植物乳杆菌α-葡萄糖基转移酶基因挖掘及表达研究

Gene Mining and Expression of α-glucosyltransferase from Lactobacillus plantarum

α-葡萄糖基转移酶(α-GTF)是一类可以利用麦芽糖,异麦芽糖,O-甲基葡萄糖苷等为底物合成低聚糖的多功能性酶,因其功能多样性和独特性而备受关注。本文利用本团队测序的植物乳杆菌(FMNP01)全基因组信息,采用生物信息学方法分析获得α-葡萄糖基转移酶基因(α-gtf),同时根据NCBI中上传的乳酸菌全基因组信息中的α-GTF的DNA序列设计引物,利用PCR技术分别从植物乳杆菌、干酪乳杆菌和短乳杆菌中克隆得到3个α-gtf基因,其编码的氨基酸序列相似性分别为42%、29%和29%;将克隆得到的3个基因分别与表达载体pET-32a连接构建成表达质粒,转入大肠杆菌BL21表达宿主菌中进行诱导表达,产物经SDS-PAGE分析表明,在75 ku处都有一明显条带,与预期蛋白分子量一致,表明这3个基因成功获得了表达。同时对诱导剂浓度、诱导时间和温度进行优化,可望为以后相关研究奠定了基础。

The α-glucosyltransferase（α-GTF） is a type of multifunctional enzyme that can synthesize oligosaccharides using maltose,isomaltose,and O-methyl glucoside as substrates,and has attracted attention because of its functional diversity and specificity.In this study,the whole genome information of Lactobacillus plantarum（FMNP01）,which was sequenced by our team,and the bioinformatics approach were used to analyze the α-gtf gene obtained fromLactobacillus.Meanwhile,the primers were designed based on the DNA sequences of the α-gtf gene in Lactobacillus,which are listed in the National Center for Biotechnology Information（NCBI） database.Three α-gtf genes were cloned by polymerase chain reaction（PCR） fromLactobacillus plantarum,Lactobacillus casei,and Lactobacillus brevis,and the similarities of the encoded amino acid sequences were 42%,29%,and 29%,respectively.The three obtained genes were cloned into vector pET-32 a to construct the recombinant pET-32a-α-gtf,which were then transformed into the expression host Escherichia coli BL21 to induce expression.The fusion proteins were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis（SDS-PAGE） and the results showed that there was an obvious band at 75 ku,consistent with the molecular weight of the predicted protein.The results indicated that three α-gtf genes were expressed successfully.Meanwhile,the inducer concentration and induction time and temperature were optimized,and this study is expected to lay the foundation for related studies in the future.