Metagenomic Library Construction And Microbial Diversity Investigation In The Rumen Of Miscanthus Feeding Cattle

There are a large number of symbiotic microorganisms in the gastrointestinal tract, however, the research for them has been subject to pure culture technology. With the rapid development of molecular biology and its application in microbiology and ecology, a new subject for uncultivated microorganism has been established and being rapidly developed-Metagenomic. Metagenomic library was constructed using DNA extracted from microorganisms of gastrointestinal mucosal surface and the contents, and genetic composition and functions of microbial communities of gastrointestinal mucosal surface and the contents are also investigated using genomics research strategy.High molecular weight DNA(HMW DNA) of the microbe in the rumen of cattle fed with miscanthus was was isolated using a combination of chemical and enzymatic lysis within a low-melting-point agarose plug in situ。Microbe metagenomic DNA could be restriction digested with Hind III in the control reactions, the partially digested HMW DNA ranged in size from50kb to150kb was size selected using pulsedfield gel electrophoresis. After electroelution and dialysis, the size-selected genomic DNA was then ligated into a commercially prepared bacterial artificial chromosome (BAC) vector, the ligation mixture was heat inactivated, dialyzed against doubledistilled water, and then electroporated into highly electrocompetent Escherichia coli cells to constructure BAC library.The BAC library consists of3,264BAC clones and the average insert size is about50kb. The capacity of inserted DNA is about163.2Mb. To screening celluase genes in the BAC library,we use two different screening culture mediums to select exocelluase and beta glucosidase gene. Finally,21clones expressing beta glucosidase activity are isolated, and laid the foundation for further study of the genetic resource about cellulose degradation like β-Glucosidase in rumen. The influence of diets on microbial structure and functions in host poorly understood. This study aimed to investigate the variation of the stomach microflora in hosts fed with different diets by correlating microbial diversity with the denaturing gradient gel electrophoresis(DGGE) and restricted fragment length polymorphisms(RFLP) methods. In this study, the experiment group was fed with miscanthus only, and the control group was fed with mixed diets. The samples were collected from the rumen, reticulum, omasum and the abomasums, respectively.Using touchdown PCR, we amplificated the16S rRNA V3of bacteria with primers341F/534R and18S rDNA of fungi with primers NS1/GCFungi from the samples. Then the amplified DNA fragments were discriminated by parallel DGGE. At the same time, the16S rDNA was amplificated with specific primer27F/1492R, and then digested with Msp Ⅰ and detected by layering native PAGE. The maps from the DGGE and RFLP were analysised by bio-rad Quantity one, and the datas were statisticsed by SPSS.The results indicated that miscanthus feeding only form environment pressure on the microorganism in cattle stomach, which affect the microbial community, especially for the bacteria community.