U.S. patent application number 13/242937 was filed with the patent office on 2012-01-12 for regulatory elements associated with cbf transcription factors of maize.
This patent application is currently assigned to PIONEER HI-BRED INTERNATIONAL, INC.. Invention is credited to Timothy G. Helentjaris, Shoba Sivasankar.
Application Number | 20120011617 13/242937 |
Document ID | / |
Family ID | 39938362 |
Filed Date | 2012-01-12 |
United States Patent
Application |
20120011617 |
Kind Code |
A1 |
Sivasankar; Shoba ; et
al. |
January 12, 2012 |
Regulatory Elements Associated with CBF Transcription Factors of
Maize
Abstract
Compositions and methods for regulating expression of a
polynucleotide of interest in a plant are provided. Compositions
include novel nucleotide sequences comprising an isolated
stress-induced promoter natively linked to the maize ZmCBF2 coding
region. A method for expressing a polynucleotide of interest in a
plant or plant cell, using a regulatory sequence described herein,
is provided. The method may comprise transforming a plant cell to
comprise a polynucleotide sequence of interest operably linked to
one or more of the regulatory sequences of the present invention
and regenerating a stably transformed plant from the transformed
plant cell.
Inventors: |
Sivasankar; Shoba;
(Urbandale, IA) ; Helentjaris; Timothy G.;
(Tucson, AZ) |
Assignee: |
PIONEER HI-BRED INTERNATIONAL,
INC.
Johnston
IA
|
Family ID: |
39938362 |
Appl. No.: |
13/242937 |
Filed: |
September 23, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12207620 |
Sep 10, 2008 |
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13242937 |
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60971278 |
Sep 11, 2007 |
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Current U.S.
Class: |
800/279 ;
435/320.1; 435/412; 435/419; 435/468; 536/24.1; 800/278; 800/281;
800/284; 800/289; 800/298; 800/302; 800/320; 800/320.1; 800/320.2;
800/320.3 |
Current CPC
Class: |
C12N 15/8237 20130101;
C07K 14/415 20130101 |
Class at
Publication: |
800/279 ;
536/24.1; 435/320.1; 435/419; 435/412; 800/298; 800/320.1; 800/320;
800/320.3; 800/320.2; 800/302; 800/278; 800/289; 800/284; 800/281;
435/468 |
International
Class: |
A01H 5/00 20060101
A01H005/00; C12N 15/82 20060101 C12N015/82; C12N 5/10 20060101
C12N005/10; A01H 5/10 20060101 A01H005/10; C12N 15/113 20100101
C12N015/113; C12N 15/63 20060101 C12N015/63 |
Claims
1. An isolated nucleic acid molecule comprising a polynucleotide
which has promoter activity and is selected from the group
consisting of: a) the polynucleotide shown as positions 1-2209 of
SEQ ID NO: 2; b) a polynucleotide comprising at least 2000
contiguous nucleotides of positions 1-2209 of SEQ ID NO: 2; and c)
a polynucleotide at least 95% identical to positions 1-2209 of SEQ
ID NO: 2 and retaining all regulatory elements identified at
positions 64-68, 318-323, 442-447, 873-877, 1160-1164, 1179-1184,
1209-1214, 1264-1269, 1483-1488, 1598-1603, 1721-1726, 1746-1751,
1957-1962, and 2081-2086 of SEQ ID NO: 2.
2. An expression cassette comprising the polynucleotide of claim 1
operably linked to a polynucleotide of interest, wherein the
polynucleotide of claim 1 is heterologous to the polynucleotide of
interest.
3. A vector comprising the expression cassette of claim 2.
4. A plant cell having stably incorporated into its genome the
expression cassette of claim 2.
5. The plant cell of claim 4, wherein said plant cell is from a
monocot.
6. The plant cell of claim 5, wherein said monocot is maize,
barley, wheat, oat, rye, sorghum or rice.
7. A plant having stably incorporated into its genome the
expression cassette of claim 2.
8. The plant of claim 7, wherein said plant is a monocot.
9. The plant of claim 8, wherein said monocot is maize, barley,
wheat, oat, rye, sorghum or rice.
10. A transgenic seed of the plant of claim 7.
11. The plant of claim 7, wherein the polynucleotide of interest
encodes a gene product that confers drought tolerance, cold
tolerance or salt tolerance.
12. The plant of claim 7, wherein the polynucleotide of interest
encodes a polypeptide involved in nutrient uptake, nitrogen use
efficiency, root strength, root lodging resistance, soil pest
management, corn rootworm resistance, carbohydrate metabolism,
protein metabolism, fatty acid metabolism or phytohormone
biosynthesis.
13. A method for expressing a polynucleotide of interest in a
plant, said method comprising introducing into the plant an
expression cassette comprising a promoter and the polynucleotide of
interest operably linked thereto, wherein said promoter comprises a
polynucleotide selected from the group consisting of: a) the
polynucleotide shown as positions 1-2209 of SEQ ID NO: 2; b) a
polynucleotide comprising at least 2000 contiguous nucleotides of
positions 1-2209 of SEQ ID NO: 2; and c) a polynucleotide at least
95% identical to positions 1-2209 of SEQ ID NO: 2 and retaining all
regulatory elements identified at positions 64-68, 318-323,
442-447, 873-877, 1160-1164, 1179-1184, 1209-1214, 1264-1269,
1483-1488, 1598-1603, 1721-1726, 1746-1751, 1957-1962, and
2081-2086 of SEQ ID NO: 2. and whereby the polynucleotide of
interest is expressed in the plant.
14. The method of claim 13, wherein said plant is a monocot.
15. The method of claim 14, wherein said monocot is maize, barley,
wheat, oat, rye, sorghum or rice.
16. The method of claim 13, wherein said polynucleotide of interest
encodes a gene product that confers drought tolerance, cold
tolerance or salt tolerance.
17. The method of claim 13, wherein said polynucleotide of interest
encodes a polypeptide involved in nutrient uptake, nitrogen use
efficiency, root strength, root lodging resistance, corn rootworm
resistance, carbohydrate metabolism, protein metabolism, fatty acid
metabolism or phytohormone biosynthesis.
18. A method for expressing a polynucleotide of interest in a plant
cell, said method comprising introducing into the plant cell an
expression cassette comprising a promoter and a polynucleotide of
interest operably linked thereto, wherein said promoter comprises a
polynucleotide selected from the group consisting of: a) the
polynucleotide shown as positions 1-2209 of SEQ ID NO: 2; b) a
polynucleotide comprising at least 2000 contiguous nucleotides of
positions 1-2209 of SEQ ID NO: 2; and (c) a polynucleotide at least
95% identical to positions 1-2209 of SEQ ID NO: 2 and retaining all
regulatory elements identified at positions 64-68, 318-323,
442-447, 873-877, 1160-1164, 1179-1184, 1209-1214, 1264-1269,
1483-1488, 1598-1603, 1721-1726, 1746-1751, 1957-1962, and
2081-2086 of SEQ ID NO: 2. and whereby the polynucleotide of
interest is expressed in the plant cell.
19. The method of claim 18, wherein said polynucleotide of interest
encodes a gene product that confers drought tolerance, cold
tolerance or salt tolerance.
20. The method of claim 18, wherein said polynucleotide of interest
encodes a polypeptide involved in nutrient uptake, nitrogen use
efficiency, root strength, root lodging resistance, corn rootworm
resistance, carbohydrate metabolism, protein metabolism, fatty acid
metabolism, or phytohormone biosynthesis.
Description
CROSS REFERENCE
[0001] This utility application is a divisional of U.S. patent
application Ser. No. 12/207,620 filed Sep. 10, 2008, pending, and
also claims the benefit of U.S. Provisional Application No.
60/971,278, filed Sep. 11, 2007, both of which are incorporated
herein by reference.
FIELD
[0002] The present invention 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 operably linked regulatory elements
that are functional within the plant host. Choice of the regulatory
element will determine when and where within the organism the
heterologous DNA sequence is expressed. Where continuous expression
is desired throughout the cells of a plant, and/or throughout
development, constitutive promoters are utilized. In contrast,
where gene expression in response to a stimulus is desired,
inducible promoters are the regulatory element of choice. Where
expression in specific tissues or organs is desired,
tissue-specific or tissue-preferred promoters may be used to drive
expression preferentially in certain tissues or organs. Such
tissue-specific or tissue-preferred promoters may be temporally
constitutive or inducible. In either case, additional regulatory
sequences upstream and/or downstream from a core promoter sequence
may be included in expression constructs of transformation vectors
to bring about varying levels of expression of heterologous
nucleotide sequences in a transgenic plant.
[0004] As this field develops and more genes become accessible, a
greater need exists for transformed plants with multiple genes, and
these multiple exogenous genes typically need to be controlled by
separate regulatory sequences. Further, some genes should be
regulated constitutively, whereas other genes should be expressed
at certain developmental stages or locations in the transgenic
organism. Accordingly, a variety of regulatory sequences having
diverse effects is needed.
[0005] Diverse regulatory sequences are also needed as undesirable
biochemical interactions can result from using the same regulatory
sequence to control more than one gene. For example, transformation
with multiple copies of a regulatory element may cause problems,
such that expression of one or more genes may be adversely
affected.
[0006] Transgenic modulation of early sensing and signaling genes
involved in abiotic stress responses requires expression of the
transgenes early upon exposure to the stress and at a moderate
level. Also, expression of such transgenes needs to be turned off
at later stages of stress exposure so as to avoid the continued
induction of downstream targets, a scenario which can easily lead
to yield penalty. The current invention provides two regulatory
sequences which can be used for early expression and tight
modulation of signaling and sensing genes, for transgenic
modulation of plant stress tolerance.
[0007] The inventors herein disclose the isolation and
characterization of promoters associated with stress-related
transcription factors that can serve as regulatory elements for
expression of isolated nucleotide sequences of interest, thereby
impacting various traits in plants. Alternatively or additionally,
the promoters may be used to drive scorable markers.
SUMMARY
[0008] The invention provides plant promoters which regulate
transcription and are induced in response to abiotic stress.
[0009] In an embodiment, the promoter drives transcription in a
stress-responsive manner, wherein said promoter comprises a
nucleotide sequence selected from the group consisting of:
[0010] a) sequences natively associated with, and that regulate
expression of, DNA coding for the CBF1 or CBF2 transcription factor
in maize (Zea mays);
[0011] b) the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ
ID NO: 2; and
[0012] c) a sequence comprising a fragment of the nucleotide
sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2.
[0013] Further embodiments are to expression cassettes,
transformation vectors, plants, plant cells and plant parts
comprising the above nucleotide sequences. The invention is further
to methods of using the sequence in plants and plant cells. An
embodiment of the invention further comprises the nucleotide
sequences described above operably linked to a detectable
marker.
BRIEF DESCRIPTION OF THE FIGURES
[0014] FIG. 1 shows the regulatory sequence of ZmCBF1 (1329 base
pairs). Putative cis-acting elements, including a TATA box (bold),
CRT/DRE consensus sequence (boxed with double lines), ABRE core
motif (single underline), and myc-binding sequences (boxed with
single lines) are marked. Bold letters, double underline and an
arrow indicate the translation start site, ATG. All features are
also indicated in the sequence listing at SEQ ID NO: 1.
[0015] FIG. 2 shows the regulatory sequence of ZmCBF2 (2209 base
pairs). Putative cis-acting elements, including a TATA box (bold),
CRT/DRE consensus sequence (boxed with double lines), and
myc-binding sequences (boxed with single line) are marked. A
putative transcription start site is indicated by a double
underline. Bold letters, double underline and an arrow indicate the
translation start site, ATG. All features are also indicated in the
sequence listing at SEQ ID NO: 2.
DETAILED DESCRIPTION
[0016] All public disclosures referred to herein are incorporated
by reference.
[0017] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Unless
mentioned otherwise, the techniques employed or contemplated herein
are standard methodologies well known to one of ordinary skill in
the art. The materials, methods and examples are illustrative only
and not limiting.
[0018] Plants adapt to environmental stresses such as cold,
drought, and salinity through modulation of gene expression.
Promoter regions of stress-inducible genes may comprise cis-acting
elements, which are DNA fragments recognized by trans-acting
factors. Transacting factors include plant hormones such as
abscisic acid (ABA) which has been shown to bind to an
ABA-responsive element (ABRE); see, for example,
Yamaguchi-Shinozaki, et al., (2005) Trends in Plant Science
10(2):88-94. Other transacting factors include nuclear proteins
capable of binding to regulatory DNA and interacting with other
molecules, notably DNA Polymerase III, to initiate transcription of
DNA operably linked to said regulatory DNA. These transcription
factors may exist as families of related proteins that share a
DNA-binding domain. The transcription factor genes may themselves
be induced by stress. Furthermore, the downstream targets of
cis-regulated genes may be transcription factors, creating a
complex network of gene response cascades.
[0019] DRE/CRT (Dehydration Response Element/C-Repeat) cis elements
function in response to stress and have been identified in numerous
plant species, including Arabidopsis, barley, Brassica, citrus,
cotton, eucalyptus, grape, maize, melon, pepper, rice, soy,
tobacco, tomato and wheat. The DRE/CRT elements comprise a core
binding site, A/GCCGAC, recognized by the trans-activating factors
known as DREB1 (DRE-Binding) and CBF (C-Repeat Binding Factor).
Secondary structure in proximity to the cis element, and/or
multiple cis factors, appear to be additional components necessary
for stress-inducible expression. (For reviews, see, Agarwal, et
al., (2006) Plant Cell Rep 25:1263-1274; Yamaguchi-Shinozaki and
Shinozaki (2005) Trends in Plant Science 10(2):88-94.) The promoter
regions of the CBF/DREB genes may comprise cis-acting elements such
as ICEr1 and ICEr2 (Zarka, et al., (2003) Plant Physiol.
133:910-918; Massari and Murre (2000) Mol. Cell. Bio.
20:429-440).
[0020] Other transcription factors include the MYC and MYC-like
proteins (see, for example, Zhu, et al., (2003) J. Biol. Chem.
278(48):47803-47811).
[0021] In accordance with the invention, nucleotide sequences are
provided that allow regulation of transcription in response to
stress. The sequences of the invention comprise regulatory elements
associated with stress-responsive polynucleotides. Thus, the
compositions of the present invention comprise novel nucleotide
sequences for plant regulatory elements natively associated with
the nucleotide sequences coding for ZmCBF1 and ZmCBF2.
[0022] ZmCBF1 and ZmCBF2 belong to the DREB1 class of transcription
factors which are induced early upon exposure to abiotic stresses
such as cold, drought, and salt. The promoter of the Arabidopsis
CBF3 gene is known to contain five myc-binding sites and to bind to
a basic helix-loop-helix protein known as the ICE1 (inducer of CBF
expression) which is an upstream regulatory protein of CBF. A
DREB1B promoter from rice (GenBank EF556551) has been isolated by
Gutha and Reddy, and was shown to be induced by abiotic stress.
(Plant Molecular Biology online 10.1007/s11103-008-9391-8, 28 Aug.
2008). Badawi, et al., (Plant Cell Physiol. 49(8):1237-1249 (2008))
reported an analysis of wheat ICE (inducer of DREB1/CBF expression)
genes and their impact on expression of cold-regulated genes of
Arabidopsis.
[0023] Identification of the regulatory regions of ZmCBF1 and
ZmCBF2 will (a) allow their use for low-level expression and tight
modulation of early sensing and signaling genes, and (b) help to
identify the maize orthologs of ICE1 transcription factor either by
yeast one-hybrid screen or to confirm the function of maize
orthologs identified by sequence homology using electrophoretic
mobility shift assays. ZmCBF1 and ZmCBF2 were previously published;
see, U.S. Pat. Nos. 7,253,000 and 7,317,141. As documented in
Example 4 of U.S. Pat. No. 7,317,141, endogenous ZmCBF2 is rapidly
induced by cold stress. Similar work with ZmCBF1 has shown that it
too is inducible by cold, with peak expression at 4 hours after
imposition of cold stress. ZmCBF1 has also been evaluated for
expression under drought stress; it is induced within 24 hours of
the withholding of water, with peak expression at 27 hours. In case
of gene expression under cold, induced levels decreased after the
peak and returned to zero upon recovery from cold over a period of
48 hours. In case of gene expression under drought, induced levels
gradually declined after the peak expression and returned to zero
after about 36 hours of withholding water. In both tests, no ZmCBF1
expression was observed prior to the stress treatment.
Stress-induced endogenous expression is consistent with the
identification of stress-responsive elements within the ZmCBF
promoter sequences as described elsewhere herein.
[0024] The ZmCBF regulatory element will be operably linked to a
sequence of interest, regulating initiation of transcription of the
operably-linked polynucleotide, which will provide for modification
of the phenotype of the plant. Such initiation of transcription may
be referred to as "promoter activity." 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. For
example, such a promoter is useful for modulation of expression of
sequences encoding stress-responsive proteins, including other
transcription factors. Additionally, linking a stress-induced
promoter with a marker, and, in particular, a visual marker, may be
useful in tracking the expression of a linked gene of interest.
[0025] A method for expressing an isolated nucleotide sequence in a
plant using a regulatory sequence disclosed herein is provided. The
method comprises transforming a plant cell with a transformation
vector that comprises an isolated nucleotide sequence operably
linked to one or more of the plant regulatory sequences of the
present invention and regenerating a stably transformed plant from
the transformed plant cell. In this manner, the regulatory
sequences are useful for controlling the expression of endogenous
as well as exogenous products in a stress-induced manner.
[0026] Frequently it is desirable to have preferential expression
of a DNA sequence in a tissue of an organism, or under certain
environmental conditions. For example, increased resistance of a
plant to insect attack might be accomplished by genetic
manipulation of the plant's genome to comprise a tissue-specific
promoter operably linked to a heterologous insecticide gene such
that the insect-deterring substances are specifically expressed in
the susceptible plant tissues. Increased tolerance to abiotic
stress might be accomplished by genetic manipulation of the plant's
genome to comprise a stress-induced promoter operably linked to a
heterologous gene encoding a plant hormone such that the hormone is
specifically expressed under the stress conditions. Preferential
expression of the heterologous nucleotide sequence in the
appropriate tissue or under the appropriate conditions reduces the
drain on the plant's resources that occurs when a constitutive
promoter initiates transcription of a heterologous nucleotide
sequence throughout the cells of the plant.
[0027] Alternatively, it might be desirable to inhibit expression
of a native DNA sequence within a plant's tissues to achieve a
desired phenotype. For example, a hairpin configuration comprising
all or a portion of the respective ZmCBF promoter may be used to
downregulate the native stress-responsive CBF1 or CBF2, or to
downregulate any other coding region to which the ZmCBF promoter is
linked. When such downregulation of a stress-responsive
polynucleotide is appropriately targeted, for example with a
reproductive-tissue-preferred promoter, certain plant tissues may
avoid detrimental effects of stress. In another example, the ZmCBF
promoter is operably linked to an antisense nucleotide sequence,
such that stress-induced expression of the antisense sequence
produces an RNA transcript that interferes with translation of the
mRNA of a second DNA sequence in a subset of the plant's cells.
DEFINITIONS
[0028] For the purposes of the present invention, unless indicated
otherwise or apparent from the context, a "subject plant" or
"subject plant cell" is one in which genetic alteration, such as
transformation, has been effected as to a gene of interest, or is a
plant or plant cell which is descended from a plant or plant cell
so altered and which comprises the alteration. A "control" or
"control plant" or "control plant cell" provides a reference point
for measuring changes in the subject plant or plant cell.
[0029] A control plant or control plant cell may comprise, for
example: (a) a wild-type plant or plant cell, i.e., of the same
genotype as the starting material for the genetic alteration which
resulted in the subject plant or subject plant cell; (b) a plant or
plant cell of the same genotype as the starting material but which
has been transformed with a null construct (i.e., with a construct
which has no known effect on the trait of interest, such as a
construct comprising a marker gene); (c) a plant or plant cell
which is a non-transformed segregant among progeny of a subject
plant or subject plant cell; (d) a plant or plant cell genetically
identical to the subject plant or subject plant cell but which is
not exposed to conditions or stimuli that would induce expression
of the gene of interest; or (e) the subject plant or subject plant
cell itself, under conditions in which the gene of interest is not
expressed.
[0030] By "stress-induced" is intended favored expression under
conditions of stress to the plant, particularly abiotic stress, for
example conditions of drought, cold, high temperature, or high
salinity.
[0031] By "regulatory element" is intended sequences responsible
for expression of the associated coding sequence including, but not
limited to, promoters, terminators, enhancers, introns, and the
like.
[0032] By "terminator" is intended a regulatory region of DNA that
causes RNA polymerase to disassociate from DNA, causing termination
of transcription.
[0033] By "promoter" is intended a regulatory region of DNA capable
of regulating the transcription of a sequence linked thereto, i.e.,
a region of DNA having promoter activity. It usually comprises a
TATA box capable of directing RNA polymerase II to initiate RNA
synthesis at the appropriate transcription initiation site for a
particular coding sequence.
[0034] A promoter may additionally comprise other recognition
sequences generally positioned upstream or 5' to the TATA box,
referred to as upstream promoter elements, which influence the
transcription initiation rate and further include elements which
impact spatial and temporal expression of the linked nucleotide
sequence. It is recognized that having identified the nucleotide
sequences for the promoter region disclosed herein, it is within
the state of the art to isolate and identify further regulatory
elements in the 5' region upstream from the particular promoter
region identified herein. Thus the promoter region disclosed herein
may comprise upstream regulatory elements such as those responsible
for tissue and temporal expression of the coding sequence, and may
include enhancers, the DNA response element for a transcriptional
regulatory protein, ribosomal binding sites, transcriptional start
and stop sequences, translational start and stop sequences,
activator sequence and the like.
[0035] In the same manner, the promoter elements which enable
expression under stress conditions can be identified, isolated, and
used with other core promoters. By core promoter is meant the
minimal sequence required to initiate transcription, such as the
sequence called the TATA box which is common to promoters in genes
encoding proteins. Thus the upstream promoter of ZmCBF1 or ZmCBF2
can optionally be used in conjunction with its own or core
promoters from other sources. The promoter may be native or
non-native to the cell in which it is found.
[0036] The isolated promoter sequence of the present invention can
be modified to provide for a range of expression levels of the
isolated nucleotide sequence. Less than the entire promoter region
can be utilized and the ability to drive stress-induced expression
retained. It is recognized that expression levels of mRNA can be
modulated with specific deletions of portions of the promoter
sequence. Thus, the promoter can be modified to be a weak or strong
promoter. Generally, by "weak promoter" is intended a promoter that
drives expression of a coding sequence at a low level. By "low
level" is intended 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. Generally, at least about 20 nucleotides
of an isolated promoter sequence will be used to drive expression
of a nucleotide sequence.
[0037] It is recognized that to increase transcription levels
enhancers can be utilized in combination with the promoter regions
of the invention. Enhancers are nucleotide sequences that act to
increase the expression of a promoter region. Enhancers are known
in the art and include the SV40 enhancer region, the 35S enhancer
element, and the like.
[0038] The promoter of the present invention can be isolated from
the 5' region of its native coding region or 5' untranslated region
(5' UTR). Likewise the terminator can be isolated from the 3'
region flanking its respective stop codon. The term "isolated"
refers to material, such as a nucleic acid or protein, which is:
(1) substantially or essentially free from components which
normally accompany or interact with the material as found in its
naturally occurring environment; or (2) if the material is in its
natural environment, the material has been altered by deliberate
human intervention to a composition and/or placed at a locus in a
cell other than the locus native to the material. Methods for
isolation of promoter regions are well known in the art.
[0039] The complete genomic sequence for maize ZmCBF1 gene has been
previously published (US Patent Application Publication Number
2006/0162027). The maize CBF1 promoter is set forth in SEQ ID NO: 1
and is 1373 nucleotides in length. The maize CBF2 promoter is set
forth in SEQ ID NO: 2 and is 2266 nucleotides in length.
[0040] Motifs identified in the ZmCBF1 or ZmCBF2 promoter are shown
in FIG. 1 and in the sequence listing.
[0041] The promoter regions of the invention may be isolated from
any plant, including, but not limited to corn (Zea mays), canola
(Brassica napus, Brassica rapa ssp.), alfalfa (Medicago sativa),
rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghum
bicolor, Sorghum vulgare), sunflower (Helianthus annuus), wheat
(Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana
tabacum), millet (Panicum spp.), potato (Solanum tuberosum),
peanuts (Arachis hypogaea), cotton (Gossypium hirsutum), sweet
potato (Ipomoea batatus), cassaya (Manihot esculenta), coffee
(Cofea 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), oats (Avena sativa), barley (Hordeum
vulgare), vegetables, ornamentals, and conifers. Preferably, plants
include corn, soybean, sunflower, safflower, canola, wheat, barley,
rye, alfalfa and sorghum.
[0042] Promoter sequences from other plants may be isolated
according to well-known techniques based on their sequence homology
to the homologous coding region of the coding sequences set forth
herein. In these techniques, all or part of the known coding
sequence is used as a probe which selectively hybridizes to other
sequences present in a population of cloned genomic DNA fragments
(i.e., genomic libraries) from a chosen organism. Methods are
readily available in the art for the hybridization of nucleic acid
sequences. An extensive guide to the hybridization of nucleic acids
is found in Tijssen, Laboratory Techniques in Biochemistry and
Molecular Biology--Hybridization with Nucleic Acid Probes, Part I,
Chapter 2 "Overview of principles of hybridization and the strategy
of nucleic acid probe assays", Elsevier, New York (1993); and
Current Protocols in Molecular Biology, Chapter 2, Ausubel, et al.,
Eds., Greene Publishing and Wiley-Interscience, New York
(1995).
[0043] "Functional variants" of the regulatory sequences are also
encompassed by the compositions of the present invention.
Functional variants include, for example, the native regulatory
sequences of the invention having one or more nucleotide
substitutions, deletions or insertions and which drive expression
of an operably-linked sequence under conditions similar to those
under which the native promoter is active. Functional variants of
the invention may be created by site-directed mutagenesis or by
induced mutation, or may occur as allelic variants
(polymorphisms).
[0044] As used herein, a "functional fragment" is a truncated
regulatory sequence formed by one or more deletions from a larger
regulatory element. For example, the 5' portion of a promoter up to
the TATA box near the transcription start site can be deleted
without abolishing promoter activity, as described by
Opsahl-Sorteberg, et al., (2004) Gene 341:49-58. Such fragments
should retain promoter activity, particularly the ability to drive
stress-induced expression. Activity can be measured by Northern
blot analysis, reporter activity measurements when using
transcriptional fusions, and the like. See, for example, Sambrook,
et al., (1989) Molecular Cloning: A Laboratory Manual (2nd ed. Cold
Spring Harbor Laboratory, Cold Spring Harbor, N.Y.), herein
incorporated by reference.
[0045] Functional fragments can be obtained by use of restriction
enzymes to cleave the naturally occurring regulatory element
nucleotide sequences disclosed herein; by synthesizing a nucleotide
sequence from the naturally occurring DNA sequence; or can be
obtained through the use of PCR technology. See particularly,
Mullis, et al., (1987) Methods Enzymol. 155:335-350 and Erlich, ed.
(1989) PCR Technology (Stockton Press, New York).
[0046] For example, a routine way to remove part of a DNA sequence
is to use an exonuclease in combination with DNA amplification to
produce unidirectional nested deletions of double stranded DNA
clones. A commercial kit for this purpose is sold under the trade
name Exo-Size.TM. (New England Biolabs, Beverly, Mass.). Briefly,
this procedure entails incubating exonuclease III with DNA to
progressively remove nucleotides in the 3' to 5' direction at 5'
overhangs, blunt ends or nicks in the DNA template. However,
exonuclease III is unable to remove nucleotides at 3', 4-base
overhangs. Timed digests of a clone with this enzyme produces
unidirectional nested deletions.
[0047] The entire promoter sequence or portions thereof can be used
as a probe capable of specifically hybridizing to corresponding
promoter sequences. To achieve specific hybridization under a
variety of conditions, such probes include sequences that are
unique and are preferably at least about 10 nucleotides in length,
and most preferably at least about 20 nucleotides in length. Such
probes can be used to amplify corresponding promoter sequences from
a chosen organism by the well-known process of polymerase chain
reaction (PCR). This technique can be used to isolate additional
promoter sequences from a desired organism or as a diagnostic assay
to determine the presence of the promoter sequence in an organism.
Examples include hybridization screening of plated DNA libraries
(either plaques or colonies; see, e.g., Innis, et al., (1990) PCR
Protocols, A Guide to Methods and Applications, eds., Academic
Press).
[0048] The stress-induced regulatory elements disclosed in the
present invention, as well as variants and fragments thereof, are
useful in the genetic manipulation of any plant when operably
linked with an isolated nucleotide sequence of interest whose
expression is to be controlled to achieve a desired phenotypic
response.
[0049] By "operably linked" is intended a functional linkage
between a promoter and a second sequence, wherein the promoter
sequence initiates and mediates transcription of the DNA sequence
corresponding to the second sequence. The expression cassette will
include a regulatory sequence of the invention operably linked to
at least one sequence of interest.
[0050] In one typical embodiment, in the context of an over
expression cassette, operably linked means that the nucleotide
sequences being linked are contiguous and, where necessary to join
two or more protein coding regions, contiguous and in the same
reading frame. In the case where an expression cassette contains
two or more protein coding regions joined in a contiguous manner in
the same reading frame, the encoded polypeptide is herein defined
as a "heterologous polypeptide" or a "chimeric polypeptide" or a
"fusion polypeptide". The cassette may additionally contain at
least one additional coding sequence to be co-transformed into the
organism. Alternatively, the additional coding sequence(s) can be
provided on multiple expression cassettes.
[0051] The regulatory elements of the invention can be operably
linked to the isolated nucleotide sequence of interest in any of
several ways known to one of skill in the art. The isolated
nucleotide sequence of interest can be inserted into a site within
the genome which is 3' to the promoter of the invention using site
specific integration as described in U.S. Pat. No. 6,187,994 herein
incorporated in its entirety by reference.
[0052] The regulatory elements of the invention can be operably
linked in expression cassettes along with isolated polynucleotide
sequences of interest for expression in the plant. Such an
expression cassette is provided with a plurality of restriction
sites for insertion of the nucleotide sequence of interest under
the transcriptional control of the regulatory elements.
[0053] The regulatory elements of the invention can be used for
directing expression of a sequence in plant tissues. This can be
achieved by increasing expression of endogenous or exogenous
products. Alternatively, the results can be achieved by providing
for a reduction of expression of one or more endogenous products,
particularly enzymes or cofactors. This down regulation can be
achieved through many different approaches known to one skilled in
the art, including antisense, cosuppression, use of hairpin
formations, or others, and discussed infra. It is recognized that
the regulatory elements may be used with their native or other
coding sequences to increase or decrease expression of an operably
linked sequence in the transformed plant or seed.
[0054] General categories of genes of interest for the purposes of
the present invention include for example, those genes involved in
information, such as zinc fingers; those involved in communication,
such as kinases; and those involved in housekeeping, such as heat
shock proteins. More specific categories of transgenes include
genes encoding important traits for agronomics, insect resistance,
disease resistance, herbicide resistance, and grain
characteristics. Still other categories of transgenes include genes
for inducing expression of exogenous products such as enzymes,
cofactors and hormones from plants and other eukaryotes as well as
prokaryotic organisms.
[0055] Modifications that affect grain traits include increasing
the content of oleic acid, or altering levels of saturated and
unsaturated fatty acids. Likewise, the level of proteins,
particularly modified proteins that improve the nutrient value of
the plant, can be increased. This is achieved by the expression of
such proteins having enhanced amino acid content.
[0056] Increasing the levels of lysine and sulfur-containing amino
acids may be desired as well as the modification of starch type and
content in the seed. Hordothionin protein modifications are
described in WO 9416078 filed Apr. 10, 1997; WO 9638562 filed Mar.
26, 1997; WO 9638563 filed Mar. 26, 1997 and U.S. Pat. No.
5,703,409 issued Dec. 30, 1997. Another example is lysine and/or
sulfur-rich root protein encoded by the soybean 2S albumin
described in WO 9735023 filed Mar. 20, 1996, and the chymotrypsin
inhibitor from barley, Williamson, et al., (1987) Eur. J. Biochem.
165:99-106.
[0057] Agronomic traits can be improved by altering expression of
genes that affect the response of root, plant or seed growth and
development during environmental stress, Cheikh-N, et al., (1994)
Plant Physiol. 106(1):45-51, and genes controlling carbohydrate
metabolism to reduce kernel abortion in maize, Zinselmeier, et al.,
(1995) Plant Physiol. 107(2):385-391.
[0058] It is recognized that any gene of interest, including the
native coding sequence, can be operably linked to the regulatory
elements of the invention and expressed in the plant.
[0059] By way of illustration, without intending to be limiting,
are examples of the types of genes which can be used in connection
with the regulatory sequences of the invention.
1. Transgenes that confer resistance to insects or disease and that
encode:
[0060] (A) Plant disease resistance genes. Plant defenses are often
activated by specific interaction between the product of a disease
resistance gene (R) in the plant and the product of a corresponding
avirulence (Avr) gene in the pathogen. A plant variety can be
transformed with cloned resistance gene to engineer plants that are
resistant to specific pathogen strains. See, for example, Jones, et
al., (1994) Science 266:789 (cloning of the tomato Cf-9 gene for
resistance to Cladosporium fulvum); Martin, et al., (1993) Science
262:1432 (tomato Pto gene for resistance to Pseudomonas syringae
pv. tomato encodes a protein kinase); Mindrinos, et al., (1994)
Cell 78:1089 (Arabidopsis RSP2 gene for resistance to Pseudomonas
syringae); McDowell and Woffenden, (2003) Trends Biotechnol.
21(4):178-83 and Toyoda, et al., (2002) Transgenic Res.
11(6):567-82. A plant resistant to a disease is one that is more
resistant to a pathogen as compared to the wild type plant.
[0061] (B) A Bacillus thuringiensis protein, a derivative thereof
or a synthetic polypeptide modeled thereon. See, for example,
Geiser, et al., (1986) Gene 48:109, who disclose the cloning and
nucleotide sequence of a Bt delta-endotoxin gene. Moreover, DNA
molecules encoding delta-endotoxin genes can be purchased from
American Type Culture Collection (Rockville, Md.), for example,
under ATCC Accession Numbers 40098, 67136, 31995 and 31998. Other
examples of Bacillus thuringiensis transgenes being genetically
engineered are given in the following patents and patent
applications and hereby are incorporated by reference for this
purpose: U.S. Pat. Nos. 5,188,960; 5,689,052; 5,880,275; PCT
Application Numbers WO 91/14778; WO 99/31248; WO 01/12731; WO
99/24581; WO 97/40162 and U.S. patent application Ser. Nos.
10/032,717; 10/414,637 and 10/606,320.
[0062] (C) An insect-specific hormone or pheromone such as an
ecdysteroid and juvenile hormone, a variant thereof, a mimetic
based thereon, or an antagonist or agonist thereof. See, for
example, the disclosure by Hammock, et al., (1990) Nature 344:458,
of baculovirus expression of cloned juvenile hormone esterase, an
inactivator of juvenile hormone.
[0063] (D) An insect-specific peptide which, upon expression,
disrupts the physiology of the affected pest. For example, see the
disclosures of Regan, (1994) J. Biol. Chem. 269:9 (expression
cloning yields DNA coding for insect diuretic hormone receptor);
Pratt, et al., (1989) Biochem. Biophys. Res. Comm. 163:1243 (an
allostatin is identified in Diploptera puntata); Chattopadhyay, et
al., (2004) Critical Reviews in Microbiology 30(1):33-54; Zjawiony,
(2004) J Nat Prod 67(2):300-310; Carlini and Grossi-de-Sa, (2002)
Toxicon 40(11):1515-1539; Ussuf, et al., (2001) Curr Sci.
80(7):847-853; and Vasconcelos and Oliveira (2004) Toxicon
44(4):385-403. See also, U.S. Pat. No. 5,266,317 to Tomalski, et
al., who disclose genes encoding insect-specific toxins.
[0064] (E) An enzyme responsible for a hyperaccumulation of a
monoterpene, a sesquiterpene, a steroid, hydroxycinnamic acid, a
phenylpropanoid derivative or another non-protein molecule with
insecticidal activity.
[0065] (F) An enzyme involved in the modification, including the
post-translational modification, of a biologically active molecule;
for example, a glycolytic enzyme, a proteolytic enzyme, a lipolytic
enzyme, a nuclease, a cyclase, a transaminase, an esterase, a
hydrolase, a phosphatase, a kinase, a phosphorylase, a polymerase,
an elastase, a chitinase or a glucanase, whether natural or
synthetic. See, PCT Application Number WO 93/02197 in the name of
Scott, et al., which discloses the nucleotide sequence of a callase
gene. DNA molecules which contain chitinase-encoding sequences can
be obtained, for example, from the ATCC under Accession Numbers
39637 and 67152. See also, Kramer, et al., (1993) Insect Biochem.
Molec. Biol. 23:691, who teach the nucleotide sequence of a cDNA
encoding tobacco hookworm chitinase; and Kawalleck, et al., (1993)
Plant Molec. Biol. 21:673, who provide the nucleotide sequence of
the parsley ubi4-2 polyubiquitin gene; U.S. application Ser. Nos.
10/389,432, 10/692,367 and U.S. Pat. No. 6,563,020.
[0066] (G) A molecule that stimulates signal transduction. For
example, see the disclosure by Botella, et al., (1994) Plant Molec.
Biol. 24:757, of nucleotide sequences for mung bean calmodulin cDNA
clones; and Griess, et al., (1994) Plant Physiol. 104:1467, who
provide the nucleotide sequence of a maize calmodulin cDNA
clone.
[0067] (H) A hydrophobic moment peptide. See, PCT Application
Number WO 95/16776 and U.S. Pat. No. 5,580,852 (disclosure of
peptide derivatives of Tachyplesin which inhibit fungal plant
pathogens) and PCT Application Number WO 95/18855 and U.S. Pat. No.
5,607,914 (teaches synthetic antimicrobial peptides that confer
disease resistance).
[0068] (I) A membrane permease, a channel former or a channel
blocker. For example, see the disclosure by Jaynes, et al., (1993)
Plant Sci. 89:43, of heterologous expression of a cecropin-beta
lytic peptide analog to render transgenic tobacco plants resistant
to Pseudomonas solanacearum.
[0069] (J) A viral-invasive protein or a complex toxin derived
therefrom. For example, the accumulation of viral coat proteins in
transformed plant cells imparts resistance to viral infection
and/or disease development effected by the virus from which the
coat protein gene is derived, as well as by related viruses. See,
Beachy, et al., (1990) Ann. Rev. Phytopathol. 28:451. Coat
protein-mediated resistance has been conferred upon transformed
plants against alfalfa mosaic virus, cucumber mosaic virus, tobacco
streak virus, potato virus X, potato virus Y, tobacco etch virus,
tobacco rattle virus and tobacco mosaic virus. Id.
[0070] (K) An insect-specific antibody or an immunotoxin derived
therefrom. Thus, an antibody targeted to a critical metabolic
function in the insect gut would inactivate an affected enzyme,
killing the insect. Cf., Taylor, et al., Abstract #497, Seventh
Intl Symposium on Molecular Plant-microbe Interactions (Edinburgh,
Scotland, 1994) (enzymatic inactivation in transgenic tobacco via
production of single-chain antibody fragments).
[0071] (L) A virus-specific antibody. See, for example,
Tavladoraki, et al. (1993), Nature 366:469, who show that
transgenic plants expressing recombinant antibody genes are
protected from virus attack.
[0072] (M) A developmental-arrestive protein produced in nature by
a pathogen or a parasite. Thus, fungal endo
alpha-1,4-D-polygalacturonases facilitate fungal colonization and
plant nutrient release by solubilizing plant cell wall
homo-alpha-1,4-D-galacturonase. See, Lamb, et al., (1992)
Bio/Technology 10:1436. The cloning and characterization of a gene
which encodes a bean endopolygalacturonase-inhibiting protein is
described by Toubart, et al., (1992) Plant J. 2:367.
[0073] (N) A developmental-arrestive protein produced in nature by
a plant. For example, Logemann, et al., (1992) Bio/Technology
10:305, have shown that transgenic plants expressing the barley
ribosome-inactivating gene have an increased resistance to fungal
disease.
[0074] (O) Genes involved in the Systemic Acquired Resistance (SAR)
Response and/or the pathogenesis related genes. Briggs, (1995)
Current Biology, 5(2):128-131; Pieterse and Van Loon, (2004) Curr.
Opin. Plant Bio. 7(4):456-64 and Somssich, (2003) Cell
113(7):815-6.
[0075] (P) Antifungal genes (Cornelissen and Melchers, (1993) Pl.
Physiol. 101:709-712; Parijs, et al., (1991) Planta 183:258-264 and
Bushnell, et al., (1998) Can. J. of Plant Path. 20(2):137-149. Also
see, U.S. patent application Ser. No. 09/950,933.
[0076] (Q) Detoxification genes, such as for fumonisin,
beauvericin, moniliformin and zearalenone and their structurally
related derivatives. For example, see, U.S. Pat. No. 5,792,931.
[0077] (R) Cystatin and cysteine proteinase inhibitors. See, U.S.
patent application Ser. No. 10/947,979.
[0078] (S) Defensin genes. See, PCT Application Number WO 03/000863
and U.S. patent application Ser. No. 10/178,213.
[0079] (T) Genes conferring resistance to nematodes. See, PCT
Application Number WO 03/033651 and Urwin, et al., (1998) Planta
204:472-479; Williamson (1999) Curr Opin Plant Bio.
2(4):327-31.
[0080] (U) Genes that confer resistance to Phytophthora Root Rot,
such as the Rps 1, Rps 1-a, Rps 1-b, Rps 1-c, Rps 1-d, Rps 1-e, Rps
1-k, Rps 2, Rps 3-a, Rps 3-b, Rps 3-c, Rps 4, Rps 5, Rps 6, Rps 7
and other Rps genes. See, for example, Shoemaker, et al.,
Phytophthora Root Rot Resistance Gene Mapping in Soybean, Plant
Genome IV Conference, San Diego, Calif. (1995).
[0081] (V) Genes that confer resistance to Brown Stem Rot, such as
described in U.S. Pat. No. 5,689,035.
2. Transgenes that confer resistance to a herbicide such as:
[0082] (A) An herbicide that inhibits the growing point or
meristem, such as an imidazolinone or a sulfonylurea. Exemplary
genes in this category code for mutant ALS and AHAS enzyme as
described, for example, by Lee, et al., (1988) EMBO J. 7:1241; and
Miki, et al., (1990) Theor. Appl. Genet. 80:449, respectively. See
also, U.S. Pat. Nos. 5,605,011; 5,013,659; 5,141,870; 5,767,361;
5,731,180; 5,304,732; 4,761,373; 5,331,107; 5,928,937 and 5,378,824
and PCT Application Number WO 96/33270.
[0083] (B) Glyphosate (resistance imparted by mutant
5-enolpyruvl-3-phosphikimate synthase (EPSP) and aroA genes,
respectively) and other phosphono compounds such as glufosinate
(phosphinothricin acetyl transferase (PAT) and Streptomyces
hygroscopicus phosphinothricin acetyl transferase (bar) genes), and
pyridinoxy or phenoxy proprionic acids and cycloshexones (ACCase
inhibitor-encoding 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. 6,566,587; 6,338,961;
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;
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
publication numbers EP1173580; WO 01/66704; EP1173581 and
EP1173582. 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. In
addition glyphosate resistance can be imparted to plants by the
over expression of genes encoding glyphosate N-acetyltransferase.
See, for example, U.S. patent application Ser. No. 10/427,692. A
DNA molecule encoding a mutant aroA gene can be obtained under ATCC
Accession Number 39256 and the nucleotide sequence of the mutant
gene is disclosed in U.S. Pat. No. 4,769,061 to Comai. European
Patent Application Number 0 333 033 to Kumada, et al., and U.S.
Pat. No. 4,975,374 to Goodman, et al., disclose nucleotide
sequences of glutamine synthetase genes which confer resistance to
herbicides such as L-phosphinothricin. The nucleotide sequence of a
phosphinothricin-acetyl-transferase gene is provided in European
Patent Number 0 242 246 and 0 242 236 to Leemans, et al. De Greef,
et al., (1989) Bio/Technology 7:61, describe the production of
transgenic plants that express chimeric bar genes coding for
phosphinothricin acetyl transferase activity. See also, U.S. Pat.
Nos. 5,969,213; 5,489,520; 5,550,318; 5,874,265; 5,919,675;
5,561,236; 5,648,477; 5,646,024; 6,177,616 and 5,879,903. Exemplary
genes conferring resistance to phenoxy proprionic acids and
cycloshexones, such as sethoxydim and haloxyfop, are the Acc1-S1,
Acc1-S2 and Acc1-S3 genes described by Marshall, et al., (1992)
Theor. Appl. Genet. 83:435.
[0084] (C) A herbicide that inhibits photosynthesis, such as a
triazine (psbA and gs+genes) and a benzonitrile (nitrilase gene).
Przibilla, et al., (1991) Plant Cell 3:169, describe the
transformation of Chlamydomonas with plasmids encoding mutant psbA
genes. Nucleotide sequences for nitrilase genes are disclosed in
U.S. Pat. No. 4,810,648 to Stalker, and DNA molecules containing
these genes are available under ATCC Accession Numbers 53435, 67441
and 67442. Cloning and expression of DNA coding for a glutathione
S-transferase is described by Hayes, et al., (1992) Biochem. J.
285:173.
[0085] (D) Acetohydroxy acid synthase, which has been found to make
plants that express this enzyme resistant to multiple types of
herbicides, has been introduced into a variety of plants (see,
e.g., Hattori, et al., (1995) Mol Gen Genet. 246:419). Other genes
that confer resistance to herbicides include: a gene encoding a
chimeric protein of rat cytochrome P4507A1 and yeast
NADPH-cytochrome P450 oxidoreductase (Shiota, et al., (1994) Plant
Physiol. 106:17), genes for glutathione reductase and superoxide
dismutase (Aono, et al., (1995) Plant Cell Physiol 36:1687, and
genes for various phosphotransferases (Datta, et al., (1992) Plant
Mol Biol 20:619).
[0086] (E) Protoporphyrinogen oxidase (protox) is necessary for the
production of chlorophyll, which is necessary for all plant
survival. The protox enzyme serves as the target for a variety of
herbicidal compounds. These herbicides also inhibit growth of all
the different species of plants present, causing their total
destruction. The development of plants containing altered protox
activity which are resistant to these herbicides are described in
U.S. Pat. Nos. 6,288,306; 6,282,837 and 5,767,373; and
international publication number WO 01/12825.
3. Transgenes That Confer Or Contribute To an Altered Grain
Characteristic, Such As:
[0087] (A) Altered fatty acids, for example, by [0088] (1)
Down-regulation of stearoyl-ACP desaturase to increase stearic acid
content of the plant. See, Knultzon, et al., (1992) Proc. Natl.
Acad. Sci. USA 89:2624 and PCT Application Number WO 99/64579
(Genes for Desaturases to Alter Lipid Profiles in Corn), [0089] (2)
Elevating oleic acid via FAD-2 gene modification and/or decreasing
linolenic acid via FAD-3 gene modification (see, U.S. Pat. Nos.
6,063,947; 6,323,392; 6,372,965 and PCT Application Number WO
93/11245), [0090] (3) Altering conjugated linolenic or linoleic
acid content, such as in PCT application Number WO 01/12800, [0091]
(4) Altering LEC1, AGP, Dek1, Superal1, mi1ps, various lpa genes
such as lpa1 lpa3, hpt or hggt. For example, see, PCT Application
Numbers WO 02/42424, WO 98/22604, WO 03/011015, U.S. Pat. No.
6,423,886, U.S. Pat. No. 6,197,561, U.S. Pat. No. 6,825,397, US
Patent Application Publication Number 2003/0079247, US Patent
Application Publication Number 2003/0204870, PCT Application
Numbers WO 02/057439, WO 03/011015 and Rivera-Madrid, et. al.,
(1995) Proc. Natl. Acad. Sci. 92:5620-5624.
[0092] (B) Altered phosphorus content, for example, by the [0093]
(1) Introduction of a phytase-encoding gene. This would enhance
breakdown of phytate, adding more free phosphate to the transformed
plant. For example, see, Van Hartingsveldt, et al., (1993) Gene
127:87, for a disclosure of the nucleotide sequence of an
Aspergillus niger phytase gene. [0094] (2) Up-regulation of a gene
that reduces phytate content. In maize, this, for example, could be
accomplished, by cloning and then re-introducing DNA associated
with one or more of the alleles, such as the LPA alleles,
identified in maize mutants characterized by low levels of phytic
acid, such as in Raboy, et al., (1990) Maydica 35:383 and/or by
altering inositol kinase activity as in PCT Application Number WO
02/059324, US Patent Application Publication Number 2003/0009011,
PCT Application Number WO 03/027243, US Patent Application
Publication Number 2003/0079247, PCT Application Number WO
99/05298, U.S. Pat. No. 6,197,561, U.S. Pat. No. 6,291,224, U.S.
Pat. No. 6,391,348, PCT Application Number WO 2002/059324, US
Patent Application Publication Number 2003/0079247, PCT Application
Numbers WO 98/45448, WO 99/55882, WO 01/04147.
[0095] (C) Altered carbohydrates effected, for example, by altering
a gene for an enzyme that affects the branching pattern of starch
or a gene altering thioredoxin. (See, U.S. Pat. No. 6,531,648).
See, Shiroza, et al., (1988) J. Bacteriol. 170:810 (nucleotide
sequence of Streptococcus mutans fructosyltransferase gene);
Steinmetz, et al., (1985) Mol. Gen. Genet. 200:220 (nucleotide
sequence of Bacillus subtilis levansucrase gene); Pen, et al.,
(1992) Bio/Technology 10:292 (production of transgenic plants that
express Bacillus licheniformis alpha-amylase); Elliot, et al.,
(1993) Plant Molec. Biol. 21 515 (nucleotide sequences of tomato
invertase genes); Sogaard, et al., (1993) J. Biol. Chem. 268:22480
(site-directed mutagenesis of barley alpha-amylase gene); and
Fisher, et al., (1993) Plant Physiol. 102:1045 (maize endosperm
starch branching enzyme II), WO Application Number 99/10498
(improved digestibility and/or starch extraction through
modification of UDP-D-xylose 4-epimerase, Fragile 1 and 2, Ref1,
HCHL, C4H); U.S. Pat. No. 6,232,529 (method of producing high oil
seed by modification of starch levels (AGP)). The fatty acid
modification genes mentioned above may also be used to affect
starch content and/or composition through the interrelationship of
the starch and oil pathways.
[0096] (D) Altered antioxidant content or composition, such as
alteration of tocopherol or tocotrienols. For example, see, U.S.
Pat. No. 6,787,683, US Patent Application Publication Number
2004/0034886 and PCT Application Number WO 00/68393 involving the
manipulation of antioxidant levels through alteration of a phytl
prenyl transferase (ppt), WO Application Number 03/082899 through
alteration of a homogentisate geranyl geranyl transferase
(hggt).
[0097] (E) Altered essential seed amino acids. For example, see,
U.S. Pat. No. 6,127,600 (method of increasing accumulation of
essential amino acids in seeds), U.S. Pat. No. 6,080,913 (binary
methods of increasing accumulation of essential amino acids in
seeds), U.S. Pat. No. 5,990,389 (high lysine), PCT Application
Number WO 99/40209 (alteration of amino acid compositions in
seeds), PCT Application Number WO 99/29882 (methods for altering
amino acid content of proteins), U.S. Pat. No. 5,850,016
(alteration of amino acid compositions in seeds), PCT Application
Number WO 98/20133 (proteins with enhanced levels of essential
amino acids), U.S. Pat. No. 5,885,802 (high methionine), U.S. Pat.
No. 5,885,801 (high threonine), U.S. Pat. No. 6,664,445 (plant
amino acid biosynthetic enzymes), U.S. Pat. No. 6,459,019
(increased lysine and threonine), U.S. Pat. No. 6,441,274 (plant
tryptophan synthase beta subunit), U.S. Pat. No. 6,346,403
(methionine metabolic enzymes), U.S. Pat. No. 5,939,599 (high
sulfur), U.S. Pat. No. 5,912,414 (increased methionine), PCT
Application Number WO 98/56935 (plant amino acid biosynthetic
enzymes), PCT Application Number WO 98/45458 (engineered seed
protein having higher percentage of essential amino acids), PCT
Application Number WO 98/42831 (increased lysine), U.S. Pat. No.
5,633,436 (increasing sulfur amino acid content), U.S. Pat. No.
5,559,223 (synthetic storage proteins with defined structure
containing programmable levels of essential amino acids for
improvement of the nutritional value of plants), PCT Application
Number WO 96/01905 (increased threonine), PCT Application
[0098] Number WO 95/15392 (increased lysine), US Patent Application
Publication Number 2003/0163838, US Patent Application Publication
Number 2003/0150014, US Patent Application Publication Number
2004/0068767, U.S. Pat. No. 6,803,498, PCT Application Number WO
01/79516, and PCT Application Number WO 00/09706 (Ces A: cellulose
synthase), U.S. Pat. No. 6,194,638 (hemicellulose), U.S. Pat. No.
6,399,859 and US Patent Application Publication Number 2004/0025203
(UDPGdH), U.S. Pat. No. 6,194,638 (RGP).
4. Genes that Control Male-sterility
[0099] There are several methods of conferring genetic male
sterility available, such as multiple mutant genes at separate
locations within the genome that confer male sterility, as
disclosed in U.S. Pat. Nos. 4,654,465 and 4,727,219 to Brar, et
al., and chromosomal translocations as described by Patterson in
U.S. Pat. Nos. 3,861,709 and 3,710,511. In addition to these
methods, Albertsen, et al., U.S. Pat. No. 5,432,068, describe a
system of nuclear male sterility which includes: identifying a gene
which is critical to male fertility; silencing this native gene
which is critical to male fertility; removing the native promoter
from the essential male fertility gene and replacing it with an
inducible promoter; inserting this genetically engineered gene back
into the plant; and thus creating a plant that is male sterile
because the inducible promoter is not "on" resulting in the male
fertility gene not being transcribed. Fertility is restored by
inducing, or turning "on", the promoter, which in turn allows the
gene that confers male fertility to be transcribed.
[0100] (A) Introduction of a deacetylase gene under the control of
a tapetum-specific promoter and with the application of the
chemical N-Ac-PPT (PCT Application Number WO 01/29237).
[0101] (B) Introduction of various stamen-specific promoters (PCT
Application Numbers WO 92/13956, WO 92/13957).
[0102] (C) Introduction of the barnase and the barstar gene (Paul,
et al., (1992) Plant Mol. Biol. 19:611-622).
[0103] For additional examples of nuclear male and female sterility
systems and genes, see also, U.S. Pat. No. 5,859,341; U.S. Pat. No.
6,297,426; U.S. Pat. No. 5,478,369; U.S. Pat. No. 5,824,524; U.S.
Pat. No. 5,850,014; and U.S. Pat. No. 6,265,640.
5. Genes that create a site for site specific DNA integration.
[0104] This includes the introduction of FRT sites that may be used
in the FLP/FRT system and/or Lox sites that may be used in the
Cre/Loxp system. For example, see, Lyznik, et al., (2003)
"Site-Specific Recombination for Genetic Engineering in Plants"
Plant Cell Rep 21:925-932 and PCT Application Number WO 99/25821,
which are hereby incorporated by reference. Other systems that may
be used include the Gin recombinase of phage Mu (Maeser, et al.,
(1991) Mol Gen Genet. 230(1-2):170-6); Vicki Chandler, The Maize
Handbook ch. 118 (Springer-Verlag 1994), the Pin recombinase of E.
coli (Enomoto, et al., 1983) and the R/RS system of the pSRi
plasmid (Araki, et al., (1992) J Mol. Biol. 225(1):25-37).
6. Genes that affect abiotic stress resistance (including but not
limited to modulation of flowering, ear and seed development,
enhancement of nitrogen utilization efficiency, altered nitrogen
responsiveness, drought resistance or tolerance, cold resistance or
tolerance, and salt resistance or tolerance) and increased yield
under stress.
[0105] For example, see, PCT Application Number WO 00/73475 where
water use efficiency is altered through alteration of malate; U.S.
Pat. No. 5,892,009, U.S. Pat. No. 5,965,705, U.S. Pat. No.
5,929,305, U.S. Pat. No. 5,891,859, U.S. Pat. No. 6,417,428, U.S.
Pat. No. 6,664,446, U.S. Pat. No. 6,706,866, U.S. Pat. No.
6,717,034, U.S. Pat. No. 6,801,104, PCT Application Numbers WO
2000/060089, WO 2001/026459, WO 2001/035725, WO 2001/034726, WO
2001/035727, WO 2001/036444, WO 2001/036597, WO 2001/036598, WO
2002/015675, WO 2002/017430, WO 2002/077185, WO 2002/079403, WO
2003/013227, WO 2003/013228, WO 2003/014327, WO 2004/031349, WO
2004/076638, WO 98/09521 and WO 99/38977 describing genes,
including CBF genes and transcription factors effective in
mitigating the negative effects of freezing, high salinity, and
drought on plants, as well as conferring other positive effects on
plant phenotype; US Patent Application Publication Number
2004/0148654 and PCT Application Number WO 01/36596 where abscisic
acid is altered in plants resulting in improved plant phenotype
such as increased yield and/or increased tolerance to abiotic
stress; PCT Application Numbers WO 2000/006341, WO 04/090143, U.S.
patent application Ser. Nos. 10/817,483 and 09/545,334 where
cytokinin expression is modified resulting in plants with increased
stress tolerance, such as drought tolerance, and/or increased
yield. Also see, PCT Application Numbers WO 02/02776, WO
2003/052063, JP 2002281975, U.S. Pat. No. 6,084,153, PCT
Application Number WO 0164898, U.S. Pat. No. 6,177,275 and U.S.
Pat. No. 6,107,547 (enhancement of nitrogen utilization and altered
nitrogen responsiveness). For ethylene alteration, see, US Patent
Application Publication Number 2004/0128719, US Patent Application
Publication Number 2003/0166197 and PCT Application Number WO
2000/32761. For plant transcription factors or transcriptional
regulators of abiotic stress, see, e.g., US Patent Application
Publication Number 2004/0098764 or US Patent Application
Publication Number 2004/0078852.
[0106] Other genes and transcription factors that affect plant
growth and agronomic traits such as yield, flowering, plant growth
and/or plant structure, nutrient uptake, especially nitrogen uptake
by plants, nitrogen use efficiency; drought tolerance and water use
efficiency; root strength, and root lodging resistance; soil pest
management, corn root worm resistance can be introduced or
introgressed into plants, see e.g., PCT Application Numbers WO
97/49811 (LHY), WO 98/56918 (ESD4), WO 97/10339 and U.S. Pat. No.
6,573,430 (TFL), U.S. Pat. No. 6,713,663 (FT), PCT Application
Numbers WO 96/14414 (CON), WO 96/38560, WO 01/21822 (VRN1), WO
00/44918 (VRN2), WO 99/49064 (GI), WO 00/46358 (FRI), WO 97/29123,
U.S. Pat. Nos. 6,794,560, 6,307,126 (GAI), PCT Application Numbers
WO 99/09174 (D8 and Rht), and WO 2004/076638 and WO 2004/031349
(transcription factors).
[0107] Commercial traits in plants can be created through the
expression of genes that alter starch or protein for the production
of paper, textiles, ethanol, polymers or other materials with
industrial uses.
[0108] Means of increasing or inhibiting a protein are well known
to one skilled in the art and, by way of example, may include,
transgenic expression, antisense suppression, co-suppression
methods including but not limited to: RNA interference, gene
activation or suppression using transcription factors and/or
repressors, mutagenesis including transposon tagging, directed and
site-specific mutagenesis, chromosome engineering (see, Nobrega,
et. al., (2004) Nature 431:988-993), homologous recombination,
TILLING (Targeting Induced Local Lesions In Genomes; McCallum, et
al., (2000) Nature Biotechnol. 18:455-457), and biosynthetic
competition to manipulate the expression of proteins.
[0109] Many techniques for gene silencing are well known to one of
skill in the art, including but not limited to knock-outs such as
by insertion of a transposable element such as Mu, Vicki Chandler,
The Maize Handbook ch. 118 (Springer-Verlag 1994) or other genetic
elements such as a FRT, Lox or other site specific integration
site; RNA interference (Napoli, et al., (1990) Plant Cell
2:279-289; U.S. Pat. No. 5,034,323, Sharp (1999) Genes Dev.
13:139-141, Zamore, et al., (2000) Cell 101:25-33; and Montgomery,
et al., (1998) PNAS USA 95:15502-15507); virus-induced gene
silencing (Burton, et al., (2000) Plant Cell 12:691-705, and
Baulcombe (1999) Curr. Op. Plant Bio. 2:109-113);
target-RNA-specific ribozymes (Haseloff, et al., (1988) Nature
334:585-591); hairpin structures (Smith, et al., (2000) Nature
407:319-320; PCT Application Numbers WO 99/53050; and WO 98/53083);
MicroRNA (Aukerman and Sakai (2003) Plant Cell 15:2730-2741);
ribozymes (Steinecke, et al., (1992) EMBO J. 11:1525, and Perriman,
et al., (1993) Antisense Res. Dev. 3:253); oligonucleotide mediated
targeted modification (e.g., PCT Application Numbers WO 03/076574
and WO 99/25853); zinc-finger targeted molecules (e.g., PCT
Application Numbers WO 01/52620; WO 03/048345; and WO 00/42219);
and other methods or combinations of the above methods known to
those of skill in the art.
[0110] Any method of increasing or inhibiting a protein can be used
in the present invention. Several examples are outlined in more
detail below for illustrative purposes.
[0111] The nucleotide sequence operably linked to the regulatory
elements disclosed herein can be an antisense sequence for a
targeted gene. (See, e.g., Sheehy, et al., (1988) PNAS USA
85:8805-8809; and U.S. Pat. Nos. 5,107,065; 5,453,566 and
5,759,829). By "antisense sequence" is intended a sequence that is
in inverse orientation to the 5'-to-3' normal orientation of that
nucleotide sequence. When delivered into a plant cell, expression
of the antisense DNA sequence prevents normal expression of the DNA
nucleotide sequence for the targeted gene. The antisense nucleotide
sequence encodes an RNA transcript that is complementary to and
capable of hybridizing with the endogenous messenger RNA (mRNA)
produced by transcription of the DNA nucleotide sequence for the
targeted gene. In this case, production of the native protein
encoded by the targeted gene is inhibited to achieve a desired
phenotypic response. Thus the regulatory sequences disclosed herein
can be operably linked to antisense DNA sequences to reduce or
inhibit expression of a native protein in the plant.
[0112] As noted, other potential approaches to impact expression of
proteins in the plant include traditional co-suppression, that is,
inhibition of expression of an endogenous gene through the
expression of an identical structural gene or gene fragment
introduced through transformation (Goring, et al., (1991) Proc.
Natl. Acad Sci. USA 88:1770-1774 co-suppression; Taylor, (1997)
Plant Cell 9:1245; Jorgensen, (1990) Trends Biotech. 8(12):340-344;
Flavell, (1994) PNAS USA 91:3490-3496; Finnegan, et al., (1994)
Bio/Technology 12:883-888; and Neuhuber, et al., (1994) Mol. Gen.
Genet. 244:230-241). In one example, co-suppression can be achieved
by linking the promoter to a DNA segment such that transcripts of
the segment are produced in the sense orientation and where the
transcripts have at least 65% sequence identity to transcripts of
the endogenous gene of interest, thereby suppressing expression of
the endogenous gene in said plant cell. (See, U.S. Pat. No.
5,283,184). The endogenous gene targeted for co-suppression may be
a gene encoding any protein that accumulates in the plant species
of interest. For example, where the endogenous gene targeted for
co-suppression is the 50 kD gamma-zein gene, co-suppression is
achieved using an expression cassette comprising the 50 kD
gamma-zein gene sequence, or variant or fragment thereof.
[0113] Additional methods of suppression are known in the art and
can be similarly applied to the instant invention. These methods
involve the silencing of a targeted gene by spliced hairpin RNA's
and similar methods also called RNA interference and promoter
silencing (see, Smith, et al., (2000) Nature 407:319-320,
Waterhouse and Helliwell (2003) Nat. Rev. Genet. 4:29-38;
Waterhouse, et al., (1998) Proc. Natl. Acad. Sci. USA
95:13959-13964; Chuang and Meyerowitz, (2000) Proc. Natl. Acad.
Sci. USA 97:4985-4990; Stoutjesdijk, et al., (2002) Plant Phystiol.
129:1723-1731; and PCT Application Numbers WO 99/53050; WO
99/49029; WO 99/61631; WO 00/49035 and U.S. Pat. No.
6,506,559).
[0114] For miRNA interference, the expression cassette is designed
to express an RNA molecule that is modeled on an endogenous gene.
The miRNA molecule encodes an RNA that forms a hairpin structure
containing a 22-nucleotide sequence that is complementary to the
endogenous gene (target sequence). miRNA molecules are highly
efficient at inhibiting the expression of endogenous genes, and the
RNA interference they induce is inherited by subsequent generations
of plants.
[0115] In one embodiment, the polynucleotide to be introduced into
the plant comprises an inhibitory sequence that encodes a zinc
finger protein that binds to a gene resulting in reduced expression
of the gene. In particular embodiments, the zinc finger protein
binds to a regulatory region of the invention. In other
embodiments, the zinc finger protein binds to a messenger RNA
encoding a protein and prevents its translation. Methods of
selecting sites for targeting by zinc finger proteins have been
described, for example, in U.S. Pat. No. 6,453,242, and methods for
using zinc finger proteins to inhibit the expression of genes in
plants are described, for example, in US Patent Application
Publication Number 2003/0037355.
[0116] The expression cassette may also include, at the 3' terminus
of the isolated nucleotide sequence of interest, a transcriptional
and translational termination region functional in plants. The
termination region can be native with the promoter nucleotide
sequence of the present invention, can be native with the DNA
sequence of interest, or can be derived from another source.
[0117] Any convenient termination regions can be used in
conjunction with the promoter of the invention, and 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; Joshi, et al., (1987) Nucleic Acid Res.
15:9627-9639.
[0118] The expression cassettes can 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. Natl. Acad.
Sci. USA 86:6126-6130; potyvirus leaders, for example, TEV leader
(Tobacco Etch Virus), Allison, et al., (1986) "The Nucleotide
Sequence of the Coding Region of Tobacco Etch Virus Genomic RNA:
Evidence for the Synthesis of a Single Polyprotein", 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. The cassette can also contain sequences that
enhance translation and/or mRNA stability such as introns.
[0119] In those instances where it is desirable to have an
expressed product of an isolated nucleotide sequence directed to a
particular organelle, particularly the plastid, amyloplast, or to
the endoplasmic reticulum, or secreted at the cell's surface or
extracellularly, the expression cassette can further comprise a
coding sequence for a transit peptide. Such transit peptides are
well known in the art and include, but are not limited to: the
transit peptide for the acyl carrier protein, the small subunit of
RUBISCO, plant EPSP synthase, and the like.
[0120] In preparing the expression cassette, the various DNA
fragments can 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 can be
employed to join the DNA fragments, or other manipulations can 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
digests, annealing, and resubstitutions such as transitions and
transversions, can be involved.
[0121] As noted herein, the present invention provides vectors
capable of expressing genes of interest under the control of the
regulatory elements of the invention. In general, the vectors
should be functional in plant cells. At times, it may be preferable
to have vectors that are functional in E. coli (e.g., production of
protein for raising antibodies, DNA sequence analysis, construction
of inserts, obtaining quantities of nucleic acids). Vectors and
procedures for cloning and expression in E. coli are discussed in
Sambrook, et al. (supra).
[0122] The transformation vector, comprising the regulatory
sequences of the present invention operably linked to an isolated
polynucleotide sequence in an expression cassette, can 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 transformation vector.
[0123] Vectors that are functional in plants can be binary plasmids
derived from Agrobacterium. Such vectors are capable of
transforming plant cells. These vectors contain left and right
border sequences that are required for integration into the host
(plant) chromosome. At a minimum, between these border sequences is
the gene to be expressed under control of the regulatory elements
of the present invention. In one embodiment, a selectable marker
and a reporter gene are also included. For ease of obtaining
sufficient quantities of vector, a bacterial origin that allows
replication in E. coli can be used.
[0124] Reporter genes can be included in the transformation
vectors. 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.
[0125] Selectable marker genes for selection of transformed cells
or tissues can be included in the transformation vectors. These can
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;
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;
phosphinothricin, DeBlock, et al., (1987) EMBO J. 6:2513-2518.
[0126] Further, when linking a promoter of the invention with a
nucleotide sequence encoding a detectable protein, stress-induced
expression of a linked sequence can be tracked, thereby providing a
useful so-called screenable or scorable markers. The expression of
the linked protein can be detected without destroying tissue. More
recently, interest has increased in utilization of screenable or
scorable markers. By way of example without limitation, the
promoter can be linked with detectable markers including a
.beta.-glucuronidase, or uidA gene (GUS), which encodes an enzyme
for which various chromogenic substrates are known (Jefferson, et
al., (1986) Proc. Natl. Acad. Sci. USA 83:8447-8451);
chloramphenicol acetyl transferase; alkaline phosphatase; a R-locus
gene, which encodes a product that regulates the production of
anthocyanin pigments (red color) in plant tissues (Dellaporta, et
al., (1988) in Chromosome Structure and Function, Kluwer Academic
Publishers, Appels and Gustafson eds., pp. 263-282; Ludwig, et al.,
(1990) Science 247:449); a p-lactamase gene (Sutcliffe, (1978)
Proc. Nat'l. Acad. Sci. U.S.A. 75:3737), which encodes an enzyme
for which various chromogenic substrates are known (e.g., PADAC, a
chromogenic cephalosporin); a xylE gene (Zukowsky, et al., (1983)
Proc. Nat'l. Acad. Sci. U.S.A. 80:1101), which encodes a catechol
dioxygenase that can convert chromogenic catechols; an
.alpha.-amylase gene (Ikuta, et al., (1990) Biotech. 8:241); a
tyrosinase gene (Katz, et al., (1983) J. Gen. Microbiol. 129:2703),
which encodes an enzyme capable of oxidizing tyrosine to DOPA and
dopaquinone, which in turn condenses to form the easily detectable
compound melanin a green fluorescent protein (GFP) gene (Sheen, et
al., (1995) Plant J. 8(5):777-84); a lux gene, which encodes a
luciferase, the presence of which may be detected using, for
example, X-ray film, scintillation counting, fluorescent
spectrophotometry, low-light video cameras, photon counting cameras
or multiwell luminometry (Teeri, et al., (1989) EMBO J. 8:343);
DS-RED EXPRESS (Matz, et al., (1999) Nature Biotech. 17:969-973,
Bevis, et al., (2002) Nature Biotech 20:83-87, Haas, et al., (1996)
Curr. Biol. 6:315-324); Zoanthus sp. yellow fluorescent protein
(ZsYellow) that has been engineered for brighter fluorescence
(Matz, et al., (1999) Nature Biotech. 17:969-973, available from BD
Biosciences Clontech, Palo Alto, Calif., USA, catalog no. K6100-1);
and cyan florescent protein (CYP) (Bolte, et al., (2004) J. Cell
Science 117:943-54 and Kato, et al., (2002) Plant Physiol
129:913-42).
[0127] A transformation vector comprising the particular regulatory
sequences of the present invention, operably linked to an isolated
polynucleotide sequence of interest in an expression cassette, can
be used to transform any plant. In this manner, genetically
modified plants, plant cells, plant tissue, and the like can be
obtained. Transformation protocols can vary depending on the type
of plant or plant cell, i.e., monocot or dicot, targeted for
transformation. Suitable methods of transforming plant cells
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, see,
for example, Townsend, et al., U.S. Pat. No. 5,563,055; direct gene
transfer, Paszkowski, et al., (1984) EMBO J. 3:2717-2722; and
ballistic particle acceleration, see, for example, Sanford, et al.,
U.S. Pat. No. 4,945,050, Tomes, et al., (1995) in Plant Cell,
Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg and
Phillips, (Springer-Verlag, Berlin); and McCabe, et al., (1988)
Biotechnology 6:923-926. Also see, Weissinger, et al., (1988)
Annual 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); Datta, et al., (1990)
Bio/Technology 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); Klein, et al., (1988) Plant
Physiol. 91:440-444 (maize); Fromm, et al., (1990) Biotechnology
8:833-839; Hooydaas-Van Slogteren, et al., (1984) Nature (London)
311:763-764; 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. G. P. 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, et al., (1995) Annals of Botany 75:407-413 (rice);
Osjoda, et al., (1996) Nature Biotechnology 14:745-750 (maize via
Agrobacterium tumefaciens).
[0128] The cells that have been transformed can be grown into
plants in accordance with conventional methods. See, for example,
McCormick, et al., (1986) Plant Cell Reports 5:81-84. These plants
can then be grown and pollinated with the same transformed strain
or different strains. The resulting plant having stress-induced
expression of the desired phenotypic characteristic can then be
identified. Two or more generations can be grown to ensure that
stress-induced expression of the desired phenotypic characteristic
is stably maintained and inherited.
[0129] The following examples are offered by way of illustration
and not by way of limitation.
EXAMPLES
[0130] As the coding regions for ZmCBF1 and ZmCBF2 had previously
been isolated, 5' regulatory regions were isolated from maize
plants via GenomeWalker.TM. (Clontech) and cloned. Isolated
promoter regions were analyzed for cis elements and compared to
Arabidopsis and rice CBF promoters.
Transformation of Maize by Particle Bombardment
[0131] Immature maize embryos from greenhouse donor plants are
bombarded with a plasmid containing an expression cassette
comprising the ZmCBF1 or ZmCBF2 promoter operably linked to a gene
of interest. The plasmid also comprises a selectable marker gene,
for example PAT (Wohlleben, et al., (1988) Gene 70:25-37), which
confers resistance to the herbicide Bialaphos. Alternatively, the
selectable marker gene is provided on a separate plasmid.
Transformation is performed as follows. Media recipes follow
below.
[0132] The ears are husked and surface sterilized in 30%
Clorox.RTM. bleach plus 0.5% Micro detergent for 20 minutes, and
rinsed two times with sterile water. The immature embryos are
excised and placed embryo axis side down (scutellum side up), 25
embryos per plate, on 560Y medium for 5 hours and then aligned
within the 2.5 cm target zone in preparation for bombardment.
[0133] A plasmid vector is made which comprises the ZmCBF1 or
ZmCBF2 promoter sequence operably linked to a gene of interest.
This plasmid DNA plus plasmid DNA containing a PAT selectable
marker is precipitated onto 1.1 .mu.m (average diameter) tungsten
pellets using a CaCl.sub.2 precipitation procedure as follows: 100
.mu.l prepared tungsten particles in water; 10 .mu.l (1 .mu.g) DNA
in Tris EDTA buffer (1 .mu.g total DNA); 100 .mu.l 2.5 M
CaCl.sub.2; and, 10 .mu.l 0.1 M spermidine.
[0134] Each reagent is added sequentially to the tungsten particle
suspension, while maintained on the multitube vortexer. The final
mixture is sonicated briefly and allowed to incubate under constant
vortexing for 10 minutes. After the precipitation period, the tubes
are centrifuged briefly, liquid removed, washed with 500 ml 100%
ethanol, and centrifuged for 30 seconds. Again the liquid is
removed, and 105 .mu.l 100% ethanol is added to the final tungsten
particle pellet. For particle gun bombardment, the tungsten/DNA
particles are briefly sonicated and 10 .mu.l spotted onto the
center of each macrocarrier and allowed to dry about 2 minutes
before bombardment.
[0135] The sample plates are bombarded at level #5 in particle gun
#HE35-1 or #HE35-2. All samples receive a single shot at 650 PSI,
with a total of ten aliquots taken from each tube of prepared
particles/DNA.
[0136] Following bombardment, the embryos are kept on 560Y medium
for 2 days, then transferred to 560R selection medium containing 3
mg/liter Bialaphos, and subcultured every 2 weeks. After
approximately 10 weeks of selection, selection-resistant callus
clones are transferred to 288J medium to initiate plant
regeneration. Following somatic embryo maturation (2-5 weeks),
well-developed somatic embryos are transferred to medium for
germination and transferred to the lighted culture room.
Approximately 7-10 days later, developing plantlets are transferred
to 272V hormone-free medium in tubes for 7-10 days until plantlets
are well established. Plants are then transferred to inserts in
flats (equivalent to 2.5'' pot) containing potting soil and grown
for 1 week in a growth chamber, subsequently grown an additional
1-2 weeks in the greenhouse, then transferred to classic 600 pots
(1.6 gallon) and grown to maturity. Plants are monitored and scored
under various conditions and compared to control plants. Marker
gene expression is observed to confirm transformation. Alterations
in phenotype, reflecting expression of the gene of interest, are
monitored.
[0137] Bombardment medium (560Y) comprises 5.0 g/l N6 basal salts
(SIGMA C-1516), 1.0 ml/l Eriksson's Vitamin Mix
(1000.times.SIGMA-1511), 0.5 mg/l thiamine HCl, 120.0 g/l sucrose,
1.0 mg/l 2.5-D, and 2.88 g/l L-proline (brought to volume with D-I
H.sub.2O following adjustment to pH 5.8 with KOH); 2.0 g/l
Gelrite.RTM. gelling agent (added after bringing to volume with D-I
H.sub.2O); and 8.5 mg/l silver nitrate (added after sterilizing the
medium and cooling to room temperature). Selection medium (560R)
comprises 5.0 g/l N6 basal salts (SIGMA C-1516), 1.0 ml/I
Eriksson's Vitamin Mix (1000.times.SIGMA-1511), 0.5 mg/l thiamine
HCl, 30.0 g/l sucrose, and 2.0 mg/l 2.5-D (brought to volume with
D-I H.sub.2O following adjustment to pH 5.8 with KOH); 3.0 g/l
Gelrite.RTM. gelling agent (added after bringing to volume with D-I
H.sub.2O); and 0.85 mg/l silver nitrate and 3.0 mg/l bialaphos
(both added after sterilizing the medium and cooling to room
temperature).
[0138] Plant regeneration medium (288J) comprises 5.3 g/l MS salts
(GIBCO 11117-075), 5.0 ml/l MS vitamins stock solution (0.100 g
nicotinic acid, 0.02 g/l thiamine HCL, 0.10 g/l pyridoxine HCL, and
0.50 g/l glycine brought to volume with polished D-I H.sub.2O)
(Murashige and Skoog, (1962) Physiol. Plant. 15:573), 100 mg/l
myo-inositol, 0.5 mg/l zeatin, 60 g/l sucrose, and 1.0 ml/l of 0.1
mM abscisic acid (brought to volume with polished D-I H.sub.2O
after adjusting to pH 5.6); 3.0 g/l Gelrite.RTM. gelling agent
(added after bringing to volume with D-I H.sub.2O); and 1.0 mg/l
indoleacetic acid and 3.0 mg/l bialaphos (added after sterilizing
the medium and cooling to 60.degree. C.). Hormone-free medium
(272V) comprises 5.3 g/l MS salts (GIBCO 11117-075), 5.0 ml/l MS
vitamins stock solution (0.100 g/l nicotinic acid, 0.02 g/l
thiamine HCL, 0.10 g/l pyridoxine HCL, and 0.50 g/l glycine brought
to volume with polished D-I H.sub.2O), 0.1 g/l myo-inositol, and
50.0 g/l sucrose (brought to volume with polished D-I H.sub.2O
after adjusting pH to 5.6); and 6 g/l Bacto.TM. Agar solidifying
agent (added after bringing to volume with polished D-I H.sub.2O),
sterilized and cooled to 60.degree. C.
Example 2
Transformation and Regeneration of Maize Callus Via
Agrobacterium
[0139] For Agrobacterium-mediated transformation of maize with the
ZmCBF1 or ZmCBF2 promoter sequence (SEQ ID NO: 1 or 2) operably
linked to a gene of interest, the method of Zhao is employed (U.S.
Pat. No. 5,981,840, and PCT Patent Publication WO98/32326, the
contents of which are hereby incorporated by reference). The
immature embryos are cultured on solid medium with a selective
agent resulting in the selective growth of transformed cells. The
callus is then regenerated into plants, and calli grown on
selective medium are cultured on solid medium to regenerate the
plants.
[0140] The plants are monitored for a modulation in phenotype when
compared to an appropriate control plant.
[0141] Details of Agrobacterium transformation may be as set out
below or as known to one of skill in the art.
Preparation of Agrobacterium Suspension
[0142] Agrobacterium is streaked out from a -80.degree. C. frozen
aliquot onto a plate containing PHI-L medium and cultured at
28.degree. C. in the dark for 3 days. PHI-L media comprises 25 ml/l
Stock Solution A, 25 ml/l Stock Solution B, 450.9 ml/l Stock
Solution C and spectinomycin (Sigma Chemicals) added to a
concentration of 50 mg/l in sterile ddH.sub.2O (stock solution A:
K.sub.2HPO.sub.4 60.0 g/l, NaH.sub.2PO.sub.4 20.0 g/l, adjust pH to
7.0 w/KOH and autoclaved; stock solution B: NH.sub.4Cl 20.0 g/l,
MgSO.sub.4.7H.sub.2O 6.0 g/l, KCl 3.0 g/l, CaCl.sub.2 0.20 g/l,
FeSO.sub.4.7H.sub.2O 50.0 mg/l, autoclaved; stock solution C:
glucose 5.56 g/l, agar 16.67 g/l (#A-7049, Sigma Chemicals, St.
Louis, Mo.) and autoclaved).
[0143] The plate can be stored at 4.degree. C. and used usually for
about 1 month. A single colony is picked from the master plate and
streaked onto a plate containing PHI-M medium [yeast extract
(Difco) 5.0 g/l; peptone (Difco) 10.0 g/l; NaCl 5.0 g/l; agar
(Difco) 15.0 g/l; pH 6.8, containing 50 mg/L spectinomycin] and
incubated at 28.degree. C. in the dark for 2 days.
[0144] Five ml of either PHI-A, [CHU(N6) basal salts (Sigma C-1416)
4.0 g/l, Eriksson's vitamin mix (1000.times., Sigma-1511) 1.0 ml/l;
thiamine.HCl 0.5 mg/l (Sigma); 2,4-dichlorophenoxyacetic acid
(2,4-D, Sigma) 1.5 mg/l; L-proline (Sigma) 0.69 g/l; sucrose
(Mallinckrodt) 68.5 g/l; glucose (Mallinckrodt) 36.0 g/l; pH 5.2]
for the PHI basic medium system, or PHI-I [MS salts (GIBCO BRL) 4.3
g/l; nicotinic acid (Sigma) 0.5 mg/l; pyridoxine.HCl (Sigma) 0.5
mg/l; thiamine.HCl 1.0 mg/l; myo-inositol (Sigma) 0.10 g/l; vitamin
assay casamino acids (Difco Lab) 1 g/l; 2,4-D 1.5 mg/l; sucrose
68.50 g/l; glucose 36.0 g/l; adjust pH to 5.2 w/KOH and
filter-sterilize] for the PHI combined medium system and 5 ml of
100 mM (3'-5'-Dimethoxy-4'-hydroxyacetophenone, Aldrich chemicals)
are added to a 14 ml Falcon tube in a hood. About 3 full loops (5
mm loop size) Agrobacterium are collected from the plate and
suspended in the tube, then the tube is vortexed to make an even
suspension. One ml of the suspension is transferred to a
spectrophotometer tube and the OD of the suspension is adjusted to
0.72 at 550 nm by adding either more Agrobacterium or more of the
same suspension medium, for an Agrobacterium concentration of
approximately 0.5.times.109 cfu/ml to 1.times.109 cfu/ml. The final
Agrobacterium suspension is aliquoted into 2 ml microcentrifuge
tubes, each containing 1 ml of the suspension. The suspensions are
then used as soon as possible.
Embryo Isolation, Infection and Co-Cultivation
[0145] About 2 ml of the same medium (here PHI-A or PHI-I) which is
used for the Agrobacterium suspension is added into a 2 ml
microcentrifuge tube. Immature embryos are isolated from a
sterilized ear with a sterile spatula (Baxter Scientific Products
S1565) and dropped directly into the medium in the tube. A total of
about 100 embryos are placed in the tube. The optimal size of the
embryos is about 1.0-1.2 mm. The cap is then closed on the tube and
the tube is vortexed with a Vortex Mixer (Baxter Scientific
Products S8223-1) for 5 sec. at maximum speed. The medium is
removed and 2 ml of fresh medium are added and the vortexing
repeated. All of the medium is drawn off and 1 ml of Agrobacterium
suspension is added to the embryos and the tube is vortexed for 30
sec. The tube is allowed to stand for 5 min. in the hood. The
suspension of Agrobacterium and embryos is poured into a Petri
plate containing either PHI-B medium [CHU(N6) basal salts (Sigma
C-1416) 4.0 g/l; Eriksson's vitamin mix (1000.times., Sigma-1511)
1.0 ml/l; thiamine.HCl 0.5 mg/l; 2.4-D 1.5 mg/l; L-proline 0.69
g/l; silver nitrate 0.85 mg/l; Gelrite.RTM. gelling agent (Sigma)
3.0 g/l; sucrose 30.0 g/l; acetosyringone 100 mM; pH 5.8], for the
PHI basic medium system, or PHI-J medium [MS Salts 4.3 g/l;
nicotinic acid 0.50 mg/l; pyridoxine HCl 0.50 mg/l; thiamine.HCl
1.0 mg/l; myo-inositol 100.0 mg/l; 2,4-D 1.5 mg/l; sucrose 20.0
g/l; glucose 10.0 g/l; L-proline 0.70 g/l; MES (Sigma) 0.50 g/l;
8.0 g/l agar (Sigma A-7049, purified) and 100 mM acetosyringone
with a final pH of 5.8 for the PHI combined medium system. Any
embryos left in the tube are transferred to the plate using a
sterile spatula. The Agrobacterium suspension is drawn off and the
embryos placed axis side down on the media. The plate is sealed
with Parafilm.RTM. tape or Pylon Vegetative Combine Tape (product
named "E.G.CUT" and available in 18 mm.times.50 m sections; Kyowa
Ltd., Japan) and is incubated in the dark at 23-25.degree. C. for
about 3 days of co-cultivation.
Resting, Selection and Regeneration Steps
[0146] For the resting step, all of the embryos are transferred to
a new plate containing PHI-C medium [CHU(N6) basal salts (Sigma
C-1416) 4.0 g/l; Eriksson's vitamin mix (1000.times. Sigma-1511)
1.0 ml/l; thiamine.HCl 0.5 mg/l; 2.4-D 1.5 mg/l; L-proline 0.69
g/l; sucrose 30.0 g/l; MES buffer (Sigma) 0.5 g/l; agar (Sigma
A-7049, purified) 8.0 g/l; silver nitrate 0.85 mg/l; carbenicillin
100 mg/l; pH 5.8]. The plate is sealed with Parafilm.RTM. or Pylon
tape and incubated in the dark at 28.degree. C. for 3-5 days.
[0147] Longer co-cultivation periods may compensate for the absence
of a resting step since the resting step, like the co-cultivation
step, provides a period of time for the embryo to be cultured in
the absence of a selective agent. Those of ordinary skill in the
art can readily test combinations of co-cultivation and resting
times to optimize or improve the transformation
[0148] For selection, all of the embryos are then transferred from
the PHI-C medium to new plates containing PHI-D medium, as a
selection medium, [CHU(N6) basal salts (SIGMA C-1416) 4.0 g/l;
Eriksson's vitamin mix (1000.times., Sigma-1511) 1.0 ml/l;
thiamine.HCl 0.5 mg/l; 2.4-D 1.5 mg/l; L-proline 0.69 g/l; sucrose
30.0 g/l; MES buffer 0.5 g/l; agar (Sigma A-7049, purified) 8.0
g/l; silver nitrate 0.85 mg/l; carbenicillin (ICN, Costa Mesa,
Calif.) 100 mg/l; bialaphos (Meiji Seika, Tokyo, Japan) 1.5 mg/l
for the first two weeks followed by 3 mg/l for the remainder of the
time; pH 5.8] putting about 20 embryos onto each plate.
[0149] The plates are sealed as described above and incubated in
the dark at 28.degree. C. for the first two weeks of selection. The
embryos are transferred to fresh selection medium at two-week
intervals. The tissue is subcultured by transferring to fresh
selection medium for a total of about 2 months. The
herbicide-resistant calli are then "bulked up" by growing on the
same medium for another two weeks until the diameter of the calli
is about 1.5-2 cm.
[0150] For regeneration, the calli are then cultured on PHI-E
medium [MS salts 4.3 g/l; myo-inositol 0.1 g/l; nicotinic acid 0.5
mg/l, thiamine.HCl 0.1 mg/l, Pyridoxine.HCl 0.5 mg/l, Glycine 2.0
mg/l, Zeatin 0.5 mg/l, sucrose 60.0 g/l, Agar (Sigma, A-7049) 8.0
g/l, Indoleacetic acid (IAA, Sigma) 1.0 mg/l, Abscisic acid (ABA,
Sigma) 0.1 mM, Bialaphos 3 mg/l, carbenicillin 100 mg/l adjusted to
pH 5.6] in the dark at 28.degree. C. for 1-3 weeks to allow somatic
embryos to mature. The calli are then cultured on PHI-F medium (MS
salts 4.3 g/l; myo-inositol 0.1 g/l; Thiamine.HCl 0.1 mg/l,
Pyridoxine.HCl 0.5 mg/l, Glycine 2.0 mg/l, nicotinic acid 0.5 mg/l;
sucrose 40.0 g/l; Gelrite.RTM. gelling agent 1.5 g/l; pH 5.6] at
25.degree. C. under a daylight schedule of 16 hrs. light (270 uE
m-2sec-1) and 8 hrs. dark until shoots and roots are developed.
Each small plantlet is then transferred to a 25.times.150 mm tube
containing PHI-F medium and is grown under the same conditions for
approximately another week. The plants are transplanted to pots
with soil mixture in a greenhouse. Transformation events are
determined at the callus stage or regenerated plant stage.
[0151] Ability of the ZmCBF1 or ZmCBF2 promoter to drive expression
in maize is confirmed by marker gene detection in plant tissue.
[0152] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it will be obvious that certain changes and
modifications may be practiced within the scope of the appended
claims. All references cited are incorporated herein by reference.
Sequence CWU 1
1
211373DNAZea maysmisc_binding(634)...(638)CRT/DRE consensus
1ccatatcgaa tccgaatcca aagttgtact attttactac tatctatcta ttaacctata
60tccatcacga atataaaagt atctaagtta ttagtgaaaa aatgtattta tttatagtat
120ctatatccgg taccctcgcc cctatctata tctcatccac gacacgagcg
gtggaccaca 180cacgacactg gggaaactgg aaaatttccg cattttcatc
ccggcttctg ctggaactga 240aagtactagt aagtaagcta cggcggttcg
tcctggatgt ggatgggcgg cccacgcgtg 300cagtgcttgt gcttcttgat
cactgacgac ccggtcgcct ggcccaaccc gggcaggcca 360cgtcgaaccc
cgtctccatc ccctcggcgg gtgatggatt cggtggggcc ggggcgggag
420gagatcgagt agcagacggc gacacccgat catcaagcgt tgagcgcgga
ggacgcaagg 480gcggacgcgc ggctacatcg aaacgagtcc ggacgaggag
gagcggaggc aggcgcctcc 540ggtcggtgtc gggggcggcg gcaggcaggt
agccccacgc gtttccgcgg gcggcgccac 600ggccctgtct ccgagctgga
aatgatcaac aagccgacgg aaaaccagtg gcctgccgcc 660gccgccgcac
cgcatggcgc atgtgccgaa gcccaccggc cgccgtcacg tgccctgccc
720acggctctga tatttttttt attattgatt ctattctacg ttgccctgct
agattttttt 780cttcttcctt tttagagatt ctctgtgtgc cctgctgatt
ctcggtgttg tcagtgaagc 840gagcgaaaga ccgaagaggt ctgccgccgc
ccaaaagcca gccgttgtcg ccatctccac 900gtggccatct cctgatcctc
tgtcgctgcc atggcggggg ccctgccgca tgtcagtccg 960cgcgactttc
cttaacggcg actgctaagc aatgggaatg ggatgggagt ggagccgacg
1020acgccacgct gacgccaacg cgtcttcccc cgccgcctcg ccaccgtccc
gcacgcagca 1080ctgtccccga ctcccccagc cagtctccag ctcacacctc
ccaagtcccg ccacgcgccc 1140gcccaactga ctgactcccg ccttcaaact
tctcccgctc ctcctgatcc acgtaccacc 1200aaatcctata tatataaacc
agaccacaac tcccgactcc atccatcgcc agccacgcca 1260tcagccatcg
cgttccgcat cgaaccagca catcgcaaga caagcagcag cagccaccac
1320tgccatcaca tggagtacgc cgccgtcggc tacggctacg ggtacgggta cga
137322266DNAZea maysmisc_binding(64)...(68)CRT/DRE consensus
2aacttcttgt ctggtcattt aggattctga acactcatac gtcagctcac atcggtttgg
60acgccgactg tacggatgtg agcagacgct ggtcgatctc cttttcctat actagctctt
120atataaatac caccttttcg cattgattgg tggccgccgc tgagttctct
actcgggtga 180acggagggga ggagcaggag acagggggga agcacctccc
acgactttgg caggccgaac 240cgaacgccac aaaagtagga tttttctccg
gttccccggg aacagtcttt ttcttctact 300ggcagaaacg gacggttcag
gtgcgggagc ttcccggctg ctgctgctgc cgccggcctt 360tttgcgatac
catactgggg gtttggcccc gggaatgctg gattgggttt tgttcgtggt
420actcttggtt tcgggactca tcacctggta atgtcgctat cgggtggatg
gatctcggcg 480acgcgagacg tacactgatt tctttttctt ttttctttat
caatcagtta tcagtacgta 540ctgctaccta gggtcctctc ttttttttcc
ccggatgcaa aatacgtact gcctgtaacc 600aatattatat ttatatagat
aaaaacaaca taaattttaa gtccctatgt ttatctgaaa 660atttagaaca
gcaatagtca tttcagtgga ccaaaggatt gggttagaat ctggacagtg
720attgcgggag aagtcctgcc atgagcttag ggtcctagtt tggcaactcc
atctttctaa 780ggccttgttc gtttgtgtcg gattacaccc gaaatcgtta
cagctaatca aagtttatat 840aaattagaga agcaatccgg ataggaatcg
ttccgaccca ccaatccgcc acaaacgaac 900aaggcctaaa gattttcatc
tttccaaaga aaaataaact aaactttctt ggaaaaaaat 960ataaatctct
taaaaaatgt ggttgccaaa ctagccctta taataaagga aaaactggag
1020gaattttaga ggaatgaaat cctatatgaa attatgttat gtggcctttg
aaacagaggc 1080tttagttcct acatatccta taaggttcct atggaatggt
ccatcgtagt gatattttag 1140agaatttcta acttaaagcc cgacctctta
gaaattgtca tttgtgcctc tctctctgca 1200ctctaattca tgtgttcttt
ctatgttgca tccaggattc tcaaaacacg gaaataggag 1260aaacacatga
ttataacgcc catgccgttg aaatcctaca aaaatttata acacggaaaa
1320gttataaaaa catgtttttg gatcatgcgc ataaaaaata taggaatgtg
agacacaaag 1380aaaataaaat atgagggtgg acctcacgct taattttccc
tccaaaatta ccatggagca 1440tgtcatttca taggattttt caaggaattg
ataggatcca atcatttgtc ccaaaaggct 1500tttatatgaa aactttctat
gcgaattgaa tcctctaaag ttcctttgtt tttcctccat 1560tccaaagtta
tctgcgtttt taattcatgt atgattacaa gtgttataca actctattcg
1620tacatttttt ttcctttcta tttttgtatt ttgtcaatcc tctgttccaa
aggaggcatc 1680taaaataggg aatgagatag ccttaatcga gtactagtag
catttgatat atcataaaaa 1740agtagcattt gatatgtgcg agaagagctg
gggttaacgt cgtaacgcaa atagttaact 1800agacggaaac cggtagcgga
aacaaaatca gccttgtggc cgcaactaca ccaaatagca 1860ccatcaagtc
actatcgaaa tcgccctgtt gcctccctac ttgcaagctg gaagctgcaa
1920cttgcgtccc cgtccagccg tccttgctct ctgacccaga tgcagccccc
ttccttcccc 1980agtgcctggt ggctcccctc ccgcgttatt cactctgcta
gtcctgctca ggtctgctga 2040ctgctgctct ctgatccaaa cctcctcctc
caccaccacc tataaatacg catcctcaaa 2100tccaccattc ccaaatcgaa
aaccactcga acacaagctc aggcaaggca aaaggaaccc 2160gcaccatccg
aggctcaagc agagaagtga tcatcatcat cagaagaaga tgtgcccaac
2220caagaagggg atgaccggag agccgagctc gccatgcagc tcggca 2266
* * * * *