【摘要】 目的:分别构建含有酿酒酵母丙酮酸脱羧酶基因pdc1和pdc5同源序列loxP-kanMX-loxP的质粒,敲除丙酮酸脱羧酶基因。方法:以两端含40 bp pdc1或pdc5的同源序列为引物,以pUG6质粒DNA为模板,通过PCR扩增loxP-kanMX-loxP基因敲除片段,将其分别插入pMD18-T、pEASY-T3,构建表达载体pTWCL-PDC1与pTWCL-PDC5并测序,构建后的质粒利用醋酸锂转化法转化酿酒酵母H5,经筛选鉴定后进行摇瓶发酵试验。结果:分别含有1693、1672 bp目的片段pdc1-loxP-kanMX、pdc5-loxP-kanMX的质粒pTWCL-PDC1与pTWCL-PDC5构建成功,发酵试验显示重组酿酒酵母H5-01与H5-02较原始菌株的乙醇产量分别下降了14.53%与17.54%,证明pdc1或pdc5基因被敲除。结论:利用Cre-loxP重组酶技术分别敲除了酿酒酵母pdc1和pdc5基因,为后续在酿酒酵母中连续敲除pdc1和pdc5奠定了良好的技术基础。更多还原

【Abstract】 Objective: To construct plasmid containing homologous loxP-kanMX-loxP fragment of Saccharomyces cerevisiae pyruvate decarboxylase gene pdc1 or pdc5 to knock out pyruvate decarboxylase gene. Methods: The loxP-kanMX-loxP gene knockout fragments were amplified by PCR using homologous sequences containing 40 bp pdc1 or pdc5 at both ends as primers from pUG6 plasmid DNA template, and were inserted into pMD18-T and pEASY-T3 to construct expression vectors pTWCL-PDC1 and pTWCL-PDC5 respectively and sequenced. The constructed plasmid was transformed into S.cerevisiae H5 by a lithium acetate conversion method, and subjected to a shake flask fermentation test after screening and identification. Results: The plasmids pTWCL-PDC1 and pTWCLPDC5 containing the 1693, 1672 bp fragment pdc1-loxP-kanMX and pdc5-loxP-kanMX respectively were successfully constructed, and the fermentation experiments showed that the ethanol production of recombinant strains S.cerevisiae H5-01 and H5-02 decreased by 14.53% and 17.54% compared with the original strain respectively, demonstrating pdc1 or pdc5 gene knockout. Conclusion: pdc1 and pdc5 in S.cerevisiae were successfully knocked out by using the Cre-loxP recombinant enzyme technology, which laid a good technical foundation for the subsequent continuous knocking out of pdc1 and pdc5 in S.cerevisiae.更多还原