Cloning And Function Analysis Of Zinc Finger Transcription Factor Ab10279 In Alternaria Brassicicola

Alternaria brassicicola is the main pathogenic fungus of cruciferous vegetable black spot,which causes a decrease in the yield and quality of these vegetables.The transcription factor is a class of proteins in an organism that regulate gene transcription.The Zn2Cys6 zinc finger protein transcription factor is unique to fungi and regulates fungal growth,development and stress response.Therefore,study on the function of the transcription factor Ab10279 gene is helpful to our knowledge about the molecular pathogenic mechanism.This work is also of great significance for the effective control of cruciferous vegetable black spot.In this study,Ab10279,a Zn2Cys6 zinc finger protein transcription factor of A.brassicicola was used as the research object.The amino acid sequence of Ab10279 gene was obtained from the fungal transcription factor database.Based on this sequence,primers were designed and the Ab10279 gene was cloned.Targeted gene disruption and functional complementation were conducted by homologous recombination principle and PEG-mediated protoplast transformation.Function of Ab10279 gene was studied by comparative analysis between the deletion mutant(?Ab10279),the complementation strain(C)and Wild-type strain(WT).The main results are as follows:(1)Ab10279 gene is 1069 bp in length which contains two introns.The full length of cDNA is 960 bp,encoding 319 amino acids,and containing a GAL4 zinc finger structure.(2)Ab10279 gene is involved in the regulation of vegetative growth and colony morphology of A.brassicicola.The colonies of the ?Ab10279 mutants are white,and the aerial hyphae are more vigorous,which differed notably from the WT strain and the complementation strain.Furthermore,growth rate of ?Ab10279 was faster than that of WT and C strains in minimal media(MM).However,the mutants grew more slow than that of WT in rich media(PDA).(3)Ab10279 gene also regulated conidiation and spore germination.The conidia yield of ?Ab10279 in PDA medium was significantly reduced.The spores became slenderer.Disruption of Ab10279 gene also had a negative effect on the germ tube branching and elongation.(4)Ab10279 gene is involved in the regulation of stress response in A.brassicicola.Under low salt stress and osmotic stress,?Ab10279 was more tolerant than the wild type and complementation strains.However,the mutants showed an opposite answer to the high salt stress and osmotic stress,which was more sensitive to the stresses than the WT.(5)Disruption of Ab10279 gene caused a dramatic decrease in fungal virulence.The lesions size in all inoculated leaves(intact and wounded leaves)was smaller than that of WT and C1 strain.Even in the intact leaves,the mycelium of mutants could still penetrate the leaves and continued to grow on the back of inoculated leaves,which indicated that disruption of Ab10279 gene may affect little of the penetrating ability of A.brassicicola.The above results indicate that Ab10279 plays an important role in vegetative growth,conidiation and spore germination,stresses response and pathogenicity of A.brassicicola.