Molecular Ecological Analysis Of Microbiota In Capra Hircus Rumen And Ctenopharyngodon Idellus Intestine And Cloning Of β-Glucosidase Genes

The gastrointestinal(GI) microbiota of animals is characterized by its high population density, wide diversity and complexity of interactions. Importantly, GI microbes have a profound influence on nutritional, physiological and immunological processes in the host. Recently, findings from culture-based methods have been supplemented with molecular ecology techniques that are based on the 16S rRNA gene. These techniques enable characterization and quantification of the microbiota, and also provide a classification scheme to predict phylogenetic relationships. Microbial community structure can be analyzed via fingerprinting techniques. Microbes inhabiting the gastrointestine of phytophagous animals conduct a multitude of biochemical reactions, including degrading of cellulose and hemicellulose. The beneficial effects of a number of fibrolytic enzymes produced by microorganisms have been investigated and confirmed in several areas of animal production. Thus, study on the diversity of microorganisms and fibrolytic enzymes in gastrointestine of phytophagous animals as well as analysis of gene function present the indispensably important significance.This thesis aimed at studying microbial community in animal gastrointestinal samples. Separation of polymerase chain reaction(PCR)-amplified 16S rDNA products using denaturing gradient gel electrophoresis(DGGE) was tested as a means. DNA extraction from the rumen of three species of goats(Copra hircus)(Boer goat, Nanjiang yellow goat, Inner Mongolia cashmere goat) was followed by PCR amplification of 16S rDNA gene, beta subunit of the RNA polymerase(rpoB) gene, and ciliate 18S rDNA gene. PCR products were analyzed by DGGE to compare the predominant bacterial and ciliate community structure. The bacteria community structures of different goats were similar to each other. The similarity of intraspecies was noticeably higher than that of interspecies. Goat species was found to be the factor influencing the rumen microbial community. In contrast, there was considerable variation in the ciliate population among individualities within the same species, and intraspecies similarities were no greater than that of interspecies. The predominant bacterial community structure in the intestine in Grass Carp(Ctenopharyngodon idellus) was also analyzed based on 16S rDNA PCR-DGGE fingerprint. The results showed that abundant bacteria were existed in the intestine. Foregut, midgut and hindgut shared the same most predominant bacteria species, the similarity of the community structures were above 77%. The bacteria species in midgut and hindgut were more than that in foregut, which might reflect the diet fed processed from the forgut to the hindgut. These results suggested that DGGE is a practicable protocol to study the complex community of gastrointestinal microbita.The study also included a correlative research work developing cellulolytic microbiology resources. Fifteen cellulose-degrading strains were isolated from the intestine of Ctenopharyngodon idellus. The species were from genus Shewanella, Aeromonas, Bacillus, Serratia, Klebsiella, Proleus, Enterobacter, and Citrobacter, according to the 16S rDNA genes analysis and BLAST. β-Glucosidase, an important glycoside hydrolase, is implicated in a wide variety of function of cellulose degrading. One set of degenerate primers was designed through homology analysis ofβ-Glucosidase amino acid sequences. A quick cloning and screening method for novelβ-Glucosidase genes was performed by PCR, and the novelty of candidate genes could be evaluated by subsequent sequence analysis. Eleven gene fragments were retrieved from the fifteen isolates. Identity of deduced amino acid sequences to each other ranged from 50% to 98%. The result confirmed the validity of the method of gene cloning and quick screening. By constructing genomic DNA library and screening the library, three full-length gene sequences coding forβ-glucosidase from Bacillus, Aeromonas, Proleus were obtained and named as bgl3, bgl5 and bgl7, respectively. The highest identity of bgl3,bgl5,bgl7 with genes coding putativeβ-glucosidase are 88%, 72% and 82%, respectively. They all have identity with BglA from Clostridium cellulovorans, which have been experimentally verified gene function, are 36.4%, 35.4%, and 34.6%. The result suggested that they are three novelβ-glucosidase genes.