Method for evaluating influence of insect-resistant transgenic crops on diversity of symbiotic bacteria in target and non-target pests
Through high-throughput sequencing and bioinformatics analysis, a method for symbiotic diversity evaluation in insect-resistant transgenic crops was established, which solved the problem of inaccurate evaluation in the existing technology, and achieved a comprehensive assessment of the diversity of symbiotic bacteria in pests, providing a scientific basis for crop safety evaluation and ecosystem protection.
Patent Information
- Application Number
- CN202510500274.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-21
- Publication Date
- 2025-07-11
AI Technical Summary
The existing evaluation methods cannot comprehensively and accurately evaluate the impact of insect-resistant genetically modified crops on the diversity of symbiotic bacteria in pests, resulting in inaccurate and incomplete evaluation results.
High-throughput sequencing technology, combined with 16S rRNA or metagenomic sequencing technology, the composition and diversity of symbiotic bacteria are analyzed through bioinformatics, and the absolute abundance of dominant bacteria is verified with absolute quantitative PCR to establish a comprehensive and scientific evaluation system.
A comprehensive and accurate assessment of the diversity of symbiotic bacteria in pests by anti-pest genetically modified crops has been achieved, providing a scientific basis for their safety evaluation, predicting potential risks and taking risk prevention and control measures to promote the sustainable development of agricultural ecosystems.
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Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of environmental safety evaluation of biotechnological products, and particularly relates to an evaluation method for the effects of insect-resistant genetically modified crops on the diversity of symbiotic bacteria in target and non-target pests. Background Art
[0002] Insect-resistant genetically modified crops are obtained by introducing exogenous insect-resistant genes (such as Bt genes) into the crop genome through genetic engineering technology, enabling them to express insecticidal proteins with insecticidal activity, thereby effectively resisting pest infestation. Since the first commercial cultivation of insect-resistant genetically modified crops in 1996, their planting area has been expanding year by year and they are currently widely used in many countries around the world. Typical insect-resistant genetically modified crops include insect-resistant cotton, insect-resistant corn, and insect-resistant rice, etc. These crops have played a significant role in reducing the use of chemical pesticides, reducing production costs, and improving crop yield and quality. However, with the expansion of the planting scale, the potential impact on the farmland ecosystem has also attracted wide attention.
[0003] There are a large number of symbiotic bacteria in pests, and a complex symbiotic relationship has been formed between these symbiotic bacteria and pests. Symbiotic bacteria play a key role in multiple physiological processes of pests such as growth, development, reproduction, nutrient metabolism, and immune defense. In addition, pest symbiotic bacteria are also closely related to the ecological adaptability of pests. They can affect the adaptability of pests to the environment and help pests survive and reproduce in different ecological environments. Therefore, changes in the diversity of pest symbiotic bacteria have a profound impact on their biological characteristics and ecological behaviors.
[0004] Currently, there is a lack of a systematic and comprehensive evaluation method for the effects of insect-resistant genetically modified crops on the diversity of symbiotic bacteria in pests. Most of the existing evaluation methods use traditional cultivation methods to analyze the diversity of symbiotic bacteria. This method can only detect symbiotic bacteria that can grow on artificial culture media and cannot identify those symbiotic bacteria that are difficult to culture or unculturable, resulting in one-sidedness in the evaluation of symbiotic bacteria diversity. In addition, the existing evaluation methods fail to fully consider the effects of various factors such as different insect-resistant genes, crop varieties, pest species, and ecological environments on the interaction between insect-resistant genetically modified crops and pest symbiotic bacteria, thus affecting the accuracy and reliability of the evaluation results.
[0005] Therefore, it is particularly important to establish a scientific, comprehensive, and accurate evaluation method to evaluate the effects of insect-resistant genetically modified crops on the diversity of symbiotic bacteria in target and non-target pests. Such a method not only helps to deeply understand the interaction between insect-resistant genetically modified crops and pest symbiotic bacteria, but also provides a scientific basis for the safety evaluation of insect-resistant genetically modified crops, and further provides a strong guarantee for the sustainable development of the agricultural ecosystem.
[0006] By utilizing modern high-throughput sequencing technology, the structural and functional changes of symbiotic bacterial communities can be more comprehensively revealed, thereby promoting the application of agricultural biotechnology and the assessment of its environmental impacts, providing important references for future biosafety supervision and sustainable agricultural development. Summary of the Invention
[0007] The present invention focuses on the evaluation of the impact of insect-resistant transgenic crops on the symbiotic bacterial diversity in target and non-target pests, aiming to solve the limitations of existing evaluation methods, such as inaccurate and incomplete evaluation results, and construct a simple, scientific and comprehensive evaluation system to provide a solid and reliable scientific basis for the safety evaluation of insect-resistant transgenic crops.
[0008] The evaluation method provided by the present invention has wide applicability, covering various types of insect-resistant transgenic crops, including but not limited to insect-resistant transgenic crops constructed by the following methods: RNAi-mediated gene silencing, gene editing technology, and exogenous gene introduction. By means of advanced techniques such as high-throughput sequencing technology, on the one hand, it overcomes the disadvantages of the limited detection range of traditional culture techniques and avoids the lack of symbiotic bacterial information caused by insufficient detection range; on the other hand, it significantly reduces the workload, shortens the evaluation cycle, and has high operational feasibility and popularization and application value.
[0009] The present invention provides an evaluation method for the impact of insect-resistant transgenic crops on the symbiotic bacterial diversity in target and non-target pests, which is specifically realized through the following technical solutions: (1) Transgenic crop planting: Plant the insect-resistant transgenic crop and its control material under controlled greenhouse conditions, preferably under greenhouse conditions with a temperature of 25±2°C and a relative humidity of 60±5%, and conduct the experiment when the crop grows to 5-8 fully expanded leaves; (2) Selection of experimental pests: Select target pests sensitive to the target traits of the insect-resistant transgenic crop and non-target pests insensitive to it, preferably pests with different mouthpart types and feeding habits of target pests and non-target pests. Further, the pests are at the insect age in the critical period of damage; (3) Pest sample treatment: For target chewing mouthpart pests, preferably 2nd instar larvae (nymphs) feed on the transgenic crop and the control material; for non-target chewing mouthpart pests, preferably 2nd instar larvae (nymphs) and the penultimate instar larvae (nymphs) feed on the transgenic crop and the control material; for target and non-target piercing-sucking mouthpart pests, preferably adults feed on the transgenic crop and the control material; within 24 hours after the pests complete molting (or molt into adults) and after 12 hours of starvation treatment, they are respectively inoculated onto the insect-resistant transgenic crop and the control material; (4) Pest sample collection: Collect pest samples of the same age 48 hours after the pests feed on the crop; The pests were inoculated onto transgenic crops and control materials respectively within 24 hours after they completed the instar molting, and samples were collected after they had fed for 48 hours. (5)Extraction of sample genomic DNA: The genomic DNA of symbiotic bacteria in pest samples was extracted using the CTAB method, preferably with a soil genomic DNA extraction kit. (6)Sequencing of sample genomic DNA: The samples were sequenced using high-throughput sequencing methods, preferably 16S rRNA or metagenomic sequencing technologies. Further, the variable region (V3-V4 region) of the 16S rRNA gene was used to obtain data on the symbiotic bacterial community composition. (7)Diversity analysis: Bioinformatics analysis was used to determine the species composition, relative abundance, and α / β diversity indices of symbiotic bacteria. For dominant bacterial groups with a relative abundance ≥ 3%, the analysis of variance method was used to compare the significance of differences in the symbiotic bacterial communities of target and non-target pests feeding on insect-resistant transgenic crops and control materials at different levels. (8)Quantitative verification of key bacterial groups: For dominant bacterial groups with a relative abundance ≥ 5% in the sequencing data, specific primers were designed to determine the absolute abundance of dominant bacterial genera or species using absolute quantitative PCR, and statistical analysis was used to verify the effect of feeding on insect-resistant transgenic crops and control materials on the absolute abundance of dominant symbiotic bacteria in pests.
[0010] The present invention uses high-throughput sequencing technology to break through the limitations of traditional cultivation techniques, enabling comprehensive and accurate detection of the diversity of symbiotic bacteria in pests, providing more reliable data support for evaluating the impact of insect-resistant transgenic crops on the diversity of symbiotic bacteria in pests. The established evaluation technical method provides a scientific basis for the safety evaluation of insect-resistant transgenic crops, can better predict the potential risks of insect-resistant transgenic crops to the agricultural ecosystem, and thus take corresponding risk prevention and control measures targeted. In addition, this method also provides a reference for evaluating the impact of other transgenic crops on the ecological environment, contributing to the healthy development of agricultural biotechnology and ecological environmental protection. Detailed implementation mode
[0011] The following is a detailed description of the method for evaluating the impact of insect-resistant transgenic crops of the present invention on the diversity of symbiotic bacteria in target and non-target pests in combination with examples. Example
[0012] This example provides a method for evaluating the impact of insect-resistant transgenic cotton on the diversity of symbiotic bacteria in the target pest Helicoverpa armigera.
[0013] 1. Operating steps: (1)Planting of transgenic crops: Insect-resistant transgenic cotton (transformed Cry2Aa )and control materials were planted under greenhouse conditions with a temperature of 25 ± 2°C and a relative humidity of 60 ± 5%. The crops grew to 5-8 fully expanded leaves. (2)Selection of experimental pests: The target chewing mouthpart pest, Helicoverpa armigera; (3)Treatment of pest samples: Within 24 hours after molting of the 2nd instar larvae of Helicoverpa armigera and after 12 hours of starvation treatment, they were respectively inoculated onto insect-resistant transgenic cotton and control materials for feeding; (4)Collection of pest samples: After Helicoverpa armigera fed on insect-resistant transgenic cotton and control materials for 48 hours respectively, 2nd instar samples of Helicoverpa armigera were collected; (5)Extraction of genomic DNA from samples: The genomic DNA of Helicoverpa armigera was extracted using a soil genomic DNA extraction kit; (6)Sequencing of genomic DNA from samples: 16S rRNA V3-V4 region sequencing was used to obtain data on the composition of the symbiotic bacterial community; (7)Diversity analysis: Through bioinformatics analysis, the species composition, relative abundance, and diversity indices of symbiotic bacteria were determined. For dominant bacterial groups with a relative abundance ≥ 3%, an independent samples T-test was used to compare the significant differences at different levels of the symbiotic bacterial community of Helicoverpa armigera feeding on insect-resistant transgenic cotton and control materials; (8)Quantitative verification of key bacterial groups: For dominant bacterial groups with a relative abundance ≥ 5% in the sequencing data, specific primers were designed respectively, and absolute quantitative PCR was used to determine the absolute abundance of dominant bacterial genera. An independent samples T-test was used to analyze and verify the effects of feeding on insect-resistant transgenic cotton and control materials on the absolute abundance of dominant symbiotic bacteria in Helicoverpa armigera.
[0014] 2. Result analysis: As can be seen from Table 1, after the target pest Helicoverpa armigera fed on insect-resistant transgenic cotton, its α-diversity indices (Chao1, Observed species, Shannon, Simpson) were significantly different from those of Helicoverpa armigera feeding on the control material. This result indicates that compared with the control material, after Helicoverpa armigera fed on insect-resistant transgenic cotton, the species richness and evenness of microorganisms in its body were significantly affected.
[0015] Table 1 Diversity indices of symbiotic bacteria in the target pest Helicoverpa armigera after feeding on insect-resistant transgenic cotton
[0016] Note: The data in the table are mean ± standard deviation; for the same index, different lowercase English letters after the data in the same row indicate significant differences between different varieties ( p < 0.05).
[0017] The relative abundance differences of microorganisms in the target pest Helicoverpa armigera that fed on insect-resistant transgenic cotton were analyzed at different taxonomic levels (Table 3). The results showed that after Helicoverpa armigera fed on insect-resistant transgenic cotton, the abundances of microorganisms with a relative abundance ≥ 3% in its symbiotic bacteria at the phylum, class, order, family, genus, and species levels all showed significant differences compared with the control materials. The absolute abundances of dominant genera with a relative abundance ≥ 5% were determined using absolute quantitative PCR technology Enterococcus , Terrisporobacter , Clostridium . The results indicated that there were significant differences in the absolute abundances of these three dominant genera in the body of Helicoverpa armigera that fed on insect-resistant transgenic cotton compared with those that fed on the control materials
[0018] Table 2 Relative abundances of symbiotic bacteria in the target pest Helicoverpa armigera after feeding on transgenic cotton (%)
[0019] Note: The data in the table are mean ± standard deviation; for the same name, different lowercase English letters after the data in the same row indicate significant differences between different varieties ( p < 0.05).
[0020] Example 2 This example provides a method for evaluating the effect of insect-resistant transgenic cotton on the symbiotic bacteria diversity in the non-target pest Aphis gossypii
[0021] 1. Operating steps: (1) Transgenic crop planting: Insect-resistant transgenic cotton (transformed Cry2Aa ) and control materials were planted under greenhouse conditions with a temperature of 25 ± 2°C and a relative humidity of 60 ± 5%. The crops grew to 5 - 8 fully expanded leaves (2) Selection of experimental pests: The non-target piercing-sucking mouthpart pest Aphis gossypii (3) Treatment of pest samples: Within 24 hours after Aphis gossypii molted into adults and after 12 hours of starvation treatment, they were respectively inoculated onto insect-resistant transgenic cotton and control materials for feeding (4) Collection of pest samples: Aphis gossypii samples were collected 48 hours after they fed on insect-resistant transgenic cotton and control materials respectively (5) Extraction of genomic DNA from samples: The genomic DNA of Aphis gossypii was extracted using a soil genomic DNA extraction kit (6) Sequencing of genomic DNA from samples: The composition data of symbiotic bacteria communities were obtained by sequencing the V3 - V4 region of 16S rRNA (7)Diversity analysis: Determine the species composition, relative abundance, and diversity indices of symbiotic bacteria through bioinformatics analysis. For dominant bacterial groups with a relative abundance ≥ 3%, an independent samples T-test was used to compare the significant differences in the symbiotic bacterial communities at different levels between cotton aphids feeding on insect-resistant transgenic cotton and the control materials. (8)Quantitative verification of key bacterial groups: For dominant bacterial groups with a relative abundance ≥ 5% in the sequencing data, specific primers were designed respectively to determine the absolute abundance of dominant bacterial genera by absolute quantitative PCR, and the independent samples T-test was used to analyze and verify the effects of feeding on insect-resistant transgenic cotton and control materials on the absolute abundance of dominant symbiotic bacteria in cotton aphids.
[0022] 2. Result analysis: As shown in Table 3, after cotton aphids fed on insect-resistant transgenic cotton, there were no significant differences in their α-diversity indices (coverage, chao1, ace, shannon, sobs, simpson) compared with those of cotton aphids feeding on the control materials. This result indicates that, compared with the control materials, the species richness and evenness of microorganisms in cotton aphids did not show obvious changes after feeding on insect-resistant transgenic cotton.
[0023] Table 3 Diversity indices of symbiotic bacteria in non-target pest cotton aphids after feeding on insect-resistant transgenic cotton
[0024] Note: The data in the table are mean ± standard deviation; for the same index, the same lowercase English letters after the data in the same row indicate significant differences between different varieties ( p ≥ 0.05).
[0025] Analysis was conducted on the significant differences in the relative abundances of microorganisms in non-target pest cotton aphids feeding on insect-resistant transgenic cotton at different taxonomic levels (Table 4). The results showed that after cotton aphids fed on insect-resistant transgenic cotton, there were no significant differences in the abundances of microorganisms with a relative abundance ≥ 3% at the phylum, class, order, family, genus, and species levels of symbiotic bacteria in their bodies compared with the control materials. The absolute abundances of dominant bacterial genera with a relative abundance ≥ 5% were determined using absolute quantitative PCR technology Acinetobacter 、 Pelomonas 、 Delftia 、 Paracidovorax . The results indicated that there were no significant differences in the absolute abundances of these 4 dominant bacterial genera in the bodies of cotton aphids feeding on insect-resistant transgenic cotton compared with those feeding on the control materials.
[0026] Table 4 Relative abundances of symbiotic bacteria in target pest cotton bollworms after feeding on transgenic cotton (%)
[0027] Note: The data in the table are mean ± standard deviation; for the same index, the same lowercase English letters after the data in the same row indicate significant differences among different varieties ( p ≥ 0.05).
[0028] Example 3 This example provides an evaluation method for the impact of insect-resistant transgenic soybeans on the diversity of symbiotic bacteria in non-target pest Helicoverpa armigera.
[0029] 1. Operating steps: (1) Transgenic crop cultivation: Cultivate insect-resistant transgenic soybeans and control materials under greenhouse conditions with a temperature of 25 ± 2°C and a relative humidity of 60 ± 5%, until the crops grow to 5 - 8 fully expanded leaves; (2) Selection of experimental pests: Non-target pest with chewing mouthparts, Helicoverpa armigera; (3) Treatment of pest samples: Within 24 hours after molting of the 2nd instar larvae and 5th instar larvae of Helicoverpa armigera and after 12 hours of starvation treatment, inoculate them onto the insect-resistant transgenic soybeans and control materials respectively for feeding; (4) Collection of pest samples: Collect Helicoverpa armigera samples 48 hours after feeding on the insect-resistant transgenic soybeans and control materials respectively; (5) Extraction of genomic DNA from samples: Extract the genomic DNA of Helicoverpa armigera using a soil genomic DNA extraction kit; (6) Sequencing of genomic DNA from samples: Obtain symbiotic bacteria community composition data by sequencing the V3 - V4 region of 16S rRNA; (7) Diversity analysis: Determine the species composition, relative abundance, and diversity indices of symbiotic bacteria through bioinformatics analysis. For the dominant bacterial groups with a relative abundance ≥ 3%, use an independent samples T - test to compare the significance differences at different levels of the symbiotic bacteria communities of Helicoverpa armigera feeding on insect-resistant transgenic soybeans and control materials; (8) Quantitative verification of key bacterial groups: For the dominant bacterial groups with a relative abundance ≥ 5% in the sequencing data, design specific primers respectively to determine the absolute abundance of the dominant bacterial genera by absolute quantitative PCR, and analyze and verify the impact of feeding on insect-resistant transgenic soybeans and control materials on the absolute abundance of the dominant symbiotic bacteria of Helicoverpa armigera through an independent samples T - test.
[0030] 2. Result analysis: (1) Impact of insect-resistant transgenic soybeans on the diversity of symbiotic bacteria in the 2nd instar larvae of Helicoverpa armigera As can be seen from Table 5, after the 2nd instar larvae of Helicoverpa armigera fed on the insect-resistant transgenic soybean, there were no significant differences in their α-diversity indices (coverage, chao1, ace, shannon, sobs, simpson) compared with those of the 2nd instar larvae of Helicoverpa armigera fed on the control material. This result indicates that, compared with the control material, the species richness and evenness of the microorganisms in the 2nd instar larvae of Helicoverpa armigera did not show obvious changes after feeding on the insect-resistant transgenic soybean.
[0031] Table 5 Diversity indices of symbiotic bacteria in the 2nd instar larvae of Helicoverpa armigera after feeding on the insect-resistant transgenic soybean
[0032] Note: The data in the table are mean ± standard deviation; for the same index, the same lowercase English letter after the data in the same row indicates significant differences between different varieties ( p ≥ 0.05).
[0033] The significant differences in the relative abundances of microorganisms in the 2nd instar larvae of the non-target pest Helicoverpa armigera fed on the insect-resistant transgenic soybean at different taxonomic levels were analyzed (Table 6). The results showed that after the 2nd instar larvae of Helicoverpa armigera fed on the insect-resistant transgenic soybean, there were no significant differences in the abundances of microorganisms with relative abundances ≥ 3% in the symbiotic bacteria in the larvae at the phylum, class, order, family, genus, and species levels compared with the control material. The absolute abundances of the dominant genera with relative abundances ≥ 5% were determined using absolute quantitative PCR technology Enterococcus , Mammaliicoccus , Enterobacter . The results indicated that there were no significant differences in the absolute abundances of these 3 dominant genera in the 2nd instar larvae of Helicoverpa armigera fed on the insect-resistant transgenic soybean compared with those fed on the control material.
[0034] Table 6 Relative abundances of symbiotic bacteria in the 2nd instar larvae of Helicoverpa armigera after feeding on the insect-resistant transgenic soybean (%)
[0035] Note: The data in the table are mean ± standard deviation; for the same index, the same lowercase English letter after the data in the same row indicates significant differences between different varieties ( p ≥ 0.05).
[0036] (2) Effects of the insect-resistant transgenic soybean on the symbiotic bacteria diversity of the 5th instar larvae of Helicoverpa armigera As can be seen from Table 7, after the 5th instar larvae of Helicoverpa armigera fed on the insect-resistant transgenic soybean, there was no significant difference in its α-diversity indices (coverage, chao1, ace, shannon, sobs, simpson) compared with those of the 5th instar larvae of Helicoverpa armigera fed on the control material. This result indicates that, compared with the control material, the species richness and evenness of the microorganisms in the 5th instar larvae of Helicoverpa armigera did not show obvious changes after feeding on the insect-resistant transgenic soybean.
[0037] Table 7 Diversity indices of symbiotic bacteria in the 5th instar larvae of Helicoverpa armigera after feeding on the insect-resistant transgenic soybean
[0038] Note: The data in the table are mean ± standard deviation; for the same index, the same lowercase English letter after the data in the same row indicates significant differences between different varieties ( p ≥ 0.05).
[0039] The significance of the differences in the relative abundances of microorganisms in the 5th instar larvae of the non-target pest Helicoverpa armigera fed on the insect-resistant transgenic soybean at different taxonomic levels was analyzed (Table 8). The results showed that after the 5th instar larvae of Helicoverpa armigera fed on the insect-resistant transgenic soybean, there were no significant differences in the abundances of microorganisms with relative abundances ≥ 3% in the symbiotic bacteria in the 5th instar larvae of Helicoverpa armigera at the phylum, class, order, family, genus, and species levels compared with the control material. The absolute abundances of the dominant genera with relative abundances ≥ 5% were determined using absolute quantitative PCR technology. Enterococcus The results showed that there was no significant difference in the absolute abundances of this dominant genus in the 5th instar larvae of Helicoverpa armigera fed on the insect-resistant transgenic soybean compared with those fed on the control material.
[0040] Table 8 Relative abundances of symbiotic bacteria in the 5th instar larvae of Helicoverpa armigera after feeding on the insect-resistant transgenic soybean (%)
[0041] Note: The data in the table are mean ± standard deviation; for the same index, the same lowercase English letter after the data in the same row indicates significant differences between different varieties ( p ≥ 0.05).
[0042] The above examples are only illustrative of the preferred embodiments of the present invention and do not constitute a limitation on the scope of the present invention. Without departing from the design concept of the present invention, all modifications, deformations, or improvements made by those skilled in the art to the technical solutions of the present invention should be included within the protection scope defined by the claims of the present invention.
Claims
1. An evaluation method for the impact of an insect-resistant transgenic crop on the diversity of symbiotic bacteria in target and non-target pests, characterized in that, The method includes the following steps: (1) Transgenic crop planting: Plant insect-resistant transgenic crops and their control materials under controllable greenhouse conditions, and conduct experiments when the crops grow to 5 - 8 fully expanded leaves; (2) Experimental pest selection: Select target pests sensitive to the target traits of the insect-resistant transgenic crops and non-target pests insensitive to them, and preferably select the insect instars at the critical period of damage; (3) Pest sample treatment: Within 24 hours after the pests complete their instar molting (or molt into adults) and after 12 hours of starvation treatment, inoculate them onto the insect-resistant transgenic crops and control materials respectively; (4) Pest sample collection: Collect pest samples of the same age after the pests feed on the crops for 48 hours; (5) Extraction of genomic DNA from samples: Extract the genomic DNA of symbiotic bacteria from the pest samples; (6) Sequencing of the genomic DNA: Use high-throughput sequencing methods to sequence and obtain data on the composition of the symbiotic bacteria community; (7) Diversity analysis: Determine the species composition, relative abundance, and α / β diversity indices of symbiotic bacteria through bioinformatics analysis. For the dominant fungal groups with a relative abundance ≥ 3%, use the analysis of variance method to compare the significance of differences in the symbiotic bacteria communities of target and non-target pests feeding on insect-resistant transgenic crops and control materials at different levels; (7) Quantitative verification of key bacterial groups: For the dominant fungal groups with a relative abundance ≥ 5% in the sequencing data, design specific primers to determine the absolute abundance of dominant bacterial genera or species using absolute quantitative PCR, and verify the effect of feeding on insect-resistant transgenic crops and control materials on the absolute abundance of dominant symbiotic bacteria in pests through statistical analysis.
2. The evaluation method according to claim 1, characterized in that The insect-resistant transgenic crops are constructed by the following methods: RNAi-mediated gene silencing, gene editing technology, and foreign gene introduction.
3. The evaluation method according to claim 1, characterized in that, In step (2), select pests with different mouthpart types and feeding habits as target pests and non-target pests.
4. The evaluation method according to claim 1, wherein In step (3), for target chewing mouthpart pests, preferably select 2nd instar larvae (nymphs) to feed on transgenic crops and control materials; for non-target chewing mouthpart pests, preferably select 2nd instar larvae (nymphs) and the second-to-last instar larvae (nymphs) to feed on transgenic crops and control materials; for target and non-target piercing-sucking mouthpart pests, preferably select adults to feed on transgenic crops and control materials.
5. The evaluation method according to claim 1, characterized in that In step (5), use the CTAB method for extracting genomic DNA from samples, and preferably use a soil genomic DNA extraction kit for extraction.
6. The evaluation method according to claim 1, wherein In step (6), preferably use 16S rRNA or metagenomic sequencing technology. Further, preferably use 16S rRNA gene variable region sequencing to obtain data on the composition of the symbiotic bacteria community.