Interaction Between Destruxin A And Five Silkworm Proteins

Destruxin A(DA)is a class of secondary compounds of hexacyclic carboxylic acid peptides and secreted by Metarhizium anisopliae,with strong insecticidal activities.DA can produce antifeedant and toxic effects on many insects through feeding,body surface contact killing,drip,blood cavity injection and so on.However,the target protein of DA remains unknown.In previous studies,several suspected DA-binding proteins were isolated and identified from Bombyx mori Bm12 cells according to the principle of Drug Affinity Responsive Target Stability(DARTS).According to the results of simulated molecular docking analysis,five representative proteins(Bm PLOD3,Bm TMEM214,Bm26SPRS 4,Bm SEC23 and Bm MOESIN)were selected for further study to verify their interaction with DA.First,we investigated the changes of the expression level of these above-mentioned genes after DA stimulated the Bm12 cells.Then,bio-layer interferometry(BLI)was used to determine the interaction between DA and proteins.And the effects of DA on protein interaction were studied by Insect two-hybrid(I2H).Finally,the temporal and spatial expression characteristics of the above genes were investigated.The main research findings are as follows:(1)The effect of DA on the expression level of five protein genesCompared with the control group,the gene expression levels of five proteins in Bm12cells varied after 200μg/m L dose of DA treatment,among which Bm PLOD3 gene expression was significantly up-regulated by 3.2 time,while the other four protein genes did not change significantly(up-regulated multiple between 1.2-1.8).(2)The interaction between DA and five proteins in vitroThe genes of five target proteins were cloned into p ET30a expression vector,and the above proteins were successfully expressed in Escherichia coli expression system by fusion of N-His tag.The soluble target proteins with high purity(all above 90%)and high concentration(ranging from 0.2 mg/m L to 0.6 mg/m L)were obtained by affinity chromatography(Ni-IDA resin).In vitro BLI test showed that Bm MOESIN protein had no interaction with DA.The other four proteins Bm TMEM214,Bm SEC23,Bm PLOD3 and Bm26SPRS4 had strong interaction with DA,and the affinity constants K_Dvalues were0.286μM,0.291μM,0.710μM and 0.899μM respectively.(3)Effect of DA on the interaction function of target proteinsIn vivo I2H results showed that the luminescence value of transfected SF-9 cells was significantly higher than that of control cells.The luminescence value ranged from 100 to20000,indicating that the four proteins had affinity with their interacting proteins.After DA treatment,the luminescence value of transfected cells was negatively correlated with the concentration of DA.When treated with DA at 0.2 ug/m L concentration,the luminescence value of four protein groups decreased to more than 50%all.Among them,DA had the most significant effect on the interaction of Bm SEC23-Bm SEC13,and the luminescence value decreased by more than 90%under the action of DA at 0.2 ug/m L dose,indicating that DA significantly affected the interaction function of Bm SEC23 protein.(4)Spatio-temporal expression characteristics of target protein genes in silkwormBm PLOD3,Bm26SPRS4,Bm SEC23 and Bm TMEM214 expressed in different degrees in eggs,3-5th instar larvae,pupae and adult stages of silkworm,and the lowest expression of four proteins were in adult stage,but the highest expression of four proteins was different.The highest expression of Bm PLOD3 and Bm26SPRS4 protein were all in the 3rd instar larval stage,and the maximum expression of Bm SEC23 and Bm TMEM214was found in the 4th instar and 5th instar larval stage respectively.These above mentioned four proteins were expressed in four tissues(epidermis,head,gut and blood cells)of silkworm larvae stage.Among them,Bm SEC23 protein had the highest expression in epidermis,followed by digestive tract;the other three proteins had all the highest expression in gut.