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Phytophthora capsici P4-ATP enzyme PcDrs2 protein and application thereof

A technology of P4-ATP, Phytophthora capsicum, applied in the field of microorganisms, can solve problems such as no reports, and achieve the effects of reducing the growth rate of mycelium, slowing down the growth rate of mycelium, and slowing down the growth rate

Pending Publication Date: 2021-12-24
HAINAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no report on the use of P4-ATPase to regulate the growth of Phytophthora capsici at present

Method used

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  • Phytophthora capsici P4-ATP enzyme PcDrs2 protein and application thereof
  • Phytophthora capsici P4-ATP enzyme PcDrs2 protein and application thereof
  • Phytophthora capsici P4-ATP enzyme PcDrs2 protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 - Obtaining Phytophthora capsici P4-ATPase PcDrs2 and its coding gene

[0034] In this example, the Phytophthora capsici PcDrs2 protein and its coding gene (or cDNA) were obtained by using the DNA (or cDNA) of the Phytophthora capsici standard strain LT1534 as a template, and using the primers listed in Table 1 to obtain it through PCR amplification. The coding gene PcDRS2 of PcDrs2 is shown in SEQ ID NO.1 in the sequence listing, and consists of 3048 nucleotides; its coding sequence is identical to the genome sequence, and is also composed of 3048 nucleotides, and SEQ ID NO.2 in the coding sequence listing The indicated protein PcDrs2. The above-mentioned proteins or genes can also be artificially synthesized.

[0035] Table 1. PcDRS2 full-length coding gene amplification primers

[0036]

Embodiment 2

[0037] Example 2 - Construction of Phytophthora capsici PcDRS2 gene knockout vector

[0038] In this example, the gene knockout vector construction method of CRISPR / Cas9 and the sequence of related vectors refer to "Fang, Y., and Tyler, B.M. (2016). Efficient disruption and replacement of an effector gene in the oomycete Phytophthora sojae using CRISPR / Cas9.Molecularplant pathology, 17(1), 127-139." and "Fang, Y., Cui, L., Gu, B., Arredondo, F., and Tyler, B.M. (2017). Efficient genome editing in the oomycete Phytophthora sojae using CRISPR / Cas9. Curr. Protoc. Microbiol. 44, 21A.1.1-21A.1.26". The sgRNA and Cas9 co-expression plasmid pYF515 used in this example and the pBluescript II SK+ homology arm vector plasmid are all stored in the green prevention and control team of tropical crop oomycete diseases of the School of Plant Protection, Hainan University.

[0039] This embodiment also includes the construction of the homology arm carrier pBS-eGFP-PcDrs2; sgRNA and Cas9 co-...

Embodiment 3

[0050] Example 3 - Obtaining Phytophthora capsici PcDRS2 Gene Knockout Mutant

[0051] Using PEG-CaCl 2 Phytophthora capsici protoplasts were prepared by the mediated protoplast transformation method, and the genetic transformation method of Phytophthora capsici was referred to "Wang, Z.W., Tyler, B.M., and Liu, X.l. (2018) Protocol of Phytophthora capsici Transformation Using the CRISPR-Cas9 System.Methods MolBiol. 1848:265-274.”.

[0052] The specific method for obtaining knockout transformants in the present invention is as follows: the homology arm carrier of the knockout gene PcDRS2 obtained in Example 2, sgRNA and Cas9 co-expression plasmid are jointly transferred into the protoplast of Phytophthora capsici strain LT1534, and the grown The transformants were screened by G418-resistant V8 solid plate culture, the DNA of the transformants was extracted for PCR verification, and the obtained mutants were sent to the company for sequencing verification to obtain a single kn...

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Abstract

The invention provides a phytophthora capsici P4-ATP enzyme PcDrs2 protein and application thereof. The DNA nucleotide sequence of the P4-ATP enzyme PcDrs2 protein is shown as SEQ ID NO.1. Experiments show that the phytophthora capsici P4-ATP enzyme PcDrs2 protein is closely related to the growth rate of phytophthora capsici hyphae and the secretion of extracellular laccase, the growth of the hyphae can be effectively slowed down and the secretion of laccase can be effectively blocked by regulating the PcDrs2 protein, and the capability of infecting hosts by phytophthora capsici is weakened, so that the aim of effectively controlling the occurrence and development of phytophthora capsici is achieved.

Description

technical field [0001] The invention relates to the technical field of microorganisms, in particular to a P4-ATPase PcDrs2 protein of Phytophthora capsici and its application. Background technique [0002] Phytophthora is an important plant pathogenic oomycete, causing severe agricultural losses and damage to natural ecosystems worldwide. For example: late blight caused by Phytophthora infestans on potatoes caused the Great Irish Famine; soybean root rot caused by Phytophthora sojae causes global annual losses of US$1-2 billion. However, Phytophthora capsici is different from most Phytophthora species with a narrow host range. Phytophthora capsici has a wide host range and can infect important agricultural vegetables such as peppers, tomatoes, gourds, eggplants, and model plants tobacco and Arabidopsis. mustard, causing huge economic losses. [0003] In view of the serious harm of Phytophthora capsici, existing scholars have classified it as one of the top ten important pa...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/00C12N15/52C12N1/15C12N15/113C12N9/22C12R1/645
CPCC12N9/00C12N15/1137C12N9/22C12N2310/20
Inventor 杨成东陈庆河贾斯蒂冯婉珍
Owner HAINAN UNIVERSITY
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