Acta Entomologica Sinica ›› 2020, Vol. 63 ›› Issue (11): 1295-1304.doi: 10.16380/j.kcxb.2020.11.002

• RESEARCH PAPERS • Previous Articles     Next Articles

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

PENG Lu1,2,3,4, QIN Yu-Dong1,2,3,4, ZOU Ming-Min1,2,3,4, DONG Shi-Jie1,2,3,4, LIU Li-Li1,2,3,4, YOU Min-Sheng1,2,3,4,*   

  1.  (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

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 HisTrapTM HP and GSTTrapTM HP columns, respectively. The JH-binding modes of PxMet-1 and PxMet-2 were analyzed using molecular docking software. 【Results】 The cloned PxMet-(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