Bacillus Thuringiensis Vip3Aa Protein Binding Assay With BBMVs Of Spodoptera Exigua

Large-scale cultivation of Bacillus thuringiensis(Bt)transgenic crops effectively controls agricultural pests such as cotton bollworm,diamondback moth,small tiger,beet armyworm,etc.The large number of GM crops have been planted,effectively improving the quality and quantity of agricultural products.However,due to the long-term high-pressure selection of Bt insecticidal proteins in agricultural pests,some insects have developed resistance to Bt insecticidal proteins,and show a trend of expansion in scale.The solution of this problem is the key to effective prevention and control.Vegetative insecticidal proteins(VIPs)and insecticidal crystalline proteins(ICPs)have distinct differences in mechanism and mode of action.With the increasing resistance of agricultural pests to insecticidal crystal proteins,the application of Vip3 Aa has become an important strategy for pest control.However,the Insecticidal Mechanism of Vip3 Aa is still unclear.In order to further clarify its insecticidal mechanism and provide theoretical basis for its pest control,the following studies are carried out.In this study,three Vip proteins,Vip3Aa11(LC50:18.399 g/m L),Vip-S686R(LC50:2.053g/m L)and Vip-Y784 N,which had different activities against Spodoptera exigua in laboratory were used as test proteins.Vip-S686 R had the highest insecticidal activity and Vip-Y784 N had no insecticidal activity.The midgut of Spodoptera exigua fed with different insecticidal active proteins was observed by transmission electron microscopy(TEM),and the pathological changes of the midgut were analyzed and compared.The saturated and competitive binding experiments in vitro were carried out with biotin labeling method and BBMVs of Spodoptera exigua.According to the observation of midgut pathological tissues,it was found that the more insecticidal activity,the more obvious and serious the midgut pathological changes were.The microvilli in the midgut of Spodoptera exigua fed with normal diet were well-structured,well-arranged,slender,numerous,densely and uniformly distributed;the mitochondria had a double-layer membrane structure with clear and complete structure,mostly elliptic,and the number of internal ridges was large and orderly arranged;the endoplasmic reticulum was well-arranged,numerous and long;the chromatin in the nucleus was evenly distributed,full-shaped,and the nuclear membrane was intact.Feeding proteins with different insecticidal activities,the greater the insecticidal activity,the more the vesicle structure,swelling deformationand abscission of microvilli in the midgut of Spodoptera exigua;the more blurred the membrane structure of mitochondria,the more distorted or even broken;the more disordered the order of endoplasmic reticulum,the more obvious the swelling;the more indistinct the boundaries of nuclei,the less and less the amount of chromatin.It was concluded that the degree of midgut lesion of Spodoptera exigua was positively correlated with the insecticidal activity,that is,the higher the insecticidal activity,the more severe the midgut lesion.Both Vip3Aa11 and Vip-S686 R proteins with insecticidal activity could bind to BBMVs in the midgut at saturated concentrations of 16 g/m L and 64 g/m L,respectively,while Vip-Y784 N proteins without insecticidal activity did not bind to BBMVs in the midgut,suggesting that the binding amount of Vip3Aa11 and BBMVs increased with the increase of insecticidal activity;Vip3Aa11 and Vip-S686 R insecticidal proteins had heterologous competition.The binding sites of insecticidal proteins may be the same.The results will lay a foundation for further study on the mechanism of interaction between insecticidal proteins and Lepidoptera pests,the interaction of midgut receptors and the mechanism of resistance.It provides a strong basis for the construction of new engineering bacteria,their industrialization and development,and the solution of the problem of pest resistance.