Phylogenetic Diversity Of Reductive Acetogenic Bacteria And Expression Of Bacterial FTHFS Genes In The Gut Of Reticulitermes Chinensis Snyder

Wood-feeding termites can degrade lignocellulose efficiently and play important roles in material recycling in nature and the improvement of soil physical and chemical properties. All these functions are closely related with termites intestinal symbiotic microorganisms. Many symbiotic microorganisms in the gut of wood-feeding termites can use CO2and H2to synthetize acetic acid, which is the most important energy source for termites. Therefore, acetogenesis can profoundly affect the nutritional metabolism in wood-feeding termites. Reticuliterm.es chinensis Snyder is a wood-feeding lower termite, which distributes in most south areas of the Yangtze River in China. It is a good model for the study of the symbiotic association between termites and their intestinal symbiotic microorganisms in cellulose degradation. Methylenetetrahydrofolate reductase (FTHFS) is the key enzyme in the metabolic pathways of acetogenesis bacteria for synthetizing acetic acid using CO2produced in cellulose degradation process, which can be used as a molecular marker. In this study, we analyzed the diversity of intestinal symbiotic acetogenic bacteria of R. chinensis by constructing DNA clone library of FTHFS genes and the expression of FTHFS genes by establishment of the cDNA clone libraries of FTHFS genes. Furthermore, the expression of FTHFS genes was studied by T-RFLP. The main results of this study are as follows:1. FTHFS gene library of symbiotic acetogenic bacteria of R. chinensis was constructed with the intestinal DNA as template. A total of32FTHFS genes of symbiotic acetogenic bacteria were obtained from216random selected clones. Phylogenetic analysis indicated that most of the acetogenic bacteria were affiliated with Spirochetes, a smaller proportion was Firmicutes. In addition to that, there were still some unidentified bacteria.2. FTHFS gene library of symbiotic acetogenic bacteria of R. chinensis was established with the intestinal cDNA as template. A total16FTHFS genes of symbiotic acetogenesis bacteria were obtained from96random selected clones. Phylogenetic analysis showed that the main groups of symbiotic acetogenic bacteria were Spirochetes, some were unidentified bacteria.3. T-RFLP analysis of FTHFS genes amplified with5’FAM labeled FTHFS-F primer based on intestinal DNA and cDNA templates showed that there were more T-RFs in the sample of DNA (7peaks) than in the sample of cDNA (3peaks), indicating that there was an obvious difference between the diversity of FTHFS genes based on DNA library and cDNA library. The results showed that only part of bacteria especially Spirochaetes with FTHFS genes can express the gene and participate in the production of acetic acid, indicating that symbiotic Spirochaetes play important roles in the metabolism of R. chinensis.