酿酒酵母遗传缺陷型菌株化学转化方法研究

Research on a Chemical-Based Yeast Transformation Method for Defective Saccharomyces Cerevisiae

目的:通过改善转化条件,提高缺陷型酿酒酵母DNA转化效率。方法:以醋酸锂化学转化法为基础,以酿酒酵母菌株fab1::KAN作为遗传转化受体,以敲除MZM1基因为转化目的,对影响遗传缺陷型菌株转化效率的参数(醋酸锂前处理、热激时间及转化后复苏时间)进行优化,确定适合缺陷型酵母DNA转化的最佳方法。结果:不同时间(30 min,60 min和120 min)醋酸锂的前处理均可以提高其转化效率,热激前使用醋酸锂处理酵母30 min转化效率最高;热激10 min是转化效率由高到低的转折点,30 min热激明显降低了转化效率;YPD培养基用于转化后酵母的复苏,随培养时间(30 min,60 min和120 min)的延长,酵母转化效率逐步增加,120 min作为热敏性菌株转化后的复苏时间较佳。结论:与野生型菌株类似,通过转化条件的优化,遗传缺陷型菌株的转化效率也可以满足大多数试验要求。

To improve the transformation efficiency of defective yeast strains by testing different transformation conditions. Methods： Based on Li Ac-transformation method, Saccharomyces cerevisiae strain fab1：：KAN was used as genetic transformation receptor, and the transformation was done by knocking out MZM1 gene, then the best conditions for transforming defective yeast strains were determined by optimizing the transformation parameters such as Li Ac pretreatment, heat shock time and post-transformation recovery time. Results： Different time（30 min, 60 min and 120 min following Li Ac treatment） could all improve the transformation efficiency, of which the highest efficiency can be achieved by 30 min pretreatment. 10 min after heat shock was the turning point from high transformation efficiency to low ones, and 30 min heat shock reduced efficiency remarkably. Transformation efficiency gradually increased along with the prolongation of culture time（30 min, 60 min and 120 min） when YPD medium was used to recover the post-transformation yeast, 120 min recovery in YPD medium should be preferred after heat shock. Conclusions： By improving transformation conditions, the transformation of defective yeast strains can satisfy the need of most purposes.