小菜蛾保幼激素受体PxMet-1和PxMet-2的原核表达、纯化及与保幼激素的结合模式

doi:10.16380/j.kcxb.2020.11.002

昆虫学报 ›› 2020, Vol. 63 ›› Issue (11): 1295-1304.doi: 10.16380/j.kcxb.2020.11.002

小菜蛾保幼激素受体PxMet-1和PxMet-2的原核表达、纯化及与保幼激素的结合模式

彭露^1,2,3,4, 秦玉冬^1,2,3,4, 邹明民^1,2,3,4, 董诗杰^1,2,3,4, 刘莉莉^1,2,3,4, 尤民生^1,2,3,4,*

(1. 福建农林大学, 闽台作物有害生物生态防控国家重点实验室, 福州 350002; 2. 福建农林大学应用生态研究所, 福州 350002; 3. 福建农林大学, 教育部害虫生态防控国际合作联合实验室, 福州 350002; 4. 福建农林大学, 福建省昆虫生态重点实验室, 福州 350002)

出版日期:2020-11-20 发布日期:2020-12-08

Prokaryotic expression, purification and juvenile hormone-binding modes of the juvenile hormone receptors PxMet-1 and PxMet-2 of Plutella xylostella (Lepidoptera: Plutellidae)

PENG Lu^1,2,3,4, QIN Yu-Dong^1,2,3,4, ZOU Ming-Min^1,2,3,4, DONG Shi-Jie^1,2,3,4, LIU Li-Li^1,2,3,4, YOU Min-Sheng^1,2,3,4,*

(1. State Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops, Fujian Agriculture and Forestry University, Fuzhou 350002, China; 2. Institute of Applied Ecology, Agriculture and Forestry University, Fuzhou 350002, China; 3. Joint International Research Laboratory of Ecological Pest Control, Ministry of Education, Agriculture and Forestry University, Fuzhou 350002, China; 4. Fujian Provincial Key Laboratory of Insect Ecology, Fujian Agriculture and Forestry University, Fuzhou 350002, China)

Online:2020-11-20 Published:2020-12-08

摘要/Abstract

摘要： 【目的】昆虫烯虫酯耐性(methoprene-tolerant, Met)蛋白属于bHLH/PAS转录因子家族成员，其与该家族其他成员形成的复合物可介导保幼激素(JH)的信号传导。本研究旨在克隆并表达小菜蛾Plutella xylostella JH受体Met蛋白基因PxMet-1和PxMet-2，纯化其蛋白，分析PxMet-1与PxMet-2与JH的结合模式，为进一步探明PxMet蛋白的功能提供依据。【方法】基于NCBI数据库，克隆验证了小菜蛾PxMet-1和PxMet-2基因的cDNA序列;把这两个基因分别与pGEX-KG载体连接构建表达载体pGEX-KG-PxMet-1和pGEX-KG-PxMet-2，导入大肠杆菌Escherichia coli Rosetta (DE3)使其表达；分别使用HisTrap^TM HP与 GSTTrap^TM HP的5 mL柱对PxMet-1和PxMet-2融合蛋白进行纯化；利用分子对接预测软件分析PxMet-1和PxMet-2蛋白与JH的结合模式。【结果】克隆获得的小菜蛾PxMet-1(GenBank登录号: MK697672)和PxMet-2(GenBank登录号: MT996234)序列与已报道的序列基本一致，仅PxMet-2基因序列存在1个非同义突变，突变位点为第41位氨基酸，由甘氨酸(Gly)突变为丙氨酸(Ala)。PxMet-1与PxMet
-2蛋白三维结构相似，均包含典型的螺旋-环-螺旋结构。体外原核表达获得具有His和GST双标签的可溶性PxMet融合蛋白，PxMet-1融合蛋白的最优表达条件为0.3 mmol/L的IPTG在16℃下诱导16 h，PxMet-2融合蛋白的为0.1 mmol/L的IPTG 在16℃下诱导16 h。PxMet-1和PxMet-2分别经150 mmol/L咪唑和20 mmol/L还原型谷胱甘肽洗脱获得高纯度的融合蛋白。SDS-PAGE和Western blot验证PxMet-1和PxMet-2融合蛋白，分别在89.2和106 kD位置获得条带，与推测大小一致。分子对接分析结果显示，PxMet-1和PxMet-2与JH存在相互作用位点，都是在PASB保守结构域，和赤拟谷盗Tribolium castaneum Met的JH结合位点相同。【结论】利用pGEX-KG原核表达系统能有效地在体外表达可溶性的PxMet-1和PxMet-2蛋白，PxMet-1和PxMet-2蛋白很可能是小菜蛾JH的分子受体。本研究为进一步解析PxMet蛋白的功能，深入阐明JH调控小菜蛾发育与生殖的分子机理奠定了基础。

关键词: 小菜蛾, 保幼激素, 烯虫酯耐性蛋白, 原核表达, 分子对接, 生殖发育

Abstract: 【Aim】 Methoprene-tolerant (Met) proteins belong to the bHLH/PAS family of transcription factors and their complexes with the other members of this family are responsible for transducing the juvenile hormone (JH) signal. This study aims to clone and express the JH receptor Met genes of Plutella xylostella (PxMet-1 and PxMet-2), to purify their proteins and to analyze the JH-binding modes of the PxMet-1 and PxMet-2, so as to provide the basis for further clarifying the function of PxMet proteins. 【Methods】 Based on the NCBI database, the cDNA sequences of PxMet-1 and PxMet-2 of P. xylostella were cloned and verified. PxMet-1 and PxMet-2 sequences were ligated with the prokaryotic expression vector pGEX-KG to construct recombinant plasmids pGEX-KG-PxMet-1 and pGEX-KG-PxMet-2, respectively, which were then introduced into Escherichia coli Rosetta (DE3) for expression. The fusion proteins of PxMet-1 and PxMet-2 were purified with 5 mL HisTrap^TM HP and GSTTrap^TM HP columns, respectively. The JH-binding modes of PxMet-1 and PxMet-2 were analyzed using molecular docking software. 【Results】 The cloned PxMet-1 (GenBank accession no.: MK697672) and PxMet-2 (GenBank accession no.: MT996234) sequences were similar to the previously reported sequences, except for one nonsynonymous mutation located in the 41st amino acid of the PxMet-2 gene, changing from glycine (Gly) to alanine (Ala). The three-dimensional structures of PxMet-1 and PxMet-2 are similar, including a typical helix-loop-helix. Soluble PxMet fusion proteins with His and GST double tags were obtained using in vitro prokaryotic expression. The optimal expression conditions of PxMet-1 were 0.3 mmol/L IPTG induction under 16℃ for 16 h, while those of PxMet-2 were 0.1 mmol/L IPTG induction under 16℃ for 16 h. The highly purified fusion proteins of PxMet-1 and PxMet-2 were obtained after elution with 150 mmol/L imidazole and 20 mmol/L reduced glutathione, respectively. Both SDS-PAGE and Western blot analysis indicated that the specific protein bands of around 89.2 and 106 kD were consistent with the predicted size of PxMet-1 and PxMet-2, respectively. The molecular docking analysis results showed that there is an interaction site with JH in the PAS-B conserved domain of PxMet-1 and PxMet-2, which is consistent with that of Tribolium castaneum Met. 【Conclusion】 Soluble proteins PxMet-1 and PxMet-2 can be effectively expressed in vitro using the pGEX-KG prokaryotic expression system, and PxMet-1 and PxMet-2 proteins are likely to be molecular receptors of JH in P. xylostella. This study provides a solid foundation for further analysis of the PxMet functions and better understanding of the molecular mechanisms that may underlie the JH-mediated development and reproduction of P. xylostella.

Key words: Plutella xylostella, juvenile hormone, methoprene-tolerant protein, prokaryotic expression, molecular docking, reproductive development

彭露, 秦玉冬, 邹明民, 董诗杰, 刘莉莉, 尤民生. 小菜蛾保幼激素受体PxMet-1和PxMet-2的原核表达、纯化及与保幼激素的结合模式[J]. 昆虫学报, 2020, 63(11): 1295-1304.

PENG Lu, QIN Yu-Dong, ZOU Ming-Min, DONG Shi-Jie, LIU Li-Li, YOU Min-Sheng. Prokaryotic expression, purification and juvenile hormone-binding modes of the juvenile hormone receptors PxMet-1 and PxMet-2 of Plutella xylostella (Lepidoptera: Plutellidae)[J]. Acta Entomologica Sinica, 2020, 63(11): 1295-1304.

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小菜蛾保幼激素受体PxMet-1和PxMet-2的原核表达、纯化及与保幼激素的结合模式

Prokaryotic expression, purification and juvenile hormone-binding modes of the juvenile hormone receptors PxMet-1 and PxMet-2 of Plutella xylostella (Lepidoptera: Plutellidae)

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