Phytophthora capsici polygalacturonase (PG) Pcipg5 gene, protein preparation method and application thereof

A technology of polygalactose and Phytophthora capsicum, applied in the field of molecular biology, can solve the problems of unseen protein production and functional research data and information, etc.

Inactive Publication Date: 2010-02-03
SHANDONG AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Before the application of the present invention, except that this research group has reported the research on the pathogenic function mechanism of Phytophthora capsici polygalac

Method used

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  • Phytophthora capsici polygalacturonase (PG) Pcipg5 gene, protein preparation method and application thereof
  • Phytophthora capsici polygalacturonase (PG) Pcipg5 gene, protein preparation method and application thereof
  • Phytophthora capsici polygalacturonase (PG) Pcipg5 gene, protein preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Embodiment 1 (joint test Phytophthora capsici genomic DNA library construction)

[0054] Phytophthora capsici strains with strong pathogenicity and high PGs activity were selected as test materials to construct a genomic DNA library. The specific steps are as follows:

[0055] (1) High-quality genomic DNA of Phytophthora capsici was extracted by CTAB method.

[0056] (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.

[0057] (3) Genomic library identification, DNA fragments of appropriate size were ligated to the vector, transformed into Escherichia coli DH5α, and 20 μL of bacterial liquid was coated on LB plates containing Amp, X-gal and IPTG, and blue-white screening was performed.

[0058] (4) 96 clones were randomly selected for colony PCR identification and sequencing analysis to ensure that the success rate of DNA fragments ...

Embodiment 2

[0059] Embodiment 2 (gene cloning and sequencing)

[0060] According to the reported polygalacturonase gene sequence design degenerate primers P710 (its sequence is shown in Seq ID No: 3) and P1150R (its sequence is shown in Seq ID No: 4), using the Pooling-PCR method Screen the genomic library and quickly isolate multiple gene members of the target gene family.

[0061] 1. Extraction of total plasmid DNA from 96 clones (alkaline lysis method)

[0062] (1) Inoculate the Escherichia coli containing the plasmid into a 96-deep-well plate with medium containing antibiotic LB, shake at 220 rpm at 37°C, and reach the late logarithmic growth stage.

[0063] (2) Transfer the cultured bacterial solution to an 80mL centrifuge tube, centrifuge at 8000rpm at 4°C for 15min, and discard the supernatant.

[0064] (3) Resuspend the bacterial cells obtained by centrifugation in a centrifuge tube filled with 5mL solution I [50mM Tris-HCl (pH8.0), 10mM EDTA (pH8.0)], shake slowly on a shaker f...

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Abstract

The invention belongs to the technical field of biology and in particular provides a polygalacturonase (PG) gene Pcipg5 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 the inoculated 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 PG 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 polygalacturonase gene of Phytophthora capsici and its coded protein. In addition, the invention also relates to the damage of the polygalacturonase encoded by the gene to the capsicum host and the degradation of the cell wall. 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 polygalacturonase (PG: EC 3.2.1.15) is a kind of enzyme secreted by various plant pathogenic oomycetes. Important pathogenic enzymes, this type of enzyme destroys the host defense system by degrading the pectin and mesogelatin of the host plant cell wall, thereby enhancing the affinity of the pathogen to the host, so this type of enzyme is an important pathogenic factor. Studies have shown that polygalacturonase is a...

Claims

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Application Information

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IPC IPC(8): C12N15/56C12P19/34C12N15/81C12N9/26C12Q1/34C12R1/84C12R1/645
Inventor 张修国
Owner SHANDONG AGRICULTURAL UNIVERSITY
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