中度嗜盐菌Halobacillus Y5甘氨酸甜菜碱转运蛋白opuD基因的克隆及功能分析

Cloning and functional identification of glycine betaine-cholinecarnitine transporter opuD gene from Halobacillus Y5

目的克隆中度嗜盐菌Halobacillus Y5甘氨酸甜菜碱转运蛋白(betaine-choline-carnitine transporter,BCCT)opu D基因,对其结构及蛋白功能进行分析,并通过耐盐碱特性试验明确其功能。方法将经过Sau3AⅠ部分酶切回收的Halobacillus Y5基因组总DNA,与用Bam HⅠ酶切并去磷酸化的线性p UC18载体连接,电转化法导入E.coli KNabc感受态细胞中,涂布在含0.2 mol/L Na Cl、Amp^(50)的LBK固体培养基上,筛选阳性克隆,提取质粒测序后,进行生物信息学分析及耐盐碱特性检测。结果已从中度嗜盐菌-喜盐芽孢杆菌Halobacillus Y5中成功克隆了1个新的耐盐碱基因,将其命名为HY5_opu D,该基因是1个新的甘氨酸甜菜碱转运蛋白基因,其编码的蛋白氨基酸序列和结构不同于以往报道的Opu D转运蛋白,且该基因能够使KNabc在0.2 mol/L Na Cl、5 mmol/L Li Cl及碱性p H 8.0环境下生长。结论成功克隆了1个新型的opu D转运蛋白基因,为开发利用中度嗜盐菌的耐盐碱基因奠定了理论基础。

Objective To clone the opuD gene of glycine betaine-choline-carnitine transporter （BCCT） from Halobacillus Y5, analyze the gene structure and protein function, and confirm the function by salt and alkaline tolerance test. Methods The total genomic DNA of Halobacillus Y5 partially digested with Sau3A I was ligated to linear pUC18 vector digested with BamH I and dephosphorylated with bacterial alkaline phosphatase, transformed to competent E. coli KNabc by electrotransformation and spread onto solid LBK medium containing 0. 2 mol/L sodium chloride, agar and 50 ~g/ml ampicilhn. The positive colonies were screened, from which recombinant plasmid was extracted, analyzed for bioin- formatics and tested for salt and alkaline tolerance. Results A new salt and alkaline tolerant gene BCCT HY5_opuD was cloned from Halobacillus Y5, which enabled KNabc to grow in LBK medium containing 0. 2 mol/L sodium chloride and 5 mmol/L lithium chloride at pH 8.0. The protein encoded by the gene showed different sequence and structure from those of OpuD transporters reported previously. Conclusion A Movel BCCT opuD gene was cloned successfully, which provided a theoretical basis for development and application of salt and alkaline tolerant gene of moderately halophilic bacteria.