| Stem rot caused by Sclerotinia sclerotiorum is an important disease on oilseedrape. At present, we can grow resistant varieties, strengthen the management ofcultivation, and use chemical fungicide treatment for the prevention and control of thedisease. But now, biological control can contribute to the sustainable development ofagriculture, and has attracted more and more attention in recent years. Elicitors canrigger the plant immune system by receptor molecules on the surface of cells, not bysterilization. Theses receptor molecules can trigger signal transduction, as well asexpression of defense genes. The elicitors have many advantages over chemicalpesticide and can be used as a new type of microbe protein pesticide to solveagricultural pest and disease problems.The SsXYN gene is a xylanase gene selected by Sclerotinia sclerotiorum gene chipinformation, which maybe play a role in plant-phytopathogen interaction. Total RNAwas extracted from Sclerotinia sclerotiorum NGA4mycelia and used as the templateto amplify the full length of cDNA of SsXYN gene by RT-PCR, and the gene fragmentwas subsequently cloned into pMD19-T vector. The results of bacterial colony PCR,enzyme analysis and sequencing analysis indicated that the S. sclerotiorum SsXYNgene was successfully cloned. SsXYN gene was digested completely from pMD19-T:SsXYN by BamH I and Sal I, and then was cloned into expression vector pET32a.The results of bacterial colony PCR and enzyme analysis showed the successfulconstruction of expression vector pET32a:SsXYN. After that, the recombinantexpression vector was carried into E. coli BL21by heat-shock transformation, and thestrain of E. coli BL21carrying SsXYN gene was obtained.The pET32a:SsXYN prokaryotic expression vector was transformed intoexpression host strain BL21, then after, IPTG was added to induce expression. Thespliced variant fusion protein was induced and expressed successfully using E. coliprokaryotic expression system and purified by affinity column chromatography.Fusion protein was carried out on SDS-PAGE, and the results showed that the targetband appeared was expectlly. Then, we infected the tobacco plants with the protein,and the results showed that it was induction of the hypersensitive response (HR) orhypersensitive cell death (HCD) in nonhost plants. diminobenzidine (DAB) was usedas selenium organic reagent to study the oxidative burst which was an early event inelicitor-induced defense responses. Elicitor induced by SsXYN can generate a lot ofreddish-brown polymer deposition in15min~3h, which showed that at the same time tobacco produced large amounts of H2O2. After6h, reddish-brown polymerdeposition was sharply reduced, which showed that oxidative bursting abilitydiminished during the later period with time. This result was in conformity with therelevant studies which have shown that the phenomenon of reactive oxygen speciesinduced by elicitor bursting with dual phase characteristics.After injecting the elicitor SsXYN into tobacco in different time periods of0h,30min and6h, the expression of different genes were detected. The accumulation oftranscripts associated with defense and cell redox was examined using semi-quantitiveqRT-PCR, and it was found that the expression of NbPR1a, NbPR2b, NbLOX, EDS1,NbWIPK, NbWRKY2, NbMEK2, NbMAPKKKa, HSR203J were in up-regulation,NbrbohA, NbrbohB, NbNR were in down-regulation. In addition, the expression ofNOA1was not significant.This study was carried out to lay a foundation for reveal the mechanisms ofelicitor induce non-host resistance molecules and the application of elicitor controlSclerotinia sclerotiorum. And it also has an important meaning on instructing practicefor the development of the new bio-pesticides that elicitor as the main component anddesign high efficiency, low toxicity of plant disease control strategy. |
