Screening For DNA-Binding Proteins Involved In DNA Interference In Plutella Xylostella

DNA interference（DNAi）is a phenomenon of gene silencing caused by homologous DNA,which has been reported in a variety of organisms.DNAi in prokaryotes is mediated by Argonaute proteins,but the mechanism of DNAi in eukaryotes has not been reported.Plutella xylostella is one of the most destructive pests in cruciferous crops and confirmed that it has DNAi phenomenon.In this study,the possible role of Argonaute in DNAi was investigated and the DNA-binding proteins involved in DNAi were explored by tanscriptomic,proteomic and RNAi techniques in P.xylostella.The main results are as follows:1.Four Argonaute family genes were identified in P.xylostella,as well as their sequence characteristics,genetic relationships and spatio-temporal expression patterns.（1）Four Argonaute family genes PxAgo1,PxAgo2,PxAgo3 and Px Piwi were cloned and identified by RACE-PCR and sequencing.The former two belonged to AGO subfamily,while the latter two belonged to PIWI subfamily.PxAgo1 had four variable spliceosomes encoding two proteins,both of which contained Argonaute linker 1 domain,N-terminal domain,PAZ domain and Piwi domain.PxAgo2 had the same structural domain as PxAgo1.PxAgo3 contained PAZ domain and Piwi domain.PxAgo2 and PxAgo3 had no variable spliceosomes.Px Piwi had two variable spliceosomes,encoding two proteins with the same structural domain as PxAgo3.（2）Phylogenetic tree showed that PxAgo1 X1,PxAgo1 X2 and PxAgo2 were on different subbranches of AGO subfamily branch.Px Piwi X1,Px Piwi X2 and PxAgo3 were on different subbranches of PIWI subfamily branch.The Argonaute proteins of P.xylostella were clustered with the corresponding Argonaute proteins of other lepidoptera.Some prokaryotic Agos（p Agos）and eukaryotic Agos（e Agos）were clustered into one branch.Insect Ago2 and other e Agos were on two different branches.Compared with Piwi of insect,PxAgo3 were more closely related to PIWI subfamily proteins in other eukaryotes.（3）The spatiotemporal expression patterns of Argonaute family showed that PxAgo1,PxAgo2,PxAgo3 and Px Piwi were all highly expressed in the pupal stage.The highest expression levels of PxAgo2,PxAgo3 and Px Piwi were in midgut,testis and testis,respectively.In the same tissue,the expression levels of PxAgo2 and Px Piwi were generally higher than those of PxAgo1 and PxAgo3.2.The phenomenon of DNAi in P.xylostella cells was confirmed,and the binding protein of DNA was captured by streptomycin-biotin method.（1）The results of cell transfection showed that the ss DNA of arginine kinase gene of P.xylostella（PxAK）and the ss DNA and long-chain PCR products of Bursαof P.xylostella（PxBursα）both inhibited the expression of the corresponding target genes in P.xylostella cells,resulting in DNAi phenomenon.（2）Analysis of the captured DNA-binding proteins revealed that no Argonaute family members were identified in the acquired proteins.A total of 197 proteins were found in the combination of the two experimental groups without their own control.These 197 proteins in the GO annotation of molecular function term mainly belonged to the three categories of binding,catalytic activity and structural molecular activity.These 197proteins in GO term were enriched in a variety of ribosomes related items and translation item.Most of these proteins were located in the translation pathway of genetic information processing pathway by KEGG pathway analysis,and significantly enriched in the two pathway,ribosome and systemic lupus erythematosus.Pfam structure analysis revealed that a total of 20 proteins containning OB-fold Pfam structure,which was necessary for ss DNA-binding protein.3.The transcriptomic analysis of P.xylostella injected with the short ds DNA fragments of PxBursαexon was performed and a total of 46candidate proteins were screened combined with DNA pull-down analysis.（1）A total of 220 differentially-expressed genes（DEGs）were identified compared with those injected with H₂O,and 261 DEGs were identified compared with those injected with short ds DNA of EGFP.These two groups of DEGs shared 32 DEGs.GO function annotation showed that DEGs screened with H₂O as the control and screened with EGFP as the control both belonged to the GO term of binding and catalytic activity of molecular function and the metabolic process of biological process;the KEGG pathway annotation showed that the most of DEGs of the two groups belonged to the two pathways of signal transduction of environmental information processing and global and overview maps of metabolism.（2）The 46 candidate proteins included 12 proteins with OB-fold Pfam and 21 proteins with typical ds DNA-binding Pfam,among which there were four RNA or DNA helicases,two RNA or DNA polymerases,three DNA replication related proteins,five translation or transcription related proteins,three histone related proteins,and three carboxylase related proteins.In conclusion,this study identified Argonaute family members of P.xylostella,and transcriptome sequencing and DNA-protein interaction method were used to screen proteins that might be involved in DNAi phenomenon,which laid a foundation for the subsequent research on DNAi mechanism.