Isolation And Functional Analysis Of BHLH Transcription Factor Family Gene Of C.gloeosporioides

Colletotrichum gloeosporioides is an important plant phytopathogenic fungus with a wide range of hosts,causing serious economic losses to agricultural production.The researches on this fungus mainly focuse on the morphological description,basic biology,genetical diversity,fungicide resistance,disease control and molecular pathogenesis.But the function of the CgbHLH transcription factor family gene of Colletotrichum gloeosporioides in mycelial growth and development,pathogenic molecular mechanism is still unclear,and there have an important production significance and academic value for disease prevention to analysis the function of CgbHLH transcription factor family gene.Based on the genomic sequences of the CgbHLH transcription factor family genes searched from whole genome database of C.gloeosporioides,the full-length cDNA sequences of CgbHLHs were cloned,gene evolutionary relationships and gene structures as well as predicted protein structures and motifs were bioinformatically analyzed,the results showed that the CgbHLHs had typical characters of bHLH family.CgbHLH gene was transiently expressed in tobacco cells and its encoding protein was localized mainly in the nucleus.The expression of CgbHLHs was quantitatively analyzed during infection process and after treatment of low or high temperature,surfactant SDS,NaCl and H2O2,using qRT-PCR method.The results showed that there were significant differences in gene expression levels among different CgbHLH genes,with CgbHLH4 and CgbHLHL6 being highly expressed during the initial stage of infection,suggesting that theses two genes might be involved in the infection process of C.gloeosporioides.Single-and double-gene-knockout mutants,single-and double-gene-RNAi mutants of CgbHLH4 and CgbHLHL6 were obtained through gene knockout method and RNA interference method.Compared with the wild-type A2 strain,the gene knockout mutant ?CgbHLH4 could not form the appressorium,its conidiation ability was weakened,mycelium became thicker and deformed,and the conidium became shorter and thicker;the mutant ?CgbHLH6 showed low appressorial formation rate,deformed hyphal,and its antioxidant capacity was reduced.The mycelium became thicker in the RNAi mutant psilent-1:CgbHLH4,which was mostly the same with the knockout mutant ?CgbHLH4,but its conidiation rate was higher than that of the?CgbHLH4.Mycelial tip of RNA interfering mutant pSlient-1:CgbHLH6 was swelled,like the knockout mutant ?CgbHLH6.Mycelium of the mutant pSlient-1:CgbHLH4:CgbHLH6 was sparse,but spread rapidly.The sensitivity test of mutants to carbendazim showed that the resistance of mutants to carbendazim was enhanced.The results of pathogenicity test showed that,when inoculated on wounded mango leaves and ripe fruits with mutant mecelia plug,the mutants were more virulent than that of the wild type,with an exception of the RNAi interfering mutant pSlient-1:CgbHLH4:CgbHLH6,ist virulence was not significantly changed.when inoculated on non-wounded leaves and ripe fruits,the virulence of mutants was largely weakened.The yeast two-hybrid assay was used to screen out the proteins interacting with bHLH transcription factor,5 proteins was initially found interacting with CgbHLH4,and 1 protein with CgbHLH6.This study initially revealed,in the aspect of bioinformatics,molecular biology,cytology,physiology and biochemistry,that the CgbHLH family of transcription factors analyzes play an important role in the regulation of C.gloeosporioides mycelial growth,development,differentiation,conidia production,appressorium formation,adaptability,carbendazim resistance and virulence..