Function Analysis Of Matrine Resistance Genes In Foreign Matter Metabolism Pathway Of Bursaphelenchus Xylophilus

Bursaphelenchus xylophilus is a plant-parasitic nematode,which is mainly spread by insects such as the Coleoptera vector.In 1971,a large number of pine trees died in Japan,which attracted worldwide attention.Pine wilt disease is a highly harmful forest disease in the forest ecosystem of the world.Since pine wood nematode was first discovery from Nanjing Zhongshan Mausoleum in 1982,Chian had made every effort to prevent,but the loss of ecological value was as high as 100 billion yuan.Chemical control is the major and effective way to prevent and control pine wood nematode.while,drug resistance and causes environmental pollution might be produced during the long-term control process.,Human beings have gradually increased their awareness of environmental protection whit the development of science and the progress of society.Therefore,searching for effective and non-polluting effective botanical agents has become the current research focus.In the present study,the pine wood nematode was treated using the matrine stress,and the appropriate concentration of pesticides was screened out.The enzyme activity detection,RNAi,and other technologies were applied.The new ways to control pine wood nematode were explored using the resistance-related functions of the essential genes BxCYP33C9,BxCYP33C4,BxCYP33D3,BxCYP33C4,BxCYP33D3,BxGST3,BxGSTl,BxPGP23.The main results are as follows:1.The virulence of pine wood nematode was determined.First,the pine wood nematode was cultivated and multiplied by Botrytis cinerea.Then,the method of Bellman funnel was used to collecte pine wood nematode(the experimental strain).Finally,compared to the control group,the survival of the experimental strain was gradually decreased with the increase of time and concentration.The concentrations of matrine were 2.308 mg/mL(LC50)and 1.581 mg/mL(LC50)at 48 h and 72 h,respectively.2.The technology of PCR was applied to clone the genes,including BxCYP33C4,BxCTP33C9,and BxCYP33D3 of P450 family genes,BxGST1 and BxGST3 of glutathione S-transferase genes,and BxPGP23 of ABC family genes in the foreign body metabolism pathway of pine wood nematode.Meanwhile,the BxPGP23 protein was cloned and analyzed by Bioinformatics.The results of the physical and chemical properties of the BxPGP23 protein indicated that the length of the Bx-pgp23 gene was 3900 bp and the encoded Bx-PGP protein was composed of 1300 amino acids.The molecular formula of the protein was C6463H10217N1733O1899S61,the molecular mass was 144.5373 kDa,and the isoelectric point was 6.56.The stability factor is 38.31,which was suggested that BxPGP23 was a stable protein.The fat coefficient was 92.97 and the total average hydrophilicity was-0.018.indicating that BxPGP23 was a hydrophilic protein.The result of protein secondary structure prediction showed that in the amino acid sequence of Bx-PGP23,there were 660 Alpha helix,214 Extended strand,66 Beta turn,and 360 Random coils,which were accounted for 50.77%,16.46%,5.08%,and 27.69%of the total secondary structure,respectively.The results of the tertiary structure prediction showed that there were multiple nucleotide-binding structural domains(NBD)and transmembrane structural domains(TMD)in this protein encoded by the gene,indicating that this protein belongs to a class of transmembrane efflux proteins.3.The cytochrome P450 and glutathione transferase enzyme activities of the pine wood nematode were assessed with different concentrations of matrine.Compared to the control group,the results indicated that the cytochrome P450 enzymes and glutathione transferase under the matrine treatment showed an upward trend.The experiment detected the main detoxification enzymes of the foreign body metabolism pathway of pine wood nematode and explored the relationship between the poisonous enzymes against exogenous toxins,which were provided a theoretical basis for the prevention and control of pine wood nematode.4.The role of the main genes in the foreign body metabolism pathway of pine wood nematode was clarified in drug stress and detoxification.In order to study the functions of related genes,in vitro synthesize dsRNA and the method of immersion was used to silence the related genes of pine wood nematode.The results showed that the immersed pine wood nematode was stained by FITC,which turned green under ultraviolet light.The phenomenon indicated that dsRNA could be absorbed by the method of immersion,the transcription level of the gene was decreased,and the effect of silence was enhanced.The RNAi was silenced,the sensitivity of matrine was increased.Compared to the control group,the survival rate was decreased with the stress of matrine.In summary,the present experiments have studied the functions of BxCYP33C9,BxCYP33C4,BxCYP33D3,BxGST3,BxGST1,and BxPGP23 genes in the foreign body metabolism pathway of pine wood nematode.The relationship of resistance to matrine was clarified.The results provided a theoretical basis for the prevention and control of pine wood nematode and a basis for the research of new control drugs.