A Priliminary Study Of Molecular Ecological Analysis Of Ciliate From The Rumen Of Buffalos

Objective: We analysis the ciliate 18S rRNA gene diversity from the rumen of buffalos, with the Polymerase Chain Reaction (PCR) and Denaturing Gradient Gel Electrophoresis (DGGE) method.Methods: 1. The effective of rumen microbial DNA extraction method we chosen is Proteinase K digesting method. 2. We optimize the PCR method. As. there are "GC clamp" with the primer, we compared the routine PCR and Touchdown PCR strategy. By the reconditioning PCR strategy, we eliminate the Heteroduplex DNA and single-stranded DNA. 3. The target gene is separated by DGGE. Then the cluster analysis and the phylogenetic analysis are carried.Results: 1. Proteinase K digesting method could obtain rumen microbial DNA efficaciously. 2. With the Touchdown PCR strategy, the aim gene fragment is best amplified, reducing mistake greatly. By the reconditioning PCR strategy eliminating the Heteroduplex DNA and single-stranded DNA, it is more suitable for Denaturing Gradient Gel Electrophoresis. 3. There are complex communities at the rumen ciliate of buffalos, but not observable distinctness at different hosts. According to the 18S rRNA gene homology analysis and the phylogenetic tree analysis, identity of all the ciliate 18S rRNA gene sequences from the rumen of buffalos is above 90%.Identity of the ciliate 18S rRNA gene sequences from the rumen of buffalos and GenBanks' is between 97% to 100%. One is nearly Epidinium caudatum, which the identity is 99.6% And others are not cultivate.