WO2025076865A1 - Flanking sequence of exogenous insert fragment of transgenic maize vb15 and use thereof - Google Patents

Abstract

A flanking sequence of an exogenous insert fragment of transgenic maize VB15 and a use thereof. On the basis of transgenic maize VB15, a flanking sequence of an exogenous insert fragment thereof is obtained, the flanking sequence comprising a 5'-end flanking sequence and/or a 3'-end flanking sequence. The 5'-end flanking sequence is the sequence shown in SEQ ID NO. 1 or a specific fragment of the sequence shown in SEQ ID NO. 1; and the 3'-end flanking sequence is the sequence shown in SEQ ID NO. 2 or a specific fragment of the sequence shown in SEQ ID NO. 2. On the basis of the flanking sequence, the transgenic maize VB15 and a related material can be specifically detected and identified, and the transgenic maize is supervised and managed.

Description

A flanking sequence of a foreign inserted fragment of transgenic corn VB15 and its application

This application claims the priority of the Chinese patent application filed with the China Patent Office on October 10, 2023, with application number CN202311302047.9 and invention name “A flanking sequence of an exogenous insertion fragment of transgenic corn VB15 and its application”, the entire contents of which are incorporated by reference into this application.

Technical Field

The invention belongs to the technical field of molecular biology, and in particular relates to a flanking sequence of a transgenic corn VB15 exogenous insertion fragment and an application thereof.

Background Art

Insect pests (especially corn borers) can cause serious reductions in corn yields. Insect-resistant transgenic corn is one of the earliest research traits in the world. The insect-resistant gene is mainly derived from the Bt insecticidal protein of Bacillus thuringiensis. Its mechanism of action is to cause perforation in the insect's gastrointestinal tract, thereby causing metabolic disorders and death of the insect. 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 borers, and the Cry3Bb expressed in MON863 has a good control effect on pests that harm the roots of corn. Syngenta Group has also developed transgenic insect-resistant corn BT11 and BT176 expressing Cry1Ab protein. Pioneer and Dow Chemical have jointly developed transgenic corn containing Cry34 and Cry35 to resist corn root pests.

So far, domestic transgenic phytase corn BVLA430101, insect-resistant and herbicide-resistant corn DBN9936, Ruifeng 125 and herbicide-resistant corn DBN9858 have been considered to meet safety standards. It is a common method to transfer exogenous insertion fragments into corn seeds to construct transgenic corn varieties. With the commercial application of transgenic corn, the upgrading of the corn industry will be accelerated. For the sustainable use of transgenic corn in the future, it is necessary to develop more resistant and excellent transformants. The flanking sequence of the exogenous insertion fragment and the detection method established based on this flanking sequence are important indicators for supervision and management. Therefore, it is necessary to obtain the flanking sequence of resistant and excellent transformed corn varieties and establish an identification system to effectively supervise insect-resistant transgenic corn varieties.

Summary of the invention

The purpose of the present invention is to provide the flanking sequence of the exogenous insertion fragment of transgenic corn VB15, so as to achieve effective supervision of transgenic corn VB15.

In order to achieve the above object, the present invention provides a flanking sequence of an exogenous insertion fragment of transgenic corn VB15, wherein the flanking sequence includes a 5' end flanking sequence and/or a 3' end flanking sequence;

The 5' flanking sequence is the sequence shown in SEQ ID NO.1 or a specific fragment of the sequence shown in SEQ ID NO.1;

The 3'-end flanking sequence is the sequence shown in SEQ ID NO.2 or a specific fragment of the sequence shown in SEQ ID NO.2;

The transgenic corn VB15 is deposited in the General Microbiological Center of China Microorganism Culture Collection Administration, with the deposit number of CGMCCNO.27565.

The present invention also provides a DNA fragment for detecting transgenic corn VB15, wherein the DNA fragment comprises DNA fragment 1 and/or DNA fragment 2;

The DNA fragment 1 is the sequence shown in SEQ ID NO.5 or a specific fragment of the sequence shown in SEQ ID NO.5;

The DNA fragment 2 is the sequence shown in SEQ ID NO.6 or a specific fragment of the sequence shown in SEQ ID NO.6.

Preferably, the specific fragment of the sequence shown in SEQ ID NO.5 is the sequence shown in SEQ ID NO.7 or SEQ ID NO.8;

The specific fragment of the sequence shown in SEQ ID NO.6 is the sequence shown in SEQ ID NO.9.

The present invention also provides primers for detecting transgenic corn VB15, the primers comprising an upstream primer and a downstream primer; The nucleotide sequence of the upstream primer is shown in any one of SEQ ID NO.11, SEQ ID NO.12 and SEQ ID NO.15; the nucleotide sequence of the downstream primer is shown in any one of SEQ ID NO.13, SEQ ID NO.14 and SEQ ID NO.16.

The present invention also provides a kit for detecting transgenic corn VB15, wherein the kit comprises the primers described in the above technical solution.

The present invention also provides the use of the flanking sequence or DNA fragment or primer or kit described in the above technical solution in detecting transgenic corn VB15 and/or transgenic corn VB15 related materials.

Preferably, the transgenic corn VB15 related materials include parents and/or offspring of transgenic corn VB15.

Preferably, the detected objects include one or more of plants, tissues, seeds, transgenic corn VB15 products and products of transgenic corn VB15 related materials.

The present invention also provides a method for detecting transgenic corn VB15 or transgenic corn VB15-related materials, comprising the following steps: extracting DNA of the material to be tested, amplifying the DNA using the primers described in the above technical solution, and if a positive amplification product is obtained, the material to be tested contains components of transgenic corn VB15.

The present invention also provides a method for planting corn resistant to insects, comprising planting corn seeds;

The genome of the corn seed contains the DNA fragment described in the above technical solution; the corn seed is transgenic corn VB15.

Beneficial effects:

The present invention constructs a transformation vector p3301UbiAbUbiVip3 with modified cry1Ab gene (patent application number CN202010553538.0) and vip3 gene (patent application number CN202010554016.2), and transfers them into the maize genome through Agrobacterium-mediated method to obtain an insect-resistant transgenic maize variety: maize VB15. The flanking sequences of the exogenous inserted fragment of the transgenic maize VB15 are obtained, including 5' flanking sequences and/or 3' flanking sequences; the 5' flanking sequence is the sequence shown in SEQ ID NO.1 or a specific fragment thereof; the 3' flanking sequence is the sequence shown in SEQ ID NO.2 or a specific fragment thereof. Based on the flanking sequences, the transgenic maize VB15 and its related materials can be specifically detected and identified, and the transgenic maize can be better supervised and managed.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

Fig. 1 is a schematic diagram of the transformation vector p3301UbiAbUbiVip3;

Figure 2 is the PCR test result in step 3 of Example 1; wherein A: cry1Ab gene; B: vip3 gene; C: bar gene; M: DNA molecular weight marker; 1: water; 2: non-transgenic corn Zong 31; 3: plasmid p3301UbiAbUbiVip3; 4-12, different generations of transgenic corn VB15;

Fig. 3 is a diagram of the insect resistance test results of Example 2;

FIG4 is a schematic diagram of the exogenous insertion sequence of transgenic corn VB15;

Fig. 5 is a diagram showing the 5' flanking sequence-specific PCR results of the exogenous inserted fragment of transgenic corn VB15 transformants;

Fig. 6 is a diagram showing the 3' flanking sequence-specific PCR results of the exogenous inserted fragment of transgenic corn VB15 transformants;

7 is a PCR result diagram of the flanking sequence of the exogenous insertion fragment of transgenic corn VB15 in Example 4;

Among them, in Figures 5 to 7, M: DNA molecular weight marker; 1: plasmid p3301UbiAbUbiVip3; 2: non-transgenic corn Zong 31; 3: water; 4-6: T2 generation transgenic corn VB15 plants; 7-9: T3 generation transgenic corn VB15 plants; 10-12: T4 generation VB15 plants.

Biological deposit information

Genetically modified corn VB15, classified and named: Zea mays, was deposited on July 25, 2023 at the General Microbiology Center of China Culture Collection Administration, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code 100101, with the deposit number CGMCC No.27565.

DETAILED DESCRIPTION

The present invention provides a flanking sequence of an exogenous insertion fragment of transgenic corn VB15, wherein the flanking sequence includes a 5' end flanking sequence and/or a 3' end flanking sequence;

The 5' flanking sequence is the sequence shown in SEQ ID NO.1 or a specific fragment of the sequence shown in SEQ ID NO.1;

The 3'-end flanking sequence is the sequence shown in SEQ ID NO.2 or a specific fragment of the sequence shown in SEQ ID NO.2;

The transgenic corn VB15 is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number CGMCC No.27565.

In the present invention, the specific fragment of the sequence shown by SEQ ID NO.1 is preferably the sequence shown by SEQ ID NO.3; the specific fragment of the sequence shown by SEQ ID NO.2 is preferably the sequence shown by SEQ ID NO.4.

The nucleotide sequences of SEQ ID NO.1 to 4 of the present invention are as follows:

The present invention constructs the transformation vector p3301UbiAbUbiVip3 with the modified cry1Ab gene (patent application number CN202010553538.0) and vip3 gene (patent application number CN202010554016.2), and transfers them into the corn genome through Agrobacterium-mediated method, thereby obtaining a new corn variety: corn VB15, which may enter commercial planting in the future. Based on the flanking sequence of the exogenous insertion fragment of transgenic corn VB15, the present invention can specifically detect transgenic corn VB15 and related materials, and can better supervise and manage transgenic corn.

The present invention also provides a DNA fragment for detecting transgenic corn VB15, wherein the DNA fragment comprises DNA fragment 1 and/or DNA fragment 2;

The DNA fragment 1 is the sequence shown in SEQ ID NO.5 or a specific fragment of the sequence shown in SEQ ID NO.5;

The DNA fragment 2 is the sequence shown in SEQ ID NO.6 or a specific fragment of the sequence shown in SEQ ID NO.6;

The transgenic corn VB15 is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number CGMCC NO.27565.

In the present invention, the specific fragment of the sequence shown in SEQ ID NO.5 is preferably SEQ ID NO.7 or SEQ ID NO.8 The specific fragment of the sequence shown in SEQ ID NO.6 is preferably the sequence shown in SEQ ID NO.9.

The nucleotide sequences of SEQ ID NO.5 to 9 of the present invention are as follows:


The underlined portion of the sequence shown is the sequence shown in SEQ ID NO.1, ie, the 5' flanking sequence, and the rest is the exogenous insertion sequence, recorded as SEQ ID NO.10.



The underlined portion of the sequence shown is the sequence shown in SEQ ID NO.2, ie, the 3' flanking sequence, and the remaining portion is the exogenous insertion sequence shown in SEQ ID NO.10.
Among them, the underlined part is the specific fragment of the sequence shown in SEQ ID NO.3, and the rest is the specific fragment of the sequence shown in SEQ ID NO.10;
Among them, the underlined part is the specific fragment of the sequence shown in SEQ ID NO.3, and the rest is the specific fragment of the sequence shown in SEQ ID NO.10;
The underlined portion is the sequence shown in SEQ ID NO.4, ie, the specific fragment of the 3'-end flanking sequence, and the remaining portion is the specific fragment of the sequence shown in SEQ ID NO.10.

The present invention also provides primers for detecting transgenic corn VB15, wherein the primers preferably include an upstream primer and a downstream primer; the nucleotide sequence of the upstream primer is preferably as shown in any one of SEQ ID NO.11, SEQ ID NO.12 and SEQ ID NO.15; the nucleotide sequence of the downstream primer is preferably as shown in any one of SEQ ID NO.13, SEQ ID NO.14 and SEQ ID NO.16;

The transgenic corn VB15 is deposited in the General Microbiology Center of China Culture Collection Administration, with the deposit number CGMCC NO.27565.

The sequence information shown in SEQ ID NO.11 to 16 of the present invention is as follows:

SEQ ID NO.11: 5'-GGTCAGCGAGCCTGTAGAAC-3';

SEQ ID NO.12: 5'-TGCACTTCTACGACGTGAGC-3';

SEQ ID NO.13: 5'-TTCTAATTCCTAAAACCAAAATCCA-3';

SEQ ID NO.14: 5'-TGCTCGATGCGTATAGCAGA-3';

SEQ ID NO.15: 5'-TCCTCGCCCAGCAGAATGTT-3';

SEQ ID NO.16: 5'-TCGCTCATGTGTTGAGCATA-3'.

The present invention utilizes the upstream primer shown in SEQ ID NO.11 and the downstream primer shown in SEQ ID NO.13 to specifically amplify the nucleotide sequence shown in SEQ ID NO.7; utilizes the upstream primer shown in SEQ ID NO.15 and the downstream primer shown in SEQ ID NO.16 to specifically amplify the nucleotide sequence shown in SEQ ID NO.8; utilizes the upstream primer shown in SEQ ID NO.12 and the downstream primer shown in SEQ ID NO.14 to specifically amplify the nucleotide sequence shown in SEQ ID NO.9; the above primer combinations can all be used to detect genetically modified corn VB15.

The present invention also provides a kit for detecting transgenic corn VB15, wherein the kit comprises the primers described in the above technical solution.

The present invention also provides the use of the DNA fragment or primer or kit described in the above technical solution in detecting transgenic corn VB15 and/or transgenic corn VB15 related materials.

In the present invention, the transgenic corn VB15 related materials preferably include parents and/or offspring of transgenic corn VB15; the offspring preferably include hybrid F1 generation; the objects of detection preferably include one or more of plants, tissues, seeds, transgenic corn VB15 products and products of transgenic corn VB15 related materials.

Since the flanking sequence of the exogenous insertion fragment of a specific transgenic event (i.e., resistant corn) is specific, the flanking sequence can be used to specifically detect the transgenic event, and a probe containing at least part of the flanking sequence and at least part of the exogenous insertion fragment is used for hybridization, or a primer containing at least part of the flanking sequence and at least part of the exogenous insertion fragment is designed for specific amplification, PCR amplification, and detection of specific bands, etc. The present invention is based on the flanking sequence and/or DNA fragment of the exogenous insertion fragment of transgenic corn VB15, and upstream specific primers are designed at the 5' flanking sequence, and downstream specific primers are designed according to the exogenous insertion fragment to amplify the specific fragment; or upstream specific primers are designed according to the exogenous insertion fragment, and downstream specific primers are designed according to the 3' flanking sequence to amplify the specific fragment, so as to realize the detection of transgenic corn VB15 and related materials.

The present invention also provides a method for detecting transgenic corn VB15 or transgenic corn VB15-related materials, comprising the following steps: extracting DNA of the material to be tested, amplifying the DNA using the primers described in the above technical solution, and if a positive amplification product is obtained, the material to be tested contains components of transgenic corn VB15.

The present invention preferably uses the upstream primer shown in SEQ ID NO.11 and the downstream primer shown in SEQ ID NO.13 to perform PCR amplification on the DNA of the test material. If the amplified product band size is 681 bp, the test material contains the components of genetically modified corn VB15. The present invention has no strict requirements on the system and conditions of the PCR amplification, and conventional operations are sufficient.

The present invention preferably uses the upstream primer shown in SEQ ID NO.15 and the downstream primer shown in SEQ ID NO.16 to perform PCR amplification on the DNA of the test material. If the amplified product band size is 376bp, the test material contains the components of genetically modified corn VB15. The present invention has no strict requirements on the system and conditions of the PCR amplification, and conventional operations are sufficient.

The present invention preferably uses the upstream primer shown in SEQ ID NO.12 and the downstream primer shown in SEQ ID NO.14 to detect The DNA of the material is amplified by PCR. If the amplified product has a band size of 549 bp, the material to be tested contains the components of transgenic corn VB15. The present invention has no strict requirements on the system and conditions of the PCR amplification, and conventional operations are sufficient.

The flanking sequences of the exogenous insertion fragment, the DNA fragment, and the PCR primers and methods for detecting the DNA fragment provided by the present invention can effectively judge whether the transgenic corn VB15 is successfully obtained, and provide a strong guarantee for the supervision of transgenic corn VB15 and its offspring.

The present invention also provides a method for planting corn resistant to insects, comprising planting corn seeds; the genome of the corn seeds comprises the DNA fragment described in the above technical solution.

In the present invention, the insects of the present invention preferably include Lepidoptera insects, and more preferably fall armyworm and/or corn borer; the corn seeds preferably include transgenic corn VB15.

To further illustrate the present invention, the flanking sequences of a transgenic corn VB15 exogenous insertion fragment provided by the present invention and its application are described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

Example 1

Obtaining transgenic corn VB15

1. Construction of transformation vector p3301UbiAbUbiVip3

The gus gene on the vector pCambia3301 (purchased from Beijing Bomade Company) was replaced with the vip3 gene (patent application number 202010554016.2), and the 35S promoter was replaced with the corn Ubiquitin promoter to construct the plant expression vector p3301UbiVip3; at the same time, the corn Ubiquitin promoter-Cry1Ab-NOS terminator expression frame was constructed into the vector p3301UbiVip3 to construct the plant expression vector p3301UbiAbUbiVip3. The structure in the T-DNA region is 35SpolyA terminator-bar gene-35S promoter-ubiquitin promoter-cry1Ab gene-NOS terminator-ubiquitin promoter-vip3 gene-NOS terminator, with a size of about 10.2kb. The vector map is shown in Figure 1.

2. Transgenic plants obtained by Agrobacterium transformation of maize embryos

The vector p3301UbiAbUbiVip3 obtained in step 1 was transformed into Agrobacterium LBA4404 by freeze-thaw method and identified by PCR. Freshly peeled 1mm corn 31 embryos were used as materials. After placing the embryos in the infection solution for one hour, they were washed once with the infection solution, and then immersed in the Agrobacterium solution with 100μM acetosyringone added, and placed for 5 minutes. Take out and blot dry with sterilized filter paper, place on D-AS medium, and co-culture at 26℃ in the dark for 3 days, and set up a control. After washing and sterilizing the embryos, place them on the screening medium containing 1.5mg/L Bialaphos, start screening and culture for two weeks, and then transfer to the screening medium containing 3mg/L Bialaphos for screening and culture, subculture every three weeks, and screen and culture for two months. Some calli grow well and are resistant calli. The resistant callus selected in the above experiment is transferred to the embryoid induction medium, and embryoids will appear in 3 weeks. Then transfer to differentiation medium for differentiation. The culture conditions are 28℃, 3000Lux light intensity per day, and 16 hours of light. Soon, regenerated seedlings will appear. When the regenerated plantlets grow to 3 leaves, the seedlings can be transplanted into cans and cultured indoors. After the seedlings grow new leaves and roots, take the seedlings out of the cans, rinse the culture medium with tap water, and transplant them into small pots mixed with nutrient soil and vermiculite (1:3). When the corn grows 2-3 new leaves again, it can be moved into the field or large pots, self-pollinated to obtain seeds, which are named corn VB15.

3. PCR identification of transgenic plants

3.1 Extraction of plant total DNA: The CTAB method was used to quickly extract the total DNA from the leaves of transgenic corn VB15 and non-transgenic corn Zong 31. The specific steps are as follows:

(1) Take a 30-50 ml centrifuge tube, add 7.5 ml CTAB extraction buffer (100 mM Tris, 1.4 M NaCl, 20 mM EDTA, 2% CTAB, 0.1% mercaptoethanol and the balance distilled water), and preheat in a 60°C constant temperature water bath for 30 min;

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

(3) Open the centrifuge tube, add 700 μl CTAB extraction buffer (100 mM Tris, 1.4 M NaCl, 20 mM EDTA, 2% CTAB, 0.1% mercaptoethanol and the balance distilled water), and keep in a 60°C water bath for 30 min, shaking gently several times;

(4) Take out the centrifuge tubes, add 1 ml of saturated phenol to each tube, shake evenly, and then add 700 μl of chloroform/isoamyl alcohol (chloroform and isoamyl alcohol). The volume ratio of alcohol was 24:1) and the mixture was lightly and thoroughly mixed and allowed to stand for more than 10 min. After the protein was denatured, the mixture was centrifuged at 12000 r/min for 10 min at room temperature and the supernatant was collected.

(5) Transfer the supernatant to a new centrifuge tube, add two-thirds of the volume of isopropanol, mix well to precipitate the nucleic acid into flocs, centrifuge at 12,000 rpm for 5 min, and discard the supernatant.

(6) Add 1 ml of 70% ethanol to the precipitate, flick it a few times with your fingers, and let it sit for more than 20 minutes;

(7) Centrifuge at 12000 r/min for 2 min and discard the supernatant;

(8) Blow dry the precipitate on a clean bench and dissolve it in an appropriate amount of sterile water (100-200 μl).

(9) Store the extracted DNA at -20°C until use.

3.2 Identification primer design

According to the sequence information of Cry1Ab gene (patent application number CN202010553538.0), the amplification primers were designed as follows: upstream primer 5’-GATCTACGCCGAGTCCTTCA-3’ (SEQ ID No.17); downstream primer 5’-ATGTTGAACGGCCTCCTGTA-3’ (SEQ ID No.18); the amplified fragment length was 795bp.

According to the sequence information of vip3 gene (patent application number CN202010554016.2), the amplification primers were designed as follows: upstream primer 5’-ATCCTCAAGAACCAGCAGCT-3’ (SEQ ID No.19); downstream primer 5’-AAGTTGTACACGTTGCCCAC-3’ (SEQ ID No.20), and the amplified fragment length was 671bp.

According to the bar gene sequence information, the amplification primers were designed as follows: upstream primer 5’-CCAGAAACCCACGTCATGCC-3’ (SEQ ID No.21); downstream primer 5’-CAGGAACCGCAGGAGTGGA-3’ (SEQ ID No.22), and the amplified fragment length was 370bp.

3.3 PCR amplification system and reaction procedure

PCR reaction system: ddH ₂ O 13.4 μl, 10× PCR buffer 2 μl, dNTP (10 mM) 0.4 μl, upstream primer (10 μM) 0.4 μl, downstream primer (10 μM) 0.4 μl, DMSO 2 μl, Taq enzyme (5 U/μl) 0.4 μl, corn total DNA 1 μl, a total of 20 μl;

PCR reaction program: pre-denaturation at 95°C for 5 minutes; denaturation at 94°C for 30 seconds, annealing at 60°C for 60 seconds, extension at 72°C for 1 minute, 35 cycles; extension at 72°C for 7 minutes; insulation at 25°C.

The PCR reaction was carried out using the above system and procedure to obtain transgenic positive plants, and the results are shown in Figure 2; wherein A: cry1Ab gene; B: vip3 gene; C: bar gene; M: DNA molecular weight marker; 1: water; 2: non-transgenic corn Zong31; 3: plasmid p3301UbiAbUbiVip3; 4-12, transgenic corn VB15 of different generations.

As can be seen from Figure 2, cry1Ab, vip3 and bar genes were integrated into the genome of transgenic corn VB15.

Example 2

Identification of insect resistance of transgenic corn VB15

When the transgenic corn VB15T3 generation planted in the greenhouse field grew to the jointing stage, the corn leaves were sampled and placed in a culture dish, and two newly hatched fall armyworm larvae were inoculated in each dish. The experiment was carried out in a culture room with a relative humidity of 70% to 80%, a temperature of 26 to 28°C, and a photoperiod of 16h:8h (L:D). The mortality of the insects was counted every 24h, and new tissues from the same source were added or replaced according to the consumption of the tissues. The experiment was repeated three times under the same conditions, and another group of parallel experiments were carried out using non-transgenic corn Zong 31 as a control. The results are shown in Table 1 and Figure 3.

Table 1 Identification of resistance of transgenic corn to fall armyworm

Note: Different lowercase letters indicate significant differences at the 0.05 level.

According to Table 1 and Figure 3, the leaves of non-GM corn Zong 31 are highly susceptible to fall armyworm, while the leaves of GM corn VB15 are highly resistant Fall armyworm, indicating that the transgenic corn VB15 provided by the present invention is an insect-resistant variety, and was deposited in the China Type Culture Collection (CGMCC) on July 25, 2023, with the deposit number CGMCC No.27565.

Example 3

Obtaining and applying the flanking sequences of the exogenous inserted fragment of transgenic maize VB15

The genomic DNA of transgenic corn VB15 was sequenced using PacBio third-generation sequencing technology to obtain the exogenous insertion sequence (SEQ ID No.10), 5’ flanking sequence (SEQ ID No.1) and 3’ flanking sequence (SEQ ID No.2). The specific sequence diagram is shown in Figure 4.

1) 5' flanking sequence-specific PCR identification

Using the 5' flanking sequence of the exogenous insertion fragment of transgenic corn VB15 and the bar gene sequence in the exogenous fragment, a pair of primers were designed for two different insertion sites, and a PCR identification method for the 5' flanking sequence of the VB15 event was established. Among them, the upstream primer designed from the corn genome at the 5' end of the integration site in an exogenous fragment is shown in SEQ ID No.11; the downstream primer designed based on the bar gene sequence is shown in SEQ ID No.13;

Extract corn genomic DNA according to step 3.1 of Example 1, and use the extracted corn genomic DNA as a template to perform PCR amplification using the upstream primer and the downstream primer;

PCR reaction system: ddH ₂ O 13.4 μl, 10× PCR buffer 2 μl, dNTP (10 mM) 0.4 μl, upstream primer (10 μM) 0.4 μl, downstream primer (10 μM) 0.4 μl, DMSO 2 μl, Taq enzyme (5 U/μl) 0.4 μl, corn total DNA 1 μl, a total of 20 μl;

PCR reaction program: pre-denaturation at 95°C for 5 minutes; denaturation at 94°C for 30 seconds, annealing at 58°C for 60 seconds, extension at 72°C for 1 minute, 35 cycles; extension at 72°C for 7 minutes.

The PCR products were detected by agarose gel electrophoresis, and the results are shown in Figure 5; wherein M: DNA molecular weight marker; 1: plasmid p3301UbiAbUbiVip3; 2: non-transgenic corn Zong 31; 3: water; 4-6: T2 generation transgenic corn VB15 plants; 7-9: T3 generation transgenic corn VB15 plants; 10-12: T4 generation VB15 plants.

As shown in Figure 5, when the upstream and downstream primers provided by the present invention were used for PCR amplification, no amplified bands were found in water, non-transgenic corn Zong 31 or plasmid p3301UbiAbUbiVip3, and only the DNA amplified sequence of transgenic corn VB15 had a specific 681bp target band, the specific sequence of which is shown in SEQ ID No. 7. Three repeated experiments were conducted based on this result, and the results reflected were consistent.

2) 3’ flanking sequence specific PCR identification

A pair of primers were designed using the 3’ flanking sequence of the exogenous insertion fragment of transgenic corn VB15 and the vip3 gene sequence in the exogenous fragment, and a PCR identification method for the 3’ flanking sequence of the VB15 event was established. The upstream primer designed based on the vip3 gene sequence is shown in SEQ ID No.15, and the downstream primer designed based on the 3’ end of the corn genome in an integration site in an exogenous fragment is shown in SEQ ID No.16.

Extract corn genomic DNA according to step 3.1 of Example 1, and use the extracted corn genomic DNA as a template to perform PCR amplification using the upstream primer and the downstream primer;

PCR reaction system: ddH ₂ O 13.4 μl, 10× PCR buffer 2 μl, dNTP (10 mM) 0.4 μl, upstream primer (10 μM) 0.4 μl, downstream primer (10 μM) 0.4 μl, DMSO 2 μl, Taq enzyme (5 U/μl) 0.4 μl, corn total DNA 1 μl, a total of 20 μl;

PCR reaction program: pre-denaturation at 95°C for 5 minutes; denaturation at 94°C for 30 seconds, annealing at 58°C for 60 seconds, extension at 72°C for 1 minute, 35 cycles; extension at 72°C for 7 minutes.

The PCR products were detected by agarose gel electrophoresis, and the results are shown in Figure 6; wherein M: DNA molecular weight marker; 1: plasmid p3301UbiAbUbiVip3; 2: non-transgenic corn Zong 31; 3: water; 4-6: T2 generation transgenic corn VB15 plants; 7-9: T3 generation transgenic corn VB15 plants; 10-12: T4 generation VB15 plants.

As can be seen from Figure 6, when the upstream and downstream primers provided by the present invention were used for PCR amplification, no amplified bands were found in water, non-transgenic plants or plasmid p3301UbiAbUbiVip3, and only the DNA amplified sequence of transgenic corn VB15 had a specific 549 bp target band, the specific sequence of which is shown in SEQ ID No. 9. Based on this result, three repeated experiments were conducted, and the results reflected were consistent.

Example 4

Application and expansion of flanking sequences of foreign inserted fragments in transgenic maize VB15

A pair of primers was randomly designed using the 5' flanking sequence of the exogenous insertion fragment of transgenic corn VB15 and the bar gene sequence in the exogenous fragment. PCR identification of the 5' flanking sequence of the VB15 event can also be performed. Among them, the upstream primer designed from the corn genome at the 5' end of the integration site in an exogenous fragment is shown in SEQ ID No. 12; the downstream primer designed based on the bar gene sequence is shown in SEQ ID No. 14;

Extract maize genomic DNA in the manner of step 3.1 of Example 1, and use the extracted genomic DNA of transgenic maize VB15 and non-transgenic maize Zong 31 as templates to perform PCR amplification using the upstream primers and downstream primers mentioned above;

PCR reaction system: ddH ₂ O 13.4 μl, 10× PCR buffer 2 μl, dNTP (10 mM) 0.4 μl, upstream primer (10 μM) 0.4 μl, downstream primer (10 μM) 0.4 μl, DMSO 2 μl, Taq enzyme (5 U/μl) 0.4 μl, corn total DNA 1 μl, a total of 20 μl;

PCR reaction program: pre-denaturation at 95°C for 5 minutes; denaturation at 94°C for 30 seconds, annealing at 58°C for 60 seconds, extension at 72°C for 1 minute, 35 cycles; extension at 72°C for 7 minutes.

The PCR products were detected by agarose gel electrophoresis, and the results are shown in Figure 7; wherein M: DNA molecular weight marker; 1: plasmid p3301UbiAbUbiVip3; 2: non-transgenic corn Zong 31; 3: water; 4-6: T2 generation transgenic corn VB15 plants; 7-9: T3 generation transgenic corn VB15 plants; 10-12: T4 generation VB15 plants.

As shown in Figure 7, when the upstream and downstream primers provided by the present invention were used for PCR amplification, no amplified bands were found in water, non-transgenic plants or plasmid p3301UbiAbUbiVip3, and only the DNA amplified sequence of transgenic corn VB15 had a specific 376bp target band, the specific sequence of which is shown in SEQ ID No. 8. Three repeated experiments were conducted based on this result, and the results reflected were consistent.

It can be seen from the above content that the technical solution provided by the present invention can specifically detect genetically modified corn VB15 and its related materials, and can better supervise and manage genetically modified corn.

Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims (10)

The flanking sequence of the exogenous insertion fragment of transgenic corn VB15 is characterized in that the flanking sequence includes a 5' flanking sequence and/or a 3' flanking sequence; The 5' flanking sequence is the sequence shown in SEQ ID NO.1 or a specific fragment of the sequence shown in SEQ ID NO.1; The 3'-end flanking sequence is the sequence shown in SEQ ID NO.2 or a specific fragment of the sequence shown in SEQ ID NO.2; The transgenic corn VB15 is deposited in the General Microbiology Center of China Culture Collection Administration, with the deposit number CGMCC NO.27565. A DNA fragment for detecting transgenic corn VB15 or transgenic corn VB15-related materials, characterized in that the DNA fragment comprises DNA fragment 1 and/or DNA fragment 2; The DNA fragment 1 is the sequence shown in SEQ ID NO.5 or a specific fragment of the sequence shown in SEQ ID NO.5; The DNA fragment 2 is the sequence shown in SEQ ID NO.6 or a specific fragment of the sequence shown in SEQ ID NO.6; The transgenic corn VB15 is deposited in the General Microbiology Center of China Culture Collection Administration, with the deposit number CGMCC NO.27565. The DNA fragment according to claim 2, characterized in that the specific fragment of the sequence shown in SEQ ID NO.5 is the sequence shown in SEQ ID NO.7 or SEQ ID NO.8; The specific fragment of the sequence shown in SEQ ID NO.6 is the sequence shown in SEQ ID NO.9. The primers for detecting transgenic corn VB15 are characterized in that the primers include an upstream primer and a downstream primer; the nucleotide sequence of the upstream primer is shown in any one of SEQ ID NO.11, SEQ ID NO.12 and SEQ ID NO.15; the nucleotide sequence of the downstream primer is shown in any one of SEQ ID NO.13, SEQ ID NO.14 and SEQ ID NO.16; The transgenic corn VB15 is deposited in the General Microbiology Center of China Culture Collection Administration, with the deposit number CGMCC NO.27565. A kit for detecting transgenic corn VB15, characterized in that the kit comprises the primers according to claim 4; The transgenic corn VB15 is deposited in the General Microbiology Center of China Culture Collection Administration, with the deposit number CGMCC NO.27565. Use of the flanking sequence of claim 1, the DNA fragment of claim 2 or 3, the primer of claim 4, or the kit of claim 5 in detecting transgenic corn VB15 and/or transgenic corn VB15 related materials; The transgenic corn VB15 is deposited in the General Microbiology Center of China Culture Collection Administration, with the deposit number CGMCC NO.27565. The use according to claim 6 is characterized in that the transgenic corn VB15 related materials include parents and/or offspring of transgenic corn VB15. The use according to claim 6 is characterized in that the objects of detection include one or more of plants, tissues, seeds, transgenic corn VB15 products and products of transgenic corn VB15 related materials. A method for detecting transgenic corn VB15 or transgenic corn VB15-related materials, characterized in that it comprises the following steps: extracting DNA of the material to be tested, amplifying the DNA using the primers described in claim 4, and if a positive amplification product is obtained, the material to be tested contains components of transgenic corn VB15; The transgenic corn VB15 is deposited in the General Microbiology Center of China Culture Collection Administration, with the deposit number CGMCC NO.27565. A method for planting corn resistant to insects, characterized by comprising planting corn seeds; The genome of the corn seed contains the DNA fragment according to claim 2 or 3; The corn seeds are seeds of transgenic corn VB15; the transgenic corn VB15 is deposited in the General Microbiological Center of China Microorganism Culture Collection Administration, with the deposit number of CGMCC NO.27565.