Method for simply and quickly preparing PCR (Polymerase Chain Reaction) template aiming at immature embryos of Chinese cabbages and application of PCR template

Abstract

The invention belongs to the technical field of biology, and particularly relates to a method for simply and rapidly preparing a PCR template for immature embryos of Chinese cabbages and application of the PCR template. Specifically, components of an extraction buffer solution are optimized, so that the interference of polysaccharides, proteins, polyphenols and lipids on later PCR amplification is effectively reduced, the extraction step is simplified, no toxic reagent needs to be used for re-purification, only one-time heating, one-time boiling and one-time centrifugation are needed, meanwhile, the process can be directly completed in a 96-deep-hole plate, and the extraction time is shortened. The obtained supernate can be directly used for PCR (Polymerase Chain Reaction) preparation, and can meet the long-time storage requirement without degradation, so that the later molecular marker detection of the Chinese cabbage and the high-throughput application requirement are facilitated.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a method for simply and quickly preparing a PCR template for immature embryos of Chinese cabbage and an application thereof. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Chinese cabbage (Brassica campestris L.ssp.pekinensis (Lour) Olsson) is an important vegetable crop, with a large amount of seeds used each year. Because it is a frequently outcrossed crop, self-pollution is serious, but hybrid vigor is obvious. In production, self-incompatible lines are mainly used for hybrid breeding. Due to the characteristics of the parents themselves and the influence of climate during the seed production process, the time of the first and last flowering periods of the father / maternal parent is inconsistent, and self-fertilized seeds are prone to appear, affecting the proportion of hybrids. Therefore, before the hybrids are sold, the hybrid purity identification is particularly important. Commonly used seed purity identification is field identification and molecular marker identification. Compared with field identification, molecular marker identification is not affected by tissue specificity and external environment, and has the characteristics of accurate, rapid, and highly repeatable identification. It has become a true and reliable method for detecting the purity of crop hybrids.

[0004] Hybrid purity identification through molecular markers generally uses mature seeds, and the seeds of the current year cannot be identified, and some may not be identified even next year, so they can only be stored in warehouses. Since the purity of seeds from different plots is unknown, in order to reduce the risk of mixing, they need to be harvested and stored separately, which is cumbersome and has high storage costs. In recent years, with the continuous increase in labor costs and seed storage costs, the seed production costs of seed companies have increased. How to identify hybrid purity before seed harvest has become a more urgent problem to be solved.

[0005] The quality of DNA extraction is a key factor in determining the success or failure of molecular marker detection technology. Traditional methods include CTAB method, SDS method, alkaline lysis method, etc. Although the extracted DNA quality is very high, the method steps are cumbersome, time-consuming, and some toxic and irritating reagents must be used, which are harmful to the operator's body. With the continuous improvement of this technology, many new extraction methods have emerged, such as kit method, magnetic bead method, etc., but the cost is high, and the extraction speed still cannot meet the requirements of high throughput. There are also some simple and rapid extraction methods, but the extracted DNA has more impurities, the amplification stability is not good, and the scope of promotion and application is limited. In particular, the DNA extraction of immature embryos of Chinese cabbage, during the development of immature embryos, accumulates more polysaccharides, proteins, polyphenols, oils and other substances, which seriously affect the PCR amplification efficiency, and there is no report on the method for extracting DNA from immature embryos of Chinese cabbage. Summary of the invention

[0006] In view of the deficiencies in the prior art, the present invention provides a method for simply and quickly preparing a PCR template for immature embryos of Chinese cabbage and application thereof.

[0007] Specifically, the present invention relates to the following technical solutions:

[0008] A first aspect of the present invention provides a method for simply and quickly preparing a PCR template for immature embryos of Chinese cabbage, the method comprising:

[0009] Immature embryos of Chinese cabbage were obtained, and total DNA was extracted using extraction buffer;

[0010] The components of the extraction buffer include: 40-60mM EDTA, 20-30mM Tris-HCL, 1-10% CHELEX-100 suspension, 0.05-0.2μg / mL proteinase K, 10%-15% sodium dodecylbenzene sulfonate, 2.0-3.0M ammonium sulfate, and the pH is alkaline.

[0011] The present invention finds through research that by reasonably adding various raw material components, especially adding ammonium sulfate and sodium dodecylbenzene sulfonate, to the extraction buffer, the interference of polysaccharides, proteins, polyphenols and lipids in immature embryos of cabbage on the later PCR can be effectively reduced, thereby simplifying the extraction steps.

[0012] In another specific embodiment of the present invention, the method comprises extracting total DNA from immature embryos of Chinese cabbage by boiling lysis method.

[0013] The second aspect of the present invention provides the above method for preparing total DNA of immature embryos of Chinese cabbage.

[0014] The third aspect of the present invention provides the use of the above method or the total DNA of immature Chinese cabbage embryos as a PCR template in the detection of molecular markers in Chinese cabbage.

[0015] Beneficial technical effects of one or more of the above technical solutions:

[0016] The above technical scheme optimizes the extraction buffer components, thereby effectively reducing the interference of polysaccharides, proteins, polyphenols and lipids on the later PCR amplification, and simplifies the extraction steps. There is no need to use toxic reagents for re-purification. Only one heating, one boiling and one centrifugation are required. It can be completed directly in a 96-deep-well plate. The obtained supernatant can be directly used for PCR preparation. At the same time, it can meet the storage requirements for a long time without degradation, which is convenient for the later molecular marker detection and high-throughput application requirements of Chinese cabbage. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0018] Figure 1 It is the amplification effect of PCR template extracted by different extracting solutions in the present invention; wherein, M is a molecular weight standard, P1 and P2 are two parents, 1 to 22 are identified hybrids, wherein A is a PCR template prepared by the extract of treatment 1, and B is a PCR template prepared by the extract of treatment 2.

[0019] Figure 2 The invention compares the amplification effects of different storage conditions and storage times of the PCR template prepared by the boiling method of the present invention; wherein, M is a molecular weight standard, A is 3 repeats of T1, B is 3 repeats of T2, C is 3 repeats of T3, D is 3 repeats of T4, a is 3 repeats of T5, b is 3 repeats of T6, c is 3 repeats of T7, and d is 3 repeats of T8.

[0020] Figure 3 The amplification effect of the PCR template of the immature embryos of the Chinese cabbage 'LHQ70' hybrid prepared by the boiling method in the present invention; wherein M is a DNA molecular weight standard, P1 and P2 are two parents, and the others are hybrids.

[0021] Figure 4 The amplification effect of the PCR template of the immature embryos of the Chinese cabbage 'Niu Pai 19' hybrid prepared by the boiling method in the present invention. DETAILED DESCRIPTION

[0022] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0023] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components and / or combinations thereof. If the experimental methods of specific conditions are not indicated in the following specific embodiments, they are generally in accordance with conventional methods and conditions in the art, and such techniques and conditions are fully explained in the literature.

[0024] The present invention is further described in conjunction with specific examples. The following examples are only for explaining the present invention and are not intended to limit the content thereof. If the specific experimental conditions are not specified in the examples, they are usually carried out under conventional conditions or under conditions recommended by the sales company; the materials and reagents used in the examples, unless otherwise specified, can be purchased through commercial channels.

[0025] In a typical embodiment of the present invention, a method for simply and quickly preparing a PCR template for immature embryos of Chinese cabbage is provided, the method comprising:

[0026] Immature embryos of Chinese cabbage were obtained, and total DNA was extracted using extraction buffer;

[0027] The components of the extraction buffer include: 40-60mM EDTA, 20-30mM Tris-HCl, 1-10% CHELEX-100 suspension, 0.05-0.2μg / mL proteinase K, 10%-15% sodium dodecylbenzene sulfonate, 2.0-3.0M ammonium sulfate, and the pH is alkaline.

[0028] In another specific embodiment of the present invention, the components of the extraction buffer include: 50 mM EDTA, 25 mM Tris-HCl, 5% CHELEX-100 suspension, 0.1 μg / mL proteinase K, 10% sodium dodecylbenzene sulfonate, 2.5 M ammonium sulfate, pH 8.0.

[0029] The present invention finds through research that by reasonably adding various raw material components, especially adding ammonium sulfate and sodium dodecylbenzene sulfonate, to the extraction buffer, the interference of polysaccharides, proteins, polyphenols and lipids in immature embryos of cabbage on the later PCR can be effectively reduced, thereby simplifying the extraction steps.

[0030] In another specific embodiment of the present invention, the method comprises extracting total DNA from immature embryos of Chinese cabbage by boiling lysis method.

[0031] In another specific embodiment of the present invention, immature Chinese cabbage embryos are separated, and an extraction buffer is added thereto for homogenization, followed by heating and centrifugation to obtain a supernatant for later use.

[0032] In order to improve the homogenization effect, zirconium oxide grinding beads can be added thereto for homogenization and grinding.

[0033] The specific conditions of the heating treatment are: heating treatment at 90-100°C for 8-10 minutes, and the specific conditions of the centrifugation are centrifugation at 3000-5000 rpm for 5-20 minutes;

[0034] In another specific embodiment of the present invention, the above method is provided for preparing total DNA of immature embryos of Chinese cabbage.

[0035] In another specific embodiment of the present invention, the above method or the use of total DNA of immature Chinese cabbage embryos as a PCR template in the detection of molecular markers in Chinese cabbage is provided.

[0036] In another specific embodiment of the present invention, the application includes using primers to perform PCR amplification; further, the nucleotide sequence of the primers is shown in SEQ ID NO.1-6.

[0037] The present invention is further explained by the following examples, but it is not intended to limit the present invention. It should be understood that the following examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0038] Example

[0039] (1) Preparation of different extracts

[0040] Preparation of 5% CHELEX-100 suspension:

[0041] Weigh 10 g CHELEX-100, add 200 ml ultrapure water, and store at 4°C.

[0042] Preparation of proteinase K mother solution: Dry powder proteinase K is dissolved in a buffer solution of 50mM Tris-HCl (pH 7.5) and 10mM CaCl2. The mother solution is diluted 1000 times, i.e. 0.1g / mL. Gently shake the solution until the proteinase K is completely dissolved. Do not vortex to mix. After filtration and sterilization, divide into small portions and store at -20℃.

[0043] Preparation of TE solution: Add 50 mM EDTA and 25 mM Tris-HCl to 1 L of purified water and adjust the pH to 8.0.

[0044] (2) Two treatments were set up to verify the amplification effect of PCR templates extracted with different extracts

[0045] Two treatments were set up. Treatment 1 (CK): After mixing equal volumes of the prepared Chelex-100 suspension and TE solution, proteinase K was added. The final composition and content of each component were: 50mM EDTA, 25mM Tris-HCL, 5% CHELEX-100 suspension, 0.1μg / mL proteinase K, pH8.0.

[0046] Treatment 2: After mixing equal volumes of the prepared Chelex-100 and TE solutions, proteinase K, sodium dodecylbenzenesulfonate and ammonium sulfate were added. The final composition and content of each component were: 50mM EDTA, 25mM Tris-HCL, 5% CHELEX-100 suspension, 0.1μg / mL proteinase K, 10% sodium dodecylbenzenesulfonate, 2.5M ammonium sulfate, pH8.0.

[0047] The true leaves of Chinese cabbage selected from the parent and the immature embryos of Chinese cabbage selected from the hybrid were used to prepare PCR templates using the extracts of treatment 1 and treatment 2, respectively. The specific preparation method is as follows: 2 4mm diameter zirconium oxide grinding beads were transferred to each well of a 96-well deep-well plate in advance, the immature embryos of Chinese cabbage were peeled off, transferred to a 96-well deep-well plate, 300μL of extraction buffer was added, homogenized using a paint mixer, heated at 90°C for 10min, centrifuged at 4000 rpm for 15min after cooling, and the supernatant was taken as a PCR template for PCR amplification. PCR (10μL) system: 2X mix 5μL, upstream and downstream primer mixture (each 10μM) 1μL (final concentration 1μM), PCR template prepared by boiling lysis method 1μL, 25mM MgCl2 solution 3μL.

[0048] in, Figure 1 The primers used were Zt240501, Figure 2 and Figure 4 The primers used were Z23125, Figure 3 The primer used was PLI-14, and the primer sequences are shown in Table 1.

[0049] Table 1 Specific primers used in the examples

[0050]

[0051]

[0052] The results are shown in the figure.

[0053] Depend on Figure 1It can be seen that for immature embryos, the PCR template prepared in treatment 2 has the best amplification effect.

[0054] (3) Verification of storage time of PCR templates prepared by boiling method: PCR templates prepared by boiling method were stored at 4°C and -20°C, respectively, with 4 time treatments, for a total of 8 treatments.

[0055] 4℃: T1: 7 days, T2: 30 days, T3: 90 days, T4: 180 days.

[0056] Repeat 3 times each, use Z23125 as the main strain of Chinese cabbage 'Niu Brand No. 19' to make a PCR 10μL system:

[0057] -20℃: T5: 7 days, T6: 30 days, T7: 90 days, T8: 180 days, with 3 replicates for each treatment.

[0058] Depend on Figure 2 It can be seen that the PCR amplification effect of the PCR template prepared by the boiling method is acceptable when stored at 4°C for 90 days, but the yield is reduced. Therefore, it can be stored at 4°C for 90 days; the PCR amplification effect does not change when stored at -20°C for 180 days. Therefore, storage at -20°C for 180 days does not affect PCR amplification.

[0059] Depend on Figure 3 , Figure 4 It can be seen that the amplification rates are 98.94% and 100% respectively, and the bands are clear and bright, indicating that the PCR template extracted by this extraction method is of good quality and can meet the needs of identifying immature embryos of hybrids.

[0060] (5) Comparative test of PCR templates prepared by extracts with ammonium sulfate and sodium dodecylbenzene sulfonate added (implementation group) and extracts without ammonium sulfate and sodium dodecylbenzene sulfonate added (control group)

[0061] 46 hybrid materials were randomly sampled under the same conditions, and three biological replicates were performed to calculate the average amplification rate of the three times. As can be seen from Table 2, 46 hybrid PCR templates of Chinese cabbage materials were prepared using the implementation group and the control group, respectively. The amplification rate of the implementation group was above 98%, the bands were clear, and there were no mixed bands, while only 22 materials in the control group had an amplification rate of above 95%, but there were many mixed bands and poor repeatability. The amplification rates of the other 24 materials were 12.76-94.68%, the bands were fuzzy, there were many mixed bands, and the results could not be determined.

[0062] Table 2 PCR amplification of 46 hybrid materials of Chinese cabbage

[0063]

[0064] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for simply and quickly preparing PCR templates for immature Chinese cabbage embryos, characterized in that: The method comprises: Immature embryos of Chinese cabbage were obtained, and total DNA was extracted using extraction buffer; The components of the extraction buffer include: 40-60mM EDTA, 20-30mM Tris-HCL, 1-10% CHELEX-100 suspension, 0.05-0.2μg / mL proteinase K, 10%-15% sodium dodecylbenzene sulfonate, 2.0-3.0M ammonium sulfate, and the pH is alkaline.

2. The method according to claim 1, characterized in that The components of the extraction buffer include: 50mM EDTA, 25mM Tris-HCL, 5% CHELEX-100 suspension, 0.1μg / mL proteinase K, 10% sodium dodecylbenzene sulfonate, 2.5M ammonium sulfate, pH8.

0.

3. The method according to claim 1, characterized in that The method comprises the steps of extracting total DNA from immature embryos of Chinese cabbage by a boiling lysis method.

4. The method according to claim 3, characterized in that The method comprises: separating immature Chinese cabbage embryos, adding extraction buffer thereto, performing homogenization treatment, and then heating and centrifuging to obtain supernatant for standby use.

5. The method according to claim 4, characterized in that: Zirconia grinding beads were added thereto for homogenization and set aside.

6. The method according to claim 4, characterized in that The specific conditions of the heating treatment are: heating treatment at 90-100° C. for 8-10 minutes, and the specific conditions of the centrifugation are centrifugation at 3000-5000 rpm for 5-20 minutes.

7. The method of any one of claims 1 to 6 for preparing total DNA of immature embryos of Chinese cabbage.

8. Use of the method according to any one of claims 1 to 6 or the total DNA of immature Chinese cabbage embryos according to claim 7 as a PCR template in the detection of molecular markers in Chinese cabbage.

9. The use according to claim 8, characterized in that The application involves using primers to perform PCR amplification.

10. The use according to claim 9, characterized in that: The primers are shown as SEQ ID NO.1-6.

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