PMID: 18720841Aug 30, 2008Paper

Molecular cloning and heterologous expression of a new xylanase gene from Verticillium dahliae

Wei sheng wu xue bao = Acta microbiologica Sinica
Guimin ZhangXian'en Zhang

Abstract

Fungus Verticillium dahliae caused greensickness of cotton and xylanase is necessary in this pathogenesis. Cloning xylanase gene from V. dahliae and heterologous expression might obtain new xylanase. By comparing the amino acid sequences of over 10 xylanases in 11 families from fungi through BLAST, we found 2 highly conserved regions, with a fragment of about 150 amino acids coding sequence in between. Degenerate primers complementary to the ends of these two conserved regions were designed to amplify the in-between sequence from V. dahliae. The whole xylanse gene containing intron was achieved by Genome-walking PCR method. A 63 bp intron was found through BLAST, the whole cDNA xynG was cloned by Dpn I -mediated PCR to delete intron. The cDNA was inserted into pHBM905 and expressed in Pichia pastoris GS115, xylanase-secreting transformants were selected on plate containing RBB-xylan. The transformant with the largest halos was selected for study the character of xylanase. The deduced amino acid sequence showed 72% identity with endo-beta-1, 4-xylanase from Cochliobolus carbonum and C. sativus in the GenBank, which means xynG is a new xylanase gene. The optimal pH of the purified recombinant enzyme was pH6. It remains over 50% r...Continue Reading

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