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A kind of cysteine ​​protease inhibitor moers1 mutant derived from rice blast fungus and application thereof

A technology of protease inhibition and rice blast fungus, applied in the fields of plant pathology and molecular biology, can solve the problems of polluted environment, decreased control effect and high cost of chemical control

Active Publication Date: 2021-12-31
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the cost of chemical control is usually high, and the continuous emergence of bacterial resistance leads to a decline in the control effect, and it is easy to cause environmental pollution

Method used

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  • A kind of cysteine ​​protease inhibitor moers1 mutant derived from rice blast fungus and application thereof
  • A kind of cysteine ​​protease inhibitor moers1 mutant derived from rice blast fungus and application thereof
  • A kind of cysteine ​​protease inhibitor moers1 mutant derived from rice blast fungus and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Example 1: MoErs1 interacts with OsRD21 in vitro and in vivo

[0020] Yeast two-hybrid experiment method: clone the full-length sequence of MoErs1 gene (without signal peptide) from the cDNA of Magnaporthe grisea wild-type strain Guy11, the full-length nucleotide sequence of MoErs1 gene is shown in SEQ ID NO: 6, and at the same time The rice gene OsRD21 was cloned from the cDNA of rice susceptible variety CO39, its amino acid is shown in SEQ ID NO: 7, its CDS sequence is shown in SEQ ID NO: 8, and the inventors found through research that OsRD21 has a cysteine ​​protease The activity was constructed into pGADT7 and pGBKT7 yeast expression vectors by NdeI / BamHI, respectively, for yeast interaction verification; the expression vectors were respectively introduced into AH109 yeast strains, and yeast transformation kits (MP Company) were used. After transformation, first grow in SD-Trp-Leu lacking medium for 3 days, then use velvet to copy onto SD-Trp-Leu-His and SD-Trp-Leu...

Embodiment 2

[0025] Example 2: MoErs1 inhibits OsRD21 cysteine ​​protease activity

[0026] Biotin-labeled DCG-04 method for measuring the activity of cysteine ​​protease: dissolve 0.5 mg of OsRD21-Flag protein expressed in tobacco in 50 mM sodium acetate solution, adjust the pH to 6.0, and then add 10 mM L-cysteine and 2 µM DCG-04. 0.2mM E-64, MoErs1-GFP protein and different mutant proteins (E3132, sigma) were added to the control, and incubated at room temperature for 5 hours. After incubation, add twice the volume of pre-cooled acetone to precipitate the protein, centrifuge at 10,000 g for 1 minute, remove the upper layer, add 70% acetone to wash twice, and dry in the air. The pellet was dissolved with TBS, added protein loading buffer, and boiled for 5 minutes. Streptavidin-coupled HRP was used to measure the activity by western hybridization, and bands could be displayed after western hybridization to prove that it had cysteine ​​protease activity.

[0027] The experimental result...

Embodiment 3

[0028] Example 3: The mutation of the interaction site cannot complement the pathogenicity defect of the MoERS1 gene deletion mutant

[0029] CM medium configuration method: Measure 20× nitrate (120g sodium nitrate, 10.4g potassium chloride, 10.4g magnesium sulfate heptahydrate, 30.4g potassium dihydrogen phosphate, add distilled water to dissolve to 1L) 50ml, 1000× trace elements ( 2.2g zinc sulfate heptahydrate, 1.1g boric acid, 0.5g manganese chloride tetrahydrate, 0.5g ferric sulfate heptahydrate, 0.17g cobalt chloride hexahydrate, 0.16g copper sulfate pentahydrate, 0.15g sodium manganate dihydrate, 5g Tetrasodium EDTA, add distilled water to dissolve to 100ml) 1ml, vitamin solution (0.01g biotin, 0.01g vitamin B6, 0.01g vitamin B1, 0.01 riboflavin, 0.01 p-aminobenzoic acid, 0.01 nicotinic acid, add distilled water to dissolve to 100ml ) 1ml, weigh 10g of glucose, 2g of peptone, 1g of yeast extract, 1g of casamino acids, and 15g of agar powder, add distilled water to quant...

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Abstract

The invention discloses a cysteine ​​protease inhibitor MoErs1 derived from Magnaporthe grisea and its mutation and application. The present invention finds for the first time that the MoErs1 protein secreted by Magnaporthe grisea has cysteine ​​protease inhibitor activity, can inhibit its activity by interacting with cysteine ​​protease in rice, interfere with the immune response of rice, thereby promoting the growth of Magnaporthe oryzae infestation.

Description

technical field [0001] The invention belongs to the fields of phytopathology and molecular biology, and in particular relates to a cysteine ​​protease inhibitor MoErs1 derived from Magnaporthe grisea and its mutation and application. Background technique [0002] As the most widely planted and most important food crop in the world, rice supplies more than half of the world's population. With the rapid increase of the global population, the demand and safety of rice production become more and more important. Magnaporthe grisea ( Magnaporthe oryzae ) caused by rice blast is a major fungal disease that affects rice cultivation and yield all over the world. The disease is regularly and repeatedly popular in rice plantations all over the country. The control of blast fungus mainly relies on disease-resistant breeding and the use of fungicides. However, the blast fungus mutates quickly in field races, and resistant varieties lose their resistance to blast after a few years of pl...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/81C12N15/31C12N15/80C12N1/15C12Q1/37C12R1/645
CPCC07K14/8139C12N15/80C12Q1/37
Inventor 张正光刘木星郑小波张海峰
Owner NANJING AGRICULTURAL UNIVERSITY
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