Panax notoginseng myb transcription factor gene pnmyb2 and its application

A transcription factor and gene technology, applied in the research fields of molecular biology and genetic engineering, can solve the problems of plant diseases, difficult to obtain disease resistance resources, huge investment, etc., and achieve broad market application prospects and breeding cycle. Shortening and cost saving effect

Active Publication Date: 2019-07-16
KUNMING UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in the process of using pesticides, it is difficult to solve the problems of human and animal safety and environmental pollution.
The traditional breeding method used to cultivate fine varieties takes a long time, slow results, huge investment, and difficult to obtain disease-resistant resources
Both of these methods are not very good at solving the problem of plant diseases

Method used

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  • Panax notoginseng myb transcription factor gene pnmyb2 and its application
  • Panax notoginseng myb transcription factor gene pnmyb2 and its application
  • Panax notoginseng myb transcription factor gene pnmyb2 and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1: PnMYB2 Full-length cDNA cloning and sequence analysis

[0023] The roots of Panax notoginseng were inoculated with Fusarium solani rot, total RNA was extracted from the roots 12 h after inoculation, the treated roots of Panax notoginseng were ground into powder with liquid nitrogen, and then transferred to a centrifuge tube, and guanidine isothiocyanate was used to Total RNA was extracted using M-MLV reverse transcriptase (promega) using total RNA as a template to synthesize the first strand of cDNA. The reaction system and operation process were as follows: take 5 μg total RNA, add 50 ngoligo (dT), 2 μL dNTP Mix ( 2.5mM each), the reaction volume was made up to 14.5μL with DEPC water; after mixing, heat denaturation at 70°C for 5 minutes, then quickly cool on ice for 5 minutes, then add 4μL 5×First-standbuffer, 0.5μL RNasin ( 200U), 1μL 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 term...

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 PnMYB2 coli plasmid pGEM-T- PnMYB2 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 Bam HI and Eco RI respectively for plasmid pGEM-T- PnMYB2 and pCAMBIA2300S for double enzyme digestion (100 μL system), the reaction system and operation process are as follows: take 20 μL pGEM-T- PnMYB2 and pCAMBIA2300S plasmid, add 10μL 10×H buffer, 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. All digested products were subjected to agarose gel electrophoresis, and then the PnMYB2 The fragments and the large fragments of the pCAMBIA2300S vector were gel-recovered separately, and 1 μL ...

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 ). 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- PnMYB2 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 mediu...

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Abstract

The invention discloses a pseudo-ginseng MYB transcription factor gene PnMYB2 and a use thereof. The gene PnMYB2 has a nucleotide sequence shown in the formula of SEQ ID NO: 1 and can code an MYB transcription factor. The molecular biology and functional genomics relation technological research proves that the gene PnMYB2 can improve fungal disease resistance of plants. The antifungal gene PnMYB2 is constructed in a plant expression carrier, is transferred into tobacco and is over-expressed. The transgenic tobacco plant has strong in-vitro anti-fungal activity. The transgenic tobacco over-expressing the gene PnMYB2 can obviously inhibit growth of Botryosphaeria dothidea, Fusarium solani, Fusarium oxysporum, Fusarium verticillioide and Alternaria panax.

Description

technical field [0001] The invention relates to molecular biology and genetic engineering related research fields, in particular to a notoginseng MYB transcription factor gene PnMYB2 and its application. Background technique [0002] During the growth and development of plants, they will continue to encounter various natural disasters and pests and diseases from the outside world, which will lead to poor growth and development of food crops, economic crops and medicinal plants, reduced production 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. The traditional methods of disease control are mainly the use of pesticides and the cultivation of superior varieties. However, in the process of u...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/29C07K14/415C12N15/82A01H5/00A01H6/82
CPCC07K14/415C12N15/8282
Inventor 刘迪秋关瑞攀曲媛葛锋杨晓艳熊吟
Owner KUNMING UNIV OF SCI & TECH
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