Application of GmbHLH transcription factor in promotion of synthesizing soy isoflavone

A technology of soybean isoflavones and transcription factors, applied in application, genetic engineering, plant genetic improvement and other directions, can solve problems such as potential safety hazards of gene metabolites, and achieve the effect of increasing soybean isoflavone content

Inactive Publication Date: 2015-07-08
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] The invention provides the application of a GmbH transcription factor in promoting the synthesis of soybean isoflavones, so as to solve the potential safety hazard of gene metabolites

Method used

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  • Application of GmbHLH transcription factor in promotion of synthesizing soy isoflavone
  • Application of GmbHLH transcription factor in promotion of synthesizing soy isoflavone
  • Application of GmbHLH transcription factor in promotion of synthesizing soy isoflavone

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Experimental program
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Effect test

Embodiment 1

[0033] Embodiment 1: Cloning and sequence analysis of GmbHHLH3a gene

[0034] Extraction of soybean RNA and synthesis of cDNA: RNAiso Plus (purchased from TaKaRa Company) was used to extract the total RNA of soybean Jilin 32 grain development for 30 days, and M-MLV reverse transcriptase (RNase H - ) (purchased from TaKaRa) for reverse transcription to synthesize the first cDNA.

[0035]Design and synthesis of primers: The sequences (completed by BGI) of immature embryo expression profiles of soybean variety Jilin 32 at three stages (20, 30 and 50 days) were input into phytozome10.1 for Blast comparison, and a sequence with The complete cDNA sequence of bHLH3-like with higher protein sequence homology of bHLH transcription factor in plants. According to the obtained cDNA sequence, its nucleotide sequence is as described in Sequence No.1, design synthetic primers, GmbHLH3a-RT-F: 5'-CGCCAAATCACAATACCC-3', GmbHLH3a-RT-R: 5'-GCCACTTAACCTACAAGACAGA-3 '. Using the above cDNA as a ...

Embodiment 2

[0037] Embodiment 2: Phylogenetic tree analysis of GmbHHLH3a gene

[0038] According to the soybean bHLH transcription factor and Arabidopsis bHLH transcription factor data published in the plantTFDB online database, the two sets of data were synthesized using ClustalW2 software to generate an unrooted evolutionary tree, and MEGA 6.0 was used to classify and mark the subfamily of the evolutionary tree (the classification scheme refers to 2001 The taxonomic content of Ralf Stracke's article) derived soybean and Arabidopsis MYC-like transcription factor families. The obtained MYC transcription factors were blasted in the NCBI database to detect relevant research information. Change the names related to Arabidopsis or soybean transcription factors to published research names to better understand the homology and functional analysis of the GmMYC gene corresponding to the AtMYC transcription factor.

[0039] After analysis, 19 soybean MYC transcription factors (such as figure 2 ...

Embodiment 3

[0040] Embodiment 3: soybean bHLH-MYC class transcription factor gene grain development period expression pattern

[0041] The sequence obtained by sequencing the expression profile of immature embryos of soybean variety Jilin 32 at three stages (20, 30 and 50 days) was used to screen out the relevant MYC transcription factors using perl language. The cluster analysis of gene expression patterns using MeV found that 9 soybean MYC transcription factors were expressed, and the expression of MYC2 transcription factor that activated DFR gene expression decreased, while the transcription factors such as GmbHLH3 and GmbHLH13 that inhibited DFR gene expression were expressed during grain development significantly increased (e.g. image 3 ).

[0042] This result is highly consistent with the result that the accumulation rate of isoflavones in the soybean grain development period is greatly increased from 30 days to 50 days.

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Abstract

The invention relates to an application of GmbHLH transcription factor in promotion of synthesizing soy isoflavone, belonging to the field of plant genetic engineering. The amino acid sequence of the GmbHLH transcription factor is shown as SEQ ID No.2, the nucleotide sequence of the encoding gene is shown as SEQ ID No.1. The selected GmbHLH transcription factor is only expressed in soy cell nucleus, and the content of the soy isoflavone can be increased by regulating the expression quantity of gene through combination with DNA sequence. No metabolites are generated during the whole process of GmbHLH as a transcription factor, so that the hidden potential hazard of the transgenic metabolism can be reduced to the greatest extent.

Description

technical field [0001] The invention relates to the field of plant genetic engineering, in particular to the application of a soybean bHLH transcription factor and its coding gene to increase the content level of soybean isoflavones in transgenic soybean tissues. Background technique [0002] Isoflavones, as a unique secondary metabolite in leguminous plants, play the role of phytoalexins in plants, especially in the protection of plants against foreign fungal invasion. At the same time, isoflavones, as analogs of human estrogen, also play an important role in promoting human health. In recent years, articles describing isoflavones in biology and medicine are common, mainly explaining the role of isoflavones in promoting human health and preventing diseases. Most of these contents focus on: isoflavones balance female estrogen levels, prevent and treat women's menopausal complications; regulate cardiovascular system, improve myocardial ischemia, and reduce blood sterol level...

Claims

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Application Information

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IPC IPC(8): C07K14/415C12N15/29C12N15/82A01H5/00
CPCC07K14/415C12N15/8243
Inventor 王庆钰刘德泉王英李景文苏连泰刘雅婧王天亮郭文云张海军张鑫生
Owner JILIN UNIV
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