| Corn stem rot occurs frequently in Shandong Province and results in serious yield reduction in recent years.Fusarium verticillioides is the main causing agent of stem rot and research of its pathogenicity mainly focused on toxins,however little is known about cell wall degrading enzyme produced by this fungus.In this study,the xylanase gene FvXyn10 was cloned from Fv7600,a standard strain of F.verticillioides.The gene was transformed into Pichia pastoris GS115 and expressed heterogeneically.The purified FvXyn10 protein was obtained by His Trap HP chromatographic column combined with AKTA protein purification system.The enzymatic properties and the role in pathogenicity were analyzed.The main results are as follows:1.Bioinformatics analysis of xylanase gene FvXyn10Bioinformatics analysis predicted that the length of the open reading frame（ORF）of FvXyn10 gene was 1377 bp,encoding 458 amino acids.The molecular formula is C2286H3476N610O675S18,and the theoretical molecular weight is 50.88k D.FvXyn10 is a hydrophilic protein with stable structure and the isoelectric point is 8.13.FvXyn10 contains20 amino acids,and there are 45 phosphorylation sites and 8 glycosylation sites.This protein contains signal peptide and belongs to secretory protein.There were 222 irregular curls,25β-corners,161 extended chains and 50α-helices in the secondary structure of FvXyn10.2.Eukaryotic expression,isolation and purification of xylanase FvXyn10Fv7600 was inoculated into stems of corn seedling,and total RNA was extracted from infected tissues 3 d postinoculation for RT-PCR to obtain the complete fragment of FvXyn10gene.After sequence determination and verification,the expression vector p PIC9K was constructed.Pichia pastoris GS115 was transformed by electric shock and strains 10-9 with high copy plasmid number were obtained.After heterologous expression of the 10-9,the crude enzyme solution was separated and purified by ammonium sulfate precipitation,dialysis and nickel column chromatography.Pure protein about 60 k Da was obtained by SDS-PAGE electrophoresis,which is larger than the predicted molecular weight of FvXyn10（50.88 k Da）.Glycoprotein electrophoresis confirmed that xylanase FvXyn10 was a glycoprotein,which increased the molecular weight of the protein.3.Study on enzymatic properties of FvXyn10Enzymatic properties were tested and the results showed that the optimum p H of xylanase FvXyn10 was 5.0,and the activity of xylanase Fvxyn10 was 87%,59%and 55%at p H8.0 to p H10.0 respectively.The optimum reaction temperature of FvXyn10 was 50℃,and the activity of FVXYN10 was 80%,76%and 74%at 60℃to 80℃.Metal ions showed different effects on the activity of FvXyn10.Mg2+at 1 m M can improve the relative enzyme activity of FvXyn10,but 5 m M Cu2+can completely lose the enzyme activity of Fvxyn10.The substrate specificity test showed that FvXyn10 could hydrolyze xylan significantly,but had no activity on dextran,pectin and other substrates,indicating that FvXyn10 had strong substrate specificity.Thin layer chromatography analysis showed that FvXyn10 could hydrolyze xylan into xylo-oligosaccharide,in which xylodisaccharide and xylotriose were the main hydrolyzed products.Methanol,ethanol,isopropyl alcohol and other organic solvents also showed inhibitory effects on the activity of FvXyn10 at different degree.4.Pathogenicity detection of FvXyn10The pathogenicity of xylanase FvXyn10 in corn was preliminarily analyzed and necrotic spots on corn leaves was observed at the injection site after 72 h,which was injected with 30μM pure enzyme.The area of necrotic spot gradually expanded with the increase of protein concentration.These results indicated that FvXyn10 may be involved in the pathogenicity of Fv7600 against corn,but the detailed mechanism needs further study. |
