Identification of anaerobic fungi from large ruminants (Bubalus bubalis L. and Bos taurus L.) and molecular cloning of rumen-associated gene
2016
Sabinay. S.G.
A modified repeated beat-beating (RBB) method was developed and evaluated by comparing it against four routinely used method designed for soil, bacteria and plant samples. The modified RBB method facilitates the concentration of microorganisms from rumen digesta and employs the use of bead-beating in the presence of high concentrations of sodium dodecyl sulfate (SDS), salt, and EDTA, with subsequent purification of ammonium acetate and isopropanol. This method resulted in a 2.2-to 4.4-fold increase in DNA yield when compared to four other widely used method. The community DNA recovered has an estimated maximum size of 9.4-to 23 kb and was pure enough for restriction digestion and PCR amplification making it deal for metagenomic library construction and microbial diversity studies. In addition, the method developed in this study does not use hazardous substances such as phenol and chloroform nor does it utilize expensive spin columns for purification. PCR - based cloning using nu-SSU-0817-5' and nu-SSU-1196-3' primers generated approx 420-bp amplicons representing 18S rDNa using genomic DNA from rumen digesta. A total of twenty-one clone sequences were generated with six clones derived from cattle and fifteen clones derived from carabao. DNA sequence analysis of the clones show high similarity with anaerobic fungi. Neocallimastix, Orpinomyces, and Piromyces were found in both ruminants. Cyllamyces was unique to cattle and two carabao clone sequences formed unidentified clades. A putative cellulase gene from metagenomic DNA of rumen digesta was detected by direct PCR. BLAST analysis revealed high similarity but low query coverage of 11-and 7.7% respectively, for fungi and bacteria. Further analysis indicated the presence of seven open reading frame (ORF). Based on MotiFinder, only ORF 2 contains a protein domain with similarity to ABC 3 transport protein. This protein is closely associated to cellulase genes of cellulolytic microorganisms.
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