【摘要】 从苏云金芽孢杆菌以色列亚种 (Bacillusthuringiensissubspisraelensis)中提取基因组DNA ,通过合成 1对特异性引物 ,用touchdownPCR的方法扩增几丁质酶ichi基因序列 (GenBank登录号 :AF5 2 6 379)。ichi序列全长为 2 5 70bp ,含有 1个 2 0 6 7bp的开放阅读框 (ORF) ,编码 6 88个氨基酸 ,推测分子量为 75 79kDa ,等电点pI=5 90的几丁质酶前体。序列和结构比较分析表明 :Ichi氨基酸序列与蜡状芽孢杆菌 (Bacilluscereus) 2 8 9几丁质酶CW、蜡状芽孢杆菌CH几丁质酶B及苏云金芽孢杆菌墨西哥亚种几丁质酶的同源性分别为 97 2 4 %、97 18%、97 6 3% ,而与苏云金芽孢杆菌巴基斯坦亚种的同源性只有 6 3 0 7%。Ichi编码区由分泌信号肽 (46AA)、催化区 (10 5AA)、粘蛋白Ⅲ型同源区 (74AA)及几丁质结合区 (40AA)组成。更多还原

【Abstract】 Chitin,a linear homopolymer of N acetyl D glucosamine,is a common constituent of fungal cell walls,exoskeletons of insects,and shells of invertebrates.Thus,chitinase,a chitin hydrolytic enzyme,has become of interest for potential use as biopesticides for controlling insect pests.Using a pair of specific primers,chitinase gene ( ichi ) was amplified by touchdown PCR from Bacillus thuringiensis subsp israelensis chromosomal DNA,and then subcloned into pGEM T easy vector. ichi sequence (GenBank Accession Number:AF526379)with a length of 2570 bp included an open reading frame (ORF) of 2067 bp,which contained 688 amino acids with Mr=75 79 kDa and pI=5 90.The amino acid sequence of ichi gene shows 97 24%,97 18%,97 63% and 63 07% identity to that of Bacillus cereus strain 28 9 chitinase CW, Bacillus cereus CH chitinase B, Bt subsp Mexican chitinase,and Bt subsp pakistani chitinase A71,respectively.It was demonstrated that Ichi contains a signal peptide (46 amino acid residues) and three functional domains:an N terminal catalytic domain (105 amino acid residues),a fibronectin type Ⅲ like domain (74 amino acid residues),and a C terminal chitin binding domain (40 amino acid residues).更多还原