| High-glucose tolerance of yeast is one of important stress resistance properties.Research on high-glucose tolerance mechanism of yeast is valuable for the high gravity ethanol fermentation and Hakka rice wine brewing.At present,the research on the high-sugar tolerance mechanism of yeast is limited to metabolic levels,the genes controlling characteristic high-glucose tolerance and corresponding pathways have not been demonstrated clearly.The research started from the sugar tolerance yeast which had been screened in honey sample can tolerate 700 g/L high sugar concentration,detected its basic performance,ribosomal 18 S rDNA,ITS,26 S rDNA D1/D2 gene region sequences and other molecular biological identification methods were used to confirm species information of high sugar tolerance yeast.Complete yeast genome information by genomic sequencing.The gene expression of high-sugar tolerance yeast under different glucose concentrations would be studied through transcriptomics and the differences of gene expression under high-glucose stress would be analyzed at RNA level.Combined with the physiological characteristics and stress-induced metabolites variation characteristics of yeast in response to different high glucose concentration,the functional genes related with high glucose tolerance were screened.Real-time quantitative PCR(q-PCR)was used to verify the accuracy of transcriptome sequencing results and to analyze the expression of key genes in metabolic pathways that may be associated with yeast high glucose toleranc,high-sugar tolerance mechanism of yeast would be discussed and elaborated on transcriptome level.The results are as follows:(1)Through the morphological characteristics and molecular biological characteristics,the screened yeast LGL-1 was comprehensively and systematically identified.Yeast LGL-1 can grow normally at a concentration of 700 g/L glucose,and the logarithmic growth period is 20-40 h.The rDNA-ITS,18 S rDNA and 26 S rDNA were used to identify the species.The result was ZygoSaccharomyces mellis.(2)The genome sequencing,assembly and quality assessment of Z.mellis LGL-1were completed using third-generation genome sequencing technologies.The total sequencing depth is approximately 130 × and the genome size is approximately 19.98 Mb.The bio-informatics software was used to annotate the Z.mellis LGL-1 genome,and a total of 9528 genes were annotated,matching 106 KEGG pathways,includingamino acid biosynthesis pathways and carbohydrate metabolism pathways.(3)The Z.mellis LGL-1 of 300 g/L,500 g/L and 700 g/L sugar concentration were selected for transcriptome sequencing.The sequenced transcripts were assembled and spliced and compared with the reference genome sequence.The ratio of each group was compared.The efficiency is between 94.26 % and 95.90 %.In the group of three different sugar concentrations,the number of differentially expressed genes of Z.mellis LGL-1 was 2155,754 and 3388,respectively;2000,678 and 3152 genes were functionally annotated in GO analysis.There were 941,1578,and 361 differentially expressed genes,respectively,which were classified by the COG database.Among the three groups,there are 103,108,and 98 different Pathways.The pathways involved in genes are antibiotic biosynthesis,carbon source metabolism,amino acid biosynthesis,yeast cell cycle,yeast MAPK signaling pathway and purine metabolism.(4)Key genes that are up-regulated and down-regulated in the three major metabolic pathways(HOG-MAPK signaling pathway,trehalose metabolic pathway,and purine metabolic pathway)associated with high glucose tolerance of Z.mellis LGL-1 were selected,among other pathways.A total of 25 genes were subjected to real-time PCR(q-PCR)verification experiments,and the expression change multiples were basically consistent with the sequencing results of the transcriptome.Among them,sho1,sln1,ssk1,gpd1,hog1;tps,otsb;amd,rpc and other key genes may be related to the high glucose tolerance mechanism of Z.mellis LGL-1. |
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