U.S. patent application number 10/626891 was filed with the patent office on 2005-05-12 for duplicated cassava vein mosaic virus enhancers and uses thereof.
Invention is credited to Nielsen, Mark T., Xu, Dongmei.
Application Number | 20050101774 10/626891 |
Document ID | / |
Family ID | 22531694 |
Filed Date | 2005-05-12 |
United States Patent
Application |
20050101774 |
Kind Code |
A1 |
Xu, Dongmei ; et
al. |
May 12, 2005 |
Duplicated cassava vein mosaic virus enhancers and uses thereof
Abstract
The invention features duplicated enhancer domains and enhancer
cassettes. The invention also features expression constructs having
one or more duplicated enhancer domains and a promoter under the
regulation of the duplicated enhancer domains. The expression
construct is useful for increasing the expression of any nucleic
acid molecule that is operably linked to the expression construct.
Accordingly, the invention also features a method for expressing a
nucleic acid molecule through the use of an expression construct of
the present invention.
Inventors: |
Xu, Dongmei; (Lexington,
KY) ; Nielsen, Mark T.; (Lexington, KY) |
Correspondence
Address: |
CLARK & ELBING LLP
101 FEDERAL STREET
BOSTON
MA
02110
US
|
Family ID: |
22531694 |
Appl. No.: |
10/626891 |
Filed: |
July 23, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10626891 |
Jul 23, 2003 |
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09641466 |
Aug 18, 2000 |
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6664384 |
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60149763 |
Aug 19, 1999 |
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Current U.S.
Class: |
536/23.72 ;
435/468; 800/280 |
Current CPC
Class: |
C12N 15/8216
20130101 |
Class at
Publication: |
536/023.72 ;
800/280; 435/468 |
International
Class: |
A01H 001/00; C12N
015/82; C07H 021/04 |
Claims
What is claimed is:
1. An enhancer cassette comprising a duplicated enhancer derived
from a cassaya vein mosaic virus.
2. The enhancer cassette of claim 1, said enhancer cassette
comprising a component having the formula (X--Y).sup.n, wherein X
corresponds to said enhancer domain derived from a cassaya vein
mosaic virus, Y is an intervening spacer domain comprising a
sequence of between zero and thirty nucleotides inclusive, and n is
an integer between 2 and 8 inclusive.
3. The enhancer cassette of claim 2, wherein X comprises
nucleotides 1 to 261 of SEQ ID NO: 1, nucleotides 1 to 332 of SEQ
ID NO: 1, or nucleotides 262 to 332 of SEQ ID NO: 1.
4. The enhancer cassette of claim 2, wherein n is 2.
5. An expression construct comprising, as a first component, an
enhancer cassette comprising a duplicated enhancer derived from a
cassaya vein mosaic virus; and, as a second component, a promoter
having an RNA polymerase binding site and an mRNA initiation
site.
6. The expression construct of claim 5, wherein said promoter is a
cassaya vein mosaic virus promoter.
7. The expression construct of claim 6, wherein said cassaya vein
mosaic virus promoter comprises nucleotides 333-444 of SEQ ID NO:
1.
8. The expression construct of claim 5, wherein said promoter is a
heterologous promoter.
9. The expression construct claim 5, further comprising, as a third
component, a nucleic acid molecule of interest, wherein said first,
second, and third components are operably linked so that the
nucleic acid molecule is transcribed.
10. The expression construct of claim 9, wherein said third
component encodes a protein providing disease or insect
resistance.
11. The expression construct of claim 9, wherein said third
component encodes an antisense RNA.
12. The expression construct of claim 9, wherein said third
component encodes a selectable marker
13. The expression construct of claim 9, wherein transcription of
said nucleic acid molecule is increased relative to transcription
of said nucleic acid molecule operably linked to an expression
construct not comprising a duplicated CsVMV enhancer domain.
14. An expression vector comprising an enhancer construct
comprising, as a first component, an enhancer cassette comprising a
duplicated enhancer derived from a cassaya vein mosaic virus; as a
second component, a promoter having an RNA polymerase binding site
and an mRNA initiation site; and, as a third component, a nucleic
acid molecule of interest, wherein said first, second, and third
components are operably linked so that the nucleic acid molecule is
transcribed.
15. A cell comprising an enhancer construct comprising, as a first
component, an enhancer cassette comprising a duplicated enhancer
derived from a cassaya vein mosaic virus; as a second component, a
promoter having an RNA polymerase binding site and an mRNA
initiation site; and, as a third component, a nucleic acid molecule
of interest, wherein said first, second, and third components are
operably linked so that the nucleic acid molecule is
transcribed.
16. The cell of claim 15, wherein said cell is a eukaryotic
cell.
17. The cell of claim 16, wherein said cell is a plant cell.
18. A transgenic plant comprising an enhancer cassette comprising a
duplicated enhancer derived from a cassaya vein mosaic virus.
19. A method for expressing a nucleic acid molecule in a cell, said
method comprising: (i) transforming said cell with an expression
construct comprising (a) a first component having the formula
(X--Y).sup.n, wherein X corresponds to an enhancer derived from a
cassaya vein mosaic virus, Y is an intervening spacer domain
comprising a sequence between zero and thirty nucleotides
inclusive, and n is an integer between 2 and 8 inclusive; (b) a
second component consisting of a promoter comprising an RNA
polymerase binding site and an mRNA initiation site; and (c) a
third component consisting of the nucleic acid molecule to be
expressed, wherein the first, second, and third components are
operably linked so that the nucleic acid molecule is transcribed,
and (ii) providing conditions that allow for expression of said
nucleic acid in said cell.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims benefit of U.S. Provisional
Application No. 60/149,763 (filed Aug. 19, 1999), now pending,
which is hereby incorporated by reference.
BACKGROUND OF THE INVENTION
[0002] The invention relates to methods of manipulating gene
expression in plants.
[0003] The ability to manipulate gene expression provides a means
of producing new characteristics in transformed plants. There are
many situations in which high or increased levels of gene
expression may be desired. For example, it is desirable to increase
production of a protein that itself maximizes the disease
resistance, yield, flavor, or any other commercially important
attribute of a plant. Similarly, the regulation of endogenous gene
expression by the exogenous expression of antisense, ribozyme RNA,
or transgene silencing may result in more valuable plants or plant
products. The enhancement of expression, through the use of the
invention disclosed herein, would facilitate these
possibilities.
SUMMARY OF THE INVENTION
[0004] We have discovered duplicated enhancer domains for use in
the enhancement of gene expression in transgenic plants. The
duplicated enhancer domains have a plurality of the repetitive
units of one or more enhancers derived from cassaya vein mosaic
virus (CsVMV). The duplicated enhancer domains are preferably
accompanied by a promoter that includes an RNA polymerase binding
site and an mRNA initiation site. Expression constructs, including
a duplicated enhancer domain and a promoter, provide for enhanced
expression of a desired trait compared to that achieved with the
promoter in the absence of the duplicated enhancer domain.
[0005] Accordingly, in a first aspect, the invention features an
enhancer cassette that includes a duplicated enhancer domain
derived from a cassaya vein mosaic virus.
[0006] Preferably, the enhancer cassette includes a component
having the formula (X--Y).sup.n, wherein X corresponds to the
enhancer domain derived from a cassaya vein mosaic virus, Y is an
intervening spacer domain having a sequence that is placed between
enhancer domains and is typically between about zero and about five
hundred nucleotides inclusive (preferably between zero and about
one hundred nucleotides and, more preferably, between zero and
thirty nucleotides), and n is an integer between 2 and 8 inclusive.
In preferred embodiments, the enhancer domain (X) has a sequence
that includes nucleotides 1 to about 261 of SEQ ID NO: 1,
nucleotides 1 to about 332 of SEQ ID NO: 1, or nucleotides of about
262 to about 332 of SEQ ID NO: 1. Preferably, in the formula of the
first aspect, (X--Y).sup.n, n is 2. The spacer domains (Y) can be
identical or different. For example, an enhancer cassette having
the formula (X--Y).sup.3 can have three different spacer
sequences.
[0007] In a related aspect, the invention features an expression
construct including the enhancer cassette of the first aspect and a
second component that includes a promoter having an RNA polymerase
binding site and an mRNA initiation site. A preferred promoter is a
cassaya vein mosaic virus promoter, such as one included in the
nucleotides from about 333 to about 444 of SEQ ID NO: 1. The
promoter can also be a heterologous promoter (for example, a
Ti-plasmid promoter such as the T-DNA gene 5 or 7 promoter). In
preferred embodiments, the expression construct includes a sequence
corresponding to SEQ ID NO: 2 (FIG. 4A), SEQ ID NO: 3 (FIG. 4B),
SEQ ID NO: 4 (FIG. 4C), SEQ ID, NO: 5 (FIG. 4D), or SEQ ID NO: 6
(FIG. 4E).
[0008] The expression construct may further include, as a third
component, a nucleic acid molecule of interest, wherein the first,
second, and third components are operably linked so that the
nucleic acid molecule is transcribed. The third component of the
construct can encode a protein providing disease or insect
resistance, an antisense RNA, a selectable marker (e.g., GUS, GFP,
and the like), a non-translatable RNA molecule, or any protein or
RNA that improves or results in a desired attribute.
[0009] This three-component expression construct of the present
invention, when placed in a transcription medium capable of
supporting transcription, typically results in increased
transcription of the nucleic acid molecule relative to
transcription of the nucleic acid molecule operably linked to an
expression construct that has only one CsVMV enhancer domain.
[0010] In related aspects, the invention also features vectors and
cells that include the enhancer cassette of the first aspect.
Preferably, the cell is a eukaryotic cell, and, more preferably, a
plant cell (e.g., from a monocotylenous plant or a dicotylenous
plant).
[0011] In another related aspect, the invention also features a
transgenic plant that includes the enhancer cassette of the first
aspect.
[0012] In yet another related aspect, the invention features a
method for expressing a nucleic acid molecule. The method includes
transforming a cell (for example, a plant cell) with an expression
construct that includes (a) a first component having the formula
(X--Y).sup.n, wherein X corresponds to the enhancer domain derived
from a cassaya vein mosaic virus, Y is an intervening spacer domain
having a sequence that is placed between enhancer domains and is
typically between about zero and about five hundred nucleotides
inclusive (preferably between zero and about one hundred
nucleotides and, more preferably, between zero and thirty
nucleotides), and n is an integer between 2 and 8 inclusive; (b) a
second component that includes a promoter (e.g., an RNA polymerase
binding site and an mRNA initiation site); and (c) a third
component that includes the nucleic acid molecule to be expressed,
wherein the first, second, and third components are operably linked
so that the nucleic acid molecule is transcribed. In preferred
embodiments, the enhancer domain (X) consists of a sequence that
includes nucleotides from about 1 to about 261 of SEQ ID NO: 1,
nucleotides from about 1 to about 332 of SEQ ID NO: 1, and
nucleotides from about 262 to about 332 of SEQ ID NO: 1.
Preferably, in the formula of the third aspect, (X--Y).sup.n, n is
2.
[0013] The promoter can be any promoter that is functional in the
transformed cell, but preferably is a cassaya vein mosaic virus
promoter (e.g., one included in nucleotides from about 333 to about
444 of SEQ ID NO: 1).
[0014] The third component of the expression construct can encode a
protein providing disease or insect resistance, an antisense RNA, a
selectable marker (e.g., GUS, GFP, and the like), or any protein or
RNA including but not limited to a nontranslatable RNA or any RNA
molecule capable of inducing transgene silencing.
[0015] As used herein, by "nucleic acid" is meant either DNA or
RNA. A "nucleic acid molecule" may be a single-stranded or
double-stranded polymer of deoxyribonucleotide or ribonucleotide
bases. Unless otherwise specified, the left hand direction of the
sequence of a single-stranded nucleic acid molecule is the 5' end,
and the left hand direction of double-stranded nucleic molecule is
referred to as the 5' direction.
[0016] By "promoter" is meant a region of nucleic acid, upstream
from a translational start codon, which is involved in recognition
and binding of RNA polymerase and other proteins to initiate
transcription. A "plant promoter" is a promoter capable of
initiating transcription in a plant cell, and may or may not be
derived from a plant cell. A "CsVMV promoter" is one derived from
the promoter region of a CsVMV genome and that, when operably
linked to a heterologous nucleic acid molecule, is capable of
initiating transcription of that molecule when present in a
transcription medium capable of supporting transcription, such as
in a plant cell, a plant, or in vitro.
[0017] Exemplary transcription media include, for example, a plant
cell, plant protoplasts, or other plant tissue culture
configurations, non-differentiated plant cells, differentiated
plant cells (such as cultured plantlets), transgenic plants, and
mature plants. Also included are in vitro expression systems such
as reconstituted expression medium composed of components required
to support transcription, as are known in the art.
[0018] By "enhancer domain" is meant a nucleic acid sequence that,
when positioned proximate to a promoter and present in a
transcription medium capable of supporting transcription, confers
increased expression relative to the expression resulting from the
promoter in the absence of the enhancer domain. By "enhancer
cassette" is meant a nucleic acid sequence that includes an
enhancer domain and, optionally, additional sequence that does not
enhance expression (e.g, intervening spacer domain).
[0019] By "duplicated enhancer domain" is meant two or more copies
of an enhancer domain. Preferably, the number of copies is between
about two and about four. The enhancer domains can be in the same
or opposite orientation, and can be contiguous or noncontiguous. In
the case of expression constructs having two duplicated enhancer
domains (e.g., domain A and domain B), the orientation and the 5'
to 3' order (e.g., 5'-AABB-3' vs. 5'-ABAB-3') are not limitations
to the invention. The enhancer domains may also be separated by
intervening spacer domains as described herein.
[0020] By "operably linked" is meant that a nucleic acid molecule
to be transcribed and an expression construct (i.e., a promoter and
an enhancer domain) are connected in such a way as to permit
transcription of the nucleic acid molecule in a suitable
transcription medium.
[0021] By "derived from" is meant that the nucleic acid molecule
was either made or designed from a second nucleic acid molecule,
the derivative retaining the functional features thereof.
[0022] By "expression construct" is meant a nucleic acid molecule
that is capable of directing transcription. An expression construct
of the present invention includes, at the least, a duplicated CsVMV
enhancer domain and a promoter. Additional domains, such as a
transcription termination signal, may also be included, as
described herein.
[0023] By "vector" or "expression vector" is meant an expression
system, a nucleic acid-based shuttle vehicle, a nucleic acid
molecule adapted for nucleic acid delivery, or an autonomous
self-replicating circular DNA (e.g., a plasmid). When a vector is
maintained in a host cell, the vector can either be stably
replicated by the cells during mitosis as an autonomous structure,
incorporated within the genome of the host cell, or maintained in
the host cell's nucleus or cytoplasm.
[0024] By "plasmid" is meant an autonomous DNA molecule capable of
replication in a cell, and includes both expression and
nonexpression types.
[0025] By "heterologous" is meant that the nucleic acid molecule
originates from a foreign source or, if from the same source, is
modified from its original form or sequence. Thus, a "heterologous
promoter" is a promoter not normally associated with the enhancer
domain that is duplicated. Similarly, a heterologous nucleic acid
molecule that is modified from its original form or is from a
source different from the source from which the promoter to which
it is operably linked was derived.
[0026] The term "plant" includes any cell having a plastid, and can
include whole plants, plant organs (e.g., stems, leaves, roots,
etc.), seeds, and cells. The class of plants that can be used in
the method of the invention is generally as broad as the class of
higher plants amenable to transformation techniques, including both
monocots and dicots.
[0027] By "transgene" is meant any piece of a nucleic acid molecule
(for example, DNA) which is inserted by artifice into a cell, and
becomes part of the organism (integrated into the genome or
maintained extrachromosomally) which develops from that cell. Such
a transgene may include a gene which is partly or entirely
heterologous (i.e., foreign) to the transgenic organism, or may
represent a gene homologous to an endogenous gene of the
organism.
[0028] By "transgenic plant" is meant a plant containing a
transgene. For example, a plant cell transformed with a vector
containing the expression construct of the present invention
operably linked to a heterologous nucleic acid molecule can be used
to produce a transgenic plant having altered phenotypic
characteristics.
[0029] Other features and advantages of the invention will be
apparent from the following description of the preferred
embodiments thereof, and from the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0030] FIG. 1 is a schematic illustration showing the sequence of
the region immediately 5' to the CsVMV translational start site.
"+1" demarcates the first nucleotide of the mRNA.
[0031] FIG. 2A is a schematic illustration showing examples of
expression constructs CV-2, CV-3, CV-4, and CV-5, each of which
having a duplicated enhancer domain. In each of these examples, the
CsVMV promoter is used, although it is understood that this could
be replaced with another promoter, as described herein. It is also
understood that additional modifications, such as the addition of
spacer sequences between the enhancer domains or the inversion of
the orientation of one or more enhancer domains, would not
substantially affect the transcriptional activity of the expression
constructs.
[0032] FIG. 2B is a schematic illustration showing the fragments
used in the ligation strategy used to generate the expression
constructs of FIG. 2A. The restriction sites have been generated
during PCR of individual fragments. These restriction sites are
included to exemplify the general strategy to ligate the fragments
together. It will be understood that other methods known in the
art, as described herein, may also be used to make the duplicated
enhancer domains and expression constructs of the present
invention.
[0033] FIG. 3 is a schematic illustration showing an exemplary
vector containing (i) a duplicated CsVMV enhancer domain (E) and a
CsVMV promoter (CsVMV) operably linked to a nucleic acid sequence
of interest (SOI), followed by a terminator (Ter); and (ii) an Act2
promoter operably linked to a gene conferring kanamycin resistance
(NPT II), also followed by a terminator.
[0034] FIGS. 4A-4D are schematic illustrations showing the
nucleotide sequences of expression constructs CV-2, CV-3, CV-4, and
CV-5, respectively.
[0035] FIG. 4E is a schematic illustration showing the nucleotide
sequence of expression construct CV-6, in which fragment CVA-5 (see
FIG. 2B) is in the opposite orientation to which it is found in
CV-5. In FIGS. 4A-4E, the nucleotides in bold represent nucleotides
added in order to create a restriction site. One skilled in the art
will recognize that these nucleotides may be omitted or replaced
with other nucleotides if desired.
[0036] FIG. 5 is a schematic illustration showing the frequency of
GUS staining in transgenic lines expressing GUS from expression
constructs containing either an unduplicated CsVMV enhancer domain
(1.times.CsVMV) or a duplicated CsVMV enhancer domain (CV-2;
2.times.CsVMV).
[0037] FIG. 6 is a schematic illustration showing GUS activity in
transgenic lines expressing GUS from expression constructs
containing unduplicated CsVMV enhancer domains ("1.times.CsVMV") or
duplicated CsVMV enhancer domains (construct CV-2;
"2.times.CsVMV").
[0038] FIG. 7 is a schematic illustration showing the frequency of
GUS staining in transgenic lines expressing GUS from expression
constructs containing either 1.times.CsVMV or 2.times.CsVMV.
DETAILED DESCRIPTION OF THE INVENTION
[0039] The present invention provides novel duplicated enhancer
domains and enhancer cassettes. The invention also provides
expression constructs having one or more duplicated enhancer
domains and a promoter under the regulation of the duplicated
enhancer domains. In the expression construct, the duplicated
enhancer domains increase the transcription efficiency, resulting
in greater expression of any nucleic acid molecule that is operably
linked to the expression construct. Of particular interest is
enhanced expression of inserted gene sequences which may be of the
same genetic origin as the host or of foreign origin, either the
naturally occurring sequences, in either sense and antisense
orientations, or synthetically prepared sequences.
[0040] Duplicated CsVMV Enhancer Domains
[0041] In one embodiment, the invention features duplicated
enhancer domains. The duplicated enhancer domains of the present
invention are derived from CsVMV, a double stranded DNA plant
pararetrovirus (Calvert et al., J. Gen. Virol. 76:1271, 1995), the
genomic sequence of which has been previously determined (GenBank
Accession Nos. U59751 and U20341), including the region containing
the promoter (Verdaguer et al., Plant Mol. Biol. 31:1129, 1996; PCT
publication WO 97/48819). Taking the first nucleotide of the mRNA
as position +1, enhancer domains are located from about -60 to
about -700 bp. Preferred enhancer domains correspond to nucleotides
from about -443 to about -183, from about -443 to about -112, and
from about -182 to about -112 (nucleotides for about 1 to about
261, nucleotides for about 1 to about 332, and nucleotides for
about 262 to about 332 of SEQ ID NO: 1, respectively), as shown in
FIGS. 2A and 2B. It will be understood that the nucleotide
positions can be altered by about five to about ten nucleotides
without substantially altering the expression-enhancing ability of
the enhancer domain. The enhancer domain that is duplicated will
usually be about 20 to about 350 bp.
[0042] Preferably, the duplicated enhancer domain is incorporated
into an enhancer cassette having the formula (X--Y).sup.n, wherein
X corresponds to an enhancer derived from CsVMV, Y is a sequence
between about zero and about thirty nucleotides inclusive, and n is
an integer between 2 and 8 inclusive. In preferred embodiments, X
has a sequence that includes nucleotides 1 to about 261,
nucleotides 1 to about 332, and nucleotides 262 to about 332 of SEQ
ID NO: 1.
[0043] Expression Constructs
[0044] In one particular embodiment of the present invention, the
duplicated enhancer domains or enhancer cassettes are placed in the
proximity of a promoter; together, these form an expression
construct. Exemplary expression constructs are shown in FIG. 2A and
FIGS. 4A-4E.
[0045] An enhancer domain is cis-acting and desirably is located
within about 5 kb, typically about 2 kb, more typically adjacent to
or within about 1 kb of a promoter to be enhanced. The combination
of the duplicated enhancer domain and the promoter is considered to
be an "expression construct." In the expression construct, the
enhancer domains may be in either orientation with respect to each
other as well as to the promoter, and can be located 5' or 3' in
relation to the promoter they enhance, usually in the 5'
direction.
[0046] A duplicated enhancer domain of the present invention finds
use with a wide variety of promoters, including promoters that are
naturally found under the control of the enhancer (i.e., adjacent
and homologous) and those not normally associated with the
particular promoter (i.e., heterologous).
[0047] The duplicated enhancer domain and promoter may be from the
same or different kingdom, family, or species. Species of interest
include viruses, prokaryotes and eukaryotes, such as bacteria,
plants, insects, and mammals. Combinations may include, for
example, (i) enhancer domains from CsVMV combined with a promoter
derived from a host for the virus; (ii) enhancer domains from CsVMV
combined with a promoter from a related virus; and (iii) enhancer
domains and a promoter, each of which is derived from CsVMV.
[0048] In addition to the aforementioned duplicated enhancer domain
and promoter, the expression constructs may also include regulatory
control regions which are generally present in the 3' regions of
plant genes (Thornburg et al., Proc. Natl. Acad. Sci. U.S.A.
84:744, 1987; An et al., Plant Cell 1:115, 1989). For example, a 3'
terminator region may be included in the expression vector to
increase stability of the mRNA. One such terminator region may be
derived from the PI-II terminator region of potato. In addition,
other commonly used terminators are derived from the octopine or
nopaline synthase signals.
[0049] Expression Vectors
[0050] An expression vector, including an expression construct, is
shown in FIG. 3. Typically, a vector containing an expression
construct of the present invention also contains a dominant
selectable marker gene used to identify those cells that have
become transformed. Useful selectable genes for plant systems
include genes encoding antibiotic resistance genes, for example,
those encoding resistance to hygromycin, kanamycin, bleomycin,
G418, streptomycin, or spectinomycin. Genes required for
photosynthesis may also be used as selectable markers in
photosynthetic-deficient strains. Genes encoding a detectable
enzyme (e.g., beta-glucuronidase (GUS); see U.S. Pat. No.
5,268,463) are also useful markers. Alternatively, the
green-fluorescent protein from the jellyfish Aequorea victoria may
be used as a selectable marker (Sheen et al., Plant J. 8:777, 1995;
Chiu et al., Curr. Biol. 6:325, 1996). Finally, genes encoding
herbicide resistance may be used as selectable markers; useful
herbicide resistance genes include the bar gene encoding the enzyme
phosphinothricin acetyltransferase and conferring resistance to the
broad spectrum herbicide Basta.RTM. (Hoechst Marion Roussel,
Frankfurt, Germany). Other selectable marker genes confer
resistance to herbicides such as glyphosate, glufosinate or
broxynil (Comai et al., Nature 317:741, 1985; Gordon-Kamm et al.,
Plant Cell 2:603, 1990; Stalker et al., Science 242:419, 1988).
[0051] Efficient use of selectable markers is facilitated by a
determination of the susceptibility of a plant cell to a particular
selectable agent and a determination of the concentration of this
agent which effectively kills most, if not all, of the
untransformed cells. Some useful concentrations of antibiotics for
plant cell transformation include, e.g., 75-100 .mu.g/ml
(kanamycin), 20-50 .mu.g/ml (hygromycin), or 5-10 .mu.g/ml
(bleomycin).
[0052] The invention also contemplates DNA constructs in which an
expression construct, including a duplicated CsVMV enhancer domain
and a promoter, is operably linked to a nucleic acid molecule one
wishes to be transcribed. The nucleic acid molecule may have a
natural open reading frame (ORF), as well as transcribed 5' and 3'
sequences flanking the ORF. Alternatively, it may be in the
"antisense" orientation in that it encodes the complement of an RNA
molecule or portion thereof. When the construct includes an ORF
(which encodes a polypeptide), an enhanced transcription initiation
rate is obtained, usually providing an increased amount of the
polypeptide. When the construct contains an antisense sequence,
complementary to the wild-type molecule, decreases the amount of
polypeptide product. In addition, antisense RNA can also function
as an inhibitor of replication of RNA (of viral genomes, for
example).
[0053] Enhanced transcription in plants may find use in enhancing
the production of proteins characteristic of the plant (endogenous)
or those proteins from other genetic sources (exogenous). For
protein production, translational initiation sequences (including a
start codon) are included in the constructs, either from the
promoter domain, from the attached coding sequences, or from a
heterologous source.
[0054] Examples of nucleic acid molecules to be expressed under the
control of the expression constructs of the present invention
include, without limitation, antisense RNAs (for gene suppression);
nutritionally important proteins; growth promoting factors;
proteins providing disease resistance; proteins providing
protection to the plant under certain environmental conditions
(e.g., proteins providing resistance to metal or other toxicity);
stress related proteins mediating tolerance to extremes of
temperature, freezing, etc.; compounds of medical importance (e.g.,
anti-microbial or anti-tumor agents); proteins of specific
commercial value; proteins that function as enzymes of metabolic
pathways; proteins of structural value to a plant host; and non
translatable RNA for the induction of transgene silencing.
[0055] For example, an expression construct having a duplicated
enhancer domain can be operably linked to a nucleic acid molecule
conferring, without limitation, herbicide resistance, fungal
disease resistance, bacterial disease resistance, or insect
resistance. Similarly, the expression construct can be operably
linked to a nucleic acid molecule, the expression of which
regulates plant ripening, degradation, color, sweetness, and the
like.
[0056] The nucleic acid molecules of interest which are transcribed
will be of at least about 8 bp, usually at least about 12 bp, more
usually at least about 20 bp, and may be about one kb or more in
length.
[0057] Methods for Making Duplicated Enhancer Domains
[0058] A variety of duplicated CsVMV enhancer domains can be
produced using standard molecular biology techniques. For example,
a duplicated enhancer can be constructed by first mapping
restriction enzyme sites in the CsVMV genomic sequence that
includes the enhancer domain of interest, then, using the
constructed map to determine the appropriate restriction enzymes,
excising the domain of interest and recombining it to form a
duplicated enhancer domain. Alternatively, a duplicated enhancer
domain or an expression construct of the present invention can be
synthesized by a variety of methods based on the sequences
described herein. Synthesis can be accomplished by chemical
synthesis methods for the production of enhancer oligonucleotides.
In addition, a nucleic acid molecule can be prepared by the
synthesis of a series of oligonucleotides which correspond to
different portions of the nucleic acid molecule, and which can be
combined by ligation to form larger nucleic acid molecules.
Finally, oligonucleotides can be used as primers in a polymerase
chain reaction (PCR) to amplify a nucleic acid molecule of
interest. The primers can further contain restriction sites to
facilitate ligation of the PCR fragments.
[0059] The expression constructs are typically prepared employing
cloning vectors, where the sequences may be naturally occurring,
mutated sequences, synthetic sequences, or combinations thereof.
The cloning vectors are well known and include prokaryotic or
eukaryotic replication systems, markers for selection of
transformed host cells, and unique dual restriction sites for
insertion or substitution of sequences.
[0060] Transgenic Plants
[0061] In one embodiment, the invention features a transgenic plant
having an expression construct operably linked to a heterologous
nucleic acid molecule of interest, the expression of which may
cause the plant to have an altered phenotype. Because the promoters
of the present invention can function in a wide variety of plants,
including both monocot plants and dicot plants, the transgenic
plant can be any type of plant that contains an expression
construct and that can express the heterologous nucleic acid
molecule.
[0062] Upon construction of the expression vector, several standard
methods are available for introduction of the vector into a plant
host, thereby generating a transgenic plant. These methods include:
(1) Agrobacterium-mediated transformation (A. tumefaciens or A.
rhizogenes); (2) a particle delivery system; (3) microinjection;
(4) polyethylene glycol (PEG) procedures; (5) liposome-mediated DNA
uptake; (6) electroporation; (7) chloroplast transformation; and
(8) vortexing. The method of transformation is not critical to the
invention. Any method which provides for efficient transformation
may be employed. As newer methods are available to transform crops
or other host cells, they may be directly applied.
[0063] One technique of transforming plants with the DNA molecules
in accordance with the present invention is by contacting the
tissue of such plants with an inoculum of a bacteria transformed
with a vector that includes a duplicated CsVMV enhancer domain.
Generally, this procedure involves inoculating the plant tissue
with a suspension of bacteria and incubating the tissue for 48 to
72 hours on regeneration medium without antibiotics at
25-28.degree. C.
[0064] Bacteria from the genus Agrobacterium can be utilized to
transform plant cells. Suitable species of such bacterium include
Agrobacterium tumefaciens and Agrobacterium rhizogenes.
Agrobacterium tumefaciens (e.g., strains C58, LBA4404, or EHA105)
is particularly useful due to its well-known ability to transform
plants.
[0065] Another approach to transforming plant cells with a gene
which imparts resistance to pathogens is particle bombardment (also
known as biolistic transformation) of the host cell. This can be
accomplished in one of several ways, such as those disclosed in
U.S. Pat. Nos. 4,945,050, 5,036,006, and 5,100,792, all to Sanford
et al., and in Emerschad et al., Plant Cell Reports, 14:6-12,
1995.
[0066] Once plant tissue is transformed in accordance with the
present invention, it is regenerated to form a transgenic plant.
Generally, regeneration is accomplished by culturing transformed
tissue on medium containing the appropriate growth regulators and
nutrients to allow for the initiation of shoot meristems.
Appropriate selection agents are added to the regeneration medium
to select for the development of transformed cells. Following shoot
initiation, shoots are allowed to develop in tissue culture and may
be screened for marker gene activity.
[0067] In general, transfer and expression of transgenes in plant
cells are now routine practices to those skilled in the art, and
have become major tools to carry out gene expression studies in
plants and to produce improved plant varieties of agricultural or
commercial interest.
EXAMPLE 1
Preparation of Duplicated CsVMV Enhancer Domains
[0068] Duplicated CsVMV enhancer domains were produced by internal
splicing and addition. The preparation of six duplicated CsVMV
enhancer domains is outlined below.
[0069] The starting plasmid was pBluscript-CsVMV, which contained
CsVMV promoter fragment extending from position -443 to +72
(nucleotides 1 to 515 of SEQ ID NO: 1). Due to the absence of
convenient restriction sites in the CsVMV promoter fragment,
polymerase chain reaction (PCR) was used to generate a set of
terminal and internal fragments.
EXAMPLE 2
Construction of Plant Expression Vectors
[0070] The pGEN plant expression vector with appropriate multiple
cloning sites was used to introduce the duplicated CsVMV enhancer
domains into tobacco plants. The different duplicated enhancer
domains generated in Example 1 were cloned into two versions of the
pGEN vectors using standard techniques.
EXAMPLE 3
Development of Transgenic Plants
[0071] The pGEN derived plasmids carrying a non-duplicated CsVMV
enhancer domain, a duplicated CsVMV enhancer domain, and a
promoterless construct were transformed separately into
Agrobacterium tumefaciens strain GV3850. Agrobacterium
tumefaciens-mediated transformations of Nicotiana tabacum cv KY14
were performed as previously described (Horsch et al., Plant
Molecular Biology Manual). Approximately 40 kanamycin resistant
transgenic lines were generated for each construct. The plants were
grown to maturity in a greenhouse.
EXAMPLE 4
Expression Analysis of CsVMV Expression Constructs
[0072] Histochemical Analysis of Expression in Calli and Young
Plants
[0073] Histochemical GUS analyses of plasmid-transformed calli and
plantlets were carried out to analyze the expression levels of
expression constructs containing the unduplicated ("1.times.")
CsVMV enhancer and the duplicated ("2.times.") CsVMV enhancer
(corresponding to expression construct CV-2; see FIGS. 2A and 4A).
Calli and the top two leaves of plantlets from each independent
transformant were collected for GUS stain analysis. Fresh tissues
were taken and incubated for three hours, six hours, and overnight
at 37.degree. C. in 2 ml reaction buffer containing 1 mM
5-bromo-4-chloro-3-indolyl glucuronide (x-gluc), 100 mM sodium
phosphate buffer pH 7.2, potassium ferrocyanide, potassium
ferricyanide, and 0.2% Triton x-100. GUS staining was scored using
a scale of 1 to 4 from light staining to heavy staining, as shown
in FIG. 5. Scoring was as follows: Score 4--all tissue stained blue
and solution was also blue; 3--most tissue stained dark blue;
2--some tissue stained light blue; 1--tissue had light blue spots;
0--no staining.
[0074] The promoterless plants and calli were all scored 0. In
contrast, none of the transgenic plants were scored 0. This
indicated that 100% of plants contained and expressed the GUS
reporter gene. The transgenic plants started to show the blue color
after 30 minutes in the stain solution. Most plants stained blue
after three hours in the solution. A higher percentage of plants
containing the double promoter showed high rating scores (80% of
plants for CV-2 were rated 3 or 4, compared with 49% for 1.times.
construct). In some high lines containing the CV-2 expression
construct, expression appeared about one hour before expression in
plants containing 1.times. expression constructs. These data thus
indicated that the CV-2 expression construct generates transgenic
plants with higher expression when compared with the 1.times.CsVMV
enhancer.
[0075] Glucuronidase Assays for the Transgenic Lines Containing
1.times. and 2.times.CsVMV Promoters
[0076] GUS activities in protein extracts prepared from leaf
tissues were quantitatively measured using a fluorometric assay
(Jefferson et al., EMBO J. 6:3901-7, 1087). The samples were
collected from interveinal tissues of young leaves from transgenic
plants generated in Example 3. Forty-one samples, 30 samples and 4
samples from independent transgenic lines were assayed for an
unduplicated enhancer construct, a duplicated enhancer construct,
and a promoterless construct, respectively. FIG. 6 depicts values
of GUS activities for different constructs. The variation among
lines containing the same expression construct can be attributed to
a combination of factors including a putative position effect
reflecting the influence of the surrounding chromatin on gene
expression, differences in copy number, and gene silencing. The
data confirmed the histochemical localization data for GUS
expression in transgenic plants. The range of the GUS expression
was 374 to 54337 pmol 4 MU/mom/mg protein for the 1.times.CsVMV
construct (mean=19596) and 2152 to 106799 for the 2.times.CsVMV
construct (mean=35678). The average value for these transgenic
plants was 331 for promoterless plants. FIG. 7 showed the frequency
of transgenic lines exhibited different level of GUS activities.
None of the plants from 2.times.CsVMV had GUS activities lower than
2000 pmol 4 MU/min/mg protein. We confirmed the foregoing results
by GUS staining the top two leaves from the same transgenic
plants.
OTHER EMBODIMENTS
[0077] All publications mentioned in this specification are herein
incorporated by reference to the same extent as if each independent
publication was specifically and individually indicated to be
incorporated by reference.
[0078] While the invention has been described in connection with
specific embodiments thereof, it will be understood that it is
capable of further modifications. This application is intended to
cover any variations, uses, or adaptations following, in general,
the principles of the invention and including such departures from
the present disclosure within known or customary practice within
the art to which the invention pertains and may be applied to the
essential features hereinbefore set forth.
* * * * *