Cloning, Subcellular Localization, And Analysis Of Tissue Expression Of Bombyx Mori Adiponectin Receptor

Adiponectin receptor (AdipoR) appears to be a membrane protein. The coaction between AdipoR and adiponectin plays a number of beneficial roles in metabolism: improved insulin sensitivity, glucose tolerance and lipid profile, decreased inflammation and atherosclerosis.Adiponectin receptor is conserved from yeast to man. However, adiponectin receptors in lepidoptera species have not been studied in the past. In this study, the in silico cloning method was used to get a new BmAdipoR gene of Bombyx mori and analyzed it with bioinformatics tools. BmAdipoR was expressed by using Bombyx mori nucleopolyhedrovirus bacmid system to detect its subcellular localization. We also determined its expression in various tissues and investigated the effect of treatment with Bombyx mori nucleopolyhedrovirus (BmNPV) at 24hpi (h post infection), 48hpi and 72hpi on the expression of BmAdipoR mRNA in the BmNPV susceptible strain (306) by real-time quantitative PCR.1. We obtained a novel cDNA sequence, which named BmAdipoR by in silico cloning approach from Bombyx mori EST database of GenBank and verified it by experiments. Structural analysis of the translated cDNA suggested it encoded membrane protein with seven transmembrane domains with its N termini as internal parts and its C termini as external parts. Phylogenetic analysis revealed that BmAdipoR closely related to AdipoR of other spieces. 2. The BmAdipoR gene was inserted in the silkworm baculovirus expression vector system transfer vector pFastBacHTb and constructed the recombinant transfer vector pFastBacHTb- BmAdipoR-EGFP. The purified pFastBacHTb-BmAdipoR-EGFP DNA was transfected DH10Bac. Homologous recombination occurred inside DH10Bac. Plasmid DNA was isolated and confirmed by PCR using puc/M13 primers. BmN-4 cells were transfected with recombinant bacmid using Lipofectamine Reagent to produce the recombinant baculovirus vBm-BmAdipoR-EGFP. BmNPV bacmid system combined with confocal microscopy revealed that BmAdipoR was targeted to cell membrane.3. BmAdipoR mRNA expression in various tissues was quantified by real-time quantitative PCR, and BmAdipoR mRNA expression was the highest in Malpighian Tubules, fat body, and testis, followed by ovary, blood, silk gland and midgut. We also investigated the effect of treatment with BmNPV at 24hpi, 48hpi and 72hpi on the expression of BmAdipoR mRNA in the 306 midgut by real-time quantitative PCR, and found that infection with BmNPV did not effect on BmAdipoR mRNA quantity in the midgut of 306 in 48 h, but significantly increased at 72 h.