Identification Of The JH Receptors And Screening Of The Interacting Proteins In Plutella Xylostella

The diamondback moth(DBM),Plutella xylostella(L.)(Lepidoptera,Plutellidae),is a proliferous,worldwide Lepidoptera pest,mainly attacking cruciferous crops.It is very difficult to control due to its high resistance,fast growth,and high fecundity.Juvenile hormone(JH),as a lipophilic sesquiterpene hormone,regulates a series of physiological processes of insect species,which involved in complicated signal transduction pathways.However,the molecular mechanisms of JH pathway are still poorly understood,and it has been gradually analyzed in some model insects until recent years.Increasing evidences show that methoprene-tolerant(Met)is the most likely to be a nuclear receptor of JH.Instead of acting alone,Met needs to form a heterodimer with other family proteins in the presence of JH to form an active transcription factor.Therefore,the identification of the key receptor of JH and its interaction protein are crucial to the in-depth analysis of the molecular mechanism of the development and reproduction of P.xylostella that regulated by JH Based on the previous research,the following experiments were carried out by the methods of molecular biology and proteomics.Firstly,the molecular characteristics of the Mets gene of P.xylostella were analyzed,and the binding characteristics of the Mets protein to JH were identified.Secondly,the prokaryotic expression and purification of Mets were performed.Finally,the interacting proteins of Mets were screened using GST pull-down.The main results were as follows:Px Met-1 and Px Met-2 contained ORFs of 1,575 bp encoding 524 aa and 2,100 bp encoding 699 aa,respectively.The predicted molecular mass of Px Met-1 and Px Met-2 were 60.5 k Da and 70.7 k Da,respectively.The three-dimensional structures of Px Met-1 and Px Met-2 were similar,including a typical helix-loop-helix.The molecular docking between JH and Mets showed that there was an interaction site in the PAS-B conserved domain of Mets,indicating that Met is a functional receptor for the JH in P.xylostella.We constructed the expression vectors p GEX-KG-Met-1 and p GEX-KG-Met-2of Px Met-1 and Px Met-2,and obtained soluble Met fusion proteins with His and GST double tags using vitro prokaryotic expression.The optimal expression conditions of Px Met-1 were 16℃,16 h,and 0.3 m M IPTG concentration,while Px Met-2 was 16℃,16 h,and 0.1 m M IPTG concentration.Px Met-1 was purified at 150 m M imidazole concentration to obtain a high-purity fusion protein,and Px Met-2 was purified at 20 m M reduced glutathione concentration.Both SDS-PAGE and Western blot analysis indicated that the purified protein was the fusion protein of Px Met-1 and Px Met-2,respectively.The purified Px Met-1 and Px Met-2 were used as the bait proteins,and the total protein of newly emerged female adults was selected as the prey protein.The interacting proteins of Px Met-1 and Px Met-2 were screened using GST pull-down,respectively.Multiple proteins were obtained for different treatment groups,including27 proteins in GST groups(controls),46 in Met-1 groups,50 in Met-1 + JH III groups,237 in Met-2 groups,230 in Met-2 + JH III groups,135 in Met-1 + Met-2 groups,and59 in Met-1 + Met-2 + JH III groups.Based on Wayne diagram analysis,27,82 and 8interacting proteins with Px Met-1,Px Met-2 and Px Met-1 and Px Met-2 were obtained,respectively,which mainly involved in cell composition,biological process and molecular function.In this study,the receptor proteins of P.xylostella JH were identified for the first time,and the interacting proteins of the receptor proteins were screened and analyzed.The results showed that there were differences on the interacting proteins of Px Met-1and Px Met-2,and previous study has shown that Px Met-1 and Px Met-2 have different stage expression preferences.It is speculated that Met-1 and Met-2 may play different roles in the development and reproduction of P.xylostella.This study provided new ideas and ways for further analysis of the interacting proteins of Met and their functions,understanding the molecular mechanisms of the development and reproduction of P.xylostella that regulated by JH,and mining the new target for effective control of P.xylostella.