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Application of positive regulatory factor nbpro19c55-1 gene and its protein in resistance to Phytophthora phytophthora

A technology of positive regulation and Phytophthora, applied in the direction of angiosperms/flowering plants, application, plant peptides, etc., can solve the problem of less research on small peptides, and achieve the effect of improving resistance

Active Publication Date: 2021-11-23
NORTHWEST A & F UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] A number of secreted small peptides involved in plant disease resistance have been identified, but there are few studies on small peptides that play an immunomodulatory role in response to Phytophthora infection in tobacco

Method used

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  • Application of positive regulatory factor nbpro19c55-1 gene and its protein in resistance to Phytophthora phytophthora
  • Application of positive regulatory factor nbpro19c55-1 gene and its protein in resistance to Phytophthora phytophthora
  • Application of positive regulatory factor nbpro19c55-1 gene and its protein in resistance to Phytophthora phytophthora

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Cloning of NbPRO19C55-1 Gene

[0028] 1. Plant material: Nicotiana benthamiana (obtained through public channels), RNA is extracted from its leaves.

[0029] Extract RNA with RNA extraction kit (OMGA, Lot#:R6827-01), identify the integrity of RNA by agarose gel electrophoresis, and then measure the purity and concentration of RNA on a spectrophotometer.

[0030] 2. Gene cloning

[0031] Use the reverse transcription kit (TaKaRa, Lot#:AHE3187A) to obtain the cDNA of Nicotiana benthamiana, and design the upstream and downstream primers NbPRO19C55-F / R according to the full-length coding sequence of NbPRO19C55 provided in the Solanaceae website,

[0032] NbPRO19C55-F: 5'-CGGGGTACCATGGCAAGATTTGTGGACTTT-3',

[0033] NbPRO19C55-R: 5'-CTAGACTAGTCTACTTATCATCATCATCCTTATAATCTTGATGATTGCCAGTGACAAC-3',

[0034] Amplification was performed using cDNA as a template. The PCR product was digested, ligated, and verified by bacterial liquid PCR to construct the vector pKannibal-AtPPR1,...

Embodiment 2

[0036] Sequence information and homology analysis of NbPRO19C55-1 gene

[0037] The full-length CDS sequence of Nicotiana benthamiana NbPRO19C55-1 gene of the present invention is 270bp. For the detailed sequence, see Sol GenomicsNetwork: Niben101Scf00803g01016.1, such as SEQ ID No: 1 of the present invention. The amino acid sequence has a total of 89 amino acids. For the detailed sequence, see Sol Genomics Network: Niben101Scf00803g01016 .1, such as SEQ ID No: 2 of the present invention.

[0038] The amino acid sequence of the protein encoded by the Nicotiana benthamiana NbPRO19C55-1 gene was searched for homology with the BLAST program, and it was found that its similarity with the amino acid sequence of a gene NbPRO19C55-2 in Nicotiana benthamiana was more than 90%. See Sol Genomics Network for the amino acid sequence: Niben101Scf03747g00005.1, such as SEQ ID No: 3 of the present invention. From the above, it can be inferred that a homologous gene has the same or similar f...

Embodiment 3

[0041]Example 3 NbPRO19C55-1 overexpression plants show resistance to Phytophthora parasitica

[0042] Design specific primers to detect the expression of NbPRO19C55-1 in overexpressed plants. About 0.2 g of tobacco leaf samples were collected to extract RNA and reverse transcribed into cDNA, then specific primers were designed to detect the expression of NbPRO19C55-1 by real-time fluorescent quantitative PCR technology. Collect common smoke safflower Dajinyuan with a seedling age of 6-7 weeks as a control, inoculate GFP-labeled Phytophthora parasitica mycelia blocks after scratching the leaves, and observe and measure the disease after cultivating in the dark at 23°C for about 48 hours. spot diameter. The result is as figure 2 shown.

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Abstract

The invention discloses the application of a positive regulatory factor NbPRO19C55‑1 gene and its protein in resisting Phytophthora phytophthora, and belongs to the field of biotechnology. The NbPRO19C55‑1 gene enhances the resistance of plants to Phytophthora parasitica by positively regulating plant immune signals Resistance; the nucleotide sequence of the NbPRO19C55‑1 gene is shown in SEQ ID No: 1, and the nucleotide sequence of the gene also includes addition, substitution, and insertion in the nucleotide sequence shown in SEQ ID No: 1 Or deletion of one or more nucleotides to generate mutants, alleles or derivatives; the use of the gene as a positive regulatory factor in improving the resistance of Phytophthora phytophthora can be used for breeding of Phytophthora resistant varieties.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to the application of a positive regulator NbPRO19C55-1 gene and its protein in resisting Phytophthora phytophthora. Background technique [0002] Plant-secreted small peptides play important regulatory functions in plant growth and development and in response to various biotic and abiotic stresses, and research on improving plant disease resistance has attracted much attention. Most of them are up-regulated in damaged or infected parts, and can quickly initiate defense reactions such as plant callose accumulation, active oxygen burst, and disease-resistant protein accumulation. Small peptides can activate plant disease resistance signals at extremely low concentrations, and can cooperate with other immune signals (such as PAMPs) and plant hormones to regulate plant disease resistance responses. If NbPRO19C55 is engineered to be induced by pathogen infection through CRISPR / Cas9-directe...

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
CPCC07K14/415C12N15/8218C12N15/8282
Inventor 单卫星文曲江
Owner NORTHWEST A & F UNIV
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