Evaluation Of Feeding Related Response Genes In Myzus Persicae As RNAi Target And Off-target Assessment To Propylaea Japonica

RNAi technology is target specific and environmentally friendly,with various application methods,thus considering as a potential green pest control method.RNAi has also been intensively used in aphid studies,however,the investigation related to target gene screening and off-target evaluation to beneficial insects is still rare.Therefore,Myzus persicae and Propylaea japonica were chosen as the research objects in this study,the responsive genes during M.persicae feeding were screened to obtain potential aphid control targets,and their off-target effects were evaluated to the ladybeetle,P.japonica.This study aimed to provide reference for RNAi-based target screening and bio-safety design in aphid control.The main findings are as follows:1.Mining of responsive genes based on transcriptome of M.persicae during feedingThe transcriptomes of Myzus persicae on artificial diet and the host plant tobacco were compared,which showed that 319 genes were differentially expressed at 24 h after the aphidswere transferred from artificial diet to tobacco,of which 140 genes were up-regulated and 179 genes were down-regulated.At 48 h after the aphidswere transferred from artificial diet to tobacco,a total of 291 genes were differentially expressed,of which 87 genes were up-regulated and 204 genes were down-regulated.Among all the up-regulated genes,there were 26 genes with FPKM value greater than20 and could be annotated.Most of these genes were related to substance and energy metabolisms.Sequence homology alignment showed that most of the differentially up-regulated genes were conserved in a variety of aphid species,which implied that these genes might have potential for design broad-spectrum RNAi-based control in aphids.2.RNAi-based targeting on responsive genes of M.persicae during feedingAmong the 26 potential candidates selected above,a total of 22 dsRNA fragments were obtained.Through artificial diet mediated RNAi,silencing of eight genes could result in significant increase of mortality or decrease of fecundity in M.persicae.Among them,silencing of five genes could also significantly affect the growth and development of M.persicae,e.g the reduced body size and weight.However,specific dsRNA in targeting these eight genes only resulted in 20～30%adjusted mortality in aphids.To further improve this,three out eight genes with higher lethality by RNAi were used to design fusion dsRNA.Compared with specific dsRNA in targeting a single gene,the fused dsRNA not only could silence the selected fusion gene separately,but also led to a higher adjusted mortality of 40%.Although the dsRNA delivery to aphidswas limited through artificial diet approach,this study still highlighted that target gene screen from feeding related response genes and the design of fusion dsRNA,could be useful in the improvement of RNAi-based aphid control.3.RNAi sensitivities among developmental stages of P.japonicaBy Pacbio sequencing technology,we obtained a full-length transcriptome of P.japonica.On this basis,the core machinery genes of RNAi pathway in P.japonica were identified,and their expression patterns upon dsRNA treatment were also analyzed.The results showed that almost all the RNAi pathway genes of P.japonica showed induced expression upon dsRNA treatment.The genes of si RNA pathway showed different expression between adults and 1 instar larvae,as response time point of si RNA pathway genes in adults was later than those in 1^stinstar larvae upon dsRNA treatment.The sensitivities to dsRNA were different among various developmental stages of P.japonica,showing the reduction of sensitivities to dsRNA as P.japonica growth.The 1^stinstar larva was the most RNAi sensitive period for P.japonica,which was also consistent with the gene responses mentioned before.By measuring the lethality of the indicator gene dsCHMP-4b at different dsRNA concentrations,the toxicological regression curve of dsCHMP-4b against the 1^stinstar larvae of P.japonica was obtained.With LC₉₉（2,713.4 ng/μL）,we assigned 3,000 ng/μL as a reference dosage to evaluate dsRNA off-target effect of M.persicae to P.japonica.4.Off-target analysis and assessment of the dsRNA in targeting M.persicae to P.japonicaThe off-target effect of dsRNAs of eight target genes and one fusion dsRNA of M.persicae on P.japonica were analyzed by using bioinformatics approach,including the off-target analysis of 16 bp perfectly matched off-target fragments（16 bp off-target fragments）,21 bp perfectly matched off-target fragments（21 bp off-target fragments）,26 bp almost perfectly matched off-target fragemnts（1～2 mismatches allowed）（26 bp off-target fragments）and long off-target fragments with sequence similarity greater than80%（80%off-target fragments）.The results showed that the distribution of off-target fragments from all genes of M.persicae was‘locality distributed’,and contained 16 bp of off-target fragments in each dsRNA.The dsRNAs also showed different off-target potential depending on the designed location.The silencing efficiency was detected by the most hits off-target fragment of the aphid dsRNA to P.japonica.Among them,only dsMPgene3013 showed serious off-target effect to P.japonica,resulting in a 75%off-target gene silencing efficiency.The parameters such as mortality,developmental duration,pupa weight and predation ability of P.japonica were further determined upon dsMPgene3013 treatment.The results showed that dsMPgene3013 reduced the pupal weight and weakened the predation function of P.japonica.The above results suggested that the off-target effects of dsRNA could be effectively predicted through dsRNA off-target analysis and evaluation,and the rational dsRNA design could reduce the off-target effect of dsRNA.In summary,this study took the M.persicae and P.japonica as the objects to analyze the dsRNA design by targeting a single gene and fusion genes.These genes were obtained from the response genes during the feeding process of the aphid.The RNAi sensitivity and off-target analysis of the important natural enemy P.japonica were also evaluated.Eight potential RNAi targets for the aphid were initially obtained,and the aphid control ability of the targets could be improved through the fusion of dsRNA design.Overall,this study provides a reference for aphid RNAi target screening as well as biosafe dsRNA design.