Isolation And Identification Of Target Genes Of Two Sexual Defective Mutants Of Sporisorium Scitamineum

Sporisorium scitamineumcauses a worldwide serious sugarcane disease,which threantens the sugarcane production in C hina.Isolation and identification of mating and pathogenicity related genes at the molecular levelprovides a theoretical basis on in-depth understanding of the interaction betweensmut fungus and sugarcane.Additionally the identification work can provide the technical support for breeding early.In this study,T-DNA random insertion mutantslibrary contained 13000 hygromycin resistant transformants ofhad establishedAgrobacterium tumefaciens-mediated transformation(ATMT)ofS.scitamineumwild type WT18 and WT17 strains.Verified with mating on the plate,12 mating-defective mutantswere screened.GenomePCRanalysis andsouthern blot showed that T-DNA had been inserted into the genome of WT17 and WT18.Besides,T-DNA was a single copy insertion in these mating-defective mutants.Mating-defective mutants33,91 and 14137 derived from WT18 and mutant 6425 from WT17 were selected by randomlymixing with the relativemating-type wild type strainrespectively andthen inoculatedto the sugarcane seedlings.Pathogenicity test resultsshowed that mobidity waswas 100% when inoculated with wild type strain,whiletwo pathogenicity-defective mutants 91 and 6425 with inoculation rate was 26.67%,mutants33 and14137 were non-pathogenic.HiTAIL-PCR assay showed that the T-DNA was inserted into the promoter region of g5893 gene in the genome of mutant 6425,which was associated with maintenance of telomere coverage,and the T-DNA was inserted into the promoter region of g4800 gene in the genome of mutant 33,which may be associated with the action of the sterol ?5,6-desaturase.Quantitive real time PCR assay showed that the expression of g5893 gene was reduced 60% in the 6425 mutant,and the expression of g4800 gene was reduced50%in mutant 33.Basing on the method of split markerandthe flanking sequences of g5893 gene,g4800 gene andhygromycin sequence,four pairs of specific primers were designed for homologous knockout fusion PCR fragment.By using the PEG mediated transformation methodand screening verification of transformants,theg5893 gene knockout mutant and the g4800 gene knockout mutant were obtained and verified.On the minimal mediumplate,the mating ability of the knockout mutant ?g5893 was weaker than that of the wild type.Further studies showed that the ?g5893 knockout mutant was less sensitive to hydrogen peroxide than the wild type strain.In addition,the sensitivity of ?g5893 knockout mutant to high salt conditions was stronger than wild type.There was no difference in mating ability between the knockout mutant ?g4800 and wild type,indicating that g4800 may not be a T-DN A insert mutation gene,or the gene was not related to mating.