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A Minjiang Lily wrky transcription factor gene lrwrky2 and its application

A technology of transcription factors and genes, applied in application, genetic engineering, plant genetic improvement, etc., can solve the problems affecting the yield and quality of lily cut flowers, scale rot and falling off, bulb quality decline, etc., and achieve broad market application prospects and breeding cycle Effect of shortening, reducing usage

Active Publication Date: 2022-06-24
KUNMING UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Fusarium infection of lily bulbs causes basal necrosis and scale rot and falls off, resulting in a decline in bulb quality; leaves turn yellow, wilt and droop after plants are infected with Fusarium, and plants wither and die early, seriously affecting the yield and quality of lily cut flowers

Method used

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  • A Minjiang Lily wrky transcription factor gene lrwrky2 and its application
  • A Minjiang Lily wrky transcription factor gene lrwrky2 and its application
  • A Minjiang Lily wrky transcription factor gene lrwrky2 and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Example 1: LrWRKY2 Full-length gene cloning and sequence analysis

[0020] Minjiang lily was inoculated with Fusarium oxysporum, and total RNA was extracted from the roots 24 h after inoculation. The inoculated roots of Minjiang lily were ground into powder with liquid nitrogen, and then transferred to a centrifuge tube, and total RNA was extracted by guanidine isothiocyanate method. RNA, using reverse transcriptase M-MLV (promega) to synthesize the first strand of cDNA with total RNA as template, the reaction system and operation process are: take 5 μg total RNA, add 50 ng oligo (dT), 2 μL dNTP (2.5 mM) in turn. each), DEPC water was added to the reaction volume of 14.5 μL; after mixing, heat denaturation at 70°C for 5 min, then quickly cool on ice for 5 min, then add 4 μL 5×First-stand buffer, 0.5 μL RNasin (200U) , 1 μL M-MLV (200U), mix well and centrifuge for a short time, incubate at 42°C for 1.5h, take out and heat at 70°C for 10 min to terminate the reaction; s...

Embodiment 2

[0023] Example 2: Plant overexpression vector construction

[0024] The insert was extracted using the SanPrep column plasmid DNA mini-extraction kit (Shanghai Shenggong). LrWRKY2 The E. coli plasmid pGEM-T- LrWRKY2 And the plasmid of the plant expression vector pCAMBIA2300s, take 1 μL for agarose gel electrophoresis to test the integrity and concentration of the extracted plasmid; use restriction endonuclease Bam HI (TaKaRa) and Xba I(TaKaRa) was applied to the plasmid pGEM-T- LrWRKY2 Double-enzyme digestion with pCAMBIA2300s (100 μL system), the reaction system and operation process are: take 20 μL pGEM-T- LrWRKY2 and pCAMBIA2300s plasmid, followed by adding 10 μL 10×Kbuffer, 4 μL Bam HI, 6 μL Xba 1. 60 μL ddH 2 O, after mixing, centrifuge for a short time, and place it at 37 °C for overnight reaction; spot all the digestion products on agarose gel for electrophoresis, and then analyze LrWRKY2 The fragment and the large fragment of the pCAMBIA2300s vector were gel...

Embodiment 3

[0027] Example 3: Agrobacterium-mediated plant genetic transformation and screening of transgenic plants

[0028] The transgenic receptor in this experiment is tobacco. Tobacco seeds were soaked in 75% alcohol for 30s, washed with sterile water, and then washed with 0.1% HgCl 2 Soak for 8 min, then wash several times with sterile water, sow on 1 / 2 MS medium, cultivate in the dark at 28 °C for 6 d, transfer to a light incubator (25 °C, 16 h / d light) after germination, and later Subculture with 1 / 2 MS medium once a month.

[0029] Remove the stored pCAMBIA2300s-containing pCAMBIA2300s- LrWRKY2 Plasmid Agrobacterium LBA4404 strain was inoculated into 5 mL of LB liquid medium containing 50 mg / L Km and 20 mg / L rifampicin, and cultured at 28°C until the medium was turbid. Pipette 1 mL of turbid bacterial solution onto LB solid medium containing 50 mg / L Km, and cultivate at 28 °C for 48 h; then scrape off an appropriate amount of Agrobacterium on the LB solid medium and inoculate i...

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Abstract

The invention discloses a Minjiang lily WRKY transcription factor gene LrWRKY2 , whose nucleotide sequence is as described in SEQ ID NO: 1, and encodes a protein with an amino acid sequence as shown in SEQ ID NO: 2, which is confirmed by functional genomics-related technical research in the present invention LrWRKY2 The gene has the function of improving the antifungal effect of the plant, and the antifungal effect of the present invention LrWRKY2 The gene is constructed on a plant expression vector and transferred to tobacco for overexpression. The transgenic tobacco plants have a strong ability to resist fungal infection. The experimental results show that the overexpression LrWRKY2 The transgenic tobacco has a high level of resistance to the infection of Nigeria oryzae, Fusarium solani, Fusarium verticillium, Vitis vinifera, and Alternaria ginseng.

Description

technical field [0001] The invention relates to the research field of molecular biology and related technologies of genetic engineering, in particular to a Minjiang lily WRKY transcription factor gene with antifungal infection ability LrWRKY2 and applications. Background technique [0002] Plants undergo large-scale reprogramming of their transcriptomes in a highly variable and temporal manner in response to pathogen invasion or other stress, and this regulatory response that confers plastic adaptation to different environmental conditions is the result of multiple transcription factors The result of network action. WRKY transcription factors are a family of regulatory protein genes in the plant defense response-related transcription factor network, which are mainly involved in the plant immune system responding to a variety of different biotic and abiotic stresses (Pandey SP, Somssich IE. The role of WRKY transcription factors in plant immunity . PlantPhysiol, 2009, 150(4...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/415C12N15/29C12N15/66C12N15/84A01H5/00A01H6/82
CPCC07K14/415C12N15/66C12N15/8282
Inventor 刘迪秋普丽梅陈虹均郑锂蕾李珊王自娥葛锋
Owner KUNMING UNIV OF SCI & TECH
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