植物乳杆菌α-半乳糖苷酶基因（melA）的克隆与序列分析

试验以L.plantarum1.557的基因组DNA为模板，通过PCR技术扩增α-半乳糖苷酶基因（melA）基因，PCR产物经纯化回收后克隆至pMD18-T载体中，转化E.coli DH5α感受态细胞，筛选阳性克隆，提取质粒进行SacⅠ和Sph Ⅰ酶切及PCR扩增鉴定，并对melA基因片段进行序列测定。结果表明，测得的melA基因序列全长2240 bp，含有酶切位点、启动子、SD序列及编码738个氨基酸的开放阅读框，理论分子质量为84 ku；4种核苷酸中GC含量为47.45%、AT含量为52.55%；序列中有3个碱基发生了变化，均为无义突变，未导致其推导的氨基酸的改变；序列测定结果与GenBank中登录的序列进行比较，核苷酸和氨基酸序列的同源性除与AY873840相比分别为96.6%和92.2%外，其余均大于99%。试验成功克隆了melA基因，为进一步构建重组表达载体奠定了基础。

Cloning and Sequence Analysis of the α-galactosidase Gene(melA)from L.plantarum

Using chromosome DNA of L. plantarum as template，melA gene was amplified by PCR and purified by purification kit in this study. The PCR product of melA gene was cloned into pMD18-T vector and transformed into DH5α competent cells. The recombinant plasmid containing melA gene was selected and the inserted melA gene was identified by cleaved with restriction endonuclease Sac Ⅰ and Sph Ⅰ, and following by PCR amplification and sequencing. The cloned complete melA gene sequence, at a length of 2240 bp, encoded 738 amino acids with a deduced molecular weight of 84 ku. The GC and AT contents were 47.45% and 52.55% respectively. Three nucleotides nonsense mutation were observed, resulting no change of deduced amino acids. The homologies of nucleotides and amino acids were more than 99% respectively (except for AY873840, 96.6% and 92.2%) compared with those in GenBank. The results showed that the whole melA gene was cloned successfully. This research proved a foundation for the construction of recombinant express vector of melA gene.