Analysis Of Three Kinds Of Antioxidase Genes Response To Hydrogen Peroxide Stress In B.Xylophilus

The pine wood nematode(Bursaphelenchus xylophilus)is a very dangerous forest pest that seriously endangers pine forests in China.The molecular mechanism of its pathogenicity remains to be further studied.In the pine wood nematode pine interaction system,overcoming the H2O2 stress produced in pine is one of the key processes for the successful infection of pine wood nematode.How pine wood nematodes respond to and overcome H2O2 stress,especially how to coordinate the antioxidant enzymes of pine wood nematodes to respond to H2O2 stress,is a key basic biological problem in pine wood nematode pine interaction system.On this basis,three genes Bx-SOD-1,Bx-sod-2 and Bx-SOD-4 encoding superoxide dismutase were cloned from pine wood nematode,and then the transcriptional response patterns of three kinds of five genes encoding antioxidant enzymes to H2O2 stress were studied.In addition,the key role of BxPrx gene in the regulation of pine wood nematode against H2O2 stress was verified by gene silencing technology.The main research contents are as follows:1.Through calculating the mortality of pine wood nematode under different concentrations of H2O2 stress,it was proved that the survival rate of pine wood nematode decreased with the increase of H2O2 concentration under H2O2 stress of 0,2.5,5.0,7.5 and 10.0 mmol·L-1 concentration.This indicated that the survival rate of pine wood nematode was negatively correlated with the concentration of H2O2.2.Three genes Bx-SOD-1 Bx-sod-2 and Bx-SOD-4encoding superoxide dismutase were cloned by searching the whole genome of pine wood nematode.Through the analysis of sequence characteristics,it was found that the three genes encoding SOD of pine wood nematode had relatively conservative sequence and molecular structure characteristics with the homologous genes of other nematodes,which indicated that the three genes encoding SOD of pine wood nematode might have similar functions with the homologous genes of other nematodes.3.The transcriptional response patterns of 5 antioxidant genes of 3 species of pine wood nematode under H2O2 stress were examined by fluorescence quantitative PCR.The results showed that the transcription level of Bxy-ctl-1 encoding catalase and BxPrx encoding peroxidase increased significantly under H2O2 stress,indicating that Bxy-ctl-1,Bx-SOD-1,Bx-sod-2 and Bx-SOD-4,which encode superoxide dismutase,showed a significant decrease in the transcription level under H2O2 stress,indicating that the three genes encoding SOD were negatively responsive to H2O2 stress.It can be concluded that these three kinds of five antioxidant genes may play key role in the resistance of pine wood nematode to H2O2 stress.4.The results showed that dsRNA of BxPrx gene produced by specific amplification primers could significantly reduce the transcription level of BxPrx gene,and the gene silencing of BxPrx significantly reduced the survival rate of pine wood nematode under H2O2 stress.These results indicate that BxPrx gene is one of the key genes to regulate the resistance of pine wood nematode to H2O2 stress.BxPrx is one of the potential targets to control pine wood nematode,and it is worth further study in the future.