恶臭假单胞菌萘质粒ⅠNL基因的克隆及酶切图谱

CLONING FOR nah I N L GENES OF NAH7 IN Pseudomonas putida AND MAPPING THE RESTRICTION ENZYMES

恶臭假单胞菌（ＰｓｅｕｄｏｍｏｎａｓｐｕｔｉｄａＧ７）携带的质粒ＮＡＨ７，经限制性内切酶ＥｃｏＲＩ切割后，产生含有萘降解酶全部基因（２４，６ｋｂ）的片段，此片段插入到载体ｐＵＣ１１９的多克隆位点，构成了ｐＫＡＯ质粒，此质粒经ＨｐａＩ，ＥｃｏＲＩ酶切获得的５．１ｋｂ片段亚屯隆到ｐＵＣ１１９中，衍生出ｐＮＮ１质粒，绘制出内切酶图谱。从ｐＮＮＩ质粒上切下含目的基因ｎａｈＩ、ｎａｈＮ和ｎａｈＬ的ｋｐｎＩ－ｋｐｎＩ片段（２．３ｋｂ）．正向插入载体ＢｌｕｅｓｃｒｉｐｔⅡＳＫ＋中获得重组质粒ｐＮＮ２，反向插入为ｐＮＮ３，将其转化到大肠杆菌ＸＬ１－Ｂｌｕｅ中，目的基因得以大量增殖。

Pseudomonas putida G7 carries plasmid NAH7. After the NAH7 was cleaved by restriction enzyme EcoRI, it produced the fragment of 24. 6 kilobase pairs (kb) which contains all of the genes for the degradation of naphthalene. The pkAO plsdmid is obtained by inserting the fragment into the vector pUC119. In order to obtain the genes of nahI nahN and nahL, we first obtained 5. 1kb HpaI-EcoRI fragment from PKAO plasmid, and then inserted it into vector pUC119, deriving the PNN1 plasmid. After that, a 2. 3kb kpnI-kpnI fragment containing the genes of nahI N L was obtained from PNNI. The fragment was cloned into vector BluescriptII SK+, so the recombinant plasmid called pNN2 and pNN3(reverse direction) was derived.