JP6472474B2 - AAD-1 event DAS-40278-9, related transgenic corn lines and their event-specific identification - Google Patents

Description

aad-1 gene (Sphingobium herbicidov
orans) is an aryloxyalkanoate dioxygenase (AAD-)
1) It encodes a protein. This trait provides resistance to 2,4-dichlorophenoxyacetic acid and aryloxyphenoxypropionate (commonly called “fop” herbicides such as quizalofop) herbicides, It can be used as a selection marker during transformation and in breeding nurseries. The aad-1 gene itself was first disclosed in WO 2005/107437 for herbicide resistance in plants (
See also US 2009-0093366).

Expression of heterologous or foreign genes in plants is affected by where the foreign gene is inserted into the chromosome. This can be due, for example, to the proximity of transcriptional regulatory elements (eg enhancers) close to the chromatin structure (eg heterochromatin) or integration sites (Weising et al., Ann. Rev. Genet 22: 421-477, 1988). The same gene in the same type of transgenic plant (or other organism) can exhibit a wide variety of expression levels between different events. There may also be differences in the spatial or temporal pattern of expression. For example, the difference in relative expression of transgenes in various plant tissues is
It may not correspond to the pattern predicted from transcriptional regulatory elements present in the introduced gene construct.

Thus, a large number of events are often created and screened to identify events that express the introduced gene of interest at a satisfactory level for a purpose. For commercial purposes, it is common to generate hundreds to thousands of different events and screen these events for a single event with the desired transgene expression level and pattern. Events having the desired level and / or pattern of transgene expression can be achieved by sexually crossing the transgene with other genetic backgrounds using conventional breeding methods (
Useful for gene transfer by sexual outcrossing). Such cross offspring maintain the transgene expression characteristic of the original transformant. This strategy ensures reliable gene expression in several varieties that are well suited to local growth conditions.

US Patent Application Publication Nos. 20020120964A1 and 20040009504A
No. 1 relates to cotton event PV-GHGT07 (1445) and compositions and methods for detecting it. WO 02/100163 relates to cotton event MONI 5985 and compositions and methods for detecting it. WO 2004/011601 relates to a corn event MON863 plant and compositions and methods for detecting it. WO 2004/072235 relates to cotton event MON 88913 and compositions and methods for detecting it.

WO 2006/098952 relates to corn event 3272. WO200
7/142840 relates to the corn event MIR162.

US Pat. No. 7,179,965 relates to cotton having a cry1F event and a cry1Ac event.

AAD-1 corn with the specific events disclosed herein has not been previously disclosed.

The present invention is registered with American Type Culture Collection (ATCC) under the accession number PTA-
It relates to the AAD-1 corn event called DAS-40278-9, with the seed deposited at 10244, and offspring derived therefrom. Other aspects of the invention include progeny plants, seeds and grains or renewable parts of plants and the corn event DAS-
40278-9 seeds and progeny, and food or feed products made from any of these. The present invention includes, but is not limited to, pollen, ovules, flowers, shoots
Also included are plant parts of the corn event DAS-40278-9, including roots and leaves and nuclei of vegetative cells, pollen cells and egg cells. The present invention relates to phenoxyauxin type and / or
Or a maize plant having resistance to aryloxyalkanoate herbicides, a novel genetic composition of maize event DAS-40278-9 and an aspect of agronomic performance of maize plant comprising maize event DAS-40278-9 Also about

The invention relates in part to plant breeding and herbicide-tolerant plants. The present invention includes a novel aad-1 transformation event in a corn plant comprising a polynucleotide sequence described herein, inserted at a specific site in the corn cell genome.

In some embodiments, the event / polynucleotide sequence can be “multiplied” with other traits including, for example, other herbicide resistance gene (s) and / or insect inhibitory proteins. However, the subject invention, as described herein,
Includes plants with a single event.

Additional traits include plant breeding, corn event DAS-40278-
Multiplication can be achieved by retransformation of transgenic plants containing 9 or addition of new traits by targeted integration by homologous recombination.

Other embodiments include, for example, a corn event DAS comprising a pat gene expression cassette
Includes excision of a polynucleotide sequence containing -40278-9. By excision of the polynucleotide sequence, the altered event is retargeted to a specific chromosomal site where additional polynucleotide sequences can be multiplied with the corn event DAS-40278-9.

In one embodiment, the present invention provides a 2008 DAS linakge map.
) On chromosome 2 at approximately 20 cM between SSR markers UMC1265 (see SEQ ID NO: 30 and SEQ ID NO: 31) and MMC0111 (see SEQ ID NO: 32 and SEQ ID NO: 33) at approximately 20 cM above A corn chromosome target site, wherein the target site comprises a heterologous nucleic acid. In another embodiment, the present invention provides SEQ ID NO: 2
Corn chromosomal target site including the location at or defined by 9 and its residues described herein as recognized by one skilled in the art.

In one embodiment, the present invention provides approximately 20 on the 2008 DAS corn chain map.
Heterologous to a position on chromosome 2 at approximately 20 cM between SSR markers UMC1265 (see SEQ ID NO: 30 and SEQ ID NO: 31) and MMC0111 (see SEQ ID NO: 32 and SEQ ID NO: 33) at cM A method of producing a transgenic corn plant comprising the step of inserting a nucleic acid is included. In yet another embodiment, the inserted heterologous nucleic acid touches 5 ′ with all or part of the 5 ′ flanking sequence defined herein with reference to SEQ ID NO: 29, see SEQ ID NO: 29 Then, it makes contact with 3 ′ or all or part of the 5 ′ flanking sequence defined in this specification.

Further, the subject invention provides an assay for detecting the presence of a subject event in a sample (eg, corn kernels). The assay is based on the DNA sequence of the recombinant construct on the genomic sequence adjacent to the insertion site, inserted into the maize genome. Kits and conditions useful for performing the assay are also provided.

Thus, the subject invention relates in part to the cloning and analysis of the DNA sequence of the entire AAD-1 insert and its border regions (in transgenic corn lines). These sequences are unique. Based on these inserts and border sequences, event specific primers were generated. PCR analysis demonstrated that these events can be identified by analysis of PCR amplicons generated using these event-specific primer sets. Thus, these and other related procedures can be used to uniquely identify corn lines containing the subject invention event.

A plasmid map of pDAS1740 is shown. The elements of the insert for DAS-40278-9 (pDAS1740) are shown. Figure 9 shows the restriction map and elements of the insert for DAS-40278-9 (pDAS1740). Amplicons, primers and cloning strategies for DNA inserts and borders for DAS-40278-9 are shown. Primer positions relative to insert and border for DAS-40278-9 are indicated. The junction area and insertion for DAS-40278-9 is shown. 10 is a breeding diagram referred to in Example 7.

DETAILED DESCRIPTION OF THE INVENTION The present invention relates in part to plant breeding and herbicide tolerant plants. The present invention relates to a novel transformation event of a maize plant (maize) comprising a subject aad-1 polynucleotide sequence described herein, inserted at a specific location in the genome of a maize cell. Including. In some embodiments, the polynucleotide sequence is “multiplied” with other trait such as, for example, other herbicide resistance gene (s) and / or gene (s) encoding insect inhibitory protein. be able to. In some embodiments, the polynucleotide sequences described above can be excised and then retargeted with additional polynucleotide sequences. However, the subject invention includes plants having a single event as described herein.

Furthermore, the subject invention provides an assay for detecting the presence of a subject event in a sample. An aspect of the subject invention is a method of designing and / or generating any diagnostic nucleic acid molecule exemplified or suggested herein, particularly based in whole or in part on the subject flanking sequences. Including.

More specifically, the subject invention, in part, is a transgenic corn event DAS-40278-9 (also known as pDAS1740-278), plant lines containing these events, and inserts and / or its It relates to the cloning and analysis of the DNA sequence of the border region. The plant lines of the subject invention can be detected using the sequences disclosed and suggested herein.

In some embodiments, the present invention relates to herbicide-tolerant corn lines and their identification. The subject invention relates, in part, to detecting the presence of a subject event to determine whether sexual cross offspring contain the event of interest. In addition, methods for detecting events are included to help comply with regulations that require, for example, pre-market approval and labeling of foods from recombinant crop plants. The presence of the subject event can be detected by any known nucleic acid detection method such as polymerase chain reaction (PCR) or DNA hybridization using nucleic acid probes. Event specific PCR
Are discussed, for example, in Windels et al. (Med. Fac. Landbouww, Univ. Gent 64 / 5b: 459462, 1999). This relates to the identification of glyphosate resistant soybean event 40-3-2 by PCR using a primer set spanning the junction between the insert and flanking DNA. More specifically, one primer contained the sequence from the insert and the second primer contained the sequence from the flanking DNA.

Corn is sphingobium herbicidovor, which encodes an aryloxyalkanoate dioxygenase (AAD-1) protein.
modified by insertion of the aad-1 gene from ans). This trait provides resistance to 2,4-dichlorophenoxyacetic acid and aryloxyphenoxypropionate (commonly called “hop” herbicides such as quizalofop) herbicides, during plant transformation and breeding It can be used as a selection marker in a tree place. Plasmid pDAS17
Transformation of maize with DNA fragments from 40 by breeding, event DAS
-40278-9 was produced.

Genomic DNA samples extracted from 20 individual corn plants from 5 generations of event DAS-40278-9 and 4 plants per generation were used for molecular characterization of AAD-1 corn event DAS-40278-9. Selected. AAD-1 protein expression was tested using an AAD-1 specific rapid test strip kit. AAD-
Only plants that tested positive for 1 protein expression were selected for subsequent molecular characterization. By Southern hybridization, the aad-1 gene is present in corn plants tested positive for AAD-1 protein expression, and the aad-1 gene is
It was confirmed that when hybridized with the aad-1 gene probe, it was inserted into these plants as a single intact copy.

Also described herein is the molecular characterization of the inserted DNA at the AAD-1 corn event DAS-40278-9. The event is F of plasmid pDAS1740.
It was generated by whisker transformation using the spI fragment. Using Southern blot analysis,
The integration pattern of the inserted DNA fragment was established and the number of insert / copy of the aad-1 gene at event DAS-40278-9 was determined. Data was generated to verify the integration and integrity of the aad-1 transgene inserted into the maize genome. Characterization of integration of non-coding regions such as promoters and terminators (designed to regulate the coding region), matrix attachment regions RB7 Mar v3 and RB7 Mar v4, and stability of transgene inserts across generations did. The stability of the inserted DNA has been demonstrated over five different generations of plants. Furthermore, the absence of a transformed plasmid backbone containing the ampicillin resistance gene (Apr) region was verified by a probe covering almost the entire backbone region in contact with the restriction site (FspI) of plasmid pDAS1740. The detailed physical map of the insertion is the event DAS-
Based on these Southern blot analyzes of 40278-9.

The level of AAD-1 protein was determined in corn tissue. In addition, a compositional analysis was performed on corn forage and kernels to determine whether an isogenic non-transformed corn line and a transgenic corn line DAS-40278-9.
(No spray, 2,4-D spray, quizalofop spray and 2,
4-D and quizalofop sprayed). The agroeconomic characteristics of isogenic non-transformed maize lines were also compared to DAS-40278-9 maize.

Non-transgenic control and aryloxyalkanoate dioxygenase-1 (A
Field expression, nutritional composition and agro-economic trials with hybrid maize lines containing AD-1) were studied by Iowa, Illinois (2 places), Indiana, Neb
The same year was done at six sites in Raska and Ontario, Canada. Expression levels are used herein for AAD-1 protein in leaves, pollen, roots, forage, whole plants and kernels, results of agro-economic determinations, and controls and DAS-402.
Summary of composition analysis of forage and kernel samples from 78-9 AAD-1 corn.

Soluble extractable AAD-1 protein was measured in corn leaves, pollen, roots, forage, whole plants and grains using a quantitative enzyme-linked immunosorbent assay (ELISA) method. Good average expression values were observed in root and pollen tissues, as discussed in more detail herein. The expression values were similar for all spray treatments and plots sprayed and unsprayed with 2,4-D and quizalofop herbicides.

A composition analysis including proximates, minerals, amino acids, fatty acids, vitamins, anti-nutrients, and secondary metabolites was performed to determine DAS-40278-9 AAD- versus control.
The equivalence of 1 maize (with or without herbicide treatment) was examined. DAS-40278-
The results for the 9 AAD-1 composition samples are all similar to the analysis of agro-economic data collected from the control and DAS-40278-9 AAD-1 corn plots, based on control lines and / or conventional corn Or better (biologically and agroeconomically).

As mentioned in the background section above, the introduction and integration of a transgene into the plant genome involves several random events (hence the name “event” for a given insertion to be expressed). That is, using many transformation techniques such as Agrobacterium transformation, “gene gun” and whiskers, it is not possible to predict where the transgene will be inserted in the genome. Thus, identifying flanking plant genomic DNA on both sides of the insert can be important for identifying plants with a given insertion event. For example, PCR primers can be designed that produce PCR amplicons that span the junction region of the insert and the host genome. This PCR amplicon can be used to identify unique or distinct types of insertion events.

Since “events” are originally random events, as part of this disclosure,
At least 2500 seeds of the maize line containing the event are American Type Culture Collection (ATCC), 10801 University Bouleva
It has been deposited with rd, Manassas, VA, 20110 and is generally available without limitation (but under patent rights). This deposit is ATCC deposit number PTA-10244 (
Yellow Dent Maze Hybrid Seeds (Zea Mays L.): D
AS-40278-9; deposited for Dow AgroSciences LLC; AT
Date of receipt of seed / strain (s) by TC: July 10, 2009; Survival confirmation date is 2009
August 17). This deposit is made and maintained under this Convention in accordance with the Budapest Treaty on Seed Deposits in Patent Proceedings. The deposit is ATCC, a public depository.
At the depositary institution, without limitation, will be maintained for 30 years or 5 years after the most recent request, or for a longer period of time, either longer than the validity period of the patent, and replaced if it does not survive during that period.

The deposited seed is part of the subject invention. Clearly, corn plants can be grown from these seeds, and such plants are part of the subject invention. The subject invention is a D contained in these corn plants useful for detecting these plants and their progeny.
It also relates to NA sequences. The subject invention detection methods and kits can be adapted to identify any one, two or even all three of these events depending on the ultimate purpose of the test.

Definitions and examples are provided herein to assist in describing the present invention and to assist one of ordinary skill in carrying out the present invention. Unless otherwise noted, terms are to be understood according to conventional conventions by those of ordinary skill in the relevant art. The nomenclature for DNA bases described in 37 CFR §1.822 is used.

As used herein, the term “offspring” refers to the AAD-1 corn event DAS-4.
Refers to the child of any generation of the parent plant, including 0278-9.

A transgenic “event” involves regenerating a population of plants obtained by transforming plant cells with a heterologous DNA, ie, a nucleic acid construct containing a transgene of interest, and inserting the transgene into the genome of the plant, to a specific genome. It is generated by selecting a specific plant that is characterized by its insertion at the location. The term “event” refers to the progeny of the original transformant and transformants containing heterologous DNA. The term “event” also refers to progeny produced by sexual crossing between a transformant and another breed containing genomic / transgene DNA. Even after repeated backcrosses with the recurrent parent, the inserted transgene DN from the transformed parent
A and flanking genomic DNA (genomic / transgene DNA) are present at the same location on the chromosome in the offspring of the cross. The term “event” can also refer to DNA from the original transformant and its progeny, which is a single parental line containing the inserted DNA (eg, the original transformant and progeny obtained by autologous reproduction). ) And the inserted DNA that is predicted to be transmitted to the offspring that will receive the inserted DNA containing the transgene of interest as a result of sexual crossing with the parental strain that does not contain the inserted DNA. Flanking genomic sequences and inserted DNA.

“Joint sequence” refers to the point at which DNA inserted into the genome is ligated with DNA from the natural maize genome that contacts the point of insertion, and the identification of one or the other junction sequence in the genetic material of the plant or Detection is sufficient to be diagnostic to the event. Included are DNA sequences spanning insertions in the corn events described herein and flanking DNA of similar length. Specific examples of such characteristic sequences are shown herein. However, the junction of the insert or other sequences that overlap the junction of the insert and the genomic sequence are also characteristic and can be used according to the subject invention.

The subject invention relates to the identification of such flanking, junction and insert sequences. Related PCR primers and amplicons are included in the present invention. In accordance with the subject invention, PCR analysis using inserted DNA and amplicons that span its boundaries is used to detect or derive lines from transgenic corn varieties on the market or transgenic corn lines that are the subject of ownership Can be identified.

The entire sequence of each of these inserts is shown herein as SEQ ID NO: 29, together with a portion of the respective flanking sequence. The insert and flanking sequence coordinates for this event with respect to SEQ ID NO: 29 (total 8557 base pairs) are listed below.
This is discussed in more detail, for example in Example 3.8.

This insertion event and its further elements are further illustrated in FIGS. These sequences (especially flanking sequences) are unique. Based on these insert and border sequences, event specific primers were generated. PCR analysis demonstrated that these maize lines can be identified in different maize genotypes by analysis of PCR amplicons generated using these event-specific primer sets.
Thus, these and other related procedures can be used to uniquely identify these maize lines. The sequences identified herein are unique. For example, G
A BLAST search against the ENBANK database includes a cloned border sequence and
It did not reveal any significant homology with the sequences in the database.

The detection technique of the subject invention is to determine which progeny plant has a given event after crossing a parent plant containing the event of interest with another plant line in an effort to provide one or more additional subject traits in the offspring. Is particularly useful in connection with plant breeding.
These PCR analysis methods benefit corn breeding programs as well as quality control, especially for transgenic corn seeds on the market. PCR detection kits for these transgenic corn lines can also be made and used this time. This can also benefit product registration and product management.

In addition, flanking maize / genomic sequences can be used to specifically identify the location of each insert in the genome. This information can be used to create a molecular marker system specific to each event. These can be used to promote breeding strategies and create linkage data.

In addition, using the flanking sequence information, the transgene integration process, the characteristics of the genomic integration site, the classification of events, the stability of the transgenes and their flanking sequences,
As well as gene expression (especially with respect to possible expression-related elements such as gene silencing, transgene methylation patterns, position effects, and MARS [matrix attachment region]).

In view of all subject disclosures, it is clear that the subject invention includes seeds that are available under ATCC Deposit Number PTA-10244. The subject invention is accession number PTA-10244.
Also included are herbicide-resistant maize plants grown from seeds deposited with the ATCC. The subject invention further includes parts of the above plants such as leaves, tissue samples, seeds produced by the above plants, pollen and the like.

Further, the subject invention is a descendant and / or progeny plant, preferably a herbicide-resistant corn plant, of a plant grown from the deposited seed, wherein said plant is herein Includes plants having a genome comprising a detectable wild type genomic DNA / insert DNA junction sequence as described. As used herein, the term “corn”
Means maize (Zea mays) and includes all varieties that can be bred with corn.

The invention also includes a process for producing a hybrid using the subject invention plant as at least one parent. For example, the subject invention includes an F ₁ hybrid plant having as one or both parents any of the plants exemplified herein. Seeds that are produced by such F ₁ hybrid of the subject invention are also included in the subject invention. The present invention relates to exemplified plants,
Different (e.g. inbred parent) was crossed with a plant, comprising the method of producing the F ₁ hybrid seed by harvesting the hybrid seed obtained. The subject invention includes exemplified plants that are either female parents or male parents. The characteristics of the resulting plant can be improved by carefully considering the parent plant.

A herbicide-tolerant corn plant first sexually crosses a first parent corn plant consisting of a corn plant grown from the seed of any one of the lines referred to herein with a second parent corn plant. Thereby producing a plurality of first progeny plants and then a first progeny plant resistant to the herbicide (or having at least one event of the subject invention)
And self-reproductive of the first progeny plant, thereby creating a plurality of second progeny plants, then from the second progeny plant, a plant resistant to the herbicide (or at least one of the events of the subject invention) Can be bred. These steps may further include backcrossing the first progeny plant or the second progeny plant with the second parent corn plant or the third parent corn plant. The corn crop or its progeny containing the corn seed of the subject invention can then be planted.

It should also be understood that two different transgenic plants can be crossed to produce offspring that contain two independently isolated additional exogenous genes. Self-reproduction of the appropriate offspring can produce homozygous plants for both additional exogenous genes. Backcrosses with parental plants and dipcrosses with non-transgenic plants are also contemplated as vegetative propagation. Other breeding methods commonly used for different traits and crops are known in the art. Backcross breeding has been used to transfer genes for simply inherited highly inherited traits to the desired homozygous cultivated varieties or inbred lines that are recurrent parents. The source of the transmitted trait is called the donor parent. The resulting plant is a recurrent parent (
For example, it is predicted to have attributes of (cultivation variety) and a desired trait transmitted from the donor parent. After the first cross, individuals with a donor parent phenotype are selected and crossed repeatedly with the recurrent parent (backcross). The resulting parent is expected to have the attributes of a recurrent parent (eg, cultivar) and the desired trait transmitted from the donor parent.

The DNA molecule of the present invention can be used as a molecular marker in a marker-assisted breeding (MAB) method. The DNA molecules of the present invention can be used in methods for identifying genetically linked agroeconomically useful traits as known in the art (eg, AFLP markers, RFLP markers, RAPD markers, SNPs and SSR). Herbicide resistance traits can be tracked using the MAB method in the offspring (or their offspring and any other corn cultivars or varieties) with the corn plant of the subject invention. The DNA molecule is a marker for this trait and is known in the art as MA
Using the B method, at least one corn line of the subject invention or its progeny can track herbicide resistance trait (s) in the parent or ancestor corn plant.
The method of the present invention can be used to identify any corn varieties having a subject event.

The subject inventive method includes a method of producing a herbicide-tolerant corn plant comprising breeding the subject inventive plant. More specifically, the above method may comprise the step of crossing two plants of the subject invention, or one plant of the subject invention with any other plant. Preferred methods further comprise the step of selecting the progeny of said hybrid by analyzing said progeny for events detectable in accordance with the subject invention.
For example, the subject invention tracks the subject event during a breeding cycle with a plant containing other desired traits such as agroeconomic traits such as those tested herein in various embodiments. Can be used for Plants containing the subject event and the desired trait are
For example, it can be detected, identified, selected, and used rapidly in further rounds of breeding. The subject event / trait can be combined by breeding with insect resistance trait (s) and / or additional herbicide tolerance traits and can be tracked according to the subject invention. One preferred embodiment of the latter is a plant comprising the subject event combined with a gene encoding resistance to the herbicide dicamba.

Thus, the subject invention is, for example, glyphosate resistant (eg, resistant plant or bacterial EPSPS, GOX, GAT), glufosinate resistant (eg, Pat, bar)
Acetolactate synthase (ALS) -inhibiting herbicide resistance (eg imidazolinones [eg imazetapyr], sulfonylureas, triazolopyrimidinesulfonanilides, pyrmidinylthiobenzoates and other chemicals [Csr1, Su
rA et al.), bromoxynyl resistance (eg Bxn), resistance to inhibitors of HPPD (4-hydroxyphenyl-pyruvate-dioxygenase) enzyme, resistance to inhibitors of phytoene desaturase (PDS), photochemical system Resistance to II-inhibiting herbicides (eg psbA), resistance to photosystem I-inhibiting herbicides, resistance to protoporphyrinogen oxidase IX (PPO) -inhibiting herbicides (eg PPO-1), to phenylurea herbicides Resistance (eg CYP76B1), dicamba-degrading enzyme (eg US2003013)
Can be combined with traits encoding 5879), others can be combined alone or in multiple combinations to effectively shift weeds and / or resistance to any herbicide of the above class. Can provide the ability to control or hinder.

With respect to additional herbicides, some additional preferred ALS (also known as AHAS) inhibitors are triazolopyrimidine sulfonanilides (eg, chloransram-methyl, diclosram, flurothram, flumethram, methotram and penoxsulam), pyrimidinylthiobenzoates (eg, Bispyribac and pyrithiobac), and full carbazone. Some preferred HPPD inhibitors include mesotrione, isoxaflutole and sulcotrione. Some preferred PPO inhibitors include flumicrolac, flumioxazin, flufenpyr, pyraflufen, fluthiaset, butaphenacyl, carfentrazone, sulfentrazone and diphenyl ethers (eg, acifluorfen, fomesafen, lactofen and oxyfluorfen) Including.

In addition, AAD-1 alone or multiplied by one or more additional HTC traits can be one or more additional inputs (such as insect resistance, fungal resistance or stress resistance) or output (such as yields). (Increased, improved oil profile, improved fiber quality, etc.). Thus, the subject invention can be used to provide a complete agro-economic package with improved crop quality that has the ability to flexibly and economically efficiently control any number of agro-economic pests .

Methods for incorporating polynucleotide sequences into specific chromosomal sites of plant cells by homologous recombination have been described in the art. For example, US Patent Application Publication No. 2009/011.
Site-specific integration as described in 1188A1 describes the use of recombinases or integrases to mediate introduction of donor polynucleotide sequences into chromosomal targets. International Patent Application No. WO 2008/021207 further describes zinc finger-mediated homologous recombination to integrate one or more donor polynucleotide sequences at specific positions in the genome. FLP described in US Pat. No. 6,720,475
The use of recombinases such as CRE / LOX as described in / FRT or US Pat. No. 5,658,772 can be used to incorporate polynucleotide sequences into specific chromosomal sites. Finally, the use of meganucleases to target donor polynucleotides to specific chromosomal locations has been described in Puchta et al., PNAS USA 93 (1996) pp. 5055-5060.

Various other methods for site-specific integration in plant cells are generally known and can be used (Kumar et al., Trands in Plant Sci. 6 (4) (2001) pp.155-159). . In addition, site-specific recombination systems that have been identified in several prokaryotes and lower eukaryotes can be applied for use in plants. Examples of such systems include, but are not limited to, the R / RS recombinase system (Araki et al. (1985) J. Mol. Biol. 182: 191-203) from the pSR1 plasmid of the yeast Zygosaccharomyces rouxii. ) And the Gin / gix system of phage Mu (Maeser and Kahlmann (1991) Mol. Gen. Genet. 230: 1
70-176).

In some embodiments of the invention, it may be desirable to incorporate or multiply the new transgene (s) in the vicinity of an existing transgenic event. Transgenic events include unique features such as single insertion sites, normal Mendelian isolation and stable expression, and agro-economic performance in and across herbicide tolerance and multiple environmental locations It can be considered a preferred genomic locus selected based on a combination of potency. The newly incorporated transgene maintains the transgene expression characteristics of existing transformants. Furthermore, the development of assays for detection and confirmation of newly incorporated events is overcome because the genomic flanking sequences and chromosomal locations of newly incorporated events have already been identified. Finally, incorporating a new transgene into a specific chromosomal location linked to an existing transgene introduces the transgene into other genetic backgrounds by sexual crossbreeding using conventional breeding methods To promote that.

In some embodiments of the invention, it may be desirable to excise a polynucleotide sequence from a transgenic event. For example, US provisional patent application 61/297.
The transgene excision as described in US Pat. No. 628 describes the use of a zinc finger nuclease to remove the polynucleotide sequence consisting of a gene expression cassette from a transgenic event integrated into a chromosome. The removed polynucleotide sequence can be a selectable marker. Upon excision and removal of the polynucleotide sequence, the modified transgenic event can be retargeted by insertion of the polynucleotide sequence. Excision of polynucleotide sequences and subsequent retargeting of modified transgenic events, such as the ability to overcome unintentional changes to the plant transcriptome due to reuse of selectable markers or expression of specific genes Provide benefits.

The subject invention discloses herein a specific site on chromosome 2 in a maize genome that is superior for the introduction of heterologous nucleic acids. Also disclosed are 5 ′ molecular markers, 3 ′ molecular markers, 5 ′ flanking sequences and 3 ′ flanking sequences useful for identifying the location of the targeting site on chromosome 2. Thus, the subject invention provides a method for introducing a heterologous nucleic acid of interest at or near the pre-established target site.
The subject invention also encompasses corn seeds and / or corn plants comprising any heterologous nucleotide sequence inserted at or near the disclosed target site. One option to achieve such targeted integration is to excise the pat expression cassette exemplified herein and / or replace it with a different insert instead. In this general respect, targeted homologous recombination can be used in accordance with the subject invention, for example and without limitation.

As used herein, “multiplying” a gene, event or trait is the combination of the desired trait into a single transgenic line. A plant breeder multiplies the transgenic traits by creating crossbreds of parents each having the desired trait and then identifying offspring that have both of these desired traits. Another way of multiplying genes is by simultaneously transferring two or more genes to the plant cell nucleus during transformation. Another way of crossing genes is by retransforming the transgenic plant with another gene of interest. For example, gene crossing can include, for example, two or more different insect traits, insect resistance trait (s) and disease resistance trait (s), two or more herbicidal resistance traits, and / or insect resistance traits ( Can be used to combine two or more different traits, including her (s) and herbicide resistant trait (s). Using a selectable marker in addition to the gene of interest can also be thought of as gene crossing.

“Homologous recombination” refers to any pair of nucleotide sequences having corresponding sites containing similar nucleotide sequences by which two nucleotide sequences can interact (recombine) to form a new recombinant DNA sequence. Refers to the reaction. Each site of similar nucleotide sequence is referred to herein as a “homologous sequence”. In general, the frequency of homologous recombination increases as the length of the homologous sequence increases. Thus, homologous recombination can occur between two nucleotide sequences that are less than identical, but the recombination frequency (or efficiency) decreases as the difference between the two sequences increases. Recombination is accomplished using one homologous sequence in each of the donor and target molecules, thereby producing a “single cross” recombination product. Alternatively, two homologous sequences can be placed on each of the target and donor nucleotide sequences. Recombination of two homologous sequences on the donor and two homologous sequences on the target results in a “double crossover” recombination product. Double homologous recombination with the target molecule was originally between the homologous sequences on the target molecule when the homologous sequence on the donor molecule touches the sequence being manipulated (eg, the sequence of interest) This results in a recombinant product that replaces the DNA sequence. Exchanging the DNA sequence between the target and donor by a double crossover recombination event,
This is called “array replacement”.

The subject AAD-1 enzyme allows for transgenic expression resulting in resistance to a combination of herbicides that control almost all broadleaf and grass weeds.
AAD-1 is, for example, other herbicide tolerant crop (HTC) traits (eg glyphosate resistance, glufosinate resistance, imidazolinone resistance, bromoxynyl resistance, etc.) and insect resistance traits (Cry1F, Cry1Ab, Cry34 / 45, etc.) ) And can serve as an excellent HTC trait. Furthermore, AAD-1 can serve as a selectable marker to assist in the selection of plant primary transformants genetically engineered with a second gene or group of genes.

The HTC trait of the subject invention can be used in novel combinations with other HTC traits, including but not limited to glyphosate resistance. These combinations of traits provide a new way of controlling weed (etc.) species through newly acquired resistance or inherent resistance to herbicides (eg glyphosate). That is, in addition to HTC traits, a novel method for controlling weeds with herbicides (herbicide resistance established by the above-described enzymes in transgenic crops) is within the scope of the present invention.

In addition, growing glyphosate-tolerant crops worldwide is widespread. When many rotations with other glyphosate-tolerant crops occur, the glyphosate-resistant native plant (voluntee
Control of rs) can be difficult in rotation crops. Thus, the use of the subject transgenic traits that have been crossed or individually transformed into crops is
Provides tools for the control of TC native crops.

Preferred plants or seeds of the subject invention have in their genome the insert sequence identified herein, along with at least 20-500 or more contiguous flanking nucleotides on either side of the insert fragment identified herein. Including. Unless indicated otherwise, references to flanking sequences refer to those identified with respect to SEQ ID NO: 29 (see table above). Again, SEQ ID NO: 29 is the heterologous D inserted into the original transformant.
Contains NA and explanatory flanking genomic sequences immediately adjacent to the inserted DNA. All or a portion of these flanking sequences can be predicted to be transmitted to offspring that receive the inserted DNA as a result of sexual crossing of the parental line containing the event.

The subject invention includes a tissue culture of renewable cells of the plant of the subject invention. Plants regenerated from such tissue culture are also included, especially the above-mentioned plants can express all morphological and physiological characteristics of the exemplified varieties. Preferred plants of the subject invention have all the physiological and morphological characteristics of plants grown from deposited seeds. The present invention further includes seeds having such seed progeny and subject qualitative traits.

The manipulation of plants or seeds or parts thereof (eg mutation, further transfection and further breeding) can lead to the creation of what may be referred to as “essentially derived” varieties. The United Nations for Protection of New Plant Varieties (UPOV) provides the following guidelines for determining whether a variety is subordinate to a protected variety:
Variety is
(I) it derives predominantly from the first variety or from a variety that itself derives predominantly from the first variety, but due to the genotype or combination of genotypes of the first variety Retains the expression of essential features,
(Ii) it can be clearly distinguished from the first variety,
(Iii) If it becomes similar to the first variety in the expression of essential characteristics due to the genotype or combination of genotypes of the first variety, except for differences due to the work of derivation, It is considered to be subordinate to the “first variety”).

UPOV, 6th meeting with international organizations, Geneva, 30 October 1992; Document produced by the Secretariat of the Union.

As used herein, a “line” is a group of plants that show little or no genetic variation between individuals for at least one trait. Such lines can be created by self-pollination and several generations of selection, or vegetative propagation using tissue or cell culture techniques from a single parent.

As used herein, the terms “cultivar” and “variety” are synonymous and refer to a line used for commercial production.

“Stability” or “stable” means that for a given element, the element is maintained from generation to generation, preferably at substantially the same level over at least three generations, eg preferably ± 15%, more preferably ± Meaning maintained at 10%, most preferably ± 5%.
Stability can be affected by planting temperature, location, stress and timing. Subsequent generation comparisons under field conditions produce the element in a similar manner.

“Commercial Utility” has good vigor and high fertility,
It is defined that oils that can be produced by farmers using conventional agricultural equipment and that have the components described can be extracted from seeds using conventional grinding and extraction equipment. To be commercially useful, seed weight, oil content and yield measured by total oil produced per acre do not have high value traits grown in the same area, otherwise compared Is within 15% of the average yield of commercial canola varieties.

“Agroeconomic Elite” means that the strain has the desired agroeconomic characteristics, such as yield, maturity, disease resistance, etc., in addition to insect resistance due to the subject event (s) Means that. As shown in the examples below, agroeconomic traits that are considered individually or in any combination in a plant containing an event of the subject invention are within the scope of the subject invention.
Any and all of these agroeconomic features and data points are used to identify such plants as points in the range of features used to define such plants, or as either one or both ends it can.

One skilled in the art will recognize, for example, that a preferred embodiment of a detection kit is a “junction sequence” in light of the present disclosure.
Alternatively, it will be appreciated that probes and / or primers directed to and / or containing a “transition sequence” (where the maize genomic flanking sequence meets the insert sequence) may be included. For example, this includes polynucleotide probes, primers and / or amplicons designed to identify one or both junction sequences (where the insert meets the flanking sequence), as shown in Table 1. . One common design is to have one primer that hybridizes in the flanking region and one primer that hybridizes in the insert. Each such primer is often at least about -15 residues in length. With this configuration, primers can be used to create / amplify detectable amplicons that indicate the presence of the subject invention event. These primers can be used to make amplicons that span (and contain) the junction sequence as described above.

About 2 "touching down" primer (s) within the flanking sequence
It is typically not designed to hybridize beyond 00 bases or beyond the junction. That is, a typical flanking primer is designed to include at least 15 residues of any strand within 200 bases within the flanking sequence from the start of the insert. That is, residues about 1674-1873 and / or about -6690-6890 of SEQ ID NO: 29.
Primers containing the appropriate size sequence in are within the scope of the subject invention. Insertion primers can also be designed anywhere on the insert, but for example from about ~ 187 residues
4-2074 and about ~ 6489-6689 can be used non-exclusively for such primer design.

One skilled in the art can design primers and probes to hybridize to a segment of SEQ ID NO: 29 (or complementary strand) and its complementary strand under a range of standard hybridization and / or PCR conditions, where And the primer or probe is
It is also recognized that it is not completely complementary to the exemplified sequence. In other words, a certain degree of mismatch is acceptable. For an approximately 20 nucleotide primer, for example, typically 1
Or a nucleotide such as 2 need not be bound to the opposite strand if the mismatch base is at the end of the primer inside or opposite the amplicon. Various suitable hybridization conditions are shown below. Synthetic nucleotide analogs such as inosine can also be used in the probe. Peptide nucleic acid (PNA) probes and DNA and RNA probes can also be used. Importantly, such probes and primers are diagnostic for the presence of the subject invention event (which can be uniquely identified and distinguished).

Note that errors in PCR amplification can occur, for example, which would result in minor sequence errors. That is, unless stated otherwise, the sequences listed herein were determined by making long amplicons from maize genomic DNA and then cloning and sequencing the amplicons. The slight differences and minor discrepancies found in sequences generated and determined in this manner are in view of the many rounds of amplification necessary to generate sufficient amplicons for sequencing from genomic DNA. It's not unusual. Those skilled in the art will recognize and be notified as such that any adjustments required by these types of general sequencing errors or inconsistencies are within the scope of the subject invention.

Note also that it is not uncommon for some genomic sequences to be deleted, for example, when inserting sequences during event creation. That is, some differences may appear between the subject flanking sequences and the genomic sequences listed, for example, in GENBANK.

Some of these differences (s) are discussed in the Examples section below. Adjustments to probes and primers can be made accordingly.

Each element of the “insert” is shown in FIGS. 1 and 2 and is discussed in more detail in the examples below. The DNA polynucleotide sequences of these elements or fragments thereof can be used as DNA primers or probes in the methods of the present invention.

In some embodiments of the invention, compositions and methods are provided for detecting the presence of a transgene / genomic insert region from a corn plant, such as in plants and seeds. The subject transgene / genome insert region junction sequence (between residues 1873-1874 and 6589-6690 of SEQ ID NO: 29), its segment, and the complementary strand of the exemplified sequence and any segment thereof, as shown herein A DNA sequence comprising is provided. The insertion region junction sequence consists of heterologous DNA inserted into the genome and D from corn cells in contact with the insertion site.
It extends to the junction with NA. Such an arrangement can be characteristic for a given event.

Based on these inserts and border sequences, event specific primers can be made. PCR analysis demonstrated that the subject invention maize lines can be identified in different maize genotypes by analysis of PCR amplicons generated using these event-specific primer sets. These and other related procedures can be used to uniquely identify these corn lines. That is, PCR amplicons derived from such primer pairs are unique and can be used to identify these maize lines.

In some embodiments, a DNA sequence comprising a contiguous fragment of the novel transgene / genomic insert region is an aspect of the present invention. A DNA sequence comprising a full length polynucleotide of the transgene insert sequence and a full length polynucleotide of a corn genomic sequence from one or more of the three corn plants described above, and / or these corn Included are sequences useful as primer sequences for the production of characteristic amplicon products for one or more of the plants.

A related embodiment is that at least 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 2
A DNA sequence comprising 4, 25 or more contiguous nucleotides, or its complementary strand,
And flanking corn DNA sequences of similar length from these sequences, or their complementary strands. Such sequences are useful as DNA primers in DNA amplification methods. The amplicons produced using these primers are characteristic for any corn event referred to herein. Therefore, the present invention provides such a DN.
Also included are amplicons generated by A and homologous primers.

The invention also includes a method of detecting in a sample the presence of DNA corresponding to the corn event referred to herein. Such methods include: (a) an amplicon that is characteristic for the event (s) described above when a sample containing DNA is used in a nucleic acid amplification reaction with DNA from at least one of these corn events. Contacting with a primer set that generates, (b) performing a nucleic acid amplification reaction, thereby generating an amplicon, and (c) detecting the amplicon.

Further detection methods of the subject invention provide DNA corresponding to at least one of the above events
In which a sample comprising DNA is hybridized under stringent hybridization conditions with DNA from at least one of the corn events described above, and a control corn plant ( Contacting DNA that is not the event of interest) with a probe that does not hybridize under stringent hybridization conditions; (b) subjecting the sample and probe to stringent hybridization conditions; (c) Detecting hybridization of the probe.

In a further embodiment, the subject invention is a method of producing a corn plant comprising the aad-1 event of the subject invention, comprising: (a) a first parent corn line (the above-mentioned herbicidal plant in the above lineage). Sexually crossing a second parental maize line (which lacks this herbicide tolerance trait), thereby producing a plurality of progeny plants, comprising an expression cassette of the present invention that confers drug resistance) (B) selecting a progeny plant using the molecular marker. Such a method may optionally include the further step of backcrossing the progeny plant with a second parental maize line to produce a true-breeding corn plant comprising the insect resistance trait described above.

According to another aspect of the invention, a method is provided for determining the mating status of a progeny of a hybrid with any one (or more) of the above three events. The above method is based on maize DN
Contacting a sample comprising A with the primer set of the subject invention may be included. The primer, when used in a nucleic acid amplification reaction with genomic DNA from at least one of the corn events, produces a first amplicon that is characteristic for at least one of the corn events. Such a method performs a nucleic acid amplification reaction,
A step of generating a first amplicon thereby, a step of detecting the first amplicon, and a sample containing corn DNA from the primer set (the primer set is a nucleic acid amplification reaction with genomic DNA from a corn plant) A second amplicon comprising a natural corn genomic DNA homologous to the corn genomic region) and a nucleic acid amplification reaction, thereby producing a second amplicon. In addition. The method includes detecting a second amplicon and comparing a first amplicon and a second amplicon in the sample, wherein the presence of both amplicons is heterozygous for the transgene insertion in the sample. And a step of indicating sex.

DNA detection kits can be developed using the compositions disclosed herein and methods known in the art of DNA detection. The kit is subject corn event DN in the sample
It is useful for identifying A and can be used in methods for breeding corn plants containing this DNA. The kit contains, for example, a DNA sequence that is homologous or complementary to a DNA sequence that is homologous or complementary to the DNA contained in the amplicon or transgene genetic element of the subject event disclosed herein. These DNA sequences can be used as probes in DNA amplification reactions or in DNA hybridization methods. The kit may also contain reagents and materials necessary for carrying out the detection method.

A “probe” is an isolated nucleic acid molecule conjugated to a conventional detectable label or reporter molecule (eg, a radioactive isotope, ligand, chemiluminescent agent or enzyme). Such a probe is in the present case a target nucleic acid strand, the genome D from one of the above corn events.
It is complementary to a strand of NA (either from a corn plant or from a sample containing DNA from an event). Probes according to the present invention include not only deoxyribonucleic acid or ribonucleic acid, but also other probe materials that bind specifically to the polyamide and the target DNA sequence and can be used to detect the presence of the target DNA sequence.

A “primer” anneals to a complementary target DNA strand by nucleic acid hybridization to form a hybrid between the primer and the target DNA strand, and is then extended along the target DNA strand by a polymerase, such as a DNA polymerase. Isolated / synthetic nucleic acid. The primer pairs of the present invention refer to their use for amplification of target nucleic acid sequences, for example, by polymerase chain reaction (PCR) or other conventional nucleic acid amplification methods.

Probes and primers are generally
Polynucleotide or longer length. Such probes and primers specifically hybridize with the target sequence under high stringency hybridization conditions. Preferably, probes and primers according to the present invention have complete sequence similarity to the target sequence, but unlike the target sequence, probes that retain the ability to hybridize with the target sequence can be designed by conventional methods.

Methods for preparing and using probes and primers include, for example, Molecular Cloning: AL
aboratory Manual, 2nd edition, 1-3 volumes, edited by Sambrook et al., Cold Spring Harbor Laboratory Pre
ss, Cold Spring Harbor, NY, 1989. PCR primer pairs can be derived from known sequences, for example by using a computer program intended for that purpose.

Primers and probes based on the flanking DNA sequences and insert sequences disclosed herein confirm the disclosed sequences (if necessary) by conventional methods such as recloning and sequencing of such sequences. Can be used to correct).

The nucleic acid probes and primers of the present invention hybridize with the target DNA sequence under stringent conditions. Any conventional nucleic acid hybridization or amplification method can be used to identify the presence of DNA from a transgenic event in a sample. A nucleic acid molecule or fragment thereof can specifically hybridize with other nucleic acid molecules under certain circumstances. As used herein, two nucleic acid molecules are said to be able to specifically hybridize to each other if the two molecules can form an antiparallel double stranded nucleic acid structure. Nucleic acid molecules are said to be the “complementary strand” of another nucleic acid molecule if they exhibit complete complementarity. As used herein, a molecule is said to exhibit “perfect complementarity” when all the nucleotides of one molecule are complementary to the other nucleotide. Two molecules are “minimally complementary” if they can hybridize to each other with sufficient stability so that they can remain annealed to each other at least under conventional “low stringency” conditions. " Similarly, molecules are said to be “complementary” if they can hybridize to each other with sufficient stability such that they can remain annealed to each other under conventional “high stringency” conditions. . Conventional stringency conditions are described in Sambrook et al., 1989.
It is described by. Deviations from perfect complementarity are thus tolerated as long as such deviations do not completely interfere with the ability of the molecule to form a double stranded structure. In order for a nucleic acid molecule to act as a primer or probe, it need only be sufficiently complementary in a sequence that can form a stable double-stranded structure under the particular solvent and salt concentration employed.

As used herein, a substantially homologous sequence is a nucleic acid sequence that specifically hybridizes with the complementary strand of the nucleic acid sequence being compared under high stringency conditions. The term “stringent conditions” is functionally defined with respect to hybridization of a nucleic acid probe to a target nucleic acid (ie, a specific nucleic acid sequence of interest) by a specific hybridization procedure discussed in Sambrook et al., 1989 at 9.52-9.55. The Sambrook et al., 1989, 9.47-9.52.
See also and 9.56-9.58. Thus, the nucleotide sequences of the present invention can be used for their ability to selectively form duplexes with complementary stretches of DNA fragments.

Depending on the application envisioned, a variety of hybridization conditions can be used to achieve varying degrees of selectivity of the probe relative to the target sequence. For applications where high selectivity is required, relatively stringent conditions are typically employed to form hybrids, such as from about 50 ° C. to about 70 ° C. with about 0.02 M to about 0.15 M NaCl. Choose relatively low salt and / or high temperature conditions as given by the temperature in ° C. For example, stringent conditions can include washing the hybridization filter at least twice with a high stringency wash buffer (0.2 × SSC, 0.1% SDS, 65 ° C.). Appropriate stringency conditions that facilitate DNA hybridization, such as washing at about 45 ° C. with 6.0 × sodium chloride / sodium citrate (SSC) followed by 2.0 × SSC at 50 ° C. Known, 6.3.1-6.3
. 6. For example, the salt concentration in the wash step can be selected from a low stringency of about 2.0 × SSC at 50 ° C. to a high stringency of about 0.2 × SSC at 50 ° C. Further, the temperature in the wash step can be increased from low stringency conditions at room temperature, about 22 ° C., to high stringency conditions at about 65 ° C. Both temperature and salt can be varied, or the other variable can be changed while either temperature or salt concentration is held constant. Such selective conditions are hardly tolerated if there is a mismatch between the probe and the template or target strand. Detection of DNA sequences by hybridization is known to those skilled in the art, and the teachings of US Pat. Nos. 4,965,188 and 5,176,995 are examples of methods for hybridization analysis.

In particularly preferred embodiments, the nucleic acids of the invention comprise one of the primers (or amplicons or other sequences) exemplified or suggested herein, including complementary strands and fragments thereof.
Alternatively, it specifically hybridizes with a plurality under high stringency conditions. In one embodiment of the present invention, the marker nucleic acid molecule of the present invention has the nucleic acid sequence shown in SEQ ID NOs: 3 to 14 or a complementary strand and / or fragment thereof.

In another aspect of the invention, a marker nucleic acid molecule of the invention comprises such a nucleic acid sequence and 8
Has sequence identity between 0% and 100% or between 90% and 100%. In a further aspect of the invention, the marker nucleic acid molecule of the invention has between 95% and 100% sequence identity with such sequences. Such sequences can be used as markers in plant breeding methods to identify offspring of genetic hybrids. Hybridization of the probe with the target DNA molecule can be detected by any number of methods known to those skilled in the art, including but not limited to fluorescent tags, radioactive tags, antibody-based tags and chemiluminescent tags. May be included.

For amplification of a target nucleic acid sequence using a particular amplification primer pair (eg by PCR), “stringent conditions” are preferably those in which the primer pair binds to a primer having the corresponding wild type sequence (or its complementary strand). Is a condition that allows hybridization with only the target nucleic acid sequence that produces the amplicon, which is a unique amplification product.

The term “specific for (target sequence)” indicates that a probe or primer hybridizes under stringent hybridization conditions with only the target sequence in a sample containing the target sequence.

As used herein, “amplified DNA” or “amplicon” refers to the product of nucleic acid amplification of a target nucleic acid sequence that is part of a nucleic acid template. For example, DN extracted from a corn plant tissue sample to determine if a corn plant resulting from sexual crossing contains transgenic event genomic DNA from the corn plant of the present invention.
A using a primer pair comprising a primer derived from a flanking sequence in the genome of a plant adjacent to the insertion site of the inserted heterologous DNA and a second primer derived from the inserted heterologous DNA, It can be subjected to nucleic acid amplification methods that produce amplicons that are characteristic of their presence. An amplicon is of a length and has a sequence that is also characteristic for events. The amplicon is derived from the combined length of primer pair + 1 nucleotide base pair and / or primer pair + approx.
Or a range of combined lengths of more nucleotide base pairs (any increment listed above + or-). Alternatively, the primer pair can be derived from flanking sequences on either side of the inserted DNA so as to generate an amplicon containing the entire inserted nucleotide sequence. The member of the primer pair derived from the plant genome sequence is the inserted DNA
It may be located at a distance from the sequence. This distance can range from 1 nucleotide base pair to about 20,000 nucleotide base pairs. The use of the term “amplicon” specifically excludes primer dimers that may be formed in a DNA thermal amplification reaction.

Nucleic acid amplification can be accomplished by any of a variety of nucleic acid amplification methods known in the art, including polymerase chain reaction (PCR). Various amplification methods are known in the art and are described in US Pat. No. 4,683,195 and US Pat. No. 4,683,202, among others. PCR amplification methods have been developed to amplify genomic DNA up to 22 kb. These methods and other methods known in the art of DNA amplification may be used in the practice of the present invention. The sequence of the heterologous transgene DNA insert or the flanking genomic sequence from the subject corn event amplifies such a sequence from the event using primers derived from the sequences presented herein and then PCR It can be confirmed (and corrected if necessary) by performing standard DNA sequencing of amplicons or cloned DNA.

Amplicons generated by these methods can be detected by multiple techniques. Agarose gel electrophoresis and staining with ethidium bromide are commonly known methods for detecting DNA amplicons. Another such method is the flanking flanking genome DN
Genetic bit analysis in which DNA oligonucleotides are designed to overlap both the A sequence and the inserted DNA sequence. Oligonucleotides are immobilized in the wells of a microwell plate. After PCR of the region of interest (using one primer in the inserted sequence and one primer in the flanking flanking genomic sequence), 1
The double-stranded PCR product can hybridize with the immobilized oligonucleotide and serves as a template for a single base extension reaction using DNA polymerase and a labeled ddNTP specific for the predicted next base. The readout can be based on fluorescence or ELISA. The signal indicates the presence of the insert / flanking sequence due to successful amplification, hybridization, and single base extension.

Another method is the pyrosequencing technique described by Winge (Innov. Pharma. Tech. 00: 18-24, 2000). In this method, the oligonucleotide is a contiguous genomic DN.
Designed to overlap with A and the inserted DNA junction. The oligonucleotide is hybridized with a single stranded PCR product (one primer in the inserted sequence and one primer in the flanking genomic sequence) from the region of interest, and DNA polymerase, ATP, sulfurylase, luciferase Incubate in the presence of apyrase, adenosine 5 ′ phosphosulfate and luciferin. DNTPs are added separately and incorporation results in a light signal that is measured. The light signal indicates the presence of the transgene insert / flanking sequence due to successful amplification, hybridization, and single or multiple base extension.

Fluorescence polarization is another method that can be used to detect the amplicons of the present invention. According to this method, oligonucleotides are designed that overlap with genomic flanking and inserted DNA junctions. Oligonucleotide is a single-stranded PCR product from the region of interest (one primer in the inserted DNA and flanking genomic DNA
One primer in the sequence) and incubated in the presence of DNA polymerase and fluorescently labeled ddNTPs. Single base extension results in the incorporation of ddNTPs. Incorporation can be measured as a change in polarization using a fluorometer. A change in polarization indicates the presence of the transgene insert / flanking sequence due to successful amplification, hybridization, and single base extension.

TAQMAN (PE Applied Biosystems, Foster Cit
y, Calif. ) Is a method for detecting and quantifying the presence of a DNA sequence. Briefly, FRET oligonucleotide probes are designed that overlap with genomic flanking and inserted DNA junctions. FRET probe and PCR primer (inserted DNA
One primer in the sequence and one primer in the flanking genomic sequence) are circulated in the presence of thermostable polymerase and dNTPs. During specific amplification, Taq D
NA polymerase cleans and releases the fluorescent moiety from the quenching portion of the FRET probe. The fluorescent signal indicates the presence of the flanking / transgene insertion sequence due to successful amplification and hybridization.

Molecular beacons have been described for use in sequence detection. Briefly, FRET oligonucleotide probes are designed that overlap the flanking genome and the inserted DNA junction. The unique structure of the FRET probe causes it to contain a secondary structure in which the fluorescent and quenching moieties are kept in close proximity. The FRET probe and PCR primer (one primer in the insert DNA sequence and one primer in the flanking genomic sequence) are circulated in the presence of a thermostable polymerase and dNTPs. After successful PCR amplification, hybridization of the FRET probe to the target sequence results in removal of the probe secondary structure and spatial separation of the fluorescent and quenching moieties. A fluorescent signal is obtained. A fluorescent signal indicates the presence of the flanking genome / transgene insert due to successful amplification and hybridization.

Although an excellent corn genome location for insertion has been disclosed, the subject invention also includes corn seeds and / or corn plants that contain at least one non-aad1 insert fragment approximately in the vicinity of the genome location. One option is to replace a different insert in place of the aad-1 insert illustrated herein. In general in these respects, for example, targeted homologous recombination can be used according to the subject invention.
This type of technology is described, for example, in WO 03/080809 A2 and the corresponding published U.S. Pat. S.
This is the subject of the application (US2003302410). Accordingly, the subject invention provides for heterologous inserts (aad-1 multiples) that border all or a recognizable portion of the flanking sequences identified herein (eg, residues 1-1873 and 6690-8557 of SEQ ID NO: 29). Plants and plant cells that contain (instead of or with a copy).

All patents, patent applications, provisional applications and publications mentioned or cited herein are:
They are incorporated by reference in their entirety to the extent that they do not conflict with the explicit teachings herein.


The present invention may include the following aspects. [1]
A transgenic corn plant comprising a genome comprising SEQ ID NO: 29.
[2]
Accession number PTA-10244 for American Type Culture Collection (ATCC)
Corn seeds containing a genome containing the AAD-1 event DAS-40278-9 present in seeds deposited under.
[3]
Corn seed comprising a genome comprising SEQ ID NO: 29.
[4]
A corn plant produced by growing seeds according to claim 2 comprising SEQ ID NO: 29.
[5]
The corn plant progeny plant of claim 4 comprising the AAD-1 event DAS-40278-9.
[6]
The herbicide-tolerant progeny plant of the corn plant of claim 1 comprising SEQ ID NO: 29.
[7]
SSR marker UMC12 which can be partially amplified by SEQ ID NO: 30 and SEQ ID NO: 31
65 and an SSR marker MM that can be partially amplified by SEQ ID NO: 32 and SEQ ID NO: 33
A transgenic corn plant comprising a transgene insert at a corn chromosome target site on chromosome 2 at approximately 20 cM between C0111, wherein the target site comprises a heterologous nucleic acid.
[8]
SSR marker UMC12 which can be partially amplified by SEQ ID NO: 30 and SEQ ID NO: 31
65 and an SSR marker MM that can be partially amplified by SEQ ID NO: 32 and SEQ ID NO: 33
A method of producing a transgenic corn plant comprising inserting a heterologous nucleic acid at a location on chromosome 2 at approximately 20 cM with C0111.
[9]
5. A plant part according to claim 4, which is selected from the group consisting of pollen, ovules, flowers, bolls, long fibers (lint), shoots, roots and leaves and comprises SEQ ID NO: 29.
[10]
The transgene insert was ligated to residues 1-1873 of SEQ ID NO: 29 and residue 66 of SEQ ID NO: 1
A transgenic corn plant comprising within or adjacent to a genomic sequence selected from the group consisting of 90-8557.
[11]
Comprising at least 15 nucleotides, residues 1 to 1873 of SEQ ID NO: 29, SEQ ID NO: 29
An isolated polynucleotide molecule that maintains hybridization under stringent wash conditions with a nucleic acid sequence selected from the group consisting of residues 6690-8557 and its complementary strand.
[12]
An isolated polynucleotide comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-28 and 30-33.
[13]
An isolated polynucleotide that hybridizes under stringent washing conditions with a nucleotide sequence selected from the group consisting of residues 1863 to 1875 of SEQ ID NO: 29, residues 6679 to 6700 of SEQ ID NO: 29, and complements thereof.
[14]
The polynucleotide according to claim 13, which is an amplicon generated by a polymerase chain reaction.
[15]
A method for detecting a corn event in a sample comprising corn DNA, said sample being received from the American Type Culture Collection (ATCC) under accession number PTA
AAD-1 corn event DAS- present in seed deposited under 10244
Contacting with at least one polynucleotide which is characteristic for 40278-9.
[16]
The sample,
a. A first primer that binds to a flanking sequence selected from the group consisting of residues 1 to 1873 of SEQ ID NO: 29, residues 6690 to 8557 of SEQ ID NO: 29, and its complementary strand; and b. Contacting with a second primer that binds to an insertion sequence comprising residues 1874-6669 of SEQ ID NO: 1 or its complementary strand, subjecting the sample to a polymerase chain reaction, and assaying for amplicons generated between the primers. The method according to claim 15, comprising: steps.
[17]
The method of claim 16, wherein the primer is selected from the group consisting of SEQ ID NOs: 1-28.
[18]
The polynucleotide comprises at least 30 nucleotides and comprises residue 1 of SEQ ID NO: 29
863 to 1875, a sequence selected from the group consisting of residues 6679 to 6700 of SEQ ID NO: 29 and its complementary strand under stringent conditions, wherein the method comprises stringently treating the sample and the polynucleotide. 16. The method of claim 15, further comprising subjecting to hybridization conditions and assaying the sample for hybridization of the polynucleotide to the DNA.
[19]
A DNA detection kit comprising the first primer and the second primer according to claim 17.
[20]
A DNA detection kit for performing the method according to claim 18.
[21]
A DNA detection kit comprising the polynucleotide according to claim 13.
[22]
A polynucleotide comprising SEQ ID NO: 29.
[23]
23. A method for producing the polynucleotide of claim 22.
[24]
A method for inserting a transgene into a DNA segment of a corn genome comprising the above D
The method wherein the NA segment comprises a 5 ′ end comprising nucleotide residues 1-1873 of SEQ ID NO: 29 and a 3 ′ end comprising nucleotide residues 6690-8557 of SEQ ID NO: 1.
[25]
A method of breeding a corn plant, comprising: crossing a first corn plant comprising SEQ ID NO: 29 with a second corn plant to produce a third corn plant comprising a genome; And assaying for the presence of SEQ ID NO: 29.
[26]
A method of introducing a herbicide-resistant trait into a corn plant, wherein the first corn plant comprising SEQ ID NO: 29 is crossed with a second corn plant to produce a third corn plant comprising a genome, Assaying three corn plants for the presence of SEQ ID NO: 29 in said genome.

The following examples are included to illustrate procedures for practicing the invention and to illustrate some preferred embodiments of the invention. These examples should not be construed as limiting. It will be appreciated by those skilled in the art that the techniques disclosed in the following examples represent specific approaches used to describe the preferred form for implementation. However, one of ordinary skill in the art, in view of this disclosure, will still be able to obtain similar or similar results without departing from the spirit and scope of the present invention while making many changes in these specific embodiments. It has recognized. Unless stated otherwise, all percentages are by weight,
All solvent mixing ratios are by volume unless stated otherwise.

Unless otherwise indicated, the following abbreviations are used.
AAD-1 Aryloxyalkanoate dioxygenase-1
bp base pair degree Celsius DNA deoxyribonucleic acid DIG digoxigenin EDTA ethylenediaminetetraacetic acid kb kilobase μg microgram μL microliter mL milliliter M molar mass OLP overlap probe PCR polymerase chain reaction PTU plant transcription unit SDS sodium dodecyl sulfate SOP standard operating procedure SSC Buffer solution containing a mixture of sodium chloride and sodium citrate, pH 7.0
Buffer solution containing a mixture of TBE Tris base, boric acid and EDTA, pH 8.3
V bolt
[Example]

Transformation and selection of AAD1 event pDAS 1740-278 The AAD1 event pDAS 1740-278 was generated by WHISKER-mediated transformation of the maize line Hi-II. The transformation method used is described in US 20090093366. Plasmid pDAS1740 (pDAB
The FspI fragment (also called 3812) (FIG. 1) was transformed into the maize line. This plasmid construct is RB7 MARv3 :: Zea mays ubiquitin 1 promoter v2 // AAD1 v3 // Zea mays PER5 3′UTR :: RB7 M
Contains a plant expression cassette containing an ARv4 plant transcription unit (PTU).

Generated a large number of events. Events that survived, were healthy, and produced haloxyhop-resistant callus tissue were assigned a unique identification code representing a putative transformation event and were continually transferred to fresh selective media. Plants regenerate from tissues originating from each unique event,
Moved to the greenhouse.

Leaf samples were taken for molecular analysis and the presence of the AAD-1 transgene was verified by Southern blot, DNA border confirmation and confirmation supported by genomic markers. Positive T0 plants were pollinated with inbred lines to obtain T1 seeds. Event pDAS1470-27
8-9 (DAS-40278-9) T1 plants were selected, self-pollinated and characterized for 5 generations. In the meantime, T1 plants were backcrossed and transfected into the elite germplasm (XHH13) for several generations with marker-assisted selection. This event
Made from independent transformed isolates. Excellent events, including single insertion site, its unique characteristics such as standard Mendelian isolation and stable expression, and herbicide tolerance and agroeconomic performance in a wide genotypic background and across multiple environmental locations Selected based on a combination of efficiencies. The following example shows the event pDAS-1740-278-9.
Contains data used to characterize.

Characterization of the pDAS 1740-278-9 event by Southern blot Southern blot analysis was used to establish the integration pattern of the inserted DNA fragment and aad-1 at event pDAS-1740-278-9 (DAS-40278-9)
The number of inserts / copy of the gene was determined. Data were obtained to verify the integration and integrity of the aad-1 transgene inserted into the maize genome.

Southern blot data is the pD at corn event DAS-40278-9.
The AS1740 / FspI fragment insert is aad-1 P from plasmid pDAS1740.
Suggested that it occurred as a simple incorporation of a single intact copy of TU. Detailed Southern blot analysis with probes specific for genes, promoters, terminators and other regulatory elements contained in the plasmid region, and cleavage sites within the plasmid that hybridize within the plasmid or maize This was done with an explanatory restriction enzyme that produces a fragment (boundary fragment) that spans the plasmid junction with genomic DNA.
The molecular weights shown from Southern hybridization for the combination of restriction enzyme and probe were unique for the event and established its identification pattern. These analyzes showed that the plasmid fragment was not transformed into maize genomic DNA without reconstitution of aad-1 PTU.
It was also shown that it was inserted into. The same hybridization fragment was observed in five different generations of transgenic maize event DAS-40278-9, indicating the stability of the inherited trait of aad-1 PTU insertions over generations. Hybridization with a mixture of three backbone probes located outside the restriction site of FspI on plasmid pDAS1740 did not detect any particular DNA / gene fragment, which indicates that the transgenic maize event DAS-40278 The absence of the ampicillin resistance gene in -9 and the absence of other vector backbone regions immediately adjacent to the FspI restriction site of plasmid pDAS1740 are shown. aad-1 corn event D
A map for explanation of the insert fragment in AS-40278-9 is shown in FIGS.

(Example 2.1)
Corn Leaf Sample Collection and Genomic DNA (gDNA) Isolation gDNA was prepared from leaves of individual plants of aad-1 corn event DAS-40278-9. gDNA, aad-1 corn event DAS-40278-9
Extracted from leaf tissue harvested from individual plants with Event DAS-40278-9
Transgenic corn seeds from 5 different generations were used. 4 per generation
Twenty individual corn plants for event DAS-40278-9, from one plant, were selected. In addition, gDNA was isolated from the traditional corn plant XHH13, which contains a genetic background that is representative of the essence lineage and the aad-1 gene is absent.

Prior to isolating gDNA, leaf punches were taken from each plant to determine aad-1 protein expression using a rapid test strip kit (American Biostica, Sw
edesboro, NJ) according to the manufacturer's recommended procedure. Each leaf punched sample was given a + or-rating for the presence or absence of aad-1, respectively. Only positive plants from five generations of event DAS-40278-9 were subjected to further characterization.

Samples of corn leaves were used for event DAS-40278-9 and conventional control XHH.
Harvested from 13 individual plants. Leaf samples were quickly frozen in liquid nitrogen and stored at approximately −80 ° C. until use.

Individual genomic DNA was extracted from frozen corn leaf tissue according to standard CTAB methods. If necessary, some genomic DNA is transferred to Qiagen Genomic-T
ip (Qiagen, Valencia, CA) was further purified using the procedure recommended by the manufacturer. After extraction, the DNA was ligated with Pico Green reagent (Invi
(Trogen, Carlsbad, Calif.). The DNA is then visualized on an agarose gel to confirm the value from the Pico Green analysis and D
The quality of NA was determined.

(Example 2.2)
DNA digestion and separation For the molecular characterization of DNA, the corn event DAS-40278-9 DN
Nine micrograms (9 μg) of genomic DNA from the A sample and the conventional control were converted to DNA1
The restriction enzyme selected per μg was digested by adding approximately 5-11 units and the corresponding reaction buffer to each DNA sample. Each sample was incubated overnight at approximately 37 ° C. Restriction enzymes EcoRI, NcoI, SacI, FseI and HindIII were used for digestion (New England Biolabs, Ipswich, MA).
. A positive hybridization control sample was obtained from plasmid DNA pDAS1740.
(PDAB3812) was prepared by combining genomic DNA from a conventional control with a ratio approximately equal to one copy of the transgene per corn genome and digested using the same procedure and restriction enzymes as the test sample. DNA from a conventional corn control (XHH13) was digested using the same procedure and restriction enzymes as the test sample to serve as a negative control.

The digested DNA sample was prepared using Quick-Precip (Edge BioSystems).
, Gaithersburg, MD) and precipitated with 1 × Blue Juice (I
nvitrogen, Carlsbad, CA) to achieve the desired volume for gel loading. DNA samples and molecular size markers were then transferred to a 0.8% agarose gel in 1 × TBE buffer (Fisher Scientific, Pitt
sburgh, PA) at 55-65 volts for approximately 18-22 hours,
Fragment separation was achieved. The gel was treated with ethidium bromide (Invitrogen, Carlsb
ad, CA) and DNA was visualized under ultraviolet (UV) light.

(Example 2.3)
Southern Transfer and Membrane Processing Southern blot analysis was performed by Memelink et al. (1994) Southern, Northern, and Western Blot An
alysis. Plant Mol. Biol. Manual F1: 1-23. Briefly, after electrophoretic separation and visualization of DNA fragments, the gel is 0.25 NH 2
Cl (Fisher Scientific, Pittsburgh, PA) approximately 1
Depurinized using 5 minutes and then denaturing solution (AccumENE, Sigma, St.
Louis, MO) for approximately 30 minutes, then neutralization solution (AccumENE, Sigma)
, St. (Louis, MO) for at least 30 minutes. Southern transfer
Nylon membrane (Roche Diagnostics, Indianapolis)
, IN) on a wicking system 10 × SSC (Sigma, St. Louis)
s, MO) and performed overnight. After transfer, the membrane is 2 x SSC
Washed in solution, the DNA was bound to the membrane by UV crosslinking. This process resulted in a Southern blot membrane that was ready for hybridization.

(Example 2.4)
DNA probe labeling and hybridization DNA fragments bound to the nylon membrane were detected using a labeled probe. The probe used for this study is a digoxigenin (DIG) labeled nucleotide [DI
G-11] -dUTP was generated from PCR-based integration from the gene element from plasmid pDAS1740 and fragments generated with primers specific for other regions. Preparation of DNA probe by PCR synthesis is performed by PCR DIG probe synthesis kit
che Diagnostics, Indianapolis, IN) was performed according to the procedure recommended by the manufacturer. A list of probes used in this study is listed in Table 1.

Labeled probes were analyzed by agarose gel electrophoresis to determine their quality and quantity. The desired amount of labeled probe is then added to the DIG Easy Hyb solution (Roche
Used for hybridization to target DNA on nylon membranes for detection of specific fragments using the procedure described for Diagnostics (Indianapolis, Ind.). Briefly, nylon membrane blots with immobilized DNA were washed briefly in 2 × SSC and 20-25 mL pre-warmed DIG Easy.
Using the Hyb solution in a hybridization bottle at approximately 50 ° C. for a minimum of 30 minutes,
Prehybridization was performed in a hybridization oven. The prehybridization solution is then decanted and 20 mL of pre-warmed DIG Easy Hy containing the desired amount of specific probe previously denatured by boiling in water for 5 minutes.
Replaced with b solution. The hybridization step is then performed at approximately 40-60 ° C
Overnight in a hybridization oven.

(Example 2.5)
Detection At the end of probe hybridization, DIG Easy H containing the probe
The yb solution was decanted into a clean tube and stored at -20 ° C. These probes could be reused 2-3 times according to the manufacturer's recommended procedure. Rinse the membrane blot briefly and place in a clean plastic container with low stringency wash buffer (2x SSC).
, 0.1% SDS) for approximately 5 minutes at room temperature, followed by high stringency wash buffer (0.1 × SSC, 0.1% SDS) for 15 minutes, each at approximately 65 ° C.
Was washed twice. The membrane blot is then transferred to another clean plastic container and washed with DIG wash and block buffer set (Roche Diagnostics).
Wash briefly with 1 × wash buffer from Indianapolis, IN) for approximately 2 minutes, block for a minimum of 30 minutes in 1 × blocking buffer, followed by anti-DIG-AP (alkaline phosphatase) in 1 × blocking buffer Antibody (1: 5
000 dilution, Roche Diagnostics, Indianapolis, IN)
And proceeded to incubation for 30 minutes at the shortest. After washing 2-3 times with 1 × wash buffer, the specific DNA probe remains bound to the membrane blot and DIG
A labeled DNA standard was added to the CDP-Star chemiluminescent nucleic acid detection system (Roche Di).
visualization, Indianapolis, Ind.) according to the manufacturer's recommendations. Blots were prepared using chemiluminescent film (Roche Diagnostics, In
dianapolis, IN) was exposed at one or more time points to detect hybridized fragments and visualize molecular size standards. The film is then all-
Develop with Pro 100 Plus film developer (Konica SRX-101),
Images were scanned for reporting. The number and size of detected bands were documented for each probe. The size of the hybridizing fragments on the Southern blot was determined using DIG-labeled DNA molecular weight marker II (MWM DIG II) visualized after DIG detection as described.

(Example 2.6)
Probe stripping
The DNA probe is removed from the membrane blot after the Southern hybridization data has been obtained, and the membrane blot is prepared according to the manufacturer's recommended procedure (DIG Appl
ication Manual for Filter Hybridization, (2003). Roche Diagnostics), different DN
Could be reused for hybridization with A probe. Briefly, after signal detection and film exposure, membrane blots are rinsed thoroughly with Milli-Q water and approximately 15 minutes in removal buffer (0.2N NaOH, 0.1% SDS) at room temperature or 37 ° C. Washed twice. The membrane blot was then washed briefly in 2 × SSC and ready for prehybridization and hybridization with another DNA probe. Expose the membrane blot to a new chemiluminescent film and remove all D
The NA probe was surely removed before proceeding to the next hybridization. The re-exposed film was saved with the previous hybridization data package in a study file for recording.

(Example 2.7)
Southern Blot Results Based on the known restriction enzyme sites of the pDAS1740 / FspI fragment, the predicted and observed fragment sizes for specific digests and probes are shown in Table 2. Two types of fragments were identified from these digests and hybridizations: an internal fragment where the known enzyme site touches the probe region and is completely contained within the pDAS1740 / FspI fragment, and a known enzyme site is one end of the probe region. A boundary fragment whose second site is predicted in the corn genome. The size of the boundary fragment varied with the event. This is because in most cases the DNA fragment integration site is unique for each event. Border fragments provide a means to determine the location of restriction enzyme sites for the integrated DNA and to assess the number of DNA insertions. Based on the Southern blot analysis completed in this study, a single copy of intact aad-1 PTU from plasmid pDAS1740 / FspI is shown in detail in the insert map, and the corn genome of event DAS-40278-9. (Figs. 2-3).

Restriction enzymes EcoRI, Nc with unique restriction sites in plasmid pDAS1740
Select oI, SacI, FseI / HindIII and select event DAS-40278-
The aad-1 gene insert in 9 was characterized. > 3382 bp,> 2764 bp
The> 4389 bp border fragment was expected to hybridize with the aad-1 gene probe after EcoRI, NcoI and SacI digestion, respectively (Table 2). About (~) 12
A single aad-1 hybridization band of 000 bp, about 4000 bp and about 16000 bp was observed when using EcoRI, NcoI and SacI, respectively, indicating that the aad in the corn genome of event DAS-40278-9
A single site of -1 gene insertion is shown. Double digestion with FseI and HindIII was selected to release a 3361 bp fragment containing the aad-1 plant transcription unit (PTU, promoter / gene / terminator) (Table 2). An expected 3361 bp fragment was observed using the aad-1 gene probe after FseI / HindIII digestion.
Digestion of DAS-40278-9 sample with all four enzyme / enzyme combinations followed by a
The results obtained with ad-1 gene probe hybridization are the results of plasmid pDAS
A single copy of the intact aad-1 PTU from 1740 is the event DAS-40
It was shown to have been inserted into the 278-9 maize genome.

Select restriction enzymes NcoI, SacI and FseI / HindIII to select event D
The promoter (ZmUbi1) region for aad-1 in AS-40278-9 was characterized. NcoI and SacI digestions are> 3472 bp and> 4, respectively, when hybridized with DNA probes specific for the ZmUbi1 promoter region.
It is predicted to produce a 389 bp boundary region fragment (Table 2). Two hybridization bands of about (~) 6300 bp and about 3600 bp are found after ZcoI digestion with ZmUb
It was detected using the i1 promoter probe. However, a band of about 3600 bp exists over the lane of all samples including the conventional control, which means that this about 3600 bp
This band suggested that the maize-derived ubiquitin promoter (ZmUbi1) probe was a non-specific signal band due to homologous binding to the maize endogenous ubi gene. In contrast, a signal band of about 6300 bp is observed for the tested DA
Detected in the S-40278-9 sample but not in the conventional control, this approximately 6300 bp band is specific for the ZmUbi1 promoter probe from plasmid pDAS1740 and is therefore shown in Table 2 Predicted NcoI / ZmUbi1
It showed that it was a band. Similarly, two hybridization bands of about 3800 bp and about 16000 bp were detected using the ZmUbi1 promoter probe after SacI digestion. An approximately 3800 bp band appears in all sample lanes, including the conventional control, and is therefore considered to be non-specific hybridization of the ZmUbi1 promoter probe to the maize endogenous ubi gene. DAS-40278
A hybridization band of approximately 16000 bp present only in the -9 sample is considered the predicted SacI / ZmUbi1 band. Double digestion with FseI / HindIII results in 3361 bp of aad-1 hybridizing with the ZmUbi1 promoter probe
It is expected to release the PTU fragment (Table 2). This 3361 bp band and about 64
A 00 bp non-specific hybridization band was detected with the ZmUbi1 promoter probe after FseI / HindIII digestion. The approximately 6400 bp band is thought to be non-specific binding of the ZmUbi1 promoter probe to the maize endogenous ubi gene. This is because this band is present in all sample lanes including conventional controls. In addition, another band very close to about 6400 bp is the conventional control, BC3S1
And several BC3S2 samples. This additional band very close to about 6400 bp is also considered non-specific. This is because it is present in the conventional control XHH13 sample lane and is likely to be associated with the genetic background of XHH13.

Same restriction enzyme / enzyme combinations NcoI, SacI and FseI / HindIII
Select Terminator at Event DAS-40278-9 (ZmPer5)
The region was characterized. NcoI digestion is a DN specific for the ZmPer5 terminator region.
When hybridized with the A probe, it is expected to produce a border region fragment of> 2764 bp (Table 2). Two hybridization bands of about (˜) 4000 bp and about 3900 bp were detected using a ZmPer5 terminator probe after NcoI digestion. An approximately 3900 bp band is present across all sample lanes, including the conventional control, indicating that the approximately 3900 bp band probably binds the maize-derived peroxidase gene terminator (ZmPer5) probe homologously to the maize endogenous per gene. It was suggested that this was a non-specific signal band. In contrast, a signal band of about 4000 bp was detected in the tested DAS-40278-9 sample but not in the conventional control, which means that this about 4000 bp band is from plasmid pDAS1740. Specific ZmPer5 terminator probes, thus indicating that this is the predicted NcoI / ZmPer5 band shown in Table 2. A> 1847 bp border fragment is expected to hybridize with the ZmPer5 terminator probe after SacI digestion. Two hybridization bands of about 1900 bp and about 9000 bp were detected using a ZmPer5 terminator probe after SacI digestion. An approximately 9000 bp band was present across all sample lanes, including the conventional control, and was therefore considered to be non-specific hybridization of the ZmPer5 terminator probe to the maize endogenous per gene. DAS-4
The approximately 1900 bp hybridization band that was present only in the 0278-9 sample is believed to be the predicted SacI / ZmPer5 band. FseI / HindIII
Double digestion at 3336b hybridizes with ZmPer5 terminator probe
It is expected to release the p aad-1 PTU fragment (Table 2). This 3361 bp band and an additional non-specific hybridization band of about 2100 bp
Detected with ZmPer5 terminator probe after HindIII digestion. An additional approximately 2100 bp band is non-specific binding of the ZmPer5 terminator probe to the maize endogenous gene. This is because this band is present in all sample lanes including the negative control. These digestions of DAS-40278-9 samples followed by Z
The results obtained from hybridization of the mUbi1 promoter and ZmPer5 terminator probe indicate that the intact aad − from plasmid pDAS1740
It was further confirmed that a single copy of 1 PTU was inserted into the corn genome of event DAS-40278-9.

Select restriction enzymes NcoI and SacI to select AAD-1 corn event DAS
The remaining elements from the pDAS1740 / FspI fragment at -40278-9 were characterized (Table 2). The DNA sequences of elements RB7 Mar v3 and RB7 Mar v4 have greater than 99.7% identity, thus RB7 Mar v3 or RB7
A DNA probe specific for Mar v4 was predicted to hybridize with DNA fragments containing either version of RB7 Mar. > 2764 bp and> 3472
The two border fragments of bp are RB7 Mar v4 and RB7 Ma after NcoI digestion.
It was predicted to hybridize with the rv3 probe (Table 2). Two hybridization bands of about (˜) 4000 bp and about 6300 bp are found in DAS-40278-9.
Observed with either RB7 Mar v4 or RB7 Mar v3 probe after NcoI digestion in the sample. Similarly,> 1847 bp and> 4389 bp 2
Two border fragments are RB7 Mar v4 and RB7 Mar v3 after SacI digestion
Expected using probes (Table 2). Hybridization bands of about 1900 bp and about 16000 bp were detected with RB7 Mar v4 or RB7 Mar v3 probes after SacI digestion in DAS-40278-9 samples.

In summary, the Southern hybridization results obtained using these elemental probes show that the DNA inserted into corn event DAS-40278-9 contains intact aad-1 PTU, the 5 ′ end of each of the inserts and It was shown to contain with 3'-end matrix attachment region RB7 Mar v3 and RB7 Mar v4.

(Example 2.8)
Absence of main chain sequence FspI main chain region of plasmid pDAS1740 (plasmid pDAS17
AAD-1 corn event DAS-40, using a combination of equimolar proportions of three DNA fragments (Table 1) covering 40 4867-7143 bp) as the backbone probe
278-9 was characterized. Since the plasmid pDAS1740 / FspI fragment was used to generate event DAS-40278-9, no specific hybridization signal was expected after any restriction enzyme digestion using the backbone probe combination (Table 2). ). In all DAS-40278-9 samples, it was confirmed that no specific hybridization signal was detected using a backbone probe after NcoI or SacI digestion. The positive control lane contained the expected hybridizing band, demonstrating that the probe could hybridize to any homologous DNA fragment if present in the sample. This data is from the corn event DAS-40278-
9 insertions did not include any vector backbone sequences outside the FspI region from plasmid pDAS1740.

Southern blot analysis for molecular characterization was performed using leaf samples from five different generations of event DAS-40278-9. The integration pattern was examined using a selected restriction enzyme digest and probe combination to insert the inserted gene, aad-1.
And the characteristics of the gene expression promoter, terminator-containing non-coding region and matrix attachment region.

Southern blot characterization of DNA inserted into event DAS-40278-9 is
Indicates that a single intact copy of ad-1 PTU has been incorporated into event DAS-40278-9. The molecular weights shown by Southern hybridization for restriction enzyme combinations and probes were unique for the event and established its identification pattern. The hybridization pattern is identical across all five generations, indicating that the insert is stable in the corn genome. Hybridization with a probe covering the backbone region beyond the pDAS1740 / FspI transformed fragment from plasmid pDAS1740 results in the vector backbone sequence being event D
It is confirmed that it is not incorporated into AS-40278-9.

Cloning and characterization of the DNA sequence in the insert and flanking border region of corn event DAS-40278-9 To characterize the inserted DNA and indicate the genomic insertion site, event DAS-40
The 278-9 insert and border region DNA sequences were determined. In total, an 8557 bp event DAS-40278-9 genomic sequence was identified, which was 1873 bp of 5 ′ flanking border sequence, 1868 bp of 3 ′ flanking border sequence and 4816 bp of D.
The NA insert was included. The 4816 bp DNA insert contains an intact aad-1 expression cassette, a 259 bp partial MAR v3 at the 5 ′ end and a 1096 bp partial MAR v4 at the 3 ′ end. Sequence analysis revealed a 21 bp insertion at the 5 ′ integration junction and a two base pair deletion from the corn genome insertion locus. A one base pair insertion was found at the 3 'integration junction between the maize genome and the DAS-40278-9 insert. In addition, a single base change (T to C) can be detected in the 3'UT
Found at position 5212 in the non-coding region of R. Both of these changes are aad-1
Does not affect the open reading frame composition of the expression cassette.

By PCR amplification based on event DAS-40278-9 insert and border sequence,
It was confirmed that the border region was of maize origin and the junction region could be used for event specific identification of DAS-40278-9. Analysis of sequences across the junction region
A new open reading frame (ORF> = 200 codons) is the event DAS-
It was not obtained from DNA insertion at 40278-9, indicating that the genomic open reading frame was not obstructed by the integration of DAS-40278-9 into the native maize genome. Overall, AAD-1 corn event DAS-40278-
Characterization of the 9 inserts and the border sequence showed that a single intact copy of the aad-1 expression cassette was integrated into the native maize genome.

(Example 3.1)
Genomic DNA extraction and quantification Genomic DNA was extracted from lyophilized or freshly ground leaf tissue using a modified CTAB method. A DNA sample was prepared using 1 × TE (10 mM Tris pH 8.0, 1 mM
EDTA) (Fluka, Sigma, St. Louis, MO)
The Green method is used by the manufacturer (Molecular Probes, Eugene,
OR) and quantified. For PCR analysis, a DNA sample is diluted with molecular biology grade water (5PRIME, Gaithersburg, MD) and 10-10
A concentration of 0 ng / μL was obtained.

(Example 3.2)
PCR primers Table 3 lists the primer sequences used to clone the DNA insert and flanking border region of event DAS-40278-9, with the positions and descriptions shown in FIG. Table 4 lists the primer sequences used to confirm the insert and border sequences. Primer positions are shown in FIGS. 4 and 5, respectively. All primers are I
ntegrated DNA Technologies, Inc. (Coralvir
le, IA). Primer water (5PRIME, Gaithersburg,
MD) was dissolved to a concentration of 100 μM for the stock solution and diluted with water to a concentration of 10 μM for the working solution.

(Example 3.3)
Genome Walking GenomeWalker (TM) Universal Kit (Clontech Labo
ratsies, Inc. , Mountain View, CA) was used to clone the 5 'and 3' flanking border sequences of corn event DAS-40278-9. AAD-1 corn event DAS-402 according to manufacturer's instructions
Approximately 2.5 μg of genomic DNA from 78-9 was added to EcoRV, StuI (both provided by the kit) or ScaI (New England Biolabs, Ipsw
ich, MA) and digested overnight. The digested DNA is converted into DNA Clean & Con
centerr ™ -25 (ZYMO Research, Orange, CA)
) And then ligated to a GenomeWalker ™ adapter to construct a GenomeWalker ™ library. Each GenomeWa
lker ™ library 1 using adapter primer AP1 provided in the kit and each construct specific primer 5End3812_A and 3End3812_C
Used as DNA template for subsequent PCR amplification. 1 of 1 microliter 1:25 dilution
The secondary PCR reaction is then secondary PCR using nested adapter primer AP2 and each nested construct specific primer 5End3812_B and 3End3812_D.
Used as a template for amplification. TaKaRa LA Taq ™ HS (Takar
a Bio Inc. Shiga, Japan) was used in PCR amplification. 50μ
During the L PCR reaction, 1 μL of DNA template, 8 μL of 2.5 mM dNTP mix, 0
. 2 μM of each primer, 2.5 units of TaKaRa LA Taq ™ HS DN
A polymerase, 5 μl of 10 × LA PCR buffer II (Mg2 + plus), and 1.5 μL of 25 mM MgCl ₂ were used. Specific PCR conditions are listed in Table 5.

(Example 3.4)
Conventional PCR
DN at corn event DAS-40278-9 using standard PCR
The A insert and border sequence were cloned and confirmed. TaKaRa LA Taq (
Trademark) (Takara Bio Inc., Shiga, Japan), HotStar
Taq DNA polymerase (Qiagen, Valencia, CA), Expand
High Fidelity PCR System (Roche Diagnostics)
, Inc. , Indianapolis, IN) or Easy-A® Hig
h-Fidelity PCR Cloning Enzyme & Master Mix (Strataage
ne, LaJolla, CA) was used for conventional PCR amplification following the manufacturer's recommended procedure. Specific PCR conditions and amplicon descriptions are listed in Table 6.

(Example 3.5)
PCR product detection, purification, PCR product subcloning and sequencing PCR products were purified according to product instructions according to product instructions 1.2% or 2% E-gel (Invit
rogen, Carlsbad, CA). Fragment size was estimated by comparison with DNA markers. If necessary, PCR fragments were purified by excising the fragments from a 1% agarose gel in 1 × TBE stained with ethidium bromide using a QIAquick gel extraction kit (Qiagen, Carlsbad, Calif.).

PCR fragments were placed in pCR®4-TOPO® in a TOPO TA Cloning® kit (Invitrogen, Carl for sequencing).
sbad, CA) was subcloned by using according to product instructions. Specifically, 2-5 microliters of TOPO® cloning reaction was performed with One
Shot chemically competent TOP10 cells were transformed according to the manufacturer's instructions. The cloned fragment was obtained by small-scale preparation of plasmid DNA (QIAprep spin miniprep kit, Qiagen, Carlsbad, Calif.) Followed by restriction digestion with EcoRI or direct colony PCR using the T3 and T7 primers provided in the kit. confirmed. Plasmid DNA or glycerol stocks of selected colonies were then sent for sequencing.

After subcloning, the putative target PCR product is first sequenced to produce the predicted D
It was confirmed that the NA fragment was cloned. Colonies containing the appropriate DNA sequence were selected for primer walking to determine the complete DNA sequence. Sequencing was performed by Cogenics (Houston, TX).

The final assembly of the insert and border sequence was performed using the Sequencher software (
Version 4.8 Gene Codes Corporation, Ann Arbo
r, MI). The insert and border sequence annotation of corn event DAS-40278-9 is Vector NTI (versions 10 and 11).
, Invitrogen, Carlsbad, CA).

Homology searches were performed using the BLAST program against the GenBank database. Open reading frame (ORF) analysis using Vector NTI (version 11, Invitrogen) was performed to identify the complete insert and ORF (> = 200 codons) in the flanking boundary sequence.

(Example 3.6)
The 5 ′ end border sequence DNA fragment was transferred to each corn event DAS-40278-9 GenomeWal.
Amplified from the ker ™ library using a specific nested primer set for the 5 ′ end of the transgene. An approximately 800 bp PCR product is generated at event DAS
-40278-9 Observed from both EcoRV and StuI GenomeWalker ™ libraries. The ScaI GenomeWalker ™ library yielded a product of approximately 2 kb. Fragments were cloned into pCR®4-TOPO® and 6 colonies from each library were randomly picked for terminal sequencing to determine the sequence from which the insert was predicted. It was confirmed to contain. Complete sequencing by primer walking of the insert resulted in a corn event DAS-40.
278-9 StuI, EcoRV and ScaI GenomeWalker ™
It was revealed that the fragments amplified from the library were 793, 822 and 2132 bp, respectively. The DNA fragments generated from the StuI and EcoRV GenomeWalker ™ libraries are 100% identical to the DNA fragments generated from the ScaI GenomeWalker ™ libraries, indicating that these DNAs
It was suggested that the fragment was amplified from the 5 ′ region of the transgene insert. 187 obtained
A BLAST search of the 3 bp maize genomic sequence showed high similarity to the sequence of the maize BAC clone. In addition, sequence analysis of the insertion junction was performed using a 917 bp MAR v3
Was cleaved at its 5 ′ end region compared to the plasmid pDAS1740 / FspI fragment, indicating that a 259 bp partial MAR v3 remained in the 5 ′ region of the aad-1 expression cassette.

(Example 3.7)
3 ′ terminal border sequence A DNA fragment of approximately 3 kb in size was ligated to a corn event DAS-40278-9.
Amplified from the StuI GenomeWalker ™ library using a specific nested primer set for the 3 ′ end of the transgene. The DNA fragment is designated as pC
Cloned into R®4-TOPO® and 10 colonies were randomly picked for terminal sequencing to confirm the expected sequence insertion. Three clones with the expected insert were fully sequenced to yield a 2997 bp DNA fragment. Sequence analysis of this DNA fragment revealed a partial MAR v4 element (lacking 70 bp of its 5 ′ region) and a 1867 bp maize genomic sequence. A BLAST search showed that the 1867 bp genomic DNA sequence was 100% identical to the sequence in the same maize BAC clone identified at the 5 'border sequence.

(Example 3.8)
DNA insert and junction sequence The DNA insert and junction region were cloned from the corn event DAS-40278-9 using a PCR-based method as previously described. Five primer pairs were designed based on the 5 ′ and 3 ′ flanking border sequences and the predicted transgene sequence. In total, five overlapping DNA fragments (1767 bp amplicon 1
1703 bp amplicon 2, 1700 bp amplicon 3, 1984 bp amplicon 4 and 1484 bp amplicon 5) were cloned and sequenced (FIG. 4). The entire insert and flanking boundary sequence was assembled based on the overlapping sequence of the five fragments. In the final sequence, the 4816 bp DNA insert from pDAS1740 / FspI, the 1873 bp 5 'flanking border sequence and 18
The presence of a 68 bp 3 'flanking boundary sequence is confirmed. The 4816 bp DNA insert is an intact aad-1 expression cassette, a 259 bp partial MAR v3 at the 5 ′ end.
And contains a 1096 bp partial MAR v4 at the 3 ′ end (SEQ ID NO: 29).

At least two clones for each primer pair were used for primer walking to obtain complete sequence information about the DNA insert and its border sequences. Sequence analysis was performed at the 5 'integration junction with maize genomic DNA and pDAS1740 / Fsp.
An insertion of 21 bp was shown between the incorporated part MAR v3 from I. The results of BLAST search and Vector NTI analysis showed that the 21 bp insert DNA showed no homology with any plant species DNA or pDAS1740 plasmid DNA. A single base pair insertion is made at the 3 'integration junction with maize genomic DNA and pDAS.
Found between the partial MAR v4 from 1740 / FspI. DNA integration
It also resulted in a 2 base pair deletion at the insertion locus of the maize genome (Figure 6). further,
A single nucleotide difference (T to C) at position 5212 bp was observed in the untranslated 3′UTR region of the DNA insert (SEQ ID NO: 29). However, none of these changes are important for aad-1 expression and appear to create no new ORF (> = 200 codons) across the junction in the insert of DAS-40278-9.

(Example 3.9)
Confirmation of Maize Genomic Sequence To confirm the insertion site of the event DAS-40278-9 transgene in the maize genome, PCR amplification was performed using different primer pairs (FIG. 4). Genomic DNA from event DAS-40278-9 and other transgenic or non-transgenic corn lines was used as a template. Using two aad-1 specific primers AI5End01 and AI5End02 and two primers 1F5End01 and 1F5End02 designed based on the 5 ′ end border sequence,
A DNA fragment in which the aad-1 gene spanned the 5 ′ end border region was amplified. Similarly, aad-1
In order to amplify a DNA fragment spanning the 3 ′ end border sequence 1
F3End05 primer and aad-1 specific AI3End01 primer were used. The DNA fragment of the expected size is the AAD-1 corn event DAS-4027.
AAD-1 corn event DAS-40278 only from 8-9 genomic DNA
Amplification was performed using each primer pair consisting of one primer at the 9 flanking boundaries and one aad-1 specific primer. The control DNA sample does not yield a PCR product using the same primer pair, which means that the cloned 5 'and 3' end border sequences are actually in each of the inserted aad-1 gene constructs. It was shown to be upstream and downstream sequences. A faint band of about 8 kb in size is shown in AAD-1 corn event DAS-40278-9, AAD-1 corn event DAS-40474.
Note that from all corn samples including the non-transgenic corn line XHH13, it was observed when the 1F5End01 and AI5End01 primer pairs were used for PCR amplification. The faint band observed (on the prepared gel) may be the result of non-specific amplification in the corn genome using this primer pair.

To further confirm DNA insertion in the maize genome, the two primers 1F5End03 and 1F5End04 at the 5 ′ end border sequence and two primers 1F3End03 and 1F3End04 at the 3 ′ end border sequence are used to span the insertion locus The DNA fragment was amplified. PCR amplification with either primer pair 1F5End03 / 1F3End03 or primer pair 1F5End04 / 1F3End04 resulted in a fragment with an expected size of approximately 8 kb from genomic DNA of AAD-1 corn event DAS-40278-9. In contrast, no PCR product was obtained from genomic DNA of AAD-1 corn event DAS-40474-7 or non-transgenic corn line XHH13. AAD-1 Corn Event DAS-40278-9 and Event DAS-40474-7 are HiII
And subsequent maize line XHH13 of the original transgenic event
The majority of the genome in each of these two events is theoretically derived from the maize line XHH13. Only the flanking border sequences close to the aad-1 transgene are carried over from the original genomic DNA and AAD-
Although preserved during the one-event gene transfer process, other regions of the genomic sequence are XHH
It is thought that it was replaced by 13 genome sequences. Thus, from genomic DNA of AAD-1 corn event DAS-40474-7 and XHH13, primer pair 1F5End03 / 1F3End03 or primer pair 1F5End04 / 1F3E
It is not surprising that no fragment was amplified using any of nd04. The approximately 3.1 and 3.3 kb fragments were transformed into the maize line HiII using the primer pairs 1F5End03 / 1F3End03 and 1F5End04 / 1F3End04, respectively.
And B73 genomic DNA, but not corn line A188. This result indicates that the border sequence originates from the genome of maize line B73.

Further cloning of maize genomic DNA from B73 / HiII,
The validity of the flanking boundary sequence was ensured. The PCR amplified fragment was sequenced and B73 /
The insert DNA region integrated at a specific location in the HiII genomic DNA was verified. Primers were designed based on the resulting sequences. The primer set Amp1F / Amp5R was used to amplify a 2212 bp fragment without insert DNA spanning the 5 ′ to 3 ′ junction from the native B73 / HiII genome. Sequence analysis revealed that there was a 2 base pair deletion from the native B73 genome at the transgene insertion locus. Analysis of the DNA sequence from the cloned native B73 genomic fragment identified that one ORF (> = 200 codons) was downstream of the 3 ′ integration junction region. Furthermore, there are no other ORFs across the original locus that incorporated the AAD-1 corn event DAS-40278-9. A BLAST search also confirmed that both 5 'and 3' end border sequences from event DAS40278-9 are next to each other on the same maize BAC clone.

In view of the uniqueness of the 5 ′ integration junction of AAD-1 corn event DAS-40278-9, two pairs of specific PCR primers, 1F5EndT1F / 1F5En
dT1R and Corn278-F / Corn278-R were designed to amplify the junction from this insert to the plant genome. As expected, the desired DNA fragment is AAD-1.
It occurred only with the genomic DNA of corn event DAS-40278-9, but not with any other transgenic or non-transgenic corn line. Therefore, these two primer pairs are AAD-1 corn event D
AS-40278-9 can be used as an event specific identifier.

Genomic characterization with DAS-40278-9 flanking SSR markers To characterize and show the genomic insertion site, a marker sequence in the vicinity of the insert was determined. A panel of polymorphic SSR markers was used to identify and map the location of the transgene.
Event pDAS 1740-278 is located on chromosome 2 at approximately 20 cM between SMC markers UMC1265 and MMC0111 at approximately 20 cM on the 2008DAS maize linkage map. Table 6 summarizes primer information for these two markers found to be in the vicinity of the transgene pDAS 1740-278.

(Example 4.1)
gDNA Isolation gDNA was extracted from leaf punches with the DNeasy 96 plant test kit (Qiagen,
(Valencia, California). The protocol was modified and adapted for automation. The isolated gDNA was purified from a molecular probe.
s, Inc. Quantified using PicoGreen® dye from (Eugene, OR). The concentration of gDNA was diluted with sterile deionized water to 5 ng / μl for all samples.

(Example 4.2)
Screening gDNA using markers The genotype of diluted gDNA was determined using a subset of simple sequence repeat (SSR) markers. The SSR marker was applied to Applied Biosystems (Foster
Citi, California) was synthesized using forward primers labeled with 6-FAM, HEX / VIC or NED (blue, green and yellow, respectively) fluorescent tags. Markers were divided into groups or panels based on their fluorescent tag and amplicon size to facilitate post-PCR multiplexing and analysis.

PCR was performed in a 384 well assay plate with 5 ng genomic DNA, 1.25
X PCR buffer (Qiagen, Valencia, California), 0.
20 μM each forward and reverse primer, 1.25 mM MgCl ₂ , 0.0
It was performed in each reaction containing 15 mM of each dNTP and 0.3 units of HotStart Taq DNA polymerase (Qiagen, Valencia, California). Amplification was performed in a GeneAmp PCR system 9700 using a 384-duplex head module (Applied Biosystems, Foster City, Cal.
ifonia). The amplification program was as follows: (1) 95
Initial activation of Taq for 12 minutes at ° C. (2) 30 seconds at 94 ° C., (3) 3 hours at 55 ° C.
0 seconds, (4) 30 seconds at 72 ° C, (5) repeat steps 2-4 for 40 cycles, and (6
) Final extension for 30 minutes at 72 ° C. The PCR product for each SSR marker panel is
2 μl of each PCR product from the same plant was multiplexed together by adding to a total volume of 60 μl of sterile deionized water. Of the multiplexed PCR product, 0.5 μl was added 1: 100
Ratios of GeneScan 500 base pair LIZ size standards and ABI HiDi formamide (Applied Biosystems, Foster City, Calif.
stamped into a 384 well loading plate containing 5 μl of loading buffer. Samples were then ABI Prism 37
30xl DNA analyzer (Applied Biosystems, Foster
(City, California) was loaded for capillary electrophoresis with a total run time of 36 minutes using manufacturer's recommendations. Marker data is from ABI Prism 373
Collected by 0xl automated sequencer data collection software version 4.0, Ge
Collected for allelic characterization and fragment size labeling with neMapper 4.0 software (Applied Biosystems).

(Example 4.3)
SSR marker results Primer data for flanking markers identified in the vicinity of the transgene,
Listed in Table 6. Two closely related markers, UMC1265 and MMC01
11 is located approximately 20 cM away from the transgene insert on chromosome 2.

Characterization of aad-1 protein at event DAS-40278-9 Recombinant aad- from transgenic maize event DAS-40278-9
The biochemical properties of one protein were characterized. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE, stained with Coomassie blue, and glycoprotein detection method), Western blot, immunodiagnostic test strip assay, matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI- TOF MS) and tandem MS
Protein sequencing analysis was used to characterize the biochemical properties of the protein.

(Example 5.1)
Immunodiagnostic strip assay The presence of aad-1 protein in leaf tissue of DAS-40278-9
Confirmed using commercially prepared immunodiagnostic test strips from ican Biostica. Strips were able to distinguish between transgenic and non-transgenic plants by testing crude leaf extracts (data not shown). The non-transgenic extract (XHH13) did not contain a detectable amount of immunoreactive protein. This result was also confirmed by Western blot analysis.

To test the expression of aad-1 protein, an immunodiagnostic strip analysis was performed. 4
Punching one leaf from each plant of XHH13 (control plant) and event DAS-40278-9 by sandwiching tissue between snap cap caps of individually labeled 1.5 mL microfuge tubes It was collected. When receiving in the laboratory, 0.5m
L aad-1 extraction buffer (American Bioostica, Sweden)
sboro, NJ) was added to each tube, and the tissue was homogenized by using a disposable pestle and then shaking the sample for about 10 seconds. After homogenization, the test strip was placed in a tube and allowed to develop for about 5 minutes. The presence or absence of aad-1 protein in the plant extract was confirmed based on the appearance (or non-occurrence) of a test line on the immunodiagnostic strip. Once the expression of aad-1 protein was confirmed for the transgenic event, maize stem tissue was harvested, lyophilized, and stored at approximately −80 ° C. until use.

(Example 5.2)
Purification of aad-1 protein from corn Immunoadjusted maize-derived aad-1 protein (molecular weight: about 33 kDa) or an aqueous crude extract from corn stalk tissue was prepared. All leaf and stem tissues were harvested and transported to the laboratory as follows. The leaves were cut from the plants with scissors, placed in a fabric bag and stored at approximately -20 ° C for future use. Separately, the stems were cut just above the soil line, placed in a cloth bag and immediately frozen at approximately -80 ° C for about 6 hours. The stem was then placed in a lyophilizer for 5 days to remove moisture. Once the tissues were completely dry, they were ground into fine powders using dry ice and stored at approximately -80 ° C until needed.

The maize-derived aad-1 protein was lyophilized from lyophilized stem tissue in phosphate-based buffer (see Table 7 for buffer components). Weighed into a glass blender and extracted by adding 500 mL extraction buffer. Tissues were blended high for 60 seconds and soluble protein was collected by centrifuging samples at 30,000 xg for 20 minutes. The pellet was re-extracted as described and the supernatants were combined and filtered through a 0.45μ filter.
The filtered supernatant was at approximately + 4 ° C; CNBr activated Sepharose 4B (GE Hea
lthcare, Piscataway, NJ)
gic Biosolution Inc. (MAb 473F1 85.1; Purified Protein A; Lot #: 609.03C-2-4; 6.5 mg / mL (total approximately 35.2 mg
)) Loaded onto an anti-adad-1 immunoaffinity column conjugated with a monoclonal antibody prepared by (Windham, ME). Unbound protein was recovered and the column was washed thoroughly with pre-cooled 20 mM ammonium bicarbonate buffer pH 8.0. Bound protein was 3.5M NaSCN (Sigma, St. Louis, MO), 50m
Eluted with M Tris (Sigma, St. Louis, MO) pH 8.0 buffer. Seven 5 mL fractions were collected and fraction number 2 → 7 was 10 m overnight at approximately + 4 ° C.
Dialyzed against M Tris, pH 8.0 buffer. Fractions were examined by SDS-PAGE and Western blot and the remaining samples were stored at approximately + 4 ° C. until used for further analysis.

Proteins bound to the immunoaffinity column were examined by SDS PAGE and the results showed that the eluted fraction contained the aad-1 protein with an approximate molecular weight of 33 kDa. In addition, a Western blot was performed, which was positive for aad-1 protein. The maize-derived aad-1 protein was isolated from approximately 30 g of lyophilized stem material.

(Example 5.3)
SDS-PAGE and Western Blot Lyophilized tissue from stems of events DAS-40278-9 and XHH13 (approximately 100
mg) into a 2 mL microfuge tube and about 1 mL PBST (Sigma) containing 10% plant protease inhibitor cocktail (Sigma, St. Louis, MO).
a, St. (Louis, MO). Extraction was facilitated by adding 4 small ball bearings and Geno grinding the sample for 1 minute. After grinding, the sample is 20,000
Centrifugation at 00 × g for 5 minutes, and supernatant was added 4 × 5 × Laemmli sample buffer (2% SDS, 50 mM Tris pH 6.8, 0.2 mg / mL bromophenol blue, 10% freshly added 2 -50% (w / w) glycerol containing mercaptoethanol) and heated at about 100 ° C for 5 minutes. After a short centrifugation, 45 μL of the supernatant was transferred to a BioRad Crite attached to a Criterion cell gel module.
It was loaded directly onto a rion SDS-PAGE gel (Bio-Rad, Hercules, CA). A positive reference standard of microorganism-derived aad-1 was PBST pH at 1 mg / mL.
Resuspended in 7.4 and further diluted with PBST. Samples were then transferred to Bio-Rad La
Mixed with emmli buffer and 5% 2-mercaptoethanol and processed as described previously. Electrophoresis was performed using Tris / Glycine / SDS buffer (Bio-Ra
d, Hercules, CA) at a voltage of 150-200 V until the front dye reached the end of the gel. After separation, the gel is cut in half and one half is
Stained using ce GelCode Blue protein stain, the other half on a nitrocellulose membrane (Bio-Rad, Hercules, Calif.) and a Mini transblot electrotransfer cell (Bio-Rad, Hercules, Calif.) 60
Electroblot using a constant voltage of 100 volts for minutes. The transfer buffer contained 20% methanol and Tris / glycine buffer from Bio-Rad. For immunodetection, the membrane was washed with aad-1 specific polyclonal rabbit antibody (Strategic Biosolution Inc., Newark,
DE, Protein A purified rabbit polyclonal antibody lot #: DAS F1 197-1
(1, 1.6 mg / mL). A conjugate of goat anti-rabbit IgG (H + L) and alkaline phosphatase (Pierce Chemical, Rockford, IL) was used as the secondary antibody. SigmaFast BCIP / NBT substrate was used to develop and visualize immunoreactive protein bands. Wash the membrane thoroughly with water to stop the reaction and record the results on a digital scanner (Hewlett Packard
(cARD, Palo Alto, CA).

In aad-1 produced by P. fluorescens, the main protein band visualized in a Coomassie-stained SDS-PAGE gel is
It was approximately 33 kDa. As expected, the corresponding maize-derived aad-1 protein (event DAS-40278-9) was the same size as the protein expressed by the microorganism. As expected, the purified fraction of plants contained minor amounts of non-immunoreactive impurities in addition to the aad-1 protein. The co-purified protein is
It was thought to be retained in the column due to weak interaction with the column matrix or to leach monoclonal antibodies from the column under severe elution conditions. Other researchers have also reported nonspecific adsorption of peptides and amino acids on cyanogen bromide activated Sepharose 4B immunosorbent (Kennedy and Barnes, 1983; Holroyde et al., 1976; Podlaski and Stern,
2008).

Pseudomonas-derived aad-1 protein showed a positive signal of the expected size by polyclonal antibody western blot analysis. This is DA
It was also observed in S-40278-9 transgenic maize stem extract. aad-
In 1 Western blot analysis, no immunoreactive protein was observed in the control XHH13 extract and no alternative size protein (aggregate or degradation product) was observed in the transgenic sample.

(Example 5.4)
Detection of post-translational glycosylation Maize-derived aad-1 protein (fraction # 3) purified by immunoaffinity chromatography was mixed 4 × with 5 × Laemmli buffer. Microbe-derived aad-1
, Soybean trypsin inhibitor, bovine serum albumin and horseradish peroxidase are diluted with Milli-Q water to an approximate concentration of plant-derived aad-1, Bio-R
Mixed with ad Laemmli buffer. The protein was then heated at about 95 ° C. for 5 minutes and centrifuged at 20000 × g for 2 minutes to obtain a clarified supernatant. The obtained supernatant was directly applied to a Bio-Rad Criterion gel and electrophoresed at about 170 V at 170 V with XT MES electrophoresis buffer (Bio-Rad, Hercules, CA).
Electrophoresis was performed essentially as described above except that it was run for 0 minutes. After electrophoresis
The gel was cut in half and one half was stained with GelCode Blue dye for total protein according to the manufacturer's protocol. After staining is complete, the gel is
A permanent dynamics densitometer was scanned to obtain a permanent visual record of the gel. The other half of the gel is a GelCode glycoprotein staining kit (Pierce Chemi).
cal, Rockford, IL) according to the manufacturer's protocol,
Glycoprotein was visualized. Glycoproteins (detection limits as low as 0.625 ng per band) were visualized as magenta bands on a light pink background. After glycoprotein staining was complete, the gel was scanned with a Hewlett Packard digital scanner to obtain a permanent visual record of the gel. After obtaining an image of the glycosylation stain, the gel is
Staining with elCode Blue was confirmed for the presence of non-glycosylated protein. The results showed that both maize-derived and microbial-derived aad-1 proteins had no detectable covalently bound carbohydrate. This result was also confirmed by peptide mass fingerprinting.

(Example 5.5)
Mass spectrometry peptide mass fingerprinting and sequencing of aad-1 from maize and Pseudomonas Mass spectrometry analysis of Padudomonas and maize derived aad-1 was performed. The aad-1 protein (event DAS-40278-9) derived from the transgenic corn stalk is subjected to solution digestion with trypsin, followed by MALDI.
-Subject to TOF MS and ESI-LC / MS. The mass of the detected peptide was compared to that estimated based on possible protease cleavage sites in the sequence of the maize-derived aad-1 protein. Theoretical cutting is in silico, Proteomet
Protein Analysis Worksheet (PA) from ricks LLC
WS) Freeware. Once denatured, the aad-1 protein is easily digested by proteases, resulting in multiple peptide peaks.

Aad-1 protein derived from transgenic maize (event DAS-40278-
In the tryptic digestion of 9), the detected peptide fragment is 1 at the C-terminus of the protein.
And F ²⁴⁸ to R ²⁵³ is a One small tryptic fragments, lacking only a single short (2 amino acids) peptide fragments, and covered nearly complete protein sequence. This analysis confirmed that the maize-derived protein amino acid sequence matched that of the microorganism-derived aad-1 protein. The results of these analyzes indicate that the amino acid sequence of the maize-derived aad-1 protein was equivalent to the P. fluorescens expressed protein.

(Example 5.5.1)
Tryptic peptide fragment sequencing In addition to peptide mass fingerprinting, maize-derived aad-1 protein (
N- and C-terminal amino acid residues of immunoaffinity purified from maize event DAS-40278-9) were sequenced and compared to the sequence of the microbial protein. Protein sequences were obtained for the first 11 residues of microbial and maize derived proteins by tandem mass spectrometry (Table 8). The amino acid sequences for both proteins are A'HAALSPL
SQR ″ (SEQ ID NO: 30), indicating that the N-terminal methionine has been removed by aminopeptidase (Table 8). The N-terminal aad-1 protein sequence is M′AHA.
ALSPLSQR ^'2 (SEQ ID NO: 31) was predicted. These results suggest that the N-terminal methionine is cleaved by methionine aminopeptidase (MAP) during or after translation in maize and P. fluorescens. M
In AP, the second residue of the protein is Gly, Ala, Ser, Cys, Thr, Pro
And in small cases like Val, methionyl residues are cleaved rapidly (Walsh, 2006).
In addition to the methionine being removed, a small portion of the N-terminal peptide of the aad-1 protein was shown to be acetylated after the N-terminal methionine was cleaved (Table 8).
). This result is often encountered with eukaryotic (plant) expressed proteins. This is because approximately 80-90% of the N-terminal residues are modified (Polevoda and Sherman, 2003).
. Also, proteins with serine and alanine at the N-terminus have been shown to be most frequently acetylated (Polevoda and Sherman, 2002). Two simultaneous translation processes,
Cleavage of the N-terminal methionine residue and N-terminal acetylation are by far the most common modification and occur in the majority of eukaryotic proteins (about 85%) (Polevoda and Sherman, 2002
). However, examples that demonstrate the biological importance associated with N-terminal acetylation are rare (Pole
voda and Sherman, 2000).

In addition to N-acetylation, slight N-terminal truncations also appeared during purification of the maize-derived aad-1 protein (Table 8). These “ragged ends” resulted in the loss of amino acids A ₂ , H ³ and A ⁴ (in various forms and amounts) from the maize-derived protein. This cleavage is believed to have occurred during the purification of the aad-1 protein. This is because the Western blot probe of the crude leaf extract contained a single distinct band at the same MW as the microbial aad-1 protein. The extraction buffer for the Western blotted sample contains excess protease inhibitor cocktail, which contains serine,
Contains a mixture of cysteine, aspartic acid and metalloproteases and protease inhibitors with broad specificity for the inhibition of aminopeptidases.

The C-terminal sequences of maize-derived and microbial-derived aad-1 proteins were determined as described above and compared with the predicted amino acid sequences (Table 9). The results showed that the measured sequence was identical to the predicted sequence, both the maize and microbial aad-1 proteins were identical and unchanged at the C-terminus.

Field expression, nutritional composition analysis and agroeconomic characteristics of a hybrid maize line containing event DAS-40278-9 The purpose of this study was to determine the level of AAD-1 protein found in corn tissue It was. In addition, a compositional analysis was performed on corn forage and grain to examine equivalence between the isogenic non-transformed corn line and the transgenic corn line DAS-40278-9 (no spray, 2, 4 Spray -D,
Spray quizalofop and spray 2,4-D and quizalofop) The agroeconomic characteristics of isogenic non-transformed maize lines were also compared to DAS-40278-9 maize. Field development, composition and agro-economic trials were conducted at six test sites within major corn-producing regions in the United States and Canada. These sites represent areas of diverse agricultural economic practices and environmental conditions. Trials are Iowa, Illinois (2 places), Indian
a, Nebraska and Ontario, Canada.

All field average values for control, no spray AAD-1, AAD-1 + quizalofop, AAD-1 +2, 4-D and AAD-1 + entry samples are within the literature for corn. there were. Although a limited number of significant differences were observed between corn and controls with no spray AAD-1, AAD-1 + quizalofop, AAD-1 + 2,4-D or both AAD-1 +, the differences were biologically I didn't think it would make sense.
Because they are small, the results were within the range found in commercial corn. A plot of the compositional results shows any biologically meaningful treatment of the non-sprayed AAD-1, AAD-1 + quizalofop, AAD-1 + 2,4-D or both AAD-1 + maize and control maize lines. It also does not show the compositional differences associated with.
As a result, no spray AAD-1, AAD-1 + quizalofop, AAD-1 +2, 4-D
And AAD-1 + corn composition results indicate that AAD-1 (event DA
S40278-9) It is confirmed that the corn is equivalent to the conventional corn line.

(Example 6.1)
Maize lines tested Hybrid seeds containing DAS-40278-9 events and control plants that are conventional hybrid seeds of the same genetic background as the test substance lines but do not contain DAS-40278-9 events are listed in Table 10.

The above corn plants were grown in locations within the major corn growth regions of the United States and Canada. 6 field trial facilities, Richland, IA; Carlyle, IL;
Wyoming, IL; Rockville, IN; York, NE; and Branch
hton, Ontario, Canada (IA, IL1, IL2, IN, NE and O
N) represents a region of diverse agricultural economic practices and environmental conditions for corn.

Test and control corn seeds were planted at approximately 24 seeds per row, with a seeding rate of approximately 10 inches (25 cm) between the seeds in each row. At each site, four replicate plots for each treatment were established, each plot consisting of 2-25 ft columns. The plots were arranged by the complete randomized block method (RCB) and randomized independently at each site. Each corn plot was bounded by two rows of non-transgenic maize hybrids of similar maturity. The entire trial site was surrounded by a minimum of 12 rows (or 30 ft) of non-transgenic maize hybrids of similar relative maturity.

Practices that manage suitable insects, weeds and disease were used to produce crops that were agriculturally acceptable. Monthly maximum and minimum temperatures, precipitation and irrigation were average for the site. These ranges are typically encountered in corn production.

(Example 6.2)
Herbicide application Herbicide treatment was performed at a spray volume of approximately 20 gallons per acre (187 L / ha). These spreads were designed to repeat the maximum label rate of commercial practice. Table 1
1 lists the herbicides used.

2,4-D (Weedar64) 3 times over the top (broadcast over-t)
he-top applications) used in test participation items 4 and 5 (seasonal total 3 lb ae
/ A). Individual sprays were pre-emergence and were approximately V4 and V8-V8.5 stages. Individual target application rates are 1.0 lb ae / A for Weedar 64, or 1120
g ae / ha. The actual spreading rate was in the range of 1096-1231 g ae / A.

Quizalofop (Assure II) was used in Study Participants 3 and 5 as a single over-the-top spray. The spraying time was approximately the V6 growth stage. The target application rate is A
It was 0.0825 lb ai / A, or 92 g ai / ha for sure II. The actual application rate was in the range of 90.8 to 103 g ai / ha.

(Example 6.3)
Agroeconomic data collection and results Agroeconomic features were recorded for all study participants in blocks 2, 3 and 4 at each location. Table 12 lists the following characteristics measured:

Analysis of agro-economic data collected from the control, no aad-1 spray, aad-1 + 2,4-D, aad-1 + quizalofop and both aad-1 + entries was performed. About cross-site analysis (across location summar)
y analysis) early population (V1 and V4), vitality, final population, crop damage, silking time, pollen fall time, lodging, root lodging, disease occurrence, insect damage, maturity days, No statistically significant differences were observed in plant height and pollen viability (shape and color) values (Table 13). Regarding greenness retention and ear height,
Significant paired t-tests were observed between controls and aad-1 + quizalofop participants, but with significant overall treatment effect or False Discovery Rates (FDR) adjusted p-value None (Table 13).

(Example 6.4)
Sample Collection Samples for expression and composition analysis were collected as listed in Table 14.

(Example 6.5)
Determination of aad-1 protein in corn samples Corn samples were analyzed for the amount of aad-1 protein. Soluble and extractable aad-1 protein was obtained from Beacon Analytical System, I
nc. (Enzyme-linked immunosorbent assay (ELISA) purchased from (Portland, ME)
) Quantify using a kit.

Samples of corn tissue are isolated from test plants, coarsely ground, lyophilized and Geno /
Prepared for expression analysis by pulverization (if necessary) using a pulverizer (Certreprep, Metuchen, New Jersey). For pollen, no additional preparation was necessary. The aad-1 protein was extracted from corn tissue using phosphate buffered saline (PBST) containing detergent Tween-20 containing 0.5% bovine serum albumin (BSA). For pollen, proteins were extracted using 0.5% PBST / BSA buffer containing 1 mg / mL sodium ascorbate and 2% protease inhibitor cocktail. Plant tissue and pollen extract were centrifuged. Collect the aqueous supernatant and dilute with appropriate buffer if necessary to obtain aa in sandwich format.
Analysis was performed using d-1 ELISA kit. The kit used the following steps. The amount of the diluted sample and the biotinylated anti-aad-1 monoclonal antibody are mixed with the immobilized anti-aad-1
Incubate in wells of microtiter plates coated with monoclonal antibodies. These antibodies bind to the aad-1 protein in the well to form a “sandwich” with the aad-1 protein bound between the soluble and immobilized antibodies. Unbound sample and conjugate are then removed from the plate by washing with PBST. Excess streptavidin-enzyme (alkaline phosphatase) conjugate
Add to wells for incubation. At the end of the incubation period, unbound reagent was removed from the plate by washing. A colored product was obtained by subsequent addition of the enzyme substrate. Since aad-1 is bound in the antibody sandwich, the level of color development is related to the concentration of aad-1 in the sample (ie, lower residual concentration results in lower color development). Absorbance at 405 nm was measured using Molecular Devices V-
Measured using a max or Spectra Max 190 plate reader. A calibration curve is generated, and the concentration of aad-1 in the unknown sample is adjusted to Soft-
Calculated from the polynomial regression equation using MAX Pro ™ software. Samples were analyzed in double wells with reported double well average concentrations.

A summary of aad-1 protein concentrations in various corn matrices (averaged between sites) is shown in Table 15. The aad-1 average protein concentration ranged from 2.87 ng / mg dry weight in the R1 stage roots to 127 ng / mg in the pollen. The expression results for the non-sprayed and sprayed plots were similar. The aad-1 protein was not detected in any of the control samples except for one control root sample from the Indiana field.

(Example 6.6)
Composition Analysis Corn forage and kernel samples were analyzed for nutrient content using various tests. Analyzes performed on forage included ash, total lipids, moisture, protein, carbohydrates, crude fibers, acidic detergent fibers, neutral detergent fibers, calcium and phosphorus. Analyzes performed on the grain are syngeneic (ash, total lipid, moisture, protein,
Carbohydrate, crude fiber, acidic detergent fiber), neutral detergent fiber (NDF), minerals, amino acids, fatty acids, vitamins, secondary metabolites and anti-nutrients. The results of nutrient analysis for corn forage and kernels were compared with values reported in the literature (Watson, 1982 (4); Watson, 1984 (5); ILSI Crop Composition Database, 2006 (6
); OECD Consensus Document on Compositional Considerations for maize, 2002 (7); and Codex Alimentarius Commission 2001 (8)).

(Example 6.6.1)
Forage composition, fiber and mineral analysis Control, unsprayed aad-1, aad-1 + quizalofop, protein, lipids in corn forage samples from both aad-1 + 2,4-D and aad-1 + entry , Ash, moisture, carbohydrates, ADF, NDF, calcium and phosphorus were analyzed. A summary of the results across all locations is shown in Table 16. For inter-site and site-specific analyses, the average values of all near-composition, fiber and minerals were within the literature. No statistical differences were observed for water, ADF, NDF, calcium and phosphorus in the inter-site analysis between controls and transgenic participants. Significant paired t-tests for protein and ash were observed for nebulized AAD-1 (protein), aad-1 + quizalofop (protein) and both aad-1 + (ash), but significant overall treatment There was no effect or FDR adjusted p-value. Both significant paired t-tests and adjusted p-values were observed for lipids compared to controls for aad-1 + quizalofop, but no significant overall treatment effect was observed. For carbohydrates, statistically significant overall treatment effects, paired t-tests and FDR adjusted p-values were observed between aad-1 + quizalofop and controls. For carbohydrates, a significant paired t-test was also observed for the no spray aad-1 participants, but no significant FDR adjusted p-value was observed. These differences are not biologically meaningful. This is because all inter-site results for these analytical items were within the literature reported for corn and the difference from the control was small (<23%).

(Example 6.6.2)
Analysis of grain proximate and fiber Results for syngeneic composition (protein, lipid, ash, moisture, cholesterol and carbohydrate) and fiber (ADF, NDF and total dietary fiber) in corn kernels across all locations Is summarized in Table 17. All results for the syngeneic composition and fiber are within the literature, and in the inter-site analysis, between the control and transgenic participants,
No significant differences were observed for lipid, ash, NDF and total dietary fiber. A significant overall treatment effect was observed for moisture, but without a significant paired t-test or FDR adjusted p-value. For ADF, a significant paired t-test was observed for both aad-1 +, but no significant overall treatment effect or FDR adjusted p-value was seen.
For both proteins and carbohydrates, significant paired tests, adjusted p-values and overall treatment effects were seen for both nebulized aad-1, aad-1 + quizalofop and aad-1 +. These differences are small (<12%) and all values were within the literature, so the differences are not considered biologically meaningful.

(Example 6.6.3)
Kernel mineral analysis Corn grains were analyzed for the minerals calcium, chromium, copper, iodine, iron, magnesium, manganese, molybdenum, phosphorus, potassium, selenium, sodium and zinc. A summary of the results across all locations is shown in Table 18. All results were within the literature reported. For inter-site analysis, no significant differences were observed for calcium, copper, iron and potassium. Average results for chromium, iodine, selenium and sodium were below the limit of quantification of the method. For magnesium and phosphorus, significant paired t-tests were observed for the nebulized aad-1 and aad-1 + quizalofop participants but with no significant overall treatment effect or FDR adjusted p-value. For manganese and molybdenum, a significant paired t-test is no spray aa
Although observed for d-1, no significant FDR adjusted p-values and overall treatment effects were seen. For both aad-1 + entries, a significant paired t-test was observed for zinc, but there was no significant FDR adjusted p-value or overall treatment effect. Furthermore, these differences from controls were small (<13%) and all values were within the literature when available.

(Example 6.6.4)
Amino acid analysis of kernels Corn samples were tested for amino acid content, control, no spray aad-1, aad-1
Table 19 shows a summary of the results from corn + quizalofop, both aad-1 + 2, 4-D and aad-1 +, and all field and individual field results. All amino acid levels were within the reported literature, and no significant differences in the field analysis were observed for arginine, lysine and tyrosine. For some amino acids, significant differences were observed in the field analysis. In these cases, the control amino acid content is lower than the aad-1 transgenic line, which
It is believed to be associated with an overall lower protein content in the control kernel compared to the d-1 line. For the nebulized aad-1 entry, significant paired t-tests and FDR adjusted p-values were found for all amino acids except arginine, glycine, lysine, tryptophan, and tyrosine with significant overall treatment effects. For the aad-1 + quizalofop entry, significant paired t-tests and FDR adjusted p-values with significant overall treatment effects were seen for all amino acids except arginine, cysteine, glycine, lysine, tryptophan and tyrosine . For aad-1 + 2,4-D participants, a significant paired t-test (with a significant FDR adjusted p-value) with a significant overall treatment effect was found for arginine, aspartic acid, glycine, histidine, lysine, tyrosine and It was found for all amino acids except valine. For both aad-1 + entries, a significant paired t-test and FDR adjusted p-value with a significant overall treatment effect was seen for all amino acids except arginine, glycine, lysine, serine, tryptophan and tyrosine It was. There were many differences observed for amino acids, but the differences were small (<15%), not observed across all sites, and all average values were within the reported literature.

(Example 6.6.5)
Fatty acid analysis of kernels Corn kernel samples were analyzed for fatty acids. A summary of the results across all locations is shown in Table 20. Controls analyzed for these fatty acids, no spray aad-1, a
All results for corn kernel samples of both ad-1 + quizalofop, aad-1 + 2,4-D and aad-1 + were within the published literature. Caprylic acid (8
: 0), capric acid (10: 0), lauric acid (12: 0), myristic acid (14: 0)
, Myristoleic acid (14: 1), pentadecanoic acid (15: 0), pentadecenoic acid (15
1), heptadecanoic acid (17: 0), heptadecenoic acid (17: 1), gamma linolenic acid (18: 3), eicosadienoic acid (20: 2), eicosatrienoic acid (20: 3) and arachidonic acid ( The results for 20: 4) were below the limit of quantification (LOQ) of the method.
In the in-situ analysis, no significant differences were observed for 16: 0 palmitic acid, 16: 1 palmitoleic acid, 18: 0 stearic acid, 18: 2 linoleic acid, 18: 3 linolenic acid and 20: 0 arachidic acid. Significant paired t-tests for 18: 1 oleic acid and 20: 1 eicoseiic acid were nebulized aad-1 (18: 1) and aad-1
+ 2,4-D (18: 1 and 20: 1) participants were observed, but with no significant overall treatment effect or FDR adjusted p-value. For 22: 0 behenic acid, a significant overall treatment effect and a significant paired t-test were found for aad-1 + 2,4-D and aad
Although seen for both -1+, there was no significant FDR adjusted p-value.

(Example 6.6.6)
Vitamin analysis of kernels Vitamin A, B1, B2, B5 in corn kernel samples from control, no spray aad-1, aad-1 + quizalofop, aad-1 + 2,4-D and both aad-1 + corn participants B6, B12, C, D, E, niacin and folic acid levels were determined. A summary of the results across all locations is shown in Table 21. Vitamin B12, D and E were not quantifiable by the analysis method used. All average results reported for vitamins were similar to reported literature values when available. Vitamins with no reported literature (vitamins B5 and C) were similar to the control values obtained (<22% difference from controls). For inter-site analysis, no statistical differences were observed except for vitamins B1, C and niacin. Significant paired t-tests for vitamin B1 were control and no spray aad-1, aad-1 + quizalofop and aad
-1+ was observed between both, but with no significant overall treatment effect or FDR adjusted p-value. For vitamin C, a significant overall treatment effect, with a significant paired t-test and FDR adjusted p-value, is aad-1 + quizalofop and aad-1 + 2,4-D
Was observed. Similarly for niacin, a significant overall processing effect, with a significant paired t-test and FDR adjusted p-value, is aad-1 + quizalofop and aa
Observed for both d-1 +. A significant paired t-test for aad-1 + 2,4-D was also found for niacin for the aad-1 + 2,4-D entry,
There was no significant overall treatment effect or FDR adjusted p-value. Differences were not observed across the field and values were within the literature (if available), so differences are not considered biologically meaningful.

(Example 6.6.7)
Analysis of kernel antinutrients and secondary metabolites Secondary in corn kernel samples from control, unsprayed aad-1, aad-1 + quizalofop, both aad-1 + 2,4-D and aad-1 + corn participants The levels of metabolites (coumaric acid, ferulic acid, furfural and inositol) and anti-nutrients (phytic acid, raffinose and trypsin inhibitors) were determined. A summary of results across all locations is shown in Tables 22 and 23. For inter-site analysis, all values were within the literature. No significant differences were observed in the in-situ analysis for inositol and trypsin inhibitor between aad-1 and control participants. The results for furfural and raffinose were below the limit of quantification of the method. Significant paired t-tests were used for coumaric acid (no spray aad-1, aad-1 + 2,4-D and aad-
1+ both) and ferulic acid (both aad-1 + quizalofop and aad-1 +) were observed. These differences were similar to controls (<10% difference) without significant overall treatment effects or FDR adjusted p-values. Significant overall treatment effects, paired t-tests and FDR adjusted p-values were found for phytic acid (no spray aad-1). All results were within the literature and were similar to the control (<11% difference), so these differences are not considered biologically meaningful.

Additional agro-economic trials Maize lines 4 compared to near-isoline maize lines 4
The agroeconomic characteristics of 0278 were evaluated across a variety of environments. The treatment included 4 genetically different hybrids tested in a total of 21 locations and their appropriate near-isogenic lineage control hybrids.

The four test hybrids were mid-late maturity hybrids in the 99-113 day relative maturity range. Experiment A tested the event DAS-40278-9 in a genetic background inbred CxBC3S1 transformation. This hybrid had a relative maturity of 109 days and was tested in 16 locations (Table 24). Experiment B tested a hybrid background inbred ExBC3S1 transformation, which is a 113 day relative maturity hybrid. This hybrid was tested at 14 locations, using a slightly different set of locations from Experiment A (Table 24).
). Experiments C and D tested hybrid background BC2S1 conversion x inbred line D and BC2S1 conversion x inbred line F, respectively. Both of these hybrids had a relative maturity of 99 days and were tested in the same 10 locations.

For each trial, the complete randomized block method was used with two iterations and two-column plots per location.
The row length was 20 feet and each row was sown with 34 seeds per row. Trials were managed using standard regional agroeconomic practices.

Data were collected and analyzed for eight agroeconomic characteristics; plant height, ear height,
Stem lodging, root lodging, final population, grain moisture, test weight and yield. The plant height and ear height parameters provide information about the appearance of the hybrid. The agro-economic characteristics of percent stem and root lodging determine the harvestability of the hybrid. Final population counts measure seed quality and seasonal growth conditions that affect yield. Percent grain moisture at harvest dictates the maturity of the hybrid, in yield (bushel / acre adjusted for humidity) and test weight (bushel corn pound adjusted to 15.5% humidity) ) Represents the fertility of the hybrid.

Analysis of variance was performed across the field using a linear model. Participating items and locations
Included in the model as a fixed effect. We searched for a mixed model that includes location and location by random participation, but the location by participation only explains a small part of the variance, and its variance component is often not significantly different from zero. It was. For stem and root lodging, logarithmic transformation was used to stabilize the variance, but the mean and range are reported on the original scale. Significant differences were obtained at the 95% confidence level. The significance of the overall treatment effect was estimated using the t test.

The results from these agroeconomic characterization trials can be found in Table 2. No statistically significant differences were found for any of the four 40278 hybrids in terms of ear height, stem lodging, root lodging, grain moisture, test weight and yield parameters compared to isogenic line controls. (At p <0.05). Final population counts and plant heights were statistically different in experiments A and B, respectively, but the other 4027 tested
Similar differences were not seen in comparison with the 8-hybrid. Some of the variability seen may be due to the low level of genetic variability remaining from the backcross of DAS-40278-9 events to elite inbred lines. The overall range of values for the measured parameters are all within the range of values obtained for traditional corn hybrids, and no conclusion can be drawn on increased weediness. In summary, agro-economic data shows that 40278 corn is biologically equivalent to conventional corn.

Use of Corn Event DAS-40278-9 Insertion Site for Targeted Integration The stable agro-economic performance of the corn event DAS-40278-9 transgene over several generations under field conditions Corn Event DAS-4027
It is suggested that these identified regions around the 8-9 insertion site provide a good genomic location for targeted integration of other transgenic genes of interest. Such targeted integration overcomes the problems of so-called “positional effects” and the risk of creating mutations in the genome when integrating the transgene into the host. Additional benefits of such targeted integration include, but are not limited to, the desired level of introduction without showing any abnormalities due to accidental insertion of the transgene at key loci in the host genome. This includes reducing the number of transformation events that must be screened and tested before obtaining transgenic plants exhibiting gene expression. In addition, such targeted integration allows cross-gene crossing, which makes breeding of elite plant lines carrying both genes more efficient.

Using the disclosed teachings, one of ordinary skill in the art can convert a subject polynucleic acid into a corn event DAS.
It can be targeted to the same insertion site on chromosome 2 as that at -40278-9 or close to the insertion site at corn event DAS-40278-9. One such method is disclosed in International Patent Application No. WO 2008/021207, which is hereby incorporated by reference in its entirety.

Briefly, genomic sequences up to 20 Kb in contact with 5 ′ of the insertion site and up to 20 Kb in contact with 3 ′ of the insertion site (parts of which are identified with reference to SEQ ID NO: 29) It is used in contact with one or a plurality of target genes to be inserted into the genomic position on the second chromosome by replacement. The gene or genes of interest can be placed exactly as in the corn event DAS-40278-9 insertion site, or anywhere in the 20 Kb region around the corn event DAS-40278-9 insertion site To provide a certain level of transgene expression without adversely affecting the plant. A DNA vector containing one or more genes of interest and flanking sequences can be produced by one of several methods known to those skilled in the art including, but not limited to, Agrobacterium-mediated transformation. Can be delivered to.
Inserting a donor DNA vector into the maize event DAS-40278-9 target site includes, but is not limited to, several methods including co-expression or upregulation of recombination enhancing genes or downregulation of endogenous recombination suppressor genes. It can be further enhanced by one of the methods. In addition, double-strand breaks in specific sequences within the genome can be used to increase the frequency of homologous recombination, thus inserting the corn event DAS-40278-9 insertion site and its flanking region into these It is known in the art that it can be enhanced by expression of natural or engineered sequence-specific endonucleases to cleave sequences. That is, using the teachings provided herein, any heterologous nucleic acid can be placed on corn chromosome 2 between the 5 ′ molecular marker discussed in Example 4 and the 3 ′ molecular marker discussed in Example 4. It can be inserted at an intervening target site, preferably within SEQ ID NO: 29, and / or that region discussed elsewhere herein.

Excision of the pat gene expression cassette from corn event DAS-40278-9 The removal of the selectable marker gene expression cassette is performed in corn event DAS-40278.
Advantageous for targeted insertion at the genomic locus of -9. Removing the pat selectable marker from corn event DAS-40278-9 allows the pat selectable marker to be reused to target and integrate polynucleic acid in chromosome 4 in subsequent generations of corn.

Using the disclosed teachings, one of ordinary skill in the art can convert a subject polynucleic acid into a corn event DAS.
It can be cut out from -40278-9. One such method is disclosed in US Provisional Patent Application 61 / 297,628, which is incorporated by reference herein in its entirety.

Briefly, a sequence-specific endonuclease such as a zinc finger nuclease that recognizes, binds and cleaves a specific DNA sequence located on the side of a gene expression cassette is designed. Zinc finger nuclease transforms the parent plant containing the transgenic zinc finger nuclease expression cassette into corn event DAS-40278.
Delivered to plant cells by crossing with a second parent plant containing -9. The resulting offspring are grown to maturity and the loss of the pat expression cassette is analyzed by painting leaves with a herbicide containing glufosinate. Progeny plants that are not resistant to herbicides are molecularly identified and self-propagated. The excision and removal of the pat expression cassette is confirmed molecularly in the offspring obtained from self-breeding. Using the teaching provided herein, any heterologous nucleic acid can be transferred from corn chromosome 2 to a target site, preferably between the 5 ′ and 3 ′ molecular markers discussed in Example 4, It can be excised at that shown in SEQ ID NO: 29, or at the indicated region.

Resistance to brittlesnap Brittle snap usually refers to the breakage of corn stalks by strong winds after the application of growth-regulating herbicides during the fast growing season. A mechanical “push” test using a bar that physically pushes corn to simulate the damage caused by strong winds was conducted at Event DAS-4027.
Hybrid corn containing 8-9 and control plants containing no event DAS-40278-9 were performed. The process was completed at four geographically different locations and repeated four times (with one exception that was repeated only three times). The plot consisted of 8 columns: 2 columns contain event DAS-40278-9, 2 columns contain no events, 2
4 rows of each of the two hybrids. Each row was 20 feet long. Corn plants are grown to V4 developmental stage and are commercially available herbicides (Weedar) containing 2,4-D
64, Nufarm Inc. , Burr Ridge, IL) 1120 g ae
/ Ha, 2240 g ae / ha and 4480 g ae / ha. A mechanical push test was performed after 7 days with the herbicide. The mechanical push test on Brittle Snap consisted of pulling down two rows of corn with a 4 foot stick to simulate wind damage. Bar height and speed of movement are set to produce low levels of stem destruction (less than 10%) using untreated plants and tested well to demonstrate differences between treatments Ensured that it was tough. The direction of Brittle Snap was used for leaning corn.

Two of the trial sites experienced strong winds and thunderstorms after 2-3 days with 2,4-D herbicides. For two consecutive days, a thunderstorm began in Huxley IA. 2 at 33 m s ^-1 at high speed
A wind speed of ˜17 m s ⁻¹ was reported in the field plot. The direction of the wind fluctuated.
A thunderstorm was reported for 1 day at Lanesboro MN. High speed winds were reported at the field plot site. In addition, both storms were accompanied by rain. The combination of rain and wind was attributed to the reported Brittle Snap damage.

An assessment of Brittle Snap damage due to mechanical push tests (and bad weather) was made by visually assessing the percentage of damage. Prior to treatment with Brittle Snap mechanical bars, plant stand counts were performed for hybrid maize containing the event DAS-40278-9 and controls. The plot standing count was re-evaluated several days after treatment with the Brittle Snap bar. The percentage of leaning and the percentage of standing trees in the plot were determined (Table 26). The data from the trial is
Hybrid corn containing event DAS-40278-9 proved less prone to brittle snaps after using 2,4-D compared to null plants.

Kernel Protein Analysis From hybrid corn containing event DAS-40278-9, kernels were produced with increased total protein content compared to control plants containing no event. Two consecutive multi-point field trials were conducted, which included no spraying and herbicide treatment in three different herbicide combinations. In 7 of 8 statistical comparisons, DAS-4027
The 8-9 event produced grains with significantly higher total protein content (Table 27)
. This data is confirmed by analysis of individual amino acids.

Additional agroeconomic traits Event DAS-402 compared to near-isoline maize
The agroeconomic characteristics of hybrid corn containing 78-9 were recovered from multiple field trials across a variety of geographical environments for the growing season. Data was collected and analyzed for agro-economic features as described in Example 7. Results for a hybrid maize line containing event DAS-40278-9 compared to null plants,
Table 28 lists. Furthermore, at the V3 stage of development, the herbicide quizalofop (280 g ae /
agroeconomic characteristics for hybrid maize lines and null plants containing event DAS-40278-9 sprayed with ha) and 2,4-D (2,240 g ae / ha) sprayed at stage V6 of development Are listed in Table 29.

SEQ ID NOs: 1-28 are primers described herein.
SEQ ID NO: 29 shows the insert and flanking sequence for the subject event DAS-40278-9.
SEQ ID NOs: 30-33 are primers for the flanking markers described in Example 4.

Claims (6)

A method for detecting AAD-1 corn event DAS-40278-9 in a sample comprising corn DNA comprising:
The sample,
a. A first primer that binds to a flanking sequence selected from the group consisting of residues 1 to 1873 of SEQ ID NO: 29, residues 6690 to 8557 of SEQ ID NO: 29, and complementary strands thereof;
b. A second primer that binds to an insertion sequence comprising residues 1874 to 6689 of SEQ ID NO: 29 or a complementary strand thereof
Contacting with
Subjecting the sample to a polymerase chain reaction and assaying for an amplicon occurring between the primers, wherein the detection of the amplicon is characteristic for the AAD-1 corn event DAS-40278-9 . A method of detecting AAD-1 corn event DAS-40278-9 in a sample comprising corn DNA comprising contacting said sample with at least one polynucleotide;
The polynucleotide comprises at least 30 nucleotides and hybridizes under stringent conditions with a sequence selected from the group consisting of residues 1863 to 1875 of SEQ ID NO: 29, residues 6679 to 6700 of SEQ ID NO: 29, and complementary strands thereof. soybean, and said method further seen including the steps of subjecting the sample and said polynucleotide to stringent hybridization conditions; and assaying the sample for hybridization of the polynucleotide with the DNA, the high The method wherein the detection of hybridization is characteristic for the AAD-1 corn event DAS-40278-9 . A first primer selected from the group consisting of SEQ ID NOs: 5, 7, 12, 14-16, 19-22, 24, 26 and 28; and SEQ ID NOs: 1-4, 6, 8-11, 13, 17, 18 A DNA detection kit for detecting AAD-1 corn event DAS-40278-9 in a sample comprising corn DNA, comprising a second primer selected from the group consisting of 23, 25 and 27. A DNA detection kit for performing the method according to claim 2 , comprising a sequence selected from the group consisting of residues 1863 to 1875 of SEQ ID NO: 29, residues 6679 to 6700 of SEQ ID NO: 29, and complementary strands thereof A kit comprising a polynucleotide that hybridizes under stringent conditions. The DNA detection kit according to claim 4 , wherein the polynucleotide comprises a sequence selected from the group consisting of residues 1863 to 1875 of SEQ ID NO: 29, residues 6679 to 6700 of SEQ ID NO: 29, and complementary strands thereof. The first primer is selected from the group consisting of SEQ ID NOs: 5, 7, 12, 14-16, 19-22, 24, 26 and 28, and the second primer is SEQ ID NOs: 1-4, 6, 8-11. The method of claim 1 , wherein the method is selected from the group consisting of: 13, 17, 18, 23, 25, and 27.