家蚕肠道产淀粉酶细菌的分离鉴定及其酶基因的克隆与表达  

Isolation and Identification of the Amylase-producing Bacteria in Silkworm Intestine and Cloning and Expression of Their Amylase Genes

作　　者：杨文静[1] 王在贵[1] 刘朝良[1] 魏国清[1] 朱保建[1] 邹昌瑞[1] 

机构地区：[1]安徽农业大学生命科学学院,安徽合肥230036

出　　处：《激光生物学报》2011年第2期219-229,共11页Acta Laser Biology Sinica

基　　金：安徽省自然科学基金项目(11040606M98);现代农业产业技术体系建设专项资金;安徽省人才专项基金项目(2008Z022)

摘　　要：从家蚕(Bombyx mori L.)5龄幼虫肠道分离鉴定产淀粉酶细菌菌株以用作微生态制剂的研究,并对该菌α-淀粉酶基因进行克隆、序列分析及在大肠杆菌中原核表达。通过含淀粉NA培养基筛选分离得到产淀粉酶菌,通过形态学观察及16S rDNA序列分析鉴定其种属,DNS法测定酶活,并设计了淀粉酶基因引物进行克隆,构建了DZ-a号菌淀粉酶基因的原核表达载体,经酶切鉴定后转化到大肠杆菌Transetta(DE3)中并诱导表达。共分离得到2株产淀粉酶细菌菌株,将其PCR扩增得到的16S rDNA序列分别与GenBank上已有序列进行比对,均与芽孢杆菌属蜡样芽孢杆菌Bacillus cereus有99%的同源性,DZ-a号菌和DZ-h号菌的产酶能力分别为20.5U/mL和24.2U/mL。产淀粉酶基因片段测序结果表明两株菌的克隆基因片段长度分别为3414bp和3378bp,均包括完整的编码区序列,可编码586个氨基酸,SDS-PAGE分析得到大小约66kD的目的蛋白。鉴定该2株菌DZ-a、DZ-h号菌均属于芽孢杆菌属蜡样芽孢杆菌,克隆测序所得序列为完整的蜡样芽孢杆菌α-淀粉酶基因序列,并成功表达。为进一步纯化和鉴定目的蛋白及研究其功能奠定了试验基础。Amylase-producing bacterias in larval guts of the Bombyx mori L. at 5th year phase were isolated and identified to application for probiotics. Ckming, sequences analysis and prokaryotic expression in Escherichia coli of this amylase gene was conducted. NA starch medium was used to isolate amylase-producing bacterias. Strains identification which was on 16S rDNA sequences analysis and morphology was carried out. Enzymatic activity of them was measured by DNS method. Primers of amylase genes were designed to clone the genes. The full-length open reading frame of the amylase gene from strain DZ-a was fused into the prokaryotie expression vector pET28a and was introduced into E. Coli Transetta （DE3） cells. As a result, two amylase-producing strains were isolated. The result of 16S rDNA analysis in Genbank suggested that the strains both have 99 % honmlogous proportion to Bacillus cereus. The enzymatic activity of amylase was strain DZ-a 20.5 U/mL and strain DZ-h 24.2 U/mL respectively. Sequences analysis of the amylase genes showed that the cloned sequences of the two strains were 3 414 bp and 3 378 bp respectively. They both contained complete open reading frames encoding 586 amino acid residues. The SDS-PAGE showed that the molecular weight of the expressed pro- tein was about 66 kD. The two strains DZ-a and DZ-h were identified as Bacillus cereus. The cloned sequences both were complete Bacillus cereus α-amylase genes. And one amylase gene of them was successfully expressed. These results will provide foundation for turther purification and identication of the target protein and for the function study of this amylase.

关 键 词：家蚕 肠道细菌 Α-淀粉酶 16S RDNA 克隆及原核表达 

分 类 号：Q786[生物学—分子生物学]