CN1328162C - Process of extracting natural silicon from plant - Google Patents

Abstract

The invention discloses a method for extracting silicon bodies from plants. The method comprises the following steps: 1) crushing plant cells in a buffer solution containing a detergent to obtain a homogenate; collecting cell wall fragments with a diameter of 2-1 mm or less in the homogenate; repeatedly rinsing the collected cell wall fragments, Until the supernatant after settling is transparent in color and free of suspended particles, a precipitate containing coarse siliceous body and silicified cell wall is obtained; 2) the precipitate containing coarse siliceous body and silicified cell wall obtained in step 1) is mixed with 0.5-3 %SDS or triton X-100 rinse solution, keep warm for 0.5-1h, and collect the precipitate; 3) Rinse the precipitate collected in step 2) with acetone or ether until no yellow substance precipitates; then use 1-8mol·L ^-1 NaCl solution or 0.1-0.5mol L ^-1 CaCl ₂ solution rinsing, the precipitate obtained is a mixture of siliceous body and silicified cell wall; 4) the mixture of siliceous body and silicified cell wall obtained in step 3 is filtered through Filter through a sieve with a diameter of 30-90 μm, and collect the filtrate to obtain silicon bodies.

Description

A kind of method of from plant, extracting natural silicon body

Technical field

The present invention relates to a kind of method of from plant, extracting natural silicon body.

Background technology

Grass is the plant that typically accumulates silicon, people did not understand the physiological function of silicon in the past, but, in recent years, a large amount of researchs proved amply, and silicon has important effect to the healthy growth that guarantees plant, silicon can improve the output of plant, increase physical strength, improve its to disease and pest, arid, uvioresistant coerce, the resistance of salt damage and heavy metal stress, so silicon is a kind of element with resistance of wide spectrum.In addition, silicon also plays an important role to human health, discovers, silicon can promote the growth of skeleton, can also reduce the absorption of people to neurotoxicity element aluminum in the food, prevents degenerative brain disorder.Because the silicon volume morphing has the kind specificity, and its form and distribution are subjected to the influence of environmental factor very little, it is Genetic Control clearly, so the silicon body is commonly used for a feature of systematics research and is used for identifying the kind classification, in recent years, because the application in archeology, the research of silicon body obtain paying attention to gradually.

Yet the accumulation of silicon in grass also reduced the digestibility of livestock to straw feed, reduced the pulp yield of paper making raw materials such as wheat straw.Therefore, in recent years, people press for the mechanism of understanding plant materials inner control siliceous deposits, on the one hand, wish to increase the content (the particularly low dicotyledons of silicon content) of silicon in the cash crop by the molecular genetic means, this both can cultivate the improved seeds with high yield and degeneration-resistant effect, also can increase the intake of human body to silicon; On the other hand, wish again by knocking out or suppress the gene of siliceous deposits, cultivate silicon content low, be suitable for the edible gramineous feed crop of livestock or the plant variety of papermaking special use.Therefore, illustrate the molecular mechanism of plant control siliceous deposits, have important application prospects at the aspects such as molecular improvement, livestock industry, paper-making industry and sanipratics of crop anti-adversity shape.

In order to disclose the synthesis mechanism of nano silicon material in the living things system, at first must obtain a large amount of silicon bodies that contains natural organic active composition, promptly natural silicon body is got the biochemical property of organic macromolecule material in the silicon body then clear.Recently, (Kr  ger N. such as the Kr  ger of Germany bio-science man, R.Deutzmann, E.Brunner, M.Sumper.2000.Species-specific polyamine from diatoms control silicamorphology.Proc.Natl.Acad.Sci.USA, 97 (26): 14133-14138; Kr  ger N., R.Deutzmann, M.Sumper.1999.Polycationic peptides from diatom biosilicathat direct silica nanosphere formation.Science, 286:1129-1132; Kr  gerN., S.Lorenz, E.Brunner, M.Sumper.2002.Self-assembly of highlyphosphorylated silaffins.Science, 298:584-586), isolate one group of pvcr protein (Silaffins) that is rich in basic aminoacids and polyamines side chain in the natural silicon body that report extracts from diatom (Cylindrotheca fusiformis) cell walls, the positively charged albumen of in vitro tests proof some of them can with soluble oligomeric silicic acid particulate generation electrostatic interaction, in several seconds, induce the sedimentary formation of silicon, and other albumen has the effect of control silicon volume morphing.Meanwhile, (ChaJ.N., D.S.Galen, D.E.Morse such as American scholar Cha, T.J.Deming.2000.Biomimetic synthesisof ordered silica structures mediated by block copolypetides.Nature, 403:289-292; Cha J.N., K.Shimizu, Y.Zhou, S.C.Christiansen, B.F.Chmelka, G.D.Stucky, D.E.Morse.1999.Silicatein filaments and subunits froma marine sponge direct the polymerization of silica and silicones in vitro.Proc.Natl.Acad.Sci.USA, 96:361-365) report, in sponge, be separated to a kind of silicon albumen (Silicatein) that can the polymerization of catalysis organo-siloxane forms nano silicon material, this albumen contains a large amount of hydroxy-amino-acids, and the hydroxyl on hydroxyl wherein and the oligomerization silicic acid molecule is by the formation of hydrogen bond action catalytic nanometer silica dioxide granule.Above important breakthrough has caused the extensive concern of international plant nutrition educational circles, medical circle and bionic nano material subject.More than all to wait marine organisms be material with simple low in research, obtaining breakthrough progress aspect the research siliceous deposits mechanism, found the protein and the gene of control siliceous deposits.Because single celled diatom or sponge cell wall structure are simple, from they quil and the cell of silication the be easy to get silicon body that contains active skull cap components of lot of pure.And for higher plant, because they are made of many cells, silicon body wherein is dispersed among the tissue of various non-silicidizes, be difficult to natural silicon body is therefrom separated, adopt of the organic component degraded of the method for processing of severe corrosive chemical reagent or high temperature sintering at present mostly, so that obtain not have other organic purified silicon body that pollutes with non-silicidize.Harrison (Harrison C.C.1996.Evidence for intramineral macromolecules containing protein from plantsilica.Phvtochem., 41:37-42) with Perry and Tucker (Perry C.C., T.K.Tucker.2003.Model studies of colloidal silica precipitation usingbiosilica extracts from Equisetum telmateia.Colloid.Polym.Sci., experiment 281:652-664) shows, the protein (it is conjugated protein to be referred to as silicon) of combining closely with the silicon body may be main " template " molecule of catalysis silicic acid polymerization, thereby think exist in the higher plant silicon body with the diatom sponge kind like the albumen of control siliceous deposits, but because they adopt the wet digestion method vitriol oil, the mixed solution divided silicon body of concentrated nitric acid and perchloric acid, caused inevitably the inner organic destruction of silicon body, failed to isolate complete protein so far with catalysis silicic acid polymerization ability.

In sum, though many researchs have proved that the formation of silicon body is subjected to Genetic Control, and, some investigators are at low protein and the gene of having found the control siliceous deposits in the marine organisms that wait, but, in higher plant, owing to lack the method for separating natural silicon body, also fail at present to find the protein and the gene of control siliceous deposits, the mechanism of higher plant control siliceous deposits still imperfectly understands.There is the concentration of soluble silicon in the data demonstration grass body to have only 2-5mmolL ^-1, and the concentration of the interior soluble silicon of frustule is up to 20-100mmolL ^-1(Kaufman P.B., P.Dayananda, Y.Takeoka, W.C.Bigelow, J.D.Jones, R.Iler.1981.Silica in shoots of higher plants.In:Simpson T.L., B.E.Volcani, eds.Silicon and siliceous structures in biological systems.NewYork:Springer-Verlag, 409-449; Epstein is Mol.Biol. E.1999.Silicon.Annu.Rev.PlantPhysiol.Plant, 50:641-664), this explanation is along with the process of biological natural evolvement, high grass has the ability of stronger synthesis of nano silicon materials than lower plant, it can make the deposition reaction of silicon carry out under very low silicon concentration, therefore has bigger bionics Study and using value.

Summary of the invention

The purpose of this invention is to provide a kind of method of from plant, extracting natural silicon body.

The method of extracting the silicon body from plant provided by the present invention may further comprise the steps:

1) broken vegetable cell in the damping fluid of SDS that contains 0.5-3% or Triton X-100 obtains homogenate; Collect the cell wall fragments of diameter below 1-2mm in the described homogenate; The collected cell wall fragments of rinsing repeatedly, the supernatant fluid color after sedimentation is transparent and do not have suspended particle, obtains containing the precipitation of thick silicon body and silicidize cell walls;

2) in SDS that containing of step 1) being obtained, being deposited in of thick silicon body and silicidize cell walls contained 0.5-3% or the damping fluid of triton X-100, be incubated and handle the back collecting precipitation; Described insulation is treated to 50-95 ℃ of insulation 0.5-1h;

3) with step 2) in the precipitation of collecting carry out rinsing with acetone or ether, separate out until no yellow substance; Use 1-8molL again ^-1NaCl solution or 0.1-0.5molL ^-1CaCl ₂The solution rinsing, the mixture that is precipitated as silicon body and silicidize cell walls that obtains;

4) the silicon body that step 3 is obtained and the mixture of silicidize cell walls are the filtration of 30-90 μ m by the filter footpath, collect filtrate, obtain the silicon body precipitation of purifying;

Described percentage composition is the quality percentage composition.

This method is suitable for lower plant and higher plant, the especially higher plant that cell contains the silicon body.

In the step 1), described broken vegetable cell is earlier with the liquid nitrogen freezing plant sample and grind plant tissue in mortar, then in containing the damping fluid that the quality percentage composition is 0.5-3%SDS or Triton X-100, with high-speed tissue mashing machine's fragmentation vegetable cell.

Step 1) and step 2) described in buffered soln be that pH is that 6.8-7.6, concentration are 0.05-0.2molL ^-1Tris-HCl or phosphate buffered saline buffer.

In the step 1), preferably be 7.2 at the pH that contains 1%Triton X-100, concentration is 0.1molL ^-1The Tris-HCl damping fluid in broken vegetable cell.

Described plant sample can be any part of plant materials, as root, and stem, blade etc.

In the step 1), the available quality percentage composition is that the SDS of 0.5-3% or the aqueous solution of Triton X-100 carry out rinsing, also availablely contains SDS that the quality percentage composition is 0.5-3% or the pH of Triton X-100 is that 6.8-7.6, concentration are 0.05-0.2molL ^-1Tris-HCl or phosphate buffered saline buffer carry out rinsing.

Step 2) in, to contain the quality percentage composition be that the pH of 2%SDS is 7.2 0.1molL to being deposited in of thick silicon body and silicidize cell walls of containing that step 1) is obtained ^-1In the damping fluid, be incubated processing in the Tris-HCl damping fluid.

Step 2) in, described insulation is treated to 90 ℃ of insulation 1h.

In the step 3), use 6 molL ^-1NaCl solution carry out rinsing.

In the step 4), described filter footpath is preferably 30-50 μ m, especially is preferably 40 μ m.

In the step 4), the filtrate natural subsidence of collection or centrifugal, the silicon body that obtains purifying precipitates.

For fear of traditional wet digestion and dry ashing method destruction to organic composition in the silicon body, the method that the invention provides a kind of pure physics is extracted natural silicon body from plant, be referred to as low temperature and smash the fractional separation method to pieces, use this method, from the fresh blade of paddy rice, isolate a large amount of silicon bodies that contains active skull cap components first, on the basis that obtains pure natural silicon body, further extracted the protein (silicon is conjugated protein) of combining closely with silicon, external checking silicon is conjugated protein to have the sedimentary function of the silicic acid of inducing, and intravital distribution and silicon have good dependency again.

Compare with the method for high temperature dry ashing divided silicon body with traditional wet digestion of strong acid, great advantage of the present invention is to keep that the organism composition is not destroyed in the silicon body, this physiological function, disclose the molecular mechanism of siliceous deposits and utilize natural silicon body, prerequisite is provided in the research in other field for further research silicon.With the silicon body that method of the present invention is extracted, be suitable for further separating and the interior conjugated protein effect in the siliceous deposits metabolism of organic macromolecule, particularly silicon of research silicon body; By to the protein-bonded research of silicon, find the metabolic gene of control siliceous deposits, not only can promote the molecular improvement of plant stress-resistance proterties, also help to understand genes involved, this will greatly promote the physiological fundamental research of plant silicon nutrient molecule, simultaneously have important application prospects in research fields such as development, the molecular improvement of crop anti-adversity shape, sanipratics, the bionic nano silicon materials of siliceous fertilizer are synthetic.

The invention will be further described below in conjunction with the drawings and specific embodiments, but be not that the present invention is limited.

Description of drawings

Figure 1A is the microphotograph (* 300) through 1mm net filtration gained filtrate

Figure 1B is the coomassie brilliant blue staining photo (* 300) through 1mm net filtration gained filtrate

Fig. 1 C is the microphotograph (* 300) of thick silicon body and silicidize cell walls

Fig. 1 D was that cell wall fragments was stayed the microphotograph (* 300) on the screen cloth after 40 μ m sieved

Fig. 1 E and Fig. 1 F are the microphotograph (* 150) of pure silicon body

Fig. 2 A is the dyeing photo (* 600) of cell wall polysaccharides in the thick silicon body

Fig. 2 B is the polysaccharide dyeing photo (* 600) of pure silicon body

Fig. 3 A is the stereoscan photograph (* 2000) of single silicon body

Fig. 3 B is the stereoscan photograph (* 2000) of single silicon body side surface

Fig. 4 A is the Laser Scanning Confocal Microscope home position observation photo (* 800) of paddy rice vein surface splayed silicon body

Fig. 4 B is autofluorescence (* 600) photo of the stripped silicon body of surface fluorescence microscopically

Fig. 4 C is the Laser Scanning Confocal Microscope light section photo (* 600) of fresh silicon body inside

The isolating silicon body of Fig. 5 A wet digestion method (differing illumination) microphotograph (* 800)

The surface fluorescence photo (* 800) of Fig. 5 B wet digestion method divided silicon body

Fig. 6 A is natural silicon body (methyl red dyeing) (* 600) photo in the intact leaf

Fig. 6 B is silicon body (methyl red dyeing) (* 150) photo of separation and purification

Fig. 7 is the conjugated protein sedimentary electromicroscopic photograph that forms with water glass of the silicon that extracts from the silicon body that extracts with method of the present invention

Fig. 8 is the SDS-PAGE collection of illustrative plates of total soluble protein in the conjugated protein and blade of silicon

Embodiment

Experimental technique among the following embodiment if no special instructions, is ordinary method.

Percentage composition among the following embodiment if no special instructions, is the quality percentage composition.

Embodiment 1, utilize method of the present invention separating natural silicon body from paddy rice

One, extracts natural silicon body

Identify the purity of silicon body with the microscopic examination method: the isolate that following steps are obtained is put on the slide glass, by the multi-functional microscope of Lecia, adopts dark-field or differs illumination and observe the purity of silicon body and take pictures.

Concrete steps are as follows:

1, thick silicon body and silicidize cell walls separates

Fresh rice leaf after the flushing with clean water is cut into the fragment of 1-2mm, grinds plant tissue with liquid nitrogen freezing and in mortar, add the 0.1molL that contains 1%Trion X-100 by 1: 3 (V/V) then ^-1Tris-HCl (pH7.2) damping fluid, with the further broken vegetable cell of high-speed tissue mashing machine, with the aperture is the screen filtration homogenate of 1mm, and with deionized water washing screen hole, containing diameter in the filtrate that obtains is following silicidize cell wall fragments and (Figure 1A of tenuigenin group of 1mm, arrow shows the silicon body, and CW is a cell wall fragments, and CY is a tenuigenin group).This filtrate is used coomassie brilliant blue staining, judge the protein pollutant of silicon surface, (Pearse A G E.1985.Histochemistry with reference to Pearse for concrete grammar, theoretical and applied.Vol2.Churchill Livingstone, London.1055p.), add 0.5% Xylene Brilliant Cyanine G R-250, reacted 3 minutes, with washed with de-ionized water 3 times, each 10 minutes; The result is shown in Figure 1B, and protein is dyed blueness (Figure 1B, arrow show the silicon body, and CW is a cell wall fragments, and CY is a tenuigenin group), and the pollution of foreign protein is described.

Have the principle of larger specific gravity according to the silicidize cell walls, earlier filtrate stired and make muddy, leave standstill then, treat natural subsidence 2-3min after, outwell supernatant liquor.With the 0.5%SDS aqueous solution rinsing and sedimentation filtrate repeatedly, the transparent no suspended particle of supernatant fluid color after sedimentation, can remove most of non-silicidize cell wall fragments and kytoplasm pollutent like this, what obtain is precipitated as thick silicon body and silicidize cell walls (Fig. 1 C, arrow shows the silicon body, and CW is a cell wall fragments).Dye the method (Periodic acid-schiff's reaction method) of polysaccharide with specificity and judge silicon surface polysaccharide pollutent, specifically (Pearse A G E.1968.Histochemistry with reference to Pearse, theoreticaland applied.Vol 1.Churchill Livingstone, London.758p.), the sodium borohydride of adding 1% is blocked endogenous aldehyde radical earlier, react after 30 minutes, with washed with de-ionized water 15 minutes, add 1% periodate oxidation again after 15 minutes, with washed with de-ionized water 15 minutes, with Schiff's reagent dyeing after 30 minutes with deionized water rinsing 15 minutes (3 times).Glycocalix is dyed redness, and the result is shown in Fig. 2 A, and showing has slight sugared contaminating impurity (among Fig. 2 A, circle shows sugared contaminating impurity).

2, the removal of contaminating protein in the silicidize cell-wall component

Thick silicon body and silicidize cell walls that step 1 obtains are precipitated as light green, contain some insoluble plasmalemma proteins, phyllochlorin and non-specific adsorption and ionic linkage bonded cytoplasmic protein, need to use washing agent (2% SDS rinsing liquid), acetone and high level salt solution successively with its wash-out, each step needs to handle twice at least.Concrete grammar is as follows:

1) SDS rinsing: thick silicon body and silicidize cell walls that step 1 is obtained precipitate and the 0.1molL that contains 2%SDS ^-1Tris-HCl (pH 7.2) damping fluid is put in (90 ℃) in the thermostat water bath by the mixed of 1: 4 (V/V), insulation 1h, during intermittent type stir, wash 4-5 time at last.Repeat once this rinse cycle.Then supernatant liquor is outwelled, remaining precipitation is for next step processing;

2) acetone rinsing: under the room temperature with step 2) in the precipitation that obtains and acetone by the mixed of 1: 2 (V/V), outwell acetone behind the magnetic agitation 30min, repeat 1-2 time again, separate out up to no yellow substance;

3) high level salt solution rinsing: under the room temperature with the precipitation and the 6molL that obtain in the step 3) ^-1NaCl solution by the mixed of 1: 4 (v/v), outwell behind the magnetic agitation 30min, wash 4-5 time, collecting precipitation contains silicon body and silicidize cell walls in this precipitation.

3, the acquisition of pure silicon body

With step 2 3) in the precipitation that obtains to continue with the aperture be that the screen filtration of 40 μ m is also used deionized water rinsing, as seen big silicidize cell wall fragments is trapped in (Fig. 1 D on the screen cloth, CW is a cell wall fragments), and the silicon body can be by filter down, again through natural subsidence can obtain purified silicon body precipitation (Fig. 1 E, F).

Should carry out polysaccharide dyeing according to the method described above by pure silicon body, the result shows that this pure silicon body does not have polysaccharide to pollute shown in Fig. 2 B.

Should carry out coomassie brilliant blue staining according to the method described above by pure silicon body, the result shows the pollution that does not have other foreign protein on the pure silicon body.

Should drip on the cover glass by pure silicon body, drying at room temperature, spray the thick golden conductive layer of 20nm then, under scanning electron microscope (Quanta of FEI Co. 200 types), observe and take pictures, the result is shown in Fig. 3 A and Fig. 3 B, the photo (Fig. 3 A and Fig. 3 B) that still amplifies from the part from whole (Fig. 1 F) no matter, the purity of silicon body all is very high, reaches more than 95%.The clear demonstration silicon body of the electron microscope photo scanning of single fan-shaped silicon body (Fig. 3 A) integral surface is smooth not to have other impurity to adhere to, prove this method separable reliability and feasibility to pure silicon body, there are a lot of circular pit (Fig. 3 B) another side of silicon body, for original and the contacted mesophyll cell of silicon body the life site, after physical separation method processing of the present invention, the mesophyll cell that links to each other with the silicon body is completely removed, only remaining its trace on the silicon body.

Method of the present invention can make isolating silicon body obtain continuous purifying, from more contaminating impurity (Figure 1A) is arranged, to slight contaminating impurity (Fig. 1 C) is arranged, arrive at last purified silicon body (Fig. 1 E, F).

The observation of the inner polyphenolic compound of silicon body that embodiment 2, the inventive method are extracted

(stone is green to issue the characteristic of yellow-green fluorescence according to phenolic compound at ultraviolet excitation, Di Ying .2000. plant polyphenol. Science Press, P134-138.), whether the inner organism of silicon body is destroyed and can be determined according to the power of its phenolic compound fluorescence, if fluorescent weakening shows that then organism is destroyed.Observe the live body blade with laser confocal microscope (Lecia TCS SP2 type), the result shows in the live body blade surface splayed silicon body to have autofluorescence (Fig. 4 A), illustrates that the silicon body contains phenolic compound, and it is the inner a kind of crude substance that exists of silicon body.With the purified silicon body of the multi-functional fluorescence microscope of Leica, under ultraviolet excitation, can see tangible yellow-green fluorescence (Fig. 4 B), further utilize the light slicing characteristics of laser confocal microscope to observe, as seen these fluorescence are from silicon body inside (Fig. 4 C), the organism of hence one can see that separation method of the present invention do not destroy silicon body inside.

The specificity dyeing of embodiment 3, silicon body

With reference to (Dayanakan P. such as Dayanakan, P.B.Kaufman, C.L.Franklin.1983.Detection of silica in Plants.Amer.J.Bot., 70 (7): dyeing process 1979-1084.) is also improved the silicon body in the rice leaf is carried out original position dyeing, and basic step is as follows:

1) decolouring: it is that the segment of 0.5cm is put into vial that blade is cut into length, the mixed solution 2-3ml that adds isopyknic ethanol and acetone, (preventing that volatilization from becoming dry) closes the lid, the room temperature lower magnetic force stirs, change new soln after solution becomes deep green, material becomes white after removing chlorophyll fully;

2) acidolysis is handled: outwell the mixed solution of ethanol and acetone, washing twice adds 50% vitriolization 5min, fully washes then 4-5 time (each 1-2min);

3) dehydration: 30% ethanol (5min) → 50% ethanol (5min) → 70% ethanol (5min) → 100% ethanol (5min) → 1/2 (volume) ethanol+1/2 (volume) dimethylbenzene (5min) → xylene solution (twice, each 5min);

4) dyeing and mounting: outwell dimethylbenzene, the xylene solution that adds a small amount of 0.1% methyl red, making it the submergence vegetable material gets final product, dyeing 20-30min, unnecessary pigment washes with xylene solution, add one on the slide glass and drip the natural gum of putting on airs, to dye good material then is put in the natural gum, covered is examined under a microscope, and the silicon body in the blade or the structure of silicidize are dyed by methyl red and be redness, the result as shown in Figure 6A, arrow shows the natural silicon body in the intact leaf, and the rice leaf surface mainly contains the silicon body of two kinds of forms, the fan-shaped silicon body (Fig. 6 A arrow 2) in the intracellular dumb-bell shape silicon of epidermis silicon body (Fig. 6 A arrow 1) and the bulliform cell.

Xylene solution with silicon body and function 0.1% methyl red of separation and purification among the embodiment 1, dyeing 20-30min, use microscopic examination, the result is shown in Fig. 6 B, the silicon body that wherein shows red and pink colour is the new silicon body that forms, and the silicon body that is not colored of part very high silicon body (the Dannayana et al. that early forms that is extent of polymerization, 1983, Dayanakan P., P.B.Kaufman, C.L.Franklin.1983.Detection of silica inPlants.Amer.J.Bot., 70 (7): 1979-1084).From these two photos as can be seen, the silicon body in the rice leaf has identical dyeing characteristic with the silicon body that separation and purification goes out.

Comparative Examples 1, dry ashing and wet digestion method divided silicon body

The difference of the silicon body that extracts for traditional method relatively and method of the present invention is extracted the silicon body with dry ashing method and wet digestion method respectively, by organic loss in the silicon body is judged in the observation of silicon body autofluorescence.

Wet digestion method is with reference to Harrison etc. (1996, Harrison C.C.1996.Evidence forintramineral macromolecules containing protein from plant silica.Phytochem., method 41:37-42), get fresh rice leaf and be cut into segment, blade is put in (V: V=4: 1) in the vitriol oil and the concentrated nitric acid mixed solution, 60-70 ℃, handled 24 hours, or with the perchloric acid of concentrated nitric acid and 60% (V: V=1: 1), 60 ℃, 30min, change extracting solution 6 times, centrifugal (800g, 5min), after the rinsed with deionized water, in 4 ℃ of preservations.Observe the fluorescence that phenolic compound sends in the silicon body, see whether organic loss takes place.The result is shown in Fig. 5 A and Fig. 5 B, show fresh paddy rice leaf with the vitriol oil and concentrated nitric acid (V: V=1: 4) appearance of the silicon body that obtains after handling of mixed solution (60-70 ℃) with separate the silicon volume morphing no significant difference that obtains with method of the present invention, all be typical fan-shaped silicon body.But under fluorescent microscope, it does not have autofluorescence (Fig. 5 B), illustrates that wherein aldehydes matter is by the strong acid oxygenolysis.

According to (Parr J F such as Parr, Lentfer C J and Boyd W E 2001 A comparativeanalysis of wet and dry ashing techniques for the extraction of phytolithsfrom plant material.J.Arch.Sci.28, dry ashing method 875-886) is extracted the silicon body, concrete steps are as follows: get fresh rice leaf and be cut into segment, be placed in the crucible, place Ma Fulu again, heat 6 hours (550 ℃), cooling back adds 10ml 10%HCl and was put in the water-bath (70 ℃) heating 20 minutes, centrifugal (3500rpm, 5min), the centrifugal (3500rpm in washing back, 5min), add 10ml 15%H ₂O ₂Be put in the water-bath (70 ℃) heating 20 minutes, centrifugal (3500rpm, 5min), the washing back is centrifugal, and (3500rpm 5min), adds 1ml ethanol and spends the night.Observe the fluorescence that phenolic compound sends in the silicon body, see whether organic loss takes place.The result shows that the dry ashing method separates in 90% the silicon body obtain and do not have autofluorescence, illustrates that this separation method also can cause organic loss in the silicon body.

Comparative Examples 2, to extract silicon from the silicon body conjugated protein

1, extraction silicon is conjugated protein

With reference to Harrison (1996, Harrison C.C.1996.Evidence for intramineralmacromolecules containing protein from plant silica.Phytochem., method 41:37-42) is extracted silicon in conjunction with egg certainly, and the concrete operations step is as follows:

1) 10molL of preparation pH5.0 ^-1NH ₄F solution will must not be used Glass Containers with clean plastic containers.Want steady and fast when transferring pH, prevent overlong time and damage pH meter;

2) prepare dialysis tubing: with molecular weight cut-off is that the dialysis tubing of 500kDa is at 2%NaCO ₃, 1mmolL ^-1EDTA boils 10min in the pH8.0 solution, and cooling is used the distilled water cleaning down, 4 ℃ of preservations;

3) NH ₄F handles: adding 8ml 10molL in the wet digestion silicon body that extracts with wet digestion method in natural silicon body that every 1g extracts with the method for embodiment 1 or the Comparative Examples 1 ^-1NH ₄F also constantly uses microscopy with magnetic stirrer under the room temperature, dissolves fully until the silicon body, filters then in Plastic Bottle, filtrate is put in 4 ℃ of refrigerators preserves.

4) dialysis: filtrate is packed in the dialysis tubing, be put in 4 ℃ the deionized water and dialyse, every 3-6h changes water one time, continues 3～4 days, reaches the pH value of deionized water up to the pH of dialyzate value, obtains NH ₄The F extracting solution;

5) concentrate: NH ₄After the lyophilize of F extracting solution, collect dry thing and be NH ₄The F extract.The result shows, obtains 1mg protein in the natural silicon body of every 1g that extracts with method of the present invention; And have to obtain 0.1mg protein in the wet digestion of the every 1g silicon body.

2, NH ₄The F extract is induced the observation of silicon precipitation ability

Get 20 μ l NH respectively ₄The F extracting solution is in Eppendorf, add 20mM water glass-phosphoric acid buffer (pH5.7) reaction 2 hours that 20 μ l newly join, the centrifuging and taking precipitation, and repeatedly wash with deionized water, remove unprecipitated water glass, the silicon precipitation is suspended in the small amount of deionized water, get hanging drop to cover glass, dry under the room temperature, spray the thick golden conductive layer of 20nm then, under the Philips scanning electron microscope, observe and take pictures.The result shows the NH that obtains from the natural silicon body that extracts with the method for embodiment 1 ₄The F extracting solution can cause the sedimentary formation of silicon within 5 minutes, be precipitated as sphere aggregates (Fig. 7); And the NH that from wet digestion silicon body, obtains ₄The F extracting solution can not be induced the sedimentary formation of silicon within 2 hours, illustrating that silicon in the wet digestion silicon body is conjugated protein to be destroyed.

Embodiment 4, SDS-polyacrylamide gel electrophoresis are analyzed the protein-bonded composition of silicon

With reference to Hermannn and Gebhard (Hermann S., V.J.Gebhard.1987.Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for theseparation of protein in the range from 1to 100 kDa.Anal.Biochem., method 166:368-379) adopts the SDS-PAGE electrophoresis to come separation and purification conjugated protein with the silicon in the silicon body of the method extraction of embodiment 1.Resolving gel concentration 8%, each sample are got 20 μ l and (are contained 20 μ g NH ₄The F extract) add 5 μ l sample buffers (5 *), boil sample 5min before the last sample, centrifugal, suct and reset and add on the running gel in the sample hole.First prerunning 15min under 30V is pressurized to 105-120V after waiting sample to run out of the sample pore chamber fully, and electrophoresis chamber is placed in the water-bath that ice bag is housed.After electrophoresis finishes, glue is prior to fixing 30min in the stationary liquid (50% methyl alcohol and 10% acetate), and poststaining (0.025% Xylene Brilliant Cyanine G G-250 and 10% acetate) 1.5h, decolouring (10% acetate) is clear to electrophoretic band again. and the result is as shown in Figure 8, show that total soluble protein of blade and silicon do not have overlapping in conjunction with total protein, the silicon that explanation is extracted from the natural silicon body that extracts with the method for embodiment 1 is conjugated protein peculiar by the silicon body, is not subjected to the pollution of cell soluble protein.NH ₄The silicon that F extracts is conjugated protein two bands, and a tangible master tape is arranged near molecular weight marker116 kDa, in addition the protein band (Fig. 8 swimming lane C) of a faint 100kDa in addition in its lower section.Among Fig. 8, swimming lane A is Marker, and molecular weight ranges is 20 to 212kDa; Swimming lane B is the blade total soluble protein; Swimming lane C is for using NH ₄The silicon that F extracts from the natural silicon body that extracts with the method for embodiment 1 is conjugated protein.

Claims (10)

1, a kind of method of extracting the silicon body from plant may further comprise the steps:

1) broken vegetable cell in the damping fluid of SDS that contains 0.5-3% or Triton X-100 obtains homogenate; Collect the cell wall fragments of diameter below 1-2mm in the described homogenate; The collected cell wall fragments of rinsing repeatedly, the supernatant fluid color after sedimentation is transparent and do not have suspended particle, obtains containing the precipitation of thick silicon body and silicidize cell walls;

2) in SDS that containing of step 1) being obtained, being deposited in of thick silicon body and silicidize cell walls contained 0.5-3% or the damping fluid of triton X-100, be incubated and handle the back collecting precipitation; Described insulation is treated to 50-95 ℃ of insulation 0.5-1h;

3) with step 2) in the precipitation of collecting carry out rinsing with acetone or ether, separate out until no yellow substance; Use 1-8 molL again ^-1NaCl solution or 0.1-0.5molL ^-1CaCl ₂The solution rinsing, the mixture that is precipitated as silicon body and silicidize cell walls that obtains;

4) the silicon body that step 3 is obtained and the mixture of silicidize cell walls are the filtration of 30-90 μ m by the filter footpath, collect filtrate, obtain the silicon body precipitation of purifying;

In the above-mentioned steps, described percentage composition is the quality percentage composition; Step 1) and step 2) in described buffered soln to be pH be that the concentration of 6.8-7.6 is 0.05-0.2molL ^-1Tris-HCl or phosphate buffered saline buffer.

2, method according to claim 1, it is characterized in that: in the step 1), described broken vegetable cell is earlier with the liquid nitrogen freezing plant sample and grind plant tissue in mortar, then in containing the damping fluid that the quality percentage composition is 0.5-3%SDS or Triton X-100, with the broken vegetable cell of high-speed tissue mashing machine.

3, method according to claim 1 and 2 is characterized in that: in the step 1), be 7.2 at the pH that contains 1%TritonX-100, concentration is 0.1molL ^-1The Tris-HCl damping fluid in broken vegetable cell.

4, method according to claim 1 and 2, it is characterized in that: in the step 1), with the quality percentage composition is that the SDS of 0.5-3% or the aqueous solution of Triton X-100 carry out rinsing, or is that 6.8-7.6, concentration are 0.05-0.2molL with containing SDS that the quality percentage composition is 0.5-3% or the pH of Triton X-100 ^-1Tris-HCl or phosphate buffered saline buffer carry out rinsing.

5, method according to claim 1 and 2 is characterized in that: step 2) in, with step 1) containing of obtaining pH that being deposited in of thick silicon body and silicidize cell walls contain 2%SDS be 7.2, concentration is 0.1molL ^-1Be incubated processing in the Tris-HCl damping fluid; Described percentage composition is the quality percentage composition.

6, according to claim l or 2 described methods, it is characterized in that: step 2) in, described insulation is treated to 90 ℃ of insulation 1h.

7, method according to claim 1 and 2 is characterized in that: in the step 3), use 6molL ^-1NaCl solution carry out rinsing.

8, method according to claim 1 and 2 is characterized in that: in the step 4), described filter footpath is 30-50 μ m.

9, method according to claim 8 is characterized in that: described filter footpath is 40 μ m.

10, method according to claim 1 and 2 is characterized in that: in the step 4), and the filtrate natural subsidence of collection or centrifugal, the silicon body that obtains purifying precipitates.

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