CN106399223B - A kind of separation of tripterygium wilfordii protoplast and transient transformation methods - Google Patents
A kind of separation of tripterygium wilfordii protoplast and transient transformation methods Download PDFInfo
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Abstract
The invention discloses a kind of separation of tripterygium wilfordii protoplast and transient transformation methods.Be experimentally confirmed: the yield of the protoplast obtained using separation method of the invention is 4.24 × 105Exogenous plasmid is successfully transformed into the protoplast that the present invention extracts by the method for PEG4000 mediated transformation and is smoothly expressed, successfully establishes thunder god vine suspending cell protoplast instantaneous conversion system by/g FW, vigor 94.5%.The present invention is to carry out the functional genomics research of tripterygium wilfordii in a deep going way to lay a good foundation, while also carrying out the researchs such as gene editing, subcellular localization, gene overexpression and interference for tripterygium wilfordii and providing material base.
Description
Technical field
The invention belongs to field of plant tissue culture, and in particular to a kind of separation and instantaneous conversion of tripterygium wilfordii protoplast
Method, the in particular to method of the isolation and purification method and foreign gene instantaneous conversion of a kind of tripterygium wilfordii protoplast.
Background technique
Chinese herb triperygium wilfordii is root class medicinal material, derives from Celastraceae plant tripterygium wilfordii (Tripterygium wilfordii
Hook.f. the xylem of root or root) has the function such as clearing heat and detoxicating, dispelling wind and removing obstruction in the meridians, relaxing tendons and activating collaterals, swelling and pain relieving, desinsection hemostasis
Effect.The main active substances of tripterygium wilfordii are alkaloids, Diterpenes, triterpenes, sesquiterpenoids and carbohydrate.With anti-inflammatory, anti-swollen
The effects of tumor, immunosupress, is mainly used in rheumatoid, antitumor, renal glomerular disease, systemic red in modern clinical treatment
The diseases such as yabbi sore.In recent years, the research of tripterygium wilfordii causes the extensive pass of the domain experts such as biomedical and agronomy both at home and abroad
Note, but blank is still belonged to for the research of tripterygium wilfordii protoplast and instantaneous conversion, tripterygium wilfordii is limited to a certain extent
The further investigation of functional gene and synthetic biology etc..
Protoplast refers to that coated " exposed " plant cell of no cell wall, outermost layer are surrounded by one layer of plasma membrane, included
Cytoplasm (including matrix, organelle and ergastic mateiral etc.) and nucleus have cellular omnipotency, can be carried out with regenerative cell's wall
Continuous division.The application prospect of protoplast is very extensive, can be used for the Study on Genetic Transformation of plant, cell fusion research,
The fields such as genetic breeding research and cell membrane research.
Summary of the invention
The first purpose of the invention is to provide a kind of separation methods of tripterygium wilfordii protoplast.
The separation method of tripterygium wilfordii protoplast provided by the invention includes the following steps:
1) tripterygium wilfordii callus suspension cell is digested with enzymolysis liquid, the thunder god vine suspending cell after being digested；
2) precipitating in thunder god vine suspending cell after collecting the enzymatic hydrolysis is to get arriving tripterygium wilfordii protoplast；
The enzymolysis liquid includes cellulase, pectase and macerozyme.
In the above method,
The mass ratio of the cellulase, the pectase and the macerozyme is 4:1:1.Wherein, 1 enzyme activity unit
Refer under specified conditions (25 DEG C, other is optimum condition), the enzyme amount of 1 micromole substrate can be converted in 1 minute, or turn
Change the 1 micromolar enzyme amount in relation to group in substrate.
In the above method,
The enzymolysis liquid further includes mannitol.
In the above method,
Concentration of the mannitol in the enzymolysis liquid is 0.3-0.7mol/L.
In the above method,
The enzymolysis liquid is by mannitol, BSA, MES, CaCl22H2O, cellulase, pectase, macerozyme and water group
At；
Concentration of the mannitol in the enzymolysis liquid is 0.6mol/L；
Mass fraction of the BSA in the enzymolysis liquid is 0.6%；
Mass fraction of the MES in the enzymolysis liquid is 0.1%；
The CaCl2·2H2Concentration of the O in the enzymolysis liquid is 0.05mol/L；
Mass fraction of the cellulase in the enzymolysis liquid is 2.0%；
Mass fraction of the pectase in the enzymolysis liquid is 0.5%；
Mass fraction of the macerozyme in the enzymolysis liquid is 0.5%.
In the above method,
Described to be collected as being centrifuged, the condition of the centrifugation is that (17-104) × g is centrifuged 5min, and the condition of the centrifugation is specific
5min is centrifuged for 67 × g.
In the above method,
The time of the enzymatic hydrolysis is 10h.
In the above method,
The tripterygium wilfordii callus suspension cell derives from tripterygium wilfordii callus.
In the above method,
It is described 1) and it is described 2) between further include the steps that filtering；The 300 mesh stainless steel cells being filtered into sterilizing
It is sieved through filter.
A second object of the present invention is to provide the protoplasts isolated by the above method.
Third object of the present invention is to provide the new applications of above-mentioned protoplast.
The present invention provides application of the above-mentioned protoplast in exogenous gene expression.
Fourth object of the present invention is to provide a kind of method of exogenous gene expression.
The method of exogenous gene expression provided by the invention includes the following steps: the above-mentioned protoplast of gene transformation
In, realize the expression of foreign gene.
In the above method, the foreign gene is gfp gene.
Fifth object of the present invention is to provide above-mentioned enzymolysis liquids.
Application of the above-mentioned enzymolysis liquid in protoplast electrofusion also belongs to protection scope of the present invention.
The present invention provides a kind of separation methods of tripterygium wilfordii protoplast.The separation of tripterygium wilfordii protoplast of the invention
The Optimum separation condition of method is: enzymolysis liquid proportion is+0.5% macerozyme of+0.5% pectase of 2.0% cellulase；Mannitol
Concentration 0.6mol/L, enzymolysis time 10h, 67 × g revolving speed are centrifuged 5min.It is experimentally confirmed: being extracted with method of the invention
The protoplast arrived is by after purification, protoplast yield is 4.24 × 105/ g FW, vigor 94.5%.The present invention also passes through
Exogenous plasmid is successfully transformed into the protoplast that the present invention extracts and smoothly expresses by the method for PEG4000 mediated transformation, at
Function establishes thunder god vine suspending cell protoplast instantaneous conversion system.The present invention is the functional gene for carrying out tripterygium wilfordii in a deep going way
Group learns research and lays a good foundation, while also carrying out gene editing, subcellular localization, gene overexpression and interference etc. for tripterygium wilfordii and grinding
Study carefully and provides material base.
Detailed description of the invention
Fig. 1 is enzymolysis liquid with the influence for comparing protoplast yield and vigor.Figure 1A is enzymolysis liquid with comparison protoplast
The influence of yield；Figure 1B is enzymolysis liquid with the influence for comparing protoplast vigor.
Fig. 2 is influence of the enzymolysis time to protoplast yield and vigor.Fig. 2A is enzymolysis time to protoplast yield
Influence；Fig. 2 B is influence of the enzymolysis time to protoplast vigor.
Fig. 3 is influence of the mannitol concentration to protoplast yield and vigor.Fig. 3 A is mannitol concentration to protoplast
The influence of yield；Fig. 3 B is influence of the mannitol concentration to protoplast vigor.
Fig. 4 is the influence for handling revolving speed to protoplast yield and vigor.Fig. 4 A is processing revolving speed to protoplast yield
Influence；Fig. 4 B is the influence for handling revolving speed to protoplast vigor.
Fig. 5 is protoplast form.Fig. 5 A is the protoplast form under visible light after purification；Fig. 5 B is Trypan Blue
Unvital protoplast form afterwards.
Fig. 6 is the 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 superimposed picture with autofluorescence for protoplast GFP fluorescence under laser confocal scanning microscope.
Specific embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
Quantitative test in following embodiments, is respectively provided with three repeated experiments, and results are averaged.
Instrument includes: centrifuge (SIGMA), laser confocal scanning microscope in following embodiments
(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 match
It is current, can at most be saved 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 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) (BSA, Roche), morpholino b acid (MES, Sai Kelin, C10035338),
CALCIUM CHLORIDE DIHYDRATE (CaCl2·2H2O, Sinopharm Chemical Reagent Co., Ltd., 20130130), polyethylene glycol (PEG4000,
Solarbio, 1226B034), sodium chloride (NaCl, Beijing modern times east fine chemicals Co., Ltd, 20140301), chlorination
Potassium (KCl, Sinopharm Chemical Reagent Co., Ltd., 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 and its Activity determination of thunder god vine suspending cell protoplast
One, the preparation of thunder god vine suspending cell
Thunder god vine suspending cell derives from callus, has a large amount of space between cells, and cell wall is easily digested, and has life
The advantages that long speed is fast, secondary metabolites accumulation is rapid, is the ideal material for preparing protoplast.It is specific the preparation method is as follows:
1, the acquisition of callus
By tripterygium 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 or so fritter is seeded in containing 0.5mg/L 2,4-D (Beijing prosperity Bioisystech Co., Ltd, ancient cooking vessel state,
DH101-1 on MS agar medium (Caisson Labs, MSP22-50LT)), 25 DEG C, dark culture induced synthesis callus group
It knits.
2, the squamous subculture of callus
The callus that selection white is glossy, quality is loose, grows fine is in 0.5mg/L 2,4-D+0.1mg/L KT
(Beijing prosperity Bioisystech Co., Ltd, ancient cooking vessel state, DH135-2)+0.5mg/L IBA (limited public affairs of Beijing ancient cooking vessel state prosperity biotechnology
Department, II172) MS agar medium on squamous subculture, 25 DEG C, dark culture.Squamous subculture 3 more than generation chooses well-grown, matter
Ground is loose, growing state is consistent or similar callus, with tweezers folder at fritter, crushing, with the callus of every bottle of 1.0g
Amount is transferred into the 250mL triangle of the MS liquid medium of the 2,4-D+0.1mg/L KT+0.5mg/L of 0.5mg/L containing 100mL IBA
It in bottle, is put on shaking table, 25 DEG C, dark, 120rpm, which suspends, to be cultivated, and subculture is primary within 20 days, obtains thunder god vine suspending cell.
Two, the separation of protoplast and its Activity determination
It chooses squamous subculture 10-12 days thunder god vine suspending cells and extracts material as protoplast.Take different enzymes
Solution liquid proportion, mannitol concentration, enzymolysis time and processing revolving speed separate tripterygium wilfordii protoplast and detect protoplast yield and
Vigor selects optimal processing conditional parameter.Specific step is as follows:
1, enzymolysis liquid is with the influence for comparing protoplast yield and vigor
(1) preparation of enzymolysis liquid: by mannitol, BSA, MES, CaCl2·2H2O, cellulase, pectase, macerozyme and
Ultrapure water mixes, solute and its concentration are as follows: 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 liquid, according to cellulase, the pectin in enzymolysis liquid
Enzymolysis liquid is divided into following 5 groups by the difference of enzyme, macerozyme mass fraction:
1 group: 1.5% (mass fraction) cellulase+0.3% (mass fraction) pectase+0.5% (mass fraction) isolation
Enzyme；
2 groups: 2.0% (mass fraction) cellulase+0.3% (mass fraction) pectase+0.5% (mass fraction) isolations
Enzyme；
3 groups: 2.0%+0.5% macerozyme of (mass fraction) cellulase+0.5% (mass fraction) pectase (quality point
Number)；
4 groups: 2.0%+0.5% macerozyme of+0.7% pectase (mass fraction) of cellulase (mass fraction) (quality point
Number)；
5 groups: 2.5%+0.5% macerozyme of+0.3% pectase (mass fraction) of cellulase (mass fraction) (quality point
Number)；
Above-mentioned 5 groups of different enzymolysis liquids are activated into 10min in 55 DEG C of water-baths respectively, 0.22 μm of filtering with microporous membrane removes
Bacterium, to test use in next step.
(2) separation of protoplast
0.5g thunder god vine suspending cell is taken, Aspirate medium crushes as far as possible, is separately added into step according to the amount of 10mL/g
(1) it is digested in 5 groups of enzymolysis liquids in, 25 DEG C, 50rpm dark shaking enzymatic hydrolysis 12h；For several times with dropper suction suspension cell suspension,
To promote the release of protoplast；It is filtered with 300 sterilized mesh stainless steel cell sieves, obtains filtrate；Filtrate is in 67 × g condition
Lower centrifugation 5 minutes, abandons supernatant, collects protoplast.
(3) purifying of protoplast
5ml W5 solution is added in isolated protoplast in step (2), cleaning protoplast, centrifugation 5 is resuspended
Minute, supernatant is abandoned, then respectively washed once with the W5 solution of 5ml, 2ml respectively.Liquid is discarded supernatant, 1ml MMG solution is added and is resuspended,
It can be obtained the protoplast of purifying.
(4) yield and viability examination of protoplast
The detection method of the yield of protoplast are as follows: with the protoplast dilution for the purifying that MMG solution obtains step (3)
5 times, by diluted Protoplast suspension drop on blood counting chamber.Counting chamber is full of to protoplast, under the microscope
It observes and counts (the protoplast number in four big lattice).Each sample repeat count is three times.
Every gram of tripterygium wilfordii Suspension Protoplasts yield (a/g FW) calculation formula: protoplast total number/(4 ×
Quality of materials) } × extension rate × 104。
The detection method (using trypan blue staining) of the vigor of protoplast: step (3) is obtained with MMG solution pure
The protoplast of change dilutes 5 times, by the trypan blue dye liquor prepared (Beijing DingGuo ChangSheng Biology Technology Co., Ltd,
5BR00130), by volume 1:10 be added to dilution after Protoplast suspension in dye, choose 4 representational views
Open country is counted.The calculation method of protoplast vigor are as follows: plasm under the visual field is not accounted for by the protoplast of Trypan Blue
The percentage of body sum.
Obtained protoplast is subjected to yield and viability examination, as a result such as Figure 1A and Figure 1B.Compare 1,2,5 group, three groups
Cellulase concentration successively increase, wherein second group of protoplast yield and vigor highest, can achieve 1.86 respectively ×
105/ g FW and 67.8%.Cellulase concentration it is too low cannot abundant dissociated cell wall, and cellulose concentration is excessively high, can make thin
After birth rupture influences protoplast activity, therefore the optium concentration of cellulose is 2.0%；Compare 2,3,4 groups, three groups of pectase
Concentration successively increases, and the 4th group of yield and vigor highest, can achieve 3.08 × 10 respectively as the result is shown5/ g FW and
80.15%, there are significant differences with other groups, therefore the optium concentration of pectase is 0.5%.To sum up, thunder god vine suspending cell
The best enzyme solution proportion of protoplast electrofusion is+0.5% macerozyme of+0.5% pectase of 2.0% cellulase.
2, enzymolysis time compares the influence of protoplast yield and vigor
(1) preparation of enzymolysis liquid: by mannitol, BSA, MES, CaCl2·2H2O, cellulase, pectase, macerozyme and
Ultrapure water mixes, and solute and its concentration are respectively as follows: 0.5mol/L mannitol, 0.6% (mass fraction) BSA, 0.1% (quality point
Number) MES, 0.05mol/L CaCl2·2H2O, 2.0% (mass fraction) cellulase, 0.5% (mass fraction) pectase,
0.5% (mass fraction) macerozyme, KOH are adjusted to pH5.6, obtain enzymolysis liquid.Above-mentioned enzymolysis liquid is activated in 55 DEG C of water-baths
10min, 0.22 μm of filtering with microporous membrane degerming, to test use in next step.
(2) separation of protoplast
0.5g thunder god vine suspending cell is taken, Aspirate medium crushes as far as possible, is separately added into step according to the amount of 10mL/g
(1) it is digested in the enzymolysis liquid in, 25 DEG C, the shaking of 50rpm dark digests 6h, 8h, 10h, 12h, 14h and 16h respectively；It is taken out with dropper
Suspension cell suspension is inhaled for several times, to promote the release of protoplast；It is filtered, is obtained with 300 sterilized mesh stainless steel cell sieves
To filtrate；Filtrate is centrifuged 5 minutes under the conditions of 67 × g, abandons supernatant, collects protoplast respectively.
(3) purifying of protoplast
5ml W5 solution is added in protoplast isolated in step (2) respectively, cleaning protoplast is resuspended, from
The heart 5 minutes, supernatant is abandoned, then respectively washed once with the W5 solution of 5ml, 2ml respectively.Liquid is discarded supernatant, 1ml MMG solution weight is added
It is outstanding, it can be obtained the protoplast of purifying.
(4) yield and viability examination of protoplast
Detection method is the same as (4) in step 1.
Obtained protoplast is subjected to yield and viability examination, as a result sees Fig. 2A and Fig. 2 B.As can be seen from the figure: with
The yield of the increase of enzymolysis time, protoplast increases accordingly, but the vigor of protoplast highest in 10h, up to 82.2%.Enzyme
Solution the time it is too short protoplast cannot be made to be kept completely separate out, extraction efficiency is low, and enzymolysis time is too long, and cellular stress is excessive,
It is easily broken, detects dead cell under mirror and fragment increases, living cells proportion is small, is unfavorable for the development of subsequent experimental.Therefore,
10h is the best enzymolysis time of tripterygium wilfordii energy Suspension Protoplasts.
3, mannitol concentration compares the influence of protoplast yield and vigor
(1) preparation of enzymolysis liquid: by mannitol, BSA, MES, CaCl2·2H2O, cellulase, pectase, macerozyme and
Ultrapure water mixes, and solute and its concentration are respectively as follows: 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 are adjusted to pH5.6, obtain enzymolysis liquid, are divided into following 5 groups according to the difference of mannitol concentration in enzymolysis liquid:
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 liquid is activated into 10min, 0.22 μm of filtering with microporous membrane degerming, to next in 55 DEG C of water-baths respectively
Step test uses.
(2) separation of protoplast
0.5g thunder god vine suspending cell is taken, Aspirate medium crushes as far as possible, is separately added into step according to the amount of 10mL/g
(1) it is digested in 5 groups of enzymolysis liquids in, 25 DEG C, 50rpm dark shaking enzymatic hydrolysis 12h；For several times with dropper suction suspension cell suspension,
To promote the release of protoplast；It is filtered with 300 sterilized mesh stainless steel cell sieves, obtains filtrate；Filtrate is in 67 × g condition
Lower centrifugation 5 minutes, abandons supernatant, collects protoplast.
(3) purifying of protoplast
5ml W5 solution is added in isolated protoplast in step (2), cleaning protoplast, centrifugation 5 is resuspended
Minute, supernatant is abandoned, then respectively washed once with the W5 solution of 5ml, 2ml respectively.Liquid is discarded supernatant, 1ml MMG solution is added and is resuspended,
It can be obtained the protoplast of purifying.
(4) yield and viability examination of protoplast
Detection method is the same as (4) in step 1.
Obtained protoplast is subjected to yield and viability examination, as a result sees 3A and Fig. 3 B.As seen from the figure, when sweet dew is pure and strong
Degree be 0.5mol/L when protoplast yield highest, and at this time vigor be not it is highest, when mannitol concentration be 0.6mol/L
Although when yield ratio 0.5mol/L it is slightly lower, protoplast vigor highest at this time.Solution osmotic pressure is lower than the infiltration of protoplast
Pressure will lead to cell and burst death；The protoplast dehydration shrinkage when mannitol concentration is excessively high, and it is unfavorable for releasing for protoplast
It puts.Although the protoplast sum survived in 0.5mol/L and two processing groups of 0.6mol/L is close, due to the original of tool survival
Raw plastid just can be carried out instantaneous conversion, and in contrast, mannitol concentration is that 0.6mol/L is to be more advantageous to opening for experiment in next step
Exhibition.So the best mannitol concentration of separated protoplast is 0.6mol/L.
4, influence of the processing revolving speed to protoplast yield and vigor
(1) preparation of enzymolysis liquid: by mannitol, BSA, MES, CaCl2·2H2O, cellulase, pectase, macerozyme and
Ultrapure water mixes, and solute and its concentration are respectively as follows: 0.5mol/L mannitol, 0.6% (mass fraction) BSA, 0.1% (quality point
Number) MES, 0.05mol/L CaCl2·2H2O, 2.0% (mass fraction) cellulase, 0.5% (mass fraction) pectase,
0.5% (mass fraction) macerozyme, KOH are adjusted to pH5.6, obtain enzymolysis liquid.Above-mentioned enzymolysis liquid is activated in 55 DEG C of water-baths
10min, 0.22 μm of filtering with microporous membrane degerming, to test use in next step.
(2) separation of protoplast
0.5g thunder god vine suspending cell is taken, Aspirate medium crushes as far as possible, is separately added into step according to the amount of 10mL/g
(1) it is digested in the enzymolysis liquid in, 25 DEG C, 50rpm dark shaking enzymatic hydrolysis 12h；Suspension cell suspension is aspirated for several times with dropper, with
Promote the release of protoplast；It is filtered with 300 sterilized mesh stainless steel cell sieves, obtains filtrate；By filtrate in different disposal
Revolving speed: being centrifuged 5 minutes under the conditions of 17 × g, 37 × g, 67 × g, 104 × g, abandons supernatant, collects protoplast respectively.
(3) purifying of protoplast
5ml W5 solution is added in protoplast isolated in step (2) respectively, cleaning protoplast is resuspended, from
The heart 5 minutes, supernatant is abandoned, then respectively washed once with the W5 solution of 5ml, 2ml respectively.Liquid is discarded supernatant, 1ml MMG solution weight is added
It is outstanding, it can be obtained the protoplast of purifying.
(4) yield and viability examination of protoplast
Detection method is the same as (4) in step 1.
Obtained protoplast is subjected to yield and viability examination, as a result sees Fig. 4 A and Fig. 4 B.Compare four groups of data, when turn
Yield can achieve 2.17 × 10 when speed is 67 × g5g/FW；Vigor reaches as high as 78.4%, and yield and vigor are highest water
It is flat.It is unfavorable for the sedimentation of protoplast when revolving speed is too low, fragment and protoplast cannot be efficiently separated；When revolving speed is excessively high
When, mechanical damage can be caused to protoplast, be equally unfavorable for isolating and purifying for protoplast.To sum up, plasm is isolated and purified
The optimum speed of body is 67 × g.
Three, the validation verification of protoplast separation purification method
1, the preparation of enzymolysis liquid: by mannitol, BSA, MES, CaCl2·2H2O, cellulase, pectase, macerozyme and super
Pure water mixes, and solute and its concentration are respectively as follows: 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 are adjusted to pH5.6, obtain enzymolysis liquid.Above-mentioned enzymolysis liquid is activated into 10min in 55 DEG C of water-baths,
0.22 μm of filtering with microporous membrane degerming, to test use in next step.
2, the separation of protoplast
0.5g thunder god vine suspending cell is taken, Aspirate medium crushes as far as possible, is added in step 1 according to the amount of 10mL/g
It is digested in enzymolysis liquid, 25 DEG C, 50rpm dark shaking enzymatic hydrolysis 10h；For several times with dropper suction suspension cell suspension, to promote original
The release of raw plastid；It is filtered with 300 sterilized mesh stainless steel cell sieves, obtains filtrate；Filtrate is centrifuged 5 under the conditions of 67 × g
Minute, supernatant is abandoned, protoplast is collected.
3, the purifying of protoplast
5ml W5 solution is added into protoplast isolated in step 2, cleaning protoplast is resuspended, is centrifuged 5 points
Clock is abandoned supernatant, then respectively be washed once with the W5 solution of 5ml, 2ml respectively.Liquid is discarded supernatant, 1ml MMG solution is added and is resuspended, i.e.,
It can get the protoplast of purifying.
4, the yield and viability examination of protoplast
Detection method is the same as (4) in the 1 of step 2.
The protoplast yield of acquisition is up to 4.24 × 105/ g FW, vigor is up to 94.5%, protoplast form and platform
Protoplast form after expecting blue dyeing is shown in Fig. 5 A and Fig. 5 B.
The application of embodiment 2, thunder god vine suspending cell protoplast in foreign gene instantaneous conversion expression
The instantaneous conversion of thunder god vine suspending cell protoplast is studied using PEG mediated transformation method, it is specific to walk
It is rapid as follows:
1, the concentration of the protoplast of the purifying obtained in the step of embodiment one three is transferred to about 5 × 105A/mL,
Obtain protoplast solution；
2, 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 the tapping mixing of 220 μ L PEG solution, transformation mixture is obtained, black
Induction Transformation mixture 20min under dark condition obtains conversion mixed liquor；
3, conversion mixed liquor is diluted with 880 μ L W5 solution at room temperature, then gentle inversion shakes centrifuge tube and is allowed to mix
Well to terminate conversion reaction.67 × g is centrifuged 2min, collects precipitating, removes supernatant；
4, the precipitating collected with W5 solution repeated washing step 3 is primary, collects precipitating, removes supernatant；
5, being deposited in porous organization's culture dish of step 4 collection is softly resuspended with 1mL WI solution, 25 DEG C dark to stand
Culture.
6, it is taken out after 18h, 67 × g is centrifuged 2min, removes part supernatant, it is concentrated, take appropriate film-making, laser is total
GFP expression is observed under confocal scanning microscopy.
As a result such as Fig. 6 A, Fig. 6 B, Fig. 6 C and Fig. 6 D.Since protoplast is thin from the thunder god vine suspending of dark culturing
Born of the same parents, therefore chlorophyll is free of in its cell, GFP green can only be observed under laser confocal scanning microscope 488nm exciting light
Fluorescence, it was demonstrated that the E3025 plasmid successful conversion for encoding GFP is expressed into protoplast, and smoothly, and thunder god vine suspending cell is primary
Plastid instantaneous conversion system is successfully established.
Sequence table
<110>Capital University of Medical Sciences
<120>a kind of separation of tripterygium 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 (5)
1. a kind of separation method of tripterygium wilfordii protoplast, includes the following steps:
1) tripterygium wilfordii callus suspension cell is digested with enzymolysis liquid, the thunder god vine suspending cell after being digested；
2) precipitating in thunder god vine suspending cell after collecting the enzymatic hydrolysis is to get arriving tripterygium wilfordii protoplast；
The time of the enzymatic hydrolysis is 10h；
The enzymolysis liquid is by mannitol, BSA, MES, CaCl2•2H2O, cellulase, pectase, macerozyme and water composition；
Concentration of the mannitol in the enzymolysis liquid is 0.6mol/L；
Mass fraction of the BSA in the enzymolysis liquid is 0.6%；
Mass fraction of the MES in the enzymolysis liquid is 0.1%；
The CaCl2•2H2Concentration of the O in the enzymolysis liquid is 0.05mol/L；
Mass fraction of the cellulase in the enzymolysis liquid is 2.0%；
Mass fraction of the pectase in the enzymolysis liquid is 0.5%；
Mass fraction of the macerozyme in the enzymolysis liquid is 0.5%.
2. according to the method described in claim 1, it is characterized by:
Described to be collected as being centrifuged, the condition of the centrifugation is that 67 × g is centrifuged 5min；
The tripterygium wilfordii callus suspension cell derives from tripterygium wilfordii callus；
It is described 1) and it is described 2) between further include the steps that filtering.
3. the protoplast being prepared by method of any of claims 1 or 2.
4. application of the protoplast as claimed in claim 3 in exogenous gene expression.
5. a kind of method of exogenous gene expression includes the following steps: gene transformation plasm as claimed in claim 3
In body, the expression of foreign gene is realized.
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| CN105670985A (en) * | 2016-04-22 | 2016-06-15 | 中国科学院华南植物园 | Camellia sinensis flower protoplast, preparation method therefor and application thereof |
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| 拟南芥叶肉原生质体分离及瞬时表达体系的建立;梁大伟;《中国优秀硕士学位论文全文数据库.基础科学辑》;20091215(第12期);A006-295 * |
| 红豆草原生质体分离与培养研究;罗玉鹏;《中国优秀硕士学位论文全文数据库.农业科技辑》;20081015(第10期);D047-106 * |
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