CN105316275A - Extraction method of halogeton mesophyll cell vacuoles - Google Patents
Extraction method of halogeton mesophyll cell vacuoles Download PDFInfo
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Abstract
The invention belongs to the technical field of extraction of plant cell vacuoles and particularly relates to an extraction method of halogeton mesophyll cell vacuoles. The extraction method of the halogeton mesophyll cell vacuoles is mainly characterized by comprising the following steps: (1) taking mesophyll; (2) enzymolysis and purification of protoplast: cutting the mesophyll obtained in the step (1) from the stalk along the mesophyll base, then putting in an enzymatic hydrolysate, carrying out enzymolysis for 2.5-3.0h at the temperature of 22-25 DEG C under the dark condition, and after the enzymolysis is finished, adding isometric washing liquid into the enzymatic hydrolysate; and (3) obtainment and purification of vacuoles: adding digitonin into the protoplast obtained in the step (2) so as to realize enrichment of pure vacuoles. The digitonin is used for dissociating the plasma membrane, thereby simplifying common complex programs of density gradient centrifugation for extracting the vacuoles. According to the invention, the process of carrying out tender mesophyll protoplast enzymolysis to obtain high-purity vacuoles only takes about 4h. The method realizes simple, efficient and fast extraction of the halogeton mesophyll cell vacuoles. The vacuoles obtained by the method are relatively high in purity, more in quantity, little in impurity contamination and good in integrality.
Description
Technical field
The invention belongs to vegetable cell vacuole extractive technique field, be specifically related to a kind of method that halophytes salt sward blade cell vacuole extracts.
Background technology
The adverse circumstance adaptation mechanisms such as research plant salt tolerance drought resisting have become the much-talked-about topic of biology at present research.Salt sward (Halogetonglomeratus) is grown on NORTHWEST CHINA drought-hit area, for Chenopodiaceae (Chenopodiaceae) Halogeton (Halogeton) annual salt-dilution halophyte, by plant root to the restricted absorption of salinity and carnification blade to effective enrichment of salinity, thus special patience adaptation mechanism is defined to salt stress, and the characteristic of its applicable artificial culture, make salt sward become the good material of research plant salt tolerance drought resistance mechanism.Vegetable cell vacuole is the organoid possessing several functions, accounts for 90% of mature cell volume, and its major function is accumulation storage metabolic substd, regulates osmotic pressure, maintains eubolism environment in cell.Early-stage Study finds, salinity is transported to leaf mesophyll cells by High-efficiency Conveyance System by salt sward, and then separating is in the middle of Cell vacuoles, makes salinity in tenuigenin remain on lower level, thus achieves the normal growth under salt stress environment.As can be seen here, salt sward must exist to separate with salinity vacuole and turns to main salt tolerant adaptation mechanism.The Cell vacuoles key that separating of research salt sward salinity vacuole is machine-processed beyond doubt that Extraction and separation purifying is complete and difficult point place.At present, little about the research of extracting halophytes vacuole skin, the method especially about the separation and purification of salt sward vacuole there is not yet any report.Therefore, current urgent need a kind of with salt sward blade for material carrys out the method for its vacuole of separation and purification, to provide theory support for the Mechanisms of Salt Resistance research of separating of vacuole and the resistance breeding of crop.
Summary of the invention
The object of the invention is to avoid the deficiencies in the prior art part and the extracting method that a kind of salt sward blade cell vacuole is provided.The method fast and effeciently can obtain that quantity is many, purity is high, the vacuole of structural integrity.
For achieving the above object, the technical scheme that the present invention takes is: a kind of extracting method of salt sward blade cell vacuole, and its principal feature is that step is:
(1) blade is drawn materials: with salt sward seedling for object, cut over-ground part, and with distilled water flushing 3-5 time, blade surface moisture dried by filter paper;
(2) enzymolysis and purifying protoplastis: the blade that step (1) obtains is cut down along blade base from stem stalk, move into the experiment table top being covered with smooth clean paper, with blade along the rip cutting of blade master pulse into two, then enzymolysis solution is put into, temperature 22-25 DEG C, under dark condition, enzymolysis 2.5-3.0h; After enzymolysis terminates, then add isopyknic washing lotion to enzymolysis solution, and be placed in dark condition, temperature 4-6 DEG C, the shaking table of rotating speed 30-40rpm shakes 10-15min, release protoplastis; Then with the liquid-transfering gun of 5ml, whole enzymolysis solution is transferred to test tube, temperature is 2-6 DEG C, and rotating speed is the centrifugal 10-15min of 2200-2500rpm, finally draws 1/2 volume supernatant liquor at the most with 5ml liquid-transfering gun, can obtain purer protoplastis;
(3) acquisition of vacuole and purifying: digitonin is added in the protoplastis that step (2) obtains, mix gently, be placed in 2-6 DEG C of standing 10min, abundant dissolved cell plasma membrane, release vacuole; Then rotating speed is 1100-1300rpm, and temperature is 2-6 DEG C, centrifugal 10-15min, draws 1/4 supernatant liquor at the most, can realize the enrichment of pure vacuole with the liquid-transfering gun of 5ml.
The extracting method of described salt sward blade cell vacuole, the blade described in its step (1) is the salt sward seedling of growth 25-40d.These young leaflet tablets not only ensure that the quality obtaining pure vacuole, more there is provided the starting material obtaining lot of pure vacuole.
The extracting method of described salt sward blade cell vacuole, protoplastis enzymolysis solution described in its step (2) consists of: cellulase 2.0-3.0% (w/v), hemicellulase 1.0-2.0% (w/v), polygalacturonase 0.5-1.0% (w/v), macerozyme 0.3-0.5% (w/v), N.F,USP MANNITOL 350-450mM, MES0.5-1.5mM, PH are 5.5-6.0; Consisting of of described washing lotion: Ficoll4001.5-3% (w/v), N.F,USP MANNITOL 250-350mM, Tris-HCl20-30mM, PH are 7.5-8.0, EDTA15-25mM.
The extracting method of described salt sward blade cell vacuole, the amount of the blade in the enzymolysis solution described in its step (2) is paved with enzymolysis solution surface with blade.Cost-saving while ensure that hydrolysis result.
The extracting method of described salt sward blade cell vacuole, in the washing lotion composition described in its step (2) N.F,USP MANNITOL the enzymolysis solution described in concentration ratio in the low 80-100mM of concentration of N.F,USP MANNITOL.Appropriate reduction osmotic pressure contributes to improving vacuole stability.
The extracting method of described salt sward blade cell vacuole, the consumption of the digitonin described in its step (3) is 30-40ul/ml.Thus while realizing whole as far as possible degradation of cell plasma membrane, reduce the injury to vacuole skin.
The extracting method of described salt sward blade cell vacuole, in step (2), (3), centrifuge tube used is centrifuge tube at the bottom of Plastic Flat, and described centrifugal centrifuge rotor used is horizontal rotor.It is pure that agents useful for same is Import Analysis, prepares with ultrapure water.5ml liquid-transfering gun rifle head tip need be cut off, to reduce to greatest extent the physical abuse in isolation and purification protoplastis and vacuole process.
Beneficial effect of the present invention is as follows:
1. directly with the seedling tender leaf growing 25-40d for material carrys out enrichment vacuole, the vacuole directly obtained from plant itself is separated than suspension cell culture the vacuole obtained and more can represents plant self character;
2. Seedling Biomass is large, and the acquisition for a large amount of vacuole provides sufficient starting material;
3. apply digitonin and carry out dissociated cell plasma membrane, thus it is good efficiently to obtain integrity rapidly, purity is high, the vacuole that quantity is many.
Accompanying drawing illustrates:
Fig. 1. the vacuole that embodiment 1 obtains;
Fig. 2. the vacuole that embodiment 2 obtains;
Fig. 3. the vacuole that embodiment 3 obtains;
Fig. 4. the vacuole that embodiment 4 obtains;
Fig. 5. the vacuole that embodiment 5 obtains;
Fig. 6. the vacuole that test example obtains.
Embodiment
Be described principle of the present invention and feature below in conjunction with embodiment, example, only for explaining the present invention, is not intended to limit scope of the present invention.Below content of the present invention is described in detail.
Embodiment 1: a kind of extracting method of salt sward blade cell vacuole, the steps include:
(1) blade is drawn materials: cut growth 30d seedling leaves, distilled water flushing 3 times, its surface is dried by filter paper;
(2) enzymolysis and purifying protoplastis: the blade that step (1) obtains is cut down along blade base from stem stalk, move into the experiment table top being covered with smooth clean paper, with blade along the rip cutting of blade master pulse into two, then enzymolysis solution is put into, under temperature 25 DEG C, dark condition, enzymolysis 2.5h; After enzymolysis terminates, then add isopyknic washing lotion to enzymolysis solution, and be placed in dark condition, temperature 6 DEG C, the shaking table of rotating speed 30rpm shakes 10min, release protoplastis; Then with the liquid-transfering gun of 5ml, whole enzymolysis solution is transferred to test tube, temperature is 4 DEG C, and rotating speed is the centrifugal 10-15min of 2500rpm, finally draws 1/2 volume supernatant liquor at the most with 5ml liquid-transfering gun, can obtain purer protoplastis;
(3) acquisition of vacuole and purifying: the digitonin adding 35ul/ml in supernatant liquor is added in the protoplastis that step (2) obtains, mix gently, be placed in 4 DEG C of standing 10min, abundant dissolved cell plasma membrane, release vacuole; Then rotating speed is 1200rpm, and temperature is 4 DEG C, centrifugal 10min, draws 1/4 supernatant liquor at the most, can realize the enrichment of pure vacuole with the liquid-transfering gun of 5ml.Add the digitonin of 35ul/ml, thus while realizing whole as far as possible degradation of cell plasma membrane, reduce the injury to vacuole skin.
Described protoplastis enzymolysis solution is: cellulase 3.0% (w/v), hemicellulase 2.0% (w/v), polygalacturonase 1.0% (w/v), macerozyme 0.5% (w/v), N.F,USP MANNITOL 450mM, MES1.0mM, PH are 5.5; Lotion prescription is: N.F,USP MANNITOL 360mM, Ficoll4002.0% (w/v), and Tris-HCl20mM, PH are 8.0, EDTA20mM.The vacuole obtained is shown in Fig. 1.
The amount of the blade in the enzymolysis solution described in step (2) is paved with enzymolysis solution surface with blade.Cost-saving while ensure that hydrolysis result.
In washing lotion composition described in step (2) N.F,USP MANNITOL the enzymolysis solution described in concentration ratio in the low 90mM of concentration of N.F,USP MANNITOL.Appropriate reduction osmotic pressure contributes to improving vacuole stability.
In step (2), (3), centrifuge tube used is centrifuge tube at the bottom of Plastic Flat, and described centrifugal centrifuge rotor used is horizontal rotor.It is pure that agents useful for same is Import Analysis, prepares with ultrapure water.5ml liquid-transfering gun rifle head tip need be cut off, to reduce to greatest extent the physical abuse in isolation and purification protoplastis and vacuole process.
Embodiment 2: a kind of extracting method of salt sward blade cell vacuole, the steps include:
(1) blade is drawn materials: cut growth 40d seedling leaves, distilled water flushing 5 times, its surface is dried by filter paper;
(2) protoplastis obtains and purifying.By blade razor blade along master pulse rip cutting, then at 22 DEG C, under dark condition after enzymolysis 2.5h, add isopyknic washing lotion, then under dark condition, 6 DEG C, the shaking table concussion 10min of 35rpm, then with the liquid-transfering gun of 5ml, enzymolysis solution is proceeded to the test tube of 10ml, then 5 DEG C, the centrifugal 10min of 2200rpm, draw the supernatant liquor of 4ml with liquid-transfering gun;
(3) vacuole obtains and purifying.In above-mentioned supernatant liquor, add the digitonin of 30ul/ml, mixing, 4 DEG C of standing 10min, 1200rpm4 DEG C of centrifugal 15min, finally collect the supernatant liquor of 2ml, observe under being placed in inverted microscope.Described protoplastis enzymolysis solution is: cellulase 2.0% (w/v), hemicellulase 1.0% (w/v), polygalacturonase 1.0% (w/v), macerozyme 0.3% (w/v), N.F,USP MANNITOL 400mM, MES1.0mM, PH are 5.8; Lotion prescription is: N.F,USP MANNITOL 300mM, Ficoll4002.0% (w/v), and Tris-HCl20mM, PH are 8.0, EDTA25mM.The vacuole obtained is shown in Fig. 2.
Embodiment 3: a kind of extracting method of salt sward blade cell vacuole, the steps include:
(1) blade is drawn materials: cut growth 25d seedling leaves, distilled water flushing 3 times, its surface is dried by filter paper;
(2) protoplastis obtains and purifying.By blade razor blade along master pulse rip cutting, then at 25 DEG C, under dark condition after enzymolysis 3.0h, add isopyknic washing lotion, then under dark condition, 2 DEG C, the shaking table concussion 15min of 40rpm, then with the liquid-transfering gun of 5ml, enzymolysis solution is proceeded to the test tube of 10ml, then 4 DEG C, the centrifugal 10min of 2200rpm, draw the supernatant liquor of 4ml with liquid-transfering gun;
(3) vacuole obtains and purifying.In above-mentioned supernatant liquor, add the digitonin of 35ul/ml, mixing, 2 DEG C of standing 15min, 1100rpm2 DEG C of centrifugal 10min, finally collect the supernatant liquor of 2ml, observe under being placed in inverted microscope.Described protoplastis enzymolysis solution is: cellulase 2.0% (w/v), hemicellulase 1.0% (w/v), polygalacturonase 0.5% (w/v), macerozyme 0.3% (w/v), N.F,USP MANNITOL 350mM, MES1.0mM, PH are 6.0; Lotion prescription is: N.F,USP MANNITOL 250mM, Ficoll4001.5% (w/v), and Tris-HCl30mM, PH are 8.0, EDTA15mM.The vacuole obtained is shown in Fig. 3.
Embodiment 4: a kind of extracting method of salt sward blade cell vacuole, the steps include:
(1) blade is drawn materials: cut growth 35d seedling leaves, distilled water flushing 5 times, its surface is dried by filter paper;
(2) protoplastis obtains and purifying.By blade razor blade along master pulse rip cutting, then at 22 DEG C, under dark condition after enzymolysis 3.0h, add isopyknic washing lotion, then under dark condition, 6 DEG C, the shaking table concussion 10min of 30rpm, then with the liquid-transfering gun of 5ml, enzymolysis solution is proceeded to the test tube of 10ml, then 2 DEG C, the centrifugal 15min of 2300rpm, draw the supernatant liquor of 4ml with liquid-transfering gun;
(3) vacuole obtains and purifying.In above-mentioned supernatant liquor, add the digitonin of 35ul/ml, mixing, 4 DEG C of standing 10min, 1300rpm6 DEG C of centrifugal 10min, finally collect the supernatant liquor of 2ml, observe under being placed in inverted microscope.Described protoplastis enzymolysis solution is: cellulase 3.0% (w/v), hemicellulase 1.0% (w/v), polygalacturonase 0.5% (w/v), macerozyme 0.3% (w/v), N.F,USP MANNITOL 350mM, MES1.0mM, PH are 5.8; Lotion prescription is: N.F,USP MANNITOL 250mM, Ficoll4002.0% (w/v), and Tris-HCl25mM, PH are 8.0, EDTA15mM.The vacuole obtained is shown in Fig. 4.
Embodiment 5: a kind of extracting method of salt sward blade cell vacuole, the steps include:
(1) blade is drawn materials: cut growth 25d seedling leaves, distilled water flushing 5 times, its surface is dried by filter paper;
(2) protoplastis obtains and purifying.By blade razor blade along master pulse rip cutting, then at 23 DEG C, under dark condition after enzymolysis 2.5h, add isopyknic washing lotion, then under dark condition, 6 DEG C, the shaking table concussion 10min of 35rpm, then with the liquid-transfering gun of 5ml, enzymolysis solution is proceeded to the test tube of 10ml, then 2 DEG C, the centrifugal 15min of 2300rpm, draw the supernatant liquor of 4ml with liquid-transfering gun;
(3) vacuole obtains and purifying.In above-mentioned supernatant liquor, add the digitonin of 30ul/ml, mixing, 4 DEG C of standing 10min, 1100rpm4 DEG C of centrifugal 15min, finally collect the supernatant liquor of 2ml, observe under being placed in inverted microscope.Described protoplastis enzymolysis solution is: cellulase 2.0% (w/v), hemicellulase 2.0% (w/v), polygalacturonase 0.1% (w/v), macerozyme 0.5% (w/v), N.F,USP MANNITOL 400mM, MES1.0mM, PH6.0; Lotion prescription is: N.F,USP MANNITOL 320mM, Ficoll4003.0% (w/v), and Tris-HCl20mM, PH are 8.0, EDTA20mM.The vacuole obtained is shown in Fig. 5.
Above embodiment all obtains that quantity is more, and purity is high, the vacuole that color is bright, sees accompanying drawing 1 respectively, 2,3, and 4 and Fig. 5.
Test example:
In the process extracting salt sward Cell vacuoles, in order to obtain pure vacuole, from Material selec-tion, enzymolysis and purifying protoplastis, finally carry out continuous exploration and optimization to the key factor in the processes such as the acquisition of vacuole and purifying, finally determine the comparatively suitable parameter of efficient rapid extraction mesophyll cell vacuole method.The vacuole obtained is shown in Fig. 6.
1. seedling is drawn materials the determination of time
Respectively with the salt sward seedling of Different growth phases for material, process A:60d, treatments B: 30d.Cut above-mentioned growth phase seedling leaves, distilled water flushing 3 times, its surface is dried by filter paper, with razor blade along master pulse rip cutting, then at 25 DEG C, under dark condition after enzymolysis 2.5h, add isopyknic test tube enzymolysis solution being proceeded to 10ml, then 4 DEG C, the centrifugal 10min of 2500rpm, draw the supernatant liquor of 4ml, observe under being placed in inverted microscope with liquid-transfering gun.Described protoplastis enzymolysis solution is: cellulase 2.0% (w/v), hemicellulase 1.0% (w/v), polygalacturonase 1.0% (w/v), macerozyme 0. washing lotion, then under dark condition, 4 DEG C, the shaking table concussion 10min of 30rpm, then uses the liquid-transfering gun 5% (w/v) of 5ml, N.F,USP MANNITOL 450mM, MES1.0mM, PH are 5.5; The formula of washing lotion is: Ficoll4002.0% (w/v), N.F,USP MANNITOL 350mM, and Tris-HCl20mM, PH are 8.0, EDTA20mM.
Found that: the seedling in above two vegetative period all can obtain a large amount of protoplastis by enzymolysis, wherein process protoplastis color in A comparatively dark, impurity is more, and treatments B protoplastis color is bright, and impurity is few, and purity is higher.Young leaflet tablet is more conducive to obtaining excellent protoplastis.
2. determine washing lotion mannitol concentration
On the basis of experiment 1, continue to carry out preferably to the concentration of washing lotion N.F,USP MANNITOL, cut growth 30d seedling leaves, distilled water flushing 3 times, its surface is dried by filter paper, with razor blade along master pulse rip cutting, then at 25 DEG C, under dark condition after enzymolysis 2.5h, add isopyknic washing lotion, then under dark condition, 4 DEG C, the shaking table concussion 10min of 30rpm, then with the liquid-transfering gun of 5ml, enzymolysis solution is proceeded to the test tube of 10ml, then 4 DEG C, the centrifugal 10min of 2500rpm, the supernatant liquor of 4ml is drawn with liquid-transfering gun, the digitonin of 35ul/ml is added again in above-mentioned supernatant liquor, mixing, 4 DEG C of standing 10min, then, 1300rpm, 4 DEG C of centrifugal 15min, finally collect the supernatant liquor of 2ml, observe under being placed in inverted microscope.Described protoplastis enzymolysis solution is: cellulase 2.0% (w/v), hemicellulase 1.0% (w/v), polygalacturonase 1.0% (w/v), macerozyme 0.5% (w/v), N.F,USP MANNITOL 450mM, MES1.0mM, PH are 5.5; The formula of washing lotion is: process A: N.F,USP MANNITOL 450MM, Ficoll4002.0% (w/v), Tris-HCl20mM, PH are 8.0, EDTA20mM; Treatments B: N.F,USP MANNITOL 350MM, Ficoll4002.0% (w/v), Tris-HCl20mM, PH are 8.0, EDTA20MM; Process C: N.F,USP MANNITOL 250MM, Ficoll4002.0% (w/v), Tris-HCl20mM, PH are 8.0, EDTA20MM.
Found that: in treatments B, complete vacuole quantity is maximum, and color is bright; Process A only finds the vacuole that seldom amount is complete, has a small amount of complete vacuole in process C.As can be seen here, the extraction that osmotic pressure contributes to vacuole is suitably reduced.
3. plasma membrane digitonin concentration of dissociating is determined
On the basis of experiment 2, continue to carry out preferably to digitonin concentration needed for plasma membrane of dissociating, cut growth 30d seedling leaves, distilled water flushing 3 times, its surface is dried by filter paper, with razor blade along master pulse rip cutting, then at 25 DEG C, under dark condition after enzymolysis 2.5h, add isopyknic washing lotion, then under dark condition, 4 DEG C, the shaking table concussion 10min of 30rpm, then with the liquid-transfering gun of 5ml, enzymolysis solution is proceeded to the test tube of 10ml, then 4 DEG C, the centrifugal 10min of 2500rpm, the supernatant liquor of 4ml is drawn with liquid-transfering gun, process A:25ul/ml is added again in above-mentioned supernatant liquor, treatments B: 35ul/ml, the digitonin of process C:45ul/ml, mixing, 4 DEG C of standing 10min, then, 1300rpm, 4 DEG C of centrifugal 15min, finally collect the supernatant liquor of 2ml, observe under being placed in inverted microscope.Described protoplastis enzymolysis solution is: cellulase 2.0% (w/v), hemicellulase 1.0% (w/v), polygalacturonase 1.0% (w/v), macerozyme 0.5% (w/v), N.F,USP MANNITOL 450mM, MES1.0mM, PH are 5.5; The formula of washing lotion is: N.F,USP MANNITOL 350MM, Ficoll4002.0% (w/v), and Tris-HCl20mM, PH are 8.0, EDTA20mM.
Observe and find: above three process all can isolate vacuole.Wherein process in A and there is a large amount of protoplastis, also with the residual body of the plasma membrane do not dissociated completely on part vacuole, the plasma membrane in process C dissociates thoroughly, and vacuole has no the existence of the residual body of plasma membrane, and purity is high, but quantity is little.It can thus be appreciated that digitonin concentration is on the low side, cause plasma membrane to degrade not exclusively, if higher meeting also dissociation solution vacuolar membrane while degraded plasma membrane, cause vacuole to break.
4. the determination of purifying vacuole centrifugal rotational speed
On the basis of experiment 3, continue to carry out preferably to vacuole purifying centrifugal rotational speed, cut growth 30d seedling leaves, distilled water flushing 3 times, its surface is dried by filter paper, with razor blade along master pulse rip cutting, then at 25 DEG C, under dark condition after enzymolysis 2.5h, add isopyknic washing lotion, then under dark condition, 4 DEG C, the shaking table concussion 10min of 30rpm, then with the liquid-transfering gun of 5ml, enzymolysis solution is proceeded to the test tube of 10ml, then 4 DEG C, the centrifugal 10min of 2500rpm, the supernatant liquor of 4ml is drawn with liquid-transfering gun, the digitonin of 35ul/ml is added again in above-mentioned supernatant liquor, mixing, 4 DEG C of standing 10min, then at process A:900rpm, treatments B: 1300rpm, process C:1600rpm, 4 DEG C of centrifugal 15min, finally collect the supernatant liquor of 2ml, observe under being placed in inverted microscope.Described protoplastis enzymolysis solution is: cellulase 2.0% (w/v), hemicellulase 1.0% (w/v), polygalacturonase 1.0% (w/v), macerozyme 0.5% (w/v), N.F,USP MANNITOL 450mM, MES1.0mM, PH are 5.5; Lotion prescription is: N.F,USP MANNITOL 350MM, Ficoll4002.0% (w/v), Tris-HCl20mM, PH are 8.0, EDTA20mM.
Found that: after process A is centrifugal, vacuole quantity is more, also impurity is more simultaneously; After process C is centrifugal, vacuole quantity is little, and impurity is also little; After treatments B is centrifugal, vacuole quantity is many, and impurity is little, and state is better.
5. the determination of purifying vacuole centrifugation time
On the basis of experiment 4, continue to carry out preferably to vacuole purifying centrifugation time, cut growth 30d seedling leaves, distilled water flushing 3 times, its surface is dried by filter paper, with razor blade along master pulse rip cutting, then at 25 DEG C, under dark condition after enzymolysis 2.5h, add isopyknic washing lotion, then under dark condition, 4 DEG C, the shaking table concussion 10min of 30rpm, then with the liquid-transfering gun of 5ml, enzymolysis solution is proceeded to the test tube of 10ml, then 4 DEG C, the centrifugal 10min of 2500rpm, the supernatant liquor of 4ml is drawn with liquid-transfering gun, the digitonin of 35ul/ml is added again in above-mentioned supernatant liquor, mixing, 4 DEG C of standing 10min, 1300rpm4 DEG C centrifugal, process A:5min, treatments B: 10min, process C:15min, process D:20min, finally collect the supernatant liquor of 2ml, observe under being placed in inverted microscope.Described protoplastis enzymolysis solution is: cellulase 2.0% (w/v), hemicellulase 1.0% (w/v), polygalacturonase 1.0% (w/v), macerozyme 0.5% (w/v), N.F,USP MANNITOL 450mM, MES1.0mM, PH are 5.5; Lotion prescription is: N.F,USP MANNITOL 350mM, Ficoll4002.0% (w/v), Tris-HCl20mM, PH are 8.0, EDTA20mM.
Found that: after process A is centrifugal, vacuole comparatively small amt, impurity is more; Treatments B and C centrifugal after, vacuole quantity is more, and impurity is also little; After process D is centrifugal, vacuole quantity is little, and impurity is also less, therefore treatments B and C are comparatively suitable centrifugation time.
Above-described embodiment is the present invention's preferably embodiment; but embodiments of the present invention are not restricted to the described embodiments; change, the modification done under other any does not deviate from spirit of the present invention and principle, substitute, combine, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.
Claims (6)
1. an extracting method for salt sward blade cell vacuole, is characterized in that step is:
(1) blade is drawn materials: with salt sward seedling for object, cut over-ground part, and with distilled water flushing 3-5 time, blade surface moisture dried by filter paper;
(2) enzymolysis and purifying protoplastis: the blade that step (1) obtains is cut down along blade base from stem stalk, move into the experiment table top being covered with smooth clean paper, with blade along the rip cutting of blade master pulse into two, then enzymolysis solution is put into, temperature 22-25 DEG C, under dark condition, enzymolysis 2.5-3.0h; After enzymolysis terminates, then add isopyknic washing lotion to enzymolysis solution, and be placed in dark condition, temperature 4-6 DEG C, the shaking table of rotating speed 30-40rpm shakes 10-15min, release protoplastis; Then with the liquid-transfering gun of 5ml, whole enzymolysis solution is transferred to test tube, temperature is 2-6 DEG C, and rotating speed is the centrifugal 10-15min of 2200-2500rpm, finally draws 1/2 volume supernatant liquor at the most with 5ml liquid-transfering gun, can obtain purer protoplastis;
(3) acquisition of vacuole and purifying: digitonin is added in the protoplastis that step (2) obtains, mix gently, be placed in 2-6 DEG C of standing 10min, abundant dissolved cell plasma membrane, release vacuole; Then rotating speed is 1100-1300rpm, and temperature is 2-6 DEG C, centrifugal 10-15min, draws 1/4 supernatant liquor at the most, can realize the enrichment of pure vacuole with the liquid-transfering gun of 5ml.
2. the extracting method of salt sward blade cell vacuole as claimed in claim 1, is characterized in that the blade described in step (1) is the salt sward seedling growing 25-40d.
3. the extracting method of salt sward blade cell vacuole as claimed in claim 1, it is characterized in that the protoplastis enzymolysis solution described in step (2) consists of: cellulase 2.0-3.0% (w/v), hemicellulase 1.0-2.0% (w/v), polygalacturonase 0.5-1.0% (w/v), macerozyme 0.3-0.5% (w/v), N.F,USP MANNITOL 350-450mM, MES0.5-1.5mM, PH are 5.5-6.0; Consisting of of described washing lotion: Ficoll4001.5-3% (w/v), N.F,USP MANNITOL 250-350mM, Tris-HCl20-30mM, PH are 7.5-8.0, EDTA15-25mM.
4. the extracting method of salt sward blade cell vacuole as claimed in claim 1, is characterized in that the amount of the blade in the enzymolysis solution described in step (2) is paved with enzymolysis solution surface with blade.
5. the extracting method of salt sward blade cell vacuole as claimed in claim 1, is characterized in that the low 80-100mM of concentration of N.F,USP MANNITOL in the enzymolysis solution described in concentration ratio of N.F,USP MANNITOL in the washing lotion composition described in step (2).
6. the extracting method of salt sward blade cell vacuole as claimed in claim 1, is characterized in that the consumption of the digitonin described in step (3) is 30-40ul/ml.
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| CN109556941A (en) * | 2019-01-27 | 2019-04-02 | 甘肃农业大学 | A kind of method that salt sward root cell plasmalemma protein extracts |
| CN113444678A (en) * | 2020-03-26 | 2021-09-28 | 武汉华大医学检验所有限公司 | Method for preparing single cell nuclear suspension, single cell sequencing method and kit |
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| CN113403253A (en) * | 2021-06-25 | 2021-09-17 | 山东农业大学 | Method for extracting woody plant apple fruit cell vacuole protein |
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