Research On Acaricidal Mechanism Of Scopoletin Targeting Voltage-gated Calcium Channels

Tetranychus cinnabarinus?Boisduval?,a carmine spider mite,is a worldwide agricultural mite that harms a variety of economic crops and poses a serious threat to China's agricultural and forestry production.At present,chemical agents are mainly used to control Tetranychus cinnabarinus,but as the"3R"problem becomes more serious,finding new and efficient green acaricides has become an urgent task.In the early stage of the laboratory,scopoletin obtained from plants such as Artemisia annua has been shown to have contact killing,evasion,and inhibit spawning effects on Tetranychus cinnabarinus.Early research in the laboratory found that scopoletin may be a nerve poison but so far,the specific acaricidal mechanism is unclear.Therefore,exploring the specific acaricidal action mechanism of scopoletin will be helpful for acaricidal modification and transformation design using scopoletin as a template,and provide theoretical guidance for the development of safe,efficient and new green acaricides.In this study,we explored the mechanism of calcium homeostasis of Tetranychus cinnabarinus caused by voltage-gated calcium channel-induced scopoletin,and then carried out molecular identification of the voltage-gated calcium channel gene and its miticide effect with scopoletin.The research on inter-relationships has achieved the following main research results:1.The effects of scopoletin on calcium homeostasis in Spodoptera frugiperda cells were investigated,and it was clarified that the plasma membrane voltage-gated calcium channel genes are involved in scopoletin stress.Calcium exoenvironment and calcium-free exoenvironment were set up,and Spodoptera frugiperda cells were treated with different concentrations of scopoletin to investigate the response of scopoletin to the intracellular free calcium ion concentration.It was found that in the presence of calcium extracellular fluid,1-100?mol/L of scopoletin can cause the increase of intracellular free calcium concentration,and within the range of 1-100?mol/L,as the concentration of scopoletin increased,the intracellular the calcium ion concentration increased accordingly,that is,the intracellular calcium ion concentration was positively correlated with the scopoletin treatment concentration.When the 1-100?mol/L concentration was increased to 1 mmol/L,scopoletin could cause the relative fluorescence intensity of intracellular calcium ions to decrease.In the absence of calcium external fluid,1-100?mol/L scopoletin can also cause an increase in intracellular free calcium concentration,but the increase is smaller than that in a calcium external fluid environment,and there is a significant difference between the two.Because scopoletin can cause increased calcium concentration in insect cells,the cellular calcium homeostasis environment is imbalanced,and this imbalance pathway is probably caused by external calcium influx mediated by plasma membrane voltage-gated calcium channels.This provides theoretical guidance for studying the effect of scopoletin on calcium homeostasis mediated by voltage-gated calcium channels of Tetranychus cinnabarinus.2.The full lengths of the subtypes of the L-type and T-type voltage-gated calcium channels TcL-VDCC and TcT-VDCC were cloned,and their expression patterns under different mite states and expression patterns under scopoletin stress were analyzed.The full length of T.cinnabarinus L-type and T-type calcium channels TcL-VDCC-?1,TcT-VDCC-?1 was obtained by segmental cloning,and their auxiliary subunits TcL-VDCC-?,TcVDCC-?2?were cloned and The sequence was submitted to NCBI,and the GenBank accession numbers were MK213369,MK256354,MK372341,and MK388861.TcL-VDCC-?1 has the typical structural characteristics of voltage-gated calcium channels,and it also has L-type calcium channel-specific domains such as IQ domains;Sequence alignment and evolution analysis show that TcL-VDCC and TcT-VDCC subunits are closest to Tetranychus urticae in evolutionary positions and cluster into a branch with arachnida.The expression pattern results of different mite states showed that these four genes were expressed in all mite states and were significantly overexpressed during the juvenile mite stage.Analysis of the stress expression patterns of TcL-VDCC-?1 and TcT-VDCC-?1 in scopoletin at different concentrations showed that their mRNA expression was up-regulated after 24h and 48h treatment,showing that TcL-VDCC,TcT-VDCC in scopoletin stress response produced an expression response.3.RNAi was used to verify the functions of TcL-VDCC and TcT-VDCC genes in the acaricidal effect of scopoletin.TcL-VDCC and TcT-VDCC dsRNA fragments?dsTc L-VDCC,dsTcT-VDCC?were synthesized and fed into the cinnabarin spider mite by feeding method.After 48 hours,the detection silencing efficiency was 55.40%and56.70%;Ester sensitivity tests showed that after feeding dsTcL-VDCC and dsTcT-VDCC with LC₃₀ scopoletin,the fatality rates after 48 hours were:11.20%,10.30%.After scopoletin treatment with LC₅₀,dsTc L-VDCC and dsTcT-VDCC after feeding,the mortality rates after 48 hours were 17.67%and 12.30,respectively.This indicates that silencing the expression of TcL-VDCC and TcT-VDCC reduces the sensitivity of cinnabarin mite to scopoletin and may be one of the potential targets for scopoletin acaricidal effect.4.Using homology modeling and molecular docking techniques to explore possible binding sites of scopoletin in TcL-VDCC.A three-dimensional structure model of T.cinnabarinus TcL-VDCC was constructed by using homology modeling technology with the crystal model of rCa_v1.1 as a template.scopoletin small molecule compound and TcL-VDCC was explored the binding conformation of the active pocket Pocket1 of the three-dimensional structure model of by molecular docking technology.The binding conformation of the active pocket Pocket1.The results show that the binding energy of scopoletin and TcL-VDCC Pocket1 is-6.771Kcal/mol,and that the scopoletin and asparagine?Asn?at position 1099 and 1171 on Pocket1 Serine?Ser?binding;at the same time,the specific calcium channel blocker nifedipine was combined with TcL-VDCC Pocket1,the results showed that the binding energy of nifedipine and TcL-VDCC Pocket1 was-7.662Kcal/mol,Nifedipine is combined with serine?Ser?at positions 1171 and 1517 on the three-dimensional structure model of TcL-VDCC.In summary,this study first discussed the effects of scopoletin on calcium homeostasis in insect cells and its possible mechanism with reference to Spodoptera frugiperda cells,and then the effect of scopoletin on voltage-gated calcium channels in the miticide mechanism of Tetranychus cinnabarinus was discussed.The role of voltage-gated calcium channels TcL-VDCC and TcT-VDCC in the acaricidal mechanism of scopolamine on Tetranychus cinnabarinus was clarified.It provided new insights and ideas for clarifying the acaricidal mechanism of scopoletin.