CN103834611A - Separation and purification method for Salvia Miltiorrhiza protoplast - Google Patents

Abstract

The invention discloses a separation and purification method for a Salvia Miltiorrhiza protoplast. The method includes the steps of: induction of a callus and subculture, protoplast separation and protoplast purification. The extraction conditions for a Salvia Miltiorrhiza suspension culture cell's protoplast include that: an enzyme solution suitable for enzymolysis of the suspension culture cell is composed of 1.5% of cellulase, 0.3% of pectinase and 0.5% of macerozyme; a suitable mannitol concentration is 0.4M; the extraction time is 12h; centrifugation is carried out at 600r/min for 5min, then collection and purification are conducted so as to obtain the protoplast with a yield of 1.1*10<6>/gFW, FDA detection shows that the vitality of the protoplast is over 95%, and the calcium ion fluorescence probe fluo-3/AM can be successfully loaded into protoplast. The protoplast extracted under the conditions of the method completely satisfies the follow-up series of study taking a single cell as the study object.

Description

A kind of separation purification method of red sage root protoplastis

Technical field

The invention belongs to technological field of biochemistry, be specifically related to a kind of separation purification method of red sage root protoplastis.

 

Background technology

Plant protoplast has been taken off cell walls, has only been had that plasma membrane surrounds, the exposed and vegetable cell of life.The effect of plasma membrane in vital movement is very important, and the biological phenomenas such as such as information transmission, energy transformation, matter transportation all have close relationship with plasma membrane.Exposed and the blodynamic characteristic of tool of protoplastis, for the research relevant with plasmalemma of plant function provides favourable condition, has disclosed the essence of a series of vital processes on cell levels.Plant carries out on cell levels the opposing reaction of elicitor, in integral level, study and be subject to various interference, can get rid of interference as material with protoplastis, highly beneficial for the phenomenon of illustrating the biological process of stress resistance of plant and occur on cell levels.As multiple external stimulus, comprise that elicitor, mechanical stimulus and low temperature stress, osmotic stress, light, oxidative stress etc. all can cause vegetable cell kytoplasm Free Ca ²⁺concentration raises.Ca ²⁺messenger function by Free Ca in regulating cell ²⁺concentration realizes.Kytoplasm Ca ²⁺the conventional laser confocal microscope of concentration detects, but because the esterase of cell walls makes to load unsuccessfully to the hydrolysis of Fluo-3/AM fluorescent probe, impact detects, and utilizes protoplastis can make probe enter smoothly the interior kytoplasm Free Ca that accurately detects of cell ²⁺.

The material that is commonly used to extract protoplastis has spire, cotyledon, root and the hypocotyl etc. of plant.In the callus of plant, having a large amount of intercellular substances, is the loose cell mass of a thin-walled, and its cell walls, easily by enzymolysis, is the ideal material of preparing protoplastis.But red sage root protoplastis separation method is gone back at present nobody and was studied.

Summary of the invention

For above-mentioned problems of the prior art and defect, the object of the present invention is to provide a kind of separation purification method of red sage root protoplastis.The protoplastis extracting under this condition can meet follow-up a series of researchs take individual cells as research object completely, for the generation, conduction and the impact on Secondary Metabolism of Plant thereof that disclose the Second messenger in cell that causes of elicitor lay the foundation.

Realizing foregoing invention object technical scheme is a kind of separation purification method of red sage root protoplastis, specifically comprises the following steps:

1) evoked callus and succeeding transfer culture

The blade of robust growth in red sage root aseptic seedling is cut into the explant of 0.5 cm × 0.5 cm size, is inoculated on MS solid medium at aseptic condition, under illumination condition, induction forms callus; By the callus inducing at equal culture condition take every 20 d succeeding transfer culture 12 d again after succeeding transfer culture one-time continuous cultivated for 3 generations, be transferred in MS liquid nutrient medium, rotating speed be 125 r/min, temperature be under the condition of 25 ℃ dark suspension culture 6 days suspension cell;

2) separation of protoplastis

Combination enzyme is added in 10 mL enzyme solvents and activate 10 min in 55 ℃ of water-baths, be placed into after normal temperature again through 0.45 μ m filtering with microporous membrane sterilizing, add the suspension cell of 1.5 g, enzymolysis mixture is placed in 40 r/min, enzymolysis 4～20 h under 25 ℃ of dark conditions;

Described combination enzyme is cellulase 1.5%, polygalacturonase 0.3%, macerozyme 0.5%;

Described enzyme solvent is the mixing solutions of the MES of KCl, 20 mM of 0.4 M N.F,USP MANNITOL, 20 mM, is adjusted to pH 5.7 with 1 M KOH;

3) protoplastis purifying

Enzymolysis mixture, again through centrifugal 1 min of 80 r/min, is filtered to enzymolysis mixed solution to centrifuge tube, add equal-volume W5 solution, under different centrifugal force, centrifugal 5 min, abandon supernatant; Add again 10 mL W5 solution centrifugal 5 min, abandon supernatant, then after adding 2 mL W5 solution resuspended after placing 30 min on ice centrifugal 3 min, abandon supernatant, add 1.5 ml containing the W5 solution of 0.4 M N.F,USP MANNITOL, resuspended preservation, obtains the protoplastis of purifying;

Described W5 solution is 154 mM NaCl, 125 mM CaCl ₂, 5 mM KCl, 2 mM MES-K, pH value 5.7.

The extraction conditions of the separation purification method of red sage root protoplastis of the present invention is: cellulase R-10 1.5%, polygalacturonase Y-23 0.3%, macerozyme R-10 0.5%, 0.4 M N.F,USP MANNITOL, extraction time is 12 h, under 600 r/min, centrifugal 5 min collect, the protoplast yield obtaining is 1.1 × 106, and vigor reaches more than 95%.The protoplastis extracting under this condition can meet follow-up a series of researchs take individual cells as research object completely, for the generation, conduction and the impact on Secondary Metabolism of Plant thereof that disclose the Second messenger in cell that causes of elicitor lay the foundation.

Accompanying drawing explanation

Figure 1A is that enzyme liquid combines the impact on protoplast yield;

Figure 1B is that enzyme liquid combines the impact on the protoplastis number that fluoresces;

Fig. 1 C is that enzyme liquid combines the impact on protoplastis vigor;

Fig. 2 A is the impact of enzymolysis time on output;

Fig. 2 B is that enzymolysis time is on the protoplastis number impact that fluoresces;

Fig. 2 C is the impact of enzymolysis time on protoplastis vigor;

Fig. 3 A is the impact of mannitol concentration on protoplast yield;

Fig. 3 B is the impact of mannitol concentration on the protoplastis number that fluoresces;

Fig. 3 C is the impact of N.F,USP MANNITOL on protoplastis vigor;

Fig. 4 A is the impact of centrifugal speed on output;

Fig. 4 B is that centrifugal speed is on several impact that fluoresces;

Fig. 4 C is the impact of centrifugal speed on vigor;

Fig. 5 A is the protoplastis after purifying under visible ray;

Fig. 5 B is the protoplastis of the green-emitting fluorescence after FDA dyeing under 488 nm exciting lights;

Fig. 5 C is FDA dyeing under fluorescent microscope oil mirror;

The full fluorescent effect figure of protoplastis 3D after the micro-lower FDA dyeing of Fig. 5 D laser co-focusing;

Fig. 5 E is the protoplastis laser capture microdissection picture under light field.

Embodiment

evoked callus and succeeding transfer culture

Salvia seeds is cultivated to aseptic seedling, then the blade of robust growth in red sage root aseptic seedling is cut into the explant of 0.5 cm × 0.5 cm size.Under aseptic condition, explant is inoculated on MS solid medium, in substratum, contain 1.0 mg/L NAA, 1.0 mg/L 6-BA, 1.0 mg/L 2,4-D, 5.5 g/L agar, 30 g/L sucrose, under illumination condition, induction forms callus.By every the callus inducing 20 d succeeding transfer culture once.Culture condition is the same.

By callus stable succeeding transfer culture 3 generation proterties succeeding transfer culture 12 d again, being transferred in the triangular flask containing 50 mL of 25 mL MS liquid nutrient mediums (without growth regulatory substance and agar), is that 125 r/min, temperature are dark suspension culture under the condition of 25 ℃ at rotating speed.The suspension culture cell of 6 days can be used for the separation of protoplastis.

the separation of protoplastis

Use respectively cellulase, polygalacturonase, macerozyme and the combined treatment material thereof of different concns.Enzyme solvent is the mixing solutions of the MES of KCl, 20 mM of 0.4 M N.F,USP MANNITOL, 20 mM, is adjusted to pH 5.7 with 1 M KOH.In 10 mL enzyme solvents, add combination in 55 ℃ of water-baths, to activate 10 min after enzyme, be placed into after normal temperature again through 0.45 μ m filtering with microporous membrane sterilizing.Add the suspension cell of 1.5 g fresh weights, enzymolysis mixture is placed in 40 r/min, and enzymolysis 4 ~ 20 h under 25 ℃ of dark conditions sample form and the vigor of examining under a microscope protoplastis, to determine best enzyme concn and enzymolysis time every 2 h.

protoplastis purifying

?utilize the enzyme concn of above-mentioned selection to combine after abundant enzymolysis material, enzymolysis mixture is again through centrifugal 1 min of 80 r/min, then uses in 600 order nylon net filter enzymolysis mixed solution to 50 mL centrifuge tubes, adds equal-volume W5 solution (154 mM NaCl, 125 mM CaCl ₂, 5 mM KCl, 2 mM MES-K, pH value 5.7), at lower centrifugal 5 min of different centrifugal force (300 r/min, 600 r/min, 1000 r/min), abandon supernatant; Add again 10 mL W5 solution centrifugal 5 min, abandon supernatant, then after adding 2 mL W5 solution resuspended after placing 30 min on ice centrifugal 3 min, abandon supernatant.Add the resuspended preservation of 1.5 ml W5 solution (containing 0.4 M N.F,USP MANNITOL), obtain the protoplastis of purifying.

the counting of protoplastis

With 5 times of W5 dilution protoplastis solution, the above-mentioned protoplastis hanging drop that takes a morsel is added on 0.1 mm blood counting chamber.When protoplastis is full of after nucleonics, at optical microphotograph Microscopic observation, and measure the concentration of protoplastis with cell counting count board: calculates the interior protoplastis number of the large lattice (totally 5 large lattice) of large lattice and central authorities on 4 angles, then press formula and calculate protoplastis number, 3 repetitions of each sample counting, finally calculate the free protoplast yield (individual/g FW) obtaining of every gram of fresh weight material.

Total protoplastis number × 10 in protoplast yield (individual/g FW)=5 large lattice ⁴× extension rate/quality (g FW).

the vigor of protoplastis detects

0.01% diacetic acid fluorescein for protoplastis vitality test (FDA) dyeing, with protoplastis number and the protoplastis sum of fluorescent microscope statistics green-emitting fluorescence, chooses 3 representational visuals field and adds up, and averages.Protoplastis vigor represents with the percentage ratio that in a visual field, great-hearted protoplastis number accounts for protoplastis sum in this visual field.

Protoplastis vigor=(protoplastis number/protoplastis sum of green-emitting fluorescence) × 100%.

²⁺ the loading of fluorescent probe Fluo-3/AM

Before utilizing laser confocal microscope to observe, protoplastis need to carry out adherent fixing.Fix protoplastis with the coated cover glass of poly-lysine (sigma) of 300,000 molecular weight, the working concentration of poly-lysine is 0.02 g/ml.Fluo-3/AM(is dissolved in anhydrous DMSO, is made into the mother liquor of 1 mmol/L, in-20 ℃ of storages, is that 20 μ mol/L are Fluo-3/AM working fluid with W5 solution (containing 0.4M N.F,USP MANNITOL) dilution mother liquor to final concentration.

Red sage root Suspension Protoplasts is incubated in to 1 h in Fluo-3/AM working fluid.After twice of W5 washing, under room temperature, leave standstill 1 h.Be fixed on afterwards on the slide of poly-lysine processing, then apply 22 mg/L SA processing.In processing 30 min, every 30 s measure first order fluorescences and also take pictures, and excitation wavelength is 488 nm, and emission wavelength is 510 nm.

the impact of enzyme on protoplast yield and vigor

Take red sage root suspended culture cell as material, with different enzyme liquid combination (0.4 M N.F,USP MANNITOL regulates osmotic pressure) processing 12 h, the centrifugal collection protoplastis of 600 r/min, result is as shown in Figure 1A～C.It is lower by (1.4 × 10 that the protoplast yield obtaining is extracted in 0.3% combination of cellulase 1.5%+ polygalacturonase ⁵individual); Cellulase 1.5%, polygalacturonase 0.3% and macerozyme 0.5% are combined use can make the output of protoplastis be increased to 1.1 × 10 ⁶individual, this may be the ability that has increased enzymolysis cell walls due to the interpolation of macerozyme; But after all being increased, three kinds of enzyme concns do not increase protoplast yield (Fig. 1 A).Get enzyme liquid material microscopy after treatment and find, rear broken cytosis is processed in enzyme liquid combination (cellulase 3.0%+ polygalacturonase 0.6%+ macerozyme 1.0%), and part protoplastis generation fragmentation causes yield reducation.Process the protoplastis obtaining with cellulase 1.5%+ polygalacturonase 0.3%+ macerozyme 0.5%, FDA hatch after under fluorescent microscope at most (Figure 1B) of fluorescigenic number, and vigor the highest (Fig. 1 C), the protoplastis that other processing obtain fluoresce number and vigor lower (Figure 1B, 1C).

the impact of enzymolysis time on protoplast yield and vigor

Output and the vigor of observing protoplastis with above-mentioned enzyme liquid combination (cellulase 1.5%+ polygalacturonase 0.3%+ macerozyme 0.5%) enzymolysis different time, result is as shown in Fig. 2 A～C.When enzymolysis 6 h, protoplast yield is very low, and after 6 h, output obviously improves, and when 12 h, protoplast yield is the highest, and (Fig. 2 A) significantly declines after 14 h.Microscopy is found, fragment showed increased in enzymolysis solution after enzymolysis 14 h illustrates that it is the result causing due to protoplastis fragmentation that protoplast yield declines.Fluoresce after the hatching Changing Pattern of protoplastis number and output of FDA is consistent, the protoplastis that enzymolysis 12 h obtain number (Fig. 2 B) at most that fluoresce.Enzymolysis 10 h are with interior protoplastis vigor lower than 90%, and the protoplastis vigor of enzymolysis 10～16 h is all in high level (> 95%), exceed after 16 h vigor sharply decline (Fig. 2 C).

the impact of mannitol concentration on protoplast yield and vigor in 2.3 enzyme liquid

When the osmotic pressure of enzymolysis solution and protoplastis can not maintain etc. while oozing, protoplasma is known from experience spalling or shrinkage, and suitable osmotic pressure is the prerequisite that obtains high quality protoplastis.The Main Function of N.F,USP MANNITOL in enzymolysis solution is to maintain osmotic pressure, and the concentration of N.F,USP MANNITOL has determined output and the vigor of protoplastis to a great extent.With enzyme combination enzymolysis 12 h of above-mentioned selection, investigate the impact of different mannitol concentrations on protoplast yield and vigor in enzyme liquid, result is as shown in Fig. 3 A～C.Under the mannitol concentration of 0.3 M, although protoplast yield height but vigor is lower, microscopy finds that smudge cells is a lot, this may be to be easy to sedimentation when the centrifugal collection because lower concentration N.F,USP MANNITOL makes protoplastis, but osmotic pressure is too low cannot make cell inside and outside wait to ooze cause protoplastis to burst, many but broken also a lot of of the protoplasma scale of construction of collection; Under the mannitol concentration of 0.4～0.5 M, the output of protoplastis is higher, and (output is higher than 10 ⁶individual); Mannitol concentration during higher than 0.5M protoplast yield sharply decline, be mainly while making the centrifugal collection of protoplastis because osmotic pressure is too high, to be difficult to sedimentation to cause low yield (Fig. 3 A).Under Hyposmolality (0.3 M N.F,USP MANNITOL) and high osmotic pressure (0.6～0.7M N.F,USP MANNITOL), the FDA protoplastis number less (Fig. 3 B) that fluoresces after hatching, protoplastis vigor lower (< 80%) (Fig. 3 C), be mainly to break because Hyposmolality can cause protoplastis, high osmotic pressure causes protoplastis shrinkage.When osmotic pressure is suitable, (0.4～0.5 M N.F,USP MANNITOL) extracts the protoplastis FDA obtaining and hatches at most (Fig. 3 B) of rear fluorescigenic number, vigor the highest (96%) (Fig. 3 C).

the impact of centrifugal speed on protoplast yield and vigor

Under the optimum combination condition of above-mentioned selection, process culturing cell, investigate the impact of different centrifugal speeds on protoplastis vigor and output, result is as shown in Fig. 4 A～4C.600 r/min are centrifugal, and the protoplast yield obtaining is the highest, the protoplast yield that rotating speed too low (300 r/min) and too high (1000 r/min) collect is all lower, be mainly that protoplastis is difficult to sedimentation during due to the slow speed of revolution, high rotating speed makes it be easy to fragmentation (Fig. 4 A).The protoplastis that the centrifugal collection of suitable rotating speed (600 r/min) obtains, number (Fig. 4 B) at most fluoresces after FDA dyeing, protoplastis vigor the highest (Fig. 4 C) be mainly because cell debris free settling not under this rotating speed, and complete protoplastis can be collected by sedimentation.

2.5 protoplastis vigor detect

FDA is a kind of lipophilic substance, can see through plasmalemma and enter cell interior, resolved into a kind of fluorescigenic non-lipophilic substance by born of the same parents' lactonase, for detection of the integrity of cytolemma, fluorescence more strong representation protoplastis vigor is higher, broken or do not fluoresce without the protoplastis of vitality.Fig. 5 A, Fig. 5 B, Fig. 5 C, Fig. 5 D, Fig. 5 E are respectively under the protoplastis after purifying under visible ray, 488 nm exciting lights the protoplastis laser capture microdissection picture under the full fluorescent effect figure of protoplastis 3D and light field after FDA dyeing under the protoplastis, fluorescent microscope oil mirror of the green-emitting fluorescence after FDA dyeing, the micro-lower FDA dyeing of laser co-focusing.Can find out, FDA can be positioned cytolemma, and the integrity of indicator cells film illustrates that the protoplastis vigor extracting under this experiment condition is higher.

Claims (1)

1. a separation purification method for red sage root protoplastis, is characterized in that, comprises the following steps:

1) evoked callus and succeeding transfer culture

The blade of robust growth in red sage root aseptic seedling is cut into the explant of 0.5 cm × 0.5 cm size, is inoculated on MS solid medium at aseptic condition, under illumination condition, induction forms callus; By the callus inducing at equal culture condition take every 20 d succeeding transfer culture 12 d again after succeeding transfer culture one-time continuous cultivated for 3 generations, be transferred in MS liquid nutrient medium, rotating speed be 125 r/min, temperature be under the condition of 25 ℃ dark suspension culture 6 days suspension cell;

2) separation of protoplastis

Combination enzyme is added in 10 mL enzyme solvents and activate 10 min in 55 ℃ of water-baths, be placed into after normal temperature again through 0.45 μ m filtering with microporous membrane sterilizing, add the suspension cell of 1.5 g, enzymolysis mixture is placed in 40 r/min, enzymolysis 4～20 h under 25 ℃ of dark conditions;

Described combination enzyme is cellulase 1.5%, polygalacturonase 0.3%, macerozyme 0.5%;

Described enzyme solvent is the mixing solutions of the MES of KCl, 20 mM of 0.4 M N.F,USP MANNITOL, 20 mM, is adjusted to pH 5.7 with 1 M KOH;

3) protoplastis purifying

Enzymolysis mixture, again through centrifugal 1 min of 80 r/min, is filtered to enzymolysis mixed solution to centrifuge tube, add equal-volume W5 solution, under different centrifugal force, centrifugal 5 min, abandon supernatant; Add again 10 mL W5 solution centrifugal 5 min, abandon supernatant, then after adding 2 mL W5 solution resuspended after placing 30 min on ice centrifugal 3 min, abandon supernatant, add 1.5 ml containing the W5 solution of 0.4 M N.F,USP MANNITOL, resuspended preservation, obtains the protoplastis of purifying;

Described W5 solution is 154 mM NaCl, 125 mM CaCl ₂, 5 mM KCl, 2 mM MES-K, pH value 5.7.

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