嗜碱细菌NTT36嗜碱基因的亚克隆及其在大肠杆菌和农杆菌中的表达

将大肠杆菌ＨＢ１０１嗜碱转化子中质粒ｐＧＣＡ所携带的嗜碱基因亚克隆至双元载体ｐＢＩ１２１质粒中，构建了植物表达载体ｐＬＧＣ重组质粒。用其转化大肠杆菌ＨＢ１０１获得了能在碱性和卡那霉素抗性平板上生长的转化子，再通过三亲交配法将亚克隆质粒ｐＬＧＣ转化进农杆菌ＬＢＡ４４０４，又获得能在碱性平板和卡那霉素及利福平双抗平板上生长的转化子，Ｓｏｕｔｈｅｒｎ杂交结果表明ＨＢ１０１转化子亚克隆质粒ｐＬＧＣ是由来自于嗜碱芽孢杆菌ＮＴＴ３６染色体ＤＮＡ和双元载体ｐＢＩ１２１组成，且农杆菌ＬＢＡ４４０４转化子含有来自大肠杆菌亚克隆转化子的ｐＬＧＣ质粒。

Subconing of Alkalophilic Gene from Alkalophilic Bacillus sp.NTT36 and Its Expression in Escherichia coli and Agrobacterium tumefaciens

The plasmid pGCA containing alkalophilic gene was partially digested with EcoR I, and the binary vecter of pBI121 was digested completly with BamH I. Both ends of fragments were blunted with Klenow fragment of E.coli DNA polymerase I, and was ligated by using T 4 DNA ligase. The recombinant plasmid pLGC isolated from HB101 transformants was introduced into E.coli and A.tumefaciens by transformation. The transformants were screened from alkaline and antibiotic plates. It was found that the alkalophilic gene was expressed in both E.coli HB101 and A.tumefaciens LBA4404. The recombinant plasmid was named as pLGC. Plasmid pLGC was proved to be composed of the DNA form plasmid pBI121 and DNA fragmant from Alkalophilic Bacillus sp. NTT36 by southern hybridization. The transformants of A. tumefaciens contain plasmid plGC.