tailieunhanh - Báo cáo khoa học: Improvement of ecdysone receptor gene switch for applications in plants: Locusta migratoria retinoid X receptor (LmRXR) mutagenesis and optimization of translation start site

Gene switches have potential applications for the regulation of transgene expression in plants and animals. Recently, we have developed a two-hybrid ecdysone receptor (EcR) gene switch using chimera 9 [CH9, a chi-mera between helices 1–8 ofHomo sapiensretinoid X receptor (HsRXR) and helices 9–12 ofLocusta migratoriaRXR (LmRXR)] as a partner for Choristoneura fumiferanaEcR (CfEcR). | Improvement of ecdysone receptor gene switch for applications in plants Locusta migratoria retinoid X receptor LmRXR mutagenesis and optimization of translation start site Ajay K. Singh1 2 Venkata S. Tavva1 2 Glenn B. Collins2 and Subba R. Palli1 1 Department of Entomology University of Kentucky Lexington KY USA 2 Plant and SoilSciences Department University of Kentucky Lexington KY USA Keywords ecdysone receptor gene switch genetically modified crops methoxyfenozide retinoid X receptor Correspondence S. R. Palli Department of Entomology S225 Ag. Science N University of Kentucky Lexington KY 40546-0091 USA Fax 1 859 323 1120 Tel 1 859 257 4962 E-mail rpalli@ Received 6 May 2010 revised 1 September 2010 accepted 8 September 2010 doi Gene switches have potential applications for the regulation of transgene expression in plants and animals. Recently we have developed a two-hybrid ecdysone receptor EcR gene switch using chimera 9 CH9 a chimera between helices 1-8 of Homo sapiens retinoid X receptor HsRXR and helices 9-12 of Locusta migratoria RXR LmRXR as a partner for Choristoneurafumiferana EcR CfEcR . As CH9 includes a region of human RXR public acceptance of this gene switch for use in genetically modified crops may be an issue. The current studies were conducted to identify an LmRXR mutant that could replace CH9 as a partner for CfEcR. The amino acid identity between LmRXR and HsRXR is fairly high. However there are a few amino acid residues that are different between these two proteins. LmRXR mutants were produced by changing the amino acids in the helices 1-8 that are different between LmRXR and HsRXR to HsRXR residues. Screening of these mutants in tobacco protoplasts identified a triple mutant A62S T81H V123I SHILmRXR that performed as well as CH9. The performance of the EcR gene switch was further improved by optimizing the translational start site Kozak sequence AACAATGG of the transgene. The EcR gene switch containing

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