Gene Expression And Regulatory Functions Of Bxy012009、Bxy005783 And Bxy003854 In Bursaphelenchus Xylophilus

Pine wilt disease is one of the most devastating epidemic diseases of Pinus plants caused by pine wood nematode,Bursaphelenchus xylophilus.Pine wilt disease currently occurs in many provinces in China,causing serious losses.Some studies have found that the pathogenicity of pine wood nematodes is closely related to the reproduction rate,and the sex ratio is an important factor affecting the reproduction rate.But the sex determination and differentiation mechanism of B.xylophilus are still unclear.Moreover,the stage of dauer larvae of B.xylophilus is suitable for adverse environments,and dauer larvae can widely spread by the feeding behaviors of Monochamus alternatus.The formation mechanism of B.xylophilus dauer larvae has not been clearly explained.The above two problems have also become a major obstacle in exploring the prevention and control measures of B.xylophilus.In this study,two genes,fem-1 and fem-2,potentially related to sex determination and daf-12 related to the formation of dauer larvae were selected,and homologous gene information in B.xylophilus,was predicted with reference torefer to C.elegans.After cloning and obtained Bxy012009,Bxy005783 and Bxy003854 fragments of pine wood nematode,Real-Time Quantitative PCR,in situ hybridization and RNAi technologies were used to study the spatiotemporal dynamic expression characteristics and regulatory functions of the three genes.The specific research results are as follows:1.Real-Time Quantitative PCR results showed that Bxy012009 and Bxy003854 genes were expressed at all developmental stages of B.xylophilus,with the highest expression in the second instar larvae and a significant decrease in the third instar larvae.2.in situ hybridization results showed that the Bxy012009 gene was expressed in multiple tissue cells such as gonads,intestines,and subcutaneous cells in the early stages of B.xylophilus development,and was concentrated in subcutaneous cells and muscles in the later stages with weak expression;Bxy005783 gene was expressed stably in the gonads of B.xylophilus;Bxy003854 gene was expressed in different developmental stages of B.xylophilus,and in the larval stage was expressed in epidermal tissues,body wall muscles,intestines and other parts,and in adult stage was highly expressed in sperm and pussy parts.3.The results of RNAi showed that the Bxy012009 and Bxy005783 genes were disturbed by double-stranded RNA and the population sex ratio of nematodes showed a downward trend,andthe sex ratio decreased most significantly after treated on the second instar larva,indicating that the Bxy012009 and Bxy005783 genes were the key genes for sex determination and the second instar stage was a critical period for sex determination;the silencing of Bxy003854 gene reduces the survival rate of nematodes in high temperature environment and delays the development of larvae,indicating that Bxy003854 gene is not only involved in the regulating of larval formation of dauer,but also related to the survival of B.xylophilus,and is a key gene for ensuring that nematodes cope with adverse environments.The above research results clarified the expression characteristics of Bxy012009,Bxy005783 and Bxy003854 and their roles on regulating sex determination and dauer formation in B.xylophilus.It provides a theoretical basis for further research on regulation mechanism of the sex determination and the dauer formation.in B.xylophilus...