【摘要】 OsAFB4基因编码一类水稻生长素受体蛋白，其缺失可使水稻对2,4-二氯苯氧乙酸（2, 4-dichlorophenoxyacetic acid, 2,4-D）和毒莠定产生抗性。为研究Os AFB4蛋白的功能、特性以及与生长素类除草剂的互作关系，需建立起Os AFB4蛋白的原核表达体系。本研究将Os AFB4基因分别克隆到原核表达载体p ET-32a、p GEX-4T和pMAL-C6T中，随后将pET-32a-OsAFB4转化至大肠埃希菌OverExpress C43(DE3)、Rosseta2(DE3)和ER2566感受态细胞，将pGEX-4T-OsAFB4转化至大肠埃希菌OverExpress C43(DE3)和Rosseta2(DE3)感受态细胞，将pMAL-C6T-OsAFB4转化至OverExpress C43(DE3)、Rosseta2(DE3)和TB1感受态细胞，经异丙基硫代-β-D-半乳糖苷（isopropylthio-β-D-galactoside, IPTG）诱导表达融合蛋白，在不同诱导时间、诱导温度和IPTG浓度下对诱导条件进行优化。蛋白质凝胶电泳结果表明，OsAFB4蛋白原核表达的最佳诱导条件为利用200μmol/L IPTG，于23℃下诱导12 h。该体系的建立为后续生长素受体的功能研究及抗生长素类除草剂位点的开发奠定了基础。更多还原

【Abstract】 OsAFB4 gene encodes a kind of rice auxin receptor protein, and its deletion can make rice resistant to 2, 4-dichlorophenoxyacetic acid(2, 4-D) and picloram. In order to study its function, characteristics and interaction with auxin herbicides, it is necessary to establish a prokaryotic expression system for OsAFB4 protein. In this study,Os AFB4 gene was cloned into prokaryotic expression vectors pET-32a, pGEX-4T, and pMAL-C6T, respectively.Then, p ET-32a-OsAFB4 was transformed into Escherichia coli strain competent cells of OverExpress C43(DE3), Rosseta2(DE3) and ER2566, pGEX-4T-OsAFB4 was transformed into E. coli strain competent cells of OverExpress C43(DE3) and Rosseta2(DE3), and pMAL-C6T-OsAFB4 was transformed into E. coli strain competent cells of OverExpress C43(DE3), Rosseta2(DE3) and TB1. The expression of fusion protein was induced by isopropylthio-β-D-galactoside(IPTG), and the induction conditions were optimized using different induction times, temperatures and IPTG concentrations. The protein gel electrophoresis results indicated that the optimal induction condition for prokaryotic expression of OsAFB4 protein was 200 μmol/L IPTG at 23 ℃ for 12 h. In conclusion, the establishment of the OsAFB4 prokaryotic expression system provides a foundation for research on auxin receptor function and the development of auxin herbicide resistance sites.更多还原