。沉默GmMYB184导致大豆毛状根异黄酮含量的显著下降。但是, 过表达GmMYB184不足以显著提高毛状根中异黄酮的含量。总之, 本研究为大豆异黄酮合成分子机制探索及大豆异黄酮品质改良提供了理论依据。

关键词: 大豆, 异黄酮含量, GmMYB184, 转录因子, 功能鉴定

Abstract:

Isoflavones comprise a group of secondary metabolites produced almost exclusively by plants in the legume family, including soybean [Glycine max (L.) Merr.]. They play vital roles in plant defense and have many beneficial effects on human health. Isoflavone content is controlled by multiple genes and complex metabolic networks. The modification of certain structural genes in the isoflavone pathway by genetic engineering has been unable to significantly improve isoflavone content. The identification and application of transcription factors specific to the isoflavone pathway may effectively resolve this problem. An R2R3-type transcription factor related to isoflavone content, GmMYB184, was cloned and its function was identified. Subcellular localization study confirmed the nuclear localization of GmMYB184. The expression profile of GmMYB184 was similar to that of IFS2 (isoflavone synthase 2), which was consistent with the isoflavone accumulation pattern. In addition, GmMYB184 and IFS2 mainly expressed in roots and mature seeds, and the expression level increased by seeds maturing. Glutathione (GSH) induction expression analysis showed that both GmMYB184 and IFS2 were induced by GSH, indicating they could be involved in similar biological processes. To examine whether GmMYB184 could regulate the expression of isoflavone biosynthesis-related genes, a dual luciferase reporter gene assay was performed, showing that GmMYB184 could increase the expression of IFS2 and CHS8 (chalcone synthase 8) to five and seven folds, respectively. Finally, to further verify the function of GmMYB184 during isoflavone biosynthesis, we constructed and then transformed overexpression vector and RNAi vector for GmMYB184 to soybean hairy roots respectively. RNAi silencing of GmMYB184 in hairy roots resulted in reduction of isoflavones. However, overexpression of GmMYB184 was not sufficient to increase isoflavone contents in hairy roots. Taken together, these results provide a theoretical foundation for the molecule mechanism explanation and genetic improvement of isoflavone content in soybean.

Key words: soybean, isoflavone content, GmMYB184, transcription factor, functional identification

表1

引物序列"

引物用途
Purpose of primer		 引物名称
Primer name		 引物序列
Primer sequence (5'-3')
荧光定量PCR引物序列 MYB184-QF TTTTGGAATAACCAAAAGTCC
RT-PCR primer sequences MYB184-QR CTGCATTCGGCATTCCGATT
IFS2-QF ATGAAGTATATAAGCCCTTC
IFS2-QR TTGGGATAAATGATGTGGCAACT
Tubulin-F GGAGTTCACAGAGGCAGAG
Tubulin-R CACTTACGCATCACATAGCA
启动子序列扩增引物 IFS2-F TTCCATCACTGTATGAAAGTC
Promoter amplification primers IFS2-R CGTGTTCTCGTCCTTGGTTTG
CHS8-F CGAGCTCTGAGCAAGTATACCAACCAT
CHS8-R CGGACTAGTCTTTCCTTCAAATTAAGTGAT

图1

双荧光素酶报告系统示意图"

图2

GmMYB184扩增结果 M: 标准分子量DL2000; MYB184: GmMYB184 cDNA产物。"

图3

胁迫响应相关R2R3-MYB转录因子序列比对不同植物中胁迫响应相关 R2R3-MYB 转录因子多序列比对。这些植物包括Arabidopsis thaliana (AtMYB2, AtMYB62, AtMYB78, AtMYB108, AtMYB112, AtMYB116), Lotus japonicas (LjMYB62, LjMYB116), Medicago truncatula (Medtr1g086530, Medtr1g086510, Medtr2g033170)以及Glycine max (GmMYB184, GmMYB84)。"

图4

胁迫响应相关R2R3-MYB转录因子系统发育分析"

图5

大豆不同组织和不同发育时期的种子中GmMYB184、IFS2的相对表达量及异黄酮含量 A: 大豆不同组织中GmMYB184的相对表达量; B: 大豆不同组织中IFS2的相对表达量; C: 大豆不同组织中异黄酮含量; D: 不同发育时期的大豆种子中GmMYB184 的相对表达量; E: 不同发育时期的大豆种子中IFS2 的相对表达量; F: 不同发育时期的大豆种子中异黄酮含量。"

图6

GmMYB184和IFS2的谷胱甘肽诱导表达模式"

图7

GmMYB184的亚细胞定位 A: 空载35S::GFP在拟南芥原生质体中表达; B: GmMYB184::GFP在拟南芥原生质体中表达。"

图8

GmMYB184对IFS2和CHS8的启动子激活活性 A: GmMYB184对IFS2的启动子激活活性; B: GmMYB184对CHS8的启动子激活活性。"

图9

毛状根中沉默和过表达GmMYB184的相对表达量 A: 毛状根中沉默GmMYB184的相对表达量; B: 毛状根中过表达GmMYB184的相对表达量。**和***分别表示转基因株系GmMYB184基因相对表达量与对照在P<0.01和P<0.001水平差异显著。"

图10

沉默和过表达GmMYB184毛状根相对异黄酮含量 A: 沉默GmMYB184毛状根中相对异黄酮含量; B: 过表达GmMYB184毛状根中相对异黄酮含量。**表示转基因株系GmMYB184基因相对表达量与对照在P<0.01水平差异显著。"

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