Method for synchronously separating chloroplast and mitochondria of high purity plant

A synchronous separation and chloroplast technology, applied in the field of cell biology and molecular biology, can solve the problems of long centrifugation time, high price, and medium osmotic pressure sensitivity, and achieve the effect of ensuring activity, simple operation and simple consumables.

Active Publication Date: 2017-07-14
INST OF OIL CROPS RES CHINESE ACAD OF AGRI SCI
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Problems solved by technology

Density gradient centrifugation method has some disadvantages: 1) mediums such as serum albumin, polysucrose, cesium chloride and Percoll are expensive; In particular, mitochondria are very sensitive to the osmotic pressure of the medium, and high concentrations will lead to its deformation or rupture; 3) The preparation of the inert gradient medium solution is cumbersome and time-consuming; 4) The prepared separation solution cannot be reused; 5) The centrifugation time is long; 6) Strict op

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  • Method for synchronously separating chloroplast and mitochondria of high purity plant
  • Method for synchronously separating chloroplast and mitochondria of high purity plant
  • Method for synchronously separating chloroplast and mitochondria of high purity plant

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Embodiment 1

[0026] The embodiment of the present invention takes rapeseed leaf tissue as an example, and this method is also applicable to the simultaneous separation of chloroplasts and mitochondria of Arabidopsis, tobacco, rice, wheat and other plants.

[0027] A method for synchronously separating high-purity plant chloroplasts and mitochondria, the steps of which are:

[0028] 1. Leaf tissue processing and grinding

[0029] During the physiological growth period of plants, the young leaf tissue of rapeseed was selected as the material for separating and purifying chloroplasts and mitochondria. After cutting the leaf tissue, put it on ice for temporary storage, rinse it several times with clean water, and clean the soil and other impurities on the leaf. Afterwards, use absorbent paper to absorb the remaining water drops on the leaves, and then use sterilized surgical scissors to cut the non-vein parts into pieces, weigh 5g and place them in a sterilized mortar, and add pre-cooled CIB ...

Embodiment 2

[0034] Example 2: Observation of chloroplasts and mitochondria

[0035] Separate 100 μL of the chloroplast and mitochondria isolated in Example 1, add 10 μL RedCMXRos was incubated at room temperature in the dark for 10 minutes, then washed twice with washing buffer, and prepared for observation under a confocal laser microscope. Observation results show that the rape leaf chloroplast purity that uses the separation of the present invention is higher ( Figure 4 A), with very little mitochondrial contamination ( Figure 4 A-3 and Figure 4 A-4), and the chloroplasts are complete and active ( Figure 4 A-1, Figure 4 A-2 and Figure 4 A-4); the mitochondria isolated synchronously have extremely high purity, no chloroplast pollution, and are active ( Figure 4 B-3 and Figure 4 B-4).

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Abstract

The present invention discloses a method for synchronously separating chloroplast and mitochondria of a high purity plant. The method comprises: A, adding leaf to a CIB buffer liquid, grinding, filtering sequentially with a 70 [mu]m filtration screen, a 35 [mu]m filtration screen and a 15 [mu]m filtration screen, and carrying out centrifugation on the filtrate for 5 min at 200*g to remove tissue residue and cell debris; B, carrying out centrifugation on the filtrate for 10 min at 1000*g, and collecting chloroplast-rich precipitate; C, collecting the supernatant after the centrifugation in the step B, filtering with a 5 [mu] filtration screen, carrying out centrifugation on the filtrate for 10 min at 2000*g, removing the precipitate, carrying out centrifugation on the supernatant for 10 min at 17000*g, and collecting mitochondria-rich precipitate; D, washing the precipitates respectively with a washing buffer liquid, and digesting with DNase I to remove background genome contamination; and E, carrying out centrifugation on the chloroplast precipitate for 10 min at 1000*g, removing the supernatant, carrying out centrifugation on the mitochondrial precipitation for 10 min at 17000*g, removing the supernatant, and washing the precipitates with a washing buffer liquid.

Description

technical field [0001] The invention belongs to the technical fields of cell biology and molecular biology, and in particular relates to a method for synchronously separating high-purity plant chloroplasts and mitochondria. Background technique [0002] The study of chloroplast and mitochondria plays a pivotal role in the fields of phylogenetic evolution, species identification, nucleocytoplasmic interaction, and genetic engineering. Isolation of high-purity chloroplast and mitochondrial organelles is the key to carry out related research. Density gradient centrifugation is currently the most commonly used method for separating these two organelles. It uses a certain medium to form a continuous or discontinuous density gradient in a centrifuge tube, puts the cell homogenate or suspension on the top of the medium, and passes through the center of gravity or centrifugation. The action of the field force stratifies and separates the cells. The density gradient centrifugation ...

Claims

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Application Information

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IPC IPC(8): C12N5/04
Inventor 华玮李俊范世航刘红芳
Owner INST OF OIL CROPS RES CHINESE ACAD OF AGRI SCI
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