Inhibition Of Juvenile Hormone Esterase (JHE) As Well As RNAi Against Its Gene In Diamondback Moth,Plutella Xylostella

The diamondback moth (DBM), Plutella xylostella (Linn.), is the most dest destructive pest on cruciferous vegetables throughout the world. Chemical pesticides is the most way to control DBM,butDBM has become highly resistant to almost all pesticides.We choose DBM as our research material,and the dsRNA for juvenile hormone esterase (JHE) was synthesized. The concentration of the DEPC solution was determined based on its toxicity on the3rd instar larvae. The effect of dsRNA on the growth and development of the3rd instar larvae as well as its JHE activity was studied. Besides, the effect of the JHE inhibitor,3-octylthio-1,1,1-trifluoro-2-propanone (OTFP), on JHE in the4th instar larvae in vivo and vitro as well as that on their pupation was investigated. The main results were as follows:(1) The synthesis of dsRNA of JHE geneThrough a column to extract total RNA of3rd instar larvae of DBM, groped DNA amplification conditions by using one-step RT-PCR, synthetized mass dsRNA. The results show that the suitable annealing temperature for RT-PCR of JHE gene fragment containing substrate binding site was ranged from60～62℃and the suitable conditions for its dsRNA synthesis were37℃for4h; for the cDNA of JHE gene fragment containing catalyzing site, the suitable annealing temperature for RT PCR was ranged from58～60℃, and the suitable conditions for its dsRNA synthesis were also37℃for4h.(2) The toxicity of DEPC solution on the3rd instar larvae of DBMFeeded3rd instar larvae of DBM with cabbage leaves dealing with DEPC treated-water, the growth of DBM become slow as the concentration of DEPC treated-water rised. DEPC solution was toxic to the3rd instar larvae of DBM at the concentration higher than0.1%(V/V),but it was safe at0.05%(V/V)or lower.(3)RNAi of JHE geneFeeded3rd instar larvae of DBM with leaves treaed by "sandwich" method, the mortality of the tested insects increased and the growth rate decreased. But the activity of JHE did not decrease, moreover, it even increased in some treatments. This indicated that up-regulation of JHE gene occurred after RNAi.(4) Effect of OTFP on the4th instar larvae of DBMFeeded4th instar larvae of DBM with OTPF of different concentrions, the growth of larvae slows, the survival rate increased. The pupation rates significantly decreased by2%、20%、40%and64%, respectively. At the same time,the pupation time was also prolonged by0.9h、9.0h、4.0h and1.2h,respectively. Meanwhile, JHE activity in DBM has been sinificantly inhibited both in vitro and vivo,the trend was especially clear in vivo.