大肠杆菌-鸭疫里默氏杆菌高效穿梭质粒pFY02的构建和应用

Construction and application of Escherichia Coli-Riemerella anatipestifer efficient shuttle plasmid pFY02

摘要:

鸭疫里默氏杆菌 (Riemerella anatipestifer，RA) 是引起鸭、鹅、火鸡等家禽传染性败血症及浆膜炎的主要病原。目前主要通过基因缺失及基因回补的方法对鸭疫里默氏杆菌的基因功能进行研究。然而，目前使用的穿梭质粒pLMF03存在结合转移效率低、酶切位点少等缺陷，不能用于所有鸭疫里默氏杆菌基因的回补。为解决这一问题，文中将结合转移位点oriT、鸭疫里默氏杆菌复制起始基因pRA0726 ori、高表达启动子基因及多种酶切位点逐一克隆至质粒pPM5，构建了新的穿梭质粒pFY02。结果表明，该质粒能够稳定存在于鸭疫里默氏杆菌，且具有较高的结合转移效率。通过回补鸭疫里默氏杆菌tonB2基因缺失株表明，该质粒可用于鸭疫里默氏杆菌基因的回补。总之，文中构建的穿梭质粒pFY02更加完善了用于鸭疫里默氏杆菌基因回补的材料。

Abstract:

Riemerella anatipestifer is a pathogen that mainly infects ducks, gooses, turkeys and other birds, causing septicemia and serositis. At present, the function of R. anatipestifer genes are studied by gene deletion and complementation. However, the shuttle plasmid pLMF03 used at present is inefficient for conjugation. Moreover, less restriction enzyme site can be used for cloning. It is not able to use for all the genes complementation. To solve this disadvantage, the conjugative transfer site, R. anatipestifer replication initiation gene, high expression promoter and a number of enzyme cutting sites were cloned into the plasmid pPM5, to generate the new shuttle plasmid pFY02. The shuttle plasmid pFY02 was stable in R. anatipestifer and had a high conjugative transfer efficiency. The R. anatipestifer tonB2 mutant strain could be complemented by shuttle plasmid pFY02 expressing tonB2, indicating that the shuttle plasmid can be used to the complementation of R. anatipestifer. Taken together, the new shuttle plasmid pFY02 constructed in this study replenishes the genetic tool for complementation.