In-situ binding method for evaluating binding of Bt toxin and midgut epithelial cells of Lepidoptera pest larvae

Abstract

The invention relates to an in-situ binding method for evaluating binding of Bt toxin Cry1Ac and midgut epithelial cells of cotton bollworm larvae. The method comprises the following steps: incubating and culturing midgut tissues of larvae of lepidoptera pests and a Bt toxin-containing Sf-900 II cell culture solution, carrying out immunostaining, observing and photographing through a laser confocal microscope, and finally carrying out quantitative processing on a fluorescence image by utilizing ImageJ software so as to calculate an average unit area fluorescence signal value. And further evaluating the difference between the binding amounts of the midgut epithelial cells and the Bt toxins of the larvae of different strains. The method can intuitively and accurately observe and evaluate the binding condition of the Bt toxin and the midgut epithelial cells on the premise of retaining the original structure and physiological environment of the midgut tissue of the larvae, provides direct data support for Bt resistance research, assists in analyzing the pest Bt resistance mechanism, has important significance in formulating resistance early warning and treatment strategies, and has a wide application prospect. The method is suitable for the biotechnical fields of Bt toxin insecticidal mechanism research, lepidoptera pest drug resistance mechanism exploration and the like.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to an in situ binding method for evaluating the binding of Bt toxin to midgut epithelial cells of lepidopteran pest larvae. Background Technology

[0002] Lepidopteran pests (such as cotton bollworms, corn borers, and diamondback moths) are a major group of pests in agricultural production. Their larvae feed on crop leaves, fruits, and stems, causing reduced yields or even crop failure. For a long time, chemical pesticides have been the main means of controlling lepidopteran pests. However, overuse has led to increased pesticide resistance in pests and increasingly prominent problems such as pesticide residues in agricultural products, making green and efficient alternative control technologies urgently needed.

[0003] Bt (Bacillus thuringiensis) toxins, as natural microbial insecticides, have become one of the core technologies for controlling lepidopteran pests due to their advantages such as high specificity, safety against non-target organisms, and good environmental compatibility. Among them, Cry1Ac and Cry1Ab are the most widely used Bt toxins, which have been transferred to crops such as cotton and corn to cultivate insect-resistant varieties, and have also been formulated into biological agents for field spraying. Their mechanism of action involves binding to specific receptors (such as aminopeptidase N and cadherin) on the microvilli (BBMV) of midgut epithelial cells of lepidopteran pest larvae, forming channels in the cell membrane, leading to cell rupture and larval death. Therefore, the binding efficiency and specificity of Bt toxins to midgut epithelial cell receptors are the key rate-limiting steps determining their insecticidal activity and are also core research targets for elucidating pest resistance mechanisms (such as receptor gene deletion or mutation leading to decreased binding capacity).

[0004] First, traditional Western blotting (WB) detection requires separating midgut BBMV using differential centrifugation, followed by SDS-PAGE electrophoresis, membrane transfer, and incubation with specific antibodies to detect the binding product. This method has three major technical bottlenecks: 1. Disruption of tissue structure leads to distortion of binding characteristics: The BBMV separation process requires repeated homogenization and high-speed centrifugation, which destroys the spatial structure (such as microvilli arrangement and intercellular connections) and physiological microenvironment (such as pH gradient and ion concentration) of midgut epithelial cells, making it impossible to reflect the natural binding site of toxins in vivo (such as whether they are specifically located at the apical microvilli) and the dynamic binding process. 2. Insufficient sensitivity in detecting low molecular weight binding products: The complex (molecular weight 200-300kDa) formed by the activated Cryotoxin active fragment (65kDa) and the receptor contains some low molecular weight binding intermediates (such as the 65kDa monomer-APN fragment complex). In Western blotting, these products are easily blurred due to diffusion effects during SDS-PAGE electrophoresis separation and efficiency loss during membrane transfer (the 0.45μm nitrocellulose membrane has a retention rate of <60% for small molecule conjugates), making it impossible to quantify low affinity binding events. 3. Receptor binding specificity cannot be verified: Western blot (WB) detection can only prove the in vitro binding of toxins to receptor proteins, but cannot rule out non-specific binding (such as hydrophobic interactions with membrane lipids), and cannot distinguish whether the binding site is located in a functional region on the cell surface (such as the glycosylation site of APN, where mutations can significantly affect binding activity), leading to a discrepancy between the detection results and the actual binding situation in vivo.

[0005] Secondly, conventional tissue staining methods (such as HE staining and DAB immunohistochemical staining) face two major technical obstacles when applied to the detection of Bt toxin binding: 1. Dehydration process leads to loss of binding signal: Routine immunohistochemistry requires gradient alcohol dehydration (70%-100%). During the dehydration process, organic solvents will cause membrane protein denaturation, further reducing the detection rate of binding signal, and the positive signal intensity will decrease by 40%-60%; 2. Insufficient resolution makes it impossible to locate binding sites: The thickness of conventional paraffin sections (5-8 μm) cannot clearly show the subcellular structures of midgut epithelial cells (such as microvilli with a length of only 1-2 μm), and the substrate deposits (0.5-1 μm in diameter) of DAB staining are prone to diffusion effects, making it impossible to distinguish whether the toxin is bound to the tip of the microvilli or the basolateral membrane, thus failing to meet the research requirements of "precisely locating binding sites".

[0006] In summary, current technologies cannot achieve precise quantitative and localized detection of the binding of Bt toxin to the midgut epithelial cells of lepidopteran pests while preserving the original structure and physiological environment of biological samples. This leads to biases in the analysis of the toxin's mechanism of action and also restricts the rapid screening of drug-resistant genes. Therefore, developing an in-situ binding detection method that can overcome the above-mentioned technical bottlenecks is of significant technical value and application importance for promoting the application of Bt toxin and controlling pesticide resistance in pests. Summary of the Invention

[0007] The purpose of this invention is to provide an in situ binding method for evaluating the binding of Bt toxin to the midgut epithelial cells of lepidopteran pest larvae. This method can intuitively and accurately observe and evaluate the binding of Bt toxin to midgut epithelial cells while preserving the original structure and physiological environment of the larval midgut tissue. This provides an effective technical means for in-depth research on the insecticidal mechanism of Bt toxin and the resistance mechanism of lepidopteran pests to Bt toxin.

[0008] To achieve the above objectives, the technical solution of the present invention is as follows: An in situ binding method for evaluating the binding of Bt toxin to midgut epithelial cells of lepidopteran pest larvae, the method comprising the following steps: (1) Preparation of midgut tissue of lepidopteran pest larvae; (2) In vitro culture of midgut tissue; (3) In situ binding reaction of Bt toxin with midgut epithelial cells; (4) Location of binding sites and quantification of binding status; The Bt toxin is Cry1Ac or Cry1Ab.

[0009] Further, (1) the preparation of midgut tissue of lepidopteran pest larvae is to obtain the midgut of the lepidopteran pest larvae to be tested. The separated midgut is immediately placed in pre-cooled PBS (0.01M, pH7.4) buffer to remove the intestinal contents.

[0010] Furthermore, (2) midgut tissue was cultured in vitro by incubating the midgut tissue in a cell culture medium containing Bt toxin for 30 min-60 min; Preferably, the Bt toxin concentration in the cell culture medium containing Bt toxin is 0.5 mg / mL; Preferably, the incubation conditions are 25°C and 550 rpm.

[0011] Preferably, the cell culture medium is an insect cell culture medium; More preferably, the cell culture medium is Sf-900IISFM cell culture medium.

[0012] Furthermore, (2) after the midgut tissue is cultured in vitro, the process also includes the removal of non-specific binding and fixation of the midgut tissue; Preferably, the removal of non-specific binding involves discarding the cell culture medium containing Bt toxin after incubation, rinsing the midgut tissue twice with the cell culture medium, and then rinsing once with 0.01M, pH 7.4 PBS buffer, each rinse lasting 5 minutes. Preferably, the midgut tissue fixation is performed by fixing the midgut tissue after removing non-specific binding in 4% paraformaldehyde-PBS solution (0.01M, pH 7.4) for 24 hours.

[0013] Furthermore, (3) the in situ binding reaction of Bt toxin with midgut epithelial cells includes the following steps: 1) Sample preparation: The fixed midgut tissue was embedded in cryosection embedding medium, frozen at -20℃ for 2 hours, and then sectioned continuously at a thickness of 4.5μm and mounted on a glass slide; 2) Fixation of tissue sections: Fix the sections in 4% paraformaldehyde-PBS solution (0.01M, pH 7.4) for 20 minutes; 3) Tissue permeabilization: Place the sections in 0.1% Triton-X100-PBS solution (0.01M, pH 7.4) for 20 minutes to permeabilize; 4) Tissue blocking: Place the sections in freshly prepared 1% BSA-PBS solution (0.01M, pH 7.4) for 1 hour to block; 5) Immunostaining: The sections were washed twice with PBS (5 minutes each time), incubated overnight at 4°C with Bt toxin polyclonal antibody (1:200 dilution), rinsed three times with PBS, and then incubated for 4 hours with a mixture of Alexa Fluor 594-labeled goat anti-rabbit IgG (1:1000 dilution) and phalloidin (1:1000 dilution). Finally, the sections were counterstained with Hoechst 33342 (1:500 dilution) for 20 minutes. After each incubation, the sections were washed three times with PBS (5 minutes each time). Furthermore, the Bt toxin polyclonal antibody is either a Cry1Ac polyclonal antibody or a Cry1Ab polyclonal antibody.

[0014] Furthermore, (4) the location of the binding site was determined by using a confocal microscope to obtain immunofluorescence images of the midgut tissue and clarify the subcellular location of the toxin binding.

[0015] Preferably, the confocal microscope is a Leica TCS SP8 laser confocal scanning microscope.

[0016] Furthermore, the combination of (4) is quantified by using ImageJ software, with a unified signal domain value of 5-255, and using the rectangular selection tool to draw a square frame with a side length of 200 pixels to define the toxin signal region, and calculating the unit area (1μm). 2 The average fluorescence signal value was calculated for each image, with the average value of three regions taken. Each treatment had six biological replicates.

[0017] Furthermore, the specific calculation of the combination case quantification in (4) is based on the Intden (Integrated Density) output by ImageJ software and the actual area of ​​the selection box. The combination case quantification calculation is as follows: per unit area (1μm 2The average fluorescence signal value is equal to Intden / the actual selected area. Beneficial effects

[0018] This invention provides an in situ binding method for evaluating the binding of Bt toxin to midgut epithelial cells of lepidopteran pest larvae. The method involves culturing midgut tissue in a cell culture medium to maintain its good activity while adding an appropriate concentration of Bt toxin, thereby achieving in situ binding of the toxin to the midgut epithelial cells.

[0019] Compared with existing methods for studying the binding of Bt toxin to the midgut epithelial cells of lepidopteran pest larvae, the advantages of using the research method of this invention are: (1) Realistically reflects the binding situation in vivo: The in situ binding method of the present invention preserves the complete structure and physiological environment of the midgut tissue of lepidopteran pest larvae, so that Bt toxin can bind to the receptor of midgut epithelial cells in its natural position, avoiding the change of receptor conformation or position migration caused by sample separation and purification, and can more realistically simulate the interaction process in the body, providing a more reliable experimental basis for studying the mechanism of toxin action.

[0020] (2) Directly locate the binding site: Through immunostaining technology, the binding site of Bt toxin on midgut epithelial cells can be directly observed under a laser confocal microscope, clarifying the binding position of the toxin and the receptor, which helps to understand the action pathway and mechanism of the toxin, and is also of great significance for studying the reasons for the resistance of lepidopteran pests to Bt toxin.

[0021] (3) More accurate quantitative assessment: Using image analysis software to quantitatively analyze fluorescence intensity and distribution can accurately assess the binding strength and binding specificity of Bt toxin to midgut epithelial cells. Compared with traditional methods, it can provide more accurate data support, which facilitates in-depth analysis and comparison of experimental results.

[0022] By clarifying the in situ binding method of Bt toxin to the midgut epithelial cells of lepidopteran pest larvae, the positive effects of this invention are: providing reliable data support for Bt resistance research, accurately assessing changes in the binding characteristics of Bt toxin to midgut epithelial cells of lepidopteran pests, providing direct evidence for elucidating the Bt resistance mechanism of lepidopteran pests (such as changes in receptor binding capacity), and assisting in the formulation of resistance early warning and control strategies. Attached Figure Description

[0023] Figure 1An immunofluorescence illustration of the cell state after culturing the midgut tissue of cotton bollworm larvae in cell culture medium, including the control group and treatment groups cultured in vitro for 10 minutes, 30 minutes, and 60 minutes; in the figure, the cell nuclei were stained with Hoechst (blue), and the cytoskeleton and basement membrane were stained with phalloidin (green); the intestinal lumen (GL) and brush border microvilli (BBM) are shown in the figure.

[0024] Figure 2 This experiment involved the in situ binding of Bt toxin (Cry1Ac) to the midgut epithelial cells of cotton bollworm larvae; among which: A is a schematic diagram of immunofluorescence localization, including the control group and the toxin treatment groups at 10 minutes, 30 minutes, and 60 minutes; in the figure, cell nuclei are stained with Hoechst (blue), the cytoskeleton and basement membrane are stained with phalloidin (green), and the binding of Bt toxin (Cry1Ac) is shown by fluorescent secondary antibody labeled with Alexa Fluor 594 (red); the intestinal lumen (GL) and brush border microvilli (BBM) are shown in the figure; B is a statistical bar chart of fluorescence intensity.

[0025] Figure 3 This diagram illustrates the selection of the Bt toxin (Cry1Ac) signal statistical region. The red area represents the immunofluorescence signal of Bt toxin (Cry1Ac), and the yellow box represents the selected Bt toxin (Cry1Ac) signal statistical region.

[0026] Figure 4 This study investigated the in situ binding of Bt toxin (Cry1Ac) to midgut epithelial cells of different strains of cotton bollworm larvae (including the susceptible strain SCD and three resistant strains SCD-r1, SCD-KI, and C2 / 3-KO); among which: A is a schematic diagram of immunofluorescence localization, including a control group of four different strains and a 30-minute toxin treatment group; in the figure, the cell nucleus is stained with Hoechst (blue), the cytoskeleton and basement membrane are stained with phalloidin (green), and the binding of Bt toxin (Cry1Ac) is shown by fluorescent secondary antibody labeled with Alexa Fluor 594 (red); the intestinal lumen (GL) and cell detachment (yellow arrows) are shown in the figure; B is a statistical bar chart of fluorescence intensity.

[0027] Figure 5 This study investigated the in situ binding of Bt toxin (Cry1Ab) to the midgut epithelial cells of different strains of corn borer larvae (including one susceptible strain and one resistant strain); among which: Figure A shows the immunofluorescence localization diagram, including the control group and two strains treated with toxins for 30 minutes. Cell nuclei were stained with Hoechst (blue), the cytoskeleton and basement membrane with phalloidin (green), and the binding of Bt toxin (Cry1Ac) was shown using Alexa Fluor 594-labeled fluorescent secondary antibody (red). The intestinal lumen (GL), brush border microvilli (BBM), and basement membrane (BM) are shown in the figure. B is a statistical bar chart of fluorescence intensity. Detailed Implementation

[0028] The present invention will be further explained below with reference to the embodiments, but the embodiments do not limit the present invention in any way.

[0029] Example 1: Determination of cell state in midgut tissue of cotton bollworm-sensitive strain SCD larvae after culturing in cell culture medium. To evaluate the cell status of midgut tissue from the susceptible cotton bollworm strain SCD (a susceptible strain collected in the 1970s from Côte d'Ivoire, Africa, and continuously raised indoors without any drug exposure for over 40 years) after culturing in cell culture medium, this experiment first selected healthy and uniformly developed SCD cotton bollworm larvae for midgut tissue preparation. Subsequently, the midgut tissue was incubated in cell culture medium, fixed, sectioned, and immunostained. Finally, immunofluorescence images of the tissue were obtained using a laser confocal microscope to observe tissue integrity and cell status.

[0030] The specific steps are as follows: 1. Sample preparation of midgut tissue from cotton bollworm larvae: 1.1 Larval Selection and Rearing Healthy, uniformly developed cotton bollworm larvae were selected and fed artificial feed until the fifth instar under conditions of 26-28℃, 30%-40% relative humidity, and 16L:8D photoperiod to ensure stable physiological state of the larvae.

[0031] 1.2 Midgut tissue acquisition The fifth-instar larvae on the second day were anesthetized in an ice bath for 5 minutes. The head and tail of the larvae were fixed with a dissecting needle, and the larvae were cut along the midline of the body wall with dissecting scissors to expose the midgut tissue. The midgut was carefully separated from the surrounding fat body, trachea and other tissues with forceps, taking care not to damage the midgut epithelium.

[0032] 1.3 Midgut tissue cleaning The isolated midgut tissue was immediately placed in pre-cooled PBS (0.01M, pH 7.4) buffer to remove intestinal contents.

[0033] 2. In vitro culture of midgut tissue 2.1 In vitro culture After cleaning the midgut tissue of each strain of cotton bollworm larvae, quickly absorb excess liquid with clean filter paper and place it in Sf-900 II SFM cell culture medium. Incubate at 25°C and 550 rpm for 10, 30 and 60 minutes respectively.

[0034] 2.2 Fixation of midgut tissue After the processed midgut tissue was quickly blotted with clean filter paper to remove excess liquid, it was fixed in 4% paraformaldehyde-PBS (0.01M, pH 7.4) solution for about 24 hours.

[0035] 3. Preparation of tissue sections 3.1 Sample Preparation After fixing, the midgut tissue was quickly blotted to remove excess liquid with clean filter paper. Then, it was embedded with cryo-section embedding medium (SAKURA, Tissue-Tek®) and frozen at -20°C for 2 hours. Finally, the tissue embedding block was serially sectioned at a thickness of 4.5 μm and mounted on a glass slide.

[0036] 3.2 Fixation of tissue sections Tissue sections were fixed in 4% paraformaldehyde-PBS (0.01M, pH 7.4) solution for 20 minutes.

[0037] 3.3 Tissue permeability Tissue sections were permeabilized in 0.1% Triton-X100-PBS (0.01M, pH 7.4) solution for 20 minutes.

[0038] 3.4 Organizational Closure Tissue sections were placed in freshly prepared 1% BSA-PBS (0.01M, pH 7.4) solution for 1 hour for blocking.

[0039] 4. Immunostaining After blocking, the tissue sections were washed twice with PBS (0.01M, pH 7.4), 5 minutes each time. Then, the tissue sections were incubated with a mixture of phalloidin (1:1000 dilution, Invitrogen) for 4 hours, and finally counterstained with Hoechst 33342 (1:500 dilution, Invitrogen) for 20 minutes. After each incubation, the tissue was washed three times with PBS (0.01M, pH 7.4), 5 minutes each time.

[0040] 5. Confocal microscopy observation Midgut epithelial cells were scanned using a laser confocal scanning microscope (Leica TCSSP8) to obtain immunofluorescence images of the midgut tissue, clarifying the subcellular localization of toxin binding (e.g., microvilli, cell membrane, etc.). The results are as follows: Figure 1As shown, the results indicated that, compared with the midgut that was not incubated after dissection, the midgut cell layer structure remained intact after in vitro culture for 10 minutes, 30 minutes, and 60 minutes. No abnormalities were observed in the cell nuclei or cytoskeleton, and the brush border membrane vesicles (BBMV) were continuous and morphologically normal. Figure 1 This indicates that the midgut tissue of cotton bollworm larvae can maintain a normal state within 60 minutes of culture in cell culture medium.

[0041] Example 2 evaluates the binding of Bt toxin Cry1Ac to midgut epithelial cells of susceptible cotton bollworm larvae (SCD) after in situ incubation for different durations. To evaluate the binding of Bt toxin Cry1Ac to midgut epithelial cells of the susceptible strain SCD (a susceptible strain collected from Côte d'Ivoire in the 1970s and continuously raised indoors without any drug exposure for over 40 years) for different durations of in situ incubation, this experiment first selected healthy and uniformly developed bollworm larvae from various strains to prepare midgut tissue. The midgut tissue was then incubated in Cry1Ac-cell culture medium at specific concentrations for different durations (10 minutes, 30 minutes, and 60 minutes). After tissue fixation, sections were prepared and immunostained. Finally, immunofluorescence images of the tissues were acquired using laser confocal microscopy, and fluorescence statistics and data processing were performed to obtain the binding data of Bt toxin Cry1Ac to midgut epithelial cells of bollworm larvae treated with gradient time.

[0042] The specific steps are as follows: 1. Sample preparation of midgut tissue from cotton bollworm larvae: Same as steps 1 and 2 in Example 1.

[0043] 2. In-situ binding reaction 2.1 Preparation of the reaction system Cry1Ac toxin was added to cell culture medium (Sf-900IISFM, Invitrogen) to prepare a 0.5 mg / ml Cry1Ac-cell culture medium mixed solution.

[0044] 2.2 Incubation reaction After cleaning, the midgut tissue of each strain of cotton bollworm larvae was quickly blotted with clean filter paper to remove excess liquid, and then placed in 0.5 mg / ml Cry1Ac- cell culture medium and cultured at 25°C and 550 rpm for 10 minutes, 30 minutes and 60 minutes respectively.

[0045] 2.3 Removal of nonspecific binding After incubation, the toxin solution was discarded, and the midgut tissue was rinsed twice with cell culture medium, followed by one rinse with PBS (0.01M, pH 7.4) buffer for 5 minutes each time to thoroughly remove unbound toxins. A control group was set up, which was supplemented with only cell culture medium (Sf-900 IISFM, Invitrogen) to exclude interference from non-specific binding.

[0046] 2.4 Fixation of midgut tissue After the processed midgut tissue was quickly blotted with clean filter paper to remove excess liquid, it was fixed in 4% paraformaldehyde-PBS (0.01M, pH 7.4) solution for about 24 hours.

[0047] 3. Preparation of tissue sections Same as Example 1.

[0048] 4. Immunostaining After blocking, the tissue sections were washed twice with PBS (0.01M, pH 7.4), 5 minutes each time. Then, the tissue sections were incubated overnight at 4°C with Cry1Ac polyclonal antibody (manufacturer: Abbexa, catalog number abx023892, 1:200), followed by washing three times with PBS (0.01M, pH 7.4) buffer. Subsequently, they were incubated for another 4 hours with a mixture of Alexa Fluor 594-labeled goatanti-rabbit IgG (1:1,000 dilution, Abcam) and phalloidin (1:1000 dilution, Invitrogen). Finally, they were counterstained with Hoechst 33342 (1:500 dilution, Invitrogen) for 20 minutes. After each incubation, the tissue sections were washed three times with PBS (0.01M, pH 7.4), 5 minutes each time.

[0049] 5. Confocal microscopy observation Midgut epithelial cells were scanned using a laser confocal scanning microscope (Leica TCSSP8) to obtain immunofluorescence images of the midgut tissue, clarifying the subcellular localization of toxin binding (e.g., microvilli, cell membrane, etc.). The results are as follows: Figure 2As shown in Figure 2A, after 10 minutes of in vitro incubation, a small amount of Cry1Ac bound to both microvilli and intracellular cells, and the midgut cells maintained their morphology intact. After 30 minutes of in vitro incubation, the amount of Cry1Ac binding to midgut cells increased significantly, but the cell state deteriorated to some extent: some stem cells at the basement membrane detached from the bottom, accompanied by nuclear deformation. After 60 minutes of in vitro incubation, a small number of midgut cells detached. It is speculated that the cell debris and toxins underwent partial degradation, and the remaining small amount of toxins were encapsulated in empty shells and fell into the intestinal lumen.

[0050] 6. Cry1Ac combined with quantization processing The fluorescence image was quantized using ImageJ software: the Cry1Ac signal was separated and a uniform signal range (5-255) was selected. Then, a rectangular marquee tool was used to draw a square with a side length of 200 pixels to define the toxin signal region. Figure 3 After obtaining the Intden (Integrated Density), the actual area of ​​the selection box is calculated, and finally the unit area (1μm) is obtained. 2 Average fluorescence signal intensity = Intden / actual area of ​​the selected box. The average value is calculated for three regions per image, with six biological replicates per treatment.

[0051] Statistical results on the binding of Bt toxin Cry1Ac to midgut epithelial cells of cotton bollworm larvae treated with gradient time are shown below. Figure 2 After 10 minutes of in vitro incubation, a small amount of Cry1Ac bound to both microvilli and intracellular cells; after 30 minutes of in vitro incubation, the amount of Cry1Ac binding to midgut cells increased significantly (approximately 2.7 times that at 10 minutes) (Figure 2B); after 60 minutes of in vitro incubation, the amount of Cry1Ac binding to midgut cells decreased by approximately 15.2% compared to 30 minutes (Figure 2B).

[0052] In summary, when the incubation time is 10 minutes, the reaction between Cry1Ac and midgut cells is insufficient, resulting in a low amount of toxin binding; while when the incubation time is 30 minutes and 60 minutes, Cry1Ac can fully react with and bind to midgut cells. Figure 2 However, the toxin binding decreased after 60 minutes of incubation, possibly because the reaction time between the toxin and midgut cells was too long, leading to partial degradation or detachment of both into the intestinal lumen. This is not conducive to accurate quantification of the fluorescence signal of Cry1Ac binding to midgut cells. Therefore, the most suitable treatment conditions are: incubation of cells in a medium containing 0.5 mg / mL Cry1Ac for 30 minutes.

[0053] Example 3 evaluates the binding of the Bt toxin Cry1Ac to the midgut epithelial cells of cotton bollworm larvae from a susceptible strain (SCD) and three resistant strains (SCD-r1, SCD-KI, C2 / 3-KO). To evaluate the Bt toxin Cry1Ac, this study compared it with a susceptible strain SCD (a susceptible strain collected in Côte d'Ivoire, Africa in the 1970s, continuously reared indoors without any pesticides for over 40 years) and three resistant strains of cotton bollworm larvae: SCD-r1 (a cadherin r1 mutant strain, approximately 438-fold resistant to Cry1Ac), SCD-KI (a TSPAN1 L31S point mutant strain, approximately 125-fold resistant to Cry1Ac), and C2 / 3-KO (ABCC2 & ABCC3 double knockout strains, more than 15,000-fold resistant to Cry1Ac). (All resistant strains were developed using CRISPR / Cas9.) This study investigated the binding of gene-edited midgut epithelial cells to the Bt toxin Cry1Ac in cotton bollworm larvae of the SCD strain. First, healthy and uniformly developed cotton bollworm larvae from various strains were selected for midgut tissue preparation. The midgut tissue was then incubated in a Cry1Ac-cell culture medium of a specific concentration. After tissue fixation, sections were prepared and immunostained. Finally, immunofluorescence images of the tissues were acquired using a laser confocal microscope, and fluorescence statistics and data processing were performed. Ultimately, data on the binding of midgut epithelial cells of four different resistant cotton bollworm larvae to the Bt toxin Cry1Ac were obtained.

[0054] The specific steps are as follows: 1. Sample preparation of midgut tissue from cotton bollworm larvae: Same as steps 1 and 2 in Example 1.

[0055] 2. In-situ binding reaction 2.1 Preparation of the reaction system Cry1Ac toxin was added to cell culture medium (Sf-900IISFM, Invitrogen) to prepare a 0.5 mg / ml Cry1Ac-cell culture medium mixed solution.

[0056] 2.2 Incubation reaction After cleaning the midgut tissue of each strain of cotton bollworm larvae, quickly absorb excess liquid with clean filter paper and place it in 0.5 mg / ml Cry1Ac- cell culture medium, and incubate at 25°C and 550 rpm for 30 minutes.

[0057] 2.3 Removal of nonspecific binding After incubation, the toxin solution was discarded, and the midgut tissue was rinsed twice with cell culture medium, followed by one rinse with PBS (0.01M, pH 7.4) buffer for 5 minutes each time to thoroughly remove unbound toxins. A control group was set up, which was supplemented with only cell culture medium (Sf-900 IISFM, Invitrogen) to exclude interference from non-specific binding.

[0058] 2.4 Fixation of midgut tissue After the processed midgut tissue was quickly blotted with clean filter paper to remove excess liquid, it was fixed in 4% paraformaldehyde-PBS (0.01M, pH 7.4) solution for about 24 hours.

[0059] 3. Preparation of tissue sections Same as Example 1.

[0060] 4. Immunostaining Same as Example 2.

[0061] 5. Confocal microscopy observation Midgut epithelial cells were scanned using a laser confocal scanning microscope (Leica TCSSP8) to obtain immunofluorescence images of the midgut tissue, clarifying the subcellular localization of toxin binding (e.g., microvilli, cell membrane, etc.). The results are as follows: Figure 4 As shown, the results revealed different cotton bollworm strains exhibited varying binding strengths and cellular responses to Cry1Ac: In the control group, almost no fluorescent signal specifically binding to Cry1Ac was detected, and cell morphology remained unchanged (Fig. 4A-a, b, c, d); the SCD strain showed the highest Cry1Ac binding affinity, accompanied by severe cell damage: numerous cells were observed to detach (presumably due to toxin-mediated destruction), and brush border membrane vesicles (BBMV) showed significant degradation (Fig. 4A-e); compared to the SCD strain, the Cry1Ac binding amount in the SCD-r1 strain was significantly reduced, and although slight cell and BBMV damage was observed, the overall midgut structure remained relatively intact (Fig. 4A-f); the Cry1Ac binding amount in the SCD-KI strain was also significantly reduced compared to the SCD strain, and the cells in this strain remained intact, with the BBMV structure largely undamaged (Fig. 4A-g); Cry1Ac was virtually undetectable in the C2 / C3-KO strain. The combination of the two cells and BBMV showed no significant difference in morphology and function compared to the control group (Figure 4A-h).

[0062] 6. Cry1Ac combined with quantization processing Same as Example 2. Statistical results of the binding of Bt toxin Cry1Ac to the midgut epithelial cells of four strains of cotton bollworm larvae are shown in Table 1 and... Figure 4.

[0063] As can be seen from the chart, the binding amount of cotton bollworm larvae with Cry1Ac in the three different resistant strains was reduced to varying degrees compared with the susceptible strain: the SCD-r1 strain was reduced by 56.1% compared with the SCD strain; the SCD-KI strain was reduced by 71.4%; and the C2 / C3-KO strain had extremely low Cry1Ac binding amount, which was reduced by 90.6% compared with the SCD strain (Figure 4B).

[0064] Previous research methods have used radiolabeled iodine or biotin to label the toxin to detect differences in the binding of BBMV to the toxin among different strains of larvae. Some studies have found that the binding amount of the toxin to the midgut receptor in resistant insects is often reduced, while others have found no significant difference in the binding amount between resistant and susceptible strains. These methods require radiolabeling and biotin, which are costly and difficult to implement. Furthermore, while studies using extracted BBMV can detect specific binding of the receptor to the toxin and even resistance phenotypes, this only reflects the essential interaction between BBMV and the toxin. Biochemical reactions within the insect may also affect toxin binding and resistance development, as evidenced by the absence of the expected reduction in binding observed earlier. Another common research method is to incubate midgut tissue sections with the toxin and then use immunofluorescence for localization. However, studies have found that the initial binding site of Cry1A toxin is concentrated on the microvilli of insect midgut cells, and that Cry1Ac has a stronger binding affinity to gypsy moth BBMV than Cry1Ab. Although Cry1Ac is about 400 times less toxic to gypsy moth larvae than Cry1Ab, this may not reflect the irreversible binding when the toxin inserts into the membrane.

[0065] In summary, this invention leverages the three major advantages of the in situ binding (ISB) method—"preservation of cell viability," "in situ visualization," and "precise quantification"—to provide a technical paradigm that more closely approximates the in vivo environment for studying the interaction between Bt toxins and the insect midgut. It offers a new tool for exploring Bt resistance mechanisms and also provides an operational approach for evaluating the target binding efficiency of novel Bt toxins and screening toxins suitable for insect-resistant crops. This is of great significance for promoting the sustainable application of Bt technology and precision pest management.

[0066] Table 1. Statistics on the binding amount of Bt toxin Cry1Ac to midgut epithelial cells of cotton bollworm larvae from four strains. Cotton bollworm strains Resistance ratio <![CDATA[Cry1Ac binding amount (a.u. / μm 2 )]]> Cry1Ac binding rate decrease (%) SCD 1 39.6 - SCD-r1 438 17.4 56.1% SCD-KI 125 11.3 71.4% C2 / 3-KO ＞15000 3.7 90.6% Example 4 evaluates the binding of the Bt toxin Cry1Ab to the midgut epithelial cells of Asian corn borer larvae in a susceptible strain (NJ-S) and a resistant strain (OfC3 & Cad-KO). To evaluate the Bt toxin Cry1Ab, a susceptible strain NJ-S (collected in Nanjing, Jiangsu Province, China in 2010 by the Insect Molecular Ecology and Evolution Laboratory of Nanjing Agricultural University, donated to the Kunzhong Molecular Toxicology Laboratory of Nanjing Agricultural University in 2017, and raised in an indoor environment without exposure to any chemical pesticides or Bt toxins and on artificial feed) and a resistant strain OfC3&Cad-KO (i.e., Cad+C2–KO, obtained using CRISPR / Cas9 gene editing technology based on the NJ-S strain, which has greater than 1264 times resistance to Cry1Ab). To investigate the binding of midgut epithelial cells to the Bt toxin Cry1Ab in corn borer larvae, this study first selected healthy, uniformly developed corn borer larvae for midgut tissue preparation. The midgut tissue was then incubated in a specific concentration of Cry1Ab-cell culture medium. After fixation, sections were prepared and immunostained. Finally, immunofluorescence images were acquired using laser confocal microscopy, and fluorescence statistics and data processing were performed. Ultimately, data on the binding of midgut epithelial cells to the Bt toxin Cry1Ab in four different resistant corn borer larvae were obtained. The specific steps are as follows: 1. Sample preparation of midgut tissue from corn borer larvae: 1.1 Larval Selection and Rearing Two healthy, uniformly developed strains of corn borer larvae were selected and fed artificial feed until the fourth instar under conditions of 26-28℃, 30%-40% relative humidity, and 16L:8D photoperiod to ensure stable physiological state of the larvae.

[0067] 1.2 Midgut tissue acquisition Anesthetize the fourth-instar larvae by placing them in an ice bath for 5 minutes. Fix the larvae's head and tail with a dissecting needle, and use dissecting scissors to cut along the midline of the larvae's body wall to expose the midgut tissue. Carefully separate the midgut from the surrounding fat body, trachea, and other tissues with forceps, avoiding damage to the midgut epithelium.

[0068] 1.3 Midgut tissue cleaning The isolated midgut tissue was immediately placed in pre-cooled PBS (0.01M, pH 7.4) buffer to remove intestinal contents.

[0069] 2. In-situ binding reaction 2.1 Preparation of the reaction system Cry1Ab toxin was added to cell culture medium (Sf-900IISFM, Invitrogen) to prepare a 0.5 mg / ml Cry1Ab-cell culture medium mixed solution.

[0070] 2.2 Incubation reaction After cleaning the midgut tissue of each strain of corn borer larvae, quickly absorb excess liquid with clean filter paper and place it in 0.5 mg / ml Cry1Ab- cell culture medium, and incubate at 25°C and 550 rpm for 30 minutes.

[0071] 2.3 Removal of nonspecific binding After incubation, the toxin solution was discarded, and the midgut tissue was rinsed twice with cell culture medium, followed by one rinse with PBS (0.01M, pH 7.4) buffer for 5 minutes each time to thoroughly remove unbound toxins. A control group was set up, which was supplemented with only cell culture medium (Sf-900 IISFM, Invitrogen) to exclude interference from non-specific binding.

[0072] 2.4 Fixation of midgut tissue After the processed midgut tissue was quickly blotted with clean filter paper to remove excess liquid, it was fixed in 4% paraformaldehyde-PBS (0.01M, pH 7.4) solution for about 24 hours.

[0073] 3. Preparation of tissue sections Same as Example 1.

[0074] 4. Immunostaining After blocking, the tissue sections were washed twice with PBS (0.01M, pH 7.4), 5 minutes each time. Then, the tissue sections were incubated overnight at 4°C with Cry1Ab polyclonal antibody (manufacturer: Abbexa, catalog number abx023892, 1:200), followed by washing three times with PBS (0.01M, pH 7.4) buffer. Subsequently, they were incubated for 4 hours with a mixture of Alexa Fluor 594-labeled goat anti-rabbit IgG (1:1,000 dilution, Abcam) and phalloidin (1:1000 dilution, Invitrogen). Finally, they were counterstained with Hoechst 33342 (1:500 dilution, Invitrogen) for 20 minutes. After each incubation, the tissue was washed three times with PBS (0.01M, pH 7.4), 5 minutes each time.

[0075] 5. Confocal microscopy observation Midgut epithelial cells were scanned using a laser confocal scanning microscope (Leica TCSSP8) to obtain immunofluorescence images of the midgut tissue, clarifying the subcellular localization of toxin binding (e.g., microvilli, cell membrane, etc.). The results are as follows: Figure 5As shown in Figure A, the results indicate that different corn borer strains exhibit varying binding strengths and cellular responses to Cry1Ab: In the control group, almost no fluorescent signal specifically binding to Cry1Ab was detected, and cell morphology remained unchanged; the NJ-S strain showed the highest affinity for Cry1Ab, accompanied by partial cell fragmentation and detachment into the intestinal lumen (presumably caused by toxin-mediated destruction); the Cry1Ab binding amount in the OfC3&Cad-KO strain was significantly lower than that in the NJ-S strain, while the cells maintained good integrity, and the BBMV structure was also relatively intact.

[0076] 6. Cry1Ab combined with quantification Same as Example 2. Statistical results regarding the binding of Bt toxin Cry1Ab to the midgut epithelial cells of the two corn borer larvae are shown in Table 2 and... Figure 5 B.

[0077] As shown in the charts, the binding amount of Cry1Ab to the resistant corn borer larvae decreased by 74.7% compared to the susceptible strain, and this significant decrease in toxin binding signal intensity can also be directly observed in the immunofluorescence images. Therefore, this invention utilizes the in situ binding method to effectively assess the binding amount of Bt toxin Cry1Ab to the midgut epithelial cells of different corn borer larvae, demonstrating that the in situ binding method can also be used to investigate the binding of Cry1Ab toxin to the midgut receptors of corn borer larvae.

[0078] Table 2. Statistics on the binding amount of Bt toxin Cry1Ab to the midgut epithelial cells of corn borer larvae from two varieties. Corn borer strains Resistance ratio <![CDATA[Cry1Ab binding amount (a.u. / μm 2 )]]> Cry1Ab binding rate decrease (%) NJ-S 1 155.5 - OfC3&Cad-KO ＞1264 39.3 74.7% The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An in situ binding method for assessing the binding of a Bt toxin to the midgut epithelial cells of a lepidopteran pest larva, characterized by, The method comprises the following steps: (1) preparing the lepidopteran pest larva midgut tissue; (2) in-vitro culturing the midgut tissue; (3) in-situ binding reaction of the Bt toxin and the midgut epithelial cells; (4) localization and quantification of the binding sites; The Bt toxin is Cry1Ac or Cry1Ab.

2. The in-situ bonding method of claim 1, wherein, (1) The preparation of the lepidopteran pest larva midgut tissue is to obtain the midgut of the lepidopteran pest larva to be tested, and immediately place the separated midgut in a pre-cooled PBS (0.01M, pH 7.4) buffer solution to remove the intestinal contents.

3. The in-situ bonding method of claim 1, wherein, (2) The in-vitro culturing of the midgut tissue is to incubate the midgut tissue in a cell culture solution containing the Bt toxin, and the incubation time is 30 min-60 min; Preferably, the concentration of the Bt toxin in the cell culture solution containing the Bt toxin is 0.5 mg / mL; Preferably, the incubation condition is 25℃, 550 rpm; Preferably, the cell culture solution is an insect cell culture solution; Further preferably, the cell culture solution is Sf-900 II SFM cell culture solution.

4. The in-situ bonding method of claim 1, wherein, (2) After the in-vitro culturing of the midgut tissue, it further comprises the steps of removing non-specific binding and fixing the midgut tissue; Preferably, the removal of non-specific binding is to discard the cell culture solution containing the Bt toxin after the incubation, rinse the midgut tissue with the cell culture solution for 2 times, and then rinse it with the PBS buffer solution (0.01M, pH 7.4) for 1 time, each time for 5 minutes; Preferably, the fixation of the midgut tissue is to place the midgut tissue after the removal of non-specific binding in a 4% paraformaldehyde-PBS solution (0.01M, pH 7.4) for fixation for 24 hours.

5. The in-situ bonding method of claim 1, wherein, (3) The in-situ binding reaction of the Bt toxin and the midgut epithelial cells comprises the following steps: 1) sample preparation: embed the fixed midgut tissue with a frozen section embedding agent, freeze it at -20℃ for 2 hours, and then continuously section it at a thickness of 4.5 μm and paste it on a glass slide; 2) fixation of the tissue section: place the section in a 4% paraformaldehyde-PBS solution for fixation for 20 minutes; 3) tissue permeation: place the section in a 0.1% Triton-X100-PBS solution for permeation for 20 minutes; 4) tissue blocking: place the section in a freshly prepared 1% BSA-PBS solution for blocking for 1 hour; 5) immunostaining: wash the section with PBS, incubate it with a Bt toxin polyclonal antibody at 4℃ overnight, wash it with PBS after the incubation, incubate it with an Alexa Fluor 594 labeled goat anti-rabbit IgG and a Texas Red®-labeled phalloidin mixture, and finally re-stain it with Hoechst 33342, and wash it with PBS after each incubation.

6. The in-situ bonding method of claim 5, wherein, The Bt toxin polyclonal antibody is a Cry1Ac polyclonal antibody or a Cry1Ab polyclonal antibody.

7. The in-situ bonding method of claim 1, wherein, (4) The localization of the binding sites is to observe by a confocal microscope, obtain the immunofluorescence image of the midgut tissue, and determine the subcellular localization of the toxin binding; preferably, the confocal microscope is a Leica TCS SP8 laser confocal scanning microscope.

8. The in-situ bonding method of claim 1, wherein, The binding of (4) was quantified by using ImageJ software, with the signal value of 5-255, and a square frame of 200 pixels was drawn to define the toxin signal area. The average fluorescence signal value per unit area (1 μm 2 ) was calculated, and 3 areas were calculated for each image to obtain the average value, and 6 biological replicates were set for each treatment.

9. The in-situ bonding method of claim 8, wherein, The binding condition quantification calculation is: the average fluorescence signal value per unit area (1 μm 2 ) = Intden (Integrated Density) / actual area of the selected frame.

Images