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Pseudo-ginseng WRKY transcription factor gene PnWRKY9 and application thereof

A transcription factor and gene technology, applied in the research fields of molecular biology and genetic engineering, can solve the problems of environmental pollution, huge investment, endanger human and animal safety, etc., and achieve broad market application prospects, shorten the breeding cycle, and reduce environmental pollution.

Active Publication Date: 2021-05-25
KUNMING UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The use of chemical pesticides often endangers the safety of humans and animals and causes environmental pollution
The cultivation of disease-resistant varieties takes a long time, slow results, huge investment, and difficult to obtain disease-resistant resources
Neither of these two methods can solve the problem of plant diseases very well; however, genetic engineering uses molecular biology techniques to clone disease-resistant genes and can quickly breed disease-resistant plant varieties with the help of transgenic technology, which is a new method to enhance plant disease resistance

Method used

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  • Pseudo-ginseng WRKY transcription factor gene PnWRKY9 and application thereof
  • Pseudo-ginseng WRKY transcription factor gene PnWRKY9 and application thereof
  • Pseudo-ginseng WRKY transcription factor gene PnWRKY9 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1: PnWRKY9 Full-length cDNA cloning and sequence analysis

[0024] The roots of Panax notoginseng were inoculated with Fusarium solani rot, total RNA was extracted from the roots 12 hours after inoculation, the treated roots of Panax notoginseng were ground into powder with liquid nitrogen, then transferred to a centrifuge tube, and total RNA was extracted by TRlzol 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 of total RNA, add 50 ngoligo (dT), 2 μL dNTP Mix (2.5mM each), Make up the reaction volume to 14.5 μL with DEPC water; after mixing, heat and denature 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 ( 200U), mix well and centrifuge briefly, incubate at 42°C for 1.5h, take it out and heat at 70°C for 10min to terminate the reac...

Embodiment 2

[0027] Embodiment 2: plant overexpression vector construction

[0028] Use the SanPrep column type plasmid DNA mini-extraction kit (Shanghai Sangong) to extract the insert PnWRKY9 coli plasmid pGEM-T- PnWRKY9 As well as the plant expression vector pCAMBIA2300S plasmid, 2 μL was used for agarose gel electrophoresis to detect the integrity and concentration of the extracted plasmid. restriction endonuclease Eco RI and Bam HI respectively for plasmid pGEM-T- PnWRKY9 and pCAMBIA2300S for double enzyme digestion (50μL system), the reaction system and operation process are as follows: take 10μL pGEM-T- PnWRKY9 and pCAMBIA2300S plasmid, add 5μL 10×Hbuffer, 2.5μL Eco RI, 2.5 μL Bam HI, 30 μL ddH 2 O, after mixing, centrifuge for a short time, and place it at 37°C for 3.5h. All digested products were subjected to agarose gel electrophoresis, and then the gel recovery kit was used for PnWRKY9 The target fragment and the large fragment of the pCAMBIA2300S vector were gel-rec...

Embodiment 3

[0031] Example 3: Plant genetic transformation mediated by Agrobacterium and screening of transgenic plants

[0032] The transgenic recipient in this experiment was tobacco ( Nicotiana tabacum ). Tobacco seeds were soaked in 75% alcohol for 30s, 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 about 14 days, transfer to light incubator (25°C, 16h / d light) after germination, and then monthly Subculture once with MS medium.

[0033] Take out the stored pCAMBIA2300S-containing pCAMBIA2300S- PnWRKY9 For the Agrobacterium LBA4404 strain of the plasmid, take 20 μL and inoculate it into 5 mL of LB liquid medium containing 50 mg / L Km and 20 mg / LRif, and culture it at 28°C until the medium is turbid. Pipette 1 mL of turbid bacterial solution and spread it on LB solid medium containing 50 mg / L Km, and incubate at 28 °C for 48 h. Then scrape off an appropriate amou...

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Abstract

The invention discloses a pseudo-ginseng WRKY transcription factor gene PnWRKY9 and the application thereof. The nucleotide sequence of the PnWRKY9 gene is as shown in SEQ ID NO: 1, and the PnWRKY9 gene encodes a WRKY transcription factor; according to the application, molecular biology and functional genomics related technical researches prove that the PnWRKY9 gene has the function of improving the fungal disease resistance of plants, the antifungal gene PnWRKY9 is constructed on a plant expression vector and transferred into tobacco for overexpression, and the transgenic tobacco plant has very strong in-vitro antifungal activity; according to the present invention, the PnWRKY9 overexpressed transgenic tobacco has the significant inhibition effect on the growth of Alternaria Compacta, Fusarium solani, and Nigrospora oryzae, and the PnWRKY9 overexpressed transgenic tobacco has the significant inhibition effect on the growth of the Alternaria Compacta, the Fusarium solani, and the Nigrospora oryzae, such that the PnWRKY9 overexpressed transgenic tobacco has the significant inhibition effect on the growth of the Nigrospora oryzae,.

Description

technical field [0001] The present invention relates to molecular biology and genetic engineering related research fields, in particular to a Panax notoginseng WRKY transcription factor gene PnWRKY9 and its application. Background technique [0002] During the growth and development of plants, they will continue to encounter various stresses from the outside world, such as pests and diseases, which will lead to poor growth and development of food crops, economic crops and medicinal plants, reduced yield or even no harvest. Plant diseases are divided into non-infestation diseases and infestation diseases. Infection diseases are caused by biological factors such as fungi, bacteria, and viruses, and are contagious, harmful, and long-lasting. There are many kinds of plant pathogenic bacteria, and their control is particularly difficult. Traditional methods of preventing and controlling plant diseases mainly include pesticide control and cultivating disease-resistant varieties...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C12N15/84A01H5/00A01H6/82
CPCC07K14/415C12N15/8205C12N15/8282
Inventor 刘迪秋邱炳玲郑锂蕾苏琳琳梁婷婷邓婕
Owner KUNMING UNIV OF SCI & TECH
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