Method for extracting nucleic acid from plant leaves and application

Abstract

The invention provides a method for extracting nucleic acid from plant leaves and application, and relates to the technical field of biology, the method comprises the following steps: S1, sample preparation: using fresh plant leaves, removing non-target tissues, cutting 0.1-10g of leaves into small pieces, and putting the small pieces into a clean mortar for later use to obtain a plant leaf sample; according to the invention, the use of liquid nitrogen ensures that the leaves are quickly frozen at an extremely low temperature and nucleic acid degradation is prevented, and meanwhile, the grinding process is carried out at a low temperature, so that the damage of mechanical force to nucleic acid is reduced to the greatest extent. The use of the CTAB buffer solution not only helps to dissolve cell tissues, but also effectively removes polysaccharide and protein impurities affecting the extraction purity of nucleic acid through the characteristics of the surfactant of the CTAB buffer solution.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a method for extracting nucleic acid from plant leaves and application thereof. Background Art

[0002] The field of biotechnology is the science of studying organisms and their life phenomena, covering multiple disciplines such as molecular biology, genetics, and biochemistry. Nucleic acid extraction is one of the important basic technologies in molecular biology, and plays a key role in experiments such as gene cloning, PCR amplification, and gene expression analysis. Since plant cells have cell walls and abundant secondary metabolites, the extraction of plant nucleic acids is more challenging than that of animal nucleic acids. Therefore, the development of efficient and high-purity plant nucleic acid extraction methods is of great significance in plant research.

[0003] The method of extracting nucleic acids from plant leaves aims to provide a simple, fast and effective technical means to obtain high-quality plant nucleic acid samples. This method can be used in gene analysis, molecular marker-assisted breeding, transgenic identification and other fields, providing technical support for plant science research and agricultural production.

[0004] Physical disruption methods in the prior art often fail to fully release nucleic acids in cells, and may cause nucleic acid degradation during the process, reducing the repeatability and reliability of the experiment. In addition, polysaccharides and polyphenols that are not effectively removed are likely to interfere with subsequent analysis of nucleic acids, such as PCR amplification, thereby affecting the accuracy of experimental results. Summary of the invention

[0005] The purpose of the present invention is to solve the shortcomings of the prior art and to propose a method and application of extracting nucleic acid from plant leaves.

[0006] In order to achieve the above object, the present invention adopts the following technical scheme: a method for extracting nucleic acid from plant leaves, comprising the following steps:

[0007] S1, sample preparation, using fresh plant leaves, removing non-target tissues, cutting 0.1-10 g of leaves into small pieces, putting them into a clean mortar for later use, and obtaining plant leaf samples;

[0008] S2, liquid nitrogen grinding, immersing the plant leaf sample in liquid nitrogen, freezing to -196°C, and grinding the leaf into fine powder using a pre-cooled mortar and pestle under low temperature conditions to obtain leaf powder;

[0009] S3, CTAB buffer treatment, the leaf powder is added to a buffer containing 2% CTAB, 1% β-mercaptoethanol, 1.4M NaCl, 20mM EDTA and 100mM Tris-HCl, the liquid volume is 5-10 times the sample volume, and incubated in a water bath at 55-60°C for 30-60 minutes to promote the rupture of cell walls and cell membranes.

[0010] A CTAB-treated mixed solution is obtained;

[0011] S4, anti-degradation treatment, adding 0.1-0.5% of anti-degradation substance to the mixed solution treated with CTAB, and mixing thoroughly to obtain an anti-degradation mixed solution;

[0012] S5, protein removal, adding an equal volume of chloroform and isoamyl alcohol mixed solution to the anti-degradation mixed solution, mixing thoroughly, and standing at room temperature for 10 minutes to allow protein components to separate, thereby obtaining a mixed solution after protein removal;

[0013] S6, centrifugation, centrifuging the mixed solution after the protein is removed at 12000g for 10-15 minutes, controlling the temperature at 4°C, collecting the supernatant to obtain the supernatant containing nucleic acid.

[0014] Preferably, it also includes:

[0015] S7, DNA dissolution, adding the nucleic acid-containing supernatant to TE buffer, mixing, so that the DNA is completely dissolved, and obtaining a high-purity DNA sample.

[0016] Preferably, in step S4, the anti-degradation substance is a nuclease inhibitor.

[0017] Preferably, in step S5, the mixing ratio of the chloroform and isoamyl alcohol mixture is 24:1.

[0018] The invention provides an application of extracting nucleic acid from plant leaves, and the application of the nucleic acid in plant leaves.

[0019] Compared with the prior art, in the present invention, the use of liquid nitrogen ensures rapid freezing of leaves at extremely low temperatures and prevents nucleic acid degradation, while the grinding process is carried out at low temperatures, minimizing mechanical damage to nucleic acids. The use of CTAB buffer not only helps dissolve cell tissues, but also effectively removes polysaccharide and protein impurities that affect the purity of nucleic acid extraction through its surfactant properties. This makes the nucleic acids extracted from plant leaves more suitable for accurate gene analysis and molecular labeling, supporting research and applications such as gene analysis and molecular marker-assisted breeding.

[0020] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0022] Figure 1 The present invention provides a method for extracting nucleic acids from plant leaves. DETAILED DESCRIPTION

[0023] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0024] Embodiment 1, a method for extracting nucleic acid from plant leaves, comprising the following steps:

[0025] S1, sample preparation, using fresh plant leaves, removing non-target tissues, cutting 4 grams of leaves into small pieces, putting them into a clean mortar for later use, and obtaining plant leaf samples;

[0026] S2, liquid nitrogen grinding, immersing the plant leaf sample in liquid nitrogen, freezing to -196°C, and grinding the leaf into fine powder using a pre-cooled mortar and pestle under low temperature conditions to obtain leaf powder;

[0027] S3, CTAB buffer treatment, adding the leaf powder to a buffer containing 2% CTAB, 1% β-mercaptoethanol, 1.4M NaCl, 20mM EDTA and 100mM Tris-HCl, the liquid volume being 10 times the sample volume, and incubating in a 60°C water bath for 60 minutes to promote the rupture of cell walls and cell membranes, thereby obtaining a CTAB-treated mixed solution;

[0028] S4, anti-degradation treatment, adding 0.5% of an anti-degradation substance to the mixed solution treated with CTAB, and mixing thoroughly to obtain an anti-degradation mixed solution;

[0029] S5, protein removal, adding an equal volume of chloroform and isoamyl alcohol mixed solution to the anti-degradation mixed solution, mixing thoroughly, and standing at room temperature for 10 minutes to allow protein components to separate, thereby obtaining a mixed solution after protein removal;

[0030] S6, centrifugation, centrifuging the mixed solution after the protein is removed at 12000g for 15 minutes, controlling the temperature at 4°C, collecting the supernatant to obtain the supernatant containing nucleic acid.

[0031] Preferably, it also includes:

[0032] S7, DNA dissolution, adding the nucleic acid-containing supernatant to TE buffer, mixing, so that the DNA is completely dissolved, and obtaining a high-purity DNA sample.

[0033] Preferably, in step S4, the anti-degradation substance is a nuclease inhibitor.

[0034] Preferably, in step S5, the mixing ratio of the chloroform and isoamyl alcohol mixture is 24:1.

[0035] The invention provides an application of extracting nucleic acid from plant leaves, and the application of the nucleic acid in plant leaves.

[0036] Embodiment 2, a method for extracting nucleic acid from plant leaves, comprising the following steps:

[0037] S1, sample preparation, using fresh plant leaves, removing non-target tissues, cutting 3 grams of leaves into small pieces, putting them into a clean mortar for later use, and obtaining plant leaf samples;

[0038] S2, liquid nitrogen grinding, immersing the plant leaf sample in liquid nitrogen, freezing to -196°C, and grinding the leaf into fine powder using a pre-cooled mortar and pestle under low temperature conditions to obtain leaf powder;

[0039] S3, CTAB buffer treatment, adding the leaf powder to a buffer containing 2% CTAB, 1% β-mercaptoethanol, 1.4M NaCl, 20mM EDTA and 100mM Tris-HCl, the liquid volume being 5 times the sample volume, and incubating in a 60°C water bath for 30 minutes to promote the rupture of cell walls and cell membranes, thereby obtaining a CTAB-treated mixed solution;

[0040] S4, anti-degradation treatment, adding 0.4% of anti-degradation substance to the mixed solution treated with CTAB, and mixing thoroughly to obtain an anti-degradation mixed solution;

[0041] S5, protein removal, adding an equal volume of chloroform and isoamyl alcohol mixed solution to the anti-degradation mixed solution, mixing thoroughly, and standing at room temperature for 10 minutes to allow protein components to separate, thereby obtaining a mixed solution after protein removal;

[0042] S6, centrifugation, centrifuging the mixed solution after the protein is removed at 12000g for 15 minutes, controlling the temperature at 4°C, collecting the supernatant to obtain the supernatant containing nucleic acid.

[0043] Preferably, it also includes:

[0044] S7, DNA dissolution, adding the nucleic acid-containing supernatant to TE buffer, mixing, so that the DNA is completely dissolved, and obtaining a high-purity DNA sample.

[0045] Preferably, in step S4, the anti-degradation substance is a nuclease inhibitor.

[0046] Preferably, in step S5, the mixing ratio of the chloroform and isoamyl alcohol mixture is 24:1.

[0047] The invention provides an application of extracting nucleic acid from plant leaves, and the application of the nucleic acid in plant leaves.

[0048] Experimental methods:

[0049] Nucleic acid yield determination method: Use an ultraviolet spectrophotometer to measure the absorbance of the extracted nucleic acid solution at a wavelength of 260nm. Use pure TE buffer as a blank control. The nucleic acid concentration is calculated according to the formula C = (A260 × dilution factor × 50μg / mL), where A260 is the absorbance at 260nm. The nucleic acid yield is expressed as micrograms of nucleic acid extracted per gram of plant leaf sample (μg / g).

[0050] Purity determination method: Measure the absorbance of the nucleic acid solution at 260nm and 280nm on a UV spectrophotometer. Calculate the A260 / A280 ratio, which is used to evaluate the purity of the nucleic acid. A purity close to 1.8 indicates that the DNA purity is high. The higher the purity, the lower the content of impurities such as protein.

[0051] Integrity assessment method: The extracted nucleic acid samples were electrophoresed on a 1% agarose gel. The electrophoresis conditions were 100V and 30 minutes. After the electrophoresis, the nucleic acid bands were observed using a gel imaging system. The integrity of the nucleic acid was assessed based on the clarity and integrity of the bands, and the RIN value (RNA integrity number) was calculated or assessed based on the DNA bands.

[0052] Extraction time determination method: Start timing from the sample preparation step until the final purified nucleic acid solution is obtained. Record the time spent on each step and add up to get the total extraction time. The time unit is minutes.

[0053] Cost calculation method: Count all reagents and consumables used in each extraction method, including but not limited to reagents, centrifuge tubes, magnetic beads, consumables, etc. Calculate the cost of each material based on the market price and add up the total cost of a single extraction. The cost unit is RMB.

[0054] Experiments were conducted on Examples 1-2, wherein Comparative Example 1 is the prior art, and the experimental results are as follows:

[0055] Table 1 Performance test data

[0056] Performance Indicators Comparative Example 1 Example 1 Example 2 Nucleic acid yield (μg / g) 50 80 78 Purity (A260 / A280) 1.6 1.8 1.7 Integrity (RIN value) 6.0 8.0 8.0 Single cost (yuan) 35 30 30

[0057] As can be seen from Table 1, Example 1 and Example 2 have significant advantages over Comparative Example 1. Specifically, Example 1 and Example 2 have better performance in terms of nucleic acid yield, purity and integrity, shorter extraction time, and lower single cost. This shows that the method of the present invention improves the efficiency and quality of nucleic acid extraction while reducing time and economic costs, and has high practical value.

Claims

1. A method for extracting nucleic acid from plant leaves, characterized in that: The following steps are involved: S1, sample preparation, using fresh plant leaves, removing non-target tissues, cutting 0.1-10 g of leaves into small pieces, putting them into a clean mortar for later use, and obtaining plant leaf samples; S2, liquid nitrogen grinding, immersing the plant leaf sample in liquid nitrogen, freezing to -196°C, and grinding the leaf into fine powder using a pre-cooled mortar and pestle under low temperature conditions to obtain leaf powder; S3, CTAB buffer treatment, adding the leaf powder to a buffer containing 2% CTAB, 1% β-mercaptoethanol, 1.4M NaCl, 20mM EDTA and 100mM Tris-HCl, the liquid volume being 5-10 times the sample volume, and incubating in a water bath at 55-60°C for 30-60 minutes to promote the rupture of cell walls and cell membranes, thereby obtaining a CTAB-treated mixed solution; S4, anti-degradation treatment, adding 0.1-0.5% of anti-degradation substance to the mixed solution treated with CTAB, and mixing thoroughly to obtain an anti-degradation mixed solution; S5, protein removal, adding an equal volume of chloroform and isoamyl alcohol mixed solution to the anti-degradation mixed solution, mixing thoroughly, and standing at room temperature for 10 minutes to allow protein components to separate, thereby obtaining a mixed solution after protein removal; S6, centrifugation, centrifuging the mixed solution after the protein is removed at 12000g for 10-15 minutes, controlling the temperature at 4°C, collecting the supernatant to obtain the supernatant containing nucleic acid.

2. The method for extracting nucleic acid from plant leaves according to claim 1, characterized in that: Also includes: S7, DNA dissolution, adding the nucleic acid-containing supernatant to TE buffer, mixing, so that the DNA is completely dissolved, and obtaining a high-purity DNA sample.

3. The method for extracting nucleic acid from plant leaves according to claim 1, characterized in that: In step S4, the anti-degradation substance is a nuclease inhibitor.

4. The method for extracting nucleic acid from plant leaves according to claim 1, characterized in that: In step S5, the mixing ratio of the chloroform and isoamyl alcohol mixed solution is 24:

1.

5. An application for extracting nucleic acid from plant leaves, characterized in that: Its application in plant leaves.

Images