Identification And Functional Analysis Of The MADS Box Transcription Factor Gene AaMcm1 In Alternaria Alternata

Tobacco brown spot is one of the main leaf fungal diseases affecting the major tobacco-growing areas in China which occurs at maturity.Once it breaks out in a large area,the yield and quality of tobacco leaves will be seriously reduced and the economic losses will be heavy.Alternaria Alternata is the pathogen of tobacco brown spot.As an important binding transcription factor in eukaryotes,MADS box transcription factor plays a critical role in the regulation of growth rate,conidia production,secondary metabolism and pathogenicity in various pathogens.In this study,the SRF type MADS box transcription factor gene AaMcm1 was cloned from A.alternata.The gene was successfully knocked out by split-marker gene knockout technology,and the mutant was complemented.The function of the gene in A.alternata was preliminarily studied.The results were as follows:（1）The AaMcm1 gene is 817 bp in length and contains two introns,which are located at242-342 bp and 477-530 bp.The full length of c DNA is 663 bp,encoding 220 amino acids.The protein sequence analysis showed that there was a MADS domain between 52-176 amino acids in the N-terminal.Phylogenetic tree analysis showed that AaMcm1 gene belonged to the SRF-Like subclass and had the closest genetic relationship with Pyrenophora tritici-repentis.（2）AaMcm1 gene is involved in the regulation of vegetative growth,colony morphology and conidia production of A.alternata.On PDA,MM and CM medium,the growth rate of AaMcm1 gene knockout mutants was seriously reduced,and the reduction rate was more than70%.The mycelium color became shallow,and it was white on MM and CM medium.On PDA medium,it was white in the first 7 days,and then gradually became gray.The aerial hyphae became less,the mycelium layer became thinner and the colony was tight.The mutant hyphae grew abnormally,the chain lattice became shorter,some chain lattice expanded,and the edge of colony grew disorderly.No conidia was produced in any medium.（3）AaMcm1 gene is involved in the regulation of melanin and chitin production and stress response process in A.alternata.The melanin content of AaMcm1 knockout mutant decreased by 20.7%and 15.8%at 7 day and 20 day respectively.The chitin content decreased by 63.3%.On salt stress media,the sensitivity of AaMcm1 gene knockout mutant was higher than that of wild type and complement,and the sensitivity of mutant was lower than that of wild type and complement on osmotic stress media.（4）AaMcm1 gene is involved in the regulation of H₂O₂ detoxification and drug resistance pathway of A.alternata.At the same time,the H₂O₂ content decomposed by AaMcm1 knockout mutant was less than that of wild type and complement,indicating that the ability of H₂O₂detoxification of mutant was reduced.On different pharmaceutical media,the AaMcm1knockout mutant showed lower sensitivity to dicopper chloride trihydroxide,prochloraz,dimethachlon,mancozeb and TIBA,but higher sensitivity to polyoxin and CHP.（5）AaMcm1 gene is involved in the regulation of cell wall degrading enzyme activity,toxin virulence and pathogenicity.The activities of pectin lyase and alkaline phosphatase in the mutant were increased,while the activities of cellulase,keratinase,acid phosphatase and lipase were decreased.The toxicity and pathogenicity of the mutant were almost lost.（6）The transcriptome study of the mutant and wild type was subjected to RNA-seq technology.1,917 and 1,322 differentially expressed genes were up-regulated and down-regulated,respectively.GO analysis showed that up-regulated genes were involved in 4,057biological processes,mainly including cellular processes,metabolic processes,biological regulation,biological process regulation and stress response,while down-regulated genes were involved in 2,825 biological processes,mainly including the establishment of cellular processes and metabolic processes,cellular processes,metabolic processes,biological regulation,positioning and positioning methods.KEGG pathway analysis showed that a total of 429 up-regulated differentially expressed genes were involved in 191 signal pathways.The genes involved in signal pathways mainly included eukaryotic ribosome synthesis,purine metabolism,cell cycle-yeast,nucleotide excision repair,DNA replication,etc.;207 down-regulated differentially expressed genes were enriched in 170 signal pathways,including carbon metabolism,starch and sucrose metabolism,glycine,serine and threonine metabolism,tyrosine metabolism.In this study,we find that AaMcm1 gene of A.alternata plays an important role in the regulation of vegetative growth,colony morphology,conidia production,melanin and chitin content,stress response,hydrogen peroxide detoxification ability,drug resistance,cell wall degrading enzyme activity,toxin virulence and pathogenicity.