Research On Influence Factors And The Mechanisms Of Insect RNAi Efficiency

RNA interference(RNAi)is a post-transcriptional gene-silencing mechanism triggered by double-stranded RNA(dsRNA).In insect research field,this technique has been widely used for identifying gene functions,meanwhile,it has been considered as a potential pest control strategy.Unfortunately,recent researches suggested that RNAi efficiency may varies among insect species,tested dsRNA length,target gene,and other experimental factors as well,which could limit further development of this bio-technique.Thus,we used the variations in RNAi efficacy as a start point to decipher the potential key factors affecting insect RNAi and their inner molecular basis.We also discovered the nanoparticle-based high efficiency RNAi technique at the same time.Here,our results would be presented as follows:1 Analysis of the relationship between in vivo dsRNA degradation and the variation in RNAi efficiency among insect speciesRNA interference(RNAi)has become an essential technique in entomology research.However,RNAi efficiency appears to vary significantly among insect species.Here,the sensitivity of four insect species from four different orders to RNAi was compared to understand the reason for this variation.A previously reported method was modified to monitor trace amounts of double-stranded RNA(dsRNA).After the administration of dsRNA,the dynamics of its content was determined in the hemolymph,in addition to the capability of its degradation in both the hemolymph and the midgut juice.The results demonstrated that our dsRNA detection method which use precise RNA isolation and real-time PCR instead of normal RNA isolation and PCR-electrophoresis could sensitively detect dsRNA in 10 ?L hemolymph sample.And our data also showed that injection of dsRNA targeting the homologous chitinase gene in Periplaneta americana,Zophobas atratus,Locusta migratoria,and Spodoptera litura,with doses(1.0,2.3,11.5,and 33.0?g,respectively)resulting in the same initial hemolymph concentration,caused 82%,78%,76%,and 20%depletion,respectively,whereas feeding doses based on body weight(24,24,36,and 30?g)accounted for 47%,28%,5%,and 1%depletion.The sensitivity of insects to RNAi was observed to be as follows:P.americana>Z.atratus>>L.migratoria>>S.litura.After dsRNA injection,similar initial hemolymph concentration(310 pg/ml)was achieved;thereafter the hemolymph concentration decreased at different speeds in these insects with the calculated relative speed index being 2.06,1.44,1.20,and 1,respectively.Thus,it implied the negative correlation(-0.9501)between the hemolymph dsRNA decreasing speed index and the RNAi effects.It also showed that the hemolymph dsRNA contents calculated using hemolymph concentration and persistence time integral were in the ratio 1,1.17,2.01,and 2.90 for S.litura,L.migratoria,Z.atratus,and P.americana,respectively.This suggested a good correlation(0.9983)with the RNAi depletions induced by feeding dsRNA.Furthermore,in vitro experiments demonstrated that the hemolymph contents after dsRNA injection were dependent on hemolymph degradation capacities,and on the degradation capabilities in the midgut juice,when dsRNA was fed.In conclusion,the RNAi efficacy in different insect species was observed to depend on the enzymatic degradation of dsRNA,which functions as the key factor determining the inner target exposure dosages.Thus,enzymatic degradation in vivo should be taken into consideration for efficient use of RNAi in insects.2 Analysis of dsRNA length affects Tribolium RNAi efficacyPrevious Researches of RNA interference(RNAi)showed that many factors could affect RNAi efficiency,double-stranded RNA(dsRNA)length is one of the experimental factors which may affect RNAi efficacy.Herein,variation in RNAi efficacy associated with dsRNA molecular length was confirmed via comparison of knockdown results following dsRNA injection into Tribolium castaneum,a RNAi sensitive species.We modified our dsRNA detection method described in Chapter 2,using probe qPCR instead of SYBR technique for detection,which shows high sensitivity and accuracy.Through in vitro experiments with T.castaneum midgut,dsRNA accumulation in the midgut,degradation by midgut homogenates and persistence in hemolymph after injection were tested to determine the causes of RNAi efficacy variation.The comparative efficacies of dsRNAs were 480 bp?240 bp>120 bp>60 bp>>21 bp.The combined midgut dsRNA accumulation and midgut homogenate-induced degradation analyses suggested cellular uptake to be the key barrier for 21 bp dsRNA functioning but was likely not the main determinant of the variation in longer dsRNAs'(?60 bp)bioactivity.In vitro RNAi experiment with T.castaneum midgut showed that long dsRNAs all significantly depleted the expression of corresponding genes,suggesting little variation in intracellular RNAi machinery's affinity for different dsRNA lengths.In vivo hemolymph content dynamics of different dsRNAs following injection indicated higher persistence of longer dsRNAs.In addition,comparison of the in vivo and in vitro RNAi efficacy also indicated the importance of hemolymph degradation.Thus,the varied efficacy of long dsRNAs resulted from their degradation by nucleases,which varied with dsRNA length.3 Research on gene self-regulation and its influence on Trbolium RNAiChoice of target gene is an important factor which may influence RNAi efficacy,unfortunately,a few reports have addressed this problem.Here,through comparison of RNAi effects between two pattering genes in the transgenic fluorescent line of red flour beetle,Tribolium castaneum,which is a RNAi sensitive Coleopteran insect,we found that mRNA levels of the pattering genes with transcriptional factor ability,including Ubx,Vg,and Nub,would go up instead of being repressed,however,mRNA levels of the genes without transcriptional factor capacity,including Eyfp,Ebony,and ChsB,could be significantly reduced by RNAi effects.Thereafter,further data on protein and phenotypic levels were recorded during the RNAi process as well.Western blotting results showed that the protein levels of both transcriptional factor genes(Ubx and Vg)and non-transcriptional factor genes(Eyfp and Ebony)were depressed in RNAi;meanwhile,we also observed the phenotypic changes in both non-and transcriptional genes.Interestingly,we found an increasing of Eyfp fluorescence in Nub RNAi beetles,which also indicated an upregulation of Eyfp expression.Typically,Nub and Eyfp genes share the same locus in this pull transgenic line,and they could be controlled by the same enhancer(s)as well,which suggested that the Nub transcription increased in Nub RNAi process too.Surprisingly,Nub protein was downregulated in RNAi,therefore,there was a negative correlation between Nub transcription and its protein level,i.e.Nub protein would downregulate its transcriptional process.Our data strongly supported that the pattering genes,Ubx,Vg,and Nub,are self-regulation genes.In order to test this theory,Ubx was chosen as a model to trace its mRNA dynamics after dsUbx injection,our results showed that the relative mRNA levels went down for 4-12 hours post dsRNA injection,after that mRNA was increasing until 48 hours post injection.On the other hands,to eliminate the noise of mRNA and protein base levels,dsVg was injected before Vg gene expression.Our data demonstrated that mRNA levels significantly increased at 2 days post dsVg injections,however,we found an obvious reduction on both mRNA levels and their phenotypes at 7 days after injections.In conclusion,the protein levels were depressed in RNAi,which contrarily enhanced their transcription and increased their mRNA during a certain time,but RNAi effects were much stronger than the feedback,hence we could still observe a phenotypic reduction.Self-regulation affects RNAi efficiency,it suggests us that evaluation should be taken seriously towards test genes and knockdown effect,but the inner machanisms towards this phenomenon is still unveil.4 Comparison of Nanoparticles Mediated RNAi in the Rice striped stem borer,Chilo suppressalisRNAi has shown a very promising prospect on insect pest control.However,low RNAi efficacy limit the further development of this biotechnology on lepidopteran insects,including the rice striped stem borer(SSB),Chilo suppressalis,one of the major destructive rice pests.In this work,the application of a variety of nanoparticles(NPs)by which dsRNA could be encapsulated was evaluated as an alternative delivery strategy for possibly increasing the bioactivity of dsRNA.Three NPs,chitosan,carbon quantum dot(CQD),and lipofectamine2000 complexed with dsRNA,to target three genes(CHSA,CHSB,and G3PDH)were tested for controlling SSB larvae.Relative mRNA expressions were quantified using qPCR to evaluate knockdown efficiency in NP-dsRNA treated larvae,and the correlated dsRNA mediated SSB larval mortality were also tested.The results demonstrated that all three NPs tested were efficient for feeding delivery by equally improving both dsRNA stability and cellular uptake.Thereafter,the in vivo content dynamics of hemolymph dsRNA after ingested different NP-dsRNA were tracked,the hemolymph dsRNA contents were in the ratio 5.67,9.43,and 1 for chitosan,CQD,and lipofactamine2000 induced samples,respectively.This showed a good correlation(0.9314-0.9854)between the hemolymph contents of dsRNA and the average RNAi depletions in non-gut tissues by feeding different genes dsRNA among the different NPs.Our results strongly suggest that due to the strong endosomal escaping ability,CQD was the most efficient carrier for inducing systemic RNAi,and thereby causing effective gene silencing and mortality in SSB.