Transgenic corn LD05 transformant specificity detection method and application thereof

Abstract

The invention provides a primer pair, a kit, a PCR (Polymerase Chain Reaction) detection method and application of the PCR detection method in detection of transgenic maize LD05.

Description

Technical Field

[0001] The invention belongs to the field of molecular biotechnology, and in particular relates to a primer pair, a PCR detection kit and a detection method for identifying a corn LD05 transformant. Background Art

[0002] Transformant LD05 is a corn transgenic material that is insect-resistant and resistant to glufosinate herbicide. It has excellent resistance to corn borer and fall armyworm, and good tolerance to glufosinate. This transformant can be used to cultivate insect-resistant and herbicide-resistant corn varieties. Establishing a transformant-specific detection method can provide an effective detection method for the identification and supervision of genetically modified organisms, and provide technical support for the safety management of agricultural genetically modified organisms.

[0003] Transformant LD05 and its detection method have been disclosed, and the patent application number is 2023101327522. The length of the PCR product between the detection primers designed by the disclosed PCR detection method is too large (554bp and 1185bp), which does not meet the requirements of the national standard "Guidelines for the Development of Qualitative PCR Methods for Detection of Components of Transgenic Plants and Their Products" (Announcement No. 2259-4-2015) that "the length of ordinary PCR products should be 120bp to 300bp", and the minimum detection limit has not been determined. Therefore, it is necessary to further establish a new high-sensitivity PCR detection system that meets national standards. Summary of the invention

[0004] To solve the above problems, the present invention provides a novel primer pair, detection kit and detection method for detecting corn LD05 transformants. The present invention can accurately identify corn LD05 transformants and separate them from conventional corn and other transgenic corn materials that do not contain LD05 by designing specific upstream primers and downstream primers. The detection method has good specificity, high sensitivity, and is easy to operate, and is convenient for completing the detection of LD05 samples.

[0005] The present invention provides a set of primer pairs, characterized in that: the primer pairs

[0006] The nucleotide sequence is 5'-AACCCTGGCGTTACCCAACTT-3' and

[0007] 5'-GTGTGATTCGTTCATAAAGGTGTG-3';

[0008] The above primer pairs were selected through software design, combination and PCR test verification, and are located on the genome and vector sequences at the downstream boundary of the exogenous insertion sequence of LD05 transformants.

[0009] The present invention also provides a detection kit, characterized in that: the detection kit comprises the above-mentioned primer pair;

[0010] The present invention also provides a PCR detection method, characterized in that: the above-mentioned detection kit is used for PCR detection, wherein the total volume of the PCR reaction system is 25 μL, the final concentrations of the upstream primer and the downstream primer are both 0.4 μM, and the annealing temperature of the PCR reaction is 58°C.

[0011] The present invention also provides the use of the above primer pair, detection kit, and detection method in detecting LD05 samples;

[0012] Wherein, the corn LD05 transformant contains a nucleic acid molecule with a sequence shown in SEQ ID NO.1.

[0013] The beneficial effects of the present invention are as follows: after software design, combination and PCR test screening, a set of primer pairs is obtained from 11 sets of primer pools, on this basis, a PCR detection method is established and optimized, and the application of the primer pairs, the detection kit and the PCR detection method can specifically detect LD05 samples with a concentration of not less than 0.1%, and can effectively distinguish LD05 materials from other corn materials that do not contain LD05, with good specificity and high sensitivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Other features, objects and advantages of the present application will become more apparent by reading the detailed description of non-limiting embodiments made with reference to the following drawings:

[0015] Figure 1 Specific PCR electrophoresis results of 11 primer pairs. Among them, M: 100bp DNA Ladder; 1: blank control water; 2-3: non-transgenic corn negative control; 4-5: 1% LD05.

[0016] Figure 2 Electrophoresis diagram of specificity test of primer pair combination I. Wherein, M: 100 bp DNA Ladder; 1: blank control water; 2: non-GM corn negative control; 3: positive control (1% LD05); 4-5: other GM corn mixtures; 6-7: GM soybean mixtures; 8-9: GM rape mixtures; 10-11: GM rice mixtures; 12-13: GM cotton mixtures.

[0017] Figure 3 Annealing temperature optimization results. Wherein, M: 100bp DNA Ladder; 1: blank control water; 2-3: non-transgenic corn negative control; 4-5: 1% LD05.

[0018] Figure 4Primer concentration optimization results. Wherein, M: 100bp DNA Ladder; 1: blank control water; 2-3: non-transgenic corn negative control; 4-5: 1% LD05.

[0019] Figure 5 Sensitivity test results. Among them, M: 100bp DNA Ladder; 1-3: 100% LD05; 4-6: 10% LD05; 7-9: 1% LD05; 10-12: 0.5% LD05; 13-15: 0.1% LD05; 16-18: 0.05% LD05; 19-21: non-GMO corn negative control; 22-24: blank control water.

[0020] Figure 6 Detection limit test results. Wherein, M: 100bp DNA Ladder; 1-60: 0.1% LD05; 61: blank control water; 62: non-GMO corn negative control; 63: positive control (1% LD05).

[0021] Figure 7 Sample test results. Among them, M: 100bp DNA Ladder; 1-3: positive control; 4-6: 1% LD05; 7-9: 0.1% LD05; 10: non-GM corn negative control; 11: non-GM rice negative control; 12: non-GM soybean negative control; 13-15: blank control water. DETAILED DESCRIPTION

[0022] The present invention will be further described below in conjunction with the accompanying drawings. The following examples are only used to illustrate the present invention but do not limit the scope of the present invention.

[0023] The term "corn" refers to maize (Zea mays) and includes all plant varieties that can be crossed with maize, including wild maize species.

[0024] The term “including” means “including but not limited to”.

[0025] The term "plant" includes whole plants, plant cells, plant organs, plant protoplasts, plant cell tissue cultures from which plants can be regenerated, plant calli, plant clumps, and intact plant cells in plants or plant parts, such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruits, stalks, roots, root tips, anthers, etc. It should be understood that the parts of transgenic plants within the scope of the present invention include, but are not limited to, plant cells, protoplasts, tissues, calli, embryos, and flowers, stems, fruits, leaves and roots, which are derived from transgenic plants or their progeny that have been previously transformed with the DNA molecules of the present invention and are therefore at least partially composed of transgenic cells.

[0026] The term "gene" refers to a nucleic acid fragment that expresses a specific protein, including regulatory sequences before the coding sequence (5' non-coding sequences) and regulatory sequences after the coding sequence (3' non-coding sequences). "Native gene" refers to a gene found in nature with its own regulatory sequences. "Chimeric gene" refers to any gene that is not a natural gene, which contains regulatory and coding sequences that are not found in nature. "Endogenous gene" refers to a natural gene that is located in its natural location in the genome of an organism. "Foreign gene" is a foreign gene that is now present in the genome of an organism and did not exist originally, and also refers to a gene introduced into a recipient cell through a transgenic step. Foreign genes can include natural genes inserted into non-natural organisms or chimeric genes. "Transgene" is a gene that has been introduced into the genome through a transformation procedure. The site in the plant genome where the recombinant DNA has been inserted can be called an "insertion site" or "target site."

[0027] Transformation procedures that cause random integration of foreign DNA can result in transformants containing different flanking regions, which are specifically contained in each transformant. When recombinant DNA is introduced into plants by traditional hybridization, its flanking regions are usually not changed. Transformants will also contain a unique junction between a segment of heterologous insert DNA and genomic DNA or between two segments of genomic DNA or between two segments of heterologous DNA. "Joint" is the point where two specific DNA fragments connect. For example, the junction is present at the position where the insert DNA connects to the flanking DNA. Junction points are also present in transformed organisms, where two DNA fragments are connected together in a manner found in natural organisms. "Joint DNA" refers to the DNA that contains a junction point.

[0028] LD05 transformants are plants and seeds comprising transgenic corn LD05 and plant cells or regenerable parts thereof, plant parts of the LD05, including but not limited to cells, pollen, ovules, flowers, shoots, roots, stems, leaves and products from corn LD05, such as corn cobs, stover, cornmeal, corn flour and biomass left in corn crop fields.

[0029] The term "primer" is a separate nucleic acid molecule that anneals to a complementary target DNA strand through nucleic acid hybridization, forms a hybrid between the primer and the target DNA strand, and then extends along the target DNA strand under the action of a polymerase (e.g., DNA polymerase). The primer pair of the present invention relates to its use in amplification of a target nucleic acid sequence, for example, by polymerase chain reaction (PCR) or other conventional nucleic acid amplification methods.

[0030] Example 1 Primer design and screening

[0031] The specific detection method of transformants requires the design of primers at the border sequences upstream and downstream of the insertion site, and the PCR amplification product needs to include the exogenous sequence and the maize genome sequence. Therefore, primers must first be designed based on the border sequences of the maize LD05 insertion site.

[0032] 1. Primer design and synthesis

[0033] A portion of the upstream (5' end) or downstream (3' end) border sequence (300-800 bp) is input as a template into the software (such as PrimerPremier 5). The template sequence must contain both the maize genome sequence (at least 150 bp in length) and the exogenous insertion sequence (at least 150 bp in length). The conditions are set in the software: ① The two primers are located in the flanking genomic region and the T-DNA region, respectively, and ② The PCR amplification product is between 120-300 bp. After the software outputs the primer combination, the 11 sets of primers ranked high in the software score and the two sets of primer pairs (X and Y) disclosed in the patent (application number 2023101327522) are selected for synthesis. Primer information is shown in Table 1.

[0034] Table 1 Primer information

[0035]

[0036]

[0037] 2. Preliminary test of primer specificity

[0038] Using 100 ng of transgenic maize LD05 genomic DNA as template, the specificity of primer combination AK was preliminarily tested by PCR experiment.

[0039] Maize genomic DNA was extracted using the CTAB method. The specific steps are as follows:

[0040] 1) Take 0.1 g of corn LD05 sample leaves, grind into powder, add 500-800 μL CTAB, and warm at 65°C for 0.5-1 h;

[0041] 2) Add 700 μL of chloroform or chloroform:isoamyl alcohol (24:1), shake slowly, centrifuge at 12000 rpm for 15 min, and take 400-700 μL of the supernatant;

[0042] 3) Add 1 mL of pre-cooled anhydrous ethanol or isopropanol, mix thoroughly, centrifuge at 12000 rpm for 10 min, and discard the supernatant;

[0043] 4) Wash the precipitate with 75% alcohol, centrifuge at 12000 rpm for 5 min, discard the alcohol, and invert to absorb water and dry;

[0044] 5) Dissolve the DNA in 50 μL ddH2O, take 3-5 μL for electrophoresis, measure the DNA concentration with an ultra-micro UV spectrophotometer and dilute to 0.2 μg / μL for later use.

[0045] PCR reaction system:

[0046]

[0047] The PCR reaction program is:

[0048] Denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 30-60 s (extension time calculated at 60 s / kb), for a total of 35 cycles; extension at 72°C for 5 min.

[0049] The PCR product was subjected to electrophoresis detection. According to the electrophoresis detection results, a primer pair with good specificity (shown as clear bands, high brightness of specific bands, and no non-specific bands) was screened as a candidate primer.

[0050]

[0051] The electrophoresis results of the specific PCR products of the 11 primer pairs are shown in Figure 1 Among them, combinations D and H did not amplify bands, and combination E amplified non-specific bands, so combinations D, E, and H were eliminated. From the brightness of specific bands, combination I had the strongest brightness, followed by combinations J and K, and the brightness of the amplified bands of combinations A, B, C, F, and G was weaker.

[0052] In addition, the combination of X and Y can also obtain brighter specific bands. The detection results are shown in the published patent (application number 2023101327522). Figure 5 shown.

[0053] 3. Preliminary test of primer sensitivity

[0054] The sensitivity test of primer combinations A, B, C, F, G, I, J, K, X, and Y was further conducted. The LD05 genomic DNA was gradiently diluted with non-transgenic maize negative control genomic DNA to obtain LD05 maize genomic DNA solutions with concentrations of 10%, 5%, 1%, and 0.5%, respectively, and used as templates to conduct preliminary sensitivity tests of the above 10 primer pairs through PCR experiments. The test results are shown in Table 2.

[0055] Table 2 Preliminary sensitivity test results

[0056]

[0057] +: indicates that there is a stripe of the expected size; -: indicates that there is no stripe of the expected size

[0058] The primer pairs of combination A, B, C, F and G can only detect LD05 with a concentration of not less than 5%, the primer pairs of combination J, K and X can detect LD05 with a concentration of not less than 1%, and the primer pairs of combination I and Y can detect LD05 with a concentration of not less than 0.5%. It can be seen that the sensitivity of combination I and Y is the highest.

[0059] Therefore, combination I and Y are the primer pair combinations with the best specificity and highest sensitivity, and are used as candidate primers for specificity screening of the next mixed sample.

[0060] IV. Specificity test of candidate combinations I and Y

[0061] Different crops and their main commercial transformants were selected for testing, and the acceptance criterion was to obtain the expected amplification only in positive samples.

[0062] The specificity verification samples are as follows:

[0063] (1) Negative control (non-GM corn);

[0064] (2) positive control (transgenic corn LD05, concentration 1%);

[0065] (3) Other genetically modified corn mixtures (Bt11, Bt176, MON810, MON863, GA21, NK603, T25, TC1507, MON89034, MON88017, 59122, MIR604, 3272, MON87460, DAS40278-9, 4114, MON87427, 5307, 1% each, with non-genetically modified corn as filler);

[0066] (4) genetically modified soybean mixture (GTS40-3-2, MON89788, A5547-127, A2704-12, 356043, 305423, CV127, MON87701, MON87708, MON87769, MON87705, FG72, DAS81419-2, 1% each, with non-genetically modified soybean as filler);

[0067] (5) GM rapeseed mixture (MS1, MS8, RF1, RF2, RF3, T45, Oxy235, Topas19 / 2, MON88302, 73496, 1% each, with non-GM rapeseed as filler);

[0068] (6) genetically modified rice mixture (TT51-1, KF-6, KMD-1, M12, KF-8, KF-2, G6H1, T1C-19, 1% each, with non-genetically modified rice as filler);

[0069] (7) A mixture of genetically modified cotton (MON1445, MON531, MON15985, LLCOTTON25, MON88913, GHB614, COT102, 1% each, with non-genetically modified cotton as filler).

[0070] The test results showed that primer pair I amplified the expected DNA fragment only from the positive control (1% LD05) sample, but did not amplify the expected size band in other mixed samples, indicating that the primer combination has a high specificity ( Figure 2 ); while primer pair Y not only amplified the expected DNA fragment from the positive control (1% LD05) sample, but also amplified a band in the transgenic cotton mixture, indicating that the specificity of primer pair Y is not as good as primer combination I. The specificity test results are shown in Table 3.

[0071] Table 3 Specificity test results of combination I and Y

[0072]

[0073] √: indicates that there are stripes of the expected size; -: indicates that there are no stripes; ×: indicates that there are stripes that do not meet expectations

[0074] In summary, combination I with a PCR product size of 226 bp is a primer pair that meets the national standard detection requirements, and its specificity and sensitivity are better than other combinations including combination X and Y.

[0075] Example 2 Reaction system optimization

[0076] In order to further improve the specificity and sensitivity of the detection method based on the combination I primer pair, the present method continues to optimize the PCR reaction system and procedure.

[0077] Using 1% transgenic maize LD05 genomic DNA as template, the annealing temperature was optimized and five annealing temperatures (54°C, 56°C, 58°C, 60°C and 62°C) were set for PCR reaction. Figure 3 ), and referring to the national standard “Qualitative PCR Method for Detection of Standard Genes in Corn for Components of Genetically Modified Plants and Their Products” (Announcement No. 1861-3-2012), 58°C, where the target band was the brightest, was selected as the optimal annealing temperature for the PCR reaction.

[0078] Using 1% transgenic corn LD05 genomic DNA as a template, the final concentration gradient of primers was set to 0.1μM, 0.2μM, 0.4μM, 0.6μM and 0.8μM, and the PCR reaction test was performed at an annealing temperature of 58°C. According to the test results ( Figure 4 ), and referring to the national standard “Qualitative PCR Method for Detection of Standard Genes in Corn in Genetically Modified Plants and Their Products” (Announcement No. 1861-3-2012), 0.4 μM, the brightest target band, was selected as the optimal final concentration of primers in the PCR system.

[0079] Based on the above experimental results, the transgenic maize LD05 transformant-specific PCR detection method selected a primer final concentration of 0.4 μM and an annealing temperature of 58°C as the final reaction conditions. The optimized PCR detection method is as follows:

[0080] PCR reaction system:

[0081]

[0082] The reaction procedure is:

[0083] Denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 30 s, for a total of 35 cycles; extension at 72°C for 5 min.

[0084] Example 3 Detection Limit

[0085] The minimum concentration of the sample that can be detected by the PCR detection method, i.e., the detection limit, was determined by the sensitivity test. Using the primer pair 1 described in Example 1 and the final reaction system optimized in Example 2, the genomic DNA of transgenic maize LD05 with a concentration of 100% was gradiently diluted with the genomic DNA of the non-transgenic maize negative control to prepare samples with concentrations of 100%, 10%, 1%, 0.5%, 0.1%, and 0.05%, respectively, and a sensitivity test was performed (calculated according to adding 100ng of DNA template to the PCR reaction system).

[0086] The test results showed that amplification products with the same expected fragment size could be stably detected in samples with a concentration of 0.05% and above (see Figure 5 ), referring to the published standards for qualitative PCR detection methods for similar genetically modified products, and taking into account the safety during the implementation of this method, it is proposed to determine the detection limit of this method at 0.1%.

[0087] In order to further verify the detection limit of this method, 60 parallel tests were performed on samples with a concentration of 0.1%. The results showed that the expected target bands could be amplified in all 60 parallel tests ( Figure 6), therefore, the detection limit of this detection method was determined to be 0.1% (calculated based on the addition of 100 ng DNA template into the PCR reaction system).

[0088] Example 4 Sample Detection

[0089] The maize transformant LD05 was extracted using the DNA extraction method (CTAB method) of Example 1 as a positive control, and wild-type maize, rice, and soybean purchased from the market were used as negative controls. The positive control, the negative control, and other genetically modified materials available in the laboratory were mixed to obtain test samples with LD05 concentrations of 1% and 0.1%, respectively. The PCR detection method optimized in Example 2 was applied, and different experimenters were assigned to use different PCR instruments to test the samples.

[0090] The test results showed that the negative control samples had no amplified bands, indicating that the method had good specificity; the positive control and 1% and 0.1% LD05 samples were amplified, and the sensitivity and detection limit met the requirements, which also showed that the method had good repeatability ( Figure 7 ).

[0091] The above is only a preferred embodiment of the present invention, and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment as above, it is not used to limit the present invention. Any technician familiar with this profession can use the technical content disclosed above to make changes or modifications to equivalent embodiments of equivalent changes without departing from the scope of the technical solution of the present invention. However, any simple modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A primer pair, characterized in that: The nucleotide sequences of the primer pair are 5'-AACCCTGGCGTTACCCAACTT-3' and 5'-GTGTGATTCGTTCATAAAGGTGTG-3'.

2. A detection kit, characterized in that: The detection kit comprises the primer pair according to claim 1.

3. A detection method, characterized in that: The detection kit according to claim 2 is used for PCR detection, wherein the total volume of the PCR reaction system is 25 μL, the final concentrations of primer 1 and primer 2 are both 0.4 μM, and the annealing temperature of the PCR reaction is 58°C.

4. Use of the primer pair according to claim 1, the detection kit according to claim 2, and the detection method according to claim 3 in detecting genetically modified corn LD05; in, The LD05 transformant contains the nucleic acid molecule shown in SEQ ID NO.1.

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