Metagenomics Methods In Environmental Microbial Ecology And Genetic Search Application

Metagenomics is a new field of research in which scientists analyze the genomes of organisms recovered directly from the environment. This thesis was based on the andvantages of metagenomic methods, and applied this method in the investigation and analysis of bacteria community in the silkworm intestine, and in the priminal research of distribution of integron-gene cassettes, information of encoding proteins, and their roles in the evolution of bacteria genomes.1. The rapid developing molecular biology has showen that the majority of microorganisms in environmental samples remain uncultured, and even undescribed. It was estimated that less than 1% of the microorganisms are readily culturable. The knowledge on microbe communities in special environments based on the traditional cuture methods is obiviously biased. To shed a light on the bacteria community in the silkworm intestine more completely, culture-dependent and culture-independent methods were used to perform the investigation. As the latter, 16s rRNA gene was amplified and a library was constructed by using the meta-genomic DNA extracted from the bacteria in the silkworm intestine as template. Restriction fragment length polymorphism (RFLP) method was used to screen recombinants originated from different bacteria, and the nucleotide sequences were determined. A BLASTn searching in the GenBank and an analysis on phylogenesis were performed. The taxonomy of these bacteria and their physiological function to silkworm were speculated. Results showed that the bacteria in silkworm intestine are mainly belonging to the genera Arthrobacter, Lactobacillus, Pseudomonas, Escherichia, Micrococcus, Bacillus, and Staphylococcus. They may play an important role to the host silkworm in the utilization of their food mulberry and disease prevention. Both the culture-dependent and culture-independent methods had the advantages and disadvantages, which complemented each others.2. Integrons are gene acquisition and expression systems through inserting to or excising from integrons by a site-specific recombination reaction catalyzed by the integron integrase, IntI. The units of DNA captured by integrons, gene cassettes, are the simplest known mobile elements. In this study, we will use special primers HS286-HS287, which was designed by the conserved regions 59-base element flanking the gene-cassette genes, to amplify the gene-cassette existed in the metagenomic DNA. And by sequencing and analysising of the amplicons, we expect to some novel information about integron-gene cassette system.We have extracted metagenomic DNA from 16 water samples collected around Southwest University of China.and 6 of them have the positive integron amplification results by using special primers HS286-HS287. The sizes of these fragments were between 300bp and 2000bp. These resulting fragments were ligated with a TA-cloning vector, and then transformed into E.coli. 25 single colonies were picked out randomly, and their plasmids were extracted to determine the nucleotide sequences of the inserted DNA fragments. Informatics analysis revealed that these fragments contained 7 different gene-cassettes, harbouring 13 deduced ORFs. These ORFs were predicted to encode proteins showing high homology to those deposited in GenBank...