Transgenic maize zms3j35 transformation event flanking sequences and uses thereof

Abstract

The present application relates to the flanking sequence of the exogenous insertion fragment of the transgenic maize ZmS3J35 transformation event and its application, and belongs to the technical field of plant genetic engineering, transgenic biological safety detection and crop breeding. The isolated nucleic acid molecule provided by the present application is the 5' flanking sequence and / or 3' flanking sequence of the exogenous insertion fragment of the transgenic maize ZmS3J35 transformation event, and the nucleotide sequences thereof are shown in SEQ ID NO. 1 and SEQ ID NO. 2. The advantage of the present application is that the obtained flanking sequence is a unique characteristic sequence of the target transgenic maize transformation event, which has strong specificity, high detection sensitivity and good stability. The flanking sequence can be used not only for qualitative and quantitative detection of transgenic maize, but also for homozygosity identification of strains, genetic stability analysis and germplasm screening. Meanwhile, the flanking sequence is suitable for full-scene applications such as field supervision, seed quality inspection, food traceability and the like, and has high industrial value.

Description

Technical Field

[0001] This invention belongs to the fields of plant genetic engineering, biosafety detection of transgenic organisms and crop breeding technology, specifically involving a flanking sequence of an exogenous inserted fragment in the transformation event of transgenic maize ZmS3J35, a specific detection reagent, a detection method and its application in the identification, germplasm screening and safety supervision of transgenic maize. Background Technology

[0002] Maize is a C4 crop with high fertilizer requirements and sensitivity to drought. Drought reduces its photosynthetic capacity, affecting quality and yield. Sucrose is a product of maize photosynthesis, and sucrose transport proteins (SUTs) play an important role in maize growth, development, stress resistance, and yield formation. Different plants have SUTs involved in drought stress responses. However, current research on the involvement of SUTs in maize drought stress responses is limited, especially lacking specific detection methods based on specific SUT gene transformation events. ZmSUT3 is a monocot-specific member of the SUT3 subfamily, and RNA-seq results show that ZmSUT3 is highly expressed in mature maize pollen. Normal development and function of maize pollen are crucial for maize reproduction. Under drought conditions, pollen development and viability can be severely affected, and gene products specifically expressed in pollen can help pollen cope with drought stress. Chinese invention patent CN110818785A, entitled "A Maize Sucrose Transporter ZmSUT3J and Its Encoding Gene and Application," discloses that overexpression of the ZmSUT3J gene in maize can improve maize's stress resistance and yield. However, this patent does not disclose any flanking sequences of specific transformation events, making it impossible to accurately identify and trace specific transformation events.

[0003] The ZmS3J35 transformation event of transgenic maize described in this invention is a stable transformation event obtained by introducing the maize sucrose transporter gene ZmSUT3J (its nucleotide sequence is shown in SEQ ID NO.1 of CN110818785A) into the maize genome via Agrobacterium-mediated transformation and screening. This transformation event exhibits tolerance to water stress, and the effective detection method helps to gain a deeper understanding of the molecular characteristics of transgenic maize, clarify the insertion location of the exogenous gene in the maize genome, the integrity of the inserted fragment, and the copy number, thereby providing key evidence for assessing the safety of transgenic maize, the genetic stability of transgenic lines, and the phenotypic expression effects.

[0004] Current detection and identification technologies for genetically modified (GM) maize are mostly based on primers designed using universal sequences of exogenous functional genes, marker genes, or vectors, which have significant technical limitations. First, universal gene sequences are widely present in various GM crops and microorganisms, resulting in poor detection specificity, a high likelihood of false positives, and an inability to accurately distinguish between different GM maize transformation events. Second, it is impossible to determine the genomic insertion site and integration status of exogenous genes, and it is impossible to distinguish between homozygous and heterozygous lines, making it difficult to meet the needs of GM maize germplasm purification and genetic stability assessment. Third, existing technologies lack specific characteristic sequences for transformation events, making it impossible to achieve precise traceability of GM maize, which is detrimental to field supervision of GM crops, seed market quality inspection, and food safety control.

[0005] The flanking sequences of the exogenous insertion fragment in the ZmS3J35 transgenic maize transformation event are the junction regions between the exogenous vector sequence and the natural maize genome sequence. These are unique and specific characteristic sequences for a single transformation event, unaffected by interference from other transgenic materials, non-transgenic maize, or microbial genomes. They are the core targets for accurate identification and targeted detection of transformation events. Currently, there is a lack of specific flanking sequences and standardized detection technologies for mainstream superior transgenic maize transformation events, hindering the industrial regulation and precision breeding applications of transgenic maize. Therefore, cloning and identifying transgenic maize-specific flanking sequences and developing supporting detection methods and application technologies have significant theoretical value and promising prospects for industrial application. Summary of the Invention

[0006] This invention provides flanking sequences of exogenous insertion fragments related to the ZmS3J35 transformation event in transgenic maize and their applications, addressing the technical problems of insufficient specificity, inability to distinguish transformation events, and inability to identify homozygous lines in traditional detection methods. It aims to fill the gaps in specific transgenic maize transformation event-specific characteristic sequences, establishing a highly specific, sensitive, and stable technology system for precise detection and germplasm screening of transgenic maize.

[0007] The technical solution adopted in this invention is an isolated nucleic acid molecule, wherein the isolated nucleic acid molecule is the 5′ flanking sequence and / or the 3′ flanking sequence of the exogenous insertion fragment of the transgenic maize ZmS3J35 transformation event; the nucleotide sequence of the 5′ flanking sequence is shown in SEQ ID NO.1, and the nucleotide sequence of the 3′ flanking sequence is shown in SEQ ID NO.2.

[0008] The isolated nucleic acid molecule of the present invention further includes the reverse complementary sequence of the sequence shown in SEQ ID NO.1, and / or the reverse complementary sequence of the sequence shown in SEQ ID NO.2.

[0009] A specific primer set for amplifying flanking sequences of exogenous inserts in the ZmS3J35 transformation event of transgenic maize, the specific primer set comprising at least one of the following primer pairs:

[0010] (1) The first primer pair for detecting the 5′ flanking sequence consists of an upstream primer F1 and a downstream primer R1, wherein the nucleotide sequence of F1 is shown in SEQ ID NO.3 and the nucleotide sequence of R1 is shown in SEQ ID NO.4;

[0011] (2) A second primer pair for detecting the 5′ flanking sequence, consisting of an upstream primer F2 and a downstream primer R2, wherein the nucleotide sequence of F2 is shown in SEQ ID NO.5 and the nucleotide sequence of R2 is shown in SEQ ID NO.6;

[0012] (3) A third primer pair for detecting the 3′ flanking sequence, consisting of an upstream primer F3 and a downstream primer R3, wherein the nucleotide sequence of F3 is shown in SEQ ID NO.7 and the nucleotide sequence of R3 is shown in SEQ ID NO.8;

[0013] (4) The fourth primer pair for real-time quantitative PCR detection consists of upstream primer F3 and downstream primer R1, wherein the nucleotide sequence of F3 is shown in SEQ ID NO.7 and the nucleotide sequence of R1 is shown in SEQ ID NO.4.

[0014] A detection kit for the transformation event of genetically modified maize ZmS3J35 includes the primer set described above, as well as PCR amplification reagents, negative control and positive control.

[0015] A PCR detection method for the transformation event of transgenic maize ZmS3J35 includes the following steps:

[0016] (1) Genomic DNA was extracted from the transgenic maize sample to be tested as an amplification template;

[0017] (2) Using the genomic DNA as a template, PCR amplification is performed using the specific primer set described above. The PCR amplification is conventional PCR or real-time quantitative PCR, used for qualitative or quantitative detection.

[0018] (3) Detect the amplification products and determine whether the transgenic corn sample to be tested is the target transgenic corn based on whether a specific target fragment is generated.

[0019] A method for identifying homozygous and heterozygous lines of transgenic maize ZmS3J35 transformation event, comprising the following steps:

[0020] (1) Extract genomic DNA from the transgenic maize sample to be tested;

[0021] (2) Using the genomic DNA as a template, PCR amplification was performed using the second primer pair and the third primer pair, and simultaneously using the genomic DNA as a template, PCR amplification was performed using the maize genomic wild-type allele-specific primer pair corresponding to the transformation event;

[0022] (3) Detect and compare the PCR amplification products of each group respectively;

[0023] (4) If only the specific amplification products of the second primer pair and the third primer pair are present, and no wild-type allele amplification product is present, the line is determined to be homozygous; if the specific amplification products of the second primer pair and the third primer pair and the wild-type allele amplification product are present at the same time, the line is determined to be heterozygous.

[0024] The genetically modified corn samples to be tested in this invention are selected from corn leaves, seeds, pollen, straw, or processed corn agricultural products.

[0025] Applications of the nucleic acid molecules described in this invention in any of the following scenarios:

[0026] (1) Identification of homozygous and heterozygous lines of the transgenic maize ZmS3J35 transformation event; (2) Germplasm purification and screening of the transgenic maize ZmS3J35 transformation event.

[0027] Application of the primer set described in this invention in any of the following scenarios:

[0028] (1) Identification of homozygous and heterozygous lines in the transgenic maize ZmS3J35 transformation event;

[0029] (2) Germplasm purification and screening of the transgenic maize ZmS3J35 transformation event.

[0030] The beneficial effects of this invention are as follows:

[0031] 1. Extremely high sequence specificity: The flanking sequences obtained by this invention are unique characteristic sequences of the target transgenic maize transformation event. They are specific binding sequences between the exogenous gene and the maize genome, which completely avoids the cross-interference problem of traditional exogenous functional gene and marker gene detection. There are no false positive or false negative results, and different transgenic maize transformation events can be accurately distinguished.

[0032] 2. High detection sensitivity and good stability: The primers designed based on the flanking sequences of this invention have strong amplification specificity and high amplification efficiency, enabling accurate detection of trace samples (seeds, leaves, agricultural product residues). The lowest detection limit can reach 0.01% of transgenic components, meeting the national standard detection requirements.

[0033] 3. Wide range of applications: The sequence of this invention can be used not only for qualitative and quantitative detection of genetically modified maize, but also for identification of homozygosity of lines, analysis of genetic stability, and germplasm screening. It is also suitable for applications in all scenarios such as field supervision, seed quality inspection, and food traceability, and has high industrialization value.

[0034] 4. Simple operation and low cost: The supporting detection methods are based on conventional PCR and fluorescent PCR technologies, which do not require complicated equipment and operating procedures. The detection cycle is short, the cost is low, and it is easy to standardize and promote large-scale application. Attached Figure Description

[0035] Figure 1 This is an electrophoresis image of 5' end-specific PCR amplification in Example 2 of the present invention;

[0036] In the figure, lane 1: Trans15K DNA Marker; lane 2: water; lane 3: non-transgenic plants; lane 4: ZmS3J35; lane 5: plants with other genes; the results show that only lane 4 amplified the specific target band.

[0037] Figure 2 This is an electrophoresis image of the 3' flanking sequence-specific amplification in Embodiment 2 of the present invention;

[0038] In the figure, lane 1: Trans2K® Plus DNA Marker; lane 2: water; lane 3: non-transgenic plant; lane 4: ZmS3J35; lane 5: other gene plants; the results show that only lane 4 amplified the specific target band.

[0039] Figure 3 This is a 5' end-specific amplification electrophoresis image of the ZmS3J35 transformation event in Example 3 of this invention;

[0040] In the figure, lane 1: Trans2K® DNA Marker; lane 2: water; lane 3: non-transgenic plant; lane 4: ZmS3J35; lane 5: other gene plants; the results show that only lane 4 amplified the specific target band.

[0041] Figure 4 This is a 3' end-specific amplification electrophoresis image of the ZmS3J35 transformation event in Example 3 of this invention;

[0042] In the figure, lane 1: Trans15K DNA Marker; lane 2: water; lane 3: non-transgenic plants; lane 4: ZmS3J35; lane 5: plants with other genes; the results show that only lane 4 amplified the specific target band.

[0043] Figure 5 This is an electrophoresis image of the 5' side-by-side sequence in Embodiment 4 of the present invention;

[0044] In the figure, lane 1: Trans2K® DNA Marker; lane 2: water; lane 3: non-transgenic plant; lane 4: ZmS3J35 homozygous plant; lane 5: ZmS3J35 heterozygous plant; the results show that only lanes 4 and 5 amplified the specific target bands.

[0045] Figure 6 This is an electrophoresis image of the 3' end side-by-side sequence in Embodiment 4 of the present invention;

[0046] In the figure, lane 1: Trans15K DNA Marker; lane 2: water; lane 3: non-transgenic plant; lane 4: ZmS3J35 homozygous plant; lane 5: ZmS3J35 heterozygous plant; the results show that only lanes 4 and 5 amplified the specific target bands.

[0047] Figure 7 This is a graph showing the qPCR detection sensitivity in Example 5 of the present invention;

[0048] In the figure: gradient dilution templates (50 - 0.005 ng / μL) all exhibited typical S-shaped amplification profiles; as the template concentration decreased, the amplification peak cycle number was gradually delayed, consistent with the concentration response law of quantitative PCR; the fluorescence signal in the baseline interval showed significant fluctuations, and the repeatability amplification curves of each group showed some dispersion; compared with the high concentration template group, the overlap of the curves of the lowest concentration (0.005 ng / μL) sample decreased, and the detection repeatability was reduced; all gradient samples could be effectively amplified and reach the amplification platform. Detailed Implementation

[0049] The present invention will be further described in detail below with reference to specific embodiments, but the present invention is not limited to the following embodiments. Unless otherwise specified, the experimental methods used in the present invention are all conventional molecular biology methods, and the reagents used are all commercially available conventional reagents.

[0050] Example 1: Cloning and Identification of Flanking Sequences in Transgenic Maize

[0051] 1. Genomic DNA of the target transgenic ZmS3J35 maize homozygous line was extracted from leaves, and a 350bp paired-end library was constructed. Non-target whole-genome resequencing was performed using the Illumina PE150 strategy.

[0052] 2. Data processing and comparison

[0053] After the raw data was filtered by quality control, the clean reads were aligned to the maize B73 reference genome V5.0 using BWA software, and the coverage and insertion sites were statistically analyzed using SAMtools.

[0054] 3. Insertion Site Determination: Exogenous T-DNA was inserted into maize chromosome 7 in single copy form, obtaining the insertion site and 5' and 3' flanking sequences of the exogenous insert fragment. Based on the insertion site sequence information obtained from sequencing, the 5' flanking sequence was amplified from transgenic maize genomic DNA by PCR using the first primer pair F1 / R1 (SEQ ID NO. 3 / 4); the 3' flanking sequence was amplified from maize genomic DNA by PCR using the third primer pair (SEQ ID NO. 7 / 8). After the amplification products were recovered and purified, they were ligated into a vector and sequenced. The sequencing results were spliced ​​and aligned to obtain the complete 5' flanking sequence (SEQ ID NO. 1) and 3' flanking sequence (SEQ ID NO. 2). Sequence analysis results showed that one end of the sequence was a conserved sequence of the maize genome, and the other end was the boundary sequence of the exogenous expression vector, which is a unique sequence of the exogenous gene-specific integration site.

[0055] 4. Sequence specificity verification: Using non-transgenic maize and other transgenic maize lines as templates, the flanking sequences were amplified and verified. The results showed that only the target transgenic maize of this invention could amplify the specific fragment, while other samples did not have amplification products, proving that the sequence has good specificity.

[0056] Example 2: Design and Screening of Specific Primer Sets

[0057] A specific primer set was used to amplify the flanking sequences of the exogenous insert fragment in the transgenic maize ZmS3J35 transformation event. This amplification of the transgenic maize ZmS3J35 transformation event refers to the use of the designed specific primers and PCR amplification techniques to detect the specificity of the transgenic maize ZmS3J35 transformation event, i.e., event-specific. The primers / probes target the flanking boundary sequences of the T-DNA insertion site: one primer falls on the flanking sequence of the maize genome, and the other falls on the inserted T-DNA sequence. Only the genome-vector linker fragment of this event will be amplified; no amplification signal will be found for non-transgenic recipients or other transformation events.

[0058] Based on the flanking sequences obtained from sequencing, specific PCR primers were designed for the binding region between the conserved region of the maize genome and the exogenous vector sequence, and two optimal primer pairs were screened out:

[0059] The first primer pair was detected at the 5' end: F1 (SEQ ID NO.3) and R1 (SEQ ID NO.4), with an amplified fragment length of 2135 bp;

[0060] The second primer pair was detected at the 5' end: F2 (SEQ ID NO.5) and R2 (SEQ ID NO.6), amplifying fragment length of 753 bp;

[0061] The third primer pair was detected at the 3' end: F3 (SEQ ID NO.7) and R3 (SEQ ID NO.8), with an amplified fragment length of 3106 bp;

[0062] The fourth primer pair for qPCR detection was F3 (SEQ ID NO.7) and R1 (SEQ ID NO.4), with an amplified fragment length of 305 bp.

[0063] Primer specificity verification: Multiple batches of repeated experiments showed that both primer pairs specifically amplified the target transgenic maize DNA only, and showed no amplification signal for non-transgenic maize or other transgenic materials. The primer specificity and stability met the detection standards. (See attached image) Figure 1 , 2 The PCR amplification conditions are shown in Table 1.

[0064] Table 1 PCR amplification conditions

[0065] 5' flanking sequence F1, R1 98℃, 1 min 98℃,10s 58℃,5s 72℃,25s 30 3' flanking sequence F3, R3 98℃, 1min 98℃,10s 66℃,5s 72℃,35s 30

[0066] Example 3: PCR-specific detection method for transgenic maize

[0067] 1. Sample DNA extraction: Take corn leaves, seeds or agricultural product samples to be tested, extract genomic DNA using the CTAB method, detect DNA purity and concentration, and dilute to 50 ng / μL for later use.

[0068] 2. PCR amplification system (20μL): 10μL of 2×TransStart® FastPfu Fly PCR SuperMix (+dye), 0.4μL each of upstream and downstream primers (10μmol / L), 0.5μL of template, and sterile ultrapure water to a final volume of 20μL.

[0069] 3. PCR primers and amplification conditions are shown in Table 2.

[0070] Table 2 PCR amplification conditions

[0071] 5' flanking sequence F2, R2 98℃, 1min 98℃,10s 58℃,5s 72℃,10s 30 3' flanking sequence F3, R3 98℃, 1min 98℃,10s 63℃,5s 72℃,35s 30

[0072] 4. Result Interpretation: The amplified products are detected by 1.5% agarose gel electrophoresis. If a specific target band of the corresponding length appears, the sample is considered a positive transgenic maize sample; if no band appears, the sample is considered negative. (See appendix) Figure 3 ,4.

[0073] Example 4: Identification of homozygous and heterozygous transgenic maize lines

[0074] The flanking sequences of this invention were used to identify the homozygosity of the transgenic maize lines: using the genomic DNA of the transgenic maize line to be tested as a template, the flanking sequences at the 5' end and 3' end were amplified respectively. The PCR primers and amplification conditions are shown in Table 3.

[0075] Table 3 PCR amplification conditions

[0076] 5' flanking sequence F2, R2 98℃, 1min 98℃,10s 58℃,5s 72℃,10s 30 3' flanking sequence F3, R3 98℃, 1min 98℃,10s 63℃,5s 72℃,35s 30

[0077] If a sample can stably amplify two sets of specific target fragments simultaneously, and there is no amplification signal at wild-type genomic alleles, it is identified as a homozygous line; if it simultaneously amplifies both transgenic specific fragments and wild-type maize genomic fragments, it is identified as a heterozygous line. This method can rapidly complete the purification and screening of transgenic maize germplasm with 100% accuracy. (See appendix) Figure 5 ,6.

[0078] Example 5: Detection Sensitivity Experiment

[0079] The target transgenic maize DNA (50 ng / uL) was mixed with non-transgenic maize DNA in a certain proportion to prepare mixed samples with transgenic component contents of 10% (5 ng / uL), 1% (0.5 ng / uL), 0.1% (0.05 ng / uL), 0.01% (0.005 ng / uL), and 0.001% (0.0005 ng / uL). The fourth primer pair (F3, R1) of this invention was used for detection. The qPCR system consisted of 10 μL of 2×PerfectStart® Green qPCR SuperMix (+Universal Passive Reference Dye), 0.4 μL each of upstream primer F3 and downstream primer R1 (10 μmol / L), 2 μL of template, and sterile ultrapure water to a final volume of 20 μL. PCR amplification procedure: Amplification cycle stage: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 15 s, 60℃ annealing and extension for 15 s, fluorescence signal acquisition, for a total of 40 cycles; Melting curve analysis stage: gradually increase the temperature from 60℃ to 95℃, acquiring fluorescence signal once every 0.5℃ increase. Results show that this method can accurately detect as little as 0.01% of transgenic components. (See attached diagram) Figure 7 Its sensitivity far exceeds that of conventional transgenic detection methods, meeting the needs for trace detection in grains and seeds.

[0080] Example 6 Assembly of the transgenic maize ZmS3J35 transformation event detection kit

[0081] This embodiment provides a transgenic maize ZmS3J35 transformation event detection kit, which contains the following components:

[0082] (1) Specific primer set: comprising at least one of the primer pairs described in claim 4, wherein each primer is individually or in combination at a concentration of 10 μmol / L;

[0083] (2) PCR amplification reagents: including DNA polymerase, dNTPs, and PCR buffer;

[0084] (3) Negative control: non-transgenic maize genomic DNA at a concentration of 50 ng / μL;

[0085] (4) Positive control: plasmid DNA containing the sequence shown in SEQ ID NO.1 and / or SEQ ID NO.2 at a concentration of 10^3 copies / μL.

[0086] The method of using the kit is described in Examples 3 and 5. The components of the kit should be stored at -20°C.

[0087] Those skilled in the art should understand that other specific primer pairs designed based on the flanking sequences (SEQ ID NO.1 and / or SEQ ID NO.2) provided in this invention, as long as they can amplify the specific target fragment of the flanking sequence, can be used in the detection method of this invention. The primer pairs and PCR amplification conditions (including annealing temperature, extension time, cycle number, etc.) given in the above embodiments are preferred schemes under specific experimental conditions. Those skilled in the art can obtain equivalent amplification conditions through conventional optimization based on the specific sequence of the primer pair used, the length of the amplified fragment, and the type of PCR platform. All of these fall within the scope of the detection method defined by this invention.

Claims

1. An isolated nucleic acid molecule, characterized in that, The isolated nucleic acid molecule is the 5′ flanking sequence and / or 3′ flanking sequence of the exogenous insertion fragment of the transgenic maize ZmS3J35 transformation event; the nucleotide sequence of the 5′ flanking sequence is shown in SEQ ID NO.1, and the nucleotide sequence of the 3′ flanking sequence is shown in SEQ ID NO.

2.

2. The isolated nucleic acid molecule according to claim 1, characterized in that, The isolated nucleic acid molecule also includes the reverse complementary sequence of the sequence shown in SEQ ID NO.1, and / or the reverse complementary sequence of the sequence shown in SEQ ID NO.

2.

3. A specific primer set, characterized in that, The specific primer set is used to amplify the flanking sequence of the exogenous insert fragment in the transgenic maize ZmS3J35 transformation event, and the specific primer set contains at least one of the following primer pairs: (1) The first primer pair for detecting the 5′ flanking sequence consists of an upstream primer F1 and a downstream primer R1, wherein the nucleotide sequence of F1 is shown in SEQ ID NO.3 and the nucleotide sequence of R1 is shown in SEQ ID NO.4; (2) A second primer pair for detecting the 5′ flanking sequence, consisting of an upstream primer F2 and a downstream primer R2, wherein the nucleotide sequence of F2 is shown in SEQ ID NO.5 and the nucleotide sequence of R2 is shown in SEQ ID NO.6; (3) A third primer pair for detecting the 3′ flanking sequence, consisting of an upstream primer F3 and a downstream primer R3, wherein the nucleotide sequence of F3 is shown in SEQ ID NO.7 and the nucleotide sequence of R3 is shown in SEQ ID NO.8; (4) The fourth primer pair for real-time quantitative PCR detection consists of upstream primer F3 and downstream primer R1, wherein the nucleotide sequence of F3 is shown in SEQ ID NO.7 and the nucleotide sequence of R1 is shown in SEQ ID NO.

4.

4. A detection kit for the transformation event of transgenic maize ZmS3J35, characterized in that, It includes the primer set as described in claim 3, as well as PCR amplification reagents, negative control and positive control.

5. A PCR detection method for the transformation event of transgenic maize ZmS3J35, characterized in that, Includes the following steps: (1) Genomic DNA was extracted from the transgenic maize sample to be tested as an amplification template; (2) Using the genomic DNA as a template, PCR amplification is performed using the specific primer set described in claim 3, wherein the PCR amplification is conventional PCR or real-time quantitative PCR, for qualitative or quantitative detection. (3) Detect the amplification products and determine whether the transgenic corn sample to be tested is the target transgenic corn based on whether a specific target fragment is generated.

6. A method for identifying homozygous and heterozygous lines of transgenic maize ZmS3J35 transformation event, characterized in that, Includes the following steps: (1) Extract genomic DNA from the transgenic maize sample to be tested; (2) Using the genomic DNA as a template, PCR amplification is performed using the second primer pair and the third primer pair in claim 3, and simultaneously using the genomic DNA as a template, PCR amplification is performed using the maize genome wild-type allele-specific primer pair corresponding to the transformation event; (3) Detect and compare the PCR amplification products of each group respectively; (4) If only the specific amplification products of the second primer pair and the third primer pair are present, and no wild-type allele amplification product is present, the line is determined to be homozygous; if the specific amplification products of the second primer pair and the third primer pair and the wild-type allele amplification product are present at the same time, the line is determined to be heterozygous.

7. The method according to claim 5 or 6, characterized in that, The genetically modified corn samples to be tested were selected from corn leaves, seeds, pollen, straw, or processed corn products.

8. The use of the nucleic acid molecule according to claim 1 or 2 in any of the following scenarios: (1) Identification of homozygous and heterozygous lines in the transgenic maize ZmS3J35 transformation event; (2) Germplasm purification and screening of the transgenic maize ZmS3J35 transformation event.

9. The application of the primer set according to claim 3 in any of the following scenarios: (1) Identification of homozygous and heterozygous lines in the transgenic maize ZmS3J35 transformation event; (2) Germplasm purification and screening of the transgenic maize ZmS3J35 transformation event.

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