【摘要】 试验以L.plantarum1.557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.coliDH5α感受态细胞,筛选阳性克隆,提取质粒进行SacⅠ和SphⅠ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me-lA基因,为进一步构建重组表达载体奠定了基础。更多还原

【Abstract】 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.更多还原