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Lilium regale Wilson WRKY transcription factor gene LrWRKY1 and application

A transcription factor and gene technology, applied in the application, genetic engineering, plant genetic improvement and other directions, can solve the problems of long period of breeding resistant varieties, time-consuming and laborious farming system, high chemical pesticide residues, and shorten the breeding period and save costs. , the effect of broad market application prospects

Active Publication Date: 2016-03-30
KUNMING UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, these methods have more or less disadvantages, such as the long period of breeding resistant varieties, high chemical pesticide residues and easy to cause environmental pollution, and time-consuming and laborious adjustment of farming system, so the traditional methods of controlling plant diseases cannot be thoroughly Solve the problem

Method used

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  • Lilium regale Wilson WRKY transcription factor gene LrWRKY1 and application
  • Lilium regale Wilson WRKY transcription factor gene LrWRKY1 and application
  • Lilium regale Wilson WRKY transcription factor gene LrWRKY1 and application

Examples

Experimental program
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Effect test

Embodiment 1

[0020] Example 1: LrWRKY1 full-length gene cloning and sequence analysis

[0021] Inoculate Minjiang lily with Fusarium oxysporum, extract total RNA from the root 24 hours after inoculation, grind the inoculated Minjiang lily root into powder with liquid nitrogen, then transfer it to a centrifuge tube, and extract total RNA by guanidine isothiocyanate method , using reverse transcriptase M-MLV (promega) to synthesize the first strand of cDNA using total RNA as a template, the reaction system and operation process are as follows: take 5 μg TotalRNA, add 50 ngoligo (dT), 2 μL dNTP (2.5 mMeach), DEPC water to the reaction volume After mixing, heat denaturation at 70°C for 5 minutes, then quickly cool on ice for 5 minutes, then add 4 μL 5×First-standbuffer, 0.5 μL RNasin (200 U), 1 μL M-MLV (200 U), mix and centrifuge for a short time. Incubate at 42°C for 1.5h, take it out and heat at 70°C for 10min to terminate the reaction. After the first strand of cDNA was synthesized, it wa...

Embodiment 2

[0024] Embodiment 2: plant overexpression vector construction

[0025] The Escherichia coli plasmid pMD-18T-LrWRKY1 inserted into LrWRKY1 and the plasmid of the plant expression vector pCAMBIA2300s were extracted using the SanPrep column plasmid DNA mini-extraction kit (Shanghai Sangong), and 1 μL was used for agarose gel electrophoresis to detect the extracted Integrity and concentration of the plasmids; Restriction enzymes BamHI (TaKaRa) and XbaI (TaKaRa) were used to double-enzyme digest the plasmids pMD-18T-LrWRKY1 and pCAMBIA2300s respectively (100 μL system), the reaction system and operation process were as follows: 20 μL pMD-18T-LrWRKY1 and pCAMBIA2300s plasmids, add 10 μL 10×Kbuffer, 4 μL BamHI, 6 μL XbaI, 60 μL ddH in sequence 2 O, mix well and 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 perform gel recovery on the LrWRKY1 fragment and the large fragment of the p...

Embodiment 3

[0028] Example 3: Plant genetic transformation mediated by Agrobacterium and screening of transgenic plants

[0029] 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 minutes, 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, 16h / d light) after germination, and use 1 / 2MS medium for subculture once.

[0030] Take out the preserved Agrobacterium LBA4404 strain containing the pCAMBIA2300s-LrWRKY1 plasmid from the -80°C refrigerator, inoculate it into 5 mL of LB liquid medium containing 50 mg / L Km and 20 mg / L rifampicin, and culture it at 28°C until the medium is turbid. Pipette 1mL of turbid bacteria solution onto LB solid medium containing 50mg / LKm, incubate at 28°C for 48h; then scrape off an appropriate amount of Agrobacterium on LB solid medium and inoculate it into ...

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Abstract

The invention discloses a lilium regale WRKY transcription factor gene LrWRKY1. The lilium regale WRKY transcription factor gene LrWRKY1 has a nucleotide sequence shown as SEQ ID NO: 1, and codes a protein with a nucleotide sequence shown as SEQ ID NO: 2. According to the lilium regale Wilson WRKY transcription factor gene LrWRKY1, functional genomics related technical research verifies that the LrWRKY1 gene has the function of increasing the fungus resistance of plants; when the antifungal LrWRKY1 gene is constructed to a plant expression vector and is transferred to tobaccos for overexpression, transgenic tobacco plants have strong in-vitro antifungal activities, and experimental results show that the LrWRKY1 overexpressed transgenic tobaccos have obvious inhibiting effects on the growth of four fungi including botryosphaeria, sclerotinite, botrytis cinerea and fusarium oxysporum.

Description

technical field [0001] The invention relates to the research fields of molecular biology and genetic engineering related technologies, in particular to a Minjiang lily WRKY transcription factor gene LrWRKY1 with antifungal activity and its application. Background technique [0002] Plant diseases are a very difficult problem in agricultural production, especially fungal diseases, which account for about 80% of the total plant diseases and seriously affect the yield and quality of crops. Traditional disease control methods have achieved certain results. One is to rely on traditional breeding methods to cultivate resistant varieties, the other is to use chemical pesticides, and the third is to adopt farming systems such as crop rotation. However, these methods have more or less disadvantages, such as the long period of breeding resistant varieties, high chemical pesticide residues and easy to cause environmental pollution, and time-consuming and laborious adjustment of farming...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C12N15/82A01H5/00
CPCC12N15/8205C12N15/8282C07K14/415
Inventor 刘迪秋王国东普丽梅李金晶杨野关瑞攀白志伟葛锋
Owner KUNMING UNIV OF SCI & TECH
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