Studies On Prokaryotic Expression And Structure Of V-ATPase Subunit A Mutant TSCA Of Mythimna Separate And Effector 8713 Of Puccinia Striformis F.sp. Tritici

The E.coli prokaryotic expression system studied earliest and becomes the most widely used for its high growth rate of host cells, simple and inexpensive bacterial cell culture. In this thesis, the refolding method of TSCA, the mutant of V-ATPase subunit A in the midgut of Mythimna Separate, as well as the expression and structural research of effector pst 8713( Puccinia Striformis f.sp. Tritici) were investigated, respectively.In the first part, the refolding and prokaryotic expression of TSCA were investigated. The vacuolar(H+)-ATPases(V-ATPases) are ATP-dependent proton pumps which are present at the plasma membrane. Recent studies have shown that the V-ATPase subunits are the targets of some small molecule medicines. We constructed designed mutation TSCA with overlap PCR, and inserted the mutation gene into pET-22b(+) vector. The TSCA mutant can not bind to ADP and prevent the feedback inhibition of ADP to the V-ATPase. In the early experiment we tried different expression temperature, time and inducer concentration. The results showed mutant TSCA proteins were expressed as inclusion bodies, and losed their native activities and structures. In this work, the refolding methods of TSCA were carried out and the corresponding results were obtained as following: By an additional of 1 mol/L Larginine in the refolding buffer at pH8.0, the soluble TSCA proteins were successfully obtained in large scale, and we finally got a 8.8 mg soluble proteins as a rate of 55.3 % once refolding cycle.The second part is the research on purification and structure of the effector 8713 of Puccinia Striformis f.sp. Tritici(pst). Pst is a biotrophic fungus which causes wheat stripe rust, one of the most destructive wheat diseases worldwide. To control it, a breeding program with rational utilization of resistance varieties is still the most efficient way. However, the resistance of wheat cultivar will be lost due to the constant pathogenicity variation of pst, and so stripe rust continue to threaten global wheat production and global food security. Therefore, understanding the mechanism of interactions between wheat and pst will greatly facilitate the developing strategies of improving wheat cultivar resistance. During infection, pst forms a specialized infection structure called haustoria to take nutrients and water from host plant tissue and establishes an intimate feeding relationship. During infection, pst releases a series of secreted proteins from haustoria to host cells, which are called effectors. Effectors are pathogen molecules that manipulate host cell structure and function thereby facilitating infection and/or triggering defense responses. But little is known about the mechanisms by which the secreted proteins enter into and function inside plant cells. Therefore, researches on the structure of pst effectors will take great effect on the study of pst infection regulation process. In this study, we performed research on structure of the effector 8713 which is great significance to explore the molecular mechanism of pst pathogenicity. The corresponding results were obtained as following :(1) Successfully constructed the recombinant plasmid pET-22b(+)-8713;(2) Optimize the expression condition;(3) Obtain the puried protein,and finally we can get 21 mg protein powder by 1 L M9 media;(4) Research on the protein structure by NMR. At present, we have completed the analysis of HSQC.