High-purity and high-quality extraction method of chloroplast DNA of strip-leaf plants

An extraction method and plant leaf technology, which is applied in the field of extracting high-purity chloroplast DNA from a large number of small strip-shaped leaf plants, can solve the problem of inability to extract high-purity chloroplast DNA, high-quality, high-concentration chloroplast DNA, and mesophyll The problem of low cells

Active Publication Date: 2017-07-21
唐山润泽粮油食品有限公司
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Problems solved by technology

[0006] On this basis, Shi Fei et al. have made a series of technological improvements and in recent years have invented a method for isolating tea chloroplast DNA (Shi Fei and Li Wei, 2013). However, due to the large and thick leaves of tea trees, it is relatively easy to extract chloroplasts, while millet, Small strip-shaped leaf plants such as wheat have thin leaves, less mesophyll cells, and less chloroplast content. Therefore, the chloroplasts of small strip-shaped leaf plants extracted directly by tea trees and other fruit trees and forest chloroplasts are not only low in concentration, but also heavily polluted. High-quality, high-concentration chloroplast DNA cannot be extracted. Moreover, this method still adopts ...

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  • High-purity and high-quality extraction method of chloroplast DNA of strip-leaf plants
  • High-purity and high-quality extraction method of chloroplast DNA of strip-leaf plants
  • High-purity and high-quality extraction method of chloroplast DNA of strip-leaf plants

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Experimental program
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Effect test

Embodiment 1

[0088] Embodiment 1, the extraction of millet chloroplast DNA.

[0089] 1. Isolation of protoplasts from millet leaves.

[0090] (1) Sow the seeds of the millet material on the seedling tray, and cut 30-40 g of fresh and healthy leaves before heading; cut the leaves into tiny thin strips horizontally and vertically with a medical scalpel, causing more traumatic surfaces, To facilitate the infiltration of the enzymatic solution.

[0091] (2) Put the treated leaves in 100 mL of enzymatic hydrolysis solution, submerge the leaves in the enzymatic hydrolysis solution, and shake for 3 h with a decolorizing shaker at 30 rpm in the dark.

[0092] (3) Dilute the enzymolyzate with 100 mL of buffer 1, filter through a 200-mesh cell sieve, collect the filtrate, and discard the residue; filtrate 4 o C, 200 g, centrifuge for 10 min, collect the precipitate, which is the protoplast ( Picture 1-1 ).

[0093] 2. Removal of millet mitochondria and rough extraction of chloroplasts.

[0094...

Embodiment 2

[0107] Embodiment 2, extraction of wheat chloroplast DNA.

[0108] 1. Isolation of protoplasts from wheat leaves.

[0109] (1) Sow the seeds of wheat materials in a petri dish, cultivate them in clear water, and cut 20-30 g of fresh and healthy leaves; cut the leaves into tiny thin strips horizontally and vertically with a medical scalpel, causing more traumatic surfaces, To facilitate the infiltration of the enzymatic solution.

[0110] (2) Place the treated leaves in 100 mL of enzymatic hydrolysis solution, submerge the leaves in the enzymatic hydrolysis solution, and shake for 3 h with a decolorizing shaker at room temperature, 30 rpm, in the dark.

[0111] (3) Dilute the enzymolyzate with 100 mL of buffer 1, filter through a 200-mesh cell sieve, collect the filtrate, and discard the residue; filtrate 4 o C, 200 g, centrifuge for 10 min, collect the precipitate, which is the protoplast ( diagram 2-1 ).

[0112] 2. Removal of wheat mitochondria and rough extraction of c...

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Abstract

The invention discloses a method for extracting chloroplast DNA of strip-leaf plants. The method comprises the following steps that A, the protoplasts of leaves are acquired by means of making mechanical injuries on the leaves and enzymolysis treatment in combination, wherein the acquired protoplasts contain chloroplasts, mitochondria and nuclei; B, impurities in the acquired products from step A are removed by differential centrifugation treatment to conduct crude extraction of the chloroplasts; C, the impurities in the acquired products from step B are removed by density gradient centrifugation to conduct purification of the chloroplasts; D, extraction is conducted on the acquired products from step C by means of a Tiangen rapid biochemical plant genome DNA extraction system to obtain high-purity chloroplast DNA. With the method for extracting the chloroplast DNA of the strip-leaf plants, high-purity and high-quality extraction can be conducted on the chloroplast DNA of the strip-leaf plants.

Description

technical field [0001] The invention relates to a plant nucleotide extraction technology, in particular to a high-purity extraction method for extracting a large amount of small strip-shaped leaf plant chloroplast DNA. Background technique [0002] Chloroplast is the organelle of green plants for photosynthesis and one of the main organelles with genetic function in the cytoplasm. As a semi-autonomous organelle, its function is the result of the interaction between the chloroplast genome and the nuclear genome (Jia Jingjing et al., 2009). Compared with the nuclear genome, organelle genomes such as chloroplasts have simple structure, small molecular weight, and multiple copies, and are not easy to recombine. Therefore, the evolutionary information of plants is preserved to a certain extent (Jing Yuxiang, 1985). Its gene content, sequence conservation and low recombination rate make the organelle genome more suitable for phylogenetic evolution and development research, and for...

Claims

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Application Information

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IPC IPC(8): C12N15/10
Inventor 刘丹陈超刘正理王丽娜冯刚李强梁丹王从磊王建贺时晓伟石斯发赵子龙吕文杰
Owner 唐山润泽粮油食品有限公司
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