CN101948796A - Method for extracting orange flesh chromoplast - Google Patents
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- CN101948796A CN101948796A CN 201010260376 CN201010260376A CN101948796A CN 101948796 A CN101948796 A CN 101948796A CN 201010260376 CN201010260376 CN 201010260376 CN 201010260376 A CN201010260376 A CN 201010260376A CN 101948796 A CN101948796 A CN 101948796A
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Abstract
The invention belongs to the field of plant biochemistry, in particular to a method for centrifugally extracting orange flesh chromoplast by applying density gradient. The method comprises the following steps: 1) squeezing and filtering sweet orange flesh with buffer solution A; centrifuging filtrate, taking supernate, centrifuging the supernate to obtain precipitate; diluting the precipitate with the buffer solution A to obtain a sample; 2) selecting a centrifuge tube, applying sterilized bovine serum to the inner wall of the centrifuge tube; diluting 30 percent of buffer solution B with the buffer solution A to prepare gradient solution, adding a sample I onto the density gradient, and centrifuging; enriching a chromoplast layer with the weight measurement ratio of 15 percent, diluting the precipitate, and centrifuging to obtain precipitate, and diluting the precipitate with the buffer solution A to obtain a sample II; 3) selecting a centrifuge tube, applying the sterilized bovine serum to the inner wall of the centrifuge tube; diluting the 30 percent of buffer solution B with the buffer solution A to prepare gradient solution, adding the sample II onto the density gradient, and centrifuging; enriching a 7.5 percent of chromoplast layer, diluting, centrifuging, removing the supernate, diluting the precipitate, and centrifuging the precipitate to obtain a sample III; and 4) selecting the sample III, repeating the step 3); and precipitating the 7.5 percent of chromoplast, diluting and centrifuging to obtain the orange flesh chromoplast.
Description
Technical field
The invention belongs to the plant biochemistry technical field, be specifically related to a kind of method of using density gradient centrifugation purification oranges and tangerines pulp chromoplastid.
Background technology
Plastid is the main organoid that exists only in plant and the alga cells.Plastid participates in photosynthesis, preserves various products, and synthetic vegetable cell basic boom and the required key molecule of function.The tunicle bag quilt the same with plastosome, that plastid is made up of a pair of concentric coat.Plastid is also similar with plastosome on half autonomy, has the more synthetic required genetic mechanism of oneself proteins.The ability that plastid has significant differentiation, dedifferentes and break up.Plastid comprises proplastid, chloroplast(id), leukoplast, chromoplastid, amyloplast.Plastid is the plant specific organoid, in biosynthesizing and metabolic function, play an important role, comprising photosynthetic C fix, amino acid, resin acid, starch and secondary metabolite synthetic.Chromoplastid is a kind of special plastid, because it is the chloroplast(id) that comes from fruit maturation or the pollen development.The major function of chromoplastid is the synthetic and storage of carotenoid, makes fruit and pollen present various colors.Chloroplast(id) is to the invariably accompany degraded of thylakoid membrane of chloroplast(id) of the transformation of chromoplastid.Though the biochemical characteristic of chromoplastid carotenoid has sizable difference plastid to have important biomolecule to learn meaning in different plants, we are quite limited to the understanding of plastid.Therefore, in order better to study the oranges and tangerines chromoplastid, top priority is to separate the purification chromoplastid.
Gradient centrifugation is that plastid is purified mainly, effectively, and the method for saving.An ideal ladder liquid will possess: chemical stability is good; High fused degree; Low-permeability; Stability in solvent; Lower optical absorption characteristics; Inexpensive etc.As: sucrose is proper gradient material.Although it high-density (＞the every number of viscosity is bigger 30%W/N) time, comparison shows that comprehensively it can widespread use.But because sucrose when low density (even also like this) when various density also has higher perviousness, need select other gradient materials when separating some, as by Ficoll of pharmacia company exploitation or the like to the organism component of perviousness sensitivity.Pair cell, the macromolecular isopycnic centrifugation of subcellular structure or other biological is separated the requirement of having nothing in common with each other.Being usually used in the isopycnic centrifugation gradient material is shown in Table 1:
Table 1 is usually used in the isopycnic centrifugation gradient material and gathers
| Title material | DNA | RNA | Nucleoprotein | Film | Subcellular organelle | Cell | Virus |
| Sucrose | Be not suitable for | Be not suitable for | Available | Better | Better | Available | Better |
| Poly-glucose | Be not suitable for | Be not suitable for | Be not suitable for | Available | Available | Best | Better |
| Cesium chloride | Best | Better | Best | Be not suitable for | Be not suitable for | Be not suitable for | Better |
| Cellular segregation liquid | Be not suitable for | Be not suitable for | Be not suitable for | Available | Better | Better | Available |
| The sea iodohydrin | Available | Available | Best | Best | Best | Better | Better |
As can be seen from Table 1, different biological sample has than big-difference the requirement of gradient material.
Though, as far back as [1] scientists such as Hadjeb in 1988 was material with the pimento, with cellular segregation liquid (percoll) is gradient liquid, separate and obtain purer chromoplastid, and at Siddique in 2006, M[2] etc. the scholar use the same method of purification, with the chromoplastid that separation obtains, the method for utilization proteomics has obtained 151 plastid albumen.It is very good to illustrate that this method is separated the chromoplastid purity that obtains on pimento.Same, Camara B[5 in 1993] be gradient liquid with sucrose, successfully isolate the chromoplastid of ripe tomato, and in 2010) chromoplastid that Cristina etc. [4] obtains separation, utilize the mass spectrum sequencing technologies to obtain 988 plastid albumen.But the applicant once attempted reference [1], [2] be gradient liquid and reference [4] [5] with sucrose with cellular segregation liquid (percoll) be the gradient liquid oranges and tangerines pulp chromoplastid of purifying, the result can not extract chromoplastid, or the chromoplastid film is damaged.
Summary of the invention
The object of the invention is to overcome the defective of prior art, and proposing a kind of is the method that gradient liquid is used density gradient centrifugation purification oranges and tangerines pulp chromoplastid with extra large iodohydrin.
Technical scheme of the present invention is as follows:
(1) get sweet orange fruit, peeling is also removed seed, and pulp is cut into bulk, and about 80 gram samples of weighing add about 300ml buffer A, squeeze the juice 8-10 second with juice extractor, will obtain the pulp biased sample and draw filter cloth to filter by two-layer rice, obtain filtrate; Filtrate is poured in the centrifuge tube, with whizzer with the centrifugal 5min of 400g, remove sediment after, obtain supernatant liquor; With supernatant liquor with the swing bucket rotor of whizzer with 6000g, centrifugal 10min obtains precipitation; The precipitation of enrichment is added the dilution of 12ml buffer A, obtain stand-by sample I, stand-by;
(2) get the centrifuge tube of 10ml, inwall is smoothened with the bovine serum of sterilizing; Is that to be diluted to weight/volume respectively be 24% for 30% buffer B with buffer A with weightmeasurement ratio, 20%, 15% gradient dilution liquid, volume is respectively 1.5ml, 2.0ml, 2.2ml, 2.0ml are mixed with discontinuous density gradient, gets the stand-by sample 2ml of step (1) and is added to above the density gradient; With whizzer under swing bucket rotor with 6750g, centrifugal 45min; Drawing the first layer is that weightmeasurement ratio is 15% chromoplastid layer, and will precipitate 10 times of dilutions, obtains precipitation with the whizzer angle rotor with the centrifugal 7min of 6000g, and will precipitate with the dilution of 2ml buffer A, obtains stand-by sample II, stand-by;
(3) get the centrifuge tube of 10ml, inwall is smoothened with the bovine serum of sterilizing; With buffer A with weightmeasurement ratio be 30% buffer B to be diluted to weight/volume respectively be 12.5%, 10%, 7.5%, 5% gradient dilution liquid, volume is respectively 1.3ml, 1.7ml, 2.5ml, 2.0ml is mixed with discontinuous density gradient; Getting stand-by sample II 2ml of (2) step is added to above the gradient gently, using the whizzer swing bucket rotor, is 6750g with the centrifugal speed, centrifugal 45min, the enrichment second layer is that weightmeasurement ratio is 7.5% chromoplastid layer, the chromoplastid layer of enrichment is diluted 10 times, with the centrifugal 7min of centrifugal speed 6000g, go supernatant to get and will precipitate and dilute 5 times with whizzer, with 600g, centrifugal 15min obtains stand-by sample III, and is stand-by.
(4) (3) step enrichment being obtained stand-by sample III is sample, repeats (3) step; The weightmeasurement ratio that enrichment is obtained is 7.5% chromoplastid precipitation, dilute 10 times, centrifugal 6000g, and 7min is to remove unnecessary extra large iodohydrin, and precipitation is diluted 10 times, is 2000g again with the centrifugal speed, and centrifugal 10min obtains final chromoplastid;
Wherein: the prescription of buffer A, B is composed as follows:
Buffer A
The 1mM tetrasodium pyrophosphate;
The 50mM hydroxyethyl piperazine ethanesulfonic acid; PH=6.8
The 330mM sorbyl alcohol;
The 2mM ethylenediamine tetraacetic acid (EDTA);
1mM MgCl
2；
2mM MnCl
2；
The 2mM DTT;
Buffer B is as follows according to the prescription of weight/volume meter:
30% (W/V) Schering AG);
The 1mM tetrasodium pyrophosphate;
The 50mM hydroxyethyl piperazine ethanesulfonic acid; PH=6.8;
The 330mM sorbyl alcohol;
The 2mM ethylenediamine tetraacetic acid (EDTA);
1mM MgCl
2；
The 2mM DTT.
Positively effect compared with prior art of the present invention is
1, the chromoplastid of Ti Chuning is typical chromoplastid spherule.
2, this chromoplastid method of purification purity height, refining effect is good.
3, the present invention is different from existing sucrose and cellular segregation liquid method (only being used in the chromoplastid that pimento, tomato etc. contain high enrichment), can the purify chromoplastid of low enrichment of method of the present invention, and density gradient liquid can reuse for the first time, improve efficient greatly, reduced reagent cost.
Description of drawings
Fig. 1: be techniqueflow chart of the present invention.
Fig. 2: be that the present invention separates the chromoplastid of purifying and obtaining, with Olympus microscope Cover-018, observations under 100 times of oily mirrors finds that chromoplastid is the spherule that membrane structure is typically arranged, and diameter is 2-5 μ m.
Fig. 3: be the west hybridization figure that the present invention separates the chromoplastid obtain of purifying.From left to right be respectively: plastid, plastosome, kytoplasm, the traget antibody of vacuole.Rbcl: the big subunit of diphosphoribulose carboxylase is a plastid sign antibody; VDAC1: voltage dependence anion channel antibody is mitochondrial outer membrane sign antibody; UDPase: UDPglucose pyrophosphorylase is the mark albumen of kytoplasm; V-ATPase: vacuole skin the 5th subunit adenosine triphosphatase is a vacuole sign antibody.C: represent red dark orange chromoplastid F: represent red dark orange pulp.
Embodiment
Embodiment 1
(1) (the sweet orange kind is red dark orange to get sweet orange, derive from Guangxi China oranges and tangerines institute) fruit, peeling is also removed seed, pulp is cut into bulk, weigh about 80 the gram samples, add about 300ml buffer A, squeeze the juice 8-10 second with juice extractor, to obtain the pulp biased sample and draw filter cloth (available from Calbiochem company) to filter, obtain filtrate by two-layer rice; Filtrate is poured in the centrifuge tube, with whizzer with the centrifugal 5min of 400g, remove sediment after, obtain supernatant liquor; With supernatant liquor with the swing bucket rotor of whizzer with 6000g, centrifugal 10min obtains precipitation; The precipitation of enrichment is added the dilution of 12ml buffer A, obtain stand-by sample I, stand-by;
(2) get the centrifuge tube of 10ml, inwall is smoothened with the bovine serum of sterilize (, following same) available from Hangzhou Sijiqing Biological Engineering Material Co., Ltd.; Is that to be diluted to weight/volume respectively be 24% for 30% buffer B with buffer A with weightmeasurement ratio, 20%, 15% gradient dilution liquid, volume is respectively 1.5ml, 2.0ml, 2.2ml, 2.0ml are mixed with discontinuous density gradient, gets the stand-by sample 2ml of step (1) and is added to above the density gradient; With whizzer under swing bucket rotor with 6750g, centrifugal 45min; Drawing the first layer is that weightmeasurement ratio is 15% chromoplastid layer, and will precipitate 10 times of dilutions, obtains precipitation with the whizzer angle rotor with the centrifugal 7min of 6000g, and will precipitate with the dilution of 2ml buffer A, obtains stand-by sample II, stand-by;
(3) get the centrifuge tube of 10ml, inwall is smoothened with the bovine serum of sterilizing; With buffer A with weightmeasurement ratio be 30% buffer B to be diluted to weight/volume respectively be 12.5%, 10%, 7.5%, 5% gradient dilution liquid, volume is respectively 1.3ml, 1.7ml, 2.5ml, 2.0ml is mixed with discontinuous density gradient; Getting stand-by sample II 2ml of (2) step is added to above the gradient gently, using the whizzer swing bucket rotor, is 6750g with the centrifugal speed, centrifugal 45min, the enrichment second layer is that weightmeasurement ratio is 7.5% chromoplastid layer, the chromoplastid layer of enrichment is diluted 10 times, with the centrifugal 7min of centrifugal speed 6000g, go supernatant to get and will precipitate and dilute 5 times with whizzer, with 600g, centrifugal 15min obtains stand-by sample III, and is stand-by.
(4) (3) step enrichment being obtained stand-by sample III is sample, repeats (3) step; The weightmeasurement ratio that enrichment is obtained is 7.5% chromoplastid precipitation, dilute 10 times, centrifugal 6000g, and 7min is to remove unnecessary extra large iodohydrin, and precipitation is diluted 10 times, is 2000g again with the centrifugal speed, and centrifugal 10min obtains final chromoplastid.
Wherein: the prescription of buffer A, B is composed as follows:
Buffer A
The 1mM tetrasodium pyrophosphate;
The 50mM hydroxyethyl piperazine ethanesulfonic acid; PH=6.8
The 330mM sorbyl alcohol;
The 2mM ethylenediamine tetraacetic acid (EDTA);
1mM MgCl
2；
2mM MnCl
2；
The 2mM DTT;
Buffer B is as follows according to the prescription of weight/volume meter:
30% (W/V) Schering AG);
The 1mM tetrasodium pyrophosphate;
The 50mM hydroxyethyl piperazine ethanesulfonic acid; PH=6.8;
The 330mM sorbyl alcohol;
The 2mM ethylenediamine tetraacetic acid (EDTA);
1mM MgCl
2；
The 2mM DTT.
To separate the chromoplastid of purifying and obtaining, with Olympus microscope Cover-018, observe under 100 times of oily mirrors, find that chromoplastid is the spherule that membrane structure is typically arranged, diameter is a 2-5 μ m (see figure 2).
Extract the albumen 30 μ g of chromoplastid and red dark orange (red dark orange is available from Guangxi oranges and tangerines institute) pulp respectively, adopt western[4] detect chromoplastid purity.The sign antibody that used four the sign antibody of Western are plastid, plastosome, kytoplasm and vacuole and as follows with the dilution step of other reagent:
(1) plastid sign antibody: the big subunit of diphosphoribulose carboxylase (Rbcl), dilution by volume: 1: 5000;
(2) mitochondrial outer membrane sign antibody: voltage dependence anion channel antibody (VDAC1), dilution by volume: 1: 2000;
(3) kytoplasm sign antibody: albumen UDPglucose pyrophosphorylase (UDPase), dilution by volume: 1: 2000;
(4) vacuole sign antibody: vacuole skin the 5th subunit adenosine triphosphatase (V-ATPase), dilution by volume: 1: 2000.
More than the described antibody of (1) (2) (3) (4) step all available from Agrisera
Company.
The result shows: plastid albumen is all arranged in pulp and the chromoplastid, but the plastid albumen of chromoplastid is obviously than high in the pulp.Do not observe plastochondria in the chromoplastid of purifying, the band of kytoplasm and vacuole illustrates that chromoplastid that we purify is not subjected to the pollution (see figure 3) of above several ubcellular organs.
Reference:
(1)Hadjeb，N.，Gounaris，I.and?Price，C.A.(1988)Chromoplast-specific?proteins?in?Capsicum?annuum.Plant?Physiol.88：42-45
(2)Siddique，M.A.，Grossmann，J.，Gruissem，W.，Baginsky，S.，Proteome?analysis?of?bell?pepper(Capsicum?annuum?L.)chromoplasts.Plant?Cell?Physiol.2006，47，1663-1673
(3)von?Zychlinski，A.，Kleffmann，T.，Krishnamurthy，N.，Sjolander，K.et?al.，Proteome?analysis?of?the?rice?etioplast?Metabolic?and?regulatory?networks?and?novel?protein?functions.Mol.Cell.Proteomics?2005，4，1072-1084.
(4)Cristina?Barsan?et?al.，Characteristics?of?the?tomato?chromoplast?revealed?by?proteomic?analysis.Jouranl?of?experimental?Botany?2010，1-19
(5)Camara?B.(1993)Plant?Phytoene?Synthase?Complex：component?enzymes，immunology，and?biogenesis.Methods?in?Enzymology，214，352-365.
Claims (1)
1. the method for oranges and tangerines pulp chromoplastid of purifying is characterized in that, the solution that is made into extra large iodohydrin is gradient liquid, carries out density gradient centrifugation three times, the chromoplastid that obtains purifying;
Its step comprises:
(1) get sweet orange fruit, peeling is also removed seed, and pulp is cut into bulk, and the 80 gram samples of weighing add the 300ml buffer A, squeeze the juice 8-10 second with juice extractor, will obtain the pulp biased sample and draw filter cloth to filter by two-layer rice, obtain filtrate; Filtrate is poured in the centrifuge tube, with whizzer with the centrifugal 5min of 400g, remove sediment after, obtain supernatant liquor; With supernatant liquor with the swing bucket rotor of whizzer with 6000g, centrifugal 10min obtains precipitation; The precipitation of enrichment is added the dilution of 12ml buffer A, obtain stand-by sample I, stand-by;
(2) get the centrifuge tube of 10ml, inwall is smoothened with the bovine serum of sterilizing; Is that to be diluted to weight/volume respectively be 24% for 30% buffer B with buffer A with weightmeasurement ratio, 20%, 15% gradient dilution liquid, volume is respectively 1.5ml, 2.0ml, 2.2ml, 2.0ml are mixed with discontinuous density gradient, gets the stand-by sample 2ml of step (1) and is added to above the density gradient; With whizzer under swing bucket rotor with 6750g, centrifugal 45min; Drawing the first layer is that weightmeasurement ratio is 15% chromoplastid layer, and will precipitate 10 times of dilutions, obtains precipitation with the whizzer angle rotor with the centrifugal 7min of 6000g, and will precipitate with the dilution of 2ml buffer A, obtains stand-by sample II, stand-by;
(3) get the centrifuge tube of 10ml, inwall is smoothened with the bovine serum of sterilizing; With buffer A with weightmeasurement ratio be 30% buffer B to be diluted to weight/volume respectively be 12.5%, 10%, 7.5%, 5% gradient dilution liquid, volume is respectively 1.3ml, 1.7ml, 2.5ml, 2.0ml is mixed with discontinuous density gradient; Getting stand-by sample II 2ml of (2) step is added to above the gradient gently, using the whizzer swing bucket rotor, is 6750g with the centrifugal speed, centrifugal 45min, the enrichment second layer is that weightmeasurement ratio is 7.5% chromoplastid layer, the chromoplastid layer of enrichment is diluted 10 times, with the centrifugal 7min of centrifugal speed 6000g, go supernatant to get and will precipitate and dilute 5 times with whizzer, with 600g, centrifugal 15min obtains stand-by sample III, and is stand-by;
(4) (3) step enrichment being obtained stand-by sample III is sample, repeats (3) step; The weightmeasurement ratio that enrichment is obtained is 7.5% chromoplastid precipitation, dilute 10 times, centrifugal 6000g, and 7min is to remove unnecessary extra large iodohydrin, and precipitation is diluted 10 times, is 2000g again with the centrifugal speed, and centrifugal 10min obtains final chromoplastid;
Wherein: the prescription of buffer A, B is composed as follows:
Buffer A
The 1mM tetrasodium pyrophosphate;
The 50mM hydroxyethyl piperazine ethanesulfonic acid; PH=6.8;
The 330mM sorbyl alcohol;
The 2mM ethylenediamine tetraacetic acid (EDTA);
1mM MgCl
2；
2mM MnCl
2；
The 2mM DTT;
Buffer B is as follows according to the prescription of weight/volume meter:
30% (W/V) Schering AG);
The 1mM tetrasodium pyrophosphate;
The 50mM hydroxyethyl piperazine ethanesulfonic acid; PH=6.8;
The 330mM sorbyl alcohol;
The 2mM ethylenediamine tetraacetic acid (EDTA);
1mM MgCl
2；
The 2mM DTT.
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|---|---|---|---|
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|---|---|---|---|
| CN 201010260376 CN101948796B (en) | 2010-08-20 | 2010-08-20 | Method for extracting orange flesh chromoplast |
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| CN101948796B CN101948796B (en) | 2012-12-19 |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103115810A (en) * | 2013-02-25 | 2013-05-22 | 中国科学院植物研究所 | Method for separating apoplast juice from plant stem tissues |
| CN106399222A (en) * | 2016-09-20 | 2017-02-15 | 华中农业大学 | Method for extracting plastoglobulus in chromoplast from citrus pulp |
| CN108728396A (en) * | 2018-06-12 | 2018-11-02 | 华中农业大学 | A kind of isolation and purification method of citrus pulp mitochondria |
| CN110218694A (en) * | 2019-05-30 | 2019-09-10 | 华中农业大学 | A kind of kiwifruit fruit amyloplaste extracting method |
-
2010
- 2010-08-20 CN CN 201010260376 patent/CN101948796B/en not_active Expired - Fee Related
Non-Patent Citations (1)
| Title |
|---|
| 《Molecular & Cellular Proteomics》 20050518 Anne von Zychlinski等 Proteome Analysis of the Rice Etioplast 第1072-1084页 1 第4卷, * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103115810A (en) * | 2013-02-25 | 2013-05-22 | 中国科学院植物研究所 | Method for separating apoplast juice from plant stem tissues |
| CN103115810B (en) * | 2013-02-25 | 2015-01-14 | 中国科学院植物研究所 | Method for separating apoplast juice from plant stem tissues |
| CN106399222A (en) * | 2016-09-20 | 2017-02-15 | 华中农业大学 | Method for extracting plastoglobulus in chromoplast from citrus pulp |
| CN106399222B (en) * | 2016-09-20 | 2019-07-30 | 华中农业大学 | A method of for extracting the plastoglobulus in citrus pulp chromoplast |
| CN108728396A (en) * | 2018-06-12 | 2018-11-02 | 华中农业大学 | A kind of isolation and purification method of citrus pulp mitochondria |
| CN108728396B (en) * | 2018-06-12 | 2020-06-30 | 华中农业大学 | Method for separating and purifying citrus pulp mitochondria |
| CN110218694A (en) * | 2019-05-30 | 2019-09-10 | 华中农业大学 | A kind of kiwifruit fruit amyloplaste extracting method |
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