Determination of the biological activity for plant hormone-related secondary metabolites—Cytological method
Some standard content:
ICS07.080
National Standard of the People's Republic of China
GB/T38484—2020
Determination of the biological activity for plant hormone-related secondarymetabolitesCytologicalmethod2020-11-19Issued
State Administration for Market Regulation
National Administration of Standardization
Issued
Implementation on 2020-11-19
This standard was drafted in accordance with the rules given in GB/T1.1-2009. This standard was proposed and managed by the China National Institute of Standardization. GB/T38484—2020
The drafting units of this standard are: China University of Metrology, China National Institute of Standardization, Zhejiang Provincial Institute of Inspection and Quarantine Science and Technology. The main drafters of this standard are: Zhang Yafen, Ye Zihong, Ma Aijin, Yu Xiaoping, Huang Chaoqun, Cui Haifeng, Xu Yipeng, Shentu Xuping, Li Yi, Chen Li, Wu Juan.
1 Scope
Cytological evaluation method for the determination of biological activity of secondary metabolites of plant hormones
This standard specifies the method for determining the biological activity of secondary metabolites of plant hormones by cytological evaluation method. GB/T38484—2020
This standard is applicable to the activity determination of secondary metabolites of plant hormones, such as auxin, cytokinin and gibberellin. Normative references
The following documents are indispensable for the application of this document. For all dated references, only the dated version applies to this document. For any undated referenced document, the latest version (including all amendments) shall apply to this document. GB/T6682 Specifications and test methods for water for analytical laboratories 3
Terms and definitions, abbreviations
3.1 Terms and definitions
The following terms and definitions apply to this document. 3.1.1
Plant hormone-related secondary metabolites Plant hormone-related secondary metabolites are active substances synthesized by plants themselves and obtained through microbial fermentation or chemical synthesis that have the ability to regulate plant growth, development and dormancy.
3.1.2
Biological activity
biology activity
The relative value of the ability of a unit concentration of a plant hormone-related secondary metabolite to promote or inhibit plant growth, development and dormancy with its corresponding standard.
2 Abbreviations
The following abbreviations apply to this document.
4-MU: 4-methylumbelliferon4-MUG: 4-methylumbelliferyl-β-D-glucuronideAuxRE::GUS: auxin-responsive gus reporter geneCTKRE::GUS: cytokinin-responsive gus reporter geneDPBS: Dulbecco's Phosphate Buffered Saline fferedSaline）GA，：gibberellic acid（gibberellic acid）GARE：：GUS：gibberellin-responsive gus reporter gene（gibberellin-responsive gus reporter gene）GUS：β-glucuronidase（β-glucuronidase）NAA：1-naphthylacetic acid（1-naphthylacetic acid）ZT：zeatin（zeatin）
GB/T38484—2020
4 Principle
Plant hormone active substances can regulate the binding of related proteins to hormone response elements in the promoter of their target genes by binding to the corresponding hormone receptors, thereby inducing or inhibiting the expression of target genes. Transgenic Arabidopsis protoplast cells carrying hormone-responsive gus reporter genes were selected as test materials, and the standard substance was used as a reference. The standard curve was obtained according to the linear regression relationship between GUS enzyme activity and the logarithm of the standard substance concentration. The ratio of the standard substance concentration to the sample concentration with the same GUS enzyme activity induced in the linear range was calculated, which represents the biological activity of the plant hormone secondary metabolites possessed by the sample at a unit concentration (mg/mL). GUS protein can hydrolyze 4-MUG to 4-MU, and the amount of 4-MU produced by hydrolyzing 4-MUG per microgram of GUS protein per minute under 37°C water bath conditions is used to indicate GUS enzyme activity.
5 Reagents or materials
Unless otherwise specified, only analytically pure reagents should be used. 5.1 Water. GB/T6682-grade.
5.2 AuxRE::GUS cell line. Arabidopsis thaliana Columbia (Col-0) ecotype, containing the gibberellin response element AuxREs, carrying a single copy of the gus gene, and the number of viable cells ≥1×10°/mL. 5.3 GARE::GUS cell line. Arabidopsis thaliana Columbia (Col-0) ecotype, containing the gibberellin response element TATC-boxs, carrying a single copy of the gus gene, and the number of viable cells ≥1×1o°/mL. 5.4CTKRE::GUS cell line. Arabidopsis thaliana Columbia (Col-0) ecotype, containing cytokinin response element ARR1AT, carrying a single copy of gus gene, viable cell count ≥1×10°/mL. 5.5 Cell line culture medium. Dissolve 4.74g of MS medium without sucrose and agar in 950mL of water, then add 30.00g sucrose, 0.75g calcium chloride, and 0.35g potassium nitrate. Mix thoroughly and adjust the pH to 5.60 with sodium hydroxide, add water to 1000mL, and filter with a 0.22um filter to sterilize. Prepare and use immediately.
5.6DPBS, pH7.4. Weigh 8.00g sodium chloride, 0.20g potassium chloride, 2.90g disodium hydrogen phosphate dodecahydrate, 0.24g potassium dihydrogen phosphate, add water to 1000mL, mix well and store at room temperature for later use. Similar commercial products can be used. 5.7 Extract. Weigh 3.72g disodium ethylenediaminetetraacetic acid dihydrate, add 700μL β-mercaptoethanol, 1mL polyethylene glycol octylphenyl ether, dissolve in DPBS and make up to 1000mL, mix well and store at room temperature for later use. 5.8 Extract containing 1mmol/L4-MUG. Weigh 105.6mg4-MUG, dissolve in extract and make up to 300mL, prepare and use immediately.
5.90.2mg/mL bovine serum albumin solution. Weigh 20.0mg bovine serum albumin standard, dissolve in DPBS and make up to 100mL, prepare and use immediately.
5.10 Reaction termination solution. Weigh 21.20g Na2COs, dissolve it in water and dilute to 1000mL. 5.11 Coomassie Brilliant Blue staining solution. Weigh 0.5g Coomassie Brilliant Blue G-250, dissolve it in 50mL 90% ethanol, add 100mL DPBS, dilute to 1000mL with water, mix well and store at room temperature. Valid for one month. 5.12 10μmol/L 4-MU mother solution. Weigh 17.6mg 4-MU, dissolve it in the reaction termination solution and dilute to 10mL, mix well to obtain 10mmol/L 4-MU solution. Take 1mL 10mmol/L 4-MU solution and add 9mL reaction termination solution, mix well to obtain 1mmol/L 4-MU solution. Take 100μL 1mmol/L 4-MU solution and add 9.9mL reaction termination solution and mix well to obtain 10μmol/L 4-MU solution. Temporarily store at 4℃ away from light.
5.134-MU working solution. Pipette 200μL of 10μmol/L 4-MU mother solution, add it to 9.8mL of reaction termination solution, mix well to obtain 200nmol/L 4-MU working solution. Then pipette 1mL of 200nmol/L 4-MU, add 1mL of reaction termination solution, mix well to obtain 100nmol/L 4-MU working solution, and dilute it twice in sequence to obtain 50nmol/L, 25nmol/L, 12.5nmol/L, and 6.25nmol/L 4-MU working solutions.
5.14NAA. Purity ≥98%.
5.15GA. Purity ≥98%.
5.16ZT. Purity ≥98%.
GB/T38484—2020
5.171mg/mLNAA standard stock solution. Weigh 10.0mgNAA standard, dissolve it with 0.5mL anhydrous ethanol, dilute to 10mL with water, mix thoroughly, refrigerate at 4℃, and store for 1 month. 5.181mg/mLGA: standard stock solution. Weigh 10.0mgGA: standard, dissolve it with 0.5mL anhydrous ethanol, dilute to 10mL with water, mix thoroughly, refrigerate at 4℃, and store for 1 month. 5.191mg/mLZT standard stock solution. Weigh 10.0mg ZT standard, dissolve it with 0.5mL anhydrous ethanol, dilute to 10mL with water, mix thoroughly, and refrigerate at 4℃ for 1 month. 5.20 NAA standard working solution. Pipette 200μL 1mg/mL NAA standard preparation solution, 800μL cell line culture medium, mix well to obtain 2×10-1mg/mL NAA solution. Dilute 10 times in sequence to obtain 2.00×10-4mg/mL 2.00×10-5mg/mL, 2.00X10-mg/ml NAA standard working solution. Pipette 632μL 1mg/mL NAA standard stock solution, 368μL cell line culture medium, mix well to obtain 6.32×10-1mg/mL NAA solution. Dilute 10 times in sequence to obtain 6.32×10-5mg/mL, 6.32×10-6mg/mL NAA standard working solution. Prepare before use.
5.21GA: standard working solution. Pipette 200μL1mg/mLGA: standard stock solution, 800μL cell line culture medium, mix well, and get 2×10-1mg/mLGA, solution. Dilute 10 times in sequence to get 2.00×10-*mg/mL, 2.00×10-5mg/mL, 2.00×10-6mg/mLGA: standard working solution. Pipette 632μL1mg/mLGA: standard stock solution, 368μL cell line culture medium, mix well, and get 6.32×10-1mg/mLGA: solution. Dilute 10 times in sequence to get 6.32×10-5mg/mL, 6.32×10-mg/mLGA, standard working solution. Prepare before use.
5.22ZT standard working solution. Pipette 200μL 1mg/mL ZT standard stock solution, 800μL cell line culture medium, mix well, and get 2×10-1mg/mL ZT solution. Dilute 10 times in sequence to get 2.00×10-3mg/mL, 2.00×10-4mg/mL, 2.00×10-5mg/mL ZT standard working solution. Pipette 632μL 1mg/mL ZT standard stock solution, 368μL cell line culture medium, mix well, and get 6.32×10-1mg/ml ZT solution. Dilute 10 times in sequence to get 6.32×10-4mg/mL, 6.32×10-5mg/mL ZT standard working solution. Prepare before use. 6 Instruments and Equipment
pH meter: accuracy 0.01.
6.11
Electronic analytical balance: accuracy 0.1mg, 0.01g, 1g. 6.3 Illumination incubator: 25℃±1℃.
6.4 Constant temperature water bath: 37℃.
Cell culture plate,
6.6 Hundred
Enzyme labeler: can detect absorbance and fluorescence intensity at the same time. 6.7
Enzyme labeling plate: black.
7 Determination steps
7.1 Sample preparation
According to the mass conversion of the effective substance (or substance to be tested) of the test sample, weigh a sufficient amount of solid sample or measure a sufficient amount of liquid sample, select appropriate reagents or water to dissolve and prepare a solution of the effective substance (or substance to be tested) as the test sample according to the product instructions or the characteristics of the substance to be tested, and the concentration is recorded as Po (mg/mL). When testing, use the corresponding buffer to dilute the test sample 5 times or 10 times in a gradient, and the concentration after dilution is recorded as, (mg/mL). Prepare at least 3 concentration gradients for the sample, and measure each concentration sample at least three times. 3
GB/T38484—2020
7.2 Determination of GUS protein induced expression level
7.2.1 Sample treatment to induce GUS protein expression and sample preparation Prepare more than 3 cell culture plates, each plate is a replicate, number each grid, select the corresponding cell line and standard according to the test object, add 1mL of AuxREs::GUS cell line to the culture well of the cell culture plate, and then add 1mL of cell line culture solution, NAA standard working solution of different concentrations and sample solution of different concentrations to each well, mark them well, and culture them in a 25℃ light incubator for 6h to induce GUS expression. Then transfer the treated cell line to a 10mL centrifuge tube, add 1mL of extraction solution to mix, and lyse on ice for 20min. Centrifuge at 4℃14000g for 30min and aspirate the supernatant as the sample solution. 7.2.2 Determination of total protein concentration in samples
Pipette 0μL, 2.0μL, 4.0μL, 6.0μL, 8.0μL, 12.0μL, 16.0μL, and 20.0μL of standard bovine serum albumin solution into the sample wells of the ELISA plate, and add DPBS solution to make up to 20.0uL (the bovine serum albumin content in each well is 0μg, 0.40μg, 0.80ug, 0.12μg, 0.16ug, 2.40ug, 3.20μg, and 4.00ug, respectively) ; According to the protein content in the sample, take an appropriate amount of sample (volume V.) and add it to the sample well of the ELISA plate, and add DPBS solution to make up to 20.0μL; each standard or sample solution is added to 3 sample wells repeatedly, and the DPBS solution is used as a blank control to adjust to zero, and then 200μL of Coomassie Brilliant Blue dye solution is added to mix with the standard or sample solution, and the absorbance is measured at a wavelength of 595nm. The standard curve is drawn with the bovine serum protein content as the horizontal axis and the absorbance as the vertical axis, and the total protein concentration P in the sample is calculated according to formula (1).
Pa=V
Where:
Pa——total protein concentration of the sample, in micrograms per microliter (μg/uL); m—protein content of the sample obtained from the standard curve, in micrograms (μug); V,——volume of the sample added when measuring protein content, in microliters (uL). The calculation result is retained to four decimal places. 7.2.3 Determination of GUS protease activity in samples
(1)
Take an appropriate amount of sample solution (to make the total protein content between 20ug and 200μg), record the volume V2, add 1mL of extract containing 1mmol/L 4-MUG, and place in a 37℃ water bath. Take out 200μL of the reaction solution at 5min, 25min, 45min, 65min and 85min in the water bath, add 800μL of the reaction stop solution to mix, and then pipette 200μL into the sample wells of the ELISA plate. Pipet 200μL of 4-MU working solution (4-MU content is 200nmol, 100nmol, 50nmol, 25nmol, 12.5nmol, 6.25nmol respectively) and add it into the sample wells of the ELISA plate. Note that each sample detection ELISA plate must be loaded with a standard. Each standard or sample solution is added to three sample wells repeatedly. The reaction stop solution is used as a blank control to adjust the zero. The fluorescence intensity is measured under the conditions of an excitation wavelength of 365 nm and an absorption wavelength of 455 nm. A standard curve is drawn with the concentration of the 4-MU standard as the horizontal axis and the fluorescence intensity as the vertical axis. The fluorescence intensity after the sample reaction is converted into the amount of 4-MU substance actually produced after the sample reaction through the standard curve. Finally, a standard curve is drawn with the sample reaction time as the horizontal axis and the amount of 4-MU substance produced after the sample reaction as the vertical axis. The amount of 4-MU produced by the hydrolysis of 4-MUG by the GUS protein per minute is calculated, and the GUS enzyme activity y in the sample is calculated according to formula (2).
Where:
y=pax
GUS enzyme activity in the sample, in pmol/(min·μg)]; (2)
n——the amount of 4-MU produced by the hydrolysis of 4-MUG by GUS protein in the sample per minute, in pmol/min; Pyi——the total protein concentration in the sample, in micrograms per microliter (ug/uL); V2——the volume of the sample added when measuring GUS enzyme activity, in microliter (μL). 4
The calculation result is retained to three decimal places. 3 Standard curve drawing
GB/T38484—2020
The induced expression level of GUS protein after treatment with the standard working solution is determined as described in 7.2, and the data is recorded in accordance with Appendix A. The logarithm value x of the concentration of the standard working solution is taken as the independent variable with a base of 10, and the GUS enzyme activity y is taken as the dependent variable to draw the standard curve. 7.4 Measurement
Press 7.2 Determine the induction expression level of GUS protein after treatment with the sample solution, and record the data with reference to Appendix A. The test with y value within the linear range of the standard curve is considered as a valid test, and the sample concentration β is recorded. The data obtained from the invalid test should be discarded. Then, according to the valid y value, the biological activity of the secondary metabolites of plant hormones in the sample is calculated according to formula (3). If there are multiple valid y values, the average value is calculated according to formula (3). E_2p×10
Where:
Biological activity of secondary metabolites of plant hormones; Concentration of the sample to be tested, in milligrams per milliliter (mg/mL); p
o——Concentration of the sample to be tested after dilution, in grams per milliliter (mg/mL); Logarithm of the concentration of the standard working solution.
（3）
The average value of the results of more than three independent experiments is used as the biological activity value of the secondary metabolites of plant hormones contained in the sample, and the calculation result is retained to three significant figures.
Repeatability
The absolute value difference of three independent determination results obtained under repeatability conditions shall not exceed 20% of the arithmetic mean. 5
GB/T38484—2020
A.1 The data records of the test of bovine growth hormone activity are shown in Table A.1. Table A.1
Control group
Test index
Cell line culture medium
Actual concentration of test solution
Protein concentration standard curve
ODs95
Total protein
Concentration blue
Enzyme activity y
Standard curve
Ea uxi
Appendix A
(Informative Appendix)
Test data record sheet
Auxin activity test data record sheet
NAA standard working solution
mg/mL
2.00×10-4
1.00×10-4
6.32×10-5
3.16×10-5
Cytokinin activity test data records are recorded in Table A.2. A.2
Table A.2
Control group
Detection index
Cell line culture medium
Actual concentration of test solution
Protein concentration standard curve
ODs9s
2.00×10
1.00×10
6.32×10-6
3.16×10- 6
Cytokinin activity detection data record table ZT standard working solution
mg/mL
2.00×10-3
1.00×10-3
6.32×10-4
3.16×10-4
2.00×10
1.00×10-4
6.32× 10-5
3.16×10-5
2.00×10-0
1.00×10-6
2.00×10-5
1.00×10-5
Sample solution
mg/mL
0./2
p./2
Sample solution
mg /mL
01/2
pu/2
Detection indicators
Total protein
Concentration pn
Enzyme activity y
Standard curve
Ecrk
Control group
Cell line culture medium
2.00×10
3The data records of gibberellin activity detection are shown in Table A.3. Table A.2 (continued)
ZT standard working solution
mg/mL
6.32×10-
2.00×10-4
6.32×10-
3 Gibberellin activity test data record table
Table A.3
Control group
Test index
Cell line culture medium
Actual concentration of test solution
Protein concentration standard curve
ODs9s
Total protein
Concentration e
Enzyme activity y
Standard curve
2.00×10-
1. 00×10
GA: Standard working solution
mg/mL
6.32×10-5
3.16×10
2.00×10-5
1.00×10
6.32×10-
3.16×10
GB/T38484—2020
Sample solution
mg/mL
2.00×10-5
2.00×10-6
Sample solution
mg/mL
1.00×10-6
0./2/200×10
3 Gibberellin activity test data record see Table A.3. Table A.2 (continued)
ZT standard working solution
mg/mL
6.32×10-
2.00×10-4
6.32×10-
3 Gibberellin activity test data record table
Table A.3
Control group
Test index
Cell line culture medium
Test solution actual concentration
Protein concentration standard curve
ODs9s
Total protein
Concentration e
Enzyme activity y
Standard curve
2.00×10-
1. 00×10
GA: Standard working solution
mg/mL
6.32×10-5
3.16×10
2.00×10-5
1.00×10
6.32×10-
3.16×10
GB/T38484—2020
Sample solution
mg/mL
2.00×10-5
2.00×10-6
Sample solution
mg/mL
1.00×10-6
0./2/200×10
3 Gibberellin activity test data record see Table A.3. Table A.2 (continued)
ZT standard working solution
mg/mL
6.32×10-
2.00×10-4
6.32×10-
3 Gibberellin activity test data record table
Table A.3
Control group
Test indexbzxZ.net
Cell line culture medium
Test solution actual concentration
Protein concentration standard curve
ODs9s
Total protein
Concentration e
Enzyme activity y
Standard curve
2.00×10-
1. 00×10
GA: Standard working solution
mg/mL
6.32×10-5
3.16×10
2.00×10-5
1.00×10
6.32×10-
3.16×10
GB/T38484—2020
Sample solution
mg/mL
2.00×10-5
2.00×10-6
Sample solution
mg/mL
1.00×10-6
0./2/2
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