Microorganism and related genereseources are valuable nature reseources. Currently，many countries start to plan some research programs which aim to protect and exploit the environmental microbiology resources in special environment. In the foreseeable future，a new round of upsurge about environmental microbiology and related gene resources exploitation will begin. The research hot points of environmental microbiology resources focused on high efficiency microorganism，degradation genes and enzyme screening. This thesis focused on the environmental problems such as persistent or ganicpollutants in petrochemical industry. We employ “omic” technologies to screen degradation genes from environmental samples in high throughput.
Through in-gel digest steps, high molecular weight genome DNA from activated sludge could be obtained. Up to 50kb genome DNA couled be isolated through in-gel digest， electrophoresis and gel recover. The productivity, purity quotient and microorganism diversity of the high molecular weight genome DNA are better than those which isolated using other techonlogies. A novel two-dimension nucleic acid electrophoresis system was set to separate ERIC-PCR fragments. This system has high resolution and could obtain about 150 nucleic acid points in one gel which was much more higher than AGE.
A novel bidirectional Substrate-induced gene-expression screening vector p18BG was obtained through gene recombination. This vector was used to screen aniline degradation genes using flow cytometry technology. Three nucleic acid fragments were obtained using p18BG.After ORF identification and BLAST, some genes which related to solute transportation, organic matter degradation and some transposons were detected.
A novel total genome DNA and total protein co-extraction technology was successful developed. Using this technology, total genome DNA and total protein could obtained from activated sludge.
This thesis developed a series of technologies which could be used in environmental microecology research. Also this study developed a novel bidirectional Substrate-induced gene-expression screening vector. These achievements will make contributions to the molecular microecology research field and supply a high throughput screening way to catch special genes and enzyme.
Keywords: Metagenome; ERIC-PCR fragments; Bidirectional Substrate-induced gene-expression screening; Aniline degradation; Metaproteome