Analysis Of The Diversity Of Termite (Macrotermes Barneyi)Gut Microorganisms And Cloning, Identification Of Theβ-glucosidase Gene

The countries all over the world are faced with the threat of energy crisis and environmental problems at present, the development of a new clean energy becomes an urgent demand. As a renewable energy leader, the cellulosic ethanol of biomass energy sources is being increasingly favored by everybody. Because of the high production costs, the relatively low and unstable hydrolytic activity of the currently commercial cellulases, the fuel ethanol from lignocellulose has not been able to truly realize industrialization. Therefore, discovery new enzymes with high lignocellulytic activity become one of the research focuses in the field of biomass energy.Cellulase is a kind of enzyme of hydrolyzing cellulose into glucose, the three main types are endo-β-1,4-glucanases (EGs), cellobiohydrolases (CBHs, a form of "exo-glucanase") and β-glucosidases (BGs). Termites are important decomposers of lignocellulose in natural ecosystems, the rich cellulose enzyme gene resources and metabolic pathways in termites and their intestinal symbiotic microorganisms may be used for cellulose ethanol exploitation. However the majority of termite gut microbes is difficult to cultivate, directly screening functional genes or gene cluster from the uncultured intestinal microorganisms by using metagenomics technology risen in recent years opens up a new way for mining and using the intestinal microbial resources.Macrotermes barneyi Light is one of the main species that hazard garden trees in Hunan province, the current domestic research mainly focused on its biological characteristics and control, and with microecology theory as the guide to study the termite gut microbes is less. Therefore, the16S rRNA gene libraries and the metagenome library of termite gut microbes were constructed in the study, and the diversity of bacteria, archaea were analyzed. In addition, a new β-glucosidase gene with the strongest enzyme activity was gotten based on functional screening and identified. The detailed results of the study were as follows.Firstly, the population structure and evolutionary relationship of intestinal bacteria and archaea were analyzed by using16S rRNA gene library cloning technology, the results were shown that the95%intestinal bacteria clones were from three phyla: Bacteroidetes, Firmicutes and Proteobacteria. In addition, a few clones belonged to Deferribacteres, Actinobacteria, Planctomycetes. The intestinal archaea mainly belonged to two phyla (Euryarchaeota and Crenarchaeota), five archaea order (Methanobacteriales, Methanomicrobiales, Methanosarcinales, Thermoplasmatales and Thermoproteales). Most of archaea clones belonged to Methanomicrobiales, followed by Thermoplasmatales, and the number of Methanobacteriales clone was least.Secondly, the plasmid and Cosmid metagenome library of gut microbes was constructed based on the pUC19and Cosmid vector. The plasmid library contained9061clones with average insert fragment length of2.4kb, which covered21.7Mb library capacity. And the Cosmid library contained more than5000clones with average insert fragment length of22kb, the library foreign DNA total capacity is about110Mb.Thirdly, there were12positive clones with beta glucoside enzyme activity by using functional screening and sequence analysis from two constructed library, and one clone named Bp6with the strongest beta glycosidase enzymes activity was cloned and identified. one DNA fragment including918bp was gained, which contain a predicted ORF with825bp named ungluBp6. ungluBp6encoded a protein (ungluBp6) with274amino acids.The results of the study were shown that the gut microbial resources in termite of Macrotermes barneyi Light were very rich, and there were a lot of unknown microbes need to be utilization.