CN105820266B - A kind of method of high efficiency extraction arabidopsis internal layer slime layer - Google Patents
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- C08B37/0057—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid beta-D-Xylans, i.e. xylosaccharide, e.g. arabinoxylan, arabinofuronan, pentosans; (beta-1,3)(beta-1,4)-D-Xylans, e.g. rhodymenans; Hemicellulose; Derivatives thereof
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Abstract
The present invention discloses a kind of method of high efficiency extraction arabidopsis internal layer slime layer, belongs to Polyose extraction field.The method of the present invention is to utilize ultrasonic extraction internal layer slime layer.The method economy, simplification work: extraction solution is water, it is not necessary to carries out desalting processing and just can get substantial amounts of seed coat slime layer, and during soda acid salt method, the salinity of residual needs to remove.Time-consuming: concussion method and acid-alkali salt method extraction time are generally 1～2h, and the later stage also needs to carry out desalination, this method can complete to extract at 20S.Extraction efficiency is high: the seed after concussion method and soda acid salt method shows a large amount of slime layer residual after aortic, and after this method is extracted, substantially remains without slime layer.The method has efficient, stable, high repeatability and other advantages, can farthest be extracted by slime layer, and the analysis that can make slime layer is more accurate, promotes the research of slime layer formation mechenism, and in chemical industry, this method can shorten extraction time, reduces cost.
Description
Technical field
The invention belongs to Polyose extraction field, particularly to a kind of method of high efficiency extraction arabidopsis internal layer slime layer.
Background technology
Angiospermous embryo, when growing, covers integument around embryo and can form seed coat, seed coat by extraneous various not
Profit factor is isolated and can regulate the sprouting of seed, and some seed plants are (such as Cruciferae, Solanaceae, flax family (Linaceae) and Plantaginaceae
Deng) seed coat when being formed, okioplast can secrete a large amount of mucus, forms one layer of gluey slime layer around seed, and this layer is glutinous
Liquid layer is to promoting the water suction of seed and being diffused with important function.Owing to slime layer is rich in pectin composition, have become as acquisition pectin
Important source material.The okioplast of arabidopsis seed coat can accumulate substantial amounts of glutinous between primary cell wall and cell membrane when differentiation
Liquid, and the Cytoplasm of cylindricality it is centrally formed at cell, one layer of secondary cell wall thickeied can be formed around this confluent monolayer cells matter,
End form becomes the structure that a kind of volcano is formed, referred to as pillar.When seed absorbs water, seed coat okioplast wall ruptures and discharges mucus,
Discharge at first for outer layer slime layer, be mainly composed of unbranched, non-methylate, non-acetylizad
rhamnogalacturonan I(RG I).One layer of mucus is also had tightly to adhere to seed coat, referred to as internal layer slime layer, this layer of mucus
Connection with seed is closely, it is very difficult to extracting, this macromolecules cross-linking being likely due in mucus or mucus are with little
Crosslinking between post, it is also possible to be okioplast broken after residue in seed coat so that stop mucus release (Western TL,
Skinner DJ,Haughn GW(2000)Differentiation of mucilage secretory cells of the
Arabidopsis seed coat.Plant physiology 122:
345-356), the composition of internal layer slime layer is also mainly RG I, can be divided into again two parts, and outside includes galactan
Sugared and hypomethylated HG, inner side includes the HG of cellulose, galactan and hyper-methylation.
The outer layer slime layer of arabidopsis is very easy to obtain, and arabidopsis seed is dipped in water concussion a few minutes, but
Internal layer slime layer is but very difficult to extract, and in scientific experiment, the extraction of arabidopsis internal layer slime layer is mainly by concussion, chemistry
Reagent such as acid-alkali salt etc. extracts (Macquet A, Ralet MC, Kronenberger J, Marion-Poll A, North HM
(2007)In situ,chemical and macromolecular study of the composition of
Arabidopsis thaliana seed coat mucilage.Plant&cell physiology 48:984-999), but
A part for the simply internal layer slime layer that these methods are extracted, the most substantial amounts of internal layer slime layer component residue is on seed coat.
Set up the extracting method of a kind of efficient arabidopsis slime layer, not only can promote the research of slime layer synthesis mechanism, it is also possible to
Chemical industry for slime layer extracts offer conveniently.
Summary of the invention
In order to overcome the shortcoming of existing arabidopsis internal layer slime layer extracting method with not enough, it is an object of the invention to provide
A kind of method of high efficiency extraction arabidopsis internal layer slime layer.The method of the present invention is to utilize ultrasonic extraction internal layer slime layer.Should
Method has efficient, stable, high repeatability and other advantages, can farthest be extracted by slime layer.
The purpose of the present invention is achieved through the following technical solutions:
A kind of method of high efficiency extraction arabidopsis slime layer, comprises the following steps:
(1) the arabidopsis seed being dried is obtained；
(2) the outer layer slime layer of arabidopsis seed is extracted；
(3) take the seed removing outer layer slime layer in step (2), utilize ultrasound wave to carry out supersound process, it is thus achieved that internal layer sticks
Liquid layer.
In order to preferably realize the present invention: also comprise the steps:
(4) the internal layer slime layer that the outer layer slime layer obtained step (2) and step (3) obtain carries out trifluoroacetic acid respectively
Hydrolysis；
(5) hydrolyzed solution obtaining step (4) carries out high speed centrifugation, takes cleer and peaceful precipitation respectively；
(6) the precipitation anthrone method obtained step (5) measures crystalline fibers cellulose content；
(7) utilize the sugared content in the hydrolyzed solution that Phenol-sulphate acid method determination step (4) obtains, reference standard curve, calculate
Obtain the sugared content in slime layer；
(8) seed after step (2), (3) process is carried out aortic.
Described in step (1) obtain be dried arabidopsis seed be realized by the following method, in order to ensure experiment can
Repeatability, is placed in next week of dry room temperature by arabidopsis seed, makes seed dehydration as far as possible, finally collects and be placed in low temperature
Low humidity seed cabinet preserves.
The Extraction solvent of the outer layer slime layer described in step (2) is water；It is preferably pure water；
The condition of the extraction described in step (2) is preferably placed on the shaking table of 150rpm concussion 5～10min；
The outer layer slime layer extracting arabidopsis seed described in step (2) is realized by the following method: weigh arabidopsis
Seed, adds pure water (arabidopsis seed: pure water=5mg:1mL), concussion, is centrifuged, takes supernatant, for outer layer slime layer；With
Pure water cleans seed 3～5 times, removes the outer layer slime layer of residual, be eventually adding pure water (arabidopsis seed: pure water=
5mg:1mL).
The condition of described concussion is preferably placed on the shaking table of 150rpm concussion 5～10min；
Described centrifugal condition is preferably 2000～5000rpm and is centrifuged 1min；
The Extraction solvent of the internal layer slime layer described in step (3) is water；It is preferably pure water；
The amplitude of the ultrasound wave described in step (3) is 20～80%；It is preferably 60%.
The extraction of the internal layer slime layer described in step (3) is realized by the following method: takes removal outer layer in step (2) and sticks
The seed of liquid layer, supersound process, stand or centrifugal, after seed sinks to bottom test tube, take supernatant, for internal layer slime layer.
Described supersound process is placed in sonicator, and (Sonics VCX130PB is equipped with 3mm probe, amplitude
60%) supersound process is carried out；
The time of described supersound process is preferably 10S～2min；It is preferably 20S～60S；More preferably 20S；
The time of described standing is preferably 1～5min；
Described centrifugal condition is preferably 2000～5000rpm and is centrifuged 1min；
The condition of the hydrolysis described in step (4) hydrolyzes 0.5～1h under the conditions of being preferably 121 DEG C.
Trifluoroacetic acid hydrolysis described in step (4) is realized by the following method: take the outer layer slime layer that step (2) obtains
The internal layer slime layer obtained with step (3), is separately added into trifluoroacetic acid, hydrolyzes 0.5～1h under the conditions of 121 DEG C.
The volume ratio of described outer layer slime layer or internal layer slime layer and trifluoroacetic acid is preferably 5:1；
Ultracentrifugal condition described in step (5) is preferably 12000rpm and is centrifuged 5～10min；
Mensuration crystalline fibers cellulose content described in step (6) is realized by the following method: obtain step (5) is heavy
Forming sediment, add 70% sulphuric acid, put and add pure water after hydrolyzing 1h at room temperature, making sulfuric acid concentration is 17.5%；Take 17.5%
Sulphuric acid gradient dilution becomes 0, the Glucose standards solution of 10,20,40,60,80,150,200 μ g/mL, adds 0.2% anthracene of pre-cooling
Ketone concentrated acid solution, shakes up immediately and is placed in cooled on ice；All samples gets out be placed on 15min in boiling water bath.Above sample
After after cooling, it is placed under spectrophotometer mensuration 620nm light absorption value, makes standard curve, calculate crystalline cellulose in sample and contain
Amount.
Phenol-sulphate acid method described in step (7) measures sugar content and is realized by the following method: take the water that step (4) obtains
Solve liquid, add 5% phenol, add concentrated sulphuric acid, shake up immediately；Take gradient dilution and become 0,10,20,40,60,80,150,200 μ g/
The Glucose standards solution of mL, adds 5% phenol, adds concentrated sulphuric acid, shakes up immediately.After 1h is placed in above sample greenhouse, it is placed in
Measure 490nm light absorption value under spectrophotometer, make standard curve, calculate sample sugar content.
Aortic described in step (8) is realized by the following method: the aortic liquid of preparation 0.01% (w/v),
Seed is carried out the 5min that dyes, washes away dyeing liquor and be placed under stereomicroscope observation.
The present invention, relative to prior art, has such advantages as and effect:
(1) economic, simplification work.Extraction solution is water, it is not necessary to carries out desalting processing and just can get substantial amounts of seed coat mucus
Layer, and during soda acid salt method, the salinity of residual needs to remove.
(2) time-consuming.Concussion method and acid-alkali salt method extraction time are generally 1～2h, and the later stage also needs to carry out desalination, this
Method can complete to extract at 20S.
(3) extraction efficiency is high.Seed after concussion method and soda acid salt method shows a large amount of glutinous after aortic
After liquid layer remains, and this method is extracted, substantially remain without slime layer.
The present invention is that high efficiency extraction arabidopsis internal layer slime layer composition provides a kind of approach reliably, in scientific research
In, the arabidopsis slime layer of high efficiency extraction can make the analysis of slime layer more accurately, promotes the research of slime layer formation mechenism,
In chemical industry, this method can shorten extraction time, reduces cost.Ultrasonic wave extraction has been applied to a lot of industry, but
In arabidopsis this certain moduli formula plant it is not yet reported that.
Accompanying drawing explanation
Fig. 1 is the aortic of arabidopsis slime layer after different extracting mode processes.
Fig. 2 is the different extracting mode extracted amounts to arabidopsis slime layer polysaccharide.
Fig. 3 is arabidopsis seed aortic after different time supersound process.
Fig. 4 is arabidopsis seed extracted amount of sugar after different time stage supersound process.
Fig. 5 is that arabidopsis seed fluorescence immunoassay is observed；Wherein, before A～D is supersound process, after E～H is supersound process.
Fig. 6 is that Phenol-sulphate acid method measures total sugar standard curve.
Fig. 7 is that anthrone method measures crystalline fibers cellulose content.
Fig. 8 is the arabidopsis each component content of seed mucilage layer.
Detailed description of the invention
Below in conjunction with embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention do not limit
In this.
The routine experimentation personnel of described technical field are according to above present disclosure and the taken scope of each parameter, all
The purpose of the present invention can be realized.
The experimental technique of unreceipted actual conditions in the following example, generally according to normal condition.
Described arabidopsis seed is wildtype Arabidopsis thaliana (Arabidopsis thaliana) Col-0 seed, is purchased from
Arabidopsis Biological Resource Center (ABRC, https: //abrc.osu.edu/).
Embodiment 1: the different extracting mode extraction efficiency researchs to arabidopsis slime layer
(1) preparation of reagent is extracted: prepare with pure water and extract reagent below: 0.05M EDTA, 0.2% ammonium oxalate (w/
V), 0.01M NaOH, 0.05M HCl.
(2) different chemical extracts the reagent extraction to arabidopsis slime layer: accurately weigh 5mg arabidopsis seed, adds 1mL
Pure water, be placed on 150rpm shaking table shake 5min, 3000rpm centrifugal after, take supernatant, for outer layer slime layer.Clean with pure water
Seed 3 times, addition 1mL correspondence reagent: pure water, 0.05M EDTA, 0.2% ammonium oxalate (w/v), 0.01M NaOH, 0.05M
HCl, on 40 DEG C of shaking tables 200rpm shake 1h, 3000rpm centrifugal after, take supernatant, for internal layer slime layer.
(3) supersound process extraction to arabidopsis slime layer: accurately weigh 5mg arabidopsis seed, adds 1mL pure water,
Be placed on 150rpm shaking table shake 5min, 3000rpm centrifugal after, take supernatant, for outer layer slime layer.Seed is cleaned 3 times with pure water,
Add 1mL pure water, supersound process 20S, after 2000rpm is centrifugal, take supernatant, for internal layer slime layer.
(4) aortic: the seed pure washing 3 times after step (2) and step (3) process, carries out aortic,
Being placed under stereomicroscope observation, result is shown in Fig. 1.As shown in Figure 1, after extracting with chemical reagent, major part slime layer also exists
On seed, and after NaOH extracts, there is the phenomenon of seed coat fragmentation in major part seed, and seed coat ruptures and is likely to result in seed
Internal saccharide is extracted, and causes result inaccurate.And the slime layer after supersound process 20S, on arabidopsis seed
Almost all disappears.
(5) carrying out total sugar determination according to the method for embodiment 4, measurement result is shown in Fig. 2.As shown in Figure 2, with various chemistry examinations
When agent is extracted, the slime layer polysaccharide ultrasonic method to be considerably less than obtained.
Embodiment 2: supersound extraction arabidopsis slime layer
(1) extraction of outer layer slime layer: accurately weigh 5mg arabidopsis seed, adds 1mL pure water, is placed in 150rpm and shakes
Shake on Chuan 5min, 3000rpm centrifugal after, take supernatant, for outer layer slime layer.
(2) slime layer of the different sonication stage of same pipe seed sample experience extracts: the seed in step (1), and accumulation is super
Sound 5S, after 10S, 20S, 40S, 1min, 2min, 4min, 6min, takes supernatant respectively, and for internal layer slime layer, each ultrasonic interval is used
Pure water cleans seed twice.
(3) slime layer change after the different sonication stage of same pipe seed sample experience: the seed in step (1), accumulation is super
Sound 0S, after 5S, 10S, 20S, 40S, 1min, 2min, 4min, 6min, takes seed respectively, and each ultrasonic interval pure water cleans
Twice of seed.
(4) seed obtaining step (1) and step (3) carries out aortic, and coloration result is shown in Fig. 3.From the figure 3, it may be seen that
When supersound process 20S, the slime layer of most seeds disappears, but when ultrasonic 4min, occurs that seed coat crushes, and illustrates super
The time of sound is very big on the impact of seed, in order to prevent seed coat from crushing, should be controlled within 2min by ultrasonic time.
(5) slime layer obtaining step (1) and step (2) carries out total sugar determination according to the method for embodiment 4 respectively, surveys
Determining result and see Fig. 4 and Fig. 8, as shown in Figure 4, when ultrasonic 20S, most slime layer polysaccharide extract, at ultrasonic 4min
After, owing to seed coat crushes, sugar content begins to ramp up again, and therefore, supersound process 20S is the time that the most reasonably processes, can by Fig. 8
Knowing, arabidopsis outer layer slime layer content is about 15.56mg/g, and internal layer slime layer content is about 21.66mg/g.
Embodiment 3: the Fluirescence observation of arabidopsis seed mucilage layer
(1) after arabidopsis seed water shakes 5min removal outer layer slime layer, with 0.1M phosphate buffer (Phosphate
Buffer Solution, PBS；1L:0.218g KH2PO4,1.463g K2HPO4,29.22g NaCl, pH 7.2) clean 2
Secondary, each 5min；
(2) soak seed with fresh 5% (w/v) skimmed milk, be placed in and shake 1h on shaking table at a slow speed；Remove defat cattle
Breast, and wash seed 5min with PBS；
(3) one anti-hatch: with little mouse-anti RG I antibody CCRC-M14 (the Complex Carbohydrate of dilution 20 times
Research Center) hatch seed 1h, resist to clean unconjugated one for 5 times with PBS washing seed；
(4) two anti-hatch: hatch with the FITC-goat anti-mouse antibody (health is century, article No. CW0113S) diluting 50 times
1h, resists to clean unconjugated two for 5 times with PBS washing seed；
(5) redye with 0.01%S4B (w/v, Direct Red 23, Sigma Aldrich prepares with 20mM NaCl)
5min, washs seed 5 times with PBS；
(6) Calcofluor dyeing: take the seed of step (3), with the Calcofluor (Sigma of 25 μ g/mL
Aldrich, prepares with PBS) dyeing 5min.
(6) sample prepared is placed in laser confocal microscope (Zeiss LSM710, RG I 490nm, S4B
552nm, Calcofluor 405nm) under observe and take pictures, result is shown in Fig. 5, as shown in Figure 5, after supersound process, arabidopsis kind epidermis
Cellulose and the RG I composition overwhelming majority in face are removed, and the most a small amount of cellulose and RG I remain (arrow).
Embodiment 4: Phenol-sulphate acid method measures total sugar
(1) Specification Curve of Increasing: accurate formulation concentration is respectively 0,10,20,40,60,80,100,150,200 μ g mL-1
Glucose solution, each standard solution takes 300 μ L, adds 5% phenol solution that 150 μ L newly prepare, and adds the dense sulfur of 1.5mL
Acid, mixes immediately, with ultra-violet and visible spectrophotometer (Shanghai U.S. spectrum reaches UV-1200) at 490nm wavelength after room temperature placement 1h
Lower measurement light absorption value, draws standard curve (Fig. 6), and obtaining regression equation is y=-0.0134+0.02991*x.
(2) sample determination: according to the method for step (1), measures the light absorption value of sample in embodiment 1,2, bent according to standard
The sugared content of each sample tried to achieve by line.
Embodiment 5: crystalline cellulose assay
(1) acquisition of slime layer crystalline cellulose: weigh 5mg seed, adds 1mL pure water, removes after shake 5min
Clearly, after cleaning twice, add 1mL water, 60% amplitude supersound process 20S, stand and make seed take supernatant after sinking to bottom.Add 200
μ L trifluoroacetic acid, 121 DEG C of hydrolysis 1h, 12000rpm take crystalline cellulose precipitation, with pure washing 3 times after being centrifuged 5min.To obtaining
The crystalline cellulose sample obtained carries out sulphuric acid hydrolysis, adds the sulphuric acid of 140 μ L 70%, hydrolyzes 1h, middle with pipettor piping and druming 5
Secondary, it is eventually adding 420 μ L pure water, making sulphuric acid ultimate density is 17.5%.
(2) crystalline cellulose assay: be respectively 0 by the sulfuric acid solution accurate formulation concentration of 17.5%, 10,20,40,
60,80,100,150,200 μ g mL-1Glucose solution, standard sample and sample take 560 μ L respectively, add the dense sulfur of 1.4mL
0.2% anthrone reagent of the pre-cooling of the new preparation of acid, puts on ice after shaking up immediately, and whole samples add and are placed in boiling water bath process
15min.Finally under 620nm wavelength, measure light absorption value with ultra-violet and visible spectrophotometer (Shanghai U.S. spectrum reaches UV-1200), paint
Standard curve processed (Fig. 7), obtaining regression equation is y=0.00736+0.02267*x.The knot in sample is tried to achieve according to standard curve
Crystalline cellulose content, result is shown in Fig. 8, and from result, arabidopsis slime layer crystalline fibers cellulose content is about 3.71mg/g.
Above-described embodiment is the present invention preferably embodiment, but embodiments of the present invention are not by above-described embodiment
Limit, the change made under other any spirit without departing from the present invention and principle, modify, substitute, combine, simplify,
All should be the substitute mode of equivalence, within being included in protection scope of the present invention.
Claims (10)
1. the method for a high efficiency extraction arabidopsis slime layer, it is characterised in that comprise the following steps:
(1) the arabidopsis seed being dried is obtained；
(2) the outer layer slime layer of arabidopsis seed is extracted；
(3) take the seed removing outer layer slime layer in step (2), utilize ultrasound wave to carry out supersound process, it is thus achieved that internal layer slime layer.
The method of high efficiency extraction arabidopsis slime layer the most according to claim 1, it is characterised in that also comprise the steps:
(4) the internal layer slime layer that the outer layer slime layer obtained step (2) and step (3) obtain carries out trifluoroacetic acid water respectively
Solve；
(5) hydrolyzed solution obtaining step (4) carries out high speed centrifugation, takes cleer and peaceful precipitation respectively；
(6) the precipitation anthrone method obtained step (5) measures crystalline fibers cellulose content；
(7) utilize the sugared content in the hydrolyzed solution that Phenol-sulphate acid method determination step (4) obtains, reference standard curve, be calculated
Sugared content in slime layer；
(8) seed after step (2), (3) process is carried out aortic.
The method of high efficiency extraction arabidopsis slime layer the most according to claim 1 and 2, it is characterised in that:
The Extraction solvent of the outer layer slime layer described in step (2) is water.
The method of high efficiency extraction arabidopsis slime layer the most according to claim 1 and 2, it is characterised in that:
The condition of the extraction described in step (2) is to be placed on the shaking table of 150rpm concussion 5～10min.
The method of high efficiency extraction arabidopsis slime layer the most according to claim 1 and 2, it is characterised in that:
The Extraction solvent of the internal layer slime layer described in step (3) is water.
The method of high efficiency extraction arabidopsis slime layer the most according to claim 1 and 2, it is characterised in that:
The amplitude of the ultrasound wave described in step (3) is 20～80%.
The method of high efficiency extraction arabidopsis slime layer the most according to claim 1 and 2, it is characterised in that:
The time of described supersound process is 10S～2min.
The method of high efficiency extraction arabidopsis slime layer the most according to claim 1 and 2, it is characterised in that:
The time of described supersound process is 20S～60S.
The method of high efficiency extraction arabidopsis slime layer the most according to claim 2, it is characterised in that:
The condition of the hydrolysis described in step (4) hydrolyzes 0.5～1h under the conditions of being 121 DEG C.
The method of high efficiency extraction arabidopsis slime layer the most according to claim 2, it is characterised in that:
Ultracentrifugal condition described in step (5) is that 12000rpm is centrifuged 5～10min.
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| CN103130866A (en) * | 2013-02-01 | 2013-06-05 | 中国科学院植物研究所 | Plant plasma membrane protein redissolution method and application in two-dimensional fluorescent difference gel electrophoresis thereof |
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Non-Patent Citations (2)
| Title |
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| In situ, Chemical and Macromolecular Study of the Composition of Arabidopsis;Audrey Macquet等;《Plant and Cell Physiology》;20070831;第48卷(第7期);第984–999页 * |
| 拟南芥籽油超声波辅助提取工艺的响应面法优化;苏青峰等;《中国油脂》;20131130;第38卷(第11期);第10-13页 * |
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