中山大学学报自然科学版 ›› 2010, Vol. 49 ›› Issue (3): 98-101.

• 研究论文 • 上一篇    下一篇

一种高效的酿酒酵母和毕赤酵母电击转化法

徐志武1,张 颖2, 王智学1, 曹 燕1, 崔 亮1, 李 盛1, 刘秋云1


  

  1. (1. 中山大学基因工程教育部重点实验室和生物工程研究中心//有害生物控制与资源利用国家重点实验室,广东 广州510275;2. 广州市疾病预防和控制中心,广东 广州 510080)
  • 收稿日期:2009-04-14 修回日期:1900-01-01 出版日期:2010-05-25 发布日期:2010-05-25
  • 通讯作者: 刘秋云

A Highly Efficient Protocol for Transformation of Saccharomyces cerevisiae and Pichia pastoris by Electroporation

XU Zhiwu 1, ZHANG Ying2, WANG Zhixue1,CAO Yan1, CUI Liang1, LI Sheng1, LIU Qiuyun1   

  1. 1. State Key Laboratory of Biocontrol, The Key Laboratory of Gene Engineering of Ministry of Education and Biotechnology Research Center, Sun Yatsen University, Guangzhou 510275, China;2. Guangzhou Center for Disease Control and Prevention, Guangzhou 510080, China)
  • Received:2009-04-14 Revised:1900-01-01 Online:2010-05-25 Published:2010-05-25

摘要: 电击转化由于其高效率而被广泛地应用于酿酒酵母的转化过程中。但是由于菌种退化或是暴露于污染的环境,很多菌种的转化效率会显著地下降2~4个数量级。探索了一种改进的电击转化方法,它借助于单链载体DNA和转化后在YPD培养基中的复苏过程,可以使转化效率比之前曾报道过的已经最优化的用醋酸锂和二硫苏糖醇进行预处理的转化方法效率提高13倍。在毕赤酵母中使用这一改进的方法,可以使转化效率提高高达114倍,在一些已经退化的酿酒酵母菌株中,此方法也可以提高转化效率将近100倍。这一方法将为几乎所有酿酒酵母和毕赤酵母的分子操作提供极大的便利。

关键词: 电击转化, 酿酒酵母, 单链载体DNA, 转化后复苏, 毕赤酵母

Abstract: Electroporation is widely used for highly efficient transformation of Saccharomyces cerevisiae. As many strains degenerate over time or over environmental exposure, their electroporation efficiencies could drop markedly by 2 to 3 orders of magnitude. Here we describe a modified electroporation procedure which included single stranded carrier DNA for electroporation as well as a postelectroporation growth period in YPD medium, which gave rise to 13 fold enhancement in transformations than an optimized lithium acetate (LiAc) and dithiothreitol (DTT) Pretreatment protocol previously reported. An enhancement of 114 fold on Pichia pastoris transformations has been observed using the modified procedure. The modified method enhanced the transformation efficiencies of some degenerated S. cerevisiae strains by nearly 100 fold. It will enable molecular manipulations in all kinds of S. cerevisiaeand P. pastoris strains by allowing propagations of all sorts of DNA libraries made from minute samples.

Key words: electroporation, Saccharomyces cerevisiae, single stranded carrier DNA, postelectroporation growth, Pichia pastoris

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