Studies On The Herbicidal Potential Target Of Natural Product Drupacine

Natural products are valuable sources of new pesticide discovery,and identification of the targets and mode of actions is the key for the development of new pesticides.Drupacine is an alkaloid with herbicidal activity isolated from Cephalotaxus sinensis.However,the target and mechanism of drupacine are still unclear.Therefore,in this study,Amaranthus retroflexus L.was used as the test weed,and Drug Affinity Responsive Target Stability(DARTS),Cellular Thermal Shift Assay(MS-CETSA),fluorescence quenching and molecular docking were used to discover and verify the target proteins of drupacine,which can provide new materials and research direction for pesticide molecular design based on target.The main results are as follows:1.The effects of drupacine on the growth of A.retroflexus seedlings were determined,and the root tips and root tip cells of A.retroflexus treated were observed.The results of in dish test showed that the activity of drupacine on A.retroflexus was strong,and the IC50 value was 38.99 mg/L on A.retroflexus root;the pot experiment showed that the fresh weight inhibition rates of 4 g/L drupacine on A.retroflexus were 87.45%and 85.26%after 14 d and 21 d of treatment,respectively.Microscopic observation showed that the root hair of A.retroflexus seedlings decreased and the root crown fell off gradually with the increase of treatment concentration and treatment time.Scanning electron microscopy(SEM)showed that the damage of root tip cells increased with the extension of treatment time.2.The effects of drupacine on defense enzyme activity,relative electrical conductivity,soluble sugar content and malondialdehyde content of A.retroflexus seedlings were determined.The α-amylase activity and soluble sugar content of treated A.retroflexus seedlings were significantly lower than that of the control,and the expression of α-amylase related genes was inhibited.The relative electrical conductivity and malondialdehyde(MDA)content of treated seedlings were also increased,while the activities of peroxidase(POD),catalase(CAT)and superoxide dismutase(SOD)of treated seedlings were significantly higher than that of the control.These results indicated that the damage of morphology and membrane structure caused by changes in physiological and biochemical reactions was one of the main reasons for drupacine to inhibit the growth of seedlings.3.DARTS and MS-CETSA were used to identify the binding proteins of drupacine.The protein gel maps of the DARTS experiment showed that distinct bands of difference appear at 30 kDa,40 kDa,and 60 kDa.In the MS-CETSA test,the optimum test temperatures of 65℃,67℃,69℃ and 71℃ were determined,showing three distinct bands around 30 kDa,60 kDa and 75 kDa,respectively.DARTS and MS-CETSA assays identified 51 and 68 major binding proteins,including 9 common binding proteins.Based on the analysis of toxic symptoms and biochemical characteristics,it is speculated that Profilin,Shikimate dehydrogenase(SkDH)and ζ-carotene desaturase(ZDS)are potential target proteins of drupacine.4.qRT-PCR was used to determine the effect of drupacine on the relative expression level of potential target genes.After treatment with 50 mg/L drupacine,the expression level of Profilin decreased gradually with the extension of time and therelative expression level at 12 h,24 h and 48 h decreased by 30.93%,75.26%and 86.60%,respectively.The relative expression level of SkDH at 3 h,6 h,12 h,24 h and 48 h decreased gradually with the increase of time,decreased by 25.53%,29.79%,65.96%,76.60%and 86.17%,respectively.The relative expression level of ZDS did not change significantly.5.Homologous modeling and molecular docking techniques were used to analyze the binding ability of Profilin,SkDH and ZDS to drupacine.The 3D protein model constructed by homology modeling met the requirements in terms of homology,ERRAT value and non-bond interaction score.Molecular docking showed that drupacine could bind SkDH better compared with Profilin and ZDS.The binding capacity of drupacine and SkDH was-7.2 kcal/mol,and it could form hydrogen bonding with ARG-344,TYR-253,ALA-250 and TYR-645.The binding energy of drupacine with Profilin and ZDS are-7.0 kcal/mol and-6.7 kcal/mol,respectively.6.The prokaryotic expression vectors of SkDH and Profilin were constructed,and the expression conditions were optimized.The two proteins were identified as soluble proteins,and the purified proteins were obtained by nickel column purification.7.Fluorescence quenching and enzyme activity assay were used to verify and determine the target protein.Fluorescence quenching results showed that the fluorescence intensity of SkDH protein decreased by about 29%,59%and 70%,respectively,after treatment with 62.5,125 and 250 mg/L drupacine.The fluorescence intensity of Profilin protein decreased to about 51.2%,36%and 35%,respectively,after treatment with 62.5,125 and 250 mg/L drupacine,however,there was no significant dose effect.These results indicated that the combination ability of drupacine and SkDH protein was strong.The enzyme activity test showed that the SkDH activity of A.retroflexus was 8.57 nmol/min/mg prot after treatment with 50 mg/L drupacine,which was 80.20%lower than that of the control group,indicating that drupacine had a strong inhibitory effect on SkDH.In conclusion,SkDH was identified as the potential herbicidal target of drupacine through physiological and biochemical analysis,candidate target screening and verification in this study.It is speculated that the mode of action is as follows:drupacine binds with SkDH,and inhibits its activity,which means the shikimic acid pathway is blocked,leads to the reduction of the production of essential amino acids such as tryptophan,tyrosine and phenylalanine,and finally weeds couldn’t get the nutrients they need to grow and die.