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

• Published in: Physiological and molecular plant pathology 1997-12, Vol.51 (6), p.377-389
• Main Authors: LÜBECK, P. S, PAULIN, L, DEGEFU, Y, LÜBECK, M, ALEKHINA, I, BULAT, S. A, COLLINGE, D. B
• Format: Article
• Language: English
• Subjects: Ascochyta pisi
• ISSN: 0885-5765; 1096-1178
• DOI: 10.1006/pmpp.1997.0126

A gene encoding a xylanase, xyl1, was isolated from the phytopathogenic fungus Ascochyta pisi Lib. by PCR cloning using degenerate primers. DNA sequence analysis revealed an open reading frame of 736 bp interrupted by an intron of 55 bp. The ORF encodes a predicted protein of 227 amino acids. The precise splicing site of the intron was identified from the sequence of a PCR product obtained using the same degenerated primers on a cDNA template. The cDNA product and a northern blot demonstrated that the gene is transcribed into mRNA when the fungus is cultured in media containing xylan as sole carbon source. The Neighbour-Joining method using the Clustal W(1.5) program demonstrated that the A. pisi xylanase is a member of the family 11 glycosyl hydrolases, and that this family represents at least five phylogenetically consistent groups. The family 11 glycosyl hydrolases can be linked with family 10 glycosyl hydrolyses through bifunctional enzymes from Ruminococcus flavefaciens and, to a lesser extent Neocallimastix patriciarum .