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Notoginseng class sweet protein gene PnTLP5 and application thereof

A kind of sweet protein and gene technology, applied in the notoginseng sweet protein gene PnTLP5 and its application field, to achieve the effect of saving cost, reducing environmental pollution, and simple operation

Active Publication Date: 2019-01-18
KUNMING UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But neither chemical sterilization nor crop rotation can fundamentally solve the problem of Panax notoginseng disease

Method used

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  • Notoginseng class sweet protein gene PnTLP5 and application thereof
  • Notoginseng class sweet protein gene PnTLP5 and application thereof
  • Notoginseng class sweet protein gene PnTLP5 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Example 1: PnTLP 5Full-length gene cloning and sequence analysis

[0020] The three-year-old Panax notoginseng leaves were used to extract total RNA, and the leaves were ground into powder with liquid nitrogen, then transferred to a centrifuge tube, and total RNA was extracted by the guanidine isothiocyanate method, and 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 (2.5mM each), and DEPC water in sequence until the reaction volume is 14.5 μL; After mixing, heat denaturation at 70°C for 5 min, then quickly cool on ice for 5 min, then add 4 μL 5×First-stand buffer, 0.5 μL RNasin (200U), 1 μL M-MLV (200U) in sequence, mix well and Centrifuge for a short time, 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 first strand of cD...

Embodiment 2

[0023] Embodiment 2: plant overexpression vector construction

[0024] Use the SanPrep column plasmid DNA mini-extraction kit (Shanghai Sangong) to extract the insert PnTLP5 Escherichia coli plasmid pMD-18T- PnTLP5 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; Bam HI (TaKaRa) and Eco RI (TaKaRa) respectively on the plasmid pMD-18T- PnTLP5 and pCAMBIA2300s for double enzyme digestion (100 μL system), the reaction system and operation process are as follows: take 20 μL pMD-18T- PnTLP5 and pCAMBIA2300s plasmid, add 10 μL 10×K buffer, 5 μL Bam HI, 5 μL Eco RI, 60 μL ddH 2 O, mix well and then centrifuge for a short time, and place it at 37°C for overnight reaction; spot all the digested products on agarose gel for electrophoresis, and then PnTLP5 The fragments and the large fragment of the pCAMBIA2300s vector were gel-recovered separately...

Embodiment 3

[0027] Example 3: Plant genetic transformation mediated by Agrobacterium and screening of transgenic plants

[0028] The transgenic recipient in this experiment is tobacco. Soak the tobacco seeds in 75% alcohol for 30s, wash them with sterile water and wash them with 0.1% HgCl 2 Soak for 8 min, 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 then Subculture once a month with 1 / 2 MS medium.

[0029] Take out the pCAMBIA2300s- containing pCAMBIA2300s- PnTLP5 The plasmid Agrobacterium LBA4404 strain was inoculated in 5 mL of LB liquid medium containing 50 mg / L Km and 20 mg / L rifampicin, 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 and inoculate it with 20 mg / L ac...

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Abstract

The invention discloses a notoginseng disease course related protein 5 family sweet protein gene PnTLP5, which has a nucleotide sequence as described in SEQ IDNO: 1 and encodes a panax notoginseng type sweet protein as shown in SEQ IDNO: 2 amino acid sequence. The invention proves that the PnTLP5 gene has the function of improving plant anti-fungus through molecular biology and genetic engineeringrelated technology research, As that antifungal PnTLP5 gene of the invention is construct on a plant expression vector and transferred into tobacco for overexpression, Results The transgenic tobaccoplants had strong antifungal activity in vitro, and the transgenic tobacco overexpressing PnTLP5 had significant inhibitory effect on the growth of Sclerotinia sclerotiorum, Fusarium solani, Fusariumverticillatum, Alternaria ginseng and Botrytis citrina.

Description

technical field [0001] The invention relates to the field of molecular biology and genetic engineering related technology research, in particular to a notoginseng sweet protein gene with antifungal activity PnTLP5 and applications. Background technique [0002] Panax notoginseng ( Panax notoginseng ) is a traditional Chinese herbal medicine with a long history in China. Panax notoginseng is mainly produced in Yanshan County, Maguan, Xichou, Guangnan, Malipo, Funing, Qiubei, Wenshan Prefecture, Yunnan, and it is also planted in Tianyang, Jingxi, Tiandong, Debao and other places in Guangxi. Panax notoginseng in Wenshan Prefecture, Yunnan Province has a long history, large output and good quality. Panax notoginseng grows in a shaded environment all the year round, and the occurrence of diseases and insect pests is relatively serious. According to statistics, there are about 20 kinds of diseases and insect pests on Panax notoginseng. Among them, the main ones are root rot, ...

Claims

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Application Information

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IPC IPC(8): C12N15/29C12N15/82A01H5/00A01H6/82
CPCC12N15/8282C07K14/415
Inventor 崔秀明刘迪秋李欣白智伟王承潇曲媛
Owner KUNMING UNIV OF SCI & TECH
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