REMI介导电转化产甘油假丝酵母

Genetic Transformation of Candida glycerinogenes by REMI and Electroporation

摘要:

为了分离耐高渗和甘油代谢相关基因，以Zeocin为选择标记，利用REMI技术电转化产甘油假丝酵母Candida glycerinogenes。考察了7种限制性内切酶对转化的影响，选择Hind III进一步优化了转化的几个条件。结果表明，在OD₆₀₀≈1.3 时收集细胞，在1.5 kV 电压下，感受态细胞浓度为2.0×10⁹个细胞/mL，100U Hind III时，能获得129个转化子/μg DNA的较高转化率，58% 的转化子稳定，表明REMI技术适合于产甘油假丝酵母的转化。

Abstract:

In order to isolate genes related with the osmoadaptation and glycerol metabolism of Candida glycerinogenes, a transformation system based on the dominant selectable marker Zeocin and restriction enzyme-mediated integration (REMI) was established. Effects of seven restriction enzymes on transformation efficiency of C.glycerinogenes were tested. Transformation conditions were optimized in the presence of Hind III. Under the optimal conditions of OD₆₀₀≈1.3, voltage of 1.5 kV, 2.0×10⁹ competent cells/mL, 100 units of Hind III added, the transformation efficiency was up to 129 trnaformants/μg DNA. 58% of transformants were stable on nonselective medium. These results suggest that REMI technique would be beneficial to the genetic transformation of C.glycerinogenes.