Flanking sequences of exogenous insert of insect-resistant transgenic maize AM11 and application thereof

Abstract

The present application relates to the technical field of molecular biology, and particularly relates to a flanking sequence of an exogenous inserted fragment of a pest-resistant transgenic corn AM11 and application thereof. The flanking sequence is a 5' end flanking sequence of the exogenous inserted fragment, specifically a sequence as shown in SEQ ID NO. 9 or a specific fragment thereof; or a 3' end flanking sequence of the exogenous inserted fragment, specifically a sequence as shown in SEQ ID NO. 10 or a specific fragment thereof. The flanking sequence is derived from a pest-resistant transgenic corn event AM11. The corn seed of the pest-resistant transgenic corn AM11 has been preserved in the China General Microbiological Culture Collection Center on September 5, 2025, with a preservation number of CGMCC NO. 46691 and a classification name of Zea mays L. Zea mays The flanking sequence of the present application can effectively determine whether the pest-resistant transgenic corn event AM11 is successfully obtained, thereby providing a strong guarantee for monitoring the pest-resistant transgenic corn AM11 and its offspring.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of molecular biology, and particularly relates to a flanking sequence of an exogenous inserted fragment of an insect-resistant transgenic maize AM11 and application thereof. BACKGROUND

[0002] Pests, especially corn borer, can cause serious yield reduction of corn. Insect-resistant transgenic corn is one of the earliest research traits in the world. The insect-resistant gene mainly comes from the Bt insecticidal protein of Bacillus thuringiensis, and its mechanism of action is to make the insect's gastrointestinal tract perforated, so that the insect's metabolism is disordered and the insect dies. Monsanto is the leader in the research and application of transgenic insect-resistant corn. Its developed insect-resistant transgenic corn MON810 and MON863 have been in commercial production for many years. The Cry1Ab protein expressed in MON810 transgenic corn can effectively control corn borer, and the Cry3Bb expressed in MON863 has good control effect on pests that harm corn roots. Syngenta also developed transgenic insect-resistant corn Bt11 and Bt176 expressing Cry1Ab protein. Pioneer and Dow jointly developed transgenic corn containing Cry34 and Cry35 to resist corn root pests. Ding Qunxing et al. (1993) first reported that Bt toxin protein gene was introduced into corn by means of ovary injection; Wang Guoying et al. (1995) used corn suspension cells, callus and immature embryos as receptors, and successfully transformed Bt toxin protein gene into corn cells by gene gun bombardment, and obtained a large number of transgenic plants; Zhou Fengyong et al. (1998) introduced Bt insecticidal protein into corn inbred line P9-10, and the exogenous gene could be stably inherited to the transgenic plant offspring. Zhang Yanzhen et al. (2002) systematically studied the introduction of Bt insecticidal gene into excellent corn inbred lines by Agrobacterium-mediated method.

[0003] Up to now, domestic transgenic phytase corn BVLA430101, insect-resistant and herbicide-tolerant corn DBN9936, Ruifeng125 and herbicide-tolerant corn DBN9858 have been considered to meet the safety standards.

[0004] With the commercial application of transgenic corn, the corn industry will be upgraded. For the sustainable use of transgenic corn in the future, it is necessary to develop more excellent transformants with resistance. The flanking sequence of the exogenous inserted fragment and the detection method established on the basis of the flanking sequence can better supervise and manage the transgenic corn. The flanking sequence of a specific transgenic event is specific, so the flanking sequence can be used to specifically detect the transgenic event.

[0005] The modified cry1Ab gene (patent application No. 202010553538.0) and cry1m7The gene (patent application number 201410591794.3) constructs a transformation vector p3301UbiAbUbiM7, and is introduced into a corn genome through an agrobacterium-mediated method, so that an insect-resistant transgenic corn event AM11 is obtained, and the event AM11 can be planted commercially in the future. Therefore, it is necessary to obtain the flanking sequence of the insect-resistant transgenic corn AM11, and to establish an identification system for supervision and management of the event. SUMMARY

[0006] The purpose of the present application is to provide a flanking sequence of an exogenous insertion fragment of an insect-resistant transgenic corn AM11 and an application thereof. The flanking sequence of the exogenous insertion fragment of the insect-resistant transgenic corn event AM11 is used to design a specific primer pair for specific detection, so that the insect-resistant transgenic corn AM11 can be better supervised and managed, and an identification system is established.

[0007] The flanking sequence of the exogenous insertion fragment of the insect-resistant transgenic corn AM11 provided by the present application is a 5' end flanking sequence of the exogenous insertion fragment of the insect-resistant transgenic corn AM11, or a 3' end flanking sequence of the exogenous insertion fragment of the insect-resistant transgenic corn AM11. The 5' end flanking sequence is a nucleotide sequence as shown in SEQ ID NO. 9 or a specific fragment thereof, and the 3' end flanking sequence is a nucleotide sequence as shown in SEQ ID NO. 10 or a specific fragment thereof.

[0008] The flanking sequence is derived from the insect-resistant transgenic corn event AM11. The corn seeds of the insect-resistant transgenic corn event AM11 have been deposited in the China General Microbiological Culture Collection Center (CGMCC) on September 5, 2025 (address: No. 1, Huixiyili, Beichen West Road, Chaoyang District, Beijing, China, postal code: 100101), and the deposit number is CGMCC NO. 46691, and the classification name is Zea mays Zea mays .

[0009] The present application provides a specific primer pair for detecting the flanking sequence of the exogenous insertion fragment of the insect-resistant transgenic corn AM11, and specifically includes any one of the following cases:

[0010] (1) a specific primer pair with the 5' end flanking sequence as a target, and the nucleotide sequences thereof are as shown in SEQ ID NO. 11-12;

[0011] (2) a specific primer pair with the 3' end flanking sequence as a target, and the nucleotide sequences thereof are as shown in SEQ ID NO. 13-14.

[0012] The present application discloses a kit containing the specific primer pair as shown in SEQ ID NO. 11-14.

[0013] This invention discloses the flanking sequences shown in SEQ ID NO. 9-10 and the specific primer pairs shown in SEQ ID NO. 11-14, and the application of the kit in detecting insect-resistant transgenic maize AM11.

[0014] This invention discloses a method for detecting insect-resistant transgenic maize AM11, which uses total DNA from the sample as a template, performs a PCR reaction using the above-mentioned specific primer pair, and determines the result based on the electrophoretic fragments of the PCR product.

[0015] Furthermore, when using the specific primer pair with nucleotide sequences as shown in SEQ ID NO. 11-12 to amplify the sample DNA by PCR, if the amplification product band size is 849 bp, then the sample to be tested contains components derived from insect-resistant transgenic maize AM11; if using the specific primer pair with nucleotide sequences as shown in SEQ ID NO. 13-14 to amplify the sample DNA by PCR, if the amplification product band size is 1474 bp, then the sample to be tested contains components derived from insect-resistant transgenic maize AM11.

[0016] This invention discloses a method for breeding corn resistant to insects, comprising planting corn seeds, wherein the genome of the corn seeds contains the sequence shown in SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.15 or SEQ ID NO.16.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The flanking sequence of the exogenous inserted fragment of insect-resistant transgenic maize AM11 and its application, as well as the specific primer pairs and methods for detecting the sequence, can effectively determine whether insect-resistant transgenic maize AM11 has been successfully obtained, providing strong protection for the supervision of insect-resistant transgenic maize AM11 and its offspring. Attached Figure Description

[0019] Figure 1 This is a schematic diagram of the transformation vector p3301UbiAbUbiM7.

[0020] Figure 2 This is a PCR image of the genomic DNA of genetically modified maize AM11.

[0021] Where A: cry1Ab Gene; B: cry1m7 Gene; C: barGene. M: DNA molecular weight marker; 1, water; 2, non-GMO maize; 3, plasmid; 4-6, T1 generation AM11 plants; 7-9, T2 generation AM11 plants; 10-12, T3 generation AM11 plants.

[0022] Figure 3 This is a diagram showing the results of the insect resistance experiment in Example 2.

[0023] Figure 4 This is a schematic diagram of the AM11 insertion sequence.

[0024] Figure 5 This is a diagram of the PCR results specific to the 5' flanking sequence of the AM11 transformant.

[0025] Wherein, M is the DNA molecular weight marker; 1 is the plasmid; 2 is the non-GMO maize; 3 is water; 4-6 are T2 generation AM11 plants; 7-9 are T3 generation AM11 plants; and 10-12 are T4 generation AM11 plants.

[0026] Figure 6 This is a diagram of PCR results for the 3' flanking sequence specificity of AM11 transformants; where M is the DNA molecular weight marker; 1 is the plasmid; 2 is non-transgenic maize; 3 is water; 4-6 are T2 generation AM11 plants; 7-9 are T3 generation AM11 plants; and 10-12 are T4 generation AM11 plants. Detailed Implementation

[0027] The technical solutions of the embodiments of this application will be further described clearly and completely below with reference to the accompanying drawings. It should be noted that the described embodiments are only some embodiments of this application, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of this application without creative effort are within the scope of protection of this application.

[0028] To make the inventive objectives, technical solutions, and advantages of this application clearer, the embodiments of this application will be further described in detail below with reference to the accompanying drawings: In order to better understand the above-mentioned objectives, features, and advantages of this invention, the advantages of this invention will be further illustrated below by comparing the embodiments with the accompanying drawings and specific implementation methods.

[0029] Example 1: Obtaining insect-resistant transgenic maize AM11

[0030] 1. Construction of transformation vector p3301UbiAbUbiM7

[0031] On the carrier pCambia3301 gus Gene replacement cry1m7Gene, 35S promoter replaced by maize Ubiquitin promoter, plant expression vector p3301 UbiM7 was constructed; at the same time, Ubiquitin promoter-Cry1Ab-NOS terminator expression frame was constructed into vector p3301 UbiM7, thereby plant expression vector p3301 UbiAbUbiM7 was constructed. The structure of T-DNA region is 35S polyA terminator- bar Gene, 35S promoter replaced by maize Ubiquitin promoter cry1Ab Gene, 35S promoter replaced by maize Ubiquitin promoter cry1m7 Gene, 35S promoter replaced by maize Ubiquitin promoter, size about 10.6 kb. Vector map is shown in Figure 1 Vector T-DNA sequence refers to SEQ ID NO. 1.

[0032] 2. Agrobacterium transformation of corn embryo to obtain transgenic plants

[0033] The vector p3301 UbiAbUbiM7 was transformed into Agrobacterium LBA4404 by freeze-thaw method, and PCR was used for identification. Freshly peeled 1 mm corn embryo of Zong 31 was used as material, and the embryo was placed in D-inf solution for one hour, then washed once with D-inf, and then immersed in Agrobacterium bacterial solution added with 100 μM acetyl-syringone, and placed for 5 minutes. Take out and dry with sterile filter paper, put on D-AS medium, incubate at 26°C in dark condition for 3 days, and set control. After washing the embryo to remove bacteria, it was placed on screening medium containing 1.5 mg / L Bialaphos, and the screening culture was started for two weeks, then transferred to screening medium containing 3 mg / L Bialaphos, and subcultured every three weeks, and the screening culture was carried out for two months. Some calli grew well, which were resistant calli. The resistant calli selected in the above experiment were transferred to induction embryoid medium, and embryoids appeared after 3 weeks. Then they were transferred to differentiation medium for differentiation, and the culture conditions were 28°C, 3000 Lux light intensity per day, and 16 hours of light. Regenerated seedlings appeared soon. When the regenerated seedlings grew to 3 leaves, the seedlings were transplanted into a jar, and cultured in a room. After the seedlings grew new leaves and roots, the seedlings were taken out of the jar, the medium was washed with tap water, and transplanted into a small flowerpot mixed with nutrient soil and vermiculite (1:3). When the corn grew 2-3 new leaves, it was transplanted into a field or a large flowerpot, and self-crossed to obtain seeds.

[0034] 3. PCR identification of transgenic plants

[0035] 3.1 Extraction of plant total DNA, CTAB method was used to quickly extract total DNA of corn leaves, and the specific steps were as follows:

[0036] (1) Take 30-50 mL centrifuge tube, add 7.5 mL CTAB extraction buffer (Tris 100 mM, NaCl 1.4 M, 20 mM EDTA, 2% CTAB, 0.1% mercaptoethanol), preheat in 60°C constant temperature water bath for 30 min;

[0037] (2) Take an appropriate amount of corn leaves and place them in a 2 mL centrifuge tube. Grind the leaves into powder using a 2000 GENO / GRINDER tissue grinder under liquid nitrogen;

[0038] (3) Open the centrifuge tube and add 700 μL of CTAB extraction buffer (Tris 100 mM, NaCl 1.4 M, 20 mM EDTA, 2% CTAB, 0.1% mercaptoethanol). Heat in a 60°C water bath for 30 min, shaking a few times during the process;

[0039] (4) Take out the centrifuge tube and add 1 mL of saturated phenol to each tube. Shake well and then add 700 μL of chloroform / isoamyl alcohol (24:1) and mix gently but thoroughly. Let stand for 10 min or more after the protein is denatured, and then centrifuge; centrifuge at 12000 r / min for 10 min at room temperature;

[0040] (5) Transfer the supernatant to a new centrifuge tube and add two-thirds volume of isopropanol. Mix well to make the nucleic acid precipitate into floccules, and centrifuge at 12000 r / min for 5 min, discarding the supernatant.

[0041] (6) Add 1 mL of 70% ethanol to the precipitate and flick it with your fingers a few times. Let stand for 20 min or more;

[0042] (7) Centrifuge at 12000 r / min for 2 min, discarding the supernatant;

[0043] (8) Blow dry the precipitate on a clean bench and dissolve it in an appropriate amount of sterile water (100-200 μL);

[0044] (9) Store the extracted DNA at -20°C for later use.

[0045] 3.2 Identification of primer design

[0046] According to the gene sequence information, the amplification primers are designed as follows Cry1Ab Upstream primer 5'-GATCTACGCCGAGTCCTTCA-3' (SEQ ID NO. 2)

[0047] Downstream primer 5'-ATGTTGAACGGCCTCCTGTA-3' (SEQ ID NO. 3)

[0048] ​

[0049] The length of the amplified fragment is 795 bp.

[0050] According to the gene sequence information, the amplification primers were designed as follows cry1m7

[0051] Upstream primer 5'-ACAACTTCTACTACCCGGGC-3' (SEQ ID NO. 4)

[0052] Downstream primer 5'-CCTCTCGATGTGGATCTGCT-3' (SEQ ID NO. 5)

[0053] The length of the amplified fragment is 848 bp.

[0054] According to the gene sequence information, the amplification primers were designed as follows bar

[0055] Upstream primer 5'-CCAGAAACCCACGTCATGCC-3' (SEQ ID NO. 6)

[0056] Downstream primer 5'-CAGGAACCGCAGGAGTGGA-3' (SEQ ID NO. 7) The length of the amplified fragment is 372 bp.

[0057] 3.3 PCR amplification system and reaction procedure

[0058] PCR reaction system:

[0059]

[0060] PCR reaction procedure:

[0061]

[0062] PCR reaction was carried out by using the above system and procedure, and transgenic positive plants were obtained, and the results are shown in Table 1. Figure 2 The results show that the genes of cry1Ab , cry1m7 and bar are integrated into the genome of the insect-resistant transgenic corn AM11.

[0063] Example 2 Insect resistance identification of transgenic corn AM11

[0064] ​​The insect-resistant transgenic maize AM11 T3 generation planted in the greenhouse field is sampled at the stage of elongation, and the maize leaves are put into culture dishes, 2 newly hatched corn borer larvae are added to each dish. The test is carried out in a culture room with a relative humidity of 70%-80%, a temperature of 26-28℃, and a light cycle of 16h:8h (L:D), the mortality rate of insects is counted every 24 h, and the same source of new tissues is added or replaced according to the tissue consumption, the test is repeated three times under the same conditions, and a group of non-transgenic maize Zong 31 is used as a control for parallel test. The results show that the non-transgenic maize Zong 31 leaf is highly susceptible to corn borer, and the transgenic maize AM11 is highly resistant to corn borer (Table 1). Figure 3

[0065] Table 1: Resistance identification of transgenic maize to corn borer

[0066]

[0067] Note: The data in the table are mean ± standard deviation, and the lower case letters after the data indicate that the differences between the treatments are significant at the 0.05 level. P <0.05 level.

[0068] Example 3: Obtaining and application of flanking sequence of insect-resistant transgenic maize AM11

[0069] Through PCR and gene separation identification of transgenic offspring, the target gene cry1Ab , cry1m7 and the selection marker gene bar in the transformation vector are integrated into the AM11 genome in a single copy and can be stably inherited in transgenic plants. Through three-generation sequencing and other methods, it is identified that the exogenous gene is integrated into the 6th chromosome of maize near 16 Mb, and the upstream and downstream flanking sequences of the insertion sequence are determined.

[0070] Through PacBio third-generation sequencing technology, the genomic DNA of the insect-resistant transgenic maize AM11 is sequenced and overlapped PCR, and the exogenous insertion sequence (SEQ ID NO. 8), the 5' end flanking sequence (SEQ ID NO. 9) and the 3' end flanking sequence (SEQ ID NO. 10) are obtained (for specific AM11 insertion sequence schematic diagram, see Figure 4 ).

[0071] 1) 5' end flanking sequence specific PCR identification

[0072] ​A pair of primers were designed according to the 5' flanking sequence of the insect-resistant transgenic corn AM11 and the 35S polyA terminator sequence in the foreign fragment, respectively, to establish a PCR identification method for the 5' flanking sequence of the AM11 event. The primer designed according to the 5' end of the corn genome in the integration site in one of the foreign fragments was 5'-GCTAGTTGATTCGGCAGGTG-3' (SEQ ID NO. 11), and the primer designed according to the 35S polyA terminator sequence was 5'-GGGTTTCGCTCATGTGTTGA-3' (SEQ ID NO. 12).

[0073] The corn genomic DNA extraction and the PCR reaction system were performed according to the method in Example 1. The PCR reaction program was 95℃ for 5 min, 35 cycles of (95℃ for 15 s, 58℃ for 15 s, 72℃ for 1 min), and 72℃ for 7 min. When the specific primers were used for PCR amplification, no amplification band was obtained for water, non-transgenic plants or plasmids, and only the transgenic plant AM11 DNA had a specific 849 bp (SEQ ID NO. 15) target band. Figure 5 Three repeated tests were performed, and the results were consistent.

[0074] 2) 3' flanking sequence-specific PCR identification

[0075] A pair of primers were designed according to the 3' flanking sequence of the insect-resistant transgenic corn AM11 and the cry1m7 gene sequence in the foreign fragment, respectively, to establish a PCR identification method for the 3' flanking sequence of the AM11 event. The primer designed according to the cry1m7 gene sequence was 5'-GCGACATCAGGGTGAACATC-3' (SEQ ID NO. 13). The primer designed according to the 3' end of the corn genome in the integration site in one of the foreign fragments was 5'-GATTCCCCTAGTAACGGCGA-3' (SEQ ID NO. 14).

[0076] The corn genomic DNA extraction and the PCR reaction system were performed according to the method in Example 1. The PCR reaction program was 95℃ for 5 min, 35 cycles of (95℃ for 15 s, 58℃ for 15 s, 72℃ for 90 s), and 72℃ for 7 min. When the specific primers were used for PCR amplification, no amplification band was obtained for water, non-transgenic plants or plasmids, and only the transgenic plant AM11 DNA had a specific 1474 bp (SEQ ID NO. 16) target band. Figure 6 Three repeated tests were performed, and the results were consistent.

[0077] While the preferred embodiments of the application have been described, additional variations and modifications can be made to these embodiments by those skilled in the art once they have the benefit of the foregoing description. Therefore, the appended claims are intended to encompass all such variations and modifications as falling within the scope of the application.

Claims

1. A flanking sequence of an exogenous insert fragment from insect-resistant transgenic maize AM11, characterized in that, The flanking sequences are the 5' and 3' flanking sequences of the exogenous insertion fragment of insect-resistant transgenic maize AM11; The 5' flanking sequence is a nucleotide sequence as shown in SEQ ID NO. 9; The 3' flanking sequence is a nucleotide sequence as shown in SEQ ID NO.10; The flanking sequence is derived from the insect-resistant transgenic maize event AM11. The maize seeds of this insect-resistant transgenic maize, AM11, were deposited on September 5, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.46691, and are classified as maize. Zea mays .

2. The application of specific primer pairs in the detection of insect-resistant transgenic maize AM11, characterized in that, The specific primer pair is used to detect the flanking sequence as described in claim 1, and the specific primer pair consists of primer pair 1 and primer pair 2: (1) Primer pair 1 with the 5' flanking sequence as the target has the nucleotide sequence shown in SEQ ID NO.11-12; (2) Primer pair 2 with the 3' flanking sequence as the target has the nucleotide sequence shown in SEQ ID NO.13-14; The seeds of the insect-resistant transgenic maize AM11 were deposited on September 5, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.46691, and classified as maize. Zea mays .

3. The application of the kit in detecting insect-resistant transgenic maize AM11, characterized in that, The kit contains the specific primer pair as described in claim 2; The seeds of the insect-resistant transgenic maize AM11 were deposited on September 5, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.46691, and classified as maize. Zea mays.

4. A method for detecting insect-resistant transgenic maize AM11, characterized in that, Using total DNA from the sample as a template, a PCR reaction was performed using the specific primer pair described in claim 2, and the results were determined based on the electrophoretic fragments of the PCR products. The PCR reaction is performed by using the specific primer pair to amplify the sample DNA. If the amplification product band size is 849 bp and 1474 bp, then the sample to be tested contains components derived from insect-resistant transgenic maize AM11. The seeds of the insect-resistant transgenic maize AM11 have been deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC NO.46691.

5. A method for breeding corn resistant to insects, characterized in that, This includes planting corn seeds, specifically genetically modified corn AM11 seeds, which have been deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.46691.

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