Protein crystal structure of magnaporthe oryzae mitogen-activated protein kinase Mpsl and application of protein crystal structure in bactericide target

A technique for activating protein kinases, crystal structures, applied in the fields of molecular biology and genetic engineering

Pending Publication Date: 2018-04-13
CHINA AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] In the study of protein kinases, although MAPK has been reported as a drug target in mammals, it has not been reported to use mitogen-activated protein kinase as a drug target in plant pathogenic fungi and related research has not been reported; Mps1 as a drug target One of the co

Method used

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  • Protein crystal structure of magnaporthe oryzae mitogen-activated protein kinase Mpsl and application of protein crystal structure in bactericide target
  • Protein crystal structure of magnaporthe oryzae mitogen-activated protein kinase Mpsl and application of protein crystal structure in bactericide target
  • Protein crystal structure of magnaporthe oryzae mitogen-activated protein kinase Mpsl and application of protein crystal structure in bactericide target

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Embodiment 1 Mps1 structure analysis

[0030] Based on the Mps1 protein crystal diffraction data collected earlier, homology modeling was carried out using the method of homologous molecular replacement, and the protein crystal structure of Mps1 was successfully resolved. High-resolution protein crystal structures, via figure 1 It can be found that the obtained structure is composed of two molecules, and the carboxy-terminal non-kinase domain of Mps1 (further experimental verification, and finally confirmed that it is composed of 15 amino acids) is close to the substrate binding site of the kinase in its structure, namely The presumed 15-amino acid carboxy-terminal non-kinase domain of Mps1 (Mps1 401-415 ) may be associated with the kinase domain (Mps1 1-360), further analysis of the analyzed crystal structure of Mps1 protein revealed that there is self-interaction, and the interaction interface is mainly concentrated in the C-terminal region of Mps1 molecule A (carb...

Embodiment 2

[0031] Example 2 Mps1 self-interaction analysis:

[0032] In order to verify that the carboxy-terminal non-kinase domain of Mps1 seen in the structure of Example 1 may interact with its own kinase domain, yeast two-hybrid technology and co-immunoprecipitation technology are used to verify and analyze it.

[0033] 1 Yeast two-hybrid, specifically including the following related reagents and steps:

[0034] Reagents: yeast two-hybrid kit, fission yeast auxotrophic strain AH109 (purchased from Clontech);

[0035] Vector: pGADT7, pGBKT7 (purchased from Clontech);

[0036] step:

[0037] (1) Yeast AH109 frozen at -80°C in the early stage of streak culture, pick a single colony and inoculate it into a 200mL sterilized Erlenmeyer flask containing 50mL YPDA liquid medium, and culture it with shaking at 30°C and 250rpm until OD 600 >1.5;

[0038] (2) Quantitatively take 200 μL of the above culture in 300 mL YPDA liquid medium and shake it at 30°C and 250 rpm until the OD 600 reach...

Embodiment 3

[0061] Example 3 Mps1 Phosphorylation Level Analysis (Western blot)

[0062] Reagents: all conventional reagents;

[0063] step:

[0064] (1) pre-purified Mps1 WT and Mps1 1-360 Prepare samples according to the condition of equal mass ratio; take 80 μL of the protein sample prepared above, add 20 μL of 5×protein loading buffer, boil for 5 minutes, and let stand on ice for 5 minutes before use;

[0065] (2) Prepare SDS-PAGE protein glue according to the formula on the Takara catalogue, and load the sample;

[0066] (3) Electrotransfer the SDS-PAGE protein gel at 15V overnight (note: apply it to the positive pole and glue to the negative pole), and place the electroporation tank on the stirrer to avoid burning out the electrotransfer instrument;

[0067] (4) First stain with Ponceau S solution for about 0.5min, and the protein transferred to the membrane can be seen; after pouring off the staining solution, use 5% skimmed milk powder (prepared with 1×TBS-T buffer) at room te...

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Abstract

The invention discloses a protein crystal structure of magnaporthe oryzae mitogen-activated protein kinase Mpsl and application of the protein crystal structure in a bactericide target. The protein crystal structure of the magnaporthe oryzae mitogen-activated protein kinase Mpsl consists of two molecules. By analysis of the protein crystal structure of the magnaporthe oryzae mitogen-activated protein kinase Mpsl, the protein crystal structure has a self-interaction effect based on structural speculateion; an experiment further verifies that the protein crystal structure has interaction betweenthe carboxyl terminal non-kinase domain (Mpsl 401-415) and the autokinase domain (Mspl 1-360), and relevant experiments verify that the carboxyl terminal non-kinase domain (Mpsl 401-415) of Mpsl hasan outstanding inhibition effect on the phosphorylation level of Mpsl, so that design, screening and optimization are performed further through the protein space structure of the carboxyl terminal non-kinase domain of Mpsl to provide a structural guidance for a small-molecular inhibitor with relatively activity of Mpsl, and a foundation is laid for development of relevant researches based on the space structure, which serves as the bactericide target, of Mpsl.

Description

technical field [0001] The invention relates to the technical fields of molecular biology and genetic engineering, in particular to a protein crystal structure of rice blast fungus mitogen-activated protein kinase Mps1 and its application in fungicide targets. Background technique [0002] Rice is one of the main food crops in the world, and about 50% of the world's population uses rice as a staple food. However, rice blast, which is caused by Magnaporthe oryzae infecting rice, is a common and extremely harmful fungal disease in various rice-producing areas around the world, and it is also one of the important threats restricting the safe production of rice and food security supply. It is reported that the annual rice yield loss caused by rice blast is about 10%-30%, which has a huge impact on world food security (Skamnioti and Gurr, 2009). At present, due to the fact that resistant varieties that can effectively resist the rice blast have not been bred, the control of rice...

Claims

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Application Information

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IPC IPC(8): C12N9/12
CPCC12N9/12C12Y207/11024
Inventor 刘俊峰周锋张国珍彭军波张圆圆
Owner CHINA AGRI UNIV
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