Subcellular Localization And Expression Analysis Of A Noval BmARM-like Gene From The Silkworm, Bombyx Mori

Using fluorescent differential display (FDD) technique in before study, we analyzeddifferential expression of genes related to BmNPV resistance among different resistantsilkworm strains and a differentially expressed fragment was obtained. Theexpressed-sequence tag (EST) database of B.mori was screened taking advantage of thespecific cDNA fragment of the result of FDD as query. The full ORF coding region was foundin this EST. In this study, the putative BmARM-like gene (GenBank accession number:JF500112) was reported for the first time. Meanwhile, the putative BmARM-like gene wascloned, expressed, characterized and subcellular localization for the first time, the results areas follows:1. The deduced amino acid sequence of the BmARM-like includes two HEAT repeatsand one Armadillo/beta-catenin-like repeat (ARM) which involved in the the canonical Wntsignalling pathway. Based on the mRNA sequence, the specific primers, were designed for theamplification of the open reading frame (ORF) of an putative BmARM-like gene. Sequencingrevealed that the open reading frame of BmARM-like gene is composed of1923bp, encoding641amino acid residues. The theoretical molecular mass and pI of the putative BmARM-likegene were evaluated to be71.29kDa and pI4.76, respectively, as calculated using theDNAStar program. The derived amino acid sequence showed32.50,24.53,22.27and19.11%identities to amino acid sequences from Apis mellifera, Drosophila melanogaster, Homosapiens and Mus musculus, respectively.2. The gene was cloned into pET-28a vector, then expressed in E coli (BL21).Recombinant fusion proteins were purified by affinity chromatography through thenickel-nitrilotriacetic acid agarose (Ni-NTA) resins. Meanwhile, anti-BmARM-like antiserumwas made.3. To investigate the BmARM-like gene messenger RNA (mRNA) express levels duringthe various stages of silkworm development and in fifth instar larvae tissues, including themidgud, fatty body, haemocyte, testis, ovary and head, Real-time PCR was employed. Theresults indicated that the BmARM-like gene shared a similar expression pattern in the fattybody, testis, ovary and head among the strains relatively susceptible P50and resistant A35,while had a highly significant difference expression level in the midgud and haemocyte.However, during different developmental stages it was higher in larva and lower in egg of thestrain P50.4. In order to study the subcellular localization of BmARM-like gene product in thecontext of BmNPV infection, we used EGFP which is fused to3’ terminus of BmARM-like as the reporter gene, and used baculovirus expression system (BEVS) constructed recombinantviruse: ODV/PH-BmARM-like-EGFP. Using confocal scanning microscope after expressingBmARM-like-EGFP fusion protein, we found that the green fluorescent signal was primarilylocalized in the nucleus of infected cells, however, a weaker signal was also observed in thecytoplasm and membrane.The above results indicated that it had a highly significant difference expression level inthe midgud and haemocyte of the two strains, respectively; this result was coincident that themain anti-viral immune tissues of silkworm are the midgud and haemocyte. BmARM-like-EGFP fusion protein was primarily localized in the nucleus of infected cells, therefore, theBmARM-like gene product may have some important role in the the canonical Wnt signallingpathway. Therefore, This study provides a theoretical basis for further reveal function ofBmARM-like gene of Bombyx mori.