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A kind of alfalfa myb transcription factor and its application of aluminum tolerance

A technology of transcription factor and alfalfa, applied in application, genetic engineering, plant gene improvement, etc., can solve problems such as unreported research data, decreased ability of plants to tolerate low temperature, and incomplete gene annotation

Active Publication Date: 2021-09-03
SHANGHAI JIAO TONG UNIV
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  • Abstract
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AI Technical Summary

Problems solved by technology

In alfalfa, the research on MYB transcription factor family genes is very limited. Although the genome sequencing of Medicago truncatula has been completed, the related gene annotation is not complete. So far, there has been no overall description of the MYB transcription factor family in Medicago truncatula Related reports
In the research published so far, the research on the function of MYB transcription factors in alfalfa mainly focuses on its regulation of the synthesis of proanthocyanidins (PA). Very beneficial antioxidant, anti-cancer, anti-inflammatory activity, etc. Recent studies have shown that phenols and flavonoids can reduce plant aluminum toxicity, but further research data has not been reported
In addition, Zhang et al. (2016) found in the study of Medicago truncatula that MtCBF4 (low temperature tolerance-related transcription factor) can directly bind to the promoter of the downstream low temperature tolerance-related gene MtCAS15, while MtMYB3 can bind to the MtCBF4 promoter to inhibit the expression of MtCBF4 , thus reducing the ability of plants to tolerate low temperature, indicating that MtMYB3 is a negative regulator

Method used

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  • A kind of alfalfa myb transcription factor and its application of aluminum tolerance
  • A kind of alfalfa myb transcription factor and its application of aluminum tolerance
  • A kind of alfalfa myb transcription factor and its application of aluminum tolerance

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Experimental program
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Effect test

Embodiment 1

[0032] Example 1: Cloning and sequence analysis of MsMYB741 gene

[0033] Extraction of RNA from alfalfa and synthesis of cDNA: Extract total RNA from the root of alfalfa WL525 with EasyPure Plant RNA Kit (purchased from Beijing Quanshijin), and use TransScript One-Step gDNA Removal and cDNASynthesis SuperMix (purchased from Beijing Quanshijin) to carry out Reverse transcription, cDNA synthesis.

[0034] Design and synthesis of primers: The seeds of the alfalfa WL525 variety were germinated for 7 days and treated with aluminum stress. After 60 hours, the whole plant was sampled and sent to Invitrogen for gene chip analysis. The gene chip used Agilent's Medicago truncatula gene chip. Through analysis, it was found that a probe whose expression was significantly up-regulated under aluminum stress compared with the control was searched in the Medicago truncatula database to obtain the cDNA sequence, and synthetic primers were designed, F: 5'-ATGTATTCAGGAATGATGGA-3', R: 5'-TTAACA...

Embodiment 2

[0036] Example 2: Expression pattern of MsMYB741 gene under adversity and hormone induction

[0037] Treatment of alfalfa variety WL525: with improved 1 / 2 Hoagland medium (Ca(NO 3 ) 2 4H 2 O 945mg / L, KNO 3 506mg / L, NH 4 NO 3 80mg / L, KH 2 PO 4 136mg / L, MgSO 4 ·7H 2 O 493mg / L, EDTA-Na 2 18.65mg / L, FeSO 4 ·7H 2 O 13.9mg / L, KI 0.83mg / L, H 3 BO 3 6.2mg / L, MnSO 4 ·H 2 O 16.9mg / L, ZnSO 4 ·7H 2 O 8.6mg / L, Na 2 MoO 4 2H 2 O 0.25mg / L, CuSO 4 ·5H 2 O 0.025mg / L, CoCl 2 ·6H 2 (00.025mg / L, pH 5.7-5.8), 28 ℃, 16h light / 8h dark conditions cultivate alfalfa hydroponic seedlings, get the hydroponic seedlings of four-leaf stage and carry out following processing:

[0038] Aluminum stress treatment: the alfalfa seedlings were exposed to 100 μmol / L AlCl 3 ·6H 2 In the 1 / 2 Hoagland culture medium of O, samples were taken after treatment for 0h, 1h, 3h, 6h, 9h, 12h and 24h, placed in liquid nitrogen quickly, and stored at -80°C for later use;

[0039] High-salt treatme...

Embodiment 3

[0046] Embodiment 3: Construction of expression vector

[0047] This embodiment according to figure 2 The schematic diagram of the expression vector structure is shown to construct the plant expression vector of MsMYB741. Specifically, the following methods can be used:

[0048] 1) Construct the plant overexpression vector of MsMYB741 by enzyme-cut ligation technology, design primers according to the target gene sequence, introduce Pst I and Bam HI restriction sites in the upstream and downstream primers, and upstream pHB-MsMYB741-F: 5′-AACTGCAGATGTATTCAGGAATGATG -3', downstream pHB-MsMYB741-R: 5'-CGCGGATCCACAAAAGGGAGCAACTAC-3', PCR amplification was performed using the pMD18-T-MsMYB741 plasmid as a template. After the amplification, the PCR product was gel-recovered, and the recovered product was connected to the cloning vector pMD18-T (purchased from Takara). After overnight incubation at 16°C, Escherichia coli (E.coli) DH5α competent cells (purchased from Shanghai Weidi...

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Abstract

The invention discloses an alfalfa MYB transcription factor and its application of aluminum toxicity resistance, and relates to the field of plant genetic engineering. The MYB transcription factor is derived from the alfalfa variety WL525 and is named MsMYB741 transcription factor. The amino acid sequence of the MsMYB741 transcription factor It is the amino acid sequence shown in the sequence SEQ ID NO: 2, or the derived amino acid sequence of the sequence SEQ ID NO: 2, and the protein encoded by the derived amino acid sequence has the characteristics of MYB transcription factor; The coding gene of transcription factor, the nucleotide sequence of the coding gene is the nucleotide sequence shown in the sequence SEQ ID NO: 1, or the homologous sequence and derivative nucleotide sequence of the sequence SEQ ID NO: 1 . The coding gene of the MsMYB741 transcription factor provided by the present invention has achieved remarkable effects in constructing expression vectors and cultivating transgenic plants resistant to aluminum toxicity, which provides a basis for improving the stress resistance of plants, especially for cultivating alfalfa varieties resistant to aluminum toxicity, It has very important application value.

Description

technical field [0001] The invention relates to the field of plant genetic engineering, in particular to an alfalfa MYB transcription factor and the application thereof for tolerance to aluminum toxicity. Background technique [0002] Medicago sativa L. (Medicago sativa L.), also known as alfalfa, is a perennial herb known as the "king of herbage". Its six advantages are as follows: first, high yield; second, good quality; The third is an important energy feed; the fourth is that alfalfa also contains some substances that are very beneficial to livestock; However, biotic and abiotic stresses have extremely adverse effects on the growth and development of alfalfa, such as aluminum toxicity. [0003] Acidic soils with a pH below 5.0 account for 30%-40% of the global arable soil area and are one of the important abiotic factors limiting crop growth. Aluminum (Al) has a content of about 7% in the earth's crust and is widely distributed. It usually exists in the form of silicat...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/415C12N15/29C12N15/82A01H5/06A01H5/10A01H6/54A01H6/20
CPCC07K14/415C12N15/8271
Inventor 安渊苏连泰周鹏于晨张钰靖王如月赵恩华
Owner SHANGHAI JIAO TONG UNIV
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