| Jujube is not only the widest species of dried fruit,but also an important medicinal plant and economic forest species in China.The yield and quality of jujube were affected seriously by fungal diseases.A.alternata is an important pathogen causing jujube fruit disease,which causing various symptoms,such as jujube black spots,jujube brown skin,jujube shrinkage,etc,We named the disease as jujube black spot disease with its typical representative symptoms.The complete genome data of A.alternata was published in 2015,laying the foundation for the research on the gene function,the interaction mechanism and the pathogenesis of the fungus.In this study,we used overlapping PCR technology,replace target genes by hygromycin resistance gene,by using the method of protoplast transformation.The four proteins in the CDK8 kinase module,SSN3,SSN8,MED12 and MED13,were knocked out.Finally,we obtained the deletion mutants of the four protein-coding genes respectively by hygromycin Screen,PCR detection,and the complementary strains of the four protein-coding gene deletion mutants.Phenotypic analysis of the above deletion mutant and its complementary strain revealed that:1.It was found that the deletion mutant of Ssn3,with inhibited growth,almost no aerial hyphae.The conidia production of the mutant decreased significantly(-98.56%),and no conidia branching,only I to 3 conidia chain.The Ssn3 deletion strain was sensitive to H2O2,NaCl and SDS stress,and it was significantly reductions in virulence on jujube and pears.However,it was interesting to note that the fruit flesh around the diseased spot of pears showed a light blue color with the deletion of Ssn3.the reason need further studies.2.The phenotype of Ssn8 deletion mutant was very similar to Ssn3 deletion mutant in terms of colony morphology,pressure stress,spore-producing morphology and spore-producing quantity.However,the growth rate of the Ssn8 deletion mutant was slightly increased in YEG culture,and the colony was gray-white.The pathogenicity of jujube and pear was decreased obviously,but,the flesh around the diseased spot of Ssn8 mutant was not light blue.3.Medl2 mutant phenotype was consistent with Ssn3 and Ssn8 mutant.But,the colony color of Medl2 mutant was very similar to that of Ssn3 mutant,with black color and slow growth,in YEG culture.The pathogenicity of jujube and pear was significantly decreased by 71%and 90%,respectively.4.The phenotype analysis of Med13 deletion strain showed that it was significantly different from the other three genes.Although its growth slowed down in PDA and YEG culture,there were relatively more aerial hyphae and the colony color was close to the wild type.Med13 mutant was less sensitive to the stress of hydrogen peroxide,sodium chloride and SDS than the other three mutants.The virulence of Med13 mutant on the fruit of jujube was stronger than that of the other three mutants,the virulence decreased by about 43%,and its virulence on the fruit of pear was not weakened.Interestingly,the phenotype of Med13 mutant was similar to that mutants of the other three gene in PCA culture medium.Almost no aerial hyphae,growth slowed down,spore production decreased significantly by 91.24%.The spore chain became shorter,there were no branches,and there were only 1 to 3 conidia.The research in this paper shows that four genes in the kinase module CDK8 of A.alternata was essential for growth,development of the conidia,elongation and branches of the conidia chain.The four genes also participate in the pathogenesis of A.alternata. |
