Evaluation method for researching risk of transgenic crops on soil springtails through litter approach based on artificial soil

Abstract

The invention discloses an evaluation method for researching the risk of transgenic crops on soil springtails through a litter approach based on artificial soil, and belongs to the technical field of agricultural production. The evaluation method comprises the following steps: uniformly mixing quartz sand, kaolin and activated carbon powder according to a mass ratio of 88: 25: 10, and adding water to prepare artificial soil; the litters of the transgenic crops and the control crops are cut into blocks and then sterilized, and the sterilized litters are obtained; respectively adding sterilized litters of a transgenic crop and a control crop into the surface layer of the artificial soil, and uniformly mixing the sterilized litters with the artificial soil after the sterilized litters absorb water to obtain a soil springtail culture medium; adding soil springtails for culture, detecting the number, body length and related functional enzyme content of the soil springtails after culture, and comparing the influence of litters of transgenic crops and control crops on the fitness of the soil springtails. The method is closer to a real natural state and can be operated repeatedly.

Description

Technical Field

[0001] The invention relates to the technical field of agricultural production, and in particular to an evaluation method for studying the risk of transgenic crops to soil springtails through litter pathways based on artificial soil. Background Art

[0002] Genetically modified insect-resistant crops are made insect-resistant by introducing foreign genes. Planting genetically modified insect-resistant crops can greatly reduce the use of chemical pesticides and labor input. However, with the promotion of genetically modified crops and the widespread application of straw return technology, foreign proteins can enter the soil ecosystem in large quantities through the rhizosphere and straw return, and may have adverse effects on the soil ecosystem.

[0003] Springtails, also known as springtails, are the common name for Collembola, a widely distributed class of small to micro arthropods with rich species and large numbers of individuals. They are one of the three major soil animals and play an important role in the nutrient cycle of the soil system. Because they are sensitive to changes in the soil environment, soil springtails are a good indicator for evaluating soil quality. Therefore, it is of great practical significance to evaluate the risk of soil springtails from transgenic crops through the litter pathway.

[0004] Previous studies on the risk assessment of transgenic litter have mainly focused on the field. However, the field environment is complex and unstable, with many uncontrollable factors. Therefore, there is no way to accurately track and investigate specific indicators of a specific soil animal group, nor is it clear whether transgenic crops will have adverse effects on soil animals due to the insertion of exogenous genes. In previous indoor biological tests on the impact of transgenic crop litter on soil springtails, the litter residues were generally crushed and placed on the surface of the culture medium as food for soil springtails. However, the powdered litter residues placed on the surface of the culture medium are quite different from the environment of the field litter buried in the soil after returning to the field, and cannot reflect the true risk to soil springtails in the field. At the same time, in previous studies on the effects of transgenic crops on soil animal functional enzymes, the selection of functional enzyme groups was relatively random and not representative. Summary of the invention

[0005] The purpose of the present invention is to provide an evaluation method for the risk of transgenic crops to soil springtails through litter fall based on artificial soil to solve the problems existing in the above-mentioned prior art. The present invention simulates litter return to the field under indoor controllable conditions, which can not only ensure the stability of experimental conditions, but also be close to the straw return to the field under natural conditions, and provide a litter risk evaluation method for transgenic crops that is close to the real natural state, repeatable and stable.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a method for evaluating the risk of transgenic crops to soil springtails through litter fall based on artificial soil, comprising the following steps:

[0008] Quartz sand, kaolin and activated carbon powder were mixed in a mass ratio of 88:25:10, and then water was added to prepare artificial soil;

[0009] The litter of the transgenic crops and the control crops is cut into pieces and sterilized to obtain sterilized litter;

[0010] Adding sterilized litter of transgenic crops and control crops to the surface of the artificial soil respectively, spraying water and leaving the litter to stand, and mixing the sterilized litter with the artificial soil after absorbing water to obtain a soil springtail culture medium;

[0011] Soil springtails are added to the soil springtail culture medium for cultivation. After the cultivation, the number, body length and related functional enzyme content of the soil springtails are detected, and the effects of litter of transgenic crops and control crops on the fitness of the soil springtails are compared.

[0012] Furthermore, the humidity of the artificial soil is 80%.

[0013] Furthermore, the litter is cut into pieces with a size of 1-2 cm×1 cm.

[0014] Furthermore, when the sterilized litter is added to the surface layer of the artificial soil, the mass ratio of the artificial soil to the sterilized litter is 153:1.

[0015] Furthermore, when spraying water, the ratio of the sterilized litter to the amount of water sprayed is 1g:15mL.

[0016] Furthermore, the amount of soil springtails added is 20 to 30 heads.

[0017] Furthermore, the soil springtails are 12 to 20 days old.

[0018] Furthermore, the soil springtail is Thalassaphorura encarpata of the family Acanthophoridae or Entomobrychidae proxima of the family Entomobryidae.

[0019] Furthermore, the culture conditions are humidity 60-70% and room temperature culture for 60 days.

[0020] Furthermore, the relevant functional enzymes include superoxide dismutase, total protease and glutathionease.

[0021] The present invention discloses the following technical effects:

[0022] The present invention uses quartz sand, kaolin and activated carbon as raw materials to prepare artificial soil. Quartz sand is the main matrix of artificial soil and is relatively loose, while kaolin has adhesiveness, and activated carbon has an adsorption effect and can loosen the soil at the same time. The three are mixed in a certain proportion, and an appropriate amount of sterile water is added to produce artificial soil with moderate looseness and stable environment. The artificial soil provided by the present invention has a stable formula, can avoid the disadvantages of complex field conditions and many uncontrollable factors, and has controllable viscosity, moderate porosity and adsorption, can more realistically simulate the state of field soil, and can more accurately simulate the return of field straw after burying litter.

[0023] The present invention cuts crop litter into blocks without crushing, thereby maintaining the original state of the straw and also avoiding the problem that the accuracy of risk assessment results is affected due to the destruction of exogenous proteins and other components in the litter during crushing.

[0024] In the prior art, when the functional enzymes of soil springtails are detected, the selected functional enzyme groups are acetylcholinesterase, superoxide dismutase and catalase, among which superoxide dismutase and catalase are both antioxidant enzymes, acetylcholinesterase is an enzyme that reacts to the state of nerve excitement, and the selected functional enzyme types are relatively single. The present invention selects a representative enzyme from digestive enzymes, antioxidant enzymes, and detoxifying enzymes (superoxide dismutase is selected as antioxidant enzyme, glutathionease is selected as detoxifying enzyme, and total protease is selected as digestive enzyme), which can avoid selecting multiple functional enzymes of the same type at the same time, causing unnecessary waste of resources, and can comprehensively reflect the response of springtails to transgenic crop litter at the physiological and biochemical levels. DETAILED DESCRIPTION

[0025] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0026] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0027] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0028] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0029] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0030] The transgenic plant used in Example 1 of the present invention is transgenic corn CM8401, the control variety is four 144, the seeds are provided by the Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, and the single soil springtail species used is Thalassaphorura encarpata of the family Echinops, which is taken from the "National Transgenic Corn, Soybean Pilot and Industrialization Base (Gongzhuling)" and the test population cultivated by the gene discovery and safety evaluation technology research team of Jilin Academy of Agricultural Sciences, and has been stably cultivated in the laboratory for 3 years; the transgenic base control crop used in Example 2 is the same as that in Implementation Case 1, and the single soil springtail species used is Entomobryaproxima of the family Longhorned Springtail, which is taken from the "National Transgenic Corn, Soybean Pilot and Industrialization Base (Gongzhuling)" and the test population cultivated by the transgenic plant environmental safety research team of Jilin Academy of Agricultural Sciences, and has been stably cultivated in the laboratory for 5 years.

[0031] Example 1

[0032] (1) Artificial soil culture medium configuration

[0033] Take 1760g of 10-mesh quartz sand, 500g of kaolin and 200g of activated carbon powder, mix the three thoroughly, then spray in 150mL of pure water, mix thoroughly, then spray 150mL of water, mix thoroughly again, repeat this process 4 times, add a total of 600mL of pure water to prepare artificial culture soil, at which time the humidity of the artificial soil culture medium is about 80%.

[0034] (2) Litter treatment of transgenic CM8401 and control crop 4144

[0035] At the late stage of crop maturity, 100 g of mature leaves of transgenic and control corn were taken, and after removing the petioles, they were cut into small pieces of length × width = 1 cm × 1 cm, mixed, and then sterilized with ultraviolet light for 2 hours. After sterilization, they were set aside.

[0036] (3) Cultivation of soil springtail T. encarpata

[0037] Cultured in a culture dish with gypsum: activated carbon = 9:1 (m / m), added with brewer's yeast as food, cultured in full darkness, culture temperature was 20±1°C, relative humidity was close to 100%, and adjusted with deionized water once a week to keep the entire culture medium moist. When synchronized, eggs laid within three days need to be collected together, and experiments are carried out at 20 days of age.

[0038] (4) Addition of artificial soil culture medium, crop litter and soil springtails

[0039] First, 153 g of the artificial soil medium in (1) was placed in a culture bottle, and then 1 g of the transgenic or control litter leaves in (2) were placed on the surface of the culture bottle with the artificial soil medium added. At this time, 5 mL of pure water was sprayed in, and the culture bottle cap was closed. After 12 hours, the litter that absorbed water was fully mixed into the culture medium, and 30 soil springtails cultured in (3) were added. Ten replicates were set for each litter species.

[0040] (5) Sampling and analysis

[0041] The culture medium with soil springtails was placed at room temperature, and pure water was added every 3 days to keep the humidity in the artificial soil culture medium between 60% and 70%. Sampling was performed 60 days after the addition of soil springtails. The soil springtails were separated from the artificial soil culture medium using a dry funnel, and then put into a -80℃ ultra-low temperature refrigerator for quick freezing and killing. The number of adults and larvae of the test springtails was obtained by manual counting under a stereoscope, and the survival rate and reproduction rate of the soil springtails were calculated. At the same time, the body length of the adults was measured, and the superoxide dismutase, total protease and glutathione enzyme in the adults were detected using an enzyme marker. The effects of litter on the fitness of soil springtails in transgenic and control materials were comprehensively compared.

[0042] The test results are shown in Table 1. The results show that at room temperature, in the artificial soil culture medium, soil springtails in the litters of CM8401 and Si 144 survived well, and there were no significant differences in their survival rate, reproduction rate, body length and functional enzymes; this indicates that compared with the control Si 144, the litters of transgenic corn CM8401 had no obvious adverse effects on the fitness of the springtail T. encarpata population.

[0043] Table 1 Survival rate, reproduction rate, body length, superoxide dismutase, total protease, and glutathionease of springtail T. encarpata in transgenic corn CM8401 and control variety 4144 (mean ± standard error)

[0044]

[0045] Note: The same lowercase letters indicate no significant difference.

[0046] Example 2

[0047] (1) Artificial soil culture medium configuration

[0048] Take 1760g 20-mesh quartz sand, 500g kaolin, and 200g activated carbon powder, mix the three thoroughly, then spray in 150mL of pure water, mix thoroughly, then spray 150mL of water, and mix thoroughly again. Repeat this process 4 times, add a total of 600mL of pure water, and prepare artificial soil. At this time, the humidity of the artificial soil culture medium is about 80%.

[0049] (2) Litter treatment of transgenic CM8401 and control crop 4144

[0050] At the late stage of crop maturity, 100 g of mature leaves of transgenic and control corn were taken, and after removing the petioles, they were cut into small pieces of length × width = 2 cm × 1 cm, mixed, and then irradiated with ultraviolet light for 3 hours, sterilized and set aside.

[0051] (3) Cultivation of soil springtail Entomobrya proxima

[0052] Cultured in a culture dish with gypsum: activated carbon = 9:1 (m / m), added with brewer's yeast as food, cultured in full darkness, culture temperature was 20±1°C, relative humidity was close to 100%, and adjusted with deionized water once a week to keep the entire culture medium moist. When synchronized, eggs laid within three days need to be collected together, and experiments are carried out at 12 days of age.

[0053] (4) Addition of culture medium, crop litter and soil springtails

[0054] First, 153 g of the artificial soil medium in (1) was placed in a culture bottle, and then 1 g of the transgenic or control litter leaves in (2) were placed on the surface of the culture bottle with the artificial soil medium added. At this time, 5 mL of pure water was sprayed in, and the culture bottle cap was closed. After 12 hours, the litter that absorbed water was fully mixed into the culture medium, and 20 soil springtails cultured in (3) were added. Ten replicates were set for each litter species.

[0055] (5) Sampling and analysis

[0056] The culture medium with soil springtails added was placed at room temperature, and purified water was added every 3 days to keep the humidity of the artificial soil culture medium between 60% and 70%. Sampling was performed 60 days after adding soil springtails, and soil springtails were separated from the artificial soil culture medium using a dry funnel. After being collected, they were put into a -80℃ ultra-low temperature refrigerator for quick freezing and killing. The number of adults and larvae of the test springtails was obtained by manual counting under a stereoscope, and the survival rate and reproduction rate of soil springtails were calculated. At the same time, the body length of adults was measured, and superoxide dismutase, total protease and glutathionease in springtails were detected using an enzyme marker. The effects of litter of transgenic and control varieties on the fitness of soil springtails were comprehensively compared.

[0057] The test results are shown in Table 2. The results show that at room temperature, in the artificial soil culture medium, the springtail E. proxima in the litters of CM8401 and Si 144 survived well, and there were no significant differences in their survival rate, reproduction rate, body length and functional enzymes; this indicates that compared with the control Si 144, the litters of transgenic corn CM8401 had no obvious adverse effects on the fitness of the springtail E. proxima population.

[0058] Table 2 Survival rate, reproduction rate, body length, superoxide dismutase, total protease, and glutathionease of E. proxima in transgenic corn CM8401 and control variety Si144 (mean ± standard error)

[0059]

[0060]

[0061] Note: The same lowercase letters indicate no significant difference.

[0062] Comparative Example

[0063] 1. Comparison of soil porosity between artificial soil and field soil

[0064] The soil porosity of the 0-5 cm layer of black soil in Gongzhuling area and the artificial soil culture medium prepared in step (1) of Example 2 were tested at 60% and 70% soil moisture, respectively, with 5 replicates for each. The test results are shown in Table 3.

[0065] Table 3 Total porosity of artificial soil and black soil in Gongzhuling area under different humidity (%)

[0066] Soil Type Soil moisture 60% Soil moisture 70% Black soil 58.71±0.47a 54.93±0.67a Artificial soil 59.03±0.62a 55.28±0.75a

[0067] Note: The same letters in the same column indicate no significant difference at the 0.05 confidence level.

[0068] The results in Table 3 show that at 60% and 70% humidity, there is no significant difference in the porosity of the artificial soil culture medium compared with the 0-5 cm layer of soil in the black soil corn field in Gongzhuling, indicating that the artificial soil culture medium is similar to the field soil state and can simulate the real field state more realistically.

[0069] 2. Effects of different treatments on litter

[0070] The CM8401 test material was crushed and not crushed (cut into 1 cm × 1 cm blocks) respectively, and after the litter treatment was completed, it was placed in an incubator, and the temperature in the incubator was maintained at 20 ± 1 ° C and the relative humidity was 60-70%. The Bt protein content of the litter with different treatments was tested on the 0th day, 20th day and 60th day in the incubator, and 5 replicates were set for each. The results are shown in Table 4.

[0071] Table 4 Bt protein content in litter under different treatments at different periods (ng / g)

[0072] Litter treatment methods Before cultivation 20 days 60 days Block 554.59±4.69a 417.18±5.69a 33.93±2.63a Powder 509.07±11.75b 290.00±6.84b 18.50±0.59b

[0073] Note: Different letters in the same column indicate significant differences at the 0.05 confidence level.

[0074] The results in Table 4 show that the content of Bt protein in powdered litter at each period is significantly lower than that in litter cut into blocks, indicating that the Bt protein in the litter is destroyed after the crushing process and is more easily degraded during the degradation process. Therefore, the litter treatment method in the present invention can better reflect the real state of the field and the results are more accurate.

[0075] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for evaluating the risk of transgenic crops to soil springtails through litter fall based on artificial soil, characterized in that: The following steps are involved: Quartz sand, kaolin and activated carbon powder were mixed in a mass ratio of 88:25:10, and then water was added to prepare artificial soil; The litter of the transgenic crops and the control crops is cut into pieces and sterilized to obtain sterilized litter; Adding sterilized litter of transgenic crops and control crops to the surface of the artificial soil respectively, spraying water and leaving the litter to stand, and mixing the sterilized litter with the artificial soil after absorbing water to obtain a soil springtail culture medium; Soil springtails are added to the soil springtail culture medium for cultivation. After the cultivation, the number, body length and related functional enzyme content of the soil springtails are detected, and the effects of litter of transgenic crops and control crops on the fitness of the soil springtails are compared.

2. The evaluation method according to claim 1, characterized in that: The humidity of the artificial soil was 80%.

3. The evaluation method according to claim 1, characterized in that: The size of the litter after cutting is 1-2 cm×1 cm.

4. The evaluation method according to claim 1, characterized in that: When the sterilized litter is added to the surface layer of the artificial soil, the mass ratio of the artificial soil to the sterilized litter is 153:

1.

5. The evaluation method according to claim 1, characterized in that: When spraying water, the ratio of the sterilized litter to the amount of water sprayed is 1g:15mL.

6. The evaluation method according to claim 1, characterized in that: The amount of soil springtails added is 20 to 30.

7. The evaluation method according to claim 6, characterized in that: The soil springtails are 12 to 20 days old.

8. The evaluation method according to claim 1, characterized in that: The soil springtails include Thalassaphorura encarpata of the family Echinopsidae or Entomobryaproxima of the family Entomobryidae.

9. The evaluation method according to claim 1, characterized in that: The culture conditions are as follows: humidity 60-70% and room temperature for 60 days.

10. The evaluation method according to claim 1, characterized in that: The relevant functional enzymes include superoxide dismutase, total protease and glutathionease.

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