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Effector protein derived from phytophthora capsici as well as coding gene and application thereof

A coding and gene technology, applied in the field of molecular biology, can solve problems such as pesticide residues threatening the human living environment

Inactive Publication Date: 2014-04-16
YUNCHENG UNIVERISTY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Extensive use of traditional chemical agents not only causes pathogens to develop drug resistance, but also causes pesticide residues to seriously threaten the living environment of human beings.

Method used

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  • Effector protein derived from phytophthora capsici as well as coding gene and application thereof
  • Effector protein derived from phytophthora capsici as well as coding gene and application thereof
  • Effector protein derived from phytophthora capsici as well as coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Embodiment 1, the acquisition of the effector protein Phcnlp1 encoding gene of Phytophthora capsici

[0047] The pathogenic Phytophthora capsici strain phy12 was selected as the test material, the genome sequence of Phytophthora capsici was analyzed according to the reported effector protein gene information, the effector protein gene in the whole genome of Phytophthora capsici was identified, and the specific primer p1 was designed and p2, use the PCR method to amplify and screen the target gene fragment, then design primers p3 and p4 according to the obtained gene fragment, and perform 3'-RACE to amplify the full length of the gene.

[0048] 1. Using the CTAB method to extract high-quality Phytophthora capsici genomic DNA

[0049] Phytophthora capsici strain phyc12 was cultured on solid oat medium (recipe: oatmeal 100g, agar 17g, water 1000ml, boiled for 30 minutes, divided into Erlenmeyer flasks, sterilized, and cooled for later use) in a 28°C biochemical incubator ...

Embodiment 2

[0097] Example 2, Phcnlp1 gene expression pattern analysis in the process of Phytophthora capsici infecting pepper host

[0098] 1. Pepper leaves inoculated with zoospores of Phytophthora capsici

[0099] 1. Zoospore preparation

[0100] (1) Transfer Phytophthora capsici strain Phyc12 to V8 medium (recipe: V8 vegetable juice 200ml, CaCO 3 3.0g, 15g of agar, add distilled water to 1000ml, boil and sterilize in a Erlenmeyer flask for later use; among them, V8 vegetable juice is commercially available) and cultivate for 4 days, pick mycelium pieces from the edge of the colony and move to fresh V8 medium for further cultivation for 4 days for later use.

[0101] (2) Pick the hyphae pieces at the edge of the colony and culture them in V8 medium, and culture them in the dark at 25°C for 5-6 days.

[0102] (3) Add sterilized water (it is better to immerse the mycelium), change the water every 1d until zoospores are produced, and adjust the zoospore concentration to 1×10 5 pieces / m...

Embodiment 3

[0135] Embodiment 3, the transient expression of Phcnlp1 gene in tobacco body

[0136] 1. Isolation of Phcnlp1 Gene Mature Peptide Sequence

[0137] According to the obtained full-length sequence of Phcnlp1 gene (sequence 2), the following primers pvxF and pvxR were designed:

[0138] pvxF: 5'-cc gctgttatcgaccacgaccaggtcgt-3' (the underlined part is the recognition sequence of ClaI, and the following sequence is the 55th-80th position of sequence 2);

[0139]pvxR: 5'-tcc ctggtaccaggcgttcgcgagct-3' (the underlined part is the recognition sequence of SmaI, and the following sequence is the reverse complementary sequence of positions 1406-1428 of sequence 2).

[0140] Using the cDNA of Phytophthora capsici strain Phyc12 as template, PCR amplification was carried out with primers pvxF and pvxR. The reaction system is 50 μL: cDNA template (2 μL), 10× buffer (5 μL), Mg 2+ (4 μL), dNTP (4 μL), 1 μL each of upstream and downstream primers, Taq enzyme (0.5 μL), ddH 2 O (32.5 μL...

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Abstract

The invention discloses an effector protein derived from phytophthora capsici as well as a coding gene and application thereof. The protein disclosed by the invention is (a) or (b) or (c): (a) protein consisting of amino acid sequences represented in the 19th-476th sites of a sequence 1 of a sequence list; (b) protein consisting of an amino acid sequence represented in the sequence 1 of the sequence list; and (c) protein obtained by substituting and / or deleting and / or adding the amino acid sequence of the protein defined by (a) or (b) through one or more amino acid residues and having an effector function. Experiments prove that the protein disclosed by the invention effectively participates in a process that phytophthora capsici infects a pepper host and has a function of inducing plant tissue cell death and even an obvious necrotic symptom on an infected part. The effector protein disclosed by the invention is significant for enriching molecular mechanism information of plant pathogenic oomycetes and host interaction and for establishing an oomycetes disease integrated control technological strategy.

Description

technical field [0001] The invention belongs to the field of molecular biology and relates to an effector protein derived from Phytophthora capsici and its coding gene and application. Background technique [0002] Effector protein (effector) is a class of molecules produced by pathogens in the process of invading the host that can overcome the host defense system. During the infection process, the pathogen uses effector proteins to overcome the host's defense system. On the one hand, it can weaken the host's resistance to facilitate its successful invasion, and on the other hand, it can use the host's nutrients for self-reproduction. Many pathogens secrete effector proteins, such as those found in bacteria, fungi, oomycetes, and nematodes. There are many types of effectors in pathogens, including NLP, Avr, CRN, PcF, ScR, glycohydrolase, pectinase, chitinase, and esterase. Among them, the NLP protein gene is ubiquitous in oomycetes, fungi, and bacteria. The number of such...

Claims

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Application Information

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IPC IPC(8): C07K14/37C12N15/31C12N15/82C12N5/10C12N1/21C12N1/15C12N1/19A01H5/00
CPCC07K14/37C12N15/8263
Inventor 冯宝珍李培谦
Owner YUNCHENG UNIVERISTY
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