CN112730284A - Method for measuring adenine content and RIP activity - Google Patents
Method for measuring adenine content and RIP activity Download PDFInfo
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- CN112730284A CN112730284A CN202011532874.3A CN202011532874A CN112730284A CN 112730284 A CN112730284 A CN 112730284A CN 202011532874 A CN202011532874 A CN 202011532874A CN 112730284 A CN112730284 A CN 112730284A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/38—Diluting, dispersing or mixing samples
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Abstract
The invention belongs to the technical field of enzyme engineering, and particularly relates to a method for measuring adenine content and RIP activity. Aiming at the defects in the existing RIP activity determination method, the invention provides a developing solution for adenine test, which consists of xanthine oxidase and BCIP. The invention also provides an adenine testing method, which is used for testing the light absorption value after a sample to be tested reacts with xanthine oxidase and BCIP. The invention also provides a RIP activity test method: reacting RIP solution with ATP or NAD+The RIP activity was calculated from the concentration of adenine in the test solution after the mixing reaction. The invention can be used for detecting the dynamic synthesis condition of RIP in a biological sample, discovering the existence of new RIP, detecting and tracking the destination of RIP in the separation and purification process, and the like.
Description
Technical Field
The invention belongs to the technical field of enzyme engineering, and particularly relates to a method for measuring adenine content and RIP activity.
Background
Ribosome-inactivating proteins (RIP) are toxic proteins having N-glycosidase activity that inactivate eukaryotic ribosomes and inhibit protein synthesis, and are widely found in angiosperms, bacteria, fungi, and algae. RIP has a plurality of biological functions of broad-spectrum anti-tumor, anti-virus, immune regulation and the like, has little toxicity to normal cells, and has been widely concerned and researched by global scientists for decades.
A serious problem in the research and development of RIP has existed for a long time-there is no simple and reliable method for determining the activity of RIP. At present, there are roughly the following methods: (ii) measuring inhibition of cell-free system protein biosynthesis by RIP in vitro. The method not only has complex operation, but also relates to radioactive isotopes, and has higher requirements on laboratory conditions; ② rRNA reacts with RIP, the product is treated with acidic aniline, and then denatured RNA electrophoresis is used for detection. This method requires that the vessel and reagent be treated with DEPC to ensure no RNase interference and only qualitative analysis; thirdly, RIP reacts with RNA, and the reaction product is detected to have free adenine content by RP-HPLC method. The method can quantitatively analyze, but only one sample can be analyzed at one time, and is very inconvenient if a large number of samples exist; fourthly, the activity of the RIP is calibrated by measuring the inhibition of the proliferation of the tumor cells in vitro, and the method needs a complicated cell culture process and has long measuring time; fifthly, RIP reacts with rRNA similar substrate, and the generated adenine is converted into a colorimetric product by coupling enzyme. However, this method requires 2 or more coupled enzymes, and is expensive.
Because the existing RIP activity determination technology has the problems of high laboratory condition requirements, no need of guaranteeing RNase interference, inconvenience in testing a large number of samples, long determination time or high cost, and the like, a quantitative, simple, convenient, rapid, low-cost and popularizable RIP activity determination method is urgently needed to be found.
Disclosure of Invention
Aiming at the defects in the existing RIP activity determination method, the invention provides a developing solution for adenine test, a test method and a RIP activity test method comprising the method, and aims to: the RIP activity is quantitatively tested by a simple, rapid and low-cost method.
A color developing solution for adenine test is prepared by dissolving enzyme and BCIP in buffer solution with pH of 6.5-7.5, wherein the enzyme is xanthine oxidase. The BCIP is 2, 6-dichlorophenolindophenol.
Preferably, in the color developing solution, the ratio of the enzyme content to the BCIP dosage is 3.0-4.5U:70-76 mu mol, preferably, 4U: 72 μmol.
Preferably, the method comprises the following steps:
(1) adding the color developing solution into a sample to be detected containing adenine for reaction;
(2) the light absorption at 600-612nm is determined, preferably at 606 nm.
Preferably, in step (1):
in a reaction system per milliliter, the adding amount of the enzyme is 1-1.5U, and the adding amount of the BCIP is 23.33-24.13 mu mol; preferably, the addition amount of the enzyme is 1.33U, and the addition amount of the BCIP is 24 mu mol;
and/or the reaction temperature is 35-39 ℃, the reaction time is 20-40min, the reaction time is 30min, and the reaction pH condition is 6.5-7.5; preferably, the pH is 7.0.
Preferably, the concentration range of the adenine contained in the sample to be detected is less than or equal to 220 MuM;
and/or the calculation formula of the concentration of adenine in the sample to be detected is as follows: Δ a ═ kx (a-a)0) Wherein, Delta A is adenine concentration, k is the change rate of absorbance along with the adenine concentration, A is the absorbance obtained by detection, A is0The absorbance was measured when the concentration of adenine in the solution was 0.
The invention also provides a method for measuring RIP activity, which comprises the following steps:
(a) mixing the RIP solution with a reaction substrate, and reacting to obtain a reaction solution containing adenine;
(b) and (b) taking the reaction liquid in the step (a) as a sample to be tested, and testing according to the measuring method to obtain the light absorption value.
Preferably, in step (a):
the reaction substrate is selected from ATP or NAD+；
And/or, in the reaction system, the concentration of the reaction substrate is 8-15mM, preferably 10 mM;
and/or the reaction temperature is 50-55 ℃, the reaction time is 30-50min, the reaction time is 30min, and the reaction pH condition is 2.0-3.5; preferably, the pH is controlled by a disodium phosphate-citric acid buffer.
Preferably, the RIP activity is calculated by the following formula:
U＝(ΔA/t)×(VtV/Ve) or U/mg ═ Δ A/t × (V)t/Ve)/C，
Wherein U is an activity unit; u/mg is specific activity unit,. DELTA.A is adenine concentration (. mu.M), VtTotal reaction volume (mL), VeRIP volume (mL); t is the reaction time (min) and C is the RIP content (mg).
A method for determining RIP activity in a plant sample comprises determining the RIP activity of the plant sample to be determined according to the method.
Preferably, the method comprises the following steps:
adding liquid nitrogen into a plant sample, grinding, adding a buffer solution with the pH value of 6.5-7.0, extracting at 4-6 ℃ overnight, and separating supernatant to obtain an extracting solution;
(II) measuring the extract obtained in step (I) as a RIP solution by the measuring method according to any one of claims 6 to 8;
preferably, the ratio of the plant sample to the buffer solution is 1 g/2 ml;
preferably, the buffer solution is 0.05-0.1M disodium hydrogen phosphate-sodium dihydrogen phosphate buffer containing 0.9% NaCI;
preferably, the process of separating the supernatant is 10000-;
preferably, the plant sample is selected from at least one of a seed, a seed embryo, a germinated seed, a stem, a root, or a leaf; and/or, the plant species is selected from corn or momordica charantia.
The technical scheme of the invention provides a novel RIP test method, which has the following principle:
ATP or NAD+As a reaction substrate, hydrolysis reaction occurs under the action of RIP to release the product adenine. Adenine and BCIP are catalyzed by XOD (xanthine oxidase) to produce hypoxanthine and reduced BCIP, and hypoxanthine and BCIP are catalyzed by XOD (yellow)Purine oxidase) to uric acid and reduced BCIP. In the above reaction process, BCIP is blue in air, and reduced BCIP obtained after the reaction is colorless, so that the decrease rate of absorbance at 606nm is measured, the content of adenine can be measured, and the RIP activity is reflected.
The determination principle of the method can be represented by the following reaction formula:
the method of the invention has the advantages that: the method is rapid and simple, only has two reaction processes, and can realize analysis automation; ATP or NAD+As a substrate for RIP, it is not interfered by RNase. The requirements on experimental equipment and conditions are low, only a common spectrophotometer is needed, and the method can be easily popularized. Has good application prospect.
Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.
The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.
Drawings
FIG. 1 shows the RIP activity changes of various tissues and organs of corn seeds;
FIG. 2 shows the RIP activity profile in seeds of Momordica charantia;
FIG. 3 is a graph showing the relationship between adenine concentration and developing solution;
FIG. 4 is a graph showing the effect of XOD activity on adenine (220. mu.M) conversion;
FIG. 5 is an analysis of the optimum temperature for α -MMC catalysis of ATP;
FIG. 6 is an analysis of the pH optimum of Ricin A catalyzed ATP.
Detailed Description
The preparation method of the main reagent and the preparation method of the sample in the embodiment of the invention comprise the following steps:
1. preparation of main reagents:
(ii) ATP (20 mM): accurately weighing 48mg ATP-Na2Dissolved in 4mL of pH 3.00.1M acetic acid-sodium acetate buffer.
(ii) buffers with different pH: and (3) storing the disodium hydrogen phosphate-citric acid buffer solution (2.2-8.0) at a low temperature.
2. The preparation method of the alpha-MMC and the Ricin A comprises the following steps:
the alpha-MMC and Ricin A involved in the method can be obtained by purchasing commercial products, and can also be prepared by adopting the methods reported in the prior literature. The embodiment of the invention adopts the following method:
alpha-MMC: crushing fresh and mature bitter gourd seeds by a crusher, sieving the crushed bitter gourd seeds by a 80-mesh sieve, and degreasing the bitter gourd seeds by acetone to obtain acetone dry powder. Adding phosphate buffer (containing 0.15M NaCl and pH 6.650 mM phosphate buffer) with a volume 5-6 times of the powder mass, stirring and extracting, centrifuging at 15000g for 20min to remove precipitate, and collecting supernatant. Adding ammonium sulfate powder into the supernatant to reach 30% saturation, standing, centrifuging at 12000g for 15min, and collecting supernatant. Ammonium sulfate was added to 85% saturation, and the mixture was allowed to stand, centrifuged at 12000g for 15min, and the precipitate was collected and dissolved in a phosphate buffer solution of pH 6.550 mM. The mixture was placed in a dialysis bag and dialyzed against a phosphate buffer solution of pH 6.550 mM. The dialyzed sample was applied to a CM-cellulose 52 column, which was washed thoroughly with the same dialysis buffer and eluted with the same buffer containing 0.12M NaCl, and the protein peak was collected. The sample obtained in the previous step is applied to a Superdex 75 chromatographic column, and eluted by using 0.15M NaCI phosphate buffer solution with pH 6.460mM as fluidity, and a protein main peak is collected.
Ricin A: pulverizing fresh semen Ricini, defatting with precooled acetone, extracting overnight with 0.15M NaCl pH 6.50.05M phosphate buffer, centrifuging at 15000g for 20min, and collecting supernatant. Adding 1M HCI to adjust pH to 3.5, centrifuging at 15000g for 15min, and collecting light yellow supernatant. Ammonium sulfate was added to 30% saturation and the supernatant was collected by centrifugation. Ammonium sulfate was added to the solution to a saturation of 65%. The pellet was collected by centrifugation, solubilized with phosphate buffer pH 6.50.05M and dialyzed against phosphate buffer pH 6.50.05M. The dialysate was applied to a CM-cellulose 52 column and washed with pH 6.50.05M phosphate buffer. The column was replaced with 0.15M NaCI pH 7.50.05M Tris-HCI buffer and passed to a Blue-Sepharose column. Washed with the same buffer, and then eluted with 0.4M NaCI buffer, pH7.50.05M Tris-HCI buffer, the protein peak was collected and concentrated.
The main characteristics of the alpha-MMC are as follows: a single protein staining band is analyzed by SDS-PAGE (under reducing and non-reducing conditions), and the relative molecular weight is 30 kD; acid PAGE analysis shows that the single protein is colored; IEF analysis pI ═ 9.1.
The main characteristics of Ricin a: SDS-PAGE (under non-reducing conditions) analyzes as a single protein coloring band, and the relative molecular weight is 65 kD; SDS-PAGE (under reducing conditions) shows two protein staining bands of 32kD and 34 kD.
Protein content determination in the examples of the invention: the Lowry method or the 280nm ultraviolet absorption method is adopted.
Example 1: the invention provides a color developing liquid
40.3mg of BCIP was weighed out accurately and 10mL, i.e., 10mM, of pH 7.00.1M phosphate buffer was added. When the BCIP is used, enzyme is added and diluted to the concentration required by the BCIP, and the BCIP is stored at low temperature.
Example 2: determination of adenine concentration
The steps of this example are as follows:
1) mixing 0.3mL of solution to be detected containing adenine with 2.7mL of developing solution, and reacting for 30min at 37 ℃;
2) the light absorption value at 606nm is measured to calculate the concentration of adenine by the formula:
ΔA＝k×(A-A0)，
wherein, Delta A is adenine concentration, k is the change rate of absorbance along with the adenine concentration, A is the detected absorbance, A is0The absorbance was measured when the concentration of adenine in the solution was 0.
Example 3: RIP Activity assay
The steps of this example are as follows:
1) reaction process of RIP with substrate: take 0.15mL RIP solution diluted appropriately and 0.15mL ATP or NAD+(20mM) were mixed, incubated at 53 ℃ and pH 3.0 for 30min with ice-bath cooling.
2) The color development reaction process comprises the following steps: 2.7mL of a developing solution was added thereto, and the reaction was carried out at 37 ℃ for 30 min. The absorbance at 606nm was measured to indicate relative activity.
Activity unit calculation formula:
U＝(ΔA/t)×(VtV/Ve) or U/mg ═ Δ A/t × (V)t/Ve)/C，
Wherein U is an activity unit; u/mg is specific activity unit,. DELTA.A is adenine concentration (. mu.M), VtTotal reaction volume (mL), VeRIP volume (mL); t is the reaction time (min) and C is the RIP content (mg).
Under the conditions of this experiment, the formation of 1. mu.M adenine on the substrate per minute is catalyzed as a unit of RIP activity (U).
Example 4: detecting changes of RIP activity of corn seeds and bitter gourd seeds in germination and development processes
The steps of this example are as follows:
1) seed culture and test Material Collection
Fresh corn seeds or bitter gourd seeds are placed into tap water to be soaked overnight, placed into a plastic box paved with disinfected sand grains and germinate in a dark place at room temperature (25-27 ℃). Rinsing with sterilized water every other day, collecting samples according to time points (collecting stem, leaf, root and embryo of semen Maydis; collecting complete germinating tissue of fructus Momordicae Charantiae seed), and storing in liquid nitrogen.
2) Preparation of crude enzyme extract
And (3) putting the sample material in a mortar, and adding liquid nitrogen for rapid grinding. A phosphate buffer solution of pH 6.50.05M was added at a ratio of 1g to 2mL, and the mixture was extracted overnight at 4 ℃. Centrifuging at 15000g for 15min, and collecting supernatant.
3) Analysis of RIP (N-glycosidase) Activity
Mixing the extractive solution 0.15mL and 0.15mL ATP (20mM), incubating at 54 deg.C and pH 3.0 for 30min, and cooling in ice bath. Then, 2.7mL of the color former prepared in example 1 was added and the reaction was carried out at 37 ℃ for 30 min. The absorbance at 606nm was measured to indicate relative activity.
The corn seeds germinate in the dark, and the RIP activity determination results of each part in the seed embryo, the germinated seed, the stem, the root and the leaf are shown in figure 1. As can be seen from FIG. 1, no significant RIP activity was detected in the embryos, shoots and stems for the first 7 days of seed germination. From day 6, no RIP activity was detected in the leaves, but in the roots, the RIP activity appeared, and then increased dramatically until day 12 and remained essentially constant. Bass et al reported that they analyzed the dynamic changes of RIP expression during development and germination of corn seeds by ELISA method, and found that the RIP expression increased rapidly and continued to day 11 from day 9 of germination of corn seeds. The results of this study are substantially consistent with the analysis results of this example.
Bitter gourd seeds germinate in the dark, and the measured RIP activity results are shown in figure 2. As can be seen from the results obtained, a higher RIP activity was detected in the germinated seeds starting at day 1 of the seed culture and continuing until day 9, whereupon the RIP activity dropped sharply and disappeared. It is now clear that there are several RIPs (e.g.MAP 30, α -MMC and β -MMC) in mature seeds of Momordica charantia, which belong to the type I RIP and exist as RNA N-glycosidase activity. During germination and development of seeds, these RIPs may be hydrolyzed by proteases and gradually lose biological activity.
The experimental results show that the method can be used for detecting the dynamic synthesis condition of the RIP in the germination and development processes of plant seeds, detecting whether the RIP exists in biological materials or not and tracking the destination of the RIP in the separation and purification processes.
The test conditions of the present invention are preferably selected by the following experimental examples.
Experimental example 1: the conditions for measurement of adenine concentration are preferably
1) Determination of optimal BCIP concentration
BCIP is prepared into 0-100 mu M by using a phosphate buffer solution with the pH of 7.00.05M, the temperature is kept in water bath at 37 ℃ for 30min, the light absorption value at 606nm is determined, and the BCIP concentration is plotted against the light absorption value.
Experimental results show that BCIP concentration and 606nm light absorption value are in a direct proportion relation. When the concentration of BCIP is more than or equal to 72 mu M, the corresponding light absorption value is more than or equal to 0.8. According to the characteristics of the spectrophotometer such as detection sensitivity, BCIP 72 mu M is determined as the optimal concentration of the reaction system.
2) Chemometric relationship between adenine and developing solution
The adenine stock solution was taken, supplemented with pH7.00.05M phosphate buffer solution to 0.3mL to make the adenine concentrations 0, 32, 64, 96, 128, 160, 200 and 220. mu.M, respectively, and 2.7mL of the color former in example 1 was added. The reaction is carried out in a water bath at 37 ℃ for 30 min. The 606nm light absorption was determined and is shown in FIG. 3 as the concentration of adenine versus light absorption.
FIG. 3 shows that the reaction rate of XOD to convert adenine to hypoxanthine and xanthine (i.e., the rate of conversion of blue DCIP to colorless DCIP) is typically stoichiometrically related to the concentration of adenine. The results show that the system reaction corresponds well to the first order reaction.
3) Determination of optimal XOD concentration
In a 3mL reaction system with pH 7.00.1M phosphate buffer, adenine was immobilized at 220. mu.M and BCIP at 72. mu. mol, XOD (0-4.0U) was varied, and the reaction was carried out at 37 ℃ for 30 min. The absorbance at 606nm was plotted against XOD activity. As can be seen from FIG. 4, when the XOD activity was 2.0U, about 90% of adenine was converted. When the XOD is 4U, adenine is completely transformed.
Experimental example 2: optimum conditions for RIP Activity assay
1) Optimum temperature measurement of RIP
0.15mL of α -MMC solution was mixed with 0.15mL of ATP (20mM) and reacted at pH3 (pH optimum) and different temperatures (30, 40, 45, 50, 55, 60 and 65) for 30 min. 2.7mL of the developing solution obtained in example 1 was immediately added, and the mixture was incubated at 37 ℃ for 30 minutes. A blank sample (alpha-MMC is inactivated by heating, and other processes are the same) is used as a control, and the light absorption value at 606nm of each sample is measured. The temperature was plotted against the relative activity (%). The experimental results obtained from FIG. 5 show that the optimum reaction temperature for converting ATP to adenine by α -MMC is 55 ℃.
2) Determination of optimum pH
0.15mL of Ricin A was incubated with 0.15mL of ATP (20mM) for 30min at different pH (disodium hydrogen phosphate-citric acid buffer) and optimum temperature (55 ℃). 2.7mL of the developing solution obtained in example 1 was added, and the mixture was incubated in a water bath at 37 ℃ for 30 minutes. The absorbance at 606nm of each tube was determined by comparison with a blank sample (Ricin A heat-inactivated, otherwise identical). The pH is plotted against the relative activity (%). From FIG. 6, it is clear that the optimum pH for catalyzing ATP hydrolysis by Ricin A is ≦ 3.
As can be seen from examples 1-4 and experimental examples 1-2, the adenine concentration test method and the RIP activity test method provided by the invention have the advantages of simple steps, short test time, low requirements on experimental instruments and conditions and test cost. And accurate measurement can be realized by optimizing the test conditions. The method has wide application prospect, and can be used for detecting the dynamic synthesis condition of the RIP in the biological sample, detecting whether the RIP exists in the biological sample, detecting and tracking the destination of the RIP in the separation and purification process, and the like.
Claims (10)
1. A color developing solution for adenine testing is characterized in that: it is prepared by dissolving enzyme and BCIP in buffer solution with pH of 6.5-7.5, wherein the enzyme is xanthine oxidase.
2. Color developing solution according to claim 1, characterized in that: in the color developing solution, the ratio of the enzyme content to the BCIP dosage is 3.0-4.5U:70-76 mu mol, preferably, 4U: 72 μmol.
3. An indirect determination method of adenine content is characterized by comprising the following steps:
(1) adding the color developing solution of claim 1 or 2 into a sample to be tested containing adenine, and reacting;
(2) the light absorption at 600-612nm is determined, preferably at 606 nm.
4. An assay method according to claim 3, wherein: in the step (1):
in a reaction system per milliliter, the adding amount of the enzyme is 1-1.5U, and the adding amount of the BCIP is 23.33-24.13 mu mol; preferably, the addition amount of the enzyme is 1.33U, and the addition amount of the BCIP is 24 mu mol;
and/or the reaction temperature is 35-39 ℃, the reaction time is 20-40min, the reaction time is 30min, and the reaction pH condition is 6.5-7.5; preferably, the pH is 7.0.
5. An assay method according to claim 3, wherein: the concentration of adenine contained in the sample to be detected is less than or equal to 220 mu M;
and/or the calculation formula of the concentration of adenine in the sample to be detected is as follows: Δ a ═ kx (a-a)0) Wherein, Delta A is adenine concentration, k is the change rate of absorbance along with the adenine concentration, A is the absorbance obtained by detection, A is0The absorbance was measured when the concentration of adenine in the solution was 0.
6. A method for determining RIP activity, comprising the steps of:
(a) mixing the RIP solution with a reaction substrate, and reacting to obtain a reaction solution containing adenine;
(b) taking the reaction solution in the step (a) as a sample to be tested, and testing according to the determination method of any one of claims 3-5 to obtain the light absorption value.
7. An assay method according to claim 6, wherein: in step (a):
the reaction substrate is selected from ATP or NAD+；
And/or, in the reaction system, the concentration of the reaction substrate is 8-15mM, preferably 10 mM;
and/or the reaction temperature is 50-55 ℃, the reaction time is 30-50min, the reaction time is 30min, and the reaction pH condition is 2.0-3.5; preferably, the pH is controlled by a disodium phosphate-citric acid buffer.
8. An assay method according to claim 6, wherein: the RIP activity is calculated by the following formula:
U＝(ΔA/t)×(VtV/Ve) or U/mg ═ Δ A/t × (V)t/Ve)/C，
Wherein U is an activity unit; u/mg is specific activity unit,. DELTA.A is adenine concentrationDegree (. mu.M), VtTotal reaction volume (mL), VeRIP volume (mL); t is the reaction time (min) and C is the RIP content (mg).
9. A method of determining RIP activity in a plant sample, comprising: the method of any one of claims 6 to 8 is used to determine the RIP activity of a plant sample to be tested.
10. An assay method according to claim 9, comprising the steps of:
adding liquid nitrogen into a plant sample, grinding, adding a buffer solution with the pH value of 6.5-7.0, extracting at 4-6 ℃ overnight, and separating supernatant to obtain an extracting solution;
(II) measuring the extract obtained in step (I) as a RIP solution by the measuring method according to any one of claims 6 to 8;
preferably, the ratio of the plant sample to the buffer solution is 1 g/2 ml;
preferably, the buffer solution is 0.05-0.1M disodium hydrogen phosphate-sodium dihydrogen phosphate buffer containing 0.9% NaCI;
preferably, the process of separating the supernatant is 10000-;
preferably, the plant sample is selected from at least one of a seed, a seed embryo, a germinated seed, a stem, a root, or a leaf; and/or, the plant species is selected from corn or momordica charantia.
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