Transcriptome Study Of The Synthesis Of Hydrogen Sulfide In Saccharomyces Cerevisiae

During wine-making,yeasts can produce a little of hydrogen sulfide.Yeasts differ greatly in production of H2S under different genetic background.H2S has strong volatility,and it can bring "rotten eggs" into wine,that will seriously affect the flavor and sensory quality of wine.In this research,the gene transcription characteristics in different fermentation periods of Saccharomyces cerevisiae were studied by RNA-Seq.Through the screening of differentially expressed genes that associate with H2S synthesis,it will provid theoretical basis for revealing the molecular mechanism of wine yeasts produce H2S and breeding of excellent Saccharomyces cerevisiae,and it will has great significance to the improvement of the flavor and sensory quality of wine.The main results were as follows:1.The H2S output of 181 Saccharomyces cerevisiae strains was studied through BIGGY agar and Triple M medium fermentation.(1)According to the color of their colonies on BIGGY agar,among the 181 strains,there were 119 high-H2S-production strains,48 moderate-H2S-production strains,9 low-H2S-production strain,and 5 none-H2S-production strains.(2)Triple M medium fermentations were proceeded using 19 selecting strains out of the 181 strains,UCD932 and UCD522 were used as control strains.According to the total H2S production of strain in the Triple M medium fermentation,the 21 strains were divided into none-H2S-production strains(0-lppm),low-H2S-production(1-200 ppm),moderate-H2S-production strains(200-1000 ppm)and high-H2S-production strains(>1000 ppm)four levels.Among them,there were 4 none-H2S-production strains,112y4,UCD932,112y14,174y1,respectively;5 low-H2S-production strains,LFP515,LFP506,44y2,LFP525-4,LFP525,respectively;8 moderate-H2S-production strains,UCD522,LFN520,LFN524-6+LFP525-4,LFN524,LFN511,32y15,3ly3,31y9,respectively;4 high-H2S-production strains,LFN524-4,LFP525-2,LFN524-6,32y12,respectively.2.18 samples of none-H2S-production strains 112y4 and high-H2S-production strains 32y12 in Triple M medium fermentation 40 h(the early stage of the H2S production,samples number were 112y4-? and 32y12-?),88 h(the highest period of H2S production,samples number were 112y4-? and 32yl2-?)and 160 h(the end of H2S production,samples number were 112y4-? and 32y12-?)was proceeded RNA-Seq,using 50 SE strategy and Illumina HiSeq 2000.(1)A large number of quality datas were acquired,Clean reads of all samples were more than 5800000,and total mapped reads of all samples to reference genes were more than 76%,unique matched reads were more than 71%;Total mapped reads of all samples to reference genome were more than 94%,unique matched reads were more than 84%.(2)Through quality assessment of reads,sequencing saturation analysis,randomness assessment and gene coverage,the results showed that randomness of all samples were very good,and sequencing depth meeted the requirements of bioinformatics analysis.(3)Clean reads were mapped to S288C reference genome,the results showed that all samples had more than 5649 genes expressed during 6500 genes of reference genome.(4)The expression of genes was calculated using RPKM,the results showed:a.Compared to none-H2S-production strains 112y4-?,there were 507 differentially expressed genes in high-H2S-production strains 32yl2-?,291 were up-regulated,216 were down-regulated;Compared to 112y4-?,there were 1193 differentially expressed genes in 32yl2-?,965 were up-regulated,228 were down-regulated;Compared to 112y4-?,there were 754 differentially expressed genes in 32yl2-?,486 were up-regulated,268 were down-regulated.b.Compared to 112y4-?,there were 1190 differentially expressed genes in 112y4-?,297 were up-regulated,893 were down-regulated;Compared to 112y4-?,there were 1303 differentially expressed genes in 112y4-?,706 were up-regulated,597 were down-regulated;Compared to 112y4-?,there were 976 differentially expressed genes in 112y4-?,881 were up-regulated,95 were down-regulated.c.Compared to 32y12-?,there were 985 differentially expressed genes in 32y12-?,522 were up-regulated,463 were down-regulated;Compared to 32yl2-?,there were 1405 differentially expressed genes in 32y12-?,840 were up-regulated,565 were down-regulated;Compared to 32yl2-?,there were 234 differentially expressed genes in 32yl2-?,112 were up-regulated,122 were down-regulated.3.Through GO function enrichment and Pathway enrichment analysis of the differentially expressed genes in sulfate metabolic pathways,the results showed that:(1)Compared to the none-H2S-production strains 112y4,MET 16 of the high-H2S-production strains 32y12 was down-regulated in 40 h;MET10 was up-regulated in 88 h;MET10,MET16 and MET14 were up-regulated in 160 h.(2)In the comparision of same strains of different fermentation periods:a.Compared to the 40 h,APA1,MET10,MET5,IRC7,MET22,MET17,MET3,MET16 and MET14 of the none-H2S-production strains 112y4 were down-regulated in 88 h;IRC7,MET22,MET10,MET5,MET17,MET3,MET16 and MET14 were down-regulated in 160 h,YML082W was up-regulated in 160 h;Compared to the 88 h,MET3 of the none-H2S-production strains 112y4 was down-regulated in 160 h,APA1,YML082W and YGR012W were up-regulated in 160 h.b.Compared to the 40 h,APA1,IRC7,MET5,MET17,MET3,MET16 and MET14 of the high-H2S-production strains 32y12 were down-regulated in 88 h;APA1,IRC7,MET5,MET17 and MET3 were down-regulated in 160 h,MET2 and YML082W were up-regulated in 160 h;Compared to the 88 h,there were no differentially expressed genes in the high-H2S-production strains 32y12 in 160 h.Through the analysis of differentially expressed genes in sulfate metabolic pathways,and other H2S synthesis related genes.MET14,MET16,MET10,SSU1,SUL1,HOM2,HOM6,SER33,MET17,CYS3,CBF1 and THI5 were preliminarily determined as the key genes that regulated the synthesis of H2S.