Plant immune activator protein secreted by plasmopara viticola, primer and application

A grape downy mildew, immune activation technology, applied in the field of plant immune activation protein, can solve environmental and human health threats and other problems, and achieve rapid and strong effects of allergic cell death

Active Publication Date: 2022-01-14
NORTHWEST A & F UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] Grape downy mildew (Plasmopara viticola) caused by grape downy mildew (Plasmopara viticola (Berk.&M.A.Curtis) Berl.&De Toni] is the most serious fungal diseas...

Method used

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  • Plant immune activator protein secreted by plasmopara viticola, primer and application
  • Plant immune activator protein secreted by plasmopara viticola, primer and application
  • Plant immune activator protein secreted by plasmopara viticola, primer and application

Examples

Experimental program
Comparison scheme
Effect test

experiment example 1

[0026] Experimental example 1 Cloning and plant transient expression of plant immune activation protein PvAvh77

[0027] (1) Genomic DNA extraction

[0028] Using the Peronospora vine strains collected from the Northwest Agriculture and Forestry University Grape Germplasm Resource Garden in Yangling, China as material, it was collected and ground into powder and added to the lysate (2% CTAB, 2% PVP40, 1% β-ME, 200mM NaCl , 0.2mM EDTA (pH 8.0 and 50mM Tris pH 8.0), incubate at 65°C for 30min, add an equal volume of chloroform-isoamyl alcohol (volume ratio 24:1), centrifuge at 16000g for 15min, extract twice, take the supernatant and use anhydrous Ethanol precipitated overnight. The precipitate was washed twice with 70% ethanol, dried and completely dissolved with 50 μLTE buffer, and the DNA content and quality were detected with a spectrophotometer.

[0029] (2) PCR amplification of the target gene

[0030] Using the extracted genomic DNA as a template, conventional PCR was ...

experiment example 2

[0055] Experimental example 2 Prokaryotic expression and purification of plant immune activation protein PvAvh77-M2

[0056] (1) Construction of prokaryotic expression vector

[0057] Using pCold-TF as the prokaryotic expression vector, since the allergic necrotic reaction caused by PvAvh77-M2 is stronger than its full-length coding sequence PvAvh77, specific primers for the gene encoding PvAvh77-M2 were designed. The primers are shown in Table 3.

[0058] Table 3 is the PvAvh77-M2 coding gene-specific primers

[0059]

[0060] The reaction system and PCR reaction program are as follows in Table 4:

[0061]

[0062]

[0063] Use the two-step method recommended by the instruction manual (KOD-Plus-Neo, TOYOBO, Japan) to carry out the PCR reaction program: pre-denaturation at 94°C for 2 minutes; denaturation at 98°C for 10 s, annealing / extension (30s / kb) at 68°C, and 35 cycles of reaction; Final extension at 72°C for 10 min; samples were stored at 12°C.

[0064] After...

experiment example 3

[0076] Experimental example 3 Plant immune activation protein causes allergic necrotic reaction on tobacco leaves

[0077] After the measured concentration of pCold-TF-PvAvh77-M2 solution was diluted to 10 μM and 15 μM, the leaves of Nicotiana benthamiana were injected, and the same concentration of pCold-TF was used as a negative control, and the lethality of the leaves was observed after 120 hours. like Figure 4 Middle D shows the schematic diagram of allergic necrosis caused by injection of purified pCold-TF and recombinant plant immune activation protein pCold-TF-PvAvh77-M2 into tobacco leaves. It is found that there is slight lethality and no significant change in the degree of lethality at different concentrations, which may be The "trigger factor" and His tag protein on the carrier affected the function of PvAvh77-M2. Therefore, thrombin is used to excise the "trigger" and His-tagged protein (such as Figure 4 Middle C, SDS-PAGE detection schematic diagram of the pur...

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Abstract

The invention discloses a plant immune activator protein secreted by peronospora viticola, a primer and application. The amino acid sequence of the plant immune activator protein is as shown in SEQ ID NO.2, the nucleotide sequence of the coding gene of the plant immune activator protein is as shown in SEQ ID NO.1, the amino acid sequence of a mutant PvAvh77-M2 of the plant immune activator protein is as shown in SEQ ID NO.4, and the nucleotide sequence of the coding gene of the plant immune activator protein is as shown in SEQ ID NO.3. The mutant PvAvh77-M2 of the plant immune activator protein can induce expression of grape disease-resistant related genes and accumulation of defensive substances, inhibit infection of plasmopara viticola and reduce harm of downy mildew to grapes, and can be used as a biopesticide for preventing downy mildew of the grapes.

Description

technical field [0001] The invention relates to a plant immune activating protein, in particular to a plant immune activating protein secreted by peronospora vine, primers and applications. Background technique [0002] Grape downy mildew (Plasmopara viticola) caused by grape downy mildew (Plasmopara viticola (Berk.&M.A.Curtis) Berl.&De Toni] is the most serious fungal disease that limits the healthy development of the grape industry. Traditional highly toxic pesticides control frost Mildew poses a serious threat to the environment and human health. In 2015, the Ministry of Agriculture and Rural Affairs proposed a zero-growth plan for chemical fertilizers and pesticides. Therefore, the development and utilization of biological pesticides is particularly important. [0003] During the process of plant-pathogen co-evolution, plants have evolved a complex and functionally diverse innate immune system to resist the invasion of potential pathogens. Pathogens can attack plants b...

Claims

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Application Information

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IPC IPC(8): C07K14/37C12N15/31C12N15/70C12N15/11C12N1/21A01N47/44A01P3/00C12R1/19
CPCC07K14/37C12N15/70A01N47/44
Inventor 徐炎付晴晴王韫镭杨静尹晓尚博兴刘国甜
Owner NORTHWEST A & F UNIV
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