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Phytophthora capsici pectate lyase (PL) Pcpel1 gene, protein preparation method and application thereof

A technology of pectin lyase and Phytophthora capsici, which is applied in the field of molecular biology, can solve the problems such as the target gene of pectin lyase of Phytophthora capsici has not been disclosed or published

Inactive Publication Date: 2010-02-03
SHANDONG AGRICULTURAL UNIVERSITY
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Problems solved by technology

Before the application of the present invention, there was no published or published research data on the role of Phytophthora capsici pectin lyase target gene in the process of pathogen-host interaction

Method used

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  • Phytophthora capsici pectate lyase (PL) Pcpel1 gene, protein preparation method and application thereof
  • Phytophthora capsici pectate lyase (PL) Pcpel1 gene, protein preparation method and application thereof
  • Phytophthora capsici pectate lyase (PL) Pcpel1 gene, protein preparation method and application thereof

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Embodiment 1

[0054] Embodiment 1 (joint test Phytophthora capsici genomic DNA library construction)

[0055] Phytophthora capsici strains with strong pathogenicity and high PELs activity were selected as test materials to construct a genomic DNA library. The specific steps are as follows:

[0056] (1) High-quality genomic DNA of Phytophthora capsici was extracted by CTAB method.

[0057] (2) Genomic DNA was interrupted by ultrasound, and the DNA fragments of 1.5Kb-3.0Kb obtained by electrophoresis detection were recovered and purified using the QIAEXII GELExtraction Kit kit.

[0058] (3) Genomic library identification, after DNA fragments of appropriate size were connected to the vector, transform Escherichia coli DH5α, take 20 μL of the bacterial liquid and smear the LB plate containing Amp, X-gal and IPTG, and blue-white screening.

[0059] (4) 96 clones were randomly selected for colony PCR identification and sequencing analysis, and the success rate of DNA fragments of required size i...

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Abstract

The invention belongs to the technical field of biology and in particular provides a pectate lyase (PL) gene Pcpel1 which is cloned from phytophthora capsici and protein preparation technology thereof. Gene and protein levels prove that the gene is effectively involved in the process that the phytophthora capsici infects hot pepper hosts and results in occurrence of the course of diseases on hot pepper leaves. Plant pathology and cytochemistry technology further prove that after the protein coded by the gene is inoculated onto the hot pepper leaves, obvious withering and shrinking occur on theinoculated parts of the leaves and the cell walls on the affected parts of the leaves are obviously degraded, namely the gene code is an important protein related to the course of diseases or is possibly an important target pathogenic gene of a phytophthora capsici PL gene cluster. The invention provides important technical reserve for further developing phytophthora capsici molecule detection technology.

Description

technical field [0001] The invention belongs to the field of molecular biology. Specifically, the invention relates to a method for isolating pectin lyase gene of Phytophthora capsici and its coded protein preparation method. In addition, the invention also relates to the destruction of pepper hosts and the degradation of cell walls by the pectin lyase encoded by the gene. Background technique [0002] During the interaction between plant pathogenic oomycetes and hosts, they secrete a variety of important cell wall degrading enzymes or pathogenic enzymes, among which pectin lyase (PEL: EC 4.2.2.2) is an important pathogenic enzyme secreted by many plant pathogenic oomycetes , Produced during the interaction between pathogens and hosts, this type of enzyme promotes the invasion and colonization of pathogens by degrading or softening the pectin, mesoplasty and pectin polymers of the cell wall. Pectin lyase often acts on the α-1,4-glucosidic bond of pectin, pectinate or low me...

Claims

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Application Information

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IPC IPC(8): C12N15/60C12P19/34C12N15/81C12N9/88C12Q1/527C12R1/84C12R1/645
Inventor 张修国
Owner SHANDONG AGRICULTURAL UNIVERSITY
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