【作者】 曾艳；

【导师】 陈强； 高俊莲；

【作者基本信息】 四川农业大学 ， 微生物学， 2009， 硕士

【摘要】 本研究同时从不可培养和可培养细菌两方面入手,从重金属污染土壤中筛选抗重金属汞和镉的基因。通过功能和序列驱动筛选相结合的方法,从已构建的土壤宏基因文库中分离鉴定抗重金属汞和镉的基因。采用传统的微生物分离培养方法,从重金属污染土壤中筛选重金属汞、镉抗性细菌菌株,然后从中分离鉴定抗重金属汞和镉的基因。研究结果如下:（1）从北京凉水河床底泥中分离得到3株抗镉菌和3株抗汞菌。在含镉（CdCl₂）为450μg/mL的LB平板上筛选得到3株抗镉菌,编号为KCd1、KCd2、KCd3;在含汞（HgCl₂）70μg/mL的LB平板上筛选得到3株抗汞菌,编号为KHg1、KHg2、KHg3。对6株抗性菌进行了形态观察、生理生化测定、脂肪酸鉴定和16S rDNA序列分析,结果表明,6株菌中有5株为芽胞杆菌属,1株为假单胞菌属。经鉴定在分类上KHg2为Bacillus silvestris,KCd1为Bacillus cereus;KCd3为Bacillus mycoides,而其它菌株的分类地位待定。（2）扩增获得了5个抗汞的merA基因,将其克隆于PET-30（a）+,并在E.coli BL21（DE3）中得以成功表达。从分离得到的3株抗汞菌的基因组DNA中扩增分别得到1个merA基因,命名为pZY1、pZY2和pZY3,从文库中得到2个merA序列,命名为pZY4和pZY5,将5个merA基因克隆到pET-30（a）+,并转入E.coliBL21（DE3）中,分别命名为E.coli BL21（DE3）pZY1至E.coli BL21（DE3）pZY5。采用对克隆质粒扩增和对克隆质粒用BamHI和HindⅢ双酶切进行验证,结果表明merA基因成功克隆于pET-30（a）+。将克隆的merA基因在E.coli BL21（DE3）中用1 mmol/L的IPTG进行诱导表达成功;采用SDS-PAGE,探讨了温度对merA基因的诱导表达效果,结果表明37℃为最佳表达温度;在不同时段加入HgCl₂,培养28h后,重组菌株生长良好,而对照不生长,表明重组质粒中含有的merA基因具有汞抗性功能。实验发现,从文库中得到的两个基因的重组菌比从抗性菌中得到的merA基因的重组菌株的生长要弱。（3）功能平板筛选得到17个在含CdCl₂（150μg/mL）平板上生长良好的抗镉阳性克隆子;从抗性菌中用Nest-PCR获得3个镉抗性基因cadA的保守序列（分别为485bp、483bp和482bp）。筛选混合基因文库,在含镉150μg/mL的平板上,以空质粒PBS为阴性对照,得到17个阳性克隆子,将其中2个测序,利用GenBank中的Blastx搜索测定的序列,结果编号为Cd15的部分蛋白序列与水解酶家族中的金属依赖酰胺水解酶（metal dependent amidohydrolase）和金属依赖性甘氨酸酶（metal dependant alycoprotease）分别有56%和53%的相似性,推测可能是新的基因,其证实工作有待于进一步实验。从3个菌中得到3个长度分别为485bp、483bp和482bp的序列,测序结果经GenBank中搜索,均为cadA中的保守序列。更多还原

【Abstract】 In this study, culture-dependent and culture-independent technology was adopted to study the heavy mental resistance genes of the soil collected from Liangshui River in Beijing. On one hand, conservative isolation methods were used to obtain desired pure strains. On the other hand, function and sequence-driven methods were used to screen the metagenomic library to get Mercury and Cadmium resistance genes. The research results are as the following:（1） By using pour plate method, three mercury-resistant strains, named KCd1, KCd2, KCd3, and three cadmium-resistant strains, named KHg1, KHg2, KHg3 were isolated. They can grow well on LB medium plate containing 450μg/mL CdCl₂ or 70μg/mL HgCl₂. Morphological observation showed the six strains were G⁺. Analysis of physiological and biochemical properties, FAME and 16S rDNA sequencing showed that one strain belonged to genus of Pseudomonas, and the others were identified to genus of Bacillus; strains KHg2, KCd1 and KCd3 belonged to Bacillus silvestris, B. cereus, B. mycoides, respectively; however, the taxonomic position of the other strains needed to further study.（2） Five merA gene （mercury resistance gene） were amplified and cloned into pET-30a （+）, and then transformed into E.coli BL21 （DE3）, the cloned gene was expressed successfully. Three merA genes were cloned from each of anti-mercury strain genome DNA, and named pZY1、pZY2 and pZY3. Two merA gene fragments, from the metagenomic library were isolated and cloned; the plasmids were named pZY4 and pZY5. These 5 merA genes were cloned into pET-30a （+） and named E.coli BL21 （DE3）.pZYl, E.coli BL21 （DE3）.pZY2, E.coli BL21 （DE3）.pZY3, E.coli BL21 （DE3）.pZY4 and E.coli BL21 （DE3）.pZY5 respectively. The recombinant clones were verified by using PCR and digestion of BamHI and HindIII successfully. The optimal temperatures was determined, the results showed that the optimal express temperature was 37℃. After cultured 28h with the addition of HgCl₂ in different growth phases, all the recombinant strains grew well, while the negative control did not grow, which showed that the recombinant plasmid contain merA gene, and possessed the mercury resistance ability; while, the negative plasmid was lack of merA gene, lead to recombinant couldn’t grow any more. At the same time, the two recombinant strains containing genes from the library grew weaker than those containing resistant genes derived from bacteria.（3）Using function-driven method, PBS as the negative control plasmid, 17 anti-cadmium positive clones, which grew well in LB medium containing 150μg/mL of Cadmium（CdCl₂） were selected, and three of cadA gene sequences were obtained from resistant bacteria by nested PCR; then, two clones （named Cd7 and Cd15）were sequenced. Analysis of gene sequence showed that there were not high similar to the sequences in the GenBank; however, Blastx search results showed that a part of Cd15 protein sequences were 56% and 53% of similarity with the family of metal dependent amidohydrolase and metal dependant alycoprotease, respectively, which suggested that they may be new genes, but needed to confirm fatherly. three sequences （respectively, 485bp、483bp and 482bp） from the anti-cadmium strains were gained, and Blast showed that they shares 99% sequences identity with partial conservative cadA gene sequences of some bacteria published in GenBank.更多还原