Studies On The Effect Of RNA Interference Targeting Tissue-Specificly Expressed Genes In Nilaparvata Lugens

RNA interference(RNAi)can specifically eliminate or shut down the expression of specific genes,and thus it is widely used to study gene functions.RNAi also offers great opportunities to manage pest populations,and reduce insect-borne infectious diseases.Although the success of RNAi has been confirmed in Nilaparvata lugens,the efficiency varied dramatically among tissues.To test the RNAi efficiency of genes in specifically-expressed different tissue,RNAi was performed to neuronally,midgut and fat body-expressed genes in N.lugens.The main research results are as follows.1.RNAi efficiency of different neuronally-specificity-expressed genes in N.lugensThree genes,prothoracicotropic hormone(NIPTTH),pigment-dispersing factor(NlPDF)and short neuropeptide F(NlsNPF)were identified.Sequence alignment revealed that NlPTTH possessed seven conserved cysteine residues.NlPDF have total amino acid residues NxE(L/I)INSLL(G/S)LPKx(L/M)NDA-NH2.NlsNPF possessed conserved structure xPxLRLRFamide.All the three genes were transcribed throughout tested developmental stages.In nymphal stage,the highest and the second highest levels of NlPTTH were at the third and fourth instar stages respectively.Similarly,the peaks of NlPDF were seen at the second and fourth instar stages,and those of NlsDPF were found at the second and fifth instar stages.In the adult stage,the mRNA levels of the three neuronally-expressed genes were lower in the females than those in the males.The tissue expression profiles of the three neuronally-transcribed genes in the fifth-instar nymphs were also tested.Their transcripts were mainly restricted to N.lugens brain-corpora cardiaca-corpora allata complex.qRT-PCR revealed that the target transcripts of NIPDF,NIPTTH and NlsNPF were significantly reduced by 67.3%-91.8%,57.4%-75.3%and 80.3%-98.7%,respectively.Knockdown of NIPTTH delayed nymph development and caused higher nymphal lethality.Moreover,silencing of three neuronally-expressed genes decreased the mRNA levels of ecdysteroidogenesis genes(NlPHM,NIDIB and NlFTZ-F1).Furthermore,RNA interference efficiency of NlsNPF is positively related to the dose of dsRNA.These results show that the neuronally-expressed genes can be successfully knocked down by dsRNA.2.RNAi efficiency of different midgut-specificity-expressed genes in N.lugensBased on the transcriptomic and genome data of N.lugens and sequences in the GenBank,two cathepsin B genes(NICB1 and NlCB2)were identified.Both proteins possessed proteinase-specific conserved domains.The temporal expression analysis revealed that NlCB1 and NlCB2 were expressed throughout all tested developmental stages.For NlCB1,the highest level was observed in the male,and followed by female,the third day of the fifth nymphal instars.For NlCB2,an expression peak occurred in each instar,most of these peaks were seen in second day.The highest and the second highest levels were at the second day of second and third instar stages.The tissue expression analysis confirmed that either NlCB1 or NlCB2 were mainly restricted to N.lugens midgut.After dsRNA injection,NlCB1 and NlCB2 expression were significantly reduced by 83.3%-94.4%and 73.5%-96.1%,respectively.Furthermore,knockdown of NlCB1 and NlCB2 caused higher nymphal lethality and deformed adults.3.RNAi efficiency of different fat body-specificity-expressed genes in N.lugensBy mining the transcriptomic and genome data of N.lugens and sequences in the GenBank,two genes encoding a cathepsin L and a cathepsin D were identified(NlCL and NlCD).NICL has conserved ERF/WNIN motif,GNFD motif and three highly conserved amino acid residues Cys,His,and Asn.NICD possesses conserved Asp-Thr/Ser-Gly(DT/SG)motif.The temporal expression profiles of NICL and NlCD were tested.They were expressed throughout all tested developmental stages.The highest level of NlCL was found in the egg stage,and followed by adult stage.In contrast,the expression of NlCL in nymphal stage was low.A expression peak occurred in eacg instar,at the third day.For NICD,the peak was in the third day of fifth instar stages and the adult stage.After dsRNA injection,NlCL and NICD expression levels were significantly downregulated by 74.3%-84.3%and 75.0%-79.3%,respectively.Furthermore,knockdown of NICL and NICD caused higher nymphal lethality and deformed adults.In conclusion,RNAi by dsRNA injection can effectively silence tissue-specifically expressed genes in the N.lugen nymphs,and dsRNA dose can influence RNAi efficiency.