Construction And Analysis Of Rumen Bacterial Artificial Chromosome Library From A Holstein Dairy Cow Rumen Microflora

It has been proved that the rumen microbial is very complex. But recent progress in molecular microbial ecology has reveled that traditional culturing methods fail to represent the scope of microbial diversity in rumen, since only less than 11% of viable microorganisms are recovered by culturing techniques. To develop methods to investigate the full extent of microbial diversity, the bacterial artificial chromosome (BAC) vector was used to construct libraries of genomic DNA isolated directly from rumen content.It is the key for constructing the BACs to gain the pure high molecular DNA is. But many polysaccharides and lipids are so hard to be removed that ruin the digestion and ligation. We developed a series of ways to clean the cell and make plugs and finally we have gotten the Rumen HWM DNA with the size in excess of 2Mb. After digested with HindIII and runed PFGE, the DNA fragments ranged from 50-100kb were collected and ligated to pCC1BA vector. The ligation mixture was transformed into E.coli EPI300 to construct library. The rumen metagenomic BAC library contains about 15360 clones and the average insert size of BAC clones was estimated to be 54.5 kb, mostly ranging from 50-70kb. Based on the data mentioned above, the capacity of this BAC library is about 837Mb. The BAC clones show good stability after cultured for 100 generations.A functional screen of the library for amylase and Cmcellulase activity was conducted using starch agar plates and Cmc-Na agar plates. Twenty-six clones with Cmcelluase activity and sixteen clones with amylase activity were detected. The clones with Cmcelluase activity were screened further for linchenase, xylanase, cellobiase activity and the result is that 25 of them have at lest one kind of other enzyme activity. The insert of two clones were sub-cloned and sequenced. Both of them have Open reading frames (ORF) that encoded putative protein with identity to cellulase by reference to GeneBank databases. Other putative genes were detected, and three genes in clone 21# have relative function to cellulose metabolism.