黑腹果蝇保幼激素反应区(JHRR)与核蛋白结合能力调控的分子机制

Molecular mechanisms regulating the binding capacity of juvenile hormone response region(JHRR) to nuclear proteins in Drosophila melanogaster

【目的】保幼激素反应区(juvenile hormone response region,JHRR)是在黑腹果蝇Drosophila melanogaster Krüppel homolog 1(Kr-h1)启动子转录调控区中鉴定获得的长120 bp的核苷酸序列。本研究旨在检测果蝇JHRR中3个类E-box序列(2个B box和1个C box)对JHRR核蛋白结合能力的影响,以及保幼激素(juvenile hormone,JH)、JH受体Met(Methoprene-tolerant)和热激蛋白Hsp83对JHRR核蛋白结合能力的调控。【方法】利用凝胶迁移阻滞(electrophoretic mobility shift assay,EMSA)技术分析果蝇JHRR中2个B box或1个C box突变后JHRR与Kc细胞中核蛋白的结合情况;提取游走早期JH滴度较高时期JH缺失果蝇(Aug21-GAL4>UAS-Grim)、Met/Gce双缺失果蝇(Met^(27)gce^(2.5k))、Met超表达果蝇(Lsp2-GAL4>UAS-Met-V5)以及Hsp83纯合突变果蝇(Hsp8308445)脂肪体组织中的核蛋白,利用EMSA技术分析它们对JHRR核蛋白结合能力的调控情况。【结果】EMSA检测结果表明,B box或C box突变后均可降低JH促进的JHRR与Kc细胞中核蛋白的结合,且C box的效果更强;JH缺失果蝇、Met/Gce双缺失果蝇脂肪体组织中核蛋白与JHRR的结合几乎检测不到,而在Hsp83纯合突变果蝇脂肪体组织中核蛋白与JHRR的结合显著下降。相反,JHRR与核蛋白的结合在Met超表达果蝇中显著增强。【结论】B box和C box对JHRR与核蛋白结合是必需的,且JHRR与核蛋白的结合依赖于JH和Met,并且受Hsp83的调控。

【Aim】 Juvenile hormone response region （JHRR） is a 120 bp nucleotide sequence identified from the promoter regulatory region of Krüppel homolog1 （Kr-h1） in Drosophila. This study aims to detect the effects of three E-box like motifs （two B boxes and one C box） on the binding capacity of JHRR to nuclear proteins, and to illustrate the regulatory effects of JH, JH receptor-Methoprene-tolerant （Met） and heat shock protein Hsp83 on the binding capacity of JHRR to nuclear proteins. 【Methods】 The electrophoretic mobility shift assay （EMSA） was used to analyze the binding capacity of JHRR with two B boxes or one C box mutants to nuclear proteins in Kc cells. Nuclear proteins of fat body tissues, which were isolated from JH deficient flies （Aug21GAL4〉UASGrim）, Met/Gce double mutant flies （Met^27gce^2.5k）,Met overexpressed flies （Lsp2-GAL4〉UAS-Met-V5） and Hsp83 homozygous mutant flies （Hsp83^08445） at the early wandering stage when the JH titer was high, were extracted, and the binding capacity of JHRR to nuclear proteins was detected by EMSA. 【Results】 EMSA results showed that both B box and C box mutation decreased the JH-induced binding capacity of JHRR to nuclear proteins in Kc cells, and the inhibitory effect of C box mutation was even more efficient. The binding complex of JHRR and nuclear proteins which were extracted from fat body tissues of JH deficient or Met/Gce double mutant larvae at the wandering stage was almost undetectable, and decreased significantly when the nuclear proteins were extracted from Hsp83 homozygous mutant larvae. In contrast, the formation of JHRR-nuclear protein complex was enhanced significantly when the nuclear proteins were extracted from fat body tissues of Met overexpressed larvae. 【Conclusion】 B box and C box are essential for JHRR to bind to nuclear proteins, and the binding activity of JHRR to nuclear proteins is dependent on JH and Met and regulated by Hsp83.