CaMKK as a Potential Target for the Natural Product Insecticide Cytisine against Megoura japonica 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 Megoura japonica 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.