Expression Analysis Of AGO Genes With RNAi In Bombyx Mori Cells

Argonaute is a highly basic protein family, containing mainly four domains, the N-terminaldomain、the PAZ domain、the middle domain and the PIWI domain. PIWI is an RNaseH domain. It cancleave the mRNA interacting with siRNA (small interfering RNA); PAZ binds unspecially thetwo-nucleotide3’overhang of an siRNA, so AGO involves in the gene regulation, and AGO is the core ofRISC (RNA-induced silencing complex). The research of AGO will make the machenism of generegulation known well, and promote the cure of diseases and the research of genetics.Bombyx mori is one of the earliest domesticating animals, it has great economic value.Meanwhile as the modle of the Lepidoptera insects, silkworm plays an important role in the study of theholometabolous development and the pest controls. At the current, we know silkworm contains four AGOproteins, they are AGO1、AGO2、AGO3and PIWI. However, their functions are not very clear. In theresearch, we chose Bombyx mori cells as the object, constructed the RNAi vector, tried to the functions ofAGO1and AGO2, meanwhile provided the basis to study the mechanism of target genes and the smallRNA(siRNA、miRNA).We identified the structure of RNAi vector and constructed the transgenic RNAi vector whichcan produce stable double-stranded hairpin RNA (hpRNA) in silkworm cells, hpRNA was cleaved intosiRNA by Dicer. Mature siRNA is further loaded into RISC, RISC identified target messages and results inendonucleolytic cleavage of targeted mRNA or translational repression. In the experiment,we used vectorpBac[A3-EGFPafm] to construct RNAi vector. RNAi vector and helper plasmid phsp were transfected intoBmN cells and interfer Ago gene expression. Meanwhile, plasmid phsp could express transposase whichcan integrate hpRNA into genomes of BmN cells. We used liposome transfection Cellfectin II Reagen to transfect RNAi vector and phsp.westarted to detect fluorescence after transfection96h.BmN cells were spindle-shaped cells at normal, butmany of BmN cells with fluorescence turned rounder and bigger comprising cells without fluorescence.After7days we collected BmN cells,extractec total RNA and did real-time PCR. Results showed that theexpression of Ago1and Ago2decrease, then the cells turned rounder and bigger96hours after transfection.