CN109844119A

CN109844119A - Plant promoter and 3#UTR for transgene expression

Info

Publication number: CN109844119A
Application number: CN201780041277.0A
Authority: CN
Inventors: M·古谱塔; S·班尼特; J·贝林格; N·萨尔德赛
Original assignee: Agrigenetics Inc
Current assignee: Agrigenetics Inc
Priority date: 2016-06-16
Filing date: 2017-08-07
Publication date: 2019-06-04
Also published as: WO2017219047A2; WO2017219047A3; EP3472326A2; UY37294A; BR102017012838A2; AR108748A1; CA3027256A1; TW201805424A

Abstract

This disclosure relates to the composition and method of the transcription and translation for promoting nucleotides sequence to be listed in plant or plant cell using the 3 ' UTR from Chlorophyll Concentration in Corn a/b binding-protein gene.Some embodiments are related to the 3 ' UTR from Chlorophyll Concentration in Corn a/b binding-protein gene, it plays the function of terminating the transcription for the nucleotide sequence being operably connected in plant.

Description

Plant promoter and 3# UTR for transgene expression

Cross Reference to Related Applications

This application claims the benefit of U.S. provisional patent application serial No. 62/350866 filed at the united states patent and trademark office at 2016, 6, 27, the disclosure of which is hereby expressly incorporated by reference in its entirety.

Technical Field

The present invention relates generally to the field of plant molecular biology and, more particularly, to expression of transgenes in plants.

Background

Many plant species can be transformed by transgenes to introduce agronomically desirable traits or characteristics. The resulting plant species are developed and/or modified to have specific desirable traits. In general, desirable traits include, for example, improving nutritional value quality, increasing yield, conferring pest or disease resistance, increasing drought and stress tolerance, improving horticultural qualities (e.g., pigmentation and growth), conferring herbicide tolerance, enabling the production of industrially useful compounds and/or materials from plants, and/or enabling the production of pharmaceuticals.

Transgenic plant species comprising multiple transgenes stacked at a single genomic locus are produced via plant transformation techniques. Plant transformation techniques allow for the introduction of a transgene into a plant cell, the recovery of a fertile transgenic plant containing a stably integrated copy of the transgene in the plant genome, and the subsequent expression of the transgene via transcription and translation of the plant genome to produce a transgenic plant having the desired trait and phenotype. However, mechanisms are needed that allow the generation of transgenic plant species to highly express multiple transgenes engineered into a trait stack.

Likewise, mechanisms that allow for expression of the transgene in specific tissues or organs of the plant are also desirable. For example, increasing the resistance of a plant to infection by a soil-borne pathogen can be achieved by transforming the plant genome with a pathogen resistance gene such that the pathogen resistance protein is robustly expressed within the plant roots. Alternatively, it may be desirable to express a transgene in plant tissue that is in a particular stage of growth or development (such as cell division or elongation). In addition, it may be desirable to express transgenes in the leaf and stem tissues of plants to provide tolerance to herbicides, or resistance to insects and pests on the ground.

Thus, there is a need for new gene regulatory elements that can drive transgenes to achieve desired levels of expression in specific plant tissues.

Disclosure of Invention

In embodiments of the disclosure, the disclosure relates to a nucleic acid vector comprising a 3' UTR operably linked to a polylinker or short polynucleotide sequence, a non-maize (Zea mays) chlorophyll a/b binding protein gene, or a combination of polylinker/polynucleotide sequence and non-maize chlorophyll a/b binding protein gene. In these aspects of this embodiment, the 3' UTR comprises a polynucleotide sequence having at least 90% sequence identity to SEQ ID No. 1. Additional embodiments include 3' UTRs comprising a polynucleotide 1000bp in length. Also included are polynucleotides sharing 80%, 85%, 90%, 92.5%, 95%, 97.5%, 99%, or 99.9% sequence identity with the 3' UTR of SEQ ID NO. 1. Various embodiments include nucleic acid vectors further comprising a sequence encoding a selectable marker. Also contemplated are embodiments of a nucleic acid vector, wherein the 3' UTR is operably linked to a transgene. Examples of such transgenes include selectable markers or gene products that confer insecticidal resistance, herbicide tolerance, nitrogen use efficiency, water use efficiency, or nutritional quality. Also contemplated are embodiments of a nucleic acid vector, wherein the 3' UTR is operably linked to an RNAi-expressing polynucleotide.

In other aspects, the disclosure relates to a nucleic acid (or polynucleotide) comprising a promoter polynucleotide sequence having at least 80%, 85%, 90%, 92.5%, 95%, 97.5%, 99%, and 99.9% sequence identity to SEQ ID NO. 2(US 005656496). Thus, such a promoter is incorporated into a nucleic acid vector comprising the 3' UTR of SEQ ID NO 1. In aspects of this embodiment, a promoter (e.g., SEQ ID NO:2) is operably linked to the 5' end of a polylinker or transgene and a 3' UTR is operably linked to the 3' end of the polylinker or transgene. Also included in this embodiment are nucleic acid vectors, wherein the promoter further comprises an intron or 5' -UTR. Subsequently, a nucleic acid vector containing the promoter of SEQ ID NO. 2 and the 3' UTR of SEQ ID NO. 1 was driven to express the transgene with constitutive tissue-specific expression.

In other aspects, the disclosure relates to plants comprising a polynucleotide sequence having at least 90% sequence identity to SEQ ID No. 1 operably linked to a transgene. Thus, the plant is a monocotyledonous or dicotyledonous plant. Specific examples of plants include maize, wheat, rice, sorghum, oats, rye, bananas, sugarcane, soybean, cotton, Arabidopsis (Arabidopsis), tobacco, sunflower, and canola. In various embodiments, such plants may be transformed, wherein the transgene is inserted into the genome of the plant. In further embodiments, the plant comprises a promoter comprising a polynucleotide sequence having at least 80%, 85%, 90%, 92.5%, 95%, 97.5%, 99%, or 99.9% sequence identity to SEQ ID No. 2. In such embodiments, SEQ ID NO 1 is 1000bp in length. In one aspect of this embodiment, the 3' UTR is operably linked to a transgene. In other embodiments, the plant comprises a 3' UTR comprising a polynucleotide sequence having at least 80%, 85%, 90%, 92.5%, 95%, 97.5%, 99%, or 99.9% sequence identity to SEQ ID No. 1. In such embodiments, SEQ ID NO 1 is 1000bp in length. In one aspect of this embodiment, the 3' UTR of SEQ ID NO. 1 is operably linked to a transgene. In addition, various embodiments relate to plants comprising the promoter of SEQ ID NO. 2 or the promoter of a maize chlorophyll a/b binding protein gene, wherein transgene expression is constitutive. Likewise, various embodiments relate to plants comprising the 3' UTR of SEQ ID NO. 1, wherein transgene expression is constitutive or tissue-specific expression, as determined by the promoter used to drive the transgene.

In other aspects, the disclosure relates to a method for producing a transgenic plant cell. Such methods utilize a gene expression cassette comprising a maize chlorophyll a/b binding protein gene 3' UTR operably linked to at least one polynucleotide sequence of interest to transform a plant cell. Next, the method discloses isolating a transformed plant cell comprising the gene expression cassette. In addition, the methods contemplate producing a transgenic plant cell comprising a maize chlorophyll a/b binding protein gene 3' UTR operably linked to at least one polynucleotide sequence of interest. Also, the method includes regenerating the transgenic plant cell into a transgenic plant. In addition, the method comprises obtaining a transgenic plant, wherein the transgenic plant comprises a gene expression cassette comprising a maize chlorophyll a/b binding protein gene 3' UTR operably linked to at least one polynucleotide sequence of interest. In such embodiments, the method of transforming a plant cell is performed using a plant transformation method. In other embodiments, the method of transforming a plant cell results in the polynucleotide sequence of interest being stably integrated into the genome of the transgenic plant cell. In aspects of such embodiments, the maize chlorophyll a/b binding protein gene 3' UTR comprises a polynucleotide of SEQ ID NO. 1.

In other aspects, the disclosure relates to an isolated polynucleotide comprising a nucleic acid sequence having at least 80%, 85%, 90%, 92.5%, 95%, 97.5%, 99%, or 99.9% sequence identity to the polynucleotide of SEQ ID No. 1. In one embodiment, the isolated polynucleotide further comprises an open reading frame polynucleotide encoding a polypeptide; and a promoter sequence. In another embodiment, the polynucleotide of SEQ ID NO. 1 is 1000bp in length.

In an embodiment of the disclosure, the disclosure relates to a nucleic acid vector comprising a 3' UTR operably linked to: short polypeptide or polylinker sequences; a non-maize chlorophyll a/b binding protein-like gene; or a combination of a polynucleotide sequence and a non-maize chlorophyll a/b binding protein-like gene, wherein the 3' UTR comprises a polynucleotide sequence having at least 90% sequence identity to SEQ ID NO. 1. In some embodiments, the 3' UTR is 1000bp in length. In further embodiments, the 3' UTR consists of a polynucleotide sequence having at least 90% sequence identity to SEQ ID No. 1. In other embodiments, the 3' UTR terminates expression of a polynucleotide encoding a selectable marker. In further embodiments, the 3' UTR is operably linked to a transgene. In aspects of this embodiment, the transgene encodes a selectable marker or gene product that confers insecticidal resistance, herbicide tolerance, nitrogen use efficiency, water use efficiency, or nutritional quality. There is provided a 3' UTR of SEQ ID No. 1 for use with a promoter, the promoter polynucleotide sequence comprising a sequence having at least 90% sequence identity to SEQ ID No. 2, wherein the promoter polynucleotide sequence is operably linked to the polylinker or the transgene. In other embodiments, the 3' UTR of SEQ ID NO. 1 is provided for use with any known plant promoter sequence comprising a sequence having at least 90% sequence identity to SEQ ID NO. 2 or to a maize chlorophyll a/b binding protein gene promoter sequence. In another embodiment, the 3' UTR of SEQ ID NO. 1 is used for constitutive or tissue-specific expression.

In yet another embodiment, the present disclosure provides a plant comprising a polynucleotide sequence having at least 90% sequence identity to SEQ ID No. 1 operably linked to a transgene or a linker sequence. According to this embodiment, the plant is selected from the group consisting of: maize, wheat, rice, sorghum, oat, rye, banana, sugarcane, soybean, cotton, Arabidopsis (Arabidopsis), tobacco, sunflower and canola. Subsequently, in some embodiments, the plant comprising a polynucleotide sequence having at least 90% sequence identity to SEQ ID No. 1 can be a maize plant. In other embodiments, a transgene operably linked to a polynucleotide sequence having at least 90% sequence identity to SEQ id No. 1 is inserted into the plant genome. In some embodiments, the polynucleotide sequence having at least 90% sequence identity to SEQ ID No. 1 is a 3'UTR and the 3' UTR is operably linked to a transgene. In other embodiments, the plant comprises a promoter sequence having SEQ ID No. 2 or a promoter sequence having at least 90% sequence identity to SEQ ID No. 2, wherein the promoter sequence is operably linked to a transgene. In another embodiment, a polynucleotide sequence having at least 90% sequence identity to SEQ ID NO. 1 is used to express a transgene in constitutive or tissue specific expression. In another embodiment, the polynucleotide sequence having at least 90% sequence identity to SEQ ID NO. 1 is 1000bp in length.

In one embodiment, the present disclosure provides a method for producing a transgenic plant cell, the method comprising the steps of: transforming a plant cell with a gene expression cassette comprising a 3' UTR of a maize chlorophyll a/b binding protein gene operably linked to at least one polynucleotide sequence of interest; isolating a transformed plant cell comprising the gene expression cassette; and generating a transgenic plant cell comprising a maize chlorophyll a/b binding protein gene 3' UTR operably linked to at least one polynucleotide sequence of interest. In other embodiments, the step of transforming the plant cell is performed using a plant transformation method. The plant transformation method may be selected from: agrobacterium-mediated transformation methods, biolistic transformation methods, silicon carbide transformation methods, protoplast transformation methods, and liposome transformation methods. In other embodiments, the polynucleotide sequence of interest is constitutively expressed throughout the transgenic plant cell. In some embodiments, the polynucleotide sequence of interest is stably integrated into the genome of the transgenic plant cell. Thus, the method for producing a transgenic plant cell may further comprise the steps of: regenerating the transgenic plant cell into a transgenic plant; and obtaining a transgenic plant, wherein the transgenic plant comprises a gene expression cassette comprising a maize chlorophyll a/b binding protein gene 3' UTR of SEQ ID NO:1 operably linked to at least one polynucleotide sequence of interest. In one embodiment, the transgenic plant cell is a monocot transgenic plant cell or a dicot transgenic plant cell. For example, a dicot transgenic plant cell can be selected from the group consisting of: arabidopsis plant cells, tobacco plant cells, soybean plant cells, canola plant cells, and cotton plant cells. In addition, the monocot transgenic plant cell is selected from the group consisting of: maize plant cells, rice plant cells and wheat plant cells. The 3' UTR of the maize chlorophyll a/b binding protein gene used in the method can comprise a polynucleotide of SEQ ID NO. 1. In various embodiments, the maize chlorophyll a/b binding protein gene 3'UTR can further comprise a first polynucleotide sequence of interest operably linked to the 3' terminus of SEQ ID NO: 1.

In one embodiment, the present disclosure provides a method for expressing a polynucleotide sequence of interest in a plant cell, the method comprising introducing into a plant cell a polynucleotide sequence of interest operably linked to the 3' UTR of a maize chlorophyll a/b binding protein gene. In some embodiments, a polynucleotide sequence of interest operably linked to the 3' UTR of a maize chlorophyll a/b binding protein gene is introduced into a plant cell by a plant transformation method. Thus, the plant transformation method may be selected from: agrobacterium-mediated transformation methods, biolistic transformation methods, silicon carbide transformation methods, protoplast transformation methods, and liposome transformation methods. In embodiments, the polynucleotide sequence of interest is constitutively expressed throughout the plant cell. In some embodiments, the polynucleotide sequence of interest is stably integrated into the genome of the plant cell. Thus, the transgenic plant cell is a monocotyledonous plant cell or a dicotyledonous plant cell. For example, the dicot cell is selected from the group consisting of: arabidopsis plant cells, tobacco plant cells, soybean plant cells, canola plant cells, and cotton plant cells. In addition, the monocot plant cell is selected from the group consisting of: maize plant cells, rice plant cells and wheat plant cells.

In one embodiment, the present disclosure provides a transgenic plant cell comprising a maize chlorophyll a/b binding protein gene 3' UTR. In some embodiments, the transgenic plant cell comprises a transgenic event. In one aspect of this embodiment, the transgenic event comprises an agronomic trait. Thus, the agronomic trait is selected from: an insecticidal resistance trait, a herbicide tolerance trait, a nitrogen use efficiency trait, a water use efficiency trait, a nutritional quality trait, a DNA binding trait, a selectable marker trait, a small RNA trait, or any combination thereof. In other embodiments, the agronomic trait comprises an herbicide tolerance trait. In one aspect of this embodiment, the herbicide tolerance trait includes an aad-1 coding sequence. In some embodiments, the transgenic plant cell produces a commodity product. The commodity is selected protein concentrate, protein isolate, grain, food, flour, oil or fiber. In one embodiment, the transgenic plant cell is selected from the group consisting of a dicot cell and a monocot cell. Thus, the monocot plant cell is a maize plant cell. In other embodiments, the 3' UTR of the maize chlorophyll a/b binding protein gene comprises a polynucleotide having at least 90% sequence identity to a polynucleotide of SEQ ID NO. 1. In yet another embodiment, the maize chlorophyll a/b binding protein gene 3' UTR is 1000bp in length. In a further embodiment, the maize chlorophyll a/b binding protein gene 3' UTR consists of SEQ ID NO: 1. In other embodiments, the maize chlorophyll a/b binding protein gene 3' UTR is used to express an agronomic trait in a constitutive or tissue-specific manner.

The present disclosure provides an isolated polynucleotide comprising a nucleic acid sequence having at least 90% sequence identity to the polynucleotide of SEQ ID No. 1. In some embodiments, the isolated polynucleotide drives constitutive or tissue-specific expression. In other embodiments, the isolated polynucleotide has expression activity within a plant cell. In various embodiments, an isolated polynucleotide comprises an open reading frame polynucleotide encoding a polypeptide; and a promoter sequence. Additional embodiments include isolated polynucleotides comprising a nucleic acid sequence having at least 90% sequence identity to the polynucleotide of SEQ ID NO. 1, wherein the polynucleotide of SEQ ID NO. 1 is 1000bp in length.

The foregoing and other features will become more apparent upon consideration of the following detailed description of several embodiments taken in conjunction with the accompanying drawings.

Drawings

FIG. 1: this figure is a schematic representation of plasmid pDAB116011 comprising the maize chlorophyll a/b binding protein gene promoter of SEQ ID NO:2 (labeled "ZMEXP 13231.1") and the maize chlorophyll a/b binding protein gene 3' UTR of SEQ ID NO:1 (labeled "ZMEXP 13363.1"). These regulatory elements are operably linked to a yellow fluorescent protein (labeled "PhiYFP") from the calix aequorea (Phialidium) species. Also included on this plasmid is an aad-1 gene expression cassette comprising a maize ubiquitin-1 promoter (labeled "ZmUbi 1 promoter") and a maize lipase 3'-UTR (labeled "Zmlip 3' UTR"). These regulatory elements are operably linked to the aad-1 gene.

FIG. 2: this figure is a schematic representation of plasmid pDAB108746, which contains the maize ubiquitin-1 promoter (labeled "ZmUbi 1 promoter") and the potato (Solanum tuberosum) protease inhibitor-II gene 3'-UTR (labeled "StPinII 3' UTR"). These regulatory elements are operably linked to a Cry34Ab1 reporter gene (labeled "Cry 34Ab 1") from bacillus thuringiensis (bacillus thuringiensis). Also included on this plasmid is an aad-1 gene expression cassette comprising a maize ubiquitin-1 promoter (labeled "ZmUbi 1 promoter") and a maize lipase 3'-UTR (labeled "ZmLip 3' UTR"). These regulatory elements are operably linked to the aad-1 gene.

Detailed Description

I. Brief summary of several embodiments

The development of transgenic plant products is becoming more and more complex. Commercially viable transgenic plants currently require the stacking of multiple transgenes at a single locus. Plant promoters and 3 'UTRs used in basic research or biotechnological applications are generally unidirectional, targeting only one gene that has been fused at the 3' end (downstream) of its promoter or at the 5 'end (upstream) of its 3' UTR. Thus, each transgene typically requires a promoter and a 3' UTR for expression, where multiple regulatory elements are required to express multiple transgenes within one gene stack. As the number of transgenes in a gene stack increases, the same promoter and/or 3' UTR are routinely used to obtain optimal levels of expression patterns for different transgenes. Obtaining optimal levels of transgene expression is necessary to produce a single polygenic trait. Unfortunately, it is known that multiple gene constructs driven by the same promoter and/or 3' UTR cause gene silencing, resulting in less efficient transgene products in the art. Repeated promoter and/or 3' UTR elements can cause homology-based gene silencing. In addition, repeated sequences within the transgene can cause homologous recombination of the gene within the locus, resulting in polynucleotide rearrangement. Silencing and rearrangement of the transgene will likely have an undesirable effect on the performance of the resulting transgenic plant to express the transgene. In addition, excess Transcription Factor (TF) binding sites due to promoter duplication can cause depletion of endogenous TF, resulting in transcriptional inactivation. Given the need to introduce multiple genes into plants for metabolic engineering and trait stacking, multiple promoters and/or 3' UTRs are needed to generate transgenic crops that drive the expression of multiple genes.

A particular problem in the identification of promoters and/or 3' UTRs is the need to identify tissue-specific promoters that are not expressed in other plant tissues in relation to a particular cell type, developmental stage and/or function in a plant. Tissue-specific (i.e., tissue-preferred) or organ-specific promoters drive gene expression in certain tissues, such as the kernels, roots, leaves, silks, or tapetum of a plant. Tissue and developmental stage specific promoters and/or 3' UTRs may be initially identified from observing expression of genes that are expressed in specific tissues or at specific time periods during plant development. These tissue-specific promoters and/or 3' UTRs are required for certain applications in the transgenic plant industry and are desirable because they allow the heterologous gene to be specifically expressed in a tissue and/or developmental stage selective manner, indicating that the heterologous gene is differentially expressed in various organs, tissues and/or times, but not in other tissues. For example, increasing the resistance of a plant to infection by a soil-borne pathogen can be achieved by transforming the plant genome with a pathogen resistance gene such that the pathogen resistance protein is robustly expressed within the plant roots. Alternatively, it may be desirable to express a transgene in plant tissue that is in a particular stage of growth or development (such as cell division or elongation). Another application is where it is desirable to use a tissue specific promoter and/or 3' UTR to limit the expression of a transgene encoding an agronomic trait in a particular tissue type, such as a developing parenchyma cell. Thus, a particular problem in the identification of promoters and/or 3' UTRs is how to identify the promoters and how to correlate the identified promoters with developmental characteristics of the cells for specific tissue expression.

Another problem with promoter identification is the need to clone all relevant cis-acting and trans-activating transcriptional control elements so that the cloned DNA fragment drives transcription in the desired specific expression pattern. Given that such control elements are located distal to the translation initiation or start site, the size of the polynucleotide selected to comprise the promoter is critical to provide for the level and pattern of expression of the promoter polynucleotide sequence. It is known that promoter length includes functional information, and that different genes are demonstrated to have longer or shorter promoters than those of other genes in the genome. Elucidating the transcription start site of a promoter and predicting functional genetic elements in the promoter region are challenging. Further adding to the challenge is the complexity, diversity and inherent degeneracy of the regulatory motifs as well as cis and trans regulatory elements (Blanchette, Mathieu et al, "Genome-wide regulatory of transcriptional regulation new instructions in human gene expression," Genome research 16.5(2006): 656-). Cis and trans regulatory elements are located in the distal portion of the promoter which regulates spatial and temporal expression of the gene to be present only at the desired site and at a specific time (Porto, Milena Silva et al, "Plant promoters: an adaptive research of structure and function" -Molecular biology technology 56.1(2014): 38-49). Existing promoter analysis tools cannot reliably identify such cis-regulatory elements in genomic sequences, thereby predicting too many false positives, as these tools generally focus only on sequence content (Fickett JW, Hatzigeoorgiou AG (1997) Eukarstic promoter recognition. genome research 7: 861-878). Thus, identification of promoter regulatory elements requires obtaining an appropriate sequence of a particular size that will result in driving expression of an operably linked transgene in a desired manner.

The present invention provides methods and compositions to overcome such problems by using maize chlorophyll a/b binding protein gene regulatory elements to express transgenes in plants.

Terms and abbreviations II

Throughout this patent application, a number of terms are used. In order to provide a clear and consistent understanding of the specification and claims, including the scope to be given such terms, the following definitions are provided.

As used herein, the term "intron" refers to any nucleic acid sequence contained in a gene (or expressed polynucleotide sequence of interest) that has been transcribed but not translated. Introns include expressed sequences of DNA as well as untranslated nucleic acid sequences within the corresponding sequences in RNA molecules transcribed therefrom. The constructs described herein may also contain sequences, such as introns, that enhance translation and/or mRNA stability. An example of one such intron is the first intron of gene II of the histone H3 variant of Arabidopsis thaliana (Arabidopsis thaliana) or any other commonly known intron sequence. Introns may be used in combination with promoter sequences to enhance translation and/or mRNA stability.

As used herein, the term "isolated" means having been removed from its natural environment, or from other compounds present when the compound was 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 molecules, proteins, and peptides).

As used herein, the term "purified" relates to the isolation of a molecule or compound in the form: substantially free of contaminants normally associated with the molecule or compound in the natural or natural environment, or substantially increases in concentration relative to other compounds present when the compound is first formed, and means increases in purity due to separation from other components of the original composition. The term "purified nucleic acid" is used herein to describe a nucleic acid sequence that is isolated, produced by isolation, 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 components (e.g., a nucleic acid can be purified from a chromosome by removing protein contaminants in the chromosome and breaking the chemical bonds connecting the nucleic acid to the remaining DNA).

As used herein, the term "synthesis" refers to a polynucleotide (i.e., DNA or RNA) molecule formed as an in vitro process via chemical synthesis. For example, synthetic DNA may be in Eppendorf during the reaction^TMFormed in a tube to enzymatically produce synthetic DNA from a natural DNA or RNA strand. Other laboratory methods may be used to synthesize polynucleotide sequences. Oligonucleotides can be chemically synthesized via solid phase synthesis using phosphoramidates on an oligonucleotide synthesizer. The synthesized oligonucleotides can be annealed to each other as a complex, thereby producing a "synthetic" polynucleotide. Other methods for chemically synthesizing polynucleotides are known in the artAre known and can be readily implemented for use in the present disclosure.

As used herein, the term "about" means 10% greater or less than the stated value or range of values, but is not intended to designate any value or range of values as only such broad definition. Each value or range of values preceded by the term "about" is also intended to encompass embodiments of the absolute value or range of values recited.

For purposes of this disclosure, "gene" includes DNA regions encoding a gene product (see below), as well as all DNA regions that regulate the production of a gene product, whether or not such regulatory sequences are adjacent to coding sequences and/or transcribed sequences. Thus, genes include, but are 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 attachment sites, and locus control regions.

As used herein, the term "natural" or "nature" defines the conditions that occur in nature. A "native DNA sequence" is a DNA sequence that occurs in nature and that has been produced by natural means or traditional breeding techniques, rather than by genetic engineering (e.g., using molecular biology/transformation techniques).

As used herein, "transgene" is defined as a nucleic acid sequence encoding a gene product, including, for example (but not limited to), mRNA. In one embodiment, a transgene is an exogenous nucleic acid, wherein the transgene sequence has been introduced by genetic engineering into a host cell (or progeny thereof) in which the transgene is not normally found. In one example, the transgene encodes an industrially or pharmaceutically useful compound, or is a gene that encodes a desired agricultural trait (e.g., a herbicide tolerance gene). In yet another example, the transgene is an antisense nucleic acid sequence, wherein expression of the antisense nucleic acid sequence inhibits expression of the target nucleic acid sequence. In one embodiment, the transgene is an endogenous nucleic acid, wherein additional genomic copies of the endogenous nucleic acid are desired; or in an antisense orientation relative to a target nucleic acid sequence in a host organism.

As used herein, the term "non-maize chlorophyll a/b binding protein transgene" or "non-ZmCAB gene" is any transgene having less than 80% sequence identity to the maize chlorophyll a/b binding protein gene coding sequence (SEQ ID NO:5, Genbank NCBI accession No. NP-001147639).

A "gene product" as defined herein is any product produced by the gene. For example, the gene product can be a direct transcription 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 mRNA. Gene products also include RNA modified by methods such as capping, polyadenylation, methylation, and editing, as well as proteins modified by, for example, methylation, acetylation, phosphorylation, ubiquitination, ADP ribosylation, myristoylation, and glycosylation. Gene expression can be affected by an external signal, such as exposure of a cell, tissue, or organism to an agent that increases or decreases gene expression. Gene expression can also be regulated anywhere in the pathway from DNA to RNA to protein. Regulation of gene expression occurs, for example, by controlling the effects on transcription, translation, RNA transport and processing, degradation of intermediate molecules (such as mRNA), or by activation, inactivation, compartmentalization, or degradation after a specific protein molecule has been produced, or a combination of these. Gene expression can be measured at the RNA level or the protein level by any method known in the art, including but not limited to Northern blotting, RT-PCR, Western blotting, or in vitro, in situ, or in vivo protein activity assays.

As used herein, the term "gene expression" relates to the process of converting the coding information of a nucleic acid transcription unit (including, for example, genomic DNA) into a functional, non-functional or structural part of a cell, which typically includes protein synthesis. Gene expression may be affected by external signals, such as exposure of a cell, tissue, or organism to an agent that increases or decreases gene expression. Gene expression can also be regulated anywhere in the pathway from DNA to RNA to protein. Regulation of gene expression occurs, for example, by controlling the effects on transcription, translation, RNA transport and processing, degradation of intermediate molecules (such as mRNA), or by activation, inactivation, compartmentalization, or degradation after a specific protein molecule has been produced, or a combination of these. Gene expression can be measured at the RNA level or the protein level by any method known in the art, including but not limited to Northern blotting, RT-PCR, Western blotting, or in vitro, in situ, or in vivo protein activity assays.

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 unlinked silencing loci can be caused by transcriptional inhibition (transcriptional gene silencing; TGS) or mRNA degradation (post-transcriptional gene silencing; PTGS) due to the production of double stranded rna (dsrna) corresponding to the promoter or transcriptional sequence, respectively. The involvement of different cellular components in each process suggests that dsRNA-induced TGS and PTGS may be caused by the diversification of ancient common mechanisms. However, a strict comparison of TGS and PTGS has been difficult to achieve because it typically relies on analysis of different silencing loci. In some cases, a single transgenic locus may 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) Planta225: 365-79. siRNA is likely to be the actual molecule that triggers TGS and PTGS on homologous sequences: sirnas will trigger silencing and methylation of homologous sequences in cis and trans in this model by extending methylation of the transgenic sequence into the endogenous promoter.

As used herein, the term "nucleic acid molecule" (or "nucleic acid" or "polynucleotide") can refer to a polymeric form of nucleotides, which can include both sense and antisense strands of RNA, cDNA, genomic DNA, as well as synthetic forms and mixed polymers of the foregoing. A nucleotide may refer to a ribonucleotide, a deoxyribonucleotide, or a modified form of either type of nucleotide. As used herein, a "nucleic acid molecule" is synonymous with "nucleic acid" and "polynucleotide". Unless otherwise indicated, nucleic acid molecules are typically at least 10 bases in length. The term may refer to RNA or DNA molecules of indefinite length. The term includes both single-stranded and double-stranded forms of DNA. Nucleic acid molecules may include either or both naturally occurring nucleotides and modified nucleotides that are linked together by naturally occurring nucleotide linkages and/or non-naturally occurring nucleotide linkages.

As will be readily understood by those skilled in the art, nucleic acid molecules may be chemically or biochemically modified, or may contain unnatural or derivatized nucleotide bases, such modifications include, for example, labels, methylation, replacement of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications (e.g., uncharged linkages such as methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, and the like, charged linkages such as phosphorothioates, phosphorodithioates, and the like, pendant moieties such as peptides, intercalators such as acridine, psoralen, and the like, chelators, alkylators, and modified linkages such as α anomeric nucleic acids, and the like).

Transcription proceeds along the DNA strand in a 5 'to 3' manner. This means that RNA is prepared by sequential addition of ribonucleotide-5 '-triphosphates to the 3' -end of the growing strand (with the necessary elimination of pyrophosphate). In a linear or circular nucleic acid molecule, a discrete element (e.g., a particular nucleotide sequence) can be referred to as "upstream" or "5 '" relative to another element if it binds in the 5' direction of the other element or will bind to the same nucleic acid. Similarly, a interspersed element may be referred to as "downstream" or "3 '" with respect to another element if it is or will bind the same nucleic acid in the 3' direction of the other element.

As used herein, a base "position" refers to the position of a given base or nucleotide residue within a given nucleic acid. A given nucleic acid can be defined by alignment with a reference nucleic acid (see below).

Hybridization involves the joining of two polynucleotide strands by hydrogen bonds. Oligonucleotides and their analogs hybridize through hydrogen bonds between complementary bases, including Watson-Crick (Watson-Crick) hydrogen bonds, Husky (Hoogsteen) hydrogen bonds, or reverse Husky hydrogen bonds. 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 the pyrimidine and the purine, and the bonding of the pyrimidine to the purine is referred to as "base pairing. More specifically, A will hydrogen bond to T or U and G will hydrogen bond to C. "complementary" means that there is base pairing between two different nucleic acid sequences or between two different regions of the same nucleic acid sequence.

"specifically hybridizable" and "specifically complementary" are terms which indicate a degree of complementarity sufficient for stable and specific binding to occur between the oligonucleotide and the DNA or RNA target. The oligonucleotide need not be 100% complementary to its target sequence to be specifically hybridizable. An oligonucleotide is specifically hybridizable when binding of the oligonucleotide to a target DNA or RNA molecule interferes with the normal function of the target DNA or RNA, and under conditions in which specific binding is desired, e.g., in the case of in vivo assays or systems, under physiological conditions, there is a sufficient degree of complementarity to avoid non-specific binding of the oligonucleotide to non-target sequences. Such binding is referred to as specific hybridization.

Hybridization conditions that result in a particular degree 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 temperature of hybridization and the ionic strength of the hybridization buffer (especially the Na + and/or Mg2+ concentration) will contribute to the stringency of hybridization, but the number of washes also affects the stringency. The calculation of hybridization conditions required to achieve a particular degree of stringency is discussed in Sambrook et al (eds.), Molecular Cloning, Arabidopsis Manual, 2 nd edition, volumes 1-3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York,1989, chapters 9 and 11.

As used herein, "stringent conditions" encompass conditions under which hybridization will only occur when there is less than 50% mismatch between the hybridizing molecule and the DNA target. "stringent conditions" include additional levels of specific stringency. Thus, as used herein, a "medium stringency" condition is one in which molecules with sequence mismatches of more than 50% do not hybridize; "high stringency" conditions are conditions under which sequences that are mismatched by more than 20% do not hybridize; whereas "very high stringency" conditions are those in which sequences that are mismatched by more than 10% do not hybridize.

In a specific embodiment, stringent conditions may comprise hybridization at 65 ℃ followed by a wash with 0.1 XSSC/0.1% SDS at 65 ℃ for 40 minutes.

Representative, non-limiting hybridization conditions are as follows:

very high stringency: hybridization in 5 XSSC buffer at 65 ℃ for 16 hours; wash twice in 2 × SSC buffer at room temperature for 15 minutes each; and washed twice in 0.5 XSSC buffer at 65 ℃ for 20 minutes each.

High stringency: hybridization in 5X-6 XSSC buffer at 65-70 ℃ for 16-20 hours; washing twice in 2 XSSC buffer at room temperature for 5-20 minutes each time; and washed twice in 1 XSSC buffer at 55-70 ℃ for 30 minutes each.

Moderate stringency: hybridization in 6 XSSC buffer at room temperature to 55 ℃ for 16-20 hours; washing at least twice in 2X-3 XSSC buffer at room temperature to 55 ℃ for 20-30 minutes each time.

In particular embodiments, specifically hybridizable nucleic acid molecules can remain bound under very high stringency hybridization conditions. In these and other embodiments, the specifically hybridizable nucleic acid molecule can remain bound under high stringency hybridization conditions. In these and other embodiments, the specifically hybridizable nucleic acid molecule can remain bound under moderate stringency hybridization conditions.

Oligonucleotide: oligonucleotides are short nucleic acid polymers. Oligonucleotides can be formed by cleaving a longer nucleic acid segment, or by polymerizing individual nucleotide precursors. Automated synthesizers allow synthesis of oligonucleotides up to several hundred bases in length. Since oligonucleotides bind to complementary nucleotide sequences, they can be used as probes for detecting DNA or RNA. Oligonucleotides (oligodeoxyribonucleotides) consisting of small DNA sequences can be used in PCR, a technique for amplifying DNA. In PCR, oligonucleotides are often referred to as "primers" which allow a DNA polymerase to extend the oligonucleotide and replicate the complementary strand.

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

As used herein, the term "percent sequence identity" can refer to a value determined by comparing two optimally aligned sequences (e.g., a nucleic acid sequence and an amino acid sequence) over a comparison window, wherein the portion of the sequences in the comparison window can comprise additions or deletions (i.e., gaps) as compared to a reference sequence (which does not comprise additions or deletions) in order to achieve 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 yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity.

Methods of sequence alignment for comparison are well known in the art. Various programs and alignment algorithms are described, for example, in the following documents: 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) FEMSMICrobiol. Lett.174: 247-50. Detailed considerations for sequence alignment methods and homology calculations can be found, for example, in Altschul et al, (1990) J.mol.biol.215: 403-10.

National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST)^TM(ii) a Altschul et al (1990)) are available from several sources, including the national center for biotechnology information (Bethesda, MD) and are available on the internet for use in connection with several sequence analysis programs. A description of how to use this program to determine sequence identity BLAST can be found on the Internet^TMThe "help" portion of (1). To compare nucleic acid sequences, BLAST can be performed using default parameters^TM(Blastn) the "Blast 2 sequence" function of the program. Nucleic acid sequences having even greater similarity to a reference sequence will show an increase in percent identity when evaluated by this method.

The term "operably linked" as used herein relates to a first nucleic acid sequence being 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 with a coding sequence when the promoter affects the transcription or expression of the coding sequence. When produced recombinantly, operably linked nucleic acid sequences are typically contiguous and, where necessary, the two protein coding regions may be joined in reading frame. However, the nucleic acids need not be contiguous in order to be operably linked.

As used herein, the term "promoter" refers to a region of DNA that is generally located upstream (toward the 5' region of a gene) of a gene and is required to initiate and drive transcription of the gene. Promoters may provide for the appropriate activation or suppression of genes they control. The promoter may contain specific sequences recognized by the transcription factor. These factors can bind to promoter DNA sequences, which lead to the recruitment of RNA polymerase (the enzyme that synthesizes RNA from the coding region of the gene). Promoters generally refer to all gene regulatory elements located upstream of a gene, including upstream promoters, 5' -UTRs, introns, and leader sequences.

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

Activation of the upstream promoter is effected by additional sequences of regulatory DNA sequence elements which are bound by various proteins and which 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 or development-specific transcription factors bind, alone or in combination, may determine the spatiotemporal expression pattern of the promoter at the transcriptional level. These cis-elements vary widely in the type of control they exert on an operably linked gene. Some elements are used to increase transcription of an operably linked gene 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) or to spatial information (e.g., tissue specificity). See, e.g., Langridge et al, (1989) Proc. Natl. Acad. Sci. USA 86: 3219-23. These cis-elements are located at different distances from the transcription start site, with some cis-elements (called proximal elements) adjacent to the smallest core promoter region, and others located several kilobases upstream or downstream of the promoter (enhancer).

The term "5 ' untranslated region" or "5 ' -UTR" as used herein is defined as an untranslated segment of the 5' end of a pre-mRNA or mature mRNA. For example, on mature mrnas, the 5' -UTR usually contains a 7-methylguanosine cap at its 5' end and is involved in many processes such as splicing, polyadenylation, mRNA export to the cytoplasm, identification of the 5' end of the mRNA by the translation machinery, and protection of the mRNA from degradation.

The term "transcription terminator" as used herein is defined as a transcribed segment at the 3' end of a pre-mRNA or mature mRNA. For example, longer stretches of DNA outside the "polyadenylation signal" site are transcribed into pre-mRNA. This DNA sequence usually contains a transcription termination signal in order to process the pre-mRNA appropriately into mature mRNA.

The term "3 ' untranslated region" or "3 ' -UTR" as used herein is defined as an untranslated segment of the 3' end of a pre-mRNA or mature mRNA. For example, on mature mrnas, this region contains a 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 polyadenylation signals and transcription terminators.

As used herein, the term "polyadenylation signal" refers to a nucleic acid sequence present in an mRNA transcript that, when a poly (a) polymerase is present, allows the transcript to be polyadenylated at a polyadenylation site located, for example, 10 to 30 bases downstream of the poly (a) signal. Many polyadenylation signals are known in the art and are suitable for use in the present invention. Exemplary sequences include AAUAAA and variants thereof, such as Loke j. et al, (2005) Plant Physiology 138 (3); 1457 and 1468.

A "DNA-binding transgene" is a polynucleotide coding sequence that encodes a DNA-binding protein. The DNA binding protein is then able to bind to another molecule. The binding protein may bind to, for example, a DNA molecule (DNA binding protein), an RNA molecule (RNA binding protein), and/or a protein molecule (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 one or more different proteins. Binding proteins may have more than one type of binding activity. For example, zinc finger proteins have DNA binding, RNA binding, and protein binding activities.

Examples of DNA binding proteins include; meganucleases, zinc fingers, CRISPRs, and TALE binding domains that can be "engineered" to bind to a predetermined nucleotide sequence. Typically, the engineered DNA binding protein (e.g., zinc finger, CRISPR, or TALE) is a non-naturally occurring protein. A non-limiting example of a method for engineering a DNA binding protein is design and selection. The designed DNA binding proteins are proteins that do not occur in nature, whose design/composition is largely generated by reasonable guidelines. Rational criteria for design include the use of substitution rules and computerized algorithms to process information in database-stored information of existing ZFP, CRISPR and/or TALE designs 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 WO03/016496 and U.S. patent publication Nos. 20110301073, 20110239315 and 20119145940.

A "zinc finger DNA binding protein" (or binding domain) is a protein or region within a larger protein that binds DNA in a sequence-specific manner through one or more zinc fingers, which are regions of amino acid sequence within the binding domain whose structure is stabilized by coordination of zinc ions. The term zinc finger DNA binding protein is often abbreviated as zinc finger protein or ZFP. A zinc finger binding domain may be "engineered" to bind to a predetermined nucleotide sequence. Non-limiting examples of methods for engineering zinc finger proteins are design and selection. The designed zinc finger proteins are proteins that do not exist in nature, and their design/composition is largely generated by rational guidelines. Reasonable criteria for design include the application of substitution rules and computerized algorithms to process existing ZFP designs and to incorporate information in the database storage information of the data. See, e.g., U.S. patent nos. 6,140,081, 6,453,242, 6,534,261, and 6,794,136; see also WO 98/53058, WO 98/53059, WO 98/53060, WO 02/016536 and WO 03/016496.

In other examples, the DNA-binding domain of the one or more nucleases comprises a naturally occurring or engineered (non-naturally occurring) TAL effector DNA-binding domain. See, e.g., U.S. patent publication No. 20110301073, which is incorporated by reference herein in its entirety. Phytopathogenic bacteria of the genus Xanthomonas (Xanthomonas) are known to cause a number of diseases in important crops. Pathogenicity of Xanthomonas depends on a conserved type III secretion (T3S) system that injects more diverse effector proteins into plant cells. Among these injected proteins are transcription activator-like (TALEN) effectors that mimic plant transcriptional activators and manipulate plant transcriptomes (see Kay et al, (2007) Science318: 648-651). These proteins contain a DNA binding domain and a transcriptional activation domain. One of the most well characterized TAL effectors is AvrBs3 from Xanthomonas campestris pepper spot disease-causing variety (Xanthomonas campestris campestgrispv. Vesicatoria) (see Bonas et al, (1989) Mol Gen Genet218:127-136 and WO 2010079430). TAL effectors contain a centralized domain of tandem repeats, each containing approximately 34 amino acids, which are critical to the DNA binding specificity of these proteins. In addition, they contain a nuclear localization sequence and an acidic transcription activation domain (for review see Schornack S et al, (2006) J plant physiol163(3): 256-272). In addition, in the plant pathogenic bacterium Ralstonia solanacearum, two genes designated brg11 and hpx17 in Ralstonia solanacearum biovar strain GMI1000 and biovar 4 strain RS1000 have been found to be homologous to the AvrBs3 family of Xanthomonas (see Heuer et al, (2007) Appl and Envimo 73(13): 4379-. The nucleotide sequences of these genes are 98.9% identical to each other, but differ by the deletion of 1,575bp in the repeat domain of hpx 17. However, both gene products have less than 40% sequence identity to the xanthomonas AvrBs3 family protein. See, e.g., U.S. patent publication No. 20110301073, which is incorporated by reference in its entirety.

The specificity of these TAL effectors depends on the sequence found in the tandem repeat. The repeat sequences comprise approximately 102bp and the repeat sequences are typically 91-100% homologous to each other (Bonas et al, supra). Polymorphisms in the repeat sequences are typically located at positions 12 and 13, and there appears to be a one-to-one correspondence between the identity of the hypervariable di-residues at positions 12 and 13 and the identity of consecutive nucleotides of the target sequence of the TAL effector (see Moscou and Bogdanove, (2009) Science326:1501 and Boch et al, (2009) Science326: 1509-. The natural coding for DNA recognition of these TAL effectors has been experimentally determined such that the HD sequences at positions 12 and 13 bind to cytosine (C), NG binds to T, NI binds to A, C, G or T, NN binds to a or G, and ING binds to T. These DNA-binding repeats have been assembled into proteins with new combinations and numbers of repeats to produce artificial transcription factors that are capable of interacting with the new sequences and activating expression of non-endogenous reporters in plant cells (Boch et al, supra). Engineered TAL proteins have been linked to fokl cleavage half-domains to generate TAL effector domain nuclease fusions (TALENs) that exhibit activity in yeast reporter gene assays (plasmid-based targets).

CRISPR (regularly clustered short palindromic repeats)/Cas (CRISPR-associated) nuclease systems are recently engineered nuclease systems based on bacterial systems that can be used for genome engineering. It is based on part of the adaptive immune response of many bacteria and archaea. When a virus or plasmid invades a bacterium, a segment of the invader's DNA is converted to CRISPR RNA (crRNA) by an "immune" response. This crRNA is then associated with another type of RNA called tracrRNA through a partially complementary region to direct the Cas9 nuclease to a region homologous to the crRNA in the target DNA called the "protospacer". Cas9 cleaves DNA at a site designated by a20 nucleotide guide sequence contained within the crRNA transcript to create a blunt end at the Double Strand Break (DSB). Cas9 requires both crRNA and tracrRNA for site-specific DNA recognition and cleavage. This system has now been engineered such that crRNA and tracrRNA can be combined into one molecule ("single guide RNA"), and the crRNA equivalent of the single guide RNA can be engineered to guide Cas9 nuclease to target any desired sequence (see Jinek et al, (2012) Science 337, p 816-821, Jinek et al, (2013), ehife 2: e00471, and DavidSegal, (2013) ehife 2: e 00563). Thus, the CRISPR/Cas system can be engineered to form a DSB at a desired target in the genome, and repair of the DSB can be affected by the use of repair inhibitors, such that error-prone repair is increased.

In other examples, the DNA-binding transgene is a site-specific nuclease comprising an engineered (non-naturally occurring) meganuclease (also known as a homing endonuclease). Recognition sequences for homing endonucleases or meganucleases such as I-SceI, I-CeuI, PI-PspI, PI-Sce, I-SceIV, I-CsmI, I-PanI, I-SceII, I-PpoI, I-SceIII, I-CreI, I-TevI, I-TevII and I-TevIII are known. See also U.S. patent numbers 5,420,032; U.S. patent nos. 6,833,252; belfort et al, (1997) Nucleic Acids Res.25: 3379-303388; dujon et al, (1989) Gene82: 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-; argast et al, (1998) J.mol.biol.280: 345-353 and New England Biolabs catalog. In addition, the DNA binding specificity of homing endonucleases and meganucleases can be engineered to bind to non-natural target sites. See, e.g., Chevalier et al, (2002) molec. Cell10: 895-905; epinat et al, (2003) Nucleic Acids Res.531: 2952-2962; ashworth et al, (2006) Nature 441: 656-; paques et al, (2007) CurrentGene Therapy7: 49-66; U.S. 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 (i.e., such that the nuclease comprises a homologous cleavage domain) or can be fused to a heterologous cleavage domain.

As used herein, the term "transformation" encompasses all techniques by which a nucleic acid molecule can be introduced into such a cell. Examples include, but are not limited to: transfection with viral vectors; transforming with a plasmid vector; electroporation; carrying out liposome transfection; microinjection (Mueller et al, (1978) Cell 15: 579-85); agrobacterium-mediated transfer; direct DNA uptake; WHISKERS^TM(ii) a mediated transformation; and particle bombardment. These techniques can be used for both stable and transient transformation of plant cells. "Stable transformation" refers to the stable inheritance of a gene resulting from the introduction of a nucleic acid fragment into the genome of a host organism. Once stably transformed, the nucleic acid fragment is stably integrated into the genome of the host organism and any progeny. 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 a host organismIn the cell nucleus or DNA-containing organelle, the gene is expressed without stable inheritance of the gene.

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

As used herein, the term transgenic "event" is generated by the following steps: transforming a plant cell with heterologous DNA (i.e., a nucleic acid construct comprising a transgene of interest), inserting the transgene into the plant genome results in regeneration of the plant population, and selecting for a particular plant characterized by insertion into a particular genomic location. The term "event" refers to the original transformant, which includes the heterologous DNA, as well as the progeny of the transformant. The term "event" also refers to progeny produced by a sexual outcross between a transformant and another variety comprising genomic/transgenic DNA. Even after repeated backcrossing with the backcross parents, the inserted transgene DNA and flanking genomic DNA (genomic/transgene DNA) from the transformed parent are still present in the progeny of the cross at the same chromosomal location. The term "event" also refers to DNA from the original transformant and its progeny, which includes the inserted DNA and flanking genomic sequences immediately adjacent to the inserted DNA, which is expected to be transferred to progeny that receive the inserted DNA including the transgene of interest, including the result of a sexual cross of a parental line including the inserted DNA (e.g., the original transformant and progeny produced by selfing) with a parental line that does not contain the inserted DNA.

As used herein, the term "polymerase chain reaction" or "PCR" defines a procedure or technique for amplifying minute amounts of nucleic acid, RNA and/or DNA, as described in U.S. patent No. 4,683,195, issued 7/28 1987. Generally, sequence information from the end of or beyond the region of interest needs to be available so that oligonucleotide primers can be designed; the sequences of these primers will be identical or similar to the opposite strands of the template to be amplified. The 5' terminal nucleotides of the two primers may coincide with the ends of the amplification material. PCR can be used to amplify specific RNA sequences, specific DNA sequences from total genomic DNA, and cDNA, phage or plasmid sequences transcribed from total cellular RNA, and the like. See generally Mullis et al, Cold Spring harbor Symp. Quant.biol.,51:263 (1987); erlich eds, PCR Technology, (Stockton Press, NY, 1989).

As used herein, the term "primer" refers to an oligonucleotide that is capable of acting as a point of initiation along complementary strand synthesis when conditions are appropriate for synthesis of a primer extension product. The synthesis conditions include the presence of four different deoxyribonucleotide triphosphates and at least one polymerization-inducing agent, such as reverse transcriptase or DNA polymerase. They are present in a suitable buffer which may contain components that act as cofactors or influence conditions such as pH at various suitable temperatures. The primers are preferably single-stranded to optimize amplification efficiency, but double-stranded sequences may also be used.

As used herein, the term "probe" refers to an oligonucleotide that hybridizes to a target sequence. In thatOrIn the type analysis procedure, a probe hybridizes to a portion of the target located between the annealing sites of two primers. The probe comprises about eight nucleotides, about ten nucleotides, about fifteen nucleotides, about twenty nucleotides, about thirty nucleotides, about forty nucleotides, or about fifty nucleotides. In some implementationsIn a version, the probe comprises from about eight nucleotides to about fifteen nucleotides. The probe may also include a detectable label, such as a fluorophore (C: (A))Fluorescein isothiocyanate, etc.). The detectable label may be covalently linked directly to the probe oligonucleotide, e.g., at the 5 'end of the probe or at the 3' end of the probe. Probes comprising a fluorophore may additionally also comprise a Quencher, e.g., Black Hole Quencher^TM、Iowa Black^TMAnd the like.

As used herein, the terms "restriction endonuclease" and "restriction enzyme" refer to bacterial enzymes, each of which cleaves 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.

As used herein, the term "vector" is used interchangeably with the terms "construct", "cloning vector" and "expression vector" and refers to a vector into which a DNA or RNA sequence (e.g., an exogenous gene) can be introduced into a host cell in order to transform the host and facilitate expression (e.g., transcription and translation) of the introduced sequence. "non-viral vector" is intended to mean any vector that does not contain a virus or retrovirus. In some embodiments, a "vector" is a DNA sequence comprising at least one DNA origin of replication and at least one selectable marker gene. Examples include, but are not limited to, plasmids, cosmids, phages, Bacterial Artificial Chromosomes (BACs) or viruses that carry exogenous DNA into cells. The vector may also include one or more genes, antisense molecules, and/or selectable marker genes, as well as other genetic elements known in the art. The vector may transduce, transform, or infect a cell, thereby causing the cell to express the nucleic acid molecule and/or protein encoded by the vector. The term "plasmid" defines a circular nucleic acid chain capable of autosomal replication in a prokaryotic or eukaryotic host cell. The term includes nucleic acids which may be DNA or RNA and which may be single-stranded or double-stranded. A plasmid with this definition may also include a sequence corresponding to a bacterial origin of replication.

As used herein, the term "selectable marker gene" as used herein defines a gene or other expression cassette that encodes a protein that aids in identifying the cell into which the selectable marker gene is inserted.A "selectable marker gene" encompasses, for example, reporter genes and genes used in plant transformation to, for example, protect plant cells from or provide resistance/tolerance to a selective agent.in one embodiment, only those cells or plants that receive a functional selectable marker can divide or grow under conditions with the selective agent.examples of selective agents can include, for example, antibiotics, including spectinomycin (spectinomycin), neomycin (neomycin), kanamycin (kanamycin), paromomycin (paromomycin), gentamycin (gentamicin) and hygromycin (hygromycin). these selectable markers include neomycin phosphotransferase (npt II), which expresses enzymes that confer resistance to antibiotics, and related neomycin, babamycin, gentamycin and G genes; or ptysomycin (gentamicin), or other selectable markers that can confer resistance to a phospho transferase (e.g., luciferase), luciferase, phospho (kanamycin), phospho, luciferase, phospho, luciferase, phospho, etc., or other selectable markers including phospho, and the like.

As used herein, the term "detectable label" refers to a detectable label such as a radioisotope, a fluorescent compound, a bioluminescent compound, a chemiluminescent compound, a metal chelator or an enzyme examples of detectable labels include, but are not limited to, fluorescent labels (e.g., FITC, rhodamine (rhodamine), lanthanide phosphors), enzyme labels (e.g., horseradish peroxidase, β -galactosidase, luciferase, alkaline phosphatase), chemiluminescent labels, biotin groups, predetermined polypeptide epitopes recognized by secondary reporters (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags). in one embodiment, detectable labels may be linked by spacer arms of various lengths to reduce potential steric hindrance.

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 a specific restriction site 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 that the cassette is inserted into the appropriate reading frame for transcription and translation. In one embodiment, an expression cassette can include a polynucleotide encoding a polypeptide of interest, and have elements other than those that facilitate transformation of a particular host cell. In one embodiment, the gene expression cassette may also include elements that allow for enhanced expression of the polynucleotide encoding the polypeptide of interest in a host cell. These elements may include, but are not limited to: promoters, minimal promoters, enhancers, response elements, terminator sequences, polyadenylation sequences, and the like.

As used herein, a "linker" or "spacer" is a bond, molecule or group of molecules that binds two separate entities to each other. The linker and spacer may provide optimal spacing for the two entities, or may also provide an unstable bond that allows the two entities to separate from each other. Labile linkages include photocleavable groups, acid labile moieties, base labile moieties, and enzymatically cleavable groups. As used herein, the term "polylinker" or "multiple cloning site" defines three or more than 10 nucleotides located within each other on a nucleic acid sequenceA plurality of clusters of type 2 restriction enzyme sites. In other instances, the term "polylinker" as used herein refers to a linker that is cloned by any known seamless cloning method (i.e., Gibson)NEBuilder HiFiDNAGolden GateAssembly、Assembly et al) are targeted to join a stretch of nucleotides of both sequences. Constructs comprising polylinkers are used for insertion and/or excision of nucleic acid sequences, such as coding regions of genes.

As used herein, the term "control" refers to a sample used in an analytical procedure for comparative purposes. Controls may be "positive" or "negative". For example, where the purpose of the analytical procedure is to detect differentially expressed transcripts or polypeptides in cells or tissues, it is often preferable to include a positive control, such as a plant sample known to exhibit the desired expression; and negative controls, such as plant samples known to lack the desired expression.

As used herein, the term "plant" includes whole plants and any progeny, cell, tissue or part of a plant. The class of plants useful in the present invention is generally as broad as the class of higher and lower plants that are subjected to mutagenesis, including angiosperms (monocotyledonous and dicotyledonous plants), gymnosperms, ferns, and multicellular algae. Thus, "plant" includes dicotyledons and monocotyledons. The term "plant part" includes any part of a plant, including, for example and without limitation: seeds (including mature seeds and immature seeds); cutting the plants; a plant cell; a plant cell culture; plant organs (e.g., pollen, embryos, flowers, fruits, buds, leaves, roots, stems, silks, and explants). The plant tissue or plant organ may be a seed, a protoplast, a callus, or any other population of plant cells organized into structural or functional units. The plant cell or tissue culture is capable of regenerating a plant having the plant physiological and morphological characteristics of the cell or tissue from which it was obtained, and is capable of regenerating a plant having substantially the same genotype as the plant. In contrast, some plant cells are not capable of regeneration to produce plants. The regenerable cells in the plant cell or tissue culture can be embryos, protoplasts, meristematic cells, callus, pollen, leaves, anthers, roots, root tips, silks, flowers, kernels, ears, cobs, husks, or stalks.

Plant parts include harvestable parts as well as parts suitable for propagation of progeny plants. Plant parts suitable for propagation include, for example and without limitation: seeds, fruits, cuttings, seedlings, tubers and rhizomes. Harvestable parts of a plant may be any useful part of a plant, including for example and without limitation: flowers, pollen, seedlings, tubers, leaves, stems, fruits, seeds, and roots.

Plant cells are the structural and physiological units of plants, including protoplasts and cell walls. Plant cells may be in the form of isolated single cells or aggregates of cells (e.g., friable callus and cultured cells), and may be part of higher tissue units (e.g., plant tissues, plant organs, and plants). Thus, a plant cell may be a protoplast, a gamete producing cell, or a cell or collection of cells that can regenerate into a whole plant. Thus, a seed comprising a plurality of plant cells and capable of regenerating into a whole plant is considered a "plant cell" in embodiments herein.

As used herein, the term "small RNA" refers to many classes of non-coding ribonucleic acids (ncrnas). The term small RNA describes short chain ncrnas produced in bacterial cells, animals, plants and fungi. These short chain ncrnas may be naturally occurring within the cell or may be generated by the introduction of exogenous sequences that express the short chain or ncRNA. Small RNA sequences do not directly encode proteins and differ from other RNAs in function because small RNA sequences are only transcribed but not translated. Small RNA sequences are involved in other cellular functions, including gene expression and modification. Small RNA molecules typically consist of about 20 to 30 nucleotides. Small RNA sequences may be derived from longer precursors. The precursors form structures that fold back on each other at self-complementary regions; they are then processed by the nuclease Dicer in animals or the nuclease DCL1 in plants.

Many types of small RNAs exist in naturally or artificially produced forms, 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 method of genetic regulation in which a gene that is normally supposed to be expressed is "turned off" by an intracellular element (in this case, a small RNA). Proteins that should normally be formed by this genetic information are not formed due to interference and the encoded information in the gene is blocked by expression.

As used herein, the term "small RNA" encompasses mRNAs designated in the literature as "microRNAs" (Storz, (2002) Science296: 1260-3; Illangasekare et al, (1999) RNA 5:1482-1489), prokaryotic "small RNAs" (sRNA) (Wassarman et al, (1999) Trends Microbiol.7:37-45), eukaryotic "non-coding RNAs (ncRNAs)", "microRNAs (miRNAs)", "small non-mRNAs (snmRNAs)", "functional RNAs (fRNAs)", "transfer RNA (tRNA)", "catalytic RNAs" [ e.g., ribozymes, including self-acylated ribozymes (Illangascarare et al, (1999) RNA 5:1482-1489) ], "small nucleolar RNAs (snoRNAs)," tmRNAs "(also designated as" 10S 88RNAs ", Muto et al, (1998) Trends Biocheci.23: 25-29; and Microbiol et al (2001) mS 889; RNAi molecules include, but are not limited to, "small interfering RNAs (siRNAs)", "endoribonuclease-produced siRNAs (e-siRNAs)", "short hairpin RNAs (shRNAs)" and "small time regulatory RNAs (stRNAs)", "cleaved siRNAs (d-siRNAs)" and aptamers; oligonucleotides and other synthetic nucleic acids comprising at least one uracil base.

Unless specifically explained otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Definitions of common terms of molecular biology can be found, for example, in: lewis, 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 (eds.), Molecular Biology and Biotechnology: acidic sensitive Desk Reference, VCH Publishers, Inc.,1995(ISBN 1-56081-.

As used herein, the terms "a" and "an" and "the" include plural referents unless the context clearly dictates otherwise.

Maize chlorophyll a/b binding protein gene regulatory elements and nucleic acids comprising same

Methods and compositions are provided for expressing a non-maize chlorophyll a/b binding protein-like transgene in a plant using a promoter or a 3' UTR from a maize chlorophyll a/b binding protein gene. In one embodiment, the 3'UTR can be the maize chlorophyll a/b binding protein gene 3' UTR of SEQ ID NO: 1.

Transgene expression can be regulated by a 3 '-untranslated gene region (i.e., 3' -UTR) located downstream of the gene coding sequence. Both the promoter and the 3' UTR can regulate transgene expression. While a promoter is necessary to drive transcription, the 3' UTR gene region can terminate transcription and initiate polyadenylation of the resulting mRNA transcript for translation and protein synthesis. The 3' UTR gene region aids in stable expression of the transgene. In one embodiment, the gene expression cassette comprises a 3' -UTR. In one embodiment, the 3'-UTR can be a maize chlorophyll a/b binding protein gene 3' -UTR. In one embodiment, the gene expression cassette comprises a 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 SEQ ID No. 1. In one embodiment, the gene expression cassette comprises a maize chlorophyll a/b binding protein gene 3' -UTR operably linked to a transgene. In an exemplary embodiment, the gene expression cassette comprises a 3' -UTR operably linked to a transgene, wherein the transgene can be an insect 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 a combination thereof.

In one embodiment, the gene expression cassette comprises a 3' UTR from a maize chlorophyll a/b binding protein gene and 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:2(US 005656496). In one embodiment, the gene expression cassette comprises a 3' UTR from a zea mays chlorophyll a/b binding protein gene, and a promoter, wherein the promoter is from a zea mays chlorophyll a/b binding protein gene. In one embodiment, the gene expression cassette comprises a 3' UTR from a maize chlorophyll a/b binding protein gene, and a promoter, wherein the promoter is derived from a plant (e.g., a maize chlorophyll a/b binding gene promoter or a maize ubiquitin 1 promoter), a virus (e.g., a cassava vein virus promoter), or a bacterium (e.g., agrobacterium tumefaciens Δ mas). In an exemplary embodiment, the gene expression cassette comprises a maize chlorophyll a/b binding protein gene 3' UTR operably linked to a transgene, wherein the transgene can be an insect resistant 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 a combination thereof.

In one embodiment, the nucleic acid vector comprises a gene expression cassette as disclosed herein. In one embodiment, the vector may be a plasmid, cosmid, Bacterial Artificial Chromosome (BAC), phage, virus, or sheared polynucleotide fragment suitable for direct transformation or gene targeting, such as to donor DNA.

According to one embodiment, there is provided a nucleic acid vector comprising a recombinant gene expression cassette, wherein the recombinant gene expression cassette comprises: a maize chlorophyll a/b binding protein gene 3' UTR, a non-maize chlorophyll a/b binding protein gene, or a combination thereof, operably linked to a polylinker sequence. In one embodiment, the recombinant gene cassette comprises a maize chlorophyll a/b binding protein gene 3' UTR operably linked to a non-maize chlorophyll a/b binding protein gene. In one embodiment, a recombinant gene cassette comprises a maize chlorophyll a/b binding protein gene 3' UTR as disclosed herein operably linked to a polylinker sequence. The polylinker is operably linked to the 3' UTR of the maize chlorophyll a/b binding protein gene in a manner such that insertion of the coding sequence into a restriction site of the polylinker will be operably linked to the coding sequence, thereby allowing expression of the coding sequence when the vector is transformed or transfected into a host cell.

According to one embodiment, a nucleic acid vector is provided comprising a gene cassette consisting of a gene promoter, a non-maize chlorophyll a/b binding protein gene, and a maize chlorophyll a/b binding protein gene 3' -UTR of SEQ ID NO: 1. In one embodiment, the maize chlorophyll a/b binding protein gene 3'-UTR of SEQ ID NO. 1 is operably linked to the 3' terminus of a non-maize chlorophyll a/b binding protein gene transgene. In another embodiment, the 3' untranslated sequence comprises SEQ ID NO 1 or a sequence having 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO 1. According to one embodiment, there is provided a nucleic acid vector comprising a gene cassette consisting of a promoter, a non-zea mays chlorophyll a/b binding protein gene and a 3'UTR, wherein the promoter is operably linked to the 5' end of the non-zea mays chlorophyll a/b binding protein gene and the 3'UTR of SEQ ID NO:1 is operably linked to the 3' end of the non-zea mays chlorophyll a/b binding protein gene. In another embodiment, the 3' untranslated sequence comprises SEQ ID NO 1 or a sequence having 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO 1. In another embodiment, the 3' untranslated sequence consists of SEQ ID NO 1 or a 1000bp sequence having 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO 1.

In one embodiment, a nucleic acid construct is provided comprising a promoter and a non-zea mays chlorophyll a/b binding protein gene and optionally one or more of the following elements:

a) a 5' untranslated region;

b) an intron; and

c) a 3' untranslated region of a non-translated region,

wherein,

the promoter consists of SEQ ID NO. 2 or known promoter sequences such as the maize chlorophyll a/b binding protein gene promoter;

the intron region consists of a known intron sequence; and

the 3' untranslated region consists of SEQ ID NO 1 or a sequence with 98% sequence identity with SEQ ID NO 1; further wherein the promoter is operably linked to the transgene, and each optional element (if present) is also operably linked to both the promoter and the transgene. In another embodiment, a transgenic cell is provided comprising the nucleic acid construct disclosed immediately above. In one embodiment, the transgenic cell is a plant cell, and in another embodiment, a plant is provided, wherein the plant comprises the transgenic cell.

In one embodiment, a nucleic acid construct is provided comprising a promoter and a non-zea mays chlorophyll a/b binding protein transgene and optionally one or more of the following elements:

a) an intron; and

b) a 3' untranslated region of a non-translated region,

wherein,

the intron region consists of a known intron sequence;

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 an Agrobacterium T-DNA border. According to one embodiment, the recombinant gene cassette further comprises a first and a second T-DNA border, wherein the first T-DNA border is operably linked to one end of the gene construct and the second T-DNA border is operably linked to the other end of the gene construct. The first and second Agrobacterium T-DNA borders may be independently selected from T-DNA border sequences derived from a bacterial strain selected from the group consisting of: a nopaline-synthesizing Agrobacterium T-DNA border, an octopine-synthesizing Agrobacterium T-DNA border, a mannopine-synthesizing Agrobacterium T-DNA border, an amber-synthesizing Agrobacterium T-border, or any combination thereof. In one embodiment, an agrobacterium strain selected from a nopaline synthetic strain, a mannopine synthetic strain, a succinine synthetic strain, or an octopine synthetic strain is provided, wherein the strain comprises a plasmid, wherein the plasmid comprises a sequence operably linked to a sequence selected from SEQ ID No. 1 or a sequence having 80, 85, 90, 95, or 99% sequence identity to SEQ ID No. 1.

Transgenes of interest suitable for use in the constructs disclosed herein include, but are not limited to, those that confer (1) resistance to a pest or disease; (2) tolerance to herbicides; (3) agronomic traits of increased value such as yield improvement, nitrogen use efficiency, water use efficiency and nutritional quality; (4) proteins bind to DNA in a site-specific manner; (5) expressing the small RNA and (6) coding sequence of the selectable marker. According to one embodiment, the transgene encodes a selectable marker or a gene product that confers insecticidal resistance, herbicide tolerance, small RNA expression, nitrogen use efficiency, water use efficiency, or nutritional quality.

1. Insect resistance

Various selectable markers, also referred to as reporter genes, can be operably linked to the maize chlorophyll a/b binding protein gene 3' UTR comprising SEQ ID NO 1 or a sequence having 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO 1. The operably linked sequences can then be incorporated into a selected vector to allow for the identification and selection of transformed plants ("transformants"). Exemplary insect resistance coding sequences are known in the art. As embodiments of insect resistance coding sequences that may be operably linked to the regulatory elements of the present disclosure, the following traits are provided. Coding sequences that provide resistance to exemplary Lepidopteran (Lepidopteran) insects include: cry1A, cry1A.105, cry1Ab, cry1Ab (truncated), cry1Ab-Ac (fusion protein), cry1Ac (toSold), cry1C, cry1F (toSold), cry1Fa2, cry2Ab2, cry2Ae, cry9C, mocry1F, pinII (protease inhibitor protein), vip3A (a), and vip3Aa 20. Coding sequences that provide exemplary Coleopteran (Coleopteran) insect resistance include: cry34Ab1 (to)Sold), cry35Ab1 (toSold), cry3A, cry3Bb1, dvsnf7, and mcry 3A. Coding sequences that provide exemplary multiple insect resistance include ecry31. ab. The above list of insect resistance genes is not meant to be limiting. The present disclosure encompasses any insect resistance gene.

2. Tolerance to herbicides

Various selectable markers, also known as reporter genes, can be operably linked to maize chlorophyll a/b knotsA polyprotein gene 3'UTR, the 3' UTR comprising SEQ ID No. 1 or a sequence having 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID No. 1. The operably linked sequences can then be incorporated into a selected vector to allow for the identification and selection of transformed plants ("transformants"). Exemplary herbicide tolerance coding sequences are known in the art. As embodiments of herbicide tolerance coding sequences that can be operably linked to the regulatory elements of the present disclosure, the following traits are provided. Glyphosate herbicides contain a mode of action by inhibiting EPSPS enzyme (5-enolpyruvylshikimate-3-phosphate synthase). This enzyme is involved in the biosynthesis of aromatic amino acids essential for plant growth and development. Various enzymatic mechanisms that can be used to inhibit the enzyme are known in the art. Genes encoding such enzymes may be operably linked to the gene regulatory elements of the present disclosure. In one embodiment, selectable marker genes include, but are not limited to, genes encoding glyphosate resistance genes including: mutant EPSPS genes such as 2mEPSPS gene, cp4EPSPS gene, mEPSPS gene, dgt-28 gene, aroA gene; and glyphosate degrading genes such as glyphosate acetyltransferase gene (gat) and glyphosate oxidase gene (gox). These traits are currently expressed in Gly-Tol^TM、 GT and RoundupAnd (5) selling. Resistance genes for glufosinate and/or pilaf (bialaphos) compounds include the dsm-2, bar and pat genes. The bar and pat traits are currently as followsAnd (5) selling. Also included are tolerance genes that provide resistance to 2,4-D, such as the aad-1 gene (note that the aad-1 gene has other activity against aryloxyphenoxypropionate herbicides) and the aad-12 gene (note that the aad-12 gene has activity against pyridyloxyacetate synthetic plantsOther activities of auxins). These traits are as followsAnd selling crop protection technologies. Resistance genes for ALS inhibitors (sulfonylureas, imidazolinones, triazolopyrimidines, pyrimidylthiobenzoates and sulfonylamino-carbonyl-triazolinones) are known in the art. These resistance genes are most often caused by point mutations in the ALS-encoding gene sequence. Other ALS inhibitor resistance genes include the hra gene, the csr1-2 gene, the Sr-HrA gene, and the surB gene. Some of the traits are trademarksAnd (5) selling. Herbicides that inhibit HPPD include pyrazolones such as pyrazoxyfen (pyrazoxyfen), topramezone (benzofenap), and topramezone (topramezone); triketones such as mesotrione (mesotrione), sulcotrione (sulcotrione), tembotrione (tembotrione), benzobicyclon (benzobicyclon); and diketonitriles such as isoxaflutole (isoxaflutole). These exemplary HPPD herbicides can be tolerated by known traits. Examples of HPPD inhibitors include the hppdPF _ W336 gene (for resistance to isoxaflutole) and the avhppd-03 gene (for resistance to mesotrione). Examples of benzonitrile herbicide tolerance trait include the bxn gene, which was shown to confer resistance to the herbicide/antibiotic bromoxynil (bromoxynil). Resistance genes for zikstroemia indica (dicamba) include the zikstroemia indica monooxygenase gene (dmo) as disclosed in international PCT publication No. WO 2008/105890. The genes for PPO or PROTOX inhibitor type herbicides (e.g. acifluorfen (acifluorfen), butafenacil (butafenacil), butafenacil (flupropazoil), pentoxazone (pentoxazone), carfentrazone (carfentrazone), isopyrafen (fluzolate), pyraflufen (pyraflufen), aclonifen (aclonifen), oxafenadine (azafenidin), flumioxazin (flumioxazin), fluroxypyr (fluiclorac), bifenox (bifenox), oxyfluorfen (oxyfluorfen), lactofen (lactofen), fomesafen (mesafen), fluoroglycofen (fluoroglycofen) and sulfentrazone (sulfazone)) are known in the art for resistance. Exemplary genes conferring resistance to PPO include wildryeThe protogenic Arabidopsis PPO enzymes (Lermontova I and Grimm B, (2000) Overexpression of plastic prototropic enzyme IX oxidase leads to resistance to the diene-ether biochemical enzyme, plant physiological 122: 75-83.), Bacillus subtilis (B.Subtilis) PPO genes (Li, X. and Nichol D.2005.development of PPO inhibitor-resistant cultures and copolymers, plant Manual. Sci.61: 277) and Choi KW, Han O, LeehJ, Yun YC, MoMK, Kim, Kuk YI, Han SU and Guh JO, (1998) amplification of biochemical enzyme, 2. promoter, DNA. Resistance genes for pyridyloxy or phenoxypropionic acid and cyclohexanone include ACCase inhibitor-encoding genes (e.g., Acc1-S1, Acc1-S2, and Acc 1-S3). Exemplary genes conferring resistance to cyclohexanedione and/or aryloxyphenoxypropionic acid include haloxyfop-p-butyl, diclofop-p-ethyl, fenoxaprop-p-ethyl (fenoxyprop), fluazifop-p-butyl (fluzifop) and quizalofop-ethyl (quizalofop). Finally, herbicides that inhibit photosynthesis (including triazines or benzonitrile) provide tolerance through the psbA gene (tolerance to triazines), the 1s + gene (tolerance to triazines), and the nitrilase gene (tolerance to benzonitrile). The above list of herbicide tolerance genes is not meant to be limiting. The present disclosure encompasses any herbicide tolerance gene.

3. Agronomic traits

Various selectable markers, also known as reporter genes, may be operably linked to the maize chlorophyll a/b binding protein gene 3'UTR, the 3' UTR comprising SEQ ID NO:1 or a sequence having 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO:1 the operably linked sequences may then be incorporated into selected vectors to allow identification and selection of transformed plants ("transformants") exemplary agronomic trait coding sequences are known in the art as embodiments of agronomic traits encoding sequences that may be operably linked to regulatory elements of the present disclosure, the following traits are provided as delayed fruit softening, as provided by the genes, inhibits the production of polygalacturonases responsible for pectin molecule breakage in the cell wall, thereby resulting in delayed fruit softening, additionally, delayed fruit ripening/aging of the acc genes is used to inhibit normal expression of the native acc synthase genes, thereby resulting in reduced ethylene production and delayed fruit ripening, however, the accd genes metabolize precursors of the fruit hormones, thereby resulting in delayed fruit ripening or the SAM-producing ethylene-alpha-beta-alpha-beta-alpha-.

DNA binding proteins

Various selectable markers, also referred to as reporter genes, can be operably linked to the maize chlorophyll a/b binding protein gene 3' UTR comprising SEQ ID NO 1 or a sequence having 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO 1. The operably linked sequences can then be incorporated into a selected vector to allow for the identification and selection of transformed plants ("transformants"). Exemplary DNA binding protein coding sequences are known in the art. As embodiments of DNA binding protein coding sequences that may be operably linked to the regulatory elements of the present disclosure, the following types of DNA binding proteins may be included: zinc fingers, Talen, CRISPR, and meganucleases. The above list of DNA binding protein coding sequences is not meant to be limiting. The present disclosure encompasses any DNA binding protein coding sequence.

5. Small RNAs

Various selectable markers, also referred to as reporter genes, can be operably linked to the maize chlorophyll a/b binding protein gene 3' UTR comprising SEQ ID NO 1 or a sequence having 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO 1. The operably linked sequences can then be incorporated into a selected vector to allow for the identification and selection of transformed plants ("transformants"). Exemplary small RNA traits are known in the art. As embodiments of small RNA coding sequences that can be operably linked to the regulatory elements of the present disclosure, the following traits are provided. For example, anti-efe small RNAs that delay fruit ripening/senescence inhibit ethylene production by silencing of ACO genes encoding ethylene forming enzymes to delay ripening. The ccomt micrornas that alter lignin production are produced by inhibiting endogenous S-adenosyl-L-methionine: trans caffeoyl-CoA 3-O-methyltransferase (CCOMT gene) to reduce the content of guaiacyl (G) lignin. In addition, black spot lesion tolerance in Solanum verrucosum can be reduced by triggering the degradation of Ppo5 transcript by Ppo5 small RNA to block the appearance of black spot lesions. Also included are dvsnf7 small RNAs that inhibit western corn rootworm together with dsRNA containing a 240bp fragment of the western corn rootworm Snf7 gene. Modified starch/carbohydrates can be produced from small RNAs such as pPhL small RNA (degrading the PhL transcript to limit the formation of reducing sugars by starch degradation) and pR1 small RNA (degrading the R1 transcript to limit the formation of reducing sugars by starch degradation). In addition, the benefits of small RNA production such as acrylamide reduction by Asn1 triggered degradation of Asn1 to attenuate asparagine formation and reduce polyacrylamide. Finally, pgas PPO inhibits the non-browning phenotype of the small RNA resulting in inhibition of PPO, thereby producing apples with a non-browning phenotype. The above list of small RNAs is not meant to be limiting. The present disclosure encompasses any small RNA coding sequence.

6. Selectable marker

Various selectable markers, also known as reporter genes, can be operably linked to the maize chlorophyll a/b binding protein gene 3'UTR, the 3' UTR comprising SEQ ID NO:1 or a sequence having 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO:1 the operably linked sequences can then be incorporated into a selected vector to allow identification and selection of transformed plant transformants ("transformants"), a number of methods can be used to confirm expression of the selectable marker in the transformed plant including, for example, DNA sequencing and PCR (polymerase chain reaction), Southern blotting, northern blotting, immunological methods for detecting proteins expressed by the vector, however, the reporter genes are typically observed by visual observation of proteins that produce colored products upon expression. 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, etc. (see Sambroror et al, incorporated by Harbourn, Inc. 2001, Inc. in its entirety.

Selection of transformed cells or tissues is performed using a selectable marker gene. Selectable marker genes include genes encoding antibiotic resistance, such as genes encoding neomycin phosphotransferase ii (neo), spectinomycin/streptomycin resistance (AAD), and hygromycin phosphotransferase (HPT or HGR), as well as genes conferring resistance to herbicidal compounds. Herbicide tolerance genes typically encode modified target proteins that are insensitive to the herbicide or enzymes that degrade or detoxify the herbicide in plants before it can function. For example, resistance to glyphosate has been obtained by using a gene encoding the mutant target enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). The genes and mutants of EPSPS are well known and are described further below. Resistance to glufosinate, bromoxynil and 2, 4-dichlorophenoxyacetate (2,4-D) has been obtained by using bacterial genes encoding PAT or DSM-2, nitrilase, AAD-1 or AAD-12, each of which is an example of a protein that detoxifies its corresponding herbicide.

In one embodiment, herbicides can inhibit the growth site or meristem, including imidazolinones or sulfonylureas, and acetohydroxyacid synthase (AHAS) and acetolactate synthase (ALS) resistance/tolerance genes for these herbicides are well known. Glyphosate tolerance genes include mutant 5-enolpyruvylshikimate-3-phosphate synthase (EPSP) and dgt-28 genes (by in vivo mutagenesis of various forms of the introduced recombinant nucleic acid and/or native EPSP gene), 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 viridochromogenes, as well as pyridyloxy or phenoxypropionic acid and cyclohexanone (accase inhibitor encoding genes). Exemplary genes conferring resistance to cyclohexanedione and/or aryloxyphenoxypropionic acid (including haloxyfop-p-butyl, diclofop-methyl, fenoxaprop-p-ethyl, fluazifop-p-butyl, quizalofop-ethyl) include the gene for acetyl-coa carboxylase (accase); acc1-S1, Acc1-S2 and Acc 1-S3. In one embodiment, the herbicide may inhibit photosynthesis, including triazines (psbA and 1s + genes) or benzonitrile (nitrilase gene). In addition, such selectable markers may include positive selectable markers such as phosphomannose isomerase (PMI).

In one embodiment, selectable marker genes include, but are not limited to, genes encoding: 2,4-D, neomycin phosphotransferase II, cyanamide hydratase, aspartokinase, dihydrodipicolinate synthase, tryptophan decarboxylase, dihydrodipicolinate synthase and desensitized aspartokinase, the bar gene, tryptophan decarboxylase, neomycin phosphotransferase (NEO), hygromycin phosphotransferase (HPT or HYG), dihydrofolate reductase (DHFR), glufosinate acetyltransferase, 2-dichloropropionate dehalogenase, acetohydroxyacid synthase, 5-enolpyruvylshikimate-phosphate synthase (aroA), haloaryl nitrilase, acetyl-coa carboxylase, dihydrofolate synthase (sul I) and 32kD photosystem II polypeptide (psbA). One embodiment further comprises a selectable marker gene encoding resistance to: chloramphenicol, methotrexate, hygromycin, spectinomycin, bromoxynil, glyphosate, and glufosinate. The above list of selectable marker genes is not meant to be limiting. The present disclosure encompasses any reporter gene or selectable marker gene.

In some embodiments, a coding sequence that is optimally expressed in a plant is synthesized. For example, in one embodiment, the coding sequence of a gene has been modified by codon optimization to enhance expression in a plant. An insect-resistant transgene, herbicide-tolerant transgene, nitrogen use efficiency transgene, water use efficiency transgene, nutritional quality transgene, DNA binding transgene, or selectable marker transgene may be optimized for expression in a particular plant species, or may be modified for optimal expression in dicotyledonous or monocotyledonous plants. Plant-preferred codons can be determined by the highest frequency codon in the protein expressed in the greatest amount in the particular plant species of interest. In one embodiment, the coding sequence, gene or transgene is designed to be expressed at higher levels in plants, resulting in higher transformation efficiencies. Methods for plant optimization of genes are well known. Guidance regarding optimization and generation of synthetic DNA sequences can be found, for example, in WO2013016546, WO2011146524, WO1997013402, U.S. patent No. 6166302, and U.S. patent No. 5380831, which are incorporated by reference herein.

Transformation of

Methods suitable for plant transformation include any method by which DNA can be introduced into a cell, such as, without limitation: electroporation (see, e.g., U.S. Pat. No. 5,384,253); microprojectile bombardment (see, e.g., U.S. Pat. nos. 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. Pat. Nos. 5,635,055, 5,824,877, 5,591,616; 5,981,840 and 6,384,301); and protoplast transformation (see, e.g., U.S. Pat. No. 5,508,184).

DNA constructs can be introduced directly into the genomic DNA of plant cells using techniques such as agitation with silicon carbide fibers (see, e.g., U.S. Pat. Nos. 5,302,523 and 5,464,765), or DNA constructs can be introduced directly into plant tissue using gene gun methods such as DNA particle bombardment (see, e.g., Klein et al (1987) Nature 327: 70-73). Alternatively, the DNA construct may be introduced into the plant cell by transformation with a nanoparticle (see, e.g., U.S. patent publication No. 20090104700, which is incorporated herein by reference in its entirety).

In addition, gene transfer can be achieved using non-Agrobacterium bacteria or viruses, such as Rhizobium (Rhizobium sp.) NGR234, Sinorhizobium meliloti (Sinorhizobium meliloti), Rhizobium loti (Mesorhizobium loti), Potato Virus X, Cauliflower mosaic virus and cassava vein mosaic virus and/or tobacco mosaic virus, see, e.g., Chung et al (2006) Trends Plant Sci.11(1): 1-4.

By applying transformation techniques, cells of virtually any plant species can be stably transformed, and these 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. Pat. nos. 5,846,797, 5,159,135, 5,004,863, and 6,624,344; techniques particularly useful for transforming brassica plants are described, for example, in us patent 5,750,871; techniques for transforming soybeans are described, for example, in U.S. Pat. nos. 6,384,301; techniques for transforming maize are described, for example, in U.S. Pat. Nos. 7,060,876 and 5,591,616 and International PCT publication No. WO 95/06722.

After efficient delivery of exogenous nucleic acid to recipient cells, transformed cells are typically identified for further culture and plant regeneration. In order to improve the ability to identify transformants, the skilled person may need to use a selectable marker gene as well as a transformation vector for generating the transformants. In an exemplary embodiment, the population of transformed cells can be analyzed by exposing the cells to one or more selective agents, or the cells can be screened for a desired marker gene trait.

Cells that survive exposure to the selection agent, or cells that have been scored positive in the screening assay, can be placed in culture in a medium that supports plant regeneration. In one embodiment, any suitable plant tissue culture medium may be modified by including other substances such as growth regulators. The tissue can be maintained on a basal medium with growth regulators until sufficient tissue is available to initiate plant regeneration work, or after repeated rounds of manual selection until the tissue morphology is suitable for regeneration (e.g., for at least 2 weeks), and then transferred to a medium conducive to shoot formation. The cultures were transferred periodically until sufficient shoot formation had occurred. Once a shoot is formed, it is transferred to a medium conducive to root formation. Once sufficient roots are formed, the plants can be transferred to soil for further growth and maturation.

Molecular validation

Transformed plants, calli, tissues or plants can be identified and isolated by selecting or screening engineered plants for traits encoded by marker genes present on the transformed DNA.

Molecular beacons for use in sequence detection have been described. Briefly, FRET oligonucleotide probes are designed to overlap flanking genomic and insert DNA junctions. The unique structure of the FRET probe allows it to contain a secondary structure that keeps the fluorescent and quenching moieties in close proximity. The FRET probe and PCR primers (one 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 to the target sequence results in removal of the probe secondary structure and spatial separation of the fluorescent moiety from the quencher moiety. The fluorescent signal indicates the presence of flanking genomic/transgene insert sequences due to successful amplification and hybridization. Such molecular beacon assays for detection in the form of an amplification reaction are embodiments of the present disclosure.

Hydrolysis Probe analysis Or(Life Technologies, Foster City, Calif.) is a method for detecting and quantifying the presence of DNA sequences. Briefly, a FRET oligonucleotide probe is designed with one oligonucleotide within the transgene, and one oligonucleotide in the flanking genomic sequence for event-specific detection. The FRET probe and PCR primers (one 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 causes cleavage and release of the fluorescent moiety on the FRET probe away from the quencher moiety. The fluorescent signal indicates the presence of flanking/transgene insert sequences due to successful amplification and hybridization. Such hydrolysis probe assays for detection in the form of an amplification reaction are embodiments of the present disclosure.

Analysis is a method of detecting and quantifying the presence of DNA sequences. In short, use is calledPolymerase Chain Reaction (PCR) -based analysis of the assay system genomic DNA samples containing integrated gene expression cassette polynucleotides are screened. For the practice of the present disclosureAssays may utilize primers containing multipleThe mixture was analyzed by PCR. Primers for PCR analysis mixtures may be included inAt 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. In addition, the primers used in the PCR analysis mixture may comprise at least one forward primer and at least one reverse primer. For example,the PCR analysis mixture may use two forward primers and one reverse primer corresponding to two different alleles. One forward primer 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. The reverse primer contains a sequence corresponding to a specific region of the genomic sequence. Such methods for detecting amplification reactionsThe assay is one embodiment of the present disclosure.

In some embodiments, the fluorescent signal or fluorescent dye is selected from the group consisting of: HEX fluorochrome, FAM fluorochrome, JOE fluorochrome, TET fluorochrome, Cy 3 fluorochrome, Cy 3.5 fluorochrome, Cy 5 fluorochrome, Cy 5.5 fluorochrome, Cy7 fluorochrome and ROX fluorochrome.

In other embodiments, the amplification reaction is performed using a suitable second fluorescent DNA dye capable of staining cellular DNA in a concentration range detectable by flow cytometry and having a fluorescence emission spectrum detectable by a real-time thermal cycler. It will 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 excitation and emission spectra may be employed, such asSYTOXSYBR Green Andin one embodiment, the second fluorescent DNA dye is used at less than 10. mu.M, less than 4. mu.M, or less than 2.7. mu.M

In other embodiments, the detection may be performed using Next Generation Sequencing (NGS). DNA sequence analysis can be used to determine the nucleotide sequence of the isolated and amplified fragments as described by Brautigma et al, 2010. The amplified fragments can be isolated and subcloned into vectors and sequenced using the chain terminator method (also known as Sanger sequencing) or dye terminator sequencing method. In addition, amplicons can be sequenced using next generation sequencing methods. NGS technology does not require a subcloning step, and multiple sequencing reads can be done in a single reaction. Three NGS platforms are commercially available, genomeSequencer FLX from 454Life Sciences/Roche^TMIllumina Genome Analyser from Solexa^TMAnd SOLID from Applied Biosystems^TM('prefix of Sequencing by extension restriction and Detection'). In addition, there are two single molecule sequencing methods currently under development. These methods include those from Helicos Bioscience^TMTrue Single Molecule Sequencing (tSMS) and Single Molecule real time from Pacific Biosciences^TMSequencing (SMRT).

Genome Sequencher FLX sold by 454Life Sciences/Roche^TMIs a long-read NGS that uses emulsion PCR and pyrosequencing to generate sequencing reads. A300-800 bp DNA fragment or a library containing 3-20kb fragments can be used. The reaction can generate over one million reads of about 250 to 400 bases per run, yielding a total of 250 to 400 megabases. This technique produces the longest read, but each runIs lower compared to other NGS technologies.

Solexa^TMIllumina Genome Analyser sold^TMIs a short read NGS that is sequenced using a synthetic method that utilizes fluorescent dye-labeled reversible terminator nucleotides and is based on solid phase bridge PCR. Paired-end sequencing libraries containing DNA fragments of up to 10kb can be used for construction. The reaction produced over 1 hundred million short reads, with reads ranging from 35 to 76 bases in length. This data can yield 3-6 gigabases per run.

Applied Biosystems^TMSequencing by the oligonucleotide ligation and detection (SOLiD) system is marketed as a short read technique. The NGS technique uses fragmented double stranded DNA up to 10kb in length. The system uses sequencing by dye-labeled oligonucleotide primer ligation and emulsion PCR to generate billions of short reads, yielding a total sequence output of up to 30 gigabases per run.

Helicos Bioscience^TMtSMS and Pacific Biosciences of^TMThe SMRT of (1) applies a different method of performing a sequence reaction using a single DNA molecule. tSMS Helicos^TMEach run of the system produced up to 8 hundred million short reads, yielding 21 gigabases. These reactions are performed using a fluorescent dye-labeled virtual terminator nucleotide, which is referred to as a "sequencing-by-synthesis" method.

Pacific Biosciences^TMThe marketed SMRT next generation sequencing system uses real-time sequencing by synthesis. This technique can produce reads up to 1,000bp in length, as it is not limited by reversible terminators. Using this technique, raw read throughput equivalent to one-fold coverage of a diploid human genome can be generated daily.

In another embodiment, detection can be accomplished using blot analysis, including Western blots, Northern blots, and Southern blots. Such blot analysis is a common technique used in biological research for the identification and quantification of biological samples. These analyses involve first separating sample components in a gel by electrophoresis, and then transferring the electrophoretically separated components from the gel to a transfer membrane made of a material such as nitrocellulose, polyvinylidene fluoride (PVDF), or Nylon (Nylon). Analytes can also be spotted directly on these carriers or directed to specific areas on the carriers by applying vacuum, capillary action or pressure without prior separation. The transfer film is then typically subjected to post-transfer processing to enhance the ability to distinguish and detect analytes from each other, either visually or by an automated reader.

In another embodiment, detection can be accomplished using an ELISA assay that uses a solid phase enzyme immunoassay to detect the presence of a substance (typically an antigen) in a liquid or wet sample. Antigens from the sample are attached to the plate surface. Then, another specific antibody is applied to the surface so that it can bind to the antigen. The antibody is linked to an enzyme and in a final step a substance containing a substrate for the enzyme is added. Subsequent reactions produce a detectable signal, most commonly a color change in the substrate.

Transgenic plants

In one embodiment, the plant, plant tissue or plant cell comprises the maize chlorophyll a/b binding protein gene 3' UTR. In one embodiment, the plant, plant tissue or plant cell comprises a maize chlorophyll a/b binding protein gene 3' UTR having a sequence selected from SEQ ID No. 1 or a sequence having 80%, 85%, 90%, 95% or 99.5% sequence identity to a sequence selected from SEQ ID No. 1. In one embodiment, the plant, plant tissue or plant cell comprises a gene expression cassette comprising a sequence selected from SEQ ID No. 1, or a sequence having 80%, 85%, 90%, 95% or 99.5% sequence identity to a sequence selected from SEQ ID No. 1 operably linked to a non-maize chlorophyll a/b binding protein gene. In an exemplary embodiment, a plant, plant tissue, or plant cell comprises a gene expression cassette comprising a maize chlorophyll a/b binding protein gene 3' UTR operably linked to a transgene, wherein the transgene can be an insect-resistant transgene, a herbicide-tolerant transgene, a nitrogen use efficiency transgene, a water use efficiency transgene, a nutritional quality transgene, a DNA binding transgene, a selectable marker transgene, or a combination thereof.

According to one embodiment, a plant, plant tissue or plant cell is provided, wherein the plant, plant tissue or plant cell comprises a gene derived from the 3'UTR of a maize chlorophyll a/b binding protein gene operably linked to a transgene, wherein the gene derived from the 3' UTR of a maize chlorophyll a/b binding protein gene comprises the sequence of SEQ ID No. 1 or a sequence having 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 operably linked to a non-maize chlorophyll a/b binding protein gene or a sequence having 80%, 85%, 90%, 95%, or 99.5% sequence identity to SEQ ID No. 1. In one embodiment, the plant, plant tissue or plant cell is a dicot or monocot or a cell or tissue derived from a dicot or monocot. In one embodiment, the plant is selected from the group consisting of maize, wheat, rice, sorghum, oat, rye, banana, sugarcane, soybean, cotton, sunflower and canola. In one embodiment, the plant is maize. According to one embodiment, the plant, plant tissue or plant cell comprises SEQ ID No. 1 operably linked to a non-maize chlorophyll a/b binding protein gene or a sequence having 80%, 85%, 90%, 95% or 99.5% sequence identity to SEQ ID No. 1. In one embodiment, the plant, plant tissue or plant cell comprises a promoter operably linked to a transgene, wherein the promoter consists of SEQ ID No. 1 or a sequence having 80%, 85%, 90%, 95% or 99.5% sequence identity to SEQ ID No. 1. According to one embodiment, a genetic construct comprising a maize chlorophyll a/b binding protein gene 3' UTR sequence operably linked to a transgene is incorporated into the genome of a plant, plant tissue or plant cell.

In one embodiment, a plant, plant tissue, or plant cell according to the methods disclosed herein can be a dicot. The dicot, plant tissue or plant cell can be, but is not limited to, alfalfa, rapeseed, canola, brassica juncea, soybean, sunflower, cotton, kidney bean, broccoli, cabbage, cauliflower, celery, cucumber, eggplant, lettuce; melon, pea, pepper, peanut, potato, pumpkin, radish, spinach, beet, sunflower, tobacco, tomato, and watermelon.

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

The present disclosure also encompasses a seed of the transgenic plant described above, wherein the seed has a transgene or a genetic construct containing a gene regulatory element of the present disclosure. The present disclosure also encompasses progeny, clones, cell lines or cells of the transgenic plants described above, wherein the progeny, clones, cell lines or cells have a transgene or a genetic construct containing the genetic regulatory elements of the present disclosure.

The present disclosure also encompasses the cultivation of a transgenic plant as described above, wherein the transgenic plant has a transgene or a genetic construct containing a gene regulatory element of the present disclosure. Thus, such transgenic plants can be engineered by transformation with a nucleic acid molecule according to the present disclosure, particularly transgenic events having one or more desired traits or containing a gene regulatory element of the present disclosure, and can be pruned or cultivated by any method known to those of skill in the art.

Method for expressing transgenes

In one embodiment, a method of expressing at least one transgene in a plant comprises growing a plant comprising a maize chlorophyll a/b binding protein gene 3' UTR operably linked to at least one transgene or polylinker sequence. In one embodiment, the maize chlorophyll a/b binding protein gene 3' UTR consists of a sequence selected from SEQ ID NO. 1 or a sequence having 80%, 85%, 90%, 95% or 99.5% sequence identity to a sequence selected from SEQ ID NO. 1. In one embodiment, a method of expressing at least one transgene in a plant comprises growing a plant comprising a maize chlorophyll a/b binding protein gene promoter and a maize chlorophyll a/b binding protein gene 3' UTR operably 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 3' UTR of a maize chlorophyll a/b binding protein gene operably linked to at least one transgene.

In one embodiment, a method of expressing at least one transgene in a plant comprises growing a plant comprising a gene expression cassette comprising a 3' UTR of a maize chlorophyll a/b binding protein gene operably linked to at least one transgene. In one embodiment, the maize chlorophyll a/b binding protein gene 3' UTR consists of a sequence selected from SEQ ID NO. 1 or a sequence having 80%, 85%, 90%, 95% or 99.5% sequence identity to a sequence selected from SEQ ID NO. 1. In one embodiment, a method of expressing at least one transgene in a plant comprises growing a plant comprising a gene expression cassette comprising a zea mays chlorophyll a/b binding protein gene promoter and a 3' UTR operably linked to a zea mays chlorophyll a/b binding protein gene of the at least one transgene. In one embodiment, a method of expressing at least one transgene in a plant comprises growing a plant comprising a gene expression cassette comprising a 3' UTR of a maize chlorophyll a/b binding protein gene operably 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 comprising a 3' UTR of a zea mays chlorophyll a/b binding protein gene operably 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 comprising a zea mays chlorophyll a/b binding protein gene promoter and a 3' UTR of a zea mays chlorophyll a/b binding protein gene operably linked to at least one transgene.

The following examples are provided to illustrate certain specific features and/or embodiments. The examples should not be construed as limiting the disclosure to the specific features or embodiments illustrated.

Examples

Example 1: novel design of combinations of optimized regulatory elements from the maize chlorophyll a/b binding protein gene

Gene-specific downstream polynucleotide sequences, termed the 3 'untranslated region (3' UTR), are generally multifunctional in vivo. RNA processing and maturation has been considered as a key control point for post-transcriptional control of eukaryotic gene expression (Szostak and Gebauer, 2012; Wilusz and Spector, 2010; Barrett et al, 2012; and Moore, 2005). These polynucleotide sequences can affect nuclear export rates, subcellular localization, transcript stability, and translation. In addition, the 3' UTR is a key target for control by small non-coding RNAs. While many of these mechanisms down-regulate gene expression, such regulation can also be used to effectively localize transcripts to specific cell types for stable accumulation and subsequent gene expression (Patel et al, 2006). Based on an assessment of contiguous chromosomal sequences associated with the maize chlorophyll a/b binding protein gene promoter, or with other known promoters, a 1000bp 3' UTR polynucleotide sequence (SEQ ID NO:1) for heterologous coding sequence expression was identified and isolated.

SEQ ID NO:1tccccggcaactaagctcaacggctatgctatgcaacttcattgtctttcggatcggagagggtgtacgtacgtggattgattgatgctgcgagatgcatgtgtgtcttttgtttcacgttgcattgcataggcaagtcgagatgatgagttggcgttgtacactaagatgaaccatgtttgtgcaatagtggtggtttttgtttcctgctggttaattgttgatatccattaatttgtttttcttctatactcctttttctctctagctctttatcttaagaaggcaagcataaatgtgcttggataaacagcagatatcaatgaaaatgaaagtagtcttataccatttaaatgtgggcaaacaaataagatatgcacttaaacagtaacgaacgaatctagagaaaatagaaagagggtatacttgtcttaacagatgcatatacttgtatatatcatatgagcagcatatatatggagaaattttaatcaaaatattttttttaaaaaaaaatcgagaatgcatttgcaccatctaatgacacagctttatcctgaccctgcatatgaatgaaatgcgtaaattcacacagtcgatcgtccatgtcttatgaccacactgtacctcttttagcgcttgttcagttacgtctggatcgaagcggattatacaggactaaatctctcactagttaaaattaaataaaaaggatttaatctctctcaatccattttgattcagacgcaaccaaactaacccttatatggtaatcggacagaatttaagtggattaaatctatctctattcaaatttgactagaaatagatttaaatcctcctcttaaattcacttctaaccgaacaaatctttcctaaagtgatcatttatgtaattctttcacaataagacacaaacaaccaacacaagaaccttctctcatcattttgttggattgtgtccacccaattcagcccagttggctacctgttggtaccg

Example 2: vector construction (pDAB116011)

The pDAB116011 vector was constructed to incorporate a new set of regulatory polynucleotide sequences flanking the transgene. The vector construct pDAB116011 contains a gene expression cassette in which the phiyfp transgene (reporter gene from a cupramma species) is driven by the maize chlorophyll a/b binding protein gene promoter of SEQ ID NO:2 (ZMEXP 13231.1-U.S. 005656496) and is flanked by the maize chlorophyll a/b binding protein gene 3' UTR of SEQ ID NO:1 (ZMEXP 13363.1). A schematic of the gene expression cassette is shown in FIG. 1 and is provided as SEQ ID NO 3. The vector also contained a selectable marker gene expression cassette containing the AAD-1 transgene (AAD-1; U.S. Pat. No.7,838,733) driven by the maize ubiquitin 1 promoter (ZmUbi1 promoter; Christensen et al, (1992) plant molecular Biology 18; 675 689) and terminated by the maize lipase 3'UTR (ZmLip 3' UTR; U.S. Pat. No.7,179,902). A schematic of the gene expression cassette is shown in FIG. 1 and is provided as SEQ ID NO. 4. This construct was constructed by: synthesis of a newly designed 3' UTR (ZMOXP 13363.1) from the maize chlorophyll a/b binding protein Gene and Cloning of the promoter into GeneArt Seamless Cloning^TM(Life technologies) entry vectors (WO 2014018512). The resulting entry vector contains the maize chlorophyll a/b binding protein gene that terminates the phiyfp transgene3' UTR, and Gateway was used^TMThe cloning system (Life Technologies) was integrated into the vector of interest and electroporated into Agrobacterium tumefaciens strain DAt13192 (International patent publication No. WO 2012016222). A clone of the resulting binary plasmid pDAB116011 was obtained and the plasmid DNA was isolated and confirmed by restriction endonuclease digestion and sequencing. The resulting construct contains a combination of regulatory elements that drive transgene expression.

A negative control construct pDAB108746 was assembled comprising the cry34Ab1 reporter (FIG. 2) and comprising the maize ubiquitin-1 promoter (Zm Ubi1 promoter) and the potato protease inhibitor-II 3'UTR (StPinII 3' UTR; An et al, (1989) Plant Cell 1; 115-22) regulatory elements. The same aad-1 expression cassette is present as in pDAB 116011. This control construct was transformed into plants using the same reagents and protocol as pDAB 116011.

Example 3: maize transformation

Transformation of Agrobacterium tumefaciens

The binary expression vector was transformed into agrobacterium tumefaciens strain DAt13192 (RecA-deficient ternary strain) (international patent publication No. WO 2012016222). Bacterial colonies were selected, binary plasmid DNA was isolated and confirmed by restriction enzyme digestion.

Agrobacterium culture initiation

Agrobacterium cultures from glycerol stocks were streaked onto AB minimal medium (Gelvin, S., 2006, Agrobacterium Virus Gene indication, Wang, K., eds., Agrobacterium Protocols, second edition, Vol.1, Humana Press, p.79; using sucrose-free 5g/L glucose and 15g/L Bacto^TMAgar preparation) and incubated at 20 ℃ for 3 days in the dark. The Agrobacterium cultures were then streaked onto YEP medium plates (Gelvin, S., 2006, Agrobacterium Virus Gene indication, by Wang, K. eds., Agrobacterium protocols, second edition, Vol.1, Humana Press, p.79) and incubated at 20 ℃ in the dark for 1 day.

On the day of the experiment, a mixture of inoculation medium (2.2g/L MS salts, 68.4g/L sucrose, 36g/L glucose, 115mg/L L-proline, 2mg/L glycine, 100mg/L inositol, 0.05mg/L nicotinic acid, 0.5mg/L pyridoxine hydrochloride, 0.5mg/L thiamine hydrochloride) and acetosyringone in a volume suitable for the experimental scale was prepared. Adding 1M acetosyringone stock solution dissolved in 100% dimethyl sulfoxide into the inoculation culture medium to obtain acetosyringone with final concentration of 200 μ M.

For each construct, 1-2 inoculating loops of agrobacterium from YEP plates were suspended in 15ml inoculation medium/acetosyringone mixture in a 50ml sterile disposable centrifuge tube and the optical density (o.d) of the solution at 600nm was determined in a spectrophotometer.₆₀₀). The suspension was then diluted to 0.25-0.35o.d using an additional inoculation medium/acetosyringone mixture.₆₀₀. The agrobacterium suspension tube was then placed horizontally on a platform shaker set at about 75rpm at room temperature for 1 to 4 hours prior to use.

Maize transformation

The experimental construct was transformed into maize by agrobacterium-mediated transformation of immature embryos isolated from inbred maize cultivar B104. The methods used are similar to those disclosed by Ishida et al, (1996) Nature Biotechnol 14: 745-. Examples of methods for producing multiple transgenic events in maize are given in U.S. patent application publication No. u.s.2013/0157369a1, which begins with a germ infection and co-cultivation step.

Example 4: at T₀Molecular confirmation of copy number at time

Putative transgenic maize plants were sampled at the V2-3 leaf stage to analyze for the presence of transgenes using cry34Ab1, phiyfp and AAD-1 quantitative PCR assays. According to the manufacturer's statementObvious useDNA extraction kit (Qiagen) total DNA was extracted from 2 leaf punch samples.

For detection of the gene of interest, a fluorescent probe containing FAM-labeled for phiyfp gene and HEX-labeled for endogenous convertase reference gene control was usedPrimer/probe sets to amplify gene-specific DNA fragments. The following primers were used for amplification of phiyfp and invertase endogenous reference genes.

PhiYFP primers/probes:

forward primer (PhiYFP v 3F): CGTGTTGGGAAAGAACTTGGA (SEQ ID NO.6)

Reverse primer: (PhiYFP v 3R): CCGTGGTTGGCTTGGTCT (SEQ ID NO.7)

And (3) probe: (PhiYFP v3 probe FAM): 5 'FAM/CACTCCCCACTGCCT/MGB _ BHQ _ 1/3' (SEQ ID NO8)

And (3) converting enzyme primer:

a forward primer: converting enzyme F: TGGCGGACGACGACTTGT (SEQ ID NO:9)

Reverse primer: converting enzyme R: AAAGTTTGGAGGCTGCCGT (SEQ ID NO:10)

Invertase probe: 5 '-/5 HEX/CGAGCAGACCGCCGTGTACTT/3BHQ _ 1/-3' (SEQ ID NO:11)

Next, the PCR reaction was performed in a reaction system having a final volume of 10. mu.l, which contained 5. mu.l of Roche480 Probe Master mix (Roche Applied Sciences, Indianapolis, IN), 0.4. mu.L of each PhiYFP V3F and PhiYFP V3R primer, invertase F and invertase R primers from 10. mu.M stock to a final concentration of 400nM0.4 μ L of each PhiYFP 3.MGB. P and invertase probe from 5 μ M stock to a final concentration of 200nM, 0.1 μ L of 10% polyvinylpyrrolidone (PVP) to a final concentration of 0.1%, 2 μ L of 10ng/μ L genomic DNA and 0.5 μ L water. DNA in RocheAmplification in 480 System under the following conditions: 1 cycle at 95 ℃ for 10 minutes; 40 cycles of the following 3 steps: 95 ℃ for 10 seconds, 58 ℃ for 35 seconds and 72 ℃ for 1 second; and a final cycle of 10 seconds at 4 ℃. Phiyfp copy number is determined by comparing the target (gene of interest)/reference (invertase gene) values of an unknown sample480 out) against phiyfp copy number.

As described above for the phiyfp gene, the AAD-1 gene was detected using an endogenous reference gene for the invertase. The AAD-1 primer sequence is as follows;

AAD1 forward primer: TGTTCGGTTCCCTCTACCAA (SEQ ID NO:12)

AAD1 reverse primer: CAACATCCATCACCTTGACTGA (SEQ ID NO:13)

AAD1 probe: 5 '-FAM/CACAGAACCGTCGCTTCAGCAACA-MGB/BHQ-3' (SEQ ID NO:14)

The cry34Ab1 gene was detected using the invertase endogenous reference gene as described above for the phiyfp gene. The sequences of Cry34Ab1 primers are as follows;

cry34Ab1 forward primer: GCCAACGACCAGATCAAGAC (SEQ ID NO:15)

Cry34Ab1 reverse primer: GCCGTTGATGGAGTAGTAGATGG (SEQ ID NO:16)

Cry34Ab1 probe: 5 '-FAM/CCGAATCCAACGGCTTCA-MGB/BHQ-3' (SEQ ID NO:17)

Finally, T containing the gene of interest was sampled at V4-5₀Plants for PhiYFP and AAD-1 leaf ELISA assay. Four leaf punch samples were collected. PhiYFP (see below) and AAD1(Acadia BioScience) ELISA assays were performed according to the manufacturer's instructions. The PhiYFP leaf ELISA and tassel ELISA results are expressed as parts per million (or ng protein per mg total plant protein). Total protein determination was performed using the Bradford assay according to the manufacturer's instructions.

T₀Plants were selfed and crossed with maize cultivar B104 non-transgenic transformation lines to obtain T₁And (4) seeds. To carry out T₁Protein studies, advancing five to six transgenic lines or events per test regulatory element construct. Thus, 30-40T's per event were seeded₁Seeds; used in the V2-3 developmental stageSeedlings were sprayed to kill non-transgenic segregants.

Example 5: molecular confirmation of protein accumulation

Next, transgenic plants were sampled for PhiYFP and AAD-1ELISA at various plant development stages as follows: leaves (V4, V12 and R3) and silks (R1). All tissues were separated and placed in tubes embedded in dry ice; (ii) a Then transferred to-80 ℃. The frozen tissues except for the leaves were lyophilized, and then the proteins were extracted for ELISA.

Putative transgenic T comprising phiyfp and aad-1 at V4, V12 and R3₁Plants were sampled for leaf ELISA assay. Four leaf punch samples were collected. Leaf punch samples were placed in tubes and a single 1/8 inch stainless steel bead (Hooverprecision Products, Cumming, GA, USA) was added to each 1.2ml tube containing 300. mu.l extraction buffer (1 XPBST supplemented with 0.05% Tween 20 and 0.5% BSA). The samples were placed in genogriner^TM(SPEXSamplePrep, Metuchen, NJ) at1,500 rpm for 4 minutes. Samples were run on a Sorvall Legend XFR^TMCentrifuge at 4,000rpm for 2 minutes. Next, an additional 300. mu.l of extraction buffer was added and the sample was incubated in Genogrinder^TMInThe treatment was carried out at1,500 rpm for a further 2 minutes. The sample was centrifuged at 4,000rpm for 7 minutes. Finally, the supernatant was collected and the ELISA assay was completed at different dilutions along with protein standards using the commercially available PhiYFP and AAD-1(Acadia BioScience) ELISA assay kit according to the manufacturer's instructions. Protein extraction for the various tissue types ELISA was performed by milling the lyophilized tissues in a paint shaker for 30 seconds in the presence of eight 0.25 inch ceramic beads (MP Biomedicals, USA, catalog number 6540-. For tissues that require further grinding, the grinding step is repeated for another 30 seconds. Garnet powder was added to the 2ml tube to cover the bent portion of the bottom of the tube. The coarsely ground tissue was transferred to a 2ml tube and filled to 0.5ml tick mark. One ceramic ball was added to each tube, as was 0.6ml of a portion extraction buffer (200. mu.l protease inhibitor cocktail, 200. mu.l 500Mm EDTA, 15.5mg DTT powder and PBST to 20 ml). All tubes were kept on ice for 10 minutes. Transferring the cold tube toIn a 2ml tube rack. The samples were ground twice for 45 seconds each. Then, 40. mu.l of 10%20 and 300. mu.l extraction buffer was added to the samples. The sample was ground for an additional 45 seconds with 5 minutes of cooling. Finally, each sample was centrifuged at13,000 rpm for 7 minutes and the supernatant was carefully transferred to a new tube to collect the extract. The extract was diluted as necessary for ELISA assay of leaf tissue. Similar assays were used for other plant tissues.

PhiYFP ELISA was performed as follows: plates were coated with capture antibody (Origene mouse anti-YFP; monoclonal (Origene # TA 150028.) the capture antibody was diluted in PBS (1. mu.g/mL) and 100. mu.l was added per well followed by incubation at +4 ℃ overnight. the plates were raised to room temperature for 20-30 min. blocked with 300. mu.l of 2% BSA in PBST per well at +37 ℃ for a minimum of 1 h. the plates were washed three times with 350. mu.l wash buffer. 100. mu.l of standard (Evagen recombinant Phi-YFP1mg/mL (Axxora EVN-FP651-C100) was added to the plates. from 2ng/ml diluted to 0.0313ng/ml at a ratio of 1: 2. 100. mu.l of sample in extraction buffer at a 1:200 dilution was added. The plates were incubated on a platform shaker at room temperature and 125rpm for 1 hour. The plate was washed 3 times with 350. mu.l of wash buffer. Add primary antibody (Evagen rabbit anti-PhiYFP; polyclonal (Axxora # EVN-AB602-C200) and incubate and wash as described above Add 1:5000 dilution of 100. mu.l secondary antibody in extraction buffer (Pierce anti-rabbit Igg (Pierce #31463)) and incubate for 30min at RT and 125rpm on a plate shaker, wash the plate three times with 350. mu.l wash buffer, add 100. mu.l substrate per well (Pierce 1Step Ultra TMBESA (Pierce #34028), shake for 10 min at 125rpm, add 100. mu.L 0.4N H₂SO₄The reaction was stopped. The absorbance was measured at 450nm by means of an optimal 650nm reference filter.

Example 6: expression profiling in crop plants of a gene operably linked to a maize chlorophyll a/b binding protein regulatory element

The maize chlorophyll a/b binding protein 3' UTR regulatory element of SEQ ID NO:1 as provided in pDAB116011 results in expression of the phifp gene in maize transgenic events. Table 1 summarizes robust expression of phiyfp transgenes in various tissue types and at different developmental stages. Little or no phiyfp leaf expression was observed or detected in plant events transformed with the negative control construct pDAB 108746. This construct pDAB108746 does not contain the phiyfp transgene. All constructs expressed the aad-1 gene in the tissues assayed.

Table 1ELISA results depicting PhiYFP and AAD-1 protein levels produced by transgene expression in various types of maize tissues. The indicated samples were obtained from the tissue type of the T1 transgenic plants.

Thus, a novel maize chlorophyll a/b binding protein gene 3' UTR gene regulatory element (SEQ ID NO:1) was identified and characterized. For the first time, novel 3' UTR regulatory elements for gene expression constructs are disclosed.

Example 7: agrobacterium-mediated transformation of a Gene operably linked to the 3' UTR of the maize chlorophyll a/b binding protein Gene

Soybeans can be transformed with a gene operably linked to the 3' UTR of the maize chlorophyll a/b binding protein gene using the same techniques previously described in patent application WO2007/053482, example #11 or example # 13.

Cotton can be transformed with a gene operably linked to the 3' UTR of the maize chlorophyll-binding protein gene using the same techniques previously described in example #14 of U.S. patent No.7,838,733 or example #12 of patent application WO2007/053482(Wright et al).

Canola flowers may be transformed with a gene operably linked to the 3' UTR of a maize chlorophyll-binding protein gene using the same technique previously described in example #26 of U.S. patent No.7,838,733 or example #22 of patent application WO2007/053482(Wright et al).

Wheat can be transformed with a gene operably linked to the 3' UTR of the maize chlorophyll a/b binding protein gene using the same technique previously described in example #23 of patent application WO 2013/116700a1(Lira et al).

Rice can be transformed with a gene operably linked to the 3' UTR of the maize chlorophyll a/b binding protein gene using the same technique previously described in example #19 of patent application WO 2013/116700A1(Lira et al).

Example 8: agrobacterium-mediated transformation of a gene operably linked to a maize chlorophyll a/b binding protein gene regulatory element

In accordance with the present disclosure, additional crops may be transformed in accordance with embodiments of the present disclosure using techniques known in the art. For agrobacterium-mediated transformation of rye see, e.g., Popelka JC, Xu J, Altpeter f., "general of eyes with low transfer gene copy number after biolistic gene transfer and transformation of (Secale center L.) -plant insenstance marker-free Transgenic eyes," Transgenic res.2003 for 10 months; 12(5):587-96. For Agrobacterium-mediated transformation of sorghum, see, e.g., Zhao et al, "Agrobacterium-mediated sorghum transformation," Plant Mol biol.2000, 12 months; 44(6):789-98. For Agrobacterium-mediated transformation of barley, 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 medial by Agrobacterium tumefaciens," plantaphysiol.1997, month 11; 115(3):971-980. For Agrobacterium-mediated Transformation of rice, see, e.g., heii et al, "Transformation of rice media by Agrobacterium tumefaciens," plantal. biol.1997, 9 months; 35(1-2):205-18.

The latin names of these and other plants are given below. It will be appreciated that other (non-agrobacterium) transformation techniques may be used to transform genes such as the 3' UTR operably linked to the zea mays chlorophyll a/b binding protein gene into these and other plants. Examples include, but are not limited to: maize (corn), wheat (Triticum spp)), rice (Oryza spp) and wild rice (Zizania spp)), barley (Hordeum spp), cotton (abaca and cotton (Gossypium spp)), soybean (Glycine max), sugar and edible sugar beets (Beta spp), sugar cane (arengainnata), tomato (tomato and other species, mucoid pulp (Physalis ixocarpa), Solanum xanthophyllum (Solanum incanum) and other species as well as tomato (cytoplasma), potato (potato and pepper), lettuce (tomato and lettuce (lettuce), lettuce (lettuce) and black pepper (lettuce), lettuce (cabbage), lettuce (lettuce) and black pepper (lettuce) respectively), rape (lettuce) and black pepper (cabbage), rape (lettuce) and black pepper (cabbage), lettuce (lettuce) and black pepper (cabbage) respectively), black pepper (lettuce) and black pepper (cabbage), black pepper (lettuce) and black pepper (lettuce) respectively), black pepper (cabbage) and black pepper (lettuce (black pepper) and black pepper (black pepper) respectively), black pepper (lettuce (black pepper) and black pepper (lettuce (black pepper) and black pepper) respectively), and black pepper (black pepper) and black pepper (black pepper) can, black pepper (black, Cabbage (brassica), Apium graveolens), eggplant (Solanum melogenna)), peanut (Arachis hypogaea), Sorghum (Sorghum spp), alfalfa (medicago sativa), carrot (Daucus carota), kidney beans (Phaseolus spp) and other genera, oat (Avena sativa) and oat (Avena strigosa), pea (Pisum, Vigna, and tragonia (tegorobus), sunflower (helyasannu), pumpkin (cusua), asparagus (Cucumis sativa), cucumber (Cucumis sativa), tobacco grass (Cucumis sativa), black grass (Cucumis sativa), Sorghum (Cucumis sativus), black grass (Cucumis sativus), Sorghum (Cucumis sativa), Sorghum (Cucumis sativus) and other genera), Cucumis (Cucumis sativus), Cucumis sativus (Cucumis sativa), Cucumis sativus (Cucumis sativus), Cucumis sativus (Cucumis sativus), Cucumis sativus) and other genera) are included in the genus, Cucumis sativus (Cucumis sativus), Cucumis sativus, Clover (clover), vetch (Vicia). For example, it is contemplated in embodiments of the present disclosure to transform such plants with a gene operably linked to the 3' UTR of the maize chlorophyll a/b binding protein gene.

The use of the 3' UTR of the maize chlorophyll a/b binding protein gene to terminate operably linked genes is useful in many deciduous and evergreen species. Such applications are also within the scope of the embodiments of the present disclosure. These species include, but are not limited to: alder (Alnus spp.), ash (Fraxinus spp.), aspen and poplar 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.).

Use of the 3' UTR of the maize chlorophyll a/b binding protein gene to terminate operably linked genes is useful for ornamental and fruiting tree species. Such applications are also within the scope of the embodiments of the present disclosure. Examples include, but are not limited to: roses (Rosa spp.), Euonymus alatus (Euonymus spp.), Petunia (Petunia spp.), Begonia (Begonia spp.), Rhododendron (Rhododendron spp.), crab apple or apple (Malus spp.), pears (Pyrus spp.), peaches (Prunus spp.) and marigold (Tagetes spp.).

While various exemplary aspects and embodiments have been discussed above, those of skill in the art will recognize certain modifications, permutations, additions and sub-combinations thereof. It is therefore intended that the following appended claims and claims hereafter introduced are interpreted to include all such modifications, permutations, additions and sub-combinations as are within their true spirit and scope.

Sequence listing

<110>Dow AgroSciences LLC

Gupta, Manju

Beringer, Jeff

Bennett, Sara

Sardesai, Nagesh

<120> plant promoter and 3' UTR for transgene expression

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tgtgcaatag tggtggtttt tgtttcctgc tggttaattg ttgatatcca ttaatttgtt 240

tttcttctat actccttttt ctctctagct ctttatctta agaaggcaag cataaatgtg 300

cttggataaa cagcagatat caatgaaaat gaaagtagtc ttataccatt taaatgtggg 360

caaacaaata agatatgcac ttaaacagta acgaacgaat ctagagaaaa tagaaagagg 420

gtatacttgt cttaacagat gcatatactt gtatatatca tatgagcagc atatatatgg 480

agaaatttta atcaaaatat tttttttaaa aaaaaatcga gaatgcattt gcaccatcta 540

atgacacagc tttatcctga ccctgcatat gaatgaaatg cgtaaattca cacagtcgat 600

cgtccatgtc ttatgaccac actgtacctc ttttagcgct tgttcagtta cgtctggatc 660

gaagcggatt atacaggact aaatctctca ctagttaaaa ttaaataaaa aggatttaat 720

ctctctcaat ccattttgat tcagacgcaa ccaaactaac ccttatatgg taatcggaca 780

gaatttaagt ggattaaatc tatctctatt caaatttgac tagaaataga tttaaatcct 840

cctcttaaat tcacttctaa ccgaacaaat ctttcctaaa gtgatcattt atgtaattct 900

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ggcaaaccac aaccacaaat ggatcaattg atctagaaca atccgaagga ggggaggcca 180

cgtcacactc acaccaaccg aaatatctgc cagtatcaga tcaaccggcc aataggacgc 240

cagcgagccc aacacctagc gacgccgcaa aattcaccgc gaggggcacc gggcacggca 300

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<223> PhiYFP transgene cassette sequence comprising ZMEXP13231.1 promoter, PhiYFP gene and ZM 13363.13' UTR; pDAB116011

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ggcaaaccac aaccacaaat ggatcaattg atctagaaca atccgaagga ggggaggcca 180

cgtcacactc acaccaaccg aaatatctgc cagtatcaga tcaaccggcc aataggacgc 240

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aaaacaaaag cccggcgcgg tgagaatatc tggcgactgg cggagacctg gtggccagcg 360

cgcggccaca tcagccaccc catccgccca cctcacctcc ggcgagccaa tggcaactcg 420

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cttcagcacc agaagacacc atgtcatctg gagcacttct ctttcatggg aagattcctt 600

acgttgtgga gatggaaggg aatgttgatg gccacacctt tagcatacgt gggaaaggct 660

acggagatgc ctcagtggga aaggtatgtt tctgcttcta cctttgatat atatataata 720

attatcacta attagtagta atatagtatt tcaagtattt ttttcaaaat aaaagaatgt 780

agtatatagc tattgctttt ctgtagttta taagtgtgta tattttaatt tataactttt 840

ctaatatatg accaaaacat ggtgatgtgc aggttgatgc acaattcatc tgtactaccg 900

gagatgttcc tgtgccttgg agcacacttg tcaccactct cacctatgga gcacagtgct 960

ttgccaagta tggtccagag ttgaaggact tctacaagtc ctgtatgcca gatggctatg 1020

tgcaagagcg cacaatcacc tttgaaggag atggcaactt caagactagg gctgaagtca 1080

cctttgagaa tgggtctgtc tacaataggg tcaaactcaa tggtcaaggc ttcaagaaag 1140

atggtcacgt gttgggaaag aacttggagt tcaacttcac tccccactgc ctctacatct 1200

ggggagacca agccaaccac ggtctcaagt cagccttcaa gatatgtcat gagattactg 1260

gcagcaaagg cgacttcata gtggctgacc acacccagat gaacactccc attggtggag 1320

gtccagttca tgttccagag tatcatcata tgtcttacca tgtgaaactt tccaaagatg 1380

tgacagacca cagagacaac atgagcttga aagaaactgt cagagctgtt gactgtcgca 1440

agacctacct ttgagtagtt agcttaatca cctagagctc tccccggcaa ctaagctcaa 1500

cggctatgct atgcaacttc attgtctttc ggatcggaga gggtgtacgt acgtggattg 1560

attgatgctg cgagatgcat gtgtgtcttt tgtttcacgt tgcattgcat aggcaagtcg 1620

agatgatgag ttggcgttgt acactaagat gaaccatgtt tgtgcaatag tggtggtttt 1680

tgtttcctgc tggttaattg ttgatatcca ttaatttgtt tttcttctat actccttttt 1740

ctctctagct ctttatctta agaaggcaag cataaatgtg cttggataaa cagcagatat 1800

caatgaaaat gaaagtagtc ttataccatt taaatgtggg caaacaaata agatatgcac 1860

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gcatatactt gtatatatca tatgagcagc atatatatgg agaaatttta atcaaaatat 1980

tttttttaaa aaaaaatcga gaatgcattt gcaccatcta atgacacagc tttatcctga 2040

ccctgcatat gaatgaaatg cgtaaattca cacagtcgat cgtccatgtc ttatgaccac 2100

actgtacctc ttttagcgct tgttcagtta cgtctggatc gaagcggatt atacaggact 2160

aaatctctca ctagttaaaa ttaaataaaa aggatttaat ctctctcaat ccattttgat 2220

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taaaaaatta ccacatattt tttttgtcac acttgtttga agtgcagttt atctatcttt 120

atacatatat ttaaacttta ctctacgaat aatataatct atagtactac aataatatca 180

gtgttttaga gaatcatata aatgaacagt tagacatggt ctaaaggaca attgagtatt 240

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caaatagctt cacctatata atacttcatc cattttatta gtacatccat ttagggttta 360

gggttaatgg tttttataga ctaatttttt tagtacatct attttattct attttagcct 420

ctaaattaag aaaactaaaa ctctatttta gtttttttat ttaatagttt agatataaaa 480

tagaataaaa taaagtgact aaaaattaaa caaataccct ttaagaaatt aaaaaaacta 540

aggaaacatt tttcttgttt cgagtagata atgccagcct gttaaacgcc gtcgacgagt 600

ctaacggaca ccaaccagcg aaccagcagc gtcgcgtcgg gccaagcgaa gcagacggca 660

cggcatctct gtcgctgcct ctggacccct ctcgagagtt ccgctccacc gttggacttg 720

ctccgctgtc ggcatccaga aattgcgtgg cggagcggca gacgtgagcc ggcacggcag 780

gcggcctcct cctcctctca cggcaccggc agctacgggg gattcctttc ccaccgctcc 840

ttcgctttcc cttcctcgcc cgccgtaata aatagacacc ccctccacac cctctttccc 900

caacctcgtg ttgttcggag cgcacacaca cacaaccaga tctcccccaa atccacccgt 960

cggcacctcc gcttcaaggt acgccgctcg tcctcccccc ccccccccct ctctaccttc 1020

tctagatcgg cgttccggtc catgcatggt tagggcccgg tagttctact tctgttcatg 1080

tttgtgttag atccgtgttt gtgttagatc cgtgctgcta gcgttcgtac acggatgcga 1140

cctgtacgtc agacacgttc tgattgctaa cttgccagtg tttctctttg gggaatcctg 1200

ggatggctct agccgttccg cagacgggat cgatttcatg attttttttg tttcgttgca 1260

tagggtttgg tttgcccttt tcctttattt caatatatgc cgtgcacttg tttgtcgggt 1320

catcttttca tgcttttttt tgtcttggtt gtgatgatgt ggtctggttg ggcggtcgtt 1380

ctagatcgga gtagaattct gtttcaaact acctggtgga tttattaatt ttggatctgt 1440

atgtgtgtgc catacatatt catagttacg aattgaagat gatggatgga aatatcgatc 1500

taggataggt atacatgttg atgcgggttt tactgatgca tatacagaga tgctttttgt 1560

tcgcttggtt gtgatgatgt ggtgtggttg ggcggtcgtt cattcgttct agatcggagt 1620

agaatactgt ttcaaactac ctggtgtatt tattaatttt ggaactgtat gtgtgtgtca 1680

tacatcttca tagttacgag tttaagatgg atggaaatat cgatctagga taggtataca 1740

tgttgatgtg ggttttactg atgcatatac atgatggcat atgcagcatc tattcatatg 1800

ctctaacctt gagtacctat ctattataat aaacaagtat gttttataat tatttcgatc 1860

ttgatatact tggatgatgg catatgcagc agctatatgt ggattttttt agccctgcct 1920

tcatacgcta tttatttgct tggtactgtt tcttttgtcg atgctcaccc tgttgtttgg 1980

tgttacttct gcaggtacag tagttagttg aggtaccgga tccacacgac accatggctc 2040

atgctgccct cagccctctc tcccaacgct ttgagagaat agctgtccag ccactcactg 2100

gtgtccttgg tgctgagatc actggagtgg acttgaggga accacttgat gacagcacct 2160

ggaatgagat attggatgcc ttccacactt accaagtcat ctactttcct ggccaagcaa 2220

tcaccaatga gcagcacatt gcattctcaa gaaggtttgg accagttgat ccagtgcctc 2280

ttctcaagag cattgaaggc tatccagagg ttcagatgat ccgcagagaa gccaatgagt 2340

ctggaagggt gattggtgat gactggcaca cagactccac tttccttgat gcacctccag 2400

ctgctgttgt gatgagggcc atagatgttc ctgagcatgg cggagacact gggttccttt 2460

caatgtacac agcttgggag accttgtctc caaccatgca agccaccatc gaagggctca 2520

acgttgtgca ctctgccaca cgtgtgttcg gttccctcta ccaagcacag aaccgtcgct 2580

tcagcaacac ctcagtcaag gtgatggatg ttgatgctgg tgacagagag acagtccatc 2640

ccttggttgt gactcatcct ggctctggaa ggaaaggcct ttatgtgaat caagtctact 2700

gtcagagaat tgagggcatg acagatgcag aatcaaagcc attgcttcag ttcctctatg 2760

agcatgccac cagatttgac ttcacttgcc gtgtgaggtg gaagaaagac caagtccttg 2820

tctgggacaa cttgtgcacc atgcaccgtg ctgttcctga ctatgctggc aagttcagat 2880

acttgactcg caccacagtt ggtggagtta ggcctgcccg ctgagtagtt agcttaatca 2940

cctagagctc ggtcgcagcg tgtgcgtgtc cgtcgtacgt tctggccggc cgggccttgg 3000

gcgcgcgatc agaagcgttg cgttggcgtg tgtgtgcttc tggtttgctt taattttacc 3060

aagtttgttt caaggtggat cgcgtggtca aggcccgtgt gctttaaaga cccaccggca 3120

ctggcagtga gtgttgctgc ttgtgtaggc tttggtacgt atgggcttta tttgcttctg 3180

gatgttgtgt actacttggg tttgttgaat tattatgagc agttgcgtat tgtaattcag 3240

ctgggctacc tggacattgt tatgtattaa taaatgcttt gctttcttct aaagatcttt 3300

aagtgct 3307

<210>5

<211>789

<212>DNA

<213> corn

<400>5

atggctgcct ccaccatggc gatctcctcc acggcgatgg ccggcacccc catcaaggtg 60

ggttccttcg gcgagggccg catcaccatg cgcaagaccg tgggcaagcc caaggtggcg120

gcgtccggca gcccctggta cggccccgac cgcgtcaagt acctcggccc cttctccggc 180

gagcccccga gctacctcac cggcgagttc cccggcgact acggctggga caccgccggg 240

ctgtccgccg accccgagac attcgccaag aaccgcgagc tggaggtgat ccactcccgc 300

tgggccatgc tcggcgcgct cggctgcgtc ttccccgagc tgctctcccg caacggcgtc 360

aagttcggcg aggccgtctg gttcaaggcc ggctcccaga tcttcagcga gggcgggctg 420

gactacctcg gcaaccccag cctgatccac gcgcagagca tcctcgccat ctgggcctgc 480

caggtcgtgc tcatgggtgc cgtcgagggc taccgcattg ccggcgggcc gctcggcgag 540

gtcgtcgacc cgctgtaccc tggcggcagc ttcgaccccc tcggcctggc cgacgacccc 600

gaggccttcg ccgagctcaa ggtgaaggag ctcaagaacg gccgcctcgc catgttttcc 660

atgttcggct tcttcgtcca ggccatcgtc accggcaagg gcccgctcga gaacctcgct 720

gaccacatcg ctgacccagt caacaacaac gcatgggcct acgccaccaa cttcgtcccc 780

ggcaactaa 789

<210>6

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> PhiYFP Forward primer

<400>6

cgtgttggga aagaacttgg a 21

<210>7

<211>18

<212>DNA

<213> Artificial sequence

<220>

<223> PhiYFP reverse primer

<400>7

ccgtggttgg cttggtct 18

<210>8

<211>15

<212>DNA

<213> Artificial sequence

<220>

<223> PhiYFP Probe

<400>8

cactccccac tgcct 15

<210>9

<211>18

<212>DNA

<213> Artificial sequence

<220>

<223> invertase forward primer

<400>9

tggcggacga cgacttgt 18

<210>10

<211>19

<212>DNA

<213> Artificial sequence

<220>

<223> invertase reverse primer

<400>10

aaagtttgga ggctgccgt 19

<210>11

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> invertase Probe

<400>11

cgagcagacc gccgtgtact t 21

<210>12

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> AAD1 Forward primer

<400>12

tgttcggttc cctctaccaa 20

<210>13

<211>22

<212>DNA

<213> Artificial sequence

<220>

<223> AAD1 reverse primer

<400>13

caacatccat caccttgact ga 22

<210>14

<211>24

<212>DNA

<213> Artificial sequence

<220>

<223> AAD1 Probe

<400>14

cacagaaccg tcgcttcagc aaca 24

<210>15

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> Cry34Ab1 Forward primer

<400>15

gccaacgacc agatcaagac 20

<210>16

<211>23

<212>DNA

<213> Artificial sequence

<220>

<223> Cry34Ab1 reverse primer

<400>16

gccgttgatg gagtagtaga tgg 23

<210>17

<211>18

<212>DNA

<213> Artificial sequence

<220>

<223> Cry34Ab1 Probe

<400>17

ccgaatccaa cggcttca 18

Claims

1.A nucleic acid vector comprising a 3' UTR operably linked to:

a) a polylinker or short polynucleotide sequence;

b) a non-maize chlorophyll a/b binding protein gene; or

c) a combination of a) and b), wherein the 3' UTR comprises a polynucleotide sequence having at least 90% sequence identity to SEQ ID NO. 1.

2. The nucleic acid vector of claim 1, wherein the 3' UTR is 1000bp in length.

3. The nucleic acid vector of claim 1, wherein the 3' UTR consists of a polynucleotide sequence having at least 90% sequence identity to SEQ ID No. 1.

4. The nucleic acid vector of any one of claims 1-3, further comprising a sequence encoding a selectable marker.

5. The nucleic acid vector of claim 1, wherein the 3' UTR is operably linked to a transgene.

6. The nucleic acid vector of claim 5, wherein the transgene encodes a selectable marker or gene product that confers insecticidal resistance, herbicide tolerance, RNAi expression, nitrogen use efficiency, water use efficiency, or nutritional quality.

7. The nucleic acid vector of any one of claims 1-3 or 5, further comprising a promoter polynucleotide sequence having at least 90% sequence identity to SEQ ID NO 2, wherein the promoter sequence is operably linked to the polylinker or the transgene.

8. The nucleic acid vector of any one of claims 1-3 or 5, further comprising an intron sequence.

9. The nucleic acid vector of claim 1, wherein the 3' UTR has constitutive or tissue-specific expression.

10. A plant comprising a polynucleotide sequence having at least 90% sequence identity to SEQ ID No. 1 operably linked to a transgene.

11. The plant of claim 10, wherein said plant is selected from the group consisting of: maize, wheat, rice, sorghum, oat, rye, banana, sugarcane, soybean, cotton, arabidopsis, tobacco, sunflower and canola.

12. The plant of claim 10, wherein said plant is maize.

13. The plant of any one of claims 10-12, wherein said transgene is inserted into the genome of said plant.

14. The plant of claim 10, wherein the 3'UTR comprises a polynucleotide sequence having at least 90% sequence identity to SEQ ID No. 1, and the 3' UTR is 1000bp in length.

15. The plant of claim 14, further comprising a promoter sequence comprising SEQ ID No. 2, wherein said promoter sequence is operably linked to said transgene.

16. The plant of claim 15, wherein said transgene has constitutive or tissue-specific expression.

17. The plant of claim 15, wherein the promoter is a maize chlorophyll a/b binding protein promoter.

18. A transgenic seed produced by the plant of claim 10.

19. A method for producing a transgenic plant cell, said method comprising the steps of:

a) transforming a plant cell with a gene expression cassette comprising a maize chlorophyll a/b binding protein 3' UTR operably linked to at least one polynucleotide sequence of interest;

b) isolating a transformed plant cell comprising the gene expression cassette; and

c) generating a transgenic plant cell comprising the maize chlorophyll a/b binding protein gene 3' UTR operably linked to at least one polynucleotide sequence of interest.

20. The method of claim 19, wherein the plant cell is transformed using a plant transformation method.

21. The method of claim 19, wherein the polynucleotide sequence of interest is stably integrated into the genome of the transgenic plant cell.

22. The method of claim 19, further comprising the steps of:

d) regenerating the transgenic plant cell into a transgenic plant; and

e) obtaining the transgenic plant, wherein the transgenic plant comprises a gene expression cassette comprising a maize chlorophyll a/b binding protein gene 3' UTR according to claim 1 operably linked to at least one polynucleotide sequence of interest.

23. The maize chlorophyll a/b binding protein gene 3' UTR of claim 19, comprising a polynucleotide of SEQ ID No. 1.

24. An isolated polynucleotide comprising a nucleic acid sequence having at least 90% sequence identity to the polynucleotide of SEQ ID No. 1.

25. The isolated polynucleotide of claim 24, further comprising an open reading frame polynucleotide encoding a polypeptide; and a promoter sequence.

26. The isolated polynucleotide of claim 24, wherein the polynucleotide of SEQ ID No. 1 is 1000bp in length.