WO2024188236A1

WO2024188236A1 - Nucleic acid sequence for detecting corn plant dbn9229 and detection method therefor

Info

Publication number: WO2024188236A1
Application number: PCT/CN2024/081178
Authority: WO
Inventors: 王利君; 刘海利; 张铭; 陶梦梦; 于彩虹; 王诚
Original assignee: 北京大北农生物技术有限公司
Priority date: 2023-03-13
Filing date: 2024-03-12
Publication date: 2024-09-19
Also published as: CN116732215A; AR132130A1

Abstract

The present invention relates to a nucleic acid sequence for detecting corn plant DBN9229 and a detection method therefor, wherein the nucleic acid sequence comprises SEQ ID NO: 1 or a complementary sequence thereof, or SEQ ID NO: 2 or a complementary sequence thereof. The corn plant DBN9229 has better resistance to Lepidopteran and better tolerance to glufosinate herbicides and has no influence on the yield, and the detection method can accurately and quickly identify whether a DNA molecule of the transgenic corn event DBN9229 is contained in a biological sample.

Description

Nucleic acid sequence for detecting corn plant DBN9229 and its detection method

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is based on the application with CN application number 202310235118.1 and application date March 13, 2023, and claims its priority. The contents of all CN applications are hereby introduced as a whole into this application.

Technical Field

The present invention relates to the field of plant molecular biology, in particular to the field of transgenic crop breeding in agricultural biotechnology research. Specifically, the present invention relates to a transgenic corn event DBN9229 with insect resistance and glufosinate herbicide tolerance, and a nucleic acid sequence and a detection method thereof for detecting whether a biological sample contains a specific transgenic corn event DBN9229.

Background Art

Maize (Zea mays L.) is a staple food crop in many parts of the world. Biotechnology has been applied to maize to improve its agronomic traits and quality. Insect resistance is an important agronomic trait in maize production, especially resistance to Lepidoptera insects, such as corn borer, cotton bollworm, oriental armyworm, cutworm, fall armyworm, etc. Maize resistance to Lepidoptera insects can be obtained by transgenic methods to express Lepidoptera insect resistance genes in maize plants. Another important agronomic trait is herbicide tolerance, such as the successful maize transformation events NK603 and GA21, which have been widely planted in major maize growing areas such as the United States. It is worth mentioning that glufosinate herbicide has a different mechanism of action from glyphosate herbicide. It is a contact herbicide that kills and can be used as an effective means of managing glyphosate-resistant weeds. Maize tolerance to glufosinate herbicide can be obtained by transgenic methods to express glufosinate herbicide tolerance genes (such as pat) in maize plants.

It is known that the expression of foreign genes in plants is affected by their chromosomal location, which may be due to chromatin structure (such as heterochromatin) or the proximity of transcriptional regulatory elements (such as enhancers) to the integration site. For this reason, it is usually necessary to screen a large number of events before it is possible to identify events that can be commercialized (i.e., events in which the introduced target gene is optimally expressed). For example, it has been observed in plants and other organisms that the expression level 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 is manifested in the fact that the actual expression pattern may be inconsistent with the expression pattern expected based on the transcriptional regulatory elements in the introduced gene construct. Therefore, it is usually necessary to generate hundreds or thousands of different events and screen them for a single event with the expected transgene expression level and expression pattern for commercialization purposes. Events with the expected transgene expression level and expression pattern can be used to introgress the transgene into other genetic backgrounds through sexual outcrossing using conventional breeding methods. Progeny generated by such crosses retain the transgene expression characteristics of the original transformant. Applying this strategy can ensure reliable gene expression in many varieties that are well adapted to local growing conditions.

It will be useful to be able to detect the presence of a specific event to determine whether the offspring of a sexual hybridization contains the target gene. In addition, the method for detecting a specific event will also help to comply with relevant regulations, such as the need to obtain formal approval and labeling of food derived from recombinant crops before being put on the market. It is possible to detect the presence of a transgenic by any well-known polynucleotide detection method, such as polymerase chain reaction (PCR) or DNA hybridization utilizing polynucleotide probes. 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, this method above can not be used to distinguish different events, particularly those events produced with the same DNA construct. Therefore, a pair of primers that often utilize the junction site of the inserted transgenic and flanking DNA to identify a transgenic specific event by PCR, specifically a first primer included in the inserted sequence and a second primer included in the inserted sequence.

Summary of the invention

The present application transforms exogenous DNA fragments into corn and obtains a transgenic corn event DBN9229 with a specific insertion position in the genome, which has good resistance to insects and good tolerance to glufosinate herbicides. Thus, the present application provides a nucleic acid sequence for detecting corn plant DBN9229 and a detection method thereof, and the detection method can accurately and quickly identify whether a biological sample contains a DNA molecule of the transgenic corn event DBN9229.

Nucleic acid molecules

To achieve the above object, in the first aspect, the present invention provides a nucleic acid molecule or a combination thereof, wherein the nucleic acid sequence thereof comprises:

At least 11 consecutive nucleotides from positions 1 to 487 of SEQ ID NO: 3 or its complementary sequence, and/or at least 11 consecutive nucleotides from positions 488 to 1105 of SEQ ID NO: 3 or its complementary sequence;

At least 11 consecutive nucleotides from positions 1 to 397 of SEQ ID NO: 4 or its complementary sequence, and/or at least 11 consecutive nucleotides from positions 398 to 860 of SEQ ID NO: 4 or its complementary sequence; and/or

At least 11 consecutive nucleotides from positions 1 to 487 of SEQ ID NO:3 or its complementary sequence, and/or at least 11 consecutive nucleotides from positions 398 to 860 of SEQ ID NO:4 or its complementary sequence.

In certain embodiments, the consecutive nucleotides are 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more consecutive nucleotides.

In certain embodiments, the complementary sequence is partially complementary or fully complementary to its parent sequence.

In certain embodiments, the nucleic acid sequence comprises 22-25 consecutive nucleotides from positions 1-487 of SEQ ID NO:3 or its complementary sequence, and 22-25 consecutive nucleotides from positions 488-1105 of SEQ ID NO:3 or its complementary sequence; and/or 22-25 consecutive nucleotides from positions 1-397 of SEQ ID NO:4 or its complementary sequence, and 22-25 consecutive nucleotides from positions 398-860 of SEQ ID NO:4 or its complementary sequence.

In some embodiments, the nucleic acid sequence comprises SEQ ID NO:1 or its complementary sequence; and/or SEQ ID NO:2 or its complementary sequence.

After the target nucleic acid fragment is inserted into the corn genome, two junction points will be generated at the 5' end and 3' end of the target nucleic acid fragment and the corn genome respectively. In this article:

SEQ ID NO:3 is a nucleic acid fragment containing the 5' junction point generated by inserting the DBN11815 construct into the corn genome. Among them, nucleotides 1-487 of SEQ ID NO:3 are the sequence of the corn genome located upstream of the 5' junction point, and nucleotides 488-1105 of SEQ ID NO:3 are the DNA sequence of the DBN11815 construct.

SEQ ID NO:1 is a nucleic acid fragment containing the 5' junction point generated by inserting the DBN11815 construct into the corn genome. Among them, nucleotides 1-11 of SEQ ID NO:1 are the sequence of the corn genome located upstream of the 5' junction point, and nucleotides 12-22 of SEQ ID NO:1 are the DNA sequence of the DBN11815 construct.

SEQ ID NO:4 is a nucleic acid fragment containing a 3' junction point generated by inserting the DBN11815 construct into the corn genome. Among them, nucleotides 1-397 of SEQ ID NO:4 are the DNA sequence of the DBN11815 construct, and nucleotides 398-860 of SEQ ID NO:4 are the sequence of the corn genome located downstream of the 3' junction point.

SEQ ID NO:2 is a nucleic acid fragment containing the 3' junction point generated by the DBN11815 construct inserted into the corn genome. Among them, the nucleotides 1-11 of SEQ ID NO:2 are the DNA sequence of the DBN11815 construct, and the nucleotides 12-22 of SEQ ID NO:2 are the sequence of the corn genome located downstream of the 3' junction point.

When the nucleic acid molecule comprises a sequence, the sequence can be a DNA template, primer or probe for detecting the presence of transgenic event DBN9229.

When the combination of nucleic acid molecules comprises two or more sequences, the sequences can be a primer pair for detecting the presence of transgenic event DBN9229.

The SEQ ID NO:1 or its complementary sequence is a 22-nucleotide sequence located near the insertion junction at the 5' end of the insertion sequence in the transgenic corn event DBN9229. The SEQ ID NO:1 or its complementary sequence spans the flanking genomic DNA sequence of the corn insertion site and the DNA sequence at the 5' end of the insertion sequence. The presence of the transgenic corn event DBN9229 can be identified by including the SEQ ID NO:1 or its complementary sequence. The SEQ ID NO:2 or its complementary sequence is a 22-nucleotide sequence located near the insertion junction at the 3' end of the insertion sequence in the transgenic corn event DBN9229. The SEQ ID NO:2 or its complementary sequence spans the DNA sequence at the 3' end of the insertion sequence and the flanking genomic DNA sequence of the corn insertion site. The SEQ ID NO:2 or its complementary sequence spans the DNA sequence at the 3' end of the insertion sequence and the flanking genomic DNA sequence of the corn insertion site. The presence of SEQ ID NO: 2 or its complementary sequence can be identified as the presence of transgenic corn event DBN9229.

In certain embodiments, the nucleic acid sequence comprises SEQ ID NO:3 or its complementary sequence, and/or SEQ ID NO:4 or its complementary sequence.

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 T-DNA insertion sequence in the SEQ ID NO:3 or its complementary sequence, or at least 11 or more continuous polynucleotides (second nucleic acid sequence) of any part of the 5' flanking corn genomic DNA region in the SEQ ID NO:3 or its complementary sequence. The nucleic acid sequence may further be homologous to or complementary to a part of the SEQ ID NO:3 that includes the complete SEQ ID NO:1. When the first nucleic acid sequence and the second nucleic acid sequence are used together, these nucleic acid sequences can be used as 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, the presence of the transgenic corn event DBN9229 or its progeny can be diagnosed. The SEQ ID NO:3 or its complementary sequence is a sequence with a length of 1105 nucleotides located near the insertion junction site at the 5' end of the T-DNA insertion sequence in the transgenic corn event DBN9229. The SEQ ID NO:3 or its complementary sequence consists of 487 nucleotides of the 5' flanking sequence of the corn genome (nucleotides 1-487 of SEQ ID NO:3), 197 nucleotides of the DBN11815 construct DNA sequence (nucleotides 488-684 of SEQ ID NO:3), 195 nucleotides of the t35S cauliflower mosaic virus transcription terminator DNA sequence (nucleotides 685-879 of SEQ ID NO:3) and 226 nucleotides of the nucleotide sequence encoding the glufosinate herbicide tolerance agent PAT protein (nucleotides 880-1105 of SEQ ID NO:3). The presence of the transgenic corn event DBN9229 can be identified by the presence of SEQ ID NO:3 or its complementary sequence.

The nucleic acid sequence may be at least 11 or more continuous polynucleotides (third nucleic acid sequence) of any part of the T-DNA insertion sequence in the SEQ ID NO:4 or its complementary sequence, or at least 11 or more continuous polynucleotides (fourth nucleic acid sequence) of any part of the 3' flanking corn genomic DNA region in the SEQ ID NO:4 or its complementary sequence. The nucleic acid sequence may further be homologous to or complementary to a portion of the SEQ ID NO:4 that includes the complete SEQ ID NO:2. When the third nucleic acid sequence and the fourth nucleic acid sequence are used together, these nucleic acid sequences can be used as DNA primer pairs in a DNA amplification method for producing an amplified product. When the amplified product produced in the DNA amplification method using the DNA primer pairs is an amplified product including SEQ ID NO:2, the presence of the transgenic corn event DBN9229 or its progeny can be diagnosed. The SEQ ID NO:4 or its complementary sequence is a sequence of 860 nucleotides located near the T-DNA insertion junction at the 3' end of the insertion sequence in the transgenic corn event DBN9229. The SEQ ID NO:4 or its complementary sequence consists of 17 nucleotides of the RB7 nuclear matrix attachment region sequence (nucleotides 1-17 of SEQ ID NO:4), 380 nucleotides of the DBN11815 construct DNA The transgenic corn event DBN9229 can be identified by comprising SEQ ID NO:4 or its complementary sequence.

In certain embodiments, one skilled in the art can determine whether the DNA of transgenic corn event DBN9229 is present by detecting the presence of the following nucleic acid molecules in corn plants or parts, seeds, cells or progeny thereof.

Therefore, in another aspect, the present invention provides a nucleic acid molecule, the nucleic acid sequence of which comprises any one selected from the following:

(1) Corresponding to nucleotide 487 of SEQ ID NO:3 or its complementary sequence and at least 11 consecutive nucleotides upstream and downstream thereof (e.g., 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more consecutive nucleotides).

In certain embodiments, the nucleic acid sequence comprises at least 3 (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) consecutive nucleotides upstream of the 487th nucleotide corresponding to SEQ ID NO:3 or its complementary sequence.

In certain embodiments, the nucleic acid sequence comprises at least 3 (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) consecutive nucleotides downstream of the 487th nucleotide corresponding to SEQ ID NO:3 or its complementary sequence.

In some embodiments, the nucleic acid sequence comprises SEQ ID NO:1 or its complementary sequence.

In some embodiments, the nucleic acid sequence comprises SEQ ID NO:6 or its complementary sequence.

In some embodiments, the nucleic acid sequence comprises SEQ ID NO:3 or its complementary sequence.

In certain embodiments, the complementarity is partial complementarity or complete complementarity.

In some embodiments, the presence of the nucleic acid molecule in a corn plant or its part, seed, cell or progeny is detected to determine whether the DNA of the transgenic corn event DBN9229 exists. In some embodiments, a representative sample of the seeds of the transgenic corn event DBN9229 has been deposited in the China General Microbiological Culture Collection Center with the deposit number CGMCC No. 45229.

(2) Corresponding to nucleotide 397 of SEQ ID NO:4 or its complementary sequence and at least 11 consecutive nucleotides upstream and downstream thereof (e.g., 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more consecutive nucleotides).

In certain embodiments, the nucleic acid sequence comprises at least 3 (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11 or more) consecutive nucleotides upstream of nucleotide 397 corresponding to SEQ ID NO: 4 or its complementary sequence. acid.

In certain embodiments, the nucleic acid sequence comprises at least 3 (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11 or more) consecutive nucleotides downstream of the 397th nucleotide corresponding to SEQ ID NO:4 or its complementary sequence.

In some embodiments, the nucleic acid sequence comprises SEQ ID NO:2 or its complementary sequence.

In some embodiments, the nucleic acid sequence comprises SEQ ID NO:7 or its complementary sequence.

In some embodiments, the nucleic acid sequence comprises SEQ ID NO:4 or its complementary sequence.

Furthermore, the above two aspects of nucleic acid sequences contain SEQ ID NO: 5 or its complementary sequence.

The SEQ ID NO: 5 or its complementary sequence is a sequence of 12446 nucleotides in length that characterizes the transgenic corn event DBN9229, and the specific genome and genetic elements contained therein are shown in Table 1. The presence of the transgenic corn event DBN9229 can be identified by the presence of the SEQ ID NO: 5 or its complementary sequence.

Table 1. Genome and genetic elements contained in SEQ ID NO: 5

Corn plants and their products

In another aspect, the present application provides a corn plant or a part, seed, cell or progeny thereof, whose genome comprises the nucleic acid molecule as described above.

In certain embodiments, representative samples of the corn seeds have been deposited in the China General Microbiological Culture Collection Center with the deposit number CGMCC No.45229.

In another aspect, the present application provides a product comprising the corn plant as described above or a part, seed, cell or progeny thereof.

In certain embodiments, the preparation comprises genomic DNA of the corn plant, or a part, seed, cell, or progeny thereof.

In certain embodiments, the product is selected from one or more of corn ears, dehusked corn, corn silk, corn pollen, corn grits, corn flour, crushed corn, corn meal, corn oil, corn starch, corn steep liquor, corn malt, corn sugar, corn syrup, margarine produced from corn oil, unsaturated corn oil, saturated corn oil, corn flakes, popcorn, ethanol and/or liquor produced from corn, distillers dried grains (DDGS) produced from corn fermentation, animal feed from corn, cosmetics, and fillers.

Detection Methods

As is well known to those skilled in the art, the first, second, third and fourth nucleic acid sequences need not consist of only DNA, but may also include RNA, a mixture of DNA and RNA, or a combination of DNA, RNA or other nucleotides or their analogs that do not serve as templates for one or more polymerases. In addition, the probe or primer of the present invention should be at least about 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 consecutive nucleotides in length, which can be selected from the nucleotides or fragments thereof described in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5. When selected from the nucleotides shown in SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5, the probe and primer can be a length of at least about 21 to about 50 or more consecutive nucleotides or fragments thereof.

The nucleic acid sequence or its complementary sequence can be used in a DNA amplification method to produce an amplicon, which is used to detect the presence of transgenic corn event DBN9229 or its progeny in a biological sample; the nucleic acid sequence or its complementary sequence can be used in a nucleotide detection method to detect the presence of transgenic corn event DBN9229 or its progeny in a biological sample.

To achieve the above object, the present invention also provides a method for detecting DNA of genetically modified corn in a sample, the method comprising:

(a) contacting the sample with primers (e.g., at least two primers) for amplifying the DNA in a nucleic acid amplification reaction or system;

(b) performing a nucleic acid amplification reaction; and

(c) detecting the presence of the DNA or its amplified product;

Wherein, the DNA or its amplified product comprises the nucleic acid sequence as described above.

In some embodiments, the target amplification product comprises SEQ ID NO:1 or its complementary sequence, SEQ ID NO:2 or its complementary sequence, SEQ ID NO:6 or its complementary sequence, and/or SEQ ID NO:7 or its complementary sequence.

In certain embodiments, in step (c), the presence of the DNA or its amplified product is detected by fluorescent quantitative PCR, sequencing, or blot hybridization.

In certain embodiments, in step (a), at least one probe is provided and contacted with the sample and primers.

In certain embodiments, the sample is a DNA sample extracted from corn.

In certain embodiments, the primer comprises the nucleic acid sequence of a nucleic acid molecule as described above.

Specifically, the two primers include the complementary sequences of SEQ ID NO:1 and SEQ ID NO:9, SEQ ID NO:8 and SEQ ID NO:9, SEQ ID NO:2 and SEQ ID NO:11, SEQ ID NO:10 and SEQ ID NO:11, or SEQ ID NO:1 and SEQ ID NO:2.

In certain embodiments, the methods are used to detect the presence of DNA from transgenic corn event DBN9229 in a sample.

(a) contacting the sample with a probe, wherein the probe comprises the nucleic acid sequence of the nucleic acid molecule as described above;

(b) hybridizing the sample to be detected and the probe under stringent hybridization conditions; and

(c) detecting the hybridization between the sample to be detected and the probe.

In certain embodiments, the sample is a DNA sample extracted from corn.

In certain embodiments, the DNA comprises the nucleic acid sequence of a nucleic acid molecule as described above.

The stringent conditions may be hybridization at 65°C in a 6×SSC (sodium citrate), 0.5% SDS (sodium dodecyl sulfate) solution, and then washing the membrane once with 2×SSC, 0.1% SDS and once with 1×SSC, 0.1% SDS.

In some embodiments, the probe comprises SEQ ID NO:1 or its complementary sequence, SEQ ID NO:2 or its complementary sequence, SEQ ID NO:6 or its complementary sequence, and/or SEQ ID NO:7 or its complementary sequence.

Optionally, at least one of said probes is labeled with at least one fluorescent group.

In certain embodiments, the detection probe comprises a fluorescent group and a quencher group, wherein the fluorescent group is capable of emitting a signal, and the quencher group is capable of absorbing or quenching the signal emitted by the fluorescent group; and The signal emitted by the probe when hybridized to its complementary sequence is different from the signal emitted when not hybridized to its complementary sequence.

In certain embodiments, the probe is selected from a TaqMan probe, a FRET probe, or any combination thereof.

(a) contacting the sample with a marker nucleic acid molecule, wherein the marker nucleic acid molecule comprises the nucleic acid sequence of the nucleic acid molecule as described above;

(b) hybridizing the sample and the marker nucleic acid molecule under stringent hybridization conditions;

(c) detecting hybridization between the sample and the marker nucleic acid molecule, and then determining through marker-assisted breeding analysis that insect resistance and/or herbicide tolerance is genetically linked to the marker nucleic acid molecule.

In some embodiments, the marker nucleic acid molecule includes at least one selected from the following: SEQ ID NO: 1 or its complementary sequence, SEQ ID NO: 2 or its complementary sequence, and/or SEQ ID NO: 6-11 or its complementary sequence.

To achieve the above object, the present invention also provides a DNA detection kit, which comprises at least one DNA molecule, and the DNA molecule comprises the nucleic acid sequence of the nucleic acid molecule as described above.

In certain embodiments, the DNA molecule can be used as one of the DNA primers or probes specific to transgenic corn event DBN9229 or its progeny.

In certain embodiments, the DNA molecule can be used as a primer and/or probe for corn plant or its part, seed, cell or offspring as described above, or for goods as described above. In certain embodiments, the primer has as described above in definition. In certain embodiments, the probe has as described above in definition.

In some embodiments, the DNA molecule comprises SEQ ID NO:1 or its complementary sequence, SEQ ID NO:2 or its complementary sequence, SEQ ID NO:6 or its complementary sequence, and/or SEQ ID NO:7 or its complementary sequence.

To achieve the above objectives, the present invention also provides a plant cell, comprising a nucleic acid sequence encoding an insect-resistant Cry1Fa2 protein, a nucleic acid sequence encoding a Cry2Ab2 protein, a nucleic acid sequence encoding a glufosinate herbicide-tolerant PAT protein and a nucleic acid sequence in a specific region, wherein the nucleic acid sequence in the specific region includes the sequence shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:6 and/or SEQ ID NO:7.

In certain embodiments, the plant cell comprises a nucleic acid sequence encoding an insect resistance Cry1Fa2 protein, The nucleic acid sequence of Cry2Ab2 protein, the nucleic acid sequence encoding the glufosinate herbicide-tolerant PAT protein and the nucleic acid sequence of a specific region, wherein the nucleic acid sequence of the specific region includes the sequence shown in SEQ ID NO:3 and/or SEQ ID NO:4.

In some embodiments, the genome of the plant cell comprises SEQ ID NO:1, SEQ ID NO:5 nucleic acid sequences at positions 685-11603 and SEQ ID NO:2, in sequence.

In some embodiments, the genome of the plant cell comprises the sequence shown in SEQ ID NO:5.

To achieve the above objectives, the present invention also provides a method for protecting corn plants from insect attack, the method comprising providing at least one transgenic corn plant cell in the diet of the target insect, the transgenic corn plant cell comprising the sequence shown in SEQ ID NO:1 and/or SEQ ID NO:2 in its genome.

In certain embodiments, target insects that feed on cells of the transgenic corn plant are inhibited from further feeding on the transgenic corn plant.

In some embodiments, the transgenic corn plant cell contains the sequence shown in SEQ ID NO:3 and/or SEQ ID NO:4 in its genome.

In some embodiments, the transgenic corn plant cell contains SEQ ID NO:1, SEQ ID NO:5 nucleic acid sequences at positions 685-11603 and SEQ ID NO:2 in sequence in its genome, or contains SEQ ID NO:5.

In certain embodiments, the transgenic corn plant cell is obtained from a corn plant as described above, or a part, seed, cell, or progeny thereof, or a preparation as described above.

In certain embodiments, the insect is a pest of the order Lepidoptera.

In some embodiments, the insect is selected from the group consisting of Spodoptera frugiperda, Mythimna seperata, Ostrinia furnacalis, Helicoverpa armiger, Conogethes punctiferalis, Spodoptera litura, Agrotis ypsilon Rottemberg, Ostrinia nubilalis, Helicoverpa zea, Diatraea grandiosella, or any combination thereof.

To achieve the above objectives, the present invention also provides a method for protecting corn plants from damage caused by herbicides or controlling weeds in a field where corn plants are planted, the method comprising applying an effective dose of glufosinate herbicide to a field where at least one transgenic corn plant is planted, wherein the transgenic corn plant contains the sequence shown in SEQ ID NO:1 and/or SEQ ID NO:2 in its genome, and the transgenic corn plant is tolerant to glufosinate herbicide.

In certain embodiments, the transgenic corn plant comprises a gene that confers resistance to the glufosinate herbicide (eg, phosphinothricin N-acetyltransferase cPAT).

In some embodiments, the transgenic corn plant contains the sequence shown in SEQ ID NO:3 and/or SEQ ID NO:4 in its genome.

In some embodiments, the transgenic corn plant contains the nucleic acid sequences of SEQ ID NO:1, SEQ ID NO:5 positions 685-11603 and SEQ ID NO:2 in sequence in its genome, or contains the sequence shown in SEQ ID NO:5.

In certain embodiments, the transgenic corn plant is or is obtained from a corn plant as described above, or a part, seed, cell or progeny thereof.

To achieve the above object, the present invention also provides a method for cultivating corn plants that are resistant to insects and/or tolerant to glufosinate-ammonium herbicides, the method comprising:

(a) planting at least one corn seed, wherein the genome of the corn seed comprises a nucleic acid sequence encoding an insect resistance Cry2Ab2 protein, a nucleic acid sequence of a Cry1Fa2 protein and/or a nucleic acid sequence encoding a glufosinate herbicide tolerance PAT protein, and a nucleic acid sequence in a specific region; or the genome of the corn seed comprises the nucleic acid sequence shown in SEQ ID NO:5;

(b) growing the corn seeds into corn plants;

(c) infesting the corn plants with target insects and/or spraying the corn plants with an effective amount of glufosinate herbicide, and harvesting corn plants having reduced plant damage compared to other plants not having the nucleic acid sequence of the specific region;

The nucleic acid sequence of the specific region comprises the sequence shown in SEQ ID NO: 1 and/or SEQ ID NO: 2. In certain embodiments, the nucleic acid sequence of the specific region comprises the sequence shown in SEQ ID NO: 3 and/or SEQ ID NO: 4.

In certain embodiments, the insect is a pest of the order Lepidoptera.

In certain embodiments, the corn seed is or is obtained from a corn plant, or a part, seed, cell, or progeny thereof, as described above.

To achieve the above object, the present invention also provides a method for producing a corn plant resistant to insects and/or tolerant to glufosinate-ammonium herbicides, the method comprising: modifying a gene encoding insect resistance contained in a first corn plant genome to generate a gene encoding insect resistance; The nucleic acid sequence of the present invention comprises a nucleic acid sequence encoding a Cry2Ab2 protein, a nucleic acid sequence of a Cry1Fa2 protein and/or a nucleic acid sequence encoding a glufosinate-tolerant PAT protein, and a nucleic acid sequence in a specific region, wherein the nucleic acid sequence in the specific region comprises at least one nucleic acid sequence selected from the group consisting of 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.

In some embodiments, the method includes introducing the nucleic acid sequence encoding the insect-resistant Cry1Fa2 protein, the nucleic acid sequence of the Cry2Ab2 protein and/or the nucleic acid sequence encoding the glufosinate-tolerant PAT protein, and the nucleic acid sequence of the specific region contained in the genome of the first corn plant, or the nucleic acid sequence shown in SEQ ID NO:5 contained in the genome of the first corn plant, into the second corn plant, thereby producing a large number of progeny plants; selecting the progeny plants having the nucleic acid sequence of the specific region, and the progeny plants are resistant to insects and/or tolerant to glufosinate-ammonium herbicides; the nucleic acid sequence of the specific region includes the sequence shown in SEQ ID NO:1 and/or SEQ ID NO:2. In some embodiments, the nucleic acid sequence of the specific region includes the sequence shown in SEQ ID NO:3 and/or SEQ ID NO:4.

In some embodiments, the method comprises sexually crossing transgenic corn event DBN9229 with another corn plant (e.g., a corn plant lacking insect resistance and/or glufosinate tolerance), thereby producing a plurality of progeny plants, and selecting the progeny plants having the nucleic acid sequence of the specific region; the nucleic acid sequence of the specific region comprises the sequence shown in SEQ ID NO: 1 and/or SEQ ID NO: 2. In some embodiments, the nucleic acid sequence of the specific region comprises the sequence shown in SEQ ID NO: 3 and/or SEQ ID NO: 4.

In certain embodiments, the method comprises selfing a first generation progeny plant, thereby producing a plurality of second generation progeny plants; infesting the progeny plants with a target insect and/or treating the progeny plants with glufosinate; and selecting the progeny plants for resistance to the insect and/or tolerance to the glufosinate herbicide.

In certain embodiments, the corn plant is or is obtained from a corn plant as described above, or a part, seed, cell or progeny thereof.

In certain embodiments, the method further comprises sexually crossing the insect-resistant and/or glufosinate-tolerant progeny plant with another corn parent and harvesting hybrid seed produced thereby.

In certain embodiments, the insect is a pest of the order Lepidoptera.

In certain embodiments, the insect is selected from the group consisting of Spodoptera frugiperda, Mythimna seperata, Ostrinia furnacalis, Helicoverpa armiger, Conogethes punctiferalis, Spodoptera litura, Agrotis ypsilon Rottemberg, Ostrinia nubilalis, Helicoverpa zea, Diatraea grandiosella, or any combination thereof.

To achieve the above objectives, the present invention also provides an agricultural product or commodity produced from the transgenic corn event DBN9229, wherein the agricultural product or commodity is corn meal, corn flour, corn oil, corn silk, corn starch, corn gluten, corn tortillas, cosmetics or fillers.

Definition of terms

In the present invention for detecting nucleic acid sequences of corn plants and their detection methods, 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.

In this text, the term "corn" refers to maize (Zea mays) and includes all plant varieties that can be crossed with maize, including wild corn species.

As used herein, the terms "comprising", "including", or "containing" mean "including but not limited to".

In the text, the term "plant" includes whole plants, plant cells, plant organs, plant protoplasts, plant cell tissue cultures from which plants can be regenerated, plant calli, plant clumps, and intact plant cells in plants or plant parts, such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruits, stalks, roots, root tips, anthers, etc. It should be understood that the parts of transgenic plants within the scope of the present invention include, but are not limited to, plant cells, protoplasts, tissues, calli, embryos, and flowers, stems, fruits, leaves and roots, which are derived from transgenic plants or their progeny that have been previously transformed with the DNA molecules of the present invention and are therefore at least partially composed of transgenic cells.

In the text, the term "gene" refers to a nucleic acid fragment that expresses a specific protein. In certain embodiments, the gene includes a regulatory sequence (5' non-coding sequence) before the coding sequence and a regulatory sequence (3' non-coding sequence) after the coding sequence. "Native gene" refers to a gene found in nature with its own regulatory sequence. "Chimeric gene" refers to any gene that is not a natural gene, which contains regulatory and coding sequences found in non-native. "Endogenous gene" refers to a natural gene, which is located in its natural position in the genome of an organism. "Foreign 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 introduced into a recipient cell through a transgenic step. Foreign genes can include natural genes or chimeric genes inserted into non-natural organisms. "Transgenic" is a gene that has been introduced into the genome through a transformation procedure. The site in the plant genome where recombinant DNA has been inserted can be called an "insertion site" or a "target site."

In the text, the term "flanking DNA" may include exogenous (heterologous) DNA naturally present in the genome of an organism such as a plant or introduced by a transformation process, such as a fragment associated with a transformation event. Therefore, flanking DNA may include a combination of natural and exogenous DNA. In the present invention, "flanking DNA" is also referred to as "flanking region" or "flanking sequence". Or "flanking genomic sequence" or "flanking genomic DNA" refers to a sequence of at least 3, 5, 10, 11, 15, 20, 50, 100, 200, 300, 400, 1000, 1500, 2000, 2500 or 5000 base pairs or longer, which is located directly upstream or downstream of the original exogenous inserted DNA molecule and is adjacent to the original exogenous inserted DNA molecule. When the flanking region is located downstream, it can also be referred to as "3'flank" or "right border flank", etc. When the flanking region is located upstream, it can also be referred to as "5'flank" or "left border flank", etc.

The transformation procedure that causes random integration of foreign DNA can result in transformants containing different flanking regions, which are specifically contained by each transformant. When recombinant DNA is introduced into plants by traditional hybridization, its flanking regions usually do not change. Transformants also can contain a unique junction between the segments of heterologous insert DNA and genomic DNA or between two sections of genomic DNA or between two sections of heterologous DNA. "Joint" is the point where two specific DNA fragments connect. For example, the junction is present at the position where the insert DNA connects to the flanking DNA. Junction points are also present in transformed organisms, where two DNA fragments are connected together in a way modified from natural organisms. "Joint region" or "junction sequence" refers to the DNA comprising a junction point.

The present invention provides a transgenic corn event called DBN9229 and its progeny, wherein the transgenic corn event DBN9229 is also called corn plant DBN9229, which includes plants and seeds of the transgenic corn event DBN9229 and plant cells or their regenerable parts, and the plant parts of the transgenic corn event DBN9229, including but not limited to cells, pollen, ovules, flowers, buds, roots, stems, silks, inflorescences, ears, leaves and products from the corn plant DBN9229, such as corn meal, corn flour, corn oil, corn steep liquor, corn silk, corn starch and biomass left in the corn crop field.

The transgenic corn event DBN9229 of the present invention comprises a DNA construct, and when it is expressed in plant cells, the transgenic corn event DBN9229 obtains resistance to insects and tolerance to glufosinate herbicides. The DNA construct comprises three expression cassettes in series, the first expression cassette comprises a suitable promoter and a suitable polyadenylation signal sequence for expression in plants, the promoter is operably connected to the nucleic acid sequence of Cry1Fa2 protein, and the nucleic acid sequence of Cry1Fa2 protein is mainly resistant to lepidopteran insects. The second expression cassette comprises a suitable promoter and a suitable polyadenylation signal sequence for expression in plants, the promoter is operably connected to the nucleic acid sequence of Cry2Ab2 protein, and the nucleic acid sequence of Cry2Ab2 protein is mainly resistant to lepidopteran insects. The third expression cassette comprises a suitable promoter and a suitable polyadenylation signal sequence for expression in plants, wherein the promoter is operably linked to a gene encoding phosphinothricin N-acetyltransferase (PAT), and the nucleic acid sequence of the PAT protein is tolerant to glufosinate herbicide.

Cry2Ab2 insecticidal protein and Cry1Fa2 insecticidal protein are two of many insecticidal proteins, which are produced by Insoluble spore-associated crystalline protein produced by Bacillus thuringiensis (Bt for short). Cry2Ab2 protein or Cry1Fa2 protein is ingested by insects and enters the midgut, and the protoxin is dissolved in the alkaline pH environment of the insect midgut, and the N- and C-termini of the protein are digested by alkaline proteases, converting the protoxin into active fragments, which bind to receptors on the surface of the insect midgut epithelial cell membrane and insert into the intestinal membrane, causing perforation lesions in the cell membrane, destroying the osmotic pressure changes and pH balance inside and outside the cell membrane, etc., disrupting the digestion process of the insect, and ultimately causing its death. The Cry2Ab2 gene and Cry1Fa2 gene can be obtained by optimizing codons or changing their nucleotide sequences in other ways to increase the stability and availability of transcripts in transformed cells. The sequences of the Cry2Ab2 gene and the Cry1Fa2 gene can be obtained from public databases (e.g., Genebank).

In the text, the term "Lepidoptera" includes two types of insects: moths and butterflies. It is the order with the most agricultural and forestry pests, such as the black cutworm, cotton bollworm, Spodoptera litura, Spodoptera two-spotted camphor, and peach borer.

The phosphinothricin N-acetyltransferase (PAT) gene can be an enzyme isolated from a strain of Streptomyces viridochromogenes, which catalyzes the conversion of L-phosphinothricin to its inactive form by acetylation to confer tolerance to glufosinate herbicides on plants. Phosphinothricin (PTC, 2-amino-4-methylphosphonobutyric acid) is an inhibitor of glutamine synthetase. PTC is the structural unit of the antibiotic 2-amino-4-methylphosphono-alanyl-alanine, and this tripeptide (PTT) has activity against Gram-positive and Gram-negative bacteria and against the fungus Botrytis cinerea. The phosphinothricin N-acetyltransferase (PAT) gene can also be used as a selective marker gene.

In the text, the term "phosphinothricin ammonium", also known as glufosinate, refers to 2-amino-4-[hydroxy(methyl)phosphonyl]butyric acid ammonium, and treatment with "phosphinothricin ammonium herbicide" refers to treatment with any herbicide formulation containing glufosinate ammonium. The selection of the use rate of a certain glufosinate ammonium formulation in order to achieve an effective biological dose is no more than the skill of ordinary agronomic technicians. The use of any herbicide formulation containing glufosinate ammonium to treat a field containing plant material derived from transgenic corn event DBN9229 will control the growth of weeds in the field and will not affect the growth or yield of plant material derived from transgenic corn event DBN9229.

In the text, the DNA construct is introduced into the plant using a transformation method, including but not limited to Agrobacterium-mediated transformation, biolistic transformation, and pollen tube pathway transformation.

The Agrobacterium-mediated transformation method is a common method for plant transformation. The foreign DNA to be introduced into the plant is cloned between the left and right border consensus sequences of the vector, i.e., the T-DNA region. The vector is transformed into Agrobacterium cells, which are then used to infect plant tissues, and the T-DNA region of the vector containing the foreign DNA is inserted into the plant genome.

The gene gun transformation method is to bombard plant cells with vectors containing foreign DNA (particle-mediated biolistic transformation).

The pollen tube channel transformation method utilizes the natural pollen tube channel (also known as pollen tube guiding tissue) formed after plant pollination to carry exogenous DNA into the embryo sac through the nucellus channel.

Following transformation, the transformed plant tissue can be regenerated into transgenic plants, and progeny harboring the foreign DNA selected using appropriate markers.

DNA construct is the combination that DNA molecule is connected to each other, and this combination provides one or more expression cassettes.DNA construct is preferably can self-replicate in bacterial cell, and contains the plasmid of different restriction endonuclease sites, and the restriction endonuclease sites contained are used to import the DNA molecule that functional gene element is provided, i.e. promoter, intron, leader sequence, encoding sequence, 3 ' terminator region and other sequences.The expression cassette contained in the DNA construct comprises the necessary gene element that provides the transcription of messenger RNA, and the expression cassette can be designed to express in prokaryotic cell or eukaryotic cell.Expression cassette of the present invention is designed to express in plant cell most preferably.

In the text, a transgenic "event" is obtained by transforming a plant cell with a heterologous DNA construct, i.e., comprising at least one nucleic acid expression cassette containing a target gene, inserted into the plant genome by a transgenic method to produce a plant population, regenerate the plant population, and select specific plants with the characteristics of insertion at a specific genomic site. The term "event" refers to the original transformant containing the heterologous DNA and the offspring of the transformant. The term "event" also refers to the offspring obtained by sexually crossing the original transformant and individuals of other varieties containing heterologous DNA, even after repeated backcrossing with the recurrent parent, the inserted DNA and flanking genomic DNA from the original transformant parent are present in the same chromosomal position in the hybrid offspring. The term "event" also refers to a DNA sequence from the original transformant, the DNA sequence comprising the inserted DNA and the flanking genomic sequence closely adjacent to the inserted DNA, the DNA sequence is expected to be transferred to the offspring, the offspring is produced by sexually crossing the parental line containing the inserted DNA (e.g., the original transformant and the offspring produced by its self-crossing) with the parental line that does not contain the inserted DNA, and the offspring receives the inserted DNA containing the target gene.

In the text, the term "recombinant" refers to a form of DNA and/or protein and/or organism that is not normally found in nature and is therefore produced by human intervention. Such human intervention can produce recombinant DNA molecules and/or recombinant plants. The "recombinant DNA molecule" is obtained by artificially combining two otherwise isolated sequence segments, for example by chemical synthesis or by genetic engineering techniques to manipulate isolated nucleic acid segments. Techniques for nucleic acid manipulation are well known.

In this text, the term "transgenic" includes any cell, cell line, callus, tissue, plant part or plant, the genotype of which is changed due to the presence of heterologous nucleic acid, and the "transgenic" includes the original transgenic body so changed and the progeny individuals generated by the original transgenic body through sexual hybridization or asexual reproduction. In the present invention, the term "transgenic" does not include plants obtained by conventional plant breeding methods or simply containing only natural mutations. of plants.

In the text, the term "heterologous" means that a first molecule is not normally found in nature in combination with a second molecule. For example, a molecule can be derived from a first species and inserted into the genome of a second species. Such a molecule is thus heterologous to the host and is artificially introduced into the genome of the host cell.

The transgenic corn event DBN9229 having resistance to lepidopteran insects and tolerance to glufosinate herbicide is cultivated by the following steps: firstly, a first parent corn plant is sexually hybridized with a second parent corn plant, thereby producing a variety of first generation progeny plants, wherein the first parent corn plant is composed of corn plants cultivated from the transgenic corn event DBN9229 and its progeny, and the transgenic corn event DBN9229 and its progeny are obtained by transformation using the expression cassette of the present invention having resistance to lepidopteran insects and tolerance to glufosinate herbicide, and the second parent corn plant lacks resistance to lepidopteran insects and/or tolerance to glufosinate herbicide; then, progeny plants having resistance to attack by lepidopteran insects and/or tolerance to glufosinate herbicide are selected; or, the first generation progeny plants are self-pollinated to produce a plurality of second generation progeny plants, the progeny plants are attacked with target insects and/or treated with glufosinate, and the progeny plants having resistance to insects and/or tolerance to glufosinate herbicide are selected. Corn plants that are resistant to lepidopteran insects and tolerant to glufosinate herbicides can be cultivated. These steps can further include backcrossing the progeny plants that are resistant to lepidopteran insects and/or tolerant to glufosinate herbicides with the second parent corn plant or the third parent corn plant, and then selecting the progeny by attacking with lepidopteran insects, applying glufosinate herbicides, or by identification of molecular markers associated with the traits (such as DNA molecules comprising the 5' end and 3' end of the insertion sequence in transgenic corn event DBN9229) to produce corn plants that are resistant to lepidopteran insects and tolerant to glufosinate herbicides.

It should also be understood that two different transgenic plants can also be mated to produce offspring containing two independent, segregating added exogenous genes. Selfing of appropriate offspring can result in offspring plants that are homozygous for both added exogenous genes. Backcrossing of parental plants and outcrossing with non-transgenic plants as described above are also contemplated, as are asexual reproduction.

In the text, the term "probe" is a separate nucleic acid molecule to which a conventional detectable label or reporter molecule, such as a radioisotope, ligand, chemiluminescent agent or enzyme is bound. Such a probe is complementary to a strand of a target nucleic acid, and in the present invention, the probe is complementary to a DNA strand from the genome of transgenic corn event DBN9229, whether the genomic DNA is from transgenic corn event DBN9229 or seeds or from plants or seeds or extracts of transgenic corn event DBN9229. The probe of the present invention includes not only deoxyribonucleic acid or ribonucleic acid, 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.

In the text, the term "primer" is a separate nucleic acid molecule that anneals to a complementary target DNA strand through nucleic acid hybridization, forms a hybrid between the primer and the target DNA strand, and then extends along the target DNA strand under the action of a polymerase (e.g., DNA polymerase). The primer pair of the present invention relates to its use in amplification of a target nucleic acid sequence, for example, by polymerase chain reaction (PCR) or other conventional nucleic acid amplification methods.

The length of probes and primers is generally 11 polynucleotides or more, for example 18 polynucleotides or more, 24 polynucleotides or more, 30 polynucleotides or more. Such probes and primers hybridize specifically with the target sequence under highly stringent hybridization conditions. Although probes that are different from the target DNA sequence and that maintain hybridization ability to the target DNA sequence can be designed by conventional methods, preferably, the probes and primers of the present invention have complete DNA sequence identity with the continuous nucleic acids of the target sequence.

Primers and probes based on the flanking genomic DNA and insertion sequences of the present invention can be determined by conventional methods, for example, by isolating the corresponding DNA molecule from plant material derived from transgenic corn event DBN9229 and determining the nucleic acid sequence of the DNA molecule. The DNA molecule comprises the transgenic insertion sequence and the corn genomic flanking sequence, and fragments of the DNA molecule can be used as primers or probes.

The nucleic acid probe and primer of the present invention hybridize with the target DNA sequence under stringent conditions. Any conventional nucleic acid hybridization or amplification method can be used to identify the presence of DNA derived from transgenic corn event DBN9229 in a sample. Nucleic acid molecules or fragments thereof can be specifically hybridized with other nucleic acid molecules under certain circumstances. As used in the present invention, if two nucleic acid molecules can form an antiparallel double-stranded nucleic acid structure, it can be said that the two nucleic acid molecules can specifically hybridize with each other. If two nucleic acid molecules show complete complementarity, then one of the nucleic acid molecules is said to be the "complement" of another nucleic acid molecule. As used in the present invention, when each nucleotide of a nucleic acid molecule is complementary to the corresponding nucleotide of another nucleic acid molecule, then the two nucleic acid molecules are said to show "complete complementarity". If two nucleic acid molecules can hybridize with sufficient stability so that they anneal and bind to each other under at least conventional "low stringency" conditions, then the two nucleic acid molecules are said to be "minimally complementary". Similarly, if two nucleic acid molecules can hybridize with sufficient stability so that they anneal and bind to each other under conventional "high stringency" conditions, then the two nucleic acid molecules are said to have "complementarity". Deviations from complete complementarity are permissible as long as such deviations do not completely prevent the two molecules from forming a double-stranded structure. In order for a nucleic acid molecule to serve as a primer or probe, it is only necessary to ensure that it has sufficient complementarity in sequence to form a stable double-stranded structure under the specific solvent and salt concentration used.

As used in the present invention, a substantially homologous sequence is a nucleic acid molecule that can specifically hybridize with the complementary strand of another matching nucleic acid molecule under highly stringent conditions. Suitable stringent conditions for promoting DNA hybridization include, for example, treatment with 6.0× sodium chloride/sodium citrate (SSC) at about 45°C and then at 50°C. The conditions are well known to those skilled in the art. For example, the salt concentration in the washing step can be selected from about 2.0×SSC, 50°C for low stringency conditions to about 0.2×SSC, 50°C for high stringency conditions. In addition, the temperature conditions in the washing step can be increased from about 22°C for low stringency conditions to about 65°C for high stringency conditions. Both the temperature conditions and the salt concentration can be changed, or one of them can remain unchanged while the other variable is changed. Preferably, a nucleic acid molecule of the present invention can specifically hybridize with one or more nucleic acid molecules or their complementary sequences 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, or any fragment of the above sequences under moderate stringency conditions, such as at about 2.0×SSC and about 65°C. More preferably, a nucleic acid molecule of the present invention specifically hybridizes with one or more nucleic acid molecules of 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 or their complementary sequences, or any fragments of the above sequences under highly stringent conditions. In the present invention, a preferred marker nucleic acid molecule has SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:6 or SEQ ID NO:7 or their complementary sequences, or any fragments of the above sequences. Another preferred marker nucleic acid molecule of the present invention has 80% to 100% or 90% to 100% sequence identity with SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:6 or SEQ ID NO:7 or their complementary sequences, or any fragments of the above sequences. SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:6 and SEQ ID NO:7 can be used as markers in plant breeding methods to identify offspring of genetic crosses. The hybridization of the probe to the target DNA molecule can be detected by any method known to those skilled in the art, including but not limited to fluorescent labeling, radioactive labeling, antibody labeling, and chemiluminescent labeling.

With respect to amplification of a target nucleic acid sequence using specific amplification primers (e.g., by PCR), "stringent conditions" refer to conditions that only allow the primers to hybridize to the target nucleic acid sequence in a DNA thermal amplification reaction, and primers having a wild-type sequence (or its complementary sequence) corresponding to the target nucleic acid sequence are able to bind to the target nucleic acid sequence and preferably produce a unique amplification product, i.e., an amplicon.

As used herein, the term "specifically binds to (a target sequence)" means that under stringent hybridization conditions, the probe or primer hybridizes only to the target sequence in a sample containing the target sequence.

As used herein, 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. For example, to determine whether a corn plant was produced by sexual hybridization containing the transgenic corn event DBN9229 of the present invention, or whether a corn sample collected from a field contains transgenic corn event DBN9229, or whether a corn extract, such as meal, flour, or oil, contains transgenic corn event DBN9229, DNA extracted from a 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 DNA of the transgenic corn event DBN9229. The primer pair includes a primer pair derived from a plant genome. A first primer for a flanking sequence adjacent to the inserted exogenous DNA insertion site, 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 DBN9229. The amplicon can range in length from the combined length of the primer pair plus one nucleotide base pair, preferably plus about 50 nucleotide base pairs, more preferably plus about 250 nucleotide base pairs, and most preferably plus about 450 nucleotide base pairs or more.

Alternatively, the primer pairs can be derived from flanking genomic sequences on both sides of the inserted DNA to produce an amplicon comprising the entire inserted nucleotide sequence. One of the primer pairs derived from the plant genomic sequence can be located at a distance from the inserted DNA sequence, the distance ranging from one nucleotide base pair to about 20,000 nucleotide base pairs. The use of the term "amplicon" specifically excludes primer dimers formed in a DNA thermal amplification reaction.

Nucleic acid amplification reaction can be achieved by any nucleic acid amplification reaction method known in the art, including polymerase chain reaction (PCR). Various nucleic acid amplification methods are well known to those skilled in the art. PCR amplification methods have been developed to amplify up to 22kb of genomic DNA and up to 42kb of phage DNA. These methods and other DNA amplification methods in the art can be used in the present invention. The inserted exogenous DNA sequence and the flanking DNA sequence from the transgenic corn event DBN9229 can be amplified by utilizing the provided primer sequences to amplify the genome of the transgenic corn event DBN9229, and after amplification, the PCR amplicon or cloned DNA is subjected to standard DNA sequencing.

A DNA detection kit based on a DNA amplification method contains DNA molecules used as primers, which specifically hybridize to the target DNA under appropriate reaction conditions and amplify diagnostic amplicons. The kit can provide an agarose gel-based detection method or many methods for detecting diagnostic amplicons known in the prior art. A kit containing DNA primers homologous or complementary to any part of the corn genome of SEQ ID NO:3 or SEQ ID NO:4, and homologous or complementary to any part of the transgenic insertion region of SEQ ID NO:5 is provided by the present invention. The primer pair particularly identified as useful in the DNA amplification method is SEQ ID NO:8 and SEQ ID NO:9, which amplify a diagnostic amplicon homologous to a portion of the 5' transgenic/genomic region of the transgenic corn event DBN9229, wherein the amplicon includes SEQ ID NO:1. Other DNA molecules used as DNA primers can be selected from SEQ ID NO:5.

The amplicons generated by these methods can be detected by a variety of techniques. One such method is Genetic Bit Analysis, in which a DNA oligonucleotide strand is designed that spans the insert DNA sequence and the adjacent flanking genomic DNA sequence. The oligonucleotide strand is immobilized in a microwell of a microplate, and after PCR amplification of the target region (using one primer each in the insert sequence and the adjacent flanking genomic sequence), the single-stranded PCR product can be hybridized to the immobilized oligonucleotide strand and used as a template for a single base extension reaction using a DNA polymerase and ddNTPs specifically labeled for the next expected base. Results are obtained using an ELISA-type method. Signals represent the presence of the insert/flanking sequence, indicating that amplification, hybridization, and single base extension reactions were successful.

Another method is pyrosequencing. This method designs an oligonucleotide chain that spans the inserted DNA sequence and the adjacent genomic DNA binding site. The oligonucleotide chain is hybridized with the single-stranded PCR product of the target region (one primer is used in the inserted sequence and one primer is used in the adjacent flanking genomic sequence), and then incubated with DNA polymerase, ATP, sulfhydryl enzyme, luciferase, apyrase, adenosine-5'-phosphosulfate and luciferin. dNTPs are added separately and the generated light signals are measured. The light signal represents the presence of the inserted/flanking sequence, which indicates that the amplification, hybridization, and single-base or multi-base extension reaction are successful.

The fluorescence polarization phenomenon described by Chen et al. (Genome Res. 9:492-498, 1999) is also a method that can be used to detect the amplicon of the present invention. Using this method requires designing an oligonucleotide chain that spans the inserted DNA sequence and the adjacent genomic DNA binding site. The oligonucleotide chain is hybridized with the single-stranded PCR product of the target region (one primer is used in the inserted sequence and one primer is used in the adjacent flanking genomic sequence), and then incubated with a DNA polymerase and a fluorescently labeled ddNTP. Single-base extension will result in the insertion of the ddNTP. This insertion can be measured by a change in its polarization using a fluorimeter. The change in polarization represents the presence of the inserted/flanking sequence, which indicates that the amplification, hybridization and single-base extension reactions are successful.

Taqman is described as a method for detecting and quantifying the presence of a DNA sequence and is described in detail in the instructions provided by the manufacturer. As briefly described below, a FRET oligonucleotide probe is designed that spans the insert DNA sequence and the adjacent genomic flanking binding site. The FRET probe and PCR primers (one primer each in the insert sequence and the adjacent flanking genomic sequence) are cycled in the presence of a thermostable polymerase and dNTPs. Hybridization of the FRET probe results in the cleavage of the fluorescent and quenching moieties on the FRET probe and the release of the fluorescent moiety. The generation of a fluorescent signal represents the presence of the insert/flanking sequence, which indicates that amplification and hybridization were successful.

Based on the principle of hybridization, suitable techniques for detecting plant materials derived from transgenic corn event DBN9229 may also include Southern blot hybridization, Northern blot hybridization, and in situ hybridization. In particular, the suitable techniques include incubating the probe and sample, washing to remove unbound probes, and detecting whether the probe has hybridized. The detection method depends on the type of label attached to the probe, for example, radiolabeled probes can be detected by X-ray exposure and development, or enzyme-labeled probes can be detected by color change through substrate conversion.

Tyangi et al. (Nature Biotech. 14:303-308, 1996) introduced the application of molecular markers in sequence detection. A brief description is as follows: a FRET oligonucleotide probe is designed that spans the inserted DNA sequence and the adjacent genomic flanking binding site. The unique structure of the FRET probe results in it containing a secondary structure. The secondary structure can keep the fluorescent part and the quenching part in close proximity. The FRET probe and PCR primers (one primer each in the insert sequence and the adjacent flanking genomic sequence) are cyclically reacted in the presence of a thermostable polymerase and dNTPs. After successful PCR amplification, hybridization of the FRET probe and the target sequence results in the loss of the probe secondary structure, thereby separating the fluorescent part and the quenching part in space and generating a fluorescent signal. The generation of the fluorescent signal represents the presence of the insert/flanking sequence, which indicates that the amplification and hybridization are successful.

Other described methods, such as microfluidics, provide methods and devices for separating and amplifying DNA samples. Optical dyes are used to detect and measure specific DNA molecules. Nanotube devices containing electronic sensors for detecting DNA molecules or nanobeads that bind specific DNA molecules and can thus be detected are useful for detecting DNA molecules of the present invention.

A DNA detection kit can be developed using the compositions of the present invention and methods described or known in the art of DNA detection. The kit is useful for identifying the presence of DNA of transgenic corn event DBN9229 in a sample, and can also be used to grow corn plants containing DNA of transgenic corn event DBN9229. The kit can contain DNA primers or probes that are homologous to or complementary to at least a portion of SEQ ID NO: 1, 2, 3, 4, or 5, or other DNA primers or probes that are homologous to or complementary to DNA contained in a transgenic genetic element of the DNA, and these DNA sequences can be used in DNA amplification reactions, or as probes in DNA hybridization methods. The DNA structure of the transgenic insertion sequence and the junction site of the corn genome contained in the corn genome and illustrated in Figure 1 and Table 1 comprises: the flanking genomic region of the corn plant DBN9229 located at the 5' end of the transgenic insertion sequence; a portion of the insertion sequence from the left border region (LB) of Agrobacterium; the first expression cassette is composed of a cauliflower mosaic virus 35S promoter (pr35S), operably linked to the glufosinate-tolerant phosphinothricin N-acetyltransferase (cPAT) of Streptomyces, and operably linked to the cauliflower mosaic virus 35S terminator (t35S); the second expression cassette is composed of an actin 1 (Actin) promoter from rice, operably linked to the maize Rubisco gene chloroplast transit peptide gene (spZmCTP2), and operably linked to the cauliflower mosaic virus 35S terminator (t35S). The first expression cassette is composed of a Cry2Ab2 protein (cCry2Ab2) of insect resistance of Bacillus thuringiensis, and is operably connected to the In2-1 gene transcription terminator (tIn2); the third expression cassette is composed of a maize ubiquitin gene 1 promoter (prZmUbi1), operably connected to the Cry1Fa2 protein (cCry1Fa2) of insect resistance of Bacillus thuringiensis, operably connected to the transcription terminator (tORF25PolyA) of mannopine synthase from the pTiA6 plasmid of Agrobacterium tumefaciens A6 strain, and operably connected to the nuclear skeleton binding sequence (eRB7); a part of the insertion sequence from the right border region (RB) of Agrobacterium, and the flanking genomic region of the maize plant DBN9229 located at the 3' end of the transgenic insertion sequence (SEQ ID NO: 5). In the DNA amplification method, the DNA molecule used as a primer can be any part of the transgenic insertion sequence derived from the transgenic maize event DBN9229, or it can be derived from the transgenic maize event DBN9229. Any portion of the DNA sequence flanking the maize genome.

Transgenic corn event DBN9229 can be combined with other transgenic corn varieties, such as transgenic corn varieties with tolerance to herbicides (such as glyphosate, dicamba, etc.), or transgenic corn varieties carrying other insect-resistant genes. Various combinations of all these different transgenic events, bred together with the transgenic corn event DBN9229 of the present invention, can provide improved hybrid transgenic corn varieties that are resistant to multiple insect pests and multiple herbicides. These varieties can show more excellent characteristics such as yield improvement compared to non-transgenic varieties and single-trait transgenic varieties.

The transgenic corn event DBN9229 of the present invention is resistant to feeding damage by lepidopteran pests and tolerates the phytotoxic effects of agricultural herbicides containing glufosinate. The corn plant DBN9229 expresses Cry2Ab2 protein and Cry1Fa2 protein from Bacillus thuringiensis, which provide resistance to feeding damage by lepidopteran pests (such as small cutworms and oriental armyworms), and expresses glufosinate-resistant phosphinothricin N-acetyltransferase (PAT) protein from Streptomyces, which confers tolerance to glufosinate. The corn plant DBN9229 has the following advantages: 1) free from economic losses caused by lepidopteran pests (such as small cutworms, oriental armyworms, cotton bollworms, fall armyworms, etc.), which are major pests in corn-growing areas; 2) the ability to apply agricultural herbicides containing glufosinate to corn crops for broad-spectrum weed control; 3) corn yield is not reduced. In addition, the transgenes encoding insect resistance and glufosinate tolerance traits are linked to the same DNA segment and are present on a single locus in the genome of transgenic corn event DBN9229, which provides enhanced breeding efficiency and enables the use of molecular markers to track transgenic inserts in breeding populations and their progeny. At the same time, SEQ ID NO: 1 or its complementary sequence, SEQ ID NO: 2 or its complementary sequence, SEQ ID NO: 3 or its complementary sequence, SEQ ID NO: 4 or its complementary sequence, SEQ ID NO: 6 or its complementary sequence, or SEQ ID NO: 7 or its complementary sequence in the detection method of the present invention can be used as a DNA primer or probe to produce an amplification product diagnosed as transgenic corn event DBN9229 or its progeny, and can quickly, accurately and stably identify the presence of plant materials derived from transgenic corn event DBN9229.

Advantageous Effects of the Invention

The present application transforms corn plant cells with exogenous DNA (i.e., a nucleic acid construct containing a target gene) and inserts it into a specific position of the corn genome. The corn plants or parts, seeds, cells or progeny of the transgenic corn event DBN9229 produced can stably and highly express proteins with insect resistance and proteins with resistance to glufosinate herbicides, and the corresponding traits can also be stably inherited.

Furthermore, experiments have confirmed that the nucleic acid molecules provided in the present application and the corn plants or parts, seeds, cells or progeny thereof have good insect resistance (particularly, the insect mortality rate in the insect resistance test reached 100%, significantly reducing the damage level of the corn plants) and glufosinate herbicide tolerance (particularly, the application of glufosinate The damage of corn plants during the vegetative period after phosphine herbicide treatment is almost zero). In addition, hybridizing corn plants or their parts, seeds, cells or progeny with other resistant corn plants can further reduce the proportion of insect-resistant refuges required in actual production and increase plant yield.

Therefore, the nucleic acid molecules of the present application and the corn plants or parts, seeds, cells or progeny thereof comprising the same have great application potential in corn planting and breeding.

Embodiments of the present invention will be described in detail below in conjunction with the accompanying drawings and examples, but it will be appreciated by those skilled in the art that the following drawings and examples are only used to illustrate the present invention, rather than to limit the scope of the present invention. Various objects and advantages of the present invention will become apparent to those skilled in the art based on the following detailed description of the accompanying drawings and preferred embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 is a schematic diagram of the structure of the transgenic insertion sequence and the junction of the corn genome for detecting the nucleic acid sequence of corn plant DBN9229 and the detection method thereof of the present invention, as well as a schematic diagram of the relative position of the nucleic acid sequence for detecting corn plant DBN9229 (refer to B73 RefGen v4 for the relative position schematic diagram). From the left, it is the 5' corn genome, LB (bNLB), multiple elements contained in the third expression cassette inserted (t35S, cPAT and pr35S), multiple elements contained in the second expression cassette inserted (prOsAct1, spZmCTP2, cCry2Ab2 and tIn2), multiple elements contained in the first expression cassette inserted (prZmUbi1, cCry1Fa2, tORF25PolyA and RB7), RB (bNRB), and 3' corn genome.

FIG. 2 is a schematic diagram of the structure of the recombinant expression vector DBN11815 for detecting the nucleic acid sequence of corn plant DBN9229 and the detection method thereof according to the present invention.

3 is an in vitro effect diagram of the transgenic corn event DBN9229 inoculated with fall armyworm, oriental armyworm and Asian corn borer for detecting the nucleic acid sequence of the corn plant DBN9229 and the detection method thereof of the present invention.

4 is a field effect diagram of the transgenic corn event DBN9229 inoculated with Asian corn borer for detecting the nucleic acid sequence of the corn plant DBN9229 and the detection method thereof of the present invention.

5 is a field effect diagram of the transgenic corn event DBN9229 under the natural occurrence conditions of fall armyworm for detecting the nucleic acid sequence of corn plant DBN9229 and the detection method thereof of the present invention.

Notes on the Deposit of Biological Materials

Seeds of the genetically modified maize event DBN9229 have been purchased from the Budapest Treaty at Beichen West Road, Chaoyang District, Beijing. The deposit is held at the China General Microbiological Culture Collection Center (CGMCC) at No. 3, Courtyard 1, with the deposit number CGMCC No. 45229 and the deposit date is July 29, 2022. The deposit will be kept at the deposit for 30 years.

Sequence information

The information of the partial sequences involved in the present invention is provided in Table 1 below.

Table 1: Description of sequences

DETAILED DESCRIPTION

The invention will now be described with reference to the following examples which are intended to illustrate the invention rather than to limit the invention.

Unless otherwise specified, the experiments and methods described in the embodiments are basically carried out according to conventional methods well known in the art and described in various references. For example, conventional techniques such as immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics and recombinant DNA used in the present invention can be found in Sambrook, Fritsch and Maniatis, MOLECULAR CLONING: A LABORATORY MANUAL, 2nd edition (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (FM Ausubel et al., ed., (1987)); METHODS IN ENZYMOLOGY series (Academic Publishing Company): PCR 2: A PRACTICAL APPROACH (MJ MacPherson, BD Black et al., ed., (1989); ... Hames (BD Hames and GR Taylor eds. (1995)), and ANIMAL CELL CULTURE (RI Freshney ed. (1987)).

In addition, if the specific conditions are not specified in the examples, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified in the reagents or instruments used, they are all conventional products that can be obtained commercially. It is known to those skilled in the art that the embodiments describe the present invention by way of example and are not intended to limit the scope of the present invention. All public cases and other references mentioned herein are incorporated herein by reference in their entirety.

Example 1. Vector cloning, transformation and screening

1.1. Vector cloning

The recombinant expression vector DBN11815 (as shown in Figure 2) was constructed using standard gene cloning technology. The vector DBN11815 contains three transgenic expression cassettes in series. The first expression cassette is composed of the maize polyubiquitin gene 1 promoter (prZmUbi1), which is operably linked to the insect-resistant Cry1Fa2 protein (cCry1Fa2) from Bacillus thuringiensis, which is operably linked to the transcription terminator (tORF25PolyA) of the mannopine synthase of the pTiA6 plasmid of Agrobacterium tumefaciens A6 strain, and which is operably linked to the nuclear skeleton binding sequence (eRB7); the second expression cassette is composed of the rice actin 1 promoter (prOsAct1), which is operably linked to the maize The Rubisco gene chloroplast localization signal peptide (spZmCTP2) is operably linked to the insect-resistant Cry2Ab2 protein (cCry2Ab2) from Bacillus thuringiensis, and is operably linked to the transcription terminator (tIn2) of the corn In2-1 gene; the third expression cassette is composed of the cauliflower mosaic virus 35S promoter (pr35S), which is operably linked to the glufosinate-tolerant phosphinothricin N-acetyltransferase (cPAT) from Streptomyces, and is operably linked to the transcription terminator (t35S) of the cauliflower mosaic virus 35S.

The vector DBN11815 was transformed into Agrobacterium LBA4404 (Invitrgen, Chicago, USA; Cat. No: 18313-015) using the liquid nitrogen method, and the transformed cells were screened using glufosinate-ammonium as a selection marker.

1.2 Plant transformation

Conventional Agrobacterium infection method was used for transformation. The immature embryos of the sterile corn variety DBN567 were co-cultured with the Agrobacterium described in Example 1.1, and the T-DNA in the constructed recombinant expression vector DBN11815 was transferred into the corn chromosome to produce the transgenic corn event DBN9229.

For Agrobacterium-mediated transformation of maize, briefly, immature embryos are separated from maize and contacted with an Agrobacterium suspension, wherein the Agrobacterium is capable of transferring the T-DNA (containing the nucleotide sequence of the Cry1Fa2 gene, the nucleotide sequence of the Cry2Ab2 gene and the nucleotide sequence of the pat gene) in the DBN11815 vector to at least one cell of one of the embryos (step 1: infection step). In this step, the embryos are preferably immersed in the Agrobacterium suspension (OD ₆₆₀ = 0.4- 0.6, infection medium (MS salts 4.3 g/L, MS vitamins, casein 300 mg/L, sucrose 68.5 g/L, glucose 36 g/L, acetosyringone (AS) 40 mg/L, 2,4-dichlorophenoxyacetic acid (2,4-D) 1 mg/L, pH 5.3)) to start the inoculation. The young embryos are co-cultivated with Agrobacterium for a period of time (3 days) (step 2: co-cultivation step). Preferably, the young embryos are cultured on solid medium (MS salts 4.3 g/L, MS vitamins, casein 300 mg/L, sucrose 20 g/L, glucose 10 g/L, AS 100 mg/L, 2,4-D 1 mg/L, agar 8 g/L, pH 5.8) after the infection step. After this co-cultivation stage, there can be an optional "recovery" step. In the "recovery" step, at least one antibiotic known to inhibit the growth of Agrobacterium (cephalosporin 150-250 mg/L) is present in the recovery medium (MS salts 4.3 g/L, MS vitamins, casein 300 mg/L, sucrose 30 g/L, 2,4-D 1 mg/L, cephalosporin 250 mg/L, phytagel 3 g/L, pH 5.8), and no selection agent for plant transformants is added (step 3: recovery step). Preferably, the young embryos are cultured on a solid medium with antibiotics but no selection agent to eliminate Agrobacterium and provide a recovery period for infected cells. Next, the inoculated young embryos are cultured on a medium containing a selection agent (glufosinate-ammonium) and growing transformed callus is selected (step 4: selection step). Preferably, the immature embryos are cultured on a screening solid medium with a selection agent (MS salts 4.3 g/L, MS vitamins, casein 300 mg/L, sucrose 30 g/L, cephalosporin 250 mg/L, glufosinate 10 mg/L, 2,4-D 1 mg/L, plant gel 3 g/L, pH 5.8), resulting in selective growth of transformed cells. Then, callus tissue is regenerated into plants (step 5: regeneration step), preferably, callus tissue grown on a medium containing a selection agent is cultured on a solid medium (MS differentiation medium and MS rooting medium) to regenerate plants.

The resistant callus obtained by screening was transferred to the MS differentiation medium (MS salt 4.3g/L, MS vitamin, casein 300mg/L, sucrose 30g/L, 6-benzyladenine 2mg/L, cephalosporin 250mg/L, 4-[hydroxy(methyl)phosphonyl]-DL-homoalanine 5mg/L, plant gel 3g/L, pH 5.8), and cultured for differentiation at 25°C. The differentiated seedlings were transferred to the MS rooting medium (MS salt 2.15g/L, MS vitamin, casein 300mg/L, sucrose 30g/L, cephalosporin 250mg/L, indole-3-acetic acid 1mg/L, plant gel 3g/L, pH 5.8), cultured at 25°C to a height of about 10cm, and moved to a greenhouse for culture until fruiting. In the greenhouse, culture was carried out at a temperature of 28°C for 16h every day and then at a temperature of 20°C for 8h.

1.3 Identification and screening of transgenic events

A total of 235 independent transgenic _T0 individuals were generated.

Since genetic transformation, gene insertion, etc. may affect the agronomic traits of corn plants (such as yellowing, death, leaf curling or poor fruiting, etc.), the above 235 independent transgenic _T0 plants were sent to the greenhouse for transplanting and cultivation to identify the agronomic traits of the transgenic _T0 plants at different stages (seedling stage-jointing stage, jointing stage-pollen shedding stage and filling stage-maturity stage). A total of 176 transgenic _T0 plants with normal agronomic traits were obtained.

The 176 ^transgenic maize plants were tested for the presence of single copies of Cry1Fa2, Cry2Ab2 and pat genes, and no vector backbone sequence, a total of 97 transgenic _T0 plants were obtained; through transgenic insertion site analysis, a total of 20 transgenic _T0 plants with complete T-DNA flanking sequences, T-DNA not inserted into important genes in the maize genome, and gene insertion without generating new open reading frames (ORFs) were screened; through evaluation and comparison of resistance to major target insects (such as black cutworms, oriental armyworms, and cotton bollworms), a total of 15 transgenic _T0 plants with good insect resistance were screened; through evaluation and comparison of tolerance to glufosinate herbicides, a total of 15 transgenic T0 plants with good tolerance to glufosinate herbicides were screened. ₀ single plant; in different generations, different geographical environments and/or different genetic background materials, the agronomic traits, molecular biology, target insect resistance, glufosinate tolerance, etc. of the transgenic corn plants were screened for stably inherited, and the transgenic corn event DBN9229 was selected as excellent, having a single copy transgene (see Example 2), good insect resistance, glufosinate herbicide tolerance and agronomic trait performance (see Examples 6 and 7).

Example 2. Detection of transgenic corn event DBN9229 using TaqMan

About 100 mg of leaves of transgenic corn event DBN9229 were taken as samples, and the genomic DNA was extracted using a plant DNA extraction kit (DNeasy Plant Maxi Kit, Qiagen). The copy numbers of Cry1Fa2 gene, Cry2Ab2 gene and pat gene were detected by Taqman probe fluorescence quantitative PCR method. At the same time, wild-type corn plants were used as controls and the above method was used for detection and analysis. The experiment was repeated 3 times and the average value was taken.

The specific method is as follows:

Step 1: Take 100 mg of leaves (after pollination) of transgenic corn event DBN9229, grind them into a homogenate using liquid nitrogen in a mortar, and take 3 replicates for each sample;

Step 2: Use a plant DNA extraction kit (DNeasy Plant Maxi Kit, Qiagen) to extract genomic DNA from the above samples. For specific methods, refer to its product manual.

Step 3: Determine the genomic DNA concentration of the above samples using an ultra-micro spectrophotometer (NanoDrop 2000, Thermo Scientific);

Step 4, adjusting the genomic DNA concentration of the above samples to the same concentration value, wherein the concentration value ranges from 80-100 ng/μL;

Step 5: Use Taqman probe fluorescence quantitative PCR method to identify the copy number of the sample, use the sample with known copy number as the standard, and use the sample of wild-type corn plant as the control, and repeat 3 times for each sample to take the average value; the sequences of fluorescence quantitative PCR primers and probes are:

The following primers and probes were used to detect the Cry1Fa2 gene sequence:

Primer 1: ttgtctgagtttgttccaggtgtg as shown in SEQ ID NO: 16 in the sequence listing;

Primer 2: ccaatcagatggagtgatgaagc as shown in SEQ ID NO: 17 in the sequence listing;

Probe 1: tgcgtttggcctcttcgacctcatc as shown in SEQ ID NO: 18 in the sequence listing;

The following primers and probes were used to detect the Cry2Ab2 gene sequence:

Primer 3: gctcctgctgccactctttg as shown in SEQ ID NO: 19 in the sequence listing;

Primer 4: gaggatcacgtcacgaatgaag as shown in SEQ ID NO: 20 in the sequence listing;

Probe 2: tcaggctgccaacctgcacctct as shown in SEQ ID NO: 21 in the sequence listing;

The following primers and probes were used to detect the pat gene sequence:

Primer 5: gagggtgttgtggctggtattg as shown in SEQ ID NO: 22 in the sequence listing;

Primer 6: tctcaactgtccaatcgtaagcg as shown in SEQ ID NO: 23 in the sequence listing;

Probe 3: cttacgctgggccctggaaggctag as shown in SEQ ID NO: 24 in the sequence listing;

The PCR reaction system is:

The 50× primer/probe mixture contained 45 μL of each primer at 1 mM concentration, 50 μL of probe at 100 μM concentration, and 860 μL 1× TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0), and was stored in an amber test tube at 4°C.

The PCR reaction conditions were:

The data were analyzed using fast real-time fluorescence quantitative PCR system software (Applied Biosystems 7900HT Fast Real-Time PCR System SDS v2.3, Applied Biosystems), and the results showed that the transgenic corn event DBN9229 obtained was a single copy.

Example 3. Analysis of the insertion site of transgenic corn event DBN9229

3.1 Genomic DNA Extraction

DNA extraction was performed according to the conventional CTAB (cetyltrimethylammonium bromide) method: 2 g of young leaves of transgenic corn event DBN9229 were ground into powder in liquid nitrogen, and then 0.5 mL of DNA extraction CTAB buffer (20 g/L CTAB, 1.4 M NaCl, 100 mM Tris-HCl, 20 mM EDTA (ethylenediaminetetraacetic acid), pH adjusted to 8.0 with NaOH) preheated at 65°C was added, mixed thoroughly, and then extracted at 65°C for 90 min; 0.5 times the volume of phenol and 0. 5 times the volume of chloroform, invert to mix; centrifuge at 12000rpm (revolutions per minute) for 10 minutes; aspirate the supernatant, add 2 times the volume of anhydrous ethanol, gently shake the centrifuge tube, and let it stand at 4°C for 30 minutes; centrifuge at 12000rpm for another 10 minutes; collect the DNA at the bottom of the tube; discard the supernatant, wash the precipitate with 1mL of 70% ethanol; centrifuge at 12000rpm for 5 minutes; vacuum dry or blow dry in a clean bench; dissolve the DNA precipitate in an appropriate amount of TE buffer and store at -20°C.

3.2 Analysis of flanking DNA sequences

The concentration of the extracted DNA samples was measured to make the concentration of the sample to be tested between 80-100 ng/μL. The genomic DNA was digested with restriction endonucleases Eco RI (5' end analysis) and Nco I (3' end analysis). 26.5 μL of genomic DNA, 0.5 μL of the above restriction endonucleases and 3 μL of digestion buffer (the restriction enzymes used were all enzymes from NEB and their matching buffers or universal buffers, now called NEBCutSmart) were added to each digestion system and digested for 1 hour. After the digestion is completed, add 70 μL of anhydrous ethanol to the digestion system, ice bath for 30 minutes, centrifuge at 12000 rpm for 7 minutes, discard the supernatant, blow dry, then add 8.5 μL of double distilled water, 1 μL of 10×T ₄ -DNA ligase buffer (NEB T4 DNA Ligase Reaction Buffer, the specific formula can be visited NEB website or refer to https://www.neb.com/products/restriction-endonucleases, https://www.neb.com/products/b0202-t4-dna-ligase-reaction-buffer) and 0.5 μL of T ₄ -DNA ligase to connect overnight at 4°C. Use a series of nested primers to perform PCR amplification to separate the 5' and 3' end genomic DNA. Specifically, the primer combination for separating the 5' end genomic DNA includes SEQ ID NO: 13 and SEQ ID NO: 30 as the first primer, SEQ ID NO: 31 and SEQ ID NO: 32 as the second primer, and SEQ ID NO: 13 as the sequencing primer. The primer combination for separating the 3' end genomic DNA includes SEQ ID NO: 15 and SEQ ID NO: 33 as the first primer, SEQ ID NO: 34 and SEQ ID NO: 35 as the second primer, and SEQ ID NO: 15 as the sequencing primer. The PCR reaction conditions are shown in Table 3.

The amplified products obtained by the above PCR amplification reaction were electrophoresed on a 2.0% agarose gel to separate the PCR amplified products, and then the target fragments were separated from the agarose matrix using a gel recovery kit (QIAquick Gel Extraction Kit, catalog #_28704, Qiagen Inc., Valencia, CA). The purified PCR amplified products were then sequenced (e.g., using ABIPrismTM 377, PE Biosystems, Foster City, CA) and analyzed (e.g., using DNASTAR sequence analysis software, DNASTAR Inc., Madison, WI).

Standard PCR methods were used to confirm the 5' and 3' flanking sequences and the junction sequence. The 5' flanking sequence and the junction sequence can be confirmed using SEQ ID NO: 8 or SEQ ID NO: 12, in combination with SEQ ID NO: 9, SEQ ID NO: 13, or SEQ ID NO: 30. The 3' flanking sequence and the junction sequence can be confirmed using SEQ ID NO: 10 or SEQ ID NO: 14, in combination with SEQ ID NO: 11, SEQ ID NO: 15, or SEQ ID NO: 33. The PCR reaction system and amplification conditions are shown in Tables 2 and 3. Those skilled in the art will appreciate that other primer sequences can also be used to confirm the flanking sequence and the junction sequence.

DNA sequencing of the PCR amplification products provides DNA that can be used to design other DNA molecules that can be used as primers and probes to identify corn plants or seeds derived from transgenic corn event DBN9229.

It was found that the nucleotides 1-487 of SEQ ID NO:5 showed the left border flank of the insertion sequence of the transgenic corn event DBN9229 of the maize genomic sequence (5' flanking sequence), and the nucleotides 11987-12446 of SEQ ID NO:5 showed the right border flank of the insertion sequence of the transgenic corn event DBN9229 of the maize genomic sequence (3' flanking sequence). The 5' junction sequence is listed in SEQ ID NO:1, and the 3' junction sequence is listed in SEQ ID NO:2.

3.3. PCR zygosity assay

The junction sequence is a relatively short polynucleotide molecule that is a novel DNA sequence that is diagnostic for the DNA of transgenic corn event DBN9229 when detected in a polynucleotide detection assay. The junction sequences in SEQ ID NO:1 and SEQ ID NO:2 are 11 polynucleotides on each side of the insertion site of the transgenic fragment in transgenic corn event DBN9229 and the corn genomic DNA. Longer or shorter polynucleotide junction sequences can be selected from SEQ ID NO:3 or SEQ ID NO:4. The junction sequences (5' junction region SEQ ID NO:1, and 3' junction region SEQ ID NO:2) are useful as DNA probes or as DNA primer molecules in DNA detection methods. The junction sequences SEQ ID NO:6 and SEQ ID NO:7 are also novel DNA sequences in transgenic corn event DBN9229, which can also be used as DNA probes or as DNA primer molecules to detect the presence of transgenic corn event DBN9229 DNA. The SEQ ID NO:6 (nucleotides 488-1105 of SEQ ID NO:3) spans the DBN11815 construct DNA sequence, the t35S transcription termination sequence and the pat gene sequence, and the SEQ ID NO:7 (nucleotides 1-397 of SEQ ID NO:4) spans the RB7 gene expression regulatory sequence and the DBN11815 construct DNA sequence.

In addition, an amplicon is produced by using at least one primer from SEQ ID NO:3 or SEQ ID NO:4, which, when used in a PCR method, produces a diagnostic amplicon of transgenic corn event DBN9229.

Specifically, a PCR amplification product is generated from the 5' end of the transgenic insertion sequence, the PCR amplification product is a PCR product containing the 5' end of the T-DNA insertion sequence flanking the genome of the plant material derived from the transgenic corn event DBN9229. This PCR amplification product contains SEQ ID NO: 3. For PCR amplification, primer 7 (SEQ ID NO: 8) was designed to hybridize with the genomic DNA sequence flanking the 5' end of the transgenic insertion sequence, and primer 8 (SEQ ID NO: 9) was paired with the pat gene sequence located in the T-DNA insertion sequence.

A PCR amplification product was generated from the 3' end of the transgenic insertion sequence, which PCR amplification product contained a portion of the genomic DNA flanking the 3' end of the T-DNA insertion sequence in the genome of the plant material from the transgenic corn event DBN9229. This PCR amplification product contained SEQ ID NO: 4. For PCR amplification, primer 9 (SEQ ID NO: 10) was designed to hybridize with the genomic DNA sequence flanking the 3' end of the transgenic insertion sequence, and primer 10 (SEQ ID NO: 11) paired with it was designed to hybridize with the RB7 gene expression regulatory sequence in the T-DNA insertion sequence.

The DNA amplification conditions described in Tables 2 and 3 can be used in the above-mentioned PCR zygosity test to generate diagnostic amplicons for transgenic corn event DBN9229. Detection of the amplicons can be performed by using a Stratagene Robocycler, MJ Engine, Perkin-Elmer 9700 or Eppendorf Mastercycler Gradient thermal cycler, etc., or by methods and equipment known to those skilled in the art.

Table 2. PCR steps and reaction mixture conditions for identification of the 5' transgenic insert/genomic junction region of transgenic maize event DBN9229

Table 3. Thermal cycler amplification conditions

Mix gently and add 1-2 drops of mineral oil over each reaction if the thermocycler does not have a cap. Perform PCR reactions on a Stratagene Robocycler (Stratagene, La Jolla, CA), MJ Engine (MJ R-Biorad, Hercules, CA), Perkin-Elmer 9700 (Perkin Elmer, Boston, MA), or Eppendorf Mastercycler Gradient (Eppendorf, Hamburg, Germany) thermocycler using the cycling parameters in Table 3. The MJ Engine or Eppendorf Mastercycler Gradient thermocycler should be run in calculated mode. The Perkin-Elmer 9700 thermocycler should be run with the ramp speed set to maximum.

The experimental results showed that: primers 7 and 8 (SEQ ID NO: 8 and 9), when used in the PCR reaction of the genomic DNA of the transgenic corn event DBN9229, produced an amplification product of a 1105bp fragment, and when used in the PCR reaction of the untransformed corn genomic DNA and the non-DBN9229 corn genomic DNA, no fragment was amplified; primers 9 and 10 (SEQ ID NO: 10 and 11), when used in the PCR reaction of the genomic DNA of the transgenic corn event DBN9229, produced an amplification product of a 860bp fragment, and when used in the PCR reaction of the untransformed corn genomic DNA and the non-DBN9229 corn genomic DNA, no fragment was amplified.

The PCR zygosity assay can also be used to identify whether material derived from transgenic corn event DBN9229 is homozygous or heterozygous. Primer 11 (SEQ ID NO: 12), Primer 12 (SEQ ID NO: 13) and Primer 13 (SEQ ID NO: 14) are used in an amplification reaction to produce a diagnostic amplicon for transgenic corn event DBN9229. The DNA amplification conditions described in Tables 4 and 5 can be used in the above zygosity assay to produce a diagnostic amplicon for transgenic corn event DBN9229.

Table 4. Reaction solution for zygosity determination

Table 5. Thermal cycler amplification conditions for zygosity assay

PCR reactions were performed on a Stratagene Robocycler (Stratagene, La Jolla, CA), MJ Engine (MJ R-Biorad, Hercules, CA), Perkin-Elmer 9700 (Perkin Elmer, Boston, MA), or Eppendorf Mastercycler Gradient (Eppendorf, Hamburg, Germany) thermocycler using the cycling parameters in Table 5. The MJ Engine or Eppendorf Mastercycler Gradient thermocycler should be run in calculated mode. The Perkin-Elmer 9700 thermocycler should be run with the ramp speed set to maximum.

In the amplification reaction, the biological sample containing template DNA contains DNA diagnostic for the presence of transgenic corn event DBN9229 in the sample. Alternatively, the amplification reaction will produce two different DNA amplicons from a biological sample containing DNA derived from a corn genome that is heterozygous for the allele corresponding to the inserted DNA present in transgenic corn event DBN9229. The two different amplicons will correspond to a first amplicon derived from a wild-type corn genomic locus (SEQ ID NO: 12 and SEQ ID NO: 14) and a second amplicon diagnostic for the presence of transgenic corn event DBN9229 DNA (SEQ ID NO: 12 and SEQ ID NO: 13). A corn DNA sample that produces only a single amplicon corresponding to the second amplicon described for a heterozygous genome can be diagnostic for the presence of transgenic corn event DBN9229 in the sample, and the sample is produced from corn seeds that are homozygous for the allele corresponding to the inserted DNA present in transgenic corn plant DBN9229.

It should be noted that the primer pairs of transgenic corn event DBN9229 were used to generate an amplicon that is diagnostic for the genomic DNA of transgenic corn event DBN9229. These primer pairs include, but are not limited to, primers 7 and 8 (SEQ The invention relates to a method for amplifying a transgenic corn plant using a primer set comprising primers 14 and 15 (SEQ ID NOs: 25 and 26) for amplifying a corn endogenous gene. The method comprises a DNA amplification reaction of ... The DNA amplification conditions described in Tables 2-5 can be used to generate diagnostic amplicons for transgenic corn event DBN9229 using appropriate primer pairs. Extracts of corn plant or seed DNA that are presumed to contain transgenic corn event DBN9229, or products derived from transgenic corn event DBN9229, that produce amplicons that are diagnostic for transgenic corn event DBN9229 when tested in a DNA amplification method can be used as templates for amplification to determine the presence or absence of transgenic corn event DBN9229.

Example 4: Detection of transgenic corn event DBN9229 using Southern blot hybridization

4.1. DNA extraction for Southern blot hybridization

Grind approximately 5-10 g of leaf tissue in liquid nitrogen using a mortar and pestle. Resuspend 4-5 g of ground leaf tissue in 20 mL CTAB lysis buffer (100 mM Tris-HCl pH 8.0, 20 mM EDTA pH 8.0, 1.4 M NaCl, 0.2% v/v β-mercaptoethanol, 2% w/v CTAB) and incubate at 65°C for 60 min. During incubation, mix the sample by inversion every 10 min. After incubation, add an equal volume of phenol/chloroform/isoamyl alcohol (25:24:1), gently invert to mix, and centrifuge at 4000 rpm for 20 min. Repeat the extraction of the aqueous phase with an equal volume of chloroform/isoamyl alcohol (24:1). The aqueous phase was collected again and an equal volume of isopropanol was added. After mixing, the mixture was placed at -20°C for 1 hour to precipitate the DNA. The DNA was then centrifuged at 4000 rpm for 5 minutes to obtain a DNA precipitate, which was then resuspended in 1 mL TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0). To degrade any RNA present, the DNA was incubated with 40 μL of 10 mg/mL RNase A at 37°C for 30 minutes, centrifuged at 4000 rpm for 5 minutes, and centrifuged at 12000 rpm for 10 minutes in the presence of 0.1 volume of 3 M sodium acetate (pH 5.2) and 2 volumes of anhydrous ethanol. After discarding the supernatant, the precipitate was washed with 1 mL of 70% (v/v) ethanol, dried at room temperature, and the DNA was redissolved in 1 mL TE buffer.

4.2 Restriction enzyme digestion

The genomic DNA concentration of the above samples was measured using an ultra-micro spectrophotometer (NanoDrop 2000, Thermo Scientific).

In a 100 μL reaction system, 5 μg of DNA was digested each time. Genomic DNA was digested with restriction endonucleases Nco I, Nhe I, Mfe I, and Spe I, respectively, using partial sequences of Cry1Fa2 gene, Cry2Ab2 gene, and pat gene on T-DNA as probes. For each enzyme, the digest was incubated overnight at the appropriate temperature. The sample was spun down to 20 μL using a speed Vacuum (Thermo Scientific).

4.3 Gel electrophoresis

Add bromophenol blue loading buffer to each sample from Example 4.2, and load each sample onto a 0.7% TAE agarose gel containing ethidium bromide, separate by electrophoresis in TAE electrophoresis buffer (40 mM Tris-acetate, 2 mM EDTA, pH 8.5), and run the gel at 20 V overnight.

After electrophoresis, the gel was treated with 0.25M HCl for 10 min to depurinate the DNA, and then the gel was treated with denaturing solution (1.5M NaCl, 0.5M NaOH) and neutralizing solution (1.5M NaCl, 0.5M Tris-HCl, pH 7.2) for 30 min each. 5×SSC (3M NaCl, 0.3M sodium citrate, pH 7.0) was poured into a porcelain dish, a glass plate was placed, and then a soaked filter paper bridge, gel, positively charged nylon membrane (Roche, Cat. No. 11417240001), three filter papers, paper tower, and weights were placed in sequence. After transferring the membrane overnight at room temperature, the nylon membrane was rinsed twice in deionized water, and the DNA was fixed on the membrane by UV crosslinker (UVP, UV Crosslinker CL-1000).

4.4 Hybridization

Use PCR to amplify a suitable DNA sequence for probe preparation. The DNA probe is SEQ ID NO: 27, SEQ ID NO: 28 or SEQ ID NO: 29, or is partially homologous or complementary to the above sequence. Use DNA Labeling and Detection Starter Kit II (Roche, Cat. No. 11585614910) to perform DIG labeling, Southern blot hybridization, membrane washing and other operations of the probe. For specific methods, refer to its product manual. Finally, use X-ray film (Roche, Cat. No. 11666916001) to detect the position where the probe binds.

Two control samples were included on each Southern run: (1) DNA from a negative (untransformed) segregant, which was used to identify any endogenous maize sequences that hybridized to the element-specific probe; and (2) DNA from a negative segregant into which Hind III-digested DBN11815 plasmid was introduced in an amount equivalent to one copy number based on probe length, which served as a positive control for hybridization and to illustrate the sensitivity of the experiment.

The hybridization data provided conclusive evidence supporting the TaqMan ^™ PCR analysis that the corn plant DBN9229 contained a single copy of the Cry1Fa2 gene, the Cry2Ab2 gene, and the pat gene. Using the Cry1Fa2 gene probe, Nco I and Nhe I digestions produced single bands of approximately 8.0 kb and 8.5 kb, respectively; using the Cry2Ab2 gene probe, Mfe I and Spe I digestions produced single bands of approximately 16 kb and 6.0 kb, respectively; using the pat gene probe, Nco I The digestion with Nhe I and Nhe I respectively produced single bands of about 5.2 kb and 5.0 kb, indicating that one copy of each of Cry1Fa2 gene, Cry2Ab2 gene and pat gene existed in the corn plant DBN9229. In addition, no hybridization bands were obtained for the backbone probe, indicating that no DBN11815 vector backbone sequence entered the genome of the corn plant DBN9229 during the transformation process.

Example 5: Screening of transgenic corn vector DBN11815

According to the basic understanding of this field, different vector designs containing the same gene will have different effects on the performance of the target trait. In order to obtain the best performance of the target trait, 10 expression vectors containing Cry1Fa2, Cry2Ab2 and pat genes, including vector DBN11815, were designed and constructed. They contain the same target gene, but the combination of the regulatory elements and expression cassettes of the target gene is different, including adjusting the arrangement order of the Cry1Fa2, Cry2Ab2, and pat gene expression cassettes; adjusting the connection direction of the Cry2Ab2 gene expression cassette with the Cry1Fa2 and pat gene expression cassettes; adjusting the promoter and/or terminator used in the Cry2Ab2 and pat gene expression cassettes; adjusting the presence of the element nucleus skeleton binding sequence (eRB7) and its position in the expression cassette. The above 10 different expression vectors were respectively transferred into corn, and the same experimental methods were used for bioassay, protein content detection and insect resistance stability evaluation to screen the optimal vector DBN11815 for product development. The experimental results of the DBN11815 vector are as follows:

5.1. Bioassay of transgenic maize vectors

The DBN11815 vector-transgenic maize plants and wild-type maize plants (non-transgenic, NGM) were subjected to bioassays against cutworms (Agrotis ypsilon Rottemberg, BCW) and oriental armyworms (Mythimna seperata, OAW) according to the following methods:

Fresh leaves (V3-V4 period) of DBN11815 transgenic corn carrier and wild-type corn plants (non-transgenic, NGM) were taken respectively, rinsed with sterile water and dried with gauze, then the veins of the corn leaves were removed and cut into strips of about 1 cm×3 cm, 1-3 leaves (the number of leaves was determined according to the insect food intake) were taken and placed on the filter paper at the bottom of a round plastic culture dish, the filter paper was moistened with distilled water, 10 artificially reared newly hatched larvae were placed in each culture dish, and the insect test culture dishes were covered and placed under the conditions of temperature 26-28°C, relative humidity 70%-80%, and photoperiod (light/dark) 16:8 for 3 days before the results were counted. The three indicators of larval development progress, mortality rate and leaf damage rate were counted to obtain the total resistance score (full score 300 points): total resistance score = 100 × mortality rate + [100 × mortality rate + 90 × (number of newly hatched insects/number of insects inoculated) + 60 × (number of newly hatched negative control insects/number of insects inoculated) + 10 × (number of negative control insects/number of insects inoculated)] + 100 × (1-leaf damage rate). Among them, the number of insects inoculated refers to the number of insects inoculated, that is, 10 per dish (depending on the feeding amount of the pests); the larval development progress has been reflected in the total resistance score formula; the leaf damage rate refers to the proportion of the leaf area eaten by the pests to the total leaf area. Three transformation events of the DBN11815 vector were selected, and each transformation event Five plants with the same growth vigour were selected from each of the two plants and the wild-type corn plant (non-transgenic, NGM) for testing, and each plant was repeated 3 times. The results are shown in Table 6.

Table 6. Average results of insect resistance bioassay of DBN11815 transgenic corn vector - mortality (%) and total resistance score (points)

5.2. Detection of insect-resistant proteins in transgenic corn

The DBN11815 transgenic corn carrier plants and wild-type corn plants (non-transgenic, NGM) were tested for the expression levels of Cry2Ab2 and Cry1Fa2 proteins in different expression vectors by ELISA according to the following method:

Fresh leaves (V3 stage) of DBN11815 corn carrier plants and wild-type corn plants (non-transgenic, NGM) were taken respectively, and after freeze-drying, 20 mg was weighed and ground with liquid nitrogen, and then 1 mL of extraction buffer (8 g/L NaCl, 0.27 g/L KH ₂ PO ₄ , 1.42 g/L Na ₂ HPO ₄ , 0.2 g/L KCl, 5.5 ml/L Tween-20, pH 7.4) was added, mixed, allowed to stand at 4°C for 30 minutes, centrifuged at 12000 g for 10 minutes, and the supernatant was diluted to an appropriate multiple with the above extraction buffer, and 80 μl of the diluted supernatant was used for ELISA detection.

The ELISA (enzyme-linked immunosorbent assay) detection kits of ENVIROLOGIX: Cry2A kit (AP005) and Cry1F kit (AP016) were used to detect and analyze the proportion of protein (Cry2Ab2 protein, Cry1Fa2 protein) content in the sample to the dry weight of the tissue. The specific method is referred to the product manual. At the same time, wild-type corn plant leaves (non-transgenic, NGM) were used as controls and detected and analyzed according to the above method. Three transformation events were selected for the DBN11815 vector, and four plants were selected for each transformation event. Four technical replicates were performed for each plant. The results are shown in Table 7.

Table 7 Average results of determination of insect-resistant gene protein levels (μg/g DWT) in tissues collected from DBN11815 transgenic corn vectors

The results of Examples 5.1 and 5.2 above show that, compared with non-transgenic control plants and the other nine expression vectors containing Cry1Fa2, Cry2Ab2 and pat genes, vector DBN11815 has the best insect resistance (to small cutworms and oriental armyworms) and protein expression.

5.3. Stability test of insect resistance of transgenic corn vector

(1) Testing the stability of anti-insect resistance of cutworms

The T2 generation plants (including homozygous and heterozygous plants) of the transgenic corn vector DBN11815 and wild-type corn plants (non-transgenic, NGM) were subjected to bioassays against Agrotis ypsilon Rottemberg (BCW). The experimental design and experimental methods were consistent with those in Example 5.1 above. The leaves of wild-type corn plants (non-transgenic, NGM) were used as controls. Three transformation events were selected for the DBN11815 vector, and five plants were selected for each transformation event. Each plant was repeated three times. The results are shown in Table 8.

Table 8. Resistance results of the T2 generation of transgenic corn vector DBN11815 to black cutworms - mortality rate (%) and total resistance score (points)

(2) Stability test of the resistance to oriental armyworm

Based on the previous experiment, the insect resistance stability of T2 generation materials under high selection pressure was further evaluated. The transgenic corn vector DBN11815 (transformation events 1 to 3) and wild-type corn plants (non-transgenic, NGM) were bioassayed against the second-instar Oriental armyworm (Mythimna seperata, OAW) according to the following method:

Take fresh leaves of corn plants (V5-V6 period), rinse them with sterile water and absorb the water on the leaves with gauze, then remove the veins of the corn leaves and cut them into strips of about 2.5 cm×3 cm, take 3 pieces of the cut strips and put them into a single hole of a 24-well plate, fill the 24-well plate in this way, put one artificially reared second-instar larvae in each single hole, cover the 24-well plate, place it at a temperature of 26-28°C, a relative humidity of 70%-80%, and a photoperiod (light/dark) of 16:8 for 7 days, and then count the results (among which, after 4 days, in order to avoid leaf rot affecting larval survival, fresh leaves need to be replaced according to the above method, the difference is that this time 6 strips of leaves are taken and placed in the 24-well plate). The wild-type corn plant leaves (non-transgenic, NGM) were used as controls, and three transformation events were selected for the transgenic corn vector DBN11815. For each transformation event, 18 plants with consistent growth were selected for testing, where every 6 plants were distributed as a whole to a 24-well plate, and the leaves of each plant were evenly distributed to 4 wells. One 24-well plate was counted as one replicate, and a total of 3 replicates were set to calculate the mortality rate. The results are shown in Table 9.

Table 9. Resistance results of the T2 generation of transgenic corn vector DBN11815 to the second-instar oriental armyworm - mortality (%)

The results of Example 5.3 show that compared with non-transgenic control plants and the other nine expression vectors containing Cry1Fa2, Cry2Ab2 and pat genes, the three transformation events tested by the transgenic corn vector DBN11815 had good resistance to black cutworms and second-instar oriental armyworms in heterozygous and homozygous plants at the transgenic sites, and the insect resistance performance of different transformation events was stable.

After comprehensive evaluation and testing in the fifth embodiment, DBN11815 performed best in terms of insect resistance bioassay, insect resistance protein expression, and insect resistance stability among different generations. The transformation event DBN9229 with excellent performance was selected for product development.

Example 6. Insect resistance detection of transgenic corn event DBN9229

6.1. Bioassay of transgenic maize event DBN9229 in the genetic background of maize inbred line DBN567

The transgenic corn event DBN9229 from the preferred transgenic corn vector DBN11815 and the wild-type corn plant (non-transgenic, NGM) were subjected to bioassays against Spodoptera frugiperda (FAW), Mythimna seperata (OAW), Ostrinia furnacalis (ACB), Helicoverpa armigera (CBW), Conogethes punctiferalis (YPM), Spodoptera litura (TCW) and Agrotis ypsilon Rottemberg (BCW), respectively. The experimental design and experimental methods were consistent with those in Example 5.1 above. The results are shown in Table 10. In addition, the transgenic corn event DBN9229 was compared with other transgenic corn plants screened in Example 1. The experimental design and experimental methods were consistent with those in Example 5.1 above. The results are shown in Table 11 and Table 12.

Table 10. Insect resistance bioassay results of leaf tissue of transgenic corn event DBN9229 - mortality (%) and total resistance score (points)

Table 11. Comparison of insect resistance (BCW) bioassays of different GM maize events - mortality

Table 12. Comparison of insect resistance (BCW) bioassays for different transgenic maize events - Total resistance score

The results showed that the leaf tissue of the transgenic corn event DBN9229 receptor background had good resistance to fall armyworm, oriental armyworm, Asian corn borer, cotton bollworm, peach borer, Spodoptera litura and black cutworm, and the test insect mortality rate and total resistance score of the transgenic corn event DBN9229 were significantly higher than those of NGM, among which the mortality rate of insects that ate the transgenic corn event DBN9229 almost all reached 100%. In addition, the insect resistance mortality rate and total resistance score of the transgenic corn event DBN9229 were significantly higher than those of other transgenic corn plants screened in Example 1.

6.2 Bioassay of transgenic maize event DBN9229 in different genetic backgrounds

The transgenic corn event DBN9229 was introduced into two genetically different corn inbred lines, MLA05 and MLB07, by backcrossing, and the generation was BC4F3. The insect resistance performance of corn event DBN9229 was evaluated under the new genetic background. The transgenic corn event DBN9229 and wild-type corn plants (non-transgenic, NGM) were bioassayed against fall armyworm (Spodoptera frugiperda, FAW), oriental armyworm (Mythimna seperata, OAW) and Asian corn borer (Ostrinia furnacalis, ACB), respectively. The experimental design and experimental methods were consistent with the above-mentioned Example 5.1. The results are shown in Table 13.

Table 13. Insect resistance bioassay results of leaf tissue of transgenic corn event DBN9229 - mortality (%) and total resistance score (points)

The results showed that the leaf tissues of transgenic corn event DBN9229 with different genetic backgrounds had good resistance to fall armyworm, oriental armyworm and Asian corn borer, and the mortality rate and total resistance score of transgenic corn event DBN9229 were significantly higher than those of NGM. Among them, the mortality rate of insects that ate leaf tissues of transgenic corn event DBN9229 with different genetic backgrounds almost reached 100%.

6.3 Bioassay of transgenic maize event DBN9229 in the genetic background of maize hybrid MZ003

The transgenic maize event DBN9229 was crossed with the conventional non-transgenic maize inbred line MLT05 to obtain the transgenic maize hybrid MZ003. The insect resistance performance of the maize event DBN9229 was evaluated in the hybrid background.

(1) Bioassay of leaf tissue of hybrid MZ003 of transgenic maize event DBN9229

Hybrid MZ003 containing corn event DBN9229 and conventional corn hybrid MZ003 (non-transgenic, NGM) were subjected to bioassays against fall armyworm (Spodoptera frugiperda, FAW) and oriental armyworm (Mythimna seperata, OAW), respectively, and the experimental design and experimental methods were consistent with the above-mentioned Example 5.1. The results are shown in Table 14.

Table 14. Insect resistance bioassay results of leaf tissue of transgenic corn event DBN9229 - mortality (%) and total resistance score (points)

The results showed that the leaf tissues of the hybrid MZ003 of the transgenic corn event DBN9229 had good resistance to both fall armyworm and oriental armyworm, and the mortality rate and total resistance score of the transgenic corn event DBN9229 were significantly higher than those of NGM. Among them, the mortality rate of insects that ate the leaf tissues of the hybrid MZ003 of the transgenic corn event DBN9229 reached 100%.

(2) The insect resistance of the hybrid MZ003 of the transgenic corn event DBN9229 in the field

The seeds of hybrid MZ003 containing corn event DBN9229 and conventional corn hybrid MZ003 (non-transgenic, NGM) were planted in a randomized block design with 3 replicates. The area of each replicate was ^30m2 (5m×6m), with a row spacing of 60cm and a plant spacing of 25cm. Conventional cultivation management was used and no pesticides were sprayed during the entire growth period. There was a 2m interval between different insect test plots to prevent the spread of insects between different plots.

(a) Cotton bollworm

Artificial inoculation was carried out during the silking period of corn, with a total of 2 inoculations. The number of artificially inoculated plants in each plot was no less than 40, and about 20 artificially reared newly hatched larvae were inoculated in the silk of each corn plant. Three days after the first inoculation, the second inoculation was carried out, with the same number of insects as the first. 14-21 days after inoculation, the damage rate of female ears, the number of surviving larvae per female ear, and the number of female ears were investigated plant by plant. Damaged length of ear. Usually the investigation starts 14 days after inoculation. If the damage level of NGM reaches susceptible or highly susceptible at this time, it is considered effective. If not, the investigation can be appropriately postponed. However, if the damage of NGM still does not reach the corresponding level 21 days after inoculation, the inoculation is considered invalid. According to the damage rate of female ears, the number of surviving larvae, and the damaged length of female ears (cm), the average damage level of cotton bollworm to female ears at the corn ear stage in each plot is calculated. The judgment criteria are shown in Table 15, and then the resistance level of corn ear stage to cotton bollworm is judged according to the standards in Table 16. The resistance results of transgenic corn event DBN9229 to cotton bollworm at the silking stage are shown in Table 17.

Table 15. Grading standards for the degree of damage caused by cotton bollworms on corn ears

Table 16. Evaluation criteria for resistance of corn ears to cotton bollworm

Table 17. Resistance results of transgenic corn event DBN9229 female ears to cotton bollworm

The results showed that under artificial inoculation conditions, the transgenic corn event DBN9229 showed a high level of resistance to cotton bollworm, while the control corn plants were more susceptible to insect infestation. The damage level of the transgenic corn event DBN9229 to cotton bollworm was significantly lower than that of non-transgenic corn (NGM), at least 3 levels lower.

(b) Asian corn borer

The test method is consistent with the above evaluation of cotton bollworm resistance. The difference is that the average value of the damage level of resistance to the female ear of the corn ear of each plot is calculated based on the damage to the female ear, the number of boreholes, the length of the borehole tunnel (cm), the age and number of surviving larvae, and the judgment criteria are shown in Table 18. Then, the resistance level to the Asian corn borer at the corn ear stage is judged according to the standards in Table 19. The resistance results of the transgenic corn event DBN9229 to the Asian corn borer at the silking stage are shown in Table 20.

Table 18. Grading standards for the degree of damage to corn ears caused by the Asian corn borer

Table 19. Evaluation criteria for resistance of corn ears to Ostrinia furnacalis

Table 20. Resistance results of transgenic corn event DBN9229 female ears to Asian corn borer

The results showed that under artificial inoculation conditions, the transgenic corn event DBN9229 showed a high level of resistance to the Asian corn borer, while the control corn plants were more susceptible to the insect. The damage level of the transgenic corn event DBN9229 to the Asian corn borer was significantly lower than that of non-transgenic corn (NGM), at least 3 levels lower.

(c) Fall Armyworm

The test method is different from that for cotton bollworm and Asian corn borer mentioned above. Fall armyworm is evaluated under natural field conditions. Field insect resistance tests were conducted on the transgenic corn event DBN9229 under natural conditions in areas where fall armyworm is more serious. Leaf tissue was evaluated around the trumpet stage. 10-15 days after the occurrence of insect infestation, and when NGM was mostly 5-6-year-old larvae, the damage of fall armyworm to corn leaves was investigated plant by plant, and the average value of the damage level of fall armyworm to corn leaves in each replicate was calculated. The judgment criteria are shown in Table 21, and then the resistance level of corn leaves to fall armyworm in the trumpet stage was judged according to the standards in Table 22. The resistance results of transgenic corn event DBN9229 to fall armyworm in the trumpet stage are shown in Table 23.

Table 21. Grading standards for the degree of damage to corn leaves by fall armyworm

Table 22. Evaluation criteria for corn leaf resistance to fall armyworm

Table 23. Resistance results of leaf tissues of transgenic corn event DBN9229 to fall armyworm under natural insect-infected conditions

The results showed that under the natural occurrence of fall armyworm, the transgenic corn event DBN9229 had a good resistance level to fall armyworm, while the control corn plants were seriously damaged. The damage level of leaf tissue of transgenic corn event DBN9229 to fall armyworm was significantly lower than that of non-transgenic corn (NGM).

6.4 Bioassay and application evaluation of transgenic maize event DBN9229×DBN9501×DBN9936

The transgenic corn event DBN9229 was genetically integrated with DBN9936 (CN104830847B) and DBN9501 (CN109868273B) and introduced into the same corn plant. Specifically, the transgenic corn event DBN9936 was first hybridized with the transgenic corn event DBN9501 to obtain a heterozygous plant of the stacked transgenic corn event DBN9501×DBN9936, and then after two generations of self-pollination, the number of target gene copies was detected by TaqMan (refer to the second embodiment) and the pure heterozygosity of the PCR zygosity detection site was detected (refer to the third embodiment), and the stacked transgenic corn event DBN9501×DBN9936 homozygous plant was obtained, which was used as the male parent to hybridize with the transgenic corn event DBN9229 (female parent) to obtain the stacked transgenic corn event DBN9229×DBN9501×DBN9936.

Those skilled in the art will appreciate that, in addition to the above examples, there are many other hybridization combinations that can be used to Genetic maize events DBN9229, DBN9501 and DBN9936 were introduced into the same maize plant.

(1) Detection of resistance of transgenic corn event DBN9229×DBN9501×DBN9936 to fall armyworm

(a) Bioassay of leaf tissue of transgenic maize event DBN9229×DBN9501×DBN9936

The transgenic maize event DBN9229×DBN9501×DBN9936 and the wild-type maize plant (non-transgenic, NGM) were subjected to bioassay against fall armyworm (Spodoptera frugiperda, FAW), and the experimental design and experimental method were consistent with the evaluation of Example 5.1 above. The results are shown in Table 24.

Table 24. Resistance results of leaf tissues of transgenic corn events DBN9229×DBN9501×DBN9936 to newly hatched fall armyworm - mortality (%) and total resistance score (points)

The results showed that the leaf tissue of the hybrid background of the transgenic corn event DBN9229×DBN9501×DBN9936 had good resistance to the fall armyworm, and the insect mortality rate in the insect resistance assay reached 100%. In addition, the test insect mortality rate and total resistance score of the transgenic corn event DBN9229×DBN9501×DBN9936 were significantly higher than those of non-transgenic corn (NGM).

(b) Field effects of transgenic corn event DBN9229×DBN9501×DBN9936

The experimental design and experimental methods are consistent with the above Example 6.3 (2) (c) evaluation of fall armyworm, and the judgment criteria are shown in Table 21 above, and the resistance evaluation criteria are shown in Table 22 above. The results of the resistance of transgenic corn event DBN9229×DBN9501×DBN9936 to fall armyworm at the trumpet stage are shown in Table 25.

Table 25. Resistance results of leaf tissues of transgenic corn event DBN9229×DBN9501×DBN9936 to fall armyworm under natural insect-infected conditions

The results showed that under the natural occurrence conditions of fall armyworm, the transgenic corn event DBN9229×DBN9501×DBN9936 had a good level of resistance to fall armyworm, and the leaf tissue damage level of the transgenic corn event DBN9229×DBN9501×DBN9936 was significantly lower than that of non-transgenic corn (NGM).

(2) Yield and quality evaluation of transgenic corn event DBN9229×DBN9501×DBN9936

The genetically stacked transgenic corn DBN9229×DBN9501×DBN9936 has multiple insect resistance mechanisms against Asian corn borer. It can further reduce the proportion of insect-resistant shelters required in actual production, better protect production benefits and delay Resistance occurs. The transgenic maize events DBN9229×DBN9501×DBN9936 (multiple insect resistance mechanisms, 5% shelter mixed planting) and DBN9936 (single insect resistance mechanism, 20% shelter drill planting) were evaluated in areas where the natural occurrence of Asian corn borer is more serious. The experimental design was to plant 20 rows per treatment, with a row length of 9m, a row spacing of 60cm, and a plant spacing of 28cm, and conventional cultivation management. According to the NY/T 1611-2017 Technical Specification for Corn Borer Monitoring and Forecasting, the number of generations of Asian corn borer, the occurrence level of each generation, and the damage rate (damage rate = number of corn plants eaten by pests/total number of plants × 100%) were investigated. The corn yield of each plot was calculated as the total corn kernel yield (weight) of each plot, and the yield difference of different products was measured in the form of yield percentage, yield percentage (%) = DBN9229 × DBN9501 × DBN9936 (5% shelter mixed planting) / DBN9936 (20% shelter drill sowing) × 100%. The yield percentage results are shown in Table 26. The moldy ear ratio of each plot was also calculated, and only the moldy ears caused by Asian corn borer were counted (moldy ear ratio = number of moldy ears/total number of plants × 100%). The moldy ear ratios of different products are shown in Table 26.

The investigation found that there were two generations of Asian corn borer in the planting area that year. The damage rate of the first generation of Asian corn borer was 57%, and the number of live worms per 100 plants of the second generation of Asian corn borer was 56. The two generations of Asian corn borer were both at the level of 4 heavy occurrence. Under the condition that the natural occurrence of Asian corn borer was relatively serious, the rate of ear mildew caused by corn borer damage was significantly lower in DBN9229×DBN9501×DBN9936 (multiple insect resistance mechanisms, 5% shelter mixed planting) than in DBN9936 (single insect resistance mechanism, 20% shelter drill sowing), and the grain quality was significantly improved (Table 26). At the same time, the yield performance was slightly improved (Table 26). It reflects the great value of the genetic superposition DBN9229×DBN9501×DBN9936 corn transformation event in production application.

Table 26. Evaluation results of transgenic corn event DBN9229×DBN9501×DBN9936 on quality and yield

Example 7. Herbicide tolerance evaluation of events

7.1. Assessment of tolerance to damage during the vegetative period and its stability

The T3, T4 and T5 generations of the transgenic corn event DBN9229 were evaluated by spraying glufosinate ammonium. The test used Basta herbicide (18% glufosinate ammonium salt solution as active ingredient) for spraying. The recommended concentration of Basta for field weed control is 400g ai/ha. In the evaluation test of each generation of materials, DBN9229 was treated with the following two herbicides: (1) Basta herbicide was sprayed at a dosage of 800g ai/ha (ai/ha means "active ingredient per hectare") at the V3 stage; (2) no herbicide was sprayed, and at the same time as the herbicide was sprayed in treatment (1), an equal volume of clean water was sprayed. Each treatment was set up with 3 replicates, and each replicate had 2 rows (row length 9m, row spacing 60cm, plant spacing Non-genetically modified corn (NGM) was used as a parallel control.

The phytotoxicity symptoms were investigated 2 weeks after the application of the drug, and the phytotoxicity symptom classification is shown in Table 27. The vegetative damage score is used as an indicator to evaluate the herbicide tolerance of the transformation event. Vegetative damage mainly refers to the phytotoxicity manifestations such as leaf burns, deformities, and wilting caused in a short period of time (several hours to more than ten days) after the glufosinate treatment; specifically, the vegetative damage score = ∑ (number of affected plants at the same level × number of levels) / (total number of plants × highest level) × 100; the vegetative damage score is determined based on the phytotoxicity investigation results 2 weeks after the glufosinate treatment. It should be noted that the conversion of glufosinate herbicides of different contents and dosage forms into the above-mentioned equivalent amount of the active ingredient glufosinate is applicable to the following conclusions. The results of the tolerance of the transgenic corn event DBN9229 to glufosinate herbicides are shown in Table 28.

Table 27. Grading standards for the degree of damage caused by glufosinate herbicides to corn during the vegetative period

Table 28. Results of tolerance of transgenic corn event DBN9229 to glufosinate herbicide

The results showed that in different generations, the damage score of the transgenic corn event DBN9229 during the vegetative period under the treatment of glufosinate herbicide (800 g a.i./ha) was 0. Therefore, the transgenic corn event DBN9229 has good tolerance to glufosinate herbicide and is stable among different generations.

7.2. Assessment of impact on production

The experiment was conducted in three different environments (A: Changchun, Jilin; B: Tangshan, Hebei; C: Jinan, Shandong). In each environment, the transgenic maize event DBN9229 was subjected to the following two treatments: (1) glufosinate treatment: maize DBN9229 plants were sprayed with the herbicide Glufosinate at a dosage of 800 g ai/ha (ai/ha means "active ingredient per hectare") at the V3 stage; (2) control treatment: (with an active ingredient of 30% benzathine) + atrazine herbicide mixed treatment; the specific operation is to use 25g ai/ha and atrazine at a dose of 945 g ai/ha Tianjin herbicide was mixed and sprayed on corn DBN9229 plants at V3 stage. Six replicates were set for each treatment. The area of each replicate plot was ^21.6m2 (9m×2.4m), with a row spacing of 60cm and a plant spacing of 28cm, and conventional cultivation management was used.

The corn yield of each plot is the total corn grain yield (weight) of each plot. The yield difference between different treatments is measured in the form of yield percentage, yield percentage = yield of the plot sprayed with glufosinate/sprayed + atrazine plot yield × 100%. The yield percentage results are shown in Table 29.

Table 29. Corn yield results of transgenic corn event DBN9229

The results showed that under different environments, there was no significant difference in the yield of the transgenic corn event DBN9229 when sprayed with basil herbicide (25 g a.i./ha) + atrazine herbicide (945 g a.i./ha) and when sprayed with glufosinate herbicide (800 g a.i./ha), which further demonstrated that the transgenic corn event DBN9229 has good tolerance to glufosinate.

Example 8. Genetically modified corn products

Agricultural products or commodities such as can be produced from transgenic corn event DBN9229. If sufficient expression is detected in the agricultural product or commodity, the agricultural product or commodity is expected to contain a nucleotide sequence that can diagnose the presence of transgenic corn event DBN9229 material in the agricultural product or commodity. The agricultural product or commodity includes, but is not limited to, corn oil, corn meal, cornmeal, corn gluten, corn tortillas, corn starch, and any other food to be consumed by animals as a food source, or otherwise used as a bulking agent or ingredient in a cosmetic composition for cosmetic purposes. Nucleic acid detection methods and/or kits based on probe or primer pairs can be developed to detect nucleotide sequences derived from transgenic corn event DBN9229 such as shown in SEQ ID NO:1 or SEQ ID NO:2 in biological samples, wherein the probe sequence or primer sequence is selected from the sequences shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5, or portions thereof, to diagnose the presence of transgenic corn event DBN9229.

In summary, the transgenic corn event DBN9229 of the present invention has good resistance to lepidopteran insects and high tolerance to glufosinate herbicides without affecting yield. The detection method can accurately and quickly identify whether the biological sample contains the DNA molecules of the transgenic corn event DBN9229.

Seeds corresponding to the biotech maize event DBN9229 have been protected under the Budapest Treaty on July 29, 2022. The deposit is deposited in the General Microbiology Center of China Microorganism Culture Collection (referred to as CGMCC, address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, zip code 100101), and the deposit number is CGMCC No. 45229. The deposit will be kept at the deposit for 30 years.

Although the specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and changes may be made to the details according to all the teachings that have been published, and these changes are within the scope of protection of the present invention. The entire invention is given by the attached claims and any equivalents thereof.

Claims

A nucleic acid molecule or a combination thereof, wherein the nucleic acid sequence comprises:

At least 11 consecutive nucleotides from positions 1 to 487 of SEQ ID NO: 3 or its complementary sequence, and/or at least 11 consecutive nucleotides from positions 488 to 1105 of SEQ ID NO: 3 or its complementary sequence;

At least 11 consecutive nucleotides from positions 1 to 397 of SEQ ID NO: 4 or its complementary sequence, and/or at least 11 consecutive nucleotides from positions 398 to 860 of SEQ ID NO: 4 or its complementary sequence; and/or

At least 11 consecutive nucleotides from positions 1 to 487 of SEQ ID NO:3 or its complementary sequence, and/or at least 11 consecutive nucleotides from positions 398 to 860 of SEQ ID NO:4 or its complementary sequence;

Preferably, the consecutive nucleotides are 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more consecutive nucleotides;

Preferably, the complementary sequence is partially complementary or fully complementary to its parent sequence;

Preferably, the nucleic acid sequence comprises 22-25 consecutive nucleotides from positions 1-487 of SEQ ID NO: 3 or its complementary sequence and 22-25 consecutive nucleotides from positions 488-1105 of SEQ ID NO: 3 or its complementary sequence; and/or 22-25 consecutive nucleotides from positions 1-397 of SEQ ID NO: 4 or its complementary sequence and 22-25 consecutive nucleotides from positions 398-860 of SEQ ID NO: 4 or its complementary sequence;

Preferably, the nucleic acid sequence comprises SEQ ID NO: 1 or its complementary sequence; and/or SEQ ID NO: 2 or its complementary sequence;

Preferably, the nucleic acid sequence comprises SEQ ID NO: 6 or its complementary sequence; and/or SEQ ID NO: 7 or its complementary sequence;

Preferably, the nucleic acid sequence comprises SEQ ID NO: 3 or its complementary sequence; and/or SEQ ID NO: 4 or its complementary sequence.
A nucleic acid molecule, the nucleic acid sequence of which comprises any one selected from the following:

(1) corresponding to nucleotide 487 of SEQ ID NO:3 or its complementary sequence and at least 11 consecutive nucleotides upstream and downstream thereof (e.g., 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more consecutive nucleotides);

Preferably, the nucleic acid sequence comprises at least 3 (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) consecutive nucleotides upstream of nucleotide 487 of SEQ ID NO: 3 or its complementary sequence;

Preferably, the nucleic acid sequence comprises at least 3 (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) consecutive nucleotides downstream of nucleotide 487 of SEQ ID NO: 3 or its complementary sequence;

Preferably, the nucleic acid sequence comprises SEQ ID NO: 1 or its complementary sequence;

Preferably, the nucleic acid sequence comprises SEQ ID NO: 6 or its complementary sequence;

Preferably, the nucleic acid sequence comprises SEQ ID NO: 3 or its complementary sequence;

Preferably, the complementarity is partial complementarity or complete complementarity;

(2) corresponding to nucleotide 397 of SEQ ID NO:4 or its complementary sequence and at least 11 consecutive nucleotides upstream and downstream thereof (e.g., 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more consecutive nucleotides);

Preferably, the nucleic acid sequence comprises at least 3 (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11 or more) consecutive nucleotides upstream of nucleotide 397 of SEQ ID NO: 4 or its complementary sequence;

Preferably, the nucleic acid sequence comprises at least 3 (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11 or more) consecutive nucleotides downstream of the 397th nucleotide of SEQ ID NO: 4 or its complementary sequence;

Preferably, the nucleic acid sequence comprises SEQ ID NO: 2 or its complementary sequence;

Preferably, the nucleic acid sequence comprises SEQ ID NO:7 or its complementary sequence;

Preferably, the nucleic acid sequence comprises SEQ ID NO: 4 or its complementary sequence;

Preferably, the complementarity is partial complementarity or complete complementarity;

Preferably, the presence of the nucleic acid molecule in a corn plant or its part, seed, cell or progeny is detected to determine whether the DNA of the transgenic corn event DBN9229 exists; preferably, representative samples of seeds of the transgenic corn event DBN9229 have been deposited in the China General Microbiological Culture Collection Center, with the deposit number CGMCC No. 45229.
The nucleic acid molecule according to claim 1 or 2 is characterized in that the nucleic acid sequence comprises SEQ ID NO: 5 or its complementary sequence.
A corn plant or part, seed, cell or progeny thereof, the genome of which comprises the nucleic acid molecule according to any one of claims 1 to 3;

Preferably, the representative sample of the corn seeds has been deposited in the China General Microbiological Culture Collection Center with the deposit number of CGMCC No.45229.
A product comprising the corn plant of claim 4 or a part, seed, cell or progeny thereof;

Preferably, the preparation comprises genomic DNA of the corn plant or a part, seed, cell or progeny thereof;

Preferably, the product is selected from one or more of corn ears, dehusked corn, corn silk, corn pollen, corn grits, corn flour, crushed corn, corn meal, corn oil, corn starch, corn steep liquor, corn malt, corn sugar, corn syrup, margarine produced from corn oil, unsaturated corn oil, saturated corn oil, corn flakes, popcorn, ethanol and/or liquor produced from corn, distillers dried grains (DDGS) produced from corn fermentation, animal feed from corn, cosmetics and fillers.
A method for detecting DNA of genetically modified corn in a sample, the method comprising:

(a) contacting the sample with primers (e.g., at least two primers) for amplifying the DNA in a nucleic acid amplification reaction or system;

(b) performing a nucleic acid amplification reaction; and

(c) detecting the presence of the DNA or its amplified product;

Wherein, the DNA or its amplified product comprises the nucleic acid sequence of the nucleic acid molecule according to any one of claims 1 to 3; preferably, the DNA or its amplified product comprises SEQ ID NO: 1 or its complementary sequence, SEQ ID NO: 2 or its complementary sequence, SEQ ID NO: 6 or its complementary sequence, and/or SEQ ID NO: 7 or its complementary sequence;

Optionally, in step (a), at least one probe is provided and contacted with the sample and primers;

Preferably, the sample is a DNA sample extracted from corn;

Preferably, the primer comprises the nucleic acid sequence of the nucleic acid molecule according to any one of claims 1 to 3;

Preferably, the two primers are selected from the complementary sequences of SEQ ID NO: 1 and SEQ ID NO: 9, SEQ ID NO: 8 and SEQ ID NO: 9, SEQ ID NO: 2 and SEQ ID NO: 11, SEQ ID NO: 10 and SEQ ID NO: 11, SEQ ID NO: 1 and SEQ ID NO: 2, or any combination thereof;

Preferably, the method is used to detect the presence of DNA from transgenic corn event DBN9229 in a sample.
A method for detecting DNA of genetically modified corn in a sample, the method comprising:

(a) contacting the sample with a probe, wherein the probe comprises the nucleic acid sequence of the nucleic acid molecule according to any one of claims 1 to 3; preferably, the probe comprises SEQ ID NO: 1 or its complementary sequence, SEQ ID NO: 2 or its complementary sequence, SEQ ID NO: 6 or its complementary sequence, and/or SEQ ID NO: 7 or its complementary sequence;

(b) hybridizing the sample to be detected and the probe under stringent hybridization conditions; and

(c) detecting the hybridization between the sample to be detected and the probe;

Preferably, the sample is a DNA sample extracted from corn;

Preferably, the DNA comprises the nucleic acid sequence of the nucleic acid molecule according to any one of claims 1 to 3;

Preferably, at least one of said probes is labeled with at least one fluorescent group;

Preferably, the detection probe comprises a fluorescent group and a quencher group, wherein the fluorescent group can emit a signal, and the quencher group can absorb or quench the signal emitted by the fluorescent group; and the signal emitted by the probe when hybridizing with its complementary sequence is different from the signal emitted when not hybridizing with its complementary sequence;

Preferably, the probe is selected from TaqMan probe, FRET probe, or any combination thereof;

Preferably, the method is used to detect the presence of DNA from transgenic corn event DBN9229 in a sample.
A method for detecting DNA of genetically modified corn in a sample, the method comprising:

(a) contacting the sample with a marker nucleic acid molecule, wherein the marker nucleic acid molecule comprises a nucleic acid sequence of the nucleic acid molecule according to any one of claims 1 to 3; preferably, the marker nucleic acid molecule comprises at least one selected from the group consisting of: SEQ ID NO: 1 or its complementary sequence, SEQ ID NO: 2 or its complementary sequence, and/or SEQ ID NO: 6-11 or its complementary sequence;

(b) hybridizing the sample and the marker nucleic acid molecule under stringent hybridization conditions;

(c) detecting hybridization between the sample and the marker nucleic acid molecule, and then determining through marker-assisted breeding analysis that insect resistance and/or herbicide tolerance is genetically linked to the marker nucleic acid molecule;

Preferably, the method is used to detect the presence of DNA from transgenic corn event DBN9229 in a sample.
A DNA detection kit, the kit comprising at least one DNA molecule, the DNA molecule comprising the nucleic acid sequence of the nucleic acid molecule according to any one of claims 1 to 3;

Preferably, the DNA molecule can be used as one of the DNA primers and/or probes specific to the transgenic maize event DBN9229 or its progeny;

Preferably, the DNA molecule can be used as a primer and/or probe specific for the corn plant or its part, seed, cell or progeny according to claim 4 or the product according to claim 5; preferably, the primer is as defined in claim 6; preferably, the probe is as defined in claim 7;

Preferably, the DNA molecule comprises SEQ ID NO: 1 or its complementary sequence, SEQ ID NO: 2 or its complementary sequence, SEQ ID NO: 6 or its complementary sequence, and/or SEQ ID NO: 7 or its complementary sequence.
A method for protecting corn plants from insect attack, the method comprising providing at least one transgenic corn plant cell in the diet of a target insect, the transgenic corn plant cell comprising in its genome the sequence shown in SEQ ID NO:1 and/or SEQ ID NO:2;

Preferably, target insects that feed on cells of the transgenic corn plant are inhibited from further feeding on the transgenic corn plant;

Preferably, the transgenic corn plant cell comprises the sequence shown in SEQ ID NO: 3 and/or SEQ ID NO: 4 in its genome;

Preferably, the transgenic corn plant cell comprises in its genome the nucleic acid sequences of SEQ ID NO:1, SEQ ID NO:5, positions 685-11603 and SEQ ID NO:2, or comprises the sequence shown in SEQ ID NO:5;

Preferably, the transgenic corn plant cell is obtained from the corn plant or part, seed, cell or progeny thereof of claim 4, or the product of claim 5;

Preferably, the insect is a lepidopteran pest;

Preferably, the insect is selected from the group consisting of fall armyworm (Spodoptera frugiperda), oriental armyworm (Mythimna seperata), Asian corn borer (Ostrinia furnacalis), cotton bollworm (Helicoverpa armiger), peach borer (Conogethes punctiferalis), fall armyworm (Spodoptera litura), cutworm (Agrotis ypsilon Rottemberg), European corn borer (Ostrinia nubilalis), corn armyworm (Helicoverpa zea), southwestern corn borer (Diatraea grandiosella), or any combination thereof.
A method for protecting corn plants from damage caused by herbicides or controlling weeds in a field where corn plants are planted, the method comprising applying an effective amount of a glufosinate herbicide to a field where at least one transgenic corn plant is planted, the transgenic corn plant comprising in its genome the sequence shown in SEQ ID NO:1 and/or SEQ ID NO:2, the transgenic corn plant being tolerant to the glufosinate herbicide;

Preferably, the transgenic corn plant comprises a gene that confers resistance to the glufosinate herbicide (e.g., phosphinothricin N-acetyltransferase cPAT);

Preferably, the transgenic corn plant comprises the sequence shown in SEQ ID NO: 3 and/or SEQ ID NO: 4 in its genome;

Preferably, the transgenic corn plant comprises the nucleic acid sequences of SEQ ID NO: 1, SEQ ID NO: 5, positions 685-11603 and SEQ ID NO: 2 in sequence in its genome, or comprises the sequence shown in SEQ ID NO: 5;

Preferably, the transgenic corn plant is or is obtained from the corn plant or part thereof, seed or plant of claim 4. offspring, cells or progeny.
A method of growing corn plants that are insect resistant and/or tolerant to glufosinate herbicides, the method comprising:

(a) planting at least one corn seed, wherein the genome of the corn seed comprises a nucleic acid sequence encoding an insect resistance Cry1Fa2 protein, a nucleic acid sequence encoding a Cry2Ab2 protein and/or a nucleic acid sequence encoding a glufosinate herbicide tolerance PAT protein, and a nucleic acid sequence in a specific region; or the genome of the corn seed comprises the nucleic acid sequence shown in SEQ ID NO:5;

(b) growing the corn seeds into corn plants;

(c) infesting the corn plants with target insects and/or spraying the corn plants with an effective amount of glufosinate herbicide, and harvesting corn plants having reduced plant damage compared to other plants not having the nucleic acid sequence of the specific region;

The nucleic acid sequence of the specific region comprises the sequence shown in SEQ ID NO: 1 and/or SEQ ID NO: 2; preferably, the nucleic acid sequence of the specific region comprises the sequence shown in SEQ ID NO: 3 and/or SEQ ID NO: 4;

Preferably, the insect is a lepidopteran pest;

Preferably, the insect is selected from the group consisting of fall armyworm (Spodoptera frugiperda), oriental armyworm (Mythimna seperata), Asian corn borer (Ostrinia furnacalis), cotton bollworm (Helicoverpa armiger), peach borer (Conogethes punctiferalis), fall armyworm (Spodoptera litura), cutworm (Agrotis ypsilon Rottemberg), European corn borer (Ostrinia nubilalis), corn armyworm (Helicoverpa zea), southwestern corn borer (Diatraea grandiosella), or any combination thereof;

Preferably, the corn seed is or is obtained from the corn plant of claim 4, or a part, seed, cell or progeny thereof.
A method for producing a corn plant that is resistant to insects and/or tolerant to glufosinate herbicides, the method comprising: introducing into the genome of the corn plant a nucleic acid sequence encoding an insect-resistant Cry1Fa2 protein, a nucleic acid sequence of a Cry2Ab2 protein and/or a nucleic acid sequence encoding a glufosinate-tolerant PAT protein, and a nucleic acid sequence in a specific region, wherein the nucleic acid sequence in the specific region comprises at least one nucleic acid sequence selected from the group consisting of 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;

Preferably, the method comprises converting the nucleic acid sequence encoding the insect resistance Cry1Fa2 protein, the nucleic acid sequence of the Cry2Ab2 protein and/or the nucleic acid sequence encoding the glufosinate-tolerant PAT protein contained in the genome of the first corn plant into and a nucleic acid sequence in a specific region, or the nucleic acid sequence shown in SEQ ID NO:5 contained in the genome of the first corn plant is introduced into a second corn plant, thereby producing a large number of progeny plants; selecting the progeny plants having the nucleic acid sequence in the specific region, and the progeny plants are resistant to insects and/or tolerant to glufosinate-ammonium herbicides; the nucleic acid sequence in the specific region comprises the sequence shown in SEQ ID NO:1 and/or SEQ ID NO:2; preferably, the nucleic acid sequence in the specific region comprises the sequence shown in SEQ ID NO:3 and/or SEQ ID NO:4;

Preferably, the method comprises crossing (e.g., sexually crossing) or selfing the transgenic corn event DBN9229 with another corn plant (e.g., a corn plant lacking insect resistance and/or glufosinate tolerance), thereby producing a plurality of progeny plants, and selecting the progeny plants having the nucleic acid sequence of the specific region;

Preferably, the method comprises selfing a first generation progeny plant, thereby producing a plurality of second generation progeny plants; infesting the progeny plants with target insects and/or treating the progeny plants with glufosinate-ammonium; selecting the progeny plants for resistance to the insects and/or tolerance to the glufosinate-ammonium herbicide;

Preferably, the corn plant is or is obtained from the corn plant of claim 4, or a part, seed, cell or progeny thereof.
The method according to claim 13, further comprising sexually crossing the insect-resistant and/or glufosinate-tolerant progeny plant with another corn parent and harvesting the hybrid seed produced thereby;

Preferably, the insect is a lepidopteran pest;

Preferably, the insect is selected from the group consisting of fall armyworm (Spodoptera frugiperda), oriental armyworm (Mythimna seperata), Asian corn borer (Ostrinia furnacalis), cotton bollworm (Helicoverpa armiger), peach borer (Conogethes punctiferalis), fall armyworm (Spodoptera litura), cutworm (Agrotis ypsilon Rottemberg), European corn borer (Ostrinia nubilalis), corn armyworm (Helicoverpa zea), southwestern corn borer (Diatraea grandiosella), or any combination thereof.
An agricultural product or commodity produced from transgenic corn event DBN9229, characterized in that the agricultural product or commodity is corn meal, corn flour, corn oil, corn silk, corn starch, corn gluten, corn tortillas, cosmetics or fillers.