| It is well known that symbiotic microorganisms in termite gut play important roles in the lignocellulose decomposition. However, due to the unculturability of most of these microorganisms, information about these microorganisms is still very limited. The advent of high-throughput sequencing technology makes it possible to get lots of information of these symbionts without pure culture. In this study, the symbiotic bacteria from the intestinal fluid of Reticulitermes chinensis Snyder were analyzed by metagenomic sequencing. A detailed analysis of the sequencing data showed metabolic pathways of dominant microorganisms, the abundance and diversity of symbiotic bacteria, the types and abundance of cellulolytic enzymes and other functional genes such as nitrogenase genes of bacteria in the gut of Reticulitermes chinensis Snyder, which formed the basis for further studies on physilogical functions of bacteria in the gut of Reticulitermes chinensis Snyder. The main results of this study are as follows:1. In total,35,943,166 reads were obtained by sequencing the bacterial DNA. Spirochaetes were the most dominant bacteria via ORFs species annotation. Firmicutes, Proteobacteria, Bacteroidetes also accounted for large proportions in bacteria components. KEGG function analysis indicated that ORFs associated with environmental information processing (membrane) were the most abundant ones among the KEGG pathways. In addition, many reads related to carbohydrate metabolism, genetic information processing (replication, translation and repair), amino acid metabolism and nucleotide metabolism.1590 glycoside hydrolase genes belonged to 80 different glycoside hydrolase families. Besides these,75 glycoside hydrolase genes can't be classified to any glycoside hydrolase families. Among them there were 49 glycoside hydrolase genes belonged to ?-glucosidase. Only 8 glycoside hydrolase genes belonged to endoglucanase. In addition, there were also many genes belonged to proteins related to glycoside hydrolase activities, such as 703 genes coding carbohydrate-binding domains that belonged to 32 families,42 genes belonging to 10 families of polysaccharide lyases, 256 genes belonging to 12 families of carbohydrate esterases and 1636 genes affiliated with 31 families of glycosyl transferases.2. In addition to the large amounts of glycoside hydrolase genes, there were also many other functional genes, such as nitrogenase genes associated with the bacteria of Reticulitermes chinensis. A broad of nitrogenase genes were present in the metagenomic sequences, including 12 nif D genes which encode molybdenum iron protein a chains,16 nif K genes that encode molybdenum iron protein ? chains,7 nif H genes that encode iron proteins. In addition, there were also 12 nif B genes and 11 nifU genes associated with nitrogen fixation.3. From the metagenomic sequencing data a ?-glucosidase (named RcBG3) originated from the intestinal bacteria, which belonged to glycoside hydrolase family 3, was chosed for further study. The expression vector of this ?-glucosidase was constructed and the enzyme was induced to expression. The optimal temperature, pH and reaction time for the activity of the purified ?-glucosidase (RcBG3) were 35?,5.2 and 10min, respectively, with p-nitrophenyl-?-D- glucoside (pNPG) as substrate. The enzyme activity of RcBG3 was 58.447±0.832U/mg protein under optimal conditions. More than 85% of its activity was retained at temperatures between 25? and 45?. However, when temperature was above 50?, RcBG3 rapidly lose its activity. No activity can be detected when the temperature increased to 55?, which means that the enzyme can not tolerate high temperature. RcBG3 had a wide pH ranges, it exhibited more than 60% of the maximum activity in pH range of 3.2 to 8.0. |
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