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A kind of notoginseng sweet protein gene pntlp2 and its application

A sweet protein-like gene technology, applied to the notoginseng-like sweet protein gene PnTLP2 and its application field

Active Publication Date: 2021-04-09
KUNMING UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The molecular size of most TLPs is 21-26 kDa, but some TLPs from coniferous and cereal plants have a smaller molecular weight of about 16-17 kDa. This type of protein lacks about a quarter of amino acids, only about 10 conserved cysteine ​​residues, but they still have antifungal activity

Method used

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  • A kind of notoginseng sweet protein gene pntlp2 and its application
  • A kind of notoginseng sweet protein gene pntlp2 and its application
  • A kind of notoginseng sweet protein gene pntlp2 and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Example 1: PnTLP2 Full-length gene cloning and sequence analysis

[0020] Three-year-old Panax notoginseng young leaves were used to extract total RNA, the leaves of Panax notoginseng were ground into powder with liquid nitrogen, and then transferred to a centrifuge tube, total RNA was extracted by guanidine isothiocyanate method, reverse transcriptase M-MLV (Promega ) using total RNA as a template to synthesize the first-strand 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 to a reaction volume of 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. The first-strand cDNA was synthesized and stored at -20...

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 PnTLP2 Escherichia coli plasmid pMD-18T- PnTLP2 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- PnTLP2 and pCAMBIA2300s for double enzyme digestion (100 μL system), the reaction system and operation process are as follows: take 20 μL pMD-18T- PnTLP2 and pCAMBIA2300s plasmid, add 10 μL 10×Kbuffer, 5 μL BamHI , 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 PnTLP2 The fragments and the large fragments of the pCAMBIA2300s vector were separately gel-recovered (...

Embodiment 3

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

[0028] The transgenic recipient in this experiment was tobacco. The tobacco seeds were soaked in 75% alcohol for 30 s, washed with sterile water and washed 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, and transfer to light incubator (25°C, 16 h / d light) after germination. Subculture once a month with 1 / 2 MS medium.

[0029] Take out the pCAMBIA2300s- containing pCAMBIA2300s-PnTLP2 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 acety...

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Abstract

The invention discloses a notoginseng sweet protein gene PnTLP2 , its nucleotide sequence is shown in SEQ ID NO: 1, encoding the sweet protein of amino acid sequence shown in SEQ ID NO: 2; PnTLP2 The gene has the function of improving the anti-fungal effect of the plant, and the anti-fungal effect of the present invention PnTLP2 The gene was constructed on a plant expression vector and transferred to tobacco for overexpression. As a result, the transgenic tobacco plants had strong antifungal activity in vitro, and the overexpression PnTLP2 The transgenic tobacco has a significant inhibitory effect on the growth of five plant pathogenic fungi, including Fusarium oxysporum, Fusarium solani, Alternaria ginseng, Sclerotinia and Nigeroryza sativa.

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 PnTLP2 and applications. Background technique [0002] Plants play an important role in human production and life. Some plants are economic crops and food crops, and many plants also have important medicinal value. During the growth process, plants are more or less under the stress of biotic or abiotic factors, which affect the formation of plant economic traits or yield traits. Among a variety of adverse stress factors, diseases caused by bacteria, fungi, and viruses are the main factors that endanger plant growth and development. Traditional disease control methods have achieved certain results. One is to rely on traditional breeding methods to cultivate resistant varieties, the other is to use chemical pesticides, and the third is to adopt farming systems such as cro...

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 刘迪秋李欣崔秀明白智伟曲媛王承潇杨晓艳
Owner KUNMING UNIV OF SCI & TECH
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