U.S. patent application number 15/502129 was filed with the patent office on 2017-08-10 for plant regulatory elements and methods of use thereof.
This patent application is currently assigned to PIONEER HI-BRED INTERNATIONAL, INC.. The applicant listed for this patent is E.I.DU PONT DE NEMOURS AND COMPANY, PIONEER HI-BRED INTERNATIONAL, INC.. Invention is credited to SCOTT DIEHN, ALBERT LU, MICHELLE VAN ALLEN.
Application Number | 20170226525 15/502129 |
Document ID | / |
Family ID | 54015169 |
Filed Date | 2017-08-10 |
United States Patent
Application |
20170226525 |
Kind Code |
A1 |
DIEHN; SCOTT ; et
al. |
August 10, 2017 |
PLANT REGULATORY ELEMENTS AND METHODS OF USE THEREOF
Abstract
The present disclosure provides compositions and methods for
regulating expression of heterologous nucleotide sequences in a
plant. Compositions include a novel nucleotide sequence for
regulatory elements from Lamium Leaf Distortion Associated Virus. A
method for expressing a heterologous nucleotide sequence in a plant
using the regulatory element sequences disclosed herein is
provided. The method comprises transforming a plant or plant cell
with a nucleotide sequence operably linked to one of the regulatory
elements of the present disclosure.
Inventors: |
DIEHN; SCOTT; (WEST DES
MOINES, IA) ; VAN ALLEN; MICHELLE; (URBANDALE,
IA) ; LU; ALBERT; (WEST DES MOINES, IA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
PIONEER HI-BRED INTERNATIONAL, INC.
E.I.DU PONT DE NEMOURS AND COMPANY |
JOHNSTON
Wilmington |
IA
DE |
US
US |
|
|
Assignee: |
PIONEER HI-BRED INTERNATIONAL,
INC.
JOHNSTON
IA
|
Family ID: |
54015169 |
Appl. No.: |
15/502129 |
Filed: |
July 30, 2015 |
PCT Filed: |
July 30, 2015 |
PCT NO: |
PCT/US2015/042842 |
371 Date: |
February 6, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62034970 |
Aug 8, 2014 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 2730/00022
20130101; A01H 1/00 20130101; C12N 15/8216 20130101 |
International
Class: |
C12N 15/82 20060101
C12N015/82; A01H 1/00 20060101 A01H001/00 |
Claims
1. A recombinant polynucleotide selected from: (a) a polynucleotide
having at least 85 percent sequence identity to the nucleic acid
sequence of SEQ ID NO: 1 or, (b) a polynucleotide comprising the
nucleic acid sequence of SEQ ID NO: 1, (c) the polynucleotide of
SEQ ID NO: 1, and (c) a fragment of SEQ ID NO: 1.
2. The recombinant polynucleotide of claim 1, wherein the
polynucleotide has at least 90 percent sequence identity to the
nucleic acid sequence of SEQ ID NO: 1.
3. The recombinant polynucleotide of claim 1, wherein the
polynucleotide has at least 95 percent sequence identity to a
nucleic acid sequence of SEQ ID NO: 1.
4. The recombinant polynucleotide of claim 1, wherein the
polynucleotide is selected from: (a) a polynucleotide having at
least 85 percent sequence identity to the nucleic acid sequence of
SEQ ID NO: 2 or, (b) a polynucleotide comprising the nucleic acid
sequence of SEQ ID NO: 2, (c) the polynucleotide of SEQ ID NO: 2,
and (c) a fragment of SEQ ID NO: 2.
5. The recombinant polynucleotide of claim 1, wherein the
polynucleotide is selected from: (a) a polynucleotide having at
least 85 percent sequence identity to the nucleic acid sequence of
SEQ ID NO: 3 or, (b) a polynucleotide comprising the nucleic acid
sequence of SEQ ID NO: 3, (c) the polynucleotide of SEQ ID NO: 3,
and (c) a fragment of SEQ ID NO: 3.
6. The recombinant polynucleotide of claim 1, wherein the
polynucleotide is selected from: (a) a polynucleotide having at
least 85 percent sequence identity to the nucleic acid sequence of
SEQ ID NO: 4 or, (b) a polynucleotide comprising the nucleic acid
sequence of SEQ ID NO: 4, (c) the polynucleotide of SEQ ID NO: 4,
and (c) a fragment of SEQ ID NO: 4.
7. The recombinant polynucleotide of claim 1, wherein the
polynucleotide is selected from: (a) a polynucleotide having at
least 85 percent sequence identity to the nucleic acid sequence of
SEQ ID NO: 5 or, (b) a polynucleotide comprising the nucleic acid
sequence of SEQ ID NO: 5, (c) the polynucleotide of SEQ ID NO: 5,
and (c) a fragment of SEQ ID NO: 5.
8. The recombinant polynucleotide of claim 1, wherein the
polynucleotide is selected from: (a) a polynucleotide having at
least 85 percent sequence identity to the nucleic acid sequence of
SEQ ID NO: 6 or, (b) a polynucleotide comprising the nucleic acid
sequence of SEQ ID NO: 6, (c) the polynucleotide of SEQ ID NO: 6,
and (c) a fragment of SEQ ID NO: 6.
9. A DNA construct; comprising (a) a regulatory element
polynucleotide selected from the group consisting of: (i) a
polynucleotide having at least 85 percent sequence identity to the
nucleic acid sequence of SEQ ID NO: 1, (ii) a polynucleotide
comprising the nucleic acid sequence of SEQ ID NO: 1 (iii) the
polynucleotide of SEQ ID NO: 1, and (iv) a fragment of SEQ ID NO:
1; and (b) a heterologous transcribable polynucleotide molecule
operably linked to the regulatory element polynucleotide.
10. The DNA construct of claim 9, wherein the regulatory element
polynucleotide has at least 90 percent sequence identity to the
nucleic acid sequence of SEQ ID NO: 1.
11. The DNA construct of claim 9, wherein the regulatory element
polynucleotide has at least 95 percent sequence identity to the
nucleic acid sequence of SEQ ID NO: 1.
12. The DNA construct of claim 9; comprising (a) a regulatory
element polynucleotide selected from the group consisting of: (i) a
polynucleotide having at least 85 percent sequence identity to the
nucleic acid sequence of SEQ ID NO: 2, (ii) a polynucleotide
comprising the nucleic acid sequence of SEQ ID NO: 2 (iii) the
polynucleotide of SEQ ID NO: 2, and (iv) a fragment of SEQ ID NO:
2; and (b) a heterologous transcribable polynucleotide molecule
operably linked to the regulatory element polynucleotide.
13. The DNA construct of claim 9; comprising (a) a regulatory
element polynucleotide selected from the group consisting of: (i) a
polynucleotide having at least 85 percent sequence identity to the
nucleic acid sequence of SEQ ID NO: 3, (ii) a polynucleotide
comprising the nucleic acid sequence of SEQ ID NO: 3 (iii) the
polynucleotide of SEQ ID NO: 3, and (iv) a fragment of SEQ ID NO:
3; and (b) a heterologous transcribable polynucleotide molecule
operably linked to the regulatory element polynucleotide.
14. The DNA construct of claim 9; comprising (a) a regulatory
element polynucleotide selected from the group consisting of: (i) a
polynucleotide having at least 85 percent sequence identity to the
nucleic acid sequence of SEQ ID NO: 4, (ii) a polynucleotide
comprising the nucleic acid sequence of SEQ ID NO: 4 (iii) the
polynucleotide of SEQ ID NO: 4, and (iv) a fragment of SEQ ID NO:
4; and (b) a heterologous transcribable polynucleotide molecule
operably linked to the regulatory element polynucleotide.
15. The DNA construct of claim 9; comprising (a) a regulatory
element polynucleotide selected from the group consisting of: (i) a
polynucleotide having at least 85 percent sequence identity to the
nucleic acid sequence of SEQ ID NO: 5, (ii) a polynucleotide
comprising the nucleic acid sequence of SEQ ID NO: 5 (iii) the
polynucleotide of SEQ ID NO: 5, and (iv) a fragment of SEQ ID NO:
5; and (b) a heterologous transcribable polynucleotide molecule
operably linked to the regulatory element polynucleotide.
16. The DNA construct of claim 9; comprising (a) a regulatory
element polynucleotide selected from the group consisting of: (i) a
polynucleotide having at least 85 percent sequence identity to the
nucleic acid sequence of SEQ ID NO: 6, (ii) a polynucleotide
comprising the nucleic acid sequence of SEQ ID NO: 6 (iii) the
polynucleotide of SEQ ID NO: 6, and (iv) a fragment of SEQ ID NO:
6; and (b) a heterologous transcribable polynucleotide molecule
operably linked to the regulatory element polynucleotide.
17. The DNA construct of claim 9, wherein the heterologous
transcribable polynucleotide molecule is a gene of agronomic
interest.
18. The DNA construct of claim 17, wherein the heterologous
transcribable polynucleotide molecule is a gene capable of
providing herbicide resistance in plants.
19. The DNA construct of claim 17, wherein the heterologous
transcribable polynucleotide molecule is a gene capable of
providing plant pest control in plants.
20. A heterologous cell stably transformed with the nucleic acid
molecule of claim 1.
21. A transgenic plant or plant cell stably transformed with the
DNA construct of claim 9.
22. The transgenic plant or plant cell of claim 21, wherein the
transgenic plant is a dicotyledon plant cell.
23. The transgenic plant or plant cell of claim 21, wherein the
transgenic plant is a monocotyledon plant cell.
24. A seed of the transgenic plant of claim 21, wherein the seed
comprises the DNA construct.
Description
REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY
[0001] A sequence listing having the file name
"5970P_Sequence_Listing.TXT" created on Jul. 18, 2014, and having a
size of 7.4 kilobytes is filed in computer readable form
concurrently with the specification. The sequence listing is part
of the specification and is herein incorporated by reference in its
entirety.
FIELD
[0002] The present disclosure relates to the field of plant
molecular biology, more particularly to regulation of gene
expression in plants.
BACKGROUND
[0003] Expression of heterologous DNA sequences in a plant host is
dependent upon the presence of an operably linked regulatory
element that is functional within the plant host. Choice of the
regulatory element sequence will determine when and where within
the organism the heterologous DNA sequence is expressed. Where
expression in specific tissues or organs is desired,
tissue-preferred regulatory elements may be used. Where gene
expression in response to a stimulus is desired, inducible
regulatory elements are the regulatory element of choice. In
contrast, where continuous expression is desired throughout the
cells of a plant, constitutive promoters are utilized. Additional
regulatory sequences upstream and/or downstream from the core
regulatory element sequence may be included in the expression
constructs of transformation vectors to bring about varying levels
of expression of heterologous nucleotide sequences in a transgenic
plant.
[0004] Frequently it is desirable to express a DNA sequence
constitutively in a plant. For example, increased resistance of a
plant to infection by soil- and air-borne pathogens might be
accomplished by genetic manipulation of the plant's genome to
comprise a constitutive regulatory element operably linked to a
heterologous pathogen-resistance gene such that pathogen-resistance
proteins are produced in the desired plant tissue.
[0005] Alternatively, it might be desirable to inhibit expression
of a native DNA sequence within a plant's tissues to achieve a
desired phenotype. In this case, such inhibition might be
accomplished with transformation of the plant to comprise a
constitutive promoter operably linked to an antisense nucleotide
sequence, such that expression of the antisense sequence produces
an RNA transcript that interferes with translation of the mRNA of
the native DNA sequence.
[0006] Genetically altering plants through the use of genetic
engineering techniques and thus producing a plant with useful
traits requires the availability of a variety of promoters. An
accumulation of promoters would enable the investigator to design
recombinant DNA molecules that are capable of being expressed at
desired levels and cellular locales. Therefore, a collection of
constitutive promoters would allow for a new trait to be expressed
at the desired level in the desired tissue. Thus, isolation and
characterization of constitutive regulatory elements that can serve
as regulatory regions for expression of heterologous nucleotide
sequences of interest in a measured constitutive manner are needed
for genetic manipulation of plants.
BRIEF SUMMARY
[0007] Compositions and methods for regulating expression of a
heterologous nucleotide sequence of interest in a plant or plant
cell are provided. DNA molecules comprising novel nucleotide
sequences for regulatory elements that initiate transcription are
provided. In some embodiments the regulatory element has promoter
activity initiating transcription in the plant cell. Embodiments of
the disclosure comprise the nucleic acid sequence set forth in SEQ
ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5,
SEQ ID NO: 6, or a complement thereof, a nucleotide sequence
comprising at least 20 contiguous nucleotides of SEQ ID NO: 1, SEQ
ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6,
wherein said sequence initiates transcription in a plant cell, and
a nucleotide sequence comprising a sequence having at least 85%
sequence identity to the sequence set forth in SEQ ID NO: 1, SEQ ID
NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6,
wherein said sequence initiates transcription in the plant
cell.
[0008] A method for expressing a heterologous nucleotide sequence
in a plant or plant cell is provided. The method comprises
introducing into a plant or a plant cell an expression cassette
comprising a heterologous nucleotide sequence of interest operably
linked to one of the regulatory elements of the present disclosure.
In this manner, the regulatory element sequences are useful for
controlling the expression of the operably linked heterologous
nucleotide sequence.
[0009] Further provided is a method for expressing a nucleotide
sequence of interest in a constitutive manner in a plant. The
method comprises introducing into a plant cell an expression
cassette comprising a regulatory element of the disclosure operably
linked to a heterologous nucleotide sequence of interest.
[0010] Expression of the nucleotide sequence of interest may
provide for modification of the phenotype of the plant. Such
modification includes modulating the production of an endogenous
product, as to amount, relative distribution, or the like, or
production of an exogenous expression product to provide for a
novel function or product in the plant. In specific methods and
compositions, the heterologous nucleotide sequence of interest
comprises a gene product that confers herbicide resistance,
pathogen resistance, insect resistance, and/or altered tolerance to
salt, cold, or drought.
[0011] Expression cassettes comprising the promoter sequences of
the disclosure operably linked to a heterologous nucleotide
sequence of interest are provided. Additionally provided are
transformed plant cells, plant tissues, seeds, and plants.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1 shows the structure of the Lamium Leaf Distortion
Associated Virus (LLDAV) regulatory region and truncations of the
regulatory region. The 1226 base pair LLDAV regulatory region (FL)
consists of a portion of the long intergenic region (LIR) upstream
of a short ORF and stem loop structure. The sequence extends 5'
into the 3' end of the last ORF of the LLDAV genome. The regulatory
region was truncated (TR) from the 5' end to segments that
consisted of 1130 bp, 904 bp, 885 bp, 443 bp, and 113 bp. The
position of the putative TATA box is depicted by the arrow.
[0013] FIG. 2 shows the nucleic acid sequence of the 1226 base pair
full-length LLDAV regulatory element (SEQ ID NO: 1). The putative
TATA box is underlined. Also indicated by inserted arrows are the
5' ends of the truncated LLDAV regulatory elements as represented
by the nucleic acid sequence of the 1130 base pair truncated
regulatory element LLDAV TR1 (SEQ ID NO: 2), the nucleic acid
sequence of the 904 base pair truncated regulatory element LLDAV
TR2 (SEQ ID NO: 3), the nucleic acid sequence of the 885 base pair
truncated regulatory element LLDAV TR3 (SEQ ID NO: 4), the nucleic
acid sequence of the 443 base pair truncated regulatory element
LLDAV TR4 (SEQ ID NO: 5), and the nucleic acid sequence of the 113
base pair truncated regulatory element LLDAV TR5 (SEQ ID NO:
6).
DETAILED DESCRIPTION OF THE INVENTION
[0014] The disclosure relates to compositions and methods drawn to
plant regulatory elements and methods of their use. The
compositions comprise nucleotide sequences for the regulatory
region of Lamium Leaf Distortion Associated Virus (LLDAV). The
compositions further comprise DNA constructs comprising a
nucleotide sequence for the regulatory region of LLDAV operably
linked to a heterologous nucleotide sequence of interest. In
particular, the present disclosure provides for isolated nucleic
acid molecules comprising the nucleotide sequence set forth in SEQ
ID NO: 1, and fragments, variants, and complements thereof.
[0015] The LLDAV regulatory element sequences of the present
disclosure include nucleotide constructs that allow initiation of
transcription in a plant. In specific embodiments, the LLDAV
regulatory element sequence allows initiation of transcription in a
constitutive manner. Such constructs of the disclosure comprise
regulated transcription initiation regions associated with plant
developmental regulation. Thus, the compositions of the present
disclosure include DNA constructs comprising a nucleotide sequence
of interest operably linked to the LLDAV regulatory element
sequence. One source for the LLDAV regulatory region sequence is
set forth in SEQ ID NO: 1.
[0016] Compositions of the disclosure include the nucleotide
sequences for LLDAV regulatory elements and fragments and variants
thereof. In specific embodiments, the regulatory element sequences
of the disclosure are useful for expressing sequences of interest
in a constitutive manner. The nucleotide sequences of the
disclosure also find use in the construction of expression vectors
for subsequent expression of a heterologous nucleotide sequence in
a plant of interest or as probes for the isolation of other
LLDAV-like regulatory elements.
Regulatory Elements
[0017] A regulatory element is a nucleic acid molecule having gene
regulatory activity, i.e. one that has the ability to affect the
transcription and/or translation of an operably linked
transcribable polynucleotide molecule. The term "gene regulatory
activity" thus refers to the ability to affect the expression of an
operably linked transcribable polynucleotide molecule by affecting
the transcription and/or translation of that operably linked
transcribable polynucleotide molecule. Gene regulatory activity may
be positive and/or negative and the effect may be characterized by
its temporal, spatial, developmental, tissue, environmental,
physiological, pathological, cell cycle, and/or chemically
responsive qualities as well as by quantitative or qualitative
indications.
[0018] Regulatory elements such as promoters, leaders, introns, and
transcription termination regions are nucleic acid molecules that
have gene regulatory activity and play an integral part in the
overall expression of genes in living cells. The term "regulatory
element" refers to a nucleic acid molecule having gene regulatory
activity, i.e. one that has the ability to affect the transcription
and/or translation of an operably linked transcribable
polynucleotide molecule. Isolated regulatory elements, such as
promoters and leaders that function in plants are therefore useful
for modifying plant phenotypes through the methods of genetic
engineering.
[0019] Regulatory elements may be characterized by their expression
pattern, i.e. as constitutive and/or by their temporal, spatial,
developmental, tissue, environmental, physiological, pathological,
cell cycle, and/or chemically responsive expression pattern, and
any combination thereof, as well as by quantitative or qualitative
indications. A promoter is useful as a regulatory element for
modulating the expression of an operably linked transcribable
polynucleotide molecule.
[0020] As used herein, a "gene expression pattern" is any pattern
of transcription of an operably linked nucleic acid molecule into a
transcribed RNA molecule. Expression may be characterized by its
temporal, spatial, developmental, tissue, environmental,
physiological, pathological, cell cycle, and/or chemically
responsive qualities as well as by quantitative or qualitative
indications. The transcribed RNA molecule may be translated to
produce a protein molecule or may provide an antisense or other
regulatory RNA molecule, such as a dsRNA, a tRNA, an rRNA, a miRNA,
and the like.
[0021] As used herein, the term "protein expression" is any pattern
of translation of a transcribed RNA molecule into a protein
molecule. Protein expression may be characterized by its temporal,
spatial, developmental, or morphological qualities as well as by
quantitative or qualitative indications.
[0022] As used herein, the term "promoter" refers generally to a
nucleic acid molecule that is involved in recognition and binding
of RNA polymerase II and other proteins (trans-acting transcription
factors) to initiate transcription. A promoter may be initially
isolated from the 5' untranslated region (5' UTR) of a genomic copy
of a gene. Alternately, promoters may be synthetically produced or
manipulated DNA molecules. Promoters may also be chimeric, that is
a promoter produced through the fusion of two or more heterologous
DNA molecules.
[0023] In one embodiment, fragments are provided of a promoter
sequence disclosed herein. Promoter fragments may exhibit promoter
activity, and may be useful alone or in combination with other
promoters and promoter fragments, such as in constructing chimeric
promoters. In specific embodiments, fragments of a promoter are
provided comprising at least about 50, 95, 150, 250, 500, or about
750 contiguous nucleotides of a polynucleotide molecule having
promoter activity disclosed herein. Such fragments may exhibit at
least about 85 percent, about 90 percent, about 95 percent, about
98 percent, or about 99 percent, or greater, identity with a
reference sequence when optimally aligned to the reference
sequence.
[0024] A promoter or promoter fragment may also be analyzed for the
presence of known promoter elements, i.e. DNA sequence
characteristics, such as a TATA-box and other known transcription
factor binding site motifs. Identification of such known promoter
elements may be used by one of skill in the art to design variants
of the promoter having a similar expression pattern to the original
promoter.
[0025] As used herein, the term "enhancer" or "enhancer element"
refers to a cis-acting transcriptional regulatory element, a.k.a.
cis-element, which confers an aspect of the overall expression
pattern, but is usually insufficient alone to drive transcription,
of an operably linked polynucleotide sequence. Unlike promoters,
enhancer elements do not usually include a transcription start site
(TSS) or TATA box. A promoter may naturally comprise one or more
enhancer elements that affect the transcription of an operably
linked polynucleotide sequence. An isolated enhancer element may
also be fused to a promoter to produce a chimeric promoter
cis-element, which confers an aspect of the overall modulation of
gene expression. A promoter or promoter fragment may comprise one
or more enhancer elements that effect the transcription of operably
linked genes. Many promoter enhancer elements are believed to bind
DNA-binding proteins and/or affect DNA topology, producing local
conformations that selectively allow or restrict access of RNA
polymerase to the DNA template or that facilitate selective opening
of the double helix at the site of transcriptional initiation. An
enhancer element may function to bind transcription factors that
regulate transcription. Some enhancer elements bind more than one
transcription factor, and transcription factors may interact with
different affinities with more than one enhancer domain. Enhancer
elements may be identified by a number of techniques, 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 using known cis-element motifs or
enhancer elements as a target sequence or target motif with
conventional DNA sequence comparison methods, such as BLAST. The
fine structure of an enhancer domain can be further studied by
mutagenesis (or substitution) of one or more nucleotides or by
other conventional methods. Enhancer elements 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. Thus, the design,
construction, and use of enhancer elements according to the methods
disclosed herein for modulating the expression of operably linked
transcribable polynucleotide molecules are encompassed by the
present disclosure.
[0026] As used herein, the term "5' untranslated flanking region"
refers to a DNA molecule isolated from the untranslated 5' region
(5' UTR) of a genomic copy of a gene and defined generally as a
nucleotide segment between the transcription start site (TSS) and
the protein coding sequence start site. These sequences, or
leaders, may be synthetically produced or manipulated DNA elements.
A leader may be used as a 5' regulatory element for modulating
expression of an operably linked transcribable polynucleotide
molecule. Leader molecules may be used with a heterologous promoter
or with their native promoter. Promoter molecules of the present
disclosure may thus be operably linked to their native leader or
may be operably linked to a heterologous leader.
[0027] As used herein, the term "chimeric" refers to a single DNA
molecule produced by fusing a first DNA molecule to a second DNA
molecule, where neither first nor second DNA molecule would
normally be found in that configuration, i.e. fused to the other.
The chimeric DNA molecule is thus a new DNA molecule not otherwise
normally found in nature. As used herein, the term "chimeric
promoter" refers to a promoter produced through such manipulation
of DNA molecules. A chimeric promoter may combine two or more DNA
fragments; an example would be the fusion of a promoter to an
enhancer element. Thus, the design, construction, and use of
chimeric promoters according to the methods disclosed herein for
modulating the expression of operably linked transcribable
polynucleotide molecules are encompassed by the present
disclosure.
[0028] It is to be understood that nucleotide sequences, located
within introns, or 3' of the coding region sequence may also
contribute to the regulation of expression of a coding region of
interest. Examples of suitable introns include, but are not limited
to, the maize IVS6 intron, or the maize actin intron. A regulatory
element may also include those elements located downstream (3') to
the site of transcription initiation, or within transcribed
regions, or both. In the context of the present disclosure a
post-transcriptional regulatory element may include elements that
are active following transcription initiation, for example
translational and transcriptional enhancers, translational and
transcriptional repressors, and mRNA stability determinants.
[0029] The regulatory elements, or variants or fragments thereof,
of the present disclosure may be operatively associated with
heterologous regulatory elements or promoters in order to modulate
the activity of the heterologous regulatory element. Such
modulation includes enhancing or repressing transcriptional
activity of the heterologous regulatory element, modulating
post-transcriptional events, or either enhancing or repressing
transcriptional activity of the heterologous regulatory element and
modulating post-transcriptional events. For example, one or more
regulatory elements, or fragments thereof, of the present
disclosure may be operatively associated with constitutive,
inducible, or tissue specific promoters or fragment thereof, to
modulate the activity of such promoters within desired tissues in
plant cells.
[0030] The disclosure encompasses isolated or recombinant nucleic
acid compositions. An "isolated" or "recombinant" nucleic acid
molecule (or DNA) is used herein to refer to a nucleic acid
sequence (or DNA) that is no longer in its natural environment, for
example in an in vitro or in a heterologous recombinant bacterial
or plant host cell. An isolated or recombinant nucleic acid
molecule, or biologically active portion thereof, is substantially
free of other cellular material or culture medium when produced by
recombinant techniques, or substantially free of chemical
precursors or other chemicals when chemically synthesized. An
isolated or recombinant nucleic acid is free of sequences
(optimally protein encoding sequences) that naturally flank the
nucleic acid (i.e., sequences located at the 5' and 3' ends of the
nucleic acid) in the genomic DNA of the organism from which the
nucleic acid is derived. For example, in various embodiments, the
isolated nucleic acid molecule can contain less than about 5 kb, 4
kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequences
that naturally flank the nucleic acid molecule in genomic DNA of
the cell from which the nucleic acid is derived. The LLDAV
regulatory element sequences of the disclosure may be isolated from
the 5' untranslated region flanking their respective transcription
initiation sites.
[0031] Fragments and variants of the disclosed promoter nucleotide
sequences are also encompassed by the present disclosure. In
particular, fragments and variants of the LLDAV regulatory element
sequence of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, SEQ ID NO:
4, SEQ ID NO: 5, and/or SEQ ID NO: 6 may be used in the DNA
constructs of the disclosure. As used herein, the term "fragment"
refers to a portion of the nucleic acid sequence. Fragments of an
LLDAV regulatory element sequence may retain the biological
activity of initiating transcription, more particularly driving
transcription in a constitutive manner. Alternatively, fragments of
a nucleotide sequence which are useful as hybridization probes may
not necessarily retain biological activity. Fragments of a
nucleotide sequence for the LLDAV regulatory region may range from
at least about 20 nucleotides, about 50 nucleotides, about 100
nucleotides, and up to the full-length nucleotide sequence of the
disclosure for the promoter region of the gene.
[0032] A biologically active portion of an LLDAV regulatory element
can be prepared by isolating a portion of the LLDAV regulatory
element sequence of the disclosure, and assessing the promoter
activity of the portion. Nucleic acid molecules that are fragments
of a LLDAV regulatory element nucleotide sequence comprise at least
about 16, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550,
600, 650, 700, 800, 900 or 1000 nucleotides, or up to the number of
nucleotides present in a full-length LLDAV regulatory element
sequence disclosed herein (for example, 1226 nucleotides for SEQ ID
NO: 1).
[0033] For nucleotide sequences, a variant comprises a deletion
and/or addition of one or more nucleotides at one or more internal
sites within the native polynucleotide and/or a substitution of one
or more nucleotides at one or more sites in the native
polynucleotide. As used herein, a "native" or "genomic" nucleotide
sequence comprises a naturally occurring nucleotide sequence. For
nucleotide sequences, naturally occurring variants can be
identified with the use of well-known molecular biology techniques,
as, for example, with polymerase chain reaction (PCR) and
hybridization techniques as outlined below. Variant nucleotide
sequences also include synthetically derived nucleotide sequences,
such as those generated, for example, by using site-directed
mutagenesis. Generally, variants of a particular nucleotide
sequence of the disclosure will have at least about 40%, 45%, 50%,
55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99% or more sequence identity to that particular
nucleotide sequence as determined by sequence alignment programs
and parameters described elsewhere herein. A biologically active
variant of a nucleotide sequence of the disclosure may differ from
that sequence by as few as 1-15 nucleic acid residues, as few as
1-10, as few as 6-10, as few as 5, as few as 4, 3, 2, or even 1
nucleic acid residue.
[0034] In some embodiments the nucleic acid molecule encoding the
regulatory region is a "non-genomic nucleic acid sequence". As used
herein a "non-genomic nucleic acid sequence" refers to a nucleic
acid molecule that has one or more changes in the nucleic acid
sequence compared to the native or genomic nucleic acid
sequence.
[0035] Variant nucleotide sequences also encompass sequences
derived from a mutagenic and recombinogenic procedure such as DNA
shuffling. With such a procedure, LLDAV regulatory element
nucleotide sequences can be manipulated to create new LLDAV
regulatory elements. In this manner, libraries of recombinant
polynucleotides are generated from a population of related sequence
polynucleotides comprising sequence regions that have substantial
sequence identity and may be homologously recombined in vitro or in
vivo. Strategies for such DNA shuffling are known in the art. See,
for example, Stemmer (1994) Proc. Natl. Acad. Sci. USA
91:10747-10751; Stemmer (1994) Nature 370:389-391; Crameri et al.
(1997) Nature Biotech. 15:436-438; Moore et al. (1997) J. Mol.
Biol. 272:336-347; Zhang et al. (1997) Proc. Natl. Acad. Sci. USA
94:4504-4509; Crameri et al. (1998) Nature 391:288-291; and U.S.
Pat. Nos. 5,605,793 and 5,837,458.
[0036] The nucleotide sequences of the disclosure can be used to
isolate corresponding sequences from other organisms, particularly
other plants, more particularly other monocots. In this manner,
methods such as PCR, hybridization, and the like can be used to
identify such sequences based on their sequence homology to the
sequences set forth herein. Sequences isolated based on their
sequence identity to the entire LLDAV regulatory element sequence
set forth herein or to fragments thereof are encompassed by the
present disclosure.
[0037] In a PCR approach, oligonucleotide primers can be designed
for use in PCR reactions to amplify corresponding DNA sequences
from genomic DNA extracted from any plant of interest. Methods for
designing PCR primers and PCR cloning are generally known in the
art and are disclosed in Sambrook et al. (1989) Molecular Cloning:
A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press,
Plainview, N.Y.), hereinafter Sambrook. See also Innis et al., eds.
(1990) PCR Protocols: A Guide to Methods and Applications (Academic
Press, New York); Innis and Gelfand, eds. (1995) PCR Strategies
(Academic Press, New York); and Innis and Gelfand, eds. (1999) PCR
Methods Manual (Academic Press, New York). Known methods of PCR
include, but are not limited to, methods using paired primers,
nested primers, single specific primers, degenerate primers,
gene-specific primers, vector-specific primers,
partially-mismatched primers, and the like.
[0038] In hybridization techniques, all or part of a known
nucleotide sequence is used as a probe that selectively hybridizes
to other corresponding nucleotide sequences present in a population
of cloned genomic DNA fragments from a chosen organism. The
hybridization probes may be labeled with a detectable group such as
P-32 or any other detectable marker. Thus, for example, probes for
hybridization can be made by labeling synthetic oligonucleotides
based on the LLDAV regulatory element sequence of the disclosure.
Methods for preparation of probes for hybridization and for
construction of genomic libraries are generally known in the art
and are disclosed in Sambrook.
[0039] For example, the entire LLDAV regulatory element sequence
disclosed herein, or one or more portions thereof, may be used as a
probe capable of specifically hybridizing to corresponding LLDAV
regulatory element sequences and messenger RNAs. To achieve
specific hybridization under a variety of conditions, such probes
include sequences that are unique among LLDAV regulatory element
sequences and are at least about 10 nucleotides in length or at
least about 20 nucleotides in length. Such probes may be used to
amplify corresponding LLDAV regulatory element sequences from a
chosen plant by PCR. This technique may be used to isolate
additional coding sequences from a desired organism, or as a
diagnostic assay to determine the presence of coding sequences in
an organism. Hybridization techniques include hybridization
screening of plated DNA libraries (either plaques or colonies; see,
for example, Sambrook).
[0040] Hybridization of such sequences may be carried out under
stringent conditions. The terms "stringent conditions" or
"stringent hybridization conditions" are intended to mean
conditions under which a probe will hybridize to its target
sequence to a detectably greater degree than to other sequences
(e.g., at least 2-fold over background). Stringent conditions are
sequence-dependent and will be different in different
circumstances. By controlling the stringency of the hybridization
and/or washing conditions, target sequences that are 100%
complementary to the probe can be identified (homologous probing).
Alternatively, stringency conditions may be adjusted to allow some
mismatching in sequences so that lower degrees of similarity are
detected (heterologous probing). Generally, a probe is less than
about 1000 nucleotides in length, optimally less than 500
nucleotides in length.
[0041] Typically, stringent conditions will be those in which the
salt concentration is less than about 1.5 M Na ion, typically about
0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to
8.3 and the temperature is at least about 30.degree. C. for short
probes (e.g., 10 to 50 nucleotides) and at least about 60.degree.
C. for long probes (e.g., greater than 50 nucleotides). Stringent
conditions may also be achieved with the addition of destabilizing
agents such as formamide. Exemplary low stringency conditions
include hybridization with a buffer solution of 30 to 35%
formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37.degree.
C., and a wash in 1.times. to 2.times.SSC (20.times.SSC=3.0 M
NaCl/0.3 M trisodium citrate) at 50 to 55.degree. C. Exemplary
moderate stringency conditions include hybridization in 40 to 45%
formamide, 1.0 M NaCl, 1% SDS at 37.degree. C., and a wash in
0.5.times. to 1.times.SSC at 55 to 60.degree. C. Exemplary high
stringency conditions include hybridization in 50% formamide, 1 M
NaCl, 1% SDS at 37.degree. C., and a final wash in 0.1.times.SSC at
60 to 65.degree. C. for a duration of at least 30 minutes. Duration
of hybridization is generally less than about 24 hours, usually
about 4 to about 12 hours. The duration of the wash time will be at
least a length of time sufficient to reach equilibrium.
[0042] Specificity is typically the function of post-hybridization
washes, the critical factors being the ionic strength and
temperature of the final wash solution. For DNA-DNA hybrids, the Tm
(thermal melting point) can be approximated from the equation of
Meinkoth and Wahl (1984) Anal. Biochem. 138:267-284:
Tm=81.5.degree. C.+16.6 (log M)+0.41 (% GC)-0.61 (% form)-500/L;
where M is the molarity of monovalent cations, % GC is the
percentage of guanosine and cytosine nucleotides in the DNA, % form
is the percentage of formamide in the hybridization solution, and L
is the length of the hybrid in base pairs. The Tm is the
temperature (under defined ionic strength and pH) at which 50% of a
complementary target sequence hybridizes to a perfectly matched
probe. Tm is reduced by about 1.degree. C. for each 1% of
mismatching; thus, Tm, hybridization, and/or wash conditions can be
adjusted to hybridize to sequences of the desired identity. For
example, if sequences with >90% identity are sought, the Tm can
be decreased 10.degree. C. Generally, stringent conditions are
selected to be about 5.degree. C. lower than the Tm for the
specific sequence and its complement at a defined ionic strength
and pH. However, severely stringent conditions can utilize a
hybridization and/or wash at 1, 2, 3, or 4.degree. C. lower than
the Tm; moderately stringent conditions can utilize a hybridization
and/or wash at 6, 7, 8, 9, or 10.degree. C. lower than the Tm; low
stringency conditions can utilize a hybridization and/or wash at
11, 12, 13, 14, 15, or 20.degree. C. lower than the Tm. Using the
equation, hybridization and wash compositions, and desired Tm,
those of ordinary skill will understand that variations in the
stringency of hybridization and/or wash solutions are inherently
described. If the desired degree of mismatching results in a Tm of
less than 45.degree. C. (aqueous solution) or 32.degree. C.
(formamide solution), it is preferred to increase the SSC
concentration so that a higher temperature can be used. An
extensive guide to the hybridization of nucleic acids is found in
Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular
Biology--Hybridization with Nucleic Acid Probes, Part I, Chapter 2
(Elsevier, New York); and Ausubel et al., eds. (1995) Current
Protocols in Molecular Biology, Chapter 2 (Greene Publishing and
Wiley-Interscience, New York). See also Sambrook.
[0043] Thus, isolated sequences that have constitutive promoter
activity and which hybridize under stringent conditions to the
LLDAV regulatory element sequences disclosed herein, or to
fragments thereof, are encompassed by the present disclosure.
[0044] The following terms are used to describe the sequence
relationships between two or more nucleic acids or polynucleotides:
(a) "reference sequence", (b) "comparison window", (c) "sequence
identity", (d) "percentage of sequence identity", and (e)
"substantial identity".
[0045] (a) As used herein, "reference sequence" is a defined
sequence used as a basis for sequence comparison. A reference
sequence may be a subset or the entirety of a specified sequence;
for example, as a segment of a full-length cDNA or gene sequence,
or the complete cDNA or gene sequence.
[0046] (b) As used herein, "comparison window" makes reference to a
contiguous and specified segment of a polynucleotide sequence,
wherein the polynucleotide sequence in the comparison window may
comprise additions or deletions (i.e., gaps) compared to the
reference sequence (which does not comprise additions or deletions)
for optimal alignment of the two sequences. Generally, the
comparison window is at least 20 contiguous nucleotides in length,
and optionally can be 30, 40, 50, 100, or longer. Those of skill in
the art understand that to avoid a high similarity to a reference
sequence due to inclusion of gaps in the polynucleotide sequence a
gap penalty is typically introduced and is subtracted from the
number of matches.
[0047] Methods of alignment of sequences for comparison are well
known in the art. Thus, the determination of percent sequence
identity between any two sequences can be accomplished using a
mathematical algorithm. Non-limiting examples of such mathematical
algorithms are the algorithm of Myers and Miller (1988) CABIOS
4:11-17; the local alignment algorithm of Smith et al. (1981) Adv.
Appl. Math. 2:482; the global alignment algorithm of Needleman and
Wunsch (1970) J. Mol. Biol. 48:443-453; the search-for-local
alignment method of Pearson and Lipman (1988) Proc. Natl. Acad.
Sci. 85:2444-2448; the algorithm of Karlin and Altschul (1990)
Proc. Natl. Acad. Sci. USA 872264, modified as in Karlin and
Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877.
[0048] Computer implementations of these mathematical algorithms
can be utilized for comparison of sequences to determine sequence
identity. Such implementations include, but are not limited to:
CLUSTAL in the PC/Gene program (available from Intelligenetics,
Mountain View, Calif.); the ALIGN program (Version 2.0) and GAP,
BESTFIT, BLAST, FASTA, and TFASTA in the GCG Wisconsin Genetics
Software Package, Version 10 (available from Accelrys Inc., 9685
Scranton Road, San Diego, Calif., USA). Alignments using these
programs can be performed using the default parameters. The CLUSTAL
program is well described by Higgins et al. (1988) Gene 73:237-244
(1988); Higgins et al. (1989) CABIOS 5:151-153; Corpet et al.
(1988) Nucleic Acids Res. 16:10881-90; Huang et al. (1992) CABIOS
8:155-65; and Pearson et al. (1994) Meth. Mol. Biol. 24:307-331.
The ALIGN program is based on the algorithm of Myers and Miller
(1988) supra. A PAM120 weight residue table, a gap length penalty
of 12, and a gap penalty of 4 can be used with the ALIGN program
when comparing amino acid sequences. The BLAST programs of Altschul
et al (1990) J. Mol. Biol. 215:403 are based on the algorithm of
Karlin and Altschul (1990) supra. BLAST nucleotide searches can be
performed with the BLASTN program, score=100, wordlength=12, to
obtain nucleotide sequences homologous to a nucleotide sequence
encoding a protein of the disclosure. BLAST protein searches can be
performed with the BLASTX program, score=50, wordlength=3, to
obtain amino acid sequences homologous to a protein or polypeptide
of the disclosure. To obtain gapped alignments for comparison
purposes, Gapped BLAST (in BLAST 2.0) can be utilized as described
in Altschul et al. (1997) Nucleic Acids Res. 25:3389.
Alternatively, PSI-BLAST (in BLAST 2.0) can be used to perform an
iterated search that detects distant relationships between
molecules. See Altschul et al. (1997) supra. When utilizing BLAST,
Gapped BLAST, PSI-BLAST, the default parameters of the respective
programs (e.g., BLASTN for nucleotide sequences, BLASTX for
proteins) can be used. See the website for the National Center for
Biotechnology Information. Alignment may also be performed manually
by inspection.
[0049] Unless otherwise stated, sequence identity/similarity values
provided herein refer to the value obtained using GAP Version 10
using the following parameters: % identity and % similarity for a
nucleotide sequence using GAP Weight of 50 and Length Weight of 3,
and the nwsgapdna.cmp scoring matrix; % identity and % similarity
for an amino acid sequence using GAP Weight of 8 and Length Weight
of 2, and the BLOSUM62 scoring matrix; or any equivalent program
thereof. An "equivalent program" is intended any sequence
comparison program that, for any two sequences in question,
generates an alignment having identical nucleotide or amino acid
residue matches and an identical percent sequence identity when
compared to the corresponding alignment generated by GAP Version
10.
[0050] GAP uses the algorithm of Needleman and Wunsch (1970) J.
Mol. Biol. 48:443-453, to find the alignment of two complete
sequences that maximizes the number of matches and minimizes the
number of gaps. GAP considers all possible alignments and gap
positions and creates the alignment with the largest number of
matched bases and the fewest gaps. It allows for the provision of a
gap creation penalty and a gap extension penalty in units of
matched bases. GAP must make a profit of gap creation penalty
number of matches for each gap it inserts. If a gap extension
penalty greater than zero is chosen, GAP must, in addition, make a
profit for each gap inserted of the length of the gap times the gap
extension penalty. Default gap creation penalty values and gap
extension penalty values in Version 10 of the GCG Wisconsin
Genetics Software Package for protein sequences are 8 and 2,
respectively. For nucleotide sequences the default gap creation
penalty is 50 while the default gap extension penalty is 3. The gap
creation and gap extension penalties can be expressed as an integer
selected from the group of integers consisting of from 0 to 200.
Thus, for example, the gap creation and gap extension penalties can
be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45,
50, 55, 60, 65 or greater.
[0051] GAP presents one member of the family of best alignments.
There may be many members of this family, but no other member has a
better quality. GAP displays four figures of merit for alignments:
Quality, Ratio, Identity, and Similarity. The Quality is the metric
maximized in order to align the sequences. Ratio is the quality
divided by the number of bases in the shorter segment. Percent
Identity is the percent of the symbols that actually match. Percent
Similarity is the percent of the symbols that are similar. Symbols
that are across from gaps are ignored. A similarity is scored when
the scoring matrix value for a pair of symbols is greater than or
equal to 0.50, the similarity threshold. The scoring matrix used in
Version 10 of the GCG Wisconsin Genetics Software Package is
BLOSUM62 (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci.
USA 89:10915).
[0052] (c) As used herein, "sequence identity" or "identity" in the
context of two nucleic acid or polypeptide sequences makes
reference to the residues in the two sequences that are the same
when aligned for maximum correspondence over a specified comparison
window. When percentage of sequence identity is used in reference
to proteins it is recognized that residue positions which are not
identical often differ by conservative amino acid substitutions,
where amino acid residues are substituted for other amino acid
residues with similar chemical properties (e.g., charge or
hydrophobicity) and therefore do not change the functional
properties of the molecule. When sequences differ in conservative
substitutions, the percent sequence identity may be adjusted
upwards to correct for the conservative nature of the substitution.
Sequences that differ by such conservative substitutions are said
to have "sequence similarity" or "similarity". Means for making
this adjustment are well known to those of skill in the art.
Typically this involves scoring a conservative substitution as a
partial rather than a full mismatch, thereby increasing the
percentage sequence identity. Thus, for example, where an identical
amino acid is given a score of 1 and a non-conservative
substitution is given a score of zero, a conservative substitution
is given a score between zero and 1. The scoring of conservative
substitutions is calculated, e.g., as implemented in the program
PC/GENE (Intelligenetics, Mountain View, Calif.).
[0053] (d) As used herein, "percentage of sequence identity" means
the value determined by comparing two optimally aligned sequences
over a comparison window, wherein 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, dividing the number of matched positions by the
total number of positions in the window of comparison, and
multiplying the result by 100 to yield the percentage of sequence
identity.
[0054] (e) The term "substantial identity" of polynucleotide
sequences means that a polynucleotide comprises a sequence that has
at least 70% sequence identity, at least 80%, at least 90%, and at
least 95%, compared to a reference sequence using one of the
alignment programs described using standard parameters.
[0055] Another indication that nucleotide sequences are
substantially identical is if two molecules hybridize to each other
under stringent conditions. Generally, stringent conditions are
selected to be about 5.degree. C. lower than the Tm for the
specific sequence at a defined ionic strength and pH. However,
stringent conditions encompass temperatures in the range of about
1.degree. C. to about 20.degree. C. lower than the Tm, depending
upon the desired degree of stringency as otherwise qualified
herein.
[0056] As used herein, the term plant includes plant cells, plant
protoplasts, plant cell tissue cultures from which plants can be
regenerated, plant calli, plant clumps, and plant cells that are
intact in plants or parts of plants such as embryos, pollen,
ovules, seeds, leaves, flowers, branches, fruit, kernels, ears,
cobs, husks, stalks, roots, root tips, anthers, and the like. Grain
is intended to mean the mature seed produced by commercial growers
for purposes other than growing or reproducing the species.
Progeny, variants, and mutants of the regenerated plants are also
included within the scope of the disclosure, provided that these
parts comprise the introduced polynucleotides.
[0057] The present disclosure may be used for transformation of any
plant species, including, but not limited to, monocots and dicots.
Examples of plant species include corn (Zea mays), Brassica sp.
(e.g., B. napus, B. rapa, B. juncea), particularly those Brassica
species useful as sources of seed oil, alfalfa (Medicago sativa),
rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghum
bicolor, Sorghum vulgare), millet (e.g., pearl millet (Pennisetum
glaucum), proso millet (Panicum miliaceum), foxtail millet (Setaria
italica), finger millet (Eleusine coracana)), sunflower (Helianthus
annuus), safflower (Carthamus tinctorius), wheat (Triticum
aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum),
potato (Solanum tuberosum), peanuts (Arachis hypogaea), cotton
(Gossypium barbadense, Gossypium hirsutum), sweet potato (Ipomoea
batatus), cassava (Manihot esculenta), coffee (Coffea spp.),
coconut (Cocos nucifera), pineapple (Ananas comosus), citrus trees
(Citrus spp.), cocoa (Theobroma cacao), tea (Camellia sinensis),
banana (Musa spp.), avocado (Persea americana), fig (Ficus casica),
guava (Psidium guajava), mango (Mangifera indica), olive (Olea
europaea), papaya (Carica papaya), cashew (Anacardium occidentale),
macadamia (Macadamia integrifolia), almond (Prunus amygdalus),
sugar beets (Beta vulgaris), sugarcane (Saccharum spp.), oats,
barley, vegetables, ornamentals, and conifers.
[0058] Vegetables include tomatoes (Lycopersicon esculentum),
lettuce (e.g., Lactuca sativa), green beans (Phaseolus vulgaris),
lima beans (Phaseolus limensis), peas (Lathyrus spp.), and members
of the genus Cucumis such as cucumber (C. sativus), cantaloupe (C.
cantalupensis), and musk melon (C. melo). Ornamentals include
azalea (Rhododendron spp.), hydrangea (Macrophylla hydrangea),
hibiscus (Hibiscus rosasanensis), roses (Rosa spp.), tulips (Tulipa
spp.), daffodils (Narcissus spp.), Eupatoriums (Eupatorium
hybrida), carnation (Dianthus caryophyllus), poinsettia (Euphorbia
pulcherrima), and chrysanthemum.
[0059] Conifers that may be employed in practicing the present
disclosure include, for example, pines such as loblolly pine (Pinus
taeda), slash pine (Pinus elliotii), ponderosa pine (Pinus
ponderosa), lodgepole pine (Pinus contorta), and Monterey pine
(Pinus radiata); Douglas fir (Pseudotsuga menziesii); Western
hemlock (Tsuga canadensis); Sitka spruce (Picea glauca); redwood
(Sequoia sempervirens); true firs such as silver fir (Abies
amabilis) and balsam fir (Abies balsamea); and cedars such as
Western red cedar (Thuja plicata) and Alaska yellow cedar
(Chamaecyparis nootkatensis). In specific embodiments, plants of
the present disclosure are crop plants (for example, corn, alfalfa,
sunflower, Brassica, soybean, cotton, safflower, peanut, sorghum,
wheat, millet, tobacco, etc.). In other embodiments, corn and
soybean plants are optimal, and in yet other embodiments corn
plants are optimal.
[0060] Other plants of interest include grain plants that provide
seeds of interest, oil-seed plants, and leguminous plants. Seeds of
interest include grain seeds, such as corn, wheat, barley, rice,
sorghum, rye, etc. Oil-seed plants include cotton, soybean,
safflower, sunflower, Brassica, maize, alfalfa, palm, coconut, etc.
Leguminous plants include beans and peas. Beans include guar,
locust bean, fenugreek, soybean, garden beans, cowpea, mung bean,
lima bean, fava bean, lentils, chickpea, etc.
[0061] As used herein, the term "transcribable polynucleotide
molecule" refers to any DNA molecule capable of being transcribed
into a RNA molecule, including, but not limited to, those having
protein coding sequences and those having sequences useful for gene
suppression. A "transgene" refers to a transcribable polynucleotide
molecule heterologous to a host cell and/or a transcribable
polynucleotide molecule artificially incorporated into a host
cell's genome.
[0062] A regulatory element of the present invention may be
operably linked to a transcribable polynucleotide molecule that is
heterologous with respect to the regulatory molecule. As used
herein, the term "heterologous" refers to the combination of two or
more polynucleotide molecules when such a combination would not
normally be found in nature. For example, the two molecules may be
derived from different species and/or the two molecules may be
derived from different genes, e.g. different genes from the same
species or the same genes from different species. A regulatory
element is thus heterologous with respect to an operably linked
transcribable polynucleotide molecule if such a combination is not
normally found in nature, i.e. that transcribable polynucleotide
molecule is not naturally occurring operably linked in combination
with that regulatory element molecule.
[0063] The transcribable polynucleotide molecule may generally be
any DNA molecule for which expression of an RNA transcript is
desired. Such expression of an RNA transcript may result in
translation of the resulting mRNA molecule and thus protein
expression. Alternatively, a transcribable polynucleotide molecule
may be designed to ultimately cause decreased expression of a
specific gene or protein. This may be accomplished by using a
transcribable polynucleotide molecule that is oriented in the
antisense direction. One of ordinary skill in the art is familiar
with using such antisense technology. Briefly, as the antisense
transcribable polynucleotide molecule is transcribed, the RNA
product hybridizes to and sequesters a complementary RNA molecule
inside the cell. This duplex RNA molecule cannot be translated into
a protein by the cell's translational machinery and is degraded in
the cell. Any gene may be negatively regulated in this manner.
[0064] Thus, one embodiment of the invention is a regulatory
element of the present invention, such as those provided as SEQ ID
NO: 1-6, operably linked to a transcribable polynucleotide molecule
so as to modulate transcription of the transcribable polynucleotide
molecule at a desired level or in a desired pattern upon
introduction of said construct into a plant cell. In one
embodiment, the transcribable polynucleotide molecule comprises a
protein-coding region of a gene, and the promoter affects the
transcription of an RNA molecule that is translated and expressed
as a protein product. In another embodiment, the transcribable
polynucleotide molecule comprises an antisense region of a gene,
and the promoter affects the transcription of an antisense RNA
molecule or other similar inhibitory RNA molecule in order to
inhibit expression of a specific RNA molecule of interest in a
target host cell.
[0065] Transcribable polynucleotide molecules expressed by the
LLDAV regulatory elements of the disclosure may be used for varying
the phenotype of a plant. Various changes in phenotype are of
interest including modifying expression of a gene, altering a
plant's pathogen or insect defense mechanism, increasing the plants
tolerance to herbicides in a plant, altering root development to
respond to environmental stress, modulating the plant's response to
salt, temperature (hot and cold), drought, and the like. These
results can be achieved by the expression of a heterologous
nucleotide sequence of interest comprising an appropriate gene
product. In specific embodiments, the heterologous nucleotide
sequence of interest is an endogenous plant sequence whose
expression level is increased in the plant or plant part.
Alternatively, the results can be achieved by providing for a
reduction of expression of one or more endogenous gene products,
particularly enzymes, transporters, or cofactors, or by affecting
nutrient uptake in the plant. These changes result in a change in
phenotype of the transformed plant.
Genes of Agronomic Interest
[0066] Transcribable polynucleotide molecules may be genes of
agronomic interest. As used herein, the term "gene of agronomic
interest" refers to a transcribable polynucleotide molecule that
when expressed in a particular plant tissue, cell, or cell type
provides a desirable characteristic associated with plant
morphology, physiology, growth, development, yield, product,
nutritional profile, disease or pest resistance, and/or
environmental or chemical tolerance. Genes of agronomic interest
include, but are not limited to, those encoding a yield protein, a
stress resistance protein, a developmental control protein, a
tissue differentiation protein, a meristem protein, an
environmentally responsive protein, a senescence protein, a hormone
responsive protein, an abscission protein, a source protein, a sink
protein, a flower control protein, a seed protein, an herbicide
resistance protein, a disease resistance protein, a fatty acid
biosynthetic enzyme, a tocopherol biosynthetic enzyme, an amino
acid biosynthetic enzyme, a pesticidal protein, or any other agent
such as an antisense or RNAi molecule targeting a particular gene
for suppression. The product of a gene of agronomic interest may
act within the plant in order to cause an effect upon the plant
physiology or metabolism or may be act as a pesticidal agent in the
diet of a pest that feeds on the plant.
[0067] In one embodiment, a regulatory element of the present
disclosure is incorporated into a construct such that the
regulatory is operably linked to a transcribable polynucleotide
molecule that is a gene of agronomic interest. The expression of
the gene of agronomic interest is desirable in order to confer an
agronomically beneficial trait. A beneficial agronomic trait may
be, for example, but not limited to, herbicide tolerance, insect
control, modified yield, fungal disease resistance, virus
resistance, nematode resistance, bacterial disease resistance,
plant growth and development, 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 and secretable peptides, improved
processing traits, improved digestibility, enzyme production,
flavor, nitrogen fixation, hybrid seed production, fiber
production, and biofuel production.
[0068] Insect resistance genes may encode resistance to pests that
damage and cause yield drag such as rootworm, cutworm, European
corn borer, and the like. Such genes include, for example, Bacillus
thuringiensis toxic protein genes (U.S. Pat. Nos. 5,366,892;
5,747,450; 5,736,514; 5,723,756; 5,593,881; and Geiser et al.
(1986) Gene 48:109); and the like.
[0069] Genes encoding disease resistance traits include
detoxification genes, such as those which detoxify fumonisin (U.S.
Pat. No. 5,792,931); avirulence (avr) and disease resistance (R)
genes (Jones et al. (1994) Science 266:789; Martin et al. (1993)
Science 262:1432; and Mindrinos et al. (1994) Cell 78:1089); and
the like.
[0070] Herbicide resistance traits may include genes coding for
resistance to herbicides that act to inhibit the action of
acetolactate synthase (ALS), in particular the sulfonylurea-type
herbicides (e.g., the acetolactate synthase (ALS) gene containing
mutations leading to such resistance, in particular the S4 and/or
Hra mutations), genes coding for resistance to herbicides that act
to inhibit action of glutamine synthase, such as phosphinothricin
or basta (e.g., the bar gene), glyphosate (e.g., the EPSPS gene and
the GAT gene; see, for example, U.S. Publication No. 20040082770
and WO 03/092360) or other such genes known in the art. The bar
gene encodes resistance to the herbicide basta, the nptll gene
encodes resistance to the antibiotics kanamycin and geneticin, and
the ALS-gene mutants encode resistance to the herbicide
chlorsulfuron.
[0071] Glyphosate resistance is imparted by mutant
5-enolpyruvl-3-phosphikimate synthase (EPSP) and aroA genes. See,
for example, U.S. Pat. No. 4,940,835 to Shah et al., which
discloses the nucleotide sequence of a form of EPSPS which can
confer glyphosate resistance. U.S. Pat. No. 5,627,061 to Barry et
al. also describes genes encoding EPSPS enzymes. See also U.S. Pat.
Nos. 6,248,876 B1; 6,040,497; 5,804,425; 5,633,435; 5,145,783;
4,971,908; 5,312,910; 5,188,642; 4,940,835; 5,866,775; 6,225,114
B1; 6,130,366; 5,310,667; 4,535,060; 4,769,061; 5,633,448;
5,510,471; Re. 36,449; RE 37,287 E; and 5,491,288; and
international publications WO 97/04103; WO 97/04114; WO 00/66746;
WO 01/66704; WO 00/66747 and WO 00/66748, which are incorporated
herein by reference for this purpose. Glyphosate resistance is also
imparted to plants that express a gene that encodes a glyphosate
oxido-reductase enzyme as described more fully in U.S. Pat. Nos.
5,776,760 and 5,463,175, which are incorporated herein by reference
for this purpose. In addition glyphosate resistance can be imparted
to plants by the over expression of genes encoding glyphosate
N-acetyltransferase. See, for example, U.S. Pat. Nos. 7,714,188 and
7,462,481.
[0072] Genes of agronomic interest with regulatory approval are
well known to one skilled in the art and can be found at the Center
for Environmental Risk Assessment
(cera-gmc.org/?action=gm_crop_database, which can be accessed using
the www prefix) and at the International Service for the
Acquisition of Agri-Biotech Applications
isaaa.org/gmapprovaldatabase/default.asp, which can be accessed
using the www prefix).
[0073] Exogenous products include plant enzymes and products as
well as those from other sources including prokaryotes and other
eukaryotes. Such products include enzymes, cofactors, hormones, and
the like.
[0074] Examples of other applicable genes and their associated
phenotype include the gene which encodes viral coat protein and/or
RNA, or other viral or plant genes that confer viral resistance;
genes that confer fungal resistance; genes that promote yield
improvement; and genes that provide for resistance to stress, such
as cold, dehydration resulting from drought, heat and salinity,
toxic metal or trace elements, or the like.
[0075] As noted, the heterologous nucleotide sequence operably
linked to the LLDAV regulatory element disclosed herein may be an
antisense sequence for a targeted gene. Thus the promoter sequences
disclosed herein may be operably linked to antisense DNA sequences
to reduce or inhibit expression of a native protein in the plant
root.
[0076] "RNAi" refers to a series of related techniques to reduce
the expression of genes (See for example U.S. Pat. No. 6,506,559).
Older techniques referred to by other names are now thought to rely
on the same mechanism, but are given different names in the
literature. These include "antisense inhibition," the production of
antisense RNA transcripts capable of suppressing the expression of
the target protein, and "co-suppression" or "sense-suppression,"
which refer to the production of sense RNA transcripts capable of
suppressing the expression of identical or substantially similar
foreign or endogenous genes (U.S. Pat. No. 5,231,020, incorporated
herein by reference). Such techniques rely on the use of constructs
resulting in the accumulation of double stranded RNA with one
strand complementary to the target gene to be silenced. The LLDAV
regulatory element of the embodiments may be used to drive
expression of constructs that will result in RNA interference
including microRNAs and siRNAs.
[0077] The regulatory element sequences of the present disclosure,
or variants or fragments thereof, when operably linked to a
heterologous nucleotide sequence of interest may drive constitutive
expression of the heterologous nucleotide sequence in the plant
expressing this construct. A "heterologous nucleotide sequence" is
a sequence that is not naturally occurring with the regulatory
element sequence of the disclosure. While this nucleotide sequence
is heterologous to the regulatory element sequence, it may be
homologous, or native, or heterologous, or foreign, to the plant
host.
[0078] The isolated regulatory element sequences of the present
disclosure may be modified to provide for a range of expression
levels of the heterologous nucleotide sequence. Thus, less than the
entire regulatory element region may be utilized and the ability to
drive expression of the nucleotide sequence of interest retained.
It is recognized that expression levels of the mRNA may be altered
in different ways with deletions of portions of the regulatory
element sequences. The mRNA expression levels may be decreased, or
alternatively, expression may be increased as a result of
regulatory element deletions if, for example, there is a negative
regulatory element (for a repressor) that is removed during the
truncation process. Generally, at least about 20 nucleotides of an
isolated regulatory element sequence will be used to drive
expression of a nucleotide sequence.
[0079] It is recognized that to increase transcription levels,
enhancers may be utilized in combination with the regulatory
element regions of the disclosure. Enhancers are nucleotide
sequences that act to increase the expression of a regulatory
element region. Enhancers are known in the art and include the SV40
enhancer region, the 35S enhancer element, and the like. Some
enhancers are also known to alter normal regulatory element
expression patterns, for example, by causing a regulatory element
to drive expression constitutively when without the enhancer, the
same regulatory element drives expression only in one specific
tissue or a few specific tissues.
[0080] Modifications of the isolated regulatory element sequences
of the present disclosure can provide for a range of expression of
the heterologous nucleotide sequence. Thus, they may be modified to
be weak promoters or strong promoters. Generally, a "weak promoter"
means a promoter that drives expression of a coding sequence at a
low level. A "low level" of expression is intended to mean
expression at levels of about 1/10,000 transcripts to about
1/100,000 transcripts to about 1/500,000 transcripts. Conversely, a
strong promoter drives expression of a coding sequence at a high
level, or at about 1/10 transcripts to about 1/100 transcripts to
about 1/1,000 transcripts.
[0081] It is recognized that the LLDAV regulatory elements of the
disclosure may be used to increase or decrease expression, thereby
resulting in a change in phenotype of the transformed plant. This
phenotypic change could further affect an increase or decrease in
levels of metal ions in tissues of the transformed plant.
[0082] The nucleotide sequences disclosed in the present
disclosure, as well as variants and fragments thereof, are useful
in the genetic manipulation of a plant. The LLDAV regulatory
element sequence is useful in this aspect when operably linked with
a heterologous nucleotide sequence whose expression is to be
controlled to achieve a desired phenotypic response. The term
"operably linked" means that the transcription or translation of
the heterologous nucleotide sequence is under the influence of the
regulatory element sequence. In this manner, the nucleotide
sequences for the regulatory elements of the disclosure may be
provided in expression cassettes along with heterologous nucleotide
sequences of interest for expression in the plant of interest, more
particularly for expression in the root of the plant.
[0083] The regulatory sequences of the embodiments are provided in
DNA constructs for expression in the organism of interest. An
"expression cassette" as used herein means a DNA construct
comprising a regulatory sequence of the embodiments operably linked
to a heterologous polynucleotide encoding a polypeptide of
interest. Such expression cassettes will comprise a transcriptional
initiation region comprising one of the regulatory element
nucleotide sequences of the present disclosure, or variants or
fragments thereof, operably linked to the heterologous nucleotide
sequence. Such an expression cassette can be provided with a
plurality of restriction sites for insertion of the nucleotide
sequence to be under the transcriptional regulation of the
regulatory regions. The expression cassette may additionally
contain selectable marker genes as well as 3' termination
regions.
[0084] The expression cassette can include, in the 5'-3' direction
of transcription, a transcriptional initiation region (i.e., a
promoter, or variant or fragment thereof, of the disclosure), a
translational initiation region, a heterologous nucleotide sequence
of interest, a translational termination region and, optionally, a
transcriptional termination region functional in the host organism.
The regulatory regions (i.e., promoters, transcriptional regulatory
regions, and translational termination regions) and/or the
polynucleotide of the embodiments may be native/analogous to the
host cell or to each other. Alternatively, the regulatory regions
and/or the polynucleotide of the embodiments may be heterologous to
the host cell or to each other. As used herein, "heterologous" in
reference to a sequence is a sequence that originates from a
foreign species, or, if from the same species, is substantially
modified from its native form in composition and/or genomic locus
by deliberate human intervention. For example, a promoter operably
linked to a heterologous polynucleotide is from a species different
from the species from which the polynucleotide was derived, or, if
from the same/analogous species, one or both are substantially
modified from their original form and/or genomic locus, or the
promoter is not the native promoter for the operably linked
polynucleotide.
[0085] The termination region may be native with the
transcriptional initiation region, may be native with the operably
linked DNA sequence of interest, may be native with the plant host,
or may be derived from another source (i.e., foreign or
heterologous to the promoter, the DNA sequence being expressed, the
plant host, or any combination thereof). Convenient termination
regions are available from the Ti-plasmid of A. tumefaciens, such
as the octopine synthase and nopaline synthase termination regions.
See also Guerineau et al. (1991) Mol. Gen. Genet. 262:141-144;
Proudfoot (1991) Cell 64:671-674; Sanfacon et al. (1991) Genes Dev.
5:141-149; Mogen et al. (1990) Plant Cell 2:1261-1272; Munroe et
al. (1990) Gene 91:151-158; Belles et al. (1989) Nucleic Acids Res.
17:7891-7903; and Joshi et al. (1987) Nucleic Acid Res.
15:9627-9639.
[0086] The expression cassette comprising the sequences of the
present disclosure may also contain at least one additional
nucleotide sequence for a gene to be cotransformed into the
organism. Alternatively, the additional sequence(s) can be provided
on another expression cassette.
[0087] Where appropriate, the nucleotide sequences whose expression
is to be under the control of the constitutive promoter sequence of
the present disclosure and any additional nucleotide sequence(s)
may be optimized for increased expression in the transformed plant.
That is, these nucleotide sequences can be synthesized using plant
preferred codons for improved expression. See, for example,
Campbell and Gowri (1990) Plant Physiol. 92:1-11 for a discussion
of host-preferred codon usage. Methods are available in the art for
synthesizing plant-preferred genes. See, for example, U.S. Pat.
Nos. 5,380,831, 5,436,391, and Murray et al. (1989) Nucleic Acids
Res. 17:477-498, herein incorporated by reference.
[0088] Additional sequence modifications are known to enhance gene
expression in a cellular host. These include elimination of
sequences encoding spurious polyadenylation signals, exon-intron
splice site signals, transposon-like repeats, and other such
well-characterized sequences that may be deleterious to gene
expression. The G-C content of the heterologous nucleotide sequence
may be adjusted to levels average for a given cellular host, as
calculated by reference to known genes expressed in the host cell.
When possible, the sequence is modified to avoid predicted hairpin
secondary mRNA structures.
[0089] The expression cassettes may additionally contain 5' leader
sequences. Such leader sequences can act to enhance translation.
Translation leaders are known in the art and include: picornavirus
leaders, for example, EMCV leader (Encephalomyocarditis 5'
noncoding region) (Elroy Stein et al. (1989) Proc. Nat. Acad. Sci.
USA 86:6126 6130); potyvirus leaders, for example, TEV leader
(Tobacco Etch Virus) (Allison et al. (1986) Virology 154:9 20);
MDMV leader (Maize Dwarf Mosaic Virus); human immunoglobulin heavy
chain binding protein (BiP) (Macejak et al. (1991) Nature 353:90
94); untranslated leader from the coat protein mRNA of alfalfa
mosaic virus (AMV RNA 4) (Jobling et al. (1987) Nature 325:622
625); tobacco mosaic virus leader (TMV) (Gallie et al. (1989)
Molecular Biology of RNA, pages 237 256); and maize chlorotic
mottle virus leader (MCMV) (Lommel et al. (1991) Virology 81:382
385). See also Della Cioppa et al. (1987) Plant Physiology 84:965
968. Methods known to enhance mRNA stability can also be utilized,
for example, introns, such as the maize Ubiquitin intron
(Christensen and Quail (1996) Transgenic Res. 5:213-218;
Christensen et al. (1992) Plant Molecular Biology 18:675-689) or
the maize Adhl intron (Kyozuka et al. (1991) Mol. Gen. Genet.
228:40-48; Kyozuka et al. (1990) Maydica 35:353-357), and the
like.
[0090] In preparing the expression cassette, the various DNA
fragments may be manipulated, so as to provide for the DNA
sequences in the proper orientation and, as appropriate, in the
proper reading frame. Toward this end, adapters or linkers may be
employed to join the DNA fragments or other manipulations may be
involved to provide for convenient restriction sites, removal of
superfluous DNA, removal of restriction sites, or the like. For
this purpose, in vitro mutagenesis, primer repair, restriction,
annealing, resubstitutions, for example, transitions and
transversions, may be involved.
[0091] Reporter genes or selectable marker genes may be included in
the expression cassettes. Examples of suitable reporter genes known
in the art can be found in, for example, Jefferson et al. (1991) in
Plant Molecular Biology Manual, ed. Gelvin et al. (Kluwer Academic
Publishers), pp. 1-33; DeWet et al. (1987) Mol. Cell. Biol.
7:725-737; Goff et al. (1990) EMBO J. 9:2517-2522; Kain et al.
(1995) BioTechniques 19:650-655; and Chiu et al. (1996) Current
Biology 6:325-330.
[0092] Selectable marker genes for selection of transformed cells
or tissues may include genes that confer antibiotic resistance or
resistance to herbicides. Examples of suitable selectable marker
genes include, but are not limited to, genes encoding resistance to
chloramphenicol (Herrera Estrella et al. (1983) EMBO J. 2:987-992);
methotrexate (Herrera Estrella et al. (1983) Nature 303:209-213;
Meijer et al. (1991) Plant Mol. Biol. 16:807-820); hygromycin
(Waldron et al. (1985) Plant Mol. Biol. 5:103-108; and Zhijian et
al. (1995) Plant Science 108:219-227); streptomycin (Jones et al.
(1987) Mol. Gen. Genet. 210:86-91); spectinomycin (Bretagne-Sagnard
et al. (1996) Transgenic Res. 5:131-137); bleomycin (Hille et al.
(1990) Plant Mol. Biol. 7:171-176); sulfonamide (Guerineau et al.
(1990) Plant Mol. Biol. 15:127-136); bromoxynil (Stalker et al.
(1988) Science 242:419-423); glyphosate (Shaw et al. (1986) Science
233:478-481; and U.S. application Ser. Nos. 10/004,357; and
10/427,692); phosphinothricin (DeBlock et al. (1987) EMBO J.
6:2513-2518).
[0093] Other genes that could serve utility in the recovery of
transgenic events but might not be required in the final product
would include, but are not limited to, examples such as GUS
(beta-glucuronidase; Jefferson (1987) Plant Mol. Biol. Rep. 5:387),
GFP (green fluorescence protein; Chalfie et al. (1994) Science
263:802), luciferase (Riggs et al. (1987) Nucleic Acids Res.
15(19):8115 and Luehrsen et al. (1992) Methods Enzymol.
216:397-414) and the maize genes encoding for anthocyanin
production (Ludwig et al. (1990) Science 247:449).
[0094] The expression cassette comprising the LLDAV regulatory
elements of the present disclosure operably linked to a nucleotide
sequence of interest may be used to transform any plant. In this
manner, genetically modified plants, plant cells, plant tissue,
seed, root, and the like can be obtained.
[0095] The methods of the disclosure involve introducing a
polypeptide or polynucleotide into a plant. "Introducing" is
intended to mean presenting to the plant the polynucleotide or
polypeptide in such a manner that the sequence gains access to the
interior of a cell of the plant. The methods of the disclosure do
not depend on a particular method for introducing a sequence into a
plant, only that the polynucleotide or polypeptides gains access to
the interior of at least one cell of the plant. Methods for
introducing polynucleotide or polypeptides into plants are known in
the art including, but not limited to, stable transformation
methods, transient transformation methods, and virus-mediated
methods.
[0096] "Stable transformation" is intended to mean that the
nucleotide construct introduced into a plant integrates into the
genome of the plant and is capable of being inherited by the
progeny thereof. "Transient transformation" is intended to mean
that a polynucleotide is introduced into the plant and does not
integrate into the genome of the plant or a polypeptide is
introduced into a plant.
[0097] Transformation protocols as well as protocols for
introducing nucleotide sequences into plants may vary depending on
the type of plant or plant cell, i.e., monocot or dicot, targeted
for transformation. Suitable methods of introducing nucleotide
sequences into plant cells and subsequent insertion into the plant
genome include microinjection (Crossway et al. (1986) Biotechniques
4:320 334), electroporation (Riggs et al. (1986) Proc. Natl. Acad.
Sci. USA 83:5602 5606), Agrobacterium-mediated transformation
(Townsend et al., U.S. Pat. No. 5,563,055 and Zhao et al., U.S.
Pat. No. 5,981,840), direct gene transfer (Paszkowski et al. (1984)
EMBO J. 3:2717 2722), and ballistic particle acceleration (see, for
example, U.S. Pat. Nos. 4,945,050; 5,879,918; 5,886,244; 5,932,782;
Tomes et al. (1995) in Plant Cell, Tissue, and Organ Culture:
Fundamental Methods, ed. Gamborg and Phillips (Springer-Verlag,
Berlin); McCabe et al. (1988) Biotechnology 6:923 926); and Lec1
transformation (WO 00/28058). Also see Weissinger et al. (1988)
Ann. Rev. Genet. 22:421 477; Sanford et al. (1987) Particulate
Science and Technology 5:27 37 (onion); Christou et al. (1988)
Plant Physiol. 87:671 674 (soybean); McCabe et al. (1988)
Bio/Technology 6:923 926 (soybean); Finer and McMullen (1991) In
Vitro Cell Dev. Biol. 27P:175-182 (soybean); Singh et al. (1998)
Theor. Appl. Genet. 96:319-324 (soybean); Datta et al. (1990)
Biotechnology 8:736 740 (rice); Klein et al. (1988) Proc. Natl.
Acad. Sci. USA 85:4305 4309 (maize); Klein et al. (1988)
Biotechnology 6:559 563 (maize); U.S. Pat. Nos. 5,240,855;
5,322,783 and 5,324,646; Klein et al. (1988) Plant Physiol. 91:440
444 (maize); Fromm et al. (1990) Biotechnology 8:833 839 (maize);
Hooykaas-Van Slogteren et al. (1984) Nature (London) 311:763-764;
U.S. Pat. No. 5,736,369 (cereals); Bytebier et al. (1987) Proc.
Natl. Acad. Sci. USA 84:5345-5349 (Liliaceae); De Wet et al. (1985)
in The Experimental Manipulation of Ovule Tissues, ed. Chapman et
al. (Longman, New York), pp. 197-209 (pollen); Kaeppler et al.
(1990) Plant Cell Reports 9:415-418 and Kaeppler et al. (1992)
Theor. Appl. Genet. 84:560-566 (whisker-mediated transformation);
D'Halluin et al. (1992) Plant Cell 4:1495-1505 (electroporation);
Li et al. (1993) Plant Cell Reports 12:250-255 and Christou and
Ford (1995) Annals of Botany 75:407-413 (rice); Osjoda et al.
(1996) Nature Biotechnology 14:745-750 (maize via Agrobacterium
tumefaciens); all of which are herein incorporated by
reference.
[0098] In specific embodiments, the DNA constructs comprising the
regulatory element sequences of the disclosure can be provided to a
plant using a variety of transient transformation methods. Such
transient transformation methods include, but are not limited to,
viral vector systems and the precipitation of the polynucleotide in
a manner that precludes subsequent release of the DNA. Thus, the
transcription from the particle-bound DNA can occur, but the
frequency with which it is released to become integrated into the
genome is greatly reduced. Such methods include the use particles
coated with polyethylimine (PEI; Sigma-Aldrich.TM. #P3143).
[0099] In other embodiments, the polynucleotide of the disclosure
may be introduced into plants by contacting plants with a virus or
viral nucleic acids. Generally, such methods involve incorporating
a nucleotide construct of the disclosure within a viral DNA or RNA
molecule. Methods for introducing polynucleotides into plants and
expressing a protein encoded therein, involving viral DNA or RNA
molecules, are known in the art. See, for example, U.S. Pat. Nos.
5,889,191, 5,889,190, 5,866,785, 5,589,367, 5,316,931, and Porta et
al. (1996) Molecular Biotechnology 5:209-221; herein incorporated
by reference.
[0100] Methods are known in the art for the targeted insertion of a
polynucleotide at a specific location in the plant genome. In one
embodiment, the insertion of the polynucleotide at a desired
genomic location is achieved using a site-specific recombination
system. See, for example, WO99/25821, WO99/25854, WO99/25840,
WO99/25855, and WO99/25853, all of which are herein incorporated by
reference. Briefly, the polynucleotide of the disclosure can be
contained in transfer cassette flanked by two non-identical
recombination sites. The transfer cassette is introduced into a
plant have stably incorporated into its genome a target site which
is flanked by two non-identical recombination sites that correspond
to the sites of the transfer cassette. An appropriate recombinase
is provided and the transfer cassette is integrated at the target
site. The polynucleotide of interest is thereby integrated at a
specific chromosomal position in the plant genome.
[0101] The cells that have been transformed may be grown into
plants in accordance with conventional ways. See, for example,
McCormick et al. (1986) Plant Cell Reports 5:81-84. These plants
may then be grown, and either pollinated with the same transformed
strain or different strains, and the resulting hybrid having
constitutive expression of the desired phenotypic characteristic
identified. Two or more generations may be grown to ensure that
expression of the desired phenotypic characteristic is stably
maintained and inherited and then seeds harvested to ensure
expression of the desired phenotypic characteristic has been
achieved. In this manner, the present disclosure provides
transformed seed (also referred to as "transgenic seed") having a
nucleotide construct of the disclosure, for example, an expression
cassette of the disclosure, stably incorporated into its
genome.
[0102] The article "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
element.
[0103] Throughout the specification 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.
[0104] All publications, patents and patent applications mentioned
in the specification are indicative of the level of those skilled
in the art to which this disclosure pertains. All publications,
patents and patent applications are herein incorporated by
reference to the same extent as if each individual publication,
patent or patent application was specifically and individually
indicated to be incorporated by reference.
[0105] The above description of various illustrated embodiments of
the disclosure is not intended to be exhaustive or to limit the
scope to the precise form disclosed. While specific embodiments of
examples are described herein for illustrative purposes, various
equivalent modifications are possible within the scope of the
disclosure, as those skilled in the relevant art will recognize.
The teachings provided herein can be applied to other purposed,
other than the examples described above. Numerous modifications and
variations are possible in light of the above teachings and,
therefore, are within the scope of the appended claims.
[0106] These and other changes may be made in light of the above
detailed description. In general, in the following claims, the
terms used should not be construed to limit the scope to the
specific embodiments disclosed in the specification and the
claims.
[0107] Efforts have been made to ensure accuracy with respect to
the numbers used (e.g. amounts, temperature, concentrations, etc.),
but some experimental errors and deviations should be allowed for.
Unless otherwise indicated, parts are parts by weight; molecular
weight is average molecular weight; temperature is in degrees
centigrade; and pressure is at or near atmospheric.
[0108] The following examples are offered by way of illustration
and not by way of limitation.
EXPERIMENTAL
Example 1: Lamium Leaf Distortion Associated Virus Regulatory
Element Sequences
[0109] The regulatory element of SEQ ID NO: 1 was obtained through
a search of Gen Bank Genomes for viral genomes that had been
sequenced and belonged to the Caulimoviridae virus family. The
search was initiated based on the well-known Cauliflower Mosaic
Virus 35S (CaMV35S) promoter. It drives constitutive expression of
heterologous genes in most tissues of most plants. Other regulatory
elements from this virus family, such as the Figwort Mosaic Virus
34S promoter also direct constitutive-like expression in plants.
Therefore, additional regulatory elements derived from the
Caulimoviridae virus family also may drive constitutive expression
in plants. The region of the genome found in what is called the
Long Intergenic Region (LIR) generally contains the regulatory
sequences necessary for promoter function in plants.
[0110] The Lamium Leaf Distortion Associated Virus (LLDAV) genome
has an LIR, so this region was targeted for functional analysis of
regulatory elements. One full-length sequence was selected to be
tested in plants. The sequence consists of 1226 bp (set forth in
SEQ ID NO: 1) and has a putative TATA box starting approximately 95
bp upstream of the 3' end of the sequence. The entire 1226 bp
sequence is referred to as the LLDAV full-length regulatory element
or LLDAV FL. Deleting segments of the 5' end of the full-length
regulatory element may alter the expression pattern and provide
insight into important sequence markers in the regulatory region.
Second, third, fourth, fifth and sixth sequences are a truncated
version of the full-length regulatory element (See FIG. 2; SEQ ID
NO: 1). LLDAV TR1, LLDAV TR2, LLDAV TR3, LLDAV TR4, and LLDAV TR5
regulatory elements respectively consist of 1130 bp, 904 bp, 885
bp, 443 bp, and 113 bp and contain the 3' end of the full-length
promoter (SEQ ID NO: 2-6).
Example 2: Expression and Deletion Analysis of the LLDAV Regulatory
Element
[0111] LLDAV FL was operably linked to the first intron of the
maize alcohol dehydrogenase gene 1 (ADH1 intron1) and the
.beta.-glucuronidase (GUS) gene, in an expression vector, to test
whether the synthesized DNA fragment would direct expression (SEQ
ID NO: 1). ADH1 intron1 was included for the purpose of increased
expression as it has been shown that in cereal plant cells the
expression of transgenes is enhanced by the presence of some 5'
proximal introns (See Callis et al. (1987) Genes and Development 1:
1183-1200; Kyozuka et al. (1990) Maydica 35:353-357).
[0112] The Ubi-1 promoter and intron from Zea mays were operably
linked to the GUS gene so that it could be used to compare the
expression pattern and expression levels of the LLDAV regulatory
elements. The Ubi-1 promoter is a strong constitutive promoter in
most tissues of Zea mays.
[0113] Regulatory elements are a collection of sequence motifs that
work together to bind transcription factors that result in the
spatial, temporal, and quantitative expression characteristics of a
promoter. Understanding the architecture and the positioning of
these motifs enhances knowledge pertaining to the regulatory
element. Segmental deletion analysis is an important tool that can
be used to begin to identify regions of a regulatory element that
contain functionally important motifs. The removal of segments from
the 5' end may change the spatial, temporal, and/or quantitative
expression patterns directed by the regulatory element. The regions
that result in a change may then be studied more closely to
evaluate the sequences and their interaction with cis and trans
factors. The truncations may also identify a minimal functional
sequence.
[0114] The restriction endonuclease recognition sites, SfuI, AccI,
Said, EcoRI and ClaI were used to remove five sequence regions of
LLDAV FL ranging in size from .about.96 to 1113 bp. LLDAV TR1,
LLDAV TR2, LLDAV TR3, LLDAV TR4, LLDAV TR5 were operably linked to
ADH1 intron1 and the GUS gene in an expression vector to test the
expression potential of the synthesized DNA fragments (SEQ ID NO:
2-6).
[0115] Stable transformed maize plants were created using
Agrobacterium protocols (detailed in Example 3) to allow for the
characterization of promoter activity, including spatial and
quantitative expression directed by the different regulatory
elements. Plants grown to V5/6 stage under greenhouse conditions
were sampled for leaf and root material to evaluate expression
pattern changes via histochemical GUS staining analysis and
quantitative fluorometric assays. Maize vegetative growth stages
are determined by the number of collared leaves on the plant.
Therefore, a plant at V5 stage has 5 fully collared leaves. Tissues
were also collected when the plants reached the reproductive growth
stages of R1-R2. R1 is noted by the emergence of silks outside the
husk and R2 is when the silks start to dry out. Results showed that
LLDAV FL drove expression in most tissues of maize similar to or
slightly better than the Ubi-1 promoter (Table 1). The exception
was pollen, where LLDAV FL expression was much lower than Ubi-1.
The 5' truncated promoters (LLDAV TR1-4) performed similarly to
LLDAV FL with the exception of LLDAV TR5 which did not function in
any of the tissues tested.
TABLE-US-00001 TABLE 1 Plant Expression Results for the LLDAV
Promoter (with ADH1 intron1 and GUS) V5-V6 R1-R2 Leaf Root Stalk
Tassel Husk Silk Pollen LLDAV FL 4 3 3 3 5 2 <0.1 LLDAV TR1 4 3
3 3 5 2 <0.1 LLDAV TR2 4 3 3 3 5 2 <0.1 LLDAV TR3 4 3.5 3.25
3 5 2 <0.1 LLDAV TR4 4 3 3 3 5 2 <0.1 LLDAV TR5 0 0 0 0 0 0 0
Ubi-1 3 3 3 3 5 2 3 untransformed 0 0 0 0 0 0 0 (negative control)
Data expressed on a 0-6 scale with the maize Ubi-1 promoter
directed expression as a comparator.
[0116] The LLDAV FL (SEQ ID NO: 1) regulatory element was operably
linked to ADH1 intron1 and an insecticidal gene (abbreviated IG2)
to test expression. The Ubi-1 promoter and intron driving the
expression of the IG2 insecticidal gene was used for comparison in
the analysis. Stable transformed maize plants were created using
Agrobacterium protocols (detailed in Example 3) and allowed to grow
to V5/6 stage when leaves were sampled. The plants were then
allowed to grow to R1-R2 stage when stalk and pollen samples were
taken. Kernels were sampled when they reached maturity.
[0117] Results from enzyme-linked immunosorbent assays (ELISA)
against IG2 showed the LLDAV FL regulatory element (SEQ ID NO: 1)
directed expression in leaf, stalk, and kernel tissues (Table 2).
Expression levels were comparable to the Ubi-1 promoter and its
intron in leaf and stalk tissues. In kernels, LLDAV FL (SEQ ID NO:
1) directed expression was lower than Ubi-1 and in pollen
expression was very low.
[0118] The expression data were supported by results from insect
consumption assays. Feeding insects dissected plant tissues
provides a rapid assessment of protein expression, as sufficient
levels are needed to protect the tissue from the insects.
Insufficient expression will result in feeding damage. Both LLDAV
FL (SEQ ID NO: 1) and Ubi-1 plants demonstrated levels of
expression that protected leaf and silk tissue against insect
damage. Tissues from negative control plants that did not have the
IG2 gene were consumed.
TABLE-US-00002 TABLE 2 Plant Expression Results for the LLDAV
Regulatory Element (with ADH1 intron1 and LLDAV: IG2) Data
expressed on a 0-6 scale with the maize Ubi-1 promoter representing
a median value. V5-V6 R1-R2 Maturity Leaf Stalk Pollen Kernels
LLDAV FL 2 3 <0.1 <0.75 Ubi-1 2 3 3 2 untransformed 0 0 0 0
(negative control) Data expressed on a 0-6 scale with the maize
Ubi-1 promoter representing a median value.
Example 3: Agrobacterium-Mediated Transformation of Maize and
Regeneration of Transgenic Plants
[0119] For Agrobacterium-mediated transformation of maize with a
regulatory element sequence of the disclosure, the method of Zhao
was employed (U.S. Pat. No. 5,981,840, and PCT patent publication
WO98/32326; the contents of which are hereby incorporated by
reference). Briefly, immature embryos were isolated from maize and
the embryos contacted with a suspension of Agrobacterium under
conditions whereby the bacteria were capable of transferring the
regulatory element sequence of the disclosure to at least one cell
of at least one of the immature embryos (step 1: the infection
step). The embryos were co-cultured with the Agrobacterium
suspension for a period of time then co-cultured on solid medium
(step 2: the co-cultivation step). An optional "resting" step was
performed following co-cultivation. In this resting step, the
embryos were incubated in the presence of at least one antibiotic
known to inhibit the growth of Agrobacterium without the addition
of a selective agent for plant transformants (step 3: resting
step). Next, the embryos were transferred and cultured on medium
containing a selective agent to recover growing, transformed calli
(step 4: the selection step). Plantlets were regenerated from calli
(step 5: the regeneration step) prior to transfer to the
greenhouse.
Example 4: Expression Analysis of the LLDAV Regulatory Element in
Canola
[0120] The LLDAV promoter was tested in canola using the GUS gene
as a reporter. Biolistic bombardment transient assays were used to
provide an initial assessment of performance. The number of GUS
staining foci and the intensity of staining was compared to the
Arabidopsis ubiqutin-10 promoter (AtUBQ10), a strong promoter in
canola tissues. Performance of the LLDAV promoter was similar to
the AtUBQ10 promoter in regard to the intensity of GUS staining.
However, a slight reduction in the number of staining foci was
observed (Table 3).
[0121] Transgenic canola plants were regenerated with both the
LLDAV:GUS and AtUBQ10:GUS vectors. Histochemical staining of
LLDAV:GUS events showed high levels of expression in leaves and
floral organs (Table 3). LLDAV directed expression was also
observed in pollen and siliques. When compared against
histochemically stained tissue from AtUBQ10:GUS plants, LLDAV was
comparable in leaves and floral organs. In pollen, LLDAV directed
expression was weaker than AtUBQ10 with about 10-15% of the pollen
grains staining. Almost all of the AtUBQ10 pollen grains stained
darkly.
TABLE-US-00003 TABLE 3 Expression Results for the LLDAV Promoter in
Canola Transient Floral assay Leaf organs Pollen LLDAV FL 2 3 3 1
AtUBQ10 3 3 3 3 Data expressed on a 0-3 scale with three indicating
strong expression.
* * * * *