AU2021104676A4 - Method for extracting peanut genome - Google Patents

Abstract

The present invention belongs to the technical field of biomacromolecule extraction, and particularly relates to a method for extracting a peanut genome. On the basis of a traditional CTAB method, through optimizing extraction reagents, steps and the like, the invention aims to integrate a novel method which is easy and rapid and can directly extract high-quality genome DNA from different tissues of peanuts so as to meet the requirement of peanut molecular biology research. Only the chloroform is used for extraction without saturated phenol or isoamyl alcohol, so that the use of toxic organic solvents is reduced, and the safety of the experiment is improved.

Description

METHOD FOR EXTRACTING PEANUT GENOME BACKGROUND OF THE INVENTION

[0001] 1. Technical Field

[0002] The invention belongs to the technical field of biomacromolecule extraction, and particularly relates to a method for extracting a peanut genome.

[0003] 2. Description of Related Art

[0004] Extraction and purification of genome DNA are the basis and key links of plant molecular biology and genetic breeding science. At present, common methods for extracting plant genome DNA include a CTAB method, an SDS method, an enzymatic method, a high-salt extraction method and the like. According to both the CTAB method and the SDS method, on the basis of lysing plant cells, organic solvents are used for extraction for multiple times, protein and the like are precipitated in an organic reagent, while nucleic acid is kept in a water phase, and therefore the purpose of separating the nucleic acid is achieved.

[0005] There are many methods for extracting plant genome DNA, but different plants have different characteristics. In practice, it needs to be improved continuously to get the ideal result.

[0006] Protein, polysaccharide, polyphenols, other secondary metabolites and the like in peanut tissue materials cause great hindrance to extraction and purification of DNA. In order to effectively prepare peanut DNA with high purity and high integrity, it has to be considered to remove proteins, lipids, saccharides, polyphenols as much as possible, and to prevent and inhibit degradation of DNA by endogenous DNAase. In several conventional methods for extracting peanut DNA, compared with an SDS method and a high-salt extraction method, a CTAB method can remove carbohydrate and polyphenol impurities in plants more effectively. The DNA yield and purity obtained by a traditional CTAB method are high, but the defects that more toxic organic reagents are used, the operation process is tedious and the like exist.

BRIEF SUMMARY OF THE INVENTION

[0007] In view of the problems existing in the prior art, the objective of the invention is to provide a method for extracting a peanut genome conveniently and rapidly which is suitable for laboratory operation.

[0008] In order to achieve the above objective, the invention adopts the following technical solutions:

[0009] A method for extracting a peanut genome includes the following steps:

[0010] (1) taking fresh peanut tissue, adding 100 L of CTAB extraction buffer solution, and grinding the mixture into homogenate;

[0011] (2) adding 100 L of CTAB extraction buffer solution again, shaking and uniformly mixing the mixture, carrying out water bath for 15 min at 65°C , and taking out and uniformly shaking the mixture every 5 min in this period;

[0012] (3) adding 100 L of chloroform into the mixture in the step (2), shaking and uniformly mixing the mixture, and centrifuging the mixture for 10 min at a rotating speed of 13000 r/min at °C;

[0013] (4) taking a supernate, adding 100 L of isopropanol, shaking and uniformly mixing the mixture, and centrifuging the mixture for 7 min at a rotating speed of 13000 r/min at 25°C;

[0014] (5) discarding the supernate, adding 150 L of 70% alcohol into a precipitate for one time of washing, discarding the supernate, carrying out uncovering and placement at room temperature to volatilize the alcohol; and

[0015] (6) finally adding water or TE to dissolve the precipitate, and storing the product for later use.

[0016] On the basis of the above solution, the CTAB extraction buffer solution described in the step (2) is prepared from the following reagents: 1.04 g of Sorbitol, 1.76 mL of 0.5 mol/L EDTA (pH 8.0, ethylenediaminetetraacetic acid), 8.8 mL of 1 mol/L Tris-HCl (pH 8.0), 1.87 g of NaCl, 3.2 mL of 10% CTAB (cetyltrimethylammonium bromide), and 4 mL of 10% Sarkosyl. The reagents are dissolved by 10 mL of ddH 20 at first, and then are subjected to volume fixing to 40 mL for sterilization.

[0017] On the basis of the above solution, the amount of the fresh peanut tissue in the step (1) is mg.

[0018] On the basis of the above solution, the adding volume of the water or TE described in the step (6) is 65 L.

[0019] The technical solution of the invention has the advantages:

[0020] The invention has the advantages of simplicity, convenience and rapidness in operation, greatly shortens the test time, saves a lot of manpower and material resources, enables tedious work to be simple, and has low cost and high extraction rate. The extracted materials have a wide range (roots, leaves and seeds), the required sample amount is small, and micro detection can be achieved. The invention has good universality, and can also be applied to extraction of other plant materials, which also achieves satisfactory results.

[0021] Peanuts are rich in lipid, protein, phenol and the like, and it is necessary to establish an efficient, simple, economical and universal method for extracting peanut genome DNA, which is suitable for the peanuts. On the basis of a traditional CTAB method, through optimizing extraction reagents, steps and the like, the invention aims to integrate a novel method which is easy and rapid and can directly extract high-quality genome DNA from different tissues of peanuts so as to meet the requirement of peanut molecular biology research. A sorbitol solution can regulate osmotic pressure and prevent premature nuclear rupture. Sodium dodecyl sarcosinate is an ionic surfactant, which can destroy cell membrane and nuclear membrane and separate tissue protein from DNA. In addition, only chloroform is used for extraction once in the extraction process, the operation process is simple, the complexity of the experiment is reduced, the time is saved, and the method is suitable for large-scale extraction. In addition, only the chloroform is used for extraction without saturated phenol or isoamyl alcohol, so that the use of toxic organic solvents is reduced, and the safety of the experiment is improved.

[0022] The invention extracts DNA in peanut tissue materials by the improved CTAB method in order to lay a foundation for further research on molecular biology of peanuts.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0023] In order to explain the embodiments of the present invention or the technical solutions in the prior art more clearly, the accompanying drawings required in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the accompanying drawings in the following description are only some embodiments of the present invention, and a person of ordinary skill in the art can still derive other drawings according to these accompanying drawings without creative efforts.

[0024] Fig. 1 is an electrophoretogram of peanut leaf genomes in Example 1 (M, Marker; 1, 2, 3, 4: serial numbers of extracted leaf genomes).

[0025] Fig. 2 is a PCR result of the peanut leaf genomes in Example 1 (M, Marker; N: Negative control; P: Positive control; 1, 2, 3: serial numbers of PCR products).

[0026] Fig. 3 is an electrophoretogram of peanut cotyledon genomes in Example 2 (M, Marker; 1, 2, 3, 4: serial numbers of extracted cotyledon genomes).

[0027] Fig. 4 is a PCR result of the peanut cotyledon genomes in Example 2 (M, Marker; N: Negative control; P: Positive control; 1, 2, 3: serial numbers of PCR products).

DETAILED DESCRIPTION OF THE INVENTION

[0028] To make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail with reference to the accompanying drawings and specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention, and are not used to limit the present invention.

[0029] Example 1

[0030] 10 mg of fresh peanut leaf tissue was taken and placed in a 1.5 mL EP tube. A small amount of liquid nitrogen was added, and the tissue was rapidly ground by using a small plastic grinding rod. 100 L of CTAB extraction buffer solution was added, and the mixture was rapidly ground into homogenate by using the small plastic grinding rod. 100 L of CTAB extraction buffer solution was added again, and the mixture was shaken and uniformly mixed. Water bath was carried out for 15 min at 65°C, and the mixture was taken out and uniformly shaken every about 5 min in this period. 100 L of chloroform was added into each 1.5 mL EP tube, and the mixture was shaken and uniformly mixed. The mixture was centrifuged for 10 min at a rotating speed of 13000 r/min at 25°C. A supernate was taken and transferred into another 1.5 mL EP tube, 100 L of isopropanol was added, and the mixture was shaken and uniformly mixed. The mixture was centrifuged for 7 min at a rotating speed of 13000 r/min at 25°C. The supernate was discarded. A precipitated was washed by 150 L of 70% alcohol for one time, and the supernate was discarded. Uncovering and placement were carried out at room temperature to volatilize the alcohol. Finally, 65 L of water or TE was added to dissolve the precipitate, and the product was stored for later use.

[0031] Detection by agarose gel electrophoresis revealed that extracted genomic fragments were intact and no protein or carbohydrate impurities remained in sample application holes (Fig. 1).

[0032] Detection by an ultraviolet spectrophotometer revealed that OD 2 60 /OD 2 80 of extracted DNA was within 1.8-2.0. With the extracted DNA as a template, an AhFAD2-2 gene was amplified through a PCR technology, and the length of the gene was 1152bp.

[0033] PCR reaction conditions are as follows: 94°C 2 min; 94°C 30 s, 68°C 30 s, 72°C 2 min, and 35 cycles; and 72°C 10 min. After agarose electrophoresis, it was found that a clear band of a target gene could be obtained through amplification, and the size of the band matched the target gene (Fig. 2).

[0034] Example 2

[0035] A thin slice of about 10 mg of fresh peanut cotyledon tissue was cut by a blade and placed in a 1.5 mL EP tube. 100 pL of CTAB extraction buffer solution was added. The tissue was rapidly ground into homogenate by using a small plastic grinding rod. 100 L of CTAB extraction buffer solution was added again, and the mixture was shaken and uniformly mixed.

Water bath was carried out for 15 min at 65°C, and the mixture was taken out and uniformly shaken every about 5 min in this period. 100 L of chloroform was added into each 1.5 mL EP tube, and the mixture was shaken and uniformly mixed. The mixture was centrifuged for 10 min at a rotating speed of 13000 r/min at 25°C. A supernate was taken and transferred into another 1.5 mL EP tube, 100 pL of isopropanol was added, and the mixture was shaken and uniformly mixed. The mixture was centrifuged for 7 min at a rotating speed of 13000 r/min at 25°C. The supernate was discarded. A precipitated was washed by 150 L of 70% alcohol for one time, and the supernate was discarded. Uncovering and placement were carried out at room temperature to volatilize the alcohol. Finally, 65 L of water or TE was added to dissolve the precipitate, and the product was stored for later use.

[0036] Detection by agarose gel electrophoresis revealed that extracted genomic fragments were intact and no protein or carbohydrate impurities remained in sample application holes (Fig. 3).

[0037] Detection by an ultraviolet spectrophotometer revealed that OD 260 /OD 280 of extracted DNA was within 1.8-2.0. With the extracted DNA as a template, an AhFAD2-1 gene was amplified through a PCR technology, and the length of the gene was 1140bp.

[0038] PCR reaction conditions are as follows: 94°C 2 min; 94°C 30 s, 68°C 30 s, 72°C 2 min, and 35 cycles; and 72°C 10 min. After agarose electrophoresis, it was found that a clear band of a target gene could be obtained through amplification, and the size of the band matched the target gene (Fig. 4).

[0039] The foregoing descriptions are only preferred implementation manners of the present invention. It should be noted that for a person of ordinary skill in the art, several improvements and modifications may further be made without departing from the principle of the present invention. These improvements and modifications should also be deemed as falling within the protection scope of the present invention.

Claims (4)

What is claimed is:

1. A method for extracting a peanut genome, including the following steps: (1) taking fresh peanut tissue, adding 100L of CTAB extraction buffer solution, and grinding the mixture into homogenate; (2) adding 100 L of CTAB extraction buffer solution again, shaking and uniformly mixing the mixture, carrying out water bath for 15 min at 65°C , and taking out and uniformly shaking the mixture every 5 min in this period; (3) adding 100 L of chloroform into the mixture in the step (2), shaking and uniformly mixing the mixture, and centrifuging the mixture for 10 min at a rotating speed of 13000 r/min at 25°C; (4) taking a supernate, adding 100 L of isopropanol, shaking and uniformly mixing the mixture, and centrifuging the mixture for 7 min at a rotating speed of 13000 r/min at 25°C; (5) discarding the supernate, adding 150 L of 70% alcohol into a precipitate for one time of washing, discarding the supernate, carrying out uncovering and placement at room temperature to volatilize the alcohol; and (6) finally adding water or TE to dissolve the precipitate, and storing the product for later use.

2. The method for extracting a peanut genome according to claim 1, the CTAB extraction buffer solution described in the step (2) is prepared from the following reagents: 1.04 g of Sorbitol, 1.76 mL of 0.5 mol/L EDTA (pH 8.0, ethylenediaminetetraacetic acid), 8.8 mL of1 mol/L Tris-HCl (pH 8.0), 1.87 g of NaCl, 3.2 mL of 10% CTAB (cetyltrimethylammonium bromide), and 4 mL of 10% Sarkosyl. The reagents are dissolved by 10 mL of ddH 20 at first, and then are subjected to volume fixing to 40 mL for sterilization.

3. The method for extracting a peanut genome according to claim 1 or 2, the amount of the fresh peanut tissue in the step (1) is 10 mg.

4. The method for extracting a peanut genome according to claim 3, the adding volume of the water or TE described in the step (6) is 65 [L.