Basic Study On Diversity Of Bacterial Community Of Intestinal Tract In Goose

Goose intestinal microbe is a complicated ecological organization, which plays an important role in health of goose and in digesting and absorbing nutrients. At present, studies on poultry intestinal microbe are rare,and there's no report on goose intestinal microbe. With the development of molecular biology, SSCP, DGGE and TGGE have been widely used. In this study, 8 methods were used to lysing bacterial cells and then the nucleic acid were purified by phenol/chloroform procedure; the optimal conditions were obtained by PCR-SSCP; intestinal microbial flora of goose had been isolated and identified by PCR-DGGE. The conclusions were drawn as follows:1. Among DNA extracted methods, it was found that the method combined antalzyme and freeze thawing for several times could successfully extract DNA from sediments. Add PVP and differential centrifugation could remove pigment and other foreign materials, then reduced the interference to the DNA extraction. The key to extract successfully was to consider the final concentration of SDS, CTAB, antalzyme and Proteinase K.2. Many factors, such as the gel-concentration, the concentration of cross-linker, the sample quantity of the PCR products, the presence of glycerol, urea and Lambda exonuclease in gels during electrophoresis, the electrophoresis temperature, voltage etc, could influence the results of SSCP. The optimal conditions were as follows: the gel-concentration was 12% (acrylamide-bisacrylamide (39:1), no urea, no glycerol), during electrophoresis, the voltage was 120V, with constant temperature.3.According to the optimal conditions,DNA was extracted from cecum and analysised the bacterial community by PCR-SSCP. Slice the clear bands and obtain an uncultured bacteria in cecum by cloning and sequencing.4. The primers designed by conservative sequences of 16S rRNA gene of E.coli was used to amplificate intestinal bacterial DNA from goose, and then amplificational products were separated directly by DGGE. More than 100 bands were obtained with DGGE in the denaturant range of 40%-60%, among which 73 bands were much brighter and 41 bands were separated at last. The results of blast analysis showed that, goose intestinal microbe has 94%-99% similarity with Pseudomonas, Bacterium, Burkholderia sp., Kocuria sp., Rothia sp., Gamma proteobacterium, Endophytic bacterium, Clostridium colinum, Bifidobacterium sp.. It showed that 16S rDNA sequence combined DGGE analysis was effective method for goose intestinal bacterial diversity study.5. The two methods obtained different band patterns, each had selectivity and limitations. Compared with SSCP, DGGE could better separate goose intestinal microbe and standardize the research of the goose intestinal microbe.