通过构建酿酒酵母沉默表达载体PURH-ADH2，使ADH2基因在PGK强启动子、CYC1终止子在特定区域内进行干扰和表达。采用BamHI和XmalI限制性内切酶对SADH2基因和PURH质粒进行双酶切，构建反义重组表达质粒PURH-SADH2，通过高效酵母转化法和电转法将重组质粒组件转化至酿酒酵母SY01细胞中，获得阳性克隆菌株JY01。酿酒酵母JY01突变菌株与出发菌株SY01和Y01发酵试验结果相比，JY01甘油脱氢酶酶活比出发菌株Y01与SY01分别下降了16.31%和13.5%；当酿酒酵母Y01、SY01和JY01菌株发酵36~60 h时，JY01菌株甘油含量相比Y01分别下降了16.34%、14.25%、14.89%；当酿酒酵母突变菌株发酵48 h时，Y01、SY01 和JY01的乙醇浓度分别为6.243 g/100 mL、7.145 g/100 mL和7.288 g/100 mL，酿酒酵母JY01发酵液乙醇量比比原始菌株Y01乙醇含量提高了14.33%。结果表明通过反义干扰ADH2基因5’UTR序列，能有效干扰酵母工程菌株ADH2转录与表达，削弱甘油积累途径，促进乙醇代谢途径。

The aim was to interfere with and repress the expression of the alcohol dehydrogenase II (ADH2) gene in a specific region by constructing a silencing expression vector PURH-ADH2. The silencing expression vector contained the ADH2 gene, a PGK strong promoter, and a CYC1 terminator. Using BamHI and XmaI restriction enzymes for the double digestion of the SADH2 and PURH plasmid, the antisense recombinant expression plasmid PURH-SADH2 was constructed. Through high-efficiency yeast transformation and electroporation, the recombinant plasmid components were transformed into Saccharomyces cerevisiae SY01 cells, and positive JY01 clone strains were obtained. Fermentation test results of the S. cerevisiae JY01 mutant strain compared with the original strains, SY01 and Y01, showed that glycerol-3-phosphate dehydrogenase activity decreased by 16.31% and 13.5%, respectively. When S. cerevisiae Y01, SY01, and JY01 were fermented for 36 h, 48 h and 60 h, the glycerol production of JY01 decreased by 16.34%, 14.25%, and 14.89%, respectively, compared with that in the original Y01 strain. After a 48-h fermentation, the ethanol concentrations of Y01, SY01, and JY01 were 6.243 g/100 mL, 7.145 g/100 mL, and 7.288 g/100 mL, respectively. The ethanol production of JY01 was 14.33% higher than that observed in the original Y01 strain. Results showed that the antisense interference of the ADH2 5′ UTR sequence can effectively interfere with ADH2 transcription and expression in engineered yeast strains, weaken glycerol accumulation pathways, and promote ethanol-metabolic pathways.

现代农业产业技术体系建设专项资金（CARS-20-4-4）