Abstract: 【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 two-hybrid 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/mL, and the insert length was 500-2 000 bp. The four bait plasmids (EcRA, EcRB1, USP1, USP2/pGBKT7) 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 DnaJ-5 (Hsp40 homolog 5, a kind of heat shock protein chaperone), MBF2 (mediator of BmFTZ-F1 type 2, a transcriptional co-activator), polyubiquitin, esr16 (ecdysteroid regulated 16 kD protein), and NEDD8-like (neural precursor cell expressed, developmentally down-regulated 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.  

Key words: Spodoptera litura, metamorphosis, ecdysone receptor, yeast two-hybrid library, interacting protein