Method for synchronously separating chloroplast and mitochondria of high purity plant

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 ...

AI Technical Summary

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 operation, difficult to master

Although commercial kits (such as the Minute developed by Invent biotechnologies TM Chloroplast Isolation Kit, Chloroplast Isolation Kit developed by SIGMA Company and Mitochondria Isolation Kit for Cultured Cells developed by ThermoFisher Company) are easy to operate and reliable in separation effect, but they are expensive, the number of times of use is limited and they cannot be used in large quantities, and the two organelles cannot be separated at the same time

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