Function Identification Of Pheromone Biosynthesis-Activating Neuropeptide And It’s Receptor By Using RNAi,in Spodoptera Exigua(H?bner)

The beet armyworm Spodoptera exigua(H?bner) is one of the most important migratory pests, of which the larvae can infest many kinds of plants and frequent outbreaks of harm. Beet armyworm is extremely easy to develop resistance to insecticides and result in the control efficiency decreased quickly in agriculture. Therefore, sex pheromone has a broad application prospect in the prevention and treatment of beet armyworm. Pheromone biosynthesis-activating neuropeptide(PBAN) and its receptor(PBANR) play an important role in regulating the sex pheromone biosynthesis in beet armyworm. We identified the function of PBAN and PBANR of beet armyworm by RNA interference(RNAi) technology in this study, in order to improve the utilizaiton of sex pheromone technology in beet armyworm. The main results are as follows:(1) Obtained 7 siRNA fragments that have effect on PBANR. A dsRNA fragment,six siRNA fragments(R001-006) and a negative control fragment(Ncontrol) were designed and synthesed according to the gene sequences of PBANR of beet armyworm. The prothorax in the female pupa which will emerge the next day and the intersegmental membrane of abdomen 8-9 somite in the newly emerged female adult were chosen as the injection sites for dsRNA and siRNA. A real-time fluorescence quantitative PCR was constructed to examine the relative expression levels of PBANR gene in the next 24, 48 and 72 hours after injection. The expression of PBANR gene increased significantly after injection of dsRNA to the pupae after 24 h and 48 h, but there was no significant difference compared with the ones inject with a Ncontrol fragment. The expression of PBANR did not have significant variation after inject with R001 to the pupae from 24-72 h, but it was significantly stimulated when injected to the adult 24 h and 72h; R002 can stimulate the PBANR expression after injected at pupae stage 48 h. R002 can inhibit the expression of PBANR after injected to the pupae 72 h, the inhibition efficiency was 22.6%, and increased the expression of target gent after injected to the adult 24 h and 48 h, but it had no significant difference at 72 h compared with the control. R003 can inhibit the expression of PBANR afer injected to the pupae 24 and 72 h, the inhibition efficiency was of 33% at 72 h. R003 can stimulate the PBANR expression after injected to the adult 48 h.The R004-R006 were injected into the intersegmental membrane of abdomen 8-9 somite in the newly emerged female moth, separately. The results showed that R004 can inhibit the expression of PBANR after injected 48 h and 72 h, and the inhibition rates were 30.57% and 29.02%, separately; PBANR expression can also be interferenced by R005 after 72 h injection, and interference rate arrived to 34.87%; R006 can inhibit the expresson of PBANR after 24 h and 72 h injection, of which the inhibitory rate was 44.3% in 72 h.These results showed that not all the interference fragment will work on the PBANR expression, but depends on the specificity of the fragment, the injection sites and the duration after the injection.(2) Obtained a siRNA fragment that have interference effect on PBAN, and revealed the influence of interference on mating and fecundity. Two siRNA fragment(N001, N002) and a negative control fragment(Ncontrol) were designed and synthesized according to the sequences PBAN. The prothorax of female pupa which will emerge the next day was chosen as the injection site. A real-time fluorescence quantitative PCR was constructed to examine the relative expression levels of PBAN gene 24, 48 and 72 hours after injection.According to the inhibitory effect of N001 on expression of PBAN, we detected the effect of N001 on the biological characteristics of beet armyworm. N001 can decrease the mating rate and mating frequency, but inhibitory effect attenuated with the increase of time after injection. From the second day of injection on there were no significant difference between the treatments and control. N001 had no effect on the fecundity of mated female. These results suggested that despite the inhibition of PBAN expression by the RNAi fragment, this inhibition effect was of short duration. Because beet armyworm will recover the expression quantity of PBAN gene expression in one day and ensure the normal mating and reproduction of female.