苏云金芽孢杆菌chi36基因的克隆、表达和酶活性分析  

Cloning,Expression and Activity of chi36 Gene from Bacillus thuringiensis

作　　者：黄必旺[1] 谢宇飞[1] 沙莉[1] 黄志鹏[1] 

机构地区：[1]福建农林大学生物农药与化学生物学教育部重点实验室,福建福州350002

出　　处：《热带作物学报》2011年第6期1106-1110,共5页Chinese Journal of Tropical Crops

基　　金：国家自然科学基金项目(No.30571257);国家自然科学基金项目(No.31071745);福建省自然科学基金项目(No.2010J01069);福建省高校服务海西建设重点项目(No.0b08b005)

摘　　要：通过PCR方法从苏云金芽孢杆菌010菌株克隆出几丁质酶基因chi36,该基因开放阅读框(ORF)的长度为1 083bp,编码360个氨基酸。氨基酸序列分析表明:Bt 010的Chi36与Bt15A3、Bc28-9、Bc6E1的Chi36相似性分别为99%、99%和96%,其N-端具有27个氨基酸的信号肽序列。该蛋白归类为18家族几丁质酶,属于一种胞外酶。将chi36基因插入到PGEX-KG原核表达载体的表达框中,构建成原核表达载体并转化到大肠杆菌E.coli BL21(DE3)进行蛋白表达,酶活性测定结果表明,表达产物对几丁质有降解作用,在pH值为5.0、温度为55℃时酶活性最高。chi36 gene was cloned from Bacillus thuringiensis strain 010 by PCR. The nucleotide sequence of chi36 included an open reading frame of 1 083 bp, encoding 360 amino acids. Analysis of its amino acid sequence showed that Chi36 of Bt 010 had 99%, 99% and 96% similarity with Chi36 of Bt 15A3, Bc 28-9 and Bc 6El respectively. ScanProsite analysis revealed that Chi36 of Bt 010 belonged to family 18 of glycosyl hydrolases, as an exochitinase with a signal peptide sequence containing 27 amino acids at N terminal. The chi36 gene was inserted into the expression plasmid pGEX-KG and expressed in E. coli BL21 （DE3）. Bioactivity assay indicated that the expressed fusion protein could digest chitin, and with the most high activity at pHS.0 and 55℃; respectively.

关 键 词：苏云金芽孢杆菌 几丁质酶 基因表达 酶活性 

分 类 号：Q78[生物学—分子生物学] S476.1[农业科学—农业昆虫与害虫防治]