Construction Of Chitinase Recombinant Of High Bacterial Locusticidal Efficiency From Pseudomonas Pseudoalcaligenes

Cloning and identification of promoters of Pseudomonas pseudoalcaligenes was reported in this thesis. Promoter-probe vector pSUPV4 was used to clone promoters of P. pseudoalcaligenes in Escherichia coli. Nine kanamycin resistant recombinants were obtained and designated as pPAl-pPA9. The pPA7, which has the highest level of kanamycin resistance, was chosen for further characterization. The PA7 fragment was sequenced and several motifs similar to prokaryotic promoter elements such as -10box,-35box and Up box were identified. The ATG site was found and SD sequence, the binding site of ribosome, was also localized just upstream from it. The PA7 fragment was analysed by blasting in Genebank and the homological gene was found.To functionally determine precise promoter sequence in PA7 fragment, the PA7 fragment was sub cloned by PCR. Two pairs of primers 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 subcloned fragments inserted at fixed direction with ORFs unchanged. All the three subcloned recombinants had the same level of kanamycin resistance (about 600 u g/ml).The result showed that the PA7-2 fragment still had 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 indicated that the 0.7Kb fragment was essential to enhancing promoter activity.Southern blotting indicated that the PA7 fragment was present in P. pseudoalcaligenes genome probably as one copy. Electroporation was used to transform P. pseudoalcaligenes by using pPA7. P. pseudoalcaligenes transformantsacquired kanamycin resistance and this indicated that pPA7 was successfully introduced into P. pseudoalcaligenes. The results further confirmed that PA7 has promoter activity in P. pseudoalcaligenes, So did PA7-2.In the thesis cloned of gene of insecticidal protein from P.pseudoalcaligene was reported. A genomic library was constructed in E.coli DH5a by using a Sau3AI digest of P. pseudoalcaligenes chromosomal DNA ligated into the BamHI site of pUC19 vector. The recombinant colonies were screened with a mixed oligodeoxynucleotide as GGNGTNTGGCARCAYCA which was coded for the amino acid sequence of the N terminus of P. pseudoalcaligenes insecticidal protein as Gly Val Trp Gin His Gin Ser His Ala Ala. Five positive clones were obtained after two rounds of screening. The smallest clone pSCl was chosen for further investigation. Hybridization with oligodeoxynucleotide suggested that the 1.4kb BamHI-Kpnl fragment from the insert of pSC 1 contained the sequence encoding for the N terminus of insecticidal protein. Sequencing of this fragment identified an open reading frame encoding a polypeptide composed of 276-amino acid residues. The deduced amino acid sequence contained a 22-amino acid signal peptide followed by a mature protein sequence. The deduced mature insecticidal protein consisted of 254-amino acid residues had a calculated molecular weight of 26.7kD, which was almost identical to that of native insecticidal protein. The 22-amino acid signal peptide had C-domain, atypic positively charged N-domain and lack a hydrophobic H-domain. The signal peptide was similar character to bacteriocin or pheromone signal peptide which are exported via ABC transporters. Signal peptides of this class of secreted proteins could not be predicted by the regular algorithms for signal peptide prediction, such as SignalP, as they consist only of N- and C-domains and completely lack a hydrophobic H-domain.The 5' -flanking region contained a possible Shine-Dalgamo sequence but had no obvious promoter consensus sequence in the -10 and -35 region similar to those in E.coli. Comparison of amino acid sequence of insecticidal protein with a range of other bacteria toxins revealed little sequence similarity.In the thesis the analysis of Bacillus circulans C-2 chitinase gene Chil sequence analysis and its domain was c...