AU2021105892A4 - Method for Determining Endogenous Hormones in Wheat - Google Patents

Abstract

The invention discloses a method for determining endogenous hormones in wheat, and belongs to the technical field of determination of plant endogenous hormones. The method for determining the endogenous hormones in wheat includes: leaching and purifying wheat leaf powder with an alcohol solution to obtain the test solution of the endogenous hormones of the wheat, and determining the content of indole-3-acetic acid, gibberellin, zeatin, kinetin, 6 benzylaminopurine and abscisic acid in the test solution of the endogenous hormones of the wheat by high performance liquid chromatography. The present invention optimizes the conditions for the determination of 6 common endogenous hormones in the leaves of wheat seedlings by high performance liquid chromatography, and the extraction and determination method of the present invention is simple in operation, high in repeatability, and stable in recovery. It is useful for analyzing the content of wheat hormones and of great significance to study the growth and development of wheat. 3W ~6-KT 12.743 300- ABA22.604 Z5 10 02567510 12. 1 17;62 2.6 rni Figure1I 4 ABA 22.453 2- G31.9 0 2, 5 7 1 12b 15 17Z 20 22.5 mnI Figure 2

Description

3W ~6-KT 12.743 300- ABA22.604 Z5

10

02567510 12. 1 17;62 2.6 rni

Figure1I

4 ABA 22.453 2- G31.9

0 2, 5 7 1 12b 15 17Z 20 22.5 mnI

Figure 2

Method for Determining Endogenous Hormones in Wheat

TECHNICAL FIELD

The invention relates to the technical field of determination of plant endogenous hormones,

in particular to a method for determining endogenous hormones in wheat.

BACKGROUND

Hormones are organic substances produced by the metabolism of plants themselves, and

move from the production site to the action site, and have obvious physiological effects at

very low concentrations. Plant gene expression, growth and development, and plant

response to certain environmental stimuli are all regulated by a variety of hormones in their

bodies. Hormones are organic compounds that naturally exist in plants. Although their

content is very low, they regulate the entire process of plant life activities. Therefore, they

are closely related to the basic laws of plant growth and development and the regulation of

metabolism. The research of plant endogenous hormones is one of the important contents

in the field of plant physiology. The accurate determination of plant endogenous hormones

content in plants is of great significance to the fields of plant life activities, crop genetic

breeding, and cultivation.

At present, the awareness of the usage of hormones in the process of wheat cultivation and

breeding has been increasing. However, due to the lack of scientific guidance, the current

situation of hormone use is rather chaotic. In order to achieve the purpose of improving

wheat yield and quality, a large number of exogenous hormones are used blindly, while

endogenous hormones are ignored. The content level leads to the imbalance of the hormone

content in the wheat body, which not only does not have a beneficial effect, but even problems such as affecting the grain filling of the wheat may occur. If you want to understand the content of relevant hormones needed in the process of wheat growth and development, only by detecting the true state of the hormones in wheat leaves and other organs, and adjusting the required hormones according to the test results, can the yield and quality of wheat be improved.

The commonly used methods for the determination of hormones include high performance

liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS), gas

chromatography-mass spectrometry (GC-MS) and enzyme-linked immunoassay (ELISA).

Since the hormone itself is not immunogenic, the ELISA method requires a coupling

reaction, which is troublesome and may cause immune cross-reactions. GC-MS and

monoclonal antibody-based immunoassays are both new technologies for modem

hormones detection. In GC-MS, selective ion detection technology is more rapid and

effective. It not only has high sensitivity but also can accurately identify the molecular

structure of hormones; however, these methods require lengthy sample purification

procedures, expensive equipment, and high cost of use and maintenance. At present, ultra

high performance liquid chromatography (UPLC) has been reported to analyze the content

of plant endogenous hormones. However, due to the fact that abscisic acid (ABA) is

sensitive to light, and the content of zeatin (ZT) in leaves, especially senescent leaves, is

extremely low, it is still not very effective, UPLC for simultaneous analysis of 6 kinds of

hormones in wheat including gibberellin (GA3), ZT, indole-3-acetic acid (IAA), kinetin

(6-KT), 6-benzylaminopurine (6-BA) and ABA can not achieve good effect.

SUMMARY

The purpose of the present invention is to provide a method for the determination of

endogenous hormones in wheat to solve the above-mentioned problems in the current

technology. Through separation, extraction, and screening of chromatographic conditions,

a method for simultaneous determination of 6 kinds of endogenous hormones including

IAA, GA3, ZT, 6-KT, 6-BA and ABA in wheat leaves has been found. The method is of

great significance for analyzing the hormone content in wheat and studying the growth and

development of wheat. In order to achieve the objectives above, the present invention

provides the following solutions:

(1) leaching wheat leaf powder with an alcohol solution to obtain a endogenous hormone

extract of wheat;

(2) purifying the wheat endogenous hormone extract to obtain test solution of endogenous

hormone of wheat;

(3) determining the content of IAA, GA3, ZT, 6-KT, 6-BA and ABA in the test solution of

the endogenous hormones of the wheat by HPLC.

Further, the wheat leaf powder is obtained by grinding the second extended leaves of wheat

seedlings; the method for cultivating the wheat seedling specifically includes: under the

conditions of light intensity of 120 pmol-m-2 -s- and temperature of 20°C, hydroponic

culture to 2 leaves and 1 heart stage.

Furthermore, the step (1) specifically comprises: adding an alcohol solution to the wheat

leaves powder, leaching for 10-15 hours at 4°C; obtaining the supernatant liquid and the

residue by centrifugation, and then washing residue with an alcohol solution, and combining the washing liquid into the supernatant liquid and obtaining the wheat endogenous hormone extract by concentration.

Further, the alcohol solution is a methanol solution pre-cooled at 4°C; the centrifugation is

specifically 6000 g / min for 15 minutes; and the concentration factor is 2 to 5 times; the

ratio of wheat-to-alcohol solution is 1 g : 10-20 mL.

Furthermore, the volume fraction of methanol in the methanol solution is 80%.

Furthermore, the step (2) specifically comprises: extracting the extract of endogenous

hormones of wheat with petroleum ether, retaining the water phase, and then adding

polyvinylpyrrolidone for ultrasonic dissolution for 30 minutes, and then adjusting pH to

3.0, extracting with ethyl acetate to retain the ethyl acetate phase, drying with nitrogen, and

adding mobile phase to dissolve the residue and then filtrating to obtain wheat endogenous

hormone test solution.

Furthermore, 0.45 m microporous membrane is used for filtration.

Further, the mobile phase is a mixed solution of methanol: water in a volume ratio of 1:1,

Furthermore, the water contains glacial acetic acid with a mass fraction of 0.07%.

Furthermore, the method for determining endogenous hormones in wheat according to

claim 1, wherein the chromatographic conditions of the HPLC method specifically include:

chromatographic column: C18; flow rate: 0.5 mL/min; column temperature: 30°C;

injection volume: 10 pL; wavelength: 254 nm; qualitative retention time; integrating to

obtain the chromatographic peak area.

Furthermore, the HPLC method for determining the content of IAA, GA3, ZT, 6-KT, 6

BA and ABA in the test solution of endogenous hormones in wheat specifically includes:

(1) Prepare the standard solution and establish the regression equation:

Prepare the standard solution: prepare the mother liquors of IAA, GA3, ZT, 6-KT, 6-BA

and ABA at a concentration of 1000 g/mL respectively; take 5 mL of each of the 6 kinds

of mother liquors and dilute to 50 mL to obtain a mixed standard solution of endogenous

hormones with a concentration of 100 [g/mL; dilute the mixed standard solution to obtain

standard solution with concentrations of 1 g/mL, 10 [g/mL, 25 g/mL and 50 [g/mL.

Establish a regression equation: determine the standard solutions of different

concentrations respectively; take the peak area obtained after determination as the ordinate,

and the concentration of the standard solution as the abscissa to draw a standard curve.

(2) Detection of endogenous hormones test solution of wheat: determine the peak area of

endogenous hormones in wheat, and calculate the content of endogenous hormones

based on the peak area and the standard curve.

The present invention discloses the following technical effects: The present invention

optimizes the HPLC method for determining the content of endogenous hormones in

wheat. The RSD and the recovery rate of standard addition of this method meet the

conditions of quantitative analysis of hormones, and the content of6 endogenous hormones

in wheat leaves can be simultaneously determined. The extraction and determination

method adopted in the present invention has simple operation, high repeatability and stable

recovery rate. It is of great significance for analysing the hormone content in wheat and

studying the growth and development of wheat.

BRIEF DESCRIPTION OF THE FIGURES

In order to more clearly illustrate the technical solutions in the embodiments of the present

invention or the prior art, the following will briefly introduce the drawings that need to be

used in the embodiments. Obviously, the drawings in the following description are merely some of the embodiments of the invention the present invention. For those of ordinary skill in the field, other drawings may be obtained based on these drawings without creative work.

Figure 1 is the chromatogram of the standard sample of 6 endogenous hormones in the

present invention;

Figure 2 is the chromatogram of the 6 endogenous hormones in the test solution of

endogenous hormones of wheat in Example 1 of the present invention.

DESCRIPTION OF THE INVENTION

Various exemplary embodiments of the present invention will now be described in detail.

The detailed description should not be considered as a limitation to the present invention,

but should be understood as a more detailed description of certain aspects, characteristics,

and embodiments of the present invention.

It should be understood that the terms described in the present invention are only used to

describe specific embodiments and are not used to limit the present invention. In addition,

for the numerical range in the present invention, it should be understood that each

intermediate value between the upper limit and the lower limit of the range is also

specifically disclosed. Each smaller range between any stated value or intermediate value

within the stated range and any other stated value or intermediate value within the stated

range is also included in the present invention. The upper and lower limits of these smaller

ranges can be independently included or excluded from the range.

Unless otherwise specified, all technical and scientific terms used herein have the same

meaning as commonly understood by those skilled in the field of the present invention.

Although the present invention only describes preferred methods and materials, any

methods and materials similar or equivalent to those described herein can also be used in

the practice or testing of the present invention. All documents mentioned in this

specification are incorporated in the form of reference to disclose and describe methods

and/or materials related to the documents. In the event of conflict with any incorporated

document, the content of this manual shall prevail. Without departing from the scope or

spirit of the present invention, various improvements and changes can be made to the

specific embodiments of the present specification, which is transparent to those skilled in

the field. Other embodiments derived from the description of the present invention will

also be transparent to the skilled person. The specification and examples of this application

are only exemplary.

As used herein, "including", "composing", "having", "containing", etc., are all open terms,

which means including but not limited to.

Example 1

A method for determining endogenous hormones in wheat:

(1) Weigh 0.01g of IAA, GA3, ZT, 6-KT, 6-BA and ABA, respectively, use mobile phase

as solvent to prepare a mother liquor with a concentration of 1000 [g/mL, take 5 mL each

of 6 kinds of mother liquors, and dilute to 50 mL to obtain a mixed standard solution of

endogenous hormones with a concentration of 100 [g/mL, and dilute the mixed standard

solution gradually to obtain standard solutions with concentration of 1 g/mL, of 10 g/mL,

[g/mL and 50 [g/mL.

(2) Instrument: Agilent 1100 HPLC (with Agilent 1100 UV detector, Agilent 1100 liquid

phase quaternary pump).

(3) Setting of chromatographic conditions:

preparation of mobile phase: add a certain amount of glacial acetic acid to the water to form

an aqueous solution of glacial acetic acid with a concentration of 0.07%, and mix the

aqueous solution of glacial acetic acid with methanol at a volume ratio of 1:1 to obtain the

mobile phase;

Chromatographic column: Shodex C18-120-54E (250 mmx4.6 mm, 5 m);

Flow rate: 0.5 mL/min;

Column temperature: 30°C;

Injection volume: 10 pL;

Wavelength: 254 nm;

Integrate to obtain the chromatographic peak area, and use the standard curve to calculate

the hormone content in the sample.

(4) Preparation for wheat powder: culture the wheat (Triticum aestivum L.) to 2 leaves and

1 heart stage under the conditions of light intensity of 1200 pmol-m-2 '-s- and temperature

of 20°C; take the second stretched leaf and add liquid nitrogen to grind it into powder to

obtain wheat powder.

(5) Preparation of endogenous hormone test solution of wheat: Take 1.0 g of wheat powder,

add 15 mL of pre-cooled methanol solution at 4C with 80% volume fraction, leach at 4°C

for 12 hours, and centrifugate at 6000 g/min for 15 min to obtain the supernatant 1 and the

residue; rinse the residue with methanol twice and centrifugate to obtain supernatant 2; the

supernatant 1 and 2 were combined and concentrated by nitrogen blowing to the remaining

1/3 volume to obtain the endogenous hormone extract of wheat; the endogenous hormone

extract was extracted 3 times with 10 mL of petroleum ether; the aqueous phase was retained, and then 0.5 g of polyvinylpyrrolidone (PVP) was added to ultrasonically dissolve for 30 minutes, filtered; the pH of the filtrate was adjusted to about 3.0, and extracted 3 times with 10 mL of ethyl acetate, and the ethyl acetate phase was retained; nitrogen blowing was carried out for drying, and 1 mL of mobile phase was added to dissolve the residue, and filtered with a 0.45 m microporous membrane to obtain a test solution of endogenous hormones of wheat.

(6) Determine the prepared standard solution using the chromatographic conditions

selected in step (3). Take the injection concentration as the ordinate y and the peak area as

the abscissa x to draw a standard curve, and calculate the linear regression equation and

correlation coefficient. The linear regression equation and correlation coefficient of each

component are shown in Table 1, and the precision is shown in Table 2.

(7) Add a certain amount of standard solution to the test solution of endogenous hormones

of wheat to prepare it as standard addition solution with a concentration of 10 g/mL;

determine the content of each hormone, and calculate recovery rate. Recovery rate of ZT,

GA3, 6-KT, IAA, 6-BA and ABA is shown in Table 3.

(8) Determine the test solution of endogenous hormones of wheat under the selected

chromatographic conditions in step (3); integrate to obtain the chromatographic peak area,

and calculate the content of endogenous hormones of wheat with the standard curve, see

Table 4.

Table 1 regression equation and correlation coefficient for standard solution

Sample Regression equation correlation coefficient r ZT y=0.0193x-0.5305 0.9900 GA3 y=0.3354x-2.8388 0.9996 6-KT y=0.0167x-1.4655 0.9990 IAA y=0.0512x-0.3592 0.9998 6-BA y=0.017x+0.41 0.9994 ABA y=0.0084x+0.5219 0.9997

Table 2 Precision

Sample RSD of retention RSD of peak area(%) time(%) ZT 0.057 1.900 GA3 0.110 0.125 6-KT 0.270 1.330 IAA 0.073 1.430 6-BA 0.018 1.670 ABA 0.031 1.350

Table 3 Recovery rate of addition

Sample Recovery rate of addition (%) ZT 92.85 GA3 95.60 6-KT 96.50 IAA 103.1 6-BA 101.3 ABA 91.01 Table 4 Endogenous hormone content in wheat leaves

Sample Content ([g/g) ZT 0.715 GA3 27.386 6-KT 5.875 IAA 5.569 6-BA 0.557 ABA 0.615

Comparative Example 1

Comparative Example 1 is the same as Example 1, except that the volume ratio of methanol

and water in the mobile phase of step (3) is 2:1.

Comparative Example 2

Comparative Example 2 is the same as Example 1, except that the volume ratio of methanol

and water in the mobile phase of step (3) is 1:2.

From Example 1 and Comparative Examples 2 to 3, it is found that as the methanol

concentration increases, the retention time of each component increases. When the

methanol: water (glacial acetic acid) ratio is greater than 1:1, the peak times of ZT and

GA3 overlap and cannot be completely separated. When the methanol: water (glacial acetic

acid) ratio is less than 1:1, although the peaks in the sample are completely separated, the

retention time increases rapidly.

Comparative Example 3

Comparative Example 3 is the same as Example 1, except that the flow rate of step (3) is

0.3 mL/min.

Comparative Example 4

Comparative Example 4 is the same as Example 1, except that the flow rate of step (3) is

0.6 mL/min.

From the results of Example 1 and Comparative Examples 2 to 3, it is found that the flow

rate in Example 1 of the present invention can completely separate the peaks of the 6

hormones without overlapping. The flow rate of Comparative Example 3 makes peak time

too long. With the flow rate of Comparative Example 4, the peaks of each hormone cannot

be completely separated from the peaks of other substances, which affects the separation effect. The flow rate in Example 1 was selected for elution based on factors such as the flow rate to completely separate the components, the relatively short retention time, and the column pressure.

The above-mentioned embodiments only describe the preferred mode of the present

invention, and do not limit the scope of the present invention. Without departing from the

design spirit of the present invention, various modifications and improvements made by

those skilled in the field to the technical solution of the present invention should fall within

the protection scope determined by the claims of the present invention.

Claims (10)

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS

1. A method for determining wheat endogenous hormones, characterized by comprising

the following steps: (1) leaching wheat leaf powder with an alcohol solution to obtain

a wheat endogenous hormone extract; (2) purifying the wheat endogenous hormone

extract to obtain test solution of wheat endogenous hormone; (3) determining the

content of IAA, GA3, ZT, 6-KT, 6-BA and ABA in the test solution of the

endogenous hormones of the wheat by HPLC.

2. The method for determining endogenous hormones in wheat according to claim 1,

wherein the wheat leaf powder is powder obtained by grinding the second extended

leaf of a wheat seedlings; the method for cultivating the wheat seedling specifically

includes: under the conditions of light intensity of 120 tmol-m-2 -s-' and temperature

of 20°C, hydroponic culture to 2 leaves and 1 heart stage.

3. The method for measuring endogenous hormones in wheat according to claim 1,

wherein the step (1) specifically comprises: adding an alcohol solution to the wheat

leaf powder, leaching for 10-15 hours at 4°C, and soaking after extraction, the

supernatant liquid and the residue are obtained by centrifugation, and then the residue

is washed with an alcohol solution, and the washing liquid is combined into the

supernatant liquid and concentrated to obtain the wheat endogenous hormone extract.

4. The method for determining endogenous hormones in wheat according to claim 3,

wherein the alcohol solution is a methanol solution pre-cooled at 4°C; the

centrifugation is specifically 6000 g/min for 15 minutes; and the concentration factor

is 2 to 5 times; the ratio of wheat-to-alcohol solution 1 g:10-20 mL.

5. The method for determining endogenous hormones in wheat according to claim 4,

wherein the volume fraction of methanol in the methanol solution is 80%.

6. The method for determining endogenous hormones in wheat according to claim 1,

characterized in that, the step (2) specifically comprises: extracting the extract of

endogenous wheat hormones with petroleum ether, retaining the water phase, and

then adding polyvinylpyrrolidone for ultrasonic dissolution for 30 minutes, and then

adjusting pH to 3.0, extracting with ethyl acetate to retain the ethyl acetate phase,

drying with nitrogen, and adding mobile phase to dissolve the residue and filtrating

to obtain wheat endogenous hormone test solution.

7. The method for determining endogenous hormones in wheat according to claim 6,

characterized in that 0.45 m microporous filter membrane is used for filtration.

8. The method for determining endogenous hormones in wheat according to claim 6,

wherein the mobile phase is a mixed solution of methanol: water in a volume ratio of

1:1.

9. The method for determining endogenous hormones according to claim 8 is

characterized in that the water contains glacial acetic acid with a mass fraction of 0.07%.

10. The method for determining endogenous hormones in wheat according to claim 1,

wherein the chromatographic conditions of the HPLC method specifically include:

chromatographic column: C18; flow rate: 0.5 mL/min; column temperature: 30 °C;

injection volume: 10 pL; wavelength: 254 nm.

-1/1- 19 Aug 2021 2021105892

Figure 1

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