| Fusarium spp.,especially F.verticillioides was reported as the main causal agent of maize stalk rot but was varied in different areas.Plant cell wall is the primary protective barrier of plant cell and pathogenic fungi produce an array of cell wall degrading enzymes enabling cell penetration and spread through plant tissue.F.verticillioides can secrete large amount of xylanase in infected corn stem,but little is konwn about the enzymatic properties and its role in pathogenesis.In this study,we firstly idenfied F.verticillioides as the main causal agent of maize stem rot in Tai’an.To learn enzymological properties of the xylanase and its role in pathogenicity we subsequently cloned and expressed xylanase gene Fv Xyl11 heterologously from standard strain of F.verticillioides Fv7600,the main results are as follows:1.Identification of Fusarium spp.as corn stalk rotSamples of stalk rot were collected from 18 corn feilds in Tai’an city,and 183 strains were isolated.Based on morphology and molecular characteristics,76 strains were identified as F.verticillioides,45 strains as F.graminearum and 20 strains as F.proliferatum with the frequencies was 41%,24%and 11%respectively.2.Cloning and eukaryotic expression of xylanase gene Fv Xyl11Based on g DNA sequence of Fv7600 from Gen Bank,a total of 15 xylanase genes were found.Using RT-PCR,expression of the xylanase gene were tested in maize stems inoculated with Fv7600 and Fv Xyl11 was found expressed highest among 15 xylanase genes.To clone Fv Xyl11,m RNA was extracted from Fv7600,c DNA was obtained by reverse transcription,and a segment about 790 bp was obtained by PCR amplification subsequently.After sequencing the segment were ligated with plasmid p PIC9K which has His-tag sequence at N-terminal.Then the recombinant plasmid was transformed into Pichia pastoris strain GS115.After PCR and G418 resistance screening,the recombinant protein Fv Xyl11 was obtained.The purified protein Fv Xyl11 about 23 k Da was obtained by protein separation and purification from engineering strain X11-48.Using PAGE electrophoresis sugar staining,the 23 k Da protein was confirmed as glycoprotein.3.Study on enzymatic properties of Fv Xyl11To investigate the enzymatic properties of Fv Xyl11,the study analyzed the effects of temperature,p H,substrate,and metal ions on enzymatic hydrolysis activity.Results indicated that the optimal reaction temperature for the xylanase was 60℃and the optimal reaction p H was 4.0.Fv Xyl11 demonstrated strong substrate specificity and utilized xylan as a specific substrate.The impact of various metal ions on enzyme activity was also evaluated.Notably,Mg2+,Na+and K+at concentrations of 1 mmol/L promoted enzyme activity,while Cu2+,Ca2+,Mn2+and Zn2+inhibited the enzyme activity.Furthermore,EDTA（1 mmol/L）,Isopropand（1%）,and SDS（1%）inhibited enzyme activity.Thin layer chromatography was utilized to analyze the hydrolyzed products of xylan degradation by Fv Xyl11.The study found that the primary hydrolyzed products produced by Fv Xyl11 during xylan degradation were oligosaccharides characterized by a low polymerization degree.Furthermore,the yield of oligosaccharides clearly increased as the reaction time was prolonged.4.Functional analysis of xylanase Fv Xyl11Upon inoculation of xylanase Fv Xyl11 into maize leaves,the necrotic degree and area of the lesion gradually increased with an increase in concentration.An effective protein concentration of 50μmol/L was used to inoculate maize leaves and the progression of maize leaf disease was observed for five days.Results revealed diseased spots at the inoculation site with initial water immersion which gradually turned yellow over time.Additionally,Fv Xyl11was found to induce overexpression of the PAL,POD,and PR1 genes.The results of Trypan blue staining showed that the Fv Xyl11 xylanase gene could induce cell death in Nicotiana benthamiana.DAB staining results showed that Fv Xyl11 protein caused active oxygen outbreak in tobacco leaves after soaking tobacco for 30 min. |
