The Pathogenesis Of CYP450 Genes From Pine Wood Nematode(Bursaphelenchus Xylophilus)

Pine wilt disease(Bursaphelenchus xylophilus)is the most serious forest disease in our country at present, but the lagging pathogenesis research and disputed results of previous studies severely retarded the propulsion of pine wilt disease basic research and the scientific prevention and control measures’ out puts. After the invasion of pine wood nematode(PWN),the terpene metabolism of pine trees were disordered causing the cavitation of pine vascular alongside with water transport disruption and ultimately led to the pine tree wilting death. Pine wood nematodes had to deal with the stress of pine secondary metabolites such as the monoterpene during the life cycle and the process of PWN catabolized the pine secondary metabolites was necessary for successful colonization and propagation in pine trees and this meant the terpene metabolism was one of the pathogenic process in PWN. The cytochrome P450 genes and their metabolic pathway were one of the important detoxificational genes to response the pinene stress in the pine wood nematode. To research the function of cytochrome P450 genes in the pathogenic processes these researches were carried out.To research the important PWN cytochrome P450 gene under the stress of pine secondary metabolites we detected the terpene metabolism in the inoculated Pinus massoniana and the express patterns of CYP450 family gene in the PWN by the gas chromatography and real-time quantitative PCR. The result showed that after inoculated with pine wood nematode the pinene synthase gene of the inoculated P. massoniana were dramatically expression at the 6th and 21 th day and then the α-pinene and β-pinene were sharply accumulation with two peaks respectively at the 9th and 27 th day and the PWN CYP33C9 expression dramatically increased at the 12 th and 15 th day and the CYP33C4 expression were highest at the 21 th day. In conclusion, after the invasion of PWN the pinene synthase gene was increased at first and then the α-pinene andβ-pinene were sharply accumulation by the biosynthesis and finally the PWN cytochrome P450(CYP450)gene expression dramatically increased responding to the accumulation of pineterpene. Therefore, we inferred that the PWN CYP450 genes might participate in the terpene metabolism and might be one of the pathogenic genes in the PWN pathogenic process.Two cytochrome P450 genes include CYP33C9 and CYP33C4 were cloned by RT-PCR technology and the CYP33C9 and CYP33C4 gene respectively were 1504 bp and 1506 bp. The CYP33C9 and CYP33C4 gene both have transferase activity and the CYP33C4 gene also have oxidordeuctase. The RNAi expression vectors were constructed and the relative expression of PWN CYP33C9 and CYP33C4 gene were inhibited by socking with matching dsRNA mixture.The result showed that after inoculated with CYP33C9 and CYP33C4 gene silencing PWN by RNAi technology the pinene synthase gene of the inoculated P. massoniana were dramatically expression respestively at the 3th and 3rd, 9th, 12 th day and then the α-pinene andβ-pinene were sharply accumulation with only one peaks respectively at the 6th and 21 th day while the pinene synthase gene with unsilenced PWN were dramatically expression at the 6th and 21 th day and then the α-pinene and β-pinene were sharply accumulation with two peaks respectively at the 9th and 27 th day. So after silenced the CYP33C9 and CYP33C4 gene the pinene metabolism of inoculated pine trees antedated the normal group and the pinene accumulation were only one peak. Therefore, the silenced CYP450 gene of pine wood nematode affected the host pinene metabolism, and these genes played an important role in the interaction between the PWN and pine trees.After inoculated with CYP33C9 and CYP33C4 gene silencing PWN the PWN population number was smaller and the reproduction rate decreased compared with control group. At the30 th day after post inoculated with the silenced PWN the inoculated pine needles hanged wilting but for green and the trees were on the verge of death with dehydrated trunkes while the pine needles wilted and yellowed and the trees were death with the withered trunkes. The reproduction rate and pathogenicity decreased after silenced CYP33C9 and CYP33C4 gene.Therefor, the CYP33C9 and CYP33C4 genes were infulenced the PWN reproduction rate and pathogenicity and may be the important genes in the pathogenic process.The α-pinene could inhibit the nematode’s reproductive rate with low sex ratio at low concentration but promote at high concentration with high sex ratio and the β-pinene might inhibit the reproduction potential with low sex ratio at high concentration and the reproductionrate was lowest with low sex ratio under the stress of α-pinene and β-pinene mixture. So the secondary metabolites of pine trees affected the reproduction rate of PWN and the PWN sex ratios are biased to females at favorite conditions and the nematodes exercise a masculinizing effect on the developing larvae in unfavorite environments and the PWN mediated the reproduction rate by changing the sex ratio to adapt the changing environment by controlling the population number.Silenced the PWN CYP33C9 and CYP33C4 gene by RNAi technology the reproduction rate decreased and the female to male ratio was smaller and under the stress of different concentration α-pinene and the mixture of α-pinene and β-pinene the reproduction rate decreased and the female to male ratio was smaller comparing with the control group. So the adaptability of PWN decreased responding the strees of pinene after Silenced the CYP33C9 and CYP33C4 genes and the two genes might be involved in the PWN terpene metabolic process andAbove all, the CYP33C9 and CYP33C4 genes might be involved in the PWN terpene metabolic process and could improve the adaptability of the PWN in the host and enhanced the reproduction rate and pathogenicity of PWN and might be the key pathogenic genes in the pathogenic process.