U.S. patent application number 12/885246 was filed with the patent office on 2011-04-07 for eukaryotic translation initiation factor gene regulatory elements for use in plants.
Invention is credited to Richard Eric Cerny, Deborah Feeney, Stanislaw Flasinski, Jesse Hart, Marianne Malven, Steven E. Screen.
Application Number | 20110082038 12/885246 |
Document ID | / |
Family ID | 35968134 |
Filed Date | 2011-04-07 |
United States Patent
Application |
20110082038 |
Kind Code |
A1 |
Flasinski; Stanislaw ; et
al. |
April 7, 2011 |
EUKARYOTIC TRANSLATION INITIATION FACTOR GENE REGULATORY ELEMENTS
FOR USE IN PLANTS
Abstract
The present invention provides eukaryotic translation initiation
factor non-coding regulatory element polynucleotide molecules
isolated from Nicotiana tabacum, Arabidopsis thaliana, and Medicago
truncatula useful for modulating transgene expression in plants.
The present invention also provides expression constructs
containing the polynucleotide molecules useful for modulating
transgene expression in plants. The present invention also provides
transgenic plants and seeds containing the polynucleotide molecules
useful for modulating transgene expression in plants.
Inventors: |
Flasinski; Stanislaw;
(Chesterfield, MO) ; Screen; Steven E.; (St.
Louis, MO) ; Feeney; Deborah; (Warrenton, MO)
; Cerny; Richard Eric; (Chesterfield, MO) ; Hart;
Jesse; (Augusta, MO) ; Malven; Marianne;
(Ellisville, MO) |
Family ID: |
35968134 |
Appl. No.: |
12/885246 |
Filed: |
September 17, 2010 |
Related U.S. Patent Documents
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Application
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Filing Date |
Patent Number |
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12349415 |
Jan 6, 2009 |
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12885246 |
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11743923 |
May 3, 2007 |
7507876 |
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12349415 |
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11207149 |
Aug 18, 2005 |
7217867 |
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11743923 |
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60602765 |
Aug 19, 2004 |
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Current U.S.
Class: |
504/206 ;
435/320.1; 435/419; 536/24.1; 800/298; 800/306; 800/312; 800/314;
800/317.2; 800/317.3; 800/317.4; 800/320; 800/320.1; 800/320.2;
800/320.3; 800/322 |
Current CPC
Class: |
C12N 15/8216 20130101;
A01N 57/20 20130101; C07K 14/415 20130101; C12N 15/8275
20130101 |
Class at
Publication: |
504/206 ;
536/24.1; 800/298; 800/320.1; 800/320; 800/320.2; 800/320.3;
800/317.4; 800/317.2; 800/312; 800/314; 800/306; 800/322;
800/317.3; 435/320.1; 435/419 |
International
Class: |
A01N 57/20 20060101
A01N057/20; C07H 21/04 20060101 C07H021/04; A01H 5/00 20060101
A01H005/00; A01H 5/10 20060101 A01H005/10; A01P 13/00 20060101
A01P013/00; C12N 15/82 20060101 C12N015/82; C12N 5/10 20060101
C12N005/10 |
Claims
1. An isolated polynucleotide molecule having gene regulatory
activity comprising a eukaryotic translation initiation factor gene
non-coding regulatory element polynucleotide molecule, or a
complement thereof, or a fragment thereof, wherein said molecule is
selected from the group consisting of: promoter, leader, and
intron.
2. An isolated polynucleotide molecule of claim 1 selected from the
group consisting of: SEQ ID NO:1 to SEQ ID NO:11.
3. An isolated polynucleic acid molecule of claim 1 comprising a
DNA molecule that a. hybridizes under stringent conditions with a
sequence selected from the group consisting of SEQ ID NO: 1 to SEQ
ID NO: 11, or any complements thereof, or any fragments thereof; or
b. exhibits an 85% or greater identity to a sequence selected from
the group consisting of SEQ ID NO: 1 to SEQ ID NO: 11, any
complements thereof, or any fragments thereof.
4. A polynucleotide construct comprising an isolated polynucleotide
molecule of claim 1, wherein said isolated polynucleotide molecule
is operably linked to a transcribable polynucleotide molecule.
5. The polynucleotide construct of claim 4, wherein the isolated
polynucleotide molecule comprises a nucleic acid sequence selected
from the group consisting of SEQ ID NOs:1-11.
6. The polynucleotide construct of claim 4, wherein said isolated
polynucleotide molecule comprises a polynucleotide sequence which
exhibits a substantial percent sequence identity greater than about
80% identity with a nucleic acid sequence selected from the group
consisting of SEQ ID NOs: 1-11.
7. The polynucleotide construct of claim 4, wherein said
transcribable polynucleotide molecule is a gene of agronomic
interest.
8. The polynucleotide construct of claim 4, wherein said
transcribable polynucleotide molecule is a herbicide tolerance
gene.
9. The polynucleotide construct of claim 8, wherein said herbicide
tolerance gene is selected from the group consisting of genes that
encode for phosphinothricin acetyltransferase, glyphosate resistant
EPSPS, hydroxyphenyl pyruvate dehydrogenase, dalapon dehalogenase,
bromoxynil resistant nitrilase, anthranilate synthase, glyphosate
oxidoreductase and glyphosate-N-acetyl transferase.
10. A transgenic plant cell stably transformed with the
polynucleotide construct of claim 4.
11. A transgenic plant stably transformed with the polynucleotide
construct of claim 4.
12. The transgenic plant of claim 11, comprising an isolated
polynucleotide molecule selected from the group consisting of SEQ
ID NOs: 1-11, wherein said isolated polynucleotide molecule is
operably linked to a transcribable polynucleotide molecule.
13. The transgenic plant of claim 11, wherein said plant is a
monocotyledonous plant selected from the group consisting of wheat,
maize, rye, rice, oat, barley, turfgrass, sorghum, millet and
sugarcane.
14. The transgenic plant of claim 11, wherein said plant is a
dicotyledonous plant selected from the group consisting of tobacco,
tomato, potato, soybean, cotton, canola, sunflower and alfalfa.
15. A seed of said transgenic plant of claim 13.
16. A seed of said transgenic plant of claim 14.
17. A method of inhibiting weed growth in a field of transgenic
glyphosate tolerant crop plants comprising: (i) planting the
transgenic plants transformed with an expression cassette
comprising (a) a eukaryotic translation initiation factor
non-coding regulatory element polynucleotide molecule active in a
plant cell and operably linked to a polynucleotide molecule
encoding a glyphosate tolerance gene and (ii) applying glyphosate
to the field at an application rate that inhibits the growth of
weeds, wherein the growth and yield of the transgenic crop plant is
not substantially affected by the glyphosate application.
18. The method of claim 17, wherein said glyphosate tolerance gene
is selected from the group consisting of a gene encoding for a
glyphosate resistant EPSPS, a glyphosate oxidoreductase and a
glyphosate-N-acetyl transferase.
19. The method of claim 17, wherein the transgenic plants are
capable of tolerating an application rate ranging up to 256
ounces/acre.
20. The method of claim 17, wherein the application of glyphosate
is at least once during the growth of the crop.
21. The transgenic plant of claim 12, wherein said plant is a
monocotyledonous plant selected from the group consisting of wheat,
maize, rye, rice, oat, barley, turfgrass, sorghum, millet and
sugarcane.
22. The transgenic plant of claim 12, wherein said plant is a
dicotyledonous plant selected from the group consisting of tobacco,
tomato, potato, soybean, cotton, canola, sunflower and alfalfa.
Description
[0001] This application claims benefit under 35USC .sctn.119(e) of
U.S. provisional application Ser. No. 60/602,765 filed Aug. 19,
2004, herein incorporated by reference in its entirety.
INCORPORATION OF SEQUENCE LISTING
[0002] Two copies of the sequence listing (Seq. Listing Copy 1 and
Seq. Listing Copy 2) and a computer-readable form of the sequence
listing, all on CD-ROMs, each containing the file named
pa.sub.--01078.rpt, which is 14,336 bytes (measured in MS-DOS) and
was created on Aug. 17, 2005 are hereby incorporated by
reference.
FIELD OF THE INVENTION
[0003] The invention relates to the fields of plant molecular
biology and plant genetic engineering, and comprises polynucleotide
molecules useful for the expression of transgenes in plants.
BACKGROUND
[0004] One of the goals of plant genetic engineering is to produce
plants with agronomically desirable characteristics or traits. The
proper expression of a desirable transgene in a transgenic plant is
one way to achieve this goal. Regulatory elements such as
promoters, leaders, and introns are non-coding polynucleotide
molecules which play an integral part in the overall expression of
genes in living cells. Isolated regulatory elements that function
in plants are therefore useful for modifying plant phenotypes
through the methods of genetic engineering.
[0005] Many regulatory elements are available and are useful for
providing good overall expression of a transgene. For example,
constitutive promoters such as P-FMV, the promoter from the 35S
transcript of the Figwort mosaic virus (U.S. Pat. No. 6,051,753);
P-CaMV 35S, the promoter from the 35S RNA transcript of the
Cauliflower mosaic virus (U.S. Pat. No. 5,530,196); P-Rice Actin 1,
the promoter from the actin 1 gene of Oryza sativa (U.S. Pat. No.
5,641,876); and P-NOS, the promoter from the nopaline synthase gene
of Agrobacterium tumefaciens are known to provide some level of
gene expression in most or all of the tissues of a plant during
most or all of the plant's lifespan. While previous work has
provided a number of regulatory elements useful to affect gene
expression in transgenic plants, there is still a great need for
novel regulatory elements with beneficial expression
characteristics. In particular, there is a need for regulatory
elements that are capable of directing expression of transgenes in
transgenic crop plants at high levels and in particular tissues,
organs, or during specific developmental stages of plant growth.
Many previously identified regulatory elements fail to provide the
patterns or levels of expression required to fully realize the
benefits of expression of selected genes in transgenic plants.
[0006] Eukaryotic translation initiation factor eIF-4A is an
ATP-dependent RNA helicase protein that is required for the binding
of mRNA to ribosomes. Members of this family have been reported in
many species including mouse, Drosophila, yeast, tobacco,
Arbabidopsis, wheat, and rice (K A Brander, et al. (1995)
Biochimica et Biophysica Acta 1261:424-444). We hypothesized that a
promoter and other non-coding regulatory elements from an eIF-4A
gene would have a constitutive expression pattern and that the
promoter and regulatory elements would be useful to direct
expression of a transgene such as a glyphosate resistant
5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) transgene to
produce a glyphosate tolerant plant. The efficient production of
glyphosate tolerant plants requires the use of a promoter and
regulatory elements capable of directing transgene expression in
all tissues including the most sensitive reproductive organs such
as anthers and meristem tissues. The present invention thus
provides such promoters and regulatory elements isolated from the
eukaryotic translation initiation factor (eIF4A) genes of Nicotiana
tabacum, Arabidopsis thaliana, and Medicago truncatula.
SUMMARY
[0007] In one embodiment the invention provides promoters and
regulatory elements isolated from Nicotiana tabacum, Arabidopsis
thaliana, and Medicago truncatula, provided as SEQ ID NO: 1-11
useful for modulating transgene expression in plants. In another
embodiment the invention provides constructs comprising the
promoter and regulatory elements useful for modulating transgene
expression in plants. In another embodiment the invention provides
a transgenic plant containing the promoter and regulatory elements
operably linked to a heterologous DNA molecule and the seed of the
transgenic plant. The transgenic plant expresses an agronomically
desirable phenotype, in particular herbicide tolerance, more
specifically, tolerance to glyphosate herbicide.
BRIEF DESCRIPTION OF THE DRAWINGS
[0008] FIG. 1 represents pMON70500.
[0009] FIG. 2 represents pMON65395.
[0010] FIG. 3 represents pMON81504.
[0011] FIG. 4 represents pMON81505.
[0012] FIG. 5 represents pMON81596.
DETAILED DESCRIPTION
[0013] The invention disclosed herein provides polynucleotide
molecules having gene regulatory activity from Nicotiana tabacum,
Arabidopsis thaliana, and Medicago truncatula. The design,
construction, and use of these polynucleotide molecules are one
object of this invention. The polynucleotide sequences of these
polynucleotide molecules are provided as SEQ ID NO: 1-11. These
polynucleotide molecules are capable of affecting the transcription
of operably linked transcribable polynucleotide molecules in both
vegetative and reproductive tissues of plants and therefore can
selectively regulate expression of transgenes in these tissues.
DEFINITIONS
[0014] The following definitions and methods are provided to better
define the present invention and to guide those of ordinary skill
in the art in the practice of the present invention. Unless
otherwise noted, terms are to be understood according to
conventional usage by those of ordinary skill in the relevant
art.
[0015] As used herein, the term "fragment" or "fragment thereof"
refers to a finite polynucleotide sequence length that comprises at
least 50, at least 75, at least 85, or at least 95 contiguous
nucleotide bases wherein its complete sequence in entirety is
identical to a contiguous component of the referenced
polynucleotide molecule.
[0016] As used herein, the term "polynucleotide molecule" refers to
the single- or double-stranded DNA or RNA molecule of genomic or
synthetic origin, i.e., a polymer of deoxyribonucleotide or
ribonucleotide bases, respectively, read from the 5' (upstream) end
to the 3' (downstream) end.
[0017] As used herein, the term "polynucleotide sequence" refers to
the sequence of a polynucleotide molecule. The nomenclature for
nucleotide bases as set forth at 37 CFR .sctn.1.822 is used
herein.
[0018] As used herein, the term "regulatory element" refers to a
polynucleotide molecule having gene regulatory activity, i.e. one
that has the ability to affect the transcription or translation of
an operably linked transcribable polynucleotide molecule.
Regulatory elements such as promoters, leaders, introns, and
transcription termination regions are non-coding polynucleotide
molecules having gene regulatory activity which play an integral
part in the overall expression of genes in living cells. Isolated
regulatory elements that function in plants are therefore useful
for modifying plant phenotypes through the methods of genetic
engineering. By "regulatory element" it is intended a series of
nucleotides that determines if, when, and at what level a
particular gene is expressed. The regulatory DNA sequences
specifically interact with regulatory proteins or other
proteins.
[0019] As used herein, the term "gene regulatory activity" refers
to a polynucleotide molecule capable of affecting transcription or
translation of an operably linked polynucleotide molecule. An
isolated polynucleotide molecule having gene regulatory activity
may provide temporal or spatial expression or modulate levels and
rates of expression of the operably linked polynucleotide molecule.
An isolated polynucleotide molecule having gene regulatory activity
may comprise a promoter, intron, leader, or 3' transcriptional
termination region.
[0020] As used herein, the term "gene expression" or "expression"
refers to the transcription of a DNA molecule into a transcribed
RNA molecule. Gene expression may be described as related to
temporal, spatial, developmental, or morphological qualities as
well as quantitative or qualitative indications. The transcribed
RNA molecule may be translated to produce a protein molecule or may
provide an antisense or other regulatory RNA molecule.
[0021] As used herein, an "expression pattern" is any pattern of
differential gene expression. In a preferred embodiment, an
expression pattern is selected from the group consisting of tissue,
temporal, spatial, developmental, stress, environmental,
physiological, pathological, cell cycle, and chemically responsive
expression patterns.
[0022] As used herein, an "enhanced expression pattern" is any
expression pattern for which an operably linked nucleic acid
sequence is expressed at a level greater than 0.01%; preferably in
a range of about 0.5% to about 20% (w/w) of the total cellular RNA
or protein.
[0023] As used herein, the term "operably linked" refers to a first
polynucleotide molecule, such as a promoter, connected with a
second transcribable polynucleotide molecule, such as a gene of
interest, where the polynucleotide molecules are so arranged that
the first polynucleotide molecule affects the function of the
second polynucleotide molecule. The two polynucleotide molecules
may or may not be part of a single contiguous polynucleotide
molecule and may or may not be adjacent. For example, a promoter is
operably linked to a gene of interest if the promoter regulates or
mediates transcription of the gene of interest in a cell.
[0024] As used herein, the term "transcribable polynucleotide
molecule" refers to any polynucleotide molecule capable of being
transcribed into a RNA molecule, including but not limited to
protein coding sequences (e.g. transgenes) and non-coding sequences
(e.g. a molecule useful for gene suppression). Methods are known in
the art for constructing and introducing constructs into a cell in
such a manner that the transcribable polynucleotide molecule is
transcribed into a functional mRNA molecule that is translated and
therefore expressed as a protein product. Methods are known in the
art for constructing and introducing constructs into a cell in such
a manner that the transcribable polynucleotide molecule is
transcribed into a molecule that is capable of causing gene
suppression. For example, posttranscriptional gene suppression
using a construct with an anti-sense oriented transcribable
polynucleotide molecule to regulate gene expression in plant cells
is disclosed in U.S. Pat. No. 5,107,065 and U.S. Pat. No.
5,759,829; posttranscriptional gene suppression using a construct
with a sense-oriented transcribable polynucleotide molecule to
regulate gene expression in plants is disclosed in U.S. Pat. No.
5,283,184 and U.S. Pat. No. 5,231,020, all of which are hereby
incorporated by reference. For the practice of the present
invention, conventional compositions and methods for preparing and
using constructs and host cells are well known to one skilled in
the art, see for example, J F Sambrook, D W Russell, and N Irwin.
(2000) Molecular Cloning: A Laboratory Manual, 3rd edition Volumes
1, 2, and 3. Cold Spring Harbor Laboratory Press, hereafter
referred to as Sambrook et al., 2000. Constructs may also be
constructed to be capable of expressing antisense RNA molecules, in
order to inhibit translation of a specific RNA molecule of
interest. For the practice of the present invention, conventional
compositions and methods for preparing and using constructs and
host cells are well known to one skilled in the art, see for
example, Molecular Cloning: A Laboratory Manual, 3rd edition
Volumes 1, 2, and 3 (2000) J. F. Sambrook, D. W. Russell, and N.
Irwin, Cold Spring Harbor Laboratory Press.
Promoters
[0025] The present invention describes the composition and utility
for eukaryotic translation Initiation Factor non-coding regulatory
element molecules from Nicotiana tabacum, Arabidopsis thaliana, and
Medicago truncatula, hereinafter referred to as eIF-NCRE molecules.
These eIF-NCRE molecules include promoters.
[0026] As used herein, the term "promoter" refers to a
polynucleotide molecule that is involved in recognition and binding
of RNA polymerase II and other proteins such as transcription
factors (trans-acting protein factors that regulate transcription)
to initiate transcription of an operably linked gene. Promoters may
themselves contain sub-elements such as cis-elements or enhancer
domains that effect the transcription of operably linked genes. A
"plant promoter" is a native or non-native promoter that is
functional in plant cells. A plant promoter can be used as a 5'
regulatory element for modulating expression of an operably linked
gene or genes. Plant promoters may be defined by their temporal,
spatial, or developmental expression pattern.
[0027] Any of the nucleic acid molecules described herein may
comprise nucleic acid sequences comprising promoters. Promoters of
the present invention can include between about 300 bp upstream and
about 10 kb upstream of the trinucleotide ATG sequence at the start
site of a protein coding region. Promoters of the present invention
can preferably include between about 300 bp upstream and about 5 kb
upstream of the trinucleotide ATG sequence at the start site of a
protein coding region. Promoters of the present invention can more
preferably include between about 300 bp upstream and about 2 kb
upstream of the trinucleotide ATG sequence at the start site of a
protein coding region. Promoters of the present invention can
include between about 300 bp upstream and about 1 kb upstream of
the trinucleotide ATG sequence at the start site of a protein
coding region. While in many circumstances a 300 bp promoter may be
sufficient for expression, additional sequences may act to further
regulate expression, for example, in response to biochemical,
developmental or environmental signals.
Promoter Activity
[0028] The activity or strength of a promoter may be measured in
terms of the amount of mRNA or protein accumulation it specifically
produces, relative to the total amount of mRNA or protein. The
promoter preferably expresses an operably linked nucleic acid
sequence at a level greater than 0.01%, preferably in a range of
about 0.5% to about 20% (w/w) of the total cellular RNA or
protein.
[0029] Alternatively, the activity or strength of a promoter may be
expressed relative to a well-characterized promoter (for which
transcriptional activity was previously assessed). For example, a
less-characterized promoter may be operably linked to a reporter
sequence (e.g., GUS) and introduced into a specific cell type. A
well-characterized promoter (e.g. the 35S promoter) is similarly
prepared and introduced into the same cellular context.
Transcriptional activity of the unknown promoter is determined by
comparing the amount of reporter expression, relative to the well
characterized promoter. In one embodiment, the activity of the
present promoter is as strong as the 35S promoter when compared in
the same cellular context. The cellular context is preferably
maize, sorghum, corn, barley, wheat, canola, soybean, or maize; and
more preferably is maize, sorghum, corn, barley, or wheat; and most
preferably is maize.
Cis Elements
[0030] Promoters of the present invention may contain one or more
of the following elements: a CAAT, a GC, or a TATA cis element.
Moreover, the promoters of the present invention may contain one or
more cis elements in addition to a GC, CAAT and/or a TATA box.
[0031] Many regulatory elements act in cis fashion ("cis elements")
and are believed to affect DNA topology, producing local
conformations that selectively allow or restrict access of RNA
polymerase to the DNA template or that facilitate selective opening
of the double helix at the site of transcriptional initiation. Cis
elements occur within, but are not limited to promoters, and
promoter modulating sequences (inducible elements). Cis elements
can be identified using known cis elements as a target sequence or
target motif in the BLAST programs of the present invention.
[0032] Promoters of the present invention may include homologues of
cis elements known to effect gene regulation and that show homology
with the promoter sequences of the present invention.
5' Non-Translated Leader and Enhancer Sequences
[0033] The present invention describes the composition and utility
for eukaryotic translation Initiation Factor non-coding regulatory
element molecules from Nicotiana tabacum, Arabidopsis thaliana, and
Medicago truncatula, hereinafter referred to as eIF-NCRE molecules.
These eIF-NCRE molecules include 5' non-translated leader
sequences.
[0034] As used herein, the term "leader" refers to a non-coding
polynucleotide molecule. A leader may be isolated from the
untranslated 5' region (5' UTR) of a genomic copy of a gene and
defined generally as a segment between the transcription start site
(TSS) and the coding sequence start site. Alternately, leaders may
be synthetically produced or manipulated non-coding DNA elements. A
"plant leader" is a native or non-native leader that is functional
in plant cells. A plant leader can be used as a 5' regulatory
element for modulating expression of an operably linked gene or
genes.
[0035] As used herein, the term "enhancer domain" refers to a
cis-acting transcriptional regulatory element, a.k.a. cis-element,
which confers an aspect of the overall control of gene expression.
An enhancer domain may function to bind transcription factors. Some
enhancer domains bind more than one transcription factor, and
transcription factors may interact with different affinities with
more than one enhancer domain. Enhancer domains can be identified
by a number of techniques, including deletion analysis, i.e.,
deleting one or more nucleotides from the 5' end or internal to a
promoter; DNA binding protein analysis using DNase I footprinting,
methylation interference, electrophoresis mobility-shift assays, in
vivo genomic footprinting by ligation-mediated PCR, and other
conventional assays; or by DNA sequence similarity analysis with
known cis-element motifs by conventional DNA sequence comparison
methods. The fine structure of an enhancer domain can be further
studied by mutagenesis (or substitution) of one or more nucleotides
or by other conventional methods. Enhancer domains can be obtained
by chemical synthesis or by isolation from promoters that include
such elements, and they can be synthesized with additional flanking
nucleotides that contain useful restriction enzyme sites to
facilitate subsequence manipulation.
[0036] Translational enhancers may also be incorporated as part of
the recombinant vector. Thus the recombinant vector may preferably
contain one or more 5' non-translated leader sequences which serve
to enhance expression of the nucleic acid sequence. Such enhancer
sequences may be desirable to increase or alter the translational
efficiency of the resultant mRNA. Examples of other non-coding
regulatory element 5' nucleic acid leader sequences include dSSU
5', PetHSP70 5', and GmHSP17.9 5'.
Introns
[0037] The present invention describes the composition and utility
for eukaryotic translation Initiation Factor non-coding regulatory
element molecules from Nicotiana tabacum, Arabidopsis thaliana, and
Medicago truncatula, hereinafter referred to as eIF-NCRE molecules.
These eIF-NCRE molecules include introns.
[0038] As used herein, the term "intron" refers to a non-coding
polynucleotide molecule. Introns may be isolated from the
intervening (non-coding) sequence of a genomic copy of a gene and
may be defined generally as a region spliced out during mRNA
processing prior to translation. Alternately, introns may be
synthetically produced or manipulated non-coding DNA elements.
Introns may themselves contain sub-elements such as cis-elements or
enhancer domains that effect the transcription of operably linked
genes. A "plant intron" is a native or non-native intron that is
functional in plant cells. A plant intron can be used as a
regulatory element for modulating expression of an operably linked
gene or genes.
[0039] The transcribable polynucleotide molecule sequence in the
recombinant vector may comprise introns. The introns may be
heterologous with respect to the transcribable polynucleotide
molecule sequence. Examples of other non-coding regulatory element
introns include the corn actin intron and the corn HSP70
intron.
Transcribable Polynucleotide Molecules
[0040] The eIF-NCRE molecules of the present invention may be
operably linked to a transcribable polynucleotide molecule sequence
that is heterologous with respect to the eIF-NCRE molecules.
[0041] The phrase "heterologous" refers to the relationship between
two or more nucleic acid or protein sequences that are derived from
different sources. For example, a promoter is heterologous with
respect to a transcribable polynucleotide sequence if such a
combination is not normally found in nature. In addition, a
particular sequence may be "heterologous" with respect to a cell or
organism into which it is inserted (i.e. does not naturally occur
in that particular cell or organism).
[0042] The transcribable polynucleotide molecule sequence may
generally be any nucleic acid sequence for which an increased level
of transcription is desired. The transcribable polynucleotide
molecule sequence preferably encodes a polypeptide that is suitable
for incorporation into the diet of a human or an animal. Suitable
transcribable polynucleotide molecule sequence include but are not
limited to those encoding a yield protein, a stress resistance
protein, a developmental control protein, a tissue differentiation
protein, a meristem protein, an environmentally responsive protein,
a senescence protein, a hormone responsive protein, an abscission
protein, a source protein, a sink protein, a flower control
protein, a seed protein, an herbicide resistance protein, a disease
resistance protein, a fatty acid biosynthetic enzyme, a tocopherol
biosynthetic enzyme, an amino acid biosynthetic enzyme, and an
insecticidal protein.
[0043] Alternatively, the eIF-NCRE molecules and transcribable
polynucleotide molecule sequence may be designed to down-regulate a
specific nucleic acid sequence. This is typically accomplished by
linking the eIF-NCRE molecules to a transcribable polynucleotide
molecule sequence that is oriented in the antisense direction. One
of ordinary skill in the art is familiar with such antisense
technology. Briefly, as the antisense nucleic acid sequence is
transcribed, it hybridizes to and sequesters a complimentary
nucleic acid sequence inside the cell. This duplex RNA molecule
cannot be translated into a protein by the cell's translational
machinery. Any nucleic acid sequence may be negatively regulated in
this manner.
Modified Transcribable Polynucleotide Molecule Sequences
[0044] The eIF-NCRE molecules of the present invention may also be
operably linked to a modified transcribable polynucleotide molecule
sequence that is heterologous with respect to the eIF-NCRE
molecules. The transcribable polynucleotide molecule sequence may
be modified to provide various desirable features. For example, a
transcribable polynucleotide molecule sequence may be modified to
increase the content of essential amino acids, enhance translation
of the amino acid sequence, alter post-translational modifications
(e.g., phosphorylation sites), transport a translated product to a
compartment inside or outside of the cell, improve protein
stability, insert or delete cell signaling motifs, etc.
Polynucleotide Molecule Isolation and Modification Methods
[0045] The present invention includes a polynucleotide molecule
having a nucleic acid sequence that: i) hybridizes under stringent
conditions with a sequence selected from the group consisting of
SEQ ID NO: 1 through SEQ ID NO: 11, or any complements thereof, or
any fragments thereof; or ii) exhibits an 85% or greater identity
to a sequences elected from the group consisting of SEQ ID NO: 1
through SEQ ID NO: 11, any complements thereof, or any fragments
thereof. The present invention also provides a nucleic acid
molecule comprising a nucleic acid sequences elected from the group
consisting of SEQ ID NO:1 through SEQ ID NO:11, any complements
thereof, or any fragments thereof. As used herein, an "isolated
polynucleotide molecule" refers to a RNA or DNA molecule that is at
least partially separated from other molecules normally associated
with it in its native state. In one embodiment, the term "isolated"
is also used herein in reference to a polynucleotide molecule that
is at least partially separated from nucleic acids which normally
flank the polynucleotide in its native state. Thus, polynucleotides
fused to regulatory or coding sequences with which they are not
normally associated, for example as the result of recombinant
techniques, are considered isolated herein. Such molecules are
considered isolated even when present, for example in the
chromosome of a host cell, or in a nucleic acid solution. The term
"isolated" as used herein is not intended to encompass molecules
present in their native state. An isolated polynucleotide is an
"isolated" molecule if it occurs as a component of a transgene in a
transgenic plant. The use of the isolated polynucleotide of the
present invention in a transgenic plant is an object of the present
invention.
[0046] The term "hybridization" refers generally to the ability of
nucleic acid molecules to join via complementary base strand
pairing. Such hybridization may occur when nucleic acid molecules
are contacted under appropriate conditions (see also, "specific
hybridization," below).
[0047] "Specifically hybridizes" refers to the ability of two
nucleic acid molecules to form an anti-parallel, double-stranded
nucleic acid structure. A nucleic acid molecule is said to be the
"complement" of another nucleic acid molecule if they exhibit
"complete complementarity," i.e., each nucleotide in one sequence
is complementary to its base pairing partner nucleotide in another
sequence. Two molecules are said to be "minimally complementary" if
they can hybridize to one another with sufficient stability to
permit them to remain annealed to one another under at least
conventional "low-stringency" conditions. Similarly, the molecules
are said to be "complementary" if they can hybridize to one another
with sufficient stability to permit them to remain annealed to one
another under conventional "high-stringency" conditions. Nucleic
acid molecules that hybridize to other nucleic acid molecules,
e.g., at least under low stringency conditions are said to be
"hybridizable cognates" of the other nucleic acid molecules.
Conventional low stringency and high stringency conditions are
described herein and by Sambrook et al., Molecular Cloning, A
Laboratory Manual, 2nd Ed., Cold Spring Harbor Press, Cold Spring
Harbor, N.Y. (1989) and by Haymes et al., Nucleic Acid
Hybridization, A Practical Approach, IRL Press, Washington, D.C.
(1985). Departures from complete complementarity are permissible,
as long as such departures do not completely preclude the capacity
of the molecules to form a double-stranded structure.
[0048] As used herein "sequence identity" refers to the extent to
which two optimally aligned polynucleotide or peptide sequences are
invariant throughout a window of alignment of components, e.g.,
nucleotides or amino acids. An "identity fraction" for aligned
segments of a test sequence and a reference sequence is the number
of identical components which are shared by the two aligned
sequences divided by the total number of components in reference
sequence segment, i.e., the entire reference sequence or a smaller
defined part of the reference sequence.
[0049] As used herein, the term "percent sequence identity" or
"percent identity" refers to the percentage of identical
nucleotides in a linear polynucleotide sequence of a reference
polynucleotide molecule (or its complementary strand) as compared
to a test polynucleotide molecule (or its complementary strand)
when the two sequences are optimally aligned (with appropriate
nucleotide insertions, deletions, or gaps totaling less than 20
percent of the reference sequence over the window of comparison).
Optimal alignment of sequences for aligning a comparison window are
well known to those skilled in the art and may be conducted by
tools such as the local homology algorithm of Smith and Waterman,
the homology alignment algorithm of Needleman and Wunsch, the
search for similarity method of Pearson and Lipman, and preferably
by computerized implementations of these algorithms such as GAP,
BESTFIT, FASTA, and TFASTA available as part of the GCG.RTM.
Wisconsin Package.RTM. (Accelrys Inc., San Diego, Calif.). An
"identity fraction" for aligned segments of a test sequence and a
reference sequence is the number of identical components which are
shared by the two aligned sequences divided by the total number of
components in the reference sequence segment, i.e., the entire
reference sequence or a smaller defined part of the reference
sequence. Percent sequence identity is represented as the identity
fraction times 100. The comparison of one or more polynucleotide
sequences may be to a full-length polynucleotide sequence or a
portion thereof, or to a longer polynucleotide sequence.
[0050] As used herein, the term "substantial percent sequence
identity" refers to a percent sequence identity of at least about
70% sequence identity, at least about 80% sequence identity, at
least about 85% sequence identity, at least about 90% sequence
identity, or even greater sequence identity, such as about 95% or
about 98% or about 99% sequence identity. Thus, one embodiment of
the invention is a polynucleotide molecule that has at least about
70% sequence identity, at least about 80% sequence identity, at
least about 85% sequence identity, at least about 90% sequence
identity, or even greater sequence identity, such as about 95% or
about 98% or about 99% sequence identity with a polynucleotide
sequence described herein. Polynucleotide molecules that are
capable of regulating or affecting the transcription of an operably
linked transcribable polynucleotide molecules and have a
substantial percent sequence identity to the polynucleotide
sequences of the polynucleotide molecules provided herein are
encompassed within the scope of this invention.
[0051] "Homology" refers to the level of similarity between two or
more nucleic acid or amino acid sequences in terms of percent of
positional identity (i.e., sequence similarity or identity).
Homology also refers to the concept of similar functional
properties among different nucleic acids or proteins.
[0052] Any number of methods well known to those skilled in the art
can be used to isolate fragments of a polynucleotide molecule
disclosed herein. For example, PCR (polymerase chain reaction)
technology can be used to amplify flanking regions from a genomic
library of a plant using publicly available sequence information. A
number of methods are known to those of skill in the art to amplify
unknown DNA sequences adjacent to a core region of known sequence.
Methods include but are not limited to inverse PCR (IPCR),
vectorette PCR, Y-shaped PCR, and genome walking approaches.
Polynucleotide molecule fragments can also be obtained by other
techniques such as by directly synthesizing the fragment by
chemical means, as is commonly practiced by using an automated
oligonucleotide synthesizer. For the present invention, the
polynucleotide molecules were isolated by designing PCR primers
based on available sequence information.
[0053] Those of skill in the art are familiar with the standard
resource materials that describe specific conditions and procedures
for the construction, manipulation, and isolation of macromolecules
(e.g., polynucleotide molecules, plasmids, etc.), as well as the
generation of recombinant organisms and the screening and isolation
of polynucleotide molecules.
Polynucleotide Constructs
[0054] Any of the eIF-NCRE molecules and transcribable
polynucleotide molecule sequences described above may be provided
in a construct. Constructs of the present invention would typically
contain a promoter, such as provided in SEQ ID NO: 1 or SEQ ID NO:
11, operably linked to a transcribable polynucleotide molecule.
[0055] As used herein, the term "construct" or refers to any
recombinant polynucleotide molecule such as a plasmid, cosmid,
virus, autonomously replicating polynucleotide molecule, phage, or
linear or circular single-stranded or double-stranded DNA or RNA
polynucleotide molecule, derived from any source, capable of
genomic integration or autonomous replication, comprising a
polynucleotide molecule where one or more polynucleotide molecule
has been linked in a functionally operative manner, i.e. operably
linked.
[0056] As used herein, the term "vector" or "vector construct"
refers to any recombinant polynucleotide construct that may be used
for the purpose of transformation, i.e. the introduction of
heterologous DNA into a host cell.
[0057] In addition, constructs may include, but are not limited to
additional polynucleotide molecules from the 3'-untranslated region
(3' UTR) of plant genes, e.g. a 3' UTR, such as the PI-II
termination region of potato, pea rubisco small subunit 3'UTR or
the octopine or nopaline synthase 3' termination regions. In
addition, constructs may include but are not limited to additional
polynucleotide molecules from the 5'-untranslated region (5' UTR)
of plant genes which can play an important role in translation
initiation and can also be a genetic component in a plant
expression construct, such as provided in SEQ ID NO: 1 through SEQ
ID NO: 11, e.g. a leader to enhance transgene expression, such as
non-translated 5' leader polynucleotide molecules derived from heat
shock protein genes which have been demonstrated to enhance gene
expression in plants (see for example, U.S. Pat. No. 5,659,122 and
U.S. Pat. No. 5,362,865, the DNA sequence of which are hereby
incorporated by reference). In addition, constructs may include but
are not limited to additional polynucleotide molecules such as
introns, e.g., the first intron of the actin 1 gene from Oryza
sativa (U.S. Pat. No. 5,641,876), the IS50L intron from the light
sensitive 1 gene of Solanum tuberosum, the intron of the heat shock
protein 70 gene of Petunia hybrida (U.S. Pat. No. 5,659,122), the
Hsp70 intron of the Heat shock protein 70 gene of Zea mays (U.S.
Pat. No. 5,593,874), the DNA sequence of which are hereby
incorporated by reference. These additional polynucleotide
molecules may be derived from a source that is native or
heterologous with respect to the other elements present in the
construct.
[0058] Means for preparing recombinant constructs are well known in
the art. Methods for making recombinant vector constructs
particularly suited to plant transformation include, without
limitation, those described in U.S. Pat. Nos. 4,971,908, 4,940,835,
4,769,061 and 4,757,011. These type of vectors have also been
reviewed (Rodriguez, et al. Vectors: A Survey of Molecular Cloning
Vectors and Their Uses, Butterworths, Boston, 1988; Glick et al.,
Methods in Plant Molecular Biology and Biotechnology, CRC Press,
Boca Raton, Fla., 1993).
[0059] Typical constructs useful for expression of nucleic acids in
higher plants are well known in the art and include vectors derived
from the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens
(Rogers, et al., Meth. In Enzymol, 153: 253-277, 1987). Other
recombinant constructs useful for plant transformation, including
the pCaMVCN transfer control vector, have also been described
(Fromm et al., Proc. Natl. Acad. Sci. USA, 82(17): 5824-5828,
1985).
Promoters in the Recombinant Constructs
[0060] The promoter used in the recombinant construct preferably
transcribes a heterologous transcribable polynucleotide molecule
sequence at a high level in a plant. More preferably, the promoter
hybridizes to a nucleic acid sequence selected from the group
consisting of SEQ ID NO:1 through SEQ ID NO:11, or any complements
thereof; or any fragments thereof. Suitable hybridization
conditions include those described above. A nucleic acid sequence
of the promoter preferably hybridizes, under low or high stringency
conditions, with SEQ ID NO:1 through SEQ ID NO:11, or any
complements thereof, or any fragments thereof. The promoter most
preferably hybridizes under high stringency conditions to a nucleic
acid sequences elected from the group consisting of SEQ ID NO:1
through SEQ ID NO:11, or any complements thereof, or any fragments
thereof.
[0061] In an alternative embodiment, the promoter comprises a
nucleic acid sequence that exhibits 85% or greater identity, and
more preferably at least 86 or greater, 87 or greater, 88 or
greater, 89 or greater, 90 or greater, 91 or greater, 92 or
greater, 93 or greater, 94 or greater, 95 or greater, 96 or
greater, 97 or greater, 98 or greater, or 99% or greater identity
to a nucleic acid sequences elected from the group consisting of
SEQ ID NO:1 through SEQ ID NO:11, or any complements thereof, or
any fragments thereof. The promoter most preferably comprises a
nucleic acid sequences elected from the group consisting of SEQ ID
NO:1 through SEQ ID NO:11, or any complements thereof, or any
fragments thereof.
Additional Promoters in the Recombinant Construct
[0062] One or more additional promoters may also be provided in the
recombinant construct. These promoters may be operably linked to
any of the transcribable polynucleotide molecule sequences
described above. Alternatively, the promoters may be operably
linked to other nucleic acid sequences, such as those encoding
transit peptides, selectable marker proteins, or antisense
sequences.
[0063] As used herein, the term "chimeric" refers to the product of
the fusion of portions of two or more different polynucleotide
molecules. As used herein, the term "chimeric promoter" refers to a
promoter produced through the manipulation of known promoters or
other polynucleotide molecules. Such chimeric promoters may combine
enhancer domains that can confer or modulate gene expression from
one or more promoters or regulatory elements, for example, by
fusing a heterologous enhancer domain from a first promoter to a
second promoter with its own partial or complete regulatory
elements. Thus, the design, construction, and use of chimeric
promoters according to the methods disclosed herein for modulating
the expression of operably linked polynucleotide sequences are
encompassed by the present invention.
[0064] Novel chimeric promoters can be designed or engineered by a
number of methods. For example, a chimeric promoter may be produced
by fusing an enhancer domain from a first promoter to a second
promoter. The resultant chimeric promoter may have novel expression
properties relative to the first or second promoters. Novel
chimeric promoters can be constructed such that the enhancer domain
from a first promoter is fused at the 5' end, at the 3' end, or at
any position internal to the second promoter. For example, one or
more caulimovirus enhancer elements fused to the promoter of the
present invention. The location of the enhancer domain fusion
relative to the second promoter may cause the resultant chimeric
promoter to have novel expression properties relative to a fusion
made at a different location. Methods for making chimeric promoters
particularly suited to plant transformation include, without
limitation, those described in U.S. Pat. No. 6,660,911.
[0065] These additional promoters may be selected on the basis of
the cell type into which the vector construct will be inserted.
Promoters which function in bacteria, yeast, and plants are all
well taught in the art. The additional promoters may also be
selected on the basis of their regulatory features. Examples of
such features include enhancement of transcriptional activity,
inducibility, tissue-specificity, and developmental
stage-specificity. In plants, promoters that are inducible, of
viral or synthetic origin, constitutively active, temporally
regulated, and spatially regulated have been described (Poszkowski,
et al., EMBO J., 3: 2719, 1989; Odell, et al., Nature, 313:810,
1985; Chau et al., Science, 244:174-181. 1989).
[0066] Often-used constitutive promoters include the CaMV 35S
promoter, the enhanced CaMV 35S promoter, the Figwort Mosaic Virus
(FMV) promoter, the mannopine synthase (mas) promoter, the nopaline
synthase (nos) promoter, and the octopine synthase (ocs)
promoter.
[0067] Useful inducible promoters include promoters induced by
salicylic acid or polyacrylic acids induced by application of
safeners (substituted benzenesulfonamide herbicides), heat-shock
promoters, a nitrate-inducible promoter derived from the spinach
nitrite reductase transcribable polynucleotide molecule sequence,
hormone-inducible promoters, and light-inducible promoters
associated with the small subunit of RuBP carboxylase and LHCP
families.
[0068] Examples of useful tissue-specific,
developmentally-regulated promoters include the .beta.-conglycinin
7S.alpha. promoter, and seed-specific promoters. Plant functional
promoters useful for preferential expression in seed plastid
include those from plant storage proteins and from proteins
involved in fatty acid biosynthesis in oilseeds. Examples of such
promoters include the 5' regulatory regions from such transcribable
polynucleotide molecule sequences as napin, phaseolin, zein,
soybean trypsin inhibitor, ACP, stearoyl-ACP desaturase, and
oleosin. Seed-specific regulation is discussed in EP 0 255 378.
Another exemplary tissue-specific promoter is the lectin promoter,
which is specific for seed tissue.
[0069] Particularly preferred additional promoters in the
recombinant construct include the nopaline synthase (nos),
mannopine synthase (mas), and octopine synthase (ocs) promoters,
which are carried on tumor-inducing plasmids of Agrobacterium
tumefaciens; the cauliflower mosaic virus (CaMV) 19S and 35S
promoters; the enhanced CaMV 35S promoter; the Figwort Mosaic Virus
(FMV) 35S promoter; the light-inducible promoter from the small
subunit of ribulose-1,5-bisphosphate carboxylase (ssRUB1SCO); the
EIF-4A promoter from tobacco (Mandel, et al., Plant Mol. Biol, 29:
995-1004, 1995); corn sucrose synthetase; corn alcohol
dehydrogenase 1; corn light harvesting complex; corn heat shock
protein; the chitinase promoter from Arabidopsis; the LTP (Lipid
Transfer Protein) promoters from broccoli; petunia chalcone
isomerase; bean glycine rich protein 1; Potato patatin; the
ubiquitin promoter from maize; and the actin promoter from
corn.
[0070] The additional promoter is preferably seed selective, tissue
selective, constitutive, or inducible. The promoter is most
preferably the nopaline synthase (NOS), octopine synthase (OCS),
mannopine synthase (MAS), cauliflower mosaic virus 19S and 35S
(CaMV19S, CaMV35S), enhanced CaMV (eCaMV), ribulose
1,5-bisphosphate carboxylase (ssRUBISCO), figwort mosaic virus
(FMV), CaMV derived AS4, tobacco RB7, wheat PDX1, tobacco EIF-4,
lectin protein (Le1), or corn RC2 promoter.
Other Elements in the Recombinant Construct
[0071] The present invention includes the composition and utility
for constructs comprising eukaryotic translation Initiation Factor
non-coding regulatory element molecules from Nicotiana tabacum,
Arabidopsis thaliana, and Medicago truncatula, hereinafter referred
to as eIF-NCRE molecules. These constructs may include 5'
non-translated leader sequences.
[0072] Various cis-acting untranslated 5' and 3' non-coding
regulatory element sequences may be included in the recombinant
nucleic acid construct. Any such regulatory sequences may be
provided in a recombinant construct with other regulatory
sequences. Such combinations can be designed or modified to produce
desirable regulatory features.
[0073] 5' non-coding regulatory element sequences typically
comprise non-translated leader sequences and/or introns.
[0074] A 3' non-translated region typically provides a
transcriptional termination signal, and a polyadenylation signal
which functions in plants to cause the addition of adenylate
nucleotides to the 3' end of the mRNA. These may be obtained from
the 3' regions to the nopaline synthase (nos) coding sequence, the
soybean 7S.alpha. storage protein coding sequence, the albumin
coding sequence, and the pea ssRUBISCO E9 coding sequence.
Particularly preferred 3' nucleic acid sequences include nos 3', E9
3', ADR12 3', 7S.alpha. 3', 11S 3', and albumin 3'.
[0075] Typically, nucleic acid sequences located a few hundred base
pairs downstream of the polyadenylation site serve to terminate
transcription. These regions are required for efficient
polyadenylation of transcribed mRNA.
[0076] The recombinant construct may further comprise a nucleic
acid sequence encoding a transit peptide. This peptide may be
useful for directing a protein to the extracellular space, a
chloroplast, or to some other compartment inside or outside of the
cell (see, e.g., European Patent Application Publication Number
0218571).
Transcribable Polynucleotide Molecule Sequences in the Recombinant
Nucleic Acid Construct
[0077] The non-coding regulatory element (NCRE) in the recombinant
construct is preferably operably linked to a transcribable
polynucleotide molecule sequence. Exemplary transcribable
polynucleotide molecule sequences, and modified forms thereof, are
described in detail above. The NCRE molecules of the present
invention may be operably linked to a transcribable polynucleotide
molecule sequence that is heterologous with respect to the NCRE. In
one aspect, the transcribable polynucleotide molecule sequence may
generally be any nucleic acid sequence for which an increased level
of transcription is desired. The transcribable polynucleotide
molecule sequence preferably encodes a polypeptide that is suitable
for incorporation into the diet of a human or an animal. Suitable
transcribable polynucleotide molecule sequences include those
encoding a yield protein, a stress resistance protein, a
developmental control protein, a tissue differentiation protein, a
meristem protein, an environmentally responsive protein, a
senescence protein, a hormone responsive protein, an abscission
protein, a source protein, a sink protein, a flower control
protein, a seed protein, an herbicide resistance protein, a disease
resistance protein, a fatty acid biosynthetic enzyme, a tocopherol
biosynthetic enzyme, an amino acid biosynthetic enzyme, and an
insecticidal protein.
[0078] Alternatively, the NCRE or the transcribable polynucleotide
molecule sequences may be designed to down-regulate a specific
nucleic acid sequence. This is typically accomplished by linking
the NCRE to a transcribable polynucleotide molecule sequence that
is oriented in the antisense direction. One of ordinary skill in
the art is familiar with such antisense technology. Using such an
approach, a cellular nucleic acid sequence is effectively down
regulated as the subsequent steps of translation are disrupted.
Nucleic acid sequences may be negatively regulated in this
manner.
[0079] Thus, one embodiment of the invention is a polynucleotide
molecule such as provided in SEQ ID NO:1 through SEQ ID NO: 11 that
is operably linked to a transcribable polynucleotide molecule so as
to direct transcription of the transcribable polynucleotide
molecule at a desired level or in a desired tissue or developmental
pattern upon introduction of the construct into a plant cell. In
some cases, the transcribable polynucleotide molecule comprises a
protein-coding region of a gene, and a non-coding regulatory
element of SEQ ID NO:1 through SEQ ID NO: 11 provides for
transcription of a functional mRNA molecule that is translated and
expressed as a protein product. Constructs may also be constructed
for transcription of antisense RNA molecules or other similar
inhibitory RNA molecules in order to suppress expression of a
specific gene of interest in a target host cell.
[0080] Exemplary transcribable polynucleotide molecules for
incorporation into constructs of the present invention include, for
example, DNA molecules or genes from a species other than the
target gene species, or even genes that originate with or are
present in the same species, but are incorporated into recipient
cells by genetic engineering methods rather than classical
reproduction or breeding techniques. Exogenous gene or genetic
element is intended to refer to any gene or polynucleotide molecule
that is introduced into a recipient cell. The type of
polynucleotide molecule included in the exogenous DNA can include
DNA that is already present in the plant cell, DNA from another
plant, DNA from a different organism, or a DNA generated
externally, such as a DNA molecule containing an antisense message
of a gene, or a DNA molecule encoding an artificial or modified
version of a gene.
Fusion Proteins
[0081] Any of the above described structural nucleic acid
sequences, and modified forms thereof, may be linked with
additional nucleic acid sequences to encode fusion proteins. The
additional nucleic acid sequence preferably encodes at least 1
amino acid, peptide, or protein. Production of fusion proteins is
routine in the art and many possible fusion combinations exist.
[0082] For instance, the fusion protein may provide a "tagged"
epitope to facilitate detection of the fusion protein, such as GST,
GFP, FLAG, or polyHIS. Such fusions preferably encode between 1 and
50 amino acids, more preferably between 5 and 30 additional amino
acids, and even more preferably between 5 and 20 amino acids.
[0083] Alternatively, the fusion may provide regulatory, enzymatic,
cell signaling, or intercellular transport functions. For example,
a sequence encoding a chloroplast transit peptide may be added to
direct a fusion protein to the chloroplasts within a plant cell.
Such fusion partners preferably encode between 1 and 1000
additional amino acids, more preferably between 5 and 500
additional amino acids, and even more preferably between 10 and 250
amino acids.
[0084] Where plastid targeting is necessary, for example, the EPSPS
enzyme functions in a plant chloroplast, therefore, DNA molecules
encoding a chloroplast transit peptide (CTP) are engineered into a
DNA molecule encoding an EPSPS protein to encode a fusion protein
of the CTP to the N terminus of an EPSPS creating a chimeric
molecule. A chimeric polynucleic acid coding sequence is comprised
of two or more open reading frames joined in-frame that encode a
chimeric protein, for example, a chloroplast transit peptide and an
EPSPS enzyme. A chimeric gene refers to the multiple genetic
elements derived from heterologous sources operably linked to
comprise a gene. In the present invention the DNA construct
expresses a chimeric CTP-EPSPS protein that directs the glyphosate
resistant EPSPS protein into the plant chloroplast. In a native
plant EPSPS gene, chloroplast transit peptide regions are contained
in the native coding sequence (for example, CTP2, Klee et al., Mol.
Gen. Genet. 210:47-442, 1987). The CTP is cleaved from the EPSPS
enzyme at the chloroplast membrane to create a "mature EPSPS or
EPSPS enzyme" that refers to the polypeptide sequence of the
processed protein product remaining after the chloroplast transit
peptide has been removed. The production of glyphosate tolerant
plants by expression of a fusion protein comprising an
amino-terminal CTP with a glyphosate resistant EPSPS enzyme is well
known by those skilled in the art, (U.S. Pat. No. 5,627,061, U.S.
Pat. No. 5,633,435, U.S. Pat. No. 5,312,910, EP 0218571, EP 189707,
EP 508909, and EP 924299). Those skilled in the art will recognize
that various chimeric constructs can be made that utilize the
functionality of a particular CTP to import glyphosate resistant
EPSPS enzymes into the plant cell chloroplast. The present
invention illustrates the utility of the combination of an eIF-NCRE
molecule operably linked to a DNA molecule that encodes for a
chloroplast transit peptide fused to an EPSP synthase.
NCRE molecules and Genes of Agronomic Interest
[0085] The recombinant construct may also contain one or more
additional transcribable polynucleotide molecule sequences. These
additional transcribable polynucleotide molecule sequences may
generally be any sequences suitable for use in a recombinant
construct. Such transcribable polynucleotide molecule sequences
include any of the transcribable polynucleotide molecule sequences,
and modified forms thereof, described above. The additional
transcribable polynucleotide molecule sequences may also be
operably linked to any of the above described NCRE sequences. The
one or more transcribable polynucleotide molecule sequences may
each be operably linked to separate NCRE sequences. Alternatively,
the transcribable polynucleotide molecule sequences may be operably
linked to a single NCRE (i.e. a single operon).
[0086] As used herein, the term "gene of agronomic interest" refers
to a transcribable polynucleotide molecule that includes but is not
limited to a gene that provides a desirable characteristic
associated with plant morphology, physiology, growth and
development, yield, nutritional enhancement, disease or pest
resistance, or environmental or chemical tolerance.
[0087] The additional transcribable polynucleotide molecule
sequences preferably encode a gene of agronomic interest, such as
but not limited to: a yield protein, a stress resistance protein, a
developmental control protein, a tissue differentiation protein, a
meristem protein, an environmentally responsive protein, a
senescence protein, a hormone responsive protein, an abscission
protein, a source protein, a sink protein, a flower control
protein, a seed protein, an herbicide resistance protein, a disease
resistance protein, a fatty acid biosynthetic enzyme, a tocopherol
biosynthetic enzyme, an amino acid biosynthetic enzyme, and an
insecticidal protein.
[0088] Alternatively, the additional transcribable polynucleotide
molecule sequences may be designed to down-regulate a specific
nucleic acid sequence. This is typically accomplished by operably
linking the second structural amino acid, in an antisense
orientation, with a promoter. One of ordinary skill in the art is
familiar with such antisense technology. The process is also
briefly described above. Any nucleic acid sequence may be
negatively regulated in this manner.
[0089] In one embodiment of the invention, a polynucleotide
molecule as shown in SEQ ID NO: 1 through SEQ ID NO: 11, or any
complements thereof or any fragments thereof comprising regulatory
elements, such as a promoter, leader, or an intron element, is
incorporated into a construct such that a polynucleotide molecule
of the present invention is operably linked to a transcribable
polynucleotide molecule that is a gene of agronomic interest.
[0090] The expression of a gene of agronomic interest is desirable
in order to confer an agronomically important trait. A gene of
agronomic interest that provides a beneficial agronomic trait to
crop plants may be, for example, including, but not limited to
genetic elements comprising herbicide resistance (U.S. Pat. No.
5,633,435 and U.S. Pat. No. 5,463,175), increased yield (U.S. Pat.
No. 5,716,837), insect control (U.S. Pat. No. 6,063,597; U.S. Pat.
No. 6,063,756; U.S. Pat. No. 6,093,695; U.S. Pat. No. 5,942,664;
and U.S. Pat. No. 6,110,464), fungal disease resistance (U.S. Pat.
No. 5,516,671; U.S. Pat. No. 5,773,696; U.S. Pat. No. 6,121,436;
U.S. Pat. No. 6,316,407, and U.S. Pat. No. 6,506,962), virus
resistance (U.S. Pat. No. 5,304,730 and U.S. Pat. No. 6,013,864),
nematode resistance (U.S. Pat. No. 6,228,992), bacterial disease
resistance (U.S. Pat. No. 5,516,671), starch production (U.S. Pat.
No. 5,750,876 and U.S. Pat. No. 6,476,295), modified oils
production (U.S. Pat. No. 6,444,876), high oil production (U.S.
Pat. No. 5,608,149 and U.S. Pat. No. 6,476,295), modified fatty
acid content (U.S. Pat. No. 6,537,750), high protein production
(U.S. Pat. No. 6,380,466), fruit ripening (U.S. Pat. No.
5,512,466), enhanced animal and human nutrition (U.S. Pat. No.
5,985,605 and U.S. Pat. No. 6,171,640), biopolymers (U.S. Pat. No.
5,958,745 and U.S. Patent Publication No. US20030028917),
environmental stress resistance (U.S. Pat. No. 6,072,103),
pharmaceutical peptides (U.S. Pat. No. 6,080,560), improved
processing traits (U.S. Pat. No. 6,476,295), improved digestibility
(U.S. Pat. No. 6,531,648) low raffinose (U.S. Pat. No. 6,166,292),
industrial enzyme production (U.S. Pat. No. 5,543,576), improved
flavor (U.S. Pat. No. 6,011,199), nitrogen fixation (U.S. Pat. No.
5,229,114), hybrid seed production (U.S. Pat. No. 5,689,041), and
biofuel production (U.S. Pat. No. 5,998,700), the genetic elements,
methods, and transgenes described in the patents listed above are
hereby incorporated by reference.
[0091] Alternatively, a transcribable polynucleotide molecule can
effect the above mentioned phenotypes by encoding a RNA molecule
that causes the targeted suppression of expression of an endogenous
gene, for example via antisense, dsRNA, or cosuppression-mediated
mechanisms. The RNA could also be a catalytic RNA molecule (i.e., a
ribozyme) engineered to cleave a desired endogenous mRNA product.
Thus, any polynucleotide molecule that encodes a protein or mRNA
that expresses a phenotype or morphology change of interest may be
useful for the practice of the present invention.
[0092] The constructs of the present invention are generally double
Ti plasmid border DNA constructs that have the right border (RB or
AGRtu.RB) and left border (LB or AGRtu.LB) regions of the Ti
plasmid isolated from Agrobacterium tumefaciens comprising a T-DNA,
that along with transfer molecules provided by the Agrobacterium
cells, permits the integration of the T-DNA into the genome of a
plant cell. The constructs also contain the plasmid backbone DNA
segments that provide replication function and antibiotic selection
in bacterial cells, for example, an E. coli origin of replication
such as ori322, a broad host range origin of replication such as
oriV, rop, or oriRi, and a coding region for a selectable marker
such as Spec/Strp that encodes for Tn7 aminoglycoside
adenyltransferase (aadA) conferring resistance to spectinomycin or
streptomycin, or a gentamicin (Gm, Gent) selectable marker gene.
For plant transformation, the host bacterial strain is often
Agrobacterium tumefaciens ABI, C58, or LBA4404, however, other
strains known to those skilled in the art of plant transformation
can function in the present invention.
Selectable Markers
[0093] The polynucleotide molecules of the present invention can be
incorporated into a DNA construct using marker genes as described
and tested in transient analyses that provide an indication of gene
expression in stable plant systems.
[0094] As used herein the term "marker gene" or "selectable marker"
refers to any transcribable polynucleotide molecule whose
expression can be screened for or scored in some way. Included
within the term "selectable markers" is also genes which encode a
secretable marker whose secretion can be detected as a means of
identifying or selecting for transformed cells.
[0095] Methods of testing for marker gene expression in transient
assays are known to those of skill in the art. Transient expression
of marker genes has been reported using a variety of plants,
tissues, and DNA delivery systems. For example, types of transient
analyses can include, but are not limited to direct gene delivery
via electroporation or particle bombardment of tissues in any
transient plant assay using any plant species of interest. Such
transient systems would include, but are not limited to
electroporation of protoplasts from a variety of tissue sources or
particle bombardment of specific tissues of interest. The present
invention encompasses the use of any transient expression system to
evaluate polynucleotide molecule operably linked to any
transcribable polynucleotide molecules, including, but not limited
to selected reporter genes, marker genes, or genes of agronomic
interest. Examples of plant tissues envisioned to test in
transients via an appropriate delivery system would include, but
are not limited to leaf base tissues, callus, cotyledons, roots,
endosperm, embryos, floral tissue, pollen, and epidermal
tissue.
[0096] The recombinant construct may further comprise a selectable
marker. The nucleic acid sequence serving as the selectable marker
functions to produce a phenotype in cells which facilitates their
identification relative to cells not containing the marker.
[0097] Examples of selectable markers include, but are not limited
to, a neo gene, which codes for kanamycin resistance and can be
selected for using kanamycin, G418, etc.; a bar gene which codes
for bialaphos resistance; a mutant EPSP synthase gene which encodes
glyphosate resistance; a nitrilase gene which confers resistance to
bromoxynil; a mutant acetolactate synthase gene (ALS) which confers
imidazolinone or sulphonylurea resistance (European Patent
Application No. 0154204); green fluorescent protein (GFP); and a
methotrexate resistant DHFR gene.
[0098] Other exemplary selectable markers include: a
.beta.-glucuronidase or uidA gene (GUS), which encodes an enzyme
for which various chromogenic substrates are known; an R-locus
gene, which encodes a product that regulates the production of
anthocyanin pigments in plant tissues (a .beta.-lactamase gene,
which encodes an enzyme for which various chromogenic substrates
are known (e.g., PADAC, a chromogenic cephalosporin); a luciferase
gene; a xy1E gene which encodes a catechol dioxygenase that can
convert chromogenic catechols; an .alpha.-amylase gene; a
tyrosinase gene, which encodes an enzyme capable of oxidizing
tyrosine to DOPA and dopaquinone (which in turn condenses to
melanin); and an .alpha.-galactosidase, which will turn a
chromogenic .alpha.-galactose substrate.
[0099] Included within the term "selectable markers" are also genes
which encode a secretable marker whose secretion can be detected as
a means of identifying or selecting for transformed cells. Examples
include markers that encode a secretable antigen that can be
identified by antibody interaction, or even secretable enzymes
which can be detected catalytically. Selectable secreted marker
proteins fall into a number of classes, including small, diffusible
proteins which are detectable, (e.g., by ELISA), small active
enzymes which are detectable in extracellular solution (e.g.,
.alpha.-amylase, .beta.-lactamase, phosphinothricin transferase),
or proteins which are inserted or trapped in the cell wall (such as
proteins which include a leader sequences such as that found in the
expression unit of extension or tobacco PR-S). Other possible
selectable marker genes will be apparent to those of skill in the
art.
[0100] The selectable marker is preferably GUS, green fluorescent
protein (GFP), neomycin phosphotransferase II (nptII), luciferase
(LUX), an antibiotic resistance coding sequence, or an herbicide
(e.g., glyphosate) resistance coding sequence. The selectable
marker is most preferably a kanamycin, hygromycin, or herbicide
resistance marker.
[0101] Any scorable or screenable marker gene can be used in a
transient assay. Exemplary marker genes for transient analyses of
the promoters or promoter fragments of the present invention
include a GUS gene (U.S. Pat. No. 5,599,670, hereby incorporated by
reference) or a GFP gene (U.S. Pat. No. 5,491,084, hereby
incorporated by reference). The constructs containing the
polynucleotide molecule operably linked to a marker gene are
delivered to the tissues and the tissues are analyzed by the
appropriate mechanism, depending on the marker. The quantitative or
qualitative analyses are used as a tool to evaluate the potential
expression profile of the polynucleotide molecule when operably
linked to genes of agronomic interest in stable plants.
[0102] Thus, in one preferred embodiment, a polynucleotide molecule
of the present invention, such as shown in SEQ ID NO:1 through SEQ
ID NO: 11, is incorporated into a DNA construct such that a
polynucleotide molecule of the present invention is operably linked
to a transcribable polynucleotide molecule that provides for a
selectable, screenable, or scorable marker. Markers for use in the
practice of the present invention include, but are not limited to
transcribable polynucleotide molecules encoding
.beta.-glucuronidase (GUS), green fluorescent protein (GFP),
luciferase (LUC), proteins that confer antibiotic resistance, or
proteins that confer herbicide tolerance. Useful antibiotic
resistance markers, including those encoding proteins conferring
resistance to kanamycin (nptII), hygromycin B (aph IV),
streptomycin or spectinomycin (aad, spec/strep) and gentamycin
(aac3 and aacC4) are known in the art.
[0103] Herbicides for which transgenic plant tolerance has been
demonstrated and the method of the present invention can be
applied, include but are not limited to: glyphosate, glufosinate,
sulfonylureas, imidazolinones, bromoxynil, delapon,
cyclohezanedione, protoporphyrionogen oxidase inhibitors, and
isoxasflutole herbicides.
[0104] "Glyphosate" refers to N-phosphonomethylglycine and its
salts, Glyphosate is the active ingredient of Roundup.RTM.
herbicide (Monsanto Co.). Plant treatments with "glyphosate" refer
to treatments with the Roundup.RTM. or Roundup Ultra.RTM. herbicide
formulation, unless otherwise stated. Glyphosate as
N-phosphonomethylglycine and its salts (not formulated Roundup.RTM.
herbicide) are components of synthetic culture media used for the
selection of bacteria and plant tolerance to glyphosate or used to
determine enzyme resistance in in vitro biochemical assays.
Examples of commercial formulations of glyphosate include, without
restriction, those sold by Monsanto Company as ROUNDUP.RTM.,
ROUNDUP.RTM. ULTRA, ROUNDUP.RTM. ULTRAMAX, ROUNDUP.RTM.,
ROUNDUP.RTM. CT, ROUNDUP.RTM. EXTRA, ROUNDUP.RTM. BIACTIVE,
ROUNDUP.RTM. BIOFORCE, RODEO.RTM., POLARIS.RTM., SPARK.RTM. and
ACCORD.RTM. herbicides, all of which contain glyphosate as its
isopropylammonium salt; WEATHERMAX.RTM., which contains glyphosate
as its potassium salt; those sold by Monsanto Company as
ROUNDUP.RTM. DRY and RIVAL.RTM. herbicides, which contain
glyphosate as its ammonium salt; that sold by Monsanto Company as
ROUNDUP.RTM. GEOFORCE, which contains glyphosate as its sodium
salt; and that sold by Zeneca Limited as TOUCHDOWNS herbicide,
which contains glyphosate as its trimethylsulfonium salt.
[0105] Polynucleotide molecules encoding proteins involved in
herbicide tolerance are known in the art, and include, but are not
limited to a polynucleotide molecule encoding
5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) described in
U.S. Pat. No. 5,627,061, U.S. Pat. No. 5,633,435, U.S. Pat. No.
6,040,497 and in U.S. Pat. No. 5,094,945 for glyphosate tolerance,
all of which are hereby incorporated by reference; polynucleotides
encoding a glyphosate oxidoreductase and a glyphosate-N-acetyl
transferase (GOX, U.S. Pat. No. 5,463,175 and GAT, U.S. Patent
publication 20030083480, herein incorporated by reference); a
polynucleotide molecule encoding bromoxynil nitrilase (Bxn)
described in U.S. Pat. No. 4,810,648 for Bromoxynil tolerance,
which is hereby incorporated by reference; a polynucleotide
molecule encoding phytoene desaturase (crtI) described in Misawa et
al, (1993) Plant J. 4:833-840 and Misawa et al, (1994) Plant J.
6:481-489 for norflurazon tolerance; a polynucleotide molecule
encoding acetohydroxyacid synthase (AHAS, aka ALS) described in
Sathasiivan et al. (1990) Nucl. Acids Res. 18:2188-2193 for
tolerance to sulfonylurea herbicides; and the bar gene described in
DeBlock, et al. (1987) EMBO J. 6:2513-2519 for glufosinate and
bialaphos tolerance; resistant hydroxyphenyl pyruvate dehydrogenase
(HPPD, U.S. Pat. No. 6,768,044). The promoter of the present
invention can express genes that encode for phosphinothricin
acetyltransferase, glyphosate resistant EPSPS, aminoglycoside
phosphotransferase, hydroxyphenyl pyruvate dehydrogenase,
hygromycin phosphotransferase, neomycin phosphotransferase, dalapon
dehalogenase, bromoxynil resistant nitrilase, anthranilate
synthase, glyphosate oxidoreductase and glyphosate-N-acetyl
transferase.
Transformed Plants And Plant Cells
[0106] The invention is also directed to a method of producing
transformed cells which comprise, in a 5' to 3' orientation, one or
more non-coding regulatory elements operably linked to a
heterologous transcribable polynucleotide molecule sequence. Other
sequences may also be introduced into the cell along with the
non-coding regulatory elements and transcribable polynucleotide
molecule sequence. These other sequences may include 3'
transcriptional terminators, 3' polyadenylation signals, other
untranslated sequences, transit or targeting sequences, selectable
markers, enhancers, and operators.
[0107] The method of transformation generally comprises the steps
of selecting a suitable host cell, transforming the host cell with
a recombinant vector, and obtaining the transformed host cell.
[0108] As used herein, the term "transformed" refers to a cell,
tissue, organ, or organism into which has been introduced a foreign
polynucleotide molecule, such as a construct. The introduced
polynucleotide molecule may be integrated into the genomic DNA of
the recipient cell, tissue, organ, or organism such that the
introduced polynucleotide molecule is inherited by subsequent
progeny. A "transgenic" or "transformed" cell or organism also
includes progeny of the cell or organism and progeny produced from
a breeding program employing such a transgenic plant as a parent in
a cross and exhibiting an altered phenotype resulting from the
presence of a foreign polynucleotide molecule.
[0109] Preferred recombinant constructs, transcribable
polynucleotide molecule sequences, non-coding regulatory elements,
and other regulatory elements are described above. The non-coding
regulatory element preferably has a nucleic acid sequence that
hybridizes under stringent conditions with SEQ ID NO:1 through SEQ
ID NO:11, or any complement thereof; or exhibits 85% or greater
identity, and more preferably at least 86 or greater, 87 or
greater, 88 or greater, 89 or greater, 90 or greater, 91 or
greater, 92 or greater, 93 or greater, 94 or greater, 95 or
greater, 96 or greater, 97 or greater, 98 or greater, or 99% or
greater identity to SEQ ID NO:1 through SEQ ID NO:11. The
recombinant construct used to transform the host cell typically
comprises, in a 5' to 3' orientation: a promoter to direct the
transcription of a transcribable polynucleotide molecule sequence,
a transcribable polynucleotide molecule sequence, a 3'
transcriptional terminator, and a 3' polyadenylation signal. The
recombinant vector may further comprise untranslated nucleic acid
sequences, transit and targeting nucleic acid sequences, selectable
markers, enhancers, or operators. Suitable recombinant vectors,
transcribable polynucleotide molecule sequences, promoters, and
other regulatory elements include, without limitation, those
described above.
[0110] Technology for introduction of DNA into cells is well known
to those of skill in the art. These methods can generally be
classified into five categories: (1) chemical methods (Graham and
Van der Eb, Virology, 54(2): 536-539, 1973; Zatloukal, et al., Ann.
N.Y. Acad. Sci., 660: 136-153, 1992); (2) physical methods such as
microinjection (Capecchi, Cell, 22(2): 479-488, 1980),
electroporation (Wong and Neumann, Biochim. Biophys. Res. Commun.,
107(2): 584-587, 1982; Fromm et al., Proc. Natl. Acad. Sci. USA,
82(17): 5824-5828, 1985; U.S. Pat. No. 5,384,253) and particle
acceleration (Johnston and Tang, Methods Cell Biol., 43(A):
353-365, 1994; Fynan et al., Proc. Natl. Acad. Sci. USA, 90(24):
11478-11482, 1993); (3) viral vectors (Clapp, Clin. Perinatol.,
20(1): 155-168, 1993; Lu, et al., J. Exp. Med., 178(6): 2089-2096,
1993; Eglitis and Anderson, Biotechniques, 6(7): 608-614, 1988);
(4) receptor-mediated mechanisms (Curiel et al., Hum. Gen. Ther.,
3(2):147-154, 1992; Wagner, et al., Proc. Natl. Acad. Sci. USA,
89(13): 6099-6103, 1992), and (5) bacterial mediated mechanisms
such as with Agrobacterium. Alternatively, nucleic acids can be
directly introduced into pollen by directly injecting a plant's
reproductive organs (Zhou, et al., Methods in Enzymology, 101: 433,
1983; Hess, Intern Rev. Cytol., 107: 367, 1987; Luo, et al., Plant
Mol. Biol. Reporter, 6: 165, 1988; Pena, et al., Nature, 325: 274,
1987). Other transformation methods include, for example,
protoplast transformation as illustrated in U.S. Pat. No.
5,508,184. The nucleic acids may also be injected into immature
embryos (Neuhaus, et al., Theor. Appl. Genet., 75: 30, 1987).
[0111] The most commonly used methods for transformation of plant
cells are the Agrobacterium-mediated DNA transfer process (Fraley
et al., Proc. Natl. Acad. Sci. U.S.A., 80: 4803, 1983) (as
illustrated in U.S. Pat. No. 5,824,877; U.S. Pat. No. 5,591,616;
U.S. Pat. No. 5,981,840; and U.S. Pat. No. 6,384,301) and the
biolistics or microprojectile bombardment mediated process (i.e.
the gene gun) (such as described in U.S. Pat. No. 5,015,580; U.S.
Pat. No. 5,550,318; U.S. Pat. No. 5,538,880; U.S. Pat. No.
6,160,208; U.S. Pat. No. 6,399,861; and U.S. Pat. No. 6,403,865).
Typically, nuclear transformation is desired but where it is
desirable to specifically transform plastids, such as chloroplasts
or amyloplasts, plant plastids may be transformed utilizing a
microprojectile mediated delivery of the desired polynucleotide for
certain plant species such as tobacco, Arabidopsis, potato and
Brassica species.
[0112] Agrobacterium-mediated transformation is achieved through
the use of a genetically engineered soil bacterium belonging to the
genus Agrobacterium. Several Agrobacterium species mediate the
transfer of a specific DNA known as "T-DNA", that can be
genetically engineered to carry any desired piece of DNA into many
plant species. The major events marking the process of T-DNA
mediated pathogenesis are: induction of virulence genes, processing
and transfer of T-DNA. This process is the subject of many reviews
(Ream, Ann. Rev. Phytopathol. 27: 583-618, 1989; Howard and
Citovsky, Bioassays, 12:103-108, 1990; Kado, Crit. Rev. Plant Sci.
10:1-32, 1991; Zambryski, Annual Rev. Plant Physiol. Plant Mol.
Biol., 43: 465-490, 1992; Gelvin, In Transgenic Plants, Kung and Wu
eds., Academic Press, San Diego, pp. 49-87, 1993; Binns and Howitz,
1994, In Bacterial Pathogenesis of Plants and Animals (Dang, ed.).
Berlin: Springer Verlag, pp. 119-138, 1994; Hooykaas and
Beijersbergen, Ann. Rev. Phytopathol. 32:157-179, 1994; Lessl and
Lanka, Cell 77:321-324, 1994; Zupan and Zambryski, Annual Rev.
Phytopathol. 27, 583-618, 1995).
[0113] With respect to microprojectile bombardment (U.S. Pat. No.
5,550,318; U.S. Pat. No. 5,538,880; U.S. Pat. No. 5,610,042; and
PCT Publication WO 95/06128; each of which is specifically
incorporated herein by reference in its entirety), particles are
coated with nucleic acids and delivered into cells by a propelling
force. Exemplary particles include those comprised of tungsten,
platinum, and preferably, gold. For the bombardment, cells in
suspension are concentrated on filters or solid culture medium.
Alternatively, immature embryos or other target cells may be
arranged on solid culture medium. The cells to be bombarded are
positioned at an appropriate distance below the microprojectile
stopping plate.
[0114] Microprojectile bombardment techniques are widely
applicable, and may be used to transform virtually any plant
species. Examples of species that have been transformed by
microprojectile bombardment include monocot species such as maize
(PCT Publication WO 95/06128), barley (Ritala et al., 1994;
Hensgens et al., 1993), wheat (U.S. Pat. No. 5,563,055,
specifically incorporated herein by reference in its entirety),
rice (Hensgens et al., 1993), oat (Torbet et al., 1995; Torbet et
al., 1998), rye (Hensgens et al., 1993), sugarcane (Bower et al.,
1992), and sorghum (Casa et al., 1993; Hagio et al., 1991); as well
as a number of dicots including tobacco (Tomes et al., 1990;
Buising and Benbow, 1994), soybean (U.S. Pat. No. 5,322,783,
specifically incorporated herein by reference in its entirety),
sunflower (Knittel et al. 1994), peanut (Singsit et al., 1997),
cotton (McCabe and Martinell, 1993), tomato (Van Eck et al. 1995),
and legumes in general (U.S. Pat. No. 5,563,055, specifically
incorporated herein by reference in its entirety).
[0115] To select or score for transformed plant cells regardless of
transformation methodology, the DNA introduced into the cell
contains a gene that functions in a regenerable plant tissue to
produce a compound that confers upon the plant tissue resistance to
an otherwise toxic compound. Genes of interest for use as a
selectable, screenable, or scorable marker will include but are not
limited to GUS, green fluorescent protein (GFP), luciferase (LUX),
antibiotic or herbicide tolerance genes. Examples of antibiotic
resistance genes include the penicillins, kanamycin (and neomycin,
G418, bleomycin); methotrexate (and trimethoprim); chloramphenicol;
kanamycin and tetracycline.
[0116] Particularly preferred selectable marker genes for use in
the present invention will include genes that confer resistance to
compounds such as antibiotics like kanamycin (nptII), hygromycin B
(aph IV) and gentamycin (aac3 and aacC4) (Dekeyser et al., Plant
Physiol., 90:217-223, 1989), and herbicides like glyphosate
(Della-Cioppa et al., Bio/Technology, 5:579-584, 1987). Other
selection devices can also be implemented including but not limited
to tolerance to phosphinothricin, bialaphos, and positive selection
mechanisms (Joersbo et al., Mol. Breed., 4:111-117, 1998) and are
considered within the scope of the present invention.
[0117] The transformed cells, identified by selection or screening
and cultured in an appropriate medium that supports regeneration,
will then be allowed to mature into plants.
[0118] The present invention can be used with any transformable
cell or tissue. By transformable as used herein is meant a cell or
tissue that is capable of further propagation to give rise to a
plant. Those of skill in the art recognize that a number of plant
cells or tissues are transformable in which after insertion of
exogenous DNA and appropriate culture conditions the plant cells or
tissues can form into a differentiated plant. Tissue suitable for
these purposes can include but is not limited to immature embryos,
scutellar tissue, suspension cell cultures, immature inflorescence,
shoot meristem, nodal explants, callus tissue, hypocotyl tissue,
cotyledons, roots, and leaves.
[0119] The regeneration, development, and cultivation of plants
from transformed plant protoplast or explants is well taught in the
art (Weissbach and Weissbach, Methods for Plant Molecular Biology,
(Eds.), Academic Press, Inc., San Diego, Calif., 1988; Horsch et
al., Science, 227: 1229-1231, 1985). This regeneration and growth
process typically includes the steps of selecting transformed cells
and culturing those individualized cells through the usual stages
of embryonic development through the rooted plantlet stage.
Transgenic embryos and seeds are similarly regenerated. In this
method, transformants are generally cultured in the presence of a
selective media which selects for the successfully transformed
cells and induces the regeneration of plant shoots (Fraley et al.,
Proc. Natl. Acad. Sci. U.S.A., 80: 4803, 1983). These shoots are
typically obtained within two to four months. The resulting
transgenic rooted shoots are thereafter planted in an appropriate
plant growth medium such as soil. Cells that survive the exposure
to the selective agent, or cells that have been scored positive in
a screening assay, may be cultured in media that supports
regeneration of plants. The shoots are then transferred to an
appropriate root-inducing medium containing the selective agent and
an antibiotic to prevent bacterial growth. Many of the shoots will
develop roots. These are then transplanted to soil or other media
to allow the continued development of roots. The method, as
outlined, will generally vary depending on the particular plant
strain employed.
[0120] The regenerated transgenic plants are self-pollinated to
provide homozygous transgenic plants. Alternatively, pollen
obtained from the regenerated transgenic plants may be crossed with
non-transgenic plants, preferably inbred lines of agronomically
important species. Conversely, pollen from non-transgenic plants
may be used to pollinate the regenerated transgenic plants.
[0121] The transgenic plant may pass along the transformed nucleic
acid sequence to its progeny. The transgenic plant is preferably
homozygous for the transformed nucleic acid sequence and transmits
that sequence to all of its offspring upon as a result of sexual
reproduction. Progeny may be grown from seeds produced by the
transgenic plant. These additional plants may then be
self-pollinated to generate a true breeding line of plants.
[0122] The progeny from these plants are evaluated, among other
things, for gene expression. The gene expression may be detected by
several common methods such as western blotting, northern blotting,
immunoprecipitation, and ELISA. The transformed plants are also
analyzed for the presence of the genes of interest and the
expression level and/or profile conferred by the non-coding
regulatory elements of the present invention. Those of skill in the
art are aware of the numerous methods available for the analysis of
transformed plants. For example, methods for plant analysis
include, but are not limited to Southern blots or northern blots,
PCR-based approaches, biochemical analyses, phenotypic screening
methods, field evaluations, and immunodiagnostic assays.
[0123] Methods for specifically transforming dicots are well known
to those skilled in the art. Transformation and plant regeneration
using these methods have been described for a number of crops
including, but not limited to, cotton (Gossypium hirsutum), soybean
(Glycine max), peanut (Arachis hypogaea), and members of the genus
Brassica. Methods for transforming dicots, primarily by use of
Agrobacterium tumefaciens and obtaining transgenic plants have been
published for cotton (U.S. Pat. No. 5,004,863; U.S. Pat. No.
5,159,135; U.S. Pat. No. 5,518,908); soybean (U.S. Pat. No.
5,569,834; U.S. Pat. No. 5,416,011; McCabe, et al., Biotechnolgy,
6: 923, 1988; Christou et al., Plant Physiol. 87:671-674 (1988));
Brassica (U.S. Pat. No. 5,463,174); peanut (Cheng et al., Plant
Cell Rep. 15:653-657 (1996), McKently et al., Plant Cell Rep.
14:699-703 (1995)); papaya; and pea (Grant et al., Plant Cell Rep.
15:254-258 (1995)).
[0124] Methods for transforming monocots are well known to those
skilled in the art. Transformation and plant regeneration using
these methods have been described for a number of crops including,
but not limited to, barley (Hordeum vulgarae); maize (Zea mays);
oats (Avena sativa); orchard grass (Dactylis glomerata); rice
(Oryza sativa, including indica and japonica varieties); sorghum
(Sorghum bicolor); sugar cane (Saccharum sp); tall fescue (Festuca
arundinacea); turfgrass species (e.g. species: Agrostis
stolonifera, Poa pratensis, Stenotaphrum secundatum); wheat
(Triticum aestivum), and alfalfa (Medicago sativa). It is apparent
to those of skill in the art that a number of transformation
methodologies can be used and modified for production of stable
transgenic plants from any number of target crops of interest.
[0125] It is apparent to those of skill in the art that a number of
transformation methodologies can be used and modified for
production of stable transgenic plants from any number of target
crops of interest.
Transgenic Plants and Transgenic Seeds
[0126] The seeds of this invention can be harvested from fertile
transgenic plants and be used to grow progeny generations of
transformed plants of this invention including hybrid plant lines
comprising the construct of this invention and expressing a gene of
agronomic interest.
[0127] The present invention also provides for parts of the plants
of the present invention. Plant parts, without limitation, include
seed, endosperm, ovule and pollen. In a particularly preferred
embodiment of the present invention, the plant part is a seed.
Utilization of Transgenic Plants
[0128] Still yet another aspect of the invention is a method of
inhibiting weed growth in a field of transgenic crop plants
comprising: (i) planting transgenic plants transformed with an
expression cassette comprising (a) a eukaryotic translation
initiation factor non-coding regulatory element polynucleotide
molecule active in the plant and operably linked to a DNA molecule
encoding a glyphosate resistant EPSPS and (ii) applying glyphosate
to the field at an application rate that inhibits the growth of
weeds, wherein the growth and yield of the transgenic crop plant is
not substantially affected by the glyphosate application. In
particular embodiments, the promoter is SEQ ID NO:1 or SEQ ID NO:2.
The glyphosate application rate is the effective rate necessary to
control weeds in a particular glyphosate tolerant crop, these rates
may range from 8 oz/A to 256 oz/A, preferably 16 oz/A to 128 oz/A,
more preferably 32 oz/A to 96 oz/A. The glyphosate is applied at
least once during the growth of the glyphosate tolerant crop and
may be applied 2, 3 or 4 times during the growth of the crop or
more as necessary to control weeds in the field. In particular
embodiments, the transgenic plants are capable of tolerating an
application rate up to 256 ounces/acre. In particular embodiments,
the transgenic plants are capable of tolerating an application rate
ranging from 8 ounces/acre to 128 ounces/acre. In particular
embodiments, the transgenic plants are capable of tolerating an
application rate ranging from 32 ounces/acre to 96 ounces/acre.
Other Transformed Organisms
[0129] Any of the above described promoters and transcribable
polynucleotide molecule sequences may be introduced into any cell
or organism such as algae cell, algae, fungal cell, fungi,
bacterial cell, or insect cell. Preferred hosts and transformants
include: fungal cells such as Aspergillus, yeasts, insects,
bacteria and algae.
[0130] The transformed cell or organism is preferably prokaryotic,
more preferably a bacterial cell, even more preferably a
Agrobacterium, Bacillus, Escherichia, Pseudomonas cell, and most
preferably is an Escherichia coli cell. Alternatively, the
transformed organism is preferably a yeast or fungal cell. The
yeast cell is preferably a Saccharomyces cerevisiae,
Schizosaccharomyces pombe, or Pichia pastoris.
[0131] Having now generally described the invention, the same will
be more readily understood through reference to the following
examples which are provided by way of illustration, and are not
intended to be limiting of the present invention, unless
specified.
[0132] Each document, patent, and reference cited herein is herein
incorporated by reference in its entirety.
[0133] The following examples are included to demonstrate preferred
embodiments of the invention. It should be appreciated by those of
skill in the art that the techniques disclosed in the examples that
follow represent techniques discovered by the inventors to function
well in the practice of the invention. However, those of skill in
the art should, in light of the present disclosure, appreciate that
many changes can be made in the specific embodiments that are
disclosed and still obtain a like or similar result without
departing from the spirit and scope of the invention, therefore all
matter set forth or shown in the accompanying drawings is to be
interpreted as illustrative and not in a limiting sense.
EXAMPLES
Example 1
Polynucleotide Molecule Identification and Cloning
[0134] Polynucleotide molecules were isolated from the eukaryotic
translation initiation factor eIF4A genes of Nicotiana tabacum,
Arabidopsis thaliana, and Medicago truncatula in order to identify
elements able to affect transgene expression in all tissues of a
dicot plant including the most sensitive reproductive organs such
as anthers and meristem tissues. All the polynucleotide molecules
were operably linked to the chloroplast transit peptide sequence,
TS-At.SHKG-CTP2; the coding sequence for the glyphosate tolerant
EPSPS gene isolated from Agrobacterium tumefaciens strain CP4,
Cr-AGRtu.aroA, (U.S. Pat. No. 5,633,435, herein incorporated by
reference); and the 3' nontranslated region of the ribulose
bisphosphate carboxylase gene from Pisum sativum, T-PS.RbcS2-E9 and
used for glyphosate tolerance characterization.
[0135] The Nicotiana tabacum eIF4A-10 gene promoter, leader, and
intron (referred to herein as P-Nt.eIF4A, L-Nt.eIF4A, and
I-Nt.eIF4A, respectively) were cloned by designing primers to the
publicly available genomic eIF4A-10 gene sequence available as
GI:475215 Accession number X79008 through NCBI. Genomic DNA from
Nicotiana tabacum was isolated and used to amplify the Nt.eIF4A-10
promoter, leader, and intron using PCR and the NteIF4A forward and
reverse primers (provided as SEQ ID NO: 12 and 13, respectively).
The sequence for the promoter P-Nt.eIF4A is provided as SEQ ID NO:
1; the sequence for the leader L-Nt.eIF4A is provided as SEQ ID NO:
2; the sequence for the intron I-Nt.eIF4A is provided as SEQ ID NO:
3. PCR products were then digested with SmaI and NcoI and cloned
into a plasmid (pMON70501) digested with the same restriction
enzymes to operably link the promoter, leader, and intron to the
AROA gene (see for example PCT publication WO 2004/009761-A2,
herein incorporated by reference). The subsequent construct
(pMON70500) was screened by restriction enzymes digestion and
sequencing to confirm that the correct sequence was present. A
double cassette construct containing a second expression cassette
was constructed by digesting pMON70500 with XmaI and sub-cloning in
a cassette containing the 7Sa' promoter operably linked to the GUS
transgene (from pMON13773 cut with NgoMIV and XmaI). The subsequent
construct (pMON65396) was screened by restriction enzymes
digestion.
[0136] The Arabidopsis thaliana eIF-4A1 gene promoter, leader, and
intron (referred to herein as P-At.eIF4A, L-At.eIF4A, and
I-At.eIF4A, respectively) were cloned by designing primers to the
publicly available genomic eIF-4A1 gene sequence available as
GI:14594801 Accession number AJ298137 through NCBI. Genomic DNA
from Arabidopsis thaliana was isolated and used to amplify the
At.eIF4A1 promoter, leader, and intron using PCR and the AteIF4A
forward and reverse primers (provided as SEQ ID NO: 14 and 15,
respectively). The sequence for the promoter P-At.eIF4A is provided
as SEQ ID NO: 4; the sequence for the leader L-At.eIF4A is provided
as SEQ ID NO: 5; the sequence for the intron I-At.eIF4A is provided
as SEQ ID NO: 6. PCR products were then digested with NotI and NcoI
and cloned into a plasmid (pMON65322) digested with the same
restriction enzymes to operably link the promoter, leader, and
intron to the AROA gene. The subsequent construct (pMON65395) was
screened by restriction enzymes digestion and sequencing to confirm
that the correct sequence was present.
[0137] The Medicago truncatula eIF-4A1 gene promoter, leader, and
intron were identified by first identifying the best putative
homologous gene in Medicago truncatula. Analysis was done using
homology based sequence searches and phylogenetic reconstructions.
The predicted protein sequences for known eIF-4A1 genes from
Nicotiana tabacum, Zea mays, Nicotinia plumbaginifolia, and
Arabidopsis thaliana were used to identify the best putative
orthologous predicted protein sequence derived from Medicago
truncatula genomic DNA. The genomic DNA sequence of this putative
ortholog was then used to predict the gene's promoter, leader, and
intron sequence for subsequent cloning.
[0138] The promoter, leader, and intron (referred to herein as
P-Mt.eIF4A, L-Mt.eIF4A, and I-Mt.eIF4A, respectively) from the
Medicago truncatula eIF-4A1 gene were cloned by designing primers
to the identified genomic DNA sequence of interest. Genomic DNA
from Medicago truncatula was isolated and used to amplify the
Mt.eIF4A promoter, leader, and intron using PCR and three sets of
MteIF4A forward and reverse primers: MtEIF4AF1 (SEQ ID NO: 16) and
MtEIF4AR (SEQ ID NO: 19); MtEIF4AF2 (SEQ ID NO: 17) and MtEIF4AR;
MtEIF4AF3 (SEQ ID NO: 18) and MtEIF4AR. Three variants of the
promoter were thus created by truncating the promoter at the 5' end
resulting in a 1941 bp promoter (referred to herein as P-Mt.eIF4A-1
and provided as SEQ ID NO: 7), a 1484 bp promoter (referred to
herein as P-Mt.eIF4A-2 and provided as SEQ ID NO: 8), and a 1029 bp
promoter (referred to herein as P-Mt.eIF4A-3 and provided as SEQ ID
NO: 9). The three promoter variants were tested with the same
leader and intron in order to directly compare their respective
expression patterns. The sequence for the leader (referred to
herein as L-Mt.eIF4A) is provided as SEQ ID NO: 10. The sequence
for the intron (referred to herein as I-Mt.eIF4A) is provided as
SEQ ID NO: 11. PCR products were then digested with NotI and PciI
and cloned into a plasmid (pMON65322) digested with NotI and NcoI
to operably link the promoter, leader, and intron to the AROA gene.
Three subsequent constructs (pMON81504, pMON81505 and pMON81506)
were screened by restriction enzymes digestion and sequencing to
confirm that the correct sequence was present. Sequences and
constructs referred to herein are summarized in Table 1 and 2,
respectively.
TABLE-US-00001 TABLE 1 Sequences Element name SEQ ID NO P-Nt.eIF4A
1 L-Nt.eIF4A 2 I-Nt.eIF4A 3 P-At.eIF4A 4 L-At.eIF4A 5 I-At.eIF4A 6
P-Mt.eIF4A-1 7 L-Mt.eIF4A 10 I-Mt.eIF4A 11 P-Mt.eIF4A-2 8
L-Mt.eIF4A 10 I-Mt.eIF4A 11 P-Mt.eIF4A-3 9 L-Mt.eIF4A 10 I-Mt.eIF4A
11
TABLE-US-00002 TABLE 2 Constructs Construct Regulatory Elements
FIG. pMON70500 P-Nt.eIF4A/L-Nt.eIF4A/I-Nt.eIF4A/aroA 1 pMON65395
P-At.eIF4A/L-At.eIF4A/I-At.eIF4A/aroA 2 pMON81504
P-Mt.eIF4A-1/L-Mt.eIF4A/I-Mt.eIF4A/aroA 3 pMON81505
P-Mt.eIF4A-2/L-Mt.eIF4A/I-Mt.eIF4A/aroA 4 pMON81506
P-Mt.eIF4A-3/L-Mt.eIF4A/I-Mt.eIF4A/aroA 5 pMON65396
P-Nt.eIF4A/L-Nt.eIF4A/I-Nt.eIF4A/aroA and -- P-Gm.7S'/GUS pMON73663
P-Gm.7S'/GUS and P-Nt.eIF4A/L-Nt.eIF4A/ -- I-Nt.eIF4A/aroA
pMON73662 P-At.eIF4A/L-At.eIF4A/I-At.eIF4A/aroA and P- --
Gm.7S'/GUS pMON81509 P-Mt.eIF4A-1/L-Mt.eIF4A/I-Mt.eIF4A/aroA and P-
-- Gm.7S'/GUS pMON81510 P-Mt.eIF4A-2/L-Mt.eIF4A/I-Mt.eIF4A/aroA and
P- -- Gm.7S'/GUS pMON8151 P-Mt.eIF4A-3/L-Mt.eIF4A/I-Mt.eIF4A/aroA
and P- -- Gm.7S'/GUS pMON26140 P-FMV/L-Ph.DnaK/aroA -- pMON20999
P-FMV/L-Ph.DnaK/aroA --
Example 2
Promoter Characterization in Arabidopsis thaliana
[0139] Each gene of interest may be amplified from a genomic or
cDNA library using primers specific to sequences upstream and
downstream of the coding region. Transformation vectors are
prepared to constitutively transcribe DNA in either sense
orientation (for enhanced protein expression) or anti-sense
orientation (for endogenous gene suppression) under the control of
an enhanced Cauliflower Mosaic Virus 35S promoter (U.S. Pat. No.
5,359,142) directly or indirectly (Moore et al. PNAS 95:376-381,
1998; Guyer et al. Genetics 149: 633-639, 1998; International
patent application NO. PCT/EP98/07577). The transformation vectors
also contain a bar gene as a selectable marker for resistance to
glufosinate herbicide.
[0140] The following abbreviations are used to describe the plants
and seeds produced:
[0141] R.sub.0=the original transgenic plant that was grown from a
single transformed plant cell
[0142] R.sub.1=the seed or plant progeny of the R.sub.0 plant
[0143] R.sub.2=the seed or plant progeny of the R.sub.1 plant
[0144] The transformation of Arabidopsis plants is carried out
using the vacuum infiltration method known in the art (Bethtold et
al. Methods Mol. Biol. 82:259-66, 1998). Seeds harvested from the
plants, named as R.sub.1 seeds, are subsequently grown in a
glufosinate-containing selective medium to select for plants which
are actually transformed and which produced R.sub.2 transgenic
seed.
[0145] A group of promoters were initially isolated and tested in
Arabidopsis thaliana for their dicot expression pattern. Selected
constructs including pMON70500 (containing P-Nt.eIF4A operably
linked to the aroA transgene) and pMON65395 (containing P-At.eIF4A
operably linked to the aroA transgene) were used to transform
Arabidopsis thaliana ecotype Columbia by the floral dip method (N
Bechtold et al. (1993) CR Acad Sci Paris Sciences di la vie/life
sciences 316: 1194-1199 and S Clough et al. (1998) Plant Journal
16(6): 735-743). Analysis of glyphosate herbicide tolerance in
transgenic Arabidopsis was used as an initial screen to identify
promising promoters for subsequent analysis in other dicots.
[0146] Seeds from R.sub.0 plants were harvested. Seeds from each
cassette construct were sterilized and placed on 50 mM glyphosate
agar media for selection of transformants. R.sub.1 seedlings that
germinated on glyphosate-containing agar were transferred to soil
after 7 days.
[0147] Events from each construct were then analyzed for vegetative
and reproductive tolerance to glyphosate. Plants transformed with
pMON26140 (containing P-FMV operably linked to the aroA transgene)
were used for comparison. This construct has been shown to provide
100% vegetative tolerance to glyphosate, but no reproductive
tolerance, i.e. the glyphosate treated plants are not fertile.
After three weeks plants in the rosette stage were sprayed with 24
ounces/acre (oz/A) or 128 ounces/acre of Roundup.RTM. Ultra
herbicide. Each glyphosate treatment was done on at least 20
separate transgenic events per construct. Plants with vegetative
damage were discarded. Plants treated with glyphosate that were
morphologically similar to non-treated plants and which also formed
siliques filled with seeds were considered tolerant. Data are
provided in Table 3 below. Measurements are provided as the
percentage of fertile glyphosate tolerant events out of the total
events analyzed for each construct at each glyphosate dosage.
TABLE-US-00003 TABLE 3 Glyphosate tolerance in Arabidopsis Fertile
Tolerant Events Fertile Tolerant Events Construct (% of events at
24 oz/A) (% of events at 128 oz/A) pMON26140 0% 0% pMON65395 85%
85% pMON70500 48% 34%
[0148] From the initial group of promoters that were tested in
Arabidopsis, the eIF4A-10 gene promoter, leader, and intron from
Nicotiana tabacum (pMON70500) and the eIF-4A1 gene promoter,
leader, and intron from Arabidopsis thaliana (pMON65395) were
selected for further investigation. These were found to perform
well in transformed Arabidopsis to provide glyphosate tolerance in
both vegetative and reproductive tissues when compared to the
control construct pMON26140.
[0149] Regulatory elements from the homologous gene in Medicago
truncatula were tested in Arabidopsis. pMON81504 (containing
P-Mt.eIF4A-1 operably linked to the aroA transgene), pMON81505
(containing P-Mt.eIF4A-2 operably linked to the aroA transgene),
and pMON81506 (containing P-Mt.eIF4A-3 operably linked to the aroA
transgene) were used to transform Arabidopsis thaliana by the
floral dip method for in planta characterization of glyphosate
tolerance. Plant transformation and glyphosate analysis were done
as described above. Data are provided in Table 4 below.
Measurements are provided as the percentage of fertile glyphosate
tolerant events out of the total events analyzed for each
construct.
TABLE-US-00004 TABLE 4 Glyphosate tolerance in Arabidopsis Tolerant
Events Tolerant Events Construct (% of events at 24 oz/A) (% of
events at 128 oz/A) pMON26140 0% 0% pMON81504 33% 30% pMON81505 32%
13% pMON81506 14% 10%
[0150] All three constructs were found to perform well in
transformed Arabidopsis to provide glyphosate tolerance in both
vegetative and reproductive tissues when compared to the control
construct pMON26140.
Example 3
Promoter Characterization in Nicotiana tabacum
[0151] pMON70500, pMON26140, pMON73663 (a double cassette construct
containing P-Gm.7S' operably linked to the GUS transgene and
P-Nt.eIF4A operably linked to the aroA transgene), pMON65396 (a
double cassette construct containing P-Nt.eIF4A operably linked to
the aroA transgene and P-Gm.7S' operably linked to the GUS
transgene), pMON73662 (a double cassette containing P-At.eIF4A
operably linked to the aroA transgene and P-Gm.7S' operably linked
to the GUS transgene), pMON81509 (a double cassette construct
containing P-Mt.eIF4A-1 operably linked to the aroA transgene and
P-Gm.7S' operably linked to the GUS transgene), pMON81510 (a double
cassette construct containing P-Mt.eIF4A-2 operably linked to the
aroA transgene and P-Gm.7S' operably linked to the GUS transgene),
and pMON81511 (a double cassette containing P-Mt.eIF4A-3 operably
linked to the aroA transgene and P-Gm.7S' operably linked to the
GUS transgene) were used to transform Nicotiana tabacum cv.
Nicotiana samsun by using the leaf disc method (R B Horsch, et al.
(1985) Science 227:1229-1231 and R B Horsch, et al. (1987) Plant
Tissue and Cell Culture pp. 317-329, Alan R. Liss, Inc.). Tobacco
shoots were rooted in MS media (R.sub.0 plants) and then
transferred to soil. Tobacco plants were analyzed for glyphosate
tolerance at the R.sub.0 stage. After 38 days of growth in soil 30
to 35 plants per construct were sprayed with 96 ounces/acre
Roundup.RTM. Ultra herbicide. Plants were scored for vegetative
tolerance to glyphosate and for fertility. Plants with vegetative
damage were discarded. Plants treated with glyphosate that were
morphologically similar to non-treated plants and which also
produced seeds were considered tolerant.
[0152] Transformation efficiency was measured and plants were
analyzed for the percentage of events having reproductive tissue
tolerance to glyphosate at 96 ounces/acre of Roundup.RTM. Ultra
herbicide. Data are provided in Table 5 below.
TABLE-US-00005 TABLE 5 Glyphosate tolerance in Tobacco Fertile
Tolerant Events Construct (% of events at 96 oz/A) pMON70500 28%
pMON65396 40% pMON73663 7% pMON73662 70% pMON26140 0% pMON81509 18%
pMON81510 9% pMON26140 0%
Example 4
Promoter Characterization in Glycine max
[0153] This example illustrates plant transformation useful in
producing the transgenic soybean plants with constructs containing
eIF-NCRE molecules of this invention, and the resultant production
and identification of transgenic seed for transgenic soybean having
an improved agronomic trait, i.e. improved nitrogen use efficiency,
improved yield, improved water use efficiency and/or improved
growth under cold stress as compared to control plants.
[0154] For Agrobacterium-mediated transformation, soybean seeds are
germinated overnight and the meristem explants excised. The
meristems and the explants are placed in a wounding vessel. Soybean
explants and induced Agrobacterium cells from a strain containing
construct plasmid DNA with the gene of interest cassette and a
plant selectable marker cassette are mixed no later than 14 hours
from the time of initiation of seed germination and wounded using
sonication. Following wounding, explants are placed in co-culture
for 2-5 days at which point they are transferred to selection media
for 6-8 weeks to allow selection and growth of transgenic shoots.
Trait positive shoots are harvested approximately 6-8 weeks post
bombardment and placed into selective rooting media for 2-3 weeks.
Shoots producing roots are transferred to the greenhouse and potted
in soil. Shoots that remain healthy on selection, but do not
produce roots are transferred to non-selective rooting media for an
additional two weeks. Roots from any shoots that produce roots off
selection are tested for expression of the plant selectable marker
before they are transferred to the greenhouse and potted in
soil.
[0155] pMON70500 and pMON20999 (containing P-FMV operably linked to
the aroA transgene) were used to transform Glycine max by an
Agrobacterium mediated method (see U.S. Pat. No. 6,384,301, herein
incorporated by reference). Transformation efficiency was measured
and then R.sub.0 plants were selfed and advanced to the R.sub.1
generation. R.sub.1 plants were tested for vegetative tolerance to
glyphosate. For the R.sub.1 evaluations, typically 48 seeds per
event were planted and an ELISA analysis was performed to identify
the positive transformants and to determine the segregation ratio.
Plants were sprayed with 52 ounces/acre of Roundup UltraMax.RTM.
herbicide at the V1 stage. Approximately one week post-spray the
events were evaluated for chlorosis. Copy number and zygosity were
also assessed. Other observations taken included: emergence,
segregation, pod set (timing of), plant height, and maturity. Those
events that had one copy of the transgene, no vector backbone, and
vegetative glyphosate tolerance were advanced to the R.sub.2
nursery to evaluate reproductive tolerance. R.sub.2 plants were
tested for reproductive tolerance to glyphosate. Plants treated
with glyphosate that were morphologically similar to non-treated
plants and which also produced seeds were considered tolerant. Data
are provided in Table 6 below.
TABLE-US-00006 TABLE 6 Glyphosate tolerance in Soybean R.sub.1
Events with Fertile Tolerant Events Construct Vegetative Tolerance
(% of total events) pMON70500 37% 4% pMON20999 55% 0%
[0156] Events containing the aroA transgene operably linked to the
P-Nt.eIF4A promoter, leader, and intron showed acceptable
transformation efficiency as well as good vegetative and
reproductive tolerance to glyphosate in soybean when compared to
the control. Four percent of the total events generated were found
to have vegetative and reproductive tolerance in soybean to
glyphosate in the R.sub.2 generation.
Example 5
Promoter Characterization in Gossypium hirsutum
[0157] Construct pMON70500 was used to transform Gossypium hirsutum
by Agrobacterium-mediated transformation methods (see PCT
publication WO 00/36911, herein incorporated by reference).
[0158] First, R.sub.0 plants were selfed to produce R.sub.1 seed.
R.sub.1 seeds of this construct were planted in the greenhouse for
multiple events. Glyphosate tolerance was analyzed using Roundup
UltraMax.RTM. herbicide at a rate of 48 ounces/acre per
application. Four applications were made at the 3, 6, 10, and 14
node stages respectively for a total of 192 ounces/acre of Roundup
UltraMax.RTM. herbicide or 5.6 pounds of the acid equivalent of
glyphosate per acre on each event. Events that demonstrated good
vegetative tolerance (and segregated 3:1 according to Mendelian
genetics) were advanced. Once the plants matured, they were mapped
by recording boll retention at the first and second positions of
the first five fruiting branches (highest possible score is 10).
Plant mapping is a schematic or map of the plant noting where the
fruit (boll) is being held or lost. It provides a good overall
picture of how the plant is responding to stressors such as insect
pressure, glyphosate sprays, or other stressors that may impact the
ability of the plant to retain bolls, see for example U.S. Pat. No.
6,660,911, herein incorporated by reference. In this study the
current commercial event mapped at 0 (n=4). Seven of the 9 events
tested mapped slightly better than the current commercial event.
Two of the events, Gh_S18179 and Gh_S18055, mapped 7.1 and 2.7
respectively (n=10), both far better than the current commercial
event.
[0159] A second set of experiments was conducted, in which thirty
R.sub.1 seeds were planted in the greenhouse for multiple events.
Glyphosate tolerance was analyzed using Roundup UltraMax.RTM.
herbicide at a rate of 48 ounces/acre per application. Four
applications were made at the 4, 8, 10 and 12-14 leaf stages. This
was designed to give the plants a good dose of glyphosate at those
growth stages we had identified as being stages or timings of
higher plant sensitivity. As the effort progressed and outstanding
tolerance from some of the events being tested was observed, the 48
oz/A rate was raised to 64 oz/A. Three experiments were performed
with events from pMON70500. In total, 36 cotton events were
screened for glyphosate tolerance from this Nelf/CP4 construct in
cotton. Reproductive tolerance was based on boll retention. Two of
these 36 events retained >50% of the first position bolls, which
corresponds to a higher rate than the current commercial event.
[0160] Having illustrated and described the principles of the
present invention, it should be apparent to persons skilled in the
art that the invention can be modified in arrangement and detail
without departing from such principles. We claim all modifications
that are within the spirit and scope of the appended claims. All
publications and published patent documents cited in this
specification are incorporated herein by reference to the same
extent as if each individual publication or patent application is
specifically and individually indicated to be incorporated by
reference.
Sequence CWU 1
1
1911054DNANicotiana tabacum 1atcatgtata tttgtgcata tccatgaaaa
tttgtgttat atatacgata tataatgtga 60tacacatagg cgtccataaa agaattgtgt
tgtatacacg atatacaaag tgatatacag 120atgtccttaa aaatatgtgt
gtgatataca ttgatgtaca caatatgcaa cgcgatatac 180acatgtcaca
gttggatttt aggtctgatg ttttacatga aatcagtcta aatcacttct
240aatcttgctc aaattttgta tatagccccg tttaggtatt ttcaaccaat
ttcactcaca 300ccactcgttc aatctaacca aaaaaaagaa gagagaagaa
aaacaaagtt gaaatgaatt 360tttctctctt agtttttgct tataattttt
ctgattacct tttcacccca ctgatttttt 420ttgcataatt tgcaaggatt
tttgctaaac tatgagagcg aaagaaaaga gatagaagaa 480gaaatacaag
gagagaaagg gggagggacg cagtgaacaa aaaaagaagt tagcggcgaa
540gagggggggg ggggggaagc agacggtttg gggccaattg tttgagagag
aatatataag 600agagtagttt ttttaggatt tggctatata atgtcaattt
tttggggcta tcttttccta 660acctaatata agactaaaaa attgtcaatt
cctgttatgt gttatcacct ggtgccattt 720tctcatagtt atacatatag
tgaaaggaaa agagggtatt agtgccaatt ttgtaaagag 780gttagaccta
aattaggccc aagaggccca atagaaaatc tagccctcaa ttttgtggaa
840tccacgtcac cgacttcttg cattaccacc cgaagcggct ccgtattgat
cctgtaactc 900ccaatttcgg gtcaaaatag gaatttcaaa tacagaagcc
aaaaaaaaaa ggaaagtaat 960ccaaaacagt attcagaaag accataaaaa
aacactagtc tcaatctttc tcttttcctc 1020tttcctgaac tcctgcggcg
tagatccgag gagt 1054294DNANicotiana tabacum 2ttcagacaaa accctaaccc
cccaatcgct tttacccact tttcatttca ctctctcttt 60cctattcctc agatcttttc
ctcatctcta tcag 943892DNANicotiana tabacum 3gttagctatg ttttttttcc
ctttaatatt ttaatgtatt tcttgtaata tttgtttgtg 60tattgaagat tgaatcttga
tgattgattg ttggtctgac tacagctggg ttttgtgtta 120tgtaactatt
tttaactatt ttggatagag gtctgtttga tgtgatgttc ttgattataa
180aaataccatc ctactttgtt atctcatatc tggttggaac atgagcaatt
tcatttctcc 240tagttcttga attaaaaacc tgaaagtatt gtgcaaaaag
atgctaggaa tgagactatc 300attgttttga tgcaatatgt tcttttaagt
aataggtgtt ttgtaagaag tctacgcagt 360tctggatgta ttttactact
cgggaaaact ggatagttgg atacttatta tgtataggaa 420gtaaatgtgg
ggattataat gcctttctct gccatctgct ctttgtattt tgtgtaaagc
480ttggcatgcc tctcgtcaga tagccatcgc taccgtacat tcttttaaga
atgaagcact 540tagacacttg ctcgtttctg cctttgtcac attgacccag
catcatataa tctgaaagat 600tggttagcag ttggctgcta tttaacttgt
atgttaaaac aattgatttt catgtgtatc 660tcctcctttt gtgctttgtg
cttcttcata aaagaaagaa aacatacatt cggttgtgct 720ctcctccttt
ttcaatggta gagaggaaga acagataatt ttattgctgc tgtaggtatt
780tgacatctgt gatattttca tagtaaggtt ttgttttttc ctttttattt
agttcaagat 840tgtttcatga atttccataa gcgtaatacc atagttcttt
tatttgctac ag 8924918DNAArabidopsis thaliana 4gcttcacaaa tttcggtttg
tactttatta tattgggccc tattattatt ctctgagctt 60ttgcgtctca ccaaaagaca
gagatcatca ggtgcgttag tgattacgta ggcatctaat 120gaacggcagg
gattagtcaa acttattaat gggcctaatc tttggcccat cgttttccct
180cgattcctgt cacacaaaaa actcctagct cttcctctac ctacacaagc
taaatacata 240ttttttgctt atcctaagca tcatgattat gttttgccct
ctccagcttt ttcttcaatg 300gcaacagatt ctaagaaagt ctcttgaggc
taaaatcaaa gcattttttg ttgaagatag 360atagcaactt gctgcttctt
catactagct agttaccttc ttcttactca atgtgttttg 420cttcgtttca
aggattcttc atatcacttg tggaacaatt acatgattaa acattcaaca
480tagagagata gatgtgttaa taagtaaaga cattttcaga taaaacgttc
ttatcagtca 540ctttattctt ctaatatcct cgttgtaatc gggaaaatgc
tttgtaacgt caaaaagcaa 600taaaagttgc aggagaaaga aagctttgga
aaagaaaata aaataaatga agaatatttt 660ctttgctagt cacaaaataa
atgaagaatt tatgttccta atttcccact agatatttgt 720ttatttattt
ttgccaaaat caagttaaga caatgagcta agtgttggaa aaccttgtcc
780gagccaaaag agtaaaaaga aagggaataa aggggtaaaa ccggaaatcc
gaaaaagaaa 840aggagaagat ttccaaagga gaaaacccta aagacggagt
atataaacaa ggtaacgcgt 900tttctctcag cctctttc 9185135DNAArabidopsis
thaliana 5ggatattcca ccagtctctc gcaatcttcg ctcttctctt tgctctctct
ctctcaacgc 60ggttcagatc cgagtttggg agattcaagc tccctgaaaa aagcccttta
ctctcgctta 120ccaactttgc tccag 1356647DNAArabidopsis thaliana
6gttcgcatcc tatcggattg tctatctatt atctcgatgt aacttttacg attcatgatt
60gtttctttac ttttggattg ttctgatttg tttttttgtg ttttccgata gatttggttt
120caactttgtt gagttgattt ggaagttttg atttttcgtt acttcaagat
cgtttttttt 180gctgtggaag tgctttcgtt tttgttctct ttattagatt
ctgagctgct gatgatgacg 240attattaaat ttaggatcta cacatctgta
gatttgttga tgggtttgta gattttgtta 300cggctaggtt agtctctcaa
tagaatgaaa tgaatcgtct tcaaagctca gatgttcttg 360tcttaacact
cacttggttt tgtctgattg aatgtttttt tttgtaattt ggagaaatag
420gttgagtggt gtctatgttt aatagataac catatgtgcc tttatacatt
ttctccgcac 480atcatctgag gcagatatta atgcttgttt tcctctcttt
atggacttcg agtttggctt 540tcttgtcaca tttcctcctt tgcaatcata
ttattaacga tatcaaaata ggtgttgtct 600ctcttccttt tgatatgaaa
tctgattgtt ttgttctttt tatgtag 64771941DNAMedicago truncatula
7cgtcttgtgt gatatctaac atccactttg ggtgcgtttg ttgtagattt caacaaaata
60gattttatgt tagaaaaatt agagattctt gtttagataa tttgaaaaaa ttttgaagct
120atttgtgttt ttttattatc caaaaaaatc gcttttttga aaaaaatgat
ttttaaatag 180tttatgtctt tcccccttat gtttaaaaaa atagattttg
agaaaagcta atcctaatag 240ttttttattt tagagtcaaa atattttttt
ttttgataaa gtaaatcttt ttaaaatcct 300aaacaaacac tcaaaactga
caagtaattt tttttaataa aaaatctaaa acaaatgtgc 360ctttatgtca
ccgtaaaaga caaaggaatt gttctcgtga gcttagttca gttggtatgg
420acaatgcata aaatatgcca gatccatggt tcaattacat tgttgtcctt
gttagccgat 480cttagttttt atagtgagaa aagcttcaat tttttttttt
aaattctctt tttctttccg 540tcattgagga atggatgatt ttaggttatt
tttgacttaa aatcattttt atgcgtcttt 600ttctcatttc tttcgatcta
aataagtgat ttttacatat atcaagtttt ttcttgtgtt 660tttcataaca
gatcaacaca gcaacacagg aggtcaaaaa atagtcccac caccaaacta
720atcattatat aataaggata ttttagaagg atgactcacg tggatcaaaa
tttgagttaa 780atagtttaac ttcagtccat tatgttctcc tgaaattcca
aataaaaaga ttaagtattt 840atcccatcag ttgaaaattg ataaaaacaa
ccaattagtt taccaaggct aaagtatgag 900ctattgaacc ccgccaaagc
taatctagtt agacaaatca acaaattcca atttacaaca 960caaccctaat
gcctccctta taaaaagtca ccccaatttt acacttcaat agaaaatgac
1020ccaatacttg gaaccaaaac acctccattg aatgagccaa tacttcaata
gaagaaagtt 1080atgtaactga aatagaataa ttaactcata aaattaatgt
aaaaccaatt caagttttct 1140acatttgaca ccttttgtac aaaatggtaa
aaccaaattg gaaagtgaca acatgaaaaa 1200tataatttga tcacatattc
ttccaataat aaaggtgatg gactgacatc attttcaaat 1260ctctctctaa
agttctagta aatgtcagtt agtaaactaa cctctgtttt tcaaatcttt
1320ctccaaaata ctcaagtgtc aatcactaaa taaaaaccaa agattttcct
acaaacatta 1380tagccaacct aaaacgaagt ataaattata cttggacaca
aacagagtgc aaaagataaa 1440ttagacaaag atccatttaa attaatgtgc
tgaataatta tgaagcaaga tatcaaaatc 1500aaattagata aaagtgtgag
tggtgaattt ctcacagttt ataaaatcat caaactcgaa 1560agcctagttt
tatataagct aaggacgatt cttaaatacc aaaaaaataa ataaaaacca
1620agaaagatta agaaatattt tctctgagtt ttacactgtt attaaggata
tttggatccg 1680atgataaaag ttttcattca acttaattat aggtccttga
tacgaatcca actttacatt 1740atgataaatc ttttaaagaa tagttttacc
atcgattatt gttccacccg gttcaagaat 1800tttagaggat actcagataa
aataaaataa aaataaaata tgaaactatt ggtattttag 1860agattaaaac
taaattttgt gttttttggg ggtgtaatca gcaaatccta atcctagtag
1920tagtaaaaat acatacgcta c 194181484DNAMedicago truncatula
8cattgttgtc cttgttagcc gatcttagtt tttatagtga gaaaagcttc aatttttttt
60tttaaattct ctttttcttt ccgtcattga ggaatggatg attttaggtt atttttgact
120taaaatcatt tttatgcgtc tttttctcat ttctttcgat ctaaataagt
gatttttaca 180tatatcaagt tttttcttgt gtttttcata acagatcaac
acagcaacac aggaggtcaa 240aaaatagtcc caccaccaaa ctaatcattg
tataataagg atattttaga aggatgactc 300acgtggatca aaatttgagt
taaatagttt aacttcagtc cattatgttc tcctgaaatt 360ccaaataaaa
agattaagta tttatcccat cagttgaaaa ttgataaaaa caaccaatta
420gtttaccaag gctaaagtat gagctattga accccgccaa agctaatcta
gttagacaaa 480tcaacaaatt ccaatttaca acacaaccct aatgcctccc
ttataaaaag tcaccccaat 540tttacacttc aatagaaaat gacccaatac
ttggaaccaa aacacctcca ttgaatgagc 600caatacttca atagaagaaa
gttatgtaac tgaaatagaa taattaactc ataaaattaa 660tgtaaaacca
attcaagttt tctacatttg acaccttttg tacaaaatgg taaaaccaaa
720ttggaaagtg acaacatgaa aaatataatt tgatcacata ttcttccaat
aataaaggtg 780atggactgac atcattttca aatctctctc taaagttcta
gtaaatgtca gttagtaaac 840taacctctgt ttttcaaatc tttctccaaa
atactcaagt gtcaatcact aaataaaaac 900caaagatttt cctacaaaca
ttatagccaa cctaaaacga agtataaatt atacttggac 960acaaacagag
tgcaaaagat aaattagaca aagatccatt taaattaatg tgctgaataa
1020ttatgaagca agatatcaaa atcaaattag ataaaagtgt gagtggtgaa
tttctcacag 1080tttataaaat catcaaactc gaaagcctag ttttatataa
gctaaggacg attcttaaat 1140accaaaaaaa taaataaaaa ccaagaaaga
ttaagaaata ttttctctga gttttacact 1200gttattaagg atatttggat
ccgatgataa aagttttcat tcaacttaat tataggtcct 1260tgatacgaat
ccaactttac attatgataa atcttttaaa gaatagtttt accatcgatt
1320attgttccac ccggttcaag aattttaggg gatactcaga taaaataaaa
taaaaataaa 1380atatgaaact attggtattt tagagattaa aactaaattt
tgtgtttttt gggggtgtaa 1440tcagcaaatc ctaatcctag tagtagtaaa
aatacatacg ctac 148491029DNAMedicago truncatula 9gccaaagcta
atctagttag acaaatcaac aaattccaat ttacaacaca accctaatgc 60ctcccttata
aaaagtcacc ccaattttac acttcaatag aaaatgaccc aatacttgga
120accaaaacac ctccattgaa tgagccaata cttcaataga agaaagttat
gtaactgaaa 180tagaataatt aactcataaa attaatgtaa aaccaattca
agttttctac atttgacacc 240ttttgtacaa aatggtaaaa ccaaattgga
aagtgacaac atgaaaaata taatttgatc 300acatattctt ccaataataa
aggtgatgga ctgacatcat tttcaaatct ctctctaaag 360ttctagtaaa
tgtcagttag taaactaacc tctgtttttc aaatctttct ccaaaatact
420caagtgtcaa tcactaaata aaaaccaaag attttcctac aaacattata
gccaacctaa 480aacgaagtat aaattatact tggacacaaa cagagtgcaa
aagataaatt aggcaaagat 540ccatttaaat taatgtgctg aataattatg
aagcaagata tcaaaatcaa attagataaa 600agtgtgagtg gtgaatttct
cacagtttat aaaatcatca aactcgaaag cctagtttta 660tataagctaa
ggacgattct taaataccaa aaaaataaat aaaaaccaag aaagattaag
720aaatattttc tctgagtttt acactgttat taaggatatt tggatccgat
gataaaagtt 780ttcattcaac ttaattatag gtccttgata cgaatccaac
tttacattat gataaatctt 840ttaaagaata gttttaccat cgattattgt
tccacccggt tcaagaattt tagaggatac 900tcagataaaa taaaataaaa
ataaaatatg aaactattgg tattttagag attaaaacta 960aattttgtgt
tttttggggg tgtaatcagc aaatcctaat cctagtagta gtaaaaatac
1020atacgctac 10291099DNAMedicago truncatula 10aaataaaaag
cacgcatata taacctaaat ctctctctca ctcttccctt caactccacg 60gcgcagatcc
gaggagtttg ggaaacccta attttcaat 9911647DNAMedicago truncatula
11gtatacgtat ctcaattctc aatttctccc aatttttcat tcttttagat tttaatctgc
60ttttttacct ttaaattatg aactttttat tttgaatcta agtttgatct gattgctttt
120ttattaataa tatattaaag ttctgatctt tgaaatcaat tatgattaac
aaaaaaaaag 180tcgattttta ttatttgttg cttatgattt gccctatgca
gttgcatata aaagttatga 240tttttgaaat cagttatgat taacataaaa
acaaaaatca atttttattg ttattttgtt 300atgattcgtt gtatagagtt
tgaatttaag atttgattca tgtcttagag gatttgttgt 360tttctgattg
gtgctttttc atgtgtttat gttatgtatt gtaatgtttg aagctgtttt
420tttatgattt atttgtgcat ctgtcttgtt cgtgaaattc tatttttttt
tttataaatc 480atgtaattaa aattcatagc tttgcttaga acagcctttg
tattatgtgt tttcttgtcg 540tgatatttgg gatgtctttt gttggtggtt
ttatttttgt tgaaaatatg ttctttttgt 600tatatatctg ttgaatttta
cctaaagtgt gttgttttgt tctatag 6471228DNAArtificial
SequenceSynthetic primer 12ctagtcccgg gatcatgtat atttgtgc
281331DNAArtificial SequenceSynthetic primer 13ccttccggtg
ccaagccagc catggctgta g 311440DNAArtificial SequenceSynthetic
primer 14gatcgatcag cggccgcttc acaaatttcg gtttgtactt
401540DNAArtificial SequenceSynthetic primer 15atcctgccat
ggatgctaca taaaaagaac aaaacaatca 401639DNAArtificial
SequenceSynthetic primer 16gatcagatct gcggccgcgt cttgtgtgat
atctaacat 391740DNAArtificial SequenceSynthetic primer 17gatcagatct
gcggccgcat tgttgtcctt gttagccgat 401837DNAArtificial
SequenceSynthetic primer 18gatcagatct gcggccgcca aagctaatct agttaga
371936DNAArtificial SequenceSynthetic primer 19aacctgacat
gtgactatag aacaaaacaa cacact 36
* * * * *