Screening And Identification Of Mercury And Cadmium Resistance Gene Of Micro-organisms From Heavy-metal Contaminated Soil

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.