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Application of LrWRKY-R2 protein and coding gene thereof in regulation and control of plant stress resistance

A technology of plant stress resistance and stress resistance, which is applied in the field of plant genetic engineering, can solve unexplained problems, reduce the use of pesticides, improve the yield and quality of lily, and improve the ability of high temperature resistance and gray mold resistance Effect

Pending Publication Date: 2022-05-10
YANGTZE NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are few reports about the WRKY family members of Lilium Minjiang's high temperature resistance, and the mechanism of WRKY regulating the high temperature resistance or disease resistance of Lily is far from elucidated.

Method used

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  • Application of LrWRKY-R2 protein and coding gene thereof in regulation and control of plant stress resistance
  • Application of LrWRKY-R2 protein and coding gene thereof in regulation and control of plant stress resistance
  • Application of LrWRKY-R2 protein and coding gene thereof in regulation and control of plant stress resistance

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1 Cloning and Sequence Analysis of LrWRKY-R2 Gene in Lilium Minjiang

[0027] Using the leaves of Lily of the Minjiang River as the material, using TRIzol TMPlus RNA Purification Kit (12183555, Invitrogen TM ), total RNA was extracted according to the instructions, and DNase I (18047019, Invitrogen TM ) to remove residual traces of DNA, and use a spectrophotometer to measure the concentration of RNA for later use.

[0028] About 2.0 μg of total RNA from leaves of Lilium Minjiang was taken, and the first-strand cDNA was synthesized according to the instructions of PrimeScript II first-strand cDNA synthesis kit (6210A, Takara).

[0029] PCR amplification system: high-fidelity amplification enzyme Prime STAR HS (R010A, TaKaRa) 0.25μL, 5×PrimeSTAR Buffer (Mg 2+ Plus) 5 μL, forward primer (LrWRKY-R2-F, 10 μM) 0.5 μL, reverse primer (LrWRKY-R2-R, 10 μM) 0.5 μL, template (DNA) 1 μL, dNTP (2.5 mM) 2 μL, sterile ddH 2 O to make up to 25 μL.

[0030] The forward and ...

Embodiment 2

[0037] Example 2 Construction of GFP fusion expression vector and analysis of subcellular localization

[0038] According to the vector sequence of pTF101-GFP and the full-length sequence of LrWRKY-R2 gene (SEQ ID NO.1), design forward primer (LrWRKY-R2-gfp-F) and reverse primer (LrWRKY-R2-gfp-R), primer Introduce seamless cloning (In-fusion) vector linker sequence and restriction site sequence. The LrWRKY-R2 gene fragment was amplified by PCR using the TA-ligated positive clone plasmid in Example 1 as a template.

[0039] The primer sequences are as follows:

[0040]

[0041]

[0042] Among them, the bold sequence of LrWRKY-R2-gfp-F is the Hind III restriction site, and the bold sequence of LrWRKY-R2-gfp-R is the Bam HI restriction site. The linker sequence of the In-fusion cloning vector is underlined.

[0043] PCR reaction system: high-fidelity amplification enzyme PrimeSTAR HS (R010A, TaKaRa) 0.5μL, 5xPrimeSTARBuffer (Mg 2+ Plus) 10 μL, forward primer (10 μM) 1 μ...

Embodiment 3

[0052] Example 3 LrWRKY-R2 Overexpression Vector Construction and Genetic Transformation of Arabidopsis

[0053] (1) Construction of pBI121-35S::LrWRKY-R2 overexpression vector

[0054] According to the pBI121 vector sequence and LrWRKY-R2 gene sequence (SEQ ID NO.1), design primers LrWRKY-R2-inf-F and LrWRKY-R2-inf-R, and introduce seamless cloning (In-fusion) vector adapter sequence into the primers and enzyme cleavage site sequences. The specific amplification of the LrWRKY-R2 gene fragment was performed using the TA-ligated positive clone plasmid in Example 1 as a template.

[0055] The primer sequences are as follows:

[0056]

[0057]

[0058] Among them, the bold sequence of the LrWRKY-R2-inf-F primer is the BamHI restriction site, and the bold sequence of the LrWRKY R2-inf-R primer is the Sac I restriction site. The linker sequence of the In-fusion cloning vector is underlined.

[0059] The PCR reaction system is: high-fidelity amplification enzyme PrimeSTAR ...

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Abstract

The invention discloses an application of an LrWRKY-R2 protein and a coding gene thereof in regulation and control of plant stress resistance, the amino acid sequence of the LrWRKY-R2 protein is shown as SEQ ID NO.2, and the nucleotide sequence of the coding gene of the LrWRKY-R2 protein is shown as SEQ ID NO.1. The invention further discloses an application of the LrWRKY-R2 protein and the coding gene thereof in regulation and control of plant stress resistance. Biochemical analysis shows that the LrWRKY-R2 is a WRKY family IId cluster member positioned in a cell nucleus. After the LrWRKY-R2 gene is overexpressed in arabidopsis thaliana or lilium brownii, a heat shock stress treatment experiment and a pathogenic bacteria bacteriostasis experiment find that the LrWRKY-R2 transcription factor is a WRKY family member with dual functions of regulating high temperature stress and gray mold resistance, a new gene resource is provided for improving the stress resistance of plants through a molecular means, and the LrWRKY-R2 transcription factor has a wide application prospect. Meanwhile, a very excellent new target is provided for genetic improvement of the lilium brownii, powerful technical support is provided for cultivation of new varieties of the lilium brownii with heat resistance and disease resistance or new materials of other plants, and important theoretical and application values are achieved for achieving high-quality and high-yield breeding in agricultural production.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering, and particularly relates to the application of LrWRKY-R2 protein and its coding gene in regulating plant stress resistance. Background technique [0002] In nature, plants are usually subjected to various biotic and abiotic stresses, and have evolved a series of defense mechanisms. As a precious flower and an important vegetable, lily has very considerable economic value. However, most lilies like cool and humid climates, and the optimum growth temperature for lilies is about 18-24°C. High temperature will cause short-term heat stress on lily plants, severe pest damage and flower abortion, directly affect the quality of cut flowers, and cause bulb degradation, etc. (Yin H, Chen Q, Yi M (2008) Effects of short-term heat stress on oxidative damage and responses of antioxidant system in Lilium longiflorum.Plant Growth Regul 54:45-54.Xin HB,Zhang H,Chen L,et al.(2010)Cloning and c...

Claims

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Application Information

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IPC IPC(8): C07K14/415C12N15/29C12N15/82A01H5/00A01H6/20A01H6/56
CPCC07K14/415C12N15/8271C12N15/8282
Inventor 符勇耀吴娇李娟刘萱姜思佳焦丽徐文姬
Owner YANGTZE NORMAL UNIVERSITY
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