副干酪乳杆菌HD1.7遗传转化体系的初步建立

Initial Establishment of Genetic Transformation System for Lactobacillus paracasei HD1.7

优化副干酪乳杆菌(Lactobacillus paracasei)HD1.7的电转化条件,为构建L.paracasei HD1.7prcR基因敲除突变株奠定基础。通过电转化方法将自杀质粒pUC18-prcR-tet导入L.paracasei HD1.7内,对影响L.paracasei HD1.7电转化的几个主要因素进行了初步研究。当L.paracasei HD1.7生长8h时,加入青霉素使终浓度为12.5μg/mL,再培养1h。经AEB1电转缓冲液(蔗糖浓度为0.3mol/L)处理后,在1.8～2.0KV电压下完成电击,并迅速加入MRS培养基中,复苏5h,成功地将自杀质粒pUC18-prcR-tet转入L.paracasei HD1.7内,并发生了部分同源重组。本研究得到了副干酪乳杆菌(Lactobacillus paracasei)HD1.7较优电转化条件,为继续研究L.paracasei HD1.7的群体感应调控因子prcR奠定了基础。

The electroporation conditions of L.paracasei HD1.7 were optimized,which provided the basis for construction of the knockout mutant strains of prcR.The suicide,pUC18-prcR-tet,was imported to the L.paracasei HD1.7 by electrotransformation and the optimized parameters affecting high-efficent electrotransforming were studied preliminarily.After L..paracasei HD1.7 had been cultivated 8 h,the recipient cells were treated by 12.5 μg/mL benzylpenicillin （final concentration）,and continued another 1 h.They were washed by electrotransformation buffer of AEB1（0.3 mol/L of sucrose）,as well as the 1.8-2.0 kv of field strength and one time of pulse,then added them in MRS culture media quickly and recovered 5 h.The high-efficient electrotransforming had been got,the suicide plasmid （pUC18-prcR-tet） was imported to the L.paracasei HD1.7 successfully,and part of the homologous recombination occurred.The optimized electroporation conditions of L.paracasei HD1.7 were obtained,which lay the foundation for studying the quorum sensing regulatory factor （prcR） of L.paracasei HD1.7.