Dynamic Of Bacterial 16S RDNA Of Tiger's Fecal Flora

Tiger gastrointestinal biota is a unknown microbial ecosystem. Unlike most microbial ecosystems, its composition had never been studied. The development of molecular biological techniques has allowed us to study microbial diversity at a new level. Experiments carried out in the first place for its necessity of exploring proper methods and optimization of PCR components that were used in the molecular ecological research. The constituents of tiger's fecal flora were revealed by establishing 16S rDNA clonal library. Development 3 Siberian tigers and 2 white bengal tigers' fecal flora observed using PCR-DGGE technique. Results indicate that, relevant stable band patterns observed during pregnant stage, simultaneously, dynamics of fecal flora might be influenced significantly by the administration of antibiotics and stress.1. Selection of Molecular Ecological Method in Fecal Flora Research of TigerFecal flora of tiger is an unknown micro biota. It was necessary to explore proper method that was used in the molecular ecological research. 5 samples selected from 3 individual Siberian tigers and 2 individual White tigers accordingly. To the same fecal sample, 10 methods were used to lysing bacterial cells and then the nucleic acid were purified by phenol/chloroform procedure, The testing process DGGE was carried out with the product of PCR, which were conducted with the primers that derived from V6-V8 conventional region of bacterial 16S rDNA gene. Results show that using different cell lysing methods obtained different band patterns, ultrasonic and homogenization are methods to use to observe more bands appearing on DGGE gel than those of any of the mechanical methods. Based on results of above, another 5 samples from same individuals were chosen, after the cell lysing procedure conducted by ultrasonic method, five purification methods were used to the same sample to extract the nucleic acid, and checked with the same testing process DGGE. Results shown that the PCR product of bacterial nucleic acid, which was purified with modified CTAB method, obtained stablethe most bands in DGGE. Experimental results can be concluded that, Using an ultrasonic technique to break up the bacterial cells, purifying the nucleic acids from the sample with modified CTAB methods, will standardize the research of the tiger's fecal flora matter.2.0ptimization of PCR Components in 16S rDNA Amplification of fecal flora oftiger(Panthera tigris)Understanding of fecal flora will shed light on intestinal microbiota, which plays an important role in their host's life. In molecular ecological research area, PCR-DGGE is one of the most important methods to investigate into such complicated environmental samples. It is necessary to optimize PCR components due to complication of samples. PCR components in 16S rDNA amplification of fecal flora was optimized in the experiment, and PCR product was visualized by gel imaging system and quantified by using Quantity One Software, and obtained the best value from multi-regression curve. Results show that, with template DNA in the range of 1-10ng, MgCL₂ (25mM) 12 μ L, dNTP8pM, primer 190pM, the yield of PCR product increased comparatively.3 The analysis of Tiger's Faecal Flora Based on Bacterial 16S rDNA SequencesBacterial 16S rDNA library of Siberian tiger fecies has been established in this research. 15 different clones of Siberian tiger were obtained Using EcoR I and Hind III in restricted fragment length polymorphism analysis. The results of Blast analysis showed that, 10 of 15 clones were belonging to Clostridium genera, among which, 6 sequences have 99% similarity with Clostridium novyi type A, and then identified as the same species of Clostridium novyi type A; 4 sequences have 97% similarity with Swine manure bacterium RT-18B, which were identified as Peptostreptococcus members; the other 16S rDNA sequences, all of which have 94-95% similarity with Clostridium pascui, Clostridium tetani E88, Clostridium sp. 14505 Clostridium perfringens and uncultured rumen bacterium clone BS28, which were also identified...