# Patent

## Title
Regulatory polynucleotides and uses thereof

## Abstract

The present disclosure provides compositions and methods for regulating expression of transcribable polynucleotides in plant cells, plant tissues, and plants. Compositions include regulatory polynucleotide molecules capable of providing expression in plant tissues and plants. Methods for expressing polynucleotides in a plant cell, plant tissue, or plants using the regulatory polynucleotide molecules disclosed herein are also provided.

## Background
This application is a §371 national phase application of International Application Serial No. PCT/US2011/043197, filed Jul. 7, 2011, designating the United States and published in English, which claims the benefit of U.S. Provisional Application No. 61/362,959, filed Jul. 9, 2010, the entire contents of which are hereby incorporated herein by reference.


FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
This invention was made with Government support under Contract No. 0810649, awarded by the National Science Foundation, STTR. The Government has certain rights in this invention


REFERENCE TO SEQUENCE LISTING SUBMITTED VIA EFS-WEB
This application is being filed electronically via EFS-Web and includes an electronically submitted Sequence Listing in .txt format. The .txt file contains a sequence listing entitled “1390416.txt” created on Jul. 5, 2011 and is 34,031 bytes in size. The sequence listing contained in this .txt file is part of the specification and is hereby incorporated by reference herein in its entirety.
FIELD
The present invention relates to polynucleotide molecules for regulating expression of transcribable polynucleotides in cells (including plant tissues and plants) and uses thereof.
BACKGROUND
The development of transgenic plants having agronomically desirable characteristics often depends on the ability to control the spatial and temporal expression of the polynucleotide responsible for the desired trait. The control of the expression is largely dependent on the availability and use of regulatory control sequences that are responsible for the expression of the operably linked polynucleotide. Where expression in specific tissues or organs is desired, tissue-preferred regulatory elements may be used. Where expression in response to a stimulus is desired, inducible regulatory polynucleotides are the regulatory element of choice. In contrast, where continuous expression is desired throughout the cells of a plant, constitutive regulatory polynucleotides are utilized.
The proper regulatory elements typically must be present and be in the proper location with respect to the polynucleotide in order to obtain expression of the newly inserted transcribable polynucleotide in the plant cell. These regulatory elements may include a promoter region, various cis-elements, regulatory introns, a 5′non-translated leader sequence and a 3′ transcription termination/polyadenylation sequence.
Since the patterns of expression of transcribable polynucleotides introduced into a plant are controlled using regulatory elements, there is an ongoing interest in the isolation and identification of novel regulatory elements which are capable of controlling expression of such transcribable polynucleotides.
SUMMARY
In one aspect, an isolated regulatory polynucleotide is provided that comprises a polynucleotide molecule selected from the group consisting of: (a) a polynucleotide molecule comprising a nucleic acid molecule having a sequence selected from the group consisting of SEQ ID NOS: 1-22 that is capable of regulating transcription of an operably linked transcribable polynucleotide molecule; (b) a polynucleotide molecule having at least about 70% sequence identity to a sequence selected from the group consisting of SEQ ID NOS:1-22 that is capable of regulating transcription of an operably linked transcribable polynucleotide molecule; and (c) a fragment of the polynucleotide molecule of (a) or (b) capable of regulating transcription of an operably linked transcribable polynucleotide molecule. In some aspects, the isolated regulatory polynucleotide is capable of regulating constitutive transcription. The isolated regulatory polynucleotide may comprise an intron.
In another aspect, a recombinant polynucleotide construct is provided comprising a regulatory polynucleotide described herein operably linked to a heterologous transcribable polynucleotide molecule. The transcribable polynucleotide molecule may encode a protein of agronomic interest.
In other aspects, such a recombinant polynucleotide construct is used to provide a transgenic host cell comprising the recombinant polynucleotide construct and to provide a transgenic plant stably transformed with the recombinant polynucleotide construct. Seed produced by such transgenic plants are also provided.
In a further aspect, a chimeric polynucleotide molecule is provided that comprises:
(1) a first polynucleotide molecule selected from the group consisting of
(a) a polynucleotide molecule comprising a nucleic acid molecule having a sequence selected from the group consisting of SEQ ID NOS: 1-22 that is capable of regulating transcription of an operably linked transcribable polynucleotide molecule;
(b) a polynucleotide molecule having at least about 70% sequence identity to a sequence selected from the group consisting of SEQ ID NOS:1-22 that is capable of regulating transcription of an operably linked transcribable polynucleotide molecule; and
(c) a fragment of the polynucleotide molecule of (a) or (b) capable of regulating transcription of an operably linked transcribable polynucleotide molecule, and
(2) a second polynucleotide molecule capable of regulating transcription of an operably linked polynucleotide molecule, wherein the first polynucleotide molecule is operably linked to the second polynucleotide molecule.
In yet a further aspect, an isolated polynucleotide molecule is provided that comprises a regulatory element derived from SEQ ID NOS: 1-22, wherein the regulatory element is capable of regulating transcription of an operably linked transcribable polynucleotide molecule.
In another aspect, a method of directing expression of a transcribable polynucleotide molecule in a host cell is provided that comprises:
(a) introducing the recombinant nucleic acid construct described herein into a host cell to produce a transgenic host cell; and
(b) selecting a transgenic host cell exhibiting expression of the transcribable polynucleotide molecule.
In a further aspect, a method of directing expression of a transcribable polynucleotide molecule in a plant is provided that comprises:
(a) introducing the recombinant nucleic acid construct described herein into a plant cell;
(b) regenerating a plant from the plant cell; and
(c) selecting a transgenic plant exhibiting expression of the transcribable polynucleotide molecule.



BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1-22 each provide the nucleotide sequence of a regulatory polynucleotide corresponding to the Arabidopsis gene having the accession number specified in the Figure. Where the regulatory polynucleotide has been modified to include the first intron from the coding sequence of the specified gene attached at the 3′ end of the 5′ UTR, the Figure indicates the gene accession number followed by the indicia “+first intron”. Where the regulatory polynucleotide has been modified to include all of, or a portion of, a 35S-minimal promoter, the Figures indicate the gene accession number followed by the indicia “+35S minimal promoter”. The figures are annotated to indicate one transcription start site (capital letter in bold), the endogenous 5′-UTR intron sequences (double underlining), the first intron from the coding sequence (single underlining), the 35S -minimal promoter from base −46 to base −1 as measured from the transcription start site (dashed underlining), and any added intron splice sequences (bold italics).
FIG. 1 provides the nucleotide sequence of a regulatory polynucleotide corresponding to the Arabidopsis gene having the accession number AT1G13440, +first intron.
FIG. 2 provides the nucleotide sequence of a regulatory polynucleotide corresponding to the Arabidopsis gene having the accession number AT1G13440.
FIG. 3 provides the nucleotide sequence of a regulatory polynucleotide corresponding to the Arabidopsis gene having the accession number AT1G22840, +first intron.
FIG. 4 provides the nucleotide sequence of a regulatory polynucleotide corresponding to the Arabidopsis gene having the accession number AT1G22840.
FIG. 5 provides the nucleotide sequence of a regulatory polynucleotide corresponding to the Arabidopsis gene having the accession number AT1G22840, +first intron, +35S minimal promoter.
FIG. 6 provides the nucleotide sequence of a regulatory polynucleotide corresponding to the Arabidopsis gene having the accession number AT1G52300, +first intron.
FIG. 7 provides the nucleotide sequence of a regulatory polynucleotide corresponding to the Arabidopsis gene having the accession number AT1G52300.
FIG. 8 provides the nucleotide sequence of a regulatory polynucleotide corresponding to the Arabidopsis gene having the accession number AT1G52300, +first intron, +35S minimal promoter.
FIG. 9 provides the nucleotide sequence of a regulatory polynucleotide corresponding to the Arabidopsis gene having the accession number AT4G37830, +first intron.
FIG. 10 provides the nucleotide sequence of a regulatory polynucleotide corresponding to the Arabidopsis gene having the accession number AT4G37830.
FIG. 11 provides the nucleotide sequence of a regulatory polynucleotide corresponding to the Arabidopsis gene having the accession number AT4G37830, +first intron, +35S minimal promoter.
FIG. 12 provides the nucleotide sequence of a regulatory polynucleotide corresponding to the Arabidopsis gene having the accession number AT3G08580.
FIG. 13 provides the nucleotide sequence of a regulatory polynucleotide corresponding to the Arabidopsis gene having the accession number AT3G08580, +35s minimal promoter.
FIG. 14 provides the nucleotide sequence of a regulatory polynucleotide corresponding to the Arabidopsis gene having the accession number AT1G51650, +first intron.
FIG. 15 provides the nucleotide sequence of a regulatory polynucleotide corresponding to the Arabidopsis gene having the accession number AT1G51650.
FIG. 16 provides the nucleotide sequence of a regulatory polynucleotide corresponding to the Arabidopsis gene having the accession number AT1G51650, +first intron, +35S minimal promoter.
FIG. 17 provides the nucleotide sequence of a regulatory polynucleotide corresponding to the Arabidopsis gene having the accession number AT3G48140, +first intron.
FIG. 18 provides the nucleotide sequence of a regulatory polynucleotide corresponding to the Arabidopsis gene having the accession number AT3G48140.
FIG. 19 provides the nucleotide sequence of a regulatory polynucleotide corresponding to the Arabidopsis gene having the accession number AT3G48140, +first intron, +35S minimal promoter.
FIG. 20 provides the nucleotide sequence of a regulatory polynucleotide corresponding to the Arabidopsis gene having the accession number AT3G08610, +first intron.
FIG. 21 provides the nucleotide sequence of a regulatory polynucleotide corresponding to the Arabidopsis gene having the accession number AT3G08610.
FIG. 22 provides the nucleotide sequence of a regulatory polynucleotide corresponding to the Arabidopsis gene having the accession number AT3G62250.
FIGS. 23A-D through 31A-D illustrate the expression data of the underlying Arabidopsis genes that correspond to the regulatory polynucleotides of FIGS. 1-22.
FIGS. 23A-23D provide a schematic representation of the endogenous expression data for the Arabidopsis gene having the accession number AT1G13440. FIG. 23A provides the expression values of this gene in different cell types which were sorted on the basis of expressing the indicated GFP markers. FIG. 23B provides the expression values of this gene from root sections along the longitudinal axis of the root. FIG. 23C provides the developmental specific expression of the gene. FIG. 23D provides the expression of the gene in response to various abiotic stresses.
FIGS. 24A-D provide a schematic representation of the endogenous expression data for the Arabidopsis gene having the accession number AT1G22840. FIG. 24A provides the expression values of this gene in different cell types which were sorted on the basis of expressing the indicated GFP markers. FIG. 24B provides the expression values of this gene from root sections along the longitudinal axis of the root. FIG. 24C provides the developmental specific expression of the gene. FIG. 24D provides the expression of the gene in response to various abiotic stresses.
FIGS. 25A-D provide a schematic representation of the endogenous expression data for the Arabidopsis gene having the accession number AT1G52300. FIG. 25A provides the expression values of this gene in different cell types which were sorted on the basis of expressing the indicated GFP markers. FIG. 25B provides the expression values of this gene from root sections along the longitudinal axis of the root. FIG. 25C provides the developmental specific expression of the gene. FIG. 25D provides the expression of the gene in response to various abiotic stresses.
FIGS. 26A-D provide a schematic representation of the endogenous expression data for the Arabidopsis gene having the accession number AT4G37830. FIG. 26A provides the expression values of this gene in different cell types which were sorted on the basis of expressing the indicated GFP markers. FIG. 26B provides the expression values of this gene from root sections along the longitudinal axis of the root. FIG. 26C provides the developmental specific expression of the gene. FIG. 26D provides the expression of the gene in response to various abiotic stresses.
FIGS. 27A-D provide a schematic representation of the endogenous expression data for the Arabidopsis gene having the accession number AT3G08580. FIG. 27A provides the expression values of this gene in different cell types which were sorted on the basis of expressing the indicated GFP markers. FIG. 27B provides the expression values of this gene from root sections along the longitudinal axis of the root. FIG. 27C provides the developmental specific expression of the gene. FIG. 27D provides the expression of the gene in response to various abiotic stresses.
FIGS. 28A-D provide a schematic representation of the endogenous expression data for the Arabidopsis gene having the accession number AT1G51650. FIG. 28A provides the expression values of this gene in different cell types which were sorted on the basis of expressing the indicated GFP markers. FIG. 28B provides the expression values of this gene from root sections along the longitudinal axis of the root. FIG. 28C provides the developmental specific expression of the gene. FIG. 28D provides the expression of the gene in response to various abiotic stresses.
FIGS. 29A-D provide a schematic representation of the endogenous expression data for the Arabidopsis gene having the accession number AT3G48140. FIG. 29A provides the expression values of this gene in different cell types which were sorted on the basis of expressing the indicated GFP markers. FIG. 29B provides the expression values of this gene from root sections along the longitudinal axis of the root. FIG. 29C provides the developmental specific expression of the gene. FIG. 289D provides the expression of the gene in response to various abiotic stresses.
FIGS. 30A-D provide a schematic representation of the endogenous expression data for the Arabidopsis gene having the accession number AT3G08610. FIG. 30A provides the expression values of this gene in different cell types which were sorted on the basis of expressing the indicated GFP markers. FIG. 30B provides the expression values of this gene from root sections along the longitudinal axis of the root. FIG. 30C provides the developmental specific expression of the gene. FIG. 30D provides the expression of the gene in response to various abiotic stresses.
FIGS. 31A-D provide a schematic representation of the endogenous expression data for the Arabidopsis gene having the accession number AT3G62250. FIG. 31A provides the expression values of this gene in different cell types which were sorted on the basis of expressing the indicated GFP markers. FIG. 31B provides the expression values of this gene from root sections along the longitudinal axis of the root. FIG. 31C provides the developmental specific expression of the gene. FIG. 31D provides the expression of the gene in response to various abiotic stresses.
FIG. 32A provides the nucleotide sequence of the regulatory polynucleotide of the Arabidopsis gene having Accession No. AT4G05320. FIGS. 32B-32E provide a schematic representation of the endogenous expression data for the Arabidopsis gene having Accession No. AT4G05320. FIG. 32B provides the expression values of this gene in different cell types which were sorted on the basis of expressing the indicated GFP markers. FIG. 32C provides the expression values of this gene from root sections along the longitudinal axis of the root. FIG. 32D provides the developmental specific expression of AT4G05320. FIG. 32E provides the expression of AT4G05320in response to various abiotic stresses.
FIGS. 33A, 33B, and 33C show average GFP Expression Index in different cell-types in 3 longitudinal zones under standard and 3 stress conditions using a regulatory polynucleotide from the Arabidopsis polyubiquitin gene UBQ10, which was identified using the methods described herein.
FIG. 34 provides representative images of GFP expression of Arabidopsis plants according to Example 4. As explained in Example 4, FIG. 34A shows GFP expression in the elongation zone and FIG. 34B shows GFP expression in the meristematic zone.
FIG. 35 provides representative images of GFP expression of Arabidopsis plants according to Example 4. As explained in Example 4, FIG. 35A shows GFP expression in the elongation zone and FIG. 35B shows GFP expression in the meristematic zone.
FIG. 36 provides representative images of GFP expression of Arabidopsis plants according to Example 4. As explained in Example 4, FIG. 36A shows GFP expression in the elongation zone and FIG. 36B shows GFP expression in the meristematic zone.
FIG. 37 provides representative images of GFP expression of Arabidopsis plants according to Example 4. As explained in Example 4, FIG. 37A shows GFP expression in the elongation zone and FIG. 37B shows GFP expression in the meristematic zone.
FIG. 38 provides representative images of GFP expression of Arabidopsis plants according to Example 4. As explained in Example 4, FIG. 38A shows GFP expression in the elongation zone and FIG. 38B shows GFP expression in the meristematic zone.
FIG. 39A shows GFP expression of construct A in the elongation zone and FIG. 39B shows GFP expression of construct A in the meristematic zone according to Example 8.



DETAILED DESCRIPTION
The present disclosure relates to regulatory polynucleotides that are capable of regulating expression of a transcribable polynucleotide in a host cell. In some embodiments, the regulatory polynucleotides are capable of regulating expression of a transcribable polynucleotide in a plant cell, plant tissue, plant, or plant seed. In other embodiments, the regulatory polynucleotides are capable of providing for constitutive expression of an operably linked polynucleotide in plants and plant tissues.
The present disclosure also provides recombinant constructs comprising such regulatory polynucleotides, as well as transgenic host cells, and organisms containing such recombinant constructs. Also provided are methods of directing expression of a transcribable polynucleotide in a host cell or organism.
Prior to describing this invention in further detail, however, the following terms will first be defined.
Definitions:
As used herein, the phrase “polynucleotide molecule” refers to a single- or double-stranded DNA or RNA of any origin (e.g., genomic or synthetic origin), i.e., a polymer of deoxyribonucleotide or ribonucleotide bases, respectively, read from the 5′ (upstream) end to the 3′ (downstream) end.
As used herein, the phrase “polynucleotide sequence” refers to the sequence of a polynucleotide molecule. The nomenclature for DNA bases as set forth at 37 CFR §1.822 is used.
As used herein, the term “transcribable polynucleotide molecule” refers to any polynucleotide molecule capable of being transcribed into a RNA molecule including, but not limited to, protein coding sequences (e.g., transgenes) and functional RNA sequences (e.g., a molecule useful for gene suppression).
As used herein, the terms “regulatory element” and “regulatory polynucleotide” refer to polynucleotide molecules having regulatory activity (i.e., one that has the ability to affect the transcription of an operably linked transcribable polynucleotide molecule). The terms refer to a polynucleotide molecule containing one or more elements such as core promoter regions, cis-elements, leaders or UTRs, enhancers, introns, and transcription termination regions, all of which have regulatory activity and may play a role in the overall expression of nucleic acid molecules in living cells. The “regulatory elements” determine if, when, and at what level a particular polynucleotide is transcribed. The regulatory elements may interact with regulatory proteins or other proteins or be involved in nucleotide interactions, for example, to provide proper folding of a regulatory polynucleotide.
As used herein, the term “core promoter” and “minimal promoter” refer to a minimal region of a regulatory polynucleotide required to properly initiate transcription. A core promoter typically contains the transcription start site (TSS), a binding site for RNA polymerase, and general transcription factor binding sites. Core promoters can include promoters produced through the manipulation of known core promoters to produce artificial, chimeric, or hybrid promoters, and can be used in combination with other regulatory elements, such as cis-elements, enhancers, or introns, for example, by adding a heterologous regulatory element to an active core promoter with its own partial or complete regulatory elements.
As used herein, the term “cis-element” refers to a cis-acting transcriptional regulatory element that confers an aspect of the overall control of the expression of an operably linked transcribable polynucleotide. A cis-element may function to bind transcription factors, which are trans-acting protein factors that regulate transcription. Some cis-elements bind more than one transcription factor, and transcription factors may interact with different affinities with more than one cis-element. Cis-elements including deletion analysis (i.e., deleting one or more nucleotides from the 5′ end or internal to a promoter), DNA binding protein analysis using DNase I footprinting, methylation interference, electrophoresis mobility-shift assays, in vivo genomic footprinting by ligation-mediated PCR, and other conventional assays; or by DNA sequence similarity analysis with known cis-element motifs by conventional DNA sequence comparison methods. The fine structure of a cis-element can be further studied by mutagenesis (or substitution) of one or more nucleotides or by other conventional methods. Cis-elements can be obtained by chemical synthesis or by isolation from regulatory polynucleotides that include such elements, and they can be synthesized with additional flanking nucleotides that contain useful restriction enzyme sites to facilitate subsequence manipulation.
As used herein, the term “enhancer” refers to a transcriptional regulatory element, typically 100-200 base pairs in length, which strongly activates transcription, for example, through the binding of one or more transcription factors. Enhancers can be identified and studied by methods such as those described above for cis-elements. Enhancer sequences can be obtained by chemical synthesis or by isolation from regulatory elements that include such elements, and they can be synthesized with additional flanking nucleotides that contain useful restriction enzyme sites to facilitate subsequence manipulation.
As used herein, the term “intron” refers to a polynucleotide molecule that may be isolated or identified from the intervening sequence of a genomic copy of a transcribed polynucleotide which is spliced out during mRNA processing prior to translation. Introns may themselves contain sub-elements such as cis-elements or enhancer domains that affect the transcription of operably linked polynucleotide molecules. Some introns are capable of increasing gene expression through a mechanism known as intron mediated enhancement (IME). IME, as distinguished from the effects of enhancers, is based on introns residing in the transcribed region of a polynucleotide. In general, IME is mediated by the first intron of a gene, which can reside in either the 5′-UTR sequence of a gene or between the first and second protein coding (CDS) exons of a gene. Without being limited by theory, IME may be particularly important in highly expressed, constitutive genes.
As used herein, the terms “leader” or “5′-UTR” refer to a polynucleotide sequence between the transcription and translation start sites of a gene. 5′-UTRs may themselves contain sub-elements such as cis-elements, enhancer domains, or introns that affect the transcription of operably linked polynucleotide molecules.
As used herein, the term “ortholog” refers to a polynucleotide from a different species that encodes a similar protein that performs the same biological function. For example, the ubiquitin genes from, for example, Arabidopsis and rice, are orthologs. Orthologs may also exhibit similar tissue expression patterns (for example, constitutive expression in plant cells or plant tissues). Typically, orthologous nucleotide sequences are characterized by significant sequence similarity. A nucleotide sequence of an ortholog in one species (for example, Arabidopsis ) can be used to isolate the nucleotide sequence of the ortholog in another species (for example, rice) using standard molecular biology techniques.
The term “expression” or “gene expression” means the transcription of an operably linked polynucleotide. The term “expression” or “gene expression” in particular refers to the transcription of an operably linked polynucleotide into structural RNA (rRNA, tRNA) or mRNA with or without subsequent translation of the latter into a protein. The process includes transcription of DNA and processing of the resulting mRNA product.
“Constitutive expression” refers to the transcription of a polynucleotide in all or substantially all tissues and stages of development and being minimally responsive to abiotic stimuli. “Constitutive plant regulatory polynucleotides” are regulatory polynucleotides that have regulatory activity in all or substantially all tissues of a plant throughout plant development. It is understood that for the terms “constitutive expression” and “constitutive plant regulatory polynucleotide” that some variation in absolute levels of expression or activity can exist among different plant tissues and stages of development.
As used herein, the term “chimeric” refers to the product of the fusion of portions of two or more different polynucleotide molecules. As used herein, the term “chimeric regulatory polynucleotide” refers to a regulatory polynucleotide produced through the manipulation of known promoters or other polynucleotide molecules, such as cis-elements. Such chimeric regulatory polynucleotides may combine enhancer domains that can confer or modulate expression from one or more regulatory polynucleotides, for example, by fusing a heterologous enhancer domain from a first regulatory polynucleotide to a promoter element (e.g. a core promoter) from a second regulatory polynucleotide with its own partial or complete regulatory elements.
As used herein, the term “operably linked” refers to a first polynucleotide molecule, such as a core promoter, connected with a second polynucleotide molecule, such as a transcribable polynucleotide (e.g., a polynucleotide encoding a protein of interest), where the polynucleotide molecules are so arranged that the first polynucleotide molecule affects the transcription of the second polynucleotide molecule. The two polynucleotide molecules may be part of a single contiguous polynucleotide molecule and may be adjacent. For example, a promoter is operably linked to a polynucleotide encoding a protein of interest if the promoter modulates transcription of the polynucleotide of interest in a cell.
An “isolated” or “purified” polynucleotide or polypeptide molecule, refers to a molecule that is not in its native environment such as, for example, a molecule not normally found in the genome of a particular host cell, or a DNA not normally found in the host genome in an identical context, or any two sequences adjacent to each other that are not normally or naturally adjacent to each other.
Regulatory Polynucleotide Molecules
The regulatory polynucleotide molecules described herein were discovered using bioinformatic screening techniques of databases containing expression and sequence data for genes in various plant species. Such bioinformatic techniques are described in more detail in the Examples set forth below.
In one embodiment, isolated regulatory polynucleotide molecules are provided. The regulatory polynucleotides provided herein include polynucleotide molecules having transcription regulatory activity in host cells, such as plant cells. In some embodiments, the regulatory polynucleotides are capable of regulating constitutive transcription of an operably linked transcribable polynucleotide molecule in transgenic plants and plant tissues.
The isolated regulatory polynucleotide molecules comprise a polynucleotide molecule selected from the group consisting of a) a polynucleotide molecule comprising a nucleic acid molecule having a sequence selected from the group consisting of SEQ ID NOs: 1-22 that is capable of regulating transcription of an operably linked transcribable polynucleotide molecule; b) a polynucleotide molecule having at least about 70% sequence identity to the sequence of SEQ ID NOs:1-22 that is capable of regulating transcription of an operably linked transcribable polynucleotide molecule; and c) a fragment of the polynucleotide molecule of a) or b) capable of regulating transcription of an operably linked transcribable polynucleotide molecule. Such fragments can be a UTR, a core promoter, an intron, an enhancer, a cis-element, or any other regulatory element.
Thus, the regulatory polynucleotide molecules include those molecules having sequences provided in SEQ ID NO: 1 through SEQ ID NO: 22. These polynucleotide molecules are capable of affecting the expression of an operably linked transcribable polynucleotide molecule in plant cells and plant tissues and therefore can regulate expression in transgenic plants. The present disclosure also provides methods of modifying, producing, and using such regulatory polynucleotides. Also included are compositions, transformed host cells, transgenic plants, and seeds containing the regulatory polynucleotides, and methods for preparing and using such regulatory polynucleotides.
The disclosed regulatory polynucleotides are capable of providing for expression of operably linked transcribable polynucleotides in any cell type, including, but not limited to plant cells. For example, the regulatory polynucleotides may be capable of providing for the expression of operably linked heterologous transcribable polynucleotides in plants and plant cells. In one embodiment, the regulatory polynucleotides are capable of directing constitutive expression in a transgenic plant, plant tissue(s), or plant cell(s).
In one embodiment, the regulatory polynucleotides may comprise multiple regulatory elements, each of which confers a different aspect to the overall control of the expression of an operably linked transcribable polynucleotide. In another embodiment, regulatory elements may be derived from the polynucleotide molecules of SEQ ID NOs:1-22. Thus, regulatory elements of the disclosed regulatory polynucleotides are also provided.
The disclosed polynucleotides include, but are not limited to, nucleic acid molecules that are between about 0.1 Kb and about 5 Kb, between about 0.1 Kb and about 4 Kb, between about 0.1 Kb and about 3 Kb, and between about 0.1 Kb and about 2 Kb, about 0.25 Kb and about 2 Kb, or between about 0.10 Kb and about 1.0 Kb.
The regulatory polynucleotides as provided herein also include fragments of SEQ ID NOs: 1-22. The fragment polynucleotides include those polynucleotides that comprise at least 50, at least 75, at least 100, at least 125, at least 150, at least 175, or at least 200 contiguous nucleotide bases where the fragment's complete sequence in its entirety is identical to a contiguous fragment of the referenced polynucleotide molecule. In some embodiments, the fragments contain one or more regulatory elements capable of regulating the transcription of an operably linked polynucleotide. Such fragments may include regulatory elements such as introns, enhancers, core promoters, leaders, and the like.
Thus also provided are regulatory elements derived from the polynucleotides having the sequences of SEQ ID NOs: 1-22. In some embodiments, the regulatory elements are capable of regulating transcription of operably linked transcribable polynucleotides in plants and plant tissues. The regulatory elements that may be derived from the polynucleotides of SEQ ID NOs:1-22 include, but are not limited to introns, enhancers, leaders, and the like. In addition, the regulatory elements may be used in recombinant constructs for the expression of operably linked transcribable polynucleotides of interest.
The present disclosure also includes regulatory polynucleotides that are substantially homologous to SEQ ID NOs:1-22. As used herein, the phrase “substantially homologous” refers to polynucleotide molecules that generally demonstrate a substantial percent sequence identity with the regulatory polynucleotides provided herein. Substantially homologous polynucleotide molecules include polynucleotide molecules that function in plants and plant cells to direct transcription and have at least about 70% sequence identity, at least about 80% sequence identity, at least about 90% sequence identity, or even greater sequence identity, specifically including about 73%, 75%, 78%, 83%, 85%, 88%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity with the regulatory polynucleotide molecules provided in SEQ ID NOs:1-22. Polynucleotide molecules that are capable of regulating transcription of operably linked transcribable polynucleotide molecules and are substantially homologous to the polynucleotide sequences of the regulatory polynucleotides provided herein are encompassed herein.
As used herein, the “percent sequence identity” is determined by comparing two optimally aligned sequences over a comparison window, where the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, divided by the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Alignment for the purposes of determining the percentage identity can be achieved in various ways that are within the skill in the art, for example, using publicly available computer software such as BLAST. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve optimal alignment over the full length of the sequences being compared.
Additional regulatory polynucleotides substantially homologous to those identified herein may be identified by a variety of methods. For example, cDNA libraries may be constructed using cells or tissues of interest and screened to identify genes having an expression pattern similar to that of the regulatory elements described herein. The cDNA sequence for the identified gene may then be used to isolate the gene's regulatory sequences for further characterization. Alternately, transcriptional profiling or electronic northern techniques may be used to identify genes having an expression pattern similar to that of the regulatory polynucleotides described herein. Once these genes have been identified, their regulatory polynucleotides may be isolated for further characterization. The electronic northern technique refers to a computer-based sequence analysis which allows sequences from multiple cDNA libraries to be compared electronically based on parameters the researcher identifies including abundance in EST populations in multiple cDNA libraries, or exclusively to EST sets from one or combinations of libraries. The transcriptional profiling technique is a high-throughput method used for the systematic monitoring of expression profiles for thousands of genes. This DNA chip-based technology arrays thousands of oligonucleotides on a support surface. These arrays are simultaneously hybridized to a population of labeled cDNA or cRNA probes prepared from RNA samples of different cell or tissue types, allowing direct comparative analysis of expression. This approach may be used for the isolation of regulatory sequences such as promoters associated with those sequences.
In some embodiments, substantially homologous polynucleotide molecules may be identified when they specifically hybridize to form a duplex molecule under certain conditions. Under these conditions, referred to as stringency conditions, one polynucleotide molecule can be used as a probe or primer to identify other polynucleotide molecules that share homology. Accordingly, the nucleotide sequences of the present invention may be used for their ability to selectively form duplex molecules with complementary stretches of polynucleotide molecule fragments. Substantially homologous polynucleotide molecules may also be determined by computer programs that align polynucleotide sequences and estimate the ability of polynucleotide molecules to form duplex molecules under certain stringency conditions or show sequence identity with a reference sequence.
In some embodiments, the regulatory polynucleotides disclosed herein can be modified from their wild-type sequences to create regulatory polynucleotides that have variations in the polynucleotide sequence. The polynucleotide sequences of the regulatory elements of SEQ ID NOs: 1-22 may be modified or altered. One method of alteration of a polynucleotide sequence includes the use of polymerase chain reactions (PCR) to modify selected nucleotides or regions of sequences. These methods are well known to those of skill in the art. Sequences can be modified, for example, by insertion, deletion, or replacement of template sequences in a PCR-based DNA modification approach. In the context of the present invention, a “variant” is a regulatory polynucleotide containing changes in which one or more nucleotides of an original regulatory polynucleotide is deleted, added, and/or substituted. In one example, a variant regulatory polynucleotide substantially maintains its regulatory function. For example, one or more base pairs may be deleted from the 5′ or 3′ end of a regulatory polynucleotide to produce a “truncated” polynucleotide. One or more base pairs can also be inserted, deleted, or substituted internally to a regulatory polynucleotide. Variant regulatory polynucleotides can be produced, for example, by standard DNA mutagenesis techniques or by chemically synthesizing the variant regulatory polynucleotide or a portion thereof.
The methods and compositions provided for herein may be used for the efficient expression of transgenes in plants. The regulatory polynucleotide molecules useful for directing expression (including constitutive expression) of transcribable polynucleotides, may provide enhancement of expression (including enhancement of constitutive expression) (e.g., through the use of IME with the introns of the regulatory polynucleotides disclosed herein), and/or may provide for increased levels of expression of transcribable polynucleotides operably linked to a regulatory polynucleotide described herein. In addition, the introns identified in the regulatory polynucleotide molecules provided herein may also be included in conjunction with any other plant promoter (or plant regulatory polynucleotide) for the enhancement of the expression of selected transcribable polynucleotides.
Also provided are chimeric regulatory polynucleotide molecules. Such chimeric regulatory polynucleotides may contain one or more regulatory elements disclosed herein in operable combination with one or more additional regulatory elements. The one or more additional regulatory elements can be any additional regulatory elements from any source, including those disclosed herein, as well as those known in the art, for example, the actin 2 intron. In addition, the chimeric regulatory polynucleotide molecules may comprise any number of regulatory elements such as, for example, 2, 3, 4, 5, or more regulatory elements.
In some embodiments, the chimeric regulatory polynucleotides contain at least one core promoter molecule provided herein operably linked to one or more additional regulatory elements, such as one or more regulatory introns and/or enhancer elements. Alternatively, the chimeric regulatory polynucleotides may contain one or more regulatory elements as provided herein in combination with a minimal promoter sequence, for example, the CaMV 35S minimal promoter. Thus, the design, construction, and use of chimeric regulatory polynucleotides according to the methods disclosed herein for modulating the expression of operably linked transcribable polynucleotide molecules are also provided.
The chimeric regulatory polynucleotides as provided herein can be designed or engineered using any method. Many regulatory regions contain elements that activate, enhance, or define the strength and/or specificity of the regulatory region. Thus, for example, chimeric regulatory polynucleotides of the present invention may comprise core promoter elements containing the site of transcription initiation (e.g., RNA polymerase II binding site) combined with heterologous cis-elements located upstream of the transcription initiation site that modulate transcription levels. Thus, in one embodiment, a chimeric regulatory polynucleotide may be produced by fusing a core promoter fragment polynucleotide described herein to a cis-element from another regulatory polynucleotide; the resultant chimeric regulatory polynucleotide may cause an increase in expression of an operably linked transcribable polynucleotide molecule. Chimeric regulatory polynucleotides can be constructed such that regulatory polynucleotide fragments or elements are operably linked, for example, by placing such a fragment upstream of a minimal promoter. The core promoter regions, regulatory elements and fragments of the present invention can be used for the construction of such chimeric regulatory polynucleotides.
Thus, also provided are chimeric regulatory polynucleotide molecules comprising (1) a first polynucleotide molecule selected from the group consisting of a) a polynucleotide molecule comprising a nucleic acid molecule having the sequence of SEQ ID NOs: 1-22 that is capable of regulating transcription of an operably linked transcribable polynucleotide molecule; b) a polynucleotide molecule having at least about 70% sequence identity to the sequence of SEQ ID NOs:1-22 that is capable of regulating transcription of an operably linked transcribable polynucleotide molecule; and c) a fragment of the polynucleotide molecule of a) or b) capable of regulating transcription of an operably linked transcribable polynucleotide molecule, and (2) a second polynucleotide molecule capable of regulating transcription of an operably linked polynucleotide molecule, wherein the first polynucleotide molecule is operably linked to the second polynucleotide molecule. The chimeric regulatory polynucleotide molecules may further comprise at least a third, fourth, fifth, or more additional polynucleotide molecules capable of regulating transcription of an operably linked polynucleotide, where the at least a third, fourth, fifth, or more additional polynucleotide molecules is/are operably linked to the first and second polynucleotide molecules.
The first and second polynucleotide molecules may be any combination of regulatory elements, including those provided herein. In one embodiment, the first polynucleotide comprises at least a core promoter element and the second polynucleotide comprises at least one additional regulatory element, including, but not limited to, an enhancer, an intron, and a leader molecule.
Methods for construction of chimeric and variant regulatory polynucleotides include, but are not limited to, combining elements of different regulatory polynucleotides or duplicating portions or regions of a regulatory polynucleotide. Those of skill in the art are familiar with the standard resource materials that describe specific conditions and procedures for the construction, manipulation, and isolation of macromolecules (e.g., polynucleotide molecules, plasmids, etc.), as well as the generation of recombinant organisms and the screening and isolation of polynucleotide molecules.
Thus, also provided are novel methods and compositions for the efficient expression of transcribable polynucleotides in plants through the use of the regulatory polynucleotides described herein. The regulatory polynucleotides described herein include constitutive promoters which may find wide utility in directing the expression of potentially any polynucleotide which one desires to have expressed in a plant. The regulatory elements disclosed herein may be used as promoters within expression constructs in order to increase the level of expression of transcribable polynucleotides operably linked to any one of the disclosed regulatory polynucleotides. Alternatively, the regulatory elements disclosed herein may be included in expression constructs in conjunction with any other plant promoter for the enhancement of the expression of one or more selected polynucleotides.
Recombinant Constructs
The disclosed regulatory polynucleotide molecules find use in the production of recombinant polynucleotide constructs, for example to express transcribable polynucleotides encoding proteins of interest in a host cell.
The recombinant constructs comprise (1) an isolated regulatory polynucleotide molecule comprising a polynucleotide molecule selected from the group consisting of a) a polynucleotide molecule comprising a nucleic acid molecule having the sequence of SEQ ID NOs: 1-22 that is capable of regulating transcription of an operably linked transcribable polynucleotide molecule; b) a polynucleotide molecule having at least about 70% sequence identity to the sequence of SEQ ID NOs:1-22 that is capable of regulating transcription of an operably linked transcribable polynucleotide molecule; and c) a fragment of the polynucleotide molecule of a) or b) capable of regulating transcription of an operably linked transcribable polynucleotide molecule operably linked to (2) a transcribable polynucleotide molecule.
The constructs provided herein may contain any recombinant polynucleotide molecule having a combination of regulatory elements linked together in a functionally operative manner. For example, the constructs may contain a regulatory polynucleotide operably linked to a transcribable polynucleotide molecule operably linked to a 3′ transcription termination polynucleotide molecule. In addition, the constructs may include, but are not limited to, additional regulatory polynucleotide molecules from the 3′-untranslated region (3′ UTR) of plant genes (e.g., a 3′ UTR to increase mRNA stability, such as the PI-II termination region of potato or the octopine or nopaline synthase 3′ termination regions). Constructs may also include but are not limited to the 5′ untranslated regions (5′ UTR) of an mRNA polynucleotide molecule which can play an important role in translation initiation and can also be a regulatory component in a plant expression construct. For example, non-translated 5′ leader polynucleotide molecules derived from heat shock protein genes have been demonstrated to enhance expression in plants. These additional upstream and downstream regulatory polynucleotide molecules may be derived from a source that is native or heterologous with respect to the other elements present on the promoter construct.
Thus, constructs generally comprise regulatory polynucleotides such as those provided herein (including modified and chimeric regulatory polynucleotides), operatively linked to a transcribable polynucleotide molecule so as to direct transcription of the transcribable polynucleotide molecule at a desired level or in a desired tissue or developmental pattern upon introduction of the construct into a plant cell. In some cases, the transcribable polynucleotide molecule comprises a protein-coding region, and the promoter provides for transcription of a functional mRNA molecule that is translated and expressed as a protein product. Constructs may also be constructed for transcription of antisense RNA molecules or other similar inhibitory RNA in order to inhibit expression of a specific RNA molecule of interest in a target host cell.
Exemplary transcribable polynucleotide molecules for incorporation into the disclosed constructs include, for example, transcribable polynucleotides from a species other than the target species, or even transcribable polynucleotides that originate with or are present in the same species, but are incorporated into recipient cells by genetic engineering methods rather than classical reproduction or breeding techniques. Exogenous polynucleotide or regulatory element is intended to refer to any polynucleotide molecule or regulatory polynucleotide that is introduced into a recipient cell. The type of polynucleotide included in the exogenous polynucleotide can include polynucleotides that are already present in the plant cell, polynucleotides from another plant, polynucleotides from a different organism, or polynucleotides generated externally, such as a polynucleotide molecule containing an antisense message of a protein-encoding molecule, or a polynucleotide molecule encoding an artificial or modified version of a protein.
The disclosed regulatory polynucleotides can be incorporated into a construct using marker genes and can be tested in transient analyses that provide an indication of expression in stable plant systems. As used herein, the term “marker gene” refers to any transcribable polynucleotide molecule whose expression can be screened for or scored in some way.
Methods of testing for marker expression in transient assays are known to those of skill in the art. Transient expression of marker genes has been reported using a variety of plants, tissues, and DNA delivery systems. For example, types of transient analyses include but are not limited to direct DNA delivery via electroporation or particle bombardment of tissues in any transient plant assay using any plant species of interest. Such transient systems would include but are not limited to electroporation of protoplasts from a variety of tissue sources or particle bombardment of specific tissues of interest. Any transient expression system may be used to evaluate regulatory polynucleotides or regulatory polynucleotide fragments operably linked to any transcribable polynucleotide molecule including, but not limited to, selected reporter genes, marker genes, or polynucleotides encoding proteins of agronomic interest. Any plant tissue may be used in the transient expression systems and include but are not limited to leaf base tissues, callus, cotyledons, roots, endosperm, embryos, floral tissue, pollen, and epidermal tissue.
Any scorable or screenable marker can be used in a transient assay as provided herein. For example, markers for transient analyses of the regulatory polynucleotides or regulatory polynucleotide fragments of the present invention include GUS or GFP. The constructs containing the regulatory polynucleotides or regulatory polynucleotide fragments of the present invention operably linked to a marker are delivered to the tissues and the tissues are analyzed by the appropriate mechanism, depending on the marker. The quantitative or qualitative analyses are used as a tool to evaluate the potential expression profile of the promoters or promoter fragments when operatively linked to polynucleotides encoding proteins of agronomic interest in stable plants.
Thus, in one embodiment, a regulatory polynucleotide molecule, or a variant, or derivative thereof, capable of regulating transcription, is operably linked to a transcribable polynucleotide molecule that provides for a selectable, screenable, or scorable marker. Markers for use in the practice of the present invention include, but are not limited to, transcribable polynucleotide molecules encoding β-glucuronidase (GUS), green fluorescent protein (GFP), luciferase (LUC), proteins that confer antibiotic resistance, or proteins that confer herbicide tolerance. Useful antibiotic resistance markers, including those encoding proteins conferring resistance to kanamycin (nptII), hygromycin B (aph IV), streptomycin or spectinomycin (aad, spec/strep), and gentamycin (aac3 and aacC4), are known in the art. Herbicides for which transgenic plant tolerance has been demonstrated and for which the methods disclosed herein can be applied include, but are not limited to, glyphosate, glufosinate, sulfonylureas, imidazolinones, bromoxynil, delapon, cyclohezanedione, protoporphyrionogen oxidase inhibitors, and isoxasflutole herbicides. Polynucleotide molecules encoding proteins involved in herbicide tolerance are known in the art, and include, but are not limited to, a polynucleotide molecule encoding 5-enolpyruvylshikimate-3-phosphate synthase (EPSP synthase); and aroA for glyphosate tolerance; a polynucleotide molecule encoding bromoxynil nitrilase (Bxn) for Bromoxynil tolerance; a polynucleotide molecule encoding phytoene desaturase (crtI) for norflurazon tolerance; a polynucleotide molecule encoding acetohydroxyacid synthase (AHAS, aka ALS) for tolerance to sulfonylurea herbicides; and the bar gene for glufosinate and bialaphos tolerance.
The regulatory polynucleotide molecules can be operably linked to any transcribable polynucleotide molecule of interest. Such transcribable polynucleotide molecules include, for example, polynucleotide molecules encoding proteins of agronomic interest. Proteins of agronomic interest can be any protein desired to be expressed in a host cell, such as, for example, proteins that provide a desirable characteristic associated with plant morphology, physiology, growth and development, yield, nutritional content, disease or pest resistance, or environmental or chemical tolerance. The expression of a protein of agronomic interest is desirable in order to confer an agronomically important trait on the plant containing the polynucleotide molecule. Proteins of agronomic interest that provide a beneficial agronomic trait to crop plants include, but are not limited to for example, proteins conferring herbicide resistance, insect control, fungal disease resistance, virus resistance, nematode resistance, bacterial disease resistance, starch production, modified oils production, high oil production, modified fatty acid content, high protein production, fruit ripening, enhanced animal and human nutrition, biopolymers, environmental stress resistance, pharmaceutical peptides, improved processing traits, improved digestibility, low raffinose, industrial enzyme production, improved flavor, nitrogen fixation, hybrid seed production, and biofuel production.
In other embodiments, the transcribable polynucleotide molecules can affect an agronomically important trait by encoding an RNA molecule that causes the targeted inhibition, or substantial inhibition, of expression of an endogenous gene (e.g., via antisense, RNAi, and/or cosuppression-mediated mechanisms). The RNA could also be a catalytic RNA molecule (i.e., a ribozyme) engineered to cleave a desired endogenous RNA product. Thus, any polynucleotide molecule that encodes a protein or mRNA that expresses a phenotype or morphology change of interest is useful for the practice of the present invention.
The constructs of the present invention may be double Ti plasmid border DNA constructs that have the right border (RB) and left border (LB) regions of the Ti plasmid isolated from Agrobacterium tumefaciens comprising a transfer DNA (T-DNA), that along with transfer molecules provided by the Agrobacterium cells, permits the integration of the T-DNA into the genome of a plant cell. The constructs also may contain the plasmid backbone DNA segments that provide replication function and antibiotic selection in bacterial cells, for example, an E. coli origin of replication such as ori322, a broad host range origin of replication such as oriV or oriRi, and a coding region for a selectable marker such as Spec/Strp that encodes for Tn7 aminoglycoside adenyltransferase (aadA) conferring resistance to spectinomycin or streptomycin, or a gentamicin (Gm, Gent) selectable marker. For plant transformation, the host bacterial strain is often Agrobacterium tumefaciens ABI, C58, or LBA4404, however, other strains known to those skilled in the art of plant transformation can function in the present invention.
Transgenic Cells, Host Cells, Plants and Plant Cells
The polynucleotides and constructs as provided herein can be used in the preparation of transgenic host cells, tissues, organs, and organisms. Thus, also provided are transgenic host cells, tissues, organs, and organisms that contain an introduced regulatory polynucleotide molecule as provided herein.
The transgenic host cells, tissues, organs, and organisms disclosed herein comprise a recombinant polynucleotide construct having (1) an isolated regulatory polynucleotide molecule comprising a polynucleotide molecule selected from the group consisting of a) a polynucleotide molecule comprising a nucleic acid molecule having the sequence of SEQ ID NOs: 1-22 that is capable of regulating transcription of an operably linked transcribable polynucleotide molecule; b) a polynucleotide molecule having at least about 70% sequence identity to the sequence of SEQ ID NOs:1-22 that is capable of regulating transcription of an operably linked transcribable polynucleotide molecule; and c) a fragment of the polynucleotide molecule of a) or b) capable of regulating transcription of an operably linked transcribable polynucleotide molecule, operably linked to (2) a transcribable polynucleotide molecule.
A plant transformation construct containing a regulatory polynucleotide as provided herein may be introduced into plants by any plant transformation method. The polynucleotide molecules and constructs provided herein may be introduced into plant cells or plants to direct transient expression of operably linked transcribable polynucleotides or be stably integrated into the host cell genome. Methods and materials for transforming plants by introducing a plant expression construct into a plant genome in the practice of this invention can include any of the well-known and demonstrated methods including electroporation; microprojectile bombardment; Agrobacterium-mediated transformation; and protoplast transformation.
Plants and plant cells for use in the production of the transgenic plants and plant cells include both monocotyledonous and dicotyledonous plants and plant cells. Methods for specifically transforming monocots and dicots are well known to those skilled in the art. Transformation and plant regeneration using these methods have been described for a number of crops including, but not limited to, soybean (Glycine max), Brassica sp., Arabidopsis thaliana, cotton (Gossypium hirsutum), peanut (Arachis hypogae), sunflower (Helianthus annuus), potato (Solanum tuberosum), tomato (Lycopersicon esculentum L.), rice, (Oryza sativa), corn (Zea mays), and alfalfa (Medicago sativa). It is apparent to those of skill in the art that a number of transformation methodologies can be used and modified for production of stable transgenic plants from any number of target crops of interest.
The transformed plants may be analyzed for the presence of the transcribable polynucleotides of interest and the expression level and/or profile conferred by the regulatory polynucleotides of the present invention. Those of skill in the art are aware of the numerous methods available for the analysis of transformed plants. For example, methods for plant analysis include, but are not limited to Southern blots or northern blots, PCR-based approaches, biochemical analyses, phenotypic screening methods, field evaluations, and immunodiagnostic assays.
The seeds of this invention can be harvested from fertile transgenic plants and be used to grow progeny generations of the transformed plants disclosed herein. The terms “seeds” and “kernels” are understood to be equivalent in meaning. In the context of the present invention, the seed refers to the mature ovule consisting of a seed coat, embryo, aleurone, and an endosperm.
Thus, also provided are methods for expressing transcribable polynucleotides in host cells, plant cells, and plants. In some embodiments, such methods comprise stably incorporating into the genome of a host cell, plant cell, or plant, a regulatory polynucleotide operably linked to a transcribable polynucleotide molecule of interest and regenerating a stably transformed plant that expresses the transcribable polynucleotide molecule. In other embodiments, such methods comprise the transient expression of a transcribable polynucleotide operably linked to a regulatory polynucleotide molecule provided herein in a host cell, plant cell, or plant.
Such methods of directing expression of a transcribable polynucleotide molecule in a host cell, such as a plant cell, include: A) introducing a recombinant nucleic acid construct into a host cell, the construct having (1) an isolated regulatory polynucleotide molecule comprising a polynucleotide molecule selected from the group consisting of a) a polynucleotide molecule comprising a nucleic acid molecule having the sequence of SEQ ID NOs: 1-22 that is capable of regulating transcription of an operably linked transcribable polynucleotide molecule; b) a polynucleotide molecule having at least about 70% sequence identity to the sequence of SEQ ID NOs:1-22 that is capable of regulating transcription of an operably linked transcribable polynucleotide molecule; and c) a fragment of the polynucleotide molecule of a) or b) capable of regulating transcription of an operably linked transcribable polynucleotide molecule, operably linked to (2) a transcribable polynucleotide molecule; and B) selecting a transgenic host cell exhibiting expression of the transcribable polynucleotide molecule.
The articles “a” and “an” are used herein to refer to one or more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one or more elements.
As used herein, the word “comprising,” or variations such as “comprises” or “comprising,” will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.
The following examples are offered by way of illustration and not by way of limitation.
EXAMPLES
Example 1
Identification of Arabidopsis Constitutive Regulatory Sequences
A bioinformatics approach was used to identify regulatory polynucleotides that have putative constitutive activity. Most plant regulatory polynucleotides (such as promoters) that are considered to have constitutive expression have been identified by their expression characteristics at the organ level (i.e., roots, shoots, leaves, seeds) and may not be truly constitutive at the cell type/tissue level. The method used to identify the regulatory polynucleotides described herein was used to identify regulatory polynucleotides having constitutive expression activity at the cell type and/or tissue level.
Using existing microarray expression data, a bioinformatics analysis method was used to identify genes from this data collection that are highly expressed in all cell types and longitudinal zones of the Arabidopsis root.
Such existing data includes microarray expression profiles of all cell-types and developmental stages within Arabidopsis root tissue (Brady et al., Science, 318:801-806 (2007)). The radial dataset comprehensively profiles expression of 14 non-overlapping cell-types in the root, while the longitudinal data set profiles developmental stages by measuring expression in 13 longitudinal sections. This detailed expression profiling has mapped the spatiotemporal expression patterns of nearly all genes in the Arabidopsis root.
The bioinformatics analysis method identified genes based on their published absolute expression level (see Brady et al, 2007, Science. 318: 801-6). This selection process used expression values that are similar to the Robust Microchip Average (RMA) expression values where a value of approximately 1.0 corresponds to the gene being expressed. The identified genes were then filtered with expression values above a certain threshold in every expression measurement. The selection resulted in Arabidopsis gene candidates that are broadly expressed in all cell-types and development stages of root tissue.
To assess expression in aerial tissue and responsiveness to abiotic stress, the expression profiles of these candidates were also analyzed in the AtGenExpress Development and Abiotic Stress datasets (available on the World Wide Web at the site weigelworld.org/resources/microarray/AtGenExpress). Candidates were further selected that showed significant expression in aerial tissue throughout development and also demonstrated little or no response to abiotic stresses according to these databases.
To identify regulatory polynucleotide molecules responsible for driving high constitutive expression of these candidate genes, upstream sequences of 1500 bp or less of the selected gene candidates were determined. Because transcription start sites are not always known, sequences upstream of the translation start site were used in all cases. Therefore, the selected regulatory polynucleotide molecules contain an endogenous 5′-UTR, and some of the endogenous 5′-UTRs contain introns. The use of such introns in expression constructs containing these regulatory sequences may increase expression through IME. Without being limited by theory, IME may be important for highly expressed constitutive genes, such as those identified here. To capture these regulatory molecules in genes that do not contain a 5′-UTR intron, chimeric regulatory polynucleotide molecules may be constructed wherein the first intron from the gene of interest is fused to the 3′-end of the 5′-UTR of the regulatory polynucleotide (which may be from the same or a different (e.g., exogenous) gene). To ensure efficient intron splicing, the introns in these chimeric molecules may be flanked by consensus splice sites.
The regulatory polynucleotides listed in Table 1 below were selected. Sequences including the regulatory polynucleotides plus the first intron from the coding region added at the 3′ end of the 5′ UTR are indicated by the corresponding gene accession number and the indicator “+first intron”. Chimeric regulatory polynucleotides prepared in which the 35S-minimal promoter sequence (5′-gcaagacccttcctctatataaggaagttcatttcatttggagagg-3′) (SEQ ID NO: 23) was substituted for the −46 to −1 endogenous minimal promoter sequence relative to the transcription start site (as annotated in the TAIR database, http://www.Arabidopsis.org/) have the indicator “+35S minimal promoter”.









TABLE 1






SEQ ID



Figure
NO:
Corresponding Gene Accession No.

















1
1
AT1G13440 + first intron


2
2
AT1G13440


3
3
AT1G22840 + first intron


4
4
AT1G22840


5
5
AT1G22840 + first intron + 35S minimal promoter


6
6
AT1G52300 + first intron


7
7
AT1G52300


8
8
AT1G52300 + first intron + 35S minimal promoter


9
9
AT4G37830 + first intron


10
10
AT4G37380


11
11
AT4G37830 + first intron + 35S minimal promoter


12
12
AT3G08580


13
13
AT3G08580 + 35S minimal promoter


14
14
AT1G51650 + first intron


15
15
AT1G51650


16
16
AT1G51650 + first intron + 35S minimal promoter


17
17
AT3G48140 + first intron


18
18
AT3G48140


19
19
AT3G48140 + first intron + 35S minimal promoter


20
20
AT3G08610 + first intron


21
21
AT3G08610


22
22
AT3G62250









The nucleic acid sequences provided in FIGS. 1 through 22 are annotated to indicate one transcription start site (Capital letter in bold), the endogenous 5′-UTR intron sequences (double underlining), the first intron from the coding sequence (single underlining), the 35S-minimal promoter from base −46 to base −1 as measured from the transcription start site (dashed underlining); and any added intron splice sequences (bold italics). All Arabidopsis genome sequences and annotations (i.e. transcription start sites, translation start sites, and introns) are from the Arabidopsis Information Resource (TAIR, available on the worldwide web at the address Arabidopsis.org/indexjsp).
Example 2
Endogenous Expression of Candidate Arabidopsis Genes
This example shows the endogenous expression data of the genes identified through the bioinformatics filtering of Example 1. Endogenous gene expression data is provided for each gene corresponding to each of the identified Arabidopsis regulatory polynucleotides is provided in FIGS. 23-31. All data shown in the figures are GC-RMA (GeneChip-RMA®) normalized expression values (log 2 scale) from Affymetrix® ATH1 microarrays which allow the detection of about 24,000 protein-encoding genes from Arabidopsis thaliana. For each gene, four plots labeled A-D are shown in the figures. Table 2 below shows the correspondence between the regulatory polynucleotides in Example 1 and the expression plots of FIGS. 23-31.








TABLE 2





Expression Figure
Regulatory Polynucleotide SEQ ID NOS


(Gene Accession No.)
(Corresponding Gene Accession No.)







23 (AT1G13440)
 1 (AT1G13440 + first intron)



 2 (AT1G13440)


24 (AT1G22840)
 3 (AT1G22840 + first intron)



 4 (AT1G22840)



 5 (AT1G22840 + first intron + 35S minimal



promoter)


25 (AT1G52300)
 6 (AT1G52300 + first intron)



 7 (AT1G52300)



 8 (AT1G52300 + first intron + 35S minimal



promoter)


26 (AT4G37830)
 9 (AT4G37830 + first intron)



10 (AT4G37830)



11 (AT4G37830 + first intron + 35S minimal



promoter)


27 (AT3G08580)
12 (AT3G08580)



13 (AT3G08580 + 35S minimal promoter)


28 (AT1G51650)
14 (AT1G51650 + first intron)



15 (AT1G51650)



16 (AT1G51650 + first intron + 35S minimal



promoter)


29 (AT3G48140)
17 (AT3G48140 + first intron)



18 (AT3G48140)



19 (AT3G48140 + first intron + 35S minimal



promoter)


30 (AT3G08610)
20 (AT3G08610 + first intron)



21 (AT3G08610)


31 (AT3G62250)
22 (AT3G62250)









Plots A and B are derived from data published by Brady et al. (Science, 318:801-806 (2007)). Plot A in each figure shows expression values from cells sorted on the basis of expressing the indicated GFP marker. Table 3 contains a key showing the specific cell types in which each marker is expressed. The table provides a description of cell types together with the associated markers. This table defines the relationship between cell-type and marker line, including which longitudinal sections of each cell-type are included. Lateral Root Primordia is included as a cell-type in this table, even though it may be a collection of multiple immature cell types. There are also no markers that differentiate between metaxylem and protoxylem or between metaphloem and protophloem, so those cell types are labeled Xylem and Phloem respectively. Together, these data provide expression information for virtually all cell-types found in the Arabidopsis root.










TABLE 3






Cell Type
Markers
Longitudinal Section








Lateral root cap
LRC
0-5 



Columella
PET111
0



Quiescent centre
AGL42
1




RM1000
1




SCR5
1



Hair cell
N/A
1-6 




COBL9
7-12



Non-hair cell
GL2
1-12



Cortex
J0571
1-12




CORTEX
6-12



Endodermis
J0571
1-12




SCR5
1-12



Xylem pole pericycle
WOL
1-8 




JO121
8-12




J2661
12 



Phloem pole pericycle
WOL
1-8 




S17
7-12




J2661
12 



Phloem
S32
1-12




WOL
1-8 



Phloem ccs
SUC2
9-12




WOL
1-8 



Xylem
S4
1-6 




S18
7-12




WOL
1-8 



Lateral root primordial
RM1000
11 



Procambium
WOL
1-8 









Plot B in each figure shows expression values from root sections along the longitudinal axis. Different regions along this axis correspond to different developmental stages of root cell development. In particular, section 0 corresponds to the columella, sections 1-6 correspond to the meristematic zone, sections 7-8 correspond to the elongation zone, and sections 9-12 correspond to the maturation zone.
Plots C and D in each figure are derived from publically available expression data of the AtGeneExpress project (available on the World Wide Web at weigelworld.org/resources/microarray/AtGenExpress). Plot C shows developmental specific expression as described by Schmid et al. (Nat. Genet., 37: 501-506 (2005)). A key for the samples in this dataset is provided in Table 4. For ease of visualization, root expression values are indicated with black bars, shoot expression with white bars, flower expression with coarse hatched bars, and seed expression with fine hatched bars.














TABLE 4







Experiment
Geno-


Photo-
Sub-


No
Sample ID
Description
type
Tissue
Age
period
strate






















1
ATGE_1
development
Wt
cotyledons
7
continuous
soil




baseline


days
light


2
ATGE_2
development
Wt
hypocotyl
7
continuous
soil




baseline


days
light


3
ATGE_3
development
Wt
roots
7
continuous
soil




baseline


days
light


4
ATGE_4
development
Wt
shoot apex,
7
continuous
soil




baseline

vegetative +
days
light






young leaves


5
ATGE_5
development
Wt
leaves 1 + 2
7
continuous
soil




baseline


days
light


6
ATGE_6
development
Wt
shoot apex,
7
continuous
soil




baseline

vegetative
days
light


7
ATGE_7
development
Wt
seedling, green
7
continuous
soil




baseline

parts
days
light


8
ATGE_8
development
Wt
shoot apex,
14
continuous
soil




baseline

transition (before
days
light






bolting)


9
ATGE_9
development
Wt
roots
17
continuous
soil




baseline


days
light


10
ATGE_10
development
Wt
rosette leaf #4, 1
10
continuous
soil




baseline

cm long
days
light


11
ATGE_11
development
gl1-T
rosette leaf #4, 1
10
continuous
soil




baseline

cm long
days
light


12
ATGE_12
development
Wt
rosette leaf # 2
17
continuous
soil




baseline


days
light


13
ATGE_13
development
Wt
rosette leaf # 4
17
continuous
soil




baseline


days
light


14
ATGE_14
development
Wt
rosette leaf # 6
17
continuous
soil




baseline


days
light


15
ATGE_15
development
Wt
rosette leaf # 8
17
continuous
soil




baseline


days
light


16
ATGE_16
development
Wt
rosette leaf # 10
17
continuous
soil




baseline


days
light


17
ATGE_17
development
Wt
rosette leaf # 12
17
continuous
soil




baseline


days
light


18
ATGE_18
development
gl1-T
rosette leaf # 12
17
continuous
soil




baseline


days
light


19
ATGE_19
development
Wt
leaf 7, petiole
17
continuous
soil




baseline


days
light


20
ATGE_20
development
Wt
leaf 7, proximal
17
continuous
soil




baseline

half
days
light


21
ATGE_21
development
Wt
leaf 7, distal half
17
continuous
soil




baseline


days
light


22
ATGE_22
development
Wt
developmental
21
continuous
soil




baseline

drift, entire
days
light






rosette after






transition to






flowering, but






before bolting


23
ATGE_23
development
Wt
as above
22
continuous
soil




baseline


days
light


24
ATGE_24
development
Wt
as above
23
continuous
soil




baseline


days
light


25
ATGE_25
development
Wt
senescing leaves
35
continuous
soil




baseline


days
light


26
ATGE_26
development
Wt
cauline leaves
21+
continuous
soil




baseline


days
light


27
ATGE_27
development
Wt
stem, 2nd
21+
continuous
soil




baseline

internode
days
light


28
ATGE_28
development
Wt
1st node
21+
continuous
soil




baseline


days
light


29
ATGE_29
development
Wt
shoot apex,
21
continuous
soil




baseline

inflorescence
days
light






(after bolting)


30
ATGE_31
development
Wt
flowers stage 9
21+
continuous
soil




baseline


days
light


31
ATGE_32
development
Wt
flowers stage
21+
continuous
soil




baseline

10/11
days
light


32
ATGE_33
development
Wt
flowers stage 12
21+
continuous
soil




baseline


days
light


33
ATGE_34
development
Wt
flowers stage 12,
21+
continuous
soil




baseline

sepals
days
light


34
ATGE_35
development
Wt
flowers stage 12,
21+
continuous
soil




baseline

petals
days
light


35
ATGE_36
development
Wt
flowers stage 12,
21+
continuous
soil




baseline

stamens
days
light


36
ATGE_37
development
Wt
flowers stage 12,
21+
continuous
soil




baseline

carpels
days
light


37
ATGE_39
development
Wt
flowers stage 15
21+
continuous
soil




baseline


days
light


38
ATGE_40
development
Wt
flowers stage 15,
21 +
continuous
soil




baseline

pedicels
days
light


39
ATGE_41
development
Wt
flowers stage 15,
21+
continuous
soil




baseline

sepals
days
light


40
ATGE_42
development
Wt
flowers stage 15,
21+
continuous
soil




baseline

petals
days
light


41
ATGE_43
development
Wt
flowers stage 15,
21+
continuous
soil




baseline

stamen
days
light


42
ATGE_45
development
Wt
flowers stage 15,
21+
continuous
soil




baseline

carpels
days
light


43
ATGE_46
development
clv3-7
shoot apex,
21+
continuous
soil




baseline

inflorescence
days
light






(after bolting)


44
ATGE_47
development
lfy-12
shoot apex,
21+
continuous
soil




baseline

inflorescence
days
light






(after bolting)


45
ATGE_48
development
ap1-15
shoot apex,
21+
continuous
soil




baseline

inflorescence
days
light






(after bolting)


46
ATGE_49
development
ap2-6
shoot apex,
21+
continuous
soil




baseline

inflorescence
days
light






(after bolting)


47
ATGE_50
development
ap3-6
shoot apex,
21+
continuous
soil




baseline

inflorescence
days
light






(after bolting)


48
ATGE_51
development
ag-12
shoot apex,
21+
continuous
soil




baseline

inflorescence
days
light






(after bolting)


49
ATGE_52
development
ufo-1
shoot apex,
21+
continuous
soil




baseline

inflorescence
days
light






(after bolting)


50
ATGE_53
development
clv3-7
flower stage 12;
21+
continuous
soil




baseline

multi-carpel
days
light






gynoeceum;






enlarged






meristem;






increased organ






number


51
ATGE_54
development
lfy-12
flower stage 12;
21+
continuous
soil




baseline

shoot
days
light






characteristics;






most organs leaf-






like


52
ATGE_55
development
ap1-15
flower stage 12;
21+
continuous
soil




baseline

sepals replaced
days
light






by leaf-like






organs, petals






mostly lacking,






2° flowers


53
ATGE_56
development
ap2-6
flower stage 12;
21+
continuous
soil




baseline

no sepals or
days
light






petals


54
ATGE_57
development
ap3-6
flower stage 12;
21+
continuous
soil




baseline

no petals or
days
light






stamens


55
ATGE_58
development
ag-12
flower stage 12;
21+
continuous
soil




baseline

no stamens or
days
light






carpels


56
ATGE_59
development
ufo-1
flower stage 12;
21+
continuous
soil




baseline

filamentous
days
light






organs in whorls






two and three


57
ATGE_73
pollen
Wt
mature pollen
6 wk
continuous
soil








light


58
ATGE_76
seed & silique
Wt
siliques, w/
8 wk
long day
soil




development

seeds stage 3;

(16/8)






mid globular to






early heart






embryos


59
ATGE_77
seed & silique
Wt
siliques, w/
8 wk
long day
soil




development

seeds stage 4;

(16/8)






early to late






heart embryos


60
ATGE_78
seed & silique
Wt
siliques, w/
8 wk
long day
soil




development

seeds stage 5;

(16/8)






late heart to mid






torpedo embryos


61
ATGE_79
seed & silique
Wt
seeds, stage 6,
8 wk
long day
soil




development

w/o siliques; mid

(16/8)






to late torpedo






embryos


62
ATGE_81
seed & silique
Wt
seeds, stage 7,
8 wk
long day
soil




development

w/o siliques; late

(16/8)






torpedo to early






walking-stick






embryos


63
ATGE_82
seed & silique
Wt
seeds, stage 8,
8 wk
long day
soil




development

w/o siliques;

(16/8)






walking-stick to






early curled






cotyledons






embryos


64
ATGE_83
seed & silique
Wt
seeds, stage 9,
8 wk
long day
soil




development

w/o siliques;

(16/8)






curled






cotyledons to






early green






cotyledons






embryos


65
ATGE_84
seed & silique
Wt
seeds, stage 10,
8 wk
long day
soil




development

w/o siliques;

(16/8)






green cotyledons






embryos


66
ATGE_87
phase change
Wt
vegetative
7
short day
soil






rosette
days
(10/14)


67
ATGE_89
phase change
Wt
vegetative
14
short day
soil






rosette
days
(10/14)


68
ATGE_90
phase change
Wt
vegetative
21
short day
soil






rosette
days
(10/14)


69
ATGE_91
comparison
Wt
leaf
15
long day
1x MS




with CAGE


days
(16/8)
agar,









1%









sucrose


70
ATGE_92
comparison
Wt
flower
28
long day
soil




with CAGE


days
(16/8)


71
ATGE_93
comparison
Wt
root
15
long day
1x MS




with CAGE


days
(16/8)
agar,









1%









sucrose


72
ATGE_94
development
Wt
root
8
continuous
1x MS




on MS agar


days
light
agar


73
ATGE_95
development
Wt
root
8
continuous
1x MS




on MS agar


days
light
agar,









1%









sucrose


74
ATGE_96
development
Wt
seedling, green
8
continuous
1x MS




on MS agar

parts
days
light
agar


75
ATGE_97
development
Wt
seedling, green
8
continuous
1x MS




on MS agar

parts
days
light
agar,









1%









sucrose


76
ATGE_98
development
Wt
root
21
continuous
1x MS




on MS agar


days
light
agar


77
ATGE_99
development
Wt
root
21
continuous
1x MS




on MS agar


days
light
agar,









1%









sucrose


78
ATGE_100
development
Wt
seedling, green
21
continuous
1x MS




on MS agar

parts
days
light
agar


79
ATGE_101
development
Wt
seedling, green
21
continuous
1x MS




on MS agar

parts
days
light
agar,









1%









sucrose









Plot D in each figure shows expression in response to abiotic stress as described by Kilian et al. (Plant J., 50: 347-363 (2007)). The data are presented as expression values from pairs of shoots (white bars) and roots (black bars) per treatment. A key for the samples in this dataset is presented in Table 5. The table identifies the codes that are used along the x-axis in plot D in each figure. The codes are presented in 4 digit format, where the first digit represents the treatment (i.e., control=0, cold=1, osmotic stress=2, etc.), the second digit represents the time point, the third digit represents the tissue (1=shoot and 2=root), and the fourth digit represents the replication number. Since the figures provide the averages of the first and second replication, the last digit is not shown in the figures.







TABLE 5







Abiotic Stress Key













Time

Sam-


Code
Treatment
point
Organ
ple





0011
Control
 0 h
Shoots
1


0012
Control
 0 h
Shoots
2


0021
Control
 0 h
Roots
1


0022
Control
 0 h
Roots
2


0711
Control
0.25 h 
Shoots
1


0712
Control
0.25 h 
Shoots
2


0721
Control
0.25 h 
Roots
1


0722
Control
0.25 h 
Roots
2


0111
Control
0.5 h
Shoots
1


0112
Control
0.5 h
Shoots
2


0121
Control
0.5 h
Roots
1


0122
Control
0.5 h
Roots
2


0211
Control
1.0 h
Shoots
1


0212
Control
1.0 h
Shoots
2


0221
Control
1.0 h
Roots
1


0222
Control
1.0 h
Roots
2


0311
Control
3.0 h
Shoots
1


0312
Control
3.0 h
Shoots
2


0321
Control
3.0 h
Roots
1


0322
Control
3.0 h
Roots
2


0811
Control
4.0 h
Shoots
1


0812
Control
4.0 h
Shoots
2


0821
Control
4.0 h
Roots
1


0822
Control
4.0 h
Roots
2


0411
Control
6.0 h
Shoots
1


0412
Control
6.0 h
Shoots
2


0421
Control
6.0 h
Roots
1


0422
Control
6.0 h
Roots
2


0511
Control
12.0 h 
Shoots
1


0512
Control
12.0 h 
Shoots
2


0521
Control
12.0 h 
Roots
1


0522
Control
12.0 h 
Roots
2


0611
Control
24.0 h 
Shoots
1


0612
Control
24.0 h 
Shoots
2


0621
Control
24.0 h 
Roots
1


0622
Control
24.0 h 
Roots
2


1111
Cold (4° C.)
0.5 h
Shoots
1


1112
Cold (4° C.)
0.5 h
Shoots
2


1121
Cold (4° C.)
0.5 h
Roots
1


1122
Cold (4° C.)
0.5 h
Roots
2


1211
Cold (4° C.)
1.0 h
Shoots
1


1212
Cold (4° C.)
1.0 h
Shoots
2


1221
Cold (4° C.)
1.0 h
Roots
1


1222
Cold (4° C.)
1.0 h
Roots
2


1311
Cold (4° C.)
3.0 h
Shoots
1


1312
Cold (4° C.)
3.0 h
Shoots
2


1321
Cold (4° C.)
3.0 h
Roots
1


1322
Cold (4° C.)
3.0 h
Roots
2


1411
Cold (4° C.)
6.0 h
Shoots
1


1412
Cold (4° C.)
6.0 h
Shoots
2


1421
Cold (4° C.)
6.0 h
Roots
1


1422
Cold (4° C.)
6.0 h
Roots
2


1511
Cold (4° C.)
12.0 h 
Shoots
1


1512
Cold (4° C.)
12.0 h 
Shoots
2


1521
Cold (4° C.)
12.0 h 
Roots
1


1522
Cold (4° C.)
12.0 h 
Roots
2


1611
Cold (4° C.)
24.0 h 
Shoots
1


1612
Cold (4° C.)
24.0 h 
Shoots
2


1621
Cold (4° C.)
24.0 h 
Roots
1


1622
Cold (4° C.)
24.0 h 
Roots
2


2111
Osmotic stress
0.5 h
Shoots
1


2112
Osmotic stress
0.5 h
Shoots
2


2121
Osmotic stress
0.5 h
Roots
1


2122
Osmotic stress
0.5 h
Roots
2


2211
Osmotic stress
 l.0 h
Shoots
1


2212
Osmotic stress
 l.0 h
Shoots
2


2221
Osmotic stress
 l.0 h
Roots
1


2222
Osmotic stress
 l.0 h
Roots
2


2311
Osmotic stress
3.0 h
Shoots
1


2312
Osmotic stress
3.0 h
Shoots
2


2321
Osmotic stress
3.0 h
Roots
1


2322
Osmotic stress
3.0 h
Roots
2


2411
Osmotic stress
6.0 h
Shoots
1


2412
Osmotic stress
6.0 h
Shoots
2


2421
Osmotic stress
6.0 h
Roots
1


2422
Osmotic stress
6.0 h
Roots
2


2511
Osmotic stress
12.0 h 
Shoots
1


2512
Osmotic stress
12.0 h 
Shoots
2


2521
Osmotic stress
12.0 h 
Roots
1


2522
Osmotic stress
12.0 h 
Roots
2


2611
Osmotic stress
24.0 h 
Shoots
1


2612
Osmotic stress
24.0 h 
Shoots
2


2621
Osmotic stress
24.0 h 
Roots
1


2622
Osmotic stress
24.0 h 
Roots
2


3111
Salt stress
0.5 h
Shoots
1


3112
Salt stress
0.5 h
Shoots
2


3121
Salt stress
0.5 h
Roots
1


3122
Salt stress
0.5 h
Roots
2


3211
Salt stress
1.0 h
Shoots
1


3212
Salt stress
1.0 h
Shoots
2


3221
Salt stress
1.0 h
Roots
1


3222
Salt stress
1.0 h
Roots
2


3311
Salt stress
3.0 h
Shoots
1


3312
Salt stress
3.0 h
Shoots
2


3321
Salt stress
3.0 h
Roots
1


3322
Salt stress
3.0 h
Roots
2


3411
Salt stress
6.0 h
Shoots
1


3412
Salt stress
6.0 h
Shoots
2


3421
Salt stress
6.0 h
Roots
1


3422
Salt stress
6.0 h
Roots
2


3511
Salt stress
12.0 h 
Shoots
1


3512
Salt stress
12.0 h 
Shoots
2


3521
Salt stress
12.0 h 
Roots
1


3522
Salt stress
12.0 h 
Roots
2


3611
Salt stress
24.0 h 
Shoots
1


3612
Salt stress
24.0 h 
Shoots
2


3621
Salt stress
24.0 h 
Roots
1


3622
Salt stress
24.0 h 
Roots
2


4711
Drought stress
0.25 h 
Shoots
1


4712
Drought stress
0.25 h 
Shoots
2


4721
Drought stress
0.25 h 
Roots
1


4722
Drought stress
0.25 h 
Roots
2


4111
Drought stress
0.5 h
Shoots
1


4112
Drought stress
0.5 h
Shoots
2


4121
Drought stress
0.5 h
Roots
1


4122
Drought stress
0.5 h
Roots
2


4211
Drought stress
1.0 h
Shoots
1


4212
Drought stress
1.0 h
Shoots
2


4221
Drought stress
1.0 h
Roots
1


4222
Drought stress
1.0 h
Roots
2


4311
Drought stress
3.0 h
Shoots
1


4312
Drought stress
3.0 h
Shoots
2


4321
Drought stress
3.0 h
Roots
1


4322
Drought stress
3.0 h
Roots
2


4411
Drought stress
6.0 h
Shoots
1


4412
Drought stress
6.0 h
Shoots
2


4421
Drought stress
6.0 h
Roots
1


4422
Drought stress
6.0 h
Roots
2


4511
Drought stress
12.0 h 
Shoots
1


4512
Drought stress
12.0 h 
Shoots
2


4521
Drought stress
12.0 h 
Roots
1


4522
Drought stress
12.0 h 
Roots
2


4611
Drought stress
24.0 h 
Shoots
1


4612
Drought stress
24.0 h 
Shoots
2


4621
Drought stress
24.0 h 
Roots
1


4622
Drought stress
24.0 h 
Roots
2


5111
Genotoxic stress
0.5 h
Shoots
1


5112
Genotoxic stress
0.5 h
Shoots
2


5121
Genotoxic stress
0.5 h
Roots
1


5122
Genotoxic stress
0.5 h
Roots
2


5211
Genotoxic stress
1.0 h
Shoots
1


5212
Genotoxic stress
1.0 h
Shoots
2


5221
Genotoxic stress
1.0 h
Roots
1


5222
Genotoxic stress
1.0 h
Roots
2


5311
Genotoxic stress
3.0 h
Shoots
1


5312
Genotoxic stress
3.0 h
Shoots
2


5321
Genotoxic stress
3.0 h
Roots
1


5322
Genotoxic stress
3.0 h
Roots
2


5411
Genotoxic stress
6.0 h
Shoots
1


5412
Genotoxic stress
6.0 h
Shoots
2


5421
Genotoxic stress
6.0 h
Roots
1


5422
Genotoxic stress
6.0 h
Roots
2


5511
Genotoxic stress
12.0 h 
Shoots
1


5512
Genotoxic stress
12.0 h 
Shoots
2


5521
Genotoxic stress
12.0 h 
Roots
1


5522
Genotoxic stress
12.0 h 
Roots
2


5611
Genotoxic stress
24.0 h 
Shoots
1


5612
Genotoxic stress
24.0 h 
Shoots
2


5621
Genotoxic stress
24.0 h 
Roots
1


5622
Genotoxic stress
24.0 h 
Roots
2


6111
Oxidative stress
0.5 h
Shoots
1


6112
Oxidative stress
0.5 h
Shoots
2


6124
Oxidative stress
0.5 h
Roots
1


6122
Oxidative stress
0.5 h
Roots
2


6211
Oxidative stress
1.0 h
Shoots
1


6212
Oxidative stress
1.0 h
Shoots
2


6223
Oxidative stress
1.0 h
Roots
1


6224
Oxidative stress
1.0 h
Roots
2


6311
Oxidative stress
3.0 h
Shoots
1


6312
Oxidative stress
3.0 h
Shoots
2


6323
Oxidative stress
3.0 h
Roots
1


6322
Oxidative stress
3.0 h
Roots
2


6411
Oxidative stress
6.0 h
Shoots
1


6412
Oxidative stress
6.0 h
Shoots
2


6421
Oxidative stress
6.0 h
Roots
1


6422
Oxidative stress
6.0 h
Roots
2


6511
Oxidative stress
12.0 h 
Shoots
1


6512
Oxidative stress
12.0 h 
Shoots
2


6523
Oxidative stress
12.0 h 
Roots
1


6524
Oxidative stress
12.0 h 
Roots
2


6611
Oxidative stress
24.0 h 
Shoots
1


6612
Oxidative stress
24.0 h 
Shoots
2


6621
Oxidative stress
24.0 h 
Roots
1


6622
Oxidative stress
24.0 h 
Roots
2


7711
UV-B stress
0.25 h 
Shoots
1


7712
UV-B stress
0.25 h 
Shoots
2


7721
UV-B stress
0.25 h 
Roots
1


7722
UV-B stress
0.25 h 
Roots
2


7111
UV-B stress
0.5 h
Shoots
1


7112
UV-B stress
0.5 h
Shoots
2


7121
UV-B stress
0.5 h
Roots
1


7122
UV-B stress
0.5 h
Roots
2


7211
UV-B stress
1.0 h
Shoots
1


7212
UV-B stress
1.0 h
Shoots
2


7221
UV-B stress
1.0 h
Roots
1


7222
UV-B stress
1.0 h
Roots
2


7311
UV-B stress
3.0 h
Shoots
1


7312
UV-B stress
3.0 h
Shoots
2


7321
UV-B stress
3.0 h
Roots
1


7322
UV-B stress
3.0 h
Roots
2


7411
UV-B stress
6.0 h
Shoots
1


7412
UV-B stress
6.0 h
Shoots
2


7421
UV-B stress
6.0 h
Roots
1


7422
UV-B stress
6.0 h
Roots
2


7511
UV-B stress
12.0 h 
Shoots
1


7512
UV-B stress
12.0 h 
Shoots
2


7521
UV-B stress
12.0 h 
Roots
1


7522
UV-B stress
12.0 h 
Roots
2


7611
UV-B stress
24.0 h 
Shoots
1


7612
UV-B stress
24.0 h 
Shoots
2


7621
UV-B stress
24.0 h 
Roots
1


7622
UV-B stress
24.0 h 
Roots
2


8715
Wounding stress
0.25 h 
Shoots
1


8712
Wounding stress
0.25 h 
Shoots
2


8723
Wounding stress
0.25 h 
Roots
1


8724
Wounding stress
0.25 h 
Roots
2


8111
Wounding stress
0.5 h
Shoots
1


8112
Wounding stress
0.5 h
Shoots
2


8124
Wounding stress
0.5 h
Roots
1


8126
Wounding stress
0.5 h
Roots
2


8211
Wounding stress
1.0 h
Shoots
1


8214
Wounding stress
1.0 h
Shoots
2


8224
Wounding stress
1.0 h
Roots
1


8225
Wounding stress
1.0 h
Roots
2


8313
Wounding stress
3.0 h
Shoots
1


8314
Wounding stress
3.0 h
Shoots
2


8324
Wounding stress
3.0 h
Roots
1


8325
Wounding stress
3.0 h
Roots
2


8411
Wounding stress
6.0 h
Shoots
1


8412
Wounding stress
6.0 h
Shoots
2


8423
Wounding stress
6.0 h
Roots
1


8424
Wounding stress
6.0 h
Roots
2


8511
Wounding stress
12.0 h 
Shoots
1


8512
Wounding stress
12.0 h 
Shoots
2


8524
Wounding stress
12.0 h 
Roots
1


8525
Wounding stress
12.0 h 
Roots
2


8611
Wounding stress
24.0 h 
Shoots
1


8612
Wounding stress
24.0 h 
Shoots
2


8624
Wounding stress
24.0 h 
Roots
1


8624_repl_8623
Wounding stress
24.0 h 
Roots
2


9711
Heat stress
0.25 h 
Shoots
1


9712
Heat stress
0.25 h 
Shoots
2


9721
Heat stress
0.25 h 
Roots
1


9722
Heat stress
0.25 h 
Roots
2


9111
Heat stress
0.5 h
Shoots
1


9112
Heat stress
0.5 h
Shoots
2


9121
Heat stress
0.5 h
Roots
1


9122
Heat stress
0.5 h
Roots
2


9211
Heat stress
1.0 h
Shoots
1


9212
Heat stress
1.0 h
Shoots
2


9221
Heat stress
1.0 h
Roots
1


9222
Heat stress
1.0 h
Roots
2


9311
Heat stress
3.0 h
Shoots
1


9312
Heat stress
3.0 h
Shoots
2


9321
Heat stress
3.0 h
Roots
1


9322
Heat stress
3.0 h
Roots
2


9811
Heat stress (3 h) + 1 h
4.0 h
Shoots
1


9812
Heat stress (3 h) + 1 h
4.0 h
Shoots
2


9821
Heat stress (3 h) + 1 h
4.0 h
Roots
1


9822
Heat stress (3 h) + 1 h
4.0 h
Roots
2


9411
Heat stress (3 h) + 3 h
6.0 h
Shoots
1


9412
Heat stress (3 h) + 3 h
6.0 h
Shoots
2


9421
Heat stress (3 h) + 3 h
6.0 h
Roots
1


9422
Heat stress (3 h) + 3 h
6.0 h
Roots
2


9511
Heat stress (3 h) + 9 h
12.0 h 
Shoots
1


9512
Heat stress (3 h) + 9 h
12.0 h 
Shoots
2


9521
Heat stress (3 h) + 9 h
12.0 h 
Roots
1


9522
Heat stress (3 h) + 9 h
12.0 h 
Roots
2


9611
Heat stress (3 h) +
24.0 h 
Shoots
1



21 h





9612
Heat stress (3 h) +
24.0 h 
Shoots
2



21 h





9621
Heat stress (3 h) +
24.0 h 
Roots
1



21 h





9622
Heat stress (3 h) +
24.0 h 
Roots
2



21 h





C0_1
Control
 0 h
Cell culture
1


C0_2
Control
 0 h
Cell culture
2


C1_1
Control
3.0 h
Cell culture
1


C1_2
Control
3.0 h
Cell culture
2


C2_1
Control
6.0 h
Cell culture
1


C2_2
Control
6.0 h
Cell culture
2


C3_1
Control
12.0 h 
Cell culture
1


C3_2
Control
12.0 h 
Cell culture
2


C4_1
Control
24.0 h 
Cell culture
1


C4_2
Control
24.0 h 
Cell culture
2


C5_1
Heat stress
0.25 h 
Cell culture
1


C5_2
Heat stress
0.25 h 
Cell culture
2


C6_1
Heat stress
0.5 h
Cell culture
1


C6_2
Heat stress
0.5 h
Cell culture
2


C7_1
Heat stress
1.0 h
Cell culture
1


C7_2
Heat stress
1.0 h
Cell culture
2


C8_1
Heat stress
3.0 h
Cell culture
1


C8_2
Heat stress
3.0 h
Cell culture
2


C9_1
Heat stress (3 h) + 1 h
4.0 h
Cell culture
1


C9_2
Heat stress (3 h) + 1 h
4.0 h
Cell culture
2


C10_1
Heat stress (3 h) + 3 h
6.0 h
Cell culture
1


C10_2
Heat stress (3 h) + 3 h
6.0 h
Cell culture
2


C11_1
Heat stress (3 h) + 9 h
12.0 h 
Cell culture
1


C11_2
Heat stress (3 h) + 9 h
12.0 h 
Cell culture
2


C12_1
Heat stress (3 h) +
24.0 h 
Cell culture
1



21 h





C12_2
Heat stress (3 h) +
24.0 h 
Cell culture
2



21 h





Treatment Codes


0—Control plants, Group Kudla


The plants were treated like the treated plants; e.g.: Transfer of Magenta boxes out of the climate chamber. Opening of the boxes and lifting the raft as long as the treatments last. Then boxes were transferred back to the climate chamber.


1—Cold stress (4° C.), Group Kudla


The Magenta boxes were placed on ice in the cold room (4° C.). The environmental light intensity was 20 μEinstein/cm2 sec. An extra light which was installed over the plants had 40 μEinstein/cm2 sec. The plants stayed there.


2—Osmotic stress, Group Kudla


Mannitol was added to a concentration of 300 mM in the Media. To add Mannitol the raft was lifted out A magnetic stir bar and a stirrer were used to mix the media and the added Mannitol. After the rafts were put back in the boxes, they were transferred back to the climate chamber.


3—Salt stress, Group Kudla


NaCl was added to a concentration of 150 mM in the Media. To add NaCl the raft was lifted out. A magnetic stir bar and a stirrer were used to mix the media and the added NaCl. After the rafts were put back in the boxes, they were transferred back to the climate chamber.


4—Drought stress, Group Kudla


The plants were stressed by 15 min. dry air stream (clean bench) until 10% loss of fresh weight; then incubation in closed vessels in the climate chamber.


5—Genotoxic stress, Group Puchta


Bleomycin + mitomycin (1.5 μg/ml bleomycin + 22 μg/ml mitomycin), were added to the indicated concentration in the Media. To add the reagents the raft was lifted out A magnetic stir bar and a stirrer were used to mix the media and the added reagents. After the rafts were put back in the boxes, they were transferred back to the climate chamber.


6—Oxidative stress, Group Bartels


Methyl Viologen was added to a final concentration of 10 μM in the Media. To add the reagent the raft was lifted out A magnetic stir bar and a stirrer were used to mix the media and the added reagent. After the rafts were put back in the boxes, they were transferred back to the climate chamber


7—UV-B stress, Group Harter


15 min. 1.18 W/m2 Philips TL40W/12


8—Wounding stress, Group Harter


Punctured with pins


9—Heat stress, Group Nover/von Koskull-Döring


38° C., samples taken at 0.25, 0.5, 1.0, 3.0 h of hs and +1, +3, +9, +21 h recovery at 25° C.


C—Heat stressed suspension culture, Group Nover/von Koskull-Döring


38° C., samples taken at 0.25, 0.5, 1.0, 3.0 h of hs and +1, +3, +9, +21 h recovery at 25° C.






Example 3
Testing Expression using Identified Regulatory Polynucleotides
Regulatory polynucleotide molecules may be tested using transient expression assays using tissue bombardment and protoplast transfections following standard protocols. Reporter constructs including the respective candidate regulatory polynucleotide molecules linked to GUS are prepared and bombarded into Arabidopsis tissue obtained from different plant organs using a PDS-1000 Gene Gun (BioRad). GUS expression is assayed to confirm expression from the candidate promoters.
To further assess the candidate regulatory polynucleotide molecules in stable transformed plants, the candidate molecules are synthesized and cloned into commercially available constructs using the manufacturer's instructions. Regulatory polynucleotide:: GFP fusions are generated in a binary vector containing a selectable marker using commercially available vectors and methods, such as those previously described (J. Y. Lee et al., Proc Natl Acad Sci USA 103, 6055 (Apr. 11, 2006)). The final constructs are transferred to Agrobacterium for transformation into Arabidopsis Columbia ecotype plants by the floral dip method (S. J. Clough, A. F. Bent, Plant J 16, 735 (December, 1998)). Transformed plants (T1) are selected by growth in the presence of the appropriate antibiotic or herbicide. Following selection, transformants are transferred to MS plates and allowed to recover.
For preliminary analysis, T1 root tips are excised, stained with propidium iodide and imaged for GFP fluorescence with a Zeiss 510 confocal microscope. Multiple T1 plants are analyzed per construct and multiple images along the longitudinal axis are taken in order to assess expression in the meristematic, elongation, and maturation zones of the root. In some cases expression may not be detectable as GFP fluorescence, but may detectable by qRT-PCR due to the higher sensitivity of the latter technique. Thus, qRT-PCR may also be used to detect the expression of GFP.
Example 4
Preparation and Expression Testing of Chimeric Regulatory Sequences
This example provides chimeric regulatory polynucleotides to test intron mediated enhancement (IME) in selected regulatory polynucleotides that lack a 5′-UTR intron (1 of the selected candidates lacks introns completely). Chimeric polynucleotide molecules were made in which the first intron from the coding sequence was fused to the 3′ end of the 5′-UTR. Consensus intron splice sites were included in these constructs to ensure efficient excision of the intron. Exemplary chimeric regulatory polynucleotide molecules prepared in this manner include those having the following nucleic acid sequences: SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, and SEQ ID NO: 20.
The identified regulatory polynucleotides can generally be subdivided into minimal or core promoters, and upstream activating sequences. To test whether the upstream activating sequences of the selected candidates could be used with different minimal promoters, chimeric regulatory polynucleotides were prepared in which the 35S-minimal promoter sequence (−46 to −1:5′-gcaagacccttcctctatataaggaagttcatttcatttggagagg) (SEQ ID NO: 23) was substituted for the −46 to −1 endogenous minimal promoter sequence relative to the transcription start site (as annotated in the TAIR database, http://www.Arabidopsis.org/) from each of the selected regulatory sequences. These substitutions were all made in the promoter cassette variants containing either an endogenous 5′-UTR intron or the first intron from the coding sequence. Exemplary chimeric regulatory polynucleotides prepared in this manner include those having the following nucleic acid sequences: SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 16, and SEQ ID NO: 19.
A summary of the all of the chimeric regulatory constructs made and tested is provided in Table 6, with each construct identified by the SEQ ID NO representing the nucleic acid sequence of the regulatory polynucleotide in the construct:







TABLE 6







Regulatory Polynucleotides












Sequence







ID of

endo-
endo-




Regulatory

genous
genous
1st intron
35S


Poly-

promoter-
5′-UTR
added
minimal


nucelotide
source
UTR seq.
intron
to A &
promoter


in Construct
gene ID
used (bp)
(bp)
C (bp)
substituted















1
AT1G13440
1107
—
 93
—


2
AT1G13440
1107
—
—
—


3
AT1G22840
293
—
479
—


4
AT1G22840
293
—
—
—


5
AT1G22840
293
—
479
Yes


6
AT1G52300
1099
—
102
—


7
AT1G52300
1099
—
—
—


8
AT1G52300
1099
—
102
Yes


9
AT4G37830
858
—
343
—


10
AT4G37830
858
—
—
—


11
AT4G37830
858
—
343
Yes


12
AT3G08580
707
493
—
—


13
AT3G08580
707
493
—
Yes


14
AT1G51650
815
—
749
—


15
AT1G51650
815
—
—
—


16
AT1G51650
815
—
749
Yes


17
AT3G48140
1043
—
157
—


18
AT3G48140
1043
—
—
—


19
AT3G48140
1043
—
157
Yes


20
AT3G08610
685
—
136
—


21
AT3G08610
685





22
AT3G62250
185
—
—
—









Candidate regulatory polynucleotide molecules were synthesized by contract (Blue Heron Biotechnology) and operably linked to a green fluorescent protein (GFP) coding sequence in a plant transformation binary vector. The final constructs were transferred to Agrobacterium and transformed into Arabidopsis Columbia ecotype plants by the floral dip method (S. J. Clough, A. F. Bent, Plant J 16, 735 (December, 1998)). Transformed plants (T1) were selected by growth on BASTA plates for 8 days. Following selection, transformants were transferred to MS plates and allowed to recover for 7-8 days. For preliminary analysis, T1 root tips were excised, stained with propidium iodide and imaged for GFP fluorescence with a Zeiss 510 confocal microscope. Five T1s were analyzed per construct and multiple images along the longitudinal axis were taken in order to assess expression in the meristematic, elongation and maturation zones of the root. The same sensitivity settings were used in all cases to provide semi-quantitative comparisons between images.
Semi-quantitative results from GFP intensity rankings are summarized in Table 7, with each construct identified by the SEQ ID NO representing the nucleic acid sequence of the regulatory polynucleotide in the construct. Some representative images of individual T1 seedlings are shown in FIGS. 34A-B through 38A-B. The images of FIGS. 34A-B through 38A-B show two channels, red and green, superimposed. The red channel shows cell wall staining and the green channel shows expression of GFP. The signal from the red channel was converted to white. Signal from the green channel was converted to grayscale such that the gray background outside of the root shows zero expression of GFP while any gray shade that is darker than the gray background outside of the root indicates







TABLE 7







GFP fluorescence in stably transformed Arabidopsis










Regulatory Polynucleotide




Sequence ID in Construct
Degree of GFP Fluorescence













1
+



2
+



3
++



4
+



5
++



6
+++++



7
+++



8
+



9
++++



10
*2



11
+



12
+++++



13
++++



14
++++



15
*2



16
+



17
+



18
+



19
+



20
+



21
−1



22
+++



23 (35S control)
+++





1− = no fluorescence detected under these conditions; no qRT-PCR data


2* = no fluorescence detected under these conditions but expression detected by qRT-PCR






GFP expression. FIGS. 34A-B through 38A-B show elongation and meristematic zones of representative plants containing constructs with the regulatory polynucleotides having the following nucleic acid sequences: SEQ ID NO: 6 (FIG. 34A: elongation zone, FIG. 34B: meristematic zone), SEQ ID NO: 9 (FIG. 35A: elongation zone, FIG. 35B: meristematic zone), SEQ ID NO: 12 (FIG. 36A: elongation zone, FIG. 36B: meristematic zone), SEQ ID NO: 13 (FIG. 37A: elongation zone, FIG. 37B: meristematic zone), and SEQ ID NO: 14 (FIG. 38A: elongation zone, FIG. 38B: meristematic zone).
All images were taken with the same microscope settings. Note that minimal differences were observed in T1s from the same construct. In some cases expression was not detectable as GFP fluorescence but was detectable by qRT-PCR due to the higher sensitivity of the latter technique.
Example 5
Generation of Derivative Regulatory Polynucleotides
This example illustrates the utility of derivatives of the native Arabidopsis regulatory polynucleotides. Derivatives of the Arabidopsis regulatory polynucleotides are generated by introducing mutations into the nucleotide sequence of the native regulatory polynucleotides. A plurality of mutagenized DNA segments derived from the Arabidopsis regulatory polynucleotides including derivatives with nucleotide deletions and modifications are generated and inserted into a plant transformation vector operably linked to a GUS marker gene. Each of the plant transformation vectors are prepared, for example, essentially as described in Example 3 above, except that the full length Arabidopsis polynucleotide is replaced by a mutagenized derivative of the Arabidopsis polynucleotide. Arabidopsis plants are transformed with each of the plant transformation vectors and analyzed for expression of the GUS marker to identify those mutagenized derivatives having regulatory activity.
Example 6
Identification of Regulatory Fragments
This example illustrates the utility of modified regulatory polynucleotides derived from the native Arabidopsis polynucleotides. Fragments of the polynucleotides are generated by designing primers to clone fragments of the native Arabidopsis regulatory polynucleotide. A plurality of cloned fragments of the polynucleotides ranging in size from 50 nucleotides up to about full length are obtained using PCR reactions with primers designed to amplify various size fragments instead of the full length polynucleotide. 3′ fragments from the 3′ end of the Arabidopsis regulatory polynucleotide comprising random fragments of about 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600 and 1650 nucleotides in length from various parts of the Arabidopsis regulatory polynucleotides are obtained and inserted into a plant transformation vector operably linked to a GUS marker gene. Each of the plant transformation vectors is prepared essentially as described, for example, in Example 3 above, except that the full length Arabidopsis polynucleotide is replaced by a fragment of the Arabidopsis regulatory polynucleotide or a combination of a 3′ fragment and a random fragment. Arabidopsis plants are transformed with each of the plant transformation vectors and analyzed for expression of the GUS marker to identify those fragments having regulatory activity.
Example 7
Identification of Orthologs
This example illustrates the identification and isolation of regulatory polynucleotides from organisms other than Arabidopsis using the native Arabidopsis polynucleotide sequences and fragments to query genomic DNA from other organisms in a publicly available nucleotide data bases including GENBANK. Orthologous genes in other organisms can be identified using reciprocal best hit BLAST methods as described in Moreno-Hagelsieb and Latimer, Bioinformatics (2008) 24:319-324. The Gramene.org database could also be queried to identify rice (Oryza sativa japonica) orthologs corresponding to the Arabidopsis genes whose regulatory elements were identified in Example 1 above. In some cases, the Arabidopsis genes may lack a rice ortholog and in other cases the Arabidopsis genes may have more than one ortholog.
Once an ortholog gene is identified, its corresponding regulatory polynucleotide sequence can be selected using methods described for Arabidopsis in Example 1. The full length polynucleotides may be cloned and inserted into a plant transformation vector which is used to transform Arabidopsis plants essentially as illustrated in Examples 3 or 4 above to verify regulatory activity and expression patterns.
Example 8
Arabidopsis Ubiquitin Regulatory Sequences
One Arabidopsis sequence identified using the technique of Example 1 was AT4g05320 (also referred to as the Arabidopsis polyubiquitin gene UBQ10). FIG. 32A provides the nucleotide sequence of the regulatory polynucleotide of the Arabidopsis gene having Accession No. AT4g05320 (SEQ ID NO: 24), with the sequence being annotated as described in Example 1. The expression pattern of the Arabidopsis ubiquitin gene was shown to be constitutive at the cell type/tissue level by the methods described in Example 1. Plots B and C (FIGS. 32B and 32C, respectively) are derived from data published by Brady et al. (Science, 318:801-806 (2007)) as discussed in Example 2 above. Plot B (FIG. 32B) provides the expression values of this gene in different cell types which were sorted on the basis of expressing the indicated GFP markers. Plot C (FIG. 32C) provides the expression values of this gene from root sections along the longitudinal axis of the root. FIG. 32D provides the developmental specific expression of AT4G05320. FIG. 32E provides the expression of AT4G05320 in response to various abiotic stresses. Plots D and E in FIG. 32 are derived from publically available expression data of the AtGeneExpress project (available on the World Wide Web at weigelworld.org/resources/microarray/AtGenExpress) also as discussed in Example 2. Plot D (FIG. 32D) shows developmental specific expression as described by Schmid et al. (Nat. Genet., 37: 501-506 (2005)). Plot E (FIG. 32E) shows expression in response to abiotic stress as described by Kilian et al. (Plant J., 50: 347-363 (2007)) as discussed above in Example 2.
A recombinant construct containing an approximately 1.2 kb fragment (including a 304 bp endogenous 5′-UTR intron) of the regulatory region from the Arabidopsis ubiquitin gene UBQ10 (corresponding to Accession No. AT4g05320) operably linked to the green fluorescence protein (GFP) coding sequence was prepared, and is referred to as construct A. A summary of the sequence used in Construct A is provided in Table 8.










TABLE 8






source
endogenous promoter-
endogenous



gene ID
UTR seq. used (bp)
5′-UTR intron (bp)








AT4G05320
1201
304









Construct A was transferred to Agrobacterium and transformed into Arabidopsis Columbia ecotype plants by the floral dip method (S. J. Clough, A. F. Bent, Plant J 16, 735 (December, 1998)). Transformed plants (T1) were selected by growth on BASTA plates for 8 days. Following selection, transformants were transferred to MS plates and allowed to recover for 7-8 days. For preliminary analysis, T1 root tips were excised, stained with propidium iodide and imaged for GFP fluorescence with a Zeiss 510 confocal microscope. Five T1s were analyzed and multiple images along the longitudinal axis were taken in order to assess expression in the meristematic, elongation and maturation zones of the root. The same sensitivity settings were used in all cases to provide semi-quantitative comparisons between images.
Two representative images are shown in FIGS. 39A-B. The images show two channels, red and green, superimposed. The red channel shows cell wall staining and the green channel shows expression of GFP. The signal from the red channel was converted to white. Signal from the green channel was converted to grayscale such that the gray background outside of the root shows zero expression of GFP while any gray shade that is darker than the gray background outside of the root indicates GFP expression. FIG. 39A shows the elongation zone and FIG. 39B shows the meristematic zone for a representative plant containing Construct A.
Additional T1 seedlings transformed with Construct A were selected, transferred to soil, and allowed to set seed. T2 seed was harvested from multiple T1 lines and single insertion lines were identified by 3:1 segregation of the selection marker in T2 seedlings. T2 seedlings from single insertion lines were grown under standard Murashige and Skoog (MS) media conditions and roots were analyzed for GFP fluorescence with a Zeiss 510 confocal microscope expression. Seedlings were then kept in MS media or transferred to high salt (MS+20 mM NaCl), low nitrogen (MS containing 0.5 mM N), or low pH (MS pH 4.6) conditions for 24 h. The roots were then again analyzed for GFP fluorescence to test expression responses to abiotic stress. The 3 stress conditions were validated to confer differential expression of known stress-responsive genes. One to seven T2 seedlings containing the transgene were analyzed per line and multiple images along the longitudinal axis were taken in order to assess expression in the meristematic, elongation and maturation zones of the root. The same sensitivity settings were used in all cases to provide quantitative comparisons between images. GFP expression in different cell-types was, determined from the images using a predefined root template. The template was calculated using a series of images manually segmented to find the root's “tissue percentage profile” (TPP), in which each region of interest in the template is a percentage of the root thickness at the specified location relative to the quiescent center (QC). Using different TPPs for each root zone, the images were segmented into different regions of interest (ROI) corresponding to different root cell-types. The average grayscale intensity of each ROI from the GFP fluorescence channel was then calculated and presented as the GFP Expression Index (GEI). The GEI varies from 0 and 1, which corresponds to no GFP expression (GEI=0) and complete saturation of GFP signal (GEI=1), respectively. FIGS. 33A, 33B, and 33C show the average GEI (±SEM) in different cell-types in 3 longitudinal zones under standard and 3 stress conditions. Note that the average GEI across all root regions for non-transgenic Arabidopsis seedlings (i.e. the background signal) is 0.0244±0.0011. These data show that the regulatory region used in construct A drives constitutive expression of GFP that was generally unresponsive to abiotic stress.
Example 9
Preparation and Quantitative Root Expression Testing of Identified Regulatory Elements in Stably Transformed Arabidopsis 
Six to sixteen additional BASTA resistant T1s, generated as described in Examples 4 and 5 and containing a construct with one of the regulatory polynucleotides represented by SEQ ID NOS: 1-22, 23, or 24, were selected per construct and allowed to set seed (T2 generation). At least two T2 lines per construct were identified that segregated 3:1 for herbicide resistance indicating they arose from single locus transgene insertion events. Plants from these lines were allowed to set seed (T3 generation) and homozygous T3 lines were identified for further characterization.
High resolution, quantitative measurements of GFP fluorescence in roots were then undertaken on two homozygous T3 lines for representative constructs. T3 seed from the two lines was grown in MS media in the RootArray, a device designed for confocal imaging of living plant roots under controlled conditions, and described in U.S. Patent Publication No. 2008/0141585 which is incorporated herein by reference in its entirety. After 5 days growth, the roots were stained with FM4-64 and imaged for GFP fluorescence in the meristematic zone, elongation zone and maturation zone with approximately 50 seedlings analyzed per line.
In order to yield quantitative results from image pixel intensities, imaging conditions and measurements were strictly controlled. The imaging normalization and calibration methods were based on two key measurements. First, on any day measurements are taken, a dilution series of an external reference fluorophore was quantitatively imaged. Second, the post objective laser intensity was directly measured before and after each RootArray experiment in order to account for variations in laser light intensity that may have occurred.
The dilution series that was imaged each day was prepared from a reference standard. The reference standard was prepared from a concentrated stock of Alexa Fluor 488® fluorophore in MES buffer (pH 6.0), with its concentration determined by spectrophotometry. Aliquots of the reference standard were stored at −20° C. as a master stock. For calibration use, a dilution series of the stock was prepared in a sealed, modified 96 well plate. The dilution series was stored at 4° C. in the dark and used for up to one month before being replaced. The Alexa Fluor® standard was verified to be stable under these conditions. The dilution series was imaged at the beginning of each day to characterize the performance of the detector and optics of the microscope as described below.
Tests have shown that laser light intensity can vary up to 10% at a given setting over the course of a RootArray experiment. To correct for this, laser power is measured before and after each RootArray experiment. The laser intensity is actively adjusted to 355±15 μW at 488 nm at the beginning of each experiment. The change in intensity measured at the end of a RootArray experiment was assumed to be due to a linear transition. Therefore, the estimated light intensity for a specific RootArray image was interpolated from that image's timestamp.
To correct for variations in laser intensity and detector response a model was developed to describe how Alexa Fluor® 488 fluorescence varied with laser intensity under the imaging conditions described herein. The laser correction model for Alexa Fluor 488® is based on the relative change of the dilution series slope versus the relative change of laser light intensity. Experiments have demonstrated that this relationship is independent of scan settings. This model was then adapted to GFP in root tissue with the addition of a GFP specific variable. This model is used to calculate a GFP expression index (GEI) as described in Equation 1 below (it is noted that the equation used to calculate the GFP Expression Index (GEI) in this example is slightly different from the equation used to calculate the GFP Expression Index in Example 8.




  
    
      
        
          GFP
          ⁢
          
            
          
          ⁢
          expression
          ⁢
          
            
          
          ⁢
          index
          ⁢
          
            
          
          ⁢
          
            (
            GEI
            )
          
        
        ⁢
        
          

        
        ⁢
        
          GEI
          =
          
            
              
                μ
                ⁡
                
                  (
                  
                    
                      roi
                      ⁡
                      
                        (
                        Img
                        )
                      
                    
                    -
                    
                      bkg
                      ⁡
                      
                        (
                        Img
                        )
                      
                    
                  
                  )
                
              
              
                
                  α
                  AF
                  DS
                
                ⁢
                
                  β
                  Sat
                
              
            
            ⁢
            
              γ
              AF
              DS
            
            ⁢
            
              γ
              AF
              Img
            
            ⁢
            
              δ
              GFP
              Img
            
          
        
      
    
    
      
        Equation
        ⁢
        
          
        
        ⁢
        1
      
    
  




roi (1 mg): The pixel population for the quantification channel (green channel) over a selected region of interest. In this case each ROI is a tissue type.
bkg (1 mg): The background pixel value for every experimental image is characterized with a novel statistics based approach.
αAFDS: Normalized slope of the dilution series standard.
γAFDS: Laser correction factor for Alexa Fluor 488® fluorophore to normalize the dilution series to the reference laser power (355 μW at 488 nm).
γAFImg: Laser correction factor for Alexa Fluor 488® fluorophore at the laser power the GFP image was taken.
δGFPImg: Relative laser correction factor for GFP fluorophore in the experimental image.
βSat: Normalization constant to prevent pixel oversaturation of the detector when the image was acquired.
The green channel image signal passes through this function to produce the GEI, a metric of fluorescent intensity that allows for comparison across RootArrays over time. The background of each experimental image was calculated as described below and subsequently subtracted from the pixel population of the region of interest. The negative values were zeroed to create an image with minimal background noise. The mean of corrected pixel intensities was divided by the slope of the dilution series to convert the pixel output to a metric of light intensity relative to the dilution series standard. The first gamma value (γAFDS) is a laser correction factor that adjusts the slope of the dilution series to what it would be if the dilution series was imaged at exactly 355 μW. The next gamma (γAFImg) and the delta values (δGFPImg) correct the GFP signal to what it would be if the root was imaged at exactly 355 μW. It is noted that all correction factors typically varied by less than 5% between experiments.
Regions of interest that have a strong signal near the point of pixel oversaturation of the detector did not exhibit a linear relationship with GFP expression. Therefore a normalization constant βSat was included to limit the scope of the dynamic bit range of the detector and the GEI is capped at 1 to preserve its linear correlation with GFP expression for all reported values <1. To calculate the background of an image(bkg (Img)), the image was first split into a grid of squares and the pixel population of each square is examined. A small number of squares was initially selected based on having the lowest percentile rankings in terms of standard deviation, 95th percentile pixel value, mean, median, and gradient magnitude. The pixel populations in the initial “seed” squares, which are assumed to be background, were then compared against the pixel populations of all other squares in a one tailed unpaired t test in order to categorize each square as “background” or “non-background”. The median pixel intensity of all squares determined to be “background” was then used as the bkg (1 mg) value in Equation 1. Tests have shown that this algorithm robustly selected background pixel populations even if there were several roots in the field of view.
The correspondence of regions of interest to different cell-types was determined from the images using a predefined root template. The template was calculated using a series of images manually segmented to find the root's “tissue percentage profile” (TPP), in which each region of interest in the template is a percentage of the root thickness at the specified location relative to the quiescent center (QC). Using different TPPs for each root zone, the images were segmented into different regions of interest (ROI) corresponding to different root cell-types. Specifically, the regions determined in all three developmental zones were the epidermis, the cortex, the endodermis, and the stele. In addition to these four regions, the root cap and the quiescent center were also determined in the meristematic zone.
To determine if a particular transgenic line exhibited significant GFP expression in an ROI, the GEI measurements for each of the 14 tissue-zone ROIs were compared to the corresponding values determined from 48 non-transgenic Arabidopsis Columbia ecotype seedlings grown under identical conditions. Significance was determined using a one-tailed Welch's t-test with a cutoff of p<0.01.
The average GEI for each of the 14 tissue-zone ROIs for 2 representative lines of each regulatory molecule that passed prescreening is shown in Tables 9-11. All values represent significant expression (p<0.01) unless indicated by bold italics. The GEIS measured from seedlings containing a 35S promoter-GFP transgene are shown for comparison. The 35S promoter is widely used in plant biotechnology and considered a standard for strong promoters. The GEIs measured from seedlings containing the regulatory region from the UBQ10 gene (see Example 8) are also shown for comparison. These data show that the regulatory polynucleotides listed in Tables 9-11 generally drive constitutive expression in the root.
Table 9 shows the GEI values of promoter sequences in regions of the meristematic zone.









TABLE 9








Gene from which




promoter is derived


Promoter
(“+ intron” is listed where


Sequence
the first intron from


(SEQ ID
coding sequence was
Meristem














NO)
added as described above)
epidermis
cortex
endodermis
stele
QC
Root cap

















23 (35S

0.396
0.282
0.236
0.229
0.957
1.000


control)


23 (35S

0.505
0.253
0.206
0.189
0.766
0.844


control)


1
AT1G13440 + intron
 
 
 
 
0.014
0.008


1
AT1G13440 + intron
0.009
0.009
0.008
0.006
0.014
0.009


2
AT1G13440
0.015
0.060
0.039
0.016
0.210
0.018


2
AT1G13440
0.013
0.015
0.012
0.009
0.047
0.014


3
AT1G22840 + intron
0.068
0.059
0.057
0.048
0.056
0.070


3
AT1G22840 + intron
0.037
0.036
0.033
0.025
0.030
0.037


4
AT1G22840
0.008
0.008
0.007
 
 
0.008


4
AT1G22840
0.009
0.008
0.007
0.006
0.008
0.008


6
AT1G52300 + intron
0.889
0.850
0.808
0.657
0.421
0.471


6
AT1G52300 + intron
0.907
0.897
0.899
0.870
0.701
0.531


7
AT1G52300
0.130
0.126
0.125
0.109
0.042
0.038


7
AT1G52300
0.322
0.320
0.326
0.304
0.089
0.062


9
AT4G37830 + intron
0.522
0.445
0.425
0.382
0.494
0.584


9
AT4G37830 + intron
0.143
0.128
0.123
0.112
0.147
0.167


10
AT4G37830
0.009
0.008
 
 
 
 


10
AT4G37830
0.009
0.008
0.008
 
 
0.008


12
AT3G08580
0.850
0.792
0.753
0.678
0.858
0.891


12
AT3G08580
0.991
0.993
0.992
0.992
0.997
0.999


14
AT1G51650 + intron
0.695
0.578
0.559
0.504
0.723
0.821


14
AT1G51650 + intron
0.645
0.466
0.435
0.381
0.603
0.646


15
AT1G51650
 
 
 
 
 
 


15
AT1G51650
 
 
 
 
 
 


17
AT3G48140 + intron
0.056
0.034
0.030
0.019
0.034
0.121


17
AT3G48140 + intron
0.027
0.019
0.016
0.013
0.021
0.054


18
AT3G48140
0.010
 
 
 
 
 


18
AT3G48140
0.011
0.008
0.007
0.006
0.011
0.037


20
AT3G08610 + intron
0.048
0.030
0.027
0.024
0.052
0.071


20
AT3G08610 + intron
0.041
0.028
0.026
0.023
0.044
0.064


21
AT3G08610
 
 
 
 
 
0.008


21
AT3G08610
 
 
 
 
 
0.008


22
AT3G62250
0.295
0.277
0.263
0.186
0.198
0.150


22
AT3G62250
0.292
0.286
0.269
0.195
0.206
0.165


24
AT4G05320 (UBQ10)
0.711
0.551
0.493
0.405
0.962
1


24
AT4G05320 (UBQ10)
0.313
0.219
0.194
0.129
0.296
0.798









Table 10 shows the GEI values of promoter sequences in regions of the elongation zone.









TABLE 10








Gene from which




promoter is derived




(“+intron” is listed



Promoter
where the first



Sequence
intron from coding
Elongation












(SEQ
sequence was added
epi-

endo-



ID NO)
as described above)
dermis
cortex
dermis
stele















23 (35S

0.240
0.083
0.084
0.195


control)







23 (35S

0.137
0.045
0.051
0.132


control)







1
AT1G13440 + intron
 
 
 
0.005


1
AT1G13440 + intron
 
 
 
 


2
AT1G13440
0.006
0.012
0.011
0.007


2
AT1G13440
 
 
 
 


3
AT1G22840 + intron
0.039
0.022
0.016
0.018


3
AT1G22840 + intron
0.022
0.013
0.011
0.011


4
AT1G22840
 
 
 
 


4
AT1G22840
0.006
 
 
 


6
AT1G52300 + intron
0.343
0.251
0.225
0.249


6
AT1G52300 + intron
0.339
0.225
0.196
0.251


7
AT1G52300
0.038
0.025
0.020
0.025


7
AT1G52300
0.078
0.050
0.044
0.057


9
AT4G37830 + intron
0.272
0.172
0.136
0.145


9
AT4G37830 + intron
0.064
0.043
0.035
0.037


10
AT4G37830
0.006
 
 
 


10
AT4G37830
 
 
 
 


12
AT3G08580
0.556
0.374
0.293
0.285


12
AT3G08580
0.987
0.863
0.687
0.693


14
AT1G51650 + intron
0.511
0.343
0.286
0.300


14
AT1G51650 + intron
0.315
0.212
0.174
0.195


15
AT1G51650
 
 
 
 


15
AT1G51650
 
 
 
 


17
AT3G48140 + intron
0.021
0.011
0.009
0.010


17
AT3G48140 + intron
0.011
0.007
0.007
0.008


18
AT3G48140
 
 
 
 


18
AT3G48140
0.007
0.005
0.005
0.005


20
At3G08610 + intron
0.034
0.019
0.014
0.014


20
At3G08610 + intron
0.027
0.016
0.012
0.013


21
At3G08610
0.007
0.005
 
 


21
At3G08610
 
 
 
 


22
AT3G62250
0.099
0.056
0.043
0.043


22
AT3G62250
0.078
0.054
0.043
0.042


24
AT4G05320 (UBQ10)
0.404
0.271
0.224
0.247


24
AT4G05320 (UBQ10)
0.223
0.171
0.131
0.116









Table 11 shows the GEI values of promoter sequences in regions of the maturation zone.









TABLE 11








Gene from which




promoter is derived




(“+intron” is listed



Promoter
where the first



Sequence
intron from coding
Maturation












(SEQ ID
sequence was added
epi-

endo-



NO)
as described above)
dermis
cortex
dermis
stele





23 (355

0.235
0.216
0.310
0.545


control)







23 (355

0.271
0.289
0.439
0.674


control)







1
AT1G13440 + intron
 
 
 
0.027


1
AT1G13440 + intron
 
 
 
0.010


2
AT1G13440
 
0.011
0.015
0.012


2
AT1G13440
 
 
0.007
0.015


3
AT1G22840 + intron
0.019
0.014
0.020
0.029


3
AT1G22840 + intron
0.014
0.010
0.014
0.019


4
AT1G22840
 
 
 
 


4
AT1G22840
 
 
 
 


6
AT1G52300 + intron
0.054
0.057
0.103
0.192


6
AT1G52300 + intron
0.074
0.052
0.076
0.211


7
AT1G52300
0.011
0.008
0.011
0.018


7
AT1G52300
0.015
0.013
0.019
0.041


9
AT4G37830 + intron
0.097
0.111
0.158
0.259


9
AT4G37830 + intron
0.027
0.030
0.042
0.067


10
AT4G37830
 
 
 
 


10
AT4G37830
 
 
 
 


12
AT3G08580
0.175
0.162
0.221
0.470


12
AT3G08580
0.327
0.327
0.457
0.846


14
AT1G51650 + intron
0.169
0.160
0.259
0.432


14
AT1G51650 + intron
0.142
0.104
0.161
0.267


15
AT1G51650
 
 
 
 


15
AT1G51650
 
 
 
 


17
AT3G48140 + intron
0.021
0.014
0.016
0.024


17
AT3G48140 + intron
0.012
0.008
0.009
0.015


18
AT3G48140
 
 
0.006
0.006


18
AT3G48140
0.010
0.006
0.006
0.007


20
At3G08610 + intron
0.014
0.011
0.015
0.023


20
At3G08610 + intron
0.014
0.010
0.013
0.020


21
At3G08610
 
 
 
 


21
At3G08610
 
 
 
 


22
AT3G62250
0.022
0.014
0.018
0.074


22
AT3G62250
0.015
0.013
0.016
0.040


24
AT4G05320 (UBQ10)
0.468
0.488
0.62 
0.819


24
AT4G05320 (UBQ10)
0.291
0.386
0.469
0.626









Example 10
Expression Testing of Regulatory Polynucleotides in Aerial Tissue of Stably Transformed Arabidopsis 
Expression of GFP in aerial tissue of stably transformed Arabidopsis was assessed by qRT-PCR in two homozygous T3 lines of some of the regulatory polynucleotides that were demonstrated to confer significant expression in all 14 tissue-zone ROIs of the root. T3 seeds from each line were grown on MS agar plates. After 7-11 days the aerial portions of approximately 10 plants were collected in triplicate for RNA extraction and cDNA synthesis. Tissue was homogenized in liquid nitrogen via bead milling and total RNA was extracted using the Allprep DNA/RNA kit (Qiagen). cDNA was generated from total RNA using the Superscript VILO cDNA synthesis kit (Invitrogen) per the manufacturer's instructions. Multiplex qPCR TaqMan® assays were conducted using either the CFX96 Real-Time PCR Detection System or the iCycler iQ Real-Time PCR Detection System (both instruments are from Bio-Rad Laboratories) with primers and probes specific for GFP and the “housekeeping” gene UBC9. Three technical qRT-PCR replicates were performed on each biological replicate, and data was processed using CFX Manager software (Bio-Rad).
To determine relative GFP expression level, PCR reaction efficiency was calculated using LinRegPCR software (Ruijter) and verified using a standard curve based method. Ct and baseline threshold values were obtained from the CFX Manager software. Data analysis was performed using the statistics package R, available at the R Project for Statistical Computing. After correcting the Ct values for reaction efficiency, the relative GFP expression was calculated by subtracting the Ct of the UBC control from that of GFP, followed by averaging across all replicates. To assess statistical significance of the data, the relative GFP expression of each line was compared to that determined from non-transgenic Arabidopsis ecotype Columbia seedlings using a one-tailed Welch's t-test. All statistical analysis was performed on the corrected Ct values, but these values were exponentiated to a linear expression scale for presentation. To normalize the linear expression scale, the data was expressed relative to a 35S-promoter control that was included in all experiments. The 35S-promoter control value was set to 100 on this scale.
Aerial expression data for regulatory polynucleotides that drove constitutive expression in Arabidopsis roots is shown in Table 12 (expression data for the regulatory region from the UBQ10 gene (see Example 8) is also shown for comparison). All expression measurements were statistically significant (p<0.01). These data show that regulatory polynucleotides that drove constitutive GFP expression in Arabidopsis roots also drove GFP expression in Arabidopsis aerial tissue.









TABLE 12





Gene from which promoter is




derived (“+intron” is listed where




the first intron from coding sequence
Promoter Sequence
Relative


was added as described above)
(SEQ ID NO)
Expression

















AT4G05320 (UBQ 10)
24
0.66


AT4G05320 (UBQ 10)
24
0.16


AT1G52300 + intron
6
0.03


AT1G52300 + intron
6
0.04


AT4G37830 + intron
9
0.10


AT4G37830 + intron
9
0.03


AT3G62250
22
0.01


AT3G62250
22
0.01









Thus, the methods disclosed herein are useful to identify regulatory polynucleotides that are capable of regulating constitutive expression of an operably linked polynucleotide.
While the invention has been described in detail and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that various changes and modifications can be made without departing from the spirit and scope of the invention.

## Claims

1. A regulatory polynucleotide
comprising a nucleic acid molecule of SEQ ID NOs: 9 wherein the nucleic acid molecule is capable of regulating transcription of an operably linked transcribable polynucleotide molecule.

2. A recombinant polynucleotide construct comprising the regulatory polynucleotide of claim 1 operably linked to a transcribable polynucleotide molecule.

3. The recombinant polynucleotide construct of claim 2, wherein the transcribable polynucleotide molecule encodes a protein of agronomic interest.

4. The recombinant polynucleotide construct of claim 2, wherein the transcribable polynucleotide molecule is operably linked to a 3′ transcription termination polynucleotide molecule.

5. A chimeric polynucleotide molecule comprising the regulatory polynucleotide of claim 1 operably linked to
a second polynucleotide molecule capable of regulating transcription of an operably linked polynucleotide molecule.

6. The chimeric polynucleotide of claim 5, wherein the second polynucleotide molecule is selected from the group consisting of a cis-element, an enhancer element, and an intron.

7. The chimeric polynucleotide of claim 6, wherein the second polynucleotide molecule comprises an intron.

8. A transgenic host cell comprising the recombinant polynucleotide construct of claim 2.

9. The transgenic host cell of claim 8, wherein the host cell is a plant cell.

10. A transgenic plant stably transformed with the recombinant polynucleotide construct of claim 2.

11. The transgenic plant of claim 10, wherein the plant is selected from the group consisting of a monocotyledonous and a dicotyledonous plant.

12. The transgenic plant of claim 11, wherein the plant is a monocotyledonous plant selected from the group consisting of wheat, corn, rice, turf grass, millet, sorghum, switchgrass, miscanthus, sugarcane, and Brachypodium.

13. transgenic plant of claim 11, wherein the plant is a dicotyledonous plant selected from the group consisting of soybean, cotton, canola, and potato.

14. A seed produced by the transgenic plant of claim 10, wherein said seed comprises said recombinant polynucleotide construct.

15. A method of directing expression of a transcribable polynucleotide molecule in a host cell comprising:
(a) introducing the recombinant polynucleotide construct of claim 2 into a host cell to produce a transgenic host cell; and
(b) selecting a transgenic host cell exhibiting expression of the transcribable polynucleotide molecule.

16. The method of claim 15, wherein the transcribable polynucleotide molecule is selected from the group consisting of a coding sequence and a functional RNA.

17. The method of claim 15, wherein the host cell is a plant cell.

18. The method of claim 17, further comprising regenerating a plant comprising the introduced recombinant polynucleotide construct.

19. A method of directing expression of a transcribable polynucleotide molecule in a plant comprising:
(a) introducing the recombinant polynucleotide construct of claim 2 into a plant cell;
(b) regenerating a plant from the plant cell; and
(c) selecting a transgenic plant exhibiting expression of the transcribable polynucleotide molecule.

20. The method of claim 19, wherein the transcribable polynucleotide molecule is selected from the group consisting of a coding sequence and a functional RNA.