Molecular cloning and characterisation of a new insect ecdysone receptor from the green vegetable bug, Nezara viridula
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NO FULL TEXT AVAILABLE. This thesis contains 3rd party copyright material. ----- The ecdysone receptor, a heterodimer of the nuclear receptors EcR and USP/RXR, binds ecdysteroids and plays a central role in controlling gene expression during the development of insects. The green vegetable bug, Nezara viridula (Nv), is a significant insect pest and a representative of three important levels of insect taxonomic classification, namely the order Hemiptera, suborder Heteroptera and infraorder Pentatomomorpha. The present study is the first to report the cloning, expression and characterisation of an ecdysone receptor from a heteropteran insect. A high-quality cDNA library was constructed and cDNA clones encoding two isoforms of NvEcR and a single NvUSP were isolated and sequenced. Phylogenetic analysis of NvEcR and NvUSP found each to group with other less evolved insect EcRs and USP/RXRs, respectively. The NvEcR isoforms, termed NvEcR10 and NvEcR11, were found to differ in the A/B domain due to use of alternative initiation of translation sites, whereas a dipeptide variation, found in their respective E domains, was attributed to alternative splicing. Co-expression of the His6-tagged ligand binding region (LBR, comprised of D and E domains) of both NvEcR isoforms with the FLAG-tagged LBR of NvUSP, was successfully achieved by infecting insect cells with appropriately constructed recombinant baculoviruses. LBR heterodimers of NvEcR10 and NvEcR11 with NvUSP, termed NvE10 and NvE11, were purified by affinity chromatography via the His6 tag of NvEcR. The dipeptide variation in NvEcR subunits of NvE10 and NvE11, predicted by conceptual translation, was confirmed by matrix assisted laser desorption ionisation mass spectrometry. NvE10 and NvE11 were demonstrated to bind the tritiated ecdysteroid, ponasterone A, with similar nanomolar affinity (Kd = 6.8 ± 0.8 nM and 7.5 ± 0.8 nM respectively) characteristic of ecdysone receptors. The ability of a range of ecdysteroids to competitively inhibit the binding of tritiated ponasterone A to NvE10 and NvE11 was determined. Both purified heterodimers showed the same affinity ranking for these ecdysteroids, viz muristerone A > ponasterone A > 20-hydroxyecdysone ~ makisterone A > inokosterone > α-ecdysone, distinguishing them from other cloned hemipteran ecdysone receptors. The Pentatomomorpha, contrary to most insects, have been suggested to utilise makisterone A instead of 20-hydroxyecdsyone as the major moulting hormone. The present study is the first to examine this postulate at the level of the ecdysone receptor. The relative affinities of makisterone A and 20-hydroxyecdysone were carefully studied. Both ecdysteroids displayed similar affinities for NvE10 and NvE11, consistent with makisterone A being a major moulting hormone in N. viridula.
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