Method for extracting mitochondrial DNA of cotton

Abstract

The invention discloses a method for extracting mitochondrial deoxyribonucleic acid (DNA) of cotton. The method comprises the following steps of: (1) homogenizing cotton, and centrifuging and collecting precipitate; (2) removing nuclear DNA, adding ethylene diamine tetraacetic acid (EDTA) solution, and centrifuging to obtain the precipitate; (3) adding buffer solution, and centrifuging to obtain mitochondria; (4) adding lysis solution and acetate solution, centrifuging, collecting supernatant, and extracting by using chloroform-isoamylol; (5) adding acetate solution and absolute ethanol, centrifuging to obtain the precipitate, and washing by using ethanol; (6) dissolving in triisopropylphenylsulfonyl (Tris)-EDTA (TE); (7) removing ribonucleic acid (RNA) and protein, extracting by using Tris saturated phenol-chloroform-isoamylol, and centrifuging and collecting the supernatant; and (8) adding the acetate solution and the ethanol, and centrifuging to obtain the precipitate; and washing by using the ethanol to obtain the mitochondrial DNA. Aiming at defects of the conventional extraction of the mitochondrial DNA, the conventional cetyltrimethylammonium bromide (CTAB) method is improved, and the extraction conditions are optimized by adjusting the time and times of centrifugation, changing reagents, increasing extraction and washing times, and the like. On the basis of ensuring that the mechanical damage degree of the mitochondria is minimum, the nuclear DNA, phenols and polysaccharides outside the mitochondria are effectively removed, pollution rate is reduced, the purity and the quality of the mitochondrial DNA are improved, and the mitochondrial DNA can meet the requirement of subsequent experiments of the gene engineering of the cotton mitochondria.

Description

technical field [0001] The invention relates to a method for extracting mitochondrial DNA of cotton. Background technique [0002] Mitochondria is the most important organelle of higher organisms and one of the cytoplasmic genetic systems. Mitochondrial genomic DNA (mtDNA) is relatively independent outside the nucleus, capable of self-replication, and controls many genetic traits. Its configuration mainly includes open ring, closed ring, superhelical, linear and multimer. Compared with higher animals, the mitochondrial genomes of higher plants are large and complex. [0003] Plant cytoplasmic male sterility is a relatively common phenomenon in nature, and the study of plant cytoplasmic male sterility is an important topic in the study of genetics. A large number of studies in the fields of genetics and molecular biology have shown that the genes that cause plant cytoplasmic male sterility are mainly distributed in the mitochondrial genome, that is, the mitochondrial genom...

AI Technical Summary

Problems solved by technology

However, the operation process of these methods is relatively complicated, the cost is high, and the purity of the extracted mtDNA is not high, which affects subsequent experiments.

In the process of extracting mtDNA from different plants, various influencing factors are extremely complex and difficult to grasp, and the requirements for the quality, purity and integrity of extracted mtDNA are getting higher and higher in the follow-up experiments of genetic engineering

Because cotton contains more phenols and polysaccharides, it is easy to oxidize during the process of isolating and purifying mtDNA, which causes certain difficulties in purifying mtDNA

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