CN108085292A - A kind of isolation and purification method of tea tree mesophyll protoplast - Google Patents
A kind of isolation and purification method of tea tree mesophyll protoplast Download PDFInfo
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Abstract
A kind of isolation and purification method of tea tree mesophyll protoplast using tea tree seed seedling tender leaf as material, is separated using enzymatic isolation method, density gradient method purifying establishes protoplast preparation system.Spire obtains easy, protoplast electrofusion and purification process simply and can carry out throughout the year, yield of protoplast after isolating and purifying is higher, integrality is good, lays a good foundation to carry out the researchs such as protein subcellular positioning, protein-interacting, gene expression regulation and subcellular organelle preparation using protoplast transient expression system.
Description
Technical field
The present invention relates to a kind of isolation and purification method, more specifically to a kind of separation of tea tree mesophyll protoplast
With purification process, belong to Skill of Plant Protoplasts field.
Background technology
Tea tree (Camellia sinensis L.) is the important leaf in China three big nothing of woody industrial crops and the world
The resource plant of one of alcoholic beverage.Since tealeaves is rich in a variety of valuable characteristics such as catechin, caffeine and theanine
Secondary metabolite, tea tree molecular biology research is increasingly deep, with a large amount of functional genes by numerous and confused identification and clone.So
And since the foundation of tea tree genetic conversion system is difficult to really break through always, lack again in model plant arabidopsis, tobacco
The metabolic pathway of the peculiar substance of tea tree, therefore the research of functional gene substantially lags.In recent years, protoplast transient expression system
It has been obtained extensively in terms of the researchs such as protein subcellular positioning, protein-interacting, gene expression regulation and subcellular organelle preparation
Using；Thus, protoplast transient expression system may be another important breakthrough mouth of tea tree functional gene research.
The premise that protoplast transient expression system is established is the separation and purifying of high yield pulp1, high vigor protoplast.Mesh
Before, using tea tree petal as the isolated protoplast of material；However, the great timeliness of the materials of tea tree petal, in very great Cheng
The application of protoplast is constrained on degree.Blade is the good material of protoplast electrofusion, however tea leaf wax is thick, polyphenol
Content is high, and protoplast is difficult to separate, and there has been no the separated reports of mesophyll protoplast at present.
The content of the invention
The present invention is for when existing tea leaf protoplast electrofusion is difficult, integrality is poor and petal protoplast extracts tool
The problems such as effect property, provide a kind of isolation and purification method of tea tree mesophyll protoplast.
To achieve the above object, technical solution of the invention is：A kind of separation of tea tree mesophyll protoplast with it is pure
Change method, which is characterized in that comprise the following steps：Step 1: the first leaf blade that acquisition tea tree seed seedling is fully deployed, distillation
Water cleans surface, and 70% ethanol disinfection 3min, then aseptic water washing 5 times, blotting paper blots, and blade then is immersed plasm
Body rinsing liquid, protoplast pre-separation 30min；Step 2: by the knife of 70% (v/v) ethanol disinfection of the blade Jing Guo step 1
Piece removes master pulse and leaf margin, is cut into 5mm2Fritter is immediately placed in sterile vial, and by material：Enzymolysis liquid=1：10 ratios add
Enter enzymolysis liquid；Step 3: the sterile vial of step 2 is placed on the sealing of Parafilm films on temperature control shaking table and in 70rpm, 25
DEG C dark condition under be incubated, during solution greening in bottle subject to sterilization, draw a small amount of solution in micro- Microscopic observation plasm
Body release conditions；Step 4: solution is passed sequentially through into 70mm stainless steels screen and 40 μm after blade enzymolysis 16-18h in step 3
Cell sieve collects filtrate in centrifuge tube, 70mm stainless steels screen and 40 μm of cell sieves is rinsed with protoplast rinsing liquid, by filtrate
Merge, filtrate centrifuges 3min in 200g, removes supernatant, soft to add in protoplast rinsing liquid suspension protoplast, 200g from
Heart 3min collects filtrate, and protoplast cleaning is repeated once, and supernatant is discarded, by 1：1 ratio adds in protoplast rinsing liquid weight
It is outstanding；Step 5: taking another sterilizing test tubes, add in and be layered liquid with the protoplast of protoplast rinsing liquid same volume, then gently
Protoplast suspension in soft sucking-off step 4 is slowly positioned on protoplast layering liquid, and 50g centrifugation 30s or standing 5min,
There is a clear, macroscopic green stripes, protoplast as after purification between Protoplast suspension and layering liquid.
Protoplast electrofusion liquid in the step 2 is formulated by following components：0.4-0.7M mannitol, 10mM chlorine
Change calcium, 20mM2- (N- morpholinoes) ethane sulfonic acid (pH5.7), 20mM potassium chloride, 0.8-3.0% (w/v) cellulase R-10,
0.6-3.0% (w/v) macerozymes R-10,0.1% (w/v) bovine serum albumin, 1% (w/v) PVPP, 2mM ascorbic acid, 0.45 μm
Filtration sterilization.
The protoplast electrofusion liquid is formulated by following components：0.6M mannitol, 10mM calcium chloride, 20mM2-
(N- morpholinoes) ethane sulfonic acid (pH5.7), 20mM potassium chloride, 1.5% (w/v) cellulase R-10,2.5% (w/v) macerozyme
R-10,0.1% (w/v) bovine serum albumin, 1% (w/v) PVPP, 2mM ascorbic acid, 0.45 μm of filtration sterilization.
Protoplast rinsing liquid in the step 4 is formulated by following components：0.4-0.7M mannitol, 10mM chlorine
Change calcium, 20mM2- (N- morpholinoes) ethane sulfonic acid (pH5.7), 20mM potassium chloride, 0.1% (w/v) bovine serum albumin, 1% (w/v)
PVPP, 2mM ascorbic acid, 0.45 μm of filtration sterilization.
Protoplast layering liquid in the step 5 is by OptidensityTMSample rate separating liquid and protoplast
25% (v/v) Iodixanol that rinsing liquid is formulated.
Compared with prior art, the beneficial effects of the invention are as follows：
For this method using tea tree seed seedling young leaflet tablet as material separation, purifying protoplast, it is easy, former that spire material obtains
Raw plastid isolation and purification is easy to operate and can carry out throughout the year, through enzymolysis separation and the yield of protoplast after density gradient purification
It is higher, integrality is good；To carry out protein subcellular positioning, protein-interacting, gene expression regulation and Asia using protoplast
The researchs such as organelle preparation are laid a good foundation.
Figure of description
Fig. 1 is the protoplast being not yet kept completely separate in the present invention.
Fig. 2 is the protoplast in being separated in the present invention.
Fig. 3 is the protoplast band being enriched with after purification in the present invention.
Fig. 4 is the protoplast being kept completely separate in the present invention.
Specific embodiment
Below in conjunction with specific embodiment, the present invention is described in further detail.
Referring to Fig. 1 to Fig. 4, a kind of isolation and purification method of tea tree mesophyll protoplast specifically includes following steps：
Step 1: the first leaf blade for being fully deployed of acquisition tea tree seed seedling, distilled water cleaning surface, then with 70% ethyl alcohol
Sterilize 3min, then aseptic water washing 5 times, then blotted with blotting paper；Then blade is immersed into protoplast rinsing liquid, plasm
Body pre-separation 30min.Blade surface sterilizes, and prevents subsequent experimental from polluting；Protoplast pre-separation can improve enzymolysis efficiency.
Step 2: the sharp cutter of 70% ethanol disinfection of the blade Jing Guo step 1 is removed master pulse and leaf margin, and cut
Into 5mm2Then fritter is immediately placed in sterile vial；And by material：Enzymolysis liquid=1：10 ratios add in enzymolysis liquid.Herein
Mechanically decoupled blade is conducive to accelerate enzymolysis, improves enzymolysis efficiency.
Step 3: the sterile vial of step 2 with Parafilm films sealing be placed on temperature control shaking table and 70rpm, 25 DEG C
Dark condition under be incubated, when jog sterile vial solution greening, it is primary in micro- Microscopic observation that pipette tips draw a small amount of solution
Plastid release conditions.Blade digests under suitable conditions, is conducive to protoplast liberation；Solution greening shows that protoplast is big
Amount release.
Step 4: in step 3 after blade enzymolysis 16-18h, enzymolysis solution is passed sequentially through into 70mm stainless steels screen and 40 μ
Then m cell sieves collect filtrate in centrifuge tube；Again 70mm stainless steels screen and 40 μm of cells are rinsed with protoplast rinsing liquid
Sieve, then filtrate is merged, and 200g centrifugations 3min；Then supernatant is carefully removed with pipette tips, then the soft protoplast that adds in floats
Washing lotion suspension protoplast, then 200g centrifugations 3min, then collects filtrate；Then protoplast cleaning is repeated once again,
And supernatant is discarded, it adds a small amount of protoplast rinsing liquid and is resuspended.This step can remove the cell mass not digested completely, clearly
Except remaining enzyme component in enzymolysis liquid, free protoplast is collected.
Step 5: taking another sterilizing test tubes, add in and be layered liquid with the protoplast of protoplast rinsing liquid same volume, so
Protoplast suspension in step 4 is softly suctioned out afterwards, is slowly positioned on protoplast layering liquid (25% (v/v) Iodixanol),
And 50g centrifugation 30s or standing 5min.There is a clear, macroscopic green bar between Protoplast suspension and layering liquid
Band, protoplast as after purification；Microscopy finds that most of protoplast greens, circular, content are enriched at this time.This step
Using density gradient purification protoplast, 0.6M mannitol and 25% (v/v) Iodixanol form boundary in Protoplast suspension
Face.
Specifically, the enzymolysis liquid in the step 2 is formulated by following components：0.4-0.7M mannitol, 10mM chlorinations
Calcium, 20mM2- (N- morpholinoes) ethane sulfonic acid (pH5.7), 20mM potassium chloride, 0.8-3.0% (w/v) cellulases R-10,0.6-
3.0% (w/v) macerozyme R-10,0.1% (v/v) bovine serum albumin, 1% (v/v) PVPP, 2mM ascorbic acid, 0.45 μm of filtering
Sterilizing, and solution matching while using.Further, the enzymolysis liquid is formulated by following components：0.6M mannitol, 10mM
Calcium chloride, 20mM2- (N- morpholinoes) ethane sulfonic acid (pH5.7), 20mM potassium chloride, 1.5% (w/v) cellulase R-10,
2.5% (w/v) macerozyme R-10,0.1% (v/v) bovine serum albumin, 1% (v/v) PVPP, 2mM ascorbic acid, 0.45 μm of filtering
Sterilizing, and solution matching while using.
Specifically, sterile vial in the step 3 be placed on the sealing of Parafilm films on temperature control shaking table and
16-18h is incubated under 70rpm, 25 DEG C of dark condition.
Specifically, the protoplast rinsing liquid in the step 4 is formulated by following components：0.4-0.7M mannitol,
10mM calcium chloride, 20mM2- (N- morpholinoes) ethane sulfonic acid (pH5.7), 20mM potassium chloride, 0.1% (v/v) bovine serum albumin,
1% (v/v) PVPP, 2mM ascorbic acid, 0.45 μm of filtration sterilization；I.e. protoplast rinsing liquid ingredient is not with adding cellulase
It is identical with the enzymolysis liquid of macerozyme.
Specifically, the protoplast layering liquid in the step 5 is by OptidensityTMSample rate separating liquid
25% (v/v) Iodixanol that (60% Iodixanol) and protoplast rinsing liquid are formulated.
Referring to Fig. 1 to Fig. 4, embodiment one,
A kind of tea tree mesophyll protoplast electrofusion and the method for purifying, its step are as follows：
Step 1: the first leaf blade for being fully deployed of acquisition tea tree seed seedling, distilled water cleaning surface, then with 70% ethyl alcohol
Sterilize 3min, then aseptic water washing 5 times, then blotted with blotting paper；Then blade is immersed into protoplast rinsing liquid, plasm
Body pre-separation 30min.
Master pulse and leaf margin are removed by the sharp cutter of 70% ethanol disinfection Step 2: using the blade Jing Guo step 1,
And it is cut into 5mm2Then fritter is immediately placed in sterile vial；And by material：Protoplast electrofusion liquid=1：10 ratios add in former
Raw plastid separating liquid.Protoplast electrofusion liquid described herein is made of following components system：0.6M mannitol, 10mM chlorinations
Calcium, 20mM2- (N- morpholinoes) ethane sulfonic acid (pH5.7), 20mM potassium chloride, 0.8-3.0% (w/v) cellulases R-10,0.6-
3.0% (w/v) macerozyme R-10,0.1% (v/v) bovine serum albumin, 1% (v/v) PVPP, 2mM ascorbic acid, 0.45 μm of filtering
Sterilizing；And the protoplast electrofusion liquid matching while using.
Step 3: the sterile vial of step 2 with Parafilm films sealing be placed on temperature control shaking table and 70rpm, 25 DEG C
Dark condition under be incubated 16-18h；During protoplast solution greening in bottle subject to sterilization, pipette tips draw a small amount of protoplast
Solution is in micro- Microscopic observation protoplast liberation situation.The result of variations of concrete fiber element enzyme and isolation enzyme concentration see the table below：
As seen from the above table, when cellulase 1.5%, macerozyme 2.0-2.5%, the separation of tea leaf mesophyll protoplast is more
And integrality is good；But further it was found that, it is short the time required to protoplast electrofusion during macerozyme 2.5%, be more advantageous to primary
The holding of plastid vigor.
Meanwhile referring to Fig. 1,2 and 4,12h is digested, cell membrane starts to degrade (Fig. 1)；14h is digested, cell wall rupture is primary
Plastid is intended to separate out (Fig. 2)；16-18h is digested, protoplasm free increases, and most of is circular, complete protoplast
(Fig. 4).
Step 4: protoplast solution is passed sequentially through 70mm stainless steels screen and 40 μm of cell sieves, filtrate is then collected
In centrifuge tube；70mm stainless steels screen and 40 μm of cell sieves are rinsed with protoplast rinsing liquid again, then filtrate is merged, and 200g
Centrifuge 3min；Then supernatant, then soft addition protoplast rinsing liquid suspension protoplast are carefully removed with pipette tips, then
200g centrifuges 3min, then collects filtrate；Then protoplast cleaning is repeated once again, and discards supernatant, added few
Protoplast rinsing liquid is measured to be resuspended.Protoplast rinsing liquid ingredient herein and addition cellulase and the protoplast of macerozyme
Separating liquid is identical.
Step 5: taking another sterilizing test tubes, add in and be layered liquid with the protoplast of protoplast rinsing liquid same volume, so
Protoplast suspension in step 4 is softly suctioned out afterwards, is slowly positioned on protoplast layering liquid (25% (v/v) Iodixanol),
And 50g centrifugation 30s or standing 5min.There is a clear, macroscopic green bar between Protoplast suspension and layering liquid
Band, referring specifically to Fig. 3, protoplast as after purification；Microscopy finds most of protoplast greens, circular, content at this time
Object enriches, referring specifically to Fig. 4.
This method is isolated using enzymatic isolation method using tea tree seed seedling young leaflet tablet as material separation, purifying protoplast
Mesophyll protoplast；Protoplast is purified using density gradient method, obtains high yield pulp1, the plasm of high vigor
Body.Compared with prior art, spire material of the present invention obtain easily, protoplast electrofusion and purification process it is simple and throughout the year can be into
Row；Through enzymolysis separation with the yield of protoplast after density gradient purification is higher, integrality is good, therefore establish efficiently primary
Plastid preparation system is laid a good foundation to carry out gene functional research using protoplast transient expression system.
The above content is a further detailed description of the present invention in conjunction with specific preferred embodiments, it is impossible to assert
The specific implementation of the present invention is confined to these explanations.For those of ordinary skill in the art to which the present invention belongs, exist
On the premise of not departing from present inventive concept, several simple deduction or replace can also be made, said structure should be all considered as belonging to
Protection scope of the present invention.
Claims (5)
- A kind of 1. isolation and purification method of tea tree mesophyll protoplast, which is characterized in that comprise the following steps：Step 1: the first leaf blade that acquisition tea tree seed seedling is fully deployed, distilled water cleaning surface, 70% ethanol disinfection 3 Min, then aseptic water washing 5 times, blotting paper blots, and blade then is immersed protoplast rinsing liquid, protoplast pre-separation 30 min；Step 2: the blade of 70% (v/v) ethanol disinfection of the blade Jing Guo step 1 is removed master pulse and leaf margin, 5 mm are cut into2 Fritter is immediately placed in sterile vial, and by material：Enzymolysis liquid=1：10 ratios add in enzymolysis liquid；Step 3: the sterile vial of step 2 be placed on temperature control shaking table with the sealing of Parafilm films and 70rpm, 25 DEG C It is incubated under dark condition, during solution greening in bottle subject to sterilization, draws a small amount of solution and released in micro- Microscopic observation protoplast To one's heart's content condition；Step 4: solution is passed sequentially through into 70 mm stainless steels screens and 40 μm of cells after blade enzymolysis 16-18h in step 3 Sieve collects filtrate in centrifuge tube, rinses 70 mm stainless steels screens and 40 μm of cell sieves with protoplast rinsing liquid, filtrate is closed And filtrate centrifuges 3 min in 200g, removes supernatant, it is soft to add in protoplast rinsing liquid suspension protoplast, 200 g from 3 min of the heart collects filtrate, and protoplast cleaning is repeated once, and supernatant is discarded, by 1：1 ratio adds in protoplast rinsing liquid It is resuspended；Step 5: taking another sterilizing test tubes, add in and be layered liquid with the protoplast of protoplast rinsing liquid same volume, then gently Protoplast suspension in soft sucking-off step 4 is slowly positioned on protoplast layering liquid, and 50 g centrifuge 30 s or standing There are a clear, macroscopic green stripes, as after purification primary between Protoplast suspension and layering liquid in 5min Plastid.
- 2. the isolation and purification method of a kind of tea tree mesophyll protoplast according to claim 1, which is characterized in that described Protoplast electrofusion liquid in step 2 is formulated by following components：0.4-0.7 M mannitol, 10 mM calcium chloride, 20 mM 2-（N- morpholinoes）Ethane sulfonic acid (pH 5.7), 20 mM potassium chloride, 0.8-3.0 % (w/v) cellulases R-10,0.6-3.0 % (w/v) macerozymes R-10,0.1 % (w/v) bovine serum albumin, 1 % (w/v) PVPP, 2 mM ascorbic acid, 0.45 μm Filtration sterilization.
- 3. the isolation and purification method of a kind of tea tree mesophyll protoplast according to claim 2, which is characterized in that described Protoplast electrofusion liquid be formulated by following components：0.6 M mannitol, 10 mM calcium chloride, 20 mM 2-（N- morpholines Generation）Ethane sulfonic acid (pH 5.7), 20 mM potassium chloride, 1.5 % (w/v) cellulases R-10,2.5 % (w/v) macerozyme R- 10,0.1 % (w/v) bovine serum albumin, 1 % (w/v) PVPP, 2 mM ascorbic acid, 0.45 μm of filtration sterilization.
- 4. the isolation and purification method of a kind of tea tree mesophyll protoplast according to claim 1, which is characterized in that described Protoplast rinsing liquid in step 4 is formulated by following components：0.4-0.7 M mannitol, 10 mM calcium chloride, 20 mM 2-（N- morpholinoes）Ethane sulfonic acid (pH 5.7), 20 mM potassium chloride, 0.1 % (w/v) bovine serum albumin, 1 % (w/v) PVPP, 2 mM ascorbic acid, 0.45 μm of filtration sterilization.
- 5. the isolation and purification method of a kind of tea tree mesophyll protoplast according to claim 1, which is characterized in that described Protoplast layering liquid in step 5 is by OptidensityTMSample rate separating liquid and protoplast rinsing liquid prepare and Into 25 % (v/v) Iodixanol.
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| CN108949665A (en) * | 2018-07-12 | 2018-12-07 | 北京林业大学 | The preparation method of lily petal protoplast |
| CN108949665B (en) * | 2018-07-12 | 2022-03-04 | 北京林业大学 | Preparation method of lily petal protoplast |
| CN111454875A (en) * | 2020-04-16 | 2020-07-28 | 中国农业科学院蔬菜花卉研究所 | Method for separating colored cell protoplast of hydrangea macrophylla |
| CN113249296A (en) * | 2021-06-30 | 2021-08-13 | 安徽农业大学 | Method for separating and purifying fresh tissue protoplast of tea tree |
| CN113249296B (en) * | 2021-06-30 | 2024-01-30 | 安徽农业大学 | Separation and purification method of fresh tissue protoplast of tea tree |
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Application publication date: 20180529 |