CN109187401B - Method for determining subcellular distribution of graphene in rice body - Google Patents

Abstract

The invention discloses a method for measuring the subcellular distribution of graphene in rice, belonging to the application and development of nanotechnology in the agricultural field. The steps are: germinating the rice seeds, and exposing the germinated rice to graphene; collecting the stem and leaf parts of the rice plants respectively, cutting them into pieces, freezing them in liquid nitrogen and then fully grinding them; adding the pre-cooled homogenization medium to the process. The ground tissue is prepared into a homogenate; the homogenate is separated into 5 parts by differential centrifugation: cell wall, chloroplast, nucleus, mitochondria and soluble part; each component is fully burned by a bio-oxidizer, and the generated carbon dioxide is collected. , and the graphene content in each component was determined by a liquid scintillation counter. To assess the possible interactions between organelles and graphene during centrifugation, three control experiments were designed to accomplish this goal. This method realizes the accurate quantification of graphene in subcellular rice plants.

Description

A method for determining the subcellular distribution of graphene in rice

technical field

The patent of the present invention relates to a method for measuring the subcellular distribution of graphene in rice, which belongs to the application and development of nanotechnology in the field of agriculture.

Background technique

Graphene is a two-dimensional honeycomb lattice structure composed of smooth single layers of carbon atoms and is the basic building block of other graphite materials. Graphene can be wrapped to form zero-dimensional fullerenes, rolled to form one-dimensional carbon nanotubes, or stacked to form three-dimensional graphite. Since graphene was first exfoliated from graphite in 2004 by Geim and Novoselov of the University of Manchester, UK, due to its many extraordinary physical and chemical properties, such as high thermal conductivity, intrinsic strength, high electrical conductivity, high light transmittance, gaseous Impermeability and easy functionalization, graphene research has developed rapidly, and graphene research has also expanded to various scientific research fields, such as electronics, optics, sensors, energy storage and conversion, and environmental pollution treatment.

In recent years, with the further development of nanotechnology, scientists gradually apply nanotechnology to the agricultural field. The main research directions involved are nano pesticides, nano fertilizers, nano vaccines, nano feeds and additives, and nano hormones. Some scientists have studied the interaction between nanoparticle antioxidants and chloroplasts extracted from plants, and found that nanoparticle antioxidants can consume reactive oxygen species produced in chloroplasts, thereby protecting the photoactivity of chloroplasts. Indirectly, it is shown that nanoparticles can protect chloroplast activity, thereby promoting photosynthesis in plants. As for carbon nanomaterials, because of its good stability and unique optical and electronic properties, once it enters the plant, it may greatly promote the photosynthesis of chloroplasts. For example, studies have shown that single-walled carbon nanotubes can absorb photons in a wider range of wavelengths, and thus absorb more solar energy, which can be stored to promote the photoelectric transfer rate in plant photosynthesis. However, the main problem at present is how to accurately quantify the subcellular distribution of graphene into plants, which is important for evaluating the influence of the presence of graphene in plants on photoelectric transfer efficiency in plant photosynthesis and plant growth. It has important research significance. In addition, unlike other studies on the subcellular distribution of soluble organic matter in plants, graphene, as a nanoparticle, may aggregate, settle or aggregate when the subcellular components are separated by differential centrifugation. Adsorbed on the surface of each component, if the regular method is used to measure the regularity of graphene in each subcellular component, a positive result may be obtained.

In order to avoid the occurrence of such positive results, the present invention sets up a plurality of control group experiments to deduct the influence of factors such as aggregation, sedimentation or adsorption of graphene in the separation process of each subcellular component. It is of great significance to accurately quantify the subcellular distribution of graphene in plants.

SUMMARY OF THE INVENTION

In view of the above problems, the present invention discloses a method for measuring the subcellular distribution of graphene in rice.

To achieve the above object, the technical scheme adopted in the present invention is:

A method for measuring the subcellular distribution of graphene in rice, the steps comprising:

(1) germination treatment is carried out to rice seeds, after the seeds are germinated, the seedlings with consistent bud and root system development are selected and transferred respectively to the mother liquor containing graphene and the mother liquor that does not contain graphene and cultivated, wherein the culturing in the mother liquor that does not contain graphene The seedling is a blank control group, wherein the development of the bud and the root system is the same, which means that the length ratio of the development of the bud and the root system is relatively balanced, and it is preferably a seedling with basically the same length of the bud and the root system;

(2) Cultivate the above-mentioned rice seedlings for 7-21 days. After the cultivation is completed, first cut them into pieces, freeze them in liquid nitrogen and then fully grind them; after the evaporation of liquid nitrogen is completed, add a pre-cooled homogenate medium to continue grinding, and then the homogenate liquid Pour it into a centrifuge tube, and then wash the mortar several times with the homogenizing medium. The cleaning solution is all poured into the centrifuge tube containing the homogenate. The liquid in the centrifuge tube is shaken up, filtered, and the filtrate is used for subsequent centrifugation;

(3) Separating the homogenate collected in step (2) into 5 parts by differential centrifugation: cell wall, chloroplast, nucleus, mitochondria and soluble part;

(4) the graphene content of 5 components of the seedling cultivated in the graphene solution obtained in the detection step (3);

(5) 5 subcellular components of the blank control group obtained in step (3) are respectively added to the centrifuge tube, then the graphene mother solution is added dropwise and shaken, and then the centrifugation conditions in step (3) of claim 1 are respectively followed. Carry out centrifugation, and finally measure the graphene content in the homogenate medium of 5 subcellular components;

(6) adding the same content of graphene mother dropwise to 5 samples of homogenizing medium of the same volume without subcellular components, shaking and shaking, then centrifuging according to the centrifugation conditions in step (3), and finally measuring Graphene content in the homogenate medium that drips graphene mother liquor;

(7) deducting the graphene content obtained in step (5) from the graphene content obtained in step (6) is the content of the graphene adsorbed by the organelle;

(8) The absolute subcellular content of graphene in the rice body is the graphene content in step (4) minus the graphene content obtained in step (7).

Preferably, the concentration of the graphene mother solution is 50-250 μg/L, and the solvent of the graphene mother solution is water.

Preferably, the homogenate medium contains 0.25mmol/L sucrose, 50mmol/L Tris-HCl (pH 7.5), and 1mmol/L dithioerythritol, the solvent is water, and each 0.2g in step (2) is cut into pieces The seedlings were triturated by adding 1 ml of homogenization medium pre-cooled to 4 °C. Preferably, in step (2), the mortar is washed with 1 ml of homogenization medium each time, and the washing is performed 4 times.

Preferably, in step (2), a nylon mesh with a pore size of 80 μm is used for filtration.

Preferably, in step (5), 1 μg of graphene mother drop is added to each subcellular fraction of 5 mL blank control group at 4°C.

Preferably, in step (6), 1 μg of graphene mother is added dropwise to 5 mL of homogenization medium at 4°C.

Preferably, the subcellular distribution state of graphene in the stem or leaf of the seedling is determined respectively.

Different from other studies on the subcellular distribution of soluble organic matter in plants, graphene, as a nanoparticle, may aggregate, settle or adsorb on the subcellular components when the subcellular components are separated by differential centrifugation. On the surface of each component, positive results may be obtained if the regularity of graphene in each subcellular component is determined according to conventional methods.

In order to avoid the occurrence of such positive results, the present invention sets up a plurality of control group experiments to deduct the influence of factors such as aggregation, sedimentation or adsorption of graphene in the separation process of each subcellular component. It is of great significance to accurately quantify the subcellular distribution of graphene in plants.

The present invention relates to a method for measuring the subcellular level distribution of graphene in rice. The method is simple and easy to operate, and different control groups are set to avoid graphene aggregation, sedimentation or subcellular distribution during the separation process. The influence of factors such as the adsorption of each component on the distribution results. Therefore, the present invention can more accurately determine the content of graphene in each subcellular component, that is, the distribution level of graphene in the cell wall, chloroplast, nucleus and mitochondria of rice tissue. It is of great significance to explore the relationship between the subcellular distribution of graphene in rice and the photosynthesis of plants.

Description of drawings

Figure 1 is a schematic diagram of the subcellular distribution and separation steps of graphene in stems and leaves.

Figure 2 shows the results of the subcellular distribution of graphene in rice stems under different exposure times.

Figure 3 shows the results of the subcellular distribution of graphene in rice leaves under different exposure times.

The X-axis label name in Figure 2 and Figure 3:

F1: cell wall

F2: Chloroplast

F3: nucleus

F4: Mitochondria

F5: Soluble fraction.

Detailed ways

Example 1 The sedimentation efficiency of graphene in the presence/absence of organelles and the enrichment efficiency of graphene by organelles

(1) Seed germination and graphene exposure: The newly harvested seeds were first soaked in a 30% H ₂ O ₂ solution for 15 min, and then washed three times with deionized water to ensure that the seed surface was sterile. The seeds were then soaked in deionized water and stored at 30°C in the dark for 48h. Finally, the soaked seeds were evenly displayed in a sterilized petri dish, and the bottom of the petri dish was cushioned with two layers of sterilized moist filter paper. The petri dish was covered and placed in a light incubator at 30°C for 7 days. After germination, it was transferred to a container containing 2 L of exposure solution with a graphene concentration of 0 μg/L.

(2) After exposure for 7 days, collect rice leaves, accurately weigh 0.2 g, and centrifuge the homogenate to obtain chloroplast fractions, and dropwise add 5 mL of homogenization medium to resuspend the obtained chloroplasts. The graphene mother was added dropwise to the resuspension homogenate with a concentration gradient of 50-250 μg/L of graphene.

(3) Since the absorption or adsorption of graphene may make the chloroplasts in the suspension heavier, two centrifugation speeds of 300g and 1500g were set, and the centrifugation time was 10min. After centrifugation, the supernatant was removed and the pellet was resuspended in homogenization medium again. Chloroplast content (λ=664nm and 647nm) was detected by UV spectrophotometer. The absorbance of 0-200 μg/L pure graphene suspension at these two wavelengths was measured simultaneously.

(4). At 4°C, five 10 mL centrifuge tubes were added to 5 mL of the five subcellular fractions obtained from rice leaves. Then, the mother solution containing 1 μg graphene was added dropwise and shaken for 3 h. Then, centrifugation was performed according to the differential centrifugation method, respectively. Finally, the graphene content in the five subcellular fractions was determined.

(5). Under the condition of 4℃, drop the mother containing 1μg graphene into 5 samples containing only 5mL of homogenization medium without subcellular components, shake for 3h, and then centrifuge according to the differential centrifugation method. Finally, the graphene content in the homogenate medium was determined.

(6). Under the condition of 4 ℃, the mother containing 1 μg graphene was added dropwise to the subcellular samples of the control plants, and shaken for 3 h. Centrifuge according to the centrifugation conditions in step (3). After centrifugation, the supernatant was poured off, and deionized water was gently added to the centrifuge tube to resuspend the subcellular fractions to avoid the suspension of free-state graphene that agglomerates and settles. Finally, the content of graphene in the suspension was determined.

Example 2 The results of the subcellular distribution of graphene in rice stems under different exposure times

(1) Seed germination and graphene exposure: The newly harvested seeds were first soaked in a 30% H ₂ O ₂ solution for 15 min, and then washed three times with deionized water to ensure that the seed surface was sterile. The seeds were then soaked in deionized water and stored at 30°C in the dark for 48h. Finally, the soaked seeds were evenly displayed in a sterilized petri dish, and the bottom of the petri dish was cushioned with two layers of sterilized moist filter paper. The petri dish was covered and placed in a light incubator at 30°C for 7 days. After germination, it was transferred to a container containing 2 L of exposure solution with a graphene concentration of 250 μg/L. At the same time, another group of blank control group was set up, that is, after germination, it was transferred to 2L of graphene-free solution.

(2) After exposure for 7 days, 14 days and 21 days, respectively, collect the stem tissue of the graphene exposure combined blank control rice, cut it into pieces, fully grind it after freezing in liquid nitrogen; secondly, add 1 mL of pre-heated tissue after the evaporation of liquid nitrogen is completed. The cold homogenization medium continued slow milling for 15 s. Subsequently, the homogenate was slowly poured into a 10 mL centrifuge tube, and then the mortar was washed four times with 1 mL of homogenization medium, and the washings were all poured into the centrifuge tube containing the homogenate. The liquid in the centrifuge tube was shaken well, filtered through a layer of nylon mesh, the filter residue was unground tissue, and the filtrate was used for subsequent centrifugation.

(3) First, centrifuge the homogenate of the graphene exposure group at 300g for 30s, and the precipitate is the cell wall, mainly including cell wall and cell wall debris. The filtrate was centrifuged at 1500g for 10min, and the precipitate was the chromosomal part. The supernatant was centrifuged at 5000g for 20min, and the pellet was the nucleus part. The supernatant was centrifuged at 15,000 g for 30 min, the pellet was the mitochondrial fraction, and the supernatant was the soluble fraction.

(4) The subcellular components of the stem obtained in the step (3) are burned with a biological oxidizer, and the generated carbon dioxide is collected, and then the graphene content in each component is measured by a liquid scintillation counter, and is set as A.

(5) Next, obtain each subcellular fraction from the homogenate in the blank control group according to the method of step (3), and then dropwise add the mother solution containing 1 μg graphene to 5 mL of each subcellular fraction and shake for 3 hours. Then centrifuge according to the differential centrifugation method, and finally measure the graphene content in the homogenate medium, which is set as B. In addition, the mother containing 1 μg of graphene was added dropwise to 5 samples containing only 5 mL of homogenization medium without subcellular components, shaken for 3 h, and then centrifuged according to the differential centrifugation method. Finally, the graphene content in the homogenate medium was measured and set as C. The adsorption amount of each subcellular component to graphene was obtained by C-B calculation.

(6) Finally, the actual content of graphene in each subcellular fraction is calculated and set as D, that is, D=A-(C-B).

Example 3 Results of subcellular distribution of graphene in rice leaves under different exposure times

(1) Seed germination and graphene exposure: The newly harvested seeds were first soaked in a 30% H ₂ O ₂ solution for 15 min, and then washed three times with deionized water to ensure that the seed surface was sterile. The seeds were then soaked in deionized water and stored at 30°C in the dark for 48h. Finally, the soaked seeds were evenly displayed in a sterilized petri dish, and the bottom of the petri dish was cushioned with two layers of sterilized moist filter paper. The petri dish was covered and placed in a light incubator at 30°C for 7 days. After germination, it was transferred to a container containing 2 L of exposure solution with a graphene concentration of 250 μg/L. At the same time, another group of blank control group was set up, that is, after germination, it was transferred to 2L of graphene-free solution.

(2) After exposure for 7 days, 14 days, and 21 days, respectively, collect the leaf tissue of the rice with graphene exposure combined with blank control, cut it into pieces, and fully grind it after freezing in liquid nitrogen; secondly, after the evaporation of liquid nitrogen is completed, add 1 mL of pre- The cold homogenization medium continued slow milling for 15 s. Then, the homogenate was slowly poured into a 10 mL centrifuge tube, and then the mortar was washed four times with 1 mL of homogenization medium, and the washings were all poured into the centrifuge tube containing the homogenate. The liquid in the centrifuge tube was shaken well, filtered through a layer of nylon mesh, the filter residue was unground tissue, and the filtrate was used for subsequent centrifugation.

(3) First, centrifuge the homogenate of the graphene exposure group at 300g for 30s, and the precipitate is the cell wall, mainly including cell wall and cell wall debris. The filtrate was centrifuged at 1500g for 10min, and the precipitate was the chromosomal part. The supernatant was centrifuged at 5000g for 20min, and the pellet was the nucleus part. The supernatant was centrifuged at 15,000 g for 30 min, the pellet was the mitochondrial fraction, and the supernatant was the soluble fraction.

(4) The subcellular components of the leaf obtained in the step (3) are burned with a biological oxidizer, and the generated carbon dioxide is collected, and then the graphene content in each component is measured by a liquid scintillation counter, and is set as A.

(5) Next, obtain each subcellular fraction from the homogenate in the blank control group according to the method of step (3), and then dropwise add the mother solution containing 1 μg graphene to 5 mL of each subcellular fraction and shake for 3 hours. Then centrifuge according to the differential centrifugation method, and finally measure the graphene content in the homogenate medium, which is set as B. In addition, the mother containing 1 μg of graphene was added dropwise to 5 samples containing only 5 mL of homogenization medium without subcellular components, shaken for 3 h, and then centrifuged according to the differential centrifugation method. Finally, the graphene content in the homogenate medium was measured and set as C. The adsorption amount of each subcellular component to graphene was obtained by C-B calculation.

(6) Finally, the actual content of graphene in each subcellular fraction is calculated and set as D, that is, D=A-(C-B).

According to the established method for measuring the subcellular level distribution of graphene in rice, the sedimentation efficiency of graphene centrifugation with/without organelles and the enrichment efficiency of graphene by organelles were firstly measured. The results are shown in Table 1. As can be seen from Table 1, under different centrifugation conditions, the sedimentation rates of pure graphene suspensions were (86.1±1.2)%, (88.4±0.7)%, (93.7±1.3)% and (95.8±1.2)%, respectively. 1.9)%. In the presence of organelles, the sedimentation rates of graphene under different centrifugation conditions were (90.1±0.5)%, (93.8±1.7)%, (96.1±0.7)% and (99.5±0.9)%, respectively. Under the corresponding centrifugation conditions, the differences between the two were 4.0%, 5.4%, 2.4% and 1.7%, respectively, and the difference was called the calculated adsorption capacity. In addition, in the presence of organelles, after centrifugation, the supernatant was removed, and deionized water was added to resuspend the subcellular fractions (graphene deposited on the bottom would not be suspended). The graphene content of , followed by (3.0±0.3)%, (4.9±0.5)%, (2.5±0.4)% and (1.1±0.2)%, that is, 1.1-4.9% of graphene will be adsorbed on the organelle. By comparing the two data, we found that the experimentally obtained graphene adsorption amount on the organelle was comparable to the calculated adsorption amount. Therefore, this method is feasible for evaluating the subcellular distribution of graphene in stems and leaves.

Based on this method, we determined the graphene content in the subcellular fractions of rice leaves exposed to 250 μg/L graphene, and found that 32.4% of the graphene was in the cell wall and cell membrane, and 43.8% of the graphene was in the chloroplast. , and 17.5% graphene in the nucleus.

Based on this method, we further measured the distribution of graphene in the subcellular subcellular of rice stems and leaves under different exposure times. The results are shown in Figures 2 and 3, respectively.

Figure 2 is a graph showing the changes in the subcellular distribution of graphene in rice stems under different exposure times. The results show that graphene is mainly distributed in the cell wall, followed by the chloroplast, and a small amount of graphene is distributed in the nucleus and mitochondria as well as in the soluble fraction. With the change of exposure time, the content of graphene in each subcellular component also changed. The graphene content in the cell wall was 0.33 μg after 7 days of exposure, and increased to 0.1 μg after 14 days of exposure. The content in the cell wall did not change significantly. For chloroplasts, the graphene content in chloroplasts was 0.04 μg after 7 days of exposure, and with the prolongation of exposure time, the graphene content in chloroplasts continued to decrease. After 21 days of exposure, the graphene content was only 0.01 μg.

Figure 3 is a graph of the subcellular distribution of graphene in rice leaves under different exposure times. The change rule is similar to the subcellular distribution rule of graphene in the stem. It is also mainly distributed in the cell wall, followed by the chloroplast, and changes with time. It is consistent with the change rule of the subcellular distribution of stems. The difference between the two is that the graphene content in each component of the leaf subcellular is less than that in the corresponding component in the stem. For example, the graphene content in the organelles in the leaf tissue is 0.198 μg after 7 days of exposure. , the graphene content was 0.039 μg after 14 days of exposure.

Table 1 Centrifugal sedimentation efficiency of graphene with/without organelles and enrichment efficiency of graphene by organelles

The difference a refers to the sedimentation ratio of graphene in the presence of organelles minus the sedimentation ratio of graphene in the absence of organelles. The difference is called the adsorption amount of graphene on the organelle.

b Deionized water was added to the centrifuge tube to suspend the organelles without suspending the graphene deposited on the substrate. The amount of graphene on the organelles in the suspension was determined as the amount of graphene adsorbed on the organelles.

c The graphene content in each subcellular organelle after exposure for 7 days under the condition of graphene exposure concentration of 250 μg/L.

references

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Claims (8)

1. a method for measuring graphene subcellular distribution in rice body, its steps comprise: (1) Germinate the rice seeds. After the seeds germinate, select seedlings with consistent bud and root system development and transfer them to the graphene-containing mother solution and the graphene-free mother solution for cultivation. The seedlings are blank control groups, and the concentration of graphene mother liquor is 50-250 g/L; (2) Cultivate the above-mentioned rice seedlings for 7-21 days. After the cultivation is completed, first cut them into pieces, freeze them in liquid nitrogen, and then fully grind them; after the evaporation of liquid nitrogen is completed, add pre-cooled homogenization medium to continue grinding, and then mix the homogenate solution Pour it into a centrifuge tube, and then wash the mortar several times with the homogenizing medium. The cleaning solution is all poured into the centrifuge tube containing the homogenate. The liquid in the centrifuge tube is shaken up, filtered, and the filtrate is used for subsequent centrifugation; (3) Separating the homogenate collected in step (2) into five subcellular components by differential centrifugation: cell wall, chloroplast, nucleus, mitochondria and soluble fraction; (4) Detecting the graphene content of the 5 subcellular components of the seedlings cultivated in the graphene solution obtained in step (3); (5) Add 5 mL of the 5 subcellular components of the blank control group obtained in step (3) into the centrifuge tube respectively, then dropwise add the graphene mother solution containing 1 μg of graphene, shake well, and then follow the steps of claim 1 ( 3) centrifugation under the conditions of centrifugation, and finally measure the graphene content in the homogenate medium of the 5 subcellular components; (6) Drop the graphene mother containing 1 μg graphene into 5 parts, each 5 mL of the homogenized medium without subcellular components, shake and shake well, and then proceed according to the centrifugation conditions in step (3) respectively. Centrifuge, finally measure the graphene content in the homogenate medium dripping the graphene mother liquor; (7) Deducting the graphene content obtained in step (5) from the graphene content obtained in step (6) is the content of graphene adsorbed by the organelle; (8) The absolute subcellular content of graphene in the rice body is the graphene content in step (4) minus the graphene content obtained in step (7). 2. the method for measuring graphene subcellular distribution in rice body according to claim 1, is characterized in that: the concentration of graphene mother liquor is 50-250 μg/L, and the solvent of graphene mother liquor is water. 3. the method for measuring graphene subcellular distribution in rice body according to claim 2, is characterized in that: in homogenizing medium, contain 0.25mmol/L sucrose, 50 mmol/L Tris-HCl, and 1mmol/L disulfide Erythritol, the solvent is water, and 1 ml of homogenization medium pre-cooled to 4° C. is added to each 0.2 g of chopped seedlings in step (2) for grinding. 4 . The method for measuring the subcellular distribution of graphene in rice according to claim 3 , wherein in step (2), 1 ml of homogenizing medium is used to clean the mortar for 4 times each time. 5 . 5. The method for measuring the subcellular distribution of graphene in rice according to claim 4, wherein in step (2), a nylon mesh with a pore size of 80 μm is used for filtration. 6. the method for measuring graphene subcellular distribution in rice body according to claim 5, is characterized in that: in step (5), under 4 DEG C of conditions, the mother drop of 1 μg graphene is added to 5 mL blank control in each subcellular fraction of the group. 7. the method for measuring graphene subcellular distribution in rice body according to claim 6, is characterized in that: in step (6), under 4 DEG C of conditions, the mother drop of 1 μg graphene is added to 5 mL homogenate in the medium. 8. The method for measuring the subcellular distribution of graphene in rice body according to any one of claims 1-7, wherein the subcellular distribution state of graphene in the stem or leaf of the seedling is determined respectively.

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