Corn event DP-023211-2 and its detection method

Abstract

Embodiments disclosed herein relate to the field of plant molecular biology, and in particular to DNA constructs for conferring insect resistance to plants. Embodiments disclosed herein relate to insect-resistant corn plants containing event DP-023211-2 and assays for detecting the presence of event DP-023211-2 in samples and compositions thereof.

Description

[0001] References to electronically submitted sequence listings

[0002] An official copy of the sequence listing is submitted electronically via EFS-Web as an ASCII formatted sequence listing, with the file name 7493_SeqList.txt, created on April 16, 2018, and having a size of 157 kilobytes, and submitted simultaneously with this specification. The sequence listing contained in the ASCII formatted file is part of this specification and is incorporated herein by reference in its entirety.

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] This application claims the benefit of U.S. Provisional Application No. 62 / 663,832, filed April 27, 2018, U.S. Provisional Application No. 62 / 678,579, filed May 31, 2018, and U.S. Provisional Application No. 62 / 776,018, filed December 6, 2018, each of which is incorporated herein by reference in its entirety. Technical Field

[0005] Embodiments disclosed herein relate to the field of plant molecular biology, including DNA constructs for conferring insect resistance to plants. Embodiments disclosed herein also include insect-resistant corn plants containing event DP-023211-2 and assays for detecting the presence of event DP-023211-2 in samples and compositions thereof. Background Art

[0006] Corn is an important crop and a major food source in many parts of the world. Despite the use of protective measures such as chemical pesticides, damage caused by insect pests is still a major factor in the loss of corn crops in the world. In view of this, in order to control insect damage and reduce the demand for traditional chemical pesticides, insect resistance has been genetically engineered into crops such as corn. One group of genes that has been used to produce transgenic insect-resistant crops is the delta-endotoxin group from Bacillus thuringiensis (Bt). Delta-endotoxins have been successfully expressed in crops such as cotton, potatoes, rice, sunflowers, and corn, and have been shown to control insect pests well in some cases. (Perlak, F. J. et al. (1990) Bio / Technology 8:939-943; Perlak, F. J. et al. (1993) Plant Mol. Biol. 22:313-321; Fujimoto, H. et al. (1993) Bio / Technology 11:1151-1155; Tu et al. (2000) Nature Biotechnology 18:1101-1104; PCT Publication WO 01 / 13731; and Bing, J. W. et al. (2000) Efficacy of Cry1F Transgenic Maize, 14 th Biennial International Plant Resistance to Insects Workshop (14th Biennial International Plant Resistance to Insects Workshop, Fort Collins, Colorado).

[0007] It is known that transgene expression in plants is influenced by many different factors, including the orientation and composition of the expression cassette driving expression of a single gene of interest, as well as the location in the plant genome, which may be due to the structure of the chromatin (e.g., heterochromatin) or the close proximity of transcriptional regulatory elements (e.g., enhancers) to the integration site (Weising et al. (1988) Ann. Rev. Genet. 22: 421-477).

[0008] It would be advantageous to be able to detect the presence of a particular event in order to determine whether the progeny of a sexual cross contain the transgene of interest.

[0009] The presence of the transgene can be detected by nucleic acid detection methods, such as by polymerase chain reaction (PCR) or Southern hybridization using nucleic acid probes. These detection methods generally focus on commonly used genetic elements, such as promoters, terminators, marker genes, etc., because for many DNA constructs, the coding regions are interchangeable. As a result, unless the DNA sequence of the flanking DNA adjacent to the inserted heterologous DNA is known, these methods may not be useful for distinguishing between different events, particularly events generated using the same DNA construct or very similar constructs. Summary of the invention

[0010] The embodiments relate to insect-resistant corn (Zea mays) plant event DP-023211-2, also referred to as "corn line DP-023211-2," "corn event DP-023211-2," and "DP-023211-2 corn," to DNA plant expression constructs of corn plant event DP-023211-2, and methods and compositions designed for detecting the transgenic constructs, flanking and insertion (target locus) regions in corn plant event DP-023211-2 and progeny thereof.

[0011] In one aspect, these compositions and methods relate to methods for producing and selecting insect-resistant monocot crop plants. Compositions include DNA constructs that confer resistance to insects when expressed in plant cells and plants. In one aspect, a DNA construct that can be introduced into a host cell and replicated in a host cell is provided, and when expressed in a plant cell and plant, the DNA construct confer resistance to insects in the plant cell and plant. Corn event DP-023211-2 was generated by Agrobacterium-mediated transformation of plasmid PHP74643. As described herein, these events include DvSSJ1 (SEQ ID NO: 6) and IPD072 (polynucleotide SEQ ID NO: 4 and amino acid SEQ ID NO: 5) boxes, which confer resistance to certain coleopteran plant pests. The insect control components have been shown to have efficacy against western corn rootworm (WCR), northern corn rootworm (NCR) and southern corn rootworm (SCR).

[0012] The first cassette is expressed as a transcript comprising two RNA fragments from the Diabrotica virgifera (Western corn rootworm) smooth wall junction protein 1 (DvSSJ1) gene separated by an intronic linker sequence derived from the intron 1 region of the maize alcohol dehydrogenase (zm-Adh1) gene to form an inverted repeat structure. The expression of the DvSSJ1 fragment is controlled by the ubiZM1 promoter, 5'UTR and a third copy of the intron together with the terminator region of the maize W64 line 27-kDa gamma zein (Z27G) gene. Two additional terminators are present to prevent transcriptional interference: the terminator region of the Arabidopsis thaliana ubiquitin 14 (UBQ14) gene (Callis et al., 1995) and the terminator region of the maize In2-1 gene (Hershev and Stoner, 1991).

[0013] The second cassette contains the insecticidal protein gene ipd072Aa (SEQ ID NO: 4) from Pseudomonas chlororaphis. The expression of the IPD072Aa protein (SEQ ID NO: 5) in plants is effective against certain coleopteran pests, including destruction of the midgut epithelium. The IPD072Aa protein is 86 amino acids in length and has a molecular weight of approximately 10 kDa. The expression of the ipd072Aa gene is controlled by the promoter region from the banana streak virus Yunnan banana (acuminata Yunnan) strain (BSV[AY]) (Zhuang et al., 2011) and the intron region from the Zea mays ortholog of the hypothetical protein (zm-HPLV9) of Oryza sativa (rice) together with the terminator region from the Arabidopsis at-T9 gene (GenBank Accession No. NM_001202984).

[0014] The third gene cassette (mo-pat gene cassette) contains the phosphinothricin acetyltransferase gene (mo-pat) from Streptomyces viridochromogenes (Wohlleben et al., 1988). The mo-pat gene expresses the phosphinothricin acetyltransferase (PAT) that confers tolerance to phosphinothricin. The PAT protein has a length of 183 amino acids and a molecular weight of approximately 21 kDa. The expression of the mo-pat gene is controlled by the promoter and intron region of the rice (rice) actin (os actin) gene (GenBank accession number CP018159) together with the third copy of the CaMV35S terminator. Two additional terminators are present to prevent transcriptional interference: the terminator regions from the Sorghum bicolor (sorghum) ubiquitin (sb-ubi) gene (Phytozome gene ID Sobic.004G049900.1) and the gamma-kafarin (sb-gkaf) gene, respectively (de Freitas et al., 1994).

[0015] The fourth gene cassette (pmi gene cassette) contains the phosphomannose isomerase (pmi) gene from Escherichia coli (Escherichiacoli) (Negrotto et al., 2000). The expression of the PMI protein in plants serves as a selective marker, which allows plant tissues to grow with mannose as a carbon source. The length of the PMI protein is 391 amino acids and the molecular weight is about 43kDa. As present in the T-DNA region of PHP74643, the pmi gene lacks a promoter, but its position is close to the flip enzyme recombination target site FRT1, and can be expressed after recombination by a properly placed promoter. The terminator of the pmi gene is the fourth copy of the pinII terminator. The additional Z19 terminator present is to prevent transcription interference between cassettes.

[0016] According to some embodiments, compositions and methods for identifying a novel corn plant designated as DP-023211-2 (ATCC deposit number PTA-124722) are provided. These methods are based on primers or probes that specifically recognize the 5' and / or 3' flanking sequences of DP-023211-2. DNA molecules comprising primer sequences are provided that, when used in a PCR reaction, will produce amplicons unique to transgenic event DP-023211-2. In one embodiment, corn plants and seeds comprising these molecules are contemplated. In addition, kits for identifying the DP-023211-2 event using these primer sequences are provided.

[0017] Some embodiments relate to specific flanking sequences of DP-023211-2 as described herein, which can be used to develop methods for identifying DP-023211-2 in biological samples. More particularly, the present disclosure relates to 5' and / or 3' flanking regions of DP-023211-2, which can be used to develop specific primers and probes. Further embodiments relate to methods for identifying the presence of DP-023211-2 in biological samples based on the use of such specific primers or probes.

[0018] According to some embodiments, a method for detecting the presence of DNA corresponding to the corn event DP-023211-2 in a sample is provided. Such methods include: (a) contacting the sample containing DNA with a DNA primer set, which is used to produce an amplicon that is diagnostic of the corn event DP-023211-2 when subjected to a nucleic acid amplification reaction with genomic DNA extracted from corn containing event DP-023211-2; (b) performing a nucleic acid amplification reaction to produce the amplicon; and (c) detecting the amplicon. In some aspects, the primer set comprises SEQ ID NOs: 7 and 8, and optionally a probe comprising SEQ ID NO: 9.

[0019] According to some embodiments, a method for detecting the presence of a DNA molecule corresponding to the DP-023211-2 event in a sample comprises: (a) contacting a sample comprising DNA extracted from a corn plant with a DNA probe molecule that hybridizes with DNA extracted from corn event DP-023211-2 under stringent hybridization conditions and does not hybridize with control corn plant DNA under the stringent hybridization conditions; (b) subjecting the sample and probe to stringent hybridization conditions; and (c) detecting hybridization of the probe with DNA extracted from corn event DP-023211-2. More specifically, a method for detecting the presence of a DNA molecule corresponding to the DP-023211-2 event in a sample, such method comprising (a) contacting a sample comprising DNA extracted from a corn plant with a DNA probe molecule consisting of a sequence unique to the event, such as a junction sequence, wherein the DNA probe molecule hybridizes to DNA extracted from the corn event DP-023211-2 under stringent hybridization conditions and does not hybridize to control corn plant DNA under the stringent hybridization conditions; (b) subjecting the sample and probe to stringent hybridization conditions; and (c) detecting hybridization of the probe to the DNA.

[0020] Additionally, kits and methods are provided for identifying event DP-023211-2 in a biological sample for detecting specific regions of DP-023211-2.

[0021] A DNA molecule is provided to comprise at least one junction sequence of DP-023211-2; wherein the junction sequence spans the junction between heterologous DNA inserted into the genome and DNA from a corn cell flanking the insertion site (i.e., flanking DNA), and is diagnostic for a DP-023211-2 event.

[0022] According to some embodiments, a method of producing an insect-resistant corn plant comprises the following steps: (a) sexually crossing a first parent corn line comprising an expression cassette disclosed herein that confers insect resistance with a second parent corn line lacking such an expression cassette, thereby producing a plurality of offspring plants; and (b) selecting offspring plants that are insect-resistant. Such methods may optionally include the further step of backcrossing the offspring plants to the second parent corn line to produce a true-breeding corn plant that is insect-resistant.

[0023] Some embodiments provide a method for producing a corn plant resistant to insects, the method comprising transforming a corn cell with a DNA construct PHP74643, growing the transformed corn cell into a corn plant, selecting a corn plant that exhibits insect resistance, and further growing it into a fertile corn plant. The fertile corn plant can be self-pollinated or crossed with a compatible corn variety to produce insect-resistant offspring.

[0024] Some embodiments further relate to a DNA detection kit for identifying corn event DP-023211-2 in a biological sample. The kit includes a first primer that specifically recognizes the 5' or 3' flanking region of DP-023211-2, and a second primer that specifically recognizes a sequence within the non-native target locus DNA or within the flanking DNA of DP-023211-2, respectively, for use in a PCR identification scheme. Further embodiments relate to a kit for identifying event DP-023211-2 in a biological sample, the kit comprising a specific probe having a sequence corresponding to or complementary to a sequence having about 80% to 100% sequence identity with a specific region of event DP-023211-2. The sequence of the probe corresponds to a specific region that includes a portion of the 5' or 3' flanking region of event DP-023211-2. In some embodiments, the first or second primer comprises any one of SEQ ID NOs: 7-8, 10-11, 13-14, 16-17, 19-20, or 22-23.

[0025] The methods and kits encompassed by the embodiments disclosed herein can be used for various purposes, such as, but not limited to, the following purposes: identifying event DP-023211-2 in plants, plant materials, or products, such as, but not limited to, food or feed products (fresh or processed) containing or derived from plant materials; additionally or alternatively, the methods and kits can be used to identify transgenic plant materials, with the purpose of separating between transgenic and non-transgenic materials; additionally or alternatively, the methods and kits can be used to determine the quality of plant materials containing corn event DP-023211-2. The kits may also contain reagents and materials required to perform the detection methods.

[0026] Further embodiments relate to the DP-023211-2 corn plant or its parts, including but not limited to pollen, ovules, pericarp, vegetative cells, pollen nuclei and egg nuclei of the corn plant DP-023211-2 and its derived progeny. In another embodiment, the DNA primer molecules targeting the DP-023211-2 corn plant and seeds provide specific amplicon products. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 - A schematic diagram of plasmid PHP74643 with the indicated genetic elements (SEQ ID NO: 1) is shown. The plasmid size is 71,116 bp.

[0028] Figure 2 . A schematic diagram of the insertion T-DNA region of plasmid PHP74643 is shown (SEQ ID NO: 2 is the T-DNA insertion sequence, SEQ ID NO: 3 is the insertion sequence T-DNA including the landing pad), indicating eight gene cassettes. The T-DNA was used to transform a pre-characterized line containing the FRT1 and FRT87 sites. The region between the FRT1 and FRT87 sites in the T-DNA containing the pmi gene, the mo-pat gene, the DvSSJ1 fragment, and the ipd072Aa gene was integrated into the corn line in a site-specific manner.

[0029] Figure 3.Shows a schematic diagram of the insertion of DP-023211-2 corn based on the SbS sequencing ("Southern-by-Sequencing") analysis. The middle box shows a single copy of the PHP74643 T-DNA integrated between the FRT1 and FRT87 sites. The site-specific landing pad sequence is shown by the outer box, and the 5' and 3' flanking corn genome is represented by horizontal black bars. Representative individual sequencing reads of FRT1 and FRT87 connections are shown as stacked lines for each connection. The FRT1 and FRT87 sequences are highlighted at each read. For the FRT1 site, the black line within each individual read to the left of the highlighted FRT1 sequence represents the adjacent site-specific landing pad sequence, while the black on the right side of the FRT1 sequence represents the integrated PHP74643 sequence. For the FRT87 site, the black line to the left of the highlighted FRT87 sequence represents the integrated PHP74643 sequence, and the black line to the right of the FRT87 sequence represents the adjacent site-specific landing pad sequence. The numbers below the map indicate the bp position of the FRT element relative to the PHP74643 T-DNA sequence ( Figure 2 ).

[0030] Figure 4 . A schematic diagram of the transformation and development of DP-023211-2 is shown.

[0031] Figure 5 is a table showing the cross performance of the five construct designs compared to the base items of non-yield agronomic traits.

[0032] Figure 6 is a table showing the hybrid performance of Event DP-023211-2 compared to basic items of non-yield agronomic traits.

[0033] Figure 7 is a table showing the inbreeding performance of the construct designs compared to a base list of all agronomic traits.

[0034] Figure 8 is a table showing the inbreeding performance of event DP-023211-2 compared to a basic list of all agronomic traits. DETAILED DESCRIPTION

[0035] As used herein, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells, and reference to "a protein" includes reference to one or more proteins and their equivalents, and so forth. All technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs unless expressly stated otherwise.

[0036] The compositions of the present disclosure include seeds and plants, plant cells and seeds derived therefrom deposited as ATCC Patent Deposit No. PTA-124722. Applicant has deposited at least 2,500 seeds of corn event DP-023211-2 (Patent Deposit No. PTA-124722) at the American Type Culture Collection (ATCC) (Manassas, Virginia 20110-2209, USA) on January 18, 2018. These deposits will be maintained under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. The seeds deposited at ATCC on January 18, 2018 were obtained from Pioneer Hi-Bred International, Inc., 7250 NW 62 Avenue, Johnston Island, Iowa 50131-1000. nd Avenue, Johnston, Iowa 50131-1000). The deposit is available during the period of application to the Commissioner of the Patent and Trademark Office and persons determined by the Commissioner to be qualified under the request. After any claim in this application is allowed, in accordance with 37 C.FR section 1.808, applicant will make available to the public a sample of at least 2,500 hybrid corn seeds at the American Type Culture Collection (ATCC), 10801 University Avenue, Manassas, Virginia 20110-2209. The deposited seeds of corn event DP-023211-2 will be maintained in the ATCC depository (which is a public depository) for 30 years, or 5 years after the most recent claim, or for the exercisable period of the patent, whichever is longer, and will be replaced if they become non-viable during this period. In addition, one or more applicants have met all of the requirements of 37 C.FR §§ 1.801-1.809, including providing an indication of the viability of the sample at the time of deposit. Applicants do not have the right to override any legal restrictions on the removal of biological material or its transportation in commerce. Applicants do not waive any infringement of rights granted by this patent or rights under the Plant Variety Protection Act (7 USC 2321 et seq.) applicable to Event DP-023211-2. Unauthorized seed propagation is prohibited. The seeds may be regulated.

[0037] As used herein, the term "corn" refers to Zea mays or maize, and includes all plant varieties that can be bred with corn, including wild corn species.

[0038] As used herein, the terms "insect resistance" and "affecting insect pests" refer to changes that affect insect feeding, growth and / or behavior at any developmental stage, including but not limited to: killing the insect; slowing growth; reducing reproductive capacity; inhibiting feeding; etc.

[0039] As used herein, the terms "pesticide activity" and "insecticide activity" are used synonymously to refer to the activity of an organism or substance (e.g., such as a protein), which activity can be measured by a number of parameters, including but not limited to pest mortality, pest weight loss, pest attraction, pest resistance, and other behavioral and physical changes in pests after ingestion and / or exposure to the organism or substance for an appropriate period of time. For example, a "pesticide protein" is a protein that exhibits pesticide activity by itself or in combination with other proteins.

[0040] As used herein, "insert DNA" refers to heterologous DNA within an expression cassette used to transform plant material, while "flanking DNA" can be genomic DNA naturally present in an organism such as a plant, or can be foreign (heterologous) DNA introduced through a transformation process unrelated to the original insert DNA molecule, such as a fragment associated with the transformation event. As used herein, "flanking region" or "flanking sequence" refers to a sequence of at least 20 bp (for some narrower embodiments, at least 50 bp, and up to 5000 bp) that is located immediately upstream and adjacent to the original non-native insert DNA molecule and / or immediately downstream and adjacent to it. The transformation procedure of the foreign DNA can result in transformants containing different flanking regions that are characteristic and unique to each transformant. When recombinant DNA is introduced into a plant by traditional hybridization, its flanking regions are generally unchanged. Through several generations of plant breeding and traditional hybridization, single nucleotide changes may occur in the flanking regions. Transformants will also contain a unique junction between a piece of heterologous insert DNA and genomic DNA, or two (2) pieces of genomic DNA or two (2) pieces of heterologous DNA. A "junction" is the point at which two (2) specific DNA fragments are joined. For example, a junction exists where the inserted DNA joins the flanking DNA. Junction points also exist in transformed organisms where two (2) DNA fragments are joined together in a manner modified from that found in the native organism. "Junction DNA" refers to the DNA that contains the junction point. The junction sequences listed in the present disclosure include the junction point between the maize genomic DNA and the 5' end of the insertion sequence, which is from at least -5 to +5 nucleotides (SEQ ID NO:31) of the junction point, from at least -10 to +10 nucleotides (SEQ ID NO:32) of the junction point, from at least -15 to +15 nucleotides (SEQ ID NO:33) of the junction point, and from at least -20 to +20 nucleotides (SEQ ID NO:34) of the junction point; and the junction point between the 3' end of the insertion sequence and the maize genomic DNA, which is from at least -5 to +5 nucleotides (SEQ ID NO:35) of the junction point, from at least -10 to +10 nucleotides (SEQ ID NO:36) of the junction point, from at least -15 to +15 nucleotides (SEQ ID NO:37) of the junction point, and from at least -20 to +20 nucleotides (SEQ ID NO:38) of the junction point. The junction sequences listed in this disclosure also include the junction point between the target locus and the 5' end of the insert sequence. In some embodiments, SEQ ID NO: 9 or 25 for DP-023211-2 represents the junction point between the target locus and the 5' end of the insert sequence.

[0041] As used herein, "heterologous" with respect to a nucleic acid sequence refers to a nucleic acid sequence that originates from a different, non-sexually compatible species or, if from the same species, is a sequence that has been substantially modified from its native form in composition and / or genomic locus by deliberate human intervention. For example, a promoter operably linked to a heterologous nucleotide sequence may be from a species different from the species from which the nucleotide sequence was derived or, if from the same species, the promoter is not naturally operably linked to the nucleotide sequence. A heterologous protein may originate from a foreign species or, if from the same species, may be substantially modified from its native form by deliberate human intervention.

[0042] The term "regulatory element" refers to a nucleic acid molecule with gene regulatory activity, that is, a nucleic acid molecule that can affect the transcription and / or translation expression pattern of an operably connected transcribable polynucleotide. Therefore, the term "gene regulatory activity" refers to the ability to affect the expression of such an operably connected transcribable polynucleotide molecule by affecting the transcription and / or translation of the operably connected transcribable polynucleotide molecule. Gene regulatory activity can be positive and / or negative, and the influence can be characterized by its following characteristics: time, space, development, tissue, environment, physiology, pathology, cell cycle and / or chemical response, and can also be characterized by quantitative indication or qualitative indication.

[0043] "Promoter" refers to a nucleotide sequence that can control the expression of a coding sequence or functional RNA. Generally speaking, the coding sequence is located at the 3' end of the promoter sequence. The promoter sequence contains a proximal element and a more distal upstream element, the latter element is usually called an enhancer. Therefore, an "enhancer" is a nucleotide sequence that can stimulate promoter activity, and can be an intrinsic element of the promoter or an inserted heterologous element to enhance the level or tissue specificity of the promoter. The promoter can be derived entirely from natural genes, or composed of different elements derived from different promoters found in nature, or even contain synthetic nucleotide segments. It should be understood by those skilled in the art that different regulatory elements may guide the expression of genes in different tissues or cell types, or at different developmental stages, or in response to different environmental conditions. In most cases, the promoter that causes the expression of nucleic acid fragments in most cell types is usually called a "constitutive promoter". It is further recognized that, since the exact boundaries of the regulatory sequence cannot be fully determined in most cases, nucleic acid fragments of different lengths can have the same promoter activity.

[0044] The "translation leader sequence" refers to a nucleotide sequence located between the promoter sequence and the coding sequence of a gene. The translation leader sequence is present in the fully processed mRNA upstream of the translation initiation sequence. The translation leader sequence can affect multiple parameters, including the processing of the primary transcript to mRNA, mRNA stability and / or translation efficiency.

[0045] "3' non-coding sequences" refer to nucleotide sequences located downstream of the coding sequence and include polyadenylation recognition sequences and other sequences encoding regulatory signals that can affect mRNA processing or gene expression. The polyadenylation signal is usually characterized by affecting the addition of polyadenylic acid tracts to the 3' end of the mRNA precursor.

[0046] DNA construct is the assembly of DNA molecules connected together, which can provide one or more expression cassettes. DNA construct can be a plasmid that can replicate itself in bacterial cells, and contains various endonuclease restriction sites, which can be used to introduce DNA molecules that provide functional genetic elements, i.e., promoters, introns, leader sequences, coding sequences, 3' terminator regions, etc.; or DNA construct can be a linear assembly of DNA molecules, such as expression cassettes. The expression cassette contained in the DNA construct comprises the genetic elements necessary for messenger RNA transcription. The expression cassette can be designed to be expressed in prokaryotic or eukaryotic cells. The expression cassette of the embodiment is designed to be expressed in plant cells.

[0047] The DNA molecules disclosed herein are provided in expression cassettes for expression in target organisms. The cassettes include 5' and 3' regulatory sequences operably linked to the coding sequence. "Operably linked" means that the nucleic acid sequences being linked are continuous, and in the case where it is necessary to link two protein coding regions, are continuous and in the same reading frame. Operably linked refers to a functional connection between a promoter and a second sequence, wherein the promoter sequence initiates and mediates transcription of a DNA sequence corresponding to the second sequence. The cassettes may additionally include at least one additional gene to be co-transformed into an organism. Alternatively, the one or more additional genes may be provided on multiple expression cassettes or multiple DNA constructs.

[0048] The expression cassette may include, in the 5' to 3' direction of transcription: a transcription and translation initiation region, a coding region, and a transcription and translation termination region that function in an organism as a host. The transcription initiation region (e.g., the promoter) may be native or similar to the host organism, or foreign or heterologous. In addition, the promoter may be a native sequence, or alternatively, a synthetic sequence. The expression cassette may additionally include a 5' leader sequence in the expression cassette construct. Such a leader sequence may serve to enhance translation.

[0049] It should be understood that the term "transgenic," as used herein, generally includes any cell, cell line, callus, tissue, plant part, or plant whose genotype has been altered by the presence of a heterologous nucleic acid, including those initially so altered as well as those generated from the initial transgene by sexual crosses or asexual propagation and retaining such heterologous nucleic acid.

[0050] Transgenic "events" are produced by transforming plant cells with one or more heterologous DNA constructs comprising a nucleic acid expression cassette comprising a transgene of interest; regenerating a plant population resulting from the insertion of the transgene into the genome of the plant; and selecting specific plants characterized by insertion into a specific genomic location. An event is phenotypically characterized as the expression of a transgene. At the genetic level, an event is part of the genetic makeup of a plant. The term "event" also refers to the offspring resulting from a sexual outcross between a transformant and another variety, wherein the offspring comprises heterologous DNA. After backcrossing with the recurrent parent, the inserted DNA and linked flanking genomic DNA from the transformed parent are also present in the offspring hybridized at the same chromosomal location. Offspring plants may contain changes in the inserted sequence due to conventional breeding techniques. The term "event" also refers to DNA from an original transformant, comprising the inserted DNA and flanking sequences immediately adjacent to the inserted DNA, which is expected to be transferred to progeny that receive the inserted DNA including the transgene of interest, resulting from a sexual cross between a parental line that includes the inserted DNA (e.g., the original transformant and progeny produced by selfing) and a parental line that does not contain the inserted DNA.

[0051] Insect-resistant DP-023211-2 corn plants can be bred by first sexually crossing a first parent corn plant with a second parent corn plant to produce a plurality of first offspring plants, wherein the first parent corn plant has a transgenic DP-023211-2 event plant derived from transformation and its offspring, wherein the transformation is performed using an expression cassette of the embodiment that confers insect resistance, wherein the second parent corn plant lacks such an expression cassette; and then selecting a first offspring plant that is resistant to insects; and selfing the first offspring plant to produce a plurality of second offspring plants; and then selecting insect-resistant plants from the second offspring plants. These steps may further include backcrossing the first insect-resistant offspring plant or the second insect-resistant offspring plant with the second parent corn plant or the third parent corn plant, thereby producing a corn plant that is resistant to insects. The term "selfing" refers to self-pollination, including the union of gametes and / or nuclei from the same organism.

[0052] As used herein, the term "plant" includes reference to whole plants, plant parts, plant organs (e.g., leaves, stems, roots, etc.), seeds, plant cells, and progeny thereof. In some embodiments, transgenic plant parts include, for example, plant cells, protoplasts, tissues, callus, embryos, and flowers, stems, fruits, leaves, and roots derived from transgenic plants previously transformed with the DNA molecules disclosed herein, or progeny thereof, and thus consist at least in part of transgenic cells.

[0053] As used herein, the term "plant cell" includes, but is not limited to, seeds, suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen, and microspores. The plant species that can be used are generally as broad as the class of higher plants suitable for transformation techniques, including monocots and dicots.

[0054] "Transformation" refers to the transfer of a nucleic acid fragment into the genome of a host organism, resulting in genetically stable inheritance. Host plants containing the transformed nucleic acid fragment are referred to as "transgenic" plants.

[0055] As used herein, the term "progeny" in the context of event DP-023211-2 refers to the offspring of any generation comprising a parent plant of corn event DP-023211-2.

[0056] The isolated polynucleotides disclosed herein can be incorporated into a recombinant construct, typically a DNA construct, which is capable of introduction into and replication in a host cell. Such a construct can be a vector, which includes a replication system and sequences capable of transcribing and translating a polypeptide coding sequence in a given host cell. Many vectors suitable for stable transfection of plant cells or for establishing transgenic plants are described in, for example, Pouwels et al., (1985; 1987 Supplement) Cloning Vectors: A Laboratory Manual, Weissbach and Weissbach (1989) Methods for Plant Molecular Biology, (Academic Press, New York); and Flevin et al., (1990) Plant Molecular Biology Manual, (Kluwer Academic Publishers). Typically, a plant expression vector includes, for example, one or more cloned plant genes under the transcriptional control of 5' and 3' regulatory sequences and a dominant selectable marker. Such plant expression vectors may also comprise a promoter regulatory region (e.g., one that controls inducible or constitutive, environmentally or developmentally regulated, or cell- or tissue-specific expression), a transcriptional initiation start site, a ribosome binding site, an RNA processing signal, a transcriptional termination site and / or a polyadenylation signal.

[0057] In the process of introducing the insertion sequence into the plant cell genome, it is not uncommon for the insertion sequence and / or the genome flanking sequence to have some deletions or other changes. Therefore, the relevant section of the plasmid sequence provided herein may include some minor variations. It is also possible for the flanking sequence provided herein. Therefore, the plant comprising the polynucleotides with a certain range of identity to the flanking and / or insertion sequence of the subject disclosure is within the scope disclosed in the subject. The identity with the sequence disclosed herein can be a polynucleotide sequence with at least 65% sequence identity, at least 70% sequence identity, at least 75% sequence identity, at least 80% identity, or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity. Hybridization and hybridization conditions as provided herein can also be used to define such plants and polynucleotide sequences of the subject disclosure. The sequence comprising the flanking sequences plus the entire insert sequence can be confirmed by reference to the deposited seeds.

[0058] In some embodiments, two different transgenic plants can also be crossed to produce offspring containing two independently segregating added exogenous genes. Selfing of appropriate progeny can produce plants that are homozygous for both added exogenous genes. Backcrossing to the parental plants and crossbreeding with non-transgenic plants, i.e., vegetative propagation, is also contemplated.

[0059] A "probe" is an isolated nucleic acid to which a conventional synthetic detectable label or reporter molecule, such as a radioisotope, ligand, chemiluminescent agent, or enzyme is attached. Such a probe is complementary to a target nucleic acid strand, such as a DNA strand isolated from corn event DP-023211-2, whether from a corn plant or from a sample containing the event DNA. Probes may include not only deoxyribonucleic acid or ribonucleic acid, but may also include polyamides and other modified nucleotides that specifically bind to a target DNA sequence and can be used to detect the presence of the target DNA sequence.

[0060] "Primers" are isolated nucleic acids that anneal to complementary target DNA strands by nucleic acid hybridization to form a hybrid between the primer and the target DNA strand, which is then extended along the target DNA strand by a polymerase (e.g., DNA polymerase). A primer pair refers to a pair of primers that are used to amplify a target nucleic acid sequence, such as by PCR or other conventional nucleic acid amplification methods. "PCR" or "polymerase chain reaction" is a technique for amplifying specific DNA segments (see U.S. Pat. Nos. 4,683,195 and 4,800,159; incorporated herein by reference).

[0061] Probe and primer have enough nucleotide length, can be under the hybridization condition or reaction condition determined by operator, specifically bind target DNA sequence.This length can be any length that is enough to be useful in the detection method of selection.Usually, the length of 11 or more nucleotides, 18 or more nucleotides and 22 or more nucleotides is used.This type of probe and primer hybridizes specifically with target sequence under high stringent hybridization condition.Although can design probe different from target DNA sequence and retain the ability of hybridizing with target DNA sequence by conventional method, probe and primer according to embodiment can have the complete DNA sequence similarity of continuous nucleotides with target sequence.Probe can be used as primer, but is usually designed to be combined with target DNA or RNA, and is not used for amplification process.

[0062] Specific primers can be used to amplify the integrated fragment to produce an amplicon that can be used as a "specific probe" to identify the event DP-023211-2 in a biological sample. When the probe hybridizes with the nucleic acid of the biological sample under conditions that allow the probe to bind to the sample, such binding can be detected, thereby indicating the presence of the event DP-023211-2 in the biological sample. In one embodiment of the present disclosure, the specific probe is a sequence that specifically hybridizes to a region within the 5' or 3' flanking region of the event under appropriate conditions, and also includes a portion of foreign DNA adjacent thereto. The specific probe may include a sequence that is at least 80%, 80% to 85%, 85% to 90%, 90% to 95%, and 95% to 100% identical (or complementary) to a specific region of the event.

[0063] Methods for making and using probes and primers are described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Vols. 1-3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York 1989 (hereinafter, "Sambrook et al., 1989"); Ausubel et al., eds., Current Protocols in Molecular Biology, Green Publishing and Wiley Scientific, New York, 1995 (including periodic updates) (hereinafter, "Ausubel et al., 1995"); and Innis et al., PCR Protocols: A Guide to Methods and Applications, Academic Press: San Diego, 1990. PCR primer pairs can be derived from known sequences, for example, by using computer programs for this purpose, such as the PCR Primer Analysis Tool in Vector NTI Version 6 (Informax Inc., Bethesda, MD); PrimerSelect (DNASTAR, Madison, WI); and Primer (Version 6). 1991, Whitehead Institute for Biomedical Research, Cambridge, MA). Alternatively, the sequences can be visually scanned and primers identified manually using guidelines known to those skilled in the art.

[0064] As used herein, "kit" refers to a set of reagents and optionally instructions for the purpose of performing the method embodiments of the present disclosure, more particularly, for the identification of event DP-023211-2 in a biological sample. Kits can be used and their components can be specifically adjusted for the purpose of quality control (e.g., purity of a seed batch), detection of event DP-023211-2 in plant material or materials containing or derived from plant material (such as but not limited to food or feed products). As used herein, "plant material" refers to material obtained from or derived from a plant.

[0065] The disclosed sequences can be confirmed (and, if necessary, corrected) by conventional methods, such as by re-cloning and sequencing such sequences using primers and probes based on the flanking DNA and insert sequences disclosed herein. The nucleic acid probes and primers hybridize to the target DNA sequence under stringent conditions. The presence of DNA from a transgenic event in a sample can be identified using any conventional nucleic acid hybridization or amplification method.

[0066] If a nucleic acid molecule exhibits complete complementarity or minimal complementarity with another nucleic acid molecule, they are referred to as the "complementary sequence" of the other nucleic acid molecule. As used herein, when each nucleotide of a molecule in a molecule is complementary to the nucleotide of another molecule, the molecules are said to exhibit "complete complementarity". If two molecules can hybridize to each other with sufficient stability so that they remain annealed to each other under at least conventional "low stringency" conditions, they are referred to as "minimum complementarity". Similarly, if the molecules can hybridize to each other with sufficient stability so that they remain annealed to each other under conventional "high stringency" conditions, they are referred to as "complementary". Conventional stringency conditions are described by Sambrook et al., 1989 and by Haymes et al. in Nucleic Acid Hybridization, a Practical Approach [nucleic acid hybridization, practical method], IRL Press, Washington, D.C. (1985), therefore, deviations from complete complementarity are permissible, as long as such deviations do not completely exclude the ability of the molecule to form a double-stranded structure. In order for a nucleic acid molecule to be useful as a primer or probe, it need only be sufficiently complementary in sequence to be able to form a stable double-stranded structure in the particular solvent and salt concentrations used.

[0067] In hybridization reactions, specificity is typically determined by the function of post-hybridization washes, with the critical factors being the ionic strength and temperature of the final wash solution. m ) is the temperature (under defined ionic strength and pH) at which 50% of the complementary target sequence hybridizes to a perfectly matched probe. For DNA-DNA hybrids, T m It can be estimated from the equation of Meinkoth and Wahl, (1984) Anal. Biochem. 138: 267-284: T m =81.5°C + 16.6 (log M) + 0.41 (% GC) - 0.61 (% form) - 500 / L; where M is the molar concentration of monovalent cations, % GC is the percentage of guanosine and cytosine nucleotides in DNA, % form is the percentage of formamide in the hybridization solution, and L is the base pair length of the hybrid. For every 1% mismatch, T m decrease by about 1°C; therefore, T m , hybridization and / or washing conditions to hybridize to sequences of the desired identity. For example, if sequences with >90% identity are sought, T m Generally, stringent conditions are selected to be greater than the T for the specific sequence and its complementary sequence under a defined ionic strength and pH. m However, in some embodiments, other stringent conditions can be used, including very stringent conditions that can use conditions lower than T mHybridization and / or washing at 1°C, 2°C, 3°C or 4°C lower; moderate stringency conditions can be used at a temperature lower than T m Hybridization and / or washing at 6°C, 7°C, 8°C, 9°C or 10°C lower; low stringency conditions can be used with a temperature lower than T m Hybridization and / or washing at 11°C, 12°C, 13°C, 14°C, 15°C or 20°C.

[0068] Using the equation, hybridization and wash compositions, and the desired T m , the skilled artisan will appreciate that variations in the stringency of the hybridization and / or wash solutions are essentially described. m If the temperature is less than 45°C (aqueous solution) or 32°C (formamide solution), the user can choose to increase the SSC concentration to allow the use of higher temperatures. A comprehensive guide to the hybridization of nucleic acids is found in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology - Hybridization with Nucleic Acid Probes, Part I, Chapter 2 (Elsevier, New York); and Ausubel et al., eds. (1995) and Sambrook et al. (1989).

[0069] In certain embodiments, the complementary sequence has the same length as the nucleic acid molecule hybridized with it. In certain embodiments, the complementary sequence is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides longer or shorter than the nucleic acid molecule hybridized with it. In certain embodiments, the complementary sequence is 1%, 2%, 3%, 4% or 5% longer or shorter than the nucleic acid molecule hybridized with it. In certain embodiments, the complementary sequence is complementary on the basis of nucleotide to nucleotide, which means that there is no mispairing nucleotide (each A is paired with T, and each G is paired with C). In certain embodiments, the complementary sequence comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or less mispairing. In certain embodiments, the complementary sequence comprises 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% or less mispairing.

[0070] "Percentage (%) of sequence identity" is determined relative to a reference sequence (subject sequence) as the percentage of amino acid residues or nucleotides in a candidate sequence (query sequence) that are identical to the corresponding amino acid residues or nucleotides in the reference sequence after aligning the sequences and introducing gaps (if necessary) to achieve maximum percentage sequence identity, and without considering any conservative substitutions of amino acids as part of sequence identity. Alignment for the purpose of determining percentage of sequence identity can be achieved in various ways within the technical scope of the art, for example, using publicly available computer software, such as BLAST, BLAST-2. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithm required to achieve maximum alignment over the full length of the sequence being compared. The percentage of identity between two sequences is a function of the number of identical positions shared by the sequences (e.g., percentage of identity of the query sequence = number of identical positions between the query sequence and the subject sequence / total number of positions of the query sequence × 100).

[0071] With respect to amplification of a target nucleic acid sequence using a specific amplification primer pair (e.g., by PCR), stringent conditions allow the primer pair to hybridize only to the target nucleic acid sequence to which a primer having a corresponding wild-type sequence (or its complement) binds and optionally to produce a unique amplification product amplicon in a DNA thermal amplification reaction.

[0072] As used herein, "amplified DNA" or "amplicon" refers to the product of nucleic acid amplification of a target nucleic acid sequence as part of a nucleic acid template. For example, in order to determine whether a corn plant produced by sexual crosses contains transgenic event genomic DNA from a corn plant disclosed herein, a DNA primer pair can be used to subject DNA extracted from a plant tissue sample to a nucleic acid amplification method, wherein the DNA primer pair includes a first primer derived from a flanking sequence adjacent to an insertion site of an inserted heterologous DNA and a second primer derived from the inserted heterologous DNA to produce an amplicon that diagnoses the presence of event DNA. Alternatively, the second primer may be derived from a surgical flanking sequence. The length and sequence of the amplicon may diagnose the event. The length of the amplicon may be any length from the total length of the primer pair plus a nucleotide base pair to an amplicon that may be produced by a DNA amplification scheme. Alternatively, the primer pair may be derived from flanking sequences on both sides of the inserted DNA to produce an amplicon that includes the entire inserted nucleotide sequence of the PHP74643 expression construct and a portion of the sequence flanking the transgenic insertion sequence. The members of the primer pair derived from the flanking sequence may be located at a distance from the inserted DNA sequence that may be from one nucleotide base pair up to the limit of the amplification reaction. The use of the term "amplicon" specifically excludes primer dimers that may be formed in a DNA thermal amplification reaction.

[0073] Nucleic acid amplification can be accomplished by any of a variety of nucleic acid amplification methods known in the art, including PCR. A variety of amplification methods are known in the art, particularly as described in U.S. Patent Nos. 4,683,195 and 4,683,202 and Innis et al., (1990) supra. PCR amplification methods have been developed to amplify up to 22 Kb of genomic DNA and up to 42 Kb of phage DNA (Cheng et al., Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States] 91: 5695-5699, 1994). These methods and other methods known in the field of DNA amplification can be used to implement the embodiments of the present disclosure. It is understood that many parameters in a particular PCR protocol may need to be adjusted for specific laboratory conditions and may be slightly modified while still allowing for the collection of similar results. These adjustments will be apparent to those skilled in the art.

[0074] The amplicons produced by these methods can be detected by a variety of techniques, including but not limited to locus analysis (Nikiforov et al., Nucleic Acid Res. 22: 4167-4175, 1994), in which DNA oligonucleotides are designed that overlap both the adjacent flanking DNA sequence and the inserted DNA sequence. The oligonucleotides are immobilized in wells of a microtiter plate. After PCR of the region of interest (e.g., using one primer in the inserted sequence and one primer in the adjacent flanking sequence), the single-stranded PCR product can be hybridized to the immobilized oligonucleotide and used as a template for a single base extension reaction using a DNA polymerase and a labeled ddNTP specific for the expected next base. The readout can be fluorescent or ELISA-based. A signal indicates the presence of the insert / flanking sequence due to successful amplification, hybridization, and single base extension.

[0075] Another detection method is the pyrophosphate sequencing technology described by Winge (2000) Innov.Pharma.Tech. [Innovative Pharmaceutical Technology] 00: 18-24. In this method, an oligonucleotide is designed that overlaps the adjacent DNA and the inserted DNA. The oligonucleotide is hybridized with a single-stranded PCR product from the target region (e.g., a primer in the inserted sequence and a primer in the flanking sequence) and incubated in the presence of DNA polymerase, ATP, sulfidase, luciferase, apyrase, adenosine 5' phosphosulfate and fluorescein. dNTPs are added separately and incorporated to produce a measurable light signal. The light signal indicates the presence of transgenic insertion / flanking sequences due to successful amplification, hybridization and single-base or multi-base extension.

[0076] Fluorescence polarization, as described by Chen et al. (1999) Genome Res. 9: 492-498, is also a method that can be used to detect amplicons. Using this method, an oligonucleotide is designed that overlaps the flanking and inserted DNA junctions. The oligonucleotide is hybridized to a single-stranded PCR product from the region of interest (e.g., one primer in the inserted DNA and one primer in the flanking DNA sequence) and incubated in the presence of a DNA polymerase and fluorescently labeled ddNTPs. Single base extension results in incorporation of the ddNTP. Incorporation can be measured using a fluorometer as a change in polarization. The change in polarization indicates the presence of the transgenic insert / flanking sequence due to successful amplification, hybridization, and single base extension.

[0077] Quantitative PCR (qPCR) is described as a method for detecting and quantifying the presence of a DNA sequence and is well understood in the instructions provided by commercially available manufacturers. Briefly, in one such qPCR method, a FRET oligonucleotide probe is designed that overlaps the flanking and insert DNA. The FRET probe and PCR primers (one primer in the insert DNA sequence and one primer in the flanking genomic sequence) are cycled in the presence of a thermostable polymerase and dNTPs. Hybridization of the FRET probe results in cleavage and release of the fluorescent moiety, moving it away from the quenching moiety on the FRET probe. The fluorescent signal indicates the presence of the flanking / transgenic insert sequence due to successful amplification and hybridization.

[0078] Molecular beacons have been described for sequence detection as described by Tyangi et al. (1996) Nature Biotech. 14: 303-308. Briefly, a FRET oligonucleotide probe is designed that overlaps the flanking and insert DNA. The unique structure of the FRET probe causes it to contain a secondary structure that holds the fluorescent and quenching moieties in close proximity. The FRET probe and PCR primers (e.g., one primer in the insert DNA sequence and one primer in the flanking sequence) are cycled in the presence of a thermostable polymerase and dNTPs. Following successful PCR amplification, hybridization of the FRET probe to the target sequence results in removal of the probe secondary structure and spatial separation of the fluorescent and quenching moieties. A fluorescent signal is generated. The fluorescent signal indicates the presence of the flanking / transgenic insert sequence due to successful amplification and hybridization.

[0079] Hybridization reactions using probes specific for sequences found within the amplicon are yet another method for detecting amplicons produced by PCR reactions.

[0080] Insect pests include insects selected from the following orders: Coleoptera, Diptera, Hymenoptera, Lepidoptera, Mallophaga, Homoptera, Hemiptera, Orthroptera, Thysanoptera, Dermaptera, Isoptera, Anoplura, Siphonaptera, Trichoptera, etc., especially Coleoptera and Lepidoptera.

[0081] Of concern are larvae and adults of the order Coleoptera, including weevils from the families Anthribidae, Bruchidae, and Curculionidae, including, but not limited to: Anthonomus grandis Boheman (boll weevil); Cylindrocopturus adspersus LeConte (sunflower stem weevil); Diaprepes abbreviatus Linnaeus (root weevil); Hyperapunctata Fabricius (clover leaf weevil); Lissorhoptrusoryzophilus Kuschel (rice water weevil); Metamasius hemipterus hemipterus Linnaeus (West Indian sugarcane weevil); cane weevil); M. hemipterus sericeus Olivier or silky cane weevil; Sitophilus granarius Linnaeus (granary weevil); S. oryzae Linnaeus (rice weevil); Smicronyx fulvus LeConte (red sunflower seedweevil); S. sordidus LeConte (gray sunflower seedweevil); Sphenophorus maidis Chittenden (maizebillbug); S.livis Vaurie (sugarcane weevil); Rhabdoscelus obscurus Boisduval (New Guinea sugarcane weevil); flea beetles, cucumber beetles, rootworms, leaf beetles, potato beetles and leaf miners of the family Chrysomelidae, including but not limited to: Chaetocnema ectypa Horn (desert cornflea beetle); C. pulicaria Melsheimer (corn flea beetle); Colaspis brunnea Fabricius (grape colaspis); Diabrotica barberi Smith & Lawrence (northern cornrootworm); D. undecimpunctata howardi (cucumber eleven-spotted leaf beetle) Barber) (southern corn rootworm); corn rootworm (D.virgifera virgifera LeConte (western corn rootworm); Leptinotarsa ​​decemlineata Say (Colorado potato beetle); Oulema melanopus Linnaeus (cerealleaf beetle); Phyllotreta cruciferae Goeze (corn flea beetle); Zygogramma exclamationis Fabricius (sunflower beetle); beetles from the family Coccinellidae, including but not limited to: Epilachna varivestis Mulsant (Mexican bean beetle); beetles and other beetles from the family Scarabaeidae, including but not limited to: Antitrogus parvulus Britton (Childers' sugarcane grub); Cyclocephala borealis Arrow (northernmasked chafer, white grub); C. immaculata Olivier (southern masked chafer, white grub); Dermolepida albohirtum Waterhouse (grey-backed sugarcane beetle); Euetheola humilis rugiceps LeConte (sugarcane beetle); Lepidiota frenchi Blackburn (French sugarcane grub); Tomarus gibbosus De Geer (carrot beetle); T. subtropicus Blatchley (sugarcane grub); Phyllophaga crinita Burmeister (white grub); P.latifrons LeConte (June beetle); Popillia japonica Newman (Japanese beetle); Rhizotrogus majalis Razoumowsky (European chafer); carpet beetles from the family Dermestidae; wireworms from the family Elateridae, Eleodes spp., Melanotus spp. (including M. communis Gyllenhal); Conoderus spp.; Limonius spp.; Agriotes spp.; Ctenicera spp.; Aeolus spp. spp.); bark beetles from the family Scolytidae; beetles from the family Tenebrionidae; beetles from the family Cerambycidae, such as but not limited to Migdolus fryanus Westwood (longhorn beetle); and beetles from the family Buprestidae, including but not limited to Aphanisticus cochinchinae seminulum Obenberger (leaf-mining buprestid beetle).

[0082] In certain embodiments, the DP-023211-2 corn event may also include a stack of additional traits. Plants comprising a stack of polynucleotide sequences may be obtained by conventional breeding methods or by one or both of genetic engineering methods. The method includes, but is not limited to, breeding a single line each comprising a target polynucleotide, transforming a transgenic plant comprising a gene disclosed herein with subsequent gene transformation, and co-converting the gene into a single plant cell. As used herein, the term "stacked" includes making multiple traits present in the same plant (i.e., two traits are incorporated into the nuclear genome, one trait is incorporated into the nuclear genome and another trait is incorporated into the genome of the plastid, or two traits are incorporated into the genome of the plastid).

[0083] In some embodiments, the DP-023211-2 corn events disclosed herein, alone or stacked with one or more additional insect resistance traits, can be stacked with one or more additional input traits (e.g., herbicide resistance, fungal resistance, viral resistance, stress tolerance, disease resistance, male sterility, stalk strength, etc.) or output traits (e.g., increased yield, modified starch, improved oil properties, balanced amino acids, high lysine or methionine, increased digestibility, improved fiber quality, drought resistance, etc.). Thus, the embodiments can be used to provide a complete agronomic program of improved crop quality with the ability to flexibly and cost-effectively control any number of agronomic pests.

[0084] In another embodiment, the DP-023211-2 corn event can be stacked with one or more additional Bt insecticidal toxins, including but not limited to the Cry3B toxins disclosed in U.S. Pat. Nos. 8,101,826, 6,551,962, 6,586,365, 6,593,273 and PCT Publication No. WO 2000 / 011185; the mCry3B toxins disclosed in U.S. Pat. Nos. 8,269,069 and 8,513,492; the mCry3A toxins disclosed in U.S. Pat. Nos. 8,269,069, 7,276,583 and 8,759,620; or the Cry34 / 35 toxins disclosed in U.S. Pat. Nos. 7,309,785, 7,524,810, 7,985,893, 7,939,651 and 6,548,291. In further embodiments, the DP-023211-2 corn event can be stacked with one or more additional transgenic events comprising these Bt insecticidal toxins and other coleopteran-active Bt insecticidal traits, for example, event MON863 disclosed in U.S. Pat. No. 7,705,216; event MIR604 disclosed in U.S. Pat. No. 8,884,102; event 5307 disclosed in U.S. Pat. No. 9,133,474; event DAS-59122 disclosed in U.S. Pat. No. 7,875,429; event DP-4114 disclosed in U.S. Pat. No. 8,575,434; event MON 87411 disclosed in U.S. Pat. No. 9,441,240; and event MON88017 disclosed in U.S. Pat. No. 8,686,230, all of which are incorporated herein by reference. In some embodiments, the DP-023211-2 corn event can be stacked with: MON87427; MON-00603-6 (NK603); MON-87460-4; LY038; DAS-06275-8; BT176; BT11; MIR162; GA21; MZDT09Y; SYN-05307-1; and DAS-40278-9.

[0085] In some embodiments, corn plants comprising a DP-023211-2 event can be treated with a seed treatment. In some embodiments, the seed treatment can be a fungicide, an insecticide, or a herbicide.

[0086] The following examples are offered by way of illustration and not by way of limitation.

[0087] Examples

[0088] Example 1. Cassette design for transgenic plants containing constructs encoding IPD072 and dsRNA targeting DvSSJ1

[0089] According to the effect and expression in the gene test transformation experiment, the IPD072 and DvSSJ1 expression cassette designs for molecular stacking were selected to produce commercial tracking events. Many different regulatory factors (promoters, introns) and other elements (terminator, RNAi hairpin design) were evaluated in the gene test experiment. A large number of different regulatory elements were used to evaluate the output and the trait effect of the expression pattern.

[0090] Three genetic testing experiments were performed to evaluate approximately 40 different IPD072 single cassettes. These experiments involved genetic design screening and two construct matrices in which multiple promoters, terminators, and subcellular targeting strategies were evaluated. Four IPD072 cassette designs were selected from these experiments to be included in the molecular stack with DvSSJ1.

[0091] A similar but more extensive approach was taken to select the three cassette designs for DvSSJ1. Approximately 100 single DvSSJ1 cassettes were evaluated in multiple T0 experiments. These included experiments designed to select the dvssj1 fragment for the hairpin stem design, the hairpin loop region, the directionality of the hairpin stem, and the promoter driving the hairpin expression.

[0092] In all cases, the DvSSJ1 hairpin was cloned upstream of the IPD072 gene. The cassettes were separated by stacks of three terminators. These combinations have not been validated in previous transformations. The genetic elements included in the T-DNA region of the selected event construct plasmid PHP74643 are described in Table 1.

[0093] Table 1: Description of genetic elements in the T-DNA region of plasmid PHP74643

[0094]

[0095]

[0096] Example 2. Transformation of Maize by Agrobacterium and Regeneration of Transgenic Plants Containing IPD072, DvSSJ1, PAT and PMI Genes

[0097] Agrobacterium-mediated SSI transformation with plasmid PHP74643 produced the DP-023211-2 corn event. Agrobacterium-mediated SSI was performed essentially as described in U.S. Patent Application Publication No. 2017 / 0240911, which is incorporated herein by reference (see, e.g., Example 3).

[0098] Infect more than 2700 immature embryos with PHP74643. Through 105 days selection and regeneration process, 46 strains of TO seedlings were regenerated in total. From all TO seedlings, sampling was performed to perform PCR analysis to verify the existence and copy number of IPD072, PMI, mo-PAT and DvSSJ1 genes inserted. In addition to this analysis, the existence of some Agrobacterium binary vector backbone sequences in the TO seedlings and the developmental genes zm-odp2 and zm-wus2 disclosed in U.S. Patents 7,579,529 and 7,256,322 were also analyzed by PCR, and these patents are incorporated herein by reference in their entirety. Selection is determined to contain a single copy of the gene inserted, there is no Agrobacterium backbone sequence and there is no plant with developmental genes for further greenhouse breeding. The sample of the TO mass event selected from those PCRs was collected for further analysis using SbS sequencing to confirm that the gene of the insertion is in correct target locus (also referred to as "landing pad") without any gene disruption. Corn event DP-023211-2 was confirmed to contain a single copy of T-DNA (see Examples 3 and 4). These selected TO plants were assayed for trait efficacy and protein expression. TO plants that met all criteria were improved and crossed with inbred lines to produce seed for further testing. A schematic overview of transformation and event development is provided in Figure 4 Given in.

[0099] Example 3. Identification of Corn Event DP-023211-2

[0100] Separate the genomic DNA from leaf tissue representing multi-generation corn event DP-023211-2, known copy number calibrator control, negative control source (DNA from non-genetically modified corn) and no template control (NTC) and use event-specific and construct-specific primers and probes to carry out quantitative real-time PCR (qPCR) amplification.Use event-specific and construct-specific mensuration to carry out real-time PCR analysis to DP-023211-2 corn DNA, confirm the stable integration and separation of the T-DNA single copy of plasmid PHP74643 in the test leaf sample, as the quantitative detection of event DP-023211-2 and IPD072, PMI, DvSSJ1 and mo-PAT transgenes in DP-023211-2 corn proved.By repeating the experiment in quadruplicate, assess the reliability of every kind of event-specific and construct-specific PCR method.Evaluate the sensitivity or detection limit (LOD) of described PCR amplification by various dilutions of genomic DNA from DP-023211-2.

[0101] Two generations of corn containing event DP-023211-2 were grown on cell division plates under typical greenhouse production conditions. Approximately 165 seeds were planted for each generation.

[0102] When plant is between V5 and V9 growth period, collect leaf sample from each healthy plant.Sample is taken from the youngest leaf that grows from the whorl of each plant.By for DP-023211-2 event and from the copy number PCR (qPCR) of IPD072, PMI, DvSSJ1 and mo-PAT transgenic in the seed grown at Pioneer Hi-Bred International Co., Ltd. (Johnston, Iowa), the genome connection of each event in three leaf punches of each plant and the copy number of PHP74643 T-DNA were analyzed.Use high alkaline extraction scheme to extract genomic DNA from leaf sample.Use standard hexadecyl trimethylammonium bromide (CTAB) extraction scheme to prepare verified laboratory control (copy number calibrator and negative control) from leaf tissue.

[0103] Using Quant-iT Genomic DNA from supporting laboratory controls was quantified using the ELISA kit (Invitrogen, Carlsbad, CA). Quantification of genomic test and control samples was estimated using a NanoDrop 2000c spectrophotometer using NanoDrop 2000 / 2000c V1.6.198 software (Thermo Scientific, Wilmington, DE).

[0104] Genomic DNA samples isolated from leaf tissue of DP-023211-2 and control samples were subjected to real-time PCR amplification using event-specific and construct-specific primers and probes spanning specific regions of the PHP74643 T-DNA and genomic junctions spanning each insertion site of event DP-023211-2. The endogenous reference gene high mobility group A (hmg-A) (Krech et al. (1999). Gene [Gene] 234: (1) 45-50) was used in duplicate with each assay to perform qualitative and quantitative assessments of each assay; demonstrating the presence of sufficient quality and quantity of DNA in the PCR reaction. The PCR target sites and expected PCR product sizes for each primer / probe set are shown in Table 3. Primer and probe sequence information supporting each target region is shown in Table 4. PCR reagents and reaction conditions are shown in Table 5. In this study, approximately 3-ng corn genomic DNA was used for all PCR reactions.

[0105] Table 3: PCR genomic DNA target sites and expected sizes of PCR products

[0106]

[0107] Table 4: Primer and probe sequences and amplicons for PCR genomic DNA target regions

[0108]

[0109]

[0110] Table 5: PCR reagents and reaction conditions

[0111]

[0112] a If using Roche 480 Complete thermal cycling, then perform 45 cycles of steps 2a and 2b.

[0113] PCR products ranging in size from 72-bp to 113-bp were amplified, representing the insertion site of event DP-023211-2 and the transgene within the T-DNA from plasmid PHP74643, and were observed in 100 individual leaf samples from event DP-023211-2 and the eight copy number calibrator genomic controls, but were absent from the eight negative genomic controls and the eight NTC controls. Each assay was performed a total of four times, and the same results were observed. C was calculated for each sample and all positive controls. T value.

[0114] Using the corn endogenous reference gene hmg-A, a 79-bp PCR product was amplified and observed in 100 individual leaf samples from event DP-023211-2 as well as eight copy number calibrators and eight negative genome controls. No amplification of the endogenous gene was observed in the eight no-template (NTC) controls tested, and no CT value was generated. For each sample, each assay was performed in duplicate, with two insertion sites and all transgenics performed four times, and the same results were observed each time. The CT values ​​for each sample and all positive and negative controls were calculated.

[0115] To evaluate the sensitivity of the construct-specific PCR assay, DP-023211-2 maize DNA was diluted into control maize genomic DNA to yield test samples containing various amounts of event DP-023211-2 DNA (5-ng, 1-ng, 100-pg, 50-pg, 20-pg, 10-pg, 5-pg, 1-pg, 0.5-pg, 0.1-pg) for a total of 5-ng maize DNA. These various amounts of DP-023211-2 maize DNA corresponded to 100%, 20%, 2%, 1%, 0.4%, 0.2%, 0.1%, 0.01% and 0.002% DP-23211-2 maize DNA in total maize genomic DNA, respectively. For the transgenic PMI, 750-pg, 500-pg and 250-pg or 15%, 10% and 5% of the DP-023211-2DNA in the total corn genomic DNA were tested. For transgenic IPD072, PMI, DvSSJ1 and mo-PAT, various amounts of DP-023211-2DNA were amplified in real time PCR. According to these analyses, the limit of detection (LOD) of 5-ng total DNA of event DP-023211-2 was determined to be about 20-pg, or 0.4% of IPD072, 500-pg, or 10% (DP-023211-2) of PMI. The sensitivity of the determination of each described assay is sufficient for many screening applications. Each concentration was tested four times in total, and the results observed each time were the same.

[0116] Real-time PCR analysis of event DP-023211-2 using event-specific and construct-specific primer / probe sets confirmed stable integration and segregation of a single copy of the T-DNA of plasmid PHP74643 of the event in the test leaf samples, as demonstrated by quantitative detection of IPD072, PMI, DvSSJ1, and mo-PAT transgenes in DP-023211-2 corn. These results were reproducible in all replicate qPCR analyses performed. The maize endogenous reference gene assay for detection of hmg-A was amplified as expected in all test samples, negative controls, and was not detected in NTC samples. Under the conditions described, the sensitivity of each assay ranged from 5-pg to 500-pg DNA, which is sufficient for many screening applications by PCR.

[0117] Example 4. Southern-by-Sequencing analysis of integrity and copy number of DP-023211-2 maize

[0118] SbS sequencing (Southern-by-Sequencing) utilizes probe-based sequence capture, next generation sequencing (NGS) technology, and bioinformatics programs to isolate, sequence, and identify inserted DNA in the corn genome. By compiling a large number of unique sequencing reads and comparing them to the transformation plasmid, unique connections due to the inserted DNA are identified in the bioinformatics analysis, and these unique connections can be used to determine the number of insertions within the plant genome. One TO plant of each DP-023211-2 corn was analyzed by SbS to determine the number of inserted copies. In addition, a sample of the control corn line was analyzed.

[0119] Genomic DNA was extracted from the T0 generation of DP-023211-2 maize and control plants.

[0120] Designed and synthesized capture probe for selecting PHP74643 plasmid sequence by Roche NimbleGen company (Madison, Wisconsin).Use a series of unique sequences comprising the plasmid sequence to design overlapping biotinylated oligonucleotides as capture probe.Design the probe group to target most sequences in the PHP74643 transformation plasmid in the enrichment process.The probe is compared with the corn genome to determine the level of the corn genome sequence that will be captured and checked simultaneously with the PHP74643 plasmid sequence.

[0121] Next generation sequencing libraries were constructed for DP-023211-2 corn plants and the control corn lines. SbS was performed as described in Zastrow-Hayes et al. Plant Genome (2015). The sequencing library was hybridized with capture probes through two rounds of hybridization to enrich the target sequence. After NGS was performed on HiSeq 2500 (Illumina, San Diego, California, USA), the sequencing reads entered the bioinformatics process for trimming and quality assurance. The reads were aligned with the corn genome and transformation constructs, and the reads containing the genome and plasmid sequences were identified as connected reads. The alignment of the connected reads with the transformation constructs showed the edges of the inserted DNA relative to the expected insertion.

[0122] In order to identify the connection including the endogenous corn sequence, the control corn genomic DNA library was captured and sequenced in the same manner as the DP-023211-2 corn plant. The average depth of sequencing of these libraries was about five times the depth of the DP-023211-2 corn plant sample. This increases the possibility of detecting the endogenous connection captured by the PHP74643 probe in the control sample, so they can be identified and removed in the DP-023211-2 corn sample.

[0123] Integration and copy number of the insertion were determined in DP-023211-2 maize derived from construct PHP74643. Figure 1 and Figure 2 A schematic diagram of the PHP74643 plasmid and the T-DNA of PHP74643 used for transformation is provided in .

[0124] SbS was performed on TO plants of DP-023211-2 maize to determine the insert copy number in the genome. Unique junctions between the genomic flanking sequences and the landing pad were detected. The FRT1 and FRT87 sites are two junction points for the integration of the target trait of the PHP74643 T-DNA into the site-specific integration line. Unique reads at the FRT1 and FRT87 junctions of the plants are shown in Figure 3 . No other connections were detected between the PHP74643 sequence and the maize genome in the plants, indicating that no other plasmid-derived insertions were present in DP-023211-2 maize. In addition, there were no connections between non-contiguous regions of the identified PHP74643 T-DNA, indicating that there were no detectable rearrangements or truncations in the inserted DNA. Furthermore, there were no connections between the maize genomic sequence and the backbone sequence of PHP74643 in the plants analyzed, demonstrating that no plasmid backbone sequence was incorporated into DP-023211-2 maize.

[0125] SbS analysis of TO plants of DP-023211-2 maize demonstrated the presence of a single insertion comprising the desired gene from the PHP74643 T-DNA in DP-023211-2 maize and the absence of additional insertions in the corresponding genome.

[0126] SbS sequencing (Southern-by-Sequencing) analysis was performed on the TO plants of DP-023211-2 corn to confirm the insertion copy number. The results showed that a single PHP74643 T-DNA insertion was present in the plants. The connection between the PHP74643 T-DNA sequence and the corn genome was not detected in the control plants, indicating that, as expected, these plants did not contain any insertion sequences derived from PHP74643. In addition, no plasmid backbone sequences were detected in the analyzed plants. SbS analysis of the TO plants of DP-023211-2 corn demonstrated that a single insertion of PHP74643 T-DNA was present in each DP-023211-2 corn, and no other insertions were present in the corresponding genomes.

[0127] Example 5. Insect Efficacy of Corn Event DP-023211-2

[0128] Efficacy data for five construct designs were generated. Each construct design consisted of three genetic backgrounds, with multiple events in each background (Table 6). 42 grain samples for each project were characterized before planting to confirm the presence of the event by event-specific PCR. Four projects required tissue sampling in the field, and all atypical plants were excluded from the experiment. Efficacy testing included: WCRW root damage at eight locations. At each location, single-row plots were planted in an incomplete block design with two replicates at each location.

[0129] Plants at approximately the V2 growth stage were artificially infested with approximately 375-750 WCRW eggs applied to the soil on both sides of the plant (approximately 750-1,500 eggs / plant in total) (depending on location). In addition, plots were planted in fields with a high probability of containing natural infestations of WCRW. Plant roots were evaluated at approximately the R2 growth stage. Two plants per plot were tagged with a unique identifier, removed from the plot and washed with pressurized water. Root damage was scored using the 0-3 node injury scale (CRWNIS) (Oleson et al. (2005) J. Econ. Entomol. 98(1): 1-8).

[0130] For the single position analysis of construct designs (Table 7), linear mixed models were applied to the node injury score model for each position separately. The construct design was considered a fixed effect. The effects of replication, replication-incomplete block, background, construct, background-construct, event, field range, field row, plot, and residual were considered independent normally distributed random variables with a mean of zero. T tests were performed to compare treatment effects. If the p value of the difference was less than 0.05, the difference was considered statistically significant. All data analyses and comparisons were performed in ASReml 3.0 (VSN International, Hemel Hempstead, UK, 2009).

[0131] For the cross-location analysis of events (Table 8), the construct design was considered a fixed effect. The effects of position, position_replicate, position_replicate_incomplete block, background, concept, background_concept, event, position_background, position_concept, position_background_concept, position_event, field range within each position, field row within each position, plot within each position, and residual within each position were considered independent normally distributed random variables with a mean of zero. T tests were performed to compare treatment effects. Differences were considered statistically significant if the P value for the differences was less than 0.05. All data analyses and comparisons were performed in ASReml 3.0 (VSN International, Hemel Hempstead, UK, 2009). Tables 6 and 7 list the estimated root damage grades for WCRW feeding, showing that some constructs performed better than others.

[0132] Table 6. Genetic background and number of events evaluated for efficacy in each construct design

[0133] Construct design Background quantity Number of events <![CDATA[SSJ72_UBI;BSV(AY) a ]]> 3 22 SSJ72_UBI;A 3 24 SSJ72_BSV(AY);A 3 15 SSJ72_3XUBI;A 3 7 SSJ72_UBI;B 3 25

[0134] a Event DP-023211-2 was included in this construct design

[0135] *A and B are different promoters

[0136] Table 7. Efficacy of construct designs against mixed populations of northern corn rootworm (NCR) and western corn rootworm (WCR) larvae in field testing.

[0137]

[0138]

[0139] a Damage ratings to root mass of individual plants were determined using a 0-3 node damage scale (Oleson et al., 2005, supra).

[0140] b Within one position, estimates with the same letter are not significantly different (T test, P > 0.05).

[0141] *A and B are different promoters

[0142] Table 8. Efficacy of events against mixed populations of NCR and WCR larvae at eight field test locations.

[0143]

[0144] a Damage ratings to root mass of individual plants were determined using a 0-3 node injury weight scale (Oleson et al., 2005, supra).

[0145] b Within one position, estimates with the same letter are not significantly different (T test, P > 0.05).

[0146] In year 3, DP-023211-2 was further field tested at 14 locations in the North American commercial corn growing zone: Benson, MN (MK_BE); Brookings, SD (BR); Fowler, IN (WN_FO); Goodland, IN (WN_GL); Janesville, WI (JV); Johnston, IA (JH and JH_D3); Mankato, MN (MK); Mansfield, IL (CI_MF); Marion, IA (MR); Redline, IA (MR); Seymour, IL (CI_SE); and York, NE (YK and YK_LI). No efficacy data were collected at CI_SE, JV, WN_FO, WN_GL, and YK because the node injury score (CRWNIS) of the negative control roots was less than 0.75.

[0147] Single row plots (10 feet long) were planted in an alpha experimental design with two replicates. Before planting, 42 kernels from each seed batch were characterized to confirm the presence of the trait by PCR. When the plants reached growth stages V2-V4, five consecutive plants were manually infested with a target infestation rate of approximately 750 eggs / plant or 1500 eggs / plant using a CRW egg infester mounted on a tractor. The eggs were infested in the soil about 4 inches deep at about 2-3 inches on both sides of each plant. 56 to 78 days after planting, the damage to the roots caused by larval feeding was evaluated. Two corn roots were tagged, manually dug out from the ground, the soil was rinsed clean with pressurized water, and the larval feeding at approximately the R2 growth stage was evaluated. Root damage was evaluated by visual rating and recording the amount of larval feeding contained on each root using the Iowa 0-3 node damage scale (Oleson et al., 2005).

[0148] The average node lesion root rating results from CRW for DP-023211-2 corn and control corn are provided in Table 9. These results indicate that corn lines containing the insect active protein IPD072Aa and the RNAi trait DvSSJ1 are effective against CRW.

[0149] Table 9. Efficacy results against corn rootworms

[0150]

[0151] a There is a statistically significant difference (P value < 0.05)

[0152] Example 6. Agronomic and yield field evaluation of corn event DP-023211-2

[0153] Agronomic field trials were conducted in the summer of 2016, comprising five molecular stacking construct designs comprising both DvSSJ1 and IPD072 used in Example 5, to generate yield data and evaluate other agronomic characteristics. Multiple events were tested for each construct design (Table 10). All inbred and hybrid materials for testing an event were generated from a single T0 plant.

[0154] Hybridization test

[0155] The hybridization trials were planted at 16 locations, each with a single replicate list of projects. Grain was harvested from 10 of the 16 locations. Each project with a common background was crossed with three test projects to generate hybrid seed for testing. The experiments were nested by test project, with each nested project randomized. Individual observations and data were collected at each planting location throughout the growing season. The following agronomic traits were analyzed for comparison to wild-type projects (WT) or projects with the same genetics but without the DvSSJ1 and IPD072 molecular stack (also known as basic comparators) (Tables 11-12 and Figure 5-6 ):

[0156] 1.) Growth Degree Units to Silking (GDUSLK): When 50% of the plants in the plot have fully silked, the total growth degree units accumulated are measured and recorded. The single-day equivalent value of this data set is approximately 2.5 growth degree units.

[0157] 2.) Gross Degree Units to Shedding (GDUSHD): When 50% of the tassels in the plot are shedding pollen, the total accumulated Gross Degree Units are measured and recorded. The single-day equivalent value of this data set is approximately 2.5 Gross Degree Units.

[0158] 3.) Ear Height (EARHT): The measurement from the ground to the point where the tallest developed ear is attached to the plant. Ear height is measured in inches.

[0159] 4.) Plant Height (PLTHT): The measurement from the ground to the base of the flag leaf. Plant height is measured in inches.

[0160] 5.) Moisture (MST): A measurement of the moisture percentage of the kernel at harvest.

[0161] 6.) Yield: Record the weight of kernels harvested from each plot. Calculate the reported bushels / acre yield by adjusting for the measured moisture of each plot.

[0162] Inbreeding test

[0163] The inbreeding trial was planted at eight locations with two replicates of the project list at each location. One replicate was nested in each location by construct design; the other replicate was planted as a randomized complete block. Agronomic data and observations from the inbreeding trial were collected and analyzed for comparison with the wild-type project (WT) or a trait-free version of the same genotype. The data generated from the inbreeding trial included the following agronomic traits ( Figure 7 and 8 ):

[0164] 1.) Growth Degree Units to Silking (GDUSLK): When 50% of the plants in the plot have fully silked, the total growth degree units accumulated are measured and recorded. The single-day equivalent value of this data set is approximately 2.5 growth degree units.

[0165] 2.) Gross Degree Units to Shedding (GDUSHD): When 50% of the tassels in the plot are shedding pollen, the total accumulated Gross Degree Units are measured and recorded. The single-day equivalent value of this data set is approximately 2.5 Gross Degree Units.

[0166] 3.) Ear Height (EARHT): The measurement from the ground to the point where the tallest developed ear is attached to the plant. Ear height is measured in inches.

[0167] 4.) Plant Height (PLTHT): The measurement from the ground to the base of the flag leaf. Plant height is measured in inches.

[0168] 5.) Ear Photometric Yield (PHTYLD): Yield estimates calculated from images of ears harvested from each plot. Values ​​are displayed in bushels per acre.

[0169] Test results

[0170] In order to evaluate the hybridization data, a mixed model framework was used to perform multi-position analysis. In the multi-position analysis, the main effect construct design was considered as a fixed effect. Factors such as position, background, test object, event, background_construct design, test object_construct design, test object_event, position_background, position_construct design, position_test object, position_background_construct design, position_test object_construct design, position_event, position_test object_event were considered as random effects. The spatial effects including the range and plots in each position were considered as random effects to eliminate external spatial noise. For each position, it was assumed that the heterogeneous residuals (heterogeneous residual) had autoregressive correlation, which was AR1*AR1. Construct design estimates and event predictions for each background were generated. T tests were performed to compare the construct design / event of WT. If the P value of the difference was less than 0.05, the difference was considered to be statistically significant. Yield analysis was performed using ASREML (VSN International Ltd.; Best Linear Unbiased Prediction; Cullis, BR et al. (1998) Biometrics 54: 1-18, Gilmour, AR et al. (2009); ASReml User Guide 3.0, Gilmour, AR, et al. (1995) Biometrics 51: 1440-50).

[0171] In order to evaluate the inbreeding data, a mixed model framework was used to perform multi-location analysis. In the multi-location analysis, the main effect construct design was considered as a fixed effect. Factors such as position, background, event, background_construct design, position_background, position_construct design, position_background_construct design, position_event and repetition within the position were considered as random effects. Spatial effects including the range and plot within each position were considered as random effects to eliminate external spatial noise. For each position, it was assumed that the heterogeneous residuals had autoregressive correlation, which was AR1*AR1. Construct design estimates and event predictions for each background were generated. T tests were performed to compare the construct design / event of WT. If the P value of the difference was less than 0.05, the difference was considered to be statistically significant. Yield analysis was performed using ASREML (VSN International Ltd.; Best Linear Unbiased Prediction; Cullis, BR et al. (1998) Biometrics 54: 1-18, Gilmour, AR et al. (2009); ASReml User Guide 3.0, Gilmour, AR, et al. (1995) Biometrics 51: 1440-50).

[0172] A similar experiment was conducted in year 2 and the results confirmed the performance data from year 1; two events were selected from the following: SSJ72_UBI; BSV(AY)_NONE construct designs.

[0173] Table 10. Number of events evaluated for each construct design

[0174]

[0175] a Event DP-023211-2 was included in this construct design

[0176] *A and B are different promoters

[0177] Table 11. Hybrid performance of construct designs compared to the basic program - yield

[0178]

[0179]

[0180] a Event DP-023211-2 was included in this construct design

[0181] *A and B are different promoters

[0182] Table 12. Hybrid Performance of Event DP-023211-2 Compared to Base Project - Yield

[0183]

[0184] Example 7. Protein expression and concentration

[0185] Protein extraction

[0186] Aliquots of treated leaf or root tissue samples were weighed into 1.2 ml tubes, with a target weight of 10 mg for leaf tissue and 20 mg for root tissue. Samples analyzed for PAT and PMI protein concentration were extracted in 0.6 ml of cold PBST, and samples for IPD072Aa protein analysis were extracted in 0.6 ml of cold PBST containing 25% Stabilzyme Select. After centrifugation, the supernatant was removed and diluted in PBST (PAT and PMI) or PBST containing 25% Stabilzyme Select (IPD072Aa) and analyzed.

[0187] Determination of IPD072Aa protein concentration

[0188] The IPD072Aa ELISA method utilizes a kit developed and produced by Pioneer Hi-Bred International Ltd. to determine the concentration of IPD072Aa protein in a sample. Standards (typically analyzed in triplicate wells) and diluted samples (typically analyzed in duplicate wells) are incubated on a plate pre-coated with an IPD072Aa-specific antibody. After incubation, unbound substances are washed off the plate. Another IPD072Aa-specific antibody conjugated to horseradish peroxidase (HRP) is added to the plate and incubated. Unbound substances are washed off the plate. The detection of the bound IPD072Aa-antibody complex is completed by adding a substrate, which generates a colored product in the presence of HRP. The reaction is terminated with an acid solution, and the optical density (OD) of each well is measured using a plate reader.

[0189] Determination of PAT protein concentration

[0190] PAT ELISA method was performed using EnviroLogix TM ) company to determine the concentration of PAT protein in the sample. The standard (typically analyzed in triplicate wells) and the diluted sample (typically analyzed in duplicate wells) are incubated together with a PAT-specific antibody conjugated to the enzyme HRP in a plate pre-coated with another PAT-specific antibody. After incubation, unbound material is washed off the plate. Detection of the bound PAT-antibody complex is completed by adding a substrate that generates a colored product in the presence of HRP. The reaction is terminated with an acid solution and the OD of each well is measured using a plate reader.

[0191] Determination of PMI protein concentration

[0192] The PMI ELISA method uses a kit developed and produced by Pioneer Hi-Bred International Ltd. to determine the concentration of PMI protein in a sample. Standards (typically analyzed in triplicate wells) and diluted samples (typically analyzed in duplicate wells) are incubated on a plate pre-coated with a PMI-specific antibody. After incubation, unbound substances are washed off the plate. Another PMI-specific antibody conjugated to an enzyme (HRP) is added to the plate and incubated. Unbound substances are washed off the plate. Detection of the bound PMI-antibody complex is completed by adding a substrate that generates a colored product in the presence of HRP. The reaction is terminated with an acid solution, and the OD of each well is measured using a plate reader.

[0193] Calculation of protein concentration

[0194] SoftMax Pro GxP (Molecular Devices) microplate data software was used to perform the calculations required to convert the OD values ​​obtained for each set of sample wells into protein concentration values.

[0195] A standard curve is included on each ELISA plate. The equation of the standard curve is derived by software that uses quadratic fitting to associate the OD values ​​obtained for each set of standard wells with the corresponding standard concentration (ng / ml). The quadratic regression equation is applied as follows:

[0196] y=Cx 2 +Bx+A

[0197] Where x = known standard concentration and y = corresponding absorbance value (OD)

[0198] Interpolation of sample concentration (ng / ml) was performed by solving the above equation for x using the A, B, and C values ​​determined for the standard curve.

[0199]

[0200] For example, given curve parameters A = 0.0476, B = 0.4556, C = -0.01910, and sample OD = 1.438

[0201]

[0202] The sample concentration values ​​were adjusted according to the dilution factor denoted as 1:N by multiplying the interpolated concentration by N.

[0203] Adjusted concentration = interpolated sample concentration x dilution factor

[0204] For example, assuming the interpolated concentration is 3.6 ng / ml, the dilution factor is 1:20

[0205] Adjusted concentration = 3.6 ng / ml x 20 = 72 ng / ml

[0206] Convert the adjusted sample concentration values ​​obtained from SoftMax Pro GxP Software from ng / ml to ng / mg sample weight as follows:

[0207]

[0208] For example, sample concentration = 72 ng / ml, extraction buffer volume = 0.60 ml, sample target weight = 10 mg

[0209]

[0210] The reportable assay lower limit of quantitation (LLOQ) in ng / ml was calculated as follows:

[0211] Reportable assay LLOQ (ng / ml) = (lowest standard concentration - 10%) x minimum dilution

[0212] For example, minimum standard concentration = 0.50 ng / ml, and minimum dilution = 10

[0213] Reportable assay LLOQ (ng / ml) = (0.50 ng / ml - (0.50 x 0.10)) x 10 = 4.5 ng / ml

[0214] The LLOQ in ng / mg sample weight was calculated as follows:

[0215]

[0216] For example, reportable assay LLOQ = 4.5 ng / ml, extraction buffer volume = 0.60 ml, sample target weight = 10 mg

[0217]

[0218] result

[0219] Table 13 provides the means, standard deviations, and ranges for IPD072Aa protein in root tissue of two generations of DP-023211-2 corn V9, and Table 14 provides the means, standard deviations, and ranges for PAT and PMI proteins in leaf tissue of two generations of DP-023211-2 corn V9.

[0220] Table 13: IPD072Aa protein concentration expressed in V9 root samples of DP-023211-2 corn

[0221]

[0222] Table 14. PAT and PMI protein concentrations expressed in V9 leaf samples of DP-023211-2 corn

[0223]

[0224]

[0225] Example 8. DvSSJ1 dsRNA expression

[0226] In 2017, different generations (BC1F1 and BC2F1) of DP-023211-2 corn were planted in 4-inch pots and organized into flat plots containing 15 pots in Johnston, Iowa, USA, using typical greenhouse production conditions.

[0227] Root samples were collected from 10 plants at approximately the V9 growth stage (i.e., when the collar of the ninth leaf became visible) and analyzed for the DP-023211-2 corn event and the presence or absence of the ipd072Aa, mo-pat, pmi, and DvSSJ1 genes using endpoint real-time PCR. Five plants that tested positive by PCR analysis were selected for further analysis.

[0228] Each root sample was obtained by removing the root from the soil and shaking it to remove excess soil. The roots were then washed thoroughly with water and removed from the plant. Above-ground supporting roots were not included in the sample. The root tissue was cut into slices 1 inch (2.5 cm) or shorter in length, a portion of the sample was collected into pre-cooled vials for QuantiGene analysis, and the rest of the sample was collected into vials for moisture analysis. All samples were stored on dry ice until transferred to a -80°C freezer.

[0229] Approximately 1.2 g of frozen V9 root tissue samples from DP-023211-2 maize plants were weighed, mixed with lysis buffer, and ground. Total RNA from 800 μl of ground tissue and lysis buffer mixture was extracted using the mirVana Total RNA Isolation Kit (Thermo Fisher Scientific, Carlsbad, CA) according to the manufacturer's instructions and eluted in 75 μl of molecular grade water. The extracted RNA was quantified using NanoDrop-8000 and stored in a -80°C freezer.

[0230] The reference standard of DvSSJ1 hairpin RNA (hpRNA) was generated by in vitro transcription. To generate a construct containing the DvSSJ1 sequence for in vitro transcription, total RNA was extracted from the transgenic plants and used to synthesize the cDNA of full-length DvSSJ1 by reverse transcription using rapid amplification of cDNA ends 5' and 3' (RACE). The resulting cDNA was cloned into the pUC57 vector under the T7 promoter. Plasmid DNA of the DvSSJ1 full-length construct was isolated from bacterial culture and used for in vitro transcription of DvSSJ1 hpRNA by SunScript RT RNase H-Kit (Sygnis, Heidelberg, Germany). The working concentration of DvSSJ1 hpRNA was 10 ng / μl. Nine points with concentrations ranging from 0.0105 to 16 pg per 40 μl were used to generate a standard curve. The measured values ​​at each point of the standard curve were generated and averaged.

[0231] 250 ng of total RNA per well was analyzed using a validated QuantiGene Plex 2.0 assay (Affymetrix Inc., Santa Clara, CA) using a standard curve created with a DvSSJ1 hpRNA reference standard at nine concentrations (0.0105 to 16 pg per 40 μl reaction volume). The probe set used in the assay was designed to specifically detect DvSSJ1 RNA transcripts. Total RNA from non-GM HC69 maize plants was used as a negative control.

[0232] QuantiGene assays were performed according to modified manufacturer's instructions. Test samples, negative control samples, and DvSSJ1 hpRNA reference standards were assayed in triplicate wells of a 96-well hybridization plate in a volume of 100 μl. In each test sample well, 250 ng of total RNA was mixed with a quarter-strength probe set and heated at 95°C. After heating for 3 minutes, the sample was cooled and kept at 54°C until use. A mixture of 40 μl RNA sample and 5 μl probe set was transferred to a hybridization plate containing 55 μl bead mixture for overnight hybridization. After signal amplification and washing, the fluorescence intensity of the assay plate was read by a MagPix analyzer (Luminex. Corp., Austin, Texas) according to the manufacturer's instructions. The net median fluorescence intensity (MFI) of each assay well is reported.

[0233] Root tissue samples were collected from five plants per generation to obtain the fresh weight to dry weight ratio. The fresh weight of each sample was recorded. The samples were then placed on dry ice, freeze-dried, and the dry weight was recorded.

[0234] Net MFI values ​​were used to calculate the mean, standard deviation, and coefficient of variation for each set of triplicate samples. A standard curve was generated on a QuantiGene assay plate and used to interpolate DvSSJ1 dsRNA concentrations based on the net MFI values. The concentration of DvSSJ1 RNA from each test sample was further converted to pg / mg fresh weight (fw) values. All fresh weight values ​​were further converted to pg / mg dry weight (dw) values. In 2 generations, the mean, standard deviation, and range of DvSSJ1 RNA levels were determined for each of the 5 plants based on fw and dw.

[0235] The reportable assay lower limit of quantitation (LLOQ) in pg / ml was calculated as follows:

[0236] Reportable assay LLOQ (pg / ml) = lowest standard concentration x 90% x lowest dilution

[0237] The lowest standard concentration was 0.0105 pg / rxn and the minimum dilution used was 0.574 rxn / mg.

[0238] Therefore, LLOQ = 0.0105 pg / rxn x 0.9 x 0.574 rxn / mg = 0.0054 pg / mg

[0239] The DvSSJ1 dsRNA expression results for DP-023211-2 corn root samples were averaged from five plants analyzed per generation, and the means, standard deviations, and ranges are summarized in Table 15.

[0240] Table 15: Summary of DvSSJ1 RNA expression levels in root tissues of DP-023211-2 maize V9

[0241]

[0242] Example 9. LC 50 and spectral analysis

[0243] Both IPD072Aa and DvSSJ1 can effectively control Diabrotica virgiferavirgifera (Western Corn Rootworm (WCR)), an insect pest of corn that feeds on the root tissue of corn plants and reduces yield. Species for IPD072Aa and DvSSJ1 testing were selected based on the following criteria: relevance of the organism to WCR, established laboratory bioassay methods, availability of laboratory-reared insects, availability of appropriate diets, and reproducibility of laboratory performance and response variables for each organism. Method development includes establishing appropriate diets and environmental conditions to achieve strong bioassay performance and establishing acceptable standards, typically less than 20% mortality for IPD072Aa within at least 7 days and for DvSSJ1 within at least 14 days. If possible, other sublethal endpoints, such as growth and development time, are also observed.

[0244] In all cases, fresh feed containing appropriate concentrations of IPD072Aa and DvSSJ1 was provided to the organisms as frequently as possible without exceeding acceptable control mortality levels or a decrease in test article stability under bioassay conditions. In most cases, fresh feed was provided at least every 3 or 4 days, or in some cases daily. Generally, acceptance criteria included ≤20% mortality in bioassay controls and ≥80% mortality observed in the individual positive controls associated with each bioassay, however, given the relatively variable performance of this organism in artificial diet laboratory bioassays, ≤30% control mortality for WCR was considered acceptable.

[0245] The LC50 for IPD072Aa was 15.9 ppm (95% confidence interval 12.6-20.6 ppm) generated using a 7-day duration bioassay. The 14-day LC50 for DvSSJ1 was 0.036 ppm (95% confidence interval 0.0066-0.065 ppm). DvSSJ1 was studied for a longer period of time as a mode of action for RNAi because it takes longer for DvSSJ1 to take effect and kill the target pest than IPD072Aa.

[0246] The activity of IPD072Aa and DvSSJ1 was evaluated by laboratory studies of organisms related to WCR or species that can be used for laboratory studies. Table 16 shows the species array used in these other bioassays, some of which represent harmful organisms of various cereals (corn, wheat, soybean, etc.), and some species are non-target organisms that provide beneficial ecosystem services in the agricultural field. Since WCR belongs to Coleoptera, special attention is paid to the organisms in Coleoptera. The other organisms selected represent three other families in Coleoptera. In addition, four different families in Lepidoptera were tested.

[0247] The no observed effect concentration (NOEC) for survival of IPD072Aa ranged from 100 ppm to greater than 1000 ppm (Table 16). No activity was observed outside of Coleoptera at the concentrations tested. The NOEC for survival of DvSSJ1 exceeded 1 ppm for all organisms tested, except for Diabrotica undecimpunctata (Southern Corn Rootworm (SCR), a close relative of WCR (Table 16). No activity of DvSSJ1 was observed against any organism other than Western (WCR) and Southern Corn Rootworm (SCR).

[0248]

[0249] The above description of the various illustrated embodiments of the present disclosure is not intended to be exhaustive or to limit the scope to the precise form disclosed. Although specific embodiments and examples are described herein for illustrative purposes, various equivalent modifications within the scope of the present disclosure are possible as will be appreciated by those skilled in the relevant art. The teachings provided herein may be applied to other purposes in addition to the above examples. Based on the above teachings, many modifications and variations are possible, and are therefore within the scope of the appended claims.

[0250] These and other changes can be made in light of the above detailed description.In general, in the following claims, the terms used should not be construed to limit the scope to the specific embodiments disclosed in the specification and the claims.

[0251] The entire disclosure of each document (including patents, patent applications, journal articles, abstracts, manuals, books, or other publications) cited in the Background, Detailed Description, and Examples is incorporated herein by reference in its entirety.

[0252] With respect to the numbers used (e.g., amounts, temperatures, concentrations, etc.), efforts have been made to ensure accuracy, but some experimental error and deviation should be allowed for. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight; temperature is in degrees Celsius; and pressure is at or near atmospheric.

Claims

1. A method for producing a corn plant comprising a genotype of corn event DP-023211-2, wherein the corn event DP-023211-2 comprises the nucleotide sequence set forth in SEQ ID NO:3, wherein the genotype comprises the nucleotide sequences set forth in SEQ ID NO:31 and SEQ ID NO:

35.

2. The method for producing a corn plant as claimed in claim 1, wherein the genotype comprises the nucleotide sequence set forth in SEQ ID NO:32 and SEQ ID NO:

36.

3. The method for producing a corn plant of claim 1, wherein the genotype comprises the nucleotide sequence set forth in SEQ ID NO:33 and SEQ ID NO:

37.

4. A method for producing a plant comprising the sequence set forth in SEQ ID NO:

3.

5. A method for producing a corn event DP-023211-2 comprising a DvSsj1 gene cassette, an IPD072Aa gene cassette, a mo-Pat gene cassette and a PMI gene cassette, wherein the corn event DP-023211-2 comprises the nucleotide sequence shown in SEQ ID NO: 3, wherein a representative sample of seeds of the corn event has been deposited with the American Type Culture Collection (ATCC) with accession number PTA-124722.

6. An isolated nucleic acid molecule, which consists of the nucleotide sequence of SEQ ID NO: 3 and its full-length complementary sequence.

7. An amplicon, which consists of the nucleic acid sequence of SEQ ID NO: 3 and its full-length complementary sequence.

8. A method for producing hybrid corn seeds, the method comprising: a) sexually crossing a first corn inbred line comprising the nucleotide sequence set forth in SEQ ID NO:3 with a second inbred line having a different genotype; b) growing progeny from said cross; and c) harvesting the hybrid seeds thus produced.

9. The method of claim 8, wherein the first corn inbred line is the female parent.

10. The method of claim 8, wherein the first corn inbred line is the male parent.

11. A method for producing a corn plant that is resistant to western corn rootworm, northern corn rootworm, and southern corn rootworm, the method comprising: a) sexually crossing a first parent corn plant with a second parent corn plant, wherein the first or second parent corn plant comprises event DP-023211-2 comprising a DvSsj1 gene cassette, an IPD072Aa gene cassette, a mo-Pat gene cassette, and a PMI gene cassette, wherein the event DP-023211-2 comprises the nucleotide sequence set forth in SEQ ID NO: 3, thereby producing a plurality of first generation progeny plants; b) selfing said first generation progeny plant, thereby producing a plurality of second generation progeny plants; and c) selecting from the second generation progeny plants that comprise said event DP-023211-2 and are resistant to the coleopteran pest.

12. A method for producing hybrid corn seeds, the method comprising: a) sexually crossing a first corn inbred line comprising corn event DP-023211-2 as defined in claim 1 with a second inbred line not comprising corn event DP-023211-2 as defined in claim 1; and b) harvesting the hybrid seeds thus produced.

13. The method of claim 12, further comprising the step of backcrossing a second generation progeny plant comprising corn event DP-023211-2 to a parent plant lacking the corn event DP-023211-2 DNA, thereby producing a backcross progeny plant that is resistant to western corn rootworm, northern corn rootworm, and southern corn rootworm.

14. A method for producing a corn plant that is resistant to corn rootworm, the method comprising: a) crossing a first parent corn plant with a second parent corn plant, wherein the first or second parent corn plant comprises event DP-023211-2 comprising a DvSsj1 gene cassette, an IPD072Aa gene cassette, a mo-Pat gene cassette, and a PMI gene cassette, wherein event DP-023211-2 comprises the nucleotide sequence set forth in SEQ ID NO: 3, thereby producing a plurality of first generation progeny plants; b) selecting a first generation progeny plant comprising said event DP-023211-2; c) backcrossing said first generation progeny plant of step (b) with a parent plant lacking said event DP-023211-2 DNA, thereby producing a plurality of backcross progeny plants; and d) selecting a plant comprising event DP-023211-2 from said backcross progeny plants; Wherein the selected backcross progeny plant of step (d) comprises SEQ ID NO: 25, 31 or 35.

15. The method of claim 14, wherein the plant of the first parent corn line is the female parent or the male parent.

16. A method for determining the zygosity of a corn plant comprising event DP-023211-2 comprising a DvSsj1 gene cassette, an IPD072Aa gene cassette, a mo-Pat gene cassette, and a PMI gene cassette in a biological sample, wherein the event DP-023211-2 comprises the nucleotide sequence set forth in SEQ ID NO: 3, the method comprising: a) contacting the sample with a first pair of DNA molecules and a second, different pair of DNA molecules such that: 1) when used in a nucleic acid amplification reaction comprising corn event DP-023211-2 DNA, produces a first amplicon diagnostic for event DP-023211-2, and 2) when used in a nucleic acid amplification reaction comprising corn genomic DNA other than DP-023211-2 DNA, produces a second amplicon diagnostic for corn genomic DNA other than DP-023211-2 DNA; b) performing a nucleic acid amplification reaction; and c) detecting the amplicons so produced, wherein detection of the presence of both amplicons indicates that the sample is heterozygous for the corn event DP-023211-2 DNA, and wherein detection of only the first amplicon indicates that the sample is homozygous for the corn event DP-023211-2 DNA.

17. The method of claim 16, wherein the first pair of DNA molecules comprises primer pair SEQ ID NO: 7 and 8.

18. The method of claim 16, wherein the first and second pairs of DNA molecules comprise a detectable label.

19. The method of claim 18, wherein the detectable label is a fluorescent label.

20. The method of claim 18, wherein the detectable label is covalently associated with one or more of the primer molecules.

21. A method for detecting the presence of a nucleic acid molecule unique to event DP-023211-2 containing a DvSsj1 gene cassette, an IPD072Aa gene cassette, a mo-Pat gene cassette, and a PMI gene cassette in a sample comprising corn nucleic acid, wherein the event DP-023211-2 comprises the nucleotide sequence set forth in SEQ ID NO: 3, the method comprising: a) contacting the sample with a pair of primers that, when used in a nucleic acid amplification reaction with genomic DNA from event DP-023211-2, produce an amplicon diagnostic for event DP-023211-2; b) performing a nucleic acid amplification reaction, thereby generating an amplicon that is diagnostic for event DP-023211-2; and c) Detection of amplicons diagnostic for event DP-023211-2.

22. The method of claim 21, wherein the nucleic acid molecule diagnostic for event DP-023211-2 is an amplicon produced by a nucleic acid amplification chain reaction.

23. A method for detecting the presence of DNA corresponding to the DP-023211-2 event containing a DvSsj1 gene cassette, an IPD072Aa gene cassette, a mo-Pat gene cassette, and a PMI gene cassette in a sample, wherein the event DP-023211-2 comprises the nucleotide sequence set forth in SEQ ID NO: 3, the method comprising: a) contacting a sample comprising corn DNA with a polynucleotide probe that hybridizes under stringent hybridization conditions to DNA from corn event DP-023211-2 and does not hybridize under said stringent hybridization conditions to non-DP-023211-2 corn plant DNA; b) subjecting the sample and probe to stringent hybridization conditions; and c) detecting hybridization of the probe with the DNA; Wherein detection of hybridization indicates the presence of the DP-023211-2 event.

24. The method of claim 23, wherein the probe comprises a detectable label.

25. The method of claim 24, wherein the detectable label is a fluorescent label.

26. The method of claim 24, wherein the detectable label is covalently associated with the probe.

27. A kit for detecting nucleic acids unique to event DP-023211-2 containing a DvSsj1 gene cassette, an IPD072Aa gene cassette, a mo-Pat gene cassette, and a PMI gene cassette, wherein the event DP-023211-2 comprises the nucleotide sequence shown in SEQ ID NO:3, the kit comprising at least one nucleic acid molecule of a contiguous polynucleotide of sufficient length to act as a primer or probe in a nucleic acid detection method, and upon amplification of a target nucleic acid sequence in a sample or hybridization with a target nucleic acid sequence in a sample and subsequent detection of the amplicon or hybridization with the target sequence, the contiguous polynucleotide is diagnostic for the presence of a nucleic acid sequence unique to event DP-023211-2 in the sample, wherein the nucleic acid molecule is a primer selected from SEQ ID NOs:7-38 and complementary sequences thereof.

28. A method for producing a corn plant comprising the genotype of corn event DP-023211-2 comprising a DvSsj1 gene cassette, an IPD072Aa gene cassette, a mo-Pat gene cassette, and a PMI gene cassette, wherein the corn event DP-023211-2 comprises the nucleotide sequence set forth in SEQ ID NO:3, wherein the genotype comprises the nucleotide sequences of SEQ ID NO:31 and SEQ ID NO:

35.

29. The method for producing a corn plant of claim 28, wherein the genotype comprises the nucleotide sequences of SEQ ID NO:32 and SEQ ID NO:

36.

30. The method for producing a corn plant of claim 28, wherein the genotype comprises the nucleotide sequence of SEQ ID NO:33 and SEQ ID NO:37.

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