RNAi In Tribolium Castaneum And The Off Target Effect Of DsRNA

RNAi is one of the most important discoveries of mordern molecular biology aera,this mechansim is conserved in a wide variety of eukaryotic organisms.In the field of insecta,RNAi opened up new avenues to pursue forward genetic investigations in non-model organisms and the potential of using it for practical purposes in novel crop protection strategies.However,the off target effect of RNAi make this technology disputable and to be one of the most serious obstacles for its application.If we applied RNAi in pest management aera,the off target effect would make it losing its specificity and leading to a controversal environmental concerns.It is necessary to evaluate and control the off target effect of RNAi in insecta.Here,we use Tribolium castaneum as target,through choosing suitable reagents to induce RNAi effect,then we used microarray to investage the off target effect triggerred by dsRNA in T.castaneum.Besides we evaluated the accuracy of"dsCheck",a major tool to predict the off target effect by dsRNA.Hence we detected the relationship between of off target effect and two sequence-related characterics of dsRNA.In addition,we also verified the existance of sequence-independment off target effect induced by dsRNA.The presence of our results provided theories basic to design efficient dsRNA and gain deeper understanding of off target effects,enhance the application of RNAi in molecular biology and pest management field.1.The comparasion of RNAi effects triggerred by dsRNA and siRNA in Tribolium castaneumBoth dsRNA and siRNA were widely used in laboratory studies to knockdown target genes.But with intact insects we found siRNA could hardly trigger phenotypic changes.In order to confirm it and select the efficient way for insect RNAi,experiments with Tribolium castaneum were carried out to compare the efficiency of dsRNA and siRNA.Four dsRNA and 10 siRNA targeting at the same 3 genes were tested by checking the suppression of target genes and the deformation of treated insects.The results showed that injection of dsRNA could reduce the expression of target genes by 55-75%for more than 7 days,and make 78.8-87.0%tested larvae deformed.Injection of their corresponding siRNA targeting the same gene did result in about 45%silence of target genes,but only in a short period from day 2 to day 4,and did not trigger any phenotypic changes.From our results,we can see the siRNA were less efficient than their corresponding dsRNA.Most of them could hardly trigger any phenotypic changes because of its temporary silence effect on target genes.Otherwise,the efficiency of different siRNA varied with their matching sequence,and careful selection were necessary for proper use.2.Detection of off target effect triggerred by dsRNA in Tribolium castaneumRNA interference has truned into a powerful tool in pest management field by GM plant and expression of dsRNA in bacterials.However,there are few researches about the off target effect induced by dsRNA in insecta.Here,in this study a beetle custom microarray based on Refseq database of T.castaneum was used to evaluate the mRNA expression level of larvae beetle treated with two dsRNA target the same gene TcChil in different site with control.We have found similar amounts of off target genes in two treatments resulted in 217 and 225 genes significantly regulated(2 times foldchange and significantly different expressed)including 141 and 151 genes down-regulated.Most of those genes were identified as being associated with metabolic process as well as cellular process,response to stimulus,biological regulation.A bioinformatics analysis was undertaken to analyise the off target genes.Here based on the sequence similarity matching,we found the off target effect in T.castaneum triggerd by dsRNA has nothing to do with the matching between the seed aera of siRNA and UTR sequence.However,we found the genes sharing more than 90%similarity of 19nt sequence with dsRNA,the possibility of off target effect was much higher than others.Subsequent,among all the off target genes,we have found the one gene related in immune response,future studies demonstrated one gene was participating in the immune response by dsRNA,hence we presumed the immune response can be induced by RNAi.From all we have discussed above,the RNAi response triggered by dsRNA in T.castanem can induce off target effect by different ways and the genes sharing high similarity with dsRNA having higher possibility in inducing off target effect.Our study would improve the design of dsRNA and gained deeper understand in off target effects triggered by dsRNA,this would lead to a better usage of RNAi as a practical tool upon experimental verification.3.Inspection of the accuracy of "dsCheck" in predicting off target effectIn order to enhance the specifity of RNAi and predict the occurance of off target effect,lots of algorithms were developed to predict the off target effect by bioinformaticians,among those the dsCheck was specific one to evaluate off target effect induced by dsRNA.Based on the two dsRNAs used in previous study and compared with the results from microarray,we want to verfiy the accuracy of dsCheck and give suggestions on modification.In the presecne of our results,we predicted 691 and 519 off target genes with the theory of dsCheck respectively,by comparing with the results in microarray,only 10 and 14 genes with down regulated were found.Here we found the accuracy of dsCheck was decreased with the decreasing of similarity between the sequence of siRNA from dsRNA and mRNA.When the mRNA shared 94%,89%or 84%similarity with dsRNA,the predicted accuracy of two dsRNA checked here was 14.3%and 33.3%,2.6%and 8.3%or 1.0%and 1.7%.Then we conducted the RNAi experiment in Drosophila melanogaster adults and S2 cell lines with dsWg,among the 23 off target genes predicted by dsCheck we detected 12 and 3 genes with depressed expression,therefore we presume the mechanism of off target effect in living insect and tissue culture was different.In presence of our data,we demonstrated that the low accuracy of off target genes predicted by dsCheck.A major problem is the software relied on the similarity between siRNAs from dsRNA and mRNA sequence without deeply understanding the mechanism of off target effect induced by dsRNA.Hence,future study should be conducted by enhancing its algorithms with up to date mechansim of off target effect.4.Key molecular characteristics of dsRNA for its off target effectThe unintended corss-reaction between dsRNA and transtripts other than target one was referred to the off target effect which make it complicate in interpreting RNAi data.Here based on the deplection level of non-target gene,we evaluated two key molecular characteristics of dsRNA for its off target effect.The results showed that in Tribolium castaneum,off' target effect become increasing when an mRNA sequence hosts a fragment shearing more than 50%similarity with dsRNA,and reaches as high as 100%as the target gene when shearing more than 80%similarity.Subsequent mosaic molecule tests showed that the dsRNA with one piece of more than 40 bp or two pieces of more than 30 bp interrupted matching sequence could trigger RNAi efficiently,but those with one 30 bp or multiple pieces of less than 20 bp fragments could not.Our results provided two molecular criterions for evaluation of the possibility if a dsRNA could induce off target effect in an intact insect pest,which will improve the design of dsRNA and gain deeper understanding in off target effects,and lead to a better usage of RNAi.5.Exploration of non specific sequence-independent off target effect in RNAiInsect immune response is the ability to defense infection from outside world.The mechanism of RNAi is evolutionary conserved defense response against virus infection or dsRNA structure molecules,however it is disputable for RNAi in inducing immune response.Here in this study,we slected 15 immune response-related genes to detect the expression levels after injected with exogenous non-related dsRNA 24 hours in Tribolium castaneum and found Defensin1 and Defensin2 were highly expressed.Then we confirmed those two up-regulated genes induced by RNAi immune response with different length and doseage dsRNA.Therefore,we provided the evidence that the sequenced-independent off target effect could be induced by immune response when applied dsRNA in T.castaneum RNAi experiment.