鲜牛奶中副干酪乳杆菌隐蔽型质粒pMA3的全序列测定及功能分析

利用乳酸乳杆菌选择性培养基SL，从鲜牛奶中分离一株携带两个天然质粒的副干酪乳杆菌MA3。通过寻找的ScaI对未知小质粒pMA3进行酶切，产物连接载体pBK-CMV进行测序，全序列为5089bp，G＋C含量为39.54%。进一步通过生物信息学分析质粒pMA3，该质粒编码四个开放阅读框，其中Rep3是复制起始蛋白，根据复制蛋白的同源性及其基因上游的连续正向重复序列证明pMA3属于pUCL-287 θ-复制型质粒，该质粒GenBank登陆号为EU255257。该质粒的发现为开发具有自主知识产权的食品级乳酸乳杆菌表达载体奠定基础。

Functional Analysis of a Cryptic Plasmid pMA3 in Lactobacillus paracasei Isolated from Fresh Milk

Lactobacillus paracasei MA3, harboring two natural plasmids, was isolated from fresh milk using selective medium SL. In order to obtain the complete sequence, the unknown small plasmid pMA3 was digested with ScaI and ligated with vector pBK-CMV. The complete sequence of pMA3 revealed to be 5809bp in length with GC content of 39.54%. The computer analysis revealed that pMA3 harbored four ORFs, one of them designated Rep3, which is replication initiation protein. Sequence analysis results determined the highly homologous similarity of Rep protein and the tandem direct repeats, which demonstrated that pMA3 belongs to the pUCL-287 subfamily of theta-type replicons. This plasmid has been deposited in GenBank under Accession No.EU255257.This research has made a basis for developing the food-grade expressing vector with independent intellectual property rights.