Identification And The Function Exploration Of The Integrin-like Protein ILP2 In Rice Endophytic Actinomycete OsiSh-2

Integrins play an important role in cell signal transduction,cell adhesion,cell movement and a lot processes.Integrins are related to many human diseases,such as in the pathological process of cancer,it related closely with the movement and proliferation of cancer cells.Therefore,the research on animal integrins has important research significance and value in animals,especially in human disease control and treatment.Integrin-like proteins similar to animal integrins also exist in plants and microorganisms.In the preliminary research work of our laboratory,through differential proteomics technology,a type of integrin-like protein(ILP)was identified in OsiSh-2,which might related to the respond of OsiSh-2 to the stimulus of Magnaporthe grisea.On this basis,this thesis takes integrin-like protein as the research object.To explore the possible functions of ILP2 in OsiSh-2,we screened and identified proteins that interact with the integrin-like protein from the total protein of OsiSh-2 by Pull-Down and LC/MS,the relevant results are as follows:1.The integrin-like protein of OsiSh-2.Through bioinformatics analysis,it was found that ILP2 protein is an asymmetric membrane protein containing a large extracellular region,a small intracellular region,and a typical transmembrane domain.At the same time,ILP2 also has two special domains of integrin protein: FG-GAP repeat sequence and four integrin alpha domains.These results indicated that ILP2 protein is a integrin-like protein that exists in OsiSh-2 and is very similar to integrins in terms of structural features and functional domains.2.Screening of proteins that interacting with ILP2.Through Pull-Down and LC/MS,71 proteins that interact with ILP2 were screened and identified from OsiSh-2 total proteins.These proteins are involved in many physiological processes,such as gene regulation,cell signal transduction,environmental regulation,carbon metabolism,etc.,indicating that ILP2 is related to many physiological processes in OsiSh-2,and has an impact on or could regulating these processes.3.Confirmation of the interaction between ILP2 protein,AraC transcriptional regulator and ectoine synase(EctC).The GST Pull-Down experiment proved that ILP2 protein has interactions with AraC and EctC1 directly,and AraC protein has interactions with EctC1 and EctC2 directly.The ILP2 protein and EctC2 might bridged by AraC,and there is an indirect relationship.4.The effect of ILP2 gene overexpression on the physiological characteristics of OsiSh-2.The ILP2 gene was successfully overexpressed in OsiSh-2 by conjugative transfer technology.The results showed that:(1)Overexpression of ILP2 gene changed the morphology of OsiSh-2 mycelium and affected its ability to produce pigment.(2)Overexpression of ILP2 gene affects the ability of OsiSh-2 strain to produce and accumulate ectoine,resulting in a decrease in the tolerance of OsiSh-2 to NaCl,which might related to the effect of ILP2 on EctC.(3)The antagonistic ability of ILP2 gene overexpression strains against Magnaporthe grisea almost completely disappeared.This might because of the overexpression of ILP2 gene affected the transcriptional regulatory factors that interact with the ILP2 protein and are related to the synthesis of antibiotics.In summary,the integrin-like protein ILP2 plays an important role in OsiSh-2 and is related to various biological processes of the strain.This study is the first in the field of streptomyces research to conduct a more detailed study on the integrin-like protein of streptomyces,revealing part of the function of the integrin-like protein ILP2.This is of great significance to the development of OsiSh-2,which has great potential for biocontrol bacteria,and provides clues and basis for follow-up research.