Study On The Methods To Improve RNAi Efficiency Through DsRNA-injection Into Bactrocera Dorsalis

Double-stranded RNA?dsRNA?triggered RNA interference?RNAi?,of which finally induce sequence-specific post-transcriptional gene silencing,has become an important tool in gene function researches and has great potential in pest control.Previous studies have shown that the dsRNA is absorbed by Bactrocera dorsalis through endocytosis.H₂O₂ can increase the ability of dsRNA uptaking to cells.Liposomes can protect dsRNA and transport into cells effectively by overcoming the electrostatic repulsion between dsRNA and cytomembrane.The nanoparticles formed by Chitosan and dsRNA can also protect dsRNA and enhance the ability of dsRNA transportation.At the same time,high temperature,low temperature and Escherichia coli can activate RNAi through increasing the expression of RNAi pathway-related genes such as Dicer-2,Agonaute-2,R2D2 and so on.At present,dsRNAs are mainly delivered into B.dorsalis by injecting or feeding.But this method has the shortcome of low silencing efficiency and poor persistance.Therefore,exploration for an improved RNAi is crucial for silence efficiency promotion of B.dorsalis.In this study,3 different kinds of genes were selected for RNAi,including a housekeeping gene Actin-2,gene with high expression in the midgut CarE-6 and gene with high expression in the central nervous system Natalisin.The RNAi efficiencies of these three genes post different treatments,such as dsRNAs mixture with different media in different proportion,high/low temperature treated B.dorsalis and E.coli treated B.dorsalis,were detected by RT-qPCR.We aim at obtaining schemes for efficient RNAi with different gens and laying foundation for researches about gene function.The mainly results are as follows:1.Clarifying the time effect of RNAi on genes silencing4000 ng/?L dsRNA in concentration were selected and the experimental group consisted of B.dorsalis injected with dsRNA+H₂O₂?100 nL,with mass fraction of 5%?,dsRNA+Lipofectamine RNAimax?with mass to volume ratio of 4:1?and dsRNA+Chitosan?with mass fraction of 0.02%?,with B.dorsalis injected with dsRNA alone as control.RNAi efficiencies were detected at 12 h,24 h,48 h and 72 h post injection.Results revealed that RNAi efficiency of Actin-2 and CarE-6 were improved at 72 h and 48 h respectively and that of Natalisin was improved at 12 h,24 h and 48 h post injection with dsRNA+H₂O₂?100 nL,with mass fraction of 5%?without significant difference.After injection with dsRNA+Lipofectamine RNAimax?with a mass to volume ratio of 4:1?,the RNAi efficiency of Actin-2 was improved at 48 h,that of CarE-6 was improved at 48 h and 72 h and that of Natalisin was significantly improved at 48 h.After injection with dsRNA+Chitosan?with mass fraction of 0.02%?,the RNAi efficiency of Actin-2 was improved at 48 h and 72 h,that of both CarE-6 was improved at 72 h and that of Natalisin was significantly improved at 48 h.All these results indicated that H₂O₂,Lipofectamine RNAimax and Chitosan could help to improve the RNAi efficiency to different extent.Based on the obtained results,we chose the time of 72 h,48 h and 24 h for the RNAi efficiency detection of Actin-2,CarE-6 and Natalisin,respectively,in the following study.2.Clarifying the effect and optimizing the dosage of the three media on RNAi efficiencySelecting 1.5?g dsRNA as the injection dose for per adult and setting B.dorsalis injected with different proportion's dsRNA+H₂O₂/Lipofectamine RNAimax/Chitosan as experimental groups with B.dorsalis injected with dsRNA alone as control.Results revealed that RNAi efficiencies of these three genes were improved post B.dorsalis injection with dsRNA+H₂O₂?100 nL,with mass fraction of 5%?without significant difference and the treatment with dsRNA+H₂O₂?100 nL,with mass fraction of 10% and15%?led to a decrease in RNAi efficiency.These results revealed that H₂O₂ is not a proper medium for efficiency improvement.RNAi efficiencies of these three genes were significantly improved post B.dorsalis injection with dsRNA+Lipofectamine RNAimax?with a mass to volume ratio of 2:1?.RNAi efficiencies of CarE-6 and Natalisin were significantly improved post B.dorsalis injection with dsCarE +Chitosan?with mass fraction of 0.02%,0.04% and 0.08%?and dsNatalisin +Chitosan?with mass fraction of 0.02%?respectively.Based on the silencing effect and cost,B.dorsalis injections with dsRNA+Chitosan?with mass fraction of 0.02%?for CarE-6 and Natalisin RNAi,and that with dsRNA+Chitosan?with mass fraction of 0.08%?for Actin-2 RNAi were recommended.3.Clarifying the effect of high/low temperature and E.coli treated B.dorsalis on RNAi efficiencyTwelve hours post injection with different volume's E.coli?OD600=1.0,100 nL,200 nL and 400 nL?,B.dorsalis was then subjected to the injection with dsRNA?1.5 ?g,4000 ng/?L?.Results revealed that treatments with 100 nL E.coli + dsActin,100 nL/200 nL E.coli + dsCarE and 200 nL E.coli + dsNatalisin led to little improvement of RNAi efficiency,and the treatments with 200 nL/400 nL E.coli + dsActin,400 nL E.coli + dsCarE and 100 nL/400 nL E.coli + dsNatalisin led to decreased RNAi efficiencies.Thus,E.coli could not be a proper treatment for RNAi efficiency improvement.B.dorsalis treated in 12°C and 42°C for 30 minutes were then injected with dsRNA.Results revealed that high and low temperature treatment could decrease the RNAi efficiency compared with room temperature treatment?27°C?.Therefore,RNAi through injection would be the best way for effective one.In summary,in order to achieve effective interference and verify gene function,schemes for RNAi with different gens were as follows:?1?For housekeeping gene,using 1.5 ?g of 4000 ng/?L dsRNA,post B.dorsalis injection 48-72 h with dsRNA+Lipofectamine RNAimax?with a mass to volume ratio of 2:1?,or dsRNA+chitosan?with mass fraction of 0.08%?at 27 °C could detect phenotype.?2?For gene with high expression in the fat body and malpighian tube,using 1.5 ?g of 4000 ng/?L dsRNA,post B.dorsalis injection 48-72 h with dsRNA+Lipofectamine RNAimax?with a mass to volume ratio of 2:1?,or dsRNA+chitosan?with mass fraction of 0.02%?at 27 °C could detect phenotype.?3?For gene with high expression in the central nervous system,using 1.5 ?g of 4000 ng/?L dsRNA,post B.dorsalis injection 12-72 h with dsRNA+Lipofectamine RNAimax?with a mass to volume ratio of 2:1?,or dsRNA+chitosan?with mass fraction of 0.02%?at 27 °C could detect phenotype.