Patent Application
20230200335
An XML formatted sequence listing having the file name “8763_SequenceListing.xml” created on Oct. 25, 2022, and having a size of 93 kilobytes is filed in computer readable form concurrently with the specification. The sequence listing comprised in this XML formatted document is part of the specification and is herein incorporated by reference in its entirety.
Embodiments disclosed herein relate to the field of plant molecular biology, including to DNA constructs for conferring insect resistance to a plant. Embodiments disclosed herein also include insect resistant corn plant containing event DP-910521-2 and assays for detecting the presence of event DP-910521-2 in a sample and compositions thereof.
Corn is an important crop and is a primary food source in many areas of the world. Damage caused by insect pests is a major factor in the loss of the world's corn crops, despite the use of protective measures such as chemical pesticides. In view of this, insect resistance has been genetically engineered into crops such as corn in order to control insect damage and to reduce the need for traditional chemical pesticides. One group of genes which have been utilized for the production of transgenic insect resistant crops is the delta-endotoxin group from Bacillus thuringiensis (Bt). Delta-endotoxins have been successfully expressed in crop plants such as cotton, potatoes, rice, sunflower, as well as corn, and in certain circumstances have proven to provide excellent control over insect pests. (Perlak, F. J et al. (1990) Bio/Technology 8:939-943; Perlak, F. J. et al. (1993) Plant Mol. Biol. 22:313-321; Fujimoto, H. et al. (1993) Bio/Technology 11:1151-1155; Tu et al. (2000) Nature Biotechnology 18:1101-1104; PCT publication WO 01/13731; and Bing, J. W. et al. (2000) Efficacy of Cry1F Transgenic Maize, 14th Biennial International Plant Resistance to Insects Workshop, Fort Collins, Colo.).
The expression of transgenes in plants is known to be influenced by many different factors, including the orientation and composition of the cassettes driving expression of the individual genes of interest, and the location in the plant genome, perhaps due to chromatin structure (e.g., heterochromatin) or the proximity of transcriptional regulatory elements (e.g., enhancers) close to the integration site (Weising et al. (1988) Ann. Rev. Genet. 22:421-477).
It would be advantageous to be able to detect the presence of a particular event in order to determine whether progeny of a sexual cross contain a transgene of interest.
It is possible to detect the presence of a transgene by a nucleic acid detection method by, e.g., a polymerase chain reaction (PCR) or DNA hybridization using nucleic acid probes. These detection methods generally focus on frequently used genetic elements, such as promoters, terminators, marker genes, etc., because for many DNA constructs, the coding region is interchangeable. As a result, such methods may not be useful for discriminating between different events, particularly those produced using the same DNA construct or very similar constructs unless the DNA sequence of the flanking DNA adjacent to the inserted heterologous DNA is known
The embodiments relate to the insect resistant corn (Zea mays) plant event DP-910521-2, also referred to as “maize line DP-910521-2,” “maize event DP-910521-2,” and “DP-910521-2 maize,” to the DNA plant expression construct of corn plant event DP-910521-2, and to methods and compositions for the detection of the transgene construct, flanking, and insertion (the target locus) regions in corn plant event DP-910521-2 and progeny thereof.
In one aspect compositions and methods relate to methods for producing and selecting an insect resistant monocot crop plant. Compositions include a DNA construct that when expressed in plant cells and plants confers resistance to insects. In one aspect, a DNA construct, capable of introduction into and replication in a host cell, is provided that when expressed in plant cells and plants confers insect resistance to the plant cells and plants. Maize event DP-910521-2 was produced by transformation with plasmid PHP79620. As described herein, these events include the cry1B.34 gene (polynucleotide SEQ ID NO: 4 and amino acid SEQ ID NO: 5) cassette (Table 1), which confers resistance to certain lepidopteran plant pests. The insect control components have demonstrated efficacy against lepidopteran insect species.
Some embodiments relate to specific flanking sequences of DP-910521-2 as described herein, which can be used to develop identification methods for DP-910521-2 in biological samples. More particularly, the disclosure relates to 5′ and/or 3′ flanking regions of DP-910521-2, which can be used for the development of specific primers and probes. Further embodiments relate to identification methods for the presence of DP-910521-2 in biological samples based on the use of such specific primers or probes.
According to some embodiments, methods of detecting the presence of DNA corresponding to the corn event DP-910521-2 in a sample are provided. Such methods comprise: (a) contacting the sample comprising DNA with a DNA primer set, that when used in a nucleic acid amplification reaction with genomic DNA extracted from corn comprising event DP-910521-2 produces an amplicon that is diagnostic for corn event DP-910521-2, respectively; (b) performing a nucleic acid amplification reaction, thereby producing the amplicon; and (c) detecting the amplicon. In some aspects, the primer set comprises SEQ ID NOs: 6 and 7, and optionally a probe comprising SEQ ID NO: 8.
According to some embodiments, methods of detecting the presence of a DNA molecule corresponding to the DP-910521-2 event in a sample comprise: (a) contacting the sample comprising DNA extracted from a corn plant with a DNA probe molecule that hybridizes under stringent hybridization conditions with DNA extracted from corn containing event DP-910521-2 and does not hybridize under the stringent hybridization conditions with a control corn plant DNA; (b) subjecting the sample and probe to stringent hybridization conditions; and (c) detecting hybridization of the probe to the DNA extracted from corn containing event DP-910521-2. More specifically, a method for detecting the presence of a DNA molecule corresponding to the DP-910521-2 event in a sample comprises (a) contacting the sample comprising DNA extracted from a corn plant with a DNA probe molecule that comprises sequences that are unique to the event, e.g. junction sequences, wherein said DNA probe molecule hybridizes under stringent hybridization conditions with DNA extracted from corn event DP-910521-2 and does not hybridize under the stringent hybridization conditions with a control corn plant DNA; (b) subjecting the sample and probe to stringent hybridization conditions; and (c) detecting hybridization of the probe to the DNA.
In addition, a kit and methods for identifying event DP-910521-2 in a biological sample which detects a DP-910521-2 specific region are provided.
DNA molecules are provided that comprise at least one junction sequence of DP-910521-2; wherein a junction sequence spans the junction located between heterologous DNA inserted into the genome and the DNA from the maize cell flanking the insertion site. Detection of the junction sequence can be diagnostic for the DP-910521-2 event.
According to some embodiments, methods of producing an insect resistant corn plant comprise the steps of: (a) sexually crossing a first parental corn line comprising the expression cassettes disclosed herein, which confer resistance to insects, and a second parental corn line that lacks such expression cassettes, thereby producing a plurality of progeny plants; and (b) selecting a progeny plant that is insect resistant. Such methods may optionally comprise the further step of back-crossing the progeny plant to the second parental corn line to produce a true-breeding corn plant that is insect resistant.
Some embodiments provide a method of producing a corn plant that is resistant to insects comprising transforming a corn cell with the DNA construct PHP79620, growing the transformed corn cell into a corn plant, selecting the corn plant that shows resistance to insects, and further growing the corn plant into a fertile corn plant. The fertile corn plant can be self-pollinated or crossed with compatible corn varieties to produce insect resistant progeny. In some embodiments, a corn plant comprises the genotype of the corn event DP-910521-2, wherein said genotype comprises a nucleotide sequence as set forth in SEQ ID NO: 26 and SEQ ID NO: 29.
Some embodiments further relate to a DNA detection kit for identifying maize event DP-910521-2 in biological samples. The kit comprises a first primer which specifically recognizes the 5′ or 3′ flanking region of DP-910521-2, and a second primer which specifically recognizes a sequence within the non-native target locus DNA of DP-910521-2, respectively, or within the flanking DNA, for use in a PCR identification protocol. A further embodiment relates to a kit for identifying event DP-910521-2 in biological samples, which kit comprises a specific probe having a sequence which corresponds or is complementary to, a sequence having between about 80% and 100% sequence identity with a specific region of event DP-910521-2. The sequence of the probe can correspond to a specific region comprising part of the 5′ or 3′ flanking region of event DP-910521-2. In some embodiments, the first or second primer comprises any one of SEQ ID NOs: 6-7, 9-10, 12-13, 15-16, or 18-19.
The methods and kits encompassed by the embodiments disclosed herein can be used for different purposes such as, but not limited to the following: to identify event DP-910521-2 in plants, plant material or in products such as, but not limited to, food or feed products (fresh or processed) comprising, or derived from plant material; additionally or alternatively, the methods and kits can be used to identify transgenic plant material for purposes of segregation between transgenic and non-transgenic material; additionally or alternatively, the methods and kits can be used to determine the quality of plant material comprising maize event DP-910521-2. The kits may also contain the reagents and materials necessary for the performance of the detection method.
A further embodiment relates to the DP-910521-2 maize plant or its parts, including, but not limited to, pollen, ovules, vegetative cells, the nuclei of pollen cells, and the nuclei of egg cells of the corn plant DP-910521-2 and the progeny derived thereof. In another embodiment, the DNA primer molecules targeting the maize plant and seed of DP-910521-2 provide a specific amplicon product
As used herein the singular forms “a”, “and”, and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a cell” includes a plurality of such cells and reference to “the protein” includes reference to one or more proteins and equivalents thereof, and so forth. All technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs unless clearly indicated otherwise. Nucleic acid sequences listed in the accompanying sequence listing and referenced herein are shown using standard letter abbreviations for nucleotide bases. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood to be included by any reference to the displayed strand.
Compositions of this disclosure include seed deposited as ATCC Patent Deposit No. PTA-127078 and plants, plant cells, and seed derived therefrom. Applicant(s) deposited at least 625 seeds of maize event DP-910521-2 (Patent Deposit No. PTA-127078) with the American Type Culture Collection (ATCC), Manassas, Va. 20110-2209 USA, on May 26, 2021. These deposits will be maintained under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. The seeds deposited with the ATCC on May 26, 2021, were taken from the deposit maintained by Pioneer Hi-Bred International, Inc., 7250 NW 62nd Avenue, Johnston, Iowa 50131-1000. Access to this deposit will be available during the pendency of the application to the Commissioner of Patents and Trademarks and persons determined by the Commissioner to be entitled thereto upon request. Upon allowance of any claims in the application, the Applicant(s) will make available to the public, pursuant to 37 C.F.R. § 1.808, sample(s) of the deposit of at least 625 seeds of hybrid maize with the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, Va. 20110-2209. This deposit of seed of maize event DP-910521-2 will be maintained in the ATCC depository, which is a public depository, for a period of 30 years, or 5 years after the most recent request, or for the enforceable life of the patent, whichever is longer, and will be replaced if it becomes nonviable during that period. Additionally, Applicant(s) have satisfied all the requirements of 37 C.F.R. §§ 1.801-1.809, including providing an indication of the viability of the sample upon deposit. Applicant(s) have no authority to waive any restrictions imposed by law on the transfer of biological material or its transportation in commerce. Applicant(s) do not waive any infringement of their rights granted under this patent or rights applicable to event DP-910521-2 under the Plant Variety Protection Act (7 USC 2321 et seq.). Unauthorized seed multiplication is prohibited. The seed may be regulated.
A first gene cassette (pmi gene cassette) contains the phosphomannose isomerase (pmi) gene from Escherichia coli (Negrotto et al., 2000). The expressed PMI protein in plant tissue serves as a selectable marker during transformation which allows for tissue growth using mannose as the carbon source. The PMI protein is 391 amino acids in length and has a molecular weight of approximately 43 kDa. As present in the recombination fragment region of PHP79620, the pmi gene lacks a promoter, but its location next to the flippase recombination target site, FRT1, allows post-recombination expression by an appropriately-placed promoter. The terminator for the pmi gene is the terminator region from the potato (Solanum tuberosum) proteinase inhibitor II (pinII) gene (Keil et al., 1986; An et al., 1989). One additional terminator is present between the first and second cassettes: the terminator region from the maize 19-kDa zein (Z19) gene (GenBank accession KX247647; Dong et al., 2016). This additional terminator element is intended to prevent any potential transcriptional interference with the downstream cassettes. Transcriptional interference is defined as the transcriptional suppression of one gene on another when both are in close proximity (Shearwin et al., 2005). The placement of one or multiple transcriptional terminators between gene cassettes has been shown to reduce the occurrence of transcriptional interference (Greger et al., 1998).
A second gene cassette (mo pat gene cassette) contains a maize-optimized version of the phosphinothricin acetyltransferase (mo-pat) gene from Streptomyces viridochromogenes (Wohlleben et al., 1988) encoding the PAT protein. The expressed PAT protein confers tolerance to phosphinothricin. The PAT protein is 183 amino acids in length and has a molecular weight of approximately 21 kDa. Expression of the mo-pat gene is controlled by the promoter and intron region of the rice (Oryza sativa) actin (os-actin) gene (GenBank accession CP018159; GenBank accession EU155408.1), in conjunction with the 35S terminator region from the cauliflower mosaic virus genome (CaMV 35S terminator; Franck et al., 1980; Guilley et al., 1982). Two additional terminators are present between the second and third cassettes to prevent transcriptional interference: the terminator regions from the sorghum (Sorghum bicolor) ubiquitin (sb-ubi) gene (Phytozome gene ID Sobic.004G049900.1; U.S. Pat. No. 9,725,731 [Abbitt et al., 2017]) and γ-kafarin (sb-gkaf) gene (de Freitas et al., 1994), respectively.
A third gene cassette (cry1B.34 gene cassette) contains the cry1B.34 gene, a chimeric gene comprised of sequences from variants of a cry1B-class gene, the cry1Ca1 gene, and the cry9db1 gene, both derived from Bacillus thuringiensis (WO Patent 2016061197 [Izumi and Yamamoto, 2016]; GenBank accession CAA30396.1; U.S. Pat. No. 7,541,517 [Flannagan et al., 2009], respectively). The expressed Cry1B.34 protein is effective against certain lepidopteran pests by causing disruption of the midgut epithelium. The Cry1B.34 protein is 1,149 amino acids in length and has a molecular weight of approximately 129 kDa. Expression of the cry1B.34 gene is controlled by two copies of the enhancer region from the mirabilis mosaic virus (MMV) genome (Dey and Maiti, 1999), the promoter region from the lamium leaf distortion-associated virus (LLDAV) genome (Zhang et al., 2008), the intron region from the maize translation initiating factor 6 (zm-i6) gene (Phytozome gene ID GRMZM2G318475; U.S. patent Ser. No. 10/344,290 [Diehen et al., 2019]), and the 5′ untranslated region (UTR) from the maize extensin (zm-extensin) gene (GenBank accession NM001111947.2; UniProt accession P14918). The terminator for the cry1B.34 gene is the terminator region from the rice (Oryza sativa) ubiquitin (os-ubi) gene (Phytozome gene ID LOC_Os06g46770.1; Wang et al., 2000).
The PHP79620 recombination fragment region contains two flippase (FLP) recombination target sequences, the FRT1 and FRT87 sites (Proteau et al., 1986; Tao et al., 2007, respectively), as well as one loxP (Dale and Ow, 1990) and two attB recombination sites, attB1 and attB3 (Hartley et al., 2000 and Katzen, 2007; Cheo et al., 2004, respectively). The presence of these sites alone does not cause any recombination, since in order to function, these sites need specific recombinase enzymes that are not naturally present in plants (Cox, 1988; Dale and Ow, 1990; Thorpe and Smith, 1998).
According to some embodiments, compositions and methods are provided for identifying a novel corn plant designated DP-910521-2 (ATCC Deposit Number PTA-127078). The methods are based on primers or probes which specifically recognize 5′ and/or 3′ flanking sequence of DP-910521-2. DNA molecules are provided that comprise primer sequences that when utilized in a PCR reaction will produce amplicons unique to the transgenic event DP-910521-2. In one embodiment, the corn plant and seed comprising these molecules is contemplated. Further, kits utilizing these primer sequences for the identification of the DP-910521-2 event are provided.
As used herein, the term “corn” means Zea mays or maize and includes all plant varieties that can be bred with corn, including wild maize species.
As used herein, the terms “insect resistant” and “impacting insect pests” refers to effecting changes in insect feeding, growth, and/or behavior at any stage of development, including but not limited to: killing the insect; retarding growth; reducing reproductive capability; inhibiting feeding; and the like.
As used herein, the terms “pesticidal activity” and “insecticidal activity” are used synonymously to refer to activity of an organism or a substance (such as, for example, a protein) that can be measured by numerous parameters including, but not limited to, pest mortality, pest weight loss, pest attraction, pest repellency, and other behavioral and physical changes of a pest after feeding on and/or exposure to the organism or substance for an appropriate length of time. For example, “pesticidal proteins” are proteins that display pesticidal activity by themselves or in combination with other proteins.
As used herein, “insert DNA” refers to the heterologous DNA within the expression cassettes used to transform the plant material while “flanking DNA” can refer to either genomic DNA naturally present in an organism such as a plant, or foreign (heterologous) DNA introduced via the transformation process which is extraneous to the original insert DNA molecule, e.g., fragments associated with the transformation event. A “flanking region” or “flanking sequence” as used herein refers to a sequence of at least 10 bp (in some narrower embodiments, at least 20 bp, at least 50 bp, and up to at least 5000 bp), which is located either immediately upstream of and contiguous with and/or immediately downstream of and contiguous with the original non-native insert DNA molecule. Transformation procedures of the foreign DNA may result in transformants containing different flanking regions characteristic and unique for each transformant. When recombinant DNA is introduced into a plant through traditional crossing, its flanking regions will generally not be changed. It may be possible for single nucleotide changes to occur in the flanking regions through generations of plant breeding and traditional crossing. Transformants will also contain unique junctions between a piece of heterologous insert DNA and genomic DNA, or two (2) pieces of genomic DNA, or two (2) pieces of heterologous DNA. A “junction” is a point where two (2) specific DNA fragments join. For example, a junction exists where insert DNA joins flanking DNA. A junction point also exists in a transformed organism where two (2) DNA fragments join together in a manner that is modified from that found in the native organism. “Junction DNA” refers to DNA that comprises a junction point. Junction sequences set forth in this disclosure include a junction point located between the maize genomic DNA and the 5′ end of the insert, which range from at least −5 to +5 nucleotides of the junction point (SEQ ID NO: 26), from at least −10 to +10 nucleotides of the junction point (SEQ ID NO: 27), and from at least −25 to +25 nucleotides of the junction point (SEQ ID NO: 28); and a junction point located between the 3′ end of the insert and maize genomic DNA, which range from at least −5 to +5 nucleotides of the junction point (SEQ ID NO: 29), from at least −10 to +10 nucleotides of the junction point (SEQ ID NO: 30), and from at least −25 to +25 nucleotides of the junction point (SEQ ID NO: 31). Junction sequences set forth in this disclosure also include a junction point located between the target locus and the 5′ end of the insert. In some embodiments, SEQ ID NOs: 8 or 21 for DP-910521-2 represent the junction point located between the target locus and the 5′ end of the insert. The complete insert with flanking regions is represented in SEQ ID NO: 3.
In one embodiment, seeds, plants, and plant parts comprising corn event DP-910521 are provided, wherein said seeds, plants, and plant parts comprise a DNA sequence chosen from SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, and SEQ ID NO: 31, or a DNA sequence chosen from a sequence having at least 95% sequence identity to SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, and SEQ ID NO: 31, wherein a representative sample of the corn event DP-910521 seed has been deposited with American Type Culture Collection (ATCC) with Accession No. PTA-127078. In another embodiment, seeds, plants, and plant parts comprising corn event DP-910521 are provided, wherein said seeds, plants, and plant parts comprise SEQ ID NO: 3 or a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 3, wherein a representative sample of the corn event DP-910521 seed has been deposited with American Type Culture Collection (ATCC) with Accession No. PTA-127078.
As used herein, “heterologous” in reference to a nucleic acid sequence is a nucleic acid sequence that originates from a different non-sexually compatible species, or, if from the same species, is substantially modified from its native form in composition and/or genomic locus by deliberate human intervention. For example, a promoter operably linked to a heterologous nucleotide sequence can be from a species different from that from which the nucleotide sequence was derived, or, if from the same species, the promoter is not naturally found operably linked to the nucleotide sequence. A heterologous protein may originate from a foreign species, or, if from the same species, is substantially modified from its original form by deliberate human intervention.
The term “regulatory element” refers to a nucleic acid molecule having gene regulatory activity, i.e., one that has the ability to affect the transcriptional and/or translational expression pattern of an operably linked transcribable polynucleotide. The term “gene regulatory activity” thus refers to the ability to affect the expression of an operably linked transcribable polynucleotide molecule by affecting the transcription and/or translation of that operably linked transcribable polynucleotide molecule. Gene regulatory activity may be positive and/or negative and the effect may be characterized by its temporal, spatial, developmental, tissue, environmental, physiological, pathological, cell cycle, and/or chemically responsive qualities as well as by quantitative or qualitative indications.
“Promoter” refers to a nucleotide sequence capable of controlling the expression of a coding sequence or functional RNA. In general, a coding sequence is located 3′ to a promoter sequence. The promoter sequence comprises proximal and more distal upstream elements, the latter elements are often referred to as enhancers. Accordingly, an “enhancer” is a nucleotide sequence that can stimulate promoter activity and may be an innate element of the promoter or a heterologous element inserted to enhance the level or tissue-specificity of a promoter. Promoters may be derived in their entirety from a native gene or be composed of different elements derived from different promoters found in nature, or even comprise synthetic nucleotide segments. It is understood by those skilled in the art that different regulatory elements may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental conditions. Promoters that cause a nucleic acid fragment to be expressed in most cell types at most times are commonly referred to as “constitutive promoters”. It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, nucleic acid fragments of different lengths may have identical or similar promoter activity.
The “translation leader sequence” refers to a nucleotide sequence located between the promoter sequence of a gene and the coding sequence. The translation leader sequence is present in the fully processed mRNA upstream of the translation start sequence. The translation leader sequence may affect numerous parameters including, processing of the primary transcript to mRNA, mRNA stability and/or translation efficiency.
The “3′ non-coding sequences” refer to nucleotide sequences located downstream of a coding sequence and include polyadenylation recognition sequences and other sequences encoding regulatory signals capable of affecting mRNA processing or gene expression. The polyadenylation signal is usually characterized by affecting the addition of polyadenylic acid tracts to the 3′ end of the mRNA precursor.
A DNA construct is an assembly of DNA molecules linked together that provide one or more expression cassettes. The DNA construct may be a plasmid that is enabled for self-replication in a bacterial cell and contains various endonuclease enzyme restriction sites that are useful for introducing DNA molecules that provide functional genetic elements, i.e., promoters, introns, leaders, coding sequences, 3′ termination regions, among others; or a DNA construct may be a linear assembly of DNA molecules, such as an expression cassette.
The expression cassette contained within a DNA construct comprises the necessary genetic elements to provide transcription of a messenger RNA. The expression cassette can be designed to express in prokaryotic cells or eukaryotic cells. Expression cassettes of the embodiments are designed to express in plant cells.
The DNA molecules disclosed herein are provided in expression cassettes for expression in an organism of interest. The cassette includes 5′ and 3′ regulatory sequences operably linked to a coding sequence. “Operably linked” means that the nucleic acid sequences being linked are contiguous and, where necessary to join two protein coding regions, contiguous and in the same reading frame. Operably linked is intended to indicate a functional linkage between a promoter and a second sequence, wherein the promoter sequence initiates and mediates transcription of the DNA sequence corresponding to the second sequence. The cassette may additionally contain at least one additional gene to be co-transformed into the organism. Alternatively, the additional gene(s) can be provided on multiple expression cassettes or multiple DNA constructs.
The expression cassette may include in the 5′ to 3′ direction of transcription: a transcriptional and translational initiation region, a coding region, and a transcriptional and translational termination region functional in the organism serving as a host. The transcriptional initiation region (e.g., the promoter) may be native or analogous, or foreign or heterologous to the host organism. Additionally, the promoter may be the natural sequence or alternatively a synthetic sequence. The expression cassettes may additionally contain 5′ leader sequences in the expression cassette construct. Such leader sequences can act to enhance translation.
It is to be understood that as used herein the term “transgenic” generally includes any cell, cell line, callus, tissue, plant part, or plant, the genotype of which has been altered by the presence of a heterologous nucleic acid including those initially so altered as well as those created by sexual crosses or asexual propagation from the initial transgenic and retains such heterologous nucleic acids.
A transgenic “event” is produced by transformation of plant cells with a heterologous DNA construct(s), including a nucleic acid expression cassette that comprises a transgene of interest, the regeneration of a population of plants resulting from the insertion of the transgene into the genome of the plant, and selection of a particular plant characterized by insertion into a particular genome location. An event is characterized phenotypically by the expression of the transgene. At the genetic level, an event is part of the genetic makeup of a plant. The term “event” also refers to progeny produced by a sexual outcross between the transformant and another variety, wherein the progeny includes the heterologous DNA. After back-crossing to a recurrent parent, the inserted DNA and the linked flanking genomic DNA from the transformed parent is present in the progeny of the cross at the same chromosomal location. A progeny plant may contain sequence changes to the insert arising as a result of conventional breeding techniques. The term “event” also refers to DNA from the original transformant comprising the inserted DNA and flanking sequence immediately adjacent to the inserted DNA that would be expected to be transferred to a progeny that receives inserted DNA including the transgene of interest as the result of a sexual cross of one parental line that includes the inserted DNA (e.g., the original transformant and progeny resulting from selfing) and a parental line that does not contain the inserted DNA.
An insect resistant DP-910521-2 corn plant may be bred by first sexually crossing a first parental corn plant having the transgenic DP-910521-2 event plant and progeny thereof derived from transformation with the expression cassettes of the embodiments that confers insect resistance, and a second parental corn plant that lacks such expression cassettes, thereby producing a plurality of first progeny plants; and then selecting a first progeny plant that is resistant to insects; and selfing the first progeny plant, thereby producing a plurality of second progeny plants; and then selecting from the second progeny plants an insect resistant plant. These steps can further include the back-crossing of the first insect resistant progeny plant or the second insect resistant progeny plant to the second parental corn plant or a third parental corn plant, thereby producing a corn plant that is resistant to insects. The term “selfing” refers to self-pollination, including the union of gametes and/or nuclei from the same organism.
As used herein, the term “plant” includes reference to whole plants, parts of plants, plant organs (e.g., leaves, stems, roots, etc.), seeds, plant cells, and progeny of same. In some embodiments, parts of transgenic plants comprise, for example, plant cells, protoplasts, tissues, callus, embryos as well as flowers, stems, fruits, leaves, and roots originating in transgenic plants or their progeny previously transformed with a DNA molecule disclosed herein, and therefore consisting at least in part of transgenic cells.
As used herein, the term “plant cell” includes, without limitation, seeds, suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen, and microspores. The class of plants that may be used is generally as broad as the class of higher plants amenable to transformation techniques, including both monocotyledonous and dicotyledonous plants.
“Transformation” refers to the transfer of a nucleic acid fragment into the genome of a host organism, resulting in genetically stable inheritance. Host plants containing the transformed nucleic acid fragments are referred to as “transgenic” plants.
As used herein, the term “progeny,” in the context of event DP-910521-2, denotes an offspring of any generation of a parent plant which comprises corn event DP-910521-2.
Isolated polynucleotides disclosed herein may be incorporated into recombinant constructs, typically DNA constructs, which are capable of introduction into and replication in a host cell. Such a construct may be a vector that includes a replication system and sequences that are capable of transcription and translation of a polypeptide-encoding sequence in a given host cell. A number of vectors suitable for stable transfection of plant cells or for the establishment of transgenic plants have been described in, e.g., Pouwels et al., (1985; Supp. 1987) Cloning Vectors: A Laboratory Manual, Weissbach and Weissbach (1989) Methods for Plant Molecular Biology, (Academic Press, New York); and Flevin et al., (1990) Plant Molecular Biology Manual, (Kluwer Academic Publishers). Typically, plant expression vectors include, for example, one or more cloned genes under the transcriptional control of 5′ and 3′ regulatory sequences and a dominant selectable marker. Such plant expression vectors also can contain a promoter regulatory region (e.g., a regulatory region controlling inducible or constitutive, environmentally- or developmentally-regulated, or cell- or tissue-specific expression), a transcription initiation start site, a ribosome binding site, an RNA processing signal, a transcription termination site, and/or a polyadenylation signal.
During the process of introducing an insert into the genome of plant cells, it is not uncommon for some deletions or other alterations of the insert and/or genomic flanking sequences to occur. Thus, the relevant segment of the plasmid sequence provided herein might comprise some minor variations, including truncations. The same is possible for the flanking sequences and junction sequences provided herein. Thus, a plant comprising a polynucleotide having some range of identity with the subject flanking and/or insert sequences is within the scope of the subject disclosure. Identity to the sequence of the present disclosure may be a polynucleotide sequence having at least 65% sequence identity, at least 70% sequence identity, at least 75% sequence identity at least 80% identity, or at least 85% 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity with a sequence exemplified or described herein. Hybridization and hybridization conditions as provided herein can also be used to define such plants and polynucleotide sequences of the subject disclosure. A sequence comprising the flanking sequences plus the full insert sequence can be confirmed with reference to the deposited seed.
In some embodiments, two different transgenic plants can also be crossed to produce offspring that contain two independently segregating added, exogenous genes. Selfing of appropriate progeny can produce plants that are homozygous for both added, exogenous genes. Back-crossing to a parental plant and out-crossing with a non-transgenic plant are also contemplated, as is vegetative propagation.
A “probe” is an isolated nucleic acid to which is attached a conventional, synthetic detectable label or reporter molecule, e.g., a radioactive isotope, ligand, chemiluminescent agent, or enzyme. Such a probe is complementary to a strand of a target nucleic acid, for example, to a strand of isolated DNA from corn event DP-910521-2 whether from a corn plant or from a sample that includes DNA from the event. Probes may include not only deoxyribonucleic or ribonucleic acids but also polyamides and other modified nucleotides that bind specifically to a target DNA sequence and can be used to detect the presence of that target DNA sequence.
“Primers” are isolated nucleic acids that anneal to a complementary target DNA strand by nucleic acid hybridization to form a hybrid between the primer and the target DNA strand, then extended along the target DNA strand by a polymerase, e.g., a DNA polymerase. Primer pairs refer to their use for amplification of a target nucleic acid sequence, e.g., by PCR or other conventional nucleic-acid amplification methods. “PCR” or “polymerase chain reaction” is a technique used for the amplification of specific DNA segments (see, U.S. Pat. Nos. 4,683,195 and 4,800,159; herein incorporated by reference).
Probes and primers are of sufficient nucleotide length to bind to the target DNA sequence specifically in the hybridization conditions or reaction conditions determined by the operator. This length may be of any length that is of sufficient length to be useful in a detection method of choice. Generally, 11 nucleotides or more in length, 18 nucleotides or more, and 22 nucleotides or more, are used. Such probes and primers hybridize specifically to a target sequence under high stringency hybridization conditions. Probes and primers according to embodiments may have complete DNA sequence similarity of contiguous nucleotides with the target sequence, although probes differing from the target DNA sequence and that retain the ability to hybridize to target DNA sequences may be designed by conventional methods. Probes can be used as primers, but are generally designed to bind to the target DNA or RNA and are not used in an amplification process.
Specific primers may be used to amplify an integration fragment to produce an amplicon that can be used as a “specific probe” for identifying event DP-910521-2 in biological samples. When the probe is hybridized with the nucleic acids of a biological sample under conditions which allow for the binding of the probe to the sample, this binding can be detected and thus allow for an indication of the presence of event DP-910521-2 in the biological sample. In an embodiment of the disclosure, the specific probe is a sequence which, under appropriate conditions, hybridizes specifically to a region within the 5′ or 3′ flanking region of the event and also comprises a part of the foreign DNA contiguous therewith. The specific probe may comprise a sequence of at least 80%, from 80 and 85%, from 85 and 90%, from 90 and 95%, and from 95 and 100% identical (or complementary) to a specific region of the event.
Methods for preparing and using probes and primers are described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1-3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. 1989 (hereinafter, “Sambrook et al., 1989”); Ausubel et al. eds., Current Protocols in Molecular Biology, Greene Publishing and Wiley-Interscience, New York, 1995 (with periodic updates) (hereinafter, “Ausubel et al., 1995”); and Innis et al., PCR Protocols: A Guide to Methods and Applications, Academic Press: San Diego, 1990. PCR primer pairs can be derived from a known sequence, for example, by using computer programs intended for that purpose such as the PCR primer analysis tool in Vector NTI version 6 (Informax Inc., Bethesda Md.); PrimerSelect (DNASTAR Inc., Madison, Wis.); and Primer (Version 0.5©, 1991, Whitehead Institute for Biomedical Research, Cambridge, Mass.). Additionally, the sequence can be visually scanned and primers manually identified using guidelines known to one of skill in the art.
A “kit” as used herein refers to a set of reagents, and optionally instructions, for the purpose of performing method embodiments of the disclosure, more particularly, the identification of event DP-910521-2 in biological samples. A kit may be used, and its components can be specifically adjusted, for purposes of quality control (e.g., purity of seed lots), detection of event DP-910521-2 in plant material, or material comprising or derived from plant material, such as but not limited to food or feed products. “Plant material” as used herein refers to material which is obtained or derived from a plant.
Primers and probes based on the flanking DNA and insert sequences disclosed herein can be used to confirm (and, if necessary, to correct) the disclosed sequences by conventional methods, e.g., by re-cloning and sequencing such sequences. The nucleic acid probes and primers hybridize under stringent conditions to a target DNA sequence. Any conventional nucleic acid hybridization or amplification method may be used to identify the presence of DNA from a transgenic event in a sample.
A nucleic acid molecule is said to be the “complement” of another nucleic acid molecule if they exhibit complete complementarity or minimal complementarity. As used herein, molecules are said to exhibit “complete complementarity” when every nucleotide of one of the molecules is complementary to a nucleotide of the other. Two molecules are said to be “minimally complementary” if they can hybridize to one another with sufficient stability to permit them to remain annealed to one another under at least conventional “low-stringency” conditions. Similarly, the molecules are said to be “complementary” if they can hybridize to one another with sufficient stability to permit them to remain annealed to one another under conventional “high-stringency” conditions. Conventional stringency conditions are described by Sambrook et al., 1989, and by Haymes et al., In: Nucleic Acid Hybridization, a Practical Approach, IRL Press, Washington, D.C. (1985), departures from complete complementarity are therefore permissible, as long as such departures do not completely preclude the capacity of the molecules to form a double-stranded structure. In order for a nucleic acid molecule to serve as a primer or probe it need only be sufficiently complementary in sequence to be able to form a stable double-stranded structure under the particular solvent and salt concentrations employed.
In hybridization reactions, specificity is typically the function of post-hybridization washes, the critical factors being the ionic strength and temperature of the final wash solution. The thermal melting point (Tm) is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly matched probe. For DNA-DNA hybrids, the Tm can be approximated from the equation of Meinkoth and Wahl (1984) Anal. Biochem. 138:267-284: Tm=81.5° C.+16.6 (log M)+0.41 (% GC)−0.61 (% form)−500/L; where M is the molarity of monovalent cations, % GC is the percentage of guanosine and cytosine nucleotides in the DNA, % form is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs. Tm is reduced by about 1° C. for each 1% of mismatching; thus, Tm, hybridization, and/or wash conditions can be adjusted to hybridize to sequences of the desired identity. For example, if sequences with >90% identity are sought, the Tm can be decreased 10° C. Generally, stringent conditions are selected to be about 5° C. lower than the Tm for the specific sequence and its complement at a defined ionic strength and pH. However, in some embodiments, other stringency conditions can be applied, including severely stringent conditions can utilize a hybridization and/or wash at 1, 2, 3, or 4° C. lower than the Tm; moderately stringent conditions can utilize a hybridization and/or wash at 6, 7, 8, 9, or 10° C. lower than the Tm; low stringency conditions can utilize a hybridization and/or wash at 11, 12, 13, 14, 15, or 20° C. lower than the Tm.
Using the equation, hybridization and wash compositions, and desired Tm, those of ordinary skill will understand that variations in the stringency of hybridization and/or wash solutions are inherently described. If the desired degree of mismatching results in a Tm of less than 45° C. (aqueous solution) or 32° C. (formamide solution), a user may choose to increase the SSC concentration so that a higher temperature can be used. An extensive guide to the hybridization of nucleic acids is found in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology Hybridization with Nucleic Acid Probes, Part I, Chapter 2 (Elsevier, New York); and Ausubel et al., eds. (1995) and Sambrook et al. (1989).
In some embodiments, a complementary sequence has the same length as the nucleic acid molecule to which it hybridizes. In some embodiments, the complementary sequence is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides longer or shorter than the nucleic acid molecule to which it hybridizes. In some embodiments, the complementary sequence is 1%, 2%, 3%, 4%, or 5% longer or shorter than the nucleic acid molecule to which it hybridizes. In some embodiments, a complementary sequence is complementary on a nucleotide-for-nucleotide basis, meaning that there are no mismatched nucleotides (each A pairs with a T and each G pairs with a C). In some embodiments, a complementary sequence comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or less mismatches. In some embodiments, the complementary sequence comprises 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% or less mismatches.
“Percent (%) sequence identity” with respect to a reference sequence (subject) is determined as the percentage of amino acid residues or nucleotides in a candidate sequence (query) that are identical with the respective amino acid residues or nucleotides in the reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any amino acid conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (e.g., percent identity of query sequence=number of identical positions between query and subject sequences/total number of positions of query sequence×100).
Regarding the amplification of a target nucleic acid sequence (e.g., by PCR) using a particular amplification primer pair, stringent conditions permit the primer pair to hybridize only to the target nucleic-acid sequence to which a primer having the corresponding wild-type sequence (or its complement) would bind and optionally to produce a unique amplification product, the amplicon, in a DNA thermal amplification reaction.
As used herein, “amplified DNA” or “amplicon” refers to the product of nucleic acid amplification of a target nucleic acid sequence that is part of a nucleic acid template. For example, to determine whether a corn plant resulting from a sexual cross contains transgenic event genomic DNA from the corn plant disclosed herein, DNA extracted from a tissue sample of a corn plant may be subjected to a nucleic acid amplification method using a DNA primer pair that includes a first primer derived from flanking sequence adjacent to the insertion site of inserted heterologous DNA, and a second primer derived from the inserted heterologous DNA to produce an amplicon that is diagnostic for the presence of the event DNA. Alternatively, the second primer may be derived from the flanking sequence. The amplicon is of a length and has a sequence that is also diagnostic for the event. The amplicon may range in length from the combined length of the primer pairs plus one nucleotide base pair to any length of amplicon producible by a DNA amplification protocol. Alternatively, primer pairs can be derived from flanking sequence on both sides of the inserted DNA so as to produce an amplicon that includes the entire insert nucleotide sequence of the PHP79620 expression construct as well as a portion of the sequence flanking the transgenic insert. A member of a primer pair derived from the flanking sequence may be located a distance from the inserted DNA sequence, this distance can range from one nucleotide base pair up to the limits of the amplification reaction. The use of the term “amplicon” specifically excludes primer dimers that may be formed in the DNA thermal amplification reaction.
Nucleic acid amplification can be accomplished by any of the various nucleic acid amplification methods known in the art, including PCR. A variety of amplification methods are known in the art and are described, inter alia, in U.S. Pat. Nos. 4,683,195 and 4,683,202 and in Innis et al., (1990) supra. PCR amplification methods have been developed to amplify up to 22 Kb of genomic DNA and up to 42 Kb of bacteriophage DNA (Cheng et al., Proc. Natl. Acad. Sci. USA 91:5695-5699, 1994). These methods as well as other methods known in the art of DNA amplification may be used in the practice of the embodiments of the present disclosure. It is understood that a number of parameters in a specific PCR protocol may need to be adjusted to specific laboratory conditions and may be slightly modified and yet allow for the collection of similar results. These adjustments will be apparent to a person skilled in the art.
The amplicon produced by these methods may be detected by a plurality of techniques, including, but not limited to, Genetic Bit Analysis (Nikiforov, et al. Nucleic Acid Res. 22:4167-4175, 1994) where a DNA oligonucleotide is designed which overlaps both the adjacent flanking DNA sequence and the inserted DNA sequence. The oligonucleotide is immobilized in wells of a microwell plate. Following PCR of the region of interest (for example, using one primer in the inserted sequence and one in the adjacent flanking sequence) a single-stranded PCR product can be hybridized to the immobilized oligonucleotide and serve as a template for a single base extension reaction using a DNA polymerase and labeled ddNTPs specific for the expected next base. Readout may be fluorescent or ELISA-based. A signal indicates presence of the insert/flanking sequence due to successful amplification, hybridization, and single base extension.
Another detection method is the pyrosequencing technique as described by Winge (2000) Innov. Pharma. Tech. 00:18-24. In this method an oligonucleotide is designed that overlaps the adjacent DNA and insert DNA junction. The oligonucleotide is hybridized to a single-stranded PCR product from the region of interest (for example, one primer in the inserted sequence and one in the flanking sequence) and incubated in the presence of a DNA polymerase, ATP, sulfurylase, luciferase, apyrase, adenosine 5′ phosphosulfate and luciferin. dNTPs are added individually and the incorporation results in a light signal which is measured. A light signal indicates the presence of the transgene insert/flanking sequence due to successful amplification, hybridization, and single or multi-base extension.
Fluorescence polarization as described by Chen et al., (1999) Genome Res. 9:492-498 is also a method that can be used to detect an amplicon. Using this method an oligonucleotide is designed which overlaps the flanking and inserted DNA junction. The oligonucleotide is hybridized to a single-stranded PCR product from the region of interest (for example, one primer in the inserted DNA and one in the flanking DNA sequence) and incubated in the presence of a DNA polymerase and a fluorescent-labeled ddNTP. Single base extension results in incorporation of the ddNTP. Incorporation can be measured as a change in polarization using a fluorometer. A change in polarization indicates the presence of the transgene insert/flanking sequence due to successful amplification, hybridization, and single base extension.
Quantitative PCR (qPCR) is described as a method of detecting and quantifying the presence of a DNA sequence and is fully understood in the instructions provided by commercially available manufacturers. Briefly, in one such qPCR method, a FRET oligonucleotide probe is designed which overlaps the flanking and insert DNA junction. The FRET probe and PCR primers (one primer in the insert DNA sequence and one in the flanking genomic sequence) are cycled in the presence of a thermostable polymerase and dNTPs. Hybridization of the FRET probe results in cleavage and release of the fluorescent moiety away from the quenching moiety on the FRET probe. A fluorescent signal indicates the presence of the flanking/transgene insert sequence due to successful amplification and hybridization.
Molecular beacons have been described for use in sequence detection as described in Tyangi et al. (1996) Nature Biotech. 14:303-308. Briefly, a FRET oligonucleotide probe is designed that overlaps the flanking and insert DNA junction. The unique structure of the FRET probe results in it containing secondary structure that keeps the fluorescent and quenching moieties in close proximity. The FRET probe and PCR primers (for example, one primer in the insert DNA sequence and one in the flanking sequence) are cycled in the presence of a thermostable polymerase and dNTPs. Following successful PCR amplification, hybridization of the FRET probe to the target sequence results in the removal of the probe secondary structure and spatial separation of the fluorescent and quenching moieties. A fluorescent signal results. A fluorescent signal indicates the presence of the flanking/transgene insert sequence due to successful amplification and hybridization.
A hybridization reaction using a probe specific to a sequence found within the amplicon is yet another method used to detect the amplicon produced by a PCR reaction.
Insect pests include insects selected from the orders Coleoptera, Diptera, Hymenoptera, Lepidoptera, Mallophaga, Homoptera, Hemiptera, Orthoptera, Thysanoptera, Dermaptera, Isoptera, Anoplura, Siphonaptera, Trichoptera, etc., particularly Lepidoptera.
Of interest are larvae and adults of the order Lepidoptera including, but not limited to, armyworms, cutworms, loopers and heliothines in the family Noctuidae Spodoptera frugiperda JE Smith (fall armyworm); S. exigua Hübner (beet armyworm); S. litura Fabricius (tobacco cutworm, cluster caterpillar); Mamestra configurata Walker (bertha armyworm); M. brassicae Linnaeus (cabbage moth); Agrotis ipsilon Hufnagel (black cutworm); A. orthogonia Morrison (western cutworm); A. subterranea Fabricius (granulate cutworm); Alabama argillacea Hübner (cotton leaf worm); Trichoplusia ni Hübner (cabbage looper); Pseudoplusia includens Walker (soybean looper); Anticarsia gemmatalis Hübner (velvetbean caterpillar); Hypena scabra Fabricius (green cloverworm); Heliothis virescens Fabricius (tobacco budworm); Pseudaletia unipuncta Haworth (armyworm); Athetis mindara Barnes and Mcdunnough (rough skinned cutworm); Euxoa messoria Harris (darksided cutworm); Earias insulana Boisduval (spiny bollworm); E. vittella Fabricius (spotted bollworm); Helicoverpa armigera Hübner (American bollworm); H. zea Boddie (corn earworm or cotton bollworm); Melanchra picta Harris (zebra caterpillar); Egira (Xylomyges) curialis Grote (citrus cutworm); borers, casebearers, webworms, coneworms, and skeletonizers from the family Pyralidae Ostrinia nubilalis Hübner (European corn borer); Amyelois transitella Walker (naval orangeworm); Anagasta kuehniella Zeller (Mediterranean flour moth); Cadra cautella Walker (almond moth); Chilo suppressalis Walker (rice stem borer); C. partellus, (sorghum borer); Corcyra cephalonica Stainton (rice moth); Crambus caliginosellus Clemens (corn root webworm); C. teterrellus Zincken (bluegrass webworm); Cnaphalocrocis medinalis Guenée (rice leaf roller); Desmia funeralis Hübner (grape leaffolder); Diaphania hyalinata Linnaeus (melon worm); D. nitidalis Stoll (pickleworm); Diatraea grandiosella Dyar (southwestern corn borer), D. saccharalis Fabricius (surgarcane borer); Eoreuma loftini Dyar (Mexican rice borer); Ephestia elutella Hübner (tobacco (cacao) moth); Galleria mellonella Linnaeus (greater wax moth); Herpetogramma licarsisalis Walker (sod webworm); Homoeosoma electellum Hulst (sunflower moth); Elasmopalpus lignosellus Zeller (lesser cornstalk borer); Achroia grisella Fabricius (lesser wax moth); Loxostege sticticalis Linnaeus (beet webworm); Orthaga thyrisalis Walker (tea tree web moth); Maruca testulalis Geyer (bean pod borer); Plodia interpunctella Hübner (Indian meal moth); Scirpophaga incertulas Walker (yellow stem borer); Udea rubigalis Guenée (celery leaftier); and leafrollers, budworms, seed worms and fruit worms in the family Tortricidae Acleris gloverana Walsingham (Western blackheaded budworm); A. variana Fernald (Eastern blackheaded budworm); Archips argyrospila Walker (fruit tree leaf roller); A. rosana Linnaeus (European leaf roller); and other Archips species, Adoxophyes orana Fischer von Rösslerstamm (summer fruit tortrix moth); Cochylis hospes Walsingham (banded sunflower moth); Cydia latiferreana Walsingham (filbertworm); C. pomonella Linnaeus (coding moth); Platynota flavedana Clemens (variegated leafroller); P. stultana Walsingham (omnivorous leafroller); Lobesia botrana Denis & Schiffermüller (European grape vine moth); Spilonota ocellana Denis & Schiffermüller (eyespotted bud moth); Endopiza viteana Clemens (grape berry moth); Eupoecilia ambiguella Hübner (vine moth); Bonagota salubricola Meyrick (Brazilian apple leafroller); Grapholita molesta Busck (oriental fruit moth); Suleima helianthana Riley (sunflower bud moth); Argyrotaenia spp.; and Choristoneura spp.
Selected other agronomic pests in the order Lepidoptera include, but are not limited to, Alsophila pometaria Harris (fall cankerworm); Anarsia lineatella Zeller (peach twig borer); Anisota senatoria J. E. Smith (orange striped oakworm); Antheraea pernyi Guérin-Méneville (Chinese Oak Tussah Moth); Bombyx mori Linnaeus (Silkworm); Bucculatrix thurberiella Busck (cotton leaf perforator); Colias eurytheme Boisduval (alfalfa caterpillar); Datana integerrima Grote & Robinson (walnut caterpillar); Dendrolimus sibiricus Tschetwerikov (Siberian silk moth), Ennomos subsignaria Hübner (elm spanworm); Erannis tiliaria Harris (linden looper); Euproctis chrysorrhoea Linnaeus (browntail moth); Harrisina americana Guérin-Méneville (grapeleaf skeletonizer); Hemileuca oliviae Cockrell (range caterpillar); Hyphantria cunea Drury (fall webworm); Keiferia lycopersicella Walsingham (tomato pinworm); Lambdina fiscellaria fiscellaria Hulst (Eastern hemlock looper); L. fiscellaria lugubrosa Hulst (Western hemlock looper); Leucoma salicis Linnaeus (satin moth); Lymantria dispar Linnaeus (gypsy moth); Manduca quinquemaculata Haworth (five spotted hawk moth, tomato hornworm); M. sexta Haworth (tomato hornworm, tobacco hornworm); Operophtera brumata Linnaeus (winter moth); Paleacrita vernata Peck (spring cankerworm); Papilio cresphontes Cramer (giant swallowtail orange dog); Phryganidia californica Packard (California oakworm); Phyllocnistis citrella Stainton (citrus leafminer); Phyllonorycter blancardella Fabricius (spotted tentiform leafminer); Pieris brassicae Linnaeus (large white butterfly); P. rapae Linnaeus (small white butterfly); P. napi Linnaeus (green veined white butterfly); Platyptilia carduidactyla Riley (artichoke plume moth); Plutella xylostella Linnaeus (diamondback moth); Pectinophora gossypiella Saunders (pink bollworm); Pontia protodice Boisduval and Leconte (Southern cabbageworm); Sabulodes aegrotata Guenée (omnivorous looper); Schizura concinna J. E. Smith (red humped caterpillar); Sitotroga cerealella Olivier (Angoumois grain moth); Thaumetopoea pityocampa Schiffermuller (pine processionary caterpillar); Tineola bisselliella Hummel (webbing clothesmoth); Tuta absoluta Meyrick (tomato leafminer); Yponomeuta padella Linnaeus (ermine moth); Heliothis subflexa Guenée; Malacosoma spp. and Orgyia spp.
In some embodiments the DP-910521-2 maize event may further comprise a stack of additional traits. Plants comprising stacks of polynucleotide sequences can be obtained by either or both of traditional breeding methods or through genetic engineering methods. These methods include, but are not limited to, breeding individual lines each comprising a polynucleotide of interest, transforming a transgenic plant comprising a gene disclosed herein with a subsequent gene and co-transformation of genes into a single plant cell. As used herein, the term “stacked” includes having the multiple traits present in the same plant (i.e., both traits are incorporated into the nuclear genome, one trait is incorporated into the nuclear genome and one trait is incorporated into the genome of a plastid or both traits are incorporated into the genome of a plastid).
In some embodiments the DP-910521-2 maize event disclosed herein, alone or stacked with one or more additional insect resistance traits can be stacked with one or more additional input traits (e.g., herbicide resistance, fungal resistance, virus resistance, stress tolerance, disease resistance, male sterility, stalk strength, and the like) or output traits (e.g., increased yield, modified starches, improved oil profile, balanced amino acids, high lysine or methionine, increased digestibility, improved fiber quality, drought resistance, and the like). Thus, the embodiments can be used to provide a complete agronomic package of improved crop quality with the ability to flexibly and cost effectively control any number of agronomic pests.
In a further embodiment, the DP-910521-2 maize event may be stacked with one or more additional insecticidal toxins, including, but not limited to, a Cry3B toxin disclosed in U.S. Pat. Nos. 8,101,826, 6,551,962, 6,586,365, 6,593,273, and PCT Publication WO 2000/011185; a mCry3B toxin disclosed in U.S. Pat. Nos. 8,269,069, and 8,513,492; a mCry3A toxin disclosed in U.S. Pat. Nos. 8,269,069, 7,276,583 and 8,759,620; or a Cry34/35 toxin disclosed in U.S. Pat. Nos. 7,309,785, 7,524,810, 7,985,893, 7,939,651 and 6,548,291. In a further embodiment, the DP-910521-2 maize event may be stacked with one or more additional transgenic events containing these Bt insecticidal toxins and other Coleopteran active Bt insecticidal traits for example, event MON863 disclosed in U.S. Pat. No. 7,705,216; event MIR604 disclosed in U.S. Pat. No. 8,884,102; event 5307 disclosed in U.S. Pat. No. 9,133,474; event DAS-59122 disclosed in U.S. Pat. No. 7,875,429; event DP-4114 disclosed in U.S. Pat. No. 8,575,434; event MON 87411 disclosed in U.S. Pat. No. 9,441,240; and event MON88017 disclosed in U.S. Pat. No. 8,686,230 all of which are incorporated herein by reference. In some embodiments, the DP-910521-2 maize event may be stacked with MON-87429-9 (MON87429 Event); MON87403; MON95379; MON87427; MON87419; MON-00603-6 (NK603); MON-87460-4; LY038; DAS-06275-8; BT176; BT11; MIR162; GA21; MZDTO9Y; SYN-05307-1; DP-915635-2; DP-23211; and DAS-40278-9.
In a further embodiment, the DP-910521-2 maize event may be stacked with one or more additional of the following provided herbicidal tolerance traits. The glyphosate herbicide contains a mode of action by inhibiting the EPSPS enzyme (5-enolpyruvylshikimate-3-phosphate synthase). This enzyme is involved in the biosynthesis of aromatic amino acids that are essential for growth and development of plants. Various enzymatic mechanisms are known in the art that can be utilized to inhibit this enzyme. The genes that encode such enzymes can be operably linked to the gene regulatory elements of the subject disclosure. In an embodiment, selectable marker genes include, but are not limited to genes encoding glyphosate resistance genes include: mutant EPSPS genes such as 2mEPSPS genes, cp4 EPSPS genes, mEPSPS genes, dgt-28 genes; aroA genes; and glyphosate degradation genes such as glyphosate acetyl transferase genes (gat) and glyphosate oxidase genes (gox). These traits are currently marketed as Gly-Tol′, Optimum® GAT®, Agrisure® GT and Roundup Ready®. Resistance genes for glufosinate and/or bialaphos compounds include dsm-2, bar and pat genes. The bar and pat traits are currently marketed as LibertyLink®. Also included are tolerance genes that provide resistance to 2,4-D such as aad-1 genes (it should be noted that aad-1 genes have further activity on arloxyphenoxypropionate herbicides) and aad-12 genes (it should be noted that aad-12 genes have further activity on pyidyloxyacetate synthetic auxins). These traits are marketed as Enlist® crop protection technology. Resistance genes for ALS inhibitors (sulfonylureas, imidazolinones, triazolopyrimidines, pyrimidinylthiobenzoates, and sulfonylamino-carbonyl-triazolinones) are known in the art. These resistance genes most commonly result from point mutations to the ALS encoding gene sequence. Other ALS inhibitor resistance genes include hra genes, the csr1-2 genes, Sr-HrA genes, and surB genes. Some of the traits are marketed under the tradename Clearfield®. Herbicides that inhibit HPPD include the pyrazolones such as pyrazoxyfen, benzofenap, and topramezone; triketones such as mesotrione, sulcotrione, tembotrione, benzobicyclon; and diketonitriles such as isoxaflutole. These exemplary HPPD herbicides can be tolerated by known traits. Examples of HPPD inhibitors include hppdPF_W336 genes (for resistance to isoxaflutole) and avhppd-03 genes (for resistance to meostrione). An example of oxynil herbicide tolerant traits include the bxn gene, which has been showed to impart resistance to the herbicide/antibiotic bromoxynil. Resistance genes for dicamba include the dicamba monooxygenase gene (dmo) as disclosed in International PCT Publication No. WO 2008/105890. Resistance genes for PPO or PROTOX inhibitor type herbicides (e.g., acifluorfen, butafenacil, flupropazil, pentoxazone, carfentrazone, fluazolate, pyraflufen, aclonifen, azafenidin, flumioxazin, flumiclorac, bifenox, oxyfluorfen, lactofen, fomesafen, fluoroglycofen, and sulfentrazone) are known in the art. Exemplary genes conferring resistance to PPO include over expression of a wild-type Arabidopsis thaliana PPO enzyme (Lermontova I and Grimm B, (2000) Overexpression of plastidic protoporphyrinogen IX oxidase leads to resistance to the diphenyl-ether herbicide acifluorfen. Plant Physiol 122:75-83), the B. subtilis PPO gene (Li, X. and Nicholl D. 2005. Development of PPO inhibitor-resistant cultures and crops. Pest Manag. Sci. 61:277-285 and Choi K W, Han O, Lee H J, Yun Y C, Moon Y H, Kim M K, Kuk Y I, Han S U and Guh J O, (1998) Generation of resistance to the diphenyl ether herbicide, oxyfluorfen, via expression of the Bacillus subtilis protoporphyrinogen oxidase gene in transgenic tobacco plants. Biosci Biotechnol Biochem 62:558-560.) Resistance genes for pyridinoxy or phenoxy proprionic acids and cyclohexones include the ACCase inhibitor-encoding genes (e.g., Acc1-S1, Acc1-S2 and Acc1-S3). Exemplary genes conferring resistance to cyclohexanediones and/or aryloxyphenoxypropanoic acid include haloxyfop, diclofop, fenoxyprop, fluazifop, and quizalofop. Finally, herbicides can inhibit photosynthesis, including triazine or benzonitrile are provided tolerance by psbA genes (tolerance to triazine), 1s+ genes (tolerance to triazine), and nitrilase genes (tolerance to benzonitrile). The above list of herbicide tolerance genes is not meant to be limiting. Any herbicide tolerance genes are encompassed by the present disclosure.
In some embodiments, the disclosed compositions can be introduced into the genome of a plant using genome editing technologies, or previously introduced polynucleotides in the genome of a plant may be edited using genome editing technologies. For example, the disclosed polynucleotides can be introduced into a desired location in the genome of a plant through the use of genome editing systems such as TALENs, meganucleases, zinc finger nucleases, CRISPR-Cas, and the like. For example, the disclosed polynucleotides can be introduced into a desired location in a genome using a CRISPR-Cas system, for the purpose of site-specific insertion. The desired location in a plant genome can be any desired target site for insertion, such as a genomic region amenable for breeding or may be a target site located in a genomic window with existing trait(s) of interest. Existing trait(s) of interest could be either endogenous traits or previously introduced traits.
In some embodiments, where the disclosed polynucleotide has previously been introduced into a genome, genome editing or genome engineering technologies may be used to alter or modify the introduced polynucleotide sequence, including flanking chromosomal genomic sequences. Site specific modifications that can be introduced into the disclosed compositions include those produced using any method for introducing site specific modification, including, but not limited to, through the use of sequence repair oligonucleotides, alone, or through the use of site-directed genome modification tools such as TALENs, meganucleases, zinc finger nucleases, CRISPR-Cas, and the like, with or without donor DNA. Site-specific modifications to the disclosed polynucleotides (including genomic flanking and junction sequences) may include, but are not limited to, changes to codon usage, changes to regulatory elements such as promoters, introns, terminators, enhancers, 5′ or 3′ untranslated regions (UTRs), or other noncoding sequences, and other regions of the polynucleotide, where the modifications do not adversely affect the phenotypic characteristics of the resulting maize plant. DP-910521-2 event plants containing modified polynucleotide sequences are also contemplated herein.
Cas polypeptides suitable for introducing site-specific modifications include, for example, Cas9, Cas12f (Cas-alpha, Cas14), Cas121 (Cas-beta), Cas12a (Cpf1), Cas12b (a C2c1 protein), Cas13 (a C2c2 protein), Cas12c (a C2c3 protein), Cas12d, Cas12e, Cas12g, Cas12h, Cas12i, Cas12j, Cas12k, Cas3, Cas3-HD, Cas 5, Cas6, Cas7, Cas8, Cas10, or combinations or complexes of these. In some aspects, transposon-associated TnpB, a programmable RNA-guided DNA endonuclease can be used.
In some aspects, a genome editing system comprises a Cas-alpha (e.g., Cas12f) endonuclease and one or more guide polynucleotides that introduce one or more site-specific modifications in a target polynucleotide sequence, resulting in a modified target sequence. As used herein, “altered target site”, “altered target sequence”, “modified target site,” and “modified target sequence” are used interchangeably and refer to a target sequence as disclosed herein that comprises at least one alteration or modification when compared to a non-altered target sequence. Such alterations or modifications include, for example: (i) replacement or substitution of at least one nucleotide, (ii) deletion of at least one nucleotide, (iii) insertion of at least one nucleotide, or (iv) any combination of (i)-(iii).
In some aspects, a genome editing system comprises a Cas-alpha endonuclease, one or more guide polynucleotides, and optionally a donor DNA. Some exemplary Cas-alpha endonucleases are described, for example, in WO2020123887.
In some aspects, a genome editing system comprises a Cas polypeptide, one or more guide polynucleotides, and optionally donor DNA, and editing a target polynucleotide sequence comprises nonhomologous end-joining (NHEJ) or homologous recombination (HR) following a Cas polypeptide-mediated double-strand break. Once a double-strand break is induced in the DNA, the cell's DNA repair mechanism is activated to repair the break. The most common repair mechanism to bring the broken ends together is the nonhomologous end-joining pathway (Bleuyard et al., (2006) DNA Repair 5:1-12). As a result, deletions, insertions, or other rearrangements are possible (Siebert and Puchta, (2002) Plant Cell 14:1121-31; Pacher et al., (2007) Genetics 175:21-9). Alternatively, the double-strand break can be repaired by homologous recombination between homologous DNA sequences. Once the sequence around the double-strand break is altered, for example, by exonuclease activities involved in the maturation of double-strand breaks, gene conversion pathways can restore the original structure if a homologous sequence is available, such as a homologous chromosome in non-dividing somatic cells, or a sister chromatid after DNA replication (Molinier et al., (2004) Plant Cell 16:342-52). Ectopic and/or epigenic DNA sequences may also serve as a DNA repair template for homologous recombination (Puchta, (1999) Genetics 152:1173-81).
In some aspects, the genome editing system comprises a Cas polypeptide, one or more guide polynucleotides, and a donor DNA. As used herein, “donor DNA” is a DNA construct that comprises a polynucleotide of interest to be inserted into the genomic target site of a Cas polypeptide. Once a double-strand break is introduced in the target site by the endonuclease, the first and second regions of homology of the donor DNA can undergo homologous recombination with their corresponding genomic regions of homology resulting in exchange of DNA between the donor DNA and the target genomic region. As such, the provided methods result in the integration of the polynucleotide of interest of the donor DNA into the double-strand break in the target site in the plant genome, thereby altering the original target site and producing an altered genomic target site.
In some aspects, a genome editing system comprises a base editing agent and a plurality of guide polynucleotides and editing a target polynucleotide sequence comprises introducing a plurality of nucleobase edits in the target polynucleotide sequence resulting in a variant nucleotide sequence. Other aspects include modified DP-910521-2 event plants produced using a genome editing system.
One or more nucleobases of a target genomic sequence can be chemically altered, in some cases to change the base from one type to another, for example from a Cytosine to a Thymine, or an Adenine to a Guanine. In some aspects, a plurality of bases, for example 2 or more, 5 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, 80 or more 90 or more, 100 or more, or even greater than 100, 200 or more, up to thousands of bases may be modified or altered, to produce a plant with a plurality of modified bases.
Any base editing complex, such as a base editing agent associated with an RNA-guided polypeptide (such as e.g., dCas associated with a deaminase), may be used to target and bind to a desired locus in the genome of an organism and chemically modify one or more nucleotides of a target genomic sequence.
Site-specific nucleotide base conversions can be achieved to engineer one or more nucleotide changes to create one or more edits into the genome. These include for example, a site-specific base edit mediated by a C·G to T·A or an A·T to G·C base editing deaminase enzymes (Gaudelli et al., Programmable base editing of A·T to G·C in genomic DNA without DNA cleavage.” Nature (2017); Nishida et al. “Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems.” Science 353 (6305) (2016); Komor et al. “Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage.” Nature 533 (7603) (2016):420-4. A catalytically “dead” or inactive Cas (dCas) polypeptide, for example an inactive Cas9 (dCas9), Cas12f (dCas12f), or another Cas polypeptide disclosed herein, fused to a cytidine deaminase or an adenine deaminase protein becomes a specific base editor that can alter DNA bases without inducing a DNA break. Base editors convert C->T (or G->A on the opposite strand) or an adenine base editor that would convert adenine to inosine, resulting in an A->G change within an editing window specified by the guide polynucleotides. Any molecule that effects a change in a nucleobase is a “base editing agent”. The dCas forms a functional complex with a guide polynucleotide that shares homology with a genomic sequence at the target site, and is further complexed with the deaminase molecule. The guided Cas polypeptide recognizes and binds to a target sequence, opening the double-strand to expose individual bases. In the case of a cytidine deaminase, the deaminase deaminates the cytosine base and creates a uracil. Uracil glycosylase inhibitor (UGI) is provided to prevent the conversion of U back to C. DNA replication or repair mechanisms then convert the Uracil to a thymine (U to T), and subsequent repair of the opposing base (formerly Gin the original G-C pair) to an Adenine, creating a T-A pair.
One or more nucleotides of the inserted event and/or the flanking genomic DNA can be modified using a prime editing technology. See e.g., Anzalone et al., Search-and-replace genome editing without double-strand breaks or donor DNA. Nature 576, 149-157 (2019). A prime editing complex includes a prime editing protein that contains an RNA-guided DNA-nicking domain, such as a Cas nickase (e.g., Cas9 nickase, Casd12f1 nickase), fused to a reverse transcriptase domain and complexed with a pegRNA. The PE-pegRNA complex is able to introduce targeted DNA edits at desired locations in the genome, by binding the target DNA and nicking the PAM-containing strand. The resulting 3′ end hybridizes to a chosen primer binding site and then primes reverse transcription of new DNA sequence containing the desired edit using the reverse transcriptase template of the pegRNA. The resulting regulatory expression elements of the disclosed recombinant expression cassette(s) may be truncated or may include a polynucleotide sequence having at least 65% sequence identity, at least 70% sequence identity, at least 75% sequence identity at least 80% identity, or at least 85% 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity with a regulatory element sequence exemplified or described herein.
Other modifications may include modifications to other portions of the DNA of the DP-910521-2 event. In some embodiments, genome engineering technologies can be used to relocate one or more expression cassettes described herein to one or more different locations of the same chromosome, or different chromosomes of maize or a different crop. In such embodiment, polynucleotides comprising one or more of the junction sequences described herein (SEQ ID NOs: 26 and/or 29) may be retained with the expression cassette(s), either partially or fully, or may be removed. Furthermore, genomic flanking sequence(s) described herein may also be retained with the expression cassette(s), either partially or fully, or may be removed.
In another embodiment, genome engineering technologies may be used to co-locate one or more transgene(s) or expression cassette(s) in physical proximity to the 5′ or 3′ junction sequence(s) described herein. With regard to physical position on a chromosome, co-located transgenes and/or expression cassettes can be separated from the 5′ or 3′ junction sequence(s), e.g., by about 1 megabase (MB; 1 million nucleotides), about 500 kilobases (Kb; 1000 nucleotides), about 400 Kb, about 300 Kb, about 200 Kb, about 100 Kb, about 50 Kb, about 25 Kb, about 10 Kb, about 5 Kb, about 4 Kb, about 3 Kb, about 2 Kb, about 1 Kb, about 500 nucleotides, about 250 nucleotides, or less. With regard to genetic distance on a chromosome, co-located transgenes and/or expression cassettes can be separated from the 5′ or 3′ junction sequence(s), e.g., by about 10 cM, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.75, 0.5, 0.25, or 0.1 cM. For example, one or more of the expression cassette(s) obtained from one or more of the additional transgenic events described above may be co-located in physical proximity to the 5′ or 3′ junction sequence(s) described herein.
In another embodiment, polynucleotides comprising one of the junction sequences (SEQ ID NOs: 26 or 29) may be introduced at either or both ends of the inserted heterologous DNA. For example, a polynucleotide comprising the 5′ junction sequence may be deleted and replaced with a polynucleotide comprising the 3′ junction sequence, or vice versa.
In another embodiment, genome editing technologies may be used to modify the previously introduced polynucleotide(s) by inverting at least one of the polynucleotide(s) of the inserted DNA of the DP-910521-2 event. Such genome editing technologies can be used to modify the previously introduced polynucleotide through the insertion, deletion and/or substitution of one or more nucleotides within the introduced polynucleotide. Alternatively, double-stranded break technologies can be used to add additional nucleotide sequences to the introduced polynucleotide. Additional sequences that may be added include, but are not limited to, additional expression elements, such as enhancer and promoter sequences. Sequences that may be deleted include, but are not limited to, regulatory elements or portions thereof that when deleted do not adversely affect function. Modifications to modulate expression patterns (e.g., reducing the expression level of the insecticidal polypeptide in certain tissue) is also contemplated by site-directed modification to the introduced expression cassette.
In another embodiment, genome engineering technologies may be used to delete or modify all or part of one or more expression cassette(s) of the DP-910521-2 event as deposited with the ATCC on May 26, 2021, having accession number PTA-127078. In this embodiment, the resulting maize plant derived from the DP-910521-2 event as deposited with the ATCC on May 26, 2021, having accession number PTA-127078 may comprise a portion of the expression cassette(s) described herein, none of the expression cassette(s) described herein, or modifications of the expression cassette(s) described herein.
In another embodiment, targeted DSB technologies may be used to position additional insecticidally-active proteins in close proximity to the disclosed compositions disclosed herein within the genome of a plant, in order to generate molecular stacks of insecticidally-active proteins.
In another embodiment, the polynucleotide sequences disclosed herein are used in a method comprising designing guide polynucleotides, such as guide RNAs (gRNAs), that recognize said polynucleotide sequences, synthesizing or obtaining said guide polynucleotides, and introducing said guide polynucleotides as part of genome engineering compositions to modify the DNA of the DP-910521-2 event as deposited with the ATCC on May 26, 2021, having accession number PTA-127078. Such resulting modifications may include a polynucleotide sequence having at least 65% sequence identity, at least 70% sequence identity, at least 75% sequence identity at least 80% identity, or at least 85% 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity with a sequence exemplified or described herein.
Embodiments include modified DP-910521-2 event plants produced using genome engineering technologies described herein.
One embodiment includes a corn plant comprising the genotype of the corn event DP-910521-2, wherein said genotype comprises a nucleotide sequence as set forth in SEQ ID NO: 26 and SEQ ID NO: 29, or a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 26 and SEQ ID NO: 29.
Another embodiment includes the corn plant comprising the genotype of the corn event DP-910521-2 of any prior embodiment, wherein said genotype comprises the nucleotide sequence set forth in SEQ ID NO: 27 and SEQ ID NO: 30, or a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 27 and SEQ ID NO: 30.
Another embodiment includes the corn plant comprising the genotype of the corn event DP-910521-2 of any prior embodiment, wherein said genotype comprises the nucleotide sequence set forth in SEQ ID NO: 28 and SEQ ID NO: 31, or a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 28 and SEQ ID NO: 31.
One embodiment includes a DNA construct comprising an operably linked first and second expression cassette, wherein said first expression cassette comprises:
1) an MMV Enhancer
2) a LLDAV Promoter;
3) a zm-i6 Intron;
4) an cry1B.34; and
5) an os-ubi Terminator.
Another embodiment includes a plant comprising the DNA construct comprising at least one operably linked expression cassette of any prior embodiment.
A further embodiment includes a plant comprising the DNA construct comprising at least one operably linked expression cassette of any prior embodiment, wherein said plant is a corn plant.
One embodiment includes a plant comprising the sequence set forth in SEQ ID NO: 21, or a sequence having at least 95% sequence identity to SEQ ID NO: 21.
One embodiment includes a corn event DP-910521-2, wherein a representative sample of seed of said corn event has been deposited with American Type Culture Collection (ATCC) with Accession No. PTA-127078.
Other embodiments include plant parts of the corn event DP-910521-2 of any prior embodiments, wherein a representative sample of seed of said corn event has been deposited with American Type Culture Collection (ATCC) with Accession No. PTA-127078.
One embodiment includes seed comprising corn event DP-910521-2, wherein said seed comprises a DNA molecule chosen from SEQ ID NO: 26 and SEQ ID NO: 29, wherein a representative sample of the corn event DP-910521-2 seed of has been deposited with American Type Culture Collection (ATCC) with Accession No. PTA-127078.
Another embodiment includes a corn plant, or part thereof, grown from the seed comprising corn event DP-910521-2 of any prior embodiment, wherein said seed comprises a DNA molecule chosen from SEQ ID NO: 26 and SEQ ID NO: 29, wherein a representative sample of the corn event DP-910521-2 seed of has been deposited with American Type Culture Collection (ATCC) with Accession No. PTA-127078.
A further embodiment includes a transgenic seed produced from the corn plant comprising corn event DP-910521-2 of any prior embodiment, wherein a representative sample of the corn event DP-910521-2 seed of has been deposited with American Type Culture Collection (ATCC) with Accession No. PTA-127078.
Other embodiments include a transgenic corn plant, or part thereof, grown from the seed corn produced from the corn plant of corn event DP-910521-2 of any prior embodiment, wherein a representative sample of the corn event DP-910521-2 seed of has been deposited with American Type Culture Collection (ATCC) with Accession No. PTA-127078.
One embodiment includes an isolated nucleic acid molecule comprising a nucleotide sequence chosen from SEQ ID NOs: 21, and 26-31, and full length complements thereof.
One embodiment includes an amplicon comprising the nucleic acid sequence chosen from SEQ ID NOs: 22-25 and full length complements thereof.
One embodiment includes a biological sample or extract derived from corn event DP-910521-2 plant, tissue, or seed, wherein said sample or extract comprises a nucleotide sequence which is or is complementary to a sequence chosen from SEQ ID NO: 26 and SEQ ID NO: 29, wherein said nucleotide sequence is detectable in said sample or extract using a nucleic acid amplification or nucleic acid hybridization method, wherein a representative sample of said corn event DP-910521-2 seed has been deposited with American Type Culture Collection (ATCC) with Accession No. PTA-127078.
Another embodiment includes the biological sample or extract derived from corn event DP-910521-2 plant, tissue, or seed, wherein said sample or extract comprises a nucleotide sequence which is or is complementary to a sequence chosen from SEQ ID NO: 26 and SEQ ID NO: 29, wherein said nucleotide sequence is detectable in said sample or extract using a nucleic acid amplification or nucleic acid hybridization method, wherein a representative sample of said corn event DP-910521-2 seed has been deposited with American Type Culture Collection (ATCC) with Accession No. PTA-127078 of any prior embodiment, wherein said biological sample comprises plant, plant tissue, or seed of transgenic corn event DP-910521-2.
Another embodiment includes the biological sample or extract derived from corn event DP-910521-2 plant, tissue, or seed, wherein said biological sample or extract comprises a nucleotide sequence which is or is complementary to a sequence chosen from SEQ ID NO: 26 and SEQ ID NO: 29, wherein said nucleotide sequence is detectable in said sample or extract using a nucleic acid amplification or nucleic acid hybridization method, wherein a representative sample of said corn event DP-910521-2 seed has been deposited with American Type Culture Collection (ATCC) with Accession No. PTA-127078 of any prior embodiment, wherein said biological sample or extract is a DNA sample extracted from the transgenic corn plant event DP-910521-2, and wherein said DNA sample comprises one or more of the nucleotide sequences chosen from SEQ ID NOs: 21-31, and the complement thereof.
Another embodiment includes the biological sample or extract derived from corn event DP-910521-2 plant, tissue, or seed, wherein said biological sample or extract comprises a nucleotide sequence which is or is complementary to a sequence chosen from SEQ ID NO: 26 and SEQ ID NO: 29, wherein said nucleotide sequence is detectable in said sample or extract using a nucleic acid amplification or nucleic acid hybridization method, wherein a representative sample of said corn event DP-910521-2 seed has been deposited with American Type Culture Collection (ATCC) with Accession No. PTA-127078 of any prior embodiment, wherein said biological sample or extract is chosen from corn flour, corn meal, corn syrup, corn oil, corn starch, and cereals manufactured in whole or in part to contain corn by-products.
One embodiment includes a method of producing hybrid corn seeds comprising:
a) sexually crossing a first inbred corn line comprising a nucleotide chosen from SEQ ID NOs: 21-31 and a second inbred line having a different genotype;
b) growing progeny from said crossing; and
c) harvesting the hybrid seed produced thereby.
Another embodiment includes the method of producing hybrid corn seeds of any prior embodiment, wherein the first inbred corn line is a female or a male parent.
One embodiment includes a method for producing a corn plant resistant to lepidopteran pests comprising:
a) sexually crossing a first parent corn plant with a second parent corn plant, wherein said first or second parent corn plant comprises event DP-910521-2 thereby producing a plurality of first generation progeny plants;
b) selfing the first generation progeny plant, thereby producing a plurality of second generation progeny plants; and
c) selecting from the second generation progeny plants that comprise the event DP-910521-2 and are resistant to a lepidopteran pest.
Another embodiment includes a method of producing hybrid corn seeds comprising:
a) sexually crossing a first inbred corn line comprising the DNA construct of claim 1 with a second inbred line not comprising the DNA construct of claim 1; and
b) harvesting the hybrid seed produced thereby.
Another embodiment includes the method of producing a corn plant resistant to lepidopteran pests of any prior embodiment, further comprising the step of backcrossing a second generation progeny plant that comprises corn event DP-910521-2 to the parent plant that lacks the corn event DP-910521-2 DNA, thereby producing a backcross progeny plant that is resistant to a lepidopteran pest.
One embodiment includes a method of determining zygosity of a corn plant comprising event DP-910521-2 in a biological sample comprising:
a) contacting said sample with a first pair of DNA molecules and a second distinct pair of DNA molecules such that:
1) when used in a nucleic acid amplification reaction comprising corn event DP-910521-2 DNA, produces a first amplicon that is diagnostic for event DP-910521-2, and
2) when used in a nucleic acid amplification reaction comprising corn genomic DNA other than DP-910521-2 DNA, produces a second amplicon that is diagnostic for corn genomic DNA other than DP-910521-2 DNA;
b) performing a nucleic acid amplification reaction; and
c) detecting the amplicons so produced, wherein detection of the presence of both amplicons indicates that said sample is heterozygous for corn event DP-910521-2 DNA, wherein detection of only the first amplicon indicates that said sample is homozygous for corn event DP-910521-2 DNA.
Another embodiment includes the method of determining zygosity of a corn plant comprising event DP-910521-2 in a biological sample in any prior embodiment, wherein the first pair of DNA molecules comprises primer pair SEQ ID NOs: 6 and 7.
A further embodiment includes the method of determining zygosity of a corn plant comprising event DP-910521-2 in a biological sample in any prior embodiment, wherein the first and second pair of DNA molecules comprise a detectable label.
Another embodiment includes the method of determining zygosity of a corn plant comprising event DP-910521-2 in a biological sample in any prior embodiment, wherein the detectable label is a fluorescent label.
A further embodiment includes the method of determining zygosity of a corn plant comprising event DP-910521-2 in a biological sample in any prior embodiment, wherein the detectable label is covalently associated with one or more of the primer molecules.
One embodiment includes a method of detecting the presence of a nucleic acid molecule that is unique to event DP-910521-2 in a sample comprising corn nucleic acids, the method comprising:
a) contacting the sample with a pair of primers that, when used in a nucleic-acid amplification reaction with genomic DNA from event DP-910521-2 produces an amplicon that is diagnostic for event DP-910521-2;
b) performing a nucleic acid amplification reaction, thereby producing the amplicon that is diagnostic for event DP-910521-2; and
c) detecting the amplicon that is diagnostic for event DP-910521-2.
Another embodiment includes the method of detecting the presence of a nucleic acid molecule that is unique to event DP-910521-2 in a sample comprising corn nucleic acids of any prior embodiment, wherein the nucleic acid molecule that is diagnostic for event DP-910521-2 is an amplicon produced by the nucleic acid amplification chain reaction.
Another embodiment includes the method of detecting the presence of a nucleic acid molecule that is unique to event DP-910521-2 in a sample comprising corn nucleic acids of any prior embodiment, wherein the method further comprises contacting the sample with a probe.
A further embodiment includes the method of detecting the presence of a nucleic acid molecule that is unique to event DP-910521-2 in a sample comprising corn nucleic acids, further comprises contacting the sample with a probe of any prior embodiment, wherein the probe comprises a detectable label.
A further embodiment includes the method of detecting the presence of a nucleic acid molecule that is unique to event DP-910521-2 in a sample comprising corn nucleic acids further comprising contacting the sample with a probe, wherein the probe comprises a detectable label of any prior embodiment, wherein the detectable label is a fluorescent label.
A further embodiment includes the method of detecting the presence of a nucleic acid molecule that is unique to event DP-910521-2 in a sample comprising corn nucleic acids further comprising contacting the sample with a probe, wherein the probe comprises a detectable label of any prior embodiment, wherein the detectable label is covalently associated with the probe.
One embodiment includes a plurality of polynucleotide primers comprising one or more polynucleotides which target event DP-910521-2 DNA template in a sample to produce an amplicon diagnostic for event DP-910521-2 as a result of a polymerase chain reaction method.
Another embodiment includes a plurality of polynucleotide primers according to any prior embodiment, wherein
a) a first polynucleotide primer comprises a nucleotide sequence as set forth in SEQ ID NO: 6, and the complements thereof; and
b) a second polynucleotide primer comprises a nucleotide sequence as set forth in SEQ ID NO: 7, and the complements thereof.
Another embodiment includes the primers of any prior embodiment, wherein said first primer and said second primer are at least 18 nucleotides.
One embodiment includes a method of detecting the presence of DNA corresponding to event DP-910521-2 in a sample, the method comprising:
a) contacting the sample comprising maize DNA with a polynucleotide probe that hybridizes under stringent hybridization conditions with DNA from maize event DP-910521-2 and does not hybridize under said stringent hybridization conditions with a non-DP-910521-2 maize plant DNA;
b) subjecting the sample and probe to stringent hybridization conditions; and
c) detecting hybridization of the probe to the DNA;
wherein detection of hybridization indicates the presence of event DP-910521-2.
One embodiment includes a kit for detecting nucleic acids that are unique to event DP-910521-2 comprising at least one nucleic acid molecule of sufficient length of contiguous polynucleotides to function as a primer or probe in a nucleic acid detection method, and which upon amplification of or hybridization to a target nucleic acid sequence in a sample followed by detection of the amplicon or hybridization to the target sequence, are diagnostic for the presence of nucleic acid sequences unique to event DP-910521-2 in the sample.
Another embodiment includes the kit for detecting nucleic acids that are unique to event DP-910521-2 comprising at least one nucleic acid molecule of sufficient length of contiguous polynucleotides to function as a primer or probe in a nucleic acid detection method, and which upon amplification of or hybridization to a target nucleic acid sequence in a sample followed by detection of the amplicon or hybridization to the target sequence, are diagnostic for the presence of nucleic acid sequences unique to event DP-910521-2 in the sample of any prior embodiment, wherein the nucleic acid molecule comprises a nucleotide sequence from SEQ ID NO: 6-31.
Another embodiment includes the kit for detecting nucleic acids that are unique to event DP-910521-2 comprising at least one nucleic acid molecule of sufficient length of contiguous polynucleotides to function as a primer or probe in a nucleic acid detection method, and which upon amplification of or hybridization to a target nucleic acid sequence in a sample followed by detection of the amplicon or hybridization to the target sequence, are diagnostic for the presence of nucleic acid sequences unique to event DP-910521-2 in the sample of any prior embodiment, wherein the nucleic acid molecule is a primer chosen from SEQ ID NOs: 6-31, and the complements thereof.
Another embodiment includes the corn plant comprising the genotype of the corn event DP-910521-2 of any prior embodiment, wherein the genotype comprises a nucleotide sequence having 1, 2, 3, 4, or 5 nucleotide changes in one of SEQ ID NO: 28 or SEQ ID NO: 31.
Another embodiment includes the corn plant comprising the genotype of the corn event DP-910521-2 of any prior embodiment, further comprising the nucleotide sequence set forth in SEQ ID NO: 3 or a nucleotide sequence having at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 3.
One embodiment includes a method of modifying the DP-910521-2 corn event, wherein a representative sample of seed of said corn event was deposited with the ATCC with accession number PTA-127078, comprising applying genome engineering technology to a DNA sequence of said DP-910521-2 corn event to modify the DNA of said corn event.
Another embodiment includes the method of modifying the DP-910521-2 corn event, wherein a representative sample of seed of said corn event was deposited with the ATCC with accession number PTA-127078, comprising applying genome engineering technology to a DNA sequence of said DP-910521-2 corn event to modify the DNA of said corn event of any prior embodiment, comprising modifying the DNA of said DP-910521-2 corn event to produce a modified DNA sequence having at least 90% sequence identity to SEQ ID NO:
3.
Another embodiment includes the method of modifying the DP-910521-2 corn event, wherein a representative sample of seed of said corn event was deposited with the ATCC with accession number PTA-127078, comprising applying genome engineering technology to a DNA sequence of said DP-910521-2 corn event to modify the DNA of said corn event of any prior embodiment, comprising modifying the DNA of said DP-910521-2 corn event to produce a modified DNA sequence having all or a portion of SEQ ID NO: 26 or SEQ ID NO: 29 duplicated in said modified DNA sequence.
Another embodiment includes the method of modifying the DP-910521-2 corn event, wherein a representative sample of seed of said corn event was deposited with the ATCC with accession number PTA-127078, comprising applying genome engineering technology to a DNA sequence of said DP-910521-2 corn event to modify the DNA of said corn event of any prior embodiment, comprising modifying the DNA of said DP-910521-2 corn event to produce a modified DNA sequence comprising an excision from SEQ ID NO: 3.
A further embodiment includes the method of modifying the DP-910521-2 corn event, wherein a representative sample of seed of said corn event was deposited with the ATCC with accession number PTA-127078, comprising applying genome engineering technology to a DNA sequence of said DP-910521-2 corn event to modify the DNA of said corn event, comprising modifying the DNA of said DP-910521-2 corn event to produce a modified DNA sequence comprising an excision from SEQ ID NO: 3 of any prior embodiment, wherein said excision comprises an excision from one or more regulatory elements of SEQ ID NO: 3 that does not substantially affect the activity of said one or more regulatory elements.
A further embodiment includes the method of modifying the DP-910521-2 corn event, wherein a representative sample of seed of said corn event was deposited with the ATCC with accession number PTA-127078, comprising applying genome engineering technology to a DNA sequence of said DP-910521-2 corn event to modify the DNA of said corn event, comprising modifying the DNA of said DP-910521-2 corn event to produce a modified DNA sequence comprising an excision from SEQ ID NO: 3 of any prior embodiment, comprising modifying the DNA of said DP-910521-2 corn event to produce a modified DNA sequence having all or a portion of SEQ ID NO: 26 or SEQ ID NO: 29 excised from said modified DNA sequence.
Another embodiment includes the method of modifying the DP-910521-2 corn event, wherein a representative sample of seed of said corn event was deposited with the ATCC with accession number PTA-127078, comprising applying genome engineering technology to a DNA sequence of said DP-910521-2 corn event to modify the DNA of said corn event, comprising modifying the DNA of said DP-910521-2 corn event to produce a modified DNA sequence comprising an excision from SEQ ID NO: 3 of any prior embodiment, comprising modifying the DNA of said DP-910521-2 corn event to produce a modified DNA sequence having at least 30% of SEQ ID NO: 3 excised from said modified DNA sequence.
A further embodiment includes the method of modifying the DP-910521-2 corn event, wherein a representative sample of seed of said corn event was deposited with the ATCC with accession number PTA-127078, comprising applying genome engineering technology to a DNA sequence of said DP-910521-2 corn event to modify the DNA of said corn event, comprising modifying the DNA of said DP-910521-2 corn event to produce a modified DNA sequence comprising an excision from SEQ ID NO: 3 of any prior embodiment, wherein at least 80% of SEQ ID NO: 3 is excised from said modified DNA sequence.
A further embodiment includes the method of modifying the DP-910521-2 corn event, wherein a representative sample of seed of said corn event was deposited with the ATCC with accession number PTA-127078, comprising applying genome engineering technology to a DNA sequence of said DP-910521-2 corn event to modify the DNA of said corn event, comprising modifying the DNA of said DP-910521-2 corn event to produce a modified DNA sequence comprising an excision from SEQ ID NO: 3 of any prior embodiment, wherein all of SEQ ID NO: 3 is excised from said modified DNA sequence.
One embodiment includes a method of generating guide polynucleotides for use with a DP-910521-2 corn event genome editing system comprising designing one or more guide polynucleotides that recognize at least a portion of SEQ ID NO: 3 and synthesizing said guide polynucleotides.
Another embodiment includes a method of modifying the DNA of the DP-910521-2 event having accession number PTA-127078 comprising introducing said one or more guide polynucleotides for use with a DP-910521-2 corn event genome editing system of any prior embodiment as part of a genome engineering composition to a DNA of the DP-910521-2 event to modify the DNA of the DP-910521-2 event.
One embodiment includes a DP-910521-2 corn event genome editing system comprising a CAS polypeptide, one or more guide polynucleotides, and DP-910521-2 corn event donor DNA.
One embodiment includes a method of modifying at least one expression cassette of the DP-910521-2 event as deposited with the ATCC having accession number PTA-127078, wherein the method comprises using genome editing technologies to modify at least one expression cassette, wherein the resulting maize plant derived from the DP-910521-2 event comprises at least one modified cassette.
Another embodiment includes the method of modifying at least one expression cassette of the DP-910521-2 event as deposited with the ATCC having accession number PTA-127078, wherein the method comprises using genome editing technologies to modify at least one expression cassette, wherein the resulting maize plant derived from the DP-910521-2 event comprises at least one modified cassette of any prior embodiment, wherein the method comprises altering expression of cry1B.34.
Another embodiment includes a method of producing a commodity plant product comprising processing grain produced from a corn event DP-910521-2 plant comprising a nucleotide sequence which is or is complementary to a sequence chosen from SEQ ID NO: 26 and SEQ ID NO: 29, wherein a representative sample of said corn event DP-910521-2 seed has been deposited with American Type Culture Collection (ATCC) with Accession No. PTA-127078, wherein said grain is processed into a commodity plant product chosen from corn flour, corn meal, corn syrup, corn oil, corn starch, and cereals manufactured in whole or in part to contain corn by-products, wherein said composition/commodity plant product comprises a detectable amount of said nucleotide sequence.
One embodiment includes a method of controlling Lepidopteran insects, comprising exposing the Lepidopteran insects to insect resistant maize plants of event DP-910521-2.
Another embodiment includes the method of controlling Lepidopteran insects, comprising exposing the Lepidopteran insects to insect resistant maize plants of event DP-910521-2, wherein the Lepidopteran insect is Fall Armyworm (Spodoptera frupperda).
Yet another embodiment includes the method of controlling Lepidopteran insects, comprising exposing the Lepidopteran insects to insect resistant maize plants of event DP-910521-2, wherein the Lepidopteran insect is Corn Earworm (Helicoverpa zea).
A further embodiment includes the method of controlling Lepidopteran insects, comprising exposing the Lepidopteran insects to insect resistant maize plants of event DP-910521-2, wherein damage from the Lepidopteran insect is controlled for maize grains or kernels from event DP-910521-2.
In some embodiments, a corn plant comprising a DP-910521-2 event may be treated with a seed treatment. In some embodiments, the seed treatment may be a fungicide, an insecticide, or a herbicide.
The following examples are offered by way of illustration and not by way of limitation.
Cassette designs for Cry1B.34 expression used in the molecular stacks to generate events was chosen based upon efficacy and expression in gene testing transformation experiments. A large number of different regulatory (promoters, introns) and other elements (terminators) were evaluated in gene testing experiments. The large number of different regulatory elements were used to evaluate expression patterns for yield and trait efficacy.
The genetic elements contained in the cry1B.34 gene cassette of T-DNA Region of the selected event construct, Plasmid PHP79620 (SEQ ID NO: 1), are described in Table 1.
thuringiensis (WO Patent 2016061197 [Izumi and
DP-910521-2 maize event was produced by particle bombardment of SSI transformation with plasmid PHP79620. Particle bombardment of SSI transformation was essentially performed as described in U.S. patent application publication number US 2019/0376073 A1, herein incorporated by reference.
PHP79620 with Cry1B34 was co-bombarded with PHP21875 containing zm-odp2, PHP73752 containing zm-wus2 and PHP5096 containing FLPM, into 6000 immature embryos of 10 SSI lines, with 600 immature embryos in each SSI line. After the 105-day selection and regeneration process, a total of 59 T0 plantlets were regenerated. Samples were taken from all T0 plantlets for PCR analysis to verify the presence and copy number of the inserted cry1B.34, pmi, and mo-pat. In addition to this analysis, the T0 plantlets were analyzed by PCR for the absence of the developmental genes, zm-odp2 and zm-wus2. Plants that were determined to contain single copy of the inserted genes, no developmental genes were selected for further greenhouse propagation. Samples from those PCR selected T0 quality events were collected for further analysis using Southern-by-Sequencing to confirm that the inserted genes were in the correct target locus without any gene disruptions. Maize events DP-910521-2 were confirmed to contain a single copy of genes (See Examples 3 and 4). These selected T0 plants were assayed for trait efficacy and protein expression. T0 plants meeting all criteria were advanced and crossed to inbred lines to produce seed for further testing. A schematic overview of the transformation and event development is presented in
For detection of the pmi, mo-pat, and cry/B.34 genes contained within DP-910521-2 maize, as well as the genomic junction spanning the insertion site for DP-910521-2 maize, regions spanning between 54-bp and 113-bp were amplified using primers and Taqman® probes specific for each unique sequence. Additionally, a 79-bp region of an endogenous reference gene, High Mobility Group A (hmg-A, GenBank accession number AF171874.1), was validated to be used in duplex with each assay for both qualitative and quantitative assessment of each assay and to demonstrate the presence of sufficient quality and quantity of DNA within the PCR reaction (Krech et al., 1999). Data from hmg-A was used in calculations regarding scoring. Data were compared to the performance of either the validated positive or copy number calibrator as well as negative genomic controls.
The real-time PCR reaction exploited the 5′ nuclease activity of the heat-activated DNA polymerase. Two primers and one probe annealed to the target DNA with the probe, which contained a 5′ fluorescent reporter dye and a 3′ quencher dye. With each PCR cycle, the reporter dye was cleaved from the annealed probe by the polymerase, emitting a fluorescent signal that intensified with each subsequent cycle. The cycle at which the emission intensity of the sample amplicon rose above the detection threshold was referred to as the CT value. When no amplification occurred, there was no CT calculated by the instrument and was assigned a CT value of 40.00.
If copy number of the test samples was to be determined, copy number calibrators (samples known to contain defined copies of the gene of interest, e.g., 1 or 2 copies) were used as controls for both the endogenous gene and gene of interest. Fold differences were used to apply a copy number for each test sample. Fold difference, or fold change, is calculated using the formula of 2−ΔCT. The ΔCT was calculated for the test samples and copy number calibrators as described above. A copy number of 1 was applied to the sample population producing a fold change between 0 and 0.7 with a maximum range of 0.75 when compared to the 2-copy calibrators. Likewise, a copy number of 2 was applied to a sample population producing a fold change ranging between 1.5 and 2.2 with a maximum range of 0.91 when compared to the single copy calibrators; and a copy number of 3 was applied to a sample population producing a fold change ranging between 1.3 and 1.5 with a maximum range of 0.35 when compared to the 2-copy calibrators.
Genomic DNA was isolated from DP-910521-2 maize leaf tissue for approximately 100 plants from each of the F1 and BC1 generations. The DNA samples were extracted using an alkaline buffer comprised of sodium hydroxide, ethylenediaminetetraacetic acid disodium salt dihydrate (Naz-EDTA) and Tris hydrochloride. Approximately 3 ng of template DNA were used per reaction.
Each assay supporting the target event and transgenes were multiplexed with the hmg-A endogenous reference assay. Reaction mixes were prepared, each comprised of all components to support both the gene of interest and the endogenous gene for the PCR reaction. The base master mix, Bioline SensiFast™ Probe Lo-ROX master mix with 30% Bovine Serum Albumin (BSA) included as an additive was used. Individual concentrations of primer varied per reaction between 300 nM and 900 nM, dependent on the optimal concentration established during analysis validation. Individual concentrations of probe per reaction were between 80 nM and 120 nM. Assay controls included no template controls (NTC) which consisted of water or Tris-EDTA (TE) buffer (10 mM Tris pH 8.0, 1 mM EDTA) as well as copy number calibrator and negative controls, all of which were validated for each assay performed. Annealing temperatures and number of cycles used during the PCR analyses are provided in Table 4. The primer and probes used for each PCR analysis are provided in Tables 2 and 3.
Genomic DNA samples isolated from collected leaf samples of 200 DP-910521-2 maize plants (100 plants from each of the F1 and BC1 generations), along with copy number calibrator, negative and NTC controls, were subjected to qPCR amplification using SensiFast™ probe Lo-ROX master mix (Bioline, London, UK) in the presence of primer pair and probes specific for the pmi and mo pat genes, and the 5′ and 3′ regions of the cry1B.34 gene, and the insertion site specific for DP-910521-2 maize which allowed for the unique identification of the PHP79620 recombinant fragment insertion in DP-910521-2 maize. For assay and DNA quality monitoring, maize hmg-A was included in duplex with each reaction as an endogenous control. Each qPCR reaction was set up in a total volume of 3 μL with approximately 3-ng (0.5 μL of volume) of the isolated genomic DNA.
The results of the qPCR copy number analyses indicate stable integration and segregation of a single copy of the genes within the T-DNA of plasmid PHP79620, with demonstrated transfer to subsequent generations.
PCR products ranging in size between 54-bp to 113-bp, representing the insertion site for DP-910521-2 maize as well as the genes within the recombinant fragment from plasmid PHP79620, were amplified and observed in leaf samples of DP-910521-2 maize as well as eight copy number calibrator genomic controls, but were absent in each of the eight negative genomic controls and eight NTC controls. Each assay was performed a total of four times with the same results observed. For each sample and all controls, CT values, ΔCT values, and copy numbers were calculated.
Using the maize endogenous reference gene hmg-A, a PCR product of 79-bp was amplified and observed in leaf samples from DP-910521-2 maize as well as eight copy number calibrator and eight negative genomic controls. Amplification of the endogenous gene was not observed in the eight NTC controls tested with no generation of CT values. For each sample, each assay was performed in duplex, analyzing for the insertion site and all genes a total of four times with the same results observed each time. For each sample, CT values, ΔCT values and copy numbers (if applicable) were calculated.
To assess the sensitivity of the construct-specific PCR assays, DP-910521-2 maize DNA was diluted in control maize genomic DNA, resulting in test samples containing various amounts of DP-910521-2 maize (5-ng, 1-ng, 500-pg, 250-pg, 100-pg, 50-pg, 20-pg, 10-pg and 5-pg) in a total of 5-ng maize DNA. These various amounts of DP910521 maize DNA correspond to 100%, 20%, 10%, 5%, 2%, 1%, 0.4%, 0.2% and 0.1% of DP-910521-2 maize DNA in total maize genomic DNA, respectively. The various amounts of DP-910521-2 maize DNA were subjected to real-time PCR amplification for pmi and mo pat genes, the 5′ and 3′ regions of the cry1B.34 gene, and the insertion site for DP-910521-2 maize. Based on these analyses, the limit of detection (LOD) in 5-ng of total DNA for DP-910521-2 maize was determined to be approximately 500-pg for pmi (10%), 250-pg for mo-pat (5%), 250-pg for cry1B.34 (5%), and 250-pg for event DP-910521-2 (5%). The determined sensitivity of each assay described is sufficient for many screening applications. Each concentration was tested a total of five times. At the point where amplification of the target tested was not detected in each replicate, the preceding concentration was determined to be the limit of sensitivity.
Real-time PCR analyses of DP-910521-2 maize using event-specific and construct-specific assays confirm the stable integration and segregation of a single copy of the recombinant fragment from plasmid PHP79620 in leaf samples tested, as demonstrated by the quantified detection of event DP-910521-2 and the pmi, mo-pat, and cry1B.34 genes in DP-910521-2 maize. These results were reproducible among all the replicate qPCR analyses conducted. The maize endogenous reference gene assay for detection of hmg-A amplified as expected in all the test samples and negative controls and was not detected in the NTC samples. The sensitivity of each assay under the conditions described ranged between 250-pg to 500-pg DNA, all sufficient for many screening applications by PCR.
CTCTTGACACTTTGTATTGGTGCTCTTGGGCTCAAGAGGGTA
CCTCACTGACTAGCTAATCGAGCTA
ACGCCCGGATCGAAGAC
AGCCTCTGCGTCGCCGAGGTCAACAACATCGACCCGTTCGTCA
TGACTGTCAAAGGCCACGGCCGTTTAGCGCGTGTTTACAACAAGCTG
TAAGAGCTTACTGAAAAAAT
CATCGTGAACCACTACATCGAGAC
CTCCACCGTGAACTTCCGCACCGAGCCCGCAGACCCCGCAGGA
GTGGATCGAC
CTGCGAAGCCGCTCAGA
CCCACGAGTTCTCCAGCTACATCAACACCGGCGACCT
aThermal cycling was cornpleted using a Roche LightCycler ® 480; 45 cycles for steps 2a and 2b were performed to obtain raw data to 40 cycles.
Genomic DNA was extracted from the T0 generation of DP-910521-2 maize. Southern-by-Sequencing (SbS) utilizes probe-based sequence capture, Next Generation Sequencing (NGS) techniques, and bioinformatics procedures to isolate, sequence, and identify inserted DNA within the maize genome. By compiling a large number of unique sequencing reads and comparing them to the transformation plasmid sequence, unique junctions resulting from inserted DNA are identified in the bioinformatics analysis and can be used to determine the number of insertions within the plant genome. The T0 plant of DP-910521-2 maize was analyzed by SbS to determine the insertion copy number.
A series of unique sequences encompassing the PHP79620 plasmid sequence was used to design overlapping biotinylated oligonucleotides as capture probes. The capture probes were designed and synthesized by Roche NimbleGen, Inc. The probe set was designed to target PHP79620 transformation plasmid sequences during the enrichment process. The probes were compared to the maize genome to determine the level of maize genomic sequence that would be captured and sequenced simultaneously with sequences derived from PHP79620.
Separate NGS libraries were constructed for DP-910521-2 maize and control maize. SbS was performed essentially as described in Zastrow-Hayes, et al (2015). The sequencing libraries were hybridized to the capture probes through two rounds of hybridization to enrich the targeted sequences. Following NGS (Illumina NextSeq), the sequencing reads entered the bioinformatics pipeline for trimming and quality assurance. Reads were aligned against the maize genome and the sequences of the intended insertion and PHP79620, and reads that contain both genomic and plasmid sequence were identified as junction reads. Alignment of the junction reads to the sequence of the intended insertion shows borders of the inserted DNA relative to the expected insertion.
To identify putative junctions that were due to endogenous maize sequences, control maize genomic DNA libraries were separately captured and sequenced in the same manner as the DP-910521-2 maize plant. These libraries were sequenced to an average depth approximately five times that of the depth for the DP-910521-2 maize plant sample. This increased the probability that the endogenous junctions captured by the probes would be detected in the control samples, so that they could be identified and removed in the DP910521 maize samples.
Integration and copy number of the insertion were determined in DP-910521-2 maize derived from construct PHP79620 (
SbS was conducted on the T0 plant of DP-910521-2 maize to determine the insertion copy number in the genome. Alignment of the SbS reads to the expected insertion region resulted in two unique junctions between the genomic flanking sequence and the landing pad. The FRT1 and FRT87 sites are the two locations where the target trait genes from PHP79620 were integrated into the site-specific integration (SSI) landing pad. There were no other junctions between the PHP79620 sequence and the maize genome detected in the plant, indicating that there are no additional plasmid-derived insertions present in DP-910521-2 maize. Additionally, there were no junctions between non-contiguous regions of PHP79620, indicating that there are no detectable rearrangements or truncations in the inserted DNA. Furthermore, there were no junctions between maize genome sequences and the backbone sequence of PHP79620 in the T0 plant, demonstrating that no plasmid backbone sequences were incorporated into DP910521 maize.
SbS analysis of the T0 plant of DP-910521-2 maize demonstrated that there is a single insertion containing the desired genes from PHP79620 in DP-910521-2 maize and that no additional insertions are present in its genome.
Southern-by-Sequencing (SbS) analysis was conducted on the T0 plant of DP-910521-2 maize to confirm insertion copy number. The results indicate a single insertion containing the desired genes from PHP79620 in the plant. No junctions between PHP79620 sequences and the maize genome were detected in control plants, indicating that, as expected, these plants did not contain any insertions derived from PHP79620. Furthermore, no plasmid backbone sequences were detected in the DP-910521-2 maize plant analyzed. SbS analysis of the T0 plant of DP-910521-2 maize demonstrated that there is a single insertion containing the desired genes from PHP79620 in DP-910521-2 maize and that no additional insertions are present in its genome.
The following junction sequences shown in Table 5 were validated through Southern-by-Sequencing (SbS) analysis.
DP-910521-2 maize was evaluated in-field for efficacy of the insect-active Cry1B.34 protein against fall armyworm, southwestern corn borer, and European corn borer. Field testing was conducted in 2020 in maize-growing regions of North America (United States and Puerto Rico; Table 6). At each field location, single-row plots were arranged in a randomized complete block experimental design with three replications. Rows were 13-15 feet (4.0-4.6 m) in length, depending on the location. Spacing between rows was 30 in. (76 cm). Seed planting rate was approximately 22 seeds per row. For all test and control plots, a conventional weed control program was implemented following normal best practices for the region, including manual weeding as needed, and no above-ground insecticide applications were applied. Agronomic practices were uniform for all plots at a given location.
Fall Armyworm (Spodoptera frugiperda) Evaluation
The Puerto Rico sites were naturally infested with fall armyworm, and therefore manual infestation was not conducted. Plots were visually evaluated for fall armyworm plant injury when plants reached the V8-V9 growth stages. Starting with the fourth plant in the plot, ten consecutive plants were individually scored for leaf injury using the Davis Scale (0-9), where a score of zero indicated no visible plant injury and a score of nine indicated plants almost totally destroyed (Table 7).
At the Stoneville location, natural infestations were supplemented with manual infestation when plants reached the V4 growth stage. Starting with the fourth plant in the row, seven consecutive plants per plot were manually infested with a mixture containing fall armyworm neonates and commercially available corn cob grits applied into the whorl of each plant. Two separate applications, a day apart, were conducted for each plant at a targeted infestation rate of 40 neonates per application. Plots were visually evaluated for fall armyworm plant injury 14 days after the last infestation date, when plants were at the V7 growth stage. The middle five infested plants were individually scored for leaf injury using the Davis Scale (0-9) described in the following table.
Southwestern Corn Borer (Diatraea grandiosella) Evaluation
For each field location, manual infestation was conducted at two separate timepoints to simulate infestations from both first and second generations. The first manual infestation was conducted when plants reached the V8 growth stage. Starting with the fourth plant in the row, at least seven consecutive plants per plot were manually infested with a mixture containing southwestern corn borer neonates and commercially available corn cob grits applied into the whorl of each plant, at a rate of 30-50 neonates per plant. The second manual infestation, intended to mimic a second generation of southwestern corn borers, was conducted at the R1 growth stage, when approximately 50% of the plants in the plots were shedding pollen. A neonate-grits mixture was applied to the leaf collar of the primary ear two times within a 7-day period, with a rate of 45-50 neonates per application.
Plots were visually evaluated for southwestern corn borer plant injury two to three weeks after the first manual infestation, when plants were at R1 growth stage. The middle five infested plants were individually scored for leaf injury using the SWCB1LF visual scoring scale (9-1), where a score of nine indicated no or very low visible plant injury and a score of one indicated plants with more than two-thirds of the leaves with lesions greater than 1-inch in size (Table 8).
Additionally, plots were visually evaluated for second generation southwestern corn borer plant injury approximately 50 days after the final infestation date, when plants were at the R5-R6 growth stages. The middle five infested plants were individually evaluated for total centimeters of stalk tunneling. For each plant, the stalk of each scored plant was split longitudinally from the upper end of the fourth inter-node above the primary ear down to the base of the plant. Tunnels or entrance holes <0.5 cm in length were not included in total tunnel length.
European Corn Borer (Ostrinia nubilalis) Evaluation
For each field location, manual infestation was conducted at two separate timepoints to simulate infestations from both first and second generations. The first manual infestation was conducted when plants were at the V5-V8 growth stages. Starting with the fourth plant in the row, at least seven consecutive plants per plot were manually infested with a mixture containing European corn borer neonates and commercially available corn cob grits applied into the whorl of each plant. At each location, plants were infested three times during a seven-day period, with an infestation rate of 20-51 neonates per application at the Reasnor, Union City, Champaign, and York sites, and 93-105 neonates per application at the Windfall site.
The second manual infestation, intended to mimic a second generation of European corn borers, was conducted when plants had reached the VT-R1 growth stage, when approximately 50% of the plants in the plots were shedding pollen. A neonate-grits mixture was applied into the whorl of each plant three times within a 9-day period, with an infestation rate of 31-72 neonates per application at the Reasnor, Union City, Champaign, and York sites, and 91-117 neonates per application at the Windfall site.
For first generation evaluations, plots were visually evaluated for European corn borer plant injury 16-33 days after the last manual infestation, when plants were at V10-R1 growth stage. The middle five infested plants were individually scored for leaf injury using the ECBLF1 visual scoring scale (9-1), where a score of nine indicated no or very low visible plant injury and a score of one indicated plants with more than two-thirds of the leaves with lesions greater than 1-inch in size (Table 9).
For second generation, plots were visually evaluated for European corn borer plant injury 40-60 days after the final second generation manual infestation date, when plants were at the R2-R5 growth stages. The middle five infested plants were individually evaluated for total centimeters of stalk tunneling. For each plant, the stalk of each scored plant was split longitudinally from the upper end of the fourth inter-node above the primary ear down to the base of the plant. Tunnels or entrance holes <0.5 cm in length were not included in total tunnel length.
Across-Locations: Statistical analyses were conducted using a linear mixed model that was applied to leaf injury ratings and stalk tunneling measurements for DP-910521-2 maize and the negative control maize across locations. The data were modeled separately for each maize pest. Data for DP-910521-2 maize (Yijmks) of location (L)i, replication (R)j, event (E)m, plot (K)k, and plant s, were modeled as a function of an overall mean μ, factors for location, location by replication, event, location by event, plot within each location (K/L)ik, and a residual within each location (ε/L)ijmks. The model can be specified as:
Y
ijmks
=μ+L
i+(L×R)ij+Em+(L×E)im+(K/L)ik+(ε/L)ijmks
where event and location treated as fixed effects, and all the other effects were treated as independent normally distributed random variables with means of zero. Treatment means were estimated from the model fitting. T-tests using standard errors from the model were conducted to compare treatment effects.
Individual Locations: Each location was modeled separately for each pest. Data for DP-910521-2 maize (Yinks) of replication (R)i, event (E)n, plot (K)k, and plant s, were modeled as a function of an overall mean μ, factors for replication, event, plot and a residual sinks. The model can be specified as:
Y
inks
=μ+R
i
+E
n
+K
k+εinks
where event was treated as fixed effect, and all the other effects were treated as independent normally distributed random variables with means of zero. Treatment means were estimated from the model fitting. T-tests using standard errors from the model were conducted to compare treatment effects.
For both the across-site and individual site analyses, a difference was considered statistically significant if the P-value of the difference was less than 0.05. All statistical data analysis and comparisons were conducted using ASReml-R 3.0 (VSN International, Hemel Hempstead, U K, 2009).
A summary of the plant injury results are provided below for fall armyworm (Table 10), southwestern corn borer (Table 11), and European corn borer (Table 12). These results indicate that under high insect pressure, as for fall armyworm, southwestern corn borer and second-generation European corn borer in these trials, the insect active Cry1B.34 protein expressed in DP-910521-2 maize provides protection from feeding compared to the negative control. Insect pressure in these trials was low for first generation European corn borer; however, the insect active Cry1B.34 protein expressed in DP-910521-2 maize provided protection from feeding compared to the negative control maize.
Agronomic field trials containing DP-910521-2 were to generate yield data and to evaluate other agronomic characteristics. All inbred and hybrid materials tested for an event were generated from a single T0 plant.
Two hybrid trials were planted at a total of 26 locations, one trial at 16 locations and the other at 10 locations, with two replicates of the entry list at each location. Grain was harvested from 10 of the 26 locations. Each entry in a common background was crossed to 5 testers within each trial, or 10 testers total, to generate hybrid seed for testing. Experiments were nested by testers, with the entries randomized within each nest. Various observations and data were collected at each planted location throughout the growing season. The following agronomic characteristics were analyzed for comparison to a wild-type entry (WT), or an entry with the same genetics but without Cry1B.34, also referred to as base comparator (Table 13):
Inbred trials were planted at 8 locations with 2 replicates of the entry list at each location. Grain was harvested from 4 locations for analysis. One replicate at each location was nested by construct design; the other replicate was planted as a randomized complete block. Agronomic data and observations were collected for the inbred trials and analyzed for comparison to a wild-type entry (WT), or untraited version of the same genotype. Data generated for the inbred trials included the following agronomic traits (Table 14):
To evaluate the hybrid data, a mixed model framework was used to perform multi location analysis. In the multi-location analysis, main effect construct design is considered as fixed effect. Factors for location, background, tester, event, background by construct design, tester by construct design, tester by event, location by background, location by construct design, location by tester, location by background by construct design, location by tester by construct design, location by event, location by tester by event, and rep within location are considered as random effects. The spatial effects including range and plot within locations were considered as random effects to remove the extraneous spatial noise. The heterogeneous residual was assumed with autoregressive correlation as AR1*AR1 for each location. The estimate of construct design and prediction of event for each background were generated. The T-tests were conducted to compare construct design/event with WT. A difference was considered statistically significant if the P-value of the difference was less than 0.05. Yield analysis was by ASREML (VSN International Ltd; Best Linear Unbiased Prediction; Cullis, B. R et. al. (1998) Biometrics 54: 1-18, Gilmour, A. R. et al (2009); ASReml User Guide 3.0, Gilmour, A. R., et al (1995) Biometrics 51: 1440-50).
To evaluate the inbred data, a mixed model framework was used to perform multi location analysis. In the multi-location analysis, main effect construct design is considered as fixed effect. Factors for location, background, event, background by construct design, location by background, location by construct design, location by background by construct design, location by event and rep within location are considered as random effects. The spatial effects including range and plot within locations were considered as random effects to remove the extraneous spatial noise. The heterogeneous residual was assumed with autoregressive correlation as AR1*AR1 for each location. The estimate of construct design and prediction of event for each background were generated. The T-tests were conducted to compare construct design/event with WT. A difference was considered statistically significant if the P-value of the difference was less than 0.05. Yield analysis was by ASREML (VSN International Ltd; Best Linear Unbiased Prediction; Cullis, B. R et. al. (1998) Biometrics 54: 1-18, Gilmour, A. R. et al (2009); ASReml User Guide 3.0, Gilmour, A. R., et al (1995) Biometrics 51: 1440-50).
For analysis of Cry1B.34 protein concentrations, processed leaf tissue sub-samples were weighed at a target weight of 10 mg. Samples were extracted with 0.60 ml of chilled phosphate-buffered saline containing polysorbate 20 (PBST). Extracted samples were centrifuged, and then supernatants were removed and prepared for analysis.
Prior to analysis, samples were diluted as applicable in PBST. Standards (typically analyzed in triplicate wells) and diluted samples (typically analyzed in duplicate wells) were incubated in a plate pre-coated with an Cry1B.34-specific antibody. Following incubation, unbound substances were washed from the plate and the bound Cry1B.34 protein was incubated with a different Cry1B.34-specific antibody conjugated to the enzyme horseradish peroxidase (HRP). Unbound substances were washed from the plate. Detection of the bound Cry1B.34-antibody complex was accomplished by the addition of substrate, which generated a colored product in the presence of HRP. The reaction was stopped with an acid solution and the optical density (OD) of each well was determined using a plate reader.
Prior to analysis, samples were diluted as applicable in PBST. Standards (typically analyzed in triplicate wells) and diluted samples (typically analyzed in duplicate wells) were co-incubated with a PAT-specific antibody conjugated to the enzyme HRP in a plate pre-coated with a different PAT-specific antibody. Following incubation, unbound substances were washed from the plate. Detection of the bound PAT-antibody complex was accomplished by the addition of substrate, which generated a colored product in the presence of HRP. The reaction was stopped with an acid solution and the OD of each well was determined using a plate reader.
Prior to analysis, samples were diluted as applicable in PBST. Standards (typically analyzed in triplicate wells) and diluted samples (typically analyzed in duplicate wells) were incubated in a plate pre-coated with a PMI-specific antibody. Following incubation, unbound substances were washed from the plate and the bound PMI protein was incubated with a different PMI-specific antibody conjugated to the enzyme HRP. Unbound substances were washed from the plate. Detection of the bound PMI-antibody complex was accomplished by the addition of substrate, which generated a colored product in the presence of HRP. The reaction was stopped with an acid solution and the OD of each well was determined using a plate reader.
SoftMax Pro GxP (Molecular Devices) microplate data software was used to perform the calculations required to convert the OD values obtained for each set of sample wells to a protein concentration value.
A standard curve was included on each ELISA plate. The equation for the standard curve was derived by the software, which used a quadratic fit to relate the OD values obtained for each set of standard wells to the respective standard concentration (ng/ml).
Adjusted Concentration=Interpolated Sample Concentration×Dilution Factor
Adjusted sample concentration values obtained from SoftMax Pro GxP software were converted from ng/ml to ng/mg sample weight as follows:
Protein concentration results (means, standard deviations, and ranges) were determined for Cry1B.34 PAT and PMI proteins in V9 leaf tissue from two generations of DP-910521-2 maize.
15 ± 1.2
14 ± 3.7
The above description of various illustrated embodiments of the disclosure is not intended to be exhaustive or to limit the scope to the precise form disclosed. While specific embodiments of and examples are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the disclosure, as those skilled in the relevant art will recognize. The teachings provided herein can be applied to other purposes, other than the examples described above. Numerous modifications and variations are possible in light of the above teachings and, therefore, are within the scope of the appended claims.
These and other changes may be made in light of the above detailed description. In general, in the following claims, the terms used should not be construed to limit the scope to the specific embodiments disclosed in the specification and the claims.
The entire disclosure of each document cited (including patents, patent applications, journal articles, abstracts, manuals, books or other disclosures) in the Background, Detailed Description, and Examples is herein incorporated by reference in their entireties.
Efforts have been made to ensure accuracy with respect to the numbers used (e.g., amounts, temperature, concentrations, etc.) but some experimental errors and deviations should be allowed for. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight; temperature is in degrees celsius; and pressure is at or near atmospheric.
This application claims the benefit of U.S. Provisional Application No. 63/264,098, filed Nov. 16, 2021, and U.S. Provisional Application No. 63/266,435, filed Jan. 5, 2022, the disclosures of which are incorporated herein by reference in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 63266435 | Jan 2022 | US | |
| 63264098 | Nov 2021 | US |
