CAJ | 학술논문

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.
이중성유당매생산균취벽극로유효모위재료,구건이kan기인위사선표기、이취벽극로유효모유당매기인상유조공구위동원비적질립pPkan.장질립pPkan전격전화취벽극로유효모,대수체균생장시기、전장강도、전격후배양시간등조건진행우화.결과표명,이OD600위0.8적취벽극로유효모위수체、전압1.5 kV、전장강도위7.5 kV·cm-1、전격후배양시간위90 min,가획득교고적전화솔,최고전화효솔위2.37×102전화자·μgDNA.재전격전화적동시가입한제성내절매10μL,대전격전화적전화솔무현저영향.경PCR사선증실87.5%적전화자위동원중조,종이건립일투유효적취벽극로유효모기인치환기술.
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 cel s, electric para-meters, 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 cels 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.