BR102017008936B1 - NUCLEIC ACID VECTOR AND USE OF A NON-ZEA MAYS C.V. B73 PLANT OR SEED - Google Patents

Abstract

PLANT PROMOTER AND 3'UTR FOR TRANSGENE EXPRESSION. Description relates to compositions and methods for promoting transcription of a nucleotide sequence in a plant or plant cell, employing a Zea mays GRMZM2G144030 promoter. Some embodiments relate to a Zea mays GRMZM2G144030 promoter that functions in plants to promote transcription of operatively linked nucleotide sequences. Other embodiments relate to a Zea mays GRMZM2G144030 3'UTR that functions in plants to terminate transcription of operatively linked nucleotide sequences.

Description

[001] This application claims priority based on provisional application 62/330527 which was filed with the U.S. Patent and Trademark Office on May 2, 2016, the disclosure of which is incorporated herein in its entirety by reference.

INCORPORATION BY REFERENCE OF MATERIAL SENT ELECTRONICALLY

[002] Incorporated by reference in its entirety is a simultaneously attached computer-readable listing of nucleotide/amino acid sequences identified below: a 26.0 KB ACII (Text) file named "78826-US-PSP-20160126-Sequence-Listing-ST25.txt" created on March 4, 2016.

DESCRIPTION FIELD

[003] The present invention relates generally to compositions and methods for promoting transcription of a nucleotide sequence in a plant or plant cell. Some embodiments relate to a novel Zea mays GRMZM2G144030 promoter and other Zea mays GRMZM2G144030 regulatory elements that function in plants to promote and/or terminate transcription of an operably linked nucleotide sequence. Particular embodiments relate to methods including a promoter (e.g., for introducing a nucleic acid molecule into a cell) and cells, cell cultures, tissues, organisms, and parts of organisms comprising a promoter, as well as products produced therefrom. Other embodiments relate to methods including a 3'UTR (e.g., for introducing a nucleic acid molecule into a cell) and cells, cell cultures, tissues, organisms, and parts of organisms comprising a promoter, as well as products produced therefrom.

BACKGROUND OF THE INVENTION

[004] Many plant species are capable of being transformed with transgenes to introduce agronomically desirable traits or characteristics. Plant species are developed and/or modified to possess desired traits. Generally, desirable traits include, for example, improving the quality of nutritional value, increasing yield, imparting resistance to pests or diseases, increasing tolerance to drought and stress, improving horticultural qualities (e.g. pigmentation and growth), imparting tolerance to herbicides, enabling the production of industrially useful compounds and/or materials from the plant, and/or enabling the production of pharmaceuticals.

[005] Transgenic plant species comprising multiple transgenes pyramided at a single genomic locus are produced by plant transformation technologies. Plant transformation technologies result in the introduction of a transgene into a plant cell, recovery of a fertile transgenic plant containing the stably integrated copy of the transgene into the plant genome, and subsequent expression of transgenes via transcription and translation of the plant genome results in transgenic plants possessing desirable traits and phenotypes. However, mechanisms that allow the production of transgenic plant species expressing high levels of multiple transgenes genetically engineered as a pyramid of traits are desirable.

[006] Similarly, mechanisms that allow expression of a transgene within particular tissues or organs of a plant are desirable. For example, increased resistance of a plant to infection by soil-borne pathogens may be achieved by transforming the plant genome with a pathogen resistance gene such that the pathogen resistance protein is robustly expressed within the roots of the plant. Alternatively, it may be desirable to express a transgene in plant tissues that are in a particular phase of growth or development, e.g., cell division or elongation. Furthermore, it may be desirable to express a transgene in the leaf or stem tissues of a plant to provide tolerance against herbicides or resistance against above-ground insects or pests.

[007] Therefore, there is a need for new gene regulatory elements that can direct desired levels of transgene expression in specific plant tissues.

BRIEF SUMMARY

[008] In embodiments of the subject matter, the subject matter relates to a nucleic acid vector comprising a promoter operably linked to a polylinker sequence, a non-GRMZM2G144030 gene; or a combination of the polylinker sequence and non-GRMZM2G144030 gene, wherein said promoter comprises a polynucleotide sequence having at least 90% sequence identity to SEQ ID NO:1. In aspects of this embodiment, the promoter is 2,082 bp in length. Additional embodiments include a promoter consisting of a polynucleotide sequence having at least 90% sequence identity to SEQ ID NO:1. In other aspects, the promoter is operably linked to a selectable marker. In additional aspects, the promoter is operably linked to a transgene. Exemplary transgenes include: a selectable marker or gene product that confers insecticide resistance, herbicide tolerance, nitrogen use efficiency, small RNA expression, site-specific nuclease, water use efficiency, nutritional quality, or DNA binding protein. In further aspects, the nucleic acid vector comprises a 3' untranslated polynucleotide sequence having at least 90% sequence identity to SEQ ID NO:5, wherein the 3' untranslated sequence is operatively linked to said polylinker or said transgene. In other aspects, the nucleic acid vector comprises a 5' untranslated polynucleotide sequence, wherein the 5' untranslated sequence is operatively linked to said polylinker or said transgene. In further aspects, the nucleic acid vector comprises an intron sequence. The promoter of the disclosure further induces transgene expression in leaf tissues.

[009] In additional embodiments of the subject matter, the description relates to a non-Zea mays c.v. B73 plant comprising a polynucleotide sequence having at least 90% sequence identity to SEQ ID NO:1 operably linked to a transgene. In one aspect of this embodiment, the plant is selected from the group consisting of wheat, rice, sorghum, oats, rye, bananas, sugarcane, cotton, Arabidopsis, tobacco, sunflower, and canola. In another aspect, the plant is a corn plant. In additional aspects, the transgene is inserted into the genome of the plant. In other embodiments, the polynucleotide sequence having at least 90% sequence identity to SEQ ID NO:1 is a promoter. In another aspect, the plant comprises a 3' untranslated sequence comprising SEQ ID NO:5, wherein the 3' untranslated sequence is operably linked to said transgene. In additional aspects, the promoter induces transgene expression in leaf tissues. In an additional aspect of these embodiments, the promoter is 2,082 bp in length.

[0010] In other embodiments of the subject matter, a method for producing a transgenic plant cell is provided. The method includes the steps of transforming a plant cell with a gene expression cassette comprising a Zea mays GRMZM2G144030 promoter operatively linked to at least one polynucleotide sequence of interest; isolating the transformed plant cell comprising the gene expression cassette; and producing a transgenic plant cell comprising the Zea mays GRMZM2G144030 promoter operatively linked to at least one polynucleotide sequence of interest. In one aspect of this embodiment, the transformation of the plant cell is carried out with a plant transformation method. The plant transformation method is selected from any of the following transformation methods: an Agrobacterium-mediated transformation method, a biolistic transformation method, a silicon carbide transformation method, a protoplast transformation method, and a liposome transformation method. In further aspects, the polynucleotide sequence of interest is preferentially expressed in leaf tissues. In further aspects, the polynucleotide sequence of interest is stably integrated into the genome of the transgenic plant cell. In further aspects, the method further includes the steps of regenerating the transgenic plant cell into a transgenic plant and obtaining the transgenic plant, wherein the transgenic plant comprises the gene expression casse comprising the Zea mays GRMZM2G144030 promoter of claim 1 operatively linked to at least one polynucleotide sequence of interest. In another aspect of the embodiment, the transgenic plant cell is a monocotyledonous transgenic plant cell or a dicotyledonous transgenic plant cell. Accordingly, the dicotyledonous transgenic plant cell can be an Arabidopsis plant cell, a tobacco plant cell, a soybean plant cell, a canola plant cell, or a cotton plant cell. Likewise, the transgenic monocot plant cell can be a corn plant cell, a rice plant cell, or a wheat plant cell. In other aspects, the Zea mays GRMZM2G144030 promoter comprising the polynucleotide of SEQ ID NO:1. In one aspect, embodiments include a first polynucleotide sequence of interest operatively linked to the 3' end of SEQ ID NO:1.

[0011] The subject matter further relates to a method for expressing a polynucleotide sequence of interest in a plant cell, the method comprising introducing into the plant cell a polynucleotide sequence of interest operatively linked to a Zea mays GRMZM2G144030 promoter. In one aspect of this embodiment, the polynucleotide sequence of interest operatively linked to the Zea mays GRMZM2G144030 promoter is introduced into the plant cell by a plant transformation method. In a further aspect, the plant transformation method is selected from an Agrobacterium-mediated transformation method, a biolistic transformation method, a silicon carbide transformation method, a protoplast transformation method, and a liposome transformation method. In further aspects, the polynucleotide sequence of interest is constitutively expressed throughout the plant cell. In still other aspects, the polynucleotide sequence of interest is stably integrated into the genome of the plant cell. In one embodiment, the transgenic plant cell is a monocotyledonous plant cell or a dicotyledonous plant cell. Accordingly, the dicotyledonous plant cell includes an Arabidopsis plant cell, a tobacco plant cell, a soybean plant cell, a canola plant cell, or a cotton plant cell. Likewise, the monocotyledonous plant cell includes a corn plant cell, a rice plant cell, or a wheat plant cell.

[0012] The subject matter further relates to a transgenic plant cell comprising a Zea mays GRMZM2G144030 promoter. In one aspect of this embodiment, the transgenic plant cell comprises a transgenic event. In other aspects, the transgenic event comprises an agronomic trait. Exemplary transgenic traits include an insecticide resistance trait, herbicide tolerance trait, nitrogen use efficiency trait, water use efficiency trait, nutritional quality trait, DNA binding trait, selectable marker trait, small RNA trait, or a combination thereof. In one embodiment, the herbicide tolerant trait comprises an aad-1 coding sequence. In other aspects, the transgenic plant cell produces a commodity product. For example, the commodity product can be protein concentrate, protein isolate, grain, powder, flour, oil, or fiber. In further aspects, the transgenic plant cell is selected from the group consisting of a dicotyledonous plant cell or a monocotyledonous plant cell. For example, the monocotyledonous plant cell can be a corn plant cell. In other aspects, the Zea mays GRMZM2G144030 promoter comprises a polynucleotide having at least 90% sequence identity to the polynucleotide of SEQ ID NO:1. In further aspects, the Zea mays GRMZM2G144030 promoter is 2,082 bp in length. In yet another aspect, the Zea mays GRMZM2G144030 promoter comprises a polynucleotide sequence having at least 90% sequence identity to SEQ ID NO:1. Further aspects include a first polynucleotide sequence of interest operatively linked to the 3' end of SEQ ID NO:1. In an additional aspect, the agronomic trait is preferentially expressed in leaf tissues.

[0013] The subject matter further relates to an isolated polynucleotide comprising a nucleic acid sequence having at least 90% sequence identity to the polynucleotide of SEQ ID NO:1. In one aspect of this embodiment, the isolated polynucleotide has preferential expression in leaf tissues. In other aspects, the isolated polynucleotide has expression activity within a plant cell. In additional aspects, the isolated polynucleotide comprises an open reading frame polynucleotide encoding a polypeptide; and a termination sequence. In a further aspect, the polynucleotide of SEQ ID NO:1 is 2,082 bp in length.

[0014] The foregoing and other features will become more apparent from the following detailed description of various embodiments which proceed with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

[0015] Figure 1: This figure is a schematic of pDAB113002 containing the Zea mays GRMZM2G144030 promoter of SEQ ID NO:1 and the Zea mays GRMZM2G144030 3'UTR of SEQ ID NO:5 in a gene expression cassette that directs expression of the cry3Ab1 transgene.

DETAILED DESCRIPTION I. Overview of the various modalities

[0016] The development of transgenic plant products is becoming increasingly complex. Commercially viable transgenic plants now require the pyramiding of multiple transgenes at a single locus. Promoters in plants used for basic research or biotechnological applications are generally unidirectional, targeting only one gene that has been fused to their 3' (downstream) end. 3'UTRs in plants used for basic research or biotechnological applications are generally unidirectional, terminating the expression of only one gene that has been fused to their 5' (downstream) end. Consequently, each transgene generally requires a promoter for expression and a 3'UTR for termination of expression, with multiple promoters and 3'UTRs being required to express multiple transgenes within a gene pyramid. With an increasing number of transgenes in gene pyramids, the same promoter and 3'UTR are routinely used to obtain similar levels of expression patterns of different transgenes. Similar levels of transgene expression are required for the production of a single polygenic trait. Unfortunately, multigene constructs driven by the same promoter and 3′UTR are known to cause gene silencing resulting in fewer effective transgene products in the field. Repeated promoter and 3′UTR elements can lead to homology-based gene silencing. Furthermore, repetitive sequences within a transgene can lead to intralocus homologous recombination of genes resulting in polynucleotide rearrangements. Transgene silencing and rearrangement are likely to have an undesirable effect on the performance of a transgenic plant engineered to express transgenes. Furthermore, excess transcription factor (TF) binding sites due to promoter repeats can cause depletion of endogenous TFs resulting in transcriptional inactivation. Given the need to introduce multiple genes into plants for metabolic genetic engineering and trait pyramiding, a variety of promoters and 3'UTRs are required to develop transgenic crops that induce multiple gene expression.

[0017] A particular problem in promoter identification is the need to identify tissue-specific promoters relating to specific cell types, developmental stages and/or functions in the plant that are not expressed in other plant tissues. Tissue-specific (e.g., tissue-preferential) promoters or organ-specific promoters induce gene expression in particular tissues such as the pulp, root, leaf or tapetum of the plant. Tissue- and developmental-stage-specific promoters can be identified initially by observing gene expression that is expressed in particular tissues or at particular time periods during plant development. Such tissue-specific promoters are necessary for certain applications in the transgenic plant industry and are desirable because they allow specific expression of heterologous genes in a tissue-selective manner and/or developmental stage of the heterologous gene differentially in various organs, tissues and/or times, but not in other tissues. For example, increased resistance of a plant to infection by soil-borne pathogens can be achieved by transforming the plant genome with a pathogen resistance gene so that the pathogen resistance protein is robustly expressed within the plant roots. Alternatively, it may be desirable to express a transgene in plant tissues that are in a particular growth or developmental phase, e.g., cell division or elongation. Another application is the convenience of using tissue-specific promoters to confine the expression of transgenes encoding an agronomic trait to specific tissue types such as developing parenchyma cells. As such, a particular problem in promoter identification is how to identify promoters and relate the promoter identifier to developmental properties of the cell for tissue-specific expression.

[0018] Another problem with respect to the identification of a promoter or 3'UTR is the requirement to clone all relevant transcriptional control elements that act in cis and activate in trans so that the cloned DNA fragment induces transcription in the specific expression pattern desired. Since the control elements are located distally from the translation initiation or stop site, the size of the polynucleotide that is selected to comprise the promoter is of importance in providing the expression level and expression patterns of the promoter polynucleotide sequence. Since the similarity elements for termination of a 3'UTR are located distally from the translation termination or stop site, the size of the polynucleotide that is selected to comprise the 3'UTR is of importance in providing termination of expression of a transgene encoded by a polynucleotide sequence. It is known that the promoter and 3'UTR portion include functional information and different genes have been shown to possess larger or smaller promoters than promoters of other genes in the genome. Elucidating the transcription start site of a promoter and predicting the functional gene elements in the promoter region is challenging. Adding to the challenge are the complexity, diversity, and inherent degenerate nature of regulatory motifs and cis- and trans-regulatory elements (Blanchette, Mathieu, et al. "Genome-wide computational prediction of transcriptional regulatory modules reveals new insights into human gene expression." Genome research 16.5 (2006): 656-668). Cis- and trans-regulatory elements are located in the distal parts of the promoter that regulate the spatial and temporal expression of a gene to occur only at required sites and at specific times (Porto, Milena Silva, et al. "Plant promoters: an approach of structure and function." Molecular biotechnology 56.1 (2014): 38-49). Existing promoter analysis tools cannot reliably identify such cis-regulatory elements in a genomic sequence, thus predicting many false positives because these tools are generally focused only on sequence content (Fickett JW, Hatzigeorgiou AG (1997) Eukaryotic promoter recognition. Genome research 7: 861-878). Consequently, identification of promoter regulatory elements requires that a suitable sequence of a specific size be obtained that will result in induction of expression of an operably linked transgene in a desirable manner. Furthermore, identification of 3'UTR regulatory elements requires that a suitable sequence of a specific size be obtained that will result in termination of expression of an operably linked transgene in a desirable manner.

[0019] Methods and compositions are provided for overcoming such problems through the use of Zea mays GRMZM2G144030 promoter elements and other Zea mays GRMZM2G144030 regulatory elements to express transgenes in planta.

II. Terms and Abbreviations

[0020] Throughout the application, numerous terms are used. In order to provide a clear and consistent understanding of the specification and claims, including the scope to be given in such terms, the following definitions are provided.

[0021] As used herein, the term "intron" refers to any nucleic acid sequence comprised in a gene (or expressed polynucleotide sequence of interest) that is transcribed but not translated. Introns include untranslated nucleic acid sequence within an expressed DNA sequence as well as the corresponding sequence in RNA molecules transcribed therefrom. A construct described herein may also contain sequences that increase translational and/or mRNA stability as well as introns. An example of such an intron is the first intron of gene II of the histone H3 variant of Arabidopsis thaliana or any other generally known intron sequence. Introns may be used in combination with a promoter sequence to increase translational and/or mRNA stability.

[0022] The term "isolated" as used herein means having been removed from its natural environment or removed from other compounds present when the compound is first formed. The term "isolated" encompasses materials isolated from natural sources as well as materials (e.g., nucleic acids and proteins) recovered after preparation by recombinant expression in a host cell or chemically synthesized compounds such as nucleic acid, protein, and peptide molecules.

[0023] The term "purified" as used herein refers to the isolation of a molecule or compound in a form that is significantly free of contaminants normally associated with the molecule or compound in a native or natural environment or significantly enriched in concentration relative to other compounds present when the compound is first formed and means having been increased in purity as a result of being separated from other compounds of the original composition. The term "purified nucleic acid" is used herein to describe a nucleic acid sequence that has been separated, produced apart from, or purified separately from other biological compounds, including, but not limited to, polypeptides, lipids, and carbohydrates, while effecting a chemical or functional change in the component (e.g., a nucleic acid can be purified from a chromosome by removing protein contaminants and breaking the chemical bonds that connect the nucleic acid to the rest of the DNA in the chromosome).

[0024] The term "synthetic" as used herein refers to a polynucleotide molecule (e.g., DNA or RNA) that has been created through chemical synthesis as an in vitro process. For example, a synthetic DNA can be created during a reaction within an Eppendorf™ tube such that the synthetic DNA is enzymatically produced from a native strand of DNA or RNA. Other laboratory methods can be used to synthesize a polynucleotide sequence. Oligonucleotides can be chemically synthesized on an oligosynthesizer through solid phase synthesis using phosphoramidites. The synthesized oligonucleotides can be annealed to one another as a complex, thereby producing a "synthetic" polynucleotide. Other methods for chemically synthesizing a polynucleotide are known in the art and can be readily implemented for use in the present disclosure.

[0025] The term "about" as used herein means greater or less than the stated value or range of values by 10 percent, but is not intended to designate any value or range of values solely for this broader definition. Each value or range of values preceded by the term "about" is also intended to encompass the modality of the stated absolute value or range of values.

[0026] For purposes of the present disclosure, a "gene" includes a region of DNA that encodes a gene product (see below) as well as regions of DNA that regulate the production of the gene product, the regulatory sequences of which are or are not adjacent to the coding and/or transcribed sequences. Accordingly, a gene includes, but is not necessarily limited to, promoter sequences, terminators, translational regulatory sequences such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, boundary elements, origins of replication, matrix binding sites, and locus control regions.

[0027] As used herein, the terms "native" or "natural" define a condition found in nature. A "native DNA sequence" is a DNA sequence present in nature that has been produced by natural means or traditional breeding techniques, but has not been generated by genetic engineering (e.g., using molecular biology/transformation techniques).

[0028] As used herein, a "transgene" is defined as a nucleic acid sequence that encodes a gene product, including for example, but not limited to, an mRNA. In one embodiment, the transgene is an exogenous nucleic acid wherein the transgene sequence has been genetically engineered into a host cell (or the progeny thereof) wherein the transgene is not normally found. In one example, a transgene encodes an industrially or pharmaceutically useful compound or a gene encodes a desirable agricultural trait (e.g., a herbicide resistance gene). In yet another example, a transgene is an antisense nucleic acid sequence wherein expression of the antisense nucleic acid sequence inhibits expression of a target nucleic acid sequence. In yet another embodiment, the transgene is an endogenous nucleic acid wherein additional genomic copies of the endogenous nucleic acid are desirable or a nucleic acid that is in the antisense orientation with respect to a target nucleic acid sequence in a host organism.

[0029] As used herein, the term "non-GRMZM2G144030 transgene" or "non-GRMZM2G144030 gene" is any transgene that has less than 80% sequence identity to the GRMZM2G144030 gene coding sequence (SEQ ID NO:4).

[0030] A "gene product" as defined herein is any product made by a gene. For example, a gene product may be a direct transcriptional product of a gene (e.g., mRNA, tRNA, rRNA, antisense RNA, interfering RNA, ribozyme, structural RNA, or any other type of RNA) or a protein produced by translation of an mRNA. Gene products also include RNAs that are modified by processes such as capping, polyadenylation, methylation, and editing, and proteins modified by, for example, methylation, acetylation, phosphorylation, ubiquitination, ADP-robosylation, myristylation, and glycosylation. Gene expression may be influenced by external signals, for example, exposure of a cell, tissue, or organism to an agent that increases or decreases gene expression. The expression of a gene may also be regulated at any site in the pathway from DNA to RNA to protein. Regulation of gene expression occurs, for example, through controls acting on transcription, translation, DNA transport and processing, degradation of intermediate molecules such as mRNA, or through activation, inactivation, compartmentalization, or degradation of specific protein molecules after they are produced, or by combinations thereof. Gene expression can be measured at the RNA level or at the protein level by any method known in the art, including, but not limited to, Northern blot, RT-PCR, Western blot, or in vitro, in situ, or in vivo protein activity assays.

[0031] As used herein, the term "gene expression" refers to the process by which the encoded information of a nucleic acid transcriptional unit (including, for example, genomic DNA) is converted into a working, non-working or structural part of a cell, generally including the synthesis of a protein. Gene expression can be influenced by external signals, for example, exposure of a cell, tissue or organism to an agent that increases or decreases gene expression. The expression of a gene can also be regulated at any site in the pathway from DNA to RNA to protein. Regulation of gene expression occurs, for example, through controls acting on transcription, translation, transport and processing of DNA, degradation of intermediate molecules such as mRNA or through activation, inactivation, compartmentalization or degradation of specific protein molecules after they are produced or by combinations thereof. Gene expression can be measured at the RNA level or at the protein level by any method known in the art, including, but not limited to, Northern blot, RT-PCR, Western blot, or in vitro, in situ, or in vivo protein activity assays.

[0032] As used herein, "homology-based gene silencing" (HBGS) is a generic term that includes both transcriptional gene silencing and post-transcriptional gene silencing. Silencing of a target locus by an unlinked silencing locus can result from inhibition of transcription (transcriptional gene silencing; TGS) or degradation of mRNA (post-transcriptional gene silencing; PTGS), due to production of double-stranded RNA (dsRNA) corresponding to the promoter or transcribed sequences, respectively. The involvement of distinct cellular components in each process suggests that dsRNA-induced TGS and PTGS likely result from diversification from a common ancestral mechanism. However, a strict comparison of TGS and PTGS has been difficult to achieve because it is generally based on the analysis of distinct silencing loci. In some instances, a single transgene locus can trigger both TGS and PTGS, due to the production of dsRNA corresponding to the promoter and transcribed sequences of different target genes. Mourrain et al. (2007) Planta 225:365-79. It is likely that siRNAs are the actual molecules that trigger TGS and PTGS at homologous sequences: siRNAs in this model would trigger silencing and methylation of homologous sequences in cis and in trans by spreading methylation of transgene sequences onto the endogenous promoter.

[0033] As used herein, the term "nucleic acid molecule" (or "nucleic acid" or "polynucleotide") may refer to a polymeric form of nucleotides that may include sense and antisense strands of RNA, cDNA, genomic DNA, and synthetic forms and mixed polymers of the foregoing. A nucleotide may refer to a ribonucleotide, deoxyribonucleotide, or a modified form of any of the nucleotide types. A "nucleic acid molecule" as used herein is synonymous with "nucleic acid" and "polynucleotide." A nucleic acid molecule is generally 10 bases in length, unless otherwise specified. The term may refer to an RNA or DNA molecule of indeterminate length. The term includes both single-stranded and double-stranded forms of DNA. A nucleic acid molecule may include either or both naturally occurring or modified nucleotides linked to each other by naturally occurring and/or non-naturally occurring nucleotide bonds.

[0034] Nucleic acid molecules may be chemically or biologically modified or may contain unnatural or derivatized nucleotide bases, as will be readily appreciated by those skilled in the art. Such modifications include, for example, labels, methylation, replacement of one or more naturally occurring nucleotides with an analog, internucleotide modifications (e.g., uncharged bonds: e.g., methylphosphonates, phosphotriesters, phosphoramidites, carbamates, etc.; charged bonds: e.g., phosphorothioates, phosphorodithioates, etc.; pendant moieties: e.g., peptides; intercalators: e.g., acridine, psoralen, etc.; chelators; alkylators; and modified bonds: e.g., alpha anomeric nucleic acids, etc.). The term "nucleic acid molecule" also includes any topological conformation, including single-stranded, double-stranded, partially duplexed, triplexed, hairpin, circular, and padlock conformations.

[0035] Transcription proceeds in a 5' to 3' manner along a DNA strand. This means that RNA is made by a sequential addition of ribonucleotide 5'-triphosphates to the 3' terminus of the progressing chain (with a requisite elimination of the pyrophosphate). In a linear or circular nucleic acid molecule, discrete elements (e.g., particular nucleotide sequences) may be referred to as being "upstream" or "5'" to an additional element if they are linked or serially linked to the same nucleic acid in the 5' direction from that element. Similarly, discrete elements may be "downstream" or "3'" to an additional element if they are or would be linked to the same nucleic acid in the 3' direction from said element.

[0036] A base "position", as used herein, refers to the location of a particular base or nucleotide residue within a designated nucleic acid. The designated nucleic acid may be defined by alignment (see below) with a reference nucleic acid.

[0037] Hybridization refers to the linking of two polynucleotide strands through hydrogen bonding. Oligonucleotides and their analogues hybridize by hydrogen bonding that includes Watson-Crick, Hoogsteen, or reverse Hoogsteen hydrogen bonding between complementary bases. Generally, nucleic acid molecules consist of nitrogenous bases that are either pyrimidines (cytosine (C), uracil (U), and thymine (T)) or purines (adenine (A) and guanine (G)). These nitrogenous bases form hydrogen bonds between a pyrimidine and a purine, and the bonding of the pyrimidine to a purine is referred to as "base pairing." More specifically, T hydrogen bonding or U and G will be bonded to C. "Complementary" refers to base pairing that occurs between two distinct nucleic acid sequences or two distinct regions of the same nucleic acid sequence.

[0038] "Specifically hybridizable" or "specifically complementary" are terms that indicate a sufficient degree of complementarity such that stable and specific binding occurs between the oligonucleotide and the target DNA or RNA. The oligonucleotide does not need to be 100% complementary to its target sequence to be specifically hybridizable. An oligonucleotide is specifically hybridizable when binding of the oligonucleotide to the target DNA or RNA molecule interferes with the normal function of the target DNA or RNA and there is a sufficient degree of complementarity to prevent nonspecific binding of the oligonucleotide to nonspecific sequences under conditions where specific binding is desirable, for example, under physiological conditions in the case of in vivo assays or systems. Such binding is referred to as specific hybridization.

[0039] Hybridization conditions resulting in particular degrees of stringency will vary depending on the nature of the hybridization method chosen and the composition and length of the hybridizing nucleic acid sequences. Generally, the hybridization temperature and the ionic strength (specifically, the Na+ and/or Mg2+ concentration) of the hybridization buffer will contribute to hybridization stringency, although cleaning times also influence stringency. Calculations regarding the hybridization conditions required to achieve specific degrees of stringency are discussed in Sambrook et al. (ed. Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1-3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, chs. 9 and 11.

[0040] As used herein, "stringent conditions" encompass conditions under which hybridization will occur only if there is less than 50% disparity between the hybridizing molecule and the target DNA. "Stringent conditions" include additional particular levels of stringency. Thus, as used herein, "moderate stringency" conditions are those under which molecules with greater than 50% sequence disparity will not hybridize; "high stringency" conditions are those under which sequences with greater than 20% disparity will not hybridize; and "very high stringency" conditions are those under which sequences with greater than 10% disparity will not hybridize.

[0041] In particular embodiments, stringent conditions may include hybridization at 65°C followed by washes at 65°C with 0.1x SSC/0.1% SDS for 40 minutes.

[0042] The following are representative non-limiting hybridization conditions:

[0043] Very high stringency: Hybridization in 5x SSC buffer at 65°C for 16 hours: washing twice in 2x SSC buffer at room temperature for 15 minutes each: and washing twice in 0.5x SSC buffer at 65°C for 20 minutes each.

[0044] High stringency: Hybridization in 5x-6x SSC buffer at 65-70°C for 16-20 hours: washing twice in 2x SSC buffer at room temperature for 5-20 minutes each: and washing twice in 1x SSC buffer at 55-70°C for 30 minutes each.

[0045] Moderate Stringency: Hybridization in 6x SSC buffer at room temperature at 55°C for 16-20 hours; washing at least twice in 2x-3x SSC buffer at room temperature at 55°C for 20-30 minutes each.

[0046] In particular embodiments, specifically hybridizable nucleic acid molecules can remain bound under very high stringency hybridization conditions. In these and additional embodiments, specifically hybridizable nucleic acid molecules can remain bound under high stringency hybridization conditions. In these and additional embodiments, specifically hybridizable nucleic acid molecules can remain bound under moderate stringency hybridization conditions.

[0047] Oligonucleotide: An oligonucleotide is a short nucleic acid polymer. Oligonucleotides can be formed by cleaving larger segments of nucleic acid or by polymerizing individual nucleotide precursors. Automated synthesizers allow the synthesis of oligonucleotides up to several hundred base pairs in length. Since oligonucleotides can bind to a complementary nucleotide sequence, they can be used as probes to detect DNA or RNA. Oligonucleotides composed of DNA (oligodeoxyribonucleotides) can be used in PCR, a technique for amplifying short DNA sequences. In PCR, the oligonucleotide is typically referred to as a "primer" that allows a DNA polymerase to extend the oligonucleotide and replicate the complementary strand.

[0048] As used herein, the term "sequence identity" in the context of two nucleic acid or polypeptide sequences may refer to residues in the two sequences that are the same when aligned for maximum match over a specified comparison window.

[0049] As used herein, the term "percent sequence identity" may refer to the value determined by comparing two optimally aligned sequences (e.g., nucleic acid sequences and amino acid sequences) over a comparison window, wherein the portion of the sequence in the comparison window may comprise additions or deletions (e.g., gaps) compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleotide or amino acid residue occurs in both sequences to produce the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to produce the percent sequence identity.

[0050] Methods for aligning sequences for comparison are well known in the art. Various alignment programs and algorithms are described, for example, in: Smith and Waterman (1981) Adv. Appl. Math. 2:482; Needleman and Wunsch (1970) J. Mol. Biol. 48:443; Pearson and Lipman (1988) Proc. Natl. Acad. Sci. U.S.A. 85:2444; Higgins and Sharp (1988) Gene 73:237-44; Higgins and Sharp (1989) CABIOS 5:151-3; Corpet et al. (1988) Nucleic Acids Res. 16:10881-90; Huang et al. (1992) Comp. Appl. Biosci. 8:155-65; Pearson et al. (1994) Methods Mol. Biol. 24:307-31; Tatiana et al. (1999) FEMS Microbiol. Lett. 174:247-50. A detailed consideration of sequence alignment methods and homology calculations can be found in, e.g., Altschul et al. (1990) J. Mol. Biol. 215:403-10.

[0051] The Basic Local Alignment Search Tool (BLAST™; Altschul et al. (1990)) from The National Center for Biotechnology Information (NCBI) is available from several sources, including the National Center for Biotechnology Information (Bethesda, MD), and on the Internet for use in connection with several sequence analysis programs. A description of how to determine sequence identity using this program is available on the Internet in the "help" section for BLAST™. For nucleic acid sequence comparisons, the "Blast 2 sequences" function of the BLAST™ (Blastn) program can be used using the default parameters. Nucleic acid sequences with ever greater similarity to reference sequences will exhibit progressive percent identity when evaluated by this method.

[0052] As used herein, the term "operably linked" refers to a first nucleic acid sequence that is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is in a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence when the promoter affects the transcription or expression of the coding sequence. When produced recombinantly, operably linked nucleic acid sequences are generally contiguous and, when necessary, join two protein coding regions in the same reading frame. However, elements need to be contiguous to be operably linked.

[0053] As used herein, the term "promoter" refers to a region of DNA that is generally located upstream (towards the 5' region of a gene) of a gene and is required to initiate and induce transcription of the gene. A promoter may allow for the appropriate activation or repression of a gene that controls it. A promoter may contain specific sequences that are recognized by transcription factors. These factors may bind to a promoter DNA sequence that results in the recruitment of DNA polymerase, an enzyme that synthesizes RNA from the coding region of the gene. The promoter generally refers to all gene regulatory elements located upstream of the gene, including, upstream promoters, 5' UTR, introns, and leader sequences.

[0054] As used herein, the term "upstream promoter" refers to a contiguous polynucleotide sequence that is sufficient to direct the initiation of transcription. As used herein, an upstream promoter encompasses the transcription initiation site with various sequence motifs that include TATA box, initiator sequence, TFIIB recognition elements, and other promoter motifs (Jennifer, E.F. et al., (2002) Genes & Dev., 16: 2583-2592). The upstream promoter provides the site of action for RNA polymerase II which is a multi-subunit enzyme with the general or basal transcription factors such as TFIIA, B, D, R, F, and H. These factors assemble into a pre-transcription initiation complex that catalyzes the synthesis of RNA from the DNA template.

[0055] Upstream promoter activation is accomplished by the additional sequence of DNA sequence regulatory elements to which various proteins bind and subsequently interact with the transcription initiation complex to activate gene expression. These gene regulatory element sequences interact with specific DNA binding factors. These sequence motifs may sometimes be referred to as cis-elements. Such cis-elements to which tissue-specific and development-specific transcription factors bind, individually or in combination, can determine the spatiotemporal expression pattern of a promoter at the transcriptional level. These cis-elements vary greatly in the type of control they exert on operably linked genes. Some elements act to increase transcription of operably linked genes in response to environmental responses (e.g., temperature, humidity, and injury). Other cis-elements may respond to developmental cues (e.g., germination, seed maturation, and flowering) and spatial information (e.g., tissue specificity). See, for example, Langridge et al., (1989) Proc. Natl. Acad. Sci. USA 86:3219-23. These cis-elements are located at varying distances from the transcription start point; some cis-elements (called proximal elements) are adjacent to a minimal core promoter region, while other elements may be positioned several kilobases upstream or downstream of the promoter (enhancers).

[0056] As used herein, the terms "5' untranslated region" or "5' UTR" or "5'UTR" are defined as the untranslated segment at the 5' terminus of pre-mRNAs or mature mRNAs. For example, in mature mRNAs, a 5' UTR typically harbors at its 5' terminus a 7-methylguanosine cap and is involved in many processes such as splicing, polyadenylation, export of mRNA toward the cytoplasm, identification of the 5' end of the mRNA by the translational machinery, and protection of mRNAs from degradation.

[0057] As used herein, the term "transcription terminator" is defined as the transcribed segment at the 3' terminus of pre-mRNAs or mature mRNAs. For example, longer stretches of DNA beyond the "polyadenylation signal" site are transcribed as a pre-mRNA. This DNA sequence usually contains a transcription termination signal for proper processing of the pre-mRNA into mature mRNA.

[0058] As used herein, the terms "3' untranslated region" or "3' UTR" or "3'UTR" are defined as the untranslated segment at the 3' terminus of pre-mRNAs or mature mRNAs. For example, in mature mRNAs, this region harbors the poly-(A) tail and is known to have many roles in mRNA stability, translation initiation, and mRNA export. In addition, the 3' UTR is considered to include the polyadenylation signal and transcription terminator.

[0059] As used herein, the term "polyadenylation signal" refers to a nucleic acid sequence present in mRNA transcripts that allows transcripts, when in the presence of a poly-(A) polymerase, to be polyadenylated at the polyadenylation site, e.g., located 10 to 30 bases downstream of the poly-(A) signal. Many polyadenylation signals are known in the art and are useful in the present invention. An exemplary sequence includes AAUAAA and variants thereof, as described in Loke J., et al., (2005) Plant Physiology 138(3); 1457-1468.

[0060] A "DNA-binding transgene" is a polynucleotide coding sequence that encodes a DNA-binding protein. The DNA-binding protein is subsequently capable of binding to another molecule. A binding protein may bind to, for example, a DNA molecule (a DNA-binding protein), an RNA molecule (an RNA-binding protein), and/or a protein molecule (a protein-binding protein). In the case of a protein-binding protein, it may bind to itself (to form homodimers, homotrimers, etc.) and/or it may bind to one or more molecules of a different protein or proteins. A binding protein may have more than one type of binding activity. For example, zinc finger proteins have DNA-binding, RNA-binding, and protein-binding activity.

[0061] Examples of DNA-binding proteins include; meganucleases, zinc fingers, CRISPRs and TALE binding domains that can be "genetically engineered" to bind to a predetermined nucleotide sequence. Typically, genetically engineered DNA-binding proteins (e.g., zinc fingers, CRISPRs or TALEs) are non-naturally occurring proteins. Non-limiting examples of methods for genetically engineered DNA-binding proteins are design and selection. An engineered DNA-binding protein is a protein that does not occur in nature whose design/composition results primarily from rational criteria. Rational criteria for design include application of substitution rules and computer algorithms to process information in a database storing existing ZFP, CRISPR and/or TALE information and binding data. See, e.g., U.S. Patents 6,140,081; 6,453,242 and 6,534,261; see also WO 98/53058; WO 98/53059; WO 98/53060; WO 02/016536 and WO 03/016496 and US Publication Nos. 20110301073, 20110239315 and 20119145940.

[0062] A "zinc finger DNA-binding protein" (or binding domain) is a protein or domain within a larger protein that binds DNA in a sequence-specific manner through one or more zinc fingers, which are regions of amino acid sequences within the binding domain whose structure is stabilized by the coordination of a zinc ion. The term zinc finger DNA-binding protein is often abbreviated as a zinc finger protein or ZFP. Zinc finger binding domains can be "genetically engineered" to bind a predetermined nucleotide sequence. Non-limiting examples of methods for genetic manipulations of zinc finger proteins are design and selection. An engineered zinc finger protein is a protein that does not occur in nature whose design/composition results primarily from rational criteria. Rational criteria for design include application of substitution rules and computer algorithms to process information in a database storing existing ZFP information and binding data. See, for example, Pat. US. No. 6,140,081; 6,453,242; 6,534,261 and 6,794,136; see also document WO 98/53058; WO 98/53059; WO 98/53060; WO 02/016536 and WO 03/016496.

[0063] In other examples, the DNA-binding domain of one or more of the nucleases comprises a naturally occurring or genetically engineered (non-naturally occurring) TAL effector DNA-binding domain. See, e.g., U.S. Patent Publication No. 20110301073, incorporated by reference in its entirety herein. Plant pathogenic bacteria of the genus Xanthomonas are known to cause many diseases in important crop plants. Xanthomonas pathogenicity depends on a conserved type III secretion system (T3S) that injects more than one different effector proteins into the plant cell. Among these injected proteins are transcription activator-like effectors (TALENs) that mimic plant transcriptional activators and manipulate the transcriptome (see Kay et al., (2007) Science 318:648-651). These proteins contain a DNA-binding domain and a transcriptional activation domain. One of the best characterized TAL effectors is AvrBs3 from Xanthomonas campestgrispv. Vesicatoria (see Bonas et al., (1989) Mol Gen Genet 218: 127-136 and WO2010079430). TAL effectors contain a central domain of tandem repeats, each repeat containing approximately 34 amino acids that are essential for the DNA-binding specificity of these proteins. In addition, they contain a nuclear localization sequence and an acidic transcriptional activation domain (for a review, see Schornack S, et al., (2006) J Plant Physiol 163(3): 256-272). Furthermore, in the plant pathogenic bacteria Ralstonia solanacearum two genes, named brg11 and hpx17, were found to be homologous to the Xanthomonas AvrBs3 family in R. solanacearum biovar 4 strain GMI1000 and R. solanacearum biovar 4 strain RS1000 (see Heuer et al. (2007) Appl and Enviro Micro 73(13): 4379-4384). These genes are 98.9% identical in nucleotide sequence to each other, but differ by a 1,575 bp deletion in the hpx17 repeat domain. However, both gene products have less than 40% sequence identity to Xanthomonas AvrBs3 family proteins. See, e.g., US Patent Publication No. 20110301073, incorporated by reference in its entirety herein.

[0064] The specificity of these TAL effectors depends on the sequences found in the tandem repeats. The repeat sequence comprises approximately 102 bp and the repeats are typically 91-100% homologous to each other (Bonas et al., ibid). The repeat polymorphism is usually located at positions 12 and 13 and appears to be a one-to-one correspondence between the identity of hypervariable di-residues at positions 12 and 13 with the identity of contiguous nucleotides in the target sequence of TAL effectors (see Moscou and Bogdanove, (2009) Science 326:1501 and Boch et al., (2009) Science 326:1509-1512). Experimentally, the natural code for DNA recognition of these TAl effectors was determined such that an HD sequence at positions 12 and 13 results in binding to cytosine (C), NG binds to T, NI to A, C, G or T, NN binds to A or G and ING binds to T. These DNA-binding repeats were assembled into proteins with novel combinations and numbers of repeats to create artificial transcription factors that are capable of interacting with novel sequences and activating the expression of a non-endogenous reporter gene in plant cells (Boch et al., ibid.). Genetically engineered TAL proteins were linked to a Fok cleavage domain half-domain to result in a TAL effector domain nuclease (TALEN) fusion exhibiting activity in a yeast reporter assay (plasmid-based target).

[0065] The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas (CRISPR-associated) nuclease system is a recently engineered nuclease system based on a bacterial system that can be used for genome engineering. It is based in part on the adaptive immune response of many bacteria and Archaea. When a virus or plasmid invades a bacterium, segments of the invading DNA are converted to CRISPR RNAs (crRNA) by the immune response. This crRNA then associates through a region of additional complementarity with another RNA type called a tracrRNA to guide the Cas9 nuclease to a region homologous to the crRNA in the target DNA called a "protospacer". Cas9 cleaves the DNA to generate blunt ends at double-strand breaks (DSBs) at sites specified by a 20-nucleotide guide sequence contained within the crRNA transcript. Cas9 requires both crRNA and tracrRNA for site-specific DNA recognition and cleavage. This system has now been genetically engineered so that crRNA and tracrRNA can be combined into one molecule (the “single guide RNA”), and the crRNA-equivalent portion of the single guide RNA can be genetically engineered to guide the Cas9 nuclease to target any desired sequence (see Jinek et al., (2012) Science 337, pp. 816–821, Jinek et al., (2013) eLife 2:e00471, and David Segal, (2013) eLife 2:e00563). Thus, the CRISPR/Cas system can be genetically engineered to create a DSB at a desired target in a genome, and repair of the DSB can be influenced by the use of repair inhibitors to cause an increase in error-prone repair.

[0066] In other examples, the DNA-binding transgene is a site-specific nuclease comprising a genetically engineered (non-naturally occurring) Meganuclease (also described as a homing endonuclease). Recognition sequences for homing endonucleases or meganucleases such as I-Sce I, I-Ceu I, PI-Psp I, PI-Sce, I-Sce IV, I-Csm I, I-Pan I, I-Sce II, I-Ppo I, I-Sce III, I-Cre I, I-Tev I, I-Tev II and I-Tev III are known. See also U.S. Patent No. 5,420,032; U.S. Patent No. 6,833,252; Belfort et al., (1997) Nucleic Acids Res. 25:3379-30 3388; Dujon et al., (1989) Gene 82:115-118; Perler et al., (1994) Nucleic Acids Res. 22, 11127; Jasin (1996) Trends Genet. 12:224-228; Gimble et al., (1996) J. Mol. Biol. 263:163-180; Argast et al., (1998) J. Mol. Biol. 280:345-353 and the New England Biolabs catalog. In addition, the DNA binding specificity of homing endonucleases and meganucleases can be genetically engineered to bind to unnatural target sites. See, for example, Chevalier et al., (2002) Molec. Cell 10:895-905; Epinat et al., (2003) Nucleic Acids Res. 5 31:2952-2962; Ashworth et al., (2006) Nature 441:656-659; Paques et al., (2007) Current Gene Therapy 7:49-66; US Patent Publication No. 20070117128. The DNA-binding domains of homing endonucleases and meganucleases can be altered in the context of the nuclease as a whole (e.g., so that the nuclease includes the cognate cleavage domain) or can be fused to a heterologous cleavage domain.

[0067] @@As used herein, the term "transformation" encompasses all techniques by which a nucleic acid molecule can be introduced into said cell. Examples include, but are not limited to: transfection with viral vectors; transformations with plasmid vectors; electroporation; lipofection; microinjection (Mueller et al., (1978) Cell 15:579-85); Agrobacterium-mediated transfer; direct DNA uptake; WHISKERS™-mediated transformation; and microprojectile bombardment. These techniques can be used for both stable transformation and transient transformation of a plant cell. "Stable transformation" refers to the introduction of a nucleic acid fragment into a genome of a host organism resulting in genetically stable inheritance. When stably transformed, the nucleic acid fragment is stably integrated into the genome of the host organism and any subsequent generation. Host organisms containing the transformed nucleic acid fragments are referred to as "transgenic" organisms. "Transient transformation" refers to the introduction of a nucleic acid fragment into the nucleus or DNA-containing organelle of a host organism resulting in genetically stable non-inherited gene expression.

[0068] An exogenous nucleic acid sequence. In one example, a transgene is a gene sequence (e.g., a herbicide resistance gene), a gene encoding an industrially or pharmaceutically useful compound, or a gene encoding a desirable agricultural trait. In yet another example, the transgene is an antisense nucleic acid sequence in which expression of the antisense nucleic acid sequence inhibits expression of a target nucleic acid sequence. A transgene may contain regulatory sequences operably linked to the transgene (e.g., a promoter). In some embodiments, a polynucleotide sequence of interest is a transgene. However, in other embodiments, a polynucleotide sequence of interest is an endogenous nucleic acid sequence in which additional genomic copies of the endogenous nucleic acid sequence are desirable or a nucleic acid sequence that is in the antisense orientation with respect to the sequence of a target nucleic acid molecule in a host organism.

[0069] As used herein, the term a transgenic "event" is produced by transformation of plant cells with heterologous DNA, i.e., a nucleic acid construct that includes a transgene of interest, regeneration of a plant population resulting from insertion of the transgene into the plant genome, and selection of a particular plant characterized by the insertion at a particular location in the genome. The term "event" refers to the original transformant and progeny of the transformant that include a heterologous DNA. The term "event" also refers to progeny produced without a sexual cross between the transformant and another variety that includes the genomic DNA/transgene. Even after repeated crossing into a recurrent parent, the inserted transgene DNA and flanking genomic DNA (genomic DNA/transgene) from the transformed parent is present in the progeny of the cross at the same chromosomal location. The term "event" also refers to the DNA of the original transformant and progeny thereof comprising the inserted DNA and flanking genomic sequence immediately adjacent to the inserted DNA that is expected to be transferred to progeny that receive inserted DNA including the transgene of interest as a result of a sexual cross of a parental line that includes the inserted DNA (e.g., the original transformant and progeny resulting from the selfing) and a parental line that does not contain the inserted DNA.

[0070] As used herein, the terms "Polymerase Chain Reaction" or "PCR" define a procedure or technique in which minute quantities of nucleic acid, RNA and/or DNA are amplified as described in U.S. Pat. No. 4,683,195 issued July 28, 1987. Generally, sequence information through or beyond the ends of the region of interest needs to be available so that oligonucleotide primers can be designed and these primers will be identical or similar in sequence to opposite strands of the template to be amplified. The 5'-terminal nucleotides of the two primers may coincide with the ends of the amplified material. PCR can also be used to amplify specific RNA sequences, specific DNA sequences from total genomic DNA and cDNA transcribed from total cellular RNA, bacteriophage or plasmid sequences, etc. See generally Mullis et al., Cold Spring Harbor Symp. Qty. Biol., 51:263 (1987); Erlich, ed., PCR Technology, (Stockton Press, NY, 1989).

[0071] As used herein, the term "primer" refers to an oligonucleotide capable of acting as a synthesis initiation point together with a complementary strand when conditions are suitable for synthesis of a primer extension product. The synthesis conditions include the presence of four different desorbonucleotide triphosphates and at least one polymerization inducing agent such as reverse transcriptase or DNA polymerase. These are present in a suitable buffer that may include constituents that are cofactors or that affect conditions such as pH and the like at various suitable temperatures. A primer is preferably a single-stranded sequence so that amplification efficiency is optimized, but double-stranded sequences may be used.

[0072] As used herein, the term "probe" refers to an oligonucleotide that hybridizes to a target sequence. In the TaqMan® or TaqMan® -style assay procedure, the probe hybridizes to a portion of the target located between the annealing site and the two primers. A probe includes about eight nucleotides, about ten nucleotides, about fifteen nucleotides, about twenty nucleotides, about thirty nucleotides, about forty nucleotides, or about fifty nucleotides. In some embodiments, a probe includes from about eight nucleotides to about fifteen nucleotides. A probe can further include a detectable label, e.g., a fluorophore (Texas-Red®, Fluorescein isothiocyanate, etc.). The detectable label may be covalently linked directly to the probe oligonucleotide, e.g., located at the 5' end of the probe or at the 3' terminus of the probe. A probe including a fluorophore may also further include a quencher, e.g., Black HoleQuencher™, Iowa Black™, etc.

[0073] As used herein, the terms "restriction endonucleases" and "restriction enzymes" refer to bacterial enzymes that cut double-stranded DNA at or near a specific nucleotide sequence. Type-2 restriction enzymes recognize and cleave DNA at the same site and include, but are not limited to, XbaI, BamHI, HindIII, EcoRI, XhoI, SalI, KpnI, AvaI, PstI, and SmaI.

[0074] As used herein, the term "vector" is used interchangeably with the terms "construct", "cloning vector", and "expression vector" and means the vehicle by which a DNA or RNA sequence (e.g., a foreign gene) can be introduced into a host cell in a manner that transforms the host and promotes expression (e.g., transcription and translation) of the introduced sequence. A "non-viral vector" is intended to mean any vector that does not comprise a virus or retrovirus. In some embodiments, a "vector" is a DNA sequence comprising at least one origin of DNA replication and at least one selectable marker gene. Examples include, but are not limited to, a plasmid, cosmid, bacteriophage, bacterial artificial chromosome (BAC), or virus that carries exogenous DNA into a cell. A vector can also include one or more genes, antisense molecules, and/or selectable marker genes, and other genetic elements known in the art. A vector can transduce, transform, or infect a cell, thereby causing the cell to express the nucleic acid molecules and/or proteins encoded by the vector. The term "plasmid" defines a circular strand of nucleic acid capable of autosomal replication in a prokaryotic or eukaryotic host cell. The term includes nucleic acid that may be DNA or RNA and may be single- or double-stranded. The plasmid of the definition may also include sequences that correspond to a bacterial origin of replication.

[0075] As used herein, the term "selectable marker gene" as used herein defines a gene or other expression cassette that encodes a protein that facilitates the identification of cells into which the selectable marker gene is inserted. For example, a "selectable marker gene" encompasses reporter genes as well as genes used in plant transformation to, for example, protect plant cells from a selective agent or provide resistance/tolerance to a selective agent. In one embodiment, only those cells or plants that receive a functional selectable marker are capable of dividing or growing under conditions with a selective agent. Examples of selective agents may include, for example, antibiotics, including spectinomycin, neomycin, kanamycin, paromomycin, gentamicin, and hygromycin. These selectable markers include neomycin phosphotransferase (npt II) which expresses an enzyme that confers resistance to the antibiotic kanamycin and genes for related antibiotics, neomycin, paromomycin, gentamicin, and G418, and the gene for hygromycin phosphotransferase (hpt) which expresses an enzyme that confers resistance to hygromycin. Other selectable marker genes may include genes encoding herbicide resistance including bar or pat (resistance against glufosinate ammonium or phosphinothricin), acetolactate synthase (ALS, resistance against inhibitors such as sulfonylureas (SUs), imidazolinones (IMIs), triazolopyrimidines (TPs), pyrimidinyl oxybenzoates (POBs), and sulfonylaminocarbonyl triazolinones that prevent the first step in the synthesis of branched-chain amino acids), glyphosate, 2,4-D, and metal resistance or sensitivity. Examples of "reporter genes" that can be used as a selectable marker gene include visual observation of expressed reporter gene proteins such as proteins encoding β-glucuronidase (GUS), luciferase, green fluorescent protein (GFP), yellow fluorescent protein (YFP), DsRed, β-galactosidase, chloramphenicol acetyltransferase (CAT), alkaline phosphatase, and the like. The phrase "positive marker" refers to plants that have been transformed to include a selectable marker gene.

[0076] As used herein, the term "detectable marker" refers to a marker capable of detection, such as, for example, a radioisotope, fluorescent compound, bioluminescent compound, chemiluminescent compound, metal chelator, or enzyme. Examples of detectable markers include, but are not limited to, the following: fluorescent markers (e.g., FITC, rhodamine, lanthanide phosphors), enzymatic markers (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminescent, biotinyl groups, predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags). In one embodiment, a detectable marker may be attached by spacer arms of various lengths to reduce potential steric hindrance.

[0077] As used herein, the terms "cassette", "expression cassette" and "gene expression cassette" refer to a segment of DNA that can be inserted into a nucleic acid or polynucleotide at specific restriction sites or by homologous recombination. As used herein, the DNA segment comprises a polynucleotide encoding a polypeptide of interest and the cassette and restriction sites are designed to ensure insertion of the cassette in the proper reading frame for transcription and translation. In one embodiment, an expression cassette can include a polynucleotide encoding a polypeptide of interest and that contains elements in addition to the polynucleotide that facilitate transformation of a particular host cell. In one embodiment, an expression gene can also include elements that allow for increased expression of a polynucleotide encoding a polypeptide of interest in a host cell. Such elements may include, but are not limited to: a promoter, a minimal promoter, an enhancer, a response element, a terminator sequence, a polyadenylation sequence, and the like.

[0078] As used herein, a "linker" or "spacer" is a bond, molecule, or group of molecules that link two separate entities to one another. Linkers and spacers can provide optimal spacing of two entities and can further provide a labile linkage that allows two entities to be separated from one another. Labile bonds include photocleavable groups, acid-labile moieties, base-labile moieties, and enzyme-cleavable groups. The terms "polylinker" or "multiple cloning site" as used herein define a group of three or more type 2 restriction enzyme sites located within 10 nucleotides of one another in a nucleic acid sequence. Constructs comprising a polylinker are used for insertion and/or excision of nucleic acid sequences such as the coding region of a gene. In other instances, the term "polylinker" as used herein refers to a stretch of nucleotides that are targeted to join two sequences via any splicing cloning method (i.e., GibsonAssembly®, NEBuilder HiFiD-NA Assembly®, Golden Gate Assembly, BioBrick® Assembly, etc.). Constructs comprising a polylinker are used for insertion and/or excision of nucleic acid sequences such as the coding region of a gene.

[0079] As used herein, the term "control" refers to a sample used in an analytical procedure for comparison purposes. A control may be "positive" or "negative". For example, where the purpose of an analytical procedure is to detect a differentially expressed transcript or polypeptide in cells or tissue, it is generally preferable to include a positive control such as a sample from a plant known to exhibit the desired expression and a negative control, such as a sample from a plant known to lack the desired expression.

[0080] As used herein, the term "plant" includes an entire plant and any descendant, cell, tissue or part of a plant. A class of plant that may be used in the present invention is generally as broad as the class of higher and lower plants amenable to mutagenesis including angiosperms (monocotyledonous and dicotyledonous plants), gymnosperms, ferns and multicellular algae. Thus, "plant" includes both dicotyledonous and monocotyledonous plants. The term "plant parts" includes any part(s) of a plant, including, for example, and without limitation: seed (including mature seed and immature seed); a plant cutting; a plant cell; a plant cell culture; a plant organ (e.g., pollen, embryo, flowers, fruits, twigs, leaves, roots, stems and explants). A plant tissue or plant organ may be a seed, protoplast, callus, or any other group of plant cells that is organized into a structural or functional unit. A plant cell or tissue culture may be capable of regenerating a plant containing the physiological and morphological characteristics of the plant from which the cell or tissue was obtained and of regenerating a plant with substantially the same genotype as the plant. In contrast, some plant cells are not capable of being regenerated to produce plants. Regenerable cells in a plant cell or tissue culture may be embryos, protoplasts, meristematic cells, callus, pollen, leaves, anthers, root tips, silks, flowers, nuclei, ears, cobs, husks, or stalks.

[0081] Plant parts include harvestable parts and parts useful for propagating progeny plants. Plant parts useful for propagation include, for example, and without limitation: seed; fruit; a cutting; a seedling; a bulb, and a rhizome. A harvestable part of a plant can be any useful part of a plant, including, for example and without limitation: flower; pollen; seedling; bulb; leaf; stem; fruit; seed, and root.

[0082] A plant cell is the structural and physiological unit of the plant, comprising a protoplast and a cell wall. A plant cell may be in the form of a single isolated cell or an aggregate of cells (e.g., a friable callus and a cultured cell), and may be part of a higher organized unit (e.g., a plant tissue, a plant organ, and a plant). Thus, a plant cell may be a protoplast, a gamete-producing cell, or a cell or collection of cells that can regenerate into an entire plant. As such, a seed that comprises multiple plant cells and is capable of regenerating into an entire plant is considered a “plant cell” in embodiments hereof.

[0083] As used herein, the term "small RNA" refers to several classes of non-coding ribonucleic acids (ncRNA). The term small RNA describes short strands of ncRNA produced in bacterial, animal, plant, and fungal cells. These short ncRNA strands may be produced naturally within the cell or may be produced by the introduction of an exogenous sequence that expresses the short strand or ncRNA. Small RNA sequences do not directly code for a protein and differ in function from other RNA in that small RNA sequences are only transcribed and not translated. Small RNA sequences are involved in other cellular functions, including gene expression and modification. Small RNA molecules are generally made of about 20 to 30 nucleotides. Small RNA sequences may be derived from larger precursors. The precursors form structures that refold upon each other into self-complementary regions that are processed by the nuclease Dicer in animals or DCL1 in plants.

[0084] Many types of small RNA exist naturally or are produced artificially, including microRNAs (miRNAs), short interfering RNAs (siRNAs), antisense RNAs, short hairpin RNAs (shRNAs), and small nucleolar RNAs (snoRNAs). Certain types of small RNAs, such as microRNAs and siRNAs, are important in gene silencing and RNA interference (RNAi). Gene silencing is a process of genetic regulation in which a gene that would normally be expressed is "turned off" by an intracellular element, in this case, small RNA. The protein that would normally be formed by this gene formation is not formed due to the interference, and the information encoded in the gene is blocked from expression.

[0085] As used herein, the term "small RNA" encompasses RNA molecules described in the literature as "mini-RNA" (Storz, (2002) Science 296:1260-3; Illangasekare et al., (1999) RNA 5:1482-1489); prokaryotic "small RNA" (sRNA) (Wassarman et al., (1999) Trends Microbiol. 7:37-45); eukaryotic "non-coding RNA" (ncRNA)"; "micro-RNA (miRNA)"; "small non-mRNA (snmRNA)"; "functional RNA (fRNA)"; "transfer RNA (tRNA)"; "catalytic RNA" [e.g., ribozymes, including self-acylating ribozymes (Illangasekare et al., (1999) RNA 5:1482-1489); "small nucleolar RNAs (snoRNAs)," "tmRNA" (a.k.a. "10S RNA," Muto et al., (1998) Trends Biochem Sci. 23:25-29; and Gillet et al., (2001) Mol Microbiol. 42:879-885); RNAi molecules including, without limitation, "small interfering RNA (siRNA)," "endoribonuclease-primed siRNA (e-siRNA)," "small hairpin RNA (shRNA)," and "small transiently regulated RNA (stRNA)," "hub siRNA (d-siRNA)," and aptamers, oligonucleotides, and other synthetic nucleic acids comprising at least one uracil base.

[0086] Unless specifically explained otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art. Definitions of common terms in molecular biology can be found in, for example: Lewin, Genes V, Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Meyers (ed.), Molecular Biology and Biotechnology: A Comprehensive Desk Reference, VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).

[0087] As used herein, "a", "an", and "the" include plural references unless the context clearly and ambiguously dictates otherwise.

III. Zea mays GRMZM2G144030 Promoter and Nucleic Acids Comprising It

[0088] Provided are methods and compositions for using a promoter and other gene regulatory elements from Zea mays GRMZM2G144030 to express non-transgenes in plants. In one embodiment, a promoter can be the Zea mays GRMZM2G144030 promoter of SEQ ID NO:1. In a further embodiment, a 3'UTR can be the Zea mays GRMZM2G144030 3'UTR of SEQ ID NO:5.

[0089] In one embodiment, a polynucleotide is provided comprising a promoter, wherein the promoter is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, or 100% identical to SEQ ID NO:1. In one embodiment, a promoter is a Zea mays GRMZM2G144030 promoter comprising a polynucleotide of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, or 100% identity to the polynucleotide of SEQ ID NO: 1. In one embodiment, an isolated polynucleotide is provided comprising at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, or 100% identity to the polynucleotide of SEQ ID NO: 1. In one embodiment, a nucleic acid vector is provided comprising a Zea mays GRMZM2G144030 promoter of SEQ ID NO:1. In one embodiment, a polynucleotide is provided comprising a Zea mays GRMZM2G144030 promoter that is operably linked to a polylinker. In one embodiment, a gene expression cassette is provided comprising a Zea mays GRMZM2G144030 promoter that is operably linked to a non-GRMZM2G144030 transgene. In one embodiment, a nucleic acid vector is provided comprising a Zea mays GRMZM2G144030 promoter that is operably linked to a non-GRMZM2G144030 transgene. In one embodiment, the promoter is SEQ ID NO:1. In an illustrative embodiment, a nucleic acid vector comprises a Zea mays GRMZM2G144030 promoter operably linked to a transgene, wherein the transgene can be an insecticide resistance transgene, a herbicide tolerance transgene, a nitrogen use efficiency transgene, a water use efficiency transgene, a nutritional quality transgene, a DNA binding transgene, a small RNA transgene, a selectable marker transgene, or combinations thereof.

[0090] Transgene expression can also be regulated by the 3' untranslated gene region (i.e., 3' UTR) located downstream of the gene coding sequence. Both a promoter and a 3' UTR can regulate transgene expression. Although a promoter is required to induce transcription, a 3' UTR gene region can terminate transcription and initiate polyadenylation of a resulting mRNA transcript for translation and protein synthesis. A 3' UTR gene region aids in the stable expression of a transgene.

[0091] In one embodiment, a nucleic acid vector is provided comprising Zea mays GRMZM2G144030 promoter as described herein and a 3' UTR. In one embodiment, the nucleic acid vector comprises a Zea mays GRMZM2G144030 3' UTR. In one embodiment, the Zea mays GRMZM2G144030 3' UTR is SEQ ID NO:5.

[0092] In one embodiment, a nucleic acid vector is provided comprising a Zea mays GRMZM2G144030 promoter as described herein and 3' UTR, wherein the 3' UTR is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, or 100% identical to the polynucleotide of SEQ ID NO:5. In one embodiment, a nucleic acid vector is provided comprising a Zea mays GRMZM2G144030 promoter as described herein and a Zea mays GRMZM2G144030 3' UTR wherein the Zea mays GRMZM2G144030 promoter and the Zea mays GRMZM2G144030 3' UTR are operatively linked to opposite ends of a polylinker. In one embodiment, a gene expression cassette is provided comprising a Zea mays GRMZM2G144030 promoter as described herein and a Zea mays GRMZM2G144030 3' UTR wherein the Zea mays GRMZM2G144030 promoter and the Zea mays GRMZM2G144030 3' UTR are operably linked to opposite ends of a non-GRMZM2G144030 transgene. In one embodiment, the 3' UTR consists of SEQ ID NO:5. In one embodiment, a gene expression cassette is provided comprising a Zea mays GRMZM2G144030 promoter as described herein and a Zea mays GRMZM2G144030 3' UTR wherein the Zea mays GRMZM2G144030 promoter comprises SEQ ID NO: 1 and the Zea mays GRMZM2G144030 3' UTR comprises SEQ ID NO: 5 wherein the promoter and 3' UTR are operably linked to opposite ends of a non-GRMZM2G144030 transgene. In one aspect of this embodiment, the 3' UTR consists of SEQ ID NO:5. In one aspect of this embodiment, the promoter consists of SEQ ID NO:1. In an illustrative embodiment, a gene expression cassette comprises a Zea mays GRMZM2G144030 3' UTR that is operably linked to a transgene, wherein the transgene can be an insecticide resistance transgene, a herbicide tolerance transgene, a nitrogen use efficiency transgene, a water use efficiency transgene, a nutritional quality transgene, a DNA or protein binding transgene, a small RNA transgene, a selectable marker transgene, or combinations thereof. In a further embodiment, the transgene is operably linked to a Zea mays GRMZM2G144030 promoter and a Zea mays GRMZM2G144030 3' UTR from the same GRMZM2G144030-like gene.

[0093] Transgene expression may also be regulated by an intron region located downstream of a promoter sequence. Either a promoter or an intron may regulate transgene expression. Although the promoter is necessary to induce transcription, the presence of an intron may increase expression levels resulting in mRNA transcript for translation and protein synthesis. An intron gene region assists in the stable expression of a transgene. In a further embodiment, an intron is operably linked to a Zea mays GRMZM2G144030 promoter.

[0094] Transgene expression may also be regulated by a 5' UTR region located downstream of a promoter sequence. Either a promoter or a 5' UTR may regulate transgene expression. Although a promoter is necessary to induce transcription, the presence of a 5' UTR may increase expression levels resulting in mRNA transcript for translation and protein synthesis. A 5' UTR gene region assists in the stable expression of a transgene. In a further embodiment, a 5' UTR is operably linked to a Zea mays GRMZM2G144030 promoter.

[0095] A Zea mays GRMZM2G144030 promoter can also comprise one or more additional sequence elements. In some embodiments, a Zea mays GRMZM2G144030 promoter can comprise an exon (e.g., a leader or signal peptide such as a chloroplast transit peptide or ER retention signal). For example and without limitation, a Zea mays GRMZM2G144030 promoter can encode an exon incorporated within the Zea mays GRMZM2G144030 promoter as an additional embodiment.

[0096] In one embodiment, a nucleic acid vector comprises a gene expression cassette as described herein. In one embodiment, a vector can be a plasmid, a cosmid, a bacterial artificial chromosome (BAC), a bacteriophage, a virus, or an excised polynucleotide fragment for use in direct transformation or gene targeting such as a donor DNA.

[0097] According to one embodiment, a nucleic acid vector is provided comprising a recombinant gene expression cassette wherein the recombinant gene expression cassette comprises a Zea mays GRMZM2G144030 promoter operably linked to a polylinker sequence, a non-GRMZM2G144030 transgene, or a combination thereof. In one embodiment, the recombinant gene expression cassette comprises a Zea mays GRMZM2G144030 promoter operably linked to a non-GRMZM2G144030 transgene. In one embodiment, the recombinant gene expression cassette comprises a Zea mays GRMZM2G144030 promoter as described herein operably linked to a polylinker sequence. The polylinker is operably linked to the Zea mays GRMZM2G144030 promoter in a manner that insertion of a coding sequence into one of the polylinker restriction sites will operably link the coding sequence allowing expression of the coding sequence when the vector is transformed or transfected into a host cell.

[0098] According to one embodiment, the Zea mays GRMZM2G144030 promoter comprises SEQ ID NO: 1, or a sequence that has at least 80, 85, 90, 95, or 99% sequence identity to SEQ ID NO: 1. According to one embodiment, the promoter sequence has a total length of greater than 1.5, 2, 2.5, 3, or 4 kb. According to one embodiment, the Zea mays GRMZM2G144030 promoter consists of SEQ ID NO: 1, or a 2,082 bp sequence that has at least 80, 85, 90, 95, or 99% sequence identity to SEQ ID NO: 1.

[0099] According to one embodiment, a nucleic acid is provided that comprises a gene cassette consisting of a Zea mays GRMZM2G144030 promoter, a non-GRMZM2G144030 transgene, and a Zea mays GRMZM2G144030 3' UTR of SEQ ID NO:5. In one embodiment, the Zea mays GRMZM2G144030 3' UTR of SEQ ID NO:5 is operably linked to the 3' end of the non-GRMZM2G144030 transgene. In another embodiment, the 3' untranslated sequence comprises SEQ ID NO:5 or a sequence that has at least 80, 85, 90, 95, 99, or 100% sequence identity to SEQ ID NO:5. According to one embodiment, a nucleic acid vector is provided comprising a gene cassette consisting of SEQ ID NO: 1, or a 2,082 bp sequence that has at least 80, 85, 90, 95, or 99% sequence identity to SEQ ID NO: 1, a non-GRMZM2G144030 transgene, and a 3' UTR of GRMZM2G144030 from Zea mays, wherein SEQ ID NO: 1 is operably linked to the 5' end of the non-GRMZM2G144030 transgene and the 3' UTR of SEQ ID NO:5 is operably linked to the 3' end of the non-GRMZM2G144030 transgene. In another embodiment, the 3' untranslated sequence comprises SEQ ID NO:5 or a sequence that has at least 80, 85, 90, 95, 99, or 100% sequence identity to SEQ ID NO:5. In another embodiment, the 3' untranslated sequence of GRMZM2G144030 from Zea mays consists of SEQ ID NO:5, or a 1,095 bp sequence that has at least 80, 85, 90, 95, or 99% sequence identity to SEQ ID NO:5.

[00100] In one embodiment, a nucleic acid construct is provided comprising a promoter and a non-GRMZM2G144030 transgene and, optionally, one or more of the following elements: a) a 5' untranslated region; b) an intron; and c) a 3' untranslated region, wherein

[00101] the promoter consists of SEQ ID NO:1 or a sequence with at least 98% sequence identity to SEQ ID NO:1; and

[00102] the 3' untranslated region consists of SEQ ID NO:5 or a sequence having at least 98% sequence identity to SEQ ID NO:5; wherein further, said promoter is operably linked to said transgene and each optional element, when present, is also operably linked to the promoter and the transgene. In another embodiment, a transgenic cell comprising the nucleic acid construct described immediately above is provided. In one embodiment, the transgenic cell is a plant cell, and in another embodiment, a plant is provided, wherein the plant comprises said transgenic cells.

[00103] In one embodiment, a nucleic acid construct is provided comprising a non-GRMZM2G144030 promoter and transgene, and optionally one or more of the following elements: a) a 5' untranslated region; and b) a 3' untranslated region,

[00104] where

[00105] the promoter consists of SEQ ID NO:1 or a sequence with at least 98% sequence identity to SEQ ID NO:1; and

[00106] the 3' untranslated region consists of SEQ ID NO:5 or a sequence having at least 98% sequence identity to SEQ ID NO:5; wherein further, said promoter is operably linked to said transgene and each optional element, when present, is also operably linked to the promoter and the transgene. In another embodiment, a transgenic cell comprising the nucleic acid construct described immediately above is provided. In one embodiment, the transgenic cell is a plant cell, and in another embodiment, a plant is provided, wherein the plant comprises said transgenic cells.

[00107] In one embodiment, a nucleic acid construct is provided comprising a promoter and a polylinker and, optionally, one of the following elements: a) a 5' untranslated region; b) an intron; and c) a 3' untranslated region.

[00108] where

[00109] the promoter consists of SEQ ID NO:1 or a sequence with at least 98% sequence identity to SEQ ID NO:1; and

[00110] the 3' untranslated region consists of SEQ ID NO:5 or a sequence having at least 98% sequence identity to SEQ ID NO:5; wherein, further, said promoter is operably linked to said polylinker and each optional element, when present, is also operably linked to the promoter and the polylinker.

[00111] According to one embodiment, the nucleic acid vector further comprises a sequence encoding a selectable marker. According to one embodiment, the recombinant gene cassette is operably linked to one edge of the Agrobacterium T-DNA. According to one embodiment, the recombinant gene cassette further comprises a first and a second edge of the T-DNA, wherein the first edge of the T-DNA is operably linked to one end of the gene construct, and the second edge of the T-DNA is operably linked to the other end of the gene construct. The first and second Agrobacterium T-DNA borders can be independently selected from T-DNA border sequences that originate from bacterial strains selected from the group consisting of an Agrobacterium T-DNA border that synthesizes nopaline, an Agrobacterium T-DNA border that synthesizes octopine, an Agrobacterium T-DNA border that synthesizes mannopine, an Agrobacterium T-DNA border that synthesizes succinamopine, or any combination thereof. In one embodiment, there is provided an Agrobacterium strain selected from the group consisting of a nopaline-synthesizing strain, a mannopine-synthesizing strain, a succinamopine-synthesizing strain, or an octopine-synthesizing strain, wherein said strain comprises a plasmid wherein the plasmid comprises a transgene operably linked to a sequence selected from SEQ ID NO: 1, or a sequence having at least 80, 85, 90, 95, or 99% sequence identity to SEQ ID NO: 1.

[00112] Transgenes of interest that are suitable for use in the presently described constructs include, but are not limited to, coding sequences that confer (1) pest or disease resistance, (2) herbicide tolerance, (3) value-added agronomic traits such as: yield improvement, nitrogen use efficiency, water use efficiency, and nutritional quality, (4) binding of a protein to DNA in a site-specific manner, (5) small RNA expression, and (6) selectable markers. According to one embodiment, the transgene encodes a selectable marker or gene product that confers insecticide resistance, herbicide tolerance, small RNA expression, nitrogen use efficiency, water use efficiency, or nutritional quality.

1. Insect Resistance

[00113] Various insect resistance coding sequences can be operably linked to the Zea mays GRMZM2G144030 promoter comprising SEQ ID NO: 1. In one embodiment, a promoter can be the Zea mays GRMZM2G144030 promoter comprising SEQ ID NO: 1, or a sequence that has at least 80, 85, 90, 95, or 99% sequence identity to SEQ ID NO: 1. In some embodiments, the sequences are operably linked to the Zea mays GRMZM2G144030 promoter comprising SEQ ID NO: 1 and a 5' UTR. The operably linked sequences can then be incorporated into a chosen vector to allow for the identification and selection of transformed plants ("transformants"). Insect resistance coding sequences are known in the art. Since embodiments of insect resistance coding sequences may be operably linked to regulatory elements of the subject matter, the following features are provided. Exemplary lepidopteran insect resistance coding sequences include: crylA; cry1A.105; crylAb; crylAb(truncated); cry1Ab-Ac (fusion protein); crylAc (marketed as Widestrike®); crylC; crylF (marketed as Widestrike®); cry1Fa2; cry2Ab2; cry2Ae; cry9C; mocrylF; pinlI (protease inhibitor protein); vip3A(a); and vip3Aa20. Coleopteran insect resistance coding sequences include: cry34Ab1 (marketed as Herculex®); cry35Ab1 (marketed as Herculex®); cry3A; cry3Bb1; dvsnf7; and mcry3A. Exemplary coding sequences that provide resistance to multiple insects include ecry31.Ab. The above list of insect resistance genes is not intended to be limiting. Any insect resistance genes are covered by this description.

2. Herbicide Tolerance

[00114] Various herbicide tolerance coding sequences can be operably linked to the Zea mays GRMZM2G144030 promoter comprising SEQ ID NO: 1, or a sequence that has 80, 85, 90, 95, or 99% sequence identity to SEQ ID NO: 1. In one embodiment, a promoter can be the Zea mays GRMZM2G144030 promoter of SEQ ID NO:1 comprising SEQ ID NO: 1, or a sequence that has at least 80, 85, 90, 95, or 99% sequence identity to SEQ ID NO: 1. In some embodiments, the sequences are operably linked to the Zea mays GRMZM2G144030 promoter comprising SEQ ID NO: 1 and a 5' UTR. The operably linked sequences can then be incorporated into a chosen vector to allow identification and selection of transformed plants ("transformants"). Exemplary herbicide tolerance coding sequences are known in the art. Since embodiments of herbicide tolerance coding sequences can be operably linked to the regulatory elements of the subject matter of the disclosure, the following features are provided. The herbicide glyphosate contains a mode of action through inhibition of the enzyme EPSPS (5-enolpyruvylshikimate-3-phosphate synthase). This enzyme is involved in the biosynthesis of aromatic amino acids that are essential for plant growth and development. Several enzymatic mechanisms that can be used to inhibit this enzyme are known in the art. The genes encoding these enzymes can be operably linked to the regulatory elements of the gene of the subject matter of the disclosure. In one embodiment, selectable marker genes include, but are not limited to, genes encoding glyphosate resistance genes which include: mutant EPSPS genes, such as the 2mEPSPS genes, cp4 EPSPS genes, mEPSPS genes, dgt-28 genes; aroA genes; and glyphosate degradation genes, such as the glyphosate acetyl transferase (gat) genes and the glyphosate oxidase (gox) genes. These traits are currently commercialized as Gly-TolTM, Optimum® GAT®, Agrisure® GT, and Roundup Ready®. Resistance genes for the glufosinate and/or bialaphos compounds include the dsm-2, bar, and pat genes. The bar and pat traits are currently commercialized as LibertyLink®. Also included are tolerance genes that provide resistance to 2,4-D, such as the aad-1 genes (it should be noted that the aad-1 genes have additional activity on aryloxyphenoxypropionate herbicides) and the aad-12 genes (it should be noted that the aad-12 genes have additional activity on synthetic pyridyloxyacetate auxins). These traits are commercialized as Enlist® crop protection technology. Resistance genes for ALS inhibitors (sulfonylureas, imidazolinones, triazolopyrimidines, pyrimidinylthiobenzoates, and sulfonylaminocarbonyltriazolinones) are well known in the art. These resistance genes most commonly result from point mutations in the ALS coding gene sequence. Other ALS inhibitor resistance genes include the hra genes, the csr1-2 genes, the Sr-HrA genes, and the surB genes. Some of the traits are marketed under the trade name Clearfield®. Herbicides that inhibit HPPD include the pyrazolones, such as pyrazoxifen, benzofenap, and topramezone; triketones, such as mesotrione, sulcotrione, tembotrione, and benzobicyclone; and diketonitriles, such as isoxaflutole. These exemplary HPPD herbicides can be tolerated by known traits. Examples of HPPD inhibitors include the hppdPF_W336 genes (for resistance to isoxaflutole) and the avhppd-03 genes (for resistance to meostrione). An example of a trait tolerant to the herbicide oxynil includes the bxn gene, which has been shown to confer resistance to the herbicide/antibiotic bromoxynil. Resistance genes for dicamba include the dicamba monooxygenase (dmo) gene as described in PCT International Publication No. WO 2008/105890. Resistance genes for PPO or PROTOX inhibitor-type herbicides (e.g., acifluorfen, butafenacil, flupropazil, pentoxazone, carfentrazone, fluazolate, pyraflufen, aclonifen, azafenidin, flumioxazine, flumiclorac, bifenox, oxyfluorfen, lactofen, fomesafen, fluoroglycofen, and sulfentrazone) are known in the art. Genes exemplares que conferem resistência a PPO incluem a supe- rexpressão de uma enzima PPO de Arabidopsis thaliana do tipo selva- gem (Lermontova I e Grimm B, (2000) Overexpression of plastidic pro-toporphyrinogen IX oxidase leads to resistance to the diphenyl-ether herbicide acifluorfen. Plant Physiol 122:75-83.), o gene da PPO de B. subtilis (Li, X. e Nicholl D. 2005. Development of PPO inhibitorresistant cultures and crops. Pest Manag. Sci. 61:277-285 e Choi KW, Han O, Lee HJ, Yun YC, Moon YH, Kim MK, Kuk YI, Han SU e Guh JO, (1998) Generation of resistance to the diphenyl ether herbicide, oxyfluorfen, via expression of the Bacillus subtilis protoporphyrinogen oxidase gene in transgenic tobacco plants. Biosci Biotechnol Biochem 62:558-560.) Resistance genes for pyridinoxy or phenoxypropionic acids and cyclohexones include the ACCase inhibitor-encoding genes (e.g., Acc1-S1, Acc1-S2, and Acc1-S3). Exemplary genes that confer resistance to cyclohexanediones and/or aryloxyphenoxypropanoic acid include haloxyfop, diclofop, fenoxyprop, fluazifop, and quizalofop. Finally, herbicides that can inhibit photosynthesis, including triazine or benzonitrile, acquire tolerance by the psbA genes (triazine tolerance), 1s+ genes (triazine tolerance), and nitrilase genes (benzonitrile tolerance). The above list of herbicide tolerance genes is not intended to be limiting. Any of the herbicide tolerance genes are encompassed by this description.

3. Agronomic Characteristics

[00115] Various agronomic trait coding sequences can be operably linked to the Zea mays GRMZM2G144030 promoter comprising SEQ ID NO: 1, or a sequence that has 80, 85, 90, 95, or 99% sequence identity to SEQ ID NO: 1. In one embodiment, a promoter can be the Zea mays GRMZM2G144030 promoter of SEQ ID NO:1 comprising SEQ ID NO: 1, or a sequence that has at least 80, 85, 90, 95, or 99% sequence identity to SEQ ID NO: 1. In some embodiments, the sequences are operably linked to the Zea mays GRMZM2G144030 promoter comprising SEQ ID NO: 1 and a 5' UTR. The operably linked sequences can then be incorporated into a chosen vector to enable identification and selection of transformed plants ("transformants"). Exemplary agronomic trait coding sequences are known in the art. Since embodiments of agronomic trait coding sequences can be operably linked to the regulatory elements of the subject matter, the following features are provided. Delayed fruit softening as provided by the pg genes inhibits the production of the polygalacturonase enzyme responsible for breaking down pectin molecules in the cell wall and thus causes delayed fruit softening. In addition, the delayed fruit ripening/senescence of the acc genes acts to suppress the normal expression of the native acc synthase gene, resulting in reduced ethylene production and delayed fruit ripening. Meanwhile, the accd genes metabolize the precursor of the fruit ripening hormone, resulting in delayed fruit ripening. Alternatively, the sam-k genes cause delayed ripening by reducing S-adenosylmethionine (SAM), a substrate for ethylene production. Drought stress tolerance phenotypes, as provided by the cspB genes, maintain normal cellular functions under water stress conditions by preserving RNA stability and translation. Another example includes the EcBetA genes that catalyze the production of the osmoprotectant compound, glycine betaine, which confers tolerance to water stress. In addition, the RmBetA genes catalyze the production of the osmoprotectant compound, glycine betaine, which confers tolerance to water stress. Enhancement of photosynthesis and yield is provided by the bbx32 gene, which expresses a protein that interacts with one or more endogenous transcription factors to regulate plant day/night physiological processes. Ethanol production can be increased by expression of the amy797E gene encoding a thermostable alpha-amylase enzyme that increases bioethanol production by increasing the thermostability of the amylase used in starch degradation. Finally, modified amino acid compositions can result from expression of the cordapA gene encoding a dihydrodipicolinate synthase enzyme that increases the production of the amino acid lysine. The above list of agronomic trait coding sequences is not intended to be limiting. Any agronomic trait coding sequence is encompassed by this description.

4. DNA Binding Proteins

[00116] Various DNA-binding protein coding sequences can be operably linked to the Zea mays GRMZM2G144030 promoter comprising SEQ ID NO: 1, or a sequence that has 80, 85, 90, 95, or 99% sequence identity to SEQ ID NO: 1. In one embodiment, a promoter can be the Zea mays GRMZM2G144030 promoter of SEQ ID NO:1 comprising SEQ ID NO: 1, or a sequence that has at least 80, 85, 90, 95, or 99% sequence identity to SEQ ID NO: 1. In some embodiments, the sequences are operably linked to the Zea mays GRMZM2G144030 promoter comprising SEQ ID NO: 1 and a 5' UTR. The operably linked sequences can then be incorporated into a chosen vector to allow identification and selection of transformed plants ("transformants"). DNA binding protein coding sequences are known in the art. Since embodiments of DNA binding protein coding sequences can be operably linked to the regulatory elements of the subject matter of the disclosure, the following types of DNA binding proteins may include zinc fingers, Talens, CRISPRs, and meganucleases. The above list of DNA binding protein coding sequences is not intended to be limiting. Any DNA binding protein coding sequences are encompassed by the present disclosure.

5. Small RNA

[00117] Various small RNAs can be operably linked to the Zea mays GRMZM2G144030 promoter comprising SEQ ID NO: 1, or a sequence that has 80, 85, 90, 95, or 99% sequence identity to SEQ ID NO: 1. In one embodiment, a promoter can be the Zea mays GRMZM2G144030 promoter of SEQ ID NO:1 comprising SEQ ID NO: 1, or a sequence that has at least 80, 85, 90, 95, or 99% sequence identity to SEQ ID NO: 1. In some embodiments, the sequences are operably linked to the Zea mays GRMZM2G144030 promoter comprising SEQ ID NO: 1 and a 5' UTR. The operably linked sequences can then be incorporated into a chosen vector to allow identification and selection of transformed plants ("transformants"). Characteristics of exemplary small RNAs are known in the art. Since embodiments of small RNA coding sequences can be operably linked to the regulatory elements of the subject matter of the disclosure, the following characteristics are provided. For example, the anti-effe small RNA delays fruit ripening/senescence by suppressing ethylene production through silencing of the ACO gene encoding an ethylene-forming enzyme. The altered lignin production of the ccomt small RNA reduces the guanacyl (G) lignin content by inhibiting endogenous S-adenosyl-L-methionine: trans-caffeoyl CoA 3-O-methyltransferase (CCOMT gene). Furthermore, black spot blight tolerance in Solanum verrucosum can be reduced by the small RNA Ppo5 that triggers degradation of Ppo5 transcripts to prevent black spot blight development. Also included is the small RNA dvsnf7 that inhibits corn rootworm larvae with dsRNA containing a 240 bp fragment of the corn rootworm Snf7 gene. Modified starch/carbohydrates can result from small RNAs such as the small RNA pPhL (degrades PhL transcripts to limit the formation of reduced sugars through starch degradation) and the small RNA pR1 (degrades R1 transcripts to limit the formation of reduced sugars through starch degradation). Additionally, benefits such as reduced acrylamide result from the small RNA asn1 triggering degradation of Asn1 to decrease asparagine formation and reduce polyacrylamide. Finally, the non-browning phenotype of the small RNA pgas suppression results in suppression of PPO to produce apples with a non-browning phenotype. The above list of small RNAs is not intended to be limiting. Any small RNA coding sequences are covered by this description.

6. Selectable Markers

[00118] Various selectable markers also described as reporter genes can be operably linked to the Zea mays GRMZM2G144030 promoter comprising SEQ ID NO: 1, or a sequence that has 80, 85, 90, 95, or 99% sequence identity to SEQ ID NO: 1. In one embodiment, a promoter can be the Zea mays GRMZM2G144030 promoter of SEQ ID NO:1 comprising SEQ ID NO: 1, or a sequence that has at least 80, 85, 90, 95, or 99% sequence identity to SEQ ID NO: 1. In some embodiments, the sequences are operably linked to the Zea mays GRMZM2G144030 promoter comprising SEQ ID NO: 1 and a 5' UTR. The operably linked sequences can then be incorporated into a chosen vector to enable identification and selection of transformed plants ("transformants"). Many methods are available to confirm the expression of selectable markers in transformed plants, including, for example, DNA sequencing and PCR (polymerase chain reaction), Southern blotting, RNA blotting, and immunological methods for detecting a protein expressed from the vector. However, reporter genes are usually detected by visual observation of proteins that, when expressed, produce a colored product. Exemplary reporter genes are known in the art and encode β-glucuronidase (GUS), luciferase, green fluorescent protein (GFP), yellow fluorescent protein (YFP, Phi-YFP), red fluorescent protein (DsRFP, RFP, etc.), β-galactosidase, and the like (see Sambrook, et al., Molecular Cloning: A Laboratory Manual, third edition, Cold Spring Harbor Press, N.Y., 2001, the contents of which are incorporated herein in their entirety by reference).

[00119] Selectable marker genes are used for selection of transformed cells or tissues. Selectable marker genes include genes encoding antibiotic resistance, such as those encoding neomycin phosphotransferase II (NEO), spectinomycin/streptinomycin resistance (AAD), and hygromycin phosphotransferase (HPT or HGR), as well as genes conferring resistance to herbicide compounds. Herbicide resistance genes generally encode a modified target protein insensitive to the herbicide or an enzyme that degrades or detoxifies the herbicide in the plant before it acts. For example, resistance to glyphosate has been obtained through the use of genes encoding mutant target enzymes, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). The genes and mutants for EPSPS are well known, and are described in more detail below. Resistance to glufosinate ammonium, bromoxynil, and 2,4-dichlorophenoxyacetate (2,4-D) has been obtained through the use of bacterial genes encoding PAT or DSM-2, a nitrilase, an AAD-1, or an AAD-12, each of which are examples of proteins that detoxify their respective herbicides.

[00120] In one embodiment, the herbicides may inhibit the growing point or meristem, including imidazolinone or sulfonylurea, and the acetohydroxyacid synthase (AH-AS) and acetolactate synthase (ALS) resistance/tolerance genes for these herbicides are well known. Glyphosate resistance genes include the mutant 5-enolpyruvylshikimate-3-phosphate synthase (EPSPs) and dgt-28 genes (via introduction of recombinant nucleic acids and/or various forms of in vivo mutagenesis of native EPSPs genes), aroA genes, and glyphosate acetyl transferase (GAT) genes, respectively). Resistance genes for other phosphono compounds include the bar and pat genes from Streptomyces species, including Streptomyces hygroscopicus and Streptomyces viridichromogenes, and pyridinoxy or phenoxy propionic acids and cyclohexones (ACCase inhibitor encoding genes). Exemplary genes that confer resistance to cyclohexanediones and/or aryloxyphenoxypropanoic acid (including haloxy-fop, diclofop, fenoxyprop, fluazifop, quizalofop) include the acetyl coenzyme A carboxylase (ACCase) genes; Acc1-S1, Acc1-S2, and Acc1-S3. In one embodiment, the herbicides can inhibit photosynthesis, including triazine (psbA and 1s+ genes) or benzonitrile (nitrilase gene). Additionally, these selectable markers may include positive selection markers such as the enzyme phosphomannose isomerase (PMI).

[00121] In one embodiment, selectable marker genes include, but are not limited to, genes encoding: 2,4-D; neomycin phosphotransferase II; cyanamide hydratase; aspartate kinase; dihydrodipicolinate synthase; tryptophan decarboxylase; dihydrodipicolinate synthase and desensitized aspartate kinase; bar gene; tryptophan decarboxylase; neomycin phosphotransferase (NEO); hygromycin phosphotransferase (HPT or HYG); dihydrofolate reductase (DHFR); phosphinothricin acetyltransferase; 2,2-dichloropropionic acid dehalogenase; acetohydroxyacid synthase; 5-enolpyruvylshikimate phosphate synthase (aroA); haloarylnitrilase; acetyl-coenzyme A carboxylase; dihydropteroate synthase (sul I); and photosystem II polypeptide of 32 kD (psbA). One embodiment also includes selectable marker genes encoding resistance to: chloramphenicol; methotrexate; hygromycin; spectinomycin; bromoxynil; glyphosate; and phosphinothricin. The above list of selectable marker genes is not intended to be limiting. Any reporter or selectable marker gene is encompassed by the present disclosure.

[00122] In some embodiments, coding sequences are synthesized for optimal expression in a plant. For example, in one embodiment, a coding sequence of a gene has been codon optimized to enhance expression in plants. An insecticide resistance transgene, a herbicide tolerance transgene, a nitrogen use efficiency transgene, a water use efficiency transgene, a nutritional quality transgene, a DNA binding transgene, or a selectable marker transgene can be optimized for expression in a specific plant species or, alternatively, can be modified for optimal expression in dicotyledonous or monocotyledonous plants. Preferred plant codons can be determined from the highest frequency codons in the proteins expressed in the highest quantity in the specific plant species of interest. In one embodiment, a coding sequence, gene, or transgene is engineered to be expressed in plants at a higher level, resulting in greater transformation efficiency. Methods for optimizing plant genes are well known. Guidance regarding the optimization and production of synthetic DNA sequences can be found, for example, in WO2013016546, WO2011146524, WO1997013402, U.S. Patent No. 6,166,302, and U.S. Patent No. 5,380,831, which are incorporated herein by reference.

TRANSFORMATION

[00123] Suitable methods for transforming plants include any method by which DNA can be introduced into a cell, for example and without limitation: electroporation (see, e.g., U.S. Patent 5,384,253); microprojectile bombardment (see, e.g., U.S. Patents 5,015,580, 5,550,318, 5,538,880, 6,160,208, 6,399,861, and 6,403,865); Agrobacterium-mediated transformation (see, e.g., U.S. Patents 5,635,055, 5,824,877, 5,591,616; 5,981,840, and 6,384,301); and protoplast transformation (see, e.g., U.S. Patent 5,508,184).

[00124] A DNA construct can be introduced directly into the genomic DNA of the plant cell using techniques such as agitation with silicon carbide fibers (see, e.g., U.S. Patents 5,302,523 and 5,464,765), or the DNA constructs can be introduced directly into the plant tissue using biolistic methods such as DNA particle bombardment (see, e.g., Klein et al. (1987) Nature 327:70-73). Alternatively, the DNA construct can be introduced into the plant cell via nanoparticle transformation (see, e.g., U.S. Patent Publication No. 20090104700, which is incorporated in its entirety herein by reference).

[00125] Furthermore, gene transfer can be achieved using non-Agrobacterium bacteria or viruses, such as Rhizobium sp. NGR234, Sinorhizoboium meliloti, Mesorhizobium loti, potato virus X, cauliflower mosaic virus and cassava streak mosaic virus and/or tobacco mosaic virus, see, for example, Chung et al. (2006) Trends Plant Sci. 11(1):1-4.

[00126] Through the application of transformation techniques, cells of virtually any plant species can be stably transformed, and such cells can be developed into transgenic plants by well-known techniques. For example, techniques that may be particularly useful in the context of cotton transformation are described in U.S. Patent Nos. 5,846,797, 5,159,135, 5,004,863, and 6,624,344; techniques for transforming Brassica plants in particular are described, for example, in U.S. Patent No. 5,750,871; techniques for transforming soybeans are described, for example, in U.S. Patent No. 6,384,301; and techniques for transforming corn are described, for example, in U.S. Patents 7,060,876 and 5,591,616, and PCT International Publication WO 95/06722.

[00127] Following efficient delivery of an exogenous nucleic acid to a recipient cell, a transformed cell is generally identified for further cultivation and plant regeneration. In order to improve the ability to identify transformants, one may desire to employ a selectable marker gene with the transformation vector used to generate the transformant. In an illustrative embodiment, a population of transformed cells may be evaluated by exposing the cells to a selective agent or agents, or the cells may be screened for the desired marker gene trait.

[00128] Cells that survive exposure to a selective agent, or cells that have been scored positive in a screening assay, can be cultured in media that support plant regeneration. In one embodiment, any suitable plant tissue culture media can be modified by the inclusion of additional substances, such as growth regulators. The tissue can be maintained in a basic medium with growth regulators until sufficient tissue is available to begin plant regeneration efforts, or following repeated rounds of manual selection, until the tissue morphology is suitable for regeneration (e.g., at least 2 weeks), and then transferred to the conductive medium for shoot formation. Cultures are transferred periodically until sufficient shoot formation has occurred. Once shoots are formed, they are transferred to a conductive medium for shoot formation. Once sufficient roots are formed, the plants can be transferred to soil for further growth and maturity.

MOLECULAR CONFIRMATION

[00129] A transformed plant cell, callus, tissue, or plant can be identified and isolated by selecting or screening the engineered plant material for traits encoded by marker genes present in the transforming DNA. For example, selection can be accomplished by culturing the engineered plant material on media containing an inhibitory amount of the antibiotic or herbicide to which the transforming gene construct confers resistance. In addition, transformed plants and plant cells can also be identified by screening for activities of any visible marker genes (e.g., the β-glucuronidase, luciferase, or gfp genes) that may be present in the recombinant nucleic acid constructs. Such selection and screening methodologies are well known to those skilled in the art. Confirmatory molecular methods that can be used to identify transgenic plants are known to those skilled in the art. Several exemplary methods are described in more detail below.

[00130] Molecular hybridization probes ("molecular beacons") have been described for use in sequence detection. Briefly, a FRET oligonucleotide probe is designed to overlap the junction of insert and flanking genomic DNA. The unique structure of the FRET probe results in it containing a secondary structure that maintains the fluorescent and quencher moieties in close proximity. The FRET probe and PCR primers (one primer in the insert DNA sequence and one in the flanking genomic sequence) are cycled in the presence of a thermostable polymerase and dNTPs. Following successful PCR amplification, hybridization of the FRET probe(s) to the target sequence results in the removal of the probe secondary structure and spatial separation of the fluorescent and quencher moieties. A fluorescent signal indicates the presence of the flanking genomic insert/transgene sequence due to successful amplification and hybridization. Such a molecular hybridization probe assay for detection of an amplification reaction is one embodiment of the subject description.

[00131] Hydrolysis probe assay, also known as TAQMAN® (Life Technologies, Foster City, California), is a method of detecting and quantifying the presence of a DNA sequence. Briefly, a FRET oligonucleotide probe is designed with one oligo within the transgene and one in the flanking genomic sequence for the detection of specific events. The FRET probe and PCR primers (one primer in the insert DNA sequence and one in the flanking genomic sequence) are cycled in the presence of a thermostable polymerase and dNTPs. Hybridization of the FRET probe results in cleavage and release of the fluorescent moiety away from the quencher moiety on the FRET probe. A fluorescent signal indicates the presence of the genomic insert/flanking sequence due to successful amplification and hybridization. Such a hydrolysis probe assay for detection of such an amplification reaction is an embodiment of the subject description.

[00132] KASPar® assays are a method of detecting and quantifying the presence of a DNA sequence. Briefly, the genomic DNA sample comprising the integrated gene expression cassette polynucleotide is screened using a well-known polymerase chain reaction (PCR)-based assay known as the KASPar® assay system. The KASPar® assay used in the practice of the subject description may utilize a KASPar® PCR assay mixture that contains multiple primers. The primers used in the PCR assay mixture may comprise at least one forward primer and at least one reverse primer. The forward primer contains a sequence corresponding to a specific region of the DNA polynucleotide and the reverse primer contains a sequence corresponding to a specific region of the genomic sequence. Furthermore, the primers used in the PCR assay mixture may comprise at least one forward primer and at least one reverse primer. For example, the KASPar® PCR assay mixture may use two forward primers corresponding to two different alleles and one reverse primer. One of the forward primers contains a sequence corresponding to a specific region of the endogenous genomic sequence. The second forward primer contains a sequence corresponding to a specific region of the DNA polynucleotide. One reverse primer contains a sequence corresponding to a specific region of the genomic sequence. Such a KASPar® assay for detection as an amplification reaction is one embodiment of the subject description.

[00133] In some embodiments the fluorescent signal or fluorescent dye is selected from the group consisting of a HEX fluorescent dye, a FAM fluorescent dye, a JOE fluorescent dye, a TET fluorescent dye, a Cy 3 fluorescent dye, a Cy 3.5 fluorescent dye, a Cy 5 fluorescent dye, a Cy 5.5 fluorescent dye, a Cy 7 fluorescent dye, and a ROX fluorescent dye.

[00134] In other embodiments the amplification reaction is performed using appropriate second fluorescent DNA dyes that are capable of labeling cellular DNA in a concentration range detectable by flow cytometry and have a fluorescent emission spectrum that is detectable by a real-time thermal cycler. It should be appreciated by those of ordinary skill in the art that other nucleic acid dyes are known and are continually being identified. Any suitable nucleic acid dye with appropriate emission and excitation spectra may be employed, such as YO-PRO-1®, SYTOX Green®, SYBR Green I®, SYTO11®, SYTO12®, SYTO13®, BOBO®, YOYO®, and TOTO®. In one embodiment, a second fluorescent DNA dye is SYTO13® used at less than 10 μM, less than 4 μM, or less than 2.7 μM.

[00135] In additional embodiments, Next Generation Sequencing (NGS) can be used for detection. As described by Brautigma et al., 2010, DNA sequence analysis can be used to determine the nucleotide sequence of the amplified and isolated fragment. The amplified fragments can be isolated and subcloned into a vector and sequenced using the chain terminator method (also known as Sanger sequencing) or dye terminator sequencing. Additionally, the amplicon can be sequenced with Next Generation Sequencing. NGS technologies do not require the subcloning step and multiple sequencing reads can be completed in a single reaction. Three NGS platforms are commercially available, the Genome Sequencer FLX™ from 454 Life Sciences/Roche, the Illumina Genome Analyser™ from Solexa and Applied Biosystems' SOLiD™ (acronym for: "Sequencing by Oligo Ligation and Detection"). In addition, there are two single molecule sequencing methods that are currently under development. These include true Single Molecule Sequencing (tSMS) from Helicos Bioscience™ and Single Molecule Real Time™ sequencing (SMRT) from Pacific Biosciences.

[00136] Genome Sequencher FLX™ which is commercially available from 454 Life Sciences/Roche is a long-read NGS that uses emulsion PCR and pyrosequencing to generate sequencing reads. DNA fragments of 300 - 800 bp or libraries containing fragments of 3 - 20 kb can be used. Reactions can produce over a million reads of approximately 250 to 400 bases per run for a total throughput of 250 to 400 megabases. This technology produces the longest reads, but the total sequence output per run is low compared to other NGS technologies.

[00137] Illumina Genome Analyser™ which is commercialized by Solexa™ is a short read NGS that uses the sequencing-by-synthesis approach with fluorescent dye-labeled reversible terminator nucleotides and is based on solid-phase PCR. The construction of paired-end sequencing libraries containing DNA fragments up to 10 kb can be used. The reactions produce over 100 million short reads that are 35 - 76 bases in length. These can produce 3 - 6 gigabases of data per run.

[00138] The Sequencing by Oligo Ligation and Detection (SOLiD) system commercialized by Applied Biosystems™ is a short-read technology. This NGS technology uses fragmented double-stranded DNA that is up to 10 kb in length. The system uses sequencing by ligation of dye-labeled oligonucleotide primers and emulsion PCR to generate one billion short reads that result in a total sequence output of up to 30 gigabases per run.

[00139] Helicos Bioscience™ tSMS and Pacific Biosciences™ SMRT apply a different approach that utilizes single DNA molecules for sequencing reactions. The Helicos™ tSMS system produces up to 800 million short reads that yield 21 gigabases per run. These reactions are completed using fluorescent dye-labeled virtual terminator nucleotides that are described as a “sequencing by synthesis” approach.

[00140] The SMRT Next Generation Sequencing system commercialized by Pacific Biosciences™ uses real-time sequence by synthesis. This technology can produce reads up to 1,000 bp in length as a result of not being limited by reversible terminators. Raw read throughput that is equivalent to one fold coverage of a diploid human genome can be produced per day using this technology.

[00141] In another embodiment, detection can be accomplished using blotting assays, including Western blots, Northern blots, and Southern blots. Such blotting assays are techniques commonly used in biological research for the identification and quantification of biological samples. These assays include first separating sample components on gels by electrophoresis, followed by transferring the electrophoretically separated components from the gels to transfer membranes that are made of materials such as nylon, polyvinylidene fluoride (PVDF), or nitrocellulose. Analytes can also be directly spotted on these supports or targeted to specific regions on the supports by applying vacuum, capillary action, or pressure, without prior separation. Transfer membranes are commonly subjected to a treatment after transfer to increase the ability of the analytes to be distinguished from each other and detected, either visually or by automated readers.

[00142] In a further embodiment detection may be accomplished using an ELISA assay, which uses a solid phase enzyme immunoassay to detect the presence of a substance, usually an antigen, in a liquid sample or wet sample. Antigens from the sample are bound to a surface of a plate. An additional specific antibody is then applied to the surface so that it can bind to the antigen. This antibody is bound to an enzyme and, in the final step, a substance containing the enzyme's substrate is added. The subsequent reaction produces a detectable signal, most commonly a color change in the substrate.

TRANSGENIC PLANTS

[00143] In one embodiment, a plant, plant tissue, or plant cell comprises a Zea mays GRMZM2G144030 promoter. In one embodiment, a plant, plant tissue, or plant cell comprises the Zea mays GRMZM2G144030 promoter of a sequence selected from SEQ ID NO:1, or a sequence of at least 80%, 85%, 90%, 95%, or 99.5% sequence identity to a sequence selected from SEQ ID NO:1. In one embodiment, a plant, plant tissue, or plant cell comprises the 3' UTR of GRMZM2G144030 from Zea mays comprises in a sequence selected from SEQ ID NO:5 or a sequence of at least 80%, 85%, 90%, 95%, or 99.5% sequence identity to a sequence selected from SEQ ID NO:5. In another embodiment, a plant, plant tissue, or plant cell comprises the Zea mays GRMZM2G144030 promoter of SEQ ID NO:1 operatively linked to a 5' UTR. In one embodiment, a plant, plant tissue, or plant cell comprises a gene expression cassette comprising a sequence selected from SEQ ID NO:1, or a sequence of at least 80%, 85%, 90%, 95%, or 99.5% sequence identity to a sequence selected from SEQ ID NO:1 operatively linked to a non-GRMZM2G144030 transgene. In an illustrative embodiment, a plant, plant tissue, or plant cell comprises a gene expression cassette that includes a Zea mays GRMZM2G144030 promoter operably linked to a transgene, wherein the transgene can be an insecticide resistance transgene, a herbicide tolerance transgene, a nitrogen use efficiency transgene, a water use efficiency transgene, a nutritional quality transgene, a DNA binding transgene, a selectable marker transgene, or combinations thereof.

[00144] According to one embodiment a plant, plant tissue or plant cell is provided wherein the plant, plant tissue or plant cell comprises a non-endogenous GRMZM2G144030 gene-derived promoter sequence operably linked to a transgene, wherein the Zea mays GRMZM2G144030 promoter-derived promoter sequence comprises a sequence of SEQ ID NO:1 or a sequence having at least 80%, 85%, 90%, 95% or 99.5% sequence identity to SEQ ID NO:1. In one embodiment, a plant, plant tissue, or plant cell is provided wherein the plant, plant tissue, or plant cell comprises SEQ ID NO: 1, or a sequence that has at least 80%, 85%, 90%, 95%, or 99.5% sequence identity to SEQ ID NO: 1 operably linked to a non-GRMZM2G144030 transgene. In one embodiment, the plant, plant tissue, or plant cell is a dicotyledonous or monocotyledonous plant or a cell or tissue derived from a dicotyledonous or monocotyledonous plant. In one embodiment, the plant is selected from the group consisting of corn, rice, sorghum, oats, rye, bananas, sugarcane, cotton, sunflower, and canola. In one embodiment, the plant is soybean. According to one embodiment the plant, plant tissue or plant cell comprises SEQ ID NO: 1 or a sequence having 80%, 85%, 90%, 95% or 99.5% sequence identity to SEQ ID NO: 1 operatively linked to a non-GRMZM2G144030 transgene. In one embodiment the plant, plant tissue or plant cell comprises a promoter operatively linked to a transgene wherein the promoter consists of SEQ ID NO: 1 or a sequence having 80%, 85%, 90%, 99.5% or 95% sequence identity to SEQ ID NO: 1. According to one embodiment the gene construct comprising Zea mays GRMZM2G144030 promoter sequence operatively linked to a transgene is incorporated into the genome of the plant, plant tissue or plant cell.

[00145] In one embodiment a non-Zea mays c.v. B73 plant, plant tissue or plant cell is provided that comprises SEQ ID NO: 1, or a sequence that has at least 80%, 85%, 90%, 95% or 99.5% sequence identity to SEQ ID NO: 1, operably linked to a transgene. According to one embodiment the non-Zea mays c.v. B73 plant, plant tissue or plant cell is a dicotyledonous or monocotyledonous plant or plant cell or tissue derived from a dicotyledonous or monocotyledonous plant. In one embodiment, the plant is selected from the group consisting of corn, rice, sorghum, oats, rye, bananas, sugarcane, cotton, sunflower, and canola. In one embodiment, the plant is soybean. According to one embodiment the promoter sequence operably linked to a transgene is incorporated into the genome of the plant, plant tissue or plant cell.

[00146] In one embodiment a non-Zea mays c.v. B73 plant, plant tissue or plant cell is provided that comprises SEQ ID NO: 1, or a sequence that has at least 80%, 85%, 90%, 95% or 99.5% sequence identity to SEQ ID NO:1, operatively linked to the 5' end of a transgene and a 3' untranslated sequence comprising SEQ ID NO:5 or a sequence that has at least 80%, 85%, 90%, 95% or 99.5% sequence identity to SEQ ID NO:5, wherein the 3' sequence is not operatively linked to said transgene. In another embodiment a non-Zea mays c.v. B73 is provided comprising SEQ ID NO: 1, or a sequence having at least 80%, 85%, 90%, 95% or 99.5% sequence identity to SEQ ID NO: 1, operably linked to the 3' end of a 5' untranslated sequence, wherein the 5' untranslated sequence is operably linked to said transgene. According to one embodiment the non-Zea mays c.v. B73 plant, plant tissue or plant cell is a dicotyledonous or monocotyledonous plant or is a plant cell or tissue derived from a dicotyledonous or monocotyledonous plant. In one embodiment, the plant is selected from the group consisting of corn, rice, sorghum, oats, rye, bananas, sugarcane, cotton, sunflower and canola. In one embodiment, the plant is soybean. According to one embodiment the promoter sequence operably linked to a transgene is incorporated into the genome of the plant, plant tissue or plant cell.

[00147] In one embodiment, a plant, plant tissue, or plant cell according to the methods described herein may be a monocotyledonous plant. The monocotyledonous plant, plant tissue, or plant cell may be, but is not limited to, corn, rice, wheat, sugarcane, barley, rye, sorghum, orchids, bamboo, bananas, cattails, lilies, oats, onions, millet, switchgrass, turf, and triticale.

[00148] In one embodiment, a plant, plant tissue, or plant cell according to the methods described herein may be a dicotyledonous plant. The dicotyledonous plant, plant tissue, or plant cell may be, but is not limited to, alfalfa, rapeseed, canola, Indian mustard, Ethiopian mustard, soybean, sunflower, cotton, beans, broccoli, cabbage, cauliflower, celery, cucumber, eggplant, lettuce; melon, pea, pepper, peanut, potato, pumpkin, radish, spinach, sugar beet, sunflower, tobacco, tomato, and watermelon.

[00149] One skilled in the art will recognize that after the stable exogenous sequence is stably incorporated into transgenic plants and confirmed to be operable, it can be introduced into other plants by sexual crossing. Any of a number of standard breeding techniques can be used, depending on the species to be crossed.

[00150] The present description also encompasses seeds of the transgenic plants described above, wherein the seeds have the transgene or gene construct containing the gene regulatory elements of the subject description. The present description further encompasses progeny, clones, cell lines or cells of the transgenic plants described above, wherein said progeny, clone, cell line or cell has the transgene or gene construct containing the gene regulatory elements of the subject description.

[00151] The present description also encompasses the cultivation of transgenic plants described above, wherein the transgenic plants have the transgene or gene construct containing the gene regulatory elements of the subject description. Accordingly, such transgenic plants may be engineered to, inter alia, have one or more desired traits or transgenic events containing the gene regulatory elements of the subject description, be transformed with nucleic acid molecules according to the invention and may be harvested or cultivated by any method known to those skilled in the art.

METHOD OF EXPRESSION OF A TRANSGENE

[00152] In one embodiment, a method of expressing at least one transgene in a plant comprises culturing a plant comprising a Zea mays GRMZM2G144030 promoter operatively linked to at least one transgene or a polylinker sequence. In one embodiment, a method of expressing at least one transgene in a plant comprises culturing a plant comprising the Zea mays GRMZM2G144030 promoter and a Zea mays GRMZM2G144030 5' UTR operatively linked to at least one transgene or a polylinker sequence. In one embodiment, a method of expressing at least one transgene in a plant comprises culturing a plant comprising a Zea mays GRMZM2G144030 3' UTR operatively linked to at least one transgene or a polylinker sequence. In one embodiment, the Zea mays GRMZM2G144030 promoter consists of a sequence selected from SEQ ID NO:1, or a sequence of at least 80%, 85%, 90%, 95%, or 99.5% sequence identity to a sequence selected from SEQ ID NO:1. In another embodiment, the Zea mays GRMZM2G144030 3' UTR consists of a sequence selected from SEQ ID NO:5, or a sequence of at least 80%, 85%, 90%, 95%, or 99.5% sequence identity to a sequence selected from SEQ ID NO:5. In one embodiment, a method of expressing at least one transgene in a plant comprises culturing a plant comprising the Zea mays GRMZM2G144030 promoter and a Zea mays GRMZM2G144030 3' UTR operatively linked to at least one transgene. In one embodiment, a method of expressing at least one transgene in a plant comprises culturing a plant comprising the Zea mays GRMZM2G144030 promoter and a Zea mays GRMZM2G144030 5' UTR operatively linked to at least one transgene. In one embodiment, a method of expressing at least one transgene in a plant tissue or plant cell comprises culturing a plant tissue or plant cell comprising a Zea mays GRMZM2G144030 promoter operatively linked to at least one transgene. In one embodiment, a method of expressing at least one transgene in a plant tissue or plant cell comprises culturing a plant tissue or plant cell comprising a Zea mays GRMZM2G144030 promoter and a Zea mays GRMZM2G144030 3' UTR operatively linked to at least one transgene. In one embodiment, a method of expressing at least one transgene in a plant tissue or plant cell comprises culturing a plant tissue or plant cell comprising a Zea mays GRMZM2G144030 promoter and a Zea mays GRMZM2G144030 5' UTR operatively linked to at least one transgene. In one embodiment, a method of expressing at least one transgene in a plant tissue or plant cell comprises culturing a plant tissue or plant cell comprising a Zea mays GRMZM2G144030 promoter and a Zea mays GRMZM2G144030 3' UTR operatively linked to at least one transgene. In one embodiment, a method of expressing at least one transgene in a plant tissue or plant cell comprises culturing a plant tissue or plant cell comprising a Zea mays GRMZM2G144030 promoter and a Zea mays GRMZM2G144030 5' UTR operatively linked to at least one transgene.

[00153] In one embodiment, a method of expressing at least one transgene in a plant comprises cultivating a plant comprising a gene expression cassette comprising a Zea mays GRMZM2G144030 promoter operably linked to at least one transgene. In one embodiment, the Zea mays GRMZM2G144030 promoter consists of a sequence selected from SEQ ID NO:1, or a sequence of at least 80%, 85%, 90%, 95%, or 99.5% sequence identity to a sequence selected from SEQ ID NO:1. In another embodiment, the 3' UTR of Zea mays GRMZM2G144030 consists of a sequence selected from SEQ ID NO:5, or a sequence of at least 80%, 85%, 90%, 95%, or 99.5% sequence identity to a sequence selected from SEQ ID NO:5. In one embodiment, a method of expressing at least one transgene in a plant comprises culturing a plant comprising a gene expression cassette comprising a Zea mays GRMZM2G144030 promoter and a Zea mays GRMZM2G144030 3' UTR operatively linked to at least one transgene. In one embodiment, a method of expressing at least one transgene in a plant comprises culturing a plant comprising a gene expression cassette comprising a Zea mays GRMZM2G144030 promoter and a Zea mays GRMZM2G144030 5' UTR operatively linked to at least one transgene. In one embodiment, a method of expressing at least one transgene in a plant comprises culturing a plant comprising a gene expression cassette comprising a Zea mays GRMZM2G144030 3' UTR operatively linked to at least one transgene. In one embodiment, a method of expressing at least one transgene in a plant comprises culturing a plant comprising a gene expression cassette comprising a Zea mays GRMZM2G144030 5' UTR operatively linked to at least one transgene. In one embodiment, a method of expressing at least one transgene in a plant tissue or plant cell comprises culturing a plant tissue or plant cell comprising a gene expression cassette containing a Zea mays GRMZM2G144030 promoter operatively linked to at least one transgene. In one embodiment, a method of expressing at least one transgene in a plant tissue or plant cell comprises culturing a plant tissue or plant cell comprising a gene expression cassette containing a Zea mays GRMZM2G144030 3' UTR operatively linked to at least one transgene. In one embodiment, a method of expressing at least one transgene in a plant tissue or plant cell comprises culturing a plant tissue or plant cell comprising a gene expression cassette containing a Zea mays GRMZM2G144030 5' UTR operatively linked to at least one transgene. In one embodiment, a method of expressing at least one transgene in a plant tissue or plant cell comprises culturing a plant tissue or plant cell comprising a gene expression cassette, a Zea mays GRMZM2G144030 promoter, and a Zea mays GRMZM2G144030 3' UTR operatively linked to at least one transgene. In one embodiment, a method of expressing at least one transgene in a plant tissue or plant cell comprises culturing a plant tissue or plant cell comprising a gene expression cassette, a Zea mays GRMZM2G144030 promoter, and a Zea mays GRMZM2G144030 5' UTR operatively linked to the at least one transgene.

[00154] The following examples are provided to illustrate certain particulars and/or embodiments. The examples should not be construed to limit the description to the particulars or embodiments exemplified.

EXAMPLES EXAMPLE 1: ISOLATION OF INNOVATIVE PROMOTER AND OTHER REGULATORY ELEMENTS

[00155] Novel gene regulatory elements of GRMZM2G144030 from Zea mays were identified through publicly available transcriptome analysis of maize seedlings. These regulatory elements were identified, isolated and cloned to characterize the expression profile of the regulatory elements for use in transgenic plants. Stable transgenic maize lines transformed with a cry3Ab1 gene isolated from Bacillus thuringiensis and an aad-1 selectable marker gene derived from Sphingobiumherbicidovorans were produced and transgene expression levels and tissue specificity were assessed. As such, novel gene regulatory elements of GRMZM2G144030 from Zea mays were identified and characterized. Promoter and 3' UTR regulatory elements for use in gene expression constructs are described.

[00156] Three data sources were considered to prioritize highly expressed maize genes in maize seedling shoots and roots: 1) 35,000 maize gene sequences and their annotations present in the public maize database from 2010, when this study was conducted; 2) gene expression data for whole maize transcriptome for V4 shoots and roots (Wang, et al., [2009], The Plant Cell; 21: 1053-1069); and, 3) full-length cDNA sequences of 9,000 genes (Alexandrov, N. et al., [2009]Plant Molecular Biology; 69:179-194). In this study, gene expression data were aligned to both the 9,000 full-length cDNA sequences and the 35,000 maize genes. Based on Fragment Mapped Kilobase exon per Million (FPKM) values, a quantitative measure of gene expression, 500 best expressed genes were identified for each of the maize shoot and root tissues.

[00157] Since transgene expression requires transgene expression throughout the life cycle of a corn plant, total mRNA was isolated from 3 different leaf stages (V4, V12, and R3), 2 different root developmental stages (V4 and V12), and one pollen stage (R1) for transgene expression analysis. The corn genotype Zea mays c.v. B73 was used for all analyses. Of the 500 prioritized genes, approximately 150 of the best expressing genes were subjected to quantitative PCR for confirmation of gene expression. The results identified a subset of these as the best expressing genes for preferential root and leaf expression.

[00158] The Zea mays GRMZM2G144030 gene regulatory element promoter (SEQ ID NO:1) is a 2,082 bp polynucleotide sequence that was identified from the genomic DNA (gDNA) sequence of Zea mays c.v. B73. From evaluation of contiguous chromosomal sequence spanning millions of base pairs, a 2,082 bp polynucleotide sequence was identified and isolated, by PCR amplification or DNA synthesis, for use in the expression of heterologous coding sequences. This novel polynucleotide sequence was analyzed for use as a regulatory sequence to direct the expression of a gene. As shown in the sequence (SEQ ID NO:2) below, the 2,082 bp Zea mays GRMZM2G144030 promoter of SEQ ID NO:1 is provided as base pairs 1 - 2,082. The native gene coding sequence of SEQ ID NO:4 is provided as base pairs 2,083 - 4,611 of SEQ ID NO:2 (the ATG start codon and the TAA stop codon are shown in capital letters). The 1,095 bp Zea mays GRMZM2G144030 3' UTR of SEQ ID NO:5 is provided as base pairs 4,612 - 5,706 of SEQ ID NO:2. ttgtcacatcgttaattcgttaggtctacatcacgctaagggttgatttggtgacaaggggatcccagga ggattggaggggattgagggtccgatcctcccgggatcccgggtcaccaaatcagctctaagggtc tgttcattttgtttcaaatctacatggattgtggggtatgaaacgaaggaggcctaaagaaccttt gacc tgacattttccccactttgatctcacgtatatgcagtactgcaatgaggtcgcaaagaagctgatcaat ggagcagatcatagtttcgttgagcctgactcagactacagattttgacctgaagcaggtcattacttc gttgggtctagctaggactaaagattttgacgtgacaaaattttcactttaatccgatgag tgagatcat agagaagctgatcaatgggctagatcatcacttcattgcgtctagctgggagtaaagattttgatgtga catttgctttcgctttgatttggtcggtgggtacttcgtacggctggctattgcttcaggttaaagtcgattgtt tagattgttacatttataatttataaacaaaatattgtagaattgataaattagtacgtattaa agct aaacgagcaaacaaagattttgatctgttgttcgcttgactttaatccatactgatttttactatttctataaa cttcaatctgattagtggatacttcgtcttttgcttatcgaatggctacttcgctgggtctggctccaactag agggtgtttaggatggctcaaggtttcagttccagtatgaagctatattttatatca atttaattagttttaag aatatttttaatataataaataaagtgacttatctaaatgcattctaaaggcttacaatttcaactcc acatttttagagcatctaatgacctattctactaacttcactaaattttctagagaaatagctatcctaaac agaatataaagattgcagcgggtatatgcctgtatattattttttgtgtattgtatttatgctccagctctg c acataagatgtataaaaaactattagggctgatttggtgaccggggatctcgaggggaagaaatcct cttgctattcaaaattgaattg caag aggatttcttcccatg g atcctctag ag atccctaatcaccaaat caaccctgatagtgagtcttggttgtgtattgaacgagatatgctaatatatttaaaacaagtattataaa ctagccttcgcctttctgacctttaaaattgatactaaacttacaaccacatactacgatgctataagga aaacttcag gtgggggtgttgcaatccaag aatagtg cgttctcacg acgaatctctcaatctg atcg gatcatcggaataagaggttcgaaaacgttcccgcacgattgttttcttatcgctgat cagcaaaggtg acgctgaaaagaaaatgaaaagtcttcgataagaaaaggaacgcaaggtcgcaagcaaagaa aaaaaaaggaaggcgagccaagggaggaggaaagatgcttttcagccgtcgatagcgtgattc aaacgcgagaagatgccttcctctgcagccgtacacgcgtccgatcgcccgacaaaaccgacggt tcagatacgctcaacatatgcacctgaaggcgtccaaccgacatgcgggcccgttttccagcaagc cccaagagtcagtgaaacacaccgtcttctctcgggttggaagcggtgggtgcacct gctgttagcg gtataaaaagcggaaaccctagcattcgccgcgagcttatcacttatcatccccatccgcacctctct ccatctccctg ctcctccg cctccttcttg atccag ccaagccag gttaaattctg acccgtcg ccgtca gatttacttccgaattcagttgatctggtttcagatttgtaatttctcttgtttcctaaaatacggggaatccg aatcgaggctaattttcggatcttgttgtttgtttctttttttttttgcagggatcgccATGtcggactcgga ggagcaccacttcgaatcgaaggccgacgccggagc gtccaagacctacccgcagcaggctgg caccgtccgtaagaacggcttcatcgtcatcaagaaccgcccctgcaaggtatatgtgtgcgcagttt tgggatcggctgtattccattgattagtgttgacccgtgtcgttgtgctgtgaactggtgatatcgagatgc agttgtctttggtcatactgtgatagtactgttaagttttgagggttgatccaatctatggaggtggaatttgt gggacttggatgctagatatacctggtggctagaaggagactttgctattgccagccggagttatatta caagtaggcttttacagaataacagttcatctgatctgactttctggagaccagaaaaatacatttaa ggcct gtttgatttgctgcctaacttgccacactttgggtaactttttcagcctatggttagttcttcaattcga gcgactaaccttagtcaaagtgtggcacagttagtcacgaaccaaacatgcccttagtcagagtag gttgcaccctg cccatataagtag ctgaaatag caaatg g gtg caatgc gggaaaactagttggcatgttgagttaattctttactcctctaaacttgtcattgaacattcaagggtactc attatttatctttg atgatg gtctg aatgtttgtaccccatgttctatg gtgatattgtccaagtg atcagaca atttatcattttaagtttcaacagagggttatccaatttccattattccaagataacgggaataagagat c acacagataatgtgttttaaggaaaataaaatacttttatctttgtcagtctgtcatacaactaaactta atgttgctgactcagtgtgatgttttcttgggagctataagctgtttaatgatgtttgaaatcaataaaaaa actaaaataacatatgccctacattttggacagagttttgatttgaccccacaatg attgtaaaacattgt tctttattattgattttttaatgagttgggttgtaattgggaagttatatttgaatattttctgtttatcaataaaac cagtttgttgtcaaaatgaagtttcttttggtctggcaatccattgacaaagagaatgtgagtacgtactt gttttgcaattaccaaacaggttatctcttctttaattctaaacctcgaaaatt ttctatgtggtattgccata ccgtattttattttaggctgtcactgtattatcataaggtgatagtaagctcatttctgtttattaacccttctatt ttatgaacttctgatggatttgagtgatatatcagcaacagttcatgatgctgtccctaaaactatttgga actttattgctcttaagtcgcagcacatacgtcgctgttttattgagtttgtttgcggaatatggttgataaaa acaacttatgctacaggttgtggaggtttctacctcaaagactggtaagcatggccatgccaaatgcc actttgttgccatagacatattcaacgggaaaagctcgaagatattgttccttcg tcacacaactgtg atgtaagttaatttctgtgttaag ctaagttcttttgatg gttcacttcatacg ccactcacaagtttg acattt ttttaagattccgcatgtgaaccgtactgagtaccagctgattgatatatcagaggatggatttgtatgtt cctctaatcattaaaactacattcttctttgaaataaatgttaacttcag tcttatattttaatttaatttgtttca ggtgagccttcttacttcagatggcaacactaaggatgatcttagactcccaactgatgagactcttgt ggcccag gtctgtttg aaaacatttttcatcttaagtcttgttg gttttcttattg acttccaaatattacag at ggtggttatctttgttag cttattagtcatatgttctttcttg gtag ctt ctcttccttgatgtttgttaattg atctg g tgcttataaggaatgtggaaagtagggccttgtagaactagacattccttatgtatttagactgtctacc attctttttaaaaatgatatgacataacaaccatgtcatataaaacatgctgaaactgcactaaaattg gttgctttaaaactgaaacgtatgcatgacctgcactttgtatgttgtctaggatacaatttatgttgattgg aagtagtatgctgcataacttatgtctgttggagagttaatttatctacctcaagtggctgcccaaactaa ataatacttctg catcttgtatgcttg gtaattcag atcaag g aagggtttg aaag c ggcaaggatcttg ttgtgactgtccagtctgctatgggggaggagcagatctgcgcgctgaaggatgttggccccaagT AActtccctgccttggaatactgtatctcaaaacctaaatcgaaaaaagaagtgtatcaaggattgct acagagacatccatctggcttgagctggcttttgctatggcaaacacaagtgcagggatcgctgggt gttgcaccgtgtctgcattatgtggttacattctggaccctgtattttctatgcttttatgcccctactgtttagt attaattatcaataaatttgtttgggacggttgaattgcatgcattgcatgaggtttctt gttcgttatttgttttc tcttgccatgcttccagtattggtgatggcagtttactcgtaattttttttgtacttgtttcgaaaactggtggg cagtttacccgcgcttgtgctccatccattgttctagtgttatccattcctcttttttcccccttcaaaatataa actataaagaagaaaatattatgacttacataaagaatcgttttagagaagcggtaccaaacatgac tcgacggaaattgctccaaaccaactattaactcgatgtttgtattttttttcttctctgccaaaacctgtc aacccgctttatggtaactttgcggtatatgttctcgaaagaaaactggtgtatagagctactcccctaa tttcaagttgtaagacattttgcccatttgatatatgtaggtagaaaagatgtcttgtaatctaaaacaga gggagtatttatgtagctttttgtagataaaatgtcttatttaaaacagagggagtatttgagtagctttttg acgattgaatgctgatttggacggagggagctacttgtgtagctttttggaacgaagt acttgagtggttt tttgaggattgatttctgtaaataaaatgtacttgacccctattataggtgtacatctggctgtgtcgggctt ggttggcatgacataatgcgtggatcgtgtctaggtcttaagcccacagattggtatggtatgacttattt agttaaggcccattaaagcctgctaaacataaacttgttgcgcagtgtgcttg tgttgtgtgggcttttatc actgtatttgcatttcgt (SEQ ID NO:2)

EXAMPLE 2: VECTOR CONSTRUCTION

[00159] The following vectors were constructed to incorporate the Zea mays GRMZM2G144030 promoter downstream of a transgene. The vector construct pDAB113002 contained a gene expression cassette in which the cry34ab1 transgene (B. thuringiensis reporter gene) was driven by the Zea mays GRMZM2G144030 promoter of SEQ ID NO:1 and was flanked by the 3' UTR of Zea mays GRMZM2G144030 of SEQ ID NO:5. A diagram of this gene expression cassette is shown in Figure 1 and is provided as SEQ ID NO:3. The vector also contained a selectable marker gene expression cassette that contained the aad-1 transgene (U.S. Pat. No. 7,838,733) driven by the Zea mays Ubiquitin 1 promoter (Christensen et al., [1992] Plant Molecular Biology 18;675-689) and was terminated by the Zea mays Lipase 3'-UTR (U.S. Pat. No. 7,179,902). A diagram of this gene expression cassette is shown in Figure 1 and is provided as SEQ ID NO:15.

[00160] This construct was constructed by synthesizing the newly designed Zea mays GRMZM2G144030 promoter and Zea mays GRMZM2G144030 3' UTR sequences by an external provider (Geneart via Life Technologies, Carlsbad, CA) and cloning the promoter into a Gateway™ donor vector (Life Technologies) using GeneArt® Seamless Cloning and Assembly Kit (Life technologies) and restriction enzymes. The resulting donor vector was integrated into a final binary destination vector using the Gateway™ cloning system (Life Technologies). Clones of pDAB113002 were obtained and confirmed by sequencing and restriction enzyme digestions. The resulting construct contained a promoter that could robustly drive expression of a transgene that was operably linked to the 3' end of the promoter.

[00161] A control construct, pDAB101556, was assembled containing the Phi-yfp transgene (Shagin et al., [2004] Mol Biol Evol 21; 841-50) driven by the Zea mays Ubiquitin-1 Promoter (Christensen et al., [1992] Plant MolecularBiology 18; 675-689) and Zea mays Peroxidase5 3'UTR regulatory elements. This control construct contained the same aad-1 expression cassette as that present in pDAB113002. This control construct was transformed into plants using the same reagents and protocols as those for pDAB113002.

EXAMPLE 3: CORN TRANSFORMATION Transformation of Agrobacterium tumefaciens:

[00162] The binary expression vector was transformed into Agrobacterium tumefacien strain DAt13192 (Re-cA deficient ternary strain) (Int. Pub. Pat. No. WO2012016222). Bacterial colonies were selected and binary plasmid DNA was isolated and confirmed by restriction enzyme digestion.

Agrobacterium Culture Initiation:

[00163] Agrobacterium cultures were streaked from glycerol stocks onto AB minimal medium (Gelvin, S., 2006, Agrobacterium Virulence Gene Induction, in Wang, K., ed. Agrobacterium Protocols Second Edition Vol. 1, Humana Press, p. 79; made without sucrose and with 5 g/L glucose and 15 g/L Bacto™ Agar) and incubated at 20 °C in the dark for 3 days. Agrobacterium cultures were then streaked onto a YEP medium plate (Gelvin, S., 2006, Agrobacterium Virulence Geneinduction, in Wang, K., ed. Agrobacterium Protocols Second Edition Vol. 1, Humana Press, p. 79) and incubated at 20 °C in the dark for 1 day.

[00164] On the day of an experiment, a mixture of inoculation medium (2.2 g/L MS salts, 68.4 g/L sucrose, 36 g/L glucose, 115 mg/L L-proline, 2 mg/L glycine, 100 mg/L Myo-Inositol, 0.05 mg/L nicotinic acid, 0.5 mg/L pyridoxine HCl, 0.5 mg/L thiamine HCl) and acetosyringone was prepared in a volume appropriate for the size of the experiment. A 1 M stock solution of acetosyringone in 100% dimethyl sulfoxide was added to the inoculation medium, to make a final acetosyringone concentration of 200 μM.

[00165] For each construct, 1-2 cycles of Agrobacterium from the YEP plate were suspended in 15 ml of the acetosyringone/inoculation medium mixture in a sterile, disposable 50 ml centrifuge tube and the optical density of the solution at 600 nm (O.D.600) was measured on a spectrophotometer. The suspension was then diluted to 0.25-0.35 O.D.600 using additional acetosyringone/inoculation medium mixture. The tube of Agrobacterium suspension was then placed horizontally on a platform shaker set to approximately 75 rpm at room temperature for between 1 and 4 hours prior to use.

Corn Transformation:

[00166] The experimental constructs were transformed into maize by Agrobacterium-mediated transformation of immature embryos isolated from the inbred line, Zea mays c.v. B104. The method used is similar to those published by Ishida et al., (1996) Nature Bio- technol; 14:745-750 and Frame et al., (2006) Plant CellRep; 25:10241034, but with several modifications and improvements to make the method amenable to high-throughput transformation. An example of a method used to produce a number of transgenic events in maize is given in U.S. Public Patent Application No. 2013/0157369 A1, beginning with the embryo infection and co-cultivation steps.

[00167] Putative T0 transgenic seedlings were transplanted from Phytatrays™ (Sigma-Aldrich; St. Louis, MO) into small 3.5" plastic pots (T. O. Plastics; Clearwater, MN) filled with growing medium (Premix BX; Premier Tech Horticulture), covered with humidomes (Arco Plastics Ltd.), and then hardened off in a growth room (28 °C day/24 °C night, 16-h photoperiod, 50-70% RH, 200 μEm-2 sec-1 light intensity). When plants reached the V3-V4 stage of development (3-4 visible leaf collars), they were transplanted into Sunshine Custom Blend 160™ soil mix and grown to flowering in the greenhouse (Light exposure type: photo or assimilation; High light threshold: 1200 PAR; duration 16-h day; 27°C day/24°C night). Plants were analyzed for transgene copy number by qPCR assays using primers designed to detect the relative copy numbers of the transgenes, and putative single-copy events selected for advancement were transplanted to 5-gallon pots.

EXAMPLE 4: MOLECULAR CONFIRMATION OF GENE COPY NUMBER AND PROTEIN EXPRESSION Presence of transgene and copy number estimate:

[00168] Corn plants were sampled at the V2-3 leaf stage and screened for transgene presence and copy number using cry34Ab1 and aad-1 quantitative PCR assays. Total DNA was extracted from leaf samples using the Qiagen™ MagAttract DNA Extraction kit™ as per the manufacturer's instructions.

[00169] The DNA fragments were then amplified with TaqMan® primer/probe sets containing a FAM-labeled fluorescent probe for the cry34Ab1 gene and a HEX-labeled fluorescent probe for the endogenous invertase reference gene. The following primers were used for the cry34Ab1 and invertase gene amplifications. Cry34Ab1 Primers/Probes: SEQ ID NO:6 (TQ.8v6.1.F): GCCATACCCTCCAGTTG SEQ ID NO:7 (TQ.8v6.1.R): GCCGTTGATGGAGTAGTAGATGG Probe: SEQ ID NO:8 (TQ.8v6.1.MGB.P): 5'-FAM/ CCGAATCCAACGGCTTCA / MGB/-3' Invertase Primers : SEQ ID NO:9 (InvertaseF): TGGCGGACGACGACTTGT SEQ ID NO:10 (InvertaseR): AAAGTTTGGAGGCTGCCGT SEQ ID NO:11 (InvertaseProbe): 5'-HEX/CGA GCA GAC CGC CGT GTA CTT /3BHQ_1/ -3'

[00170] PCR reactions were performed in a final volume of 10 μl containing 5 μl of Roche LightCycler 480 Probes Master Mix™ (Roche Applied Sciences, Indianapolis, IN; Catalog 04887301001); 0.4 μl each of the primers TQ.8v6.1.F, TQ.8v6.1.R, InvertaseF, and InvertaseR from 10 μM stocks to a final concentration of 400 nM; 0.4 μl each of the probes, TQ.8v6.1.MGB.P and InvertaseProbe, from 5 μM stocks to a final concentration of 200 nM, 0.1 μl of 10% polyvinylpyrrolidone (PVP) at a final concentration of 0.1%; 2 μl of 10 ng/μl genomic DNA and 0.5 μl water. DNA was amplified on a Roche LightCycler 480 System™ under the following conditions: 1 cycle of 95 °C for 10 min; 40 cycles of the following 3 steps: 95 °C for 10 seconds; 58 °C for 35 seconds and 72 °C for 1 second and a final cycle of 4 °C for 10 seconds. The cry34Ab1 copy number was determined by comparing Reference/Target values for the unknown samples (emitted by LightCycler 480) to reference target values of cry34Ab1 copy number controls.

[00171] Aad-1 gene detection was performed as described above for the cry34Ab1 gene using the endogenous reference gene invertase. The Aad-1 primer sequences were as follows; the PCR cycles remain the same: SEQ ID NO:12 (AAD1 Forward Primer): TGTTCGGTTCCCTCTACCAA SEQ ID NO:13 (AAD1 Reverse Primer): CAACATCCATCACCTTGACTGA SEQ ID NO:14 (AAD1 Probe): 5'- FAM/CACAGAACCGTCGCTTCAGCAACA-MGB/BHQ3'

T0 plants selected for transgene expression:

[00172] T0 plants containing cry34Ab1 and aad-1 transgenes and growing in the greenhouse were sampled at the V45 developmental stage for the leaf ELISA assay. Four leaf holes were sampled. Protein extracts for ELISA assays were prepared by adding a 1/8" stainless steel microsphere (Hoover Precision Products, Cumming, GA, USA) to each 1.2 ml tube containing foil holes and 300 μl of extraction buffer (1X PBST [Fischer Scientific, St. Louis, MO] supplemented with 0.05% Tween 20 and 0.5% BSA). Samples were processed in a Genogrinder™ (SPEX SamplePrep, Metuchen, NJ) at 1,500 rpm for 4 min. Samples were centrifuged at 4,000 rpm for 2 min in a Sorvall Legend XFR™ centrifuge. After this step, an additional 300 μl of extraction buffer was added to the samples and they were processed once more in the Genogrinder™ at 1,500 rpm for 2 min. Samples were centrifuged at 4,000 rpm for 7 minutes. The supernatant was collected and ELISA completed at different dilutions along with Cry34Ab1 and AAD-1 protein standards. Cry34Ab1 (Agdia, Inc.; Cat No 04500/4800) and AAD- 1 (Acadia BioScience, LLC; Cat No ABS-041) ELISA assays were performed as per manufacturer's instructions and ELISA results were expressed as ng/cm2 of leaf surface area or as parts per million (or ng of target protein per mg of total plant protein).

[00173] Another set of plants were sampled at V4-5 for the entire root mass. Samples were immediately frozen and freeze-dried for one week and then ground. ELISA was performed as described above for leaf samples. Total root protein estimation was performed with the Bradford detection method as per the manufacturer's instructions (Thermo Scientific/Pierce, USA). Root ELISA results were expressed in parts per million (or ng target protein per mg total plant protein).

T1 plant selected for gene expression and gene expression detection:

[00174] T0 plants were backcrossed with Zea mays c.v. B104 to obtain T1 seeds. Three to five T1 lines (or events) of each of the regulatory element constructs were advanced for protein expression studies. Approximately forty T1 seeds of each of the events were sown, seedlings at the V2-3 development stage were sprayed with assure® II to eliminate null plants. All surviving plants were sampled for transgene copy number assays, which were performed as described above.

[00175] For transgene gene expression analysis, plants were sampled at multiple growth and developmental stages as follows: leaf (V4, V12, and R3); root (V4); stem, pollen, silk (all at R1), and seed and spike (all at R3). All tissues were sampled in tubes embedded in dry ice; which were transferred to -80 °C immediately after sampling was completed. Frozen tissues were lyophilized prior to protein extraction for ELISA.

[00176] Protein extraction for leaf ELISA was performed as described for the T0 samples, as described in the previous section. For example, protein extraction tubes for various tissue type ELISAs were performed by grinding the lyophilized tissue in 50 ml tubes in a paint mixer for 30 seconds in the presence of eight 0.25" ceramic microspheres (MP Biomedicals, USA). The step was repeated for certain tissues requiring additional grinding for an additional 30 seconds. Protein was then extracted into 2 ml polypropylene tubes containing enough coarse powder to cover the curved bottom portion of the tubes. The coarsely ground tissue was transferred to 2 ml tubes to fill to the 0.3 ml mark. One 0.25" ceramic bead was then added to each tube and 0.6 ml of extraction buffer (200 μl of protease inhibitor cocktail [Research Products International Corp., Solon, OH, USA], 200 μl of 500 mM EDTA, 15.5 mg of 100 mM EDTA, and 100 mg of 100 mM EDTA). TDT and PBST at 20 ml). All tubes were kept on ice for 10 minutes and then processed for 45 seconds in the Genogrinder™. Then, 40 μL of 10% Tween-20 was added and another 300 μL of extraction buffer to the tubes and the samples were ground for an additional 45 seconds. The tubes were centrifuged at 13,000 rpm for 7–14 minutes. The supernatant was carefully transferred to a fresh tube. For ELISA assays, the extract was diluted in the extraction buffer as needed. ELISA results were expressed as ng/cm2 of leaf surface area or as parts per million (or ng protein per mg total plant protein).

EXAMPLE 5: GENE EXPRESSION OPERATELY LINKED TO THE ZEA MAYS GRMZM2G144030 PROMOTER AND 3' UTR REGULATORY ELEMENTS

[00177] Corn plants were transformed with a gene expression construct containing the Zea mays GRMZM2G144030 promoter and Zea mays GRMZM2G144030 as described above. ELISA analysis confirmed that the novel promoter drove expression of a transgene, and that the novel 3' UTR effectively terminated transgene expression. Quantitative measurements of Cry34Ab1 protein obtained from transgenic plants comprising novel promoter constructs are shown in Table 1. The data showed that Cry34Ab1 protein in plants containing the Zea mays GRMZM2G144030 promoter and the Zea mays GRMZM2G144030 3' UTR (i.e., pDAB113002) is preferentially expressed in root, stem, cob, seed, and silk tissues compared to marginally low expression in tissues such as leaf. TABLE 1: T0 Expression of Zea mays GRMZM2G144030 Promoters from Cry34Ab1 and AAD-1. The leaf ELISA was measured in ng of target protein per cm2 of leaf surface, while the remaining tissue type ELISA was measured in ng of target protein per mg of total protein. *Only qualitative testing was completed ND = not determined

[00178] Cry34Ab1 ELISA results indicated that the Zea mays GRMZM2G144030 promoter regulatory element (SEQ ID NO:1) and Zea mays GRMZM2G144030 3' UTR (SEQ ID NO:5) drove the preferred expression of Cry34Ab1 raqzis, stem, spike, seed, and silk in T1 events that were transformed with the pDAB113002 construct. Events produced from the transformation also expressed AAD-1 protein in both leaf and root tissues, as expected. In summary, the Zea mays GRMZM2G144030 promoter was designed to show high levels of expression of a transgene in leaf tissues in a plant species.

EXAMPLE 6: CULTURE-MEDIATED TRANSFORMATION OF GENES OPERATELY LINKED TO THE ZEA MAYS GRMZM2G144030 PROMOTER

[00179] Soybean can be transformed with genes operably linked to the Zea mays GRMZM2G144030 promoter using the same techniques previously described in Example 11 or 13 of patent application WO 2007/053482.

[00180] Cotton can be transformed with genes operably linked to the Zea mays GRMZM2G144030 promoter using the same techniques previously described in Examples 14 of U.S. Patent No. 7,838,733 or Example 12 of patent application WO 2007/053482 (Wright et al.).

[00181] Canola can be transformed with genes operably linked to the Zea mays GRMZM2G144030 promoter using the same techniques previously described in Example 26 of U.S. Patent No. 7,838,733 or Example 22 of patent application WO 2007/053482 (Wright et al.).

[00182] Wheat can be transformed with genes operably linked to the Zea mays GRMZM2G144030 promoter using the same techniques previously described in Example 23 of patent application WO 2013/116700A1 (Lira et al.).

[00183] Rice can be transformed with genes operably linked to the Zea mays GRMZM2G144030 promoter using the same techniques previously described in Example 19 of patent application WO 2013/116700A1 (Lira et al.).

EXAMPLE 7 AGROBACTERIUM-MEDIATED TRANSFORMATION OF GENES OPERATELY LINKED TO THE ZEA MAYS GRMZM2G144030 PROMOTER

[00184] In light of the subject description, additional crops may be transformed according to embodiments of the subject description using techniques that are known in the art. For Agrobacterium-mediated transformation of rye, see, e.g., Popelka JC, Xu J, Altpeter F., "Generation of rye with low transgene copy number after biolistic gene transfer and production of (Secale cereale L.) plants instantly marker-free transgenic rye," Transgenic Res. 2003 Oct;12(5):587-96.). For Agrobacterium-mediated transformation of sorghum, see, e.g., Zhao et al., "Agrobacterium-mediated sorghum transformation," Plant Mol Biol. 2000 Dec;44(6):789-98. For Agrobacterium-mediated transformation, see, e.g., Tingay et al, " Agrobacterium tumefaciens-mediated barley transformation", The Plant Journal, (1997) 11: 1369-1376. For Agrobacterium-mediated transformation of wheat, see, e.g., Cheng et al, "Genetic Transformation of Wheat Mediated by Agrobacterium tumefaciens", Plant Physiol. 1997 Nov;115(3):971-980. For Agrobacterium-mediated transformation of rice, consult, e.g., Hiei et al, "Transformation of rice mediated by Agrobacterium tumefaciens", Plant Mol. Biol. 1997 Sep;35(1-2):205-18.

[00185] The Latin names for these and other plants are given below. It should be clear that other (non-Agrobacterium) transformation techniques can be used to transform the genes operably linked to the f promoter of GRMZM2G144030 from Zea mays, for example, into these and other plants. Examples include, but are not limited to; Corn (Zea mays), Wheat (Triticum spp.), Rice (Oryza spp. and Zizania spp.), Barley (Hordeum spp.), Cotton (Abroma augusta and Gossypium spp.), Soybean (Glycine max), Table and sugar beet (Beta spp.), Sugarcane (Saccharum officinarum and other spp.), Cactus pear (Arenga pinnata), Tomato (Lycopersicon esculentum and other spp., Physalis ixocarpa, Solanum incanum and other spp., and Cyphomandra betacea), Potato (Solanum tuberosum), Sweet potato (Ipomoea batatas), Rye (Secale spp.), Peppers (Capsicum annuum, chinense and frutescens), Lettuce (Lactuca sativa, perennis, and pulchella), Cabbage (Brassica spp.), Celery (Apium graveolens), Eggplant (Solanum melongena), Peanut (Arachis hypogea), Sorghum (Sorghum spp.), Alfalfa (Medicago sativa), Carrot (Daucus carota), Beans (Phaseolus spp. and other genera), Oats (Avena sativa and strigosa), Peas (Pisum, Vigna, and Tetragonolobus spp.), Sunflower (Helianthus annuus), Pumpkin (Cucurbita spp.), Cucumber (Cucumis sativa), Tobacco (Nicotiana spp.), Arabidopsis (Arabidopsis thaliana), Lawn (Lolium, Agrostis, Poa, Cynodon, and other genera), Clover (Trifolium), Vetch (Vicia). The transformation of these plants, with genes operationally linked to the GRMZM2G144030 promoter of Zea mays, for example, is contemplated in modalities of the subject description.

[00186] Use of the Zea mays GRMZM2G144030 promoter to drive operably linked genes may be employed in many deciduous and evergreen hardwood species. Such applications are also within the scope of embodiments of this disclosure. Such species include, but are not limited to; alder (Alnus spp.), ash (Fraxinus spp.), poplar and aspen species (Populus spp.), beech (Fagus spp.), birch (Betula spp.), cherry (Prunus spp.), eucalyptus (Eucalyptus spp.), hickory (Carya spp.), maple (Acer spp.), oak (Quercus spp.), and pine (Pinus spp.).

[00187] Use of the Zea mays GRMZM2G144030 promoter to drive operably linked genes can be implemented in ornamental and fruit species. Such applications are also within the scope of embodiments of this disclosure. Examples include, but are not limited to; rose (Rosa spp.), burning bush (Euonymus spp.), petunia (Petunia spp.), begonia (Begonia spp.), rhododendron (Rhododendron spp.), crabapple (Malus spp.), pear (Pyrus spp.), peach (Prunus spp.), and marigold (Tagetes spp.).

[00188] While a variety of exemplary aspects and embodiments have been discussed above, those skilled in the art will recognize certain modifications, permutations, additions, and subcombinations thereof. It is therefore intended that the following appended claims and claims hereinafter be construed to include all such modifications, permutations, additions, and subcombinations as are within their true spirit and scope.

Claims (12)

1. A nucleic acid vector comprising a promoter operably linked to a heterologous polynucleotide sequence of: a) a polylinker sequence; b) a non-GRMZM2G144030 gene; or c) a combination of a) and b) wherein said promoter comprises a polynucleotide sequence as set forth by SEQ ID NO:1. 2. The nucleic acid vector of claim 1, wherein said promoter is 2,082 bp in length. 3. Nucleic acid vector according to claim 1, characterized in that said promoter consists of a polynucleotide sequence as established by SEQ ID NO:1. 4. Nucleic acid vector according to any one of claims 1 to 3, characterized in that it further comprises a coding sequence for a selectable marker. 5. The nucleic acid vector of claim 1, wherein said promoter is operably linked to a transgene. 6. The nucleic acid vector of claim 5, wherein the transgene encodes a selectable marker or gene product conferring insecticide resistance, herbicide tolerance, nitrogen use efficiency, small RNA expression, site-specific nuclease, water use efficiency, nutritional quality, or expression of DNA binding proteins. 7. The nucleic acid vector of any one of claims 1 to 3 or 5, further comprising a 3' untranslated polynucleotide sequence having the sequence as set forth by SEQ ID NO:5, wherein the 3' untranslated sequence is operably linked to said polylinker or said non-GRMZM2G144030 gene. 8. The nucleic acid vector of any one of claims 1 to 3 or 5, further comprising a 5' untranslated polynucleotide sequence, wherein the 5' untranslated sequence is operably linked to said polylinker or said non-GRMZM2G144030 gene. 9. Nucleic acid vector according to any one of claims 1 to 3 or 5, characterized in that it further comprises an intron sequence. 10. Nucleic acid vector according to claim 1, characterized in that said promoter drives transgene expression in leaf, root, stem, spike, grain, pollen and silk tissues. 11. Method for producing a transgenic plant cell, characterized by the fact that the method comprises the steps of: a) transforming a plant cell with a gene expression cassette comprising a Zea mays GRMZM2G144030 promoter, wherein said Zea mays GRMZM2G144030 promoter comprises a polynucleotide sequence as set forth by SEQ ID NO: 1, operatively linked to at least one polynucleotide sequence of interest; b) isolating the transformed plant cell comprising the gene expression cassette; and, c) producing a transgenic plant cell comprising the Zea mays GRMZM2G144030 promoter operatively linked to at least one polynucleotide sequence of interest. 12. Method for expressing a polynucleotide sequence of interest in a plant cell, characterized by the fact that the method comprises introducing into the plant cell a polynucleotide sequence of interest operably linked to a Zea mays GRMZM2G144030 promoter, wherein said Zea mays GRMZM2G144030 promoter comprises a polynucleotide sequence as set forth by SEQ ID NO: 1.