Method for separating and purifying citrus pulp mitochondria

A technology for separation and purification of mitochondria, applied in the field of plant biochemistry, can solve the problems of inability to effectively extract mitochondria, difficulty in separation and purification of mitochondria, damage to mitochondrial membrane, etc., and achieve the effect of complete double-layer membrane structure, good purification effect and high purity.

Active Publication Date: 2018-11-02
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, for citrus pulp juice cell tissue, because it contains a large amount of organic acids, pectin, polysaccharides, phenols and pigments, these all bring difficulties to the separation and purification of mitochondria
The separation and purification of citrus pulp mitochondria by the prior art methods cannot effectively extract the mitochondria, or the mitochondrial membrane has been damaged

Method used

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  • Method for separating and purifying citrus pulp mitochondria
  • Method for separating and purifying citrus pulp mitochondria
  • Method for separating and purifying citrus pulp mitochondria

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preparation example Construction

[0047] After the filtrate is obtained, the present invention performs differential centrifugation, and the precipitate obtained after the differential centrifugation is suspended with buffer B to obtain sample I; the buffer B is an aqueous solution comprising the following components: 0.3-0.5M sorbic acid Alcohol and 20-50mM 3-(N-marin generation)propanesulfonic acid. The aqueous solution of buffer B in the present invention includes sorbitol, and the molar concentration of said sorbitol is preferably 0.31-0.4M, more preferably 0.32-0.35M, most preferably 0.33M. The aqueous solution of the buffer solution B of the present invention includes 3-(N-marinyl)propanesulfonic acid, the molar concentration of the 3-(N-marinyl)propanesulfonic acid is preferably 30-58mM, more preferably 45mM -52 mM, most preferably 50 mM. The present invention has no special limitation on the preparation method and raw material source of the buffer B. When the buffer B of the present invention is used...

Embodiment 1

[0064] (1) Preparation the day before the extraction: prepare buffers A, B, C and gradient dilutions according to Table 2-5 respectively, and store them in a refrigerator at 4°C after preparation; assemble the juicer and prepare the simple density gradient device (such as figure 2 shown); prepare a chopping board, knife, gauze, funnel, 500mL plastic measuring cup, and plastic foam box;

[0065] (2) Preparations on the day of extraction: Add dithiothreitol to the required concentration in buffer A, mix thoroughly to dissolve it completely; pre-cool the required laboratory utensils on ice, turn on the centrifuge and Pre-cool after installing the required rotor, all the following experimental operations are performed on ice unless otherwise specified;

[0066] (3) Take two 13mL centrifuge tubes, each tube is sequentially added with buffer C diluted Percoll volume fractions of 35%, 22.5% and 18% gradient dilution solution, the volumes were 3mL, 6mL and 2mL, prepared as Discontinu...

Embodiment 2

[0089] Get Wenzhou Satsuma fruit, and all other steps and the solution used are the same as in Example 1.

[0090] Utilize the method of the present invention, the mitochondrion of the extracted Satsuma citrus is stained with the mitochondria-specific vital dye Jianna Green B at room temperature for 20 minutes, and observed under a 100-fold oil lens with an Olympus microscope DP70, and it is found that evenly distributed blue-green mitochondria, such as Figure 4 shown;

[0091] The purified mitochondria were stained with MitoTracker Red, a mitochondria-specific fluorescent dye, at room temperature in the dark for 20 minutes, and observed with a Leica laser scanning confocal microscope SP8 at an excitation wavelength of 552nm and an emission wavelength of 560-630 nm, and mitochondria emitting red fluorescence were found ,Such as Figure 5 shown;

[0092] Observing the purified mitochondria under a transmission electron microscope, it can be seen that the purified mitochondr...

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Abstract

The invention provides a method for separating and purifying citrus pulp mitochondria, and relates to the technical field of plant biochemistry. Percoll is used as a gradient medium, and the citrus pulp mitochondria are separated and purified by means of differential centrifugation and density gradient differentiation by the aid of the method. The method includes steps of 1), mixing citrus pulp and buffer solution A with each other, then squeezing juice and filtering the juice to obtain filtered fluid; 2), carrying out differential centrifugation on the obtained filtered fluid, and suspendingobtained precipitates by buffer solution B to obtain samples I; 3), carrying out density gradient centrifugation on the obtained samples I to obtain samples II; 4), mixing the obtained samples II andbuffer solution B with one another and centrifugally collecting precipitates to obtain samples III; 5), mixing the obtained samples III and buffer solution B with one another and centrifugally collecting precipitates to obtain the citrus pulp mitochondria. The method has the advantages that the citrus pulp mitochondria obtained by the aid of the method are in the typical shapes of ellipsoids, double-layer membrane structures are complete, and cristae are clearly visible; the citrus pulp mitochondria purified by the aid of the method are high in purity, and good purification effects can be realized by the aid of the method.

Description

technical field [0001] The invention belongs to the technical field of plant biochemistry, and in particular relates to a method for separating and purifying citrus pulp mitochondria. Background technique [0002] Mitochondria, as an important organelle, is the power factory of cell metabolism, in which electron transfer, tricarboxylic acid cycle and oxidative phosphorylation are continuously carried out, and adenosine triphosphate (ATP) and metabolic intermediates are provided for other metabolisms, so it is related to the respiration of fruits The effect is closely related to energy metabolism. Citric acid is an important product of the tricarboxylic acid cycle and the most important organic acid in citrus fruits. Its content directly determines the flavor quality of the fruit. Mitochondrial electron transport chain complexes Ⅰ and Ⅲ are the main sites of reactive oxygen species (ROS) generation, which in turn affect the postharvest physiology and senescence process of fr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/04
CPCC12N5/04
Inventor 程运江李欣柴应芳许让伟王鹏蔚田真曾云流邓秀新
Owner HUAZHONG AGRI UNIV
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