U.S. patent application number 11/544940 was filed with the patent office on 2007-04-19 for recombinant dna constructs and methods for controlling gene expression.
Invention is credited to Edwards Allen, Alessandra Frizzi, Larry A. Gilbertson, Liang Guo, Sara E. Heisel, Nancy Houmard, Shihshieh Huang, David K. Kovalic, Michael H. Luethy, Linda Lutfiyya, Thomas Malvar, Philip W. Miller, James K. Roberts, Yuanji Zhang.
Application Number | 20070089184 11/544940 |
Document ID | / |
Family ID | 37669306 |
Filed Date | 2007-04-19 |
United States Patent
Application |
20070089184 |
Kind Code |
A1 |
Huang; Shihshieh ; et
al. |
April 19, 2007 |
Recombinant DNA constructs and methods for controlling gene
expression
Abstract
The present invention provides molecular constructs and methods
for use thereof, including constructs including heterologous miRNA
recognition sites, constructs for gene suppression including a gene
suppression element embedded within an intron flanked on one or on
both sides by non-protein-coding sequence, constructs containing
engineered miRNA or miRNA precursors, and constructs for
suppression of production of mature microRNA in a cell. Also
provided are transgenic plant cells, plants, and seeds containing
such constructs, and methods for their use. The invention further
provides transgenic plant cells, plants, and seeds containing
recombinant DNA for the ligand-controlled expression of a target
sequence, which may be endogenous or exogenous. Also disclosed are
novel miRNAs and miRNA precursors from crop plants including maize
and soy.
Inventors: |
Huang; Shihshieh;
(Stonington, CT) ; Malvar; Thomas; (North
Stonington, CT) ; Luethy; Michael H.; (Webster
Groves, MO) ; Miller; Philip W.; (O'Fallon, MO)
; Gilbertson; Larry A.; (Chesterfield, MO) ;
Allen; Edwards; (O'Fallon, MO) ; Heisel; Sara E.;
(St. Louis, MO) ; Kovalic; David K.; (Clayton,
MO) ; Roberts; James K.; (Chesterfield, MO) ;
Houmard; Nancy; (N. Stonington, CT) ; Frizzi;
Alessandra; (Westerly, RI) ; Zhang; Yuanji;
(St. Charles, MO) ; Guo; Liang; (St. Louis,
MO) ; Lutfiyya; Linda; (St. Louis, MO) |
Correspondence
Address: |
MONSANTO COMPANY
800 N. LINDBERGH BLVD.
ATTENTION: GAIL P. WUELLNER, IP PARALEGAL, (E2NA)
ST. LOUIS
MO
63167
US
|
Family ID: |
37669306 |
Appl. No.: |
11/544940 |
Filed: |
October 10, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11303745 |
Dec 15, 2005 |
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11544940 |
Oct 10, 2006 |
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60638256 |
Dec 21, 2004 |
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60639094 |
Dec 24, 2004 |
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60701124 |
Jul 19, 2005 |
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60711834 |
Aug 26, 2005 |
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60720005 |
Sep 24, 2005 |
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60726106 |
Oct 13, 2005 |
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60736525 |
Nov 14, 2005 |
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Current U.S.
Class: |
800/278 |
Current CPC
Class: |
C12N 15/113 20130101;
C12N 15/8217 20130101; C12N 15/8218 20130101; C12N 15/8286
20130101; C12N 15/8216 20130101; Y02A 40/146 20180101; C12N
2310/113 20130101 |
Class at
Publication: |
800/278 |
International
Class: |
A01H 1/00 20060101
A01H001/00; C12N 15/82 20060101 C12N015/82 |
Claims
1-61. (canceled)
62. A recombinant DNA construct comprising a transcribable
engineered miRNA precursor designed to suppress a target sequence,
wherein said transcribable engineered miRNA precursor is derived
from the fold-back structure of a maize or soybean MIR sequence
selected from the group consisting of the MIR sequences identified
in Tables 3, 4, 5, 6, 9, and 10 and their complements.
63. A method to suppress expression of a target sequence in a plant
cell, comprising transcribing in a plant cell the recombinant DNA
construct of claim 62, whereby expression of said target sequence
is suppressed relative to its expression in the absence of
transcription of said recombinant DNA construct.
64-85. (canceled)
86. A transgenic seed having in its genome the recombinant DNA
construct of claim 62.
87. A transgenic plant grown from the transgenic seed of claim
86.
88. The transgenic seed of claim 86, wherein said transgenic seed
has modified primary metabolite, trace element, carotenoid,
vitamin, or secondary metabolite composition, or improved storage
or processing quality.
89. The transgenic plant of claim 87, wherein said transgenic plant
has at least one altered trait, relative to a plant lacking said
recombinant DNA construct, selected from the group of traits
consisting of: (a) improved abiotic stress tolerance; (b) improved
biotic stress tolerance; (c) improved resistance to a pest or
pathogen of said plant; (d) modified primary metabolite
composition; (e) modified secondary metabolite composition; (f)
modified trace element, carotenoid, or vitamin composition; (g)
improved yield; (h) improved ability to use nitrogen or other
nutrients; (i) modified agronomic characteristics; (j) modified
growth or reproductive characteristics; and (k) improved harvest,
storage, or processing quality.
90. A transgenic plant cell having in its genome a recombinant DNA
construct comprising a transcribable engineered miRNA precursor
designed to suppress a target sequence, wherein said transcribable
engineered miRNA precursor is derived from the fold-back structure
of a maize or soybean MIR sequence selected from the group
consisting of the MIR sequences identified in Tables 3, 4, 5, 6, 9,
and 10 and their complements.
91. The transgenic plant cell of claim 90, wherein said recombinant
DNA construct further comprises a gene expression element for
expressing at least one gene of interest.
92. A regenerated transgenic plant or seed prepared from the
transgenic plant cell of claim 90.
93. A method of providing at least one altered plant tissue,
comprising: (a) providing a transgenic plant comprising a
regenerated plant prepared from a transgenic plant cell of claim
90, or a progeny plant of said regenerated plant; and (b)
transcribing said recombinant DNA construct in at least one tissue
of said transgenic plant, whereby an altered trait in said at least
one tissue results, relative to tissue wherein said recombinant DNA
construct is not transcribed, said altered trait being selected
from: (i) improved abiotic stress tolerance; (ii) improved biotic
stress tolerance; (iii) improved resistance to a pest or pathogen
of said plant; (iv) modified primary metabolite composition; (v)
modified secondary metabolite composition; (vi) modified trace
element, carotenoid, or vitamin composition; (vii) improved yield;
(viii) improved ability to use nitrogen or other nutrients; (ix)
modified agronomic characteristics; (x) modified growth or
reproductive characteristics; and (xi) improved harvest, storage,
or processing quality.
94. The method of claim 93, wherein said transgenic plant is a
transgenic crop plant.
Description
PRIORITY CLAIMS AND INCORPORATION OF SEQUENCE LISTINGS
[0001] This is a divisional application of U.S. patent application
Ser. No. 11/303,745, filed 15 Dec. 2005, which claims the benefit
of priority to U.S. Provisional Patent Applications 60/638,256,
which was filed on 21 Dec. 2004, 60/639,094, which was filed on 24
Dec. 2004, 60/701,124, which was filed on 19 Jul. 2005, 60/711,834,
which was filed on 26 Aug. 2005, 60/720,005, which was filed on 24
Sep. 2005, 60/726,106, which was filed on 13 Oct. 2005, and
60/736,525, which was filed on 14 Nov. 2005, incorporated by
reference in their entirety herein. The sequence listing that is
contained in the file named "38-15(53429)100.rpt" which is 97
kilobytes (measured in operating system MS-Windows), created on 29
Sep. 2006, and located in computer readable form on a compact disk
(CD-R), is filed herewith and incorporated herein by reference. The
sequence listing contained in the file named "38-15(53429)C.rpt",
which is 97 kilobytes (measured in MS-Windows), located in computer
readable form on a compact disk created on 28 Sep. 2006, and filed
on 29 Sep. 2006 as a replacement sequence listing for U.S. patent
application Ser. No. 11/303,745, which was filed on 15 Dec. 2005,
is incorporated by reference in its entirety herein. The sequence
listings contained in the files "53429A.ST25.txt" (file size of 15
kilobytes, recorded on 21 Dec. 2004, and filed with U.S.
Provisional Application 60/638,256 on 21 Dec. 2004),
"38-21(53709)B.ST25.txt" (file size of 4 kilobytes, recorded on 23
Dec. 2004, and filed with U.S. Provisional Application 60/639,094
on 24 Dec. 2004), "38-15(53429)B.rpt" (file size of 7 kilobytes,
recorded on 19 Jul. 2005, filed with U.S. Provisional Application
60/701,124 on 19 Jul. 2005), "38-15(54068)A.rpt" (file size of 6
kilobytes, recorded on 26 Aug. 2005, filed with U.S. Provisional
Application 60/711,834 on 26 Aug. 2005), "38-21(54176)A.rpt" (file
size of 29 kilobytes, recorded on 23 Sep. 2005, and filed with U.S.
Provisional Application 60/720,005 on 24 Sep. 2005), and
"38-21(54232)A.rpt" (file size of 61 kilobytes, recorded on 12 Oct.
2005, and filed with U.S. Provisional Application 60/726,106 on 13
Oct. 2005) are incorporated by reference in their entirety
herein.
FIELD OF THE INVENTION
[0002] The present invention discloses molecular constructs and
methods for the control of gene expression, for example, gene
suppression in plants or in plant pests or pathogens or suppressing
expression of a target RNA in a specific cell. Also disclosed are
transgenic eukaryotes, including transgenic plant cells, plants,
and seeds, whose genome includes molecular constructs for
controlling expression of an endogenous or an exogenous gene.
BACKGROUND OF THE INVENTION
[0003] Nucleic acid aptamers include DNA or RNA sequences that can
recognize and specifically bind, often with high affinity, a
particular molecule or ligand. See, for example, reports describing
in vitro aptamer selection by Tuerk and Gold (1990) Science,
249:505-510, Ellington and Szostak (1990) Nature, 346:818-822, and
Ellington and Szostak (1992) Nature, 355:850-852, as well as
Jenison et al. (1994) Science, 263:1425-1429, which demonstrated
the ability of an RNA aptamer to distinguish between theophylline
and caffeine (which differ by a single methyl group) by four orders
of magnitude. Similar to antibodies that bind specific antigens or
receptors that bind specific molecules, aptamers are useful alone,
to bind to a specific ligand (see, for example, Shi et al. (1999)
Proc. Natl. Acad. Sci. USA, 96:10033-10038, which describes a
multivalent RNA aptamer effective as a protein antagonist), and in
combination, e.g., as a molecular "escort" for delivery of an agent
to a specific location, cell, or tissue (see, for example, Hicke
and Stephens (2000) J. Clin. Investigation, 106:923-928) or as part
of a riboswitch. Riboswitches are complex folded RNA sequences
including an aptamer domain for a specific ligand. Naturally
occurring riboswitches have been found mainly in bacteria, and more
recently in fungi (Kubodera et al. (2003) FEBS Lett., 555:516-520)
and plants (Sudarsan et al. (2003) RNA, 9:644-647, which is
incorporated by reference). Many riboswitches contain conserved
domains within species (Barrick et al., (2004) Proc. Natl. Acad.
Sci. USA, 101:6421-6426, which is incorporated by reference).
Riboswitches that act in a "cis" fashion (i.e., that control
expression of an operably linked sequence) are known to occur in
the non-coding regions of mRNAs in prokaryotes, where they control
gene expression by harnessing allosteric structural changes caused
by ligand binding. For a review of "cis" riboswitches, see Mandal
and Breaker (2004a) Nature Rev. Mol. Cell. Biol., 5:451-463, which
is incorporated by reference. Riboswitches that act in a "trans"
fashion (i.e., that control expression of a sequence not operably
linked to the riboswitch) have also been designed, see, for
example, Bayer and Smolke (2005) Nature Biotechnol., 23:337-343,
which is incorporated by reference.
[0004] Most known naturally occurring riboswitches are "off"
switches, wherein the default state is "on" (i.e., the gene under
the riboswitch's control is expressed), and ligand binding turns
the gene "off". In prokaryotes, these riboswitches have been found
mainly in the 5' untranslated region (5' UTR) of mRNAs encoding
biosynthesis genes; in eukaryotes, riboswitches have been found in
the 3' untranslated region (3' UTR) or within introns (Sudarsan et
al. (2003) RNA, 9:644-647; Templeton and Moorhead (2004) Plant
Cell, 16:2252-2257). When an increased concentration of a
particular metabolite or ligand is "sensed" by the riboswitch
(bound by the aptamer domain), the riboswitch "switches off" gene
expression through transcription termination and/or translation
attenuation; see, for example, FIG. 2 in Mandal and Breaker (2004a)
Nature Rev. Mol. Cell. Biol., 5:451-463 and FIG. 4 in Sudarsan et
al. (2003) RNA, 9:644-647.
[0005] At least two types of "on" riboswitches have been reported,
wherein the default state is "off" and ligand binding turns the
gene "on". Expression of ydhL, encoding a purine exporter, is
turned on by adenine binding to the ydhL aptamer; see Mandal and
Breaker (2004b) Nature Struct. Mol. Biol., 11:29-35). Similarly
lysine "on" riboswitches have been proposed to activate the
expression of lysine exporter or degradation genes; see Rodionov et
al. (2003) Nucleic Acids Res., 31:6748-6757. There are also lysine
"off" riboswitches that control the expression of lysine
biosynthesis genes; see Sudarsan et al (2003) Genes Dev.,
17:2688-2697.
[0006] A typical riboswitch is composed of an aptamer domain that
remains largely conserved, and a regulatory domain that can vary
more widely during evolution. In a non-limiting example, the
coenzyme-B.sub.12 riboswitch controls gene expression by two main
mechanisms, as dictated by the architecture of the regulatory
domain (see FIG. 2 in Mandal and Breaker (2004a) Nature Rev. Mol.
Cell. Biol., 5:451-463). If the regulatory domain contains a
"terminator stem", the binding of coenzyme-B.sub.12 to its aptamer
triggers transcriptional termination. If the expression platform
contains an "anti-ribosome binding site stem", the binding of
coenzyme-B.sub.12 to its aptamer triggers translational
attenuation. In some instances, it is believed that transcription
and translation can be controlled simultaneously.
[0007] The present invention provides a novel transgenic plant
having in its genome recombinant DNA that transcribes to at least
one RNA aptamer to which a ligand binds, and can further include at
least one regulatory RNA domain capable of regulating the target
sequence. Depending on the design of the recombinant DNA, the
regulatory RNA can act "in trans" or "in cis" in the transgenic
plants to control expression of an endogenous or of an exogenous
target sequence, and the ligand can be exogenous or endogenous.
Transgenic plants of the invention are preferably stably transgenic
plants in which a desired trait, or an altered trait, is achieved
in the transgenic plant (or in a seed or progeny plant of the
transgenic plant) according to whether or not the ligand is bound
to the aptamer and the resulting expression (or suppression) of the
target sequence.
[0008] Current methods to suppress a gene include, for example, the
use of antisense, co-suppression, and RNA interference. Anti-sense
gene suppression in plants is described by Shewmaker et al. in U.S.
Pat. Nos. 5,107,065, 5453,566, and 5,759,829. Gene suppression in
bacteria using DNA which is complementary to mRNA encoding the gene
to be suppressed is disclosed by Inouye et al. in U.S. Pat. Nos.
5,190,931, 5,208,149, and 5,272,065. RNA interference or
double-stranded RNA-mediated gene suppression has been described
by, e.g., Redenbaugh et al. in "Safety Assessment of Genetically
Engineered Fruits and Vegetables", CRC Press, 1992; Chuang et al.
(2000) PNAS, 97:4985-4990; Wesley et al. (2001) Plant J.,
27:581-590.
[0009] The efficiency of anti-sense gene suppression is typically
low. Redenbaugh et al. in "Safety Assessment of Genetically
Engineered Fruits and Vegetables", CRC Press, 1992, report a
transformation efficiency ranging from 1% to 20% (page 113) for
tomato transformed with a construct designed for anti-sense
suppression of the polygalacturonase gene. Chuang et al. reported
in PNAS, (2000) 97:4985-4990 that anti-sense constructs, sense
constructs, and constructs where anti-sense and sense DNA are
driven by separate promoters had either no, or weak, genetic
interference effects as compared to potent and specific genetic
interference effects from dsRNA constructs (see FIG. 1 and Table 1,
PNAS, (2000) 97:4985-4990). See also Wesley et al. who report in
The Plant Journal, (2001) 27:581-590, e.g., at Table 1, the
comparative efficiency of hairpin RNA, sense constructs, and
anti-sense constructs at silencing a range of genes in a range of
plant species with a clear indication that the efficiency for
anti-sense constructs is typically about an order of magnitude
lower than the efficiency for hairpin RNA.
[0010] Matzke et al. in Chapter 3 ("Regulation of the Genome by
double-stranded RNA") of "RNAi--A Guide to Gene Silencing", edited
by Hannon, Cold Spring Harbor Laboratory Press, 2003, discuss the
use of polyadenylation signals in promoter inverted repeat
constructs. At page 58, they state that "the issue of whether to
put polyadenylation signals in promoter inverted repeat constructs
is unsettled because the nature of the RNA triggering RdDM
[RNA-directed DNA methylation] is unresolved. Depending on whether
short RNA or dsRNA is involved in RdDM, the decision to include a
polyadenylation site might differ depending on the experimental
system used. If dsRNA is involved in RdDM, then a polyadenylation
signal is not required because dsRNA forms rapidly by
intramolecular folding when the entire inverted repeat is
transcribed. Indeed, nonpolyadenylated dsRNAs might be retained in
the nucleus and induce RdDM more efficiently than polyadenylated
dsRNAs. Matzke et al. continue: "If short RNAs guide homologous DNA
methylation, then the situation in plants and mammals differ. In
plants, which probably possess a nuclear form of Dicer,
non-polyadenylated dsRNAs would still be optimal because they
should feed preferentially into a nuclear pathway for dsRNA
processing."
[0011] Carmichael et al. in U.S. Pat. Nos. 5,908,779 and 6,265,167
disclose methods and constructs for expressing and accumulating
anti-sense RNA in the nucleus using a construct that comprises a
promoter, anti-sense sequences, and sequences encoding a cis- or
trans-ribozyme. The cis-ribozyme is incorporated into the
anti-sense construct in order to generate 3' ends independently of
the polyadenylation machinery and thereby inhibit transport of the
RNA molecule to the cytoplasm. Carmichael demonstrated the use of
the construct in mouse NIH 3T3 cells.
[0012] Various other nucleic acid constructs and methods for gene
suppression have been described in recent publications. Shewmaker
et al. (U.S. Pat. No. 5,107,565) disclose constructs for gene
silencing that can contain two or more repetitive anti-sense
sequence in tandem for modulating one or more genes. Resistance to
a virus was achieved in a transgenic plant by use of a transgene
containing a direct repeat of the virus's movement protein (Sijen
et al. (1996) Plant Cell, 8:2277-2294). Another report demonstrated
that nucleic acid constructs containing a promoter, a terminator,
and direct or interrupted tandem repeats of either sense or
anti-sense sequences, could induce gene silencing in plants (Ma and
Mitra (2002) Plant J, 31:37-49. The expression of
1-aminocyclopropane-1-carboxylic acid (ACC) oxidase was
downregulated in transgenic tomatoes containing a nucleic acid
construct including a direct repeat of the ACC oxidase 5'
untranslated region sequence in the anti-sense orientation
(Hamilton et al. (1998) Plant J., 15:737-346). Waterhouse and Wang
(U.S. Patent Application Publication 2003/0165894) disclose a
method for reducing phenotypic expression using nucleic acid
constructs that transcribe to aberrant RNAs including
unpolyadenylated RNAs. Clemente et al. disclose nucleic acid
constructs including sense or anti-sense sequences lacking a normal
3' untranslated region and optionally including a ribozyme, that
transcribe to unpolyadenylated RNA. All of the patents cited in
this paragraph are incorporated by reference in their entirety
herein.
[0013] DNA is either coding (protein-coding) DNA or non-coding DNA.
Non-coding DNA includes many kinds of non-translatable
(non-protein-coding) sequence, including 5' untranslated regions,
promoters, enhancers, or other non-coding transcriptional regions,
3' untranslated regions, terminators, and introns. The term
"intron" is generally applied to segments of DNA (or the RNA
transcribed from such segments) that are located between exons
(protein-encoding segments of the DNA), wherein, during maturation
of the messenger RNA, the introns present are enzymatically
"spliced out" or removed from the RNA strand by a cleavage/ligation
process that occurs in the nucleus in eukaryotes. Lin et al. (2003)
Biochem. Biophys. Res. Comm., 310:754-760, and Lin et al. U.S.
Patent Application Publications 2004/0106566 and 2004/0253604,
which are incorporated by reference in their entirety herein,
disclose methods for inducing gene silencing using nucleic acid
constructs containing a gene silencing molecule (sense or
anti-sense or both) within an intron flanked by multiple
protein-coding exons, wherein, upon splicing and removal of the
intron, the protein-coding exons are linked to form a mature mRNA
encoding a protein with desired function and the gene silencing
molecule is released.
[0014] However, apart from introns found between protein-encoding
exons, there are other non-coding DNA sequences that can be spliced
out of a maturing messenger RNA. One example of these are
spliceable sequences that that have the ability to enhance
expression in plants (in some cases, especially in monocots) of the
downstream coding sequence; these spliceable sequences are
naturally located in the 5' untranslated region of some plant
genes, as well as in some viral genes (e.g., the tobacco mosaic
virus 5' leader sequence or "omega" leader described as enhancing
expression in plant genes by Gallie and Walbot (1992) Nucleic Acids
Res., 20:4631-4638). These spliceable sequences or
"expression-enhancing introns" can be artificially inserted in the
5' untranslated region of a plant gene between the promoter but
before any protein-coding exons. For example, it was reported that
inserting a maize alcohol dehydrogenase (Zm-Adh1) or Bronze-1
expression-enhancing intron 3' to a promoter (e.g., Adh1,
cauliflower mosaic virus 35S, or nopaline synthase promoters) but
5' to a protein-coding sequence (e.g., chloramphenicol
acetyltransferase, luciferase, or neomycin phosphotransferase II)
greatly stimulated expression of the protein (Callis et al. (1987)
Genes Dev., 1:1183-1200). The Adhlintron greatly stimulated
expression of a reporter gene (Mascarenkas et al. (1990) Plant Mol.
Biol., 15:913-920). Cis-acting elements that increase transcription
of a downstream coding sequence in transformed plant cells were
reported to occur in the 5' untranslated region of the rice actin 1
(Os-Act1) gene (Wang et al. (1992) Mol. Cell. Biol., 12:3399-3406).
The rice Act1 gene was further characterized to contain a 5'
expression-enhancing intron that is located upstream of the first
protein-coding exon and that is essential for efficient expression
of coding sequence under the control of the Act1 promoter (McElroy
et al. (1990) Plant Cell, 2:163-171). The Shrunken-1 (Sh-1) intron
was reported to give about 10 times higher expression than
constructs containing the Adh-1 intron (Vasil et al. (1989) Plant
Physiol., 91:1575-1579). The maize sucrose synthase intron, when
placed between a promoter and the firs protein-coding exon, also
increases expression of the encoded protein, and splicing of the
intron is required for this enhanced expression to occur (Clancy
and Hannah (2002) Plant Physiol., 130:918-929).
Expression-enhancing introns have also been characterized for heat
shock protein 18 (hsp18) (Silva et al. (1987) J. Cell Biol.,
105:245) and the 82 kilodalton heat shock protein (hsp82) (Semrau
et al. (1989) J. Cell Biol., 109, p. 39A, and Mettler et al. (May
1990) N.A.T.O. Advanced Studies Institute on Molecular Biology,
Elmer, Bavaria). U.S. Pat. Nos. 5,593,874 and 5,859,347 describe
improved recombinant plant genes including a chimeric plant gene
with an expression-enhancing intron derived from the 70 kilodalton
maize heat shock protein (hsp70) in the non-translated leader
positioned 3' from the gene promoter and 5' from the first
protein-coding exon. All of the patents and publications cited in
this paragraph are incorporated by reference herein.
[0015] The present inventors have found that, unexpectedly, introns
can be utilized to deliver a gene suppression element in the
absence of any protein-coding exons (coding sequence). In the
present invention, an intron, such as an expression-enhancing
intron (preferred in certain embodiments), is interrupted by
embedding within the intron a gene suppression element, wherein,
upon transcription, the gene suppression element is excised from
the intron to function in suppressing a target gene. Thus, no
protein-coding exons are required to provide the gene suppressing
function of the recombinant DNA constructs disclosed herein.
[0016] MicroRNAs (miRNAs) are non-protein coding RNAs, generally of
between about 19 to about 25 nucleotides (commonly about 20-24
nucleotides in plants), that guide cleavage in trans of target
transcripts, negatively regulating the expression of genes involved
in various regulation and development pathways (Bartel (2004) Cell,
116:281-297). In some cases, miRNAs serve to guide in-phase
processing of siRNA primary transcripts (see Allen et al. (2005)
Cell, 121:207-221, which is incorporated herein by reference).
[0017] Some microRNA genes (MIR genes) have been identified and
made publicly available in a database ("miRBase", available on line
at microrna.sanger.ac.uk/sequences). Additional MIR genes and
mature miRNAs are also described in U.S. Patent Application
Publications 2005/0120415 and 2005/144669A1, which is incorporated
by reference herein. MIR genes have been reported to occur in
inter-genic regions, both isolated and in clusters in the genome,
but can also be located entirely or partially within introns of
other genes (both protein-coding and non-protein-coding). For a
recent review of miRNA biogenesis, see Kim (2005) Nature Rev. Mol.
Cell. Biol., 6:376-385. Transcription of MIR genes can be, at least
in some cases, under promotional control of a MIR gene's own
promoter. MIR gene transcription is probably generally mediated by
RNA polymerase II (see, e.g., Aukerman. and Sakai (2003) Plant
Cell, 15:2730-2741; Parizotto et al. (2004) Genes Dev.,
18:2237-2242), and therefore could be amenable to gene silencing
approaches that have been used in other polymerase II-transcribed
genes. The primary transcript (which can be polycistronic) termed a
"pri-miRNA", a miRNA precursor molecule that can be quite large
(several kilobases) and contains one or more local double-stranded
or "hairpin" regions as well as the usual 5' "cap" and
polyadenylated tail of an mRNA. See, for example, FIG. 1 in Kim
(2005) Nature Rev. Mol. Cell. Biol., 6:376-385.
[0018] In animal cells, this pri-miRNA is believed to be "cropped"
by the nuclear RNase III Drosha to produce a shorter miRNA
precursor molecule known as a "pre-miRNA". Following nuclear
processing by Drosha, pre-miRNAs are exported to the nucleus where
the enzyme Dicer generates the short, mature miRNAs. See, for
example, Lee et al. (2002) EMBO Journal, 21:4663-4670; Reinhart et
al. (2002) Genes & Dev., 16:161611626; Lund et al. (2004)
Science, 303:95-98; and Millar and Waterhouse (2005) Funct. Integr
Genomics, 5:129-135, which are incorporated by reference herein. In
contrast, in plant cells, microRNA precursor molecules are believed
to be largely processed in the nucleus. Whereas in animals both
miRNAs and siRNAs are believed to result from activity of the same
DICER enzyme, in plants miRNAs and siRNAs are formed by distinct
DICER-like (DCL) enzymes, and in Arabidopsis a nuclear DCL enzyme
is believed to be required for mature miRNA formation (Xie et al.
(2004) PLoS Biol., 2:642-652, which is incorporated by reference
herein). Additional reviews on microRNA biogenesis and function are
found, for example, in Bartel (2004) Cell, 116:281-297; Murchison
and Hannon (2004) Curr. Opin. Cell Biol., 16:223-229; and Dugas and
Bartel (2004) Curr. Opin. Plant Biol., 7:512-520. MicroRNAs can
thus be described in terms of RNA (e.g., RNA sequence of a mature
miRNA or a miRNA precursor RNA molecule), or in terms of DNA (e.g.,
DNA sequence corresponding to a mature miRNA RNA sequence or DNA
sequence encoding a MIR gene or fragment of a MIR gene or a miRNA
precursor).
[0019] MIR gene families appear to be substantial, estimated to
account for 1% of at least some genomes and capable of influencing
or regulating expression of about a third of all genes (see, for
example, Tomari et al. (2005) Curr. Biol., 15:R61-64; G. Tang
(2005) Trends Biochem. Sci., 30:106-14; Kim (2005) Nature Rev. Mol.
Cell. Biol., 6:376-385). Because miRNAs are important regulatory
elements in eukaryotes, including animals and plants, transgenic
suppression of miRNAs could, for example, lead to the understanding
of important biological processes or allow the manipulation of
certain pathways useful, for example, in biotechnological
applications. For example, miRNAs are involved in regulation of
cellular differentiation, proliferation and apoptosis, and are
probably involved in the pathology of at least some diseases,
including cancer, where miRNAs may function variously as oncogenes
or as tumor suppressors. See, for example, O'Donnell et al. (2005)
Nature, 435:839-843; Cai et al. (2005) Proc. Natl. Acad. Sci. USA,
102:5570-5575; Morris and McManus (2005) Sci. STKE, pe41 (available
online at
stke.sciencemag.org/cgi/reprint/sigtrans;2005/297/pe41.pdf).
MicroRNA (MIR) genes have identifying characteristics, including
conservation among plant species, a stable foldback structure, and
processing of a specific miRNA/miRNA* duplex by Dicer-like enzymes
(Ambros et al. (2003) RNA, 9:277-279). These characteristics have
been used to identify miRNAs and their corresponding genes in
plants (Xie et al. (2005) Plant Physiol., 138:2145-2154;
Jones-Rhoades and Bartel (2004) Mol. Cell, 14:787-799; Reinhart et
al. (2002) Genes Dev., 16:1616-1626; Sunkar and Zhu (2004) Plant
Cell, 16:2001-2019). Publicly available microRNA genes are
catalogued at miRBase (Griffiths-Jones et al. (2003) Nucleic Acids
Res., 31:439-441).
[0020] MiRNAs have been found to be expressed in very specific cell
types in Arabidopsis (see, for example, Kidner and Martienssen
(2004) Nature, 428:81-84, Millar and Gubler (2005) Plant Cell,
17:705-721). Suppression can be limited to a side, edge, or other
division between cell types, and is believed to be required for
proper cell type patterning and specification (see, for example,
Palatnik et al. (2003) Nature, 425:257-263). Suppression of a GFP
reporter gene containing an endogenous miR171 recognition site was
found to limit expression to specific cells in transgenic
Arabidopsis (Parizotto et al. (2004) Genes Dev., 18:2237-2242).
Recognition sites of miRNAs have been validated in all regions of
an mRNA, including the 5' untranslated region, coding region, and
3' untranslated region, indicating that the position of the miRNA
target site relative to the coding sequence may not necessarily
affect suppression (see, for example, Jones-Rhoades and Bartel
(2004). Mol. Cell, 14:787-799, Rhoades et al. (2002) Cell,
110:513-520, Allen et al. (2004) Nat. Genet., 36:1282-1290, Sunkar
and Zhu (2004) Plant Cell, 16:2001-2019).
[0021] The invention provides novel recombinant DNA constructs and
methods for use thereof for suppression of production of mature
miRNA in a cell, where the constructs are designed to target at
least one miRNA precursor or at least one promoter of a miRNA
precursor. Using constructs of the invention, suppression of
production of mature miRNA can occur in the nucleus or in the
cytoplasm or in both. In plants, microRNA precursor molecules are
believed to be largely processed in the nucleus. Thus, in many
preferred embodiments of the recombinant DNA construct of the
invention, particularly (but not limited to) embodiments where the
suppression occurs in a plant cell, suppression preferably occurs
wholly or substantially in the nucleus. Another potential advantage
of the invention is that miRNA precursors (especially pri-miRNAs,
and to a lesser extent pre-miRNAs) offer substantially larger
target sequences than does a mature miRNA.
[0022] In a preferred embodiment, the constructs and methods of the
invention are designed to target nuclear-localized miRNA precursors
(such as pri-miRNAs and pre-miRNA) prior to their export from the
nucleus; such embodiments provide an advantage over conventional
gene suppression constructs (e.g., containing inverted repeats)
that typically result in accumulation of dsRNA in the cytoplasm. In
such embodiments, recombinant DNA constructs of the invention
include a gene suppression element designed to remain in the
nucleus after transcription, for example, a gene suppression
element that is transcribed to RNA lacking functional nuclear
export signals. Such embodiments are particularly preferred for
use, e.g., in plants, where processing of miRNA is believed to
occur largely in the nucleus. In one preferred embodiment of the
invention, the recombinant DNA construct includes a suppression
element (e.g., one or more inverted repeats, anti-sense sequence,
tandem repeats, or other suppression elements) embedded within a
spliceable intron. The resulting suppression transcript remains in
the nucleus, preferably resulting in the nuclear degradation of the
target pri-miRNA or pre-miRNA, or alternatively, resulting in
transcriptional silencing of a target MIR gene promoter, which, in
turn, reduces the accumulation of the mature miRNA.
[0023] In other embodiments, recombinant DNA constructs of the
invention include a suppression element transcribable to RNA that
is exported from the nucleus to the cytoplasm, where, for example,
the transcribed and exported RNA targets a cytoplasmic pre-miRNA.
Such embodiments are particularly useful where miRNA processing at
least partly occurs in the cytoplasm, e.g., in animal cells. In
such embodiments, the suppression element is preferably transcribed
to RNA including functional nuclear export signals.
[0024] In multicellular eukaryotes, including plants, microRNAs
(miRNAs) regulate endogenous genes by a post-transcriptional
cleavage mechanism in a cell-type specific manner. The invention
further provides a recombinant DNA construct, and methods for the
use thereof, wherein the construct includes transcribable DNA that
transcribes to RNA including (a) at least one exogenous miRNA
recognition site recognizable by a mature miRNA expressed in a
specific cell, and (b) target RNA to be suppressed in the specific
cell, whereby said target RNA is expressed in cells other than said
specific cell. These constructs are useful for suppressing
expression of a target RNA in a specific cell of a multicellular
eukaryote (but allowing expression in other cells), including
transcribing in the multicellular eukaryote a recombinant DNA
construct including a promoter operably linked to DNA that
transcribes to RNA including: (a) at least one exogenous miRNA
recognition site recognizable by a mature miRNA expressed in a
specific cell, and (b) target RNA to be suppressed in the specific
cell, wherein the mature miRNA guides cleavage of target RNA in the
specific cell, whereby expression of the target RNA is suppressed
in the specific cell relative to its expression in cells lacking
expression of the mature miRNA.
[0025] The present invention further provides novel mature miRNA
sequences and MIR gene sequences from crop plants, including maize
and soybean. The mature miRNAs processed from these genes belong to
canonical families conserved across distantly related plant
species. These MIR genes and their encoded mature miRNAs are
useful, e.g., for modifying developmental pathways, e.g., by
affecting cell differentiation or morphogenesis (see, for example,
Palatnik et al. (2003) Nature, 425:257-263; Mallory et al. (2004)
Curr. Biol., 14:1035-1046), to serve as sequence sources for
engineered (non-naturally occurring) miRNAs that are designed to
target sequences other than the transcripts targetted by the
naturally occurring miRNA sequence (see, for example, Parizotto et
al. (2004) Genes Dev., 18:2237-2242, and U.S. Patent Application
Publications 2004/3411A1, 2005/0120415, which are incorporated by
reference herein), and to stabilize dsRNA. A MIR gene itself (or
its native 5' or 3' untranslated regions, or its native promoter or
other elements involved in its transcription) is useful as a target
sequence for gene suppression (e.g., by methods of the present
invention), where suppression of the miRNA encoded by the MIR gene
is desired. Promoters of MIR genes can have very specific
expression patterns (e.g., cell-specific, tissue-specific, or
temporally specific), and thus are useful in recombinant constructs
to induce such specific transcription of a DNA sequence to which
they are operably linked.
SUMMARY OF THE INVENTION
[0026] The present invention discloses a transgenic plant cell, as
well as transgenic plants and transgenic seed of such plants,
having in its genome recombinant DNA for the ligand-controlled
expression of a target sequence. One aspect of this invention
provides a transgenic plant cell having in its genome recombinant
DNA including transcribable DNA including DNA that transcribes to
an RNA aptamer capable of binding to a ligand. In some embodiments
of the invention, the recombinant DNA further includes at least one
T-DNA border. In many embodiments, the transcribable DNA further
includes DNA that transcribes to regulatory RNA capable of
regulating expression of a target sequence, wherein the regulation
of the target sequence 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.
[0027] Another aspect of the invention provides a method of
reducing damage to a plant by an invertebrate pest or by a
bacterial, fungal, or viral pathogen of said plant, including
transcribing in the plant a recombinant DNA construct including
transcribable DNA including DNA that transcribes to an RNA aptamer
capable of binding to a ligand, wherein the ligand comprises at
least part of a molecule endogenous to the pest or pathogen, and
whereby binding of the RNA aptamer to the ligand reduces damage to
the plant by the pest or pathogen, relative to damage in the
absence of transcription of the recombinant DNA construct. In
particularly preferred embodiments, the pest or pathogen is an
invertebrate pest of the plant, and the ligand includes at least
part of a molecule of the digestive tract lining of the
invertebrate pest.
[0028] Another aspect of the invention provides a recombinant DNA
construct including: (a) transcribable DNA including DNA that
transcribes to an RNA aptamer capable of binding to a ligand; and
(b) DNA sequence that transcribes to double-stranded RNA flanking
said transcribable DNA. In some embodiments, the recombinant DNA
construct further includes DNA that transcribes to regulatory RNA
capable of regulating expression of a target sequence, 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.
[0029] The present invention discloses recombinant DNA constructs
for suppression of at least one target gene, as well as methods for
their use. In one aspect, the present invention provides a
recombinant DNA construct for plant transformation including a
first gene suppression element for suppressing at least one first
target gene, wherein the gene suppression element is embedded in an
intron, and wherein the intron is located adjacent to at least one
element selected from the group consisting of a promoter element
and a terminator element. The construct can optionally include at
least one T-DNA border, a second gene suppression element, a gene
expression element, or both. The invention further provides
transgenic plant cells and transgenic plants and seeds derived
therefrom, containing such a recombinant DNA construct, and a
method for effecting gene suppression by expressing such a
recombinant DNA construct in a transgenic plant.
[0030] In another aspect, the present invention provides a
transgenic seed having in its genome a recombinant DNA construct
for suppressing at least one first target gene, including DNA
capable of initiating transcription in a plant and operably linked
to a first transcribable heterologous DNA, wherein said first
transcribable heterologous DNA is embedded in an intron. The
invention further provides a transgenic plant grown from the
transgenic seed, and methods for gene suppression or for concurrent
gene suppression and gene expression, that include growing such
transgenic plants. A potential advantage of the use of constructs
of this invention is avoidance of unintentional systemic spreading
of gene suppression.
[0031] Another aspect of the invention discloses recombinant DNA
constructs and methods for suppression of production of mature
microRNA in a cell, for example, by targetting for suppression a
miRNA precursor or a promoter of a miRNA gene
[0032] In one aspect, the present invention provides a recombinant
DNA construct for suppressing production of mature microRNA (miRNA)
in a cell, including a promoter element operably linked to a
suppression element for suppression of at least one target microRNA
precursor. The recombinant DNA constructs include at least one
suppression element for suppression of at least one target microRNA
precursor. The suppression element suppresses at least one target
sequence selected from a target sequence of the at least one target
microRNA precursor, or a target sequence of a promoter of the at
least one target microRNA precursor, or both.
[0033] In another aspect, the present invention provides a
transgenic plant having in its genome the recombinant DNA construct
of the invention (i.e., a recombinant DNA construct for suppressing
production of mature microRNA (miRNA) in a cell, including a
promoter element operably linked to a suppression element for
suppression of at least one target microRNA precursor), as well as
seed and progeny of such transgenic plants.
[0034] In still another aspect, the present invention provides a
method to suppress expression of a target sequence in a plant cell,
including transcribing in a plant cell a recombinant DNA construct
including a transcribable engineered miRNA precursor, derived from
the fold-back structure of a maize or soybean MIR sequence or their
complements, designed to suppress a target sequence, whereby
expression of the target sequence is suppressed relative to its
expression in the absence of transcription of the recombinant DNA
construct.
[0035] In a further aspect, the present invention provides a
recombinant DNA construct including a promoter operably linked to
DNA that transcribes to RNA including (a) at least one exogenous
miRNA recognition site recognizable by a mature miRNA expressed in
a specific cell, and (b) target RNA to be suppressed in the
specific cell, whereby said target RNA is expressed in cells other
than said specific cell.
[0036] In yet another aspect, the present invention provides
methods for suppressing expression of a target RNA in a specific
cell of a multicellular eukaryote, including transcribing in the
multicellular eukaryote a recombinant DNA construct including a
promoter operably linked to DNA that transcribes to RNA including:
(a) at least one exogenous miRNA recognition site recognizable by a
mature miRNA expressed in a specific cell, and (b) target RNA to be
suppressed in the specific cell, wherein the mature miRNA guides
cleavage of target RNA in the specific cell, whereby expression of
the target RNA is suppressed in the specific cell relative to its
expression in cells lacking expression of the mature miRNA.
[0037] Other specific embodiments of the invention are disclosed in
the following detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0038] FIG. 1 schematically illustrates DNA vectors as described in
Example 1. Legend: pale grey regions labelled "e35X-Hsp70": a
chimeric promoter element including an enhanced CaMV35S promoter
linked to an enhancer element (an intron from heat shock protein 70
of Zea mays, Pe35S-Hsp70 intron); medium grey regions labeled
"LUC": DNA coding for firefly luciferase; dark grey regions labeled
"3"nos": a 3'UTR DNA from Agrobacterium tumefaciens nopaline
synthase gene. Vectors are conventionally depicted as transcribing
from left (5') to right (3'). Arrows indicate orientation of the
luciferase segments as sense (arrowhead to right) or anti-sense
(arrowhead to left).
[0039] FIG. 2 schematically illustrates DNA vectors as described in
Example 2. Legend: pale grey regions labelled "e35s": a chimeric
promoter including an enhanced CaMV35S promoter linked to an
enhancer element (an intron from heat shock protein 70 of Zea mays,
Pe35S-Hsp70 intron); medium grey regions labeled "GUS": DNA coding
for beta-glucuronidase; medium grey regions labeled "LUC": DNA
coding for firefly luciferase; dark grey regions labeled "3' nos":
a 3'UTR DNA from Agrobacterium tumefaciens nopaline synthase gene.
Vectors are conventionally depicted as transcribing from left (5')
to right (3'). Arrows indicate orientation of the luciferase
segments as sense (arrowhead to right) or anti-sense (arrowhead to
left).
[0040] FIG. 3 depicts results of the experiments described in
Example 2. X-axis indicates the vectors (see FIG. 2) used. Y-axis
values are given as the logarithm of the ratio of logarithm of the
ratio of firefly luciferase to Renilla luciferase,
"log(Fluc/Rluc)"; error bars are 95% confidence intervals.
[0041] FIG. 4 is a schematic map of a plasmid including an enhanced
anti-sense construct as described in Example 3.
[0042] FIG. 5A is a schematic map of a vector including an enhanced
anti-sense construct and described in Example 4. The plasmid
includes an aroA gene as an herbicidal selectable marker, and a
recombinant DNA construct for enhanced anti-sense gene suppression,
consisting of a seed-specific maize L3 oleosin promoter operably
linked to transcribable DNA consisting of about 300 base pairs of a
maize lysine ketoglutarate reductase (LKR) gene (LKR region of the
lysine ketoglutarate reductase/saccharopine dehydrogenase gene,
LKR/SDH) in an anti-sense orientation, wherein a functional
polyadenylation site is absent in this transcribable DNA, and left
T-DNA border (LB) and right T-DNA border (RB) elements. FIG. 5B
depicts a recombinant DNA construct of the present invention for
gene suppression and described in Example 4, including left T-DNA
border (LB) and right T-DNA border (RB) elements, and a promoter
element operably linked to an intron (maize heat shock protein 70
intron, I-Zm-hsp70) within which is embedded a first gene
suppression element for suppressing at least one first target gene
(in this example, maize lysine ketoglutarate reductase/saccharopine
dehydrogenase gene (LKR/SDH)). The first gene suppression element
can include any gene suppression element as described above under
the heading "Gene Suppression Elements" wherein the intron is
located adjacent to the promoter element. In the specific,
non-limiting embodiment depicted in FIG. 5B, the promoter element
is an endosperm-specific maize B32 promoter (nucleotides 848
through 1259 of GenBank accession number X70153, see also Hartings
et al. (1990) Plant Mol. Biol., 14:1031-1040, which is incorporated
herein by reference), although other promoter elements could be
used. This specific embodiment also includes an aroA gene as an
herbicidal selectable marker; other selectable marker or reporter
genes can be used. As shown in the lower part of FIG. 5B, the
intron-embedded gene suppression element ("GSE") can include any
one or more gene suppression elements as described under "Gene
Suppression Elements".
[0043] FIG. 6A is a schematic map of a vector including an enhanced
anti-sense construct as described in Example 5. The vector includes
an aroA gene as an herbicidal selectable marker and a recombinant
DNA construct for enhanced anti-sense gene suppression, consisting
of a TUB-1 root specific promoter from Arabidopsis thaliana
operably linked to transcribable DNA consisting of anti-sense
oriented DNA of a nematode major sperm protein (msp) of a soybean
cyst nematode, wherein a functional polyadenylation site is absent
in this transcribable DNA. The plasmid also includes left T-DNA
border (LB) and right T-DNA border (RB) elements. FIG. 6B is a
schematic map of a recombinant DNA construct of the present
invention as described in Example 5, which includes an aroA gene as
an herbicidal selectable marker and a recombinant DNA construct of
the present invention for gene suppression, including left T-DNA
border (LB) and right T-DNA border (RB) elements, and a TUB-1 root
specific promoter from Arabidopsis thaliana operably linked to an
intron (maize alcohol dehydrogenase intron, I-Zm-adh1) within which
is embedded a first transcribable heterologous DNA that includes an
anti-sense DNA segment that is anti-sense to the target gene,
nematode major sperm protein of a soybean cyst nematode, wherein a
functional polyadenylation site is absent in this transcribable
heterologous DNA.
[0044] FIG. 7A depicts a gene suppression element useful in a
recombinant DNA construct of the invention, including
intron-embedded tandem repeats for enhancing nuclear-localized gene
silencing as described in Example 6. Such an element can be
combined with at least one T-DNA border in the construct for
Agrobacterium-mediated transformation of a plant cell. The
constructs optionally include a gene expression element, which can
be upstream (5') or downstream (3') of the intron. In a variation
of this embodiment (not shown), the intron-embedded tandem repeats
are located 3' to the terminator. FIG. 7B shows another vector
useful for nuclear-localized gene silencing by tandem repeats,
wherein the vector includes tandem repeats transcribed from
constructs lacking a functional terminator. In a variation of this
embodiment (not shown), the tandem repeats are located 3' to the
terminator. FIG. 7C shows yet another vector useful for
nuclear-localized gene silencing by tandem repeats, wherein the
vector includes tandem repeats under transcriptional control of two
opposing promoters.
[0045] FIG. 8A schematically depicts non-limiting recombinant DNA
constructs of the invention as described in Example 8. For use in
Agrobacterium-mediated transformation of plant cells, at least one
T-DNA border is generally included in each construct (not shown).
These constructs include a promoter element ("pro"), an intron
flanked on one or on both sides by non-protein-coding DNA, an
optional terminator element ("ter"), at least one first gene
suppression element ("GSE" or "GSE1") for suppressing at least one
first target gene, and can optionally include at least one second
gene suppression element ("GSE2") for suppressing at least one
second target gene, at least one gene expression element ("GEE")
for expressing at least one gene of interest, or both. In
embodiments containing a gene expression element, the gene
expression element can be located adjacent to (outside of) the
intron. In one variation of this embodiment (not shown), the gene
suppression element (embedded in an intron flanked on one or on
both sides by non-protein-coding DNA) is located 3' to the
terminator. In other constructs of the invention (not shown), a
gene suppression element (not intron-embedded) is located 3' to the
terminator (see Example 22). FIG. 8B schematically depicts examples
of recombinant DNA constructs distinct from those of the present
invention. These constructs can contain a gene suppression element
that is located adjacent to an intron or between two discrete
introns (that is to say, not embedded within a single intron), or
can include a gene expression element including a gene suppression
element embedded within an intron which is flanked on both sides by
protein-coding DNA (e.g., protein-coding exons that make up a gene
expression element).
[0046] FIG. 9 depicts various non-limiting examples of gene
suppression elements and transcribable exogenous DNAs useful in the
recombinant DNA constructs of the invention. Where drawn as a
single strand (FIGS. 9A through 9E), these are conventionally
depicted in 5' to 3' (left to right) transcriptional direction,
where the arrows indicate anti-sense sequence (arrowhead pointing
to the left), or sense sequence (arrowhead pointing to the right).
Where drawn as double-stranded (anti-parallel) transcripts (FIGS.
9F and 9G), the 5' and 3' transcriptional directionality is as
shown. Solid lines, dashed lines, and dotted lines indicate
sequences that target different target genes.
[0047] These gene suppression elements and transcribable exogenous
DNAs can include: 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, or 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 (FIG. 9A); DNA that
includes at least one sense DNA segment that is at least one
segment of the at least one first target gene, or 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 (FIG. 9B); 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 (FIG. 9C); 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
(FIG. 9D); 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 said multiple anti-sense
DNA segments and the multiple sense DNA segments are arranged in a
series of inverted repeats (FIG. 9E); and DNA that includes
nucleotides derived from a miRNA (see also FIG. 5B), or DNA that
includes nucleotides of a siRNA (FIG. 9F). FIG. 9F depicts various
non-limiting arrangements of double-stranded RNA (dsRNA) that can
be transcribed from embodiments of the gene suppression elements
and transcribable exogenous DNAs useful in the recombinant DNA
constructs of the invention. When such dsRNA is formed, it can
suppress one or more target genes, and can form a single
double-stranded RNA or multiple double strands of RNA, or a single
dsRNA "stem" or multiple "stems". Where multiple dsRNA "stems" are
formed, they can be arranged in "hammerheads" or "cloverleaf"
arrangements. Spacer DNA is optional and can include sequence that
transcribes to an RNA (e.g., a large loop of antisense sequence of
the target gene or an aptamer) that assumes a secondary structure
or three-dimensional configuration that confers on the transcript a
desired characteristic, such as increased stability, increased
half-life in vivo, or cell or tissue specificity.
[0048] FIG. 10A depicts a non-limiting gene suppression element
("GSE") useful in recombinant DNA constructs of the invention, as
described in Example 10. FIG. 10B depicts a representation of the
type of RNA double hairpin molecule that it would be expected to
produce. In this example, orientations of the sequences are
anti-sense followed by sense for sequence 1, then sense followed by
anti-sense for sequence 2 (FIG. 10A). Analogous recombinant DNA
constructs could be designed to provide RNA molecules containing
more than 2 double-stranded "stems", as shown in FIG. 10C, which
depicts an RNA molecule containing 3 "stems".
[0049] FIG. 11 depicts fold-back structures of maize and soyMIR
sequences, as described in detail in Example 14. Nucleotides
corresponding to the mature miRNA are indicated by bold font,
Watson-Crick base-pairing by a vertical line, and base-pairing
mismatches by a dot.
[0050] FIG. 12 depicts fold-back structures of maize and soy MIR
sequences, as described in detail in Example 15. Nucleotides
corresponding to the mature miRNA are indicated by bold font,
Watson-Crick base-pairing by a vertical line, and base-pairing
mismatches by a dot.
[0051] FIG. 13 depicts a miR166 consensus fold-back structure
(Griffiths-Jones (2004) Nucleic Acids Res., 32, Database Issue,
D109-D111, which is incorporated by reference herein) with the
nucleotides corresponding to the mature miRNA indicated by the
shaded nucleotides, as described in Example 16.
[0052] FIG. 14 depicts expression levels of the indicated mature
miRNAs in various tissues from maize, as described in detail in
Example 17.
[0053] FIG. 15 depicts a non-limiting example of transcribable DNA
sequence including an exogenous miRNA recognition site,
chloroplast-targeted TIC809 with a miRNA162 recognition site (in
bold text) located in the 3' untranslated region (SEQ ID NO. 220),
as described in detail in Example 18. The translated amino acid
sequence is also shown.
[0054] FIG. 16 depicts a non-limiting example of transcribable DNA
sequence including an exogenous miRNA recognition site,
non-chloroplast-targeted TIC809 with a miRNA164 recognition site
(in bold text) located in the 3' untranslated region (SEQ ID NO.
221), as described in detail in Example 18. The translated amino
acid sequence is also shown.
[0055] FIG. 17 depicts the strong and specific endosperm expression
of the miR167g microRNA (SEQ ID NO. 4) cloned from maize endosperm,
as described in detail in Example 19. Northern blots of RNA from
maize (LH59) tissues probed with an end-labeled mature miR167
22-mer LNA probe specific for SEQ ID NO. 4 (FIG. 17A) or with a
.about.400 bp miR167g gene-specific probe (FIG. 17B). Transcription
profiling of maize tissues corroborated the Northern blot results
(FIG. 17C); the transcript corresponding to miR167g was abundantly
and specifically expressed in endosperm tissue (abundances are
categorized as follows: >5000, high abundance, 97.sup.th
percentile; 700-5000, moderate abundance, 20.sup.th percentile;
400-700, average abundance; 200-400, low abundance; <200, not
detected). Selected abbreviations: "DAP" or "DA", days after
pollination; "WK", whole kernel, "endo", endosperm.
[0056] FIG. 18 depicts a partial annotation map, including
locations of the miR167a and miR167g genes and mature miRNAs, and
promoter elements (e.g., TATA boxes), of the genomic cluster within
which was identified the miR167g promoter sequences as described in
detail in Example 19. Abbreviations: "PBF", prolamin box binding
factor; "ARF" auxin-responsive (auxin binding) factor; "NIT2",
activator of nitrogen-related genes; "LYS14", element that binds to
UASLYS, an upstream activating element conferring Lys14- and
adipate semialdehyde-dependent activation and apparent repression;
"GLN3", element that binds the nitrogen upstream activation
sequence of glutamine synthetase.
[0057] FIG. 19 depicts Northern blots from a transient expression
assay in Nicotiana benthamiana. Small RNA blots were hybridized to
probes specific for the mature miRNAs predicted to be processed
from miR164e (SEQ ID NO. 228) (FIG. 19A) and from an miRNA
engineered to target Colorado potato beetle vacuolar ATPase (SEQ ID
NO. 229) (FIG. 19B), as described in detail in Example 20. Results
show that the predicted mature miRNAs were processed efficiently in
vivo.
[0058] FIG. 20 depicts results described in detail in Example 21.
FIG. 20A depicts the fold-back structure of SEQ ID NO. 236, the
predicted miRNA precursor for SEQ ID NO. 234; the mature miRNA is
located at bases 106-126, the corresponding miRNA* at bases
156-175, and another abundant miRNA was also found to be located at
bases 100-120 in the stem of the fold-back structure. "Count"
refers to the number of occurrences of a small RNA in the filtered
set of 381,633 putative miRNA sequences that was analyzed. FIG. 20B
depicts a transcription profile in soy tissues for the miRNA
precursor SEQ ID NO. 236. FIG. 20C depicts a transcription profile
in soy tissues for a predicted target, polyphenol oxidase (SEQ ID
NO. 250) for the mature miRNA (SEQ ID NO. 234).
[0059] FIG. 21 depicts results described in detail in Example 21.
FIG. 21A depicts the fold-back structure of SEQ ID NO. 239, the
predicted miRNA precursor for SEQ ID NO. 237; the mature miRNA is
located at bases 163-183, and the miRNA* at bases 18-63. "Count"
refers to the number of occurrences of a small RNA in the filtered
set of 381,633 putative miRNA sequences that was analyzed. FIG. 21B
depicts a transcription profile in soy tissues for a predicted
target, polyphenol oxidase (SEQ ID NO. 251) for the mature miRNA
(SEQ ID NO. 237).
[0060] FIG. 22 depicts results described in detail in Example 21.
FIG. 22A depicts the fold-back structure of SEQ ID NO. 242, the
predicted miRNA precursor for SEQ ID NO. 240; the mature miRNA is
located at bases 87-107, and the miRNA* at bases 150-169. "Count"
refers to the number of occurrences of a small RNA in the filtered
set of 381,633 putative miRNA sequences that was analyzed.
[0061] FIG. 23 depicts results described in detail in Example 21.
FIG. 23A depicts the fold-back structure of SEQ ID NO. 245, the
predicted miRNA precursor for SEQ ID NO. 243; the mature miRNA is
located at bases 61-81, and the miRNA* at bases 109-129. "Count"
refers to the number of occurrences of a small RNA in the filtered
set of 381,633 putative miRNA sequences that was analyzed.
[0062] FIG. 24 depicts results described in detail in Example 21.
FIG. 24A (top) depicts the fold-back structure of SEQ ID NO. 248,
one of the predicted miRNA precursors for SEQ ID NO. 246; the
mature miRNA is located at bases 157-178, and the miRNA* at bases
72-93. FIG. 24A (bottom) depicts the fold-back structure of SEQ ID
NO. 249, another predicted miRNA precursors for SEQ ID NO. 246; the
mature miRNA is located at bases 123-144, and the miRNA* at bases
58-79. "Count" refers to the number of occurrences of a small RNA
in the filtered set of 381,633 putative miRNA sequences that was
analyzed. FIG. 24B (top) depicts a transcription profile in soy
tissues for the miRNA precursor SEQ ID NO. 248. FIG. 24B (bottom)
depicts a transcription profile in soy tissues for the miRNA
precursor SEQ ID NO. 249.
[0063] FIG. 25 depicts various embodiments of recombinant DNA
constructs including a gene suppression element 3' to a terminator,
as described in detail in Example 22.
[0064] FIG. 26 depicts constructs and results described in detail
in Example 22. FIG. 26A depicts a recombinant DNA construct
(pMON100552) for suppressing a target gene (luciferase), containing
a gene suppression element 3' to a terminator. FIG. 26A depicts a
control construct (pMON100553). Y-axis values are given as the
logarithm of the ratio of logarithm of the ratio of firefly
luciferase to Renilla luciferase, "log(Fluc/Rluc)"; error bars are
95% confidence intervals.
[0065] FIG. 27 schematically depicts non-limiting embodiments of
the recombinant DNA useful in making transgenic plants of the
invention. The transcribable DNA includes DNA that transcribes to
at least one RNA aptamer domain, and can further include DNA that
transcribes to an RNA regulatory domain (which can act "in cis" or
"in trans"). Useful promoters include any promoter capable of
transcribing the transcribable DNA in a transgenic plant of the
invention, e.g., a pol II promoter or a pol III promoter. Various
embodiments can include introns, double-stranded RNA-forming
regions, and/or microRNA recognition sites. Some embodiments can
further include one or more separate gene expression elements or
gene suppression elements (shown here as a gene of interest, "GOI",
which can be positioned upstream or downstream of the transcribable
DNA).
[0066] FIG. 28 depicts non-limiting embodiments of recombinant DNA
useful in making transgenic plants of the invention, as described
in Example 24. Abbreviations: "TS", target sequence; "Ter",
terminator; a gene expression element represented by a non-limiting
gene of interest "dapA", cordapA; "TSs.sub.up", a gene suppression
element. FIG. 24F depicts one mechanism for an "on" riboswitch
acting in cis. "RB", right T-DNA border element; "LB", left T-DNA
border element.
[0067] FIG. 29, top panel, depicts different systems of controlling
expression of a target sequence (in this non-limiting example, of
green fluorescent protein, "GFP") as described in Example 28. The
bottom panel depicts a non-limiting example of a riboswitch
autoinduced by its own ligand (lysine), as described in Example
28.
[0068] FIG. 30 depicts a non-limiting example of a riboswitch in a
binary vector useful in making a transgenic plant of the invention,
as described in Example 29. "RB", right T-DNA border element; "LB",
left T-DNA border element; "Nos Ter", Nos terminator.
DETAILED DESCRIPTION OF THE INVENTION
[0069] Unless defined otherwise, all technical and scientific terms
used have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs.
Generally, the nomenclature used and the manufacture or laboratory
procedures described below are well known and commonly employed in
the art. Conventional methods are used for these procedures, such
as those provided in the art and various general references. Unless
otherwise stated, nucleic acid sequences in the text of this
specification are given, when read from left to right, in the 5' to
3' direction. Where a term is provided in the singular, the
inventors also contemplate aspects of the invention described by
the plural of that term. The nomenclature used and the laboratory
procedures described below are those well known and commonly
employed in the art. Where there are discrepancies in terms and
definitions used in references that are incorporated by reference,
the terms used in this application shall have the definitions
given. Other technical terms used have their ordinary meaning in
the art that they are used, as exemplified by a variety of
technical dictionaries. The inventors do not intend to be limited
to a mechanism or mode of action. Reference thereto is provided for
illustrative purposes only.
I. Selective Expression of a Target Sequence in Transgenic Plant
Cells, Plants, and Seeds
[0070] The present invention provides a transgenic plant cell
having in its genome recombinant DNA including transcribable DNA
including DNA that transcribes to an RNA aptamer capable of binding
to a ligand. In some embodiments of the invention, for example, in
transgenic plant cells made transgenic by Agrobacterium-mediated
transformation, the recombinant DNA further includes at least one
T-DNA border. In many embodiments, the transcribable DNA further
includes DNA that transcribes to regulatory RNA capable of
regulating expression of a target sequence, wherein the regulation
of the target sequence 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.
[0071] Further provided by the invention is a transgenic plant
including a regenerated plant prepared from a transgenic plant cell
having in its genome recombinant DNA including transcribable DNA
including DNA that transcribes to an RNA aptamer capable of binding
to a ligand, or a progeny plant (which may be a hybrid progeny
plant) of the regenerated plant. Such transgenic plants may be
plants of any developmental stage, including seed, and include
transgenic plants grown from such seed. Also claimed are plant
tissues regenerated from the transgenic plant cell of the
invention.
[0072] In preferred embodiments, the transgenic plant cell or plant
having in its genome recombinant DNA including transcribable DNA
including DNA that transcribes to an RNA aptamer capable of binding
to a ligand has at least one altered trait, relative to a plant
lacking the recombinant DNA, as described in detail under the
heading "Making and Using Transgenic Plant Cells and Plants". In
these embodiments, the altered trait is typically obtained by
providing the ligand to at least some cells or tissues of the
transgenic plant. In one preferred embodiment, the altered trait is
provided by contacting the transgenic plant with an exogenous
ligand that binds to the aptamer. In some of these embodiments, the
exogenous ligand is physically applied to the plant (e.g., a
synthetic or natural ligand applied to the plant as a foliar spray
or root solution), or applied (e.g., as a coating or soak) to
transgenic seed of the transgenic plant. For example, the altered
trait may be obtained by contacting the transgenic plant with an
herbicide (e.g., glyphosate or dicamba) that binds to an aptamer
specific for the herbicide, thus turning "on" or "off" the
regulatory RNA. In other embodiments, the ligand is an exogenous
ligand produced by or found in a pest or pathogen of the transgenic
plant, or a ligand (e.g., an allelochemical) produced by adjacent
plants of the same or different species as the transgenic plant. In
another preferred embodiment, the altered trait is obtained through
the binding of an endogenous ligand to the aptamer. In such
embodiments, the ligand is endogenous to the transgenic plant,
e.g., a ligand produced constitutively, or in a specific cell or
tissue, or under biotic or abiotic stress, or at a particular
developmental or seasonal time. In a non-limiting example, the
altered trait is obtained during a period of stress (biotic or
abiotic), wherein a ligand, such as a stress-responsive molecule or
hormone (e.g., salicylic acid, jasmonic acid, ethylene,
glutathione, ascorbate, auxins, cytokinins), is endogenously
produced by the transgenic plant, and binds to an aptamer specific
for the stress-responsive molecule. In yet another example, the
altered trait may be obtained in response to a pest or pathogen of
the transgenic plant, wherein the aptamer is specific for a ligand
produced by the plant in response to the pest or pathogen.
[0073] Transcribable DNA: The transcribable DNA includes DNA that
transcribes to an RNA aptamer capable of binding to a ligand. By
"transcribable" is meant that the DNA is capable of being
transcribed to RNA. Thus, in preferred embodiments, the recombinant
DNA further includes a promoter operably linked to the
transcribable DNA. Promoters of use in the invention are preferably
promoters functional in a plant cells, as described under the
heading "Promoter Elements". Suitable promoters can be constitutive
or non-constitutive promoters. In various embodiments, the promoter
element can include a promoter selected from the group consisting
of a constitutive promoter, a spatially specific promoter, a
temporally specific promoter, a developmentally specific promoter,
and an inducible promoter. In one embodiment of the invention, the
promoter is a pol II promoter. In another embodiment, the promoter
is a pol III promoter (see, for example, Eckstein (2005) Trends
Biochem. Sci., 30:445-452).
[0074] In many preferred embodiments, the transcribable DNA further
includes DNA that transcribes to regulatory RNA capable of
regulating expression of a target sequence, 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, that is to say, the
conformation of the regulatory RNA is allosterically influenced by
the conformation of the RNA aptamer, which in turn is determined by
whether the RNA aptamer is occupied or unoccupied by the specific
ligand.
[0075] In some embodiments, the transcribable DNA is optionally
flanked on one or both sides by a ribozyme (e.g., a self-cleaving
ribozyme or a hairpin ribozyme) (see, e.g., FIG. 27A). See, for
example, Esteban et al. (1997) J. Biol. Chem., 272:13629-13639,
which describes the effects of conformation on hairpin ribozyme
kinetics and provides guidelines for hairpin ribozyme sequence
modification, and Najafi-Shoushtari et al. (2004) Nucleic Acids
Res., 32:3212-3219, which describes conformationally controlled
hairpin ribozymes. In other embodiments, the transcribable DNA is
optionally embedded within a spliceable intron (see, e.g., FIG.
27C). Introns suitable for use in the invention are preferably
introns that are spliceable in planta; plant-sourced introns are
especially preferred. Non-limiting examples of especially preferred
plant introns include a rice actin 1 intron (1-Os-Act1), a maize
heat shock protein intron (1-Zm-hsp70), and a maize alcohol
dehydrogenase intron (1-Zm-adh1). Embodiments where the
transcribable DNA is flanked by intron splicing sites can further
include additional sequence to allow cleavage of the transcript,
e.g., DNA that transcribes to RNA including at least one microRNA
recognition site or DNA that transcribes to RNA capable of forming
double-stranded RNA (dsRNA) (see, e.g., FIG. 27D). In other
embodiments, the transcribable DNA includes DNA that transcribes to
RNA sequence that can be processed in an RNAi pathway (i.e., to
produce small interfering RNAs or microRNAs, see, for example, Xie
et al. (2004) PLoSBiol., 2:642-652; Bartel (2004) Cell,
116:281-297; Murchison and Hannon (2004) Curr. Opin. Cell Biol.,
16:223-229; and Dugas and Bartel (2004) Curr. Opin. Plant Biol.,
7:512-520, which are incorporated by reference). In non-limiting
examples, the transcribable DNA is optionally flanked by DNA that
transcribes to RNA including at least one microRNA recognition site
(see, e.g., FIG. 27E). In these embodiments, the miRNA recognition
site is preferably a miRNA recognition site recognized by a miRNA
endogenous to the plant in which transcription occurs. In a
non-limiting example, the transcribable DNA is flanked on both
sides by a miRNA recognition site that is recognized by a mature
miRNA that is expressed in an inducible or a spatially or
temporally specific manner. In yet other embodiments, the
transcribable DNA is optionally flanked on one or both sides by DNA
that transcribes to RNA capable of forming double-stranded RNA
(dsRNA) (see, e.g., FIG. 27E), for example, by forming an inverted
repeat where the transcribable DNA is located in the middle
"spacer" or "loop" region, or by forming separate dsRNA regions on
one or both sides of the transcribable DNA, which may be processed
to small interfering RNAs or to mature microRNAs. In certain
embodiments, the transcribable DNA can further include at least one
gene expression (or suppression) element for the expression of any
gene or genes or interest (including coding or non-coding
sequence), as described under the heading "Gene Expression
Elements" (see, e.g., FIG. 27C and FIG. 1D, where a gene expression
element is represented by a gene of interest, "GOI", and FIG. 28C
and FIG. 28D, where a gene expression element is represented by a
specific gene of interest, cordapA, "dapA", and FIG. 28E, where a
gene suppression element is represented by "TS.sub.sup").
[0076] RNA Aptamers: Nucleic acid aptamers are nucleic acid
molecules that bind to a ligand through binding mechanism that is
not primarily based on Watson-Crick base-pairing (in contrast, for
example, to the base-pairing that occurs between complementary,
anti-parallel nucleic acid strands to form a double-stranded
nucleic acid structure). See, for example, Ellington and Szostak
(1990) Nature, 346:818-822. A nucleic acid aptamer generally
includes a primary nucleotide sequence that allows the aptamer to
form a secondary structure (e.g., by forming stem-loop structures)
that allows the aptamer to bind to its ligand. Binding of the
aptamer to its ligand is preferably specific, allowing the aptamer
to distinguish between two or more molecules that are structurally
similar (see, for example, Bayer and Smolke (2005) Nature
Biotechnol., 23:337-343). Aptamers useful in the invention can,
however, be monovalent (binding a single ligand) or multivalent
(binding more than one individual ligand, e.g., binding one unit of
two or more different ligands). See, for example, Di Giusto and
King (2004) J. Biol. Chem., 279:46483-46489, describing the design
and construction of multivalent, circular DNA aptamers, which is
incorporated by reference.
[0077] Aptamers useful in the invention can include DNA, RNA,
nucleic acid analogues (e.g., peptide nucleic acids), locked
nucleic acids, chemically modified nucleic acids, or combinations
thereof. See, for example, Schmidt et al. (2004) Nucleic Acids
Res., 32:5757-5765, who describe locked nucleic acid aptamers. In
one preferred embodiment of the invention, the aptamer is an RNA
aptamer. In a particularly preferred embodiment, the aptamer is
produced by transcription in planta. Examples of aptamers can be
found, for example, in the public Aptamer Database, available on
line at aptamer.icmb.utexas.edu (Lee et al. (2004) Nucleic Acids
Res., 32(1):D95-100).
[0078] Aptamers can be designed for a given ligand by various
procedures known in the art, including in vitro selection or
directed evolution techniques. See, for example, "SELEX"
("systematic evolution of ligands by exponential enrichment"), as
described in Tuerk and Gold (1990) Science, 249:505-510, Ellington
and Szostak (1990) Nature, 346:818-822, Ellington and Szostak
(1992) Nature, 355:850-852, selection of bifunctional RNA aptamers
by chimeric SELEX, as described by Burke and Willis (1998), RNA,
4:1165-1175, selection using ligands bound to magnetic particles as
described by Murphy et al. (2003) Nucleic Acids Res., 31:e110, an
automated SELEX technique described by Eulberg et al. (2005)
Nucleic Acids Res., 33(4):e45, and a SELEX-type technique for
obtaining aptamers raised against recombinant molecules expressed
on cell surfaces, as descried by Ohuchi et al (2005) Nucleic Acid
Symposium Series, 49:351-352 Selection can begin with a random pool
of RNAs, from a partially structured pool of RNAs (see, for
example, Davis and Szostak (2002) Proc. Natl. Acad. Sci. USA, 99:
11616-11621), or from a pool of degenerate RNAs (see, for example,
Geiger et al. (1996) Nucleic Acids Res., 24: 1029-1036). Secondary
structure models, folding, and hybridization behavior for a given
RNA sequence can be predicted using algorithms, e.g., as described
by Zuker (2003) Nucleic Acids Res., 31: 3406-3415. Thus, aptamers
for a given ligand can be designed de novo using suitable
selection. One non-limiting example of aptamer design and selection
is described in detail in Weill et al. (2004) Nucleic Acids Res.,
32:5045-5058, which describes isolation of various ATP-binding
aptamers and secondary selection of aptamers that bind cordycepin
(3' deoxyadenosine). Another non-limiting example of aptamer design
is given in Huang and Szostak (2003) RNA, 9:1456-1463, which
describes the in vitro evolution of novel aptamers with new
specificities and new secondary structures from a starting aptamer.
All citations in this paragraph are specifically incorporated by
reference.
[0079] Ligands useful in the invention can include amino acids or
their biosynthetic or catabolic intermediates, peptides, proteins,
glycoproteins, lipoproteins, carbohydrates, fatty acids and other
lipids, steroids, terpenoids, hormones, nucleic acids, aromatics,
alkaloids, natural products or synthetic compounds (e.g., dyes,
pharmaceuticals, antibiotics, herbicides), inorganic ions, and
metals, in short, any molecule (or part of a molecule) that can be
recognized and be bound by a nucleic acid secondary structure by a
mechanism not primarily based on Watson-Crick base pairing. In this
way, the recognition and binding of ligand and aptamer is analogous
to that of antigen and antibody, or of biological effector and
receptor. Ligands can include single molecules (or part of a
molecule), or a combination of two or more molecules (or parts of a
molecule), and can include one or more macromolecular complexes
(e.g., polymers, lipid bilayers, liposomes, cellular membranes or
other cellular structures, or cell surfaces). See, for example,
Plummer et al. (2005) Nucleic Acids Res., 33:5602-5610, which
describes selection of aptamers that bind to a composite small
molecule-protein surface; Zhuang et al. (2002) J. Biol. Chem.,
277:13863-13872, which describes the association of insect mid-gut
receptor proteins with lipid rafts, which affects the binding of
Bacillus thuringiensis insecticidal endotoxins; and Homann and
Goringer (1999) Nucleic Acids Res., 27:2006-2014, which describes
aptamers that bind to live trypanosomes; these citations are
incorporated by reference.
[0080] Non-limiting examples of specific ligands include vitamins
such as coenzyme B.sub.12 and thiamine pyrophosphate, flavin
mononucleotide, guanine, adenosine, S-adenosylmethionine,
S-adenosylhomocysteine, coenzyme A, lysine, tyrosine, dopamine,
glucosamine-6-phosphate, caffeine, theophylline, antibiotics such
as chloramphenicol and neomycin, herbicides such as glyphosate and
dicamba, proteins including viral or phage coat proteins and
invertebrate epidermal or digestive tract surface proteins, and
RNAs including viral RNA, transfer-RNAs (t-RNAs), ribosomal RNA
(rRNA), and RNA polymerases such as RNA-dependent RNA polymerase
(RdRP). One class of RNA aptamers useful in the invention are
"thermoswitches" that do not bind a ligand but are thermally
responsive, that is to say, the aptamer's conformation is
determined by temperature. See, for example, Box 3 in Mandal and
Breaker (2004) Nature Rev. Mol. Cell. Biol., 5:451-463, which is
incorporated by reference.
[0081] An aptamer can be described by its binding state, that is,
whether the aptamer is bound (or unbound) to its respective ligand.
The binding site (or three-dimensional binding domain or domains)
of an aptamer can be described as occupied or unoccupied by the
ligand. Similarly, a population of a given aptamer can be described
by the fraction of the population that is bound or unbound to the
ligand. The affinity of an aptamer for its ligand can be described
in terms of the rate of association (binding) of the aptamer with
the ligand and the rate of dissociation of the ligand from the
aptamer, e.g., by the equilibrium association constant (K) or by
its reciprocal, the affinity constant (K.sub.a) as is well known in
the art. These rates can be determined by methods similar to those
commonly used for determining binding kinetics of ligands and
receptors or antigens and antibodies, such as, but not limited to,
equilibrium assays, competition assays, surface plasmon resonance,
and predictive models. The affinity of an aptamer for its ligand
can be selected, e.g., during in vitro evolution of the aptamer, or
further modified by changes to the aptamer's primary sequence,
where such changes can be guided by calculations of binding energy
or by algorithms, e.g., as described by Zuker (2003) Nucleic Acids
Res., 31:3406-3415 or Bayer and Smolke (2005) Nature Biotechnol.,
23:337-343.
[0082] The binding state of an aptamer preferably at least
partially determines the secondary structure (e.g., the formation
of double-stranded or single stranded regions) and the
three-dimensional conformation of the aptamer. In embodiments where
the transcribable DNA further includes DNA that transcribes to
regulatory RNA capable of regulating expression of a target
sequence, the binding state of the aptamer allosterically affects
the conformation of the regulatory RNA and thus the ability of the
regulatory RNA to regulate expression of the target sequence.
[0083] In one preferred embodiments, the aptamer (transcribed RNA)
is flanked by DNA that transcribes to RNA capable of forming
double-stranded RNA (dsRNA) (FIG. 27E). In some of these
embodiments, the dsRNA is processed by an RNAi (siRNA or miRNA)
mechanism, whereby the aptamer is cleaved from the rest of the
transcript. In other, particularly preferred embodiments, the two
transcribed RNA regions flanking the aptamer form at least
partially double-stranded RNA "stem" between themselves, wherein
the aptamer serves as a "spacer" or "loop" in a stem-loop
structure; such an arrangement is expected to enhance the stability
or half-life of the transcript in a manner analogous to that
observed for DNA (see, for example, Di Giusto and King (2004) J.
Biol. Chem., 279:46483-46489, which is incorporated by reference).
Transgenic plants having in their genome DNA that transcribes to
such aptamers having enhanced stability are particularly desirable,
e.g., where the aptamer functions to inhibit or kill a pathogen or
pest of the transgenic plant.
[0084] Target Sequence: The regulatory RNA is capable of regulating
expression of a target sequence, wherein the regulation of the
target sequence 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. Any target
sequence may be chosen, including one or more target sequences
selected from a gene native to the transgenic plant of the
invention, a transgene in the transgenic plant, and a gene native
to a pest or pathogen of the transgenic plant. The target sequence
can include a sequence that expresses a gene of interest (e.g., an
RNA encoding a protein), or a sequence that suppresses a gene of
interest (e.g., an RNA that is processed to an siRNA or miRNA that
in turn suppresses the gene of interest).
[0085] The regulatory RNA can regulate the transcription and/or
translation of any target nucleic acid sequence or sequences of
interest. In some embodiments, the recombinant DNA further includes
a second gene regulatory element for regulating (i.e., suppressing
or expressing) at least one second target sequence that is in
addition to the target sequence regulated by the regulatory RNA.
Whether a first target sequence or a second target sequence, the
target sequence can include a single sequence or part of a single
sequence that is targetted for regulation, or can include, for
example, multiple consecutive segments of a target sequence,
multiple non-consecutive segments of a target sequence, multiple
alleles of a target sequence, or multiple target sequences from one
or more species.
[0086] The target sequence can be translatable (coding) sequence,
or can be non-coding sequence (such as non-coding regulatory
sequence), or both. The target sequence can include at least one
eukaryotic target sequence, at least one non-eukaryotic target
sequence, or both. A target sequence can include any sequence from
any species (including, but not limited to, non-eukaryotes such as
bacteria, and viruses; fungi; plants, including monocots and
dicots, such as crop plants, ornamental plants, and
non-domesticated or wild plants; invertebrates such as arthropods,
annelids, nematodes, and molluscs; and vertebrates such as
amphibians, fish, birds, domestic or wild mammals, and even humans.
Suitable target sequences are further described as "target genes"
under the heading "Target Genes".
[0087] Non-limiting examples of a target sequence include
non-translatable (non-coding) sequence, such as, but not limited
to, 5' untranslated regions, promoters, enhancers, or other
non-coding transcriptional regions, 3' untranslated regions,
terminators, and introns. Target sequences can also include genes
encoding microRNAs, small interfering RNAs, RNA components of
ribosomes or ribozymes, small nucleolar RNAs, and other non-coding
RNAs (see, for example, non-coding RNA sequences provided publicly
at rfam.wustl.edu; Erdmann et al. (2001) Nucleic Acids Res.,
29:189-193; Gottesman (2005) Trends Genet., 21:399-404;
Griffiths-Jones et al. (2005) Nucleic Acids Res., 33:121-124, which
are incorporated by reference). One specific example of a target
sequence includes a microRNA recognition site (that is, the site on
an RNA strand to which a mature miRNA binds and induces cleavage).
Another specific example of a target sequence includes a microRNA
precursor sequence, that is, the primary transcript encoding a
microRNA, or the RNA intermediates processed from this primary
transcript (e.g., a nuclear-limited pri-miRNA or a pre-miRNA which
can be exported from the nucleus into the cytoplasm). See, for
example, Lee et al. (2002) EMBO Journal, 21:4663-4670; Reinhart et
al. (2002) Genes & Dev., 16:161611626; Lund et al. (2004)
Science, 303:95-98; and Millar and Waterhouse (2005) Funct. Integr
Genomics, 5:129-135, which are incorporated by reference. Target
microRNA precursor DNA sequences can be native to the transgenic
plant of the invention, or can be native to a pest or pathogen of
the transgenic plant. Target sequences can also include
translatable (coding) sequence for genes encoding transcription
factors and genes encoding enzymes involved in the biosynthesis or
catabolism of molecules of interest (such as, but not limited to,
amino acids, fatty acids and other lipids, sugars and other
carbohydrates, biological polymers, and secondary metabolites
including alkaloids, terpenoids, polyketides, non-ribosomal
peptides, and secondary metabolites of mixed biosynthetic origin).
A target sequence can be a native gene targetted for expression
control (e.g., suppression), with or without concurrent expression
(or suppression) of an exogenous transgene, for example, by
including a gene expression (or suppression) element in the same or
in a separate recombinant DNA construct. For example, it can be
desirable to replace a native gene with an exogenous transgene
homologue.
[0088] One preferred embodiment of the invention provides
transgenic plant cells (or transgenic plants, progeny plants, or
seeds derived from the transgenic plant cells) having in their
genome a recombinant DNA including transcribable DNA including DNA
that transcribes to an RNA aptamer capable of binding to a ligand,
for suppressing a plant pest or pathogen (e.g., viruses, bacteria,
fungi, and invertebrates such as insects, nematodes, and
molluscs).
[0089] Examples of such embodiments include transgenic plant cells
(or transgenic plants, progeny plants, or seeds derived from the
transgenic plant cells) having in their genome a recombinant DNA
including transcribable DNA including DNA that transcribes to one
or more RNA aptamers that bind to one or more ligands involved in a
pest or pathogen's ability to recognize, invade, or feed on a
plant, or in the pest or pathogen's ability to recruit additional
individuals of its species, or in the pest or pathogen's ability to
grow, metamorphose, or reproduce. Non-limiting examples of ligands
suitable for this approach include the insect mid-gut brush border
receptor proteins that are recognized by Bacillus thuringiensis
insecticidal endotoxins. See, for example, Knight et al. (1995) J.
Biol. Chem., 270:17765-17770, and Gill et al. (1995) J. Biol.
Chem., 270:27277-27282, which describe the isolation,
identification, and cloning of examples of such receptor proteins;
Gomez et al. (2001) J. Biol. Chem., 276:28906-28912, and Daniel et
al. (2002) Appl. Env. Microbiol., 68:2106-2112, which describe
techniques for identifying binding epitopes of such receptor
proteins and for studying their binding affinities; Jurat-Fuentes
and Adang (2001) Appl. Env. Microbiol., 67:323-329, and
Jurat-Fuentes et al. (2001), Appl. Env. Microbiol., 67:872-879,
which describe endotoxin-receptor binding assays involving either
membrane blots or surface plasmon resonance measured binding of
brush border membrane vesicles to endotoxin; all of these are
incorporated by reference. Other examples of suitable ligands to
which RNA aptamers of the invention bind include steroid receptors,
such as estrogen receptors, androgen receptors, retinoid receptors,
and ecdysone receptors (see, for example, Saez et al. (2000) Proc.
Natl. Acad. Sci. USA, 97:14512-14517. Where ligands are receptor
molecules or receptor complexes, RNA aptamers of the invention can
optionally act as antagonists or as agonists.
[0090] One aspect of the invention provides transgenic plants
wherein the target sequence is selected to provide resistance to a
plant pest or pathogen, for example, resistance to a nematode such
as soybean cyst nematode or root knot nematode or to a pest insect.
Thus, target sequences (i.e., "target genes") of interest can also
include endogenous genes of plant pests and pathogens as described
in detail under "Target Genes". Pests and pathogens of interest
include invertebrates (including nematodes, molluscs, and insects),
fungi, bacteria, mollicute, and viruses, as described in detail
under "Target Genes". Thus, a target sequence need not be
endogenous to the plant in which the recombinant DNA is
transcribed. It is envisioned that recombinant DNA of the invention
can be transcribed in a plant and used to control expression of a
target sequence endogenous to a pathogen or pest that may infest
the plant.
[0091] Regulatory RNA: In many embodiments, the transcribable DNA
further includes DNA that transcribes to regulatory RNA capable of
regulating expression of a target sequence, wherein the regulation
of the target sequence 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.
Such combinations of an aptamer with a regulator RNA domain are
commonly known as riboswitches. The regulatory RNA is typically
downstream of the aptamer but the two domains may overlap; see,
e.g., Najafi-Shoushtari and Famulok (2005) RNA, 111:1514-1520,
which is incorporated by reference and describes a hairpin ribozyme
that includes an aptamer domain and is competitively regulated by
flavin mononucleotide and an oligonucleotide complementary to the
aptamer domain. In some embodiments, the regulatory RNA is operably
linked to the target sequence, and acts "in cis". In other
embodiments, the regulatory RNA is not operably linked to the
target sequence, and acts "in trans".
[0092] In riboswitch embodiments including an aptamer and a
regulatory RNA, the riboswitch regulates expression of the target
sequence by any suitable mechanism. One non-limiting mechanism is
transcriptional regulation by the ligand-dependent formation of an
intrinsic terminator stem (an extended stem-loop structure
typically followed by a run of 6 or more U residues) that causes
RNA polymerase to abort transcription, e.g., before a complete mRNA
is formed. In "off" riboswitches, in the absence of sufficient
ligand, the unbound aptamer domain permits formation of an
"antiterminator stem", which prevents formation of the intrinsic
terminator stem and thus allows transcription to proceed; thus, the
default state of the riboswitch is "on" (i.e., transcription
normally proceeds) and the ligand must be added to turn the
riboswitch off. In "on" riboswitches that use this mechanism, the
aptamer domain must be in the bound (ligand-occupied) conformation
to permit formation of the "antiterminator stem" and allow
transcription. Another mechanism is translation regulation, where
ligand binding causes structural changes in full-length mRNAs and
thereby permits (or prevents) ribosomes from binding to the
ribosomal binding site (RBS); the formation of an "anti-anti-RBS"
stem and an "anti-RBS" stem is also mutually exclusive. In "on"
riboswitches that use this mechanism, absence of the ligand allows
formation of an anti-anti-RBS, and thus a structurally unencumbered
RBS to which the ribosome can bind. A combination of both
transcriptional and translational regulation is also possible. For
a detailed discussion of regulation mechanisms, see Mandal and
Breaker (2004) Nature Rev. Mol. Cell Biol., 5:451-463, which is
incorporated by reference.
[0093] In some embodiments, the regulatory RNA includes a ribozyme,
e.g., a self-cleaving ribozyme, a hammerhead ribozyme, or a hairpin
ribozyme. Certain embodiments of the regulatory RNA include RNA
sequence that is complementary or substantially complementary to
the target sequence. One non-limiting example is where the
regulatory RNA includes an anti-sense segment that is complementary
or substantially complementary to the target sequence. See, for
example, Bayer and Smolke (2005) Nature Biotechnol., 23:337-343,
where the regulatory RNA includes both an anti-sense segment
complementary to the target sequence, and a sense segment
complementary to the anti-sense segment, wherein the anti-sense
segment and sense segment are capable of hybridizing to each other
to form an intramolecular double-stranded RNA.
[0094] In embodiments where regulation of a target sequence
involves Watson-Crick base-pairing of the regulatory RNA to the
target sequence (e.g., in trans-acting embodiments, see, e.g.,
Bayer and Smolke (2005) Nature Biotechnol., 23:337-343), the target
sequence of interest can be more specifically targetted by
designing the regulatory RNA to include regions substantially
non-identical to a non-target sequence sequence. Non-target
sequences can include any gene for which the expression is
preferably not modified, either in a plant transcribing the
recombinant DNA construct or in organisms that may come into
contact with RNA transcribed from the recombinant DNA construct. A
non-target sequence can include any sequence from any species
(including, but not limited to, non-eukaryotes such as bacteria,
and viruses; fungi; plants, including monocots and dicots, such as
crop plants, ornamental plants, and non-domesticated or wild
plants; invertebrates such as arthropods, annelids, nematodes, and
molluscs; and vertebrates such as amphibians, fish, birds, domestic
or wild mammals, and even humans).
[0095] In one embodiment of the invention, the target sequence is a
gene endogenous to a given species, such as a given plant (such as,
but not limited to, agriculturally or commercially important
plants, including monocots and dicots), and the non-target sequence
can be, for example, a gene of a non-target species, such as
another plant species or a gene of a virus, fungus, bacterium,
invertebrate, or vertebrate, even a human. One non-limiting example
is where it is desirable to design either the aptamer, or the
regulatory RNA, or both, in order to modify the expression of a
target sequence that is a gene endogenous to a single species
(e.g., Western corn rootworm, Diabrotica virgifera virgifera
LeConte) but to not modify the expression of a non-target sequence
such as genes from related, even closely related, species (e.g.,
Northern corn rootworm, Diabrotica barberi Smith and Lawrence, or
Southern corn rootworm, Diabrotica undecimpunctata).
[0096] In other embodiments (e.g., where it is desirable to modify
the expression of a target sequence across multiple species), it
may be desirable to design the aptamer, or the regulatory RNA, or
both, to modify the expression of a target sequence common to the
multiple species in which the expression of the target sequence is
to be modified. Thus, the aptamer, or the regulatory RNA, or both,
can be selected to be specific for one taxon (e.g., specific to a
genus, family, or even a larger taxon such as a phylum, e.g.,
arthropoda) but not for other taxa (for example, plants or
vertebrates or mammals). In one non-limiting example of this
embodiment, a regulatory RNA can be selected so as to target
pathogenic fungi (e.g., a Fusarium spp.) but not target any gene
sequence from beneficial fungi (e.g., beneficial soil mycorrhizal
fungi).
[0097] In another non-limiting example of this embodiment, the
aptamer, or the regulatory RNA, or both, to regulate gene
expression in corn rootworm can be selected to be specific to all
members of the genus Diabrotica. For example, a regulatory RNA
including a Diabrotica-targetted suppression element (e.g.,
anti-sense RNA, double-stranded RNA, microRNA, or tandem RNA
repeats) can be selected so as to not target any gene sequence from
beneficial coleopterans (for example, predatory coccinellid
beetles, commonly known as ladybugs or ladybirds) or other
beneficial insect species.
[0098] The required degree of specificity of a regulatory RNA that
includes a gene suppression element (e.g., anti-sense RNA,
double-stranded RNA, microRNA, or tandem RNA repeats) for
suppression of a target sequence depends on various factors. For
example, where the gene suppression element includes
double-stranded RNA (dsRNA), factors can include the size of the
smaller dsRNA fragments that are expected to be produced by the
action of Dicer, and the relative importance of decreasing the
dsRNA's potential to suppress non-target sequences. For example,
where the dsRNA fragments are expected to be 21 base pairs in size,
one particularly preferred embodiment can be to include in the
regulatory RNA a sequence capable of forming dsRNA and encoding
regions substantially non-identical to a non-target sequence, such
as regions within which every contiguous fragment including at
least 21 nucleotides matches fewer than 21 (e.g., fewer than 21, or
fewer than 20, or fewer than 19, or fewer than 18, or fewer than
17) out of 21 contiguous nucleotides of a non-target sequence. In
another embodiment, regions substantially non-identical to a
non-target sequence include regions within which every contiguous
fragment including at least 19 nucleotides matches fewer than 19
(e.g., fewer than 19, or fewer than 18, or fewer than 17, or fewer
than 16) out of 19 contiguous nucleotides of a non-target
sequence.
[0099] In some embodiments, it may be desirable to design the
aptamer, the regulatory RNA, or both, to include regions predicted
to not generate undesirable polypeptides, for example, by screening
the aptamer, the regulatory RNA, or both, for sequences that may
encode known undesirable polypeptides or close homologues of these.
Undesirable polypeptides include, but are not limited to,
polypeptides homologous to known allergenic polypeptides and
polypeptides homologous to known polypeptide toxins. Publicly
available sequences encoding such undesirable potentially
allergenic peptides are available, for example, the Food Allergy
Research and Resource Program (FARRP) allergen database (available
at allergenonline.com) or the Biotechnology Information for Food
Safety Databases (available at www.iit.edu/.about.sgendel/fa.htm)
(see also, for example, Gendel (1998) Adv. Food Nutr. Res.,
42:63-92, which is incorporated by reference). Undesirable
sequences can also include, for example, those polypeptide
sequences annotated as known toxins or as potential or known
allergens and contained in publicly available databases such as
GenBank, EMBL, SwissProt, and others, which are searchable by the
Entrez system (www.ncbi.nih.gov/Entrez). Non-limiting examples of
undesirable, potentially allergenic peptide sequences include
glycinin from soybean, oleosin and agglutinin from peanut,
glutenins from wheat, casein, lactalbumin, and lactoglobulin from
bovine milk, and tropomyosin from various shellfish
(allergenonline.com). Non-limiting examples of undesirable,
potentially toxic peptides include tetanus toxin tetA from
Clostridium tetani, diarrheal toxins from Staphylococcus aureus,
and venoms such as conotoxins from Conus spp. and neurotoxins from
arthropods and reptiles (www.ncbi.nih.gov/Entrez).
[0100] In one non-limiting example, a proposed aptamer, regulatory
RNA, or both, can be screened to eliminate those transcribable
sequences encoding polypeptides with perfect homology to a known
allergen or toxin over 8 contiguous amino acids, or with at least
35% identity over at least 80 amino acids; such screens can be
performed on any and all possible reading frames in both
directions, on potential open reading frames that begin with ATG,
or on all possible reading frames, regardless of whether they start
with an ATG or not. When a "hit" or match is made, that is, when a
sequence that encodes a potential polypeptide with perfect homology
to a known allergen or toxin over 8 contiguous amino acids (or at
least about 35% identity over at least about 80 amino acids), is
identified, the DNA sequences corresponding to the hit can be
avoided, eliminated, or modified when selecting sequences to be
used in the aptamer, the regulatory RNA, or both.
[0101] Avoiding, elimination of, or modification of, an undesired
sequence can be achieved by any of a number of methods known to
those skilled in the art. In some cases, the result can be novel
sequences that are believed to not exist naturally. For example,
avoiding certain sequences can be accomplished by joining together
"clean" sequences into novel chimeric sequences to be used in a
gene suppression element.
[0102] Where the regulatory RNA includes double-stranded RNA
(dsRNA) for silencing a target gene, applicants recognize that in
some dsRNA-mediated gene silencing, it is possible for imperfectly
matching dsRNA sequences to be effective at gene silencing. For
example, it has been shown that mismatches near the center of a
miRNA complementary site has stronger effects on the miRNA's gene
silencing than do more distally located mismatches. See, for
example, FIG. 4 in Mallory et al. (2004) EMBO J., 23:3356-3364,
which is incorporated by reference. In another example, it has been
reported that, both the position of a mismatched base pair and the
identity of the nucleotides forming the mismatch influence the
ability of a given siRNA to silence a target sequence, and that
adenine-cytosine mismatches, in addition to the G:U wobble base
pair, were well tolerated (see Du et al. (2005) Nucleic Acids Res.,
33:1671-1677, which is incorporated by reference). Thus, a
regulatory RNA that includes double-stranded RNA need not always
have 100% sequence identity with the intended target sequence, but
generally would preferably have substantial sequence identity with
the intended target sequence, such as about 95%, about 90%, about
85%, or about 80% sequence identity with the intended target
sequence. One skilled in the art would be capable of judging the
importance given to screening for regions predicted to be more
highly specific to the first target sequence or predicted to not
generate undesirable polypeptides, relative to the importance given
to other criteria, such as, but not limited to, the percent
sequence identity with the intended first target sequence or the
predicted gene silencing efficiency of a given sequence. For
example, it may be desirable for a given regulatory RNA that
includes double-stranded RNA for gene silencing to be active across
several species, and therefore one skilled in the art can determine
that it is more important to include in the regulatory RNA regions
specific to the several species of interest, but less important to
screen for regions predicted to have higher gene silencing
efficiency or for regions predicted to generate undesirable
polypeptides.
[0103] In many embodiments, the transgenic plant cell has in its
genome recombinant DNA including transcribable DNA including (a)
DNA that transcribes to an RNA aptamer capable of binding to a
ligand, and (b) DNA that transcribes to regulatory RNA capable of
regulating expression of a target sequence, wherein the regulation
is dependent on the conformation of the regulatory RNA, and the
conformation of said regulatory RNA is allosterically affected by
the binding state of said RNA aptamer. In these embodiments,
binding of the aptamer to its ligand results in a specific change
in the expression of the target sequence, which may be an increase
or a decrease in expression, depending on the design of the
recombinant DNA.
[0104] In one embodiment, binding of the ligand to the RNA aptamer
results in an increase of expression of the target sequence
relative to expression in the absence of the binding. In another
embodiment, binding of the ligand to the RNA aptamer results in a
decrease of expression of the target sequence relative to
expression in the absence of the binding.
[0105] Some embodiments are characterized by "autoinducibility". In
one such embodiment, binding of the ligand to the RNA aptamer
results in an increase of expression of the target sequence
relative to expression in the absence of the binding, wherein the
increase of expression results in a level of the ligand sufficient
to maintain the increase of expression. In another embodiment,
binding of the ligand to the RNA aptamer results in a decrease of
expression of the target sequence relative to expression in the
absence of the binding, the decrease of expression resulting in a
level of the ligand sufficient to maintain the increase of
expression.
[0106] Thus, another aspect of the invention is a method of
modifying expression of a gene of interest in a plant cell,
including transcribing in a transgenic plant cell of the invention,
or a plant, progeny plant, or seed or other plant tissue derived
from such a transgenic plant cell, recombinant or heterologous DNA
that transcribes to (a) an RNA aptamer capable of binding to a
ligand, and (b) regulatory RNA capable of regulating expression of
a target sequence, wherein the regulation is dependent on the
conformation of the regulatory RNA, and wherein the conformation of
the regulatory RNA is allosterically affected by the binding state
of the RNA aptamer, whereby expression of the gene of interest is
modified relative to its expression in the absence of transcription
of the recombinant DNA construct.
[0107] Method of Reducing Invertebrate Pest Damage to a Plant: The
present invention also provides a method of reducing damage to a
plant by a pest or pathogen of the plant, including transcribing in
the plant a recombinant DNA construct including transcribable DNA
including DNA that transcribes to an RNA aptamer capable of binding
to a ligand, wherein the ligand includes at least part of a
molecule endogenous to the pest or pathogen, and whereby binding of
the RNA aptamer to the ligand reduces damage to the plant by the
pest or pathogen, relative to damage in the absence of
transcription of the recombinant DNA construct. The ligand can
include at least part of any molecule that is part of a pest's
anatomy (e.g., a coat or surface protein or macromolecular
structure), or is produced or secreted by the pest or pathogen
(e.g., an enzyme secreted by a pathogen in invasion of a plant
cell)
[0108] In particularly preferred embodiments, the pest or pathogen
is an invertebrate pest of the transgenic plant, and the ligand
includes at least part of a molecule of the digestive tract lining
of the invertebrate pest, e.g., insect mid-gut brush border
receptor proteins that are recognized by Bacillus thuringiensis
insecticidal endotoxins (see discussion above under the heading
"RNA Aptamers"). Invertebrate pests of interest are listed above
under the heading "Target Sequences".
[0109] The invention also contemplates and claims an analogous
method for improving resistance in a transgenic plant to bacterial,
fungal, or viral pathogens. The method reduces damage to a
transgenic plant by a bacterial, fungal, or viral pathogen of the
plant, including the step of transcribing in the plant a
recombinant DNA construct including transcribable DNA including DNA
that transcribes to an RNA aptamer capable of binding to a ligand,
wherein the ligand includes at least part of a molecule endogenous
to the bacterial, fungal, or viral pathogen, and whereby binding of
the RNA aptamer to the ligand reduces damage to the plant by the
bacterial, fungal, or viral pathogen, relative to damage in the
absence of transcription of the recombinant DNA construct.
Bacterial, fungal, and viral pathogens of interest are provided
under the heading "Target Genes".
[0110] Recombinant DNA Constructs: The present invention further
provides a recombinant DNA construct including: (a) transcribable
DNA including DNA that transcribes to an RNA aptamer capable of
binding to a ligand; and (b) DNA sequence that transcribes to
double-stranded RNA flanking said transcribable DNA. In some
embodiments, the recombinant DNA construct further includes DNA
that transcribes to regulatory RNA capable of regulating expression
of a target sequence, 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. The transcribable DNA is DNA that is capable of
being transcribed in a eukaryotic cell, preferably an animal cell
or a plant cell.
[0111] The double-stranded RNA (dsRNA) is preferably RNA that is
capable of being processed through an RNAi pathway (i.e., to
produce small interfering RNAs or microRNAs, see, for example, Xie
et al. (2004) PLoSBiol., 2:642-652; Bartel (2004) Cell,
116:281-297; Murchison and Hannon (2004) Curr. Opin. Cell Biol.,
16:223-229; and Dugas and Bartel (2004) Curr. Opin. Plant Biol.,
7:512-520, which are incorporated by reference). The RNAi pathway
can be that found in animals or that found in plants. See, e.g.,
Lee et al. (2002) EMBO Journal, 21:4663-4670; Reinhart et al.
(2002) Genes & Dev., 16:161611626; Lund et al. (2004) Science,
303:95-98; and Millar and Waterhouse (2005) Funct. Integr Genomics,
5:129-135, which are incorporated by reference. Whereas in animals
both miRNAs and siRNAs are believed to result from activity of the
same DICER enzyme, in plants miRNAs and siRNAs are formed by
distinct DICER-like (DCL) enzymes, and in Arabidopsis a nuclear DCL
enzyme is believed to be required for mature miRNA formation (Xie
et al. (2004) PLoS Biol., 2:642-652, which is incorporated by
reference).
[0112] In non-limiting examples, the transcribable DNA is
optionally flanked on one or both sides by DNA that transcribes to
RNA capable of forming double-stranded RNA (dsRNA) (for example, by
forming an inverted repeat where the transcribable DNA is located
in the middle "spacer" region, or by forming separate dsRNA regions
on one or both sides of the transcribable DNA, which may be
processed to small interfering RNAs, to microRNA precursors such as
pre-miRNAs, or to mature microRNAs). In yet other embodiments, the
transcribable DNA is optionally flanked by DNA that transcribes to
RNA including at least one microRNA recognition site. In these
embodiments, the miRNA recognition site is preferably a miRNA
recognition site recognized by a miRNA endogenous to the plant in
which transcription occurs. In a non-limiting example, the
transcribable DNA is flanked on both sides by a miRNA recognition
site that is recognized by a mature miRNA that is expressed in an
inducible or a spatially or temporally specific manner. The
transcribable DNA can further include at least one gene expression
element.
[0113] The invention further provides a transgenic eukaryotic cell
including in its genome a recombinant DNA construct including: (a)
transcribable DNA including DNA that transcribes to an RNA aptamer
capable of binding to a ligand; and (b) DNA sequence that
transcribes to double-stranded RNA flanking said transcribable DNA.
Such cells may be animal cells or plant cells. Also provided is a
transgenic plant having in its genome a recombinant DNA construct
including: (a) transcribable DNA including DNA that transcribes to
an RNA aptamer capable of binding to a ligand; and (b) DNA sequence
that transcribes to double-stranded RNA flanking said transcribable
DNA Methods for preparing and using the recombinant DNA constructs,
and for making transgenic cells and transgenic plants, are
described under the headings "Making and Using Recombinant DNA
Constructs" and "Making and Using Transgenic Plant Cells and
Transgenic Plants".
II. Recombinant DNA Constructs Containing Introns and Gene
Suppression Elements
[0114] The present invention provides a recombinant DNA construct
for plant transformation including a promoter operably linked to a
first gene suppression element for suppressing at least one first
target gene, wherein said first gene suppression element is
embedded in an intron flanked on one or on both sides by
non-protein-coding DNA. In some embodiments, the recombinant DNA
construct consists entirely of non-protein-coding DNA (e.g., a
promoter, a gene suppression element that is embedded in an intron
and that transcribes to a non-coding RNA, and an optional
terminator). Thus, the invention includes the use of an intron to
deliver a gene suppression element in the absence of any
protein-coding exons.
[0115] In some embodiments, the intron is located adjacent to at
least one element selected from the group consisting of the
promoter and a terminator, that is to say, directly contiguous (or
essentially directly, with no substantial intervening sequence)
with the promoter or with a terminator or with both. In one
specific embodiment, the intron is directly (or essentially
directly) 3' to the promoter. The intron can also optionally be
directly (or essentially directly) 5' to a terminator, if a
terminator is present in the recombinant DNA construct. Where the
intron is adjacent to a terminator element, any intervening
sequence preferably does not include a self-splicing ribozyme. In
one preferred embodiment, the intron containing the gene
suppression element is flanked directly (on the 5' end) by the
promoter element, and (on the 3' end) by the terminator element if
one is present.
[0116] The inventors have unexpectedly found that transcription can
continue downstream of a terminator at least sufficiently to allow
transcription of a gene suppression element located 3' to the
terminator (downstream of a polyadenylation sequence). Thus another
aspect of the invention is a recombinant DNA construct including a
promoter, a terminator, transcribable sequence (which can include
coding or non-coding sequence or both, and can include, e.g., a
gene expression element, a gene suppression element, an aptamer, or
a riboswitch) between the promoter and the terminator, and at least
one gene suppression element that is 3' to the terminator. In
various embodiments, at least one gene suppression element (such as
any one or more of those described under "Gene Suppression
Elements"), whether embedded in an intron or not, is located
downstream of a terminator and sufficiently proximate to the
terminator to permit transcription of the gene suppression element.
In a specific embodiment, the intron is located downstream of a
terminator and sufficiently proximate to the terminator to permit
transcription of the intron. In one preferred but non-limiting
embodiment, the intron is directly (or essentially directly) 3' to
a terminator. Introns can affect the expression of adjacent
sequences (e.g., depending on the intron's splicing efficiency),
and thus one advantage of placing a gene suppression element (or
intron containing a gene suppression element) 3' to a terminator
includes allowing expression of a sequence between the promoter and
the terminator, wherein the expression is not influenced by in the
manner that it may be if the gene suppression element (or intron
containing a gene suppression element) was also located between the
promoter and the terminator. Another advantage includes the
likelihood that a gene suppression element 3' to a terminator will
be processed as an aberrant transcript (e.g., converted to
double-stranded RNA in an RNA-dependent RNA polymerase manner even
in the absence of inverted repeat sequences), which can increase
the efficiency of gene suppression (see Examples 1, 2, and 3, which
illustrate that lack of sequences necessary for polyadenylation
enhanced the efficiency of a gene suppression element). Yet another
advantage is that this approach reduces the need for multiple
promoter elements, especially useful when stacking multiple genetic
constructs to be expressed in a single cell.
[0117] The recombinant DNA construct contains one or more first
gene suppression element for suppressing at least one first target
gene and embedded in an intron flanked on one or on both sides by
non-protein-coding DNA. Suitable gene suppression elements are
described under the heading "Gene Suppression Elements". Where the
recombinant DNA construct contains more than one first gene
suppression element, each of these first gene suppression elements
can include one or more elements as described herein. The first
target gene can include a single gene or part of a single gene that
is targetted for suppression, or can include, for example, multiple
consecutive segments of a first target gene, multiple
non-consecutive segments of a first target gene, multiple alleles
of a first target gene, or multiple first target genes from one or
more species. Suitable first target genes are described under the
heading "Target Genes".
[0118] Introns of use in the recombinant DNA construct are
described under the heading "Introns". The intron is located
adjacent to at least one element selected from the group consisting
of a promoter element and a terminator element, as described under
the headings "Promoter Elements" and "Terminator Elements"
respectively. Preferably, upon transcription of the recombinant DNA
construct, the first gene suppression element is spliced out of the
intron. In some embodiments, the recombinant DNA construct is
designed so that the RNA transcribed from the first gene
suppression element, when spliced out of the intron, lacks at least
one of a functional polyadenylation signal or a functional
polyadenylation site (or any other element that facilitates
transport of a transcribed RNA into the cytoplasm), or lacks a 3'
untranslated region; the resulting transcribed RNA (and gene
suppression by the transcribed RNA) is preferably localized in the
nucleus. In other embodiments, the recombinant DNA construct is
designed so that the RNA transcribed from the first gene
suppression element, when spliced out of the intron, is transported
out of the nucleus for gene suppression in the cytoplasm.
[0119] In various embodiments of the invention, the recombinant DNA
constructs are optionally characterized by any one or more of the
following. The recombinant DNA construct can further include at
least one of: (a) at least one T-DNA border region, as described
under "T-DNA Borders"; (b) spacer DNA, as described under "Spacer
DNA"; (c) a gene expression element for expressing at least one
gene of interest, wherein the gene expression element is located
adjacent to the intron; (d) a gene expression element for
expressing at least one gene of interest, wherein said gene
expression element is located adjacent to said first gene
suppression element and within said intron; and (e) a second gene
suppression element for suppressing at least one second target
gene, wherein the second gene suppression element is located
outside of (e.g., adjacent to) the intron. These further aspects
are described in more detail below.
[0120] In some embodiments, the recombinant DNA construct further
includes a gene expression element for expressing at least one gene
of interest, wherein the gene expression element is located
adjacent to the intron. The gene of interest can include a single
gene or multiple genes. Gene expression elements are further
described under the heading "Gene Expression Elements".
[0121] In yet other embodiments, the recombinant DNA construct
further includes a second gene suppression element for suppressing
at least one second target gene, wherein the second gene
suppression element is located outside of, e.g., adjacent to, the
intron. The at least one second target gene can include a single
gene or part of a single gene that is targetted for suppression, or
can include, for example, multiple consecutive segments of a second
target gene, multiple non-consecutive segments of a second target
gene, multiple alleles of a second target gene, or multiple second
target genes from one or more species. Suitable second target genes
are described under the heading "Target Genes".
[0122] Gene Suppression Elements: The gene suppression element can
be transcribable DNA of any suitable length, and will generally
include at least about 19 to about 27 nucleotides (for example 19,
20, 21, 22, 23, or 24 nucleotides) for every target gene that the
recombinant DNA construct is intended to suppress. In many
embodiments the gene suppression element includes more than 23
nucleotides (for example, more than about 30, about 50, about 100,
about 200, about 300, about 500, about 1000, about 1500, about
2000, about 3000, about 4000, or about 5000 nucleotides) for every
target gene that the recombinant DNA construct is intended to
suppress.
[0123] Suitable gene suppression elements useful in the recombinant
DNA constructs of the invention include at least one element (and,
in some embodiments, multiple elements) 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;
[0124] (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;
[0125] (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;
[0126] (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 said 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 miRNA, preferably
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
[0127] (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.
[0128] Any of these gene suppression elements, whether transcribing
to a single double-stranded RNA or to multiple double-stranded
RNAs, can be designed to suppress more than one target gene,
including, for example, more than one allele of a target gene,
multiple target genes (or multiple segments of at least one target
gene) from a single species, or target genes from different
species.
[0129] Anti-Sense DNA Segments: In one embodiment, the at least one
anti-sense DNA segment that is anti-sense to at least one segment
of the at least one first target gene includes DNA sequence that is
anti-sense or complementary to at least a segment of the at least
one first target gene, and can include multiple anti-sense DNA
segments, that is, 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. Multiple anti-sense DNA segments can include
DNA sequence that is anti-sense or complementary to multiple
segments of the at least one first target gene, or to multiple
copies of a segment of the at least one first target gene, or to
segments of multiple first target genes, or to any combination of
these. Multiple anti-sense DNA segments can be fused into a
chimera, e.g., including DNA sequences that are anti-sense to
multiple segments of one or more first target genes and fused
together.
[0130] The anti-sense DNA sequence that is anti-sense or
complementary to (that is, can form Watson-Crick base-pairs with)
at least a segment of the at least one first target gene has
preferably at least about 80%, or at least about 85%, or at least
about 90%, or at least about 95% complementarity to at least a
segment of the at least one first target gene. In one preferred
embodiment, the DNA sequence that is anti-sense or complementary to
at least a segment of the at least one first target gene has
between about 95% to about 100% complementarity to at least a
segment of the at least one first target gene. Where the at least
one anti-sense DNA segment includes multiple anti-sense DNA
segments, the degree of complementarity can be, but need not be,
identical for all of the multiple anti-sense DNA segments.
[0131] Sense DNA Segments: In another embodiment, the at least one
sense DNA segment that is at least one segment of the at least one
first target gene includes DNA sequence that corresponds to (that
is, has a sequence that is identical or substantially identical to)
at least a segment of the at least one first target gene, and can
include multiple sense DNA segments, that is, multiple copies of at
least one sense DNA segment that corresponds to (that is, has the
nucleotide sequence of) at least one segment of the at least one
first target gene. Multiple sense DNA segments can include DNA
sequence that is or that corresponds to multiple segments of the at
least one first target gene, or to multiple copies of a segment of
the at least one first target gene, or to segments of multiple
first target genes, or to any combination of these. Multiple sense
DNA segments can be fused into a chimera, that is, can include DNA
sequences corresponding to multiple segments of one or more first
target genes and fused together.
[0132] The sense DNA sequence that corresponds to at least a
segment of the target gene has preferably at least about 80%, or at
least about 85%, or at least about 90%, or at least about 95%
sequence identity to at least a segment of the target gene. In one
preferred embodiment, the DNA sequence that corresponds to at least
a segment of the target gene has between about 95% to about 100%
sequence identity to at least a segment of the target gene. Where
the at least one sense DNA segment includes multiple sense DNA
segments, the degree of sequence identity can be, but need not be,
identical for all of the multiple sense DNA segments.
[0133] Multiple Copies: Where the gene suppression element includes
multiple copies of anti-sense or multiple copies of sense DNA
sequence, these multiple copies can be arranged serially in tandem
repeats. In some embodiments, these multiple copies can be arranged
serially end-to-end, that is, in directly connected tandem repeats.
In some embodiments, these multiple copies can be arranged serially
in interrupted tandem repeats, where one or more spacer DNA segment
can be located adjacent to one or more of the multiple copies.
Tandem repeats, whether directly connected or interrupted or a
combination of both, can include multiple copies of a single
anti-sense or multiple copies of a single sense DNA sequence in a
serial arrangement or can include multiple copies of more than one
anti-sense DNA sequence or of more than one sense DNA sequence in a
serial arrangement.
[0134] Double-stranded RNA: In those embodiments wherein the gene
suppression element includes either at least one anti-sense DNA
segment that is anti-sense to at least one segment of the at least
one target gene or at least one sense DNA segment that is at least
one segment of the at least one target gene, RNA transcribed from
either the at least one anti-sense or at least one sense DNA may
become double-stranded by the action of an RNA-dependent RNA
polymerase. See, for example, U.S. Pat. No. 5,283,184, which is
incorporated by reference herein.
[0135] In yet other embodiments, the gene suppression element can
include 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 (as described above
under the heading "Anti-sense DNA Segments") and at least one sense
DNA segment that is at least one segment of the at least one first
target gene (as described above under the heading "Sense DNA
Segments"). Such a gene suppression element can further include
spacer DNA segments. Each at least one anti-sense DNA segment is
complementary to at least part of a sense DNA segment in order to
permit formation of double-stranded RNA by intramolecular
hybridization of the at least one anti-sense DNA segment and the at
least one sense DNA segment. Such complementarity between an
anti-sense DNA segment and a sense DNA segment can be, but need not
be, 100% complementarity; in some embodiments, this complementarity
can be preferably at least about 80%, or at least about 85%, or at
least about 90%, or at least about 95% complementarity.
[0136] The double-stranded RNA can be in the form of a single dsRNA
"stem" (region of base-pairing between sense and anti-sense
strands), or can have multiple dsRNA "stems". In one embodiment,
the gene suppression element can include DNA that transcribes to
RNA for suppressing the at least one first target gene by forming
essentially 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; the multiple serial anti-sense and multiple
serial sense segments can form a single double-stranded RNA "stem"
or multiple "stems" in a serial arrangement (with or without
non-base paired spacer DNA separating the multiple "stems"). In
another embodiment, the gene suppression element includes DNA that
transcribes to RNA for suppressing the at least one first target
gene by forming multiple dsRNA "stems" 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 said multiple anti-sense DNA segments and
the multiple sense DNA segments are arranged in a series of dsRNA
"stems" (such as, but not limited to inverted repeats"). Such
multiple dsRNA "stems" can further be arranged in series or
clusters to form tandem inverted repeats, or structures resembling
"hammerhead" or "cloverleaf" shapes. Any of these gene suppression
elements can further include spacer DNA segments found within a
dsRNA "stem" (for example, as a spacer between multiple anti-sense
or sense DNA segments or as a spacer between a base-pairing
anti-sense DNA segment and a sense DNA segment) or outside of a
double-stranded RNA "stem" (for example, as a loop region
separating a pair of inverted repeats). In cases where base-pairing
anti-sense and sense DNA segment are of unequal length, the longer
segment can act as a spacer. FIGS. 5B and 9 depict illustrations of
possible embodiments of these gene suppression constructs.
[0137] miRNAs: In a further embodiment, the gene suppression
element can include DNA that includes nucleotides derived from a
miRNA (microRNA), that is, a DNA sequence that corresponds to a
miRNA native to a virus or a eukaryote of interest (including
plants and animals, especially invertebrates), or a DNA sequence
derived from such a native miRNA but modified to include nucleotide
sequences that do not correspond to the native miRNA. While miRNAs
have not to date been reported in fungi, fungal miRNAs, should they
exist, are also suitable for use in the invention. A particularly
preferred embodiment includes a gene suppression element containing
DNA that includes nucleotides derived from a viral or plant
miRNA.
[0138] In a non-limiting example, the nucleotides derived from a
miRNA can include DNA that includes nucleotides corresponding to
the loop region of a native miRNA and nucleotides that are selected
from a target gene sequence. In another non-limiting example, the
nucleotides derived from a miRNA can include DNA derived from a
miRNA precursor sequence, such as a native pri-miRNA or pre-miRNA
sequence, or nucleotides corresponding to the regions of a native
miRNA and nucleotides that are selected from a target gene sequence
number such that the overall structure (e.g., the placement of
mismatches in the stem structure of the pre-miRNA) is preserved to
permit the pre-miRNA to be processed into a mature miRNA. In yet
another embodiment, the gene suppression element can include DNA
that includes nucleotides derived from a miRNA and capable of
inducing or guiding in-phase cleavage of an endogenous transcript
into trans-acting siRNAs, as described by Allen et al. (2005) Cell,
121:207-221, which is incorporated by reference in its entirety
herein. Thus, the DNA that includes nucleotides derived from a
miRNA can include sequence naturally occurring in a miRNA or a
miRNA precursor molecule, synthetic sequence, or both.
[0139] siRNAs: In yet another embodiment, the gene suppression
element can include DNA that includes nucleotides of a small
interfering RNA (siRNA). The siRNA can be one or more native siRNAs
(such as siRNAs isolated from a non-transgenic eukaryote or from a
transgenic eukaryote), or can be one or more DNA sequences
predicted to have siRNA activity (such as by use of predictive
tools known in the art, see, for example, Reynolds et al (2004)
Nature Biotechnol., 22:326-330, which is incorporated by reference
in its entirety herein). Multiple native or predicted siRNA
sequences can be joined in a chimeric siRNA sequence for gene
suppression. Such a DNA that includes nucleotides of a siRNA
preferably includes at least 19 nucleotides, and in some
embodiments preferably includes at least 21, at least 22, at least
23, or at least 24 nucleotides. In other embodiments, the DNA that
includes nucleotides of a siRNA can contain substantially more than
21 nucleotides, for example, more than about 50, about 100, about
300, about 500, about 1000, about 3000, or about 5000 nucleotides
or greater.
[0140] Introns: As used herein, "intron" or "intron sequence"
generally means non-coding DNA sequence from a natural gene, which
retains in the recombinant DNA constructs of this invention its
native capability to be excised from pre-mRNA transcripts, e.g.,
native intron sequences found with associated protein coding RNA
regions, wherein the native introns are spliced, allowing exons to
be assembled into mature mRNAs before the RNA leaves the nucleus.
Such an excisable intron has a 5' splice site and a 3' splice site.
Introns can be self-splicing or non-self-splicing (that is,
requiring enzymes or a spliceosome for splicing to occur) and can
be selected for different splicing efficiency.
[0141] Introns suitable for use in constructs of the invention can
be viral introns (e.g., Yamada et al. (1994) Nucleic Acids Res.,
22:2532-2537), eukaryotic introns (including animal, fungal, and
plant introns), archeal or bacterial introns (e.g., Belfort et al.
(1995) J. Bacteriol., 177:3897-3903), or any naturally occurring or
artificial (e.g., Yoshimatsu and Nagawa (1989) Science,
244:1346-1348) DNA sequences with intron-like functionality in the
plant in which the recombinant DNA construct of the invention is to
be transcribed. While essentially any intron can be used in the
practice of this invention as a host for embedded DNA, particularly
preferred are introns that are introns that enhance expression in a
plant or introns that are derived from a 5' untranslated leader
sequence. Where a recombinant DNA construct of the invention is
used to transform a plant, plant-sourced introns can be especially
preferred. Examples of especially preferred plant introns include a
rice actin 1 intron (I-Os-Act1) (Wang et al. (1992) Mol. Cell.
Biol., 12:3399-3406; McElroy et al. (1990) Plant Cell, 2:163-171),
a maize heat shock protein intron (I-Zm-hsp70) (U.S. Pat. Nos.
5,593,874 and 5,859,347), and a maize alcohol dehydrogenase intron
(I-Zm-adh1) (Callis et al. (1987) Genes Dev., 1:1183-1200). Other
examples of introns suitable for use in the invention include the
tobacco mosaic virus 5' leader sequence or "omega" leader (Gallie
and Walbot (1992) Nucleic Acids Res., 20:4631-4638), the Shrunken-1
(Sh-1) intron (Vasil et al. (1989) Plant Physiol., 91:1575-1579),
the maize sucrose synthase intron (Clancy and Hannah (2002) Plant
Physiol., 130:918-929), the heat shock protein 18 (hsp18) intron
(Silva et al. (1987) J. Cell Biol., 105:245), and the 82 kilodalton
heat shock protein (hsp82) intron (Semrau et al. (1989) J. Cell
Biol., 109, p. 39A, and Mettler et al. (May 1990) N.A.T.O. Advanced
Studies Institute on Molecular Biology, Elmer, Bavaria).
[0142] Promoter Elements: Where the recombinant DNA construct is to
be transcribed in an animal cell, the promoter element is
functional in an animal. Where the recombinant DNA construct is to
be transcribed in an plant cell, the promoter element is functional
in a plant. Preferred promoter elements include promoters that have
promoter activity in a plant transformed with the recombinant DNA
constructs of the invention. Suitable promoters can be constitutive
or non-constitutive promoters. In various embodiments, the promoter
element can include a promoter selected from the group consisting
of a constitutive promoter, a spatially specific promoter, a
temporally specific promoter, a developmentally specific promoter,
and an inducible promoter.
[0143] Non-constitutive promoters suitable for use with the
recombinant DNA constructs of the invention include spatially
specific promoters, temporally specific promoters, and inducible
promoters. Where transcription of the construct is to occur in a
plant cell, spatially specific promoters can include organelle-,
cell-, tissue-, or organ-specific promoters functional in a plant
(e.g., a plastid-specific, a root-specific, a pollen-specific, or a
seed-specific promoter for suppressing expression of the first
target RNA in plastids, roots, pollen, or seeds, respectively). In
many cases a seed-specific, embryo-specific, aleurone-specific, or
endospenn-specific promoter is especially useful. Where
transcription of the construct is to occur in an animal cell,
spatially specific promoters include promoters that have enhanced
activity in a particular animal cell or tissue (e.g., enhanced or
specific promoter activity in nervous tissue, liver, muscle, eye,
blood, marrow, breast, prostate, gonads, or other tissues).
Temporally specific promoters can include promoters that tend to
promote expression during certain developmental stages in an animal
or plant's growth or reproductive cycle, or during different times
of day or night, or at different seasons in a year. Inducible
promoters include promoters induced by chemicals (e.g., exogenous
or synthetic chemicals as well as endogenous pheromones and other
signaling molecules) or by environmental conditions such as, but
not limited to, biotic or abiotic stress (e.g., water deficit or
drought, heat, cold, high or low nutrient or salt levels, high or
low light levels, or pest or pathogen infection). An
expression-specific promoter can also include promoters that are
generally constitutively expressed but at differing degrees or
"strengths" of expression, including promoters commonly regarded as
"strong promoters" or as "weak promoters".
[0144] In one particularly preferred embodiment, the promoter
element includes a promoter element functional in a plant
transformed with a recombinant DNA construct of the invention.
Non-limiting specific examples include an opaline synthase promoter
isolated from T-DNA of Agrobacterium, and a cauliflower mosaic
virus 35S promoter, among others, as well as enhanced promoter
elements or chimeric promoter elements, e.g., an enhanced
cauliflower mosaic virus (CaMV) 35S promoter linked to an enhancer
element (an intron from heat shock protein 70 of Zea mays). Many
expression-specific promoters functional in plants and useful in
the method of the invention are known in the art. For example, U.S.
Pat. Nos. 5,837,848; 6,437,217 and 6,426,446 disclose root specific
promoters; U.S. Pat. No. 6,433,252 discloses a maize L3 oleosin
promoter; U.S. Patent Application Publication 2004/0216189
discloses a promoter for a plant nuclear gene encoding a
plastid-localized aldolase; U.S. Pat. No. 6,084,089 discloses
cold-inducible promoters; U.S. Pat. No. 6,140,078 discloses salt
inducible promoters; U.S. Pat. No. 6,294,714 discloses
light-inducible promoters; U.S. Pat. No. 6,252,138 discloses
pathogen-inducible promoters; and U.S. Patent Application
Publication 2004/0123347 A1 discloses water deficit-inducible
promoters. All of the above-described patents and patent
publications disclosing promoters and their use, especially in
recombinant DNA constructs functional in plants, are incorporated
herein by reference.
[0145] The promoter element can include nucleic acid sequences that
are not naturally occurring promoters or promoter elements or
homologues thereof but that can regulate expression of a gene.
Examples of such "gene independent" regulatory sequences include
naturally occurring or artificially designed RNA sequences that
include a ligand-binding region or aptamer and a regulatory region
(which can be cis-acting). See, for example, Isaacs et al. (2004)
Nat. Biotechnol., 22:841-847, Bayer and Smolke (2005) Nature
Biotechnol., 23:337-343, Mandal and Breaker (2004) Nature Rev. Mol
Cell Biol., 5:451-463, Davidson and Ellington (2005) Trends
Biotechnol., 23:109-112, Winkler et al. (2002) Nature, 419:952-956,
Sudarsan et al. (2003) RNA, 9:644-647, and Mandal and Breaker
(2004) Nature Struct. Mol. Biol., 11:29-35, all of which are
incorporated by reference herein. Such "riboregulators" could be
selected or designed for specific spatial or temporal specificity,
for example, to regulate translation of the exogenous gene only in
the presence (or absence) of a given concentration of the
appropriate ligand.
[0146] Terminator Elements: In some embodiments, the recombinant
DNA construct includes both a promoter element and a functional
terminator element. Where it is functional, the terminator element
includes a functional polyadenylation signal and polyadenylation
site, allowing RNA transcribed from the recombinant DNA construct
to be polyadenylated and processed for transport into the
cytoplasm.
[0147] In other embodiments, a functional terminator element is
absent. In some embodiments where a functional terminator element
is absent, at least one of a functional polyadenylation signal and
a functional polyadenylation site is absent. In other embodiments,
a 3' untranslated region is absent. In these cases, the recombinant
DNA construct is transcribed as unpolyadenylated RNA and is
preferably not transported into the cytoplasm.
[0148] T-DNA Borders: T-DNA borders refer to the DNA sequences or
regions of DNA that define the start and end of an Agrobacterium
T-DNA (tumor DNA) and function in cis for transfer of T-DNA into a
plant genome by Agrobacterium-mediated transformation (see, e.g.,
Hooykaas and Schilperoort (1992) Plant Mol. Biol., 19:15-38). In
one preferred embodiment of the recombinant DNA construct of the
invention, the intron in which is embedded the gene suppression
element is located between a pair of T-DNA borders, which can be a
set of left and right T-DNA borders, a set of two left T-DNA
borders, or a set of two right T-DNA borders. In another
embodiment, the recombinant DNA construct includes a single T-DNA
border and an intron-embedded gene suppression element.
[0149] Spacer DNA: Spacer DNA segments can include virtually any
DNA (such as, but not limited to, translatable DNA sequence
encoding a gene of interest, translatable DNA sequence encoding a
marker or reporter gene; transcribable DNA derived from an intron,
which upon transcription can be excised from the resulting
transcribed RNA; transcribable DNA sequence encoding RNA that forms
a structure such as a loop or stem or an aptamer capable of binding
to a specific ligand; spliceable DNA such as introns and
self-splicing ribozymes; transcribable DNA encoding a sequence for
detection by nucleic acid hybridization, amplification, or
sequencing; and a combination of these). Spacer DNA can be found,
for example, between parts of a gene suppression element, or
between different gene suppression elements. In some embodiments,
spacer DNA is itself sense or anti-sense sequence of the target
gene. In some preferred embodiments, the RNA transcribed from the
spacer DNA (e.g., a large loop of antisense sequence of the target
gene or an aptamer) assumes a secondary structure or
three-dimensional configuration that confers on the transcript a
desired characteristic, such as increased stability, increased
half-life in vivo, or cell or tissue specificity.
[0150] Target Genes: The recombinant DNA construct can be designed
to suppress any first target gene. In some embodiments, the
construct further includes a second gene suppression element for
suppressing at least one second target gene, wherein the second
gene suppression element is located adjacent to the intron. Whether
a first or a second target gene, the target gene can include a
single gene or part of a single gene that is targetted for
suppression, or can include, e.g., multiple consecutive segments of
a target gene, multiple non-consecutive segments of a target gene,
multiple alleles of a target gene, or multiple target genes from
one or more species.
[0151] The target gene can be translatable (coding) sequence, or
can be non-coding sequence (such as non-coding regulatory
sequence), or both, and can include at least one gene selected from
the group consisting of a eukaryotic target gene, a non-eukaryotic
target gene, a microRNA precursor DNA sequence, and a microRNA
promoter. The target gene can be native (endogenous) to the cell
(e.g., a cell of a plant or animal) in which the recombinant DNA
construct of the invention is transcribed, or can be native to a
pest or pathogen of the plant or animal in which the construct is
transcribed. The target gene can be an exogenous gene, such as a
transgene in a plant.
[0152] The target gene can include a single gene or part of a
single gene that is targetted for suppression, or can include, for
example, multiple consecutive segments of a target gene, multiple
non-consecutive segments of a target gene, multiple alleles of a
target gene, or multiple target genes from one or more species. A
target gene sequence can include any sequence from any species
(including, but not limited to, non-eukaryotes such as bacteria,
and viruses; fungi; plants, including monocots and dicots, such as
crop plants, ornamental plants, and non-domesticated or wild
plants; invertebrates such as arthropods, annelids, nematodes, and
molluscs; and vertebrates such as amphibians, fish, birds, domestic
or wild mammals, and even humans.
[0153] Non-limiting examples of a target gene include
non-translatable (non-coding) sequence, such as, but not limited
to, 5' untranslated regions, promoters, enhancers, or other
non-coding transcriptional regions, 3' untranslated regions,
terminators, and introns. Target genes can also include genes
encoding microRNAs, small interfering RNAs, RNA components of
ribosomes or ribozymes, small nucleolar RNAs, and other non-coding
RNAs (see, for example, non-coding RNA sequences provided publicly
at rfam.wustl.edu; Erdmann et al. (2001) Nucleic Acids Res.,
29:189-193; Gottesman (2005) Trends Genet., 21:399-404;
Griffiths-Jones et al. (2005) Nucleic Acids Res., 33:121-124, which
are incorporated by reference herein). One specific example of a
target gene includes a microRNA precursor DNA sequence, that is,
the primary DNA transcript encoding a microRNA, or the RNA
intermediates processed from this primary transcript (e.g., a
nuclear-limited pri-miRNA or a pre-miRNA which can be exported from
the nucleus into the cytoplasm), or a microRNA promoter. See, for
example, Lee et al. (2002) EMBO Journal, 21:4663-4670; Reinhart et
al. (2002) Genes & Dev., 16:161611626; Lund et al. (2004)
Science, 303:95-98; and Millar and Waterhouse (2005) Funct. Integr
Genomics, 5:129-135, which are incorporated by reference herein. In
one non-limiting embodiment, the target gene includes nucleotides
of a loop region of at least one target microRNA precursor. In
plants, microRNA precursor molecules (e.g., primary miRNA
transcripts) are believed to be largely processed in the nucleus,
and thus recombinant DNA constructs of the invention that are
transcribed to non-polyadenylated suppression transcripts are
expected to suppress these and other nuclear-localized target genes
in plants more effectively than conventional gene suppression
constructs that result in, e.g., double-stranded RNA molecules
localized in the cytoplasm. Target microRNA precursor DNA sequences
can be native to the transgenic plant in which the recombinant DNA
construct of the invention is transcribed, or can be native to a
pest or pathogen of the transgenic plant. Target genes can also
include translatable (coding) sequence for genes encoding
transcription factors and genes encoding enzymes involved in the
biosynthesis or catabolism of molecules of interest (such as, but
not limited to, amino acids, fatty acids and other lipids, sugars
and other carbohydrates, biological polymers, and secondary
metabolites including alkaloids, terpenoids, polyketides,
non-ribosomal peptides, and secondary metabolites of mixed
biosynthetic origin). A target gene can be a native gene targetted
for suppression, with or without concurrent expression of an
exogenous transgene, for example, by including a gene expression
element in the same or in a separate recombinant DNA construct. For
example, it can be desirable to replace a native gene with an
exogenous transgene homologue.
[0154] It can be useful to provide transgenic plants having in
their genome a DNA construct for suppressing a gene which is
exogenous to the host plant but endogenous to a plant pest or
pathogen (e.g., viruses, bacteria, fungi, and invertebrates such as
insects, nematodes, and molluscs). Thus, one aspect of the
invention provides recombinant DNA constructs wherein the target
gene is selected to provide resistance to a plant pest or pathogen,
for example, resistance to a nematode such as soybean cyst nematode
or root knot nematode or to a pest insect. Thus, target genes of
interest can also include endogenous genes of plant pests and
pathogens. Pest invertebrates include, but are not limited to, pest
nematodes (e.g., cyst nematodes Heterodera spp. especially soybean
cyst nematode Heterodera glycines, root knot nematodes Meloidogyne
spp., lance nematodes Hoplolaimus spp., stunt nematodes
Tylenchorhynchus spp., spiral nematodes Helicotylenchus spp.,
lesion nematodes Pratylenchus spp., ring nematodes Criconema spp.,
and foliar nematodes Aphelenchus spp. or Aphelenchoides spp.), pest
molluscs (slugs and snails), and pest insects (e.g., corn
rootworms, Lygus spp., aphids, corn borers, cutworms, armyworms,
leafhoppers, Japanese beetles, grasshoppers, and other pest
coelepterans, dipterans, and lepidopterans). Plant pathogens of
interest include fungi (e.g., the fungi that cause powdery mildew,
rust, leaf spot and blight, damping-off, root rot, crown rot,
cotton boll rot, stem canker, twig canker, vascular wilt, smut, or
mold, including, but not limited to, Fusarium spp., Phakospora
spp., Rhizoctonia spp., Aspergillus spp., Gibberella spp.,
Pyricularia spp., Alternaria spp., and Phytophthora spp.), bacteria
(e.g., the bacteria that cause leaf spotting, fireblight, crown
gall, and bacterial wilt), mollicutes (e.g., the mycoplasmas that
cause yellows disease and spiroplasmas such as Spiroplasma
kunkelii, which causes corn stunt), and viruses (e.g., the viruses
that cause mosaics, vein banding, flecking, spotting, or abnormal
growth). See also G. N. Agrios, "Plant Pathology" (Fourth Edition),
Academic Press, San Diego, 1997, 635 pp., which is incorporated by
reference herein, for descriptions of fungi, bacteria, mollicutes
(including mycoplasmas and spiroplasmas), viruses, nematodes,
parasitic higher plants, and flagellate protozoans, all of which
are plant pests or pathogens of interest. See also the continually
updated compilation of plant pests and pathogens and the diseases
caused by such on the American Phytopathological Society's "Common
Names of Plant Diseases", compiled by the Committee on
Standardization of Common Names for Plant Diseases of The American
Phytopathological Society, 1978-2005, available online at
www.apsnet.org/online/common/top.asp, which is incorporated by
reference herein.
[0155] Non-limiting examples of fungal plant pathogens of
particular interest include Phakospora pachirhizi (Asian soy rust),
Puccinia sorghi (corn common rust), Puccinia polysora (corn
Southern rust), Fusarium oxysporum and other Fusarium spp.,
Alternaria spp., Penicillium spp., Pythium aphanidermatum and other
Pythium spp., Rhizoctonia solani, Exserohilum turcicum (Northern
corn leaf blight), Bipolaris maydis (Southern corn leaf blight),
Ustilago maydis (corn smut), Fusarium graminearum (Gibberella
zeae), Fusarium verticilliodes (Gibberella moniliformis), F.
proliferatum (G. fujikuroi var. intermedia), F. subglutinans (G.
subglutinans), Diplodia maydis, Sporisorium holci-sorghi,
Colletotrichum graminicola, Setosphaeria turcica, Aureobasidium
zeae, Phytophthora infestans, Phytophthora sojae, Sclerotinia
sclerotiorum, and the numerous fungal species provided in Tables 4
and 5 of U.S. Pat. No. 6,194,636, which is incorporated in its
entirety by reference herein.
[0156] Non-limiting examples of bacterial pathogens include
Pseudomonas avenae, Pseudomonas andropogonis, Erwinia stewartii,
Pseudomonas syringae pv. syringae, and the numerous bacterial
species listed in Table 3 of U.S. Pat. No. 6,194,636, which is
incorporated in its entirety by reference herein.
[0157] Non-limiting examples of viral plant pathogens of particular
interest include maize dwarf mosaic virus (MDMV), sugarcane mosaic
virus (SCMV, formerly MDMV strain B), wheat streak mosaic virus
(WSMV), maize chlorotic dwarf virus (MCDV), barley yellow dwarf
virus (BYDV), banana bunchy top virus (BBTV), and the numerous
viruses listed in Table 2 of U.S. Pat. No. 6,194,636, which is
incorporated in its entirety by reference herein.
[0158] Non-limiting examples of invertebrate pests include pests
capable of infesting the root systems of crop plants, e.g.,
northern corn rootworm (Diabrotica barberi), southern corn rootworm
(Diabrotica undecimpunctata), Western corn rootworm (Diabrotica
virgifera), corn root aphid (Anuraphis maidiradicis), black cutworm
(Agrotis ipsilon), glassy cutworm (Crymodes devastator), dingy
cutworm (Feltia ducens), claybacked cutworm (Agrotis gladiaria),
wireworm (Melanotus spp., Aeolus mellillus), wheat wireworm (Aeolus
mancus), sand wireworm (Horistonotus uhlerii), maize bilibug
(Sphenophorus maidis), timothy bilibug (Sphenophorus zeae),
bluegrass billbug (Sphenophorus parvulus), southern corn billbug
(Sphenophorus callosus), white grubs (Phyllophaga spp.), seedcorn
maggot (Delia platura), grape colaspis (Colaspis brunnea), seedcorn
beetle (Stenolophus lecontei), and slender seedcorn beetle
(Clivinia impressifrons), as well as the parasitic nematodes listed
in Table 6 of U.S. Pat. No. 6,194,636, which is incorporated in its
entirety by reference herein.
[0159] Target genes from pests can include invertebrate genes for
major sperm protein, alpha tubulin, beta tubulin, vacuolar ATPase,
glyceraldehyde-3-phosphate dehydrogenase, RNA polymerase II, chitin
synthase, cytochromes, miRNAs, miRNA precursor molecules, miRNA
promoters, as well as other genes such as those disclosed in Table
II of United States Patent Application Publication 2004/0098761 A1,
which is incorporated by reference herein. Target genes from
pathogens can include genes for viral translation initiation
factors, viral replicases, miRNAs, miRNA precursor molecules,
fungal tubulin, fungal vacuolar ATPase, fungal chitin synthase,
enzymes involved in fungal cell wall biosynthesis, cutinases,
melanin biosynthetic enzymes, polygalacturonases, pectinases,
pectin lyases, cellulases, proteases, and other genes involved in
invasion and replication of the pathogen in the infected plant.
Thus, a target gene need not be endogenous to the plant in which
the recombinant DNA construct is transcribed. A recombinant DNA
construct of the invention can be transcribed in a plant and used
to suppress a gene of a pathogen or pest that may infest the
plant.
[0160] Specific, non-limiting examples of suitable target genes
also include amino acid catabolic genes (such as, but not limited
to, the maize LKR/SDH gene encoding lysine-ketoglutarate reductase
(LKR) and saccharopine dehydrogenase (SDH), and its homologues),
maize zein genes, genes involved in fatty acid synthesis (e.g.,
plant microsomal fatty acid desaturases and plant acyl-ACP
thioesterases, such as, but not limited to, those disclosed in U.S.
Pat. Nos. 6,426,448, 6,372,965, and 6,872,872), genes involved in
multi-step biosynthesis pathways, where it may be of interest to
regulate the level of one or more intermediates, such as genes
encoding enzymes for polyhydroxyalkanoate biosynthesis (see, for
example, U.S. Pat. No. 5,750,848); and genes encoding cell-cycle
control proteins, such as proteins with cyclin-dependent kinase
(CDK) inhibitor-like activity (see, for example, genes disclosed in
International Patent Application Publication Number WO 05007829A2).
Target genes can include genes encoding undesirable proteins (e.g.,
allergens or toxins) or the enzymes for the biosynthesis of
undesirable compounds (e.g., undesirable flavor or odor
components). Thus, one embodiment of the invention is a transgenic
plant or tissue of such a plant that is improved by the suppression
of allergenic proteins or toxins, e.g., a peanut, soybean, or wheat
kernel with decreased allergenicity. Target genes can include genes
involved in fruit ripening, such as polygalacturonase. Target genes
can include genes where expression is preferably limited to a
particular cell or tissue or developmental stage, or where
expression is preferably transient, that is to say, where
constitutive or general suppression, or suppression that spreads
through many tissues, is not necessarily desired. Thus, other
examples of suitable target genes include genes encoding proteins
that, when expressed in transgenic plants, make the transgenic
plants resistant to pests or pathogens (see, for example, genes for
cholesterol oxidase as disclosed in U.S. Pat. No. 5,763,245); genes
where expression is pest- or pathogen-induced; and genes which can
induce or restore fertility (see, for example, the barstar/barnase
genes described in U.S. Pat. No. 6,759,575); all the publications
and patents cited in this paragraph are incorporated by reference
in their entirety herein.
[0161] The recombinant DNA constructs of the invention can be
designed to be more specifically suppress the target gene, by
designing the gene suppression element or elements to include
regions substantially non-identical to a non-target gene sequence.
Non-target genes can include any gene not intended to be silenced
or suppressed, either in a plant transcribing the recombinant DNA
construct or in organisms that may come into contact with RNA
transcribed from the recombinant DNA construct. A non-target gene
sequence can include any sequence from any species (including, but
not limited to, non-eukaryotes such as bacteria, and viruses;
fungi; plants, including monocots and dicots, such as crop plants,
ornamental plants, and non-domesticated or wild plants;
invertebrates such as arthropods, annelids, nematodes, and
molluscs; and vertebrates such as amphibians, fish, birds, domestic
or wild mammals, and even humans).
[0162] In one embodiment, the target gene is a gene endogenous to a
given species, such as a given plant (such as, but not limited to,
agriculturally or commercially important plants, including monocots
and dicots), and the non-target gene can be, e.g., a gene of a
non-target species, such as another plant species or a gene of a
virus, fungus, bacterium, invertebrate, or vertebrate, even a
human. One non-limiting example is where the gene suppression
element is designed to suppress a target gene that is a gene
endogenous to a single species (e.g., Western corn rootworm,
Diabrotica virgifera virgifera LeConte) but to not suppress a
non-target gene such as genes from related, even closely related,
species (e.g., Northern corn rootworm, Diabrotica barberi Smith and
Lawrence, or Southern corn rootworm, Diabrotica
undecimpunctata).
[0163] In other embodiments (e.g., where it is desirable to
suppress a target gene across multiple species), it may be
desirable to design the gene suppression element to suppress a
target gene sequence common to the multiple species in which the
target gene is to be silenced. Thus, a gene suppression element can
be selected to be specific for one taxon (for example, specific to
a genus, family, or even a larger taxon such as a phylum, e.g.,
arthropoda) but not for other taxa (e.g., plants or vertebrates or
mammals). In one non-limiting example of this embodiment, a gene
suppression element for gene silencing can be selected so as to
target pathogenic fungi (e.g., a Fusarium spp.) but not target any
gene sequence from beneficial fungi.
[0164] In another non-limiting example of this embodiment, a gene
suppression element for gene silencing in corn rootworm can be
selected to be specific to all members of the genus Diabrotica. In
a further example of this embodiment, such a Diabrotica-targetted
gene suppression element can be selected so as to not target any
gene sequence from beneficial coleopterans (for example, predatory
coccinellid beetles, commonly known as ladybugs or ladybirds) or
other beneficial insect species.
[0165] The required degree of specificity of a gene suppression
element for suppression of a target gene depends on various
factors. For example, where the gene suppression element contains
DNA that transcribes to RNA for suppressing a target gene by
forming double-stranded RNA (dsRNA), factors can include the size
of the smaller dsRNA fragments that are expected to be produced by
the action of Dicer, and the relative importance of decreasing the
dsRNA's potential to suppress non-target genes. For example, where
the dsRNA fragments are expected to be 21 base pairs in size, one
particularly preferred embodiment can be to include in the gene
suppression element DNA that transcribes to dsRNA and that encodes
regions substantially non-identical to a non-target gene sequence,
such as regions within which every contiguous fragment including at
least 21 nucleotides matches fewer than 21 (e.g., fewer than 21, or
fewer than 20, or fewer than 19, or fewer than 18, or fewer than
17) out of 21 contiguous nucleotides of a non-target gene sequence.
In another embodiment, regions substantially non-identical to a
non-target gene sequence include regions within which every
contiguous fragment including at least 19 nucleotides matches fewer
than 19 (e.g., fewer than 19, or fewer than 18, or fewer than 17,
or fewer than 16) out of 19 contiguous nucleotides of a non-target
gene sequence.
[0166] In some embodiments, it may be desirable to design the gene
suppression element to include regions predicted to not generate
undesirable polypeptides, for example, by screening the gene
suppression element for sequences that may encode known undesirable
polypeptides or close homologues of these. Undesirable polypeptides
include, but are not limited to, polypeptides homologous to known
allergenic polypeptides and polypeptides homologous to known
polypeptide toxins. Publicly available sequences encoding such
undesirable potentially allergenic peptides are available, for
example, the Food Allergy Research and Resource Program (FARRP)
allergen database (available at allergenonline.com) or the
Biotechnology Information for Food Safety Databases (available at
www.iit.edu/.about.sgendel/fa.htm) (see also, for example, Gendel
(1998) Adv. Food Nutr. Res., 42:63-92, which is incorporated by
reference herein). Undesirable sequences can also include, for
example, those polypeptide sequences annotated as known toxins or
as potential or known allergens and contained in publicly available
databases such as GenBank, EMBL, SwissProt, and others, which are
searchable by the Entrez system (www.ncbi.nih.gov/Entrez).
Non-limiting examples of undesirable, potentially allergenic
peptide sequences include glycinin from soybean, oleosin and
agglutinin from peanut, glutenins from wheat, casein, lactalbumin,
and lactoglobulin from bovine milk, and tropomyosin from various
shellfish (allergenonline.com). Non-limiting examples of
undesirable, potentially toxic peptides include tetanus toxin tetA
from Clostridium tetani, diarrheal toxins from Staphylococcus
aureus, and venoms such as conotoxins from Conus spp. and
neurotoxins from arthropods and reptiles
(www.ncbi.nih.gov/Entrez).
[0167] In one non-limiting example, a gene suppression element is
screened to eliminate those transcribable sequences encoding
polypeptides with perfect homology to a known allergen or toxin
over 8 contiguous amino acids, or with at least 35% identity over
at least 80 amino acids; such screens can be performed on any and
all possible reading frames in both directions, on potential open
reading frames that begin with ATG, or on all possible reading
frames, regardless of whether they start with an ATG or not. When a
"hit" or match is made, that is, when a sequence that encodes a
potential polypeptide with perfect homology to a known allergen or
toxin over 8 contiguous amino acids (or at least about 35% identity
over at least about 80 amino acids), is identified, the DNA
sequences corresponding to the hit can be avoided, eliminated, or
modified when selecting sequences to be used in a gene suppression
element.
[0168] Avoiding, elimination of, or modification of, an undesired
sequence can be achieved by any of a number of methods known to
those skilled in the art. In some cases, the result can be novel
sequences that are believed to not exist naturally. For example,
avoiding certain sequences can be accomplished by joining together
"clean" sequences into novel chimeric sequences to be used in a
gene suppression element.
[0169] Where the gene suppression element contains DNA that
transcribes to RNA for suppressing a target gene by forming
double-stranded RNA (dsRNA), applicants recognize that in some
dsRNA-mediated gene silencing, it is possible for imperfectly
matching dsRNA sequences to be effective at gene silencing. For
example, it has been shown that mismatches near the center of a
miRNA complementary site has stronger effects on the miRNA's gene
silencing than do more distally located mismatches. See, for
example, FIG. 4 in Mallory et al. (2004) EMBO J., 23:3356-3364,
which is incorporated by reference herein. In another example, it
has been reported that, both the position of a mismatched base pair
and the identity of the nucleotides forming the mismatch influence
the ability of a given siRNA to silence a target gene, and that
adenine-cytosine mismatches, in addition to the G:U wobble base
pair, were well tolerated (see Du et al. (2005) Nucleic Acids Res.,
33:1671-1677, which is incorporated by reference herein). Thus, the
DNA that transcribes to RNA for suppressing a target gene by
forming double-stranded RNA need not always have 100% sequence
identity with the intended target gene, but generally would
preferably have substantial sequence identity with the intended
target gene, such as about 95%, about 90%, about 85%, or about 80%
sequence identity with the intended target gene. One skilled in the
art would be capable of judging the importance given to screening
for regions predicted to be more highly specific to the first
target gene or predicted to not generate undesirable polypeptides,
relative to the importance given to other criteria, such as, but
not limited to, the percent sequence identity with the intended
first target gene or the predicted gene silencing efficiency of a
given sequence. For example, it may be desirable for a given DNA
sequence for dsRNA-mediated gene silencing to be active across
several species, and therefore one skilled in the art can determine
that it is more important to include in the gene suppression
element regions specific to the several species of interest, but
less important to screen for regions predicted to have higher gene
silencing efficiency or for regions predicted to generate
undesirable polypeptides.
[0170] Gene Expression Element: The recombinant DNA constructs of
the invention can further include a gene expression element. Any
gene or genes of interest can be expressed by the gene expression
element, including coding or non-coding sequence or both, and can
include naturally occurring sequences or artificial or chimeric
sequences or both. Where the gene expression element encodes a
protein, such constructs preferably include a functional terminator
element to permit transcription and translation of the gene
expression element.
[0171] In some embodiments, the recombinant DNA construct further
includes a gene expression element for expressing at least one gene
of interest, wherein the gene expression element is located
adjacent to the intron. In other embodiments, the recombinant DNA
construct further includes a gene expression element for expressing
at least one gene of interest, wherein the gene expression element
is located adjacent to the first gene suppression element and
within the intron; in such cases, the gene expression element can
be operably linked to a functional terminator element that is
itself also within the intron. The gene of interest to be expressed
by the gene expression element can include at least one gene
selected from the group consisting of a eukaryotic target gene, a
non-eukaryotic target gene, and a microRNA precursor DNA sequence.
The gene of interest can include a single gene or multiple genes
(such as multiple copies of a single gene, multiple alleles of a
single gene, or multiple genes including genes from multiple
species). In one embodiment, the gene expression element can
include self-hydrolyzing peptide sequences, e.g., located between
multiple sequences coding for one or more polypeptides (see, for
example, the 2A and "2A-like" self-cleaving sequences from various
species, including viruses, trypanosomes, and bacteria, disclosed
by Donnelly et al. (2001), J. Gen. Virol., 82:1027-1041, which is
incorporated herein by reference). In another embodiment, the gene
expression element can include ribosomal "skip" sequences, e.g.,
located between multiple sequences coding for one or more
polypeptides (see, for example, the aphthovirus foot-and-mouth
disease virus (FMDV) 2A ribosomal "skip" sequences disclosed by
Donnelly et al. (2001), J. Gen. Virol., 82:1013-1025, which is
incorporated herein by reference).
[0172] A gene of interest can include any coding or non-coding
sequence from any species (including, but not limited to,
non-eukaryotes such as bacteria, and viruses; fungi; plants,
including monocots and dicots, such as crop plants, ornamental
plants, and non-domesticated or wild plants; invertebrates such as
arthropods, annelids, nematodes, and molluscs; and vertebrates such
as amphibians, fish, birds, and mammals. Non-limiting examples of a
non-coding sequence to be expressed by a gene expression element
include, but not limited to, 5' untranslated regions, promoters,
enhancers, or other non-coding transcriptional regions, 3'
untranslated regions, terminators, intron, microRNAs, microRNA
precursor DNA sequences, small interfering RNAs, RNA components of
ribosomes or ribozymes, small nucleolar RNAs, and other non-coding
RNAs. Non-limiting examples of a gene of interest further include,
but are not limited to, translatable (coding) sequence, such as
genes encoding transcription factors and genes encoding enzymes
involved in the biosynthesis or catabolism of molecules of interest
(such as amino acids, fatty acids and other lipids, sugars and
other carbohydrates, biological polymers, and secondary metabolites
including alkaloids, terpenoids, polyketides, non-ribosomal
peptides, and secondary metabolites of mixed biosynthetic origin).
A gene of interest can be a gene native to the plant in which the
recombinant DNA construct of the invention is to be transcribed, or
can be a non-native gene. A gene of interest can be a marker gene,
for example, a selectable marker gene encoding antibiotic,
antifungal, or herbicide resistance, or a marker gene encoding an
easily detectable trait (e.g., phytoene synthase or other genes
imparting a particular pigment to the plant), or a gene encoding a
detectable molecule, such as a fluorescent protein, luciferase, or
a unique polypeptide or nucleic acid "tag" detectable by protein or
nucleic acid detection methods, respectively). Selectable markers
are genes of interest of particular utility in identifying
successful processing of constructs of the invention.
[0173] In some embodiments of the invention, the recombinant DNA
constructs are designed to suppress at least one endogenous gene
and to simultaneously express at least one exogenous gene. In one
non-limiting example, the recombinant DNA construct includes a gene
suppression element for suppressing a endogenous (maize) lysine
ketoglutarate reductase/saccharopine dehydrogenase (LKR/SDH) gene
and a gene expression element for expressing an exogenous
(bacterial) dihydrodipicolinic acid synthase protein, where the
construct is transcribed in a maize (Zea mays) plant; such a
construct would be especially useful for providing maize with
enhanced levels of lysine. In another non-limiting example, the
recombinant DNA construct includes a gene suppression element for
suppressing at least one endogenous (maize) zein gene and a gene
expression element for expressing an exogenous or modified zein
protein, where the construct is transcribed in a maize (Zea mays)
plant; such a construct would be especially useful for providing
maize with modified zein content, e.g., zeins with modified amino
acid composition.
[0174] Second Gene Suppression Element: In some embodiments, the
recombinant DNA construct further includes a second gene
suppression element for suppressing at least one second target
gene, wherein the second gene suppression element is located
adjacent to the intron. The second gene suppression element can
include any element as described above under "Gene Suppression
Elements". In these embodiments, where the construct includes a
functional terminator element, the construct can be designed so
that the first gene suppression element, which is embedded in the
intron, preferably causes nuclear suppression of the first target
gene, whereas the second gene suppression element preferably causes
extra-nuclear or cytoplasmic suppression of the second target gene.
The second target gene can be any gene or genes as described above
under the heading "Target Genes", and can include coding or
non-coding sequence or both. The second target gene or genes can be
endogenous or exogenous to the plant in which the recombinant DNA
construct is transcribed, and can include multiple target
genes.
[0175] Methods of Gene Suppression and Methods for Screening for
Traits: The present invention provides a method of effecting gene
suppression, including (a) providing a transgenic plant comprising
a regenerated plant prepared from a transgenic plant cell
containing a recombinant DNA construct for plant transformation
including a promoter operably linked to a first gene suppression
element for suppressing at least one first target gene, wherein
said first gene suppression element is embedded in an intron
flanked on one or on both sides by non-protein-coding DNA, or a
progeny plant of said regenerated plant; and (b) transcribing said
recombinant DNA construct in said transgenic plant; wherein said
transcribing produces RNA that is capable of suppressing said at
least one first target gene in said transgenic plant, whereby said
at least one first target gene is suppressed relative to its
expression in the absence of transcription of said recombinant DNA
construct.
[0176] In some embodiments, the at least one first target gene is
at least one gene selected from the group consisting of a gene
native to said transgenic plant, a transgene in said transgenic
plant, a gene native to a viral, a bacterial, a fungal, or an
invertebrate pest or pathogen of said transgenic seed or of said
transgenic plant, a microRNA precursor sequence, and a microRNA
promoter. The at least one first target gene can be multiple target
genes. In other embodiments, the gene suppression is nuclear
suppression of a microRNA precursor sequence or a microRNA
promoter. Gene suppression by the method of the invention can be
spatially specific, temporally specific, developmentally specific,
or inducible gene suppression. In another embodiment of the method,
the recombinant DNA construct further includes a gene expression
element for expressing at least one gene of interest, wherein the
gene expression element is located outside of (e.g., adjacent to)
the intron, and wherein the gene suppression is effected with
concurrent expression of the at least one gene of interest in the
transgenic plant.
[0177] In one preferred embodiment of the method, the resulting
gene suppression is non-systemic suppression of a gene native to
the transgenic plant or a transgene in the transgenic plant, for
example, to provide non-systemic, tissue-specific suppression of at
least one target gene in the transgenic plant, which can be useful,
for example, for limiting gene suppression to specific tissue, such
as in seeds or roots in plants, wherein the target gene can be
native to the transgenic plant in which the construct is
transcribed or native to a pest or pathogen of said plant. In such
embodiments, it is preferred that the transcribable heterologous
DNA is transcribed to RNA that remains in the nucleus, for example,
to a messenger RNA (mRNA) that lacks processing signals such as
polyadenylation for transport of the mRNA to the cytoplasm. In one
particular example of this embodiment, the gene suppression is
non-systemic, nuclear suppression of a microRNA precursor DNA
sequence or of a microRNA promoter. The method can employ the
recombinant DNA constructs of this invention to modify the lipid,
protein, carbohydrate, or amino acid composition or content of
plant seeds by non-systemically suppressing enzymes in biosynthetic
pathways for such components. In a non-limiting specific example,
transgenic maize having recombinant DNA for suppressing lysine
ketoglutarate reductase (LKR/SDR) can be produced using a
recombinant DNA construct of this invention consisting of an
endosperm-specific or a seed-specific promoter operably linked to,
an intron containing, for example, tandem copies of anti-sense
oriented DNA from the maize endogenous gene encoding LKR/SDH.
Suppression of LKR/SDH is non-systemic (depending on the promoter,
limited to the endosperm or to the seed), and seed from such a
transgenic maize plant with the recombinant DNA construct will have
increased lysine as compared to seed of substantially equivalent
genotype but without the recombinant DNA.
[0178] The present invention further provides a method of
concurrently effecting gene suppression of at least one target gene
and gene expression of at least one gene of interest, including
growing a transgenic plant from a transgenic seed having in its
genome a recombinant DNA construct for suppressing at least one
first target gene, including DNA capable of initiating
transcription in a plant and operably linked to a first
transcribable heterologous DNA, wherein the first transcribable
heterologous DNA is embedded in an intron, and wherein the
recombinant DNA construct further includes a gene expression
element for expressing the at least one gene of interest, the gene
expression element being located adjacent to the intron, and
wherein, when the recombinant DNA construct is transcribed in the
transgenic plant, transcribed RNA that is capable of suppressing
the at least one first target gene and transcribed RNA encoding the
at least one gene of interest are produced, whereby the at least
one first target gene is suppressed relative to its expression in
the absence of transcription of the recombinant DNA construct and
the at least one gene of interest is expressed. The transcribed RNA
that is capable of suppressing the at least one first target gene
is transcribed from the intron-embedded first transcribable
heterologous DNA. The transcribed RNA encoding the at least one
gene of interest is transcribed from the gene expression element.
Where the transcribed RNA encoding the at least one gene of
interest includes coding region for a protein to be expressed, it
is preferably transcribed as RNA capable of transport into the
cytoplasm for translation. The intron-embedded first transcribable
heterologous DNA can be designed to suppress a single or multiple
target genes. The gene expression element can be designed to
express a single or multiple target genes. Optionally, the
recombinant DNA construct can include a second transcribable
heterologous DNA for suppression at least one second target gene,
wherein the second transcribable heterologous DNA is transcribed
into RNA capable of transport into the cytoplasm; in such
embodiments of the method, the at least one first target gene is
preferably suppressed by nuclear suppression, and the at least one
second target gene is preferably suppressed by cytoplasmic
suppression.
[0179] In one embodiment of the method, transgenic plants are
produced that have a modified nutritional content, or that produce
seed having a modified nutritional content. In particularly
preferred embodiment, the method is useful for providing transgenic
maize producing seed with enhanced levels of lysine, tryptophan,
methionine, oil, or a combination of any of these. In one
non-limiting example, the method makes use of a recombinant DNA
construct that includes (a) a gene suppression element (embedded in
an intron flanked on one or both sides by non-protein-coding DNA)
for suppressing a endogenous (maize) lysine ketoglutarate
reductase/saccharopine dehydrogenase (LKR/SDH) gene, and,
optionally, (b) a gene expression element for expressing an
exogenous (e.g., a bacterial) dihydrodipicolinic acid synthase
protein, where the construct is transcribed in a maize (Zea mays)
plant. This method preferably provides transgenic maize producing
seed with enhanced levels of lysine (free or protein-bound or
both). In another non-limiting example of the method, the
recombinant DNA construct includes a gene suppression element
(embedded in an intron flanked on one or both sides by
non-protein-coding DNA) for suppressing at least one endogenous
(maize) zein synthesis gene (e.g., an alpha-zein, such as a
19-kiloDalton alpha-zein or a 22-kiloDalton alpha-zein, or a gene
encoding any one or more of the alpha-, beta-, gamma-, and
delta-zeins) and optionally for suppressing an endogenous (maize)
lysine catabolic enzyme gene (lysine ketoglutarate
reductase/saccharopine dehydrogenase or LKR/SDH), and a gene
expression element for expressing an exogenous lysine synthesis
gene sequence encoding enzymes for synthesis of lysine or its
precursors (e.g., aspartate kinase (AK) and dihydrodipicolinic acid
synthase (DHDPS), and homologues of these genes). This method
preferably provides transgenic maize producing seed with enhanced
levels of lysine (free or protein-bound or both), and more
preferably provides transgenic maize producing seed with enhanced
levels of two or more of lysine, tryptophan, and oil. Also
preferred are methods using similar recombinant DNA constructs to
transform maize, where, for example, the gene expression element is
used to express other biosynthetic genes of interest, such as
asparagine synthase or a modified zein or other storage protein,
wherein the resulting transgenic maize produces seed containing
modified free amino acid or protein content, preferably with
enhanced levels of lysine, tryptophan, methionine, oil, or a
combination of these.
[0180] The present invention further provides a method of
concurrently effecting gene suppression of at least one target gene
and gene expression of at least one gene of interest, including:
(a) providing a transgenic plant comprising a regenerated plant
prepared from a transgenic plant cell containing a recombinant DNA
construct for plant transformation including a promoter operably
linked to a first gene suppression element for suppressing at least
one first target gene, wherein the first gene suppression element
is embedded in an intron flanked on one or on both sides by
non-protein-coding DNA, or a progeny plant of the regenerated
plant, wherein the recombinant DNA construct further includes a
gene expression element for expressing the at least one gene of
interest and the gene expression element is located adjacent to the
intron; and (b) transcribing the recombinant DNA construct in the
transgenic plant, wherein, when the recombinant DNA construct is
transcribed in the transgenic plant, transcribed RNA that is
capable of suppressing the at least one first target gene and
transcribed RNA encoding the at least one gene of interest are
produced, whereby the at least one first target gene is suppressed
relative to its expression in the absence of transcription of the
recombinant DNA construct and the at least one gene of interest is
concurrently expressed.
[0181] The present invention also provides a method of screening
for traits in a transgenic plant resulting from suppression of an
endogenous gene, wherein the method includes: (a) providing a
transgenic plant includes a regenerated plant prepared from a
transgenic plant cell containing a recombinant DNA construct for
plant transformation including a promoter operably linked to a
first gene suppression element for suppressing at least one first
target gene, wherein the first gene suppression element is embedded
in an intron flanked on one or on both sides by non-protein-coding
DNA, or a progeny plant of the regenerated plant; (b) transcribing
the recombinant DNA construct in said transgenic plant; and (c)
analyzing the transgenic plant for the traits. The method can
optionally further include screening for transcription of the gene
suppression element. In some embodiments of the method wherein the
recombinant DNA construct further includes at least one gene
expression element, the screening can optionally further include
detection of expression of a gene encoded by the gene expression
element.
[0182] The methods of the invention make use of procedures to
introduce the recombinant DNA constructs into a transgenic plant
cell, and the production of transgenic plants or progeny plants
from such cells. Such procedures are described under the heading
"Making and Using Transgenic Plant Cells and Plants". Detecting or
measuring the gene suppression (or concurrent gene expression)
obtained by transcription of the construct can be achieved by any
suitable methods, including protein detection methods (e.g.,
western blots, ELISAs, and other immunochemical methods),
measurements of enzymatic activity, or nucleic acid detection
methods (e.g., Southern blots, northern blots, PCR, RT-PCR,
fluorescent in situ hybridization,). Such methods are well known to
those of ordinary skill in the art as evidenced by the numerous
handbooks available; see, for example, Joseph Sambrook and David W.
Russell, "Molecular Cloning: A Laboratory Manual" (third edition),
Cold Spring Harbor Laboratory Press, NY, 2001; Frederick M. Ausubel
et al. (editors) "Short Protocols in Molecular Biology" (fifth
edition), John Wiley and Sons, 2002; John M. Walker (editor)
"Protein Protocols Handbook" (second edition), Humana Press, 2002;
and Leandro Pena (editor) "Transgenic Plants: Methods and
Protocols", Humana Press, 2004, which are incorporated by reference
herein.
[0183] Other suitable methods for detecting or measuring gene
suppression (or concurrent gene expression) include measurement of
any other trait that is a direct or proxy indication of gene
suppression (or concurrent gene expression) in the plant in which
the construct is transcribed, relative to one in which the
construct is not transcribed, e.g., gross or microscopic
morphological traits, growth rates, yield, reproductive or
recruitment rates, resistance to pests or pathogens, or resistance
to biotic or abiotic stress (e.g., water deficit stress, salt
stress, nutrient stress, heat or cold stress). Such methods can use
direct measurements of a phenotypic trait or proxy assays (e.g.,
plant part assays such as leaf or root assays to determine
tolerance of abiotic stress).
III. Recombinant DNA Constructs for Suppressing Production of
Mature miRNA and Methods of Use Thereof
[0184] Another aspect of the invention provides a recombinant DNA
construct for suppressing production of mature microRNA in a cell,
including a promoter element operably linked to a gene suppression
element for suppression of at least one target sequence selected
from the at least one target microRNA precursor or a promoter of
the at least one target microRNA precursor or both. In one
non-limiting embodiment, the target sequence includes nucleotides
of a loop region of at least one target microRNA precursor (that
is, at least some nucleotides in any single-stranded region forming
a loop-like or gap-like domain in a stem-loop RNA structure of a
pri-miRNA or a pre-miRNA). Target microRNA precursor DNA sequences
can be native (endogenous) to the cell (e.g., a cell of a plant or
animal) in which the recombinant DNA construct of the invention is
transcribed, or can be native to a pest or pathogen of the plant or
animal in which the recombinant DNA construct of the invention is
transcribed.
[0185] Using constructs of the invention, suppression of production
of mature miRNA can occur in the nucleus or in the cytoplasm or in
both. In many preferred embodiments, particularly (but not limited
to) embodiments where the suppression occurs in a plant cell,
suppression preferably occurs wholly or substantially in the
nucleus, and the gene suppression element is preferably transcribed
to RNA lacking functional nuclear export signals. In these
embodiments, the RNA transcribed from such a gene suppression
element preferably remains in the nucleus and results in enhanced
nuclear suppression of production of mature miRNA; such a gene
suppression element is preferably characterized by at least one of
the following: (a) at least one of a functional polyadenylation
signal and a functional polyadenylation site is absent; (b) a 3'
untranslated region is absent; (c) a self-splicing ribozyme is
located adjacent to and 3' to the suppression element; and/or (d)
the suppression element is embedded in an intron, preferably an
intron flanked on one or on both sides by non-protein-coding
DNA.
[0186] The recombinant DNA construct for suppressing production of
mature microRNA in a cell includes at least one gene suppression
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 target sequence; (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 target sequence; (c) DNA that includes at least one sense DNA
segment that is at least one segment of the at least one target
sequence; (d) DNA that includes multiple copies of at least one
sense DNA segment that is at least one segment of the at least one
target sequence; (e) DNA that transcribes to RNA for suppressing
the at least one first target sequence 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
sequence and at least one sense DNA segment that is at least one
segment of the at least one target sequence; (f) DNA that
transcribes to RNA for suppressing the at least one first target
sequence 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 target sequence and multiple
serial sense DNA segments that are at least one segment of the at
least one target sequence; (g) DNA that transcribes to RNA for
suppressing the at least one first target sequence 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 target sequence and multiple sense DNA segments that are
at least one segment of the at least one target sequence, 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 miRNA (which can be an
animal, plant, or viral miRNA and is preferably a viral or an
animal miRNA where the construct is to be transcribed in an animal
cell, and preferably a viral or a plant miRNA where the construct
is to be transcribed in a plant cell); and (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. In some embodiments, the gene suppression element
suppresses multiple target microRNA precursors or multiple microRNA
promoters or a combination of both. In some embodiments, the target
sequence includes nucleotides of a loop region of the at least one
target microRNA precursor.
[0187] Where the recombinant DNA construct is to be transcribed in
an animal cell, the promoter includes a promoter element functional
in an animal, and the at least one target microRNA precursor is
endogenous to the animal or a eukaryotic pest or pathogen of the
animal. Where the recombinant DNA construct is to be transcribed in
a plant cell, the promoter element is functional in a plant, and
the at least one target microRNA precursor is endogenous to the
plant or to a eukaryotic pest or eukaryotic pathogen of the plant.
In various embodiments, the recombinant DNA construct includes a
promoter element which can be selected from the group consisting of
a constitutive promoter, a spatially specific promoter, a
temporally specific promoter, a developmentally specific promoter,
and an inducible promoter.
[0188] In various embodiments, the recombinant DNA construct for
suppressing production of mature microRNA in a cell optionally
includes at least one of: (a) at least one T-DNA border; (b) spacer
DNA; (c) a gene expression element for expressing at least one gene
of interest; and (d) a second gene suppression element for
suppressing at least one second target gene, wherein the second
gene suppression element is located adjacent to the intron. In
various embodiments, the recombinant DNA construct is further
characterized by any of the following conditions: (a) the
terminator element includes a functional polyadenylation signal and
polyadenylation site; or (b) at least one of a functional
polyadenylation signal and a functional polyadenylation site is
absent in the terminator element; or (c) a 3' untranslated region
is absent.
[0189] The invention further provides a method of effecting
suppression of mature microRNA production in a eukaryotic cell,
including transcribing in a eukaryotic cell a recombinant DNA
construct for suppressing production of mature microRNA in a cell,
including a promoter element operably linked to a gene suppression
element for suppression of at least one target sequence selected
from the at least one target microRNA precursor or a promoter of
the at least one target microRNA precursor or both, whereby mature
microRNA production is suppressed relative to its production in the
absence of transcription of the recombinant DNA construct. In one
preferred embodiment of the method, the suppression is nuclear
suppression, and the suppression element is transcribed in the cell
to RNA lacking functional nuclear export signals. The suppression
element suppresses at least one target sequence selected from at
least one target microRNA precursor molecule or a promoter of the
at least one microRNA precursor molecule, or both. The method can
include transcription of the recombinant DNA construct in a cell of
an animal, wherein the at least one target microRNA precursor is
endogenous to the animal or a eukaryotic or viral pest or pathogen
of the animal. The method can include transcription of the
recombinant DNA construct in a cell of a plant, wherein the at
least one target microRNA precursor is endogenous to the plant or a
eukaryotic or viral pest or pathogen of the plant. In various
embodiments, the recombinant DNA construct further includes a gene
expression element for expressing at least one gene of interest,
wherein the suppression of mature microRNA production is effected
with concurrent expression of the at least one gene of interest in
the cell.
[0190] In preferred embodiments, the mature miRNA to be suppressed
is a plant miRNA in a plant cell. Suppression can be of a consensus
sequence of multiple mature miRNAs or multiple miRNA precursors, or
of a miRNA promoter that promotes transcription of multiple miRNAs,
or of a consensus sequence of multiple miRNA promoters. In
preferred embodiments, the mature miRNA is a miRNA of a crop plant,
such as, but not limited to, a miRNA of any of the plant species
enumerated under the heading "Transgenic Plants". Especially
preferred are methods where the mature miRNA to be suppressed is a
maize or soybean mature microRNA. In preferred embodiments, the
target microRNA precursor molecule is derived from the fold-back
structure of a crop plant mature miRNA, such as a maize or soybean
MIR sequence selected from the MIR sequences identified in Tables
3, 4, 5, 6, 9, and 10, and their complements. In specifically
claimed embodiments, the target microRNA precursor molecule is
derived from the fold-back structure of a maize or soybean MIR
sequence selected from the group consisting of SEQ ID NO. 6, 7, 8,
9, 10, 12, 14, 16, 18, 20, 22, 24, 28, 30, 32, 34, 38, 39, 43, 44,
227, 228, 236, 239, 242, 245, 248, and 249, and their
complements.
[0191] Promoters and other elements useful in the recombinant DNA
constructs for suppressing production of mature microRNA in a cell
are described in detail under the headings "Gene Suppression
Elements", "Promoter Elements", "Introns", "Terminator Elements",
"T-DNA Borders", "Spacer DNA", and "Gene Expression Elements", and
elsewhere in this disclosure. Techniques for making and using
recombinant DNA constructs of the invention, for making transgenic
plant cells and transgenic plants, seeds, and progeny plants, and
for assaying the effects of transcribing the recombinant DNA
constructs, are described in detail under the headings "Making and
Using Recombinant DNA Constructs", "Making and Using Transgenic
Plant Cells and Transgenic Plants", and elsewhere in this
disclosure.
IV. Engineered Heterologous miRNA for Controlling Gene
Expression
[0192] Engineered miRNAs and trans-acting siRNAs (ta-siRNAs) are
useful for gene suppression with increased specificity. The
invention provides a recombinant DNA construct including a
transcribable engineered miRNA precursor designed to suppress a
target sequence, wherein the transcribable engineered miRNA
precursor is derived from the fold-back structure of a MIR gene,
preferably a maize or soybean MIR sequence selected from the group
consisting of the MIR sequences identified in Tables 3, 4, 5, 6, 9,
and 10, and their complements. In specifically claimed embodiments,
the transcribable engineered miRNA precursor is derived from the
fold-back structure of a maize or soybean MIR sequence selected
from the group consisting of SEQ ID NO. 6, 7, 8, 9, 10, 12, 14, 16,
18, 20, 22, 24, 28, 30, 32, 34, 38, 39, 43, 44, 227, 228, 236, 239,
242, 245, 248, and 249, and their complements. These miRNA
precursors are also useful for directing in-phase production of
siRNAs (e.g., heterologous sequence designed to be processed in a
trans-acting siRNA suppression mechanism in planta). The invention
further provides a method to suppress expression of a target
sequence in a plant cell, including transcribing in a plant cell a
recombinant DNA construct including a transcribable engineered
miRNA precursor designed to suppress a target sequence, wherein the
transcribable engineered miRNA precursor is derived from the
fold-back structure of a MIR gene, preferably a maize or soybean
MIR sequence selected from the group consisting of the MIR
sequences identified in Tables 2, 3, and 4, and their complements,
whereby expression of the target sequence is suppressed relative to
its expression in the absence of transcription of the recombinant
DNA construct. In specifically claimed embodiments, the
transcribable engineered miRNA precursor is derived from the
fold-back structure of a maize or soybean MIR sequence selected
from the group consisting of SEQ ID NO. 6, 7, 8, 9, 10, 12, 14, 16,
18, 20, 22, 24, 28, 30, 32, 34, 38, 39, 43, 44, 227, 228, 236, 239,
242, 245, 248, and 249, and their complements.
[0193] The mature miRNAs produced, or predicted to be produced,
from these miRNA precursors may be engineered for use in
suppression of a target gene, e.g., in transcriptional suppression
by the miRNA, or to direct in-phase production of siRNAs in a
trans-acting siRNA suppression mechanism (see Allen et al. (2005)
Cell, 121:207-221, Vaucheret (2005) Science STKE, 2005:pe43, and
Yoshikawa et al. (2005) Genes Dev., 19:2164-2175, all of which are
incorporated by reference herein). Plant miRNAs generally have
near-perfect complementarity to their target sequences (see, for
example, Llave et al. (2002) Science, 297:2053-2056, Rhoades et al.
(2002) Cell, 110:513-520, Jones-Rhoades and Bartel (2004) Mol.
Cell, 14:787-799, all of which are incorporated by reference
herein). Thus, the mature miRNAs can be engineered to serve as
sequences useful for gene suppression of a target sequence, by
replacing nucleotides of the mature miRNA sequence with nucleotides
of the sequence that is targetted for suppression; see, for
example, methods disclosed by Parizotto et al. (2004) Genes Dev.,
18:2237-2242 and especially U.S. Patent Application Publications
2004/0053411A1, 2004/0268441A1, 2005/0144669, and 2005/0037988 all
of which are incorporated by reference herein. When engineering a
novel miRNA to target a specific sequence, one strategy is to
select within the target sequence a region with sequence that is as
similar as possible to the native miRNA sequence. Alternatively,
the native miRNA sequence can be replaced with a region of the
target sequence, preferably a region that meets structural and
thermodynamic criteria believed to be important for miRNA function
(see, for example, U.S. Patent Application Publication
2005/0037988). Sequences are preferably engineered such that the
number and placement of mismatches in the stem structure of the
fold-back region or pre-miRNA is preserved. Thus, an engineered
miRNA or engineered miRNA precursor can be derived from any of the
mature miRNA sequences, or their corresponding miRNA precursors
(including the fold-back portions of the corresponding MIR genes)
disclosed herein. The engineered miRNA precursor can be cloned and
expressed (transiently or stably) in a plant cell or tissue or
intact plant.
[0194] Promoters and other elements useful in the recombinant DNA
constructs including a transcribable engineered miRNA precursor
designed to suppress a target sequence are described in detail
under the headings "Gene Suppression Elements", "Promoter
Elements", "Introns", "Terminator Elements", "T-DNA Borders",
"Spacer DNA", and "Gene Expression Elements", and elsewhere in this
disclosure. Techniques for making and using recombinant DNA
constructs of the invention, for making transgenic plant cells
containing the recombinant DNA constructs and transgenic plants,
seeds, and progeny plants derived therefrom, and for assaying the
effects of transcribing the recombinant DNA constructs, are
described in detail under the headings "Making and Using
Recombinant DNA Constructs", "Making and Using Transgenic Plant
Cells and Transgenic Plants", and elsewhere in this disclosure.
V. Recombinant DNA Constructs Including Exogenous miRNA Recognition
Sites and Methods for Use Thereof
[0195] One aspect of the invention provides a recombinant DNA
construct including transcribable DNA that transcribes to RNA
including (a) at least one exogenous miRNA recognition site
recognizable by a mature miRNA expressed in a specific cell of a
multicellular eukaryote, and (b) target RNA to be suppressed in the
specific cell, whereby said target RNA is expressed in cells other
than said specific cell. The multicellular eukaryote can be any
multicellular eukaryote (e.g., plant, animal, or fungus), and is
preferably a plant or an animal. The constructs are prepared by
methods known in the art, for example, as disclosed below under the
heading "Making and Using Recombinant DNA Constructs of the
Invention".
[0196] Generally, the recombinant DNA construct includes a promoter
operably linked to the transcribable DNA. Suitable promoters
include any promoter that is capable of transcribing DNA in the
cell where transcription is desired, and are generally promoters
functional in a eukaryotic cell, e.g., the promoters listed below
under the heading "Promoter Elements". Where the specific cell is
an animal cell, the promoter is a promoter functional in the animal
cell. Where the specific cell is a plant cell, the promoter is a
promoter functional in the plant cell. In one embodiment of the
invention, the promoter is preferably a constitutive promoter or a
promoter that allows expression in cells not limited to the
specific cell in which expression of the target RNA is to be
suppressed. The recombinant DNA construct can optionally include a
terminator, e.g., a functional terminator that allows
polyadenylation of the transcript.
[0197] Mature miRNA: By mature miRNA is meant the small RNA
processed from a miRNA precursor (e.g., pri-miRNA or pre-miRNA),
that is capable of recognizing and binding to a specific sequence
("miRNA recognition site") within an RNA transcript, and guiding
the cleavage of that transcript. In one preferred, non-limiting
embodiment of the invention, the mature miRNA is a crop plant
miRNA, such as a maize miRNA or a soy miRNA. Non-limiting examples
of specific miRNAs are provided in the Examples.
[0198] Target RNA: The target RNA is any RNA of interest, and can
include at least one of non-coding RNA, a suppression element; and
a gene expression element, or any combination of these. Non-coding
RNA can include RNA that functions as a suppression element (such
as those described under the heading "Gene Suppression Elements")
as well as RNA with a secondary structure conferring upon it a
desired function, e.g., RNA ribozymes or RNA aptamers that can bind
a specific ligand. The target RNA can include a gene expression
element (described under the heading "Gene Expression Elements")
and can include coding or non-coding sequence from any species.
[0199] miRNA Recognition Site: The at least one miRNA recognition
site is exogenous, that is, occurring in other than a naturally
occurring or native context. One or more (identical or different)
exogenous miRNA recognition sites can be variously located in the
recombinant DNA construct: (a) in the 5' untranslated region of the
target RNA, or (b) in the 3' untranslated region of the target RNA,
or (c) within the target RNA. Inclusion of the exogenous miRNA
recognition site within a coding region may be constrained by the
requirements of the amino acid sequence, but is possible if the
inclusion does not produce translated polypeptides with undesirable
characteristics (e.g., loss or decrease of function). Any miRNA
recognition site may be used in carrying out the invention;
particularly preferred are any of the miRNA recognition sites
provided in Tables 8, 11, and 12, and specifically claimed are the
miRNA recognition sites having SEQ ID NOS. 64-219 and 250-346.
[0200] In one non-limiting embodiment, it may be desirable to
express the target RNA under a non-specific (e.g., a "strong"
constitutive promoter) throughout most cells, but not in specific
cells, of a multicellular eukaryote such as a plant. Thus, the at
least one exogenous miRNA recognition site is generally chosen
according to knowledge of spatial or temporal expression of the
corresponding mature miRNA that recognizes and binds to the miRNA
recognition site.
[0201] Cleavage of a target RNA transcript and the subsequent
suppression of the target RNA is dependent on base pairing between
the mature miRNA and its cognate miRNA recognition site. Thus, the
at least one exogenous miRNA recognition site is designed to have
sufficient sequence complementarity to the mature miRNA to allow
recognition and binding by the mature miRNA. In plants, sequence
complementarity of a miRNA and its recognition site is typically
high, e.g., perfect complementarity between 19, 20, or 21 out of 21
nucleotides (in the case of a mature miRNA that is 21 nucleotides
in length), that is, complementarity of about 90% or greater. A
similar degree of complementarity is preferable for recognition
sites for plant miRNAs of any length (e.g., 20, 21, 22, 23, and 24
nucleotides). The sequence requirements for mature miRNA binding to
a recognition site, and methods for predicting miRNA binding to a
given sequence, are discussed, for example, in Llave et al. (2002)
Science, 297:2053-2056, Rhoades et al. (2002) Cell, 110:513-520,
Jones-Rhoades and Bartel (2004) Mol. Cell, 14:787-799, Schwab et al
(2005) Developmental Cell, 8:517-527, and Xie et al. (2005) Plant
Physiol., 138:2145-2154, all of which are incorporated by reference
herein. When designing a miRNA recognition site as well as its
exact location in or adjacent to a target RNA, it is also
preferable to avoid sequences that have undesirable
characteristics, such sequences encoding undesirable polypeptides,
as described under the heading "Target Genes". When designing
target RNA as a transgene to be expressed, the unintentional
introduction of an exogenous miRNA recognition site is preferably
avoided where suppression by a mature miRNA is not desired.
[0202] One preferred aspect of the invention includes a transgenic
plant cell or a transgenic plant containing in its genome the
recombinant DNA construct including at least one exogenous miRNA
recognition site and target RNA. Suitable transgenic plants include
a regenerated plant prepared from a transgenic plant cell having in
its genome the recombinant DNA construct including at least one
exogenous miRNA recognition site and target RNA, or a progeny plant
of such a regenerated plant; progeny plants include plants of any
developmental stage (including seeds) and include hybrid progeny
plants. One preferred embodiment is a transgenic crop plant wherein
the mature miRNA that recognizes the exogenous miRNA recognition
site is a maize or soybean miRNA (e.g., a miRNA derived from the
fold-back structure of a maize or soybean MIR sequence selected
from the MIR sequences identified in Tables 3, 4, 5, 6, 9, and 10,
and their complements, or more specifically, a MIR sequence
selected from SEQ ID NO. 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22,
24, 28, 30, 32, 34, 38, 39, 43, 44, 227, 228, 236, 239, 242, 245,
248, and 249, and their complements).
[0203] These constructs are useful in methods, as disclosed and
claimed herein, for suppressing expression of a target RNA in a
specific cell of a multicellular eukaryote, including transcribing
in the multicellular eukaryote a recombinant DNA construct
including a promoter operably linked to DNA that transcribes to RNA
including: (a) at least one exogenous miRNA recognition site
recognizable by a mature miRNA expressed in a specific cell, and
(b) target RNA to be suppressed in the specific cell, wherein the
mature miRNA guides cleavage of target RNA in the specific cell,
whereby expression of the target RNA is suppressed in the specific
cell relative to its expression in cells lacking expression of the
mature miRNA. Suitable multicellular eukaryotes include plants
(e.g., mosses, ferns, monocots, and dicots) and animals (including
mammals and other vertebrates). Where the multicellular eukaryote
is a plant, the mature miRNA is preferably a plant mature miRNA; in
some embodiments, the mature miRNA is preferably a mature miRNA
from a crop plant such as, but not limited to, maize or soy (e.g.,
a miRNA derived from the fold-back structure of a maize or soybean
MIR sequence selected from the MIR sequences identified in Tables
3, 4, 5, 6, 9, and 10, and their complements, or more specifically,
a MIR sequence selected from SEQ ID NO. 6, 7, 8, 9, 10, 12, 14, 16,
18, 20, 22, 24, 28, 30, 32, 34, 38, 39, 43, 44, 227, 228, 236, 239,
242, 245, 248, and 249, and their complements).
[0204] In some embodiments, the recombinant DNA construct further
includes a gene expression element for expressing at least one gene
of interest (as described in detail below under "Gene Expression
Element"), wherein the expression of the target RNA is suppressed
with concurrent expression of the at least one gene of interest in
the specific cell. In other embodiments, the target RNA includes a
gene suppression element embedded in an intron, preferably an
intron flanked on one or on both sides by non-protein-coding DNA,
as described under "II. Recombinant DNA Constructs Containing
Introns and Gene Suppression Elements".
[0205] Promoters and other elements useful in the recombinant DNA
constructs including at least one exogenous miRNA recognition site
and target RNA are described in detail under the headings "Gene
Suppression Elements", "Promoter Elements", "Introns", "Terminator
Elements", "T-DNA Borders", "Spacer DNA", and "Gene Expression
Elements", and elsewhere in this disclosure. Techniques for making
and using recombinant DNA constructs of the invention, for making
transgenic plant cells containing the recombinant DNA constructs
and transgenic plants, seeds, and progeny plants derived therefrom,
and for assaying the effects of transcribing the recombinant DNA
constructs, are described in detail under the headings "Making and
Using Recombinant DNA Constructs", "Making and Using Transgenic
Plant Cells and Transgenic Plants", and elsewhere in this
disclosure.
[0206] Making and Using Recombinant DNA Constructs: The recombinant
DNA constructs of the present invention can be made by any method
suitable to the intended application, taking into account, for
example, the type of expression desired and convenience of use in
the plant in which the construct is to be transcribed. General
methods for making and using DNA constructs and vectors are well
known in the art and described in detail in, for example, handbooks
and laboratory manuals including Sambrook and Russell, "Molecular
Cloning: A Laboratory Manual" (third edition), Cold Spring Harbor
Laboratory Press, NY, 2001, which is incorporated herein by
reference. An example of useful technology for building DNA
constructs and vectors for transformation is disclosed in US Patent
Application Publication 2004/0115642 A1, incorporated herein by
reference. DNA constructs can also be built using the GATEWAY.TM.
cloning technology (available from Invitrogen Life Technologies,
Carlsbad, Calif.), which 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, and in U.S. Patent Application
Publications 2001/283529, 2001/282319 and 2002/0007051, all of
which are incorporated herein by reference. The GATEWAY.TM. Cloning
Technology Instruction Manual, which is also supplied by
Invitrogen, provides concise directions for routine cloning of any
desired DNA into a vector comprising operable plant expression
elements. Another alternative vector fabrication method employs
ligation-independent cloning as disclosed by Aslandis et al. (1990)
Nucleic Acids Res., 18:6069-6074 and Rashtchian et al. (1992)
Biochem., 206:91-97, where a DNA fragment with single-stranded 5'
and 3' ends is ligated into a desired vector which can then be
amplified in vivo.
[0207] In certain embodiments, the DNA sequence of the recombinant
DNA construct includes sequence that has been codon-optimized for
the plant in which the recombinant DNA construct is to be
expressed. For example, a recombinant DNA construct to be expressed
in a plant can have all or parts of its sequence (e.g., the first
gene suppression element or the gene expression element)
codon-optimized for expression in a plant. See, e.g., U.S. Pat. No.
5,500,365; De Amicis and Marchetti (2000) Nucleic Acid Res.,
28:3339-3346, which are incorporated by reference herein.
[0208] In certain embodiments, the DNA sequence of the recombinant
DNA construct includes sequence that has been codon-optimized for
the cell (e.g., an animal, plant, or fungal cell) in which the
construct is to be expressed. For example, a construct to be
expressed in a plant cell can have all or parts of its sequence
(e.g., the first gene suppression element or the gene expression
element) codon-optimized for expression in a plant. See, for
example, U.S. Pat. No. 5,500,365; De Amicis and Marchetti (2000)
Nucleic Acid Res., 28:3339-3346, which are incorporated by
reference herein.
[0209] Making and Using Transgenic Plant Cells and Transgenic
Plants: The invention provides and claims a transgenic plant cell
having in its genome any of the recombinant DNA constructs
presently disclosed. The transgenic plant cell can be an isolated
plant cell (e.g., individual plant cells or cells grown in or on an
artificial culture medium), or can be a plant cell in
undifferentiated tissue (e.g., callus or any aggregation of plant
cells). The transgenic plant cell can be a plant cell in at least
one differentiated tissue selected from the group consisting of
leaf (e.g., petiole and blade), root, stem (e.g., tuber, rhizome,
stolon, bulb, and corm) stalk (e.g., xylem, phloem), wood, seed,
fruit (e.g., nut, grain, fleshy fruits), and flower (e.g., stamen,
filament, anther, pollen, carpel, pistil, ovary, ovules). The
invention further provides a transgenic plant having in its genome
any of the recombinant DNA constructs presently disclosed,
including a regenerated plant prepared from the transgenic plant
cells claimed herein, or a progeny plant (which can be a hybrid
progeny plant) of the regenerated plant, or seed of such a
transgenic plant. Also provided is a transgenic seed having in its
genome any of the recombinant DNA constructs presently disclosed,
and a transgenic plant grown from such transgenic seed.
[0210] The transgenic plant cell or plant of the invention can be
any suitable plant cell or plant of interest. Stably transformed
transgenic plants are particularly preferred. In many preferred
embodiments, the transgenic plant is a fertile transgenic plant
from which seed can be harvested, and thus the invention further
claims seed of such transgenic plants, wherein the seed is
preferably also transgenic, that is, preferably contains the
recombinant construct of the invention.
[0211] Where a recombinant DNA construct is used to produce a
transgenic plant cell or transgenic plant of the invention, the
transformation can include any of the well-known and demonstrated
methods and compositions. Suitable methods for plant transformation
include virtually any method by which DNA can be introduced into a
cell, such as by direct delivery of DNA (e.g., by PEG-mediated
transformation of protoplasts, by electroporation, by agitation
with silicon carbide fibers, and by acceleration of DNA coated
particles), by Agrobacterium-mediated transformation, by viral or
other vectors, etc. One preferred method of plant transformation is
microprojectile bombardment, for example, as illustrated in U.S.
Pat. No. 5,015,580 (soy), U.S. Pat. No. 5,550,318 (maize), U.S.
Pat. No. 5,538,880 (maize), U.S. Pat. No. 6,153,812 (wheat), U.S.
Pat. No. 6,160,208 (maize), U.S. Pat. No. 6,288,312 (rice) and U.S.
Pat. No. 6,399,861 (maize), and U.S. Pat. No. 6,403,865 (maize),
all of which are incorporated by reference.
[0212] Another preferred method of plant transformation is
Agrobacterium-mediated transformation. In one preferred embodiment
of the invention, the transgenic plant cell of the invention is
obtained by transformation by means of Agrobacterium containing a
binary Ti plasmid system, wherein the Agrobacterium carries a first
Ti plasmid and a second, chimeric plasmid containing at least one
T-DNA border of a wild-type Ti plasmid, a promoter functional in
the transformed plant cell and operably linked to a gene
suppression construct of the invention. See, for example, U.S. Pat.
No. 5,159,135; De Framond (1983) Biotechnology, 1:262-269; and
Hoekema et al., (1983) Nature, 303:179, which are incorporated by
reference. In such a binary system, the smaller plasmid, containing
the T-DNA border or borders, can be conveniently constructed and
manipulated in a suitable alternative host, such as E. coli, and
then transferred into Agrobacterium.
[0213] Detailed procedures for Agrobacterium-mediated
transformation of plants, especially crop plants, include, for
example, procedures disclosed in U.S. Pat. Nos. 5,004,863,
5,159,135, and 5,518,908 (cotton); U.S. Pat. Nos. 5,416,011,
5,569,834, 5,824,877 and 6,384,301 (soy); U.S. Pat. No. 5,591,616
(maize); U.S. Pat. No. 5,981,840 (maize); U.S. Pat. No. 5,463,174
(brassicas), all of which are incorporated by reference. Similar
methods have been reported for, among others, peanut (Cheng et al.
(1996) Plant Cell Rep., 15: 653); asparagus (Bytebier et al. (1987)
Proc. Natl. Acad. Sci. U.S.A., 84:5345); barley (Wan and Lemaux
(1994) Plant Physiol., 104:37); rice (Toriyama et al. (1988)
Bio/Technology, 6:10; Zhang et al. (1988) Plant Cell Rep., 7:379;
wheat (Vasil et al. (1992) Bio/Technology, 10:667; Becker et al.
(1994) Plant J., 5:299), and alfalfa (Masoud et al. (1996)
Transgen. Res., 5:313). See also U.S. Patent Application
Publication 2003/0167537 A1, incorporated by reference, for a
description of vectors, transformation methods, and production of
transformed Arabidopsis thaliana plants where transcription factors
are constitutively expressed by a CaMV35S promoter. Transgenic
plant cells and transgenic plants can also be obtained by
transformation with other vectors, such as, but not limited to,
viral vectors (e.g., tobacco etch potyvirus (TEV), barley stripe
mosaic virus (BSMV), and the viruses referenced in Edwardson and
Christie, "The Potyvirus Group: Monograph No. 16, 1991, Agric. Exp.
Station, Univ. of Florida, which is incorporated by reference),
plasmids, cosmids, YACs (yeast artificial chromosomes), BACs
(bacterial artificial chromosomes) or any other suitable cloning
vector, when used with an appropriate transformation protocol,
e.g., bacterial infection (e.g., with Agrobacterium as described
above), binary bacterial artificial chromosome constructs, direct
delivery of DNA (e.g., via PEG-mediated transformation,
desiccation/inhibition-mediated DNA uptake, electroporation,
agitation with silicon carbide fibers, and microprojectile
bombardment). It would be clear to one of skill in the art that
various transformation methodologies can be used and modified for
production of stable transgenic plants from any number of plant
species of interest. All of the above-described patents and
publications disclosing materials and methods for plant
transformation are incorporated by reference in their entirety.
[0214] Transformation methods to provide transgenic plant cells and
transgenic plants containing stably integrated recombinant DNA 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 or parts of
embryos, and gametic cells such as microspores, pollen, sperm, and
egg cells. It is contemplated that any cell from which a fertile
plant can be regenerated can be useful as a recipient cell for
practice of the invention. Callus can be initiated from various
tissue sources, including, but not limited to, immature embryos or
parts of embryos, seedling apical meristems, microspores, and the
like. Those cells which are capable of proliferating as callus can
serve as 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, for example, in U.S. Pat.
Nos. 6,194,636 and 6,232,526 and U.S. Application Publication
2004/0216189, which are incorporated by reference.
[0215] In general transformation practice, DNA is introduced into
only a small percentage of target cells in any one transformation
experiment. Marker genes are generally 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 antibiotics or herbicides to
which a plant cell may be resistant can be a useful agent for
selection. 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 can be tested further to confirm stable integration
of the recombinant DNA. Commonly used selective marker genes
include those conferring resistance to antibiotics such as
kanamycin or paromomycin (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 useful
selective marker genes and selection agents 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 by reference. Screenable markers or
reporters, such as markers that provide an ability to visually
identify transformants can also be employed. Non-limiting examples
of useful screenable markers include, for example, a gene
expressing a protein that produces a detectable color by acting on
a chromogenic substrate (e.g., beta-glucuronidase (GUS) (uidA) or
luciferase (luc)) or that itself is detectable, such as green
fluorescent protein (GFP) (gfp) or an immunogenic molecule. Those
of skill in the art will recognize that many other useful markers
or reporters are available for use.
[0216] Detecting or measuring the resulting change in expression of
the target gene (or concurrent expression of a gene of interest)
obtained by transcription of the recombinant construct in the
transgenic plant of the invention can be achieved by any suitable
methods, including protein detection methods (e.g., western blots,
ELISAs, and other immunochemical methods), measurements of
enzymatic activity, or nucleic acid detection methods (e.g.,
Southern blots, northern blots, PCR, RT-PCR, fluorescent in situ
hybridization). Such methods are well known to those of ordinary
skill in the art as evidenced by the numerous handbooks available;
see, for example, Joseph Sambrook and David W. Russell, "Molecular
Cloning: A Laboratory Manual" (third edition), Cold Spring Harbor
Laboratory Press, NY, 2001; Frederick M. Ausubel et al. (editors)
"Short Protocols in Molecular Biology" (fifth edition), John Wiley
and Sons, 2002; John M. Walker (editor) "Protein Protocols
Handbook" (second edition), Humana Press, 2002; and Leandro Pena
(editor) "Transgenic Plants: Methods and Protocols", Humana Press,
2004, which are incorporated by reference.
[0217] Other suitable methods for detecting or measuring the
resulting change in expression of the target gene (or concurrent
expression of a gene of interest) obtained by transcription of the
recombinant DNA in the transgenic plant of the invention include
measurement of any other trait that is a direct or proxy indication
of expression of the target gene (or concurrent expression of a
gene of interest) in the transgenic plant in which the recombinant
DNA is transcribed, relative to one in which the recombinant DNA is
not transcribed, e.g., gross or microscopic morphological traits,
growth rates, yield, reproductive or recruitment rates, resistance
to pests or pathogens, or resistance to biotic or abiotic stress
(e.g., water deficit stress, salt stress, nutrient stress, heat or
cold stress). Such methods can use direct measurements of a
phenotypic trait or proxy assays (e.g., in plants, these assays
include plant part assays such as leaf or root assays to determine
tolerance of abiotic stress).
[0218] The recombinant DNA constructs of the invention can be
stacked with other recombinant DNA for imparting additional traits
(e.g., in the case of transformed plants, traits including
herbicide resistance, pest resistance, cold germination tolerance,
water deficit tolerance, and the like) for example, by expressing
or suppressing other genes. Constructs for coordinated decrease and
increase of gene expression are disclosed in U.S. Patent
Application Publication 2004/0126845 A1, incorporated by
reference.
[0219] Seeds of transgenic, fertile plants can be harvested and
used to grow progeny generations, including hybrid generations, of
transgenic plants of this invention that include the recombinant
DNA construct in their genome. Thus, in addition to direct
transformation of a plant with a recombinant DNA construct,
transgenic plants of the invention can be prepared by crossing a
first plant having the recombinant DNA with a second plant lacking
the construct. For example, the recombinant DNA can be introduced
into a 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 into the resulting progeny. A
transgenic plant of the invention with one recombinant DNA
(effecting change in expression of a target gene) can be crossed
with a plant line having other recombinant DNA that confers one or
more additional trait(s) (such as, but not limited to, herbicide
resistance, pest or disease resistance, environmental stress
resistance, modified nutrient content, and yield improvement) to
produce progeny plants having recombinant DNA that confers both the
desired target sequence expression behavior and the additional
trait(s).
[0220] 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 segregate such that some of the plant
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.
[0221] Yet another aspect of the invention is a transgenic plant
grown from the transgenic seed of the invention. This invention
contemplates transgenic plants grown directly from transgenic seed
containing the recombinant DNA as well as progeny generations of
plants, including inbred or hybrid plant lines, made by crossing a
transgenic plant grown directly from transgenic seed to a second
plant not grown from the same transgenic seed.
[0222] Crossing can include, for example, the following steps:
(a) plant seeds of the first parent plant (e.g., non-transgenic or
a transgenic) and a second parent plant that is transgenic
according to the invention;
(b) grow the seeds of the first and second parent plants into
plants that bear flowers;
(c) pollinate a flower from the first parent with pollen from the
second parent; and
(d) harvest seeds produced on the parent plant bearing the
fertilized flower.
[0223] It is often desirable to introgress recombinant DNA into
elite varieties, e.g., by backcrossing, to transfer a specific
desirable trait from one source to an inbred or other plant that
lacks that trait. This can be accomplished, for example, by first
crossing a superior inbred ("A") (recurrent parent) to a donor
inbred ("B") (non-recurrent parent), which carries the appropriate
gene(s) for the trait in question, for example, a construct
prepared in accordance with the current invention. The progeny of
this cross first are selected in the resultant progeny for the
desired trait to be transferred from the non-recurrent parent "B",
and then the selected progeny are mated back to the superior
recurrent parent "A". After five or more backcross generations with
selection for the desired trait, the progeny are hemizygous for
loci controlling the characteristic being transferred, but are like
the superior parent for most or almost all other genes. The last
backcross generation would be selfed to give progeny which are pure
breeding for the gene(s) being transferred, i.e., one or more
transformation events.
[0224] Through a series of breeding manipulations, a selected DNA
construct can be moved from one line into an entirely different
line without the need for further recombinant manipulation. One can
thus produce inbred plants which are true breeding for one or more
DNA constructs. By crossing different inbred plants, one can
produce a large number of different hybrids with different
combinations of DNA constructs. In this way, plants can be produced
which have the desirable agronomic properties frequently associated
with hybrids ("hybrid vigor"), as well as the desirable
characteristics imparted by one or more DNA constructs.
[0225] Genetic markers can be used to assist in the introgression
of one or more DNA constructs of the invention from one genetic
background into another. Marker assisted selection offers
advantages relative to conventional breeding in that it can be used
to avoid errors caused by phenotypic variations. Further, genetic
markers can provide data regarding the relative degree of elite
germplasm in the individual progeny of a particular cross. For
example, when a plant with a desired trait which otherwise has a
non-agronomically desirable genetic background is crossed to an
elite parent, genetic markers can be used to select progeny which
not only possess the trait of interest, but also have a relatively
large proportion of the desired germplasm. In this way, the number
of generations required to introgress one or more traits into a
particular genetic background is minimized. The usefulness of
marker assisted selection in breeding transgenic plants of the
current invention, as well as types of useful molecular markers,
such as but not limited to SSRs and SNPs, are discussed in PCT
Application Publication WO 02/062129 and U.S. Patent Application
Publications Nos. 2002/0133852, 2003/0049612, and 2003/0005491,
each of which is incorporated by reference in their entirety.
[0226] In certain transgenic plant cells and transgenic plants of
the invention, it may be desirable to concurrently express (or
suppress) a gene of interest while also regulating expression of a
target gene. Thus, in some embodiments, the transgenic plant
contains recombinant DNA further including a gene expression (or
suppression) element for expressing at least one gene of interest,
and regulation of expression of a target gene is preferably
effected with concurrent expression (or suppression) of the at
least one gene of interest in the transgenic plant.
[0227] Thus, as described herein, the transgenic plant cells or
transgenic plants of the invention can be obtained by use of any
appropriate transient or stable, integrative or non-integrative
transformation method known in the art or presently disclosed. The
recombinant DNA constructs can be transcribed in any plant cell or
tissue or in a whole plant of any developmental stage. Transgenic
plants can be derived from any monocot or dicot plant, such as, but
not limited to, plants of commercial or agricultural interest, such
as crop plants (especially crop plants used for human food or
animal feed), wood- or pulp-producing trees, vegetable plants,
fruit plants, and ornamental plants. Non-limiting examples of
plants of interest include grain crop plants (such as wheat, oat,
barley, maize, rye, triticale, rice, millet, sorghum, quinoa,
amaranth, and buckwheat); forage crop plants (such as forage
grasses and forage dicots including alfalfa, vetch, clover, and the
like); oilseed crop plants (such as cotton, safflower, sunflower,
soybean, canola, rapeseed, flax, peanuts, and oil palm); tree nuts
(such as walnut, cashew, hazelnut, pecan, almond, and the like);
sugarcane, coconut, date palm, olive, sugarbeet, tea, and coffee;
wood- or pulp-producing trees; vegetable crop plants such as
legumes (for example, beans, peas, lentils, alfalfa, peanut),
lettuce, asparagus, artichoke, celery, carrot, radish, the
brassicas (for example, cabbages, kales, mustards, and other leafy
brassicas, broccoli, cauliflower, Brussels sprouts, turnip,
kohlrabi), edible cucurbits (for example, cucumbers, melons, summer
squashes, winter squashes), edible alliums (for example, onions,
garlic, leeks, shallots, chives), edible members of the Solanaceae
(for example, tomatoes, eggplants, potatoes, peppers,
groundcherries), and edible members of the Chenopodiaceae (for
example, beet, chard, spinach, quinoa, amaranth); fruit crop plants
such as apple, pear, citrus fruits (for example, orange, lime,
lemon, grapefruit, and others), stone fruits (for example, apricot,
peach, plum, nectarine), banana, pineapple, grape, kiwifruit,
papaya, avocado, and berries; and ornamental plants including
ornamental flowering plants, ornamental trees and shrubs,
ornamental groundcovers, and ornamental grasses. Preferred dicot
plants include, but are not limited to, canola, cotton, potato,
quinoa, amaranth, buckwheat, safflower, soybean, sugarbeet, and
sunflower, more preferably soybean, canola, and cotton. Preferred
monocots include, but are not limited to, wheat, oat, barley,
maize, rye, triticale, rice, ornamental and forage grasses,
sorghum, millet, and sugarcane, more preferably maize, wheat, and
rice.
[0228] The ultimate goal in plant transformation is to produce
plants which are useful to man. In this respect, transgenic plants
of the invention can be used for virtually any purpose deemed of
value to the grower or to the consumer. For example, one may wish
to harvest the transgenic plant itself, or harvest transgenic seed
of the transgenic plant for planting purposes, or products can be
made from the transgenic plant or its seed such as oil, starch,
ethanol or other fermentation products, animal feed or human food,
pharmaceuticals, and various industrial products. For example,
maize is used extensively in the food and feed industries, as well
as in industrial applications. Further discussion of the uses of
maize can be found, for example, in U.S. Pat. Nos. 6,194,636,
6,207,879, 6,232,526, 6,426,446, 6,429,357, 6,433,252, 6,437,217,
and 6,583,338 and PCT Publications WO 95/06128 and WO 02/057471,
each of which is incorporated by reference in its entirety.
[0229] Thus, in preferred embodiments, a transgenic plant of the
invention has at least one altered trait, relative to a plant
lacking said recombinant DNA construct, selected from the group of
traits consisting of:
(a) improved abiotic stress tolerance;
(b) improved biotic stress tolerance;
(c) improved resistance to a pest or pathogen of the plant;
(d) modified primary metabolite composition;
(e) modified secondary metabolite composition;
(f) modified trace element, carotenoid, or vitamin composition;
(g) improved yield;
(h) improved ability to use nitrogen or other nutrients;
(i) modified agronomic characteristics;
(j) modified growth or reproductive characteristics; and
(k) improved harvest, storage, or processing quality.
[0230] The invention further provides a method of providing at
least one altered plant tissue, including: (a) providing a
transgenic plant including a regenerated plant prepared from a
transgenic plant cell having in its genome any of the recombinant
DNA constructs presently disclosed, or a progeny plant of the
regenerated plant; and (b) transcribing the recombinant DNA
construct in at least one tissue of the transgenic plant, whereby
an altered trait in the at least one tissue results, relative to
tissue wherein the recombinant DNA construct is not transcribed,
the altered trait being selected from:
(i) improved abiotic stress tolerance;
(ii) improved biotic stress tolerance;
(iii) improved resistance to a pest or pathogen of the plant;
(iv) modified primary metabolite composition;
(v) modified secondary metabolite composition;
(vi) modified trace element, carotenoid, or vitamin
composition;
(vii) improved yield;
(viii) improved ability to use nitrogen or other nutrients;
(ix) modified agronomic characteristics;
(x) modified growth or reproductive characteristics; and
(xi) improved harvest, storage, or processing quality.
In preferred embodiments of the method of providing at least one
altered plant tissue, the transgenic plant from which such tissue
is obtained is a crop plant as described herein.
[0231] In particularly preferred embodiments, the transgenic plant
is characterized by: improved tolerance of abiotic stress (e.g.,
tolerance of water deficit or drought, heat, cold, non-optimal
nutrient or salt levels, non-optimal light levels) or of biotic
stress (e.g., crowding, allelopathy, or wounding); by improved
resistance to a pest or pathogen (e.g., insect, nematode, fungal,
bacterial, or viral pest or pathogen) of the plant; by a modified
primary metabolite (e.g., fatty acid, oil, amino acid, protein,
sugar, or carbohydrate) composition; a modified secondary
metabolite (e.g., alkaloids, terpenoids, polyketides, non-ribosomal
peptides, and secondary metabolites of mixed biosynthetic origin)
composition; a modified trace element (e.g., iron, zinc),
carotenoid (e.g., beta-carotene, lycopene, lutein, zeaxanthin, or
other carotenoids and xanthophylls), or vitamin (e.g., tocopherols)
composition; improved yield (e.g., improved yield under non-stress
conditions or improved yield under biotic or abiotic stress);
improved ability to use nitrogen or other nutrients; modified
agronomic characteristics (e.g., delayed ripening; delayed
senescence; earlier or later maturity; improved shade tolerance;
improved resistance to root or stalk lodging; improved resistance
to "green snap" of stems; modified photoperiod response); modified
growth or reproductive characteristics (e.g., intentional dwarfing;
intentional male sterility, useful, e.g., in improved hybridization
procedures; improved vegetative growth rate; improved germination;
improved male or female fertility); improved harvest, storage, or
processing quality (e.g., improved resistance to pests during
storage, improved resistance to breakage, improved appeal to
consumers); or any combination of these traits.
[0232] In one preferred embodiment, transgenic seed, or seed
produced by the transgenic plant, has modified primary metabolite
(e.g., fatty acid, oil, amino acid, protein, sugar, or
carbohydrate) composition, a modified secondary metabolite (e.g.,
alkaloids, terpenoids, polyketides, non-ribosomal peptides, and
secondary metabolites of mixed biosynthetic origin) composition, a
modified trace element (e.g., iron, zinc), carotenoid (e.g.,
beta-carotene, lycopene, lutein, zeaxanthin, or other carotenoids
and xanthophylls), or vitamin (e.g., tocopherols,) composition, an
improved harvest, storage, or processing quality, or a combination
of these. For example, it can be desirable to modify the amino acid
(e.g., lysine, methionine, tryptophan, or total protein), oil
(e.g., fatty acid composition or total oil), carbohydrate (e.g.,
simple sugars or starches), trace element, carotenoid, or vitamin
content of seeds of crop plants (e.g., canola, cotton, safflower,
soybean, sugarbeet, sunflower, wheat, maize, or rice), preferably
in combination with improved seed harvest, storage, or processing
quality, and thus provide improved seed for use in animal feeds or
human foods. In another instance, it can be desirable to change
levels of native components of the transgenic plant or seed of a
transgenic plant, for example, to decrease levels of proteins with
low levels of lysine, methionine, or tryptophan, or to increase the
levels of a desired amino acid or fatty acid, or to decrease levels
of an allergenic protein or glycoprotein (e.g., peanut allergens
including ara h 1, wheat allergens including gliadins and
glutenins, soy allergens including P34 allergen, globulins,
glycinins, and conglycinins) or of a toxic metabolite (e.g.,
cyanogenic glycosides in cassaya, solanum alkaloids in members of
the Solanaceae).
EXAMPLES
Example 1
[0233] This example illustrates the construction and use of vectors
designed for double-stranded RNAi suppression or for anti-sense
suppression of a luciferase gene. The gene suppression experiments
used were similar to a dual luciferase assay described by Horstmann
et al. (2004) BMC Biotechnol., 4:13, which is incorporated by
reference herein. A prior art vector, "vector 1A", designed for
double-stranded RNAi suppression of a luciferase gene was
constructed as depicted in FIG. 1A with an RNAi transcription unit
with a polyadenylation site including (a) a chimeric promoter
including an enhanced CaMV35S promoter linked to an enhancer
element (an intron from heat shock protein 70 of Zea mays,
Pe35S-Hsp intron), (b) an inverted repeat of DNA coding for firefly
luciferase (LUC) with anti-sense oriented DNA followed by a sense
oriented DNA, and (c) a 3'UTR DNA from Agrobacterium tumefaciens
nopaline synthase gene (3'NOS) which provides a polyadenylation
(polyA) site. Elements of the plasmid comprising the RNAi
transcription unit had a DNA sequence of SEQ ID NO. 1. See Table 1
for a description of the elements within SEQ ID NO. 1.
TABLE-US-00001 TABLE 1 Nucleotide position Element in SEQ ID NO. 1
CaMV e35S promoter 1-614 Hsp 70 intron 645-1448 Firefly luciferase
anti-sense 1455-1025 Firefly luciferase sense 2082-2502 3' UTR from
nopaline synthase 2515-2767
[0234] A prior art vector, "vector 1B", designed for anti-sense
suppression of a luciferase gene and containing a polyA site was
constructed as depicted in FIG. 1B with an anti-sense transcription
unit including (a) the CaMV e35S-Hsp 70 intron chimeric promoter as
described in Table 1, (b) the firefly luciferase anti-sense
sequence described in Table 2, and (c) the 3' UTR from nopaline
synthase as described in Table 1.
[0235] A novel vector, "vector 1C", designed for double-stranded
RNAi suppression of a luciferase gene was constructed as depicted
in FIG. 1C with an RNAi transcription unit without a
polyadenylation site and including (a) the CaMV e35S-Hsp 70 intron
chimeric promoter as described in Table 1, and (b) an inverted
repeat of DNA coding for firefly luciferase, including the firefly
luciferase anti-sense and firefly luciferase sense sequences
described in Table 1. The RNAi transcription unit did not have
3'UTR DNA sequence providing a functional polyadenylation site.
[0236] Another novel vector, "vector 1D", designed for anti-sense
suppression of a luciferase gene and without a functional
polyadenylation site was constructed as depicted in FIG. 1D with an
anti-sense transcription unit without polyadenylation site and
including (a) the CaMV e35S-Hsp 70 intron chimeric promoter and (b)
the firefly luciferase anti-sense sequence described in Table 1.
The RNAi transcription unit did not have 3'UTR DNA sequence
providing a functional polyadenylation site.
[0237] Maize protoplasts were prepared as previously described by
Sheen (1990) Plant Cell, 2:1027-1038, which is incorporated by
reference herein. Each of the four vectors 1A through 1D was
electroporated together with reporter vectors for firefly
luciferase and Renilla luciferase into three separate volumes of
maize protoplasts. Two sets of firefly luciferase suppression
experiments were performed to confirm the enhanced ability for gene
suppression exhibited by the constructs without a functional
polyadenylation site (vectors 1C and 1D) relative to the anti-sense
construct with a functional polyadenylation site (vector 1B). The
relative level of suppression of the target gene, firefly
luciferase, was indicated by the ratio of firefly luciferase to
Renilla luciferase "ffLUC/rLUC", and the results of the two
experiments are given in Table 2. TABLE-US-00002 TABLE 2 Average
ffLUC/rLUC First Second Vector Description of Construct experiment
experiment 1A RNAi with polyA site 1862 2387 1B anti-sense with
polyA site 6089 13988 1C RNAi without polyA site 3620 5879 1D
anti-sense without polyA site 2238 4762
Example 2
[0238] This example further illustrates the construction and use of
vectors designed for double-stranded RNAi suppression or for
anti-sense suppression of a luciferase gene. The gene suppression
experiments used were similar to a dual luciferase assay described
by Horstmann et al. (2004) BMC Biotechnol., 4:13. The vectors
illustrated in FIG. 2 were constructed. Vector 2A (FIG. 2A), a
control vector not encoding anti-sense or double-stranded RNA for
the target gene (firefly luciferase), consisted of (a) the CaMV
e35S-Hsp 70 intron chimeric promoter as described in Example 1 and
Table 1, (b) an inverted repeat of DNA coding for
beta-glucuronidase (GUS) (uidA) with anti-sense oriented DNA
followed by a sense oriented DNA, and (c) a 3'UTR DNA from
Agrobacterium tumefaciens nopaline synthase gene (3'NOS) as
described in Example 1 and Table 1, which provides a
polyadenylation (polyA) site. Vector 2B (FIG. 2B), a prior art
vector designed for double-stranded RNAi suppression of a
luciferase gene, consisted of (a) the CaMV e35S-Hsp 70 intron
chimeric promoter as described in Example 1 and Table 1, (b) an
inverted repeat of DNA coding for firefly luciferase (LUC) with
anti-sense oriented DNA followed by a sense oriented DNA, as
described in Example 1 and Table 1, and (c) a 3'UTR DNA from
Agrobacterium tumefaciens nopaline synthase gene (3'NOS) as
described in Example 1 and Table 1, which provides a
polyadenylation (polyA) site. Vector 2C (FIG. 2C), a novel vector,
consisted of (a) the CaMV e35S-Hsp 70 intron chimeric promoter as
described in Example 1 and Table 1, (b) the firefly luciferase
anti-sense sequence, as described in Example 1 and Table 1, (c)
spacer DNA consisting of a 3'UTR DNA from Agrobacterium tumefaciens
nopaline synthase gene (3'NOS) as described in Example 1 and Table
1, and (d) the firefly luciferase sense sequence, as described in
Example 1 and Table 1. Vector 2D (FIG. 2D), a novel vector,
consisted of (a) the CaMV e35S-Hsp 70 intron chimeric promoter as
described in Example 1 and Table 1, (b) a first copy of the firefly
luciferase anti-sense sequence, as described in Example 1 and Table
1, (c) spacer DNA consisting of a 3'UTR DNA from Agrobacterium
lumefaciens nopaline synthase gene (3'NOS) as described in Example
1 and Table 1, and (d) a second copy of the firefly luciferase
anti-sense sequence. Vector 2E (FIG. 2E), a prior art vector
designed for anti-sense RNA suppression of a luciferase gene,
consisted of (a) the CaMV e35S-Hsp 70 intron chimeric promoter as
described in Example 1 and Table 1, (b) the firefly luciferase
anti-sense sequence, as described in Example 1 and Table 1, and (c)
a 3'UTR DNA from Agrobacterium tumefaciens nopaline synthase gene
(3'NOS) as described in Example 1 and Table 1, which provides a
polyadenylation (polyA) site.
[0239] Each of the four vectors was electroporated together with
reporter vectors for firefly luciferase and Renilla luciferase into
three separate volumes of maize protoplasts prepared as previously
described by Sheen (1990) Plant Cell, 2:1027-1038. Firefly
luciferase suppression experiments were performed, and the relative
level of suppression of the target gene, firefly luciferase, was
indicated by the logarithm of the ratio of firefly luciferase to
Renilla luciferase, "log(Fluc/Rluc)", as depicted in FIG. 3.
Example 3
[0240] This example describes transformation of a crop plant
(maize) with an enhanced anti-sense construct. A plasmid for binary
vector Agrobacterium-mediated transformation of maize is
constructed including the elements shown in FIG. 4. Specifically,
the plasmid includes an nptII gene as an antibiotic selectable
marker and a recombinant DNA construct for enhanced anti-sense gene
suppression, consisting of a CaMV35S promoter operably linked to
transcribable DNA consisting of about 300 base pairs of a green
fluorescent protein (gfp) gene in an anti-sense orientation,
wherein a functional polyadenylation site is absent in this
transcribable DNA. The plasmid also includes left T-DNA border (LB)
and right T-DNA border (RB) elements. A control plasmid for RNAi
suppression of green fluorescent protein (GFP) is constructed by
adding to the enhanced anti-sense construct shown in FIG. 4 a
repeat of the gfp DNA in the sense orientation followed by a 3' NOS
element including a functional polyadenylation site. Maize callus
for transformation is selected from a transgenic maize line
expressing GFP. Both the plasmid with the enhanced anti-sense
construct and the control plasmid with the RNAi construct are
inserted into maize callus by Agrobacterium-mediated
transformation. Events are selected as being resistant to
kanamycin. The efficiency of suppression with enhanced anti-sense
constructs is substantially the same as with the RNAi
constructs.
Example 4
[0241] This example illustrates the use of a recombinant DNA
construct for non-systemic suppression of a target gene in specific
tissue of a transgenic plant. Specifically, this example describes
transformation of a crop plant (maize) with an enhanced anti-sense
construct. A plasmid for binary vector Agrobacterium-mediated
transformation of corn is constructed including the elements shown
in FIG. 5A. Specifically, the plasmid includes an aroA gene as an
herbicidal selectable marker and a recombinant DNA construct for
enhanced anti-sense gene suppression, consisting of a seed-specific
maize L3 oleosin promoter (as disclosed in U.S. Pat. No. 6,433,252,
incorporated herein by reference) operably linked to transcribable
DNA consisting of about 300 base pairs of the LKR domain of a maize
lysine ketoglutarate reductase/saccharopine dehydrogenase gene
(LKR/SDH) in an anti-sense orientation, wherein a functional
polyadenylation site is absent in this transcribable DNA. The
plasmid also includes left T-DNA border (LB) and right T-DNA border
(RB) elements. The plasmid with the enhanced anti-sense construct
is inserted into maize callus by Agrobacterium-mediated
transformation. Events are 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/SDH. The
mature transgenic seeds are dissected to extract protein for
Western analysis. Seed from transgenic maize plants shows reduction
in LKR/SDH and increased lysine as compared to wild type.
[0242] In a further development of this approach, a recombinant DNA
construct of the present invention is constructed as follows. A
plasmid for binary vector Agrobacterium-mediated transformation of
corn is constructed as shown in FIG. 5B, which includes a
recombinant DNA construct of the present invention for gene
suppression, including left and right T-DNA borders containing
between them a promoter element operably linked to an intron (maize
heat shock protein 70 intron, I-Zm-hsp70) within which is embedded
a first gene suppression element for suppressing at least one first
target gene (in this non-limiting example, the at least one first
target gene includes coding sequence from the LKR domain, coding
sequence from the SDH domain, or non-coding sequence of the maize
lysine ketoglutarate reductase/saccharopine dehydrogenase gene
(LKR/SDH), or any combination of these). The first gene suppression
element can include any gene suppression element as described above
under the heading "Gene Suppression Elements" wherein the intron is
located adjacent to the promoter element. In the specific,
non-limiting embodiment depicted in FIG. 5B, the promoter element
is an endosperm-specific maize B32 promoter (nucleotides 848
through 1259 of GenBank accession number X70153, see also Hartings
et al. (1990) Plant Mol. Biol., 14:1031-1040, which is incorporated
herein by reference), although other promoter elements could be
used. This specific embodiment also includes an aroA gene as an
herbicidal selectable marker; other selectable marker or reporter
genes can be used, e.g., a selectable marker conferring glyphosate
resistance, epsps-cp4 (5-enolpyruvylshikimate-3-phosphate synthase
from Agrobacterium tumefaciens strain CP4). The intron-embedded
gene suppression element includes any one or more gene suppression
elements, including, for example, single or multiple copies of
sense or anti-sense, tandem or interrupted repeats, single or
multiple sense/anti-sense pairs able to form dsRNA for gene
suppression, gene suppression sequences derived from an miRNA,
sequences including siRNAs, or combinations of any of these. The
construct optionally includes a gene expression element (e.g.,
transcribable or translatable DNA outside of the intron), a second
gene suppression element, or both.
[0243] In one non-limiting example, the gene suppression element
includes an about 300 base-pair anti-sense DNA segment that is
anti-sense to the target gene, maize lysine ketoglutarate
reductase/saccharopine dehydrogenase gene (LKR/SDH), wherein a
functional polyadenylation site is absent in this transcribable
heterologous DNA. The plasmid also includes left T-DNA border (LB)
and right T-DNA border (RB) elements. The plasmid with the
intron-embedded transcribable heterologous DNA is inserted into
maize callus by Agrobacterium-mediated transformation. Events are
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/SDH. The mature transgenic seeds are dissected
to extract protein for Western analysis. Seed from transgenic maize
plants shows endosperm-specific reduction in LKR/SDH and increased
lysine as compared to wild type.
Example 5
[0244] This example illustrates use of recombinant DNA constructs
for pest control in plants producing by means of gene suppression
in a specific tissue of a transgenic plant. Specifically, this
example describes transformation of a crop plant (soybean) with an
enhanced anti-sense construct. A plasmid for binary vector
Agrobacterium-mediated transformation of soybean is constructed
including the elements shown in FIG. 6. Specifically, the plasmid
includes an aroA gene as an herbicidal selectable marker and a
recombinant DNA construct for enhanced anti-sense gene suppression,
consisting of a TUB-1 root specific promoter from Arabidopsis
thaliana (disclosed in FIG. 1 of U.S. Patent Application
Publication 2004/078841 A1, incorporated by reference herein)
operably linked to transcribable DNA consisting of anti-sense
oriented DNA of a nematode major sperm protein (msp) of a soybean
cyst nematode (disclosed as SEQ ID NO:5 in U.S. Patent Application
Publication 2004/0098761 A1, incorporated herein by reference),
wherein a functional polyadenylation site is absent in this
transcribable DNA. The plasmid also includes left T-DNA border (LB)
and right T-DNA border (RB) elements. The plasmid with the enhanced
anti-sense construct is inserted into soybean callus by
Agrobacterium-mediated transformation. Events are selected as being
resistance to glyphosate herbicide. Reduction in soybean cyst
nematode infestation as compared to wild type is observed.
[0245] In a further development of this approach, a recombinant DNA
construct of the present invention is constructed as follows. A
plasmid for binary vector Agrobacterium-mediated transformation of
corn is constructed, which includes an aroA gene as an herbicidal
selectable marker and a recombinant DNA construct of the present
invention for gene suppression, consisting of a TUB-1 root specific
promoter from Arabidopsis thaliana (disclosed in FIG. 1 of U.S.
Patent Application Publication 2004/078841 A1, incorporated by
reference herein) operably linked to an intron (maize alcohol
dehydrogenase intron, I-Zm-adh1) within which is embedded a first
gene suppression element for suppression of an endogenous gene of a
crop plant pest (soybean cyst nematode); in this specific,
non-limiting example, the gene suppression element is transcribable
heterologous DNA that includes an anti-sense DNA segment that is
anti-sense to the target gene, nematode major sperm protein of a
soybean cyst nematode (disclosed as SEQ ID NO:5 in U.S. Patent
Application Publication 2004/0098761 A1, incorporated herein by
reference), wherein the resulting transcribed RNA is
unpolyadenylated. As a selectable marker, the plasmid alternatively
uses a gene conferring glyphosate resistance, epsps-cp4
(5-enolpyruvylshikimate-3-phosphate synthase from Agrobacterium
tumefaciens strain CP4). Other promoters, first transcribable
heterologous DNAs, or introns can be substituted; the construct
optionally includes a gene expression element, a second
transcribable heterologous DNA for suppressing a second target
gene, or both. The plasmid optionally contains a transcribable or
translatable gene expression element outside of the intron. The
plasmid also includes left T-DNA border (LB) and right T-DNA border
(RB) elements. The plasmid with the enhanced anti-sense construct
is inserted into soybean callus by Agrobacterium-mediated
transformation. Events are selected as being resistance to
glyphosate herbicide. Reduction in soybean cyst nematode
infestation as compared to wild type is observed.
Example 6
[0246] This example illustrates a recombinant DNA construct of the
invention, specifically, a construct including a gene suppression
element that contains intron-embedded tandem repeats. More
specifically, this illustrates a construct including a suppression
element that contains intron-embedded tandem repeats for
suppression of at least one target microRNA precursor. The tandem
repeats are designed to suppress at least one target sequence
selected from said at least one target microRNA precursor or a
promoter of said at least one target microRNA precursor or both.
This example also describes methods for testing recombinant DNA
constructs for their ability to silence a target gene, and
optionally for their ability to concurrently express a gene of
interest.
[0247] Gene silencing by tandem repeats may operate through a
nuclear-localized heterochromatin-associated RNAi pathway. See, for
example, Sijen et al. (1996) Plant Cell, 8:2277-2294, Ma and Mitra
(2002) Plant J, 31:37-49, Zilberman et al. (2003) Science,
299:716-719, and Martienssen (2003) Nat. Genet., 35:213-214, which
are incorporated by reference herein. The present invention
provides recombinant DNA constructs for enhanced nuclear-localized
gene silencing (e.g., suppression of production of mature
microRNA). Non-limiting examples of such constructs are constructs
with one or more suppression elements including tandem repeats,
where the tandem repeats are embedded in an intron; such constructs
can optionally include a gene expression element (FIG. 7A), which
can be upstream (5', not shown) or downstream (3', as shown) of the
intron. Two other approaches for enhancing nuclear-localized gene
silencing by tandem repeats are tandem repeats that are transcribed
but not processed for transport into the cytoplasm, e.g.,
transcribed from constructs lacking a functional terminator, as
shown in FIG. 7B, and tandem repeats under transcriptional control
of two opposing promoters, as shown in FIG. 7C. By embedding tandem
repeats in an intron (e.g., FIG. 7A), transgenic transcripts splice
out the tandem repeat containing intron in the nucleus. By removing
or omitting a functional terminator of a transgene cassette (e.g.,
FIG. 7B), the resulting RNA transcripts containing tandem repeats
are without a polyA signal and more likely to accumulate in the
nucleus. In a construct where tandem repeats are flanked by
opposing or convergent promoters (e.g., FIG. 7C), one promoter will
transcribe the sense strand, and the other will transcribe the
antisense strand; these two complementary strands can form a dsRNA.
The purpose for this is to provide the initial dsRNA substrate for
a Dicer or a Dicer-like enzyme. Thus, for example, Dicer produces
siRNAs, and RDR2-dependent amplification of dsRNA and siRNAs,
facilitated by the tandem repeat configuration, maintains the
silencing pathway for these sequences.
[0248] In the non-limiting examples shown in FIG. 7, there are two
copies in the tandem repeat. Also encompassed by the invention are
embodiments with the copy number of the tandem repeat ranging from
2 to about 100, as well as embodiments with tandem or interrupted
repeats of one or more sequences (in non-limiting examples, these
could include, e.g., AABB, AABAA, AABAABAA, AABAABB, ABBBBAA,
AAABBB, AABBAA, and other arrangements, where A and B represent
discrete sequences, each of which can be repeated). The size of
each repeat is preferably at least about 19, or at least about 21,
or at least about 50, at least about 100, at least about 200, or at
least about 500 nucleotides in length. Preferably, at least two of
the repeats are in the tandem repeat orientation. Unique or
non-repeated sequences, including repeats of a second sequence, can
optionally occur as "spacers" between some or all of the repeated
units. Such spacers are preferably at least about 4, at least about
10, or at least about 20 nucleotides in length. Having unique
sequences between facilitate assembly and/or verification of the
tandem repeats. The repeats can be arranged in either the sense or
antisense orientation, or, for example, where there are repeats of
more than one sequence, each sequence can independently be in an
arrangement of tandem sense repeats or tandem anti-sense
repeats.
Example 7
[0249] This example describes non-limiting methods for testing any
of the recombinant DNA constructs of the invention for their
ability to silence a target gene, and optionally for their ability
to concurrently express a gene of interest. Constructs can be
designed and tested in transient assays by various means known to
one skilled in the art, for example, protoplast transient
transformation and Agrobacterium infiltration assays. For example,
constructs can be designed where the target gene is a gene easily
assayed for suppression (e.g., green fluorescent protein or GFP,
luciferase or luc, or other reporter or marker genes commonly
used). Such transient assays can generally be used to test any
recombinant DNA constructs, e.g., constructs containing
intron-embedded gene suppression elements (including gene
suppression elements other than tandem repeats) for their ability
to suppress a target gene.
[0250] In one non-limiting example, experiments to assay for gene
suppression of a target gene (the reporter gene, luciferase) are
carried out with a maize protoplast model system. Maize protoplasts
are prepared as previously described by Sheen (1990) Plant Cell,
2:1027-1038, which is incorporated by reference herein.
Polyethylene glycol (PEG)-mediated transformations (see, for
example, Armstrong et al. (1990), Plant Cell Rep., 9:335-339, which
is incorporated by reference herein) are performed in deep well (2
milliliters/well) 96-well plates. Separate vectors containing
either firefly luciferase or Renilla luciferase are employed as
reporters. The firefly luciferase reporter vector includes a
chimeric promoter including a chimeric promoter including an
enhanced cauliflower mosaic virus (CaMV) 35S promoter linked to an
enhancer element (an intron from heat shock protein 70 of Zea
mays), the coding sequence of the firefly luciferase gene luc, and
a 3'untranslated region (3' UTR) DNA from Agrobacterium tumefaciens
nopaline synthase gene (3'NOS) which provides a polyadenylation
(polyA) site. The Renilla luciferase reporter vector includes the
same chimeric promoter, the coding sequence of the Renilla
luciferase gene luc, and the same 3'NOS UTR terminator. Generally,
1.3 micrograms firefly luciferase reporter vector DNA, 0.6
micrograms Renilla luciferase reporter vector DNA, and additional
plasmid (pUC18) DNA are added to each well in order to maintain the
total amount of RNA plus DNA constant at 12.5 micrograms per well.
To each well is added 160 microliters (2.times.10.sup.6 protoplasts
per milliliter) of maize protoplasts. Protoplasts are made
transformation-competent by treatment with a solution containing 4
grams PEG 4000, 2 milliliters water, 3 milliliters 0.8 molar
mannitol, and 1 milliliter Ca(NO.sub.3).sub.2. The protoplasts are
co-transformed with the test recombinant DNA constructs of the
invention, where the target gene is firefly luciferase, together
with the reporter vectors for firefly luciferase and Renilla
luciferase, into 4 separate volumes of maize protoplasts; the test
constructs can be delivered in a vector. The relative level of
suppression of the target gene, firefly luciferase, is indicated by
the intensity of firefly luciferase emission ("Fluc") normalized to
Renilla luciferase emission (Rluc). A negative control test vector
is, for example, one similar to the test vectors containing the
gene suppression elements but containing a gene suppression element
targetting a non-relevant gene such as beta-glucuronidase (GUS)
(uidA). A positive control test vector is, for example, one similar
to the test vector but containing, for example, the full-length
firefly luc gene. The relative level of suppression of the target
gene, firefly luciferase, is given as the logarithm of the ratio of
firefly luciferase emission to Renilla luciferase emission,
"log(Fluc/Rluc)".
[0251] Transient assays such as the one described in the preceding
paragraph can be designed to optionally simultaneously assay for
expression of a gene of interest. For example, a model gene of
interest can include GFP. The experiments are carried out as in the
preceding paragraph, where the test recombinant DNA constructs can
contain both a first gene suppression element for suppressing the
target gene and a gene expression element for expressing a gene of
interest such as GFP. The expression of GFP can be simultaneously
monitored by spectrophotometry as is the firefly and Renilla
luciferase emission.
Example 8
[0252] This example describes various non-limiting embodiments of
recombinant DNA constructs of the invention and useful in making
transgenic eukaryotes (including transgenic plant cells, plants,
and seeds) of the invention. One non-limiting application of these
constructs is, for example, suppression of at least one target
miRNA precursor or miRNA promoter, or non-systemic gene suppression
of a gene endogenous to a plant or to a pest or pathogen of the
plant.
[0253] FIG. 8A schematically depicts non-limiting examples of
recombinant DNA constructs of the invention for suppression of at
least one target gene. These constructs include at least one first
gene suppression element ("GSE" or "GSE1") for suppressing at least
one first target gene, wherein the first gene suppression element
is embedded in an intron flanked on one or on both sides by
non-protein-coding DNA. These constructs utilize an intron (in many
embodiments, an intron derived from a 5' untranslated region or an
expression-enhancing intron is preferred) to deliver a gene
suppression element without requiring the presence of any
protein-coding exons (coding sequence). The constructs can
optionally include at least one second gene suppression element
("GSE2") for suppressing at least one second target gene, at least
one gene expression element ("GEE") for expressing at least one
gene of interest (which can be coding or non-coding sequence or
both), or both. In embodiments containing an optional gene
expression element, the gene expression element can be located
outside of (e.g., adjacent to) the intron. In some embodiments, the
intron containing the first gene suppression element is 3' to a
terminator.
[0254] To more clearly differentiate recombinant DNA constructs of
the invention (containing at least one gene suppression element
embedded within a single intron flanked on one or on both sides by
non-protein-coding DNA) from the prior art, FIG. 8B schematically
depicts examples of prior art recombinant DNA constructs. These
constructs can contain a gene suppression element that is located
adjacent to an intron flanked by protein-coding sequence, or
between two discrete introns (wherein the gene suppression element
is not embedded in either of the two discrete introns), or can
include a gene expression element including a gene suppression
element embedded within an intron which is flanked by multiple
exons (e.g., exons including the coding sequence of a protein).
Example 9
[0255] This example describes various non-limiting embodiments of
gene suppression constructs of the invention. FIG. 9 depicts
various non-limiting examples of gene suppression elements and
transcribable exogenous DNAs useful in the recombinant DNA
constructs of the invention. Where drawn as a single strand (FIGS.
9A through 9E), these are conventionally depicted in 5' to 3' (left
to right) transcriptional direction; the arrows indicate anti-sense
sequence (arrowhead pointing to the left), or sense sequence
(arrowhead pointing to the right). These gene suppression elements
and transcribable exogenous DNAs can include: 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, or 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 (FIG. 9A); DNA that includes at least one sense DNA segment
that is at least one segment of the at least one first target gene,
or 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 (FIG. 9B); 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 (FIG. 9C); 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
(FIG. 9D); 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 said multiple anti-sense
DNA segments and the multiple sense DNA segments are arranged in a
series of inverted repeats (FIG. 9E); and DNA that includes
nucleotides derived from a miRNA, or DNA that includes nucleotides
of a siRNA (FIG. 9F).
[0256] FIG. 9F depicts various non-limiting arrangements of
double-stranded RNA that can be transcribed from embodiments of the
gene suppression elements and transcribable exogenous DNAs useful
in the recombinant DNA constructs of the invention. When such
double-stranded RNA is formed, it can suppress one or more target
genes, and can form a single double-stranded RNA or multiple double
strands of RNA, or a single double-stranded RNA "stem" or multiple
"stems". Where multiple double-stranded RNA "stems" are formed,
they can be arranged in "hammerheads" or "cloverleaf"
arrangements.
Example 10
[0257] This example describes various non-limiting embodiments of
recombinant DNA constructs of the invention and useful in making
transgenic eukaryotes (including transgenic plant cells, plants,
and seeds) of the invention. More specifically, this example
describes embodiments of gene suppression constructs that
transcribe to RNA capable of forming multiple double-stranded
"stems" and suppress one or more target genes.
[0258] To form a double "hairpin" molecule or a double-stranded RNA
structure resembling a "hammerhead", a recombinant DNA construct is
designed to include a single-stranded, contiguous DNA sequence
including two non-identical pairs of self-complementary sequences
is used, wherein the DNA can transcribe to RNA also including two
non-identical pairs of self-complementary sequences that can form
two separate double-stranded RNA "stems". Each member of a
non-identical pair of self-complementary sequences preferably
includes at least about 19 to about 27 nucleotides (for example 19,
20, 21, 22, 23, or 24 nucleotides) for every target gene that the
recombinant DNA construct is intended to suppress; in many
embodiments the pair of self-complementary sequence can be larger
than at least about 19 to about 27 base pairs (for example, more
than about 30, about 50, about 100, about 200, about 300, about
500, about 1000, about 1500, about 2000, about 3000, about 4000, or
about 5000 base pairs) for every target gene that the recombinant
DNA construct is intended to suppress. Each non-identical pair of
self-complementary sequences can be separated by spacer DNA, for
example, additional nucleotides that can form a loop connecting the
two strands of RNA forming a double-stranded hairpin, or that can
separate adjacent double-stranded RNA "stems". Spacer DNA can
include nucleotides that are located at the distal end of one or
both members of the pair the self-complementary sequences, for
example, where inclusion of these nucleotides as "spacer" sequence
facilitates the formation of the double-stranded RNA structures, or
facilitates the assembly and maintenance of these sequences in
plasmids. Spacer DNA can include sequence encoding an aptamer. The
non-identical pair of self-complementary sequences can include
sequence derived from a single segment of a single target gene,
multiple copies of a single segment of a single target gene,
multiple segments of a single target gene, segments of multiple
target genes, or any combination of these, with or without spacer
DNA. Multiple "hairpins" can be formed in an analogous fashion by
including more than two non-identical pairs of self-complementary
sequences that can form two separate double-stranded RNA
"stems".
[0259] A specific, non-limiting example of this configuration of
sequences is shown in FIG. 10, which depicts a gene suppression
element ("GSE", FIG. 10A) useful in recombinant DNA constructs of
the invention, and a representation of the type of RNA double
hairpin molecule that it would be expected to produce (FIG. 10B).
The double hairpin molecule is depicted with a 3' untranslated
region including a polyadenylated tail; however, embodiments of the
invention also include analogous constructs that produce a double
hairpin molecule lacking a polyadenylated tail or a 3' untranslated
region. In this example, orientations of the sequences are
anti-sense followed by sense for sequence 1, then sense followed by
anti-sense for sequence 2 (FIG. 10A). This arrangement may be
convenient, e.g., when both sequence 1 and 2 are derived from the
same target gene, in which cases the sense sequences can represent
sequences that are contiguous in the native target gene. However,
any order of sense and anti-sense sequences can be used in the
recombinant DNA construct, as long as the transcribed RNA is
capable of forming two separate double-stranded RNA "stems".
Analogous recombinant DNA constructs could be designed to provide
RNA molecules containing more than 2 double-stranded "stems", as
shown in FIG. 10C, which depicts an RNA molecule containing 3
"stems".
Example 11
[0260] This example describes a non-limiting embodiment of the
recombinant DNA construct of the invention, and methods for its
use. More particularly, this example describes a recombinant DNA
construct containing a gene suppression construct that transcribes
to RNA capable of forming multiple double-stranded "stems" and that
suppresses a first target gene, wherein the recombinant DNA
construct can be transcribed in a transgenic plant, and the first
target gene is a gene native to a pest or pathogen of the
transgenic plant.
[0261] In this non-limiting example, an RNA molecule that is
capable of generating a double hairpin structure is designed to be
transcribed from a recombinant DNA construct containing a gene
suppression element similar to that shown in FIG. 10A. In this
specific example, the gene suppression element ("GSE") contains a
first sense sequence and second sense sequence (as depicted in FIG.
10A), which are contiguous sequences from SEQ ID NO. 2 (a 872
nucleotide segment of the cDNA sequence of the corn root worm
vacuolar ATPase gene). However, this method can be used for
noncontiguous sequences, including sequences from different genes.
The complete gene suppression element given as SEQ ID NO. 3
contains DNA sequences of SEQ ID NO. 2 arranged as follows: the
reverse complement of the DNA segment starting at nucleotide 1 and
ending at nucleotide 300 of SEQ ID NO. 2, followed by the DNA
segment starting at nucleotide 100 and ending at nucleotide 600 of
SEQ ID NO. 2, followed by the reverse complement of the DNA segment
staring at nucleotide 300 and ending at nucleotide 500 of SEQ ID
NO. 2. This gene suppression element (SEQ ID NO. 3) is embedded in
a suitable intron (as described above under the heading "Introns")
that is operably linked to a suitable promoter element (as
described above under the heading "Promoter Elements"). Where it is
desirable to transcribe RNA that is transported out of the nucleus,
a terminator element can be included either embedded in the intron
containing the GSE and operably linked to (5' to) the gene
suppression element, or outside of and 5' to the intron containing
the GSE.
Example 12
[0262] This example describes a non-limiting embodiment of the
recombinant DNA construct of the invention, and methods for its
use. More particularly, this example describes a recombinant DNA
construct containing a gene suppression construct that suppresses a
miRNA precursor molecule, e.g., a pri-miRNA.
[0263] The primary transcript of a miRNA gene (MIR gene), termed a
pri-miRNA, is believed to be hundreds to thousands nucleotides in
length and largely processed in the nucleus to a smaller (generally
less than 100 nucleotides) stem-loop structure, which is then
exported to the cytoplasm for further processing into a mature
miRNA. By embedding a gene suppression element for suppressing a
miRNA precursor molecule (for example, DNA that transcribes to RNA
for suppressing a pri-miRNA by forming double-stranded RNA,
preferably double-stranded RNA that lacks polyadenylation) into a
spliceable intron, the resulting double-stranded RNA is expected to
remain in the nucleus due to the absence of cis-acting nuclear
export signals, resulting in suppression of the miRNA that is more
efficient than that achieved by constructs that produce cytoplasmic
dsRNA. Another potential advantage of this approach is that the
miRNA precursors offer larger target sequences for suppression than
does a mature miRNA. Alternatively, an intron-embedded gene
suppression element can be designed to target the promoter
sequences of the miRNA precursor, resulting in transcriptional gene
silencing. See, for example, Matzke and Birchler (2005) Nat. Rev.
Genet., 6:24-35, Matzke et al. (2004), Biochim. Biophys. Acta,
1677:129-141, and Papp et al. (2003) Plant Physiol., 132:1382-1390,
all of which are incorporated by reference herein.
[0264] One general, non-limiting design for a recombinant DNA
construct includes a suppression element for suppressing production
of a mature miRNA and preferably designed to target the pri-miRNA
sequence of a targeted MIR gene, wherein the gene suppression
element is embedded in an intron (e.g., a heat shock 70, actin 1,
or alcohol dehydrogenase intron) flanked on one or on both sides by
non-protein-coding DNA, which is fused to a reporter gene (e.g.,
beta-glucuronidase GUS, or green fluorescent protein GFP) and
driven by a constitutive (e.g., 35S) or tissue specific (e.g., B32)
promoter. Such a construct generally resembles that shown in FIG.
7A, where the reporter gene can be upstream or downstream of the
intron. The recombinant DNA construct can be transformed into
Arabidopsis by standard techniques. Expression of the optional
reporter gene confirms the proper processing of the intron in the
transgenic Arabidopsis in which the construct is transcribed.
[0265] In a non-limiting specific example of this approach, a
recombinant DNA construct of the invention is designed to suppress
a specific allele, MIR164c, of the Arabidopsis thaliana microRNA
gene MIR164. Loss-of-function of this allele, eep1, caused by T-DNA
insertion, has been shown to increase the number of petals of early
flowers in Arabidopsis (see Baker et al. (2005) Curr. Biol.,
15:303-315, which is incorporated by reference herein). One
specific, non-limiting construct includes a heat shock 70 intron,
within which is embedded a suppression element including DNA that
transcribes to a sense/anti-sense double-stranded RNA for
suppressing the pri-miRNA of MIR164c sequence, fused to GFP and
driven by a 35S promoter. GFP expression confirms transcription and
proper splicing of the construct in Arabidopsis plants transformed
with the construct. The "early extra petal" phenotype of eep1 is
used to score for the miRNA164c suppression.
[0266] In another non-limiting, specific example of this approach,
a recombinant DNA construct of the invention is designed to
suppress the Arabidopsis thaliana microRNA gene MIR172, which
regulates the mRNA of a floral homeotic gene, APETALA2 (X. Chen
(2004) Science, 303:2022-2025). Elevated miRNA172 accumulation
results in floral organ identity defects similar to those in
loss-of-function apetala2 mutants. On the other hand, the
expression of mutant APETALA2 mRNA resistant to miRNA172 causes
different floral patterning defects. One specific, non-limiting
construct includes a heat shock 70 intron, within which is embedded
a suppression element (for example, DNA that transcribes to a
sense/anti-sense double-stranded RNA for suppressing the pri-miRNA
of MIR162 sequence), fused to GFP and driven by a 35S promoter. GFP
expression confirms transcription and proper splicing of the
construct in Arabidopsis plants transformed with the construct. The
floral patterning defect phenotype is used to score for the
miRNA172 suppression.
Example 13
[0267] This example describes a non-limiting embodiment of a
recombinant DNA construct of the invention, and methods for its
use. More particularly, this example describes identifying a MIR
gene in maize and, further, making and using a recombinant DNA
construct containing a gene suppression element that suppresses
production of the mature miRNA transcribed from the identified MIR
gene in maize.
[0268] A single small RNA was isolated and cloned using procedures
based on published protocols (Llave et al. (2002) Plant Cell,
14:1605-1619, and Lau et al. (2001) Science, 294:858-862). Low
molecular weight RNA was isolated from developing maize endosperm.
Adaptors were ligated followed by RT-PCR for conversion of RNA to
DNA. Additional PCR amplification followed by TA cloning and
sequencing led to the identification of a highly abundant 22-mer in
maize endosperm corresponding to the DNA sequence
TGAAGCTGCCAGCATGATCTGG (SEQ ID NO. 4). Sequence alignment analysis
showed that the isolated 22-mer sequence is homologous to a rice
sequence annotated as "Oryza sativa precursor microRNA 167g gene,
complete sequence" (GenBank accession number AY551238, gi:45593912)
and having the sequence,
GAAGATATTAGTTCTTGCTGGTGTGAGAGGCTGAAGCTGCCAGCATGATCTGGTCCATGAGTTGCACT
GCTGAATATATTGAATTCAGCCAGGAGCTGCTACTGCAGTTCTGATCTCGATCTGCATTCGTTGTTCTGA
GCTATGTATGGATAATGATCGGTTTGAAGGCATCCATGTCTTTAATTTCATCGATCAGATCATGTTGCAGC
TTCACTCTCTCACTACCAGCAAAACCATCTCA (SEQ ID NO. 5, with the homologous
nucleotides indicated by bold, underlined text). A proprietary
maize genomic DNA sequence database was searched for sequences
containing 22-mer segments identical to SEQ ID NO. 4 or to its
complement. The sequences thus identified included overlapping SEQ
ID NO. 6, SEQ ID NO. 7, and SEQ ID NO. 8, as given in Table 3, with
the location of the 22-mer indicated by underlined text.
TABLE-US-00003 TABLE 3 SEQ ID NO. Sequence 6
GTTTTGGCTTGTTCACCCCTCATGTGCACATGCTGTTACTCCGAAG
CTTGCGCTTTTGTATTCGTTGTTGCATTGCAACCATCCCCGCCGAA
GGTGAGCCGAAGGTAATCTTGGGTATTCTACCTGCAACACTTATTA
ATTCAAGCTACAAAACAGTTGTCGAGTTAGTTTTTTTTTTACCTTC
GAAAAGAAGACTTCCGGCAATGCACAACTTCCCATCTGCATTATCG
TGAGCAGGATTGTAGGCACACAGTGATGACGAAGACAGAGACAGCA
ATATACACAACCGAACCAAGAGAGAAGCAAAGGCATAATAATAAAA
AAGAGAGAGGAAACTAGATCGACAAGGCCATTATTATCACGGATAA
TTATCAACGTCGTCAACGGCGGAAATAAGCTAGCTTGACTGGTGGT
CTCTGGCGAGTGCAGCATGGATATGAATTGCAGGAGGGTGAGCTAG
CTAGGGTTTTCGATGTGGGGCCACCAGCAGATGAAACTACAGCATG
ACCTGGTCCTGGTGCTCATTATTACCCTCTCTCTCTCTCCCTTCCC
CTCTGATCTTGGATTCGTCGATCCATATATGACAGTCAGGGACGGG
GGAGAGAGAGAGAGTGACAGGGGCCGGTAGTAGTATAGATTACATC
CATTTTACATATACCACCACCATCATAACCAGATCATGCTGGCAGC
TTCACCAACTCGTGGTGCACCACTACATACCCTCTCGTCTGATCCA AACGGAGGAAGGAGGAAGAA
7 TTGGCTTGTTCACCCCTCATGTGCACATGCTGTTACTCCGAAGCTT
GCGCTTTTGTATTCGTTGTTGCATTGCAACCATCCGCGCGGAAGGT
GAGCCGAAGGTAATCTTGGGTATTCTACCTGCAACACTTATTAATT
CAAGCTACAAAACAGTTGTCGAGTTAGTTTTTTTTTTACCTTCGAA
AAGAAGACTTCCGGCAATGCACAACTTCCCATCTGCATfATCGTGA
GCAGGATTGTAGGCACACAGTGATGACGAAGACAGAGACAGCAATA
TACACAACCGAACCAAGAGAGAAGCAAAGGCATAATAATAAAAAAA
GAGAGAGGAAACTAGATCGACAAGGCCATTATTATCACGGATAATT
AATCAACGTCGTCAACGGCGGAAATAAGCTAGCTTGACTGGTGGTC
TCTGGCGAGTGCAGCATGGATATGAATTGCAGGAGGGTGAGCTAGC
TAGGGTTTTCGATGTGCGGCCACCAGCAGATGAAACTACAGCATGA
CCTGGTCCTGGTGCTCATTAATTACCCTCTCTCTCTCTCCCTTCCC
CTCTCATCTTGGATTCGTCGATCCATATATGACAGTCAGGGACGGG
GGAGAGAGAGAGAGTGACAGGGGCCGGTAGTAGTATAGATTACATC
CATCTTACATATACCACCACCATCATAACCAGATCATGCTGGCAGC
TTCACCAACTCGTGGTGCACCACTACATACCCTCTCGTCTGATCCA
AACGGAGGAAGGAGGAAGAAGAGCTAGCTATCCGAGAGAGAGGGAG
AGGGTAGAGAGATGGAGAGAGCGAGGAATGAATTGAAGAACCGAGG
GATAGCTATAGCTATATATATATGGGGATGGGGAGGCCAACGTCTC GCTCACTCGC 8
TATTCTACCTGCAACACTTATTAATTCAAGCTACAAAACAGTTGTC
GAGTTAGTTTTTTTTTTACCTTCGAAAAGAAGACTTCCGGCAATGC
ACAACTTCCCATCTGCATTATCGTGAGCAGGATTGTAGGCACACAG
TGATGACGAAGACAGAGACAGCAATATACACAACCGAACCAAGAGA
GAAGCAAAGGCATAATAATAAAAAAAGAGAGAGGAAACTAGATCGA
CAAGGCCATTATTATCACGGATAATTAATCAACGTCGTCAACGGCG
GAAATAAGCTAGCTTGACTGGTGGTCTCTGGCGAGTGCAGCATGGA
TATGAATTGCAGGAGGGTGAGCTAGCTAGGGTTTTCGATGTGCGGC
CACCAGCAGATGAAACTACAGCATGACCTGGTCCTGGTGCTCATTA
ATTACCCTCTCTCTCTCTCCCTTCCCCTCTCATCTTGGATTCGTCG
ATCCATATATGACAGTCAGGGACGGGGGAGAGAGAGAGAGTGACAG
GGGCCGGTAGTAGTATAGATTACATCCATCTTACATATACCACCAC
CATCATAACCAGATCATGCTGGCAGCTTCACCAACTCGTGGTGCAC
CACTACATACCCTCTCGTCTGATCCAAACGGAGGAAGGAGGAAGAA
GAGCTAGCTATCCGAGAGAGAGGGAGAGGGTAGAGAGATGGAGAGA
GCGAGGAATGAATTGAAGAACCGAGGGATAGCTATAGCTATATATA
TATGGGATGGGGAGGCCAACGTCTCGCTCACTCGCAGCGTATTTTG
ATGCCCTTTTTTATTTGTTGCATTTCGATCCATTTTCTTTTGTCCT
GCGCTTTTTTCGTACGATGTTTGTTGCAAGGATAAGCCTTTCGG
[0269] These three sequences (SEQ ID NO. 6, SEQ ID NO. 7, and SEQ
ID NO. 8) overlapped to give a single contiguous sequence SEQ ID
NO. 9,
GTTTTGGCTTGTTCACCCCTCATGTGCACATGCTGTTACTCCGAAGCTTGCGCTTTTGTATTCGTTGTTGC
ATTGCAACCATCCCCGCCGAAGGTGAGCCGAAGGTAATCTTGGGTATTCTACCTGCAACACTTATTAATT
CAAGCTACAAAACAGTTGTCGAGTTAGTTTTTTTTTTACCTTCGAAAAGAAGACTTCCGGCAATGCACAA
CTTCCCATCTGCATTATCGTGAGCAGGATTGTAGGCACACAGTGATGACGAAGACAGAGACAGCAATAT
ACACAACCGAACCAAGAGAGAAGCAAAGGCATAATAATAAAAAAAGAGAGAGGAAACTAGATCGACA
AGGCCATTATTATCACGGATAATTAATCAACGTCGTCAACGGCGGAAATAAGCTAGCTTGACTGGTGGT
CTCTGGCGAGTGCAGCATGGATATGAATTGCAGGAGGGTGAGCTAGCTAGGGTTTTCGATGTGCGGCCA
CCAGCAGATGAAACTACAGCATGACCTGGTCCTGGTGCTCATTAATTACCCTCTCTCTCTCTCCCTTCCC
CTCTCATCTTGGATTCGTCGATCCATATATGACAGTCAGGGACGGGGGAGAGAGAGAGAGTGACAGGG
GCCGGTAGTAGTATAGATTACATCCATCTTACATATACCACCACCATCATAACCAGATCATGCTGGCA
GCTTCACCAACTCGTGGTGCACCACTACATACCCTCTCGTCTGATCCAAACGGAGGAAGGAGGAAGAA
GAGCTAGCTATCCGAGAGAGAGGGAGAGGGTAGAGAGATGGAGAGAGCGAGGAATGAATTGAAGAAC
CGAGGGATAGCTATAGCTATATATATATGGGGATGGGGAGGCCAACGTCTCGCTCACTCGCAGCGTATT
TTGATGCCCTTTTTTATTTGTTGCATTTCGATCCATTTTCTTTTGTCCTGCGCTTTTTTCGTACGATGTTTGT
TGCAAGGATAAGCCTTTCGG (with the location of the 22-mer indicated by
bold, underlined text). This was identified as a maize MIR167
sequence which transcribes to a pri-miRNA. Recombinant DNA
constructs of the invention, containing one or more suppression
elements for suppressing the identified MIR167 pri-miRNA (or a
pre-miRNA) are designed and transformed into maize plants by
procedures such as those described above under the heading
"Recombinant DNA Constructs for Suppressing Production of Mature
miRNA and Methods of Use Thereof" and elsewhere in this disclosure
(e.g., by Agrobacterium-mediated transformation). One non-limiting
suppression element is an inverted repeat containing one or more
sense and anti-sense pairs of SEQ ID NO. 4, embedded in an intron.
Suppression of production of the mature miRNA corresponding to the
identified MIR gene is detected by analysis of low molecular weight
RNA from resulting transgenic maize endosperm and other tissues
(e.g., embryo, leaf, root, flower) for example, by using a labelled
oligoprobe corresponding to the 22-mer (SEQ ID NO. 4 or its
complement). Transgenic suppression of production of the mature
miRNA encoded by a MIR167 gene, is useful, for example, for
identifying related genetic elements or to manipulate the pathways
that are controlled by MIR167, e.g., by identifying target genes
suppressed by a mature miRNA encoded by a MIR167 gene. Thus, the
transgenic tissues are also analyzed for morphological and
compositional changes (such as, but not limited to, changes in
primary metabolite, secondary metabolite, trace element,
carotenoid, or vitamin composition or modified responses to biotic
or abiotic stress, or modified yield) to assess the function of the
maize MIR167.
Example 14
[0270] This example describes novel mature miRNAs and MIR genes
identified in crop plants (maize and soy). Novel MIR sequences were
identified in proprietary expressed sequence tag (EST) sequence
databases from crop plants. The criteria that were used for
identifying MIR genes included a conserved miRNA sequence of at
least 19 nucleotides, a stable predicted fold-back structure
encompassing the miRNA in one arm, and the absence of a significant
open reading frame (ORF). Seven MIR sequences were identified in
maize (Zea mays): SEQ ID NO. 10 (Zm-MIR164e, including the DNA
sequence SEQ ID NO. 11 corresponding to the conserved mature miRNA
miR164e), SEQ ID NO. 12 (Zm-MIR319-like, including the DNA sequence
SEQ ID NO. 13 corresponding to the conserved mature miRNA
miR319-like), SEQ ID NO. 14 (Zm-MIR393b, including the DNA sequence
SEQ ID NO. 15 corresponding to the conserved mature miRNA miR393b),
SEQ ID NO. 16 (Zm-MIR399g, including the DNA sequence SEQ ID NO. 17
corresponding to the conserved mature miRNA miR399g), SEQ ID NO. 18
(Zm-MIR408b, including the DNA sequence SEQ ID NO. 19 corresponding
to the conserved mature miRNA miR408b), SEQ ID NO. 20 (Zm-MIR398,
including the DNA sequence SEQ ID NO. 21 corresponding to the
conserved mature miRNA miR398), and SEQ ID NO. 22 (Zm-MIR397,
including the DNA sequence SEQ ID NO. 23 corresponding to the
conserved mature miRNA miR397). Six MIR sequences were identified
in soybean (Glycine max): SEQ ID NO. 24 (Gm-MIR393a, including the
DNA sequence SEQ ID NO. 25 corresponding to the conserved mature
miRNA miR393a), SEQ ID NO. 26 (Gm-MIR393b, including the DNA
sequence SEQ ID NO. 27 corresponding to the conserved mature miRNA
miR393b), SEQ ID NO. 28 (Gm-MIR399, including the DNA sequence SEQ
ID NO. 29 corresponding to the conserved mature miRNA miR399), SEQ
ID NO. 30 (Gm-MIR164a, including the DNA sequence SEQ ID NO. 31
corresponding to the conserved mature miRNA miR164a), SEQ ID NO. 32
(Gm-MIR164b, including the DNA sequence SEQ ID NO. 33 corresponding
to the conserved mature miRNA miR164b), and SEQ ID NO. 34
(Gm-MIR164c, including the DNA sequence SEQ ID NO. 35 corresponding
to the conserved mature miRNA miR164c). The novel MIR sequences are
given in Table 4, with the location of nucleotides corresponding to
the mature miRNA indicated by underlined text. TABLE-US-00004 TABLE
4 SEQ ID NO. Sequence 10
GTATGTTCTCCGCTCACTCCCCCATTCCACTCTCATCCATCTCTCA
AGCTACACACATATAAAAAAAAAAGAGTAGAGAAGGACCGCCGTTA
GAGCACTTGATGCATGCGTACGTCGATCCGGCGGACCGATCTGCTT
TTGCTTGTGTGCTTGGTGAGAAGGTCCCTGTTGGAGAAGCAGGGCA
CGTGCAGAGACACGCCGGAGCACGGCCGCCGCCGATCTACCGACCT
CCCACACCTGCCTTGTGGTGTGGGGGTGGAGGTCNNNNNNCGNAGC
GAGAGCTGNCGNTGNTGNTTNGATGCTGNTNGCTCCTCCTGCNCGT
GCTCCCCTTCTCCACCACGGCCTTCTCACCACCCTCCTGCCCCGGC
GGCGGCGGCGGCGGACCGCCCTTGCCGCGATCAATAATGAAACCAA
AAGCCGACAGTGTTTGAGCAGGAAACACAAAAGGCGGATATCCCAC
TGNTAGCACTTCTGCGTTGATCATGGTCATCTGGAACAAAATAATA
CTTGGGGACTTTACAGCGAGTGCAGCATGCTTAAGCTAGTTC 12
TTCGGTCCAAGTAGTGGTGGTCATAATATGCTCCAAATAAAAGAAA
GGTGGAGGAGCATCTCACAGACGACACAGCTGCTATGCTAGCACAC
GTCGAATCAATAGCTAGTTGCATGCAAAGTTCCAAAGCAAATAAAC
AGTGAGATCGAAAGACGTTTCGCTGTTGCACGACACGACGAATCGA
TCGAACGAAAGTGTGTTTTTATGATTCCACAGATTCTCGTTTATAT
ATAATGCTAGCTAGCTAATCTAGAACGTACAGTGCACACCATCTTC
TTCCACAGATCACAGAAAGACAGCAGAAACCTGCATGGATCGGATC
CGGTCCTGTCCTGTAAGATCTACACACATGCAAAGCAAATCAATTT
CTTCCTTTTCTTTTCTTCAGAAACTGGGATAACTTTTTGGAAGAGA
TCGAACAGTATATAGATTCAGGGAGCAGATCAAGGATTATATATAT
AGCTAGTATGTGTACATATCAAAAGGGCAAGAAAAGTACAAAAAAG
CATCGGATCTCCATTATATATATACAACAGCTATATAACAACCACA
GAAGAACAGTAAGCACGCACATGGTAAAATTAAAATAGCCTGGCAG
CTGCTATGGATGTATGCATCAGATGCCTAATATATATGCAAGATAA
TAATTAATAAGCAGCTCAAGCAAAGACAGATCAAGAGTTCGAGACA
GCAGGTTGGAAAATAAAATACAGATCATATGAAGTAAAACCTTGAC
TTGAGATACGAATGATGAAGCTGCATGGGTAAAGTAAACAAGGAAA
GGATCGGAGGGAGCACCCTTCAGTCCAAGCAAAGACGGTGCGAGAT
CGAAGCTTTTACCTCCCGCTTCATTCACTCATCTGCGAAGCTCGTT
TCCATGGCCGTTTGCTTGGCATGTGGGTGAATGAGTCGGCAGCTAA
TCCGACCCTAGCACCGCCCCTGAGTGGACTGAAGGACGCTCTCTTC
CATCCGGCCGGCGACCATCGATCACAACCATGACGCCGCGCCCGGC
GGCAAATATATTAACAAGAAATGAAATCAAAAGAGAGAGGAAGAAC
AAACATGATGCGCAGCTGCGCTAGCTAGTGCTTGATCTGTCTGACC
ACCTCATGGCGCGCAGTGTTTAGTTTTCTCCCTGGATCTTGCGAAG
AAGGCGATGGATTTTTCGATGGTTGCAAGGAGGAGCGACCGACAAA
GGGTTTATATAATATGTAGACGGC 14
GCCGGCCGGGTCGGGATGCCGCCTACTAGCAGGAAGCTAGTGGAGG
ACTCCAAAGGGATCGCATTGATCTAACCTGCCGATCGACGCCGACG
TACGTACGTGCCCGAGGACAAGCAGATCAGTCAGTGCAATCCCTTT
GGAATTCTCCACTTAGCGCCTCCATCCCCGCGCCGCCCTCCAGGTT
TCGCTTCGATCCATCCATGTTTCCTTCGTTTAAATTAGTTCGTTTG
TTTTTTTTTTATTATTTATTTGATTCGCCGCCGCCGGTCTATCTAC
TCTGTTTGCAACGCCTTTCGATCCATCGGCTTCTACTGTATGCTAT
AAAGGGTTTTTTTACATTGGTCCGATGCATGAGAGGAGCTGTGCAG
ACCAACATGGCAACCAATTACATCGATCTTGAGGACTCTTATGGAC
CAACATGCCAAGTTCTTCATTGCTTGTACTACCATTCAAGTTGTCA
AACAATTACCAAACTCAAGTATTCGAGAGAAGCATATATGTTAGTC
AAATAGCAAATTCTTTACTAACTGATCTATGTACCGACATGTCAAC
TTCTTGCATACCAACGTGGCAAGAAGGTAATCATTGTTCATGAATA AGATTATCACTA 16
CTAGGAATGGTACGGTGCTGGCTAAGCTAGCTAGATCATCGTCCTG
GAGCTGAGAGCAGCAGCTACCTATATATCTAGCTGGTTTTCTAACG
ACGATGACGAACGACCGCGGGACTAGCATGATGCAGCTAGCTGAAG
ACAGTTGTAGGCAGCTCTCCTCTGGCAGGCAGGCGCGCGGTCATCG
TGGCCATCGACGACGGTTGCTTGGCTCTGCTATGCTGTGTTCGTTC
GGCCATGGTGTGCTAGCTAGCCGTGCATGCGTTGCAGTGTAACATG
CGTGCATGCACGCGCGTACGTCCTGCCAAAGGAGAGTTGCCCTGCG
ACTGTCTTCAGCTCGAACAAGATCGACCGGCCCGGACAGGAATGTT
GGGCGTACGTTGTCATCAGGGTTTAAGCTCCACGATTCCAAATATT
CACCACTTCTGGGAGGAGTTTTGAAGCTGCTCGAAAGCATATTGTG
TCTGAGTGTAATAAATCGGCGGGGAATCATATGTTCATGTTCTCAC
TGCAAGAATAAGCTFGTCAAAGAGGGTGGTGAAGTAAAAAATCTCA
CCTGATCAGCGGCACAGGTGCTCCTAGCGACGGGTGTAAGTCATGG
AGGACAAGCAACAGGAAGTCCAGTGGCAAGTGCTTCCATCGTCGTC
AAATCACAGGTCAGGGGTTAATTATATGGGGGAAGAGGCCATTATC
ATCAGGTACGCGTGGTTCTCACACAGTCGGGGCCACGTTCGTTGAT
GATCTGCCTCTTAATCGGCATCTCAAACTCTTGTTGTGCTCTCTAC
ATCAGTAGAGAAGGTGTGTTCACAAGTCGTTTCTTCTTAAGACTAT
GTTTTGGTTGATCTTGATCTATAGAACTATTTTATTGTAGAACTAC
TGAACCCTTTCGAAGTGTTGTACTCAATTTGTGTAGAACAATGCAT
GATTAATTTCTACCAATAGTCTACGGTAGCCGGTAGTTGTTTTATC
CTACTAGAAATTGTTGCATGGTTAATTGGTTAATTTGTGTAGGATG
TGCCAAAAGAAGAGGAAGAGAACACCATCAATATGAATGGTGAATT
ATTCGTAAGCTTATCTTCCACTAATGGTGCTGGAAGCCAGAAGGAG
AAAGAGGAGGATGGAGATCATGTGTCAAGGCTCAGGAGATAAATCG
AGGAAGAAAAAGATCGAAGGGTGGTGTTTAGTTGTATCCTTCCAAG
TTCCAAGTTCACGGTAAAGAGAGGAAAGTGTGCTAGTCAAGAGAGT
ATGGGATGGAGATAGGCACCATTGGACTTGGAGTGGAGGACAAGAT
GTTACCATTTTGCATTTCCATGGAGCGTGGAGACTTGAGTGCTTCA
ATCTTTTTATTAAATCAGTCTGAGCGATGATGAGTCTAAAGAGACT
AAGACTATATCATAATCTACGATGGATTTAATCTATAAGGTGGATA
TATCACATATGGTTGCCAATCTTGTATATTTCATATTTGCATGGTT
GGTAGTTGCACTGTTGCAATCTTAAGACCTGTATAGTTGCATATTT
GATTGTGTTTTTAGAATGTTGATTTGTGGTTGTGCTCGCTTCTTTC T 18
GGTACCTTTAGCGTTAGCACAGACACACACAGGTAAGGAGAGCGAG
AGGTGGGTTGGGTTTGATCGGAGACAGGGACGAGGCAGAGCATGGG
TAGGGGGCCATCAACAGAATTCCAAATTTGATTTCTGTTTGCTCGC
TCACAAAATGGAGGGACTCACCACAAAACACACTCAGGCGTTGTTG
CTCCCTCCCCTGCACTGCCTCTTCCCTGGCTCCTCACCGTCTCCCA
TCCACCTATCCTCTCTCTTTCTCTCTCTCGTTATGGTTTTGTATAA
TTTTTTTTCCTGCATTCTTTTTCTCAGTACAAGTCCTACACTAATT
TGGCTGTCTTTGCACCAGTACTAATAAACACCGCAGGTCCCTGCAA
TAGGGTTTACAACAATTCTATTGTAATGACTGCTGTAAAACATCCG
CATCATTTAATTCAACTTTCCGGTTTCAGTCAGCCCTGCAAAAGTG
CTCCTCCGTTCGTCCGCGTTTGGTGTTGGCTTCTGCGGCTCCGGTG
CCCAGAGTTGCTGCCGGCGGAGGCCGAGCAGGAGCGCAACTAACAA
GAGCGGCCAAGGCGCCAGTGATCCTCACCATGGACAGGAGATCGAT
GGAGATGAGCGTGAGCTTCCGATGCTTCGGTACCCGAAGAAAAGAA
CGGGAACAAAGGCGAGAAACATGATCCACCTCTATGCTTTTTTGGC
AACATATCCTATGCTTAAACAGTTATGGTGTTCAAATGTACACATT
AATAGAGCGTTTGGTTTGAAGAATCACACCATCTAAATTGAGGTGG
TGCATCATGAATTTATTCCTTAAAAAAAAAAAAAAAAAAAAAAAA 20
GCCGGCCGGGTCGGGTGTGTTCTCAGGTCGCCCCCGATCACAGCCA
ACGCGGGCGACCGCGCGCCATTATAGCACACGGGGCACGGCACGCC
TTCGGCCTCCCACTAACTGCACAAGAGGACGACGCGGCAGCGAGGA
GGGAGCAAAGGAAAGGGGATATGTCGAGGCCGCCCAACAGGAGCGA
CGCGCACCTGTCCGCCGAGGACGAGGCGGCGCTGGAGGCCGAGGTG
CGGGAGTACTACGACGACGCGGCGCCAAAGCGCCACACCAAGCCCT
CCCGCAGCGAGCACTCCGCCGTGTACGTCGACGCGCTCGTCCCGGA
CGTCGGCGGCAACTCCCACCCGGAGCTGGACAAGTTCCAAGAGCTG
GAAGCCCACACCGAGAGGTTGGTGTACGAGGGCGCCAATGTGGGAG
ATGAGTTCGTAGAGACGGAGTACTACAAGGACCTCGGCGGCGTCGG
CGAGCAGCACCACACGACCGGAACGGGCTTCATCAAGATGGACAAA
GCTAAAGGCGCCCCCTTCAAACTGTCTGAAGATCCCAATGCAGAGG
AGCGACATGCTTCTTGCAGGGGAAACCCTGCTACCAACGAGTGGAT
CCCGTCAGCTGACACGGTAAGACTGGGGGAGCACAGTCCAGTTTAT
CCTATGCAGGTGCAGGGTCGGCTCCAATCGGCGTCTCTACTGACGA
ACGCATCGTTAGCTTGTACCCAGCGTCAGACAAGCCAAGCAGAAGC
GACAGCTGAGGGACTGTATATCTCAAGCCATGAGAATTCAGACGAG
TGCTTTCCGCCATTAGAATAAGGAACCACACTGGTTGTCCACCGTA
TCTTCACTGTTCTGCGTCGAGATTCTTGTGATTCTTACGTGGAACA
AATTAAGCGTGCTACGAGTTAGACCTCTGTGTTCTGGCTGTAAATG
GCAAGGAATGAAGTTCTAATCGTGGTTCAGCAGTCAATCAATTACT
GTGTTTCTGATCCTAAGGCTCTAGAAACAATCGGACCTTCAAAATA
AACTAGGCGAAAATTCTATGTCGTTTCG 22
GAGCGGGGTCTTGAAACTGGCTGCGCAGAAGGAAGGGATGAAGGGG
TTCCTGGAGCTCGACGCCGAGGTTTTCGAGCTTGCCCCTTCGTTCT
TTCTGGTCGAGCTGAAGAAGGCCAGCGGTGACACCATTGAGTACCA
AAGGCTCGTGAGGGAAGAAGTGCGGCCTGCGCTGAAGGATATGGTC
TGGGCTTGGCAGAGCGACCGGCACCAGCAGCAGCAGCAGCGGTGCG
AGCAGTCTGTGCAAGGAGAGGACCAGCAGCAGCCGTTGTCGTCTTT
GCCGACGCAGCAGTAGTCACTGCACCACCAGTTGCGACCGCCATAA
CCAGATCACGTCAAAACTGCACCAAGCCGCACAGGACTAGTAACTC
CCACTTGCATCGACGCTTATGTGATTGCGGAATTGTGTTTCAGGTT
ACCTGCCTGCTGCGGTAGGACCTAAAACGCCTACCTGCCTACCATT
TGGCATTTTTTTGTATACTGTACGTACATTAGAGTAATAAACAAAC
ATGCTTAACTTTTCAGCTTTCGATTGGAATGTGCTTTTCGATGTAA
CTCTGTAACCAGTGTAGGTACGAAGTCGATTAGCCACAGGGTCTGG
CCATGTTGACCTCACGTAGCCCTGGTTCATTGGTGTAACAGTTTGT
TGGCTGCGGCTTTACATTATTTTGTCTCTATGGATTACGGCTGCGA
CTATGTGTAGCTGAACAAGCTGGTATATGATGAGCCCTGGAAACGT
GTGTTTACTGCAGCTATTTGCAGCCAGTGACTGTTGATACAAACGA
CGAAGTAGAGTTGGTTGTTTATGTAGGCACGCAGCATGACCATAAT
ATCCATGAATCATGGATAGATGCACAATGTTTAGGAAACAGGTGTG
TGTGGCTGGCTGGTGGTGCGAGAAGAGATGCGCTGCCTTGATGTAC
TGTAGTGGGACTGGGAGGGATGCGTCTCGCAGTACAGTCTGTACTA
TCATCTCTACACGCACGCACGGAGGCTCGACGTGTCGGCGGCGGCG
GTCCAGACTCCATATGGATCCGTAGTAGTACAACCTGTTGGCGGGT
AGTACAGGTTGGAGCACGCCTCTTCTTCAGTCTTCCTTCCTGAGAT
GAGGAGTCACTCACCAGCAAACGCTTGCAGTACACCCCGCTCGCGG
GCGTTGTTTATAGTGATCGGTAGCGTGAGCACAGAGCGCCATCAGA
AGATGCAAAGAGAAAGAGAAGCAAAGGCATCATTGAGCGCAGCGTT
GATGAGCCAGCCGCCGTGCCTCCCCTGTCGGCTGCGGCGGCTCACC
AGCGCTGCACTCAATTACGCCTTTGCTTTCTCCCGCTGGCCGCGTG
TGTGCAGAGCGGGCGGGCGTTCGGCATCATTCATCAGGTTTGCTTC
ATTTATTATGCACTCATCGAAGGCTTCTCCTTCGACACTGTCTAGG
TGGCGCAGGATCTGAATCAGATGGGTGTCGTCTTCTTCCTCCATCT
GCACTCCTGCCCCGTATGATGTCGGTGTCCTAGGACGGCCAGTTGT
CTGCGTTCTGGTTAACCCAATTACCTGACGGGGCGGACGACGCTGA
TAATGATCAGAGAGAGCATGAGGCCATATGCAAGCCTAGACCTAGC
TCCCAAACTATTAAAGGTTGCTTCGAGCCCTGGCTGTCATATCAAC
TACCAACCAGTTTATGTCGATTATCAGTTCCTATCTATCACAACGC
TCCACTGCACAACCTTAACCTTTACTGTAAACCTATAGTCACCTCA
TCGCTTACATCGGGTTTTTCCCCCTCTTTCGTAGACTTTTAGTTAA
CATCAAACAATGCATTTTATTGAAATCCAAAATACATCTGACTGCG
TAATTGAGTAGATTTATCCCAAAATTTAATTAGCATGCCGCTGTGA
GCTAGGAGAGCGACACTAGTTTACAATATGACAGTGTTTGTGTTCG
GCCAAACCATTTTTGTTGATGGGTAAGGGGACACGACCCCCAAATA
GACGCTCTCATTTTAATGAAGAATTAGTTGTGGACTAATTGATAAT
TCCCATTACAATCGGATTGCACGCATTAAATCTTAGTGCTAAGGAG
GTGTTACAAATGAACCTAAAAAAGAAAAGATAATTGTTGAWTTAAT
GTGGGTCTGGTCCATATTAATATTCAATAATTGTCAATGCTAGTTG
TCACTTTATGCTACGGTGTACTAGTACTTACAAACTAGAAGTTTAA
GGGACAATTCACTYAACTTAAATAGGTGGACTATTGGTGCATCTAT
TGAGAAGCTGAGAAAAGGATGAAGGACTGTCACGCGTGCGCGCACC
CTGATCTGTTGAGAAGCTGAGATCGTAGGAACAAGAATCACTAAAT
TCGGAGTTACAGATTTCAAGTTATGATTTTTCGAAGGTTTTATGTG
TTTGGTACGGAATTGATTGTGATCAATTTTAATATGGGTTTTCATG
CTAAAACTGAGGTACTAAGTGGTAAACAAAATTATAGAAATTGGAA
TGGGTTAAAAAGGAGTTTGCATGATTTTCCTATGAATTATACAAGA
TTATGGATTTATTTTAATACCAAAATCACTTTTTATATTTATTTTA
CCCTGGTTTTCTATCCACTAGACTGCGCCCAAGATTATACTAAAGT
TTAGGGGCAACTGCATAAAAAAACTAAGACTTAGGGCCCGTTTGTG
ATGGACTGCGGGTTGATAACTTAGAAACAGAGGGTCTCTTATGTAA
ACTGTATGTGCTGAAGGGGTATGAAGCATCTACGATCGTCAGATTA
CAATTCCACGGCCAAGATTAAATCGCCAGTGCGATGAACCGTTACG
TAACAGCCATCATCCGATCTGAGATCTACGACCCTGATTCTATGCC
CTAAAACCTCCCAGATCCACTCCCTTTGTCCGAATCGGTACGCATC
GGATTAAATCGCAGCCGCACTCTGATGGATCTACGGCCCACGCAGA
TCATCCCCCATACCAACGGCGAACGGGCGCCGCCGCCCGTAAACAC
GGCGGTGGCCATGGCCGTGGATGGCCAACTCGACTTCGAGGCCGTA
ATCCTCTAGTCTAAGACGTGCTACGTGGTAAGTGGATGAAGACGAT
TTCCATGGGTTCAGTACTTACCGAGGGCAAGGTCGTGCACAAGCTG
TTCACGGCGAAGCGCGGCCGTAGCAAAAATTGAAAGGGAAATGTGA
CTTTGGGCTATTTCTATAAATGTTTTGGTGATTAGATGCCCAACAC
ATATTGTTTTAGTTCATATGTGCTAAGTGATTGAGAAGTGCAAATC
AAGAATCAAGGTATATTTCTAGCCCTAGTAAATTTCTTTTGGATAC
TAACATATCTCTCTAAGTGCTAGGGACACTACCAAGAAAAAGTGGA
AATGAACTGGAGAAGAAGGCAGAGT
24 ACCATTACACTCTTAGTGAATATTCATAAAATATAAAGTTCCTCCT
GGGCGAGAAACATCTCCATGTTAAGGAACAGTGCGAAGAATTATTA
CACCAGACATATTCAAGGCAACTAGTGGAATCCAATAAGGAATGCT
GGCCCACTGCGGAAATATTTCGGGTTGAATGATAGGGAAGGGGCTC
ATTCAACAAAAATCTTAATTTTCTCGGAGATTGGCAAATCTACATT
GACAAGATAAATAAATAATTTATGAAAACAATAAAAAAATGATAAT
GGAAACAGGGCTTATAATATAAGCACTACTAAGCTAGTTTGTTTCT
CCTACGCTAAAAGCCTAATCTCAAACCTACCCACTTCCTACAAGAG
AGAAAGGGGGGGATAGTGTATAATACCCTCAACTTCGAACCAATAT
TCATCAGAAGTAGAGGTGTGGGTATTCTTCCACTGCAACTGGAGGA
GGCATCCAAAGGGATCGCATTGATCCCAAATCCAAGCTTTAATATT
TTTCTCTCTTCTCACTCAATAATATTAATTTATTTGGGATCATGCT
ATCCCTTTGGATTTCTCCTTTAATGGCTTCTATAATGATGGCTCTC
TCATGGATTCTGCTTGCTGCACCACAACACAAACACTTTCATATAC GCCTCTAATGCT 26
CACAATACAATTAAGCTCATCATACTGGTCCTGAAATTGGTGAATA
AAGTTGTTTTGTGGTGGATGAGTACTGAGTAGTGGTGCCTTATTGT
GGGTGGAGAGTTCCAAAGGGATCGCATTGATCTAATTCTTGTAGAT
GTTTACACTTGCAAGCTTTGCAATTCCTGGATTCAGATGTTATTCA
GTGGTTCACTTATTGGATCATGCGATCCCTTAGGAACTTTCCATCA
ACTCTAAACATCTTGTTGATCCATTTGAGGAATTAATTTCATAGGT
TCATATAATGGCGACTGATTTCTTCTAATGGTAATGGACATCACCA
AACAACAACAAAGCAACCTTCTTTGTCGTCTACACTGCGCTTATCC
AAATTTTTTCAGTCCAACATGAGAATCTTGGTTACTGGAGGAGCTG
GATTCATTGCGTCTTACTTAGTTGACAGATTGATGGAAAATGAAAA
AAATGAGGTTATTGTCGTTGCATAGGTGCTTTCATTTTACGTTCTT
CAACATTCCGAATTGAACTTCAGTGGTCCTTGCAATGGCAACGAAT
TCTTCTGATGTACTATCGCCGAAGCAACCTCCCTTGCCATCTCCCT
TGCGTTTCTCCAAATCTATCAGTCTAACATGAGAATCTTGATTACG
GGAGGAGCTGGATTCATTGGTTCTCACCTAGTTGATAGATTGATGG
AAAATGAAAAAAATGAGGTCATTGTTGCTGACAACTACTTCACTGG
ATCAAAGGACAACCTCAAAAAATGGATTGGTCATCCAAGATTTGAG
CTTATCCGTCATGATGTCACTGAACCTTTGACGATTGAGGTTGATC
AGATCTACCATCTTGCATGCCCCGCATCTCCTATTTTCTACAAATA
TAATCCTGTGAAGACAATAAAGACAAATGTGATTGGCACACTGAAA
CATGCTTGGGCTTGCAAAACGAGTTGGGGCAAGGATTTTACTCACA
TCAACATCTGAGGTATATGGGGATCCTCTTGTGCATCCCCAACCTG
AAGGCTATTGGGGCAATGTGAACCCTATTGGAGTTCGTAGTTGCTA
TGATGAGGGGAAACGTGTGGCTGAAACTTTGATGTTTGATTATCAT
AGGCAGCATGGAATAGAAATACGTGTTGCAAGAATCTTTAACACAT
ATGGGCCGCGCATGAATATTGATGATGGACGTGTTGTCAGCAACTT
CATTGCTCAAGCAATTCGTGGTGAACCCTTGACAGTCCAGTCTCCA
GGAACACAAACTCGCAGTTTCTGCTATGTCTCTGATCTGGTTGATG
GACTTATCCGTCTCATGGAAGGATCCGACACTGGACCAATCAACCT
TGGAAATCCAGGTGAATTACAATGCTAGAACTTGCTGAGACAGTGA
AGGAGCTTATTAATCCAGATGTGGAGATAAAGGTAGTGGAGAACAC
TCCTGATGATCCGCGACAGAGAAAACCAATCATAACAAAAGCAATG
GAATTGCTTGGCTGGGAACCAAAGGTTAAGCTGCGAGATGGGCTTC
CTCTTATGGAAGAGGATTTTCGTTTGAGGCTTGGATTTGACAAAAA
AAATTAACTTATTTTCGCTCCTTTTATATCTAGTCAAAATATTCAG
ATAATAAGTGGGATGGATTATTCTATTAAGTTTTCCTATTTTTCCT
TTTCATAATTATGATACTTAGGAAGTAGGGGTGCCTGTATTTTGGC
TTCCTCAATCAAGATCGTACTCTTGTTTTCACAAAGCACTGCAGGA
ATCATGCCTTTGCAAATTTTGCCGGTAAAATTACTACTGAGTTAAA ATTTTCCTATAG 28
TGAAAATTACGTTTTCCCTTTTCCTTTTGTTGCCGGTTAGCACTTC
AATGTAAAAATTAATTCACCATAAAGGATGGTTCGCATACAAAAGA
ATAAAACCTTATGAAAGGACACATGCAACGCAAAATAAAGGCATCG
TTCCATAGGATATGCCGATCCTAGTGAGCCATAAATAACGTTCCCA
AAGGCATTCCTCTATGTGTGTGGATCTTCCCAGTTGCAGCTGCATT
ACAGGGCAAGTTCTCCATTGGCAGGTAGCCACTATGATATGCATCT
CATAAATATTTGCAACTTTCTTAATGTGCAATCTGCCAAAGGAGAT
TTGCCCAGCGATTCTCCTGCAACATCTGCTTCATGAAAACAGTATT
CGTTAGTTTCTTCAATCATTCATTAGAAACATTTCTTGTACTGGTT
GAAATGTTGCATCTCGAACCATTCATATGCCATATTTCCCTTGTTT
TGTATTTTGGTAAAAACCATTTTCCC 30
CTGGAGAGTAAGACCTGAATTTCACTCATTGTTCCTGCCAATGTCC
TTAGTTAGATAAATCTAATTTTTTCTCTCTCTAAAGTTGCATCTAT
AAATATGAGCCTTTCCCTTGGTGCAGATCAATTTGAGCTTTCATTA
CCGTTCTCATGAAGCTTAGGGTGCATGCAACGGTCTCTACTTAGTA
CTGGTTGAGAAGCTCCTTGTTGGAGAAGCAGGGCACGTGCAAGTCT
CTTGGATCTCAAATGCCACTGAACCCTTTGCACGTGCTCCCCTTCT
CCAACACGGGTTTCTCCCCTTGCTTTTCTCCTAACCAATTGTGTCC
AGCACTTATGAGGTAATCGCTTTCCTCCTATGTCTTAATTTGGTCC
TACGTAAAGATCTACAATATGCATCTTCTTTGAGATACGGGCTGAA
GCATGGTACTTTTAAATTGAAGGCTTCAATAACTATATTTAGAGGG
AAAATTCAACATACAAAGAAGGAAGAAGTGTTATGCATACAATATT
TTACCGATGTTCTATGCGTATCAAACATA 32
TCTATATAATTTTTTTCCTATTTTATTTTTTATTTTATTTTGTATC
ATATCACTTATACATCTTTTACTTTCACTCATACACTAAATTTTCG
GGTGTAGGAATACTCCGGCAAAGAGAGAATAGGTTTGCTTATTTCC
TAATTCTGAAGTTAGGGTACGTGCGTAATTTACTGTGTGTTCTGTG
ATGATGAGTTAAGTGGTCCTATTTTACATGTAACTTTTGACAATCT
GTTTGGGTTGAGAATACAAATTAAGGCCCCACACCCAACTAAGCTT
AGCTCTCTCCCATTTTTAGCACCCATCCCGCACCCAACTTTAAAAG
CACCCTCAATTGCCTCTTCTATTATAGGAGAGTAGGCTTCAAAGCA
CACAAGAATATGATAAGATGAAGAAGTTCAGTGTCTCAAAATTCAC
CACTTCTCTTAAAACCTCCCTCATTTGTTTTTTCACACTTTCCTTT
CCCTCACCACTCTCTCTATTACCTCTTGTTTGTTGTTAAGAGTACT
CAGAAGAATAACTCCTCCAAACCCACTTAGCATGTGGCAAAGGTGC
ATGCTGAGCAAGATGGAGAAGCAGGGCACGTGCAATTCTAACTCAT
GAAACCATAGAATCATCTTGTTTTTTCTTCTTTTCACTCTAACCAA ATAGATTCCTCTACCTGCAG
34 ACTCAAGCTTGAAGCACCAAAGTTGCAGTCGGAGGAGTCACAGATT
AAATTCTTCGCTTCTTTAACCTTTGTGTTTCTCTTTTCATACCATT
GTTTCTTTCCCTATAGCTGCTTTAATTTTCTTGTGAGAGTCAGAAA
AGTATCACTATATCAAGTGACATGATCATCAGAATTGAATTATGTG
CATGTTGTGCAAGATGGAGAAGCAGGGCACGTGCAATACTAACTCA
TGAACACTACACGGNGCGTGAACTCGGAGAATCATATTCTCTTCTG
CTTCATTTCACCAACAAGAGAGATCCTATTAGTTAGTTCTTCATGT
GCCCCTCTTTCCCATCATGACAACAGCACCTTATATATATTGCATT
TGGAAATGTTGAACGATGAAGTTCGCTTGGCTTCTGCTCATAAATC
AGCACCGAGNTTTATAGGTTATGCTCCAT
[0271] The fold-back structure of the pri-miRNA was identified in
each of these crop plant MIR sequences using the program EINVERTED
(Rice et al (2000) Trends Genet., 16:276-277), and the results
depicted in FIG. 11, which shows the fold-back portion of the
sequences, with the nucleotide positions indicated by numbers. The
fold-back portion of the MIR sequences is included in the pre-miRNA
precursors processed from these MIR genes.
[0272] The MIR sequences, the complete MIR genes which include
these, and the miRNA precursors (i.e., pri-miRNAs and pre-miRNAs)
processed from these, are useful as target sequences for gene
suppression (e.g., for nuclear suppression of the production of
mature miRNAs encoded by these MIR genes) and as a source of primer
or probe sequences (e.g., for primer sequences for cloning and
sequencing the promoters of these MIR genes). The fold-back portion
of the sequences has been proposed to be sufficient for miRNA
processing (Parizotto et al. (2004) Genes Dev., 18:2237-2242), and
thus in many embodiments the region of the sequence that contains
the fold-back portion is preferably targetted for suppression, or,
alternatively, serves as the source of a sequence for suppressing a
target gene.
[0273] The mature miRNAs produced from these miRNA precursors may
be engineered for use in suppression of a target gene, e.g., in
transcriptional suppression by the miRNA, or to direct in-phase
production of siRNAs in a trans-acting siRNA suppression mechanism
(see Allen et al. (2005) Cell, 121:207-221, Vaucheret (2005)
Science STKE, 2005:pe43, and Yoshikawa et al. (2005) Genes Dev.,
19:2164-2175, all of which are incorporated by reference herein).
Plant miRNAs generally have near-perfect complementarity to their
target sequences (see, for example, Llave et al. (2002) Science,
297:2053-2056, Rhoades et al. (2002) Cell, 110:513-520,
Jones-Rhoades and Bartel (2004) Mol. Cell, 14:787-799, all of which
are incorporated by reference herein). Thus, the mature miRNAs can
be engineered to serve as sequences useful for gene suppression of
a target sequence, by replacing nucleotides of the mature miRNA
sequence with nucleotides of the sequence that is targetted for
suppression; see, e.g, methods disclosed by Parizotto et al. (2004)
Genes Dev., 18:2237-2242 and especially U.S. Patent Application
Publications 2004/0053411A1, 2004/0268441A1, 2005/0144669, and
2005/0037988 all of which are incorporated by reference herein.
When engineering a novel miRNA to target a specific sequence, one
strategy is to select within the target sequence a region with
sequence that is as similar as possible to the native miRNA
sequence. Alternatively, the native miRNA sequence can be replaced
with a region of the target sequence, preferably a region that
meets structural and thermodynamic criteria believed to be
important for miRNA function (see, e.g., U.S. Patent Application
Publication 2005/0037988). Sequences are preferably engineered such
that the number and placement of mismatches in the stem structure
of the fold-back region or pre-miRNA is preserved. Thus, an
engineered miRNA or engineered miRNA precursor can be derived from
any of the mature miRNA sequences, or their corresponding miRNA
precursors (including the fold-back portions of the corresponding
MIR genes) disclosed herein.
[0274] An engineered miRNA precursor based on a mature miRNA (e.g.,
a mature miRNA corresponding to SEQ ID NO. 11, 13, 15, 17, 19, 21,
23, 25, 27, 29, 31, 33, or 35), preferably including the fold-back
portion (e.g. as depicted in FIG. 11) of the corresponding MIR
sequences (e.g., SEQ ID NO. 10, 12, 14, 16, 18, 22, 24, 26, 28, 30,
or 34), is cloned and used to evaluate engineered miRNAs in
transient plant (e.g., tobacco, maize, soy, potato, and
Arabidopsis) assays. Successful constructs are moved to stable
transformation into a plant of interest, including maize, soybean,
or potato. The sequence targetted for suppression can be endogenous
or exogenous to the plant cell in which the engineered miRNA
construct is expressed.
[0275] In a non-limiting example, engineered miRNA sequences based
on the fold-back portion of SEQ ID NO. 10, 12, 14, 16, 18, 22, 24,
26, 28, 30, or 34 are engineered to target green fluorescent
protein (GFP), with nucleotides of the native sequence replaced
with nucleotides to match a targetted portion of the GFP sequence,
while maintaining the position and number of mismatches in the stem
portion of the fold-back structure, by altering as needed the
opposite strand of the stem of the fold-back structure or
pre-miRNA. The engineered miRNA sequence is placed in an expression
cassette including a suitable promoter (e.g., e35S) and terminator
(e.g., Nos 3' transcriptional terminator). As a control, a similar
gene cassette that expresses the native (non-engineered fold-back
portion of SEQ ID NO. 10, 12, 14, 16, 18, 22, 24, 26, 28, 30, or
34) is used. A third cassette is designed to express the target
sequence (GFP) and used for co-transformation with either of the
miRNA cassettes. These three cassettes are inserted into binary
vectors for use in Agrobacterium-mediated transformation.
Constructs are tested for their ability to suppress the expression
of GFP in a transient co-transformation experiment in which leaves
are transformed in planta on wild-type maize, soybean, potato,
Arabidopsis, or Nicotiana spp. plants. After four days, leaf
punches corresponding to the regions infiltrated with Agrobacterium
containing the plasmids are assayed for GFP fluorescence, which is
normalized to total protein content. Constructs that express a
miRNA that has been engineered to suppress the GFP gene have lower
GFP expressed than the unengineered control.
Example 15
[0276] This example describes identifying novel mature miRNAs and
the corresponding MIR sequences in soy. A single small RNA was
isolated and cloned using procedures based on published protocols
(Llave et al. (2002) Plant Cell, 14:1605-1619, and Lau et al.
(2001) Science, 294:858-862). In summary, low molecular weight RNA
was isolated from soy (Glycine max) leaf tissue. Adaptors were
ligated followed by RT-PCR for conversion of RNA to DNA. Additional
PCR amplification followed by TA cloning and sequencing led to the
identification of novel mature miRNA 21-mers corresponding to the
DNA sequence TGAGACCAAATGAGCAGCTGA (SEQ ID NO. 36) or
ATGCACTGCCTCTTCCCTGGC (SEQ ID NO. 37).
[0277] A soy cDNA contig sequence database was searched for
sequences containing 21-mer segments identical to SEQ ID NO. 36 or
SEQ ID NO. 37 or to their respective complements. The sequences
thus identified included SEQ ID NO. 38 (including the DNA sequence
SEQ ID NO. 36 corresponding to a non-conserved mature miRNA) and
SEQ ID NO. 39 (Gm-MIR408, including the DNA sequence SEQ ID NO. 37
corresponding to the conserved mature miRNA miR408). The novel MIR
sequences are given in Table 5, with the location of nucleotides
corresponding to the mature miRNA indicated by underlined text.
TABLE-US-00005 TABLE 5 SEQ ID NO. Sequence 38
AAAATTCATTACATTGATAAAACACAATTCAAAAGATCAATGTTCC
ACTTCATGCAAAGACATTTCCAAAATATGTGTAGGTAGAGGGGTTT
TACAGGATCGTCCTGAGACCAAATGAGCAGCTGACCACATGATGCA
GCTATGTTTGCTATTCAGCTGCTCATCTGTTCTCAGGTCGCCCTTG
TTGGACTGTCCAACTCCTACTGATTGCGGATGCACTTGCCACAAAT
GAAAATCAAAGCGAGGGGAAAAGAATGTAGAGTGTGACTACGATTG
CATGCATGTGATTTAGGTAATTAAGTTACATGATTGTCTAATTGTG
TTTATGGAATTGTATATTTTCAGACCAGGCACCTGTAACTAATTAT
AGGTACCATACCTTAAAATAAGTCCAACTAAGTCCATGTCTGTGAT
TTTTTAGTGTCACAAATCACAATCCATTGCCATTGGTTTTTTAATT
TTTCATTGTCTGTTGTTTAACTAACTCTAGCTTTTTAGCTGCTTCA
AGTACAGATTCCTCAAAGTGGAAAATGTTGTTTGAAGTCAATAAAA
AGAGCTTTGATGATCATCTGCATTGTCTAAGTTGGATAAACTAATT
AGAGAGAACTTTTGAACTTTGTCTACCAAATATCTGTCAGTGTCAT
CTGTCAGTTCTGCAAGCTGAAGTGTTGAATCCACGAGGTGCTTGTT
GCAAAGTTGTGATATTAAAAGACATCTACGAAGAAGTTCAAGCAAA ACTCTTTTTGGC 39
CCGTGGTGGGCGAAGGGAATTAACGCCTATCGCGTGGCGAGAGAAG
GAGCAGAACGGCAGGGGGGGGCCGGCTCCGGGGGGGCGCCCCGGTA
CGGACCGCGCTCTCCGAGTCCCTGGGGTCCCCCCCCCAGAACATCC
TAATCGAAAAATTCAAGAGTGCATTTTGTGCGTAATGTAGTTAATT
AGACAAATTTCTAATGTGAGAATCTTTCTGAGAATGAGATGTTGCT
AAATATTTCGGATGTTGTCGACAAGGATGAGGTAATAATAGTTAGA
GACAGGACAAAGCAGGGGAACAGGCAGAGCATGGATGGAGCTATCA
ACACAATATTGTCAAGAAACTGAGAGTGAGAGGAGAAATATGTTGT
GGTTCTGCTCATGCACTGCCTCTTCCCTGGCTCTGTCTCCATTTCT
CCTTCCCTTATTTATTTTTTGATTTATTGAGTATGATCTGTTTTCA
AATGTGTTCATAGGTTCAACTTATTAAGGTACGAACATACTCTGGG
CATTGAAAACTGGTTTGACTCTTGAACATATTCCGCACCACTAATC
TTTCTTGTAATCCAGGCTCACGCACGATCACTATAAGGTCCCACAT
TCTTAGTGGCCTAATCGTTGGAAAATGCTACTTTGGCACTACTTGA
TGAATTGTATGGCTGGGATTTTTTTCCCCTTGCTTGTAGAATCCTC
TCAATTTATGTAACCATCGTGTACTCATTTACATGTCATCATTTTT
GAATGAGATGTGATATACATAGAGCAAAAAAAAAAAAAAAATTGTA
TGACCTCATTTTCTGTGTTTATTTCTCTCCATCAATATCATTTTCT
AAATCTCAAAATTCTCTCTTTTTTCTTAGTTGTAGAAGTTATTGTT
TACTCGACTCCTCGCCTCACATCCCTCTCACCCCTCTCCCCACTAC
TGCCCCGCCAGCGTCACCGATGCTCTCCTTTGTGGCCGGT
[0278] The fold-back structure of the pri-miRNA was identified in
these MIR sequences using the program EINVERTED (Rice et al. (2000)
Trends Genet., 16:276-277), and the results shown in FIG. 12, with
nucleotide positions indicated by numbers in the fold-back portion
of the sequences. The fold-back portion of the MIR sequences is
included in the pre-miRNA precursors processed from these MIR
genes.
[0279] A family of related miRNAs was cloned from the soy leaf
tissue, including the abundant miRNA described above and
corresponding to the DNA sequence TGAGACCAAATGAGCAGCTGA (SEQ ID NO.
36), and in lower abundances mature miRNA 21-mers corresponding to
the DNA sequence TGAGATCAAATGAGCAGCTGA (SEQ ID NO. 40),
TGAGACCAAATGAGCAGCTGT (SEQ ID NO. 41), and TGAGACCAAATGACCAGCTGA
(SEQ ID NO. 42), respectively, each of which differs from SEQ ID
NO. 36 at only one nucleotide position.
[0280] The MIR sequences, the complete MIR genes which include
these, and the miRNA precursors (i.e., pri-miRNAs and pre-miRNAs)
processed from these, the mature miRNAs transcribed from these, and
miRNA recognition sites of the mature miRNAs have various utilities
as described above in Examples 12, 13, and 14 and elsewhere in this
disclosure.
Example 16
[0281] This example describes identifying novel MIR sequences in
maize. Public and proprietary maize (Zea mays) genomic datasets
were searched for novel microRNA precursor sequences, starting with
all pre-miRNA sequences known at the time (April 2004) using blastn
and a very permissive cutoff (e<=10,000). Hits matching a
minimum length criteria were extracted and tested (cmsearch)
against all known miRNA covariance models (Rfam v5.1). Sequences
showing significant similarity (>15 bits) to Rfam models were
folded (mfold) and putative miRNAs identified. Two microRNA
precursors in the miR66 family were thus identified, and are listed
in Table 6. These novel MIR sequences contained the consensus
fold-back structure indicated by the shaded nucleotides depicted in
FIG. 13 (Griffiths-Jones (2004) Nucleic Acids Res., 32, Database
Issue, D109-D111, which is incorporated by reference herein).
TABLE-US-00006 TABLE 6 MIR gene Sequence SEQ ID NO. 43
GTTAAGGGGTCTGTTGTCTGGTTCAAGGTCGCCACAG
CAGGCAAATAAAGCCCATTTCGCGCTTAGCATGCACC
ATGCATGATGGGTGTACCTGTTGGTGATCTCGGACCA GGCTTCAATCCCTTTAAC SEQ ID NO.
44 GTCGAGGGGAATGACGTCCGGTCCGAACGAGCCACGG
CTGCTGCTGCGCCGCCGCGGGCTTCGGACCAGGCTTC ATTCCCCGTGAC
[0282] The MIR sequences, the complete MIR genes which include
these, the miRNA precursors (i.e., pri-miRNAs and pre-miRNAs)
processed from these, and the mature miRNAs transcribed from these,
and miRNA recognition sites of the mature miRNAs have various
utilities as described above in Examples 12, 13, and 14 and
elsewhere in this disclosure.
Example 17
[0283] This non-limiting example describes the distribution of
miRNAs in specific cells or tissues of a multicellular eukaryote (a
plant). Knowledge of the spatial or temporal distribution of a
given miRNA's expression is useful, e.g., in designing recombinant
constructs to be expressed in a spatially or temporally specific
manner. This example discloses mature miRNA expression patterns in
maize and provides sequences of recognition sites for these miRNAs
that are suitable for inclusion in recombinant DNA constructs
useful in maize and other plants.
[0284] Total RNA was isolated from LH244 maize plants using Trizol
(Invitrogen, Carlsbad, Calif.). Seven developmental stages were
used, including roots and shoot meristems from germinating
seedlings, juvenile (V1 to V2) and adult leaves (V7 to V8), stalk
internode, tassel before shedding, and immature (approximately 1")
ears. Five micrograms total RNA was resolved on 17% PAGE-Urea as
described by Allen et al. (2004) Nat. Genet., 36:1282-1290, which
is incorporated by reference herein. Blots were probed with DNA
oligonucleotides that were antisense to the small RNA sequence and
end-labelled with gamma .sup.32P-ATP using Optikinase (USB). The
probes used, and their respective sequences, are given in Table 7.
TABLE-US-00007 TABLE 7 SEQ ID NO. Sequence miRNA 45
GTGCTCACTCTCTTCTGTCA miR156 46 TAGAGCTCCCTTCAATCCAAA miR159 47
TGGCATCCAGGGAGCCAGGCA miR160 48 CTGGATGCAGAGGTTTATCGA miR162 49
TGCACGTGCCCTGCTTCTCCA miR164 50 GGGGAATGAAGCCTGGTCCGA miR166 51
TAGATCATGCTGGCAGCTTCA miR167 52 TTCCCGACCTGCACCAAGCGA miR168 53
TCGGCAAGTCATCCTTGGCTG miR169 54 GATATTGGCGCGGCTCAATCA miR171 55
CTGCAGCATCATCAAGATTCT miR172 56 GGCGCTATCCCTCCTGAGCTT miR390 57
GATCAATGCGATCCCTTTGGA miR393 58 TGGGGTCCTTACAAGGTCAAGA TAS3 5'D7(+)
59 GGAGGTGGACAGAATGCCAA miR394 60 GAGTTCCCCCAAACACTTCAC miR395 61
CATCAACGCTGCGCTCAATGA miR397 62 CGGGGGCGACCTGAGAACACA miR398 63
AGCCAGGGAAGAGGCAGTGCA miR408
[0285] The results are shown in FIG. 14. Individual mature miRNAs
were expressed at differing levels in specific cells or tissues.
For example, Zm-miR390 was not expressed, or expressed only at low
levels, in root and adult leaf.
Example 18
[0286] This example describes recombinant DNA constructs of the
invention, useful for suppressing expression of a target RNA in a
specific cell of or derived from a multicellular eukaryote such as
a plant cell or an animal cell, and methods for their use. The
constructs include a promoter operably linked to DNA that
transcribes to RNA including at least one exogenous miRNA
recognition site recognizable by a mature miRNA expressed in a
specific cell of a multicellular eukaryote, and target RNA to be
suppressed in the specific cell, wherein said target RNA is to be
expressed in cells of the multicellular eukaryote other than the
specific cell.
[0287] Strong constitutive promoters that are expressed in nearly
all plant cells have been identified (e.g., CaMC 35S, OsAct), but
strong spatially specific (cell- or tissue-specific) and temporally
specific promoters have been less well characterized. To limit
target RNA or transgene expression to a specific cell or tissue
type in the absence of a strong cell- or tissue-specific promoter,
it may be desirable to suppress in selected cells or tissues the
expression of a transcript under the control of a constitutive
promoter. The invention provides methods that use recognition
sequences of endogenous miRNAs to suppress expression of a
constitutively expressed target RNA in specific cells.
[0288] Methods of the invention allow spatially or temporally
specific post-transcriptional control of expression of a target RNA
wherein transcription is driven by a non-specific (e.g.,
constitutive) promoter. The methods of the invention allow, for
example, the restricted expression of a gene transcribed by a
constitutive promoter or a promoter with expression beyond the
desired cell or tissue type(s). Restricted expression may be
spatially or temporally restricted, e.g., restricted to specific
tissues or cell types or files, or to specific developmental,
reproductive, growth, or seasonal stages. Where a miRNA is
expressed under particular conditions (e.g., under biotic stress
such as crowding, allelopathic interactions or pest or pathogen
infestation, or abiotic stress such as heat or cold stress, drought
stress, nutrient stress, heavy metal or salt stress), the
corresponding miRNA recognition site can be used for conditionally
specific suppression, i.e., to suppress a target RNA under the
particular condition.
[0289] For example, Zm-miR162 is poorly expressed in maize roots
(see Example 17 and FIG. 14), therefore, designing an expression
construct to include an exogenous miRNA162 recognition site
adjacent to, or within, a constitutively expressed target RNA, may
limit target RNA transcript accumulation in all cells of a maize
plant with the exception of roots. This method has utility for all
gene expression applications in multicellular eukaryotes (plants
and animals), where restricted expression is desired in cells
wherein the given mature miRNA is expressed.
[0290] In multicellular eukaryotes, including plants, microRNAs
(miRNAs) regulate endogenous genes by a post-transcriptional
cleavage mechanism, which can spatially or temporally specific. The
present invention provides methods by which the addition of a miRNA
recognition site to a constitutively expressed transgene could be
used to limit expression of the transgene to cells lacking, or
distant to those expressing, the complementary mature miRNA either
spatially or temporally (including conditionally). Manipulation of
these miRNA recognition sites in new transcripts introduced into
transgenic plant cells and transgenic plants derived from these
cells, is useful for altering expression patterns for the new
transgene.
[0291] In an alternative approach, an existing (native or
endogenous) miRNA recognition site is mutated (e.g., by chemical
mutagenesis) sufficiently to reduce or prevent cleavage (see
Mallory et al. (2004) Curr. Biol., 14:1035-1046, incorporated by
reference herein). In this way a target RNA sequence with desirable
effects, e.g., increased leaf or seed size, can be expressed at
levels higher than when the native or endogeous miRNA recognition
site was present. One embodiment is to replace a native gene with
an engineered homologue, wherein a native miRNA has been mutated or
even deleted, that is less susceptible to cleavage by a given
miRNA.
[0292] One embodiment of the method is the introduction of at least
one exogenous miRNA recognition site (typically a 21 nucleotide
sequence) into the 5' or into the 3' untranslated regions of a
target RNA, or within the target RNA. Where the target RNA includes
coding sequence, the at least one exogenous miRNA recognition site
can be introduced into the coding region of the target RNA. This
results in the reduced expression of the target RNA in tissues or
cell types that express the corresponding mature miRNA. By
including a recognition site corresponding to a mature miRNA in a
target RNA transcript, it is possible to modulate the target RNA's
expression in such a way that even under the control of a
constitutive promoter, the target RNA is expressed only in selected
cells or tissues or during selected temporal periods. This allows
both the high levels of expression obtainable with strong
constitutive promoters, and spatial or temporal limiting of such
expression.
[0293] Any miRNA recognition site may be used, preferably where the
expression of the corresponding mature miRNA has been determined to
suit the desired expression or suppression of the target RNA.
Numerous miRNA recognition sequences are known. See, for example,
Jones-Rhoades and Bartel (2004). Mol. Cell, 14:787-799, Rhoades et
al. (2002) Cell, 110:513-520, Allen et al. (2004) Nat. Genet.,
36:1282-1290, which are incorporated by reference herein). Also see
the ASRP database online (Gustafson et al. (2005) Nucleic Acids
Res., 33:D6379-D640). Non-limiting examples of miRNA recognition
sites useful in constructs and methods of the invention include
those provided in Table 8, which gives the recognition site
sequences for the indicated miRNA family and indicates the
distribution among "all plants" (i.e., lower plants, monocots, and
dicots), monocots and/or dicots. The plant species from which the
miRNA was identified and the abbreviations used were: Arabidopsis
thaliana (At), Glycine max (Gm), Gossypium hirsutum (Gh), Hordeum
vulgare (Hv), Lycopersicum esculentum (Le), Lotus corniculatus var.
japonicus (synonymous with "Lotus japonicus") (Lj), Medicago
truncatula (Mt), Mesembryanthemum crystallinum (Mc), Oryza sativa
(Os), Pennisetum glaucum (Pg), Phaseolus vulgaris (Pv), Populus
tremula (Pt), Saccharum officinarum (So), Sorghum bicolor (Sb),
Theobroma cacao (Tc), Triticum aestivum (Ta), Vitis vinifera (Vv),
and Zea mays (Zm). TABLE-US-00008 TABLE 8 miRNA Recognition
Recognition Site SEQ ID NO. Site Sequence miR156 family recognition
sequence-all plants 64 At1g27370 GUGCUCUCUCUCUUGUGUCA 65 At1g53160
CUGCUCUCUCUCUUCUGUCA 66 At2g33810 UUGCUUACUCUCUUCUGUCA 67 At3g15270
CCGCUCUCUCUCUUCUGUCA miR159 family recognition sequence-all plants
68 At5g06100 UGGAGCUCCCUUCAUUCCAAU 69 At2g26960
UCGAGUUCCCUUCAUUCCAAU 70 At4g26930 AUGAGGUCUCUUCAAACCAAA 71
At2g26950 UGGAGCUCCCUUCAUUCCAAG 72 At2g32460 UAGAGCUUCCUUCAAACCAAA
73 At3g60460 UGGAGCUCCAUUCGAUCCAAA 74 At5g55020
AGCAGCUCCCUUCAAACCAAA 75 PvMYB CAGAGCUCCCUUCACUCCAAU 76 VvMYB
UGGAGCUCCCUUCACUCCAAU 77 HvMYB33 UGGAGCUCCCUUCACUCCAAG 78 OsMYB33
UGGAGCUCCCUUUAAUCCAAU miR160 familly target sequences-all plants 79
At1g77850 UGGCAUGCAGGGAGCCAGGCA 80 At2g28350 AGGAAUACAGGGAGCCAGGCA
81 At4g30080 GGGUUUACAGGGAGCCAGGCA 82 OsARF AGGCAUACAGGGAGCCAGGCA
83 LjARF AAGCAUACAGGGAGCCAGGCA miR161 family target
sequences-Arabidopsis 84 At5g41170 ACCUGAUGUAAUCACUUUCAA 85
At1g06580 CCCGGAUGUAAUCACUUUCAG 86 At1g63150 UUGUUACUUUCAAUGCAUUGA
87 At5g16640 CCCUGAUGUAUUUACUUUCAA 88 At1g62590
UAGUCACGUUCAAUGCAUUGA 89 At1g62670 CCCUGAUGUAUUCACUUUCAG 90
At1g62860 CCCUGAUGUUGUUACUUUCAG 91 At1g62910 UAGUCACUUUCAGCGCAUUGA
92 At1g62930 UCCAAAUGUAGUCACUUUCAG 93 At1g63080
UCCAAAUGUAGUCACUUUCAA 94 At1g63130 UCCAAAUGUAGUCACUUUCAG 95
At1g63400 UCCAAAUGUAGUCACUUUCAA 96 At1g63230 UUGUAACUUUCAGUGCAUUGA
97 At1g63330 UAGUCACGUUCAAUGCAUUGA 98 At1g63630
UUGUUACUUUCAGUGCAUUGA 99 At1g64580 CCCUGAUGUUGUCACUUUCAC 100
At2g41720 UUGUUACUUACAAUGCAUUGA 101 At1g63070 UAGUCUUUUUCAACGCAUUGA
miR162 family target sequences-monocots and dicots 102 At1g01040
CUGGAUGCAGAGGUAUUAUCGA 103 PtDCL1 CUGGAUGCAGAGGUCUUAUCGA 104 OsDCL1
CUGGAUGCAGAGGUUUUAUCGA miR163 family target sequences-Arabidopsis
105 At1g66700 AUCGAGUUCCAAGUCCUCUUCAA 106 At1g66720
AUCGAGUUCCAGGUCCUCUUCAA 107 At3g44860 AUCGAGUUCCAAGUUUUCUUCAA
miR164 family target sequences-monocots and dicots 108 At1g56010
AGCACGUACCCUGCUUCUCCA 109 At5g07680 UUUACGUGCCCUGCUUCUCCA 110
At5g53950 AGCACGUGUCCUGUUUCUCCA 111 At5g61430 UCUACGUGCCCUGCUUCUCCA
112 At5g39610 CUCACGUGACCUGCUUCUCCG 113 OsNAC1
CGCACGUGACCUGCUUCUCCA 114 MtNAC CUUACGUGUCCUGCUUCUCCA 115 GmNAC
CUUACGUGCCCUGCUUCUCCA 116 LeNAC GCCACGUGCACUGCUUCUCCA miR165/166
family target sequences-all plants 117 At1g30490
UUGGGAUGAAGCCUGGUCCGG 118 At5g60690 CUGGGAUGAAGCCUGGUCCGG 119
At1g52150 CUGGAAUGAAGCCUGGUCCGG 120 PtHDZIPIII
CCGGGAUGAAGCCUGGUCCGG miR167 family target sequences-monocots and
dicots 121 At1g30330 GAGAUCAGGCUGGCAGCUUGU 122 At5g37020
UAGAUCAGGCUGGCAGCUUGU 123 OsARF6 AAGAUCAGGCUGGCAGCUUGU miR168
family target sequences-all plants 124 At1g48410
UUCCCGAGCUGCAUCAAGCUA miR169 family target sequences-all plants 125
At1g17590 AAGGGAAGUCAUCCUUGGCUG 126 At1g54160 ACGGGAAGUCAUCCUUGGCUA
127 At1g72830 AGGGGAAGUCAUCCUUGGCUA 128 At3g05690
AGGCAAAUCAUCUUUGGCUCA 129 At3g20910 GCGGCAAUUCAUUCUUGGCUU 130
At5g12840 CCGGCAAAUCAUUCUUGGCUU 131 At3g14020 AAGGGAAGUCAUCCUUGGCUA
132 ZmHAP2 GUGGCAACUCAUCCUUGGCUC 133 VvHAP2 UGGGCAAUUCAUCCUUGGCUU
134 OsHAP2 AUGGCAAAUCAUCCUUGGCUU 135 GmHAP2 UAGGGAAGUCAUCCUUGGCUC
136 GhHAP2 CUGGGAAGUCAUCCUUGGCUC miR170/171 family target
sequences-all plants 137 At2g45160 GAUAUUGGCGCGGCUCAAUCA miR172
family target sequences-all plants 138 At4g36920
CUGCAGCAUCAUCAGGAUUCU 139 At2g28550 CAGCAGCAUCAUCAGGAUUCU 140
At5g60120 AUGCAGCAUCAUCAGGAUUCU 141 At5g67180 UGGCAGCAUCAUCAGGAUUCU
142 At2g39250 UUGUAGCAUCAUCAGGAUUCC 143 At3g54990
UUGCAGCAUCAUCAGGAUUCC miR319 family target sequences-all plants 144
At4g18390 CAGGGGGACCCUUCAGUCCAA 145 At1g53230 GAGGGGUCCCCUUCAGUCCAU
146 At3g15030 GAGGGGUCCCCUUCAGUCCAG 147 At2g31070
AAGGGGUACCCUUCAGUCCAG 148 At1g30210 UAGGGGGACCCUUCAGUCCAA 149
OsPCF5 GAGGGGACCCCUUCAGUCCAG 150 OsPCF8 UCGGGGCACACUUCAGUCCAA
miR393 family target sequences-monocots and dicots 151 At1g12820
AAACAAUGCGAUCCCUUUGGA 152 At4g03190 AGACCAUGCGAUCCCUUUGGA 153
At3g23690 GGUCAGAGCGAUCCCUUUGGC 154 At3g62980 AGACAAUGCGAUCCCUUUGGA
miR394 family target sequences-monocots and dicots 155 At1g27340
GGAGGUUGACAGAAUGCCAAA miR395 family target sequences-monocots and
dicots 156 At5g43780 GAGUUCCUCCAAACACUUCAU 157 At3g22890
GAGUUCCUCCAAACUCUUCAU 158 At5g10180 AAGUUCUCCCAAACACUUCAA miR396
family target sequences-monocots and dicots 159 At2g22840
UCGUUCAAGAAAGCCUGUGGAA 160 At2g36400 CCGUUCAAGAAAGCCUGUGGAA 161
At4g24150 UCGUUCAAGAAAGCAUGUGGAA 162 At2g45480
ACGUUCAAGAAAGCUUGUGGAA 163 At3g52910 CCGUUCAAGAAAGCCUGUGGAA miR397
family target sequences-monocots and dicots 164 At2g29130
AAUCAAUGCUGCACUCAAUGA 165 At2g38080 AGUCAACGCUGCACUUAAUGA
166 At2g60020 AAUCAAUGCUGCACUUAAUGA miR398 family target
sequences-monocots and dicots 167 At1g08830 AAGGGGUUUCCUGAGAUCACA
168 At2g28190 UGCGGGUGACCUGGGAAACAUA 169 At3g15640
AAGGUGUGACCUGAGAAUCACA miR173 family target sequences-Arabidopsis
170 AtTAS1a GUGAUUUUUCUCAACAAGCGAA 171 AtTAS1c
GUGAUUUUUCUCUACAAGCGAA 172 AtTAS2 GUGAUUUUUCUCUCCAAGCGAA miR399
family target sequences-monocots and dicots 173 At2g33770
UAGGGCAUAUCUCCUUUGGCA 174 At2g33770 UUGGGCAAAUCUCCUUUGGCA 175
At2g33770 UCGAGCAAAUCUCCUUUGGCA 176 At2g33770 UAGAGCAAAUCUCCUUUGGCA
177 At2g33770 UAGGGCAAAUCUUCUUUGGCA 178 OsE2UBC
UAGGGCAAAUCUCCUUUGGCA 179 OsE2UBC CUGGGCAAAUCUCCUUUGGCA 180 OsE2UBC
UCGGGCAAAUCUCCUUUGGCA 181 OsE2UBC CCGGGCAAAUCUCCUUUGGCA 182 PtE2UBC
GCGGGCAAAUCUUCUUUGGCA 183 MtE2UBC AAGGGCAAAUCUCCUUUGGCA 184 TaE2UBC
UAGGGCAAAUCUCCUUUGGCG 185 TaE2UBC CUGGGCAAAUCUCCUUUGGCG 186 TaE2UBC
UUCGGCAAAUCUCCUUUGGCA miR403 family target sequences-dicots 187
At1g31280 GGAGUUUGUGCGUGAAUCUAAU miR390 family target sequences-all
plants 188 At3g17185 CUUGUCUAUCCCUCCUGAGCUA 189 SbTAS3
UAUGUCUAUCCCUUCUGAGCUG 190 SoTAS3 UAUGUCUAUCCCUUCUGAGCUA 191 ZmTAS3
UAUGUCUAUCCCUUCUGAGCUG 192 OsTAS3 UCGGUCUAUCCCUCCUGAGCUG 193 PgTAS3
UUAGUCUAUCCCUCGUGAGCUA 194 VvTAS3 AUUGCCUAUCCCUCCUGAGCUG 195 TcTAS3
CCUUGCUAUCCCUCCUGAGCUG 196 LeASR CUUGUCUAUCCCUCCUGAGCUG 197 ZmTAS3
CCCUUCUAUCCCUCCUGAGCUA 198 PtTAS3 CUUGUCUAUCCCUCCUGAGCUA 199 OsTAS3
GCCUUCUAUCCCUCCUGAGCUA 200 TaTAS3 GCCUUCUAUCCCUCCUGAGCUA 201 HvTAS3
CCUIUCUAUCCCUCCUGAGCUA 202 PtTAS3 GCUGUCUAUCCCUCCUGAGCUA 203 McTAS3
UGUGUCUAUCCCUCCUGAGCUA miR447 family target sequences-Arabidopsis
204 At5g60760 UGACAAACAUCUCGUCCCCAA 205 At3g45090
UGACAAACAUCUCGUUCCUAA miR408 family target sequences-monocots and
dicots 206 At2g02850 CCAAGGGAAGAGGCAGUGCAU 207 At2g30210
ACCAGUGAAGAGGCUGUGCAG 208 At2g47020 GCCAGGGAAGAGGCAGUGCAU 209
At5g05390 GCCGGUGAAGAGGCUGUGCAA 210 At5g07130 GCCGGUGAAGAGGCUGUGCAG
TAS3 ta-siRNA target sequences-monocots and dicots 211 At2g33860a
AGGGUCUUGCAAGGUCAAGAA 212 At5g60450a AAGGUCUUGCAAGGUCAAGAA 213
OsARE3-like GAGGUCUUGCAAGGUCAAGAA 214 OsARF2-like
ACGGUCUUGCAAGGUCAAGAA TAS1/TAS2 target sequences-Arabidopsis
thaliana 215 Atg12770 AGAACUAGAGAAAGCAUUGGA 216 Atg12770
AGAGUAAGAUGGAGCUUGAUA 217 At1g63130 AGAUGGUGGAAAUGGGAUAUC 218
At1g63230 UUGUUGAUCGUAUGGUAGAAG 219 At1g62930
GGUAUUCGAGUAUCUGCAAAA
[0294] Thus, a transgenic plant expressing a recombinant DNA
construct that, under the control of a constitutive promoter (e.g.,
a 35S promoter) transcribes to RNA containing a Zm-miR390
recognition site and a target RNA would be expected to show
suppression of the target RNA expression in root and adult leaf,
relative to expression in other tissues.
[0295] In another example, Zm-miR172 was expressed at high levels
in stalk, and not expressed, or expressed only at low levels, in
other tissues. A transgenic plant expressing a construct that,
under the control of a strong constitutive promoter (e.g., a CaMV
35S promoter) transcribes to RNA containing a Zm-miR172 recognition
site and a target RNA would be expected to express that target RNA
at higher levels in tissues other than stalk (where expression of
the target RNA would be suppressed).
[0296] To illustrate use of the constructs and methods of the
invention to control expression of a gene of interest, a reporter
gene is used as the gene of interest itself, or as a surrogate for
the gene of interest. For example, where expression of a reporter
gene (e.g., green fluorescent protein, GFP) is desired in maize
stalk and immature ear tissue, a miR156 target site is included in
a GFP expression cassette and expressed in a stably transgenic
maize plant under the control of the CaMV 35S promoter. In other
tissues (e.g., roots, leaves, and tassel), GFP expression is
suppressed. The suppression phenotype may be limited to very
specific cell types within the suppressed tissues, with neighboring
cells showing expression or a gradient of expression of GFP
adjacent to those cells expressing the mature miR156.
[0297] In another example, a strong constitutive promoter is used
to drive expression of a Bacillus thuringiensis insecticidal
protein or protein fragment ("Bt"), where a recognition site for a
miRNA expressed in pollen is included in the construct, resulting
in strong expression in tissues of the plant except for the
pollen.
[0298] One specific, non-limiting example of the method is the
inclusion of the recognition site for a miRNA that is not expressed
in roots to a recombinant DNA construct including a target RNA of
which expression is desired only in the roots. A strong
constitutive promoter (e.g., enhanced 35S) can still be used, but
the target RNA's expression is now restricted to the cells that
that do not express the corresponding mature miRNA. A specific
example of this approach is the inclusion of a maize miRNA162,
maize miRNA164, or maize miRNA390 recognition site in a recombinant
DNA construct for the expression of a Bacillus thuringiensis
insecticidal protein or protein fragment ("Bt", see, for example,
the Bacillus thuringiensis insecticidal sequences and methods of
use thereof disclosed in U.S. Pat. No. 6,953,835 and in U.S.
Provisional Patent Application No. 60/713,111, filed on 31 Aug.
2005, which are incorporated by reference herein) as the target
RNA, e.g., in a construct including the expression cassette
e35S/Bt/hsp17. These miRNAs (e.g., miRNA162, miRNA164, or miRNA390)
are not substantially expressed in maize roots but are expressed in
most other tissues. Including one or more of these recognition
sites within the expression cassette reduces the expression of
transcripts in most tissues other than root, but maintains high Bt
target RNA expression levels in roots, such as is desirable for
control of pests such as corn rootworm. In one embodiment,
combinations of different miRNA recognition sites are included in
the construct in order to achieve the desired expression
pattern.
[0299] Non-limiting specific examples of transcribable DNA sequence
including an exogenous miRNA recognition site are depicted in FIG.
15 and FIG. 16. FIG. 15 depicts chloroplast-targeted TIC809 with a
miRNA162 recognition site (in bold text) located in the 3'
untranslated region (SEQ ID NO. 220). FIG. 16 depicts non-targeted
TIC809 with a miRNA164 recognition site (in bold text) located in
the 3' untranslated region (SEQ ID NO. 221).
Example 19
[0300] This example describes a crop plant miRNA gene with
tissue-specific expression, and identification of the miR gene
promoter. More particularly, this example describes identification
of a maize miR167 promoter sequence with endosperm-specific
expression. A member of the miR167 family (SEQ ID NO. 4) was found
to represent about a quarter of the small RNA population cloned
from developing maize endosperm as described in Example 13. To
determine whether a single miR167 gene family member is responsible
for the observed strong endosperm expression, several miR167 genes
were analyzed by RT-PCR. Nine Zea mays miR167 stem-loop sequences
were found in the public miRNA registry ("miRBase", available on
line at microma.sanger.ac.uk/sequences), listed as miR167a through
miR167i. Tissue-specific RT-PCR was performed for several of the Z.
mays miR167 sequences using gene-specific primers for first strand
cDNA synthesis followed by PCR with gene-specific primer pairs.
Expression of miR167g was strong and tissue-specific for endosperm
(15, 20 days after pollination).
[0301] To determine whether miR167g is abundantly expressed in
endosperm, Northern blots of maize (LH59) were prepared. The blot
was probed with an end-labeled mature miR167 22-mer LNA probe (FIG.
17A), stripped, and re-probed with a .about.400 bp miR167g
gene-specific probe (FIG. 17B). The strong endosperm signal
observed indicated that miR167g is largely responsible for
endosperm-enhanced expression. Transcription profiling of maize
tissues corroborated the Northern blot results (FIG. 17C); the
transcript corresponding to miR167g was abundantly and specifically
expressed in endosperm tissue.
[0302] A GenBank publicly available 804 base pair cDNA sequence
(annotated as "ZM_BFb0071I20.r ZM_BFb Zea mays cDNA 5', mRNA
sequence") and having the accession number DR827873.1 (GI:71446823)
is incorporated here by reference. This sequence includes a segment
corresponding to the mature miR167g (SEQ ID NO. 4). Using the
public sequence, bioinformatic analysis was performed on
proprietary maize genomic sequence. A 4.75 kilobase genomic cluster
including sequence from maize inbred line B73 was identified as
containing predicted gene sequences for miR167a and miR167g. A 486
base pair region between the two miR167 genes was identified as
having homology to an expressed sequence tag (EST) sequence.
Promoter motifs were identified in the upstream sequences of both
(miR167a and miR167g) predicted transcripts. A region of 1682 base
pairs (SEQ ID NO. 222) between the predicted miR167a and miR167g
transcripts, and a smaller region of 674 base pairs (SEQ ID NO.
223) between the EST and the predicted miR167g transcript was
identified as miR167g promoter sequences. Subsets of these
sequences (e.g., at least about 50, about 100, about 150, about
200, about 250, or about 300 nucleotides of SEQ ID NO. 222 or SEQ
ID NO. 223, or fragments of at least about 50, about 100, about
150, and about 200 contiguous nucleotides having at least 85%, at
least 90%, or at least 95% identity to a segment of SEQ ID NO. 222
or SEQ ID NO. 223) are also useful as promoters; their promoter
effects are demonstrable by procedures well known in the art (e.g.,
to drive expression of a reporter gene such as luciferase or green
fluorescent protein). The annotation map, including locations of
the miR167a and miR167g genes and mature miRNAs, and promoter
elements (e.g., TATA boxes), of this genomic cluster is shown in
FIG. 18. The annotation map also shows the location of
auxin-responsive factor (ARF) motifs or auxin response elements
with the sequence TGTCTC (SEQ ID NO. 224), which indicates that
auxin may regulate expression of miR167g. Mature miR167 miRNAs are
complementary to ARF6 and ARF8 (which encode activating ARFs) and
have been proposed to regulate auxin homeostasis; see, for example,
Rhoades et al. (2002) Cell, 110:513-520, Bartel and Bartel (2003)
Plant Physiol., 132:709-717, Ulmasov et al. (1999) Proc. Natl.
Acad. Sci. USA, 96:5844-5849, and Mallory et al. (2005) Plant Cell,
17:1360-1375, all of which are incorporated by reference
herein.
[0303] In addition to the miR167g promoter sequences (SEQ ID NO.
222 and SEQ ID NO. 223) identified from maize inbred line B73, two
additional miR167g promoter sequences (SEQ ID NO. 225 and SEQ ID
NO. 226) were amplified from the maize inbred line LH244. The 3'
ends of SEQ ID NO. 225 and SEQ ID NO. 226 were determined
experimentally by 5' RACE (rapid amplification of cDNA ends,
Invitrogen Corporation, Carlsbad, Calif.) of miR167g. The 5' end of
the 768 base pairs sequence (SEQ ID NO. 225) corresponds to the end
of a GenBank publicly available 481 base pair cDNA sequence
(annotated as "QCG17c03.yg QCG Zea mays cDNA clone QCG17c03, mRNA
sequence") and having the accession number CF035345.1
(GI:32930533). The 5' end of the 407 base pairs sequence (SEQ ID
NO. 226) corresponds to the end of a GenBank publicly available 746
base pair cDNA sequence (annotated as "MEST991_A06.T7-1
UGA-ZmSAM-XZ2 Zea mays cDNA, mRNA sequence") and having the
accession number DN214085.1 (GI:60347112).
[0304] The miR167g promoter sequences, miR167g gene, mature miR167g
microRNA, and miR167g recognition site described herein have
various utilities as described in Examples 12, 13, 14, and 18, and
elsewhere in this disclosure. In particular, a miR167g promoter is
useful as an endosperm-specific promoter, and can be used, for
example to replace the maize B32 promoter used in the recombinant
DNA construct described in Example 4 (also see FIG. 5B). In another
utility, the miR167g sequence or mature miR167g (or a precursor
thereof) is engineered to suppress a target gene, especially where
suppression is to be endosperm-specific. The miR167g recognition
site is useful, e.g., in constructs for gene expression where the
gene is to be expressed in tissues other than endosperm.
Example 20
[0305] This example describes a recombinant DNA construct including
a transcribable engineered miRNA precursor designed to suppress a
target sequence, wherein the transcribable engineered miRNA
precursor is derived from the fold-back structure of a MIR gene,
preferably a maize or soybean MIR sequence.
[0306] MicroRNA genes were cloned essentially as described in
Example 15 from maize. These included a ZmMIR159a sequence (SEQ ID
NO. 227) and a ZmMIR164e (SEQ ID NO. 228); the sequences are
provided in Table 9, with the location of nucleotides corresponding
to the mature miRNA indicated by underlined text. TABLE-US-00009
TABLE 9 Zea mays MIR sequence MIR159a
GCATCTGCTGTTCTTTATTTCTATACATACATAT (SEQ ID NO. 227)
ATACTATCACCGGTTATTTGCTTCTCTATTCTGT
CCGAGTACTTTACGGTGTTCCGCACATAGATCTC
GTGGCCGGCGGTTTTGCGCTTTCGCTTGCGTTTC
TTGGCCCTGCTGGTGTTTGACCGGACCGAACGGG
GGCAGATCGATGCTTTGGGTTTGAAGCGGAGCTC
CTATCATTCCAATGAAGGGTCGTTCCGAAGGGCT
GGTTCCGCTGCTCGTTCATGGTTCCCACTATCCT
ATCTCATCATGTGTATATATGTATTCCATGGGGG
AGGGTTTCTCTCGTCTTTGAGATAGGCTTGTGGT
TTGCATGACCGAGGAGCTGCACCGCCCCCTTGCT
GGCCGCTCTTTGGATTGAAGGGAGCTCTGCATCC
TGATGCACCCCTCCATTTTTTTTTGCTTGTTGTG
TCCTTCCTGGGACCTGAGATCTGAGGCTCGTGGT GGCTCACTG MIR164e
CCTTGTATGTTCTCCGCTCACTCCCCCAUCCACT (SEQ ID NO. 228)
CTCATCCATCTCTCAAGCTACACACATATAAAAA
AAAAAGAGTAGAGAAGGACCGCCGTTAGAGCACT
TGATGCATGCGTACGTCGATCCGGCGGACCGATC
TGCTTTTGCTTGTGTGCTTGGTGAGAAGGTCCCT
GTTGGAGAAGCAGGGCACGTGCAGAGACACGCCG
GAGCACGGCCGCCGCCGATCTACCGACCTCCCAC
ACCTGCCTTGTGGTGTGGGGGTGGAGGTCGTCGG
TGGAAGCGATAGCTGTCGTTGTTGCTTCGATGTT
GTTAGCTCCTCCTGCACGTGCTCCCCTTCTCCAC
CACGGCCTTCTCACCACCCTCCTCCCCCGGCGGC
GGCGGCGGCGGACCGCCCTTGCCGCGATCAATAA
TGAACCAAAAGCCGACAGTATTTGAGCAGGAAAT
ACAAGAGGCGGATATCCCACTGCTAGCACTTCTG
CGTTGATCATGtTCATCTGGAACAAAATAATACT CGGCGACTTTACAGCGAGTGCAGCATG
[0307] An engineered miRNA, "MIR159a-CPB.miR1", based on cloned SEQ
ID NO. 227, was designed to target a vacuolar ATPase sequence from
Colorado potato beetle and had the sequence
GCATCTGCTGTTCTTTATTTCTATACATACATATATACTATCACCGGTTATTTGCTTCTCTATTCTGTCCGA
GTACTTTACGGTGTTCCGCACATAGATCTCGTGGCCGGCGGTTTTGCGCTTTCGCTTGCGTTTCTTGGCCC
TGCTGGTGMGACCGGACCGAACGGGGGCAGATCGATGCTTTGGGTTTGAAGatacGtggCaAaacTaggAAT
GAAGGGTCGTTCCGAAGGGCTGGTTCCGCTGCTCGTTCATGGTTCCCACTATCCTATCTCATCATGTGTA
TATATGTATTCCATGGGGGAGGGTTTCTCTCGTCTTTGAGATAGGCTTGTGGTTTGCATGACCGAGGAGC
TGCACCGCCCCCTTGCTGGCCGCTCTTTCCTGGTTCTGCCACGTATCATCCTGATCCACCCCTCCATTTT
TTTTTGCTTGTTGTGTCCTTCCTGGGACCTGAGATCTGAGGCTCGTGGTGGCTCACTG (SEQ ID
NO. 229, where the nucleotides corresponding to the engineered
mature miRNA are indicated by bold underlined text, and the
nucleotides included in the complementary strand of the miRNA
hairpin are indicated by lower-case text). A recombinant DNA
construct containing this engineered miRNA (SEQ ID NO. 229), was
made and expressed in tobacco (N. benthamiana) using a transient in
planta expression assay as in Llave et al. (2002) Plant Cell,
14:1605-1619 and Palatnik et al. (2003) Nature, 425:257-263, which
are incorporated by reference herein. Briefly, Agrobacterium
tumefaciens containing a binary expression vector was grown to late
log phase, VIR genes induced, and all desired combinations of
expression vectors mixed to a final optical density (600
nanometers) of 0.5. A GFP expression vector was used to equalize
all mixes to the same optical density. Agrobacterium mixes were
infiltrated into N. benthamiana using a syringe applied with slight
pressure to the bottom surface of two to three leaves per plant
leaf. Inoculated leaves were harvested 48 hours after infiltration.
All assays were performed in triplicate, with a single plant per
replicate. The predicted mature engineered miRNA processed from the
precursor sequence SEQ ID NO. 229 has the sequence
UUUCCUGGUUCUGCCACGUAU (SEQ ID NO. 230), which has a Reynolds score
of 4 (where values range from -1 to 10 and a higher score is
predictive of efficacy; see Reynolds et al. (2004) Nature
Biotechnol., 22:326-330, which is incorporated by reference in its
entirety herein), a functional asymmetry score of -1.1 (where a
negative value predicts incorporation into the RISC complex, see
Khvorova et al. (2003) Cell, 115:209-216, which is incorporated by
reference herein), and was observed to be efficiently processed
(FIG. 19B).
[0308] This approach is useful with other plant mature miRNA and
miRNA precursor sequences, which can be engineered to silence
various target genes of the plant or of a pest or pathogen of the
plant. Thus, another engineered miRNA, "MIR159a-CRW.miR1", also
based on cloned SEQ ID NO. 227, is designed to target a vacuolar
ATPase sequence from corn rootworm and had the sequence
GCATCTGCTGTTCTTTATTTCTATACATACATATATACTATCACCGGTTATTTGCTTCTCTATTCTGTCCGA
GTACTTTACGGTGTTCCGCACATAGATCTCGTGGCCGGCGGTTTTGCGCTTTCGCTTGCGTTTCTTGGCCC
TGCTGGTGTTTGACCGGACCGAACGGGGGCAGATCGATGCTTTGGGTTTGAAGTCTCTGGCAGTAACTG
ACAATGAAGGGTCGTTCCGAAGGGCTGGTTCCGCTGCTCGTTCATGGTTCCCACTATCCTATCTCATCAT
GTGTATATATGTATTCCATGGGGGAGGGTTTCTCTCGTCTTTGAGATAGGCTTGTGGTTTGCATGACCGA
GGAGCTGCACCGCCCCCTTGCTGGCCGCTCTTTGTCCGTTTCTGCCAGAGACATCCTGATCCACCCCTC
CATTTTTTTTTGCTTGTTGTGTCCTTCCTGGGACCTGAGATCTGAGGCTCGTGGTGGCTCACTG
(SEQ ID NO. 231, where the nucleotides corresponding to the
engineered mature miRNA are indicated by bold underlined text). The
Western corn rootworm (Diabrotica virgifera) vacuolar ATPase
sequence selected for suppression has the sequence
AGAAGCCTGGCAATTTCCAAGGTGATTTTGTCCGTTTCTGCCAGAGATGCTTTACCTACCAGCTGCACAA
TTTCGGCTAGATCATCTTCTTCCTGAAGAATTTCCTTAACTTGGTTCTAAGAGGAATAAACTCTTGGAA
GTTTTTGTCATAAAAGTCGTCCAATGCTCTTAAATATTTGGAATATGATCCAAGCCAGTCTACTGAAGGG
AAGTGCTTACGTTGGGCAAG (SEQ ID NO. 232). The predicted mature
engineered miRNA processed from the precursor sequence SEQ ID NO.
229 has the sequence UUUGUCCGUUUCUGCCAGAGA (SEQ ID NO. 233), which
has a Reynolds score of 6 and a functional asymmetry score of -3.2.
This gene suppression element is tested in Agrobacterium-mediated
transient assays in tobacco for expression of the engineered miRNA,
and then stably transformed into maize to test for efficacy in
controlling corn rootworm. These engineered miRNAs or miRNA
precursors can be included in various recombinant DNA constructs of
the invention, e.g., in a construct including the engineered miRNA
or miRNA precursor embedded within an intron flanked on one or on
both sides by non-protein-coding DNA, or in combination with a
miRNA recognition site, or with an aptamer.
Example 21
[0309] Current criteria for miRNA identification have emphasized
phylogenetic conservation of miRNAs across species, and thus few
non-conserved or species-specific miRNAs in plants have been
characterized in plants. This example describes identifying five
novel non-conserved miRNAs and the corresponding MIR sequences from
a size-fractionized cDNA library constructed from soybean leaves.
Criteria for miRNA identification included: (1) a cloned 21-nt
small RNA, and possible miRNA* (strand corresponding to the miRNA)
at a lower abundance, (2) containment of the miRNA/miRNA* duplex
wholly within a short, imperfect foldback structure, (3) derivation
of the miRNA from an RNA Pol II non-protein-coding transcript, and
(4) presence of a complementary target site in a coding gene; see
Ambros et al. (2003) RNA, 9: 277-279, which is incorporated by
reference herein.
[0310] Small RNAs were extracted from adaptor-containing raw
sequences and their strands were determined. This sequence set was
filtered to remove small RNA sequences that were virus, tRNA, rRNA,
chloroplast and mitochondria RNAs, and transgene, resulting in a
filtered set of 381,633 putative miRNA sequences. Small RNAs not
originating from the above sources and not homologous to known
miRNAs were mapped to reference soybean cDNA sequences. For the
mapped cDNA sequences with low protein-coding content, a cDNA
sequence fragment of about 250 nucleotides, containing the putative
miRNA, was folded using RNA Folder. The foldback structure was
examined to check if the small RNA was located in the stem, and if
an extensively (but not perfectly) complementary small RNA with
lower abundance was located in the opposite side of the stem. The
potential targets of the small RNA are predicted based on rules
modified from Jones-Rhoades and Bartel (2004) Mol. Cell,
14:787-799, and Zhang (2005) Nucleic Acids Res., 33:W701-704, which
are incorporated by reference herein. Table 10 lists the five novel
non-conserved miRNAs cloned from soy leaf tissue, and for each the
corresponding miRNA* and precursor pri-miRNA(s); abundance
("abund") is given as the number of times the sequence occurred in
a total of 381,633 sequences. TABLE-US-00010 TABLE 10 miRNA miRNA*
miRNA SEQ SEQ precursor ID ID SEQ ID NO. sequence abund NO.
sequence abund NO. 234 UGAGACCAAAUGAGCAGCUGA 94123 235
GCUGCUCAUCUGUUCUCAGG 26 236 237 UAGAAGCUCCCCAUGUUCUCA 7259 238
GAGCAUGGGUAACUUCUAU 24 239 240 UGUUGCGGGUAUCUUUGCCUC 4127 241
GGCGUAGAUCCCCACAACAG 9 242 243 UGCGAGUGUCUUCGCCUCUGA 3778 244
GGAGGCGUAGAUACUCACACC 70 245 246 UUGCCGAUUCCACCGAUUCCUA 3733 247
GCUGCUCAUCUGUUCUCAGG 93 248, 249
[0311] For each novel soy miRNA, the fold-back structure of the
miRNA precursor sequence(s) was predicted by an algorithm
("RNAFolder", based on RNAfold, publicly available at
www.tbi.univie.ac.at/.about.ivo/RNA/RNAfold.html), and the miRNA
precursor transcription profile obtained when available, as listed
in Table 11. Examples of predicted targets (recognition sites) in
soybean and their expression pattern identified were identified for
two of the miRNAs (SEQ ID NO: 234 and SEQ ID NO. 237).
TABLE-US-00011 TABLE 11 miRNA predicted predicted precursor G. max
target target miRNA miRNA miRNA precursor transcription
(recognition site) expression SEQ ID NO. precursor fold-back
profile sequence pattern 234 236 see FIG. 20A see FIG. 20B
polyphenol oxidase see FIG. (SEQ ID NO. 250) 20C 237 239 see FIG.
21A -- polyphenol oxidase see FIG. (SEQ ID NO. 251) 21B 240 242 see
FIG. 22 -- -- -- 243 245 see FIG. 23 -- -- -- 246 248, 249 see FIG.
24A see FIG. 24B -- --
[0312] In addition, target (recognition site) sequences for each
novel soy miRNA were identified from in-house ("MRTC") soy
databases, as listed in Table 12. TABLE-US-00012 TABLE 12 miRNA SEQ
ID NO. 234 Glycine max target Location of miRNA sequence (3'
.fwdarw. 5') (recognition target AGUCGACGAGUAAACCAGAGU site) SEQ ID
(recognition target (recognition site) NO. MRTC designation site)
sequence score mismatch 252 MRT3847_253879C.2 153-173
ucagcugcucaucuguucuca 2.5 2 253 MRT3847_54392C.5 402-422
ccagcugcucauuuggucacu 2.5 3 254 MRT3847_41382C.3 118-138
ucagcucuucuuuuggucucu 2.5 4 255 MRT3847_319840C.1 408-428
ucagcuacugaucuggucuca 3 3 256 MRT3847_326146C.1 117-137
ucagcuguuccuuuguucucu 3 4 257 MRT3847_39543C.6 768-788
ucagcuguuccuuuguucucu 3 4 258 MRT3847_253942C.4 1837-1857
guagcuucucacuuggucuua 3 5 259 MRT3847_260486C.4 124-144
uuagcugcuucuucggucucu 3 5 260 MRT3847_210520C.2 357-377
uuagaugcuuguuuggucuuu 3 6 miRNA SEQ ID NO. 237 Glycine max target
Location of miRNA sequence (3' .fwdarw. 5') (recognition target
ACUCUUGUACCCCUCGAAGAU site) SEQ ID (recognition target (recognition
site) NO. MRTC designation site) sequence score mismatch 261
MRT3847_303349C.1 435-455 ugagaacauggggagccucua 1.5 1 262
MRT3847_14593C.6 1133-1153 agaggacauggggagauucua 2 3 263
MRT3847_241913C.3 1111-1131 agaggacauggggagguucua 2 3 264
MRT3847_32439C.4 1142-1162 ugagaacaugggaaucuucua 2.5 2 265
MRT3847_187197C.5 689-709 aaagaacauggggagccucua 2.5 3 266
MRT3847_33448C.5 1047-1067 ugagaacaugggggauuucua 2.5 3 267
MRT3847_39693C.6 305-325 ugugaagguggggagcuucuu 2.5 4 268
MRT3847_50432C.5 89-109 ggagaacaugcagagcuucug 2.5 4 269
MRT3847_95417C.1 308-328 ugagaaacuggggagcuuuuc 2.5 4 270
MRT3847_115705C.2 82-101 ugagaac-uggugagcuucug 3 3 271
MRT3847_182667C.1 143-162 ugaguac-uggggagcuucuc 3 3 272
MRT3847_184995C.1 16-36 ugagagcauggguaacuucua 3 3 273
MRT3847_253437C.4 141-160 ugagcac-uggggagcuucuc 3 3 274
MRT3847_293395C.2 294-313 ugagcac-uggggagcuucuc 3 3 275
MRT3847_63512C.6 321-340 ugagcac-uggggagcuucuc 3 3 276
MRT3847_64829C.6 1087-1107 ugagaacaugggaacuuucua 3 3 277
MRT3847_80470C.3 15-35 ugagagcauggguaacuucua 3 3 278
MRT3847_136444C.5 312-332 ugagaaccugguaagcuucug 3 4 279
MRT3847_231576C.1 360-380 ugagaacaucgaaagcuucuu 3 4 280
MRT3847_263317C.1 90-110 ugaggacaaggggagcuuaug 3 4 281
MRT3847_304409C.1 217-237 cuaaaacauggggagcuucuu 3 4 282
MRT3847_247682C.3 1287-1307 ugaggaaauagggaguuucug 3 5 283
MRT3847_251048C.2 280-300 ugagaacauagugaguuuuuu 3 5 284
MRT3847_270705C.2 575-595 uaggaucguggggagcuucuc 3 5 285
MRT3847_304509C.2 592-612 uaggaucguggggagcuucuc 3 5 286
MRT3847_62576C.4 540-560 uaggaucguggggagcuucuc 3 5 287
MRT3847_67153C.3 661-681 gaugaauauggggaguuucua 3 5 miRNA SEQ ID NO.
240 Glycine max target Location of miRNA sequence (3' .fwdarw. 5')
(recognition target CUCCGUUUCUAUGGGCGUUGU site) SEQ ID (recognition
target (recognition site) NO. MRTC designation site) sequence score
mismatch 288 MRT3847_106868C.2 318-338 ggggcaaggacauccgcaacg 2.5 5
289 MRT3847_307036C.1 171-191 aaggcaaaguugcccgcgacg 2.5 5 290
MRT3847_308816C.2 719-739 gaggcaaagaugcgagcaacg 3 4 291
MRT3847_6248C.3 584-604 gcggcaaagauacucacaacc 3 4 292
MRT3847_104943C.2 177-197 aacgcaaagagaccuguaaca 3 5 293
MRT3847_290510C.2 181-201 aaggcaaagaugccagcgacg 3 5 294
MRT3847_294184C.2 1090-1110 gagccaaagagacccgugacg 3 5 295
MRT3847_321797C.1 847-867 aaggcauagauagucgcagca 3 5 296
MRT3847_63653C.5 1096-1116 aaggcaaagaugccagcaaug 3 5 297
MRT3847_9362C.2 481-501 uagggaaagauacauguaaca 3 5 298
MRT3847_112761C.3 331-351 gaggcaaaguuguucgcaaug 3 6 299
MRT3847_249731C.3 515-535 caggcaaagaugucugcaauu 3 6 300
MRT3847_313052C.1 253-273 uagguauggauacuugcaaca 3 6 301
MRT3847_318082C.1 123-143 aaggcaaagcugcccgcgaug 3 6 miRNA SEQ ID
NO. 243 Glycine max target Location of miRNA sequence (3' .fwdarw.
5') (recognition target AGUCUCCGCUUCUGUGAGCGU site) SEQ ID
(recognition target (recognition site) NO. MRTC designation site)
sequence score mismatch 302 MRT3847_160536C.3 182-202
ucaggggaggagacacucgca 2 3 303 MRT3847_290017C.2 304-324
uuagaggcaaagacacucguc 2 4 304 MRT3847_97323C.1 55-75
ucagaggagaagauacucgug 2 4 305 MRT3847_182887C.1 43-63
ucagaggagaagacacgcgca 2.5 2 306 MRT3847_290275C.2 177-197
ucagaggggaagacacacgcu 2.5 3 307 MRT3847_296312C.2 155-175
ucagaggggaagacacacgcu 2.5 3 308 MRT3847_292252C.2 171-191
ucagaggugaggacacacgcu 2.5 4 309 MRT3847_206250C.1 306-326
ccagaggcggaugcauucgca 2.5 5 310 MRT3847_240825C.3 436-456
acagaggcagggacacuugca 2.5 5 311 MRT3847_250458C.2 776-796
gcagaggugaagaagcuugca 2.5 5 312 MRT3847_36461C.4 87-107
uuagaggagaggauacucgcg 2.5 5 313 MRT3847_48749C.4 715-735
gcagaggugaagaagcuugca 2.5 5 314 MRT3847_97362C.3 566-586
ucagaggcaaagauacccgca 3 3 315 MRT3847_20647C.2 143-163
uuagaggggaagacacgcgcu 3 4 316 MRT3847_219382C.1 147-167
ucagaggggaagacacccgug 3 4 317 MRT3847_243196C.3 73-93
ucagaggcuaagagacuugua 3 4 318 MRT3847_248880C.3 760-780
ucagaggggaagacacgcgug 3 4 319 MRT3847_25201C.4 173-193
ucagaggggaagacacccgug 3 4 320 MRT3847_264555C.4 212-232
ucagaggggaagacacacguu 3 4 321 MRT3847_28447C.6 142-162
ucagaggggaagacacacguu 3 4 322 MRT3847_32431C.4 59-79
ucaggggugaagacacacgua 3 4 323 MRT3847_99342C.1 116-136
ucagaggggaagacacccgug 3 4 324 MRT3847_210811C.2 273-293
ucagaaacgaagacgcucguu 3 5 325 MRT3847_240622C.2 92-112
uccgaggggaagauacucguu 3 5 326 MRT3847_254863C.2 175-195
uccgaggggaagauacucguc 3 5 327 MRT3847_255345C.3 113-133
uccgaggggaagauacucguc 3 5 328 MRT3847_257424C.1 378-398
gcagaggcuguggcacucgca 3 5 329 MRT3847_38012C.4 56-76
uuagaggcgaggacacacguu 3 5 330 MRT3847_6951C.6 306-326
uccgaggagaagauacucguu 3 5 331 MRT3847_263266C.4 163-183
ucaguggcgaaggcguucguc 3 6 332 MRT3847_272810C.2 502-522
uuagaggugauggcacucgug 3 6 miRNA SEQ ID NO. 246 Glycine max target
Location of miRNA sequence (3' .fwdarw. 5') (recognition target
AUCCUUACCCACCUUAGCCGUU site) SEQ ID (recognition target
(recognition site) NO. MRTC designation site) sequence score
mismatch 333 MRT3847_302750C.1 259-280 ggggaauggguggaaacggcaa 1.5 3
334 MRT3847_136115C.3 661-682 ugggaaugggugggauggguaa 2.5 4 335
MRT3847_235247C.2 694-715 ugggaaugggugggauggguaa 2.5 4 336
MRT3847_21031C.3 1364-1385 auggaacugguggaauuggcaa 2.5 5 337
MRT3847_297070C.2 280-301 cgggaaagguuggaauuggcaa 2.5 5 338
MRT3847_248343C.3 392-413 uaggaauggguggauuuugcaa 3 3 339
MRT3847_207469C.2 1-20 ggaauggguggcgugggcaa 3 5 340
MRT3847_216295C.4 537-558 caggaaaggggggaguuggcaa 3 5 341
MRT3847_287795C.2 141-162 uagcaauggguuggaucgguga 3 5 342
MRT3847_302511C.2 35-56 guugaauggguggaauuggaaa 3 5 343
MRT3847_312620C.1 46-67 guugaauggguggaauuggaaa 3 5 344
MRT3847_20416C.2 679-700 aaggaauugggggaauugguac 3 6 345
MRT3847_297209C.1 289-310 cacgaguggggggaaucggcgg 3 6 346
MRT3847_6639C.4 195-216 guggaauggguggucuugguaa 3 6
Example 22
[0313] This example describes a recombinant DNA construct of the
invention, including a promoter, a terminator, transcribable
sequence between the promoter and the terminator, and at least one
gene suppression element that is 3' to the terminator. More
specifically, this example demonstrates that a gene suppression
element 3' to a terminator was transcribed and silenced a target
gene in a plant cell.
[0314] Most expression cassettes include both a promoter and a
terminator (i.e., a genetic element containing sequences necessary
for polyadenylation of the primary transcript), between which is
contained the sequence(s) to be expressed in a cell. Nonetheless,
it is likely that the primary transcript extends beyond the
terminator element. In plants, it is believed that transcription
continues some distance beyond the polyadenylation signal and site.
In one of the few studies to examine transcription termination in
plants, transcripts terminated downstream of the polyA site by as
much as 300 bp; no single transcriptional termination sites were
found, but rather a series of potential termination sites that
corresponded with T-rich sequences; see Hasegawa et al (2003) Plant
J, 33:1063-1072. It is believed that polyadenylation pathway genes
are conserved from animals to plants; see Yao et al. (2002) J. Exp.
Bot., 53:2277-2278. Plant mRNAs analogous sequences are found in
positions similar to those of animal AAUAAA and U-rish sequences,
suggesting an equivalent regulatory mechanisms in plants; see
Graber et al. (1999) Proc. Natl. Acad. Sci. USA, 96:14055-14060. In
yeast and animals, transcripts have been shown to extend over 1
kilobase downstream of the polyadenylation signal and site; see
Proudfoot (2004) Curr. Opin. Cell Biol., 16:272-278. The 3' end of
a mature RNA transcript is formed by cleavage and polyadenylation
at the polyA site. Although the primary transcript extends well
beyond the polyA site, most current models for transcriptional
termination invoke a coupling between polyadenylation and
termination; see Proudfoot (2004) Curr. Opin. Cell Biol.,
16:272-278. For example, some evidence indicates that the presence
of PolII "pause sites" downstream of the polyadenylation site.
Removal of such pause sites is expected to allow transcription to
extend even further downstream of the polyadenylation site. Thus, a
single RNA transcript can be used to both express a gene (with
sequence upstream of the terminator) and suppress a gene (with RNA
downstream of the terminator), and furthermore allows the
expression and suppression to be temporally and spatially coupled.
In one non-limiting example, the coordinated expression of a
bacterial cordapA gene and suppression of the endogenous LKR-SDH
gene has been shown to result in elevation of lysine levels in the
maize kernel. Another example is the expression in a transgenic
plant of a gene encoding a Bacillus thuringiensis insecticidal
protein and the production of dsRNA targetting an essential corn
rootworm (CRW) gene, the combination of which provides enhanced
control of CRW.
[0315] Various non-limiting embodiments are depicted in FIG. 25,
where gene suppression elements can be any of those disclosed
herein, e.g., the gene suppression elements depicted in FIG. 8, as
well as aptamers or riboswitches. In one embodiment, an inverted
repeat of at least 21 base pairs is positioned 3' to a terminator,
e.g., downstream of a typical gene expression cassette that
includes a promoter, a sequence to be expressed, and a terminator.
In other embodiments, tandem repeats of anti-sense or sense
sequence of the target gene are used as the gene suppression
element. In some embodiments, the gene suppression element is
embedded in an intron directly or substantially directly 3' to the
terminator. The downstream sequence can contain a gene suppression
element designed to be processed by a trans-acting siRNA mechanism,
e.g., sequences corresponding to a target gene fused to a miRNA
target sequence, such that miRNA-triggered dsRNA production occurs
resulting in silencing of a target gene. A second terminator can be
included as shown in FIG. 26A, or can be omitted, as the absence of
a polyadenylation signal downstream of a gene suppression element
does not reduce suppression efficiency (see Example 1) and can
enhance it. Where two terminators are included, it is preferable
that the two terminators be unrelated to reduce the possibility of
recombination between them.
[0316] The constructs depicted in FIG. 26A (suppression construct)
and FIG. 26B (control construct) were tested in a maize protoplast
assay as described in Examples 1 and 2. Firefly luciferase
suppression experiments were performed, and the target gene,
firefly luciferase, was suppressed by an inverted repeat 3' to the
terminator, as indicated by the logarithm of the ratio of firefly
luciferase to Renilla luciferase, "log(Fluc/Rluc)", as depicted in
FIG. 26C.
Example 23
[0317] This non-limiting example illustrates the transgenic plants
of the invention, which have in their genome recombinant DNA
including transcribable DNA including DNA that transcribes to an
RNA aptamer capable of binding to a ligand. One application of the
invention is to provide a ligand-activated, herbicide-resistant
system for gene identity preservation ("gene lock") as well as to
maintain herbicide-resistant volunteer control.
[0318] In one embodiment, the DNA sequence encoding an "on"
riboswitch is inserted into an expression cassette containing as
the target sequence "CP4", a selectable marker conferring
glyphosate resistance, epsps-cp4
(5-enolpyruvylshikimate-3-phosphate synthase from Agrobacterium
tumefaciens strain CP4), to conditionally express CP4 in transgenic
plants. See the construct depicted in FIG. 28A, where CP4 is the
target sequence ("TS"), and FIG. 28F, which depicts a non-limiting
example of a CP4 expression cassette useful for
Agrobacterium-mediated transformation of maize and other crop
plants, and the expected "ligand A"-controlled CP4 expression.
Transgenic plants harboring the riboswitch-controlled CP4 cassette
express CP4 only in the presence of the ligand, which is applied
(e.g., by a foliar spray) to the plant by means of a proprietary
glyphosate formulation containing the ligand. Upon application, the
formulated glyphosate herbicide activates CP4
transcription/translation and renders the transgenic plant
resistant to glyphosate. Transgenic plants are susceptible to
generic glyphosate formulations that do not contain the ligand.
Similarly, this approach can be applied to any other
herbicide-resistance gene/herbicide combinations, for example,
dicamba-degrading-oxygenase/dicamba, or antibiotic-resistance
gene/antibiotic combination.
[0319] Ligand-activated herbicide resistance riboswitches allow
formulation of crop-specific herbicides, by using a riboswitch that
binds to a different ligand for selected plant species. For
example, where an adenine-binding riboswitch is used for soybeans
and a lysine-binding riboswitch is used for corn, a
lysine-containing glyphosate formulation will control
non-transgenic weeds as well as glyphosate-resistant soybean
volunteers (e.g., from a previous crop).
[0320] In another embodiment, an autoinduced riboswitch is used to
treat seeds. If the residual herbicide lasts longer than the ligand
in plant tissues after the ligand-containing herbicide formulation
is applied, it could cause crop damage due to the shut down of the
herbicide resistance gene. One approach to prevent this is to
choose a ligand that is an endogenously produced metabolite and to
include a mechanism for the ligand's production with the
riboswitch. This makes it possible to engineer an autoinduced
riboswitch to maintain expression of the herbicide resistance gene.
Using a lysine-autoinduced riboswitch for glyphosate resistance as
an example (FIG. 28C), the addition of a second gene,
Corynebacterium DHDPS or cordapA ("dapA") (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), maintains a
persistent lysine level sufficient to maintain expression of both
CP4 and dapA. This autoinduced system also allows the ligand to be
applied by seed treatment as an alternative to including the ligand
in the glyphosate formulation. Untreated seeds are viable but
require treatment with the appropriate ligand prior to planting in
order for the resulting plants to be resistant to the
herbicide.
Example 24
[0321] This non-limiting example further illustrates the transgenic
plants of the invention. One embodiment of the invention is to use
an herbicide such as glyphosate as a chemical hybridization agent.
This embodiment entails transgenic plants having lower CP4
expression in male tissues relative to the rest of plants, whereby,
when the transgenic plants are exposed to glyphosate, male
sterility ensues. One approach is to combine a transcriptional
control riboswitch with tissue specific control of expression of
that riboswitch. An example is depicted in FIG. 28E, where
"Promoter1" is a constitutive promoter driving expression of the
target gene ("TS") CP4, and "Promoter 2" is a male-specific
promoter driving lysine-induced, riboswitch controlled expression
of a gene suppression construct for suppressing CP4 ("TS.sub.sup").
Application of lysine and glyphosate (e.g. as a spray) results in
male sterility. Alternatively, using the construct shown in FIG.
28D, where "Promoter I" is constitutive, "Promoter 2" is
male-specific, and the target gene ("TS") is CP4, initial lysine
application reduces overall CP4 expression, but CP4 expression is
enhanced in male tissues, thereby causing male tissues to be more
susceptible to glyphosate.
Example 25
[0322] This non-limiting example further illustrates the transgenic
plants of the invention. One embodiment of the invention is induced
expression of a trait gene under the control of a constitutive
promoter. The insertion of a riboswitch allows the trait genes,
though under the control of a constitutive promoter, to be
expressed only upon selected conditions. This makes it possible to
avoid yield penalty (e.g., loss of yield due to non-selective
expression of the trait gene), transgene silencing, or other
concerns caused by constitutive expression of the transgenes.
Non-limiting examples of such riboswitches include a glyphosate
"on" riboswitch for CP4 expression, a salicylic acid "on"
riboswitch for disease resistance genes, a jasmonic acid "on"
riboswitch for insect resistance genes, an ascorbate "on"
riboswitch for oxidative stress tolerance genes, and a proline or
glycine betaine or mannitol riboswitch for drought tolerance
genes.
Example 26
[0323] This example further illustrates the transgenic plants of
the invention. One embodiment is chemically inducible or
suppressible male sterility or fertility for hybridization.
Preferred examples use a riboswitch containing an aptamer that
binds a ligand that is an already registered substance, e.g., an
approved herbicide. In a non-limiting example, a transgenic plant
harboring a male sterility gene under the control of a
male-specific promoter and a glyphosate "off" riboswitch is
male-sterile unless glyphosate is applied. In contrast, a
transgenic plant harboring a male sterility gene under the control
of a male-specific promoter and a glyphosate "on" riboswitch is
male-sterile only when glyphosate is applied.
Example 27
[0324] This non-limiting example further illustrates the transgenic
plants of the invention. One embodiment of the invention includes
artificial riboswitches that are engineered in vitro to permit
expression (or suppression) of a target sequence under inducible
conditions or in response to biotic or abiotic stress. Such
riboswitches use novel aptamers designed for a specific ligand by
means well known in the art. See, for example, the detailed
discussion above under the heading "Aptamers". Especially useful
riboswitches are designed to be triggered by registered
agricultural chemicals (e.g., glyphosate, dicamba), disease-induced
compounds (e.g., salicylic acid), invertebrate pest-induced or
wounding-induced compounds (e.g., jasmonic acid), water
stress-induced compounds (e.g., proline, glycine betaine, mannitol)
and oxidative stress-induced compounds (e.g., ascorbate).
Example 28
[0325] This non-limiting example further illustrates the transgenic
plants of the invention. Riboswitches useful in transgenic plants
of the invention are designed to function at a given concentration
of the ligand. One embodiment is a lysine riboswitch engineered to
function in a transgenic plant.
[0326] Naturally occurring bacterial lysine riboswitches exist as
both "on" and "off" riboswitches, and have a K.sub.d.about.1
millimolar (128 ppm) in vitro (see Sudarsan et al. (2003) Genes
Dev., 17:2688-2697, which provides individual and consensus
sequences of prokaryotic lysine-responsive riboswitches, and is
incorporated by reference). However, maize tissues generally have a
lysine content of less than 50 ppm, which is thus a concentration
useful as the default state for novel lysine riboswitches. Using
bacterial lysine riboswitches as an example, a series of constructs
(FIG. 28) is transformed into maize callus, producing transgenic
maize callus lines or transgenic maize plants useful for studying
riboswitch efficacy in plants and plant cells. In some embodiments,
a non-lysine-feedback-inhibited lysine biosynthetic gene, cordapA,
is co-expressed in order to obtain autoinducible control of gene
expression. As shown in FIG. 28A and FIG. 28B, transcribable DNA
fragments of .about.150 base pairs, encoding lysine "on" (or "off")
riboswitches, are inserted between the promoter and the target
sequence ("TS"), in this example a green fluorescent protein (GFP)
reporter gene. Other reporter genes or marker genes, as well as any
gene of interest, can be used as the target sequence. The callus
lines transformed with these constructs display a lysine inducible
(or lysine-suppressible) GFP expression phenotype (FIG. 29, top
panel, A and B). In some embodiments, a second cassette containing
cordapA ("dapA") under the control of a lysine "on" riboswitch, is
added (FIG. 28C and FIG. 28D); these callus lines or transgenic
plants become autoinducible or autosupressible (FIG. 29, top panel,
C and D). FIG. 29, lower panel, schematically depicts an expression
cassette, useful in Agrobacterium-mediated transformation of maize
and other plants, containing a lysine "on" riboswitch, whereby
binding of lysine to the aptamer of the riboswitch induces
expression of CP4 (for glyphosate resistance) as well as expression
of Corynebacterium DHDPS or cordapA ("DHDPS"). The resulting
endogenous synthesis of additional lysine maintains expression of
the transgenes.
Example 29
[0327] This example further illustrates the transgenic plants of
the invention. One preferred embodiment is a transgenic plant
including in its genome transcribable DNA that transcribes to a
"trans"-acting riboswitch, i.e., a riboswitch that affects
expression of a target sequence to which it is not operably
linked.
[0328] In some embodiments, the "trans" riboswitch is flanked by
ribozymes (e.g., self-splicing or hammerhead ribozymes) and is
transcribed under the control of a pol II promoter (FIG. 27A); see,
for example, Bayer and Smolke (2005) Nature Biotechnol.,
23:337-343, which is incorporated by reference. In other
embodiments, the "trans" riboswitch is transcribed under the
control of a pol III promoter (FIG. 27B), whereby transcription is
terminated at a poly-T region. In other embodiments, the "trans"
riboswitch is flanked by intron-splicing junctions (FIG. 27C),
whereby the riboswitch is spliced out after transcription; such
embodiments can optionally include DNA that transcribes to a
microRNA recognition site or DNA that transcribes to RNA capable of
forming double-stranded RNA (dsRNA) (FIG. 27D). Embodiments
containing intron-embedded transcribable DNA can optionally include
one or more gene expression (or suppression) elements ("GOI" in
FIG. 27C and FIG. 27D). Alternatively, the transcribed riboswitch
can be flanked by double-stranded RNA that can be cleaved through
an RNAi (siRNA or miRNA) processing mechanism (FIG. 27E). In yet
other embodiments, the "trans" riboswitch is flanked by DNA that
transcribes to a microRNA recognition site (FIG. 27E), whereby
cleavage of the transcribed riboswitch occurs after binding of the
corresponding mature miRNA to the miRNA recognition site. These
approaches enable the creation of noncoding riboregulators with
defined 5' and 3' ends that are free of potentially interfering
flanking sequences. In still other embodiments, the "trans"
riboswitch is flanked by DNA that transcribes to RNA capable of
forming double-stranded RNA (dsRNA) (FIG. 27E). In some of these
cases, the dsRNA is processed by an RNAi (siRNA or miRNA)
mechanism, whereby the transcribed riboswitch is cleaved from the
rest of the transcript. In other cases, the two transcribed RNA
regions flanking the "trans" riboswitch form at least partially
double-stranded RNA "stem" between themselves, wherein the "trans"
riboswitch serves as a "spacer" or "loop" in a stem-loop
structure.
[0329] In one example, the transgenic plant has in its genome an
expression cassette using pol II promoters to express a "trans"
riboswitch flanked by self-cleaving hammerhead ribozyme sequences,
resulting in a riboswitch with defined 5' and 3' ends, free of
potentially interfering flanking sequences (FIG. 27A; also see
Bayer and Smolke (2005) Nature Biotechnol., 23:337-343). An
alternative approach uses expression cassettes under the control of
pol III promoters to produce non-coding RNAs with minimal 5' and 3'
flanking sequences; RNA polymerase II transcribes structural or
catalytic RNAs that are usually shorter than 400 nucleotides in
length, and recognizes a simple run of T residues as a termination
signal; it has been used to transcribe siRNA duplexes (Lu et al.
(2004) Nucleic Acids Res., 32:e171, which is incorporated by
reference). Riboregulators expressed by Pol III are expected to
generate transcripts with relatively defined 5' and 3' ends (FIG.
27B). It has been used to transcribe siRNA duplexes. Alternatively,
a "trans" riboswitch is fused to the minimal sequences required for
splicing, and endogenous intron splicing mechanisms are used to
release the riboregulator (FIG. 27C). This intron-embedded
configuration provides the advantage of allowing concurrent
expression of a gene of interest (GOI).
[0330] One specific application of "trans" riboswitch is their use
in generating transgenic plants with inducible male sterility or
fertility. Hybrid plant varieties have a significant yield
advantage over their inbred counterparts, but can be more costly to
produce. Reversible male sterility/fertility is one of the most
cost-effective ways to produce hybrids. In this application,
"trans" riboswitches are designed to target endogenous genes
required for male development. Suppression of any of these genes
results in male sterility. "Trans" riboswitches driven by
male-specific pol II promoters (FIG. 27A) can be used to control
the expression of any target sequence or gene that leads to cell
death (apoptosis) or growth arrest. Alternatively "trans"
riboswitches transcribed under the control of pol III promoters
(FIG. 27B), which are constitutive, are designed to be male
specific to avoid undesirable phenotypes. In an inducible male
fertility system, the "trans" riboswitch used is an "off" switch
(where the riboswitch is bound to its target sequence by default
and is released from the target sequence when bound by ligand),
supply of the ligand (by endogenous biosynthesis or exogenous
application, e.g., by spraying) restores fertility. In an inducible
male sterility system, the "trans" riboswitch used is an "on"
switch, and binding of the ligand results in the "trans" riboswitch
binding to its target sequence and inducing male sterility.
[0331] Another specific application of "trans" riboswitches is
their use in generating transgenic plants displaying "gene lock".
Seeds containing an "off" "trans" riboswitch designed to target
endogenous genes required for germination will not be able to
germinate. When under the control of a pol II promoter (FIG. 1A),
any gene functioning in cell death or growth arrest can be
targetted. Alternatively Pol III driven "trans" riboswitches (FIG.
1B) would have to target genes that are specific to germination to
avoid undesirable phenotypes. The germination restoration could be
seed treatment and illegally copied seeds without seed treatment
would not be able to germinated.
[0332] "Trans" riboswitches, similarly to the "cis" riboswitches
described in Examples 23 through 2, are useful in regulating
transgenes. In a specific example, a transgenic plant including a
"trans" riboswitch designed to regulate the glyphosate-resistance
transgene CP4 as the target sequence is particularly useful in
"trans" riboswitch-controlled applications parallel to that
described in Example 23 (glyphosate as a ligand for
ligand-activated herbicide resistance, or for control of herbicide
resistant volunteers) and Example 24 (glyphosate as a chemical
hybridization agent). To illustrate this approach, CP4 expression
is suppressed in stably transformed maize callus. A modified
transcribable DNA encoding an "off" "trans" riboswitch with
theophylline as its ligand (Bayer and Smolke (2005) Nature
Biotechnol., 23:337-343) is designed to target CP4 as a target
sequence. Transcription of the theophylline riboswitch can be
driven either by a pol II promoter (e.g., FIG. 27A) or by pol III
promoter (e.g., FIG. 27B). The transcribable DNA is inserted into a
binary vector (FIG. 30) and co-transformed into maize callus under
nptIII selection, generating stably transformed maize callus lines.
CP4 expression is assayed in the transformed cells, where CP4
expression is observed to be suppressed in transformed cells that
are treated with theophylline.
[0333] Similarly, a "trans" riboswitch is used to control
expression of an endogenous target sequence (lysine ketoglutarate
reductase/saccharopine dehydrogenase gene, LKR/SDH) in stably
transformed maize plants. A modified transcribable DNA encoding an
"off" "trans" riboswitch with theophylline as its ligand (Bayer and
Smolke (2005) Nature Biotechnol., 23:337-343) is designed to target
at least one region of the LKR/SDH sequence, and co-transformed
into maize callus. LKR/SDH expression is assayed in the resulting
transformed cells, where LKR/SDH expression is observed to be
suppressed in transformed cells that are treated with
theophylline.
[0334] All of the materials and methods disclosed and claimed
herein can be made and used without undue experimentation as
instructed by the above disclosure. Although the materials and
methods of this invention have been described in terms of preferred
embodiments and illustrative examples, it will be apparent to those
of skill in the art that variations can be applied to the materials
and methods described herein without departing from the concept,
spirit and scope of the invention. All such similar substitutes and
modifications apparent to those skilled in the art are deemed to be
within the spirit, scope and concept of the invention as defined by
the appended claims.
Sequence CWU 1
1
346 1 2767 DNA Artificial sequence Synthetic construct 1 ggtccgatgt
gagacttttc aacaaagggt aatatccgga aacctcctcg gattccattg 60
cccagctatc tgtcacttta ttgtgaagat agtggaaaag gaaggtggct cctacaaatg
120 ccatcattgc gataaaggaa aggccatcgt tgaagatgcc tctgccgaca
gtggtcccaa 180 agatggaccc ccacccacga ggagcatcgt ggaaaaagaa
gacgttccaa ccacgtcttc 240 aaagcaagtg gattgatgtg atggtccgat
gtgagacttt tcaacaaagg gtaatatccg 300 gaaacctcct cggattccat
tgcccagcta tctgtcactt tattgtgaag atagtggaaa 360 aggaaggtgg
ctcctacaaa tgccatcatt gcgataaagg aaaggccatc gttgaagatg 420
cctctgccga cagtggtccc aaagatggac ccccacccac gaggagcatc gtggaaaaag
480 aagacgttcc aaccacgtct tcaaagcaag tggattgatg tgatatctcc
actgacgtaa 540 gggatgacgc acaatcccac tatccttcgc aagacccttc
ctctatataa ggaagttcat 600 ttcatttgga gaggacacgc tgacaagctg
actctagcag atctaccgtc ttcggtacgc 660 gctcactccg ccctctgcct
ttgttactgc cacgtttctc tgaatgctct cttgtgtggt 720 gattgctgag
agtggtttag ctggatctag aattacactc tgaaatcgtg ttctgcctgt 780
gctgattact tgccgtcctt tgtagcagca aaatataggg acatggtagt acgaaacgaa
840 gatagaacct acacagcaat acgagaaatg tgtaatttgg tgcttagcgg
tatttattta 900 agcacatgtt ggtgttatag ggcacttgga ttcagaagtt
tgctgttaat ttaggcacag 960 gcttcatact acatgggtca atagtatagg
gattcatatt ataggcgata ctataataat 1020 ttgttcgtct gcagagctta
ttatttgcca aaattagata ttcctattct gtttttgttt 1080 gtgtgctgtt
aaattgttaa cgcctgaagg aataaatata aatgacgaaa ttttgatgtt 1140
tatctctgct cctttattgt gaccataagt caagatcaga tgcacttgtt ttaaatattg
1200 ttgtctgaag aaataagtac tgacagtatt ttgatgcatt gatctgcttg
tttgttgtaa 1260 caaaatttaa aaataaagag tttccttttt gttgctctcc
ttacctcctg atggtatcta 1320 gtatctacca actgacacta tattgcttct
ctttacatac gtatcttgct cgatgccttc 1380 tccctagtgt tgaccagtgt
tactcacata gtctttgctc atttcattgt aatgcagata 1440 ccaagcggcc
atggcacacc cttaggtaac ccagtagatc cagaggaatt cattatcagt 1500
gcaattgttt tgtcacgatc aaaggactct ggtacaaaat cgtattcatt aaaaccggga
1560 ggtagatgag atgtgacgaa cgtgtacatc gactgaaatc cctggtaatc
cgttttagaa 1620 tccatgataa taattttctg gattattggt aatttttttt
gcacgttcaa aattttttgc 1680 aacccctttt tggaaacaaa cactacggta
ggctgcgaaa tgttcatact gttgagcaat 1740 tcacgttcat tataaatgtc
gttcgcgggc gcaactgcaa ctccgataaa taacgcgccc 1800 aacaccggca
taaagaattg aagagagttt tcactgcata cgacgattct gtgatttgta 1860
ttcagcccat atcgtttcat agcttctgcc aaccgaacgg acatttcgaa gtattccgcg
1920 tacgtgatgt tcacctcgat atgtgcatct gtaaaagcaa ttgttccagg
aaccagggcg 1980 tatctcttca tagccttatg cagttgctct ccagcggttc
catcctctag aggatagaat 2040 ggcgccgggc ctttctttat gtttttggcg
tcttcacgcg tcgatatggg ctgaatacaa 2100 atcacagaat cgtcgtatgc
agtgaaaact ctcttcaatt ctttatgccg gtgttgggcg 2160 cgttatttat
cggagttgca gttgcgcccg cgaacgacat ttataatgaa cgtgaattgc 2220
tcaacagtat gaacatttcg cagcctaccg tagtgtttgt ttccaaaaag gggttgcaaa
2280 aaattttgaa cgtgcaaaaa aaattaccaa taatccagaa aattattatc
atggattcta 2340 aaacggatta ccagggattt cagtcgatgt acacgttcgt
cacatctcat ctacctcccg 2400 gttttaatga atacgatttt gtaccagagt
cctttgatcg tgacaaaaca attgcactga 2460 taatgaattc ctctggatct
actgggttac ctaagggtgt gggatccaat tcccgatcgt 2520 tcaaacattt
ggcaataaag tttcttaaga ttgaatcctg ttgccggtct tgcgatgatt 2580
atcatataat ttctgttgaa ttacgttaag catgtaataa ttaacatgta atgcatgacg
2640 ttatttatga gatgggtttt tatgattaga gtcccgcaat tatacattta
atacgcgata 2700 gaaaacaaaa tatagcgcgc aaactaggat aaattatcgc
gcgcggtgtc atctatgtta 2760 ctagatc 2767 2 872 DNA Artificial
sequence Synthetic construct 2 gcaagtatgg cctgtacgtc aagcaaggcc
agtcagtgaa aaattacctg ccaaccatcc 60 tctgcttaca ggacagcgtg
tacttgatgc tcttttccca tgtgtacagg gtggtactac 120 tgccattccc
ggagctttcg gttgtggaaa aactgtaatt tcacaatctc tttccaaata 180
ttccaactct gatgtcatta tctacgtcgg ttgcggagaa agaggtaacg aaatgtctga
240 agtattgaga gatttccctg aattgactgt tgaaattgac gggcacactg
aatctattat 300 gaaacgtacc gcattggtcg ccaacacatc taacatgcct
gtagctgctc gtgaagcttc 360 tatctatact ggtattactc tttctgaata
cttccgtgat atgggttaca acgtatctat 420 gatggctgac tcgacatcac
gttgggccga agctttgaga gaaatttcag gtcgtttggc 480 tgaaatgcct
gccgattccg gttatccggc ttacttaggt gcccgtttgg cttccttcta 540
cgaacgtgct ggtcgcgtta aatgtttagg taatccagac agagaaggat ccgtttcaat
600 tgtaggagcc gtatcacctc ctggtggtga tttctcagat cctgttacca
ctgctactct 660 tggtattgta caggtgttct ggggtttgga caagaaactt
gcccaacgta agcacttccc 720 ttcagtagac tggcttggat catattccaa
atatttaaga gcattggacg acttttatga 780 caaaaacttc caagagttta
ttcctcttag aaccaaagtt aaggaaattc ttcaggaaga 840 agatgatcta
gccgaaattg tgcagctggt ag 872 3 1000 DNA Artificial sequence
Synthetic construct 3 ataatagatt cagtgtgccc gtcaatttca acagtcaatt
cagggaaatc tctcaatact 60 tcagacattt cgttacctct ttctccgcaa
ccgacgtaga taatgacatc agagttggaa 120 tatttggaaa gagattgtga
aattacagtt tttccacaac cgaaagctcc gggaatggca 180 gtagtaccac
cctgtacaca tgggaaaaga gcatcaagta cacgctgtcc tgtaagcaga 240
ggatggttgg caggtaattt ttcactgact ggccttgctt gacgtacagg ccatacttgc
300 tgtgtacagg gtggtactac tgccattccc ggagctttcg gttgtggaaa
aactgtaatt 360 tcacaatctc tttccaaata ttccaactct gatgtcatta
tctacgtcgg ttgcggagaa 420 agaggtaacg aaatgtctga agtattgaga
gatttccctg aattgactgt tgaaattgac 480 gggcacactg aatctattat
gaaacgtacc gcattggtcg ccaacacatc taacatgcct 540 gtagctgctc
gtgaagcttc tatctatact ggtattactc tttctgaata cttccgtgat 600
atgggttaca acgtatctat gatggctgac tcgacatcac gttgggccga agctttgaga
660 gaaatttcag gtcgtttggc tgaaatgcct gccgattccg gttatccggc
ttacttaggt 720 gcccgtttgg cttccttcta cgaacgtgct ggtcgcgtta
aatgtttagg taatccagac 780 agagaaggat ccgtttcaat cggaatcggc
aggcatttca gccaaacgac ctgaaatttc 840 tctcaaagct tcggcccaac
gtgatgtcga gtcagccatc atagatacgt tgtaacccat 900 atcacggaag
tattcagaaa gagtaatacc agtatagata gaagcttcac gagcagctac 960
aggcatgtta gatgtgttgg cgaccaatgc ggtacgtttc 1000 4 22 DNA Zea mays
4 tgaagctgcc agcatgatct gg 22 5 240 DNA Oryza sativa 5 gaagatatta
gttcttgctg gtgtgagagg ctgaagctgc cagcatgatc tggtccatga 60
gttgcactgc tgaatatatt gaattcagcc aggagctgct actgcagttc tgatctcgat
120 ctgcattcgt tgttctgagc tatgtatgga tttgatcggt ttgaaggcat
ccatgtcttt 180 aatttcatcg atcagatcat gttgcagctt cactctctca
ctaccagcaa aaccatctca 240 6 759 DNA Zea mays 6 gttttggctt
gttcacccct catgtgcaca tgctgttact ccgaagcttg cgcttttgta 60
ttcgttgttg cattgcaacc atccccgccg aaggtgagcc gaaggtaatc ttgggtattc
120 tacctgcaac acttattaat tcaagctaca aaacagttgt cgagttagtt
ttttttttac 180 cttcgaaaag aagacttccg gcaatgcaca acttcccatc
tgcattatcg tgagcaggat 240 tgtaggcaca cagtgatgac gaagacagag
acagcaatat acacaaccga accaagagag 300 aagcaaaggc ataataataa
aaaaagagag aggaaactag atcgacaagg ccattattat 360 cacggataat
taatcaacgt cgtcaacggc ggaaataagc tagcttgact ggtggtctct 420
ggcgagtgca gcatggatat gaattgcagg agggtgagct agctagggtt ttcgatgtgc
480 ggccaccagc agatgaaact acagcatgac ctggtcctgg tgctcattaa
ttaccctctc 540 tctctctccc ttcccctctc atcttggatt cgtcgatcca
tatatgacag tcagggacgg 600 gggagagaga gagagtgaca ggggccggta
gtagtataga ttacatccat tttacatata 660 ccaccaccat cataaccaga
tcatgctggc agcttcacca actcgtggtg caccactaca 720 taccctctcg
tctgatccaa acggaggaag gaggaagaa 759 7 884 DNA Zea mays 7 ttggcttgtt
cacccctcat gtgcacatgc tgttactccg aagcttgcgc ttttgtattc 60
gttgttgcat tgcaaccatc cccgccgaag gtgagccgaa ggtaatcttg ggtattctac
120 ctgcaacact tattaattca agctacaaaa cagttgtcga gttagttttt
tttttacctt 180 cgaaaagaag acttccggca atgcacaact tcccatctgc
attatcgtga gcaggattgt 240 aggcacacag tgatgacgaa gacagagaca
gcaatataca caaccgaacc aagagagaag 300 caaaggcata ataataaaaa
aagagagagg aaactagatc gacaaggcca ttattatcac 360 ggataattaa
tcaacgtcgt caacggcgga aataagctag cttgactggt ggtctctggc 420
gagtgcagca tggatatgaa ttgcaggagg gtgagctagc tagggttttc gatgtgcggc
480 caccagcaga tgaaactaca gcatgacctg gtcctggtgc tcattaatta
ccctctctct 540 ctctcccttc ccctctcatc ttggattcgt cgatccatat
atgacagtca gggacggggg 600 agagagagag agtgacaggg gccggtagta
gtatagatta catccatctt acatatacca 660 ccaccatcat aaccagatca
tgctggcagc ttcaccaact cgtggtgcac cactacatac 720 cctctcgtct
gatccaaacg gaggaaggag gaagaagagc tagctatccg agagagaggg 780
agagggtaga gagatggaga gagcgaggaa tgaattgaag aaccgaggga tagctatagc
840 tatatatata tggggatggg gaggccaacg tctcgctcac tcgc 884 8 872 DNA
Zea mays 8 tattctacct gcaacactta ttaattcaag ctacaaaaca gttgtcgagt
tagttttttt 60 tttaccttcg aaaagaagac ttccggcaat gcacaacttc
ccatctgcat tatcgtgagc 120 aggattgtag gcacacagtg atgacgaaga
cagagacagc aatatacaca accgaaccaa 180 gagagaagca aaggcataat
aataaaaaaa gagagaggaa actagatcga caaggccatt 240 attatcacgg
ataattaatc aacgtcgtca acggcggaaa taagctagct tgactggtgg 300
tctctggcga gtgcagcatg gatatgaatt gcaggagggt gagctagcta gggttttcga
360 tgtgcggcca ccagcagatg aaactacagc atgacctggt cctggtgctc
attaattacc 420 ctctctctct ctcccttccc ctctcatctt ggattcgtcg
atccatatat gacagtcagg 480 gacgggggag agagagagag tgacaggggc
cggtagtagt atagattaca tccatcttac 540 atataccacc accatcataa
ccagatcatg ctggcagctt caccaactcg tggtgcacca 600 ctacataccc
tctcgtctga tccaaacgga ggaaggagga agaagagcta gctatccgag 660
agagagggag agggtagaga gatggagaga gcgaggaatg aattgaagaa ccgagggata
720 gctatagcta tatatatatg ggatggggag gccaacgtct cgctcactcg
cagcgtattt 780 tgatgccctt ttttatttgt tgcatttcga tccattttct
tttgtcctgc gcttttttcg 840 tacgatgttt gttgcaagga taagcctttc gg 872 9
988 DNA Zea mays 9 gttttggctt gttcacccct catgtgcaca tgctgttact
ccgaagcttg cgcttttgta 60 ttcgttgttg cattgcaacc atccccgccg
aaggtgagcc gaaggtaatc ttgggtattc 120 tacctgcaac acttattaat
tcaagctaca aaacagttgt cgagttagtt ttttttttac 180 cttcgaaaag
aagacttccg gcaatgcaca acttcccatc tgcattatcg tgagcaggat 240
tgtaggcaca cagtgatgac gaagacagag acagcaatat acacaaccga accaagagag
300 aagcaaaggc ataataataa aaaaagagag aggaaactag atcgacaagg
ccattattat 360 cacggataat taatcaacgt cgtcaacggc ggaaataagc
tagcttgact ggtggtctct 420 ggcgagtgca gcatggatat gaattgcagg
agggtgagct agctagggtt ttcgatgtgc 480 ggccaccagc agatgaaact
acagcatgac ctggtcctgg tgctcattaa ttaccctctc 540 tctctctccc
ttcccctctc atcttggatt cgtcgatcca tatatgacag tcagggacgg 600
gggagagaga gagagtgaca ggggccggta gtagtataga ttacatccat cttacatata
660 ccaccaccat cataaccaga tcatgctggc agcttcacca actcgtggtg
caccactaca 720 taccctctcg tctgatccaa acggaggaag gaggaagaag
agctagctat ccgagagaga 780 gggagagggt agagagatgg agagagcgag
gaatgaattg aagaaccgag ggatagctat 840 agctatatat atatggggat
ggggaggcca acgtctcgct cactcgcagc gtattttgat 900 gccctttttt
atttgttgca tttcgatcca ttttcttttg tcctgcgctt ttttcgtacg 960
atgtttgttg caaggataag cctttcgg 988 10 548 DNA Zea mays unsure
(1)..(548) unsure at all n locations 10 gtatgttctc cgctcactcc
cccattccac tctcatccat ctctcaagct acacacatat 60 aaaaaaaaaa
gagtagagaa ggaccgccgt tagagcactt gatgcatgcg tacgtcgatc 120
cggcggaccg atctgctttt gcttgtgtgc ttggtgagaa ggtccctgtt ggagaagcag
180 ggcacgtgca gagacacgcc ggagcacggc cgccgccgat ctaccgacct
cccacacctg 240 ccttgtggtg tgggggtgga ggtcnnnnnn cgnagcgaga
gctgncgntg ntgnttngat 300 gctgntngct cctcctgcnc gtgctcccct
tctccaccac ggccttctca ccaccctcct 360 cccccggcgg cggcggcggc
ggaccgccct tgccgcgatc aataatgaaa ccaaaagccg 420 acagtgtttg
agcaggaaac acaaaaggcg gatatcccac tgntagcact tctgcgttga 480
tcatggtcat ctggaacaaa ataatacttg gggactttac agcgagtgca gcatgcttaa
540 gctagttc 548 11 22 DNA Zea mays 11 tggagaagca gggcacgtgc ag 22
12 1219 DNA Zea mays 12 ttcggtccaa gtagtggtgg tcataatatg ctccaaataa
aagaaaggtg gaggagcatc 60 tcacagacga cacagctgct atgctagcac
acgtcgaatc aatagctagt tgcatgcaaa 120 gttccaaagc aaataaacag
tgagatcgaa agacgtttcg ctgttgcacg acacgacgaa 180 tcgatcgaac
gaaagtgtgt ttttatgatt ccacagattc tcgtttatat ataatcctag 240
ctagctaatc tagaacgtac agtgcacacc atcttcttcc acagatcaca gaaagacagc
300 agaaacctgc atggatcgga tccggtcctg tcctgtaaga tctacacaca
tgcaaagcaa 360 atcaatttct tccttttctt ttcttcagaa actgggataa
ctttttggaa gagatcgaac 420 agtatataga ttcagggagc agatcaagga
ttatatatat agctagtatg tgtacatatc 480 aaaagggcaa gaaaagtaca
aaaaagcatc ggatctccat tatatatata caacagctat 540 ataacaacca
cagaagaaca gtaagcacgc acatggtaaa attaaaatag cctggcagct 600
gctatggatg tatgcatcag atgcctaata tatatgcaag ataataatta ataagcagct
660 caagcaaaga cagatcaaga gttcgagaca gcaggttgga aaataaaata
cagatcatat 720 gaagtaaaac cttgacttga gatacgaatg atgaagctgc
atgggtaaag taaacaagga 780 aaggatcgga gggagcaccc ttcagtccaa
gcaaagacgg tgcgagatcg aagcttttac 840 ctcccgcttc attcactcat
ctgcgaagct cgtttccatg gccgtttgct tggcatgtgg 900 gtgaatgagt
cggcagctaa tccgacccta gcaccgcccc tgagtggact gaaggacgct 960
ctcttccatc cggccggcga ccatcgatca caaccatgac gccgcgcccg gcggcaaata
1020 tattaacaag aaatgaaatc aaaagagaga ggaagaacaa acatgatgcg
cagctgcgct 1080 agctagtgct tgatctgtct gaccacctca tggcgcgcag
tgtttagttt tctccctgga 1140 tcttgcgaag aaggcgatgg attttcgatg
gttgcaagga ggagcgaccg acaaagggtt 1200 tatataatat gtagacggc 1219 13
23 DNA Zea mays 13 gtggactgaa ggacgctctc ttc 23 14 616 DNA Zea mays
14 gccggccggg tcgggatgcc gcctactagc aggaagctag tggaggactc
caaagggatc 60 gcattgatct aacctgccga tcgacgccga cgtacgtacg
tgcccgagga caagcagatc 120 agtcagtgca atccctttgg aattctccac
ttagcgcctc catccccgcg ccgccctcca 180 ggtttcgctt cgatccatcc
atgtttcctt cgtttaaatt agttcgtttg tttttttttt 240 attatttatt
tgattcgccg ccgccggtct atctactctg tttgcaacgc ctttcgatcc 300
atcggcttct actgtatgct ataattaagg gtttttttac attggtccga tgcatgagag
360 gagctgtgca gaccaacatg gcaaccaatt acatcgatct tgaggactct
tatggaccaa 420 catgccaagt tcttcattgc ttgtactacc attcaagttg
tcaaacaatt accaattaac 480 tcaagtattc gagagaagca tatatgttag
tcaaatagca aattctttac taactgatct 540 atgtaccgac atgtcaactt
cttgcatacc aacgtggcaa gaaggtaatc attgttcatg 600 aataagatta tcacta
616 15 21 DNA Zea mays 15 tccaaaggga tcgcattgat c 21 16 1568 DNA
Zea mays 16 ctaggaatgg tacggtgctg gctaagctag ctagatcatc gtcctggagc
tgagagcagc 60 agctacctat atatctagct ggttttctaa cgacgatgac
gaacgaccgc gggactagca 120 tgatgcagct agctgaagac agttgtaggc
agctctcctc tggcaggcag gcgcgcggtc 180 atcgtcgcca tcgacgacgg
ttgcttggct ctgctatgct gtgttcgttc ggccatggtg 240 tgctagctag
ccgtgcatgc gttgcagtgt aacatgcgtg catgcacgcg cgtacgtcct 300
gccaaaggag agttgccctg cgactgtctt cagctcgaac aagatcgacc ggcccggaca
360 ggaatgttgg gcgtacgttg tcatcagggt ttaagctcca cgattccaaa
tattcaccac 420 ttctgggagg agttttgaag ctgctcgaaa gcatattgtg
tctgagtgta ataaatcggc 480 ggggaatcat atgttcatgt tctcactgca
agaataagct tgtcaaagag ggtggtgaag 540 taaaatctca cctgatcagc
ggcacaggtg ctcctagcga cgggtgtaag tcatggagga 600 caagcaacag
gaagtccact gccaagtgct tccatcgtcg tcaaatcaca ggtcaggggt 660
taattatatg ggggaagagg ccattatcat caggtacgcg tggttctcac acagtcgggg
720 ccacgttcgt tgatgatctg cctcttaatc ggcatctcaa actcttgttg
tgctctctac 780 atcagtagag aaggtgtgtt cacaagtcgt ttcttcttaa
gactatgttt tggttgatct 840 tgatctatag aactatttta ttgtagaact
actgaaccct ttcgaagtgt tgtactcaat 900 ttgtgtagaa caatgcatga
ttaatttcta ccaatagtct acggtagccg gtagttgttt 960 tatcctacta
gaaattgttg catggttaat tggttaattt gtgtaggatg tgccaaaaga 1020
agaggaagag aacaccatca atatgaatgg tgaattattc gtaagcttat cttccactaa
1080 tggtgctgga agccagaagg agaaagagga ggatggagat catgtgtcaa
ggctcaggag 1140 ataaatcgag gaagaaaaag atcgaagggt ggtgtttagt
tgtatccttc caagttccaa 1200 gttcacggta aagagaggaa agtgtgctag
ttcaagagag tatgggatgg agataggcac 1260 cattggactt ggagtggagg
acaagatgtt accattttgc atttccatgg agcgtggaga 1320 cttctgagtg
cttcaatctt tttattaaaa atcagtctga gcgatgatga gtctaaagag 1380
actaagacta tatcataatc tacgatggat ttaatctata aggtggatat atcacatatg
1440 gttgccaatc ttgtatattt catatttgca tggttggtag ttgcactgtt
gcaatcttaa 1500 gacctgtata gttgcatatt tgattgtgtt tttagaatgt
tgatttgtgg ttgtgctcgc 1560 ttctttct 1568 17 21 DNA Zea mays 17
tgccaaagga gagttgccct g 21 18 825 DNA Zea mays 18 ggtaccttta
gcgttagcac agacacacac aggtaaggag agcgagaggt gggttgggtt 60
tgatcggaga cagggacgag gcagagcatg ggtagggggc catcaacaga attccaaatt
120 tgatttctgt ttgctcgctc acaaaatgga gggactcacc acaaacacac
tcaggcgttg 180 ttgctccctc ccctgcactg cctcttccct ggctcctcac
cgtctcccat ccacctatcc 240 tctctctttc tctctctcgt tatggttttg
tataattttt tttcctgcat tcttttctca 300 gtacaagtcc tacactaatt
tggctgtctt tgcaccagta ctaataaaca ccgcaggtcc 360 ctgcaatagg
gtttacaaca attctattgt aatgactgct gtaaaacatc cgcatcattt 420
aattcaactt tccggtttca gtcagccctg caaaagtgct cctccgttcg tccgcgtttg
480 gtgttggctt ctgcggctcc ggtgcccaga gttgctgccg gcggaggccg
agcaggagcg 540 caactaacaa gagcggccaa ggcgccagtg atcctcacca
tggacaggag atcgatggag 600 atgagcgtga gcttccgatg cttcggtacc
cgaagaaaag aacgggaaca aaggcgagaa 660 acatgatcca cctctatgct
tttttggcaa catatcctat gcttaaacag ttatggtgtt 720 caaatgtaca
cattaataga gcgtttggtt tgaagaatca caccatctaa attgaggtgg 780
tgcatcatga atttattcct taaaaaaaaa aaaaaaaaaa aaaaa 825 19 20 DNA Zea
mays 19 tgcactgcct cttccctggc 20 20 1040 DNA Zea mays 20 gccggccggg
tcgggtgtgt tctcaggtcg cccccgatca cagccaacgc gggcgaccgc 60
gcgccattat agcacacggg gcacggcacg ccttcggcct cccactaact gcacaagagg
120 acgacgcggc agcgaggagg gagcaaagga aaggggatat gtcgaggccg
cccaacagga 180 gcgacgcgca cctctccgcc gaggacgagg cggcgctgga
ggccgaggtg cgggagtact 240 acgacgacgc ggcgccaaag cgccacacca
agccctcccg cagcgagcac tccgccgtgt 300 acgtcgacgc gctcgtcccg
gacgtcggcg gcaactccca cccggagctg gacaagttcc 360 aagagctgga
agcccacacc gagaggttgg tgtacgaggg cgccaatgtg ggagatgagt 420
tcgtagagac ggagtactac aaggacctcg gcggcgtcgg cgagcagcac cacacgaccg
480 gaacgggctt catcaagatg gacaaagcta aaggcgcccc cttcaaactg
tctgaagatc 540 ccaatgcaga ggagcgacat gcttcttgca ggggaaaccc
tgctaccaac gagtggatcc 600
cgtcagctga cacggtaaga ctgggggagc acagtccagt ttatcctatg caggtgcagg
660 gtcggctcca atcggcgtct ctactgacga acgcatcgtt agcttgtacc
cagcgtcaga 720 caagccaagc agaagcgaca gctgagggac tgtatatctc
aagccatgag aattcagacg 780 agtgctttcc gccattagaa taaggaacca
cactggttgt ccaccgtatc ttcactgttc 840 tgcgtcgaga ttcttgtgat
tcttacgtgg aacaaattaa gcgtgctacg agttagacct 900 ctgtgttctg
gctgtaaatg gcaaggaatg aagttctaat cgtggttcag cagtcaatca 960
attactgtgt ttctgatcct aaggctctag aaacaatcgg accttcaaaa taaactaggc
1020 gaaaattcta tgtcgtttcg 1040 21 21 DNA Zea mays 21 tgtgttctca
ggtcgccccc g 21 22 3389 DNA Zea mays 22 gagcggggtc ttgaaactgg
ctgcgcagaa ggaagggatg aaggggttcc tggagctcga 60 cgccgaggtt
ttcgagcttg ccccttcgtt ctttctggtc gagctgaaga aggccagcgg 120
tgacaccatt gagtaccaaa ggctcgtgag ggaagaagtg cggcctgcgc tgaaggatat
180 ggtctgggct tggcagagcg accggcacca gcagcagcag cagcggtgcg
agcagtctgt 240 gcaaggagag gaccagcagc agccgttgtc gtctttgccg
acgcagcagt agtcactgca 300 ccaccagttg cgaccgccat aaccagatca
cgtcaaaact gcaccaagcc gcacaggact 360 agtaactccc acttgcatcg
acgcttatgt gattgcggaa ttgtgtttca ggttacctgc 420 ctgctgcggt
aggacctaaa acgcctacct gcctaccatt tggcattttt ttgtatactg 480
tacgtacatt agagtaataa acaaacatgc ttaacttttc agctttcgat tggaatgtgc
540 ttttcgatgt aactctgtaa ccagtgtagg tacgaagtcg attagccaca
gggtctggcc 600 atgttgacct cacgtagccc tggttcattg gtgtaacagt
ttgttggctg cggctttaca 660 ttattttgtc tctatggatt acggctgcga
ctatgtgtag ctgaacaagc tggtatatga 720 tgagccctgg aaacgtgtgt
ttactgcagc tatttgcagc cagtgactgt tgatacaaac 780 gacgaagtag
agttggttgt ttatgtaggc acgcagcatg accataatta tccatgaatc 840
atggatagat gcacaatgtt taggaaacag gtgtgtgtgg ctggctggtg gtgcgagaag
900 agatgcgctg ccttgatgta ctgtactggg actgggaggg atgcgtctcg
cagtacagtc 960 tgtactatca tctctacacg cacgcacgca ggctcgacgt
gtcggcggcg gcggtccaga 1020 ctccatatgg atccgtagta gtacaacctg
ttggcgggta gtacaggttg gagcacgcct 1080 cttcttcagt cttccttcct
gagatgagga gtcactcacc agcaaaacgc ttgcagtaca 1140 ccccgctcgc
gggcgttgtt tatagtgatc ggtagcgtga gcacagagcg ccatcagaag 1200
atgcaaagag aaagagaagc aaaggcatca ttgagcgcag cgttgatgag ccagccgccg
1260 tgcctcccct gtcggctgcg gcggctcacc agcgctgcac tcaattacgc
ctttgctttc 1320 tcccgctggc cgcgtgtgtg cagagcgggc gggcgttcgg
catcattcat caggtttgct 1380 tcatttatta tgcactcatc gaaggcttct
ccttcgacac tgtctaggtg gcgcaggatc 1440 tgaatcagat gggtgtcgtc
ttcttcctcc atctgcactc ctgccccgta tgatgtcggt 1500 gtcctaggac
ggccagttgt ctgcgttctg gttaacccaa ttacctgacg gggcggacga 1560
cgctgataat gatcagagag agcatgaggc catatgcaag cctagaccta gctcccaaac
1620 tattaaaggt tgcttcgagc cctggctgtc atatcaacta ccaaccagtt
tatgtcgatt 1680 atcagttcct atctatcaca acgctccact gcacaacctt
aacctttact gtaaacctat 1740 agtcacctca tcgcttacat cgggtttttc
cccctctttc gtagactttt agttaacatc 1800 aaacaatgca ttttattgaa
atccaaaata catctgactg cgtaattgag tagatttatc 1860 ccaaaattta
attagcatgc cgctgtgagc taggagagcg acactagttt acaatatgac 1920
agtgtttgtg ttcggccaaa ccatttttgt tgatgggtaa ggggacacga cccccaaata
1980 gacgctctca ttttaatgaa gaattagttg tggactaatt gataattccc
attacaatcg 2040 gattgcacgc attaaatctt agtgctaagg aggtgttaca
aatgaaccta aaaaagaaaa 2100 gataattgtt gawttaatgt gggtctggtc
catattaata ttcaataatt gtcaatgcta 2160 gttgtcactt tatgctacgg
tgtactagta cttaccaaac tagaagttta agggacaatt 2220 cactyaactt
aaataggtgg actattggtg catctattga gaagctgaga aaaggatgaa 2280
ggactgtcac gcgtgcgcgc accctgatct gttgagaagc tgagatcgta ggaacaagaa
2340 tcactaaatt cggagttaca gatttcaagt tatgattttt cgaaggtttt
atgtgtttgg 2400 tacggaattg attaagtgat caattttaat atgggtttca
tgctaaaact gaggtactaa 2460 gtggtaaaca aaattataga aattggaatg
ggttaaaaag gagtttgcat gattttccta 2520 tgaattatac aagattatgg
atttatttta ataccaaaat cactttttat atttatttta 2580 ccctggtttt
ctatccacta gactgcgccc aagattatac taaagtttag gggcaactgc 2640
ataaaaaaac taagacttag ggcccgtttg tgatggactg cgggttgata acttagaaac
2700 agagggtctc ttatgtaaac tgtatgtgct gaaggggtat gaagcatcta
cgatcgtcag 2760 attacaattc cacggccaag attaaatcgc cagtgcgatg
aaccgttacg taacagccat 2820 catccgatct gagatctacg accctgattc
taaatgccct aaaacctccc agatccactc 2880 cctttgtccg aatcggtacg
catcggatta aatcgcagcc gcactctgat ggatctacgg 2940 cccacgcaga
tcatccccca taccaacggc gaacgggcgc cgccgcccgt aaacacggcg 3000
gtggccatgg ccgtggatgg ccaactcgac ttcgaggccg taatcctcta gtctaagacg
3060 tgctacgtgg taagtggatg aagacgattt ccatgggttc agtacttacc
gagggcaagg 3120 tcgtgcacaa gctgttcacg gcgaagcgcg gccgtagcaa
aaattgaaag ggaaatgtga 3180 ctttgggcta tttctataaa tgttttggtg
attagatgcc caacacatat tgttttagtt 3240 catatgtgct aagtgattga
gaagtgcaaa tcaagaatca aggtatattt ctagccctag 3300 taaatttctt
ttggatacta acatatctct ctaagtgcta gggacactac caagaaaagt 3360
ggaaatgaac tggagaagtt tggcagagt 3389 23 21 DNA Zea mays 23
tcattgagcg cagcgttgat g 21 24 660 DNA Glycine max 24 accattacac
tcttagtgaa tatttcataa aatataaagt tcctcctggg cgagaaacat 60
ctccatgttt aaggaaacag tgcgaagaat tattacacca gacatattca aggcaactag
120 tggaatccaa taaggaatgc tggcccactg cggaaaatat ttcgggttga
atgataggga 180 aggggctcat tcaacaaaaa tcttaatttt ctcggagatt
ggcaaatcta cattgacaag 240 ataaataaat aatttatgaa aacaataaaa
aaatgataat ggaaacaggg cttataatat 300 aagcactact aagctagttt
gtttctccta cgctaaaagc ctaatctcaa acctacccac 360 ttcctacaag
agagaaaggg ggggatagtg tataataccc tcaacttcga accaatattc 420
atcagaagta gaggtgtggg tattcttcca ctgcaactgg aggaggcatc caaagggatc
480 gcattgatcc caaatccaag ctttaatatt tttctctctt ctcactcaat
aatattaatt 540 tatttgggat catgctatcc ctttggattt ctcctttaat
ggcttctata atgatggctc 600 tctcatggat tctgcttgct gcaccacaac
acaaacactt tcatatacgc ctctaatgct 660 25 21 DNA Glycine max 25
tccaaaggga tcgcattgat c 21 26 1809 DNA Glycine max 26 cacaatacaa
ttaagctcat catactggtc ctgaaattgg tgaataaagt tgttttgtgg 60
tggatgagta ctgagtagtg gtgccttatt gtgggtggag agttccaaag ggatcgcatt
120 gatctaattc ttgtagatgt ttacacttgc aagctttgca tgcaattcct
ggattcagat 180 gttattcagt ggttcactta ttggatcatg cgatccctta
ggaactttcc atcaactcta 240 aacatcttgt tgatccattt gaggaattaa
tttcataggt tcatataatg gcgactgatt 300 cttctaatgg taatggacat
caccaaacaa caacaaagca accttctttg tcgtctacac 360 tgcgcttatc
caaatttttt cagtccaaca tgagaatctt ggttactgga ggagctggat 420
tcattgcgtc ttacttagtt gacagattga tggaaaatga aaaaaatgag gttattgtcg
480 ttgcataggt gctttcattt tacgttcttc aacattccga attgaacttc
agtggtcctt 540 gcaatggcaa cgaattcttc tgatgtacta tcgccgaagc
aacctccctt gccatctccc 600 ttgcgtttct ccaaattcta tcagtctaac
atgagaatct tgattacggg aggagctgga 660 ttcattggtt ctcacctagt
tgatagattg atggaaaatg aaaaaaatga ggtcattgtt 720 gctgacaact
acttcactgg atcaaaggac aacctcaaaa aatggattgg tcatccaaga 780
tttgagctta tccgtcatga tgtcactgaa cctttgacga ttgaggttga tcagatctac
840 catcttgcat gccccgcatc tcctattttc tacaaatata atcctgtgaa
gacaataaag 900 acaaatgtga ttggcacact gaacatgctt gggcttgcaa
aacgagttgg ggcaaggatt 960 ttactcacat caacatctga ggtatatggg
gatcctcttg tgcatcccca acctgaaggc 1020 tattggggca atgtgaaccc
tattggagtt cgtagttgct atgatgaggg gaaacgtgtg 1080 gctgaaactt
tgatgtttga ttatcatagg cagcatggaa tagaaatacg tgttgcaaga 1140
atctttaaca catatgggcc gcgcatgaat attgatgatg gacgtgttgt cagcaacttc
1200 attgctcaag caattcgtgg tgaacccttg acagtccagt ctccaggaac
acaaactcgc 1260 agtttctgct atgtctctga tctggttgat ggacttatcc
gtctcatgga aggatccgac 1320 actggaccaa tcaaccttgg aaatccaggt
gaatttacaa tgctagaact tgctgagaca 1380 gtgaaggagc ttattaatcc
agatgtggag ataaaggtag tggagaacac tcctgatgat 1440 ccgcgacaga
gaaaaccaat cataacaaaa gcaatggaat tgcttggctg ggaaccaaag 1500
gttaagctgc gagatgggct tcctcttatg gaagaggatt ttcgtttgag gcttggattt
1560 gacaaaaaaa attaacttat tttcgctcct tttatatcta gtcaaaatat
tcagataata 1620 agtgggatgg attattctat taagttttcc tatttttcct
tttcataatt atgatactta 1680 ggaagtaggg gtgcctgtat tttggcttcc
tcaatcaaga tcgtactctt gtttcacaaa 1740 gcactgcagc aatcatgcct
ttgcaaattt tgccggtaaa attactactg agttaaaatt 1800 ttcctatag 1809 27
21 DNA Glycine max 27 tccaaaggga tcgcattgat c 21 28 487 DNA Glycine
max 28 tgaaaattac gttttccctt ttccttttgt tgccggttag cacttcaatg
taaaaattaa 60 ttcaccataa aggatggttc gcatacaaaa gaataaaacc
ttatgaaagg acacatgcaa 120 cgcaaaataa aggcatcgtt ccataggata
tgccgatcct agtgagccat aaataacgtt 180 cccaaaggca ttcctctatg
tgtgtggatc ttcccagttg cagctgcatt acagggcaag 240 ttctccattg
gcaggtagcc actatgatat gcatctcata aatatttgca actttcttaa 300
tgtgcaatct gccaaaggag atttgcccag cgattctcct gcaacatctg cttcatgaaa
360 acagtattcg ttagtttctt caatcattca ttagaaacat ttcttgtact
ggttgaaatg 420 ttgcatctcg aaccattcat atgccatatt tcccttgttt
tgtattttgg taaaaaccat 480 ttttccc 487 29 21 DNA Glycine max 29
tgccaaagga gatttgccca g 21 30 535 DNA Glycine max 30 ctgcagagta
agacctgaat ttcactcatt gttcctgcca atgtccttag ttagataaat 60
ctaatttttt ctctctctaa agttgcatct ataaatatga gcctttccct tggtgcagat
120 caatttgagc tttcattacc gttctcatga agcttagggt gcatgcaacg
gtctctactt 180 actactggtt gagaagctcc ttgttggaga agcagggcac
gtgcaagtct cttggatctc 240 aaatgccact gaaccctttg cacgtgctcc
ccttctccaa cacgggtttc tccccttgct 300 tttctcctaa ccaattgtgt
ccagcactta tgaggtaatc gctttcctcc tatgtcttaa 360 tttggtccta
cgtaaagatc tacaatatgc atcttctttg agatacgggc tgaagcatgg 420
tacttttaaa ttgaaggctt caataactat atttagaggg aaaattcaac atacaaagaa
480 ggaagaagtg ttatgcatac aatattttac cgatgttcta tgcgtatcaa acata
535 31 21 DNA Glycine max 31 tggagaagca gggcacgtgc a 21 32 663 DNA
Glycine max 32 tctatataat ttttttccta ttttattttt tattttattt
tgtatcatat cacttataca 60 tcttttactt tcactcatac actaaatttt
cgggtgtagg aatactccgg caaagagaga 120 ataggtttgc ttatttccta
attctgaagt tagggtacgt gcgtaattta ctgtgtgttc 180 tgtgatgatg
agttaagtgg tcctatttta catgtaactt ttgacaatct gtttgggttg 240
agaatacaaa ttaaggcccc acacccaact aagcttagct ctctcccatt tttagcaccc
300 atcccgcacc caactttaaa agcaccctca attgcctctt ctattatagg
agagtaggct 360 tcaaagcaca caagaatatg ataagatgaa gaagttcagt
gtctcaaaat tcaccacttc 420 tcttaaaacc tccctcattt gttttttcac
actttccttt ccctcaccac tctctctatt 480 acctcttgtt tgttgttaag
agtactcaga agaataactc ctccaaccca cttagcatgt 540 ggcaaaggtg
catgctgagc aagatggaga agcagggcac gtgcaattct aactcatgaa 600
accatagaat catcttgttt tttcttcttt tcactctaac caaatagatt cctctacctg
660 cag 663 33 21 DNA Glycine max 33 tggagaagca gggcacgtgc a 21 34
443 DNA Glycine max unsure (1)..(443) unsure at all n locations 34
actcaagctt gaagcaccaa agttgcagtc ggaggagtca cagattaaat tcttcgcttc
60 tttaaccttt gtgtttctct tttcatacca ttgtttcttt ccctatagct
gctttaattt 120 tcttgtgaga gtcagaaaag tatcactata tcaagtgaca
tgatcatcag aattgaatta 180 tgtgcatgtt gtgcaagatg gagaagcagg
gcacgtgcaa tactaactca tgaacactac 240 acggngcgtg aactcggaga
atcatattct cttctgcttc atttcaccaa caagagagat 300 cctattagtt
agttcttcat gtgcccctct ttcccatcat gacaacagca ccttatatat 360
attgcatttg gaaatgttga acgatgaagt tcgcttggct tctgctcata aatcagcacc
420 gagntttata ggttatgctc cat 443 35 21 DNA Glycine max 35
tggagaagca gggcacgtgc a 21 36 21 DNA Glycine max 36 tgagaccaaa
tgagcagctg a 21 37 21 DNA Glycine max 37 atgcactgcc tcttccctgg c 21
38 748 DNA Glycine max 38 aaaattcatt acattgataa aacacaattc
aaaagatcaa tgttccactt catgcaaaga 60 catttccaaa atatgtgtag
gtagaggggt tttacaggat cgtcctgaga ccaaatgagc 120 agctgaccac
atgatgcagc tatgtttgct attcagctgc tcatctgttc tcaggtcgcc 180
cttgttggac tgtccaactc ctactgattg cggatgcact tgccacaaat gaaaatcaaa
240 gcgaggggaa aagaatgtag agtgtgacta cgattgcatg catgtgattt
aggtaattaa 300 gttacatgat tgtctaattg tgtttatgga attgtatatt
ttcagaccag gcacctgtaa 360 ctaattatag gtaccatacc ttaaaataag
tccaactaag tccatgtctg tgatttttta 420 gtgtcacaaa tcacaatcca
ttgccattgg ttttttaatt tttcattgtc tgttgtttaa 480 ctaactctag
ctttttagct gcttcaagta cagattcctc aaagtggaaa atgttctttg 540
aagtcaataa aaagagcttt gatgatcatc tgcattgtct aagttggata aactaattag
600 agagaacttt tgaactttgt ctaccaaata tctgtcagtg tcatctgtca
gttctgcaag 660 ctgaagtgtt gaatccacga ggtgcttgtt gcaaagttgt
gatattaaaa gacatctacg 720 aagaagttca agcaaaactc tttttggc 748 39 960
DNA Glycine max 39 ccgtggtggg cgaagggaat taacgcctat cgcgtggcga
gagaaggagc agaacggcag 60 gggggggccg gctccggggg ggcgccccgg
tacgcaccgc gctctccgag tccctggggt 120 ccccccccca gaacatccta
atcgaaaaat tcaagagtgc attttgtgcg taatgtagtt 180 aattagacaa
atttctaatg tgagaatctt tctgagaatg agatgttgct aaatatttcg 240
gatgttgtcg acaaggatga ggtaataata gttagagaca ggacaaagca ggggaacagg
300 cagagcatgg atggagctat caacacaata ttgtcaagaa actgagagtg
agaggagaaa 360 tatgttgtgg ttctgctcat gcactgcctc ttccctggct
ctgtctccat ttctccttcc 420 cttatttatt ttttgattta ttgagtatga
tctgttttca aatgtgttca taggttcaac 480 ttattaaggt acgaacatac
tctgggcatt gaaaactggt ttgactcttg aacatattcc 540 gcaccactaa
tctttcttgt aatccaggct cacgcacgat cactataagg tcccacattc 600
ttagtggcct aatcgttgga aaatgctact ttggcactac ttgatgaatt gtatggctgg
660 gatttttttc cccttgcttg tagaatcctc tcaatttatg taaccatcgt
gtactcattt 720 acatgtcatc atttttgaat gagatgtgat atacatagag
caaaaaaaaa aaaaaaattg 780 tatgacctca ttttctgtgt ttatttctct
ccatcaatat cattttctaa atctcaaaat 840 tctctctttt ttcttagttg
tagaagttat tgtttactcg actcctcgcc tcacatccct 900 ctcacccctc
tccccactac tgccccgcca gcgtcaccga tgctctcctt tgtggccggt 960 40 21
DNA Glycine max 40 tgagatcaaa tgagcagctg a 21 41 21 DNA Glycine max
41 tgagaccaaa tgagcagctg t 21 42 21 DNA Glycine max 42 tgagaccaaa
tgaccagctg a 21 43 129 DNA Zea mays 43 gttaaggggt ctgttgtctg
gttcaaggtc gccacagcag gcaaataaag cccatttcgc 60 gcttagcatg
caccatgcat gatgggtgta cctgttggtg atctcggacc aggcttcaat 120
ccctttaac 129 44 86 DNA Zea mays 44 gtcgagggga atgacgtccg
gtccgaacga gccacggctg ctgctgcgcc gccgcgggct 60 tcggaccagg
cttcattccc cgtgac 86 45 20 DNA artificial sequence Synthetic
construct 45 gtgctcactc tcttctgtca 20 46 21 DNA artificial sequence
Synthetic construct 46 tagagctccc ttcaatccaa a 21 47 21 DNA
artificial sequence Synthetic construct 47 tggcatccag ggagccaggc a
21 48 21 DNA artificial sequence Synthetic construct 48 ctggatgcag
aggtttatcg a 21 49 21 DNA artificial sequence Synthetic construct
49 tgcacgtgcc ctgcttctcc a 21 50 21 DNA artificial sequence
Synthetic construct 50 ggggaatgaa gcctggtccg a 21 51 21 DNA
artificial sequence Synthetic construct 51 tagatcatgc tggcagcttc a
21 52 21 DNA artificial sequence Synthetic construct 52 ttcccgacct
gcaccaagcg a 21 53 21 DNA artificial sequence Synthetic construct
53 tcggcaagtc atccttggct g 21 54 21 DNA artificial sequence
Synthetic construct 54 gatattggcg cggctcaatc a 21 55 21 DNA
artificial sequence Synthetic construct 55 ctgcagcatc atcaagattc t
21 56 21 DNA artificial sequence Synthetic construct 56 ggcgctatcc
ctcctgagct t 21 57 21 DNA artificial sequence Synthetic construct
57 gatcaatgcg atccctttgg a 21 58 22 DNA artificial sequence
Synthetic construct 58 tggggtcctt acaaggtcaa ga 22 59 20 DNA
artificial sequence Synthetic construct 59 ggaggtggac agaatgccaa 20
60 21 DNA artificial sequence Synthetic construct 60 gagttccccc
aaacacttca c 21 61 21 DNA artificial sequence Synthetic construct
61 catcaacgct gcgctcaatg a 21 62 21 DNA artificial sequence
Synthetic construct 62 cgggggcgac ctgagaacac a 21 63 21 DNA
artificial sequence Synthetic construct 63 agccagggaa gaggcagtgc a
21 64 20 RNA Arabidopsis thaliana 64 gugcucucuc ucuucuguca 20 65 20
RNA Arabidopsis thaliana 65 cugcucucuc ucuucuguca 20 66 20 RNA
Arabidopsis thaliana 66 uugcuuacuc ucuucuguca 20 67 20 RNA
Arabidopsis thaliana 67 ccgcucucuc ucuucuguca 20 68 21 RNA
Arabidopsis thaliana 68 uggagcuccc uucauuccaa u 21 69 21 RNA
Arabidopsis thaliana 69 ucgaguuccc uucauuccaa u 21 70 21 RNA
Arabidopsis thaliana 70 augagcucuc uucaaaccaa a 21 71 21 RNA
Arabidopsis thaliana 71 uggagcuccc uucauuccaa g 21 72 21 RNA
Arabidopsis thaliana 72 uagagcuucc uucaaaccaa a 21 73 21 RNA
Arabidopsis thaliana 73 uggagcucca uucgauccaa a 21 74 21 RNA
Arabidopsis thaliana 74 agcagcuccc uucaaaccaa a
21 75 21 RNA Phaseolus vulgaris 75 cagagcuccc uucacuccaa u 21 76 21
RNA Vitis vinifera 76 uggagcuccc uucacuccaa u 21 77 21 RNA Hordeum
vulgare 77 uggagcuccc uucacuccaa g 21 78 21 RNA Oryza sativa 78
uggagcuccc uuuaauccaa u 21 79 21 RNA Arabidopsis thaliana 79
uggcaugcag ggagccaggc a 21 80 21 RNA Arabidopsis thaliana 80
aggaauacag ggagccaggc a 21 81 21 RNA Arabidopsis thaliana 81
ggguuuacag ggagccaggc a 21 82 21 RNA Oryza sativa 82 aggcauacag
ggagccaggc a 21 83 21 RNA Lotus corniculatus var. japonicus 83
aagcauacag ggagccaggc a 21 84 21 RNA Arabidopsis thaliana 84
accugaugua aucacuuuca a 21 85 21 RNA Arabidopsis thaliana 85
cccggaugua aucacuuuca g 21 86 21 RNA Arabidopsis thaliana 86
uuguuacuuu caaugcauug a 21 87 21 RNA Arabidopsis thaliana 87
cccugaugua uuuacuuuca a 21 88 21 RNA Arabidopsis thaliana 88
uagucacguu caaugcauug a 21 89 21 RNA Arabidopsis thaliana 89
cccugaugua uucacuuuca g 21 90 21 RNA Arabidopsis thaliana 90
cccugauguu guuacuuuca g 21 91 21 RNA Arabidopsis thaliana 91
uagucacuuu cagcgcauug a 21 92 21 RNA Arabidopsis thaliana 92
uccaaaugua gucacuuuca g 21 93 21 RNA Arabidopsis thaliana 93
uccaaaugua gucacuuuca a 21 94 21 RNA Arabidopsis thaliana 94
uccaaaugua gucacuuuca g 21 95 21 RNA Arabidopsis thaliana 95
uccaaaugua gucacuuuca a 21 96 21 RNA Arabidopsis thaliana 96
uuguaacuuu cagugcauug a 21 97 21 RNA Arabidopsis thaliana 97
uagucacguu caaugcauug a 21 98 21 RNA Arabidopsis thaliana 98
uuguuacuuu cagugcauug a 21 99 21 RNA Arabidopsis thaliana 99
cccugauguu gucacuuuca c 21 100 21 RNA Arabidopsis thaliana 100
uuguuacuua caaugcauug a 21 101 21 RNA Arabidopsis thaliana 101
uagucuuuuu caacgcauug a 21 102 22 RNA Arabidopsis thaliana 102
cuggaugcag agguauuauc ga 22 103 22 RNA Populus tremula 103
cuggaugcag aggucuuauc ga 22 104 22 RNA Oryza sativa 104 cuggaugcag
agguuuuauc ga 22 105 23 RNA Arabidopsis thaliana 105 aucgaguucc
aaguccucuu caa 23 106 23 RNA Arabidopsis thaliana 106 aucgaguucc
agguccucuu caa 23 107 23 RNA Arabidopsis thaliana 107 aucgaguucc
aaguuuucuu caa 23 108 21 RNA Arabidopsis thaliana 108 agcacguacc
cugcuucucc a 21 109 21 RNA Arabidopsis thaliana 109 uuuacgugcc
cugcuucucc a 21 110 21 RNA Arabidopsis thaliana 110 agcacguguc
cuguuucucc a 21 111 21 RNA Arabidopsis thaliana 111 ucuacgugcc
cugcuucucc a 21 112 21 RNA Arabidopsis thaliana 112 cucacgugac
cugcuucucc g 21 113 21 RNA Oryza sativa 113 cgcacgugac cugcuucucc a
21 114 21 RNA Medicago truncatula 114 cuuacguguc cugcuucucc a 21
115 21 RNA Glycine max 115 cuuacgugcc cugcuucucc a 21 116 21 RNA
Lycopersicum esculentum 116 gccacgugca cugcuucucc a 21 117 21 RNA
Arabidopsis thaliana 117 uugggaugaa gccugguccg g 21 118 21 RNA
Arabidopsis thaliana 118 cugggaugaa gccugguccg g 21 119 21 RNA
Arabidopsis thaliana 119 cuggaaugaa gccugguccg g 21 120 21 RNA
Populus tremula 120 ccgggaugaa gccugguccg g 21 121 21 RNA
Arabidopsis thaliana 121 gagaucaggc uggcagcuug u 21 122 21 RNA
Arabidopsis thaliana 122 uagaucaggc uggcagcuug u 21 123 21 RNA
Oryza sativa 123 aagaucaggc uggcagcuug u 21 124 21 RNA Arabidopsis
thaliana 124 uucccgagcu gcaucaagcu a 21 125 21 RNA Arabidopsis
thaliana 125 aagggaaguc auccuuggcu g 21 126 21 RNA Arabidopsis
thaliana 126 acgggaaguc auccuuggcu a 21 127 21 RNA Arabidopsis
thaliana 127 aggggaaguc auccuuggcu a 21 128 21 RNA Arabidopsis
thaliana 128 aggcaaauca ucuuuggcuc a 21 129 21 RNA Arabidopsis
thaliana 129 gcggcaauuc auucuuggcu u 21 130 21 RNA Arabidopsis
thaliana 130 ccggcaaauc auucuuggcu u 21 131 21 RNA Arabidopsis
thaliana 131 aagggaaguc auccuuggcu a 21 132 21 RNA Zea mays 132
guggcaacuc auccuuggcu c 21 133 21 RNA Vitis vinifera 133 ugggcaauuc
auccuuggcu u 21 134 21 RNA Oryza sativa 134 auggcaaauc auccuuggcu u
21 135 21 RNA Glycine max 135 uagggaaguc auccuuggcu c 21 136 21 RNA
Gossypium hirsutum 136 cugggaaguc auccuuggcu c 21 137 21 RNA
Arabidopsis thaliana 137 gauauuggcg cggcucaauc a 21 138 21 RNA
Arabidopsis thaliana 138 cugcagcauc aucaggauuc u 21 139 21 RNA
Arabidopsis thaliana 139 cagcagcauc aucaggauuc u 21 140 21 RNA
Arabidopsis thaliana 140 augcagcauc aucaggauuc u 21 141 21 RNA
Arabidopsis thaliana 141 uggcagcauc aucaggauuc u 21 142 21 RNA
Arabidopsis thaliana 142 uuguagcauc aucaggauuc c 21 143 21 RNA
Arabidopsis thaliana 143 uugcagcauc aucaggauuc c 21 144 21 RNA
Arabidopsis thaliana 144 cagggggacc cuucagucca a 21 145 21 RNA
Arabidopsis thaliana 145 gagggguccc cuucagucca u 21 146 21 RNA
Arabidopsis thaliana 146 gagggguccc cuucagucca g 21 147 21 RNA
Arabidopsis thaliana 147 aagggguacc cuucagucca g 21 148 21 RNA
Arabidopsis thaliana 148 uagggggacc cuucagucca a 21 149 21 RNA
Oryza sativa 149 gaggggaccc cuucagucca g 21 150 21 RNA Oryza sativa
150 ucggggcaca cuucagucca a 21 151 21 RNA Arabidopsis thaliana 151
aaacaaugcg aucccuuugg a 21 152 21 RNA Arabidopsis thaliana 152
agaccaugcg aucccuuugg a 21 153 21 RNA Arabidopsis thaliana 153
ggucagagcg aucccuuugg c 21 154 21 RNA Arabidopsis thaliana 154
agacaaugcg aucccuuugg a 21 155 21 RNA Arabidopsis thaliana 155
ggagguugac agaaugccaa a 21 156 21 RNA Arabidopsis thaliana 156
gaguuccucc aaacacuuca u 21 157 21 RNA Arabidopsis thaliana 157
gaguuccucc aaacucuuca u 21 158 21 RNA Arabidopsis thaliana 158
aaguucuccc aaacacuuca a 21 159 22 RNA Arabidopsis thaliana 159
ucguucaaga aagccugugg aa 22 160 22 RNA Arabidopsis thaliana 160
ccguucaaga aagccugugg aa 22 161 22 RNA Arabidopsis thaliana 161
ucguucaaga aagcaugugg aa 22 162 22 RNA Arabidopsis thaliana 162
acguucaaga aagcuugugg aa 22 163 22 RNA Arabidopsis thaliana 163
ccguucaaga aagccugugg aa 22 164 21 RNA Arabidopsis thaliana 164
aaucaaugcu gcacucaaug a 21 165 21 RNA Arabidopsis thaliana 165
agucaacgcu gcacuuaaug a 21 166 21 RNA Arabidopsis thaliana 166
aaucaaugcu gcacuuaaug a 21 167 21 RNA Arabidopsis thaliana 167
aagggguuuc cugagaucac a 21 168 22 RNA Arabidopsis thaliana 168
ugcgggugac cugggaaaca ua 22 169 22 RNA Arabidopsis thaliana 169
aaggugugac cugagaauca ca 22 170 22 RNA Arabidopsis thaliana 170
gugauuuuuc ucaacaagcg aa 22 171 22 RNA Arabidopsis thaliana 171
gugauuuuuc ucuacaagcg aa 22 172 22 RNA Arabidopsis thaliana 172
gugauuuuuc ucuccaagcg aa 22 173 21 RNA Arabidopsis thaliana 173
uagggcauau cuccuuuggc a 21 174 21 RNA Arabidopsis thaliana 174
uugggcaaau cuccuuuggc a 21 175 21 RNA Arabidopsis thaliana 175
ucgagcaaau cuccuuuggc a 21 176 21 RNA Arabidopsis thaliana 176
uagagcaaau cuccuuuggc a 21 177 21 RNA Arabidopsis thaliana 177
uagggcaaau cuucuuuggc a 21 178 21 RNA Oryza sativa 178 uagggcaaau
cuccuuuggc a 21 179 21 RNA Oryza sativa 179 cugggcaaau cuccuuuggc a
21 180 21 RNA Oryza sativa 180 ucgggcaaau cuccuuuggc a 21 181 21
RNA Oryza sativa 181 ccgggcaaau cuccuuuggc a 21 182 21 RNA Populus
tremula 182 gcgggcaaau cuucuuuggc a 21 183 21 RNA Medicago
truncatula 183 aagggcaaau cuccuuuggc a 21 184 21 RNA Triticum
aestivum 184 uagggcaaau cuccuuuggc g 21 185 21 RNA Triticum
aestivum 185 cugggcaaau cuccuuuggc g 21 186 21 RNA Triticum
aestivum 186 uucggcaaau cuccuuuggc a 21 187 22 RNA Arabidopsis
thaliana 187 ggaguuugug cgugaaucua au 22 188 22 RNA Arabidopsis
thaliana 188 cuugucuauc ccuccugagc ua 22 189 22 RNA Sorghum bicolor
189 uaugucuauc ccuucugagc ug 22 190 22 RNA Saccharum officinarum
190 uaugucuauc ccuucugagc ua 22 191 22 RNA Zea mays 191 uaugucuauc
ccuucugagc ug 22 192 22 RNA Oryza sativa 192 ucggucuauc ccuccugagc
ug 22 193 22 RNA Pennisetum glaucum 193 uuagucuauc ccuccugagc ua 22
194 22 RNA Vitis vinifera 194 auugccuauc ccuccugagc ug 22 195 22
RNA Theobroma cacao 195 ccuugcuauc ccuccugagc ug 22 196 22 RNA
Lycopersicum esculentum 196 cuugucuauc ccuccugagc ug 22 197 22 RNA
Zea mays 197 cccuucuauc ccuccugagc ua 22 198 22 RNA Populus tremula
198 cuugucuauc ccuccugagc ua 22 199 22 RNA Oryza sativa 199
cccuucuauc ccuccugagc ua 22 200 22 RNA Triticum aestivum 200
cccuucuauc ccuccugagc ua 22 201 22 RNA Hordeum vulgare 201
ccuuucuauc ccuccugagc ua 22 202 22 RNA Populus tremula 202
ccugucuauc ccuccugagc ua 22 203 22 RNA Mesembryanthemum
crystallinum 203 ugugucuauc ccuccugagc ua 22 204 21 RNA Arabidopsis
thaliana 204 ugacaaacau cucgucccca a 21 205 21 RNA Arabidopsis
thaliana 205 ugacaaacau cucguuccua a 21 206 21 RNA Arabidopsis
thaliana 206 ccaagggaag aggcagugca u 21 207 21 RNA Arabidopsis
thaliana 207 accagugaag aggcugugca g 21 208 21 RNA Arabidopsis
thaliana 208 gccagggaag aggcagugca u 21 209 21 RNA Arabidopsis
thaliana 209 gccggugaag aggcugugca a 21 210 21 RNA Arabidopsis
thaliana 210 gccggugaag aggcugugca g 21 211 21 RNA Arabidopsis
thaliana 211 agggucuugc aaggucaaga a 21 212 21 RNA Arabidopsis
thaliana 212 aaggucuugc aaggucaaga a 21 213 21 RNA Oryza sativa 213
gaggucuugc aaggucaaga a 21 214 21 RNA Oryza sativa 214 acggucuugc
aaggucaaga a 21 215 21 RNA Arabidopsis thaliana 215 agaacuagag
aaagcauugg a 21 216 21 RNA Arabidopsis thaliana 216 agaguaagau
ggagcuugau a 21 217 21 RNA Arabidopsis thaliana 217 agauggugga
aaugggauau c 21 218 21 RNA Arabidopsis thaliana 218 uuguugaucg
uaugguagaa g 21 219 21 RNA Arabidopsis thaliana 219 gguauucgag
uaucugcaaa a 21 220 1242 DNA artificial sequence Synthetic
construct 220 acacgctgaa accatcttcc acacactcaa gccacactat
tggagaacac acagggacaa 60 cacaccataa ccgccgccgc cggtagaaga
tggcgcccac cgtgatgatg gcctcgtcgg 120 ccaccgccgt cgctccgttc
caggggctca agtccaccgc cagcctcccc gtcgcccgcc 180 gctcctccag
aagcctcggc aacgtcagca acggcggaag gatccggtgc atgcaggtgt 240
ggccggccta cggcaacaag aagttcgaga cgctgtcgta cctgccgccg ctgtcgaccg
300 gcgggcgcat ccgctgcatg caggccatgg ccttcttcaa ccgggtgatc
accctcacgg 360 tgccgtcgtc agacgtggtc aactactcgg agatctacca
ggtggctcct cagtatgtca 420 accaggccct gaccctggcc aagtacttcc
agggcgccat cgacggcagc accctgaggt 480 tcgacttcga gaaggcgtta
cagatcgcca acgacatccc gcaggccgcg gtggtcaaca 540 ccctgaacca
gaccgtccag caggggaccg tccaggtcag cgtcatgatc gacaagatcg 600
tggacatcat gaagaatgtc ctgtccatcg tgatagacaa caagaagttt tgggatcagg
660 tcacggctgc catcaccaac accttcacga acctgaacag ccaggagtcg
gaggcctgga 720 tcttctatta caaggaggac gcccacaaga cgtcctacta
ttacaacatc ctcttcgcca 780 tccaggacga agagacgggt ggcgtgatgg
ccacgctgcc catcgccttc gacatcagtg 840 tggacatcga gaaggagaag
gtcctgttcg tgaccatcaa ggacactgag aattacgccg 900 tcaccgtcaa
ggcgatcaac gtggtccagg cactccagtc tagcagggat tctaaggtgg 960
ttgatgcgtt caaatcgcca cggcacttac cccggaagag gcataagatt tgctctaact
1020 cgtgatgact gctggatgca gaggtattat cgatgcgttt ggacgtatgc
tcattcaggt 1080 tggagccaat ttggttgatg tgtgtgcgag ttcttgcgag
tctgatgaga catctctgta 1140 ttgtgtttct ttccccagtg ttttctgtac
ttgtgtaatc ggctaatcgc caacagattc 1200 ggcgatgaat aaatgagaaa
taaattgttc tgattttgag tg 1242 221 956 DNA artificial sequence
Synthetic construct 221 acacgctgac aagctgactc tagcagatcc tctagaacca
tcttccacac actcaagcca 60 cactattgga gaacacacag ggacaacaca
ccataagatc caagggaggc ctccgccgcc 120 gccggtagaa gtgatcaacc
atggccttct tcaaccgggt gatcaccctc acggtgccgt 180 cgtcagacgt
ggtcaactac tcggagatct accaggtggc tcctcagtat gtcaaccagg 240
ccctgaccct ggccaagtac ttccagggcg ccatcgacgg cagcaccctg aggttcgact
300 tcgagaaggc gttacagatc gccaacgaca tcccgcaggc cgcggtggtc
aacaccctga 360 accagaccgt ccagcagggg accgtccagg tcagcgtcat
gatcgacaag atcgtggaca 420 tcatgaagaa tgtcctgtcc atcgtgatag
acaacaagaa gttttgggat caggtcacgg 480 ctgccatcac caacaccttc
acgaacctga acagccagga gtcggaggcc tggatcttct 540 attacaagga
ggacgcccac aagacgtcct actattacaa catcctcttc gccatccagg 600
acgaagagac gggtggcgtg atggccacgc tgcccatcgc cttcgacatc agtgtggaca
660 tcgagaagga gaaggtcctg ttcgtgacca tcaaggacac tgagaattac
gccgtcaccg 720 tcaaggcgat caacgtggtc
caggcactcc agtctagcag ggattctaag gtggttgatg 780 cgttcaaatc
gccacggcac ttaccccgga agaggcataa gatttgctct aactcgtgat 840
gaatgtacgt gccctgcttc tccatctgca tgcgtttgga cgtatgctca ttcaggttgg
900 agccaatttg gttgatgtgt gtgcgagttc ttgcgagtct gatgagacat ctctgt
956 222 1683 DNA Zea mays 222 atgtattgct gatgacgctg cgccttcttg
tttttttgct gcaactttga gaaagataga 60 tccatctgca tgcttttttc
cgctgctatg gtgtatggtt gtgtgctgca gattttggat 120 ctgacttgtg
agaaccgtcg acggacccct gcacacagta cgtgagacga tcgaggagga 180
tggaggcacg cagtacgttt tgttctcgat ctctgctaca gcatgtcatc ttaattaagc
240 ctactggttg catgcatggg tgaggattat tcatcgctaa gtttccatgt
acgtagcata 300 ctatcacatg tacaatgaaa taggcaaata gcctagacgt
tcttctcatg acgaccatgt 360 ctgccaatta aatatattgc aggtagtaaa
cctcaagtac tgatagccat taattcttgg 420 ttggagttcg acagagaaga
tcgaaaagac atgtatagaa tactgatcgt ctgatcatat 480 cgtccctacc
tatctgtctg tctctaccaa agtgggctac agtacgttag ctagctgtct 540
cttcgaagac actgatagga tgtttgatta caagtccaac ggaaccactt gactgcatac
600 ggttaccact tactcatgca agaaaaaaaa ttgcattttc aaattcgaac
cccaagtcgt 660 ctagtgtagg gtcttatgtt cataaccagg tgggataaca
aacatattaa tcgatctgca 720 tatatatata tatacacaaa agggctacac
agattacaga tgcagtgcat agaacctaat 780 tgcaggtggg ggaccccggc
cctcccccgg tggacaataa aaaaaatcca gtttccaagc 840 ccaagctata
ggtaggcagt ccagagcggt ggtcttgtca ctttcttact tccaaaacca 900
ggccactgtt gatgtaggct ggctggctgg ctcatgtgcc acagttgctg ttcgttatta
960 actgtagtaa acatcagtgt ggacgggcgc cagaatttca gatctcggta
cgtatgctgt 1020 gtggattcag cgttatttga acaccgtaat aatgctctcc
agcagattgt gaattgtgaa 1080 tacagttcgt agagaacact atttataatg
cagacgttat gtttacatag tttagtttaa 1140 aatgggagat aagatagaag
agatagaatg agtaattggc tggagatcaa atcgtgcata 1200 ttattgtgca
aaacactgtt tttccatata gtggagtttt aaagtatggg acgagagagc 1260
agatagcaaa tcgtgtatat ggtcgtgcaa atattatata tgtggttgtg caaaactccg
1320 aaatttgaaa taggagacac agttgataat tctacgctct acgcacgggc
ggcggactgc 1380 actactagtt catcggatgc gttagcgtgc cactcctcat
cttgtttcct tgtacgtact 1440 agtgcaatcc gtcagccgca cggctccagt
ccactccagt ccagcaacag cgtcacctcc 1500 agctccgaaa ggcttatcct
tgcaacaaac atcgtacgaa aaaggcgcag gacaaaagaa 1560 aatggatcga
aatgcaacaa ataaaaaagg gcatcaaaat acgctgcgag tgagcgagac 1620
gttggcctcc ccatcccata tatatatagc tatagctatc cctcggttct tcaattcatt
1680 cct 1683 223 674 DNA Zea mays 223 acgtatgctg tgtggattca
gcgttatttg aacaccgtaa taatgctctc cagcagattg 60 tgaattgtga
atacagttcg tagagaacac tatttataat gcagacgtta tgtttacata 120
gtttagttta aaatgggaga taagatagaa gagatagaat gagtaattgg ctggagatca
180 aatcgtgcat attattgtgc aaaacactgt ttttccatat agtggagttt
taaagtatgg 240 gacgagagag cagatagcaa atcgtgtata tggtcgtgca
aatattatat atgtggttgt 300 gcaaaactcc gaaatttgaa ataggagaca
cagttgataa ttctacgctc tacgcacggg 360 cggcggactg cactactagt
tcatcggatg cgttagcgtg ccactcctca tcttgtttcc 420 ttgtacgtac
tagtgcaatc cgtcagccgc acggctccag tccactccag tccagcaaca 480
gcgtcacctc cagctccgaa aggcttatcc ttgcaacaaa catcgtacga aaaaggcgca
540 ggacaaaaga aaatggatcg aaatgcaaca aataaaaaag ggcatcaaaa
tacgctgcga 600 gtgagcgaga cgttggcctc cccatcccat atatatatag
ctatagctat ccctcggttc 660 ttcaattcat tcct 674 224 6 DNA artificial
sequence Synthetic construct 224 tgtctc 6 225 768 DNA Zea mays 225
tcagcgttat ttgaacaccg taaagcctct ccggcagatt gtgaatacac agttgtggag
60 aacgctattt ataacgcaga cactatttat aatgcagatg tgtaaaagtg
aaatttaaaa 120 tagtagatga gataggagag atagaatgag taaactgctg
gagagcaaat cgtgcatatg 180 atcgtgcaaa acaccgtttt tcgtagagtg
aagtttaaaa tagcaggtga gagagtagat 240 aggatgagta agctgatgga
gagcaaatat tgtatatacg tggtcggtgc aatagagtga 300 aatttgaaat
aactgacaca gttttggtgc gtggaaatag acgaggataa ttctagtgca 360
atccgcactg ccagtggacc ccgcccgacg ataattctac gcacgggcgg cgcactgcac
420 tactagttca tcgatcggat gcgttagcgt gcccctcctc atattgtttc
cttgtacgta 480 ctagtgcaat ccgtcagccg cacggctcca gtccactcca
gtccagcaac agcgtcacct 540 ccagctccga aaggcttatc cttgcaacaa
acatcgtacg aaaaaggcgc aggaaaarga 600 aaagtgtcga aatacgacat
aaaaaaagca tcaaaatacg ctgcgagtga gygagacatt 660 ggcctcccca
tcccatatat atatagctat agctayccct cggttcttca attcatctat 720
cccccgctct ctccatctct ctaccctttc tctctctcgg atagctag 768 226 407
DNA Zea mays 226 tttgaaataa ctgacacagt tttggtgcgt ggaaatagac
gaggataatt ctagtgcaat 60 ccgcactgcc agtggacccc gcccgacgat
aattctacgc acgggcggcg cactgcacta 120 ctagttcatc gatcggatgc
gttagcgtgc ccctcctcat attgtttcct tgtacgtact 180 agtgcaatcc
gtcagccgca cggctccagt ccactccagt ccagcaacag cgtcacctcc 240
agctccgaaa ggcttatcct tgcaacaaac atcgtacgaa aaaggcgcag gaaaargaaa
300 agtgtcgaaa tacgacataa aaaaagcatc aaaatacgct gcgagtgagy
gagacattgg 360 cctccccatc ccatatatat atagctatag ctayccctcg gttcttc
407 227 485 DNA Zea mays 227 gcatctgctg ttctttattt ctatacatac
atatatacta tcaccggtta tttgcttctc 60 tattctgtcc gagtacttta
cggtgttccg cacatagatc tcgtggccgg cggttttgcg 120 ctttcgcttg
cgtttcttgg ccctgctggt gtttgaccgg accgaacggg ggcagatcga 180
tgctttgggt ttgaagcgga gctcctatca ttccaatgaa gggtcgttcc gaagggctgg
240 ttccgctgct cgttcatggt tcccactatc ctatctcatc atgtgtatat
atgtattcca 300 tgggggaggg tttctctcgt ctttgagata ggcttgtggt
ttgcatgacc gaggagctgc 360 accgccccct tgctggccgc tctttggatt
gaagggagct ctgcatcctg atccacccct 420 ccattttttt ttgcttgttg
tgtccttcct gggacctgag atctgaggct cgtggtggct 480 cactg 485 228 539
DNA Zea mays 228 ccttgtatgt tctccgctca ctcccccatt ccactctcat
ccatctctca agctacacac 60 atataaaaaa aaaagagtag agaaggaccg
ccgttagagc acttgatgca tgcgtacgtc 120 gatccggcgg accgatctgc
ttttgcttgt gtgcttggtg agaaggtccc tgttggagaa 180 gcagggcacg
tgcagagaca cgccggagca cggccgccgc cgatctaccg acctcccaca 240
cctgccttgt ggtgtggggg tggaggtcgt cggtggaagc gatagctgtc gttgttgctt
300 cgatgttgtt agctcctcct gcacgtgctc cccttctcca ccacggcctt
ctcaccaccc 360 tcctcccccg gcggcggcgg cggcggaccg cccttgccgc
gatcaataat gaaaccaaaa 420 gccgacagta tttgagcagg aaatacaaga
ggcggatatc ccactgctag cacttctgcg 480 ttgatcatgt tcatctggaa
caaaataata ctcggcgact ttacagcgag tgcagcatg 539 229 485 DNA
artificial sequence Synthetic construct 229 gcatctgctg ttctttattt
ctatacatac atatatacta tcaccggtta tttgcttctc 60 tattctgtcc
gagtacttta cggtgttccg cacatagatc tcgtggccgg cggttttgcg 120
ctttcgcttg cgtttcttgg ccctgctggt gtttgaccgg accgaacggg ggcagatcga
180 tgctttgggt ttgaagatac gtggcaaaac taggaatgaa gggtcgttcc
gaagggctgg 240 ttccgctgct cgttcatggt tcccactatc ctatctcatc
atgtgtatat atgtattcca 300 tgggggaggg tttctctcgt ctttgagata
ggcttgtggt ttgcatgacc gaggagctgc 360 accgccccct tgctggccgc
tctttcctgg ttctgccacg tatcatcctg atccacccct 420 ccattttttt
ttgcttgttg tgtccttcct gggacctgag atctgaggct cgtggtggct 480 cactg
485 230 21 RNA artificial sequence Synthetic construct 230
uuuccugguu cugccacgua u 21 231 485 DNA artificial sequence
Synthetic construct 231 gcatctgctg ttctttattt ctatacatac atatatacta
tcaccggtta tttgcttctc 60 tattctgtcc gagtacttta cggtgttccg
cacatagatc tcgtggccgg cggttttgcg 120 ctttcgcttg cgtttcttgg
ccctgctggt gtttgaccgg accgaacggg ggcagatcga 180 tgctttgggt
ttgaagtctc tggcagtaac tgacaatgaa gggtcgttcc gaagggctgg 240
ttccgctgct cgttcatggt tcccactatc ctatctcatc atgtgtatat atgtattcca
300 tgggggaggg tttctctcgt ctttgagata ggcttgtggt ttgcatgacc
gaggagctgc 360 accgccccct tgctggccgc tctttgtccg tttctgccag
agacatcctg atccacccct 420 ccattttttt ttgcttgttg tgtccttcct
gggacctgag atctgaggct cgtggtggct 480 cactg 485 232 230 DNA
Diabrotica virgifera 232 agaagcctgg caatttccaa ggtgattttg
tccgtttctg ccagagatgc tttacctacc 60 agctgcacaa tttcggctag
atcatcttct tcctgaagaa tttccttaac tttggttcta 120 agaggaataa
actcttggaa gtttttgtca taaaagtcgt ccaatgctct taaatatttg 180
gaatatgatc caagccagtc tactgaaggg aagtgcttac gttgggcaag 230 233 21
RNA artificial sequence Synthetic construct 233 uuuguccguu
ucugccagag a 21 234 21 RNA Glycine max 234 ugagaccaaa ugagcagcug a
21 235 20 RNA Glycine max 235 gcugcucauc uguucucagg 20 236 762 DNA
Glycine max 236 aaaattcatt acattgataa aacacaattc aaaagatcaa
tgttccactt catgcaaaga 60 catttccaaa atatgtgtag gtagaggggt
tttacaggat cgtcctgaga ccaaatgagc 120 agctgaccac atgatgcagc
tatgtttgct attcagctgc tcatctgttc tcaggtcgcc 180 cttgttggac
tgtccaactc ctactgattg cggatgcact tgccacaaat gaaaatcaaa 240
gcgaggggaa aagaatgtag agtgtgacta cgattgcatg catgtgattt aggtaattaa
300 gttacatgat tgtctaattg tgtttatgga attgtatatt ttcagaccag
gcacctgtaa 360 ctaattatag gtaccatacc ttaaaataag tccaactaag
tccatgtctg tgatttttta 420 gtgtcacaaa tcacaatcca ttgccattgg
ttttttaatt tttcattgtc tgttgtttaa 480 ctaactctag ctttttagct
gcttcaagta cagattcctc aaagtggaaa atgttctttg 540 aagtcaataa
aaagagcttt gatgatcatc tgcattgtct aagttggata aactaattag 600
agagaacttt tgaactttgt ctaccaaata tctgtcagtg tcatctgtca gttctgcaag
660 ctgaagtgtt gaatccacga ggtgcttgtt gcaaagttgt gatattaaaa
gacatctacg 720 aagaagttca agcaaaactc tttttggcaa aaaaaaaaaa aa 762
237 21 RNA Glycine max 237 uagaagcucc ccauguucuc a 21 238 19 RNA
Glycine max 238 gagcaugggu aacuucuau 19 239 346 DNA Glycine max
unsure (1)..(346) unsure at all n locations 239 ttttctccta
actttgagag catgggtaac ttctattttt atctctgncc ccntttcctt 60
catcttttct tcaaccttta ttcgttcctt tttcaactgt taaaaggcct gactatgttg
120 aggaaattaa gaaaatgggt ttgttgccga tgccagcaga atagaagctc
cccatgttct 180 caccgttagc agaaaacggg tgttatctgg ataagaccgc
caggttccat tcccttgttt 240 gccagcacca ccatcacttc ttcactctag
tatccgattt ttttaaagga tcgttgtccc 300 ttgccttctg gtggacttct
atggagaagt ttcttcacac cctatg 346 240 21 RNA Glycine max 240
uguugcgggu aucuuugccu c 21 241 20 RNA Glycine max 241 ggcguagauc
cccacaacag 20 242 434 RNA Glycine max 242 aacacuaggg uuugcauucc
uccuuuagcc gcaacccaua uucuaagcuu ccuuucuccu 60 acuguucugu
gucgggccag caaaacuguu gcggguaucu uugccucuga aggaaaguug 120
ugccuauuau uauggcuuau ugcuuuagug gcguagaucc ccacaacagu uaugcuugca
180 cugccuuuug ucuccgagac uaacaaauuu gauuguaugu ucucuuguug
cuaaacuuuu 240 gauuuugacc cgaacugcau gaggcaugaa aguuucauag
ugguucaacc acaguaaaau 300 aggaugguca guuuaugucu ggguuuuaua
agaauuuuua gaucugucuu gauuacugga 360 ccauuggaug aacacccugu
ugguguugaa aaaguagcuu cagccuucug gaugugguua 420 ugagcuuucg augc 434
243 21 RNA Glycine max 243 ugcgaguguc uucgccucug a 21 244 21 RNA
Glycine max 244 ggaggcguag auacucacac c 21 245 439 DNA Glycine max
245 aattagggtt ctctggtcct cccgcccccg gtgctggcat tctcaagcca
gtgaaatcgg 60 tgcgagtgtc ttcgcctctg agagagatac tatgagatct
caagcctcgg aggcgtagat 120 actcacacct ctttttctgg ctatctcacc
actgctcttt tccgccgggg cacgaaggtc 180 cttcgcctca ccaaatttcg
ttctttaaat ttcacctata tatgtgtata tttttaataa 240 taataattag
gtttaaaggg aaaaaaagtc gcttcttaat cctttattca ttgtatgcag 300
aacgatttga ttcgtgatga taatgtatgg atctgtatag tcggttgctc tagtatccaa
360 taatttgatt tctaacaaat taatatgtag tggccttctt ggacatgaaa
aaaattcttg 420 aaattgggtt gttaggtta 439 246 22 RNA Glycine max 246
uugccgauuc cacccauucc ua 22 247 20 RNA Glycine max 247 gcugcucauc
uguucucagg 20 248 495 DNA Glycine max 248 gactagatgt gaatgtgatt
catattcata gagagagaaa gggaaaggga gaagagcttg 60 aggaagtgat
gggagatggg agggtcggta aagaatatat ctgagactcg actcaatctc 120
gatctctctc agtgttgtgt tgttttgttt atccttttgc cgattccacc cattcctatg
180 atttccttcg gttcctctct ttccactctc ctctccgctc tttcctcttg
ttatggtaag 240 cacctttctt cttcagatct gctctttata ccatacactt
attatagatc taagttttta 300 tggaccttaa ctatcttcct tgatctctta
ttaattttaa ccgctctctc tttgttgctg 360 gacatgttac ttcaagataa
caaattgctt ttttattttt catcttttct ctcgttctct 420 tgtttaaggt
ttctataaat catcgatgag atacctataa taatatactt attacagaca 480
aaaaaaaaaa aaaaa 495 249 825 DNA Glycine max 249 ctgttctata
ttgatagagt gagaaaggga aaggaagaag agcttgagga agtgatggga 60
gatgggaggg tcggtaaagg ataacagcgt ctctatgatt aattgttgtg ttgtttattc
120 ttttgccgat tccacccatt cctatgattt tctttggttc ctttctttcc
actctcctct 180 ccgctctcca caatctgtta tggtcatgaa gctgccggtc
tgactgggtt acattcaaga 240 caagaaaaaa caacaaatcg cttctttctt
ttttcgtctt ttctttcctt tttcttgttt 300 aaggcttaac aaattatctc
agctctagta gatgtagatt attacagaca gatgctagtt 360 aattagctag
ctccacaaga tgtttaaaaa tgtgatctat ccatatcaag ctggaccaaa 420
tccaaataat tttactggag cttttctttc ttggtaaaag ctggacattt ttaaaggttt
480 gggtgccact ataacgccac caaagttttc tttcttgatt tttaaccaag
ttacattttt 540 ttccctaaat tattccagcg ctaataaata ctgaattttc
tgtttttttt ataaaaagaa 600 ttttacatta aattctttga aaataatttc
attttggttg ttaatatttt tttttagttc 660 ataacaaatt aacgaatttt
gtatttattt ttttataaaa aattattaga aaataacata 720 ttaaatcaag
caaaaaaatg tattttatta aaataaaaaa gtgaaggaaa aaattattta 780
aaaccaacac accaacatat tttaactttt ttattaaact aaacg 825 250 21 RNA
Glycine max 250 ccagcugcuc auuuggucac u 21 251 21 RNA Glycine max
251 agaggacaug gggagguucu a 21 252 21 RNA Glycine max 252
ucagcugcuc aucuguucuc a 21 253 21 RNA Glycine max 253 ccagcugcuc
auuuggucac u 21 254 21 RNA Glycine max 254 ucagcucuuc uuuuggucuc u
21 255 21 RNA Glycine max 255 ucagcuacug aucuggucuc a 21 256 21 RNA
Glycine max 256 ucagcuguuc cuuuguucuc u 21 257 21 RNA Glycine max
257 ucagcuguuc cuuuguucuc u 21 258 21 RNA Glycine max 258
guagcuucuc acuuggucuu a 21 259 21 RNA Glycine max 259 uuagcugcuu
cuucggucuc u 21 260 21 RNA Glycine max 260 uuagaugcuu guuuggucuu u
21 261 21 RNA Glycine max 261 ugagaacaug gggagccucu a 21 262 21 RNA
Glycine max 262 agaggacaug gggagauucu a 21 263 21 RNA Glycine max
263 agaggacaug gggagguucu a 21 264 21 RNA Glycine max 264
ugagaacaug ggaaucuucu a 21 265 21 RNA Glycine max 265 aaagaacaug
gggagccucu a 21 266 21 RNA Glycine max 266 ugagaacaug ggggauuucu a
21 267 21 RNA Glycine max 267 ugugaaggug gggagcuucu u 21 268 21 RNA
Glycine max 268 ggagaacaug cagagcuucu g 21 269 21 RNA Glycine max
269 ugagaaacug gggagcuuuu c 21 270 20 RNA Glycine max 270
ugagaacugg ugagcuucug 20 271 20 RNA Glycine max 271 ugaguacugg
ggagcuucuc 20 272 21 RNA Glycine max 272 ugagagcaug gguaacuucu a 21
273 20 RNA Glycine max 273 ugagcacugg ggagcuucuc 20 274 20 RNA
Glycine max 274 ugagcacugg ggagcuucuc 20 275 20 RNA Glycine max 275
ugagcacugg ggagcuucuc 20 276 21 RNA Glycine max 276 ugagaacaug
ggaacuuucu a 21 277 21 RNA Glycine max 277 ugagagcaug gguaacuucu a
21 278 21 RNA Glycine max 278 ugagaaccug guaagcuucu g 21 279 21 RNA
Glycine max 279 ugagaacauc gaaagcuucu u 21 280 21 RNA Glycine max
280 ugaggacaag gggagcuuau g 21 281 21 RNA Glycine max 281
cuaaaacaug gggagcuucu u 21 282 21 RNA Glycine max 282 ugaggaaaua
gggaguuucu g 21 283 21 RNA Glycine max 283 ugagaacaua gugaguuuuu u
21 284 21 RNA Glycine max 284 uaggaucgug gggagcuucu c 21 285 21 RNA
Glycine max 285 uaggaucgug gggagcuucu c 21 286 21 RNA Glycine max
286 uaggaucgug gggagcuucu c 21 287 21 RNA Glycine max 287
gaugaauaug gggaguuucu a 21 288 21 RNA Glycine max 288 ggggcaagga
cauccgcaac g 21 289 21 RNA Glycine max 289 aaggcaaagu ugcccgcgac g
21 290 21 RNA Glycine max 290 gaggcaaaga ugcgagcaac g 21 291 21 RNA
Glycine max 291 gcggcaaaga uacucacaac c 21 292 21 RNA Glycine max
292 aacgcaaaga gaccuguaac a 21 293 21 RNA Glycine max 293
aaggcaaaga ugccagcgac g
21 294 21 RNA Glycine max 294 gagccaaaga gacccgugac g 21 295 21 RNA
Glycine max 295 aaggcauaga uagucgcagc a 21 296 21 RNA Glycine max
296 aaggcaaaga ugccagcaau g 21 297 21 RNA Glycine max 297
uagggaaaga uacauguaac a 21 298 21 RNA Glycine max 298 gaggcaaagu
uguucgcaau g 21 299 21 RNA Glycine max 299 caggcaaaga ugucugcaau u
21 300 21 RNA Glycine max 300 uagguaugga uacuugcaac a 21 301 21 RNA
Glycine max 301 aaggcaaagc ugcccgcgau g 21 302 21 RNA Glycine max
302 ucaggggagg agacacucgc a 21 303 21 RNA Glycine max 303
uuagaggcaa agacacucgu c 21 304 21 RNA Glycine max 304 ucagaggaga
agauacucgu g 21 305 21 RNA Glycine max 305 ucagaggaga agacacgcgc a
21 306 21 RNA Glycine max 306 ucagagggga agacacacgc u 21 307 21 RNA
Glycine max 307 ucagagggga agacacacgc u 21 308 21 RNA Glycine max
308 ucagagguga ggacacacgc u 21 309 21 RNA Glycine max 309
ccagaggcgg augcauucgc a 21 310 21 RNA Glycine max 310 acagaggcag
ggacacuugc a 21 311 21 RNA Glycine max 311 gcagagguga agaagcuugc a
21 312 21 RNA Glycine max 312 uuagaggaga ggauacucgc g 21 313 21 RNA
Glycine max 313 gcagagguga agaagcuugc a 21 314 21 RNA Glycine max
314 ucagaggcaa agauacccgc a 21 315 21 RNA Glycine max 315
uuagagggga agacacgcgc u 21 316 21 RNA Glycine max 316 ucagagggga
agacacccgu g 21 317 21 RNA Glycine max 317 ucagaggcua agagacuugu a
21 318 21 RNA Glycine max 318 ucagagggga agacacgcgu g 21 319 21 RNA
Glycine max 319 ucagagggga agacacccgu g 21 320 21 RNA Glycine max
320 ucagagggga agacacacgu u 21 321 21 RNA Glycine max 321
ucagagggga agacacacgu u 21 322 21 RNA Glycine max 322 ucagggguga
agacacacgu a 21 323 21 RNA Glycine max 323 ucagagggga agacacccgu g
21 324 21 RNA Glycine max 324 ucagaaacga agacgcucgu u 21 325 21 RNA
Glycine max 325 uccgagggga agauacucgu u 21 326 21 RNA Glycine max
326 uccgagggga agauacucgu c 21 327 21 RNA Glycine max 327
uccgagggga agauacucgu c 21 328 21 RNA Glycine max 328 gcagaggcug
uggcacucgc a 21 329 21 RNA Glycine max 329 uuagaggcga ggacacacgu u
21 330 21 RNA Glycine max 330 uccgaggaga agauacucgu u 21 331 21 RNA
Glycine max 331 ucaguggcga aggcguucgu c 21 332 21 RNA Glycine max
332 uuagagguga uggcacucgu g 21 333 22 RNA Glycine max 333
ggggaauggg uggaaacggc aa 22 334 22 RNA Glycine max 334 ugggaauggg
ugggaugggu aa 22 335 22 RNA Glycine max 335 ugggaauggg ugggaugggu
aa 22 336 22 RNA Glycine max 336 auggaacugg uggaauuggc aa 22 337 22
RNA Glycine max 337 cgggaaaggu uggaauuggc aa 22 338 22 RNA Glycine
max 338 uaggaauggg uggauuuugc aa 22 339 20 RNA Glycine max 339
ggaaugggug gcgugggcaa 20 340 22 RNA Glycine max 340 caggaaaggg
gggaguuggc aa 22 341 22 RNA Glycine max 341 uagcaauggg uuggaucggu
ga 22 342 22 RNA Glycine max 342 guugaauggg uggaauugga aa 22 343 22
RNA Glycine max 343 guugaauggg uggaauugga aa 22 344 22 RNA Glycine
max 344 aaggaauugg gggaauuggu ac 22 345 22 RNA Glycine max 345
cacgaguggg gggaaucggc gg 22 346 22 RNA Glycine max 346 guggaauggg
uggucuuggu aa 22
* * * * *
References