| Alternaria brassicicola is widely distributed in nature because of its strong adaptability to the environment and host,often causing black spot disease of cruciferous vegetables and serious economic losses.Moreover,A.brassicicola is also a source of airborne pathogens in many parts of the world and has become an increasingly recognized risk factor for asthma and fatal asthma exacerbations.Early studies have shown that the FUS3/KSS1-MAPK signaling pathway plays an important role in the pathogenicity of A.brassicicola,but for AbSte12,its downstream transcription factor,its function,mechanism and regulatory genes are not clear.This study was mainly based on A.brassicicola and then came to understand deeply of the biological function,downstream target genes and regulatory mechanism of transcription factor AbSte12 by building AbSte12 knock-out mutants and Complementary and comparing with Wild-type strain.The main results are as follows:1.Analysis of AbSte12 gene in Bioinformatics.Queried homologous gene of A.brassicicola and named AbSte12 based on Saccharomyces cerevisiae Ste12（NP011952.1）and a homologous gene of Pyrenophora teres f.teres（XP003303640.1）comparing with the constructed local genome database of A.brassicicola.The specific primers were used to amplify the DNA and cDNA of AbSte12 gene from A.brassicicola.The DNA was 2364 bp in length containing three introns and cDNA was 2091 bp encoding 696 amino acids.It showed that the amino acid sequences of AbSte12 have strong homology to other different Ste12-like genes by DNAMAN analysis.The amino acid sequences of A.brassicicola Ste12 was analyzed by SMART and the results suggested that different STE12 proteins contain a typical Ste-homolog domain in the N terminal region and two C2H2 zinc fingers in the C terminal region,which are lacking in yeast.The phylogeny of different Ste12-like gene was analyzed by MEGA 6.0 and the results showed that the AbSte12 of A.brassicicola had the closest relationship with Ste12 of Botrytis cinerea and Ste12 of Sclerotinia borealis.2.Construction of AbSte12 knock-out mutants.It’s need to construct of AbSte12 targeting vector A + M + B and obtain protoplasts of A.brassicicola.Then carried out transformation by PEG-mediated and selected transformants from the medium which containing hygromycin.The specific primers were designed and validated to obtain AbSte12 knock-out mutants by ordinary PCR.In addition,it was validated that a single copy of the hygromycin phosphotransferase gene has been inserted into the AbSte12 gene deletion mutant by Southern blotting.3.Functional study of AbSte12.ΔAbSte12 mutant was compared with Wild-type strain between colony phenotype,conidial formation,pathogenicity,penetrating ability of mycelial and other aspects.The results showed that the growth rate of ΔAbSte12 mutant on PDA medium was slowed down and ΔAbSte12 mutant couldn’t produce mature conidia.We inoculated detached cabbage leaves with mycelial plugs of A.brassicicola WT and ΔAbSte12 mutants and found that the ΔAbSte12 mutant could not infect leaf tissue and lose pathogenicity completely.In addition,the ΔAbSte12 mutant couldn’t penetrate through the cellophane and grow again on P DA medium.These results suggest that AbSte12 is involved in the regulation of mycelial growth,conidial formation,penetrating ability and pathogenicity in A.brassicicola.4.Identification of the regulatory genes of AbSte12 in the downstream.ΔAbSte12 mutant was compared with Wild-type strain by Real-time PCR.The results This result confirmed preliminarily that the downstream regulatory genes of AbSte12 were AbBud8,AbBem1,AbPck1,AbChs1,AbSho1 and AbPrm2. |
