Eukaryotic Expression And Functional Analysis Of Three Polygalacturonases From Fusarium Oxysporum F.Sp.Nelumbicola

Lotus is one of the important aquatic economic crops in our country,after continous growing of the same crop for a long time,the increased soil born pathogenic fungus causing them to wilt,and the rhizome rot of lotus is particularly serious.The rhizome rot of lotus was caused by the fungus Fusarium oxysporum f.sp.nelumbicola.The pathogenic fungus could infect lotus through the wounded root and stem,and then spread through the vascular bundle up to block xylem conduit.In this study,we investigated the pathogenic role of pg1,pg5 and pgb genes by Pichia pastoris expression system,gene knock-out techniques and subcellular localization.1.In this study,we fabricated eukaryotic expression vectors of PG1,PG5 and PGb to research the enzymatic characteristics and pathogenic analysis of PG isozyme by Pichia pastoris expression system.The result showed the protein molecular weight of PG1,PG5 and PGb were 38 KDa,37 KDa and 53 KDa by SDS-PAGE and Western blot detection.They were consistent with the predictied molecular weight.The optimal pH and temperature for PG activity of the recombinant proteins were investigated.The optimal pH value and temperature of recombinant PG1 activity were found to be pH9.0 and 40?,and the PG1 activity was 3.268 U/mg.The pH value and the optimal temperature of recombinant PG5 activity were pH6.0 and 50?,and the PG5 activity was 3.028 U/mg.The pH value and the optimal temperature of recombinant PGb activity were shown to be pH10.0 and 50?.2.The inoculation of recombinant protein could induce the death of lotus.The lotus plant inoculated with pPICZ?A-pg1 and pPICZ?A-pgb were dead after 4 days,and the lotus plant inoculated with pPICZ?A-pg5 were dead after 7 days.The lotus stem and leaf of diseased and healthy had been to make biopsy,then morphological change of cells was observed.The color of the diseased cells turn to brown,and the cell wall were degradation serious.3.Using eGFP-fusions in transient expression assays in Nicotiana benthamiana,we observed that pg1,pg5 and pgb targeted a wide range of plant cell structures.we used Gateway cloning technology to constructed fusion expression vector pCNH1-eGFP-pg1,pCNH1-eGFP-pg5 and pCNH1-eGFP-pgb.PGb was localized specifically in the nucleus and cytoplasm,but was excluded from the nucleus.PG1 and PG5 were localized in the cytoplasm and were often seen surrounding the nucleus.4.The gene replacement vectors were constructed to produce mutants of F23 a that lacked PG1,PG5 and PGb,respectively.Lotus was inoculated with the 3 PG gene knockout strains and F23 a.Lotus inoculated with mutant F23a?pg1 also all dead,but it slower than inoculated with F23 a.A small part of lotus inoculated with mutant F23a?pg5died while,others started to show disease.Lotus inoculated with mutant F23a?pgb were similar with F23 a.According to our results,knockout of pg1 significantly affected F.oxysporum f.sp.nelumbicola ability to cause disease,while no or less effects were observed when pg5 and pgb were knockedout,suggesting that the function of pg1,pg5,and pgb are redundant.These results suggested that pg1 might play a primary role throughout the pathosenesis role of F.oxysporum f.sp.nelumbicola.