The Impact Of Silencing Of DsRNase On The Efficiency Of Plastid-mediated RNAi For Control Of 28-spotted Ladybird Beetle,Henosepilachna Vigintioctopunctata(Fabricius)

After ingested by pests,exogenous double-stranded RNA（dsRNA）can induce RNA interference（RNAi）through small interfering RNA（si RNA）pathway and down-regulate the expression of pest target genes,resulting in growth retardation or even death of pests,so as to effectively protect crops.In recent years,the plastid transformation technology has been successfully achieved in a variety of plants,and it has become a hot topic to express dsRNA targeting the essential genes of target pests by plastid-mediated RNAi（PM-RNAi）for pest control.The dsRNA targeting the essential gene of coccinellid 28‐spotted potato ladybird（Henosepilachna vigintioctopunctata,HV）in the transpalstomic potato plants can cause 100%mortality of the second instar HV larvae after feeding the transpalstomic plants to the leaf beetle.However,the resistance of late instar larvae to transpalstomic plants was higher than that of early instar larvae.Double-stranded ribonuclease（dsRNase）in pests can degrade dsRNA,which leads to reduced insect resistance efficiency of RNAi.This may be one of the reasons why the late instar larvae are relatively insensitive to PM-RNAi.Knockdown of dsRNase by RNAi method can improve the resistance of transpalstomic potato plants with low or medium insect resistance efficiency to Colorado potato beetle（CPB,Leptinotarsa decemlineata）.The two beetles belong to the order Coleoptera.Whether dsRNase is also present in HV,and if so,whether the RNAi resistance efficiency of transplastomic plants can be improved after silencing dsRNase becomes the subject of this study.To solve the above problems,the following experiments were carried out in this study,and the experimental contents and results were as follows:1.RNAi target genes of HV were screened in vitro,soluble NSF-attachment proteins（Snap）andβ-actin（ACT）were selected,and dsRNA was synthesized in vitro.Ds RNA specific to green fluorescent protein（ds Gfp）and H₂O as the control.It was found that both of the two target genes were effectively prevents HV,and the insect resistance level was ds Actin>ds Snap.2.According to the above results,plastid transformation vectors for ds Actin and ds Snap expression were constructed.The vectors were bombarded to potato plastids by Biolistic Particle Delivery System.The leaves of transplastomic plants were fed to the second instar HV larvae,and the resistance of the transplastomic plants to HV was consistent with the results of in vitro bioassay:St-ds Actin>St-ds Snap.Considering that the silencing of dsRNase of CPB could only improve the resistance of transplastomic plants having weak or moderate strength of the insecticidal RNAi effect,St-ds Snap showing relatively weak resistant level to HV was selected to carry out follow-up experiments.3.Total RNA of H.vigintioctopunctata was extracted for transcripsome analyses.Two orthologs of dsRNase were found in H.vigintioctopunctata,namely HvdsRNase1 and HvdsRNase2,respectively.Subsequently,we conducted analysis of the developmental stage-specific and tissue-specific expression profile of HvdsRNase genes,the expression of HvdsRNase1 was significantly higher in the 4^th instar,while HvdsRNase2 were highly expressed in adults,and both HvdsRNase1 and HvdsRNase2 were highly expressed in the midgut of larvae.4.DsdsRNase1,dsdsRNase2,and the fusion fragment dsdsRNase1/2 were synthesized in vitro.In order to determine the effeciency of down-regulation of HvdsRNase gene after feeding on dsdsRNase,potato leaves coated with dsdsRNase1,dsdsRNase2,dsdsRNase1+dsdsRNase2,dsdsRNase1/2 were fed to HV.At the 2^nd,3^rd and 4^th day,the expression of HvdsRNase gene in the samples was analyzed,and it was found that dsdsRNase1+dsdsRNase2 and dsdsRNase1/2treatment could significantly down-regulate the expression of HvdsRNase1 and HvdsRNase2genes simultaneously.5.RNAi-of-RNAi.In the following bioassay,third-instar HV larvae were fed with St-ds Snap leaves painted with dsdsRNase1/2,St-wt leaves painted with H₂O were applied as control.It was found that there was no significant change between the two groups of St-wt+dsdsRNase1/2 and St-wt+H₂O.The survival rate,body weight,leaf area consumed by HV and pupation rate of St-ds Snap+dsdsRNase1/2 group were significantly lower than those of St-ds Snap+H₂O group.The vertiginous drop of survival rate of HV larvae in St-ds Snap+H₂O group occurred on the 7^th day,while in St-ds Snap+dsdsRNase1/2 group,it occurred in advance,on the 6^th day.However,when analyzing the gene expression of the test HV larvae on the 5^thday,it was found that the Snap gene of St-ds Snap+dsdsRNase1/2 and St-ds Snap+H₂O had no significant difference.In conclusion,silencing the expression of HvdsRNase genes can further improve the efficiency of insect resistance by PM-RNAi.