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

Abstract:

[Objective] 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. [Methods] 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 DpnⅠ-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. [Results] The deduced amino acid sequence showed 72% identity with endo-b-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% relative activity at pH5-9. The optimal temperature was 45℃. The most favorable substrate for the xylanase (XYNG) is Beechwood xylan. Mg2+ and Ca2+ improve the enzyme activity by 33.7% and 16.6%, respectively. EDTA, b- Mercap-toethanol and NaN3 don’t affect the enzyme activity. Tween-80 and DMSO activated enzyme activity by 28.4% and 12.8%. Hg+, in concentration of 5 mmol/L, also inhibited the enzyme activity. [Conclusion] The xylanase gene xynG was firstly cloned from the fungi that caused greensickness of cotton. The xylanase genes containing one intron can be efficiently cloned from plant pathogens and white-rot fungi using strategy in this research. It is unnecessary to explore enzyme ex-pression condition and measure enzyme activity of the original strain. Enzyme character analysis showed that the XynG have potential application in the production of xylo-oligosaccharide and the improvement of bread quality.