枯草杆菌ylyA基因的绿色荧光蛋白标记

Green fluorescent protein labeling of ylyA gene in Bacillus subtilis

摘要:

【目的】本研究对枯草杆菌ylyA基因进行荧光标记以便对其产物YlyA在菌体中的位置进行初步观察。【方法】以不同菌株基因组DNA为模板，对ylyA基因进行PCR扩增和序列分析；重新设计引物扩增全长的ylyA并将其克隆到载体pSG1729中，形成gfpmut1-ylyA融合而构建重组载体pNG426；将pNG426转化枯草杆菌168菌株，双交换使gfpmut1-ylyA插入染色体的amyE位点，用碘染色法和菌落PCR对阳性转化子BS363进行鉴定。NA固体培养基上生长的BS363经0.5%木糖诱导表达后，利用表面荧光显微镜技术进行观察。【结果】通过对多个PCR产物的序列分析确定了ylyA基因的正确序列以及正确的翻译起始位点；成功将重组载体pNG426转化枯草杆菌得到了BS363菌株；荧光检测结果表明GFP标记的YlyA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】生长缓慢的BS363菌体，在0.5%木糖诱导下产生的荧光标记YlyA蛋白分布在细胞外周，可能在膜生物学中发挥作用。

Abstract:

[Objective] In this study we labeled the ylyA gene of Bacillus subtilis with green fluorescent protein (GFP) to investigate the subcellular localization of YlyA protein. [Methods] Chromosomal DNA was prepared from different strains of Bacillus subtilis, ylyA amplified by PCR and the products sequenced. Full-length ylyA was then amplified and inserted into the GFP plasmid vector pSG1729, to give pNG426 containing a gfpmut1-ylyA fusion. Finally, Bacillus subtilis 168 was transformed with pNG426, resulting in insertion of the gfpmut1-ylyA fusion into the chromosome at the amyE locus. Double crossover integrants (subsequently named BS363) were identified by their inability to hydrolyse starch and verified by colony-PCR. Following induction of gfpmut1-ylyA expression with 0.5% xylose, localization of the fusion protein was determined by epifluorescence microscopy. [Results] The correct sequence and translation start site of ylyA was identified from sequence analysis of the several amplified PCR products permitting construction of a gfpmut1-ylyA fusion. Microscopic observation of strain BS363 showed that the GFP labeled YlyA was distributed around the cell periphery, closely juxtaposed with the cytoplasmic membrane. [Conclusion] GFP labeled YlyA produced by BS363 cultured on the nutrient agar solid medium distributed around the cell periphery.This suggests it may play a role in membrane biology.