# Patent

## Title
Corn event MIR162

## Abstract

A novel transgenic corn event designated MIR162 is disclosed. The invention relates to nucleic acids that are unique to event MIR162 and to methods for detecting the presence of the MIR162 event based on DNA sequences of the recombinant constructs inserted into the corn genome that resulted in the MIR162 event and of genomic sequences flanking the insertion site. The invention further relates to corn plants comprising the transgenic genotype of MIR162 and to methods for producing a corn plant by crossing a corn plant comprising the MIR162 genotype with itself or another corn variety. Seeds of corn plants comprising the MIR162 genotype are also objects of the present invention. The invention also relates to methods of controlling insects using MIR162 corn plants.

## Background
This application is a divisional of U.S. patent application Ser. No. 13/534,202, filed Jun. 27, 2012, which is a divisional of U.S. patent application Ser. No. 12/301,824, filed Jul. 15, 2009, now U.S. Pat. No. 8,232,456, which is a §371 of PCT/US2007/012301, filed May 24, 2007, and published Dec. 13, 2007 as WO 2007/142840, which claims priority from U.S. Provisional Application No. 60/810,499, filed Jun. 3, 2006. These documents are incorporated herein by reference in their entirety.


BACKGROUND
The present invention relates generally to the field of plant molecular biology, plant transformation, and plant breeding. More specifically, the invention relates to insect resistant transgenic corn plants comprising a novel transgenic genotype and to methods of detecting the presence of nucleic acids that are unique to the transgenic corn plants in a sample and compositions thereof.
Plant pests are a major factor in the loss of the world's important agricultural crops. About $8 billion are lost every year in the U.S. alone due to infestations of non-mammalian pests including insects. In addition to losses in field crops, insect pests are also a burden to vegetable and fruit growers, to producers of ornamental flowers, and to home gardeners.
Insect pests are mainly controlled by intensive applications of chemical pesticides, which are active through inhibition of insect growth, prevention of insect feeding or reproduction, or cause death. Good insect control can thus be reached, but these chemicals can sometimes also affect other, beneficial insects. Another problem resulting from the wide use of chemical pesticides is the appearance of resistant insect varieties. This has been partially alleviated by various resistance management practices, but there is an increasing need for alternative pest control agents. Biological pest control agents, such as Bacillus thuringiensis (Bt) strains expressing pesticidal toxins like δ-endotoxins, have also been applied to crop plants with satisfactory results, offering an alternative or compliment to chemical pesticides. The genes coding for some of these δ-endotoxins have been isolated and their expression in heterologous hosts have been shown to provide another tool for the control of economically important insect pests. In particular, the expression of Bt δ-endotoxins has provided efficient protection against selected insect pests, and transgenic plants expressing such toxins have been commercialized, allowing farmers to reduce applications of chemical insect control agents.
Another family of insecticidal proteins produced by Bacillus species during the vegetative stage of growth (vegetative insecticidal proteins (Vip)) has also been identified. U.S. Pat. Nos. 5,877,012, 6,107,279, and 6,137,033, herein incorporated by reference, describe a new class of insecticidal proteins called Vip3. Other disclosures, including WO 98/18932, WO 98/33991, WO 98/00546, and WO 99/57282, have also now identified homologues of the Vip3 class of proteins. Vip3 coding sequences encode approximately 88 kDa proteins that possess insecticidal activity against a wide spectrum of lepidopteran pests, including, but not limited to, black cutworm (BCW, Agrotis ipsilon), fall armyworm (FAW, Spodoptera frugiperda), tobacco budworm (TBW, Heliothis virescens), sugarcane borer, (SCB, Diatraea saccharalis), lesser cornstalk borer (LCB, Elasmopalpus lignosellus), and corn earworm (CEW, Helicoverpa zea), and when expressed in transgenic plants, for example corn (Zea mays), confer protection to the plant from insect feeding damage.
Present plant transformation methods generally lead to the random integration of transgenes like vip3 into a host-plant genome. This random insertion of introduced DNA into the plant's genome can be lethal if the foreign DNA happens to insert into, and thus mutate, a critically important native gene. In addition, even if a random insertion event does not impair the functioning of a host cell gene, the expression of an inserted foreign gene may be influenced by “position effects” caused by the surrounding genomic DNA. In some cases, the gene is inserted into sites where the position effects are strong enough to prevent the synthesis of an effective amount of product from the introduced gene. For example, it has been observed in plants that there may be wide variations in levels of expression of a heterologous gene introduced into a plant's chromosome among individually selected events. There may also be differences in spatial or temporal patterns of expression, for example, differences in the relative expression of a transgene in various plant tissues, that may not correspond to the patterns expected from transcriptional regulatory elements present in the introduced gene construct. In other instances, overproduction of the gene product has deleterious effects on the cell. Because of these potential problems, it is common to produce hundreds of different events and screen those events for a single event that has desired transgene expression patterns and levels for commercial purposes. However, once a commercially viable site within the plant's genome is identified it would be advantageous to target genes of interest to that non-detrimental site.
Several methods for the targeted insertion of a nucleotide sequence of interest into a specific chromosomal site within a plant cell have been described. Site-specific recombination systems have been identified in several prokaryotic and lower eukaryotic organisms. Such systems typically comprise one or more proteins that recognize two copies of a specific nucleotide sequence, cleave and ligate those nucleotide sequences, and thereby provide a precise, site-specific exchange of genetic information. Several site-specific recombinases are known in the art. These include, but are not limited to, e.g., the bacteriophage P1 Cre/lox system (Austin et al. (1981) Cell 25: 729-736), the R/RS recombinase system from the pSRi plasmid of the yeast Zygosaccharomyces rouxii (Araki et al. (1985) J. Mol. Biol. 182: 191-203), the Gin/gix system of phage Mu (Maeser and Kahlmann (1991) Mol. Gen. Genet. 230: 170-176), the FLP/FRT recombinase system from the 2 .mu.m plasmid of the yeast Saccharomyces cerevisiae (Broach et al. (1982) Cell 29: 227-234), and the Int recombinase from bacteriophage Lambda (Landy (1989) Annu. Rev. Biochem. 58: 912-949; Landy (1993) Curr. Opin. Genet. Dev. 3: 699-707; Lorbach et al. (2000) J. Mol. Biol. 296: 1175-1181; and WO 01/16345). One particularly useful site-specific targeting approach, disclosed in US Patent Application Publication No. 2006/0130179, herein incorporated by reference, uses lambda integrase mediated recombination. The method comprises introducing into a plant cell a target nucleotide sequence comprising a first Integrase Recognition Site; introducing into the plant cell a donor nucleotide sequence comprising a second Integrase Recognition Site; and introducing into the plant cell an Integrase or Integrase complex. Another useful site-specific targeting approach is disclosed in US Patent Application Publication No. 2006/0253918, herein incorporated by reference, which uses homologous recombination to integrate one or more genes (gene stacking) at specific locations in the genome.
An event that has desired levels or patterns of transgene expression is useful for introgressing the transgene into other genetic backgrounds by sexual out-crossing using conventional breeding methods. Progeny of such crosses maintain the transgene expression characteristics of the original transformant. This strategy is used to ensure reliable gene expression in a number of varieties that are well adapted to local growing conditions. It would also 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. In addition, a method for detecting a particular event would be helpful for complying with regulations requiring the pre-market approval and labeling of foods derived from recombinant crop plants, for example. It is possible to detect the presence of a transgene by any well-known nucleic acid detection method including but not limited to thermal amplification (polymerase chain reaction (PCR)) using polynucleotide primers or DNA hybridization using nucleic acid probes. Typically, for the sake of simplicity and uniformity of reagents and methodologies for use in detecting a particular DNA construct that has been used for transforming various plant varieties, these detection methods generally focus on frequently used genetic elements, for example, promoters, terminators, and marker genes, because for many DNA constructs, the coding sequence region is interchangeable. As a result, such methods may not be useful for discriminating between constructs that differ only with reference to the coding sequence. In addition, such methods may not be useful for discriminating between different events, particularly those produced using the same DNA construct unless the sequence of chromosomal DNA adjacent to the inserted heterologous DNA (“flanking DNA”) is known.
For the foregoing reasons, there is a need for insect resistant transgenic corn events comprising novel nucleic acid sequences which are unique to the transgenic corn event, useful for identifying the transgenic corn event and for detecting nucleic acids from the transgenic corn event in a biological sample, as well as kits comprising the reagents necessary for use in detecting these nucleic acids in a biological sample. There is a further need to provide specific target sites within the maize genome to allow for targeting and control of insertion of nucleotide sequences to be integrated into the corn genome.
SUMMARY
The present invention relates to a transformed corn (Zea mays) event, designated MIR162 comprising a novel transgenic genotype that comprises a vip3Aa20 coding sequence, which is unique to event MIR162. The vip3Aa20 coding sequence encodes a Vip3Aa20 insecticidal protein that confers insect resistance to MIR162 corn plants. The MIR162 event also comprises a pmi coding sequence encoding a PMI protein that confers upon corn cells the ability to utilize mannose as a carbon source. In addition to the vip3A20 coding sequence, the present invention also provides other nucleic acids that are unique to MIR162. The invention also provides transgenic corn plants comprising the nucleic acids unique to MIR162, seed from the transgenic corn plants, and to methods for producing a transgenic corn plant comprising the unique nucleic acids of the invention by crossing a corn inbred comprising the nucleic acids unique to MIR162 with itself or another corn line of a different genotype. An example of seed, and hence corn plants grown from the seed, comprising nucleic acids unique to MIR162 was deposited at the American Type Culture Collection as accession No. PTA-8166. The transgenic corn plants of the invention may have essentially all of the morphological and physiological characteristics of corresponding isogenic non-transgenic corn plants in addition to those conferred upon the corn plants by the novel genotype of the invention. Biological samples and extracts from MIR162 corn plants, tissues and seeds are also provided by the present invention. The present invention also provides compositions and methods for detecting the presence of nucleic acids unique to MIR162 in biological samples based on the DNA sequence of the recombinant expression cassettes inserted into the corn genome that resulted in the MIR162 event and of genomic sequences flanking the insertion site. The present invention also provides a non-detrimental insertion target site on a maize chromosome useful for inserting genes of interest to a specific location on the chromosome and to methods of altering a maize genome by inserting heterologous nucleic acids at the disclosed insertion site or in the vicinity of the disclosed insertion site. The MIR162 event can be further characterized by analyzing expression levels of the Vip3Aa20 and PMI proteins as well as by testing MIR162 for efficacy against lepidopteran insect pests. The present invention also provides methods of producing transgenic corn plants resistant to a broader spectrum of insect pests by stacking the Vip3Aa20 insect resistant trait with insect resistance traits different than Vip3Aa20.
The foregoing and other aspects of the invention will become more apparent from the following detailed description.
DESCRIPTION OF THE SEQUENCES IN THE SEQUENCE LISTING
SEQ ID NO: 1 is the Vip3Aa20 coding sequence in MIR162.
SEQ ID NO: 2 is the Vip3Aa20 amino acid sequence.
SEQ ID NO: 3 is the sequence of plasmid pNOV1300.
SEQ ID Nos: 4-12 are primers and probes useful in a TAQMAN assay.
SEQ ID NO: 13 is the sequence of a vip3Aa20 probe.
SEQ ID NO: 14 is the sequence of a pmi probe.
SEQ ID Nos: 15-37 are primers useful in the present invention.
SEQ ID No: 38 is the sequence of a vip3Aa20 amplicon.
SEQ ID Nos: 39-40 are primers useful in the present invention.
SEQ ID No: 41 is the sequence of the CJ134/179 5′ amplicon.
SEQ ID Nos: 42-43 are primers useful in the present invention.
SEQ ID NO: 44 is a vip3Aa20 3′ amplicon.
SEQ ID NO: 45 is the 5′ genome-insert junction.
SEQ ID NO: 46 is corn genome sequence flanking 5′ to insert.
SEQ ID NO: 47 is the 3′ insert-genome junction.
SEQ ID NO: 48 is corn genome flanking 3′ to insert.
SEQ ID NO: 49 is the MIR162 insert and flanking sequences.
SEQ ID Nos. 50-54 are primers useful in the present invention.
SEQ ID NO: 55 is a 5′ PCR amplicon
SEQ ID Nos. 56-58 are primers useful in the present invention.
SEQ ID NO: 59 is a 3′ PCR amplicon.
SEQ ID Nos. 60-105 are primers useful in the present invention.
SEQ ID NO: 106 is the sequence of the region of maize chromosome 5 comprising the disclosed chromosomal target site.
SEQ ID NO: 107 is the maize genomic sequence that was displaced by the insertion of heterologous DNA in MIR162.


DETAILED DESCRIPTION
The following definitions and methods are provided to better define the present invention and to guide those of ordinary skill in the art in the practice of the present invention. Unless otherwise noted, terms used herein are to be understood according to conventional usage by those of ordinary skill in the relevant art. Definitions of common terms in molecular biology may also be found in Rieger et al., Glossary of Genetics: Classical and Molecular, 5th edition, Springer-Verlag: New York, 1994. The nomenclature for DNA bases and amino acids as set forth in 37 C.F.R. §1.822 is used herein.
As used herein, the term “amplified” means the construction of multiple copies of a nucleic acid molecule or multiple copies complementary to the nucleic acid molecule using at least one of the nucleic acid molecules as a template. Amplification systems include, but not limited to the polymerase chain reaction (PCR) system, ligase chain reaction (LCR) system, nucleic acid sequence based amplification (NASBA, Cangene, Mississauga, Ontario), Q-Beta Replicase systems, transcription-based amplification system (TAS), and strand displacement amplification (SDA). See, e.g., Diagnostic Molecular Microbiology Principles and Applications, D. H. Persing et al., Ed., American Society for Microbiology, Washington, D.C. (1993). The product of amplification is termed an amplicon.
A “coding sequence” is a nucleic acid sequence that is transcribed into RNA such as mRNA, rRNA, tRNA, snRNA, sense RNA or antisense RNA. Preferably the RNA is then translated in an organism to produce a protein.
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.
“Detection kit” as used herein refers to a kit of parts useful in detecting the presence or absence of DNA unique to MIR162 plants in a sample, wherein the kit comprises nucleic acid probes and/or primers of the present invention, which hybridize specifically under high stringency conditions to a target DNA sequence, and other materials necessary to enable nucleic acid hybridization or amplification methods.
As used herein the term transgenic “event” refers to a recombinant plant produced by transformation and regeneration of a plant cell or tissue with heterologous DNA, for example, an expression cassette that includes a gene of interest. The term “event” refers to the original transformant and/or progeny of the transformant that include the heterologous DNA. The term “event” also refers to progeny produced by a sexual outcross between the transformant and another corn line. Even after repeated backcrossing to a recurrent parent, the inserted DNA and the flanking DNA from the transformed parent is present in the progeny of the cross at the same chromosomal location. The term “event” also refers to DNA from the original transformant comprising the inserted DNA and flanking genomic 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. Normally, transformation of plant tissue produces multiple events, each of which represent insertion of a DNA construct into a different location in the genome of a plant cell. Based on the expression of the transgene or other desirable characteristics, a particular event is selected. Thus, “event MIR162”, “MIR162” or “MIR162 event” may be used interchangeably.
An insect resistant MIR162 corn plant can be bred by first sexually crossing a first parental corn plant consisting of a corn plant grown from a transgenic MIR162 corn plant, such as a MIR162 corn plant grown from the seed deposited at the ATCC under accession No. PTA-6188, and progeny thereof derived from transformation with the expression cassettes of the embodiments of the present invention that confers insect resistance, and a second parental corn plant that lacks insect resistance, 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.
“Expression cassette” as used herein means a nucleic acid molecule capable of directing expression of a particular nucleotide sequence in an appropriate host cell, comprising a promoter operably linked to the nucleotide sequence of interest which is operably linked to termination signals. It also typically comprises sequences required for proper translation of the nucleotide sequence. The expression cassette may also comprise sequences not necessary in the direct expression of a nucleotide sequence of interest but which are present due to convenient restriction sites for removal of the cassette from an expression vector. The expression cassette comprising the nucleotide sequence of interest may be chimeric, meaning that at least one of its components is heterologous with respect to at least one of its other components. The expression cassette may also be one that is naturally occurring but has been obtained in a recombinant form useful for heterologous expression. Typically, however, the expression cassette is heterologous with respect to the host, i.e., the particular nucleic acid sequence of the expression cassette does not occur naturally in the host cell and must have been introduced into the host cell or an ancestor of the host cell by a transformation process known in the art. The expression of the nucleotide sequence in the expression cassette may be under the control of a constitutive promoter or of an inducible promoter that initiates transcription only when the host cell is exposed to some particular external stimulus. In the case of a multicellular organism, such as a plant, the promoter can also be specific to a particular tissue, or organ, or stage of development. An expression cassette, or fragment thereof, can also be referred to as “inserted sequence” or “insertion sequence” when transformed into a plant.
A “gene” is a defined region that is located within a genome and that, besides the aforementioned coding sequence, may comprise other, primarily regulatory, nucleic acid sequences responsible for the control of the expression, that is to say the transcription and translation, of the coding portion. A gene may also comprise other 5′ and 3′ untranslated sequences and termination sequences. Further elements that may be present are, for example, introns.
“Gene of interest” refers to any gene which, when transferred to a plant, confers upon the plant a desired characteristic such as antibiotic resistance, virus resistance, insect resistance, disease resistance, or resistance to other pests, herbicide tolerance, improved nutritional value, improved performance in an industrial process or altered reproductive capability.
“Genotype” as used herein is the genetic material inherited from parent corn plants not all of which is necessarily expressed in the descendant corn plants. The MIR162 genotype refers to the heterologous genetic material transformed into the genome of a plant as well as the genetic material flanking the inserted sequence.
A “heterologous” nucleic acid sequence is a nucleic acid sequence not naturally associated with a host cell into which it is introduced, including non-naturally occurring multiple copies of a naturally occurring nucleic acid sequence.
A “homologous” nucleic acid sequence is a nucleic acid sequence naturally associated with a host cell into which it is introduced.
“Operably-linked” refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one affects the function of the other. For example, a promoter is operably-linked with a coding sequence or functional RNA when it is capable of affecting the expression of that coding sequence or functional RNA (i.e., that the coding sequence or functional RNA is under the transcriptional control of the promoter). Coding sequences in sense or antisense orientation can be operably-linked to regulatory sequences.
“Primers” as used herein are isolated nucleic acids that are annealed to a complimentary target DNA strand by nucleic acid hybridization to form a hybrid between the primer and the target DNA strand, and then extended along the target DNA strand by a polymerase, such as DNA polymerase. Primer pairs or sets can be used for amplification of a nucleic acid molecule, for example, by the polymerase chain reaction (PCR) or other conventional nucleic-acid amplification methods.
A “probe” is an isolated nucleic acid to which is attached a conventional detectable label or reporter molecule, such as a radioactive isotope, ligand, chemiluminescent agent, or enzyme. Such a probe is complimentary to a strand of a target nucleic acid, in the case of the present invention, to a strand of genomic DNA from corn event MIR162. The DNA of MIR162 can be from a corn plant or from a sample that includes DNA from MIR162. Probes according to the present invention include not only deoxyribonucleic or ribonucleic acids but also polyamides and other probe materials that bind specifically to a target DNA sequence and can be used to detect the presence of that target DNA sequence.
Primers and probes are generally between 10 and 15 nucleotides or more in length. Primers and probes can also be at least 20 nucleotides or more in length, or at least 25 nucleotides or more, or at least 30 nucleotides or more in length. Such primers and probes hybridize specifically to a target sequence under high stringency hybridization conditions. Primers and probes according to the present invention may have complete sequence complementarity with the target sequence, although probes differing from the target sequence and which retain the ability to hybridize to target sequences may be designed by conventional methods.
As used herein gene or trait “stacking” is combining desired traits into one transgenic line. Plant breeders stack transgenic traits by making crosses between parents that each have a desired trait and then identifying offspring that have both of these desired traits. Another way to stack genes is by transferring two or more genes into the cell nucleus of a plant at the same time during transformation. Another way to stack genes is by re-transforming a transgenic plant with another gene of interest. For example, gene stacking can be used to combine two different insect resistance traits, an insect resistance trait and a disease resistance trait, or a herbicide resistance trait. The use of a selectable marker in addition to a gene of interest would also be considered gene stacking.
“Stringent conditions” or “stringent hybridization conditions” include reference to conditions under which a probe will hybridize to its target sequence, to a detectably greater degree than to other sequences. Stringent conditions are target-sequence-dependent and will differ depending on the structure of the polynucleotide. By controlling the stringency of the hybridization and/or wash conditions, target sequences can be identified which are 100% complementary to the probe (homologous probing). Alternatively, stringency conditions can be adjusted to allow some mismatching in sequences so that lower degrees of similarity are detected (heterologous probing). Longer sequences hybridize specifically at higher temperatures. 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 “Overview of principles of hybridization and the strategy of nucleic acid probe assays”, Elsevier: New York; and Current Protocols in Molecular Biology, Chapter 2, Ausubel et al., Eds., Greene Publishing and Wiley-Interscience: New York (1995), and also Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual (5th Ed. Cols Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).
Specificity is typically the function of post-hybridization washes, the critical factors being the ionic strength and temperature of the final wash solution. Generally, high stringency hybridization and wash conditions are selected to be about 5° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Typically, under high stringency conditions a probe will hybridize to its target subsequence, but to no other sequences.
An example of high stringency hybridization conditions for hybridization of complementary nucleic acids which have more than 100 complementary residues on a filter in a Southern or northern blot is 50% formamide with 1 mg of heparin at 42° C., with the hybridization being carried out overnight. An example of very high stringency wash conditions is 0.15M NaCl at 72° C. for about 15 minutes. An example of high stringency wash conditions is a 0.2×SSC wash at 65° C. for 15 minutes (see, Sambrook, infra, for a description of SSC buffer).
Exemplary hybridization conditions for the present invention include hybridization in 7% SDS, 0.25 M NaPO4 pH 7.2 at 67° C. overnight, followed by two washings in 5% SDS, 0.20 M NaPO4 pH7.2 at 65° C. for 30 minutes each wash, and two washings in 1% SDS, 0.20 M NaPO4 pH7.2 at 65° C. for 30 minutes each wash. An exemplary medium stringency wash for a duplex of, e.g., more than 100 nucleotides, is 1×SSC at 45° C. for 15 minutes. An exemplary low stringency wash for a duplex of, e.g., more than 100 nucleotides, is 4-6×SSC at 40° C. for 15 minutes.
For probes of about 10 to 50 nucleotides, high stringency conditions typically involve salt concentrations of less than about 1.0 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3, and the temperature is typically at least about 30° C. High stringency conditions can also be achieved with the addition of destabilizing agents such as formamide. In general, a signal to noise ratio of 2× (or higher) than that observed for an unrelated probe in the particular hybridization assay indicates detection of a specific hybridization. Nucleic acids that do not hybridize to each other under high stringency conditions are still substantially identical if the proteins that they encode are substantially identical. This occurs, e.g., when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code.
The following are exemplary sets of hybridization/wash conditions that may be used to hybridize nucleotide sequences that are substantially identical to reference nucleotide sequences of the present invention: a reference nucleotide sequence preferably hybridizes to the reference nucleotide sequence in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, 1 mM EDTA at 50° C. with washing in 2×SSC, 0.1% SDS at 50° C., more desirably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, 1 mM EDTA at 50° C. with washing in 1×SSC, 0.1% SDS at 50° C., more desirably still in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, 1 mM EDTA at 50° C. with washing in 0.5×SSC, 0.1% SDS at 50° C., preferably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, 1 mM EDTA at 50° C. with washing in 0.1×SSC, 0.1% SDS at 50° C., more preferably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, 1 mM EDTA at 50° C. with washing in 0.1×SSC, 0.1% SDS at 65° C. The sequences of the present invention may be detected using all the above conditions. For the purposes of defining the invention, the high stringency conditions are used.
“Transformation” is a process for introducing heterologous nucleic acid into a host cell or organism. In particular, “transformation” means the stable integration of a DNA molecule into the genome of an organism of interest.
“Transformed/transgenic/recombinant” refer to a host organism such as a bacterium or a plant into which a heterologous nucleic acid molecule has been introduced. The nucleic acid molecule can be stably integrated into the genome of the host or the nucleic acid molecule can also be present as an extrachromosomal molecule. Such an extrachromosomal molecule can be auto-replicating. Transformed cells, tissues, or plants are understood to encompass not only the end product of a transformation process, but also transgenic progeny thereof. A “non-transformed”, “non-transgenic”, or “non-recombinant” host refers to a wild-type organism, e.g., a bacterium or plant, which does not contain the heterologous nucleic acid molecule. As used herein, “transgenic” refers to a plant, plant cell, or multitude of structured or unstructured plant cells having integrated, via well known techniques of genetic manipulation and gene insertion, a sequence of nucleic acid representing a gene of interest into the plant genome, and typically into a chromosome of a cell nucleus, mitochondria or other organelle containing chromosomes, at a locus different to, or in a number of copies greater than, that normally present in the native plant or plant cell. Transgenic plants result from the manipulation and insertion of such nucleic acid sequences, as opposed to naturally occurring mutations, to produce a non-naturally occurring plant or a plant with a non-naturally occurring genotype. Techniques for transformation of plants and plant cells are well known in the art and may comprise for example electroporation, microinjection, Agrobacterium-mediated transformation, and ballistic transformation.
As used herein, the term “unique” to MIR162 means distinctively characteristic of MIR162. Therefore, nucleic acids unique to event MIR162 are not found in other non-MIR162 corn plants.
The “Vip3” class of proteins comprises, for example, Vip3Aa, Vip3Ab, Vip3Ac, Vip3Ad, Vip3Ae, VipAf, Vip3Ag, Vip3Ba, and Vip3Bb, and their homologues. “Homologue” means that the indicated protein or polypeptide bears a defined relationship to other members of the Vip3 class of proteins. “Vip3Aa20” is a Vip3 homologue unique to event MIR162. It was generated by spontaneous mutations introduced into the maize-optimized vip3Aa19 gene comprised in pNOV1300 (SEQ ID NO: 3) during the plant transformation process.
This invention relates to a genetically improved line of corn that produces an insect control protein, Vip3Aa20, and a phosphomannose isomerase enzyme (PMI) that allows the plant to utilize mannose as a carbon source. The invention is particularly drawn to a transgenic corn event designated MIR162 comprising a novel genotype, as well as to compositions and methods for detecting nucleic acids unique to MIR162 in a biological sample. The invention is further drawn to corn plants comprising the MIR162 genotype, to transgenic seed from the corn plants, and to methods for producing a corn plant comprising the MIR162 genotype by crossing a corn inbred comprising the MIR162 genotype with itself or another corn line. Corn plants comprising the MIR162 genotype of the invention are useful in controlling lepidopteran insect pests including, but not limited to, black cutworm (BCW, Agrotis ipsilon), fall armyworm (FAW, Spodoptera frugiperda), tobacco budworm (TBW, Heliothis virescens), sugarcane borer (SCB, Diatraea saccharalis), lesser cornstalk borer (LCB, Elasmopalpus lignosellus), corn earworm (CEW, Helicoverpa zea), and western bean cutworm (WBCW, Striacosta albicosta). The invention is further drawn to a method of protecting transgenic corn from feeding damage whereby stacking the insect resistance trait of MIR162 with a different insect resistance trait in the same transgenic plant results is a corn plant that is protected from feeding damage to a greater degree than the insect resistance traits alone.
In one embodiment, the present invention encompasses an isolated nucleic acid molecule comprising a nucleotide sequence that is unique to event MIR162.
In another embodiment, the present invention encompasses an isolated nucleic acid molecule that links a heterologous DNA molecule inserted into the genome of MIR162 to genome DNA in MIR162 comprising at least 10 or more (for example 15, 20, 25, 50 or more) contiguous nucleotides of the heterologous DNA molecule and at least 10 or more (for example 15, 20, 25, 50, or more) contiguous nucleotides of the genome DNA flanking the point of insertion of the heterologous DNA molecule. Also included are nucleotide sequences that comprise 10 or more nucleotides of contiguous insert sequence from event MIR162 and at least one nucleotide of flanking DNA from event MIR162 adjacent to the insert sequence. Such nucleotide sequences are unique to and diagnostic for event MIR162. Nucleic acid amplification or hybridization of genomic DNA from MIR162 produces an amplicon comprising such unique sequences and is diagnostic for event MIR162. In one aspect of this embodiment, the nucleotide sequence is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 38, SEQ ID NO: 41, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 55, SEQ ID NO: 59, and the complements thereof.
In another embodiment, the invention encompasses an isolated nucleic acid molecule comprising a nucleotide sequence which comprises at least one junction sequence of event MIR162, wherein a junction sequence spans the junction between a heterologous expression cassette inserted into the corn genome and DNA from the corn genome flanking the insertion site and is diagnostic for the event. In one aspect of this embodiment, the junction sequence is selected from the group consisting of SEQ ID NO: 45, SEQ ID NO: 47, and the complements thereof.
In another embodiment, the present invention encompasses an isolated nucleic acid molecule linking a heterologous DNA molecule to the corn plant genome in event MIR162 comprising a sequence of from about 11 to about 20 contiguous nucleotides selected from the group consisting of SEQ ID NO: 45, SEQ ID NO: 47, and the complements thereof.
In another embodiment, the invention encompasses an isolated nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 38, SEQ ID NO: 41, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 55, SEQ ID NO: 59, and the complements thereof. In one aspect of this embodiment, the isolated nucleic acid molecule is comprised in a corn seed deposited at the American Type Culture Collection under the accession No. PTA-8166, or in plants grown from the seed.
In one embodiment of the present invention, an amplicon comprising a nucleotide sequence unique to event MIR162 is provided. In one aspect of this embodiment, the nucleotide sequence is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 38, SEQ ID NO: 41, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 55, SEQ ID NO: 59, and the complements thereof.
In another embodiment, the present invention encompasses flanking sequence primers for detecting event MIR162. Such flanking sequence primers comprise a nucleotide sequence of at least 10-15 contiguous nucleotides from the 5′ or the 3′ flanking sequence. In one aspect of this embodiment, the contiguous nucleotides are selected from nucleotides 1-1088 (inclusive) of SEQ ID NO: 49 (arbitrarily designated herein as the 5′ flanking sequence), or the complements thereof. In another aspect of this embodiment, the 5′ flanking sequence primers are selected from the group consisting of SEQ ID NO: 36, SEQ ID NO: 39, SEQ ID NO: 53, SEQ ID NOs: 68-80, and the complements thereof. In another aspect of this embodiment, the contiguous nucleotides are selected from nucleotides 9391-10579 (inclusive) of SEQ ID NO: 49 (arbitrarily designated herein as the 3′ flanking sequence), or the complements thereof. In yet another aspect of this embodiment, the 3′ flanking sequence primers are selected from the group consisting of SEQ ID NO: 58, SEQ ID NOs: 97-105, and the complements thereof.
In still another embodiment, the present invention encompasses a pair of polynucleotide primers comprising a first polynucleotide primer and a second polynucleotide primer that function together in the presence of a event MIR162 DNA template in a sample to produce an amplicon diagnostic for event MIR162. In one aspect of this embodiment, the first primer and/or the second primer is chosen from SEQ ID NO: 1 or the compliment thereof. In another aspect of this embodiment, the first primer and/or the second primer is selected from the group consisting of SEQ ID NOs: 15-35, SEQ ID NO: 37, SEQ ID NO: 42, and the complements thereof. In yet another aspect of this embodiment, the amplicon that is produced by the pair of primers comprises SEQ ID NO: 1, SEQ ID NO: 38, SEQ ID NO: 44, or the complements thereof.
In another embodiment, the present invention encompasses a pair of polynucleotide primers comprising a first polynucleotide primer and a second polynucleotide primer which function together in the presence of a event MIR162 DNA template in a sample to produce an amplicon diagnostic for event MIR162, wherein the first primer is or is complementary to a corn plant genome sequence flanking the point of insertion of a heterologous DNA sequence inserted into the genome of event MIR162, and the second polynucleotide primer sequence is or is complementary to the heterologous DNA sequence inserted into the genome of event MIR162.
In one aspect of this embodiment, the first polynucleotide primer comprises at least 10 contiguous nucleotides from a nucleotide sequence selected from the group consisting of nucleotides 1-1088 of SEQ ID NO: 49, nucleotides 9391-10579 of SEQ ID NO: 49, and the complements thereof. In a further aspect of this embodiment, the first primer is selected from the group consisting of SEQ ID NO: 36, SEQ ID NO: 39, SEQ ID NO: 53, SEQ ID NO: 57, SEQ ID NOs: 68-72, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NOs: 97-105, and the complements thereof. In another aspect of this embodiment, the second polynucleotide primer comprises at least 10 contiguous nucleotides from position 1089-9390 of SEQ ID NO: 49, or complements thereof. In still a further aspect of this embodiment, the second polynucleotide primer is selected from the group consisting of SEQ ID NOs: 15-35, SEQ ID NO: 37, SEQ ID NO: 40, SEQ ID NOs: 50-52, SEQ ID NOs: 54, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 63, SEQ ID NO: 73, SEQ ID NO: 82, SEQ ID NO: 96, and the complements thereof.
In another aspect of this embodiment, the first polynucleotide primer, which is set forth in SEQ ID NO: 36, and the second polynucleotide primer which is set forth in SEQ ID NO: 37, function together in the presence of a event MIR162 DNA template in a sample to produce an amplicon diagnostic for event MIR162 as described in Example 4. In one embodiment of this aspect, the amplicon comprises the nucleotide sequence set forth in SEQ ID NO: 38.
In yet another aspect of this embodiment, the first polynucleotide primer, which is set forth in SEQ ID NO: 39, and the second polynucleotide primer, which is set forth in SEQ ID NO: 40, function together in the presence of a corn event MIR162 DNA template in a sample to produce an amplicon diagnostic for the corn event MIR162 as described in Example 4. In one embodiment of this aspect, the amplicon comprises the nucleotide sequence set forth in SEQ ID NO: 41.
In another aspect of this embodiment, the first polynucleotide primer, which is set forth in SEQ ID NO: 53, and the second polynucleotide primer, which is set forth in SEQ ID NO: 54, function together in the presence of a corn event MIR162 DNA template in a sample to produce an amplicon diagnostic for the corn event MIR162 as described in Example 5. In one embodiment, the amplicon comprises the nucleotide sequence set forth in SEQ ID NO: 55.
In a still a further aspect of this embodiment, the first polynucleotide primer, which is set forth in SEQ ID NO: 58, and the second polynucleotide primer, which is set forth in SEQ ID NO: 56, function together in the presence of a corn event MIR162 DNA template in a sample to produce an amplicon diagnostic for the corn event MIR162 as described in Example 5. In one embodiment, the amplicon comprises the nucleotide sequence set forth in SEQ ID NO: 59.
Of course, it is well within the skill in the art to obtain additional sequence further out into the genome sequence flanking either end of the inserted heterologous DNA sequences for use as a primer sequence that can be used in such primer pairs for amplifying the sequences that are diagnostic for the MIR162 event. For the purposes of this disclosure, the phrase “further out into the genome sequence flanking either end of the inserted heterologous DNA sequences” refers specifically to a sequential movement away from the ends of the inserted heterologous DNA sequences, the points at which the inserted DNA sequences are adjacent to native genomic DNA sequence, and out into the genomic DNA of the particular chromosome into which the heterologous DNA sequences were inserted. Preferably, a primer sequence corresponding to or complementary to a part of the insert sequence should prime the transcriptional extension of a nascent strand of DNA or RNA toward the nearest flanking sequence junction. Consequently, a primer sequence corresponding to or complementary to a part of the genomic flanking sequence should prime the transcriptional extension of a nascent strand of DNA or RNA toward the nearest flanking sequence junction. A primer sequence can be, or can be complementary to, a heterologous DNA sequence inserted into the chromosome of the plant, or a genomic flanking sequence. One skilled in the art would readily recognize the benefit of whether a primer sequence would need to be, or would need to be complementary to, the sequence as set forth within the inserted heterologous DNA sequence or as set forth SEQ ID NO: 38 depending upon the nature of the product desired to be obtained through the use of the nested set of primers intended for use in amplifying a particular flanking sequence containing the junction between the genomic DNA sequence and the inserted heterologous DNA sequence.
In another embodiment, the present invention encompasses an isolated insecticidal protein comprising SEQ ID NO: 2 and a nucleic acid molecule encoding SEQ ID NO: 2. In one aspect of this embodiment, the nucleic acid molecule is SEQ ID NO: 1. The present invention also encompasses a chimeric gene comprising a heterologous promoter operably linked to the nucleic acid molecule, and to recombinant vectors and host cells comprising the chimeric gene.
In yet another embodiment, the present invention encompasses a method of detecting the presence of a nucleic acid molecule that is unique to event MIR162 in a sample comprising corn nucleic acids, wherein the method comprises: (a) contacting the sample with a pair of polynucleotide primers that, when used in a nucleic acid amplification reaction with genomic DNA from event MIR162 produces an amplicon that is diagnostic for event MIR162; (b) performing a nucleic acid amplification reaction, thereby producing the amplicon; and (c) detecting the amplicon. In one aspect of this embodiment, the amplicon comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 38, SEQ ID NO: 41, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 55, SEQ ID NO: 59, and the compliments thereof.
In another embodiment, the present invention encompasses a method of detecting the presence of a nucleic acid molecule that is unique to event MIR162 in a sample comprising corn nucleic acids, wherein the method comprises: (a) contacting the sample with a probe that hybridizes under high stringency conditions with genomic DNA from event MIR162 and does not hybridize under high stringency conditions with DNA from a control corn plant; (b) subjecting the sample and probe to high stringency hybridization conditions; and (c) detecting hybridization of the probe to the DNA. Detection can be by any means well known in the art including fluorescent, chemiluminescent, radiological, immunological, and the like. In the case in which hybridization is intended to be used as a means for amplification of a particular sequence to produce an amplicon which is diagnostic for the MIR162 event, the production and detection by any means well known in the art of the amplicon is intended to be indicative of the intended hybridization to the target sequence where one probe or primer is utilized, or sequences where two or more probes or primers are utilized. The term “biological sample” is intended to comprise a sample that contains or is suspected of containing a nucleic acid comprising from between five and ten nucleotides either side of the point at which one or the other of the two terminal ends of the inserted heterologous DNA sequence contacts the genomic DNA sequence within the chromosome into which the heterologous DNA sequence was inserted, herein also known as the junction sequences. In addition, the junction sequence comprises as little as two nucleotides: those being the first nucleotide within the flanking genomic DNA adjacent to and covalently linked to the first nucleotide within the inserted heterologous DNA sequence. In one aspect of this embodiment, the probe comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 38, SEQ ID NO: 41, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 55, SEQ ID NO: 59, and the complements thereof.
In yet another embodiment, the present invention encompasses a kit for the detection of nucleic acids that are unique to event MIR162 in biological sample. The kit includes 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 MIR162 in the sample. The kit further includes other materials necessary to enable nucleic acid hybridization or amplification methods. In one aspect of this embodiment, a nucleic acid molecule contained in the kit comprises a nucleotide sequence from SEQ ID NO: 1 or SEQ ID NO: 49. In another aspect of this embodiment, the nucleic acid molecule is a primer selected from the group consisting of SEQ ID NOs: 15-37, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NOs: 50-54, SEQ ID NOs: 56-58, SEQ ID NOs: 60-105, and the complements thereof. In yet another aspect of this embodiment, the amplicon comprises SEQ ID NO: 1, SEQ ID NO: 38, SEQ ID NO: 41, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 55, SEQ ID NO: 59, or the complements thereof. A variety of detection methods can be used including, but not limited to TAQMAN (Perkin Elmer), thermal amplification, ligase chain reaction, southern hybridization, ELISA methods, and colorimetric and fluorescent detection methods. In particular the present invention provides for kits for detecting the presence of the target sequence, i.e., at least the vip3Aa20 sequence or a junction sequence, in a sample containing genomic nucleic acid from MIR162. The kit is comprised of at least one polynucleotide capable of binding to the target site or substantially adjacent to the target site and at least one means for detecting the binding of the polynucleotide to the target site. The detecting means can be fluorescent, chemiluminescent, colorimetric, or isotopic and can be coupled at least with immunological methods for detecting the binding. A kit is also envisioned which can detect the presence of the target site in a sample, i.e., at least the vip3Aa20 sequence or a junction sequence of MIR162, taking advantage of two or more polynucleotide sequences which together are capable of binding to nucleotide sequences adjacent to or within about 100 base pairs, or within about 200 base pairs, or within about 500 base pairs or within about 1000 base pairs of the target sequence and which can be extended toward each other to form an amplicon which contains at least the target site.
In another embodiment, the present invention encompasses a method of detecting Vip3Aa20 protein in a biological sample, the method comprising: (a) extracting protein from event MIR162 tissue; (b) assaying the extracted protein using an immunological method comprising antibody specific for the Vip3Aa20 protein produced by the MIR162 event; and (c) detecting the binding of said antibody to the Vip3Aa20 protein.
In yet another embodiment, the present invention encompasses a biological sample derived from a event MIR162 corn plant, tissue, or seed, wherein the sample comprises a nucleotide sequence which is or is complementary to a sequence that is unique to event MIR162, and wherein the sequence is detectable in the sample using a nucleic acid amplification or nucleic acid hybridization method. In one aspect of this embodiment, the nucleotide sequence is or is complementary to SEQ ID NO: 1, SEQ ID NO: 38, SEQ ID NO: 41, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 55, or SEQ ID NO: 59. In another aspect of this embodiment, the sample is selected from the group consisting of corn flour, corn meal, corn syrup, corn oil, cornstarch, and cereals manufactured in whole or in part to contain corn by-products.
In another embodiment, the present invention encompasses an extract of a biological sample derived from a MIR162 corn plant, tissue, or seed comprising a nucleotide sequence which is or is complementary to a sequence that is unique to MIR162. In one aspect of this embodiment, the sequence is detectable in the extract using a nucleic acid amplification or nucleic acid hybridization method. In another aspect of this embodiment, the sequence is or is complementary to SEQ ID NO: 1, SEQ ID NO: 38, SEQ ID NO: 41, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 55, or SEQ ID NO: 59. In yet another aspect of this embodiment, the sample is selected from the group consisting of corn flour, corn meal, corn syrup, corn oil, cornstarch, and cereals manufactured in whole or in part to contain corn by-products.
Another embodiment of the present invention encompasses a corn plant, or parts thereof, and seed from a corn plant comprising the genotype of the transgenic event MIR162, wherein the genotype comprises a nucleotide sequence set forth in SEQ ID NO: 1, SEQ ID NO: 38, SEQ ID NO: 41, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 55, SEQ ID NO: 59, or the complements thereof. One example of corn seed comprising the nucleic acid molecules of the invention was deposited 23 Jan. 2007 and assigned the ATCC Accession No. PTA-8166. In one aspect of this embodiment, the corn plant is from the inbred corn lines CG5NA58, CG5NA58A, CG3ND97, CG5NA01, CG5NF22, CG4NU15, CG00685, CG00526, CG00716, NP904, NP911, NP948, NP934, NP982, NP991, NP993, NP2010, NP2013, NP2015, NP2017, NP2029, NP2031, NP2034, NP2045, NP2052, NP2138, NP2151, NP2166, NP2161, NP2171, NP2174, NP2208, NP2213, NP2222, NP2275, NP2276, NP2316, BCTT609, AF031, NPH8431, 894, BUTT201, R327H, 2044BT, and 2070BT. One skilled in the art will recognize however, that the MIR162 genotype can be introgressed into any plant variety that can be bred with corn, including wild maize species, and thus the list of inbred lines of this embodiment are not meant to be limiting.
In another embodiment, the present invention encompasses a corn plant comprising at least a first and a second DNA sequence linked together to form a contiguous nucleotide sequence, wherein the first DNA sequence is within a junction sequence and comprises at least about 11 contiguous nucleotides selected from the group consisting of nucleotides 1079-1098 of SEQ ID NO: 49, nucleotides 9381-9400, and the complements thereof, wherein the second DNA sequence is within the heterologous insert DNA sequence set forth in SEQ ID NO: 49, and the complements thereof; and wherein the first and the second DNA sequences are useful as nucleotide primers or probes for detecting the presence of corn event MIR162 nucleic acid sequences in a biological sample. In one aspect of this embodiment, the nucleotide primers are used in a DNA amplification method to amplify a target DNA sequence from template DNA extracted from the corn plant and the corn plant is identifiable from other corn plants by the production of an amplicon corresponding to a DNA sequence comprising SEQ ID NO: 45 or SEQ ID NO: 47.
Corn plants of the invention can be further characterized in that simultaneously digesting the plant's genomic DNA with the restriction endonucleases KpnI, EcoRV or NcoI results in an about a 8 kb, a 13 kb or 4.6 kb vip3Aa20 hybridizing band, respectively, using a vip3Aa20 probe under high stringency conditions. Exemplified herein is a vip3Aa20 probe comprising the nucleotide sequence set forth in SEQ ID NO: 13.
Corn plants of the invention can be further characterized in that digesting the plant's genomic DNA with the restriction endonuclease Acc65I or BamHI results in a single pmi hybridizing band using a pmi probe under high stringency conditions. Exemplified herein is a pmi probe comprising the nucleotide sequence set forth in SEQ ID NO: 14.
In one embodiment, the present invention provides a corn plant, wherein the MIR162 genotype confers upon the corn plant insect resistance or ability to utilize mannose as a carbon source, or both insect resistance and the ability to utilize mannose as a carbon source. In one aspect of this embodiment, the transgenic genotype conferring insect resistance upon the corn plant of the invention comprises a vip3Aa20 gene and the transgenic genotype conferring the ability to utilize mannose as a carbon source upon the maize plant of the invention comprises a pmi gene.
In yet another embodiment, the present invention provides 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 MIR162 DNA, thereby producing a plurality of first generation progeny plants; (b) selecting a first generation progeny plant that is resistant to one or more lepidopteran pests; (c) selfing the first generation progeny plant, thereby producing a plurality of second generation progeny plants; and (d) selecting from the second generation progeny plants, a plant that is resistant to one or more lepidopteran pests; wherein the second generation progeny plants comprise a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 55, and SEQ ID NO: 59.
In another embodiment, the present invention provides a method of producing hybrid corn seeds comprising: (a) planting seeds of a first inbred corn line comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 55, and SEQ ID NO: 59, and seeds of a second inbred line having a different genotype; (b) cultivating corn plants resulting from said planting until time of flowering; (c) emasculating said flowers of plants of one of the corn inbred lines; (d) sexually crossing the two different inbred lines with each other; and (e) harvesting the hybrid seed produced thereby. In one aspect of this embodiment, the first inbred corn line provides the female parents. In another aspect of this embodiment, the first inbred corn line provides the male parents. The present invention also encompasses the hybrid seed produced by the embodied method and hybrid plants grown from the seed.
One skilled in the art will recognize that the transgenic genotype of MIR162 can be introgressed by breeding into other corn lines comprising different transgenic genotypes. For example, a MIR162 corn inbred can be crossed with a corn inbred comprising the transgenic genotype of the lepidopteran resistant Bt11 event (U.S. Pat. Nos. 6,114,608 and 6,342,660, herein incorporated by reference). The resulting seed and progeny plants have the stacked insect resistance traits and the combined spectrum of activity of Cry1Ab and Vip3Aa20. Another trait stack encompassed by the present invention includes combining the MIR162 insect resistance trait and the MIR604 insect resistance trait (US Patent Application publication No. 2005/0216970, published Sep. 29, 2005, herein incorporated by reference). The stacked traits in the resulting seed and progeny confer upon the plants an increased spectrum of activity; i.e. the plants are active against both lepidopteran and coleopteran insect pests.
Therefore, the present invention encompasses a method of protecting a transgenic corn plant from feeding damage by one or more insect pests wherein the method comprises stacking in the same transgenic corn plant a Vip3Aa20 insect resistance trait with another insect resistance trait that is different from Vip3Aa20, whereby the stacked traits protect the corn plant against feeding damage by one or more insect pests to a greater degree than would be expected due to the insect resistance traits alone. In one aspect of this embodiment, the Vip3Aa20 insect resistance trait comprised in event MIR162 is stacked with the Cry3A055 insect resistance trait comprised in event MIR604 in the same transgenic corn plant by sexually crossing event MIR162 with event MIR604 or by transforming the traits together into the same plant.
Examples of other transgenic events which can be crossed with a MIR162 inbred include, the glyphosate tolerant GA21 event, the glyphosate tolerant/lepidopteran insect resistant MON802 event, the lepidopteran resistant DBT418 event, the male sterile event MS3, the phosphinothricin tolerant event B16, the lepidopteran insect resistant event MON 80100, the phosphinothricin tolerant events T14 and T25, the lepidopteran insect resistant event 176, and the coleopteran resistant event MON863, all of which are known in the art. It will be further recognized that other combinations or stacks can be made with the transgenic genotype of the invention and thus these examples should not be viewed as limiting.
One skilled in the art will also recognize that transgenic corn seed comprising the MIR162 genotype can be treated with various seed-treatment chemicals, including insecticides, to augment or syngergize the insecticidal activity of the Vip3Aa20 protein.
The subject invention discloses herein a specific site on chromosome 5 in the maize genome that is excellent for insertion of heterologous nucleic acids. Also disclosed is a 5′ molecular marker (opie2; nucleotides 1680-3338 of SEQ ID NO: 106) and a 3′ molecular marker (gag; nucleotides 43,275-45,086 of SEQ ID NO: 106) useful in identifying the location of a targeting site on chromosome 5. Thus, the subject invention provides methods to introduce heterologous nucleic acids of interest into this pre-established target site or in the vicinity of this target site. The subject invention also encompasses a corn seed and/or a corn plant comprising any heterologous nucleotide sequence inserted at the disclosed target site or in the general vicinity of such site. One option to accomplish such targeted integration is to substitute a different insert in place of the vip3Aa20 expression cassette exemplified herein. In this general regard, targeted homologous recombination, for example without limitation, can be used according to the subject invention. “Homologous recombination” refers to a reaction between any pair of nucleotide sequences having corresponding sites containing a similar nucleotide sequence (i.e., homologous sequences) through which the two molecules can interact (recombine) to form a new, recombinant DNA sequence. The sites of similar nucleotide sequence are each referred to herein as a “homology sequence”. Generally, the frequency of homologous recombination increases as the length of the homology sequence increases. Thus, while homologous recombination can occur between two nucleotide sequences that are less than identical, the recombination frequency (or efficiency) declines as the divergence between the two sequences increases. Recombination may be accomplished using one homology sequence on each of the donor and target molecules, thereby generating a “single-crossover” recombination product. Alternatively, two homology sequences may be placed on each of the target and donor nucleotide sequences. Recombination between two homology sequences on the donor with two homology sequences on the target generates a “double-crossover” recombination product. If the homology sequences on the donor molecule flank a sequence that is to be manipulated (e.g., a sequence of interest), the double-crossover recombination with the target molecule will result in a recombination product wherein the sequence of interest replaces a DNA sequence that was originally between the homology sequences on the target molecule. The exchange of DNA sequence between the target and donor through a double-crossover recombination event is termed “sequence replacement.” This type of technology is the subject of, for example, US patent Application Publication No. 2006/0253918, herein incorporated by reference. With the disclosed target site now being identified and with the sequences surrounding the identified target site, the skilled person will recognize that other methods for targeted integration of heterologous nucleic acids may be used. Such methods, for example without limitation, are disclosed in US Patent Application Publication No. 2007/0039074 and US Patent Application Publication No. 2006/0130179.
In one embodiment, the present invention encompasses a maize chromosomal target site located on chromosome 5 between a opie2 molecular marker set forth as nucleotides 1680-3338 of SEQ ID NO: 106 and a gag molecular marker set forth as nucleotides 43,275-45,086 of SEQ ID NO: 106, wherein the target site comprises a heterologous nucleic acid. In another embodiment, the maize chromosomal target site is located on chromosome 5 between nucleotides 25,454 and 25,513 of SEQ ID NO: 106. In yet another embodiment, the chromosomal target site is flanked 5′ by nucleotides 5,454 to 25,454 of SEQ ID NO: 106 and flanked 3′ by nucleotides 25,513 to 45,513 of SEQ ID NO: 106.
In one embodiment, the present invention encompasses a method of making a transgenic maize plant comprising inserting a heterologous nucleic acid at a position on chromosome 5 located between a opie2 molecular marker set forth as nucleotides 1680-3338 of SEQ ID NO: 106 and a gag molecular marker set forth as nucleotides 43,275-45,086 of SEQ ID NO: 106. In another embodiment, the heterologous nucleic acid is inserted on chromosome 5 between nucleotides 25,454 and 25,513 of SEQ ID NO: 106. In still another embodiment, the inserted heterologous nucleic acid is flanked 5′ by nucleotides 5,454 to 25,454 of SEQ ID NO: 106 and flanked 3′ by nucleotides 25,513 to 45,513 of SEQ ID NO: 106
The transgenic genotype of the present invention can be introgressed in any corn inbred or hybrid using art recognized breeding techniques. The goal of plant breeding is to combine in a single variety or hybrid various desirable traits. For field crops, these traits may include resistance to insects and diseases, tolerance to herbicides, tolerance to heat and drought, reducing the time to crop maturity, greater yield, and better agronomic quality. With mechanical harvesting of many crops, uniformity of plant characteristics such as germination and stand establishment, growth rate, maturity, and plant and ear height, is important.
Field crops are bred through techniques that take advantage of the plant's method of pollination. A plant is self-pollinated if pollen from one flower is transferred to the same or another flower of the same plant. A plant is cross-pollinated if the pollen comes from a flower on a different plant.
Plants that have been self-pollinated and selected for type for many generations become homozygous at almost all gene loci and produce a uniform population of true breeding progeny. A cross between two different homozygous lines produces a uniform population of hybrid plants that may be heterozygous for many gene loci. A cross of two plants each heterozygous at a number of gene loci will produce a population of hybrid plants that differ genetically and will not be uniform.
Corn (Zea mays L.), can be bred by both self-pollination and cross-pollination techniques. Corn has separate male and female flowers on the same plant, located on the tassel and the ear, respectively. Natural pollination occurs in corn when wind blows pollen from the tassels to the silks that protrude from the tops of the ears.
A reliable method of controlling male fertility in plants offers the opportunity for improved plant breeding. This is especially true for development of corn hybrids, which relies upon some sort of male sterility system. There are several options for controlling male fertility available to breeders, such as: manual or mechanical emasculation (or detasseling), cytoplasmic male sterility, genetic male sterility, gametocides and the like.
Hybrid corn seed is typically produced by a male sterility system incorporating manual or mechanical detasseling. Alternate strips of two corn inbreds are planted in a field, and the pollen-bearing tassels are removed from one of the inbreds (female). Providing that there is sufficient isolation from sources of foreign corn pollen, the ears of the detasseled inbred will be fertilized only from the other inbred (male), and the resulting seed is therefore hybrid and will form hybrid plants.
The laborious, and occasionally unreliable, detasseling process can be avoided by using one of many methods of conferring genetic male sterility in the art, each with its own benefits and drawbacks. These methods use a variety of approaches such as delivering into the plant a gene encoding a cytotoxic substance associated with a male tissue specific promoter or an antisense system in which a gene critical to fertility is identified and an antisense to that gene is inserted in the plant (see: Fabinjanski, et al. EPO 89/3010153.8 publication no. 329,308 and PCT application PCT/CA90/00037 published as WO 90/08828).
The use of male sterile inbreds is but one factor in the production of corn hybrids. Plant breeding techniques known in the art and used in a corn plant breeding program include, but are not limited to, recurrent selection, backcrossing, pedigree breeding, restriction length polymorphism enhanced selection, genetic marker enhanced selection and transformation. The development of corn hybrids in a corn plant breeding program requires, in general, the development of homozygous inbred lines, the crossing of these lines, and the evaluation of the crosses. Pedigree breeding and recurrent selection breeding methods are used to develop inbred lines from breeding populations. Corn plant breeding programs combine the genetic backgrounds from two or more inbred lines or various other germplasm sources into breeding pools from which new inbred lines are developed by selfing and selection of desired phenotypes. The new inbreds are crossed with other inbred lines and the hybrids from these crosses are evaluated to determine which of those have commercial potential. Plant breeding and hybrid development, as practiced in a corn plant-breeding program, are expensive and time-consuming processes.
Pedigree breeding starts with the crossing of two genotypes, each of which may have one or more desirable characteristics that is lacking in the other or which complements the other. If the two original parents do not provide all the desired characteristics, other sources can be included in the breeding population. In the pedigree method, superior plants are selfed and selected in successive generations. In the succeeding generations the heterozygous condition gives way to homogeneous lines as a result of self-pollination and selection. Typically in the pedigree method of breeding five or more generations of selfing and selection is practiced: F1→F2; F2→F3; F3→F4; F4→F.5; etc.
Recurrent selection breeding, backcrossing for example, can be used to improve an inbred line and a hybrid that is made using those inbreds. Backcrossing can be used to transfer a specific desirable trait from one inbred or source to an inbred that lacks that trait. This can be accomplished, for example, by first crossing a superior inbred (recurrent parent) to a donor inbred (non-recurrent parent), that carries the appropriate gene(s) for the trait in question. The progeny of this cross is then mated back to the superior recurrent parent followed by selection in the resultant progeny for the desired trait to be transferred from the non-recurrent parent. After five or more backcross generations with selection for the desired trait, the progeny will be homozygous for loci controlling the characteristic being transferred, but will be like the superior parent for essentially all other genes. The last backcross generation is then selfed to give pure breeding progeny for the gene(s) being transferred. A hybrid developed from inbreds containing the transferred gene(s) is essentially the same as a hybrid developed from the same inbreds without the transferred gene(s).
Elite inbred lines, that is, pure breeding, homozygous inbred lines, can also be used as starting materials for breeding or source populations from which to develop other inbred lines. These inbred lines derived from elite inbred lines can be developed using the pedigree breeding and recurrent selection breeding methods described earlier. As an example, when backcross breeding is used to create these derived lines in a corn plant-breeding program, elite inbreds can be used as a parental line or starting material or source population and can serve as either the donor or recurrent parent.
A single cross corn hybrid results from the cross of two inbred lines, each of which has a genotype that complements the genotype of the other. The hybrid progeny of the first generation is designated F1. In the development of commercial hybrids in a corn plant-breeding program, only the F1 hybrid plants are sought. Preferred F1 hybrids are more vigorous than their inbred parents. This hybrid vigor, or heterosis, can be manifested in many polygenic traits, including increased vegetative growth and increased yield.
The development of a corn hybrid in a corn plant breeding program involves three steps: (1) the selection of plants from various germplasm pools for initial breeding crosses; (2) the selfing of the selected plants from the breeding crosses for several generations to produce a series of inbred lines, which, although different from each other, breed true and are highly uniform; and (3) crossing the selected inbred lines with different inbred lines to produce the hybrid progeny (F1). During the inbreeding process in corn, the vigor of the lines decreases. Vigor is restored when two different inbred lines are crossed to produce the hybrid progeny (F1). An important consequence of the homozygosity and homogeneity of the inbred lines is that the hybrid between a defined pair of inbreds will always be the same. Once the inbreds that give a superior hybrid have been identified, the hybrid seed can be reproduced indefinitely as long as the homogeneity of the inbred parents is maintained.
A single cross hybrid is produced when two inbred lines are crossed to produce the F1 progeny. A double cross hybrid is produced from four inbred lines crossed in pairs (A×B and C×D) and then the two F1 hybrids are crossed again (A×B)×(C×D). A three-way cross hybrid is produced from three inbred lines where two of the inbred lines are crossed (A×B) and then the resulting F1 hybrid is crossed with the third inbred (A×B)×C. Much of the hybrid vigor exhibited by F1 hybrids is lost in the next generation (F2). Consequently, seed from hybrids is not used for planting stock.
Hybrid seed production requires elimination or inactivation of pollen produced by the female parent. Incomplete removal or inactivation of the pollen provides the potential for self-pollination. This inadvertently self-pollinated seed may be unintentionally harvested and packaged with hybrid seed.
Once the seed is planted, it is possible to identify and select these self-pollinated plants. These self-pollinated plants will be genetically equivalent to the female inbred line used to produce the hybrid.
Typically these self-pollinated plants can be identified and selected due to their decreased vigor. Female selfs are identified by their less vigorous appearance for vegetative and/or reproductive characteristics, including shorter plant height, small ear size, ear and kernel shape, cob color, or other characteristics.
Identification of these self-pollinated lines can also be accomplished through molecular marker analyses. See, “The Identification of Female Selfs in Hybrid Maize: A Comparison Using Electrophoresis and Morphology”, Smith, J. S. C. and Wych, R. D., Seed Science and Technology 14, pp. 1-8 (1995), the disclosure of which is expressly incorporated herein by reference. Through these technologies, the homozygosity of the self-pollinated line can be verified by analyzing allelic composition at various loci along the genome. Those methods allow for rapid identification of the invention disclosed herein. See also, “Identification of Atypical Plants in Hybrid Maize Seed by Postcontrol and Electrophoresis” Sarca, V. et al., Probleme de Genetica Teoritica si Aplicata Vol. 20 (1) p. 29-42.
As is readily apparent to one skilled in the art, the foregoing are only some of the various ways by which the inbred of the present invention can be obtained by those looking to introgress the transgenic genotype of the invention into other corn lines. Other means are available, and the above examples are illustrative only.
The following examples are intended solely to illustrate one or more preferred embodiments of the invention and are not to be construed as limiting the scope of the invention.
EXAMPLES
Example 1. Transformation and Selection of the MIR162 Event
The MIR604 event was produced by Agrobacterium-mediated transformation of a proprietary corn (Zea mays) line Immature embryos were transformed essentially as described in Negrotto et al. (Plant Cell Reports 19: 798-803, 2000), incorporated herein by reference, using a DNA fragment from plasmid pNOV1300 (SEQ ID NO: 3). pNOV1300 contains a nucleotide sequence comprising tandem expression cassettes. The first expression cassette comprises a ZmUbiInt promoter region from a Zea mays polyubiquitin gene, which contains the first intron (GenBank® Accession number S94464) operably linked to a vip3Aa19 coding sequence further operably linked to PEPC Intron #9 from the phosphoenolpyruvate carboxylase gene (GenBank® Accession Number X15239) from Zea mays (Matsuoka and Minami, 1989. European J. Of Biochem. 181:593-598) and a 35S terminator sequence from the 35S RNA from the cauliflower mosaic virus genome (Similar to GenBank® Accession Number AF140604). Its function is to provide a polyadenylation sequence (Franck et al., 1980. Cell 21:285-294). The vip3Aa19 gene in pNOV1300 comprises a synthetic maize-optimized vip3Aa coding sequence (Estruch, et al., 1999.) which was synthesized to accommodate the preferred codon usage for maize (Murray et al., 1989). The synthetic vip3Aa19 coding sequence used in plant transformations encodes the identical amino acid sequence as the native vip3Aa1 coding sequence found in the soil bacterium Bacillus thuringiensis strain AB88 (U.S. Pat. No. 5,877,012), with the exception of a single amino acid difference at position 284; the native vip3Aa1 coding sequence encodes lysine, whereas the synthetic vip3Aa19 coding sequence encodes glutamine at this position. The vip3Aa19 coding sequence encodes an insect control protein, Vip3Aa19 that provides resistance to lepidopteran insects. The second expression cassette is comprised of a ZmUbiInt promoter operably linked to a pmi coding sequence (also known as E. coli manA) encoding phosphomannose isomerase (GenBank® Accession number M15380), which catalyzes the isomerization of mannose-6-phosphate to fructose-6-phosphate (Negrotto et al., 2000). The pmi coding sequence is further operably linked to a nopaline synthase 3′ end transcription termination and polyadenylation sequence.
Immature embryos were excised from 8-12 day old ears and rinsed with fresh medium in preparation for transformation. Embryos were mixed with the suspension of Agrobacterium cells harboring the transformation vector pNOV1300, vortexed for 30 seconds, and allowed to incubate for an additional 5 minutes. Excess solution containing Agrobacterium was aspirated and embryos were then moved to plates containing a non-selective culture medium. Embryos were co-cultured with the remaining Agrobacterium at 22° C. for 2-3 days in the dark. Embryos were transferred to culture medium supplemented with ticarcillin (100 mg/ml) and silver nitrate (1.6 mg/l) and incubated in the dark for 10 days. Embryos producing embryogenic callus were transferred to cell culture medium containing mannose.
Regenerated plantlets were tested by TAQMAN® PCR analysis (see Example 2) for the presence of both the pmi and vip3Aa19 genes, as well as for the absence of the antibiotic resistance spectinomycin (spec) gene. It was later discovered (See Example 4 below) that during the transformation process two mutations were introduced into the vip3Aa19 coding sequence, one of which resulted in an amino acid change in the Vip3Aa19 protein. Therefore, this new vip3Aa coding sequence, which is unique to event MIR162, was designated vip3Aa20. The vip3Aa20 coding sequence encodes isoleucine at position 129 in place of the methionine residue encoded by the vip3Aa19 gene.
Plants positive for both transgenes, and negative for the spec gene, were transferred to the greenhouse for further propagation. Positive events were identified and screened using insect bioassays against fall armyworm. Insecticidal events were characterized for copy number by TAQMAN analysis. MIR162 was chosen for further analysis based on having a single copy of the transgenes, good protein expression as identified by ELISA, and good insecticidal activity against fall armyworm.
The breeding pedigree of the MIR162 event was as follows: T0 MIR162 plant (x NPH8431)→→NPH8431 (MIR162) F1 (x NP2161)→NP2161(MIR162) F1 (x NP2161)→NP2161 (MIR162) BC1F1 (x B9620)→F1 (x B9620)→BC1F1 (x B9620)→BC2F1 (x B9620)→BC3F1 (x B9620)→BC4F1 (x B9620). Plant material from the BC4 generation was used for the Southern analysis, copy number determination and sequencing of the insert DNA. Negative controls for the experiments consisted of 10 negative segregant plants from the BC4 generation.
Example 2. MIR162 Detection by TAQMAN PCR
TAQMAN analysis was essentially carried out as described in Ingham et al. (Biotechniques, 31:132-140, 2001) herein incorporated by reference. Briefly, genomic DNA was isolated from leaves of transgenic and non-transgenic corn plants using the Puregene® Genomic DNA Extraction kit (Gentra Systems, Minneapolis, Minn.) essentially according to the manufacturer's instruction, except all steps were conducted in 1.2 ml 96-well plates. The dried DNA pellet was resuspended in TE buffer (10 Mm Tris-HCl, pH 8.0, 1 mM EDTA).
TAQMAN PCR reactions were carried out in 96-well plates. For the endogenous corn gene control, primers and probes were designed specific to the Zea mays alcohol dehydrogenase (adhI) coding sequence (Genbank accession no. AF044295). It will be recognized by the skilled person that other corn genes can be used as endogenous controls. Reactions were multiplexed to simultaneously amplify vip3Aa and adhI or pmi and adhI. For each sample, a master mixture was generated by combining 20 μL extracted genomic DNA with 35 μL 2× TAQMAN Universal PCR Master Mix (Applied Biosystems) supplemented with primers to a final concentration of 900 nM each, probes to a final concentration of 100 nM each, and water to a 70 μL final volume. This mixture was distributed into three replicates of 20 μL each in 96-well amplification plates and sealed with optically clear heat seal film (Marsh Bio Products). PCR was run in the ABI Prism 7700 instrument using the following amplification parameters: 2 min at 50° C. and 10 min at 95° C., followed by 35 cycles of 15 s at 95° C. and 1 min at 60° C.
Results of the TAQMAN analysis demonstrated that event MIR162 had one copy of the vip3Aa20 gene and one copy of the pmi gene.
Primers and probes that were used in the TAQMAN PCR reactions are shown in Table 1.







TABLE 1







Primers used in TAQMAN Assay.









Primer Name
Primer Sequence
Sequence No:





Vip3Aa-forward
5′CACCTTCAGCAACCCGAACTA3′
SEQ ID NO: 4


Vip3Aa-reverse
5′GCTTAGCCTCCACGATCATCTT3′
SEQ ID NO: 5


Vip3Aa-probe
5′GTCCTCGTCGCTGCCCTTCACCT3′
SEQ ID NO: 6



(5′ label = FAM, 3′ label = TAMRA)


 


PMI-forward
5′CCGGGTGAATCAGCGTTT3′
SEQ ID NO: 7


PMI-reverse
5′GCCGTGGCCTTTGACAGT3′
SEQ ID NO: 8


PMI-probe
5′TGCCGCCAACGAATCACCGG3′
SEQ ID NO: 9



(5′ label = FAM, 3′label = TAMRA)


 


ZmADH-267forward
5′GAACGTGTGTTGGGTTTGCAT3′
SEQ ID NO: 10


ZmADH-337 reverse
5′TCCAGCAATCCTTGCACCTT3′
SEQ ID NO: 11


ZmADH-316 probe
5′TGCAGCCTAACCATGCGCAGGGTA3′
SEQ ID NO: 12



(5′label = TET, 3′ label = TAMRA)









Example 3. MIR162 Detection by Southern Blot
Genomic DNA used for southern analysis was isolated from pooled leaf tissue of 10 plants representing the BC4 generation of MIR162 using essentially the method of Thomas et al. (Theor. Appl. Genet. 86:173-180, 1993), incorporated herein by reference. All plants used for DNA isolation were individually analyzed using TAQMAN PCR (as described in Example 2) to confirm the presence of a single copy of the vip3Aa20 gene and the pmi gene. For the negative segregant controls, DNA was isolated from pooled leaf tissue of negative segregants from the BC4 generation. These negative segregant plants were individually analyzed using TAQMAN PCR to confirm the absence of the vip3Aa20 and pmi genes, but were, as expected, positive for the endogenous maize adhI gene.
Southern analysis was carried out using conventional molecular biology techniques. (See Chomczynski, P. 1992. Analytical Biochemistry 201:34-139) Genomic DNA (7.5 μg) was digested with restriction enzymes that digest within the event MIR162 insert, but not within the coding sequence that corresponds to the specific probe used in the experiment. This approach allowed for determination of the number of copies of each gene, corresponding to the specific probe used for each Southern analysis, which was incorporated into event MIR162.
Another series of restriction digests was performed in which the insert was digested with restriction enzymes that would release a fragment of known size from the insert. This approach provided additional evidence for the presence of a single copy of each coding sequence present in MIR162 and allowed for the detection of partial copies of the insert that may be closely linked to the MIR162 insert. Following agarose gel electrophoresis and alkaline transfer to a ZetaProbe® GT membrane (Bio-Rad, Cat. No. 162-0195), hybridizations were carried out using full-length PCR generated element probes. The probes were labeled with 32P via random priming using the MegaPrimeTM system (Amersham Biosciences, Cat. No. RPN1607). Hybridization was carried out at 65° C., followed by multiple washes in 2×SSC, 0.1% SDS and then 0.1×SSC and 0.1% SDS. The membranes were then subjected to autoradiography.
Included in each Southern analysis were three control samples: (1) DNA from a negative (non-transformed) segregant used to identify any endogenous Zea mays sequences that may cross-hybridize with the element-specific probe; (2) DNA from a negative segregant into which is introduced an amount of digested pNOV1300 that is equal to one copy number based on plasmid size was introduced, to demonstrate the sensitivity of the experiment in detecting a single gene copy within the Zea mays genome; and (3) Digested pNOV1300 plasmid equal to one copy number based on plasmid size, to act as a positive control for hybridization as well as to demonstrate the sensitivity of the experiment.
The results of Southern analyses demonstrated that the MIR162 insert contains a single copy of the vip3Aa20 gene and pmi gene and contains no pNOV1300 backbone sequences. A vip3Aa19 probe (SEQ ID NO: 13) was used for the vip3Aa20 Southern analysis. The nucleotide sequences of vip3Aa19 and vip3Aa20 differ by two nucleotides and are 99.9% identical. Therefore, the vip3Aa19 probe hybridized to the vip3Aa20 sequence present in MIR162 under stringent conditions. Using the vip3Aa19 probe, a KpnI and an EcoRV digest resulted in single hybridization bands approximately 8 kb and 13 kb in size, respectively. In addition, an NcoI double digest resulted in a single hybridization band consistent with the expected size of 4.6 kb. Using the pmi probe (SEQ ID NO: 14), a Acc65I and a BamHI digest resulted in single hybridization bands of approximately 4 kb and 6 kb in size, respectively. In addition, an XmaI+HindIII double digest resulted in a single hybridization band consistent with the expected size of 8.1 kb. The 8.1 kb XmaI+HindIII pNOV1300 band (positive control) also hybridized with the vip3Aa19 and pmi probes as expected. Some cross-hybridization in the plasmid-only lanes with the DNA ladder probe was detected. Typically commercially available DNA ladders may contain some vector sequences that can cross-hybridize with the plasmid control sequences as observed in these experiments, but, this does not impact the findings of this study. Finally, a pNOV1300 backbone probe did not hybridize demonstrating the absence of incorporation of any pNOV1300 vector backbone sequences into MIR162 during the transformation process.
Example 4. Heterologous DNA Insert Sequencing
The nucleotide sequence of the vip3Aa and pmi coding sequences in the heterologous DNA molecule inserted in MIR162 was determined to demonstrate overall integrity of the insert, contiguousness of the functional elements and to detect any individual basepair changes. The coding sequences were amplified from DNA derived from the BC4 generation. PCR amplification was carried out using either Expand High Fidelity PCR system (Roche, Cat. No. 1732650) or PfuUltraTM Hotstart High-Fidelity DNA polymerase (Stratagene, Cat. No. 600390). Each PCR product was individually cloned into either pCR®-XL-TOPO vector (Invitrogen, Cat. No. K4700-20) or pCR®-BluntII-TOPO vector (Invitrogen, Cat. No. K2800-20) and three separate clones for each PCR product were identified and sequenced. Sequencing was carried out using the ABI3730XL analyzer using ABI BigDye® 1.1 or Big Dye 3.1 dGTP (for GC-rich templates) chemistry. The sequence analysis was done using the Phred, Phrap, and Consed package from the University of Washington and was carried out to an error rate of less than 1 in 10,000 bases (Ewing & Green, 1998. Genome Research 8:186-194). The final consensus sequence for each gene was determined by combining the sequence data from the three individual clones to generate one consensus sequence for each gene. Sequence alignment was performed using the ClustalW program with the following parameters: scoring matrix blosum55, gap opening penalty 15, gap extension penalty 6.66 (Thompson et al, 1994. Nucleic Acids Research 22:4673-4680).
The full vip3Aa20 coding sequence was PCR amplified using primers MOV3Aa-01-5′: 5′ATGAACAAGAACAACACCAA3′ (SEQ ID NO: 15) and MOV3Aa-01-3′: 5′CTACTTGATGCTCACGTCGTAG3′ (SEQ ID NO: 16) and PfuUltra Hotstart enzyme generating a 2370 bp product. The PCR amplicon was sequenced using the primers shown in Table 2.









TABLE 2





Primer Name
quence (5′→3′)
Sequence No.







b03503b
ACGAGCAGAACCAGGTGC
SEQ ID NO: 17


 


b03503c
GGTGAAGAAGGACGGCAG
SEQ ID NO: 18


 


b03503d
ACCTGTCGCAAGCTGCTGGG
SEQ ID NO: 19


 


b03503e
TGGACAAGCTGCTGTGTC
SEQ ID NO: 20


 


b03503f
TGCAGGCCGACGAGAACAG
SEQ ID NO: 21


 


b03503g
TGATCCAGTACACCGTGAA
SEQ ID NO: 22


 


b03503h
ACCCTGACCCTGTACCAG
SEQ ID NO: 23


 


b03504b
GTGTTGCCGCTGATGTTG
SEQ ID NO: 24


 


b03504c
CGTACTCGGTCTTCGGCT
SEQ ID NO: 25


 


b03504d
CTGCAGGCCAAAGCCGTT
SEQ ID NO: 26


 


b03504e
TCGCCGTAGATCACCTCG
SEQ ID NO: 27


 


b03504f
GCTTGCGACAGGTGGTCA
SEQ ID NO: 28


 


b03504g
TTGCTGCTGGTCTCGGTGG
SEQ ID NO: 29


 


b03504h
CGTTGGCGATCTTAAGGAT
SEQ ID NO: 30


 


b00203c
GCAAGCCATCGATTCAC
SEQ ID NO: 31


 


b00203d
GCAACACCCTGACCCTG
SEQ ID NO: 32


 


b00203e
TCTACGACGTGAGCATCAAG
SEQ ID NO: 33


 


b00203f
GTAGAAGTGCACGATCGGG
SEQ ID NO: 34


 


b00203g
CGGTGCTGGTCCAGTTG
SEQ ID NO: 35









Two other PCR reactions overlapped the full vip3Aa20 coding sequence. The 5′ end of vip3Aa20 was covered with a PCR amplification using primers 162INSERT-F2: 5′ACACCAATGATGCAAATAGGC3′ (SEQ ID NO: 36) and VIP_R4 5′GAAGGTGTTCAGGTAGAACTCGAAG3′ (SEQ ID NO: 37) and Expand High Fidelity enzyme. The second reaction covered the 3′ end of vip3Aa20; the product was amplified with primers VIP-F3: 5′GGTGCTGTTCGAGAAGAGGT3′ (SEQ ID NO: 42) and PMI_REV1: 5′CGATTTATCACTCTCAATCACAT3′ (SEQ ID NO: 43) and Expand High Fidelity enzyme. The amplicons generated by these reactions comprised a 2946 bp nucleotide sequence (SEQ ID NO: 38) and a 2577 bp nucleotide sequence (SEQ ID NO: 44), respectively.
The consensus sequence data revealed two nucleotide changes in the vip3Aa coding sequence in MIR162 (designated vip3Aa20; SEQ ID NO: 1) compared to the vip3Aa coding sequence in pNOV1300 (designated vip3Aa19), which was used to transform MIR162. The first nucleotide change, a G to T mutation, occurred at position 387 of the vip3Aa19 coding sequence (SEQ ID NO: 3). This mutation resulted in the methionine at position 129 of Vip3Aa19 being changed to isoleucine in Vip3Aa20 (M129I), SEQ ID NO: 2. The second nucleotide change occurred at position 1683 of the coding sequence, a G to C mutation, but did not result in an amino acid change. Therefore, the vip3Aa20 coding sequence (SEQ ID NO: 1) and the Vip3Aa20 protein (SEQ ID NO: 2) are unique to the MIR162 event and can be used to identify any plant comprising the MIR162 transgenic genotype. The pmi coding sequence MIR162 was identical to that in the transformation plasmid pNOV 1300. An alignment of the Vip3Aa20 and Vip3Aa19 insecticidal proteins is shown in Table 3.







TABLE 3







Comparison of Vip3Aa20 and Vip3Aa19 amino acid sequences.








Name
Sequence Alignment





Vip3Aa20
  (1) MNKNNTKLSTRALPSFIDYFNGIYGFATGIKDIMNMIFKTDTGGDLTLDE


Vip3Aa19
  (1) MNKNNTKLSTRALPSFIDYFNGIYGFATGIKDIMNMIFKTDTGGDLTLDE


 


Vip3Aa20
 (51) ILKNQQLLNDISGKLDGVNGSLNDLIAQGNLNTELSKEILKIANEQNQVL


Vip3Aa19
 (51) ILKNQQLLNDISGKLDGVNGSLNDLIAQGNLNTELSKEILKIANEQNQVL


 


Vip3Aa20 Vip3Aa19






Vip3Aa20
(151) DKLDIINVNVLINSTLTEITPAYQRIKYVNEKFEELTFATETSSKVKKDG


Vip3Aa19
(151) DKLDIINVNVLINSTLTEITPAYQRIKYVNEKFEELTFATETSSKVKKDG


 


Vip3Aa20
(201) SPADILDELTELTELAKSVTKNDVDGFEFYLNTFHDVMVGNNLFGRSALK


Vip3Aa19
(201) SPADILDELTELTELAKSVTKNDVDGFEFYLNTFHDVMVGNNLFGRSALK


 


Vip3Aa20
(251) TASELITKENVKTSGSEVGNVYNFLIVLTALQAQAFLTLTTCRKLLGLAD


Vip3Aa19
(251) TASELITKENVKTSGSEVGNVYNFLIVLTALQAQAFLTLTTCRKLLGLAD


 


Vip3Aa20
(301) IDYTSIMNEHLNKEKEEFRVNILPTLSNTFSNPNYAKVKGSDEDAKMIVE


Vip3Aa19
(301) IDYTSIMNEHLNKEKEEFRVNILPTLSNTFSNPNYAKVKGSDEDAKMIVE


 


Vip3Aa20
(351) AKPGHALIGFEISNDSITVLKVYEAKLKQNYQVDKDSLSEVIYGDMDKLL


Vip3Aa19
(351) AKPGHALIGFEISNDSITVLKVYEAKLKQNYQVDKDSLSEVIYGDMDKLL


 


Vip3Aa20
(401) CPDQSEQIYYTNNIVFPNEYVITKIDFTKKMKTLRYEVTANFYDSSTGEI


Vip3Aa19
(401) CPDQSEQIYYTNNIVFPNEYVITKIDFTKKMKTLRYEVTANFYDSSTGEI


 


Vip3Aa20
(451) DLNKKKVESSEAEYRTLSANDDGVYMPLGVISETFLTPINGFGLQADENS


Vip3Aa19
(451) DLNKKKVESSEAEYRTLSANDDGVYMPLGVISETFLTPINGFGLQADENS


 


Vip3Aa20
(501) RLITLTCKSYLRELLLATDLSNKETKLIVPPSGFISNIVENGSIEEDNLE


Vip3Aa19
(501) RLITLTCKSYLRELLLATDLSNKETKLIVPPSGFISNIVENGSIEEDNLE


 


Vip3Aa20
(551) PWKANNKNAYVDHTGGVNGTKALYVHKDGGISQFIGDKLKPKTEYVIQYT


Vip3Aa19
(551) PWKANNKNAYVDHTGGVNGTKALYVHKDGGISQFIGDKLKPKTEYVIQYT


 


Vip3Aa20
(601) VKGKPSIHLKDENTGYIHYEDTNNNLEDYQTINKRFTTGTDLKGVYLILK


Vip3Aa19
(601) VKGKPSIHLKDENTGYIHYEDTNNNLEDYQTINKRFTTGTDLKGVYLILK


 


Vip3Aa20
(651) SQNGDEAWGDNFIILEISPSEKLLSPELINTNNWTSTGSTNISGNTLTLY


Vip3Aa19
(651) SQNGDEAWGDNFIILEISPSEKLLSPELINTNNWTSTGSTNISGNTLTLY


 


Vip3Aa20
(701) QGGRGILKQNLQLDSFSTYRVYFSVSGDANVRIRNSREVLFEKRYMSGAK


Vip3Aa19
(701) QGGRGILKQNLQLDSFSTYRVYFSVSGDANVRIRNSREVLFEKRYMSGAK


 


Vip3Aa20
(751) DVSEMFTTKFEKDNFYIELSQGNNLYGGPIVHFYDVSIK


Vip3Aa19
(751) DVSEMFTTKFEKDNFYIELSQGNNLYGGPIVHFYDVSIK





The shaded box indicates the amino acid change.






Example 5. Analysis of Flanking DNA Sequence
A number of methods are known to those of skill in the art to amplify unknown DNA sequences adjacent to a core region of known sequence. Those methods include, but are not limited to, inverse PCR (iPCR) [Ochman et. al., Genetics 120:621-623 (1988); Triglia et. al., Nucleic Acids Res. 16:8186 (1988)], panhandle PCR [Jones and Winistorfer, Nucleic Acids Res. 20:595-600 (1992); Jones and Winistorfer, Biotechniques 23:132-138 (1997)], cassette ligation-anchored PCR [Mueller and Wold, Science 246:780-786 (1989)], vectorette-PCR [Riley et. al., Nucleic Acids Res. 18:2887-2890 (1990)], novel-Alu-PCR [Puskas et. al., Nucleic Acids Res. 22:3251-3252 (1994)] and Thermal Asymmetric Interlaced PCR (TAIL-PCR) [Liu and Whittier, Genomics 25:673-681 (1995)].
One method used to amplify corn genome DNA sequence flanking the heterologous DNA inserted into event MIR162 was vectorette PCR essentially as described by Riley et al., Nucleic Acids Res. 18:2887-2890 (1990), incorporated herein by reference.
The 5′ flanking sequence and junction sequence was confirmed using standard PCR procedures. The following primer pairs, or complements thereof, were used to confirm the sequence: 162INSERT-F2: 5′ACACCAATGATGCAAATAGGC3′ (SEQ ID NO: 36)/VIP_R4: 5′GAAGGTGTTCAGGTAGAACTCGAAG3′ (SEQ ID NO: 37) and CJB 179: 5′ATGCAAATAGGCTGGGAATAGTC3′ (SEQ ID NO: 39)/CJB 134 5′GTACCAGCTTGCTGAGTGGCT3′ (SEQ ID NO: 40). The resulting amplicon has the sequence shown is SEQ ID NO: 41 and comprises the 5′ junction sequence of SEQ ID NO: 45. It will be recognized that other primer sequences can be used to confirm the flanking and junction sequences. Using this method, the MIR162 insert was found to be flanked 5′ by nucleotides 1040-1088 of the corn genomic sequence shown in SEQ ID NO: 46.
A larger region of the 5′ flanking sequence from event MIR162 was generated using the Seegene DNA Walking SpeedUpTM Premix kit following the manufacturer's instructions.
A first PCR reaction was performed independently in four individual tubes using primer FE1002: 5′CGTGACTCCCTTAATTCTCCGCT3′ (SEQ ID NO: 50) with one of the DW-ACP 1, 2, 3, or 4 primers supplied by the manufacturer. The following reagents were mixed in a PCR tube on ice: 100 μg MIR162 genomic DNA, 4 μl 2.5 μM DW-ACP (one each with DW-ACP 1, 2, 3, or 4), 4 μl 2.5 μM FE1002, 19 μl distilled water, and 25 μl 2× SeeAmpTM ACPTM Master Mix II. The tubes were placed in a preheated (94° C.) thermal cycler. PCR was completed using the following program: one cycle at 94° C. for five minutes, 42° C. for one minute, and 72° C. for two minutes, 30 cycles of 94° C. for 40 seconds, 55° C. for 40 seconds, and 72° C. for 90 seconds, and one cycle at 72° C. for seven minutes. The PCR products were purified using Exonuclease I and Shrimp Alkaline Phosphatase.
A second PCR reaction was performed independently in four individual tubes using primer FE1003: 5′GATCAGATTGTCGTTTCCCGCCTT3′ (SEQ ID NO: 51) with the DW-ACPN primer supplied by the manufacturer of the kit. The following reagents were mixed in a PCR tube on ice: 3 μl purified PCR product, 1 μl 10 μM DW-ACPN, 1 μl 10 μM FE1003, 5 μl distilled water, and 10 μl 2× SeeAmpTM ACPTM Master Mix II. The tubes were placed in a preheated (94° C.) thermal cycler. PCR was completed using the following program: one cycle at 94° C. for five minutes, 35 cycles of 94° C. for 40 seconds, 60° C. for 40 seconds, and 72° C. for 90 seconds, and one cycle at 72° C. for seven minutes.
A third PCR reaction was performed independently in four individual tubes using primer FE1004: 5′GATTGTCGTTTCCCGCCTTCAGTT3′ (SEQ ID NO: 52) with the Universal primer supplied by the manufacturer. The following reagents were mixed in a PCR tube on ice: 2 μl purified PCR product, 1 μl 10 μM Universal primer, 1 μl 10 μM FE1004, 6 μl distilled water, and 10 μl 2× SeeAmpTM ACPTM Master Mix II. The tubes were placed in a preheated (94° C.) thermal cycler. PCR was completed using the following program: one cycle at 94° C. for five minutes, 35 cycles of 94° C. for 40 seconds, 60° C. for 40 seconds, and 72° C. for 90 seconds, and one cycle at 72° C. for seven minutes.
Ten μl of the PCR products were run on a 1% agarose gel containing ethidium bromide. The appropriate band was extracted from the agarose gel and purified using a Qiagen Qiaquick Gel Extraction Kit according to the manufacturer's instructions. The extracted DNA was cloned into an Invitrogen TOPO-XL cloning vector according to the manufacturer's instructions. This clone was transformed into E. coli, and the plasmid DNA was extracted from the cells after overnight growth with a Qiagen Miniprep kit according to the manufacturer's instructions. This plasmid was used for end run sequencing.
A new primer was designed within the new, previously unknown sequence to be used with a primer in the heterologous DNA insert to amplify the full 1 kb of flanking sequence out of the genomic DNA. Flanking sequence primer 162DWConf3: 5′CCTGTGTTGTTGGAACAGACTTCTGTC3′ (SEQ ID NO: 53) and insert DNA primer FE0900: 5′GGCTCCTTCAACGTTGCGGTTCTGTC3′ (SEQ ID NO: 54) were used to amplify a nucleic acid molecule comprising the 5′ flanking sequence for confirmation. The sequence of the resulting amplicon is set forth in SEQ ID NO: 55. This 5′ amplicon comprises the 5′ junction sequence set forth in SEQ ID NO: 45. Ten μl of the PCR product (amplicon) was run on a 1% agarose gel containing ethidium bromide. The appropriate band was extracted from the agarose gel and purified using a Qiagen Qiaquick Gel Extraction Kit according to the manufacturer's instructions. The extracted DNA was cloned into an Invitrogen TOPO-XL cloning vector according to the manufacturer's instructions. This clone was transformed into E. coli. Plasmid DNA was extracted from the cells after overnight growth in media with a Qiagen Miniprep kit according to the manufacturer's instructions. Three plasmids were completely sequenced using the primers shown in Table 4. The plasmid sequences were aligned to generate the complete confirmed 5′ flanking sequence. Using this method, approximately 1 kb of the 5′ flanking sequence (SEQ ID NO: 46) was determined







TABLE 4







Primer sequences.









Primer Name
Sequence (5′→3′)
Sequence No.





b00201h
TTCACGGGAGACTTTATCTG
SEQ ID NO: 60


 


b00605a
CCGATTCATTAATGCAG
SEQ ID NO: 61


 


b00701b
ACGTAAAACGGCTTGTC
SEQ ID NO: 62


 


b00702b
GTTTAAACTGAAGGCGG
SEQ ID NO: 63


 


b00704h
AATAATATCACTCTGTACATCC
SEQ ID NO: 64


 


b01106f
GTTGTAAAACGACGG
SEQ ID NO: 65


 


b01709f
TAGGCACCCCAGGCTTTA
SEQ ID NO: 66


 


b03504a
AATTGAATTTAGCGGCCG
SEQ ID NO: 67


 


b05102f
GGTCCCTACAACATAAATAG
SEQ ID NO: 68


 


b05102g
TTCGTCCCTACTATCAACGC
SEQ ID NO: 69


 


b05102h
CTTTAGGCATCAGCGGGT
SEQ ID NO: 70


 


b05103a
AGCATCTGCGTAAGCACA
SEQ ID NO: 71


 


b05103b
CTGATGACACCAATGATGC
SEQ ID NO: 72


 


b05103c
GATCAGATTGTCGTTTCCC
SEQ ID NO: 73


 


b05103d
GCATCATTGGTGTCATCAG
SEQ ID NO: 74


 


b05103e
TGTGCTTACGCAGATGCT
SEQ ID NO: 75


 


b05103f
ACCCGCTGATGCCTAAAG
SEQ ID NO: 76


 


b05103g
GCGTTGATAGTAGGGACGAA
SEQ ID NO: 77


 


b05103h
CTATTTATGTTGTAGGGACC
SEQ ID NO: 78


 


b05210a
CTAGACTGGAAAGCGGAG
SEQ ID NO: 79


 


b05210b
CCACTTTCATCCCTAGTTG
SEQ ID NO: 80









The 3′ flanking sequence from event MIR162 was generated using the Clonetech GenomeWalkerTM Universal kit (Clonetech Laboratories, Inc.) following the manufacturer's instructions.
First, pools of uncloned, adaptor-ligated genomic DNA fragments, known as GenomeWalker “libraries” were constructed. Each library was constructed by digesting the MIR162 genomic DNA with a restriction enzyme (DraI, EcoRV, PvuII, StuI, and XmnI) as follows: For example, 25 μl MIR162 genomic DNA (0.1 μg/μl), 8 μl restriction enzyme (10 units/μl), 10 μl restriction enzyme buffer (10×), and 57 μl distilled H2O were mixed in a tube and incubated at 37° C. overnight.
DNA was then purified by using several rounds of phenol/chloroform extraction. Finally, the DNA was precipitated and washed with ethanol, dried and dissolved into 20 μl of TE buffer.
To ligate the GenomeWalker Adapter ends to the MIR162 genomic DNA, 4 μl of the digested, purified genomic DNA was mixed with 1.9 μl of GenomeWalker Adapter (25 μM), 1.6 μl 10× Ligation Buffer, and 0.5 μl T4 DNA Ligase (6 units/μl). These reactions were incubated overnight at 16° C. The reactions were stopped with incubation at 70° C. for five minutes. After the reaction was stopped, 72 μl of TE was added to each tube, and the contents were mixed thoroughly.
A first PCR reaction was performed using primer AP1, supplied by the manufacturer, with different primers designed within the known heterologous insert DNA sequence (Round 1 “gene specific primers” or “GSP1”). The following reagents were mixed in a PCR tube on ice: 1 μl of the appropriate MIR162 DNA library, 1 μl 10 μM AP1, 1 μl 10 μM GSP1, 1 μl 10 mM dNTPs, 5 μl 10× Advantage 2 PCR Buffer, 1 μl of BD Advantage 2 Polymerase, and 40 μl distilled water. PCR was completed using the following program: seven cycles at 94° C. for 25 seconds and 72° C. for four minutes, 32 cycles at 94° C. for 25 seconds and 67° C. for four minutes, and one cycle at 67° C. for four minutes. Each primary PCR reaction was diluted 50-fold by adding 1 μl of the primary PCR product with 49 μl of distilled water. The reactions that worked were (1) the DraI and the XmnI libraries with the GSP1 primer 162GW3F1: 5′TCTCTTGCTAAGCTGGGAGCTCGATCCG3′ (SEQ ID NO: 56) and primer AP1.
A second PCR reaction was performed independently using primer AP2, supplied by the manufacturer, with different primers designed within the known heterologous insert DNA sequence (Round 2 “gene specific primers” or “GSP2”). The following reagents were mixed in a PCR tube on ice: 1 μl of the appropriate diluted primary PCR product, 1 μl 10 μM AP2, 1 μl 10 μM GSP2, 1 μl 10 mM dNTPs, 5 μl 10× Advantage 2 PCR Buffer, 1 μl of BD Advantage 2 Polymerase, and 40 μl distilled water. PCR was completed using the following program: five cycles at 94° C. for 25 seconds and 72° C. for four minutes, 20 cycles at 94° C. for 25 seconds and 67° C. for four minutes, and one cycle at 67° C. for four minutes. The reactions that worked were (1) the DraI and the XmnI libraries with the GSP2 primer 162GW3F2: 5′AAGATTGAATCCTGTTGCCGGTCTTGCG3′ (SEQ ID NO: 57) and primer AP2.
Ten μl of the PCR products were run on a 1% agarose gel containing ethidium bromide. The appropriate band was extracted from the agarose gel and purified using a Qiagen Qiaquick Gel Extraction Kit according to the manufacturer's instructions. The extracted DNA was cloned into an Invitrogen TOPO-XL cloning vector according to the manufacturer's instructions. This clone was transformed into E. coli, and the plasmid DNA was extracted from the cells after overnight growth with a Qiagen Miniprep kit according to the manufacturer's instructions. This plasmid was sequenced using end run sequencing.
A new primer was designed within the new, previously unknown sequence to be used with a primer in the insert DNA to amplify approximately 1 kb of 3′ flanking sequence out of the genomic DNA. The insert DNA primer 162GW3F1: 5′TCTCTTGCTAAGCTGGGAGCTCGATCCG3′ (SEQ ID NO: 56) and a 3′ flanking sequence primer 1623′GWR1: 5CTGGTGAACCGATTTTTACGGAGG3′ (SEQ ID NO: 58) were used to amplify a nucleic acid molecule comprising the 3′ flanking sequence for confirmation. The sequence of the resulting amplicon is set forth in SEQ ID NO: 59. This 3′ amplicon comprises the 3′ junction sequence set forth in SEQ ID NO: 47. Ten μl of the PCR amplicon was run on a 1% agarose gel containing ethidium bromide. The appropriate band was extracted from the agarose gel and purified using a Qiagen Qiaquick Gel Extraction Kit according to the manufacturer's instructions. The extracted DNA was cloned into an Invitrogen TOPO-XL cloning vector according to the manufacturer's instructions. This clone was transformed into E. coli. Plasmid DNA was extracted from the cells after overnight growth in media with a Qiagen Miniprep kit according to the manufacturer's instructions. Three plasmids were completely sequenced using the primers shown in Table 5. The plasmid sequences were aligned to generate the complete confirmed 3′ flanking sequence (SEQ ID NO: 48).







TABLE 5







Primer sequences.









Primer Name
Sequence (5′→3′)
Sequence No.





b00106a
GATTGAATCCTGTTGCC
SEQ ID NO: 81


 


b00106b
TCTCATAAATAACGTCATGC
SEQ ID NO: 82


 


b00108a
TCTGTGGATAACCGTATTAC
SEQ ID NO: 83


 


b00201h
TTCACGGGAGACTTTATCTG
SEQ ID NO: 60


 


b00605a
CCGATTCATTAATGCAG
SEQ ID NO: 61


 


b00704h
AATAATATCACTCTGTACATCC
SEQ ID NO: 64


 


b00712e
AGTAACATAGATGACACCGC
SEQ ID NO: 84


 


b01106a
CCAGTGTGCTGGAATTCG
SEQ ID NO: 85


 


b01106f
GTTGTAAAACGACGG
SEQ ID NO: 65


 


b01107h
CCAGTGTGATGGATATCTGC
SEQ ID NO: 86


 


b01108e
CCAGTGTGCTGGAATTCG
SEQ ID NO: 87


 


b01111f
CCAGTGTGATGGATATCTGC
SEQ ID NO: 88


 


b01709f
TAGGCACCCCAGGCTTTA
SEQ ID NO: 66


 


b02701a
GTGTGCTGGAATTCGCCCTT
SEQ ID NO: 89


 


b02701e
TATCTGCAGAATTCGCCCTT
SEQ ID NO: 90


 


b02702a
GTGTGCTGGAATTCGCCCTT
SEQ ID NO: 91


 


b02702e
TATCTGCAGAATTCGCCCTT
SEQ ID NO: 92


 


b02703a
GTGTGCTGGAATTCGCCCTT
SEQ ID NO: 93


 


b02703e
TATCTGCAGAATTCGCCCTT
SEQ ID NO: 94


 


b02704a
GTGTGCTGGAATTCGCCCTT
SEQ ID NO: 95


 


b02811a
GGTCTTGCGATGATTATC
SEQ ID NO: 96


 


b05104c
GAGAGGAATGGCAGCAGA
SEQ ID NO: 97


 


b05104d
CATGACGGGTTTGAGATT
SEQ ID NO: 98


 


b05104e
AATCTCAAACCCGTCATG
SEQ ID NO: 99


 


b05104f
TCTGCTGCCATTCCTCTC
SEQ ID NO: 100


 


b05104g
GATCAACCCGGAGAGGAAT
SEQ ID NO: 101


 


b05104h
CCATGACGGGTTTGAGAT
SEQ ID NO: 102


 


b05105c
CAACCGACCTGACAAGTGAC
SEQ ID NO: 103


 


b05105e
ATCTCAAACCCGTCATGG
SEQ ID NO: 104


 


b05105f
ATTCCTCTCCGGGTTGATC
SEQ ID NO: 105









Example 6. Detection of Vip3Aa20 Protein in MIR162 by ELISA
Extracts were prepared from MIR162 leaves, roots, pith, kernels, silk, pollen and whole plants. They were quantitatively analyzed for Vip3Aa20 by ELISA using immunoaffinity purified goat anti-Vip3A and Protein A-purified rabbit anti-Vip3A polyclonal antibodies using art recognized ELISA procedures. Vip3Aa20 was detected in all tissues analyzed across all growth stages. The mean level of Vip3Aa20 protein detected in the whole plant at anthesis and seed maturity was 10 μg/g fresh weight and 16 μg/g fresh weight, respectively. The mean level of Vip3Aa20 protein in leaves at anthesis was 22 μg/g fresh weight.
Example 7. Field Efficacy of MIR162
The MIR162 event was tested in the field for efficacy against fall armyworm (FAW, Spodoptera frugiperda), corn earworm (CEW, Helicoverpa zea), black cutworm (BCW, Agrotis ipsilon), and European corn borer (ECB, Ostrinia nubilalis). Performance of the MIR162 event was compared with that of Warrior (Syngenta, Inc.), a conventional insecticide standard applied at a rate of 11.2 g a.i./acre, the transgenic corn event Bt11, comprising a cry1Ab gene, and a Bt11×MIR162 hybrid, produced by crossing a Bt11 inbred line with a MIR162 inbred line.
Twenty-eight trials were planted in 13 states that represented the major corn growing regions of the continental United States. Trials were planted in a randomized complete block design with four replicated plots per block. Plots were 17.5 row feet per treatment per replication. Planting density was targeted at approximately 30,000 plants/acre. Immuno-diagnostic strips were used to confirm the presence or absence of the Vip3Aa20 and Cry1Ab proteins in the different treatment groups.
Natural pest infestations were utilized in trials where populations were sufficiently high; where they were not, artificial infestations were carried out. Artificial infestation with two 2nd- to 3rd-instar larvae at V1-V2 was utilized in the BCW trials. Plots were rated at 3, 7, and 14 days post-infestation. BCW damage was recorded as partially damaged plants and fully cut plants. FAW plots were rated 7 and 14 days post-infestation or after 3rd-instar larvae were observed in control plants. The following scale was used to evaluate FAW and CEW leaf damage:

    
    
        0.01—No visible leaf damage
        1—Pin-hole damage on a few leaves
        2—Small amount of shot-hole damage on a few leaves
        3—Shot-hole damage on several leaves
        4—Shot-hole damage and lesions on a few leaves
        5—Lesions on several leaves
        6—Large lesions on several leaves
        7—Large lesions and portions eaten away on a few leaves
        8—Large lesions and portions eaten away on several leaves
        9—Large lesions and portions eaten away on most leaves
    
    


Plant damage was assessed for both first and second generation ECB. The following scale was used for rating first generation damage, typically when larvae were in the 3rd to 4th instar:

    
    
        1—No visible leaf damage
        2—Small amount of shot-hole injury on a few leaves
        3-Shot-hole injury common on several leaves
        4—Several leaves with shot-holes and elongated lesions
        5—Several leaves with elongated lesions
        6—Several leaves with elongated lesions about 2.5 cm
        7—Long lesions common on about one half of the leaves
        8—Long lesions common on about two-thirds of the leaves
        9—Most leaves with long lesions
    
    


Second generation ECB damage was assessed three to four weeks after artificial infestation or the end of the peak egg laying period. The following measurements were taken: number live larvae/stalk, number live larvae/shank, number live larvae/ear, number of tunnels/stalk, cumulative tunnel length (cm)/stalk, cumulative tunnel length (cm)/shank, number tunnels/ear, cumulative tunnel length kernel damage (cm)/ear, and % infested plants.
CEW trials were generally planted late to increase natural infestation levels. Feeding damage to ears was evaluated when CEW larvae on control plants were at the L5-L6 growth stage. Ear ratings included recording the number of larvae observed per ear and length of visible kernel feeding measured from the ear tip to the average lowest kernel destroyed.
Results of the BCW field trial are shown in Table 6. Less than 3% of the MIR162 plants and Bt11×MIR162 plants were cut by BCW larvae. Significant numbers of Bt11 and control plants were cut. Plants comprising the MIR162 genotype had less BCW feeding damage than the conventional insecticide treated plants.







TABLE 6







Stalk damage ratings from five trials with BCW at 21 days after


infestation. Damage was measured as percent of total plants cut.










Treatment
% Cut Plants














MIR162
2



Bt11
42



Bt11 X MIR162
3



Warrior Insecticide
12



Negative Control
40










The FAW field trial results are shown in Table 7. FAW feeding damage was measured on a scale of 0.01 to 9. Mean feeding damage in the MIR162 hybrids was very low (<1) and significantly lower than average damage observed in the Bt11 and conventional insecticide treatments. Insect pressure in these trials was heavy with approximately 50 to 100 neonate larvae/plant. Bt11 provided some protection from damage, whereas the conventional insecticide treatment provided no protection, sustaining the same amount of damage as the control.







TABLE 7







Leaf feeding damage ratings from five trials for FAW. Mean damage


ratings at 14 days after infestation are presented for each treatment.










Treatment
Mean Leaf Damage Rating (0.01-9)














MIR162
0.90



Bt11
2.52



Bt11 X MIR162
0.84



Warrior Insecticide
3.60



Negative Control
3.78










Results of the trials to assess first generation ECB damage are presented in Table 8. ECB feeding damage was rated on a scale of 1-9. In these trials, MIR162 conferred minimal protection against ECB feeding damage. Bt11 fully protected the plants from ECB feeding damage. The Bt11×MIR162 plants had the same level of protection as the Bt11 plants. The conventional insecticide treatment provided better protection than the MIR162 trait but significantly less protection than that provided by Bt11.







TABLE 8







Leaf feeding damage field trial. Mean damage ratings


at 14 days after infestation.










Treatment
Mean Leaf Damage Rating (1-9)














MIR162
2.95



Bt11
1.00



Bt11 X MIR162
1.00



Warrior Insecticide
2.05



Negative Control
3.88










Second generation ECB damage results are presented in Table 9. Feeding damaged was measured as cumulative tunnel length in each corn stalk (if more than one tunnel was found, tunnel lengths were summed). The Bt11 and Bt11×MIR162 treatments provided strong protection against stalk boring, whereas no protection against tunneling was provided by MIR162 alone or the insecticide treatment.







TABLE 9







Stalk damage ratings from seven trials for second generation


ECB larvae measured in tunnel length (cm) per stalk. Measurements


were taken three to four weeks after artificial infestation.










Treatment
Mean Tunnel Length (cm)














MIR162
5.46



Bt11
0.37



Bt11 X MIR162
0.48



Warrior Insecticide
5.06



Negative Control
5.04










Results of trials to assess CEW damage are presented in Table 10. Feeding damage was rated as length of kernel damage per ear, measured from the ear tip to the average lowest kernel destroyed. Significant ear damage was observed in the Bt11, insecticide, and check plots. Bt11 provided some level of protection compared to untreated check and was comparable to the protection provided by the conventional insecticide treatment. MIR162 and Bt11×MIR162 provided almost complete protection of the ears from CEW larval feeding damage.







TABLE 10







Ear damage ratings from six trials for CEW measured as


average length of feeding damage. Measurements were


taken when CEW larvae were L5-L6 in check plants.










Treatment
Mean Ear Damage (cm)














MIR162
0.17



Bt11
2.24



Bt11 X MIR162
0.02



Warrior Insecticide
2.20



Negative Control
3.42










Example 8. Efficacy of MIR162 Against Western Bean Cutworm
Current commercial transgenic events producing Cry1Ab protein have not provided acceptable levels of protection against the western bean cutworm (WBCW, Striacosta albicosta). Therefore, MIR162 alone and stacked with other transgenic genotypes was tested for efficacy against WBCW.
WBCW eggs were collected from wild caught female moths. Larvae were fed on a meridic black cutworm diet until use in the experiments. Corn plants were field grown.
The following treatments were tested: MIR162, Bt11, MIR604, MIR162×Bt11, MIR162×MIR604, MIR604×Bt11, Force® (Syngenta, Inc.), a conventional insecticide applied at planting to a negative isoline, and two negative control isolines. MIR604 is a novel transgenic corn event that comprises a cry3A055 gene encoding a protein that is active against corn rootworm (Diabrotica spp.) larvae and is disclosed in US Patent Application publication No. 2005/0216970, published Sep. 29, 2005, herein incorporated by reference.
For the experiments, a two-inch piece of green silks and husk was cut from ears from field grown corn plants in each treatment and replication. The terminal brown ends of the silks were removed and the husk discarded. Approximately 1.5 inches of silks were placed in individual 14 ml plastic cups. One larva was then placed in each cup and the cups sealed. Several different stages of larvae were tested ranging from 3rd to 6th-instars. Cups containing silks and larvae were held at natural day length and room temperature for the duration of the experiments. Larval survival was recorded after eight days. Treatments were replicated four times per experiment.
Results of the WBCW experiments are presented in Table 11. Survival of WBCW on silks from the negative isolines and the conventional insecticide treatment were nearly 100%. Survival of WBCW larvae on Bt11 and MIR604 silks, tested either alone or in combination in the same plant, was not different from survival on the negative isolines. Survival of WBCW larvae was reduced when larvae were fed silks from MIR162. The combination of MIR162×Bt11 in the same plant did not decrease survival any further than MIR162 alone. However, surprisingly, when the MIR162 transgenic genotype was stacked with the MIR604 transgenic genotype in the same plant, larval mortality significantly increased compared to MIR162 or MIR604 alone.







TABLE 11







Percent (±SE) survival of WBCW larvae on corn silks.









Experiment Number














Treatment
1
2
3
4
5
6
7





Bt11
75 (25)
75 (25)
100 (0)
100 (0)
100 (0) 
100 (0) 
100 (0)


MIR162
25 (25)
0 (0)
 0 (0)
 50 (29)
50 (29)
50 (29)
 0 (0)


MIR604




100 (0) 
100 (0) 
100 (0)


MIR162xBt11
25 (25)
25 (25)
 0 (0)
 0 (0)
25 (25)
25 (25)
 25 (25)


MIR162xMIR604




0 (0)
25 (25)
 50 (29)


MIR604xBt11






 75 (25)


Force




100 (0) 
100 (0) 
100 (0)


Neg. Control #1
100 (0) 
75 (25)
100 (0)
100 (0)
75 (25)
100 (0) 
100 (0)


Neg. Control #2
100 (0) 
100 (0) 
100 (0)
100 (0)
100 (0) 
100 (0) 
100 (0)









Example 9. Use of Event Mir162 Insertion Site for Targeted Integration in Maize
The MIR162 flanking sequences disclosed in SEQ ID NO: 46 and SEQ ID NO: 48 were used to search maize genomic databases. Identical matches to both flanking sequences where found on a BAC clone, CH201-307P5, of chromosome 5 (NCBI Accession No. AC185313) on Contig 13 (SEQ ID NO: 106). More specifically, the MIR162 insert is on chromosome 5 between a 5′ molecular marker, designated herein as the Opie2 marker (nucleotides 1680-3338 of SEQ ID NO: 106), and a 3′ molecular marker, designated herein as the gag marker (nucleotides 43,275-45,086 of SEQ ID NO: 106). Using this information, it was determined that the heterologous DNA inserted into MIR162 displaced 58 nucleotides of maize genomic DNA, nucleotides 25,455 to 25,512 of SEQ ID NO: 106 (also shown as SEQ ID NO: 107), which is between the 5′ flanking sequence (nucleotides 1-25,454 of SEQ ID NO: 106) and the 3′ flanking sequence (nucleotides 25,513-51,328 of SEQ ID NO: 106).
Consistent agronomic performance of the transgene of event MIR162 over several generations under field conditions suggests that these identified regions around the MIR162 insertion site provide good genomic locations for the targeted integration of other transgenic genes of interest. Such targeted integration overcomes the problems with so-called “positions effects,” and the risk of creating a mutation in the genome upon integration of the transgene into the host. Further advantages of such targeted integration include, but are not limited to, reducing the large number of transformation events that must be screened and tested before obtaining a transgenic plant that exhibits the desired level of transgene expression without also exhibiting abnormalities resulting from the inadvertent insertion of the transgene into an important locus in the host genome. Moreover, such targeted integration allows for stacking transgenes rendering the breeding of elite plant lines with both genes more efficient.
Using the above disclosed teaching, the skilled person is able to use methods know in the art to target heterologous nucleic acids of interest to the same insertion site on chromosome 5 as that in MIR162 or to a site in close proximity to the insertion site in MIR162. One such method is disclosed in US Patent Application Publication No. 20060253918, herein incorporated by reference in its entirety. Briefly, up to 20 Kb of the genomic sequence flanking 5′ to the insertion site (nucleotides 5,454 to 25,454 of SEQ ID NO: 106) and up to 20 Kb of the genomic sequence flanking 3′ to the insertion site (nucleotides 25,513 to 45,513 of SEQ OD NO: 106) are used to flank the gene or genes of interest that are intended to be inserted into a genomic location on Chromosome 5 via homologous recombination. These sequences can be further flanked by T-DNA border repeats such as the left border (LB) and right border (RB) repeat sequences and other booster sequences for enhancing T-DNA delivery efficiency. The gene or genes of interest can be placed exactly as in the MIR162 insertion site or can be placed anywhere within the 20 Kb regions around the MIR162 insertion sites to confer consistent level of transgene expression without detrimental effects on the plant. The DNA vectors containing the gene or genes of interest and flanking sequences can be delivered into plant cells via one of the several methods known to those skilled in the art, including but not limited to Agrobacterium-mediated transformation. The insertion of the DNA vector into the MIR162 target site can be further enhanced by one of the several methods, including but not limited to the co-expression or up-regulation of recombination enhancing genes or down-regulation of endogenous recombination suppression genes. Furthermore, it is known in the art that cleavage of specific sequences in the genome can be used to increase homologous recombination frequency, therefore insertion into the MIR162 insertion site and its flanking regions can be enhanced by expression of natural or designed sequence-specific endonucleases for cleaving these sequences. Thus, using the teaching provided herein, any heterologous nucleic acid can be inserted on maize chromosome 5 at a target site located between nucleotides 25,454 and 45,513 of SEQ ID NO: 106 or a target site in the vicinity to this site.
Example 10. Use of Event MIR162 Insertion Site and Flanking Sequences for Stabilization of Gene Expression
The genomic sequences flanking the MIR162 insertion site may also be used to stabilize expression of other gene(s) of interest when inserted as a transgene in other genomic locations in maize and other crops. Specifically, up to 20 Kb of the genomic sequence flanking 5′ to the insertion site (nucleotides 5,454 to 25,454 of SEQ ID NO: 106) and up to 20 Kb of the genomic sequence flanking 3′ to the insertion site (nucleotides 25,513 to 45,513 of SEQ OD NO: 106) are used to flank the gene or genes of interest that are intended to be inserted into the genome of plants. These sequences can be further flanked by T-DNA border repeats such as the left border (LB) and right border (RB) repeat sequences and other booster sequences for enhancing T-DNA delivery efficiency. The gene or genes of interest can be placed exactly as in the MIR162 insertion site or can be placed anywhere within the 20 Kb regions around the MIR162 insertion sites to confer consistent level of transgene expression. The DNA vectors containing the gene or genes of interest and MIR162 insertion site flanking sequence can be delivered into plant cells via one of the several methods known to those skilled in the art, including but not limited to protoplast transformation, biolistic bombardment and Agrobacterium-mediated transformation. The delivered DNA can be integrated randomly into a plant genome or can also be present as part of the independently segregating genetic units such as artificial chromosome or mini-chromosome. The DNA vectors containing the gene(s) of interest and the MIR162 insertion site flanking sequences can be delivered into plant cells. Thus, by surrounding a gene or genes of interest with the genomic sequence flanking the MIR162 insertion site, the expression of such genes are stabilized in a transgenic host plant such as a dicot plant or a monocot plant like corn.
DEPOSIT
Applicants have made a deposit of corn seed of event MIR162 disclosed above on 23 Jan. 2007 in accordance with the Budapest Treaty at the American Type Culture Collection (ATCC), 1801 University Boulevard, Manassas, Va. 20110 under ATCC Accession No. PTA-8166. The deposit will be maintained in the depositary for a period of 30 years, or 5 years after the last request, or the effective life of the patent, whichever is longer, and will be replaced as necessary during that period. Applicants impose no restrictions on the availability of the deposited material from the ATCC; however, Applicants have no authority to waive any restrictions imposed by law on the transfer of biological material or its transportation in commerce. Applicants do not waive any infringement of their rights granted under this patent or under the Plant Variety Protection Act (7 USC 2321 et seq.).
All publications and published patent documents cited in this specification are incorporated herein by reference to the same extent as if each individual publication or patent document was specifically and individually indicated to be incorporated by reference.

## Claims

1. A method of detecting the presence of a nucleic acid molecule that is unique to event MIR162 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 MIR162 produces an amplicon that is diagnostic for event MIR162;
(b) performing a nucleic acid amplification reaction, thereby producing the amplicon; and
(c) detecting the amplicon.

2. A method of detecting the presence of a nucleic acid molecule that is unique to event MIR162 in a sample comprising corn nucleic acids, the method comprising:
(a) contacting the sample with a probe that hybridizes under high stringency conditions with genomic DNA from event MIR162 and does not hybridize under high stringency conditions with DNA of a control corn plant, wherein the probe comprises the nucleotide sequence of SEQ ID NO: 45 or SEQ ID NO: 47;
(b) subjecting the sample and probe to high stringency hybridization conditions; and
(c) detecting hybridization of the probe to the nucleic acid molecule.

3. An amplicon diagnostic for event MIR162, wherein said amplicon is at least 20 nucleotides in length and comprises the nucleic acid sequence of SEQ ID NO: 45, SEQ ID NO: 47, the nucleotide of position 387 of SEQ ID NO: 1 or the nucleotide of position 1683 of SEQ ID NO: 1.

4. A pair of polynucleotide primers comprising a first polynucleotide primer and a second polynucleotide primer which function together in the presence of an event MIR162 DNA template in a sample to produce an amplicon diagnostic for event MIR162, wherein said first primer and second primer comprise at least 10 contiguous nucleotides of SEQ ID NO: 1 and wherein said amplicon is at least 20 nucleotides in length and comprises the nucleotide of position 387 of SEQ ID NO: 1 and/or position 1683 of SEQ ID NO: 1.

5. A method of detecting the presence of a nucleic acid molecule that is unique to event MIR162 in a sample comprising corn nucleic acids, the method comprising:
(a) contacting the sample with a pair of primers of claim 4 that, when used in a nucleic-acid amplification reaction with genomic DNA from event MIR162 produces an amplicon that is diagnostic for event MIR162;
(b) performing a nucleic acid amplification reaction, thereby producing the amplicon; and
(c) detecting the amplicon.