Molecular Characteristic Of Thermoaphilic Cellulolytic Anaerobic Bacteria And Cloning Of β-glucosidases Gene Of B2 Strains

30 to 50 percent of dry weight of plants is Cellulose which is the most widely distributed carbohydrate in the world. It is reported that there are 1.55×109 tons of cellulose produced by plants' photosynthesis every year in the world and 89 percent of them can't be used by human. The ways of degradation are mainly acidolysis, oxygenolysis, microbial degradation etc. Microbial degadation , the center of natural carbon cycle, is one of the important basic which keep all lives living. The recent years , the research of cellulase genes has been gotten rapid development. Some of cellulase genes which was cloned from many kinds of bacteria and fungi (There are more than forty bacteria' cellulose genes was cloned now) ,was expressed in E. coli and other receptor cell.The enzyme system of thermophilic cellulolytic anaerobic bacteria is different from cellulolytic aerobic bacteria's. The techniques of use cellulase in many industries need hyperthermal condition. We got several extremely thermoaphilic cellulolytic anaerobic bacteria from Biogas Institute of the Ministry ofAgriculture. The genetic diversity and genetic relation of several stains of Bang was done with statistical data of RAPD. The power of cellulolytic of Strain B2 was the strongest in these bacteria . It' s cellulase was the peak and more stabilization . The phylogenetic analysis based on 16S rDNA suggested strain B2 was the closest relative of Thermoanaerobacter ethanalicus with 99.8% sequence similarity. The strain Bang2 was initially identified Thermoanaerobacter according to foregone reseach (biochemical and physiological results) & sequence analysis of 16S rDNA.Genomic DNA of Bang2 was extracted and was partially digested by restriction enzyme HinⅢ, and ligated with plasmid pUC18 by T4DNA ligase. After transformation E. coli JM109, the genomic library was constructed and 5278 recombinants was obtained. The ratio of the recombinants which the strains turn black on the esculin plates was about 0. 26%. The results after digested by restriction enzyme HindⅢ revealed that the recombinants which had been picked out contained foreign DNA fragments. The results showed that the β -glucosidases gene has been cloned. No plasmid was detected in Bang2' s genomic DNA. The recombinants which contained β -glucosidases fragments was digested by restriction enzyme Hind Ⅲ　and revealed that there is about 700bp foreign fragment in it.