Study On Potential Targets Of Cytisine On The Nervous System Of Megoura Japonica Matsumural

Natural products have been the important sources for the creation of new pesticides.They not only provide lead compounds for the creation of new pesticides,but also can be used as probes to find new targets.It has been found in the laboratory that cytisine has strong contact activity on a variety of aphids such as Megoura japonica Matsumura,showing typical symptoms of nervous system such as excitement,and has a significant effect on the nervous system enzyme system.It is preliminary speculated that cytisine acts on the nervous system of aphids,but its target needs to be further studied.In this study,the potential target proteins of cytisine were identified by a combination of drug affinity based target stability（DARTS）,cell thermal shift analysis and mass spectrometry（MS-CETSA）and transcriptomics,and the potential target proteins were verified by fluorescence quenching and molecular docking.The main results are as follows:1.Cytisine had strong aphicidal activity against Mjaponica,and the LD50 treated for 24 h and 48 h were 0.037 μg/head and 0.011 μg/head,respectively.2.DARTS and MS-CETSA were used to search for the binding proteins of cytisine.The SDS-PAGE gel map of DARTS test showed 3 distinct protein bands in the molecular weight range of 35～45 kDa,45～60 kDa and 60～75 kDa.In the MS-CETSA test,it was determined that the incubation temperature of the optimum binding protein of cytisine was 60～66℃,and 2 clear bands were shown in the molecular weight range of 45～60 kDa and 100～140 kDa respectively.68 and 51 major binding proteins were identified through DARTS and MS-CETSA tests,among which 3 co-binding proteins Ca2+/calregulin-dependent protein kinase（CaMKK）,Serine/threonine protein phosphatase（PPP2R3B）and Endophilinil-A were recognised.3.Transcriptomics was used to analyze potential targets of cytisine on M.japonica.The transcriptome sequencing showed 125 up-regulated gene and 68 down-regulated genes.The virulence symptoms,physiological and biochemical characteristics of M.japonica were analyzed by cytisine,and 14 candidate target genes were screened.The candidate genes were verified by RT-qPCR,and the results were consistent with the transcriptome.By combining the candidate target genes of omics with the potential target proteins of DARTS and MS-CETSA,CaMKK and PPP2R3B were speculated as potential target proteins of cytisine preliminatively.4.RT-qPCR was used to determine the influence of cytisine on the relative expression level of potential target genes.After treatment with 1000 mg/L cytisine,the expression level of CaMKK increased gradually with the extension of time,and the relative expression level of CaMKK at 12 h,24 h and 48 h was 0.64,1.15 and 1.21,respectively.The relative expression of PPP2R3B increased first and then decreased with the increase of time after treatment with 1000 mg/L cytisine,and the relative expression of PPP2R3B at 12 h,24 h and 48 h were 0.20,1.69 and 0.37,respectively.The relative gene expressions of CaMKK and PPP2R3B increased with the increase of cytisine concentration after 24 h treatment.The relative expression of CaMKK was 0.36,1.15 and 1.49 under the treatment of 500,1000 and 1500 mg/L cytisine,respectively.The relative expression of PPP2R3B was 0.35,1.69 and 3.77 under the treatment of 500,1000 and 1500 mg/L cytisine,respectively.5.Homology modeling and molecular docking techniques were used to analyze the binding ability and binding mode of cytisine with potential target proteins.SWISS-MODEL online server was used to conduct homology modeling for CaMKK and PPP2R3B proteins.SAVES online server evaluated the constructed protein models by PROCHECK,ERRAT and Verify₃D.The 3D models of CaMKK and PPP2R3B proteins successfully constructed can be used for subsequent tests.AutoDock software was used for molecular docking and PyMOL software was used to visualize the docking results.It was found that the binding energy of cytisine and CaMKK was-6.61 kcal/mol,in which Leu 338 residues interacted with each other in the way of hydrogen bond.The binding energy of cytisine and PPP2R3B is-6.53 kcal/mol,where Lys 655 residues interact by hydrogen bonding.6.Prokaryotic expression and fluorescence quenching techniques were used to verify and identify potential target proteins of cytisine.The prokaryotic expression vector of CaMKK was constructed,and the protein expression conditions were optimized.All CaMKK proteins were identified as soluble proteins,and the concentration of purified proteins was 0.39 mg/mL by nickel column purification.Fluorescence quenching results showed that the fluorescence intensity of CaMKK protein decreased by 28.5%,34.86%,39.68%,51.00%,55.16%,73.99%and 83.29%,after treatment with the concentration of 31.25,62.5,125,250,500,1000 and 2000 mg/L,respectively.In summary,this study proved that CaMKK is the potential target of cytisine through target searching,analysis and verification.Combined with toxicological symptoms and physiological and biochemical analysis,the mechanism of cytisine was preliminarily speculated as follows:cytisine affects the activity of CaMKK,leading to AMP-activated protein kinase（AMPK）activity was changed,and energy metabolism of aphid cells was furtherdisrupted,resulting in aphid death.