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Cloning and prokaryotic expression of a xylanase gene of Valsa mali var.mali

Xiangpeng Shi;Baohua Li;Caixia Wang

  Valsa mali var.mali(Vmm),a destructive pathogen of apple tree,secretes a series of cell wall-degrading enzymes(CWDEs) in the infection process,in which the xylanase showed the highest activities and most powerful macerating ability to apple tissues.To better understand the role of xylanase in the pathogenicity of Vmm,in this study,a gene encoding xylanase,Xylanase Ⅱ,was cloned and identified.The full-length cDNA is 969 bp containing a 5 -untranslated region(5 -UTR) of 135 bp and a 3 -untranslated region(3 -UTR) of 153 bp.The open reading frame(ORF) of681 bp encodes a protein of 226 amino acid residues with a predicted molecular weight of 23.8 kD and an isoelectric point of 5.29,and its predicted molecular formula is G_(1052)H_(1620)N_(272)O_(352)S_4.No putative N-glycosylation site(Asn-Xaa-Thr/Ser) was found.It was identified as a family 11 of glycosyl hydrolase by running a Conserved Domain Search at the National Center for Biotechnology Information(NCBI) website.Amino acid sequences showed the presence of conserved residues at the active site,Glul22 and Glu213 may be catalytic residues,and Trpll3 and Trpl24 may be involved in substrate binding.Prokaryotic expression results showed that efficient expression of pETXylanase Ⅱ protein could be realized after induced for 10 h at 15℃ with 0.1 mmol/L IPTG in Escherichia coil Rosetta.Solubility analysis showed that the fused protein mainly existed in the soluble fractions.Western blotting confirmed that the molecular weight of the recombinant pET-Xylanase Ⅱ was appropriately 42 kDa,consistent with the predicted result.The enzyme activity of the recombinant protein was performed,and high xylanase activates were observed when reaction temperature was50℃ and reaction time was 30 min.In the present study,Xylanase Ⅱ of Vmm was successfully identified,and prokaryotic expression protein with high activity was achieved,which provide a basis for better elucidating Vmm pathogenicity mechanisms and lay a foundation for developing more effective disease-management strategies.……