CN106399223A - Tripterygium wilfordii protoplast separation and instantaneous conversion method - Google Patents
Tripterygium wilfordii protoplast separation and instantaneous conversion method Download PDFInfo
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Abstract
The invention discloses a Tripterygium wilfordii protoplast separation and instantaneous conversion method. Experiments prove that the yield of protoplasts obtained by the separation method is 4.24*10<5>/g FW, and the activity is 94.5%; and exogenous plasmids are successfully converted into the protoplasts extracted by the invention through a PEG 4000 mediated conversion method and are smoothly expressed, thereby successfully establishing a Tripterygium wilfordii suspension cell protoplast instantaneous conversion system. The invention lays a foundation for carrying out functional genomics research of Tripterygium wilfordii in a deep-going way, and simultaneously provides a material basis for research of Tripterygium wilfordii such as gene editing, subcellular localization, gene overexpression and interference and the like.
Description
Technical field
The invention belongs to field of plant tissue culture is and in particular to a kind of separation of Radix Tripterygii Wilfordii protoplast and instantaneous conversion
Method, particularly to a kind of method of isolation and purification method of Radix Tripterygii Wilfordii protoplast and exogenous gene instantaneous conversion.
Background technology
Chinese herb triperygium wilfordii is root class medical material, from Celastraceae plant Radix Tripterygii Wilfordii (Tripterygium wilfordii
Hook.f. root) or the xylem of root, have the work(such as heat-clearing and toxic substances removing, dispelling wind and removing obstruction in the collateral, relaxing muscles and tendons to promote blood circulation, reducing swelling and alleviating pain, parasite killing hemostasis
Effect.The main active substances of Radix Tripterygii Wilfordii are alkaloidss, Diterpeneses, triterpeness, sesquiterpenoidss and saccharide.There is antiinflammatory, resist and swell
Tumor, immunosuppressant etc. act on, and are mainly used in rheumatoid, antitumor, renal glomerular disease, systemic red in modern clinical treatment
The diseases such as yabbi skin ulcer.In recent years, the research of Radix Tripterygii Wilfordii causes the biomedical extensive pass with domain experts such as agronomy both at home and abroad
Note, but the research for Radix Tripterygii Wilfordii protoplast and instantaneous conversion still belongs to blank, limits Radix Tripterygii Wilfordii to a certain extent
The further investigation of the aspect such as functional gene and synthetic biology.
Protoplast refers to do not have cell wall coated " exposed " plant cell, and outermost layer is surrounded by one layer of plasma membrane, includes
Cytoplasm (including substrate, organelle and ergastic mateiral etc.) and nucleus, are had cellular omnipotency, can be carried out with regenerative cell's wall
Continuous division.The application prospect of protoplast widely, can be used for the Study on Genetic Transformation of plant, cell fusion research,
The field such as genetic breeding research and cell membrane research.
Content of the invention
First purpose of the present invention is to provide a kind of separation method of Radix Tripterygii Wilfordii protoplast.
The separation method of the Radix Tripterygii Wilfordii protoplast that the present invention provides comprises the steps：
1) Radix Tripterygii Wilfordii calluss suspension cell, the thunder god vine suspending cell after being digested are digested with enzymolysis solution；
2) collect the precipitation in the thunder god vine suspending cell after described enzymolysis, that is, obtain Radix Tripterygii Wilfordii protoplast；
Described enzymolysis solution includes cellulase, pectase and macerozyme.
In said method,
The mass ratio of described cellulase, described pectase and described macerozyme is 4:1:1.Wherein, 1 enzyme activity unit
Refer to, under specified conditions (25 DEG C, other is optimal condition), the enzyme amount of 1 micromole substrate can be converted in 1 minute, or turn
Change the 1 micromolar enzyme amount about group in substrate.
In said method,
Described enzymolysis solution also includes Mannitol.
In said method,
Concentration in described enzymolysis solution for the described Mannitol is 0.3-0.7mol/L.
In said method,
Described enzymolysis solution is by Mannitol, BSA, MES, CaCl2 2H2O, cellulase, pectase, macerozyme and water group
Become；
Concentration in described enzymolysis solution for the described Mannitol is 0.6mol/L；
Mass fraction in described enzymolysis solution for the described BSA is 0.6%；
Mass fraction in described enzymolysis solution for the described MES is 0.1%；
Described CaCl2·2H2Concentration in described enzymolysis solution for the O is 0.05mol/L；
Mass fraction in described enzymolysis solution for the described cellulase is 2.0%；
Mass fraction in described enzymolysis solution for the described pectase is 0.5%；
Mass fraction in described enzymolysis solution for the described macerozyme is 0.5%.
In said method,
Described be collected as being centrifuged, the condition of described centrifugation is that (17-104) × g is centrifuged 5min, and the condition of described centrifugation is concrete
It is centrifuged 5min for 67 × g.
In said method,
The time of described enzymolysis is 10h.
In said method,
Described Radix Tripterygii Wilfordii calluss suspension cell derives from Radix Tripterygii Wilfordii calluss.
In said method,
The step filtering also is included between described 1) and described 2)；Described 300 mesh rustless steel cells being filtered into sterilizing
It is sieved through filter.
Second object of the present invention is to provide to separate, by said method, the protoplast obtaining.
Third object of the present invention is to provide the new application of above-mentioned protoplast.
The invention provides application in exogenous gene expression for the above-mentioned protoplast.
Fourth object of the present invention is to provide a kind of method of exogenous gene expression.
The method of the exogenous gene expression that the present invention provides comprises the steps：By above-mentioned for gene transformation protoplast
In, realize the expression of exogenous gene.
In said method, described exogenous gene is gfp gene.
5th purpose of the present invention is to provide above-mentioned enzymolysis solution.
Application in protoplast electrofusion for the above-mentioned enzymolysis solution falls within protection scope of the present invention.
The invention provides a kind of separation method of Radix Tripterygii Wilfordii protoplast.The separation of the Radix Tripterygii Wilfordii protoplast of the present invention
The Optimum separation condition of method is：Enzymolysis solution proportioning is 2.0% cellulase+0.5% pectase+0.5% macerozyme；Mannitol
Concentration 0.6mol/L, enzymolysis time 10h, 67 × g rotating speed is centrifuged 5min.It is experimentally confirmed：Extracted with the method for the present invention
The protoplast arriving is through after purification, protoplast yield is 4.24 × 105/ g FW, vigor is 94.5%.The present invention also by
Exogenous plasmid is successfully transformed in the protoplast of present invention extraction and smoothly expresses by the method for PEG4000 mediated transformation, becomes
Work(establish thunder god vine suspending cell protoplast instantaneous conversion system.The present invention is to carry out the functional gene of Radix Tripterygii Wilfordii in a deep going way
Group is learned research and is laid a good foundation, and also carries out gene editing, Subcellular Localization, gene overexpression and interference etc. for Radix Tripterygii Wilfordii simultaneously and grinds
Study carefully and provide material base.
Brief description
Fig. 1 is the impact to protoplast yield and vigor for the enzymolysis solution proportioning.Figure 1A is enzymolysis solution proportioning to protoplast
The impact of yield；Figure 1B is the impact to protoplast vigor for the enzymolysis solution proportioning.
Fig. 2 is the impact to protoplast yield and vigor for the enzymolysis time.Fig. 2A is enzymolysis time to protoplast yield
Impact；Fig. 2 B is the impact to protoplast vigor for the enzymolysis time.
Fig. 3 is the impact to protoplast yield and vigor for the mannitol concentration.Fig. 3 A is mannitol concentration to protoplast
The impact of yield；Fig. 3 B is the impact to protoplast vigor for the mannitol concentration.
Fig. 4 is to process the impact to protoplast yield and vigor for the rotating speed.Fig. 4 A is to process rotating speed to protoplast yield
Impact；Fig. 4 B is to process the impact to protoplast vigor for the rotating speed.
Fig. 5 is protoplast form.Fig. 5 A is protoplast form after purification under visible ray；Fig. 5 B is Trypan Blue
Unvital protoplast form afterwards.
Fig. 6 is protoplast and GFP picture under laser confocal scanning microscope light field.Fig. 6 A sweeps for laser co-focusing
Retouch the protoplast under microscope light field；Fig. 6 B is the GFP of protoplast under laser confocal scanning microscope 488nm exciting light
Fluorescence picture；Fig. 6 C is protoplast Chloroplast auto-fluorescence picture under laser confocal scanning microscope 488nm exciting light；Figure
6D is that under laser confocal scanning microscope, protoplast GFP fluorescence is superimposed picture with autofluorescence.
Specific embodiment
Experimental technique used in following embodiments if no special instructions, is conventional method.
Material used, reagent etc. in following embodiments, if no special instructions, all commercially obtain.
Quantitative test in following embodiments, is respectively provided with three times and repeats to test, results averaged.
In following embodiments, instrument includes：Centrifuge (SIGMA), laser confocal scanning microscope
(VisiTech), superclean bench, microscope, constant temperature culture oscillator, multiplex vavuum pump of circulating water type, electronic balance, high temperature
High-pressure sterilizing pot.
W5 solution in following embodiments is 0.125mol/L CaCl2、0.154mol/L NaCl、5mmol/L KCl、
2mmol/L MES, KOH are adjusted to pH5.6, autoclave sterilization.
MMG solution in following embodiments is 15mmol/L MgCl2, 0.1%MES, 0.4mol/L Mannitol, KOH is adjusted to
PH5.6, autoclave sterilization.
PEG solution in following embodiments is 40%PEG4000,0.2mol/L Mannitol, 0.1mol/L CaCl2(now join
Now use, at most can preserve five days in 4 DEG C).
WI solution in following embodiments is 0.5mol/L Mannitol, 4mmol/L MES, 20mmol/L KCl, and KOH is adjusted to
PH5.6, autoclave sterilization.
At the purchase of the reagent in following embodiments and catalog number is as follows：Cellulase (R-10, YAKULT,
150703-02), pectase (Y-23, YAKULT), macerozyme (R-10, YAKULT), Mannitol (D-Mannitol,
Solarbio, 1126F034), bovine serum albumin (BSA, Roche), morpholino b acid (MES, Sai Kelin, C10035338),
CALCIUM CHLORIDE DIHYDRATE (CaCl2·2H2O, Chemical Reagent Co., Ltd., Sinopharm Group, 20130130), Polyethylene Glycol (PEG4000,
Solarbio, 1226B034), and sodium chloride (NaCl, the modern east fine chemicals company limited in Beijing, 20140301), chlorination
Potassium (KCl, Chemical Reagent Co., Ltd., Sinopharm Group, 20120925), phosphate buffer (PBS, gibco, 1760235), platform
Expect blue dye liquor (Beijing DingGuo ChangSheng Biology Technology Co., Ltd, 5BR00130).
Embodiment 1, a kind of isolation and purification method of thunder god vine suspending cell protoplast and its Activity determination
First, the preparation of thunder god vine suspending cell
Thunder god vine suspending cell derives from calluss, has substantial amounts of intercellular substance, cell wall is easily digested, and has life
The advantages of long speed is fast, secondary metabolitess accumulation is rapid, is the ideal material preparing protoplast.Concrete preparation method is as follows：
1st, the acquisition of calluss
By Radix Tripterygii Wilfordii edible tender branch sterilization treatment, cottage breeding goes out aseptic seedling, then takes the Newborn Leaves of aseptic seedling, is cut into
0.5cm × 0.5cm about fritter, be seeded in containing 0.5mg/L 2,4-D (prosperity Bioisystech Co., Ltd of Beijing ancient cooking vessel state,
DH101-1 on MS agar culture medium (Caisson Labs, MSP22-50LT)), 25 DEG C, light culture induced synthesis wound healing group
Knit.
2nd, the successive transfer culture of calluss
Choose the calluss that white is glossy, quality is loose, grow fine in 0.5mg/L 2,4-D+0.1mg/L KT
(prosperity Bioisystech Co., Ltd of Beijing ancient cooking vessel state, DH135-2)+0.5mg/L IBA (Beijing ancient cooking vessel state prosperity limited public affairs of biotechnology
Department, II172) MS agar culture medium on successive transfer culture, 25 DEG C, light culture.Successive transfer culture 3 more than generation, chooses well-grown, matter
The calluss that ground is loose, growing state is consistent or similar, press from both sides into fritter with tweezers, and crushing, with the calluss of every bottle of 1.0g
Amount transfer into the 2,4-D+0.1mg/L KT+0.5mg/L IBA of 0.5mg/L containing 100mL MS liquid medium 250mL triangle
In bottle, it is put on shaking table, 25 DEG C, dark, 120rpm suspension culture, 20 days subcultures once, obtain thunder god vine suspending cell.
2nd, the separation of protoplast and its Activity determination
Choose the successive transfer culture thunder god vine suspending cell of 10-12 days and extract material as protoplast.Take different enzymes
Solution liquid proportioning, mannitol concentration, enzymolysis time and process rotating speed separate Radix Tripterygii Wilfordii protoplast and detect protoplast yield and
Vigor, selects optimal processing conditional parameter.Comprise the following steps that：
1st, the impact to protoplast yield and vigor for the enzymolysis solution proportioning
(1) preparation of enzymolysis solution：By Mannitol, BSA, MES, CaCl2·2H2O, cellulase, pectase, macerozyme and
Ultra-pure water mixes, and solute and its concentration are：0.5mol/L Mannitol, 0.6% (mass fraction) BSA, 0.1% (mass fraction)
MES、0.05mol/L CaCl2·2H2O, KOH are adjusted to pH5.6, obtain enzymolysis solution, according to the cellulase in enzymolysis solution, pectin
Enzyme, the difference of macerozyme mass fraction, enzymolysis solution is divided into following 5 groups：
1 group：1.5% (mass fraction) cellulase+0.3% (mass fraction) pectase+0.5% (mass fraction) isolates
Enzyme；
2 groups：2.0% (mass fraction) cellulase+0.3% (mass fraction) pectase+0.5% (mass fraction) isolates
Enzyme；
3 groups：2.0% (mass fraction) cellulase+0.5% (mass fraction) pectase+0.5% macerozyme (divide by quality
Number)；
4 groups：2.0% cellulase (mass fraction)+0.7% pectase (mass fraction)+0.5% macerozyme (divide by quality
Number)；
5 groups：2.5% cellulase (mass fraction)+0.3% pectase (mass fraction)+0.5% macerozyme (divide by quality
Number)；
Above-mentioned 5 groups of different enzymolysis solutions are activated respectively in 55 DEG C of water-baths 10min, 0.22 μm of filtering with microporous membrane removes
Bacterium, treats that next step test uses.
(2) separation of protoplast
Take 0.5g thunder god vine suspending cell, Aspirate medium, crush as far as possible, the amount according to 10mL/g is separately added into step
(1) digest in 5 groups of enzymolysis solutions in, 25 DEG C, 50rpm dark shaking enzymolysis 12h；With dropper suction suspension cell suspension for several times,
To promote the release of protoplast；Filtered with 300 sterilized mesh rustless steel cell sieves, obtain filtrate；Filtrate is in 67 × g condition
Lower centrifugation 5 minutes, abandons supernatant, collects protoplast.
(3) purification of protoplast
Separate in step (2) in the protoplast obtaining and add 5ml W5 solution, resuspended cleaning protoplast, it is centrifuged 5
Minute, abandon supernatant, respectively be washed once with the W5 solution of 5ml, 2ml respectively.Abandoning supernatant, adds 1ml MMG solution resuspended,
The protoplast of purification can be obtained.
(4) yield of protoplast and viability examination
The detection method of the yield of protoplast is：The protoplast dilution of purification step (3) being obtained with MMG solution
5 times, the Protoplast suspension of dilution is dropped on blood counting chamber.Treat that counting chamber is full of by protoplast, under the microscope
Observe and count (the protoplast numbers in four big lattice).Each sample repeat count three times.
Every Cray Tripterygium wilfordii Suspension Protoplasts yield (individual/g FW) computing formula：Protoplast total number/(4 ×
Quality of materials) } × extension rate × 104.
The detection method (adopting trypan blue staining) of the vigor of protoplast：With MMG solution by step (3) obtain pure
Change protoplast dilute 5 times, by the trypan blue preparing dye liquor (Beijing DingGuo ChangSheng Biology Technology Co., Ltd,
5BR00130), by volume 1：10 are added to dyeing in the Protoplast suspension after dilution, and selection 4 is representational to be regarded
Open country is counted.The computational methods of protoplast vigor are：Protoplasm under this visual field is not accounted for by the protoplast of Trypan Blue
The percentage ratio of body sum.
The protoplast obtaining is carried out yield and viability examination, result such as Figure 1A and Figure 1B.Relatively 1,2,5 groups, three groups
Cellulase concentration increase successively, wherein second group of protoplast yield and vigor highest, can reach 1.86 respectively ×
105/ g FW and 67.8%.Cellulase concentration too low can not abundant dissociated cell wall, and cellulose concentration is too high, can make thin
After birth ruptures, impact protoplast activity, therefore the optium concentration of cellulose is 2.0%；Relatively 2,3,4 groups, three groups of pectase
Concentration increases successively, and result shows the 4th group of yield and vigor highest, can reach 3.08 × 10 respectively5/ g FW and
80.15%, there is significant difference with other groups, therefore the optium concentration of pectase is 0.5%.To sum up, thunder god vine suspending cell
The optimal enzyme liquid proportioning of protoplast electrofusion is 2.0% cellulase+0.5% pectase+0.5% macerozyme.
2nd, enzymolysis time compares the impact of protoplast yield and vigor
(1) preparation of enzymolysis solution：By Mannitol, BSA, MES, CaCl2·2H2O, cellulase, pectase, macerozyme and
Ultra-pure water mixes, and solute and its concentration are respectively：0.5mol/L Mannitol, 0.6% (mass fraction) BSA, 0.1% (quality is divided
Number) MES, 0.05mol/L CaCl2·2H2O, 2.0% (mass fraction) cellulase, 0.5% (mass fraction) pectase,
0.5% (mass fraction) macerozyme, KOH is adjusted to pH5.6, obtains enzymolysis solution.Above-mentioned enzymolysis solution is activated in 55 DEG C of water-baths
10min, 0.22 μm of filtering with microporous membrane is degerming, treats that next step test uses.
(2) separation of protoplast
Take 0.5g thunder god vine suspending cell, Aspirate medium, crush as far as possible, the amount according to 10mL/g is separately added into step
(1) digest in the enzymolysis solution in, 25 DEG C, the dark shaking of 50rpm digests 6h, 8h, 10h, 12h, 14h and 16h respectively；Taken out with dropper
Inhale suspension cell suspension for several times, to promote the release of protoplast；Filtered with 300 sterilized mesh rustless steel cell sieves, obtain
To filtrate；Filtrate is centrifuged 5 minutes under the conditions of 67 × g, abandons supernatant, collects protoplast respectively.
(3) purification of protoplast
Separate in step (2) in the protoplast obtaining respectively and add 5ml W5 solution, resuspended cleaning protoplast, from
The heart 5 minutes, abandons supernatant, respectively be washed once with the W5 solution of 5ml, 2ml respectively.Abandoning supernatant, adds 1ml MMG solution weight
Outstanding, you can to obtain the protoplast of purification.
(4) yield of protoplast and viability examination
Detection method is with (4) in step 1.
The protoplast obtaining is carried out yield and viability examination, result is shown in Fig. 2A and Fig. 2 B.As can be seen from the figure：With
The increase of enzymolysis time, the yield of protoplast improves therewith, but the vigor of protoplast highest in 10h, reach 82.2%.Enzyme
The solution time is too short can not to make protoplast be kept completely separate out, and extraction efficiency is low, and enzymolysis time is long, and cellular stress is excessive,
It is easily broken, under mirror, detection dead cell and fragment increase, and living cells proportion is little, is unfavorable for the development of subsequent experimental.Therefore,
10h is the optimal enzymolysis time of Radix Tripterygii Wilfordii energy Suspension Protoplasts.
3rd, mannitol concentration compares the impact of protoplast yield and vigor
(1) preparation of enzymolysis solution：By Mannitol, BSA, MES, CaCl2·2H2O, cellulase, pectase, macerozyme and
Ultra-pure water mixes, and solute and its concentration are respectively：0.6% (mass fraction) BSA, 0.1% (mass fraction) MES, 0.05mol/L
CaCl2·2H2O, 2.0% (mass fraction) cellulase, 0.5% (mass fraction) pectase, 0.5% (mass fraction) isolation
Enzyme, KOH is adjusted to pH5.6, obtains enzymolysis solution, is divided into following 5 groups according to the difference of mannitol concentration in enzymolysis solution：
1 group：0.3mol/L Mannitol；
2 groups：0.4mol/L Mannitol；
3 groups：0.5mol/L Mannitol；
4 groups：0.5mol/L Mannitol；
5 groups：0.7mol/L Mannitol；
Above-mentioned enzymolysis solution is activated respectively in 55 DEG C of water-baths 10min, 0.22 μm of filtering with microporous membrane is degerming, treats next
Step test uses.
(2) separation of protoplast
Take 0.5g thunder god vine suspending cell, Aspirate medium, crush as far as possible, the amount according to 10mL/g is separately added into step
(1) digest in 5 groups of enzymolysis solutions in, 25 DEG C, 50rpm dark shaking enzymolysis 12h；With dropper suction suspension cell suspension for several times,
To promote the release of protoplast；Filtered with 300 sterilized mesh rustless steel cell sieves, obtain filtrate；Filtrate is in 67 × g condition
Lower centrifugation 5 minutes, abandons supernatant, collects protoplast.
(3) purification of protoplast
Separate in step (2) in the protoplast obtaining and add 5ml W5 solution, resuspended cleaning protoplast, it is centrifuged 5
Minute, abandon supernatant, respectively be washed once with the W5 solution of 5ml, 2ml respectively.Abandoning supernatant, adds 1ml MMG solution resuspended,
The protoplast of purification can be obtained.
(4) yield of protoplast and viability examination
Detection method is with (4) in step 1.
The protoplast obtaining is carried out yield and viability examination, result is shown in 3A and Fig. 3 B.As seen from the figure, when manna is pure and strong
Spend for protoplast yield highest during 0.5mol/L, and now vigor is not highest, when mannitol concentration is 0.6mol/L
Although when yield more slightly lower than 0.5mol/L, now protoplast vigor highest.Solution osmotic pressure is less than the infiltration of protoplast
Pressure can lead to cell to burst death；The protoplast dehydration shrinkage when mannitol concentration is too high, and it is unfavorable for releasing of protoplast
Put.Although the protoplast sum of survival is close in two treatment groups of 0.5mol/L and 0.6mol/L, due to having the former of survival
Raw plastid just can carry out instantaneous conversion, and in contrast, mannitol concentration is to be more beneficial for opening of next step experiment for 0.6mol/L
Exhibition.So, the optimal mannitol concentration of separated protoplast is 0.6mol/L.
4th, process the impact to protoplast yield and vigor for the rotating speed
(1) preparation of enzymolysis solution：By Mannitol, BSA, MES, CaCl2·2H2O, cellulase, pectase, macerozyme and
Ultra-pure water mixes, and solute and its concentration are respectively：0.5mol/L Mannitol, 0.6% (mass fraction) BSA, 0.1% (quality is divided
Number) MES, 0.05mol/L CaCl2·2H2O, 2.0% (mass fraction) cellulase, 0.5% (mass fraction) pectase,
0.5% (mass fraction) macerozyme, KOH is adjusted to pH5.6, obtains enzymolysis solution.Above-mentioned enzymolysis solution is activated in 55 DEG C of water-baths
10min, 0.22 μm of filtering with microporous membrane is degerming, treats that next step test uses.
(2) separation of protoplast
Take 0.5g thunder god vine suspending cell, Aspirate medium, crush as far as possible, the amount according to 10mL/g is separately added into step
(1) digest in the enzymolysis solution in, 25 DEG C, 50rpm dark shaking enzymolysis 12h；Aspirate suspension cell suspension with dropper for several times, with
Promote the release of protoplast；Filtered with 300 sterilized mesh rustless steel cell sieves, obtain filtrate；By filtrate in different disposal
Rotating speed：It is centrifuged 5 minutes under the conditions of 17 × g, 37 × g, 67 × g, 104 × g, abandons supernatant, collect protoplast respectively.
(3) purification of protoplast
Separate in step (2) in the protoplast obtaining respectively and add 5ml W5 solution, resuspended cleaning protoplast, from
The heart 5 minutes, abandons supernatant, respectively be washed once with the W5 solution of 5ml, 2ml respectively.Abandoning supernatant, adds 1ml MMG solution weight
Outstanding, you can to obtain the protoplast of purification.
(4) yield of protoplast and viability examination
Detection method is with (4) in step 1.
The protoplast obtaining is carried out yield and viability examination, result is shown in Fig. 4 A and Fig. 4 B.Relatively four groups of data, when turning
Speed can reach 2.17 × 10 for yield during 67 × g5g/FW；Vigor reaches as high as 78.4%, and yield and vigor are highest water
Flat.It is unfavorable for that when rotating speed is too low the sedimentation of protoplast, fragment and protoplast can not be efficiently separated；When rotating speed is too high
When, protoplast can be caused with mechanical damage, equally be unfavorable for isolating and purifying of protoplast.To sum up, isolate and purify protoplasm
The optimum speed of body is 67 × g.
3rd, the validation verification of protoplast separation purification method
1st, the preparation of enzymolysis solution：By Mannitol, BSA, MES, CaCl2·2H2O, cellulase, pectase, macerozyme and super
Pure water mixes, and solute and its concentration are respectively：0.6mol/L Mannitol, 0.6% (mass fraction) BSA, 0.1% (mass fraction)
MES、0.05mol/L CaCl2·2H2O, 2.0% (mass fraction) cellulase, 0.5% (mass fraction) pectase, 0.5%
(mass fraction) macerozyme, KOH is adjusted to pH5.6, obtains enzymolysis solution.Above-mentioned enzymolysis solution is activated 10min in 55 DEG C of water-baths,
0.22 μm of filtering with microporous membrane is degerming, treats that next step test uses.
2nd, the separation of protoplast
Take 0.5g thunder god vine suspending cell, Aspirate medium, crush as far as possible, the amount according to 10mL/g adds in step 1
Digest in enzymolysis solution, 25 DEG C, 50rpm dark shaking enzymolysis 10h；With dropper suction suspension cell suspension for several times, former to promote
The release of raw plastid；Filtered with 300 sterilized mesh rustless steel cell sieves, obtain filtrate；Filtrate is centrifuged 5 under the conditions of 67 × g
Minute, abandon supernatant, collect protoplast.
3rd, the purification of protoplast
Separate in step 2 in the protoplast obtaining and add 5ml W5 solution, resuspended cleaning protoplast, it is centrifuged 5 points
Clock, abandons supernatant, respectively be washed once with the W5 solution of 5ml, 2ml respectively.Abandoning supernatant, adds 1ml MMG solution resuspended, that is,
The protoplast of purification can be obtained.
4th, the yield of protoplast and viability examination
Detection method is with (4) in the 1 of step 2.
The protoplast yield obtaining up to 4.24 × 105/ g FW, vigor up to 94.5%, protoplast form and platform
Expect that the protoplast form after blue dyeing is shown in Fig. 5 A and Fig. 5 B.
The application in exogenous gene instantaneous conversion expression of embodiment 2, thunder god vine suspending cell protoplast
Using PEG mediated transformation method, the instantaneous conversion of thunder god vine suspending cell protoplast is studied, concrete step
Suddenly as follows：
1st, the concentration of the protoplast of the purification obtaining in the step 3 of embodiment one is transferred to about 5 × 105Individual/mL,
Obtain protoplast solution；
2nd, by the protoplast solution of 200 μ l, 20 μ g E3025 plasmid (E3025 plasmid carries GFP coded sequence,
The nucleotide sequence of E3025 plasmid is as shown in sequence 1) and 220 μ L PEG solution rap mixing, obtain transformation mixture, black
Induction Transformation mixture 20min under dark condition, obtains converting mixed liquor；
3rd, mixed liquors are converted with 880 μ L W5 solution dilutions under room temperature, then gentle inversion shake centrifuge tube is allowed to mix
Well to terminate conversion reaction.67 × g is centrifuged 2min, collects precipitation, removes supernatant；
4th, use the precipitation that W5 solution repeated washing step 3 is collected once, collect precipitation, remove supernatant；
5th, with 1mL WI solution softly being deposited in porous organization's culture dish of collecting of resuspended step 4,25 DEG C of dark standings
Culture.
6th, take out after 18h, 67 × g is centrifuged 2min, removes part supernatant, it is concentrated, take appropriate film-making, laser is common
GFP expression is observed under confocal scanning microscopy.
Result such as Fig. 6 A, Fig. 6 B, Fig. 6 C and Fig. 6 D.Because protoplast is thin from the thunder god vine suspending of dark culturing
Do not contain chlorophyll in born of the same parents, therefore its cell, GFP green under laser confocal scanning microscope 488nm exciting light, can only be observed
Fluorescence is it was demonstrated that the E3025 plasmid successful conversion of coding GFP is in protoplast, and smoothly expresses, and thunder god vine suspending cell is primary
Plastid instantaneous conversion system is successfully established.
Sequence table
<110>The Capital University of Medical Sciences
<120>A kind of separation of Radix Tripterygii Wilfordii protoplast and transient transformation methods
<160>1
<210>1
<211>4612bp
<212>DNA
<213>Artificial sequence
<220>
<223>
<400>1
tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60
cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120
ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180
accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240
attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300
tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360
tttcccagtc acgacgttgt aaaacgacgg ccagtgccac ccataatacc cataatagct 420
gtttgccaac cggtcaacat gtggagcacg acacacttgt ctactccaaa aatatcaaag 480
atacagtctc agaagaccaa agggcaattg agacttttca acaaagggta atatccggaa 540
acctcctcgg attccattgc ccagctatct gtcactttat tgtgaagata gtggaaaagg 600
aaggtggctc ctacaaatgc catcattgcg ataaaggaaa ggccatcgtt gaagatgcct 660
ctgccgacag tggtcccaaa gatggacccc cacccacgag gagcatcgtg gaaaaagaag 720
acgttccaac cacgtcttca aagcaagtgg attgatgtga taacatggtg gagcacgaca 780
cacttgtcta ctccaaaaat atcaaagata cagtctcaga agaccaaagg gcaattgaga 840
cttttcaaca aagggtaata tccggaaacc tcctcggatt ccattgccca gctatctgtc 900
actttattgt gaagatagtg gaaaaggaag gtggctccta caaatgccat cattgcgata 960
aaggaaaggc catcgttgaa gatgcctctg ccgacagtgg tcccaaagat ggacccccac 1020
ccacgaggag catcgtggaa aaagaagacg ttccaaccac gtcttcaaag caagtggatt 1080
gatgtgatat ctccactgac gtaagggatg acgcacaatc ccactatcct tcgcaagacc 1140
cttcctctat ataaggaagt tcatttcatt tggagaggac gtcgagagtt ctcaacacaa 1200
catatacaaa acaaacgaat ctcaagcaat caagcattct acttctattg cagcaattta 1260
aatcatttct tttaaagcaa aagcaatttt ctgaaaattt tcaccattta cgaacgatag 1320
ccatggtccg gactcagatc tcgagctcaa gcttcgaatt ctgcagtcga cggtaccgcg 1380
ggcccgggat ggtagatctg actagtaaag gagaagaact tttcactgga gttgtcccaa 1440
ttcttgttga attagatggt gatgttaatg ggcacaaatt ttctgtcagt ggagagggtg 1500
aaggtgatgc aacatacgga aaacttaccc ttaaatttat ttgcactact ggaaaactac 1560
ctgttccgtg gccaacactt gtcactactt tctcttatgg tgttcaatgc ttttcaagat 1620
acccagatca tatgaagcgg cacgacttct tcaagagcgc catgcctgag ggatacgtgc 1680
aggagaggac catcttcttc aaggacgacg ggaactacaa gacacgtgct gaagtcaagt 1740
ttgagggaga caccctcgtc aacaggatcg agcttaaggg aatcgatttc aaggaggacg 1800
gaaacatcct cggccacaag ttggaataca actacaactc ccacaacgta tacatcatgg 1860
ccgacaagca aaagaacggc atcaaagcca acttcaagac ccgccacaac atcgaagacg 1920
gcggcgtgca actcgctgat cattatcaac aaaatactcc aattggcgat ggccctgtcc 1980
ttttaccaga caaccattac ctgtccacac aatctgccct ttcgaaagat cccaacgaaa 2040
agagagacca catggtcctt cttgagtttg taacagctgc tgggattaca catggcatgg 2100
atgaactata caaagctagc cactaatcta gagtccgcaa aaatcaccag tctctctcta 2160
caaatctatc tctctctatt tttctccaga ataatgtgtg agtagttccc agataaggga 2220
attagggttc ttatagggtt tcgctcatgt gttgagcata taagaaaccc ttagtatgta 2280
tttgtatttg taaaatactt ctatcaataa aatttctaat tcctaaaacc aaaatccagt 2340
gacgcggccg cacccataat acccataata gctgtttgcc agtaatcatg gtcatagctg 2400
tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata 2460
aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca 2520
ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc 2580
gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg 2640
cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta 2700
tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc 2760
aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag 2820
catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac 2880
caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc 2940
ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcaatg ctcacgctgt 3000
aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc 3060
gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga 3120
cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta 3180
ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta 3240
tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga 3300
tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg 3360
cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag 3420
tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc 3480
tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact 3540
tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt 3600
cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta 3660
ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta 3720
tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc 3780
gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat 3840
agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt 3900
atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg 3960
tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca 4020
gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta 4080
agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg 4140
cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact 4200
ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg 4260
ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt 4320
actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga 4380
ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc 4440
atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa 4500
caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcta agaaaccatt 4560
attatcatga cattaaccta taaaaatagg cgtatcacga ggccctttcg tc 4612
Claims (10)
1. a kind of separation method of Radix Tripterygii Wilfordii protoplast, comprises the steps：
1) Radix Tripterygii Wilfordii calluss suspension cell, the thunder god vine suspending cell after being digested are digested with enzymolysis solution；
2) collect the precipitation in the thunder god vine suspending cell after described enzymolysis, that is, obtain Radix Tripterygii Wilfordii protoplast；
Described enzymolysis solution includes cellulase, pectase and macerozyme.
2. method according to claim 1 it is characterised in that：
The mass ratio of described cellulase, described pectase and described macerozyme is 4:1:1.
3. method according to claim 1 and 2 it is characterised in that：
Described enzymolysis solution also includes Mannitol.
4. method according to claim 3 it is characterised in that：
Concentration in described enzymolysis solution for the described Mannitol is 0.3-0.7mol/L.
5. according to described method arbitrary in claim 1-4 it is characterised in that：
Described enzymolysis solution is by Mannitol, BSA, MES, CaCl2·2H2O, cellulase, pectase, macerozyme and water composition；
Concentration in described enzymolysis solution for the described Mannitol is 0.6mol/L；
Mass fraction in described enzymolysis solution for the described BSA is 0.6%；
Mass fraction in described enzymolysis solution for the described MES is 0.1%；
Described CaCl2·2H2Concentration in described enzymolysis solution for the O is 0.05mol/L；
Mass fraction in described enzymolysis solution for the described cellulase is 2.0%；
Mass fraction in described enzymolysis solution for the described pectase is 0.5%；
Mass fraction in described enzymolysis solution for the described macerozyme is 0.5%.
6. according to described method arbitrary in claim 1-5 it is characterised in that：
Described be collected as being centrifuged, the condition of described centrifugation is that (17-104) × g is centrifuged 5min, and the condition of described centrifugation is specially 67
× g is centrifuged 5min；
And/or, the time of described enzymolysis is 10h；
And/or, described Radix Tripterygii Wilfordii calluss suspension cell derives from Radix Tripterygii Wilfordii calluss；
And/or, also include the step filtering between described 1) and described 2).
7. the protoplast being prepared by described method arbitrary in claim 1-6.
8. application in exogenous gene expression for the protoplast described in claim 7.
9. a kind of method of exogenous gene expression, comprises the steps：By the protoplasm described in gene transformation claim 7
In body, realize the expression of exogenous gene.
10. the enzymolysis solution described in claim 1-6；
And/or, application in protoplast electrofusion for the enzymolysis solution described in claim 1-6.
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| CN105062949A (en) * | 2015-06-26 | 2015-11-18 | 上海交通大学 | Preparation method of Calibrachoa protoplast |
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| CN112111443A (en) * | 2020-09-24 | 2020-12-22 | 中国林业科学研究院林业研究所 | Method for separating and transforming catalpa bungei xylem protoplast |
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