Method for evaluating safety of transgenic insect-resistant corn to non-target organisms in soil and application of method

Abstract

The invention belongs to the technical field of biological safety evaluation, and particularly relates to a method for evaluating non-target biological safety of transgenic insect-resistant corn in soil and application of the method. According to the method, an artificial soil method is adopted to simulate natural exposure conditions, and leaves of transgenic insect-resistant corn and non-transgenic control corn in the elongation stage are freeze-dried and then added into artificial soil for culturing eisenia fetida. After in-vivo DNA of eisenia fetida in different culture periods is extracted respectively, PCR amplification is carried out on bacterial 16S rRNA genes and fungus ITS regions, a library is constructed, and amplicon sequencing is carried out. The safety of the transgenic insect-resistant corn to a non-target organism eisenia fetida is comprehensively evaluated by analyzing the change of the diversity of Alpha and Beta of microorganisms in eisenia fetida. According to the invention, a scientific, accurate and efficient evaluation method is successfully established, and the method has important significance for perfecting an evaluation system for non-target biological safety of transgenic crops.

Description

Technical Field

[0001] The present invention belongs to the technical field of biosafety evaluation, and in particular relates to a method for evaluating the safety of transgenic insect-resistant corn on non-target organisms in soil and an application thereof. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance understanding of the overall background of the invention and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to those skilled in the art.

[0003] Genetically modified insect-resistant crops may enter the soil through pollen and debris, potentially affecting non-target organisms. Earthworms, as keystone species in soil ecosystems (accounting for 60%-80% of soil biomass), play a vital role in biorecycling and soil improvement, making them ideal indicators of ecological safety. Traditional assessment methods rely primarily on phenotypic indicators such as weight change, growth and development, and reproductive capacity, making them difficult to detect sublethal effects from low-dose, long-term exposure.

[0004] The animal microbiome is an important indicator of host health. Changes in the microbiome can precede macroscopic biological changes, making it a valuable early warning indicator. Due to its high sensitivity, functional relevance, and ecological value, earthworm microbial diversity is emerging as an innovative biomarker for assessing the impacts of genetically modified crops on earthworms. The earthworm's intestinal and coelomic microbiome forms a close symbiotic relationship with its host, and its community structure is highly sensitive to changes in the host's internal and external environments.

[0005] In the early days, people used culture methods to detect only culturable microorganisms (about 1% of microorganisms are culturable), and molecular fingerprinting technology (such as DGGE / T-RFLP) can only reflect the dominant bacterial community, and low-abundance species are easily overlooked. Therefore, the evaluation results of microbial diversity using traditional methods are not accurate.

[0006] The 16S rRNA gene (approximately 1500 bp) is an ideal marker for bacterial classification because it is ubiquitous in bacteria and contains nine hypervariable regions (V1-V9) and conserved regions. High-throughput sequencing technology can be used to analyze the microbial diversity in earthworms, detecting the vast majority of microorganisms, including difficult-to-cultivate and low-abundance species, significantly improving detection sensitivity. Combined with functional prediction tools such as PICRUSt2, it can also analyze the functional characteristics of microbial communities. This method, by monitoring changes in the earthworm microbiome, can not only sensitively detect the sublethal effects of genetically modified crops but also elucidate the mechanisms of microbe-host interactions at the molecular level, providing a scientific basis for ecological risk assessment of genetically modified crops. Therefore, establishing an earthworm microbiome analysis method based on amplicon sequencing is of great significance for the safety assessment of genetically modified crops. Summary of the Invention

[0007] In view of the deficiencies of the prior art, the present application provides a method for evaluating the safety of transgenic insect-resistant corn to non-target organisms in soil and its application. Specifically, the present application simulates natural exposure conditions by using artificial soil method, and adds the leaf blades of transgenic insect-resistant corn and its non-transgenic control corn at the jointing stage into the artificial soil after freeze-drying, for culturing earthworms. The DNA in the earthworms at different culture periods is extracted, and then the bacterial 16S rRNA gene and fungal ITS region PCR amplification are performed and a library is constructed, and amplicon sequencing is performed. By analyzing the changes of the Alpha diversity and Beta diversity of the microorganisms in the earthworms, the safety of the transgenic insect-resistant corn to the non-target organism earthworms is comprehensively evaluated, so as to be used for the evaluation system of the safety of transgenic crops to non-target organisms. Based on the above research results, the present application is completed.

[0008] Specifically, the present application relates to the following technical solutions: In a first aspect of the present application, a method for evaluating the safety of transgenic insect-resistant corn to non-target organisms in soil is provided, which comprises: S1, adding the leaf blades of transgenic insect-resistant corn and its non-transgenic control corn into artificial soil after drying, for culturing non-target organisms in the artificial soil; S2, extracting the DNA in the non-target organisms at different culture periods for PCR amplification of microorganisms and constructing a library, and performing amplicon sequencing; S3, analyzing the changes of the Alpha diversity and Beta diversity of the microorganisms in the non-target organisms; The non-target organism can be earthworms.

[0009] In a second aspect of the present application, the above method is applied in the ecological safety evaluation of transgenic insect-resistant corn. The present application proves by experiments that the transgenic insect-resistant corn has no significant effect on the diversity of microorganisms in the earthworms in soil, indicating that it has good ecological safety.

[0010] The beneficial technical effects of the one or more technical solutions above are: The above technical solution provides a method based on amplicon sequencing for analyzing the change of the diversity of microorganisms in earthworms in soil, which is a transgenic crop environmental safety evaluation method, so as to be used for the evaluation system of the safety of transgenic crops to non-target organisms, and thus protects the safety of agricultural products and public health, and therefore has good practical application value. BRIEF DESCRIPTION OF DRAWINGS

[0011] The drawings accompanying the specification of the present application serve to provide a further understanding of the present application, and the schematic embodiments of the present application and the description thereof serve to explain the present application, and do not constitute an improper limitation on the present application.

[0012] Figure 1 为本发明实施例中样品稀释曲线。

[0013] Figure 2 This is a histogram of sample alpha diversity in an embodiment of the present invention.

[0014] Figure 3 This is a principal coordinate analysis diagram of the sample in the embodiment of the present invention. DETAILED DESCRIPTION

[0015] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present application belongs.

[0016] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, they indicate the presence of features, steps, operations, devices, components and / or combinations thereof.

[0017] The present invention will be further described with reference to specific examples. The following examples are intended only to illustrate the present invention and are not intended to limit its contents. Experimental conditions not specified in the examples are generally based on conventional conditions or those recommended by the sales company. Materials and reagents used in the examples are commercially available unless otherwise specified.

[0018] In a typical embodiment of the present invention, a method for evaluating the safety of transgenic insect-resistant corn to non-target organisms in soil is provided, the method comprising: S1. Drying leaves of transgenic insect-resistant corn and its non-transgenic control corn and adding them to artificial soil to cultivate non-target organisms in the artificial soil; S2. Extract DNA from non-target organisms at different culture stages, perform PCR amplification of the microorganisms, construct libraries, and perform amplicon sequencing; S3. Analyze changes in microbial alpha diversity and beta diversity within non-target organisms; 所述非靶标生物可以为赤子爱胜蚓。

[0019] In step S1, the leaves are selected from corn grown to the jointing stage. Since the expression of exogenous proteins in various tissues and organs of transgenic corn throughout its growth period is generally highest in leaves at the jointing stage, the present application previously tested the expression of exogenous proteins in various tissues and organs of transgenic insect-resistant corn at various growth stages, thus selecting the period and organ with the highest expression. The drying may be freeze-drying, thereby maximizing the preservation of protein activity in the leaves.

[0020] The specific components and usage of the artificial soil are as follows: peat moss: 5-15 parts, kaolin: 10-30 parts, industrial sand: 60-70 parts, and calcium carbonate: 1-5 parts. Furthermore, the specific components and usage of the artificial soil are as follows: peat moss: 10 parts, kaolin: 20 parts, industrial sand: 68 parts, and calcium carbonate: 2 parts. This artificial soil can effectively simulate natural exposure conditions while avoiding the variability of natural soil, ensuring comparability of experimental results.

[0021] In another specific embodiment of the present invention, in step S2, Different culture periods can be selected from the 7th day, 14th day, 21st day and 28th day of culture.

[0022] 所述微生物具体可以为细菌和真菌; The PCR amplification of the microorganism is specifically PCR amplification of the bacterial 16S rRNA gene and the fungal ITS region; in another specific embodiment of the present invention, the amplification primers for the bacterial 16S rRNA gene are: V3-V4 region primer 341F (5′-CCTAYGGGRBGCASCAG-3′, SEQ ID NO.1) / 805R (5′-GACTACHVGGGTATCTAATCC-3′, SEQID NO.2); the amplification primers for PCR amplification of the fungal ITS region are: ITS1F (5′-CTTGGTCATTTAGAGGAAGTAA-3′, SEQ ID NO.3) / ITS2R (5′-GCTGCGTTCTTCATCGATGC-3′, SEQ ID NO.4).

[0023] Furthermore, the present invention uses Nextera XT for library construction and Qubit® to quantify library concentration to ensure balanced concentration of each sample; uses Bioanalyzer or LabChip GX to confirm library fragment size; and after library construction, uses Illumina MiSeq for amplicon sequencing.

[0024] In step S3, the specific method includes: using FastQC and Trimmomatic for quality control and removing low-quality sequences (Q30 or below); clustering 16S rRNA (bacteria) and ITS (fungi) sequences using USEARCH (97% similarity threshold, i.e., sequences with a similarity of 97% or higher are considered to be the same operational taxonomic unit (OTU); normalizing the OTU table using the CSS method (normalize_table.py); calculating the alpha diversity of bacteria and fungi based on the normalized OTU table; and analyzing the beta diversity of bacteria and fungi using CSS normalization (beta_diversity.py). In this paper, OTUs present in all samples are defined as core taxa.

[0025] In another embodiment of the present invention, the above method is used to evaluate the ecological safety of transgenic insect-resistant corn. Experiments conducted by the present invention demonstrate that transgenic insect-resistant corn has no significant effect on the microbial diversity of the non-target organism Eisenia fetida in soil, demonstrating its good ecological safety.

[0026] The present invention is further explained by the following examples, but is not intended to limit the present invention. It should be understood that these examples are only intended to illustrate the present invention and are not intended to limit the scope of the present invention.

[0027] Example 一、供试材料 The test corn consisted of genetically modified insect-resistant corn and its non-genetically modified control. At the jointing stage, 150 g of leaves from each corn variety were collected four times. These leaves were crushed in the field and frozen in liquid nitrogen. After return to the laboratory, they were freeze-dried for 24-48 hours and then frozen at -80°C until ready for use. Using freeze-dried leaves maximized protein activity.

[0028] Earthworms: Before the experiment, remove the required number of earthworms, clean them with deionized water, place them in a beaker, seal the beaker with white gauze, and place the beaker in an incubator (20 ± 2°C) to allow the earthworms to defecate for 24 hours. During this time, change the filter paper at least once to drain the intestinal contents and reduce environmental microbial interference. Weigh the defecation-free earthworms to ensure that their average weight meets the experimental requirements.

[0029] 人工土壤组成成份及配比如下: 表1 人工土壤组成成份及配比

[0030] 二、试验方法 1. 生物测定方法 An artificial soil method was used to simulate natural exposure conditions. Freeze-dried leaf powder was added to the artificial soil and thoroughly stirred. Distilled water was added to adjust the soil moisture content to 30%–35%. 500 g of the prepared soil (soil thickness in a 1000 mL beaker should be at least 8 cm) was placed in a beaker, along with 10 earthworms. The beaker was then tied with gauze. The beaker was placed in an artificial climate chamber at (20 ± 2)°C, 80%–85% humidity, and continuous light intensity of 400–800 lx. On the 7th, 14th, 21st, and 28th day of incubation, one earthworm was randomly selected and frozen in liquid nitrogen and stored at −80°C.

[0031] 2. DNA提取与建库 Earthworms were removed, surface disinfected with 75% ethanol, and then homogenized. Earthworm DNA was extracted using the QIAGEN DNeasy Blood & Tissue Kit (suitable for host-microbe mixed DNA extraction) according to the manufacturer's instructions. DNA quality was assessed using the Qubit® dsDNA HS Assay and NanoDrop assay. The bacterial 16S rRNA gene was amplified using primers 341F (5′-CCTAYGGGRBGCASCAG-3′, SEQ ID NO. 1) and 805R (5′-GACTACHVGGGTATCTAATCC-3′, SEQ ID NO. 2) in the V3-V4 region. The fungal ITS region was amplified using primers ITS1F (5′-CTTGGTCATTTAGAGGAAGTAA-3′, SEQ ID NO. 3) and ITS2R (5′-GCTGCGTTCTTCATCGATGC-3′, SEQ ID NO. 4). The amplification system consists of a high-fidelity enzyme (such as KAPA HiFi HotStart ReadyMix), primers (10 μM each in 0.2 μL), and template DNA. The amplification program is 95°C for 3 minutes → denaturation: 95°C for 30 seconds → annealing: 55°C for 30 seconds (bacteria) or 50°C for 30 seconds (fungi) → extension: 72°C for 30 seconds (25-35 cycles) → final extension: 72°C for 5 minutes. Libraries are constructed using the Nextera XT Index Kit, and library concentration is quantified using Qubit® to ensure uniform concentration across samples. Library fragment size is confirmed using a Gilent 2100 Bioanalyzer or LabChip GX. After library construction, amplicon sequencing is performed using the Illumina MiSeq (2×250 / 300 bp, suitable for mid-throughput) platform.

[0032] 3. 数据分析 Bacterial 16S rRNA gene and fungal ITS sequences were processed using QIIME v1.9 and USEARCH v10. In brief, read quality was assessed using FastQC v0.11.5. Low-quality reads with a score below Q30 were pairwise trimmed using Trimmomatic v0.39. Trimmed sequences were clustered, and sequences with a similarity of ≥97% were considered to be the same operational taxonomic unit (OTU). Sequences were classified as either bacterial or fungal using the SILVA v138.1 and UNITE v8.2 databases. The OTU tables for bacteria and fungi were normalized for alpha diversity estimation using the normalize_table.py script in QIIME according to the CSS method. For beta diversity analysis of bacteria and fungi, the cumulative sum scaling (CSS) normalization method was applied using beta_diversity. OTUs present in all samples were defined as core taxa.

[0033] 3. Results Analysis 1. Dilution curve The dilution curve randomly extracts a certain number of sequences from the sample, counts the number of species represented by these sequences, and constructs a curve with the number of sequences and the number of species to verify whether the amount of sequencing data is sufficient to reflect the species diversity in the sample, and indirectly reflects the richness of species in the sample. The figure below reflects the rate of emergence of new features (new species) under continuous sampling: within a certain range, as the number of sequencing increases, if the curve shows a sharp rise, it means that a large number of species have been discovered in the community; when the curve tends to be flat, it means that the species in this environment will not increase significantly with the increase in the number of sequencing. The dilution curve can be used as a judgment on whether the sequencing volume of each sample is sufficient. A sharp rise in the curve indicates that the sequencing volume is insufficient and the number of sequences needs to be increased; otherwise, it indicates that the sample sequence is sufficient and data analysis can be performed. Figure 1 As shown in the figure, the curves of all samples eventually tend to be flat, indicating that the sequencing quantity has met the requirements for evaluating microbial diversity, which is the prerequisite for subsequent evaluation.

[0034] 2. Alpha Diversity Analysis Alpha diversity reflects the species richness and species diversity of a single sample. The Shannon index is used to measure species diversity and is affected by the species richness and species evenness of the sample community. Under the same species richness, the greater the evenness of each species in the community, the greater the diversity of the community. The larger the Shannon index and Simpson index values, the higher the species diversity of the sample. Figure 2 As shown in the figure, there was no significant difference in the Shannon diversity index among all samples, indicating that transgenic insect-resistant corn had no significant effect on the microbial diversity in the non-target organism Eisenia fetida in the soil.

[0035] 3. Beta diversity analysis based on Bray Curtis algorithm PCoA (Principal Coordinates Analysis) sorts a series of eigenvalues ​​and eigenvectors, selects the top eigenvalues, and uses the idea of ​​dimensionality reduction to find the most important coordinates in the distance matrix, thereby observing the differences between individuals or groups. Bray Curtis's algorithm compares species differences based on the sequence features contained in the sample (i.e., comparison based on features). Figure 3 As shown, the transgenic group and the control group samples were mixed together and not completely separated, which once again shows that transgenic insect-resistant corn has no significant effect on the microbial diversity in the non-target organism Eisenia fetida in the soil.

[0036] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A method for evaluating the safety of transgenic insect-resistant corn against non-target organisms in soil, the method comprising: S1. Dried leaves of transgenic insect-resistant corn and its non-transgenic control corn were added to artificial soil to cultivate non-target organisms in the soil; S2. Extract DNA from non-target organisms at different culture stages, perform PCR amplification of the microorganisms, construct libraries, and perform amplicon sequencing; S3. Analyze changes in microbial alpha diversity and beta diversity within non-target organisms; The non-target organism is the earthworm Eisenia fetida.

2. The method according to claim 1, wherein In the step S1, the leaves are selected from corn leaves obtained when the corn grows to the jointing stage; and the drying is freeze-drying.

3. The method according to claim 1, wherein In step S1, the specific components and usage of the artificial soil are as follows: peat moss: 5-15 parts, kaolin: 10-30 parts, industrial sand: 60-70 parts, and calcium carbonate: 1-5 parts; further, the specific components and usage of the artificial soil are as follows: peat moss: 10 parts, kaolin: 20 parts, industrial sand: 68 parts, and calcium carbonate: 2 parts.

4. The method according to claim 1, wherein In the step S2, Different culture periods can be selected from the 7th day, 14th day, 21st day and 28th day of culture.

5. The method according to claim 1, wherein In the step S2, The microorganisms are specifically bacteria and fungi; The PCR amplification of the microorganism is specifically PCR amplification of the bacterial 16S rRNA gene and the fungal ITS region.

6. The method according to claim 5, wherein The amplification primers for bacterial 16S rRNA gene are shown in SEQ ID NO. 1-2; the amplification primers for PCR amplification of fungal ITS region are shown in SEQ ID NO. 3-4.

7. The method according to claim 1, wherein In step S2, Nextera XT was used for library construction, and Qubit® was used to quantify the library concentration to ensure balanced concentrations of each sample. A Bioanalyzer or LabChip GX was used to confirm the library fragment size. After library construction, amplicon sequencing was performed using Illumina MiSeq.

8. The method according to claim 1, wherein In step S3, the specific method includes: using FastQC and Trimmomatic for quality control and removing low-quality sequences; using USEARCH to cluster 16S rRNA (bacteria) and ITS (fungi) sequences; using the CSS method to normalize the OTU table (normalize_table.py); calculating the alpha diversity of bacteria and fungi based on the normalized OTU table; and analyzing the beta diversity of bacteria and fungi based on CSS normalization (beta_diversity.py).

9. The method according to claim 8, wherein The low-quality sequences were those with scores lower than Q30; the similarity threshold in clustering was 97%.

10. Use of the method according to any one of claims 1 to 9 in the ecological safety assessment of transgenic insect-resistant corn.

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