嗜麦芽假单胞菌质粒pSH1的限制图谱及km^r标记的克隆

RESTRICTION MAP OF PLASMID pSH1 OF PSEUDOMONAS MALTOPHILIA AND ITS DEVELOPMENT

对嗜麦芽假单胞菌Ｐ２菌株（ＰｓｅｕｄｏｍｏｎａｓｍａｌｔｏｏｈｉｌｉａＰ２）的质粒ｐＳＨ１进行了限制酶切分析，确定了ＢｇｌⅡ、ＥｃｏＲⅠ、ＰｓｔⅠ、ＸｂａⅠ、ＢａｍＨⅠ、ＢｇｌⅠ、及ＰｖｕⅡ共７种限制性内切酶在ｐＳＨ１、质粒上的切割位点，前４种酶均为单一切点；后３种依次为２、７、５个切点．通过双酶切和部分酶切的方法绘制了ｐＳＨ１质粒的限制酶切图谱．将ｐＧＰ１－２质粒上的卡那霉素抗性基因（ｋｍｒ）片段插入ｐＳＨ１，获得了重组质粒ｐＳＨ２．ｐＳＨ２是由ｐＧＰ１－２质粒上大小为２．９０ｋｂ的ＤＮＡ片段（含ｋｍｒ）和ｇｈＨ１经ＢａｍＨⅠ－ＰｓｔⅠ双酶切产生５．７０ｋｂ大片段组成，它能够重新转化到喀麦芽假单胞菌受体（ｋｍｒ）中，其ｋｍｒ标记能够得到稳定的表达．

In this paper , we analyzed plasmid pSH1 of Pseudomonas maltophilia P2 strain with restriction endonucleases and determined the sites of seven endonucleases(Bg1 Ⅰ, EcoR Ⅰ,PSt Ⅰ,Xba Ⅰ,BamH Ⅰ, Bg1 Ⅰ and Pvu Ⅰ). Four of them(Bg1 Ⅱ,EcoR Ⅰ, Pst Ⅰ,Xba Ⅰ) have a single restriction site ,two for BamH Ⅰ,five for Pvu Ⅱ,Seven for Bg1 Ⅰ. The restriction map of PSH1 was determined.Recombinant plasmid pSH2 has been obtained by insertiong the kanamycin-resistance gene fromplasmid pGP1- 2 into pSH1. Plasmid pSH2 consists of the 2. 90 kb fragment (including the Km'gene) and the 5. 70 kb BamH Ⅰ-Pst Ⅰ restriction fragment from pSH1. It could be transformed into P.maltophilia P27 strain and the cloned Km' gene coule be expressed. The resulls showed that the constructed plasmid pSH2 is a useful cloning vector for genetic study and gene clone in P. maltophilia.