Cloning Of StTPS1 And Establishing Genetic Transformation System Of Setosphaeria Turcica

Northern Corn Leaf Blight is one of the most serious corn diease,and in the past few years in the northeastern region was high trend and cause serious yield loss.The main reason is that the cold weather here is benefit to occurrence and disease,and in recent years the planted maize variaties are more single in the Northeast maize main producing areas.These factors contribute to the prevail of Northern Corn Leaf Blight in the northeasten.For a long time,people do not understand the pathogenesis of Northern Corn Leaf Blight in depth,and limit the development of new prevention and control technology,so there is a urgent need for an in-depth study of its pathogenesis.There is a report that TPS1 gene control development and pathogenesis of rice blast fungus,but there is no report about TPS1’s functions in Setosphaeira turcica,here were try to establish a technology platform for gene knockout in Setosphaeira turcica and try to elucidate the role of StTPS1 in fungus development and formation of pathogenesis.And this research may provide a steady cornerstone for the following research and control of Northern Corn Leaf Blight disease.(1)Cloning of TPS1 in S.turcica.A homologous gene in the genome of S.turcica was successfully identified using the BLAST tool and designated as StTPS1.Designed specific primer pair using Primer Premier 5 software to clone StTPS1 gene with polymerase chain reaction.The StTPS1 gene consists of 2090 base pairs and encodes 517 amino acids,has four exons and thress introns.StTPS1 gene has untranslated regions in both ends.According to the highly homology with MgTPSl in Magnaporthe grisea,I inferred that StTPS1 may fulfill same functions as MgTPS1.(2)Bioinformatics analysis of TPS1 in S.turcica.The molecular weight,theoretical pI,instable index,and grand average of hydropathicity were 58376.29,5.36,42.05,and-0.248 suggesting that it is a hydropathic and unstable protein.A search of the pfam database showed the StTPS1 coding protein to share features with those of the glycosyltransferase family.The phylogenetic tree showed StTPS1 with a high degree of homology with MgTPS1.These results indicated that StTPS1 may fulfill the same functions in S.turcica as MgTPSl in M.grisea.(3)Construction of StTPS1 gene knockout plasmid.The hygromycin resistance gene was amplified by PCR using primers hph-F and hph-R from pSilent-1 plasmid(Fungal Genetics Stock Center,Kansas State University,KS,U.S.).PCR was performed using PrimeSTAR Max DNA Polymerase(Takara,Dalian,China).HindⅢ/SmaⅠ restriction enzyme recognition sites were added to the 5’ ends of the primers hph-F and hph-R.After double enzyme digestion,the PCR product was ligated to pBlueScript Ⅱ SK(+)(Stratagene,La Jolla,CA,U.S.)to generate pBS-HPH.Fragments of StTPS1 gene upstream and downstream flanking sequence 1125 bp and 895 bp were amplified by PCR using primer combinations up-F/R and down-F/R with PrimeSTAR Max DNA Polymerase(Takara,Dalian,China).XhoⅠ/HindⅢ restriction enzyme recognition sites were added to the 5’ ends of the up-F/R primers,and SmaⅠ/NotⅠ restriction enzyme recognition sites were added to the 5’ ends of down-F/R primers,respectively.These PCR products were then constructed into the pBS-HPH plasmid to generate StTPSl gene knockout vector named pBS-StTPS1.(4)Generation of gene deletion mutants.In this chapter,my research try to establish genetic transforming system and gene knock out technology platform of Setosphaeira turcica.Firstly,my work comfirmed that Setosphaeira turcica grown for forteen days can produce more conidia when it growing on the V8 vegetable juice medium at 23℃ with full darkness.Secondly,I developed two new methods to harvest conidia of Setosphaeira turcica.Thirdly,I confirmed that the ratio of lywallzyme,driselasw,and snailase in the enzymesolution.The enzymatice conditions are 28℃,80 rpm,and 3-5 hours to release protoplasts.After optimizing the genetic transforming system and several attemps,I obtained several transformants that are putative gene knockout mutants,and there are being testing for their correctness and pathogenecity.(5)Response of host resistance enzymes during S.turcica infection.In order to understand the mechanism behind the host resistance,in this chapter I measured the enzymes activities during the pathogen infection to the change of maize biochemical traits.According to the previously work,the activities of PAL,PPO,CAT,and POD had changed when host paint treated with pytopathogenetic fungal.Inoculated corn species Huangzaosi with Setosphaeira turcica strain SYY1303,the activites of POD,CAT,and PPO was upregulated initially and then down to the normal codition.In contrast,the activity of PAL was down regulated initially and then back to normal condition.These data coincident with previous researches about other pathogen-host system,and this was the first report of how maize defese against Setosphaeira turcica in resistance enzymes way.