The Cloning Of McrA Gene And Its Application In The Analyzing Methanogen Diversity In Biogas Digesters

The traditional methods on environmental methanogen research are time-consuming and need enormous efforts. Modern molecular biotechnology which is developed recently,has avoided these shortages and play an increasingly important role in the study of environmental microbiology.Our work improved the DNA extraction method.Through the test,it can meet our requirement. This research use the method of RFLP analysis depending on the mcrA gene which is specific to Methanogenic archaea, the real-time quantitative PCR analysis of mcrA gene and DGGE analysis of 16S rDNA to analysis the methanogenic archaea's community structure and quantity in the rural biogas digesters. We amplifyed the methyl coenzyme M reductaseαsubunit(mcrA)gene, established its gene library and did the RFLP analysis of the positive clones digesting by restriction enzyme PstⅠ. We got 3,4,4,2 species operational taxonomic units (OTU) in the No.1,No.2,No.3 and No.4 biogas digester respectively. After sequencing the selected positive clone in every unique OTU, we did its phylogenetic analysis. As a result, there are different methanogenic archaea populations exist in the rural biogas digesters.In No.1 biogas digester there are Methanogenium,Methanocorpusculum and Methanosarcina. In No.2 biogas digester there are Methanogenium,Methanocorpusculum,Methanobacterium and uncultured methanogen;In No.3 biogas digester there are Methanospirillum,Methanobacterium and uncultured methanogen. In No.4 biogas digester there are Methanogenium and Methanobacterium.We did DGGE analysis of methanogenic archaea 16S rDNA partial gene through Touch-down PCR. The results showed that there are different methanogen populations exist in the normal biogas digesters and varying degrees. However Methanosaeta and Methanoculleus existing in all biogas digesters, also there exists uncultured euryarchaeote spp.. Although DGGE and RFLP analysis are all aimed at methanogenic specific DNA sequences, the results from comparative analysis between the two methods showed the mcrA gene seems to be more powerful in this study.In addition, we used the real-time quantitative PCR to analysis the methanogenic archaea's quantity. Designed the specific primers qMCR-F/qMCR-R to the methanogen mcrA gene. Construct standard curve according to the standard plasmid. Quantify the absolute number of methanogens appeared in the biogas digesters. The results showed that the number of methanogen in these biogas digesters were varies from 105 /mL to 108/mL.