RNA Interference Of Paederus Fuscipes Based On Oral Delivery

In recent years,RNA interference（RNAi）technology has provided a new idea for RNAi-mediated Transgenic Crops.However,the current safety evaluation system is mainly applicable to transgenic Bt crops.The safety evaluation system of RNAi-mediated Transgenic Crops is different from the current transgenic Bt crops,and the current safety evaluation system needs to be adjusted and optimized appropriately.In this study,the important predatory natural enemy of paddy field,Rove beetle（Paederus fuscipes）,was used as the research object.Two methods,ds RNA was entrapped in chitosan-based nanoparticles feeding and ds RNA direct feeding,were used to introduce ds RNA into the midgut of Rove beetle by artificial diet.A RNAi safety evaluation system for feeding Rove beetle was established to provide technical support for the safety evaluation of RNAi-mediated transgenic rice.The results are as follows:（1）The V-ATPase-A gene sequence was obtained by transcriptome sequencing.A total of 57944158 Sequenced Reads were obtained by high-throughput sequencing of the gene library of Rove beetle,Quality control filtering of Sequenced Reads to get clean reads of 55911022.For transcriptome sequencing without reference genome,the longest Cluster sequence was obtained by clustering at Corset level for subsequent analysis.The length of transcripts and clustering sequences were counted,and the number of transcripts and genes was 117734 and 92 540 respectively.Seven databases of Nr,Nt,Pfam,KOG/COG,Swiss-prot,KEGG and GO are used to annotate gene function.There are 52 Pf V-ATPase-A in the transcriptome that can be annotated.The length of V-ATPase-A（Pf V-ATPase-A）gene sequence is 2442 bp,of which 5’UTR length is 152 bp,3’UTR length is 451 bp and coding region is 1839 bp.The complete protein sequence contains 613 amino acids.（2）Feeding method of chitosan encapsulated ds RNA mixed into artificial diet.Chitosan/ds RNA nanoparticles（ds RNA/CS）were prepared and the related indicators of ds RNA/CS nanoparticles detected.The size of nanoparticles observed by transmission electron microscope.The entrapment efficiency of ds RNA/CS nanoparticles detected by centrifugation and gel electrophoresis.The ds RNA release rate of ds RNA/CS nanoparticles detected by DEPC H2O dissociation 48h.The results show that:The diameter of ds RNA/CS nanoparticles is less than 200nm,the encapsulation efficiency is more than 95%,and the dissociation rate of ds RNA is about 12.38%at 48h.The ds Pf V-ATPase-A/CS and artificial feed were mixed evenly,dried to dry powder by freeze-drying,Feeding the 1^stinstar larvae of Rove beetle and replacing the diet every 24 hours.To detect the expression level of Pf V-ATPase A in Rove beetle.The results showed that:The expression Level of Pf V-ATPase-A decreased significantly by 41.3%and 45.3%on the 3rd and 6th day,respectively.ds Pf V-ATPase-A/CS had no lethal effect on Rove beetle larvae.（3）Feeding method of direct incorporation of ds RNA into artificial diet.Artificial feed was prepared by sterilizing the components of pig liver powder.After dissolving ds Pf V-ATPase-A in DEPC H₂O,it is quickly mixed with artificial feed,and then freeze-dried to dry powder.The mixture of ds Pf V-ATPase-A in artificial feed is uniform,and the degradation of ds Pf V-ATPase-A can be effectively avoided.The 1^stinstar larvae were fed with artificial diet containing ds Pf V-ATPase-A,and the diet was replaced every 24 hours.Detection of stability of ds Pf V-ATPase-A in artificial diet after 24 hours.At the same time,the ds Pf V-ATPase-A in the feed was re-extracted and microinjected into the adult to test whether the ds Pf V-ATPase-A in the artificial feed was kept biological activity.The results showed that:There was no significant degradation of ds RNA in artificial diet within 24 hours,and the expression of ds Pf V-ATPase-A extracted from artificial diet could be strongly interfered with by injecting ds Pf V-ATPase-A into adults,and the expression level decreased by more than 90%.This indicated that the ds Pf V-ATPase-A added into the feed maintained good biological activity.Finally,the interference efficiency of ds Pf V-ATPase-A in artificial diet to Pf V-ATPase-A in the larvae was tested.The results showed that the expression of endogenous Pf V-ATPase-A decreased significantly by 75.8%and 72.5%on the 3rd and 6th day of feeding with ds Pf V-ATPase-A.ds Pf V-ATPase-A had a strong lethal effect on the larvae,and the survival rate of larvae fed with ds Pf V-ATPase-A artificial diet was 86.11%lower than that fed with ds GFP.In this study,nanoparticles were added into artificial diet and ds RNA was directly added into artificial diet.Both methods could significantly interfere with the expression of target genes,but the latter had higher interference efficiency and simpler and faster operation.In the future,new materials of RNAi-mediated Transgenic Rice were cultivated.If indoor experiments were carried out to evaluate the safety of RNAi-mediated Transgenic Rice to rove beetle.both technical systems could be selected,but the latter was more efficient and fast.In this study,we established a standardized feeding method for RNAi evaluation using V-ATPase-A as the target gene.It provides a meaningful theoretical reference and necessary basic data for the future safety evaluation technology system of RNAi-mediated Transgenic Rice and the research of rove beetle.