| Microbial diversity plays an integral and unique role in variety of ecosystem, for example, this has been proven in agricultural ecosystem by many recent research. Microorganisms play a key role in global cycle of carbon, nitrogen, sulfur, and heavy metal and so on, thus it affects the material balance, the composition of the atmosphere and geochemistry procedure in the global scale. Understanding the structure and composition of microbial communities and their responses and adaptations to environmental perturbations such as toxic contaminants, climate change, and agricultural and industrial practices is critical in maintaining or restoring desirable ecosystem functions. However, because less than 1 % of microorganisms can be cultured, characterization and detection of microbial populations in natural environments present a great challenge to microbial ecologists. Current methods for analyzing microbial communities, especially their key functions, are too cumbersome. Rapid, simple, reliable, quantitative, and cost-effective tools that can be operated in real-time and in heterogeneous field-scale environments are needed.Microarrays (or microchips) are a recently developed genomic technology that has been shown to be a powerful tool for studying gene expression and regulation on a genomic scale and detecting genetic polymorphisms in both eukaryotes and prokaryotes. Compared to conventional membrane-based hybridization, glass slide-based microarrays offer the additional advantages of high sensitivity, rapid detection, lower cost, automation, and low background levels. Although microchip-based genomic technology is potentially an extremely powerful tool for characterizing microbial communities and their biological functions, the concept and performance of microarray hybridization have not been rigorously tested and validated with complex environmental samples.A recent discovery is that some functional genes, such as nitrite reductase gene can be used as marker genes to analyze special functional group of microorganisms in replacing 16S rRNA gene. In addition, quantitative Reverse Sample Genome Probing (RSGP), which permits the identification of bacteria based on genomic DNA hybridization, has been employed in many environments to monitor bacterial population in response to environmental change.In this study, new denitrifiers were isolated and new nir genes were cloned from marine sediments samples to enrich the sequence information of the functional gene in denitrifying bacteria. Based on this, two kinds of microarraies: functional gene microarray (FGA) and community genomic microarray (CGA), were constructed andtested. And the application of these microarraies were explored using both surface soil and marine sediment samples. The key results obtained from this study are as bellow.1. Isolation of denitrifying isolates, cloning of nir genes and the diversity investigation of denitrifying bacteria in Pacific Northwest Marine Sediment communities30 denitrifiers were isolated from the marine sediment sample from Puget Sound and two sites of Washington margin by enriching in nutrient broth and plating on agar anaerobically under denitrifying conditions, screening using rep-PCR finger print analysis and nitrate depletion testing. 12 nirS gene and 5 nirK genes were amplified by PCR and sequenced from these isolates, some of which were new for the genebank. The phylogenetic analysis indicated that these isolates are very diverse that they are distributed to 7 genera or 9 species.nirK and nirS genes were cloned from the marine sediment samples from Puget Sound and two sites on the Washington continental margin by PCR approaches. nirS sequences could be amplified from samples of both sampling sites, whereas nirK sequences were detected only in samples from the Washington margin. To assess the underlying nir gene structure, PCR products of both genes were cloned and screened by restriction fragment length polymorphism (RFLP). Rarefraction analysis revealed a high level of diversity especially...
|
PDF Full Text Request