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Effector protein Avh87 derived from phytophthora cinnamomi, and coding gene and application thereof

An effector protein, Phytophthora technology, applied in the field of molecular biology, can solve problems such as environmental pollution, difficulty in preventing and controlling Phytophthora cinnamomyces, and increasing the burden on farmers, so as to reduce plant mortality, eliminate or reduce the impact of epidemics, and significantly reduce plant mortality. The effect of applying value

Active Publication Date: 2019-06-21
NANJING FORESTRY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition to causing huge economic losses in agriculture, forestry and horticulture, the inadvertent introduction of this pathogen has disastrous consequences for natural ecosystems and biodiversity
At home and abroad, spraying pesticides is mainly used to control the prevalence and harm of Phytophthora camphora, but the ideal control effect has not been achieved, which not only increases the burden on farmers, but also causes environmental pollution.
Although the growth of Phytophthora camphora can be inhibited by using environmentally friendly methods such as mulching and composting to allow other microorganisms to proliferate and secrete cellulase in the mulch, the distribution scale of Phytophthora camphora and the ability to survive in soil or asymptomatic plants for several years , so that the control of Phytophthora camphora disease still faces great challenges and difficulties

Method used

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  • Effector protein Avh87 derived from phytophthora cinnamomi, and coding gene and application thereof
  • Effector protein Avh87 derived from phytophthora cinnamomi, and coding gene and application thereof
  • Effector protein Avh87 derived from phytophthora cinnamomi, and coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1: Acquisition of the gene encoding the effector protein Avh87 of Phytophthora camphora

[0038] The Phytophthora camphora strain was selected as the test material, and the genome sequence of Phytophthora camphora was analyzed according to the reported effector protein gene information, and the effector protein genes in the whole genome of Phytophthora camphora were obtained. Then design primers p1 and p2 according to the obtained gene fragments, and amplify and screen the obtained target gene fragments.

[0039] 1. Using CTAB-SDS method to extract high-quality Phytophthora camphora genomic DNA

[0040] Cultivate Phytophthora camphora strains on solid V8 medium plates (recipe: 170ml V8 vegetable juice plus 1.6g calcium carbonate, mix well, centrifuge at 2000rpm for 5min to get supernatant, add pure water to 1L, add 15g agar powder, autoclave After 3 days of culturing in a biochemical incubator at 25°C, three bacterial blocks with a diameter of 4mm were transpla...

Embodiment 2

[0054] Example 2: Analysis of the expression pattern of the Avh87 gene during the infection of apple by Phytophthora camphora

[0055] 1. Phytophthora camphora strains infecting apples

[0056] The mycelium blocks stored in the refrigerator at 4°C were placed on V8 solid medium for activation, and cultured at 25°C for 24-36h. Place a piece of sterilized filter paper of appropriate size on the bottom of the sterilized Petri dish (diameter 9cm) and moisten it with sterilized water. Sterilize the sterile operating table. Wipe the surface of the apples with alcohol cotton to sterilize them. Use the outer flame of the alcohol lamp to sterilize the edge of the cutter, and cut about 1cm on the four sides of the apple 2 cubes, remove the pulp. Pick out the colony and put it on the sterilized filter paper and stuff it into the incision on the surface of the apple. Only three sides are stuffed, and the other side is used as a control of blank solid medium. Plug the incision with ab...

Embodiment 3

[0064] Example 3: Transient expression of Avh87 gene in tobacco

[0065] 1. Construction of PVX recombinant expression vector

[0066] (1) The target gene fragment Avh87 was amplified by Sma I restriction PCR, and the inserted fragment was recovered, with a size of about 327 bp.

[0067] Reaction system: ddH 2 O (33 μL), Sma I (2 μL), plasmid (10 μL), 10×cut Buffer (5 μL). 37°C, 30min.

[0068] (2) Ligate with pGR107 vector cut with the same restriction enzymes, and transform Escherichia coli DH5α.

[0069] (3) The transformed DH5α was screened for Kan resistance, the obtained colonies were shaken overnight at 37°C, and the plasmid was extracted.

[0070] (4) Recombinant plasmid was digested and identified with restriction endonuclease Sma I. The recombinant plasmids that were initially identified correctly after enzyme digestion (two target bands with a size of about 10098bp and 327bp) were sent to GenScript Biotechnology Co., Ltd. for sequencing. Show through sequencin...

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PUM

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Abstract

The invention discloses effector protein Avh87 derived from phytophthora cinnamomi, and a coding gene and application thereof. The protein has the amino acid sequence of SEQ ID No. 1 in the sequence table. The nucleotide sequence expressing the protein is represented by SEQ ID No. 2 in the sequence table. Experiments show that the gene can inhibit plant cell hypersensitive reactions induced by apoptotic precursor protein Bax, or inhibit plant cell death induced by pathogenic phytophthora elicitors INF1. The effector protein Avh87 is of great significance in enriching molecular mechanism information of the interaction between the phytopathogenic oomycetes and hosts, and in establishing a comprehensive prevention and control technology strategy for phytopathogenic oomycete diseases.

Description

technical field [0001] The invention belongs to the field of molecular biology, and more specifically relates to an effector protein derived from Phytophthora camphora and its coding gene and application. Background technique [0002] Effector proteins are a class of exocrine protein molecules secreted by pathogens that can change the cell structure and metabolic pathways of host plants, thereby promoting successful infection of host plants or triggering host defense responses. Many plant pathogens secrete effector proteins, such as those found in bacteria, fungi, oomycetes, and nematodes. There are many types of effectors in plant pathogens, including RxLR, Avr, CRN, PcF, ScR, glycohydrolase, pectinase, chitinase, and esterase. [0003] During the long-term co-evolution process, plants have formed two layers of defense mechanisms to resist the invasion of pathogenic bacteria. The first layer is the immune PTI (PAMP-triggered immunity, PTI) triggered by pathogen-associated...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/37C12N15/31C12N15/82C12N15/74A01H5/00A01H6/82
Inventor 戴婷婷徐月李中言焦彬彬
Owner NANJING FORESTRY UNIV
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