PCR cloning, DNA sequencing and phylogenetic analysis of a xylanase gene from the phytopathogenic fungus Ascochyta pisi Lib.

摘要

PCR cloning with degenerative primers was used to isolate a xylanase encoding gene, xyl1. DNA sequence analysis revealed an open reading frame of 736 bp interrupted by an intron of 55 bp. The ORF encoded a predicted protein of 227 amino acids. The sequence of a PCR product obtained using the same degenerated primers on a cDNA template was used to identify the precise splicing site of the intron. The cDNA product and northern blotting suggested that the gene was transcribed into mRNA when A. pisi was cultured in media containing xylan as the sole C source. Neighbour-Joining analysis using the Clustal W(1.5) programme demonstrated that the A. pisi xylanase is a member of the family 11 glycosyl hydrolases, and that this family represents at least 5 phylogenetically consistent groups. The family 11 glycosyl hydrolases are linked with family 10 glycosyl hydrolyses through bifunctional enzymes from Ruminococcus flavefaciens, and to a lesser extent Neocallimastix patriciarum.