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Chestnut Phytophthora pathogenicity gene p5cdh and its application

A chestnut Phytophthora bacterium, pathogenicity technology, applied in the direction of application, genetic engineering, plant genetic improvement, etc., can solve environmental pollution, ecological damage and other problems

Active Publication Date: 2015-10-07
GUANGXI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Medication mainly relies on chemical agents, but the extensive use of chemical pesticides will bring negative impacts such as environmental pollution and ecological damage, and biological control has received more and more attention

Method used

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  • Chestnut Phytophthora pathogenicity gene p5cdh and its application
  • Chestnut Phytophthora pathogenicity gene p5cdh and its application
  • Chestnut Phytophthora pathogenicity gene p5cdh and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Cluster analysis of homologous proteins in embodiment 1, P5Cdh and other fungi

[0038] Enter the amino acid sequence of P5Cdh into GenBank (http: / / www.ncbi.nlm.nih.gov) for BlastP search, and obtain the amino acid sequences of homologous proteins in multiple species. JGI344979 is the P5Cdh protein of Phytophthora chestnut, and the homologous proteins of P5Cdh protein in other fungi are: XP_003718931 from Magnaporthe oryzae, EGU80836 from Fusarium oxysporum, XP_750764 from Aspergillus fumigatus, CAP80517 is from Penicillium chrysogenum and XP_001596907 is from Sclerotinia sclerotiorum. The phylogenetic tree was constructed using the Neighbor-joining method of the MEGA4.0 software, and the bootstrap test was selected for 1000 times during the tree building process, and displayed in TREEVIEW. as attached figure 1 As shown, the clustering of P5Cdh and homologous proteins in other plant pathogenic fungi analyzed the evolutionary conservation and kinship of this protein ( ...

Embodiment 2

[0039] Example 2, Prokaryotic expression and enzyme activity detection of P5Cdh

[0040] 1) Prokaryotic expression of P5Cdh: use the software MitoPro II to predict the P5Cdh signal peptide to be 1-45aa, prepare the total RNA of EP155 as in 1) and 3) of Example 4, synthesize the first strand of cDNA, and use the primer P5Cdh-f-Hind III (5 '-gcgaagcttatggcttctcgcagggtcagtctcc-3') and P5Cdh-r-XbaⅠ (5'-gcctctagagacctcattgctcggatactcgac-3') amplified cDNA sequence of P5Cdh deletion signal peptide (1-45aa) was cloned into prokaryotic expression vector pGEX-4T-1 containing GST tag The recombinant expression plasmid pGEX-4T-P5CdhΔ45 was obtained in the HindⅢ / SalⅠ cloning site. The recombinant plasmid was introduced into the competent cells of Escherichia coli expression host BL21 by chemical transformation method, and spread on the LA plate containing 100 μg / mL ampicillin for recombinant screening. After the obtained transformants were identified by electrophoresis analysis and enzym...

Embodiment 3

[0042] Example 3, Functional Research of P5Cdh Gene in Phytophthora Chestnut

[0043] 1) Construction of knockout cassette

[0044] Gene knockout adopts the method of homologous recombination, and replaces the coding region of the P5Cdh gene in the high-efficiency homologous recombination system of Phytophthora chestnut with the hygromycin phosphotransferase gene. The specific strategy is shown in the appendix figure 2a. The left arm forward and reverse primers are P5Cdh-A (5'-atttagccgtcgtgacatcttcctc-3') and P5Cdh-3 (5'-tctttctagaggatccccgggtaccggattggatggattgagctgt-3'), and the right arm forward and reverse primers are P5Cdh-2 (5' -atatcatcttctgtcgacctgcaggctatccgagcaatgaggtctg-3') and P5Cdh-B (5'-ggtgtgagagagaaggtggtcgttc-3'), the amplified template is EP155 total DNA, and the amplified target fragment sizes are 929bp and 1058bp respectively. The forward and reverse primers of hygromycin phosphotransferase gene (hph) were hph-F (5’-cggtacccggggatcctctag-3’) and hph-R (5...

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Abstract

The invention discloses a pathogenic gene P5Cdh of cryphonectria parasitica and an application thereof. The pathogenic gene affects the pathogenicity and sporulation of fungi. The gene contains three exons and two introns, has DNA (deoxyribonucleic acid) total length of 2270bp and cDNA (complementary DNA) total length of 1782bp and codes 594 amino acids. A protein expressed by the gene is shown in SEQ.ID.No.3 and is used for regulating and controlling pathogenesis of cryphonectria parasitica. Experiments prove that pathogenic scabs formed by a deletion mutant obtained after the gene is replaced by a hygromycin resistance gene hph on in-vitro Chinese chestnut tree branches are obviously smaller than the pathogenic scabs produced by a wild type strain, thus loss of the pathogenicity of cryphonectria parasitica is caused by deletion of the gene P5Cdh. The gene P5Cdh as well as an expression vector, a host and the like thereof have important significance in plant disease control drug and cryphonectria parasitica control study. Transcription of the gene P5Cdh is inhibited after cryphonectria parasitica is infected with a hypovirus. The protein coded by the gene is probably a target after infection with the hypovirus. The mutual effects of the hypovirus and the gene have important significance in studying a molecular mechanism of virus-host interaction.

Description

technical field [0001] The invention belongs to the field of microbial genetic engineering and plant protection, and in particular relates to a pathogenicity gene P5Cdh of Phytophthora chestnut affecting fungal pathogenicity and sporulation and application thereof. Background technique [0002] Chestnut blight (Cryphonectria parasitica) is the pathogen that causes chestnut blight. It belongs to filamentous fungi in Ascomycetes in classification, and has now become a model fungus for studying the pathogenic mechanism of plant pathogenic fungi. [0003] At present, about 70% to 80% of plant diseases are caused by plant pathogenic fungi. Studying the infection process of pathogenic fungi is the basis for in-depth study of the pathogenic molecular mechanism of pathogenic fungi. According to the process of pathogenic fungi infecting the host, it is known that the pathogenic factors and genes of pathogenic fungi mainly include: (1) related to the adhesion of plant pathogenic fungi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/53C12N9/06C12N15/80C12Q1/68C12Q1/32G01N33/573A01N61/00A01P3/00C12R1/645
Inventor 陈保善姚姿婷邹承武周辉王金子卢立丹李洋
Owner GUANGXI UNIV