Molecular Cloning,Expression Profiling And Functional Studies Of BmAGO1 Gene In Silkworm,Bombyx Mori

RNA-induced silencing complex（RISC）is a ribonucleoprotein complexes,which contains Argonaute（AGO）protein.RISC can bind mature microRNA（miRNA）and mRNA of its target gene,resulting in gene silence or degradation in post-transcriptional level,and thus participate in regulation of gene expression.AGO family proteins are the core of RISC complex and the key protein of functional miRNA pathway,which can bind miRNA and cut its binding target genes and play an important role in the biological process of growth and development.Presently,the isolation of AGO-binding RNA by immunprecipitation is becoming the main method for the isolation and identification of miRNA target genes.A new gene was screened from the cDNA library of silkworm pupa in our laboratory,which contains an open reading frame（ORF）of 2739 bp encoding 912 amino acid residues with a predicted molecular mass of 101.5 kDa and a isoelectric point of 9.26.The predicted protein contains two conserved domains:PAZ and PIWI,and has high homology with AGO1 protein of many other species;so we named it BmAGO1（Accession number in Genbank is NM₀01102461）.The high antigenicity domain named Kago-1 of BmAGO1 is analysed by DNAstar and amplified by PCR,which was cloned into the prokaryotic expression vector pET-28a and the recombinant plasmid was transformed into E.coli BL21（DE3）.After being induced with IPTG,this gene was successfully expressed.The molecular weight of this fusion protein was about 27 kDa by SDS-PAGE（His tag was weight of 3.56 kDa,and Kago-1 was weight of 23.99 kDa）and was identified by mass-spectrum.After being purified with Ni2+ aiffinity chromatography,the purified fusion protein was used to immunize a male New Zealand rabbit.The titer of the polyclonal antibody reaches 1:25600 measured by ELISA.Total proteins of the different developmental stages of silkworm and the different tissues of the fifth instar larvae were extracted for expression profiling.The results showed that the distributing of BmAGO1 in four stages of silkworm is different with the highest in the pupa and the lowest in the egg.In the fifth instar larvae,BmAGO1 protein was only found in four different larva’s tissues:the head,skin,silk gland and markov.Subcellular localization of BmAGO1 was shown to be mainly presented in cytoplasm of the BmN cells,while less in nucleus.Further down-regulating BmAGO1 expression by RNA interference in BmN cells,the cell viability was increased after RNAi by MTT assay;flow cytometry cytometry analysis showed that the number of cells in S phase after RNAi was increased compared with the control group,indicating BmAGO1 and its binding miRNA may be involved in cell cycle regulation.AGO binding RNA contain miRNA and its target gene mRNA,so the miRNA target mRNA isolation by AGO immuneprecipitation is the effective method for identifying miRNA target genes in large-scale.We used two methods to separate AGO binding RNA:（1）Using the above prepared antibody,AGO1 binding RNA were isolated by immunoprecipitation from silkworm tissues.As BmAGO1 was expressed in head and skin of silkworm in a high level,the BmAGO1 binding RNA were co-immunoprecipitated using BmAGO1 antibody from head and skin of silkworm.Three potential miRNA target genes,BmEm4,Bm（spl）-like and BmEmc,were identified in BmAGO1 binding RNA by qRT-PCR.Compared with the control,BmEm4,Bm（spl）-like and BmEmc in BmAGO binding RNA were enriched 3.52,1.56 and 0.75 times,respectively;（2）the BmAGO1 can be expressed in BmN cells with HIS tags.Using anti-HIS monoclonal antibody,the HIS-AGO binding RNA could be co-immunoprecipitated.The ORF of BmAGO1 was cloned into the eukaryotic expression vector pIEx-1,and the recombinant plasmids was transfected into BmN cells by liposome-mediation.BmAGO1 is expressed in the cells,successfully,then HIS-BmAGO1 binding RNA was immunoprecipitated using anti-HIS monoclonal antibody.The same genes,BmEm4,Bm（spl）-like and BmEmc,was identified in HIS-BmAGO1 binding RNA by qRT-PCR.Compared with the control,BmEm4,Bm（spl）-like and BmEmc in HIS-BmAGO binding RNA were enriched 5.04,1.08 and 0.63 times,respectively.These results indicated that BmAGO1 binding RNA could contain a high level of BmEm4 and a small amount of Bm（spl）-like.Previous studies have shown that miR-7 and miR-79 binding sites were existed in the 3’UTR of BmEm4,which further confirmed that the BmEm4 is the target gene of miRNA.Presently,there was little related work about identification of miRNA target genes in silkworm.This work lays the foundation for the large-scale identifying miRNA target genes in silkworm,Bombyx mori.