金黄色葡萄球菌核酸酶基因缺失突变株RN4220Δnuc1的构建

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。本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。经过了七轮培养和筛选，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。

Construction of Staphylococcal nuclease mutant-S.aureus 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, pBT2Δnuc1 was successfully constructed. The recombinant plasmid pBT2?nuc1 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 nuc1-deleted mutant of S. aureus strain RN4220 (termed RN4220Δnuc1) was successfully constructed and conformed by PCR and RT-PCR.