Preliminary Study On Substrate Type And Active Binding Domain Of Rice CCCH Type Protein C3H12

The disease is one of the main reasons for the reduction of rice yield,and the research on the related genes of rice disease resistance can provide a new way for the breeding of resistant varieties.CCCH type zinc finger proteins are widespread in eukaryotes and play an important role in plant growth and development.Studies have found that C3H12 is the first CCCH type zinc finger protein involved in disease resistance in rice,but there is a lack of studies on biochemical and molecular level structure mechanism,combined with the substrate type,the active site and substrate binding domain.In this study,firstly the prokaryotic expression vector of C3H12 gene was constructed,and the C3H12(WT)was obtained by purification.Secondly,the RNA substrate ARE19,nonamer and microRNA mimics N.C,which may bind to the target protein,was synthesized and used to study the interaction between C3H12 and substrate by EMSA.Thirdly,the second structure of C3H12 sequence was analysed,and according to the arrangement of the relative position of tandem zinc finger gene,truncated protein such as C3H12?C201-439,C3H12?N1-329,C3H12?201-329 and C3H12?N1-77C408-439 were constructed to study the active domain to interact with RNA substrate.The results obtained in the experiment were as follows:(1)Using PCR and overlap PCR method to construct the prokaryotic expression vector of C3H12(WT)protein and its truncated mutant protein C3H12?C201-439,C3H12?N1-329,C3H12?201-329,C3H12?N1-77C408-439 construction,we obtained pMAL and pET32 prokaryotic expression vector with MBP tag and His tag respectively and the sequenci ng results were correct.(2)We explored the natural inducing condition and purification scheme of C3H12,found that the protein can be expressed in the normal 30 degrees centigrade and E.Coli can get zinc from natural medium without additional zinc ion.Conventional affinity method that proteins passed through the column in real time can not purify protein with MBP tag and His tag.The method of incubating protein with the column material more than 2 hours at low temperatures was needed.Conventional methods such as affinity,ion exchange,gel filtration and hydrophobic chromatography,can separate the tag and the target protein,but can not remove the other useless protein,suggesting that there may be a binding between the uselessprotein and the target protein.A single band protein was obtained when 0.3% concentration of surfactant sarkosyl was used.(3)RNA substrates that may bind to target proteins were synthesized and labeled,and then substrates ARE19,nonamer and microRNA mimics N.C were labeled and purified.Dot blotting experiment showed that substrates were biotinylated successfully.(4)Using RNA-EMSA method to study on the interaction between C3H12 and RNA substrates,we found that C3H12 combined with ARE19 type substrate.The deletion mutant protein EMSA test found that C3H12 ? C201-439 was not bound to substrate,whereas C3H12?N1-329,C3H12?201-329,and C3H12?N1-77C408-439 were bound to the substrate ARE19.C3H12 substrate binding site was protein C3H12?N1-329.The binding of C3H12?201-329 to substrate showed that 141 amino acids were not directly involved in substrate binding,suggesting that their presence may be related to the interaction with other proteins.The above results reveals the substrate binding type and the active site of C3H12,which lays a foundation for the further research of the binding mode on residues and atomic level,to support the research of molecular mechanisms of disease resistance of rice.