CaMKK as a Potential Target for the Natural Product Insecticide Cytisine against <i>Megoura japonica</i> Matsumura

Cytisine, a botanical compound, has strong contact activity against a variety of aphids. However, the target and mechanism of its aphidicidal action remain unclear. In this study, the biochemical characteristics of cytisine against <i>Megoura japonica</i> were tested, and the potential target proteins of cytisine were identified, and further verified by fluorescence quenching and molecular docking. Cytisine can affect the activity of Ca²⁺, Mg²⁺-ATPase, and Na⁺, K⁺-ATPase, with inhibitory rates of 50.00% and 65.22%, respectively. Sixty-eight and fifty-one major binding proteins were identified by drug affinity responsive target stability (DARTS) and cellular thermal shift assay and mass spectrometry (MS-CETSA), respectively. 125 up-regulated and 68 down-regulated genes were obtained by transcriptome sequencing. By combining the candidate target genes of transcriptomics with the potential target proteins of DARTS and MS-CETSA, CaMKK and PPP2R3B were speculated as potential target proteins. The molecular docking results showed that the binding energies of cytisine to CaMKK and PPP2R3B were -6.61 kcal/mol and -6.53 kcal/mol. The fluorescence intensity of CaMKK protein decreased by 28.50, 34.86, 39.68, 51.00, 55.16, 73.99, and 83.29% after different concentrations of cytisine treatment. This research proved that CaMKK is the potential target of cytisine, providing a new target resource for the creation of new pesticides.