中文摘要：

【目的】蜕皮激素在昆虫变态发育中起着关键的调控作用。蜕皮激素活化为20-羟基蜕皮酮（20-hydroxyecdysone,20E）后与蜕皮激素受体二聚体〔蜕皮激素受体（ecdysone receptor,EcR）和超气门蛋白（ultraspiracle protein,USP）〕结合,启动20E诱导的级联反应。本研究旨在从互作蛋白角度探究EcR/USP自身调控的分子机理。【方法】以重要农业害虫斜纹夜蛾Spodoptera litura为研究对象,通过构建酵母双杂交筛选系统分别筛选了与EcR和USP相互作用的蛋白。【结果】文库转化效率达到3.0×106cfu/μg,文库滴度达到1.3×108cfu/m L,文库插入片段大小在500~2 000 bp之间。4种诱饵质粒（EcRA/p GBKT7,EcRB1/p GBKT7,USP1/p GBKT7和USP2/p GBKT7）对酵母细胞无毒性,并且无自激活性,说明构建的酵母双杂交文库质量可靠。用以上4种诱饵质粒筛选酵母双杂交文库,共得到110个互作蛋白,其中与EcRB1,USP1和USP2互作的蛋白分别有26,52和32个,未筛选到与EcRA互作的蛋白。随后,从中挑选了Dna J-5（Hsp40 homolog 5,一种热激蛋白分子伴侣）,MBF2（mediator of Bm FTZ-F1 type 2,一种转录共激活子）,polyubiquitin（多聚泛素类蛋白）,esr16（ecdysteroid regulated 16k Da protein,一种蜕皮激素调控蛋白）和NEDD8-like（neural precursor cell expressed,developmentally down-regulated protein 8,一种泛素调节相关蛋白）5种蛋白,利用酵母双杂交和Far-Western印迹法进一步验证了蛋白间的互作关系。【结论】分子伴侣和泛素化修饰等在蜕皮激素受体调控中可能起着重要作用。本研究对深入理解昆虫变态发育的分子机理具有重要意义。

英文摘要：

【Aim】Ecdysone plays a key role in insect metamorphosis. It is firstly activated into 20-hydroxyecdysone（ 20E） and then binds to its dimer-receptor composed of ecdysone receptor（ EcR） and ultraspiracle protein（ USP） to initiate 20E-induced cascade reaction. This study aims to explore the regulation mechanisms of EcR / USP from the aspects of protein interaction. 【Methods 】 A yeast twohybrid system was constructed to screen the interacting proteins of EcR and USP from Spodoptera litura,an important agricultural pest. 【Results】The transformation efficiency of yeast library was 3. 0 × 106 cfu /μg,the library titer was 1. 3 × 108 cfu / m L,and the insert length was 500- 2 000 bp. The four bait plasmids（ EcRA,EcRB1,USP1,USP2 / p GBKT7） showed no obvious toxicity to yeast cells and had no autoactivation ability,suggesting that the yeast library is qualified for the next screening of interacting proteins. After screening the library using four bait plasmids,a total of 110 interacting proteins were obtained, including 26 EcRB1-interacting proteins, 52 USP1-interacting proteins, and 32 USP2-interacting proteins. However,no protein was identified to interact with EcRA. Subsequently,five proteins,including Dna J-5（ Hsp40 homolog 5,a kind of heat shock protein chaperone）, MBF2（ mediator of Bm FTZ-F1 type 2,a transcriptional co-activator）,polyubiquitin,esr16（ ecdysteroid regulated 16 k Da protein）,and NEDD8-like（ neural precursor cell expressed,developmentally downregulated protein 8,a ubiquitylation-related protein）,were selected and their interactive relationship with EcR / USP was further confirmed by both yeast two-hybrid and Far-Western blotting. 【Conclusion】Our data suggest that heat shock protein chaperone and ubiquitylation may play roles in the regulation of EcR /USP,and this will contribute to the understanding of insect metamorphosis.