| Eurotium cristatum is the dominant fungus involved in the fermentation of Chinese Fuzhuan brick tea which also has been known as “gold flower fungi”,the asexual morph of the fungus is Aspergillus cristatus.Our study results showed that the sporulation mode of the fungi could be regulated by osmotic pressure,it`s different with other mode of regulated sporulation.Moreover,the sexual development mechanism of fungus is considered as an important fact regarding to the sporulation mode,and it still not clear in this group.It is necessary that we study the sexual sporulaiton genes and regulation mechanism of E.cristatum.This study analyzed the distribution of sporulation genes in genome of Eurotium cristatum using genome sequencing,and several methods were followed: 1)the change the expression levels of the sporulation genes from the fungus were analyzed using transcriptome sequencing;2)these results were verified by RT-q PCR;3)the significantly different expressed MAT gene were carried out by gene knockout and bioinformatics analysis,subsequently,we analyzed the arrangement and expression of mycotoxin gene cluster in the the E.cristatum genome and the mycotoxin were detected by HPLC.Based on the data and analysis,the following conclusions could be given:(1)The genome sequencing and analysis of Eurotium cristatumThe genomic DNA of Eurotium cristatum was extracted and sequenced by Illumina sequencing,the result showed the genome of E.cristatum was 27.9 Mb and had 10,136 genes,the gene density was 2.98 kb per gene,the annotated coding regions accounted for 51.92% of the genome,with an average coding length of 1,571 nt and 2.98 exons per gene;the average exon length was 479 nt.(2)Evolution analysis of mating-type lociComparison of the mating-type gene loci among Aspergillus species indicated that the mode in Eurotium cristatum differs from those in Aspergillus species,this model supports the evolution of homothallism from heterothallism.(3)Genes component and expression of HOG pathwayHOG pathway genes in Saccharomyces cerevisiae have homologs in the Eurotium cristatum genome,with the exception of Sln1.at low and high osmolarity,there was no significant change in the level of expression of most key genes in the HOG pathway,and the results indicated that E.cristatum had other mechanism to deal with the change in osmotic pressure(4)Collinearity analysis of mycotoxin genes clusters and mycotoxin detectionWe analyzed the collinearity of six kinds of mycotoxin gene clusters by using Blastp and In Paranoid,the result showed there were no known mycotoxin gene cluters in Eurotium cristatum genome.Meanwhile,we assayed six mycotoxins by HPLC,the result suggested that the strain is safe under low-and high-osmolarity conditions.(5)Transcriptom analysis of sporulation genes under different osmolarityIn this study,the expression level of 107 sporulation genes under different osmolarity were analyzed,the result indicated that high osmolarity is beneficial for asexual development and inhibit sexual development,there are 2/3 asexual sporulation genes with up-regulation and 4/5 sexual sporulation genes with down-regulation.Moreover,we obtained MAT genes with the differentially expression and sexual sporulation correlation by this analysis.(6)Bioinformatics analysis of MAT genesAmino acid and nucleotide sequence of MAT genes were analyzed by the bioinformatics methods,the analysis results indicated that the sequence and structure of MAT genes is conservative,it supported that the MAT1-1-1 gene is developed from MAT1-2-1 gene.(7)RT-qPCR detection expression level of MAT genes under different development stagesCompared with mycelium stage,RT-q PCR detection expression level of MAT genes under asexual and sexual development stages,the results showed that the expression level of MAT genes under sexual development is the highest and the expression level of two MAT genes is relatively consistent.(8)Function study of E.cristatum MAT genesThe function of Eurotium cristatum MAT genes were studied by gene knockout technology,the results indicated that the formation and mature of cleistothecia,ascus and ascospore were controlled by MAT genes in E.cristatum.Meanwhile,it could regulate mycelium development and it has no infection to the asexual development and black exudates.The study reported whole genome sequencing of E.cristatum for the first time,and got a new model of the mating-type locus,this study verified that function and evolution model of MAT gene had no relation;secondly,the paper analysed the regulation of osmolarity to spore formation of E.cristatum using transcriptome sequencing for the first time,the result indicated osmolarity regulated expression level of spore formation gene for regulation spore formation style;thirdly,gene expression analysis demonstrated that the HOG pathway of E.cristatum was not involved in the response to high osmotic pressure,E.cristatum may respond to high osmolarity stress via mechanisms other than the HOG pathway.Lastly,a mycotoxin gene cluster collinearity analysis and an HPLC assay indicated that the locations of the mycotoxin genes have no obvious relation with mycotoxin production,the fungus is considered to be safe under low-and high-osmolarity conditions. |
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