Method for Simultaneously Separating and Extracting Maize Nuclei and Chloroplasts

A technology of simultaneous separation and nucleus, applied in the field of plant biochemistry, can solve the problem of not being able to obtain high-purity active nucleus and chloroplast at the same time, and achieve the effect of low permeability, high density, and non-toxic density

Active Publication Date: 2021-01-15
HENAN AGRICULTURAL UNIVERSITY
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Problems solved by technology

[0007] The technical problem to be solved by the present invention is to provide a method for simultaneously separating and extracting corn nuclei and chloroplasts to solve the current technical problem of being unable to simultaneously obtain a large number of high-purity nuclei and chloroplasts that can maintain activity

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  • Method for Simultaneously Separating and Extracting Maize Nuclei and Chloroplasts
  • Method for Simultaneously Separating and Extracting Maize Nuclei and Chloroplasts

Examples

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Embodiment 1

[0037] Simultaneously separate and extract the method for corn leaf nucleus and chloroplast, see figure 1 , including the following steps:

[0038] (1) Take the leaves of corn seedlings that have been hydrocultured to the three-leaf stage under the conditions of 16h light / 8h dark, 28°C / 25°C (light / dark) and relative humidity of 60%, take 4g of the top 4cm of corn seedlings, and put the leaves Roughly cut into small pieces of 5×5 mm, put them into a juicer, add 75mL of crushing buffer, put them in the refrigerator at 4°C, grind and crush at intervals, pause for 6 seconds every 5 seconds, repeat 4 times, and then filter with a 300-mesh sieve Put the homogenate in a 150 mL Erlenmeyer flask, centrifuge at 4°C and 1000g for 5 minutes in a low-temperature centrifuge, discard the supernatant, and the obtained precipitate is a crude extract containing chloroplasts and nuclei;

[0039] (2) Add 0.5 mL of separation buffer to the precipitate obtained in step (1) to obtain a resuspension...

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Abstract

The invention discloses a method for simultaneously separating and extracting cell nucleuses and chloroplasts. The method aims at solving the technical problem that existing separation and extractionmethods cannot separate high-activity cell nucleuses and chloroplasts at the same time. The method comprises the following main steps: (1) taking and cutting plant seedlings or leaves into pieces, added a crushing buffer solution, performing grinding, crushing and filtering to obtain homogenate, performing centrifugal separation, and discarding supernatant to obtain precipitate; (2) adding a separation buffer solution into the precipitate obtained in the previous step, performing uniform mixing to obtain a resuspension, adding the resuspension onto a Percoll density gradient separation solution, and carrying out gradient centrifugal separation; and (3) respectively extracting chloroplast and bottom precipitate at the gradient interface of the separation solution, respectively adding a purification buffer solution for centrifugal purification and separation, and removing supernatant to obtain corresponding chloroplast and cell nucleus. According to the invention, different buffer solutions are used for separating and purifying the cell nucleus and the chloroplast, operation is easy and convenient, the extraction rate of the obtained purified cell nucleus reaches up to 80%, and the extraction rate of the chloroplast reaches up to 90%.

Description

technical field [0001] The invention relates to the technical field of plant biochemistry, in particular to a method for simultaneously separating and extracting corn nuclei and chloroplasts. Background technique [0002] The nucleus contains almost all genetic information and is extremely important for gene expression and regulation. With the development of genome sequencing, DNA fiber fluorescence in situ hybridization and the construction of large fragment genome libraries play an important role in promoting plant genome sequencing. The extracted and purified nuclei are the basis for making DNA fibers and constructing large fragment genome libraries. [0003] And chloroplast is a kind of plastid, which is the unique energy converter of higher plants and some algae; the double-layer membrane structure makes it separate from the cytoplasm, because it contains chlorophyll, so it is called chloroplast, and the research on chloroplast will be useful. It will help us reveal t...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/04
CPCC12N5/04C12N2509/10
Inventor 杨浩苏磊尤晨王亮李雪王志飞
Owner HENAN AGRICULTURAL UNIVERSITY
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