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Pseudo-ginseng WRKY transcription factor gene PnWRKY15 and application

A transcription factor and gene technology, applied in application, genetic engineering, plant genetic improvement, etc., can solve the problems of serious diseases, damage to the yield and quality of medicinal materials, and long growth cycle of Panax notoginseng, and achieve a shortened breeding cycle and a broad market. Application prospect, cost saving effect

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

However, Panax notoginseng has a long growth cycle, prefers warmth and humidity, and serious diseases, especially fungal diseases, seriously endanger the yield of Panax notoginseng and the quality of medicinal materials.

Method used

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  • Pseudo-ginseng WRKY transcription factor gene PnWRKY15 and application
  • Pseudo-ginseng WRKY transcription factor gene PnWRKY15 and application
  • Pseudo-ginseng WRKY transcription factor gene PnWRKY15 and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Example 1: PnWRKY15 Full-length gene cloning and sequence analysis

[0021] Total RNA was extracted from the roots of annual Panax notoginseng, ground into powder with liquid nitrogen, then transferred to a centrifuge tube, total RNA was extracted by guanidine isothiocyanate method, reverse transcriptase M-MLV (promega) was used to The total RNA is used as the template to synthesize the first strand of cDNA. The reaction system and operation process are as follows: take 5 μg Total RNA, add 50 ng oligo (dT), 2 μL dNTP (2.5 mM each), and DEPC water in sequence to a reaction volume of 14.5 μL; mix well Afterwards, heat denaturation at 70°C for 5 minutes, then rapidly cool on ice for 5 minutes, then add 4 μL 5×First-stand buffer, 0.5 μL RNasin (200 U), 1 μL M-MLV (200 U), mix well and centrifuge for a short time, 42°C Incubate for 1.5 hours, take it out and heat at 70°C for 10 minutes to terminate the reaction; after the first strand of cDNA is synthesized, store it at -20...

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 PnWRKY15 coli plasmid pGEM-T- PnWRKY15 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; Eco RI (TaKaRa) and Bam HI (TaKaRa) for plasmid pGEM-T- PnWRKY15 and pCAMBIA2300S for double enzyme digestion (100μL system), the reaction system and operation process are as follows: take 20μL pGEM-T- PnWRKY15 and pCAMBIA2300S plasmid, add 10μL 10×Hbuffer, 5μL Eco RI, 5 μL Bam HI, 60 μL ddH 2 O, after mixing, centrifuge for a short time, and place at 37°C for overnight reaction. Spot all digested products on agarose gel for electrophoresis, and then PnWRKY15 The fragments and the large fragments of the pCAMBIA2300S vector were gel-recovered separately, and the SanPrep column DNA gel reco...

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 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 MS every month thereafter The medium was subcultured once.

[0030] Take out the stored pCAMBIA2300S-containing pCAMBIA2300S- PnWRKY15 The Agrobacterium LBA4404 strain of the plasmid was inoculated in 5 mL of LB liquid medium containing 50 mg / L Km and 20 mg / L rifampicin, and cultivated at 28°C until the medium was cloudy. Pipette 1mL of turbid bacterial solution onto LB solid medium containing 50mg / L Km, and incubate at 28°C for 48h; then scrape off an appropriate amount of Agrobac...

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Abstract

The invention discloses a pseudo-ginseng WRKY transcription factor gene PnWRKY15. The nucleotide sequence of which is as shown in SEQ ID NO: 1, the PnWRKY15 gene is proved to have a function of improving the antifungal function of plants through functional genomics related technical research, and the antifungal PnWRKY15 gene is constructed to a plant expression vector and transferred into tobacco for overexpression, transgenic tobacco plants have very strong fungal infection resistance, and experimental results show that the PnWRKY15 overexpression transgenic tobacco leaf total protein obviously inhibits mycelial growth of fusarium chlamydosporum and alternaria compacta.

Description

technical field [0001] The invention relates to the field of molecular biology and genetic engineering related technology research, in particular to a Panax notoginseng WRKY transcription factor gene with the ability to resist fungal infection PnWRKY15 and applications. Background technique [0002] During the growth and development of plants, they often encounter various natural disasters and pests and diseases from the outside world, resulting in stunted growth, stunted growth, reduced yield and even death. When plants face pathogen invasion or other stresses, they massively reprogram the transcriptome in a highly variable and temporal manner in response to stress, and this regulation endows plants with plastic adaptation to different environmental conditions. The response is the result of a network of multiple transcription factors. Transcription factors (transcription factor, TF) are a class of proteins that can bind to specific sequences upstream of genes and regulate...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C12N15/84C07K14/415A01H5/00A01H6/82
CPCC07K14/415C12N15/8282
Inventor 刘迪秋郑锂蕾苏琳琳梁婷婷曲媛崔秀明葛锋
Owner KUNMING UNIV OF SCI & TECH
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