Nucleic acid molecules of maize transformation event MN85N-E6 and their detection methods

Abstract

The present invention relates to a nucleic acid molecule and a detection method of a maize transformation event MN85N-E6, wherein the nucleic acid molecule of the maize MN85N-E6 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 maize transformation event MN85N-E6 of the present invention has insect resistance and glufosinate-ammonium herbicide resistance characteristics, and the detection method can accurately and quickly identify whether a biological sample contains a DNA molecule of a transgenic maize event MN85N-E6.

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 corn transformation event MN85N-E6, and in particular to a transgenic corn event MN85N-E6 that is resistant to insects and tolerant to the application of glufosinate herbicide, and a nucleic acid molecule and a detection method thereof for detecting whether a biological sample contains a specific transgenic corn event MN85N-E6. Background Art

[0002] Corn is one of the most widely planted crops in my country. The entire growth period of corn will be affected by a variety of pests. Among them, corn borer is the main pest in corn production, which can cause about 10% reduction in production each year. The damage rate of Asian corn borer in my country's spring corn is about 30%, and the damage rate of summer corn is 20%-30%, which can reach 90% in severe cases, with a reduction of more than 30%. In recent years, with global warming, the damage of corn borer has further increased. Fall armyworm is a new agricultural pest that invaded my country from Yunnan at the end of 2018. There are 19 host plants, of which the occurrence area of ​​corn accounts for 98.1% of the total crop occurrence area, and there is a trend of gradually developing into a major corn pest. Therefore, cultivating insect-resistant and herbicide-resistant transgenic corn varieties and promoting transgenic corn will be an effective means for us to increase yields, achieve supply and demand balance, and ensure food security.

[0003] The use of a single Bt protein control strategy can easily cause target pests to develop resistance, so it is necessary to develop new insecticidal proteins to improve insecticidal efficiency and prolong control effects. LC85-Ac is a new insecticidal protein that can be used to control lepidopteran pests including fall armyworm, corn borer, Asian corn borer, oriental armyworm, beet armyworm, armyworm, cutworm, and two-spotted armyworm (patent application number 202410332785.6).

[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 for crop pathogens and pests, and are one of the important biological limiting factors for crop yield. According to statistics from the Food and Agriculture Organization of the United Nations, the global food production loss caused by weeds is as high as US$95 billion per year, equivalent to the loss of 380 million tons of wheat, which is about more than half of the global wheat production in 2009. In addition, with the acceleration of the migration of rural population to cities in my country, the scale and mechanization of agricultural planting is a foreseeable trend, which makes traditional manual weeding methods unrealistic. The promotion and use of herbicides can greatly reduce the difficulty of field management and reduce labor intensity. The development of new high-efficiency, low-toxic, and residue-free herbicide products is expensive, time-consuming and difficult. This problem can be overcome by cultivating corn resistant to lethal herbicides through genetic engineering technology. Spraying 1-2 times during the growth period of corn can effectively solve the weed problem, reducing the amount of herbicides used and input costs. Therefore, herbicide-resistant transgenic corn has very broad application value and market potential.

[0005] The present invention connects an insect-resistant gene expression cassette and a herbicide-resistant gene expression cassette in series, so that they are efficiently expressed in transgenic corn, have both insect-resistant and herbicide-resistant traits, and further enhance 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 location, which may be due to chromatin structure (e.g., heterochromatin) or the proximity of transcriptional regulatory elements (e.g., enhancers) to the integration site. For this reason, it is often necessary to screen a large number of events before it is possible to identify events that are commercially viable (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 amount of expression of the introduced gene can vary greatly between events; there may also be differences in the spatial or temporal pattern of expression, such as differences in the relative expression of the transgene between different plant tissues, which can be manifested in the actual expression pattern being inconsistent with the expression pattern expected based on the transcriptional regulatory elements in the introduced gene construct, resulting in differences in the trait expression of the transformation events. Therefore, it is often necessary to generate hundreds or thousands of different events and screen these events to find a single event with the expected transgene expression amount and expression pattern for commercial purposes. Events with the expected transgene expression amount and expression pattern can be used to introgress the transgene into other genetic backgrounds using conventional breeding methods through sexual outcrossing. The progeny produced by this cross retain the transgene expression characteristics of the original transformation event. Applying this strategy model can ensure reliable gene expression in many varieties that are well adapted to local growing conditions. Therefore, more transformation events need to be identified and screened to obtain excellent transformation events with excellent comprehensive trait 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 contains the target gene. In addition, methods for detecting specific events will also help to comply with relevant regulations, such as the need to obtain 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 transgenics 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 method cannot be used to distinguish different events, especially those generated with the same DNA construct. Therefore, a pair of primers that span the junction of the inserted transgenic and flanking DNA are often used to identify transgenic specific events by PCR, specifically a first primer containing a flanking sequence and a second primer containing an inserted sequence. Summary of the invention

[0008] The purpose of the present invention is to provide a corn transformation event (also called transformant) with excellent insect resistance and herbicide resistance, as well as a nucleic acid molecule and a detection method for detecting corn MN85N-E6. The transgenic corn event MN85N-E6 has excellent insect resistance and good tolerance to glufosinate herbicide, and the detection method can accurately and quickly identify whether a biological sample contains a DNA molecule of a specific transgenic corn event MN85N-E6.

[0009] To achieve the above object, the present invention uses the pBWA(V)HS-bar-LC85AcA1 expression vector to transform corn B104 by Agrobacterium-mediated method, and obtains 51 positive transformation events. Through the identification of insect resistance and herbicide resistance traits, it was found that the transformation event MN85N-E6 is a transformant with excellent herbicide tolerance and insect resistance, which can be used to improve the insect resistance and herbicide resistance traits of corn.

[0010] In order to characterize the identity characteristics of MN85N-E6, 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] Furthermore, 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] Furthermore, 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 corn transformation events, characterized in that it comprises a 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 thereof or a variant thereof or a reverse complementary sequence thereof.

[0015] The present invention also provides a primer pair for detecting corn transformation events, 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 thereof or a variant thereof or a reverse complementary sequence thereof.

[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 corn 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 corn transformation events, characterized in that it comprises 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 the sample to be tested.

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

[0020] 1) obtaining corn containing the above nucleic acid molecule;

[0021] 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,

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

[0023] Furthermore, the present invention also provides products made from corn 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-E6, and the SEQ ID NO: 1 spans the DNA sequence at the left flank 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-E6 can be identified by including 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-E6, and the SEQ ID NO: 2 spans the DNA sequence at the 3' end of the right border of the insertion sequence and the DNA sequence at the right flank of the maize insertion site. The presence of the transgenic maize event MN85N-E6 can be identified by including 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 continuous polynucleotides (first nucleic acid sequence) of any part of the transgenic insertion sequence in the SEQ ID NO: 3 or its reverse complementary sequence, or at least 11 or more continuous polynucleotides (second nucleic acid sequence) of any part of the 5' left flank corn genomic DNA region in the SEQ ID NO: 3 or its reverse complementary sequence. The nucleic acid sequence may further be homologous to or reverse complementary to a part of the SEQ ID NO: 3 that includes 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 producing an amplification product. When the amplification product produced in the DNA amplification method using the DNA primer pair 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 corn event MN85N-E6 or its progeny can be diagnosed.

[0026] The SEQ ID NO: 3 is a sequence with a length of 1577 nucleotides located near the insertion junction site at the 5' end of the insertion sequence in the transgenic corn event MN85N-E6. The SEQ ID NO: 3 consists of a 476-nucleotide corn left flank genomic DNA sequence (nucleotides 1-476 of SEQ ID NO: 3), a 68-nucleotide pBWA(V)HS-bar-LC85AcA1 construct left border DNA sequence (nucleotides 477-544 of SEQ ID NO: 3), and a 1033-nucleotide 5' end DNA sequence of the first expression cassette of the glufosinate-resistant gene (nucleotides 545-1577 of SEQ ID NO: 3). The presence of the transgenic corn event MN85N-E6 can be identified by the presence of the SEQ ID NO: 3 or its reverse complementary sequence.

[0027] The nucleic acid sequence may be at least 11 or more continuous polynucleotides (third nucleic acid sequence) of any part of the transgenic insertion sequence in the SEQ ID NO: 4 or its reverse complementary sequence, or at least 11 or more continuous polynucleotides (fourth nucleic acid sequence) of any part of the 3' right flank corn genomic DNA region in the SEQ ID NO: 4 or its reverse complementary sequence. The nucleic acid sequence may further be homologous to or reverse complementary to a portion of the SEQ ID NO: 4 that includes the complete SEQ ID NO: 2 or SEQ ID NO: 7. 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 producing an amplification product. When the amplification product produced in the DNA amplification method using the DNA primer pair is an amplification product including SEQ ID NO: 2 or SEQ ID NO: 4 or SEQ ID NO: 7 or its reverse complementary sequence, the presence of the transgenic corn event MN85N-E6 or its progeny can be diagnosed.

[0028] The SEQ ID NO: 4 is a sequence of 1394 nucleotides in length located near the insertion junction at the 3' end of the insertion sequence in the transgenic corn event MN85N-E6. The SEQ ID NO: 4 consists of a 424-nucleotide 3' end DNA sequence of the second expression cassette of the insect-resistant gene (nucleotides 1-424 of SEQ ID NO: 4), a 462-nucleotide pBWA(V)HS-bar-LC85AcA1 construct right border DNA sequence (nucleotides 425-886 of SEQ ID NO: 4), and a 508-nucleotide right flank genomic DNA sequence of the corn integration site (nucleotides 887-1394 of SEQ ID NO: 4). The presence of the transgenic corn event MN85N-E6 can be identified by the presence of the SEQ ID NO: 4 or its reverse complementary sequence.

[0029] The SEQ ID NO: 5 is a sequence of 7144 nucleotides in length that characterizes the transgenic maize event MN85N-E6. The presence of the transgenic maize event MN85N-E6 can be identified by including the SEQ ID NO: 5 or its reverse complementary sequence.

[0030] The present invention also provides a method for protecting corn plants from damage caused by herbicides, characterized in that it includes 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, SEQ ID NO: 5 nucleic acid sequences at positions 477-6636 and SEQ ID NO: 2 in its genome, or the transgenic corn plant comprises SEQ ID NO: 5 in its genome; and the transgenic corn plant has tolerance to glufosinate herbicide.

[0031] The present invention also provides a method for protecting corn plants from insect attack, characterized in that it includes 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, SEQ ID NO: 5 nucleic acid sequences at positions 477-6636 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 feeds on the transgenic corn plant cell is inhibited from further feeding on the corn plant.

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

[0033] The invention provides a nucleic acid molecule for detecting corn plants and a detection method thereof. The transgenic corn event MN85N-E6 has insect resistance and tolerance to glufosinate herbicides. The corn plant with the trait expresses LC85-Ac protein and phosphinothricin acetyltransferase (PAT) protein, which endows the plant with insect resistance and tolerance to glufosinate. At the same time, in the detection method of the 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 a DNA primer or probe to generate an amplification product diagnosed as the transgenic corn event MN85N-E6 or its progeny, and the presence of plant materials derived from the transgenic corn event MN85N-E6 can be identified quickly, accurately and stably.

[0034] The transgenic corn event MN85N-E6 has a strong tolerance to glufosinate and outstanding insect resistance. These characteristics allow the MN85N-E6 transformant to be used to improve the glufosinate tolerance and insect resistance of corn, thereby breeding new corn varieties that are resistant to insects and herbicides.

[0035] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0037] Figure 2 Indoor bioassay results of fall armyworm.

[0038] A: B104 (filament); B, C: MN85N-E6 (filament);

[0039] D: B104 (blade); E, F: MN85N-E6 (blade).

[0040] Figure 3 Corn borer indoor test results.

[0041] A: B104 (filament); B, C: MN85N-E6 (filament);

[0042] D: B104 (blade); E, F: MN85N-E6 (blade).

[0043] Figure 4 MN85N-E6 transformation event-specific PCR validation results.

[0044] M: DNA Marker, with size marked next to it (unit: bp);

[0045] N: blank control water;

[0046] C: receptor control B104 genomic DNA;

[0047] 1: genomic DNA of transformant MN85N-E6;

[0048] 2: Sample containing MN85N-E6 genomic DNA.

[0049] A: The expected size of the left border PCR fragment is 1063 bp; B: The expected size of the right border PCR fragment is 708 bp. DETAILED DESCRIPTION

[0050] The transformation event MN85N-E6 involved in this application refers to a corn plant in which a foreign gene insert (T-DNA insert) is inserted between specific genomic sequences after genetic transformation using corn B104 as a recipient. In a specific embodiment, the expression vector used for transgenic has Figure 1 The physical map shown in FIG. 5 , and the resulting T-DNA insert has the sequence shown in nucleotides 477-6636 of SEQ ID NO: 5. Transformation event MN85N-E6 can refer to this transgenic process, or can refer to the T-DNA insert in the genome obtained by this process, or a combination of the T-DNA insert and flanking sequences, or can refer to the corn plant obtained by this transgenic process. In a specific example, the event is also applicable to plants obtained by transforming other recipient varieties with the same expression vector, thereby inserting the T-DNA insert into the same genomic position. Transformation event MN85N-E6 can also refer to offspring plants obtained by asexual reproduction, sexual reproduction, doubling or doubling reproduction of the above-mentioned plants, or a combination of the above.

[0051] Example 1 Acquisition of transformation events and character identification

[0052] LC85-AcA1 The gene encodes a protein called LC85-Ac, which has a significant control effect on pests such as fall armyworm and corn borer; bar The 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 (the physical map of the vector is shown in Figure 1 ,Include LC85-AcA1 Gene expression cassette and barGene expression cassette), transformed into receptor B104 by Agrobacterium-mediated method, and 51 positive transformation events were obtained. The herbicide tolerance, insect resistance and related agronomic traits of the T1 generation plants of these transformed seedlings were identified and screened.

[0053] 1. Herbicide resistance screening

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

[0055] Table 1 Herbicide tolerance performance

[0056]

[0057] The values ​​are the mean ± standard deviation of three biological replicates. LSD was used for multiple comparisons (α = 0.05) in statistical analysis, and different letters indicate significant differences in the same column of data at the same herbicide concentration.

[0058] 2. Insect resistance

[0059] Using B104 as a reference, the transformants with better insect resistance were screened from the above 15 transformation events by leaf bioassay. The insect resistance of the materials was evaluated by feeding the newly hatched larvae of fall armyworm and corn borer with detached corn leaves.

[0060] The results of indoor bioassays are shown in Table 2. Among the 15 transformants, MN85N-E1 and MN85N-E6 were highly resistant to fall armyworm, and the others were moderately resistant, lowly resistant, or susceptible. MN85N-E1 and MN85N-E6 were highly resistant to corn borer, and the others were resistant, moderately resistant, lowly resistant, or susceptible. At the same time, the transformants with high resistance to fall armyworm and corn borer were MN85N-E1 and MN85N-E6.

[0061] Figure 2 and Figure 3 The indoor bioassay effects of the leaves of the transformant MN85N-E6 on the fall armyworm and corn borer were shown respectively.

[0062] Table 2 Indoor bio-test

[0063]

[0064] The data were expressed as the mean ± standard deviation of 4 biological replicates, and the significance of the differences in the same column was analyzed using the LSD method (α = 0.05).

[0065] In summary, MN85N-E1 and MN85N-E6 are transformants with excellent herbicide resistance and insect resistance, which can be used to improve the glufosinate herbicide tolerance and insect resistance of corn, thereby cultivating new insect-resistant and herbicide-resistant corn varieties.

[0066] Example 2 Molecular Characterization of Transformation Event MN85N-E6

[0067] In order to further clarify the identity characteristics of the transformation event MN85N-E6, the present invention analyzed the insertion site of the MN85N-E6 exogenous sequence on the maize genome and the insertion structure of the exogenous sequence.

[0068] 1. Analysis of flanking sequences of the insertion site of exogenous sequences in the maize genome

[0069] 100 mg of plant leaves were taken, quickly ground with liquid nitrogen, and total DNA was extracted using the CTAB method. After the concentration of genomic DNA was measured, the total amount of DNA was guaranteed to be >2 mg. Using genome resequencing, each Read was 150 bp long, at least 20 Gb of data was obtained, and the data quality indicator Q30 was guaranteed to be ≥80% (i.e., the proportion of bases with a sequencing error rate greater than 0.1% was less than 20%). According to the genome resequencing results, the BWA software was used to compare and screen the sequence homology with all the sequences obtained by sequencing using the transgenic vector T-DNA sequence as a template (BWA, http: / / bio-bwa.sourceforge.net / , default settings). The screened sequences were further assembled and screened, and the Reads whose sequences were all vector sequences were removed. Finally, a type of Read sequence was obtained, which was characterized by half of the genome sequence and the other half of the vector sequence. The obtained genome sequence was compared with the maize reference genome (MaizeGDB database https: / / www.maizegdb.org / blast, with Zm-B73-REFERENCE-NAM-5.0 as the reference sequence) to obtain the specific position of the genome sequence on the genome, which is the possible insertion site.

[0070] The sequencing results were compared with the reference genome and exogenous T-DNA sequences to obtain the insertion position information of the exogenous insertion fragment, which was located at Chr 8:75933155-75933164 bp in the maize genome. Subsequently, the forward and reverse primers were designed on the genome flanking sequences and exogenous insertion sequences at the left and right borders of the insertion site using the Primerblast software on the NCBI website (https: / / blast.ncbi.nlm.nih.gov / Blast) (as shown in Table 3). The insertion site was verified by PCR amplification using the genomic DNA of the transgenic maize line as a template. The amplified product fused a part of the maize genome sequence and a part of the T-DNA sequence, and the PCR product was sequenced and analyzed.

[0071] Table 3 Primer information

[0072]

[0073] The PCR reaction system specifically includes 1.0 μL genomic DNA, 10.0 μL 2×PCR Mixture, 0.5 μL forward primer (10 μM) and 0.5 μL reverse primer (10 μM), and water is added to make up to 20 μL.

[0074] The PCR amplification program was as follows: 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; and extension at 72°C for 7 min.

[0075] PCR amplification results are shown in Figure 4 The PCR amplification products of the left and right borders were sequenced to obtain the T-DNA insertion border sequence and flanking sequences of the MN85N-E6 transformant, which were assembled as follows (capital letters represent maize genome sequences, lowercase letters represent T-DNA sequences, and shading indicates the positions of transformant-specific PCR primers):

[0076] Left border:

[0077] GCTTTCTGTA TTGCCCTCTT CCAAATTTGT GTCTTTTTCA ACGTCATCTT CATTGGCATC 60

[0078] TGGCGTTGAT GAACCTTGCA TGTTAGCACT CATGGAAGGG GCTTCAACAT CTGTTCCCTC 120

[0079] TGCTGAAGGT AACATATCAA GTGAGGAAGC ATCTTTAGAT ACAGAGTGCA CCTCATCGGC 180

[0080] AACATTTGGC AATTTATCTG CTGCACCATG GCCTACTTCT GATGCAAATA CAGTTTGGTT 240

[0081] GTATTCATTG GGCGCAAGAT TTGGTGTATT GGGAATGGCT GAATCACCAA TAGCTGGCAT 300

[0082] GACAATGCTA CTTGCAACAa gacaacttaa taacacattg cggacgtttt taatgttaga 360

[0083] ctgaattaac gccgaattaa ttcgggggat ctggatttta gtactggatt ttggttttag 420

[0084] gaattagaaa ttttattgat agaagtattt tacaaataca aatacatact aagggtttct 480

[0085] tatatgctca acacatgagc gaaaccctat aggaacccta attcccttat ctgggaacta 540

[0086] ctcacacatt attatggaga aagcgatcgc tcaaatctcg gtgacgggca ggaccggacg 600

[0087] gggcggtacc ggcaggctga agtccagctg ccagaaaccc acgtcatgcc agttcccgtg 660

[0088] cttgaagccg gccgcccgca gcatgccgcg gggggcatat ccgagcgcct cgtgcatgcg 720

[0089] cacgctcggg tcgttgggca gcccgatgac agcgaccacg ctcttgaagc cctgtgcctc 780

[0090] cagggacttc agcaggtggg tgtagagcgt ggagcccagt cccgtccgct ggtggcgggg 840

[0091] ggagacgtac acggtcgact cggccgtcca gtcgtaggcg ttgcgtgcct tccaggggcc 900

[0092] cgcgtaggcg atgccggcga cctcgccgtc cacctcggcg acgagccagg gatagcgctc 960

[0093] ccgcagacgg acgaggtcgt ccgtccactc ctgcggttcc tgcggctcgg tacggaagtt 1020

[0094] gaccgtgctt gtctcgatgt agtggttgac gatggtgcag acc 1063

[0095] Right boundary:

[0096] gcgagcaagc tctaagagga gtgtcgacaa gcttggcact ggccgtcgtt ttacaacgtc 60

[0097] gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg 120

[0098] ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc 180

[0099] tgaatggcga atgctagagc agcttgagct tggatcagat tgtcgtttcc cgccttcagt 240

[0100] ttaaactatc agtgtttgac aggaCCCAAC TTTAGCTTGA GGCCGGCCCA TCTTCACTAT 300

[0101] TTCAGCCATG GAAGGGCGAC CTGGAACACC ACCCCAACCA TGTTTCACTT GAGATGGAGG 360

[0102] CTGGAAAGTG TTCTTAGCAT CAGCCTGGGC AAATTGCTG CAAACATAAT AAGCCTAAAA 420

[0103] AAATTAGGAA GCCTGCCTAG GAAACCTTGA AGCTACATTT ACAAATTACC AGATGAAGTG 480

[0104] AACATATTTA ATTTGCATGA GCGAAGCTAT ACATTGTCAA AGTTAAAGAG AGTGGCAGTT 540

[0105] 600

[0106] GATGATGCAA AAATGAAGAA AACAAAGTAT ATGAAACTCT GAGACTGCAA AAGATGATCC 660

[0107] CTTCATAATA TTCAAGCTAG AAAGTATCAG GTCCTTCAAT GCCGTAAC 708

[0108] The results of sequencing analysis showed that the exogenous T-DNA sequence of MN85N-E6 was reversely inserted from RB-LB into the maize genome at Chr8:75933155-75933164 bp.

[0109] The recipient maize genome sequence at the position of the transformant insertion (the deletion line marks the genome deletion sequence caused by the insertion of the transformant T-DNA):

[0110] GCTTTCTGTA TTGCCCTCTT CCAAATTTGT GTCTTTTTCA ACGTCATCTT CATTGGCATC 60

[0111] TGGCGTTGAT GAACCTTGCA TGTTAGCACT CATGGAAGGG GCTTCAACAT CTGTTCCCTC 120

[0112] TGCTGAAGGT AACATATCAA GTGAGGAAGC ATCTTTAGAT ACAGAGTGCA CCTCATCGGC 180

[0113] AACATTTGGC AATTTATCTG CTGCACCATG GCCTACTTCT GATGCAAATA CAGTTTGGTT 240

[0114] GTATTCATTG GGCGCAAGAT TTGGTGTATT GGGAATGGCT GAATCACCAA TAGCTGGCAT 300

[0115] GACAATGCTA CTTGCAACA G ATCTGCT CCC AACTTTAGCT TGAGGCCGGC CCATCTTCAC 360

[0116] TATTTCAGCC ATGGAAGGGC GACCTGGAAC ACCACCCCAA CCATGTTTCA CTTGAGATGG 420

[0117] AGGCTGGAAA GTGTTCTTAG CATCAGCCTG GGCAACATTG CTGCAAACAT AATAAGCCTA 480

[0118] AAAAATTAG GAAGCCTGCC TAGAAACCT TGAGCTACA TTTACAAATT ACCATTGA 540

[0119] GTGAACATAT TTAATTTGCA TGAGCGAAGC TATACATTGT CAAAGTTAAA GAGAGTGGCA 600

[0120] GTTTGCCCTG GAAATGGTT AGTTACT ATTACKGG CAAAATACA QUESTION ATC 660

[0121] ACAGATGATG CAAAAATGAA GAAAACAAAG TATATGAAAC TCTGAGACTG CAAAAGATGA 720

[0122] TCCCTTCATA ATATTCAAGC TAGAAAGTAT CAGGTCCTTC AATGCCGTAA C 771

[0123] Analysis of the left and right border sequences showed that the insertion of the exogenous sequence caused an 8 bp deletion mutation in the recipient corn genome; at the same time, the T-DNA region LB deleted 37 bp and RB deleted 17 bp.

[0124] 2. Analysis of full-length insert sequences in vitro

[0125] Using SEQ ID NO: 8 and SEQ ID NO: 11 as primers and the genomic DNA of the transformant MN85N-E6 as a template, PCR amplification was performed, and the obtained PCR product was the exogenous full-length insertion sequence. The PCR product was subjected to second-generation sequencing analysis and sequence splicing to obtain the actual insertion sequence of the MN85N-E6 transformant and the left and right flanking maize genome sequences, as shown in SEQ ID NO: 5.

[0126] Example 3 Detection method of transformation event MN85N-E6

[0127] New varieties can be bred from transgenic corn event MN85N-E6 and agricultural products or commodities can be produced. If sufficient amounts are detected in the agricultural products or commodities, the agricultural products or commodities are expected to contain nucleotide sequences that can diagnose the presence of transgenic corn event MN85N-E6 materials in the agricultural products or commodities. The agricultural products or commodities include, but are not limited to, corn oil, cornmeal, corn flour, corn paste, starch and other condiments or any other food consumed by animals as a food source, or cosmetics, industrial products, etc. Nucleic acid detection methods and / or kits based on probe or primer pairs can be developed to detect transgenic corn event MN85N-E6 nucleotide sequences such as those shown in SEQ ID NO: 1 or SEQ ID NO: 2 in biological samples, 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 transgenic corn event MN85N-E6.

[0128] One of the detection methods is: using the PCR method to detect samples containing MN85N-E6, 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).

[0129] PCR reaction system:

[0130] 2×PCR mix 10 μL Forward primer (10 μM) 0.5 μL Reverse primer (10 μM) 0.5 μL Genomic DNA 1 μL <![CDATA[ddH2O]]> To a final volume of 20 μL

[0131] The reaction procedure is:

[0132] 95℃, 5 min; (95℃, 30 sec; 55℃, 30 sec; 72℃, 60 sec) × 30 cycles; 72℃, 7 min.

[0133] The PCR product was electrophoresed in 1% (w / v) 1×TAE agarose gel. Figure 4 shown.

[0134] The expected target bands (SEQ ID NO: 6 and SEQ ID NO: 7, respectively) can be amplified in the MN85N-E6 transformation event. Moreover, the PCR method can track the existence of the transformation event and thus be applied to breeding work.

[0135] In summary, the transgenic corn event MN85N-E6 of the present invention can improve plant insect resistance and has a high tolerance to glufosinate herbicide, and can be used to improve other corn germplasms and create new corn hybrid combinations. Its detection method can accurately and quickly identify whether the biological sample contains the DNA molecule of the transgenic corn event MN85N-E6.

[0136] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the spirit and scope of the technical solution of the present invention.

Claims

1. A nucleic acid molecule for detecting maize transformation event MN85N-E6, characterized in that: The nucleic acid molecule sequence is any one of the following groups: i) the sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2, or their reverse complementary sequences; ii) the sequences shown in SEQ ID NO: 3 and SEQ ID NO: 4, or their reverse complementary sequences; iii) the sequences shown in SEQ ID NO: 6 and SEQ ID NO: 7, or their reverse complementary sequences; iv) the sequence shown in SEQ ID NO: 5, or its reverse complementary sequence; The maize transformation event MN85N-E6 refers to using Zm-B73-REFERENCE-NAM-5.0 as the reference genome and inserting the exogenous fragment into the Chr8:75933155-75933164bp position; the exogenous fragment has the sequence shown in the 477th-6636th nucleotides of SEQ ID NO:

5.

2. A primer pair for detecting the maize transformation event MN85N-E6 according to claim 1, characterized in that: The primer pair is the sequence shown in SEQ ID NO:8 and SEQ ID NO:9; and the sequence shown in SEQ ID NO:10 and SEQ ID NO:

11.

3. A kit or microarray for detecting the maize transformation event MN85N-E6 according to claim 1, characterized in that: Comprising the nucleic acid molecule according to claim 1 and / or the primer pair according to claim 2.

4. A method for detecting the maize transformation event MN85N-E6 according to claim 1, 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 nucleic acid molecule according to claim 1; ii) the primer pair according to claim 2; iii) the nucleic acid molecule according to claim 1 and the primer pair according to claim 2; iv) The kit or microarray according to claim 3.

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