【作者】 林宇岚；

【导师】 陈贻锴；

【作者基本信息】 福建医科大学 ， 病原生物学， 2006， 硕士

【摘要】 【目的】利用氯化汞(HgCl2)进行选择性培养,从自然界的土壤中分离耐汞菌。从中筛选出可以高效还原无机汞离子的优良菌株进行研究,以实际应用于工业汞污水的处理和汞污染土壤和水体的修复。【方法】①耐汞菌株的分离:采集日光灯厂厂区汞污染的土壤,用常规方法在含HgCl2的培养基上培养,长出的单菌落进一步分离纯化、观察菌落的形态、革兰氏染色、细菌生化分类鉴定及抗生素耐药性分析;②耐汞水平测定:用含Hg2+浓度不同的培养液进行培养,观察菌株的生长情况;③高耐菌株生长能力的研究:包括观察菌株在含汞培养液中的生存趋势;在不同pH条件下的生长情况;在灭菌自来水及农田土中的生存能力;④高耐菌株对Hg2+的还原能力:将待测菌接种于含汞浓度不同的LB培养液中,培养48h后取上清,用双道原子荧光光度计测定残余的汞含量,得出汞还原率;⑤16SrRNA分类鉴定:提取待测菌的基因组DNA,根据细菌16SrRNA基因中的保守序列设计并合成引物,PCR扩增产物,测序结果通过BLASTN数据库及同源性比较分析而明确待测菌的分类鉴定。⑥merA基因片段的克隆和鉴定:根据Gene Bank数据库提供的已知的merA基因序列及同源性比较分析,在高度保守的DNA序列区设计PCR引物,对待测菌的基因组DNA进行扩增,测序结果通过BLASTN进行同源性比较分析,鉴定待测菌是否含有merA基因以及其与已知的merA基因的同源性。同时尝试将所扩增的基因片段转化合适的受体菌。更多还原

【Abstract】 【Objective】: Through HgCl2-selected media, some special bacterial stains would be isolated from mercury- polluted soil, and then the best one should be selected on the basis of the phenotypic, physiological, biochemical tests and genic sequence analysis. All these results will help to apply the selected one into bioremediation of mercury-contaminated soil and water actually.【Methods】:①Isolation of high mercury-resistant bacteria: use HgCl2-selected media to incubate the bacteria out of the soil which we collect from fluorescent lamp’s manufactory by normal process. Different colonies would be incubated alone, and then we will discriminate them by tests including: the Gram’s staining; phenotypic, phy-siological and biochemical test; antibiotic resistance test, and so on.②Hg2+ toleration test: incubate the bacteria by using the media that have different Hg2+ concentrations, and then observe the growing of those bacteria.③Researches on the growing of the selected high mercury- degrading bacterium: including monitor the bacterium’s growth under different Hg2+ concentrations or pH conditions; observing its ability of living in the soil and natural water.④Testing the level of transforming Hg2+ into Hg0: inoculate the growing bacteria which are just on logarithmic growth phase into several LB liquid media that contain different definite Hg2+ concentrations. After 48 hours, collect the liquid by centrifugation to determine the rudimental Hg2+ concentration on AFS-230a double-tracts atomic fluorescence absorptiometer.⑤Classification and identification: according to the reserved 16SrRNA sequences by comparing the same bacterial strains in更多还原