Nucleic acid molecule of corn transformation event MN85N-E1 and detection method thereof

Abstract

The invention relates to a nucleic acid molecule of a corn transformation event MN85N-E1 and a detection method. The nucleic acid molecule of the corn transformation event MN85N-E1 comprises a sequence as shown in SEQ ID NO: 1 or a reverse complementary sequence of the sequence as shown in SEQ ID NO: 1, or a sequence as shown in SEQ ID NO: 2 or a reverse complementary sequence of the sequence as shown in SEQ ID NO: 2. The corn transformation event MN85N-E1 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 MN85N-E1 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 MN85N-E1, in particular to a transgenic maize event MN85N-E1 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 MN85N-E1 and its detection method. Background Art

[0002] Maize is one of the crops with the widest planting area in China. The whole 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. The damage rate of spring maize by the Asian corn borer in China is about 30%, and the damage rate of summer maize is 20%-30%, which can reach 90% in severe cases, resulting in a reduction in yield of more than 30%. In recent years, with the global warming, the damage of the Asian corn borer has been further aggravated. The fall armyworm is a new agricultural pest that invaded China from Yunnan at the end of 2018. The host plants have reached 19 species, among which the occurrence area of maize accounts for 98.1% of the total occurrence area of crops, and there is a trend of gradually becoming the main pest of maize. Cultivating insect-resistant and herbicide-tolerant transgenic maize varieties and promoting transgenic maize will be an effective means for us to increase unit yield, achieve supply-demand balance, and ensure food security.

[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. The LC85-Ac protein is a new insecticidal protein that can be used to control lepidopteran pests including the fall armyworm, Asian corn borer, Oriental armyworm, beet armyworm, Spodoptera litura, cutworm, and Athetis lepigone (Patent Application No. 202410332785.6).

[0004] Field weeds compete with crops for water, fertilizer, light energy and growth space, directly affecting the yield and quality of crops. 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. According to statistics from the Food and Agriculture Organization of the United Nations, the global food production loss caused by weeds reaches up to $95 billion annually, equivalent to a loss of 380 million tons of wheat, accounting for more than half of the global wheat production in 2009. Among the economic losses of $95 billion, poor developing countries bear approximately $70 billion. Therefore, effectively controlling field weeds is one of the important measures to promote food production increase. In addition, with the accelerating speed of rural population migration 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 herbicide dosage and input cost. Therefore, herbicide-tolerant transgenic maize has very broad application value and market potential.

[0005] In the present invention, the insect-resistant gene expression cassette and the herbicide-tolerant gene expression cassette are connected in series to enable their highly efficient expression in transgenic maize, possessing both insect-resistant and herbicide-tolerant traits, and 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 proximity of chromatin structures (such as heterochromatin) or transcriptional regulatory elements (such as enhancers) to the integration site. For this reason, it is usually necessary to screen a large number of events 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 among 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 out 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 generated by this hybridization method retain the transgene expression characteristics of the original transformation event. Applying this strategic model can ensure reliable gene expression in many varieties, and these varieties can well adapt to local growth conditions. Therefore, it is necessary to identify and screen more transformation events 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, such as the need for formal approval and labeling of foods derived from recombinant crops before they are 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, these 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 of the inserted transgene and the flanking DNA is often used by PCR to identify specific transgenic 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, as well as a nucleic acid molecule for detecting maize MN85N-E1 and a detection method thereof. The transgenic maize event MN85N-E1 has excellent insect resistance 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 MN85N-E1.

[0009] To achieve the above object, the present invention uses the pBWA(V)HS-bar-LC85AcA1 expression vector to transform maize B104 by the Agrobacterium-mediated method, and 51 positive transformation events are obtained. Through the identification of insect resistance and herbicide tolerance traits, it is found that the transformation event MN85N-E1 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 MN85N-E1, 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] Still 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] Still 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 by comprising the above-mentioned probe and / or primer pair.

[0018] The present invention also provides a method for detecting maize transformation events, characterized by including 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) through 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 resistance trait 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 located near the insertion junction at the 5' end of the insertion sequence in the transgenic maize event MN85N-E1. 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 insertion sequence. The presence of the transgenic maize event MN85N-E1 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 located near the insertion junction at the 3' end of the insertion sequence in the transgenic maize event MN85N-E1. The SEQ ID NO:2 spans the DNA sequence at the 3' end of the right border of the insertion sequence and the right flanking genomic DNA sequence of the maize insertion site. The presence of the transgenic maize event MN85N-E1 can be identified by containing the SEQ ID NO:2 or its reverse complementary sequence.

[0025] In the present invention, the nucleic acid sequence may be at least 11 or more consecutive polynucleotides (first nucleic acid sequence) of any part of the transgene insertion sequence in SEQ ID NO:3 or its reverse complementary sequence, or at least 11 or more consecutive polynucleotides (second nucleic acid sequence) of any part of the 5'-left flanking maize genomic DNA region in SEQ ID NO:3 or its reverse complementary sequence. The nucleic acid sequence may further be a part of 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 SEQ ID NO:1 or SEQ ID NO:3 or SEQ ID NO:6 or its reverse complementary sequence, the presence of the transgenic maize event MN85N-E1 or its progeny can be diagnosed.

[0026] SEQ ID NO:3 is a sequence with a length of 758 nucleotides located near the insertion junction at the 5'-end of the insertion sequence in the transgenic maize event MN85N-E1. SEQ ID NO:3 consists of a 363-nucleotide maize left flanking genomic DNA sequence (nucleotides 1-363 of SEQ ID NO:3) and a 5'-end DNA sequence of the first expression cassette of the glufosinate-tolerant gene with 395 nucleotides (nucleotides 364-758 of SEQ ID NO:3). The presence of the transgenic maize event MN85N-E1 can be identified by including SEQ ID NO:3 or its reverse complementary sequence.

[0027] The nucleic acid sequence may be at least 11 or more consecutive polynucleotides (third nucleic acid sequence) of any part of the transgenic 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 may 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 that includes SEQ ID NO: 2 or SEQ ID NO: 4 or SEQ ID NO: 7 or its reverse complementary sequence, the presence of transgenic maize event MN85N-E1 or its progeny can be diagnosed.

[0028] SEQ ID NO: 4 is a sequence with a length of 718 nucleotides located near the insertion junction at the 3'-end of the insertion sequence in transgenic maize event MN85N-E1. SEQ ID NO: 4 consists of the 3'-end DNA sequence of the second expression cassette of the insect-resistant gene with 44 nucleotides (nucleotides 1-44 of SEQ ID NO: 4), the right border DNA sequence of the pBWA(V)HS-bar-LC85AcA1 construct with 458 nucleotides (nucleotides 45-502 of SEQ ID NO: 4), and the genomic DNA sequence of the right flanking of the maize integration site with 216 nucleotides (nucleotides 503-718 of SEQ ID NO: 4). The presence of transgenic maize event MN85N-E1 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 6689 nucleotides that characterizes transgenic maize event MN85N-E1. The presence of transgenic maize event MN85N-E1 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 injury 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 379-6416 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 379-6416 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 feeding on the transgenic maize plant cell is inhibited from further feeding on the maize plant.

[0032] In the nucleic acid molecule for detecting maize plants and its detection method of 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 its detection method. The transgenic maize event MN85N-E1 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 MN85N-E1 or its progeny, and the presence of plant materials derived from the transgenic maize event MN85N-E1 can be identified quickly, accurately and stably.

[0034] The transgenic maize event MN85N-E1 has strong glufosinate tolerance and prominent insect-resistant traits. These characteristics enable the MN85N-E1 transformant to be used to improve the glufosinate 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 embodiments. Description of the Drawings

[0036] Figure 1 Physical map of the recombinant expression vector pBWA(V)HS-bar-LC85AcA1.

[0037] Figure 2 Indoor bioassay effect of Spodoptera frugiperda.

[0038] A: B104 (silk); B, C: MN85N-E1 (silk);

[0039] D: B104 (leaf); E, F: MN85N-E1 (leaf).

[0040] Figure 3 Indoor bioassay effect of Ostrinia furnacalis.

[0041] A: B104 (silk); B, C: MN85N-E1 (silk);

[0042] D: B104 (leaf); E, F: MN85N-E1 (leaf).

[0043] Figure 4 Verification results of event-specific PCR for the MN85N-E1 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 MN85N-E1;

[0048] 3: Sample containing genomic DNA of MN85N-E1.

[0049] A: Expected size of the left border PCR fragment is 474 bp; B: Expected size of the right border PCR fragment is 286 bp. Detailed Embodiments

[0050] The transformation event MN85N-E1 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 purposes has Figure 1 the physical map shown, and the obtained T-DNA insert has the sequence shown by nucleotides 379-6416 of SEQ ID NO:5. The transformation event MN85N-E1 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 other receptor varieties transformed with the same expression vector, so as to obtain plants with the T-DNA insert inserted into the same genomic position. The transformation event MN85N-E1 can also refer to the progeny plants obtained by asexual reproduction, sexual reproduction, haploid or diploid reproduction or a combination of the above of the above plants.

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

[0052] The LC85-AcA1 gene encodes the 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. The present invention uses the pBWA(V)HS-bar-LC85AcA1 expression vector (see the physical map of the vector in Figure 1 , which contains the LC85-AcA1 gene expression cassette and the bar gene expression cassette), and transforms the receptor B104 by the Agrobacterium-mediated method to obtain 51 positive transformation events, and the herbicide tolerance and insect resistance of the T1 generation plants of these transformed seedlings are identified and screened.

[0053] 1. Herbicide Resistance Screening

[0054] Using B104 as a reference, transformants with better herbicide tolerance were screened by spraying glufosinate at twice the medium recommended concentration in the field at the seedling stage. The results showed that the tolerance of 15 transformation events to glufosinate herbicide was significantly higher than that of the control (Table 1), and they were named MN85N-E1 to MN85N-E15.

[0055] Table 1 Herbicide Tolerance Performance

[0056]

[0057]

[0058] The values are the mean ± standard deviation from 3 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 15 transformation events by the method of in - vitro leaf bioassay. The 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 the in - vitro bioassay are shown in Table 2. Among these 15 transformants, MN85N - E1 and MN85N - E6 showed high resistance to Spodoptera frugiperda, and the rest were moderately resistant, lowly resistant or susceptible; MN85N - E1 and MN85N - E6 showed high resistance to Ostrinia furnacalis, and the rest were resistant, moderately resistant, lowly resistant or susceptible. The transformants that were highly resistant to both Spodoptera frugiperda and Ostrinia furnacalis were MN85N - E1 and MN85N - E6.

[0062] Figure 2 and Figure 3 respectively show the in - vitro bioassay effects of the leaves of transformant MN85N - E1 on Spodoptera frugiperda and Ostrinia furnacalis.

[0063] Table 2 In - vitro bioassay

[0064]

[0065] The values are expressed as the mean ± standard deviation from 4 biological replicates, and the significance of the difference in the same column of data was analyzed by the LSD method (α = 0.05).

[0066] Overall, MN85N - E1 and MN85N - E6 are transformants with excellent herbicide tolerance and insect resistance, which can 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.

[0067] Example 2 Molecular characterization of transformation event MN85N - E1

[0068] To further clarify the identity characteristics of transformation event MN85N - E1, the present invention analyzed the insertion site of the exogenous sequence of MN85N - E1 on the maize genome and the insertion structure of the exogenous sequence.

[0069] 1. Analysis of the flanking sequences of the insertion site of the exogenous sequence on the maize genome

[0070] 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 genomic 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 genomic resequencing results, use the BWA software (BWA, http: / / bio-bwa.sourceforge.net / , default settings) with the transgenic vector T-DNA sequence as a template to perform sequence homology comparison and screening with all the sequenced sequences. 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. Compare 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.

[0071] Align the sequencing results with the reference genome and the exogenous T-DNA sequence respectively to obtain the insertion position information of the exogenous inserted fragment, which is at the position of the maize genome chr04: 124884119 - 124884156bp . 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 inserted sequences at the left and right boundaries of the insertion site (as shown in Table 3). Using the genomic DNA of the transgenic maize line as a template, verify the insertion site by PCR amplification. The amplified product fuses a part of the maize genomic sequence and a part of the T-DNA sequence, and sequence analysis is performed on the PCR product.

[0072] Table 3 Primer information table

[0073]

[0074] 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.

[0075] The PCR amplification program is: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing 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.

[0076] 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 sequences and the flanking sequences on both sides of the MN85N-E1 transformant, which 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):

[0077] Left boundary:

[0078] AGCTCCGACA GCTGTCCCTC CGCCAGGCTC CAGCACTCCT CCAACGGCCA CGACATCACA 60

[0079] CCAGCTGGGT GCCAAAATCT CTCCGGCTTC CACGACGGCA TGTACTTAGG GCGCTAGCTC 120

[0080] TCCTCCGCTA GACACGTAGC ACTCTGCTAC ACCCCCCATT TTACACCTGG ATCCTCTCCT 180

[0081] TACGCCTATA AAAGGAAGGA CCAGGGCCCT CTTAGAGAAG GTGGGCCGCG CAGGGACAAG 240

[0082] GACGAGACAG GCGCTCGCGT GAGGCCGCTC GCTCCCTCTC CCGTGTGGAC GCTTGTAACC 300

[0083] CCCTACTGCA AGCGCACCCg atagaagtat tttacaaata caaatacata ctaagggttt 360

[0084] cttatatgct caacacatga gcgaaaccct ataggaaccc taattccctt atctgggaac 420

[0085] tactcacaca ttattatgga gaaagcgatc gctcaaatct cggtgacggg cagg 474

[0086] Right boundary:

[0087] aacttaatcg ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc 60

[0088] gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg cgaatgctag agcagcttga 120

[0089] gcttggatca gattgtcgtt tcccgccttc agtttaaact atcagtgttt gacTTCCACC 180

[0090] TCTCTCACGC CCATCTCCGG CCACCTCACT TCCCCCCTTC GCGCTCGGCC TCGCGTCGAC 240

[0091] CCATCTGGGC TGGGGCACGC GGCGACATTC ACTCGTCGGC TTAGGG 286

[0092] Sequencing analysis results showed that the exogenous fragment of MN85N-E1 was stably inserted into the maize genome at the position of chr04: 124884119-124884156bp.

[0093] Receptor maize genome sequence display at the insertion position of the transformant (the deleted genomic sequences caused by the insertion of the transformant T-DNA are marked with strikethrough):

[0094]

[0095] 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.

[0096] 2. Analysis of the full-length exogenous insertion sequence of the transformant

[0097] Using SEQ ID NO:8 and SEQ ID NO:11 as primers and the genomic DNA of the transformant MN85N-E1 as a template, PCR amplification was performed, and the obtained PCR product was the full-length exogenous insertion sequence. The PCR product was subjected to second-generation sequencing analysis and sequence splicing to obtain the actual insertion sequence of the MN85N-E1 transformant and the genomic sequences of the left and right flanks of maize, as shown in SEQ ID NO:5.

[0098] Example 3 Detection method for transformation event MN85N-E1

[0099] New varieties developed from the genetically modified maize event MN85N-E1 and the production of 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 a nucleotide sequence capable of diagnosing the presence of the genetically modified maize event MN85N-E1 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 products for animal consumption as a food source, or cosmetics, industrial products, etc. Nucleic acid detection methods and / or kits based on probes or primer pairs can be developed to detect the nucleotide sequence of the genetically modified maize event MN85N-E1 such as those shown in SEQ ID NO:1 or SEQ ID NO:2 in a biological sample, wherein the probe sequence or primer amplification sequence is 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 MN85N-E1.

[0100] One of the detection methods is: using the PCR method to detect a sample containing MN85N-E1, 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).

[0101] PCR reaction system:

[0102]

[0103] The reaction program is:

[0104] 95°C, 5 min; (95°C, 30 sec; 55°C, 30 sec; 72°C, 30 sec) × 30 cycles; 72°C, 7 min.

[0105] Take the PCR product and electrophorese it in a 1% (w / v) 1×TAE agarose gel for detection, and the results are as Figure 4 shown.

[0106] In the MN85N-E1 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. In summary, the transgenic maize event MN85N-E1 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 quickly identify whether the DNA molecule of the transgenic maize event MN85N-E1 is contained in a biological sample.

[0107] 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 a glufosinate herbicide to a field planted with at least one transgenic corn plant, wherein the transgenic corn plant sequentially comprises the nucleic acid sequences of SEQ ID NO:1, SEQ ID NO:5 at positions 379 to 6416, and SEQ ID NO:2 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 379-6416 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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