Safety evaluation method of transgenic insect-resistant plant to non-target predatory natural enemies
By studying the binding potential of Bt protein and non-target predator midgut protein, using Pull-down detection and ligand hybridization methods, the problem of safety assessment of non-target predator natural enemies was solved, and the problem of transgenic insect-resistant plants was achieved. Efficient and accurate safety evaluation.
Patent Information
- Application Number
- CN202411055364.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-08-02
- Publication Date
- 2025-02-18
AI Technical Summary
The prior art is difficult to effectively evaluate the safety of transgenic insect-resistant plants against non-target predatory natural enemies, especially the lack of accurate and efficient detection methods at the molecular level.
By studying the binding potential of Bt protein to non-target predator midgut protein, the safety of transgenic insect-resistant plants against non-target predator natural enemies was evaluated by using methods such as Pull-down detection and ligand hybridization.
It provides a safe, efficient and specific safety evaluation method, which can accurately reflect the safety of genetically modified insect-resistant plants against non-target predatory natural enemies, and has important scientific and practical significance.
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Figure CN119470901A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to ecological risk assessment of transgenic insect-resistant plants, and in particular to a method for evaluating the safety of transgenic insect-resistant plants against non-target predatory natural enemies. Background Art
[0002] The commercial planting of transgenic insect-resistant cotton has effectively suppressed the outbreak of target pests and brought significant economic benefits, but its environmental safety has always attracted the attention of scholars at home and abroad and has become the focus of research. Therefore, it is very important to evaluate the safety of transgenic crops, among which the impact on non-target insects is an important part of evaluating the safety of transgenic crops.
[0003] Predatory natural enemies play an important role in controlling the number of pests. They control the number of pests by directly preying on them. They have the characteristics of large appetite and strong adaptability to the environment. They are important natural enemies of pests in nature and are of great significance to maintaining ecological balance and reducing the use of chemical pesticides. By feeding on herbivorous insects, the Bt protein expressed by transgenic crops can be transmitted to predatory natural enemies through the food chain. Therefore, it is very important to explore the impact of transgenic insect-resistant plants on non-target predatory natural enemies.
[0004] At present, the Bt genes of transgenic insect-resistant crops mainly include Cyt, Cry and Vip genes, and the proteins encoded by them have insecticidal effects on certain pests. Bt protein binds to insect midgut proteins to cause insect midgut perforation or cell osmotic pressure imbalance, thereby causing insect death. Therefore, determining whether Bt protein binds to the insect midgut is the first condition to be considered for Bt protein to exert its toxic effect. At present, research on the potential impact of transgenic insect-resistant crops on non-target natural enemies mainly focuses on biological level detection such as survival rate, litter size, and body weight. However, with the widespread application of molecular level technologies such as genes and proteins, it also guides the risk assessment of transgenic insect-resistant plants to non-target organisms. Therefore, it is particularly important to explore an accurate, efficient and scientific molecular detection method for evaluating the safety of transgenic insect-resistant plants against non-target predatory natural enemies. Summary of the invention
[0005] The purpose of the present invention is to address the above-mentioned situation and, by studying the binding potential of Bt protein with the midgut protein of non-target predatory natural enemies, provide a safe, efficient and highly specific method for evaluating the safety of transgenic insect-resistant plants against non-target predatory natural enemies, aiming to accurately reflect the safety of transgenic insect-resistant plants against non-target predatory natural enemies.
[0006] The object of the present invention is achieved by providing a method for evaluating the safety of transgenic insect-resistant plants against non-target predatory natural enemies, the method comprising the following steps:
[0007] (1) Obtaining samples of target insects and non-target predatory natural enemies of transgenic insect-resistant plants.
[0008] Furthermore, the target insects in step (1) are the target insects (Lepidoptera) directly targeted by the Bt protein, which serve as the positive control of the test, and the non-target predatory natural enemies are natural enemies that feed on the target insects.
[0009] Preferably, the target insects are Lepidoptera insects of about 4th instar.
[0010] (2) Dissect the midguts of target insects and non-target predatory natural enemies and extract the midgut proteins of target insects and non-target predatory natural enemies.
[0011] Further, the midguts of target insects and non-target predatory natural enemies were dissected under a microscope and rinsed three times with sterile water after use. The midguts of target insects and non-target predatory natural enemies were collected into 1.5 mL centrifuge tubes for extraction of midgut brush border membrane vesicle proteins (BBMVs).
[0012] (3) Pull-down assay to detect the binding ability of Bt protein to the midgut proteins of target insects and non-target predatory natural enemies.
[0013] Furthermore, the reagents required for the reaction system are: target insect midgut BBMV, non-target predatory natural enemy midgut BBMV, Bt protein, biotin-labeled Bt protein, BSA solution;
[0014] Pull-down test samples:
[0015] a) 20 μg of target insect midgut BBMV protein + 0.1 μg of biotin-labeled Cry protein;
[0016] b) 20 μg of target insect midgut BBMV protein + 0.1 μg of biotin-labeled Cry protein + 10 μg of unlabeled Cry protein;
[0017] c) BBMV 20 μg in target insect midgut (negative control).
[0018] d) 20ug of BBMV protein from the midgut of non-target predatory natural enemy insects + 0.1μg of biotin-labeled Cry protein;
[0019] e) 20 μg of BBMV protein from the midgut of non-target predatory natural enemy insects + 0.1 μg of biotin-labeled Cry protein + 10 μg of unlabeled Cry protein;
[0020] f) 20 μg of BBMV in the midgut of non-target predatory natural enemy insects (negative control).
[0021] By testing the binding ability of the above samples with Bt protein, the safety of transgenic insect-resistant plants against non-target predatory natural enemies is evaluated.
[0022] (4) Ligand hybridization method was used to detect the binding ability of midgut proteins of target insects and non-target predatory natural enemies.
[0023] Furthermore, the reagents required for the reaction system include: target insect midgut BBMV, non-target predatory natural enemy midgut BBMV, Bt protein, Bt protein polyclonal antibody, and BSA solution; the binding ability of target insect midgut BBMV and non-target predatory natural enemy midgut BBMV with Bt protein is verified by ligand hybridization method, and the safety of transgenic insect-resistant plants to non-target predatory natural enemies is further evaluated.
[0024] The beneficial effects of the present invention are as follows: the present invention provides a method for evaluating the safety of transgenic insect-resistant plants on non-target predatory natural enemies, and evaluates the safety of transgenic plants on non-target predatory natural enemies by detecting the binding potential of Bt protein with the midgut protein of non-target predatory natural enemies. The samples involved in the present invention are not limited by many factors, and samples at any stage in the field or laboratory can be tested, which can better reflect the potential impact of transgenic insect-resistant plants on non-target predatory natural enemies in the field. The present invention establishes an accurate and scientific evaluation method, which can more efficiently and accurately reflect the safety of transgenic insect-resistant plants on non-target predatory natural enemies, and is of great significance to improving the safety of transgenic crop planting. BRIEF DESCRIPTION OF THE DRAWINGS
[0025] Figure 1 The competitive binding method was used to detect the binding ability of Cry1Ac protein with the BBMV protein in the midgut of the big-eyed cicada stink bug. Beet armyworm was used as a positive control. Biotin Cry1Ac: insect BBMV protein bio Cry1Ac protein; biotin Cry1Ac+unabledCry1Ac insect BBMV protein biotin-labeled Cry1Ac protein + 100 times the volume of biotin-unlabeled Cry1Ac protein; the negative control was insect BBMV protein.
[0026] Figure 2 The ligand hybridization method was used to detect the binding ability of Cry1Ac protein to the BBMV protein in the midgut of the big-eyed cicada bug. a): Spodoptera exigua was used as a positive control, with two binding bands (approximately 110 and 120 kDa), and no binding bands were found between the BBMV protein of the nymphs and adults of the big-eyed cicada bug and the Cry1Ac protein. (b): The negative control PVDF membrane was not incubated with Cry1Ac protein, and no binding bands were detected for the insect BBMV protein in the figure.
[0027] Figure 3Binding ability of biotin-labeled Cry2Aa protein to BBMV protein from nymphs (a) and adults (b) of Cotton stink bugs. Each 100 μL system contained 20 μg BBMV protein and 0.1 μg biotin-labeled Cry2Aa protein. 100-fold unlabeled Cry2Aa protein was added to the competitive binding assay. BBMV protein from the midgut of cotton bollworm was used as a positive control.
[0028] Figure 4 Binding ability of biotin-labeled Cry1Ac protein to BBMV protein from nymphs and adults of Cotton stink bugs. Each 100 μL system contained 20 μg BBMV protein and 0.1 μg biotin-labeled Cry1Ac protein. 100-fold unlabeled Cry1Ac protein was added to the competitive binding assay. BBMV protein from the midgut of cotton bollworm was used as a positive control. DETAILED DESCRIPTION
[0029] The following examples are used to illustrate the present invention but are not intended to limit the scope of the present invention.
[0030] Example 1 Safety evaluation of Cry1Ac-transgenic cotton against non-target predatory natural enemy, the big-eyed cicada stink bug
[0031] (1) Obtaining samples of the target insect beet armyworm and the non-target predatory natural enemy, the big-eyed cicada stink bug, of Cry1Ac-transfected insect-resistant cotton.
[0032] Furthermore, the target insect of the Cry1Ac-resistant cotton in step (1) is the beet armyworm, which serves as a positive control of the test, and the non-target predatory natural enemy is the big-eyed cicada stink bug that feeds on the beet armyworm.
[0033] Preferably, the target insect Spodoptera exigua is preferably selected at around 4th instar.
[0034] (2) The midguts of the target insect Spodoptera exigua and the predatory natural enemy Mylophobia spp. were dissected, and the midgut proteins of the target insect Spodoptera exigua and the non-target predatory natural enemy Mylophobia spp. were extracted.
[0035] Further, the midguts of the target insect beet armyworm and the non-target predatory natural enemy, the big-eyed cicada long stink bug, were dissected under a microscope and rinsed three times with sterile water after use. The midguts of the target insect beet armyworm and the non-target predatory natural enemy, the big-eyed cicada long stink bug, were collected into 1.5 ml centrifuge tubes respectively for the extraction of midgut brush border membrane vesicle proteins (BBMV).
[0036] (3) Pull-down test to detect the binding ability of Bt protein with the midgut protein of the target insect Spodoptera exigua and the non-target predatory natural enemy Mylophobia fasciatus.
[0037] Furthermore, the reagents required for the reaction system are: target insect Spodoptera exigua midgut BBMV, non-target predatory natural enemy Macropus ceratoptera midgut BBMV, Cry1Ac protein, biotin-labeled Cry1Ac protein, BSA solution;
[0038] Pull-down test samples:
[0039] a) 20 μg of BBMV protein in the midgut of target insect Spodoptera exigua + 0.1 μg of biotin-labeled Cry1Ac protein;
[0040] b) 20 μg of BBMV protein from the midgut of target insect Spodoptera exigua + 0.1 μg of biotin-labeled Cry1Ac protein + 10 μg of unlabeled Cry1Ac protein;
[0041] c) 20 μg of BBMV from the midgut of target insect Spodoptera exigua (negative control).
[0042] d) 20ug of BBMV protein in the midgut of the non-target predatory natural enemy, the big-eyed cicada bug, + 0.1μg of biotin-labeled Cry1Ac protein;
[0043] e) 20 μg of BBMV protein in the midgut of the non-target predatory natural enemy, the big-eyed cicada bug, + 0.1 μg of biotin-labeled Cry1Ac protein + 10 μg of unlabeled Cry1Ac protein;
[0044] f) 20 μg of BBMV in the midgut of the non-target predatory natural enemy, Mylosoma macrophylla (negative control).
[0045] By testing the binding ability of the above samples with Bt protein, the safety of transgenic insect-resistant plants against non-target predatory natural enemies is evaluated.
[0046] (4) The ligand hybridization method was used to detect the binding ability of the midgut proteins of the target insect Spodoptera exigua and the non-target predatory natural enemy Mylos nudibranch.
[0047] Furthermore, the reagents required for the reaction system include: BBMV in the midgut of the target insect Spodoptera exigua, BBMV in the midgut of the non-target predatory natural enemy Mylopidae, Cry1Ac protein, polyclonal antibody to Cry1Ac protein, and BSA solution; the binding ability of the target insect midgut BBMV and the non-target predatory natural enemy Mylopidae to Cry1Ac protein was verified by ligand hybridization method, and the safety of transgenic insect-resistant plants to non-target predatory natural enemies was further evaluated.
[0048] The results showed that no binding band was detected in the negative control (only insect BBMV protein). After adding 100 times the volume of unlabeled biotin Cry1Ac protein to the target insect beet armyworm BBMV protein, the binding reaction between biotin-labeled Cry1Ac protein and beet armyworm BBMV was replaced ( Figure 1 ), indicating that Cry1Ac protein forms a specific binding with the BBMV protein of beet armyworm. In the BBMV protein of the big-eyed cicada bug, the binding amount with Cry1Ac protein is very low. In addition, after adding 100 times the volume of unlabeled Cry1Ac protein to the reaction solution of the BBMV protein of the big-eyed cicada bug and the biotin-labeled Cry1Ac protein, the binding amount did not change significantly, indicating that Cry1Ac protein cannot form a high specific binding with the BBMV protein of the big-eyed cicada bug ( Figure 1 ).
[0049] The ligand hybridization method was used to detect the binding ability of the BBMV protein of the big-eyed cicada bug and the Cry1Ac protein. The results showed that the Cry1Ac protein could form a specific binding with the BBMV protein of the beet armyworm larvae ( Figure 2 ). However, the BBMV protein of the nymphs and adults of the big-eyed cicada bug did not produce binding bands with the Cry1Ac protein ( Figure 2 No binding bands were found in the control membranes of Spodoptera exigua or BBMV proteins incubated with Cry1Ac protein, which proved the reliability of the experimental process. The experimental results showed that the midgut BBMV proteins of nymphs and adults of nymphs could not form specific binding with Cry1Ac protein.
[0050] Example 2 Safety evaluation of Cry2Aa transgenic cotton against non-target predatory natural enemies of stink bugs
[0051] (1) Obtaining samples of target insect cotton bollworm and non-target predatory natural enemy stink bugs in Cry2Aa-transformed insect-resistant cotton.
[0052] Furthermore, the target insect of the Cry2Aa-transformed insect-resistant cotton in step (1) is cotton bollworm, which serves as a positive control of the test, and the non-target predatory natural enemy is a stink bug that feeds on cotton bollworm.
[0053] Preferably, the target insect cotton bollworm is preferably an insect of about 4th instar.
[0054] (2) Dissect the midguts of the target insect cotton bollworm and the predatory natural enemy, the caterpillar, and extract the midgut proteins of the target insect cotton bollworm and the non-target predatory natural enemy, the caterpillar.
[0055] Further, the midguts of the target insect cotton bollworm and the non-target predatory natural enemy gnats were dissected under a microscope and rinsed three times with sterile water after use. The midguts of the target insect cotton bollworm and the non-target predatory natural enemy gnats were collected into 1.5 ml centrifuge tubes for the extraction of midgut brush border membrane vesicle proteins (BBMV).
[0056] (3) Pull-down assay was used to detect the binding ability of Bt protein to the midgut protein of target insect cotton bollworm and non-target predatory natural enemy cobweb stink bug.
[0057] Furthermore, the reagents required for the reaction system are: target insect cotton bollworm midgut BBMV, non-target predatory natural enemy midgut BBMV, Cry2Aa protein, biotin-labeled Cry2Aa protein, BSA solution;
[0058] Pull-down test samples:
[0059] a) 20 μg of BBMV protein in the midgut of target insect cotton bollworm + 0.1 μg of biotin-labeled Cry2Aa protein;
[0060] b) 20 μg of BBMV protein in the midgut of target insect cotton bollworm + 0.1 μg of biotin-labeled Cry2Aa protein + 10 μg of unlabeled Cry2Aa protein;
[0061] c) 20 μg of BBMV from the midgut of target insect Helicoverpa armigera (negative control).
[0062] d) 20ug of BBMV protein from the midgut of non-target predatory natural enemy stink bugs + 0.1μg of biotin-labeled Cry2Aa protein;
[0063] e) 20 μg of BBMV protein from the midgut of non-target predatory natural enemy stink bugs + 0.1 μg of biotin-labeled Cry2Aa protein + 10 μg of unlabeled Cry2Aa protein;
[0064] f) 20 μg of BBMV in the midgut of non-target predatory natural enemy stink bug (negative control).
[0065] By testing the binding ability of the above samples with Bt protein, the safety of transgenic insect-resistant plants against non-target predatory natural enemies is evaluated.
[0066] (4) The ligand hybridization method was used to detect the binding ability of the midgut proteins of the target insect, cotton bollworm, and the non-target predatory natural enemy, the stink bug.
[0067] Furthermore, the reagents required for the reaction system include: target insect cotton bollworm midgut BBMV, non-target predatory natural enemy midgut BBMV, Cry2Aa protein, Cry2Aa protein polyclonal antibody, and BSA solution; the binding ability of target insect cotton bollworm midgut BBMV and non-target predatory natural enemy midgut BBMV with Cry2Aa protein was verified by ligand hybridization method, and the safety of transgenic insect-resistant plants to non-target predatory natural enemies was further evaluated.
[0068] The results showed that the negative control BBMV protein showed no binding bands. The positive control cotton bollworm had obvious protein binding bands with biotin-labeled Cry2Aa toxin. When 100 times the unlabeled Cry2Aa toxin (unlabeled-Cry2Aa) was added to the binding reaction solution, the brightness of the band was significantly weakened, indicating that the binding of Bio-Cry2Aa toxin to the BBMV protein in the midgut of cotton bollworm was replaced. In contrast, the binding level of Cry2Aa toxin to the BBMV protein in the midgut of caterpillars was much lower, and when 100 times the unlabeled Cry2Aa toxin was added, the binding band of the BBMV protein in the midgut of caterpillars and the Cry2Aa protein did not show a significant weakening ( Figure 3 ), indicating that the BBMV protein in the midgut of the stink bug cannot specifically bind to the Cry2Aa toxin.
[0069] The results of ligand hybridization showed that the membrane that had not been incubated with Cry2Aa toxin did not show any bands, proving the feasibility and reliability of the experiment. Figure 4 The results showed that the positive control cotton bollworm midgut BBMV protein had multiple obvious protein binding bands with Cry2Aa, indicating that the cotton bollworm midgut BBMV protein had binding proteins with Cry2Aa toxin, while the midgut BBMV protein of the nymph and adult caterpillar stink bug did not show obvious protein binding bands, indicating that the midgut BBMV protein of the caterpillar stink bug did not have binding proteins with Cry2Aa toxin.
[0070] Although the present invention has been described in detail above with general explanations and specific implementation methods, it is obvious to those skilled in the art that some modifications or improvements can be made on the basis of the present invention. Therefore, these modifications or improvements made on the basis of not departing from the spirit of the present invention all belong to the scope of protection claimed by the present invention.
Claims
1. A method for evaluating the safety of transgenic insect-resistant plants against non-target predatory natural enemies, characterized in that: The following steps are involved: (1) Obtaining samples of target insects and non-target predatory natural enemies of transgenic insect-resistant plants; (2) Dissect the midguts of target insects and non-target predatory natural enemies and extract midgut proteins of target insects and non-target predatory natural enemies; (3) Pull-down test to detect the binding ability of Bt protein to midgut proteins of target insects and non-target predatory natural enemies; (4) Ligand hybridization method is used to detect the binding ability of midgut proteins of target insects and non-target predatory natural enemies.
2. The method according to claim 1, characterized in that The target insects are the target insects (Lepidoptera) directly targeted by the Bt protein, which serve as the positive control in the test, and the non-target predatory natural enemies are the natural enemies that feed on the target insects.
3. The method according to claim 2, characterized in that The target insects are Lepidoptera insects of about 4th instar.
4. The method according to claim 1, characterized in that: The midgut protein is a brush border membrane vesicle protein (BBMV).