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Key proteins for regulating and controlling secretion of extracellular vesicles from phytophthora capsici as well as coding gene and application thereof

A key protein, coding gene technology, applied in application, genetic engineering, plant genetic improvement and other directions, can solve the problems of limited varieties of resistant varieties and poor resistance stability.

Inactive Publication Date: 2021-10-01
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] However, the types of resistant varieties in commercially cultivated crops are limited and the resistance stability is not good, and chemical control is still the main control method for growth.

Method used

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  • Key proteins for regulating and controlling secretion of extracellular vesicles from phytophthora capsici as well as coding gene and application thereof
  • Key proteins for regulating and controlling secretion of extracellular vesicles from phytophthora capsici as well as coding gene and application thereof
  • Key proteins for regulating and controlling secretion of extracellular vesicles from phytophthora capsici as well as coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0090] Embodiment 1, acquisition of Phytophthora capsici Pcsec4-1, Pcsec4-2 proteins and their coding genes

[0091] In this embodiment, Phytophthora capsici Sec4 protein Pcsec4-1, Pcsec4-2 and its coding gene (or cDNA) can use the DNA (or cDNA) of Phytophthora capsici standard strain LT1534 as a template, obtained by amplification of the primers listed in Table 1 . Wherein, the material for DNA or RNA extraction can be the mycelium of Phytophthora capsici standard strain LT1534. Wherein, the coding gene Pcsec4-1 of Pcsec4-1 is shown as sequence 1 in the sequence listing, and the sequence 1 in the sequence listing is composed of 624 nucleotides; The sequence encodes the protein Pcsec4-1 shown in Sequence 2 in the Sequence Listing. The above-mentioned proteins or genes can also be artificially synthesized. The coding gene Pcsec4-2 of Pcsec4-2 is shown in the sequence 3 in the sequence listing, and the sequence 3 in the sequence listing is composed of 609 nucleotides; the 1st...

Embodiment 2

[0094] Embodiment 2, the construction of Phytophthora capsici Pcsec4-1 and Pcsec4-2 gene knockout vector

[0095] In this example, the CRISPR / Cas9-based gene knockout vector construction method and the sequence of the related vector and the NPT II gene sequence are described in the document "Fang, Y., and Tyler, B.M. (2016). Efficient disruption and replacement of an effector gene in the oomycete Phytophthora sojae using CRISPR / Cas9. Molecular plant 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 pBluescript II SK+ homology arm vector plasmid (Donor vector), the sgRNA expression vector pYF2.3G-Ribo-sgRNA and the Cas9 expression vector pYF2-PsNLS-hSpCas9 used in this example were all donated by Professor Brett M. Tyler of Oregon State University, USA .

[0096] Donor vectors pBS-NPTII-Pcsec4-1 and pBS-...

Embodiment 3

[0104] Embodiment 3, acquisition of Phytophthora capsici Pcsec4-1 gene and Pcsec4-2 gene knockout transformants

[0105] Using CaCl 2 -PEG-mediated protoplast transformation method to prepare Pcsec4-1 and Pcsec4-2 gene knockout transformants, the method of oomycete genetic transformation is described in the literature "Fang, Y., and Tyler, B.M. (2016). Efficient disruption and replacement of an effector gene in the oomycete Phytophthora sojae using CRISPR / Cas9. Molecular plant pathology, 17(1), 127-139."

[0106] The knockout transformant was specifically obtained by combining the knockout gene Pcsec4-1 Donor vector pBS-NPTII-Pcsec4-1 obtained in Example 1 and its own sgRNA recombinant vector pYF2.3G-Pcsec4-1 and the Cas9 expression vector pYF2-PsNLS- hSpCas9; Pcsec4-2's Donor vector pBS-NPTII-Pcsec4-1 and its own sgRNA recombinant vector pYF2.3G-Pcsec4-2 and Cas9 expression vector pYF2-PsNLS-hSpCas9 (the respective homology arms and sgRNA vector and Cas9 expression After mi...

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Abstract

The invention discloses key proteins Pcsec4-1 and Pcsec4-2 for regulating and controlling secretion of extracellular vesicles from phytophthora capsici as well as a coding gene and application thereof. The key protein sequence for regulating and controlling vesicle secretion provided by the invention is shown as a sequence 2 and a sequence 4; the coding gene is shown as a sequence 1 and a sequence 3. Experiments prove that the protein provided by the invention plays an important role in the growth and development process of Phytophthora capsici, specifically, after the protein is deleted, the growth of phytophthora capsici hyphae is slowed down, the number of zoospores is reduced, the pathogenicity is reduced, the secretion amount of extracellular vesicles is reduced, and the like. The conclusions provide a technical basis for exploring phytophthora capsici development and a pathogenic molecular mechanism, and provide a potential molecular target for research and development of novel bactericides in the future.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to the homologous proteins Pcsec4-1 and Pcsec4-2 of the key protein Sec4 (Small GTPase Family Proteins) for regulating the secretion of extracellular vesicles from Phytophthora capsici and their coding genes and application. Background technique [0002] Phytophthora capsici is an important class of plant pathogens in oomycetes. The pathogen was first isolated from peppers in 1918. In 1922, it was named P. capsici Leonian by American scholar Leon H. Leonian (Hausbeck and Lamour, 2004). In taxonomic status, Phytophthora capsici belongs to Kingdom Chromista, Oomycetes, Peronosporales, Peronosporaceae, and Phytophthora. Phytophthora capsici is a worldwide-distributed pathogen with a wide range of hosts and serious damage. It can infect more than 70 kinds of plants including Solanaceae, Cucurbitaceae, and Fabaceae (Granke et al., 2012). After Phytophthora capsici infects peppers, it ser...

Claims

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Application Information

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IPC IPC(8): C07K14/37C12N15/31C12N15/80C12N1/15C12Q1/04C12R1/645
CPCC07K14/37C12N15/80C12Q1/04Y02A50/30
Inventor 刘西莉方媛王治文张思聪张博瑞张灿刘鹏飞
Owner CHINA AGRI UNIV
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