Method for extracting leaf blade dna of catalpa genus plant

Abstract

The application provides a DNA extraction method for leaves of a plant of the genus Catalpa, and relates to the technical field of DNA extraction of plant leaves. The method is based on the CTAB method, and a lysis solution is optimized so that the extraction method is suitable for the plant of the genus Catalpa. The DNA extraction method for leaves of the plant of the genus Catalpa has the beneficial effect of high extraction rate.

Description

Technical Field

[0001] This invention provides a method for extracting DNA from the leaves of plants in the genus *Catella*, and relates to the field of plant leaf DNA extraction technology. Background Technology

[0002] Currently, commonly used methods for plant DNA extraction include: CTAB (full name), mCTAB (full name), SDS (full name), urea method, high-salt method, and kit method. Among these, column chromatography based on the mCTAB method is the most commonly used. Extracting DNA from young plant leaves is relatively easy, while extracting DNA from mature plant leaves is more difficult.

[0003] In studies related to Catalpa trees, DNA is often extracted from young leaves. However, due to differences in research content and needs, extraction from mature leaves is required. Zheng Guangshun modified the CTAB method to extract DNA from mature Catalpa leaves, but only performed agarose gel electrophoresis to detect its integrity, failing to detect the removal of polysaccharides and proteins. Summary of the Invention

[0004] This invention provides a method for extracting DNA from leaves of the genus *Catalpa*, which solves the technical problems of existing technologies that use universal DNA extraction to extract DNA from *Catalpa* leaves, resulting in high degradation rates and protein content, making it impossible to participate in Southern hybridization to determine telomere length.

[0005] This invention is implemented by including the following steps: (1) Take 1-2 g of mature leaves, grind them thoroughly in liquid nitrogen, and quickly transfer them into a 10 ml centrifuge tube. Add 4 ml of CTAB extract and 20 μl of DTT (1.0 mol / L), shake well, and then incubate in a 65 ℃ water bath for 30 min. (2) Remove the centrifuge tube, cool it, add an equal volume of chloroform / isoamyl alcohol (24:1) mixture, shake well for 10 min, centrifuge at 10000 r / min for 10 min at 4℃; transfer the supernatant to a new 10 ml centrifuge tube; (3) Add 2 times the volume of anhydrous ethanol and shake gently. Place in a -20 ℃ refrigerator to cool for 30 min, centrifuge at 10000 r / min for 10 min at 4 ℃, and remove the supernatant. (4) Add 3 ml of 75% ethanol to the precipitate and quickly invert the precipitate to wash it. Centrifuge at 10000 r / min for 10 min at 4 ℃ and remove the supernatant. (5) After repeating (4), place the centrifuge tube in a clean bench to dry the remaining ethanol; (6) Dissolve the precipitate with 1 ml TE buffer and transfer it to a 2 ml centrifuge tube; (7) Add an equal volume of Tris saturated phenol / chloroform / isoamyl alcohol (25:24:1) mixture, mix well, and after forming an emulsion, let it stand on ice for 10 min, centrifuge at 4 ℃ and 10000 r / min for 10 min, and aspirate the supernatant into a new centrifuge tube. (8) Add an equal volume of chloroform / isoamyl alcohol mixture, mix well, centrifuge at 10000 r / min for 10 min at 4℃, and transfer the supernatant to a new centrifuge tube. If there is protein in the solution, repeat steps 6 and 7. (9) Add 2 volumes of anhydrous ethanol and shake gently. Place in a -20 ℃ refrigerator to cool for 30 min, and centrifuge at 10000 r / min for 10 min at 4℃. (10) Add 1 ml of 75% ethanol to the precipitate and quickly invert the precipitate to wash it. Centrifuge at 10000 r / min for 10 min at 4 ℃ and remove the supernatant. (11) After repeating (10), place the centrifuge tube in a clean bench to dry the remaining ethanol; (12) Dissolve it in 50 μl ddH2O and add a small amount of RNase to digest the RNA. Detailed Implementation Example

[0006] 1. Material preparation The samples were 5-year-old grafted seedlings free of pests and diseases, preserved in the Gangou Conservation Bank of the Shandong Provincial Forestry and Grassland Germplasm Resources Center (36°37.5′N, 117°10′E, located in Licheng District, Jinan City, Shandong Province, at an altitude of 200 m, belonging to the temperate monsoon climate zone, with an average annual temperature of about 15.0 ℃ and an annual precipitation of about 660 mm). These seedlings included *Catalpa bungei* (2-8), *Catalpa hainanensis* (GQS-WH004), and *Catalpa bungei 'Zinnia'*. Mature leaves from the south-facing central part of these *Catalpa* species were collected and immediately frozen in liquid nitrogen for treatment and preservation for DNA extraction. Each sample was collected three times as a biological replicate.

[0007] Based on three commonly used DNA extraction methods—mCTAB, CTAB, and SDS—a three-factor, three-level orthogonal experiment was designed (see Table 1) to screen DNA extraction methods, precipitation methods, and additives for Catalpa species, and the results were obtained by agarose gel electrophoresis.

[0008] Table 1. Three-factor, three-level orthogonal experiment table

[0009] mCTAB method: (1) Take 200 mg of leaves, grind them thoroughly in liquid nitrogen, and quickly transfer them into a 2 ml centrifuge tube. Add 750 μl of preheated CATB extract without PVP, shake well, and then incubate in a water bath at 65 ℃ for 30 min. (2) Remove the centrifuge tube, cool it, add an equal volume of Tris saturated phenol / chloroform / isoamyl alcohol (25:24:1) mixture, shake well for 10 min, centrifuge at 10000 r / min for 10 min; transfer the supernatant to a new centrifuge tube; (3) Add an equal volume of chloroform / isoamyl alcohol (24:1) mixture to the centrifuge tube, shake well for 10 min, centrifuge at 10000 r / min for 10 min at 4℃; transfer the supernatant to a new centrifuge tube; (4) Add 2 / 3 volume of isopropanol and shake gently. Place in a -20 ℃ refrigerator to cool for 30 min, centrifuge at 10000 r / min for 10 min at 4 ℃, and discard the supernatant. (5) Add 1 ml of 75% ethanol to the precipitate and quickly invert the precipitate to wash it. Centrifuge at 10000 r / min for 10 min at 4 ℃ and remove the supernatant. (6) After repeating (5), place the centrifuge tube in a clean bench to dry the remaining ethanol; (7) Dissolve it in 50 μl ddH2O and add a small amount of 0.5 μl RNase to digest the RNA.

[0010] CTAB method: (1) Take 1-2 g of mature leaves, grind them thoroughly in liquid nitrogen, and quickly transfer them to a 10 ml centrifuge tube. Quickly add 4 ml of CTAB extraction solution and 20 μl of DTT (1.0 mol / L), shake well, and incubate at 65 ℃ for 30 min. The concentrations of β-mercaptoethanol and PVP in the CTAB extraction solution are 1%β and 4%, respectively, and the rest are standard components. β-mercaptoethanol can destroy disulfide bonds in proteins, causing them to denature and precipitate. It also inhibits the activity of nucleases, as the nuclease activity in detached mature leaves is strong. PVP is an antioxidant that can reduce the interference of phenolic oxidation on DNA.

[0011] (2) Remove the centrifuge tube, cool it, add an equal volume of chloroform / isoamyl alcohol (24:1) mixture, shake well for 10 min, centrifuge at 10000 r / min for 10 min at 4℃; transfer the supernatant to a new 10 ml centrifuge tube; (3) Add 2 times the volume of anhydrous ethanol and shake gently. Place in a -20 ℃ refrigerator to cool for 30 min, centrifuge at 10000 r / min for 10 min at 4 ℃, and remove the supernatant. (4) Add 3 ml of 75% ethanol to the precipitate and quickly invert the precipitate to wash it. Centrifuge at 10000 r / min for 10 min at 4 ℃ and remove the supernatant. (5) After repeating (4), place the centrifuge tube in a clean bench to dry the remaining ethanol; (6) Dissolve the precipitate with 1 ml TE buffer and transfer it to a 2 ml centrifuge tube; (7) Add an equal volume of Tris saturated phenol / chloroform / isoamyl alcohol (25:24:1) mixture, mix well, and after forming an emulsion, let it stand on ice for 10 min, centrifuge at 4 ℃ and 10000 r / min for 10 min, and aspirate the supernatant into a new centrifuge tube. (8) Add an equal volume of chloroform / isoamyl alcohol (24:1) mixture, mix well, centrifuge at 10000 r / min for 10 min at 4℃, and transfer the supernatant to a new centrifuge tube. If there is protein in the solution, repeat steps 6 and 7. (9) Add 2 volumes of anhydrous ethanol and shake gently. Place in a -20 ℃ refrigerator to cool for 30 min, and centrifuge at 10000 r / min for 10 min at 4℃. (10) Same as mCTAB method (5), (6), (7).

[0012] SDS method: (1) Take 200 mg of leaves and grind them thoroughly in liquid nitrogen. Then quickly transfer them into a 2 ml centrifuge tube and add 700 μl of preheated SDS extraction solution, which includes 500 mmol / L NaCl, 100 mmol / L Tris-HCl ph8, 50 mmol / L EDTA ph8, and 120 μl 20% SDS. Shake well and then incubate in a water bath at 65 ℃ for 20 min. (2) Add 0.45 ml of 5M potassium acetate (1 / 10V), mix well, incubate on ice for 20 minutes, and centrifuge at 10000 rpm for 20 minutes.

[0013] (3) Remove the centrifuge tube, cool it, add an equal volume of Tris saturated phenol / chloroform / isoamyl alcohol (25:24:1) mixture, vortex to mix, let stand at 4℃ for 10 min, centrifuge at 10000 r / min at 4℃ for 10 min; aspirate the supernatant into a new centrifuge tube; (4) Add 2 / 3 volume of isopropanol to the tube, place it at -20 ℃ for 30 min to precipitate, and then centrifuge at 10000 r / min for 10 min at 4 ℃. (5) Same as (5), (6), and (7) of the mCTAB method.

[0014] DNA was extracted from the leaves of the three species of Catalpa collected using mCTAB, CTAB, and SDS methods, respectively. A three-factor, three-level orthogonal experiment was conducted to evaluate the extraction method, precipitation method, and added substances. The results are shown in Table 2.

[0015] Table 2 Orthogonal Experiment Table for Example 1

[0016] See Table 2. A260 is the absorbance peak of pure nucleic acids, the absorbance of proteins is A230~280, A280 is the absorption peak of proteins, and the absorbance peak of salts and most organic matter is A230.

[0017] In ①, the DNA concentration of *Catalpa bungei* was the highest: 746.8 ng / μl, followed by *Catalpa macrocarpa*: 746.8 ng / μl, and then *Catalpa bungei*: 328.433 ng / μl; the A260 / A280 ratio from high to low was: *Catalpa bungei* 2.033, *Catalpa macrocarpa* 2.027, and *Catalpa bungei* 1.963; the A260 / 230 ratio from high to low was: *Catalpa bungei* 6, *Catalpa macrocarpa* 2.48, and *Catalpa bungei* 2.4.

[0018] In ②, the highest concentration of DNA was found in *Catalpa bungei* 'Zinnia': 1437.733 ng / μl, followed by *Catalpa bungei*: 1127.967 ng / μl, and then *Catalpa hainanensis*: 800.733 ng / μl; the A260 / 280 ratio from highest to lowest was: *Catalpa bungei* 1.907, *Catalpa hainanensis* 1.88, and *Catalpa bungei* 'Zinnia' 1.823; the A260 / 230 ratio from highest to lowest was: *Catalpa bungei* 1.477, *Catalpa bungei* 'Zinnia' 1.117, and *Catalpa hainanensis* 1.01.

[0019] In category ③, the highest DNA concentration was found in *Catalpa bungei* (993.1 ng / μl), followed by *Catalpa argentea* (683.067 ng / μl), and then *Catalpa macrocarpa* (556.9 ng / μl). The A260 / 280 ratio from highest to lowest was: *Catalpa macrocarpa* 2.003, *Catalpa bungei* (1.913), and *Catalpa argentea* 1.75. The A260 / 230 ratio from highest to lowest was: *Catalpa macrocarpa* 2.64, *Catalpa bungei* (2.39), and *Catalpa argentea* 1.707.

[0020] In category ④, the highest DNA concentration was found in *Catalpa bungei* (114.233 ng / μl), followed by *Catalpa macrocarpa* (53.867 ng / μl), and then *Catalpa bungei* (46 ng / μl). The A260 / 280 ratio from highest to lowest was: *Catalpa bungei* 1.723, *Catalpa bungei* (1.663), and *Catalpa macrocarpa* (1.647). The A260 / 230 ratio from highest to lowest was: *Catalpa bungei* 1.843, *Catalpa macrocarpa* (1.837), and *Catalpa bungei* (1.723).

[0021] In category ⑤, the DNA concentration of *Catalpa bungei* 'Zinnia' was the highest: 131.3 ng / μl, followed by *Catalpa bungei*: 61.033 ng / μl, and then *Catalpa hainanensis*: 59.067 ng / μl; the A260 / 280 from highest to lowest was: *Catalpa bungei* 1.777, *Catalpa hainanensis* 1.743, and *Catalpa bungei* 1.71; the A260 / 230 from highest to lowest was: *Catalpa hainanensis* 1.7, *Catalpa bungei* 1.047, and *Catalpa bungei* 'Zinnia' 1.01.

[0022] In section ⑥, the highest DNA concentration was found in *Cathaya argyrophylla* (162.5 ng / μl), followed by *Catalpa bungei* (132.533 ng / μl), and then *Catalpa bungei 'Zinnia'* (131.3 ng / μl). The A260 / 280 ratio from highest to lowest was: *Cathaya argyrophylla* (1.74), *Catalpa bungei* (1.693), and *Catalpa bungei 'Zinnia'* (1.677). The A260 / 230 ratio from highest to lowest was: *Cathaya argyrophylla* (1.85), *Catalpa bungei 'Zinnia'* (1.697), and *Catalpa bungei 'Zinnia'* (1.663).

[0023] In section ⑦, the DNA concentration of *Catalpa bungei* was the highest: 355.733 ng / μl, followed by *Catalpa bungei*: 156.833 ng / μl, and then *Catalpa hainanensis*: -315.7 ng / μl; the A260 / 280 from highest to lowest was: *Catalpa bungei* 1.03, *Catalpa hainanensis* 0.757, and *Catalpa bungei* 0.573; the A260 / 230 from highest to lowest was: *Catalpa hainanensis* 0.14, *Catalpa bungei* -0.037, and *Catalpa bungei* -0.093.

[0024] In category ⑧, the highest DNA concentration was found in *Catalpa bungei* (948.3 ng / μl), followed by *Catalpa hainanensis* (380.633 ng / μl), and then *Catalpa davidii* (349.5 ng / μl). The A260 / 280 ratio, from highest to lowest, was: *Catalpa davidii* (1.03), *Catalpa hainanensis* (0.757), and *Catalpa bungei* (0.573). The A260 / 230 ratio, from highest to lowest, was: *Catalpa bungei* (0.453), hybrid *Catalpa bungei* (0.11), and *Catalpa hainanensis* (0.16). In category 9, the highest DNA concentration was found in *Cathaya argyrophylla* (853.2 ng / μl), followed by *Cathaya argyrophylla* (522.267 ng / μl), and then *Cathaya zinnia* (516.4 ng / μl). The A260 / 280 ratio, from highest to lowest, was: *Cathaya zinnia* (1.59), *Cathaya argyrophylla* (0.977), and *Cathaya argyrophylla* (0.857). The A260 / 230 ratio, from highest to lowest, was: *Cathaya argyrophylla* (0.217), *Cathaya argyrophylla* (0.28), and *Cathaya zinnia* (1.07).

[0025] The A260 / 280 of pure DNA is 1.8. Therefore, the closer the A260 / 280 is to 1.8, the purer the DNA is. The presence of protein will lower the A260 / 280. The A260 / 280 of pure RNA is 2.0. Therefore, it is necessary to analyze the agarose gel to determine whether RNA or degraded RNA is present when the A260 / 280 is greater than 1.8. When the A260 / 280 is less than 1.8, it is also necessary to consider whether there is a large amount of protein and DNA degradation at the same time, which makes the absorbance close to 1.8.

[0026] The A260 / 280 values ​​for ①②③ are all above 1.8, indicating DNA degradation. The higher the value, the more severe the degradation. For ⑦⑧⑨, only ⑦ and ⑨, extracted from the 'Zinnia elegans' tree, have A260 / 280 values ​​above 1.0, but all are below 1.7, indicating extremely high protein content. The highest concentration is 1127.967 ng / μl for ②, followed by 948.3 ng / μl for ⑧, and the lowest is 46 ng / μl for ④. However, the integrity and A260 / 280 values ​​detected by agarose gel electrophoresis indicate that the high concentrations obtained by the mCTAB and SDS methods are due to the color enhancement effect caused by DNA degradation.

[0027] A three-factor, three-level orthogonal experiment was conducted to screen the extraction method, precipitation method, and added substances. The DNA extraction method for *Catella* species was determined as follows: 1% β-mercaptoethanol and 1.5% PVP were added to CTAB lysis buffer, and chloroform:isoamyl alcohol = 24:1 was used to remove proteins. Then, 10% PEG 8000 and 2.0 mol / L NaCl were added to precipitate the DNA with two times the volume of anhydrous ethanol. The precipitate was washed with 75% ethanol, dried in a clean bench, and resuspended in 500 μl of RNase A TE buffer containing 0.04 mmol / L. The precipitate was then placed in a 4°C refrigerator for 10 min. Proteins were removed using Tris-saturated phenol:chloroform:isoamyl alcohol = 25:24:1 and chloroform:isoamyl alcohol = 24:1. Then, 10% sodium acetate and 2.5 times the volume of anhydrous ethanol were added to precipitate the DNA, and the precipitate was dried in a clean bench again. Finally, the DNA was dissolved in 50 mL of ddH2O to obtain pure whole-genome DNA.

[0028] This application employs a modified CTAB method to replace the conventional CTAB method, and adds PVP K30 as a reducing agent to prevent phenols from oxidizing to quinones, thereby enhancing the polarity of polysaccharides and preventing the browning of phenols. The addition of β-mercaptoethanol allows it to bind to DNA, protecting its integrity. By measuring A260, A280, and A230 using an ultra-micro spectrophotometer, and calculating the impurities of proteins, polysaccharides, and other organic matter in the extract using the ratios of A260 / 280 and A230 / A260, DNA quantification can be achieved efficiently and rapidly.

[0029] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for extracting DNA from leaves of plants in the genus *Catalpa*, characterized in that, Includes the following steps: (1) Take the sample leaf and grind it in liquid nitrogen. After grinding, put it into a centrifuge tube, add the extraction solution and DTT, shake well and then bathe in water for a period of time. The extraction solution includes CTAB extraction solution, 1% β-mercaptoethanol and 4% PVP. (2) Add an equal volume of chloroform / isoamyl alcohol mixture to a centrifuge tube, shake well, centrifuge, and take the supernatant; (3) Add 2 times the volume of anhydrous ethanol to the supernatant from step (2), cool at low temperature and centrifuge to remove the supernatant; (4) Add 75% ethanol to the precipitate and quickly invert the container to wash the precipitate. Centrifuge to remove the supernatant. (5) Repeat step (4) to dry the remaining ethanol; (6) After adding TE buffer to the precipitate, transfer it to a centrifuge tube; (7) Add an equal volume of Tris-saturated phenol / chloroform / isoamyl alcohol mixture to a centrifuge tube, mix well to form an emulsion, let stand on ice, centrifuge, and take the supernatant. (8) Add an equal volume of chloroform / isoamyl alcohol mixture, mix well, centrifuge and take the supernatant; (9) Add 2 times the volume of anhydrous ethanol, shake gently, cool at low temperature, centrifuge, and remove the supernatant; (10) Add 75% ethanol to the precipitate and quickly invert the precipitate to wash it. Centrifuge and remove the supernatant. (11) Repeat step (10) and blow dry the remaining ethanol; (12) Add ddH2O and ribonuclease to the precipitate.

2. The method for extracting DNA from leaves of *Catalpa* plants according to claim 1, characterized in that, Centrifugation conditions were: 4℃, 10000 r / min, 10 min.

3. The method for extracting DNA from leaves of *Catalpa* plants according to claim 1, characterized in that, The low-temperature cooling conditions are: -20 ℃, 30 min.

4. The method for extracting DNA from leaves of *Catalpa* plants according to claim 1, characterized in that, In step (8), the ratio of chloroform to isoamyl alcohol is 24:1.

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