Expression analysis and immunohistochemical localization of putative tumor suppressor QM homologue from the cabbage butterfly, Pieris rapae

Abstract

The tumor suppressor, QM has been cloned and characterized from eukaryotic organisms including humans, vertebrates, invertebrates, plants and yeast. However, no study on Pieris rapaeQM (PrQM) has been reported to date. In this study, cDNA encoding a putative QM protein (PrQM) was obtained from expressed sequence tags (ESTs) of the Pieris rapaecDNA library. Phylogenetic analysis of PrQM showed high similarity of amino acid sequence with other putative homologues identified from Heliothis virescens (95%), Bombyx mori (92%), Plutella xylostella (92%), Drosophila melanogaster (89%) and Polyrhachis vicina (85%), indicating that QM is highly conserved among insects. Semi-quantitative PCR datasets revealed that PrQM transcripts are highly expressed in head, silk gland, integument and fat body, with pronounced expression observed during the egg stage. The coding region of the PrQM gene was cloned into the pET28 (+) expression vector and the recombinant protein purified by His-tag affinity chromatography was used for antibody production. Western blotting with the anti-PrQM antibody detected a single band corresponding to the expected molecular weight of both endogenous (26kDa) and recombinant (29kDa) PrQM. Furthermore, immunohistochemical and confocal microscopic analysis with the anti-PrQM antibody showed that the QM gene is highly expressed in the cytoplasm of fat body, gut and Malpighian tubules in virus-uninfected control larvae, but down-regulated at 4 days post Pieris rapae granulovirus (PiraGV) infection. These data show that PrQM is negatively regulated upon virus infection and suggests a putative immunomodulatory function.