CN102174464A - Complete chloroplast long-time isolated culture method - Google Patents
Complete chloroplast long-time isolated culture method Download PDFInfo
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- CN102174464A CN102174464A CN 201110038865 CN201110038865A CN102174464A CN 102174464 A CN102174464 A CN 102174464A CN 201110038865 CN201110038865 CN 201110038865 CN 201110038865 A CN201110038865 A CN 201110038865A CN 102174464 A CN102174464 A CN 102174464A
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Abstract
The invention discloses a complete chloroplast long-time isolated culture method. Sterile seedling cotyledons of cucumber, tobacco and spinach are used as materials respectively; citric acid, NaCl, Tris and ethylene diamine tetraacetic acid (EDTA) are used as chloroplast extraction reagents of main components; complete chloroplast (figure 1) is separated by the operations of grinding, gauze filtration, differential centrifugation and the like of the plant materials; and MnCl2, MgSO4, NaCl, sorbitol and the like are used as culture reagents of the main components, and the isolated chloroplast is subjected to liquid culture. After being cultured in vitro for about 20 days, the chloroplast still keeps green and is microscopically normal (figure 2), and the membrane structure is complete (figure 3). In two weeks of isolated culture, the chloroplast is split and propagated to reach relatively stable number (figure 4). Proved by isolated transformation and exogenous gene detection of the chloroplast, the DNA in the chloroplast cultured for long time is not degraded (figure 5). The method can provide convenience and a new path for basic theories and application research of chloroplast function, genetic transformation, cell engineering and the like.
Description
[technical field]:
The invention belongs to the plant gene engineering technology field, relate generally to the technology and the enforcement of complete chloroplast(id) separation of plant and vitro culture, comprise that chloroplast(id) separates and the preparation of vitro culture reagent and related schedule of operation.
[background technology]:
Chloroplast(id) is the important organelle that plant becomes conversion of solar energy chemical energy, it is the source of the tellurian main picked-up energy of surviving, therefore, the structure of chloroplast(id), function, genome and gene expression regulation etc. all become the problem that life science receives much concern, and human existence such as basic theory that it is relevant and applied research and grain, the energy, environment are closely related.Though the chloroplast(id) that is in the eukaryotic cells has independently genome and double-deck microbial film bag quilt, but belong to semiautonomous organelle, its generation, growth and function not only are subjected to the regulation and control of cell nucleus gene group, also influence each other synergy with other organoid.The observation that morphocytology behind chloroplast mutation and the genetic transformation is changed or the like can provide important method for chloroplast gene group and gene expression regulation research.At present, the technology of foreign gene importing chloroplast(id) still is confined to main particle gun method in the world, chloroplast mutation body and transformant screening are still relatively more difficult, these become the critical bottleneck of correlative studys such as restricting the chloroplast(id) conversion and application development process, and the chloroplast(id) isolated operation is an important breach, exsomatize chloroplast(id) if can be kept perfectly the long period, will provide convenient and open up new way for the operation of chloroplast gene engineering.
[summary of the invention]:
The purpose of this invention is to provide complete chloroplast(id) extracts and the external long-time cultivation related reagent of chloroplast(id) and extraction and cultural method.
Complete chloroplast(id) provided by the invention extracts relevant reagent set and becomes to comprise:
Above all ingredients is dissolved in respectively in the deionized water, preserves separately in 4 ℃ of refrigerators behind the autoclaving.
Complete chloroplast(id) isolated culture related reagent provided by the invention is formed and is comprised the following moiety that shows concentration:
EDTA(PH8.0) 2mM
MnCl
2 1mM
MgSO
4 1mM
NaCl 10mM
MES 50mM
Sorbyl alcohol 0.3M;
Above reagent is dissolved in the deionized water, transfers pH to 6.2, preserves in 4 ℃ of refrigerators behind the autoclaving.
Complete chloroplast(id) of the present invention extracts and the concrete steps of long-time isolated culture method comprise:
1st, take by weighing 4 ℃ of fresh plant cotyledon 8～10g that spend the night, shred, place the mortar of precooling on ice;
2nd, the BufferA40ml in the described extraction reagent is added in the mortar at twice, and adds 1g quartz sand, grind after 6 layers of filtered through gauze to the 50ml centrifuge tube;
3rd, with the 2nd the step in lapping liquid in 4 ℃ of centrifugal 6min of 800g, abandon supernatant;
4th, the BufferB 25ml in the described extraction reagent of adding precooling in the 3rd step adds the 17.5ul beta-mercaptoethanol, the suspension chloroplast(id) then;
5th, with the 4th the step in suspension in 4 ℃ of centrifugal 8min of 800g, abandon supernatant;
6th, the BufferC 12ml in the described extraction reagent of adding in the 5th step, the suspension chloroplast(id);
7th, with the 6th the step in suspension in 4 ℃ of centrifugal 8min of 1000g, abandon supernatant, obtain complete chloroplast(id);
8th, get the described isolated culture reagent of the 10ml complete chloroplast(id) that the 7th step obtained that suspends, place the 25 ℃ of following lucifuges or the low light level (long-time exsomatizing) to cultivate.
Advantage of the present invention and beneficial effect:
The present invention is main experiment material with cucumber, tobacco, set up the vitro culture technology that is suitable for the functional study of higher plant chloroplast(id), utilize this operative technique that stripped chloroplast(id) vitro culture was reached about 20 days, broken through the longest in the world report time of having only about 10 days, and through the methods such as quantity that the chloroplast(id) micromorphology is observed, complete chloroplast(id) is identified and write down to the biofilm structure integrity, prove through the chloroplast(id) microscopic morphology under the long-time isolated culture state normally and structure be kept perfectly.Because chloroplast(id) is a plant to carry out photosynthesis sun power is transformed into the place of bioenergy, be the tellurian synthetic source of the main energy of active that earns a bare living, so the correlative study of chloroplast(id) function and Application Areas people's attention extremely always.Still perplexed by food shortage, environmental pollution and energy shortage or the like problem the new millennium mankind, therefore, people focus on sight on the chloroplast(id) of green plants and representative thereof once more, and chloroplast gene group and gene expression research also become a new focus of life science in recent years.To the chloroplast(id) that exsomatizes carry out that the relevant rudimentary theory and application research saves time, easy handling, observation and advantage such as easy to detect, so the present invention provides extremely important technology platform for chloroplast(id) function, chloroplast(id) genetic transformation and cell engineering operation.
[description of drawings]:
The stripped chloroplast(id) (2000 times amplification) of Fig. 1 for cultivating as yet after separating.
Fig. 2 is that the microscopic morphology of chloroplast(id) after the isolated culture is observed (1500 times of amplifications), and wherein A～C is respectively and cultivated 5 days, 11 days and 18 days.
Fig. 3 expects that for the isolated culture chloroplast(id) being carried out platform blue dyeing detects its film integrality (1000 times of amplifications), and A～C is respectively and cultivated 0 day, 2 days and 12 days.
The counting of Fig. 4 under dark condition, the chloroplast(id) of isolated culture different time being carried out.
Fig. 5 detects figure for gene amplification in the chloroplast(id) that exsomatizes, and A is for cultivating 11 days, and B is for cultivating 20 days.
[embodiment]:
Embodiment 1: complete chloroplast(id) extracts relevant reagent set and becomes to comprise:
Above all ingredients is dissolved in respectively in the deionized water, preserves separately in 4 ℃ of refrigerators behind the autoclaving.
Embodiment 2: complete plant chloroplast isolated culture related reagent is formed and is comprised:
Above reagent is dissolved in the deionized water, transfers pH to 6.2, preserves in 4 ℃ of refrigerators behind the autoclaving.
Embodiment 3: complete chloroplast(id) extracts and long-time isolated culture schedule of operation.
(1) takes by weighing 4 ℃ of fresh plant cotyledon 8～10g that spend the night, shred, place the mortar of precooling on ice.
(2) 40ml BufferA adds in the mortar at twice, and adds the quartz sand about 1g, grinds after 6 layers of filtered through gauze (operation on ice) to the 50ml centrifuge tube.
(3) 4 ℃ of centrifugal 6min of 800g abandon supernatant.
(4) BufferB (time spent adds the 17.5ul beta-mercaptoethanol) of adding 25ml precooling, the suspension chloroplast(id).
(5) 4 ℃ of centrifugal 8min of 800g abandon supernatant.
(6) add 12ml BufferC, suspension chloroplast(id).
(7) 4 ℃ of centrifugal 8min of 1000g abandon supernatant, are precipitated as the complete chloroplast(id) that extraction obtains.
(8) the complete chloroplast(id) precipitation that obtains with 10ml isolated culture reagent suspension step (7) places the 25 ℃ of following lucifuges or the low light level to cultivate (isolated culture for a long time).
Embodiment 4: the chloroplast(id) counting of isolated culture.
Utilize blood counting chamber to calculate the number (diluting 100 times) of the chloroplast(id) that extracts.
| On | Down | |
| Upper left | 12 | 14 |
| The lower-left | 17 | 19 |
| Upper right | 11 | 8 |
| The bottom right | 3 | 10 |
| In | 13 | 20 |
Each lattice volume is 0.1/400=1/4000mm
3=1/4000,000ml;
The average bacterium number of every little lattice=(A
1+ A
2+ A
3+ A
4+ A
5)/80
Bacterium number/ml=[(A in the sample
1+ A
2+ A
3+ A
4+ A
5)/80] * 4000,000 * extension rate
Calculate: about 3 * 10
8
Embodiment 5: platform is expected blue staining and microscopic examination evaluation chloroplast(id) form and membrane structure integrity, and Fig. 1 is for cultivating 0 hour chloroplast(id) microscopic morphology, and its texture ratio is more complete; Observed isolated culture respectively 5 days, 11 days and the microscopic morphology of 18 days chloroplast(id)s, its structure is still more complete shown in figure A～C; Isolated culture 0 day, 2 days and 12 days chloroplast(id)s are carried out platform expect blue dyeing, the result shows still complete (Fig. 3 A～C) of its membrane structure.
The chloroplast(id) that exsomatizes is carried out electricity transform, cultivate with aforesaid method then.Isolated culture chloroplast DNA with conversion is a masterplate, pcr amplification external source GFP (green protein) gene, and result (Fig. 5) shows that the chloroplast(id) that exsomatizes transforms back 20 days external source GFP genes of cultivation amplified production is still arranged.
Claims (3)
1. a complete chloroplast(id) extracts related reagent, it is characterized in that this reagent composition comprises:
Above all ingredients is dissolved in respectively in the deionized water, preserves separately in 4 ℃ of refrigerators behind the autoclaving.
2. complete chloroplast(id) isolated culture related reagent is characterized in that this reagent comprises the following moiety that shows concentration:
EDTA(PH8.0) 2mM
MnCl
2 1mM
MgSO
4 1mM
NaCl 10mM
MES 50mM
Sorbyl alcohol 0.3M;
Above reagent is dissolved in the deionized water, transfers pH to 6.2, preserves in 4 ℃ of refrigerators behind the autoclaving.
3. long-time isolated culture method of complete chloroplast(id) is characterized in that the concrete steps of this method comprise:
1st, take by weighing 4 ℃ of fresh plant cotyledon 8～10g that spend the night, shred, place the mortar of precooling on ice;
2nd, the BufferA40ml in the described reagent of claim 1 is added in the mortar at twice, and adds 1g quartz sand, grind after 6 layers of filtered through gauze to the 50ml centrifuge tube;
3rd, with the 2nd the step in lapping liquid in 4 ℃ of centrifugal 6min of 800g, abandon supernatant;
4th, the BufferB 25ml in the described reagent of claim 1 of adding precooling in the 3rd step adds the 17.5ul beta-mercaptoethanol, the suspension chloroplast(id) then;
5th, with the 4th the step in suspension in 4 ℃ of centrifugal 8min of 800g, abandon supernatant;
6th, the BufferC 12ml in the described reagent of adding claim 1 in the 5th step, the suspension chloroplast(id);
7th, with the 6th the step in suspension in 4 ℃ of centrifugal 8min of 1000g, abandon supernatant, obtain complete chloroplast(id);
8th, get the described isolated culture reagent of the 10ml claim 2 complete chloroplast(id) that the 7th step obtained that suspends, place the 25 ℃ of following lucifuges or the low light level to cultivate.
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| CN 201110038865 CN102174464A (en) | 2011-02-16 | 2011-02-16 | Complete chloroplast long-time isolated culture method |
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| CN 201110038865 CN102174464A (en) | 2011-02-16 | 2011-02-16 | Complete chloroplast long-time isolated culture method |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103232972A (en) * | 2013-04-15 | 2013-08-07 | 广州白云山拜迪生物医药有限公司 | Medium freeze-drying powder production process for lymphocyte culture |
| CN104513856A (en) * | 2014-12-24 | 2015-04-15 | 湖南文理学院 | Method for detecting chloroplast DNAs to identify cytoplasmic male sterile line of hot pepper |
| CN105907697A (en) * | 2016-06-15 | 2016-08-31 | 河南农业大学 | Preparation method of wheat complete chloroplasts |
| 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 |
| CN114136930A (en) * | 2021-01-14 | 2022-03-04 | 北京林业大学 | Method for rapidly identifying integrity of plant chloroplast |
-
2011
- 2011-02-16 CN CN 201110038865 patent/CN102174464A/en active Pending
Non-Patent Citations (5)
| Title |
|---|
| 《中国油料》 19950630 梁明山 等 油菜叶绿体DNA的提取和纯化 第57页第15-25行 3 第17卷, 第2期 * |
| 《中国糖料》 20050930 崔杰 等 甜菜叶绿体DNA分离纯化方法 第29页第22-26行 1、3 , 第3期 * |
| 《安徽农业科学》 20070531 程霞英 等 菠菜离体叶绿体培养研究 第4419页左栏第17-20行、、右栏第3-7行、图2，第4464页左栏第2-4行 2、3 第35卷, 第15期 * |
| 《现代植物生理学实验指南》 19991231 中国科学院上海植物生理研究所 等 现代植物生理学实验指南 第2页第10-25行 2、3 , * |
| 《生物生产力和光合作用测定技术》 19860930 J·库姆斯 等 生物生产力和光合作用测定技术 第129-131页 1-3 , * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103232972A (en) * | 2013-04-15 | 2013-08-07 | 广州白云山拜迪生物医药有限公司 | Medium freeze-drying powder production process for lymphocyte culture |
| CN104513856A (en) * | 2014-12-24 | 2015-04-15 | 湖南文理学院 | Method for detecting chloroplast DNAs to identify cytoplasmic male sterile line of hot pepper |
| CN105907697A (en) * | 2016-06-15 | 2016-08-31 | 河南农业大学 | Preparation method of wheat complete chloroplasts |
| CN105907697B (en) * | 2016-06-15 | 2019-07-26 | 河南农业大学 | A kind of preparation method of wheat complete excision |
| 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 |
| CN114136930A (en) * | 2021-01-14 | 2022-03-04 | 北京林业大学 | Method for rapidly identifying integrity of plant chloroplast |
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Application publication date: 20110907 |