Synergistic Effect Of Nano-Mg（OH）₂ On Anti-Ultraviolet Ability And Bioactivity Of Bt Cry Protein

Bacillus thuringiensis(Bt)is a gram-positive and spore-forming bacterium.Bt formulation is a microbial bioinsecticide,which is the largest produced and the most widely used.But the Bt formulation was sensitive to Ultraviolet light(UV)in the application process.UV shortened the duration of activity of Bt,which would limit the application of Bt formulation.Nano-Mg(OH)2 was selected as the carrier in this study,then Bt insecticidal crystal protein was loaded with nano-Mg(OH)2,in order to understand the resistance to UV,the biological insecticidal activity and the synergistic mechanism of complexes.The nano-Mg(OH)2 was prepared by co-precipitation of magnesium chloride hexahydrate and sodium hydroxide,X-ray powder diffraction(XRD)was applied to analyze the size of synthesized nano-Mg(OH)2,and the size of nano-Mg(OH)2 at(101)direction was 12 nm according to the Scherrer equation.Scanning electron microscope(SEM)analysis showed that the sample contained a lot of small nano-flakes structure with uniform dispersion,and the morphology of Mg(OH)2 particles had certain changes after loading with Cry toxin,but it still with flakes structure.Energy Dispersive Spectroscopy(EDS)indicated that C,N,S,Mg and O elements existed in the complexes,indicating that the Cry protein was loaded on Mg(OH)2.Zeta potential analysis elucidated that nano-Mg(OH)2 could absorb Cry protein by electrostatic adsorption.The result from sodium dodecyl sulfonate-polyacrylamide gelelectrophoresis(SDS-PAGE)indicated that the protein band was obviously lighter after loading,and the protein concentration was significantly decreased,which demonstrated the adsorption between nano-Mg(OH)2 and Cry protein does occurred.Bt LLP29 strain with high mosquitocidal activity was selected as a target strain,and the Bt insecticidal protein was obtained by alkali soluble method.The optimal loading conditions and adsorption characteristics between nano-Mg(OH)2 and crystal protein were studied.The optimum parameters for loading efficiency of protein were as follows:the saturated adsorption capacity could achieve as high as 75.62 mg/g,pH:7,adsorption time:40 minutes,temperature:32.3℃.The adsorption behavior fitted with the Langmuir isotherm,the adsorption kinetics followed the pseudo-second-order rate model,and the adsorption is spontaneous and exothermic process.Results from SDS-PAGE and bioassay of Culex quinquefasciatus suggested that the mortality rate has increased by more than four times after loading with nano-Mg(OH)2 when the concentration of insecticidal protein was 300 μg/mL after the UV-resistance.This demonstrates that the insecticidal activity of protein was effectivively improved after loading with nano-Mg(OH)2,and it is also confirmed the UV-resistance of loaded protein was enhanced,which could effectively protect the protein from destructing.In order to further study of the synergistic effect of nano-Mg(OH)2 on insecticidal activity,Bt insecticidal crystal protein Cry11 Aa was used as model protein which also obtained by alkali soluble method.Fourier transform infrared spectroscopy(FI-IR)analysis suggested that the intensity of Mg-O absorption peak(3699 cm-1)was reduced,it indicated that Cry11Aa was adsorbed on nano-Mg(OH)2.Transmission electron microscopy(TEM)was used to observe the intestinal epithelial cells and microvilli of C.quinquefasciatus larvae.It was found that nano-Mg(OH)2 loaded Cry11Aa could increase the damage of gut epithelial cells of C.quinquefasciatus,and this might lead to the high insecticidal activity.The present work provides the fundamental information to gain biocompatible and environmental-friendly nano-formulation of Bt bioinsecticide.