Construction and analysis of rumen bacterial artificial chromosome library from a dairy cow rumen microflora

Abstract:

The high molecular weight DNA was extracted and purified directly from rumen samples in the study by using culture-independent and pulsed field gel electrophoresis approaches. After digestion with HindⅢ, DNA fragments ranging from 50-100kb was collected and ligated to pCC1BAC vector. The ligation mixture was transformed into E.coli EPI300 and a rumen metagenomic BAC library with about 15360 clones was constructed. The average insert size is about 54.5kb, mostly ranging from 50-70kb, and the capacity of this BAC library is about 837Mb. Several BAC clones with activity of amylase,Cmcellulase had been screened from the BAC library.The clones with Cmcelluase activity were screened further for linchenase, xylanase, cellobioase activity and the result is that 25 of them have at least one kind of other enzyme activity.