Nucleic acid sequence for detecting the QY2569-42 transgenic maize event, DNA structure, DNA molecule, methods for detecting the presence of DNA of the QY2569-42 transgenic maize event, preparations, agricultural products or produce manufactured from the QY2569-42 transgenic maize event.

Abstract

The invention relates to the transgenic maize event, QY2569-42, and also relates to a nucleic acid sequence for the detection of the maize plant QY2569-42 and a method for its detection. The nucleic acid sequence of the maize plant includes SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11 and / or SEQ ID NO:12, or their complementary nucleic acid sequences. The maize plant QY2569-42 of the invention exhibits good resistance to lepidopteran insects and good tolerance to the herbicide glufosinate without affecting yield. In addition, the detection method can accurately and quickly identify whether a biological sample contains DNA molecules of the QY2569-42 transgenic corn event.

Description

Genetically modified corn event QY2569-42 and its detection method Technical Field

[0001] The present invention relates to a transgenic corn event QY2569-42, and also relates to a nucleic acid sequence for detecting the corn plant QY2569-42 and a detection method thereof. Background Art

[0002] Agricultural pests are the primary factor impacting crop production. Currently, chemical pesticides remain the primary method of pest control, playing a crucial role in maintaining the stable development of agricultural production. Pesticides employ non-specific toxicity, killing target pests while also harming beneficial insects and birds. Long-term use can cause severe environmental pollution, disrupting ecological balance, and posing a direct threat to human and livestock safety due to their accumulation in the food chain. Weeds are the second most important factor impacting crop production, competing with crops for water, fertilizer, and light, reducing yields and diminishing the quality of agricultural products. Traditional manual weeding is time-consuming, labor-intensive, and inefficient, while mechanical weeding is expensive. Chemical weed control requires high technical skills and is prone to plant damage, impacting crop growth and development. Chemicals can also affect sensitive neighboring crops.

[0003] Corn (Zea mays L.) is a major food crop in many parts of the world. Currently, transgenic breeding technology has been widely used in corn to improve its agronomic traits and quality. Insect resistance is an important agronomic trait in corn production, especially resistance to Lepidoptera insects, such as the Asian corn borer, fall armyworm, cotton bollworm, and oriental armyworm. Corn resistance to Lepidoptera insects can be obtained by expressing Lepidoptera insect resistance genes (such as mvip3Aa20 and cry1Ab) in corn plants through transgenic methods. Another important agronomic trait is herbicide tolerance, such as tolerance to glufosinate herbicide. Corn tolerance to glufosinate herbicide can be obtained by expressing glufosinate herbicide tolerance genes (such as pat) in corn plants through transgenic methods.

[0004] Insect resistance traits and / or herbicide tolerance traits can be used alone or in combination with other traits (such as tolerance to other herbicides or resistance to other pests or pathogens). Trait combinations can be achieved by breeding each individual trait together, but breeding individual traits together in corn and maintaining these combinations with multiple elite germplasms during the breeding process is a time-consuming and expensive process. However, combining multiple insect resistance traits and / or herbicide tolerance traits at a single location or locus in the corn genome can also achieve trait combinations and simplify the breeding process, making it an economical and effective transgenic breeding approach.

[0005] Transgene expression in plants is influenced by numerous factors, including the site of insertion into the recipient plant genome, the elements used in the expression cassettes, and interactions between these elements. This is further complicated for transgene insertions containing two or more expression cassettes, each harboring a transgene conferring a separate trait, also known as multi-gene transgenic events. The expression levels and patterns of the exogenous genes can vary significantly between events. Therefore, it is essential to first select the optimal expression cassettes for each trait, assemble constructs, and generate a large number of transgenic events. Through rigorous molecular characterization, greenhouse testing, and multi-year field trials at multiple locations and under various conditions, extensive agronomic, phenotypic, and molecular data are collected to screen for superior transgenic events that exhibit the desired transgene expression levels and patterns for commercialization.

[0006] Summary of the Invention

[0007] The purpose of the present invention is to provide a transgenic corn event QY2569-42 and a nucleic acid sequence and a detection method for detecting the transgenic corn event QY2569-42.

[0008] The technical solution adopted in the present invention is as follows:

[0009] The present invention provides a nucleic acid sequence comprising:

[0010] (a) SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11 and / or SEQ ID NO:12; or

[0011] (b) a nucleic acid sequence complementary to (a);

[0012] The nucleic acid sequence is derived from transgenic maize event QY2569-42.

[0013] In a specific embodiment, the nucleic acid sequence is an amplicon diagnostic for the presence of corn event QY2569-42.

[0014] The present invention also provides a DNA construct comprising three expression cassettes, wherein:

[0015] a) the first expression cassette comprises, in operable connection, a promoter OsUbi2 promoter as shown in SEQ ID NO:29, a nucleic acid sequence as shown in SEQ ID NO:30, a coding region of the mVip3Aa20 gene, a nucleic acid sequence as shown in SEQ ID NO:31, a chloroplast leader peptide CTP-TS-SSU, and a terminator T-Ara5 as shown in SEQ ID NO:32 for terminating expression of the gene;

[0016] b) the second expression cassette comprises, in operable connection, a promoter P-FMV as shown in SEQ ID NO:33, a cry1Ab gene coding region as shown in SEQ ID NO:34, an intron I-HSP70 as shown in SEQ ID NO:35, a chloroplast localization peptide CTP2 as shown in SEQ ID NO:36, and a terminator T-Tr7 as shown in SEQ ID NO:37 for terminating gene expression;

[0017] c) The third expression cassette comprises, in operable connection, a promoter P-CaMV35S as shown in SEQ ID NO: 38, a pat gene coding region as shown in SEQ ID NO: 39, and a termination sequence T-35S polyA for terminating gene expression as shown in SEQ ID NO: 40.

[0018] The present invention also provides a DNA molecule having a sufficient length of consecutive nucleotides of SEQ ID NO: 10 to be used as a DNA probe specific for SEQ ID NO: 10 in a DNA sample derived from a corn plant, corn seed or corn cell, wherein the DNA probe comprises SEQ ID NO: 1 or SEQ ID NO: 2.

[0019] In a specific embodiment, the probe is SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:11 or SEQ ID NO:12.

[0020] The present invention also provides a method for detecting the presence of DNA of the transgenic maize event QY2569-42 in a sample, comprising:

[0021] contacting a sample to be tested with at least two primers in a nucleic acid amplification reaction;

[0022] performing nucleic acid amplification reactions;

[0023] detecting the presence of amplification products;

[0024] The amplification product comprises (a) SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and / or SEQ ID NO: 10; or (b) a nucleic acid sequence complementary to (a);

[0025] The nucleic acid sequence is derived from transgenic maize event QY2569-42.

[0026] In a specific embodiment, the amplified product further comprises SEQ ID NO: 11 or its complementary sequence and / or SEQ ID NO: 12 or its complementary sequence.

[0027] In another specific embodiment, the two primers include SEQ ID NO:13 and SEQ ID NO:14, or SEQ ID NO:15 and SEQ ID NO:16.

[0028] The present invention also provides a method for detecting the presence of DNA of the transgenic maize event QY2569-42 in a sample, comprising:

[0029] contacting the sample to be detected with a probe comprising SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8 or a complementary sequence thereof;

[0030] hybridizing the sample to be detected and the probe under stringent hybridization conditions;

[0031] Detecting the hybridization between the sample to be detected and the probe.

[0032] In a specific embodiment, the probe further comprises SEQ ID NO: 11 or its complementary sequence, or SEQ ID NO: 12 or its complementary sequence.

[0033] In another embodiment, at least one of said probes is labeled with at least one fluorescent group.

[0034] The present invention also provides a method for detecting the presence of DNA of the transgenic maize event QY2569-42 in a sample, comprising:

[0035] contacting the sample to be tested with a marker nucleic acid molecule comprising SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8 or a complementary sequence thereof;

[0036] hybridizing the sample to be detected and the marker nucleic acid molecule under stringent hybridization conditions;

[0037] The hybridization between the sample to be tested and the marker nucleic acid molecule is detected, and then marker-assisted breeding analysis is performed to determine whether insect resistance and / or herbicide tolerance is genetically linked to the marker nucleic acid molecule.

[0038] In a specific embodiment, the marker nucleic acid molecule further comprises SEQ ID NO: 11 or its complementary sequence, or SEQ ID NO: 12 or its complementary sequence.

[0039] The present invention also provides a DNA detection kit, which includes at least one DNA molecule, wherein the DNA molecule includes SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8 or its complementary sequence, which can be used as one of the DNA primers or probes specific for the transgenic corn event QY2569-42 or its progeny.

[0040] In a specific embodiment, the DNA molecule used as a probe further comprises SEQ ID NO: 11 or its complementary sequence, or SEQ ID NO: 12 or its complementary sequence.

[0041] The present invention also provides a method for protecting corn plants from insect infestation, comprising providing at least one transgenic corn plant cell in the diet of a target insect, wherein the transgenic corn plant cell comprises in its genome SEQ ID NO:1, the nucleic acid sequence at positions 777-11945 of SEQ ID NO:10 and SEQ ID NO:2, or SEQ ID NO:3, the nucleic acid sequence at positions 777-11945 of SEQ ID NO:10 and SEQ ID NO:4, or SEQ ID NO:5, the nucleic acid sequence at positions 777-11945 of SEQ ID NO:10 and SEQ ID NO:6, or the genome of the transgenic corn plant cell comprises SEQ ID NO:10; and target insects that ingest the transgenic corn plant cell are inhibited from further ingesting the corn plant.

[0042] The present invention also provides a method for protecting corn plants from damage caused by herbicides, which 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 comprises in its genome SEQ ID NO:1, the nucleic acid sequence at positions 777 to 11945 of SEQ ID NO:10 and SEQ ID NO:2, or SEQ ID NO:3, the nucleic acid sequence at positions 777 to 11945 of SEQ ID NO:10 and SEQ ID NO:4, or SEQ ID NO:5, the nucleic acid sequence at positions 777 to 11945 of SEQ ID NO:10 and SEQ ID NO:6, or the genome of the transgenic corn plant comprises SEQ ID NO:10; the transgenic corn plant has tolerance to the glufosinate herbicide.

[0043] The present invention also provides a method for controlling weeds in a field where corn plants are planted, comprising applying an effective dose of a glufosinate herbicide to a field where at least one transgenic corn plant is planted, wherein the transgenic corn plant comprises in its genome SEQ ID NO:1, the nucleic acid sequence at positions 777 to 11945 of SEQ ID NO:10, and SEQ ID NO:2, or SEQ ID NO:3, the nucleic acid sequence at positions 777 to 11945 of SEQ ID NO:10, and SEQ ID NO:4, or SEQ ID NO:5, the nucleic acid sequence at positions 777 to 11945 of SEQ ID NO:10, and SEQ ID NO:6, or the genome of the transgenic corn plant comprises SEQ ID NO:10; and the transgenic corn plant is tolerant to the glufosinate herbicide.

[0044] The present invention also provides a method for cultivating a corn plant that is resistant to insects, comprising: planting at least one corn seed, wherein the genome of the corn seed comprises a nucleic acid sequence in a specific region, wherein the nucleic acid sequence in the specific region sequentially comprises SEQ ID NO:1, the nucleic acid sequence at positions 777 to 11945 of SEQ ID NO:10, and SEQ ID NO:2, or SEQ ID NO:3, the nucleic acid sequence at positions 777 to 11945 of SEQ ID NO:10, and SEQ ID NO:4, or SEQ ID NO:5, the nucleic acid sequence at positions 777 to 11945 of SEQ ID NO:10, and SEQ ID NO:6, or the nucleic acid sequence in the specific region comprises SEQ ID NO:10; and allowing the corn seed to grow into a corn plant;

[0045] The corn plants are infested with target insects and the plants are harvested having reduced plant injury compared to other plants not having the nucleic acid sequence of the specific region.

[0046] The present invention also provides a method for cultivating a corn plant tolerant to glufosinate-ammonium herbicide, comprising: planting at least one corn seed, wherein the genome of the corn seed comprises a nucleic acid sequence of a specific region, wherein the nucleic acid sequence of the specific region sequentially comprises SEQ ID NO:1, the nucleic acid sequence at positions 777 to 11945 of SEQ ID NO:10, and SEQ ID NO:2, or SEQ ID NO:3, the nucleic acid sequence at positions 777 to 11945 of SEQ ID NO:10, and SEQ ID NO:4, or SEQ ID NO:5, the nucleic acid sequence at positions 777 to 11945 of SEQ ID NO:10, and SEQ ID NO:6, or the nucleic acid sequence of the specific region comprises SEQ ID NO:10;

[0047] allowing the corn seeds to grow into corn plants;

[0048] The corn plants are sprayed with an effective amount of glufosinate herbicide, and the plants are harvested having reduced plant damage compared to other plants not having the nucleic acid sequence of the specific region.

[0049] The present invention also provides a method for cultivating corn plants that are resistant to insects and tolerate glufosinate herbicides, comprising: planting at least one corn seed, wherein the genome of the corn seed comprises a nucleic acid sequence of a specific region, wherein the nucleic acid sequence of the specific region sequentially comprises SEQ ID NO:1, the nucleic acid sequence at positions 777 to 11945 of SEQ ID NO:10, and SEQ ID NO:2, or SEQ ID NO:3, the nucleic acid sequence at positions 777 to 11945 of SEQ ID NO:10, and SEQ ID NO:4, or SEQ ID NO:5, the nucleic acid sequence at positions 777 to 11945 of SEQ ID NO:10, and SEQ ID NO:6, or the nucleic acid sequence of the specific region comprises SEQ ID NO:10;

[0050] allowing the corn seeds to grow into corn plants;

[0051] The corn plants are sprayed with an effective amount of glufosinate herbicide, and the plants are harvested to have reduced plant damage compared to other plants that do not have the nucleic acid sequence of the specific region. The plants with reduced plant damage are also resistant to insect feeding damage.

[0052] The present invention also provides a method for producing a corn plant resistant to insects, comprising hybridizing a corn plant whose genome sequentially comprises the nucleic acid sequence of SEQ ID NO: 1, SEQ ID NO: 10, positions 777 to 11945, and SEQ ID NO: 2, or SEQ ID NO: 3, SEQ ID NO: 10, positions 777 to 11945, and SEQ ID NO: 4, or SEQ ID NO: 5, SEQ ID NO: 10, positions 777 to 11945, and SEQ ID NO: 6, with another corn plant, thereby producing a plurality of progeny plants; and selecting a progeny plant whose genome comprises a nucleic acid sequence of a specific region, wherein the nucleic acid sequence of the specific region sequentially comprises the nucleic acid sequence of SEQ ID NO: 1, SEQ ID NO: 10, positions 777 to 11945, and SEQ ID NO: 2, or SEQ ID NO: 3, SEQ ID NO: 10, positions 777 to 11945, and SEQ ID NO: 4, or SEQ ID NO: 5, SEQ ID NO: 10, positions 777 to 11945, and SEQ ID NO: 6. NO:10 nucleic acid sequence at positions 777-11945 and SEQ ID NO:6, or the nucleic acid sequence of the specific region comprises SEQ ID NO:10, and the progeny plants have reduced plant damage to insect feeding.

[0053] In one embodiment, the method comprises: sexually crossing a first parent corn plant of transgenic corn event QY2569-42 that is resistant to insects with a second parent corn plant that lacks insect resistance, thereby producing a plurality of progeny plants;

[0054] infesting the progeny plants with target insects;

[0055] The progeny plants are selected for having reduced plant injury compared to other plants not having the nucleic acid sequence of the specific region.

[0056] The present invention also provides a method for producing a corn plant tolerant to glufosinate-ammonium herbicide, comprising hybridizing a corn plant whose genome sequentially comprises the nucleic acid sequence of SEQ ID NO: 1, SEQ ID NO: 10, positions 777 to 11945, and SEQ ID NO: 2, or SEQ ID NO: 3, SEQ ID NO: 10, positions 777 to 11945, and SEQ ID NO: 4, or SEQ ID NO: 5, SEQ ID NO: 10, positions 777 to 11945, and SEQ ID NO: 6, with another corn plant, thereby producing a large number of progeny plants; and selecting a progeny plant whose genome comprises a nucleic acid sequence of a specific region, wherein the nucleic acid sequence of the specific region sequentially comprises the nucleic acid sequence of SEQ ID NO: 1, SEQ ID NO: 10, positions 777 to 11945, and SEQ ID NO: 2, or SEQ ID NO: 3, SEQ ID NO: 10, positions 777 to 11945, and SEQ ID NO: 4, or SEQ ID NO: 5, SEQ ID NO: 10, positions 777 to 11945, and SEQ ID NO: 6. NO:10 nucleic acid sequence at positions 777-11945 and SEQ ID NO:6, or the nucleic acid sequence of the specific region contains SEQ ID NO:10, and the progeny plant has glufosinate tolerance.

[0057] In one embodiment, the method comprises: sexually crossing a first parent corn plant of transgenic corn event QY2569-42 that is tolerant to glufosinate herbicide with a second parent corn plant that lacks glufosinate tolerance, thereby producing a plurality of progeny plants;

[0058] treating the progeny plants with a glufosinate-ammonium herbicide;

[0059] The progeny plants are selected to be tolerant to glufosinate ammonium.

[0060] The present invention also provides a method for producing a corn plant that is resistant to insects and tolerates the application of a glufosinate herbicide, comprising: sexually crossing a first parent corn plant of a transgenic corn event QY2569-42 that is glufosinate-tolerant and insect-resistant with a second parent corn plant that lacks glufosinate-tolerant and / or insect-resistant, thereby producing a plurality of progeny plants;

[0061] treating the progeny plants with glufosinate-ammonium;

[0062] The progeny plants are selected to be tolerant to glufosinate ammonium, and the progeny plants tolerant to glufosinate ammonium are also resistant to feeding damage by insects.

[0063] The present invention also provides a method for improving tolerance of corn plants, the method comprising:

[0064] a) constructing a DNA construct as described above;

[0065] b) inserting the DNA construct into the genome of a maize cell;

[0066] c) regenerating the corn cell into a corn plant; and

[0067] d) selecting maize plants comprising said DNA construct.

[0068] In one embodiment, improving the tolerance of corn plants comprises their resistance to an effective amount of at least one herbicide, preferably glufosinate-ammonium; and / or

[0069] Improving corn plant tolerance includes resistance to at least one insect, preferably the Asian corn borer, the oriental armyworm, the fall armyworm, and / or the cotton bollworm.

[0070] The present invention also provides a composition produced from the transgenic corn event QY2569-42, wherein the composition is corn flour, corn meal, corn oil, corn silk or corn starch.

[0071] The present invention also provides an agricultural product or commodity produced from the transgenic corn event QY2569-42, wherein the agricultural product or commodity is corn flour, corn meal, corn oil, corn starch, corn gluten, corn tortilla, cosmetics or fillers.

[0072] The present invention also provides a plant cell, plant part, plant or seed comprising the aforementioned nucleic acid sequence.

[0073] The present invention also provides a non-living plant material comprising the aforementioned nucleic acid sequence.

[0074] Some terms used in this specification are defined below.

[0075] The term "corn" as used herein refers to Zea mays and includes all plant species that can be crossed with corn, including wild corn species.

[0076] The term "comprising" as used herein means "including but not limited to".

[0077] As used herein, "insecticide" or "insect-resistant" refers to being toxic to crop pests, thereby achieving "control" and / or "prevention" of crop pests. Preferably, "insecticide" or "insect-resistant" refers to killing crop pests. Such pests include members of the order Lepidoptera.

[0078] The "Lepidoptera", scientifically known as Lepidoptera, includes two types of insects: moths and butterflies. It is the order with the most agricultural and forestry pests, such as the corn borer, fall armyworm, cotton bollworm, oriental armyworm, two-spotted armyworm, and peach borer.

[0079] As used herein, "herbicide" refers to an active ingredient that kills, controls, or otherwise inhibits the growth of plants. "Herbicide tolerance," "herbicide resistance," "herbicide tolerance," or "herbicide resistance" as used herein refers to the ability of a plant to continue growing despite exposure to a herbicide that kills, inhibits, or reduces the growth of common or wild plants, or even halts their growth compared to wild plants. Such herbicides include glutamine synthetase inhibitors, such as glufosinate.

[0080] The term "gene" refers to a nucleic acid fragment that expresses a specific protein, including regulatory sequences preceding the coding sequence (5' non-coding sequences) and following the coding sequence (3' non-coding sequences). A "native gene" refers to a gene found in nature that has its own regulatory sequences. A "chimeric gene" refers to any gene that is not a natural gene and contains regulatory and coding sequences that are not found in nature. An "endogenous gene" refers to a natural gene that is located in its natural location in the genome of an organism. An "exogenous gene" is a foreign gene that is now present in the genome of an organism and did not originally exist, and also refers to a gene that is introduced into a recipient cell through a transgenic step. Exogenous genes can include natural genes inserted into non-natural organisms or chimeric genes. A "transgene" is a gene that has been introduced into the genome through a transformation procedure. The site in the plant genome where the recombinant DNA has been inserted can be called an "insertion site" or "target site."

[0081] The terms "nucleotide sequence" or "nucleic acid sequence" are used interchangeably and refer to oligonucleotides, nucleotides or polynucleotides and fragments or portions thereof, which may be single-stranded or double-stranded and represent the sense or antisense strand. Nucleic acids include DNA, RNA or hybrids thereof and may be of natural or synthetic origin. For example, a nucleic acid may include mRNA or cDNA. A nucleic acid may include a nucleic acid that has been amplified (e.g., using the polymerase chain reaction). The single-letter codes for nucleotides are as described in Table 1 of Section 2422 of the U.S. Patent Office Manual of Patent Examining Procedure.

[0082] The term "transgenic" plant refers to a plant comprising a heterologous polynucleotide. Preferably, the heterologous polynucleotide is stably integrated in the genome so that the polynucleotide is passed to successive generations. The heterologous polynucleotide can be integrated into the genome separately or integrated as a part of a recombinant expression cassette." transgenic" is used herein to refer to any cell, cell line, callus, tissue, plant part or plant, whose genotype is changed due to the presence of heterologous nucleic acids, including those initially changed transgenic organisms or cells, and those produced from initial transgenic organisms or cell hybridization or asexual reproduction. As used herein, the term "transgenic" is not intended to include changing genomes (chromosomes or chromosomes) by conventional plant breeding methods (for example, hybridization) or by naturally occurring events (such as, self-fertilization, random cross fertilization, non-recombinant virus infection, non-recombinant bacterial transformation, non-recombinant transposition or spontaneous mutation).

[0083] A transgenic "event" is produced by transforming plant cells with heterologous DNA (i.e., a nucleic acid construct containing a transgene of interest), regenerating a plant population resulting from the insertion of the transgene into the plant genome, and screening for specific plants characterized by an insertion at a specific genomic location. The term "event" refers to the initial transformant and the progeny of the transformant that contain the heterologous DNA. The term "event" also refers to the progeny produced by a sexual outcross between a transformant and another variety containing the genomic / transgenic DNA. Even after repeated backcrossing with the recurrent parent, the inserted transgenic DNA and flanking genomic DNA (genomic / transgenic DNA) from the transformed parent remain at the same chromosomal location in the hybrid progeny. The term "event" also refers to DNA from the initial transformant and its progeny that contain the inserted DNA and flanking genomic sequences immediately adjacent to the inserted DNA, which DNA will be transferred to the progeny. By sexually crossing a parental line containing the inserted DNA (e.g., the initial transformant and the progeny produced by selfing) with a parental line that does not contain the inserted DNA, the progeny are obtained to contain the DNA of the desired transgene.

[0084] The term "insert DNA" or "insert sequence" refers to heterologous DNA within an expression cassette used to transform plant material. The insert DNA is derived from the T-DNA contained in the binary vector used in Agrobacterium-mediated plant transformation.

[0085] " flanking DNA " or " flanking sequence " described in the present invention can comprise the exogenous (heterologous) DNA that is naturally present in the organism of for example plant or by the transformation process introducing, for example the fragment relevant to transformation event.Therefore, flanking DNA can comprise the combination of natural and exogenous DNA.In the present invention, " flanking region " or " flanking sequence " or " genome border zone " or " genome border sequence " refer to at least 3,5,10,11,15,20,50,100,200,300,400,1000,1500,2000,2500 or 5000 base pairs or longer sequence, and it is positioned at the direct upstream or downstream of initial exogenous insertion DNA molecule and is adjacent to initial exogenous insertion DNA molecule.When this flanking region was positioned at downstream, it also can be called " left border flank " or " 3 ' flank " or " 3 ' genome border zone " or " genome 3 ' border sequence " etc. When the flanking region is located upstream, it may also be referred to as the "right border flank" or the "5' flank" or the "5' genomic border region" or the "genomic 5' border sequence" or the like.

[0086] The term "junction" refers to the point where two specific DNA fragments are connected. For example, a junction occurs where the insert DNA joins the flanking DNA. A junction point also occurs in transformed organisms where two DNA fragments are joined together in a manner modified from that found in the native organism. "Junction DNA" or "junction sequence" refers to the DNA comprising the junction point.

[0087] A "junction sequence" spans the point at which DNA inserted into the genome joins DNA from the native maize genome flanking the insertion point, wherein identification or detection of one or the other junction sequence in the plant genetic material is sufficient to be diagnostic for the event. DNA sequences spanning the insertion and similar lengths of flanking DNA in the maize events described herein are included. Specific examples of such diagnostic sequences are provided herein; however, other sequences that overlap the junction of the insertion or the junction of the insertion and the genomic sequence are also diagnostic and can be used according to the present invention.

[0088] The term "probe" refers to an isolated nucleic acid molecule to which is bound a conventional detectable label or reporter molecule, such as a radioisotope, ligand, chemiluminescent agent, or enzyme. Such a probe is complementary to a strand of a target nucleic acid. In the present invention, the probe is complementary to a strand of genomic DNA from transgenic maize event QY2569-42, whether the genomic DNA is from transgenic maize event QY2569-42 or seeds, or from plants, seeds, or extracts thereof. The probes of the present invention include not only deoxyribonucleic acids (DNAs) or RNAs, but also polyamides and other probe materials that specifically bind to a target DNA sequence and can be used to detect the presence of the target DNA sequence.

[0089] The term "primer" refers to an isolated nucleic acid molecule that anneals to a complementary target DNA strand through nucleic acid hybridization, forming a hybrid between the primer and the target DNA strand, which is then extended along the target DNA strand by a polymerase (e.g., DNA polymerase). The primer pairs of the present invention relate to their use in amplifying a target nucleic acid sequence, for example, by polymerase chain reaction (PCR) or other conventional nucleic acid amplification methods.

[0090] The nucleic acid probes and primers of the present invention hybridize to target DNA molecules under stringent conditions. Any conventional nucleic acid hybridization or amplification method can be used to identify the presence of DNA from transgenic plants in a sample. Polynucleic acid molecules, also referred to as nucleic acid segments, or fragments thereof, are capable of specific hybridization with other nucleic acid molecules in certain circumstances.

[0091] As used herein, if two polynucleotide molecules can form an antiparallel double-stranded nucleic acid structure, it is said that the two molecules can specifically hybridize to each other.If two nucleic acid molecules show complete complementarity, then one nucleic acid molecule is said to be "complementary" to another nucleic acid molecule.As used herein, when each nucleotide of one of the molecules is complementary to the nucleotide of another molecule, the molecule is said to show "complete complementarity".If two molecules can hybridize to each other with enough stability so that they remain bound to each other under at least conventional "low stringency" conditions, they are said to be "minimum complementary".Similarly, if molecules can hybridize to each other with enough stability so that they remain bound to each other under conventional "high stringency" conditions, they are said to be "complementary".Sambrook et al., 1989 and Hames et al., Nucleic Acid Hybridization, A Practical Approach, IRL Press, Washington, DC (1985) describe conventional stringency conditions.Therefore, deviation from complete complementarity is allowed, as long as such deviation does not completely exclude the ability of molecules to form double-stranded structures. In order for a nucleic acid molecule to function as a primer or probe, it is only necessary that the nucleic acid molecules be sufficiently complementary in sequence to form a stable double-stranded structure under the particular solvent and salt concentrations employed.

[0092] The term "amplicon" refers to the product of nucleic acid amplification of a target nucleic acid sequence that is part of a nucleic acid template. "Amplicon" and "amplification product" are used interchangeably herein. For example, to determine whether a corn plant was produced by sexual crosses containing the transgenic corn event QY2569-42 of the present invention, whether a corn sample collected from a field contains transgenic corn event QY2569-42, or whether a corn extract, such as meal, flour, or oil, contains transgenic corn event QY2569-42, DNA extracted from the corn plant tissue sample or extract can be subjected to a nucleic acid amplification method using a primer pair to produce an amplicon that is diagnostic for the presence of transgenic corn event QY2569-42 DNA. The primer pair includes a first primer derived from flanking sequences adjacent to the insertion site of the inserted exogenous DNA in the plant genome, and a second primer derived from the inserted exogenous DNA. The amplicon has a length and sequence that is also diagnostic for the transgenic corn event QY2569-42. The length of the amplicon can range from the combined length of the primer pair plus one nucleotide base pair, preferably plus about fifty nucleotide base pairs, more preferably plus about two hundred and fifty nucleotide base pairs, and most preferably plus about four hundred and fifty nucleotide base pairs or more.

[0093] The term "plant" is used in its broadest sense as it relates to organic matter and is intended to encompass eukaryotic organisms belonging to the kingdom Plantae, examples of which include, but are not limited to, vascular plants, vegetables, seeds, flowers, trees, herbs, shrubs, grasses, vines, ferns, mosses, fungi and algae, as well as clones, offsets and plant parts used for asexual propagation (e.g., cuttings, tubes, shoots, rhizomes, underground stems, clumps, crowns, bulbs, corms, tubers, rhizomes, plants / tissues produced in tissue culture, etc.). The term "plant" also encompasses complete plants, ancestors and descendants of plants and plant parts, including seeds, seedlings, stems, leaves, roots (including tubers), flowers, florets, fruits, pedicels, stalks, stamens, anthers, stigmas, styles, ovaries, petals, sepals, carpels, root tips, root caps, root hairs, leaf hairs, seed hairs, pollen grains, microspores, cotyledons, hypocotyls, epicotyls, xylem, phloem, parenchyma, endosperm, companion cells, guard cells, and any other known organs, tissues, and cells of plants, and tissues and organs each of which comprises the gene / nucleic acid of interest. The term "plant" also encompasses plant cells, suspension cultures, callus, embryos, meristematic regions, gametophytes, sporophytes, pollen, and microspores, again wherein each of the foregoing comprises the gene / nucleic acid of interest.

[0094] The term "plant tissue" or "plant part" includes plant cells, protoplasts, plant tissue cultures, plant callus, plant pieces, as well as plant embryos, pollen, ovules, seeds, leaves, stems, flowers, branches, seedlings, fruits, kernels, ears, roots, root tips, anthers, and the like.

[0095] The term "plant cell" is to be understood as any cell from or found in a plant, which is capable of forming, for example, undifferentiated tissue such as callus, differentiated tissue such as embryos, plant components, plants or seeds.

[0096] The genome of a plant, plant tissue or plant cell mentioned in the present invention refers to any genetic material in a plant, plant tissue or plant cell, and includes the genomes of the cell nucleus, plastids and mitochondria.

[0097] The term "expression cassette" refers to a complete element required for expressing a gene, which contains a nucleotide sequence that can encode a target protein and has a start codon and a stop codon.

[0098] The term "kit" refers to any article of manufacture (eg, a package or container) comprising at least one device, and the kit may further comprise instructions for use, supplementary reagents and / or components or assemblies used in the methods described herein or steps thereof.

[0099] In some embodiments, the kit further comprises one or more of nucleic acid extraction reagents, nucleic acid amplification reagents, positive controls, and negative controls.

[0100] The terms "DNA construct" and "recombinant vector" mean a vector containing a heterologous or recombinant nucleotide sequence. The term "vector" refers to a nucleic acid fragment or polynucleotide fragment used to introduce or transfer one or more nucleic acids or one or more polynucleotides into a target cell or tissue.

[0101] Vector transformation methods include Agrobacterium-mediated transformation, electroporation, microparticle bombardment, polyethylene glycol-medium absorption, and the like to introduce recombinant plasmids into plants.

[0102] The plant transformation recipients in the present invention include plant cells (including suspension culture cells), protoplasts, callus tissues, hypocotyls, seeds, cotyledons, buds and mature plants.

[0103] The scope of transgenic plants includes not only the plants obtained at the time of gene introduction, but also its clones and offspring (T1 generation, T2 generation or subsequent generations). The scope of the present invention also includes all mutants and variants of the above-mentioned transgenic plants that show the characteristics of the first generation transgenic plants after hybridization and fusion. The scope of the present invention also includes plant parts, such as seeds, flowers, stems, fruits, leaves, roots, tubers, and tuberous stems, which are derived from plants that have been genetically modified in advance by the methods mentioned in the present invention, or their offspring, and are composed of at least a portion of genetically modified cells.

[0104] The present invention relates to the identification of such flanking, junction, and insertion sequences. The present invention also includes PCR primers and amplicons designed based on the aforementioned sequences. The present invention's PCR analysis of amplicons spanning the inserted DNA and its boundaries can be used to detect or identify commercial transgenic corn varieties or lines derived from the proprietary transgenic corn of the present invention.

[0105] Sequence Description

[0106] SEQ ID NO: 1 The insertion site of the 5' transgenic fragment in transgenic maize event QY2569-42 and 10 nucleotides on each side of the maize genomic DNA;

[0107] SEQ ID NO: 2: insertion site of the 3' transgenic fragment in transgenic maize event QY2569-42 and 10 nucleotides on each side of maize genomic DNA;

[0108] SEQ ID NO: 3 The insertion site of the 5' transgene fragment in transgenic maize event QY2569-42 and 20 nucleotides on each side of the maize genomic DNA;

[0109] SEQ ID NO:4: the insertion site of the 3' transgene fragment in transgenic maize event QY2569-42 and 20 nucleotides on each side of the maize genomic DNA;

[0110] SEQ ID NO:5 The insertion site of the 5' transgenic fragment in transgenic maize event QY2569-42 and 50 nucleotides on each side of the maize genomic DNA;

[0111] SEQ ID NO:6 The insertion site of the 3' transgene fragment in transgenic maize event QY2569-42 and 50 nucleotides on each side of the maize genomic DNA;

[0112] SEQ ID NO: 7 is a 1269 nucleotide sequence located near the insertion junction at the 5' end of the insertion sequence of transgenic maize event QY2569-42;

[0113] SEQ ID NO: 8 is a 3057 nucleotide sequence located near the insertion junction at the 3' end of the insertion sequence of transgenic maize event QY2569-42;

[0114] SEQ ID NO:9 The insertion sequence of transgenic maize event QY2569-42 from the T-DNA region of the pQY002569 vector;

[0115] SEQ ID NO: 10 entire T-DNA sequence, 5' and 3' flanking maize genomic sequence;

[0116] SEQ ID NO:11 is a sequence located within SEQ ID NO:7 and is the amplicon of primers SEQ ID NO:13 and SEQ ID NO:14;

[0117] SEQ ID NO:12 is a sequence located within SEQ ID NO:8 and is the amplicon of primers SEQ ID NO:15 and SEQ ID NO:16;

[0118] SEQ ID NO: 13 primer on the 5' flanking genomic sequence;

[0119] SEQ ID NO: 14: a primer located on the T-DNA that pairs with SEQ ID NO: 13;

[0120] SEQ ID NO: 15 primer on the 3' flanking genomic sequence;

[0121] SEQ ID NO: 16: a primer located on the T-DNA that pairs with SEQ ID NO: 15;

[0122] SEQ ID NO: 17 Primer 1 for PCR detection of mvip3Aa20;

[0123] SEQ ID NO: 18 Primer 2 for PCR detection of mvip3Aa20;

[0124] SEQ ID NO: 19 Primer 1 for PCR detection of Cry1Ab;

[0125] SEQ ID NO: 20 Primer 2 for PCR detection of Cry1Ab;

[0126] SEQ ID NO:21 primer 1 for PCR detection of pat;

[0127] SEQ ID NO:22 primer 2 for PCR detection of pat;

[0128] SEQ ID NO: 23 probe primer 1 for mvip3Aa20 in Southern hybridization detection;

[0129] SEQ ID NO: 24 probe primer 2 for mvip3Aa20 in Southern hybridization detection;

[0130] SEQ ID NO: 25 Probe primer 1 for Cry1Ab in Southern hybridization detection;

[0131] SEQ ID NO:26 Probe primer 2 for Cry1Ab in Southern hybridization detection;

[0132] SEQ ID NO: 27 probe primer 1 for pat in Southern hybridization detection;

[0133] SEQ ID NO: 28 probe primer 2 for pat in Southern hybridization detection;

[0134] SEQ ID NO:29 is the nucleic acid sequence of the promoter OsUbi2 promoter;

[0135] SEQ ID NO:30 is the nucleic acid sequence of the coding region of the mVip3Aa20 gene;

[0136] SEQ ID NO:31 is the nucleic acid sequence of chloroplast leader peptide CTP-TS-SSU;

[0137] SEQ ID NO:32 is the nucleic acid sequence of terminator T-Ara5;

[0138] SEQ ID NO:33 is the nucleic acid sequence of promoter P-FMV;

[0139] SEQ ID NO:34 is the nucleic acid sequence of the cry1Ab gene coding region;

[0140] SEQ ID NO:35 is the nucleic acid sequence of intron I-HSP70;

[0141] SEQ ID NO:36 is the nucleic acid sequence of chloroplast localization peptide CTP2;

[0142] SEQ ID NO:37 is the nucleic acid sequence of terminator T-Tr7;

[0143] SEQ ID NO:38 is the nucleic acid sequence of promoter P-CaMV35S;

[0144] SEQ ID NO:39 is the nucleic acid sequence of the coding region of the pat gene;

[0145] SEQ ID NO: 40 is the nucleic acid sequence of the termination sequence T-35S polyA. BRIEF DESCRIPTION OF THE DRAWINGS

[0146] Figure 1 is a schematic diagram of the pQY002569 vector.

[0147] FIG2 is a schematic diagram showing the structure of the binding site between the transgenic insertion sequence and the corn genome for detecting the nucleic acid sequence of the corn plant QY2569-42 and the detection method thereof.

[0148] Figure 3 shows the target gene amplification results of QY2569-42 T2-T4 generations. M: Marker; 1: QY2569-42 T2 generation individual plant; 2: QY2569-42 T3 generation individual plant; 3: QY2569-42 T4 generation individual plant; P: plasmid pQY002569; N: non-transgenic maize ZM11.

[0149] Figure 4 shows the analysis of restriction enzyme cutting sites in the inserted sequence.

[0150] Figure 5 shows the results of plant-specific PCR amplification of QY2569-42 T2-T4 generations. M: Marker; 1: QY2569-42 T2 generation individual plant; 2: QY2569-42 T3 generation individual plant; 3: QY2569-42 T4 generation individual plant; P: plasmid pQY002569; N: non-transgenic maize ZM11.

[0151] Fig. 6 Schematic diagram of the chromosome location of the QY2569-42 insertion sequence.

[0152] Figure 7: Field effects of transgenic maize event QY2569-42 inoculated with Spodoptera frugiperda at the heart leaf and ear stages; CK is ZM11.

[0153] Figure 8 shows the field effects of transgenic maize event QY2569-42 inoculated with Asian corn borer at the heart leaf stage and ear stage; CK is ZM11.

[0154] Figure 9: Field effects of transgenic maize event QY2569-42 inoculated with Oriental armyworm at the heartleaf stage; CK is ZM11.

[0155] Figure 10: Field effects of transgenic maize event QY2569-42 inoculated with cotton bollworm at the ear stage; CK is ZM11. DETAILED DESCRIPTION

[0156] The following examples are presented so as to provide those skilled in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent or imply that the experiments below are all or the only experiments performed. It should be understood by those skilled in the art that many variations and / or modifications may be made to the invention shown in the specific aspects without departing from the spirit or scope of the invention as broadly described. Therefore, the aspects herein are to be considered in all respects as illustrative and not restrictive.

[0157] Example 1: Construction of transgenic vector pQY002569

[0158] The pQY002569 plasmid is 17,632 bp in size and was artificially constructed based on pCAMBIA1300. pCAMBIA1300 is a commonly used plant expression vector. It lacks a gus reporter gene in its backbone and instead contains an E. coli origin of replication and a pUC18 backbone. It also contains the T-DNA left border (LB), right border (RB), a kanamycin resistance gene (KmR), a pBR322 origin of replication, a pVS1 replicon (pVS1 rep), and a pVS1 staA region. The hygromycin resistance gene on the pCAMBIA1300 plasmid was removed by enzyme digestion, and then three artificially synthesized target gene expression cassettes were introduced by the Golden Gate method: OsUbi2 promoter::CTP-TS-SSU::mvip3Aa20::T-Ara5; P-FMV::I-HSP70::CTP2::cry1Ab::T-Tr7; P-CaMV35S::pat::T-35S polyA to form the final vector pQY002569 (as shown in Figure 1).

[0159] Example 2: Transformation of pQY002569 vector and screening of transgenic plants

[0160] QY2569 insect-resistant and herbicide-tolerant corn was created through Agrobacterium-mediated immature embryo transformation, introducing the insect-resistant genes mvip3Aa20 and cry1Ab, and the herbicide-tolerant gene pat, into the non-transgenic corn line ZM11. The ZM11 inbred line was hybridized with Qi 319 (Q319) via HiII, then backcrossed with Q319 for two generations. Progeny susceptible to genetic transformation were selected and then selfed, resulting in the line retaining the disease-resistant properties of Q319.

[0161] Transgenic maize plants were tested for the presence of mvip3Aa20, cry1Ab, and the herbicide-tolerant gene pat by PCR and Southern hybridization analysis, and transgene copy number was characterized for insect-resistant and glufosinate-tolerant lines. Through screening, maize event QY2569-42 was selected as superior, demonstrating a single transgene copy, good insect resistance, glufosinate-tolerant herbicide performance, and agronomic performance.

[0162] Example 3: Genetic Elements and Specific Detection Methods of Transgenic Maize Event QY2569-42

[0163] The transgenic maize event QY2569-42 insert sequence is from the T-DNA region of the pQY002569 vector and is 11,283 bp (SEQ ID NO: 9). The insert sequence includes a T-DNA fragment with a missing portion of RB and a missing portion of LB. Its structure is shown in Figure 2, and the genetic elements contained in the insert sequence are shown in Table 1.

[0164] Table 1. Genetic elements in the QY2569-42 insertion sequence

[0165] Based on the insertion fragment and the genomic sequences on both sides of the insertion fragment, relevant primers were designed and the insertion element detection method and specific detection method of the transgenic maize event QY2569-42 were developed.

[0166] 1. PCR detection of exogenous genes

[0167] DNA was extracted and purified according to the NY / T674 agricultural industry standard of the People's Republic of China. Genomic DNA from leaves of transgenic maize QY2569-42 T2 to T4 generations was used as a template to amplify the exogenous genes mvip3Aa20, cry1Ab, and pat to determine if the exogenous genes had integrated into the maize genome. Amplification primers are shown in Table 2:

[0168] Table 2. PCR primer information of exogenous genes in transgenic maize event QY2569-42

[0169] Amplification results are shown in Figure 3. Using genomic DNA from QY2569-42 T2 to T4 plants and ZM11 (negative control) as templates, the expected fragments of the exogenous genes mvip3Aa20, cry1Ab, and pat were amplified from all QY2569-42 T2-T4 generations, with sizes of 585 bp, 565 bp, and 794 bp, respectively. No exogenous gene bands were amplified from the negative control; however, the exogenous gene bands were amplified from the positive control (transformation vector).

[0170] 2. Southern Detection of Exogenous Genes

[0171] By analyzing the sequence of the inserted element in the vector for restriction sites, we determined the restriction sites selected for Southern analysis. Based on the restriction sites and probe positions (Figure 4), we accurately predicted the sizes of the hybridizing fragments. The expected and actual hybridizing fragment sizes were perfectly consistent.

[0172] Using the pQY002569 plasmid as a template, specific primers for the mvip3Aa20, cry1Ab, and pat genes were used, respectively, using the Roche DIG probe labeling kit. Digoxigenin-labeled specific probes were prepared according to the instructions and hybridized with enzyme-digested maize genomic DNA for detection. The primer sequences are as follows:

[0173] Table 3. Southern probe PCR primer information

[0174] Southern hybridization results showed that the mvip3Aa20, cry1Ab and pat gene probes all obtained hybridization results consistent with expectations, and there was only one copy of the insertion in the maize genome, with no other unexpected fragments inserted.

[0175] 3. Specific detection of transgenic corn event QY2569-42

[0176] Genome Walking was performed on transgenic maize event QY2569-42, resulting in a 720-bp 5' flanking sequence located within SEQ ID NO: 7 and a 2757-bp 3' flanking sequence located within SEQ ID NO: 8. Amplicons were generated using at least one primer from SEQ ID NO: 7 or SEQ ID NO: 8, which were used to generate diagnostic amplicons for transgenic maize event QY2569-42 using PCR (Table 4).

[0177] Table 4. Specific PCR primer information for flanking sequences of transgenic maize QY2569-42

[0178] Specifically, a PCR product was generated from the 5' end of the transgene insert sequence. A primer (SEQ ID NO: 13) was designed to hybridize to the genomic DNA sequence flanking the 5' end of the transgene insert sequence, and a primer (SEQ ID NO: 14) was paired with the primer located at the transcription start sequence of the transgene OsUbi2. The PCR product is located within SEQ ID NO: 7, and the product sequence is shown in SEQ ID NO: 11.

[0179] Simultaneously, a PCR product was generated from the 3' end of the transgene insert sequence. A primer (SEQ ID NO: 15) was designed to hybridize to the genomic DNA sequence flanking the 3' end of the transgene insert sequence, and a primer (SEQ ID NO: 16) was designed to pair with the primer located on the T-DNA. The PCR product is located within SEQ ID NO: 8, and the product sequence is shown in SEQ ID NO: 12.

[0180] Junction sequences are relatively short polynucleotide molecules that are novel DNA sequences that, when detected in a polynucleic acid detection assay, are diagnostic for the DNA of transgenic maize event QY2569-42. The 5' junction sequences are set forth in SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 5, and the 3' junction sequences are set forth in SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 6. Longer or shorter polynucleotide junction sequences can be selected from SEQ ID NO: 7 or SEQ ID NO: 8. The junction sequences (5' junction region SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 5, and 3' junction region SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 6) are useful as DNA probes or as DNA primer molecules in DNA detection methods.

[0181] The results in Figure 5 show that using genomic DNA from T2 to T4 generations of transgenic maize event QY2569-42 as a template, specific PCR fragments of 1068 bp and 798 bp were amplified for the 5' and 3' flanking regions of the exogenous insert in all QY2569-42 T2-T4 generations. No bands were amplified from the transformed recipient or vector.

[0182] The DNA amplification conditions shown in the PCR detection results of the exogenous gene can be used in the above-mentioned PCR zygosity test to generate a diagnostic amplicon for transgenic maize event QY2569-42.

[0183] 4. Localization of the QY2569-42 insertion sequence in plant cells

[0184] Southern hybridization, flanking sequence analysis, and transformant-specific PCR confirmed that the insert sequence of transgenic maize QY2569-42 was integrated into the maize chromosome. The T-DNA insertion occurred as a single copy at a single locus. Using the sequenced maize variety Mo17-CAU as the reference genome (https: / / download.maizegdb.org / Zm-Mo17-REFERENCE-CAU-1.0 / Zm-Mo17-REFERENCE-CAU-1.0.fa.gz), the T-DNA insertion site of QY2569-42 was located at chromosome 3, chr3:186,051,461-186,051,875. During T-DNA integration, a random 2-bp insertion occurred between the LB end and the genome, along with a 415-bp duplication of the 5' flanking sequence. A schematic diagram of the insert sequence's location within the genome is shown in Figure 6.

[0185] Example 4: Detection of mVip3Aa20, Cry1Ab, and PAT Protein Expression in Transgenic Maize Event QY2569-42

[0186] ELISA (enzyme-linked immunosorbent assay) was used to analyze the expression of the inserted sequence in various organs and tissues. Protein expression of the inserted sequence was measured in young leaves and seeds of QY2569-42 maize. Following conventional planting procedures, sampling was performed at the following locations and stages: V2 (leaves, roots), V6 (leaves, stalks, roots), R1 (leaves, stalks, roots, pollen, tassels, filaments, ears), and R6 (leaves, stalks, roots, kernels). Using the same non-transgenic maize strain, ZM11, as a control, the expression levels of mVip3Aa20, Cry1Ab, and PAT proteins were determined in various tissues and organs of QY2569-42 from T2 to T4 generations.

[0187] ELISA results from T2 to T4 generations of QY2569-42 showed that mVip3Aa20, Cry1Ab, and PAT proteins were detectable in all tissue samples of transgenic maize. The content of each target protein per gram of tissue varied. The content of mVip3Aa20 protein in T4 generation was the highest in R6 leaves (481.62±88.35μg / g fwt) and the lowest in V2 and V6 roots (36.74±4.36 and 36.19±6.35μg / g fwt, respectively). The content of Cry1Ab protein was the highest in R6 leaves (8.01±2.55μg / g fwt) and the lowest in pollen (0.03±0.01μg / g fwt). The content of PAT protein was the highest in R6 leaves (6.17±1.21μg / g The target protein was detected in fwt plants, with the lowest levels in pollen and grains, at 0.63±0.02 and 0.48±0.08 μg / g fwt, respectively. In contrast, no target protein was detected in any tissue of non-transgenic plants at different stages. These results indicate that exogenous protein expression in QY2569-42 is stable.

[0188] Example 5: Insect Resistance Detection of Transgenic Corn Event QY2569-42

[0189] 1. Indoor bioassay of corn plant QY2569-42

[0190] Test materials were grown in a greenhouse, with 3 to 30 plants per material planted, depending on seed quantity. Conventional cultivation and management were followed, with no pesticides applied throughout the entire growth period. Young and tender parts of plants were collected at the 6- to 8-leaf stage, silking stage, and grain filling stage, and inoculated with the Asian corn borer, fall armyworm, and cotton bollworm, respectively. Young and tender parts of oriental armyworms were collected at the 4- to 6-leaf stage to be fed, and resistance levels were evaluated.

[0191] When corn reached the appropriate growth stage, heart leaves, pistils, and ears were collected from QY2569-42 corn fields and corresponding non-transgenic corn fields for in vitro tissue resistance testing. Twenty samples were randomly collected from each plot for each treatment. An appropriate amount of leaves were cut and placed in plastic boxes. Ten second-instar larvae were then inoculated, with each box forming one replicate, and each treatment was replicated 10 times. Test materials from the same batch were added based on the larvae's feeding behavior, and the number of surviving larvae was counted and recorded. The experimental period lasted five days. Indoor bioassays of QY2569-42 against Asian corn borer, Oriental armyworm, fall armyworm, and cotton bollworm over four generations showed stable resistance to Asian corn borer and fall armyworm in leaves, silks, and seeds of the T3 and T4 generations of QY2569-42 plants. With the exception of a small number of T4 leaves (less than 3%), all other survival rates were zero. Resistance to Oriental armyworm in leaves was stable, with zero survival in the T3 generation and less than 3% in the T4 generation. Resistance to cotton bollworm in silks and seeds was stable, with zero survival in both the T3 and T4 generations. Survival rates of various target insects in non-transgenic and susceptible controls were all above 70%, indicating that these plant tissues maintained high levels of resistance, significantly different from those of the non-transgenic and susceptible controls.

[0192] 2. Field resistance efficacy of QY2569-42 against target pests

[0193] Referring to relevant standards such as Ministry of Agriculture Announcement No. 953-10.1-2007 and NY / T 1248.5, the Duncan's new multiple range method (DPS) analysis software was used to analyze and compare the resistance of transgenic maize QY2569-42 to the major lepidopteran pests, including Asian corn borer, oriental armyworm, fall armyworm, and cotton bollworm, to determine the resistance level of transgenic maize QY2569-42 to these pests.

[0194] Pilot trials in Shandong and Hainan provinces tested the resistance of QY2569-42 T2-T4 generations to these target pests through artificial inoculation in the field. The results showed that QY2569-42 exhibited high resistance to these three pests. In contrast, the recipient corn ZM11 and the susceptible control were highly susceptible or susceptible. Plant height was suppressed by the Asian corn borer, a significant difference from QY2569-42. Specific field test results are shown in Figures 7-10.

[0195] Example 6: Tolerance of transgenic corn event QY2569-42 to target herbicides

[0196] The tolerance of the transgenic maize event QY2569-42 to target herbicides was assessed in accordance with "Environmental Safety Testing of Transgenic Plants and Their Products - Herbicide-Resistant Maize Part 1: Herbicide Tolerance" (Ministry of Agriculture Announcement No. 953, November 1, 2007). The target herbicide for the PAT protein expressed by the QY2569-42 transformants was glufosinate. Glufosinate was sprayed on the stems and leaves of maize plants at the 4-5 leaf stage. Plant height and damage were measured and recorded 7, 14, and 28 days after application. The results showed no significant difference in seedling emergence between the transgenic maize event QY2569-42 and its non-transgenic counterpart. The QY2569-42 transformants were tolerant to a 4-fold dose of glufosinate. Seven, 14, and 28 days after spraying with a 4-fold dose of glufosinate, the seedling emergence rate, damage level, and plant height showed no significant differences compared to the unsprayed control or to those sprayed with 1-fold or 2-fold doses of glufosinate. The non-transgenic receptor ZM11 had poor tolerance to glufosinate ammonium and all died 7 days after application of 1 times the dose of glufosinate ammonium herbicide.

[0197] Example 7. Observation and Analysis of the Main Agronomic Traits of Transgenic Event QY2569-42

[0198] The transgenic events QY2569-42 and ZM11 (control) were investigated for their growth period and agronomic traits, including plant height, ear height, anther color, filament color, plant type, kernel color, and rachis color. The results showed that the agronomic traits of the transgenic maize and recipient varieties were essentially identical, with no significant changes observed.

[0199] In summary, the transgenic corn event QY2569-42 is genetically stable and has good resistance to lepidopteran pests such as the Asian corn borer, oriental armyworm, fall armyworm and cotton bollworm. It also has a high tolerance to herbicides such as glufosinate, has no effect on yield, and the detection method can accurately and quickly identify whether biological samples contain DNA molecules of the transgenic corn event QY2569-42.

[0200] Seeds corresponding to transgenic maize event QY2569-42 were deposited with the China Center for Type Culture Collection (CCTCC) on August 22, 2023. Address: 299 Bayi Road, Wuchang District, Wuhan University, opposite the First Affiliated Primary School, 430072, Hubei Province, China. The seeds are designated Zea mays L. QY2569-42 and assigned the CCTCC accession number P202336. The deposit will remain at the depository for 30 years.

[0201] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not limiting. 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 solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. A nucleic acid sequence, characterized in that The nucleic acid sequence comprises: (a) SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11 and / or SEQ ID NO:12; or (b) a nucleic acid sequence complementary to (a); The nucleic acid sequence is derived from the transgenic maize event QY2569-42, which is deposited in the China Center for Type Culture Collection in the form of seeds with a deposit number of CCTCC NO: P202336; Preferably, the nucleic acid sequence is an amplicon diagnostic for the presence of corn event QY2569-42.

2. A DNA construct comprising three expression cassettes, wherein: a) the first expression cassette comprises in operable connection a promoter OsUbi2 promoter as shown in SEQ ID NO:29, a mVip3Aa20 gene coding region as shown in SEQ ID NO:30, a chloroplast leader peptide CTP-TS-SSU as shown in SEQ ID NO:31, and a terminator T-Ara5 for terminating the expression of the gene as shown in SEQ ID NO:32; b) the second expression cassette comprises in operable connection a promoter P-FMV as shown in SEQ ID NO:33, a cry1Ab gene coding region as shown in SEQ ID NO:34, an intron I-HSP70 as shown in SEQ ID NO:35, a chloroplast localization peptide CTP2 as shown in SEQ ID NO:36, and a terminator T-Tr7 as shown in SEQ ID NO:37 for terminating the expression of the gene; c) The third expression cassette comprises in operable connection a promoter P-CaMV35S as shown in SEQ ID NO:38, a pat gene coding region as shown in SEQ ID NO:39, and a termination sequence T-35S polyA for terminating the expression of the gene as shown in SEQ ID NO:

40.

3. A DNA molecule having a sufficient length of contiguous nucleotides of SEQ ID NO: 10 to be used as a DNA probe specific for SEQ ID NO: 10 in a DNA sample derived from a corn plant, corn seed or corn cell, wherein the DNA probe comprises SEQ ID NO: 1 or SEQ ID NO: 2; Preferably, the probe is SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:11 or SEQ ID NO:

12.

4. A method for detecting the presence of DNA of transgenic maize event QY2569-42 in a sample, characterized in that: include: contacting the sample to be tested with at least two primers in a nucleic acid amplification reaction; performing nucleic acid amplification reactions; detecting the presence of the amplification product; The amplification product comprises (a) SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and / or SEQ ID NO: 10; or (b) a nucleic acid sequence complementary to (a); The nucleic acid sequence is derived from the transgenic maize event QY2569-42, which is deposited in the China Center for Type Culture Collection in the form of seeds with a deposit number of CCTCC NO: P202336; Preferably, the amplification product further comprises SEQ ID NO: 11 or its complementary sequence and / or SEQ ID NO: 12 or its complementary sequence; More preferably, the two primers include SEQ ID NO: 13 and SEQ ID NO: 14, or SEQ ID NO: 15 and SEQ ID NO:

16.

5. A method for detecting the presence of DNA of transgenic maize event QY2569-42 in a sample, characterized in that: include: Contacting the sample to be detected with a probe, wherein the probe comprises SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8 or a complementary sequence thereof; Hybridizing the sample to be detected and the probe under stringent hybridization conditions; Detecting the hybridization between the sample to be detected and the probe; The transgenic maize event QY2569-42 is deposited in the China Center for Type Culture Collection in the form of seeds, with the deposit number CCTCC NO: P202336; Preferably, the probe further comprises SEQ ID NO: 11 or its complementary sequence, or SEQ ID NO: 12 or its complementary sequence; More preferably, at least one of said probes is labeled with at least one fluorescent group.

6. A method for detecting the presence of DNA of transgenic maize event QY2569-42 in a sample, characterized in that: include: Contacting the sample to be detected with a marker nucleic acid molecule, wherein the marker nucleic acid molecule includes SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8 or a complementary sequence thereof; Hybridizing the sample to be detected and the marker nucleic acid molecule under stringent hybridization conditions; Detecting the hybridization between the sample to be tested and the marker nucleic acid molecule, and then determining through marker-assisted breeding analysis that insect resistance and / or herbicide tolerance are genetically linked to the marker nucleic acid molecule; The transgenic maize event QY2569-42 is deposited in the China Center for Type Culture Collection in the form of seeds, with the deposit number CCTCC NO: P202336; Preferably, the marker nucleic acid molecule further comprises SEQ ID NO: 11 or its complementary sequence, or SEQ ID NO: 12 or its complementary sequence.

7. A DNA detection kit, characterized in that: comprising at least one DNA molecule comprising SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8 or a complementary sequence thereof, which can be used as one of the DNA primers or probe specific for transgenic maize event QY2569-42 or its progeny; The transgenic maize event QY2569-42 is deposited in the China Center for Type Culture Collection in the form of seeds, with the deposit number CCTCC NO: P202336; Preferably, the DNA molecule used as a probe further comprises SEQ ID NO: 11 or its complementary sequence, or SEQ ID NO: 12 or its complementary sequence.

8. 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 a target insect, wherein the transgenic corn plant cell comprises in its genome SEQ ID NO:1, the nucleic acid sequence at positions 777-11945 of SEQ ID NO:10 and SEQ ID NO:2, or SEQ ID NO:3, the nucleic acid sequence at positions 777-11945 of SEQ ID NO:10 and SEQ ID NO:4, or SEQ ID NO:5, the nucleic acid sequence at positions 777-11945 of SEQ ID NO:10 and SEQ ID NO:6, or the genome of the transgenic corn plant cell comprises SEQ ID NO:10; and target insects that ingest the transgenic corn plant cell are inhibited from further ingesting the corn plant.

9. 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 comprises in its genome SEQ ID NO:1, a nucleic acid sequence at positions 777 to 11945 of SEQ ID NO:10 and SEQ ID NO:2, or SEQ ID NO:3, a nucleic acid sequence at positions 777 to 11945 of SEQ ID NO:10 and SEQ ID NO:4, or SEQ ID NO:5, a nucleic acid sequence at positions 777 to 11945 of SEQ ID NO:10 and SEQ ID NO:6, or the genome of the transgenic corn plant comprises SEQ ID NO:10; the transgenic corn plant has tolerance to the glufosinate herbicide.

10. A method for controlling weeds in a field growing corn plants, 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 comprises in its genome SEQ ID NO:1, a nucleic acid sequence at positions 777 to 11945 of SEQ ID NO:10 and SEQ ID NO:2, or SEQ ID NO:3, a nucleic acid sequence at positions 777 to 11945 of SEQ ID NO:10 and SEQ ID NO:4, or SEQ ID NO:5, a nucleic acid sequence at positions 777 to 11945 of SEQ ID NO:10 and SEQ ID NO:6, or the genome of the transgenic corn plant comprises SEQ ID NO:10; the transgenic corn plant has tolerance to the glufosinate herbicide.

11. A method for growing corn plants resistant to insects, characterized in that: include: Planting at least one corn seed, wherein the genome of the corn seed comprises a nucleic acid sequence of a specific region, wherein the nucleic acid sequence of the specific region sequentially comprises SEQ ID NO:1, the nucleic acid sequence of positions 777-11945 of SEQ ID NO:10, and SEQ ID NO:2, or SEQ ID NO:3, the nucleic acid sequence of positions 777-11945 of SEQ ID NO:10, and SEQ ID NO:4, or SEQ ID NO:5, the nucleic acid sequence of positions 777-11945 of SEQ ID NO:10, and SEQ ID NO:6, or the nucleic acid sequence of the specific region comprises SEQ ID NO:10; allowing the corn seed to grow into a corn plant; The corn plants are infested with target insects and the plants are harvested having reduced plant injury compared to other plants not having the nucleic acid sequence of the specific region.

12. A method for growing corn plants tolerant to glufosinate-ammonium herbicides, characterized in that: include: Planting at least one corn seed, wherein the genome of the corn seed comprises a nucleic acid sequence of a specific region, wherein the nucleic acid sequence of the specific region comprises, in sequence, SEQ ID NO:1, the nucleic acid sequence of positions 777 to 11945 of SEQ ID NO:10, and SEQ ID NO:2, or SEQ ID NO:3, the nucleic acid sequence of positions 777 to 11945 of SEQ ID NO:10, and SEQ ID NO:4, or SEQ ID NO:5, the nucleic acid sequence of positions 777 to 11945 of SEQ ID NO:10, and SEQ ID NO:6, or the nucleic acid sequence of the specific region comprises SEQ ID NO:10; allowing the corn seeds to grow into corn plants; The corn plants are sprayed with an effective amount of glufosinate herbicide, and the plants having reduced plant damage compared to other plants not having the nucleic acid sequence of the specific region are harvested.

13. A method of growing corn plants that are resistant to insects and tolerate glufosinate herbicides, characterized in that: include: Planting at least one corn seed, wherein the genome of the corn seed comprises a nucleic acid sequence of a specific region, wherein the nucleic acid sequence of the specific region comprises, in sequence, SEQ ID NO:1, the nucleic acid sequence of positions 777 to 11945 of SEQ ID NO:10, and SEQ ID NO:2, or SEQ ID NO:3, the nucleic acid sequence of positions 777 to 11945 of SEQ ID NO:10, and SEQ ID NO:4, or SEQ ID NO:5, the nucleic acid sequence of positions 777 to 11945 of SEQ ID NO:10, and SEQ ID NO:6, or the nucleic acid sequence of the specific region comprises SEQ ID NO:10; allowing the corn seeds to grow into corn plants; The corn plants are sprayed with an effective dose of glufosinate-ammonium herbicide, and the corn plants and other plants not having the nucleic acid sequence of the specific region are harvested. The plants in the series have reduced plant damage compared to the plants having reduced plant damage, and the plants having reduced plant damage are also resistant to insect feeding damage.

14. A method for producing corn plants resistant to insects, characterized in that: The method comprises hybridizing a corn plant whose genome sequentially comprises the nucleic acid sequence of SEQ ID NO:1, SEQ ID NO:10, positions 777 to 11945 and SEQ ID NO:2, or SEQ ID NO:3, SEQ ID NO:10, positions 777 to 11945 and SEQ ID NO:4, or SEQ ID NO:5, SEQ ID NO:10, positions 777 to 11945 and SEQ ID NO:6 with another corn plant, thereby producing a large number of progeny plants; and selecting a progeny plant whose genome comprises a nucleic acid sequence of a specific region, wherein the nucleic acid sequence of the specific region sequentially comprises the nucleic acid sequence of SEQ ID NO:1, SEQ ID NO:10, positions 777 to 11945 and SEQ ID NO:2, or SEQ ID NO:3, SEQ ID NO:10, positions 777 to 11945 and SEQ ID NO:4, or SEQ ID NO:5, SEQ ID NO:10, positions 777 to 11945 and SEQ ID NO:

6. NO:6, or the nucleic acid sequence of the specific region comprises SEQ ID NO:10, and the progeny plants have reduced plant damage to insect feeding; Preferably, the method comprises: sexually crossing a first parent corn plant of transgenic corn event QY2569-42 that is resistant to insects with a second parent corn plant that lacks insect resistance, thereby producing a plurality of progeny plants; infesting the progeny plants with target insects; Selecting the progeny plants having reduced plant injury compared to other plants not having the nucleic acid sequence of the specific region; The transgenic corn event QY2569-42 is deposited in the China Center for Type Culture Collection in the form of seeds, with the deposit number CCTCC NO: P202336.

15. A method for producing corn plants tolerant to glufosinate herbicide, characterized in that: The method comprises hybridizing a corn plant whose genome sequentially comprises the nucleic acid sequence of SEQ ID NO:1, SEQ ID NO:10, positions 777 to 11945 and SEQ ID NO:2, or SEQ ID NO:3, SEQ ID NO:10, positions 777 to 11945 and SEQ ID NO:4, or SEQ ID NO:5, SEQ ID NO:10, positions 777 to 11945 and SEQ ID NO:6 with another corn plant, thereby producing a large number of progeny plants; and selecting a progeny plant whose genome comprises a nucleic acid sequence of a specific region, wherein the nucleic acid sequence of the specific region sequentially comprises the nucleic acid sequence of SEQ ID NO:1, SEQ ID NO:10, positions 777 to 11945 and SEQ ID NO:2, or SEQ ID NO:3, SEQ ID NO:10, positions 777 to 11945 and SEQ ID NO:4, or SEQ ID NO:5, SEQ ID NO:10, positions 777 to 11945 and SEQ ID NO:

6. NO:6, or the nucleic acid sequence of the specific region comprises SEQ ID NO:10, and the progeny plant has glufosinate-ammonium tolerance; Preferably, the method comprises: sexually crossing a first parent corn plant of a transgenic corn event QY2569-42 that is tolerant to glufosinate herbicide with a second parent corn plant that lacks glufosinate tolerance, thereby producing a plurality of progeny plants; treating the progeny plants with a glufosinate-ammonium herbicide; Selecting the progeny plants that are tolerant to glufosinate-ammonium; The transgenic corn event QY2569-42 is deposited in the China Center for Type Culture Collection in the form of seeds, with the deposit number CCTCC NO: P202336.

16. A method of producing corn plants that are resistant to insects and tolerate the application of glufosinate herbicides, characterized in that: include: sexually crossing a first parent corn plant of a transgenic corn event QY2569-42 that is glufosinate-tolerant and insect-resistant with a second parent corn plant that lacks glufosinate-tolerant and / or insect-resistant, thereby producing a plurality of progeny plants; Treating the progeny plants with glufosinate-ammonium; Selecting the progeny plants that are tolerant to glufosinate ammonium, wherein the progeny plants that are tolerant to glufosinate ammonium are also resistant to insect feeding damage; The transgenic corn event QY2569-42 is deposited in the China Center for Type Culture Collection in the form of seeds, with the deposit number CCTCC NO: P202336.

17. A method for improving tolerance in corn plants, the method comprising: a) constructing a DNA construct as claimed in claim 3; b) inserting the DNA construct into the genome of a corn cell; c) regenerating the corn cell into a corn plant; and d) selecting a corn plant comprising said DNA construct; Preferably, improving the tolerance of the corn plant comprises its resistance to an effective amount of at least one herbicide, preferably glufosinate-ammonium; and / or Improving corn plant tolerance includes resistance to at least one insect, preferably the Asian corn borer, the oriental armyworm, the fall armyworm, and / or the cotton bollworm.

18. A composition produced from transgenic corn event QY2569-42, characterized in that: The composition is corn flour, corn meal, corn oil, corn silk or corn starch; the transgenic corn event QY2569-42 is preserved in the China Center for Type Culture Collection in the form of seeds, with the preservation number CCTCC NO: P202336.

19. An agricultural product or commodity produced from transgenic corn event QY2569-42, characterized in that: The agricultural product or commodity is corn flour, corn meal, corn oil, corn starch, corn gluten, corn tortilla, cosmetics or fillers; the transgenic corn event QY2569-42 is deposited in the China Center for Type Culture Collection in the form of seeds, with the deposit number CCTCC NO: P202336.

20. A plant cell, plant part, plant, seed or non-living plant material comprising the nucleic acid sequence of claim 1.