CN106676054A - Rapid and simple method for extracting complete arabidopis thaliana chloroplast - Google Patents
Rapid and simple method for extracting complete arabidopis thaliana chloroplast Download PDFInfo
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- CN106676054A CN106676054A CN201611010468.4A CN201611010468A CN106676054A CN 106676054 A CN106676054 A CN 106676054A CN 201611010468 A CN201611010468 A CN 201611010468A CN 106676054 A CN106676054 A CN 106676054A
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Abstract
The invention belongs to the technical fields of cytobiology, biochemistry, molecular biology and the like, and discloses a rapid and simple method for extracting complete arabidopis thaliana chloroplast. The method comprises the following operating steps: (1) putting arabidopis thaliana leaves into a Medium I, and homogenizing at a low speed; (2) filtering, and then horizontally centrifuging 1500g of a filtered product for 5min at the temperature of 4 DEG C; (3) suspending chloroplast precipitate by using a Medium II, and then horizontally centrifuging 3000g of the suspended product for 30min at the temperature of 4 DEG C in 40%/80% percoll gradient; (4) absorbing the complete chloroplast, and washing the absorbed chloroplast with SH buffer for twice; (5) suspending the chloroplast precipitate by using the SH buffer, and then quantifying by using pigment.
Description
Technical field
It is more particularly to a kind of quick the invention belongs to technical fields such as cell biology, biochemistry and molecular biology
The extracting method of easy arabidopsis complete excision.
Background technology
Arabidopsis (Arabidopsis thaliana), also known as Arabidopsis thaliana, are currently known minimum in Plant Genome,
It is also a kind of quite varied model organism of application.And chloroplaset is the distinctive organelle of plant cell, it is photosynthetic to be that plant is carried out
The place of effect, in addition to photosynthesis, also participates in the synthesis of amino acid, aliphatic acid, plant growth substance, vitamin etc..Cause
And, development and functional study extremely people's concern always of plant chloroplast.Meanwhile, the chloroplaset for obtaining high-purity is used for albumen
The research of matter function turns into prerequisite.At present, the method extracted on arabidopsis chloroplaset, it is most of by extracting spinach and pea
The method of beans chloroplaset is improved.The separation method of Riet in 1985 et al. has only used a kind of percoll gradients of concentration, obtains
The chloroplaset purity for arriving is inadequate；The extracting method of Daniel Salvi in 2008 needs to use the high speed horizontal centrifugal of higher strip part
Machine.The formula of also certain methods extract solution is complicated, spends the time long.It is green the invention provides a kind of fast simple complete leaf
Body extracting method, extraction time is short, and formula is simple, and centrifugation requires low, after being quantitative determined, can directly apply to chloroplaset
The various follow-up studies such as the extraction of memebrane protein, stromatin and chloroplast DNA.
The content of the invention
In order to overcome the shortcoming and deficiency of prior art, it is an object of the invention to provide a kind of fast simple complete leaf
The extracting method of green body.
The purpose of the present invention is achieved through the following technical solutions：
A kind of extracting method of fast simple arabidopsis complete excision, it is characterised in that including following operating procedure：
(1) apparatus precooling；
(2) when the incubator photoperiod 2-3h is started, the Arabidopsis leaf of 3-4 weeks is taken, is placed on ice；
(3) Medium I (buffer solution I) of 200ml, low speed homogenate and then in refiner are added；
(4) using miracloth (magical filter cloth) or filtered through gauze, 1500g horizontal centrifugal 5min, it is precipitated as blackish green
Thick chloroplaset, supernatant is light green color；
(5) try one's best removal supernatant, precipitation is softly suspended with Medium II (buffer solution II) of about 5ml；
(6) 40%/80%percoll (cell separation liquid) gradient is configured, by sample loading to percoll gradients upper strata,
3000g horizontal centrifugals 30min；
(7) complete excision is located at the intersection of upper and lower two percoll components, and complete excision is suctioned out；Use SH
Buffer (SH buffer solutions) dilutes 3 times, and 1500g horizontal centrifugal 2min, precipitation is resuspended in 25ml SH buffer, 1000g centrifugations
1min；
(8) precipitation is suspended (about 5-10ml) with appropriate SH buffer, can observe chloroplaset by microscopy immediately, and pigment is fixed
Amount.
More specifically, a kind of fast simple complete excision extracting method, including following operating procedure：
(1) all apparatus of this method (tissue mashing refiner, beaker, test tube, reagent etc.) need to be on ice or -20 DEG C pre-
It is cold, it is centrifuged under the conditions of 4 DEG C.
(2) when the incubator photoperiod 2-3h is started, the common 20g of Arabidopsis leaf of 3-4 weeks is taken, is placed on ice.
(3) Medium I (buffer solution the I) (330mM sorbitol (sorboses of 200ml and then in refiner are added
Alcohol), 30mM Tricine-KOH (trimethylglycine-potassium hydroxide) pH 8.4,5mM EGTA, 5mM EDTA, 10mM
NaHCO3), low speed homogenate, the time is no more than 5min.
(4) in using 3 layers of miracloth (magical filter cloth) or 8 layers of filtered through gauze to 4 50ml round bottom centrifuge tubes,
1500g horizontal centrifugal 5min, are precipitated as blackish green thick chloroplaset, and supernatant is light green color.
(5) try one's best removal supernatant, precipitation Medium II (buffer solution the II) (300mM sorbitol (sorbs of about 5ml
Sugar alcohol), 20mM Hepes-KOH (4- HEPESs-potassium hydroxide) pH 7.6,5mM MgCl2, 2.5mM EDTA)
It is soft to suspend (pipette tips cut big mouth).
(6) 23ml 40%/80%percoll (cell separation liquid) gradient is configured in 50ml centrifuge tubes, by sample loading
To 4 percoll gradients upper stratas, 3000g horizontal centrifugals 30min.
(7) complete excision is located at the intersection (5ml graduation marks) of upper and lower two percoll (cell separation liquid) component,
Carefully complete excision is suctioned out, each percoll intersection can suction out 5-10ml chloroplasets.With SH buffer, (SH is buffered
Liquid) (330mM sorbitol, 50mM Hepes-KOH pH 8.0,2mM DTT, DTT now need to be added with existing) 3 times of dilution, 1500g
Horizontal centrifugal 2min, precipitation is resuspended in 25ml SH buffer, 1000g centrifugations 1min.
(8) precipitation appropriate SH buffer (SH buffer solutions) (330mM sorbitol, 50mM Hepes-KOH pH
8.0,2mM DTT, DTT need to show and be added with existing) suspend (about 5-10ml), chloroplaset can be observed by microscopy immediately, and pigment is quantitative.
(Percoll (cell separation liquid) gradient is prepared：Prepare first containing 6% (w/v) sorbitol (D-sorbite)
100%Percoll (cell separation liquid), then prepares Percoll (cell separation liquid) gradient.40% component, 7.2ml 100%
Percoll+10.8ml SH buffer；80% component, 4ml 100%Percoll+1ml SH buffer.)
Compared with prior art, the present invention has advantages below and beneficial effect：
(1) present invention need not save step and time by arabidopsis dark treatment overnight.
(2) three extract recipes of the invention are simple, but can extract a large amount of great-hearted chloroplasets.
(3) present invention improves over step 4, the size of 6 centrifugal force, the requirement to centrifuge is greatly reduced.
(4) present invention improves over the time of step 6 centrifugation so that obtain complete excision as much as possible.
Brief description of the drawings
Fig. 1 is the arabidopsis complete excision figure extracted using the present invention, (note：Bar=5 μm).
Specific embodiment
With reference to embodiment, the present invention is described in further detail, but embodiments of the present invention not limited to this.
A kind of extracting method of fast simple arabidopsis complete excision of embodiment 1
(1) all apparatus of this method (tissue mashing refiner, beaker, test tube, reagent etc.) need to be on ice or -20 DEG C pre-
It is cold, it is centrifuged under the conditions of 4 DEG C.
(2) when the incubator photoperiod 2-3h is started, the common 20g of Arabidopsis leaf of 3-4 weeks is taken, is placed on ice.
(3) Medium I (330mM sorbitol, the 30mM Tricine-KOH of 200ml and then in refiner are added
PH 8.4,5mM EGTA, 5mM EDTA, 10mM NaHCO3), low speed homogenate, the time is no more than 5min.
(4) in use 3 layers of miracloth or 8 layer of filtered through gauze to 4 50ml round bottom centrifuge tubes, 1500g horizontal centrifugals
5min, is precipitated as blackish green thick chloroplaset, and supernatant is light green color.
(5) try one's best removal supernatant, precipitation Medium II (300mM sorbitol, the 20mM Hepes-KOH of about 5ml
PH 7.6,5mM MgCl2, 2.5mM EDTA) and soft suspension (pipette tips cut big mouth).
(6) 23ml 40%/80%percoll gradients are configured in 50ml centrifuge tubes, by sample loading to 4 percoll
Gradient upper strata, 3000g horizontal centrifugals 30min.
(7) complete excision is located at the intersection (5ml graduation marks) of upper and lower two percoll components, carefully by complete leaf
Green body is suctioned out, and each percoll intersection can suction out 5-10ml chloroplasets.With SH buffer (330mM sorbitol,
50mM Hepes-KOH pH 8.0,2mM DTT, DTT now need to be added with existing) 3 times of dilution, 1500g horizontal centrifugal 2min, precipitation weight
It is suspended from 25ml SH buffer, 1000g centrifugations 1min.
(8) precipitation is suspended (about 5-10ml) with appropriate SH buffer, can observe chloroplaset by microscopy immediately, and pigment is fixed
Amount.
(Percoll gradients are prepared：The 100%Percoll containing 6% (w/v) sorbitol is prepared first, is then prepared
Percoll gradients.40% component is 7.2ml 100%Percoll+10.8ml SH buffer, and 80% component is 4ml 100%
80% component of Percoll+1ml SH buffer, 5ml is slowly expelled to 40% component bottom with syringe needle long.)
Above-described embodiment is the present invention preferably implementation method, but embodiments of the present invention are not by above-described embodiment
Limitation, it is other it is any without departing from Spirit Essence of the invention and the change, modification, replacement made under principle, combine, simplification,
Equivalent substitute mode is should be, is included within protection scope of the present invention.
Claims (8)
1. a kind of extracting method of fast simple arabidopsis complete excision, it is characterised in that including following operating procedure：
(1) apparatus precooling；
(2) when the incubator photoperiod 2-3h is started, the Arabidopsis leaf of 3-4 weeks is taken, is placed on ice；
(3) Medium I (buffer solution I) of 200ml, low speed homogenate and then in refiner are added；
(4) using miracloth (magical filter cloth) or filtered through gauze, 1500g horizontal centrifugal 5min, it is precipitated as blackish green thick leaf
Green body, supernatant is light green color.
(5) try one's best removal supernatant, precipitation is softly suspended with Medium II (buffer solution II) of about 5ml；
(6) 40%/80%percoll (cell separation liquid) gradient is configured, by sample loading to percoll gradients upper strata, 3000g
Horizontal centrifugal 30min；
(7) complete excision is located at the intersection of upper and lower two percoll components, and complete excision is suctioned out；Use SH buffer
(SH buffer solutions) dilutes 3 times, and 1500g horizontal centrifugal 2min, precipitation is resuspended in 25ml SH buffer, 1000g centrifugations 1min；
(8) precipitation is suspended (about 5-10ml) with appropriate SH buffer, can observe chloroplaset by microscopy immediately, and pigment is quantitative.
2. extracting method according to claim 1, it is characterised in that apparatus precooling includes tissue mashing refiner, burns
Cup, test tube, reagent are on ice or -20 DEG C of precoolings.
3. extracting method according to claim 1, it is characterised in that the formula of the Medium I is：330mM
Sorbitol, 30mM Tricine-KOH pH 8.4,5mM EGTA, 5mM EDTA, 10mM NaHCO3。
4. extracting method according to claim 1, it is characterised in that the formula of the Medium II is：300mM
Sorbitol, 20mM Hepes-KOH pH 7.6,5mM MgCl2, 2.5mM EDTA.
5. extracting method according to claim 1, it is characterised in that the preparation side of the SH buffer is：330mM
Sorbitol, 50mM Hepes-KOH pH 8.0,2mM DTT, DTT now need to be added with existing.
6. extracting method according to claim 1, it is characterised in that Percoll gradient compound methods are：Prepare first and contain
The 100%Percoll of 6% (w/v) sorbitol, then prepares Percoll gradients；40% component is 7.2ml 100%
Percoll+10.8ml SH buffer, 80% component is 80% group of 4ml 100%Percoll+1ml SH buffer, 5ml
Divide and be slowly expelled to 40% component bottom with syringe needle long.
7. the extracting method of a kind of fast simple arabidopsis complete excision according to claim 1, it is characterised in that：
Using in 3 layers of miracloth or 8 layer of filtered through gauze to 50ml round bottom centrifuge tubes, 1500g horizontal centrifugals 5min.
8. the extracting method of a kind of fast simple arabidopsis complete excision according to claim 1, it is characterised in that：
23ml 40%/80%percoll gradients are configured in 50ml centrifuge tubes, by sample loading to percoll gradients upper strata, 3000g
Horizontal centrifugal 30min.
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| CN201611010468.4A CN106676054A (en) | 2016-11-17 | 2016-11-17 | Rapid and simple method for extracting complete arabidopis thaliana chloroplast |
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| CN201611010468.4A CN106676054A (en) | 2016-11-17 | 2016-11-17 | Rapid and simple method for extracting complete arabidopis thaliana chloroplast |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107456952A (en) * | 2017-08-28 | 2017-12-12 | 陕西师范大学 | A kind of preparation method for catching light oxygen release papery coating material |
| CN113061567A (en) * | 2021-04-06 | 2021-07-02 | 石河子大学 | Method for extracting chloroplast of kochia scoparia |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102533729A (en) * | 2012-01-11 | 2012-07-04 | 福建农林大学 | Method for extracting deoxyribonucleic acid (DNA) from mature kenaf laminas |
| CN104277974A (en) * | 2013-07-03 | 2015-01-14 | 中国科学院植物研究所 | Hydrogen production chlamydomonas chloroplast separation method |
-
2016
- 2016-11-17 CN CN201611010468.4A patent/CN106676054A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102533729A (en) * | 2012-01-11 | 2012-07-04 | 福建农林大学 | Method for extracting deoxyribonucleic acid (DNA) from mature kenaf laminas |
| CN104277974A (en) * | 2013-07-03 | 2015-01-14 | 中国科学院植物研究所 | Hydrogen production chlamydomonas chloroplast separation method |
Non-Patent Citations (1)
| Title |
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| 宗学凤: "《植物生理研究技术》", 30 September 2011, 西南师范大学出版社 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107456952A (en) * | 2017-08-28 | 2017-12-12 | 陕西师范大学 | A kind of preparation method for catching light oxygen release papery coating material |
| CN107456952B (en) * | 2017-08-28 | 2019-12-03 | 陕西师范大学 | A kind of preparation method for catching light oxygen release papery coating material |
| CN113061567A (en) * | 2021-04-06 | 2021-07-02 | 石河子大学 | Method for extracting chloroplast of kochia scoparia |
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Application publication date: 20170517 |