Interaction Of Detoxification Enzyme Genes In Brown Planthopper(Nilaparvata Lugens)with Gramine And Ferulic Acid In Rice,and Their Potential Application

Numerous plant compounds play an important role in defensing herbivore,but herbivore's detoxification enzymes has traditionally been thought that they can be effectively dealt with plant toxic compounds to keep these herbivores to survive on the host plant.However,about the molecular mechanism between plants toxic compound and detoxification enzyme in herbivore and the potential application of its control pest population is not much.Therefore,we regard the important detoxification enzymes,glutathione-S-transferase(GST),carboxylesterase(CarE)in the brown planthopper(BPH,Nilaparvata lugens)and rice secondary compounds,gramine and ferulic acid as the research object,explore the interaction between them from the toxicity of compound to BPH,GST and CarE activity and induced gene expression in insect,and effect of related genes using RNA interference(RNAi)on BPH sensitivity to the compound in this study.Meanwhile,plant mediated GST gene interference further confirmed the continued interference of BPH detoxification enzyme genes have a protective effect on the regulation of BPH population and rice plants.Our results indicated that after BPH fed with gramine or ferulic acid diet at 3 d,BPH nymph mortalities were in dose-dependent fashions with gramine and ferulic acid concentration,respectively;the LC50 of gramine was 14.99 ?g/ml,and the LC25 and LC50 of ferulic acid were 5.81 and 23.0 pg/ml,respectively.The low gramine and ferulic acid concentrations(<LC50)significantly induced GST activity in BPH,and the induction of BPH GST activity by gramine was in a dose-dependent fashion with gramine concentration.In addation,the low ferulic acid concentrations(<LC50)can also significantly induce CarE activity in BPH.Two kinds of rice compounds have a significant impact on GST or CarE gene expression in BPH.Non-lethal dose of gramine(2.0 and 3.0?g/ml)can significantly induce the transcriptional levels of seven GST genes(NlGSTO1,NlGSTT1,NlGSTE1,NlGSTS1,NlGSTS2,NlGSTD1,NlGSTD2,NIGST1-1,except for NIGSTD1).The low ferulic acid concentrations(2.0,4.0 and 8.0 ?g/ml)can significantly induce the transcripts of NlGSTD1 and NlGSTE1,but 4.0 and 8.0 ferulic acid can significantly suppress the expression level of NICE in BPH.In the study on the relationship between the GST gene interference and enzyme activity in BPH,for the studied eight GST genes,the interfered transcripts of three genes(NIGST1-1,NlGSTD2 and NIGSTE1)significantly suppressed the insect GST activity,and the GST activity in dsRNA-treated BPH was only correlated with the transcriptional levels of NlGST1-1,NIGSTD2 and NlGSTE1.Meanwhile,the interference of NlCE can significantly reduce the expression level of NICE and CarE activity in BPH.The interference of BPH detoxification enzyme gene affects BPH sensitivity to toxic compounds.Except for NIGSTO1,interference of other seven genes significantly increased the nymph sensitivity to gramine,and the mortalities of the nymphs that pre-fed on dsRNAs of NIGST1-1,NlGSTD1,NIGSTD2,NIGSTE1,NIGSTS1,NIGSTS2 and NlGSTT1 were 3.28,2.44,2.51,2.99,2.15,2.95 and 2.62 folds as that of the nymphs only fed on gramine,respectively.Meanwhile,compared with the BPH just feeding ferulic acid,the silencing of three genes(NlGSTD1,NlGSTE1 and NlCE)significantly increased the nymph sensitivity to ferulic acid,and the mortalities of dsRNA pre-fed nymphs that fed on ferulic acid diets were increased by 92.85%,119.85%,or 124.58%,respectively.We constructed NlGST1-1 from GST family interference and expression vector,and the recombinant vector successfully expressed in transgenic rice strain important organs and tissues.Northern blot analysis revealed the presence of NlGST1-1 dsRNA in transgenic rice lines.The bioassay using the T1 generation of gst651 and gst426 rice lines showed that compared with the control(Ctrl),after BPH nymphs fed on the transgenic gst651,gst426 rice lines,NlGST1-1 expression level,GST activity,relative growth rate and its adult fecundity in BPH were significantly reduced by 22.92%-44.97%,11.56%-26.08%,12.23%-21.11%and 25.20%-26.13%,respectively.When gst651,gst426 and Ctrl rice plants were inoculated first instar BPH(20 head/strains)at 21 days post BPH feeding,gst651,gst426 rice lines still keep growth,but most leaves and stems of the control plants emerged dry rot.Thus,rice mediated GST gene interference can effectively inhibit the growth and the development of BPH population,and protects the host plant against the dangers of pests.In conclusion,the study showed that the rice gramine and ferulic acid are plant compounds that are toxic to BPH,and these compounds can induce the expression of GST and CarE family genes and enzyme activities.GST activity is closely related with the expression level of most genes from GST family in BPH.The knockdown of some GST and CarE genes can enhance the sensibility of plant toxic compound to BPH.Rice mediated the interference of BPH GST gene not only can effectively inhibit the growth and the development of BPH population,but also can protect the host plant from insect attack.Therefore,our research provided a scientific basis for the adaptive mechanism of herbivore to host plants,and also provided a new way on the development of environment friendly strategy for sucking mouthpart pest control.