Structural Biology Of The Arabidopsis Protein BAM1 In Complex With Tomato Yellow Leaf Curl Virus Protein C4

At present,the agricultural planting,processing and export of tomato products in China are in a continuous growth trend.As an important cash crop in China,the agricultural safety production of tomato is the top priority in our work.The occurrence of tomato yellow leaf curl virus disease has seriously affected the progress and development of tomato industry,and caused great losses to agricultural economy,so it is of great significance to study how to control tomato yellow leaf curl virus disease.RNAi is an important mechanism of antiviral immunity in plants,and effector factors produced by viruses can inhibit RNAi in the relative evolution of the plant-pathogen arms race.The purpose of this study is to understand the molecular mechanism of BAM1 protein promoting intercellular transport of RNAi and the interaction between virus C4 protein and plant host BAM1 protein at the molecular level by analyzing the crystal structure of the complex of Tomato yellow leaf curl virus C4 protein and Arabidopsis thaliana protein BAM1,the inhibitor of RNAi.It provides theoretical basis for further understanding the mechanism of antiviral immunity and prevention and treatment of tomato yellow leaf curve virus.In this study,a common recombinant expression vector was constructed by gene cloning,and a plasmid co-expressing Arabidopsis BAM1 and Tomato yellow leaf curl virus C4 was successfully constructed by the method of co-expression vector construction.The co-expression plasmid was transformed into the Rosetta receptor of Escherichia coli prokaryotic expression system for heterologous expression of Arabidopsis BAM1 protein and tomato yellow leaf curl virus C4 protein.Primarily using nickel column affinity chromatography separation and miscellaneous protein target protein,anion exchange chromatography separation and purification further bad purpose protein,protein and state the purpose of the protein,TEV enzyme label removal of fusion,again using anion exchange chromatography separation of target protein and label,then through target protein gel filtration chromatography purification,and testing protein aggregation state;In this study,a series of schemes were explored for the expression and purification of BAM1 and C4 proteins,such as buffer p H,ion strength,protein label selection and truncation body selection,etc.,and finally BAM1 and C4 protein complex with high purity and good homogeneity was obtained.The C4 protein was isolated and purified by Escherichia coli.C4 was in a highly polymerized state,lost its biochemical function and could not interact with BAM1.Build two protein expression vector,after expressing purification process,found no longer in C4 aggregation state,by the high aggregation state position,migrated to monomer location,and C4 protein and BAM1 altogether elution on gel filtration chromatography column,which shows that we are identified by using the method of gel filtration chromatography C4 and BAM1 interactions in vitro.In this study,crystals of BAM1 and C4 protein complex were obtained through initial crystal screening,which promoted the crystal structure analysis of BAM1 and C4 protein complex,and provided a solid foundation for the successful completion of the follow-up project.