Cloning And Functional Analysis Of BHLH Transcription Factor Family Genes From Alternaria Alternata

Basic helix-loop-helix(bHLH)transcription factors are highly conserved transcription factors in Eukaryotes,which regulate the conidia production and development,mycelial morphology and pathogenicity of many phytopathogenic fungi.Alternaria alternata is one of the most important diseases of tobacco leaves in tobacco production.Under suitable conditions,Alternaria alternata may cause fulminant damage,resulting in the decline of quality of tobacco leaves,the decrease of aroma quality and quantity,the increase of irritability and impurity,it has become a major obstacle to the production of high-quality tobacco leaves.The bHLH transcription factor family was cloned from Alternaria alternata and 12 genes named AabHLH1~AabHLH12 were obtained.It was found that the amino acid length of these 12 bHLH transcription factor genes was quite different and their homology was very low,but they all have a common bHLH domain,and the location of the domain was not fixed,some of them were distributed at the amino end,some are in the middle,others are closer to the carboxyl end.The insertion mutant M1~M4 of AabHLH1~AabHLH4 gene was obtained by Linear Minimum Element Method and PEG mediated protoplast transformation.The primary functional analysis of AabHLH1~AabHLH4 gene was carried out by comparing the differences between the mutants and wild type and complement.The results are as follows:(1)AabHLH1 gene is 1650 bp in length and contains no introns and encodes a protein of 549 amino acids;AabHLH2 gene is 396 bp in length and contains no introns and encodes a 131 amino acid protein;AabHLH3 gene contains a 99 bp intron and encodes a 549 amino acid protein;AabHLH4 gene is 1,317 bp in length and has no introns and encodes a 438 amino acid protein.(2)AabHLH1~AabHLH4 genes are involved in regulating the colony phenotype and growth of Alternaria alternata.The colony of M1 becomes darker,the aerial hypha becomes weaker,and the growth rate decreases;the M2 colony becomes thicker on the PDA,the growth rate increases,the mycelium layer becomes significantly thinner on the MM,and the growth rate is lower than that of wild type;the M3 colony color becomes lighter,the mycelium layer becomes thinner,and the growth rate is slightly lower;the M4 colony becomes darker on the PDA,the growth rate increases,and the color is lighter on the MM,and the growth rate decreases.(3)AabHLH1~AabHLH4 genes are involved in the sporulation process of Alternaria alternata,but the effect stage is different.AabHLH1 gene affected the sporulation and germination of Alternaria alternata,which showed that the conidia yield was decreased,the germination time was delayed,the germination rate was decreased,and partial spore beak becomes longer.AabHLH2 gene affected the germination of Alternaria alternata,the results showed that the germination time of conidia was slightly delayed,AabHLH3 gene affected the sporulation and germination of Alternaria alternata,the conidia yield was decreased,the germination time was delayed,and the germination stopped after 8 hours,the length of bud tube was shortened and the germination rate was decreased.AabHLH4 gene influenced the sporulation and germination of Alternaria alternata,which showed the increase of conidia yield and the shortening and enlargement of internodes.(4)AabHLH1~AabHLH4 are involved in the regulation of melanin content in Alternaria alternata.The Melanin content of M1~M3 colony decreased in different degree,while that of M4 colony increased significantly.(5)AabHLH1~AabHLH4 genes are involved in regulating the utilization of nonfermentable carbon sources.The growth of M1~M4 colony was inhibited on 1% ethanol nonfermentable carbon source medium,and the growth of M1~M4 colony was promoted on 1% LArabinose and 1% D-Xylose non-fermentable carbon source medium.(6)AabHLH1~AabHLH4 genes are involved in the regulation of toxin toxicity and pathogenicity.The virulence of M1~M4 was lower than that of wild type,and the virulence of M4 was lower than that of wild type,and the pathogenicity of M2 was lower than that of wild type,and almost all of M3 was lost.(7)AabHLH1~AabHLH4 genes are involved in the regulation of stress response.The tolerance of M1 colony to salt stress and osmotic stress was higher than that of wild type and complement,the tolerance of M2 colony to salt stress was lower than that of osmotic stress,and the tolerance of M3 colony to salt stress was lower than that of wild type,the tolerance of M4 colony to salt stress and osmotic stress was lower than that of wild type.It can be inferred from this that,AabHLH1~AabHLH4 gene is involved in the regulation of pigments,mycelium morphology,conidia production and germination,vegetative growth,utilization of non-fermentable carbon sources,stress response,toxin toxicity and pathogenicity of Alternaria alternata.