Method for extracting genome DNA (Deoxyribose Nucleic Acid) of single dry celery seed

Abstract

The invention discloses a method for extracting genome DNA (Deoxyribose Nucleic Acid) of a single dry celery seed by adopting a specific primer pair. Comprising the steps of genome DNA extraction, PCR amplification and detection, PCR amplification product detection through agarose gel electrophoresis and a fluorescence imager and the like. Experimental results show that according to the method for extracting the genome DNA from the single dry celery seed by using the alkali lysis method, extraction of the genome DNA of 48 seeds only needs 30 min, and the obtained genome can meet the PCR amplification requirement, can be stored for 17 days or more at 4 DEG C, has high stability and can be used for molecular identification work of the purity of celery commodity seeds in the later period.

Description

Technical Field

[0001] The invention belongs to the technical field of vegetable planting and relates to a method for extracting genome DNA of single-grain dry celery seeds and application thereof. Background Art

[0002] Celery (Apiumgraveolens L.) is a biennial herbaceous plant belonging to the genus Apiaceae. It is an important leafy vegetable widely grown worldwide and one of the top ten most important vegetables in my country. Celery petioles and leaves are not only rich in nutrients such as vitamins, cellulose and carotenoids, but its seeds also contain abundant volatile physiologically active substances. Many studies at home and abroad have shown that celery contains a variety of medicinal components such as apigenin and bergamotolactone, and is considered to be a food with potential medicinal and edible effects.

[0003] The extraction of crop genomic DNA is a prerequisite for molecular marker identification. Directly extracting genomic DNA from seeds omits the germination and growth processes of seeds, greatly improving the timeliness of molecular marker identification. The extraction of genomic DNA from seeds of field crops such as corn has relatively mature methods from the formulation of lysis solution, lysis time and temperature, and the amount of magnetic beads, and has been widely used in actual breeding processes. In crops such as vegetables, many researchers have studied the rapid extraction of genomic DNA directly from seeds. Yang Fan et al. used the magnetic bead method to extract and detect genomic DNA from single seeds of seven vegetables of different shapes and sizes (cucumber, cabbage, kale, mustard, tomato, pepper and onion, etc.), and established a genomic DNA extraction method for single seeds of various vegetable crops, which can meet the needs of KASP detection. Through the use of the PlantZol kit, Tiangen quick plant genomic DNA extraction system, TPS method and CTAB method, the genomic DNA of onion seeds was extracted and the purity of seeds was identified by molecular markers. It was determined that the CTAB method was the best for extracting the total genomic DNA of onion dry seeds, which could meet the needs of molecular marker identification of onion hybrids. Zhang Hong et al. used five different methods, including the conventional CTAB method, sucrose extraction method, improved alkaline lysis method, improved alkaline boiling method and rapid DNA release method, to extract genomic DNA from seeds of cruciferous crops such as Chinese cabbage and radish, and evaluated their extraction quality and storage time. It was found that after 1-2 days of seed germination, the genomic DNA extracted by the rapid DNA release method could meet the needs of molecular marker detection of Chinese cabbage and radish, and had good stability, which greatly shortened the genome extraction time.

[0004] Since celery seeds are extremely small, with a thousand-grain weight of only about 0.4-0.5 g, it is very difficult to perform various operations on single seeds. So far, there have been no reports on the direct use of single dry celery seeds for genomic DNA extraction, which greatly limits the application of molecular marker technology in the rapid identification of celery seed purity. Summary of the invention

[0005] This patent establishes a method for rapid extraction of genomic DNA from single-grain dry celery seeds. The obtained genome can meet the requirements of PCR amplification and has high stability. It can be used for large-scale molecular identification of celery seed purity and improve celery breeding efficiency.

[0006] To achieve the above purpose, the present invention discloses the following technical solution: The invention firstly discloses specific primers for extracting single-grain dry seed genes of celery, which have nucleotide sequence genes shown as SEQ ID NO.1 and SEQ ID NO.2.

[0007] The present invention further discloses a method for extracting genomic DNA from single-grain dry seeds of celery using the nucleotide sequence gene, which is characterized by the following steps: (1) Extraction of genomic DNA: Take a single dried celery seed and place it in a centrifuge tube; grind the seed with a grinding rod; add 100 μL of NaOH to the centrifuge tube and continue to grind the seeds in the centrifuge tube with the grinding rod until they are completely ground; then heat the centrifuge tube at 95°C for 3-5 min and centrifuge at 12,000 rpm for 1 min; transfer 10 μL of the supernatant to a new centrifuge tube and add 300 μL of Tris-HCl to obtain the celery genomic DNA for later use; (2) PCR amplification and detection: The genomic DNA extracted as above was used as template, and PCR amplification was performed with AgActin-F: 5`-CTTCCTGCCATATATGATTGG-3`; AgActin-R: 5`- GCCAGCACCTCGATCTTCATG-3` as Actin-specific primers. The PCR amplification products were detected by 1% agarose gel electrophoresis and fluorescence imaging. It was found that all genomic DNAs were able to correctly amplify specific PCR products, which proved that the genomic DNA extracted from single-grain dry celery seeds could meet the needs of PCR amplification and be used for the subsequent molecular marker identification of celery seed purity.

[0008] (3) Identification of the stability of the obtained genomic DNA: The alkaline lysis method was used to extract genomic DNA from single-grain dry celery seeds. It only took 30 minutes to extract genomic DNA from 48 seeds. The obtained genome could meet the needs of PCR amplification and could be stored at 4°C for more than 17 days, showing high stability.

[0009] The present invention is described in more detail as follows: 1. Extraction of genomic DNA: Take a single dried celery seed and place it in a 1.5 mL centrifuge tube; grind the seed with a grinding rod; add 100 μL NaOH (0.4 mol / L) to the centrifuge tube, and continue to grind the seeds in the centrifuge tube with a grinding rod until they are completely ground; then heat the centrifuge tube at 95°C for 3 min and centrifuge at 12000 rpm for 1 min; take 10 μL of the supernatant and transfer it to a new 1.5 mL centrifuge tube, and then add 300 μL Tris-HCl (0.1 mol / L, pH 8.0) to prepare the celery genomic DNA.

[0010] 2. PCR amplification and detection: The genomic DNA extracted above was used as template, and AgActin-F: 5`-CTTCCTGCCATATATGATTGG-3`; AgActin-R: 5`- GCCAGCACCTCGATCTTCATG-3` were used as Actin Specific primers were used for PCR amplification. The amplification reaction system was: 2 μL genomic DNA, 0.15 μL primers AgActin-F / R, 5 μL 2×Taq mix, 2.7 μL ddH2O. The amplification reaction procedure was: 94℃, 5min; 94℃, 20s, 56℃, 20s, 72℃, 45s, 30 Cycles; 72℃, 5min. The PCR amplification products were detected by 1% agarose gel electrophoresis and fluorescence imaging. It was found that all genomic DNA could correctly amplify specific PCR products, proving that the genomic DNA extracted from single-grain dry seeds of celery can meet the needs of PCR amplification and can be used for subsequent molecular marker identification of celery seed purity.

[0011] The genomic DNA of 48 dried celery seeds was extracted by alkaline lysis method and stored at 4°C for 7 days. ActinSpecific primers were used for PCR amplification, and agarose gel electrophoresis was used to detect that almost all samples could amplify the corresponding specific bands. The extracted genomic DNA was further stored at 4°C for 10 days, and PCR amplification and electrophoresis were performed. It was also found that almost all genomic DNA could still amplify the corresponding specific bands. This proves that the genomic DNA extracted from single-grain dry celery seeds in this patent can be stored at 4°C for at least 14 days and can still meet the needs of PCR amplification, and has good stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 Flow chart of genomic DNA extraction from single-grain dry celery seeds: A. Prepare celery seeds; B. Prepare 1.5mL centrifuge tubes; C. 0.3mm grinding rods and centrifuge tubes; D. Grinding rods inserted into centrifuge tubes for grinding; E. Add NaOH to centrifuge tubes and continue grinding; F. Supernatant in centrifuge tubes; Figure 2 PCR amplification test results of genomic DNA of single-grain dry seeds of celery; Figure 3 Comparison of PCR amplification results of genomic DNA extracted from celery seeds after storage for different time periods; A. 7 days; B. 17 days. DETAILED DESCRIPTION

[0013] The present invention is described below by specific embodiments. Unless otherwise specified, the technical means used in the present invention are methods known to those skilled in the art. In addition, the embodiments should be understood to be illustrative rather than limiting the scope of the present invention, and the essence and scope of the present invention are limited only by the claims. For those skilled in the art, various changes or modifications to the material ingredients and dosages in these embodiments, without departing from the essence and scope of the present invention, also belong to the protection scope of the present invention. Celery seeds, raw materials and reagents used in the present invention are all commercially available. Example

[0014] 1. Extraction of genomic DNA: Take the single dried celery seeds of the celery variety "Uventus" bred by the Vegetable Research Institute of Tianjin Academy of Agricultural Sciences ( Figure 1 A) in a 1.5 mL centrifuge tube ( Figure 1 B); Select a 0.3 mm coarse grinding rod to grind the seeds ( Figure 1 C and 1D); add 100 μL NaOH (0.4 mol / L) to the centrifuge tube and continue to grind the seeds in the centrifuge tube with a grinding rod until they are completely ground ( Figure 1E); then heat the centrifuge tube at 95°C for 3 min and centrifuge at 12000 rpm for 1 min; take 10 μL of the supernatant and transfer it to a new 1.5 mL centrifuge tube, then add 300 μL Tris-HCl (0.1 mol / L, pH 8.0) to prepare the celery genomic DNA ( Figure 1 F).

[0015] 2. PCR amplification and detection: The genomic DNA (No. 1-5) extracted from the above-mentioned single-seed celery was used as a template, and AgActin-F: 5`-CTTCCTGCCATATATGATTGG-3` and AgActin-R: 5`- GCCAGCACCTCGATCTTCATG-3` were used as Actin-specific primers for PCR amplification. The amplification reaction system was: 2 μL of genomic DNA, 0.15 μL of primers AgActin-F / R, 5 μL of 2×Taq mix, and 2.7 μL of ddH2O. Amplification reaction procedure: 94℃, 5min; 94℃, 20s, 56℃, 20s, 72℃, 45s, 30 Cycles; 72℃, 5min. PCR amplification products were detected using 1% agarose gel electrophoresis and fluorescence imaging. It was found that all genomic DNAs were able to correctly amplify specific PCR products ( Figure 2 ), which proved that the genomic DNA extracted from single-grain dry seeds of celery can meet the needs of PCR amplification and can be used for subsequent molecular marker identification of celery seed purity.

[0016] 3. Stability evaluation of the obtained genomic DNA The genomic DNA was extracted from 48 dried celery seeds by alkaline lysis method and stored at 4°C for 7 days. Then, the seeds were used as templates for PCR amplification using Actin-specific primers. Agarose gel electrophoresis showed that all samples could amplify the corresponding specific bands ( Figure 3 A). The extracted genomic DNA was stored at 4°C for 10 days, and PCR amplification and electrophoresis were performed. It was also found that almost all genomic DNA could still amplify the corresponding specific bands ( Figure 3 B). It is proved that the genomic DNA extracted from single-grain dry seeds of celery in this application can be stored for at least 17 days at 4°C and still meet the needs of PCR amplification, and has good stability. Example

[0017] Celery different extraction samples Experimental cycle Timeliness Comprehensive cost Extraction conditions Single leaf 60-70d slow 400 NTD Loose Single seed 30min Fast and efficient 50 NTD strict Conclusion: From obtaining a seed of celery breeding material to extracting its genomic DNA, if celery leaves are used as materials to extract the genome, it takes at least 60-70 days from sowing to leaf growth, while using a single dry seed as a sample can be completed within 30 minutes, which not only has good timeliness, but also can greatly reduce the overall cost. However, a single dry celery seed is small and hard, and the grinding conditions are high when extracting DNA, and the grinding is difficult. Example

[0018] Different vegetable seed sizes Thousand-grain weight Grinding method Degree of grinding Large seeds (such as beans) 300-700 g Oscillating grinder or grinding rod More than 50% Medium seeds (such as cucumber) 20-40 g Oscillating grinder or grinding rod More than 50% Celery (small seeds) 0.4-0.5 g Grinding rod More than 90% Conclusion: When extracting DNA from large or medium-sized vegetable seeds, the machine oscillation grinding method or the grinding rod method is often used, and the grinding rod should be relatively coarse; celery seeds are extremely small, with a thousand-grain weight of only 0.4-0.5g. The machine oscillation grinding method is difficult to effectively grind single-grain dry celery seeds. The grinding rod should not be too coarse, preferably 0.3 mm, and the degree of grinding should be much higher than that of large or medium-sized vegetable seeds to ensure that the grinding obtains sufficient yield to meet the needs of DNA extraction.

Claims

1. A specific primer for extracting a gene from single-grain dry celery seeds, having a nucleotide sequence gene shown in SEQ ID NO.1 and SEQ ID NO.

2.

2. A method for extracting genomic DNA from single-grain dry celery seeds using the nucleotide sequence gene of claim 1, characterized in that Proceed as follows: (1) Extraction of genomic DNA: Take a single dried celery seed and place it in a centrifuge tube; grind the seed with a grinding rod; add 100 μL of NaOH to the centrifuge tube and continue to grind the seeds in the centrifuge tube with the grinding rod until they are completely ground; then heat the centrifuge tube at 95°C for 3-5 min and centrifuge at 12,000 rpm for 1 min; transfer 10 μL of the supernatant to a new centrifuge tube and add 300 μL of Tris-HCl to obtain the celery genomic DNA for later use; (2) PCR amplification and detection: The genomic DNA extracted above was used as a template and AgActin-F: 5`-CTTCCTGCCATATATGATTGG-3`; SEQ ID NO.1 AgActin-R: 5`- GCCAGCACCTCGATCTTCATG-3`SEQ ID NO.2 As Actin PCR amplification was performed using specific primers; (3) PCR amplification products were detected by 1% agarose gel electrophoresis and fluorescence imaging. It was found that all genomic DNA could correctly amplify specific PCR products, which proved that the genomic DNA extracted from single-grain dry celery seeds could meet the needs of PCR amplification and be used for subsequent molecular marker identification of celery seed purity.

3. Application of the method for extracting genomic DNA from single-grain dry seeds of celery as described in claim 2 in rapid identification of celery seed purity; the application refers to: improving the stability of celery seeds and facilitating the identification of celery seed molecular markers.

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