The Expression Analysis And Cloning Of StSNF1 In Setosphaeria Turcica

Northern leaf blight,caused by the infection of Exserohilum turcicumPass.)Leonard&Suggs,is one of the major leaf diseases in maize producing areas in the world,and especially in the Northeast,northern North,Northwest of China and Southern mountainous areas.The production of maize was threated by this disease seriously,and the reduction was more than 20?30%in the epidemic years.Currently,the study of pathogenic factors in Setosphaeria turcica(Luttrell)Leonard&Suggs mainly focused on cell signal transduction pathway,cell wall degrading enzymes,oxidoreductase,melanin and toxins.The research of cell signaling pathways including cAMP pathway,MAPK pathway,Ca2 pathway is extensive,but there are few studies on AMPK pathway.StSNF1,as a new cutting-in point and was cloned in S.turcica.In this study,the physicochemical properties and structure characteristics of SNF1 protein were analyzed by bioinformatics technique.The expression level of StSNFl at different infection stages in maize,different stages in the conidial invasive growth of S.turcica and at the different carbon sources were studied by Real-time PCR.Furthermore,StSNF1 gene knockout vector was constructed and successfully obtained the mutant.1.The location of SNF1 in the genome of S.turcica was identified by NCBI and JGI database.The primers(SNF1 F1 and SNF1 R1)of SNF1 gene was designed by S.turcica genomic database.The homology analysis showed that the gene was a single copy in the S.turcica,which was named StSNF1.Sequence analysis showed that StSNF1 was 3046 bp,contained with three exons and two introns,ORF 2634 bp,moreover,the structure was highly similar to the CcSNF1 of Cochliobolus carbonum.2.StSNFl gene encoding protein that analyzed by bioinformatics technique.The result showed the protein is hydrophilic with the molecular weight of the protein was 97.94 kD,isoelectric point was 8.29,instability index was 53.24,and the grand average of hydropathicity(GRAVY)was-0.638.The StSNF1 neither the membrane proteins nor the secretory proteins for the reason that the transmembrane region and signal peptide were not detected in the amino acid sequence of the protein.The result of subcellular localization showed that the percentage of K-NN in the nucleus was 78.3%,and it was speculated that the main site of the function of the protein was nuclear The protein has two functional domains,one at the carbon terminal and the other at the nitrogen end.Specifically,one protein kinase domain and one carbon catabolite-derepressing protein kinase,ubiquitin-associated domain(SNF1_UBA)are located in nitrogen end,5 kinase associated domain 1(KA1)combinations together as a series connection on the opposite end.Structural analysis showed that random coil was the principal secondary structureal,followed by a-helix.Homology analysis showed that there was high consistency(90.92%)between StSNFl and CcSNFl,but the homology with Saccharomyces cerevisiae and other pathogenic fungi was on the low side.3.The expression level of StSNFl in S.turcica by the technology of Real-time PCR.The result indicated that the expression of StSNF1 was significantly up-regulated in late stage of infection,especially at 72 hours,after that the expressions of StSNFl decreased gradually until 96 hours,while the expression of StSNF1 was enhanced again during 120 hours The expression trends of StSNF1 was up-regulated during the conidia germination,24 hours(the mature period of the invading filament)become maximum,and the expression of StSNFl is more than four-fold increase than 6 hours(the initial stage of appressorium formation).It can be concluded that StSNFl is related to the infection of conidia germination.The highest expression of StSNFl was found in the medium that sucrose as the sole carbon source followed by pectin.On the contrary,StSNF1 showed the lowest expression level in the glycerol medium.The expression level of StSNFl gene in glucose and fructose medium was consistent.That was a half of the expressed amount in pectin medium and one third of the expression level in sucrose.It can be concluded that the StSNFl gene was related to the utilization of sucrose and pectin.To summarize,the StSNFl gene was highly expressed in the late stage of infection,and was up-regulated in the process of spore infection,in addition,it was closely related to carbon source metabolism.4.The StSNFl gene mutant was successfully obtained through optimization of genetic transformation system.This study found that the protoplast release more from the spore of S.turcica use the enzymolysis of Lywallzyme,Driselase,Snailase under the optimum temperature 280C and between the optimum enzymolysis in 6?8 hours.