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Acta Agronomica Sinica ›› 2018, Vol. 44 ›› Issue (02): 185-196.doi: 10.3724/SP.J.1006.2018.00185

• Orginal Article • Previous Articles     Next Articles

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 Online:2018-02-12 Published: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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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

Table 1

Primer sequences"

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

Fig. 1

Diagram for dual luciferase report system"

Fig. 2

Amplification result for GmMYB184 M: DNA marker DL2000; MYB184: cDNA of GmMYB184."

Fig. 3

Sequence alignment for stress responsive R2R3-MYB transcription factors Multiple alignment of putative stress responsive R2R3-MYB transcript factors in various plant species: Arabidopsis thaliana (AtMYB2, AtMYB62, AtMYB78, AtMYB108, AtMYB112, AtMYB116), Lotus japonicas (LjMYB62, LjMYB116), Medicago truncatula (Medtr1g086530, Medtr1g086510, Medtr2g033170), and Glycine max (GmMYB184, GmMYB84)."

Fig. 4

Phylogenetic analysis for stress responsive R2R3-MYB transcription factors"

Fig. 5

Relative expression levels of GmMYB184 and IFS2, and isoflavone contents in different tissues at different developing stages of soybean seed A: relative expression levels of GmMYB184 in different tissues of soybean; B: relative expression levels of IFS2 in different tissues of soybean; C: isoflavone contents in different tissues of soybean; D: relative expression levels of GmMYB184 in different developing stages of soybean seed; E: relative expression levels of IFS2 in different developing stages of soybean seed; F: isoflavone contents in different developing stages of soybean seed."

Fig. 6

GSH induced expression pattern of GmMYB184 and IFS2"

Fig. 7

Subcellular localization analysis of GmMYB184 A: expression of 35S::GFP in Arabidopsis protoplasts; B: expression of GmMYB184::GFP in Arabidopsis protoplasts."

Fig. 8

GmMYB184 induce the promoter activity of IFS2 and CHS8 A: GmMYB184 induces the promoter activity of IFS2; B: GmMYB184 induces the promoter activity of CHS8."

Fig. 9

Relative expression levels of RNAi and over expression of GmMYB184 in hairy roots A: relative expression levels of RNAi of GmMYB184 in hairy roots; B: relative expression levels of over expression of GmMYB184 in hairy roots. ** and *** represent the relative expression levels of GmMYB184 between the transgenic lines and control lines were significantly different at the 0.01 and 0.001 probability levels, respectively."

Fig. 10

Relative isoflavone contents of RNAi and over expression of GmMYB184 in hairy roots A: relative isoflavone contents of RNAi of GmMYB184 in hairy roots; B: relative isoflavone contents of over expression of GmMYB184 in hairy roots. ** represents the relative expression levels of GmMYB184 between the transgenic lines and control lines were significantly different at the 0.01 probability level."

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