CN109136321A - A kind of plant sub-cellular location reagent box and its application - Google Patents
A kind of plant sub-cellular location reagent box and its application Download PDFInfo
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- CN109136321A CN109136321A CN201810991595.XA CN201810991595A CN109136321A CN 109136321 A CN109136321 A CN 109136321A CN 201810991595 A CN201810991595 A CN 201810991595A CN 109136321 A CN109136321 A CN 109136321A
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Abstract
The present invention relates to field of biotechnology, concretely it is related to a kind of plant sub-cellular location reagent box, consists of the following reagents: 5~20ml of enzymolysis liquid, 5~25ml of WI solution, 30~50ml of W5 solution, MMG solution 5~25ml and PEG-Calcium conversion 5~20ml of solution；3. the application of the kit, preparation including 1. Plasmid DNA, 2. conversion stop reaction, protoplast are 4. resuspended, is 5. incubated for, 6. signal observation and protoplast storage；The subcellular localization of destination gene expression product can be rapidly completed in this kit, pass through the culture to protoplast, the exogenous genetic material being transferred to inside it can be expressed in vivo, to create good condition to study certain functions of these inhereditary materials.
Description
Technical field
The present invention relates to field of biotechnology, are concretely related to a kind of plant sub-cellular location reagent box and its application.
Background technique
It is more and more new with the development of the development of biotechnology, especially genomics and the progress of sequencing technologies
Gene is found, and has formd mass data.The function of specifying unknown gene inquires into its potential application value, is gene
Group learns the ultimate aim of research.Protein be the expression product of gene, the subcellular localization of protein and the structure of protein and
Functional relationship is close, and protein necessarily is in suitable its function of position competence exertion.For a new gene, its expression is specified
Product, that is, protein subcellular localization can provide important clue to study the function of the gene.Currently, determining that protein is sub-
There are mainly three types of the methods of cellular localization: Cell Fractionation method, electron-microscopic analysis, laser confocal, and wherein laser is copolymerized
Burnt method is most widely used.Laser confocal is stimulated the fluorescence signal of generation using mark fluorescent albumen, can be intuitive
Observe the subcellular location where target protein.When the subcellular localization to target protein is studied, it is positive right to need
According to, i.e., with special subcellular status the albumen with detectable label, prove the positioning of agnoprotein.
Summary of the invention
Therefore the present invention proposes a kind of plant sub-cellular location reagent box and its application, is testing goal gene expression product
Whether it is positioned at plant born of the same parents and instruction is provided.
The technical scheme of the present invention is realized as follows: a kind of plant sub-cellular location reagent box, consists of the following reagents:
5~20ml of enzymolysis liquid, 5~25ml of WI solution, 30~50ml of W5 solution, MMG solution 5~25ml and PEG-Calcium conversion are molten
5~20ml of liquid.
Further, the enzymolysis liquid includes 0.1~0.2g of cellulase, 0.02~0.06g of pectase, 10mg/ml
The μ l of BSA60~120, the μ l of 0.2M MES800~1200,1M CaCl2μ l of 80~110 μ l, 2M KCl85~120,0.8M mannose
4800~5300 μ l of alcohol.
Further, the WI solution includes 0.2M, MES 150~220 μ l, 2M, KCl 80~120 μ l, 0.8M,
6000~7000 μ l of Mannitol.
Further, the W5 solution includes 0.2M, 400~550 μ l, 1M NaCl of MES 7000~7800 μ l, 1M
CaCl26000~6800 μ l, 2M KCl, 100~140 μ l.
Further, the MMG solution includes 180~240 μ l, 0.8M Mannitol of 0.2M MES, 4600~5200 μ
L, 2M MgCl230~90 μ l.
Further, the PEG-Calcium conversion solution includes 2300~2800 μ l, 1M of 0.8M Mannitol
CaCl2800~1100 μ l, PEG4000,2~5g.
A kind of application of plant sub-cellular location reagent box, including the following steps:
1. the preparation of Plasmid DNA: method of measuring in use extracts the Plasmid DNA to be converted, and detects on horizontal glue, to guarantee
Super spirial plasmid ratio is high, takes 5-10 μ l plasmid spare in 2ml centrifuge tube, in bimolecular fluorescence complementary experiment, every kind of plasmid
For 10 μ g, while plasmid control and label control are set；
2. conversion: the protoplast of 100 μ l is added in each centrifuge tube, it is even with the 200 light persorptions of μ l pipette tips, 110 μ are added
The PEG-Calcium solution of l, with index finger, gently attack tube bottom places 10mins at room temperature and completes conversion instead to mix well
It answers；
3. stopping reaction: taking the W5 solution under 420 μ l room temperature states to be added in above-mentioned conversion fluid, gently attack and reverse
Solution is centrifuged 2mins to stop conversion reaction, with desktop desk centrifuge, removes supernatant；
4. protoplast is resuspended, it is slowly added to 500 μ lWI solution and protoplast is resuspended；
5. being incubated for: in room temperature 100RPM shaker overnight culture protoplast, centrifuge tube needs horizontal positioned to guarantee most
The carboxylic card Benzylpenicillin sodium salt of 50 μ g/ml is added if discovery has bacteria breed in being incubated overnight liquid in big liquid level product；
6. signal observation and protoplast storage: before observation protoplast, first mixing gently test tube, taking out 20 μ l is
Can, it is observed under above-mentioned fluorescence or CLSM, with ultraviolet excitation, plastid can be observed at the absorbing wavelength of RFP and GFP
With the signal for being transferred to plasmid, 100g centrifugation 2mins collects unspent protoplast and is stored in spare in -80 DEG C of refrigerators；Such as
Fruit needs to extract protoplast albumen, can 6000g centrifugation 2mins collect all protoplasts, extract total protein and simultaneously utilize
Western hybridization check.
Further, the plasmid is extracted to protoplasts of Arabidopsis thaliana broken by ultrasonic, and the inspection concrete operations of protoplasts of Arabidopsis thaliana broken by ultrasonic
Are as follows: it is stained with transparent rubberized fabric on glass slide, digs out a 8x8mm with blade2Square, gently shake up enzymolysis liquid, be taken out
20 μ l are placed in square, covered, and the size of protoplast is checked under microscope bright field.
Further, the protoplasts of Arabidopsis thaliana broken by ultrasonic is diluted and filters enzymolysis liquid, concrete operations after checking
Are as follows: the W5 solution of isometric (5ml) is added in enzymolysis liquid, shakes gently, takes out 75- from the culture dish of soaked in absolute ethyl alcohol
The nylon filter of μm hole, removes alcohol with distilled water flushing, it is extra to be removed later with a small amount of W5 solution rinse filter membrane
Filter membrane is placed on funnel by water, filters the blade residue in enzymolysis liquid, collects protoplast in the round bottom sterile tube of 15ml.
It further, further include collection step, 100-150g is centrifuged 1-2mins, and protoplast can be deposited on test tube bottom,
Carefully and supernatant is poured out, soft rotating tubular body is dispersed in protoplast again in remaining liquid.
By above disclosure, the invention has the benefit that destination gene expression can be rapidly completed in this kit
The subcellular localization of product, by the culture to protoplast, inside the exogenous genetic material that is transferred to can in vivo carry out
Expression, thus to study certain functions (such as signal transduction, metabolic pathway, transcription and translation mechanism) of these inhereditary materials wound
Good condition is made.
Specific embodiment
Below in conjunction with the embodiment of the present invention, the technical scheme in the embodiment of the invention is clearly and completely described,
Obviously, described embodiment is only a part of the embodiments of the present invention, instead of all the embodiments.Based on of the invention
Embodiment, every other embodiment obtained by those of ordinary skill in the art without making creative efforts, all
Belong to the scope of protection of the invention.
Embodiment 1
One, reagent prepares:
(1), the preparation of enzymolysis liquid；
| Ingredient | Dosage | Final concentration |
| Cellulase | 0.1g | 1.8% |
| Pectase | 0.03g | 0.45% |
| 10mg/ml BSA | 80μl | 0.1% |
| 0.2M MES | 800μl | 16mM |
| 1M CaCl2 | 90μl | 12mM |
| 2M KCl | 100μl | 20mM |
| 0.8M mannitol | 5200μl | 0.4M |
It should be pointed out that first weighing enzyme powder, the MES solution of 70 DEG C of processing 3-5 minutes is added, adds except CaCl2
With the room temperature reagent except BSA, NDA enzyme and proteinase activity a small amount of in 55 DEG C of water-bath 15 minutes inactivation enzyme solutions, cold after concussion
But to after room temperature, CaCl is added2And BSA, it is then sterilized, is placed in brown reagent bottle with 0.45 μm of filter membrane.
(2), the preparation of WI solution；
| Ingredient | Dosage | Final concentration |
| 0.2M, MES | 180μl | 3mM |
| 2M,KCl | 100μl | 18mM |
| 0.8M, Mannitol | 6500μl | 0.5M |
Without individually sterilizing, room temperature preservation is now matched.
(3), the preparation of W5 solution；
| Ingredient | Dosage | Final concentration |
| 0.2M, MES | 450μl | 2mM |
| 1M, NaCl | 7000μl | 142mM |
| 1M, CaCl2 | 6200μl | 115mM |
| 2M, KCl | 110μl | 4mM |
Without individually sterilizing, room temperature preservation is now matched.
(4), the preparation of MMG solution；
| Ingredient | Dosage | Final concentration |
| 0.2M, MES | 210μl | 3.5mM |
| 0.8M, Mannitol | 4800μl | 0.4M |
| 2M MgCl2 | 80μl | 20mM |
Without individually sterilizing, room temperature preservation is now matched.
(5), PEG-Calcium converts the preparation of solution；
| Ingredient | Dosage | Final concentration |
| 0.8M, Mannitol | 2600μl | 0.21M |
| 1M CaCl2 | 1000μl | 100mM |
| PEG4000 | 4g | 40% |
It is noted that 6 milliliters or so of distilled water and other solution are added after weighing PEG4000, vibrate 3 minutes
Afterwards, in constant volume, since PEG4000 is difficult to be completely dissolved, it is therefore necessary to be initially configured at least 1 hour before using the reagent.
The solution room temperature preservation, now matches, without sterilizing.
Two, protoplast electrofusion:
S1, arabidopsis plantation；
The condition of arabidopsis plantation are as follows: the lower level conditions of illumination in 12 hours (23 DEG C) and 12 hours dark (18 DEG C)
Under, relative humidity 55-65%.
S2, blade are selected and are cut；
It is chosen from the plant in 3-4 week of growth and stretches good blade (1-1.5cm), as the printing paper of bilayer tiling
On, using fresh sharp blade along blade to be disposably cut into the strip of 1-1.5mm with main lobe arteries and veins vertical direction, and stand
I.e. soft putting into enzymolysis liquid and gently rotate is allowed to submerge.
S3, vacuum processing；
Vaned enzymolysis liquid will be filled and be placed in vacuscope about 0.6hr, to guarantee that enzymolysis liquid can preferably go deep into mesophyll group
It knits, continues to digest 3hr later in shading environment.
S4, protoplast inspection；
It is stained with transparent rubberized fabric on glass slide, digs out a 8x8mm with blade2Square, gently shake up enzymolysis liquid, from
20 μ l of middle taking-up are placed in square, covered, and the size of protoplast is checked under microscope bright field.(arabidopsis mesophyll
Cell protoplast is generally 30-50 μm, spherical shape, it is seen that plastid abundant)
S5, dilution and filtering enzymolysis liquid；
The W5 solution of isometric (6ml) is added in enzymolysis liquid, shakes gently, is taken from the culture dish of soaked in absolute ethyl alcohol
The nylon filter of 75- μm of hole out removes alcohol with distilled water flushing, is removed later with a small amount of W5 solution rinse filter membrane more
Remaining water, filter membrane is placed on funnel, filters the blade residue in enzymolysis liquid, collects protoplast in the round bottom sterile tube of 15ml
In.
S6, protoplast is collected；
100-150g is centrifuged 1-2mins, and protoplast can be deposited on test tube bottom, supernatant is poured out carefully and as far as possible
Liquid, soft rotating tubular body are dispersed in protoplast again in remaining liquid.
S7, protoplast is resuspended；
It takes the W5 solution of 5ml that protoplast is resuspended, protoplast is placed in 30 minutes on ice later, gravity precipitinogen
Protoplast is resuspended using 1-1.5mlMMG solution later with the W5 in pipette tips removal supernatant in raw plastid 15min.
Three, DNA-PEG-Ca is converted
1. the preparation of Plasmid DNA；Method is measured in use and extracts the Plasmid DNA to be converted, and is detected on horizontal glue, to guarantee
Super spirial plasmid ratio is high, takes 5-10 μ l plasmid spare in 2ml centrifuge tube, in bimolecular fluorescence complementary experiment, every kind of plasmid
For 10 μ g；
2. converting
The protoplast of 100 μ l is added in each centrifuge tube, it is even with the 200 light persorptions of μ l pipette tips, the PEG of 110 μ l is added
Solution, with index finger, gently attack tube bottom places 10mins at room temperature and completes conversion reaction to mix well.
3. stopping reaction
The W5 solution under 420 μ l room temperature states is taken to be added in above-mentioned conversion fluid, gently attack and reverse solution are to stop
Conversion reaction is centrifuged 2mins with desktop desk centrifuge, removes supernatant.
4. protoplast is resuspended, it is slowly added to 500 μ lWI solution and protoplast is resuspended.
Protoplast cuhnre and harvest
Be incubated for: in room temperature 100RPM shaker overnight culture protoplast, centrifuge tube needs horizontal positioned to guarantee maximum
Liquid level product the carboxylic card Benzylpenicillin sodium salt of 50 μ g/ml is added if discovery has a bacteria breed in being incubated overnight liquid.
Signal observation and protoplast storage
It observes before protoplast, first mixes gently test tube, take out 20 μ l, seen under above-mentioned fluorescence or CLSM
It surveys, with ultraviolet excitation, plastid and the signal for being transferred to plasmid, 100g centrifugation can be observed at the absorbing wavelength of RFP and GFP
2mins collects unspent protoplast and is stored in spare in -80 DEG C of refrigerators；It, can if necessary to extract protoplast albumen
6000g is centrifuged 2mins and collects all protoplasts, extracts total protein and utilizes western hybridization check.
Embodiment 2
One, reagent prepares:
(1), the preparation of enzymolysis liquid；
| Ingredient | Dosage | Final concentration |
| Cellulase | 0.14g | 1.4% |
| Pectase | 0.05g | 0.5% |
| 10mg/ml BSA | 100μl | 0.1% |
| 0.2M MES | 1000μl | 20mM |
| 1M CaCl2 | 100μl | 10mM |
| 2M KCl | 100μl | 20mM |
| 0.8M mannitol | 5000μl | 0.4M |
It should be pointed out that first weighing enzyme powder, the MES solution of 70 DEG C of processing 3-5 minutes is added, adds except CaCl2
With the room temperature reagent except BSA, NDA enzyme and proteinase activity a small amount of in 55 DEG C of water-bath 10 minutes inactivation enzyme solutions, cold after concussion
But to after room temperature, CaCl is added2And BSA, it is then sterilized, is placed in brown reagent bottle with 0.45 μm of filter membrane.
(2), the preparation of WI solution；
| Ingredient | Dosage | Final concentration |
| 0.2M, MES | 200μl | 4mM |
| 2M,KCl | 100μl | 20mM |
| 0.8M, Mannitol | 6250μl | 0.5M |
Without individually sterilizing, room temperature preservation is now matched.
(3), the preparation of W5 solution；
| Ingredient | Dosage | Final concentration |
| 0.2M, MES | 550μl | 2.4mM |
| 1M, NaCl | 7600μl | 154mM |
| 1M, CaCl2 | 6200μl | 122mM |
| 2M, KCl | 125μl | 5mM |
Without individually sterilizing, room temperature preservation is now matched.
(4), the preparation of MMG solution；
| Ingredient | Dosage | Final concentration |
| 0.2M, MES | 200μl | 4mM |
| 0.8M, Mannitol | 5000μl | 0.4M |
| 2M MgCl2 | 75μl | 15mM |
Without individually sterilizing, room temperature preservation is now matched.
(5), PEG-Calcium converts the preparation of solution；
| Ingredient | Dosage | Final concentration |
| 0.8M, Mannitol | 2500μl | 0.2M |
| 1M CaCl2 | 1000μl | 100mM |
| PEG4000 | 3g | 30% |
It is noted that 5 milliliters or so of distilled water and other solution are added after weighing PEG4000, vibrate 2 minutes
Afterwards, in constant volume, since PEG4000 is difficult to be completely dissolved, it is therefore necessary to be initially configured at least 1 hour before using the reagent.
The solution room temperature preservation, now matches, without sterilizing.
Two, protoplast electrofusion:
S1, arabidopsis plantation；
The condition of arabidopsis plantation are as follows: the lower level conditions of illumination in 13 hours (23 DEG C) and 11 hours dark (20 DEG C)
Under, relative humidity 40-65%.
S2, blade are selected and are cut；
It is chosen from the plant in 3-4 week of growth and stretches good blade (1-1.5cm), as the printing paper of bilayer tiling
On, using fresh sharp blade along blade to be disposably cut into the strip of 1-1.5mm with main lobe arteries and veins vertical direction, and stand
I.e. soft putting into enzymolysis liquid and gently rotate is allowed to submerge.
S3, vacuum processing；
Vaned enzymolysis liquid will be filled and be placed in vacuscope about 1hr, to guarantee that enzymolysis liquid can preferably go deep into mesophyll tissue,
Continue to digest 3.5hr in shading environment later.
S4, protoplast inspection；
It is stained with transparent rubberized fabric on glass slide, digs out a 8x8mm with blade2Square, gently shake up enzymolysis liquid, from
20 μ l of middle taking-up are placed in square, covered, and the size of protoplast is checked under microscope bright field.(arabidopsis mesophyll
Cell protoplast is generally 30-50 μm, spherical shape, it is seen that plastid abundant)
S5, dilution and filtering enzymolysis liquid；
The W5 solution of isometric (5ml) is added in enzymolysis liquid, shakes gently, is taken from the culture dish of soaked in absolute ethyl alcohol
The nylon filter of 75- μm of hole out removes alcohol with distilled water flushing, is removed later with a small amount of W5 solution rinse filter membrane more
Remaining water, filter membrane is placed on funnel, filters the blade residue in enzymolysis liquid, collects protoplast in the round bottom sterile tube of 15ml
In.
S6, protoplast is collected；
100-150g is centrifuged 1-2mins, and protoplast can be deposited on test tube bottom, supernatant is poured out carefully and as far as possible
Liquid, soft rotating tubular body are dispersed in protoplast again in remaining liquid.
S7, protoplast is resuspended；
It takes the W5 solution of 5ml that protoplast is resuspended, protoplast is placed in 30 minutes on ice later, gravity precipitinogen
Protoplast is resuspended using 1-1.5mlMMG solution later with the W5 in pipette tips removal supernatant in raw plastid 15min.
Three, DNA-PEG-Ca is converted
1. the preparation of Plasmid DNA；Method is measured in use and extracts the Plasmid DNA to be converted, and is detected on horizontal glue, to guarantee
Super spirial plasmid ratio is high, takes 5-10 μ l plasmid spare in 2ml centrifuge tube, in bimolecular fluorescence complementary experiment, every kind of plasmid
For 10 μ g；
2. converting
The protoplast of 100 μ l is added in each centrifuge tube, it is even with the 200 light persorptions of μ l pipette tips, the PEG of 110 μ l is added
Solution, with index finger, gently attack tube bottom places 10mins at room temperature and completes conversion reaction to mix well.
3. stopping reaction
The W5 solution under 420 μ l room temperature states is taken to be added in above-mentioned conversion fluid, gently attack and reverse solution are to stop
Conversion reaction is centrifuged 2mins with desktop desk centrifuge, removes supernatant.
4. protoplast is resuspended, it is slowly added to 500 μ lWI solution and protoplast is resuspended.
Protoplast cuhnre and harvest
Be incubated for: in room temperature 100RPM shaker overnight culture protoplast, centrifuge tube needs horizontal positioned to guarantee maximum
Liquid level product the carboxylic card Benzylpenicillin sodium salt of 50 μ g/ml is added if discovery has a bacteria breed in being incubated overnight liquid.
Signal observation and protoplast storage
It observes before protoplast, first mixes gently test tube, take out 20 μ l, seen under above-mentioned fluorescence or CLSM
It surveys, with ultraviolet excitation, plastid and the signal for being transferred to plasmid, 100g centrifugation can be observed at the absorbing wavelength of RFP and GFP
2mins collects unspent protoplast and is stored in spare in -80 DEG C of refrigerators；It, can if necessary to extract protoplast albumen
6000g is centrifuged 2mins and collects all protoplasts, extracts total protein and utilizes western hybridization check.
Embodiment 3
One, reagent prepares:
(1), the preparation of enzymolysis liquid；
| Ingredient | Dosage | Final concentration |
| Cellulase | 0.2g | 1.7% |
| Pectase | 0.06g | 0.4% |
| 10mg/ml BSA | 120μl | 0.1% |
| 0.2M MES | 1100μl | 20mM |
| 1M CaCl2 | 110μl | 10mM |
| 2M KCl | 120μl | 25mM |
| 0.8M mannitol | 5300μl | 0.3M |
It should be pointed out that first weighing enzyme powder, the MES solution of 70 DEG C of processing 3-5 minutes is added, adds except CaCl2
With the room temperature reagent except BSA, NDA enzyme and proteinase activity a small amount of in 55 DEG C of water-bath 10 minutes inactivation enzyme solutions, cold after concussion
But to after room temperature, CaCl is added2And BSA, it is then sterilized, is placed in brown reagent bottle with 0.45 μm of filter membrane.
(2), the preparation of WI solution；
| Ingredient | Dosage | Final concentration |
| 0.2M, MES | 220μl | 5mM |
| 2M,KCl | 120μl | 22mM |
| 0.8M, Mannitol | 7000μl | 0.6M |
Without individually sterilizing, room temperature preservation is now matched.
(3), the preparation of W5 solution；
| Ingredient | Dosage | Final concentration |
| 0.2M, MES | 550μl | 3mM |
| 1M, NaCl | 7800μl | 156mM |
| 1M, CaCl2 | 6800μl | 118mM |
| 2M, KCl | 140μl | 4.5mM |
Without individually sterilizing, room temperature preservation is now matched.
(4), the preparation of MMG solution；
| Ingredient | Dosage | Final concentration |
| 0.2M, MES | 240μl | 4.5mM |
| 0.8M, Mannitol | 5200μl | 0.5M |
| 2M MgCl2 | 90μl | 15mM |
Without individually sterilizing, room temperature preservation is now matched.
(5), PEG-Calcium converts the preparation of solution；
| Ingredient | Dosage | Final concentration |
| 0.8M, Mannitol | 2500μl | 0.2M |
| 1M CaCl2 | 1000μl | 100mM |
| PEG4000 | 3g | 30% |
It is noted that 5 milliliters or so of distilled water and other solution are added after weighing PEG4000, vibrate 2 minutes
Afterwards, in constant volume, since PEG4000 is difficult to be completely dissolved, it is therefore necessary to be initially configured at least 1 hour before using the reagent.
The solution room temperature preservation, now matches, without sterilizing.
Two, protoplast electrofusion:
S1, arabidopsis plantation；
The condition of arabidopsis plantation are as follows: the lower level conditions of illumination in 13 hours (23 DEG C) and 11 hours dark (20 DEG C)
Under, relative humidity 40-65%.
S2, blade are selected and are cut；
It is chosen from the plant in 3-4 week of growth and stretches good blade (1-1.5cm), as the printing paper of bilayer tiling
On, using fresh sharp blade along blade to be disposably cut into the strip of 1-1.5mm with main lobe arteries and veins vertical direction, and stand
I.e. soft putting into enzymolysis liquid and gently rotate is allowed to submerge.
S3, vacuum processing；
Vaned enzymolysis liquid will be filled and be placed in vacuscope about 1hr, to guarantee that enzymolysis liquid can preferably go deep into mesophyll tissue,
Continue to digest 3.5hr in shading environment later.
S4, protoplast inspection；
It is stained with transparent rubberized fabric on glass slide, digs out a 8x8mm with blade2Square, gently shake up enzymolysis liquid, from
20 μ l of middle taking-up are placed in square, covered, and the size of protoplast is checked under microscope bright field.(arabidopsis mesophyll
Cell protoplast is generally 30-50 μm, spherical shape, it is seen that plastid abundant)
S5, dilution and filtering enzymolysis liquid；
The W5 solution of isometric (5ml) is added in enzymolysis liquid, shakes gently, is taken from the culture dish of soaked in absolute ethyl alcohol
The nylon filter of 75- μm of hole out removes alcohol with distilled water flushing, is removed later with a small amount of W5 solution rinse filter membrane more
Remaining water, filter membrane is placed on funnel, filters the blade residue in enzymolysis liquid, collects protoplast in the round bottom sterile tube of 15ml
In.
S6, protoplast is collected；
100-150g is centrifuged 1-2mins, and protoplast can be deposited on test tube bottom, supernatant is poured out carefully and as far as possible
Liquid, soft rotating tubular body are dispersed in protoplast again in remaining liquid.
S7, protoplast is resuspended；
It takes the W5 solution of 5ml that protoplast is resuspended, protoplast is placed in 30 minutes on ice later, gravity precipitinogen
Protoplast is resuspended using 1-1.5mlMMG solution later with the W5 in pipette tips removal supernatant in raw plastid 15min.
Three, DNA-PEG-Ca is converted
1. the preparation of Plasmid DNA；Method is measured in use and extracts the Plasmid DNA to be converted, and is detected on horizontal glue, to guarantee
Super spirial plasmid ratio is high, takes 5-10 μ l plasmid spare in 2ml centrifuge tube, in bimolecular fluorescence complementary experiment, every kind of plasmid
For 10 μ g；
2. converting
The protoplast of 100 μ l is added in each centrifuge tube, it is even with the 200 light persorptions of μ l pipette tips, the PEG of 110 μ l is added
Solution, with index finger, gently attack tube bottom places 10mins at room temperature and completes conversion reaction to mix well.
3. stopping reaction
The W5 solution under 420 μ l room temperature states is taken to be added in above-mentioned conversion fluid, gently attack and reverse solution are to stop
Conversion reaction is centrifuged 2mins with desktop desk centrifuge, removes supernatant.
4. protoplast is resuspended, it is slowly added to 500 μ lWI solution and protoplast is resuspended.
Protoplast cuhnre and harvest
Be incubated for: in room temperature 100RPM shaker overnight culture protoplast, centrifuge tube needs horizontal positioned to guarantee maximum
Liquid level product the carboxylic card Benzylpenicillin sodium salt of 50 μ g/ml is added if discovery has a bacteria breed in being incubated overnight liquid.
Signal observation and protoplast storage
It observes before protoplast, first mixes gently test tube, take out 20 μ l, seen under above-mentioned fluorescence or CLSM
It surveys, with ultraviolet excitation, plastid and the signal for being transferred to plasmid, 100g centrifugation can be observed at the absorbing wavelength of RFP and GFP
2mins collects unspent protoplast and is stored in spare in -80 DEG C of refrigerators；It, can if necessary to extract protoplast albumen
6000g is centrifuged 2mins and collects all protoplasts, extracts total protein and utilizes western hybridization check.
Finally, it is stated that the above examples are only used to illustrate the technical scheme of the present invention and are not limiting, although referring to compared with
Good embodiment describes the invention in detail, those skilled in the art should understand that, it can be to skill of the invention
Art scheme is modified or replaced equivalently, and without departing from the objective and range of technical solution of the present invention, should all be covered at this
In the scope of the claims of invention.
Claims (10)
1. a kind of plant sub-cellular location reagent box, which is characterized in that consist of the following reagents: 5~20ml of enzymolysis liquid, WI solution
5~25ml, 30~50ml of W5 solution, MMG solution 5~25ml and PEG-Calcium convert 5~20ml of solution.
2. a kind of plant sub-cellular location reagent box according to claim 1, it is characterised in that: the enzymolysis liquid includes fibre
Tie up 0.1~0.2g of plain enzyme, 0.02~0.06g of pectase, the μ l of 10mg/ml BSA60~120, μ l of 0.2M MES800~1200,1M
CaCl2μ l of 80~110 μ l, 2M KCl85~120,4800~5300 μ l of 0.8M mannitol.
3. a kind of plant sub-cellular location reagent box according to claim 2, it is characterised in that: the WI solution includes
150~220 80~120 6000~7000 μ l of μ l, 0.8M, Mannitol of μ l, 2M, KCl of 0.2M, MES.
4. a kind of plant sub-cellular location reagent box according to claim 3, it is characterised in that: the W5 solution includes
400~550 μ l, 1M NaCl of 0.2M, MES, 7000~7800 μ l, 1M CaCl26000~6800 μ l, 2M KCl 100~
140μl。
5. a kind of plant sub-cellular location reagent box according to claim 4, it is characterised in that: the MMG solution includes
180~240 μ l, 0.8M Mannitol of 0.2M MES, 4600~5200 μ l, 2M MgCl230~90 μ l.
6. a kind of plant sub-cellular location reagent box according to claim 5, it is characterised in that: the PEG-Calcium
Converting solution includes 2300~2800 μ l, 1M CaCl of 0.8M Mannitol2800~1100 μ l, PEG4000,2~5g.
7. a kind of application of plant sub-cellular location reagent box as described in claim 1, it is characterised in that: including following
Step:
1. the preparation of Plasmid DNA: method of measuring in use extracts the Plasmid DNA to be converted, and detects on horizontal glue, to guarantee super spiral shell
It is high to revolve plasmid ratio, takes 5-10 μ l plasmid spare in 2ml centrifuge tube, in bimolecular fluorescence complementary experiment, every kind of plasmid is 10 μ
G, while plasmid control and label control are set；
2. conversion: the protoplast of 100 μ l is added in each centrifuge tube, it is even with the 200 light persorptions of μ l pipette tips, it is added 110 μ l's
PEG-Calcium solution, with index finger, gently attack tube bottom places 10mins at room temperature and completes conversion reaction to mix well；
3. stopping reaction: taking the W5 solution under 420 μ l room temperature states to be added in above-mentioned conversion fluid, gently attack and reverse solution
To stop conversion reaction, it is centrifuged 2mins with desktop desk centrifuge, removes supernatant；
4. protoplast is resuspended, it is slowly added to 500 μ lWI solution and protoplast is resuspended；
5. being incubated for: in room temperature 100RPM shaker overnight culture protoplast, centrifuge tube needs horizontal positioned maximum to guarantee
The carboxylic card Benzylpenicillin sodium salt of 50 μ g/ml is added if discovery has bacteria breed in being incubated overnight liquid in liquid level product；
6. signal observation and protoplast storage: before observation protoplast, test tube is first mixed gently, takes out 20 μ l,
It is observed under above-mentioned fluorescence or CLSM, with ultraviolet excitation, plastid can be observed at the absorbing wavelength of RFP and GFP and is turned
Enter the signal of plasmid, 100g centrifugation 2mins collects unspent protoplast and is stored in spare in -80 DEG C of refrigerators；If needed
Extract protoplast albumen, can 6000g centrifugation 2mins collect all protoplasts, it is simultaneously miscellaneous using western to extract total protein
Hand over detection.
8. a kind of application of plant sub-cellular location reagent box according to claim 7, it is characterised in that: the plasmid mentions
It takes to protoplasts of Arabidopsis thaliana broken by ultrasonic, and the inspection concrete operations of protoplasts of Arabidopsis thaliana broken by ultrasonic are as follows: be stained with transparent rubberized fabric on glass slide, use
Blade digs out a 8x8mm2Square, gently shake up enzymolysis liquid, be taken out 20 μ l and be placed in square, covered,
The size of protoplast is checked under microscope bright field.
9. a kind of application of plant sub-cellular location reagent box according to claim 7, it is characterised in that: the arabidopsis
Protoplast through inspection after, be diluted and filter enzymolysis liquid, concrete operations are as follows: be added in equal volume (5ml) W5 solution in
It in enzymolysis liquid, shakes gently, the nylon filter of 75- μm of hole is taken out from the culture dish of soaked in absolute ethyl alcohol, uses distilled water
Alcohol is removed in flushing, removes extra water with a small amount of W5 solution rinse filter membrane later, filter membrane is placed on funnel, filtering enzymatic hydrolysis
Blade residue in liquid collects protoplast in the round bottom sterile tube of 15ml.
10. a kind of application of plant sub-cellular location reagent box according to claim 8, it is characterised in that: further include receiving
Collect step, 100-150g is centrifuged 1-2mins, and protoplast can be deposited on test tube bottom, carefully and supernatant is poured out, soft to revolve
Tube body is dispersed in protoplast again in remaining liquid.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112680473A (en) * | 2021-01-15 | 2021-04-20 | 浙江省农业科学院 | Establishment and application of melon transient expression system |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003010507A2 (en) * | 2001-07-16 | 2003-02-06 | Ahram Biosystems Inc. | System for intracellular process monitoring and in vivo drug screening |
| CN101402938A (en) * | 2008-11-17 | 2009-04-08 | 中国科学院植物研究所 | Method for producing protoplast |
| CN102373235A (en) * | 2011-10-17 | 2012-03-14 | 南京林业大学 | Method for performing transient expression by introducing foreign gene into poplar bioplast |
| WO2014199358A1 (en) * | 2013-06-14 | 2014-12-18 | Cellectis | Methods for non-transgenic genome editing in plants |
-
2018
- 2018-08-29 CN CN201810991595.XA patent/CN109136321A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003010507A2 (en) * | 2001-07-16 | 2003-02-06 | Ahram Biosystems Inc. | System for intracellular process monitoring and in vivo drug screening |
| CN101402938A (en) * | 2008-11-17 | 2009-04-08 | 中国科学院植物研究所 | Method for producing protoplast |
| CN102373235A (en) * | 2011-10-17 | 2012-03-14 | 南京林业大学 | Method for performing transient expression by introducing foreign gene into poplar bioplast |
| WO2014199358A1 (en) * | 2013-06-14 | 2014-12-18 | Cellectis | Methods for non-transgenic genome editing in plants |
Non-Patent Citations (3)
| Title |
|---|
| SANG-DONG YOO等: "Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis", 《NATURE PROTOCOLS》 * |
| 张晓慧等: "两种瞬时表达体系研究拟南芥Profilin-1 的亚细胞定位", 《生物技术通报》 * |
| 蒋琦: "拟南芥FATB蛋白的亚细胞定位及其缺失对内质网和高尔基体的影响", 《中国优秀硕士学位论文全文数据库 基础科学辑》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112680473A (en) * | 2021-01-15 | 2021-04-20 | 浙江省农业科学院 | Establishment and application of melon transient expression system |
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