Visualized investigation of yeast transformation induced with Li+ and polyethylene glycol
Visualized investigation of yeast transformation induced with Li+ and polyethylene glycol
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Li和聚乙二醇诱导酵母转化的可视化研究
DOI：
10.1016/j.talanta.2008.06.018
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发表时间：
2008-10-15
期刊：
影响因子：
6.1
通讯作者：
Tong, Hua
中科院分区：
文献类型：
--
作者：
Chen, Ping;Liu, Hui-Hui;Tong, Hua
采用荧光显微镜（FM）观察YOYO-1标记的质粒DNA与酵母细胞表面的结合，扫描电镜（SEM）和原子力显微镜（AFM）观察酵母细胞表面形貌的变化，结合转化频率实验，研究Li+和聚乙二醇（PEG）对酿酒酵母遗传转化的影响。结果表明，在相同条件下，酵母原生质体的转化率远高于完整酵母细胞的转化率。PEG是DNA与完整酵母细胞或酵母原生质体表面结合所必需的，并且对完整酵母细胞或酵母原生质体的表面形貌没有影响。在PEG存在下，Li+可显著增强质粒DNA与完整酵母细胞表面的结合，提高其转化频率，并影响其表面形貌。另一方面，在用Li+处理原生质体时，没有发现对DNA结合到原生质体表面的影响，没有发现转化体数目的增加和表面形貌的改变。本研究直接观察了Li+和PEG对酵母遗传转化的影响，而不是从转化频率的结果推断。这些结果表明，细胞壁可能是质粒DNA摄取的屏障。Li+可增加酵母细胞壁的通透性，从而增加完整酵母细胞上DNA结合的暴露位点。PEG的主要作用是诱导DNA与细胞表面结合。(c)2008 Elsevier B. V.保留所有权利。
The effects of Li+ and polyethylene glycol (PEG) on the genetic transformation of Saccharomyces cerevisiae were investigated by using fluorescence microscopy (FM) to visualize the binding of plasmid DNA labeled with YOYO-1 to the surface of yeast cells, scanning electron microscopy (SEM)and atomic force microscopy (AFM) to image the change in surface topography of yeast cells, coupled with transformation frequency experiments. The results showed that under the same conditions, the transformation frequencies of yeast protoplasts were much higher than those of intact yeast cells. PEG was absolutely required for the binding of DNA to the surface of intact yeast cells or yeast protoplasts, and had no effect on the surface topography of intact yeast cells or yeast protoplasts. In the presence of PEG, Li+ could greatly enhance the binding of plasmid DNA to the surface of intact yeast cells, increase their transformation frequency, and affect their surface topography. On the other hand, no effect on the DNA binding to the surface of protoplasts and no increase in the number of transformants and no surface topography changes were found upon the treatment with Li+ to protoplasts. In the present work, the effects of Li+ and PEG on yeast genetic transformation were directly visualized, rather than those deduced from the results of transformation frequencies. These results indicate that cell wall might be a barrier for the uptake of plasmid DNA. Li+ could increase the permeability of yeast cell wall, then increase the exposed sites of DNA binding on intact yeast cells. The main role of PEG was to induce DNA binding to cell surface. (c) 2008 Elsevier B.V. All rights reserved.