The Selection Of Candidate Gene Controlling Tribolium Castaneum By Bacteria-derived DsRNA And Its Assessment Of Non-target Effect

In recent years,the research on insect RNA interference（RNAi）technology has achieved unprecedented development,and the pest control methodsbased on RNAi have drawn widely attention.At present,the main factors restricting the application of dsRNA in pest control are efficient and low-cost dsRNA synthesis methodsand the safety risk of dsRNA.Based on this,this paper firstly analyzed and summarized the research hotspots of global insect RNAi studies by Cite Space software,determined to take Tribolium castaneum as the research object,established the method of feeding T.castaneum with bacteria-derived dsRNA,and screened out the dsRNA fragments to control Tribolium castaneum.Also,the non target effect of the dsRNA was evaluated.This study will provide a new method for the integrated management of T.castaneum,and provide a certain data basis for the evaluation of non target effect of RNAi of T.castaneum,and provide a theoretical basis for the safety application of dsRNA in pest control.The main results are as follows.1.Screening of dsRNA for controlling T.castaneum expressed by bacteria.The recombinant expression vectors of four candidate genes dsRNA of T.castaneum were constructed and expressed in E.coli.It was found that dsTcCht10 and dsTc VATP-C had significant effects on the survival rate and gene transcription level of T.castaneum larvae when fed with RNA bulk mixture of E.coli.Then the concentration gradient of dsTcCht10 was tested,and the LC₅₀of dsTcCht10 was calculated to be 105.5 ng/mg.The dsRNA fragment may have a certain application potential in the control of T.castaneum.2.Analysis of the transcriptome of T.castaneum fed with dsTcCht10Through the transcriptome sequencing,the gene expression of T.castaneum fed with dsTcCht10 was analyzed.The results showed that after effective silencing of TcCht10,besides the significant decrease of the expression of TcCht10,the expression of the remaining 52 genes related to the epidermis also changed significantly.In addition,dsTcCht10 may also affect the transcription level of other non-target genes of T.castaneum.In the future,it can be combined with new sequencing methodssuch as degradome and in-depth analysis and verification based on the whole genome to better understand the off-target effects of dsRNA or si RNA.3.Evaluation of the effects of dsTcCht10 on two non-target organisms.Different concentrations of dsTcCht10 were fed to two non-target organisms,Nasonia vitripennis and Habrobracon hebetor,and the dsRNA of the homologous genes of the non-target organisms was used as a positive control.The results showed that dsTcCht10 feeding had no significant effect on the survival rate of these two non-target organisms,but 1000 ng/μL of dsTcCht10 had a significant effect on the transcription level of Cht10 gene in N.vitripennis.This may be related to the sequence similarity between dsTcCht10 fragment and the homologous gene of N.vitripennis,and there is a continuous nucleotide sequence match between them.