摘要
为从分子水平上阐释产甘油假丝酵母(Candida glycerinogenes)高产甘油机理,建立一种方便可行的遗传转化系统是十分必要的。与G418和潮霉素等抗生素相比,Zeocin抗生素对C.glycerinogenes具有较低的致死浓度。以pGAPZb作为构建整合载体的骨架,以Zeocin抗性基因作为选择标记,以URA3基因作为整合位点,构建了C.glycerinogenes整合载体pGA-CU。整合载体经过限制酶线性化后用作转化载体,基于电击转化的方法成功获得了抗Zeocin的转化子并经过PCR分析进一步确证。通过优化电击转化的参数,获得了较为稳定的转化效率,基于这一技术的转化效率每微克DNA可获得120个转化子。为进一步研究该菌株的遗传背景和代谢机理奠定了基础。
In order to facilitate mechanism of high glycerol production by C. glycerinogenes in molecular and genetic level, it is increasingly necessary to develop an efficient and conventional genetic transformation system of C. glycerinogenes. Compared to some antibiotics used for the selection of yeast transformants such as G418, hygromycin B et al, C. glycerinogenes has a higher sensitivity to Zeocin. Using vector pGAPZb as backbone, Zeocin resistance gene for selection marker and URA3 for integrative locus, the integrative plasmid pGA-CU was constructed. After linearized by restriction enzyme, plasmid was transformed into C. glycerinogenes by eletroporation. Transformants were selected and identified by PCR amplification for an unique fragment. With some optimized parameters for eleetroporation, the maximal transformation frequencies of 120 transformants/μg DNA were obtained for an linearized integrative plasmid. This opened a venue for studying the biological character and biosynthesis glycerol of this species at the molecular level.
出处
《激光生物学报》
CAS
CSCD
2008年第2期176-180,共5页
Acta Laser Biology Sinica
基金
国家自然科学基金项目(30570142)
关键词
产甘油假丝酵母
遗传转化系统
电击转化
整合质粒
C. glycerinogenes
genetic transformation system
electroporation
integrative plasmid
