脆壁克鲁维酵母基因置换技术的建立

以中性乳糖酶生产菌脆壁克鲁维酵母为材料,构建以kan基因为筛选标记、以脆壁克鲁维酵母乳糖酶基因上游调控区为同源臂的质粒pPkan。将质粒pPkan电击转化脆壁克鲁维酵母,对受体菌生长时期、电场强度、电击后培养时间等条件进行优化。结果表明,以OD600为0.8的脆壁克鲁维酵母为受体、电压1.5 kV、电场强度为7.5 kV.cm-1、电击后培养时间为90 min,可获得较高的转化率,最高转化效率为2.37×102转化子.μgDNA。在电击转化的同时加入限制性内切酶10μL,对电击转化的转化率无显著影响。经PCR筛选证实87.5%的转化子为同源重组,从而建立一套有效的脆壁克鲁维酵母基因置换技术。

Estabishment of techniques for gene replacement in Kluyveromyces fragilis

To estibish the homologous recombination and electroporation system in Kluyverom-yces fragilis,we constructed the plasmid pPkan was coustucted which with Homologous arms of the UAS of lactase gene from lactase product strain.Paper studied the effects of growth phase of cells,electric parameters,cultured time after electroporation on transformation efficiency.At the same time,we studied the relation that combined the electroporation with REMI.The result indicated that an efficient and stable system of electroporation in Kluyveromyces fragilis had been developed,providing a powerful tool for the transgenic research of Kluyveromyces fragilis.The results showed that a high transformation efficiency was achieved in Kluyveromyces fragilis cells at mid-log growth phase electroporated with voltage,electric intensity 7.5 kV·cm-1 and cultured time after electroporation was 90 min.However,when restriction enzyme was added to electroporation system at 10 μL,the transformation efficiency was not increased.And 87.5% of positive transformants was homologous recombination.