Cloning,Sequencing And Expression Of α-Amylase Gene Rrom Xanthomonsa Campestris Pv.Campestris

Alpha-amylase was one of important enzymes in industry field, and the activist fields in enzyme studying .Genomic library of Xanthomonas campestris pv. campestris was constructed, and alpha- amylase gene was cloned> sequenced and analysed.The total DNA of Xanthomonas campestris pv. campestris and pUCIS plasmid were extracted respectively by different methods. The genome DNA was partially digested by 0.5U Mobl restriction enzyme . The time of digestion must be controlled. The resμlt of digestion woμld be effected whether longer or shorter. Agarose gel electrophorase coμld be used to detect the digestion resμlt and control the time .20 minutes were selected. Pieces of 2-7 Kb DNA was extracted. pUCIS plasmid was totally digested with BamHl enzyme and extracted by gel purification. To prevent the self ligation , CLAP was used to dephosphorylate totally . 2-7 Kb DNA and pUCIS plasmid were mixed.. T4 DNA ligase , ligase buffer and deioned water were added to 20 μl reaction volume . Because the ligation efficiency was very low, after ligation for 16-18h at 14℃ to 16℃,the reaction volume was enlarged to 50μl in the same condition for 16 to 18 h again. The ligation product was transformed with high efficiency competent cells, and more than 20000 transformants were gotten. LBSP plate screening was first used , then Idione- starch method was taken, and positive cloning of alpha- amylase gene were identified. After checking the length of positive cloning plasmid , digesting it with the restriction enzymes and testing of its ruction ,alpha- amylase gene of about 2Kb was cloned andits restriction enzyme map was made.The piece of DNA sequenced by Sangon was an 1899 bp fragment. Sequence analysis revealed a potential ORF encoding a protein of 475 amino acid residues. The deduced amino acid sequence had 45% homology to that of the alpha-amylase of Pseudomonas sp. KFCC10818. It also contained all four homology sequences highly conserved in the alpha-amylase from a wide range of organisms.The expression of the Amy gene in Escherichia coli was poor, and some of the expression were secreted. To extract the total protein, the cells were broken, the germination liguid was enriched, and TCA and Acetone A.R. were used. SDS-PAGE (12%W/V) showed that the molecular weight of alpha-amylase was 45 KD, which was the same as the result deduced from the nucleotide sequence.