CN112014501B - Research method for migration process of steroid estrogen in wheat - Google Patents
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Abstract
The invention discloses a method for researching the migration process of steroid estrogen in wheat, which belongs to the technical field of restoration of polluted plants in water or soil, and comprises the following steps: 1) adding nutrient solution containing 17 beta-E2 and nutrient solution not containing 17 beta-E2 into different chambers of the plant culture device respectively; 2) dividing the root system of the mature root seedling into two parts, and transplanting the two parts to a chamber of a plant culture device respectively; 3) and respectively measuring the contents of 17 beta-E2 in the overground part, the root part and the nutrient solution of the seedlings at different culture times. According to the invention, the accumulation amount of 17 beta-E2 in different positions of the plant along with the time can be visually seen by sampling and measuring at different time points, so that the migration process of 17 beta-E2 in the plant body is judged. The invention has simple process, convenient operation, no need of complex experimental equipment and low design cost, and provides an important technical support for researching the migration and accumulation processes of steroid estrogen and other pollutants in plants.
Description
Technical Field
The invention belongs to the technical field of remediation of polluted plants in water or soil, and particularly relates to a research method for a migration process of steroid estrogen in wheat.
Background
Steroid estrogen is mainly hormone substances produced by human or animal ovarian cells and the like, and after the steroid estrogen enters an animal body as exogenous estrogen, the steroid estrogen can simulate the action of endogenous hormones of cells or change the activity of the endogenous hormones of the cells, so that the sexual differentiation and the normal metabolic function in the body of the human or animal are influenced, the normal growth, development, reproduction and other functions of organisms are interfered, even the effects of killing, carcinogenesis, teratogenesis and the like are produced, and the steroid estrogen is a novel micropollutant which is paid much attention in the 21 st century. Studies have shown that steroid estrogens are present at very low concentration levels (e.g., 1 ng/L)-1) Can cause harm to the organism. Effluent water of a sewage treatment plant and feces of a livestock and poultry farm are two major pollution sources of steroid estrogen, and the steroid estrogen enters a plant body through soil due to land utilization of the effluent water and the feces, and finally enters a human body through a food chain, so that the health and growth of the human body are seriously threatened. There has been no report on the study of whether steroid estrogens can migrate from aerial parts to roots. By studying the distribution and migration process of steroid estrogen in plants, the potential accumulation possibility of steroid estrogen in different parts of edible crops can be judged, and the estrogen exposure risk of human body caused by food chain can be further evaluated. Therefore, the research method for effectively, intuitively and simply researching the migration process of the steroid estrogen in the plant body has important significance for fully knowing the residual accumulation rule of the steroid estrogen in the plant body and the potential health risk caused by the residual accumulation rule, and can provide an extremely important technical support for the development of the plant restoration method polluted by the steroid estrogen and other organic pollutants in water or soil.
Disclosure of Invention
In order to solve the problems, the invention provides a method for researching the migration process of steroid estrogen in wheat, which comprises the following steps:
1) respectively adding a nutrient solution containing 17 beta-E2 and a nutrient solution not containing 17 beta-E2 into different chambers of the plant culture device;
2) equally dividing the root system of the mature root seedling into two parts, and respectively transplanting the two parts into a chamber of the plant culture device in the step 1);
3) and respectively measuring the contents of 17 beta-E2 in the overground part, the root part and the residual nutrient solution of the wheat seedlings at different culture times.
The number of true leaves of the mature root system seedling is more than or equal to 4.
The concentration of 17 beta-E2 in the nutrient solution in the step 1) is 100-2000 mu g/L, and further, the concentration of 17 beta-E2 is 1000 mu g/L.
The Hoagland concentration in the nutrient solution in the step 1) is 1/4-1/2 times of that of the standard Hoagland nutrient solution. In the step 3), the culture time is more than or equal to 48 hours.
The culture process of the mature root seedlings is as follows:
1) placing the seeds on wet filter paper, germinating in dark, and keeping the filter paper wet during germination to obtain seedlings;
2) and (3) culturing the seedlings obtained in the step 1) in a Hoagland nutrient diluent to obtain mature root seedlings.
In the culture process of the mature root seedlings, in the step 1), the germination temperature is 22-25 ℃, and the germination time is 5-6 days.
The illumination period of the culture process in the step 2) of the culture process of the mature root seedlings is 16h, the dark period is 8h, the relative humidity is 75% +/-2%, the temperature is 20-25 ℃, and the illumination intensity is 200-400 mu mol/m2S; further, the light intensity was 300. mu. mol/m2·s。
The concentration of the Hoagland nutrient diluent in the step 2) of the culture process of the mature root seedlings is 1/4-1/2 times that of the standard Hoagland nutrient solution.
The culture process of the mature root seedlings, namely the seeds in the step 1), further comprise a pretreatment process, wherein the wheat seeds are soaked in 10-20 wt% of hydrogen peroxide solution for 10 min; removing the seeds floating on the upper layer of the hydrogen peroxide solution and the residual seeds, thoroughly washing the residual seeds with deionized water, and selecting uniform and full seeds.
The plant culture device consists of 2 transparent chambers and 1 cover plate, wherein the middle of the cover plate is provided with a round hole which ensures that a wheat root system enters a glass chamber and keeps wheat seedlings to grow vertically; 2 glass chambers below the cover plate are provided with gaps to prevent the culture solution from being polluted mutually.
The invention has the beneficial effects that:
1. the invention develops an effective, intuitive and simple research method for the migration process of steroid estrogen in plants, has important significance for fully knowing the residual accumulation rule of steroid estrogen in plants and potential health risks brought by the residual accumulation rule, and provides an extremely important technical support for the development of a phytoremediation method for water or soil polluted by steroid estrogen and other organic pollutants.
2. According to the invention, through ingenious experimental design, the cumulant of the 17 beta-E2 in different positions of the plant along with the time can be visually seen only by sampling and measuring at different time points, so that the migration process of the 17 beta-E2 in the plant body is judged. The invention has simple process, convenient operation, no need of complex experimental equipment and low design cost, and provides an important technical support for researching the migration and accumulation processes of steroid estrogen and other organic pollutants in plants. In addition, the method can provide possibility for accurately evaluating the risk of accumulation of organic pollutants in crops.
Drawings
FIG. 1 is a photograph of wheat obtained from wheat seeds hydroponically cultured until the root system thereof is mature before rooting culture;
FIG. 2 is a photograph of wheat during rooting culture;
FIG. 3 is a graph of 17 β -E2 concentration profiles of 17 β -E2 in nutrient solution and roots soaked therein at different times in example 3;
FIG. 4 is a 17 β -E2 concentration profile of the roots and aerial parts of the nutrient solution without 17 β -E2 soaked therein at different times in example 3;
FIG. 5 is a graph showing the variation of the 17 β -E2 content in the wheat roots according to the concentration of 17 β -E2 in the nutrient solution soaked in the wheat roots in example 4;
FIG. 6 is a graph showing the variation of the 17 β -E2 content in the aerial parts of wheat and barley in example 4 according to the concentration of 17 β -E2 in the nutrient solution;
Detailed Description
The invention is described in further detail below with reference to the following figures and specific examples:
a method for researching the migration process of steroid estrogen in crop wheat bodies comprises the following specific steps:
1) soaking the wheat seeds in 10-20% hydrogen peroxide solution for 10 min;
2) removing the seeds floating on the upper layer of the hydrogen peroxide solution and the residual seeds in the step 1), thoroughly washing the residual seeds with deionized water, selecting uniform and full seeds, placing the seeds on wet filter paper, germinating for 5 days in the dark at the temperature of 22-25 ℃, and keeping the filter paper wet all the time during germination;
3) preparing Hoagland nutrient solution with standard concentration by using a Hoagland nutrient solution reagent, and diluting the Hoagland nutrient solution with different degrees to obtain nutrient solution with concentration 1/4-1/2 times of that of the standard Hoagland nutrient solution;
4) selecting wheat seedlings with similar growth vigor after germination obtained after water culture till the root system is mature as shown in figure 1, placing the wheat seedlings in a culture dish containing a proper amount of nutrient solution prepared in the step 3), and culturing at the illumination period of 16h, the dark period of 8h, the relative humidity of 75 +/-2%, the temperature of 20-25 ℃ and the illumination intensity of 200-400 mu mol/m2S, culturing the wheat seedlings until the wheat seedlings have mature roots;
5) taking a self-made plant culture device, and respectively adding a nutrient solution which is not polluted by 17 beta-E2 and a 17 beta-E2 nutrient solution with the concentration of 100-2000 mu g/L into 2 chambers carried by the device;
6) taking wheat seedlings with mature roots and similar growth vigor in the step 4), uniformly dividing the roots into two parts, and transplanting the two parts into the culture device in the step 5), wherein the two parts are shown in the figure 2;
7) and (3) taking the overground parts of the wheat seedlings, the root systems in different containers and the residual nutrient solution in the corresponding containers at different culture times, and measuring the content of 17 beta-E2 by using a liquid chromatography-tandem mass spectrometry.
Preferably, in step 1), the concentration of the hydrogen peroxide solution is 15%;
preferably, in the step 4), when the number of true leaves is more than or equal to 4, the wheat seedlings are required seedlings;
preferably, in the step 4), the illumination intensity for culturing the wheat seedlings is 300 mu mol/m2·s；
Preferably, in the step 5), the concentration of the 17 beta-E2 is 1000 mu g/L;
preferably, in the step 5), the self-made plant cultivation device consists of 2 150mL glass chambers and 1 cover plate, wherein the cover plate is a paper plate with a hole with the diameter of 1cm in the middle and the thickness of 3cm, the hole can ensure that the root system of wheat enters the 2 glass chambers under the cover plate, and the wheat seedling can be kept to grow vertically with a certain thickness; suitable gaps are left in 2 glass chambers below the cover plate, so that the culture solution is prevented from being polluted mutually;
preferably, in the step 7), the culture time of the transplanted wheat is more than or equal to 48 hours.
Example 1
Soaking and sterilizing wheat seeds in 10%, 15% and 20% hydrogen peroxide for 10min, thoroughly cleaning with deionized water, selecting uniform and plump seeds, placing on wet filter paper, and germinating in dark for 5d while keeping the filter paper wet all the time. After the seeds germinate, the seeds are placed in a culture dish containing 1/4 Hoagland nutrient solution with standard concentration and cultured under set culture conditions until seedlings grow until a fourth true leaf appears. Then placing the wheat in a culture dish containing 1000 mu g/L of 17 beta-E2 solution, and simultaneously adding nutrient solution into the solution to ensure that the concentration of the nutrient solution is 1/2 of the standard Hoagland nutrient solution concentration so as to ensure the normal growth of wheat. Destructive sampling is carried out after 7d of culture, and the root of the wheat seedling is taken to measure the content of 17 beta-E2. During the culture, the wheat seedlings were cultured under conditions except for the light intensity (set at 200, 300 and 400. mu. mol/m, respectively)2S) other culture conditions were controlled as follows: the illumination period is 16 hours, the dark period is 8 hours, the relative humidity is 75% +/-2%, and the temperature is 20-25 ℃. The results are shown in Table 1.
TABLE 1 content of 17 beta-E2 in wheat seedlings roots in different hydrogen peroxide solutions and under different lighting conditions
As shown in the table, the absorption of the wheat to 17 beta-E2 is influenced by hydrogen peroxide and light intensity, wherein the hydrogen peroxide is 15 percent and the light intensity is 300 mu mol/m2S is the maximum uptake of 17 β -E2 by wheat.
Example 2
The plant culture device consists of 2 150mL glass chambers and 1 cover plate, wherein the cover plate is a paperboard with a hole with the diameter of 1cm in the middle and the thickness of 3cm, the hole can ensure that root systems of the rooted wheat which are evenly divided into two parts enter the 2 glass chambers under the cover plate respectively, and the wheat seedlings can grow vertically with a certain thickness; suitable gaps are left in 2 glass chambers below the cover plate, and the culture solution is prevented from being polluted mutually.
Example 3
Mixing wheat seeds with 15% H2O2Soaking and sterilizing for 10min, thoroughly cleaning with deionized water, selecting uniform and plump seeds, placing on wet filter paper, germinating in dark for 5d, and keeping the filter paper wet all the time during germination. After the seeds germinate, the seeds are placed in a culture dish containing 1/4 Hoagland nutrient solution with standard concentration and cultured under the set culture condition until seedlings grow until a fourth true leaf appears. 50mL of deionized water and 50mL of 1000 mu g/L17 beta-E2 solution are respectively added into 2 chambers of the plant culture device, and the same volume of Hoagland nutrient solution with standard concentration is added into two containers to ensure that the volume of the Hoagland nutrient solution is 1/2 with the concentration of the standard Hoagland nutrient solution so as to ensure the normal growth of wheat. Taking wheat seedlings with similar growth vigor after being cultured by the culture solution, uniformly dividing the root part of the wheat seedlings into two parts, respectively placing the two parts in two containers, then carrying out destructive sampling after culturing for 4, 12, 24, 48 and 72 hours, and measuring the contents of the overground part of the wheat seedlings, the root systems in different containers and the residual solution 17 beta-E2 in the corresponding containers. The culture conditions of the wheat seedlings in the culture process are controlled as follows: the illumination period is 16h, the dark period is 8h, the relative humidity is 75% +/-2%, the temperature is 20-25 ℃, and the illumination intensity is 300 mu mol/m2·s。
The concentration distribution of 17 beta-E2 in different parts of wheat and in solution at different times is shown in figure 3 and figure 4, and the graph shows that the concentration of 17 beta-E2 in the nutrient solution containing estrogen decreases along with the increase of time; and the content of 17 beta-E2 in the overground part of the wheat seedling and the root system in different containers is increased and then reduced along with the time, wherein the time (24h) for the overground part of the wheat seedling and the root system in the nutrient solution without estrogen to reach the peak value is delayed by 12h compared with the time (12h) for the concentration of the 17 beta-E2 in the nutrient solution with estrogen to reach the peak value. In addition, the nutrient solution without estrogen starts to detect 17 beta-E2 after 24 hours. From the above results, it is presumed that the migration pathway of 17 β -E2 in plants is: the 17 beta-E2 in the estrogen nutrient solution is absorbed by the root system of the wheat, then enters the overground part through further migration and mass transfer, then migrates from the overground part to the root system without the estrogen nutrient solution, is discharged out of the wheat body through the root system, and is finally released to the nutrient solution. Meanwhile, it can be also found that the time required for observing the migration path of 17 beta-E2 in wheat seedlings is at least 48 h.
Example 4
Mixing wheat seeds with 15% H2O2Soaking and sterilizing for 10min, thoroughly cleaning with deionized water, selecting uniform and plump seeds, placing on wet filter paper, germinating in dark for 5d, and keeping the filter paper wet all the time during germination. After the seeds germinate, the seeds are placed in a culture dish containing 1/4 Hoagland nutrient solution with standard concentration and cultured under the set culture condition until seedlings grow until a fourth true leaf appears. Wheat with similar growth vigor is taken and transplanted into a culture dish containing a series of 17 beta-E2 solutions with concentration gradients (100, 500, 1000 and 2000 mu g/L), and meanwhile, a nutrient solution is added into the solutions to ensure that the concentration of the nutrient solution is 1/2 of the standard Hoagland nutrient solution so as to ensure the normal growth of the wheat. Destructive sampling was carried out after 7d of cultivation, and wheat seedlings were divided into roots and aerial parts, in which the content of 17 β -E2 was determined, respectively. The culture conditions of the wheat seedlings in the culture process are controlled as follows: the illumination period is 16h, the dark period is 8h, the relative humidity is 75% +/-2%, the temperature is 20-25 ℃, and the illumination intensity is 300 mu mol/m2·s。
The change of the content of the wheat root system 17 beta-E2 along with the exposure concentration is shown in figure 5 and figure 6, and the graph shows that the concentration of the wheat root and the overground part 17 beta-E2 increases along with the increase of the exposure concentration when the concentration of the 17 beta-E2 is in the range of 100-1000 mug/L, and the cumulative amount of the wheat root and the overground part to the 17 beta-E2 decreases along with the increase of the concentration when the concentration of the 17 beta-E2 is more than 1000 mug/L. Wherein, when the concentration of the 17 beta-E2 is 1000 mug/L, the accumulation of the root parts and the overground parts of the wheat is the largest.
Claims (7)
1. A method for researching the migration process of steroid estrogen in wheat is characterized by comprising the following steps:
1) respectively adding a nutrient solution containing 17 beta-E2 and a nutrient solution not containing 17 beta-E2 into different chambers of the plant culture device; the concentration of 17 beta-E2 in the nutrient solution is 100-2000 mug/L;
2) equally dividing the root system of the mature root seedling into two parts, and respectively transplanting the two parts into a chamber of the plant culture device in the step 1);
3) respectively measuring the contents of the overground part, the root part and the residual nutrient solution of the wheat seedling in 17 beta-E2 at different culture times;
the plant culture device consists of 2 transparent chambers and 1 cover plate, wherein the middle of the cover plate is provided with a round hole which ensures that a wheat root system enters a glass chamber and keeps wheat seedlings to grow vertically; gaps are reserved in 2 glass chambers below the cover plate, so that the culture solution is prevented from being polluted mutually;
the culture process of the mature root seedlings is as follows:
pre-treating the seeds, and soaking the wheat seeds in 10-20 wt% hydrogen peroxide solution for 10 min; removing the seeds floating on the upper layer of the hydrogen peroxide solution and the residual seeds, thoroughly cleaning the residual seeds with deionized water, and selecting uniform and full seeds;
a) placing the seeds on wet filter paper, germinating in dark, and keeping the filter paper wet during germination to obtain seedlings;
b) culturing the seedlings obtained in the step a) in a Hoagland nutrient diluent to obtain mature root seedlings; the illumination period of the culture process is 16h, the dark period is 8h, the relative humidity is 75% +/-2%, the temperature is 20-25 ℃, and the illumination intensity is 200-400 mu mol/m2·s。
2. The method as claimed in claim 1, wherein the number of true leaves of the mature root system seedling is not less than 4.
3. The method as claimed in claim 1, wherein the concentration of 17 β -E2 in the nutrient solution of step 1) is 1000 μ g/L.
4. The method as claimed in claim 1, wherein the concentration of Hoagland in the nutrient solution in the step 1) is 1/4-1/2 times of that of a standard Hoagland nutrient solution; in the step 3), the culture time is more than or equal to 48 hours.
5. The method according to claim 1, wherein the germination temperature in step a) is 22-25 ℃ and the germination time is 5-6 days.
6. The method according to claim 1, wherein the light intensity during the culturing in step b) is 300. mu. mol/m2·s。
7. The method of claim 1, wherein the concentration of the Hoagland nutrient diluent in step b) is 1/4-1/2 times that of the standard Hoagland nutrient diluent.
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| CN101803541A (en) * | 2009-02-13 | 2010-08-18 | 中国科学院沈阳应用生态研究所 | Test device for researching accumulation and migration of heavy metal in plants and use thereof |
| WO2018064323A1 (en) * | 2016-09-28 | 2018-04-05 | Organovo, Inc. | Use of engineered renal tissues in assays |
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