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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- 239000003153 chemical reaction reagent Substances 0.000 title claims abstract description 32
- 210000001938 protoplast Anatomy 0.000 claims abstract description 102
- 239000007788 liquid Substances 0.000 claims abstract description 56
- 239000013612 plasmid Substances 0.000 claims abstract description 35
- 238000002360 preparation method Methods 0.000 claims abstract description 21
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 21
- 238000006243 chemical reaction Methods 0.000 claims abstract description 18
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 15
- 239000011575 calcium Substances 0.000 claims abstract description 13
- 229910052791 calcium Inorganic materials 0.000 claims abstract description 10
- 238000003860 storage Methods 0.000 claims abstract description 6
- 241000196324 Embryophyta Species 0.000 claims description 19
- 239000000594 mannitol Substances 0.000 claims description 19
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 claims description 18
- 229930195725 Mannitol Natural products 0.000 claims description 18
- 235000010355 mannitol Nutrition 0.000 claims description 18
- 239000001110 calcium chloride Substances 0.000 claims description 17
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 17
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 16
- 239000012528 membrane Substances 0.000 claims description 13
- 239000006228 supernatant Substances 0.000 claims description 13
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 11
- 210000002706 plastid Anatomy 0.000 claims description 11
- 238000012360 testing method Methods 0.000 claims description 11
- 241000219194 Arabidopsis Species 0.000 claims description 10
- 108090000790 Enzymes Proteins 0.000 claims description 10
- 102000004190 Enzymes Human genes 0.000 claims description 10
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 10
- 239000000284 extract Substances 0.000 claims description 10
- 238000000034 method Methods 0.000 claims description 9
- 239000000047 product Substances 0.000 claims description 9
- 239000012153 distilled water Substances 0.000 claims description 8
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L magnesium chloride Substances [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 claims description 8
- 238000005119 centrifugation Methods 0.000 claims description 7
- 235000019441 ethanol Nutrition 0.000 claims description 6
- 238000007689 inspection Methods 0.000 claims description 6
- 241000219195 Arabidopsis thaliana Species 0.000 claims description 5
- 241000894006 Bacteria Species 0.000 claims description 5
- 239000004677 Nylon Substances 0.000 claims description 5
- FCPVYOBCFFNJFS-LQDWTQKMSA-M benzylpenicillin sodium Chemical compound [Na+].N([C@H]1[C@H]2SC([C@@H](N2C1=O)C([O-])=O)(C)C)C(=O)CC1=CC=CC=C1 FCPVYOBCFFNJFS-LQDWTQKMSA-M 0.000 claims description 5
- 230000000295 complement effect Effects 0.000 claims description 5
- 238000001218 confocal laser scanning microscopy Methods 0.000 claims description 5
- 230000005284 excitation Effects 0.000 claims description 5
- 238000002474 experimental method Methods 0.000 claims description 5
- 239000004744 fabric Substances 0.000 claims description 5
- 239000012530 fluid Substances 0.000 claims description 5
- 238000011010 flushing procedure Methods 0.000 claims description 5
- 239000011521 glass Substances 0.000 claims description 5
- 239000003292 glue Substances 0.000 claims description 5
- 229910001629 magnesium chloride Inorganic materials 0.000 claims description 5
- 229920001778 nylon Polymers 0.000 claims description 5
- 239000011780 sodium chloride Substances 0.000 claims description 5
- 238000001914 filtration Methods 0.000 claims description 4
- 238000001514 detection method Methods 0.000 claims 1
- 230000007071 enzymatic hydrolysis Effects 0.000 claims 1
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 claims 1
- 239000000835 fiber Substances 0.000 claims 1
- 230000014509 gene expression Effects 0.000 abstract description 6
- 230000004960 subcellular localization Effects 0.000 abstract description 5
- 238000001727 in vivo Methods 0.000 abstract description 2
- 239000000463 material Substances 0.000 abstract description 2
- 239000000243 solution Substances 0.000 description 62
- MTHSVFCYNBDYFN-UHFFFAOYSA-N diethylene glycol Chemical compound OCCOCCO MTHSVFCYNBDYFN-UHFFFAOYSA-N 0.000 description 25
- 239000004615 ingredient Substances 0.000 description 15
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 12
- 238000004321 preservation Methods 0.000 description 12
- 230000001954 sterilising effect Effects 0.000 description 12
- 229920001030 Polyethylene Glycol 4000 Polymers 0.000 description 9
- 229940088598 enzyme Drugs 0.000 description 9
- 238000012545 processing Methods 0.000 description 6
- 238000005303 weighing Methods 0.000 description 6
- 108010059892 Cellulase Proteins 0.000 description 4
- 229940106157 cellulase Drugs 0.000 description 4
- 238000009396 hybridization Methods 0.000 description 4
- 102000035195 Peptidases Human genes 0.000 description 3
- 108091005804 Peptidases Proteins 0.000 description 3
- 210000001367 artery Anatomy 0.000 description 3
- 230000009514 concussion Effects 0.000 description 3
- 238000010790 dilution Methods 0.000 description 3
- 239000012895 dilution Substances 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 230000005484 gravity Effects 0.000 description 3
- 238000003306 harvesting Methods 0.000 description 3
- 238000005286 illumination Methods 0.000 description 3
- 230000002779 inactivation Effects 0.000 description 3
- 210000000473 mesophyll cell Anatomy 0.000 description 3
- 229920001223 polyethylene glycol Polymers 0.000 description 3
- 239000000843 powder Substances 0.000 description 3
- 235000019833 protease Nutrition 0.000 description 3
- 210000003462 vein Anatomy 0.000 description 3
- 238000011161 development Methods 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 101710134784 Agnoprotein Proteins 0.000 description 1
- WQZGKKKJIJFFOK-QTVWNMPRSA-N D-mannopyranose Chemical compound OC[C@H]1OC(O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-QTVWNMPRSA-N 0.000 description 1
- 101710124584 Probable DNA-binding protein Proteins 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 230000030570 cellular localization Effects 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 238000010218 electron microscopic analysis Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000005194 fractionation Methods 0.000 description 1
- 230000037353 metabolic pathway Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 230000014616 translation Effects 0.000 description 1
Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5097—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving plant cells
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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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