Nucleic acid molecule of maize transformation event MN85zN-Ex and detection method thereof

Abstract

The invention relates to a corn transformation event MN85zN-Ex nucleic acid molecule and a detection method. The corn transformation event MN85zN-Ex nucleic acid molecule comprises a sequence shown in SEQ ID NO: 1 or a reverse complementary sequence thereof, or a sequence shown in SEQ ID NO: 2 or a reverse complementary sequence thereof. The corn transformation event MN85zN-Ex has the characteristics of insect resistance and glufosinate-ammonium herbicide resistance, and the detection method can accurately and quickly identify whether a biological sample contains the DNA molecule of the transgenic corn event MN85zN-Ex or not.

Description

Technical Field

[0001] The present invention relates to the field of plant biotechnology. Specifically, it relates to a nucleic acid molecule and a detection method of a maize transformation event MN85zN-Ex, in particular to a transgenic maize event MN85zN-Ex with insect resistance and tolerance to glufosinate herbicide application, and a nucleic acid molecule for detecting whether a biological sample contains the specific transgenic maize event MN85zN-Ex and its detection method. Background Art

[0002] Maize is one of the most widely planted crops in China. The entire growth period of maize is affected by various insect pests. Among them, the Asian corn borer is the main pest in maize production, which can cause a reduction in yield of about 10% every year. Therefore, cultivating transgenic maize varieties with insect resistance and herbicide tolerance and promoting the popularization of transgenic maize will be an effective means for us to increase unit yield and achieve the balance between supply and demand.

[0003] Using a single Bt protein control strategy is likely to cause target pests to develop resistance. Therefore, it is necessary to develop new insecticidal proteins to improve insecticidal efficiency and extend the control effect. LC85-Ac is a novel insecticidal protein that can be used to control Lepidoptera pests including Spodoptera frugiperda, Ostrinia furnacalis, Ostrinia nubilalis, Mythimna separata, Spodoptera exigua, Prodenia litura, Agrotis ypsilon, and Athetis lepigone (Patent Application No. 2024103327856).

[0004] Field weeds compete with crops for water, fertilizer, light energy, and growth space, directly affecting crop yield and quality. At the same time, many weeds are intermediate hosts of crop pathogens and pests, and are one of the important biological limiting factors for crop yield increase. Therefore, effectively controlling field weeds is one of the important measures to promote food production. In addition, with the accelerating speed of the migration of rural population to cities in China, the large-scale and mechanized agricultural planting is a foreseeable trend, which makes the traditional manual weeding method unrealistic. The popularization and use of herbicides can greatly reduce the difficulty of field management and lower the labor intensity. Developing new herbicide products with high efficiency, low toxicity, and no residue is costly, time-consuming, and difficult. Cultivating herbicide-tolerant maize through transgenic technology can overcome this problem. Spraying 1-2 times during the maize growth period can effectively solve the weed problem, reducing the amount of herbicide used and the input cost. Therefore, herbicide-tolerant transgenic maize has very broad application value and market potential.

[0005] The present invention concatenates an insect-resistant gene expression cassette and a herbicide-tolerant gene expression cassette to enable their efficient expression in transgenic maize, with both insect resistance and herbicide tolerance traits, further enhancing the application and economic value of the product.

[0006] It is known that the expression of foreign genes in plants is affected by their chromosomal positions, possibly due to the chromatin structure (such as heterochromatin) or the proximity of transcriptional regulatory elements (such as enhancers) to the integration site. For this reason, it is usually necessary to screen a large number of events in order to identify events that can be commercialized (i.e., events in which the introduced target gene is optimally expressed). For example, it has been observed in plants and other organisms that the expression levels of introduced genes can vary widely between events; there may also be differences in the spatial or temporal patterns of expression, such as differences in the relative expression of transgenes between different plant tissues, and this difference is manifested in that the actual expression pattern may not be consistent with the expression pattern expected based on the transcriptional regulatory elements in the introduced gene construct, resulting in differences in the phenotypic performance of transformation events. Therefore, it is usually necessary to generate hundreds or thousands of different events and screen from these events a single event with the expected transgene expression level and expression pattern for commercial purposes. Events with the expected transgene expression level and expression pattern can be used to introgress the transgene into other genetic backgrounds through sexual outcrossing using conventional breeding methods. The offspring produced by this type of cross maintain the transgene expression characteristics of the original transformation event. Applying this strategic model can ensure reliable gene expression in many varieties that are well adapted to local growing conditions. Therefore, more transformation events need to be characterized and screened to obtain excellent transformation events with excellent comprehensive phenotypic performance and commercial prospects.

[0007] It would be beneficial to be able to detect the presence of a specific event to determine whether the offspring of a sexual cross contain the gene of interest. In addition, methods for detecting specific events will also help to comply with relevant regulations, for example, foods derived from recombinant crops require official approval and labeling before being put on the market. It is possible to detect the presence of transgenes by any well-known polynucleotide detection method, such as polymerase chain reaction (PCR). These detection methods usually focus on commonly used genetic elements, such as promoters, terminators, marker genes, etc. Therefore, unless the sequence of the chromosomal DNA ("flanking DNA") adjacent to the inserted transgenic DNA is known, the above methods cannot be used to distinguish different events, especially those generated with the same DNA construct. Therefore, currently, a pair of primers spanning the junction between the inserted transgene and the flanking DNA is often used by PCR to identify specific transgene events, specifically, a first primer containing the flanking sequence and a second primer containing the inserted sequence. Summary of the Invention

[0008] The object of the present invention is to provide a maize transformation event (also called transformant) with excellent insect resistance and herbicide tolerance traits, as well as nucleic acid molecules for detecting maize MN85zN-Ex and a detection method thereof. The transgenic maize event MN85zN-Ex has excellent insect resistance traits and good tolerance to glufosinate herbicide, and the detection method can accurately and rapidly identify whether a biological sample contains the DNA molecule of a specific transgenic maize event MN85zN-Ex.

[0009] To achieve the above object, the present invention uses the pBWA(V)HS-bar-LC85AcA5 expression vector to transform maize B104 by an Agrobacterium-mediated method, and 40 positive transformation events are obtained. Through the identification of insect resistance and herbicide tolerance traits, it is found that the transformation event MN85zN-Ex is a transformant with excellent herbicide tolerance and insect resistance, and can be used to improve the insect resistance and herbicide tolerance traits of maize.

[0010] To characterize the identity characteristics of MN85zN-Ex, the present invention provides a nucleic acid molecule, which comprises the sequence shown in SEQ ID NO:1 and / or SEQ ID NO:2, or its reverse complementary sequence.

[0011] Furthermore, the nucleic acid molecule comprises the sequence shown in SEQ ID NO:3 and / or SEQ ID NO:4, or its reverse complementary sequence.

[0012] Even further, the nucleic acid molecule comprises the sequence shown in SEQ ID NO:6 and / or SEQ ID NO:7, or its reverse complementary sequence.

[0013] Even further, the nucleic acid molecule comprises the sequence shown in SEQ ID NO:5 or its reverse complementary sequence.

[0014] The present invention also provides a probe for detecting a maize transformation event, which is characterized by comprising the sequence shown in SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:3 or SEQ ID NO:4 or SEQ ID NO:6 or SEQ ID NO:7, or a fragment or a variant thereof, or its reverse complementary sequence.

[0015] The present invention also provides a primer pair for detecting a maize transformation event, which is characterized in that the amplification product of the primer pair comprises the sequence shown in SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:3 or SEQ ID NO:4 or SEQ ID NO:6 or SEQ ID NO:7, or a fragment or a variant thereof, or its reverse complementary sequence.

[0016] In some embodiments, the primer pair is the sequence shown in SEQ ID NO:8 and SEQ ID NO:9; or the sequence shown in SEQ ID NO:10 and SEQ ID NO:11.

[0017] The present invention also provides a kit or microarray for detecting maize transformation events, characterized in that it comprises the above-mentioned probe and / or primer pair.

[0018] The present invention also provides a method for detecting maize transformation events, characterized in that it includes using the above-mentioned probe or the above-mentioned primer pair or the above-mentioned probe and primer pair or the above-mentioned kit or microarray to detect whether the transformation event exists in a sample to be tested.

[0019] The present invention also provides a method for breeding maize, characterized in that the method comprises the following steps:

[0020] 1) Obtaining maize containing the above nucleic acid molecule;

[0021] 2) Obtaining maize plants, seeds, plant cells, progeny plants or plant parts from the maize obtained in step 1) by pollen culture, unfertilized embryo culture, doubling culture, cell culture, tissue culture, self-crossing or hybridization or a combination of the above; and optionally,

[0022] 3) Identifying the insect-resistant traits and / or herbicide resistance of the progeny plants obtained in step 2), and using the above method to detect whether the transformation event exists therein.

[0023] Furthermore, the present invention also provides products made from the maize plants, seeds, plant cells, progeny plants or plant parts obtained by the above method, including food, feed or industrial raw materials.

[0024] The SEQ ID NO:1 is a 22-nucleotide sequence near the insertion junction at the 5'-end of the inserted sequence in the transgenic maize event MN85zN-Ex. The SEQ ID NO:1 spans the left flanking genomic DNA sequence of the maize insertion site and the DNA sequence at the 5'-end of the left border of the inserted sequence. The presence of the transgenic maize event MN85zN-Ex can be identified by containing the SEQ ID NO:1 or its reverse complementary sequence. The SEQ ID NO:2 is a 22-nucleotide sequence near the insertion junction at the 3'-end of the inserted sequence in the transgenic maize event MN85zN-Ex. The SEQ ID NO:2 spans the DNA sequence at the 3'-end of the right border of the inserted sequence and the right flanking genomic DNA sequence of the maize insertion site. The presence of the transgenic maize event MN85zN-Ex can be identified by containing the SEQ ID NO:2 or its reverse complementary sequence.

[0025] In the present invention, the nucleic acid sequence can be at least 11 or more consecutive polynucleotides (the first nucleic acid sequence) of any part of the transgenic inserted sequence in the SEQ ID NO:3 or its reverse complementary sequence, or at least 11 or more consecutive polynucleotides (the second nucleic acid sequence) of any part of the 5'-left flanking maize genomic DNA region in the SEQ ID NO:3 or its reverse complementary sequence. The nucleic acid sequence can further be a part of the SEQ ID NO:3 that is homologous to or reverse complementary to the complete SEQ ID NO:1 or SEQ ID NO:6. When the first nucleic acid sequence and the second nucleic acid sequence are used together, these nucleic acid sequences include a DNA primer pair in a DNA amplification method for generating an amplification product. When the amplification product generated by using the DNA primer pair in the DNA amplification method is an amplification product including the SEQ ID NO:1 or SEQ ID NO:3 or SEQ ID NO:6 or their reverse complementary sequences, the presence of the transgenic maize event MN85ZN-EX or its progeny can be diagnosed.

[0026] The SEQ ID NO:3 is a 1420-nucleotide sequence near the insertion junction at the 5'-end of the inserted sequence in the transgenic maize event MN85zN-Ex. The SEQ ID NO:3 consists of an 850-nucleotide maize left flanking genomic DNA sequence (nucleotides 1-850 of the SEQ ID NO:3) and a 570-nucleotide DNA sequence at the 5'-end of the first expression cassette of the glufosinate-resistant gene (nucleotides 851-1420 of the SEQ ID NO:3). The presence of the transgenic maize event MN85zN-Ex can be identified by containing the SEQ ID NO:3 or its reverse complementary sequence.

[0027] The nucleic acid sequence can be at least 11 or more consecutive polynucleotides (third nucleic acid sequence) of any part of the transgene insertion sequence in SEQ ID NO:4 or its reverse complementary sequence, or at least 11 or more consecutive polynucleotides (fourth nucleic acid sequence) of any part of the 3'-right flanking maize genomic DNA region in SEQ ID NO:4 or its reverse complementary sequence. The nucleic acid sequence can further be a part of SEQ ID NO:4 that is homologous to or reverse complementary to SEQ ID NO:2 or SEQ ID NO:7 in its entirety. When the third nucleic acid sequence and the fourth nucleic acid sequence are used together, these nucleic acid sequences include a DNA primer set in a DNA amplification method for generating an amplification product. When the amplification product generated by using a DNA primer pair in a DNA amplification method is an amplification product including SEQ ID NO:2 or SEQ ID NO:4 or SEQ ID NO:7 or their reverse complementary sequences, the presence of transgenic maize event MN85zN-Ex or its progeny can be diagnosed.

[0028] SEQ ID NO:4 is a sequence with a length of 1074 nucleotides located near the insertion junction at the 3'-end of the insertion sequence in transgenic maize event MN85zN-Ex. SEQ ID NO:4 consists of the 3'-end DNA sequence (nucleotides 1-55 of SEQ ID NO:4) of the second expression cassette of the insect-resistant gene with 55 nucleotides, the right border DNA sequence of the pBWA(V)HS-bar-LC85AcA5 construct with 436 nucleotides (nucleotides 56-491 of SEQ ID NO:4), and the genomic DNA sequence of the right flanking of the maize integration site with 583 nucleotides (nucleotides 492-1074 of SEQ ID NO:4). The presence of transgenic maize event MN85zN-Ex can be identified by including SEQ ID NO:4 or its reverse complementary sequence.

[0029] SEQ ID NO:5 is a sequence with a length of 7528 nucleotides characterizing transgenic maize event MN85zN-Ex. The presence of transgenic maize event MN85zN-Ex can be identified by including SEQ ID NO:5 or its reverse complementary sequence.

[0030] The present invention also provides a method for protecting maize plants from damage caused by herbicides, which is characterized by comprising applying an effective dose of glufosinate herbicide to a field planted with at least one transgenic maize plant, wherein the transgenic maize plant sequentially comprises SEQ ID NO:1, the nucleic acid sequence at positions 851-6907 of SEQ ID NO:5, and SEQ ID NO:2 in its genome, or the genome of the transgenic maize plant comprises SEQ ID NO:5; and the transgenic maize plant has tolerance to glufosinate herbicide.

[0031] The present invention also provides a method for protecting maize plants from insect infestation, which is characterized by comprising providing at least one transgenic maize plant cell in the diet of a target insect, wherein the transgenic maize plant cell sequentially comprises SEQ ID NO:1, the nucleic acid sequence at positions 851-6907 of SEQ ID NO:5, and SEQ ID NO:2 in its genome, or the genome of the transgenic maize plant cell comprises SEQ ID NO:5; and the target insect that ingests the transgenic maize plant cell is inhibited from further ingesting the maize plant.

[0032] In the nucleic acid molecules and detection methods for detecting maize plants according to the present invention, the following definitions and methods can better define the present invention and guide those of ordinary skill in the art to implement the present invention. Unless otherwise specified, the terms are understood according to the conventional usage of those of ordinary skill in the art.

[0033] The present invention provides a nucleic acid molecule for detecting maize plants and a detection method thereof. The transgenic maize event MN85zN-Ex has insect-resistant traits and the effect of tolerating glufosinate herbicide. Maize plants with such traits express LC85-Ac protein and phosphinothricin acetyltransferase (PAT) protein, which confer insect resistance and tolerance to glufosinate on the plants. At the same time, in the detection method of the present invention, SEQ ID NO:1 or its reverse complementary sequence, SEQ ID NO:2 or its reverse complementary sequence, SEQ ID NO:3 or its reverse complementary sequence, SEQ ID NO:4 or its reverse complementary sequence, SEQ ID NO:6 or its reverse complementary sequence, or SEQ ID NO:7 or its reverse complementary sequence can be used as DNA primers or probes to generate amplification products diagnostic of the transgenic maize event MN85zN-Ex or its progeny, and the presence of plant materials derived from the transgenic maize event MN85zN-Ex can be identified quickly, accurately, and stably.

[0034] The transgenic maize event MN85zN-Ex has strong tolerance to glufosinate ammonium and prominent insect-resistant traits. These characteristics enable the MN85zN-Ex transformant to be used to improve the glufosinate ammonium herbicide tolerance and insect-resistant traits of maize, thereby cultivating new maize varieties with insect resistance and herbicide tolerance.

[0035] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and examples. Description of the Drawings

[0036] Figure 1 Physical map of the recombinant expression vector pBWA(V)HS-bar-LC85AcA5.

[0037] Figure 2 Results of indoor bioassays against Spodoptera frugiperda.

[0038] A: B104 (silk); B, C: MN85zN-Ex (silk);

[0039] D: B104 (leaf); E, F: MN85zN-Ex (leaf).

[0040] Figure 3 Results of indoor bioassays against Ostrinia furnacalis.

[0041] A: B104 (silk); B, C: MN85zN-Ex (silk);

[0042] D: B104 (leaf); E, F: MN85zN-Ex (leaf).

[0043] Figure 4 Results of event-specific PCR verification for the MN85zN-Ex transformation.

[0044] M: DNA Marker, size marked beside (unit: bp);

[0045] N: Blank control water;

[0046] C: Genomic DNA of the receptor control B104;

[0047] 1, 2: Genomic DNA of the transformant MN85zN-Ex;

[0048] 3: Sample containing genomic DNA of MN85zN-Ex.

[0049] LB: Expected size of the left border PCR fragment is 830 bp; RB: Expected size of the right border PCR fragment is 271 bp. Detailed Embodiments

[0050] The transformation event MN85zN-Ex involved in this application refers to a maize plant obtained by genetic transformation using maize B104 as the receptor, into which an exogenous gene insert (T-DNA insert) is inserted between specific genomic sequences. In a specific embodiment, the expression vector used for transgenic transformation has Figure 1 the physical map shown, and the obtained T-DNA insert has the sequence shown by nucleotides 851-6907 of SEQ ID NO:5. The transformation event MN85zN-Ex can refer to this transgenic process, or the T-DNA insert in the genome obtained by this process, or the combination of the T-DNA insert and the flanking sequences, or can refer to the maize plant obtained by this transgenic process. In a specific example, this event is also applicable to the transformation of other receptor varieties with the same expression vector, so as to obtain plants with the T-DNA insert inserted into the same genomic position. The transformation event MN85zN-Ex can also refer to the progeny plants obtained by asexual reproduction, sexual reproduction, haploidization or doubling of the above plants, or a combination of the above.

[0051] Example 1 Obtaining and Character Identification of Transformation Events

[0052] The LC85-AcA5 gene encodes the insecticidal protein LC85-Ac, which has a significant control effect on pests such as Spodoptera frugiperda and Ostrinia furnacalis; the bar gene encodes phosphinothricin acetyltransferase, which can improve the tolerance of plants to glufosinate herbicides. In the present invention, the pBWA(V)HS-bar-LC85AcA5 expression vector (see the physical map of the vector Figure 1 , which contains the LC85-AcA5 gene expression cassette and the bar gene expression cassette) was used to transform the receptor B104 by the Agrobacterium-mediated method, and 40 positive transformation events were obtained, and the herbicide tolerance, insect resistance and related agronomic traits of the T1 generation plants of these transformed seedlings were identified and screened.

[0053] 1. Screening for Herbicide Resistance

[0054] Taking B104 as a reference, the transformants with better herbicide tolerance were screened by spraying glufosinate at twice the medium recommended concentration at the seedling stage. The results showed that only 12 transformation events had significantly higher tolerance to glufosinate herbicide than the control (Table 1).

[0055] Table 1 Performance of Herbicide Tolerance

[0056]

[0057]

[0058] The values are the means ± standard deviations from three biological replicates. Statistical analysis was performed using LSD for multiple comparisons (α = 0.05), and different letters indicate significant differences in the same column of data at the same herbicide concentration.

[0059] (2) Insect resistance identification

[0060] Using B104 as a reference, transformants with better insect resistance were screened from the above 12 transformation events by the method of in - vitro leaf bioassay. Newly hatched larvae of Spodoptera frugiperda and Ostrinia furnacalis were fed with detached maize leaves to evaluate the insect resistance of the materials.

[0061] The results of in - vitro bioassay showed that the larval mortality rates caused by the leaves of these 12 transformants were all significantly higher than those of the control (Table 2). Among them, MN85zN - Ed, MN85zN - Ef, MN85zN - Ej, MN85zN - Ek, MN85zN - Em, MN85zN - Eq, MN85zN - Ev, MN85zN - Ex, and MN85zN - Ey had high resistance to Spodoptera frugiperda, and the rest had medium resistance or resistance; MN85zN - Ec, MN85zN - Ed, MN85zN - Ef, MN85zN - Eh, MN85zN - Ek, MN85zN - Eq, MN85zN - Ev, MN85zN - Ex, and MN85zN - Ey had high resistance to Ostrinia furnacalis, and the rest had medium resistance or resistance. At the same time, the transformants with high resistance to both Spodoptera frugiperda and Ostrinia furnacalis were MN85zN - Ed, MN85zN - Ef, MN85zN - Ek, MN85zN - Eq, MN85zN - Ev, MN85zN - Ex, and MN85zN - Ey.

[0062] Figure 2 and Figure 3 respectively show the in - vitro bioassay effects of the leaves of transformant MN85zN - Ex on Spodoptera frugiperda and Ostrinia furnacalis.

[0063] Table 2 In - vitro bioassay

[0064]

[0065] The values are expressed as the means ± standard deviations from three biological replicates, and the significance of differences in the same column of data was analyzed by the LSD method (α = 0.05).

[0066] (3) Agronomic trait investigation

[0067] The agronomic traits (such as plant height and grain weight) of the transformants MN85zN-Ed, MN85zN-Ef, MN85zN-Ek, MN85zN-Eq, MN85zN-Ev, MN85zN-Ex and MN85zN-Ey were further investigated and recorded in detail, as shown in Table 3. The results showed that the plant heights and 100-grain weights of the transformants MN85zN-Ed, MN85zN-Ef, MN85zN-Ek, MN85zN-Eq and MN85zN-Ev were significantly lower than those of the control B104, while the agronomic traits (plant height and 100-grain weight) of the transformants MN85zN-Ex and MN85zN-Ey were better than those of the control.

[0068] Table 3 Results of investigation on partial agronomic traits

[0069]

[0070] The values are the means ± standard deviations from 3 biological replicates. Statistical analysis was performed using LSD for multiple comparisons (α = 0.05), and different letters indicate significant differences in the data in the same column at the same time period.

[0071] Overall, MN85zN-Ex and MN85zN-Ey are transformants with excellent herbicide tolerance, insect resistance and good agronomic traits, which can be used to improve the glufosinate herbicide tolerance and insect resistance traits of maize, so as to cultivate new maize varieties with insect resistance and herbicide tolerance.

[0072] Example 2 Molecular characterization of the transformation event MN85zN-Ex

[0073] In order to further clarify the identity characteristics of the transformation event MN85zN-Ex, the present invention analyzed the insertion site of the exogenous sequence of MN85zN-Ex on the maize genome and the insertion structure of the exogenous sequence.

[0074] 1. Analysis of the flanking sequences of the insertion site of the exogenous sequence on the maize genome

[0075] Take 100 mg of plant leaves, quickly grind them in liquid nitrogen, and extract total DNA using the CTAB method. After measuring the concentration of genomic DNA, ensure that the total amount of DNA > 2 μg. Using genome resequencing, with each read length of 150 bp, obtain at least 20 Gb of data, and ensure that the data quality index Q30 ≥ 80% (i.e., the proportion of bases with a sequencing error rate greater than 0.1% is less than 20%). According to the genome resequencing results, use the BWA software with the transgenic vector T-DNA sequence as the template to perform sequence homology comparison and screening with all the sequenced sequences (BWA, http: / / bio-bwa.sourceforge.net / , default settings). Further assemble and screen the selected sequences, remove the reads with all vector sequences, and finally obtain a type of read sequence, which is characterized by half being genomic sequence and the other half being vector sequence. Align the obtained genomic sequence with the maize reference genome (MaizeGDB database https: / / www.maizegdb.org / blast , with Zm-B73-REFERENCE-NAM-5.0 as the reference sequence) for homologous alignment analysis to obtain the specific position of this genomic sequence on the genome, which is the possible insertion site.

[0076] Align the sequencing results with the reference genome and the exogenous T-DNA sequence respectively to obtain the insertion position information of the exogenous insertion fragment, which is at the position of maize genome chr02:121775269 - 121775347 bp. Subsequently, use the Primerblast software on the NCBI website (https: / / blast.ncbi.nlm.nih.gov / Blast) to design forward and reverse primers respectively on the genomic flanking sequences and exogenous insertion sequences at the left and right boundaries of the insertion site (as shown in Table 4). Using the genomic DNA of the transgenic maize line as the template, verify the insertion site by PCR amplification. The amplified product incorporates a part of the maize genomic sequence and a part of the T-DNA sequence, and sequence analysis of the PCR product is carried out.

[0077] Table 4 Primer information table

[0078]

[0079] The specific PCR reaction system contains 1.0 μL of genomic DNA, 10.0 μL of 2×PCR Mixture, 0.5 μL of forward primer (10 μM), and 0.5 μL of reverse primer (10 μM), and the volume is made up to 20 μL with water.

[0080] The PCR amplification procedure was as follows: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for a certain time (set at 60 s / kb), 30 cycles; extension at 72°C for 7 min.

[0081] The results of PCR amplification are shown in Figure 4 . The PCR amplification products at the left and right boundaries were sequenced to obtain the T-DNA insertion boundary sequence and the flanking sequences on both sides of the MN85zN-Ex transformant, and were assembled as follows (capital letters are maize genomic sequences, lowercase letters are T-DNA sequences, and the shaded areas indicate the positions of the transformant-specific PCR primers):

[0082] Left boundary:

[0083]

[0084] Right boundary:

[0085]

[0086] The results of sequencing analysis showed that the exogenous fragment of MN85zN-Ex was stably inserted into the position of maize genome chr02:121775269 - 121775347 bp.

[0087] Display of the receptor maize genomic sequence at the insertion site of the transformant (the deleted genomic sequences caused by the insertion of the transformant T-DNA are marked with strikethrough):

[0088]

[0089]

[0090] Using BLASTN in the MaizeGDB database to analyze the genomic sequences upstream and downstream of the insertion site, the results showed that the insertion site of the exogenous sequence was located in the gene intergenic region of the maize genome and there were no known functional genes.

[0091] 2. Analysis of the full-length inserted exogenous sequence of the transformant

[0092] Using SEQ ID NO:8 and SEQ ID NO:11 as primers and the genomic DNA of the transformant MN85zN-Ex as the template, PCR amplification was carried out, and the obtained PCR product was the full-length inserted exogenous sequence. The PCR product was subjected to second-generation sequencing analysis and sequence splicing to obtain the actual inserted sequence of the MN85zN-Ex transformant and the flanking maize genomic sequences, as shown in SEQ ID NO:5.

[0093] Example 3 Detection method for the transformation event MN85zN-Ex

[0094] New varieties can be developed from the genetically modified maize event MN85zN-Ex and used to produce agricultural products or commodities. If a sufficient amount is detected in the agricultural product or commodity, the agricultural product or commodity is expected to contain nucleotide sequences capable of diagnosing the presence of genetically modified maize event MN85zN-Ex material in the agricultural product or commodity. The agricultural products or commodities include, but are not limited to, corn oil, cornmeal, corn flour, corn paste, starch, and other seasonings or any other food used as a food source for animal consumption, or cosmetics, industrial products, etc. Nucleic acid detection methods and / or kits based on probes or primer pairs can be developed to detect, in biological samples, such as

[0095] the nucleotide sequences of the genetically modified maize event MN85zN-Ex shown in SEQ ID NO:1 or SEQ ID NO:2, wherein the probe sequences or primer amplification sequences are selected from the sequences shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7 to diagnose the presence of the genetically modified maize event MN85zN-Ex.

[0096] One of the detection methods is: using the PCR method to detect samples containing MN85zN-Ex, and the PCR primer pairs used are SEQ ID NO:8 (forward primer), SEQ ID NO:9 (reverse primer) or SEQ ID NO:10 (forward primer), SEQ ID NO:11 (reverse primer).

[0097] PCR reaction system:

[0098]

[0099] The reaction program is:

[0100] Pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for a certain time (set at 60 s / kb), 30 cycles; extension at 72°C for 7 min.

[0101] Take the PCR product and perform electrophoresis detection in 1% (w / v) 1×TAE agarose gel, and the results are as Figure 4 shown.

[0102] In the MN85zN-Ex transformation event, the expected target bands (SEQ ID NO:6 and SEQ ID NO:7 respectively) can be amplified. Moreover, this PCR method can trace the presence of the transformation event and thus be applied to breeding work.

[0103] In summary, the transgenic maize event MN85zN-Ex of the present invention can improve plant insect resistance and has high tolerance to glufosinate herbicide, and can be used to improve other maize germplasms and create new maize hybrid combinations. Its detection method can accurately and rapidly identify whether the DNA molecule of the transgenic maize event MN85zN-Ex is contained in a biological sample.

[0104] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. A nucleic acid molecule, characterized in that Includes any of the following: i) comprising the sequence shown in SEQ ID NO: 1 and / or SEQ ID NO: 2, or the reverse complementary sequence thereof; ii) comprising the sequence shown in SEQ ID NO: 3 and / or SEQ ID NO: 4, or the reverse complementary sequence thereof; iii) comprising the sequence shown in SEQ ID NO: 6 and / or SEQ ID NO: 7, or the reverse complementary sequence thereof; iv) comprising the sequence shown in SEQ ID NO: 5, or its reverse complementary sequence.

2. A probe for detecting a maize transformation event, characterized in that: It includes the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4 or SEQ ID NO: 6 or SEQ ID NO: 7 or its fragment or variant or its reverse complementary sequence.

3. A primer pair for detecting a maize transformation event, characterized in that: The amplification product of the primer pair comprises the sequence of claim 2; Optionally, the primer pair is the sequence shown by SEQ ID NO:8 and SEQ ID NO:9; or the sequence shown by SEQ ID NO:10 and SEQ ID NO:

11.

4. A kit or microarray for detecting maize transformation events, characterized in that: Comprising the probe according to claim 2 and / or the primer pair according to claim 3.

5. A method for detecting a corn transformation event, characterized in that The method comprises using any of the following to detect whether the conversion event exists in the sample to be tested: i) the probe according to claim 2; ii) the primer pair according to claim 3; iii) the probe according to claim 2 and the primer pair according to claim 3; iv) The kit or microarray according to claim 4.

6. A method for breeding corn, characterized in that: The method comprises the following steps: 1) obtaining corn containing the nucleic acid molecule according to claim 1; 2) subjecting the corn obtained in step 1) to pollen culture, unfertilized embryo culture, doubling culture, cell culture, tissue culture, selfing or hybridization or a combination thereof to obtain corn plants, seeds, plant cells, offspring plants or plant parts; and optionally, 3) evaluating the insect resistance traits and / or identifying the herbicide resistance of the offspring plants obtained in step 2), and detecting the presence of the transformation event therein using the method of claim 5.

7. Products made from the corn plants, seeds, plant cells, progeny plants or plant parts obtained by the method of claim 6, including food, feed or industrial raw materials.

8. A method for protecting corn plants from damage caused by herbicides, characterized in that The method comprises applying an effective dose of glufosinate herbicide to a field planted with at least one transgenic corn plant, wherein the transgenic corn plant comprises SEQ ID NO:1, the nucleic acid sequence of positions 851-6907 of SEQ ID NO:5 and SEQ ID NO:2 in sequence in its genome, or the genome of the transgenic corn plant comprises SEQ ID NO:5; and the transgenic corn plant has tolerance to the glufosinate herbicide.

9. A method for protecting corn plants from insect attack, characterized in that: The method comprises providing at least one transgenic corn plant cell in the diet of the target insect, wherein the transgenic corn plant cell comprises SEQ ID NO:1, the nucleic acid sequence of positions 851-6907 of SEQ ID NO:5 and SEQ ID NO:2 in sequence in its genome, or the genome of the transgenic corn plant cell comprises SEQ ID NO:5; the target insect that ingests the transgenic corn plant cell is inhibited from further ingesting the corn plant.

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