Ion-beam-induced gene transfer in Saccharomyces cerevisiae
Ion-beam-induced gene transfer in Saccharomyces cerevisiae
复制标题
DOI：
10.1016/j.surfcoat.2009.02.067
复制
发表时间：
2009-06
影响因子：
5.4
通讯作者：
S. Anuntalabhochai;R. Chandej;M. Sanguansermsri;S. Ladpala;R. Cutler;T. Vilaithong
中科院分区：
文献类型：
--
作者：
S. Anuntalabhochai;R. Chandej;M. Sanguansermsri;S. Ladpala;R. Cutler;T. Vilaithong
本文提出了一种利用低能离子束诱导酵母（Saccharomyces cerevisiae）基因转移的方法。通过以1-100×1015ions /cm2的离子效应将氮离子加速到50-60 keV的能量范围，对酵母细胞进行轰击，以表征随着能量水平的增加，细胞存活率的下降。使用优化的酵母细胞存活轰击条件（50 keV，离子影响为1和2×1015ions/cm2），将兼容质粒载体（pYES2）成功导入酵母细胞，质粒浓度更高，质粒转移效果更好。为了研究外来基因在酵母中的表达，我们从地衣芽孢杆菌中选择两个标记基因——绿色荧光蛋白（GFP）和硫辛酸合成酶（lipoic acid syntase）——亚克隆到pYES2（分别设计pYGFP和pYlip）中，然后转化到细胞中。诱导10 h后，分析这些基因的表达水平。经sds -聚丙烯酰胺凝胶电泳检测，经pYGFP转化的酵母细胞中显示出高强度的GFP蛋白，并且pYLip在34 kDa处显示出预期的额外蛋白带。作为一种转化酵母的方法，低能离子束轰击不仅效率高，而且在不到10分钟的时间内就能转化酵母，比其他酵母转化方法快得多。
In this paper we present a novel method to induce gene transfer in yeast (Saccharomyces cerevisiae) using a low-energy ion beam. By accelerating nitrogen ions to the 50–60 keV energy range with ions fluences of 1–100×1015ions/cm2, yeast cells were bombarded to characterize the decrease in survival with increasing energy level. Using bombardment conditions optimized for yeast cell survival (50 keV with ion fluences of 1 and 2×1015ions/cm2), a compatible plasmid vector (pYES2) was successfully introduced into the yeast cell with higher concentrations of the plasmid providing improved plasmid transfer. To investigate expression of exotic genes in yeast, two marker genes – GFP and lipoic acid synthetase from Bacillus licheniformis – were chosen to subclone into pYES2 (designed pYGFP and pYlip respectively), and subsequently transformed into the cells. After 10 h of induction, the expression levels of these genes were analyzed. The pYGFP transformed yeast exhibited a high intensity of GFP protein in the yeast cells and the pYLip showed the expected additional protein-band at 34 kDa detected by SDS-polyacrylamide gel electrophoresis. As a method to transform yeast, low-energy ion beam bombardment is both highly efficient and since yeast can be transformed in less than 10 min, much more rapid than other yeast transformation methods.