Burkholderia cepaciaからクローニングしたキチナーゼ遺伝子の塩基配列決定

概要

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[Synopsis] In this study, we cloned a chitinase gene from Burkholderia cepacia and identified a nucleotide sequence. A genomic library was constructed by the use of λEMBL3, and then phage clones producing chitinase were selected by using 4-MU chitotrioside as a substrate. The inserted DNA was subcloned into a pBluescript vector with restriction enzymes to make pBlueCAK12 (4.4 kb) clone including a chitinase gene. The DNA fragment was deleted by exonuclease III and the chitinase-coding region was determined by chitinase activity detected by the use of 4-MU chitotrioside. The nucleotide sequence of chitinase gene (2.98 kb) was determined by primer walking method, in which ORF of chitinase gene was amplified by specific primers including the expected start and stop codons. The amplified DNA was inserted into a pBluescript vector, and then chitinase activity was detected by the use of 4-MU chitotrioside.