Lilium regale WRKY transcription factor gene LrWRKY3 and application thereof

A transcription factor and gene technology, applied in application, genetic engineering, plant genetic improvement and other directions, can solve the problems affecting the yield and quality of cut lily flowers, scale rot and shedding, and the quality of seed bulbs, and achieve broad market application prospects and breeding cycles. The 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 LrWRKY3 and application thereof
  • Lilium regale WRKY transcription factor gene LrWRKY3 and application thereof
  • Lilium regale WRKY transcription factor gene LrWRKY3 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Example 1: LrWRKY3 Full-length gene cloning and sequence analysis

[0021] 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), 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 termin...

Embodiment 2

[0024] Embodiment 2: plant overexpression vector construction

[0025] Use the SanPrep column plasmid DNA mini-extraction kit (Shanghai Sangong) to extract the insert LrWRKY3 coli plasmid pGEM-T- LrWRKY3 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; Xba I (TaKaRa) and EcoR I(TaKaRa) and respectively on the plasmid pGEM-T- LrWRKY3 and pCAMBIA2300s for double enzyme digestion (100 μL system), the reaction system and operation process are as follows: Take 20 μL pGEM-T- LrWRKY3 and pCAMBIA2300s plasmid, add 10 μL 10×M buffer, 4 μL Xba I, 6 μL EcoR 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 LrWRKY3 The fragments and the large fragments of the pCAMBIA2300s vector were gel-recovered separately, and the ...

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, 16 h / d light) after germination, and then monthly Subculture once with 1 / 2 MS medium.

[0030] Take out the pCAMBIA2300s- containing pCAMBIA2300s- LrWRKY3 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 m...

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Abstract

The invention discloses a lilium regale WRKY transcription factor gene LrWRKY3 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 LrWRKY3 gene has the function of improving the antifungal performance of plants; the antifungal LrWRKY3 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 fungus infection resistance, and experimental results show that transgenic tobacco leaves with over-expression of LrWRKY3 have very strong resistance to infection of five pathogenicfungi such as alternaria oryzae, Botryosphaeria dothidea, fusarium graminearum, Fusarium rotunda and fusarium solani.

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 LrWRKY3 and applications. 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. Plants have an innate immune system to avoid the invasion of pathogenic bacteria, including a complex network that strictly regulates defense responses. Transcription factors regulate genes as signal pathways and play an important role in plant disease resistance responses, especially the WRKY transcription factor family, which participates in the defense of biotic and abiotic Regulation of defense (Agarwal P, Patel K, Agarwal PK. Ectopic expression of wxya confers enhanced r...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/415C12N15/29C12N15/84A01H5/00A01H6/82
CPCC07K14/415C12N15/8282
Inventor 刘迪秋普丽梅王自娥郑锂蕾陈虹均李珊葛锋
Owner KUNMING UNIV OF SCI & TECH
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