Flowerlike Mg(OH)2 Cross-Nanosheets for Controlling Cry1Ac Protein

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Agricultural and Environmental Chemistry

Flower-like Mg(OH)2 Cross Nano-sheets for Controlling Cry1Ac Protein Loss: Evaluates Its Insecticidal activity and Biosecurity Wenhua Rao, Yating Zhan, Saili Chen, Zhangyan Xu, Tengzhou Huang, Xianxian Hong, Yilin Zheng, Xiaohong Pan, and Xiong Guan J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b00575 • Publication Date (Web): 27 Mar 2018 Downloaded from http://pubs.acs.org on March 30, 2018

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Journal of Agricultural and Food Chemistry

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Flower-like Mg(OH)2 Cross Nano-Sheets for Controlling

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Cry1Ac Protein Loss: Evaluates Its Insecticidal Activity

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and Biosecurity

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Wenhua Rao,† Yating Zhan,† Saili Chen,† Zhangyan Xu,† Tengzhou Huang,†

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Xianxian Hong,† Yilin Zheng,† Xiaohong Pan,*,† and Xiong Guan*,†

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†

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of Plant Protection & Key Lab of Biopesticide and Chemical Biology, Ministry of

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Education, Fujian Agriculture and Forestry University, Fuzhou, Fujian 350002, People’s

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Republic of China

State Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops, College

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Corresponding Authors

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*Tel. & Fax: +086-591-83789258. E-mail: [email protected] (Xiaohong Pan).

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*Tel. & Fax: +086-591-83789258. E-mail: [email protected] (Xiong Guan).

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1 ACS Paragon Plus Environment


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ABSTRACT: Bacillus thuringiensis (Bt) can produce Cry proteins during the sporulation

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phase, and Cry protein is effective against lepidopteran, coleopteran, dipteran insects and

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nematodes. However, Cry protein tends to be discharged into soil and non-target plants

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through rainwater runoff, leading to reduced effective period toward target insects. In the

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present study, nano-Mg(OH)2 (MHNPs) were synthesized to control the loss of Cry1Ac

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protein and deliver protein to Helicoverpa armigera (Lepidoptera: Noctuidae). The

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results showed that Cry1Ac protein could be loaded onto MHNPs through electrostatic

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adsorption, and both MHNPs and Cry protein were stable during the adsorption process.

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Meanwhile, the Cry1Ac-loaded MHNPs (designated as Cry1Ac-MHNPs) could retain on

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the surface of cotton leaves, resulting in enhanced adhesion amount of Cry1Ac protein by

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59.50% and increased pest mortality by 75.00%. Additionally, MHNPs could be slowly

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decomposed by acid medium and MHNPs showed no obvious influence on the cotton, Bt,

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Escherichia coli and H. armigera. Therefore, MHNPs could serve as an efficient

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nanocarrier for delivery of Cry1Ac protein and be used as a potential adjuvant for

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biopesticide in agricultural applications.

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KEYWORDS: Bacillus thuringiensis, Cry1Ac protein, nano-Mg(OH)2, Helicoverpa

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armigera, loss control, insecticidal activity, biosafety

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1. INTRODUCTION

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Pesticides have been extensively applied in agricultural production. However, about 90%

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pesticide goes to soil through rainwater runoff, resulting in reduced effective period to

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target insects and adverse effects on non-target organism.1,2 As one type of biopesticides,

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Bacillus thuringiensis (Bt) is widely used for biological control of pest worldwide, it

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produces Cry protein during the sporulation phase,3,4 and Cry protein is effective against

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lepidopteran, coleopteran, dipteran insects and nematodes.5-7 Similar to the chemical

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pesticides, Cry protein may be lost because of rainwater and other environmental factors,

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leading to reduced efficacy and economy loss. Therefore, controlling of biopesticide loss

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would be a promising countermeasure to ensure adequate effect.

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Nanomaterials can enhance stability of drug and promote drug utilization rate as well

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as control pesticide loss since they possess high surface area, strong adsorption capacity

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and electrical properties.8-13 Additionally, Cry protein can be easily loaded onto

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nanomaterials, and the bioactivity of Cry protein is not affected by the nanomaterials.14-19

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However, some nanomaterials are expensive and even show potential risks to human

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health and ecosystem, resulting in restricted practical application of nanomaterials in

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agriculture.20-22 Therefore, selection of suitable materials would be a key process to

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prevent loss of biopesticides.

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As a low cost material, nano-Mg(OH)2 (MHNPs) have already been widely applied as

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a green adsorbent and antibacterial agent.23-25 However, only few studies have used



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MHNPs to prevent loss of biopesticides. Additionally, the safety of nanoproducts in

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agriculture and the potential of adverse ecosystem responses from nanoparticles are not

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clearly understood. Therefore, in the present study, we aimed to (i) assess the adsorption

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process between Cry1Ac protein and MHNPs as well as the stability of MHNPs, (ii)

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evaluate the retention ability of Cry1Ac-MHNP complex on the surface of cotton leaf and

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the bioactivity of Cry1Ac-MHNPs to Helicoverpa armigera (H. armigera), and (iii)

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investigate the biosafety of MHNPs to cotton, Bt, E. coli and H. armigera. Our results

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provided a promising method to control the loss of biopesticides in agricultural

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applications.

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2. MATERIALS AND METHODS

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2.1. Materials. Light magnesium oxide with a purity of 99% was provided by

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Sinopharm Chemical Reagent Company (Shanghai, China). Pageruler pre-stained protein

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ladder was purchased from Thermo Scientific Company (USA). MES was obtained from

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Shanghai Macklin Biochemical Company (Shanghai, China). Tryptone and yeast extract

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were supplied from Oxid Company (England). Cotton (Ji228) was provided by Hebei

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Jinfa Seed Company (Hebei, China). Other chemicals were of analytical grade and

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purchased from Sinopharm Chemical Reagent Company (Shanghai, China).

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2.2. Insect Culture. The H. armigera larvae were reared in an environmentally

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controlled laboratory (T = 27 ± 1 °C, RH = 60 ± 5%, L:D = 16:8) at Fujian Agriculture

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and Forestry University, China.


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2.3. Preparation of Cry1Ac Protein. Bt (HD-73) was preserved in the Key

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Laboratory of Biopesticide and Chemical Biology, Fujian Agriculture and Forestry

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University, China. The preparation of Cry1Ac protein was conducted according to a

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previously described method26 with minor modifications. Briefly, Bt was inoculated on

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solid half strength Luria-Bertani (LB) medium and cultured at 30 °C for 48 h. After

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washing with 1 M sodium chloride (NaCl) and double distilled water (ddH2O) twice, the

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crystals and spores were resuspended in solubilization buffer (50 mM Na2CO3, 50 mM

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EDTA, 5% β-mercaptoethanol, pH 9.5) for at least 4 h, and centrifuged at 16737 g for 20

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min at 4 °C. The supernatant was then harvested, and the pH was adjusted with 4 M

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HAc-NaAc (pH 4.5) to 4.5. The supernatant was kept on ice for 1 h, and the proteins

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were collected by centrifugation at 16737 g for 20 min at 1 °C. The obtained proteins

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were dissolved in 50 mM Na2CO3 (pH 9.5) and stored at -80 °C prior to further test.

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2.4. Synthesis of MHNPs. MHNPs were synthesized according to the previous

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studies with minor modifications.23,27,28 Briefly, 3.5 g light magnesium oxide was slowly

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added to ddH2O with continuous stirring in a water bath at 80 °C for 1 h. The mixture

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was centrifuged at 12298 g in Eppendorf centrifuge for 5 min and washed with ddH2O

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twice for further characterization.

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2.5. Loading of Cry1Ac Protein onto MHNPs. To investigate optimal loading of

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Cry1Ac protein to MHNPs, 1 mL Cry1Ac protein (1.52 mg/mL) in 50 mM Na2CO3 was

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loaded onto MHNPs [Cry1Ac-MHNPs (w:w)] at various ratios (1:3.28, 1:6.56, 1:9.84,



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1:13.12 and 1:19.68), and then the mixture was incubated in 2-mL centrifuge tube at

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room temperature with a agitation in Standard rotary mixing instrument (Scilogex Co.,

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USA) for 50 min [50 min was enough for adsorption according to the adsorption kinetic

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analysis (Figure S2)]. The suspension was collected and centrifuged at 16737 g for 10

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min at 4 °C. The adsorption rate was determined based on the protein concentration in the

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supernatant using bovine serum albumin (BSA) as standard protein (Bradford assay).29

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Additionally, to assess the stability of Cry1Ac protein during the loading process, the

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residual Cry1Ac protein in the supernatant was analyzed by SDS-PAGE.

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2.6. Characterization. The Braumer-Emmet-Teller (BET) surface area of MHNPs

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was determined with a Micromeritics’ ASAP 2000 (Micromeritics Co., USA). Cry1Ac

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protein, MHNPs and Cry1Ac-MHNPs were dried at ultra-low temperature in vacuum

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desiccator (Beijing Songyuanhuaxing Technology Develop Co., China). Zeta potential

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and size distribution were determined by Malvern’s Zetasizer Nano ZS90 (Malvern Co.,

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England).28 MHNPs and Cry1Ac-MHNPs were characterized on a JSM-6700F scanning

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electron microscope (SEM) (JEOL Co., Japan) equipped with an Oxford-INCA

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energy-dispersive X-ray spectroscopy (EDS). The crystal structure and interaction

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analyses of MHNPs and Cry1Ac-MHNPs were performed on Tecnai G2 F20 S-TWIN

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(200 kV) (TEM) (FEI Co., USA) and PANalytical X’ Pert PRO diffractometer (XRD)

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(PANalytical Co., Netherlands). The thermal gravimetric analysis (TGA) of MHNPs was

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recorded by the simultaneous thermal analyzer STA449C (Netzsch Co., Germany) in


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argon atmosphere within the temperature range of 25−800 °C and a heating rate of

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10 °C/min. FT-IR measurement was conducted within the range of 500–4000 cm−1 using

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Nicolet IS10 (Thermo Scientific Co., USA).

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2.7. Investigation of Adhesion Performance of Cry1Ac-MHNPs on Cotton

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Leaf. Briefly, 1 mL Cry1Ac protein (1.98 mg/mL) and 0.03 g MHNPs were mixed by

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Standard rotary mixing instrument for 50 min at room temperature [Cry1Ac-MHNPs

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(w:w) at 1:15.15, this ratio was between 1:19.68 and 1:13.12, thus the spraying

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Cry1Ac-MHNP complex might release small amount of Cry1Ac protein immediately for

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pest control]. Cotton leaves were divided into two groups with same leaf size and stage.

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Cotton leaves in the first group were placed on the petri dish with 30° on the ground at

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room temperature, and the mixture (Cry1Ac-MHNPs) was evenly sprayed onto the leaf

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surface. After air-drying, 3 mL ddH2O was evenly sprayed onto the leaf surface to

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simulate the rainwater. Subsequently, the leaves were air-dried and immersed into 3 mL

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50 mM dissolved medium (Na2CO3, pH 9.5) and shaken at room temperature for 10 min.

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The pH of suspensions was adjust to 4.5 with 4 M HAc-NaAc (pH 4.5), and the

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suspensions were kept on ice for 1 h and then centrifuged at 16737 g for 20 min at 4 °C.

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MHNPs could be resolved by 4 M HAc-NaAc medium (pH 4.5), and the Cry1Ac protein

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could be precipitated at pH 4.5.26 The precipitation was collected and washed with ddH2O

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for three times. Finally, 3 mL dissolved medium (50 mM Na2CO3, pH 9.5) was added to

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dissolve the protein, and protein concentration was quantified using BSA as described



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above.

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After spraying with ddH2O, MHNPs, Cry1Ac protein and Cry1Ac-MHNPs

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respectively, another group of leaves was treated with ddH2O runoff treatments and

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air-dried as described above. Subsequently, the leaves were punched into wafer (d = 6

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mm), stored at -80 °C in ultra-low temperature freezer (Thermo Scientific Co., USA) for

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12 h, and then dried in vacuum desiccator overnight for further test. The leaves were

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characterized by SEM.

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2.8. Pest Bioassays. Bioassays were conducted according to a previously described

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protocol by Liao30 with minor modifications. Briefly, artificial diet was added into

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24-well tissue culture plates, and then 50 µL toxin (Cry1Ac protein concentrations were

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3.12, 6.25, 12.5, 25, 50 and 100 µg/mL) or control [ddH2O, MHNPs (0.03 g/mL)] were

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evenly transferred onto the surface of the diet and allowed to dry before addition of the

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larvae. Subsequently, one second-instar H. armigera was added to each well (n = 24 per

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treatment). Finally, after incubation in the environmentally controlled room (T = 27 ±

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1 °C, RH = 60 ± 5%, L:D = 16:8) for 7 days, larval mortality was scored by gentle

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probing. All the treatments were carried out in triplicate.

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To further investigate the adhesion performance, leaves were divided into four groups

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and treated with different water runoff treatments according to the Section 2.7. Next, the

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leaves were punched into wafer (d = 6 mm) and randomly divided into three groups, with

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six replications in each group. Leaves were then added into 12-well tissue culture plates,


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and one larva was added to each well (n = 6 per treatment). Larval mortality was scored

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by gentle probing.

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2.9. Acid Hydrolysis of MHNPs. To simulate the acid hydrolysis of MHNPs in the

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field, MHNPs and Cry1Ac-MHNPs (the loading ratio was 1:15.15) were mixed in 2-mL

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centrifuge tube containing 1 mL 30 mM MES medium (pH 5.6) at room temperature with

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mild agitation. The supernatant was replaced with fresh medium everyday. The weight of

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MHNPs was recorded. The control group was set as ddH2O (pH 6.8).

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2.10. Biosafety Evaluation of MHNPs. 2.10.1. Biosafety evaluation on cotton.

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Biosafety evaluation of MHNPs on cotton (plant) was conducted with seed germination

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assays.31,32 Briefly, an 11-cm filter paper (Whatman) was placed in a 150-mm plastic petri

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dish. Then 5 mL of test solution was added onto the filter paper. Test solutions were

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ddH2O, NaOH (pH 10.6), 12.5-500 mg/L Mg2+ (MgSO4·7H2O) or 12.5-500 mg/L

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MHNPs. A total of 12 cotton seeds were placed on the filter paper. After growing for 8

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days, roots and shoots of germinated seeds were measured using digital calipers, and the

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germination rate was recorded. Additionally, to evaluate the influence of the MHNPs to

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the cotton leaf, one milliliter 0.03 g/mL MHNPs or ddH2O were sprayed evenly in the

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surface of the cotton leaf (n = 6 per treatment). After 15 days, the total chlorophyll and

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carotenoid of the leaves were extracted according to a previously described method.33

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The total chlorophyll and carotenoid of the leaves in different treatments were measured

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by UV–Vis absorptiometry (UV-1600, Japan). All tests were carried out in triplicate.



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2.10.2. Biosafety evaluation on Bt and E. coli. Bt (HD73) (Gram-positive

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bacterium) was added into the LB containing 12.5-500 mg/L Mg2+ (MgSO4·7H2O),

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12.5-500 mg/L MHNPs, ddH2O or NaOH (pH 10.6) and cultured at 30 °C for 24 h. The

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OD600 of the resulting solution was recorded by UV–Vis absorptiometry (UV-1600, Japan)

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to reflect the growth of Bt. Additionally, biosafety evaluation on E. coli (Gram-negative

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bacterium) was also conducted at 37 °C. All tests were carried out in triplicate.

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2.10.3. Biosafety evaluation on pest. Biosafety evaluation on pest was conducted

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according to the Section 2.8. Second-instar H. armigera was selected for the test. All tests

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were carried out in triplicate.

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2.11. Statistical Analysis. All results were expressed as means ± standard deviation

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(SD). Data were arranged with Microsoft Excel 2016 and analyzed with SPSS 18.0 in

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independent t-test and Probit algorithm. A p value of < 0.05 was considered as

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statistically significant.

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3. RESULTS AND DISCUSSION

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3.1. Synthesis and Morphology Investigation. MHNPs were synthesized in

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order to fabricate a carrier for Cry1Ac protein. SEM images demonstrated that MHNPs

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had the flower-like self-supported structure consisting of cross nano-sheets (Figure S1a),

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which was similar to the reported morphology of MHNPs.23,34 The morphology of

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Cry1Ac-MHNPs had no obvious changes (Figure S1b). Additionally, the EDS revealed

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that the compound contained C, N, P and S elements (Figure S3), confirming that Cry1Ac


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protein was loaded onto MHNPs. Besides, we also found that the morphology of MHNPs

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was not changed according to the TEM images (Figure S1c and d), indicating the

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structural stability of MHNPs. Moreover, the physical size and distribution of particle

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size were determined, and a (Z average) particle dimension of 712 nm (MHNPs) and 825

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nm (Cry1Ac-MHNPs) was observed, indicating that MHNPs and Cry1Ac-MHNPs had a

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little agglomeration in water (Figure S4).

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3.2. Stable Property and Interaction Analysis. We performed XRD, TGA, zeta

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potential measurements and FT-IR analyses in order to investigate stability of MHNPs as

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well as the interactions between MHNPs and Cry1Ac protein. XRD patterns of MHNPs

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showed that no obvious new peak or peak shift was found before and after loading of

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Cry1Ac protein (Figure 1a), indicating that no obvious chemical reaction occurred

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between Cry1Ac protein and MHNPs and the adsorption process might be induced by

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physical modification. The physical adsorption process might be beneficial to the stability

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of Cry1Ac protein, suggesting that MHNPs did not affect the biological activity of

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Cry1Ac protein. Additionally, the size of MHNPs at the (101) direction was 18.53 nm

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according to the Scherrer equation, close to the reported value for MHNPs (15.60 nm),23

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indicating that MHNPs in some dimensions were at nano level and they could be

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embedded into the groove of the leaf surface.

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The TGA measurement showed that the weight loss of MHNPs at 300 °C might be

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attributed to the H2O loss from MHNPs. Moreover, the weight loss of MHNPs within the



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temperature range of 300−450 °C might be attributed to the decomposition of Mg(OH)2

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and subsequent transformation into MgO (Figure 1b).35 The TGA pattern of MHNPs

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demonstrated that MHNPs could be a perfect nanocarrier for delivery of Cry1Ac protein.

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The zeta potential measurement was also performed to investigate the interface binding

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between MHNPs and Cry1Ac protein. The results showed that Cry1Ac protein carried

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negatively charges, while MHNPs exhibited a positively charged surface, and

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Cry1Ac-MHNPs exhibited a negatively charged surface (Figure 1c). This result indicated

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that the binding between MHNPs and Cry1Ac protein could be driven by the electrostatic

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interactions.

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Besides, FTIR measurement was employed to further investigate the interaction

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between MHNPs and Cry1Ac protein. Figure 1d shows that the intense peaks at 3699.8

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cm−1 and 448.9 cm−1 were the characteristic peaks of Mg–O.36 A new peak at about

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1647.2 cm−1 might belong to the –NH2 groups in Cry1Ac-MHNPs, indicating that the Cry

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protein was adsorbed onto MHNPs. Additionally, the intensity of the hydroxyl group

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(-OH) absorption peak (3448 cm−1) was significantly increased. These results showed that

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the protein was adsorbed onto MHNPs. BET measurement showed that MHNPs

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possessed a high surface area at a value of 100.1 m²/g, confirming that MHNPs had large

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capacity to absorb Cry1Ac protein.

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3.3. Loading of Cry1Ac Protein onto MHNPs. To construct the Cry1Ac-MHNP

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complexes and investigate the optimal and complete loading process of Cry1Ac protein


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onto MHNPs, Cry1Ac protein was loaded onto MHNPs at different loading ratios. The

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Cry1Ac protein was extracted from Bt (HD73), and the SDS-PAGE revealed a molecular

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mass of 130 kDa for Cry1Ac protein (Figure 2a, lane 1).26 The Cry1Ac protein was

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completely loaded at a Cry1Ac/MHNP mass ratio of 1:19.68 (w:w, mg) (Figure 2a, lane

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6), and the adsorption process did not affect the size of Cry1Ac protein, which was

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consistent with previous study.37 Moreover, the concentration measurement of the

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Cry1Ac protein in the supernatant at this mass ratio showed that 97.16% Cry1Ac protein

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was loaded onto MHNPs (Figure 2b), suggesting that MHNPs had high adhesive strength

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toward Cry1Ac protein.

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3.4. Binding Performance between Cry1Ac-MHNPs and Cotton Leaf.

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According to the adsorption results between Cry1Ac protein and MHNPs, we

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investigated the adhesion performance between Cry1Ac-MHNPs and cotton leaf. There

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were lots of micro/nano-grooves on the surface of cotton leaf, resulting in a rough surface

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and a high adhesion ability for MHNPs (Figure 3a). When Cry1Ac protein was loaded,

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there was no obvious change in leaf surface (Figure 3c). When MHNPs and

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Cry1Ac-MHNPs were sprayed onto the leaf surface, lots of micro/nano-sheets were

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embedded into the groove of the leaf surface (Figure 3b). Therefore, after ddH2O runoff

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treatments, there were still a large amount of MHNPs distributed on the leaf surface

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(Figure 3d). This result confirmed the strong adhesion ability of MHNPs on the leaf

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surface.



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To further certify the adsorption performance between Cry1Ac-MHNPs and leaf, the

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Cry1Ac protein on the leaf surface was extracted, and the concentration measurement

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confirmed the anti-washing ability of MHNPs (Figure 3e). The retained protein amount

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was increased along with the mass of MHNPs, and retained protein biomass in groups of

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15, 20 and 30 mg was significantly higher compared with Cry1Ac protein along group (p

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< 0.01). The significant increase of the adhesion in Cry1Ac protein biomass was mainly

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attributed to the high adsorption and retaining capacities of MHNPs on the surface of

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cotton leaf.

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3.5. Acid Hydrolysis of MHNPs. To investigate the residual period of

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Cry1Ac-MHNPs, 30 mM MES medium (pH 5.6) was added to MHNPs and

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Cry1Ac-MHNPs to simulate the acid hydrolysis of MHNPs in the nature environment.

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The weight of MHNPs was not changed obviously when MHNPs were mixed with

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ddH2O. However, the weight of MHNPs was slowly decreased, and finally an equilibrium

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was established when MHNPs were rinsed with the MES medium for 27 times.

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Additionally, the residual period of Cry1Ac-MHNPs was same as MHNPs (Figure S5a).

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This result indicated that loading of Cry1Ac protein onto MHNPs did not affect the acid

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hydrolysis process of MHNPs. Meanwhile, the dry weight of final samples confirmed

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that MHNPs could be slowly resolved by acid medium, while they remained stable in the

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neutral environment (Figure S5b). The result demonstrated that Cry1Ac-MHNPs had a

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balance between long persistence and biodegradability, which was beneficial for the


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economy and environment.

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3.6. Pest Bioassay. Bioassays were conducted on the artificial diet. Table S1 shows

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that Cry1Ac protein adsorbed and non-adsorbed MHNPs had comparable LC50 values

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[LC50 (Cry1Ac) = 0.67 (0.57–0.79) µg/cm2 and LC50 (Cry1Ac-MHNPs) = 0.71 (0.60–0.86) µg/

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cm2]. The larvae showed abnormal size reduction after the treatment with Cry1Ac protein,

285

and the size of larvae was negatively correlated with the concentration of Cry1Ac protein

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(Figure S6). However, there was no obvious difference in size before and after the

287

treatment of MHNPs, suggesting that MHNPs did not affect the toxicity of Cry1Ac

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protein. This finding was consistent with a previous report that silica particles do not

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change the biological activity of Cry1Ab.15

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In addition, cotton leaves with different treatments were prepared to feed H. armigera

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larvae. At the end of 72 h, the larval mortality after treatment of Cry1Ac-MHNPs was

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significantly higher compared with other treatments (p < 0.001) (Figure 4a). Figure 4b

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reveals that the larvae treated with Cry1Ac-MHNPs-leaf showed abnormal size reduction

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compared with others. This finding might be attributed to the high adhesion ability of

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Cry1Ac-MHNPs on the cotton leaves. These results further confirmed the high adsorption

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and retaining capacities of MHNPs on the leaf, indicating that MHNPs could be used as a

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potential adjuvant for biopesticide in agricultural applications.

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3.7. Biosafety of MHNPs. In order to assess the biosafety of MHNPs, we

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investigated the effects of different concentrations of Mg2+ and MHNPs on cotton, Bt, E.



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coli and H. armigera. With increasing the concentration of Mg2+ and MHNPs from 12.5

301

to 500 mg/mL, the cotton seed germination and the shoot length and the root length had

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no significant difference with the treatment of ddH2O (p > 0.05) (Figure 5 a and b).

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Additionally, with spraying the MHNPs on the surface of cotton leave, the total

304

chlorophyll and carotenoid had no significant difference with the treatment of ddH2O (p >

305

0.05) (Figure S7). Meanwhile, the OD600 value of Bt and E. coli after adding with Mg2+

306

and MHNPs (12.5-500 mg/mL) exhibited no significant difference with the control group

307

(p > 0.05), suggesting the MHNPs did not affect the growth of Bt and E. coli (Figure 5c).

308

Additionally, the mortality of H. armigera with treatments of Mg2+ and MHNPs

309

(12.5-500 mg/mL) had no significant difference with ddH2O group (p > 0.05) (Figure 5

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d). The results above suggesting that MHNPs could be used as a safe adjuvant for

311

preparation of Bt formulation.

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Supporting Information

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Additional figures and table depicting the data in SEM and TEM images of MHNPs and

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Cry1Ac-MHNPs, adsorption kinetic analysis, EDS patern of Cry1Ac-MHNPs, size

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distribution of MHNPs and Cry1Ac-MHNPs particles, acid hydrolysis of MHNPs,

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mortality and the digital photographs of second instar larvae of H. armigera treated with

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Cry1Ac protein and Cry1Ac-MHNPs on the artificial diet and the amount of the

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chlorophyll and carotenoid of the cotton leave before and after treating with MHNPs.

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This material is available free of charge via the Internet at http://pubs.acs.org.


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Funding

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This work was supported by the National Natural Science Foundation of China

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(31601686), Natural Science Foundation of Fujian Province, China (2016J01112),

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Science Fund for Distinguished Young Scholars of Fujian Agriculture and Forestry

324

University (Grant xjq201719), and the Special Fund for Scientific and Technological

325

Innovation of Fujian Agriculture and Forestry University (Grants CXZX2017214,

326

KF2015063-065).

327

Notes

328

The authors declare no competing financial interest.

329 330

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Figure 1. (a) XRD pattern, (b) TGA, (c) zeta potential measurements and (d) FTIR of the

438

samples.

439 440



441 442

Figure 2. (a) SDS-PAGE analysis of the adsorption between Cry1Ac protein and MHNPs

443

[M: Marker; 1: Cry1Ac protein; 2–6: Cry1Ac-MHNPs (w:w)] at various ratios of 1:3.23,

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MHNPs.

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448 449

Figure 3. (a–d) SEM images of cotton leaves treated with ddH2O, MHNPs, Cry1Ac

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protein or Cry1Ac-MHNPs, respectively. (e) Adhesion performance of Cry1Ac protein

451

after loading onto different mass of MHNPs. Compared with the control group, statistical

452

significance is shown with **p < 0.01.

453 454 455 456 457 458 459



460 461

Figure 4. (a) Mortality of the pest after feeding leaves with different treatments for 72 h.

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Compared with the Cry1Ac-MHNP group, statistical significance is shown with ***p

Flowerlike Mg(OH)2 Cross-Nanosheets for Controlling Cry1Ac Protein

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