Primer group for homozygosity and heterozygosity identification of transgenic maize ND207 and detection method

Abstract

The invention discloses a primer group for homozygosity and heterozygosity identification of transgenic maize ND207 and a detection method. The invention provides a primer pair group. The primer pair group consists of a primer pair 1 (SEQ ID No.1 and SEQ ID No.2) and a primer pair 2 (SEQ ID No.3 and SEQ ID No.4); the primer pair 1 is used for specifically detecting whether to-be-detected corn is transgenic corn ND207 or not; the primer pair 2 is used for detecting whether the insertion of an exogenous target gene in the transgenic maize ND207 is heterozygous or homozygous. Two pairs of primers are designed for the transgenic maize ND207, a set of PCR detection method for homozygosis and heterozygosis of the transgenic maize ND207 is established, and the PCR detection method is of great significance to screening of homozygous transformants and improvement of seed purity and quality in the breeding process.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a primer set and a detection method for identifying pure heterozygous transgenic corn ND207. Background Art

[0002] In 1990, Monsanto and Decathlon in the United States took the lead in the research of transgenic insect-resistant corn, using gene gun technology to introduce exogenous Bt genes into corn varieties, and successfully obtained transgenic corn with insect-resistant traits. Subsequently, Bt genes were widely used in the development of transgenic insect-resistant corn varieties. In 1995, Syngenta successively cultivated BT176 and BT11 transgenic events; Monsanto, Pioneer, Bayer and other companies subsequently launched a large number of transgenic insect-resistant corn with Cry protein and Vip3A protein as the core, MON810, DBT418, TC1507, MON863, MIR162, 5307, etc., and based on these materials, hybrid combinations were carried out to successfully obtain transgenic corn with multiple insect-resistant gene aggregation traits (https: / / www.isaaa.org / gmapprovaldatabase / eventslist / default.asp). Since 2008, with the support of the National Major Transgenic Project, China has also launched eight excellent transformants of transgenic insect-resistant and herbicide-resistant corn, including ND207 from China National Forestry Seed Group Co., Ltd. and China Agricultural University, DBN9936, DBN9501, DBN3601T from Beijing Dabeinong Biotechnology Co., Ltd., Ruifeng 125 and Ruifeng 8 from Hangzhou Ruifeng Biotechnology Co., Ltd. and Zhejiang University, and Bt11×GA21 and Bt11×MIR162×GA21 from China Seed Group Co., Ltd. The promotion of insect-resistant and herbicide-resistant transgenic corn not only reduces the use of pesticides and production costs, but also increases the yield and quality of crops due to the reduction of pest damage; by planting transgenic corn, the traditional farming model has been changed on a limited arable land area, the farming method has been simplified, manpower and material resources have been saved, and the dual improvement of ecological and economic benefits has been achieved, which is of great significance to ensuring national food security.

[0003] Transgenic corn ND207 is a transgenic insect-resistant corn variety developed by China Agricultural University. This variety uses Agrobacterium infection to transfer the codon-optimized lepidopteran pest resistance genes mCry1Ab and mCry2Ab into the hybrid combination of corn inbred lines HiIIA and HiIIB with high transformation efficiency, and is screened with the screening marker gene bar. Then it is backcrossed and introduced into the Zheng 58 inbred line, and an excellent transformant ND207 is obtained through multiple tests such as glufosinate herbicide screening, insect resistance identification, and target protein expression in each generation. At present, transgenic corn ND207 has obtained the agricultural genetically modified biosafety certificate and has important industrial application prospects (Chang Lijuan et al. Qualitative PCR detection method and standardization of transformation events of transgenic corn ND207. Acta Agronomica Sinica, 2023, Issue 07).

[0004] It is known that the expression of exogenous genes in plants is affected by their chromosomal location, which may be due to the proximity of chromatin structure (such as heterochromatin) or transcriptional regulatory elements (such as enhancers) to the integration site. For this reason, it is usually necessary to screen a large number of events before it is possible to identify events that can be commercialized (i.e., events in which the introduced target gene is optimally expressed). At present, the detection methods for transgenic insertion elements can be divided into screening methods, gene-specific methods, construction-specific methods, and transformant-specific detection methods. The establishment of a transformant-specific PCR detection method must be cloned into the flanking sequence of the insertion site, in which one PCR primer is designed on the genome of the transformed recipient and the other is designed on the exogenous insertion fragment. Since the insertion site of the transformant is determined to be unique, the specific information of the insertion position of the exogenous gene in the transgenic plant can be determined by transformant-specific PCR detection.

[0005] In the process of transgenic breeding, obtaining homozygous plants is a very critical step and an important factor in ensuring seed purity. In addition to transformant-specific detection, identification of pure heterozygous transgenic plants at the molecular level can also be done by amplifying the transgenic insertion site sequence and the genome sequences at both ends using the PCR method. Rapid and efficient screening of homozygous plants can significantly accelerate the process of transgenic breeding, so the method of identifying pure heterozygous transgenic plants has important application value. Summary of the invention

[0006] The purpose of the present invention is to provide a rapid and accurate detection method for identifying the homozygous transgenic corn ND207.

[0007] In a first aspect, the invention claims a set of primer pairs.

[0008] The primer pair set claimed in the present invention consists of primer pair 1 and primer pair 2;

[0009] The primer pair 1 consists of two primers shown in SEQ ID No.1 and SEQ ID No.2;

[0010] The primer pair 2 consists of two primers shown in SEQ ID No.3 and SEQ ID No.4;

[0011] The primer pair 1 is used to specifically detect whether the tested corn is the transgenic corn ND207; the primer pair 2 is used to detect whether the insertion of the exogenous target gene in the transgenic corn ND207 is heterozygous or homozygous.

[0012] In transgenic corn ND207, the exogenous target genes are insect-resistant genes mcry1Ab and mcry2Ab, and herbicide-resistant gene (selection marker gene) bar. The specific information is as follows: Figure 1 Same as below.

[0013] In a second aspect, the present invention claims the use of the primer pair set described in the first aspect above in the following aspects: specifically detecting whether the tested corn is transgenic corn ND207, and detecting whether the insertion of the exogenous target gene in the transgenic corn ND207 is heterozygous or homozygous.

[0014] In a third aspect, the present invention claims a method that can both specifically detect whether the tested corn is the transgenic corn ND207 and detect whether the insertion of the exogenous target gene in the transgenic corn ND207 is heterozygous or homozygous.

[0015] The method claimed in the present invention can not only specifically detect whether the tested corn is the transgenic corn ND207, but also detect whether the insertion of the exogenous target gene in the transgenic corn ND207 is heterozygous or homozygous, and can include the following steps:

[0016] (A1) using the genomic DNA of the corn to be tested as a template and using the primer pair 1 described in the first aspect above to perform PCR amplification;

[0017] If a band of 396 bp appears in the electrophoresis result, the maize to be tested is or is a candidate for transgenic maize ND207, and the (A2) test is continued; otherwise (no amplified band), the maize to be tested is not or is not a candidate for transgenic maize ND207, and the (A2) test is not required;

[0018] (A2) using the genomic DNA of the test corn detected as or a candidate for transgenic corn ND207 in (A1) as a template, and using the primer pair 2 in the first aspect above to perform PCR amplification;

[0019] If a band of 891 bp appears in the electrophoresis result, the insertion of the exogenous target gene in the tested corn is heterozygous; otherwise (no amplified band), the insertion of the exogenous target gene in the tested corn is homozygous.

[0020] In a fourth aspect, the present invention claims a primer pair.

[0021] The primer pair claimed in the present invention is primer pair 1 or primer pair 2;

[0022] The primer pair 1 consists of two primers shown in SEQ ID No.1 and SEQ ID No.2;

[0023] The primer pair 2 consists of two primers shown in SEQ ID No.3 and SEQ ID No.4;

[0024] The primer pair 1 is used to specifically detect whether the tested corn is the transgenic corn ND207; the primer pair 2 is used to detect whether the insertion of the exogenous target gene in the transgenic corn ND207 is heterozygous or homozygous.

[0025] In a fifth aspect, the present invention claims protection for any of the following uses of the primer pair described in the fourth aspect above:

[0026] (B1) Use of the primer pair 1 described in the fourth aspect above in specifically detecting whether the tested corn is transgenic corn ND207;

[0027] (B2) Use of the primer pair 2 described in the fourth aspect above in detecting whether the insertion of the exogenous target gene in the transgenic corn ND207 is heterozygous or homozygous.

[0028] In a sixth aspect, the present invention claims protection for any of the following methods:

[0029] Method I: A method for specifically detecting whether a tested corn is a transgenic corn ND207, which may include the following steps: using the genomic DNA of the tested corn as a template, and using the primer pair 1 in the fourth aspect above to perform PCR amplification;

[0030] If a 396 bp band appears in the electrophoresis result, the corn to be tested is or is a candidate for transgenic corn ND207; otherwise (no amplified band), the corn to be tested is not or is not a candidate for transgenic corn ND207.

[0031] Method II: A method for detecting whether the insertion of an exogenous target gene in transgenic corn ND207 is heterozygous or homozygous, which may include the following steps: using the genomic DNA of the transgenic corn ND207 to be tested as a template, and using the primer pair 2 in the fourth aspect above to perform PCR amplification;

[0032] If a band of 891 bp appears in the electrophoresis result, the insertion of the exogenous target gene in the transgenic corn ND207 to be tested is heterozygous; otherwise (no amplified band), the insertion of the exogenous target gene in the transgenic corn ND207 to be tested is homozygous.

[0033] In the above-mentioned related aspects, the corn to be tested may be a sexual hybrid progeny or a self-pollinated progeny of the transgenic corn ND207.

[0034] In the above-mentioned related aspects, the reaction conditions for performing the PCR amplification are any of the following:

[0035] (1) Pre-denaturation at 95°C for 3 min; 32 cycles of denaturation at 95°C for 15 s, annealing at 56°C for 15 s, and extension at 72°C for 30 s; final extension at 72°C for 5 min.

[0036] (2) Pre-denaturation at 95°C for 3 min; 32 cycles of denaturation at 95°C for 15 s, annealing at 56°C for 15 s, and extension at 72°C for 60 s; final extension at 72°C for 5 min.

[0037] Among them, the primer pair 1 is preferably applicable to the reaction conditions shown in (1); the primer pair 2 is preferably applicable to the reaction conditions shown in (2).

[0038] In the above-mentioned related aspects, the final concentrations of the upstream and downstream primers in the reaction system (25 μL) during the PCR amplification are both 0.4 μM, and the amount of the DNA template is less than 0.25 μg / 25 μL.

[0039] In the seventh aspect, the present invention claims protection for the use of the primer pair group described in the first aspect above or the primer pair described in the fourth aspect above or the method described in the second aspect above or the third aspect above in the following: screening for homozygous strains for the insertion of an exogenous target gene in the sexual hybrid offspring or self-pollinated offspring of transgenic corn ND207.

[0040] In the eighth aspect, the present invention claims protection for the use of the primer pair group described in the first aspect above or the primer pair described in the fourth aspect above or the method described in the second aspect above or the third aspect above in the following applications: improving seed purity and / or seed quality during the production of transgenic insect-resistant and herbicide-resistant corn seeds.

[0041] In the present invention, the transgenic corn ND207 in which the exogenous target gene is inserted into both chromosome sets is homozygous, and the transgenic corn ND207 in which the exogenous target gene is inserted into only one chromosome set is heterozygous. Figure 1 shown.

[0042] The present invention designs a first primer located at a flanking sequence and a second primer located at an insertion sequence for transgenic corn ND207, and on this basis, further introduces a third primer and a fourth primer of the flanking sequences at both ends of the insertion site, and establishes a set of PCR detection methods for homozygous transgenic corn ND207. The present invention is of great significance for screening homozygous transformants in the breeding process and improving seed purity and quality.

[0043] The transgenic corn ND207 of the present invention has good resistance to lepidopteran insects and good tolerance to glufosinate herbicide, and has no effect on yield. By identifying the pure and heterozygous insertion sites of its sexual hybrid offspring, the seed purity and seed quality in the seed production process can be effectively improved, and it will also help to comply with relevant laws and regulations and ensure national food security. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 This is a schematic diagram of the vector information of the exogenous target genes (mcry1Ab, mcry2Ab, bar) in transgenic corn ND207 and the primer positions for PCR detection.

[0045] Figure 2 The figure is the result of ND207-F0 / ND207-R0 amplification. In the figure, NOD207-1 to ND207-31 are 31 transgenic maize ND207 plants tested.

[0046] Figure 3 The figure is the result of ND207-F1 / ND207-R1 amplification. In the figure, NOD207-1 to ND207-31 are 31 transgenic maize ND207 plants tested. DETAILED DESCRIPTION

[0047] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0048] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0049] Transgenic corn ND207: the "transgenic corn ND207" recorded in the article "Chang Lijuan et al. Specific qualitative PCR detection method and standardization of transformation events of transgenic corn ND207. Acta Agronomica Sinica, Issue 07, 2023", which can be obtained by the public from China Agricultural University and can only be used to repeat the experiment of the present invention and cannot be used for other purposes. ND207 is a transgenic insect-resistant corn variety developed by China Agricultural University. The codon-optimized anti-lepidopteran pest genes mCry1Ab and mCry2Ab were transferred into the hybrid combination of corn inbred lines HiIIA and HiIIB with high transformation efficiency by Agrobacterium infection, and then screened with the screening marker gene bar, and then introduced into the Zheng 58 inbred line through backcrossing and breeding. An excellent transformant ND207 was obtained through multiple tests such as herbicide resistance screening, insect resistance identification, target protein expression, etc. in each generation. At present, transgenic corn ND207 has obtained the agricultural transgenic biosafety certificate and has important industrial application prospects.

[0050] Example 1: Detection of pure heterozygosity of transgenic maize ND207 material by PCR reaction

[0051] 1. Establishment of a method for detecting pure heterozygosity of transgenic maize ND207 material by PCR reaction

[0052] 1. Primer design

[0053] For information on the vectors of exogenous target genes (mcry1Ab, mcry2Ab, bar) in transgenic corn ND207, see Figure 1 Both chromosome sets Figure 1 The transgenic maize ND207 with the insertion of the exogenous target gene is homozygous, with only one chromosome set occurring. Figure 1 The transgenic maize ND207 with the insertion of the exogenous target gene shown is heterozygous.

[0054] A total of two pairs of primers were designed and synthesized:

[0055] Primer pair 1: primer ND207-F0 located in the flanking sequence and primer ND207-R0 located in the insertion sequence;

[0056] Primer pair 2: Primer ND207-F1 and primer ND207-R1 located at the flanking sequences at both ends of the insertion site.

[0057] The positions of the two pairs of primers are Figure 1 The specific primer sequence information is shown in Table 1 and Table 2.

[0058] Table 1. Primer pair 1 related information

[0059]

[0060] Table 2. Primer pair 2 related information

[0061]

[0062] 2. Extraction of genomic DNA

[0063] Take leaves of the transgenic plants to be tested and non-transgenic plants to extract genomic DNA. The specific method is as follows:

[0064] Weigh a small amount of leaves and place them in a 2mL centrifuge tube. Quickly freeze them in liquid nitrogen and then grind them into powder in a sample grinder. Add 650μL 1×CTAB to the centrifuge tube, shake thoroughly and mix well, place in a 65℃ water bath for 40min, and mix every 10min. After cooling to room temperature, add 650μL chloroform:isoamyl alcohol (volume ratio is 24:1), shake slowly for 10min, and centrifuge at 12000rpm for 10min. Pipette 400μL of supernatant into a new 1.5mL centrifuge tube, add an equal volume of isopropanol, turn it upside down, and let it stand at -20℃ for 2h or overnight. Centrifuge at 12000rpm for 10min, discard the supernatant, and keep the precipitate. Add 75% ethanol to wash twice, centrifuge at 7500rpm for 5min, discard the ethanol, and dry. Dissolve the DNA in ddH2O 80μL, and determine the DNA quality and concentration using NanoDrop2000 ultra-micro spectrophotometer.

[0065] 3. PCR amplification system

[0066] The PCR amplification system is shown in Table 3.

[0067] Table 3. PCR amplification system

[0068] Reagents Volume (μL) Final concentration 2×Phanta Max Master Mix(Dye Plus) 12.5 1× Forward primer 10 μmol / L 1.0 0.4μM Reverse primer 10 μmol / L 1.0 0.4μM DNA template <0.25μg <0.25μg / 25μL <![CDATA[ddH2O]]> X Make up to 25 μL Total volume 25.0 -

[0069] Note: The target fragment amplified by primer pair 2 is a high GC content sequence, and PCR enhancer can be added for amplification, that is, 5 μL of 5×PCR enhancer is added to the above amplification system.

[0070] 4. PCR reaction conditions

[0071] The PCR reaction conditions are shown in Table 4.

[0072] Table 4. PCR reaction conditions

[0073]

[0074] 5. Test result examples

[0075] Primer pair 1 in Table 1 (SEQ ID No. 1 and SEQ ID No. 2) produces an amplification product of a 396 bp fragment (SEQ ID No. 5) when PCR amplification is performed using genomic DNA of transgenic corn ND207 (either homozygous or heterozygous) as a template according to the system and conditions shown in steps 3 and 4. When PCR amplification is performed using genomic DNA of non-transgenic corn as a template according to the system and conditions shown in steps 3 and 4, no fragment is amplified.

[0076] When the primer pair 2 in Table 2 (SEQ ID No. 3 and SEQ ID No. 4) was PCR amplified using the genomic DNA of homozygous transgenic corn ND207 as a template according to the system and conditions shown in steps 3 and 4, no fragment was amplified (because the inserted fragment exceeded 6 kb and could not be amplified under the above amplification conditions). When the primer pair 2 was PCR amplified using the genomic DNA of heterozygous transgenic corn ND207 and the genomic DNA of non-transgenic corn as templates according to the system and conditions shown in steps 3 and 4, an amplification product of 891 bp fragment (SEQ ID No. 6) was generated.

[0077] 2. Application case of pure heterozygous method for detecting transgenic maize ND207 material by PCR reaction

[0078] Using the two primer pairs in step 1, and following the reaction systems and conditions in steps 1, 3 and 4, 31 leaf samples of single transgenic maize ND207 collected from Hainan Province in 2024 were randomly selected for testing. Non-transgenic maize (ND207 transgenic recipient) was used as a negative control.

[0079] The detection results of primer pair 1 in Table 1 are as follows Figure 2 As shown in the figure, the 396 bp target band was amplified from all 31 transgenic maize ND207 plants tested, confirming that all 31 plants tested were transgenic maize ND207 (including homozygous and heterozygous). No amplified band was found in non-transgenic maize.

[0080] The detection results of primer pair 2 in Table 2 are as follows Figure 3 As shown. Among the 31 transgenic maize ND207 plants tested, 8 plants numbered ND207-1, ND207-2, ND207-3, ND207-4, ND207-6, ND207-7, ND207-13 and ND207-14 amplified the target band of 891 bp. Figure 2 This indicates that these plants are heterozygous transgenic maize ND207; the other plants have no amplified bands, indicating that these plants are homozygous transgenic maize ND207. Non-transgenic maize has an 891bp amplified band.

[0081] III. Verification of the Identification Accuracy of the Primer Set / Detection Method of the Present Invention

[0082] This experimental example verifies the accuracy of the primer set / detection method of the present invention in the identification of pure heterozygous transgenic corn ND207, and is carried out in the following manner: on the basis of completing step 2, 31 transgenic corn ND207 are randomly divided into three groups, and the two primer pairs in step 1 are used to perform PCR reaction detection according to the reaction system and conditions in steps 3 and 4, and the amplification product (such as Figure 2 and Figure 3 ) were used for Sanger sequencing, and the sequences were aligned using NCBI (https: / / www.ncbi.nlm.nih.gov / ).

[0083] The specific operation is as follows: 31 transgenic maize ND207 were randomly divided into three groups, and the two primer pairs in step 1 were used to perform PCR reaction detection according to the reaction system and conditions in steps 3 and 4. The electrophoresis results corresponding to each single plant sample were compared with Figure 2 and Figure 3 The amplified products corresponding to the target bands obtained above were subjected to Sanger sequencing using NCBI_NucleotideBLAST. The sequencing results of primer pair 1, i.e. Figure 2 The sequencing results of the amplified products corresponding to the target band in the middle were compared with the sequence SEQ ID No.5. The sequence SEQ ID No.5 consists of two parts: the first 143 bp is the 5' end flanking sequence of the junction, and the last 253 bp is the repair sequence and part of the insertion sequence. The comparison result is 100%, indicating that primer pair 1 can perform transformant-specific amplification of transgenic corn ND207, that is, all 31 plants to be tested are transgenic corn ND207; the sequencing results of primer pair 2, that is, Figure 3 The sequencing results of the amplified product corresponding to the target band in the middle were compared with the sequence SEQ ID No.6, which is the genomic sequence of the exogenous target gene insertion site. The comparison result was 100%, indicating that the individual plant with an 891bp band in the electrophoresis result and a 100% sequencing result comparison was the heterozygous transgenic corn ND207, and the other individual plants were homozygous.

[0084] Repeated experiments have shown that the accuracy of the primer set and detection method for identifying pure heterozygous transgenic corn ND207 of the present invention is 100%.

[0085] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. A primer pair group, consisting of primer pair 1 and primer pair 2; The primer pair 1 consists of two primers shown in SEQ ID No.1 and SEQ ID No.2; The primer pair 2 consists of two primers shown in SEQ ID No.3 and SEQ ID No.4; The primer pair 1 is used to specifically detect whether the tested corn is the transgenic corn ND207; the primer pair 2 is used to detect whether the insertion of the exogenous target gene in the transgenic corn ND207 is heterozygous or homozygous.

2. Use of the primer pair set according to claim 1 in the following: specifically detecting whether the tested corn is transgenic corn ND207, and detecting whether the insertion of the exogenous target gene in the transgenic corn ND207 is heterozygous or homozygous.

3. A method for specifically detecting whether the tested corn is transgenic corn ND207 and detecting whether the insertion of the exogenous target gene in the transgenic corn ND207 is heterozygous or homozygous, comprising the following steps: (A1) using the genomic DNA of the corn to be tested as a template and using the primer pair 1 in claim 1 to perform PCR amplification; If a band of 396 bp appears in the electrophoresis result, the corn to be tested is or is a candidate for transgenic corn ND207; otherwise, the corn to be tested is not or is not a candidate for transgenic corn ND207; (A2) using the genomic DNA of the test corn detected as or a candidate for transgenic corn ND207 in (A1) as a template, and using the primer pair 2 in claim 1 to perform PCR amplification; If a band of 891 bp appears in the electrophoresis result, the insertion of the exogenous target gene in the tested corn is heterozygous; Otherwise, the insertion of the exogenous target gene in the tested corn is homozygous.

4. Primer pair, which is primer pair 1 or primer pair 2; The primer pair 1 consists of two primers shown in SEQ ID No.1 and SEQ ID No.2; The primer pair 2 consists of two primers shown in SEQ ID No.3 and SEQ ID No.4; The primer pair 1 is used to specifically detect whether the tested corn is the transgenic corn ND207; the primer pair 2 is used to detect whether the insertion of the exogenous target gene in the transgenic corn ND207 is heterozygous or homozygous.

5. Any of the following uses of the primer pair according to claim 4: (B1) Use of the primer pair 1 of claim 4 in specifically detecting whether the tested corn is genetically modified corn ND207; (B2) Use of the primer pair 2 described in claim 4 in detecting whether the insertion of the exogenous target gene in the transgenic corn ND207 is heterozygous or homozygous.

6. Either of the following methods: Method I: A method for specifically detecting whether a tested corn is a genetically modified corn ND207, comprising the following steps: using the genomic DNA of the tested corn as a template, and performing PCR amplification using the primer pair 1 in claim 4; If a band of 396 bp appears in the electrophoresis result, the corn to be tested is or is a candidate for the transgenic corn ND207; otherwise, the corn to be tested is not or is not a candidate for the transgenic corn ND207; Method II: A method for detecting whether the insertion of an exogenous target gene in transgenic corn ND207 is heterozygous or homozygous, comprising the following steps: using the genomic DNA of the transgenic corn ND207 to be tested as a template, and using the primer pair 2 in claim 4 to perform PCR amplification; If a band of 891 bp appears in the electrophoresis result, the insertion of the exogenous target gene in the transgenic corn ND207 to be tested is heterozygous; otherwise, the insertion of the exogenous target gene in the transgenic corn ND207 to be tested is homozygous.

7. The primer pair set, use, method or primer pair according to any one of claims 1 to 6, characterized in that: The corn to be tested is the sexual hybrid offspring or self-pollinated offspring of the transgenic corn ND207.

8. Use of the primer pair set according to claim 1 or the primer pair according to claim 4 or the method according to claim 3 or 6 in the following: screening for homozygous lines for the insertion of an exogenous target gene in sexual hybrid offspring or self-pollinated offspring of transgenic maize ND207.

9. Use of the primer pair set according to claim 1 or the primer pair according to claim 4 or the method according to claim 3 or 6 in the following application: improving seed purity and / or seed quality in the process of transgenic insect-resistant and herbicide-resistant corn seed production.

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