tailieunhanh - Báo cáo khoa học: Molecular cloning, expression analysis and functional confirmation of ecdysone receptor and ultraspiracle from the Colorado potato beetle Leptinotarsa decemlineata

cDNA cloning of ecdysone receptor (EcR) and ultraspiracle (USP) of the coleopteran Colorado potato beetleLeptinotarsa decemlineata(LdEcR and LdUSP) was conducted. Amino-acid sequences of the proteins deduced from cDNA sequences showed striking homology to those of other insects, especially the coleopteran yellow mealworm Tenebrio molitor. | iFEBS Journal Molecular cloning expression analysis and functional confirmation of ecdysone receptor and ultraspiracle from the Colorado potato beetle Leptinotarsa decemlineata Takehiko Ogura1 Chieka Minakuchi1 Yoshiaki Nakagawa1 Guy Smagghe2 and Hisashi Miyagawa1 1 Division of Applied Life Sciences Graduate Schoolof Agriculture Kyoto University Japan 2 Laboratory of Agrozoology Department of Crop Protection Faculty of Bioscience Engineering Ghent University Belgium Keywords ecdysone receptor EcR Leptinotarsa decemlineata ponasterone A ultraspiracle USP 20-Hydroxyecdysone Correspondence Y. Nakagawa Division of Applied Life Sciences Graduate Schoolof Agriculture Kyoto University Kyoto 606-8502 Japan Fax 81 75 7536123 Tel 81 75 7536117 E-mail naka@ Present address Department of Biology University of Washington Seattle WA 98195-1800 USA Received 27 April 2005 revised 13 June 2005 accepted 16 June 2005 doi cDNA cloning of ecdysone receptor EcR and ultraspiracle USP of the coleopteran Colorado potato beetle Leptinotarsa decemlineata LdEcR and LdUSP was conducted. Amino-acid sequences of the proteins deduced from cDNA sequences showed striking homology to those of other insects especially the coleopteran yellow mealworm Tenebrio molitor. Northern hybridization analysis showed a message for the LdEcR A-isoform a message for the LdEcR B1-isoform and a message for the LdUSP in fat body gut integument testis and ovaries. In developmental profile studies expression of both the LdEcR and LdUSP transcript in integument changed dramatically. In gel mobility shift assays in vitro translated LdEcR alone bound weakly to the pal1 ecdysone response element although LdUSP alone did not and this binding was dramatically enhanced by the addition of LdUSP. LdEcR LdUSP complex also showed significant binding to an ecdysone agonist ponasterone A KD nM while LdEcR alone showed only weak binding KD nM and .

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