PCR cloning, DNA sequencing and phylogenetic analysis of a xylanase gene from the phytopathogenic fungusAscochyta pisiLib. |
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| Authors: | PS Lübeck L Paulin Y Degefu M Lübeck I Alekhina SA Bulat DB Collinge |
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| Institution: | aPlant Pathology Section, Department of Plant Biology, The Royal Veterinary and Agricultural University, Thorvaldsensvej 40, 1871 Frederiksberg C, Copenhagen, Denmark;bInstitute of Biotechnology, DNA Synthesis and Sequencing Laboratory, P.O. Box 56, FIN-00014, University of Helsinki, Finland;cDepartment of Plant Biology, Plant and Forest Pathology, P.O. Box 28, FIN-00014, University of Helsinki, Finland;dKomarov Botanical Institute, Laboratory of Fungal Biochemistry, St Petersburg, 197376, Russia;ePetersburg Nuclear Physics Institute (PNPI), Gatchina, 188350, Russia |
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| Abstract: | A gene encoding a xylanase,xyl1, was isolated from the phytopathogenic fungusAscochyta pisiLib. 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 theA. pisixylanase 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 fromRuminococcus flavefaciensand, to a lesser extentNeocallimastix patriciarum. |
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