U.S. patent application number 11/394567 was filed with the patent office on 2006-10-26 for dissimilar promoters for gene suppression.
Invention is credited to Larry Gilbertson, Shihshieh Huang, Thomas Malvar.
Application Number | 20060242736 11/394567 |
Document ID | / |
Family ID | 38188995 |
Filed Date | 2006-10-26 |
United States Patent
Application |
20060242736 |
Kind Code |
A1 |
Huang; Shihshieh ; et
al. |
October 26, 2006 |
Dissimilar promoters for gene suppression
Abstract
Methods of gene suppression comprise transforming eukaryotic
cells with recombinant DNA constructs including promoters with
dissimilar expression patterns operably linked to one or more gene
suppression elements and, optionally, one or more gene expression
elements.
Inventors: |
Huang; Shihshieh;
(Stonington, CT) ; Malvar; Thomas; (North
Stonington, CT) ; Gilbertson; Larry; (Chesterfield,
MO) |
Correspondence
Address: |
MONSANTO COMPANY
800 N. LINDBERGH BLVD.
ATTENTION: GAIL P. WUELLNER, IP PARALEGAL, (E2NA)
ST. LOUIS
MO
63167
US
|
Family ID: |
38188995 |
Appl. No.: |
11/394567 |
Filed: |
March 31, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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11311892 |
Dec 19, 2005 |
|
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11394567 |
Mar 31, 2006 |
|
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60638491 |
Dec 23, 2004 |
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Current U.S.
Class: |
800/285 ;
435/419; 435/468; 536/23.6 |
Current CPC
Class: |
C12N 15/8218 20130101;
C12N 15/8254 20130101 |
Class at
Publication: |
800/285 ;
435/419; 435/468; 536/023.6 |
International
Class: |
A01H 1/00 20060101
A01H001/00; C07H 21/04 20060101 C07H021/04; C12N 15/82 20060101
C12N015/82; C12N 5/04 20060101 C12N005/04 |
Claims
1. Transgenic seed having in its genome a recombinant DNA construct
comprising: (a) a plant endosperm-specific promoter operably linked
to at least one first gene suppression element, and (b) a plant
embryo-specific promoter in the opposite orientation to said plant
endosperm-specific promoter and located 3' to said at least one
first gene suppression element.
2. The transgenic seed of claim 1, wherein said at least one first
gene suppression element comprises a gene suppression element for
silencing an amino acid catabolism gene.
3. The transgenic seed of claim 2, wherein said recombinant DNA
construct further comprises one or more elements selected from: (a)
at least one second gene suppression element operably linked to
said plant embryo-specific promoter; (b) an amino acid biosynthesis
gene operably linked to either said plant endosperm-specific
promoter or plant embryo-specific promoter; and (c) a selectable
marker gene.
4. The transgenic seed of claim 2, wherein said transgenic seed is
transgenic maize seed, and said amino acid catabolism gene is a
lysine catabolism gene.
5. The transgenic seed of claim 4, wherein said recombinant DNA
construct further comprises one or more elements selected from: (a)
at least one second gene suppression element for silencing a lysine
catabolism gene operably linked to said plant embryo-specific
promoter; (b) a lysine biosynthesis gene operably linked to said
plant endosperm-specific promoter; (c) an aspartate kinase gene
operably linked to either said plant endosperm-specific promoter or
plant embryo-specific promoter; and (d) a selectable marker
gene.
6. The transgenic seed of claim 2, wherein: (a) said recombinant
DNA construct comprises: (i) a plant endosperm-specific promoter
operably linked to at least one first gene suppression element
comprising DNA that transcribes to RNA for silencing a lysine
catabolism gene by forming double-stranded RNA, and (ii) a plant
embryo-specific promoter in the opposite orientation to said first
promoter and operably linked to said at least one first gene
suppression element; or (b) said recombinant DNA construct
comprises: (i) a plant endosperm-specific promoter operably linked
to at least one first gene suppression element comprising DNA that
transcribes to RNA for silencing a lysine catabolism gene by
forming double-stranded RNA, (ii) a plant embryo-specific promoter
in the opposite orientation to said first promoter and operably
linked to said at least one first gene suppression element, and
(iii) at least one terminator operably linked to either said first
or second promoters; or (c) said recombinant DNA construct
comprises: (i) a plant endosperm-specific promoter operably linked
to at least one first intron-embedded gene suppression element for
silencing a lysine catabolism gene, at least one lysine
biosynthesis gene, and a first terminator, (ii) a plant
embryo-specific promoter in the opposite orientation to said first
promoter and operably linked to at least one second gene
suppression element for silencing a lysine catabolism gene; or (d)
said recombinant DNA construct comprises: (i) a first gene
suppression cassette comprising a plant endosperm-specific promoter
operably linked to at least one first intron-embedded gene
suppression element for silencing a lysine catabolism gene, at
least one lysine biosynthesis gene, and a first terminator, and
(ii) a second gene suppression cassette comprising a plant
embryo-specific promoter operably linked to at least one second
gene suppression element for silencing a lysine catabolism gene,
and a second terminator, wherein said first and second gene
suppression cassettes are in opposite orientations; or (e) said
recombinant DNA construct comprises: (i) a first gene suppression
cassette comprising a plant endosperm-specific promoter operably
linked to at least one first intron-embedded gene suppression
element for silencing a lysine catabolism gene, at least one lysine
biosynthesis gene and a first terminator, and (ii) a second gene
suppression cassette comprising a plant embryo-specific promoter
operably linked to at least one intron-embedded second gene
suppression element for silencing a lysine catabolism gene, at
least one lysine biosynthesis gene and a second terminator, wherein
said first and second gene suppression cassettes are in opposite
orientations; or (f) said recombinant DNA construct comprises: (i)
a first gene suppression cassette comprising a plant
endosperm-specific promoter operably linked to at least one first
gene suppression element for silencing a lysine catabolism gene,
and a first terminator, and (ii) a second gene suppression cassette
comprising a plant embryo-specific promoter operably linked to at
least one second gene suppression element for silencing a lysine
catabolism gene, and a second terminator, wherein said first and
second gene suppression cassettes are in opposite orientations.
7. Stably transgenic plant cells having in their genome a
recombinant DNA construct comprising: (a) a first promoter operably
linked to at least one first gene suppression element for silencing
at least one first target gene, and (b) a second promoter that is
in the opposite orientation to said first promoter and is located
3' to said at least one first gene suppression element, wherein
said first and said second promoters have dissimilar expression
patterns, and wherein transcription of said recombinant DNA
construct in a plant cell results in silencing of said at least one
first target gene.
8. The stably transgenic plant cells of claim 7, wherein said first
and said second promoters comprise a plant embryo-specific promoter
and a plant endosperm-specific promoter and said stably transgenic
plant cells comprise seed embryo and endosperm cells of a crop
plant.
9. A recombinant DNA construct for transformation of a plant cell,
comprising: (a) a first promoter operably linked to at least one
first gene suppression element for silencing at least one first
target gene, and (b) a second promoter that is in the opposite
orientation to said first promoter and is located 3' to said at
least one first gene suppression element, wherein said first and
said second promoters have dissimilar expression patterns, and
wherein transcription of said recombinant DNA construct in a plant
cell results in silencing of said at least one first target
gene.
10. The recombinant DNA construct of claim 9, wherein first and
second promoters have dissimilar spatial expression patterns, and
said silencing occurs in at least two distinct spatial
locations.
11. The recombinant DNA construct of claim 9, wherein first and
second promoters have dissimilar temporal expression patterns, and
said silencing occurs in at least two distinct times or
developmental stages.
12. The recombinant DNA construct of claim 9, wherein said at least
one gene suppression element is under transcriptional control of
both said first and said second promoters.
13. The recombinant DNA construct of claim 9, further comprising
one or more of: (a) a second gene suppression element operably
linked to said second promoter; (b) at least one gene expression
element for expressing at least one exogenous gene, (c) at least
one terminator, and (d) at least one T-DNA border.
14. The recombinant DNA construct of claim 9, wherein said at least
one first gene suppression element comprises at least one element
selected from the group consisting of: (a) DNA that comprises at
least one anti-sense DNA segment that is anti-sense to at least one
segment of said at least one first target gene; (b) DNA that
comprises multiple copies of at least one anti-sense DNA segment
that is anti-sense to at least one segment of said at least one
first target gene; (c) DNA that comprises at least one sense DNA
segment that is at least one segment of said at least one first
target gene; (d) DNA that comprises multiple copies of at least one
sense DNA segment that is at least one segment of said at least one
first target gene; (e) DNA that transcribes to RNA for suppressing
said at least one first target gene by forming double-stranded RNA
and comprises at least one anti-sense DNA segment that is
anti-sense to at least one segment of said at least one target gene
and at least one sense DNA segment that is at least one segment of
said at least one first target gene; (f) DNA that transcribes to
RNA for suppressing said at least one first target gene by forming
a single double-stranded RNA and comprises multiple serial
anti-sense DNA segments that are anti-sense to at least one segment
of said at least one first target gene and multiple serial sense
DNA segments that are at least one segment of said at least one
first target gene; (g) DNA that transcribes to RNA for suppressing
said at least one first target gene by forming multiple double
strands of RNA and comprises multiple anti-sense DNA segments that
are anti-sense to at least one segment of said at least one first
target gene and multiple sense DNA segments that are at least one
segment of said at least one first target gene, and wherein said
multiple anti-sense DNA segments and said multiple sense DNA
segments are arranged in a series of inverted repeats; (h) DNA that
comprises nucleotides derived from a plant miRNA; (i) DNA that
comprises nucleotides of a siRNA; (j) DNA that transcribes to an
RNA aptamer capable of binding to a ligand; and (k) DNA that
transcribes to an RNA aptamer capable of binding to a ligand, and
DNA that transcribes to regulatory RNA capable of regulating
expression of said first target gene, wherein said regulation is
dependent on the conformation of said regulatory RNA, and said
conformation of said regulatory RNA is allosterically affected by
the binding state of said RNA aptamer.
15. The recombinant DNA construct of claim 9, wherein said first
gene suppression element is embedded in an intron.
16. The recombinant DNA construct of claim 9, further comprising a
second gene suppression element operably linked to said second
promoter, wherein said first and second gene suppression elements
are embedded in an intron.
17. The recombinant DNA construct of claim 9, wherein said first
and said second promoters comprise a plant embryo-specific promoter
and a plant endosperm-specific promoter.
18. A method of gene silencing in a plant, comprising: (a)
transforming a plant cell with the recombinant DNA construct of
claim 9, thereby providing a transgenic plant cell; (b) preparing a
regenerated transgenic plant from said transgenic plant cell, or a
transgenic progeny seed or plant of said regenerated transgenic
plant; (c) transcribing said recombinant DNA construct in said
regenerated transgenic plant or said transgenic progeny seed or
plant whereby said at least one first target gene is silenced in
said regenerated transgenic plant or said transgenic progeny seed
or plant.
19. The method of claim 18, wherein said plant is a crop plant.
20. The method of claim 18, wherein said recombinant DNA construct
is transcribed in a transgenic progeny seed having substantial
endosperm, and said first and said second promoters comprise a
plant embryo-specific promoter and a plant endosperm-specific
promoter.
21. The method of claim 20, wherein said transgenic progeny seed is
transgenic progeny maize seed, said at least one first target gene
is at least one lysine catabolism gene, and said at least one
lysine catabolism gene is silenced in embryo and endosperm cells of
said transgenic progeny seed.
22. The method of claim 21, wherein said recombinant DNA construct
further comprises at least one lysine biosynthesis gene operably
linked to said endosperm-specific promoter.
Description
REFERENCE TO RELATED APPLICATIONS
[0001] This application is a Continuation-In-Part of U.S. patent
application Ser. No. 11/311,892, "Gene Suppression in Transgenic
Plants Using Multiple Constructs", filed 19 Dec. 2005, incorporated
herein by reference.
FIELD OF THE INVENTION
[0002] Disclosed herein are recombinant DNA constructs and methods
useful in gene suppression and transgenic plant cells, transgenic
plants, and transgenic seeds containing DNA transferred using such
recombinant DNA constructs and methods.
BACKGROUND
[0003] Redenbaugh et al. in "Safety Assessment of Genetically
Engineered Fruits and Vegetables--A Case Study of the Flavr
Savr.TM. Tomato", CRC Press, Inc. (1992) disclosed introducing an
anti-sense DNA construct into a tomato genome by Agrobacterium
transformation to produce gene silencing of the polygalacturonase
(PG) gene. A common characteristic of transferred DNA (T-DNA) in
transgenic plants exhibiting the desired trait was two or more
T-DNA regions or fragments inserted in a head to head and/or tail
to tail arrangement consistent with a report by Jorgensen et al.
Mol. Gen. Genet. 207:471-477 (1987) that multiple copies of the
T-DNA are often transferred to and integrated into the genome of a
single cell; and, when this occurs, the T-DNAs are predominately
organized in inverted repeat structures in plants transformed with
Agrobacterium. With reference to FIG. 1 and Table 1 tomato was
transformed with a plasmid containing the anti-sense construct
(FIG. 1a) comprising a full-length PG cDNA in the anti-sense
orientation between an "enhanced" 35S CaMV promoter and the 3'
region of the Agrobacterium tml gene and an artificial kan marker
gene. TABLE-US-00001 TABLE 1 Element Reference Left border from
T-DNA of pTiA6, Barker et al., Plant Mol. Biol. 2: 335-350 (1983)
Mas 5' from mannopine synthase gene, Barker et al., ibid promoter
Npt II neomycin phosphotransferase gene from transposon Tn5,
Jorgenson, Mol. Gen. 177: 65 (1979) Mas 3' polyadenylation region
from mannopine synthase gene Barker et al., ibid Double Gardner et
al., Nucl. Acids Res. 9: 2871-2888 (1981) CaMV35S promoter
Anti-sense full length of polygalacturonase cDNA in anti-sense PG
orientation, Sheehy et al. Proc. Natl. Acad. Sci. USA. 85:
8805-8809 Tml 3' polyadenylation region of tml gene from pTiA6,
Barker et al. ibid Right border with overdrive t-strand enhancer
element, McBride et al. Plant, Mol. Biol. 14: 269-276 (1990)
[0004] This construct was used for commercial-scale transformations
of several inbred tomato lines as part of the development and
marketing of Flavr Savr.TM. tomatoes by Calgene in 1994. Tomato
lines denoted 501, 502, 7B, 22B and 28B were transformed with
pCGN1436 using disarmed Agrobacterium tumefaciens. Events were
selected based primarily on phenotype, i.e. low PG enzyme activity
in ripe fruit. Approximately 150 transgenic event plants were
produced for each inbred and 573 plants with ripe fruit were
assayed for PG levels. Between 14-25% of those events across all
tomato lines had PG activity lowered by 95% or greater and resulted
in a total of 103 events. Of those plants, 84 had enough seed for
kanamycin germination assays to determine segregation ratios and 27
events (representing between 3-10 events for each inbred)
segregated 3:1 for the kan gene. Based on preliminary southern
analysis, only about 40% of the 27 events with 3:1 segregation
ratios clearly appeared to have the PGAS gene and kan gene inserted
at a single physical locus. Eight of those events were chosen for
detailed molecular analysis of T-DNA insert structures based on the
availability of homozygous lines. The results of those analyses are
shown in FIG. 1b-d, with the finding that all 8 events across the
inbred lines had T-DNA inserts containing inverted repeat elements.
The data were consistent with event 501-1001 having only a single
T-DNA insert, but with the tml 3' region present as an inverted
repeat as illustrated in FIG. 1b. Six events appeared to contain
two T-DNA regions in a "tail to tail" arrangement as illustrated in
FIG. 1c and event 501-1035 had 3 inserts integrated in a manner
illustrated in FIG. 1d.
[0005] Northern analysis of the 8 selected events demonstrated no
correlation between PG anti-sense RNA levels and the efficacy of PG
gene silencing. A range of PG anti-sense RNA levels were observed,
ranging from easily detected amounts in one event to undetectable
levels in multiple events, all of which produced the gene silenced
trait of delayed ripening. Potential read-through transcripts
larger in size than expected were detected for the marker kan gene
and for the PG anti-sense gene. The observation that inverted
repeat elements in T-DNA inserts were likely transcribed as larger
than expected RNAs, albeit at low levels, supports the thesis that
PG mRNA reductions were due to RNAi induced by the production of
RNA capable of forming dsRNA. The structure of anti-sense insert
illustrated in FIG. 1b with inverted repeat of 3' tml (sense
followed by anti-sense) is very similar to the sense construct
utilized for gene silencing by Brummell et al. as disclosed in
Plant Journal, 33, 793-800 (2003) using 3' nos element (anti-sense
followed by sense) as an inverted repeat. In each case a 3' hairpin
loop could be formed and used as primer for RNA-dependent RNA
polymerase and the formation of dsRNA sequences of the target
RNA.
[0006] The discovery of inverted repeats of inserted T-DNA
illustrated in FIG. 1c and as an element of FIG. 1c, suggested
increasing the efficacy of transformation with anti-sense DNA
constructs by directly transforming with the inverted repeat in the
plasmid. Yet, the presence of inverted repeats in plasmids has been
believed to be problematic when inside bacteria, e.g. E. coli,
which interfere with plasmid maintenance, resulting in plasmid
instability. The following described invention provides the
potential advantages of employing inverted repeat elements in a
transformation construct without the disadvantage of adjacent
inverted repeats in bacteria.
[0007] A single expression cassette containing inverted repeats of
sequences from a target gene may not be effective for gene
suppression in desired plant tissue. For instance, the CaMV 35S
promoter is typically denoted as "constitutive", but is does not
express well in pollen. The "constitutive" rice actin 1 promoter
expresses well in pollen but not as well in leaves. The following
described invention provides advantages of gene suppression in
multiple plant tissues not afforded by use of a single cassette
with a single promoter.
SUMMARY OF THE INVENTION
[0008] This invention provides an improved method of gene
suppression comprising transforming eukaryotic cells with multiple
gene suppression constructs located adjacent to each other on a
plasmid. In one aspect of the invention the multiple gene
suppression constructs can be multiple adjacent anti-sense gene
suppression constructs; in another aspect they can be multiple
adjacent sense (co-suppression) gene suppression constructs. In a
further aspect, they can be multiple adjacent sense and anti-sense
gene suppression constructs. The multiple adjacent gene suppression
constructs can be overlapping or non-overlapping. More particularly
the method comprises inserting into a plasmid for
Agrobacterium-mediated transformation a cassette for expressing
sense (or anti-sense) DNA from a gene targeted for suppression
adjacent to a second cassette for expressing the same sense (or
anti-sense) DNA.
[0009] The invention further provides transgenic seed having in its
genome a recombinant DNA construct comprising: (a) a plant
endosperm-specific promoter operably linked to at least one first
gene suppression element, and (b) a plant embryo-specific promoter
in the opposite orientation to the plant endosperm-specific
promoter and located 3' to the at least one first gene suppression
element.
[0010] The invention further provides stably transgenic plant cells
having in their genome a recombinant DNA construct comprising: (a)
a first promoter operably linked to at least one first gene
suppression element for silencing at least one first target gene,
and (b) a second promoter that is in the opposite orientation to
the first promoter and is located 3' to the at least one first gene
suppression element, wherein the first and the second promoters
have dissimilar expression patterns, and wherein transcription of
the recombinant DNA construct in a plant cell results in silencing
of the at least one first target gene.
[0011] This invention further provides constructs for
transformation of eukaryotic cells (such as plant cells), methods
for their use, and stably transformed transgenic plant cells
containing such constructs. These constructs, include (a) a first
promoter operably linked to at least one first gene suppression
element for silencing at least one first target gene, and (b) a
second promoter that is in the opposite orientation to the first
promoter and is located 3' to the at least one first gene
suppression element, wherein the first and said second promoters
have dissimilar expression patterns, and wherein transcription of
the recombinant DNA construct in a eukaryotic cell (such as a plant
cell) results in silencing of the at least one first target gene.
The dissimilar expression patterns include spatially or temporally
dissimilar expression patterns, as well as inducible expression
patterns.
[0012] A characteristic of the invention is variation in regulatory
elements in the cassettes, i.e. the promoter regulatory elements
and/or the polyadenylation regulatory elements. In embodiments
using anti-sense cassettes, the first anti-sense expression
cassette comprises a first promoter operably linked to DNA of a
gene targeted for suppression in an anti-sense orientation
optionally followed by a first 3' element (e.g. comprising a
polyadenylation signal and polyadenylation site); and, the second
anti-sense RNA expression cassette comprises a second promoter
operably linked to said DNA of a gene targeted for suppression in
an anti-sense orientation optionally followed by a second 3'
element. The first and second cassettes are assembled into a DNA
construct in a tail-to-tail configuration so that the promoters are
at the ends of the assembled construct bounding transcribable DNA
of the gene targeted for suppression and, when 3' elements are
used, the 3' elements are (a) contiguous or (b) adjacent to the
promoters either between the promoters and the transcribable DNA or
at the extreme regions of the assembly. At a minimum the first and
second promoters are different. First and second 3' elements can
also be different.
[0013] The method further comprises transforming eukaryotic cells
by transferring a DNA construct with such assembled first and
second cassettes from a plasmid by Agrobacterium-mediated
transformation. A transgenic organism is regenerated from cells
transformed with the first and second cassettes; and, a trait
resulting from suppression of the level of protein encoded by said
DSA of a gene targeted for suppression is measured in the
transgenic organism.
[0014] In aspects of the method promoters can include well-known
promoters that are functional in plants including Agrobacterium
nopaline synthase (nos) promoter, Agrobacterium octopine synthase
(ocs) promoter, the cauliflower mosaic virus promoter (CaMV 35S),
figwort mosaic virus promoter (FMV), maize RS81 promoter, rice
actin promoter, maize RS324 promoter, maize PR-1 promoter, maize A3
promoter, gamma coixin B32 endosperm-specific promoter, maize L3
oleosin embryo-specific promoter, rd29a promoter, and any of the
other well-know promoters useful in plant gene expression.
[0015] In aspects of the method the intron is any spliceable
intron. In some embodiments, the intron is preferably a
transcription-enhancing intron, e.g., "enhancers" such as 5'
introns of the rice actin 1 and rice actin 2 genes, the maize
alcohol dehydrogenase gene, the maize heat shock protein 70 gene,
and the maize shrunken 1 gene.
[0016] In aspects of the method the 3' elements are selected from
the group consisting of the well-known 3' elements, e.g.
Agrobacterium gene 3' elements such as nos 3', tml 3', tmr 3', tms
3', ocs 3', tr7 3' and plant gene 3' elements such as wheat
(Triticum aestivum) heat shock protein 17 (Hsp17) 3', a wheat
ubiquitin gene 3', a wheat fructose-1,6-biphosphatase gene 3', a
rice glutelin gene 3', a rice lactate dehydrogenase gene 3', a rice
beta-tubulin gene 3', a pea (Pisum sativum) ribulose bisphosphate
carboxylase gene (rbs) 3', and 3' elements from other genes within
the host plant.
[0017] In other aspects of the method at least one of the multiple
cassettes comprises a marker gene, e.g. an herbicide marker gene
that provides resistance to glyphosate (aroA or EPSPS) or
glufosinate (pat or bar); a bacteriocide marker gene that provides
resistance to kanamycin (npt II), gentamycin (aac 3), hygromycin
(aph IV), streptomycin and spectinomycin (aadA), or ampicillin
(amp); or a screenable marker such as a luciferase (luc) or a
fluorescent protein (gfp) or a beta-glucuronidase (uidA). The
length of the DNA of a gene targeted for suppression can be any
length, but preferably at least 21 nucleotides in length.
[0018] Another aspect of the invention provides a plasmid for
Agrobacterium-mediated transformation comprising such a first
cassette for expressing sense (or anti-sense) DNA from a gene
targeted for suppression adjacent to such a second cassette for
expressing the same DNA, where the cassettes are assembled so that
the different 3' untranslated regions are contiguous. In many cases
the cassettes and at least one marker cassette are located between
left and right T-DNA borders on the plasmid.
[0019] In a preferred aspect of the invention a transgenic corn
plant contains a DNA construct with adjacent cassettes for
anti-sense suppression of the lysine ketoglutarate reductase gene
using an endosperm specific promoter in one cassette and an embryo
specific promoter in the other cassette.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] FIGS. 1 and 2 illustrate DNA constructs.
[0021] FIG. 3 depicts non-limiting examples of constructs of the
invention, e.g., as described in Example 3. The endosperm-specific
promoter is indicated by "pB32", the embryo-specific promoter by
"pL3", the gene suppression element(s) by "SUP-LKR/SDH" (which
represents a stabilized anti-sense suppression element targetting
endogenous lysine ketoglutarate reductase/saccharopine
dehydrogenase), "GSE1", and "GSE2", and terminators by "tHsp17",
"tGlb1", "ter1", and "ter2".
DETAILED DESCRIPTION OF THE INVENTION
[0022] As used herein "cassette" means a combination of DNA
elements normally associated with the expression of protein from a
gene and comprises at least (a) DNA for initiating transcription
such as a promoter element, (b) DNA coding for a protein such as
cDNA or genomic DNA comprising exons and introns, and (c) DNA for
splicing 3' RNA from transcribed RNA after coding sequence and
adding a polyA tail such as a 3' element containing a
polyadenylation site. Typically, when the DNA coding for a protein
is in a sense orientation, the transcribed RNA can be translated to
express protein or, in some cases, for sense co-suppression. When
the DNA coding for protein is in an anti-sense orientation, the
transcribed RNA can be involved in a gene suppression mechanism.
For instance, to promote gene suppression anti-sense DNA typically
corresponds to DNA that is transcribed to mRNA upstream of a
polyadenylation site. Thus, an "anti-sense cassette" means a
combination of DNA elements comprising a promoter operably linked
to anti-sense oriented DNA from a gene targeted for suppression and
a 3' element. Although common, it is not critical that the 3'
element contain a polyadenylation site. What is important in either
adjacent sense cassettes or adjacent anti-sense cassettes is that
adjacent 3' elements are distinct, i.e. transcribed RNA from
adjacent 3' elements is are not capable of hybridizing to from
double-stranded RNA or being readily excised from a plasmid in E.
coli.
[0023] Recombinant DNA constructs, e.g. the cassettes of this
invention, can be readily prepared by those skilled in the art
using commercially available materials and well-known, published
methods. When multiple genes are targeted for suppression,
polycistronic DNA elements can be fabricated as illustrated and
disclosed in U.S. application Ser. No. 10/465,800, incorporated
herein by reference. A useful technology for building DNA
constructs and vectors for transformation is the GATEWAY.TM.
cloning technology (available from Invitrogen Life Technologies,
Carlsbad, Calif.) uses the site specific recombinase LR cloning
reaction of the Integrase att system from bacteriophage lambda
vector construction, instead of restriction endonucleases and
ligases. The LR cloning reaction is disclosed in U.S. Pat. Nos.
5,888,732 and 6,277,608, U.S. Patent Application Publications
2001283529, 2001282319, 20020007051, and 20040115642, all of which
are incorporated herein by reference. The GATEWAY.TM. Cloning
Technology Instruction Manual which is also supplied by Invitrogen
also provides concise directions for routine cloning of any desired
DNA into a vector comprising operable plant expression
elements.
[0024] An alternative vector fabrication method employs
ligation-independent cloning as disclosed by Aslandis, C. et al.,
Nucleic Acids Res., 18, 6069-6074, 1990 and Rashtchian, A. et al.,
Biochem., 206, 91-97, 1992 where a DNA fragment with
single-stranded 5' and 3' ends are ligated into a desired vector
which can then be amplified in vivo.
[0025] Numerous promoters that are active in plant cells have been
described in the literature. These include promoters present in
plant genomes as well as promoters from other sources, including
nopaline synthase (NOS) promoter and octopine synthase (OCS)
promoters carried on tumor-inducing plasmids of Agrobacterium
tumefaciens, caulimovirus promoters such as the cauliflower mosaic
virus or figwort mosaic virus promoters. For instance, see U.S.
Pat. Nos. 5,858,742 and 5,322,938 which disclose versions of the
constitutive promoter derived from cauliflower mosaic virus
(CaMV35S), U.S. Pat. No. 5,378,619 which discloses a Figwort Mosaic
Virus (FMV) 35S promoter, U.S. Pat. No. 6,437,217 which discloses a
maize RS81 promoter, U.S. Pat. No. 5,641,876 which discloses a rice
actin promoter, U.S. Pat. No. 6,426,446 which discloses a maize
RS324 promoter, U.S. Pat. No. 6,429,362 which discloses a maize
PR-1 promoter, U.S. Pat. No. 6,232,526 which discloses a maize A3
promoter, U.S. Pat. No. 6,177,611 which discloses constitutive
maize promoters, U.S. Pat. No. 6,433,252 which discloses a maize L3
oleosin promoter, U.S. Pat. No. 6,429,357 which discloses a rice
actin 2 promoter and intron, U.S. Pat. No. 5,837,848 which
discloses a root specific promoter, U.S. Pat. No. 6,084,089 which
discloses cold inducible promoters, U.S. Pat. No. 6,294,714 which
discloses light inducible promoters, U.S. Pat. No. 6,140,078 which
discloses salt inducible promoters, U.S. Pat. No. 6,252,138 which
discloses pathogen inducible promoters, U.S. Pat. No. 6,175,060
which discloses phosphorus deficiency inducible promoters, U.S.
Patent Application Publication 2002/0192813A1 which discloses 5',
3' and intron elements useful in the design of effective plant
expression vectors, U.S. patent application Ser. No. 09/078,972
which discloses a coixin promoter, U.S. patent application Ser. No.
09/757,089 which discloses a maize chloroplast aldolase promoter,
and U.S. patent application Ser. No. 10/739,565 which discloses
water-deficit inducible promoters, all of which are incorporated
herein by reference. These and numerous other promoters that
function in plant cells are known to those skilled in the art and
available for use in recombinant polynucleotides of the present
invention to provide for expression of desired genes in transgenic
plant cells.
[0026] In aspects of the method the 3' elements are selected from
the group consisting of the well-known 3' elements from
Agrobacterium tumefaciens genes such as nos 3', tml 3', tmr 3', tms
3', ocs 3', tr7 3', e.g. disclosed in U.S. Pat. No. 6,090,627,
incorporated herein by reference; 3' elements from plant genes such
as wheat (Triticum aestivum) heat shock protein 17 (Hsp17 3'), a
wheat ubiquitin gene, a wheat fructose-1,6-biphosphatase gene, a
rice glutelin gene a rice lactate dehydrogenase gene and a rice
beta-tubulin gene, all of which are disclosed in U.S. published
patent application 2002/0192813 A1, incorporated herein by
reference; and the pea (Pisum sativum) ribulose bisphosphate
carboxylase gene (rbs 3'), and 3' elements from the genes within
the host plant.
[0027] Furthermore, the promoters may be altered to contain
multiple "enhancer sequences" to assist in elevating gene
expression. Such enhancers are known in the art. By including an
enhancer sequence with such constructs, the expression of the
selected protein may be enhanced. These enhancers often are found
5' to the start of transcription in a promoter that functions in
eukaryotic cells, but can often be inserted in the forward or
reverse orientation 5' or 3' to the coding sequence. In some
instances, these 5' enhancing elements are introns. Particularly
useful enhancers are the 5' introns of the rice actin 1 (see U.S.
Pat. No. 5,641,876) and rice actin 2 genes, the maize alcohol
dehydrogenase gene, the maize heat shock protein 70 gene (see U.S.
Pat. No. 5,593,874) and the maize shrunken 1 gene.
[0028] In some aspects of the invention it is preferred that the
promoter element in the DNA construct be capable of causing
sufficient expression to result in the production of an effective
amount of a polypeptide in water deficit conditions. Such promoters
can be identified and isolated from the regulatory region of plant
genes that are over expressed in water deficit conditions. Specific
water-deficit-inducible promoters for use in this invention are
derived from the 5' regulatory region of genes identified as a heat
shock protein 17.5 gene (HSP17.5), an HVA22 gene (HVA22), a Rab17
gene and a cinnamic acid 4-hydroxylase (CA4H) gene (CA4H) of Zea
mays, or derived from the 5' regulatory region of genes identified
as a rab17 gene (RAB17), a cinnamic acid 4-hydroxylase (CA4H) gene
(CA4H), an HVA22 gene (HVA22), and genes for heat shock proteins
17.5 (HSP17.5), 22 (HSP22) and 16.9 (HSP16.9) of Oryza sativa. Such
water-deficit-inducible promoters are disclosed in U.S. application
Ser. No. 10/739,565 and Ser. No. 11/066,911, incorporated herein by
reference.
[0029] In other aspects of the invention, sufficient expression in
plant seed tissues is desired to effect improvements in seed
composition. Exemplary promoters for use for seed composition
modification include promoters from seed genes such as napin (U.S.
Pat. No. 5,420,034), maize L3 oleosin (U.S. Pat. No. 6,433,252),
zein Z27 (Russell et al. (1997) Transgenic Res. 6(2): 157-166),
globulin 1 (Belanger et al (1991) Genetics 129:863-872), glutelin 1
(Russell (1997) supra), and peroxiredoxin antioxidant (Per1) (Stacy
et al. (1996) Plant Mol Biol. 31(6):1205-1216).
[0030] In still other aspects of the invention, preferential
expression in plant green tissues is desired. Promoters of interest
for such uses include those from genes such as SSU (Fischhoff et
al. (1992) Plant Mol Biol. 20:81-93), aldolase and pyruvate
orthophosphate dikinase (PPDK) (Taniguchi et al. (2000) Plant Cell
Physiol. 41(1):42-48).
[0031] In practice DNA is introduced into only a small percentage
of target cells in any one transformation experiment. Marker genes
are used to provide an efficient system for identification of those
cells that are stably transformed by receiving and integrating a
transgenic DNA construct into their genomes. Preferred marker genes
provide selective markers which confer resistance to a selective
agent, such as an antibiotic or herbicide. Any of the herbicides to
which plants of this invention may be resistant are useful agents
for selective markers. Potentially transformed cells are exposed to
the selective agent. In the population of surviving cells will be
those cells where, generally, the resistance-conferring gene is
integrated and expressed at sufficient levels to permit cell
survival. Cells may be tested further to confirm stable integration
of the exogenous DNA. Commonly used selective marker genes include
those conferring resistance to antibiotics such as kanamycin
(nptII), hygromycin B (aph IV) and gentamycin (aac3 and aacC4) or
resistance to herbicides such as glufosinate (bar or pat) and
glyphosate (EPSPS). Examples of such selectable markers are
illustrated in U.S. Pat. Nos. 5,550,318; 5,633,435; 5,780,708 and
6,118,047, all of which are incorporated herein by reference.
Screenable markers which provide an ability to visually identify
transformants can also be employed, e.g., a gene expressing a
colored or fluorescent protein such as a luciferase or green
fluorescent protein (GFP) or a gene expressing a beta-glucuronidase
or uidA gene (GUS) for which various chromogenic substrates are
known.
[0032] The invention provides transgenic seed having in its genome
a recombinant DNA construct comprising: (a) a plant
endosperm-specific promoter operably linked to at least one first
gene suppression element, and (b) a plant embryo-specific promoter
in the opposite orientation to the plant endosperm-specific
promoter and located 3' to the at least one first gene suppression
element. In some embodiments, the plant embryo-specific promoter
can transcribe the at least one first gene suppression element. In
other embodiments, the plant embryo-specific promoter can
transcribe at least one second gene suppression element (e.g., a
second gene suppression element for silencing the same gene
targetted by the endosperm-specific promoter, or for silencing a
different gene).
[0033] In one embodiment of the transgenic seed, the at least one
first gene suppression element includes a gene suppression element
for silencing a catabolism gene of an amino acid (or of an amino
acid's biosynthetic intermediates), such as, but not limited to, a
lysine catabolism gene. Other catabolism genes can be silenced,
such as genes involved in catabolism of lipids or carbohydrates or
of their biosynthetic intermediates. In one specifically claimed
embodiment, the transgenic seed is transgenic maize seed, and the
amino acid catabolism gene is a lysine catabolism gene, such as the
endogenous maize LKR/SDH gene.
[0034] In some embodiments of the transgenic seed, the recombinant
DNA construct further includes one or more elements selected from:
(a) at least one second gene suppression element operably linked to
the plant embryo-specific promoter; (b) an amino acid biosynthesis
gene operably linked to either the plant endosperm-specific
promoter or plant embryo-specific promoter; and (c) a selectable
marker gene. In some embodiments where both a gene suppression
element and an expression element for another gene (e.g., an amino
acid biosynthesis gene) are operably linked to one promoter, the
gene suppression element can be embedded in an intron, which in
many embodiments is preferably a transcription-enhancing intron
(e.g., "enhancers" such as 5' introns of the rice actin 1 and rice
actin 2 genes, the maize alcohol dehydrogenase gene, the maize heat
shock protein 70 gene, and the maize shrunken 1 gene). In some
preferred embodiments, the recombinant DNA construct further
comprises one or more elements selected from: (a) at least one
second gene suppression element for silencing a lysine catabolism
gene operably linked to the plant embryo-specific promoter; (b) a
lysine biosynthesis (e.g., an exogenous DHDPS or CordapA gene)
biosynthesis gene operably linked to the plant endosperm-specific
promoter; (c) an aspartate kinase gene (e.g., a lysC gene) operably
linked to either the plant endosperm-specific promoter or plant
embryo-specific promoter; and (d) a selectable marker gene. In
certain preferred embodiments, the construct includes an aspartate
kinase gene (operably linked to either the embryo- or the
endosperm-specific promoter) and a gene suppression element for
silencing endogenous LKR/SDH (preferably operably linked to the
endosperm-specific promoter or to both the embryo- and the
endosperm-specific promoters), and preferably also includes an
exogenous DHDPS or CordapA gene (operably linked to the
endosperm-specific promoter). Marker genes include selectable
markers (such as are commonly used to select transformed cells,
e.g., antibiotic or herbicide resistance genes), detectable markers
(e.g., luciferase, green fluorescent protein, GUS), and can include
coding sequence or non-coding sequence (for example a suppression
element that suppresses an endogenous gene resulting in an
observable phenotype, e.g., a suppression element for silencing a
gene involved in plant pigment production).
[0035] FIG. 3 depicts non-limiting embodiments of recombinant DNA
constructs useful for providing transgenic seeds of the
invention:
[0036] (a) (see FIG. 3A) the recombinant DNA construct includes:
(i) a plant endosperm-specific promoter operably linked to at least
one first gene suppression element including DNA that transcribes
to RNA for silencing a lysine catabolism gene by forming
double-stranded RNA (e.g., DNA that includes at least one
anti-sense DNA segment that is anti-sense to at least one segment
of the at least one target gene and at least one sense DNA segment
that is at least one segment of the at least one first target gene;
or DNA that encodes at least one trans-acting miRNA and that
transcribes to double-stranded RNA targetted to the target gene in
both transcriptional directions), and (ii) a plant embryo-specific
promoter in the opposite orientation to the first promoter and
operably linked to the at least one first gene suppression
element;
[0037] (b) (see FIG. 3B) the recombinant DNA construct includes:
(i) a plant endosperm-specific promoter operably linked to at least
one first gene suppression element including DNA that transcribes
to RNA for silencing a lysine catabolism gene by forming
double-stranded RNA (e.g., DNA that includes at least one
anti-sense DNA segment that is anti-sense to at least one segment
of the at least one target gene and at least one sense DNA segment
that is at least one segment of the at least one first target gene;
or DNA that encodes a trans-acting miRNA in both transcriptional
directions), (ii) a plant embryo-specific promoter in the opposite
orientation to the first promoter and operably linked to the at
least one first gene suppression element, and (iii) at least one
terminator operably linked to either the first or second promoters
(wherein each terminator can be on either side of the oppositely
oriented promoter); or
[0038] (c) (see FIG. 3C) the recombinant DNA construct includes:
(i) a plant endosperm-specific promoter operably linked to at least
one first intron-embedded gene suppression element for silencing a
lysine catabolism gene, at least one lysine biosynthesis gene
(preferably cordapA or lysC or both), and a first terminator, (ii)
a plant embryo-specific promoter in the opposite orientation to the
first promoter and operably linked to at least one second gene
suppression element (which is optionally embedded in an intron,
preferably a transcription-enhancing intron) for silencing a lysine
catabolism gene; or
[0039] (d) (see FIG. 3D) the recombinant DNA construct includes:
(i) a first gene suppression cassette including a plant
endosperm-specific promoter operably linked to at least one first
intron-embedded gene suppression element for silencing a lysine
catabolism gene, at least one lysine biosynthesis gene (preferably
cordapA or lysC or both), and a first terminator, and (ii) a second
gene suppression cassette including a plant embryo-specific
promoter operably linked to at least one second gene suppression
element for silencing a lysine catabolism gene, and a second
terminator, wherein the first and second gene suppression cassettes
are in opposite orientations (optionally assembled so that the
promoters are at the ends of the construct); or
[0040] (e) (see FIG. 3E) the recombinant DNA construct includes:
(i) a first gene suppression cassette including a plant
endosperm-specific promoter operably linked to at least one first
intron-embedded gene suppression element for silencing a lysine
catabolism gene, at least one lysine biosynthesis gene (preferably
cordapA or lysC or both), and a first terminator, and (ii) a second
gene suppression cassette including a plant embryo-specific
promoter operably linked to at least one intron-embedded second
gene suppression element for silencing a lysine catabolism gene, at
least one lysine biosynthesis gene (preferably cordapA or lysC or
both) and a second terminator, wherein the first and second gene
suppression cassettes are in opposite orientations (optionally
assembled so that the promoters are at the ends of the construct);
or
[0041] (f) (see FIG. 3F) the recombinant DNA construct includes:
(i) a first gene suppression cassette including a plant
endosperm-specific promoter operably linked to at least one first
gene suppression element for silencing a lysine catabolism gene,
and a first terminator, and (ii) a second gene suppression cassette
including a plant embryo-specific promoter operably linked to at
least one second gene suppression element for silencing a lysine
catabolism gene, and a second terminator, wherein the first and
second gene suppression cassettes are in opposite orientations
(optionally assembled so that the promoters are at the ends of the
construct).
[0042] FIG. 4 depicts other specific embodiments of constructs of
the invention. While FIGS. 3 and 4 depict some gene suppression
elements as including sense and anti-sense sequence in the form of
a stabilized anti-sense element ("SUP-LKR/SDH"), other gene
suppression elements are useful, providing that they are
transcribed by the appropriate promoter to an RNA molecule or
molecules capable of suppressing the target gene(s). Where
constructs include two non-overlapping expression "cassettes" (see,
for example, FIGS. 3D, 3E, and 3F), an alternative arrangement is
for the two promoters to be located adjacent to each other and
oppositely oriented (resulting in "divergent" transcription).
[0043] Generally, it is preferable to prevent "read through" of
terminators and unintentional silencing of, e.g., an opposing
promoter or sequence operably linked to an opposing promoter. Thus,
in some embodiments, an intron or other spliceable element such as
a ribozyme can be optionally inserted (see, for example, the bottom
construct of FIG. 3B) to prevent "read through" of any downstream
sequence. In some embodiments where a transcript of a gene
suppression element need not be polyadenylated (e.g., where the
target is localized in the nucleus), an intron or other spliceable
element such as a ribozyme can be inserted to prevent "read
through" of the opposing promoter (see, for example, the bottom
construct of FIG. 3A). In other embodiments, an intron can be
arranged to include a gene suppression element embedded within it,
and further to prevent "read through" of any downstream sequence
(see, for example, the bottom construct of FIG. 3E).
[0044] The invention further provides stably transgenic plant cells
having in their genome a recombinant DNA construct including: (a) a
first promoter operably linked to at least one first gene
suppression element for silencing at least one first target gene,
and (b) a second promoter that is in the opposite orientation to
the first promoter and is located 3' to the at least one first gene
suppression element, wherein the first and the second promoters
have dissimilar expression patterns, and wherein transcription of
the recombinant DNA construct in a plant cell results in silencing
of the at least one first target gene. By "stably transgenic plant
cells" is meant plant cells that have stably integrated an
exogenous gene (transgene) into their genome. In many preferred
embodiments, such stably transgenic plant cells are homozygous for
the transgene. In particularly preferred embodiments, the
integrated transgene is heritable, that is, transferable to progeny
plants. The dissimilar expression patterns include spatially or
temporally dissimilar expression patterns, as well as inducible
expression patterns. Non-limiting examples of suitable first and
second promoters include first and second promoters that control
transcription in different organelles, cells, or tissues, or first
and second promoters that control transcription under different
times (e.g., at different points of a circadian cycle) or
developmental periods, or first and second promoters that are
induced differently by an inducer or are induced by different
inducers. The stably transgenic plant cells can be isolated
transgenic plant cells or can be in a transgenic plant regenerated
from the transgenic plant cell, or a transgenic progeny seed or
transgenic progeny plant of such a regenerated transgenic plant. In
one preferred embodiment of the stably transgenic plant cells, the
first and the second promoters comprise a plant embryo-specific
promoter and a plant endosperm-specific promoter and the stably
transgenic plant cells comprise seed embryo and endosperm cells of
a crop plant (e.g., maize, rice, or other crop plants that have
seed containing substantial endosperm).
[0045] This invention further provides constructs for
transformation of eukaryotic cells (such as plant cells and animal
cells), methods for their use, and stably transgenic plant cells
containing such constructs. These constructs include (a) a first
promoter operably linked to at least one first gene suppression
element for silencing at least one first target gene, and (b) a
second promoter that is in the opposite orientation to the first
promoter and is located 3' to the at least one first gene
suppression element, wherein the first and said second promoters
have dissimilar expression patterns, and wherein transcription of
the recombinant DNA construct in a eukaryotic cell (such as a plant
cell or animal cell) results in silencing of the at least one first
target gene. The dissimilar expression patterns include spatially
or temporally dissimilar expression patterns, as well as inducible
expression patterns. Thus, in some embodiments, the first and
second promoters have dissimilar spatial expression patterns, and
the silencing occurs in at least two distinct spatial locations. In
other embodiments, the first and second promoters have dissimilar
temporal expression patterns, and the silencing occurs in at least
two distinct times or developmental stages (either non-overlapping
or overlapping periods of time). Suitable promoters include, for
example, first and second promoters that control transcription in
different organelles (e.g., plastids, nucleus, mitochondria),
cells, or tissues, or first and second promoters that control
transcription under different times (e.g., at different points of a
circadian cycle) or developmental periods, or first and second
promoters that are induced differently by an inducer or are induced
by different inducers.
[0046] In some embodiments of the recombinant DNA construct, the at
least one gene suppression element is under transcriptional control
of both the first and the second promoters. In these embodiments,
the at least one gene suppression element is transcribed in both
directions and suppresses the at least one target gene in two
locations (or at two distinct times or developmental stages).
[0047] In some embodiments, the recombinant DNA construct further
includes one or more of: (a) a second gene suppression element
operably linked to the second promoter; (b) at least one gene
expression element for expressing at least one exogenous gene; (c)
at least one terminator, and (d) at least one T-DNA border. The
second gene suppression element is arranged such that transcription
of the second gene suppression element results in the intended
silencing of the gene it targets; thus, in many embodiments, the
second gene suppression element is oriented opposite to the first
promoter. The at least one exogenous gene expressed by the at least
on gene expression element can be any gene or genes to be expressed
out of native context, and can include, e.g., a marker gene, a
codon-optimized gene, an allelic replacement of a native gene.
[0048] In some embodiments of the recombinant DNA construct, the at
least one first gene suppression element includes at least one
element selected from the group consisting of: (a) DNA that
includes at least one anti-sense DNA segment that is anti-sense to
at least one segment of the at least one first target gene; (b) DNA
that includes multiple copies of at least one anti-sense DNA
segment that is anti-sense to at least one segment of the at least
one first target gene; (c) DNA that includes at least one sense DNA
segment that is at least one segment of the at least one first
target gene; (d) DNA that includes multiple copies of at least one
sense DNA segment that is at least one segment of the at least one
first target gene; (e) DNA that transcribes to RNA for suppressing
the at least one first target gene by forming double-stranded RNA
and includes at least one anti-sense DNA segment that is anti-sense
to at least one segment of the at least one target gene and at
least one sense DNA segment that is at least one segment of the at
least one first target gene; (f) DNA that transcribes to RNA for
suppressing the at least one first target gene by forming a single
double-stranded RNA and includes multiple serial anti-sense DNA
segments that are anti-sense to at least one segment of the at
least one first target gene and multiple serial sense DNA segments
that are at least one segment of the at least one first target
gene; (g) DNA that transcribes to RNA for suppressing the at least
one first target gene by forming multiple double strands of RNA and
includes multiple anti-sense DNA segments that are anti-sense to at
least one segment of the at least one first target gene and
multiple sense DNA segments that are at least one segment of the at
least one first target gene, and wherein the multiple anti-sense
DNA segments and the multiple sense DNA segments are arranged in a
series of inverted repeats; (h) DNA that includes nucleotides
derived from a plant miRNA; (i) DNA that includes nucleotides of a
siRNA; (j) DNA that transcribes to an RNA aptamer capable of
binding to a ligand; and (k) DNA that transcribes to an RNA aptamer
capable of binding to a ligand, and DNA that transcribes to
regulatory RNA capable of regulating expression of the first target
gene, wherein the regulation is dependent on the conformation of
the regulatory RNA, and the conformation of the regulatory RNA is
allosterically affected by the binding state of the RNA aptamer.
Suitable gene suppression elements are further described in U.S.
patent application Ser. No. 11/303,745, which is incorporated
herein by reference.
[0049] In some embodiments of the recombinant DNA construct, the
first gene suppression element is embedded in an intron. In
preferred embodiments, the intron is flanked on one or on both
sides by non-protein-coding DNA, and more preferably is a
transcription-enhancing intron (e.g., "enhancers" such as 5'
introns of the rice actin 1 and rice actin 2 genes, the maize
alcohol dehydrogenase gene, the maize heat shock protein 70 gene,
and the maize shrunken 1 gene).
[0050] In some embodiments, the recombinant DNA construct further
includes a second gene suppression element operably linked to the
second promoter, wherein the first and second gene suppression
elements are embedded in an intron (either individually in separate
introns or together in a single intron). The second gene
suppression element is arranged such that transcription of the
second gene suppression element results in the intended silencing
of the gene it targets; thus, in many embodiments, the second gene
suppression element is oriented opposite to the first promoter.
[0051] In one particularly preferred embodiment of the recombinant
DNA construct, the first and the second promoters include a plant
embryo-specific promoter and a plant endosperm-specific
promoter.
[0052] Further provided by this invention is a method of gene
silencing in a plant, including: (a) transforming a plant cell with
the recombinant DNA construct including (i) a first promoter
operably linked to at least one first gene suppression element for
silencing at least one first target gene, and (ii) a second
promoter that is in the opposite orientation to the first promoter
and is located 3' to the at least one first gene suppression
element, wherein the first and said second promoters have
dissimilar expression patterns, and wherein transcription of the
recombinant DNA construct in a eukaryotic cell (such as a plant
cell or animal cell) results in silencing of the at least one first
target gene, thereby providing a transgenic plant cell; (b)
preparing a regenerated transgenic plant from the transgenic plant
cell, or a transgenic progeny seed or transgenic progeny plant of
the regenerated transgenic plant; (c) transcribing the recombinant
DNA construct in the regenerated transgenic plant or the transgenic
progeny seed or transgenic progeny plant, whereby the at least one
first target gene is silenced in the regenerated transgenic plant
or the transgenic progeny seed or transgenic progeny plant.
[0053] In preferred embodiments of the method, the plant is a crop
plant, for example, grain crops (e.g., maize, rice, wheat, barley,
rye), legumes (e.g., soybean, alfalfa, beans, peanuts), oilseeds
(e.g., rape, canola, soybean, nuts), and fruit or vegetable crop
plants. In one preferred embodiment, the recombinant DNA construct
is transcribed in a transgenic progeny seed having substantial
endosperm (e.g., a transgenic maize or rice seed or other cereal
grain seed), and the first and the second promoters include a plant
embryo-specific promoter and a plant endosperm-specific promoter.
Particularly preferred is the method wherein the transgenic progeny
seed is transgenic progeny maize seed, the at least one first
target gene is at least one lysine catabolism gene, and the at
least one lysine catabolism gene is silenced in embryo and
endosperm cells of the transgenic progeny seed. In another
particularly preferred embodiment of the method, the transgenic
progeny seed is transgenic progeny maize seed, the at least one
first target gene is at least one lysine catabolism gene, the at
least one lysine catabolism gene is silenced in embryo and
endosperm cells of the transgenic progeny seed, and the recombinant
DNA construct further includes at least one lysine biosynthesis
gene operably linked to the endosperm-specific promoter.
[0054] This invention further provides a method for manufacturing
transgenic maize seed having an increased level of a nutrient, the
method comprising: (a) selecting a first transgenic maize plant
comprising a recombinant DNA construct including (i) a first
promoter operably linked to at least one first gene suppression
element for silencing at least one first target gene, wherein the
at least one first target gene is a catabolism gene of a nutrient
selected from an amino acid, a lipid, or a carbohydrate, and (ii) a
second promoter that is in the opposite orientation to the first
promoter and is located 3' to the at least one first gene
suppression element, wherein the first and said second promoters
have dissimilar expression patterns, and wherein transcription of
the recombinant DNA construct in a eukaryotic cell (such as a plant
cell or animal cell) results in silencing of the at least one first
target gene; (b) introgressing the recombinant DNA construct into a
second maize plant; (c) growing seed from the second maize plant to
produce a population of progeny maize plants; (d) screening the
population of progeny maize plants for progeny maize plants that
produce maize seed having an increased level of the nutrient,
relative to non-transgenic maize plants; (e) selecting from the
population one or more progeny maize plants that produce maize seed
having an increased level of the nutrient, relative to
non-transgenic maize plants; (f) verifying that the recombinant DNA
construct is stably integrated in the selected progeny maize
plants; (g) verifying that the catabolism gene of the nutrient is
silenced in the selected progeny maize plants, relative to maize
plants lacking the recombinant DNA construct; (h) collecting
transgenic maize seed from the selected progeny maize plants. The
recombinant DNA construct optionally includes a gene expression
element. In various embodiments of the method, the nutrient to be
increased is an amino acid (e.g., lysine, methionine, or
tryptophan), a lipid (e.g., a fatty acid or fatty acid ester), or a
carbohydrate (e.g., a simple sugar or a complex carbohydrate). In
one preferred embodiment of the method, the nutrient is lysine, the
catabolism gene is a lysine catabolism gene (e.g., maize lysine
ketoglutarate reductase/saccharopine dehydrogenase), and the first
and the second promoters include a plant embryo-specific promoter
and a plant endosperm-specific promoter; optionally, the
recombinant DNA construct also includes a gene expression element
for expression of a lysine biosynthesis gene (e.g., cordapA or
lysC).
Plant Transformation Methods
[0055] Numerous methods for transforming plant cells with
recombinant DNA are known in the art and may be used in the present
invention. Two commonly used methods for plant transformation are
Agrobacterium-mediated transformation and microprojectile
bombardment. Microprojectile bombardment methods are illustrated in
U.S. Pat. No. 5,015,580 (soybean); U.S. Pat. No. 5,550,318 (corn);
U.S. Pat. No. 5,538,880 (corn); U.S. Pat. No. 5,914,451 (soybean);
U.S. Pat. No. 6,160,208 (corn); U.S. Pat. No. 6,399,861 (corn) and
U.S. Pat. No. 6,153,812 (wheat) and Agrobacterium-mediated
transformation is described in U.S. Pat. No. 5,159,135 (cotton);
U.S. Pat. No. 5,824,877 (soybean); U.S. Pat. No. 5,591,616 (corn);
and U.S. Pat. No. 6,384,301 (soybean), all of which are
incorporated herein by reference. For Agrobacterium tumefaciens
based plant transformation systems, additional elements present on
transformation constructs include T-DNA left and/or right border
sequences (generally both left and right border sequences, but
preferably at least one border sequence, e.g. at least a right
border sequence) to facilitate incorporation of the recombinant
polynucleotide into the plant genome.
[0056] In general it is useful to introduce recombinant DNA
randomly, i.e. at a non-specific location, in the genome of a
target plant line. In special cases it may be useful to target
recombinant DNA insertion in order to achieve site-specific
integration, e.g. to replace an existing gene in the genome, to use
an existing promoter in the plant genome, or to insert a
recombinant polynucleotide at a predetermined site known to be
active for gene expression. Several site specific recombination
systems exist which are known to function implants include
cre-10.times. as disclosed in U.S. Pat. No. 4,959,317 and FLP-FRT
as disclosed in U.S. Pat. No. 5,527,695, both incorporated herein
by reference.
[0057] Transformation methods of this invention are preferably
practiced in tissue culture on media and in a controlled
environment. "Media" refers to the numerous nutrient mixtures that
are used to grow cells in vitro, that is, outside of the intact
living organism. Recipient cell targets include, but are not
limited to, meristem cells, callus, immature embryos and gametic
cells such as microspores, pollen, sperm and egg cells. It is
contemplated that any cell from which a fertile plant may be
regenerated is useful as a recipient cell. Callus may be initiated
from tissue sources including, but not limited to, immature
embryos, seedling apical meristems, microspores and the like. Cells
capable of proliferating as callus are also recipient cells for
genetic transformation. Practical transformation methods and
materials for making transgenic plants of this invention, e.g.
various media and recipient target cells, transformation of
immature embryos and subsequent regeneration of fertile transgenic
plants are disclosed in U.S. Pat. Nos. 6,194,636 and 6,232,526 and
U.S. patent application Ser. No. 09/757,089, which are incorporated
herein by reference.
[0058] The seeds of transgenic plants can be harvested from fertile
transgenic plants and be used to grow progeny generations of
transformed plants of this invention including hybrid plants line
comprising the recombinant DNA construct expressing an agent for
genes suppression.
[0059] In addition to direct transformation of a plant with a
recombinant DNA construct, transgenic plants can be prepared by
crossing a first plant having a recombinant DNA construct with a
second plant lacking the construct. For example, recombinant DNA
for gene suppression can be introduced into a first plant line that
is amenable to transformation to produce a transgenic plant which
can be crossed with a second plant line to introgress the
recombinant DNA for gene suppression into the second plant
line.
[0060] A transgenic plant with recombinant DNA effecting gene
suppression can be crossed with transgenic plant line having other
recombinant DNA that confers another trait, e.g. yield improvement,
herbicide resistance or pest resistance to produce progeny plants
having recombinant DNA that confers both gene suppression and the
other trait. Typically, in such breeding for combining traits the
transgenic plant donating the additional trait is a male line and
the transgenic plant carrying the base traits is the female line.
The progeny of this cross will segregate such that some of the
plants will carry the DNA for both parental traits and some will
carry DNA for one parental trait; such plants can be identified by
markers associated with parental recombinant DNA Progeny plants
carrying DNA for both parental traits can be crossed back into the
female parent line multiple times, e.g. usually 6 to 8 generations,
to produce a progeny plant with substantially the same genotype as
one original transgenic parental line but for the recombinant DNA
of the other transgenic parental line.
EXAMPLES
Example 1
[0061] This example illustrates a method of this invention. With
reference to FIG. 2 two cassettes are prepared for anti-sense
suppression of luciferase in an organism expressing luciferase. A
first luciferase anti-sense cassette comprises CaMV 35S promoter
(35S 3') operably linked to an anti-sense segment of firefly
luciferase coding DNA (anti-sense LUC) and nos 3' element. A second
luciferase anti-sense cassette comprises a FMV promoter (FMV 5')
operably linked to the same anti-sense segment of firefly
luciferase coding DNA and a wheat heat shock protein 3' element
(hsp 3'). The anti-sense cassettes are assembled in an
transformation plasmid inverted with respect to each other with the
respective 3' elements being contiguous. Surprisingly, the
assembled cassettes are not prone to excision when the plasmid is
inserted into common strains of E. coli. The plasmid is
co-transformed into a plant cell along with a two plasmids capable
of expressing the firefly luciferase and Renilla luciferase genes,
the latter serving as a baseline control against which firefly
luciferase expression is normalized. Thus, the ratio of firefly
luciferase to Renilla luciferase expression is a measurement of the
level of suppression of the firefly luciferase gene. As compared to
plant cells transformed with a single copy of either of the firefly
luciferase anti-sense cassettes, the multiple cassettes exhibit a
higher level of firefly luciferase suppression in transgenic plant
cells.
Example 2
[0062] This example illustrates a construct useful for selective
gene suppression in plant tissues. A first anti-sense gene
suppression construct was prepared comprising a corn plant
endosperm specific promoter B32 (nucleotides 848 through 1259 of
GenBank accession number X70153, see also Hartings et al. (1990)
Plant Mol. Biol., 14:1031-1040) operably linked to transcribable
DNA consisting of about 500 base pairs of the LKR domain of a maize
lysine ketoglutarate reductase/saccharopine dehydrogenase gene
(LKR/SDH) in first segment in an anti-sense orientation linked to a
second segment in a sense orientation. Because LKR is a lysine
catabolism enzyme, its suppression resulted in increased lysine. A
second anti-sense gene suppression construct was prepared
essentially the same as the first anti-sense gene suppression
construct except that the promoter was replaced with a corn plant
embryo specific promoter L3 oleosin (see U.S. Pat. No. 6,433,252).
A third gene suppression construct according to this invention was
prepared by linking a B32 promoter that used in the first construct
to the 3' end of the second construct providing a construct with
opposing promoters operably linked to an anti-sense oriented
segment of DNA from the gene targeted for suppression. In one
alternative embodiment the gene suppression construct of this
invention is prepared from the second anti-sense gene suppression
construct by replacing the 3' regulatory region that provides a
polyadenylation signal and site with the B32 promoter inserted in
an opposite orientation to the L3 promoter at the opposing end of
the construct (see FIG. 3A). In another alternative embodiment, the
construct of this invention is prepared by adding the B32 promoter
downstream of the 3' regulatory region and in an opposite
orientation to the L3 promoter at the opposing end of the
construct; optionally a second 3' regulatory region is inserted
between the L3 promoter and the transcribable DNA. In yet another
embodiment the construct of this invention is prepared by locating
3' regulatory regions at the external regions of the construct
where each 3' regulatory region is oriented to the promoters at the
opposing end of the construct. In still another embodiment two
anti-sense constructs are assembled in a tail-to-tail orientation
providing a construct bounded by the respective promoters.
[0063] Plasmids suitable for Agrobacterium-mediated plant
transformation were prepared using each of (a) the first anti-sense
gene suppression construct with the B32 promoter, (b) the second
ant-sense gene suppression construct with the L3 promote and (c) a
gene suppression construct of this invention with a B32 and an L3
promoter at opposing ends of the construct and in opposite
orientations. Each construct was inserted into a plasmid for binary
vector of an Agrobacterium-mediated transformation system between
left and right T-DNA borders and next to a selectable marker
cassette for expressing an aroA gene from A. tumefaciens. Each
plasmid was inserted into maize callus by Agrobacterium-mediated
transformation. Events were selected as being resistance to
glyphosate herbicide and grown into transgenic maize plants to
produce F1 seed. Mature seeds from each event are analyzed to
determine success of transformation and suppression of LKR. The
mature transgenic seeds are dissected to extract protein for
Western analysis. Seed from transgenic maize plants shows reduction
in LKR and increased lysine as compared to wild type. The first
construct with the endosperm specific promoter provides seed with
about 1000 ppm of free lysine; LKR reduction is essentially
observed only in endosperm tissue. The second construct with the
embryo specific promoter provides seed with about 300 ppm of free
lysine; LKR reduction is essentially observed only in embryo
tissue. Because lysine is believed to travel between embryo and
endosperm, concurrent suppression of LKR in both embryo and
endosperm tissues using the construct of this invention provides
seed with higher values of free lysine than the additive effect
from suppression in one tissue alone, e.g. greater than 1300
ppm.
Example 3
[0064] This non-limiting example illustrates constructs for
transforming plant cells and methods for use thereof, and
transgenic maize seed of the invention. In this specific example, a
recombinant DNA construct including a plant embryo-specific
promoter and a plant endosperm-specific promoter, each operably
linked to at least one gene suppression element for silencing a
lysine catabolism gene, is used to provide transgenic plant cells,
and transgenic progeny maize plants and seeds derived from such
transgenic plant cells, wherein the transgenic progeny seed have
increased lysine.
[0065] One non-limiting embodiment of a recombinant DNA constructs
useful, e.g., for providing transgenic plant cells, transgenic
plants, and transgenic seeds of the invention, is illustrated in
FIG. 3B and includes: (i) a plant endosperm-specific promoter
operably linked to at least one first gene suppression element
including DNA that transcribes to RNA for silencing a lysine
catabolism gene by forming double-stranded RNA (e.g., DNA that
includes at least one anti-sense DNA segment that is anti-sense to
at least one segment of the at least one target gene and at least
one sense DNA segment that is at least one segment of the at least
one first target gene; or DNA that encodes a trans-acting miRNA in
both transcriptional directions), (ii) a plant embryo-specific
promoter in the opposite orientation to the first promoter and
operably linked to the at least one first gene suppression element,
and (iii) at least one terminator operably linked to either the
first or second promoters (wherein each terminator can be on either
side of the oppositely oriented promoter.
[0066] In a specific example, a recombinant DNA construct
(illustrated in FIG. 4, third construct from top) is stably
introduced by Agrobacterium-mediated transformation into maize
plant cell and progeny maize plants are regenerated as described
under "Plant Transformation Methods" above. This construct includes
(a) a plant endosperm-specific promoter ("pB32") operably linked to
a stabilized anti-sense gene suppression element targetting
endogenous lysine ketoglutarate reductase/saccharopine
dehydrogenase ("SUP-LKR/SDH") embedded in an intron ("intron 1",
e.g., an hsp70 intron) and a gene expression element for expressing
a lysine-insensitive Corynebacterium DHDPS or cordapA ("cordap A")
(see U.S. Pat. Nos. 6,459,019 and 5,773,691 and U.S. Patent
Application Publication No. 2003/0056242, which are incorporated by
reference), and a first terminator; and (b) a plant embryo-specific
promoter ("pL3") in the opposite orientation to the
endosperm-specific promoter and operably linked to a stabilized
anti-sense gene suppression element targetting endogenous lysine
ketoglutarate reductase/saccharopine dehydrogenase ("SUP-LKR/SDH"),
optionally a selectable marker, and a second terminator. Optionally
an intron or ribozyme is positioned to prevent "read-through" of
the opposite promoter. Transgenic seed from the regenerated plants
show reduction in LKR in both embryo and endosperm seed tissues,
relative to seed in which the recombinant DNA construct is absent
or is not transcribed, and increased levels of lysine in the
transgenic seed. Levels of cordapA are increased, relative to seed
in which the recombinant DNA construct is absent or is not
transcribed, resulting in a further increased lysine level in the
transgenic seed. Overall, levels of lysine in the transgenic seed
are increased relative to transgenic seed in which the expression
of endogenous lysine ketoglutarate reductase/saccharopine
dehydrogenase is silenced in either embryo or endosperm tissues but
not in both.
* * * * *