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

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...

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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 the homogenization method for cell disruption, and the simple differential centrifugation method is used to remove nuclei and mitochondria, which cannot be applied to the extraction of chloroplasts from small strip-shaped leaf plants.

[0007] In view of this, Yuan Jincheng, Wang Xiaochun, etc. proposed a rapid extraction method of millet chloroplast DNA and a preparation method of wheat intact chloroplasts (Yuan Jincheng et al., 2008; Wang Xiaochun et al., 2016), but these methods also use the homogenate grinding method to break the cells And single differential centrifugation or density gradient to remove nuclear or mitochondrial contamination, therefore, it is still impossible to extract high-purity pollution-free chloroplast DNA

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