Primer group for homozygosity and heterozygosity identification of transgenic maize CC-2 and detection method

Abstract

The invention discloses a primer group for homozygosity and heterozygosity identification of transgenic maize CC-2 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.2), the primer pair 1 is used for specifically detecting whether to-be-detected corn is transgenic corn CC-2 or not; the primer pair 2 is used for detecting whether the insertion of an exogenous target gene maroACC in the transgenic maize CC-2 is heterozygous or homozygous. Two pairs of primers are designed for the transgenic maize CC-2, a set of PCR detection method for homozygosis and heterozygosis of the transgenic maize CC-2 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 homozygous transgenic corn CC-2. Background Art

[0002] The herbicide-resistant genes used commercially worldwide include glyphosate-resistant genes epsps (5-enolpyruvylshikimate-3-phosphate synthase gene), gat (glyphosate N-acetyltransferase gene), goxv247 (glyphosate oxidoreductase gene), glufosinate-resistant genes pat and bar (glufosinate acetyltransferase gene), sulfonylurea herbicide-resistant genes als (acetolactate synthase gene), etc. Glyphosate is a systemic non-selective herbicide, and its main target is EPSPS (5-enolpyruvylshikimate-3-phosphate synthase). In 1983, researchers from Monsanto and the University of Washington isolated the highly glyphosate-tolerant CP4 strain from soil Agrobacterium, and successfully inserted its epsps gene into the plant genome in 1986 to obtain glyphosate-resistant plants. Subsequently, five countries including the United States, Canada, and Argentina began to commercially grow glyphosate-resistant soybeans, corn, and rapeseed. In 2022 and 2023, five herbicide-resistant transformants were successively launched in China, namely, pyrazosulfuron-methyl / glyphosate-resistant corn 'nCX-1' (with CdP450 and cp4 epsps genes introduced), glyphosate-resistant corn 'GA21' (with mepsps gene introduced), insect-resistant / glyphosate-tolerant corn 'Bt11×GA21' (with polymerized cry1Ab, pat and mepsps genes), insect-resistant / glyphosate-tolerant / glufosinate-tolerant corn 'Bt11×MIR162×GA2' (with polymerized cry1Ab, pat, vip3Aa20 and mepsps genes), insect-resistant / herbicide-tolerant corn 'BFL4-2' (with cry1Ab, cry1F and cp4 epsps genes introduced) and glyphosate-tolerant corn 'CC-2' (with maroACC gene introduced).

[0003] Transgenic corn CC-2 is a hybrid combination of maize inbred lines HiIIA and HiIIB with high transformation efficiency that was transformed by gene gun co-transformation at China Agricultural University. More than 500 transformants with positive maroACC protein were obtained by screening with glyphosate, and finally an excellent transformant CC-2 was obtained through systematic screening such as copy number detection and expression detection. In the cultivation environment, transgenic corn CC-2 and its corresponding non-transgenic corn are consistent in growth period, plant height, yield, etc., and can grow normally; in the wasteland environment, transgenic corn CC-2 and its corresponding non-transgenic corn have no competitive advantage over weeds and no risk of weeding (Song Xinyuan et al. Study on the survival and competitiveness of transgenic herbicide-resistant corn CC-2. Crop Journal, Issue 06, 2014).

[0004] The commercialization of genetically modified herbicide-tolerant crops will be a new and efficient means to effectively solve the many problems that arise in the use of herbicides in my country, and it is also an effective measure to improve the international competitiveness of the grain industry. The large-scale promotion of herbicide-tolerant crops will be of great benefit in increasing crop yields, saving farming costs, and reducing crop damage. At the same time, it will play a positive role in improving product competitiveness, promoting the adjustment of planting structure, reducing carbon emissions from mechanical operations, and improving environmental biosafety.

[0005] 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.

[0006] 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, the 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 through 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

[0007] The purpose of the present invention is to provide a rapid and accurate detection method for identifying homozygous transgenic corn CC-2.

[0008] In a first aspect, the invention claims a set of primer pairs.

[0009] The primer pair set claimed in the present invention consists of primer pair 1 and primer pair 2;

[0010] The primer pair 1 consists of two primers shown in SEQ ID No.1 and SEQ ID No.2;

[0011] The primer pair 2 consists of two primers shown in SEQ ID No.3 and SEQ ID No.2;

[0012] The primer pair 1: SEQ ID No. 1 and SEQ ID No. 2 are designed on the insertion sequence and the 3' end flanking sequence respectively, and are used for specific detection of transgenic corn CC-2;

[0013] The primer pair 2: SEQ ID No. 3 and SEQ ID No. 2 are designed on the 5' end and 3' end flanking sequences respectively, and are used to detect whether the insertion of the exogenous target gene maroACC in the transgenic corn CC-2 is heterozygous or homozygous.

[0014] 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 CC-2, and detecting whether the insertion of the exogenous target gene maroACC in the transgenic corn CC-2 is heterozygous or homozygous.

[0015] In a third aspect, the present invention claims a method for specifically detecting whether the tested corn is the transgenic corn CC-2, and detecting whether the insertion of the exogenous target gene maroACC in the transgenic corn CC-2 is heterozygous or homozygous.

[0016] The method claimed in the present invention can not only specifically detect whether the tested corn is the transgenic corn CC-2, but also detect whether the insertion of the exogenous target gene maroACC in the transgenic corn CC-2 is heterozygous or homozygous, and can include the following steps:

[0017] (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;

[0018] If a band of 368 bp appears in the electrophoresis result, the maize to be tested is or is a candidate for transgenic maize CC-2, 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 CC-2, and the (A2) test is not required;

[0019] (A2) using the genomic DNA of the test corn detected as or a candidate for transgenic corn CC-2 in (A1) as a template, and using the primer pair 2 in the first aspect above to perform PCR amplification;

[0020] If a band of 3196 bp appears in the electrophoresis result, the insertion of the exogenous target gene maroACC in the tested maize is heterozygous; otherwise (no amplified band), the insertion of the exogenous target gene maroACC in the tested maize is homozygous.

[0021] In a fourth aspect, the present invention claims a primer pair.

[0022] The primer pair claimed in the present invention is primer pair 1 or primer pair 2;

[0023] The primer pair 1 consists of two primers shown in SEQ ID No.1 and SEQ ID No.2;

[0024] The primer pair 2 consists of two primers shown in SEQ ID No.3 and SEQ ID No.2;

[0025] The primer pair 1 is used for specific detection of transgenic corn CC-2; the primer pair 2 is used for detecting whether the insertion of the exogenous target gene maroACC in the transgenic corn CC-2 is heterozygous or homozygous.

[0026] 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:

[0027] (B1) Use of the primer pair 1 described in the fourth aspect above in the specific detection of transgenic corn CC-2;

[0028] (B2) Use of the primer pair 2 described in the fourth aspect above in detecting whether the insertion of the exogenous target gene maroACC in transgenic corn CC-2 is heterozygous or homozygous.

[0029] In a sixth aspect, the present invention claims protection for any of the following methods:

[0030] Method I: A method for specifically detecting whether a tested corn is a transgenic corn CC-2, 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;

[0031] If a 368 bp band appears in the electrophoresis result, the corn to be tested is or is a candidate for transgenic corn CC-2. Otherwise (no amplified band), the corn to be tested is not or is not a candidate for transgenic corn CC-2.

[0032] Method II: A method for detecting whether the insertion of the exogenous target gene maroACC in transgenic corn CC-2 is heterozygous or homozygous, which may include the following steps: using the genomic DNA of the transgenic corn CC-2 to be tested as a template, and using the primer pair 2 in the fourth aspect above to perform PCR amplification;

[0033] If a band of 3196 bp appears in the electrophoresis result, the insertion of the exogenous target gene maroACC in the transgenic corn CC-2 to be tested is heterozygous; otherwise (no amplified band), the insertion of the exogenous target gene maroACC in the transgenic corn CC-2 to be tested is homozygous.

[0034] 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 CC-2.

[0035] In the above-mentioned related aspects, the reaction conditions for performing the PCR amplification are any of the following:

[0036] (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.

[0037] (2) Pre-denaturation at 95°C for 3 min; 1 cycle of denaturation at 95°C for 15 s, annealing at 58°C for 15 s, and extension at 72°C for 3 min 15 s, for a total of 32 cycles; final extension at 72°C for 5 min.

[0038] 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).

[0039] 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.

[0040] In the seventh aspect, the present invention claims the use of the primer pair group described in the first aspect above, 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 lines for the insertion of the exogenous target gene maroACC in sexual hybrid offspring or self-pollinated offspring of transgenic corn CC-2.

[0041] 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 herbicide-resistant transgenic corn seeds.

[0042] In the present invention, the transgenic corn CC-2 with the maroACC gene inserted in both chromosome sets is homozygous, and the transgenic corn CC-2 with the maroACC gene inserted in only one chromosome set is heterozygous. Figure 1 shown.

[0043] The present invention designs a first primer contained in a flanking sequence and a second primer containing an insertion sequence for transgenic corn CC-2, 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 transgenic corn CC-2 homozygous PCR detection methods. The present invention is of great significance for screening homozygous transformants in the breeding process and improving seed purity and quality.

[0044] The transgenic corn CC-2 of the present invention has good tolerance to glyphosate herbicides 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

[0045] Figure 1 This is a schematic diagram of the vector information and primer positions for PCR detection of the exogenous target gene maroACC in transgenic corn CC-2.

[0046] Figure 2 The figure is the CC-2-F0 / CC-2-R amplification result. In the figure, CC-1 to CC-26 are 26 transgenic corn CC-2 plants tested.

[0047] Figure 3 The figure is the CC-2-F1 / CC-2-R amplification result. In the figure, CC-1 to CC-26 are 26 transgenic corn CC-2 plants tested. DETAILED DESCRIPTION

[0048] 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.

[0049] 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.

[0050] Transgenic corn CC-2: The "transgenic herbicide-resistant corn CC-2" recorded in the article "Song Xinyuan et al. Study on the Survival Competitiveness of Transgenic Herbicide-Resistant Corn CC-2. Crop Journal, Issue 06, 2014" is available to 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. Although the maroACC gene can confer resistance to glyphosate herbicides on the transformed corn, there are obvious differences in resistance among different transformants. The growth of some transformants will be inhibited when sprayed with 200ml / mu of glyphosate. The excellent transformant CC-2 obtained through many years of resistance test screening will not be affected when sprayed with 1800ml / mu of glyphosate compared to the control corn that was not sprayed.

[0051] Example 1: Detection of pure heterozygosity of transgenic corn CC-2 material by PCR reaction

[0052] 1. Establishment of a method for detecting pure heterozygosity of transgenic maize CC-2 material by PCR reaction

[0053] 1. Primer design

[0054] For information on the vector of the exogenous target gene maroACC in transgenic corn CC-2, see Figure 1 Both chromosome sets Figure 1 The transgenic maize CC-2 with the insertion of the maroACC gene is homozygous, with only one chromosome set occurring. Figure 1 Transgenic maize CC-2 is heterozygous for the insertion of the indicated maroACC gene.

[0055] The present invention designs primers on the genome on both sides of the transgenic corn CC-2 insertion site, and designs primers on the insertion sequence. The two pairs of primers (three in total) are as follows:

[0056] Primer pair 1: primer CC-2-R located in the flanking sequence and primer CC-2-F0 located in the insert sequence;

[0057] Primer pair 2: primer CC-2-R and primer CC-2-F1 located at the flanking sequences at both ends of the insertion site.

[0058] CC-2-F0: 5'-CACTGGCCGTCGTTTTACAA-3' (SEQ ID No. 1);

[0059] CC-2-R: 5'-GATGTGCCGCCTCAACATTT-3' (SEQ ID No. 2);

[0060] CC-2-F1: 5'-GGTTTATGGTTCTCCCCGTG-3' (SEQ ID No. 3).

[0061] As shown in Table 1 and Table 2.

[0062] Among them, the CC-2-F0 / CC-2-R amplified region is the junction region between the 3' end transgenic sequence of transgenic corn CC-2 and the corn genome, and the amplified sequence is 368bp, as shown in SEQ ID No.4; the CC-2-F1 / CC-2-R amplified fragment is the corn genome sequence, and the amplified sequence is 3196bp. In the genome of homozygous transgenic corn CC-2, the fragment between the two primers CC-2-F1 and CC-2-R is the region including the complete inserted fragment, which is expected to be more than 8kb. Such a long fragment cannot be amplified under ordinary PCR conditions; therefore, the 3196bp amplified product of the corn genome sequence can only be obtained in non-transgenic samples (not containing the exogenous target gene maroACC gene insertion) and samples with heterozygous maroACC gene insertion sites, as shown in SEQ ID No.5.

[0063] Table 1. Primer pair 1 related information

[0064]

[0065]

[0066] Table 2. Primer pair 2 related information

[0067]

[0068] 2. Extraction of genomic DNA

[0069] Take leaves of the transgenic plants to be tested and non-transgenic plants to extract genomic DNA. The specific method is as follows:

[0070] 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.

[0071] 3. PCR amplification system

[0072] The PCR amplification system is shown in Table 3.

[0073] Table 3. PCR amplification system

[0074] Reagents Volume (μL) Final concentration 2×Phanta Max Master Mix(Dye Plus) 12.5 1× <![CDATA[Forward primer 10 μmol L -1 > 1.0 0.4μM <![CDATA[Reverse primer 10 μmol L -1 > 1.0 0.4μM DNA template <0.25μg <0.25μg / 25μL <![CDATA[ddH2O]]> X Make up to 25 μL Total volume 25.0 -

[0075] 4. PCR reaction conditions

[0076] The PCR reaction conditions are shown in Table 4.

[0077] Table 4. PCR reaction conditions

[0078]

[0079]

[0080] 5. Test result examples

[0081] Primer pair 1 in Table 1 (SEQ ID No. 1 and SEQ ID No. 2) produces an amplification product of a 368 bp fragment (SEQ ID No. 4) when PCR amplification is performed using genomic DNA of transgenic corn CC-2 (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.

[0082] When the primer pair 2 in Table 2 (SEQ ID No. 3 and SEQ ID No. 2) was PCR amplified using the genomic DNA of homozygous transgenic corn CC-2 as a template according to the system and conditions shown in steps 3 and 4, no fragment was amplified (because the inserted fragment exceeded 8 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 CC-2 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 a 3196 bp fragment (SEQ ID No. 5) was generated.

[0083] 2. Application case of pure heterozygous method for detecting transgenic corn CC-2 materials by PCR reaction

[0084] Using the two primer pairs in step 1, and following the reaction systems and conditions in steps 1, 3 and 4, 26 leaf samples of single transgenic corn CC-2 collected from Hainan Province in 2024 were randomly selected for testing. Non-transgenic corn (transformation receptor of CC-2) was used as a negative control.

[0085] The detection results of primer pair 1 in Table 1 are as follows Figure 2As shown in the figure, the 368bp target band was amplified from all 26 transgenic corn CC-2 plants tested, confirming that all 26 plants tested were transgenic corn CC-2 (including homozygous and heterozygous). No amplified band was found in non-transgenic corn.

[0086] The detection results of primer pair 2 in Table 2 are as follows Figure 3 As shown. Among the 26 transgenic maize CC-2 plants tested, 10 plants numbered CC-3, CC-4, CC-7, CC-8, CC-11, CC-15, CC-19, CC-20, CC-23 and CC-24 amplified the target band of 3196 bp. Figure 2 The explanation indicates that these plants are heterozygous transgenic corn CC-2; the other plants have no amplified bands, indicating that these plants are homozygous transgenic corn CC-2. Non-transgenic corn has a 3196bp amplified band.

[0087] III. Verification of the Identification Accuracy of the Primer Set / Detection Method of the Present Invention

[0088] This experimental example verifies the accuracy of the primer set / detection method of the present invention in the identification of pure heterozygous transgenic corn CC-2, and is carried out in the following manner: on the basis of completing step 2, 26 transgenic corn CC-2 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 / ).

[0089] The specific operation is as follows: 26 transgenic corn CC-2 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.4. The sequence SEQ ID No.4 consists of two parts: the first 302 bp is a partial insertion sequence, and the last 66 bp is a 3'-end flanking sequence at the junction. The comparison result is 100%, indicating that primer pair 1 can perform transformant-specific amplification of transgenic corn CC-2, that is, all 26 plants to be tested are transgenic corn CC-2; the sequencing results of primer pair 2, that is, Figure 3The sequencing results of the amplified product corresponding to the target band were compared with the sequence SEQ ID No. 5, and the comparison result was 100%, indicating that the individual plant with a 3196 bp band in the electrophoresis result and a 100% sequencing result comparison was a heterozygous transgenic corn CC-2, and the other individual plants were homozygous.

[0090] Repeated experiments have shown that the accuracy of the primer set and detection method for identifying homozygous transgenic corn CC-2 of the present invention is 100%.

[0091] 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.2; The primer pair 1 is used to specifically detect whether the tested corn is transgenic corn CC-2; the primer pair 2 is used to detect whether the insertion of the exogenous target gene maroACC in the transgenic corn CC-2 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 CC-2, and detecting whether the insertion of the exogenous target gene maroACC in the transgenic corn CC-2 is heterozygous or homozygous.

3. A method for specifically detecting whether the tested corn is transgenic corn CC-2 and detecting whether the insertion of the exogenous target gene maroACC in the transgenic corn CC-2 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 368 bp appears in the electrophoresis result, the corn to be tested is or is a candidate for transgenic corn CC-2; otherwise, the corn to be tested is not or is not a candidate for transgenic corn CC-2; (A2) using the genomic DNA of the test corn detected as or a candidate for transgenic corn CC-2 in (A1) as a template, and using the primer pair 2 in claim 1 to perform PCR amplification; If a band of 3196 bp appears in the electrophoresis result, the insertion of the exogenous target gene maroACC in the tested corn is heterozygous; Otherwise, the insertion of the exogenous target gene maroACC 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.2; The primer pair 1 is used to specifically detect whether the tested corn is transgenic corn CC-2; the primer pair 2 is used to detect whether the insertion of the exogenous target gene maroACC in the transgenic corn CC-2 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 CC-2; (B2) Use of the primer pair 2 described in claim 4 in detecting whether the insertion of the exogenous target gene maroACC in transgenic corn CC-2 is heterozygous or homozygous.

6. Either of the following methods: Method I: A method for specifically detecting whether a tested corn is a transgenic corn CC-2, comprising the following steps: using the genomic DNA of the tested corn as a template, and using the primer pair 1 in claim 4 to perform PCR amplification; If a band of 368 bp appears in the electrophoresis result, the corn to be tested is or is a candidate for transgenic corn CC-2; otherwise, the corn to be tested is not or is not a candidate for transgenic corn CC-2; Method II: A method for detecting whether the insertion of the exogenous target gene maroACC in transgenic corn CC-2 is heterozygous or homozygous, comprising the following steps: using the genomic DNA of the transgenic corn CC-2 to be tested as a template, and using the primer pair 2 in claim 4 to perform PCR amplification; If a band of 3196 bp appears in the electrophoresis result, the insertion of the exogenous target gene maroACC in the transgenic corn CC-2 to be tested is heterozygous; Otherwise, the insertion of the exogenous target gene maroACC in the transgenic corn CC-2 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 a sexual hybrid offspring or a self-pollinated offspring of the transgenic corn CC-2.

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 screening homozygous lines for the insertion of the exogenous target gene maroACC in sexual hybrid progenies or self-pollinated progenies of transgenic corn CC-2.

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 herbicide-resistant transgenic corn seed production.

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