CN106399222B - A method of for extracting the plastoglobulus in citrus pulp chromoplast - Google Patents
A method of for extracting the plastoglobulus in citrus pulp chromoplast Download PDFInfo
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- CN106399222B CN106399222B CN201610845699.0A CN201610845699A CN106399222B CN 106399222 B CN106399222 B CN 106399222B CN 201610845699 A CN201610845699 A CN 201610845699A CN 106399222 B CN106399222 B CN 106399222B
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Abstract
The present invention relates to a kind of methods for extracting the plastoglobulus in citrus pulp chromoplast comprising following steps: S1: extracting chromoplast from citrus pulp；S2: chromoplast described in ultrasonication obtains chromoplast lysate；S3: the chromoplast lysate is centrifuged in 150000g Gradient using the serial solution containing Iohexol concentration gradient, the supernatant liquid after layering is plastoglobulus.Chromoplast plastoglobulus can effectively be extracted from citrus pulp by using method of the invention, layered effect is obvious, and the purity is high of obtained plastoglobulus, refining effect is good, and maintains integrality of the protein in plastoglobulus in type and structure.
Description
Technical field
The present invention relates to plant biochemistry fields, more specifically it relates to a kind of matter for extracting in citrus pulp chromoplast
The method of body bead.
Background technique
Plastid is distinctive organelle in plant, including chloroplaset, leucoplast and chromoplast.Plastoglobulus is to be widely present
Hdl particle (the citrus pulp electromicroscopic photograph of such as Fig. 4, black region is plastoglobulus) in plastid.In chloroplaset,
Plastoglobulus is connected in structure with thylakoid outer membrane, and thylakoid generates plastoglobulus in the form blistered.Plastoglobulus conduct
Micro- domain on thylakoid plays a significant role on participating in thylakoid synthesis, reparation, degradation and metabolism.In citrus fruit
There is also a large amount of plastoglobulus in meat chromoplast (a kind of form of plastid), however its function is not known.Plastoglobulus
It is mainly made of monofilm, the lipid material wrapped up by the film and about 30 kinds of protein being embedded on the film, wherein lipid
Matter includes plastoquinone, chlorophyll quinone, tocopherol, triacylglycerol, carotenoid etc..The protein being embedded on film can be divided into three
Major class: (1) the highest fibrillin of abundance (FBNs)；(2) enzyme for participating in lipid metabolism object, for example, ABC1K kinases and portion
Divide the enzyme for participating in carotenoid and vitamin E metabolic；(3) other protein.
The protein inlayed on the film of plastoglobulus is related with its function, and changing protein on plastoglobulus can cause to be metabolized
The variation of object.Function in arabidopsis about the albumen composition of chloroplaset plastoglobulus and part-structure gene has obtained tentatively
Verifying, but the origin for the plastoglobulus in non-green tissue, development, protein composition and regulatory mechanism are not known.
In order to study function of the plastoglobulus in the non-green tissue such as citrus pulp, it is necessary first to isolated plastoglobulus.China
Patent 201010260376.8 discloses a kind of method of extracting orange flesh chromoplast, however still not specifically for citrus fruit
The extracting method of the plastoglobulus of meat chromoplast.
The density gradient centrifugation that exceeds the speed limit is most common plastoglobulus method of purification.Ideal ladder liquid will have excellent chemistry
Stability, high fused degree, low-permeability, this material of the characteristics such as stability and lower light absorption is best in good solvent
Or it is cheap.For example, sucrose is exactly a kind of proper functionally gradient material (FGM).Although it is sticky at high density (> 30%W/N)
Coefficient is larger, but is comprehensively compared and shows that it can be widely applied.But since sucrose all has higher infiltration under various density
Permeability (even also such when low-density) therefore when separating the biological components of some permeability sensitivities, needs to select other ladders
Spend material, such as the Ficoll developed by pharmacia company.To cell, subcellular structure or other biological macromolecular
Isopycnic centrifugation separates requirement of also having nothing in common with each other.It is usually used in isopycnic centrifugation functionally gradient material (FGM) to be shown in Table 1:
The application of 1 isopycnic centrifugation gradient of table
| Title material | DNA | RNA | Nucleoprotein | Film | Subcellular organelle | Cell | Virus |
| Sucrose | Be not suitable for | Be not suitable for | It can use | Preferably | Preferably | It can use | Preferably |
| Poly- glucose | Be not suitable for | Be not suitable for | Be not suitable for | It can use | It can use | Most preferably | Preferably |
| Cesium chloride | Most preferably | Preferably | Most preferably | Be not suitable for | Be not suitable for | Be not suitable for | Preferably |
| Cell separating liquid | Be not suitable for | Be not suitable for | Be not suitable for | It can use | Preferably | Preferably | It can use |
| Extra large iodohydrin | It can use | It can use | Most preferably | Most preferably | Most preferably | Preferably | Preferably |
As it can be seen from table 1 requirement of the different biological samples to functionally gradient material (FGM) differs greatly.
Ultracentrifugation is carried out using cell separating liquid (Percoll) in arabidopsis within Ytterberg etc. 2006, separate
To plastoglobulus, and pass through Mass Spectrometric Identification 33 kinds of protein therein.Peter K.Lundquist in 2012 etc. is basic herein
On advanced optimize scheme and remove the not high albumen of part confidence level using new technology, while three kinds of albumen of Authenticate afresh
Matter may be located on plastoglobulus, so that the protein on plastoglobulus is determined as 30 kinds.It is separated from capsicum chromoplast
Have that 27 kinds of protein are identified on obtained plastoglobulus, wherein have 12 it is consistent with arabidopsis plastoglobulus albumen
(Ytterberg etc. 2006).Above description of test this method is obvious to separating plastid bead effect.
However, the citrus fruit meat plastoglobulus of non-green tissue exists obviously on source and function with arabidopsis and capsicum
Specificity.After attempting the above method, plastoglobulus can not be layered or layered effect is unobvious, therefore, the above method
It is unsuitable for the purification & isolation of citrus fruit meat plastoglobulus.
Summary of the invention
In order to solve the above technical problems, the present invention provides a kind of plastoglobulus for extracting in citrus pulp chromoplast
Method comprising following steps:
S1: chromoplast is extracted from citrus pulp；
S2: chromoplast described in ultrasonication obtains chromoplast lysate；
S3: using the serial solution containing Iohexol concentration gradient to the chromoplast lysate in 150000g Gradient
Centrifugation, the supernatant liquid after layering is plastoglobulus.
Further, the serial solution containing Iohexol concentration gradient is successively respectively by Iohexol mass volume ratio
20%, 10%, 7.5% and 5% Iohexol solution composition.
Preferably, further comprised the steps of: before ultrasonication to S1 obtain in the chromoplast add protease inhibitors and
Inhibitors of phosphatases.
Preferably, further comprised the steps of: before ultrasonication the chromoplast is quick-frozen, maintain curdled appearance for a period of time
Afterwards, melt on ice.
Preferably, centrifugation inside pipe wall used in gradient centrifugation is smoothened with sterile cow's serum in advance in S3.
Preferably, it the described method comprises the following steps:
S1: extracting chromoplast from 1600 grams of citrus pulp, and the chromoplast is centrifuged 7min under 6000g and is sunk
It forms sediment；
S2: chromoplast described in ultrasonication obtains chromoplast lysate, comprising the following steps:
S2.1: the precipitating is resuspended with 4ml buffer solution B, and adds 100 μ l protease inhibitors and 100 μ l phosphorus thereto
Sour enzyme inhibitor obtains chromoplast mixed liquor；
S2.2: by the chromoplast mixed liquor liquid nitrogen flash freezer, and 1.5h is placed at -80 DEG C, then melted on ice；
S2.3: the chromoplast mixed liquor of freeze thawing treatment is broken with Ultrasonic Cell Disruptor under ice bath state, obtains coloured
Body lysate；
S3: the chromoplast lysate is centrifuged in 150000g Gradient using the solution containing Iohexol, including following
Step:
S3.1: taking 13.2ml centrifuge tube, is uniformly applied on the centrifugation inside pipe wall with sterile cow's serum；
S3.2: using buffer solution A dilution buffer B, obtain Iohexol content be respectively mass volume ratio 20%, 10%,
7.5% and 5% Iohexol solution sequentially adds chromoplast lysate described in 4ml in Xiang Suoshu centrifuge tube and the centrifugation is added
Pipe, the Iohexol solution of 2ml 20%, the Iohexol solution of 1ml 10%, the Iohexol solution of 2ml 7.5% and 3ml 5%
Iohexol solution；
S3.3: by added with the centrifuge tube of the chromoplast lysate and Iohexol density gradient solution under 4 DEG C of 150000g
It is centrifuged 15h, after liquid layered, top layer's liquid is plastoglobulus.
Wherein, the formula of the buffer solution A is as follows:
The formula of the buffer solution B is as follows:
Chromoplast plastoglobulus can effectively be extracted from citrus pulp by using method of the invention, layered effect is bright
Aobvious, the purity is high of obtained plastoglobulus, refining effect is good, and maintains the protein in plastoglobulus in type and knot
Integrality on structure.
Detailed description of the invention
Fig. 1 is the flow chart of method of the invention；
Fig. 2 is the chromoplast photo under light microscopic；
Fig. 3 is the protein immunoblot photo of the plastoglobulus extracted using method of the invention；
Fig. 4 is the electron micrograph of citrus pulp.
Specific embodiment
Principles and features of the present invention are described below in conjunction with example and attached drawing, example is served only for explaining this hair
It is bright, it is not intended to limit the scope of the present invention.
As shown in Figure 1, extracting plastoglobulus by following steps:
S1: chromoplast (form of the chromoplast under light microscopic is as shown in Figure 2) is extracted from 1600 grams of citrus pulp, and by institute
It states chromoplast and is centrifuged 7min under 6000g and precipitated；
S2.1: being resuspended the precipitating with 4ml buffer solution B, and add thereto 100 μ l protease inhibitors (Roche,
REF.05892791001) and 100 μ l inhibitors of phosphatases (Roche, REF.04906837001), chromoplast mixed liquor is obtained；
S2.2: by the chromoplast mixed liquor liquid nitrogen flash freezer, and 1.5h is placed at -80 DEG C, then melted on ice；
S2.3: the chromoplast mixed liquor of freeze thawing treatment is broken with Ultrasonic Cell Disruptor under ice bath state, 30% function
Rate, 5 seconds every time, ultrasound 6 times totally 30 seconds, obtained chromoplast lysate；
S3.1: taking 13.2ml centrifuge tube, is uniformly applied on the centrifugation inside pipe wall with sterile cow's serum；
S3.2: using buffer solution A dilution buffer B, obtain Iohexol content be respectively mass volume ratio 20%, 10%,
7.5% and 5% Iohexol solution sequentially adds chromoplast lysate described in 4ml in Xiang Suoshu centrifuge tube and the centrifugation is added
Pipe, the Iohexol solution of 2ml 20%, the Iohexol solution of 1ml 10%, the Iohexol solution of 2ml 7.5% and 3ml 5%
Iohexol solution；
S3.3:, will be close added with the chromoplast lysate and Iohexol using rotary head BECKMAN SW41Ti after trim
The centrifuge tube of degree gradient solution is centrifuged 15h under 4 DEG C of 150000g, and after liquid layered, top layer's liquid is plastoglobulus.
Wherein, the formula of the buffer solution A is as follows:
The formula of the buffer solution B is as follows:
Use the special of the specific antibody PGL35 of plastoglobulus, the specific antibody Toc75 of plastid film and plastid matrix
The purity for the plastoglobulus that property antibody RuBL is identified by the method for protein immunoblot, obtained result such as Fig. 3 institute
Show, the plastoglobulus purity is high extracted by means of the present invention, impurity is few.
The foregoing is merely presently preferred embodiments of the present invention, is not intended to limit the invention, it is all in spirit of the invention and
Within principle, any modification, equivalent replacement, improvement and so on be should all be included in the protection scope of the present invention.
Claims (1)
1. a kind of method for extracting the plastoglobulus in citrus pulp chromoplast, which comprises the following steps:
S1: extracting chromoplast from 1600 grams of citrus pulp, and the chromoplast is centrifuged 7min under 6000g and is precipitated；
S2: chromoplast described in ultrasonication obtains chromoplast lysate, comprising the following steps:
S2.1: the precipitating is resuspended with 4ml buffer solution B, and adds 100 μ l protease inhibitors and 100 μ l phosphatases thereto
Inhibitor obtains chromoplast mixed liquor；
S2.2: by the chromoplast mixed liquor liquid nitrogen flash freezer, and 1.5h is placed at -80 DEG C, then melted on ice；
S2.3: the chromoplast mixed liquor of freeze thawing treatment is broken with Ultrasonic Cell Disruptor under ice bath state, obtains chromoplast and splits
Solve liquid；
S3: being centrifuged the chromoplast lysate in 150000g Gradient using the serial solution containing Iohexol concentration gradient,
Supernatant liquid after layering is plastoglobulus, comprising the following steps:
S3.1: taking 13.2ml centrifuge tube, is uniformly applied on the centrifugation inside pipe wall with sterile cow's serum；
S3.2: buffer solution A dilution buffer B is used, obtaining Iohexol content is respectively 20%, 10%, 7.5% and of mass volume ratio
It is molten to sequentially add chromoplast lysate, the Iohexol of 2ml20% described in 4ml in Xiang Suoshu centrifuge tube for 5% Iohexol solution
Liquid, the Iohexol solution of 1ml10%, the Iohexol solution of 2ml7.5% and 3ml5% Iohexol solution；
S3.3: it will be centrifuged under 4 DEG C of 150000g added with the centrifuge tube of the chromoplast lysate and Iohexol density gradient solution
15h, after liquid layered, top layer's liquid is plastoglobulus；
Wherein, the formula of the buffer solution A is as follows:
The formula of the buffer solution B is as follows:
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| CN201610845699.0A CN106399222B (en) | 2016-09-20 | 2016-09-20 | A method of for extracting the plastoglobulus in citrus pulp chromoplast |
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| CN115725615B (en) * | 2022-08-17 | 2023-06-06 | 甘肃农业大学 | Upland cotton GhABC1K14-A09 gene and application thereof in drought resistance and salt tolerance of upland cotton |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101948796A (en) * | 2010-08-20 | 2011-01-19 | 华中农业大学 | Method for extracting orange flesh chromoplast |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101948796A (en) * | 2010-08-20 | 2011-01-19 | 华中农业大学 | Method for extracting orange flesh chromoplast |
Non-Patent Citations (4)
| Title |
|---|
| Iodixanol梯度离心法分离脂蛋白;党美林等;《第七届海峡两岸心血管研讨会》;20091231;54 |
| Plant organelle proteomics;Kathryn S Lilley et al.;《Current Opinion in Plant Biology》;20071231;594–599 |
| Protein Profiling of Plastoglobules in Chloroplasts and Chromoplasts. A Surprising Site for Differential Accumulation of Metabolic Enzymes;A. Jimmy Ytterberg et al.;《Plant Physiology》;20060331;984–997 |
| 柑橘果实有色体结构及其分化机理研究;曾云流;《中国博士学位论文全文数据库 农业科技辑》;20160315;全文 |
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