The Construction Of Metagenomic Library Of Heavy-metal Contaminated Soil In Beijing, And The Screening Of Resistant Bacteria From The Soil

The heavy metal contamination in soil is becoming a significant problem of globle environment. Not only can it recede the fertility of soil, degradate the yield and quality of produces, even more deteriorate the nature of water, but also have negative effects to human beings through food cycle. The southeast area of Beijing is one of the five biggest pollution water releasing areas in China. It is reported that some places have the problem of heavy metal contamination. Biological remediation is one of effective methods to slove the problem of pollutions. With the development of Molecular Biology, it has the possibility to obtain the heavy metal resistant genes through metagenomic libraries from soil microbes which play a key role in the bio-remediation. It is a new way to explore the microbes' resources without cultivation steps.The purposes of the research are to explore an effective procedure to extract and purify the metagenomes in the heavy metal contaminated soil, construct a metagenomic library of it, and screen the library for the Hg²⁺ and Cd²⁺ resistant clones. Moreover, isolate the Hg²⁺ and Cd²⁺ resistant bacteria in the soil in traditional cultivation method to get the potential heavy metal genes in another way.We compare the different processes of extraction and purification of total soil DNA and explore an efficient procedure to obtain pure DNA. The procedure is easy to operate and the amount of purified DNA is about 40～50μg DNA/g soil, moreover, it can be operated in a series of molecular biology experiments. We construct a metagenomic library with pBluescript SK(+) and the recovery of the digested soil DNA. It has 10080 clones in the library, and the average size of the inserts is 4.5kb. It contains about 45Mb soil DNA. The percentage of the inserts which are more than 3kb is about 54.2%, which are between 2～4kb is about 50%, which is less than 2kb is only about 8.3%. Screening the library through the functional driven method, we find out a positive clone which can survive in the 150ug/mL Cd²⁺ contaminated medium.Determining and analyzing the sequence, the insert of the clone is 2.47kb, but has little similarity with the known sequences through BlastX. The orgin and functional identification of the sequence are waiting for further research.Screening 4 microbes from heavy metal contaminated soil though the isolation and culture methods. They are KG-1, KG-2, KG-3 and KG-4 which can grow in 22.2μg/mL HgCl₂ medium and they are identified as Bacillus by the construction of 16S rDNA phylogenetic trees of them. Moreover, we isolate 2 microbes which can grow in 350μg/mL Cd²⁺ medium.