斜纹夜蛾酵母双杂交文库的构建及其在蜕皮激素受体互作蛋白筛选中的应用

doi:10.16380/j.kcxb.2016.08.001

昆虫学报 ›› 2016, Vol. 59 ›› Issue (8): 801-811.doi: 10.16380/j.kcxb.2016.08.001

• 研究论文 • 下一篇

斜纹夜蛾酵母双杂交文库的构建及其在蜕皮激素受体互作蛋白筛选中的应用

范琳芳^1,#, 何文婧^1,#, 苏智仁¹, 朱家明¹, 罗威¹, 张纯², 黄立华^1,*

（1. 华南师范大学生命科学学院, 广州市昆虫发育调控与应用研究重点实验室, 广州510631; 2. 广东省农业科学研究院植物保护研究所, 广东省植物保护新技术重点实验室, 广州510640）

出版日期:2016-08-20 发布日期:2016-08-20

Construction of yeast two-hybrid system and its application in screening the interacting proteins of ecdysone receptors in Spodoptera litura (Lepidoptera: Noctuidae)

FAN Lin-Fang^1,#, HE Wen-Jing^1,#, SU Zhi-Ren¹, ZHU Jia-Ming¹, LUO Wei¹, ZHANG Chun², HUANG Li-Hua^1,*

(1. Guangzhou Key Laboratory of Insect Development Regulation and Application Research, School of Life Sciences, South China Normal University, Guangzhou 510631, China; 2. Guangdong Provincial Key Laboratory of High Technology for Plant Protection, Institute of Plant Protection, Guangdong Academy of Agricultural Sciences, Guangzhou 510640, China)

Online:2016-08-20 Published:2016-08-20

摘要/Abstract

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3.0×10⁶ cfu/μg，文库滴度达到1.3×10⁸ cfu/mL，文库插入片段大小在500~2 000 bp之间。4种诱饵质粒（EcRA/pGBKT7, EcRB1/pGBKT7, USP1/pGBKT7和USP2/pGBKT7）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DnaJ-5（Hsp40 homolog 5, 一种热激蛋白分子伴侣），MBF2（mediator of BmFTZ-F1 type 2, 一种转录共激活子），polyubiquitin（多聚泛素类蛋白），esr16（ecdysteroid regulated 16kDa 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。

关键词: 斜纹夜蛾, 变态发育, 蜕皮激素受体, 酵母双杂交文库, 互作蛋白

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

范琳芳, 何文婧, 苏智仁, 朱家明, 罗威, 张纯, 黄立华. 斜纹夜蛾酵母双杂交文库的构建及其在蜕皮激素受体互作蛋白筛选中的应用[J]. 昆虫学报, 2016, 59(8): 801-811.

FAN Lin-Fang, HE Wen-Jing, SU Zhi-Ren, ZHU Jia-Ming, LUO Wei, ZHANG Chun, HUANG Li-Hua. Construction of yeast two-hybrid system and its application in screening the interacting proteins of ecdysone receptors in Spodoptera litura (Lepidoptera: Noctuidae)[J]. Acta Entomologica Sinica, 2016, 59(8): 801-811.

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链接本文: http://www.insect.org.cn/CN/10.16380/j.kcxb.2016.08.001

http://www.insect.org.cn/CN/Y2016/V59/I8/801

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斜纹夜蛾酵母双杂交文库的构建及其在蜕皮激素受体互作蛋白筛选中的应用

Construction of yeast two-hybrid system and its application in screening the interacting proteins of ecdysone receptors in Spodoptera litura (Lepidoptera: Noctuidae)

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