Molecular Cloning And Functional Study Of Methoprene-tolerant(Met) From Nilaparvata Lugens (Sta)

The brown planthopper Nilaparvata lugens St?l is an insect of imcomplete metamorphosis which has two types of wings. As it feeds specifically on rice, it has been one of the most destructive ri ce pests in Asia and threatens the rice production. Juvenile hormone(JH) plays an important role in regulating the development of insect. However, the mechanism of JH signaling pathways has not been clearly elucidated till now. Methoprene-tolerant( Met)is the receptor of JH, which belongs to b HLH-PAS family. After recepted JH signal, Met forms a dimer with Tai/SRC/FISC which have the same DNA-binding domains. Then, the dimer recognizes JH response elements to promote the expression of genes such as downstream transcription facter Kr-h1.In this study, the cloned N. lugens Met protein contains 517 amino acids and has a typical basic helix-loop-helix/Per-Arnt-Sim(b HLH-PAS), including b HLH, Per-Arnt-Sim-A(PAS-A), Per-Arnt-Sim-B(PAS-B) and PAS-associated C-terminal(PAC) domains. PAS-B and PAC are highly conserved and the rest two have relatively lower homology. We explored the expression levels of Met in different developmental stages, tissues and under methoprene-induction. Nl Met expressed in all tissues and high est in embryonic stage measured by q RT-PCR. The expression levels varied in nymph stages while was relatively constant in adults(as shown in Figure 2.5). After methoprene-induction, the expression level of Nl Met increased. The results demonstrated that Nl Met, which is a homologous gene with Tribolium castaneum and Drosophila melanogaster, as a receptor to get involved in the JH signal transmission and regulate the growth and development of N. lugens.We observed and analyzed the characteristics of ecdysis, the ratio of genital malformation by RNA interference in the 4th and 5th nymphs. Comparing with normal phenotypic females, we further explored the RNA interference effect on ovary development in order to provide a theoretical foundation for the mechanism of JH signal transmission. We observed different mortality when Nl Met and Nl Kr-h1 genes were silencing in brown planthopper development. The mortality was highest in nymph, followed by pharate brachypterous female, third was pharate macropterous male and ma le brachypterous. Interestingly, it was zero in female pharate macropterous. Furthermore, RNAi caused different degrees of external genitalia malformations in both male and female. After injecting ds GFP and ds Nl Met to 4th and 5th nymph, there was no significant difference in external genitalia. There was obvious difference in female genitals after injecting ds Nl Kr-h1 to 4th nymph. The ovarian development was effected and varied significantly in Nl Met/Nl Kr-h1- silenced female adults. These results suggested that the juvenile hormone receptor and its downstream transcription factor Kr-h1 were required in ovariole development and egg maturation in N. lugens.We found methoprene treatment had no effect on ovarian tube number, but reduced the development of brachypterous ovarian. We noticed that after silencing of Nl Met, Nl Krh-1 or Nl Met+Nl Kr-h1, there were no difference on ovarian tube number of 4th nymph female adults comparing with control group, but the emergence of 5th nymph female adults ovarian tubules drastically decreased(P=0.05). The development of ovary also decreased significantly(P=0.05). The ovary develop slower when Nl Met+Nl Kr-h1-ds RNA was injected into 5th nymph, which indicated Nl Met and Nl Kr-h1 probably interact with each other. RNAi on 5th nymph, i.e., ds RNA interference on Nl Met, Nl Kr-h1 or Nl Met+Nl Kr-h1, the number of oviposition decreased significantly. The results showed that JH receptor Nl Met and its downstream transcription factor Kr-h1 were indispensable in the process of N. lugens ovary development and egg maturity.JH plays an important role in N. lugens metamorphosis and ovary development. To transmit its signal normally, it is necessary for N. lugens metamorphosis, external genitals and ovary development. Consequently, our research provides a theoretical foundation of JH signal transmission mechanism, which can be used in N. lugens prophylaxis and treatment to achieve ultimately population control.