Study On The Effect Of Sublethal Dose Of Imidacloprid On OBP17 Gene Expression And In Vitro And In Vivo Function Of Apis Cerana Cerana

The Apis cerana cerana is a unique native bee species in China.It has excellent characteristics such as cold resistance,resistance to mites,and good use of sporadic nectar sources.It is very important to the agricultural system and ecological environment of China.Many physiological behaviors of Apis cerana cerana,such as foraging,feeding and population management,are related to their sensitive olfactory system.Odorant-binding proteins(OBPs)are one of the important protein families in the insect olfactory system.It has complex and diverse physiological functions such as combining and transmitting chemical odor molecules,anti-inflammatory and improving insect resistance.At present,the widely used neonicotinoid insecticide imidacloprid can cause a variety of physiological and behavioral abnormalities including olfactory damage in bees at sublethal doses.However,the molecular mechanism of its olfactory damage to Apis cerana cerana has not been reported so far.In view of this,this study firstly used transcriptome technology to study the effects of sublethal doses of imidacloprid on the sensory system of Apis cerana cerana,and found that the odorant-binding protein OBP17 had a significant response to imidacloprid treatment Then analyzed the temporal and spatial expression profile of Acer OBP17gene in different tissues of Apis cerana cerana by q PCR technology,and analyzed the binding profile of Acer OBP17 gene with 24 kinds of chemical ligands by using the prokaryotic expression of Acer OBP17 recombinant protein.Meanwhile,we used thermodynamic technology to study its binding with imidacloprid andβ-ionone respectively,so as to analyze the in vitro function of Acer OBP17 recombinant protein.Finally,RNAi and EAG technology are used to further study the function of OBP17protein in the propodium of Apis cerana cerana worker bees,which provides a theoretical basis for a deeper understanding of the physiological functions of Acer OBP17 protein under imidacloprid stress.The main results obtained in this study are as follows:1.Transcriptome sequencing of Apis cerana cerana worker bees treated with sublethal dose of imidacloprid,a total of 571 differentially expressed genes were identified.The GO and KEGG analysis found that DEGs are mainly related to multiple pathways such as protein translation,redox,oxidative phosphorylation and ribosomes.This indicates that the sublethal dose of imidacloprid affects the expression of proteins related to protein translation,redox,oxidative phosphorylation and other physiological processes and many proteins related to the formation of nucleus and ribosomes in Apis cerana cerana,as well as the nucleus and ribosome composition.It affects cellular respiration,nerve signal transduction,immune response,the maintenance of homeostasis and detoxification of Apis cerana cerana.2.The Acer OBP17 gene was cloned.Its ORF is 408 bp in length,encoding 135amino acids,including 4 conserved cysteines(Cys),with a molecular weight of 15.17KDa and an isoelectric point of 4.47,indicating that the Acer OBP17 protein conforms to the basic characteristics of insect minus-C OBPs protein,the N-terminal contains a signal peptide of 16 amino acids.It is an acidic small molecule protein.The phylogenetic tree shows that the sequence of Acer OBP17 has the highest similarity with a PBP-related protein of Apis cerana cerana.The predicted 3D structure of Acer OBP17protein has 6α-helices.The analysis of expression profile showed that the OBP17 gene had a high expression level in the three pairs of feet of worker bees,and the expression level reached the highest value in the mesopodium of bees.The prokaryotic expression vector of OBP17 gene was successfully constructed,and the Acer OBP17 recombinant protein with biological activity was successfully induced and expressed and purified.3.Fluorescence competition binding experiments showed that Acer OBP17protein only had a strong binding ability with imidacloprid andβ-ionone among the 24ligands,and the dissociation constant K_D values were 3.78μmol/L and 16.86μmol/L,respectively.Fluorescence direct binding experiments show that the quenching mechanism of the binding of Acer OBP17 protein to imidacloprid orβ-ionone is dynamic quenching at low temperature,and changes to static quenching as the temperature rises.The binding constants between the two and Acer OBP17 protein reached the maximum at 285.15 K and 295.15 K,respectively,which were 6.94×10~4L/mol and 9.05×10~4 L/mol.However,the binding ability of both of them to Acer OBP17protein decreased significantly at a temperature of 300.15 K,and K_A reached the lowest value,and the temperature had a more obvious effect on the ability of Acer OBP17protein to bindβ-ionone than imidacloprid.Thermodynamic analysis found that the main forces of both binding to Acer OBP17 protein under higher temperature conditions are both hydrogen bonds and van der Waals forces,but under low temperature conditions the main force ofβ-ionone and Acer OBP17 protein is hydrophobic interaction.Molecular docking showed that Phe107 contributed the most energy in the process of binding imidacloprid andβ-ionone.4.Injecting ds GFP and ds OBP17 into the abdomen of worker bees,successfully inhibited the expression of OBP17 gene in the feet of worker bees,and the expression was the lowest when ds RNA was injected for 24 h.With the prolongation of the injection time,the silence effect appears to be weakened to a certain extent.The EAG response to the propodium of the worker bee 24 h after injection of ds RNA showed that the intensity of the EAG response increased with the increase of the stimulus concentration,and the silencing of the OBP17 gene would enhance the response of the propodium of the bee to imidacloprid andβ-ionone.In summary,treatment with sublethal dose of imidacloprid increased the expression of OBP17 gene in Apis cerana cerana.Fluorescence binding experiments show that among the 24 ligands,Acer OBP17 has the strongest binding ability to imidacloprid,and the K_A value reaches the maximum under low temperature conditions.RNAi results show that OBP17 gene silencing can enhance the EAG response of the propodium to imidacloprid.It is speculated that the OBP17 protein has certain detoxification and body protection functions,but the specific principle and process of action need to be further studied.This result lays a foundation for in-depth exploration of the physiological functions of the Acer OBP17 protein,and also provides a reference for understanding the mechanism of imidacloprid and the olfactory-related proteins of the Apis cerana cerana.