Peanut MYB transcription factor AhMYB32 and application thereof

A technology of transcription factor, R2R3-MYB, applied in application, genetic engineering, plant genetic improvement, etc., can solve problems such as negative regulation

Inactive Publication Date: 2016-09-21
JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, some MYB proteins may negati

Method used

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  • Peanut MYB transcription factor AhMYB32 and application thereof
  • Peanut MYB transcription factor AhMYB32 and application thereof
  • Peanut MYB transcription factor AhMYB32 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Embodiment 1: Acquisition of AhMYB32 gene

[0022] Select the relatively drought-resistant peanut variety: Fenghua No. 1 (publicly known and public, purchased in the market), germinate and grow in the light incubator, and wait for the seedlings to grow to the 3-leaf stage, carry out drought (20% PEG6000) and high-salt (1% NaCl) treatment, After 24 hours of treatment, equal amounts of leaves were taken and mixed into a mortar, quickly ground into powder under liquid nitrogen, and RNA extraction kit (E.Z.N.A. TM Plant RNAKit (purchased from OMEGA Company) was used to extract the total RNA of the sample, and digested with DNAseI. The integrity of the total RNA was detected by 1% agarose gel electrophoresis, and the concentration and purity of the total RNA were detected by a NanoDrop-2000 spectrophotometer. The total RNA was reversed into cDNA by a reverse transcription kit.

[0023] According to the transcriptome sequencing results of peanut drought and salt tolerance, ...

Embodiment 2

[0024] Embodiment 2: Expression analysis of AhMYB32 gene

[0025] The peanut variety: Fenghua No. 1 was selected and germinated and grown in a light incubator. After the seedlings grew to the 3-leaf stage, they were treated with drought (20% PEG6000) and high salt (1% NaCl) respectively. The 12h leaf samples were frozen in liquid nitrogen and stored in a -80°C refrigerator. The total RNA of the samples was respectively extracted with an RNA extraction kit (E.Z.N.A.TM Plant RNA Kit, purchased from OMEGA), and reversed into cDNA.

[0026] Quantitative primers P3 and P4 (Table 1) were designed based on the AhMYB32 sequence, and the peanut 18S RNA gene primer was used as an internal reference control. The reaction system was prepared according to the operation instructions of the Real Master Mix (SYBR Green) kit (TaKaRa), and each reaction was repeated 3 times; The reaction program was: denaturation at 94°C for 5 min; denaturation at 94°C for 20 s, annealing at 55°C for 30 s, ext...

Embodiment 3

[0027] Example 3: Analysis of transcriptional self-activation activity of AhMYB32 gene

[0028]According to the AhMYB32 sequence, primers P5 and P6 (Table 1) with EcoR1 and BamH1 restriction sites were designed respectively, and the target fragment was obtained by PCR amplification. (purchased from Shanghai Jierui Bioengineering Co., Ltd.) After the reaction, transform Escherichia coli Trans1-T1 competent cells (purchased from Beijing Quanshijin Biotechnology Co., Ltd.), and the positive clones were sequenced and analyzed to ensure the reading frame of the coding region in the expression vector Correct, thus obtaining the pGBKT7-AhMYB32 bait vector. Then the bait carrier plasmid was transformed into yeast Y2H competent (purchased from Shanghai Maiqi Biotechnology Co., Ltd.), and the transformation bacteria liquid was divided into 1, 10, 10 2 、10 3 Gradiently dilute, and then take 100 μL of the bacterial solution and spread the transformed bacterial solution on the SD / -Trp an...

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Abstract

The invention discloses a peanut R2R3-MYB transcription factor AhMYB32 and application thereof and belongs to the field of gene engineering. The cDNA sequence of the gene is SEQ ID NO.1, and the coding amino acid sequence of the gene is SEQ ID NO.2. The peanut gene AhMYB32 is reported for the first time, fluorescent quantitation expression analysis shows that the gene resists drought and is high in salt inducible expression, functional verification is carried out through transgenic arabidopsis, and results show that compared with wild plants, overexpression transgenic plants have enhanced drought tolerance and salt tolerance. Thus, AhMYB32 can serve as a target gene to be guided into plants, and the drought tolerance and the salt tolerance of the plants are improved.

Description

technical field [0001] The invention discloses the peanut MYB transcription factor AhMYB32 and its application, which belongs to the field of biological genetic engineering. Background technique [0002] As one of the largest transcription factor families in plants, MYB transcription factors play an important regulatory role in the process of plant stress response. Paz-Ares et al. (1987) cloned the COLORED1 (C1) gene from corn grain aleurone for the first time, encoding a protein with a MYB domain, involved in anthocyanin synthesis. After the Arabidopsis genome was successfully sequenced, the plant MYB gene family was described and classified for the first time. According to the number of MYB domains contained, they can be roughly divided into four categories: R1 subclass with only one MYB domain; R2R3 subclass with two MYB domains; R1R2R3 subclass members with three MYB domains; and finally A subgroup is atypical MYB, which contains 4 or 5 MYB domains, and the number in p...

Claims

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Application Information

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IPC IPC(8): C07K14/415C12N15/29A01H5/00
CPCC07K14/415C12N15/8273
Inventor 刘永惠陈志德沈一沈悦
Owner JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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