《基因敲除对黄酒酵母高级醇生成量的影响》-毕业论文（设计）.doc

1 前言 摘 要 高级醇是影响黄酒风味的重要物质之一，本文通过同源重组技术分别对支链氨基酸转氨酶的编码基因BAT1和BAT2，以及天冬氨酸β-半醛脱氢酶的编码基因HOM2进行敲除，研究这些基因敲除对黄酒酵母高级醇生成量的影响。主要研究内容及结果如下： （1）构建重组质粒pUC-BABK，PCR扩增重组质粒pUC-BABK上的重组盒BA-KanMX-BB并将其转化黄酒酵母单倍体，筛选获得BAT1基因敲除突变株。将BAT1基因敲除突变株与出发菌株进行黄酒发酵实验，发酵结果显示BAT1基因敲除突变株的高级醇生成量和基本发酵性能均未发生明显变化。 （2）将重组盒BA-KanMX-BB转化BAT2基因敲除黄酒酵母单倍体突变株，筛选获得BAT1、BAT2基因双敲除突变株。将BAT1、BAT2双敲除突变株及其单敲除突变株与出发菌株同时进行黄酒发酵实验，发酵结果显示：BAT1、BAT2双敲除突变株的生长繁殖受到了抑制，CO2总失重平均降低了2.8 g，酒精度平均下降了1.5度。同时，BAT1、BAT2双敲除突变株产正丙醇、异丁醇和异戊醇含量较出发菌株分别平均降低了10.46%、52.16%、25.31%，较BAT1突变株分别平均降低了10.12%、51.82%、25.28%，较BAT2突变株分别平均降低了10.76%、24.71%、15.72%。 （3）构建重组质粒pUC-HABK，PCR扩增重组质粒pUC-HABK上的重组盒HA-KanMX-HB并将其转化黄酒酵母二倍体，筛选获得HOM2基因一个等位基因敲除的突变株RY1-1。将突变株RY1-1与出发菌株RY1同时进行黄酒发酵实验，发酵结果显示：RY1-1产异戊醇的含量明显下降，比RY1降低了30.39%，并且其基本发酵性能无明显变化。RY1-1去除KanMX抗性基因后，得到的突变株RY1-2的高级醇生成量和基本发酵性能与RY1-1相比基本保持不变。 （4）构建重组质粒pUC-HB1A1K，在突变株RY1-2的基础上再次敲除HOM2基因，获得HOM2基因两个等位基因敲除的突变株RY1-3。将突变株RY1-3与突变株RY1-2及出发菌株RY1同时进行黄酒发酵实验，发酵结果显示：与RY1和RY1-2相比，RY1-3的CO2总失重平均降低了5.0 g，酒精度平均下降了3.5度。同时，RY1-3产正丙醇、异丁醇和异戊醇含量较RY1分别降低63.27%、46.80%和66.98%，较RY1-2分别降低63.75%、42.09%和53.52%。 关键词：黄酒酵母；支链氨基酸转氨酶编码基因；天冬氨酸β-半醛脱氢酶编码基因；同源重组；黄酒发酵；高级醇 ABSTRACT In this paper, higher alcohols as the main byproducts during yellow wine yeast fermentation were studied. BAT1 and BAT2 genes encoding branched-chain amino acid transaminase, HOM2 gene encoding aspartic β semi-aldehyde dehydrogenase were knocked out by the double homologous recombination, respectively. Then we studied the effect of gene deletion on the basic fermentation performances and higher alcohols production. (1) Recombinant plasmid pUC-BABK were constructed. The disruption cassette BA-KanMX-BB from the recombinant plasmid pUC-BABK was transformed into yellow wine yeast haploid and the BAT1 deletion strains were successfully obtained. The yellow rice wine fermentation test of BAT1 deletion strains and parental strains showed that the higher alcohols production and the basic fermentation performances were not affected by the deletion of BAT1 gene. (2) The disrupti