根癌农杆菌介导工业化产甘油假丝酵母的遗传转化.pdfVIP

应用与环境生物学报 2007，13(6)：868—871 ChinJApplEnvironBiol=ISSN1006．687X 根癌农杆菌介导工业化产甘油假丝酵母的遗传转化 饶志明 张君胜 2 沈微 方慧英 诸葛健 ( 江南大学工业生物技术教育部重点实验室和工业微生物中心 江苏无锡 214036) ( 江苏畜牧兽医职业技术学院 江苏泰州 225300) 摘 要 通过构建质粒pCAM 3300-zeocin，将其电击转化到根癌农杆菌LBA4404中．将含有目的质粒pCAM 3300-zeocin 的根癌农杆菌和工业化产甘油假丝酵母(Candida glycerinogenes)WL2002-5共培养，利用zeocin抗性基因为筛选标记， 实现了pCAM 3300-zeocin对产甘油假丝酵母的转化．并根据产甘油假丝酵母的生长特性，对转化条件进行了优化，在共 培养时间为24 h，产甘油假丝酵母和根癌农杆菌的细胞比例为1：(500～lOOO)时最高转化率达到2个转化子／10 个 酵母细胞．初步建立了根癌农杆菌介导转化产甘油假丝酵母的转化方法．图3表2参12 关键词 产甘油假丝酵母；转化；根癌农杆菌；geocin基因 CLC Q75：TQ923 Genetic Transformation of Industrialized Strain Candida glycerinogenes by Agrobacterium tumefaciens RAO Zhiming 一 ，ZHANG Junsheng 一，SHEN Wei ，FANG Huiying ZHUGE Jian ( Key Laboratory ofIndustrialBiotechnology，MinistryofEducation，Research CenterofIndustrialMicroorganisms， Jiangnan University，Wuxi 214122，Jiangsu，China) ( Jiangsu Animal Husbandry 0nd Veterinary College，Taizh0u 225300，Jiangsu．China) Abstract In this research，the plasmid pCAM3300-zeocin was constructed and transformed into Agrobacterium tumefaciens． The A．tumefaciens containing the binary vector pCAM3300-zeocin Was CO—cultivated with industrialized strain Candida glycerologenesis WL2002—5．Then，transformants were screened on a plate containing 150 mg／L Zeocin．PCR results demon· strated that zeocin gene Was integrated into nuclear genome of C glycerologenesis．Research results indicated that zeocin gene could be utilized as selection marker and the Zeocin COuld be used as selection pressure in transform ation of C glycerologene． sis．According to the growth characteristics of C glycerologenesis．the transform ation conditions were further optimized．The re· suhs showed that the tra