Use of aspartic endopeptidase gene

A technology of aspartic acid and endopeptidase, applied in the field of gene function and application, can solve problems such as unpublished literature reports

Active Publication Date: 2018-06-15
KUNMING UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

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  • Use of aspartic endopeptidase gene
  • Use of aspartic endopeptidase gene

Examples

Experimental program
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Embodiment 1

[0021] Example 1: Alternaria longiferium Pep1 Gene cloning and sequence analysis

[0022] According to the transcriptome data of Alternaria longiferium infection, a differentially expressed gene fragment was selected as the research object; DNAWalking SpeedUp was used to TM Premix kit (Seegene, Korea) designed specific primers to amplify unknown sequences at the 5' end (TSP1: 5'-GCAGTGTCGGTGTAGATGAGA-3', TSP2: 5'-AACAGTTGCCTGAGTGGTAGTGG-3', TSP3: 5'-GCGTTGATGTAGGTGATGGGAC-3') And specific primers for amplifying the unknown sequence at the 3' end (TSP1°: 5'-CTACTCAGGGTGGTTACATCTTC-3', TSP2°: 5'-CTTCCCGACTTCTCCATCACTG-3', TSP3°: 5'-ACTGTCCCTGGCAAGTACCTCG-3'), using Trizol (Invitrogen , USA) to extract the total RNA of the wild-type strain C-00 of Alternaria longiferium (the deposit number is CGMCC 3.17854), using the reverse-transcribed cDNA as a template, using the above primers and DNAWalking SpeedUp TM The primers in the Premix kit were used for PCR amplification, and th...

Embodiment 2

[0025] Example 2: Alternaria longhandle Pep1 gene knockout

[0026] 1. Gene knockout cassette construction

[0027] The gene knockout cassette includes 3 parts of the sequence, Pep1 5' homology arm, hygromycin expression cassette (hph) and Pep1 The 3' end homology arm and gene knockout cassette are constructed as follows: the 5' end and 3' end homology arms are respectively amplified with primers pep1-5f+pep1-5r and pep1-3f+ pep1-3r; primers pep1- The 5' end of 5r and pep1-3f contains 29 bases homologous to the hygromycin expression cassette; it was amplified using the genome of Alternaria longiferium wild-type strain C-00 as a template Pep1 Homology arms at the 5' and 3' ends; the hygromycin expression cassette was amplified using the plasmid pCSN44 as a template. Using the PCR product of the homology arm at the 5' end, the homology arm at the 3' end, and the hygromycin expression cassette as a template, the gene knockout cassette was obtained by fusion PCR amplifi...

Embodiment 3

[0042] Embodiment 3: Alternaria longiferium infects tobacco leaf experiment

[0043] Several pieces of fresh, disease-free, and mature K326 tobacco leaves were collected in the field, washed with sterile distilled water, sterilized with 1% sodium hypochlorite for 2 minutes, rinsed with sterile distilled water, and dried in the air, and then put into a container containing two layers of sterile filter paper. tablet. Infection experiments were carried out at two levels, one of which was bacterial block infection: Alternaria elongata (wild-type strain C-00 and knockout strains Pep1∆3, Pep1∆3, Pep1∆ 5) On the PDA plate, use a sterilized puncher with a diameter of 10 mm to punch a hole on the edge of the colony to obtain a 10 mm diameter bacterial block, and immediately inoculate the bacterial block on the treated K326 leaves; the second is spore infection: Add 10 mL of sterile water dropwise to the 7-day-cultivated Alternaria longiferium PDA plate, gently scrape off the conidia o...

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Abstract

The invention discloses a novel use of an aspartic endopeptidase gene. The use is an application of the aspartic endopeptidase gene as a drug target in preventing and/or controlling tobacco brown spot. The nucleotide sequence of the spartic endopeptidase gene is shown in SEQ ID NO:1. According to the invention, a wild-type strain of A. longipes and a Pep1 gene knockout strain are taken as researchobjects for carrying out a tobacco leaf infection experiment in vitro, and the result shows that the pathogenicity of the Pep1 gene knockout strain to tobacco leaves is obviously reduced, so that theaspartic endopeptidase gene is a key pathogenic factor in the A. longipes, and can be used as a drug target for screening drugs for preventing and/or controlling the tobacco brown spot. An aspartic endopeptidase inhibitor can be used to prepare the drugs for preventing and controlling the tobacco brown spot.

Description

technical field [0001] The invention belongs to the field of gene function and application, and in particular relates to the application of an aspartic endopeptidase (Pep1) gene in preventing and / or controlling tobacco red spot disease. Background technique [0002] Tobacco red spot disease is a disease caused by Alternaria ( Alternaria longipes , A. alternate and A. nicotiana ) caused by fungal leaf spot disease. The disease occurs in every tobacco producing area in the world, and is one of the most threatening diseases in tobacco production. Tobacco red spot disease reduces the yield and appearance quality of tobacco leaves, reduces the grade of tobacco leaves, and increases the loss rate of output value by almost multiples relative to the loss rate of yield, resulting in a serious decline in the economic benefits of tobacco leaf production. [0003] Alternaria longiferium ( A. longipes ) belongs to the half-known Mycelia, Hyphospora, and Pleuromycetes. Its mycel...

Claims

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Application Information

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IPC IPC(8): A01N57/16A01P3/00
CPCA01N57/16
Inventor 罗义勇张柯
Owner KUNMING UNIV OF SCI & TECH
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