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Medicago sativa L. WRKY transcription factor and application thereof in aluminum toxicity resistance and salt stress resistance

A technology of transcription factor and alfalfa, applied in application, genetic engineering, plant genetic improvement, etc., can solve complex genetic background and other problems, achieve the effect of improving aluminum toxicity and salt tolerance, and improving stress resistance

Active Publication Date: 2021-04-09
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Since alfalfa is a perennial, cross-pollinated autotetraploid with a complex genetic background, traditional breeding methods still face great challenges in the selection of aluminum tolerance and salt-tolerant varieties

Method used

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  • Medicago sativa L. WRKY transcription factor and application thereof in aluminum toxicity resistance and salt stress resistance
  • Medicago sativa L. WRKY transcription factor and application thereof in aluminum toxicity resistance and salt stress resistance
  • Medicago sativa L. WRKY transcription factor and application thereof in aluminum toxicity resistance and salt stress resistance

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1: Cloning and sequence analysis of MsWRKY22 gene

[0046] Extraction of total RNA from alfalfa and synthesis of cDNA: Total RNA from leaves of alfalfa WL525 was extracted with EasyPure Plant RNA Kit (purchased from Quanshijin), and reversed with TransScript One-Step gDNA Removal and cDNASynthesis SuperMix (purchased from Quanshijin). Transcription, synthesis of cDNA.

[0047] Design and synthesis of primers: According to the gene chip data of Medicago sativa L. WL525 under Al and control treatments, differentially expressed WRKY gene fragments were selected, compared in NCBI, and Medicago truncatula (Medicago truncatula ) in the WRKY gene with higher homology with this gene, primers were designed according to the gene sequence, and homologous cloning was carried out in Medicago sativa WL525. The upstream primer sequence and downstream primer sequence are shown in SEQ ID No.3 and SEQ ID No.4. Show.

[0048] (F: 5'-TACAGACTTTGTCCCTTTTACCTATG-3', R: 5'-TAGTTCC...

Embodiment 2

[0051] Example 2: Expression patterns of MsWRKY22 gene induced by Al, NaCl and hormones

[0052] Cultivation and treatment of alfalfa WL525 plants: remove the coating, wash the seeds and evenly distribute them in the growth trays with filter paper, keep them moist, and when they germinate and grow the first true leaves, select seedlings with the same growth Transplant in 1 / 2 Hoagland nutrient solution for hydroponics. The formula of Hoagland culture medium is as follows: Ca(NO 3 ) 2 4H 2 O 0.62g / L, KNO 3 0.34g / L, KH 2 PO 4 0.06g / L, NH 4 NO 3 0.053g / L, MgSO 4 0.24g / L, MgCl 2 0.67mg / L, H 3 BO 3 0.38mg / L, MnSO 4 0.2mg / L, ZnSO 4 ·7H 2 O 0.29mg / L, CuSO 4 0.01mg / L, FeSO 4 ·7H 2 O0.02785g / L, EDTA-Na 2 0.0373g / L, PH 5.7-5.8), 28°C, 16h light / 8h dark condition culture, after one week, carry out the following treatment: add 0, 100μM AlCl to the nutrient solution respectively 3 , 200mM NaCl and 100μM ABA, MeJA, SA, take the underground and aerial parts of alfa...

Embodiment 3

[0058] Example 3: Subcellular localization of MsWRKY22 in tobacco epidermal cells.

[0059] Plant expression vector construction: the cloned MsWRKY22 gene was introduced into a restriction site and linked to the pMD18-T vector, followed by double digestion. After the gel was cut and recovered, it was linked with the linearized pHB-YFP vector with the same restriction enzyme site, transformed into Escherichia coli, and the plasmid was extracted after the bacterial liquid PCR verification, and transformed into Agrobacterium competent GV3101.

[0060] Tobacco seedling cultivation: Sprinkle tobacco seeds evenly in a 1:1 mixed substrate with vermiculite. After they germinate, transplant them into flowerpots and pour them into 1 / 2 Hoagland nutrient solution every two days.

[0061] Agrobacterium preparation: Streak activation of Agrobacterium glycerol strains containing pHB-MsWRKY22-YFP and empty load, pick a single clone into YEB liquid medium supplemented with antibiotics Kan50 ...

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Abstract

The invention relates to an application of a medicago sativa L. WRKY transcription factor in improving aluminum toxicity resistance and salt stress resistance of transgenic plants. 1, the WRKY transcription factor protein disclosed by the invention is a protein composed of an amino acid sequence shown in SEQ ID NO. 2; 2, the invention also provides a nucleotide sequence for encoding the protein, which is shown in SEQ ID NO.1; 3, the invention provides an application of the medicago sativa L. WRKY transcription factor protein and an encoding gene thereof in improving aluminum toxicity resistance and salt stress resistance of transgenic plants; 4, the invention provides a host cell containing the MsWRKY22 transcription factor gene and transformed by genetic engineering; and 5, the invention provides an application of an MsWRKY22 nucleotide sequence or an amino acid sequence or a cloning vector or an expression vector or a host cell in genetic engineering. The invention provides a theoretical basis for molecular breeding for improving aluminum toxicity resistance and salt stress resistance of medicago sativa L. by using a genetic engineering technology, and has very important application value.

Description

technical field [0001] The invention relates to the field of plant genetic engineering, in particular to an alfalfa WRKY transcription factor and its application in tolerance to aluminum toxicity and salt stress. Background technique [0002] Alfalfa (Medicago sativa L.) is an important legume forage. Because of its high yield, excellent grass quality and good palatability, it enjoys the reputation of "the king of pastures". In acidic soil (pH<5.0), aluminum is mostly in the form of ions (Al 3+ or Al[OH] 4- ) can be toxic to plants. Soil salinization will seriously affect the yield and quality of alfalfa, causing serious economic losses. [0003] WRKY transcription factors are widely involved in the process of plant growth, development, signal transduction, biotic stress and abiotic stress, and its N-terminal contains a highly conserved WRKYGQK core motif and a zinc finger structure of CX4-5CX22-23HXH. In Arabidopsis, AtWRKY46 can directly specifically bind to the W-b...

Claims

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Application Information

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IPC IPC(8): C07K14/415C12N15/29C12N15/82C12N15/84A01H5/00A01H6/82A01H6/54
CPCC07K14/415C12N15/8271C12N15/8273
Inventor 安渊文武武周鹏王如月赵恩华樊娜娜
Owner SHANGHAI JIAO TONG UNIV
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