Proteins And Genes Expression Difference Of Induced Systemic Acquired Resistance In Wheat Against Powdery Mildew

The powdery mildew disease caused by Blumeria gramminis.DC.f.sp.tritici is one of themost important diseases of wheat. Accompanying with the resistance loss of cultivarcontaining Pm resistance gene, wheat may face epidemic risk of powdery mildew in China.Now people are looking for new methods for the disease control, the most important advanceis systemic acquired resistance(SAR),SAR is defined as an increase in the resistance levelwithout any change of genetic constitution, only due to the first inoculation or chemicalinduction. In essence, the change of resistance level reflects the expression differences ofgenes and proteins. Study on the protein and gene expression profiles during SAR can revealthe molecular mechanism of systemic acquired resistance. In this research, we examined the genes and proteins expression profiles of wheat leavesobtaining systemic acquired resistance. the susceptible wheat cultivar XiaoYan.No.6 was usedas host plant, the pathogen (Blumeria gramminis.DC.f.sp.tritici)was a mixture strains. Wecompared the induction effects between Benzothiadiazole(BTH) , β -aminobutyricacid(BABA),salicylic acid(SA),potassium chloride(KCl),Oxalic acid against the powderymildew. The results showed BABA and BTH had the stronge inducing effects against thepathogen. 0.4mmol/L BTH and 10mmol/L BABAcan induce the systemic acquired resistancemore effectively, the relative defense effects was about 60% and 50% respectively. The mostefficient induction interval for BTH and BABA were 2 days and 2-4days respectively. Study on inducing effects on defense-related enzymes showed that the first and secondleaf treatment with BTH and BABA can systematically induce the increase ofperoxidase(POD), phenylalanine ammonia-lyase(PAL),Endo-chitinase,Exo-chitinase and β-1,3-glucanase activity in the third leaf, inoculation also can induce the increase of thesedefense-related enzymes activity. This may be indicate the leaves have the same reactionmechanism for the induction and inoculation, defense-related enzymes were involving in theexpression of induced resistance, Different activity profiles of defense-related enzymesinduced by BTH and BABA reflected that the two inducers had some difference in inductionsystemic acquired resistance. In this research we established a novel method for preparationof the chitinase substrate －colloidal chitin(patent pending). In further research BTH was used as resistance inducer, the protein profiles werestudied by sodium dodecylsulfate polyacrylamide gel electrophoresis(SDS-PAGE). Theresults showed that BTH-treated leaves and inoculated leaves can both express four newproteins and increase one kind of protein content in intercellular washing fluid, two proteinswas purified by preparative electrophoresis through potassium chloride negative staining, gelcut and electro-elution. The purified proteins were analyzed by SDS-PAGE, only single bandwas presented for each protein, their molecular weight was 19.2kD and 35.7kD respectively.This method laid the techniques basis for plant pathogenesis-related proteins research. Theprofiles of SDS-PAGE showed there were no difference expressed protein existing inintracellular protein of wheat leaves, this may be the result of low resolution of onedimensional electrophoresis. The gene expression differences were assayed by suppression subtractivehybridization(SSH), the third BTH-treated leaves after inoculation 96 hours was served astester, the third non-treated leaves after inoculation 96 hours was served as driver. The totalRNA was isolated by Qiagen RNA mini Kit, the double stranded cDNA was synthesized byClontech Smart cDNA systhesis techniques. RsaⅠ digestion, adaptor ligation, liquidhybridization and nested PCR were performed according to the protocol of PCR select cDNAsubtraction kit with some modifications. A subtractive library was constructed by ligated thenested PCR products into pGEM T easy vector(Promega),transforming the ligat...