Molecular cloning, expression analysis and functional confirmation of ecdysone receptor and ultraspiracle from the Colorado potato beetle Leptinotarsa decemlineata

Abstract

The growth of insects progresses via unique physiological events such as molting and metamorphosis. Those processes are strictly regulated by two peripheral hormones, molting hormone (20-hydroxyecdysone; 20E) and juvenile hormone (JH). 20E controls transcription of target genes by interacting with molting hormone receptor proteins, which bind to ecdysone response elements (EcREs) located upstream of the target genes. The transcriptional activation by 20E triggers signal cascades, and the development is accomplished via complex regulatory mechanisms [1]. The heterodimer of two nuclear receptors, ecdysone receptor (EcR) and ultraspiracle (USP), functions as a molting hormone receptor, and 20E is known to be a ligand for EcR. USP is the homologue of vertebrate RXR [2,3]. Amino-acid sequences of EcR and USP were first determined in the dipteran fruit fly Drosophila melanogaster [4][5][6], and subsequently determined in other insects [7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24][25], as well as a crustacean [26] and a tick [27,28]. These receptor proteins consist of regions

Edysone receptor isoforms play distinct roles in controlling molting and metamorphosis in the red flour beetle, Tribolium castaneum

“…The EcRA specific sequence from T. castaneum also showed a similarity with EcRA isoform-specific sequences identified in Choristoneura fumiferana (Perera et al 1999), Bombyx mori (Kamimura et al, 1997), Aedes aegypti (Wang et al, 2002), Drosophila melanogaster (Talbot et al, 1993) and Locusta migratoria (Saleh et al, 1998). The EcRB isoform-specific region contained 96 amino acids and these amino acids showed the highest similarity with the amino acids identified in the EcRB isoform of Leptinotarsa decemlineata (Ogura et al 2005). As shown in Table 1, the amino acids identified in the EcRB isoform-specific region of T. castaneum also showed a similarity with the amino acids identified in the EcRB isoforms of Tenebrio molitor (Mouillet et al, 1997), Celuca pugilator (Chung et al, 1998) and Daphnia magna (Accession # AB274824.1).…”

“…Analysis of sequences showed that EcR gene from T. castaneum codes for two isoforms that contain different amino terminal A/B domain and a common region containing CDEF domains. EcRA specific region contained 160 amino acids and showed the highest similarity with EcRA isoform cloned form Leptinotarsa decemlineata (Table 1) (Ogura et al 2005). The EcRA specific sequence from T. castaneum also showed a similarity with EcRA isoform-specific sequences identified in Choristoneura fumiferana (Perera et al 1999), Bombyx mori (Kamimura et al, 1997), Aedes aegypti (Wang et al, 2002), Drosophila melanogaster (Talbot et al, 1993) and Locusta migratoria (Saleh et al, 1998).…”

cDNA cloning of <i>ecdysone receptor</i> (<i>EcR</i>) and <i>ultraspiracle</i> (<i>USP</i>) from <i>Harmonia axyridis</i> and <i>Epilachna vigintioctopunctata</i> and the evaluation of the binding affinity of ecdysone agonists to the <i>in vitro</i> translated EcR/USP heterodimers

“…Total RNA was extracted using TRIzol (Life Technologies) as described previously. 27) cDNAs of EcRs and USPs were synthesized from total RNA with a Ready-To-Go T-Primed FirstStrand Kit (GE Healthcare). Reverse transcription polymerase chain reaction (RT-PCR) was performed with degenerate primers designed in our previous study.…”

“…Reverse transcription polymerase chain reaction (RT-PCR) was performed with degenerate primers designed in our previous study. 27,28) To amplify EcR from H. axyridis, the first PCR was performed using EcR-F1 and EcR-R2 primers with the annealing temperature of 48°C. To conduct the second and the third PCRs (nested PCR), EcR-F2/ R2 and EcR-F3/R3 primers were used with the annealing temperatures of 52°C and 46°C, respectively.…”

“…The ligand binding assay was performed as described previously. 27,29) Briefly, in vitro translated EcR and USP proteins from H. axyridis and E. vigintioctopunctata were incubated with [ 3 H] PonA and a test compound for 90 min at 25°C. A 1000-fold excess of unlabeled PonA was added to measure nonspecific binding, and total binding was obtained for the treatment with a carrier.…”