| Promoter-probe vector pSUPV4 was used to clone promoters of Pseudomonas pseudoalcaligenes in Escherichia coli. Nine kanamycin resistant recombinants were obtained and designated as pPAl-pPA9. The pPA7 ,which has the highest kanamycin resistance ,was chose for further characterization . the PA7 fragment was sequenced and several motifs similar to prokaryotic promoter elements such as - lObox,-35box and Up box were found . The SD sequence which was bound by ribosome and ATG site were also found. The PAT fragment was blast in Genebank ,but there is no its' homological sequence . Southern blotting indicated that the PA7 fragment was present in P. pseudoalcaligenes genome probably as one copy .Dot blotting indicated that the PA4,PA7,PAS,PA9 were highly homologous .The PA7 fragment was sub cloned by PCR . Two pair of primer were designed . The forward primers were FPPA1 and FPPA2 starting at 899bp and 764bp (from 5' end) ,and the reserve primers were RPPA2 and RPPA1 starting at 1933bp and 1120bp .These primers also ensure the sub cloned fragments directional inserting with ORFs unchanged . Three sub cloned recombinants were gained and named as pPA7-1 pPA7-2 pPA7-3 each has different inserting fragments .All the three sub cloned recombinants have the same kanamycin resistance about 600u g/ml .Theresult shows that the PA7-2 fragment still has promoter activity .The absence of 0.9Kb from 3' end had no influence on the promoter activity,while the absence of 0.7Kb from 5' end led the decrease of kanamycin resistance,which indicates that this 0. 7Kb fragment is important to promoter activity.Electroporation was used to transform pseudomonas pseudoaligenes with pPA7 and X2 analysis indicated that pPA7 had transformed into p seudomonas pseudoaligenes successfully. The kanamycin resistance of transformants increase obviously. According to the result it is sure that PA7 also has promoter activity in pseudomonas pseudoaligenes. PA7-2 was also proved having promoter activity in the same way.
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