CN108535374A - A kind of method of auxin in measurement plant tissue - Google Patents
A kind of method of auxin in measurement plant tissue Download PDFInfo
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- CN108535374A CN108535374A CN201810246613.1A CN201810246613A CN108535374A CN 108535374 A CN108535374 A CN 108535374A CN 201810246613 A CN201810246613 A CN 201810246613A CN 108535374 A CN108535374 A CN 108535374A
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- auxin
- plant tissue
- methanol
- hormone
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N2030/062—Preparation extracting sample from raw material
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Abstract
The invention discloses a kind of methods of auxin in measurement plant tissue, are related to technical field of biological.The method of the present invention includes：Hormone extraction process and hormone test process；The hormone extraction process includes the following steps：Fresh plant sample is ground to broken in liquid nitrogen, obtains sample to be extracted；It is added into the sample to be extracted after the D5 IAA standard specimens of methanol, the mixed extract of propyl alcohol and acetic acid and 0.5ppm and be ultrasonically treated and centrifugal treating, obtained supernatant, which is placed in nitrogen in Brown Glass Brown glass bottles and jars only, to be blown to dry, obtains processed material；Membrane filtration is carried out again after carrying out methanol resuspension to the processed material, and obtained filtrate is extract, and LC MS detections are carried out to the extract.The present invention in vegetable material only with a kind of extracting solution (methanol, propyl alcohol and acetic acid mixed extract, and design three's reasonable volume ratio) direct extraction auxin (IAA), it is easy to operate and at low cost without using extracting small column material.
Description
Technical field
The present invention relates to technical field of biological more particularly to a kind of methods measuring auxin in plant tissue.
Background technology
Plant hormone is a series of natural small molecule compounds generated in plant, and the plant type for participating in regulation and control plant is built
The important physiology course such as utilize at, photosynthesis and water nutrition.Plant hormone is main according to their structure and physiological function
It is divided into following a few classes, including auxin, the basic element of cell division, abscisic acid, gibberellin, five major class of ethylene.Auxin is so far
One group of plant hormone of most study, this is because they are first plant hormones being found, they are widely used
In plant propagation draft.It adjusts plant cell division, elongation, differentiation, and in a manner of concentration dependent.Auxin is being planted
Each aspect of object growth and development is essential.Plant hormone is extremely low in plant in-vivo content, usually in fresh weight
In the range of 0.1-50ug/kg, and classes of compounds complexity is various in plant, therefore, qualitative and quantitative point of plant hormone
Analysis is always one of the difficult point of plant hormone research field.
Plant hormone detection method mainly has radioimmunology, enzyme-linked immunization, gas chromatography, high performance liquid chromatography
Method, gas chromatography-mass spectrometry and Liquid Chromatography-Mass Spectrometry etc..With the development of highly sensitive mass-spectrometric technique, increasingly
Method mostly based on one mass spectrometric hyphenated technique of high performance liquid chromatography establishes.One mass spectrometric hyphenated technique of high performance liquid chromatography can
To provide the retention time and molecular structure information of target hormone simultaneously, there is high sensitivity, be suitble to multicomponent analysis, reliably
The advantages that stablizing, may be implemented to complete while qualitative and quantitative analysis.
It needs to extract the hormone in material before plant hormone detection.Solvent extraction process is that current plant hormone is ground
Most common method in studying carefully.Target plant hormone to be measured is fully extracted from vegetable material, is mainly determined by two aspects:
Plant sample is adequately ground under conditions of liquid nitrogen frozen；Suitable solvent extraction object.It is carried by Primary organic solvent
Contain a large amount of interfering substance in the plant crude extract taken, the substance of these interference measurements can influence the accurate qualitative of plant hormone
Quantitative analysis.Therefore it needs to pre-process plant crude extract, purification enrichment is carried out to target plant hormone to be analyzed, to the greatest extent
It possibly reduces or eliminates background interference complicated in sample, to make sample be suitble to qualitative and quantitative analysis.Plant hormone
Method for purifying and enriching includes gold-liquid extraction, Solid Phase Extraction, and thin-layer chromatography, half preparative high-performance liquid chromatographic are purified and sent out in recent years
The new technology that exhibition is got up, such as solid phase microextraction and membrane extraction technology etc..Wherein there are a large amount of phosphorus in gold-liquid extraction matrix
The chaff interferents such as fat generate emulsion；Solid Phase Extraction can reduce sample substrate interference, improve the sensitivity of detection, but used
Extraction pillar higher price；The fiber head that spe medium is coated with used in solid phase microextraction is expensive, frangibility, and by
Its limited accumulation ability is determined in its limited volume and surface area.
Invention content
In view of this, an embodiment of the present invention provides a kind of method of auxin in measurement plant tissue, liquid is mainly utilized
The triple level four bars of phase chromatography accurately measure auxin IAA in plant tissue, solve the problems, such as testing cost height and cumbersome.
In order to achieve the above objectives, invention broadly provides following technical solutions：
On the one hand, an embodiment of the present invention provides a kind of methods of auxin in measurement plant tissue, including hormone extraction
Process and hormone test process；It is characterized in that, the hormone extraction process includes the following steps：
Fresh plant sample is ground to broken in liquid nitrogen, obtains sample to be extracted；
Methanol, propyl alcohol and the mixed extract of acetic acid and the D5-IAA standard specimens of 0.5ppm are added into the sample to be extracted
After be ultrasonically treated and centrifugal treating, obtained supernatant is placed in nitrogen in Brown Glass Brown glass bottles and jars only and is blown to dry, obtains processed material；
Membrane filtration is carried out again after carrying out methanol resuspension to the processed material, and obtained filtrate is extract, to the extraction
Object carries out LC-MS detections.
Preferably, the ratio of the fresh plant, the mixed extract and the D5-IAA is 100mg：1mL：20μ
L。
Preferably, the volume ratio of the methanol, the propyl alcohol and the acetic acid is 20:79:1.
Preferably, the process of the supersound process is：4 DEG C of ultrasound 15min.
Preferably, the process of the centrifugal treating is：Under the conditions of 4 DEG C, 15000rpm centrifuges 15min.
Preferably, the membrane filtration selects 0.22 μm of filter membrane.
Preferably, the hormone test process is to use liquid phase chromatogram-mass spectrometry combination method.
Preferably, the liquid chromatographic detection condition：
Chromatographic column：Thermo HYPERSIL GOLD C18 chromatographic columns (2.1 × 100,3um)；
Column temperature：35℃；
Mobile phase：A:0.1% acetic acid water, B:Methanol；
Gradient：0-0.2min, A=90%；0.2-6min, A are decremented to 10%；6-8min, A=10%；8.1min
A is incremented to 90%；8.1-10min, A=90%
Sampling volume：7μl.
Preferably, the Mass Spectrometer Method condition：
Spray voltage：3000v；
Sheath gas：35Arb；
Assist gas：10Arb；
Atomization temperature：275℃；
Ion transfer tube temperature：325℃.
Compared with prior art, the beneficial effects of the invention are as follows：
The present invention in vegetable material only with a kind of extracting solution (methanol, propyl alcohol and acetic acid mixed extract, and design
Three's reasonable volume ratio) auxin (IAA) is directly extracted, it is easy to operate and at low cost without using the small column material of extraction.The present invention
Combine the said extracted method of the present invention that can accurately measure auxin in plant tissue using the triple level Four pole technologies of liquid chromatogram
IAA。
Specific implementation mode
For further illustrate the present invention to reach the technological means and effect that predetermined goal of the invention is taken, below with compared with
Specific implementation mode, technical solution, feature and its effect applied according to the present invention is described in detail as after in good embodiment.Under
State it is bright in multiple embodiments in special characteristic, structure or feature can be combined by any suitable form.
Embodiment 1
Hormone extracts：Precise about 100mg fresh plant samples, are ground to crushing in liquid nitrogen；To grinding rear sample
Middle addition 1ml methanol/propyl alcohol/acetic acid (20/79/1V/V/V) extracting solution, the D5-IAA standard specimens of 20ul0.5ppm, 4 DEG C of ultrasounds
15min；Under the conditions of 4 DEG C, 15000rpm centrifuges 15min；Supernatant is taken to move into amber glass vials, nitrogen is blown to dry；60ul methanol
It is resuspended；0.22 μm of filter membrane is crossed, LC-MS detections are carried out to the filtrate being obtained by filtration；
Liquid-phase condition：
Chromatographic column：Thermo HYPERSIL GOLD C18 chromatographic columns (2.1 × 100,3um)；
Column temperature：35℃；
Mobile phase：A:0.1% acetic acid water, B:Methanol；
Gradient：0-0.2min, A=90%；0.2-6min, A are decremented to 10%；6-8min, A=10%；8.1min
A is incremented to 90%；8.1-10min, A=90%
Sampling volume：7μl；
Mass Spectrometry Conditions：
Spray voltage：3000v；
Sheath gas：35Arb；
Assist gas：10Arb；
Atomization temperature：275℃；
Ion transfer tube temperature：325℃；
Detecting IAA using the method for the embodiment of the present invention 1, the results are shown in Table 1.
Table 1.IAA and D5-IAA mass spectrometry parameters list
Place, those skilled in the art can not select from the prior art to the greatest extent in the embodiment of the present invention.
Disclosed above is only the specific implementation mode of the present invention, but scope of protection of the present invention is not limited thereto, is appointed
What those familiar with the art in the technical scope disclosed by the present invention, can easily think of the change or the replacement, answer
It is included within the scope of the present invention.Therefore, protection scope of the present invention should be with above-mentioned scope of the claims
It is accurate.
Claims (9)
1. a kind of method measuring auxin in plant tissue, including hormone extraction process and hormone test process；Its feature exists
In the hormone extraction process includes the following steps：
Fresh plant sample is ground to broken in liquid nitrogen, obtains sample to be extracted；
Methanol, propyl alcohol and the mixed extract of acetic acid is added into the sample to be extracted and the D5-IAA standard specimens of 0.5ppm are laggard
Row is ultrasonically treated and centrifugal treating, and obtained supernatant, which is placed in nitrogen in Brown Glass Brown glass bottles and jars only, to be blown to dry, obtains processed material；
To the processed material carry out methanol resuspension after carry out membrane filtration again, obtained filtrate be extract, to the extract into
Row LC-MS detections.
2. a kind of method measuring auxin in plant tissue as described in claim 1, which is characterized in that the fresh plant
The ratio of object, the mixed extract and the D5-IAA is 100mg：1mL：20μL.
3. a kind of method measuring auxin in plant tissue as claimed in claim 2, which is characterized in that the methanol, institute
The volume ratio for stating propyl alcohol and the acetic acid is 20:79:1.
4. a kind of method measuring auxin in plant tissue as claimed in claim 3, which is characterized in that the supersound process
Process be：4 DEG C of ultrasound 15min.
5. a kind of method measuring auxin in plant tissue as claimed in claim 4, which is characterized in that the centrifugal treating
Process be：Under the conditions of 4 DEG C, 15000rpm centrifuges 15min.
6. a kind of method measuring auxin in plant tissue as described in claim 1, which is characterized in that the membrane filtration choosing
With 0.22 μm of filter membrane.
7. a kind of method measuring auxin in plant tissue as described in claim 1, which is characterized in that the hormone test
Process is to use liquid phase chromatogram-mass spectrometry combination method.
8. a kind of method measuring auxin in plant tissue as claimed in claim 7, which is characterized in that the liquid chromatogram
Testing conditions：
Chromatographic column：Thermo HYPERSIL GOLD C18 chromatographic columns (2.1 × 100,3um)；
Column temperature：35℃；
Mobile phase：A:0.1% acetic acid water, B:Methanol；
Gradient：0-0.2min, A=90%；0.2-6min, A are decremented to 10%；6-8min, A=10%；8.1min, A are passed
Increase to 90%；8.1-10min, A=90%
Sampling volume：7μl.
9. a kind of method measuring auxin in plant tissue as claimed in claim 8, which is characterized in that the Mass Spectrometer Method
Condition：
Spray voltage：3000v；
Sheath gas：35Arb；
Assist gas：10Arb；
Atomization temperature：275℃；
Ion transfer tube temperature：325℃.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103353490A (en) * | 2013-06-24 | 2013-10-16 | 浙江大学 | Method for detecting pear endogenous hormone by using ultra-high performance liquid chromatography electrospray tandem mass spectrum |
| CN106501427A (en) * | 2016-10-26 | 2017-03-15 | 武汉绿剑可瑞信科技有限公司 | The pre-treating method and simultaneous quantitative determination of multiple endogenous plant hormones in a kind of plant sample |
| CN106706826A (en) * | 2015-11-18 | 2017-05-24 | 中国科学院大连化学物理研究所 | Analysis method of plant hormones in milligram-grade plants |
| CN107024552A (en) * | 2017-03-28 | 2017-08-08 | 北京林业大学 | A kind of method of plant hormone in measure Subgenus Yulania Species Based |
| CN107543746A (en) * | 2016-11-16 | 2018-01-05 | 武汉绿剑可瑞信科技有限公司 | The pre-treating method and quantitative detecting method of the endogenous plant hormone of three class difference chemical property in a kind of plant sample |
-
2018
- 2018-03-23 CN CN201810246613.1A patent/CN108535374A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103353490A (en) * | 2013-06-24 | 2013-10-16 | 浙江大学 | Method for detecting pear endogenous hormone by using ultra-high performance liquid chromatography electrospray tandem mass spectrum |
| CN106706826A (en) * | 2015-11-18 | 2017-05-24 | 中国科学院大连化学物理研究所 | Analysis method of plant hormones in milligram-grade plants |
| CN106501427A (en) * | 2016-10-26 | 2017-03-15 | 武汉绿剑可瑞信科技有限公司 | The pre-treating method and simultaneous quantitative determination of multiple endogenous plant hormones in a kind of plant sample |
| CN107543746A (en) * | 2016-11-16 | 2018-01-05 | 武汉绿剑可瑞信科技有限公司 | The pre-treating method and quantitative detecting method of the endogenous plant hormone of three class difference chemical property in a kind of plant sample |
| CN107024552A (en) * | 2017-03-28 | 2017-08-08 | 北京林业大学 | A kind of method of plant hormone in measure Subgenus Yulania Species Based |
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Application publication date: 20180914 |