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Lilium regale WRKY transcription factor gene LrWRKY2 and application thereof

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

Active Publication Date: 2020-02-21
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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  • Lilium regale WRKY transcription factor gene LrWRKY2 and application thereof
  • Lilium regale WRKY transcription factor gene LrWRKY2 and application thereof
  • Lilium regale WRKY transcription factor gene LrWRKY2 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

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

Embodiment 2

[0023] Embodiment 2: plant overexpression vector construction

[0024] Use the SanPrep column plasmid DNA mini-extraction kit (Shanghai Sangong) to extract the insert LrWRKY2 coli plasmid pGEM-T- LrWRKY2 As well as the plasmid of the plant expression vector pCAMBIA2300s, take 1 μL for agarose gel electrophoresis to detect the integrity and concentration of the extracted plasmid; Bam HI (TaKaRa) and Xba I(TaKaRa) respectively on the plasmid pGEM-T- LrWRKY2 and pCAMBIA2300s for double enzyme digestion (100 μL system), the reaction system and operation process are as follows: Take 20 μL pGEM-T- LrWRKY2 and pCAMBIA2300s plasmid, add 10 μL 10×K buffer, 4 μL Bam HI, 6 μL Xba I. 60 μL ddH 2 O, mix well and then centrifuge for a short time, and place it at 37°C for overnight reaction; spot all the digested products on agarose gel for electrophoresis, and then LrWRKY2 The fragments and the large fragments of the pCAMBIA2300s vector were gel-recovered separately, and the SanP...

Embodiment 3

[0027] Example 3: Plant genetic transformation mediated by Agrobacterium and screening of transgenic plants

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

[0029] Take out the pCAMBIA2300s- containing pCAMBIA2300s- LrWRKY2 The plasmid Agrobacterium LBA4404 strain was inoculated in 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 incubate at 28°C for 48 h; then scrape off an appropriate amount of Agrobacterium on LB solid medium and inoculate it with 20 mg / L ...

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Abstract

The invention discloses a lilium regale WRKY transcription factor gene LrWRKY2 and a preparation method thereof. A nucleotide sequence of the gene is shown as SEQ ID NO: 1, and a protein with an aminoacid sequence shown as shown in SEQ ID NO: 2; according to the invention, functional genomics related technical researches prove that the LrWRKY2 gene has the function of improving the antifungal performance of plants; the antifungal LrWRKY2 gene disclosed by the invention is constructed on a plant expression vector and is transferred into tobacco for overexpression; the transgenic tobacco plantshave very strong fungal infection resistance, and experimental results show that the transgenic tobacco with over-expression of LrWRKY2 has high-level resistance to infection of alternaria oryzae, fusarium solani, fusarium verticillatum, Botryosphaeria dothidea and Alternaria panax.

Description

technical field [0001] The invention relates to the field of molecular biology and genetic engineering related technology research, in particular to a Minjiang lily WRKY transcription factor gene with the ability to resist fungal infection LrWRKY2 and applications. Background technique [0002] Plants massively reprogram their transcriptomes in a highly variable and temporal manner in response to pathogen invasion or other adversity stresses, and this regulatory response that endows plants with plastic adaptation to different environmental conditions is the result of a variety of transcription factors The result of network action. WRKY transcription factor is a regulatory protein gene family in the plant defense response-related transcription factor network, which is mainly involved in the response of plant immune system to a variety of different biotic and abiotic stresses (Pandey SP, Somssich IE. The role of WRKY transcription factors in plant immunity Bakshi M, Oelmülle...

Claims

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Application Information

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IPC IPC(8): C07K14/415C12N15/29C12N15/66C12N15/82A01H5/00A01H6/82
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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