摘要
【目的】建立圆红冬孢酵母的高效电转化方法,为利用圆红冬孢酵母重组表达功能性基因提供方法基础。【方法】以圆红冬孢酵母作为受体菌,将具有潮霉素抗性标记的改造质粒p CAMBIA1301-KCS通过电转化的方式导入到受体菌中,研究酵母细胞培养时间(2、8、14、20、26 h)、电击电压(1、1.5、2.0、2.5、3.0 kV)、前处理剂种类(DTT、LiAc、CaCl2、H2O)对转化效率的影响。【结果】当培养时间为20 h [培养液D(600 nm)约为1.6]、电击电压为2.0 kV、前处理试剂为DTT时,该系统转化率最高可达到20.8×104 cfu/μg。【结论】采用电击法将环状质粒转化入圆红冬孢酵母,能够成功实现外源基因的遗传转化,采用优化的转化条件可以获得较高的转化效率,是过去相关报道最高转化率的100倍。
【Objective】The aim of this study is to optimize the conditions of transformation system of Rhodosporidium toruloides.【Methods】A modified plasmid pCAMBIA1301-KCS, carrying hygromycin phosphotransferase gene, was transformed into R. toruloide by electroporation method. The primary conditions affecting the efficiency of transformation were investigated and optimized which included the yeast cell cultivation time(2, 8, 14, 20, 26 h), shock voltage(1, 1.5, 2, 2.5, 3.0 kV), and the type of pretreating agent(DTT, Li Ac, CaCl2, H2 O)(30 mmol/L). 【Results】The results indicated that cultivation time of 20 h [D(600 nm) of cultivation broth was about 1.6], shock voltage of 2.0 kV and the application of DTT were the optimal transformation conditions.【Conclusion】This is a study on the electroporation gene transformation of R. toruloide with circular plasmid. This work could provide a higher effective technique for gene transformation and lay the foundation of gene modification and application of R.toruloides.
作者
裴祎
李玉环
朱宏波
胡雪琼
钟敏
李雁群
PEI Yi;LI Yu-huan;ZHU Hong-bo;HU Xue-qiong;ZHONG Min;LI Yan-qun(Colleage of food science and techonology,Guangdong Ocean University,Zhanjiang,524088,China;The Institute for Marine Drugs and Nutrition,Guangdong Ocean University,Zhanjiang,524088,China;Guangdong provincial key laboratory of aquatic product processing and safety,Guangdong Ocean University,Zhanjiang,524088,China;Colleage of agriculture,Guangdong Ocean University,Zhanjiang,524088,China)
出处
《广东海洋大学学报》
CAS
2018年第6期48-54,共7页
Journal of Guangdong Ocean University
基金
广东省自然科学基金(2015A030313616)
广东省国际合作项目(2017A050501038)
湛江市科技计划项目(2015A03026)
关键词
圆红冬孢酵母
电转化
转化效率
转基因
Rhodosporidium toruloides
electroporation
transformation efficiency
genetic transformation
