Promoter-probe vector pSUPV4 is used to clone the promoter of Pseudomonas pseudoalcaligenes directly in E. coli, and the recombinant pPA7, which has the highest kanamycin resistance, is obtained. Sequencing the PA7 fr...Promoter-probe vector pSUPV4 is used to clone the promoter of Pseudomonas pseudoalcaligenes directly in E. coli, and the recombinant pPA7, which has the highest kanamycin resistance, is obtained. Sequencing the PA7 fragment discloses several motifs similar to the conservative domains of prokaryotic promoters, including -10 box, -35 box, parallel SD fragment essential to transcription initiation, and the translation initiation site ATG. Southern blotting of PA7 indicates that the PA7 fragment comes from P. pseudoalcaligenes genome and has probably one copy. The PA7 fragment is subcloned by PCR, and the result shows that the 5’-flanking fragment from 889 to 1120 bp has promoter activity, which can be enhanced by the 0.7Kb fragment at 5’ end. The fragments of pPA7 and pPA7-2 are transferred into pseudomonas pseudoaligenes by electroporation, and the significant higher kanamycin resistance of transformants than that of control indicates that the PA7 fragment has the promoter activity in P. pseudoaligene.展开更多
文摘Promoter-probe vector pSUPV4 is used to clone the promoter of Pseudomonas pseudoalcaligenes directly in E. coli, and the recombinant pPA7, which has the highest kanamycin resistance, is obtained. Sequencing the PA7 fragment discloses several motifs similar to the conservative domains of prokaryotic promoters, including -10 box, -35 box, parallel SD fragment essential to transcription initiation, and the translation initiation site ATG. Southern blotting of PA7 indicates that the PA7 fragment comes from P. pseudoalcaligenes genome and has probably one copy. The PA7 fragment is subcloned by PCR, and the result shows that the 5’-flanking fragment from 889 to 1120 bp has promoter activity, which can be enhanced by the 0.7Kb fragment at 5’ end. The fragments of pPA7 and pPA7-2 are transferred into pseudomonas pseudoaligenes by electroporation, and the significant higher kanamycin resistance of transformants than that of control indicates that the PA7 fragment has the promoter activity in P. pseudoaligene.
