CN103589678A - Method for extraction of Crassulaceae plant cell vacuoles - Google Patents
Method for extraction of Crassulaceae plant cell vacuoles Download PDFInfo
- Publication number
- CN103589678A CN103589678A CN201310532489.2A CN201310532489A CN103589678A CN 103589678 A CN103589678 A CN 103589678A CN 201310532489 A CN201310532489 A CN 201310532489A CN 103589678 A CN103589678 A CN 103589678A
- Authority
- CN
- China
- Prior art keywords
- vacuole
- density gradient
- protoplastis
- gradient medium
- solution
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Abstract
The invention discloses a method for extraction of Crassulaceae plant cell vacuoles. The method includes protoplast extraction, vacuole coarse extraction, and vacuole purification. The vacuole purification includes: resuspending the vacuoles obtained by coarse extraction in a density gradient medium solution with a mass/volume concentration of 0-5%, adding a density gradient medium solution with a mass/volume concentration of 7-10% into the lower layer of the resuspension solution, and carrying out density gradient centrifugation, thus obtaining purified vacuoles. In the invention, during vacuole purification, firstly the density gradient medium solution with a small concentration is utilized to resuspend the coarsely extracted vacuoles in a centrifuge tube, then the density gradient medium solution with a large concentration is slowly added into the centrifuge tube bottom along the centrifuge tube wall, the solution in the centrifuge tube falls into two layers at the moment, and then density gradient centrifugation is performed. The obtained vacuoles have the advantages of high membrane integrity, large quantity, and high purity.
Description
Technical field
The invention belongs to vegetable cell vacuole extractive technique field, be specifically related to a kind of method of extracting crassulaceae plants Cell vacuoles.
Background technology
Sedum alfredii Hance is one of a few Cd hyperaccumulator of China domestic discovery, belongs to Crassulaceae, to zinc lead, also has very strong patience and accumulation ability ， China heavy-metal contaminated soil phytoremediation field to have great application prospect.Research to Sedum alfredii Hance Hyperaccumulation and physiological mechanism, can not only promote this plant in contaminate environment, to repair the direct application in field, more contribute to verify tired this phenomenon of heavy metal of plant ultraproduct, for promoting improvement and the optimization of phytoremediation technology of polluted soil, provide reliable theoretical basis.Given this, Sedum alfredii Hance is as the special plant of a kind of many Metal-accumulations of Asia, and its correlative study has caused many external colleagues' concern.
Early-stage Study finds, Cd hyperaccumulator Sedum alfredii Hance root system can active absorption cadmium, by efficient haulage system, is transported to plant shoot a large amount of accumulation.As a kind of biological nonessential element, cadmium too much accumulation in common vegetable cell can produce serious toxic action conventionally.Visible, Sedum alfredii Hance overground part is certainly existing a set of mechanism of the homeostasis for a large amount of cadmium elements.
It has been generally acknowledged that, heavy metal depends on the effect that separates (Sequestration) of specific cells or organoid (as cell walls, vacuole etc.) and the sequestering action (Chelation) of some organic compound at the stable state network regulation of plant shoot.
Vacuole skin in vegetable cell has the saturating property of special selection, makes vacuole have hypertonicity matter, causes that moisture moves in vacuole, exchanges ganglion cell's osmotic pressure, maintains turgescence and have much relations, and can make many kinds of substance in vacuole, store and accumulate.The early-stage Study result hint of relevant Sedum alfredii Hance, Sedum alfredii Hance overground part may be the effect that separates of vacuole to the super Accumulation Mechanism key of cadmium.By LA-ICP-MS and two kinds of different analytical technologies of synchrotron radiation X RF, the distribution characteristics of cadmium in Sedum alfredii Hance cauline leaf is carried out to original position analysis, all show that the place that mainly separates of cadmium is the parenchymas such as cortex ,Sui He mesophyll tissue.Visible, containing the parenchyma cell of a large amount of vacuoles, at Sedum alfredii Hance, occupy an leading position in to the separating process of cadmium, this is mainly present in epidermic cell greatly different from the cadmium of finding on other hyperaccumulative plants, but need further to studies confirm that, and the extraction of vacuole acts on separating of cadmium in hyperaccumulative plant further clear and definite vacuole and Related Mechanism is particularly important.
The method that vacuole extracts generally comprises lysing cell and extracts protoplastis, then cracking protoplastis extracts vacuole.Because the cellularstructure of different vegetable cells is variant, and the membrane structure of vacuole is very easily affected by the external environment and breaks, and the method for therefore extracting vacuole is also not quite similar.Existing relevant report (the Ma of extraction for the vacuole of penny cress blade, J.F., et al., Subcellular localisation of Cd and Zn in the leaves of a Cd-hyperaccumulating ecotype of Thlaspi caerulescens.Planta, 2005.220 (5): 731-736).But the method is in vacuole purge process, first crude extract bubble is resuspended in the ficoll solution that concentration is larger, on the upper strata of this resuspended liquid, add again the ficoll solution that one deck concentration is less and carry out density gradient centrifugation, the efficiency of extracting vacuole is lower, even cannot obtain the vacuole complete, purity is higher.
Summary of the invention
The invention provides a kind of method of extracting crassulaceae plants Cell vacuoles, utilize the method can be simple and effectively from crassulaceae plants cell extraction to vacuole complete and that purity is higher.
Extract a method for crassulaceae plants Cell vacuoles, comprise that protoplastis extracts, vacuole is slightly carried and vacuole purifying, described vacuole purifying comprises:
It is in 0～5% density gradient medium solution that the vacuole of slightly carrying is resuspended in to mass volume ratio concentration, obtains resuspended liquid; Toward this resuspended liquid lower floor, adding mass volume ratio concentration is 7～10% density gradient medium solution, carries out density gradient centrifugation, obtains the vacuole of purifying.
Density gradient centrifugation is generally carried out in the less centrifuge tube of volume.Applicant utilizes the resuspended crude extract bubble of density gradient medium solution that mass volume ratio concentration is less, along centrifugal tube wall toward centrifuge tube, bottom slowly adds the density gradient medium solution that concentration is larger again, now in centrifuge tube, solution is divided into two-layer, and then carry out density gradient centrifugation, in two layers of solution, there will be a pink chromatograph, in this pink chromatograph, be the vacuole of purifying.
Compare with the density gradient medium solution (lower floor, viscosity is large) of high density, the vacuole being resuspended in the density gradient medium solution (upper strata, viscosity is little) of lower concentration is more easily assembled toward the separation surface of two-layer density gradient medium solution; And in the density gradient medium solution of high density, more difficult removal protoplastis sticks to the protoplast membrane on vacuole surface while breaking, be difficult to obtain the vacuole that purity is higher.
Because density gradient centrifugation is carried out in centrifuge tube, when slowly adding lower floor's density gradient medium solution along centrifugal tube wall toward bottom, the contact surface between this solution and resuspended liquid changes from small to big, and makes separation surface between the two comparatively clear; If first at centrifuge tube Zhong Jia lower floor density gradient medium solution, then increase the weight of suspension on upper strata, contact surface is between the two larger from the beginning, after liquid feeding completes, separation surface between the two slightly dislikes fuzzy, is unfavorable for the purifying of vacuole.
As not making specified otherwise, in the present invention, mass volume ratio concentration be take g/100mL as unit, and 0～5% is equivalent to 0～5g/100mL(0～50g/L), 7～10% be equivalent to 7～10g/100mL(70～100g/L).
In the present invention, described crassulaceae plants cell is preferably parenchymatous cell, more preferably mesophyll tissue's cell.Parenchyma keratinization degree is lower, and pre-treatment is comparatively convenient.Described crassulaceae plants is preferably Sedum alfredii Hance.Being convenient to provides technique means for the super Accumulation Mechanism of cadmium in Sedum alfredii Hance.
Particularly, the method that the present invention extracts crassulaceae plants Cell vacuoles comprises:
(1) protoplastis extracts: get Sedum alfredii Hance blade, immerse in cell pyrolysis liquid after being cut into the fine strip shape that width is 1～2mm, after cracking, filtration, eccentric cleaning, obtain protoplastis;
Preferably take young leaflet tablet and cut into slices, before section, first tear the upper epidermis of blade off or/and lower epidermis, then cut into slices with the direction vertical with vein, to improve the extraction efficiency of protoplastis.
Described cell pyrolysis liquid is preferably the aqueous solution consisting of the following composition: 1-1.5%(w/v) cellulase, 0.2-0.4%(w/v) macerozyme, 0.35～0.45M N.F,USP MANNITOL, 18～22mM Repone K, 18～22mM, mono-water morpholino b acid, 8～12mM CaCl
2calcium chloride, 4～6mM beta-mercaptoethanol; The aqueous solution more preferably consisting of the following composition: 1-1.5%(w/v) cellulase, 0.2-0.4%(w/v) macerozyme, 0.4M N.F,USP MANNITOL, 20mM Repone K, 20mM mono-water morpholino b acid, 10mM CaCl
2calcium chloride, 5mM beta-mercaptoethanol.
Macerozyme is for emanating mesophyll tissue for unicellular, cellulase is for degradation of cell wall, and beta-mercaptoethanol is for the damage of anti-block to protoplastis, and other materials are as osmotic pressure regulator, maintain osmotic pressure balance in protoplastis, prevent that protoplastis from breaking.
Cracking, by removing by filter mesophyll tissue, obtains the protoplastis of slightly carrying and it is carried out to eccentric cleaning.The rotating speed of eccentric cleaning is preferably 80～100g, and the time is preferably 8～12min, and temperature is preferably 4～10 ℃; Eccentric cleaning can optionally repeat 1～2 time, and the centrifugal condition of low speed low temperature is conducive to make protoplast membrane to keep complete.
Eccentric cleaning step should be protected in liquid and carry out at protoplastis, and this protoplastis protection liquid can adopt following one-tenth assignment system: 150～155mM sodium-chlor, 120～125mM calcium chloride, 4～6mM Repone K, 1～2mM, mono-water morpholino b acid.
(2) vacuole is slightly carried: adopt protoplastis lysate cracking protoplastis, and the vacuole that centrifugal rear acquisition is slightly carried, described centrifugal rotating speed is 300～600g, the time is 5～8min.
Protoplastis pyrolysis time is preferably 30～60min, more preferably 30min.
Described protoplastis lysate is preferably the aqueous solution consisting of the following composition: 0.45～0.46M N.F,USP MANNITOL, 0.45～0.46mM ethylene glycol bis (2-amino-ethyl ether tetraacethyl), 0.90～0.91mM3-[(3-cholesterol aminopropyl) dimethylamino]-1-propanesulfonic acid and 18.18～18.19mM, mono-water morpholino b acid, 113.60～113.70mM calcium chloride, 4.50～4.60mM Repone K; PH is 8.0; More preferably: 0.455M N.F,USP MANNITOL, 0.455mM ethylene glycol bis (2-amino-ethyl ether tetraacethyl), 0.909mM3-[(3-cholesterol aminopropyl) dimethylamino]-1-propanesulfonic acid and 18.182mM mono-water morpholino b acid, 113.64mM calcium chloride, 4.505mM Repone K; PH is 8.0.
3-[(3-cholesterol aminopropyl) dimethylamino]-1-propanesulfonic acid as tensio-active agent so that protoplastis break, ethylene glycol bis (2-amino-ethyl ether tetraacethyl) is used as and calcium ion chelating, keep the interior calcium ion concn of vacuole in proper level, other materials, for maintaining the osmotic pressure balance of vacuole, prevent that vacuole from breaking.
As preferably, described centrifugal rotating speed is 500～600g, and the time is 5～6min.Crude extract bubble is carried out when centrifugal also should under low temperature (4～10 ℃) condition, carrying out.For obtaining the higher vacuole of purity, before carrying out vacuole purifying, available vacuole protection liquid carries out 1～2 cleaning to crude extract bubble, described vacuole protection liquid is preferably the aqueous solution consisting of the following composition: 18～22mM mono-water morpholino b acid, 0.6～1.0M N.F,USP MANNITOL, 113.60～113.70mM calcium chloride, 4.50～4.60mM Repone K; PH8.0; The aqueous solution more preferably consisting of the following composition: 20mM mono-water morpholino b acid, 0.8M N.F,USP MANNITOL, 113.64mM calcium chloride, 4.55mM Repone K; PH8.0.
(3) vacuole purifying: it is in 0～5% density gradient medium solution that the vacuole of slightly carrying is resuspended in to mass volume ratio concentration, toward this resuspended liquid lower floor, adding mass volume ratio concentration is 7～10% density gradient medium solution, carry out density gradient centrifugation, obtain the vacuole of purifying.
In above-mentioned concentration range, in upper strata density gradient medium solution and lower floor's density gradient medium solution, the mass volume ratio concentration difference of density gradient medium differs larger, purification effect to vacuole is better, preferably upper strata density gradient medium solution does not contain 10% density gradient medium containing density gradient medium ，Er lower floor density gradient medium solution.
The volume ratio of upper strata density gradient medium solution and lower floor's density gradient medium solution does not require, as long as double-layer separate interface is high-visible.
In the present invention, described density gradient medium solution is the vacuole protection liquid that is dissolved with density gradient medium, and the composition of described vacuole protection liquid is same as above.
Described density gradient medium can be selected ficoll, cesium chloride or sucrose.
Preferably, the rotating speed of described density gradient centrifugation is 800～1500g, and the time is 5～45min; More preferably, the rotating speed of described density gradient centrifugation is 1000～1500g, and the time is 10～30min.General 10min left and right, when centrifugal effect is not good enough, can proper extension centrifugation time.
If without specified otherwise, in the present invention, each solution all adopts ultrapure water preparation.
Compared with prior art, beneficial effect of the present invention is embodied in:
The present invention is when carrying out vacuole purifying, first in centrifuge tube, utilize the resuspended crude extract bubble of density gradient medium solution that concentration is less, along centrifugal tube wall toward centrifuge tube, bottom slowly adds the density gradient medium solution that concentration is larger again, now in centrifuge tube, solution is divided into two-layer, and then carry out density gradient centrifugation, thus obtained vacuole skin integrity is good, and vacuole quantity is many, and purity is high.
Accompanying drawing explanation
Fig. 1 is the state graph after Sedum alfredii Hance vanes cell pyrolysis liquid is processed;
The protoplastis aspect graph of Fig. 2 for extracting;
Fig. 3 is the aspect graph that in protoplastis, vacuole is colored;
Fig. 4 is the aspect graph of crude extract bubble;
Fig. 5 is embodiment 1 vacuole purification result figure;
Fig. 6 is embodiment 2 vacuole purification result figure;
Fig. 7 is embodiment 3 vacuole purification result figure;
Fig. 8 is embodiment 4 vacuole purification result figure;
Fig. 9 is embodiment 5 vacuole purification result figure;
Figure 10 is the result figure that embodiment 6 utilizes the protoplastis lysate cracking protoplastis of document record;
Figure 11 is the result figure that embodiment 6 utilizes 6/5 times of diluent cracking protoplastis of the protoplastis lysate that document records;
Figure 12 is the result figure that embodiment 7 utilizes the protoplastis lysate cracking protoplastis of the concentration that contains 2/3 times of CHAPS;
Figure 13 is the result figure that embodiment 7 utilizes the protoplastis lysate liquid cracking protoplastis of the concentration that contains 4/3 times of CHAPS.
Embodiment
Embodiment 1
1 solution preparation
(1) cell pyrolysis liquid: 15g cellulase, 4g macerozyme, 72.88g N.F,USP MANNITOL, 1.49g Repone K, 4.264g mono-water morpholino b acid, 1.11g calcium chloride, 0.39065g beta-mercaptoethanol, is dissolved in ultrapure water and is settled to 1L;
(2) protoplastis protection liquid: 0.1543g sodium-chlor, 13.875g calcium chloride, 0.373g Repone K, 0.426g mono-water morpholino b acid, is dissolved in ultrapure water and is settled to 1L;
(3) protoplastis lysate: 0.083g N.F,USP MANNITOL, 0.173g ethylene glycol bis 2-amino-ethyl ether (EGTA), 0.559g3-[(3-cholesterol aminopropyl) dimethylamino]-1-propanesulfonic acid (CHAPS), a 3.876g water morpholino b acid, 12.614g calcium chloride, 0.339g Repone K, is dissolved in 950mL ultrapure water, with the Tris solution of 2M, regulate pH to 8.0, then be settled to 1L; ;
(4) vacuole protection liquid: 4.264g mono-water morpholino b acid, 145.76g N.F,USP MANNITOL, 12.614g calcium chloride, 0.339g Repone K, is dissolved in 950mL ultrapure water, regulates pH to 8.0, then be settled to 1L with the Tris solution of 2M; ;
(5) 10% ficoll solution: 100g ficoll-400,4.264g mono-water morpholino b acid, 145.76g N.F,USP MANNITOL, 12.614g calcium chloride, 0.339g Repone K, is dissolved in 950mL ultrapure water, regulates pH to 8.0, then be settled to 1L with the Tris solution of 2M; ;
(6) neutral red solution: 0.1g toluylene red, the acetic acid of 200 μ L 1%, the chloroform of 50 μ L, ultrapure water is settled to 100mL, and 4 ℃ keep in Dark Place.
2 vacuoles extract
(1) preparation of samples
Get the tender Sedum alfredii Hance blade 4g of children, with tweezers, tear its upper epidermis or lower epidermis off, with thin blade, take the direction vertical with vein and be cut into the fine strip shape that width is 1～2mm, standby.
(2) protoplastis extracts
1. get 30mL cell pyrolysis liquid in 50mL centrifuge tube, 50 ℃ of preheating 10min, are transferred in 250mL vacuum-pumping flask cooling;
2. the fine strip shape blade cutting is immersed and is cooled in the cell pyrolysis liquid of room temperature (25 ℃), be positioned over isothermal vibration case (28-30 ℃) left and right after suction filtration 10min, 45-65r/min shakes 2-2.5h;
3. until fine strip shape blade, be substantially white in color and transparently while swimming on liquid level (Fig. 1), with 150 order filtering membranes, protoplastis be filled in 50mL centrifuge tube, and with twice of 5mL protoplastis buffer solution for cleaning flask and filter membrane;
4. the centrifuge tube that protoplastis is housed is placed in whizzer (model: beckman j2-hs) centrifugal, rotating speed 80g, 10 ℃ of temperature, time 10min, abandons supernatant;
5. get the rear recentrifuge of 30mL protoplastis protection liquid resuspension precipitation, rotating speed 80g, 10 ℃ of temperature, time 10min, abandons supernatant; Gained precipitation is the protoplastis that concentration is higher (Fig. 2).
(3) vacuole is slightly carried
1. get the protoplastis that 15mL protoplastis lysate resuspension step (2) obtains, add 75 μ L neutral red solution, slightly put upside down and mix 3min, to vacuole dye (Fig. 3);
2. the rear standing centrifuge tube that dyeed, makes protoplastis cracking 30min, during with 5mL pipettor, repeatedly blow and beat liquid level 5～8 times;
3. centrifuge tube is placed in whizzer (model: Thermo-S00264) centrifugal, select slow slow 3 grades or 4 grades of slowing down that start, rotating speed 600g, time 5min, 10 ℃ of temperature, abandon supernatant;
4. get 15mL vacuole protection liquid resuspension precipitation recentrifuge, select slow slow 3 grades or 4 grades of slowing down that start, rotating speed 600g, time 5min, 10 ℃ of temperature, abandon supernatant; Gained precipitation is the vacuole (Fig. 4) of slightly carrying.
Vacuole in protoplastis is dyed redness by toluylene red as seen from Figure 3, the peripheral parcel of red vacuole chloroplast(id); As seen from Figure 4, most of protoplastis cracking after centrifugal, the transparent red vacuole in visible edge, some protoplast membrane breaking still sticks to vacuole surface.
(4) vacuole purifying
1. get the vacuole that 10mL vacuole protection liquid resuspension is slightly carried, along centrifugal tube wall toward bottom, slowly add 10% ficoll solution 2mL, at this moment in centrifuge tube, formed 2 layerings;
2. centrifuge tube is placed in to whizzer (model: Thermo-S00264), carry out density gradient centrifugation, rotating speed 1500g, time 10min, 10 ℃ of temperature; At resuspended liquid and the pink chromatograph of 10% ficoll solution intermediate formation one, get pink chromatograph liquid mirror inspection (Fig. 5).
As seen from Figure 5, the transparent red vacuole in rarely seen edge in the visual field, has no protoplastis and other impurity.
Embodiment 2
Utilize method and the reagent identical with embodiment 1 to carry out vacuole extraction, but in centrifuge tube bottom, add 7% ficoll solution during purifying vacuole, carry out density gradient centrifugation, get middle transition layer (not forming obvious pink chromatograph) the liquid microscopy of resuspended liquid and 7% ficoll solution, microscopy the results are shown in Figure 6.
As seen from Figure 6, only have the red vacuole that a small amount of edge is transparent, most is the vacuole that protoplastis or surface adhesion have the protoplast membrane that breaks, and vacuole degree of purification is not high.
Embodiment 3
Utilize method and the reagent identical with embodiment 1 to carry out vacuole extraction, but in centrifuge tube bottom, add 4% ficoll solution during purifying vacuole, carry out density gradient centrifugation, get middle transition layer (this layer is extremely not obvious) the liquid microscopy of resuspended liquid and 4% ficoll solution, microscopy the results are shown in Figure 7.
In Fig. 7, only see that raw plastid or surface adhesion have the vacuole of the protoplast membrane that breaks, have no the red vacuole that edge is transparent.
Embodiment 4
Utilize method and the reagent identical with embodiment 1 to carry out vacuole extraction; but during purifying vacuole; adopt document (Ma; J.F.; et al.; Subcellular localisation of Cd and Zn in the leaves of a Cd-hyperaccumulating ecotype of Thlaspi caerulescens.Planta; 2005.220 (5): 731-736), record; the vacuole of slightly carrying with 2mL10% ficoll solution resuspension; on resuspended liquid upper strata, add again 10mL vacuole protection liquid, then carry out density gradient centrifugation.To appearing at pink chromatograph (this layer not obvious) the liquid microscopy between resuspended liquid and vacuole protection liquid, microscopy the results are shown in Figure 8.
Visible minority protoplastis in Fig. 8, most of for surface adhesion has the vacuole of the protoplast membrane that breaks, have no the red vacuole that edge is transparent.
Embodiment 5
Utilize method and the reagent identical with embodiment 1 to carry out vacuole extraction; but during purifying vacuole, first in centrifuge tube, add 2mL10% ficoll solution, then get the crude extract bubble of vacuole protection liquid resuspension for 10mL; be added to 10% ficoll solution upper strata, then carry out density gradient centrifugation.To appearing at the pink chromatograph liquid mirror inspection between resuspended liquid and vacuole protection liquid, microscopy the results are shown in Figure 9.
Visible minority protoplastis in Fig. 9, most of for surface adhesion has the vacuole of the protoplast membrane that breaks, have no the red vacuole that edge is transparent.
Embodiment 6
Utilize method and the reagent identical with embodiment 1 to carry out vacuole extraction, but during crude extract bubble, adopt document (Ma, J.F., et al., Subcellular localisation of Cd and Zn in the leaves of a Cd-hyperaccumulating ecotype of Thlaspi caerulescens.Planta, 2005.220 (5): (formula is the protoplastis lysate of recording 731-736): 0.5M N.F,USP MANNITOL, 1mM ethylene glycol bis 2-amino-ethyl ether, 0.5mM3-[(3-cholesterol aminopropyl) dimethylamino]-1-propanesulfonic acid, 20mM mono-water morpholino b acid, pH8.0), 6/5 times of diluent of this protoplastis lysate carries out cracking to protoplastis, the crude extract bubble obtaining is carried out to microscopy, microscopy the results are shown in Figure 10, Figure 11.
As seen from Figure 10, utilize the crude extract bubble of the protoplastis lysate cracking protoplastis acquisition of document record, vacuole skin has shrinkage, illustrates that also this protoplastis lysate concentration is excessive.
As seen from Figure 11, the crude extract bubble that utilizes 6/5 times of diluent cracking protoplastis of the protoplastis lysate that document records to obtain, its comparatively small amt, illustrates that cracking degree is inadequate.
In embodiment 1, protoplastis lysate is equivalent to 11/10 times of diluent that document is recorded formula, and cracking degree is suitable, and vacuole can not produce shrinkage.
Embodiment 7
Utilize method and the reagent identical with embodiment 1 to carry out vacuole extraction, but during crude extract bubble, in the protoplastis lysate adopting, the concentration of CHAPS is respectively 2/3 times and 4/3 times of embodiment 1 protoplastis lysate, and the crude extract obtaining is steeped and carries out microscopy, and microscopy the results are shown in Figure 12, Figure 13.
As seen from Figure 12, utilize the protoplastis lysate of the concentration that contains 2/3 times of CHAPS not good to the lytic effect of protoplastis, the not cracking of most protoplastiss.
As seen from Figure 13, utilize the protoplastis lysate of the concentration that contains 4/3 times of CHAPS too strong to the splitting action of protoplastis, protoplastis breaks substantially, and vacuole is also broken a lot.
Claims (8)
1. extract a method for crassulaceae plants Cell vacuoles, comprise that protoplastis extracts, vacuole is slightly carried and vacuole purifying, it is characterized in that, described vacuole purifying comprises:
It is in 0～5% density gradient medium solution that the vacuole of slightly carrying is resuspended in to mass volume ratio concentration, obtains resuspended liquid; Toward this resuspended liquid lower floor, adding mass volume ratio concentration is 7～10% density gradient medium solution, carries out density gradient centrifugation, obtains the vacuole of purifying.
2. the method for claim 1, it is characterized in that, it is in 0% density gradient medium solution that the vacuole of slightly carrying is resuspended in to mass volume ratio concentration, and toward this resuspended liquid lower floor, adding mass volume ratio concentration is 10% density gradient medium solution, carries out density gradient centrifugation.
3. the method for claim 1, it is characterized in that, described density gradient medium solution is the vacuole protection liquid that is dissolved with density gradient medium, described vacuole protection liquid is the aqueous solution consisting of the following composition: 18～22mM mono-water morpholino b acid, 0.6～1.0M N.F,USP MANNITOL, 113.60～113.70mM calcium chloride, 4.50～4.60mM Repone K; PH8.0.
4. method as claimed in claim 3, is characterized in that, described density gradient medium is ficoll, cesium chloride or sucrose.
5. the method for claim 1, is characterized in that, the rotating speed of described density gradient centrifugation is 800～1500g, and the time is 5～45min.
6. the method as described in as arbitrary in claim 1～5, is characterized in that, adopts protoplastis lysate cracking protoplastis, the vacuole that centrifugal rear acquisition is slightly carried, and described centrifugal rotating speed is 300～600g, the time is 5～8min.
7. method as claimed in claim 6, it is characterized in that, described protoplastis lysate is the aqueous solution consisting of the following composition: 0.45～0.46M N.F,USP MANNITOL, 0.45～0.46mM ethylene glycol bis (2-amino-ethyl ether tetraacethyl), 0.90～0.91mM3-[(3-cholesterol aminopropyl) dimethylamino]-1-propanesulfonic acid and 18.18～18.19mM, mono-water morpholino b acid, 113.60～113.70mM calcium chloride, 4.50～4.60mM Repone K; PH is 8.0.
8. method as claimed in claim 6, is characterized in that, protoplastis pyrolysis time is 30～60min.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310532489.2A CN103589678B (en) | 2013-10-31 | 2013-10-31 | A kind of method extracting crassulaceae plants Cell vacuoles |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310532489.2A CN103589678B (en) | 2013-10-31 | 2013-10-31 | A kind of method extracting crassulaceae plants Cell vacuoles |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103589678A true CN103589678A (en) | 2014-02-19 |
| CN103589678B CN103589678B (en) | 2015-09-16 |
Family
ID=50079996
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310532489.2A Active CN103589678B (en) | 2013-10-31 | 2013-10-31 | A kind of method extracting crassulaceae plants Cell vacuoles |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103589678B (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104388380A (en) * | 2014-12-10 | 2015-03-04 | 南京农业大学 | Method for extracting pear pollen tube vacuoles |
| CN104498585A (en) * | 2014-11-28 | 2015-04-08 | 湖南农业大学 | Method for determining content of nitrate nitrogen inside and outside vacuoles in plant protoplasts |
| CN105316275A (en) * | 2015-11-27 | 2016-02-10 | 甘肃农业大学 | Extraction method of halogeton mesophyll cell vacuoles |
| CN106244513A (en) * | 2016-07-26 | 2016-12-21 | 北京林业大学 | A kind of vacuole that separates is distributed and the method for motion with albumen on observation tonoplast |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101792730A (en) * | 2010-03-03 | 2010-08-04 | 中国科学院植物研究所 | Method for detecting tonoplast membrane proton stream |
-
2013
- 2013-10-31 CN CN201310532489.2A patent/CN103589678B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101792730A (en) * | 2010-03-03 | 2010-08-04 | 中国科学院植物研究所 | Method for detecting tonoplast membrane proton stream |
Non-Patent Citations (2)
| Title |
|---|
| 夏朝晖等: "盐和干旱胁迫对燕子掌(Crassula agenten Thunb.)叶片液泡膜H+-ATPase活性的影响", 《植物生理学报》 * |
| 李廷强等: "锌在超积累植物东南景天叶片细胞水平的吸收积累特征", 《植物营养与肥料学》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104498585A (en) * | 2014-11-28 | 2015-04-08 | 湖南农业大学 | Method for determining content of nitrate nitrogen inside and outside vacuoles in plant protoplasts |
| CN104388380A (en) * | 2014-12-10 | 2015-03-04 | 南京农业大学 | Method for extracting pear pollen tube vacuoles |
| CN105316275A (en) * | 2015-11-27 | 2016-02-10 | 甘肃农业大学 | Extraction method of halogeton mesophyll cell vacuoles |
| CN106244513A (en) * | 2016-07-26 | 2016-12-21 | 北京林业大学 | A kind of vacuole that separates is distributed and the method for motion with albumen on observation tonoplast |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103589678B (en) | 2015-09-16 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103589678B (en) | A kind of method extracting crassulaceae plants Cell vacuoles | |
| Collett Jr et al. | Cloud chemistry at Mt Rigi, Switzerland: dependence on drop size and relationship to precipitation chemistry | |
| Andersson et al. | The importance of colloids for the behavior of uranium isotopes in the low-salinity zone of a stable estuary | |
| CN102145256B (en) | Method for separating boron isotopes by simulated moving bed chromatography | |
| CN103333068B (en) | Extraction optimization method of Helianthus tuberosus L straw chlorogenic acid by response surface methodology | |
| CN103013984B (en) | Method of extracting chloroplast total RNA (Ribose Nucleic Acid) together with total protein from woody plant material, chloroplast total protein and application | |
| CN103163001B (en) | A kind of method of extracting purifying microcysin LR, RR taking Dian Chi blue-green alga bloom as raw material | |
| CN104004928B (en) | A kind of purifying method for concentration of plutonium | |
| CN105316275A (en) | Extraction method of halogeton mesophyll cell vacuoles | |
| CN102285982A (en) | Method for separating and purifying monomer compounds from Chinese medicinal herb golden thread | |
| CN101838300B (en) | Method for extracting residual abamectin | |
| Loh et al. | Potential source and diagenetic signatures of oceanic dissolved and particulate organic matter as distinguished by lipid biomarker distributions | |
| CN105985315A (en) | Method for extracting nicotine from tobacco waste | |
| CN103709152B (en) | A kind of separation purification method of puerarin | |
| CN103901143B (en) | A kind of pre-treating method analyzed for tetrabromobisphenol A in a small amount of biological anteserum | |
| CN102309576A (en) | Preparation method of uncaria total alkaloids | |
| CN102899318B (en) | Method for extracting nanmu RNA (Ribonucleic Acid) | |
| CN104231015A (en) | Preparation method of lobetyolin | |
| CN101747275B (en) | Method for separating Huperzine A from Huperziaserrata by foamet | |
| CN201823480U (en) | Concentration-separation device for di methyl carbonate and methanol | |
| CN105198900A (en) | Pure yessotoxin (YTX) extracting and preparing method | |
| CN204910875U (en) | A transformer oil water separator for photovoltaic power plant | |
| He et al. | Study on separation and purification of resveratrol in wine grape residue with aqueous two phase extraction method | |
| CN103467297B (en) | Method for separating and purifying methyl p-hydroxybenzoate and 3-indolylformaldehyde from Trichosanthes kirilowii Maxim stem and leaf | |
| CN104448913A (en) | Process for extracting pigment of dragon fruit peel |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |