部分酶解酵母高效电击转化研究

HIGH-EFFICIENCY TRANSFORMATION OF PARTIAL DIGESTED YEAST BY ELECTROPORATION

以酵母质粒ＹＣｐ５０为外源ＤＮＡ，电击转化部分酶解酵母宿主菌ＡＢ１３８０，转化效率稳定在１０＾６转化子／μｇ质粒ＤＮＡ左右，比不酶解酵母或酵母原生质球作受体的电击转化效率高一个数量级以上，也比ＰＥＧ介导的酵母原生质球转化高３￣５倍，而且适合于大片段ＤＮＡ如水稻ＹＡＣ分子的转化。达最佳转化时的有关技术参数为：新接菌种通气培养至细胞密度１×１０＾８￣１．５×１０＾８个／ｍｌ；

Partial digested yeast can be transformed in a very high frequency by electroporation, About 1 × 106 transformants /μg DNA have been stably obtained with yeast strain AB1380 and plasmid YCp50. The efficiency is one order of magnitude higher than that obtained with intact yeast or spheroplast by electropolation, and also 3-5 fold over that of yeast spheroplast transformation mediated by PEG. Furthermore this procedure is suitable for macromolecule transformation such as rice YAC moleculars. The process is highly dependent on several aspects; culture yeast cells to a density of 1 ×108-1.5× 108cells / ml; add 300u of lyticase into 20ml of cell suspension and digest at 30℃ for 5min to achieve partial digestion; adjust the cells density for electric pulse to 1×109-1.5 × 109cells/ml; set the electric field strength and the capacitance .to 6.25kV/cm and 25μF respectively; spread transformed cells on plates just after electroporation.