Safety evaluation method for diversity of transgenic crops on soil microorganisms
By calculating the alpha diversity index of soil microbial communities through high-throughput sequencing technology, the standardization problem of soil microbial diversity assessment of genetically modified crops was solved, the comparability and reliability of research results were improved, and the safety assessment of soil microbial communities was ensured.
Patent Information
- Application Number
- CN202510863223.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-25
- Publication Date
- 2025-09-16
AI Technical Summary
Existing technologies lack unified standard methods to evaluate the long-term impacts of genetically modified crops on soil microbial diversity, resulting in poor comparability of research results and insufficient long-term monitoring of soil microbial community structure and its ecological functions.
High-throughput sequencing technology was used to extract DNA from rhizospheric soil samples of transgenic crops and control crops. PCR amplification and calculation of the alpha diversity index of the microbial community, including sobs, Chaol index, ACE index, Shannon index and Simpson index, were performed to establish a standardized safety assessment method.
It has achieved an accurate evaluation of the impact of genetically modified crops on soil microbial diversity, improved the comparability and reliability of research results, and ensured the safety assessment of soil microbial communities.
Smart Images
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Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of safety evaluation of transgenic crops on soil microbial diversity, and more particularly to a method for safety evaluation of transgenic crops on soil microorganisms. Background Art
[0002] The cultivation of genetically modified plants may affect the associated soil microbial communities. These microorganisms play a vital role in soil ecosystems, including decomposing organic matter, promoting nutrient cycling, and maintaining soil structure. Studies have shown that genetically modified crops may lead to changes in soil invertebrate and microbial communities, which may affect the overall health and function of the soil. Recent research progress in the safety assessment of genetically modified plants, especially their effects on soil microorganisms, has attracted widespread attention. The widespread use of genetically modified plants has made it particularly important to conduct a comprehensive assessment of their environmental impacts, especially their potential impacts on soil microbial communities.
[0003] While some studies have explored the effects of genetically modified plants on soil microbes, this area remains relatively underrepresented. Many studies focus on plant-microbe interactions, while long-term monitoring of microbial community structure and its ecological functions remains systematic. Some studies have suggested that the cultivation of genetically modified crops may lead to changes in the abundance of specific microbial communities, thereby affecting soil biodiversity and ecological balance.
[0004] Currently, studies on the effects of genetically modified crops on soil microbial diversity focus primarily on the short term, with a lack of in-depth research on long-term effects. Current methods for evaluating soil microbial communities lack unified standards, resulting in poor comparability of results. Therefore, establishing a standardized approach to assessing changes in soil microbial communities is crucial to improve the comparability and reliability of research results. Summary of the Invention
[0005] In view of this, the present invention is proposed.
[0006] The method of this invention differs from traditional methods that utilize soil microorganisms for cultivation, such as isolation, identification, counting, and microscopic evaluation. Soil microbial communities are more sensitive to environmental factors than the physical and chemical characteristics of the soil. This method can provide microscopic insights into the adaptive changes of transgenic plants to their environments, making it more valuable for research.
[0007] In order to achieve the above object, the present invention adopts the following technical solutions:
[0008] In one aspect, an embodiment of the present invention provides a method for evaluating the safety of transgenic crops to soil microorganisms, the process comprising:
[0009] (1) Rhizosphere soil samples of transgenic and non-transgenic crops were collected during the critical growth period. A five-point diagonal sampling method was used for each plot, with two plants of normal growth and consistent vigor being sampled at each point.
[0010] (2) DNA was extracted from the collected soil samples, amplified by PCR, and subjected to high-throughput sequencing. The alpha diversity index of microbial community diversity was calculated using bioinformatics. If the soil richness of the transgenic crops was not significantly different from that of the non-transgenic control crops, then the transgenic crops posed no safety risk to soil microbial diversity.
[0011] In a preferred embodiment, in step 1), if the soil sample is taken before sowing, special locations such as ground piles, water hoses, roadsides, ridges, and composting sites should be avoided as much as possible. If the sampling period is the seedling stage, the complete root system is dug out and the root soil is collected with a sampling tool. If the sampling period is after the seedling stage, without digging out the entire plant, the lateral roots 15 to 20 cm underground near the main root are dug out with a shovel (previously disinfected and sterilized), and the rhizosphere soil is collected with a soil collection tool;
[0012] In a preferred embodiment, in step 1), before sampling the soil sample, the topsoil of about 1 cm from the ground surface should be removed, and the soil 1 cm from the root system should be collected using a sampling tool and placed in a 2 mL sterile centrifuge tube, with about 0.1 to 0.5 g of soil sample taken per tube;
[0013] In a preferred embodiment, in step 1), the soil samples are collected from three plots for each treatment, and rhizosphere soil samples of 10 crop plants at 5 points in each plot are mixed and placed in a sealed bag as one sample. Two samples are collected from each plot, for a total of six samples per treatment in each period.
[0014] In a preferred embodiment, in step 1), in the PCR method, the primers are universal primers 338F / 806R, such as 338F-ACTCCTACGGGAGGCAGCAG (SEQ ID NO. 1) and 806R-GGACTACHVGGGTWTCTAAT (SEQ ID NO. 2).
[0015] In a preferred embodiment, in step 2), the alpha diversity index mainly includes sobs (the number of species actually observed), Chaol index, ACE index, Shannon index, and Simpson index.
[0016] The above technical solution demonstrates that, compared to existing technologies, a method for assessing the safety of transgenic crops on soil microorganisms involves collecting soil samples from transgenic crops grown simultaneously, extracting DNA, amplifying the 16s rRNA gene, and performing high-throughput sequencing. The data from each sample at each time period is then used to calculate the alpha diversity index of the microbial community. This method establishes a precise and scientific evaluation method that more efficiently and accurately reflects the safety of transgenic crops on soil microorganisms, and is of great significance for improving the safety of transgenic crop cultivation. DETAILED DESCRIPTION
[0017] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.
[0018] Example 1 Safety evaluation results of transgenic cotton on soil microbial diversity
[0019] Rhizosphere soil samples of transgenic cotton and corresponding non-transgenic cotton were collected before sowing, at the seedling stage, at the bud stage, at the boll stage, and at the boll opening stage. A diagonal 5-point sampling method was used in each plot, with two normal and uniformly growing cotton plants sampled at each point. The specific sampling method was as follows:
[0020] (1) Sampling before sowing: The sampling point should be selected at the later sowing location, and special locations such as ground piles, water hoses, roadsides, ridges, and composting sites should be avoided as much as possible. First, remove the surface soil about 1 cm above the ground surface, and use a pre-sterilized sampling tool to take soil samples between 15 and 20 cm underground. Place the samples in a 2 mL sterile centrifuge tube, taking about 0.1 to 0.5 g of soil samples per tube, and place them in an ice pack insulation box.
[0021] (2) Sampling at the seedling stage: First, remove the surface soil about 1 cm from the ground surface, dig out the complete root system, use a sampling tool to collect the soil 1 cm from the root system, and put it into a 2 mL sterile centrifuge tube. Take about 0.1-0.5 g of soil sample from each tube and put it into an ice pack insulation box.
[0022] (3) Sampling during the bud, boll, and boll stages: First, remove the topsoil about 1 cm below the surface. Without digging out the entire plant, use a shovel (sterilized in advance) to dig out the lateral roots 15 to 20 cm below the taproot. Use a sampling tool to collect the soil 1 cm below the root system and place it in a 2 mL sterile centrifuge tube. Take about 0.1 to 0.5 g of soil sample per tube and place it in an ice pack incubator. After sampling, backfill the soil and mark the plant to avoid sampling the same plant during the next growth period.
[0023] (4) Three plots (GM and non-GM cotton) were collected for each treatment. Rhizosphere soil samples from 10 cotton plants at 5 sites in each plot were mixed and placed in a sealed bag as one sample. Two samples were collected from each plot, for a total of 6 samples per treatment per period. The samples were immediately placed in an ice pack incubator or liquid nitrogen and transported to the laboratory for storage at -80°C for microbial analysis.
[0024] (5) Microbial community analysis was performed using the 16S sequencing method. The method is as follows: Total DNA from soil samples was extracted using a soil DNA extraction kit according to the instructions. Qualified genomic DNA samples and corresponding 16S rDNA fusion primers were used to configure the PCR reaction system, and the PCR reaction parameters were set for PCR amplification. The universal primers for the V3-V4 region used were 338F / 806R: 338F-ACTCCTACGGGAGGCAGCAG, such as SEQ ID NO.1, and 806R-GGACTACHVGGGTWTCTAAT, such as SEQ ID NO.2. The PCR amplification products were purified using Agencourt AMPure XP magnetic beads and dissolved in Elution Buffer, labeled, and the library was constructed. The fragment range and concentration of the library were tested using an Agilent 2100 Bioanalyzer. Qualified libraries were sequenced on the HiSeq platform based on the size of the inserted fragments.
[0025] The raw sequencing data was filtered, leaving high-quality clean data for later analysis. Reads were spliced into tags based on overlap between reads. Tags were clustered into OTUs (ontally encoded transcriptomes) and compared with databases and annotated. Based on the OTUs and annotations, sample species complexity analysis, intergroup species differences analysis, association analysis, and model prediction were performed. Based on OTU abundance and taxonomic annotation, the relative abundance distribution of OTUs at the phylum to genus level was summarized, and bacterial diversity analysis was performed based on the OTU table.
[0026] The alpha diversity index of the microbial community was calculated using data from each sample at each time period. These indices include the sobs (number of observed species), Chaol index, ACE index, Shannon index, and Simpson index. These indices were calculated using the R programming language.
[0027] The experimental results are shown in Table 1. There was no significant difference in the soil microbial diversity index between the transgenic cotton and the control plants. This result indicates that the transgenic cotton plant used in this experiment does not pose a safety risk to soil microbial diversity.
[0028] Table 1 Microbial diversity index of transgenic cotton and non-transgenic cotton
[0029]
[0030] Note: The data in the table are average values for the same growth period, and there is no significant difference between different varieties.
[0031] Example 2 Safety evaluation results of transgenic corn on soil microbial diversity
[0032] Rhizosphere soil samples of transgenic corn and corresponding non-transgenic corn were collected before sowing, at the seedling stage, at the jointing stage, at the silking stage, and at the maturity stage. A five-point diagonal sampling method was used for each plot, with two normal and uniformly growing corn plants sampled at each point. The specific sampling method was as follows:
[0033] (1) Sampling before sowing: The sampling point should be selected at the later sowing location, and special locations such as ground piles, water hoses, roadsides, ridges, and composting sites should be avoided as much as possible. First, remove the surface soil about 1 cm above the ground surface, and use a pre-sterilized sampling tool to take soil samples between 15 and 20 cm underground. Place the samples in a 2 mL sterile centrifuge tube, taking about 0.1 to 0.5 g of soil samples per tube, and place them in an ice pack insulation box.
[0034] (2) Sampling at the seedling stage: First, remove the surface soil about 1 cm from the ground surface, dig out the complete root system, use a sampling tool to collect the soil 1 cm from the root system, and put it into a 2 mL sterile centrifuge tube. Take about 0.1-0.5 g of soil sample from each tube and put it into an ice pack insulation box.
[0035] (3) Sampling at the jointing, silking, and maturity stages: First, remove the topsoil about 1 cm below the surface. Without digging out the entire plant, use a shovel (pre-sterilized) to dig out the lateral roots 15 to 20 cm below the taproot. Use a sampling tool to collect the soil 1 cm below the root system and place it in a 2 mL sterile centrifuge tube. Take about 0.1 to 0.5 g of soil sample per tube and place it in an ice pack incubator. After sampling, backfill the soil and mark the plant to avoid sampling the same plant during the next growth period.
[0036] (4) Three plots (GM and non-GM corn) were collected for each treatment. Rhizosphere soil samples from 10 corn plants at 5 sites in each plot were mixed and placed in a sealed bag as one sample. Two samples were collected from each plot, for a total of 6 samples per treatment per period. The samples were immediately placed in an ice pack incubator or liquid nitrogen and transported to the laboratory for storage at -80°C for microbial analysis.
[0037] (5) Microbial community analysis was performed using the 16S sequencing method. The method is as follows: Total DNA from soil samples was extracted using a soil DNA extraction kit according to the instructions. Qualified genomic DNA samples and corresponding 16S rDNA fusion primers were used to configure the PCR reaction system, and the PCR reaction parameters were set for PCR amplification. The universal primers for the V3-V4 region used were 338F / 806R: 338F-ACTCCTACGGGAGGCAGCAG, such as SEQ ID NO.1, and 806R-GGACTACHVGGGTWTCTAAT, such as SEQ ID NO.2. The PCR amplification products were purified using Agencourt AMPure XP magnetic beads and dissolved in Elution Buffer, labeled, and the library was constructed. The fragment range and concentration of the library were tested using an Agilent 2100 Bioanalyzer. Qualified libraries were sequenced on the HiSeq platform based on the size of the inserted fragments.
[0038] The raw sequencing data was filtered, leaving high-quality clean data for later analysis. Reads were spliced into tags based on overlap between reads. Tags were clustered into OTUs (ontally encoded transcriptomes) and compared with databases and annotated. Based on the OTUs and annotations, sample species complexity analysis, intergroup species differences analysis, association analysis, and model prediction were performed. Based on OTU abundance and taxonomic annotation, the relative abundance distribution of OTUs at the phylum to genus level was summarized, and bacterial diversity analysis was performed based on the OTU table.
[0039] The alpha diversity index of the microbial community was calculated using data from each sample at each time period. These indices include the sobs (number of observed species), Chaol index, ACE index, Shannon index, and Simpson index. These indices were calculated using the R programming language.
[0040] The experimental results are shown in Table 2. There was no significant difference in the soil microbial diversity index between the transgenic corn and the control plants. This result indicates that the transgenic corn used in this experiment does not pose a safety risk to soil microbial diversity.
[0041] Table 2 Microbial diversity index of transgenic corn and non-transgenic corn
[0042]
[0043] Note: The data in the table are average values for the same growth period, and there is no significant difference between different varieties.
[0044] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.
[0045] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not limited to the embodiments shown herein but is intended to conform to the widest scope consistent with the principles and novel features disclosed herein.
Claims
1. A method for safety assessment of transgenic crops on soil microbial diversity, characterized in that the process include: (1) Rhizosphere soil samples were collected at different stages of crop growth, and non-transgenic crops of the same period served as controls; (2) DNA was extracted from the collected soil samples, amplified by PCR and subjected to high-throughput sequencing, and the alpha diversity index of the microbial community diversity was calculated using bioinformatics.
2. The method for safety assessment of transgenic crops on soil microbial diversity according to claim 1, characterized in that: In step 1), the soil samples are collected during the key growth and development periods of crops, including before sowing, seedling stage, flowering stage, and maturity stage.
3. The method for safety assessment of transgenic crops on soil microbial diversity according to claim 1, characterized in that: In step 1), if soil sampling is done before sowing, avoid specific locations such as ground piles, water hoses, roadsides, ridges, and composting sites. If sampling is done during the seedling stage, remove the entire root system and collect the root soil using a sampling tool. If sampling is done after the seedling stage, use a shovel (previously disinfected and sterilized) to remove the lateral roots 15-20 cm below the taproot, without removing the entire plant. Use a soil collection tool to collect the rhizosphere soil.
4. The method for safety assessment of transgenic crops on soil microbial diversity according to claim 1, characterized in that: In step 1), before sampling the soil sample, the surface soil of about 1 cm should be removed. The soil 1 cm below the root system should be collected with a sampling tool and placed in a 2 mL sterile centrifuge tube. Approximately 0.1 to 0.5 g of soil sample should be taken in each tube.
5. The method for safety assessment of transgenic crops on soil microbial diversity according to claim 1, characterized in that: In step 1), the soil samples were collected from 3 plots for each treatment. Rhizosphere soil samples of 10 crops from 5 points in each plot were mixed and placed in a sealed bag as one sample. Two samples were collected from each plot, for a total of 6 samples per treatment in each period.
6. The method for safety assessment of transgenic crops on soil microbial diversity according to claim 1, characterized in that: In step 2), in the PCR method, the primers are universal primers 338F / 806R, specifically 338F-ACTCCTACGGGAGGCAGCAG and 806R-GGACTACHVGGGTWTCTAAT.
7. The method for safety assessment of transgenic crops on soil microbial diversity according to claim 1, characterized in that: In step 2), the alpha diversity index mainly includes sobs (the number of species actually observed), Chaol index, ACE index, Shannon index, and Simpson index.