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A kind of Panax notoginseng plant hormone binding protein gene pnphbp1 and application

A plant hormone and protein-binding technology, applied in the field of molecular biology and genetic engineering related technology research, can solve the problems of weak research foundation of disease-resistant genetic breeding, long growth cycle of Panax notoginseng, frequent occurrence of diseases, etc., and achieve broad market application prospects, Effect of shortening breeding cycle and reducing environmental pollution

Active Publication Date: 2020-11-17
KUNMING UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although the demand for Panax notoginseng is increasing year by year, the output of Panax notoginseng has not increased significantly. One of the main reasons is the long growth cycle and frequent occurrence of diseases of Panax notoginseng.
Panax notoginseng's disease-resistant genetic breeding research foundation is weak, which has become a limiting factor for the healthy development of the Panax notoginseng industry

Method used

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  • A kind of Panax notoginseng plant hormone binding protein gene <i>pnphbp1</i> and application
  • A kind of Panax notoginseng plant hormone binding protein gene <i>pnphbp1</i> and application
  • A kind of Panax notoginseng plant hormone binding protein gene <i>pnphbp1</i> and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Example 1: PnPhBP1 Full-length gene cloning and sequence analysis

[0021] Fusarium solani was used to inoculate Panax notoginseng, and the roots 24 hours after inoculation were used to extract total RNA. The inoculated roots of Panax notoginseng were ground into powder with liquid nitrogen, then transferred to a centrifuge tube, and the total RNA was extracted by guanidine isothiocyanate method. For RNA, reverse transcriptase M-MLV (promega) was used to synthesize the first strand of cDNA using total RNA as a template. The reaction system and operation process were as follows: take 5 μg total RNA, add 50 ng oligo (dT), 2 μL dNTP (2.5 mM each), DEPC water to a reaction volume of 14.5 μL; after mixing, heat denaturation at 70°C for 5 min, then rapidly cool on ice for 5 min, then add 4 μL 5×First-stand buffer, 0.5 μL RNasin (200U) in sequence , 1 μL 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 termi...

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 PnPhBP1 Escherichia coli plasmid pMD-18T- PnPhBP1 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; Pst I (TaKaRa) and EcoR I(TaKaRa) against plasmid pMD-18T- PnPhBP1 and pCAMBIA2300s for double enzyme digestion (100 μL system), the reaction system and operation process are as follows: take 20 μL pMD-18T- PnPhBP1 and pCAMBIA2300s plasmid, add 10 μL 10×Kbuffer, 4 μL Pst I, 6 μL EcoR I. 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 PnPhBP1 The fragments and the large fragment of the pCAMBIA2300s vector were gel-recovered separately; 1 μL of the...

Embodiment 3

[0028] Example 3: Plant genetic transformation mediated by Agrobacterium and screening of transgenic plants

[0029] The transgenic recipient in this experiment is tobacco, the tobacco seeds are soaked in 75% alcohol for 30s, 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, transfer to light incubator (25°C, 16h / d light) after germination, and then Subculture once a month with 1 / 2 MS medium.

[0030] Take out the pCAMBIA2300s- containing pCAMBIA2300s- PnPhBP1 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 a...

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Abstract

The invention discloses a panax notoginseng phytohormone conjugated protein gene PnPhBP1. The PnPhBP1 has a nucleotide sequence shown in SEQ ID NO: 1 and encodes protein with an amino acid sequence shown in SEQ ID NO: 2. According to the application, proven by functional genomics related technical studies, the gene PnPhBP1 has the function of improving fungal resistance of plants; the antifungal gene PnPhBP1 is constructed into a plant expression vector and is transferred into tobacco for overexpression, thus, transgenic nicotiana tabacum plants have very high in-vitro antifungal activity, andexperiment results display that transgenic nicotiana tabacum with PnPhBP1 overexpression plays an obvious role in inhibiting the growth of three kinds of fungi such as colletotrichum gloeosporioides,botrytis cinerea and fusarium solanum.

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 plant hormone binding protein gene PnPhBP1 and applications. Background technique [0002] Plant diseases are a very difficult problem in agricultural production, especially fungal diseases, which account for about 80% of the total plant diseases and seriously affect the yield and quality of crops. 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 crop rotation. However, these methods have more or less disadvantages, such as the long period of breeding resistant varieties, high chemical pesticide residues and easy to cause environmental pollution, and time-consuming and laborious adjustment of farming system, so the tradit...

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 刘迪秋普丽梅唐笔锋王倩赵秦李欣葛锋
Owner KUNMING UNIV OF SCI & TECH
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