Eukaryotic Expression Of Dicer2 And Its Interacting Proteins In Plutella Xylostella

Dicer is an endoribonuclease that belongs to the RNase III family.It is highly conserved in evolution and is one of the important elements in RNA interference.In Drosophila melanogaster,Dicer1 and Dicer2 have cofactors interacting with them in the mi RNA pathway and si RNA pathway,separately,such as Dicer1 / R3D1-L,Dicer2 / R2D2.In contrast,Dicer and its interacting proteins of Plutella xylostella have not been studied.Therefore,this experiment focuses on the research of Px Dicer2,using the Bac-to-Bac baculovirus expression system to express the protein of interest in vitro,and to obtain possible interacting proteins by co-immunoprecipitation.The RNAi of RNAi technology was used to analyze the effect of possible interaction proteins on the RNAi efficiency in P.xylostellaThe full-length sequence of Px Dicer2 c DNA was obtained by two-step PCR amplification.The ORF of Px Dicer2 was of 5085 bp,encoding 1694 amino acids.Px Dicer2 enzymes contain a helicase domain,Dicer dimer domain,PAZ domain,and two RNase Ⅲ domains,but lacking a ds RBD domain(double-stranded RNA binding domain).Using the Bac-to-Bac baculovirus expression system,the fusion protein EGFP-Dicer2 was successfully expressed with a protein size of about 230 KD.Co-immunoprecipitation was performed using GFP-Trap?＿A beads in the lysates of P.xylostella cells expressing the fusion protein EGFP-Dicer2 and 48 proteins that may interact with Dicer2 were identified from the co-precipitated proteins.The 48 possible proteins interacting with Dicer2 were annotated with Gene ontology(GO analysis),and the results showed that in terms of molecular function,42% and 39% of the proteins were involved in catalytic function and binding,respectively;in cells In terms of composition,33%,17%,and 15% of the proteins are classified as cell parts,protein inclusion complexes,and membrane parts,respectively;in terms of participating biological processes,39%,30%,and 15% proteins are involved in cellular processes,metabolic processes,and biological regulation.Among the 48 proteins,8 candidate genes were selected for RNAi experimentusing Px Cht as the reporter gene.The experiment showed that after simultaneous ds RNA injection of one of four candidate genes(0356,4974,3445,4479)and Px Cht,the expression level of Px Cht was significantly increased at different time points,indicating that these four candidate genes may participate in the RNAi pathway through interacting with Dicer2,affecting the efficiency of RNAi.The interaction between Dicer2 and four genes needs to be further studied at the protein level.