# Patent

## Title
Constructs for expressing transgenes using regulatory elements from setaria ubiquitin genes

## Abstract

Provided are constructs and methods for expressing a transgene in plant cells and/or plant tissues using the regulatory elements, including the promoters and/or 3′-UTRs, isolated from Setaria italica ubiquitin genes.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims priority to U.S. Provisional Patent Application No. 61/872,134, filed Aug. 30, 2013, which is hereby incorporated by reference in its entirety.


INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY
Incorporated by reference in its entirety is a computer-readable nucleotide/amino acid sequence listing submitted concurrently herewith and identified as follows: One 68 KB ASCII (Text) file named 231407SubSeqListing.txt, created on Sep. 7, 2016.
IN THE SEQUENCE LISTING
Please replace the Sequence Listing currently of record with the computer-readable form of the Substitute Sequence Listing (filename: 231407SubSeqListing.txt, created on Sep. 7, 2016) submitted herewith.
BACKGROUND
Plant transformation is an attractive technology for use in introducing agronomically desirable traits or characteristics into different crop plant species. Plant species are developed and/or modified to have particular desirable traits. Generally, 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), imparting herbicide resistance, enabling the production of industrially useful compounds and/or materials from the plant, and/or enabling the production of pharmaceuticals.
Transgenic plants comprising multiple transgenes stacked at a single genomic locus are produced via plant transformation technologies. Plant transformation technologies result in the introduction of transgene into a plant cell, recovery of a fertile transgenic plant that contains the stably integrated copy of the transgene in the plant genome, and subsequent transgene expression via transcription and translation of the transgene(s) results in transgenic plants that possess desirable traits and phenotypes. Each transgene in a stack typically requires an independent promoter for gene expression, and thus multiple promoters are used in a transgene stack.
The need for co-expression of multiple transgenes for regulating the same trait frequently results in the repeated use of the same promoter to drive expression of the multiple transgenes. However, the repeated use of promoters comprising sequences that share a high level of sequence identity may lead to homology-based gene silencing (HBGS). HBGS has been observed to occur frequently in transgenic plants (Peremarti et al., 2010) when repetitive DNA sequences are used within a transgene. In addition, repeated use of similar DNA sequences in transgene constructs has proven to be challenging in Agrobacterium due to recombination and instability of the plasmid.
Described herein are ubiquitin regulatory elements (e.g., promoters and 3′-UTR) that share low levels of sequence identity or homology with the Maize ubiquitin) promoter. Further described are constructs and methods utilizing ubiquitin regulatory elements.
SUMMARY
Disclosed herein are constructs and methods for expressing a transgene in plant cells and/or plant tissues. In one embodiment regulatory elements of a ubiquitin gene are purified from Panicum virgatum, Brachypodium distachyon, or Setaria italica genomes and recombined with sequences not natively linked to said regulatory elements to create an expression vector for expressing transgenes in plant cells not native to the ubiquitin regulatory sequences. In one embodiment an expression vector is provided wherein the regulatory elements of a ubiquitin gene are operably linked to a polylinker sequence. Such an expression vector eases the insertion of a gene or gene cassette into the vector in an operably linked state with the ubiquitin gene regulatory sequences.
In an embodiment, a construct is provided comprising a Panicum virgatum, Brachypodium distachyon, or Setaria italica ubiquitin promoter. In an embodiment, a gene expression cassette is provided comprising a Panicum virgatum, Brachypodium distachyon or Setaria italica ubiquitin promoter operably linked to a transgene. In an embodiment, a gene expression cassette includes a Panicum virgatum, Brachypodium distachyon or Setaria italica ubiquitin 5′-UTR operably linked to a transgene. In an embodiment, a gene expression cassette includes a Panicum virgatum, Brachypodium distachyon or Setaria italica ubiquitin 5′-UTR operably linked to a promoter. In an embodiment, a gene expression cassette includes a Panicum virgatum, Brachypodium distachyon or Setaria italica ubiquitin intron operably linked to a transgene. In an embodiment, a gene expression cassette includes a Panicum virgatum, Brachypodium distachyon or Setaria italica ubiquitin intron operably linked to a promoter. In an embodiment, a construct includes a gene expression cassette comprising Panicum virgatum, Brachypodium distachyon or Setaria italica ubiquitin 3′-UTR. In an embodiment, a gene expression cassette includes Panicum virgatum, Brachypodium distachyon or Setaria italica ubiquitin 3′-UTR operably linked to a transgene. In an embodiment, a gene expression cassette includes at least one, two, three, five, six, seven, eight, nine, ten, or more transgenes.
In an embodiment, a gene expression cassette includes independently a) a Panicum virgatum, Brachypodium distachyon, or Setaria italica ubiquitin promoter, b) a Panicum virgatum, Brachypodium distachyon or Setaria italica ubiquitin intron, c) a Panicum virgatum, Brachypodium distachyon or Setaria italica ubiquitin 5′-UTR, and d) a Panicum virgatum, Brachypodium distachyon or Setaria italica ubiquitin 3′-UTR.
In accordance with one embodiment a nucleic acid vector is provided comprising a promoter operably linked to a non-ubiquitin transgene, wherein the promoter consists of SEQ ID NO: 17 or 41 or a sequence having 90% sequence identity with SEQ ID NO: 17 or 41. In a further embodiment the nucleic acid vector comprises a gene cassette, wherein the gene cassette comprises a promoter, a non-ubiquitin transgene and a 3′ untranslated region, wherein the promoter consists of SEQ ID NO: 17 or 41 operably linked to a first end of a transgene, wherein the second end of the transgene is operably linked to a 3′ untranslated sequence consisting of SEQ ID NO: 6.
Methods of growing plants expressing a transgene using the Panicum virgatum, Brachypodium distachyon, or Setaria italica promoters, 5′-UTRs, introns, and 3′-UTRs are disclosed herein. Methods of culturing plant tissues and cells expressing a transgene using the Panicum virgatum, Brachypodium distachyon or Setaria italica promoters, 5′-UTRs, introns, and 3′-UTRs are also disclosed herein.
In accordance with one embodiment a plant, plant tissue, or plant cell is provided comprising a promoter operably linked to a non-ubiquitin transgene, wherein the promoter comprises SEQ ID NO: 3. In accordance with one embodiment a non-Setaria plant or plant cell is provided comprising SEQ ID NO: 3, or a sequence that has 90% sequence identity with SEQ ID NO: 3 operably linked to a transgene. In one embodiment the plant is a corn variety. In one embodiment a plant, plant tissue, or plant cell is provided comprising a promoter operably linked to a non-ubiquitin transgene, wherein the promoter consists of SEQ ID NO: 17, 40, 41 or 42. In one embodiment a non-Setaria plant or plant cell is provided comprising a gene cassette, wherein the gene cassette comprises a promoter operably linked to a transgene, further wherein the promoter consists SEQ ID NO: 17. In one embodiment a non-Setaria plant or plant cell is provided comprising a gene cassette, wherein the gene cassette comprises a promoter operably linked to a transgene, further wherein the promoter consists SEQ ID NO: 41. In a further embodiment the promoter is operably linked to a first end of a transgene, wherein the second end of the transgene is operably linked to a 3′ untranslated sequence consisting of SEQ ID NO: 6.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the protein alignment of Zea mays ubiquitin (ZM Ubi1) protein sequence to Brachypodium distachyon and Setaria italic ubiquitin sequences used for promoter identification. The Zm Ubi1 protein sequence is disclosed herein as SEQ ID NO:22. The S. italica Ubi2 protein sequence is disclosed herein as SEQ ID NO:23. The B. distachyon Ubi1 promoter sequence is disclosed herein as SEQ ID NO:24. The B. distachyon Ubi1C protein sequence is disclosed herein as SEQ ID NO:25. The consensus sequence is disclosed herein as SEQ ID NO:26.
FIG. 2 shows the alignment of Zea mays ubiquitin (ZM Ubi1) promoter polynucleotide sequence to Brachypodium distachyon and Setaria italica ubiquitin promoter polynucleotides identified herein. The Zea may Ubiquitin 1 (Zm-Ubi) promoter sequence is disclosed herein as SEQ ID NO:27. The B. distachyon Ubi1 promoter sequence is disclosed herein as SEQ ID NO:16. The B. distachyon Ubi1-C promoter sequence is disclosed herein as SEQ ID NO:15. The S. italica Ubi2 promoter sequence is disclosed herein as SEQ ID NO:17.
FIG. 3 is a plasmid map showing the synthesized Setaria italica Ubiquitin2 promoter genetic element.
FIG. 4 is a plasmid map showing the synthesized Brachypodium distachyon Ubiquitin1 C promoter genetic element and flanking seamless cloning overhang location.
FIG. 5 is a plasmid map showing the synthesized Brachypodium distachyon Ubiquitin1 promoter genetic element and flanking seamless cloning overhang location.
FIG. 6 is a plasmid map showing the expression vector containing Setaria italica ubiquitin2 (SI-Ubi2) promoter fused to PhiYFP reporter gene.
FIG. 7 is a plasmid map showing the expression vector containing Brachypodium distachyon Ubiquitin1 C promoter fused to PhiYFP reporter gene.
FIG. 8 is a plasmid map showing the expression vector containing Brachypodium distachyon Ubiquitin1 promoter fused to PhiYFP reporter gene.
FIG. 9 is a plasmid map showing the expression vector containing OS Act1 (Rice Actin1) promoter fused to PhiYFP reporter gene.
FIG. 10 is a plasmid map showing the expression vector containing ZM Ubi1 promoter fused to PhiYFP reporter gene.
FIG. 11 is a plasmid map showing the binary destination vector used to build binary expression vectors using Gateway technology.
FIG. 12 is a plasmid map showing the binary expression vector containing Setaria italica Ubiquitin2 (SI-Ubi2) promoter fused to yellow fluorescent protein (Phi YFP) marker gene coding region containing ST-LS1 intron followed by fragment comprising a StPinII 3′UTR from potato.
FIG. 13 is a plasmid map showing the binary expression vector containing Brachypodium distachyon Ubiquitin1 C promoter fused to yellow fluorescent protein (Phi YFP) marker gene coding region containing ST-LS1 intron followed by fragment comprising a StPinII 3′UTR from potato.
FIG. 14 is a plasmid map showing the binary expression vector containing Brachypodium distachyon Ubiquitin1 promoter fused to yellow fluorescent protein (Phi YFP) marker gene coding region containing ST-LS1 intron followed by fragment comprising a StPinII 3′UTR from potato.
FIG. 15 is a plasmid map showing the binary expression vector containing OS Act1 promoter fused to yellow fluorescent protein (Phi YFP) marker gene coding region containing ST-LS1 intron followed by fragment comprising a StPinII 3′UTR from potato.
FIG. 16 is a plasmid map showing the binary expression vector containing ZM Ubi1 promoter fused to yellow fluorescent protein (Phi YFP) marker gene coding region containing ST-LS 1 intron followed by fragment comprising a StPinII 3′UTR from potato.
FIG. 17 shows YFP expression in a T0 leaf where YFP is driven by the cross species ubiquitin and Os Act 1 promoters as depicted in FIGS. 12, 13, 14, 15, and 16.
FIG. 18 shows AAD1 expression in a T0 leaf where AAD1 is driven by the Zm Ubi 1 promoter as depicted in FIGS. 12, 13, 14, 15, and 16.
FIG. 19 shows transient YFP expression driven by the Brachypodium distachyon and Setaria italica novel promoters as compared to YFP expression driven by the ZM Ubi1 and OS Act1 promoters.
FIG. 20 shows YFP expression in calli tissues driven by the novel Brachypodium distachyon and Setaria italica promoters as compared to YFP expression driven by the ZM Ubi1 and OS Act1 promoters.
FIG. 21 shows YFP expression in root tissue driven by the novel Brachypodium distachyon and Setaria italica promoters as compared to YFP expression driven by the ZM Ubi1 and OS Act1 promoters.
FIG. 22 is a plasmid map showing the synthesized Panicum virgatum Ubiquitin1 promoter genetic element and flanking seamless cloning overhang location.
FIG. 23 is plasmid map showing the synthesized Panicum virgatum Ubiquitin1 3′UTR genetic element and flanking seamless cloning overhang location.
FIG. 24 is plasmid map showing the synthesized Brachypodium distachyon Ubiquitin1C 3′UTR genetic element and flanking seamless cloning overhang location.
FIG. 25 is plasmid map showing the synthesized Brachypodium distachyon Ubiquitin1 3′UTR genetic element and flanking seamless cloning overhang location.
FIG. 26 is plasmid map showing the synthesized Setaria italica ubiquitin2 (SI-Ubi2) 3′UTR genetic element and flanking seamless cloning overhang location.
FIG. 27 is plasmid map showing the expression vector containing Panicum virgatum Ubiquitin1 promoter and 3′UTR fused to PhiYFP reporter gene.
FIG. 28 is plasmid map showing the expression vector containing Brachypodium distachyon Ubiquitin1 C promoter and 3′UTR fused to PhiYFP reporter gene.
FIG. 29 is a plasmid map showing the expression vector containing Setaria italica ubiquitin2 promoter and 3′UTR fused to PhiYFP reporter gene.
FIG. 30 is plasmid map showing the expression vector containing Brachypodium distachyon Ubiquitin1 promoter and 3′UTR fused to PhiYFP reporter gene.
FIG. 31 is a plasmid map showing the binary expression vector containing Brachypodium distachyon Ubiquitin1 C promoter fused to yellow fluorescent protein (Phi YFP) marker gene coding region containing ST-LS1 intron followed by fragment comprising a Brachypodium distachyon Ubiquitin1 C 3′UTR.
FIG. 32 is a plasmid map showing the binary expression vector containing Panicum virgatum Ubiquitin1 promoter fused to yellow fluorescent protein (Phi YFP) marker gene coding region containing ST-LS1 intron followed by fragment comprising a Panicum virgatum Ubiquitin1 3′UTR.
FIG. 33 is a plasmid map showing the binary expression vector containing Setaria italica ubiquitin2 promoter fused to yellow fluorescent protein (Phi YFP) marker gene coding region containing ST-LS1 intron followed by fragment comprising a Setaria italica ubiquitin2 3′UTR.
FIG. 34 is a plasmid map showing the binary expression vector containing Brachypodium distachyon Ubiquitin1 promoter fused to yellow fluorescent protein (Phi YFP) marker gene coding region containing ST-LS1 intron followed by fragment comprising a Brachypodium distachyon Ubiquitin1 3′UTR.
FIG. 35 presents the Brachypodium distachyon Ubiquitin1 C coding sequence and putative promoter (upstream sequence of ATG). The upstream promoter sequence is underlined, the 5′-UTR sequence is presented in uppercase, the intron is boxed, the Ubi1 CDS is in italics, the 3′-UTR and the transcription termination sequence is downstream of TAA (Translational Stop Codon).
FIG. 36 presents the Brachypodium distachyon Ubiquitin 1 coding sequence and putative promoter. The upstream promoter is underlined, the 5′UTR sequence is in uppercase, the intron is boxed, the CDS is in italics, the 3′-UTR and transcription termination sequence is downstream of TAA (Translational Stop Codon).
FIG. 37 presents the Setaria italica Ubiquitin2 coding sequence and putative promoter. The upstream promoter is underlined, the 5′UTR sequence is in uppercase, the intron is boxed, the CDS is in italics, the 3′-UTR and transcription termination sequence is downstream of TAA (Translational Stop Codon).
FIG. 38 presents the Panicum virgatum (Switchgrass)Ubiquitin 1 coding sequence and putative promoter. The upstream promoter is underlined, the 5′UTR sequence is in uppercase, the intron is boxed, the CDS is in italics, the 3′-UTR and transcription termination sequence is downstream of TAA (Translational Stop Codon).



DETAILED DESCRIPTION
Definitions
In describing and claiming the invention, the following terminology will be used in accordance with the definitions set forth below.
The term “about” as used herein means greater or lesser than the value or range of values stated by 10 percent, but is not intended to designate any value or range of values to only this broader definition. Each value or range of values preceded by the term “about” is also intended to encompass the embodiment of the stated absolute value or range of values.
As used herein, the term “backcrossing” refers to a process in which a breeder crosses hybrid progeny back to one of the parents, for example, a first generation hybrid F1 with one of the parental genotypes of the F1 hybrid.
A “promoter” is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3′ direction) coding sequence. A promoter may contain specific sequences that are recognized by transcription factors. These factors may bind to a promoter DNA sequence, which results in the recruitment of RNA polymerase. For purposes of defining the present invention, the promoter sequence is bounded at its 3′ terminus by the transcription initiation site and extends upstream (5′ direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Within the promoter sequence will be found a transcription initiation site (conveniently defined for example, by mapping with nuclease S1), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase. The promoter may be operatively associated with other expression control sequences, including enhancer and repressor sequences.
For the purposes of the present disclosure, a “gene,” includes a DNA region encoding a gene product (see infra), as well as all DNA regions which regulate the production of the gene product, whether or not such regulatory sequences are adjacent to coding and/or transcribed sequences. Accordingly, a gene includes, but is not necessarily limited to, promoter sequences, terminators, translational regulatory sequences such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, boundary elements, replication origins, matrix attachment sites and locus control regions.
As used herein the terms “native” or “natural” define a condition found in nature. A “native DNA sequence” is a DNA sequence present in nature that was produced by natural means or traditional breeding techniques but not generated by genetic engineering (e.g., using molecular biology/transformation techniques).
As used herein a “transgene” is defined to be a nucleic acid sequence that encodes a gene product, including for example, but not limited to, an mRNA. In one embodiment the transgene is an exogenous nucleic acid, where the transgene sequence has been introduced into a host cell by genetic engineering (or the progeny thereof) where the transgene is not normally found. In one example, a transgene encodes an industrially or pharmaceutically useful compound, or a gene encoding a desirable agricultural trait (e.g., an herbicide-resistance gene). In yet another example, a transgene is an antisense nucleic acid sequence, wherein expression of the antisense nucleic acid sequence inhibits expression of a target nucleic acid sequence. In one embodiment the transgene is an endogenous nucleic acid, wherein additional genomic copies of the endogenous nucleic acid are desired, or a nucleic acid that is in the antisense orientation with respect to the sequence of a target nucleic acid in a host organism.
As used herein the term “non-ubiquitin transgene” is any transgene that has less than 80% sequence identity with the Zea may Ubiquitin 1 coding sequence (SEQ ID NO:27).
“Gene expression” as defined herein is the conversion of the information, contained in a gene, into a gene product.
A “gene product” as defined herein is any product produced by the gene. For example the gene product can be the direct transcriptional product of a gene (e.g., mRNA, tRNA, rRNA, antisense RNA, interfering RNA, ribozyme, structural RNA or any other type of RNA) or a protein produced by translation of a mRNA. Gene products also include RNAs which are modified, by processes such as capping, polyadenylation, methylation, and editing, and proteins modified by, for example, methylation, acetylation, phosphorylation, ubiquitination, ADP-ribosylation, myristilation, and glycosylation. Gene expression can be influenced by external signals, for example, exposure of a cell, tissue, or organism to an agent that increases or decreases gene expression. Expression of a gene can also be regulated anywhere in the pathway from DNA to RNA to protein. Regulation of gene expression occurs, for example, through controls acting on transcription, translation, RNA transport and processing, degradation of intermediary molecules such as mRNA, or through activation, inactivation, compartmentalization, or degradation of specific protein molecules after they have been made, or by combinations thereof. Gene expression can be measured at the RNA level or the protein level by any method known in the art, including, without limitation, Northern blot, RT-PCR, Western blot, or in vitro, in situ, or in vivo protein activity assay(s).
As used herein, the term “intron” is defined as any nucleic acid sequence comprised in a gene (or expressed nucleotide sequence of interest) that is transcribed but not translated. Introns include untranslated nucleic acid sequence within an expressed sequence of DNA, as well as corresponding sequence in RNA molecules transcribed therefrom. A construct described herein can also contain sequences that enhance translation and/or mRNA stability such as introns. An example of one such intron is the first intron of gene II of the histone H3 variant of Arabidopsis thaliana or any other commonly known intron sequence. Introns can be used in combination with a promoter sequence to enhance translation and/or mRNA stability.
As used herein, the terms “5′ untranslated region” or “5′-UTR” is defined as the untranslated segment in the 5′ terminus of pre-mRNAs or mature mRNAs. For example, on mature mRNAs, a 5′-UTR typically harbors on its 5′ end a 7-methylguanosine cap and is involved in many processes such as splicing, polyadenylation, mRNA export towards the cytoplasm, identification of the 5′ end of the mRNA by the translational machinery, and protection of the mRNAs against degradation.
As used herein, the terms “transcription terminator” is defined as the transcribed segment in the 3′ terminus of pre-mRNAs or mature mRNAs. For example, longer stretches of DNA beyond “polyadenylation signal” site is transcribed as a pre-mRNA. This DNA sequence usually contains one or more transcription termination signals for the proper processing of the pre-mRNA into mature mRNA.
As used herein, the term “3′ untranslated region” or “3′-UTR” is defined as the untranslated segment in a 3′ terminus of the pre-mRNAs or mature mRNAs. For example, on mature mRNAs this region harbors the poly-(A) tail and is known to have many roles in mRNA stability, translation initiation, and mRNA export.
As used herein, the term “polyadenylation signal” designates a nucleic acid sequence present in mRNA transcripts that allows for transcripts, when in the presence of a poly-(A) polymerase, to be polyadenylated on the polyadenylation site, for example, located 10 to 30 bases downstream of the poly-(A) signal. Many polyadenylation signals are known in the art and are useful for the present invention. An exemplary sequence includes AAUAAA and variants thereof, as described in Loke J., et al., (2005) Plant Physiology 138(3); 1457-1468.
The term “isolated” as used herein means having been removed from its natural environment, or removed from other compounds present when the compound is first formed. The term “isolated” embraces 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.
The term “purified,” as used herein relates to the isolation of a molecule or compound in a form that is substantially free of contaminants normally associated with the molecule or compound in a native or natural environment, or substantially enriched in concentration relative to other compounds present when the compound is first formed, and means having been increased in purity as a result of being separated from other components of the original composition. The term “purified nucleic acid” is used herein to describe a nucleic acid sequence which has been separated, produced apart from, or purified away from other biological compounds including, but not limited to polypeptides, lipids and carbohydrates, while effecting a chemical or functional change in the component (e.g., a nucleic acid may be purified from a chromosome by removing protein contaminants and breaking chemical bonds connecting the nucleic acid to the remaining DNA in the chromosome).
As used herein, the terms “homology-based gene silencing” or “HBGS” are generic terms that include both transcriptional gene silencing and posttranscriptional gene silencing. Silencing of a target locus by an unlinked silencing locus can result from transcription inhibition (transcriptional gene silencing; TGS) or mRNA degradation (post-transcriptional gene silencing; PTGS), owing to the production of double-stranded RNA (dsRNA) corresponding to promoter or transcribed sequences, respectively. Involvement of distinct cellular components in each process suggests that dsRNA-induced TGS and PTGS likely result from the diversification of an ancient common mechanism. However, a strict comparison of TGS and PTGS has been difficult to achieve because it generally relies on the analysis of distinct silencing loci. A single transgene locus can be described to trigger both TGS and PTGS, owing to the production of dsRNA corresponding to promoter and transcribed sequences of different target genes.
As used herein, the terms “nucleic acid molecule”, “nucleic acid”, or “polynucleotide” (all three terms are synonymous with one another) refer to a polymeric form of nucleotides, which may include both sense and anti-sense strands of RNA, cDNA, genomic DNA, and synthetic forms, and mixed polymers thereof. “A nucleotide” may refer to a ribonucleotide, deoxyribonucleotide, or a modified form of either type of nucleotide. A nucleic acid molecule is usually at least 10 bases in length, unless otherwise specified. The terms may refer to a molecule of RNA or DNA of indeterminate length. The terms include single- and double-stranded forms of DNA. A nucleic acid molecule may include either or both naturally-occurring and modified nucleotides linked together by naturally occurring and/or non-naturally occurring nucleotide linkages.
Nucleic acid molecules may be modified chemically or biochemically, or may contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those of skill in the art. Such modifications include, for example, labels, methylation, substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications (e.g., uncharged linkages: for example, methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.; charged linkages: for example, phosphorothioates, phosphorodithioates, etc.; pendent moieties: for example, peptides; intercalators: for example, acridine, psoralen, etc.; chelators; alkylators; and modified linkages: for example, alpha anomeric nucleic acids, etc.). The term “nucleic acid molecule” also includes any topological conformation, including single-stranded, double-stranded, partially duplexed, triplexed, hairpinned, circular, and padlocked conformations.
Transcription proceeds in a 5′ to 3′ manner along a DNA strand. This means that RNA is made by sequential addition of ribonucleotide-5′-triphosphates to the 3′ terminus of the growing chain (with a requisite elimination of the pyrophosphate). In either a linear or circular nucleic acid molecule, discrete elements (e.g., particular nucleotide sequences) may be referred to as being “upstream” relative to a further element if they are bonded or would be bonded to the same nucleic acid in the 5′ direction from that element. Similarly, discrete elements may be “downstream” relative to a further element if they are or would be bonded to the same nucleic acid in the 3′ direction from that element.
As used herein, the term “base position,” refers to the location of a given base or nucleotide residue within a designated nucleic acid. A designated nucleic acid may be defined by alignment with a reference nucleic acid.
As used herein, the term “hybridization” refers to a process where oligonucleotides and their analogs hybridize by hydrogen bonding, which includes Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary bases. Generally, nucleic acid molecules consist of nitrogenous bases that are either pyrimidines (cytosine (C), uracil (U), and thymine (T)) or purines (adenine (A) and guanine (G)). These nitrogenous bases form hydrogen bonds between a pyrimidine and a purine, and bonding of a pyrimidine to a purine is referred to as “base pairing.” More specifically, A will hydrogen bond to T or U, and G will bond to C. “Complementary” refers to the base pairing that occurs between two distinct nucleic acid sequences or two distinct regions of the same nucleic acid sequence.
As used herein, the terms “specifically hybridizable” and “specifically complementary” refers to a sufficient degree of complementarity such that stable and specific binding occurs between an oligonucleotide and the DNA or RNA target. Oligonucleotides need not be 100% complementary to its target sequence to specifically hybridize. An oligonucleotide is specifically hybridizable when binding of the oligonucleotide to the target DNA or RNA molecule interferes with the normal function of the target DNA or RNA, and there is sufficient degree of complementarity to avoid non-specific binding of an oligonucleotide to non-target sequences under conditions where specific binding is desired, for example under physiological conditions in the case of in vivo assays or systems. Such binding is referred to as specific hybridization. Hybridization conditions resulting in particular degrees of stringency will vary depending upon the nature of the chosen hybridization method and the composition and length of the hybridizing nucleic acid sequences. Generally, the temperature of hybridization and the ionic strength (especially Na+ and/or Mg2+ concentration) of a hybridization buffer will contribute to the stringency of hybridization, though wash times also influence stringency. Calculations regarding hybridization conditions required for attaining particular degrees of stringency are discussed in Sambrook et al. (ed.), Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1-3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, chs. 9 and 11.
As used herein, the term “stringent conditions” encompasses conditions under which hybridization will only occur if there is less than 50% mismatch between the hybridization molecule and the DNA target. “Stringent conditions” include further particular levels of stringency. Thus, as used herein, “moderate stringency” conditions are those under which molecules with more than 50% sequence mismatch will not hybridize; conditions of “high stringency” are those under which sequences with more than 20% mismatch will not hybridize; and conditions of “very high stringency” are those under which sequences with more than 10% mismatch will not hybridize. In particular embodiments, stringent conditions can include hybridization at 65° C., followed by washes at 65° C. with 0.1×SSC/0.1% SDS for 40 minutes. The following are representative, non-limiting hybridization conditions:

    
    
        Very High Stringency: hybridization in 5×SSC buffer at 65° C. for 16 hours; wash twice in 2×SSC buffer at room temperature for 15 minutes each; and wash twice in 0.5×SSC buffer at 65° C. for 20 minutes each.
        High Stringency: Hybridization in 5-6×SSC buffer at 65-70° C. for 16-20 hours; wash twice in 2×SSC buffer at room temperature for 5-20 minutes each; and wash twice in 1×SSC buffer at 55-70° C. for 30 minutes each.
        Moderate Stringency: Hybridization in 6×SSC buffer at room temperature to 55° C. for 16-20 hours; wash at least twice in 2×-3×SSC buffer at room temperature to 55° C. for 20-30 minutes each.

In an embodiment, specifically hybridizable nucleic acid molecules can remain bound under very high stringency hybridization conditions. In an embodiment, specifically hybridizable nucleic acid molecules can remain bound under high stringency hybridization conditions. In an embodiment, specifically hybridizable nucleic acid molecules can remain bound under moderate stringency hybridization conditions.

    
    


As used herein, the term “oligonucleotide” refers to a short nucleic acid polymer. Oligonucleotides may be formed by cleavage of longer nucleic acid segments, or by polymerizing individual nucleotide precursors. Automated synthesizers allow the synthesis of oligonucleotides up to several hundred base pairs in length. Because oligonucleotides may bind to a complementary nucleotide sequence, they may be used as probes for detecting DNA or RNA. Oligonucleotides composed of DNA (oligodeoxyribonucleotides) may be used in PCR, a technique for the amplification of small DNA sequences. In PCR, an oligonucleotide is typically referred to as a “primer,” which allows a DNA polymerase to extend the oligonucleotide and replicate the complementary strand.
As used herein, the terms “Polymerase chain reaction” or “PCR” define a procedure or technique in which minute amounts of nucleic acid, RNA and/or DNA, are amplified as described in U.S. Pat. No. 4,683,195 issued Jul. 28, 1987. Generally, sequence information from the ends of the region of interest or beyond needs to be available, such that oligonucleotide primers can be designed; these primers will be identical or similar in sequence to opposite strands of the template to be amplified. The 5′ terminal nucleotides of the two primers may coincide with the ends of the amplified material. PCR can be used to amplify specific RNA sequences, specific DNA sequences from total genomic DNA, and cDNA transcribed from total cellular RNA, bacteriophage or plasmid sequences, etc. See generally Mullis et al., Cold Spring Harbor Symp. Quant. Biol., 51:263 (1987); Erlich, ed., PCR Technology, (Stockton Press, NY, 1989).
As used herein, the term “primer” refers to an oligonucleotide capable of acting as a point of initiation of synthesis along a complementary strand when conditions are suitable for synthesis of a primer extension product. The synthesizing conditions include the presence of four different deoxyribonucleotide triphosphates and at least one polymerization-inducing agent such as reverse transcriptase or DNA polymerase. These are present in a suitable buffer, which may include constituents which are co-factors or which affect conditions such as pH and the like at various suitable temperatures. A primer is preferably a single strand sequence, such that amplification efficiency is optimized, but double stranded sequences can be utilized.
As used herein, the term “probe” refers to an oligonucleotide that hybridizes to a target sequence. In the TaqMan® or TaqMan®-style assay procedure, the probe hybridizes to a portion of the target situated between the annealing site of the two primers. A probe includes about eight nucleotides, about ten nucleotides, about fifteen nucleotides, about twenty nucleotides, about thirty nucleotides, about forty nucleotides, or about fifty nucleotides. In some embodiments, a probe includes from about eight nucleotides to about fifteen nucleotides. A probe can further include a detectable label, e.g., a fluorophore (Texas-Red®, Fluorescein isothiocyanate, etc.). The detectable label can be covalently attached directly to the probe oligonucleotide, e.g., located at the probe's 5′ end or at the probe's 3′ end. A probe including a fluorophore may also further include a quencher, e.g., Black Hole QuencherTM, Iowa BlackTM, etc.
As used herein, the terms “sequence identity” or “identity” can be used interchangeably and refer to nucleic acid residues in two sequences that are the same when aligned for maximum correspondence over a specified comparison window.
As used herein, the term “percentage of sequence identity” refers to a value determined by comparing two optimally aligned sequences (e.g., nucleic acid sequences or amino acid sequences) over a comparison window, wherein the portion of a sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to a reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. A percentage is calculated by determining the number of positions at which an identical nucleic acid 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 comparison window, and multiplying the result by 100 to yield the percentage of sequence identity. Methods for aligning sequences for comparison are well known. Various programs and alignment algorithms are described in, for example: Smith and Waterman (1981) Adv. Appl. Math. 2:482; Needleman and Wunsch (1970) J. Mol. Biol. 48:443; Pearson and Lipman (1988) Proc. Natl. Acad. Sci. U.S.A. 85:2444; Higgins and Sharp (1988) Gene 73:237-44; Higgins and Sharp (1989) CABIOS 5:151-3; Corpet et al. (1988) Nucleic Acids Res. 16:10881-90; Huang et al. (1992) Comp. Appl. Biosci. 8:155-65; Pearson et al. (1994) Methods Mol. Biol. 24:307-31; Tatiana et al. (1999) FEMS Microbiol. Lett. 174:247-50.
The National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLASTTM; Altschul et al. (1990) J. Mol. Biol. 215:403-10) is available from several sources, including the National Center for Biotechnology Information (Bethesda, Md.), and on the internet, for use in connection with several sequence analysis programs. A description of how to determine sequence identity using this program is available on the internet under the “help” section for BLASTTM. For comparisons of nucleic acid sequences, the “Blast 2 sequences” function of the BLASTTM (Blastn) program may be employed using the default parameters. Nucleic acid sequences with even greater similarity to the reference sequences will show increasing percentage identity when assessed by this method.
As used herein, the term “operably linked” refers to two components that have been placed into a functional relationship with one another. The term, “operably linked,” when used in reference to a regulatory sequence and a coding sequence, means that the regulatory sequence affects the expression of the linked coding sequence. “Regulatory sequences,” “regulatory elements”, or “control elements,” refer to nucleic acid sequences that influence the timing and level/amount of transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences may include promoters; translation leader sequences; 5′ and 3′ untranslated regions, introns; enhancers; stem-loop structures; repressor binding sequences; termination sequences; polyadenylation recognition sequences; etc. Particular regulatory sequences may be located upstream and/or downstream of a coding sequence operably linked thereto. Also, particular regulatory sequences operably linked to a coding sequence may be located on the associated complementary strand of a double-stranded nucleic acid molecule. Linking can be accomplished by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice. However, elements need not be contiguous to be operably linked.
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; transformation with plasmid vectors; electroporation; lipofection; microinjection (Mueller et al. (1978) Cell 15:579-85); Agrobacterium-mediated transfer; direct DNA uptake; whiskers-mediated transformation; and microprojectile bombardment.
As used herein, the term “transduce” refers to a process where a virus transfers nucleic acid into a cell.
The terms “polylinker” or “multiple cloning site” as used herein defines a cluster of three or more Type-2 restriction enzyme sites located within 10 nucleotides of one another on a nucleic acid sequence. Constructs comprising a polylinker are utilized for the insertion and/or excision of nucleic acid sequences such as the coding region of a gene.
As used herein, the terms “restriction endonucleases” and “restriction enzymes” refer to bacterial enzymes, each of which cut double-stranded DNA at or near a specific nucleotide sequence. Type-2 restriction enzymes recognize and cleave DNA at the same site, and include but are not limited to XbaI, BamHI, HindIII, EcoRI, XhoI, SalI, KpnI, AvaI, PstI and SmaI.
The term “vector” is used interchangeably with the terms “construct”, “cloning vector” and “expression vector” and means the vehicle by which a DNA or RNA sequence (e.g. a foreign gene) can be introduced into a host cell, so as to transform the host and promote expression (e.g. transcription and translation) of the introduced sequence. A “non-viral vector” is intended to mean any vector that does not comprise a virus or retrovirus. In some embodiments a “vector” is a sequence of DNA comprising at least one origin of DNA replication and at least one selectable marker gene. Examples include, but are not limited to, a plasmid, cosmid, bacteriophage, bacterial artificial chromosome (BAC), or virus that carries exogenous DNA into a cell. A vector can also include one or more genes, antisense molecules, and/or selectable marker genes and other genetic elements known in the art. A vector may transduce, transform, or infect a cell, thereby causing the cell to express the nucleic acid molecules and/or proteins encoded by the vector.
The term “plasmid” defines a circular strand of nucleic acid capable of autosomal replication in either a prokaryotic or a eukaryotic host cell. The term includes nucleic acid which may be either DNA or RNA and may be single- or double-stranded. The plasmid of the definition may also include the sequences which correspond to a bacterial origin of replication.
The term “selectable marker gene” as used herein defines a gene or other expression cassette which encodes a protein which facilitates identification of cells into which the selectable marker gene is inserted. For example a “selectable marker gene” encompasses reporter genes as well as genes used in plant transformation to, for example, protect plant cells from a selective agent or provide resistance/tolerance to a selective agent. In one embodiment only those cells or plants that receive a functional selectable marker are capable of dividing or growing under conditions having a selective agent. Examples of selective agents can include, for example, antibiotics, including spectinomycin, neomycin, kanamycin, paromomycin, gentamicin, and hygromycin. These selectable markers include neomycin phosphotransferase (npt II), which expresses an enzyme conferring resistance to the antibiotic kanamycin, and genes for the related antibiotics neomycin, paromomycin, gentamicin, and G418, or the gene for hygromycin phosphotransferase (hpt), which expresses an enzyme conferring resistance to hygromycin. Other selectable marker genes can include genes encoding herbicide resistance including bar or pat (resistance against glufosinate ammonium or phosphinothricin), acetolactate synthase (ALS, resistance against inhibitors such as sulfonylureas (SUs), imidazolinones (IMIs), triazolopyrimidines (TPs), pyrimidinyl oxybenzoates (POBs), and sulfonylamino carbonyl triazolinones that prevent the first step in the synthesis of the branched-chain amino acids), glyphosate, 2,4-D, and metal resistance or sensitivity. Examples of “reporter genes” that can be used as a selectable marker gene include the visual observation of expressed reporter gene proteins such as proteins encoding β-glucuronidase (GUS), luciferase, green fluorescent protein (GFP), yellow fluorescent protein (YFP), DsRed, β-galactosidase, chloramphenicol acetyltransferase (CAT), alkaline phosphatase, and the like. The phrase “marker-positive” refers to plants that have been transformed to include a selectable marker gene.
As used herein, the term “detectable marker” refers to a label capable of detection, such as, for example, a radioisotope, fluorescent compound, bioluminescent compound, a chemiluminescent compound, metal chelator, or enzyme. Examples of detectable markers include, but are not limited to, the following: fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors), enzymatic labels (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminescent, biotinyl groups, predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags). In an embodiment, a detectable marker can be attached by spacer arms of various lengths to reduce potential steric hindrance.
As used herein, the term “detecting” is used in the broadest sense to include both qualitative and quantitative measurements of a specific molecule, for example, measurements of a specific polypeptide.
As used herein, the terms “cassette”, “expression cassette” and “gene expression cassette” refer to a segment of DNA that can be inserted into a nucleic acid or polynucleotide at specific restriction sites or by homologous recombination. As used herein the segment of DNA comprises a polynucleotide that encodes a polypeptide of interest, and the cassette and restriction sites are designed to ensure insertion of the cassette in the proper reading frame for transcription and translation. In an embodiment, an expression cassette can include a polynucleotide that encodes a polypeptide of interest and having elements in addition to the polynucleotide that facilitate transformation of a particular host cell. In an embodiment, a gene expression cassette may also include elements that allow for enhanced expression of a polynucleotide encoding a polypeptide of interest in a host cell. These elements may include, but are not limited to: a promoter, a minimal promoter, an enhancer, a response element, a terminator sequence, a polyadenylation sequence, and the like.
As used herein a “linker” or “spacer” is a bond, molecule or group of molecules that binds two separate entities to one another. Linkers and spacers may provide for optimal spacing of the two entities or may further supply a labile linkage that allows the two entities to be separated from each other. Labile linkages include photocleavable groups, acid-labile moieties, base-labile moieties and enzyme-cleavable groups.
As used herein, the term “control” refers to a sample used in an analytical procedure for comparison purposes. A control can be “positive” or “negative”. For example, where the purpose of an analytical procedure is to detect a differentially expressed transcript or polypeptide in cells or tissue, it is generally preferable to include a positive control, such as a sample from a known plant exhibiting the desired expression, and a negative control, such as a sample from a known plant lacking the desired expression.
As used herein, the term “plant” includes a whole plant and any descendant, cell, tissue, or part of a plant. A class of plant that can be used in the present invention is generally as broad as the class of higher and lower plants amenable to mutagenesis including angiosperms (monocotyledonous and dicotyledonous plants), gymnosperms, ferns and multicellular algae. Thus, “plant” includes dicot and monocot plants. The term “plant parts” include any part(s) of a plant, including, for example and without limitation: seed (including mature seed and immature seed); a plant cutting; a plant cell; a plant cell culture; a plant organ (e.g., pollen, embryos, flowers, fruits, shoots, leaves, roots, stems, and explants). A plant tissue or plant organ may be a seed, protoplast, callus, or any other group of plant cells that is organized into a structural or functional unit. A plant cell or tissue culture may be capable of regenerating a plant having the physiological and morphological characteristics of the plant from which the cell or tissue was obtained, and of regenerating a plant having substantially the same genotype as the plant. In contrast, some plant cells are not capable of being regenerated to produce plants. Regenerable cells in a plant cell or tissue culture may be embryos, protoplasts, meristematic cells, callus, pollen, leaves, anthers, roots, root tips, silk, flowers, kernels, ears, cobs, husks, or stalks.
Plant parts include harvestable parts and parts useful for propagation of progeny plants. Plant parts useful for propagation include, for example and without limitation: seed; fruit; a cutting; a seedling; a tuber; and a rootstock. A harvestable part of a plant may be any useful part of a plant, including, for example and without limitation: flower; pollen; seedling; tuber; leaf; stem; fruit; seed; and root.
A plant cell is the structural and physiological unit of the plant, comprising a protoplast and a cell wall. A plant cell may be in the form of an isolated single cell, or an aggregate of cells (e.g., a friable callus and a cultured cell), and may be part of a higher organized unit (e.g., a plant tissue, plant organ, and plant). 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. As such, a seed, which comprises multiple plant cells and is capable of regenerating into a whole plant, is considered a “plant cell” in embodiments herein.
The term “protoplast,” as used herein, refers to a plant cell that had its cell wall completely or partially removed, with the lipid bilayer membrane thereof naked, and thus includes protoplasts, which have their cell wall entirely removed, and spheroplasts, which have their cell wall only partially removed, but is not limited thereto. Typically, a protoplast is an isolated plant cell without cell walls which has the potency for regeneration into cell culture or a whole plant.
Unless otherwise specifically explained, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this disclosure belongs. Definitions of common terms in molecular biology can be found in, for example: Lewin, Genes V, Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Meyers (ed.), Molecular Biology and Biotechnology: A Comprehensive Desk Reference, VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).
Embodiments
As disclosed herein novel recombinant constructs are provided for expressing a non-ubiquitin transgene using the regulatory sequences of a ubiquitin gene from Panicum virgatum, Brachypodium distachyon, or Setaria italica. These constructs can be used to transform cells, including plant cells, to produce complete organisms that express the transgene gene product in their cells.
Regulatory Elements
Plant promoters used for basic research or biotechnological application are generally unidirectional, directing only one gene that has been fused at its 3′ end (downstream). It is often necessary to introduce multiple genes into plants for metabolic engineering and trait stacking and therefore, multiple promoters are typically required in transgenic crops to drive the expression of multiple genes.
Development of transgenic products is becoming increasingly complex, which requires stacking multiple transgenes into a single locus. Traditionally, each transgene usually requires a promoter for expression wherein multiple promoters are required to express different transgenes within one gene stack. This frequently leads to repetitive use of the same promoter within one transgene stack to obtain similar levels of expression patterns of different transgenes for expression of a single polygenic trait. Multi-gene constructs driven by the same promoter are known to cause gene silencing resulting in less efficacious transgenic products in the field. Excess of transcription factor (TF)-binding sites due to promoter repetition can cause depletion of endogenous TFs leading to transcriptional inactivation. The silencing of transgenes will likely undesirably affect performance of a transgenic plant produced to express transgenes. Repetitive sequences within a transgene may lead to gene intra locus homologous recombination resulting in polynucleotide rearrangements.
It is desirable to use diversified promoters for the expression of different transgenes in a gene stack. In an embodiment, diversified constitutive ubiquitin obtained from different plant species can drive transcription of multiple transcription units, including RNAi, artificial miRNA, or hairpin-loop RNA sequences.
Provided are methods and constructs using a constitutive ubiquitin (Ubi1) promoter to express non-ubiquitin transgenes in plant. In an embodiment, a promoter can be the Brachypodium distachyon ubiquitin1 C (Ubi1C) promoter.







(SEQ ID NO: 1)







CTGCTCGTTCAGCCCACAGTAACACGCCGTGCGACATGCAGATGCCC


 


TCCACCACGCCGACCAACCCCAAGTCCGCCGCGCTCGTCCACGGCGC


 


CATCCGCATCCGCGCGTCAACGTCATCCGGAGGAGGCGAGCGCGATG


 


TCGACGGCCACGGCGGCGGCGGACACGACGGCGACGCCCCGACTCC


 


GCGCGCGCGTCAAGGCTGCAGTGGCGTCGTGGTGGCCGTCCGCCTGC


 


ACGAGATCCCCGCGTGGACGAGCGCCGCCTCCACCCAGCCCCTATAT


 


CGAGAAATCAACGGTGGGCTCGAGCTCCTCAGCAACCTCCCCACCCC


 


CCCTTCCGACCACGCTCCCTTCCCCCGTGCCCCTCTTCTCCGTAAACC


 


CGAGCCGCCGAGAACAACACCAACGAAAGGGCGAAGAGAATCGCCA


 


TAGAGAGGAGATGGGCGGAGGCGGATAGTTTCAGCCATTCACGGAG


 


AAATGGGGAGGAGAGAACACGACATCATACGGACGCGACCCTCTAG


 


CTGGCTGGCTGTCCTAAAGAATCGAACGGAATCGCTGCGCCAGGAGA


 


AAACGAACGGTCCTGAAGCATGTGCGCCCGGTTCTTCCAAAACACTT


 


ATCTTTAAGATTGAAGTAGTATATATGACTGAAATTTTTACAAGGTTT


 


TTCCCCATAAAACAGGTGAGCTTATCTCATCCTTTTGTTTAGGATGTA


 


CGTATTATATATGACTGAATATTTTTTATTTTCATTGAATGAAGATTTT


 


CGACCCCCCAAAAATAAAAAACGGAGGGAGTACCTTTGTGCCGTGTA


 


TATGGACTAGAGCCATCGGGACGTTTCCGGAGACTGCGTGGTGGGGG


 


CGATGGACGCACAACGACCGCATTTTCGGTTGCCGACTCGCCGTTCG


 


CATCTGGTAGGCACGACTCGTCGGGTTCGGCTCTTGCGTGAGCCGTG


 


ACGTAACAGACCCGTTCTCTTCCCCCGTCTGGCCATCCATAAATCCCC


 


CCTCCATCGGCTTCCCTTTCCTCAATCCAGCACCCTGATT






In an embodiment, a promoter can be the Brachypodium distachyon ubiquitin 1 (Ubi1) promoter.








(SEQ ID NO: 2)







GGCGTCAGGACTGGCGAAGTCTGGACTCTGCAGGGCCGAACTGCTGA


 


AGACGAAGCAGAGGAAGAGAAAGGGAAGTGTTCGACTTGTAATTTG


 


TAGGGGTTTTTTTTAGAGGAACTTGTAATTTGTAGGTGGGCTGGCCTC


 


GTTGGAAAAACGATGCTGGCTGGTTGGGCTGGGCCGATGTACGCTTG


 


CAAACAACTTGTGGCGGCCCGTTCTGGACGAGCAGGAGTTTCTTTTTT


 


GTTCTCACTTTTCTGGTCTTCTTTAGTTACGGAGTACCTTTTGTTTTTT


 


AAAGGAGTTACCTTTTTTTTAGGAATTCTTTAGTTACCTTTCGCTTGCT


 


CTCAAAAAATATTTAACTTTCGCTTTTTTTCATTTTAATTTTTGCAACT


 


ATTTACGAGTTTCATGAATGCTTATTTTCCAGCATATCATTATTTGCA


 


AGTATTTTTATGCCGTATGTATTGGACGAGAGCCATCGGGACTGTTCC


 


AGAGACTGCGTGGTGGGGACGGCTCCCAACCGCCTTTTCTATCTCTGT


 


TCGCATCCGGTGGCCGACTTGGCTCGCGCGTGAGCCGTGACGTAACA


 


GACTTGGTCTCTTCCCCATCTGGCCATCTATAAATTCCCCCATCGATC


 


GACCCTCCCTTTCC






In an embodiment, a promoter can be the Setaria italica ubiquitin 2 (Ubi2) promoter.








(SEQ ID NO: 3)







TGCGTCTGGACGCACAAGTCATAGCATTATCGGCTAAAATTTCTTAAT


 


TTCTAAATTAGTCATATCGGCTAAGAAAGTGGGGAGCACTATCATTT


 


CGTAGAACAAGAACAAGGTATCATATATATATATATATATAATATTT


 


AAACTTTGTTAAGTGGAATCAAAGTGCTAGTATTAATGGAGTTTCAT


 


GTGCATTAAATTTTATGTCACATCAGCAATTTTGTTGACTTGGCAAGG


 


TCATTTAGGGTGTGTTTGGAAGACAGGGGCTATTAGGAGTATTAAAC


 


ATAGTCTAATTACAAAACTAATTGCACAACCGCTAAGCTGAATCGCG


 


AGATGGATCTATTAAGCTTAATTAGTCCATGATTTGACAATGTGGTGC


 


TACAATAACCATTTGCTAATGATGGATTACTTAGGTTTAATAGATTCG


 


TCTCGTGATTTAGCCTATGGGTTCTGCTATTAATTTTGTAATTAGCTCA


 


TATTTAGTTCTTATAATTAGTATCCGAACATCCAATGTGACATGCTAA


 


AGTTTAACCCTGGTATCCAAATGAAGTCTTATGAGAGTTTCATCACTC


 


CGGTGGTATATGTACTTAGGCTCCGTTTTCTTCCACCGACTTATTTTTA


 


GCACCCGTCACATTGAATGTTTAGATACTAATTAGAAGTATTAAACG


 


TAGACTATTTACAAAATCCATTACATAAGACGAATCTAAACGGCGAG


 


ACGAATCTATTAAACCTAATTAGTCCATGATTTGACAATGTGTTGCTA


 


CAGTAAACATTTGCTAATGATGGATTAATTAGGCTTAATAGATTCGTC


 


TCGCCGTTTAGCCTCCACTTATGTAATGGGTTTTCTAAACAATCTACG


 


TTTAATACTCCTAATTAGTATCTAAATATTCAATGTGACACGTGCTAA


 


AAATAAGTCAGTGGAAGGAAGAGAACGTCCCCTTAGTTTTCCATCTT


 


ATTAATTGTACGATGAAACTGTGCAGCCAGATGATTGACAATCGCAA


 


TACTTCAACTAGTGGGCCATGCACATCAGCGACGTGTAACGTCGTGA


 


GTTGCTGTTCCCGTAG






In an embodiment, a promoter can be the Panicum virgatum (Switchgrass) ubiquitin 1 promoter.








(SEQ ID NO: 35)







TTGAATTTTAATTTCAAATTTTGCAGGGTAGTAGTGGACATCACAATA


 


CATATTTAGAAAAAGTTTTATAATTTTCCTCCGTTAGTTTTCATATAAT


 


TTTGAACTCCAACGATTAATCTATTATTAAATATCCCGATCTATCAAA


 


ATAATGATAAAAATTTATGATTAATTTTTCTAACATGTGTTATGGTGT


 


GTACTATCGTCTTATAAAATTTCAACTTAAAACTCCACCTATACATGG


 


AGAAATGAAAAAGACGAATTACAGTAGGGAGTAATTTGAACCAAAT


 


GGAATAGTTTGAGGGTAAAATGAACTAAACAATAGTTTAGGAGGTTA


 


TTCAGATTTTAGTTATAGTTGAGAGGAGTAATTTAGACTTTTTCCTAT


 


CTTGAATTGTTGACGGCTCTCCTATCGGATATCGGATGGAGTCTTTCA


 


GCCCAACATAACTTCATTCGGGCCCAAACGTTCGTCCATCCAGCCTA


 


GGGAGAACATTTTGCCCATGATATCTGTTTTTCTTTTTTTCTATTTTCA


 


CTGGTATTATAGGAGGGAAATATACAACGTGTTCACCTTTGGTTTCAT


 


TCTTGTTCCATCTGAATTTATCTAAAACTGTGTTTGAACTTCGTAAGA


 


ATTTTGTTCGATCTGTCCGGTACATCGTGTTGATAGGTGGCCTCCGAG


 


ATTCTTCTTTTTAACCGGCAAAGTAAAATAATCTCAGCTCCAGCCTAA


 


CGTCAATTATCAGAGAGAGAAAAAAATATTTTTTTATGATTGATCGG


 


AAACCAACCGCCTTACGTGTCGATCCTGGTTCCTGGCCGGCACGGCG


 


GAGGAAAGCGACCGACCTCGCAACGCCGGCGCACGGCGCCGCCGTG


 


TTGGACTTGGTCTCCCGCGACTCCGTGGGCCTCGGCTTATCGCCGCCG


 


CTCCATCTCAACCGTCCGCTTGGACACGTGGAAGTTGATCCGTCGCGC


 


ACCAGCCTCGGAGGTAACCTAACTGCCCGTACTATAAATCCGGGATC


 


CGGCCTCTCCAATCCCCATCGCCA






In an embodiment, a nucleic acid construct is provided comprising a ubiquitin promoter. In an embodiment, the ubiquitin promoter is a Panicum virgatum, Brachypodium distachyon or Setaria italica ubiquitin promoter. In an embodiment, a nucleic acid construct is provided comprising a promoter, wherein the promoter is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, or 100% identical to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:35. In an embodiment, a nucleic acid construct is provided comprising a ubiquitin promoter that is operably linked to a polylinker. In an embodiment, a gene expression cassette is provided comprising a ubiquitin promoter that is operably linked to a non-ubiquitin transgene. In one embodiment the promoter consists of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:35. In an illustrative embodiment, a gene expression cassette comprises a ubiquitin promoter that is operably linked to the 5′ end of a transgene, wherein the transgene can be an insecticidal resistance transgene, an herbicide tolerance transgene, a nitrogen use efficiency transgene, a water us efficiency transgene, a nutritional quality transgene, a DNA binding transgene, a selectable marker transgene, or combinations thereof.
In addition to a promoter, a 3′-untranslated gene region (i.e., 3′UTR) or terminator is needed for transcription termination and polyadenylation of the mRNA. Proper transcription termination and polyadenylation of mRNA is important for stable expression of transgene. The transcription termination becomes more critical for multigene stacks to avoid transcription read-through into next transgene. Similarly, non-polyadenylated aberrant RNA (aRNA) is a substrate for plant RNA-dependent RNA polymerases (RdRPs) to convert aRNA into double stranded RNA (dsRNA) leading to small RNA production and transgene silencing. Strong transcription terminators therefore are very useful both for single gene and multiple gene stacks. While a promoter is necessary to drive transcription, a 3′-UTR gene region can terminate transcription and initiate polyadenylation of a resulting mRNA transcript for translation and protein synthesis. A 3′-UTR gene region aids stable expression of a transgene.
In accordance with one embodiment a nucleic acid construct is provided comprising a ubiquitin transcription terminator. In an embodiment, the ubiquitin transcription terminator is a Panicum virgatum, Brachypodium distachyon or Setaria italica ubiquitin transcription terminator. In an embodiment, a nucleic acid construct is provided comprising a transcription terminator, wherein the transcription terminator 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:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:36. In an embodiment, a nucleic acid construct is provided comprising a ubiquitin transcription terminator that is operably linked to a polylinker. In an embodiment, a gene expression cassette is provided comprising a ubiquitin transcription terminator that is operably linked to the 3′ end of a non-ubiquitin transgene. In one embodiment the transcription terminator consists of SEQ ID NO: 4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:36. In an illustrative embodiment, a gene expression cassette comprises a ubiquitin transcription terminator that is operably linked to a transgene, wherein the transgene can be an insecticidal resistance transgene, an herbicide tolerance transgene, a nitrogen use efficiency transgene, a water us efficiency transgene, a nutritional quality transgene, a DNA binding transgene, a selectable marker transgene, or combinations thereof. In one embodiment a nucleic acid vector is provided comprising a transcription terminator operably linked to either a polylinker sequence, a non-ubiquitin transgene or a combination of both, wherein the transcription terminator comprises SEQ ID NO: 6 or a sequence that has 90% sequence identity with SEQ ID NO: 6. In one embodiment the transcription terminator is less than 1 kb in length, and in a further embodiment the transcription terminator consists of the 3′UTR sequence of SEQ ID NO: 6.
In an embodiment, a nucleic acid construct is provided comprising a ubiquitin promoter as described herein and a 3′-UTR. In an embodiment, the nucleic acid construct comprises a ubiquitin 3′-UTR. In an embodiment, the ubiquitin 3′-UTR is a Panicum virgatum, Brachypodium distachyon or Setaria italica ubiquitin 3′-UTR. In an embodiment, a 3′-UTR can be the Brachypodium distachyon ubiquitin1 C (Ubi1C) 3′-UTR.







(SEQ ID NO: 4)







GTTTGTCAAAAACTGGCCTACAGTCTGCTGCCCCTGTTGGTCTGCCCC


 


TTGGAAGTAGTCGTGTCTATGGTTATGTGAGAAGTCGTTGTGTTCTTT


 


CTAATCCCGTACTGTTTGTGTGAACATCTGCTGCTGTCGTATTGCATC


 


GTGAAGAATCCTGTTATGAATAAGTGAACATGAACCTTGTTCTGTGA


 


TTACGGCTTCGTGGTTATGCGAACGTTCTTACAAACGCAATTGCACCT


 


GATGTAAAATCGTTTTTGCTAGCTGTATGGAACAAGTGCTCATGATGT


 


TCATGCAAGATGCAATTCCAGCTTTTGTTGGTTTGTCATCTTTGTACT


 


GTGCTTACCGCACATAAAGATTGCATCTTGCTTATTGCTTTGTTGCTTT


 


GGTGCTCGTCCGCTTCTCCTTGCACCTTATCAAACCTTTGTTTAGATTC


 


TCTTCTTATAGCACTTGGTAACTCTCAGCTTTACAACGCCAGTACTGT


 


TTCTGAAATTTCATGACTGATAAAGCTGATAGATGGAGTACTAATAT


 


ATGACATCTTTCCATAAATGTTCGGGTGCAGAGATATGGAGGCCCCA


 


GGATCCTATTTACAGGATGAACCTACCTGGGCCGCTGTACGCATGAC


 


ATCCGCGAGCAAGTCTGAGGTTCTCAATGTACACATGAAATTGATTTT


 


TGCTGCGTTTGGCTTGGCTGATCGTTGCATTTGTTCTGATTCATCAGA


 


GTTAAATAACGGATATATCAGCAAATATCCGCAGCATCCACACCGAC


 


CACACGTCCGGTTAACAGAGTCCCCCTGCCTTGCTTTAATTATTACGG


 


AGTACTCCGCTATTAATCCTTAGATATGTTTCGAAGGAACTCAAACCT


 


TCCTCCATCTGCAAATCTCAGTGCTTCAAAACTGGAATTAGATAATTG


 


AAACCTTCATTCGGTTGCAATTCACAACTGCAAATTGAACAGCACTG


 


TCAATTTCAATTTCGGGTTCACGATTCCACCGATAGGTTGACATGATC


 


CATGATCCACCCATTGTACAAC






In an embodiment, a 3′-UTR can be the Brachypodium distachyon ubiquitin 1 (Ubi1) 3′-UTR.








(SEQ ID NO: 5)







GCTTCTGCCGAACTGGTTCACAGTCTGCTGCCCTTGGTGGTCTGCCCC


 


TTAGTGGTCATGCCTTTTGTTATGTGTCTTGCGTCCCAATCCTGTATCG


 


TTTGTGTGAACATCTCTGCTGCTGTATAGCAGCTTGAATCCTGTTATG


 


AATTTGTGAACCTGAACCTTGTTCCGTGAATCATGTTATGAATAAGTG


 


AACCTGAACCTTGTTCCGTGATTATTGTTACAATCTGTTGTGCCGTAT


 


GGTTGGTCGTGTGTGATTTATGTTGAACTGGAGAACCAAGTTCGTTCC


 


AGGACATATTGCAACCTAAGCTAAACCATGTAGAACTACTTGTTCTG


 


GGAGACATAAAACGTCATTTTTATGCATTCGTAACATTTAAGCATACT


 


ACAATAATTGTATTGTCCTTTTCCTACTCATCCTTGAAACCATATGCC


 


TCTTCTCAGCGCCTCTACATGCAGTGTGCTCAGAACAAACAGGCCCT


 


GCCAGCTGCTTTTCAATTTTCCAATTAATAACCACAATAGTCGGACTA


 


TGGCATCTGTGGGTGACTATGCAAGATGTTGCTGTCAGGTCTCTGAA


 


ACTTTTCCCATGTATCTGTTGAAATTACCCAGTAAATTCATGCCTCTA


 


TTTAATCTGGCATGGTTGATTTTCAAACAGAATGTGTTTTTTTTTGTTC


 


TGGAAGCTATATTGGTAAATAAATACAAAGCTGGAGTGTGATTATAT


 


TTCCAACAGATATTCAAGAAAATCTCAGTTGATTTATTTACTACTGTA


 


GTATATATATATATCTTACAGTTGACTTCTCATATTTCAAACGACATG


 


TGAGCACATTGTTCAGTTTCTTAGGATGTGTTGTGTGCTCAAAGGTGT


 


AATTTTGCATTCTGCCCTCCGAGTAAACACTACACGTATTTTTTTGAG


 


TGGCAGTGCATTTGATTACAAGGCAACAACAACAAAAACCTATGGCA


 


AGATATCCTTCTTAGAGGCTGCCAGGATCATTTTGACTGAACTATGTA


 


AGGCTGAAGAAAAGG






In an embodiment, a 3′-UTR can be the Setaria italica ubiquitin 2 (Ubi2) 3′-UTR.








(SEQ ID NO: 6)







GCCCATCGGTCATGGATGCTTCTACTGTACCTGGGTCGTCTGGTCTCT


 


GCCTGTGTCACCTTTGAAGTACCTGTGTCGGGATTGTGTTTGGTCATG


 


AACTGCAGTTTGTCTTTGATGTTCTTTTGTCTGGTCTTATGAACTGGTT


 


GTATCTGTATGTTTACTGTAAACTGTTGTTGCGGTGCAGCAGTATGGC


 


ATCCGAATGAATAAATGATGTTTGGACTTAAATCTGTACTCTGTTTGT


 


TTTCGGTTATGCCAGTTCTATATTGCCTGAGATCAGAATGTTTAGCTT


 


TTGAGTTCTGTTTGGCTTGTGGTCGACTCCTGTTTCTTACTTGAGGCGT


 


AACTCTGTTCTGGCAAACTCAAATGTCTAACTGAATGTTTTAGGACTT


 


AATTGTTGGACAGATTAACGTGTTTGGTTTGTTTCTAGATTGTGATTC


 


GGAAGGCTTGTTAGTTGTGGAATCAAGGAGAGCAGCTAGGTCTGTGC


 


AGAACGTTATTTTGGATTTAAGCCTTCTCAGATTATGCCATTACTCTA


 


AACCTAATGATATCATATTTCACTCGGGGATGTTGGAGTAGTCTTTTC


 


TTTCTCCTGCAGACAAAATGATTTTGCTTTCGTGTGTGTACATGATTTT


 


GTGCAACTGTTGCAACAACTGAAGTAGACAAGTTTTGACCTCACCAG


 


AAGAATGAAAAAGATTTTGGAATTTGTTACATCGACAAACCATTGTA


 


ACTTGGCCCATCAGAATGCACAGAAGAGCGGCTACAAATTGACATGC


 


GTTGCAAACTTTGCAATAGTTGATGCACATGTTTGCCATTGCCTGCCA


 


GTCTTAGGAAAAGTGTGTGGTTCGAGAAATCTAAGCATATGTGCTCT


 


GCTCACATTGCGTGGAACCCACACAGCTTTGTCACACTCTTGTCCACT


 


CCAGAAGTCATTCCTGGCGCTGTTTACCCCTGGTAAAAGGTAACCGA


 


AAACTTCTCAAGGCTGTACCCAAAACTGGAAGGAAATTTGGAGGAAA


 


TCTTTGCTTTTGATCGGCTCACTCTTTC






In an embodiment, a 3′-UTR can be the Panicum virgatum (Switchgrass) ubiquitin 1 3′-UTR.








(SEQ ID NO: 36)







GCCTAGTGCTCCTGAGTTGCCTTTTGTCGTTATGGTCAACCTCTGGTTT


 


AAGTCGTGTGAACTCTCTGCATTGCGTTGCTAGTGTCTGGTTGTGGTT


 


GTAATAAGAACATGAAGAACATGTTGCTGTGGATCACATGACTTTTT


 


TTTTTGAACCGGAAGATCACATGACTTTCATGGCTTTAAGTTCCTGAA


 


CTCTGAAATCTGGACCCCTTTTTAAGCTCTGAACTCATCATTCTTGCA


 


TTTACATCTGGTGTTGATCTTATTGATGTGATGCAGTCCTGCTGAAAT


 


AGTCAATGTAGATTCATGACTGACTGATTGCGTTTATGGTGTGTATGT


 


TGTTAACAAGCTGAAGGTCGTGTGGTGTCTTTCCAGTTAGACGAAGT


 


GTGCTTTATTGTAGCGTGTAGTGCTGCTGGATGATTGATGAACTGAAA


 


CATTCTGCATTTAGCAACTAGCGAGCCAAAGGTGATGACTGAGTTTC


 


TGTAGACCTGTTTTTTTATGCCCATGGTCGTTCTTCAATTGCACTTGAT


 


TTTCACATTAGCTGGATCATAATCTGAGCAGACTACTCAAAAGTACA


 


AAGTTCATCTTCGCTATGACGCTTTGCCACTAGGATTTTCTTTGTATG


 


ATTTGTTTACAAATCCTGTAATCTAGTCAAAAGAAAAGCCAAAATTTT


 


TCTTTGTATGATTTGTTTACAAATCCTCTAATCTAGTCAAAGAAAAGC


 


CAAATTTATCCCTCCTGGTCCCCTACATCACGTAGCTATGTGGCCCGC


 


AAGCAGATGAAAGCAGCCCCGTCAGCCGACGCCGACGCCGACGCCA


 


ACACATCCTGCTCCTCCCTCGCCGGCGCCGGCGCCGGCGAGGCCGCA


 


CCGCCGCTGCCCCGTGGCCGCAGGCACACGGTGCCGCACTGCCGCCG


 


CCCCGTGGCCGCAGGCACACGGTGCCGCACTGCCGCCGCCTCCCCTT


 


CCGGCATTGCCGGACGGCTGGGCTACTGTCCCCGCCGCCTTCCCAAT






In an embodiment, a nucleic acid construct is provided comprising a ubiquitin promoter as described herein and 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:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:36. In an embodiment, a nucleic acid construct is provided comprising a ubiquitin promoter as described herein and the 3′-UTR wherein the ubiquitin promoter and 3′-UTR are both operably linked to opposite ends of a polylinker. In an embodiment, a gene expression cassette is provided comprising a ubiquitin promoter as described herein and a 3′-UTR, wherein the ubiquitin promoter and 3′-UTR are both operably linked to opposite ends of a non-ubiquitin transgene. In one embodiment the a 3′-UTR, consists of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:36. In one embodiment, a gene expression cassette is provided comprising a ubiquitin promoter as described herein and a 3′-UTR, wherein the ubiquitin promoter comprises SEQ ID NO: 3 and the 3′-UTR comprises SEQ ID NO: 6 wherein the promoter and 3′-UTR are operably linked to opposite ends of a non-ubiquitin transgene. In one embodiment the a 3′-UTR, consists of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:36. In one embodiment the promoter consists of SEQ ID NO: 3, 17 or 40 and the 3′-UTR, consists of SEQ ID NO:6. In an illustrative embodiment, a gene expression cassette comprises a ubiquitin 3′-UTR that is operably linked to a transgene, wherein the transgene can be an insecticidal resistance transgene, an herbicide tolerance transgene, a nitrogen use efficiency transgene, a water us efficiency transgene, a nutritional quality transgene, a DNA binding transgene, a selectable marker transgene, or combinations thereof. In a further embodiment the transgene is operably linked to a ubiquitin promoter and a 3′-UTR from the same ubiquitin gene isolated from Panicum virgatum, Brachypodium distachyon, or Setaria italica. 
In one embodiment a vector is provided comprising a first transgene and/or polylinker and a second transgene and/or polylinker wherein the first transgene and/or polylinker is operably linked to a promoter comprising a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:35 and operably linked to a 3′-UTR, comprising a sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:36 and the second transgene and/or polylinker is operably linked to a promoter comprising a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:35 and operably linked to a 3′-UTR, comprising a sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:36, further wherein the promoter of the first transgene and/or polylinker and second transgene and/or polylinker are derived from Ubi genes from different plant species. In a further embodiment the vector is provided with a third transgene and/or polylinker wherein the third transgene and/or polylinker polylinker is operably linked to a promoter comprising a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:35 and operably linked to a 3′-UTR, comprising a sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:36, further wherein the promoter of the third transgene and/or polylinker is derived from Ubi genes from a different plant species from the promoter of the first and second transgene and/or polylinker.
Transgene expression may also be regulated by an intron region located downstream of the promoter sequence. Both a promoter and an intron can regulate transgene expression. While a promoter is necessary to drive transcription, the presence of an intron can increase expression levels resulting in mRNA transcript for translation and protein synthesis. An intron gene region aids stable expression of a transgene.
In an embodiment, a nucleic acid construct is provided comprising a ubiquitin promoter as described herein and an intron. In one embodiment the intron is operably linked to the 3′ end of the promoter. In an embodiment, a nucleic acid construct is provided comprising a ubiquitin intron operably linked to the 3′ end of a ubiquitin promoter isolated from Panicum virgatum, Brachypodium distachyon or Setaria italica or a derivative of such promoter sequence. In an embodiment, the ubiquitin intron is a Panicum virgatum, Brachypodium distachyon or Setaria italica ubiquitin intron, or a derivative of such intron sequence.
In an embodiment, an intron can be the Brachypodium distachyon ubiquitin1 C intron.







(SEQ ID NO: 7)







GTATGCAGCCTCGCTTCCTCCTCGCTACCGTTTCAATTCTGGAGTAGGTC


 


GTAGAGGATACCATGTTGATTTGACAGAGGGAGTAGATTAGATACTTGTA


 


GATCGAAGTGCGGATGTTCCATGGTAGATGATACCATGTTGATTTCGATT


 


AGATCGGATTAAATCTTTGTAGATCGAAGTGCGCATGTTCCATGAATTGC


 


CTGTTACCAGTAGATTCAAGTTTTTCTGTGTTATAGAGGTGGGATCTACT


 


CGTTGAGATGATTAGCTCCTAGAGGACACCATGCCGTTTTGGAAAATAGA


 


TCAGAACCGTGTAGATCGATGTGAGCATGTGTTCCTGTAGATCCAAGTTC


 


TTTCGCATGTTACTAGTTGTGATCTATTGTTTGTGTAATACGCTCTCGAT


 


CTATCCGTGTAGATTTCACTCGATTACTGTTACTGTGGCTTGATCGTTCA


 


TAGTTGTTCGTTAGGTTTGATCGAACAGTGTCTGAACCTAATTGGATATG


 


TATTCTTGATCTATCAACGTGTAGGTTTCAGTCATGTATTTATGTACTCC


 


CTCCGTCCCAAATTAACTGACGTGGATTTTGTATAAGAATCTATACAAAT


 


CCATGTCAGTTAATTCGGGATGGAGTACCATATTCAATAATTTGTTTATT


 


GCTGTCCACTTATGTACCATATGTTTGTTGTTCCTCATGTGGATTCTACT


 


AATTATCATTGATTGGTGATCTTCTATTTTGCTAGTTTCCTAGCTCAATC


 


TGGTTATTCATGTAGATGTGTTGTTGAAATCGGAGACCATGCTTGTTATT


 


AGATAGTTTATTGCTTATCAGTTTCATGTTCTGGTTGATGCAACACATAT


 


TCATGTTCGCTATCTGGTTGCTGCTTGATATTCTCTGATTTACATTCATT


 


ATAAGAATATATTCTGCTCTGGTTGTTGCTTCTCATGACTTTACCTACTC


 


GGTAGGTGACTTACCTTTTGGTTTACAATTGTCAACTATGCAG






In an embodiment, an intron can be the Brachypodium distachyon ubiquitin 1 (Ubi1) intron








(SEQ ID NO: 8)







GTATGTAGCCTCTCGATTCCTCCTCAGCCCTGCCCTCGATTTGGTGTACG


 


CGTTGAGATGATGATCTCGTAGATGTCTAGATGACACCATGTCGATTTGA


 


AATAGATCAGATCCGTGTAGATCGATGAGCTCCTGTGTACCTGTGGATTC


 


AAGTTATTTTCGCATGCTATTGTTGTGATCTACTAGATCTAGTGTGTGTA


 


TTCTATGCTATCGATTTCTCCGTGTAGATTTCACTCGATTACTGTTACTG


 


TGGCTTGATCGGCCATAGATGTTGGTTAAGGTTTGATCGGTTAGTGTTTG


 


AACCTGCGTGGATATCTAGCATCCATCTATTATCGTGTAGGTTTCGAACA


 


AACAAGCACTATTATTGTACTGATGGTTCGTCTATGGTTGGTTTTGACCG


 


TTTTAGTGTTGAACGAGCCTTCTGTATTTGTTTATTGCTGTCCAGTGATG


 


TACCATGTTCGTTGAGTGTCGGATTATACTAATTATTGTTGATTGATAAT


 


CTTGTAGTTTGCTTTTCCTAATTTATTTATCGTAGTCCTGATTTGCCTCA


 


GCTGTGCCTCACCCGTGCGATGGTCAATCAACTTGTTAGCCCAATCTGCT


 


TAATCATGTACATTTGTTGTTAGAATCAGAGATCAAGCCAATTAGCTATC


 


TTATTGCTTATCTGTTCCATGTTCTGATCGATGTAACAGTCTACACTTTT


 


GCTCTGTGCTACTTGATTAAAACATTCTGACTTAAATTCATGATTGGAAG


 


TTTCAGATCTGATTGTTGCCTTACTTGACTAATATCTATTCATGTGACAC


 


CTCTCTGTCTTGGTAACTTACCGCTGTTTGTTTGTAATTTCTGACTATGC


 


AG






In an embodiment, an intron can be the Setaria italica ubiquitin 2 (Ubi2) intron 1








(SEQ ID NO: 9)







GTCACGGGTTCCTTCCCCACCTCTCCTCTTCCCCACCGCCATAAATAG






In an embodiment, an intron can be the Setaria italica ubiquitin 2 (Ubi2) intron 2








(SEQ ID NO: 10)







GTACGGCGATCGTCTTCCTCCTCTAGATCGGCGTGATCTGCAAGTAGTTG


 


ATTTGGTAGATGGTTAGGATCTGTGCACTGAAGAAATCATGTTAGATCCG


 


CGATGTTTCTGTTCGTAGATGGCTGGGAGGTGGAATTTTTGTGTAGATCT


 


GATATGTTCTCCTGTTTATCTTGTCACGCTCCTGCGATTTGTGGGGATTT


 


TAGGTCGTTGATCTGGGAATCGTGGGGTTGCTTCTAGGCTGTTCGTAGAT


 


GAGGTCGTTCTCACGGTTTACTGGATCATTGCCTAGTAGATCAGCTCGGG


 


CTTTCGTCTTTGTATATGGTGCCCATACTTGCATCTATGATCTGGTTCCG


 


TGGTGTTACCTAGGTTTCTGCGCCTGATTCGTCCGATCGATTTTGTTAGC


 


ATGTGGTAAACGTTTGGTCATGGTCTGATTTAGATTAGAGTCGAATAGGA


 


TGATCTCGATCTAGCTCTTGGGATTAATATGCATGTGTCACCAATCTGTT


 


CCGTGGTTAAGATGATGAATCTATGCTTAGTTAATGGGTGTAGATATATA


 


TGCTGCTGTTCCTCAATGATGCCGTAGCTTTTACCTGAGCAGCATGGATC


 


CTCCTGTTACTTAGGTAGATGCACATGCTTATAGATCAAGATATGTACTG


 


CTACTGTTGGAATTCTTTAGTATACCTGATGATCATCCATGCTCTTGTTA


 


CTTGTTTTGGTATACTTGGATGATGGCATGCTGCTGCTTTTTGTTGATTT


 


GAGCCCATCCATATCTGCATATGTCACATGATTAAGATGATTACGCTGTT


 


TCTGTATGATGCCATAGCTTTTATGTGAGCAACATGCATCCTCCTGGTTA


 


TATGCATTAATAGATGGAAGATATCTATTGCTACAATTTGATGATTATTT


 


TGTACATACGATGATCAAGCATGCTCTTCATACTTTGTTGATATACTTGG


 


ATAATGAAATGCTGCTGCACGTTCATTCTATAGCACTAATGATGTGATGA


 


ACACGCACGACCTGTTTGTGGCATCTGTTTGAATGTGTTGTTGCTGTTCA


 


CTAGAGACTGTTTTATTAACCTACTGCTAGATACTTACCCTTCTGTCTGT


 


TTATTCTTTTGCAG






In an embodiment, an intron can be the Panicum virgatum (Switchgrass) ubiquitin intron.








(SEQ ID NO: 37)







GTACTCCTACCTAATCCTCCTTAACTGATCTCTCCTCTATCACGTTGGT


 


AATCTTCGAATGATCTGCTGCCTGGCTCGCTGTTCCCCCTCGTTATGCA


 


CTGTTTCCATCACGAGTTTTTTTTTTCATCATCTAATCTATGCGGTTGC


 


GGAAGAATTGTGGCTAGTGGAGTAGTTTTCTGTGCTTGATCGGTAGATT


 


CGATGTGTGGGTGTATGGATGTTTTCTGAAAAGTTGCTGGATTAGTTTA


 


CGCTTTCAGGCCGCAGGTCTGTTCGAAATTGATTATGAAGTCTATATGC


 


TTTGGATCTATCGATTTCCAGTTTTATTCAGATGTAGGCCAAAAAATTG


 


TCGGCATTTGTGTGGAATTAGTTGGCCTTTAGGTCTGCACATTCATGGT


 


GACGGCACAGTTGCTGCTGGCTGTTGCGTGGGACGAGTTATTATAGTTG


 


TTTTTGTTTTTCCCTGATTGATTCACATTTTCAATGATAACTAGCCTTT


 


GTCACCTAACCAAGTCCAGGTTGATCCTATCTGTGTTCTTCAGCTACCA


 


GTTTGCATAGATGATGGTGTATTCGATTGCTTTAGTAGGCCTTCTGATT


 


TCACATCTAATTCTGTCATGAATATAGATAACTTTACATGCTTTTGATA


 


TACTTTATATTTGAACTGTTCACTGTCCAGCCTATTTTGGATAATTGAG


 


TGCATTGGCTTTTGATGCCTGAATTATTCACATGTTCCTGGATAATTGA


 


CCTGTGTCACCTAGTTGACTGTTTTTTGAGGTGCCACCCGTCTGTTCAG


 


CTGATTTGTGTATTCGATTGCTCTAGTTAATCTTTTGATTATGCAGCTA


 


GTGCTTTGTCATATGTAGCTTTATAGGCTTCTGATGTCCTTGGATATAG


 


TTCAGTCTACTTGTCAAGTTGCTTTACAAGTAGTAGCTCTGATTCTATT


 


TGGCTTCCTGAGTCAGAGCTTTGCAAATTGCTTGTTGTTACATTACATC


 


ATATTACTTGAATTGCAGTTATTTAATGGTTGGATTGTTGCTGTTTACTT


 


CTACATTTTTTGCTGTTTTATATTATACTAAAATGTTTGTGTTGCTGCT


 


TTTCAG






In an embodiment, a nucleic acid construct is provided comprising a ubiquitin promoter as described herein and an intron, wherein the intron 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:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:37. In an embodiment, a nucleic acid construct is provided comprising a ubiquitin promoter as described herein, an intron sequence and a polylinker wherein the promoter and intron are operably linked to a polylinker. In an embodiment, a gene expression cassette is provided comprising a ubiquitin promoter as described herein, an intron sequence and a non-ubiquitin transgene wherein the promoter and intron are operably linked to the 5′ end of the transgene. Optionally the construct further comprises a 3′-UTR that is operably linked to the 3′ end of the non-ubiquitin transgene or polylinker. In one embodiment the promoter and 3′-UTR sequences are selected from those described herein and the intron sequence consists of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:37. In an embodiment, a gene expression cassette comprises a ubiquitin intron that is operably linked to a promoter, wherein the promoter is a Panicum virgatum, Brachypodium distachyon or Setaria italica ubiquitin promoter, or a promoter that originates from a plant (e.g., Zea mays ubiquitin 1 promoter), a virus (e.g., Cassava vein mosaic virus promoter) or a bacteria (e.g., Agrobacterium tumefaciens delta mas). In an illustrative embodiment, a gene expression cassette comprises a ubiquitin intron that is operably linked to a transgene, wherein the transgene can be an insecticidal resistance transgene, an herbicide tolerance transgene, a nitrogen use efficiency transgene, a water us efficiency transgene, a nutritional quality transgene, a DNA binding transgene, a selectable marker transgene, or combinations thereof.
Transgene expression may also be regulated by a 5′-UTR region located downstream of the promoter sequence. Both a promoter and a 5′-UTR can regulate transgene expression. While a promoter is necessary to drive transcription, the presence of a 5′-UTR can increase expression levels resulting in mRNA transcript for translation and protein synthesis. A 5′-UTR gene region aids stable expression of a transgene.
In an embodiment, a nucleic acid construct is provided comprising a ubiquitin promoter as described herein and a 5′-UTR. In one embodiment the 5′-UTR is operably linked to the 3′ end of the promoter. In an embodiment, a nucleic acid construct is provided comprising a ubiquitin a 5′-UTR operably linked to the 3′ end of a ubiquitin promoter isolated from Panicum virgatum, Brachypodium distachyon or Setaria italica or a derivative of such promoter sequence. In a further embodiment the 3′ end of the 5′-UTR is operably linked to the 5′ end of a ubiquitin intron from Panicum virgatum, Brachypodium distachyon or Setaria italica, as described herein.
In an embodiment, a 5′-UTR can be the Brachypodium distachyon ubiquitin1 C (Ubi1C) 5′-UTR.







(SEQ ID NO: 11)







CCGATCGAAAAGTCCCCGCAAGAGCAAGCGACCGATCTCGTGAATCTCC


 


GTCAAG






In an embodiment, a 5′-UTR can be the Brachypodium distachyon ubiquitin 1 (Ubi1) 5′-UTR.







(SEQ ID NO: 12)







CCAATCCAGCACCCCCGATCCCGATCGAAAATTCTCCGCAACAGCAAGC


 


GATCGATCTAGCGAATCCCCGTCAAG






In an embodiment, a 5′-UTR can be the Setaria italica ubiquitin 2 (Ubi2) 5′-UTR1







(SEQ ID NO: 13)







AGAAATATCAACTGGTGGGCCACGCACATCAGCGTCGTGTAACGTGGAC


 


GGAGGAGCCCCGTGACGGCGTCGACATCGAACGGCCACCAACCACGGAA


 


CCACCCGTCCCCACCTCTCGGAAGCTCCGCTCCACGGCGTCGACATCTA


 


ACGGCTACCAGCAGGCGTACGGGTTGGAGTGGACTCCTTGCCTCTTTGC


 


GCTGGCGGCTTCCGGAAATTGCGTGGCGGAGACGAGGCGGGCTCGTCTC


 


ACACGGCACGGAAGAC






In an embodiment, a 5′-UTR can be the Setaria italica ubiquitin 2 (Ubi2) 5′-UTR2







(SEQ ID NO: 14)







CCGACCCCCTCGCCTTTCTCCCCAATCTCATCTCGTCTCGTGTTGTTCG


 


GAGCACACCACCCGCCCCAAATCGTTCTTCCCGCAAGCCTCGGCGATCC


 


TTCACCCGCTTCAAG






In an embodiment, a 5′-UTR can be the Panicum virgatum (Switchgrass) ubiquitin 5′-UTR.







(SEQ ID NO: 38)







CAAGTTCGCGATCTCTCGATTTCACAAATCGCCGAGAAGACCCGAGCAG


 


AGAAGTTCCCTCCGATCGCCTTGCCAAG.






In an embodiment, a nucleic acid construct is provided comprising a ubiquitin promoter as disclosed herein and a 5′-UTR, wherein the 5′-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:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, or SEQ ID NO:38. In an embodiment, a nucleic acid construct is provided comprising ubiquitin promoter, wherein the promoter is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, or 100% identical to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:35, and a 5′-UTR operably linked to a polylinker. In an embodiment, a gene expression cassette is provided comprising a ubiquitin promoter, wherein the promoter is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, or 100% identical to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:35, and a 5′-UTR sequences operably linked to a non-ubiquitin transgene. Optionally, the construct can further comprise a ubiquitin intron as disclosed herein operably linked to the 3′ end of the 5′-UTR and the 5′ end of the non-ubiquitin transgene, and optionally further comprising a 3′-UTR that is operably linked to the 3′ end of the non-ubiquitin transgene. In one embodiment the promoter, intron and 3′-UTR sequences are selected from those described herein and the 5′-UTR sequence consists of SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, or SEQ ID NO:38. In one embodiment the 3′-UTR consists of SEQ ID NO:13 or SEQ ID NO: 14.
In an embodiment, a gene expression cassette comprises a ubiquitin 5′-UTR that is operably linked to a promoter, wherein the promoter is a Panicum virgatum, Brachypodium distachyon or Setaria italica ubiquitin promoter, or a promoter that originates from a plant (e.g., Zea mays ubiquitin 1 promoter), a virus (e.g., Cassava vein mosaic virus promoter) or a bacteria (e.g., Agrobacterium tumefaciens delta mas). In an illustrative embodiment, a gene expression cassette comprises a ubiquitin 5′-UTR that is operably linked to a transgene, wherein the transgene can be an insecticidal resistance transgene, an herbicide tolerance transgene, a nitrogen use efficiency transgene, a water us efficiency transgene, a nutritional quality transgene, a DNA binding transgene, a selectable marker transgene, or combinations thereof.
In one embodiment a nucleic acid construct is provided comprising a promoter and a polylinker and optionally one or more of the following elements:
a) a 5′ untranslated region;
b) an intron; and
c) a 3′ untranslated region,
wherein
the promoter consists of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:35 or a sequence having 98% sequence identity with SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:35;
the 5′ untranslated region consists of SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, or SEQ ID NO:38 or a sequence having 98% sequence identity with SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, or SEQ ID NO:38
the intron consists of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:37 or a sequence having 98% sequence identity with SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:37
the 3′ untranslated region consists of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:36 or a sequence having 98% sequence identity with SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:36; further wherein said promoter is operably linked to said polylinker and each optional element, when present, is also operably linked to both the promoter and the polylinker.
In one embodiment a nucleic acid construct is provided comprising a promoter and a non-ubiquitin transgene and optionally one or more of the following elements:
a) a 5′ untranslated region;
b) an intron; and
c) a 3′ untranslated region,
wherein
the promoter consists of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:35 or a sequence having 98% sequence identity with SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:35;
the 5′ untranslated region consists of SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, or SEQ ID NO:38 or a sequence having 98% sequence identity with SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, or SEQ ID NO:38
the intron consists of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:37 or a sequence having 98% sequence identity with SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:37
the 3′ untranslated region consists of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:36 or a sequence having 98% sequence identity with SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:36; further wherein said promoter is operably linked to said transgene and each optional element, when present, is also operably linked to both the promoter and the transgene. In a further 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 a further embodiment a plant is provided wherein the plant comprises said transgenic cells.
In accordance with one embodiment transgene expression is regulated by a promoter operably linked to an intron and 5′-UTR region, wherein the intron and 5′-UTR region are located downstream of the promoter sequence. A promoter operably linked to an intron and 5′-UTR region can be used to drive transgene expression. While a promoter is necessary to drive transcription, the presence of the intron and 5′-UTR can increase expression levels resulting in mRNA transcript for translation and protein synthesis.
In an embodiment, a gene expression cassette comprises a promoter operably linked to a 5′-UTR and intron region. In an embodiment, a gene expression cassette comprises a ubiquitin promoter operably linked to a ubiquitin 5′-UTR and ubiquitin intron. In an embodiment, the ubiquitin promoter operably linked to a 5′-UTR and intron region is a Panicum virgatum, Brachypodium distachyon or Setaria italica ubiquitin promoter operably linked to an intron and 5′-UTR.
In an embodiment, a promoter operably linked to a 5′-UTR and intron can be the Brachypodium distachyon ubiquitin1 C (Ubi1C) promoter operably linked to an intron and 5′-UTR. In one embodiment the promoter comprises or consists of the sequence of SEQ ID NO: 15:







(SEQ ID NO: 15)







CTGCTCGTTCAGCCCACAGTAACACGCCGTGCGACATGCAGATGCCCTC


 


CACCACGCCGACCAACCCCAAGTCCGCCGCGCTCGTCCACGGCGCCATC


 


CGCATCCGCGCGTCAACGTCATCCGGAGGAGGCGAGCGCGATGTCGACG


 


GCCACGGCGGCGGCGGACACGACGGCGACGCCCCGACTCCGCGCGCGCG


 


TCAAGGCTGCAGTGGCGTCGTGGTGGCCGTCCGCCTGCACGAGATCCCC


 


GCGTGGACGAGCGCCGCCTCCACCCAGCCCCTATATCGAGAAATCAACG


 


GTGGGCTCGAGCTCCTCAGCAACCTCCCCACCCCCCCTTCCGACCACGC


 


TCCCTTCCCCCGTGCCCCTCTTCTCCGTAAACCCGAGCCGCCGAGAACA


 


ACACCAACGAAAGGGCGAAGAGAATCGCCATAGAGAGGAGATGGGCGGA


 


GGCGGATAGTTTCAGCCATTCACGGAGAAATGGGGAGGAGAGAACACGA


 


CATCATACGGACGCGACCCTCTAGCTGGCTGGCTGTCCTAAAGAATCGA


 


ACGGAATCGCTGCGCCAGGAGAAAACGAACGGTCCTGAAGCATGTGCGC


 


CCGGTTCTTCCAAAACACTTATCTTTAAGATTGAAGTAGTATATATGAC


 


TGAAATTTTTACAAGGTTTTTCCCCATAAAACAGGTGAGCTTATCTCAT


 


CCTTTTGTTTAGGATGTACGTATTATATATGACTGAATATTTTTTATTT


 


TCATTGAATGAAGATTTTCGACCCCCCAAAAATAAAAAACGGAGGGAGT


 


ACCTTTGTGCCGTGTATATGGACTAGAGCCATCGGGACGTTTCCGGAGA


 


CTGCGTGGTGGGGGCGATGGACGCACAACGACCGCATTTTCGGTTGCCG


 


ACTCGCCGTTCGCATCTGGTAGGCACGACTCGTCGGGTTCGGCTCTTGC


 


GTGAGCCGTGACGTAACAGACCCGTTCTCTTCCCCCGTCTGGCCATCCA


 


TAAATCCCCCCTCCATCGGCTTCCCTTTCCTCAATCCAGCACCCTGATT


 


CCGATCGAAAAGTCCCCGCAAGAGCAAGCGACCGATCTCGTGAATCTCC


 


GTCAAGGTATGCAGCCTCGCTTCCTCCTCGCTACCGTTTCAATTCTGGA


 


GTAGGTCGTAGAGGATACCATGTTGATTTGACAGAGGGAGTAGATTAGA


 


TACTTGTAGATCGAAGTGCGGATGTTCCATGGTAGATGATACCATGTTG


 


ATTTCGATTAGATCGGATTAAATCTTTGTAGATCGAAGTGCGCATGTTC


 


CATGAATTGCCTGTTACCAGTAGATTCAAGTTTTTCTGTGTTATAGAGG


 


TGGGATCTACTCGTTGAGATGATTAGCTCCTAGAGGACACCATGCCGTT


 


TTGGAAAATAGATCAGAACCGTGTAGATCGATGTGAGCATGTGTTCCTG


 


TAGATCCAAGTTCTTTCGCATGTTACTAGTTGTGATCTATTGTTTGTGT


 


AATACGCTCTCGATCTATCCGTGTAGATTTCACTCGATTACTGTTACTG


 


TGGCTTGATCGTTCATAGTTGTTCGTTAGGTTTGATCGAACAGTGTCTG


 


AACCTAATTGGATATGTATTCTTGATCTATCAACGTGTAGGTTTCAGTC


 


ATGTATTTATGTACTCCCTCCGTCCCAAATTAACTGACGTGGATTTTGT


 


ATAAGAATCTATACAAATCCATGTCAGTTAATTCGGGATGGAGTACCAT


 


ATTCAATAATTTGTTTATTGCTGTCCACTTATGTACCATATGTTTGTTG


 


TTCCTCATGTGGATTCTACTAATTATCATTGATTGGTGATCTTCTATTT


 


TGCTAGTTTCCTAGCTCAATCTGGTTATTCATGTAGATGTGTTGTTGAA


 


ATCGGAGACCATGCTTGTTATTAGATAGTTTATTGCTTATCAGTTTCAT


 


GTTCTGGTTGATGCAACACATATTCATGTTCGCTATCTGGTTGCTGCTT


 


GATATTCTCTGATTTACATTCATTATAAGAATATATTCTGCTCTGGTTG


 


TTGCTTCTCATGACTTTACCTACTCGGTAGGTGACTTACCTTTTGGTTT


 


ACAATTGTCAACTATGCAG






In an embodiment, a promoter operably linked to 5′-UTR and intron can be the Brachypodium distachyon ubiquitin 1 (Ubi1) promoter operably linked to a 5′-UTR and intron. In one embodiment the promoter comprises or consists of the sequence of SEQ ID NO: 16:







(SEQ ID NO: 16)







GGCGTCAGGACTGGCGAAGTCTGGACTCTGCAGGGCCGAACTGCTGAAG


 


ACGAAGCAGAGGAAGAGAAAGGGAAGTGTTCGACTTGTAATTTGTAGGG


 


GTTTTTTTTAGAGGAACTTGTAATTTGTAGGTGGGCTGGCCTCGTTGGA


 


AAAACGATGCTGGCTGGTTGGGCTGGGCCGATGTACGCTTGCAAACAAC


 


TTGTGGCGGCCCGTTCTGGACGAGCAGGAGTTTCTTTTTTGTTCTCACT


 


TTTCTGGTCTTCTTTAGTTACGGAGTACCTTTTGTTTTTTAAAGGAGTT


 


ACCTTTTTTTTAGGAATTCTTTAGTTACCTTTCGCTTGCTCTCAAAAAA


 


TATTTAACTTTCGCTTTTTTTCATTTTAATTTTTGCAACTATTTACGAG


 


TTTCATGAATGCTTATTTTCCAGCATATCATTATTTGCAAGTATTTTTA


 


TGCCGTATGTATTGGACGAGAGCCATCGGGACTGTTCCAGAGACTGCGT


 


GGTGGGGACGGCTCCCAACCGCCTTTTCTATCTCTGTTCGCATCCGGTG


 


GCCGACTTGGCTCGCGCGTGAGCCGTGACGTAACAGACTTGGTCTCTTC


 


CCCATCTGGCCATCTATAAATTCCCCCATCGATCGACCCTCCCTTTCCC


 


CAATCCAGCACCCCCGATCCCGATCGAAAATTCTCCGCAACAGCAAGCG


 


ATCGATCTAGCGAATCCCCGTCAAGGTATGTAGCCTCTCGATTCCTCCT


 


CAGCCCTGCCCTCGATTTGGTGTACGCGTTGAGATGATGATCTCGTAGA


 


TGTCTAGATGACACCATGTCGATTTGAAATAGATCAGATCCGTGTAGAT


 


CGATGAGCTCCTGTGTACCTGTGGATTCAAGTTATTTTCGCATGCTATT


 


GTTGTGATCTACTAGATCTAGTGTGTGTATTCTATGCTATCGATTTCTC


 


CGTGTAGATTTCACTCGATTACTGTTACTGTGGCTTGATCGGCCATAGA


 


TGTTGGTTAAGGTTTGATCGGTTAGTGTTTGAACCTGCGTGGATATCTA


 


GCATCCATCTATTATCGTGTAGGTTTCGAACAAACAAGCACTATTATTG


 


TACTGATGGTTCGTCTATGGTTGGTTTTGACCGTTTTAGTGTTGAACGA


 


GCCTTCTGTATTTGTTTATTGCTGTCCAGTGATGTACCATGTTCGTTGA


 


GTGTCGGATTATACTAATTATTGTTGATTGATAATCTTGTAGTTTGCTT


 


TTCCTAATTTATTTATCGTAGTCCTGATTTGCCTCAGCTGTGCCTCACC


 


CGTGCGATGGTCAATCAACTTGTTAGCCCAATCTGCTTAATCATGTACA


 


TTTGTTGTTAGAATCAGAGATCAAGCCAATTAGCTATCTTATTGCTTAT


 


CTGTTCCATGTTCTGATCGATGTAACAGTCTACACTTTTGCTCTGTGCT


 


ACTTGATTAAAACATTCTGACTTAAATTCATGATTGGAAGTTTCAGATC


 


TGATTGTTGCCTTACTTGACTAATATCTATTCATGTGACACCTCTCTGT


 


CTTGGTAACTTACCGCTGTTTGTTTGTAATTTCTGACTATGCAG






In an embodiment, a promoter operably linked to a 5′-UTR and intron can be the Setaria italica ubiquitin 2 (Ubi2) promoter operably linked to a 5′-UTR and intron. In one embodiment the promoter comprises or consists of the sequence of SEQ ID NO: 17:







(SEQ ID NO: 17)







TGCGTCTGGACGCACAAGTCATAGCATTATCGGCTAAAATTTCTTAATT


 


TCTAAATTAGTCATATCGGCTAAGAAAGTGGGGAGCACTATCATTTCGT


 


AGAACAAGAACAAGGTATCATATATATATATATATATAATATTTAAACT


 


TTGTTAAGTGGAATCAAAGTGCTAGTATTAATGGAGTTTCATGTGCATT


 


AAATTTTATGTCACATCAGCAATTTTGTTGACTTGGCAAGGTCATTTAG


 


GGTGTGTTTGGAAGACAGGGGCTATTAGGAGTATTAAACATAGTCTAAT


 


TACAAAACTAATTGCACAACCGCTAAGCTGAATCGCGAGATGGATCTAT


 


TAAGCTTAATTAGTCCATGATTTGACAATGTGGTGCTACAATAACCATT


 


TGCTAATGATGGATTACTTAGGTTTAATAGATTCGTCTCGTGATTTAGC


 


CTATGGGTTCTGCTATTAATTTTGTAATTAGCTCATATTTAGTTCTTAT


 


AATTAGTATCCGAACATCCAATGTGACATGCTAAAGTTTAACCCTGGTA


 


TCCAAATGAAGTCTTATGAGAGTTTCATCACTCCGGTGGTATATGTACT


 


TAGGCTCCGTTTTCTTCCACCGACTTATTTTTAGCACCCGTCACATTGA


 


ATGTTTAGATACTAATTAGAAGTATTAAACGTAGACTATTTACAAAATC


 


CATTACATAAGACGAATCTAAACGGCGAGACGAATCTATTAAACCTAAT


 


TAGTCCATGATTTGACAATGTGTTGCTACAGTAAACATTTGCTAATGAT


 


GGATTAATTAGGCTTAATAGATTCGTCTCGCCGTTTAGCCTCCACTTAT


 


GTAATGGGTTTTCTAAACAATCTACGTTTAATACTCCTAATTAGTATCT


 


AAATATTCAATGTGACACGTGCTAAAAATAAGTCAGTGGAAGGAAGAGA


 


ACGTCCCCTTAGTTTTCCATCTTATTAATTGTACGATGAAACTGTGCAG


 


CCAGATGATTGACAATCGCAATACTTCAACTAGTGGGCCATGCACATCA


 


GCGACGTGTAACGTCGTGAGTTGCTGTTCCCGTAGAGAAATATCAACTG


 


GTGGGCCACGCACATCAGCGTCGTGTAACGTGGACGGAGGAGCCCCGTG


 


ACGGCGTCGACATCGAACGGCCACCAACCACGGAACCACCCGTCCCCAC


 


CTCTCGGAAGCTCCGCTCCACGGCGTCGACATCTAACGGCTACCAGCAG


 


GCGTACGGGTTGGAGTGGACTCCTTGCCTCTTTGCGCTGGCGGCTTCCG


 


GAAATTGCGTGGCGGAGACGAGGCGGGCTCGTCTCACACGGCACGGAAG


 


ACGTCACGGGTTCCTTCCCCACCTCTCCTCTTCCCCACCGCCATAAATA


 


GCCGACCCCCTCGCCTTTCTCCCCAATCTCATCTCGTCTCGTGTTGTTC


 


GGAGCACACCACCCGCCCCAAATCGTTCTTCCCGCAAGCCTCGGCGATC


 


CTTCACCCGCTTCAAGGTACGGCGATCGTCTTCCTCCTCTAGATCGGCG


 


TGATCTGCAAGTAGTTGATTTGGTAGATGGTTAGGATCTGTGCACTGAA


 


GAAATCATGTTAGATCCGCGATGTTTCTGTTCGTAGATGGCTGGGAGGT


 


GGAATTTTTGTGTAGATCTGATATGTTCTCCTGTTTATCTTGTCACGCT


 


CCTGCGATTTGTGGGGATTTTAGGTCGTTGATCTGGGAATCGTGGGGTT


 


GCTTCTAGGCTGTTCGTAGATGAGGTCGTTCTCACGGTTTACTGGATCA


 


TTGCCTAGTAGATCAGCTCGGGCTTTCGTCTTTGTATATGGTGCCCATA


 


CTTGCATCTATGATCTGGTTCCGTGGTGTTACCTAGGTTTCTGCGCCTG


 


ATTCGTCCGATCGATTTTGTTAGCATGTGGTAAACGTTTGGTCATGGTC


 


TGATTTAGATTAGAGTCGAATAGGATGATCTCGATCTAGCTCTTGGGAT


 


TAATATGCATGTGTCACCAATCTGTTCCGTGGTTAAGATGATGAATCTA


 


TGCTTAGTTAATGGGTGTAGATATATATGCTGCTGTTCCTCAATGATGC


 


CGTAGCTTTTACCTGAGCAGCATGGATCCTCCTGTTACTTAGGTAGATG


 


CACATGCTTATAGATCAAGATATGTACTGCTACTGTTGGAATTCTTTAG


 


TATACCTGATGATCATCCATGCTCTTGTTACTTGTTTTGGTATACTTGG


 


ATGATGGCATGCTGCTGCTTTTTGTTGATTTGAGCCCATCCATATCTGC


 


ATATGTCACATGATTAAGATGATTACGCTGTTTCTGTATGATGCCATAGC


 


TTTTATGTGAGCAACATGCATCCTCCTGGTTATATGCATTAATAGATGG


 


AAGATATCTATTGCTACAATTTGATGATTATTTTGTACATACGATGATC


 


AAGCATGCTCTTCATACTTTGTTGATATACTTGGATAATGAAATGCTGC


 


TGCACGTTCATTCTATAGCACTAATGATGTGATGAACACGCACGACCTG


 


TTTGTGGCATCTGTTTGAATGTGTTGTTGCTGTTCACTAGAGACTGTTT


 


TATTAACCTACTGCTAGATACTTACCCTTCTGTCTGTTTATTCTTTTGC


 


AG






In an embodiment, a promoter operably linked to a 5′-utr and intron can be the Setaria italica ubiquitin 2 (ubi2) promoter operably linked to a 5′-utr and intron. In one embodiment the promoter comprises or consists of the sequence of SEQ ID NO: 41:







(SEQ ID NO: 41)







TGCGTCTGGACGCACAAGTCATAGCATTATCGGCTAAAATTTCTTAATT


 


TCTAAATTAGTCATATCGGCTAAGAAAGTGGGGAGCACTATCATTTCGT


 


AGAACAAGAACAAGGTATCATATATATATATATATATAATATTTAAACT


 


TTGTTAAGTGGAATCAAAGTGCTAGTATTAATGGAGTTTCATGTGCATT


 


AAATTTTATGTCACATCAGCAATTTTGTTGACTTGGCAAGGTCATTTAG


 


GGTGTGTTTGGAAGACAGGGGCTATTAGGAGTATTAAACATAGTCTAAT


 


TACAAAACTAATTGCACAACCGCTAAGCTGAATCGCGAGATGGATCTAT


 


TAAGCTTAATTAGTCCATGATTTGACAATGTGGTGCTACAATAACCATT


 


TGCTAATGATGGATTACTTAGGTTTAATAGATTCGTCTCGTGATTTAGC


 


CTATGGGTTCTGCTATTAATTTTGTAATTAGCTCATATTTAGTTCTTAT


 


AATTAGTATCCGAACATCCAATGTGACATGCTAAAGTTTAACCCTGGTA


 


TCCAAATGAAGTCTTATGAGAGTTTCATCACTCCGGTGGTATATGTACT


 


TAGGCTCCGTTTTCTTCCACCGACTTATTTTTAGCACCCGTCACATTGA


 


ATGTTTAGATACTAATTAGAAGTATTAAACGTAGACTATTTACAAAATC


 


CATTACATAAGACGAATCTAAACGGCGAGACGAATCTATTAAACCTAAT


 


TAGTCCATGATTTGACAATGTGTTGCTACAGTAAACATTTGCTAATGAT


 


GGATTAATTAGGCTTAATAGATTCGTCTCGCCGTTTAGCCTCCACTTAT


 


GTAATGGGTTTTCTAAACAATCTACGTTTAATACTCCTAATTAGTATCT


 


AAATATTCAATGTGACACGTGCTAAAAATAAGTCAGTGGAAGGAAGAGA


 


ACGTCCCCTTAGTTTTCCATCTTATTAATTGTACGATGAAACTGTGCAG


 


CCAGATGATTGACAATCGCAATACTTCAACTAGTGGGCCATGCACATCA


 


GCGACGTGTAACGTCGTGAGTTGCTGTTCCCGTAGAGAAATATCAACTG


 


GTGGGCCACGCACATCAGCGTCGTGTAACGTGGACGGAGGAGCCCCGTG


 


ACGGCGTCGACATCGAACGGCCACCAACCACGGAACCACCCGTCCCCAC


 


CTCTCGGAAGCTCCGCTCCACGGCGTCGACATCTAACGGCTACCAGCAG


 


GCGTACGGGTTGGAGTGGACTCCTTGCCTCTTTGCGCTGGCGGCTTCCG


 


GAAATTGCGTGGCGGAGACGAGGCGGGCTCGTCTCACACGGCACGGAAG


 


ACGTCACGGGTTCCTTCCCCACCTCTCCTCTTCCCCACCGCCATAAATAG






In an embodiment, a promoter operably linked to a 5′-UTR and intron can be the Panicum virgatum (Switchgrass) ubiquitin promoter operably linked to a 5′-UTR and intron. In one embodiment the promoter comprises or consists of the sequence of SEQ ID NO: 39:







(SEQ ID NO: 39)







TTGAATTTTAATTTCAAATTTTGCAGGGTAGTAGTGGACATCACAATAC


 


ATATTTAGAAAAAGTTTTATAATTTTCCTCCGTTAGTTTTCATATAATT


 


TTGAACTCCAACGATTAATCTATTATTAAATATCCCGATCTATCAAAAT


 


AATGATAAAAATTTATGATTAATTTTTCTAACATGTGTTATGGTGTGTA


 


CTATCGTCTTATAAAATTTCAACTTAAAACTCCACCTATACATGGAGAA


 


ATGAAAAAGACGAATTACAGTAGGGAGTAATTTGAACCAAATGGAATAG


 


TTTGAGGGTAAAATGAACTAAACAATAGTTTAGGAGGTTATTCAGATTT


 


TAGTTATAGTTGAGAGGAGTAATTTAGACTTTTTCCTATCTTGAATTGT


 


TGACGGCTCTCCTATCGGATATCGGATGGAGTCTTTCAGCCCAACATAA


 


CTTCATTCGGGCCCAAACGTTCGTCCATCCAGCCTAGGGAGAACATTTT


 


GCCCATGATATCTGTTTTTCTTTTTTTCTATTTTCACTGGTATTATAGG


 


AGGGAAATATACAACGTGTTCACCTTTGGTTTCATTCTTGTTCCATCTG


 


AATTTATCTAAAACTGTGTTTGAACTTCGTAAGAATTTTGTTCGATCTG


 


TCCGGTACATCGTGTTGATAGGTGGCCTCCGAGATTCTTCTTTTTAACC


 


GGCAAAGTAAAATAATCTCAGCTCCAGCCTAACGTCAATTATCAGAGAG


 


AGAAAAAAATATTTTTTTATGATTGATCGGAAACCAACCGCCTTACGTG


 


TCGATCCTGGTTCCTGGCCGGCACGGCGGAGGAAAGCGACCGACCTCGC


 


AACGCCGGCGCACGGCGCCGCCGTGTTGGACTTGGTCTCCCGCGACTCC


 


GTGGGCCTCGGCTTATCGCCGCCGCTCCATCTCAACCGTCCGCTTGGAC


 


ACGTGGAAGTTGATCCGTCGCGCACCAGCCTCGGAGGTAACCTAACTGC


 


CCGTACTATAAATCCGGGATCCGGCCTCTCCAATCCCCATCGCCACAAG


 


TTCGCGATCTCTCGATTTCACAAATCGCCGAGAAGACCCGAGCAGAGAA


 


GTTCCCTCCGATCGCCTTGCCAAGGTACTCCTACCTAATCCTCCTTAAC


 


TGATCTCTCCTCTATCACGTTGGTAATCTTCGAATGATCTGCTGCCTGG


 


CTCGCTGTTCCCCCTCGTTATGCACTGTTTCCATCACGAGTTTTTTTTT


 


TCATCATCTAATCTATGCGGTTGCGGAAGAATTGTGGCTAGTGGAGTAG


 


TTTTCTGTGCTTGATCGGTAGATTCGATGTGTGGGTGTATGGATGTTTT


 


CTGAAAAGTTGCTGGATTAGTTTACGCTTTCAGGCCGCAGGTCTGTTCG


 


AAATTGATTATGAAGTCTATATGCTTTGGATCTATCGATTTCCAGTTTT


 


ATTCAGATGTAGGCCAAAAAATTGTCGGCATTTGTGTGGAATTAGTTGG


 


CCTTTAGGTCTGCACATTCATGGTGACGGCACAGTTGCTGCTGGCTGTT


 


GCGTGGGACGAGTTATTATAGTTGTTTTTGTTTTTCCCTGATTGATTCA


 


CATTTTCAATGATAACTAGCCTTTGTCACCTAACCAAGTCCAGGTTGAT


 


CCTATCTGTGTTCTTCAGCTACCAGTTTGCATAGATGATGGTGTATTCG


 


ATTGCTTTAGTAGGCCTTCTGATTTCACATCTAATTCTGTCATGAATAT


 


AGATAACTTTACATGCTTTTGATATACTTTATATTTGAACTGTTCACTG


 


TCCAGCCTATTTTGGATAATTGAGTGCATTGGCTTTTGATGCCTGAATT


 


ATTCACATGTTCCTGGATAATTGACCTGTGTCACCTAGTTGACTGTTTT


 


TTGAGGTGCCACCCGTCTGTTCAGCTGATTTGTGTATTCGATTGCTCTA


 


GTTAATCTTTTGATTATGCAGCTAGTGCTTTGTCATATGTAGCTTTATA


 


GGCTTCTGATGTCCTTGGATATAGTTCAGTCTACTTGTCAAGTTGCTTT


 


ACAAGTAGTAGCTCTGATTCTATTTGGCTTCCTGAGTCAGAGCTTTGCA


 


AATTGCTTGTTGTTACATTACATCATATTACTTGAATTGCAGTTATTTA


 


ATGGTTGGATTGTTGCTGTTTACTTCTACATTTTTTGCTGTTTTATATT


 


ATACTAAAATGTTTGTGTTGCTGCTTTTCAG






In an embodiment, a nucleic acid construct is provided comprising a promoter operably linked to an intron and 5′-UTR. In one embodiment the construct comprises 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:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:41 or SEQ ID NO:39. In one embodiment, a nucleic acid construct is provided comprising a ubiquitin promoter sequence comprising or consisting of a sequence 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:15, SEQ ID NO:16, SEQ ID NO:17, or SEQ ID NO:39 operably linked to a polylinker. Optionally, the construct can further comprise 3′-UTR that is operably linked to the 3′ end of the polylinker. In an embodiment, a gene expression cassette is provided comprising a ubiquitin promoter sequence wherein the promoter sequence comprises or consists of a sequence 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:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO: 41 or SEQ ID NO:39. operably linked to a non-ubiquitin transgene. Optionally, the construct can further comprise 3′-UTR that is operably linked to the 3′ end of the non-ubiquitin transgene. In one embodiment the 3′-UTR sequence consists of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:36. In an illustrative embodiment, the transgene can be an insecticidal resistance transgene, an herbicide tolerance transgene, a nitrogen use efficiency transgene, a water us efficiency transgene, a nutritional quality transgene, a DNA binding transgene, a selectable marker transgene, or combinations thereof. In one embodiment the transgene is an herbicide resistance gene. In one embodiment a vector is provided comprising 1, 2, 3 or 4 promoter sequences independently selected from the group consisting of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:36.
In an embodiment, a gene expression cassette comprises a ubiquitin promoter, a ubiquitin 5′-UTR, a ubiquitin intron, and a ubiquitin 3′-UTR. In an embodiment, a ubiquitin promoter, a ubiquitin 5′-UTR, a ubiquitin intron, and a ubiquitin 3′-UTR can each be independently a Panicum virgatum, Brachypodium distachyon or Setaria italica ubiquitin promoter; Panicum virgatum Brachypodium distachyon or Setaria italica ubiquitin 5′-UTR; Panicum virgatum, Brachypodium distachyon or Setaria italica ubiquitin intron; and, a Panicum virgatum, Brachypodium distachyon or Setaria italica ubiquitin 3′-UTR. In an embodiment, a gene expression cassette comprises: a) a promoter, wherein the promoter is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, or 100% identical to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:36; b) 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:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:37; c) a 5′-UTR, wherein the 5′-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:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, or SEQ ID NO:38; or, d) an intron, wherein the intron 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:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO: 41 or SEQ ID NO:39.
For example, a gene expression cassette may include both a promoter, an intron, and a 5′-UTR wherein the promoter is a polynucleotide of SEQ ID NO:3, the intron is a polynucleotide of SEQ ID NO:9 or 10, and the 5′-UTR is a polynucleotide of SEQ ID NO:13 or 14. Likewise, a gene expression cassette may include both a promoter, an intron, and a 5′-UTR wherein the promoter is a polynucleotide of SEQ ID NO:2, the intron is a polynucleotide of SEQ ID NO:8, and the 5′-UTR is a polynucleotide of SEQ ID NO:12. Furthermore, a gene expression cassette may include both a promoter, an intron, and a 5′-UTR wherein the promoter is a polynucleotide of SEQ ID NO:3, the intron is a polynucleotide of SEQ ID NO:9 and/or SEQ ID NO:10, and the 5′-UTR is a polynucleotide of SEQ ID NO:13 or 14. In addition, a gene expression cassette may include both a promoter, an intron, and a 5′-UTR wherein the promoter is a polynucleotide of SEQ ID NO:3, the intron is a polynucleotide of SEQ ID NO:9 or 10, and the 5′-UTR is a polynucleotide of SEQ ID NO:13.
For example, a gene expression cassette may include both a promoter, an intron, a 5′-UTR, and a 3′-UTR wherein the promoter is a polynucleotide of SEQ ID NO:3, the intron is a polynucleotide of SEQ ID NO:9 or 10, the 5′-UTR is a polynucleotide of SEQ ID NO:13 or 14, and the 3′-UTR is a polynucleotide of SEQ ID NO: 6. Likewise, a gene expression cassette may include both a promoter, an intron, a 5′-UTR, and a 3′-UTR wherein the promoter is a polynucleotide of SEQ ID NO:3, the intron is a polynucleotide of SEQ ID NO:9 or 10, the 5′-UTR is a polynucleotide of SEQ ID NO:13 or 14 and the 3′-UTR is a polynucleotide of SEQ ID NO:6. Furthermore, a gene expression cassette may include both a promoter, an intron, a 5′-UTR, and a 3′-UTR wherein the promoter is a polynucleotide of SEQ ID NO:3, the intron is a polynucleotide of SEQ ID NO:9 and/or SEQ ID NO:10, the 5′-UTR is a polynucleotide of SEQ ID NO:13 or 14, and the 3′-UTR is a polynucleotide of SEQ ID NO:6. In addition, a gene expression cassette may include both a promoter, an intron, a 5′-UTR, and a 3′-UTR wherein the promoter is a polynucleotide of SEQ ID NO:35, the intron is a polynucleotide of SEQ ID NO:37, the 5′-UTR is a polynucleotide of SEQ ID NO:38, and the 3′-UTR is a polynucleotide of SEQ ID NO:36.
In addition, a gene expression cassette may include both a promoter, and a 3′-UTR wherein the promoter is a polynucleotide of SEQ ID NO:3 and a 3′-UTR of SEQ ID NO:6. In an embodiment, a gene expression cassette may include both a promoter and a 3′-UTR wherein the promoter is a polynucleotide of SEQ ID NO:3 and a 3′-UTR of SEQ ID NO:5. In an embodiment, a gene expression cassette may include both a promoter and a 3′-UTR wherein the promoter is a polynucleotide of SEQ ID NO:3 and a 3′-UTR of SEQ ID NO:6. In an embodiment, a gene expression cassette may include both a promoter and a 3′-UTR wherein the promoter is a polynucleotide of SEQ ID NO:35 and a 3′-UTR of SEQ ID NO:36.
In an embodiment, a gene expression cassette comprises a ubiquitin promoter, ubiquitin 5′-UTR, and a ubiquitin 3′-UTR that are operably linked to a non-ubiquitin transgene. In an embodiment, a gene expression cassette comprises a ubiquitin promoter, a ubiquitin intron, ubiquitin 5′-UTR, and a ubiquitin 3′-UTR that are operably linked to a non-ubiquitin transgene.
A promoter, an intron, a 5′-UTR, and 3′-UTR can be operably linked to different transgenes within a gene expression cassette when a gene expression cassette includes one or more transgenes. In an illustrative embodiment, a gene expression cassette comprises a ubiquitin promoter that is operably linked to a transgene, wherein the transgene can be an insecticidal resistance transgene, an herbicide tolerance transgene, a nitrogen use efficiency transgene, a water use efficiency transgene, a nutritional quality transgene, a DNA binding transgene, a selectable marker transgene, or combinations thereof. In an illustrative embodiment, a gene expression cassette comprises a ubiquitin promoter, an intron, and a 5′-UTR that are operably linked to a transgene, wherein the transgene can be an insecticidal resistance transgene, an herbicide tolerance transgene, a nitrogen use efficiency transgene, a water use efficiency transgene, a nutritional quality transgene, a DNA binding transgene, a selectable marker transgene, or combinations thereof. In an illustrative embodiment, a gene expression cassette comprises a ubiquitin 3′-UTR that is operably linked to a transgene, wherein the transgene encodes for a gene product that enhances insecticidal resistance, herbicide tolerance, nitrogen use efficiency, water us efficiency, nutritional quality or combinations thereof.
A ubiquitin intron and a 5′-UTR can be operably linked to different promoters within a gene expression cassette. In an illustrative embodiment, the promoters originate from a plant (e.g., Zea mays ubiquitin 1 promoter), a virus (e.g., Cassava vein mosaic virus promoter) or a bacteria (e.g., Agrobacterium tumefaciens delta mas). In an illustrative embodiment, a gene expression cassette comprises a ubiquitin promoter that is operably linked to a transgene, wherein the transgene can be an insecticidal resistance transgene, an herbicide tolerance transgene, a nitrogen use efficiency transgene, a water use efficiency transgene, a nutritional quality transgene, a DNA binding transgene, a selectable marker transgene, or combinations thereof.
In an embodiment, a vector comprises a gene expression cassette as disclosed herein. In an embodiment, a vector can be a plasmid, a cosmid, a bacterial artificial chromosome (BAC), a bacteriophage, a virus, or an excised polynucleotide fragment for use in direct transformation or gene targeting such as a donor DNA.
In accordance with one embodiment a nucleic acid vector is provided comprising a recombinant gene cassette wherein the recombinant gene cassette comprises a ubiquitin based promoter operably linked to a polylinker sequence, a non-ubiquitin transgene or combination thereof. In one embodiment the recombinant gene cassette comprises a ubiquitin based promoter operably linked to a non-ubiquitin transgene. In one embodiment the recombinant gene cassette comprises a ubiquitin based promoter as disclosed herein operably linked to a polylinker sequence. The polylinker is operably linked to the ubiquitin based promoter in a manner such that insertion of a coding sequence into one of the restriction sites of the polylinker will operably link the coding sequence allowing for expression of the coding sequence when the vector is transfected into a host cell.
In accordance with one embodiment the ubiquitin based promoter comprises SEQ ID NO: 3 or a sequence that has 90, 95 or 99% sequence identity with SEQ ID NO: 3. In accordance with one embodiment the promoter sequence has a total length of no more than 1.5, 2, 2.5, 3 or 4 kb. In accordance with one embodiment the ubiquitin based promoter consists of SEQ ID NO: 3 or a 1064 bp sequence that has 90, 95 or 99% sequence identity with SEQ ID NO: 3.
In accordance with one embodiment a nucleic acid vector is provided comprising a gene cassette that consists of SEQ ID NO: 17, a non-ubiquitin transgene and a 3′-UTR, wherein SEQ ID NO: 17 is operably linked to the 5′ end of the non-ubiquitin transgene and the 3′-UTR is operably linked to the 3′ end of the non-ubiquitin transgene. In a further embodiment the 3′ untranslated sequence comprises SEQ ID NO: 6 or a sequence that has 90, 95, 99 or 100% sequence identity with SEQ ID NO: 6. In accordance with one embodiment a nucleic acid vector is provided comprising a gene cassette that consists of SEQ ID NO: 17, or a 2600 bp sequence that has 90, 95, or 99% sequence identity with SEQ ID NO: 17, a non-ubiquitin transgene and a 3′-UTR, wherein SEQ ID NO: 17 is operably linked to the 5′ end of the non-ubiquitin transgene and the 3′-UTR is operably linked to the 3′ end of the non-ubiquitin transgene. In a further embodiment the 3′ untranslated sequence comprises SEQ ID NO: 6 or a sequence that has 90, 95, 99 or 100% sequence identity with SEQ ID NO: 6. I a further embodiment the 3′ untranslated sequence consists of SEQ ID NO: 6, or a 1032 bp sequence that has 90, 95, or 99% sequence identity with SEQ ID NO: 6.
In accordance with one embodiment the nucleic acid vector further comprises a sequence encoding a selectable maker. In accordance with one embodiment the recombinant gene cassette is operably linked to an Agrobacterium T-DNA border. In accordance with one embodiment the recombinant gene cassette further comprises a first and second T-DNA border, wherein first T-DNA border is operably linked to one end of the gene construct, and said second T-DNA border is operably linked to the other end of the gene construct. The first and second Agrobacterium T-DNA borders can be independently selected from T-DNA border sequences originating from bacterial strains selected from the group consisting of a nopaline synthesizing Agrobacterium T-DNA border, an ocotopine synthesizing Agrobacterium T-DNA border, a succinamopine synthesizing Agrobacterium T-DNA border, or any combination thereof. In one embodiment an Agrobacterium strain selected from the group consisting of a nopaline synthesizing strain, a mannopine synthesizing strain, a succinamopine synthesizing strain, or an octopine synthesizing strain is provided, wherein said strain comprises a plasmid wherein the plasmid comprises a transgene operably linked to a sequence selected from SEQ ID NO: 3, SEQ ID NO: 17 or a sequence having 90, 95, or 99% sequence identity with SEQ ID NO: 3 or SEQ ID NO: 17.
Transgenes of interest and suitable for use in the present disclosed constructs include, but are not limited to, coding sequences that confer (1) resistance to pests or disease, (2) resistance to herbicides, and (3) value added traits as disclosed in WO2013116700 (DGT-28), US20110107455 (DSM-2), U.S. Pat. No. 8,283,522 (AAD-12); U.S. Pat. No. 7,838,733 (AAD-1); U.S. Pat. Nos. 5,188,960; 5,691,308; 6,096,708; and 6,573,240 (Cry1F); U.S. Pat. Nos. 6,114,138; 5,710,020; and 6,251,656 (Cry1Ac); U.S. Pat. Nos. 6,127,180; 6,624,145 and 6,340,593 (Cry34Ab1); U.S. Pat. Nos. 6,083,499; 6,548,291 and 6,340,593 (Cry35Ab1), the disclosures of which are incorporated herein. In accordance with one embodiment the transgene encodes a selectable marker or a gene product conferring insecticidal resistance, herbicide tolerance, nitrogen use efficiency, water use efficiency, or nutritional quality.
In accordance with one embodiment a nucleic acid vector is provided comprising a gene cassette wherein the gene cassette comprises a promoter region operably linked to the 5′ end of a transgene wherein the 3′ end of the transgene is linked to a 3′ untranslated region. In one embodiment the promoter region comprises SEQ ID NO: 3 or a sequence that has 90, 95 or 99% sequence identity with SEQ ID NO: 3. In accordance with one embodiment the promoter region consists of SEQ ID NO: 3 or SEQ ID NO: 17. In one embodiment the 3′ untranslated sequence comprises SEQ ID NO: 6 or a sequence that has 90, 95 or 99% sequence identity with SEQ ID NO: 6, and in one embodiment the 3′ untranslated sequence consists of SEQ ID NO: 6 or a 1032 bp sequence having 90, 95 or 99% sequence identity with SEQ ID NO: 6.
In accordance with one embodiment a nucleic acid vector is provided comprising a gene cassette wherein the gene cassette comprises a promoter region operably linked to the 5′ end of a 5′ untranslated sequence, wherein the 3′ end of the 5′ untranslated sequence is operably linked to the 5′ end of the transgene wherein the 3′ end of the transgene is linked to a 3′ untranslated region. In one embodiment the promoter region comprises or consists of SEQ ID NO: 3 or a sequence that has 90, 95 or 99% sequence identity with SEQ ID NO: 3. In one embodiment the promoter region consists of SEQ ID NO: 3 or a 1032 bp sequence that has 90, 95 or 99% sequence identity with SEQ ID NO: 3. In accordance with one embodiment the 5′ untranslated sequence comprises or consists of SEQ ID NO: 13 or a sequence that has 90% sequence identity with SEQ ID NO: 13. In accordance with one embodiment the 5′ untranslated sequence comprises or consists of SEQ ID NO: 14 or a sequence that has 90% sequence identity with SEQ ID NO: 14. In accordance with one embodiment the 5′ untranslated sequence consists of SEQ ID NO: 13 or a 261 bp sequence that has 90% sequence identity with SEQ ID NO: 13. In accordance with one embodiment the 5′ untranslated sequence consists of SEQ ID NO: 14 or a 113 bp sequence that has 90% sequence identity with SEQ ID NO: 14. In one embodiment the 3′ untranslated sequence comprises or consists of SEQ ID NO: 6 or a sequence that has 90, 95 or 99% sequence identity with SEQ ID NO: 6. In one embodiment the 3′ untranslated sequence consists of SEQ ID NO: 6 or a 1032 bp sequence that has 90, 95 or 99% sequence identity with SEQ ID NO: 6. In a further embodiment the nucleic acid vector further comprises a ubiquitin intron inserted between the 5′ untranslated region and the transgene, and operably linked to the promoter and transgene. In one embodiment the ubiquitin intron comprises or consists of SEQ ID NO: 9 or 10 or a sequence that has 90, 95 or 99% sequence identity with SEQ ID NO: 9 or 10. In one embodiment the ubiquitin intron consists of SEQ ID NO: 9 or a 48 bp sequence that has 90, 95 or 99% sequence identity with SEQ ID NO: 9. In one embodiment the ubiquitin intron consists of SEQ ID NO: 10 or a 1114 bp sequence that has 90, 95 or 99% sequence identity with SEQ ID NO: 10
In accordance with one embodiment a nucleic acid vector is provided comprising a gene cassette wherein the gene cassette comprises a promoter region operably linked to the 5′ end of a transgene wherein the 3′ end of the transgene is linked to a 3′ untranslated region. In one embodiment the promoter region comprises SEQ ID NO: 40 or a sequence that has 90, 95 or 99% sequence identity with SEQ ID NO: 40.







(SEQ ID NO: 40)







TGCGTCTGGACGCACAAGTCATAGCATTATCGGCTAAAATTTCTTAATT


 


TCTAAATTAGTCATATCGGCTAAGAAAGTGGGGAGCACTATCATTTCGT


 


AGAACAAGAACAAGGTATCATATATATATATATATATAATATTTAAACT


 


TTGTTAAGTGGAATCAAAGTGCTAGTATTAATGGAGTTTCATGTGCATT


 


AAATTTTATGTCACATCAGCAATTTTGTTGACTTGGCAAGGTCATTTAG


 


GGTGTGTTTGGAAGACAGGGGCTATTAGGAGTATTAAACATAGTCTAAT


 


TACAAAACTAATTGCACAACCGCTAAGCTGAATCGCGAGATGGATCTAT


 


TAAGCTTAATTAGTCCATGATTTGACAATGTGGTGCTACAATAACCATT


 


TGCTAATGATGGATTACTTAGGTTTAATAGATTCGTCTCGTGATTTAGC


 


CTATGGGTTCTGCTATTAATTTTGTAATTAGCTCATATTTAGTTCTTAT


 


AATTAGTATCCGAACATCCAATGTGACATGCTAAAGTTTAACCCTGGTA


 


TCCAAATGAAGTCTTATGAGAGTTTCATCACTCCGGTGGTATATGTACT


 


TAGGCTCCGTTTTCTTCCACCGACTTATTTTTAGCACCCGTCACATTGA


 


ATGTTTAGATACTAATTAGAAGTATTAAACGTAGACTATTTACAAAATC


 


CATTACATAAGACGAATCTAAACGGCGAGACGAATCTATTAAACCTAAT


 


TAGTCCATGATTTGACAATGTGTTGCTACAGTAAACATTTGCTAATGAT


 


GGATTAATTAGGCTTAATAGATTCGTCTCGCCGTTTAGCCTCCACTTAT


 


GTAATGGGTTTTCTAAACAATCTACGTTTAATACTCCTAATTAGTATCT


 


AAATATTCAATGTGACACGTGCTAAAAATAAGTCAGTGGAAGGAAGAGA


 


ACGTCCCCTTAGTTTTCCATCTTATTAATTGTACGATGAAACTGTGCAG


 


CCAGATGATTGACAATCGCAATACTTCAACTAGTGGGCCATGCACATCA


 


GCGACGTGTAACGTCGTGAGTTGCTGTTCCCGTAGCCGACCCCCTCGCC


 


TTTCTCCCCAATCTCATCTCGTCTCGTGTTGTTCGGAGCACACCACCCG


 


CCCCAAATCGTTCTTCCCGCAAGCCTCGGCGATCCTTCACCCGCTTCAA


 


G.






In one embodiment the promoter region comprises SEQ ID NO: 42 or a sequence that has 90, 95 or 99% sequence identity with SEQ ID NO: 42.







(SEQ ID NO: 42)







TGCGTCTGGACGCACAAGTCATAGCATTATCGGCTAAAATTTCTTAATT


 


TCTAAATTAGTCATATCGGCTAAGAAAGTGGGGAGCACTATCATTTCGT


 


AGAACAAGAACAAGGTATCATATATATATATATATATAATATTTAAACT


 


TTGTTAAGTGGAATCAAAGTGCTAGTATTAATGGAGTTTCATGTGCATT


 


AAATTTTATGTCACATCAGCAATTTTGTTGACTTGGCAAGGTCATTTAG


 


GGTGTGTTTGGAAGACAGGGGCTATTAGGAGTATTAAACATAGTCTAAT


 


TACAAAACTAATTGCACAACCGCTAAGCTGAATCGCGAGATGGATCTAT


 


TAAGCTTAATTAGTCCATGATTTGACAATGTGGTGCTACAATAACCATT


 


TGCTAATGATGGATTACTTAGGTTTAATAGATTCGTCTCGTGATTTAGC


 


CTATGGGTTCTGCTATTAATTTTGTAATTAGCTCATATTTAGTTCTTAT


 


AATTAGTATCCGAACATCCAATGTGACATGCTAAAGTTTAACCCTGGTA


 


TCCAAATGAAGTCTTATGAGAGTTTCATCACTCCGGTGGTATATGTACT


 


TAGGCTCCGTTTTCTTCCACCGACTTATTTTTAGCACCCGTCACATTGA


 


ATGTTTAGATACTAATTAGAAGTATTAAACGTAGACTATTTACAAAATC


 


CATTACATAAGACGAATCTAAACGGCGAGACGAATCTATTAAACCTAAT


 


TAGTCCATGATTTGACAATGTGTTGCTACAGTAAACATTTGCTAATGAT


 


GGATTAATTAGGCTTAATAGATTCGTCTCGCCGTTTAGCCTCCACTTAT


 


GTAATGGGTTTTCTAAACAATCTACGTTTAATACTCCTAATTAGTATCT


 


AAATATTCAATGTGACACGTGCTAAAAATAAGTCAGTGGAAGGAAGAGA


 


ACGTCCCCTTAGTTTTCCATCTTATTAATTGTACGATGAAACTGTGCAG


 


CCAGATGATTGACAATCGCAATACTTCAACTAGTGGGCCATGCACATCA


 


GCGACGTGTAACGTCGTGAGTTGCTGTTCCCGTAGAGAAATATCAACTG


 


GTGGGCCACGCACATCAGCGTCGTGTAACGTGGACGGAGGAGCCCCGTG


 


ACGGCGTCGACATCGAACGGCCACCAACCACGGAACCACCCGTCCCCAC


 


CTCTCGGAAGCTCCGCTCCACGGCGTCGACATCTAACGGCTACCAGCAG


 


GCGTACGGGTTGGAGTGGACTCCTTGCCTCTTTGCGCTGGCGGCTTCCG


 


GAAATTGCGTGGCGGAGACGAGGCGGGCTCGTCTCACACGGCACGGAAG


 


AC






In accordance with one embodiment the promoter region consists of SEQ ID NO: 40 or a 1177 bp sequence having 90, 95 or 99% sequence identity with SEQ ID NO: 40. In accordance with one embodiment the promoter region consists of SEQ ID NO: 40. In accordance with one embodiment the promoter region consists of SEQ ID NO: 42 or a 1325 bp sequence having 90, 95 or 99% sequence identity with SEQ ID NO: 42. In accordance with one embodiment the promoter region consists of SEQ ID NO: 42. In one embodiment the 3′ untranslated sequence consists of SEQ ID NO: 6 or a 1032 bp sequence that has 90, 95 or 99% sequence identity with SEQ ID NO: 6, and in one embodiment the 3′ untranslated sequence consists of SEQ ID NO: 6.
In an embodiment, a cell or plant is provided comprising a gene expression cassette as disclosed herein. In an embodiment, a cell or plant comprises a vector comprising a gene expression cassette as disclosed herein. In an embodiment, a vector can be a plasmid, a cosmid, a bacterial artificial chromosome (BAC), a bacteriophage, or a virus. Thereby, a cell or plant comprising a gene expression cassette as disclosed herein is a transgenic cell or transgenic plant, respectively. In an embodiment, a transgenic plant can be a monocotyledonous plant. In an embodiment, a transgenic monocotyledonous plant can be, but is not limited to maize, wheat, rice, sorghum, oats, rye, bananas, sugar cane, and millet. In an embodiment, a transgenic plant can be a dicotyledonous plant. In an embodiment, a transgenic dicotyledonous plant can be, but is not limited to soybean, cotton, sunflower, and canola. An embodiment also includes a transgenic seed from a transgenic plant as disclosed herein.
In an embodiment, a gene expression cassette includes two or more transgenes. The two or more transgenes may not be operably linked to the same promoter, intron, or 5′-UTR or 3′-UTR as disclosed herein. In an embodiment, a gene expression cassette includes one or more transgenes. In an embodiment with one or more transgenes, at least one transgene is operably linked to a promoter, intron, 5′-UTR, or 3′-UTR or the subject disclosure.
Selectable Markers
Various selectable markers also described as reporter genes can be incorporated into a chosen expression vector to allow for identification and selectable of transformed plants (“transformants”). Many methods are available to confirm expression of selectable markers in transformed plants, including for example DNA sequencing and PCR (polymerase chain reaction), Southern blotting, RNA blotting, immunological methods for detection of a protein expressed from the vector, e g., precipitated protein that mediates phosphinothricin resistance, or visual observation of other proteins such as reporter genes encoding β-glucuronidase (GUS), luciferase, green fluorescent protein (GFP), yellow fluorescent protein (YFP), DsRed, β-galactosidase, chloramphenicol acetyltransferase (CAT), alkaline phosphatase, and the like (See Sambrook, et al., Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Press, N.Y., 2001, the content of which is incorporated herein by reference in its entirety).
Selectable marker genes are utilized for selection of transformed cells or tissues. Selectable marker genes include genes encoding antibiotic resistance, such as those encoding neomycin phosphotransferase II (NEO) and hygromycin phosphotransferase (HPT) as well as genes conferring resistance to herbicidal compounds. Herbicide resistance genes generally code for a modified target protein insensitive to the herbicide or for an enzyme that degrades or detoxifies the herbicide in the plant before it can act. For example, resistance to glyphosate has been obtained by using genes coding for mutant target enzymes, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). Genes and mutants for EPSPS are well known, and further described below. Resistance to glufosinate ammonium, bromoxynil, and 2,4-dichlorophenoxyacetate (2,4-D) have been obtained by using bacterial genes encoding pat or DSM-2, a nitrilase, an aad-1, or an aad-12 gene, which detoxifies the respective herbicides.
In an embodiment, herbicides can inhibit the growing point or meristem, including imidazolinone or sulfonylurea, and genes for resistance/tolerance of acetohydroxyacid synthase (AHAS) and acetolactate synthase (ALS) for these herbicides are well known. Glyphosate resistance genes include mutant 5-enolpyruvylshikimate-3-phosphate synthase (EPSPs) and dgt-28 genes (via the introduction of recombinant nucleic acids and/or various forms of in vivo mutagenesis of native EPSPs genes), aroA genes and glyphosate acetyl transferase (GAT) genes, respectively). Resistance genes for other phosphono compounds include bar genes from Streptomyces species, including Streptomyces hygroscopicus and Streptomyces viridichromogenes, and pyridinoxy or phenoxy proprionic acids and cyclohexones (ACCase inhibitor-encoding genes). Exemplary genes conferring resistance to cyclohexanediones and/or aryloxyphenoxypropanoic acid (including Haloxyfop, Diclofop, Fenoxyprop, Fluazifop, Quizalofop) include genes of acetyl coenzyme A carboxylase (ACCase)—Acc1-S1, Acc1-S2 and Acc1-S3. In an embodiment, herbicides can inhibit photosynthesis, including triazine (psbA and 1s+ genes) or benzonitrile (nitrilase gene).
In an embodiment, selectable marker genes include, but are not limited to genes encoding: neomycin phosphotransferase II; cyanamide hydratase; aspartate kinase; dihydrodipicolinate synthase; tryptophan decarboxylase; dihydrodipicolinate synthase and desensitized aspartate kinase; bar gene; tryptophan decarboxylase; neomycin phosphotransferase (NEO); hygromycin phosphotransferase (HPT or HYG); dihydrofolate reductase (DHFR); phosphinothricin acetyltransferase; 2,2-dichloropropionic acid dehalogenase; acetohydroxyacid synthase; 5-enolpyruvyl-shikimate-phosphate synthase (aroA); haloarylnitrilase; acetyl-coenzyme A carboxylase; dihydropteroate synthase (sul I); and 32 kD photosystem II polypeptide (psbA).
An embodiment also includes genes encoding resistance to: chloramphenicol; methotrexate; hygromycin; spectinomycin; bromoxynil; glyphosate; and phosphinothricin.
The above list of selectable marker genes is not meant to be limiting. Any reporter or selectable marker gene are encompassed by the present invention.
Selectable marker genes are synthesized for optimal expression in a plant. For example, in an embodiment, a coding sequence of a gene has been modified by codon optimization to enhance expression in plants. A selectable marker gene can be optimized for expression in a particular plant species or alternatively can be modified for optimal expression in dicotyledonous or monocotyledonous plants. Plant preferred codons may be determined from the codons of highest frequency in the proteins expressed in the largest amount in the particular plant species of interest. In an embodiment, a selectable marker gene is designed to be expressed in plants at a higher level resulting in higher transformation efficiency. Methods for plant optimization of genes are well known. Guidance regarding the optimization and production of synthetic DNA sequences can be found in, for example, WO2013016546, WO2011146524, WO1997013402, U.S. Pat. No. 6,166,302, and U.S. Pat. No. 5,380,831, herein incorporated by reference.
Transformation
Suitable methods for transformation of plants include any method by which DNA can be introduced into a cell, for example and without limitation: electroporation (see, e.g., U.S. Pat. No. 5,384,253); micro-projectile 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).
A DNA construct may be introduced directly into the genomic DNA of the plant cell using techniques such as agitation with silicon carbide fibers (See, e.g., U.S. Pat. Nos. 5,302,523 and 5,464,765), or the DNA constructs can be introduced directly to plant tissue using biolistic methods, such as DNA particle bombardment (see, e.g., Klein et al. (1987) Nature 327:70-73). Alternatively, the DNA construct can be introduced into the plant cell via nanoparticle transformation (see, e.g., US Patent Publication No. 20090104700, which is incorporated herein by reference in its entirety).
In addition, gene transfer may be achieved using non-Agrobacterium bacteria or viruses such as Rhizobium sp. NGR234, Sinorhizoboium meliloti, Mesorhizobium loti, potato virus X, cauliflower mosaic virus and cassava vein mosaic virus and/or tobacco mosaic virus, See, e.g., Chung et al. (2006) Trends Plant Sci. 11(1):1-4.
Through the application of transformation techniques, cells of virtually any plant species may be stably transformed, and these cells may 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 for transforming Brassica plants in particular are described, for example, in U.S. Pat. No. 5,750,871; techniques for transforming soy bean are described, for example, in U.S. Pat. No. 6,384,301; and techniques for transforming maize are described, for example, in U.S. Pat. Nos. 7,060,876 and 5,591,616, and International PCT Publication WO 95/06722.
After effecting delivery of an exogenous nucleic acid to a recipient cell, a transformed cell is generally identified for further culturing and plant regeneration. In order to improve the ability to identify transformants, one may desire to employ a selectable marker gene with the transformation vector used to generate the transformant. In an illustrative embodiment, a transformed cell population can be assayed by exposing the cells to a selective agent or agents, or the cells can be screened for the desired marker gene trait.
Cells that survive exposure to a selective agent, or cells that have been scored positive in a screening assay, may be cultured in media that supports regeneration of plants. In an embodiment, any suitable plant tissue culture media may be modified by including further substances, such as growth regulators. Tissue may be maintained on a basic media with growth regulators until sufficient tissue is available to begin plant regeneration efforts, or following repeated rounds of manual selection, until the morphology of the tissue is suitable for regeneration (e.g., at least 2 weeks), then transferred to media conducive to shoot formation. Cultures are transferred periodically until sufficient shoot formation has occurred. Once shoots are formed, they are transferred to media conducive to root formation. Once sufficient roots are formed, plants can be transferred to soil for further growth and maturity.
To confirm the presence of a desired nucleic acid comprising constructs provided in regenerating plants, a variety of assays may be performed. Such assays may include: molecular biological assays, such as Southern and northern blotting and PCR; biochemical assays, such as detecting the presence of a protein product, e.g., by immunological means (ELISA, western blots, and/or LC-MS MS spectrophotometry) or by enzymatic function; plant part assays, such as leaf or root assays; and/or analysis of the phenotype of the whole regenerated plant.
Transgenic events may be screened, for example, by PCR amplification using, e.g., oligonucleotide primers specific for nucleic acid molecules of interest. PCR genotyping is understood to include, but not be limited to, polymerase-chain reaction (PCR) amplification of genomic DNA derived from isolated host plant callus tissue predicted to contain a nucleic acid molecule of interest integrated into the genome, followed by standard cloning and sequence analysis of PCR amplification products. Methods of PCR genotyping have been well described (see, e.g., Rios et al. (2002) Plant J. 32:243-53), and may be applied to genomic DNA derived from any plant species or tissue type, including cell cultures. Combinations of oligonucleotide primers that bind to both target sequence and introduced sequence may be used sequentially or multiplexed in PCR amplification reactions. Oligonucleotide primers designed to anneal to the target site, introduced nucleic acid sequences, and/or combinations of the two may be produced. Thus, PCR genotyping strategies may include, for example and without limitation: amplification of specific sequences in the plant genome; amplification of multiple specific sequences in the plant genome; amplification of non-specific sequences in the plant genome; and combinations of any of the foregoing. One skilled in the art may devise additional combinations of primers and amplification reactions to interrogate the genome. For example, a set of forward and reverse oligonucleotide primers may be designed to anneal to nucleic acid sequence(s) specific for the target outside the boundaries of the introduced nucleic acid sequence.
Forward and reverse oligonucleotide primers may be designed to anneal specifically to an introduced nucleic acid molecule, for example, at a sequence corresponding to a coding region within a nucleotide sequence of interest comprised therein, or other parts of the nucleic acid molecule. Primers may be used in conjunction with primers described herein. Oligonucleotide primers may be synthesized according to a desired sequence and are commercially available (e.g., from Integrated DNA Technologies, Inc., Coralville, Iowa). Amplification may be followed by cloning and sequencing, or by direct sequence analysis of amplification products. In an embodiment, oligonucleotide primers specific for the gene target are employed in PCR amplifications.
Method of Expressing a Transgene
In an embodiment, a method of expressing at least one transgene in a plant comprises growing a plant comprising a ubiquitin promoter operably linked to at least one transgene. In an embodiment, a method of expressing at least one transgene in a plant comprising growing a plant comprising a ubiquitin 5′-UTR operably linked to at least one transgene. In an embodiment, a method of expressing at least one transgene in a plant comprising growing a plant comprising a ubiquitin intron operably linked to at least one transgene. In an embodiment, a method of expressing at least one transgene in a plant comprising growing a plant comprising a ubiquitin promoter, a ubiquitin 5′-UTR, and a ubiquitin intron operably linked to at least one transgene. In an embodiment, a method of expressing at least one transgene in a plant comprising growing a plant comprising a ubiquitin 3′-UTR operably linked to at least one transgene. In an embodiment, a method of expressing at least one transgene in a plant tissue or plant cell comprising culturing a plant tissue or plant cell comprising a ubiquitin promoter operably linked to at least one transgene. In an embodiment, a method of expressing at least one transgene in a plant tissue or plant cell comprising culturing a plant tissue or plant cell comprising a ubiquitin 5′-UTR operably linked to at least one transgene. In an embodiment, a method of expressing at least one transgene in a plant tissue or plant cell comprising culturing a plant tissue or plant cell comprising a ubiquitin intron operably linked to at least one transgene. In an embodiment, a method of expressing at least one transgene in a plant tissue or plant cell comprising culturing a plant tissue or plant cell comprising a ubiquitin promoter, a ubiquitin 5′-UTR, and a ubiquitin intron operably linked to at least one transgene. In an embodiment, a method of expressing at least one transgene in a plant tissue or plant cell comprising culturing a plant tissue or plant cell comprising a ubiquitin 3′-UTR operably linked to at least one transgene.
In an embodiment, a method of expressing at least one transgene in a plant comprises growing a plant comprising a gene expression cassette comprising a ubiquitin promoter operably linked to at least one transgene. In one embodiment the ubiquitin promoter consists of a sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:35, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, and SEQ ID NO:39 or a sequence that has 90, 95 or 995 sequence identity with a sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:35, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, and SEQ ID NO:39. In an embodiment, a method of expressing at least one transgene in a plant comprises growing a plant comprising a gene expression cassette comprising a ubiquitin intron operably linked to at least one transgene. In an embodiment, a method of expressing at least one transgene in a plant comprises growing a plant comprising a gene expression cassette comprising a ubiquitin 5′-UTR operably linked to at least one transgene. In an embodiment, a method of expressing at least one transgene in a plant comprises growing a plant comprising a gene expression cassette comprising a ubiquitin promoter, a ubiquitin 5′-UTR, and a ubiquitin intron operably linked to at least one transgene. In an embodiment, a method of expressing at least one transgene in a plant comprises growing a plant comprising a gene expression cassette comprising a ubiquitin 3′-UTR operably linked to at least one transgene. In an embodiment, a method of expressing at least one transgene in a plant tissue or plant cell comprises culturing a plant tissue or plant cell comprising a gene expression cassette a ubiquitin promoter operably linked to at least one transgene. In an embodiment, a method of expressing at least one transgene in a plant tissue or plant cell comprises culturing a plant tissue or plant cell comprising a gene expression cassette a ubiquitin intron operably linked to at least one transgene. In an embodiment, a method of expressing at least one transgene in a plant tissue or plant cell comprises culturing a plant tissue or plant cell comprising a gene expression cassette a ubiquitin 5′-UTR operably linked to at least one transgene. In an embodiment, a method of expressing at least one transgene in a plant tissue or plant cell comprises culturing a plant tissue or plant cell comprising a gene expression cassette a ubiquitin promoter, a ubiquitin 5′-UTR, and a ubiquitin intron operably linked to at least one transgene. In an 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 ubiquitin 3′-UTR operably linked to at least one transgene.
Transgenic Plants
In an embodiment, a plant, plant tissue, or plant cell comprises a ubiquitin promoter. In an embodiment, a ubiquitin promoter can be a Panicum virgatum, Brachypodium distachyon or Setaria italica ubiquitin promoter. In an embodiment, a plant, plant tissue, or plant cell comprises a gene expression cassette comprises a promoter, wherein the promoter is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, or 100% identical to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:35 wherein the promoter is operably linked to a non-ubiquitin transgene. In an embodiment, a plant, plant tissue, or plant cell comprises a gene expression cassette comprising a sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:35, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, and SEQ ID NO:39 or a sequence that has 90, 95 or 995 sequence identity with a sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:35, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, and SEQ ID NO:39 that is operably linked to a non-ubiquitin transgene. In an illustrative embodiment, a plant, plant tissue, or plant cell comprises a gene expression cassette comprising a ubiquitin promoter that is operably linked to a transgene, wherein the transgene can be an insecticidal resistance transgene, an herbicide tolerance transgene, a nitrogen use efficiency transgene, a water us efficiency transgene, a nutritional quality transgene, a DNA binding transgene, a selectable marker transgene, or combinations thereof.
In an embodiment, a plant, plant tissue, or plant cell comprises a gene expression cassette comprising a 3′-UTR. In an embodiment, a plant, plant tissue, or plant cell comprises a gene expression cassette comprising a ubiquitin 3′-UTR. In an embodiment, the ubiquitin 3′-UTR is a Panicum virgatum, Brachypodium distachyon or Setaria italica ubiquitin 3′-UTR. In an embodiment, a 3′-UTR can be the Brachypodium distachyon ubiquitin1 C (Ubi1C) 3′-UTR, Brachypodium distachyon ubiquitin1 3′-UTR, or Setaria italica ubiquitin 3′-UTR.
In an embodiment, a plant, plant tissue, or plant cell comprises a gene expression cassette comprising an intron, wherein the intron 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:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:37. In an embodiment, a gene expression cassette comprises a ubiquitin intron that is operably linked to a promoter, wherein the promoter is a Panicum virgatum, Brachypodium distachyon or Setaria italica ubiquitin promoter, or a promoter that originates from a plant (e.g., Zea mays ubiquitin 1 promoter), a virus (e.g., Cassava vein mosaic virus promoter) or a bacteria (e.g., Agrobacterium tumefaciens delta mas). In an embodiment, a plant, plant tissue, or plant cell comprises a gene expression cassette comprising a ubiquitin intron that is operably linked to a transgene. In an illustrative embodiment, a plant, plant tissue, or plant cell comprising a gene expression cassette comprising a ubiquitin intron that is operably linked to a transgene, wherein the transgene can be an insecticidal resistance transgene, an herbicide tolerance transgene, a nitrogen use efficiency transgene, a water us efficiency transgene, a nutritional quality transgene, a DNA binding transgene, a selectable marker transgene, or combinations thereof.
In an embodiment, a plant, plant tissue, or plant cell comprises a gene expression cassette comprising a 5′-UTR, wherein the 5′-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:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, or SEQ ID NO:38. In an embodiment, a gene expression cassette comprises a ubiquitin intron that is operably linked to a promoter, wherein the promoter is a Panicum virgatum, Brachypodium distachyon or Setaria italica ubiquitin promoter, or a promoter that originates from a plant (e.g., Zea mays ubiquitin 1 promoter), a virus (e.g., Cassava vein mosaic virus promoter) or a bacteria (e.g., Agrobacterium tumefaciens delta mas). In an embodiment, a plant, plant tissue, or plant cell comprises a gene expression cassette comprising a ubiquitin 5′-UTR that is operably linked to a transgene. In an illustrative embodiment, a plant, plant tissue, or plant cell comprising a gene expression cassette comprising a ubiquitin 5′-UTR that is operably linked to a transgene, wherein the transgene can be an insecticidal resistance transgene, an herbicide tolerance transgene, a nitrogen use efficiency transgene, a water us efficiency transgene, a nutritional quality transgene, a DNA binding transgene, a selectable marker transgene, or combinations thereof.
In an embodiment, a plant, plant tissue, or plant cell comprises a gene expression cassette comprising a ubiquitin promoter and a ubiquitin 3′-UTR. In an embodiment, a plant, plant tissue, or plant cell comprises a ubiquitin promoter and 3′-UTR can each be independently a Panicum virgatum, Brachypodium distachyon or Setaria italica ubiquitin promoter and a Panicum virgatum, Brachypodium distachyon or Setaria italica ubiquitin promoter. In an embodiment, a plant, plant tissue, or plant cell comprises a gene expression cassette comprising a) a promoter, wherein the promoter is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, or 100% identical to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:35 and b) 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:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:36.
In an embodiment, a plant, plant tissue, or plant cell comprises a gene expression cassette comprising a ubiquitin promoter, ubiquitin 5′-UTR, ubiquitin intron, and a ubiquitin 3′-UTR that are operably linked to a transgene. The promoter, intron, 5′-UTR, and 3′-UTR can be operably linked to different transgenes within a gene expression cassette when a gene expression cassette includes two or more transgenes. In an illustrative embodiment, a gene expression cassette comprises a ubiquitin promoter that is operably linked to a transgene, wherein the transgene can be an insecticidal resistance transgene, an herbicide tolerance transgene, a nitrogen use efficiency transgene, a water us efficiency transgene, a nutritional quality transgene, a DNA binding transgene, a selectable marker transgene, or combinations thereof. In an illustrative embodiment, a gene expression cassette comprises a ubiquitin intron that is operably linked to a transgene, wherein the transgene can be an insecticidal resistance transgene, an herbicide tolerance transgene, a nitrogen use efficiency transgene, a water us efficiency transgene, a nutritional quality transgene, a DNA binding transgene, a selectable marker transgene, or combinations thereof. In an embodiment, a gene expression cassette comprises a ubiquitin intron that is operably linked to a promoter, wherein the promoter is a Panicum virgatum, Brachypodium distachyon or Setaria italica ubiquitin promoter, or a promoter that originates from a plant (e.g., Zea mays ubiquitin 1 promoter), a virus (e.g., Cassava vein mosaic virus promoter) or a bacteria (e.g., Agrobacterium tumefaciens delta mas). In an illustrative embodiment, a gene expression cassette comprises a ubiquitin 5′-UTR that is operably linked to a transgene, wherein the transgene can be an insecticidal resistance transgene, an herbicide tolerance transgene, a nitrogen use efficiency transgene, a water us efficiency transgene, a nutritional quality transgene, a DNA binding transgene, a selectable marker transgene, or combinations thereof. In an embodiment, a gene expression cassette comprises a ubiquitin 5′-UTR that is operably linked to a promoter, wherein the promoter is a Panicum virgatum, Brachypodium distachyon or Setaria italica ubiquitin promoter, or a promoter that originates from a plant (e.g., Zea mays ubiquitin 1 promoter), a virus (e.g., Cassava vein mosaic virus promoter) or a bacteria (e.g., Agrobacterium tumefaciens delta mas). In an illustrative embodiment, a gene expression cassette comprises a ubiquitin 3′-UTR that is operably linked to a transgene, wherein the 3′-UTR can be an insecticidal resistance transgene, an herbicide tolerance transgene, a nitrogen use efficiency transgene, a water us efficiency transgene, a nutritional quality transgene, a DNA binding transgene, a selectable marker transgene, or combinations thereof.
In an embodiment, a plant, plant tissue, or plant cell comprises a vector comprising a ubiquitin promoter, 5′-UTR, intron, and/or 3′-UTR as disclosed herein. In an embodiment, a plant, plant tissue, or plant cell comprises a vector comprising a ubiquitin promoter, 5′-UTR, intron, and/or 3′-UTR as disclosed herein operably linked to a non-ubiquitin transgene. In an embodiment, a plant, plant tissue, or plant cell comprises a vector comprising a gene expression cassette as disclosed herein. In an embodiment, a vector can be a plasmid, a cosmid, a bacterial artificial chromosome (BAC), a bacteriophage, or a virus.
In accordance with one embodiment a plant, plant tissue, or plant cell is provided wherein the plant, plant tissue, or plant cell comprises a non-endogenous ubiquitin derived promoter sequence operably linked to a transgene, wherein the ubiquitin derived promoter sequence comprises a sequence SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:35 or a sequence having 90. 95, 98 or 99% sequence identity with SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:35. In one embodiment a plant, plant tissue, or plant cell is provided wherein the plant, plant tissue, or plant cell comprises SEQ ID NO: 3, or a sequence that has 90% sequence identity with SEQ ID NO: 3 operably linked to a non-ubiquitin transgene. In one embodiment the plant, plant tissue, or plant cell is a dicotyledonous or monocotyledonous plant or a cell or tissue derived from a dicotyledonous or monocotyledonous plant. In one embodiment the plant is selected from the group consisting of maize, wheat, rice, sorghum, oats, rye, bananas, sugar cane, soybean, cotton, sunflower, and canola. In one embodiment the plant is Zea mays. In accordance with one embodiment the plant, plant tissue, or plant cell comprises SEQ ID NO: 3, SEQ ID NO: 17 or a sequence having 90. 95, 98 or 99% sequence identity with SEQ ID NO: 3 or SEQ ID NO: 17 operably linked to a non-ubiquitin transgene. 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: 3, SEQ ID NO: 17 or a sequence having 90. 95, 98 or 99% sequence identity with SEQ ID NO: 3 or SEQ ID NO: 17. In accordance with one embodiment the gene construct comprising non-endogenous ubiquitin derived promoter sequence operably linked to a transgene is incorporated into the genome of the plant, plant tissue, or plant cell.
In one embodiment a non-Setaria plant, plant tissue, or plant cell is provided comprising SEQ ID NO: 3, or a sequence that has 90, 95, 98 or 99% sequence identity with SEQ ID NO: 3, operably linked to a transgene. In accordance with one embodiment the non-Setaria plant, plant tissue, or plant cell is a dicotyledonous or monocotyledonous plant or plant cell or tissue derived from a dicotyledonous or monocotyledonous plant. In one embodiment the plant is selected from the group consisting of maize, wheat, rice, sorghum, oats, rye, bananas, sugar cane, soybean, cotton, sunflower, and canola. In one embodiment the plant is Zea mays. In accordance with one embodiment the promoter sequence operably linked to a transgene is incorporated into the genome of the plant, plant tissue, or plant cell. In one embodiment the plant, plant tissue, or plant cell further comprises a 5′ untranslated sequence comprising SEQ ID NO: 13 or a sequence that has 90% sequence identity with SEQ ID NO: 13, wherein the 5′ untranslated sequence is inserted between, and operably linked to, said promoter and said transgene. In one embodiment the plant, plant tissue, or plant cell further comprises a 5′ untranslated sequence comprising SEQ ID NO: 14 or a sequence that has 90% sequence identity with SEQ ID NO: 14, wherein the 5′ untranslated sequence is inserted between, and operably linked to, said promoter and said transgene. In a further embodiment the plant, plant tissue, or plant cell further comprises an intron sequence inserted after the 5′ untranslated sequence. In one embodiment the intron sequence is an intron sequence isolated from a ubiquitin gene of Panicum virgatum, Brachypodium distachyon, or Setaria italica. In one embodiment the sequence comprises or consists of SEQ ID NO: 9. In one embodiment the sequence comprises or consists of SEQ ID NO: 10.
In one embodiment a non-Setaria plant, plant tissue, or plant cell is provided that comprises SEQ ID NO: 3, or a sequence that has 90. 95, 98 or 99% sequence identity with SEQ ID NO: 3, operably linked to the 5′ end of a transgene and a 3′ untranslated sequence comprising SEQ ID NO: 6 or a sequence that has 90% sequence identity with SEQ ID NO: 6, wherein the 3′ untranslated sequence is operably linked to said transgene. In accordance with one embodiment the non-Setaria plant, plant tissue, or plant cell is a dicotyledonous or monocotyledonous plant or is a plant issue or cell derived from a dicotyledonous or monocotyledonous plant. In one embodiment the plant is selected from the group consisting of maize, wheat, rice, sorghum, oats, rye, bananas, sugar cane, soybean, cotton, sunflower, and canola. In one embodiment the plant is Zea mays. In accordance with one embodiment the promoter sequence operably linked to a transgene is incorporated into the genome of the plant, plant tissue, or plant cell. In one embodiment the plant, plant tissue, or plant cell further comprises a 5′ untranslated sequence comprising SEQ ID NO: 13 or 14 or a sequence that has 90% sequence identity with SEQ ID NO: 13 or 14, wherein the 5′ untranslated sequence is inserted between, and operably linked to, said promoter and said transgene. In a further embodiment the plant, plant tissue, or plant cell further comprises an intron sequence inserted after the 5′ untranslated sequence. In one embodiment the intron sequence is an intron sequence isolated from a ubiquitin gene of Panicum virgatum, Brachypodium distachyon, or Setaria italica. In one embodiment the 5′ untranslated sequence consists of SEQ ID NO: 13. In one embodiment the 5′ untranslated sequence consists of SEQ ID NO: 13.
In one embodiment a non-Setaria plant, plant tissue, or plant cell is provided that comprises SEQ ID NO: 17, or a sequence having 90% sequence identity with SEQ ID NO: 17 operably linked to a transgene. In one embodiment a non-Setaria plant, plant tissue, or plant cell is provided that comprises SEQ ID NO: 40, or a sequence having 90% sequence identity with SEQ ID NO: 40 operably linked to a transgene. In one embodiment a non-Setaria plant, plant tissue, or plant cell is provided that comprises SEQ ID NO: 41, or a sequence having 90% sequence identity with SEQ ID NO: 41 operably linked to a transgene. In one embodiment a non-Setaria plant, plant tissue, or plant cell is provided that comprises SEQ ID NO: 42, or a sequence having 90% sequence identity with SEQ ID NO: 42 operably linked to a transgene. In one embodiment a non-Setaria plant, plant tissue, or plant cell is provided that comprises a promoter operably linked to a transgene, wherein the promoter consists of SEQ ID NO: 17, or a sequence having 90% sequence identity with SEQ ID NO: 17. In a further embodiment non-Setaria plant, plant tissue, or plant cell further comprises a 3′ untranslated sequence of a ubiquitin gene of Panicum virgatum, Brachypodium distachyon, or Setaria italica. In one embodiment the 3′ untranslated sequence comprises or consists of SEQ ID NO: 6 or a sequence that has 90% sequence identity with SEQ ID NO: 6, wherein the 3′ untranslated sequence is operably linked to 3′ end of the transgene.
In an embodiment, a plant, plant tissue, or plant cell according to the methods disclosed herein can be a monocotyledonous plant. The monocotyledonous plant, plant tissue, or plant cell can be, but not limited to corn, rice, wheat, sugarcane, barley, rye, sorghum, orchids, bamboo, banana, cattails, lilies, oat, onion, millet, and triticale.
In an embodiment, a plant, plant tissue, or plant cell according to the methods disclosed herein can be a dicotyledonous plant. The dicotyledonous plant, plant tissue, or plant cell can be, but not limited to rapeseed, canola, indian mustard, ethiopian mustard, soybean, sunflower, and cotton.
With regard to the production of genetically modified plants, methods for the genetic engineering of plants are well known in the art. For instance, numerous methods for plant transformation have been developed, including biological and physical transformation protocols for dicotyledonous plants as well as monocotyledonous plants (e.g., Goto-Fumiyuki et al., Nature Biotech 17:282-286 (1999); Miki et al., Methods in Plant Molecular Biology and Biotechnology, Glick, B. R. and Thompson, J. E. Eds., CRC Press, Inc., Boca Raton, pp. 67-88 (1993)). In addition, vectors and in vitro culture methods for plant cell or tissue transformation and regeneration of plants are available, for example, in Gruber et al., Methods in Plant Molecular Biology and Biotechnology, Glick, B. R. and Thompson, J. E. Eds., CRC Press, Inc., Boca Raton, pp. 89-119 (1993).
One of skill in the art will recognize that after the exogenous sequence is stably incorporated in transgenic plants and confirmed to be operable, it can be introduced into other plants by sexual crossing. Any of a number of standard breeding techniques can be used, depending upon the species to be crossed.
A transformed plant cell, callus, tissue or plant may be identified and isolated by selecting or screening the engineered plant material for traits encoded by the marker genes present on the transforming DNA. For instance, selection can be performed by growing the engineered plant material on media containing an inhibitory amount of the antibiotic or herbicide to which the transforming gene construct confers resistance. Further, transformed cells can also be identified by screening for the activities of any visible marker genes (e.g., the yfp, gfp, β-glucuronidase, luciferase, B or Cl genes) that may be present on the recombinant nucleic acid constructs. Such selection and screening methodologies are well known to those skilled in the art.
Physical and biochemical methods also may be used to identify plant or plant cell transformants containing inserted gene constructs. These methods include but are not limited to: 1) Southern analysis or PCR amplification for detecting and determining the structure of the recombinant DNA insert; 2) Northern blot, S1 RNase protection, primer-extension or reverse transcriptase-PCR amplification for detecting and examining RNA transcripts of the gene constructs; 3) enzymatic assays for detecting enzyme or ribozyme activity, where such gene products are encoded by the gene construct; 4) Next Generation Sequencing analysis; 5) protein gel electrophoresis, Western blot techniques, immunoprecipitation, or enzyme-linked immunoassays (ELISA), where the gene construct products are proteins. Additional techniques, such as in situ hybridization, enzyme staining, and immunostaining, also may be used to detect the presence or expression of the recombinant construct in specific plant organs and tissues. The methods for doing all these assays are well known to those skilled in the art.
Effects of gene manipulation using the methods disclosed herein can be observed by, for example, northern blots of the RNA (e.g., mRNA) isolated from the tissues of interest. Typically, if the mRNA is present or the amount of mRNA has increased, it can be assumed that the corresponding transgene is being expressed. Other methods of measuring gene and/or encoded polypeptide activity can be used. Different types of enzymatic assays can be used, depending on the substrate used and the method of detecting the increase or decrease of a reaction product or by-product. In addition, the levels of polypeptide expressed can be measured immunochemically, i.e., ELISA, RIA, EIA and other antibody based assays well known to those of skill in the art, such as by electrophoretic detection assays (either with staining or western blotting). As one non-limiting example, the detection of the AAD-1 (aryloxyalkanoate dioxygenase; see WO 2005/107437) and PAT (phosphinothricin-N-acetyl-transferase), EC 2.3.1.183) proteins using an ELISA assay is described in U.S. Patent Publication No. 20090093366 which is herein incorporated by reference in its entirety. The transgene may be selectively expressed in some cell types or tissues of the plant or at some developmental stages, or the transgene may be expressed in substantially all plant tissues, substantially along its entire life cycle. However, any combinatorial expression mode is also applicable.
The present disclosure also encompasses seeds of the transgenic plants described above wherein the seed has the transgene or gene construct. The present disclosure further encompasses the progeny, clones, cell lines or cells of the transgenic plants described above wherein said progeny, clone, cell line or cell has the transgene or gene construct.
While the invention has been described with reference to specific methods and embodiments, it will be appreciated that various modifications and changes may be made without departing from the invention.
EXAMPLE 1
Transformation of Agrobacterium tumefaciens 
The binary expression vectors were transformed into Agrobacterium tumefaciens strain DAt13192 (RecA minus ternary strain) (Int'l. Pat. Pub. No. WO2012016222). Bacterial colonies were isolated, and binary plasmid DNA was isolated and confirmed via restriction enzyme digestion.
Corn Transformation
Agrobacterium Culture Initiation. Agrobacterium cultures were streaked from glycerol stocks onto Agrobacterium (AB) minimal medium (as disclosed in WO 2013090734, the disclosure of which is incorporated herein by reference) and incubated at 20° C. in the dark for 3 days. Agrobacterium cultures were then streaked onto a plate of YEP (see WO 2013090734) medium and incubated at 20° C. in the dark for 1 day.
On the day of the experiment, a mixture of inoculation medium (see WO 2013090734) and acetosyringone were prepared in a volume appropriate to the number of bacterial strains comprising plant transformation constructs in the experiment. Inoculation medium was pipetted into a sterile, disposable 250 ml flask. Next, a 1 M stock solution of acetosyringone in 100% dimethyl sulfoxide was added to the flask containing inoculation medium in a volume appropriate to make a final acetosyringone concentration of 200 μM. The required volumes of Inoculation medium and 1 M acetosyringone stock solution are listed in TABLE 1.







TABLE 1







The amount of inoculation medium/acetosyringone mixture to


make according to the number of constructs being prepared









Number of




constructs to
Inoculation
1M acetosyringone


prepare
medium (mL)
stock (μL)












1
50
10


2
100
20


3
150
30


4
200
40


5
250
50









For each construct, 1-2 loops of Agrobacterium from the YEP plate were suspended in 15 ml of the inoculation medium/acetosyringone mixture inside a sterile, disposable 50 ml centrifuge tube, and the optical density of the solution at 600 nm (OD600) was measured in a spectrophotometer. The suspension was then diluted down to 0.25-0.35 OD600 using additional inoculation medium/acetosyringone mixture. The tube of Agrobacterium suspension was then placed horizontally on a platform shaker set at about 75 rpm at room temperature and incubated between 1 and 4 hours before use.
Ear sterilization and embryo isolation. Ears from Zea mays cultivar B104 were harvested 10-12 days post pollination. Harvested ears were de-husked and surface-sterilized by immersion in a 20% solution of commercial bleach (Ultra Clorox® Germicidal Bleach, 6.15% sodium hypochlorite) and two drops of Tween® 20, for 20 minutes, followed by three rinses in sterile, deionized water inside a laminar flow hood. Immature zygotic embryos (1.8-2.2 mm long) were aseptically excised from each ear and distributed into one or more micro-centrifuge tubes containing 2.0 ml of Agrobacterium suspension into which 2 μl of 10% Break-Thru® S233 surfactant had been added.
Agrobacterium co-cultivation. Upon completion of the embryo isolation activity, the tube of embryos was closed and placed on a rocker platform for 5 minutes. The contents of the tube were then poured out onto a plate of co-cultivation medium, and the liquid Agrobacterium suspension was removed with a sterile, disposable transfer pipette. The co-cultivation plate containing embryos was placed at the back of the laminar flow hood with the lid ajar for 30 minutes; after which time the embryos were oriented with the scutellum facing up using a microscope. The co-cultivation plate with embryos was then returned to the back of the laminar flow hood with the lid ajar for a further 15 minutes. The plate was then closed, sealed with 3M® Micropore® tape, and placed in an incubator at 25° C. with 24 hours/day light at approximately 60 μmol m−2 s−1 light intensity
Callus Selection and Regeneration of Transgenic Events. Following the co-cultivation period, embryos were transferred to Resting medium (see WO 2013090734). No more than 36 embryos were moved to each plate. The plates were placed in clear boxes and incubated at 27° C. with 24 hours/day light at approximately 50 μmol m−2 s−1 light intensity for 7-10 days. Callused embryos were then transferred onto Selection I medium (see WO 2013090734). No more than 18 callused embryos were moved to each plate of Selection I. The plates were placed in clear boxes and incubated at 27° C. with 24 hours/day light at approximately 50 μmol m−2 s−1 light intensity for 7 days. Callused embryos were then transferred to Selection II medium (see WO 2013090734). No more than 12 callused embryos were moved to each plate of Selection II media. The plates were placed in clear boxes and incubated at 27° C. with 24 hours/day light at approximately 50 μmol m−2 s−1 light intensity for 14 days.
At this stage resistant calli were moved to Pre-Regeneration medium (see WO 2013090734). No more than 9 calli were moved to each plate of Pre-Regeneration media. The plates were placed in clear boxes and incubated at 27° C. with 24 hours/day light at approximately 50 μmol m−2 s−1 light intensity for 7 days. Regenerating calli were then transferred to Regeneration medium in PhytatraysTM (see WO 2013090734). and incubated at 28° C. with 16 hours light/8 hours dark per day at approximately 150 μmol m−2 s−1 light intensity for 7-14 days or until shoots develop. No more than 5 calli were placed in each PhytatrayTM. Small shoots with primary roots were then isolated and transferred to Shoot Elongation medium (see WO 2013090734). Rooted plantlets about 6 cm or taller were transplanted into soil and moved out to a growth chamber for hardening off.
YFP Transient expression. Transient YFP expression was observed in transformed embryos and after 3 days of co-cultivation with Agrobacterium. The embryos were observed under a stereomicroscope (Leica Microsystems, Buffalo Grove, Ill.) using a YFP filter and 500 nm light source.
Transfer and Establishment of T0 Plants in the Greenhouse. Transgenic plants were transferred on a regular basis to the greenhouse. Plants were transplanted from PhytatraysTM to small pots (T. O. Plastics, 3.5′′ SVD, 700022C) filled with growing media (Premier Tech Horticulture, ProMix BX, 0581 P) and covered with humidomes to help acclimate the plants. Plants were placed in a Conviron growth chamber (28° C./24° C., 16-hour photoperiod, 50-70% RH, 200 μmol m−2 s−1 light intensity) until reaching V3-V4 stage. This aided in acclimating the plants to soil and harsher temperatures. Plants were then moved to the greenhouse (Light Exposure Type: Photo or Assimilation; High Light Limit: 1200 μmol m−2 s−1 photosynthetically active radiation (PAR); 16-hour day length; 27° C. Day/24° C. Night) and transplanted from the small pots to 5.5 inch pots. Approximately 1-2 weeks after transplanting to larger pots plants were sampled for bioassay. One plant per event was assayed.
EXAMPLE 2
Identification of the Promoters
The maize ubiquitin coding sequence was BLASTx searched in the Phytozome (Goodstein et al., 2012) database using Brachypodium distachyon and Setaria italica as target genomes.







Maize Ubiquitin (ZM Ubi1) Coding Sequence







(SEQ ID NO: 18)







ATGCAGATCTTTGTGAAAACCCTGACTGGCAAGACTATCACCCTCGAGG


 


TGGAGTCGTCTGACACCATTGACAACGTTAAGGCCAAGATCCAGGACAA


 


GGAGGGCATCCCCCCAGACCAGCAGCGGCTCATCTTTGCTGGCAAACA


 


GCTTGAGGACGGGCGCACGCTTGCTGACTACAACATCCAGAAGGAGAGC


 


ACCCTCCACCTTGTGCTCCGTCTCAGGGGAGGCATGCAGATCTTTGTGA


 


AAACCCTGACCGGCAAGACTATCACCCTCGAGGTGGAGTCCTCTGACAC


 


CATTGACAACGTCAAGGCCAAGATCCAGGACAAGGAGGGCATCCCTCCA


 


GACCAGCAGCGGCTCATCTTTGCTGGGAAGCAGCTTGAGGACGGGCGCA


 


CGCTTGCCGACTACAACATCCAGAAGGAGAGCACCCTCCACTTGGTGCT


 


GCGCCTCAGGGGAGGCATGCAGATCTTCGTGAAGACCCTGACCGGCAAG


 


ACTATCACCCTCGAGGTGGAGTCTTCAGACACCATCGACAACGTCAAGG


 


CCAAGATCCAGGACAAGGAGGGCATTCCCCCAGACCAGCAGCGGCTCAT


 


CTTTGCTGGAAAGCAGCTTGAGGACGGGCGCACGCTTGCCGACTACAAC


 


ATCCAGAAGGAGAGCACCCTCCACTTGGTGCTGCGCCTCAGGGGAGGCA


 


TGCAGATCTTCGTGAAGACCCTGACCGGCAAGACTATCACCCTCGAGGT


 


GGAGTCTTCAGACACCATCGACAATGTCAAGGCCAAGATCCAGGACAAG


 


GAGGGCATCCCACCGGACCAGCAGCGTTTGATCTTCGCTGGCAAGCAGC


 


TGGAGGATGGCCGCACCCTTGCGGATTACAACATCCAGAAGGAGAGCAC


 


CCTCCACCTGGTGCTCCGTCTCAGGGGTGGTATGCAGATCTTTGTGAAG


 


ACACTCACTGGCAAGACAATCACCCTTGAGGTGGAGTCTTCGGATACCA


 


TTGACAATGTCAAGGCCAAGATCCAGGACAAGGAGGGCATCCCACCCGA


 


CCAGCAGCGCCTCATCTTCGCCGGCAAGCAGCTGGAGGATGGCCGCACC


 


CTGGCGGATTACAACATCCAGAAGGAGAGCACTCTCCACCTGGTGCTCC


 


GCCTCAGGGGTGGCATGCAGATTTTTGTGAAGACATTGACTGGCAAGAC


 


CATCACCTTGGAGGTGGAGAGCTCTGACACCATTGACAATGTGAAGGCC


 


AAGATCCAGGACAAGGAGGGCATTCCCCCAGACCAGCAGCGTCTGATCT


 


TTGCGGGCAAGCAGCTGGAGGATGGCCGCACTCTCGCGGACTACAACAT


 


CCAGAAGGAGAGCACCCTTCACCTTGTTCTCCGCCTCAGAGGTGGTATG


 


CAGATCTTTGTAAAGACCCTGACTGGAAAAACCATAACCCTGGAGGTTG


 


AGAGCTCGGACACCATCGACAATGTGAAGGCGAAGATCCAGGACAAGGA


 


GGGCATCCCCCCGGACCAGCAGCGTCTGATCTTCGCCGGCAAACAGCTG


 


GAGGATGGCCGCACCCTAGCAGACTACAACATCCAAAAGGAGAGCACCC


 


TCCACCTTGTGCTCCGTCTCCGTGGTGGTCAGTAA






The protein alignments are shown in FIG. 1. Two sequences that aligned with the Zea mays Ubiquitin 1 protein were identified from Brachypodium distachyon. Only one sequence that aligned with the Zea mays Ubiquitin 1 protein was identified each from Setaria italic and Panicum virgatum. An approximately 2 kb DNA sequence upstream from a predicted translational start site (ATG) was determined to be the beginning of the putative promoter sequence and used for expression characterization. The polynucleotide sequence alignments of the novel promoters that were isolated from Panicum virgatum, Brachypodium distachyon and Setaria italica were aligned to the ZM Ubi1 promoter and found to share low levels of sequence similarity across the 2 kb DNA region (FIGS. 2A-C).
The UBI coding sequence and putative promoter for the Panicum virgatum, Brachypodium distachyon and Setaria italica ubiquitin genes are indicated in FIGS. 35-38.
EXAMPLE 3
Vector Construction
The four promoter sequences were commercially synthesized and incorporated into plasmid vectors as depicted in FIG. 3 (pDAB113091), FIG. 4 (pDAB113092), FIG. 5 (pDAB113066) and FIG. 22 (pDAB118238). Similarly four 3′UTR/transcription termination sequences were commercially synthesized and incorporated into plasmid vectors as depicted in FIG. 23 (pDAB118237), FIG. 24 (pDAB118207), FIG. 25 (pDAB118208) and FIG. 26 (pDAB118209). The sequences were flanked by 15-18 nucleotide homology fragments on both ends for seamless cloning (GeneArt® Seamless Cloning and Assembly Kit, Invitrogen, Carlsbad, Calif.) and type II restriction enzyme sites inserted for the isolation of promoter fragments. Seamless cloning compatible Zea mays Ubi1 promoter (Christensen and Quail (1996) Transgenic Research. 5; 213-218; Christensen et al., (1992) Plant Molecular Biology. 18; 675-689) or Oryzae sativa Actin promoter (McElroy et al., (1990) Plant Cell. 2; 163-71), and PhiYFP (Shagin et al., (2004) Mol Biol Evol. 21; 841-50) coding sequence comprising the ST-LS1 intron (Vancanneyt et al., (1990) Mol Gen Genet. 220; 245-50), and St PinII or native 3′-UTR (An et al., 1989 Plant Cell. 1; 115-22) fragments were obtained using PCR or typeII restriction enzymes. Finally, the promoter::PhiYFP::St PinII 3′-UTR fragments were assembled using seamless cloning to create transient expression vectors (FIG. 6, pDAB113103; FIG. 7, pDAB113104; FIG. 8, pDAB113105; FIG. 9, pDAB113106; and, FIG. 10, pDAB113107; FIG. 27, pDAB120403; FIG. 28, pDAB118234, FIG. 29, pDAB118235; and FIG. 30, pDAB118236) for transient expression testing. These transient expression vectors were integrated into a binary vector containing the Zm Ubi 1 promoter and AAD-1 coding sequence (International Patent Publication No. 2005107437) and Zm Lip 3′UTR (Paek et al., (1998) Molecules and Cells, 8(3): 336-342). The resulting binaries were confirmed via restriction enzyme digestion and sequencing reaction (FIG. 12, pDAB113117; FIG. 13, pDAB113118; FIG. 14, pDAB113119; FIG. 15, pDAB113120; FIG. 16, pDAB113121; FIG. 31, pDAB120400; FIG. 32, pDAB120404; FIG. 33, pDAB120401; and, FIG. 34, pDAB120402).
EXAMPLE 4
Transient Expression Testing
Transient expression was tested using particle bombardment of immature maize (B104) embryos. Forty embryos were used per treatment in a Petri plate for bombardment. YFP image analysis was done after overnight incubation of particle bombardment. FIG. 19 shows YFP expression levels obtained from the novel promoters. The data show that YFP expression levels obtained from the novel promoters (pDAB113103, pDAB113104, and pDAB113105) is comparable to the YFP expression levels obtained from the ZM Ubi1 promoter (pDAB113106) and the OS Act 1 promoter (pDAB113107) as visually observed under the microscope. Plant tissues were imaged on a Leica EL6000-mercury metal HalideTM microscope. Confocal and Differential Interference Contrast (DIC) images were captured using Chroma 42003-ZsYellow 1TM filters.
EXAMPLE 5
Transgene Copy Number Estimation Using Real Time TaqManTM PCR
The stable integration of the yfp transgene within the genome of the transgenic Z. mays plants was confirmed via a hydrolysis probe assay. Stably-transformed transgenic Z. mays plantlets that developed from the callus were obtained and analyzed to identify events that contained a low copy number (1-2 copies) of full-length T-strand inserts. Identified plantlets were advanced to the green house and grown.
The Roche Light Cycler480TM system was used to determine the transgene copy number. The method utilized a biplex TaqMan® reaction that employed oligonucleotides specific to the yfp gene and to the endogenous Z. mays reference gene, invertase (Genbank Accession No: U16123.1), in a single assay. Copy number and zygosity were determined by measuring the intensity of yfp-specific fluorescence, relative to the invertase-specific fluorescence, as compared to known copy number standards.
A yfp gene-specific DNA fragment was amplified with one TaqMan® primer/probe set containing a probe labeled with FAMTM fluorescent dye, and invertase was amplified with a second TaqMan® primer/probe set containing a probe labeled with HEXTM fluorescence (TABLE 2). The PCR reaction mixture was prepared as set forth in TABLE 3, and the gene-specific DNA fragments were amplified according to the conditions set forth in TABLE 4. Copy number and zygosity of the samples were determined by measuring the relative intensity of fluorescence specific for the reporter gene, yfp, to fluorescence specific for the reference gene, invertase, as compared to known copy number standards.







TABLE 2







Forward and reverse nucleotide primer


and fluorescent probes.










Primer/Probe
Sequence













PhiYFP v3
Forward Primer
(SEQ ID NO: 28)




CGTGTTGGGAAAGAACTTGGA


PhiYFP v3
Reverse Primer
(SEQ ID NO: 29)




CCGTGGTTGGCTTGGTCT


PhiYFP v3
Probe
(SEQ ID NO: 30)




5′FAM/CACTCCCCACTGCCT/




MGB_BHQ_1/3′


 


Invertase
Forward Primer
(SEQ ID NO: 31)




TGGCGGACGACGACTTGT


Invertase
Reverse Primer
(SEQ ID NO: 32)




AAAGTTTGGAGGCTGCCGT


Invertase
Probe
(SEQ ID NO: 33) 5′HEX/




CGAGCAGACCGCCGTGTACTT/




3BHQ_1/3′





(synthesized by Integrated DNA Technologies, Coralville, IA).













TABLE 3







Taqman ® PCR reaction mixture.











Working
Final
Volume


Component
Concentration
Concentration
(μl)













Water
—
—
0.5


Roche LightCyler
2X
1X
5


480 Probes Master


Mix











PhiYFP v3 F
10 μM
400
nM
0.4


PhiYFP v3 R
10 μM
400
nM
0.4


PhiYFP v3 Probe-
 5 μM
200
nM
0.4


FAM


Invertase F
10 μM
400
nM
0.4


Invertase R
10 μM
400
nM
0.4


Invertase Probe—
 5 μM
200
nM
0.4


Hex










Polyvinylpyrrolidone
10%
0.1%
0.1


(PVP)











Genomic DNA
Diluted BioCel
~10
ng/uL
2


template
DNA (~5 nglul)











Total reaction
—
—
10.0


volume
















TABLE 4







Thermocycler conditions for PCR amplification.












PCR Steps
Temp (° C.)
Time
No. of cycles
















Step-1
95
10 minutes
1



Step-2
95
10 seconds
40




58
35 seconds




72
 1 second



Step-3
40
seconds
1










Standards were created by diluting the vector, pDAB108706, into Z. mays B104 genomic DNA (gDNA) to obtain standards with a known relationship of pDAB108706:gDNA. For example, samples having one; two; and four cop(ies) of vector DNA per one copy of the Z. mays B104 gDNA were prepared. One and two copy dilutions of the pDAB108706 mixed with the Z. mays B104 gDNA standard were validated against a control Z. mays event that was known to be hemizygous, and a control Z. mays event that was known to be homozygous (Z. mays event 278; see PCT International Patent Publication No. WO 2011/022469 A2). A TaqMan® biplex assay that utilizes oligonucleotides specific to the AAD1 gene and oligonucleotides specific to the endogenous Z. mays reference gene, invertase, was performed by amplifying and detecting a gene-specific DNA fragment for AAD1 with one TaqMan® primer/probe set containing a probe labeled with FAM fluorescent dye, and by amplifying and detecting a gene-specific DNA fragment for invertase with a second TaqMan® primer/probe set containing a probe labeled with HEXTM fluorescence (TABLE 2). The AAD1 TaqMan® reaction mixture was prepared as set forth in TABLE 3, and the specific fragments were amplified according to the conditions set forth in TABLE 4.
The level of fluorescence that was generated for each reaction was analyzed using the Roche LightCycler® 480 Thermocycler according to the manufacturer's directions. The FAMTM fluorescent moiety was excited at an optical density of 465/510 nm, and the HEXTM fluorescent moiety was excited at an optical density of 533/580 nm. The copy number was determined by comparison of Target/Reference values for unknown samples (output by the LightCycler® 480) to Target/Reference values of four known copy number standards (Null, 1-Copy (hemi), 2-Copy (homo) and 4-Copy). Results from the transgene copy number analysis of transgenic plants obtained via transformation with different promoter constructs are shown in TABLE 5. Only plants with 1-2 copies of the yfp transgene were transferred to the greenhouse for further expression analyses.







TABLE 5







Transgene copy number estimation of the transgenic plants obtained


from promoter construct described herein and control constructs.












Number of Positive




Construct
Events
1-2 Copies of yfp







pDAB113117
32
17



pDAB113118
26
13



pDAB113119
30
16



pDAB113120
43
10



pDAB113121
36
19










EXAMPLE 6
Expression of Genes Operably Linked to Ubiquitin Promoters Protein Extraction
T0 plants were sampled at V4-5 using a leaf ELISA assays. Sample were collected in 96-well collection tube plate, and 4 leaf disks (paper hole punch size) were taken for each sample. Two 4.5 mm BBs and 200 μL extraction buffer [1×PBS supplemented with 0.05% Tween®-20 and 0.05% BSA (Millipore Probumin®, EMD Millipore Corp., Billerica, Mass.)] were added to each tube. For AAD1 extraction, the concentration of BSA was increased to 0.5%. Plates were processed in a KLECO bead mill at full speed for 3 minutes. Additional 200 μL of extraction buffer was added to each tube followed by inversion to mix. Plates were spun for 5 minutes at 3000 rpm. Supernatant was transferred to corresponding wells in a deep well 96 stored on ice.
YFP and AAD1 ELISA Procedure
Nunc® 96-well Maxi-Sorp Plates (Thermo Fisher Scientific Inc., Rockford, Ill.) were used for ELISA. Plates were coated with mouse monoclonal anti-YFP capture antibody (OriGene Technologies Inc., Rockville, Md.). The antibody was diluted in PBS (1 μg/mL) and 150 μL of diluted PBS was added per well. The plates were incubated overnight at 4° C. The overnight plates were kept at room temperature for 20-30 minutes before washing 4× with 350 μL of wash buffer [1×PBS supplemented with 0.05% Tween®-20 (Sigma-Aldrich, St. Louis, Mo.)]. Plates were blocked with 200 μL per well of blocking buffer [1×PBS supplemented with 0.05% Tween®-20 plus 0.5% BSA (Millipore Probumin®)] for a minimum of 1 hr at +37° C. followed by 4× washing with 350 μL of wash buffer (Tomtec QuadraWashTM 2, Tomtec, Inc., Hamden, Conn.).
For the YFP ELISA, Evrogen recombinant Phi-YFP 1 mg/mL (Axxora LLC, Farmingdale, N.Y.) was used as a standard. A 5-parameter fit standard curve (between the 1 ng/ml and 0.125 ng/ml Standards) was used to ensure all data fall in the linear portion of the curve. 100 μL of standard or sample was added to the well. A minimum 1:4 dilution of sample in the Assay Buffer was used. Plates were incubated for 1 hr at RT on plate shaker (250 rpm; Titer Plate shaker) followed by 4× washing with 350 μL of wash buffer (Tomtec QuadraWashTM 2). About 100 μL of 1 μg/mL Evrogen rabbit polyclonal anti-PhiYFP primary antibody (Axxora) was added to each well. Plates were incubated for 1 hr at room temperature on a plate shaker at 250 rpm followed by 4× washing with 350 μL of wash buffer (Tomtec QuadraWashTM 2). Next, 100 μL of anti-rabbit IgG HRP secondary antibody (Thermo Scientific) diluted 1:5000 in Blocking/Assay buffer, which was added to each well. Plates were incubated for 1 hr at room temperature on plate shaker at 250 rpm followed by 4× washes with 350 μL of wash buffer (Tomtec QuadraWashTM 2). 100 μL of Pierce 1 Step Ultra TMB ELISA (Thermo Scientific) substrate was added in the well with gentle shaking for 10 minutes. Reaction was stopped by adding 50 μL of 0.4N H2SO4. Absorbance was read at 450 nm with a 650 nm reference filter.
AAD1 expression levels were determined by ELISAs using kits from Acadia BioSciences (Portland, Me.). The ELISAs were performed using multiple dilutions of the extracts and using the reagents and instructions provided by the supplier. The protein levels were normalized using total soluble protein assay, performed using the 660 nm protein assay reagent supplied by Thermo Scientific and following the supplier's instructions.
EXAMPLE 7
Whole Plant YFP Image Analysis Exemplifying Stable Expression of Genes Operably Linked to Ubiquitin Promoters
Whole plants that contained a low copy number of the binary plasmid were grown in a greenhouse. Plant tissues were imaged on a Leica EL6000-mercury metal HalideTM microscope. Confocal and Differential Interference Contrast (DIC) images were captured using Chroma 42003-ZsYellow 1TM filters. Representative examples of stable expression of YFP in callus and root tissue of transgenic T0 maize plants obtained from Z. mays embryos transformed with the Brachypodium distachyon Ubiquitin1 C, Brachypodium distachyon Ubiquitin1, and Setaria italica ubiquitin 2 promoters described herein are presented in FIG. 20 to FIG. 21, respectively. The promoters drove robust expression of the yfp coding sequences both in callus (FIG. 20) and root (FIG. 21) plant tissues.
EXAMPLE 8
Whole Plant T0 Stable Expression of Genes Operably Linked to Ubiquitin Promoters
Additional data was produced from an ELISA analysis of the expressed YFP protein. The ELISA analysis further confirmed that the novel promoters drove robust expression of a transgene. The quantitative measurements of YFP protein obtained from transgenic plants comprising novel promoter constructs are shown in FIG. 17 and TABLE 6. The data show that expression of YFP protein in the plants containing the novel promoters (pDAB113117, pDAB113118, and pDAB113119) is several fold higher that YFP expression obtained from the Os Act1 (Rice Actin1) promoter (pDAB113120). Comparatively, FIG. 18 and TABLE 7 show that similar level of AAD1 expression was obtained from all the constructs. This is expected because AAD1 is driven by the Zm Ubi1 promoter for all of the constructs.







TABLE 6







Cross Species Ubiquitin Promoter T0 Leaf YFP expression











Construct
Mean (ng/mg TSP)
Statistical significance















pDAB113121
144.00173
A



pDAB113118
92.37256
AB



pDAB113119
65.30393
B



pDAB113117
55.24345
B



pDAB113120
12.77181
B







Levels not connected by same letter are significantly different













TABLE 7







Cross Species Ubiquitin Promoter T0 Leaf AAD1 expression











Construct
Mean (ng/mg TSP)
Statistical significance















pDAB113121
119.06932
A



pDAB113118
109.19796
A



pDAB113119
96.29021
A



pDAB113117
85.40412
A



pDAB113120
83.81594
A







Levels not connected by same letter are significantly different






EXAMPLE 9
Whole Plant T1 Stable Expression of Genes Operable Linked to Ubiquitin Promoters and 3′UTRs
T0 single transgene copy plants were backcrossed to wild type B104 corn plants to obtain T1 seed. Hemizygous T1 plants were used for analysis. Five events per construct and 5-10 plants per event for V4 and V12 leaf expression. Three events per construct and 3 plants per event were used for the other tissue type expression. Zygosity analysis was done for AAD1/YFP.
The quantitative measurements of YFP protein obtained from leaf tissue of T1 transgenic plants comprising novel promoter constructs are shown in TABLE 8. The data confirmed the T0 leaf expression results and further showed that consistent high expression of YFP protein was obtained in the V4, V12 and R3 leaf tissue of the plants containing the novel promoters (pDAB113117, pDAB113118, and pDAB113119). TABLE 8 also shows that there was several fold increase in the expression of YFP protein when this novel promoters were used in combination with their native 3′UTRs (pDAB120400, pDAB120401, and pDAB120402) instead of PinII 3′UTR (pDAB113117, pDAB113118, and pDAB113119). YFP protein expression was detected from the plants containing construct pDAB120404 confirming that novel promoter and 3′UTR used in this construct drive expression of a transgene.







TABLE 8







Cross Species Ubiquitin Promoter and 3′UTR T1 Leaf Expression









Mean YFP (ng/mg TSP)














V12



Construct
Event
V4 Leaf
Leaf
R3 Leaf














pDAB113117
pDAB113117[1]-006
44.0
169.4
2108.3


pDAB113117
pDAB113117[1]-007
44.8
181.4
2582.7


pDAB113117
pDAB113117[1]-008
79.6
322.8
4096.3


pDAB113117
pDAB113117[1]-019
74.3
369.1
3420.3


pDAB113117
pDAB113117[1]-028
34.2
168.2
2164.1


pDAB113118
pDAB113118[1]-005
33.6
148.8
2094.7


pDAB113118
pDAB113118[1]-007
54.9
180.1
2171.4


pDAB113118
pDAB113118[1]-010

138.0
2748.2


pDAB113118
pDAB113118[1]-023
46.2
156.6
2216.8


pDAB113118
pDAB113118[1]-025
41.7
132.9
2071.4


pDAB113119
pDAB113119[1]-001
133.1
436.0
6744.0


pDAB113119
pDAB113119[1]-005
49.2
138.6
1772.9


pDAB113119
pDAB113119[1]-011
54.6
133.9
1415.5


pDAB113119
pDAB113119[1]-013

129.1
1807.7


pDAB113119
pDAB113119[1]-028
38.5
129.9
1632.8


pDAB113120
pDAB113120[1]-005
9.8
69.6
493.5


pDAB113120
pDAB113120[1]-010
24.3
74.5
638.3


pDAB113120
pDAB113120[1]-014
17.2
79.7
552.4


pDAB113120
pDAB113120[1]-023
13.2
55.4
372.2


pDAB113120
pDAB113120[1]-032
12.5
69.6
233.7


pDAB113121
pDAB113121[1]-008
327.9


pDAB113121
pDAB113121[1]-011
166.2
271.2
4472.6


pDAB113121
pDAB113121[1]-018
128.2
362.0
7116.3


pDAB113121
pDAB113121[1]-023
112.2
309.1
6813.7


pDAB113121
pDAB113121[1]-026
118.7
311.7
6300.7


pDAB120400
pDAB120400[1]-001
640.8182


pDAB120400
pDAB120400[1]-002
339.24463


pDAB120400
pDAB120400[1]-004
943.96511


pDAB120400
pDAB120400[1]-007
1653.7402


pDAB120400
pDAB120400[1]-024
466.01906


pDAB120401
pDAB120401[1]-001
833.04373


pDAB120401
pDAB120401[1]-011
471.9103


pDAB120401
pDAB120401[1]-019
795.08285


pDAB120401
pDAB120401[1]-022
721.58288


pDAB120401
pDAB120401[1]-025
696.94286


pDAB120402
pDAB120402[1]-010
750.82185


pDAB120402
pDAB120402[1]-011
619.38603


pDAB120402
pDAB120402[1]-014
618.98144


pDAB120402
pDAB120402[1]-030
625.84385


pDAB120404
pDAB120404[1]-003
44.088479


pDAB120404
pDAB120404[1]-013
47.464389


pDAB120404
pDAB120404[1]-014
52.204801


pDAB120404
pDAB120404[1]-016
45.397854


pDAB120404
pDAB120404[1]-020
46.913279









High YFP protein expression was found in different tissue types including cob, husk, kernel, pollen, root, silk and stem sampled from the transgenic corn plants containing novel Ubiquitin Promoters driving YFP (Table 9). These data demonstrate that the novel promoters and 3′UTRs claimed here drive high constitutive expression of transgene in plants and would be useful for biotechnological applications.







TABLE 9







Cross Species Ubiquitin Promoter T1 Expression in Different Tissue Type









Mean YFP (ng/mg TSP)















Construct
Event
Cob
Husk
Kernel
Pollen
V12 Root
Silk
Stem


















pDAB113117
pDAB113117[1]-006
3452.1
1164.3
1341.2
397.1
2292.7
1405.0
7279.8


pDAB113117
pDAB113117[1]-007
2519.6
954.7
1410.9
414.3
2245.6
1974.3
6179.0


pDAB113117
pDAB113117[1]-019
8362.3
2280.8
2829.6
749.5
7112.4
4790.2
13044.1


pDAB113118
pDAB113118[1]-005
2801.6
620.9
886.3
782.2
1136.1
636.7
1953.6


pDAB113118
pDAB113118[1]-007
2339.2
524.9
725.1
376.1
1495.6
1271.0
2806.9


pDAB113118
pDAB113118[1]-023
1302.1
491.8
716.8
435.3
1193.0
829.1
1522.9


pDAB113119
pDAB113119[1]-011



399.7
1025.9
942.2
2475.6


pDAB113119
pDAB113119[1]-013
2238.1
572.0
1050.5
438.1
1311.2
539.7
2235.2


pDAB113119
pDAB113119[1]-028
2013.9
536.4
1061.4
450.0
1166.3
826.9
1912.5


pDAB113120
pDAB113120[1]-005
1166.8
310.5
514.0
1704.1
169.7
322.1
739.3


pDAB113120
pDAB113120[1]-023
1096.4
531.9
845.8
1433.9
268.4
572.2
877.9


pDAB113120
pDAB113120[1]-032
1344.1
587.8
985.1
1252.3
187.6
472.4
694.0


pDAB113121
pDAB113121[1]-011
6779.1
2942.3
3452.6
2022.6
5834.0
2881.7
7445.5


pDAB113121
pDAB113121[1]-023
4830.0
2689.8
1913.7
1641.8
2547.7
2453.8
8295.9


pDAB113121
pDAB113121[1]-026
8186.2
3889.3
4432.3
1432.0
2521.9
2182.5
7760.7









All references, including publications, patents, and patent applications, cited herein are hereby incorporated by reference to the extent they are not inconsistent with the explicit details of this disclosure, and are so incorporated to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein. The references discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention. The following examples are provided to illustrate certain particular features and/or embodiments. The examples should not be construed to limit the disclosure to the particular features or embodiments exemplified.

## Claims

1. A nucleic acid vector comprising a promoter, wherein
i) said promoter is operably linked to a polylinker sequence;
ii) said promoter is operably linked to a non-ubiquitin-coding sequence or
iii) said promoter is operably linked to a sequence comprising a polylinker sequence and a non-ubiquitin-coding sequence, wherein said promoter comprises SEQ ID NO: 3 or a sequence that has at least 99% sequence identity with SEQ ID NO: 3 and can initiate transcription of an operably linked coding sequence.

2. The nucleic acid vector of claim 1 wherein said promoter is less than 3kb in length.

3. The nucleic acid vector of claim 1 wherein said promoter consists of SEQ ID NO: 3.

4. The nucleic acid vector of claim 1 further comprising a sequence encoding a selectable marker.

5. The nucleic acid vector of claim 4 wherein said promoter is operably linked to a transgene.

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

7. The nucleic acid vector of claim 1 further comprising a 3′ untranslated sequence comprising SEQ ID NO: 6 or a sequence that has at least 99% sequence identity with SEQ ID NO: 6, wherein the 3′ untranslated sequence is operably linked to said polylinker or said coding sequence.

8. The nucleic acid vector of claim 1 further comprising a 5′ untranslated sequence comprising SEQ ID NO: 13 or a sequence that has at least 99% sequence identity with SEQ ID NO: 13, wherein the 5′ untranslated sequence is inserted between, and operably linked to, said promoter sequence and said polylinker or said coding sequence.

9. The nucleic acid vector of claim 1 further comprising a 5′ untranslated sequence comprising SEQ ID NO: 14 or a sequence that has at least 99% sequence identity with SEQ ID NO: 14, wherein the 5′ untranslated sequence is inserted between, and operably linked to, said promoter sequence and said polylinker or said coding sequence.

10. The nucleic acid vector of claim 9 further comprising an intron sequence inserted after the 5′ untranslated sequence.

11. The nucleic acid vector of claim 10 wherein the intron sequence comprises SEQ ID NO: 9 or SEQ ID NO: 10.

12. The nucleic acid vector of claim 1 wherein the promoter consists of a sequence selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 40, SEQ ID NO: 41 and SEQ ID NO: 42 or a sequence having at least 99% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 40, SEQ ID NO: 41 and SEQ ID NO: 42, said promoter being operably linked to said polylinker or said coding sequence, wherein said promoter can initiate transcription of the operably linked coding sequence.

13. The nucleic acid vector of claim 1 wherein the promoter consists of SEQ ID NO: 40 or SEQ ID NOL 42 wherein said promoter is operably linked to said polylinker or said coding sequence.

14. The nucleic acid vector of claim 1 wherein the promoter consists of SEQ ID NO: 17 or a sequence having at least 99% sequence identity with SEQ ID NO: 17, said promoter being operably linked to said polylinker or said coding sequence, wherein said promoter can initiate transcription of the operably linked coding sequence.

15. The nucleic acid vector of claim 14 further comprising a 3′ untranslated sequence comprising SEQ ID NO: 6 or a sequence that has at least 99% sequence identity with SEQ ID NO: 6, wherein the 3′ untranslated sequence is operably linked to said polylinker or said coding sequence.

16. A non-Setaria plant comprising a Setariapromoter, wherein said Setaria promoter comprises SEQ ID NO: 3, or a sequence that has at least 99% sequence identity with SEQ ID NO: 3 operably linked to a coding sequence wherein said Setaria promoter can initiate transcription of the operably linked coding sequence.

17. The plant of claim 16 wherein said plant is selected from the group consisting of maize, wheat, rice, sorghum, oats, rye, bananas, sugar cane, soybean, cotton, sunflower, and canola.

18. The plant of claim 16 wherein said plant is Zea mays.

19. The plant of claim 18 wherein the transgene is inserted into the genome of said plant.

20. The plant of claim 16 further comprising a 5′ untranslated sequence comprising SEQ ID NO: 13 or a sequence that has at least 99% sequence identity with SEQ ID NO: 13, wherein the 5′ untranslated sequence is inserted between, and operably linked to, said Setaria promoter and said transgene.

21. The plant of claim 16 further comprising a 5′ untranslated sequence comprising SEQ ID NO: 14 or a sequence that has at least 99% sequence identity with SEQ ID NO: 14, wherein the 5′ untranslated sequence is inserted between, and operably linked to, said Setaria promoter and said transgene.

22. The plant of claim 21 further comprising an operably linked intron sequence inserted after the 5′ untranslated sequence and before said transgene.

23. The plant of claim 22 wherein the intron sequence comprises SEQ ID NO: 9 or SEQ ID NO: 10.

24. The plant of claim 20 further comprising a 3′ untranslated sequence comprising SEQ ID NO: 6 or a sequence that has at least 99% sequence identity with SEQ ID NO: 6, wherein the 3′ untranslated sequence is operably linked to said transgene.

25. The plant of claim 16 wherein the Setaria promoter consists of SEQ ID NO: 40, SEQ ID NO: 42 or a sequence having at least 99% sequence identity with SEQ ID NO: 40 or SEQ ID NO: 42, wherein said Setaria promoter is operably linked to said transgene and can initiate transcription of said transgene.

26. The plant of claim 16 wherein the Setaria promoter consists of SEQ ID NO: 17, SEQ ID NO: 41 or a sequence having at least 99% sequence identity with SEQ ID NO: 17 or SEQ ID NO: 41 wherein said Setaria promoter is operably linked to said transgene.

27. The plant of claim 26 further comprising a 3′ untranslated sequence comprising SEQ ID NO: 6 or a sequence that has at least 99% sequence identity with SEQ ID NO: 6, wherein the 3′ untranslated sequence is operably linked to said transgene.

28. A nucleic acid vector comprising a transcription terminator, wherein
i) said terminator is operably linked to a polylinker sequence;
ii) said terminator is operably linked to a non-ubiquitin-encoding transgene; or
iii) said terminator is operably linked to a sequence comprising a polylinker sequence and a non-ubiquitin-encoding transgene, wherein said transcription terminator comprises SEQ ID NO: 6 or a sequence that has at least 99% sequence identity with SEQ ID NO: 6.

29. The nucleic acid vector of claim 28 wherein said transcription terminator is less than 1kb in length.

30. The nucleic acid vector of claim 29 wherein said transcription terminator consists of SEQ ID NO: 6.