Application of Yangbi big-bubble walnut transcription factor gene JsWRKY1

A technology of Yangbi big bubble walnut and transcription factors, which is applied in the research fields of molecular biology and genetic engineering, can solve the problems affecting crop yield and quality, crop yield reduction, high disease incidence, etc., achieve broad market application prospects, shorten the breeding cycle, The effect of reduced use

Active Publication Date: 2015-09-02
KUNMING UNIV OF SCI & TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, plant diseases seriously affect the yield and quality of crops. Among them, fungal diseases are the most important type of plant diseases, accounting for about 70%-80%.
The traditional methods of controlling fungal diseases are mainly breeding resistant new varieties, adopting reasonable farming systems and using chemical pesticides. Although these methods have achieved certain results, they cannot fundamentally solve the problem of fungal diseases

Method used

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  • Application of Yangbi big-bubble walnut transcription factor gene JsWRKY1
  • Application of Yangbi big-bubble walnut transcription factor gene JsWRKY1
  • Application of Yangbi big-bubble walnut transcription factor gene JsWRKY1

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1: JsWRKY1 Full-length cDNA cloning and sequence analysis

[0023] The young leaves of walnut spp. were inoculated with G. anthracnose, and the total RNA was extracted from the leaves 4 h after inoculation. The treated leaves of walnut spp. were ground into powder with liquid nitrogen, and then transferred to a centrifuge tube. Total RNA was extracted by guanidine isothiocyanate method. 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 (2.5 mM 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 ( 200 U), 1 μL M-MLV (200 U), mix well and centrifuge briefly, incubate at 42°C for 1.5 h, take it out and heat at 70°C for 10 min t...

Embodiment 2

[0026] Embodiment 2: plant overexpression vector construction

[0027] Use the SanPrep column type plasmid DNA mini-extraction kit (Shanghai Sangong) to extract the insert JsWRKY1 coli plasmid pMD18-T- JsWRKY1 As well as the plant expression vector pCAMBIA2300S plasmid, 1 μL was used for agarose gel electrophoresis to detect the integrity and concentration of the extracted plasmid. restriction endonuclease Eco RI (TaKaRa) and Bam HI (TaKaRa) against plasmid pMD18-T- JsWRKY1 and pCAMBIA2300S for double enzyme digestion (100 μL system), the reaction system and operation process are as follows: take 20 μL pMD18-T- JsWRKY1 and pCAMBIA2300S plasmid, add 10 μL 10×H buffer, 5 μL EcoRI , 5 μL BamHI , 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 SanPrep Column DNA Gel Extraction Kit (Shanghai Shenggong) was used for JsWRKY1 The fragments and ...

Embodiment 3

[0030] Example 3: Plant genetic transformation mediated by Agrobacterium and screening of transgenic plants

[0031] The transgenic recipient in this experiment was tobacco ( Nicotiana tabacum L.). Tobacco seeds were soaked in 75% alcohol for 30 s, washed with sterile water and washed 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 5-8 days, transfer to light incubator after germination (25°C, 16h / d light), Subculture once a month with MS medium.

[0032] Take out the stored pCAMBIA2300S-containing pCAMBIA2300S- JsWRKY1 Agrobacterium LBA4404 strain of the plasmid was inoculated in 5 mL of LB liquid medium containing 50 mg / L Km and 20 mg / L rifampin in 20 μL, 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 m...

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Abstract

The invention discloses application of a Yangbi big-bubble walnut transcription factor gene JsWRKY1. The JsWRKY1 gene is proved to have a function of enhancing the fungal disease resistance of a plant through technical research related to functional genomics. The antifungal gene JsWRKY1 is established to a plant expression vector and transferred into a tobacco for overexpression, and a transgenic tobacco plant has very high in-vitro antifungal activity, and a transgenic tobacco subjected to JsWRKY1 overexpression achieves obvious inhibiting effect on the growth of botryosphaeria dothidea, moniliform gibberella, colletotrichum gloeosporioides and fusarium oxysporum.

Description

technical field [0001] The present invention relates to molecular biology and genetic engineering related research field, especially the transcription factor gene of walnut pachyrhiza with antifungal activity JsWRKY1 Applications. Background technique [0002] In the 21st century, human beings are faced with three irreversible situations: decrease of cultivated land, increase of population and continuous improvement of people's living standards. Increasing crop yield per unit area is an important measure to solve these problems. However, plant diseases seriously affect the yield and quality of crops. Among them, fungal diseases are the most important type of plant diseases, accounting for about 70%-80%. The traditional methods of controlling fungal diseases are mainly breeding resistant new varieties, adopting reasonable farming systems and using chemical pesticides. Although these methods have achieved certain results, they cannot fundamentally solve the problem of fungal ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/84C12N15/29A01H5/00
Inventor 刘迪秋陈瑞王国东陈朝银韩青葛锋
Owner KUNMING UNIV OF SCI & TECH
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