金黄色葡萄球菌核酸酶基因缺失突变株RN4220 Δnuc1的构建

Construction of Staphylococcal Nuclease Mutant—S.aureus RN4220Δnuc1

金黄色葡萄球菌存在两个核酸酶编码基因,一个是葡萄球菌核酸酶(Staphylococcal nuclease,SNase),命名为nuc1,另一个是耐热核酸酶(Thermonuclease,TNase),命名为nuc2,nuc2是一个新的候选基因,以往认为金黄色葡萄球菌中的核酸酶只源于一个编码基因nuc1,为了进一步研究nuc2基因的功能,首先要将金黄色葡萄球菌nuc1基因缺失。研究目的就是通过构建同源重组质粒pBT2Δnuc1,将其电转入金黄色葡萄球菌菌株RN4220中,获得nuc1基因缺失突变株。经过了7轮培养和筛选,同源重组几率为2%(7/345),筛选出的nuc1突变株用PCR方法和RT-PCR进行了验证,从而获得了nuc1基因缺失突变株RN4220Δnuc1。

Two ORFs in Staphylococcus aureus genomes were predicted to encode nucleases. One is encoded the nuclease A （SNase） without a gene name （termed nuc1） ; the other is encoded a thermonuclease （TNase） with a name of nuc （termed nuc2 ）. To further investigate the function of nuc2 in S. aureus, the resulting plasmid, pBT2 △nucl was successfully constructed. The recombinant plasmid pBT2 △nucl was introduced into S. aureus RN4220 by electroporation. Using the resistant marker for seven passages of culture, the recombination rate was about 2% （7/345）. A nucl-deleted mutant of S. aureus strain RN4220 （termed RN4220△nucl ） was successfully constructed and conformed by PCR and RT-PCR.