Nucleic acid molecules, transgenic cells, plants and seeds of transgenic maize event ME240913 expressing Cry1Da protein, uses thereof, plant products, methods, kits and amplicons for detecting said event, and methods of producing transgenic plants and controlling lepidopteran pests

Abstract

The invention relates to a new transgenic maize event expressing a truncated or modified form of the Cry1Da insecticidal protein, designated event ME240913. The invention relates to nucleic acids specific for event ME240913. Primers, amplicons, methods and kits for detecting the presence of event ME240913 are also defined. The invention also relates to maize plants containing said event, uses thereof, methods and compositions for the control of lepidopteran pests. The invention describes a maize event that shows high levels of plant protection against damage caused by feeding by lepidopterans, including Spodoptera frugiperda, and Cry1F resistant insects. The event of the invention shows high toxicity to Spodoptera frugiperda.

Description

Technical Field

[0001] This invention relates to the fields of plant molecular biology, plant transformation and plant propagation, and pest control. More specifically, this invention relates to transgenic maize (Zeamays) plants containing novel transgenic genotypes that are resistant to lepidopteran pests, their uses, methods for controlling lepidopteran pests, and methods for detecting the presence of transgenic maize plant-specific nucleic acids in plant products and compositions thereof. Background Technology

[0002] Continued advancements in genetic engineering technology have enabled the development of commercially significant transgenic plants containing heterologous genes of interest that can confer desired traits. Among these genes of interest are those that confer resistance to herbicides, environmental stresses, diseases, and invertebrate pests.

[0003] Within the context of genes encoding proteins used to control invertebrate pests, the cry gene from the Gram-positive bacterium Bacillus thuringiensis (Bt) can be mentioned. This bacterium, naturally occurring in several habitats (including soil, leaves, grain residues, dust, water, plant matter, and insects), possesses the inherent characteristic of forming protein crystals during its quiescent and / or sporulation phases. Protein crystals, or δ-endotoxins, comprising 20-30% of total cellular protein (Boucias & Pendland, 1998), can possess specific insecticidal properties and can exhibit various shapes, such as bipyramidal, spherical, rectangular, cubic, and irregular. Bipyramidal crystals have a higher toxicity frequency than other crystal shapes, particularly against lepidopteran insects.

[0004] The mechanism of action of insecticidal Cry proteins typically involves the dissolution of crystals in the midgut of the target insect, the digestion of the protoxin by proteases present in the insect's gut, the adhesion of the active toxin to midgut receptors, and the insertion of the active Cry toxin into the apical cell membrane, creating ion channels or pores (cell lysis). Once these channels are formed, they alter the insect's feeding habits and disrupt gut integrity, leading to reduced growth and even death.

[0005] One advantage of using Cry proteins in agriculture is that each of these proteins exhibits selective toxicity to a variety of pest species and is non-toxic to other vertebrate organisms such as fish, amphibians, reptiles, birds, and mammals. Another advantage of Cry proteins is their relative specificity to pests from different crops. Several cry genes are known. Cry1, cry2, and cry9 genes are generally active against lepidopterans; cry2, cry4A, cry10, cry11, cry17, cry19, cry24, cry25, cry27, cry29, cry30, cry32, cry39, and cry40 genes are generally active against dipterans; cry3, cry7, and cry8 genes are generally active against coleopterans; and cry5, cry12, cry13, and cry14 genes are generally active against nematodes. Commercial products containing insecticidal Bt-Cry proteins are currently being produced and used as biopesticides. These products account for a high percentage of sales and have been primarily used for the control of Lepidoptera and Diptera pests for over 60 years. Another use of this Cry protein is its expression in transgenic plants.

[0006] Although specific Bt proteins are used to control pests in genetically modified maize plants, their use has led to the emergence of Lepidoptera and Coleoptera populations resistant to some of these Bt proteins (Tabashnik, BE; Brévault, T.; Carrière, Y. Insect resistance to genetically engineered crops: successes and failures. ISB News Report: Agricultural and Environmental Biotechnology, January 2014.). One specific example is the emergence of populations of the fall armyworm (Spodoptera frugiperda) with specific resistance to the Cry1F and Cry1A genes, most common in tropical conditions. The genus Spodoptera is a major pest found in maize-growing regions worldwide, and the development of resistance to the Cry1F and Cry1A proteins poses a significant challenge to maize production in these regions.Populations of the genus *Noctuidae* resistant to the Cry1F protein were found in the field: (1) in Puerto Rico in 2010 (Storer, NP; Babcock, JM; Schlenz, M.; Meade, T.; Thompson, GD; Bing, JW; Huckaba, RM. Discovery and characterization of field resistance to *Noctuidae*: *S. frugiperda* (Lepidoptera: Noctuidae) in Puerto Rico. Journal of Economic Entomology, v. 103, p. 1031-1038, 2010.), (2) in the United States (Huang, F.; Qureshi, JA; Meagher JR, RL; Reisig, DD; Head, GP; Andow, DA; NI, X.; Kerns, D.; Buntin, GD; Niu, Y.; Yang, F.; Dangal, V. Cry1F resistance in fall armyworm). Field-evolved resistance to Cry1F maize by S.frugiperda (Lepidoptera: Noctuidae) in Brazil. Crop Protection, v.64, p.150-158, 2014). In Brazil, a population of fall armyworms resistant to Cry1Ab was also discovered (Omoto, C.; Berbardi, O. Salmeron, E.; Sorgatto, RJ; Dourado, PM; Crivellari, A.; Carvalho, RA; Willse, A.; Martinelli, S.; Head, GP. Field-evolved resistance to Cry1Ab maize by S. frugiperda in Brazil. Pest Management Science, Chichester, 2016).Due to selection pressures from products based on transgenic plants containing a single cry1 gene active against fall armyworm, some of these Cry1F-resistant fall armyworm populations have rapidly developed.

[0007] Due to the significant challenge of developing resistance to a single Cry1F expression gene in maize plants, commercial varieties containing combinations of two or more insecticidal genes, such as cry1Ab, Cry1F, vip3A, cry1A.105, cry2Ab, cry3Bb, cry34Ab, cry35Ab, mcry3A, ecry3.1Ab, and dvsnf7, are available.

[0008] Therefore, the use of proteins exhibiting different modes of action in so-called pyramiding is crucial, as this slows the rate of resistance development. Thus, for example, there is great interest in the continued discovery of new proteins active against the fall armyworm, which does not show cross-resistance with those already resistant to Cry1F.

[0009] The dvsnf7 gene is another pest control technique that uses double-stranded RNA to suppress genes important for insect survival. However, it has little effect on lepidopterans, which are extremely voracious and rapidly cause defoliation.

[0010] Therefore, there is a strong need to develop novel candidate proteins that are toxic to both wild-type insect populations and Cry1F / Cry1A resistant populations. These new proteins should be highly valuable for use in transgenic strategies, suitable for transgenic technology, and capable of controlling lepidopteran pests in transgenic crops.

[0011] One such protein is Cry1Da, produced by certain Bt strains, and is known to be toxic to the fall armyworm (Costa, ML, Lana, UG, Barros, EC, Paiva, LV and Valicente, FH, J of Agricultural Science, 2014, vol 6, pp128-136). However, Cry1Da proteins have been previously reported to have a limited toxicity spectrum against lepidopteran pests (von Frankenhuyzen, 2009) and varying toxicity to different populations of lepidopteran species (e.g., the fall armyworm). For example, Monnerat et al. (2006) found that Cry1Da proteins were toxic to fall armyworms collected in Colombia and Mexico, but non-toxic to populations collected in Brazil (Applied and Environmental Microbiology, 2006, vol 72, pp. 7029-7035).

[0012] The relatively small activity spectrum against lepidopteran pests and the variability in toxicity have been significant limitations to the use of Cry1Da. Specifically, Yon Frankenhuyzen (Frankenhuyzen, K. 2009. Minireview: Insecticidal activity of Bacillus thuringiensis crystal proteins. Journal of Invertebrate Pathology. 101: 1-16) reported that, compared to the major tested Cry1A and 2Aa proteins (active against >80% of tested species), Cry1Da has the lowest activity spectrum against a wide range of lepidopteran species (toxic only to 44% of tested species). Due to the limitations of the natural Cry1Da protein in terms of its activity spectrum and variable toxicity levels, researchers have been working to improve the structure of the natural Cry1Da protein to make it more effective against a wider range of lepidopteran pests (more specifically, the corn earworm (Helicoverpa zea)).

[0013] WO2007107302 describes the use of Cry1C, Cry1D, or Cry1Da sequences to generate novel chimeric proteins with potential activity against the corn ear borer. Similarly, WO2015143311 and WO2016061377 describe structural modifications of the Cry1Da protein in an attempt to broaden its spectrum of activity against other lepidopteran pests, particularly the corn ear borer, including modifications to the natural Cry1Da amino acid sequence or fusion with other insecticidal proteins to increase insecticidal activity against a wider range of lepidopteran pests. Because these experimental and research findings have focused on broadening the spectrum of Cry1Da's activity against other lepidopterans, virtually no data are available regarding the toxicity of Cry1Da or its naturally occurring variants against fall armyworm populations, including those resistant to Cry1F. The only study involving the evaluation of Cry1Da expression in transgenic maize plants indicates that this protein provides small to moderate protection against leaf damage caused by the fall armyworm (a population not specified in this work). Furthermore, no specific data were provided regarding the toxicity of this protein to this pest. Given the extent of damage caused in the aforementioned studies, it is reasonable to conclude that the genetically modified plants have only minor toxic effects on insects.

[0014] For genetically modified plants, a DNA construct encoding the desired protein is inserted individually into the plant genome via genetic transformation. Current plant transformation methods primarily use *Agrobacterium tumefaciens*, which typically results in low copy numbers of the gene construct in the host plant genome. The gene construct contains a promoter, coding region, and terminator. The promoter is a temporal and, more importantly, gene-specific element that determines regulation and expression. Typically, for sufficiently large expression, such as the production of insecticidal proteins to control lepidopteran pests, constitutive promoters are used, such as the promoter controlling ubiquitin gene expression in maize.

[0015] The integration of DNA constructs into the host genome is random, and this random insertion into the plant genomic DNA may affect genes whose products are crucial for plant survival, thus rendering the resulting plants ineffective. Furthermore, random insertion can target host genomic regions that may negatively impact the expression of genes of interest, regardless of the use of constitutive promoters. In other instances, excessive production of construct gene products has detrimental effects on cells, primarily leading to reduced yields. Due to these potential problems, dozens (and in some cases hundreds) of different events are typically generated, and screening is performed on individual events with the desired transgene expression patterns and levels for commercial purposes.

[0016] Events exhibiting desired transgenic expression levels or patterns can be used to transfer genetic material into other genetic backgrounds within the same species via conventional sexual hybridization. There are reports of genes that, even at high constitutive expression levels in the parental genotype, do not necessarily show the same response in other genetic backgrounds. Therefore, it is desirable that, in addition to adequate spatiotemporal expression, transformation events should also result in low gene expression changes during hybridization with other genotypes (different backgrounds).

[0017] In this scenario, the offspring of such a hybrid retain the transgenic expression characteristics of the initial transformed host plant. This strategy is used to ensure reliable gene expression in multiple varieties highly adapted to local growing conditions. This is influenced by inserting the integrated DNA into the optimal location within the genome, thereby providing optimal levels of spatiotemporal expression, stability across generations and across multiple genetic origins. Therefore, the physical genomic location of the inserted DNA becomes a key characteristic of the validity of the resulting product, and is thus novel.

[0018] It is also of great significance to establish a method capable of detecting the presence of specific transformation events in plants or processed samples for testing seed quality and field release. This method can also be used to identify and monitor gene segregation in sexually hybridized progeny, screen for events through hybridization, and detect them in food derived from recombinant plants. Well-known nucleic acid detection methods include (but are not limited to) in vitro DNA amplification PCR (polymerase chain reaction) using polynucleotide primers. Another method is DNA hybridization using nucleic acid probes. Detection methods can use primers or probes based on common elements between different gene constructs or on specific regions of the constructs.

[0019] For the reasons mentioned above, there is a need for novel nucleic acid sequences specific to genetically modified maize events, detection methods for identifying genetically modified maize events and detecting nucleic acids from genetically modified maize events in plant products, and kits containing the reagents required for detecting these nucleic acids in plant products. Summary of the Invention

[0020] This invention relates to a transgenic maize event, designated ME240913, comprising a novel transgenic genotype containing a cry1Da nucleic acid sequence optimized for expression in maize. The cry1Da coding sequence encodes a truncated variant of the natural Cry1Da protein (SEQ ID NO: 3), which surprisingly confers a high level of protection to the plant against leaf damage caused by several naturally occurring fall armyworm populations in Brazil (both wild-type and resistant to Cry1F). Importantly, the leaf tissue of event ME240913 exhibits unexpectedly high toxicity to lepidopteran pests, such as Noctuidae insects, particularly the fall armyworm. In addition to the cry1Da coding sequence, this invention further provides other nucleic acids specific to event ME240913, namely amplicon sequences generated by PCR reactions using specific primers to confirm the 5' conjugate sequence, the 3' conjugate sequence, the 5' and 3' flanking sequences, and / or their complements. The present invention further provides an amplicon containing the specific nucleic acid of event ME240913, a transgenic maize plant containing the specific nucleic acid of event ME240913, and seeds of the transgenic maize plant.

[0021] The present invention also relates to a method for producing transgenic maize plants containing nucleic acids specific to the present invention by sexually hybridizing a first parent maize plant with a second parent maize plant to produce a plurality of first-generation offspring plants, wherein at least one of the parent plants contains nucleic acids specific to event ME240913, selecting first-generation offspring plants resistant to lepidopteran pests, self-pollinating the first-generation offspring plants to produce a plurality of second-generation offspring plants, and selecting plants resistant to lepidopteran pests from the second-generation offspring plants.

[0022] This invention further describes methods for controlling lepidopteran pests, such as those in the Noctuidae family, particularly the fall armyworm. The invention includes maize plants and methods described in event ME240913 that can effectively control specific lepidopteran pest populations, such as the fall armyworm, which becomes resistant to plants expressing the Cry1F protein.

[0023] A method for producing hybrid maize seeds has also been disclosed. This method includes the following steps: planting seeds of a first innate maize cultivar containing at least one nucleotide sequence specific to event ME240913 and seeds of a second innate cultivar with a different genotype; allowing maize plants produced from the planted seeds to grow until flowering time; emasculating the flowers of one of the innate maize cultivars; sexually hybridizing the two different innate cultivars with each other; and harvesting the resulting hybrid seeds.

[0024] Seed samples containing nucleic acids specific to event ME240913, and corresponding maize plants grown from those seeds, are deposited at the American Type Culture Collection (ATCC) under accession number PTA-126224. Except for the morphological and physiological characteristics conferred by the novel genotypes of this invention on the maize plants, the transgenic maize plants of this invention substantially exhibit all the corresponding morphological and physiological characteristics of the non-transgenic syngeneic maize plants. This invention also provides plant products and extracts from ME240913 maize plants, tissues, and seeds.

[0025] The present invention further provides a method for introducing event ME240913 into a maize line by sexually hybridizing a plant containing event ME240913, such as (but not limited to) a plant obtained from seeds deposited at the American Center for Type Culture Collection (ATCC) with accession number PTA-126224.

[0026] According to the present invention, event ME240913 can be combined with other transgenic maize events using methods known in the art, such as gene aggregation. The teachings of ME240913 regarding high levels of control over plant damage caused by various Brazilian fall armyworm populations (including Cry1F-resistant insects) establish that the new event is toxic to Brazilian Cry1F-resistant fall armyworm populations. These results also indicate that event ME240913 acts through a different mechanism of action than Cry1F, and therefore, maize events will be highly effective when used in combination with other insecticidal genes integrated into the plant using gene aggregation. Event ME240913 also expresses the herbicide resistance pat(bar) gene. Therefore, event ME240913 can also be used to reduce herbicide resistance rates using gene aggregation.

[0027] Examples of gene aggregation include (but are not limited to) herbicide and pest resistance events. Using this method, event ME240913 can, for example, be combined with an event containing one or more genes selected from the following: pat gene, cp4 epsps (5-enolpyruvyl-shikimate-3-phosphate synthase) gene, cry1Ab gene, cry1F gene, vip3A gene, cry1A.105 gene, cry2Ab gene, cry3Bb gene, cry34Ab gene, cry35Ab gene, mcry3A gene, ecry3.1Ab, dvsnf7 gene, and amy797E gene.

[0028] The present invention further provides a pair of polynucleotide primers comprising a first polynucleotide primer and a second polynucleotide primer, which work together to generate a diagnostic amplicon for event ME240913 in the presence of a DNA template of event ME240913 in a sample, wherein the first polynucleotide primer comprises a portion of a 5' flanking sequence, a portion of a 3' flanking sequence and / or a complement thereof, wherein the second polynucleotide primer comprises a portion of a specific insert sequence or a complement thereof, and wherein the maize event ME240913 has a specific sequence containing an event insert and a flanking sequence from the maize genome.

[0029] This invention relates to transgenic maize plants and their cells and tissues, which contain the nucleic acid molecules of this invention.

[0030] In one embodiment, the transgenic maize plant is resistant to lepidopteran pests. In a most preferred embodiment, the transgenic maize plant is highly resistant to leaf damage caused by naturally occurring fall armyworm populations found in maize-producing regions of Brazil.

[0031] In another embodiment, the transgenic maize plant is highly toxic to the fall armyworm, causing 100% mortality when fed fresh leaf tissue. In yet another embodiment, the transgenic maize plant is a plant highly toxic to the fall armyworm, as demonstrated by the high mortality and morbidity rates of the fall armyworm after lyophilizing the freeze-dried leaves of event ME240913 at a ratio of 1:25 (weight / weight) with artificial diet. The high toxicity of the freeze-dried plant leaf tissue from event ME240913 indicates that the event is highly effective in controlling the fall armyworm and can be used to aggregate several insect control genes.

[0032] Maize seeds containing the nucleotide molecules of the present invention have also been disclosed. In one embodiment, the maize seeds are deposited at the U.S. Center for Type Culture Collection (UCC) under accession number PTA-126224 and are used to produce transgenic maize plants.

[0033] Furthermore, the present invention relates to maize plant products, tissues or seeds of event ME240913, which contain the nucleotide sequence of the present invention or its complement, and wherein the sequence can be detected in the plant product by nucleic acid amplification or nucleic acid hybridization methods.

[0034] The present invention further considers plant products, such as (but not limited to) corn cereals, feed, cornmeal, corn flour, corn syrup, corn oil, corn starch, and cereal foods made wholly or partially from corn derivatives.

[0035] The present invention further provides a kit for detecting nucleic acids specific to event ME240913, comprising at least one nucleic acid molecule, said nucleic acid molecule being a primer or probe, said primer or probe comprising a nucleic acid sequence containing a specific sequence and its complement, wherein said primer or probe diagnoses the presence of a nucleic acid sequence specific to event ME240913 in a sample by amplifying or hybridizing a target nucleic acid sequence in a sample and then detecting the amplicon or hybridization with the target sequence.

[0036] Further disclosed is a method for detecting the presence of at least one nucleic acid molecule specific to event ME240913 in a sample containing maize nucleic acid, wherein the method comprises the steps of: contacting the sample with a pair of specific primers of the present invention, performing a nucleic acid amplification reaction to generate an amplicon, and detecting the amplicon, wherein the amplicon contains a nucleic acid sequence specific to event ME240913 or its complement, and wherein the maize event ME240913 has a specific sequence containing an event insert and a flanking sequence from the maize genome.

[0037] Another method for detecting the presence of nucleic acid molecules specific to event ME240913 in a sample containing nucleic acids from maize includes the following steps: contacting the sample with a probe that hybridizes with DNA of event ME240913 under highly stringent conditions and does not hybridize with DNA of control maize plants under highly stringent conditions, wherein the probe contains a nucleotide sequence specific to event ME240913 and its complement, subjecting both the sample and the probe to highly stringent hybridization conditions, and detecting hybridization of the probe with the nucleic acid molecule, wherein the maize event ME240913 has a specific sequence containing event inserts and flanking sequences from the maize genome.

[0038] Furthermore, a method for controlling lepidopteran pests in maize plants is disclosed, wherein the maize plants contain nucleic acid molecules containing a nucleic acid sequence or its complement specific to event ME240913, wherein the method includes the step of planting seeds obtained from plants containing the nucleic acid sequence specific to event ME240913 in the growing area of ​​maize plants susceptible to lepidopteran pests.

[0039] Furthermore, a method for controlling lepidopteran pests in maize plants containing a truncated and modified Cry1Da protein produced by expressing a unique sequence of nucleic acid specific to event ME240913, resulting in increased expression levels of the truncated and modified Cry1Da protein in leaves. These levels are highly protective against leaf damage caused by naturally occurring populations in Brazil and are also highly toxic to the fall armyworm, as evidenced by the high mortality rates observed in the studies conducted.

[0040] The use of plants, plant cells, plant parts or seeds, including those mentioned in event ME240913, for hybridization with a second plant, regeneration of plants, planting or growing plants in fields or producing plant products is further disclosed.

[0041] These and other aspects of the invention will become more apparent from the following detailed description.

[0042] Sequence descriptions in the sequence list SEQ ID NO: 1 is the nucleotide sequence encoding a truncated Cry1Da protein for expression optimization in maize, present in event ME240913.

[0043] SEQ ID NO: 2 is the nucleic acid sequence of the full-length transgenic construct of event ME240913, which includes, in the following order: the 3'UTR terminator region of the Tvsp gene; the coding region (bar) and translation enhancer region (tev) of the phosphinic acid acetyltransferase gene; the repeating promoter region of the CaMV 35S gene and the promoter region of the ubiquitin gene; the codon-optimized nucleic acid sequence of the cry1Da gene (SEQ-ID-NO: 1); and the 3'UTR terminator region of the carmine synthase gene.

[0044] SEQ ID NO: 3 is the amino acid sequence of the truncated Cry1Da protein expressed by event ME240913.

[0045] SEQ ID NO: 4 is the 5' conjugation sequence.

[0046] SEQ ID NO: 5 is a 3' conjugation sequence.

[0047] SEQ ID NO: 6 is the 5' flanking sequence.

[0048] SEQ ID NO: 7 is the 3' flanking sequence.

[0049] SEQ ID NO: 8 is a nucleotide sequence containing the 5' flanking sequence (nucleotides 1-116), the full-length insertion sequence (nucleotides 117-6306), and the 3' flanking sequence (nucleotides 6307-6424) of event ME240913.

[0050] SEQ ID NO: 9 is the forward primer sequence used to amplify the 5' conjugate sequence.

[0051] SEQ ID NO: 10 is the reverse primer sequence used to amplify the 5' conjugate sequence.

[0052] SEQ ID N: 11 is an illustrative PCR amplicon sequence obtained using primers for confirming the specificity of the 3' conjugation sequence.

[0053] SEQ ID NO: 12 is a probe sequence for detecting the 5' conjugate sequence.

[0054] SEQ ID NO: 13 is the forward primer sequence used to amplify the 3' conjugate sequence.

[0055] SEQ ID N: 14 is the reverse primer sequence used to amplify the 3' conjugate sequence.

[0056] SEQ ID NO: 15 is an illustrative PCR amplicon sequence obtained using primers for confirming the specificity of the 5' conjugation sequence.

[0057] SEQ ID NO: 16 is a probe sequence for detecting the 3' conjugate sequence. Attached Figure Description

[0058] Figure 1 A representative diagram of the insert for event ME240913 and the corresponding nucleotide sequence (including the 5' and 3' junctions and flanking sequences of the maize genome) defining the event.

[0059] Figure 2 A diagram depicting the gene construct Ubi::cry1Da::NOS e 2x35S::bar::Tvsp of the binary vector pTF101.1 inserted into the Hind III and EcoRI enzyme sites between the left and right boundaries of TDNA.

[0060] Figure 3 This shows the results of a 5-day assessment of fall armyworm control using fresh maize leaves in event ME240913. Fresh leaf samples are shown for non-GMO maize (a) and maize genetically modified with Cry1Da (b).

[0061] Figure 4The results of assays on fresh leaf tissues of fall armyworm exposed to two genetic backgrounds containing event ME240913 are shown. The survival rate (%) of fall armyworm was evaluated and displayed up to 3 days after exposing newly hatched larvae to fresh control maize leaves and maize leaves expressing event ME240913 under two different genetic backgrounds (heterozygotes and RC1F1).

[0062] Figure 5 The survival rate of newly hatched larvae 14 days after exposure to freeze-dried leaves at a 1:25 dilution in artificial control diet and event ME240913 was shown.

[0063] Figure 6 shows the size of surviving caterpillars after exposure to freeze-dried leaf tissue of event ME240913 diluted 1:25 in an artificial diet for 7 (A) and 10 (B) days, compared with the control diet.

[0064] Figure 7 The results show the damage to leaves of control maize plants (conv) and event ME240913 (GMO) in six different fall armyworm colonies.

[0065] Figure 8 shows photographs of damage to maize plants (left) and maize plants from event ME240913 in a field infested by fall armyworm colonies from Palotina / PR (8A), Rondonópolis / MT (8B), Rondonópolis+CampoVerde / MT (8C), Paracatu / MG (8D), Sete Lagoas / MG (8E), and Ivatuba / PR (8F), comparing the damage to maize plants (right) in the control group.

[0066] Figure 9 This refers to a chart displaying the damage score (±CI, P=0.05) caused by fall armyworm infestation in the 1970 Oak scale. Treatment 1: Transgenic maize containing the codon-optimized cry1Da nucleic acid sequence (SEQ ID NO: 1) of this invention + Cry1F resistant caterpillar population; Treatment 2: Non-transgenic L3 maize line + Cry1F resistant caterpillar population; Treatment 3: Transgenic maize containing the codon-optimized cry1Da nucleic acid molecule (SEQ ID NO: 1) of this invention + susceptible caterpillar population; Treatment 4: Non-transgenic L3 maize line + susceptible caterpillar population. Detailed Implementation

[0067] Unless otherwise defined, all terms used in this art, notes and other scientific terms used herein are intended to have the meaning commonly understood by those skilled in the art in this invention. In some instances, terms with their commonly understood meanings are defined in this document for clarity and / or for timely reference, and the inclusion of such definitions in this document should not necessarily be construed as representing a substantial difference relative to the common understanding in the prior art.

[0068] The techniques and procedures described or referenced in this document are generally well understood and employed by those skilled in the art using conventional methods. Where appropriate, procedures involving the use of commercially available kits and reagents are generally performed according to manufacturer-defined protocols and / or parameters, unless otherwise specified.

[0069] It is worth noting that, where appropriate, the present invention is not limited to the methods, schemes, cell lines, genus or animal species, constructs and specific reagents as described, which can obviously be varied. Furthermore, the terminology used in this document is for the purpose of describing examples of specific embodiments only and is not intended to limit the scope of the invention.

[0070] Throughout this document, the singular forms “a” and “the” or any term or expression in the singular form include reference to the plural, unless the context clearly indicates otherwise.

[0071] Throughout this document, the word “contains” and any variations thereof, such as “containing” or “including,” shall be interpreted as an “open-ended term,” which may mean that it includes additional elements or groups of elements that are not explicitly mentioned and do not have restrictive characteristics.

[0072] Throughout this document, the phrase “consists” and any variations such as “consist” or “consisting” should be interpreted as “closed terms” and may not imply the inclusion of additional elements or groups of elements with restrictive characteristics that are not explicitly described.

[0073] Throughout this document, any precise value or range of precise values ​​provided for a particular factor, quantity, concentration, or preference shall be interpreted as providing a corresponding value or range that is also approximate, for example, by expressing “about”.

[0074] Throughout this document, words and expressions such as “preferred,” “particularly,” “for example,” “like,” “more particularly,” and variations thereof must be interpreted as entirely optional features, preferred embodiments, or possible non-exhaustive examples, without limiting the scope of the invention.

[0075] Throughout this document, terms and expressions such as “nucleic acid” and “nucleotide” shall be interpreted as naturally occurring, synthetic, or artificial nucleic acids or nucleotides. This includes single-stranded or double-stranded deoxyribonucleotides (DNA) or ribonucleotides (RNA) in sense or antisense configurations, or any nucleotide analogues, polymers, or hybrids thereof. Unless otherwise stated, a particular nucleic acid sequence also implicitly includes variants of its conserved modifications (e.g., degenerate codon substitutions) and complementary sequences, as well as explicitly specified sequences. The term “nucleic acid” in this document may be used interchangeably with the terms “gene,” “cDNA,” “mRNA,” “oligonucleotide,” “nucleic acid molecule,” or “primer.”

[0076] The terms "nucleic acid molecule" and "nucleic acid sequence" refer to polymers of single-stranded or double-stranded DNA or RNA bases read from the 5' to 3' ends. These include chromosomal DNA, self-replicating plasmids, and infectious DNA or RNA polymers that play a major structural role. They also refer to a continuous list of abbreviations, letters, characters, or words representing nucleotides or genes, as commonly used in the technical field of this invention.

[0077] As used herein, the term "amplified" means the construction of multiple copies of a nucleic acid molecule or multiple copies complementary to a nucleic acid molecule using at least one nucleic acid molecule as a template. Amplification systems include (but are not limited to) polymerase chain reaction (PCR) systems, ligase chain reaction (LCR) systems, nucleic acid sequence-based amplification (NASBA, Cangene, Mississauga, Ontario), Q-β replicase systems, transcription-based amplification systems (TAS), and strand displacement amplification (SDA). See, for example: Diagnostic Molecular Microbiology: Principles and Applications, DHPersing et al., Ed., American Society for Microbiology, Washington, DC (1993). Amplification products are described as amplicones.

[0078] The "coding sequence" is a nucleic acid sequence that is transcribed into RNA, such as mRNA, rRNA, tRNA, snRNA, sense RNA, or antisense RNA. Preferably, the RNA is then translated in the organism to produce a protein.

[0079] Throughout this document, terms and expressions such as “sequence similarity” or “identity” relative to another sequence should be interpreted as the percentage of nucleotides in a sequence that are identical to those in another sequence after sequence alignment and, where necessary, the introduction of gaps to achieve the maximum percentage of sequence identity. According to the invention, the phrase “at least 70% similarity” is defined, for example, as 70%–100% similarity or identity. Preferably, the similarity percentage is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%.

[0080] A "gene" is a defined region located within the genome, and although it contains the coding sequence mentioned above, it may contain other sequences, primarily regulatory nucleic acid sequences responsible for controlling expression, i.e., transcription and translation of the coding region. Genes may also contain other 5' and 3' untranslated sequences and termination sequences. Other possible elements include, for example, introns.

[0081] "Gene of interest" refers to any gene that, when transferred into a plant, imparts a desired trait to the plant, such as antibiotic resistance, virus resistance, insect resistance, disease resistance, or resistance to other pests, herbicide tolerance, improved nutritional value, enhanced performance in industrial processes, or altered reproductive capacity.

[0082] As used in this article, "genotype" refers to genetic material inherited from the parent maize plant. Genotype ME240913 refers to heterologous genetic material transformed within the plant genome, as well as genetic material flanking inserted sequences.

[0083] "Heterologous" nucleic acid sequences are nucleic acid sequences that are not naturally associated with the host cell into which they are introduced, including multiple copies of the nucleic acid sequence that are not naturally present.

[0084] "Homologous" nucleic acid sequences are nucleic acid sequences that are naturally associated with the host cell in which they are introduced.

[0085] "High toxicity" of maize leaf tissue refers to the ability of a leaf tissue sample to cause 100% mortality in lepidopteran species such as the fall armyworm within 7 days of exposure to the leaf tissue. Another part of the definition of "high toxicity" is the ability of dried leaf tissue diluted 1:25 with standard maize leaf tissue to cause >95% mortality and morbidity within 14 days of exposure to a leaf tissue diet.

[0086] "Operationally ligated" refers to the association of nucleic acid sequences into a single nucleic acid fragment, such that the function of one affects the function of the other. For example, when a promoter can influence the expression of a coding sequence or functional RNA (i.e., when the coding sequence or functional RNA is under the transcriptional control of the promoter), the promoter is operationally ligated to the coding sequence or functional RNA. Coding sequences in the sense or antisense direction can be operationally ligated to regulatory sequences.

[0087] As used herein, “plant protection” refers to the ability of intact maize plants to resist leaf damage caused by susceptible lepidopteran pests (including, but not limited to, protection against leaf damage caused by the fall armyworm). Protection can be assessed by examining plants or photographs of plants to compare damage found in control maize plants with damage observed in maize plants expressing Cry1Da. As used herein, “plant protection” also refers to the ability of intact plants to resist damage from susceptible lepidopteran pests, including, but not limited to, the use of standard and recognized methods for classifying plant damage.

[0088] As used in this article, "primers" are isolated nucleic acids that undergo hybridization annealing to a complementary target DNA strand to form a hybrid between the primer and the target DNA strand, and are then extended along the target DNA strand by a polymerase (such as DNA polymerase). Primer pairs or primer sets can be used to amplify nucleic acid molecules, for example, via polymerase chain reaction (PCR) or other conventional nucleic acid amplification methods.

[0089] The "probe" is an isolated nucleic acid that binds to a conventionally detectable marker or reporter molecule, such as a radioisotope, ligand, chemiluminescent agent, or enzyme. This probe is complementary to the target nucleic acid strand, in the case of this invention, to the genomic DNA strand from the maize event ME240913. The DNA from event ME240913 can be derived from maize plants or from samples containing DNA from event ME240913. The probes of this invention comprise not only ribonucleic acid or deoxyribonucleic acid, but also polyamides and other probe materials that specifically bind to the target DNA sequence and can be used to detect the presence of that target DNA sequence.

[0090] Primers and probes are typically 10-15 nucleotides long or longer. They can also be at least 20 nucleotides long, or at least 25 nucleotides long, or at least 30 nucleotides long. These primers and probes specifically hybridize to the target sequence under highly stringent hybridization conditions. The primers and probes of this invention can have full-length sequences complementary to the target sequence, although probes that differ from the target sequence but retain the ability to hybridize to the target sequence can be designed using conventional methods.

[0091] "Strong conditions" or "strong hybridization conditions" refer to conditions under which the probe will hybridize with its target sequence to a higher degree of detectability than other sequences. Strict conditions depend on the target sequence and will vary depending on the polynucleotide structure. By controlling hybridization strictness and / or washing conditions, target sequences that are 100% complementary to the probe (homologous probe) can be identified. Alternatively, strict conditions can be adjusted to allow for some mismatches in the sequence, resulting in the detection of a lower degree of similarity (heterologous probe). Longer sequences hybridize specifically at higher temperatures. Detailed guidelines on nucleic acid hybridization can be found in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology - Hybridization with Nucleic Acid Probes, Part I, Chapter 2, "Overview of principles of hybridization and the strategy of nucleic acid probe assays", Elsevier: New York; and Current Protocols in Molecular Biology, Chapter 2, Ausubel et al., Eds., Greene Publishing and Wiley-Interscience: New York (1995) and Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual (5 th Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

[0092] Specificity is generally a function of post-hybridization washing, with the ionic strength and temperature of the final washing solution being key factors. Typically, highly stringent hybridization and washing conditions are chosen to be below the pyrolysis point (T0) of the specific sequence at a given ionic strength and pH. m Approximately 5℃. m The temperature at which 50% of the target sequence hybridizes with a perfectly matched probe (at a defined ionic strength and pH). Generally, under highly stringent conditions, the probe will hybridize with its target sequence but not with other sequences.

[0093] An example of highly stringent hybridization conditions for hybridizing complementary nucleic acids with more than 100 complementary residues in Southern blotting or Northern blotting filters is overnight hybridization at 42°C with 50% formamide and 1 mg heparin. An example of very highly stringent washing conditions is 0.15 M NaCl at 72°C for approximately 15 minutes. An example of highly stringent washing conditions is washing with 0.2x SSC at 65°C for 15 minutes (see Sambrook below for a description of the SSC buffer).

[0094] A good example of hybridization conditions used in this invention includes overnight hybridization at 67°C in 7% SDS, 0.25M NaPO4, pH 7.2, followed by two washes at 65°C for 30 minutes each in 5% SDS, 0.20M NaPO4, pH 7.2, and two washes at 65°C for 30 minutes each in 1% SDS, 0.20M NaPO4, pH 7.2. A good example of moderately stringent washing for duplexes, for example, exceeding 100 nucleotides, is 1x SSC for 15 minutes at 45°C. A good example of low-stringent washing for duplexes, for example, exceeding 100 nucleotides, is 4-6x SSC for 15 minutes at 40°C.

[0095] For probes of approximately 10–50 nucleotides, highly stringent conditions generally involve a salt concentration of less than approximately 1.0 M Na ions at pH 7.0–8.3, typically approximately 0.01–1.0 M Na ions (or other salts), and a temperature typically at least approximately 30 °C. Highly stringent conditions can also be achieved by adding destabilizing agents such as formamide. Typically, a signal-to-noise ratio of 2x (or higher) observed for unrelated probes in a specific hybridization assay indicates that specific hybridization has been detected. If nucleic acids that do not hybridize to each other under highly stringent conditions encode substantially identical proteins, they remain substantially identical. This occurs, for example, when nucleic acid copies are created using the maximum codon degeneracy allowed by the genetic code.

[0096] The following are good examples of hybridization / washing conditions that can be used to hybridize nucleotide sequences substantially identical to the reference nucleotide sequence of the present invention: The reference nucleotide sequence is preferably hybridized with the nucleotide sequence in 7% sodium dodecyl sulfate (SDS), 0.5M NaPO4, and 1mM EDTA at 50°C and washed in 2X SSC and 0.1% SDS at 50°C; more preferably, hybridized in 7% sodium dodecyl sulfate (SDS), 0.5M NaPO4, and 1mM EDTA at 50°C and washed in 1X SSC and 0.1% SDS at 50°C; most preferably, hybridized in 7% sodium dodecyl sulfate (SDS), 0.5M NaPO4, and 1mM EDTA at 50°C and washed in 0.5X SSC and 0.1% SDS at 50°C; and preferably hybridized in 7% sodium dodecyl sulfate (SDS), 0.5M NaPO4, and 1mM EDTA at 50°C and washed in 0.1X SSC and 0.1% SDS at 50°C. Washing in SSC and 0.1% SDS at 50°C, more preferably hybridizing in 7% sodium dodecyl sulfate (SDS), 0.5M NaPO4, and 1mM EDTA at 50°C and washing in 0.1X SSC and 0.1% SDS at 65°C. All of the above conditions can be used to detect the sequences of the present invention. Highly stringent conditions have been used for the purposes of defining the invention.

[0097] Throughout this document, terms and expressions such as “promoter,” “promoter sequence,” etc., shall be interpreted as DNA sequences that, once operatively linked to a nucleotide sequence of interest, control the transcription of that nucleotide sequence into RNA. A promoter is located 5' (or upstream) of the transcription start site of the nucleotide sequence of interest to which it controls the transcription of its mRNA and provides specific binding sites for RNA polymerases and other transcription factors for transcription initiation. It may include other regulatory sequences known to those skilled in the art. According to the invention, a promoter may be heterologous or homologous to the corresponding cell or host. If the nucleic acid sequence originates from a different species, or if it originates from the same species and is modified from its initial form, it is “heterologous” to the organism or the second nucleic acid sequence.

[0098] As used in this paper, the term "specific to event ME240913" refers to the unique characteristics of event ME240913. Therefore, nucleic acids specific to event ME240913 were not found in maize plants different from ME240913.

[0099] As used in this article, the term "maize" refers to the species maize and includes all plant species that can reproduce with maize, including wild-type maize species.

[0100] As used herein, “detection kit” refers to a component kit for detecting the presence of plant-specific nucleic acids in a sample, wherein the kit includes the nucleic acid probes and / or primers of the present invention that specifically hybridize with the target DNA sequence under highly stringent conditions, as well as other materials required to realize the nucleic acid amplification or hybridization method.

[0101] Throughout this document, the term "transformation," etc., should be interpreted as the process of introducing heterologous DNA into cells, plant tissues, or plants. This can occur under natural or artificial conditions, such as using several methods well-known in the art, in prokaryotic or eukaryotic host cells. The method is typically based on the selection of the host cells to be transformed and may include (but is not limited to) viral infection, electroporation, lipid transfection, particle bombardment (bio-ejection), and Agrobacterium-mediated methods.

[0102] Throughout this document, the term "transgenic" should be interpreted as any nucleic acid sequence introduced into a cell (whether or not it is integrated into the genome) through experimental manipulation. A transgenic sequence can be an "endogenous DNA sequence" or an "exogenous DNA sequence" (i.e., "heterologous"). The term "endogenous DNA sequence" refers to a nucleotide sequence that is naturally present in the cell to which it is introduced. The term "exogenous DNA sequence" refers to a nucleotide sequence that is not naturally present in the cell to which it is introduced. Regarding the transformed organism, the term "transgenic" means an organism transformed with a recombinant DNA molecule, which preferably contains a suitable promoter operatively linked to the DNA sequence of interest.

[0103] Throughout this document, the term "vector" should be interpreted as a construct containing a DNA sequence operatively linked to one or more suitable control sequences capable of causing the DNA sequence to be expressed in a suitable host. Such control sequences include, for example, promoters for transcription, optional operon sequences for controlling such transcription, sequences encoding suitable mRNA binding sites against ribosomes, and sequences controlling the termination of transcription and translation.

[0104] Several vectors are suitable for carrying out this invention. These vectors can replicate autonomously in a host organism or by chromosome replication. The vector may also be a plasmid. According to this document, the terms "plasmid" and "vector" are sometimes used interchangeably. Preferably, the vector of this invention comprises a cry1Da nucleic acid molecule containing the nucleic acid sequence as defined herein as SEQ ID NO: 1.

[0105] As used herein, the term "transgenic event" refers to a recombinant plant produced by transforming and regenerating plant tissues or cells with heterologous DNA (e.g., an expression cassette containing the gene of interest). The term "event" refers to the initial transformant and / or its progeny containing heterologous DNA. The term "event" also refers to progeny produced by sexual hybridization between a transformant and another maize variety. Furthermore, even after repeated backcrosses with a recurrent parent, the inserted DNA and flanking DNA from the transformant parent remain at the same chromosomal locations in the hybrid progeny. The term "event" also refers to DNA from the initial transformant, containing the inserted DNA and flanking sequences immediately adjacent to the inserted DNA, which is expected to be transferred to progeny receiving the inserted DNA (including the transgene of interest) due to sexual hybridization between a parental line containing the inserted DNA (e.g., the initial transformant and its progeny produced by self-pollination) and a parental line not containing the inserted DNA. Typically, plant tissue transformation produces multiple events, each representing the insertion of a DNA construct into a different location in the plant cell genome. Specific events are selected based on transgene expression or other desired traits. Therefore, the terms "event ME240913" and "event" are used interchangeably.

[0106] Insect-resistant ME240913 maize plants can be propagated by: first sexually crossing a first parent maize plant, consisting of maize plants grown from transgenic ME240913 maize plants, such as those grown from seeds deposited at ATCC with accession number PTA-126224, and their offspring derived from transformations using the expression cassette of an embodiment of the present invention that confers resistance to lepidopteran pests, with a second parent maize plant that may or may not exhibit resistance to lepidopteran pests, thereby producing multiple first-generation progeny plants; then selecting the first-generation progeny plants resistant to lepidopteran pests; and self-pollinating the first-generation progeny plants to produce multiple second-generation progeny plants; and then selecting those plants resistant to lepidopteran pests from the second-generation progeny plants. These steps may further include backcrossing the first-generation or second-generation lepidopteran pest-resistant plants with the second or third parent maize plants to produce maize plants resistant to lepidopteran pests. This method can be used to introduce the ME240913 event gene into maize lines and to aggregate the ME240913 event with other transgenic events.

[0107] Throughout this document, expressions such as "host cell," "host organism," etc., should be interpreted as referring to a specific host organism or a specific target cell, but also as the offspring or potential offspring of these organisms or cells. Because certain modifications may occur in successive generations due to mutations or environmental effects, these offspring do not necessarily need to be identical to the parent cell. However, they are still included within the scope of protection of this invention. According to the invention, the host cell can be prokaryotic or eukaryotic. Preferably, the host cell of the invention is a plant host cell. Preferably, it contains a nucleic acid sequence specific to event ME240913, selected from SEQ ID NO: 4, SEQ ID NO: 5, and their complements.

[0108] Throughout this document, terms and expressions such as “transgenic plant cell” and “transgenic plant” should be interpreted as cells or plants that have and preferably express transgenes through experimental manipulation, and further refer to the offspring of transgenic plants and subsequent generations of plants as described above.

[0109] Throughout this document, the term "plant," etc., shall be interpreted as all or part of a plant organism. In this context, "part" means plant cells and tissues, organs, and plant parts in all their forms, such as seeds, leaves, anthers, fibers, tubers, roots, root hairs, stems, embryos, callus, cotyledons, petioles, collected material, plant tissues, regenerated tissues, and cell cultures. The transgenic plants of this invention can be generated and self-pollinated or hybridized with other individuals to obtain further transgenic plants. Transgenic plants can also be obtained through vegetative propagation of transgenic plant cells.

[0110] Throughout this document, terms and expressions such as "pests" and "lepidopteran pests" should be interpreted as insects of the order Lepidoptera, including (but not limited to) the families Papilionidae, Pieridae, Lycaenidae, Nymphalidae, Danidae, Satyridae, Hesperiidae, Sphingidae, Saturniidae, Geometridae, Arctiidae, Noctuidae, Lymantriidae, Sesiidae, Crombidae, and Tineidae, and more specifically, species of the genus *Spodoptera* within the family Noctuidae. (sp.), especially the fall armyworm (S. frugiperda) (Noctuidae), and species of the genus Diatraea (Cambididae), especially the small sugarcane borer (D. saccharalis) (Cambididae).

[0111] One embodiment of the present invention relates to a method for controlling lepidopteran pests (including, but not limited to, caterpillars) in crops. Any method for controlling lepidopteran pests in crops is included within the scope of the present invention and is not particularly associated with embodiments of the present invention, provided that the crop of the present invention contains at least one nucleic acid sequence specific to event ME240913 selected from SEQ ID NO: 4, SEQ ID NO: 5 and their complements, wherein the method preferably includes planting seeds obtained from plants containing at least one nucleic acid sequence specific to event ME240913 as defined herein in crop cultivation areas susceptible to lepidopteran pests.

[0112] The “Cry1Da” class of proteins further includes its homologs. “Homologous” means that the protein or polypeptide has a defined relationship with other members of the Cry1Da class of proteins.

[0113] This invention relates to genetically improved maize lines that produce modified Cry1Da proteins for the control of lepidopteran pests. The invention is specifically designed for a transgenic maize event named ME240913 containing a novel genotype, and for compositions and methods for detecting nucleic acids specific to the ME240913 event in biological samples. The invention is further designed for maize plants containing the ME240913 genotype, transgenic seeds of maize plants, and methods for producing maize plants containing the ME240913 genotype by hybridizing self-pollinated maize containing the ME240913 genotype or another maize line. Maize plants containing the ME240913 genotype of this invention can be used to control lepidopteran pests, including (but not limited to) Noctuidae and / or Pyralidae, preferably the fall armyworm and the sugarcane borer. Maize plants from the ME240913 event exhibit phytoprotection against susceptible lepidopteran pests, including (but not limited to) wild-type Cry1F-resistant and Cry1A-resistant fall armyworms.

[0114] In another implementation, maize plants exhibit high toxicity to susceptible lepidopteran pests, including (but not limited to) the fall armyworm.

[0115] In one embodiment, the present invention relates to an isolated nucleic acid molecule comprising a nucleotide sequence specific to event ME240913.

[0116] In another embodiment, the present invention relates to an isolated nucleic acid molecule that binds a heterologous DNA molecule introduced into the genome of event ME240913 to the genomic DNA of event ME240913, comprising at least 10 or more (e.g., 15, 20, 25, 30 or more) consecutive nucleotides of the heterologous DNA molecule and at least 10 or more (e.g., 15, 20, 25, 30 or more) consecutive nucleotides of the genomic DNA flanking the insertion site of the heterologous DNA molecule. It also includes a nucleotide sequence comprising 10 or more nucleotides of the consecutive insertion sequence of event ME240913 and at least one nucleotide of the flanking DNA of event ME240913 adjacent to the insertion sequence. This nucleotide sequence is specific to event ME240913 and is diagnostic for event ME240913. Nucleic acid hybridization or amplification of the genomic DNA of event ME240913 produces an amplicon containing this specific sequence, thereby enabling the diagnosis of event ME240913. In one aspect of this implementation, the nucleotide sequences are selected from SEQ ID NO: 4, 5 and 8 and their complements.

[0117] In another embodiment, the present invention relates to an isolated nucleic acid molecule comprising a nucleotide sequence containing at least one conjugating sequence from event ME240913, wherein the conjugating sequence crosses the conjugation between a heterologous expression cassette inserted into the maize genome and maize genomic DNA by pairing an insertion site specific to and diagnosing event ME240913. In one aspect of this embodiment, the conjugating sequence is selected from SEQ ID NO: 4 and 5 and their complements.

[0118] In another embodiment, the present invention relates to an isolated nucleic acid molecule for linking a heterologous DNA molecule to the genome of a maize plant in event ME240913, comprising at least one sequence selected from ID NO: 4, 5 and their complements.

[0119] In another embodiment, the present invention relates to an isolated nucleic acid molecule comprising a nucleotide sequence specific to event ME240913, wherein said nucleotide sequence encodes a protein comprising the amino acid sequence of SEQ ID NO: 3. In one aspect of this embodiment, the nucleotide sequence is SEQ ID NO: 8 and / or its complement.

[0120] In another embodiment, the present invention relates to an isolated nucleic acid molecule comprising a nucleotide sequence selected from 4, 5, and 8 and their complements. In one aspect of this embodiment, the isolated nucleic acid molecule is present in maize seeds or plants grown from said seeds, deposited at the U.S. Center for Type Culture Collection (UCC) with accession number PTA-126224.

[0121] In one embodiment of the invention, an amplicon comprising a nucleotide sequence specific to event ME240913 is provided. In one aspect of this embodiment, the nucleotide sequence is selected from SEQ ID NO: 11 and 15 and their complements.

[0122] In another embodiment, the invention includes a flanking sequence primer for detecting event ME240913. This flanking sequence primer comprises a nucleotide sequence of at least 10 consecutive nucleotides from a 5' or 3' flanking sequence. In one aspect of this embodiment, the consecutive nucleotides are selected from at least 10 consecutive nucleotides of SEQ ID NO: 6 (5' flanking sequence) or its complement. In another aspect of this embodiment, the primer for the 5' flanking sequence has the sequence of SEQ ID NO: 9 or its complement. In another aspect of this embodiment, the consecutive nucleotides are selected from at least 10 consecutive nucleotides of SEQ ID NO: 7 (3' flanking sequence) or its complement. In yet another aspect of this embodiment, the primer for the 3' flanking sequence has the sequence of SEQ ID NO: 12 or its complement.

[0123] In yet another embodiment, the invention comprises a pair of polynucleotide primers comprising a first polynucleotide primer and a second polynucleotide primer, which work together to generate a diagnostic amplicon for event ME240913 in the presence of a DNA template from event ME240913 in the sample. In one aspect of this embodiment, the first and / or second primers are selected from SEQ ID NO: 9, 10, or their complements. In another aspect of this embodiment, the first and / or second primers are selected from SEQ ID NO: 13, 14, and their complements. In yet another aspect of this embodiment, the amplicon generated by the primer pair comprises SEQ ID NO: 11, 15, or their complements.

[0124] In another embodiment, the invention includes a pair of polynucleotide primers comprising a first polynucleotide primer and a second polynucleotide primer, which work together to generate a diagnostic amplicon for event ME240913 in the presence of a DNA template from event ME240913 in the sample. The first primer is sequence-equivalent or complementary to the insertion site of a heterologous DNA sequence from the maize plant genome that is inserted into the genome of event ME240913, and the second polynucleotide primer sequence is sequence-equivalent or complementary to the heterologous DNA sequence inserted into the genome of event ME240913.

[0125] In one aspect of this embodiment, the first polynucleotide primer comprises at least 10 consecutive nucleotides from positions 1-116 and 6307-6424 of SEQ ID NO: 8 and their complements. In another aspect of this embodiment, the first primer is selected from SEQ ID NO: 9, 13 and their complements. In another aspect of this embodiment, the second polynucleotide primer comprises at least 10 consecutive nucleotides from positions 117-6306 of SEQ ID NO: 8 or their complements. In yet another aspect of this embodiment, the second polynucleotide primer is selected from SEQ ID NO: 10, 14 and their complements.

[0126] In another aspect of this embodiment, the first polynucleotide primer as shown in SEQ ID NO: 9 and the second polynucleotide primer as shown in SEQ ID NO: 10 work together to generate a diagnostic amplicon for event ME240913 in the presence of a DNA template for event ME240913 in the sample. In one embodiment of this aspect, the amplicon comprises a nucleotide sequence as shown in SEQ ID NO: 11.

[0127] In yet another embodiment, the present invention relates to a method for detecting the presence of a nucleic acid molecule specific to event ME240913 in a sample containing maize nucleic acid, wherein the method comprises: (a) contacting the sample with a pair of primers, (b) performing a nucleic acid amplification reaction to generate an amplicon, and (c) detecting the amplicon.

[0128] In another embodiment, the present invention relates to a method for detecting the presence of a nucleic acid molecule specific to event ME240913 in a sample containing maize nucleic acid, wherein the method comprises: (a) contacting the sample with a probe that hybridizes under highly stringent conditions with genomic DNA from event ME240913 and does not hybridize under highly stringent conditions with DNA from control maize plants, wherein the probe comprises at least 10 consecutive nucleotides selected from SEQ ID NO: 4, SEQ ID NO: 5 and their complements; (b) subjecting the sample and probe to highly stringent hybridization conditions; and (c) detecting the hybridization of the probe with the nucleic acid molecule. Detection can be performed by any means well known in the art, including fluorescence, chemiluminescence, radioactivity, immunology, etc. Where hybridization is used as a means of amplifying a specific sequence to generate an amplicon for diagnosing event ME240913, the generation and detection of the amplicon by any means well known in the art indicates hybridization with the target sequence, wherein at least one probe or primer is used.

[0129] The term "biological sample" is defined as a sample of maize plant containing or suspected of containing nucleic acids, wherein the nucleic acids are attached at one or the other end of an inserted heterologous DNA sequence to either side of a point on a genomic DNA sequence within a chromosome in which the heterologous DNA sequence is inserted, and comprise 5-10 nucleotides, also referred to herein as a conjugation sequence. Furthermore, the conjugation sequence comprises as few as two nucleotides: either flanking a nucleotide adjacent to the first nucleotide covalently linked to the first nucleotide within the inserted heterologous DNA sequence, or the first nucleotide within the genomic DNA. In one aspect of this embodiment, the probe comprises a nucleotide sequence of at least 10 consecutive nucleotides containing SEQ ID NO: 4, 5 and their complements.

[0130] In yet another embodiment, the present invention relates to a kit for detecting nucleic acids specific to event ME240913 in biological samples. The kit comprises at least one nucleic acid molecule of sufficient length as a primer or probe in methods for detecting nucleic acids. Amplification or hybridization of the target nucleic acid sequence in the sample, followed by detection of the amplicon or hybridization with the target sequence, diagnosing the presence of an event ME240913-specific nucleic acid sequence in the sample. The kit further comprises other materials necessary to allow nucleic acid amplification or hybridization. In one aspect of this embodiment, the nucleic acid molecule present in the kit comprises a nucleotide sequence selected from SEQ ID NO: 9, 10, 12, 13, 14, 16 and their complements. In another aspect of this embodiment, the nucleic acid molecule is a primer selected from SEQ ID NO: 9, 10, 13, 14 and their complements. In yet another aspect of this embodiment, the amplicon comprises SEQ ID NO: 11, 15 or their complements. Various detection methods can be used, including (but not limited to) TAQMAN, thermal amplification, ligase chain reaction, Southern blotting, ELISA, and colorimetric and fluorescence detection methods. In particular, the present invention provides a kit for detecting the presence of a target sequence (i.e., at least SEQ ID NO: 4, 5, or a conjugation sequence) in a sample containing genomic nucleic acid from ME240913. The kit comprises at least two polynucleotides capable of binding to or substantially adjacent to the target site and at least one means for detecting the binding of the polynucleotides to the target site. The detection means may be fluorescence, chemiluminescence, colorimetry, or isotopic assay, and may be coupled at least with an immunological method to detect binding. The kit may also utilize two or more polynucleotide sequences to detect the presence of a target site (i.e., at least SEQ ID NO: 4, 5, or a conjugation sequence of event ME240913) in a sample, said polynucleotide sequences being capable together of binding to a nucleotide sequence adjacent to or within about 100 base pairs of the target sequence, and being able to extend together to form an amplicon containing at least the target site.

[0131] In another embodiment, the present invention relates to a method for detecting Cry1Da protein in a biological sample, the method comprising: (a) extracting tissue protein from event ME240913; (b) analyzing the extracted protein using an immunological method comprising an antibody specific to Cry1Da protein generated by event ME240913; and (c) detecting the binding of said antibody to Cry1Da protein.

[0132] In yet another embodiment, the present invention relates to a plant product derived from corn plants, tissues, or seeds of event ME240913, wherein the plant product comprises a nucleotide sequence that is equivalent to or complementary to a sequence specific to event ME240913, and wherein the sequence can be detected in the plant product using nucleic acid amplification or hybridization methods. In one aspect of this embodiment, the nucleotide sequence is equivalent to or complementary to at least one of SEQ ID NO: 4, 5, and their complements. In another aspect of this embodiment, the plant product is selected from cornmeal, corn flour, corn syrup, corn oil, corn starch, and cereal foods wholly or partially manufactured containing corn-based products.

[0133] In another embodiment, the present invention relates to an extract of a plant product derived from the ME240913 corn plant, tissue, or seed, comprising a nucleotide sequence that is equivalent to or complementary to a sequence specific to ME240913. In one aspect of this embodiment, the sequence can be detected in the extract using nucleic acid amplification or hybridization methods. In another aspect of this embodiment, the sequence is equivalent to or complementary to at least one of SEQ ID NO: 4 and 5. Furthermore, in another aspect of this embodiment, the plant product is selected from cornmeal, corn flour, corn syrup, corn oil, corn starch, and cereal foods wholly or partially manufactured containing corn-based products.

[0134] Another embodiment of the invention relates to a maize plant or a portion thereof comprising the genotype ME240913, and seeds of the maize plant, wherein the genotype comprises at least one nucleotide sequence of SEQ ID NO: 4, 5, or their complement. One example of maize seeds comprises the nucleic acid molecule of the invention deposited on 10 / 28 / 2019 and assigned accession number PTA-126224. In one aspect of this embodiment, the maize plant is derived from the Hill maize strain. However, those skilled in the art will recognize that the ME240913 genotype can be introduced into any plant variety that can be bred with maize (including wild-type maize species), and therefore the list of strains bred in this manner should not be limited.

[0135] In another embodiment, the present invention relates to a maize plant comprising at least first and second DNA sequences (these linked to form a continuous nucleotide sequence), wherein the first DNA sequence is located within a conjugation sequence and comprises at least about 10 continuous nucleotides selected from nucleotides 1-116 and 6307-6424 of SEQ ID NO: 8 and their complements, wherein the second DNA sequence is located within an inserted heterologous DNA sequence and comprises at least about 10 continuous nucleotides selected from nucleotides 117-6306 of SEQ ID NO: 8 and their complements; and wherein the first and second DNA sequences can be used as probes or nucleotide primers to detect the presence of the maize event ME240913 nucleic acid sequence in a biological sample. In one aspect of this embodiment, nucleotide primers are used in a DNA amplification method to amplify a target DNA sequence from standard DNA extracted from a maize plant, and the maize plant can be identified from other maize plants by generating an amplicon corresponding to a DNA sequence comprising SEQ ID NO: 11, 15 and their complements.

[0136] In one embodiment, the present invention relates to a maize plant wherein the genotype ME240913 confers resistance to lepidopteran pests. In one aspect of this embodiment, the transgenic genotype conferring resistance to lepidopteran pests to the maize plant of the present invention comprises a truncated or modified cry1Da gene.

[0137] In another embodiment, maize plants express appropriate leaf concentrations of a truncated cry1Da protein, which is modified to provide a high level of protection to the plant leaves against damage by the fall armyworm. In another embodiment, high levels of plant leaf protection have been found in several fall armyworm populations in Brazil, including populations known to have a high frequency of cry1F-resistant fall armyworms. In another embodiment, the insertion of the cry1Da gene from the maize plant ME20913 produces adequate expression of a truncated and modified cry1Da protein in leaf tissue, thereby generating high toxicity against susceptible lepidopteran species, including the fall armyworm. In yet another embodiment, the maize plant ME20913 is highly toxic to the Cry1F-resistant fall armyworm.

[0138] In yet another embodiment, the present invention provides a method for producing maize plants resistant to lepidopteran pests, comprising the steps of: sexually hybridizing a first parent maize plant with a second parent maize plant, wherein the first or second parent maize plant contains DNA of event ME240913, to produce a plurality of first-generation progeny plants; and selecting the first-generation progeny plants containing event ME240913. Preferably, the method further comprises self-pollinating the first-generation progeny plants to produce a plurality of second-generation progeny plants; and selecting one plant from the second-generation progeny plants containing event ME240913. In one embodiment, the selection step may be based on an evaluation of resistance to lepidopteran pests, detecting event ME240913 DNA using the methods taught in the present invention, or treating with a herbicide and selecting herbicide-resistant plants promoted by the herbicide resistance gene PAT(bar) present in event ME240913. In a preferred embodiment, the method for producing transgenic maize plants containing the specific nucleic acids of the present invention includes sexually hybridizing a first parent maize plant containing event ME240913 with a second non-transgenic parent maize plant to produce offspring plants, selecting first-generation offspring plants resistant to lepidopteran pests, repeating the backcrossing cycle 4 times, and self-pollinating the parent plants containing event ME240913 to obtain homozygous plants.

[0139] In another embodiment, the present invention provides a method for producing hybrid maize seeds, comprising the steps of: planting seeds of a first innate maize line containing event ME240913 and seeds of a second innate line with a different genotype; sexually hybridizing the two different innate lines; and harvesting the resulting hybrid seeds. In a preferred embodiment, the method comprises at least one step of: cultivating the maize plants produced by the planting until flowering season, and emasculating the flowers of one of the innate maize lines. In one aspect of this embodiment, the first breeding maize line provides female offspring. In another aspect of this embodiment, the first breeding maize line provides male offspring. The present invention further relates to hybrid seeds produced by the method of the present invention and hybrid plants grown from such seeds.

[0140] The sole purpose of the following embodiments is to illustrate one or more preferred embodiments of the present invention and should not be construed as limiting the scope of the invention. Example

[0141] Example 1 - Gene Construct A gene construct containing a ubiquitin promoter, the nucleotide sequence of SEQ ID NO: 1 (encoding the truncated Cry1Da insecticidal protein of SEQ ID NO: 3) optimized for expression in maize, and the 3' region of the Agrobacterium tumefaciens carmine synthase gene, was synthesized in the pUC vector with HindIII and EcoRI restriction sites flanking it at the DNA Cloning Service (http: / / www.dna-cloning.com / ). Following the manufacturer's instructions (LifeTech), the construct was transferred from the pUC vector to the pTF101 vector using EcoRI and HindIII restriction enzymes and T4 ligase (Paz et al., 2004). Selection of the recombinant pTF101 UBI::cry1Da::NOS plasmid was performed by transformation of *E. coli* DH5α using spectinomycin, and the clone was confirmed by sequencing and digestion with HindIII and BamHI enzymes. For sequencing, the commercial kit BigDyeTerminator v3.1 (Applied Biosystems) was used. Plasmid DNA from two bacterial colonies containing gene constructs was sequenced and compared with the sequence of interest, and they were found to be identical.

[0142] Once the UBI::cry1Da::NOS gene was confirmed to be cloned into plasmid pTF101, this gene construct was used to transform Agrobacterium tumefaciens strain EHA101 using electroporation (BioRad / MicroPulser). The same procedure as the confirmation transformation was performed to demonstrate the presence of a binary vector containing the cry1Da gene in Agrobacterium tumefaciens. Plasmid DNA was isolated from Agrobacterium tumefaciens colonies and amplified using primers to detect the bar gene.

[0143] Agrobacterium tumefaciens EHA101 containing the gene constructs of interest (UBI::cry1Da::NOST and 35S::bar::35T) was used for genetic transformation of maize.

[0144] Example 2 - Genetic transformation of immature HiII maize embryos by Agrobacterium tumefaciens This transformation protocol, slightly modified from the scheme of Frame et al. (2002), uses the HiII maize genotype (Armstrong et al., 1991). In short, for this genotype, immature embryos between 1.8 and 2.0 mm in length (10-12 days after pollination) are collected. The ears used for embryo collection are immersed in a 1:1 solution of commercial bleach (2.5% sodium hypochlorite) and distilled H₂O containing 1-2 drops of Tween 20 for 20 minutes. They are then rinsed twice with sterile distilled water for 5 minutes each time.

[0145] Immature embryos were collected from shallow cuts in the grains using a scraper. For transferring the gene construct to maize, *Agrobacterium tumefaciens* EHA101 was used. From a stock culture of *Agrobacterium tumefaciens* containing the gene construct of interest, stored in glycerol at -80°C, the embryos were transferred to maize using the necessary antibiotics (100 mg / L). -1 Spectinomycin and 50 mg / L -1 Kanamycin in YEP medium (5 g / L) -1 Yeast extract; 10 g / L -1 Peptone; 5g / L -1 NaCl; 15 g / L -1 Prepare streaks in *Agrobacterium* (parental plates) and incubate at 28°C for 2-3 days. For genetic transformation, streak *Agrobacterium* colonies isolated from parental plates in YEP medium containing necessary antibiotics. Incubate the plates at 19°C for 2-5 days. Then, resuspend the *Agrobacterium* in infection medium (4.0 g / L) supplemented with 100 μM acetylsyleugenol. -1 N6 salt; 68.4 g / L -1 Sucrose; 36.0 g / L -1 Glucose; 0.7g / L -1 Proline; 1.5 mg / L -1 2,4-D; 1.0 mL / L -1 N6 Vitamin (1000X = 1.0g / L) -1 Thiamine HCl; 0.5 g / L -1 Pyridoxine HCl; 0.5 g / L -1 In a solution of nicotinic acid (pH 5.2), until an OD550 of 0.3-0.4 is reached, and the solution is incubated for 2 hours at 150 rpm and 23°C in a shaker.

[0146] For infection of immature maize embryos, collect 50-100 embryos in 1 mL of infection medium supplemented with acetylsuccinyl syringone. After collection, rinse the embryos twice, add 1 mL of bacterial culture, and incubate the suspension at 23°C for 5 minutes. After infection, transfer the embryos to co-culture medium (4.0 g / L). -1 N6 salt; 1.5 mg / L -1 2,4-D; 30.0 g / L -1 Sucrose; 0.7g / L -1 Proline; 1.0 mL / L -1 N6 Vitamin (1000X); 0.85 mg / L -1 AgNO3; 100 μM acetylsuccinone; 300 mg / L -1 L-cysteine; 3.0 g / L -1Plant gel (pH 5.8) with the scutellaria side facing up. Incubate the plates in the dark at 20°C for 3–5 days. After co-culture, transfer the embryos to a dormant medium (4.0 g / L) at 28°C (dark). -1 N6 salt; 1.5 mg / L -1 2,4-D; 30.0 g / L -1 Sucrose; 0.5g / L -1 MES; 0.7gL -1 Proline; 1.0 mL / L -1 Vitamin N6 (1000X); 0.85 mg / L - 1 AgNO3; 100 mg / L -1 Tioxin; 3.0gL -1 Plant gel (pH 5.8) was applied for 7-15 days. Then, the embryos were transferred to a selective medium (4.0 g / L). -1 N6 salt; 1.5 mg / L -1 2,4-D; 30.0 g / L -1 Sucrose; 0.5g / L -1 MES; 0.7gL -1 Proline; 1.0 mL / L -1 Vitamin N6 (1000X); 0.85 mg / L -1 AgNO3; 100 mg / L -1 Tioxin; 1.5 and 3.0 mg / L Ialaphos; 3.0 g / L -1 Plant gel (pH 5.8) (25 embryos / plate). These embryos were passaged in a selective medium every 15 days to select for vigorous callus growth.

[0147] Selected callus tissue was transferred to regeneration medium (4.62 g / L). -1 MS salt; 60.0 g / L -1 Sucrose; 100 mg / L -1 Inositol; 1.0 mL / L -1 MS Vitamin (1000X); 1.5 mg / L Bialaphos; 4.0 g / L -1 Plant gel (pH 5.8) was incubated at 26±2℃ (in the dark) for 15–21 days. Callus tissue with a dry appearance and opaque white color, ready for germination, was transferred to germination medium (4.62 g / L). -1 MS salt; 30.0 g / L -1 Sucrose; 100 mg / L -1 Inositol; 1.0 mL / L -1MS Vitamin (1000X = 0.5g / L) -1 Thiamine HCl; 0.5 g / L -1 Pyridoxine HCl; 0.05 g / L -1 Nicotinic acid); 3.0 g / L -1 Plant gel; pH 5.8 (12 callus / plate), 25℃, 80-100 μE / m 2 (Light intensity per second, 16-hour light cycle).

[0148] Seedlings with well-developed root and leaf structures, measuring approximately 5 cm in length (14-20 days), were transplanted into pots in a greenhouse containing a commercially available mixture of soil and organic matter (2 / 3 soil and 1 / 3 organic matter (TDP 30 / 15)) for an intermediate adaptation period.

[0149] After obtaining the Hill genotype carrying event ME240913, molecular marker-assisted selection was used to introgress the event from the HiII genotype into the tropical L3 strain.

[0150] Example 3 - Bioassay To evaluate the susceptibility of transgenic maize expressing truncated Cry1Da protein to fall armyworm, bioassays were performed in the laboratory.

[0151] Bioassays were performed as follows: newly hatched fall armyworm caterpillars were used to infect the leaves of transgenic cry1Da maize plants and non-transgenic isolines (5 caterpillars per plant). The maize used were at developmental stages V7 and V8, and the experiments were conducted in plastic containers and incubated in a suitable growth chamber (28°C and 60% humidity, 12 hours light). Damage scores were assessed after 5 days. In each case, the experimental design consisted of an "experimental group" (transgenic maize containing the truncated cry1Da construct in event ME240913) and a "control group" (non-transgenic maize).

[0152] The parameters assessed were: damage scores using the scale proposed by Carvalho in 1970 (0: no damage to plant leaves; 1: thinning of plant leaves; 2: perforation of plant leaves; 3: tearing of plant leaves; 4: plant showing column damage; and 5: plant showing column destruction); caterpillar survival rate (counting the number of surviving caterpillars in each pot); and caterpillar biomass (using a precision level of four decimal places).

[0153] Example 4 - Bioassay for controlling fall armyworm using transgenic maize event ME240913 Fall armyworm assay: First, the control effect of event ME240913 on this pest was tested. Seeds from the event were germinated in a greenhouse, and when the plants reached the stage between V10 and V12 leaf stages, two of the youngest leaves from each plant were used for fall armyworm bioassay. Three replicates were performed, with five caterpillars per replicate. HiII and L3 maize leaves were used as negative controls (caterpillars grew normally), and Corn leaves were used as a positive control (caterpillars could not grow). In this first test, event ME240913 was found to have a good ability to control caterpillar development, achieving a 100% mortality rate (Table 1).

[0154] Table 1 - Assessment of Event ME240913 in Fall Armyworm Control Bioassays using this event were repeated four times, with 20 caterpillars per replicate, and the results confirmed that the event had the ability to control the development of fall armyworm (Table 2). Figure 3 Bioassays of fall armyworm feeding in non-GMO maize and GMO maize of this invention were performed. No treatments using the Viptera maize genotype were used in this experiment.

[0155] Table 2 - Comparative assessment of the impact of transgenic maize events on fall armyworm control (bioassay 2) ME240913(cry1Da) / 1 0 20 0 ME240913(cry1Da) / 2 0 20 0 ME240913(cry1Da) / 3 0 20 0 ME240913(cry1Da) / 4 0 20 0 HiII / 1 18 02 176.7 HiII / 2 18 02 131.7 HiII / 3 15 05 137.0 HiII / 4 17 03 212.7 L3 / 1 19 01 131.9 L3 / 2 19 01 132.5 L3 / 3 19 01 133.3 L3 / 4 18 02 115.6 Example 5 - Exposure to fresh leaf tissue from two genetic backgrounds containing event ME240913 In this experiment, heterozygote 1 (leaves V8 and V9) of the Helix L85 strain X Embrapa L3 ME240913 strain was used. Heterozygote 2 was the HiII ME240913 X Embrapa L3 strain (leaves V5 and V6) and the control was the Helix L85 X Embrapa L3 strain heterozygote (leaves V8 and V9). Both heterozygotes were heterozygous for event ME240913.

[0156] Use a metal cutter to cut discs of fresh leaves with a diameter of 1.8 cm and place them in 2.0% agar (1 mL / well) in a 128-well plastic bioassay tray (Bio-Ba-128, CD International, Pitman, NJ, USA).

[0157] The susceptibility standard laboratory population of fall armyworm (Spodoptera frugiperda) from Embrapa Milho e Sorgo, the same population used by Omoto et al. 2016 (Omoto, C., Bernardi, O., Salmeron, E., Sorgatto, RJ, Dourado, PM, Crivellari, A., Carvalho, RA, Willse, A., Martinelli, S., & Head, GP (2016). Field-evolved resistance to Cry1Ab maize by Spodoptera frugiperda in Brazil. Pest Management Science, 72(9), 1727-1736.), was used as the susceptibility standard in the test, maintained on an artificial diet without any insecticide or Bt selection pressure.

[0158] Newly hatched larvae (0-24 hours) were placed into each well containing a leaf disc using a fine brush. Plates were sealed with Bio-CV-16 adhesive (CD International, Pitman, NJ, USA) and placed in an acclimatization chamber (temperature 26±1℃; relative humidity 60±10%; 14:10h light: dark cycle). 120 larvae were used per treatment.

[0159] Mortality was assessed 24 hours after exposure, and then daily until 100% mortality was achieved. Dead caterpillars were considered those that did not respond to brush touch.

[0160] Fresh maize leaves expressing leaf tissue from event ME240913 caused mortality in fall armyworms starting the day after feeding, and reaching 100% mortality after 3 days. The same rapid and complete mortality pattern was observed in both heterozygotes tested. Results are as follows: Figure 4 As shown.

[0161] Example 6 - Lyophilized leaf tissue exposed to an artificial diet diluted 1:25 in the form of event ME240913 This experiment evaluated heterozygotes (leaf V8 and V9) bred between the Helix L85 X Embrapa L3 line containing event ME240913 and the control Helix L85 X Embrapa L3 line. Results were compared with those obtained using leaves from the control heterozygote Helix L85 X Embrapa L3, also harvested at V8 and V9 stages.

[0162] Approximately seven plants from the two maize hybrids were harvested after 27 days of growth. Leaves from the V8 and V9 developmental stages were placed in plastic bags frozen in liquid nitrogen and transferred to an ultra-low temperature freezer at -80°C. The leaf tissue was then freeze-dried. After freeze-drying, the material was ground using a tissue homogenizer (IKA A11 Basic). The freeze-dried and ground leaf samples were stored at room temperature in plastic cups with sealed lids.

[0163] Lyophilized transgenic maize leaves were prepared at a ratio of 1:25 in a 4% (w / w) artificial diet for fall armyworm. A negative control contained 4% lyophilized non-transgenic tissue. Approximately 1 L of the fall armyworm artificial diet was prepared according to a modified protocol containing only 56% of the total agar content of the conventional protocol. The diet was cooled as needed and maintained at 55°C in a water bath. For each treatment, 160 g of the fall armyworm diet was added to a plastic cup containing pre-weighed lyophilized tissue. The flaky tissue was mixed with a spatula until visually homogeneous. The mixture was transferred to a perforated plastic bag and added to each well of a bioassay tray (128-well CD-International tray) by pressing the diet through the entire bag (as a piping bag). For each transgenic and non-transgenic material, approximately 0.8 ml of the diet / leaf powder mixture was dispensed into each of 128 individual wells (256 in total).

[0164] As described in the tests above, a standard susceptible population of fall armyworm was used.

[0165] Newly hatched larvae (0-24 hours) were placed in each well using a fine brush. The plates were sealed with Bio-CV-16 adhesive (CD International, Pitman, NJ, USA) and placed in an adaptation chamber (temperature 26±1℃; relative humidity 60±10%; 14:10h light:dark cycle).

[0166] Mortality was recorded on days 3, 6–10, and 13–14 after exposure. Larvae that were noticeably inactive and did not move when touched with a fine brush were considered dead.

[0167] In this experiment, a mortality rate of 67% was observed on day 7 and 97% (125 / 128 larvae) on day 14. The remaining three larvae showed significant growth inhibition compared to the larvae that fed on the control leaf tissue.

[0168] Figure 5 The survival rate (%) of newly hatched fall armyworm caterpillars is described, as evaluated up to 14 days after exposure to freeze-dried leaves at a ratio of 1:25 in an artificial diet obtained from leaves obtained from event ME240913 and controls.

[0169] Example 7 - Protection against leaf damage in maize plants affected by event ME240913 was achieved in a field infested by six different fall armyworm populations from various sources. Control plants were obtained from heterozygotes between the Helix L85 x Embrapa L3 lines. Plants containing the event were obtained from heterozygotes between the Helix L85 x Embrapa L3 ME240913 lines.

[0170] As described, naturally occurring fall armyworm populations at the larval stage were collected from six different maize production sites in Brazil: two populations were collected in Paraná (Palotina and Ivatuba), two in Mato Grosso (Rondonópolis and Campo Verde), and two in Minas Gerais (Paracatu and Sete Lagoas). The larvae were fed artificial diets under laboratory conditions until adulthood (a cycle of approximately ~30 days) without any selective pressure from pesticides or Bt. The newly hatched larvae were then allowed to infect plants at the V4 growth stage under field conditions.

[0171] The treatment consisted of combinations of two heterozygotes and six insect populations, with three replicates. Five-meter-long plots, each planted in five rows, were used to assess infection in three central rows. The remaining two rows served as buffer zones (boundaries).

[0172] According to Davis et al. (1992), damage caused by caterpillars feeding on maize plants was evaluated. In summary, a scale from 0 to 9 was used, where 0 represents no damage to the plant and 9 represents the plant with extensive leaf damage. Therefore, an increase in the scale unit represents a higher level of leaf damage.

[0173] Field scores were recorded at 7, 14, and 21 days post-exposure.

[0174] The mean visual damage of leaves treated with the maize heterozygote L85XL3 resulted in infection scores ranging from 3.4 to 5.19 for six different fall armyworm populations in the control treatment, while the infection score was close to zero in the Cry1Da-expressing heterozygotes. In other words, the mean plant score evaluated in heterozygotes containing Cry1Da events was much lower (<0.1).

[0175] Figure 7This study describes the damage scores (from 0 to 9) induced by different fall armyworm populations (± confidence interval, probability 5%) feeding on conventional heterozygotes (conv) and maize heterozygotes ME240913 in the field (Davis et al., 1992 scale). Evaluation was conducted 14 days after infection.

[0176] Figures 8A-8F The image shows a single control (conventional) heterozygote (left side of the photo) obtained from breeding between the Helix-L85 x Embrapa-L3 lines, and its corresponding syngeneic version of Helix-L85 x Embrapa-L3 containing event ME240913 (right side of the photo). Each photo shows a conventional version and a version containing event ME240913 from each of six different fall armyworm populations collected at the following locations in Brazil: populations collected in Palotina-PR (8A), Rondonópolis-MT (8B), Rondonópolis+Campo Verde-MT (8C), Paracatu-MG (8D), Sete Lagoas-MG (8E), and Ivatuba-PR (8F). No leaf damage was observed in the control on the left side of each photo, while the heterozygote containing event ME240913 (the heterozygote on the right side of each photo) showed no damage.

[0177] Example 8 - Bioassay using a population of caterpillars resistant to transgenic maize containing the Cry1F gene Measurements were conducted to verify the potential of event ME240913 in controlling fall armyworm populations resistant to the Cry1F protein.

[0178] Experiments were conducted in a greenhouse using transgenic maize containing event ME240913 and non-transgenic maize (negative control). Newly hatched larvae belonging to two different fall armyworm populations (one resistant to the Cry1F gene and the other susceptible to the same gene) were inoculated onto maize plants at stages V7 and V8 (15 caterpillars per plant). After infection, the pots were isolated using vacuum cages, and damage was assessed at 7, 14, and 21 days. The experimental design consisted of four treatments, each with five pots containing 2–3 maize plants: Treatment 1: According to Leite et al., 2016, a population of fall armyworm resistant to the Cry1F protein was used to infect transgenic event ME240913.

[0179] Treatment 2: According to Leite et al. (2016), Cry1F-resistant fall armyworm populations were used to infect non-transgenic syngeneic L3 lines.

[0180] Treatment 3: Transgenic event ME240913, in which susceptible caterpillar populations were bred and maintained in the Embrapa Milho e Sorgo Entomology Laboratory.

[0181] Treatment 4: Infecting non-GMO homologous strains with susceptible caterpillar populations raised and maintained in the Embrapa Milho e Sorgo Entomology Laboratory.

[0182] The results showed that transgenic plants containing event ME240913 were able to inhibit their development ( Figure 9 This study effectively controlled the infestation of Cry1F protein-resistant fall armyworm populations and protected plants from this pest, as well as susceptible populations, as observed by damage scores (±CI, P = 0.05). As assessed 21 days after release of the caterpillars under different treatments, the percentage of fall armyworm survival was 0% for treatments 1 and 3, and approximately 65% ​​and 35% for treatments 2 and 4, respectively. As assessed 21 days after placing the caterpillars under different treatments, the fall armyworm biomass was 0% for treatments 1 and 3, and approximately 260 mg and 300 mg for treatments 2 and 4, respectively. The mean non-overlapping CIs differed from each other in both cases (P = 0.05).

[0183] Example 8 describes the use of a codon-optimized Cry1Da sequence to produce maize plants expressing a truncated Cry1Da sequence, which exhibits high toxicity (100% mortality) to both wild-type and Cry1F-resistant fall armyworm populations. The fact that 100% mortality was identified in Cry1F-resistant fall armyworm populations when these populations were fed fresh leaves from event ME240913 confirms that the truncated and codon-modified protein expressed from the Cry1Da gene functions through a mechanism different from that present in commercial events containing the Cry1F gene.

[0184] Preservation of biological materials The maize seeds from the aforementioned event ME240913 were deposited under the Budapest Treaty with accession number PTA-126224 at the American Center for Type Culture Collection (ATCC), 1801 University Boulevard, Manassas, VA 20110, on October 28, 2019. <110> Herlis Seedling Co., Ltd. Embrapa, a Brazilian agricultural research company <120> Nucleic acid molecules, cells, plants, and transgenic seeds expressing the Cry1Da protein in the transgenic maize ME240913 event, their uses, plant products, methods, kits, and amplicon for detecting the event, and methods for producing transgenic plants and controlling lepidopteran pests. <130> Event ME240913 <140> PCT / BR2020 / 050407 <141> 2020-10-13 <150> BR102019023319-2 <151> 2019-11-06 <160> 16 <170> PatentIn version 3.5 <210> 1 <211> 1878 <212> DNA <213> Artificial sequence <220> <223> The nucleotide sequence encoding a truncated Cry1Da protein in maize, optimized for expression, was found in event ME240913. <400> 1 atggagatca acaaccagaa ccagtgcgtg ccgtacaact gcctgtccaa cccgaaggag 60 atcatcctgg gcgaggagag gctggagacc ggcaacaccg tggccgacat ctccctgggc 120 ctgatcaact tcctgtactc caacttcgtg ccgggcggcg gcttcatcgt gggcctgctg 180 gagctgatct ggggcttcat cggcccgtcc cagtgggaca tcttcctggc ccagatcgag 240 cagctgatct cccagaggat cgaggagttc gccaggaacc aggccatctc caggctggag 300 ggcctgtcca acctgtacaa ggtgtacgtg agggccttct ccgactggga gaaggacccg 360 accaacccgg ccctgaggga ggagatgagg atacagttca acgacatgaa ctccgccctg 420 atcaccgcca tcccgctgtt cagggtgcag aactacgagg tggccctgct gtccgtgtac 480 gtgcaggccg ccaacctgca cctgtccatc ctgagggacg tgtccgtgtt cggcgagagg 540 tggggctacg acaccgccac catcaacaac aggtactccg acctgacctc cctgatccac 600 gtgtacacca accactgcgt ggacacctac aaccagggcc tgaggaggct ggagggcagg 660 ttcctgtccg actggatcgt gtacaacagg ttcaggaggc agctgaccat ctccgtgctg 720 gacatcgtgg ccttcttccc gaactacgac atcaggacct acccgatcca gaccgccacc 780 cagctgacca gggaggtgta cctggacctg ccgttcatca acgagaacct gtccccggcc 840 gcctcctacc cgaccttctc cgccgccgag tccgccatca tcaggtcccc gcacctggtg 900 gacttcctga actccttcac catctacacc gactccctgg ccaggtacgc ctactggggc 960 ggccacctgg tgaactcctt caggaccggc accaccacca acctgatcag gtccccgctg 1020 tacggcaggg agggcaacac cgagaggccg gtgaccatca ccgcctcccc gtccgtgccg 1080 atcttcagga ccctgtccta catcaccggc ctggacaact ccaacccggt ggccggcatc 1140 gagggcgtgg agttccagaa caccatctcc aggtccatct acaggaagtc cggcccgatc 1200 gactccttct ccgagctgcc gccgcaggac gcctccgtgt ccccggccat cggctactcc 1260 cacaggctgt gccacgccac cttcctggag aggatctccg gcccgaggat cgccggcacc 1320 gtgttctcct ggacccacag gtccgcctcc ccgaccaacg aggtgtcccc gtccaggatc 1380 acccagatcc cgtgggtgaa ggcccacacc ctggcctccg gcgcctccgt gatcaagggc 1440 ccgggcttca ccggcggcga catcctgacc aggaactcca tgggcgagct gggcaccctg 1500 agggtgacct tcaccggcag gctgccgcag tcctactaca tcaggttcag gtacgctcc 1560 gtggccaaca ggtccggcac cttcaggtac tcccagccgc cgtcctacgg catctccttc 1620 ccgaagacca tggacgccgg cgagccgctg acctccaggt ccttcgccca caccaccctg 1680 ttcaccccga tcacctttc cagggcccag gaggagttcg acctgtacat ccagtccggc 1740 gtgtacatcg acaggatcga gttcatcccg gtgaccgcca ccttcgaggc cgagtacgac 1800 ctggagaggg cccagaaggt ggtgaacgcc ctgttcacct ccaccaacca gctgggcctg 1860 aagaccgacg tgacctaa 1878 <210> 2 <211> 5647 <212> DNA <213> Artificial Sequence <220> <223> Complete construct of event ME240913 transgenic <400> 2 tattgcactc ccttttaact gttttttatt acaaaaatgc cctggaaaat gcactccctt 60 tttgtgtttg tttttttgtg aaacgatgtt gtcaggtaat ttatttgtca gtctactatg 120 gtggcccatt atattaatag caactgtcgg tccaatagac gacgtcgatt ttctgcattt 180 gtttaaccac gtggatttta tgacatttta tattagttaa tttgtaaaac ctacccaatt 240 aaagacctca tatgttctaa agactaatac ttaatgataa caattttctt ttagtgaaga 300 aagggataat tagtaaatat ggaacaaggg cagaagattt attaaagccg cgtaagagac 360 aacaagtagg tacgtggagt gtcttaggtg acttacccac ataacataaa gtgacattaa 420 caaacatagc taatgctcct atttgaatag tgcatatcag cataccttat tacatataga 480 taggagcaaa ctctagctag attgttgagc agatctcggt gacgggcagg accggacggg 540 gcggtaccgg caggctgaag tccagctgcc agaaacccac gtcatgccag ttcccgtgct 600 tgaagccggc cgcccgcagc atgccgcggg gggcatatcc gagcgcctcg tgcatgcgca 660 cgctcgggtc gttgggcagc ccgatgacag cgaccacgct cttgaagccc tgtgcctcca 720 gggacttcag caggtgggtg tagagcgtgg agcccagtcc cgtccgctgg tggcgggggg 780 agacgtacac ggtcgactcg gccgtccagt cgtaggcgtt gcgtgccttc caggggcccg 840 cgtaggcgat gccggcgacc tcgccgtcca cctcggcgac gagccaggga tagcgctccc 900 gcagacggac gaggtcgtcc gtccactcct gcggttcctg cggctcggta cggaagttga 960 ccgtgcttgt ctcgatgtag tggttgacga tggtgcagac cgccggcatg tccgctcgg 1020 tggcacggcg gatgtcggcc gggcgtcgtt ctgggctcat ggtagatccc ccgttcgtaa 1080 atggtgaaaa ttttcagaaa attgcttttg ctttaaaaga aatgatttaa attgctgcaa 1140 tagaagtaga atgcttgatt gcttgagatt cgtttgtttt gtatatgttg tgttgagaat 1200 taattctcga ggtcctctcc aaatgaaatg aacttcctta tatagaggaa gggtcttgcg 1260 aaggatagtg ggattgtgcg tcatccctta cgtcagtgga gatatcacat caatccactt 1320 gctttgaaga cgtggttgga acgtcttcttt tttccacgat gctcctcgtg ggtgggggtc 1380 catctttggg accactgtcg gcagaggcat cttcaacgat ggcctttcct ttatcgcaat 1440 gatggcattt gtaggagcca ccttcctttt ccactatctt cacaataaag tgacagatag 1500 ctgggcaatg gaatccgagg aggtttccgg atattaccct ttgttgaaaa gtctcaattg 1560 cccttggtc ttctgagact gtatctttga tatttttgga gtagacaagt gtgtcgtgct 1620 ccaccatgtt atcacatcaa tccacttgct ttgaagacgt ggttggaacg tcttcttttt 1680 ccacgatgct cctcgtgggt gggggtccat ctttgggacc actgtcggca gaggcatctt 1740 caacgatggc ctttccttta tcgcaatgat ggcatttgta ggagccacct tccttttcca 1800 ctatcttcac aataaagtga cagatagctg ggcaatggaa tccgaggagg tttccggata 1860 ttaccctttg ttgaaaagtc tcaattgccc tttggtcttc tgagactgta tctttgatat 1920 ttttggagta gatagtgtg tcgtgctcca ccatgttgac ctgcaggcat gcaagcttgt 1980 taacctgcag tgcagcgtga cccggtcgtg cccctctta gagataatga gcattgcatg 2040 tctaagttat aaaaaattac cacatatttt tttgtcaca cttgtttgaa gtgcagttta 2100 tctatcttta tacatatatt taacttac tcacgaata ataatcta tagtactaca 2160 atatatcag tgttttagag atcatataa atgacagtt agacatggtc taaggacaa 2220 ttgagtattt tgacaacagg actctacagt ttatcttt tagtgtgcat gtgttctcct 2280 ttttttgc aaatagcttc acctatata tacttcatcc attttag tacatccatt 2340 taggtttag ggttaatggt ttttagac taatttttt agtacatcta ttttattcta 2400 ttttagccctc taaattaaga aaactaaaac tctattttag ttttttatt taataattta 2460 gatataaat agaataat aagtgacta aaatttaac aaataccctt taagaatta 2520 aaaaaactaa ggaaacattt ttctgtttc gagtagataa tgccagcctg ttaaacgccg 2580 tcgatcgacg agtctaacgg acaccaacca gcgaaccagc agcgtcgcgt cgggccaagc 2640 gaagcagacg gcacggcatc tctgtcgctg cctctggacc cctctcgaga gttccgctcc 2700 accgttggac ttgctccgct gtcggcatcc agaaattgcg tggcggagcg gcagacgtga 2760 gccggcacgg caggcggcct cctcctcctc tcacggcacc ggcagctacg ggggattcct 2820 ttcccaccgc tccttcgctt tcccttcctc gccccgccgta ataaatagac accccctcca 2880 caccctcttt ccccaacctc gtgttgttcg gagcgcacac acacacaacc agatctcccc 2940 caaatccacc cgtcggcacc tccgcttcaa ggtacgccgc tcgtcctccc cccccccccc 3000 tctctacctt ctctagatcg gcgttccggt ccatggttag ggcccggtag ttctacttct 3060 gttcatgttt gtgttagatc cgtgtttgtg ttagatccgt gctgctagcg ttcgtacacg 3120 gatgcgacct gtacgtcaga cacgttctga ttgctaactt gccagtgttt ctctttgggg 3180 aatcctggga tggctctagc cgttccgcag acgggatcga tctaggatag gtatacatgt 3240 tgatgtgggt tttactgatg catatacatg atggcatatg cagcatctat tcatatgctc 3300 taaccttgag tacctatcta ttataataaa caagtatgtt ttataattat tttgatcttg 3360 atatacttgg atgatggcat atgcagcagc tatatgtgga tttttttagc cctgccttca 3420 tacgctattt atttgcttgg tactgtttct tttgtcgatg ctcaccctgt tgtttggtgt 3480 tacttctgca ggggatccga gctcatggag atcaacaacc agaaccagtg cgtgccgtac 3540 aactgcctgt ccaacccgaa ggagatcatc ctgggcgagg agaggctgga gaccggcaac 3600 accgtggccg acatctccct gggcctgatc aacttcctgt actccaactt cgtgccgggc 3660 ggcggcttca tcgtgggcct gctggagctg atctggggct tcatcggccc gtcccagtgg 3720 gacatcttcc tggcccagat cgagcagctg atctcccaga ggatcgagga gttcgccagg 3780 aaccaggcca tctccaggct ggagggcctg tccaacctgt acaaggtgta cgtgagggcc 3840 ttctccgact gggagaagga cccgaccaac ccggccctga gggaggagat gaggatacag 3900 ttcaacgaca tgaactccgc cctgatcacc gccatcccgc tgttcagggt gcagaactac 3960 gaggtggccc tgctgtccgt gtacgtgcag gccgccaacc tgcacctgtc catcctgagg 4020 gacgtgtccg tgttcggcga gaggtggggc tacgacaccg ccaccatcaa caacaggtac 4080 tccgacctga cctccctgat ccacgtgtac accaaccact gcgtggacac ctacaaccag 4140 ggcctgagga ggctggaggg caggttcctg tccgactgga tcgtgtacaa caggttcagg 4200 aggcagctga ccatctccgt gctggacatc gtggccttct tcccgaacta cgacatcagg 4260 acctacccga tccagaccgc cacccagctg accagggagg tgtacctgga cctgccgttc 4320 atcaacgaga acctgtcccc ggccgcctcc tacccgacct tctccgccgc cgagtccgcc 4380 atcatcaggt ccccgcacct ggtggacttc ctgaactcct tcaccatcta caccgactcc 4440 ctggccaggt acgcctactg gggcggccac ctggtgaact ccttcaggac cggcaccacc 4500 accaacctga tcaggtcccc gctgtacggc agggagggca acaccgagag gccggtgacc 4560 atcaccgcct ccccgtccgt gccgatcttc aggaccctgt cctacatcac cggcctggac 4620 aactccaacc cggtggccgg catcgagggc gtggagttcc agaacaccat ctccaggtcc 4680 atctacagga agtccggccc gatcgactcc ttctccgagc tgccgccgca ggacgcctcc 4740 gtgtccccgg ccatcggcta ctcccacagg ctgtgccacg ccaccttcct ggagaggatc 4800 tccggcccga ggatcgccgg caccgtgttc tcctggaccc acaggtccgc ctccccgacc 4860 aacgaggtgt ccccgtccag gatcacccag atcccgtggg tgaaggccca caccctggcc 4920 tccggcgcct ccgtgatcaa gggcccgggc ttcaccggcg gcgacatcct gaccaggaac 4980 tccatgggcg agctgggcac cctgagggtg accttcaccg gcaggctgcc gcagtcctac 5040 tacatcaggt tcaggtacgc ctccgtggcc aacaggtccg gcaccttcag gtactcccag 5100 ccgccgtcct acggcatctc cttcccgaag accatggacg ccggcgagcc gctgacctcc 5160 aggtccttcg cccacaccac cctgttcacc ccgatcacct tctccagggc ccaggaggag 5220 ttcgacctgt acatccagtc cggcgtgtac atcgacagga tcgagttcat cccggtgacc 5280 gccaccttcg aggccgagta cgacctggag agggcccaga aggtggtgaa cgccctgttc 5340 acctccacca accagctggg cctgaagacc gacgtgacct aagtcgacgg atccgatcgt 5400 tcaaacattt ggcaataaag tttcttaaga ttgaatcctg ttgccggtct tgcgatgatt 5460 atcatataat ttctgttgaa ttacgttaag catgtaataa ttaacatgta atgcatgacg 5520 ttatttatga gatgggtttt tatgattaga gtcccgcaat tatacattta atacgcgata 5580 gaaaacaaaa tatagcgcgc aaactaggat aaattatcgc gcgcggtgtc atctatgtta 5640 ctagatc 5647 <210> 3 <211> 625 <212> PRT <213> Artificial sequence <220> <223> The truncated Cry1Da protein expressed by event ME240913 <400> 3 Met Glu Ile Asn Asn Gln Asn Gln Cys Val Pro Tyr Asn Cys Leu Ser 1 5 10 15 Asn Pro Lys Glu Ile Ile Leu Gly Glu Glu Arg Leu Glu Thr Gly Asn 20 25 30 Thr Val Ala Asp Ile Ser Leu Gly Leu Ile Asn Phe Leu Tyr Ser Asn 35 40 45 Phe Val Pro Gly Gly Gly Phe Ile Val Gly Leu Leu Glu Leu Ile Trp 50 55 60 Gly Phe Ile Gly Pro Ser Gln Trp Asp Ile Phe Leu Ala Gln Ile Glu 65 70 75 80 Gln Leu Ile Ser Gln Arg Ile Glu Glu Phe Ala Arg Asn Gln Ala Ile 85 90 95 Ser Arg Leu Glu Gly Leu Ser Asn Leu Tyr Lys Val Tyr Val Arg Ala 100 105 110 Phe Ser Asp Trp Glu Lys Asp Pro Thr Asn Pro Ala Leu Arg Glu Glu 115 120 125 Met Arg Ile Gln Phe Asn Asp Met Asn Ser Ala Leu Ile Thr Ala Ile 130 135 140 Pro Leu Phe Arg Val Gln Asn Tyr Glu Val Ala Leu Leu Ser Val Tyr 145 150 155 160 Val Gln Ala Ala Asn Leu His Leu Ser Ile Leu Arg Asp Val Ser Val 165 170 175 Phe Gly Glu Arg Trp Gly Tyr Asp Thr Ala Thr Ile Asn Asn Arg Tyr 180 185 190 Ser Asp Leu Thr Ser Leu Ile His Val Tyr Thr Asn His Cys Val Asp 195 200 205 Thr Tyr Asn Gln Gly Leu Arg Arg Leu Glu Gly Arg Phe Leu Ser Asp 210 215 220 Trp Ile Val Tyr Asn Arg Phe Arg Arg Gln Leu Thr Ile Ser Val Leu 225 230 235 240 Asp Ile Val Ala Phe Phe Pro Asn Tyr Asp Ile Arg Thr Tyr Pro Ile 245 250 255 Gln Thr Ala Thr Gln Leu Thr Arg Glu Val Tyr Leu Asp Leu Pro Phe 260 265 270 Ile Asn Glu Asn Leu Ser Pro Ala Ala Ser Tyr Pro Thr Phe Ser Ala 275 280 285 Ala Glu Ser Ala Ile Ile Arg Ser Pro His Leu Val Asp Phe Leu Asn 290 295 300 Ser Phe Thr Ile Tyr Thr Asp Ser Leu Ala Arg Tyr Ala Tyr Trp Gly 305 310 315 320 Gly His Leu Val Asn Ser Phe Arg Thr Gly Thr Thr Thr Asn Leu Ile 325 330 335 Arg Ser Pro Leu Tyr Gly Arg Glu Gly Asn Thr Glu Arg Pro Val Thr 340 345 350 Ile Thr Ala Ser Pro Ser Val Pro Ile Phe Arg Thr Leu Ser Tyr Ile 355 360 365 Thr Gly Leu Asp Asn Ser Asn Pro Val Ala Gly Ile Glu Gly Val Glu 370 375 380 Phe Gln Asn Thr Ile Ser Arg Ser Ile Tyr Arg Lys Ser Gly Pro Ile 385 390 395 400 Asp Ser Phe Ser Glu Leu Pro Pro Gln Asp Ala Ser Val Ser Pro Ala 405 410 415 Ile Gly Tyr Ser His Arg Leu Cys His Ala Thr Phe Leu Glu Arg Ile 420 425 430 Ser Gly Pro Arg Ile Ala Gly Thr Val Phe Ser Trp Thr His Arg Ser 435 440 445 Ala Ser Pro Thr Asn Glu Val Ser Pro Ser Arg Ile Thr Gln Ile Pro 450 455 460 Trp Val Lys Ala His Thr Leu Ala Ser Gly Ala Ser Val Ile Lys Gly 465 470 475 480 Pro Gly Phe Thr Gly Gly Asp Ile Leu Thr Arg Asn Ser Met Gly Glu 485 490 495 Leu Gly Thr Leu Arg Val Thr Phe Thr Gly Arg Leu Pro Gln Ser Tyr 500 505 510 Tyr Ile Arg Phe Arg Tyr Ala Ser Val Ala Asn Arg Ser Gly Thr Phe 515 520 525 Arg Tyr Ser Gln Pro Pro Ser Tyr Gly Ile Ser Phe Pro Lys Thr Met 530 535 540 Asp Ala Gly Glu Pro Leu Thr Ser Arg Ser Phe Ala His Thr Thr Leu 545 550 555 560 Phe Thr Pro Ile Thr Phe Ser Arg Ala Gln Glu Glu Phe Asp Leu Tyr 565 570 575 Ile Gln Ser Gly Val Tyr Ile Asp Arg Ile Glu Phe Ile Pro Val Thr 580 585 590 Ala Thr Phe Glu Ala Glu Tyr Asp Leu Glu Arg Ala Gln Lys Val Val 595 600 605 Asn Ala Leu Phe Thr Ser Thr Asn Gln Leu Gly Leu Lys Thr Asp Val 610 615 620 Thr 625 <210> 4 <211> twenty four <212> DNA <213> Artificial sequence <220> <223> 5' conjugate sequence <400> 4 aaaaatgccg cgacttgcgg acgt 24 <210> 5 <211> 34 <212> DNA <213> Artificial sequence <220> <223> 3' conjugation sequence <400> 5 cccgccttca gtttaaactt tggcatttgt ggaa 34 <210> 6 <211> 65 <212> DNA <213> Artificial sequence <220> <223> 5' Flanking Sequence <400> 6 gaggtcgacg gttcgaatac taacaaccgc gtagcgcgcg aattttgcgc gaaaaatgcc 60 gcgac 65 <210> 7 <211> 67 <212> DNA <213> Artificial sequence <220> <223> 3' Flanking Sequence <400> 7 ttggcatttg tggaatagat cccgagtctc ctacaacaca aggcaagccc cggtgcattt 60 acccttt 67 <210> 8 <211> 6322 <212> DNA <213> Artificial sequence <220> <223> Nucleotide sequence containing the 5' flanking sequence, the complete insertion sequence, and the 3' flanking sequence of event ME240913 <220> <221> misc_feature <222> (1)..(65) <223> 5' Flanking Sequence <220> <221> misc_feature <222> (66)..(6255) <223> Full-length insertion sequence <220> <221> misc_feature <222> (6256)...(6322) <223> 3' Flanking Sequence <400> 8 gaggtcgacg gttcgaatac taacaaccgc gtagcgcgcg aattttgcgc gaaaaatgcc 60 gcgacttgcg gacgttttta atgtactgaa ttaacgccga attgctctag cattcgccat 120 tcaggctgcg caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc 180 tggcgaaagg gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt 240 cacgacgttg taaaacgacg gccagtgcca agctaattcg cttcaagacg tgctcaaatc 300 actatttcca cacccctata tttctattgc actcccttt aactgttttt tattacaaaa 360 atgccctgga aaatgcactc cctttttgtg tttgttttt tgtgaaacga tgttgtcagg 420 taatttattt gtcagtctac tatggtggcc cattatatta atagcaactg tcggtccaat 480 agacgacgtc gattttctgc atttgtttaa ccacgtggat tttatgacat tttatattag 540 ttaatttgta aaacctaccc aattaaagac ctcatatgtt ctaaagacta atacttaatg 600 ataacaattt tcttttagtg aagaaaggga taattagtaa atatggaaca agggcagaag 660 atttattaaa gccgcgtaag agacaacaag taggtacgtg gagtgtctta ggtgacttac 720 ccacataaca taaagtgaca ttaacaaaca tagctaatgc tcctatttga atagtgcata 780 tcagcatacc ttattacata tagataggag caaactctag ctagattgtt gagcagatct 840 cggtgacggg caggaccgga cggggcggta ccggcaggct gaagtccagc tgccagaaac 900 ccacgtcatg ccagttcccg tgcttgaagc cggccgcccg cagcatgccg cggggggcat 960 atccgagcgc ctcgtgcatg cgcacgctcg ggtcgttggg cagcccgatg acagcgacca 1020 cgctcttgaa gccctgtgcc tccagggact tcagcaggtg ggtgtagagc gtggagccca 1080 gtcccgtccg ctggtggcgg ggggagacgt acacggtcga ctcggccgtc cagtcgtagg 1140 cgttgcgtgc cttccagggg cccgcgtagg cgatgccggc gacctcgccg tccacctcgg 1200 cgacgagcca gggatagcgc tcccgcagac ggacgaggtc gtccgtccac tcctgcggtt 1260 cctgcggctc ggtacggaag ttgaccgtgc ttgtctcgat gtagtggttg acgatggtgc 1320 agaccgccgg catgtccgcc tcggtggcac ggcggatgtc ggccgggcgt cgttctgggc 1380 tcatggtaga tcccccgttc gtaaatggtg aaaattttca gaaaattgct tttgctttaa 1440 aagaaatgat ttaaattgct gcaatagaag tagaatgctt gattgcttga gattcgtttg 1500 ttttgtatat gttgtgttga gaattaattc tcgaggtcct ctccaaatga aatgaacttc 1560 cttatataga ggaagggtct tgcgaaggat agtgggattg tgcgtcatcc cttacgtcag 1620 tggagatatc acatcaatcc acttgctttg aagacgtggt tggaacgtct tctttttcca 1680. cgatgctcct cgtgggtggg ggtccatctt tgggaccact gtcggcagag gcatcttcaa 1740. cgatggcctt tcctttatcg caatgatggc atttgtagga gccaccttcc ttttccacta tcttcacaat aaagtgacag atagctgggc aatggaatcc gaggaggttt ccggatatta ccctttgttg aaaagtctca attgcccttt ggtcttctga gactgtatct ttgatatttt tggagtagac aagtgtgtcg tgctccacca tgttatcaca tcaatccact tgctttgaag acgtggttgg aacgtcttct ttttccacga tgctcctcgt gggtgggggt ccatctttgg gaccactgtc ggcagaggca tcttcaacga tggccttttcc tttatcgcaa tgatggcatt tgtaggagcc accttccttt tccactatct tcacaataaa gtgacagata gctgggcaat ggaatccgag gaggtttccg gatattaccc tttgttgaaa agtctcaatt gccctttggt cttctgagac tgtatctttg atatttttgg agtagacaag tgtgtcgtgc tccaccatgt 2280 2340. tgacctgcag gcatgcagc ttgttaacct gcagtgcagc gtgaccccgt cgtgcccctc tctagagata atgagcattg catgtctaag ttataaaaaa ttaccacata ttttttttgt 2400 cacacttgtt tgaagtgcag tttatctatc tttatacata tatttaaact ttactctacg 2460 aataatataa tctatagtac tacaataata tcagtgtttt agagaatcat ataaatgaac 2520 agttagacat ggtctaaagg acaattgagt atttgacaa caggactcta cagttttatc 2580 ttttagtgt gcatgtgttc tcctttttt ttgcaaatag cttcacctat ataatacttc 2640 atccatttta ttagtacatc catttagggt ttagggttaa tggtttttat agactaattt 2700 ttttagtaca tctattttat tctattttag cctctaaatt aagaaaacta aaactctatt 2760 ttagtttttt tatttaataa tttagatata aatagaata aaataaagtg actaaaaatt 2820 aaacaaatac cctttaagaa attaaaaaaa ctaaggaaac atttttcttg tttcgagtag 2880 ataatgccag cctgttaaac gccgtcgatc gacgagcta acggacacca accagcgaac 2940 cagcagcgtc gcgtcgggcc aagcgaagca gacggcacgg catctctgtc gctgcctctg 3000 gaccctctc gagagtccg ctccaccgtt ggacttgctc cgctgtcggc atccagaaat 3060 tgcgtggcgg agcggcagac gtgagccggc acggcaggcg gcctcctcct cctctcacgg 3120 caccggcagc tacgggggat tcctttccca ccgctccttc gctttccctt cctcgcccgc 3180 cgtaataaat agacaccccc tccacaccct ctttccccaa cctcgtgttg ttcggagcgc 3240 acacacacac aaccagatct cccccaaatc cacccgtcgg cacctccgct tcaaggtacg 3300 ccgctcgtcc tccccccccc cccctctcta ccttctctag atcggcgttc cggtccatgg 3360 ttagggcccg gtagttctac ttctgttcat gtttgtgtta gatccgtgtt tgtgttagat 3420 ccgtgctgct agcgttcgta cacggatgcg acctgtacgt cagacacgtt ctgattgcta 3480 acttgccagt gtttctcttt ggggaatcct gggatggctc tagccgttcc gcagacggga 3540 tcgatctagg ataggtatac atgttgatgt gggttttact gatgcatata catgatggca 3600 tatgcagcat ctattcatat gctctaacct tgagtaccta tctattataa taaacaagta 3660 tgttttataa ttattttgat cttgatatac ttggatgatg gcatatgcag cagctatatg 3720 tggatttttt tagccctgcc ttcatacgct atttatttgc ttggtactgt ttcttttgtc 3780 gatgctcacc ctgttgtttg gtgttacttc tgcaggggat ccgagctcat ggagatcaac 3840 aaccagaacc agtgcgtgcc gtacaactgc ctgtccaacc cgaaggagat catcctgggc 3900 gaggagaggc tggagaccgg caacaccgtg gccgacatct ccctgggcct gatcaacttc 3960 ctgtactcca acttcgtgcc gggcggcggc ttcatcgtgg gcctgctgga gctgatctgg 4020 ggcttcatcg gcccgtccca gtgggacatc ttcctggccc agatcgagca gctgatctcc 4080 cagaggatcg aggagttcgc caggaaccag gccatctcca ggctggaggg cctgtccaac 4140 ctgtacaagg tgtacgtgag ggccttctcc gactgggaga aggacccgac caacccggcc 4200 ctgagggagg agatgaggat acagttcaac gacatgaact ccgccctgat caccgccatc 4260 ccgctgttca gggtgcagaa ctacgaggtg gccctgctgt ccgtgtacgt gcaggccgcc 4320 aacctgcacc tgtccatcct gagggacgtg tccgtgttcg gcgagaggtg gggctacgac 4380 accgccacca tcaacaacag gtactccgac ctgacctccc tgatccacgt gtacaccaac 4440 cactgcgtgg acacctacaa ccagggcctg aggaggctgg agggcaggtt cctgtccgac 4500 tggatcgtgt acaacaggtt caggaggcag ctgaccatct ccgtgctgga catcgtggcc 4560 ttcttcccga actacgacat caggacctac ccgatccaga ccgccaccca gctgaccagg 4620 gaggtgtacc tggacctgcc gttcatcaac gagaacctgt ccccggccgc ctcctacccg 4680 accttctccg ccgccgagtc cgccatcatc aggtccccgc acctggtgga cttcctgaac 4740 tccttcacca tctacaccga ctccctggcc aggtacgcct actggggcgg ccacctggtg 4800 aactccttca ggaccggcac caccaccaac ctgatcaggt ccccgctgta cggcagggag 4860 ggcaacaccg agaggccggt gaccatcacc gcctccccgt ccgtgccgat cttcaggacc 4920 ctgtcctaca tcaccggcct ggacaactcc aacccggtgg ccggcatcga gggcgtggag 4980 ttccagaaca ccatctccag gtccatctac aggaagtccg gcccgatcga ctccttctcc 5040 gagctgccgc cgcaggacgc ctccgtgtcc ccggccatcg gctactccca caggctgtgc 5100 cacgccacct tcctggagag gatctccggc ccgaggatcg ccggcaccgt gttctcctgg 5160 acccacaggt ccgcctcccc gaccaacgag gtgtccccgt ccaggatcac ccagatcccg 5220 tgggtgaag gggctccggc gcctccgtga tcaagggccc gggcttcacc 5280 ggcggcgaca tcctgaccag gaactccatg ggcgagctgg gcacctgag ggtgaccttc 5340 accggcaggc tgccgcagtc ctactacatc aggttcaggt acgcctccgt ggccacagg 5400 tccggcacct tcaggtactc ccagccgccg tcctacggca tctccttccc gaagaccatg 5460 gacgccggcg agccgctgac ctccaggtcc tcgcccaca ccaccgtt caccccgatc 5520 accttctcca gggcccagga ggagttcgac ctgtacatcc agtccggcgt gtacatcgac 5580 aggatcgagt tcatcccggt gaccgccacc tcgaggccg agtacgacct gaggaggggcc 5640 cagaaggtgg tgaacgccct gttcacctcc accaccacc tgggctga gaccgacgtg 5700 acctaagtcg acggatccga tcgttcaac atttggcaat aaagttttctt agattgaat 5760 cctgttgccg gtcttgcgat gattatcata taatttctgt tgaattacgt taagcatgta 5820 ataattaaca tgtaatgcat gacgttattt atgagatgggg ttttgat tagagtcccg 5880 caattataca tttaatacgc gatagaaaac aaaatatagc gcgcaacta ggataatta 5940 tcgcgcgcgg tgtcatctat gttatactagat cggttaacga attcgtaatc atgtcatagc 6000 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca 6060 taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct 6120 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac 6180 gcgcggggag aggcggtttg cgtattggag cttgagcttg gatcagattg tcgtttcccg 6240 ccttcagttt aaactttggc atttgtggaa tagatcccga gtctcctaca acacaaggca 6300 agccccggtg catttaccct tt 6322 <210> 9 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Forward primer sequence for amplifying the 5' conjugate sequence <400> 9 aggtcgacgg ttcgaatact 20 <210> 10 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Reverse primer sequence for amplifying the 5' conjugation sequence <400> 10 gctagagcaa ttcggcgtta a 21 <210> 11 <211> 110 <212> DNA <213> Artificial sequence <220> <223> The explanatory amplicon sequence was obtained by PCR using primers specific to the 5' conjugate sequence. <400> 11 aggtcgacgg ttcgaatact aacaaccgcg tagcgcgcga attttgcgcg aaaaatgccg 60 cgacttgcgg acgtttttaa tgtactgaat taacgccgaa ttgctctagc 110 <210> 12 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Probe sequence for detecting the 5' junction sequence <400> 12 acaaccgcgt agcgcgcgaa 20 <210> 13 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Forward primer sequence for amplifying the 3' conjugation sequence <400> 13 cggtttgcgt attggagctt 20 <210> 14 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Reverse primer sequence for amplifying the 3' conjugation sequence <400> 14 ggcttgcctt gtgttgtagg 20 <210> 15 <211> 111 <212> DNA <213> Artificial sequence <220> <223> The explanatory amplicon sequence was obtained by PCR using primers specific to the 3' conjugate sequence. <400> 15 cggtttgcgt attggagctt gagcttggat cagattgtcg tttcccgcct tcagtttaaa 60 ctttggcatt tgtggaatag atcccgagtc tcctacaaca caaggcaagc c 111 <210> 16 <211> 25 <212> DNA <213> Artificial sequence <220> <223> Probe sequence for detecting the 3' junction sequence <400> 16 tggatcagat tgtcgtttcc cgcct 25

Claims

1. A nucleic acid molecule, characterized in that... It consists of the nucleotide sequence of SEQ ID NO: 8, which is contained in maize seeds deposited at the U.S. Center for Type Culture Collection with accession number PTA-126224 and is specific to event ME240913.

2. The nucleic acid molecule of claim 1, characterized in that... The nucleotide sequence encodes a truncated Cry1Da protein consisting of the amino acid sequence of SEQ ID NO:

3.

3. A method for producing maize plants resistant to lepidopteran pests, including event ME240913, characterized in that... include: Sexual hybridization of a first parent maize plant with a second parent maize plant to produce multiple first-generation offspring plants, wherein the first or second parent maize plant contains the nucleic acid molecule of event ME240913 as defined in claim 1; Plants containing the DNA of event ME240913 were selected from the progeny plants.

4. A method for producing hybrid maize seeds, characterized in that... include: Seeds of a first innate maize line containing the nucleic acid molecule of event ME240913 as defined in claim 1 and seeds of a second innate line with a different genotype are planted. The two different innate strains were sexually hybridized; and Harvest the hybrid seeds produced as a result.

5. The method of claim 4, characterized in that... The first congenital maize strain is the parent plant.

6. The method of claim 4, characterized in that... The first congenital maize strain is the male parent.

7. A method for controlling lepidopteran pests on maize plants, wherein the maize plants contain a nucleic acid molecule of event ME240913 as defined in claim 1, the method being characterized by planting seeds obtained from plants containing the nucleic acid molecule in the growth area of ​​maize plants susceptible to lepidopteran pests.

8. The method of claim 7, characterized in that... The lepidopteran pest in question is the fall armyworm.

9. The method of claim 8, characterized in that... The fall armyworm is resistant to Cry1F and / or Cry1A.

10. The method of claim 8 or 9, characterized in that... Fresh plant leaves containing nucleic acid molecules of event ME240913 as defined in claim 1 are highly toxic to the fall armyworm.

11. Use of a plant, plant cell, plant part, or seed comprising a nucleic acid molecule as defined in any one of claims 1 or 2, characterized in that... It is used to propagate or regenerate plants with a second plant, to plant or grow plant fields, or to produce plant products.

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