Identification And Expression Pattern Analysis Of BHLH Transcription Factor Family And The Function Analysis Of StRTG3 In Setosphaeria Turcia

The bHLH transcription factor family is a highly conserved transcription factor in eukaryotes.The family members are widely found in animals,plants and fungi and play important role in regulating the growth and development of eukaryotes.So far,the research on bHLH transcription factors has mainly focused on plants and animals,and there have been relatively few studies on fungal bHLH transcription factors.For example,studies on Magnaporthe oryzae and Aspergillus indicate that bHLH transcription factors are involved in important growth stages such as mycelial growth and development,conidia production and appressorium development.However,there have not been systematic reports on Setosphaeria turcica.Therefore,in the present study,the bHLH protein transcription factor family was identified in the genome of S.turcica.The structure characteristics and physicochemical properties were analyzed.The expression patterns of bHLH protein family members in pathogen growth and development were clarified by RNA-Seq technique.Further cloning the homologous gene of S.cerevisiaeScRTG3,that is,the family member gene StbHLH10,named it StRTG3,and created silent mutants of StRTG3 gene to preliminary explore the function of this gene.This study can clarify the basic characteristics of the StbHLH protein family of S.turcica as a whole,and initially explore the role of the protein fam ily in the growth and development of the pathogen,laying a foundation for further research on the function of each member of the StbHLH protein transcription factor family.In addition,screening and identification of StRTG3 gene silencing transformants provides a material basis for furthering analysis of the function of this gene.The main results are as follows:1.A total of 14 members of the bHLH protein transcription factor family were identified in S.turcica,and their gene structure analysis indicated that 7 genes without introns.and the other 7 genes are broken genes;physical and chemical properties analysis showed that most of the members of this family were weakly acidic;phylogenetic trees of three species of S.turcica,S.cerevisiae and M.oryzae were constructed,and analysis revealed that most members of S.turcica bHLH family were on the same branch as M.oryzae,indicating that these species were closer evolutionarily to each other than to S.cerevisiae.2.By analyzing gene expression patterns at 5 developmental stages of the pathogen including hypha,conidium,germ tube,appressorium and penetration peg.These results showed that except StbHLH1was not expressed,other family member genes had different levels of expression,Randomly select three genes of thoseand used the total RNA of the 3 developmental stages as a template for real time PCR analysis.The results showed that the changes in the expression levels of those genes were consistent with the results of RNA-Seq data,indicating that these genes are involving in the abovementioned development process of the pathogen.3.By comparing with the S.cerevisiaeScRTG3 gene.the RTG3 homologous gene was identified in S.turcica database.This gene is a member of the bHLH transcription factor family,StbHLH10,which we named StRTG3.The gene was cloned and found to be 1659 bp in length with no introns.4.Based on the pSilent-1 vector,a StRTG3 gene silencing vector pSilent-1:StRTG3 containing hygromycin resistance was constructed.Through the PEG-mediated protoplast transformation method,the silent vector was transformed into the protoplast of the bacteria,and through hygromycin resistance screening and qRT-PCR verification,two StRTG3 gene silent transformants R3-1 and R3-3 were obtained,The expression levels of StRTG3 were 43%and 51%of those in wild-type strain,respectively.5.Compared with the wild type,the growth rate of the two transformants was reduced,the colony color is off-white,and the aerial hyphae were denser.Under hyperosmotic stress conditions,the growth rate of the two transformants was significantly lower than that of the wild type.Both transformants had a reduced ability to penetrate cellophane.It is preliminarily speculated that this gene is involved in the regulation of hyphae development and penetration.6.Based on the pET32a vector,a prokaryotic expression vector pET32a-StRTG3 was constructed to express and purify the StRtg3-6×His recombinant protein in the prokaryotic system.Further,EMSA technology was used to prove that StRtg3 could directly bind to the STRE element(5’-GGTACC-3’)in the promoter region of the Hyperosmotic stress-related gene StMSN2,preliminary verification of StRtg3 on the transcriptional regulation of the target gene StMSN2 in vitro.