| RNA interference(RNAi)technology has been widely employed for pest control since its discovery.When feeding dsRNA targeting pest essential gene,suppression of these genes by the RNAi pathway may result in growth retardation or even death of the pest.There are several factors that can influence the efficacy of RNAi responses in insects,such as sequene and length of dsRNA,administered dose of dsRNA and delivery method.Plant-mediated RNAi shows great potential in the field of crop pretection.Due to the lack of RNAi mechanism in plastids(chloroplasts),high-level dsRNAs can stably accumulate in this organelle,therefore a potent insecticidal activity may achieve.The Colorado potato beetle is one of the most important quarantine pests in the world,it causes great economic and productive damage to the potato industry after its invasion in China since 1990 s.Previous studies indicated that plastid-mediated RNAi was an effective approach to control Colorado potato beetle,which is sensitive to RNAi.However,the factors that could influence the RNAi efficiency in Colorado potato beetle remain unclear.This study focused on three factors impacting on the RNAi efficiency in Colorado potato beetles,including the length and location of dsRNA,as well as nucleotide mismatch tolerance between dsRNA and target mRNA.To achieve this goal,dsRNAs of different lengths and different sequences,and mutated dsRNA with up to 3% nucleotide mismatches were synthesized in vitro to feed Colorado potato beetles.Simultaneously,different-length dsRNAs expressed from potato plastid genome were fed to Colorado potato beetle to RNAi efficiency examination.The research results are as following:1)The length of dsRNA affects RNAi efficiency.When feeding Colorado potato beetle with in vitro-synthesized dsRNA targeting its β-actin gene(ds ACT),200 bp or longer(297 bp,400 bp and 700 bp)dsRNA showed stronger insecticidal activity compared with that of 60 bp dsRNA.However,no significant difference of RNAi effects was observed between 200 bp and longer dsRNA,including post-feeding survival,weight change and down-regulation of target gene.Namely,the RNAi efficiency of dsRNA of different lengths in vitro was ds ACT60<ds ACT200≈ds ACT297≈ds ACT400≈ds ACT700 of length ranged2)RNAi efficiency is influenced by the location of dsRNA.The survival rate of Colorado potato beetles was markedly different when they fed with in vitro-synthesized dsRNA targeting different locations with respect to the mRNA sequence.Deep analysis suggested that higher RNAi efficiency might be attributed to more siRNA populations derived from dsRNA.3)RNAi system can tolerante 3% nucleotide mismatches in Colorado potato beetle.In vitro-synthesized dsRNA with up to 3% nucleotide mismathches did impair RNAi effects in Colorado potato beetle.These included survival rate,weight change and suppression of target gene.4)The length of dsRNA affects the production of dsRNA in plastids thus insecticidal effect.By using Northern blot analysis,we found that the accumulation of dsRNA negatively correlated with the lengths of dsRNA ranging from 200 to 700 bp.Remarkly,dsRNA accumulated to as much as 0.6% of the total cellular RNA in transplastomic potato expressing200-bp ds ACT(St-ACT200).In agreement with high-level dsRNA,the insecticidal activity against Colorado potato beetle was strongest in St-ACT200,followed by St-ACT297,StACT400 and St-ACT700.Taken together,multiple factors were found to affect RNAi efficiency in Colorado potato beetles,including a negative correlationship between length and accumulation of dsRNA produced in plastids,the optimal length for expressing high-level dsRNA in plastids,and the importance of high-level expression of dsRNAs for pest control.Our data laid a foundation for the subsequent dsRNA designing for plastid-mediated RNAi against pests. |
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