乳酸菌隐蔽性质粒pEV105复制子的鉴定

Characterization of the Replicon of the Cryptic Plasmid pEV105 from LAB

将Tn1541转座子的红霉素抗性rRNA甲基化酶基因克隆至pUC19的EcoO109I位点,构建成能够确定乳酸菌质粒复制子所在片段的复制检测载体pUB380。将乳酸菌隐蔽性质粒pEV1053个酶切片断(4.5、3.5、3kb)分别连接至此载体,并电转化至原宿主菌。试验结果显示,连接了3.5kb片段的载体能够使原宿主菌表达红霉素抗性的片段,即复制子所在片段。以Southern杂交对质粒pEV105复制模式进行分析,结果显示pEV105为θ型复制。用400μg/mL吖啶橙对菌株KLDS6.0718处理50代后没有质粒丢失,说明pEV105在宿主菌内十分稳定。该复制子在构建乳酸菌食品级克隆表达载体中可作为稳定的复制子使用。

A replication probe vector （pUB380） was constructed by insertion of the erythromycin resistance rRNA methylase（erm） gene from Tn1545 into pUC19, pEV105 is a cryptic plasmid of lactic acid bacteria KLDS6.0718 of relatively small size （10.5 kb）. After three restriction endonuclease fragments （about 4.5 kb, 3.5 kb, 3 kb） of pEV105 was separately ligased to pUB380, the recombinant plasmids were electrotransformated to host KLDS6.0718 respectively. After three restriction endonuclease fragments （about 4.5 kb, 3.5 kb, 3 kb） of pEV105 was separately ligased to pUB380,the recombinant plasmids were electrotransformated to host KLDS6.0718 respectively, and found that the recombinant plasmid which carried 3 570 bp fragment gave strain KLDS6.0718 the resistance to erythromycin. The result indicates that the replicon of pEV105 wad located on a 3 570bp fragment. And the result of Southern blot indicates that pEV105 is thetatype replication. Treated strain KLDS6.0718 with 400 μg/mL acridine orange for 50 generation, none of the native plasmids was lost which indicates pEV105 is very stable in its natural host. The 3.5 kb fragment of minimal replicon can be used for consructing LAB food-grade cloning and expressing vector as stable replicon.