转新疆雪莲去饱和酶基因sikSAD重组酵母低温和酒精耐受性分析

根据GenBank收录的sikSAD基因序列, 采用反转录PCR技术从新疆雪莲(Sasussured involucrata Kar. et Kir)中克隆了sikSAD基因, 并构建了pYES2-sikSAD大肠杆菌/酵母穿梭表达载体, 通过电击法转化酿酒酵母288C菌株, 并利用PCR和SDS-PAGE对转化酿酒酵母进行鉴定, 最后通过低温胁迫和酒精胁迫进行抗性初步分析及方差分析。结果表明: 低温胁迫实验中, 转sikSAD基因酿酒酵母在低温条件下仍能存活, 并且在温度恢复到28 °C时能迅速生长, 生长状态良好, 不饱和脂肪酸油酸的含量有明显的变化。酒精胁迫实验中, 其能耐受一定浓度的酒精, 并且耐受能力比非转基因酿酒酵母提高了十几个百分点。可见, 在低温胁迫和高浓度酒精条件下, 转新疆雪莲sikSAD基因酿酒酵母表现出了优良的活性和生长优势, 显示出较强的抗性特征, 用分子手段改造酿酒酵母, 为工业生产提供高质量的酿酒酵母奠定实验基础。

Low temperature and alcohol tolerance traits of the recombinant yeast with Sasussured involucrata Kar. et Kir de-saturase sikSAD

According to the sequence of sikSAD gene from GenBank, we cloned sikSAD gene from Sasussured involucrata Kar. et Kir though RT-PCR, and constructed the recombinant E. coli/Yeast shuttle vector pYES2-sikSAD. Then it was transformed into 288C yeast by electroportion method, PCR and SDS-PAGE analysis indicated that sikSAD had been integrated into the genome. That the recombinant vector had been transformed into yeast. Finally, we made preliminary analysis on the resistance and analysis of variance under low temperature stress and ethanol tolerance. The results suggest that the recombinant yeast pYES2-sikSAD can not only survive in low temperature but also grow well at 28 °C in the experiment of low temperature stress, the percentage of oleic acid have obvious change. In the experiment of ethanol tolerance, the recombinant yeast pYES2-sikSAD can tolerant certain concentra-tion of ethanol, also the ability of tolerance increased more than 10 percentage compared with non-transformed yeast. The above testify that the recombinant yeast pYES2-sikSAD have more toler-ance and excellent activity and growth advantage under the low temperature and high ethanol. This ex-periment using molecular technology to reconstruct yeast which can provide the basement for producing Saccharomyces cerevisiae with higher quality in industry.