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A Minjiang lily wrky transcription factor gene lrwrky4 and its application

A technology of transcription factors and genes, applied in the fields of application, genetic engineering, plant genetic improvement, etc., to achieve the effects of reduced use, shortened breeding cycle, and broad market application prospects

Active Publication Date: 2022-06-14
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 lrwrky4 and its application
  • A Minjiang lily wrky transcription factor gene lrwrky4 and its application
  • A Minjiang lily wrky transcription factor gene lrwrky4 and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Example 1: LrWRKY4 Full-length gene cloning and sequence analysis

[0021] The roots of lily were inoculated with Fusarium oxysporum, total RNA was extracted from the root 24 h after inoculation, the treated root of lily was ground into powder with liquid nitrogen, then transferred into a centrifuge tube, and extracted by guanidine isothiocyanate method Total RNA; M-MLV reverse transcriptase (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 Mix ( 2.5mM each), the reaction volume was made up to 14.5 μL with DEPC water; after mixing, heated and denatured at 70°C for 5 min, then rapidly cooled on ice for 5 min, then added 4 μL 5×First-stand buffer, 0.5 μL RNasin (200U), 1 μL M-MLV (200U), mix well and centrifuge briefly, incubate at 42°C for 1.5 h, take it out and heat at 70°C for 10 min to terminate the reaction; after the...

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 LrWRKY4 coli plasmid pGEM-T- LrWRKY4 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 and Eco RI respectively for plasmid pGEM-T- LrWRKY4 and pCAMBIA2300S for double enzyme digestion (100 μL system), the reaction system and operation process are as follows: take 20 μL pGEM-T- LrWRKY4 and pCAMBIA2300S plasmid, add 10μL 10×M buffer, 5μL Xba I, 5 μL Eco RI, 60 μL ddH 2 O, after mixing, centrifuge for a short time, and place at 37°C for overnight reaction. All digested products were subjected to agarose gel electrophoresis, and then the LrWRKY4 The fragments and the large fragments of the pCAMBIA2300S vector were gel-recovered separately, and 1 μL of the recovered product ...

Embodiment 3

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

[0029] The transgenic recipient in this experiment was tobacco ( Nicotiana tabacum ), the tobacco seeds were soaked in 75% alcohol for 30s, washed with sterile water and washed with 0.1% HgCl 2 Soak for 8 min, then wash several times with sterile water, sow on 1 / 2MS medium, culture in dark at 28°C for 5-8d, transfer to light incubator (25°C, 16h / d light) after germination, and then Subculture once a month with 1 / 2 MS medium.

[0030] Take out the stored pCAMBIA2300S-containing pCAMBIA2300S- LrWRKY4 Agrobacterium LBA4404 strain of the plasmid, inoculated 20 μL into 5 mL LB liquid medium containing 50 mg / L Km and 20 mg / L rifampin, 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...

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Abstract

The invention discloses a Minjiang lily WRKY transcription factor gene LrWRKY4 , its nucleotide sequence is as described in SEQ ID NO: 1, and the protein encoding the amino acid sequence as shown in SEQ ID NO: 2 is confirmed by the present invention through functional genomics-related technical research LrWRKY4 The gene has the function of improving the anti-fungal effect of the plant, and the anti-fungal effect of the present invention LrWRKY4 The gene is constructed on a plant expression vector and transferred to tobacco for overexpression. The transgenic tobacco has strong antifungal activity, and the experimental results show that the overexpression LrWRKY4 The transgenic tobacco has a high level of resistance to the infection of Nigeria oryzae, Fusarium graminearum, Verticillium verticillium, Vitis vinifera, 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 LrWRKY4 and applications. Background technique [0002] Plants are subject to various abiotic or biotic stresses during their growth and development. Abiotic stresses include drought, waterlogging, salinity, lack of nutrients, low temperature, radiation, etc. Biological stresses include fungi, bacteria, viruses, nematodes or parasitic Seed plants etc. Among the biotic stresses, the number of plant diseases caused by fungi is the largest, accounting for more than 70%, seriously endangering the growth of plants and their economic value. Fungal vitality is tenacious, and many pathogenic fungi can form special tissues or spores to overwinter, and are harmful all year round. Fungi are easy to spread, mainly through airflow and water flow in the field; in addition, wind, rain, and inse...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/415C12N15/29C12N15/84A01H5/00A01H6/82
CPCC07K14/415C12N15/8282
Inventor 刘迪秋赵秦郑锂蕾陈虹均王自娥李珊葛锋
Owner KUNMING UNIV OF SCI & TECH
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