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Panax notoginseng thaumatin-like protein gene PnTLP2 and application thereof

A kind of sweet protein and gene technology, which is applied to the Panax notoginseng sweet protein gene PnTLP2 and application fields, and achieves the effects of reduced use, shortened breeding cycle and simple operation.

Active Publication Date: 2019-02-01
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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  • Panax notoginseng thaumatin-like protein gene PnTLP2 and application thereof
  • Panax notoginseng thaumatin-like protein gene PnTLP2 and application thereof
  • Panax notoginseng thaumatin-like protein gene PnTLP2 and application thereof

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×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 PnTLP2 The fragments and the large fragments of the pCAMBIA2300s vector were separately gel-recovere...

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 acet...

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Abstract

The invention discloses a panax notoginseng thaumatin-like protein gene PnTLP2, which is the thaumatin-like protein encoding as an amino acid sequence shown in SEQ ID NO: 2 and having a nucleotide sequence shown in SEQ ID NO: 1. The applicaition adopts molecular biology and reverse genetics related techniques to confirm that the PnTLP2 gene has the function of improving fungal resistance of plants, the antifungal PnTLP2 gene of the invention is constructed on a plant expression vector and transferred into tobacco for overexpression, the transgenic tobacco plants have strong in-vitro antifungalactivity, the transgenic tobacco overexpressing PnTLP2 has significant inhibitory effects on five plant pathogenic fungi of fusarium oxysporum, fusarium solani, alternaria panax, sclerotinia sclerotiorum and nigrospora oryzae.

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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IPC IPC(8): C12N15/29C07K14/415C12N15/82A01H5/00A01H6/82
CPCC12N15/8282C07K14/415
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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