枯草杆菌ylmK基因的荧光标记及Ylmk蛋白的亚细胞定位

目的：对枯草杆菌ylmK基因进行3’端荧光标记以便对其产物YlmK蛋白在菌体中的位置进行初步观察。方法：以BS168菌株基因组DNA为模板，PCR扩增ylmK基因的3’端序列，并将其克隆到载体pSGll64中，形成ylmK’-gfpmutl融合，构建重组载体pNC-424；将pNG424转化枯草杆菌168菌株，单交换形成完整的功能性3’端gfpmutl标记的ylmK基因，菌落PCR对阳性转化子BS362进行鉴定，用表面荧光显微镜技术对BS362进行观察。结果：荧光检测结果表明GFP标记的YlmK分布于菌体的外周，在位置上靠近细胞膜并与之平行排列。结论：YlmK是一种膜蛋白。

Green Fluorescence Labeling of ylmK Gene and the Subcellular Localization of YlmK in Bacillus subtilis

Objective： ylmK gene of Bacillus subtilis was labeled with the green fluorescence to investigate the subcellular localization of YlmK protein. Methods： Chromosomal DNA was prepared from Bacillus subtilis 168, 3＇ terminal sequence ofylmK gene （ylmK＇） was amplified by PCR. ylmK＇ was then inserted into the plasmid vector pSGlI64, to give pNG424 containing a ylmK＇-gfpmutl fusion. Finally, Bacillus subtilis 168 was transformed with pNG424, single crossover occurred and formed the full-length functional fluorescence labeled ylmK gene. The integrant （ subsequently named BS362） was identified by colony-PCR. Localization of the fusion protein was determined by epifluorescence microscopy. Results ： Microscopic observation of strain BS362 showed that the green fluorescence labeled YlmK was distributed around the cell periphery, closely juxtaposed with the cytoplasmic membrane. Conclusion：YlmK was a membrane tethered protein.