U.S. patent application number 14/627543 was filed with the patent office on 2015-06-11 for micrornas.
This patent application is currently assigned to THE ROCKEFELLER UNIVERSITY. The applicant listed for this patent is THE ROCKEFELLER UNIVERSITY. Invention is credited to Nam-Hai CHUA, Shih-Shun LIN, Qi-Wen NIU, Jose L. REYES-TABOADA, Takashi SOYANO, Xiuren ZHANG.
Application Number | 20150159157 14/627543 |
Document ID | / |
Family ID | 36203437 |
Filed Date | 2015-06-11 |
United States Patent
Application |
20150159157 |
Kind Code |
A1 |
REYES-TABOADA; Jose L. ; et
al. |
June 11, 2015 |
MicroRNAs
Abstract
The invention provides methods and compositions useful in target
sequence suppression, target sequence validation and target
sequence down regulation. The invention provides polynucleotide
constructs useful for gene silencing or RNA down regulation, as
well as cells, plants and seeds comprising the polynucleotides. The
invention also provides a method for using microRNA to silence a
target sequence or to down regulate RNA.
Inventors: |
REYES-TABOADA; Jose L.;
(Mexico City, MX) ; ZHANG; Xiuren; (New York,
NY) ; SOYANO; Takashi; (New York, NY) ; CHUA;
Nam-Hai; (New York, NY) ; NIU; Qi-Wen; (New
York, NY) ; LIN; Shih-Shun; (New York, NY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
THE ROCKEFELLER UNIVERSITY |
New York |
NY |
US |
|
|
Assignee: |
THE ROCKEFELLER UNIVERSITY
New York
NY
|
Family ID: |
36203437 |
Appl. No.: |
14/627543 |
Filed: |
February 20, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11247587 |
Oct 12, 2005 |
8975471 |
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14627543 |
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PCT/US2004/033370 |
Oct 12, 2004 |
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11247587 |
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60671089 |
Apr 14, 2005 |
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Current U.S.
Class: |
800/285 ;
435/412; 435/414; 435/415; 435/416; 435/419; 435/468; 536/24.5;
800/298; 800/306; 800/312; 800/314; 800/317.3; 800/320; 800/320.1;
800/320.2; 800/320.3; 800/322 |
Current CPC
Class: |
C12N 15/8283 20130101;
C12N 2310/141 20130101; C12N 15/113 20130101; Y02A 40/146 20180101;
C12N 15/8218 20130101; C12N 15/827 20130101; C12N 15/8261 20130101;
C12N 15/8216 20130101 |
International
Class: |
C12N 15/113 20060101
C12N015/113 |
Claims
1. A nucleic acid construct comprising a promoter functional in a
plant cell operatively linked to a polynucleotide comprising two or
more modified miRNA precursors operatively linked together, wherein
each modified miRNA precursor comprises a fragment of a plant miRNA
primary transcript that is modified in the miRNA sequence and the
miRNA complementary sequence, further wherein each modified miRNA
precursor is capable of forming a double-stranded RNA or an RNA
hairpin, wherein each modified miRNA is an miRNA modified to be (i)
fully complementary to a target sequence, (ii) fully complementary
to a target sequence except for GU base pairing or (iii) fully
complementary to a target sequence in the first ten nucleotides
counting from the 5' end of the miRNA, and wherein the target
sequence of each modified miRNA is different.
2. The nucleic acid contruct of claim 1, wherein the plant miRNA
transcripts are selected from the group consisting of Arabidopsis,
tomato, soybean, rice and corn primary miRNA transcripts.
3. The nucleic acid contruct of claim 1, wherein at least two of
the plant miRNA primary transcripts are the same.
4. The nucleic acid contruct of claim 1, wherein at least two of
the plant miRNA primary transcripts are different.
5. The nucleic acid contruct of claim 1, wherein a target sequence
is in a non-coding region of RNA.
6. The nucleic acid contruct of claim 1, wherein a target sequence
is in a coding region of RNA.
7. The nucleic acid contruct of claim 1, wherein a target sequence
contains a splice site of RNA.
8. The nucleic acid contruct of claim 1, wherein at least one of
the plant miRNA primary transcripts is a plant miR159 primary
transcript.
9. The nucleic acid contruct of claim 1, wherein at least one of
the plant miRNA primary transcripts is a plant miR169 primary
transcript.
10. A plant cell comprising the nucleic acid construct of claim
1.
11. The plant cell of claim 10, wherein the plant is selected from
the group consisting of corn, wheat, rice, barley, oats, sorghum,
millet, sunflower, safflower, cotton, soy, canola, alfalfa,
Arabidopsis, and tobacco.
12. The plant cell of claim 10, wherein the nucleic acid construct
is inserted in an intron of a gene or transgene of the plant
cell.
13. A transgenic plant comprising the nucleic acid construct of
claim 1.
14. The transgenic plant of claim 13, wherein the plant is selected
from the group consisting of corn, wheat, rice, barley, oats,
sorghum, millet, sunflower, safflower, cotton, soy, canola,
alfalfa, Arabidopsis, and tobacco.
15. The transgenic plant of claim 13, wherein the nucleic acid
construct is inserted into an intron of a gene or transgene of the
transgenic plant.
16. A seed of the transgenic plant of of claim 13.
17. A seed of the transgenic plant of of claim 15.
18. A method for down regulating two or more different target
sequences in a plant cell comprising: (a) transforming a plant cell
with a nucleic acid construct comprising a promoter functional in a
plant cell operatively linked to a polynucleotide comprising two or
more modified miRNA precursors operatively linked together, wherein
each modified miRNA precursor comprises a fragment of a plant miRNA
primary transcript that is modified in the miRNA sequence and the
miRNA complementary sequence, further wherein each modified miRNA
precursor is capable of forming a double-stranded RNA or an RNA
hairpin, wherein each modified miRNA is an miRNA modified to be (i)
fully complementary to a target sequence, (ii) fully complementary
to a target sequence except for GU base pairing or (iii) fully
complementary to a target sequence in the first ten nucleotides
counting from the 5' end of the miRNA, and wherein the target
sequence of each modified miRNA is different; and (b) expressing
the nucleic acid construct for a time sufficient to produce each
modified miRNA, wherein each modified miRNA down regulates a
different target sequence.
19. The method of claim 18, wherein each modified miRNA binds to
its target sequence and the double-stranded RNA is cleaved.
20. The method of claim 18, wherein the plant miRNA transcripts are
selected from the group consisting of Arabidopsis, tomato, soybean,
rice and corn primary miRNA transcripts.
21. The method of claim 18, wherein at least two of the plant miRNA
primary transcripts are the same.
22. The method of claim 18, wherein at least two of the plant miRNA
primary transcripts are different.
23. The method of claim 18, wherein a target sequence is in a
non-coding region of RNA.
24. The method of claim 18, wherein a target sequence is in a
coding region of RNA.
25. The method of claim 18, wherein a target sequence contains a
splice site of RNA.
26. The method of claim 18, wherein at least one of the plant miRNA
primary transcripts is a plant miR159 primary transcript.
27. The method of claim 18, wherein at least one of the plant miRNA
primary transcripts is a plant miR169 primary transcript.
28. The method of claim 18, wherein the nucleic acid construct is
inserted in an intron of a gene or transgene of the plant cell.
29. A method for down regulating two or more different target
sequences in a plant, the method comprises obtaining a transgenic
plant from the transformed plant cell of step (a) of claim 18 and
expressing the nucleic acid construct of step (b) in the transgenic
plant.
30. The method of claim 29, wherein each modified miRNA binds to
its target sequence and the double-stranded RNA is cleaved.
31. The method of claim 29, wherein the plant miRNA transcripts are
selected from the group consisting of Arabidopsis, tomato, soybean,
rice and corn primary miRNA transcripts.
32. The method of claim 29, wherein at least two of the plant miRNA
primary transcripts are the same.
33. The method of claim 29, wherein at least two of the plant miRNA
primary transcripts are different.
34. The method of claim 29, wherein a target sequence is in a
non-coding region of RNA.
35. The method of claim 29, wherein a target sequence is in a
coding region of RNA.
36. The method of claim 29, wherein a target sequence contains a
splice site of RNA.
37. The method of claim 29, wherein at least one of the plant miRNA
primary transcripts is a plant miR159 primary transcript.
38. The method of claim 29, wherein at least one of the plant miRNA
primary transcripts is a plant miR169 primary transcript.
39. The method of claim 29, wherein the nucleic acid construct is
inserted in an intron of a gene or transgene of the plant cell.
40. The method of claim 29, further comprising harvesting seed from
the transgenic plant.
41. The method of claim 40, further comprising growing a plant from
the seed and expressing the nucleic acid construct in the growing
plant.
42. The method of claim 40, further comprising growing a plant from
the seed and crossing the growing plant with a different plant to
produce progeny seed comprising the nucleic acid construct.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] The present application is a division of U.S. patent
application Ser. No. 11/247,587 filed on 12 Oct. 2005, which in
turn is a continuation-in-part of PCT international application No.
PCT/US2004/033379, filed on 12 Oct. 2004. U.S. patent application
Ser. No. 11/247,587 is also related to and claims priority under 35
U.S.C. .sctn.119(e) to U.S. provisional patent application Ser. No.
60/671,089, filed on 14 Apr. 2005. Each application is incorporated
herein by reference.
SEQUENCE SUBMISSION
[0002] The present application is being filed along with a Sequence
Listing in electronic format. The Sequence Listing is entitled
2312-136SequenceListing.txt, created on 5 Feb. 2015 and is 69 kb in
size. The information in the electronic format of the Sequence
Listing is incorporated herein by reference in its entirety.
FIELD OF THE INVENTION
[0003] The field of the present invention relates generally to
plant molecular biology and plant biotechnology. More specifically
it relates to constructs and methods to suppress the expression of
targeted genes or to down regulate targeted genes.
BACKGROUND OF THE INVENTION
[0004] RNA interference refers to the process of sequence-specific
post-transcriptional gene silencing in animals mediated by short
interfering RNAs (siRNAs) (Fire et al. (1998) Nature 391:806-811).
The corresponding process in plants is commonly referred to as
post-transcriptional gene silencing (PTGS) or RNA silencing and is
also referred to as quelling in fungi. The process of
post-transcriptional gene silencing is thought to be an
evolutionarily-conserved cellular defense mechanism used to prevent
the expression of foreign genes and is commonly shared by diverse
flora and phyla (Fire (1999) Trends Genet. 15:358-363). Such
protection from foreign gene expression may have evolved in
response to the production of double-stranded RNAs (dsRNAs) derived
from viral infection or from the random integration of transposon
elements into a host genome via a cellular response that
specifically destroys homologous single-stranded RNA of viral
genomic RNA. The presence of dsRNA in cells triggers the RNAi
response through a mechanism that has yet to be fully
characterized.
[0005] A new class of small RNA molecules is involved in regulating
gene expression in a number of eukaryotic organisms ranging from
animals to plants. These short RNAs or microRNAs (miRNAs; miRs) are
20-22 nucleotide-long molecules that specifically base-pair to
target messenger-RNAs to repress their translation or to induce
their degradation. Recent reports have identified numerous miRNAs
from vertebrates, Caenorhabditis elegans, Drosophila and
Arabidopsis thaliana (Bartel (2004) Cell 116:281-297; He and Hannon
(2004) Nature Reviews Genetics 5:522-531).
[0006] Viruses such as Turnip Mosaic Virus (TuMV) and Turnip Yellow
Mosaic Virus (TYMV) cause considerable crop loss world-wide and
have serious economic impact on agriculture (Morch et al. (1998)
Nucleic Acids Res 16:6157-6173; Skotnicki et al. (1992) Arch Virol
127:25-35; Tomlinson (1987) Ann Appl Biol 110:661-681). Most if not
all plant viruses encode one or more proteins that are able to
suppress the host's post-transcriptional gene silencing (PTGS)
mechanism so as to ensure their successful replication in host
cells. The PTGS is a mechanism that a plant host uses to defend
against viruses by triggering breakdown of double stranded RNAs
which are produced as intermediates in viral genome replication
(Bernstein et al. (2001) Nature 409:363-366; Hamilton and Baulcombe
(1999) Science 286:950-952; Zamore et al. (2000) Cell
31:25-33).
[0007] Reduction of the activity of specific genes (also known as
gene silencing, or gene suppression), including virus genes, is
desirable for several aspects of genetic engineering in plants.
There is still a need for methods and constructs that induce gene
suppression against a wide selection of target genes, and that
result in effective silencing of the target gene at high
efficiency.
SUMMARY OF THE INVENTION
[0008] According to one aspect, the present invention provides a
method of down regulating a target sequence in a cell and a nucleic
acid construct for use in this method, as well as a polynucleotide
for use in the nucleic acid construct. The method comprises
introducing into the cell a nucleic acid construct capable of
producing miRNA and expressing the nucleic acid construct for a
time sufficient to produce the miRNA, wherein the miRNA inhibits
expression of the target sequence. The nucleic acid construct
comprises a polynucleotide encoding a modified miRNA precursor
capable of forming a double-stranded RNA or a hairpin, wherein the
modified miRNA precursor comprises a modified miRNA and a sequence
complementary to the modified miRNA, wherein the modified miRNA is
a miRNA modified to be (i) fully complementary to the target
sequence or (ii) fully complementary to the target sequence except
for GU base pairing. As is well known in the art, the pre-miRNA
forms a hairpin which in some cases the double-stranded region may
be very short, e.g., not exceeding 21-25 by in length. The nucleic
acid construct may further comprise a promoter operably linked to
the polynucleotide. The cell may be a plant cell, either monocot or
dicot, including, but not limited to, corn, wheat, rice, barley,
oats, sorghum, millet, sunflower, safflower, cotton, soy, canola,
alfalfa, Arabidopsis, and tobacco. The promoter may be a
pathogen-inducible promoter or other inducible promoters. The
binding of the modified miRNA to the target RNA leads to cleavage
of the target RNA. The target sequence of a target RNA may be a
coding sequence, an intron or a splice site.
[0009] According to another aspect, the present invention provides
an isolated polynucleotide encoding a modified plant miRNA
precursor, the modified precursor is capable of forming a
double-stranded RNA or a hairpin and comprises a modified miRNA and
a sequence complementary to the modified miRNA, wherein the
modified miRNA is a miRNA modified to be (i) fully complementary to
the target sequence or (ii) fully complementary to the target
sequence except for GU base pairing. Expression of the
polynucleotide produces a miRNA precursor which is processed in a
host cell to provide a mature miRNA which inhibits expression of
the target sequence. The polynucleotide may be a nucleic acid
construct or may be the modified plant miRNA precursor. The nucleic
acid construct may further comprise a promoter operably linked to
the polynucleotide. The promoter may be a pathogen-inducible
promoter or other inducible promoter. The binding of the modified
miRNA to the target RNA leads to cleavage of the target RNA. The
target sequence of a target RNA may be a coding sequence, a
non-coding sequence or a splice site.
[0010] According to another aspect, the present invention provides
a nucleic acid construct for suppressing a multiple number of
target sequences. The nucleic acid construct comprises at least two
and up to 45 or more polynucleotides, each of which encodes a miRNA
precursor capable of forming a double-stranded RNA or a hairpin.
Each miRNA is substantially complementary to a target or is
modified to be complementary to a target as described herein. In
some embodiments, each of the polynucleotides encoding precursor
miRNAs in the construct is individually placed under control of a
single promoter. In some embodiments, the multiple polynucleotides
encoding precursor miRNAs are operably linked together such that
they can be placed under the control of a single promoter. The
promoter may be operably linked to the construct of multiple miRNAs
or the construct of multiple miRNAs may be inserted into a host
genome such that it is operably linked to a single promoter. The
promoter may be a pathogen-inducible promoter or other inducible
promoter. In some embodiments, the multiple polynucleotides are
linked one to another so as to form a single transcript when
expressed. Expression of the polynucleotides in the nucleic acid
construct produces multiple miRNA precursors which are processed in
a host cell to provide multiple mature miRNAs, each of which
inhibits expression of a target sequence. In one embodiment, the
binding of each of the mature miRNA to each of the target RNA leads
to cleavage of each of the target RNA. The target sequence of a
target RNA may be a coding sequence, a non-coding sequence or a
splice site.
[0011] According to another aspect, the present invention provides
a method of down regulating a multiple number of target sequences
in a cell. The method comprises introducing into the cell a nucleic
acid construct capable of producing multiple miRNAs and expressing
the nucleic acid construct for a time sufficient to produce the
multiple miRNAs, wherein each of the miRNAs inhibits expression of
a target sequence. The nucleic acid construct comprises at least
two and up to 45 or more polynucleotides, each of which encodes a
miRNA precursor capable of forming a double-stranded RNA or a
hairpin. Each miRNA is substantially complementary to a target or
is modified to be complementary to a target as described herein. In
some embodiments, each of the polynucleotides encoding precursor
miRNAs in the construct is individually placed under control of a
single promoter. In some embodiments, the multiple polynucleotides
encoding precursor miRNAs are linked together such that they can be
under the control of a single promoter as described herein. In some
embodiments, the multiple polynucleotides are linked one to another
so as to form a single transcript when expressed. In some
embodiments, the construct may be a hetero-polymeric pre-miRNA or a
homo-polymeric pre-miRNA. Expression of the polynucleotides in the
nucleic acid construct produces multiple miRNA precursors which are
processed in a host cell to provide multiple mature miRNAs, each of
which inhibits expression of a target sequence. In one embodiment,
the binding of each of the mature miRNA to each of the target RNA
leads to cleavage of each of the target RNA. The target sequence of
a target RNA may be a coding sequence, a non-coding sequence or a
splice site.
[0012] According to a further aspect, the present invention
provides a cell comprising the isolated polynucleotide or nucleic
acid construct of the present invention. In some embodiments, the
isolated polynucleotide or nucleic acid construct of the present
invention may be inserted into an intron of a gene or a transgene
of the cell. The cell may be a plant cell, either a monocot or a
dicot, including, but not limited to, corn, wheat, rice, barley,
oats, sorghum, millet, sunflower, safflower, cotton, soy, canola,
alfalfa, Arabidopsis, and tobacco.
[0013] According to another aspect, the present invention provides
a transgenic plant comprising the isolated polynucleotide or
nucleic acid construct. In some embodiments, the isolated
polynucleotide or nucleic acid construct of the present invention
may be inserted into an intron of a gene or a transgene of the
transgenic plant. The transgenic plant may be either a monocot or a
dicot, including, but not limited to, corn, wheat, rice, barley,
oats, sorghum, millet, sunflower, safflower, cotton, soy, canola,
alfalfa, Arabidopsis, and tobacco.
[0014] According to a further aspect, the present invention
provides a method of inhibiting expression of a target sequence in
a cell comprising: (a) introducing into the cell a nucleic acid
construct comprising a modified plant miRNA precursor comprising a
first and a second oligonucleotide, wherein at least one of the
first or the second oligonucleotides is heterologous to the
precursor, wherein the first oligonucleotide encodes an RNA
sequence substantially identical to the target sequence, and the
second oligonucleotide encodes a miRNA substantially complementary
to the target sequence, whereby the precursor encodes a miRNA; and
(b) expressing the nucleic acid construct for a time sufficient to
produce the miRNA, wherein the miRNA inhibits expression of the
target sequence.
[0015] According to another aspect, the present invention provides
an isolated polynucleotide comprising a modified plant miRNA
precursor, the modified precursor comprising a first and a second
oligonucleotide, wherein at least one of the first or the second
oligonucleotides is heterologous to the precursor, wherein the
first oligonucleotide encodes an RNA sequence substantially
identical to a target sequence, and the second oligonucleotide
comprises a miRNA substantially complementary to the target
sequence, wherein expression of the polynucleotide produces the
miRNA which inhibits expression of the target sequence. The present
invention also relates to a cell comprising this isolated
polynucleotide. The cell may be a plant cell, either monocot or
dicot, including, but not limited to, corn, wheat, rice, barley,
oats, sorghum, millet, sunflower, safflower, cotton, soy, canola,
alfalfa, Arabidopsis, and tobacco.
[0016] According to a further aspect, the present invention
provides for a method of inhibiting expression of a target sequence
in a cell, such as any of those herein described that further
comprises producing a transformed plant, wherein the plant
comprises the nucleic acid construct which encodes the miRNA. The
present invention also relates to a plant produced by such methods.
The plant may a monocot or a dicot, including, but not limited to,
corn, wheat, rice, barley, oats, sorghum, millet, sunflower,
safflower, cotton, soy, canola, alfalfa, Arabidopsis, and
tobacco.
BRIEF DESCRIPTION OF THE FIGURES
[0017] FIG. 1 shows the predicted hairpin structure formed by the
sequence (SEQ ID NO:175) surrounding miR172a-2. The mature microRNA
is indicated by a grey box.
[0018] FIG. 2 shows the miR172a-2 overexpression phenotype. a, Wild
type (Columbia ecotype) plant, 3.5 weeks old. b, EAT-D plant, 3.5
weeks old. c, Wild type flower. d, EAT-D flower. Note absence of
second whorl organs (petals). Arrow indicates sepal with ovules
along the margins and stigmatic papillae at the tip. e, Cauline
leaf margin from a 35S-EAT plant. Arrows indicate bundles of
stigmatic papillae projecting from the margin. f, Solitary
gynoecium (arrow) emerging from the axil of a cauline leaf of a
35S-EAT plant.
[0019] FIG. 3 shows the EAT gene contains a miRNA that is
complementary to APETALA2 (AP2). a, Location of the EAT gene on
chromosome 5. The T-DNA insertion and orientation of the 35S
enhancers is indicated. The 21-nt sequence corresponding to
miR172a-2 is shown below the EAT gene (SEQ ID NO:86). b, Putative
21-nt miR172a-2/AP2 RNA duplex is shown below the gene structure of
AP2. The GU wobble in the duplex is underlined. The sequence for
miR172a-2 is SEQ ID NO:48, and the sequence for APT2 RNA is SEQ ID
NO:47. c, Alignment of AP2 21-nt region (black bar) and surrounding
sequence with three other Arabidopsis AP2 family members, and with
two maize AP2 genes (IDS1 and GL15). The sequences are set forth in
SEQ ID NO:49 (AP2), SEQ ID NO:50 (At5g60120), SEQ ID NO:51
(At2g28550), SEQ ID NO:52 (At5g67180), SEQ ID NO:53 (IDS1) and SEQ
ID NO:54 (GL15). d, Alignment of miR172a-2 miRNA (black bar) and
surrounding sequence with miR172-like sequences from Arabidopsis,
tomato, soybean, potato and rice. The sequences are set forth in
SEQ ID NO:176 (mir172-2), SEQ ID NO:177 (mir172a-1), SEQ ID NO:178
(mir172d), SEQ ID NO:179 (mir172b), SEQ ID NO:180 (mir172c), SEQ ID
NO:181 (AI484737), SEQ ID NO:182 (BI320499), SEQ ID NO:183 (BQ1),
SEQ ID NO:184 (AP003277), SEQ ID NO:185 (AP004048), SEQ ID NO:186
(AP005247), SEQ ID NO:187 (ctg7420).
[0020] FIG. 4 shows the miR172a-2 miRNA expression. a, Northern
blot of total RNA from wild type (lanes 3 and 7) and EAT-D (lanes 4
and 8). Blots were probed with sense (lanes 1-4) or antisense
(lanes 5-8) oligo to miR172a-2 miRNA. 100 .mu.g of sense oligo
(lanes 2 and 6) and antisense oligo (lanes 1 and 5) were loaded as
hybridization controls. Nucleotide size markers are indicated on
the left. b, 51 nuclease mapping of miR172a-2 miRNA. A
5'-end-labeled probe undigested (lane 1) or digested after
hybridization to total RNA from wild-type (lane 2), EAT-D (lane 3),
or tRNA (lane 4).
[0021] FIG. 5 shows the developmental expression pattern of miR172
family members. a, RT-PCR of total RNA from wild type seedlings
harvested at 2, 5, 12, and 21 days after germination (lanes 1-4,
respectively), or from mature leaves (lane 5) and floral buds (lane
6). Primers for PCR are indicated on the left. b, Northern analysis
of mirR172 expression in the indicated mutants, relative to wild
type (Col). Blot was probed with an oligo to miR172a-2; however,
all miR172 members should cross hybridize.
[0022] FIG. 6 shows the expression analysis of putative EAT target
genes. a, Northern blot analysis of polyA+RNA isolated from wild
type (Col) or EAT-D floral buds. Probes for hybridization are
indicated on the right. b, Western blot of proteins from wild type
or EAT-D floral buds, probed with AP2 antibody. RbcL, large subunit
of ribulose 1,5-bisphosphate carboxylase as loading control.
[0023] FIG. 7 shows the identification of LAT-D. a, Location of the
T-DNA insert in LAT-D, in between At2g28550 and At2g28560. The
4.times. 35S enhancers are approximately 5 kb from At2g28550. b,
RT-PCR analysis of At2g28550 expression in wild type versus LAT-D
plants.
[0024] FIG. 8 shows that EAT-D is epistatic to LAT-D. Genetic cross
between EAT-D and LAT-D plants, with the resultant F1 plants shown,
along with their flowering time (measured as rosette leaf
number).
[0025] FIG. 9 shows the loss-of-function At2g28550 (2-28550) and
At5g60120 (6-60120) mutants. Location of T-DNA in each line is
indicated, along with intron/exon structure.
[0026] FIG. 10 shows the potential function of the miR172 miRNA
family. a, Temporal expression of miR172a-2 and its relatives may
cause temporal downregulation of AP2 targets (e.g. At2g28550 and
At5g60120), which may trigger flowering once the target proteins
drop below a critical threshold (dotted line). b, Dicer cleavage at
various positions may generate at least four distinct miRNAs from
the miR172 family (indicated as a single hairpin with a miRNA
consensus sequence). Sequences at the 5' and 3' ends of each miRNA
are indicated, with the invariant middle 15 nt shown as ellipses.
The putative targets recognized by the individual miRNAs are in
parentheses below each.
[0027] FIGS. 11A-11C show an artificial microRNA (miRNA) designed
to cleave the phytoene desaturase (PDS) mRNAs of Nicotiana
benthamiana. FIG. 11A shows the structure of the pre-miR159a
sequence construct under the control of the CaMV 35S promoter (35S)
and NOS terminator (Tnos). The orientation and position of the
mature miRNA is indicated by an arrow. FIG. 11B shows that point
mutations in miR159a (SEQ ID NO:141) (indicated by arrows) turn it
into miR-PDS.sup.159a (SEQ ID NO:142) to become fully complementary
to a region in N. benthamiana PDS mRNA. FIG. 11C shows that
Northern blot analysis of Agrobacterium infiltrated N. benthamiana
leaves shows expression of miR-PDS.sup.159a, miR-PDS.sup.159a* and
miR159 in samples infiltrated with an empty vector (vector) or the
artificial miRNA (miR-PDS.sup.159a) 1, 2, 3 days post infiltration
(d.p.i).
[0028] FIGS. 12A-12B show that miR-PDS.sup.159a (SEQ ID NO:142)
causes PDS mRNA (SEQ ID NO:143) cleavage. FIG. 12 A shows Northern
blot analysis of PDS mRNA from samples infiltrated with empty
vector or miR-PDS.sup.159a after 1 or 2 days post infiltration
(d.p.i.) (upper panel). The bottom panel shows the EtBr-stained
agarose gel from the same samples. FIG. 12B shows the site of
cleavage of the miRNA. 5'RACE analysis was conducted on samples
infiltrated with miR-PDS.sup.159a constructs and the 5'-end
sequence of 5 out of 6 clones indicated the site of cleavage of the
miRNA as indicated by an arrow.
[0029] FIGS. 13A-13E show that the expression of miR-PDS.sup.169g
results in cleavage of the PDS mRNA. FIG. 13A shows the point
mutations (underlined nucleotides) in miR169g (SEQ ID NO:144) that
it turn it into miR-PDSa.sup.169g (SEQ ID NO:145) or
miR-PDSb.sup.169g (SEQ ID NO:146) to become fully complementary to
two different regions in N. benthamiana PDS mRNA. FIG. 13B shows
Northern blot analysis of two different miR169g expression
constructs. Total RNA was extracted from non-infiltrated leaves (C)
or from leaves infiltrated with Agrobacterium containing the
pre-miR169g sequence in the context of a 0.3 kb (0.3 kb) or 2.0 kb
(2.0 kb) fragment, or from control Arabidopsis leaves (+). The
arrow indicates the position of the miR169 signal. FIG. 13C shows
Northern blot showing the expression of miR-PDSa.sup.169g (a) and
miR-PDSb.sup.169g (b) in infiltrated leaves containing the 0.3 kb
construct but not in control using the empty plasmid (vector). FIG.
13D shows the sites of cleavage of the miRNA. 5'RACE analysis was
conducted on samples infiltrated with miR-PDS.sup.169g a (SEQ ID
NO:145) and b (SEQ ID NO:146) constructs and the 5'-end sequence
identified from independent clones is indicated by an arrow
together with the number of clones analyzed. The PDS mRNAs are SEQ
ID NO:147 and SEQ ID NO:148. FIG. 13E shows a Northern blot
analysis to detect PDS mRNA levels in plants infiltrated with
Agrobacterium strains carrying the empty vector (C) or constructs
expressing miR-PDSa.sup.169g (a) or miR-PDSb.sup.169g (b).
[0030] FIGS. 14A-14C show the microRNA-directed cleavage of
Nicotiana benthamiana rbcS mRNAs. FIG. 14A shows that point
mutations in miR159a (SEQ ID NO:141) (indicated by arrows) turn it
into miR-rbcS.sup.159a-A (SEQ ID NO:149) to become complementary to
a region common to all N. benthamiana rbcS mRNAs (shown as rbcS
mRNA; SEQ ID NO:150). miRNA:mRNA base-pairs are indicated by
vertical lines and G:U wobble base-pairs by colons. FIG. 14B shows
that Northern blot analysis of Agrobacterium infiltrated N.
benthamiana leaves shows expression of miR-rbcS.sup.159a-A in
samples infiltrated with an empty vector (C) or the artificial
miRNA (A) 2 days post infiltration (d.p.i). FIG. 14C shows that
RT-PCR analysis was used to detect rbcS mRNA abundance for all six
genes in the same samples shown in B. Amplification of EF1.alpha.
mRNA served as a loading control.
[0031] FIGS. 15A-15B show the schematic representation of the genes
and relevant sequences used in the work shown in FIGS. 11-14. FIG.
15A shows the PDS gene from Lycopersicum esculetum that was used as
reference sequence since the complete PDS gene from N. benthamiana
is not known (segments missing are shown as a dashed line). Large
grey arrows indicate positions targeted by the miR-PDS constructs
described in the text. Small arrowheads indicate primers used for
5'RACE analysis. Known N. benthamiana PDS fragments are indicated
along with the origin of the sequences. FIG. 15B shows the
different reported sequences that were used to assemble the rbcS
gene sequence schematized here. The grey arrow indicates the
position of the sequence targeted by miR-rbcS.sup.159a-A, the
arrowheads indicate the position of primers used in RT-PCR
experiments shown in FIGS. 14A-14C.
[0032] FIGS. 16A-16B show a summary of changes introduced to
Arabidopsis miR159a and miR169g. FIG. 16A shows sequences of
miR-PDS.sup.159a (SEQ ID NO:142) and miR-rbcS.sup.159a-A (SEQ ID
NO:149) as compared to miR159a (SEQ ID NO:141). The base-changes in
each case are underlined while unmodified positions are marked with
an asterisk. FIG. 16B shows sequences of miR-PDSa.sup.169g (SEQ ID
NO:145) and miR-PDSb.sup.169g (SEQ ID NO:146) as compared to
miR169g (SEQ ID NO:144). The base-changes in each case are
underlined while unmodified positions are marked with an
asterisk.
[0033] FIG. 17 shows development of Arabidopsis root hairs in
wildtype, mutant and transgenic plants. Panel A: Wild type root
shows many root hair structures. Panel B: Very few root hair in cpc
mutant. Panel C: 35S::CPC plants show more root hairs. Panel D:
More root hair in g12 mutant. This figure is taken from Wada et al.
((2002) Development 129:5409-5419).
[0034] FIG. 18 shows Arabidopsis root hair development in
transgenic plants. Panel a: XVE::pre-miRCPC1.sup.159a without
inducer (estradiol). Panel b: XVE::pre-miRCPC1.sup.159a with
inducer (estradiol). Panel c: XVE::pre-miR159a without inducer
(estradiol). Panel d: XVE::pre-miR159a with inducer
(estradiol).
[0035] FIG. 19 shows Arabidopsis root hair development in
transgenic plants. Panel a: 35S::pre-miR159. Panel b:
35S::pre-miRCPC1.sup.159a. Panel c: 35S::pre-miRP69.sup.159a.
[0036] FIG. 20 shows Arabidopsis root hair development in
transgenic plants. Panel a: 35S::pre-miR159. Panel b:
35S::pre-miRCPC1.sup.159a.
[0037] FIGS. 21A-21E represent a diagram for a process for
designing a polymeric pre-miRNA. FIG. 21A: The products of
amplification of three different pre-miRNAs (pre-miR A, pre-miR B
and pre-miR C) in which AvrII, SpeI and XhoI sites have been added
by amplification. FIG. 21B: Pre-miR A is digested with SpeI and
XhoI and pre-miR B is digested with AvrII and XhoI. FIG. 21C: The
digested pre-miR A and pre-miR B are ligated to form a dimeric
pre-miRNA. FIG. 21D: Pre-miR A-B is digested with SpeI and XhoI and
pre-miR C is digested with AvrII and XhoI. FIG. 21E: The digested
pre-miR A-B and pre-miR C are ligated to form a trimeric
pre-miRNA.
[0038] FIG. 22 is a diagram of a dimeric construct containing
pre-miRPDS1.sup.169g and pre-miRCPC3.sup.159a.
[0039] FIGS. 23A and 23B show that mature miRPDS1.sup.169g (FIG.
23A) and miRCPC3.sup.159a (FIG. 23B) was successfully produced from
the dimeric construct. Lane 1 is 35S::pre-miRPDS1.sup.169g, lane 2
is 35S::CPC3.sup.159a and lane 3 is
35S::pre-miRPDS1.sup.169g-CPC3.sup.159a.
[0040] FIG. 24 shows the structure of the miR159a precursor (SEQ ID
NO:161).
[0041] FIG. 25 shows Northern blot analysis of miR-HC-Pro.sup.159a
were performed with three different treatments: (1) Agrobacterial
cells with 35S::pre-miR-HC-Pro.sup.159a, (2) Agrobacterial cells
with 35S::HC-Pro, and (3) Agrobacterial cells with
35S::pre-miR-HC-Pro.sup.159a and 35S::HC-Pro.
[0042] FIG. 26 shows shows Northern blot analysis of miR-P69, 4
different treatments were performed: (1) Agrobacterial cells
carrying 35S:: pre-miR-P69.sup.159a, (2) Agrobacterial cells
XVE::pre-miR-P69.sup.159a, (3) Agrobacterial cells carrying
35S::P69, and (4) Agrobacterial cells carrying
35S::pre-miR-P69.sup.159a and 35S::P-69.
[0043] FIG. 27 shows Northern blot analysis of mature artificial
miRNA levels for randomly picked T.sub.2
35S::pre-miRHC-Pro.sup.159a transgenic lines (plants). The T.sub.2
plants are known to be transgenic because they were first selected
on Kan-containing medium to remove WT. The T.sub.2 plants are
either heterozygous (one copy) or homozygous (two copies), and the
ratio should be about 2:1.
[0044] FIG. 28 shows Northern blot analysis of mature artificial
miRNA levels for randomly picked T.sub.2 35S::pre-miR-P69.sup.159a
transgenic lines (plants).
[0045] FIG. 29 shows that T.sub.2 transgenic plants expressing
miR-HC-Pro.sup.159a artificial miRNA are resistant to TuMV
infection. Photographs were taken 2 weeks (14 days after infection)
after inoculation. T.sub.2 transgenic plants expressing
miR-HC-Pro.sup.159a (line #11; FIG. 33B) developed normal
inflorescences whereas WT plants and T.sub.2 transgenic plants
expressing miR-P69.sup.159a (line #1; FIG. 33B) showed viral
infection symptoms. The bar represents 3 cm.
[0046] FIG. 30 shows symptoms of inflorescences caused by TuMV
infection. (Top panel) Fourteen days after TuMV infection, T.sub.2
transgenic miR-P69.sup.159a plants (line #1) and col-0 plants
showed shorter internodes between flowers in inflorescences,
whereas T.sub.2 miR-HC-Pro.sup.159a transgenic plant (line #11)
displayed normal inflorescences development. The bar represents 1
cm. (Bottom panel) Close-up views of inflorescences on
TuMV-infected Arabidopsis plants. T.sub.2 transgenic
miR-P69.sup.159a plants (line #1) and col-0 plants showed
senescence and pollination defects whereas T.sub.2 transgenic
miR-HC-Pro.sup.159a plants (line #11) showed normal flower and
silique development. For mock-infection, plants were inoculated
with buffer only. The bar represents 0.2 cm.
[0047] FIG. 31 shows symptoms of siliques caused by TuMV infection.
In TuMV-infected T.sub.2 transgenic miR-P69.sup.159a plants (line
#1) and WT (col-0) plants, siliques were small and mal-developed.
T.sub.2 transgenic miR-HC-Pro.sup.159a plants (line #11) were
resistant to TuMV infection and showed normal silique development.
Buffer-inoculated plants (mock-inculated) were used as controls.
The bar represents 0.5 cm.
[0048] FIG. 32 shows Western blot analysis of TuMV coat protein
(CP) levels in leaves of different transgenic and WT plants.
[0049] FIG. 33 shows (A) Western blot analysis of representative
plants of 35S:: miR-HC-Pro.sup.159a, 35S:: miR-P69.sup.159a, and WT
(Col-o) and (B) Northern blot analysis of miRNAs produced by the
transgenic plants.
[0050] FIG. 34 shows ELISA detection of TuMV in different
transgenic and non-transgenic Arabidopsis.
[0051] FIG. 35 shows Northern blot analysis of
pre-miR-P69.sup.159a, pre-miR-HC-Pro.sup.159a and
pre-miR-P69.sup.159a-HC-Pro.sup.159a demonstrating that
homo-dimeric miRNA precursor, pre-miR-P69.sup.159a-HC-Pro.sup.159a,
can produce mature miR-P69.sup.159a and miR-HC-Pro.sup.159a.
[0052] FIG. 36 shows constructs in which miR-HC-Pro.sup.159a is
placed in either intron 1 or intron 2 of the CPC gene.
[0053] FIG. 37 shows Northern blot analysis of the constructs of
FIG. 36 and demonstrates that intron 1 and intron 2 of the CPC
transcript can be used to produce artificial miRNAs.
[0054] FIG. 38 shows constructs in which pre-miR-HC-Pro.sup.159a is
placed in either intron 1 or intron 2 and pre-miR-P69.sup.159a is
placed in either intron 2 or intron 1 of the CPC gene.
[0055] FIG. 39 shows Northern blot analysis of the constructs of
FIG. 38 and demonstrates that it is possible to use CPC introns to
produce two different artificial miRNAs simultaneously in one
transcript.
DETAILED DESCRIPTION
[0056] Recently discovered small RNAs play an important role in
controlling gene expression. Regulation of many developmental
processes including flowering is controlled by small RNAs. It is
now possible to engineer changes in gene expression of plant genes
by using transgenic constructs which produce small RNAs in the
plant.
[0057] The invention provides methods and compositions useful for
suppressing targeted sequences. The compositions can be employed in
any type of plant cell, and in other cells which comprise the
appropriate processing components (e.g., RNA interference
components), including invertebrate and vertebrate animal cells.
The compositions and methods are based on an endogenous miRNA
silencing process discovered in Arabidopsis, a similar strategy can
be used to extend the number of compositions and the organisms in
which the methods are used. The methods can be adapted to work in
any eukaryotic cell system. Additionally, the compositions and
methods described herein can be used in individual cells, cells or
tissue in culture, or in vivo in organisms, or in organs or other
portions of organisms.
[0058] The compositions selectively suppress the target sequence by
encoding a miRNA having substantial complementarity to a region of
the target sequence. The miRNA is provided in a nucleic acid
construct which, when transcribed into RNA, is predicted to form a
hairpin structure which is processed by the cell to generate the
miRNA, which then suppresses expression of the target sequence.
[0059] A nucleic acid construct is provided to encode the miRNA for
any specific target sequence. Any miRNA can be inserted into the
construct, such that the encoded miRNA selectively targets and
suppresses the target sequence.
[0060] A method for suppressing a target sequence is provided. The
method employs the constructs above, in which a miRNA is designed
to a region of the target sequence, and inserted into the
construct. Upon introduction into a cell, the miRNA produced
suppresses expression of the targeted sequence. The target sequence
can be an endogenous plant sequence, or a heterologous transgene in
the plant. The target gene may also be a gene from a plant
pathogen, such as a pathogenic virus, nematode, insect, or mold or
fungus.
[0061] A plant, cell, and seed comprising the construct and/or the
miRNA is provided. Typically, the cell will be a cell from a plant,
but other eukaryotic cells are also contemplated, including but not
limited to yeast, insect, nematode, or animal cells. Plant cells
include cells from monocots and dicots. The invention also provides
plants and seeds comprising the construct and/or the miRNA. Viruses
and prokaryotic cells comprising the construct are also
provided.
[0062] Units, prefixes, and symbols may be denoted in their SI
accepted form. Unless otherwise indicated, nucleic acids are
written left to right in 5' to 3' orientation; amino acid sequences
are written left to right in amino to carboxyl orientation,
respectively. Numeric ranges recited within the specification are
inclusive of the numbers defining the range and include each
integer within the defined range. Amino acids may be referred to
herein by either commonly known three letter symbols or by the
one-letter symbols recommended by the IUPAC-IUB Biochemical
Nomenclature Commission. Nucleotides, likewise, may be referred to
by their commonly accepted single-letter codes. Unless otherwise
provided for, software, electrical, and electronics terms as used
herein are as defined in The New IEEE Standard Dictionary of
Electrical and Electronics Terms (5.sup.th edition, 1993). The
terms defined below are more fully defined by reference to the
specification as a whole.
[0063] As used herein, "nucleic acid construct" or "construct"
refers to an isolated polynucleotide which is introduced into a
host cell. This construct may comprise any combination of
deoxyribonucleotides, ribonucleotides, and/or modified nucleotides.
The construct may be transcribed to form an RNA, wherein the RNA
may be capable of forming a double-stranded RNA and/or hairpin
structure. This construct may be expressed in the cell, or isolated
or synthetically produced. The construct may further comprise a
promoter, or other sequences which facilitate manipulation or
expression of the construct.
[0064] As used here "suppression" or "silencing" or "inhibition"
are used interchangeably to denote the down-regulation of the
expression of the product of a target sequence relative to its
normal expression level in a wild type organism. Suppression
includes expression that is decreased by about 10%, 15%, 20%, 25%,
30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,
95%, or 100% relative to the wild type expression level.
[0065] As used herein, "encodes" or "encoding" refers to a DNA
sequence which can be processed to generate an RNA and/or
polypeptide.
[0066] As used herein, "expression" or "expressing" refers to the
generation of an RNA transcript from an introduced construct, an
endogenous DNA sequence, or a stably incorporated heterologous DNA
sequence. The term may also refer to a polypeptide produced from an
mRNA generated from any of the above DNA precursors.
[0067] As used herein, "heterologous" in reference to a nucleic
acid is a nucleic acid that originates from a foreign species, or
is synthetically designed, or, if from the same species, is
substantially modified from its native form in composition and/or
genomic locus by deliberate human intervention. A heterologous
protein may originate from a foreign species or, if from the same
species, is substantially modified from its original form by
deliberate human intervention.
[0068] By "host cell" is meant a cell which contains an introduced
nucleic acid construct and supports the replication and/or
expression of the construct. Host cells may be prokaryotic cells
such as E. coli, or eukaryotic cells such as fungi, yeast, insect,
amphibian, nematode, or mammalian cells. Alternatively, the host
cells are monocotyledonous or dicotyledonous plant cells. An
example of a monocotyledonous host cell is a maize host cell.
[0069] The term "introduced" means providing a nucleic acid or
protein into a cell. Introduced includes reference to the
incorporation of a nucleic acid into a eukaryotic or prokaryotic
cell where the nucleic acid may be incorporated into the genome of
the cell, and includes reference to the transient provision of a
nucleic acid or protein to the cell. Introduced includes reference
to stable or transient transformation methods, as well as sexually
crossing.
[0070] The term "isolated" refers to material, such as a nucleic
acid or a protein, which is: (1) substantially or essentially free
from components which normally accompany or interact with the
material as found in its naturally occurring environment or (2) if
the material is in its natural environment, the material has been
altered by deliberate human intervention to a composition and/or
placed at a locus in the cell other than the locus native to the
material.
[0071] As used herein, "miRNA" refers to an oligoribonucleic acid,
which suppresses expression of a polynucleotide comprising the
target sequence transcript or down regulates a target RNA. A "miRNA
precursor" refers to a larger polynucleotide which is processed to
produce a mature miRNA, and includes a DNA which encodes an RNA
precursor, and an RNA transcript comprising the miRNA. A "mature
miRNA" refers to the miRNA generated from the processing of a miRNA
precursor. A "miRNA template" is an oligonucleotide region, or
regions, in a nucleic acid construct which encodes the miRNA. The
"backside" region of a miRNA is a portion of a polynucleotide
construct which is substantially complementary to the miRNA
template and is predicted to base pair with the miRNA template. The
miRNA template and backside may form a double-stranded
polynucleotide, including a hairpin structure. As is known for
natural miRNAs, the mature miRNA and its complements may contain
mismatches and form bulges and thus do not need to be fully
complementary.
[0072] As used herein, the phrases "target sequence" and "sequence
of interest" are used interchangeably. Target sequence is used to
mean the nucleic acid sequence that is selected for suppression of
expression, and is not limited to polynucleotides encoding
polypeptides. The target sequence comprises a sequence that is
substantially or completely complementary to the miRNA. The target
sequence can be RNA or DNA, and may also refer to a polynucleotide
comprising the target sequence.
[0073] As used herein, "nucleic acid" means a polynucleotide and
includes single or double-stranded polymer of deoxyribonucleotide
or ribonucleotide bases. Nucleic acids may also include fragments
and modified nucleotides.
[0074] By "nucleic acid library" is meant a collection of isolated
DNA or RNA molecules which comprise and substantially represent the
entire transcribed fraction of a genome of a specified organism or
of a tissue from that organism. Construction of exemplary nucleic
acid libraries, such as genomic and cDNA libraries, is taught in
standard molecular biology references such as Berger and Kimmel,
Guide to Molecular Cloning Techniques, Methods in Enzymology, Vol.
152, Academic Press, Inc., San Diego, Calif. (Berger); Sambrook et
al., Molecular Cloning--A Laboratory Manual, 2nd ed., Vol. 1-3
(1989); and Current Protocols in Molecular Biology, F. M. Ausubel
et al., Eds., Current Protocols, a joint venture between Greene
Publishing Associates, Inc. and John Wiley & Sons, Inc.
(1994).
[0075] As used herein "operably linked" includes reference to a
functional linkage of at least two sequences. Operably linked
includes linkage between a promoter and a second sequence, wherein
the promoter sequence initiates and mediates transcription of the
DNA sequence corresponding to the second sequence.
[0076] As used herein, "plant" includes plants and plant parts
including but not limited to plant cells, plant tissue such as
leaves, stems, roots, flowers, and seeds.
[0077] As used herein, "polypeptide" means proteins, protein
fragments, modified proteins, amino acid sequences and synthetic
amino acid sequences. The polypeptide can be glycosylated or
not.
[0078] As used herein, "promoter" includes reference to a region of
DNA that is involved in recognition and binding of an RNA
polymerase and other proteins to initiate transcription.
[0079] The term "selectively hybridizes" includes reference to
hybridization, under stringent hybridization conditions, of a
nucleic acid sequence to a specified nucleic acid target sequence
to a detectably greater degree (e.g., at least 2-fold over
background) than its hybridization to non-target nucleic acid
sequences and to the substantial exclusion of non-target nucleic
acids. Selectively hybridizing sequences typically have about at
least 80% sequence identity, or 90% sequence identity, up to and
including 100% sequence identity (i.e., fully complementary) with
each other.
[0080] The term "stringent conditions" or "stringent hybridization
conditions" includes reference to conditions under which a probe
will selectively hybridize to its target sequence. Stringent
conditions are sequence-dependent and will be different in
different circumstances. By controlling the stringency of the
hybridization and/or washing conditions, target sequences can be
identified which are 100% complementary to the probe (homologous
probing). Alternatively, stringency conditions can be adjusted to
allow some mismatching in sequences so that lower degrees of
similarity are detected (heterologous probing). Generally, a probe
is less than about 1000 nucleotides in length, optionally less than
500 nucleotides in length.
[0081] Typically, stringent conditions will be those in which the
salt concentration is less than about 1.5 M Na ion, typically about
0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to
8.3 and the temperature is at least about 30.degree. C. for short
probes (e.g., 10 to 50 nucleotides) and at least about 60.degree.
C. for long probes (e.g., greater than 50 nucleotides). Stringent
conditions may also be achieved with the addition of destabilizing
agents such as formamide. Exemplary low stringency conditions
include hybridization with a buffer solution of 30 to 35%
formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37.degree.
C., and a wash in 1.times. to 2.times.SSC (20.times.SSC=3.0 M
NaCl/0.3 M trisodium citrate) at 50 to 55.degree. C. Exemplary
moderate stringency conditions include hybridization in 40 to 45%
formamide, 1 M NaCl, 1% SDS at 37.degree. C., and a wash in
0.5.times. to 1.times. SSC at 55 to 60.degree. C. Exemplary high
stringency conditions include hybridization in 50% formamide, 1 M
NaCl, 1% SDS at 37.degree. C., and a wash in 0.1.times.SSC at 60 to
65.degree. C.
[0082] Specificity is typically the function of post-hybridization
washes, the critical factors being the ionic strength and
temperature of the final wash solution. For DNA-DNA hybrids, the
T.sub.m can be approximated from the equation of Meinkoth and Wahl
((1984) Anal Biochem 138:267-284): T.sub.m=81.5.degree. C.+16.6
(log M)+0.41 (%GC)-0.61 (% form)-500/L; where M is the molarity of
monovalent cations, % GC is the percentage of guanosine and
cytosine nucleotides in the DNA, % form is the percentage of
formamide in the hybridization solution, and L is the length of the
hybrid in base pairs. The T.sub.m is the temperature (under defined
ionic strength and pH) at which 50% of a complementary target
sequence hybridizes to a perfectly matched probe. T.sub.m is
reduced by about 1.degree. C. for each 1% of mismatching; thus,
T.sub.m, hybridization and/or wash conditions can be adjusted to
hybridize to sequences of the desired identity. For example, if
sequences with >90% identity are sought, the T.sub.m can be
decreased 10.degree. C. Generally, stringent conditions are
selected to be about 5.degree. C. lower than the thermal melting
point (T.sub.m) for the specific sequence and its complement at a
defined ionic strength and pH. However, severely stringent
conditions can utilize a hybridization and/or wash at 1, 2, 3, or
4.degree. C.. lower than the thermal melting point (T.sub.m);
moderately stringent conditions can utilize a hybridization and/or
wash at 6, 7, 8, 9, or 10.degree. C.. lower than the thermal
melting point (T.sub.m); low stringency conditions can utilize a
hybridization and/or wash at 11, 12, 13, 14, 15, or 20.degree. C..
lower than the thermal melting point (T.sub.m). Using the equation,
hybridization and wash compositions, and desired T.sub.m, those of
ordinary skill will understand that variations in the stringency of
hybridization and/or wash solutions are inherently described. If
the desired degree of mismatching results in a T.sub.m of less than
45.degree. C.. (aqueous solution) or 32.degree. C.. (formamide
solution) it is preferred to increase the SSC concentration so that
a higher temperature can be used. An extensive guide to the
hybridization of nucleic acids is found in Tijssen, Laboratory
Techniques in Biochemistry and Molecular Biology--Hybridization
with Nucleic Acid Probes, Part I, Chapter 2 "Overview of principles
of hybridization and the strategy of nucleic acid probe assays",
Elsevier, N.Y. (1993); and Current Protocols in Molecular Biology,
Chapter 2, Ausubel et al., Eds., Greene Publishing and
Wiley-Interscience, New York (1995). Hybridization and/or wash
conditions can be applied for at least 10, 30, 60, 90, 120, or 240
minutes.
[0083] As used herein, "transgenic" includes reference to a plant
or a cell which comprises a heterologous polynucleotide. Generally,
the heterologous polynucleotide is stably integrated within the
genome such that the polynucleotide is passed on to successive
generations. Transgenic is used herein to include any cell, cell
line, callus, tissue, plant part or plant, the genotype of which
has been altered by the presence of heterologous nucleic acid
including those transgenics initially so altered as well as those
created by sexual crosses or asexual propagation from the initial
transgenic. The term "transgenic" as used herein does not encompass
the alteration of the genome (chromosomal or extra-chromosomal) by
conventional plant breeding methods or by naturally occurring
events such as random cross-fertilization, non-recombinant viral
infection, non-recombinant bacterial transformation,
non-recombinant transposition, or spontaneous mutation.
[0084] As used herein, "vector" includes reference to a nucleic
acid used in introduction of a polynucleotide of the invention into
a host cell. Expression vectors permit transcription of a nucleic
acid inserted therein.
[0085] Polynucleotide sequences may have substantial identity,
substantial homology, or substantial complementarity to the
selected region of the target gene. As used herein "substantial
identity" and "substantial homology" indicate sequences that have
sequence identity or homology to each other. Generally, sequences
that are substantially identical or substantially homologous will
have about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99%, or 100% sequence identity wherein the percent sequence
identity is based on the entire sequence and is determined by GAP
alignment using default parameters (GCG, GAP version 10, Accelrys,
San Diego, Calif.). GAP uses the algorithm of Needleman and Wunsch
((1970) J Mol Biol 48:443-453) to find the alignment of two
complete sequences that maximizes the number of matches and
minimizes the number of sequence gaps. Sequences which have 100%
identity are identical. "Substantial complementarity" refers to
sequences that are complementary to each other, and are able to
base pair with each other. In describing complementary sequences,
if all the nucleotides in the first sequence will base pair to the
second sequence, these sequences are fully complementary.
[0086] Through a forward genetics approach, a microRNA that confers
a developmental phenotype in Arabidopsis was identified. This
miRNA, miR172a-2 (Park et al. (2002) Curr Biol 12:1484-1495),
causes early flowering and defects in floral organ identity when
overexpressed. The predicted target of miR172a-2 is a small
subfamily of APETALA2-like transcription factors (Okamuro et al.
(1997) Proc Natl Acad Sci USA 94:7076-7081). Overexpression of
miR172a-2 downregulates at least one member of this family. In
addition, overexpression of one of the AP2-like target genes,
At2g28550, causes late flowering. This result, in conjunction with
loss-of-function analyses of At2g28550 and another target gene,
At5g60120, indicates that at least some of the AP2-like genes
targeted by miR172a-2 normally function as floral repressors. The
EAT-D line overexpressing miR172-a2 has a wild-type response to
photoperiod. The genomic region encoding the miRNA was also
identified (SEQ ID NO:1) and used to produce a cassette into which
other miRNAs to target sequences can be inserted (SEQ ID NO:3), and
to produce an expression vector (SEQ ID NO:44) useful for cloning
the cassettes and expressing the miRNA. The expression vector
comprises the 1.4 kb region encoding the miRNA. Expression of this
region is processed in the cell to produce the miRNA which
suppresses expression of the target gene. Alternatively, the miRNA
may be synthetically produced and introduced to the cell
directly.
[0087] In one embodiment, there is provided a method for the
suppression of a target sequence comprising introducing into a cell
a nucleic acid construct encoding a miRNA substantially
complementary to the target. In some embodiments the miRNA
comprises about 10-200 nucleotides, about 10-15, 15-20, 19, 20, 21,
22, 23, 24, 25, 26, 27, 25-30, 30-50, 50-100, 100-150, or about
150-200 nucleotides. In some embodiments the nucleic acid construct
encodes the miRNA. In some embodiments the nucleic acid construct
encodes a polynucleotide precursor which may form a double-stranded
RNA, or hairpin structure comprising the miRNA. In some
embodiments, nucleotides 39-59 and 107-127 of SEQ ID NO:3 are
replaced by the backside of the miRNA template and the miRNA
template respectively. In some embodiments, this new sequence
replaces the equivalent region of SEQ ID NO:1. In further
embodiments, this new sequence replaces the equivalent region of
SEQ ID NO:44.
[0088] In some embodiments, the nucleic acid construct comprises a
modified endogenous plant miRNA precursor, wherein the precursor
has been modified to replace the endogenous miRNA encoding regions
with sequences designed to produce a miRNA directed to the target
sequence. In some embodiments the miRNA precursor template is a
miR172a miRNA precursor. In some embodiments, the miR172a precursor
is from a dicot or a monocot. In some embodiments the miR172a
precursor is from Arabidopsis thaliana, tomato, soybean, rice, or
corn. In some embodiments the miRNA precursor is SEQ ID NO:1, SEQ
ID NO:3, or SEQ ID NO:44.
[0089] In another embodiment the method comprises:
[0090] A method of inhibiting expression of a target sequence in a
cell comprising:
[0091] (a) introducing into the cell a nucleic acid construct
comprising a promoter operably linked to a polynucleotide, wherein
the polynucleotide comprises in the following order: [0092] (i) at
least about 20 and up to 38 contiguous nucleotides in the region of
nucleotides 1-38 of SEQ ID NO:3, [0093] (ii) a first
oligonucleotide of 10 to about 50 contiguous nucleotides, wherein
the first oligonucleotide is substantially complementary to a
second oligonucleotide [0094] (iii) at least about 20 and up to 47
contiguous nucleotides in the region of nucleotides 60-106 of SEQ
ID NO:3, [0095] (iv) the second oligonucleotide of about 10 to
about 50 contiguous nucleotides, wherein the second oligonucleotide
encodes a miRNA, and the second oligonucleotide is substantially
complementary to the target sequence, and [0096] (v) at least about
20 and up to 32 contiguous nucleotides in the region of nucleotides
128-159 of SEQ ID NO:3; wherein the polynucleotide encodes an RNA
precursor capable of forming a hairpin, and
[0097] (b) expressing the nucleic acid construct for a time
sufficient to produce the miRNA, wherein the miRNA inhibits
expression of the target sequence.
[0098] In another embodiment, the method comprises selecting a
target sequence of a gene, and designing a nucleic acid construct
comprising polynucleotide encoding a miRNA substantially
complementary to the target sequence. In some embodiments, the
target sequence is selected from any region of the gene. In some
embodiments, the target sequence is selected from an untranslated
region. In some embodiments, the target sequence is selected from a
coding region of the gene. In some embodiments, the target sequence
is selected from a region about 50 to about 200 nucleotides
upstream from the stop codon, including regions from about 50-75,
75-100, 100-125, 125-150, or 150-200 upstream from the stop codon.
In further embodiments, the target sequence and/or the miRNA is
based on the polynucleotides and process of EAT suppression of
Apetela2-like genes in Arabidopsis thaliana. In some embodiments,
nucleotides 39-59 and 107-127 of SEQ ID NO:3 are replaced by the
backside of the miRNA template (first oligonucleotide) and the
miRNA template (second oligonucleotide) respectively. In some
embodiments, this new sequence replaces the equivalent region of
SEQ ID NO:1. In further embodiments, this new sequence replaces the
equivalent region of SEQ ID NO:44.
[0099] In some embodiments, the miRNA template, (i.e. the
polynucleotide encoding the miRNA), and thereby the miRNA, may
comprise some mismatches relative to the target sequence. In some
embodiments the miRNA template has >1 nucleotide mismatch as
compared to the target sequence, for example, the miRNA template
can have 1, 2, 3, 4, 5, or more mismatches as compared to the
target sequence. This degree of mismatch may also be described by
determining the percent identity of the miRNA template to the
complement of the target sequence. For example, the miRNA template
may have a percent identity including about at least 70%, 75%, 77%,
78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% as compared to
the complement of the target sequence.
[0100] In some embodiments, the miRNA template, (i.e. the
polynucleotide encoding the miRNA) and thereby the miRNA, may
comprise some mismatches relative to the miRNA backside. In some
embodiments the miRNA template has >1 nucleotide mismatch as
compared to the miRNA backside, for example, the miRNA template can
have 1, 2, 3, 4, 5, or more mismatches as compared to the miRNA
backside. This degree of mismatch may also be described by
determining the percent identity of the miRNA template to the
complement of the miRNA backside. For example, the miRNA template
may have a percent identity including about at least 70%, 75%, 77%,
78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% as compared to
the complement of the miRNA backside.
[0101] In some embodiments, the target sequence is selected from a
plant pathogen. Plants or cells comprising a miRNA directed to the
target sequence of the pathogen are expected to have decreased
sensitivity and/or increased resistance to the pathogen. In some
embodiments, the miRNA is encoded by a nucleic acid construct
further comprising an operably linked promoter. In some
embodiments, the promoter is a pathogen-inducible promoter.
[0102] In another embodiment, the method comprises replacing the
miRNA encoding sequence in the polynucleotide of SEQ ID NO:3 with a
sequence encoding a miRNA substantially complementary to the target
region of the target gene.
[0103] In another embodiment a method is provided comprising a
method of inhibiting expression of a target sequence in a cell
comprising:
[0104] (a) introducing into the cell a nucleic acid construct
comprising a promoter operably linked to a polynucleotide encoding
a modified plant miRNA precursor comprising a first and a second
oligonucleotide, wherein at least one of the first or the second
oligonucleotides is heterologous to the precursor, wherein the
first oligonucleotide is substantially complementary to the second
oligonucleotide, and the second oligonucleotide encodes a miRNA
substantially complementary to the target sequence, wherein the
precursor is capable of forming a hairpin; and
[0105] (b) expressing the nucleic acid construct for a time
sufficient to produce the miRNA, wherein the miRNA inhibits
expression of the target sequence.
[0106] In another embodiment a method is provided comprising a
method of inhibiting expression of a target sequence in a cell
comprising:
[0107] (a) introducing into the cell a nucleic acid construct
comprising a promoter operably linked to a polynucleotide encoding
a modified plant miR172 miRNA precursor comprising a first and a
second oligonucleotide, wherein at least one of the first or the
second oligonucleotides is heterologous to the precursor, wherein
the first oligonucleotide is substantially complementary to the
second oligonucleotide, and the second oligonucleotide encodes a
miRNA substantially complementary to the target sequence, wherein
the precursor is capable of forming a hairpin; and
[0108] (b) expressing the nucleic acid construct for a time
sufficient to produce the miRNA, wherein the miRNA inhibits
expression of the target sequence.
[0109] In some embodiments, the modified plant miR172 miRNA
precursor is a modified Arabidopsis miR172 miRNA precursor, or a
modified corn miR172 miRNA precursor or the like.
[0110] In another embodiment, there is provided a nucleic acid
construct for suppressing a target sequence. The nucleic acid
construct encodes a miRNA substantially complementary to the
target. In some embodiments, the nucleic acid construct further
comprises a promoter operably linked to the polynucleotide encoding
the miRNA. In some embodiments, the nucleic acid construct lacking
a promoter is designed and introduced in such a way that it becomes
operably linked to a promoter upon integration in the host genome.
In some embodiments, the nucleic acid construct is integrated using
recombination, including site-specific recombination. See, for
example, PCT International published application No. WO 99/25821,
herein incorporated by reference. In some embodiments, the nucleic
acid construct is an RNA. In some embodiments, the nucleic acid
construct comprises at least one recombination site, including
site-specific recombination sites. In some embodiments the nucleic
acid construct comprises at least one recombination site in order
to facilitate integration, modification, or cloning of the
construct. In some embodiments the nucleic acid construct comprises
two site-specific recombination sites flanking the miRNA precursor.
In some embodiments the site-specific recombination sites include
FRT sites, lox sites, or att sites, including attB, attL, attP or
attR sites. See, for example, PCT International published
application No. WO 99/25821, and U.S. Pat. Nos. 5,888,732,
6,143,557, 6,171,861, 6,270,969, and 6,277,608, herein incorporated
by reference.
[0111] In some embodiments, the nucleic acid construct comprises a
modified endogenous plant miRNA precursor, wherein the precursor
has been modified to replace the miRNA encoding region with a
sequence designed to produce a miRNA directed to the target
sequence. In some embodiments the miRNA precursor template is a
miR172a miRNA precursor. In some embodiments, the miR172a precursor
is from a dicot or a monocot. In some embodiments the miR172a
precursor is from Arabidopsis thaliana, tomato, soybean, rice, or
corn. In some embodiments the miRNA precursor is SEQ ID NO:1, SEQ
ID NO:3, or SEQ ID NO:44.
[0112] In another embodiment, the nucleic acid construct comprises
an isolated polynucleotide comprising a polynucleotide which
encodes a modified plant miRNA precursor, the modified precursor
comprising a first and a second oligonucleotide, wherein at least
one of the first or the second oligonucleotides is heterologous to
the precursor, wherein the first oligonucleotide i is substantially
complementary to the second oligonucleotide, and the second
oligonucleotide comprises a miRNA substantially complementary to
the target sequence, wherein the precursor is capable of forming a
hairpin.
[0113] In another embodiment, the nucleic acid construct comprises
an isolated polynucleotide comprising a polynucleotide which
encodes a modified plant miR172 miRNA precursor, the modified
precursor comprising a first and a second oligonucleotide, wherein
at least one of the first or the second oligonucleotides is
heterologous to the precursor, wherein the first oligonucleotide is
substantially complementary to the second oligonucleotide, and the
second oligonucleotide comprises a miRNA substantially
complementary to the target sequence, wherein the precursor is
capable of forming a hairpin. In some embodiments, the modified
plant miR172 miRNA precursor is a modified Arabidopsis miR172 miRNA
precursor, or a modified corn miR172 miRNA precursor, or the
like.
[0114] In some embodiments the miRNA comprises about 10-200
nucleotides, about 10-15, 15-20, 19, 20, 21, 22, 23, 24, 25, 26,
27, 25-30, 30-50, 50-100, 100-150, or about 150-200 nucleotides. In
some embodiments the nucleic acid construct encodes the miRNA. In
some embodiments the nucleic acid construct encodes a
polynucleotide precursor which may form a double-stranded RNA, or
hairpin structure comprising the miRNA. In some embodiments,
nucleotides 39-59 and/or 107-127 of SEQ ID NO:3 are replaced by the
backside of the miRNA template and the miRNA template respectively.
In some embodiments, this new sequence replaces the equivalent
region of SEQ ID NO:1. In further embodiments, this new sequence
replaces the equivalent region of SEQ ID NO:44. In some
embodiments, the target region is selected from any region of the
target sequence. In some embodiments, the target region is selected
from a untranslated region. In some embodiments, the target region
is selected from a coding region of the target sequence. In some
embodiments, the target region is selected from a region about 50
to about 200 nucleotides upstream from the stop codon, including
regions from about 50-75, 75-100, 100-125, 125-150, or 150-200
upstream from the stop codon. In further embodiments, the target
region and/or the miRNA is based on the polynucleotides and process
of EAT suppression of Apetela2-like sequences in Arabidopsis
thaliana.
[0115] In another embodiment the nucleic acid construct comprises
an isolated polynucleotide comprising in the following order at
least 20 and up to 38 contiguous nucleotides in the region from
nucleotides 1-38 of SEQ ID NO:3, a first oligonucleotide of about
10 to about 50 contiguous nucleotides, wherein the first
oligonucleotide is substantially complementary to a second
oligonucleotide, at least about 20 and up to 47 contiguous
nucleotides in the region from nucleotides 60-106 of SEQ ID NO:3, a
second oligonucleotide of about 10 to about 50 contiguous
nucleotides, wherein the second oligonucleotide encodes a miRNA,
and the second oligonucleotide is substantially complementary to
the target sequence, and at least about 20 and up to 32 contiguous
nucleotides in the region from nucleotides 128-159 of SEQ ID NO:3,
wherein the polynucleotide encodes an RNA precursor capable of
forming a hairpin structure.
[0116] In some embodiments there are provided cells, plants, and
seeds comprising the introduced polynucleotides, and/or produced by
the methods of the invention. The cells include prokaryotic and
eukaryotic cells, including but not limited to bacteria, yeast,
fungi, viral, invertebrate, vertebrate, and plant cells. Plants,
plant cells, and seeds of the invention include gynosperms,
monocots and dicots, including but not limited to, for example,
rice, wheat, oats, barley, millet, sorghum, soy, sunflower,
safflower, canola, alfalfa, cotton, Arabidopsis, and tobacco.
[0117] In some embodiments, the cells, plants, and/or seeds
comprise a nucleic acid construct comprising a modified plant miRNA
precursor, wherein the precursor has been modified to replace the
endogenous miRNA encoding regions with sequences designed to
produce a miRNA directed to the target sequence. In some
embodiments the miRNA precursor template is a miR172a miRNA
precursor. In some embodiments, the miR172a precursor is from a
dicot or a monocot. In some embodiments the miR172a precursor is
from Arabidopsis thaliana, tomato, soybean, rice, or corn. In some
embodiments the miRNA precursor is SEQ ID NO:1, SEQ ID NO:3, or SEQ
ID NO:44. In some embodiments the miRNA precursor is encoded by SEQ
ID NO:1, SEQ ID NO:3, or SEQ ID NO:44. In some embodiments, the
nucleic acid construct comprises at least one recombination site,
including site-specific recombination sites. In some embodiments
the nucleic acid construct comprises at least one recombination
site in order to facilitate modification or cloning of the
construct. In some embodiments the nucleic acid construct comprises
two site-specific recombination sites flanking the miRNA precursor.
In some embodiments the site-specific recombination sites include
FRT sites, lox sites, or att sites, including attB, attL, attP or
attR sites. See, for example, PCT International published
application No. WO 99/25821, and U.S. Pat. Nos. 5,888,732,
6,143,557, 6,171,861, 6,270,969, and 6,277,608, herein incorporated
by reference.
[0118] In a further embodiment, there is provided a method for down
regulating a target RNA comprising introducing into a cell a
nucleic acid construct that encodes a miRNA that is complementary
to a region of the target RNA. In some embodiments, the miRNA is
fully complementary to the region of the target RNA. In some
embodiments, the miRNA is complementary and includes the use of G-U
base pairing, i.e. the GU wobble, to otherwise be fully
complementary. In some embodiments, the first ten nucleotides of
the miRNA (counting from the 5' end of the miRNA) are fully
complementary to a region of the target RNA and the remaining
nucleotides may include mismatches and/or bulges with the target
RNA. In some embodiments the miRNA comprises about 10-200
nucleotides, about 10-15, 15-20, 19, 20, 21, 22, 23, 24, 25, 26,
27, 25-30, 30-50, 50-100, 100-150, or about 150-200 nucleotides.
The binding of the miRNA to the complementary sequence in the
target RNA results in cleavage of the target RNA. In some
embodiments, the miRNA is a miRNA that has been modified such that
the miRNA is fully complementary to the target sequence of the
target RNA. In some embodiments, the miRNA is an endogenous plant
miRNA that has been modified such that the miRNA is fully
complementary to the target sequence of the target RNA. In some
embodiments, the polynucleotide encoding the miRNA is operably
linked to a promoter. In some embodiments, the nucleic acid
construct comprises a promoter operably linked to the miRNA.
[0119] In some embodiments, the nucleic acid construct encodes the
miRNA. In some embodiments, the nucleic acid construct comprises a
promoter operably linked to the miRNA. In some embodiments, the
nucleic acid construct encodes a polynucleotide which may form a
double-stranded RNA, or hairpin structure comprising the miRNA. In
some embodiments, the nucleic acid construct comprises a promoter
operably linked to the polynucleotide which may form a
double-stranded RNA, or hairpin structure comprising the miRNA. In
some embodiments, the nucleic acid construct comprises an
endogenous plant miRNA precursor that has been modified such that
the miRNA is fully complementary to the target sequence of the
target RNA. In some embodiments, the nucleic acid construct
comprises a promoter operably linked to the miRNA precursor. In
some embodiments, the nucleic acid construct comprises about 50
nucleotides to about 3000 nucleotides, about 50-100, 100-150,
150-200, 200-250, 250-300, 300-350, 350-400, 400-450, 450-500,
500-600, 600-700, 700-800, 800-900, 900-1000, 1000-1100, 1100-1200,
1200-1300, 1300-1400, 1400-1500, 1500-1600, 1600-1700, 1700-1800,
1800-1900, 1900-2000, 2000-2100, 2100-2200, 2200-2300, 2300-2400,
2400-2500, 2500-2600, 2600-2700, 2700-2800, 2800-2900 or about
2900-3000 nucleotides.
[0120] In some embodiments, the nucleic acid construct lacking a
promoter is designed and introduced in such a way that it becomes
operably linked to a promoter upon integration in the host genome.
In some embodiments, the nucleic acid construct is integrated using
recombination, including site-specific recombination. In some
embodiments, the nucleic acid construct is an RNA. In some
embodiments, the nucleic acid construct comprises at least one
recombination site, including site-specific recombination sites. In
some embodiments the nucleic acid construct comprises at least one
recombination site in order to facilitate integration,
modification, or cloning of the construct. In some embodiments the
nucleic acid construct comprises two site-specific recombination
sites flanking the miRNA precursor.
[0121] In another embodiment, the method comprises a method for
down regulating a target RNA in a cell comprising introducing into
the cell a nucleic acid construct that encodes a miRNA that is
complementary to a region of the target RNA and expressing the
nucleic acid construct for a time sufficient to produce miRNA,
wherein the miRNA down regulates the target RNA. In some
embodiments, the miRNA is fully complementary to the region of the
target RNA. In some embodiments, the miRNA is complementary and
includes the use of G-U base pairing, i.e. the GU wobble, to
otherwise be fully complementary.
[0122] In another embodiment, the method comprises selecting a
target RNA, selecting a miRNA, comparing the sequence of the target
RNA (or its DNA) with the sequence of the miRNA, identifying a
region of the target RNA (or its DNA) in which the nucleotide
sequence is similar to the nucleotide sequence of the miRNA,
modifying the nucleotide sequence of the miRNA so that it is
complementary to the nucleotide sequence of the identified region
of the target RNA and preparing a nucleic acid construct comprising
the modified miRNA. In some embodiments, the miRNA is fully
complementary to the identified region of the target RNA. In some
embodiments, the miRNA is complementary and includes the use of G-U
base pairing, i.e. the GU wobble, to otherwise be fully
complementary. In some embodiments, a nucleic acid construct
encodes a polynucleotide which may form a double-stranded RNA, or
hairpin structure comprising the miRNA. In some embodiments, a
nucleic acid construct comprises a precursor of the miRNA, i.e., a
pre-miRNA that has been modified in accordance with this
embodiment.
[0123] In another embodiment, the method comprises selecting a
target RNA, selecting a nucleotide sequence within the target RNA,
selecting a miRNA, modifying the sequence of the miRNA so that it
is complementary to the nucleotide sequence of the identified
region of the target RNA and preparing a nucleic acid construct
comprising the modified miRNA. In some embodiments, the miRNA is
fully complementary to the identified region of the target RNA. In
some embodiments, the miRNA is complementary and includes the use
of G-U base pairing, i.e. the GU wobble, to otherwise be fully
complementary. In some embodiments, a nucleic acid construct
encodes a polynucleotide which may form a double-stranded RNA, or
hairpin structure comprising the miRNA. In some embodiments, a
nucleic acid construct comprises a precursor of the miRNA, i.e., a
pre-miRNA that has been modified in accordance with this
embodiment.
[0124] In some embodiments, the miRNA is a miRNA disclosed in the
microRNA registry, now also known as the miRBase Sequence Database
(Griffiths-Jones (2004) Nucl Acids Res 32, Database
issue:D109-D111; http:colon//micrornadotsangerdotacdotuk/). In some
embodiments, the miRNA is ath-MIR156a, ath-MIR156b, ath-MIR156c,
ath-MIR156d, ath-MIR156e, ath-MIR156f, ath-MIR156g, ath-MIR156h,
ath-MIR157a, ath-MIR157b, ath-MIR157c, ath-MIR157d, ath-MIR158a,
ath-MIR158b, ath-MIR159a, ath-MIR159b, ath-MIR159c, ath-MIR160a,
ath-MIR160b, ath-MIR160c, ath-MIR161, ath-MIR162a, ath-MIR162b,
ath-MIR163, ath-MIR164a, ath-MIR164b, ath-MIR164c, ath-MIR165a,
ath-MIR165b, ath-MIR166a, ath-MIR166b, ath-MIR166c, ath-MIR166d,
ath-MIR166e, ath-MIR166f, ath-MIR166g, ath-MIR167a, ath-MIR167b,
ath-MIR167c, ath-MIR167d, ath-MIR168a, ath-MIR168b, ath-MIR169a,
ath-MIR169b, ath-MIR169c, ath-MIR169d, ath-MIR169e, ath-MIR169f,
ath-MIR169g, ath-MIR169h, ath-MIR169i, ath-MIR169j, ath-MIR169k,
ath-MIR169l, ath-MIR169m, ath-MIR169n, ath-MIR170, ath-MIR171a,
ath-MIR171b, ath-MIR171c, ath-MIR172a, ath-MIR172b, ath-MIR172c,
ath-MIR172d, ath-MIR172e, ath-MIR173, ath-MIR319a, ath-MIR319b,
ath-MIR319c, ath-MIR390a, ath-MIR390b, ath-MIR393a, ath-MIR393b,
ath-MIR394a, ath-MIR394b, ath-MIR395 a, ath-MIR395b, ath-MIR395c,
ath-MIR395d, ath-MIR395e, ath-MIR395f, ath-MIR396a, ath-MIR396b,
ath-MIR397a, ath-MIR397b, ath-MIR398a, ath-MIR398b, ath-MIR398c,
ath-MIR399a, ath-MIR399b, ath-MIR399c, ath-MIR399d, ath-MIR399e,
ath-MIR399f, ath-MIR400, ath-MIR401, ath-MIR402, ath-MIR403,
ath-MIR404, ath-MIR405a, ath-MIR405b, ath-MIR405d, ath-MIR406,
ath-MIR407, ath-MIR408, ath-MIR413, ath-MIR414, ath-MIR415,
ath-MIR416, ath-MIR417, ath-MIR418, ath-MIR419, ath-MIR420,
ath-MIR426, ath-MIR447a, ath-MIR447b, ath-MIR447c, osa-MIR156a,
osa-MIR156b, osa-MIR156c, osa-MIR156d, osa-MIR156e, osa-MIR156f,
osa-MIR156g, osa-MIR156h, osa-MIR156i, osa-MIR156j, osa-MIR156k,
osa-MIR156l, osa-MIR159a, osa-MIR159b, osa-MIR159c, osa-MIR159d,
osa-MIR159e, osa-MIR159f, osa-MIR160a, osa-MIR160b, osa-MIR160c,
osa-MIR160d, osa-MIR160e, osa-MIR160f, osa-MIR162a, osa-MIR162b,
osa-MIR164a, osa-MIR164b, osa-MIR164c, osa-MIR164d, osa-MIR164e,
osa-MIR166a, osa-MIR166b, osa-MIR166c, osa-MIR166d, osa-MIR166e,
osa-MIR166f, osa-MIR166j, osa-MIR166k, osa-MIR166l, osa-MIR166g,
osa-MIR166h, osa-MIR166i, osa-MIR166m, osa-MIR166n, osa-MIR167a,
osa-MIR167b, osa-MIR167c, osa-MIR167d, osa-MIR167e, osa-MIR167f,
osa-MIR167g, osa-MIR167h, osa-MIR167i, osa-MIR167j, osa-MIR168a,
osa-MIR168b, osa-MIR169a, osa-MIR169b, osa-MIR169c, osa-MIR169d,
osa-MIR169e, osa-MIR169f, osa-MIR169g, osa-MIR169h, osa-MIR169i,
osa-MIR169j, osa-MIR169k, osa-MIR169l, osa-MIR169m, osa-MIR169n,
osa-MIR169o, osa-MIR169p, osa-MIR169q, osa-IR171a, osa-MIR171b,
osa-MIR171c, osa-MIR171d, osa-MIR171e, osa-MIR171f, osa-MIR171g,
osa-MIR171h, osa-MIR171i, osa-MIR172a, osa-MIR172b, osa-MIR172c,
osa-MIR173d, osa-MIR390, osa-MIR319a, osa-MIR319b, osa-MIR393,
osa-MIR393b, osa-MIR394, osa-MIR395b, osa-MIR395c, osa-MIR395d,
osa-MIR395e, osa-MIR395g, osa-MIR395h, osa-MIR395i, osa-MIR395j,
osa-MIR395k, osa-MIR395l, osa-MIR395m, osa-MIR395n, osa-MIR395o,
osa-MIR395r, osa-MIR395q, osa-MIR395c, osa-MIR395a, osa-MIR395f,
osa-MIR395p, osa-MIR396a, osa-MIR396b, osa-MIR396c, osa-MIR397a,
osa-MIR397b, osa-MIR398a, osa-MIR398b, osa-MIR399a, osa-MIR399b,
osa-MIR399c, osa-MIR399d, osa-MIR399e, osa-MIR399f, osa-MIR399g,
osa-MIR399h, osa-MIR399i, osa-MIR399j, osa-MIR399k, osa-MIR408,
osa-MIR413, osa-MIR414, osa-MIR415, osa-MIR416, osa-MIR 417,
osa-MIR418, osa-MIR419, osa-MIR426, osa-MIR437, osa-MIR439,
osa-MIR439c, osa-MIR439d, osa-MIR438e, osa-MIR439f, osa-MIR439g,
osa-MIR439h, osa-MIR440, osa-MIR441a, osa-MIR441c, osa-MIR442,
osa-MIR443, osa-MIR445d, osa-MIR446, zma-MIR156a, zma-MIR156b,
zma-MIR156c, zma-MIR156d, zma-MIR156e, zma-MIR156f, zma-MIR156g,
zma-MIR156h, zma-MIR156i, zma-MIR156j, zma-MIR156k, zma-MIR159a,
zma-MIR159b, zma-MIR159c, zma-MIR159d, zma-MIR160a, zma-MIR160b,
zma-MIR160c, zma-MIR160d, zma-MIR160e, zma-MIR160f, zma-MIR 161l,
zma-MIR162, zma-MIR164a, zma-MIR164b, zma-MIR164c, zma-MIR164d,
zma-MIR166a, zma-MIR166b, zma-MIR166c, zma-MIR166d, zma-MIR166e,
zma-MIR166e, zma-MIR166f, zma-MIR166g, zma-MIR166h, zma-MIR166i,
zma-MIR166j, zma-MIR166k, zma-MIR166m, zma-MIR167a, zma-MIR167b,
zma-MIR167c, zma-MIR167d, zma-MIR 167e, zma-MIR167f, zma-MIR167g,
zma-MIR167h, zma-MIR168a, zma-MIR168b, zma-MIR169a, zma-MIR169b,
zma-MIR169c, zma-MIR169d, zma-MIR169e, zma-MIR169f, zma-MIR169g,
zma-MIR169i, zma-MIR169j, zma-MIR169k, zma-MIR171a, zma-MIR171b,
zma-MIR171c, zma-MIR171d, zma-MIR171e, zma-MIR171f, zma-MIR171g,
zma-MIR171h, zma-MIR171i, zma-MIR171j, zma-MIR171k, zma-MIR172a,
zma-MIR172b, zma-MIR172c or zma-MIR172d, zma-MIR172e, zma-MIR319a,
zma-MIR319b, zma-MIR319d, zma-MIR393, zma-MIR394a, zma-MIR394b,
zma-MIR395a, zma-MIR395b, zma-MIR395c, zma-MIR395d, zma-MIR396a,
zma-MIR396b, zma-MIR399a, zma-MIR399b, zma-MIR399c, zma-MIR399d,
zma-MIR399e, zma-MIR399f, zma-MIR408.
[0125] In some embodiments, the miRNA is a miRNA disclosed in
Genbank (USA), EMBL (Europe) or DDBJ (Japan). In some embodiments,
the miRNA is selected from one of the following Genbank accession
numbers: AJ505003, AJ505002, AJ505001, AJ496805, AJ496804,
AJ496803, AJ496802, AJ496801, AJ505004, AJ493656, AJ493655,
AJ493654, AJ493653, AJ493652, AJ493651, AJ493650, AJ493649,
AJ493648, AJ493647, AJ493646, AJ493645, AJ493644, AJ493643,
AJ493642, AJ493641, AJ493640, AJ493639, AJ493638, AJ493637,
AJ493636, AJ493635, AJ493634, AJ493633, AJ493632, AJ493631,
AJ493630, AJ493629, AJ493628, AJ493627, AJ493626, AJ493625,
AJ493624, AJ493623, AJ493622, AJ493621, AJ493620, AY615374,
AY615373, AY730704, AY730703, AY730702, AY730701, AY730700,
AY730699, AY730698, AY599420, AY551259, AY551258, AY551257,
AY551256, AY551255, AY551254, AY551253, AY551252, AY551251,
AY551250, AY551249, AY551248, AY551247, AY551246, AY551245,
AY551244, AY551243, AY551242, AY551241, AY551240, AY551239,
AY551238, AY551237, AY551236, AY551235, AY551234, AY551233,
AY551232, AY551231, AY551230, AY551229, AY551228, AY551227,
AY551226, AY551225, AY551224, AY551223, AY551222, AY551221,
AY551220, AY551219, AY551218, AY551217, AY551216, AY551215,
AY551214, AY551213, AY551212, AY551211, AY551210, AY551209,
AY551208, AY551207, AY551206, AY551205, AY551204, AY551203,
AY551202, AY551201, AY551200, AY551199, AY551198, AY551197,
AY551196, AY551195, AY551194, AY551193, AY551192, AY551191,
AY551190, AY551189, AY551188, AY501434, AY501433, AY501432,
AY501431, AY498859, AY376459, AY376458, AY884233, AY884232,
AY884231, AY884230, AY884229, AY884228, AY884227, AY884226,
AY884225, AY884224, AY884223, AY884222, AY884221, AY884220,
AY884219, AY884218, AY884217, AY884216, AY728475, AY728474,
AY728473, AY728472, AY728471, AY728470, AY728469, AY728468,
AY728467, AY728466, AY728465, AY728464, AY728463, AY728462,
AY728461, AY728460, AY728459, AY728458, AY728457, AY728456,
AY728455, AY728454, AY728453, AY728452, AY728451, AY728450,
AY728449, AY728448, AY728447, AY728446, AY728445, AY728444,
AY728443, AY728442, AY728441, AY728440, AY728439, AY728438,
AY728437, AY728436, AY728435, AY728434, AY728433, AY728432,
AY728431, AY728430, AY728429, AY728428, AY728427, AY728426,
AY728425, AY728424, AY728423, AY728422, AY728421, AY728420,
AY728419, AY728418, AY728417, AY728416, AY728415, AY728414,
AY728413, AY728412, AY728411, AY728410, AY728409, AY728408,
AY728407, AY728406, AY728405, AY728404, AY728403, AY728402,
AY728401, AY728400, AY728399, AY728398, AY728397, AY728396,
AY728395, AY728394, AY728393, AY728392, AY728391, AY728390,
AY728389, AY728388, AY851149, AY851148, AY851147, AY851146,
AY851145, AY851144 or AY599420.
[0126] In some embodiments, the miRNA is selected from one of the
sequences disclosed in U.S. published patent application No.
2005/0144669 Al, incorporated herein by reference.
[0127] In some embodiments, the above miRNAs, as well as those
disclosed herein, have been modified to be directed to a specific
target as described herein.
[0128] In some embodiments the target RNA is an RNA of a plant
pathogen, such as a plant virus or plant viroid. In some
embodiments, the miRNA directed against the plant pathogen RNA is
operably linked to a pathogen-inducible promoter. In some
embodiments, the target RNA is an mRNA. The target sequence in an
mRNA may be a non-coding sequence (such as an intron sequence, 5'
untranslated region and 3' untranslated regeion), a coding sequence
or a sequence involved in mRNA splicing. Targeting the miRNA to an
intron sequence compromises the maturation of the mRNA. Targeting
the miRNA to a sequence involved in mRNA splicing influences the
maturation of alternative splice forms providing different protein
isoforms.
[0129] In some embodiments there are provided cells, plants, and
seeds comprising the polynucleotides of the invention, and/or
produced by the methods of the invention. In some embodiments, the
cells, plants, and/or seeds comprise a nucleic acid construct
comprising a modified plant miRNA precursor, as described herein.
In some embodiments, the modified plant miRNA precursor in the
nucleic acid construct is operably linked to a promoter. The
promoter may be any well known promoter, including constitutive
promoters, inducible promoters, derepressible promoters, and the
like, such as described below. The cells include prokaryotic and
eukaryotic cells, including but not limited to bacteria, yeast,
fungi, viral, invertebrate, vertebrate, and plant cells. Plants,
plant cells, and seeds of the invention include gynosperms,
monocots and dicots, including but not limited to, rice, wheat,
oats, barley, millet, sorghum, soy, sunflower, safflower, canola,
alfalfa, cotton, Arabidopsis, and tobacco.
[0130] In another embodiment, there is provided a method for down
regulating multiple target RNAs comprising introducing into a cell
a nucleic acid construct encoding a multiple number of miRNAs. One
miRNA in the multiple miRNAs is complementary to a region of one of
the target RNAs. In some embodiments, a miRNA is fully
complementary to the region of the target RNA. In some embodiments,
a miRNA is complementary and includes the use of G-U base pairing,
i.e. the GU wobble, to otherwise be fully complementary. In some
embodiments, the first ten nucleotides of the miRNA (counting from
the 5' end of the miRNA) are fully complementary to a region of the
target RNA and the remaining nucleotides may include mismatches
and/or bulges with the target RNA. In some embodiments a miRNA
comprises about 10-200 nucleotides, about 10-15, 15-20, 19, 20, 21,
22, 23, 24, 25, 26, 27, 25-30, 30-50, 50-100, 100-150, or about
150-200 nucleotides. The binding of a miRNA to its complementary
sequence in the target RNA results in cleavage of the target RNA.
In some embodiments, the miRNA is a miRNA that has been modified
such that the miRNA is fully complementary to the target sequence
of the target RNA. In some embodiments, the miRNA is an endogenous
plant miRNA that has been modified such that the miRNA is fully
complementary to the target sequence of the target RNA. In some
embodiments, the miRNA is operably linked to a promoter. In some
embodiments, the multiple miRNAs are linked one to another so as to
form a single transcript when expressed. In some embodiments, the
nucleic acid construct comprises a promoter operably linked to the
miRNA.
[0131] In some embodiments, the nucleic acid construct encodes
miRNAs for suppressing a multiple number of target sequences. The
nucleic acid construct encodes at least two miRNAs. In some
embodiments, each miRNA is substantially complementary to a target
or which is modified to be complementary to a target as described
herein. In some embodiments, the nucleic acid construct encodes for
2-30 or more miRNAs, for example 3-40 or more miRNAs, for example
3-45 or more miRNAs, and for further example, multimers of 2, 3, 4,
5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,
23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,
40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or more miRNAs. In some
embodiments, the multiple miRNAs are linked one to another so as to
form a single transcript when expressed.
[0132] In some embodiments, polymeric pre-miRNAs that are
artificial miRNA precursors consisting of more than one miRNA
precursor units are provided. The polymeric pre-miRNAs can either
be hetero-polymeric with different miRNA precursors, or
homo-polymeric containing several units of the same miRNA
precursor. The Examples demonstrate that hetero-polymeric
pre-miRNAs are able to produce different mature artificial miRNAs.
For example, pre-miR-PDS1.sup.169g-CPC3.sup.159a, which is a dimer
comprising of pre-miR-CPC3.sup.159a and pre-miR-PDS1.sup.169g can
produce mature miR-PDS1.sup.169g and miR-CPC3.sup.159a when
expressed in plant cells. The Examples also demonstrate that
homo-polymeric miRNA precursors are able to produce different
mature artificial miRNAs. For example,
pre-miR-P69.sup.159a-HC-Pro.sup.159a, which is a dimer comprising
pre-miR-P69.sup.159a and pre-miR-HC-Pro.sup.159a, can produce
mature miR-P69.sup.159a and miR-HC-Pro.sup.159a. In a similar
manner, hetero- or homo-polymeric pre-miRNAs are produced that
contain any number of monomer units, such as described herein. An
exemplary method for preparing a nucleic acid construct comprising
multiple pre-miRNAs under the control of a single promoter is shown
in Examples 21 and 27. Each mature miRNA is properly processed from
the nucleic acid construct as demonstrated in Examples 22 and
27.
[0133] In some embodiments, the nucleic acid construct comprises
multiple polynucleotides, each polynucleotide encoding a separate
miRNA precursor, i.e., a separate pre-miRNA. The polynucleotides
are operably linked one to another such that they may be placed
under the control of a single promoter. In some embodiments, the
multiple polynucleotides are linked one to another so as to form a
single transcript containing the multiple pre-miRNAs when
expressed. The single transcript is processed in the host cells to
produce multiple mature miRNAs, each capable of downregulating its
target gene. As many polynucleotides encoding the pre-miRNAs as
desired can be linked together, with the only limitation being the
ultimate size of the transcript. It is well known that transcripts
of 8-10 kb can be produced in plants. Thus, it is possible to form
a nucleic acid construct comprising multimeric polynucleotides
encoding 2-30 or more pre-miRNAs, for example 3-40 or more
pre-miRNAs, for example 3-45 or more pre-miRNAs, and for further
example, multimers of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,
32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,
49, or more pre-miRNAs.
[0134] In some embodiments, the nucleic acid construct further
comprises a promoter operably linked to the polynucleotide encoding
the multiple number of miRNAs. In some embodiments, the nucleic
acid construct lacking a promoter is designed and introduced in
such a way that it becomes operably linked to a promoter upon
integration in the host genome. In some embodiments, the nucleic
acid construct is integrated using recombination, including
site-specific recombination. See, for example, PCT International
published application No. WO 99/25821, herein incorporated by
reference. In some embodiments, the nucleic acid construct is an
RNA. In some embodiments, the nucleic acid construct comprises at
least one recombination site, including site-specific recombination
sites. In some embodiments the nucleic acid construct comprises at
least one recombination site in order to facilitate integration,
modification, or cloning of the construct. In some embodiments the
nucleic acid construct comprises two site-specific recombination
sites flanking the miRNA precursor. In some embodiments the
site-specific recombination sites include FRT sites, lox sites, or
att sites, including attB, attL, attP or attR sites. See, for
example, PCT International published application No. WO 99/25821,
and U.S. Pat. Nos. 5,888,732, 6,143,557, 6,171,861, 6,270,969, and
6,277,608, herein incorporated by reference.
[0135] In some embodiments, the pre-miRNA is inserted into an
intron in a gene or a transgene of a cell or plant. If the gene has
multiple introns, a pre-miRNA, which can be the same or different,
can be inserted into each intron. In some embodiments the pre-miRNA
inserted into an intron is a polymeric pre-miRNA, such as described
herein. During RNA splicing, introns are released from primary RNA
transcripts and therefore, as illustrated herein, can serve as
precursors for miRNAs. Most introns contain a splicing donor site
at the 5' end, splicing acceptor site at the 3' end and a branch
site within the intron. The branch site is important for intron
maturation--without it, an intron can not be excised and released
from the primary RNA transcript. A branch site is usually located
20-50 nt upstream of the splicing acceptor site, whereas distances
between the splice donor site and the branch site are largely
variable among different introns. Thus, in some embodiments, the
pre-miRNA is inserted into an intron between the splicing donor
site and the branch site.
[0136] In some embodiments the target RNA is an RNA of a plant
pathogen, such as a plant virus or plant viroid. In some
embodiments, the miRNA directed against the plant pathogen RNA is
operably linked to a pathogen-inducible promoter. In some
embodiments, the target RNA is an mRNA. The target sequence in an
mRNA may be an intron sequence, a coding sequence or a sequence
involved in mRNA splicing. Targeting the miRNA to an intron
sequence compromises the maturation of the mRNA. Targeting the
miRNA to a sequence involved in mRNA splicing influences the
maturation of alternative splice forms providing different protein
isoforms. In some embodiments, the target includes genes affecting
agronomic traits, insect resistance, disease resistance, herbicide
resistance, sterility, grain characteristics, and commercial
products.
[0137] In some embodiments there are provided cells, plants, and
seeds comprising the nucleic acid construct encoding multiple
miRNAs of the invention, and/or produced by the methods of the
invention. In some embodiments, the cells, plants, and/or seeds
comprise a nucleic acid construct comprising multiple
polynucleotides, each encoding a plant miRNA precursor, as
described herein. In some embodiments, the multiple polynucleotides
are operably linked to a promoter. The promoter may be any well
known promoter, including constitutive promoters, inducible
promoters, derepressible promoters, and the like, such as described
below. The polynucleotides encoding the miRNA precursors are linked
together. In some embodiments, the multiple polynucleotides are
linked one to another so as to form a single transcript containing
the multiple pre-miRNAs when expressed in the cells, plants or
seeds. The cells include prokaryotic and eukaryotic cells,
including but not limited to bacteria, yeast, fungi, viral,
invertebrate, vertebrate, and plant cells. Plants, plant cells, and
seeds of the invention include gynosperms, monocots and dicots,
including but not limited to, rice, wheat, oats, barley, millet,
sorghum, soy, sunflower, safflower, canola, alfalfa, cotton,
Arabidopsis, and tobacco.
[0138] The present invention concerns methods and compositions
useful in suppression of a target sequence and/or validation of
function. The invention also relates to a method for using microRNA
(miRNA) mediated RNA interference (RNAi) to silence or suppress a
target sequence to evaluate function, or to validate a target
sequence for phenotypic effect and/or trait development.
Specifically, the invention relates to constructs comprising small
nucleic acid molecules, miRNAs, capable of inducing silencing, and
methods of using these miRNAs to selectively silence target
sequences.
[0139] RNA interference refers to the process of sequence-specific
post-transcriptional gene silencing in animals mediated by short
interfering RNAs (siRNAs) (Fire et al. (1998) Nature 391:806-810).
The corresponding process in plants is commonly referred to as
post-transcriptional gene silencing (PTGS) or RNA silencing and is
also referred to as quelling in fungi. The process of
post-transcriptional gene silencing is thought to be an
evolutionarily-conserved cellular defense mechanism used to prevent
the expression of foreign genes and is commonly shared by diverse
flora and phyla (Fire et al. (1999) Trends Genet. 15:358-363). Such
protection from foreign gene expression may have evolved in
response to the production of double-stranded RNAs (dsRNAs) derived
from viral infection or from the random integration of transposon
elements into a host genome via a cellular response that
specifically destroys homologous single-stranded RNA of viral
genomic RNA. The presence of dsRNA in cells triggers the RNAi
response through a mechanism that has yet to be fully
characterized.
[0140] The presence of long dsRNAs in cells stimulates the activity
of a ribonuclease III enzyme referred to as dicer. Dicer is
involved in the processing of the dsRNA into short pieces of dsRNA
known as short interfering RNAs (siRNAs) (Bernstein et al. (2001)
Nature 409:363-366). Short interfering RNAs derived from dicer
activity are typically about 21 to about 23 nucleotides in length
and comprise about 19 base pair duplexes (Elbashir et al. (2001)
Genes Dev 15:188-200). Dicer has also been implicated in the
excision of 21- and 22-nucleotide small temporal RNAs (stRNAs) from
precursor RNA of conserved structure that are implicated in
translational control (Hutvagner et al. (2001) Science
293:834-838). The RNAi response also features an endonuclease
complex, commonly referred to as an RNA-induced silencing complex
(RISC), which mediates cleavage of single-stranded RNA having
sequence complementarity to the antisense strand of the siRNA
duplex. Cleavage of the target RNA takes place in the middle of the
region complementary to the antisense strand of the siRNA duplex
(Elbashir et al. (2001) Genes Dev 15:188-200). In addition, RNA
interference can also involve small RNA (e.g., microRNA, or miRNA)
mediated gene silencing, presumably through cellular mechanisms
that regulate chromatin structure and thereby prevent transcription
of target gene sequences (see, e.g., Allshire, Science
297:1818-1819 2002; Volpe et al. (2002) Science 297:1833-1837;
Jenuwein (2002) Science 297:2215-2218; Hall et al. (2002) Science
297:2232-2237). As such, miRNA molecules of the invention can be
used to mediate gene silencing via interaction with RNA transcripts
or alternately by interaction with particular gene sequences,
wherein such interaction results in gene silencing either at the
transcriptional or post-transcriptional level.
[0141] RNAi has been studied in a variety of systems. Fire et al.
((1998) Nature 391:806-811) were the first to observe RNAi in C.
elegans. Wianny and Goetz ((1999) Nature Cell Biol 2:70) describe
RNAi mediated by dsRNA in mouse embryos. Hammond et al. ((2000)
Nature 404:293-296) describe RNAi in Drosophila cells transfected
with dsRNA. Elbashir et al. ((2001) Nature 411:494-498) describe
RNAi induced by introduction of duplexes of synthetic 21-nucleotide
RNAs in cultured mammalian cells including human embryonic kidney
and HeLa cells.
[0142] Small RNAs play an important role in controlling gene
expression. Regulation of many developmental processes, including
flowering, is controlled by small RNAs. It is now possible to
engineer changes in gene expression of plant genes by using
transgenic constructs which produce small RNAs in the plant.
[0143] Small RNAs appear to function by base-pairing to
complementary RNA or DNA target sequences. When bound to RNA, small
RNAs trigger either RNA cleavage or translational inhibition of the
target sequence. When bound to DNA target sequences, it is thought
that small RNAs can mediate DNA methylation of the target sequence.
The consequence of these events, regardless of the specific
mechanism, is that gene expression is inhibited.
[0144] It is thought that sequence complementarity between small
RNAs and their RNA targets helps to determine which mechanism, RNA
cleavage or translational inhibition, is employed. It is believed
that siRNAs, which are perfectly complementary with their targets,
work by RNA cleavage. Some miRNAs have perfect or near-perfect
complementarity with their targets, and RNA cleavage has been
demonstrated for at least a few of these miRNAs. Other miRNAs have
several mismatches with their targets, and apparently inhibit their
targets at the translational level. Again, without being held to a
particular theory on the mechanism of action, a general rule is
emerging that perfect or near-perfect complementarity favors RNA
cleavage, especially within the first ten nucleotides (counting
from the 5'end of the miRNA), whereas translational inhibition is
favored when the miRNA/target duplex contains many mismatches. The
apparent exception to this is microRNA 172 (miR172) in plants. One
of the targets of miR172 is APETALA2 (AP2), and although miR172
shares near-perfect complementarity with AP2 it appears to cause
translational inhibition of AP2 rather than RNA cleavage.
[0145] MicroRNAs (miRNAs) are noncoding RNAs of about 19 to about
24 nucleotides (nt) in length that have been identified in both
animals and plants (Lagos-Quintana et al. (2001) Science
294:853-858, Lagos-Quintana et al. (2002) Curr Biol 12:735-739; Lau
et al. (2002) Science 294:858-862; Lee and Ambros (2001) Science
294:862-864; Llave et al. (2002) Plant Cell 14:1605-1619;
Mourelatos et al. (2002) Genes Dev 16:720-728; Park et al. (2002)
Curr Biol 12:1484-1495; Reinhart et al. (2002) Genes Dev
16:1616-1626). They are processed from longer precursor transcripts
that range in size from approximately 70 to 200 nt, and these
precursor transcripts have the ability to form stable hairpin
structures. In animals, the enzyme involved in processing miRNA
precursors is called Dicer, an RNAse III-like protein (Grishok et
al. (2001) Cell 106:23-34; Hutvagner et al. (2001) Science
293:834-838; Ketting et al. (2001) Genes Dev 15:2654-2659). Plants
also have a Dicer-like enzyme, DCL1 (previously named CARPEL
FACTORY/SHORT INTEGUMENTS1/SUSPENSOR1), and recent evidence
indicates that it, like Dicer, is involved in processing the
hairpin precursors to generate mature miRNAs (Park et al. (2002)
Curr Biol 12:1484-1495; Reinhart et al. (2002) Genes Dev
16:1616-1626). Furthermore, it is becoming clear from recent work
that at least some miRNA hairpin precursors originate as longer
polyadenylated transcripts, and several different miRNAs and
associated hairpins can be present in a single transcript
(Lagos-Quintana et al. (2001) Science 294:853-858; Lee et al.
(2002) EMBO J 21:4663-4670). Recent work has also examined the
selection of the miRNA strand from the dsRNA product arising from
processing of the hairpin by DICER (Schwartz et al. (2003) Cell
115:199-208). It appears that the stability (i.e. G:C vs. A:U
content, and/or mismatches) of the two ends of the processed dsRNA
affects the strand selection, with the low stability end being
easier to unwind by a helicase activity. The 5' end strand at the
low stability end is incorporated into the RISC complex, while the
other strand is degraded.
[0146] In animals, there is direct evidence indicating a role for
specific miRNAs in development. The lin-4 and let-7 miRNAs in C.
elegans have been found to control temporal development, based on
the phenotypes generated when the genes producing the lin-4 and
let-7 miRNAs are mutated (Lee et al. (1993) Cell 75:843-854;
Reinhart et al. (2000) Nature 403-901-906). In addition, both
miRNAs display a temporal expression pattern consistent with their
roles in developmental timing Other animal miRNAs display
developmentally regulated patterns of expression, both temporal and
tissue-specific (Lagos-Quintana et al. (2001) Science 294:853-853,
Lagos-Quintana et al. (2002) Curr Biol 12:735-739; Lau et al.
(2001) Science 294:858-862; Lee and Ambros (2001) Science
294:862-864), leading to the hypothesis that miRNAs may, in many
cases, be involved in the regulation of important developmental
processes. Likewise, in plants, the differential expression
patterns of many miRNAs suggests a role in development (Llave et
al. (2002) Plant Cell 14:1605-1619; Park et al. (2002) Curr Biol
12:1484-1495; Reinhart et al. (2002) Genes Dev 16:1616-1626), which
has now been shown (e.g., Guo et al. (2005) Plant Cell
17:1376-1386).
[0147] MicroRNAs appear to regulate target genes by binding to
complementary sequences located in the transcripts produced by
these genes. In the case of lin-4 and let-7, the target sites are
located in the 3' UTRs of the target mRNAs (Lee et al. (1993) Cell
75:843-854; Wightman et al. (1993) Cell 75:855-862; Reinhart et al.
(2000) Nature 403:901-906; Slack et al. (2000) Mol Cell 5:659-669),
and there are several mismatches between the lin-4 and let-7 miRNAs
and their target sites. Binding of the lin-4 or let-7 miRNA appears
to cause downregulation of steady-state levels of the protein
encoded by the target mRNA without affecting the transcript itself
(Olsen and Ambros (1999) Dev Biol 216:671-680). On the other hand,
recent evidence suggests that miRNAs can, in some cases, cause
specific RNA cleavage of the target transcript within the target
site, and this cleavage step appears to require 100%
complementarity between the miRNA and the target transcript
(Hutvagner and Zamore (2002) Science 297:2056-2060; Llave et al.
(2002) Plant Cell 14:1605-1619), especially within the first ten
nucleotides (counting from the 5' end of the miRNA). It seems
likely that miRNAs can enter at least two pathways of target gene
regulation. Protein downregulation when target complementarity is
<100%, and RNA cleavage when target complementarity is 100%.
MicroRNAs entering the RNA cleavage pathway are analogous to the
21-25 nt short interfering RNAs (siRNAs) generated during RNA
interference (RNAi) in animals and posttranscriptional gene
silencing (PTGS) in plants (Hamilton and Baulcombe (1999) Science
286:950-952; Hammond et al., (2000) Nature 404:293-296; Zamore et
al., (2000) Cell 31:25-33; Elbashir et al., (2001) Nature
411:494-498), and likely are incorporated into an RNA-induced
silencing complex (RISC) that is similar or identical to that seen
for RNAi.
[0148] Identifying the targets of miRNAs with bioinformatics has
not been successful in animals, and this is probably due to the
fact that animal miRNAs have a low degree of complementarity with
their targets. On the other hand, bioinformatic approaches have
been successfully used to predict targets for plant miRNAs (Llave
et al. (2002) Plant Cell 14:1605-1619; Park et al. (2002) Curr Biol
12:1484-1495; Rhoades et al. (2002) Cell 110:513-520), and thus it
appears that plant miRNAs have higher overall complementarity with
their putative targets than do animal miRNAs. Most of these
predicted target transcripts of plant miRNAs encode members of
transcription factor families implicated in plant developmental
patterning or cell differentiation. Nonetheless, biological
function has not been directly demonstrated for any plant miRNA.
Although Llave et al. ((2002) Science 297:2053-2056) have shown
that a transcript for a SCARECROW-like transcription factor is a
target of the Arabidopsis miRNA mir171, these studies were
performed in a heterologous species and no plant phenotype
associated with mir171 was reported.
[0149] The methods provided can be practiced in any organism in
which a method of transformation is available, and for which there
is at least some sequence information for the target sequence, or
for a region flanking the target sequence of interest. It is also
understood that two or more sequences could be targeted by
sequential transformation, co-transformation with more than one
targeting vector, or the construction of a DNA construct comprising
more than one miRNA sequence. The methods of the invention may also
be implemented by a combinatorial nucleic acid library construction
in order to generate a library of miRNAs directed to random target
sequences. The library of miRNAs could be used for high-throughput
screening for gene function validation.
[0150] General categories of sequences of interest include, for
example, those genes involved in regulation or information, such as
zinc fingers, transcription factors, homeotic genes, or cell cycle
and cell death modulators, those involved in communication, such as
kinases, and those involved in housekeeping, such as heat shock
proteins.
[0151] Target sequences further include coding regions and
non-coding regions such as promoters, enhancers, terminators,
introns and the like, which may be modified in order to alter the
expression of a gene of interest. For example, an intron sequence
can be added to the 5' region to increase the amount of mature
message that accumulates (see for example Buchman and Berg (1988)
Mol Cell Biol 8:4395-4405); and Callis et al. (1987) Genes Dev
1:1183-1200).
[0152] The target sequence may be an endogenous sequence, or may be
an introduced heterologous sequence, or transgene. For example, the
methods may be used to alter the regulation or expression of a
transgene, or to remove a transgene or other introduced sequence
such as an introduced site-specific recombination site. The target
sequence may also be a sequence from a pathogen, for example, the
target sequence may be from a plant pathogen such as a virus, a
mold or fungus, an insect, or a nematode. A miRNA can be expressed
in a plant which, upon infection or infestation, would target the
pathogen and confer some degree of resistance to the plant. The
Examples herein demonstrate the techniques to design artificial
miRNAs to confer virus resistance/tolerance to plants. In some
embodiments, two or more artificial miRNA sequences directed
against different seqeuences of the virus can be used to prevent
the target virus from mutating and thus evading the resistance
mechanism. In some embodiments, sequences of artifical miRNAs can
be selected so that they target a critical region of the viral RNA
(e.g. active site of a silencing gene suppressor). In this case,
mutation of the virus in this selected region may render the
encoded protein inactive, thus preventing mutation of the virus as
a way to escape the resistance mechanism. In some embodiments, an
artifical miRNA directed towards a conserved sequence of a family
of viruses would confer resistance to members of the entire family.
In some embodiments, an artifical miRNA directed towards a sequence
conserved amongst members of would confer resistance to members of
the different viral families (e.g., see Table 1).
TABLE-US-00001 TABLE 1 Conserved Viral Genome Sequence of TuMV for
Artificial miRNA Design TuMV CY5 No Region.sup.a Gene
Sequence.sup.b (SEQ ID NO:) length 1 3207 to 3229 P3
5'-cgatttaggcggcagatacagcg-3'(167) 23 2 9151 to 9185 CP
5'-attctcaatggtttaatggtctg 35 gtgcattgagaa-3'(168) 3 9222 to 9227
CP 5'-ataaacggaatgtgggtgatgatgga-3'(169) 26 4 9235 to 9255 CP
5'-gatcaggtggaattcccgatc-3'(170) 21 5 9270 to 9302 CP
5'-cacgccaaacccacatttaggcaaa 32 taatggc-3'(171) 6 9319 to 9386 CP
5'-gctgaagcgtacattgaaaagcgtaacca 68
agaccgaccatacatgccacgatatggtcttc agcgcaa-3'(172) 7 9430 to 9509 CP
5'-gaaatgacttctagaactccaatacgtgcga 80
gagaagcacacatccagatgaaagcagcagca ctgcgtggcgcaaataa-3'(173) 8 9541
to 9566 CP 5'-acaacggtagagaacacggagaggca-3'(174) 26 .sup.aThe
region of genome sequence is according to TuMV CY5 strain
(AF530055). .sup.bThe highly conserved of TuMV sequence from 21
different TuMV strains was alignment by Vector NTI Advance 10.0.1
software (Invitrogen Corp). The full-length sequence of 21
different TuMV strains were obtained from the GenBank database
under the following accession numbers including AB093596, AB093598,
AB093599, AB093600, AB093615, AB093616, AB093617, AB093618,
AB093619, AB093611, AB093612, AY227024, AB093609, AF394601,
AF169561, AF530055, AF394602, AB093623, AB093624, AY090660,
D83184.
[0153] In plants, other categories of target sequences include
genes affecting agronomic traits, insect resistance, disease
resistance, herbicide resistance, sterility, grain characteristics,
and commercial products. Genes of interest also include those
involved in oil, starch, carbohydrate, or nutrient metabolism as
well as those affecting, for example, kernel size, sucrose loading,
and the like. The quality of grain is reflected in traits such as
levels and types of oils, saturated and unsaturated, quality and
quantity of essential amino acids, and levels of cellulose. For
example, genes of the phytic acid biosynthetic pathway could be
suppressed to generate a high available phosphorous phenotype. See,
for example, phytic acid biosynthetic enzymes including inositol
polyphosphate kinase-2 polynucleotides, disclosed in WO 02/059324,
inositol 1,3,4-trisphosphate 5/6-kinase polynucleotides, disclosed
in WO 03/027243, and myo-inositol 1-phosphate synthase and other
phytate biosynthetic polynucleotides, disclosed in WO 99/05298, all
of which are herein incorporated by reference. Genes in the
lignification pathway could be suppressed to enhance digestibility
or energy availability. Genes affecting cell cycle or cell death
could be suppressed to affect growth or stress response. Genes
affecting DNA repair and/or recombination could be suppressed to
increase genetic variability. Genes affecting flowering time could
be suppressed, as well as genes affecting fertility. Any target
sequence could be suppressed in order to evaluate or confirm its
role in a particular trait or phenotype, or to dissect a molecular,
regulatory, biochemical, or proteomic pathway or network.
[0154] A number of promoters can be used, these promoters can be
selected based on the desired outcome. It is recognized that
different applications will be enhanced by the use of different
promoters in plant expression cassettes to modulate the timing,
location and/or level of expression of the miRNA. Such plant
expression cassettes may also contain, if desired, a promoter
regulatory region (e.g., one conferring inducible, constitutive,
environmentally- or developmentally-regulated, or cell- or
tissue-specific/selective expression), a transcription initiation
start site, a ribosome binding site, an RNA processing signal, a
transcription termination site, and/or a polyadenylation
signal.
[0155] Constitutive, tissue-preferred or inducible promoters can be
employed. Examples of constitutive promoters include the
cauliflower mosaic virus (CaMV) 35S transcription initiation
region, the 1'- or 2'- promoter derived from T-DNA of Agrobacterium
tumefaciens, the ubiquitin 1 promoter, the Smas promoter, the
cinnamyl alcohol dehydrogenase promoter (U.S. Pat. No. 5,683,439),
the Nos promoter, the pEmu promoter, the rubisco promoter, the
GRP1-8 promoter and other transcription initiation regions from
various plant genes known to those of skill. If low level
expression is desired, weak promoter(s) may be used. Weak
constitutive promoters include, for example, the core promoter of
the Rsyn7 promoter (WO 99/43838 and U.S. Pat. No. 6,072,050), the
core 35S CaMV promoter, and the like. Other constitutive promoters
include, for example, U.S. Pat. Nos. 5,608,149; 5,608,144;
5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463; and
5,608,142. See also, U.S. Pat. No. 6,177,611, herein incorporated
by reference.
[0156] Examples of inducible promoters are the Adhl promoter which
is inducible by hypoxia or cold stress, the Hsp70 promoter which is
inducible by heat stress, the PPDK promoter and the PEP (phophoenol
pyruvate) carboxylase promoter which are both inducible by light.
Also useful are promoters which are chemically inducible, such as
the In2-2 promoter which is safener induced (U.S. Pat. No.
5,364,780), the ERE promoter which is estrogen induced, and the
Axigl promoter which is auxin induced and tapetum specific but also
active in callus (PCT International published application No. WO
02/04699). Other examples of inducible promoters include the GVG
and XVE promoters, which are induced by glucocorticoids and
estrogen, respectively (U.S. Pat. No. 6,452,068).
[0157] Examples of promoters under developmental control include
promoters that initiate transcription preferentially in certain
tissues, such as leaves, roots, fruit, seeds, or flowers. An
exemplary promoter is the anther specific promoter 5126 (U.S. Pat.
Nos. 5,689,049 and 5,689,051). Examples of seed-preferred promoters
include, but are not limited to, 27 kD gamma zein promoter and waxy
promoter (Boronat et al. (1986) Plant Sci 47:95-102; Reina et al.
(1990) Nucl Acids Res 18(21):6426; Kloesgen et al. (1986) Mol. Gen.
Genet. 203:237-244). Promoters that express in the embryo,
pericarp, and endosperm are disclosed in U.S. Pat. No. 6,225,529
and PCT International published application No. WO 00/12733. The
disclosures of each of these are incorporated herein by reference
in their entirety.
[0158] In some embodiments it will be beneficial to express the
gene from an inducible promoter, particularly from a
pathogen-inducible promoter. Such promoters include those from
pathogenesis-related proteins (PR proteins), which are induced
following infection by a pathogen; e.g., PR proteins, SAR proteins,
beta-1,3-glucanase, chitinase, etc. See, for example, Redolfi et
al. (1983) Neth. J. Plant Pathol. 89:245-254; Uknes et al. (1992)
Plant Cell 4:645-656; and Van Loon (1985) Plant Mol. Virol.
4:111-116. See also PCT International published application No. WO
99/43819, herein incorporated by reference.
[0159] Of interest are promoters that are expressed locally at or
near the site of pathogen infection. See, for example, Marineau et
al. (1987) Plant Mol Biol 9:335-342; Matton et al. (1989) Molecular
Plant-Microbe Interactions 2:325-331; Somsisch et al. (1986) Proc
Natl Acad Sci USA 83:2427-2430; Somsisch et al. (1988) Mol Gen
Genet 2:93-98; and Yang (1996) Proc Natl Acad Sci USA
93:14972-14977. See also, Chen et al. (1996) Plant J 10:955-966;
Zhang et al. (1994) Proc Natl Acad Sci USA 91:2507-2511; Warner et
al. (1993) Plant J 3:191-201; Siebertz et al. (1989) Plant Cell
1:961-968; U.S. Pat. No. 5,750,386 (nematode-inducible); and the
references cited therein. Of particular interest is the inducible
promoter for the maize PRms gene, whose expression is induced by
the pathogen Fusarium moniliforme (see, for example, Cordero et al.
(1992) Physiol Mol Plant Path 41:189-200).
[0160] Additionally, as pathogens find entry into plants through
wounds or insect damage, a wound-inducible promoter may be used in
the constructions of the polynucleotides. Such wound-inducible
promoters include potato proteinase inhibitor (pin II) gene (Ryan
(1990) Ann Rev Phytopath 28:425-449; Duan et al. (1996) Nature
Biotech 14:494-498); wun1 and wun2, U.S. Pat. No. 5,428,148; win1
and win2 (Stanford et al. (1989) Mol Gen Genet 215:200-208);
systemin (McGurl et al. (1992) Science 225:1570-1573); WIP1
(Rohmeier et al. (1993) Plant Mol Biol 22:783-792; Eckelkamp et al.
(1993) FEBS Lett 323:73-76); MPI gene (Corderok et al. (1994) Plant
J 6(2):141-150); and the like, herein incorporated by
reference.
[0161] Chemical-regulated promoters can be used to modulate the
expression of a gene in a plant through the application of an
exogenous chemical regulator. Depending upon the objective, the
promoter may be a chemical-inducible promoter, where application of
the chemical induces gene expression, or a chemical-repressible
promoter, where application of the chemical represses gene
expression. Chemical-inducible promoters are known in the art and
include, but are not limited to, the maize In2-2 promoter, which is
activated by benzenesulfonamide herbicide safeners, the maize GST
promoter, which is activated by hydrophobic electrophilic compounds
that are used as pre-emergent herbicides, and the tobacco PR-la
promoter, which is activated by salicylic acid. Other
chemical-regulated promoters of interest include steroid-responsive
promoters (see, for example, the glucocorticoid-inducible promoter
in Schena et al. (1991) Proc Natl Acad Sci USA 88:10421-10425 and
McNellis et al. (1998) Plant J 14(2):247-257) and
tetracycline-inducible and tetracycline-repressible promoters (see,
for example, Gatz et al. (1991) Mol Gen Genet 227:229-237, and U.S.
Pat. Nos. 5,814,618 and 5,789,156), herein incorporated by
reference.
[0162] Tissue-preferred promoters can be utilized to target
enhanced expression of a sequence of interest within a particular
plant tissue. Tissue-preferred promoters include Yamamoto et al.
(1997) Plant J 12(2):255-265; Kawamata et al. (1997) Plant Cell
Physiol 38(7):792-803; Hansen et al. (1997) Mol Gen Genet
254(3):337-343; Russell et al. (1997) Transgenic Res 6(2):157-168;
Rinehart et al. (1996) Plant Physiol 112(3):1331-1341; Van Camp et
al. (1996) Plant Physiol 112(2):525-535; Canevascini et al. (1996)
Plant Physiol 112(2):513-524; Yamamoto et al. (1994) Plant Cell
Physiol 35(5):773-778; Lam (1994) Results Probl Cell Differ
20:181-196; Orozco et al. (1993) Plant Mol Biol 23(6):1129-1138;
Matsuoka et al. (1993) Proc Natl Acad Sci USA 90(20):9586-9590; and
Guevara-Garcia et al. (1993) Plant J 4(3):495-505. Such promoters
can be modified, if necessary, for weak expression.
[0163] Leaf-preferred promoters are known in the art. See, for
example, Yamamoto et al. (1997) Plant J 12(2):255-265; Kwon et al.
(1994) Plant Physiol 105:357-67; Yamamoto et al. (1994) Plant Cell
Physiol 35(5):773-778; Gotor et al. (1993) Plant J 3:509-18; Orozco
et al. (1993) Plant Mol Biol 23(6):1129-1138; and Matsuoka et al.
(1993) Proc Natl Acad Sci USA 90(20):9586-9590. In addition, the
promoters of cab and RUBISCO can also be used. See, for example,
Simpson et al. (1958) EMBO J 4:2723-2729 and Timko et al. (1988)
Nature 318:57-58.
[0164] Root-preferred promoters are known and can be selected from
the many available from the literature or isolated de novo from
various compatible species. See, for example, Hire et al. (1992)
Plant Mol Biol 20(2):207-218 (soybean root-specific glutamine
synthetase gene); Keller and Baumgartner (1991) Plant Cell
3(10):1051-1061 (root-specific control element in the GRP 1.8 gene
of French bean); Sanger et al. (1990) Plant Mol Biol 14(3):433-443
(root-specific promoter of the mannopine synthase (MAS) gene of
Agrobacterium tumefaciens); and Miao et al. (1991) Plant Cell
3(1):11-22 (full-length cDNA clone encoding cytosolic glutamine
synthetase (GS), which is expressed in roots and root nodules of
soybean). See also Bogusz et al. (1990) Plant Cell 2(7):633-641,
where two root-specific promoters isolated from hemoglobin genes
from the nitrogen-fixing nonlegume Parasponia andersonii and the
related non-nitrogen-fixing nonlegume Trema tomentosa are
described. The promoters of these genes were linked to a
.beta.-glucuronidase reporter gene and introduced into both the
nonlegume Nicotiana tabacum and the legume Lotus corniculatus, and
in both instances root-specific promoter activity was preserved.
Leach and Aoyagi ((1991) Plant Science (Limerick) 79(1):69-76)
describe their analysis of the promoters of the highly expressed
rolC and rolD root-inducing genes of Agrobacterium rhizogens. They
concluded that enhancer and tissue-preferred DNA determinants are
dissociated in those promoters. Teeri et al. ((1989) EMBO J
8(2):343-350) used gene fusion to lacZ to show that the
Agrobacterium T-DNA gene encoding octopine synthase is especially
active in the epidermis of the root tip and that the TR2' gene is
root specific in the intact plant and stimulated by wounding in
leaf tissue, an especially desirable combination of characteristics
for use with an insecticidal or larvicidal gene. The TR1' gene,
fused to nptII (neomycin phosphotransferase II) showed similar
characteristics. Additional root-preferred promoters include the
VfENOD-GRP3 gene promoter (Kuster et al. (1995) Plant Mol Biol
29(4):759-772); and rolB promoter (Capana et al. (1994) Plant Mol
Biol 25(4):681-691. See also U.S. Pat. Nos. 5,837,876; 5,750,386;
5,633,363; 5,459,252; 5,401,836; 5,110,732; and 5,023,179. The
phaseolin gene (Murai et al. (1983) Science 23:476-482 and
Sengopta-Gopalen et al. (1988) Proc Natl Acad Sci USA
82:3320-3324.
[0165] Transformation protocols as well as protocols for
introducing nucleotide sequences into plants may vary depending on
the type of plant or plant cell, i.e., monocot or dicot, targeted
for transformation. Suitable methods of introducing the DNA
construct include microinjection (Crossway et al. (1986)
Biotechniques 4:320-334; and U.S. Pat. No. 6,300,543), sexual
crossing, electroporation (Riggs et al. (1986) Proc Natl Acad Sci
USA 83:5602-5606), Agrobacterium-mediated transformation (Townsend
et al., U.S. Pat No. 5,563,055; and U.S. Pat. No. 5,981,840),
direct gene transfer (Paszkowski et al. (1984) EMBO J 3:2717-2722),
and ballistic particle acceleration (see, for example, U.S. Pat.
No. 4,945,050; U.S. Pat. No. 5,879,918; U.S. Pat. No. 5,886,244;
U.S. Pat. No. 5,932,782; Tomes et al. (1995) "Direct DNA Transfer
into Intact Plant Cells via Microprojectile Bombardment," in Plant
Cell, Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg
and Phillips (Springer-Verlag, Berlin); and McCabe et al. (1988)
Biotechnology 6:923-926). Also see Weissinger et al. (1988) Ann Rev
Genet 22:421-477; Sanford et al. (1987) Particulate Science and
Technology 5:27-37 (onion); Christou et al. (1988) Plant Physiol
87:671-674 (soybean); Finer and McMullen (1991) In Vitro Cell Dev
Biol 27P:175-182 (soybean); Singh et al. (1998) Theor Appl Genet
96:319-324 (soybean); Datta et al. (1990) Biotechnology 8:736-740
(rice); Klein et al. (1988) Proc Natl Acad Sci USA 85:4305-4309
(maize); Klein et al. (1988) Biotechnology 6:559-563 (maize); U.S.
Pat. No. 5,240,855; U.S. Pat. No. 5,322,783; U.S. Pat. No.
5,324,646; Klein et al. (1988) Plant Physiol 91:440-444 (maize);
Fromm et al. (1990) Biotechnology 8:833-839 (maize); Hooykaas-Van
Slogteren et al. (1984) Nature 311:763-764; U.S. Pat. No. 5,736,369
(cereals); Bytebier et al. (1987) Proc Natl Acad Sci USA
84:5345-5349 (Liliaceae); De Wet et al. (1985) in The Experimental
Manipulation of Ovule Tissues, ed. Chapman et al. (Longman, N.Y.),
pp. 197-209 (pollen); Kaeppler et al. (1990) Plant Cell Reports
9:415-418 and Kaeppler et al. (1992) Theor Appl Genet 84:560-566
(whisker-mediated transformation); D'Halluin et al. (1992) Plant
Cell 4:1495-1505 (electroporation); Li et al. (1993) Plant Cell
Reports 12:250-255 and Christou and Ford (1995) Annals of Botany
75:407-413 (rice); Osjoda et al. (1996) Nature Biotechnology
14:745-750 (maize via Agrobacterium tumefaciens); and U.S. Pat. No.
5,736,369 (meristem transformation), all of which are herein
incorporated by reference.
[0166] The nucleotide constructs may be introduced into plants by
contacting plants with a virus or viral nucleic acids. Generally,
such methods involve incorporating a nucleotide construct of the
invention within a viral DNA or RNA molecule. Further, it is
recognized that useful promoters encompass promoters utilized for
transcription by viral RNA polymerases. Methods for introducing
nucleotide constructs into plants and expressing a protein encoded
therein, involving viral DNA or RNA molecules, are known in the
art. See, for example, U.S. Pat. Nos. 5,889,191, 5,889,190,
5,866,785, 5,589,367 and 5,316,931; herein incorporated by
reference.
[0167] In some embodiments, transient expression may be desired. In
those cases, standard transient transformation techniques may be
used. Such methods include, but are not limited to viral
transformation methods, and microinjection of DNA or RNA, as well
other methods well known in the art.
[0168] The cells from the plants that have stably incorporated the
nucleotide sequence may be grown into plants in accordance with
conventional ways. See, for example, McCormick et al. (1986) Plant
Cell Reports 5:81-84. These plants may then be grown, and either
pollinated with the same transformed strain or different strains,
and the resulting hybrid having constitutive expression of the
desired phenotypic characteristic imparted by the nucleotide
sequence of interest and/or the genetic markers contained within
the target site or transfer cassette. Two or more generations may
be grown to ensure that expression of the desired phenotypic
characteristic is stably maintained and inherited and then seeds
harvested to ensure expression of the desired phenotypic
characteristic has been achieved.
[0169] Initial identification and selection of cells and/or plants
comprising the DNA constructs may be facilitated by the use of
marker genes. Gene targeting can be performed without selection if
there is a sensitive method for identifying recombinants, for
example if the targeted gene modification can be easily detected by
PCR analysis, or if it results in a certain phenotype. However, in
most cases, identification of gene targeting events will be
facilitated by the use of markers. Useful markers include positive
and negative selectable markers as well as markers that facilitate
screening, such as visual markers. Selectable markers include genes
carrying resistance to an antibiotic such as spectinomycin (e.g.
the aada gene, Svab et al. (1990) Plant Mol Biol 14:197-205),
streptomycin (e.g., aada, or SPT, Svab et al. (1990) Plant Mol Biol
14:197-205; Jones et al. (1987) Mol Gen Genet 210:86), kanamycin
(e.g., nptII, Fraley et al. (1983) Proc Natl Acad Sci USA
80:4803-4807), hygromycin (e.g., HPT, Vanden Elzen et al. (1985)
Plant Mol Biol 5:299), gentamycin (Hayford et al. (1988) Plant
Physiol 86:1216), phleomycin, zeocin, or bleomycin (Hille et al.
(1986) Plant Mol Biol 7:171), or resistance to a herbicide such as
phosphinothricin (bar gene), or sulfonylurea (acetolactate synthase
(ALS)) (Charest et al. (1990) Plant Cell Rep 8:643), genes that
fulfill a growth requirement on an incomplete media such as HIS3,
LEU2, URA3, LYS2, and TRP1 genes in yeast, and other such genes
known in the art. Negative selectable markers include cytosine
deaminase (codA) (Stougaard (1993) Plant J. 3:755-761), tms2
(DePicker et al. (1988) Plant Cell Rep 7:63-66), nitrate reductase
(Nussame et al. (1991) Plant J 1:267-274), SU1 (O'Keefe et al.
(1994) Plant Physiol. 105:473-482), aux-2 from the Ti plasmid of
Agrobacterium, and thymidine kinase. Screenable markers include
fluorescent proteins such as green fluorescent protein (GFP)
(Chalfie et al. (1994) Science 263:802; U.S. Pat. No. 6,146,826;
U.S. Pat. No. 5,491,084; and WO 97/41228), reporter enzymes such as
.beta.-glucuronidase (GUS) (Jefferson (1987) Plant Mol Biol Rep
5:387; U.S. Pat. No. 5,599,670; U.S. Pat. No. 5,432,081),
.beta.-galactosidase (lacZ), alkaline phosphatase (AP), glutathione
S-transferase (GST) and luciferase (U.S. Pat. No. 5,674,713; Ow et
al. (1986) Science 234:856-859), visual markers like anthocyanins
such as CRC (Ludwig et al. (1990) Science 247:449-450) R gene
family (e.g. Lc, P, S), A, C, R-nj, body and/or eye color genes in
Drosophila, coat color genes in mammalian systems, and others known
in the art.
[0170] One or more markers may be used in order to select and
screen for gene targeting events. One common strategy for gene
disruption involves using a target modifying polynucleotide in
which the target is disrupted by a promoterless selectable marker.
Since the selectable marker lacks a promoter, random integration
events are unlikely to lead to transcription of the gene. Gene
targeting events will put the selectable marker under control of
the promoter for the target gene. Gene targeting events are
identified by selection for expression of the selectable marker.
Another common strategy utilizes a positive-negative selection
scheme. This scheme utilizes two selectable markers, one that
confers resistance (R+) coupled with one that confers a sensitivity
(S+), each with a promoter. When this polynucleotide is randomly
inserted, the resulting phenotype is R+/S+. When a gene targeting
event is generated, the two markers are uncoupled and the resulting
phenotype is R+/S-. Examples of using positive-negative selection
are found in Thykjar et al. (1997) Plant Mol Biol 35:523-530; and
PCT International published application No. WO 01/66717, which are
herein incorporated by reference.
[0171] The practice of the present invention employs, unless
otherwise indicated, conventional techniques of chemistry,
molecular biology, microbiology, recombinant DNA, genetics,
immunology, cell biology, cell culture and transgenic biology,
which are within the skill of the art. See, e.g., Maniatis et al.
(1982) Molecular Cloning (Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, N.Y.); Sambrook et al. (1989) Molecular Cloning, 2nd
Ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
N.Y.); Sambrook and Russell (2001) Molecular Cloning, 3rd Ed. (Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.); Ausubel
et al. (1992) Current Protocols in Molecular Biology (John Wiley
& Sons, including periodic updates); Glover (1985) DNA Cloning
(IRL Press, Oxford); Anand (1992) Techniques for the Analysis of
Complex Genomes (Academic Press).; Guthrie and Fink (1991) Guide to
Yeast Genetics and Molecular Biology (Academic Press).; Harlow and
Lane (1988) Antibodies, (Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, N.Y.); Jakoby and Pastan, eds. (1979) "Cell Culture"
Methods in Enzymology Vol. 58 (Academic Press, Inc., Harcourt Brace
Jovanovich (NY)).; Nucleic Acid Hybridization (B. D. Hames & S.
J. Higgins eds. 1984); Transcription And Translation (B. D. Hames
& S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I.
Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes
(IRL Press, 1986); B. Perbal, A Practical Guide To Molecular
Cloning (1984); the treatise, Methods In Enzymology (Academic
Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J.
H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor
Laboratory); Methods In Enzymology, Vols. 154 and 155 (Wu et al.
eds.), Immunochemical Methods In Cell And Molecular Biology (Mayer
and Walker, eds., Academic Press, London, 1987); Handbook Of
Experimental Immunology, Volumes I-IV (D. M. Weir and C. C.
Blackwell, eds., 1986); Riott, Essential Immunology, 6th Edition,
Blackwell Scientific Publications, Oxford, 1988; Hogan et al.,
Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y., 1986); Westerfield, M., The
zebrafish book. A guide for the laboratory use of zebrafish (Danio
rerio), (4th Ed., Univ. of Oregon Press, Eugene, 2000); Methods in
Arabidopsis Research (C. Koncz et al., eds, World Scientific Press,
Co., Inc., River Edge, Minnesota, 1992); Arabidopsis: A Laboratory
Manual (D. Weigel and J. Glazebrook, eds., Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., 2002).
EXAMPLES
[0172] The following are non-limiting examples intended to
illustrate the invention. Although the present invention has been
described in some detail by way of illustration and example for
purposes of clarity of understanding, it will be obvious that
certain changes and modifications may be practiced within the scope
of the appended claims. For example, any of the pre-miRNAs and
miRNAs described herein can be used in place of the pre-miRNAs and
miRNAs used in the examples. Examples 1-15 are derived from PCT
International published application Nos. WO 2005/052170 and WO
2005/035769 and from U.S. published application Nos. US
2005/0138689 and US 2005/0120415, each incorporated herein by
reference.
Example 1
[0173] The example describes the identification of a microRNA
[0174] The following experiments are carried out on the Arabidopsis
thaliana Col-0 ecotype. Plants are grown in long days (16 h light,
8 h dark) under cool white light at 22.degree. C.
[0175] Arabidopsis plants are transformed by a modified version of
the floral dip method, in which Agrobacterium cell suspension is
applied to plants by direct watering from above. The T-DNA vector
used, pHSbarENDs, contained four copies of the CAMV 35S enhancer
adjacent to the right border, an arrangement similar to that
described by Weigel et al. (Plant Physiol. 122:1003-1013, 2000).
Transformed plants are selected with glufosinate (BASTA) and
screened for flowering time, which resulted in the identification
of the early-flowering EAT-D mutant. A single T-DNA cosegregating
with early flowering is identified in EAT-D, and TAIL-PCR is
performed to amplify sequences adjacent to the left and right
borders of the T-DNA. To identify transcripts upregulated in the
EAT-D mutant, Northern blots containing RNA extracted from wild
type (Col-0) and EAT-D plants is probed. Probes for the genes
At5g04270 and At5g04280 (GenBank NC.sub.--003076) do not detect any
difference between wild type and EAT-D, whereas a probe from the
intergenic region identifies an .about.1.4 kb transcript that is
expressed at significantly higher levels in EAT-D than in wild
type.
[0176] To isolate the full-length EAT cDNA, 5'- and 3'-RACE-PCR is
performed with a GeneRacer kit (Invitrogen) that selects for
5'-capped mRNAs. Reverse transcription is carried out using an
oligo-dT primer, and PCR utilized a gene-specific primer (SEQ ID
NO:45 5'-CTGTGCTCACGATCTTGTTGTTCTTGATC-3') paired with the 5' kit
primer, or a second gene-specific primer (SEQ ID NO:46
5'-GTCGGCGGATCCATGGAAGAAAGCTCATC-5') paired with the 3' kit
primer.
[0177] The Arabidopsis EAT-D (Early Activation Tagged--Dominant)
mutant is identified in an activation tagging population (Weigel et
al. (2000) Plant Physiol 122:1003-1013). As evidenced by visual
inspection and by measuring rosette leaf number (Table 2), the
EAT-D mutant flowers extremely early. In addition, EAT-D displays
floral defects that are virtually identical to those observed for
strong apetala2 (ap2) mutant alleles (Bowman et al. (1991)
Development 112:1-20), including the complete absence of petals and
the transformation of sepals to carpels. This ap2-like phenotype is
only observed in EAT-D homozygotes, whereas both EAT-D
heterozygotes and homozygotes are early flowering, indicating that
the flowering time phenotype is more sensitive to EAT-D dosage than
the ap2-like floral phenotype.
TABLE-US-00002 TABLE 2 Rosette leaf numbers for Arabidopsis lines
Genotype rosette leaf no. floral phenotype Col-0 11.4 +/- 1.2 wild
type EAT-D 3.1 +/- 0.8 ap2 EAT-OX 2.0 +/- 0.2 ap2 + additional
eatdel 11.1 +/- 1.1 wild type miR172a1-OX 2.1 +/- 0.3 ap2 +
additional LAT-D 22.5 +/- 2.1 wild type At2g28550-OX 28.6 +/- 3.6
wild type 5-60120 10.2 +/- 1.4 wild type 2-28550 8.7 +/- 0.6 wild
type 5-60120; 2-28550 6.0 +/- 0.8 wild type
[0178] The activation-tagged T-DNA insert in EAT-D is mapped to
chromosome 5, in between the annotated genes At5g04270 and
At5g04280. 5'- and 3'-RACE PCR is then used with primers located
within this region to identify a 1.4 kb transcript (SEQ ID NO:1),
which is named EAT, that is upregulated in EAT-D. When the 1.4 kb
EAT cDNA is fused to the constitutive CAMV 35S promoter and the
resultant 35S::EAT construct is introduced into wild type (Col-0)
plants by Agrobacterium-mediated transformation (Clough and Bent
(1998) Plant J 16:735-743), the 35S::EAT transformants display the
identical early-flowering and ap2-like phenotypes seen for EAT-D
(Table 1). Many of the 35S::EAT transformants occasionally display
additional defects, including stigmatic papillae on cauline leaf
margins and the formation of a complete or partial flower rather
than a secondary inflorescence in the axils of cauline leaves.
Ectopic expression of the EAT gene in 35S::EAT plants, therefore,
affects both flowering time and the specification of floral organ
identity.
[0179] The EAT gene produces a 1417-nucleotide noncoding RNA that
is predicted to be 5'-capped and polyadenylated, based on the
RACE-PCR methodology. BLASTN and BLASTX searches of several
databases with the EAT cDNA do not reveal extensive nucleotide or
predicted amino acid sequence identity between EAT and any other
gene. However, a 21-nucleotide (nt) (SEQ ID NO:4) stretch in the
middle of the EAT transcript is identified that is identical to
miR172a-2, a recently identified miRNA (Park et al. (2002) Curr
Biol 12:1484-1495). To confirm the functional importance of the
miR172a-2 sequence within the EAT cDNA, a mutant form of EAT is
generated in which the miR172a-2 sequence is deleted, and a
construct consisting of this mutant EAT cDNA, eatdel, is made
driven by the 35S promoter. Transgenic plants carrying this
35S::eatdel construct flower with the same number of leaves as
wild-type and had normal flowers (Table 1), indicating that the
miR172a-2 sequence is necessary to confer both the flowering time
and floral organ identity phenotypes seen in EAT-overexpressing
lines.
[0180] As noted by Park et al. (2002) Curr Biol 12:1484-1495), the
21-nt miR172a-2 miRNA has the potential to form an RNA duplex with
a sequence near the 3' end of the coding region of AP2 (Table
3).
TABLE-US-00003 TABLE 3 Putative 21-nt miR172a-2/AP2 RNA duplex
Sequence Duplex SEQ ID NO: AP2 RNA 5'-CUGCAGCAUCAUCAGGAUUCU-3' 47
EAT miRNA 3'-UACGUCGUAGUAGUUCUAAGA-5' 48
[0181] The GU wobble in the duplex is underlined.
[0182] This particular region of the AP2 gene is poorly conserved
at the nucleotide level among the AP2 family; nevertheless, the AP2
sequence (SEQ ID NO:49) that is complementary to miR172a-2 is found
in a similar location in three other Arabidopsis AP2 family
members, At5g60120 (SEQ ID NO:50), At2g28550 (SEQ ID NO:51),
At5g67180 (SEQ ID NO:52). In addition, the sequence can be found at
the corresponding positions of the maize AP2 genes indeterminate
spikelet1 (Chuck et al. (1998) Genes Dev 12:1145-1154) (IDS1 (SEQ
ID NO:53)) and glossy15 (Moose and Sisco (1996) Genes Dev
10:3018-3027) (GL15 (SEQ ID NO:54)), and in AP2 family members from
many other plant species, including soybean, rice, wheat, tomato
and pea (not shown). The alignment of three Arabidopsis and two
maize AP2 family members is shown in Table 4 below.
TABLE-US-00004 TABLE 4 Alignment of AP2 21-nt region (black bar)
and surrounding sequence (SEQ ID NO:) AP2
ACCAAGTGTTGACAAATGCTGCAGCATCAT (49) CAGGATTCTCTCCTCATCATCACAATCAG
At 5g60120 CACCGCCACTGTTTTCAAATGCAGCATCAT (50)
CAGGATTCTCACTCTCAGCTACACGCCCT At 2g28550
CACCATTGTTCTCAGTTGCAGCAGCATCAT (51) CAGGATTCTCACATTTCCGGCCACAACCT
At 5g67180 GAAATCGAGTGGTGGGAATGGCAGCATCAT (52)
CAGGATTCTCTCCTCAACCTTCCCCTTAC IDS1 ACGTGCCGTTGCACCACTCTGCAGCATCAT
(53) CAGGATTCTCTACCGCCGCCGGGGCCAAC GL15
ACGCCAGCAGCGCCGCCGCTGCAGCATCAT (54)
CAGGATTCCCACTGTGGCAGCTGGGTGCG
[0183] There is an additional copy of the miR172a-2 miRNA in the
Arabidopsis genome on chromosome 2 (miR172a-1, FIG. 2d), and
miR172a-2 is highly similar to three other Arabidopsis loci. Like
the miR172a-2 miRNA, all four reiterations of the sequence are in
intergenic regions, i.e. in between the Arabidopsis genes currently
annotated in GenBank. In addition, the sequence is found in ESTs
from tomato, potato and soybean, and four copies were found in the
genomic sequence of rice.
Example 2
[0184] This example describes the construction of expression
vectors
[0185] To overexpress the EAT gene, primers containing XhoI sites
(SEQ ID NO:55 5'-GACTACTCGAGCACCTCTCACTCCCTTTCTCTAAC-3' and SEQ ID
NO:56 5'-GACTACTCGAGGTTCTCAAGTTGAGCACTTGAAAAC-3') are designed to
amplify the entire EAT gene from Col-0 DNA. The PCR product is
digested with XhoI and inserted into a modified pBluescriptSK+
vector (Stratagene, La Jolla, Calif.) that lacked BamHI and HindIII
sites, to generate EATX4 (SEQ ID NO:44). To generate the 35S::EAT
transformants, the XhoI-cut EAT gene is inserted into the binary
vector pBE851 in between a CAMV 35S promoter and b-phaseolin
terminator, and Col-0 was transformed by floral dip. To generate
the eatdel construct, two oligonucleotides are synthesized (SEQ ID
NO:57 5'-GATCCATGGAAGAAAGCTCAT
CTGTCGTTGTTTGTAGGCGCAGCACCATTAAGATTCACATGGAAATTGATAAATAC-3' and SEQ
ID NO:58 5'-CCTAAATTAGGGTTTTGATATGTATATTCAACAATCGACG
GCTACAAATACCTAA-3') that completely recreated the BamHI/HindIII
fragment of the EAT cDNA except that it lacked the 21 nt miR172a-2
sequence located within the fragment. These two oligos are annealed
to their synthesized complementary strands (SEQ ID NO:59 5'-TAG
GGTATTTATCAATTTCCATGTGAATCTTAATGGTGCTGCGCCTACAAACAACGACAG
ATGAGCTTTCTTCCATG-3' and SEQ ID NO:60 5'-AGCTTTAGGTATTTGTAGCCGTC
GATTGTTGAATATACATATCAAAACCCTAATT-3') and ligated to EATX4 that had
been digested with BamHI and HindIII, in a trimolecular ligation
reaction. This resulted in the replacement of 159 by of wild-type
EAT sequence with the 138 by mutant sequence. The eatdel cDNA is
then subcloned into pBE851 and transformed as described above.
BASTA is used to select in plants for both the EAT and eatdel
overexpression constructs.
[0186] To test whether another member of the miR172 family,
miR172a-1, would confer a phenotype similar to that of miR172a-2, a
construct containing the 35S promoter fused to the genomic region
surrounding miR172a-1 is generated. Plants containing the
35S::miR172a-1 construct flower early and display an ap2 phenotype
(Table 1), indicating that miR172a-1 behaves in an identical manner
to miR172a-2 when overexpressed.
[0187] All of the miR172 miRNA family members are located within a
sequence context that allows an RNA hairpin to form (FIG. 1).
Presumably this hairpin is the substrate which is subsequently
cleaved by a plant Dicer homolog to generate the mature miRNA. The
location of the miRNA within the hairpin, i.e. on the 3' side of
the stem, is conserved amongst all the members of the miR172
family, and this may reflect a structural requirement for
processing of this particular miRNA family. The 21-nt miR172a-2
miRNA, therefore, is predicted to be a member of a family of miRNAs
that have the capacity to regulate a subset of AP2 genes by forming
an RNA duplex with a 21-nt cognate sequence in these genes.
Example 3
[0188] The example describes the analysis of microRNA expression
and AP2 expression
[0189] Total RNA is isolated from wild type and EAT-D whole plants
that had already flowered, using TRIZOL reagent (Sigma). 50 mg of
each RNA is subjected to electrophoresis on a 15% TBE-Urea
Criterion gel (BioRad), electroblotted onto Hybond-N+ filter paper
(Amersham) using a TransBlot-SD apparatus (BioRad). The filter is
then hybridized at 37.degree. C. overnight in UltraHyb-Oligo buffer
(Ambion) with 32P-labeled oligos. The oligos are 30-mers that
corresponded to either the sense or antisense strands of the
miR172a-2 miRNA, with 4-5 nt of flanking sequence on each side. The
filter is washed twice at 37.degree. C., in buffer containing
2.times. SSC and 0.5% SDS. For S1 analysis, probe is made by
end-labeling an oligo (SEQ ID NO:61)
(5'-ATGCAGCATCATCAAGATTCTCATATACAT-3') with T4 polynucleotide
kinase and 32P. Hybridization and processing of 51 reactions are
carried out using standard protocols. For developmental analysis of
miR172a-2 and miR172a-1, total RNA is isolated from plants at the
various stages and tissues indicated in Example 4, using an Rneasy
kit (Qiagen). RT-PCR is carried out using standard protocols, and
utilized oligos specific for sequences adjacent to miR172a-2 (SEQ
ID NO:62) (5'-GTCGGCGGATCCATGG AAGAAAGCTCATC-3' and (SEQ ID NO:63)
5'-CAAAGATCGATCCAGACTTCAATCAA TATC-3') or sequences adjacent to
miR172a-1 (SEQ ID NO:64) (5'-TAATTTCCGGAGCCAC GGTCGTTGTTG-3' and
(SEQ ID NO:65) 5'-AATAGTCGTTGATTGCCGATGCAGCATC-3'). Oligos used to
amplify the ACT11 (Actin) transcript were: (SEQ ID NO:66)
5'-ATGGCAGATGGTGAAGACATTCAG-3', and (SEQ ID NO:67)
5'-GAAGCACTTCCTGTG GACTATTGATG-3'. RT-PCR analysis of AP2 is
performed on RNA from floral buds, and utilized the following
oligos: (SEQ ID NO:68) 5'-TTTCCGGGCAGCAGCAACATTGGTAG-3', and (SEQ
ID NO:69) 5'-GTTCGCCTAAGTTAACAAGAGGATTTAGG-3'. Oligos used to
amplify the ANT transcript are: (SEQ ID NO:70)
5'-GATCAACTTCAATGACTAACTCTG GTTTTC-3', and (SEQ ID NO:71)
5'-GTTATAGAGAGATTCATTCTGTTTCACATG-3'.
[0190] Immunoblot analysis of AP2 is performed on proteins
extracted from floral buds. Following electrophoresis on a 10%
SDS-PAGE gel, proteins are transferred to a Hybond-P membrane
(Amersham) and incubated with an antibody specific for AP2 protein
(aA-20, Santa Cruz Biotechnology). The blot is processed using an
ECL-plus kit (Amersham).
[0191] Northern analysis using probes both sense and antisense to
the miR172a-2 miRNA identifies a small single-stranded RNA of 21-25
nucleotides accumulating to much higher levels in EAT-D mutant
plants relative to wild type. The small amount of transcript seen
in wild type presumably represents endogenous levels of not only
the miR172a-2 miRNA but also its family members, which are similar
enough to cross-hybridize with the probe. The predicted miR172a-2
hairpin is 117 nt in length (FIG. 1), a small amount of an -100 nt
transcript accumulating is detected in EAT-D, this likely
represents partially processed miR172a-2 hairpin precursor. 51
nuclease mapping of the miR172a-2 miRNA provides independent
confirmation of the 5' end of miR172a-2 reported by Park et al.
((2002) Curr Biol 12:1484-1495).
Example 4
[0192] The example describes the developmental pattern of EAT miRNA
expression.
[0193] To address the wild-type expression pattern of miR172a-2
separate from its other Arabidopsis family members, RT-PCR is used
to specifically detect a fragment of the 1.4 kb EAT full-length
precursor transcript containing miR172a-2. EAT precursor transcript
expression is temporally regulated, with little or no transcript
detected two days after germination, and progressively more
steady-state transcript accumulation seen as the plant approaches
flowering. The precursor transcript of miR172a-1 shows a similar
temporal pattern of expression. Both miR172a-2 and miR172a-1
precursor transcripts continue to be expressed after flowering has
occurred, and accumulate in both leaves and floral buds. Expression
of the precursors for the other miR172 family members is not
detected, perhaps due to their exclusive expression in tissue types
not included in this analysis, or because their precursor
transcripts are too transient to detect. The temporal expression
pattern seen for miR172a-2 and miR172a-1 is reminiscent of that
observed for let-7 and lin-4, two miRNAs that control developmental
timing in C. elegans (Feinbaum and Ambros (1999) Dev Biol
210:87-95; Reinhart et al. (2000) Nature 403:901-906).
Example 5
[0194] The levels of miR172 in various flowering time mutants are
assessed, in an attempt to position miR172 within the known
flowering time pathways. The levels of miR172 are not altered in
any of the mutants tested, and the levels of the EAT transcript are
identical in plants grown in long days versus plants grown in short
days.
Example 6
[0195] The example describes evaluation of protein expression
[0196] Immunoblot analysis indicates that AP2 protein is reduced
3.5-fold in the EAT-D mutant relative to wild type, whereas the AP2
transcript is unaffected. This data suggests that the miR172a-2
miRNA negatively regulates AP2 by translational inhibition. The
predicted near-perfect complementarity between the miR172a-2 miRNA
and the AP2 target site would be predicted to trigger AP2 mRNA
cleavage by the RNA interference (RNAi) pathway (Llave et al.
(2002) Plant Cell 14:1605-1619; Hutvagner and Zamore (2002) Science
297:2056-2060). Indeed, others have proposed that many plant miRNAs
enter the RNAi pathway exclusively due to their near-perfect
complementarity to putative targets (Rhoades et al. (2002) Cell
110:513-520). While there is no evidence regarding the GU wobble
base pair in the predicted miR172a-2/AP2 RNA duplex, it is
conserved in all predicted duplexes between miR172 family members
and their AP2 targets. Regardless of the mechanism, it is apparent
from the AP2 expression data and the observed phenotype of EAT-D
that AP2 is a target of negative regulation by miR172a-2, at least
when miR172a-2 is overexpressed.
Example 7
[0197] In the same genetic screen that identified the
early-flowering EAT-D mutant, an activation-tagged late-flowering
mutant, called LAT-D, is identified. The LAT-D mutant displays no
additional phenotypes besides late flowering (Table 1), and the
late-flowering phenotype cosegregates with a single T-DNA
insertion. Sequence analysis of the T-DNA insert in LAT-D indicates
that the 4.times. 35S enhancer is located approximately 5 kb
upstream of At2g28550, which is one of the AP2-like target genes
that are potentially regulated by miR172. RT-PCR analysis using
primers specific for At2g28550 indicates that the transcript
corresponding to this gene is indeed expressed at higher levels in
the LAT-D mutant relative to wild type. To confirm that
overexpression of At2g28550 causes late flowering, a genomic region
containing the entire At2g28550 coding region (from start to stop
codon) is fused to the 35S promoter, and transgenic plants
containing this construct are created. Transgenic 35S::At2g28550
plants flower later than wild type plants, and are slightly later
than the LAT-D mutant (Table 1). This late flowering phenotype is
observed in multiple independent transformants.
[0198] The fact that overexpression of At2g28550 causes late
flowering suggests that miR172 promotes flowering in part by
downregulating At2g28550. However, because miR172 appears to affect
protein rather than transcript accumulation of its target genes,
and because there is not an antibody to the At2g28550 gene product,
this regulation is tested indirectly via a genetic cross. A plant
heterozygous for LAT-D is crossed to a plant homozygous for EAT-D,
such that all F1 progeny would contain one copy of EAT-D and 50% of
the F1 progeny would also have one copy of LAT-D. F1 progeny are
scored for the presence or absence of the LAT-D allele by PCR, and
also are scored for flowering time. All of the F1 plants are early
flowering, regardless of whether or not they contained a copy of
the LAT-D allele, indicating that EAT-D is epistatic to LAT-D. This
result is consistent with the idea that miR172a-2, which is
overexpressed in EAT-D, directly downregulates At2g28550, which is
overexpressed in LAT-D.
Example 8
[0199] To assess the effects of reducing At2g28550 function, plants
containing a T-DNA insertion in the At2g28550 gene are identified.
In addition, a T-DNA mutant for At2g60120, a closely related
AP2-like gene that also contains the miR172 target sequence, is
identified. Plants homozygous for either the At2g28550 insert or
the At5g60120 insert are slightly early flowering relative to wild
type (Table 1). The two mutants are crossed, and the double mutant
is isolated by PCR genotyping. The At2g28550/At5g60120 double
mutant is earlier flowering than either individual mutant (Table
1), suggesting that the genes have overlapping function. The early
flowering phenotype of the At2g28550/At5g60120 double mutant is
consistent with the idea that the early flowering phenotype of
miR172-overexpressing lines is due to downregulation of several
AP2-like genes, including At2g28550 and At5g60120. Interestingly,
the At2g28550/At5g60120 double mutant is not as early as
miR172-overexpressing lines (c.f. EAT-OX, Table 1), which suggests
that other AP2-like targets of miR172, for example AP2 itself or
At5g67180, also contribute to flowering time control. Because ap2
mutants are not early flowering, any potential negative regulation
of flowering by AP2 must be normally masked by genetic
redundancy.
Example 9
[0200] This example describes a method of target selection and
method to design DNA constructs to generate miRNAs using the
constructs of SEQ ID NOS: 3 and 44. Any sequence of interest can be
selected for silencing by miRNA generated using the following
method:
[0201] 1. Choose a region from the coding strand in a gene of
interest to be the target sequence. Typically, choose a region of
about 10-50 nucleotides found in a similar location to the region
targeted by EAT in AP2-like genes, which are regions about 100 nt
upstream of the stop codon. The exact location of the target,
however, does not appear to be critical. It is recommended to
choose a region that has .about.50% GC and is of high sequence
complexity, i.e. no repeats or long polynucleotide tracts. It is
also recommended that the chosen region ends with a T or A, such
that the complementary miRNA will start with an A or U. This is to
help ensure a lower stability at the 5' end of the miRNA in its
double-stranded Dicer product form (Schwartz, et al. (2003) Cell
115:199-208). For example, in the miR172a-2 precursor, the miRNA
sequence starts with an A, and many other miRNAs start with a
U.
[0202] 2. To use the construct of SEQ ID NO:3, create a 21
nucleotide sequence complementary to the 21 nt target region
(miRNA). Optionally, change a C in the miRNA to a T, which will
generate a GU wobble with the target sequence, which mimics the GU
wobble seen in EAT.
[0203] 3. Create the 21 nucleotide "backside" sequence of the
hairpin. This will be substantially complementary to the miRNA from
step 2. Note, this backside sequence will also be substantially
identical to the target sequence. Typically, introduce a few
mismatches to make some bulges in the stem of the hairpin that are
similar to the bulges in the original EAT hairpin. Optionally,
introduce an A at the 3' end of the backside, to create mismatch at
the 5' end of the miRNA. This last step may help ensure lower
stability at the 5' end of the miRNA in its double-stranded Dicer
product form (Schwartz et al. (2003) Cell 115:199-208).
[0204] 4. Replace the 21 nucleotide miRNA sequence and the 21
nucleotide "backside" sequence in the EAT BamHI/HindIII DNA
construct (SEQ ID NO:3) with the new miRNA and "backside" sequences
from steps 2 and 3.
[0205] 5. Use MFOLD (GCG, Accelrys, San Diego, Calif.), or an
equivalent program, to compare the new hairpin from Step 4 with the
original hairpin. Generally, the sequence substantially replicate
the structure of the original hairpin (FIG. 1). It is predicted
that the introduced bulges need not be exactly identical in length,
sequence or position to the original. Examine the miRNA sequence in
the hairpin for the relative stability of the 5' and 3' ends of the
predicted dsRNA product of Dicer.
[0206] 6. Generate four synthetic oligonucleotides of 76-77
nucleotides in length to produce two double-stranded fragments
which comprise the BamHI and HindIll restriction sites, and a 4
nucleotide overhang to facilitate directional ligation which will
recreate the BamHI/HindIII fragment. Design of the overhang can be
done by one of skill in the art, the current example uses the 4
nucleotide region of positions 79-82 (CCTA) of SEQ ID NO:3. Hence,
for example:
[0207] Oligo 1 will have an unpaired BamHI site at the 5' end, and
will end with the nucleotide at position 78 of SEQ ID NO:3.
[0208] Oligo 2 will have the nucleotides of position 79-82 (CCTA)
unpaired at the 5' end, and will terminate just before the HindIII
site (or positions 151-154 in SEQ ID NO:3).
[0209] Oligo 3 will be essentially complementary to Oligo 1,
(nucleotides 5-78 of SEQ ID NO:3), and will terminate with 4
nucleotides complementary to nucleotides 1-4 (CCTA) of Oligo 2.
[0210] Oligo 4 will be essentially complementary to Oligo 2
beginning at the nucleotide of position 5, and will terminate with
the HindIII site at the 3' end.
[0211] Anneal the oligonucleotides to generate two fragments to be
used in a subsequence ligation reaction with the plasmid
sequence.
[0212] Optionally, two synthetic oligonucleotides comprising attB
sequences can be synthesized and annealed to create an attB-flanked
miRNA precursor that is then integrated into a vector using
recombinational cloning (GATEWAY, InVitrogen Corp., Carlsbad,
Calif.).
[0213] 7. Ligate the two DNA fragments from Step 6 in a
trimolecular ligation reaction with a plasmid cut with
BamHI/HindIII. The current example uses the modified pBluescript
SK+ plasmid of SEQ ID NO:44, which comprises the 1.4 kb EAT
sequence of SEQ ID NO:1, digested with BamHI/HindIII and gel
purified away from the small fragment using standard molecular
biological techniques. The new designed miRNA to the gene of
interest has replaced the previous miRNA.
[0214] If an attB-flanked sequence is used from Step 6, the BP and
LR recombination reactions (GATEWAY, InVitrogen Corp., Carlsbad,
Calif.) can be used to insert the modified hairpin into a
destination vector comprising the full-length miR172a-2
precursor.
[0215] 8. The plasmid from Step 7, subject to any other
preparations or modifications as needed, is used to transform the
target organism using techniques appropriate for the target.
[0216] 9. Silencing of the target gene can be assessed using
techniques well-known in the art, for example, Northern blot
analysis, immunoblot analysis if the target gene of interest
encodes a polypeptide, and any phenotypic screens relevant to the
target gene, for example flowering time, or floral morphology.
Example 10
[0217] Described in this example are methods one may use for
introduction of a polynucleotide or polypeptide into a plant
cell.
[0218] A. Maize particle-mediated DNA delivery
[0219] A DNA construct can be introduced into maize cells capable
of growth on suitable maize culture medium. Such competent cells
can be from maize suspension culture, callus culture on solid
medium, freshly isolated immature embryos or meristem cells.
Immature embryos of the Hi-II genotype can be used as the target
cells. Ears are harvested at approximately 10 days
post-pollination, and 1.2-1 5 mm immature embryos are isolated from
the kernels, and placed scutellum-side down on maize culture
medium.
[0220] The immature embryos are bombarded from 18-72 hours after
being harvested from the ear. Between 6 and 18 hours prior to
bombardment, the immature embryos are placed on medium with
additional osmoticum (MS basal medium, Musashige and Skoog (1962)
Physiol Plant 15:473-497, with 0.25 M sorbitol). The embryos on the
high-osmotic medium are used as the bombardment target, and are
left on this medium for an additional 18 hours after
bombardment.
[0221] For particle bombardment, plasmid DNA (described above) is
precipitated onto 1.8 mm tungsten particles using standard
CaCl2-spermidine chemistry (see, for example, Klein et al. (1987)
Nature 327:70-73). Each plate is bombarded once at 600 PSI, using a
DuPont Helium Gun (Lowe et al. (1995) Bio/Technol 13:677-682). For
typical media formulations used for maize immature embryo
isolation, callus initiation, callus proliferation and regeneration
of plants, see Armstrong (1994) In The Maize Handbook, M. Freeling
and V. Walbot, eds. Springer Verlag, N.Y., pp 663-671.
[0222] Within 1-7 days after particle bombardment, the embryos are
moved onto N6-based culture medium containing 3 mg/l of the
selective agent bialaphos. Embryos, and later callus, are
transferred to fresh selection plates every 2 weeks. The calli
developing from the immature embryos are screened for the desired
phenotype. After 6-8 weeks, transformed calli are recovered.
[0223] B. Soybean transformation
[0224] Soybean embryogenic suspension cultures are maintained in 35
ml liquid media SB 196 or SB 172 in 250 ml Erlenmeyer flasks on a
rotary shaker, 150 rpm, 26C with cool white fluorescent lights on
16:8 hr day/night photoperiod at light intensity of 30-35 uE/m2s.
Cultures are subcultured every two weeks by inoculating
approximately 35 mg of tissue into 35 ml of fresh liquid media.
Alternatively, cultures are initiated and maintained in 6-well
Costar plates.
[0225] SB 172 media is prepared as follows: (per liter), 1 bottle
Murashige and Skoog Medium (Duchefa # M 0240), 1 ml B5 vitamins
1000.times. stock, 1 ml 2,4-D stock (Gibco 11215-019), 60 g
sucrose, 2 g MES, 0.667 g L-Asparagine anhydrous (GibcoBRL
11013-026), pH 5.7. SB 196 media is prepared as follows: (per
liter) 10 ml MS FeEDTA, 10 ml MS Sulfate, 10 ml FN-Lite Halides, 10
ml FN-Lite P,B,Mo, 1 ml B5 vitamins 1000.times. stock, 1 ml 2,4-D,
(Gibco 11215-019), 2.83 g KNO3, 0.463 g (NH4)2504, 2 g MES, 1 g
Asparagine Anhydrous, Powder (Gibco 11013-026), 10 g Sucrose, pH
5.8. 2,4-D stock concentration 10 mg/ml is prepared as follows:
2,4-D is solubilized in 0.1 N NaOH, filter-sterilized, and stored
at -20.degree. C. B5 vitamins 1000.times. stock is prepared as
follows: (per 100 ml)--store aliquots at -20.degree. C., 10 g
myo-inositol, 100 mg nicotinic acid, 100 mg pyridoxine HCl, 1 g
thiamin.
[0226] Soybean embryogenic suspension cultures are transformed with
various plasmids by the method of particle gun bombardment (Klein
et al. (1987) Nature 327:70). To prepare tissue for bombardment,
approximately two flasks of suspension culture tissue that has had
approximately 1 to 2 weeks to recover since its most recent
subculture is placed in a sterile 60.times.20 mm petri dish
containing 1 sterile filter paper in the bottom to help absorb
moisture. Tissue (i.e. suspension clusters approximately 3-5 mm in
size) is spread evenly across each petri plate. Residual liquid is
removed from the tissue with a pipette, or allowed to evaporate to
remove excess moisture prior to bombardment. Per experiment, 4-6
plates of tissue are bombarded. Each plate is made from two
flasks.
[0227] To prepare gold particles for bombardment, 30 mg gold is
washed in ethanol, centrifuged and resuspended in 0.5 ml of sterile
water. For each plasmid combination (treatments) to be used for
bombardment, a separate micro-centrifuge tube is prepared, starting
with 50 .mu.l of the gold particles prepared above. Into each tube,
the following are also added; 5 .mu.l of plasmid DNA (at 1
.mu.g/.mu.l), 50 .mu.l CaCl2, and 20 .mu.l 1 0.1 M spermidine. This
mixture is agitated on a vortex shaker for 3 minutes, and then
centrifuged using a microcentrifuge set at 14,000 RPM for 10
seconds. The supernatant is decanted and the gold particles with
attached, precipitated DNA are washed twice with 400 .mu.l aliquots
of ethanol (with a brief centrifugation as above between each
washing). The final volume of 100% ethanol per each tube is
adjusted to 40 .mu.l, and this particle/DNA suspension is kept on
ice until being used for bombardment.
[0228] Immediately before applying the particle/DNA suspension, the
tube is briefly dipped into a sonicator bath to disperse the
particles, and then 5 .mu.L of DNA prep is pipetted onto each
flying disk and allowed to dry. The flying disk is then placed into
the DuPont Biolistics PDS1000/HE. Using the DuPont Biolistic
PDS1000/HE instrument for particle-mediated DNA delivery into
soybean suspension clusters, the following settings are used. The
membrane rupture pressure is 1100 psi. The chamber is evacuated to
a vacuum of 27-28 inches of mercury. The tissue is placed
approximately 3.5 inches from the retaining/stopping screen (3rd
shelf from the bottom). Each plate is bombarded twice, and the
tissue clusters are rearranged using a sterile spatula between
shots.
[0229] Following bombardment, the tissue is re-suspended in liquid
culture medium, each plate being divided between 2 flasks with
fresh SB 196 or SB 172 media and cultured as described above. Four
to seven days post-bombardment, the medium is replaced with fresh
medium containing a selection agent. The selection media is
refreshed weekly for 4 weeks and once again at 6 weeks. Weekly
replacement after 4 weeks may be necessary if cell density and
media turbidity is high.
[0230] Four to eight weeks post-bombardment, green, transformed
tissue may be observed growing from untransformed, necrotic
embryogenic clusters. Isolated, green tissue is removed and
inoculated into 6-well microtiter plates with liquid medium to
generate clonally-propagated, transformed embryogenic suspension
cultures.
[0231] Each embryogenic cluster is placed into one well of a Costar
6-well plate with 5 mls fresh SB196 media with selection agent.
Cultures are maintained for 2-6 weeks with fresh media changes
every 2 weeks. When enough tissue is available, a portion of
surviving transformed clones are subcultured to a second 6-well
plate as a back-up to protect against contamination.
[0232] To promote in vitro maturation, transformed embryogenic
clusters are removed from liquid SB 196 and placed on solid agar
media, SB 166, for 2 weeks. Tissue clumps of 2-4 mm size are plated
at a tissue density of 10 to 15 clusters per plate. Plates are
incubated in diffuse, low light (<10 .mu.E) at 26+/-1.degree. C.
After two weeks, clusters are subcultured to SB 103 media for 3-4
weeks.
[0233] SB 166 is prepared as follows: (per liter), 1 pkg. MS salts
(Gibco/ BRL-Cat #11117-017), 1 ml B5 vitamins 1000.times. stock, 60
g maltose, 750 mg MgCl2 hexahydrate, 5 g activated charcoal, pH
5.7, 2 g gelrite. SB 103 media is prepared as follows: (per liter),
1 pkg. MS salts (Gibco/BRL-Cat #11117-017), 1 ml B5 vitamins
1000.times. stock, 60 g maltose, 750 mg MgCl2 hexahydrate, pH 5.7,
2 g gelrite. After 5-6 week maturation, individual embryos are
desiccated by placing embryos into a 100.times.15 petri dish with a
1 cm.sup.2 portion of the SB 103 media to create a chamber with
enough humidity to promote partial desiccation, but not death.
[0234] Approximately 25 embryos are desiccated per plate. Plates
are sealed with several layers of parafilm and again are placed in
a lower light condition. The duration of the desiccation step is
best determined empirically, and depends on size and quantity of
embryos placed per plate. For example, small embryos or few
embryos/plate require a shorter drying period, while large embryos
or many embryos/plate require a longer drying period. It is best to
check on the embryos after about 3 days, but proper desiccation
will most likely take 5 to 7 days. Embryos will decrease in size
during this process.
[0235] Desiccated embryos are planted in SB 71-1 or MSO medium
where they are left to germinate under the same culture conditions
described for the suspension cultures. When the plantlets have two
fully-expanded trifoliate leaves, germinated and rooted embryos are
transferred to sterile soil and watered with MS fertilizer. Plants
are grown to maturity for seed collection and analysis. Healthy,
fertile transgenic plants are grown in the greenhouse.
[0236] SB 71-1 is prepared as follows: 1 bottle Gamborg's B5 salts
w/ sucrose (Gibco/BRL-Cat #21153-036), 10 g sucrose, 750 mg MgCl2
hexahydrate, pH 5.7, 2 g gelrite. MSO media is prepared as follows:
1 pkg Murashige and Skoog salts (Gibco 11117-066), 1 ml B5 vitamins
1000.times. stock, 30 g sucrose, pH 5.8, 2 g Gelrite.
Example 11
[0237] This example describes the design and synthesis of miRNA
targets and hairpins directed to various gene targets found in
maize, soy, and/or Arabidopsis, using the method described in
Example 9.
[0238] A. Targeting Arabidopsis AGAMOUS, At4g18960
[0239] The miRNA sequence of SEQ ID NO:4 is selected and designed.
The sequence is put into the BamHI/HindIII hairpin cassette by
annealing the synthetic oligonucleotides of SEQ ID NOS: 12-15, and
ligating them into the BamHI/HindIII backbone fragment of SEQ ID
NO:44.
[0240] Arabidopsis thaliana Col-0 is transformed and grown as
described in Example 1. After transformation with a vector
comprising the miRNA of SEQ ID NO:4, 88% of the transformants
exhibit a mutant AGAMOUS (ag) floral phenotype, characterized by
the conversion of stamens to petals in whorl 3, and carpels to
another ag flower in whorl 4 (Bowman, et al. (1991) The Plant Cell
3:749-758). The mutant phenotype varies between transformants, with
approximately 1/3 exhibiting a strong ag phenotype, 1/3 exhibiting
an intermediate ag phenotype, and 1/3 exhibiting a weak ag
phenotype. Gel electrophoresis and Northern Blot analysis of small
RNAs isolated from the transformants demonstrates that the degree
of the mutant ag phenotype is directly related to the level of
antiAG miRNA, with the strongest phenotype having the highest
accumulation of the processed miRNA (.about.21 nt).
[0241] B. Targeting Arabidopsis Apetela3 (AP3), At3g54340
[0242] Two miRNA targets from AP3 are selected and oligonucleotides
designed.
[0243] The miRNA sequence of SEQ ID NO:5 is selected and designed.
The sequence is put into the BamHI/HindIII hairpin cassette by
annealing the synthetic oligonucleotides of SEQ ID NOS: 16-19, and
ligating them into the BamHI/HindIII backbone fragment of SEQ ID
NO:44.
[0244] The miRNA sequence of SEQ ID NO:6 is selected and designed.
The sequence is put into the BamHI/HindIII hairpin cassette by
annealing the synthetic oligonucleotides of SEQ ID NOS: 20-23, and
ligating them into the BamHI/HindIII backbone fragment of SEQ ID
NO:44.
[0245] Arabidopsis thaliana Col-0 is transformed and grown as
described in Example 1. After transformation with a vector
comprising the miRNA of SEQ ID NO:5, the transformants have novel
leaf and floral phenotypes, but do not exhibit any mutant AP3
phenotype. Gel electrophoresis and Northern analysis of RNA
isolated from 2 week old rosette leaf tissue from the transformants
demonstrates that the highest accumulation of the processed miRNA
(-21 nt) corresponds to the "backside" strand of the precursor,
which evidently silences a different target sequence to produce the
novel leaf and floral phenotypes.
[0246] A new target sequence is selected, with the correct
asymmetry in order for the miRNA target strand to be selected
during incorporation into RISC (Schwartz et al. (2003) Cell
115:199-208). The miRNA sequence of SEQ ID NO:6 is selected and
designed. The sequence is put into the BamHI/HindIII hairpin
cassette by annealing the synthetic oligonucleotides of SEQ ID NOS:
20-23, and ligating them into the BamHI/HindIII backbone fragment
of SEQ ID NO:44. Greater than 90% of the transformants show
silencing for the AP3 gene, as demonstrated by floral phenotype and
electrophoretic analysis. An approximately 21 nt miRNA (antiAP3b)
is detected at high levels in the transgenic plants, and not in
wild type control plants. RT-PCR analysis confirmed that the amount
of AP3 transcript is reduced in the transformants, as compared to
wild type control plants.
[0247] C. Targeting Maize Phytoene Desaturase
[0248] Two miRNA targets from phytoene desaturase (PDS) are
selected and oligonucleotides designed.
[0249] The miRNA sequence of SEQ ID NO:7 is selected and designed.
The sequence is put into the BamHI/HindIII hairpin cassette by
annealing the synthetic oligonucleotides of SEQ ID NOS: 24-27, and
ligating them into the BamHI/HindIII backbone fragment of SEQ ID
NO:44.
[0250] The miRNA sequence of SEQ ID NO:8 is selected and designed.
The sequence is put into the BamHI/HindIII hairpin cassette by
annealing the synthetic oligonucleotides of SEQ ID NOS: 28-31, and
ligating them into the BamHI/HindIII backbone fragment of SEQ ID
NO:44.
[0251] D. Targeting Maize Phytic Acid biosynthetic enzymes
[0252] Three maize phytic acid biosynthetic enzyme gene targets are
selected and miRNA and oligonucleotides designed. Inositol
polyphosphate kinase-2 polynucleotides are disclosed in PCT
International published application No. WO 02/059324, herein
incorporated by reference. Inositol 1,3,4-trisphosphate 5/6-kinase
polynucleotides are disclosed in PCT International published
application No. WO 03/027243, herein incorporated by reference.
Myo-inositol 1-phosphate synthase polynucleotides are disclosed in
PCT International published application No. WO 99/05298, herein
incorporated by reference.
[0253] Inositol polyphosphate kinase-2 (IPPK2)
[0254] The miRNA sequence of SEQ ID NO:9 is selected and designed.
The sequence is put into the BamHI/HindIII hairpin cassette by
annealing the synthetic oligonucleotides of SEQ ID NOS: 32-35, and
ligating them into the BamHI/HindIII backbone fragment of SEQ ID
NO:44.
[0255] Inositol 1,3,4-trisphosphate 5/6-kinase-5 (ITPK5)
[0256] The miRNA sequence of SEQ ID NO:10 is selected and designed.
The sequence is put into the BamHI/HindIII hairpin cassette by
annealing the synthetic oligonucleotides of SEQ ID NOS: 36-39, and
ligating them into the BamHI/HindIII backbone fragment of SEQ ID
NO:44.
[0257] Myo-inositol 1-phosphate synthase (mi1ps)
[0258] The miRNA sequence of SEQ ID NO:11 is selected and designed.
The sequence is put into the BamHI/HindIII hairpin cassette by
annealing the synthetic oligonucleotides of SEQ ID NOS: 40-43, and
ligating them into the BamHI/HindIII backbone fragment of SEQ ID
NO:44.
[0259] E. Targeting Soy Apetela2-like sequences (AP2)
[0260] The same EAT (miR172a-2) construct, comprising SEQ ID NO:1,
used for Arabidopsis transformation is used to transform soybean.
This construct has a miRNA template sequence which encodes the
miRNA of SEQ ID NO:48. The construct is created using a PCR
amplification of miR172a-2 precursor sequence from Arabidopsis,
restriction digestion, and ligation as described in Example 2.
[0261] Soybean tissue is transformed and grown essentially as
described in Example 10. After transformation, 42% of the
transformants exhibit a mutant phenotype, characterized by the
conversion of sepals to leaves. Plants exhibiting the strongest
phenotypes are sterile, and produce no seed. Both the homeotic
conversion of the organs and the effects on fertility are similar
to that seen for ap2 mutant alleles in Arabidopsis. Small RNA gel
electrophoresis and Northern analysis, probed with an
oligonucleotide probe antisense to miR172, shows accumulation of
miR172 in the transgenic lines. A small amount of endogenous soy
miR172 is also detected in the soy control line. The degree of the
mutant phenotype is directly related to the level of miRNA, with
the strongest phenotype having the highest accumulation of the
processed miRNA (.about.21 nt).
[0262] F. Targeting Arabidopsis AP2-like genes
[0263] The miRNA sequence of SEQ ID NO:72 is selected and designed.
The sequence is put into the attB hairpin cassette by annealing the
synthetic oligonucleotides of SEQ ID NOS: 73-74, and performing the
BP recombination reaction (GATEWAY) to generate the attL
intermediate. This intermediate is used in the LR reaction to
recombine with the destination vector, generally described in
Example 12, comprising the EAT full-length precursor containing
attR sites, and negative selection markers in place of the hairpin.
The product of this reaction comprises the miR172a-2 precursor
hairpin cassette flanked by attR sites (i.e., the hairpin replaces
the marker cassette).
[0264] G. Targeting Arabidopsis Fatty Acid Desaturase (FAD2)
[0265] The miRNA sequence of SEQ ID NO:75 is selected and designed
based on the sequence of NM.sub.--112047 (At3g12120). The sequence
is put into the attB hairpin cassette by annealing the synthetic
oligonucleotides of SEQ ID NOS: 76-77, and performing the BP
recombination reaction (GATEWAY) to generate the attL intermediate.
This intermediate is used in the LR reaction to recombine with the
destination vector, generally described in Example 12, comprising
the EAT full-length precursor containing attR sites, and negative
selection markers in place of the hairpin. The product of this
reaction comprises the FAD2 miRNA precursor hairpin cassette
flanked by attR sites (i.e., the hairpin replaces the marker
cassette). The effect of the anti-FAD2 miRNA can be determined by
fatty acid analysis to determine the change in the fatty acid
profile, for example, see Wu et al. (1997) Plant Physiol.
113:347-356, herein incorporated by reference.
[0266] H. Targeting Arabidopsis Phytoene Desaturase (PDS)
[0267] The miRNA sequence of SEQ ID NO:78 is selected and designed
based on the sequence of NM.sub.--202816 (At4g14210). The sequence
is put into the attB hairpin cassette by annealing the synthetic
oligonucleotides of SEQ ID NOS: 79-80, and performing the BP
recombination reaction (GATEWAY) to generate the attL intermediate.
This intermediate is used in the LR reaction to recombine with the
destination vector, generally described in Example 12, comprising
the EAT full-length precursor containing attR sites, and negative
selection markers in place of the hairpin. The product of this
reaction comprises the PDS miRNA precursor hairpin cassette flanked
by attR sites (i.e., the hairpin replaces the marker cassette).
Transgenic plants containing the antiPDS construct are
photobleached upon germination in greater than about 90% of the
lines, indicating silencing of PDS.
Example 12
[0268] This example describes the construction of expression
vectors using recombinational cloning technology.
[0269] The vector described in Example 2 (SEQ ID NO:44) is modified
to incorporate att recombination sites to facilitate
recombinational cloning using GATEWAY technology (InVitrogen,
Carlsbad, Calif.). The BamHI/HindIII segment is replaced with a
sequence comprising in the following order: attR1-CAM-ccdB-attR2.
Upon recombination (BP+LR) with oligos containing attB sites
flanking the miRNA hairpin precursor construct, the selectable
markers are replaced by the miRNA hairpin precursor.
Example 13
[0270] This example, particularly Table 5, summarizes the target
sequences and oligos used for miRNA silencing constructs as
described in the examples.
TABLE-US-00005 TABLE 5 Precursor oligos miRNA miRNA SEQ ID Organism
Target gene name template NOS Arabidopsis AP2-like miR172-a2 SEQ ID
NO: 86 55-56 (PCR) none EATdel none 57-60 AGAMOUS antiAG SEQ ID NO:
4 12-15 APETELA3 (a) antiAP3a SEQ ID NO: 5 16-19 APETELA3 (b)
antiAP3b SEQ ID NO: 6 20-23 Corn PDS1 antiPDS1 SEQ ID NO: 7 24-27
PDS2 antiPDS1 SEQ ID NO: 8 28-31 IPPK2 antiIPPK2 SEQ ID NO: 9 32-35
ITPK5 antiITPK5 SEQ ID NO: 10 36-39 MI1PS antiMI1PS SEQ ID NO: 11
40-43 Soybean AP2-like miR172a-2 SEQ ID NO: 86 55-56 (PCR)
Arabidopsis AP2-like miR172a-2 SEQ ID NO: 72 73-74 FAD2 antiFAD2
SEQ ID NO: 75 76-77 PDS antiAtPDS SEQ ID NO: 78 79-80 Corn miR172b
miR172 SEQ ID NO: 92 91 PDS antiZmPDS SEQ ID NO: 95 94
Example 14
[0271] This example describes the identification and isolation of
genomic corn miR172 precursors.
[0272] The Genome Survey Sequence (GSS) database of the National
Center for Biotechnology Information (NCBI) is searched using the
21nt miR172a-2 sequence in order to identify genomic corn sequences
containing miR172 precursor sequence. Several corn miR172
precursors are identified, and named miR172a-miR172e (SEQ ID NOS:
81-85) as summarized in Table 6. Each sequence is imported into
Vector NTI (InVitrogen, Carlsbad, Calif.) and contig analyses done.
The analysis identifies four distinct loci, each with a unique
consensus sequence. A region of about 200 nucleotides surrounding
the miRNA sequence from each locus is examined for secondary
structure folding using RNA Structure software (Mathews et al.
(2004) Proc Natl Acad Sci USA 101:7287-7292, herein incorporated by
reference). The results of this analysis identifies the hairpin
precursors of each of the corn sequences miR172a-e.
TABLE-US-00006 TABLE 6 Corn miR172 precursors and positions of
hairpin, & miRNA duplex components Precursor NCBI ID Corn Line
SEQ ID NO: Length Hairpin Backside miRNA miR172a CG090465 B73 81
907 508-598 512-532 574-594 miR172b BZ401521 B73 82 1128 551-654
567-587 620-640 and (both) BZ4011525 miR172c CG247934 B73 83 912
230-400 250-270 364-384 miR172d CG097860 B73 84 1063 351-520
361-381 466-486 and BZ972414 miR172e CG065885 B73 85 1738 913-1072
931-951 1033-1053 and (both) CC334589
[0273] Oligonucleotides are designed in order amplify miR172a or
miR172b from a B73 genomic corn library, these primers also add
restriction enzyme recognition sites in order to facilitate cloning
(BamHI or EcoRV). Alternatively, PCR primers designed to create att
sites for recombinational cloning could be used. After PCR
amplification, the products are isolated, purified, and confirmed
by sequence analysis. Once confirmed, these sequences are inserted
into a construct comprising the corn ubiquitin (UBI) promoter. This
construct can be used for further transformation vector
construction, for example, with the addition of att sites, the
GATEWAY system can be used.
[0274] The following PCR primers are used to amplify a sequence
comprising the hairpin precursor of corn miR172a
TABLE-US-00007 Forward primer (SEQ ID NO: 87) 5'
GGATCCTCTGCACTAGTGGGGTTATT 3' Reverse primer (SEQ ID NO: 88) 5'
GATATCTGCAACAGTTTACAGGCGTT 3'
[0275] The following PCR primers are used to amplify a sequence
comprising the hairpin precursor of corn miR172b
TABLE-US-00008 Forward primer (SEQ ID NO: 89) 5'
GGATCCCATGATATAGATGATGCTTG 3' Reverse primer (SEQ ID NO: 90) 5'
GATATCAAGAGCTGAGGACAAGTTTT 3'
Example 15
[0276] This example describes the design and synthesis of miRNA
targets and hairpins directed to various gene targets found in
maize, for use with the corn miR172b miRNA precursor.
[0277] A. miR172b Target in Corn
[0278] Similar to the Arabidopsis EAT examples, the corn miR172b
hairpin precursor will be tested by overexpression in corn. The
precursor sequence comprising the miRNA template is shown in SEQ ID
NO:91. The miRNA is shown in SEQ ID NO:92, and the backside of the
miRNA duplex is shown in SEQ ID NO:93. A double-stranded DNA
molecule comprising the miRNA precursor and restriction enzyme
overhangs, for BamHI and KpnI, is created by annealing the
oligonucleotides of SEQ ID NOS: 97 and 98.
[0279] B. Phytoene Desaturase (PDS)
[0280] An oligonucleotide comprising the the miRNA template is
shown in SEQ ID NO:94. The miRNA directed to PDS is shown in SEQ ID
NO:92, and the backside of the miRNA duplex is shown in SEQ ID
NO:93. A double-stranded DNA molecule comprising the miRNA
precursor and restriction enzyme overhangs, for BamHI and KpnI, is
created by annealing the oligonucleotides of SEQ ID NOS: 99 and
100.
[0281] The oligonucleotides of this example can be inserted into
vectors for transformation of corn using standard cloning
techniques, including restriction digestion and ligation, and/or
recombinational cloning such as GATEWAY.
Example 16
[0282] This example describes the materials and methods used for
Examples 17-19.
[0283] Plasmid constructs
[0284] A fragment of 276 base pairs containing the entire sequence
of Arabidopsis miR159a (see below) was cloned by PCR amplification
using primers CACC-miR159a-prec: 5' CACCACAGTTTGCTTATGTCGGATCC 3'
(SEQ ID NO:101) and miR159a-Xma: 5' TGACCCGGGATGTAGAGCTCCCTTCAATCC
3' (SEQ ID NO:102). The miR159a-Xma contains 18 of 21 nucleotides
of the mature miR159a (bold) and an introduced XmaI site (italic).
The PCR fragment was cloned in the pENTR/SD/D-TOPO vector
(Invitrogen) according to manufacturers directions to obtain
pENTR-miR159a-prec.
[0285] The Gateway recombination system was used to transfer the
pre-miR159a sequence to the plant binary vector pK2GW7, which
contains two copies of the 35S promoter and a NOS terminator to
generate pK2-pre-miR159a.
[0286] Mutagenesis of pre-miR159a was performed by PCR with the
following oligonucleotides.
[0287] 5'-miR-PDS.sup.159a: 5'
ATAGATCTTGATCTGACGATGGAAGAAGAGATCCTAAC T TTTCAAA 3' (SEQ ID NO:103;
This oligonucleotide contains a natural Bgl II site (italic) and
the miR-PDS.sup.159a* sequence (bold)).
[0288] 3'-miR-PDS.sup.159a: 5' TGACCCGGGATGAAGAGATCCCATATTTCCAAA 3'
SEQ ID NO:104; This oligonucleotide contains point mutations in the
miR159a sequence (bold) to increase its complementarity to the PDS
mRNA sequence, based on available N. benthamiana PDS mRNA partial
sequence (Genbank AJ571700, see below)).
[0289] PCR amplification of the miR159a precursor using the above
primers and pENTR-miR159a-prec DNA as template generated a DNA
fragment that was digested with BglII and XmaI to be re-inserted
into pENTR-pre-miR159a, to generate pENTR-pre-miR-PDS.sup.159a.
Gateway system procedures were used again to transfer the
miR-PDS.sup.159a precursor to pK2GW7 and generate
pK2-pre-miR-PDS.sup.159a.
[0290] The miR-PDS.sup.169g was cloned as follows. An Arabidopsis
genomic fragment of 222 base pairs containing the miR169g sequence
(see below) was amplified using primers miR169g-For 5'
CACCAATGATGATTACGATGATGAGAGTC 3' (SEQ ID NO:105), and miR169g-Rev
5' CAAAGTTTGATCACGATTCATGA 3' (SEQ ID NO:106). The resulting PCR
fragment was introduced into pENTR/D-TOPO vector (Invitrogen) to
obtain pENTR-pre-miR169g. The pre-miR169g sequence was then
transferred into binary vectors pBADC and pB2GW7 using the Gateway
system to generate pBA-pre-miR169g and pB2-pre-miR169g.
[0291] Two miR-PDS.sup.169g precursors were created using
pENTR-pre-miR169g as template and the Quick-change Mutagenesis kit
from Stratagene. pENTR-pre-miR-PDSa.sup.169g was made by using the
following oligonucleotides:
TABLE-US-00009 miR169.sup.PDsa: SEQ ID NO: 107) 5'
GAGAATGAGGTTGAGTTTAGTCT GACTTGGCCAGTTTTTTTACCAATG 3', and
miR169.sup.PDsa*: (SEQ ID NO:108) 5' CTGATTCTGGTGTTGGCCAAGTC
AGACTAAACTCTGTTTCCTTCTC 3'.
[0292] pENTR-pre-miR-PDSb.sup.169g was produced by using the
oligonucleotides:
TABLE-US-00010 miR169.sup.PDsb: (SEQ ID NO: 109) 5'
GAGAATGAGGTTGATCTCTTTCCAGTC TTCAGGGTTTTTTTACCAATG 3', and
miR169.sup.PDsb*: (SEQ ID NO: 110) 5' GATTCTGGTGTCCTGAAGACTGGAAAG
AGATCTGTTTCCTTCTCTTC 3'.
[0293] The two mutagenized miR-PDS.sup.169g precursors above were
then transferred into plant binary vectors pBADC and pB2GW7 to
generate pBA-pre-miR-PDSa.sup.169g, pB2-pre-miR-PDSa.sup.169g;
pBA-pre-miR-PDSb.sup.169g, and pB2-pre-miR-PDSb.sup.169g.
[0294] Precursors for artificial miRNAs that target N. benthamiana
rbcS transcripts (pENTR-pre-miR-rbcS.sup.159a-A) were produced
using similar procedures as those described for
pENTR-miR-PDS.sup.159a using the following primers and cloned into
pK2GW7:
TABLE-US-00011 MrbcSA-S: (SEQ ID NO: 111) 5'
TCTGACGATGGAAGTTCCTCGCCCG ACATTCGAAAATGAGTTGA 3', and MrbcSA-R:
(SEQ ID NO: 112) 5' AAACCCGGGATGTTCCTCGCCCGGAA TTCGAAAGAGAGTAAAAG
3'.
[0295] All cloned sequences were confirmed by DNA sequencing.
[0296] Precursor sequences used
TABLE-US-00012 miR159a precursor template sequence (276bp) (SEQ ID
NO: 113) ACAGTTTGCTTATGTCGGATCCATAATATATTTGACAAGATACTTTGTT
TTTCGATAGATCTTGATCTGACGATGGAAGTAGAGCTCCTTAAAGTTCA
AACATGAGTTGAGCAGGGTAAAGAAAAGCTGCTAAGCTATGGATCCCAT
AAGCCCTAATCCTTGTAAAGTAAAAAAGGATTTGGTTATATGGATTGCAT
ATCTCAGGAGCTTTAACTTGCCCTTTAATGGCTTTTACTCTTC CATCCCGGGTC (Sequence
of the pre-miR159a cloned. Sequences of miR159a* and miR159a
(italic) are shown in bold. Nucleotides changed in miR-PDS.sup.159a
are underlined.) miR-159a mature template (SEQ ID NO: 114) 5'
TTTGGATTGAAGGGAGCTCTA 3' miR-PDS.sup.159a mature template (SEQ ID
NO: 115) 5' TTTGGA a a t A t GGGAt CTCT t 3' miR169g precursor
template sequence 0.3 kb (222bp) (SEQ ID NO: 116)
AATGATGATTACGATGATGAGAGTCTCTAGTTGTATCAGAGGGTCTTGCA
TGGAAGAATAGAGAATGAGGTT GTTT
TTTTACCAATGAATCTAATTAACTGATTCTGGTGTCCGGCAAGTTGACCT
TGGCTCTGTTTCCTTCTCTTCTTTTGGATGTCAGACTCCAAGATATCTAT
CATCATGAATCGTGATCAAACTTTG (Sequence of the pre-miR169g fragment
(0.3kb) cloned. Sequences of miR169g (italic) and miR169g* are
shown in bold. Nucleotides changed in miR- PDS.sup.169g are
underlined.) miR169g mature template (SEQ ID NO: 117) 5'
GAGCCAAGGATGACTTGCCGG 3' miR-PDSa.sup.169g mature template (SEQ ID
NO: 118) 5' GAG t t t AG t c TGACTTG gC c a 3' miR169g mature
template (SEQ ID NO: 119) 5' GAGCCAAGGATGACTTGCCGG 3'
miR-PDSb.sup.169g mature template (SEQ ID NO: 120) 5' GA tC t c t t
t c c a g t c T tC aGG 3' miR169g precursor template sequence 2.0kb
(2474bp) (SEQ ID NO: 121)
AAGCTTTGATCTTTAGCTCTTTGCCAAAGCTTCTTTTGATTTTTCTATT
TCTCTAATCTATCCATTGACCATTTGGGGTGATGATATTCTTCAATTTA
TGTTGTTGTTTATTGCCCATCCACAGACCCACGTTTGATTTGTTTAATC
AAAATATATAAACTGACAGTTGTGCCACTAGTCACTTGCCAATTAAGCA
TTCCAAAGCTCCTTCCTTTACATTAGTATCAAGTGAGACTAGCACAAGC
TTTTAAGTCCAGATAAAAAGCCCCATGGAAGGGAAGCTTTCAAGAACGA
GATTTAACCGTAAAACCCAATTTCGATTTCCGCTAATAATTTGGATCCA
AAAATCTAGACAAAATCTGATAAAATTAGACAAAGAAATGGATAAAACC
CCAAAACCCATAATCGTCGTTGTTCTTGTTTGCTTCAATATCACTCTTT
CCCCTCCAACGAGTTAGTTAGAGTGACGTGGCAGCTGAACTAGATTTGG
AGTAACGGGATAGATTACCCATAAAGCCCAATAATGATCATTACGTGAG
ACATAACTTGCTTAGATAACCTCATTTTATGGGCTTAGATGGGGTCTCT
AGTGTTAGTCATAAGCTCTTAAATACCATTTCTAGTTATATATCAATCT
TTAGCTTGGAATTGGATCGTTGTCCTATAGTAAAAAAACTTTTACTATT
TTATGTTAGCAATCCCACTTAACATTCAATATGTTTAAAATGAAAGAGT
TTACCAAAAGGAAAGAAAAAAAGGTTGGTAATGAATTTATCTAATCGGA
TACGATATTTCATAATCTAATGATGGGATCTATCAATAAATAGAATCAA
AGTTAACTTTAACGCTTTTGTTACCTGTTTTCTTTCTTTAGCAATTAAT
ATTAAACGAGTTTTAGTAATATAAATATGTTTCCAGTTATATACCAAAC
TTTATGTAATATTCATAAGCTTGCCAAAATTTACAAGAGTTTTTGGAAC
GCGCACAAAATTCTCATATATTTCTTACCCAAAAATAAATTTTTTTTTT
TTTTTTACTTGTTTATAATCCTATATGAACATTGCTCATCTTCCCCATT
TGATGGTAATTTTTCTATTCCTATATGTAATTAAATCCTAACTAATGAA
ATTGAAAACATAATTTGAAGATAATCAATCCTAATATCTCCCGTCTTAG
ATCTATTTAAATGGTCTTATTTAATTTCCTATATTTTGGCCTAATTATT
TATTTGATATAGTGAATTTATGGAAGCTTCATGTTGATGGAATAAAACC
GGCTTATCCCAATTAATCGATCGGGAGCTATAACACAAATCGAAACTCT
AGTAGCTATAAAGAGTGTGTAATAGCTTTGGATCACATGTATTACTATT
TATTTACTAGCTCGTGCAACAATTGGCTTTGGGAAAAAATTTATTTACT
AGTACTCCCCCTTCACAATGTGATGAGTCTCCAAATGATATATTCTCAA
CCCAAAGGACAATCTGAAATTTTCAATATATATTCCATTTTATCCGCAA
CATTTGAAATTTGTGGCAATGTTTTTAAAAAGACTATTTATAAAGAATC
TTTCTAAATTGTTTCTACGACAATCGATAACACCTTTTGTTGATCAACC
CCACACAAGACTATGATTCCAATCCTAAGAAACATACGACACGTGGATT
TTTATGTCACACTAGTACGATGCGTCGATGCCTTCAGAGTACGAATATT
ATTCACATAAAATTCTTATTCGAATTTGATAATATAAGGTCAGCCAATC
TTTTAAAGTAATTATATTCTTCAATATACGGTTGTGGTCAAAATTCCAT
TTTATTTTGTAGCTTGCATGCACTACTAGTTTAAAACCATGCATGGATT
TATTGCATATAATAACATTATATGAATTTTCAATTAAATTAATCCACAC
ATTTCCCATTTCAATATGCCTATAAATACCTTCATCACGAGTATGACAA
GATCACAAGACAAGAAAAGAAAGGTAGAGAAAACATGATAATGATGATT
ACGATGATGAGAGTCTCTAGTTGTATCAGAGGGTCTTGCATGGAAGAAT AGAGAATGAGGT
GTTTTTTTACCAA TGAATCTAATTAACTGATTCTGGTGTCCGGCAAGTTGACCTTGGCTCT
GTTTCCTTCTCTTCTTTTGGATGTCAGACTCCAAGATATCTATCATCA
TGAATCGTGATCAAACTTTGTAATTTCATTGAAATGTGTTTTTCTTGA
TGCGAATTTTTTGGCTTACGGTTTTTCGATTTGAATGATCAGATTTTT
GTTTTTGCACTCAAACTATAGTTTCACTTAGGTTCTATTTTTTTCAGGT
TTATGAATGATAAAACAAGTAAGATTTTATGCTAGTTTTAGTTCATTTT
TCGATTCAAATTCAAACATCTTGGTTTTGGTTTAGTTAAGTTTGATTTT
TCAAGTCAAATGCTATGTTTTCTTGT (Sequence of the pre-miR169g fragment
(2.0kb) cloned. Sequences of miR169g (italic) and miR169g* are
shown in bold.)
[0297] Target gene sequences used:
[0298] Nicotiana benthamiana PDS sequences:
[0299] 5'end probe sequence (corresponding to Le-PDS pos.1-268, see
FIG. 15A):
TABLE-US-00013 (SEQ ID NO: 122)
ATGCCTCAAATTGGACTTGTTTCTGCTGTTAACTTGAGAGTCCAAGGTAG
TTCAGCTTATCTTTGGAGCTCGAGGTCGTCTTCTTTGGGAACTGAAAGTC
GAGATGGTTGCTTGCAAAGGAATTCGTTATGTTTTGCTGGTAGCGAATCA
ATGGGTCATAAGTTAAAGATTCGTACTCCCCATGCCACGACCAGAAGATT
GGTTAAGGACTTGGGGCCTTTAAAGGTCGTATGCATTGATTATCCAAGAC
CAGAGCTGGACAATACAG
[0300] Partial+5'RACE fragment. Assembled sequence from partial
Nicotiana benthamiana PDS sequence (Genbank AJ571700) and 5'RACE
experiments (corresponding to Le-PDS pos. 858-1514, see FIG.
15A).
TABLE-US-00014 (SEQ ID NO: 123)
GGCACTCAACTTTATAAACCCTGACGAGCTTTCGATGCAGTGCATTTTGA
TTGCTTTGAACAGATTTCTTCAGGAGAAACATGGTTCAAAAATGGCCTTT
TTAGATGGTAACCCTCCTGAGAGACTTTGCATGCCGATTGTGGAACATAT
TGAGTCAAAAGGTGGCCAAGTCAGACTAAACTCACGAATAAAAAAGATCG
AGCTGAATGAGGATGGAAGTGTCAAATGTTTTATACTGAATAATGGCAGT
ACAATTAAAGGAGATGCTTTTGTGTTTGCCACTCCAGTGGATATCTTGAA GCTTCTTTTG
CCCATATTTCCAA AAGTTGGAGAAGCTAGTGGGAGTTCCTGTGATAAATGTCCATATATGGTT
TGACAGAAAACTGAAGAACACATCTGATAATCTGCTCTTCAGCAGAAGCC
CGTTGCTCAGTGTGTACGCTGACATGTCTGTTACATGTAAGGAATATTAC
AACCCCAATCAGTCTATGTTGGAATTGGTATTTGCACCCGCAGAAGAGTG
GATAAATCGTAGTGACTCAGAAATTATTGATGCTACAATGAAGGAACTAG
GCAAGCTTTTCCCTGATGAAATTTCGGCAGATCAGAGCAAAGCAAAAATA TTGAAGTACCATGT
(Sequences targeted by miR-PDSa.sup.169g (bold), miR-PDSb.sup.169g
(bold and italic)and miR-PDS.sup.159a (underlined) are
indicated.)
[0301] Nicotiana benthamiana rbcS sequences (Bolded nucleotides in
all six rbcS gene sequences correspond to the sequence targeted by
miR-rbcS.sup.159a-A.):
TABLE-US-00015 rbcS1 (Genbank accessions: CN748904: 56-633bp,
CN748069: 419-end) (SEQ ID NO: 124)
GGAGAAAGAGAAACTTTCTGTCTTAAGAGTAATTAGCAATGGCTTCCTCA
GTTCTTTCCTCAGCAGCAGTTGCCACCCGCAGCAATGTTGCTCAAGCTAA
CATGGTTGCACCTTTCACAGGTCTTAAGTCTGCTGCCTCATTCCCTGTTT
CAAGAAAGCAAAACCTTGACATCACTTCCATTGCCAGCAACGGCGGAAGA
GTGCAATGCATGCAGGTGTGGCCACCAATTAACATGAAGAAGTATGAGAC
TCTCTCATACCTTCCCGATTTGAGCCAGGAGCAATTGCTCTCCGAAATTG
AGTACCTTTTGAAAAATGGATGGGTTCCTTGCTTGGAATTCGAGACTGAG
AAAGGATTTGTCTACCGTGAACACCACAAGTCACCAGGATACTATGATGG
CAGATACTGGACCATGTGGAAGCTACCTATGTTCGGATGCACTGATGCCA
CCCAAGTGTTGGCTGAGGTGGGAGAGGCGAAGAAGGAATACCCACAGGCC
TGGGTCCGTATCATTGGATTTGACAACGTGCGTCAAGTGCAGTGCATCAG
TTTCATTGCCTCCAAGCCTGACGGCTACTGAGTTTCATATTAGGACAACT
TACCCTATTGTCTGTCTTTAGGGGCAGTTTGTTTGAAATGTTACTTAGCT
TCTTTTTTTTCCTTCCCATAAAAACTGTTTATGTTCCTTCTTTTTATTCG
GTGTATGTTTTGGATTCCTACCAAGTTATGAGACCTAATAATTATGATTT
TGTGCTTTGTTTGTAAAAAAAAAAAAAAAAA rbcS2 (Genbank accessions:
CN748495: 3-552 b, CN748945: 364-575 b) (SEQ ID NO: 125)
TCTTTCTGTCTTAAGTGTAATTAACAATGGCTTCCTCAGTTCTTTCCTCA
GCAGCAGTTGCCACCCGCAGCAATGTTGCTCAAGCTAACATGGTTGCACC
TTTCACTGGTCTTAAGTCAGCTGCCTCGTTCCCTGTTTCAAGGAAGCAAA
ACCTTGACATCACTTCCATTGCCAGCAACGGCGGAAGAGTGCAATGCATG
CAGGTGTGGCCACCAATTAACAAGAAGAAGTACGAGACTCTCTCATACCT
TCCTGATCTGAGCGTGGAGCAATTGCTTAGCGAAATTGAGTACCTCTTGA
AAAATGGATGGGTTCCTTGCTTGGAATTCGAGACTGAGCGCGGATTTGTC
TACCGTGAACACCACAAGTCACCGGGATACTATGACGGCAGATACTGGAC
CATGTGGAAGTTGCCTATGTTCGGATGCACTGATGCCACCCAAGTGTTGG
CCGAGGTGGAAGAGGCGAAGAAGGCATACCCACAGGCCTGGATCCGTATT
ATTGGATTCGACAACGTGCGTCAAGTGCAGTGCATCAGTTTCATTGCCTA
CAAGCCAGAAGGCTACTAAGTTTCATATTAGGACAACTTACCCTATTGTC
CGACTTTAGGGGCAATTTGTTTGAAATGTTACTTGGCTTCTTTTTTTTTT
AATTTTCCCACAAAAACTGTTTATGTTTCCTACTTTCTATTCGGTGTATG
TTTTTGCATTCCTACCAAGTTATGAGACCTAATAACTATGATTTGGTGCT TTGTTTGTAAAT
rbcS3(Genbank accessions: CN746374: 22-108 b, CN748757: 156-175 b,
CN748929: 158-309 b, CN748913: 319-489 b, CN748777: 485-603 b,
CN748188: 453-529 b) (SEQ ID NO: 126)
TAGCAATAGCTTTAAGCTTAGAAATTATTTTCAGAAATGGCTTCCTCAGT
TATGTCCTCAGCAGCTGCTGTTGCGACCGGCGCCAATGCTGCTCAAGCCA
ACATGGTTGCACCCTTCACTGGCCTCAAGTCCGCCTCCTCCTTCCCTGTT
ACCAGGAAACAAAACCTTGACATTACCTCCATTGCTAGCAATGGTGGAAG
AGTTCAATGCATGCAGGTGTGGCCACCAATTAACATGAAGAAGTACGAGA
CACTCTCATACCTTCCTGATTTGAGCCAGGAGCAATTGCTTAGTGAAGTT
GAGTACCTTTTGAAAAATGGATGGGTTCCTTGCTTGGAATTCGAGACTGA
GCGTGGATTCGTCTACCGTGAACACCACAACTCACCAGGATACTACGATG
GCAGATACTGGACCATGTGGAAGTTGCCCATGTTCGGGTGCACTGATGCC
ACTCAGGTGTTGGCTGAGGTCGAGGAGGCAAAGAAGGCTTACCCACAAGC
CTGGGTTAGAATCATTGGATTCGACAACGTCCGTCAAGTGCAATGCATCA
GTTTTATCGCCTCCAAGCCAGAAGGCTACTAAAATCTCCATTTTTAAGGC
AACTTATCGTATGTGTTCCCCGGAGAAACTGTTTTGGTTTTCCTGCTTCC
TTATATTATTCAATGTATGTTTTTGAATTCCAA rbcS4 (Genbank accessions
CN748906: 9-607 b, CN747257: 629-709b) (SEQ ID NO: 127)
AATGGCTTCCTCAGTTATGTCCTCAGCTGCCGCTGTTGCCACCGGCGCCA
ATGCTGCTCAAGCCAGTATGGTTGCACCTTTCACTGGCCTCAAGTCCGCA
ACCTCCTTCCCTGTTTCCAGAAAACAAAACCTTGACATTACTTCCATTGC
TAGCAACGGCGGAAGAGTTCAATGCATGCAGGTGTGGCCACCAATTAACA
AGAAGAAGTACGAGACACTCTCATACCTTCCCGATTTGAGCCAGGAGCAA
TTGCTTAGTGAAGTTGAGTACCTGTTGAAAAATGGATGGGTTCCTTGCTT
GGAATTCGAGACTGAGCGTGGATTCGTCTACCGTGAACACCACAGCTCAC
CAGGATATTATGATGGCAGATACTGGACCATGTGGAAGTTGCCCATGTTC
GGGTGCACTGATGCCACTCAGGTGTTGGCTGAGGTCGAGGAGGCAAAGAA
GGCTTACCCACAAGCCTGGGTTAGAATCATTGGATTCGACAATGTCCGTC
AAGTGCAATGCATCAGTTTCATCGCCTACAAGCCAGAAGGCTACTAGAAT
CTCCATTTTTAAGGCAACTTATCGTATGTGTTCCCCGGAGAAACTGTTTT
GGTTTTTCCTGCTTCATTATATTATTCAATGTATGTTTTTGAATTCCAAT
CAAGGTTATGAGAACTAATAATGACATTTAATTTGTTTCTTTTCTATATA rbcS5 (Genbank
accession: CN744712: 16-713 b) (SEQ ID NO: 128)
TAAATAATTAATTGCAACAATGGCTTCCTCTGTGATTTCCTCAGCTGCTG
CCGTTGCCACCGGCGCTAATGCTGCTCAAGCCAGCATGGTTGCACCCTTC
ACTGGCCTCAAATCTGCTTCCTCCTTCCCTGTTACCAGAAAACAAAACCT
TGACATTACATCCATTGCTAGCAATGGTGGAAGAGTCCAATGCATGCAGG
TGTGGCCACCAATTAACATGAAGAAGTACGAGACACTCTCATACCTTCCT
GATTTGAGCCAGGAGCAATTGCTTAGTGAAGTTGAGTATCTTTTGAAAAA
TGGATGGGTTCCTTGCTTGGAATTCGAGACTGAGCGTGGATTTGTCTACC
GTGAACATCACAGCTCACCAGGATACTACGATGGCAGATACTGGACCATG
TGGAAGTTGCCCATGTTCGGGTGCACTGATGCCACTCAGGTGTTGGCTGA
GGTCGAGGAGGCAAAGAAGGCTTACCCACAAGCCTGGGTTAGAATCATTG
GATTCGACAACGTCCGTCAAGTGCAATGCATCAGTTTTATCGCCTCCAAG
CCAGAAGGCTACTAAAATCTCCATTTTTAAGGCAACTTATCGTATGTGTT
CCCCGGAGAAACTGTTTTGGTTTTCCTGCTTCATTATATTATTCAATGTA
TGTTTTTGAATTCCAATCAAGGTTATGAGAACTAATAATGACATTTAA rbcS6 (Genbank
accessions: CN745030: 14-123 b, CN748077: 1-523 b) (SEQ ID NO: 129)
GCACGAGGCTTCCTCAGTTATGTCCTCAGCTGCCGCTGTTTCCACCGGCG
CCAATGCTGTTCAAGCCAGCATGGTCGCACCCTTCACTGGCCTCAAGGCC
GCCTCCTCCTTCCCGGTTTCCAGGAAACAAAACCTTGACATTACTTCCAT
TGCTAGAAATGGTGGAAGAGTCCAATGCATGCAGGTGTGGCCGCCAATTA
ACAAGAAGAAGTACGAGACACTCTCATACCTTCCTGATTTGAGCGTGGAG
CAATTGCTTAGCGAAATTGAGTACCTTTTGAAAAATGGATGGGTTCCTTG
CTTGGAATTCGAGACTGAGCATGGATTCGTCTACCGTGAACACCACCACT
CACCAGGATACTACGATGGCAGATACTGGACGATGTGGAAGTTGCCCATG
TTCGGGTGCACCGATGCCACTCAGGTCTTGGCTGAGGTAGAGGAGGCCAA
GAAGGCTTACCCACAAGCCTGGGTCAGAATCATTGGATTCGACAACGTCC
GTCAAGTGCAATGCATCAGTTTCATCGCCTACAAGCCCGAAGGCTATTAA
AATCTCCATTTTTAGGACAGCTTACCCTATGTATTCAGGGGAAGTTTGTT
TGAATTCTCCTGGAGAAACTGTTTTGGTTTTCCTTTGTTTTAATCTTCTT
TCTATTATATTTTTGGATTTTACTCAAGTTTATAAGAACTAATAATAATC
ATTTGTTTCGTTACTAAAAAAAAAAAA
[0302] Infiltration of N. benthamiana with Agrobacterium
tumefaciens
[0303] Infiltration with A. tumefaciens carrying appropriate
plasmids was carried out as follows. Cells were grown to
exponential phase in the presence of appropriate antibiotics and 40
.mu.M acetosyringone. They were harvested by centrifugation,
resuspended in 10 mM MgCl.sub.2 containing 150 .mu.M acetosyringone
and incubated at room temperature for 2 hrs without agitation.
Infiltration was performed by using a syringe without needle
applied to the abaxial side of leaves. After 1, 2, or 3 days leaf
tissue was collected, frozen and ground in liquid nitrogen before
RNA extraction.
[0304] Northern Blot Hybridizations
[0305] Leaves from Nicotiana benthamiana were used to extract total
RNA using the Trizol reagent (Invitrogen). 10-20 .mu.g total RNA
were resolved in a 15% polyacrylamide/1.times.TBE (8.9 mM Tris, 8.9
mM Boric Acid, 20 mM EDTA)/8 M urea gel and blotted to a
Hybond-N+membrane (Amersham). DNA oligonucleotides with the exact
reverse-complementary sequence to miRNAs were end-labeled with
.sup.32P-.gamma.-ATP and T4 polynucleotide kinase (New England
Biolabs) to generate high specific activity probes. Hybridization
was carried out using the ULTRAHyb-Oligo solution according to the
manufacturer's directions (Ambion, Tex.), and signals were detected
by autoradiography. In each case, the probe contained the exact
antisense sequence of the expected miRNA to be detected.
[0306] Northern blot hybridizations to detect PDS mRNA abundance
were performed according to standard procedures. The 5' end probe
corresponded to a fragment of N. benthamiana PDS gene reported
before (Guo et al. (2003) Plant J 34:383-392) equivalent to the
tomato PDS gene sequence positions 1-268 (Genbank X59948, see
above). The 3'end probe corresponded to a fragment obtained by
5'RACE and equivalent to the tomato PDS gene sequence positions
1192-1514.
[0307] 5' RACE
[0308] To identify the products of miRNA-directed cleavage the
First Choice RLM-RACE Kit (Ambion) was used in 5' RACE experiments,
except that total RNA (2 .mu.g) was used for direct ligation to the
RNA adapter without further processing of the RNA sample.
Subsequent steps were according to the manufacturer's directions.
Oligonucleotide sequences for nested PCR amplification of PDS
cleavage fragment were:
TABLE-US-00016 3'Nb-PDS1 (SEQ ID NO: 130) 5'
CCACTCTTCTGCAGGTGCAAAAACC 3' 3'Nb-PDS2 (SEQ ID NO: 131) 5'
ACATGGTACTTCAATATTTTTGCTTTGC 3' 3'Nb-PDS3 (SEQ ID NO: 132) 5'
GATCTTTGTAAAGGCCGACAGGGTTCAC 3'
[0309] All three primers were designed based on available sequence
information for the tomato PDS gene since the complete N.
bethamiana PDS gene sequence has not been published.
[0310] PCR fragments obtained from 5' RACE experiments were cloned
in the pCR4 vector (Invitrogen) and analyzed by DNA sequencing of
individual clones.
[0311] RT-PCR
[0312] First strand cDNA was synthesized form 5 .mu.g total RNA
using an oligo-dT primer (Sigma) and Ready-To-Go You-Prime
First-strand beads (Amersham Biosciences). Amounts of first strand
cDNA were normalized by PCR using primers for EF1.alpha. (Nishihama
et al. (2002) Cell 109:87-99). To amplify DNA fragments of rbcS
cDNAs, the following primers were used.
TABLE-US-00017 NBrbcs5: 1/2-F: (SEQ ID NO: 133) 5'
TTCCTCAGTTCTTTCCTCAGCAGCAGTTG 3' rbcS3-F: (SEQ ID NO: 134) 5'
CTCAGTTATGTCCTCAGCAGCTGC 3' rbcS4/6-F: (SEQ ID NO: 135) 5'
TCCTCAGTTATGTCCTCAGCTGCC 3' NBrbcS5-F: (SEQ ID NO: 136) 5'
TGTGATTTCCTCAGCTGCTGCC 3' NBrbcs1 rev2: (SEQ ID NO: 137) 5'
AACTCAGTAGCCGTCAGGCTTGG 3' NBrbcs2 rev2: (SEQ ID NO: 138) 5'
AATATGAAACTTAGTAGCCTTCTGGCTTGT 3' NBrbcs3/4/5 rev1: (SEQ ID NO:
139) 5' GTTTCTCCGGGGAACACATACGA 3' NBrbcs6 rev1: (SEQ ID NO: 140)
5' AAACAAACTTCCCCTGAATACATAGGG 3'
Example 17
[0313] This example describes the design of an artificial microRNA
to cleave the phytoene desaturase (PDS) mRNAs of Nicotiana
benthamiana.
[0314] Arabidopsis miRNAs identified so far have been shown to
target different mRNAs, and a significant number encodes
transcription factors (Bartel (2004) Cell 116:281-297; Wang et al.
(2004) Genome Biol 5:R65; Rhoades et al. (2002) Cell 110:513-520).
Base-pairing of plant miRNAs to their target mRNAs is almost
perfect and results in cleavage of the RNA molecule as has been
shown for several examples (Jones-Rhoades and Bartel (2004) Mol
Cell 14:787-799), resulting in silencing of gene expression.
Alternatively, miRNA interaction with the target mRNA can result in
inhibition of translation rather than mRNA cleavage as shown for
miR172 of Arabidopsis (Aukerman and Sakai (2003) Plant Cell
15:2730-2741; Chen (2004) Science 303:2022-2025).
[0315] In an effort to design artificial miRNAs that can inhibit
the expression of particular genes, we sought to modify the
sequence of a known miRNA to target an mRNA of choice.
[0316] The Arabidopsis miR159 has been shown to target a set of MYB
transcription factors. Base-pairing of miR159 to its target mRNAs
is almost perfect and results in cleavage of the RNA molecule
(Achard et al. (2004) Development 131:3357-3365; Palatnik et al.
(2003) Nature 425:257-263). There are three genomic sequences
(MIR159a, MIR159b, MIR159c) with the potential to encode miR159.
The natural promoter and precise precursor sequence of miR159 are
not known, nor is it known whether microRNA genes are transcribed
by DNA polymerase II or III. We decided to use as precursor
sequence a DNA fragment of 276 by that contains the Arabidopsis
miR159a. This precursor sequence, which is called pre-miR159a was
placed downstream of a 35S promoter and flanked at the 3' end by a
polyA addition sequence of the nopaline synthase gene (FIG. 11A).
We decided to use the N. benthamiana phytoene desaturase (PDS) gene
as a target to see whether we can design an artificial microRNA to
cleave its mRNA and thereby compromise its expression. We compared
the sequence of At-miR159a to that of PDS to find the best match
between the two sequences. For one particular region of the PDS
mRNA we found that only 6 base changes are sufficient to convert
miR159a into a miRNA capable to perfectly base-pair to PDS mRNA
(FIG. 11B). We called this sequence miR-PDS.sup.159a.
[0317] To generate pre-miR-PDS.sup.159a, PCR techniques were used
to introduce point mutations in both miR159a and the miR159a*
sequence (the RNA sequence located in the opposite arm to the miRNA
within the precursor sequence) in the context of the Arabidopsis
pre-miR159a. The resulting precursor was placed under the control
of the strong cauliflower mosaic virus (CaMV) 35S promoter and
expressed in N. benthamiana by infiltration of Agrobacterium
tumefaciens containing the appropriate constructs.
[0318] Expression of the Arabidopsis pre-miR-PDS.sup.159a in N.
benthamiana was first analyzed to confirm that the mutations
introduced in its sequence did not affect its processing and
maturation of miR-PDS.sup.159a. Northern blot analysis showed that
2 to 3 days after infiltration miR-PDS.sup.159a is clearly
expressed (FIG. 11C), accumulating to levels comparable to
endogenous miR159. Biogenesis of known miRNAs includes the
generation of the almost complementary miRNA* which is short-lived
and accumulates to very low levels when compared to those of the
actual miRNA. Consistently, the presence of miR-PDS.sup.159a* was
detected but its abundance was significantly lower than that of
miR-PDS.sup.159a (FIG. 11C, middle panel). Expression of endogenous
miR159 was unchanged under these conditions and served as both a
loading and probe-specificity control (FIG. 11B, bottom panel). In
addition, this result indicates that expression of an artificial
miRNA based on the Arabidopsis miRNA precursor does not affect
expression of the endogenous N. benthamiana miR159. Finally, these
findings imply that the enzymatic machinery for processing of
natural microRNA precursors is not rate limiting and can process
artificial precursors with great efficiency.
[0319] We next determined whether expression of miR-PDS.sup.159a
resulted in the expected cleavage of the endogenous PDS mRNA.
Northern blot hybridization of the samples expressing
miR-PDS.sup.159a showed a clear reduction in PDS mRNA levels (FIG.
12A). To further establish the mechanism of PDS mRNA reduction we
set to define: (1) whether the PDS mRNA is cleaved by
miR-PDS.sup.159a and contains a diagnostic 5' phosphate, and (2)
whether the cleavage point corresponds to the predicted site, based
on the PDS mRNA:miR-PDS.sup.159a base-pairing interaction. To this
end, 5'RACE experiments were performed. We found that the 5'-end
sequence of 5 out of 6 independent clones mapped the site of
cleavage after the tenth nucleotide counting from the 5' end of
miR-PDS.sup.159a. The location of the cleavage site correlates
perfectly with published work with other miRNA targets
(Jones-Rhoades and Bartel (2004) Mol Cell 14:787-799).
[0320] The results demonstrate that the reduction of PDS mRNA
levels was caused by accurate cleavage directed by
miR-PDS.sup.159a.
Example 18
[0321] This example demonstrates that microRNA-directed cleavage of
PDS mRNA can be produced from a different microRNA precursor.
[0322] To show that expression of artificial miRNAs is not
restricted to the use of pre-miR159a we have designed a different
miR-PDS based on a putative precursor sequence containing the
Arabidopsis miR169g to generate two different miR-PDS.sup.169g
(FIG. 13A). During the design of the expression vector for miR169g,
we noticed that a construct containing only the stem-loop precursor
of 222 by resulted in higher accumulation of the mature miRNA than
a construct containing the entire 2.0 kb intergenic region
including the miR169g gene (FIG. 13B). Based on this result we
decided to continue our mutagenesis of miRNA sequences using
exclusively short precursor vectors. Examination of the PDS mRNA
with the miR169g sequence revealed a region in the mRNA sequence
susceptible for miRNA cleavage, different from that found for
miR-PDS.sup.159a. Seven point mutations turn miR169g into a
microRNA capable of base-pairing perfectly to the PDS mRNA
(miR-PDSa.sup.169g, FIG. 13A). As shown before for the
miR159a-based miR-PDS, transient expression of miR-PDSa.sup.169g in
N. benthamiana is easily detected (FIG. 13C). In addition, to test
whether the entire miRNA can be changed independently of its
original sequence, we have generated miR-PDSb.sup.169g (FIG. 13A
and FIG. 13C), which targets a different region in the PDS mRNA
selected irrespective of its homology to the original miR169g.
Using both miR-PDSa.sup.169g and miR-PDSb.sup.169g we could detect
cleavage of the PDS mRNA, as determined by 5'RACE analysis (FIG.
13D) and a reduction in PDS mRNA levels as determined by Northern
blot analysis (FIG. 13E).
[0323] These results show that a different miRNA precursor can be
used to target degradation of PDS mRNA and importantly, that the
sequence of the original miRNA can be extensively changed to design
an artificial one.
Example 19
[0324] This example demonstrates microRNA-directed specific
cleavage of Nicotiana benthamiana rbcS mRNAs.
[0325] To show that this approach can be used to target other genes
different from PDS, we have introduced point mutations in miR159a
to target the different members of the Rubisco small subunit (rbcS)
gene family of N. benthamiana. We searched for rbcS EST transcripts
present in publicly available databases and found that at least 6
different rbcS transcripts are expressed in N. benthamiana.
Nucleotide sequences of the coding region of these rbcS transcripts
were over 90% identical to each other, and allowed us to design
miR-rbsS.sup.159a-A, which targets all members of the gene family.
Here, the sequence introduced in miR159a was not guided by the
minimal number of changes that would target rbsS but reflected the
need to target a specific region common to all rbsS mRNAs and thus
included several changes. In this way, we have generated one miRNA
that targets all six rbcS mRNAs (miR-rbcS.sup.159a-A, FIG.
14A).
[0326] As in the previous examples, we have detected efficient
expression of the miR-rbsS.sup.159a-A (FIG. 14B), but due to the
high degree of homology among the members of this family, distinct
rbsS mRNAs have been difficult to detect by Northern blot analysis.
Instead, we have used semi-quantitative RT-PCR to determine the
levels of mRNAs in plants infiltrated with Agrobacterium strains
containing the miR-rbcS.sup.159a-A construct. Compared to leaves
infiltrated with the empty binary vector (C in FIG. 14C), mRNA
accumulation for rbcS genes 1, 2 and 3 was reduced while for rbcS
genes 4, 5 and 6 it could not be detected in samples infiltrated
with a miR-rbcS.sup.159a-A construct (A in FIG. 14C). These results
indicate that the artificial miRNA targeted all the rbcS mRNAs it
was intended for, although the efficiency in each case varied.
Finally, the presence of the artificial miRNA did not interfere
with expression of other plant genes such as EF1a (FIG. 14C, bottom
panel).
[0327] The artificial miRNAs presented here are distributed along
three different locations in PDS mRNA (summarized in FIG. 15A), and
have been used to target 2 different genes (PDS and rbcS, FIG. 15A
and FIG. 15B). This range of use is also reflected in the
flexibility of the miRNA sequences, as the artificial miRNAs show
that almost every nucleotide position can be changed (FIG. 16).
Changes in miR159a to create two artificial miRNAs retained only 8
positions unchanged (FIG. 16A). In the case of miR169g this number
was reduced to only three positions (FIG. 16B). Moreover, when the
mutations in both miRNAs are analyzed together, only the first two
nucleotide positions remain untouched. This suggests that every
position along the miRNA sequence can be changed, adding to the
advantages of using artificial miRNAs for gene silencing.
Example 20
[0328] This example demonstrates that artificial miRNACPC.sup.159a
inhibits root hair development in Arabidopsis.
[0329] Root epidermal cells differentiate root-hair cells and
hairless cells. Only root-hair epidermal cells are able to develop
into root hair. In Arabidopsis roots, among a total of 16-22 cell
files, 8 symmetrically positioned cell files are root-hair cells
and all others are hairless cell files. CAPRICE (CPC), a MYB like
protein, positively regulates root hair development by negatively
regulating GLABRA2 (GL2), which promotes root epidermal cells
differentiation into hairless cells. In cpc mutant, GL2 causes most
epidermal cells to differentiate into root hairless cells, and
consequently, very few cells are able develop root hair. Roots of
the g12 mutant or wild type transgenic plants over-expressing CPC,
produce more root hairs compared to wild type roots (FIG. 17; Wada
et al. (2002) Development 129:5409-5419).
[0330] CPC is a good candidate for investigations on the utility of
artificial miRNAs to silence or suppress gene function because the
loss-of-function phenotype of CPC appears at a very early stage
during seedling development, does not cause lethality and is easy
to observe. Using pre-miRNA159 as a backbone two artificial
pre-miRNAs, pre-miRCPC1.sup.159a and pre-miRCPC3.sup.159a were
designed to target different regions of the CPC mRNA. Mature
miRCPC1.sup.159a and miRCPC3.sup.159a are complementary to the
sequences located in nt 233-253 and nt 310-330, respectively, of
the CPC messenger RNA. The nucleotide sequences for the precursor
and mature miRNAs are as follows.
TABLE-US-00018 miRCPC1.sup.159a precursor template: (SEQ ID NO:
151) 5' acagtttgcttatgtcggatccataatatatttgacaagatactttg
tttttcgatagatcttgatctgacgatggaagaagaggtgagtaatgttg
aaacatgagttgagcagggtaaagaaaagctgctaagctatggatcccat
aagccctaatccttgtaaagtaaaaaaggatttggttatatggattgcat
atctcaggagctttaacttgccctttaatggcttttactcttctttcgat
actactcacctcttcatcccgggtca 3'. miRCPC1.sup.159a mature template:
(SEQ ID NO: 152) 5'tttcgatactactcacctctt 3'. miRCPC3.sup.159a
precursor template: (SEQ ID NO: 153) 5'
acagtttgcttatgtcggatccataatatatttgacaagatactttg
tttttcgatagatcttgatctgacgatggaagctcgttggcgacaggtgg
gagcatgagttgagcagggtaaagaaaagctgctaagctatggatcccat
aagccctaatccttgtaaagtaaaaaaggatttggttatatggattgcat
atctcaggagctttaacttgccctttaatggcttttactcttcctcccac
ctgacgccaacgagcatcccgggtca 3'. miRCPC3.sup.159a mature template:
(SEQ ID NO: 154) 5' ctcccacctgacgccaacgag 3'.
[0331] These two artificial pre-miRNAs were cloned into a vector
which contains a constitutive 35S promoter for expression of these
precursors. Northern blot analysis of Nicotinana benthamiana leaves
infiltrated by Agrobacteria carrying 35S::per-miRCPC1.sup.159a or
35S:: pre-miRCPC3.sup.159a constructs indicated successful
production of mature miRCPC1.sup.159a and miRCPC3.sup.159a.
[0332] Arabidopsis thaliana plants were transformed by Agrobacteria
carrying XVE::pre-miRCPC1.sup.159a or 35S::pre-miRCPC1.sup.159a,
and many transgenic lines were obtained. T.sub.1 seeds of
XVE::pre-miRCPC1.sup.159a plants were geminated on antibiotic
selection medium containing kanamycin and resistant transgenic
seedlings were transferred to MS medium with or without
.beta.-estradiol, an inducer of the XVE system. T.sub.1 transgenic
lines carrying XVE::pre-miR159 were used as a control. Pre-miR159
is the backbone used to construct the artificial
pre-miRCPC1.sup.159a.
[0333] No difference in root hair development between
XVE::pre-miR159 seedlings grown on medium with or without inducer
(FIG. 18, panels c and d) was seen. By contrast,
XVE::pre-miRCPC1.sup.159a seedlings grown on medium with
.beta.-estradiol clearly developed fewer root hairs (FIG. 18, panel
b) than those grown without inducer (FIG. 18, panel a).
[0334] T.sub.1 seedlings of transgenic Arabidopsis seedlings
carrying 35S::pre-miRCPC1.sup.159a, 35S::pre-miR159 and
35S::pre-miRP69.sup.159a were investigated and similar results were
obtained as the XVE inducible lines. T.sub.1 seeds of transgenic
lines were geminated on a BASTA-selective medium and two-week old
seedlings were transferred to MS medium plates placed vertically in
a tissue culture room. In this experiment, two negative controls
were used: transgenic lines carrying 35S::pre-miR159 and those
carrying 35S::pre-miRP69.sup.159a. The latter was designed using
pre-miR159 as a backbone to produce an artificial
pre-miRP69.sup.159a targeting nt 214-234 of the P69 mRNA of turnip
yellow mosaic virus (TYMV; Bozarth et al. (1992) Virology
187:124-130). The nucleotide sequences for the precursor and mature
miRNAs are as follows.
TABLE-US-00019 miRP69.sup.159a precursor template: (SEQ ID NO: 155)
5' acagtttgcttatgtcggatccataatatatttgacaagatactttg
tttttcgatagatcttgatctgacgatggaagccacaagacaatcgagac
tttcatgagttgagcagggtaaagaaaagctgctaagctatggatcccat
aagccctaatccttgtaaagtaaaaaaggatttggttatatggattgcat
atctcaggagctttaacttgccctttaatggcttttactcttcaaagtct
cgattgtcttgtggcatcccgggtca 3' miRP69.sup.159a mature template: (SEQ
ID NO: 156) 5' aaagtctcgattgtcttgtgg 3'.
[0335] Seedlings of both types of transgenic plants developed
abundant root hair as wild type plants (FIG. 19, panels a and c).
By contrast, among 30 independent 35S::pre-miRCPC1.sup.159a lines,
18 lines showed clearly fewer root hair (FIG. 19, panel b) compared
to negative control plants (FIG. 19 panels a and c).
[0336] In negative control transgenic plants (35S::pre-miR159 and
pre-miRP69.sup.159a), all root-hair file cells in the epidermis of
the root tip region were able to develop root hairs (FIG. 20, panel
a; see arrows). However, in transgenic lines carrying
35S::pre-miRCPC1.sup.159a many cells in root-hair files were unable
to produce root hairs (FIG. 20, panel b; see arrows). These results
indicate that the artificial miRCPCl.sup.159a is able to induce
cleavage of the endogenous CPC mRNA to cause a loss function of the
CPC gene function and inhibit root hair development.
Example 21
[0337] This example describes one embodiment of a process for the
designing a polymeric pre-miRNA.
[0338] Step 1: Different pre-miRNAs are amplified by PCR to include
an AvrII site in the 5' end and to include an SpeI site and an XhoI
site in the 3' end Each pre-miRNA is then cloned into a vector,
such as pENTR/SD/D-TOPO (Invitrogen) to produce, for example,
pENTR/pre-miRA, pENTR/pre-miRB and pENTR/pre-miRC (FIG. 21A).
[0339] Step 2: The pENTR/pre-miRA is digested with the restriction
enzymes SpeI and XhoI. The restriction enzymes AvrII and XhoI are
used to digest the pENTR/pre-miRB vector (FIG. 21B). Opened vector
pENTR/pre-miRA and DNA fragment of pre-miRB are collected and
purified for further steps.
[0340] Step 3: The opened vector pENTR/pre-miRA and DNA fragment of
pre-miRB from step 2 are ligated to generate dimeric pre-miRA-B
(FIG. 21C). Because of compatible cohesive ends of AvrII and SpeI,
the pre-miRB fragment can be inserted into the opened
pENTR/pre-miRA and both AvrII and SpeI sites will disappear after
ligation (FIG. 21C).
[0341] Step 4: The pENTR/pre-miRA-B is digested by with the
restriction enzymes SpeI and XhoI, and pENTR/pre-miRC is digested
with the restriction enzymes AvrII and XhoI (FIG. 21D). Opened
vector pENTR/pre-miRA-B and DNA fragment of pre-miRC are collected
and purified for further steps.
[0342] Step 5: The opened vector pENTR/pre-miRA-B and DNA fragment
of pre-miRC from step 4 are ligated to generated triple
pre-miRNA-B-C (FIG. 21E).
[0343] In this manner, or using functionally equivalent restriction
enzymes polymeric pre-miRNAs containing more pre-miRNA units can be
prepared. As many pre-miRNAs as desired can be linked together in
this fashion, with the only limitation being the ultimate size of
the transcript. It is well known that transcripts of 8-10 kb can be
produced in plants. Thus, it is possible to form a multimeric
pre-miRNA molecule containing from 2-30 or more, for example from
3-40 or more, for example from 3-45 and more, and for further
example, multimers of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,
33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,
or more pre-miRNAs.
Example 22
[0344] This example demonstrates the successful processing of a
dimeric pre-iRNA to two mature miRNAs.
[0345] Artificial pre-miRPDS1.sup.169g and pre-miRCPC3.sup.159a
were linked to form dimeric precursor,
pre-miRPDS.sup.169g-CPC3.sup.159a as described in Example 21. This
dimeric miRNA precursor was cloned into a vector in which 35S
promoter drives expression of the
pre-miRPDS1.sup.169g-CPC3.sup.159a (FIG. 22). The nucleotide
sequences for the precursor and mature miRNAs are as follows.
TABLE-US-00020 miRPDS1.sup.169g precursor template: (SEQ ID NO:
157) 5' aatgatgattacgatgatgagagtctctagttgtatcagagggtctt
gcatggaagaatagagaatgaggttgagtttagtctgacttggccagttt
ttttaccaatgaatctaattaactgattctggtgttggccaagtcagact
aaactctgtttccttctcttcttttggatgtcagactccaagatatctat
catcatgaatcgtgatcaaactttg 3'. miRPDS1.sup.169g mature template:
(SEQ ID NO: 158) 5' gagtttagtctgacttggcca 3'.
miRPDS1.sup.169g-CPC3.sup.159a precursor template: (SEQ ID NO: 159)
5' cacctaggaatgatgattacgatgatgagagtctctagttgtatcag
agggtcttgcatggaagaatagagaatgaggttgagtttagtctgacttg
gccagtttttttaccaatgaatctaattaactgattctggtgttggccaa
gtcagactaaactctgtttccttctcttcttttggatgtcagactccaag
atatctatcatcatgaatcgtgatcaaactttgaagggtgggcgactagg
acagtttgcttatgtcggatccataatatatttgacaagatactttgttt
ttcgatagatcttgatctgacgatggaagctcgttggcgacaggtgggag
catgagttgagcagggtaaagaaaagctgctaagctatggatcccataag
ccctaatccttgtaaagtaaaaaaggatttggttatatggattgcatatc
tcaggagctttaacttgccctttaatggcttttactcttcctcccacctg
acgccaacgagcatcccgggtcaaagggtgggcgactagtctagactcga gtatt 3'.
[0346] Northern blotting analysis of tobacco Nicotiana benthamiana
levies, infiltrated by Agrobacteria carrying different constructs
of 35S::pre-miRPDS1.sup.169g, 35S::pre-miRCPC3.sup.159a and
35S::pre-miRPDS.sup.169g-CPC3.sup.159a, indicates that mature
miRPDS.sup.169g and CPC3.sup.159a were successfully produced from
the dimeric miRNA precursor (FIGS. 23A and 23B). In this
experiment, treatment 1 is 35S::pre-miRPDS1.sup.169g, treatment 2
is 35S::miRCPC3.sup.159a and treatment 3 is 35S::
pre-miRPDS.sup.169g-CPC3.sup.159a. When miRPDS1.sup.169g anti sense
DNA oligo as probe, both land 3 treatments showed signals that
proved the dimeric precursor was able to produce matured
miRPDS1.sup.169g. When the probe is miRCPC3.sup.159a anti sense DNA
oligo, signal in treatment 3 confirmed the ability of
pre-miRPDS.sup.169g-CPC3.sup.159a to generate mature
miRCPC3.sup.159a.
Example 23
Design of Anti-viral miRNAs
[0347] Since viral gene silencing suppressors are used to
counteract host defense, we reasoned that compromising the
production of these suppressors by the expression of specific
miRNAs would be an effective mechanism to confer resistance or
tolerance to plant viruses (Roth et al. (2004) Virus Res
102:97-108). This principle is demonstrated by using TuMV as an
example.
[0348] HC-Pro and P69 are plant PTGS suppressors encoded by TuMV
and TYMV, respectively (Anandalakshmi et al. (1998) Proc Natl Acad
Sci USA 95:13079-13084; Chen et al. (2004) Plant Cell 16:1302-1313;
Kasschau and Carrington (1998) Cell 95:461-470). Using these two
viral suppressor genes as targets, artificial miRNAs were designed
with sequence complementarity to their coding sequences. The coding
sequence for HC-Pro is SEQ ID NO:188, and the coding sequence for
P69 is SEQ ID NO:189.
[0349] At-miR159a is strongly expressed in most Arabidopsis organs
and at high levels. Similar high level expression was also found in
other plants species such as corn and tobacco. For these reasons,
the miR159a precursor (pre-miR159a) was used as a backbone to
generate artificial miRNAs. Pre-miR159a, a 184nt stem-loop RNA,
produces mature miR159a (5'-uuuggauugaagggagcucua-3'; SEQ ID
NO:160) from the base of its stem near the 3'end. This base stem
sequence is the miR159a sequence and the complementary strand is
called miR159a* sequence (FIG. 24; SEQ ID NO:161). To design
artificial miRNA, the miR159a sequence was replaced by a sequence
5'-acuugcucacgcacucgacug-3' (SEQ ID NO:162), which is complementary
to the viral sequence encoding HC-P from 2045 to 2065 of the TuMV
genome sequence. The miR159a* sequence was also altered to maintain
the stem structure. For more efficient miRNA processing and
convenient manipulation of the artificial miRNA precursor, a 78bp
sequence cloned from the genome sequence upstream of pre-miR159 was
added to the 5'end of this artificial miRNA precursor. This primary
miRNA-like artificial miRNA precursor was called
pre-miRHC-P.sup.159a. Its DNA sequence follows.
TABLE-US-00021 Pre-miRHC-P.sup.159a (SEQ ID NO: 163) 5'
CAGTTTGCTTATGTCGGATCCATAATATATTTGACAAGATACTTTGT
TTTTCGATAGATCTTGATCTGACGATGGAAGCAGTCGAGTGCGTGAGCAA
GTCATGAGTTGAGCAGGGTAAAGAAAAGCTGCTAAGCTATGGATCCCATA
AGCCCTAATCCTTGTAAAGTAAAAAAGGATTTGGTTATATGGATTGCATA
TCTCAGGAGCTTTAACTTGCCCTTTAATGGCTTTTACTCTTCACTTGCTC ACGCACTCGACTGC
3'
[0350] Using the same method, pre-miRP69.sup.159a was also
constructed. Pre- miRP69.sup.159a was predicted to generate mature
artificial miRNA P69.sup.159a, 5'-aaagucucgauugucuugugg-3' (SEQ ID
NO:164), to target the P69 gene of TYMV. Its DNA sequence
follows.
TABLE-US-00022 Pre-miR P69.sup.159a (SEQ ID NO: 165) 5'
CAGTTTGCTTATGTCGGATCCATAATATATTTGACAAGATACTTTGT
TTTTCGATAGATCTTGATCTGACGATGGAAGCCACAAGACAATCGAGACT
TTCATGAGTTGAGCAGGGTAAAGAAAAGCTGCTAAGCTATGGATCCCATA
AGCCCTAATCCTTGTAAAGTAAAAAAGGATTTGGTTATATGGATTGCATA
TCTCAGGAGCTTTAACTTGCCCTTTAATGGCTTTTACTCTTCAAAGTCTC GATTGTCTTGTGGC
3'
Example 24
Expression of pre-miRHC-P.sup.159a and pre-miRP69.sup.159a in
Nicotiana benthamiana
[0351] Replacement of the miR159 and miR159* sequences in the
pre-miR159 may possibly effect RNA folding structure which is
believed to be important for miRNA biosynthesis. A tobacco
transient expression system was used to check whether these two
artificial miRNA precursors can produce the desired miRNAs.
Agrobacterial cells containing plasmids with
35S::pre-miR-HC-Pro.sup.159a, 35::pre-miR-P69.sup.159a,
35S::HC-Pro, 35S::P69, and XVE::pre-miR-P69.sup.159a were used to
infiltrate N. benthamiana leaves (Llave et al. (2000) Proc Natl
Acad Sci USA 97:13401-13406; Voinnet et al. (2000) Cell
103:157-167).
[0352] One ml of stationary phase growth culture of Agrobacteria
tumefaciens carrying different constructs were cultured overnight
in 50 ml LB medium containing 100 mg/l spectinomycin and 50 mg/l
kanamycin and cells were collected by centrifugation at 4,000 rpm
for 10 minutes. Bacterial pellets were re-suspended in 50 ml 10 mM
MgCl.sub.2 solution with 75 .mu.l of 100 mM acetosyringone. After
incubation at room temperature for 3 hr without shaking, the
Agrobacterial suspensions were infiltrated into leaves of N.
bethamiana by a syringe. Two days later, total RNA was extracted
from the infiltrated leaves using the trizol reagent (Invitogen)
and analyzed by northern blot hybridizations (Guo et al. (2005)
Plant Cell 17:1376-1386; Wang et al. (2004) Genome Biol 5:R65).
Samples of 20 .mu.g total RNA were analyzed by electrophoresis on a
15% polyacrylamide gel and blotted to a Hybond-N+ membrane
(Amersham). DNA oligonucleotides with exact complementary sequence
to miR-HC-Pro.sup.159a or pre-miR-P69.sup.159a were end-labeled
with [.gamma.-.sup.32P]-ATP and T4 polynucleotide kinase to
generate high specific activity probe. Hybridization was carried
out using the ULTRA-Hyb Oligo solution according to the
manufacturer's directions (Ambion) and signals were detected by
autoradiography.
[0353] Northern blot analyses of miR-HC-Pro.sup.159a were performed
with three different treatments: (1) Agrobacterial cells with
35S::pre-miR-HC-Pro.sup.159a, (2) Agrobacterial cells with
35S::HC-Pro, and (3) Agrobacterial cells with
35S::pre-miR-HC-Pro.sup.159a and 35S::HC-Pro. The results are shown
in FIG. 25.
[0354] Note that mature miR-HC-Pro.sup.159a signals were detected
in all treatments with 35S::pre-miR-HC-Pro.sup.159a (column 1, 2,
5, 6 of FIG. 25). No signal was detected when leaves were
infiltrated with the 35S::HC-Pro construct only (column 3 and 4 of
FIG. 25). This result indicates that the artificial
pre-miR-HC-Pro.sup.159a can generate mature miR-HC-Pro.sup.159a in
the plant cell.
[0355] In the case of miR-P69, 4 different treatments were
performed: (1) Agrobacterial cells carrying 35S::
pre-miR-P69.sup.159a, (2) Agrobacterial cells carrying
XVE::pre-miR-P69.sup.159a, (3) Agrobacterial cells carrying
35S::P69, and (4) Agrobacterial cells carrying
35S::pre-miR-P69.sup.159a and 35S::P-69. Note that the XVE system
is a transcriptional inducible system responsive to
.beta.-estradiol (Zuo et al. (2001) Nature Biotechnol
19(2):157-61).
[0356] The Northern blot results (FIG. 26) showed that mature
miR-P69.sup.159a was detectable only in leaves infiltrated with
35S::pre-miR-P69.sup.159a and XVE::pre-miR-P69.sup.159a plus
inducer (column 1, 2, 4, 6, 8, and 9 of FIG. 26). Leaves
infiltrated with 35S::P69 and XVE::pre-miRP69.sup.159a without
inducer can not produce miR-P69.sup.159a (column 3, 5, and 7 of
FIG. 26). Together, these results indicate that artificial
pre-miR-P69.sup.159a can be successfully used to generate mature
miR-P69.sup.159a.
Example 25
Stable Arabidopsis Transgenic Lines with High Artificial miRNAs
Expression Levels
[0357] Constructs containing 35S::pre-miR-HC-Pro.sup.159a or
35S::pre-miR-P69.sup.159a was transformed into Arabidopsis Col-0
ecotype mediated by Agrobacteria using the floral dip method
(Clough and Bent (1998) Plant J 16:735-43).
[0358] Transgenic seedlings were selected on selection medium (MS
salts 4.3 g/l+Sucrose 10 g/l+Basta 10 mg/l+Carbenicilin 200
mg/l+Agar 8 g/l). Twelve different 35S::pre-miR-HC-Pro.sup.159a or
35S::pre-miR-P69.sup.159a T.sub.2 transgenic lines were randomly
picked and used to analyze mature artificial miRNA levels by
northern blots. Among 12 transgenic 35S::pre-miRHC-Pro.sup.159a
lines, 11 lines showed high levels of expression of
miRHC-Pro.sup.159a (FIG. 27). In Arabidopsis transgenic
35S::pre-miR-P69.sup.159a plants, all T.sub.2 lines tested showed
miR-P69.sup.159a signals and 10 lines showed high expression levels
(FIG. 28).
Example 26
TuMV Virus Challenge of WT and Transgenic Plants
[0359] Inoculation of WT and transgenic Arabidopsis lines with the
TuMV
[0360] N. benthamiana leaves were inoculated with Turnip mosaic
virus (TuMV) (Chen et al. (2003) Plant Dis 87:901-905) and two
weeks later tissues were extracted in 1:20 (wt/vol) dilution in
0.05 M potassium phosphate buffer (pH 7.0). This extract was used
as a viral inoculum. T.sub.2 plants of 35S::miR-HC-Pro.sup.159a
transgenic Arabidopsis lines were grown in a greenhouse for 4 weeks
(5 to 6 leaves stage) before inoculation. Plants were dusted with
600-mesh Carborundum on the first to fourth leaf and gently rubbed
with 200 .mu.l inoculum. Wild type Arabidopsis thaliana (col-0)
plants and transgenic plants expressing 35S::miR-P69.sup.159a were
used as controls. Inoculated plants were kept in a
temperature-controlled greenhouse (23.degree. C. to 28.degree. C.)
and symptom development was monitored daily for 2 weeks.
[0361] Enzyme-Linked Immunosorbent Assay (ELISA)
[0362] Leaf disks (a total of 0.01 g) from different systemic
leaves of each plant infected with TuMV were taken 14 dpi (days
post infection), and assayed by indirect enzyme-linked
immunosorbent assay (ELISA) using a polyclonal antiserum to TuMV
coat protein (CP) (Chen et al. (2003) Plant Dis 87:901-905) and
goat anti-rabbit immunoglobulin G conjugated with alkaline
phosphatase. The substrate p-nitrophenyl phosphate was used for
color development. Results were recorded by measuring absorbance at
405 nm using Tunable Microplate Reader (VersaMax, Molecular Devices
Co., CA).
[0363] Western Blot Analysis
[0364] Western blot analysis was conducted using the rabbit
antiserum to TuMV CP (Chen et al. (2003) Plant Dis 87:901-905) and
goat anti-rabbit immunoglobulin G conjugated with alkaline
phosphatase. Systemic leaves from Arabidopsis plants were
homogenized in 20 volumes (wt/vol) of denaturation buffer (50 mM
Tris-HCl, pH 6.8, 4% SDS, 2% 2-mercaptoethanol, 10% glycerol, and
0.001% bromophenol blue). Extracts were heated at 100.degree. C.
for 5 min and centrifuged at 8,000.times.g for 3 min to pellet
plant debris. Total protein of each sample (15 .mu.l) was loaded on
12% polyacryamide gels, separated by SDS-polyacrylamide gel
electrophoresis, and subsequently transferred onto PVDF membrane
(immobilon-P, Millipore, Bedford, Mass.) using an electro transfer
apparatus (BioRad). The membranes were incubated with polyclonal
rabbit antiserum to TuMV CP as primary antibodies and
peroxidase-conjugated secondary antibodies (Amersham Biosciences)
before visualization of immunoreactive proteins using ECL kits
(Amersham Biosciences). Gels were stained with coomassie-blue R250
and levels of the large subunit of RUBISCO (55 kd) were used as
loading controls.
[0365] It was found that transgenic plants expressing
miR-HC-Pro.sup.159a artificial miRNA are resistant to TuMV
infection (FIG. 29). Photographs were taken 2 weeks (14 days after
infection) after inoculation. Plants expressing miR-HC-Pro.sup.159a
(line #11; FIG. 33B) developed normal inflorescences whereas WT
plants and transgenic plants expressing miR-P69.sup.159a (line #1;
FIG. 33B) showed viral infection symptoms.
[0366] Fourteen days after TuMV infection, miR-P69.sup.159a (line
#1) and col-0 plants showed shorter internodes between flowers in
inflorescences, whereas miR-HC-Pro.sup.159a transgenic plant (line
#11) displayed normal inflorescences development (FIG. 30, upper
panel). Close-up views of inflorescences on TuMV-infected
Arabidopsis plants. miR-P69.sup.159a (line #1) and col-0 plants
showed senescence and pollination defects whereas
miR-HC-Pro.sup.159a plants (line #11) showed normal flower and
silique development (FIG. 30, bottom panel). For mock-infection,
plants were inoculated with buffer only.
[0367] In TuMV-infected miR-P69.sup.159a (line #1) and WT (col-0)
plants, siliques were small and mal-developed. miR-HC-Pro.sup.159a
plants (line #11) were resistant to TuMV infection and showed
normal silique development (FIG. 31). Buffer-inoculated plants
(mock-inculated) were used as controls.
[0368] Two independent experiments were performed to examine the
resistance of various transgenic miR-HC-Pro.sup.159a and WT plants
to TuMV infection. Experiment 1: Sixteen individual plants of a
T.sub.2 transgenic line (line #11 of miR-HC-Pro.sup.159a plant and
line #1 of miR-P69.sup.159a plant) were used. Twelve individual
plants were inoculated with virus whereas 4 individual plants were
inoculated with buffer as control (MOCK). After 2 weeks system
leaves were collected for western blot analyses using an antibody
against TuMV CP. No TuMV CP was detected in miR-HC-Pro.sup.159a
transgenic plants, whereas, TuMV CP was highly expressed in
miR-P69.sup.159a and WT col-0 plants (FIG. 32). The large subunit
(55 kd) of RUBISCO was used as a loading control. Note that no CP
was detected in lane 6 (top panel) and lane 4 (middle panel) likely
due to failed virus inoculation. These plants had no symptoms. The
results of the infectivity assay are shown in Table 7.
TABLE-US-00023 TABLE 7 Infectivity Assay of Transgenic Arabidopsis
of the miR-HC-Pro.sup.159a and miR-P69.sup.159a Challenged with
TuMV Inocula Number of seedlings Transgenic line Resistant
Susceptible Total Resistant rate (%) miR-HC-Pro.sup.159a #11 12 0
12 100 miR-P69.sup.159a #1 1 11 12 8.3 col-0 1 11 12 8.3
[0369] Experiment 2: The following transgenic lines and WT plants
were used. (1) 35S::miR-HC-Pro.sup.159a plants: line #10 (12 plants
inoculated with TuMV and 4 with buffer;); line #11 (12 plants
inoculated with TuMV and 4 with buffer); line #12 (9 plants
inoculated with TuMV and 4 with buffer); line #13 (10 plants
inoculated with TuMV and 4 with buffer). (2) 35S::miR-P69.sup.159a
plants: line #1 (8 plants inoculated with TuMV and 4 with buffer);
line #2 (7 plants inoculated with TuMV and 4 with buffer); line #3
(9 plants inoculated with TuMV and 4 with buffer); line 7 (5 plants
inoculated with TuMV and 4 with buffer).
[0370] Western blot results of a representative plant from each
transgenic line are shown in FIG. 33, panel A. Levels of the large
subunit (55kd) of RUBISCO were used as loading controls. All plants
expressing 35S::miR-HC-Pro.sup.159a were resistant to the virus and
did not show any visible symptoms nor expressed any TuMV CP. All WT
plants and 35S::miR-P69.sup.159a plants showed TuMV infection
symptoms and expressed high levels of TuMV CP. All mock-infected
plants were normal and did not express any TuMV CP. Expression of
artificial miRNA in miR-HC-Pro.sup.159a and miR-P69.sup.159a
transgenic Arabidopsis is shown in FIG. 33, panel B. The results of
the infectivity assay are shown in Table 8.
TABLE-US-00024 TABLE 8 Infectivity Assay of Transgenic Arabidopsis
of the miR-HC-Pro.sup.159a and miR-P69.sup.159a Challenged with
TuMV Inocula Number of seedlings Transgenic line Resistant
Susceptible Total Resistant rate (%) miR-HC-Pro.sup.159a #10 12 0
12 100 miR-HC-Pro.sup.159a #11 12 0 12 100 miR-HC-Pro.sup.159a #12
9 0 9 100 miR-HC-Pro.sup.159a #13 10 0 10 100 miR-P69.sup.159a #1 0
8 8 0 col-0 1 11 12 8.3
[0371] Fourteen days after infection with TuMV, samples of systemic
leaves were collected and extracts assayed by ELISA. The results
are means of ELISA readings of 9 or 12 plants from two different
experiments. The results (FIG. 34) show that the
miR-HC-Pro.sup.159a plants were completely resistant to TuMV
infection. The readings were taken after 30 min of substrate
hydrolysis.
Example 27
[0372] Production of More Than One Synthetic miRNAs from Same
Transcript Using Homo-Polymeric pre-miRNAs
[0373] Polymeric pre-miRNAs are artificial miRNA precursors
consisting of more than one miRNA precursor units. They can either
be hetero-polymeric with different miRNA precursors, or
homo-polymeric containing several units of the same miRNA
precursor. In previous Examples, it has been demonstrated that
hetero-polymeric pre-miRNAs are able to produce different mature
artificial miRNAs. For example,
pre-miR-PDS1.sup.169g-CPC3.sup.159a, which is a dimer comprising of
pre-miR-CPC3.sup.159a and pre-miR-PDS1.sup.169g can produce mature
miR-PDS1.sup.169g and miR-CPC3.sup.159a when expressed in plant
cells. Here, the use of homo-polymeric miRNA precursors to produce
different mature artificial miRNAs is described.
[0374] Pre-miR-P69.sup.159a and pre-miR-HC-Pro.sup.159a were
generated from the pre-miR159a backbone. They are derived from the
same miRNA precursor. They were linked together to form a
homo-dimeric pre-miRNA, pre-miR-P69.sup.159a-HC-Pro.sup.159a. The
DNA sequence follows.
TABLE-US-00025 Pre-miRP69159a-HC-P.sup.159a (SEQ ID NO: 166) 5'
cagtttgcttatgtcggatccataatatatttgacaagatactttgt
ttttcgatagatcttgatctgacgatggaagccacaagacaatcgagact
ttcatgagttgagcagggtaaagaaaagctgctaagctatggatcccata
agccctaatccttgtaaagtaaaaaaggatttggttatatggattgcata
tctcaggagcttttaacttgccctttaatggcttttactcttc AAAGTCTCGATTGTCTTGTGGc
cagtttgcttatgtcggatccataatatatttgacaagatactttgtttt
tcgatagatcttgatctgacgatggaagcagtcgagtgcgtgagcaagtc
atgagttgagcagggtaaagaaaagctgctaagctatggatcccataagc
cctaatccttgtaaagtaaaaaaggatttggttatatggattgcatatct
caggagctttaacttgccctttaatggcttttactcttcACTTGCTCACG CACTCGACTGc
3'
[0375] The sequences in lower case text are At-miR159 backbone. The
sequence in bold text is miR-P69.sup.159a. The sequence in italic
text is miR-HC-Pro.sup.159a. The sequence in bold italic text is
the linker sequence.
[0376] A tobacco transient expression system was used to check
whether this homo-dimeric miRNA precursor can produce the desired
mature miR-P69.sup.159a and miR-HC-Pro.sup.159a. In this
experiment, three treatments were performed: (1) Agrobacteria with
35S::pre-miR-P69.sup.159a, (2) Agrobacteria with
35S::pre-miR-HC-Pro.sup.159a, and (3) Agrobacteria with
35::pre-miR-P69.sup.159a-HC-Pro.sup.159a. Northern analysis
indicated that homo-dimeric miRNA precursor,
pre-miR-P69.sup.159a-HC-Pro.sup.159a, can produce mature
miR-P69.sup.159a and miR-HC-Pro.sup.159a (FIG. 35).
Example 28
Expression of miRNAs from pre-miRNAs Inserted in Intronic
Sequences
[0377] During RNA splicing, introns are released from primary RNA
transcripts and therefore can potentially serve as precursors for
miRNAs. In this example, the insertion of pre-miRNAs into such
intronic sequences to produce artificial miRNAs is described.
[0378] Most introns begin with the sequence 5'-GU-3' and end with
the sequence 5'-AG-3'. These sequences are referred to as the
splicing donor and splicing acceptor site, respectively. In
addition to these sequences, the branch site which is located
within introns is also important for intron maturation. Without the
branch site, an intron can not be excised and released from the
primary RNA transcript. A branch site is located 20-50 nt upstream
of the splicing acceptor site. Distances between the splice donor
site and the branch site are largely variable among different
introns. For this reason, it was decided to insert artificial
pre-miRNAs in between these two sites, i.e., the splice donor site
and the branch site, of introns.
[0379] The Arabidopsis CARPRICE (CPC) gene contains three exons and
two introns. Following the consensus sequence of the branch site
5'-CU(A/G)A(C/U)-3', where A is conserved in all transcripts, two
branch sites located in 128 to132 nt (intron 1) and 722 to 726 nt
(intron 2) downstream of the start codon are predicted. Sequences
from 111 to 114 nt and from 272 to 697 nt, located in intron 1 and
in intron 2, respectively, were replaced by artificial miRNA
precursors containing the miR159a backbone. The DNA sequence
follows.
TABLE-US-00026 CPC genome sequence (SEQ ID NO: 167)
atgtttcgttcagacaaggcggaaaaaatggataaacgacgacggagaca
gagcaaagccaaggcttcttgttccgaagGTCTGATTTCTCTTTGTTTCT
CTCTATATCTTTTTGATCGGTTTGAGT TTTGTATGTTTGTTTC
GCAGaggtgagtagtatcgaatgggaagctgtgaagatgtcagaagaaga
agaagatctcatttctcggatgtataaactcgttggcgacagGTTAGAGA
CTCTTTCTCTCTCGATCCATCTTGTTGCTTTCTCTTTTTTTTGGTCTTTC
GATTTTTGTCGAATCTGCTTAGATTTTGATCTCAAAGTCGGTCGTTTATT
TATGCATTTTCTTGGTTTTTCTATTATATTATTGGGTCTAACTTACCGAG
CTGTCAATGACTGTGTTCAGCCTGATTTTTGATCTTGTTATTATTCTCTG
TTTTTTGTTTTAGTTGTTCAAATAGCAAAACCTAATCAAGATTTCGTTTT
CAGTTTCTTTTTTTATATATGATTCTTTAGCAAAACATATTCTTAATTTA
TGTCAGAACTCACTTTGGCTAGTTTGGTTCAATTTTGATTACAGCATGTT
TGTATGAAGTCAAAGTGTAAATTACGATTTTGGTTCGGTTCCATAGAATT
TTAACCGAATTACAAACTTTATGCGGTTTTTATCGGAATAAAAGGTATTT
TGGTAAGTGTAAGTTCCTCAACA TGTTAGCCTATCCTACGTGG
CGCGTAGgtgggagttgatcgccggaaggatcccgggacggacgccggag
gagatagagagatattggcttatgaaacacggcgtcgtattgccaacaga
cgaagagactntttaggaaatga
[0380] The sequences in lower case are exons. The sequences in bold
italic text are branch sites. The sequences in bold were replaced
by artificial pre-miRNAs. Intron sequences include sequences in
normal text, bold text and bold italic text.
[0381] Constructs 35S::CPC-A and 35S:: CPC-B were generated to
check whether intron 1 or intron 2 of the unspliced CPC transcript
can be used to insert artificial pre-miRNA for the production of
artificial miRNAs. In the CPC-A construct, pre-miR-HC-Pro.sup.159a
was inserted into intron 1 with no change in intron 2. In CPC-B,
pre-miR-HC-Pro.sup.159a was inserted into intron 2 with no change
in intron 1 (FIG. 36). Agrobacterial cells carrying 35S:: CPC-A,
35S:: CPC-B, 35S::pre-miR-HC-Pro.sup.159a, and 35S::pre-miR159a
were infiltrated into N. benthamiana leaves for transient
expression. Northern blot hybridizations using a probe
complementary to miR-HC-Pro.sup.159a showed that in 4 separate
experiments leaf samples infiltrated with CPC-A and CPC-B expressed
miR-HC-Pro.sup.159a (FIG. 37). This result demonstrates that both
intron 1 and intron 2 of the CPC transcript can be used to produce
artificial miRNAs.
[0382] Constructs 35S::CPC-C and 35S::CPC-D were generated to
determine the possibility of producing miRNAs in both introns. In
CPC-C, pre-miR-HC-Pro.sup.159a was inserted into intron 1 and
pre-miR-P69.sup.159a into intron 2. In CPC-D, pre-miR-P69.sup.159a
was inserted into intron 1 and pre-miR-HC-Pro.sup.159a into intron
2 (FIG. 38). Agrobacterial cells carrying 35S:: CPC-C, 35S::CPC-D,
35S::pre-miR-HC-P.sup.159a, and 35S::pre-miR-P69.sup.159a were
infiltrated into N. benthamiana leaves for transient expression.
FIG. 39 shows northern blot results of four independent
experiments. Note that all of the four samples show signals
corresponding to miR-HC-Pro.sup.159a miRNA and miR-P69.sup.159a,
although the signal in sample 1 is weak (FIG. 39, 1 of 35S::CPC-C).
This weak signal could be due to a lower transient expression
efficiency in this particular sample. A similar situation was
encountered in sample 4 of the 35S::CPC-D experiment. These results
demonstrate that it is possible to use CPC introns to produce two
different artificial miRNAs simultaneously in one transcript.
[0383] The above examples are provided to illustrate the invention
but not to limit its scope. Other variants of the invention will be
readily apparent to one of ordinary skill in the art and are
encompassed by the appended claims. For example, in the Examples
described above, pre-miR159a and pre-miR169g were used to generate
artifical pre-miRNAs. However, other pre-miRNAs, such as described
herein, could be used in place of pre-miR159a and pre-miR169g. All
publications, patents, patent applications, and computer programs
cited herein are hereby incorporated by reference. It will also be
appreciated that in this specification and the appended claims, the
singular forms of "a," "an" and "the" include plural reference
unless the context clearly dictates otherwise. It will further be
appreciated that in this specification and the appended claims, The
term "comprising" or "comprises" is intended to be open-ended,
including not only the cited elements or steps, but further
encompassing any additional elements or steps.
Sequence CWU 1
1
18911417DNAArabidopsis thaliana 1gcacctctca ctccctttct ctaactagtc
ttgtgtgcac ccatttatgt gtacgtacta 60ttatctcata aataaatatt tttaaaatta
gatgcattta ttgatatgaa aaagttacaa 120gattagtttg ttgtgtgtga
gactttggat cgacagatcg aaaaattaac taaccggtca 180gtattgaata
tcaactatta tatgctccat gcattcgctt atagtttcac acaatttgtt
240ttcttcacgg tctaaaatca gaagattcca tatattttct tatgacgtaa
aaggaccact 300tataagttga cacgtcagcc cttggattcg tgaggttttt
ctctctactt cacctatcta 360cttttcctca tatcccactg cttttctcct
tcttgttctt gtttttctcg tttttttctt 420cttcttctcc aagaaaatag
agatcgaaaa gattagatct attttgtgta gcaagaaatt 480atcattttcg
tttcttcatt catatattgt tctattatgt tgtacaataa tagatactcg
540atctcttgtg cgtgcgtaaa ttttatacaa gttgtcggcg gatccatgga
agaaagctca 600tctgtcgttg tttgtaggcg cagcaccatt aagattcaca
tggaaattga taaataccct 660aaattagggt tttgatatgt atatgagaat
cttgatgatg ctgcatcaac aatcgacggc 720tacaaatacc taaagcttga
gaaagaaact tgaagatatt gattgaagtc tggatcgatc 780tttggtaaat
ctctctcttg attagtttta agaatcactt ttttttttct gtgtttgaac
840atgtttacat atatcatcta tgtctcaata tatatatttt cttaatctag
ggtcaatgac 900ggattagggc gttaattaca atgaatatgg aaaaactatt
ttgcctttga tcttgacttg 960agtgttgatg aacagatgta taatgttatg
tagtatgtac tgtatttttt ctagaatcat 1020tctttagtct ccaactctcc
attaatcaaa tgaggtcctt ataggtaatg ctatgatcaa 1080gaacaacaag
atcgtgagca cagatcggcc agttcggtca ctttttaaaa gagagatgtt
1140atattgttaa tttgttatta tcaggtataa taaatacaga atagttcgtc
cagagaccag 1200acattttata gtttcaattt tatgacagtc ttgtaataat
atttgtttaa tagtgtgtca 1260ccttctattt ctgggttatt acttggtccc
gaaattttct tattgttcta attttgtaat 1320attagaaatt tggttttctt
gccaaatcaa atcaaacatt acggtgtgtt gtacattgta 1380ccagaacttt
tgttttcaag tgctcaactt gagaacc 1417295DNAArabidopsis thaliana
2aggcgcagca ccattaagat tcacatggaa attgataaat accctaaatt agggttttga
60tatgtatatg agaatcttga tgatgctgca tcaac 953159DNAArtificial
SequencemiRNA template cassette 3ggatccatgg aagaaagctc atctgtcgtt
gtttgtaggc gcagcaccat taagattcac 60atggaaattg ataaataccc taaattaggg
ttttgatatg tatatgagaa tcttgatgat 120gctgcatcaa caatcgacgg
ctacaaatac ctaaagctt 159421DNAArtificial SequencemiRNA template to
target At4g18960 4taggttgtaa tgccgcgact t 21521DNAArtificial
SequencemiRNA template to target At3g54340 5ggtggaaatg aagagcgtaa g
21621DNAArtificial SequencemiRNA template to target At3g54340
6agagcgtaag cacgtgaccc t 21721DNAArtificial SequencemiRNA template
to target maize phytoene desaturase 7tgctggcaga agtccgattg c
21821DNAArtificial SequencemiRNA template to target maize phytoene
desaturase 8agcttcctgg ataggactgc a 21921DNAArtificial
SequencemiRNA template to target is maize IPPK2 9aagttgtggt
taatcacccc a 211021DNAArtificial SequencemiRNA template to target
is maize ITPK5 10gaggacagtt tcgtatcctg g 211121DNAArtificial
SequencemiRNA template to target is maize Mi1ps3 11gagcgtttac
caccggtgtg c 211277DNAArtificial SequenceSynthetic oligo1/4 for
At4g18960 target 12gatccatgga agaaagctca tctgtcgttg tttgtaggca
gtcgcggcac tacaaccaaa 60tggaaattga taaatac 771377DNAArtificial
SequenceSynthetic oligo2/4 for At4g18960 target 13tagggtattt
atcaatttcc atttggttgt agtgccgcga ctgcctacaa acaacgacag 60atgagctttc
ttccatg 771476DNAArtificial SequenceSynthetic oligo3/4 for
At4g18960 target 14cctaaattag ggttttgata tgtatattag gttgtaatgc
cgcgactttc aacaatcgac 60ggctacaaat acctaa 761576DNAArtificial
SequenceSynthetic oligo4/4 for At4g18960 target 15agctttaggt
atttgtagcc gtcgattgtt gaaagtcgcg gcattacaac ctaatataca 60tatcaaaacc
ctaatt 761677DNAArtificial SequenceSynthetic oligo1/4 for At3g54340
target 16gatccatgga agaaagctca tctgtcgttg tttgtaggat tacgcccttc
attaccacca 60tggaaattga taaatac 771777DNAArtificial
SequenceSynthetic oligo2/4 for At3g54340 target 17tagggtattt
atcaatttcc atggtggtaa tgaagggcgt aatcctacaa acaacgacag 60atgagctttc
ttccatg 771876DNAArtificial SequenceSynthetic oligo3/4 for
At3g54340 target 18cctaaattag ggttttgata tgtatatggt ggaaatgaag
agcgtaagtc aacaatcgac 60ggctacaaat acctaa 761976DNAArtificial
SequenceSynthetic oligo4/4 for At3g54340 target 19agctttaggt
atttgtagcc gtcgattgtt gacttacgct cttcatttcc accatataca 60tatcaaaacc
ctaatt 762077DNAArtificial SequenceSynthetic oligo1/4 for At3g54340
target 20gatccatgga agaaagctca tctgtcgttg tttgtaggcg gtcacgcgct
tacgctcaca 60tggaaattga taaatac 772177DNAArtificial
SequenceSynthetic oligo2/4 for At3g54340 target 21tagggtattt
atcaatttcc atgtgagcgt aagcgcgtga ccgcctacaa acaacgacag 60atgagctttc
ttccatg 772276DNAArtificial SequenceSynthetic oligo3/4 for
At3g54340 target 22cctaaattag ggttttgata tgtatatgag agcgtaagca
cgtgaccctc aacaatcgac 60ggctacaaat acctaa 762376DNAArtificial
SequenceSynthetic oligo4/4 for At3g54340 target 23agctttaggt
atttgtagcc gtcgattgtt gagggtcacg tgcttacgct ctcatataca 60tatcaaaacc
ctaatt 762477DNAArtificial SequenceSynthetic oligo1/4 for phytoene
desaturase target 24gatccatgga agaaagctca tctgtcgttg tttgtaggca
atcggacttc tgccagcaca 60tggaaattga taaatac 772577DNAArtificial
SequenceSynthetic oligo2/4 for phytoene desaturase target
25tagggtattt atcaatttcc atgtgctggc agaagtccga ttgcctacaa acaacgacag
60atgagctttc ttccatg 772676DNAArtificial SequenceSynthetic oligo3/4
for phytoene desaturase target 26cctaaattag ggttttgata tgtatatgtg
ctggcagaag tccgattgcc aacaatcgac 60ggctacaaat acctaa
762776DNAArtificial SequenceSynthetic oligo4/4 for phytoene
desaturase target 27agctttaggt atttgtagcc gtcgattgtt ggcaatcgga
cttctgccag cacatataca 60tatcaaaacc ctaatt 762877DNAArtificial
SequenceSynthetic oligo1/4 for phytoene desaturase target
28gatccatgga agaaagctca tctgtcgttg tttgtagtac agtcccatcc aggaagcaca
60tggaaattga taaatac 772977DNAArtificial SequenceSynthetic oligo2/4
for phytoene desaturase target 29tagggtattt atcaatttcc atgtgcttcc
tggatgggac tgtactacaa acaacgacag 60atgagctttc ttccatg
773076DNAArtificial SequenceSynthetic oligo3/4 for phytoene
desaturase target 30cctaaattag ggttttgata tgtatatgag cttcctggat
aggactgcac aacaatcgac 60ggctacaaat acctaa 763176DNAArtificial
SequenceSynthetic oligo4/4 for phytoene desaturase target
31agctttaggt atttgtagcc gtcgattgtt gtgcagtcct atccaggaag ctcatataca
60tatcaaaacc ctaatt 763277DNAArtificial SequenceSynthetic oligo1/4
for IPPK2 target 32gatccatgga agaaagctca tctgtcgttg tttgtaggcg
gggtgataaa ccacaacata 60tggaaattga taaatac 773377DNAArtificial
SequenceSynthetic oligo2/4 for IPPK2 target 33tagggtattt atcaatttcc
atatgttgtg gtttatcacc ccgcctacaa acaacgacag 60atgagctttc ttccatg
773476DNAArtificial SequenceSynthetic oligo3/4 for IPPK2 target
34cctaaattag ggttttgata tgtatataag ttgtggttaa tcaccccatc aacaatcgac
60ggctacaaat acctaa 763576DNAArtificial SequenceSynthetic oligo4/4
for IPPK2 target 35agctttaggt atttgtagcc gtcgattgtt gatggggtga
ttaaccacaa cttatataca 60tatcaaaacc ctaatt 763677DNAArtificial
SequenceSynthetic oligo1/4 for ITPK5 target 36gatccatgga agaaagctca
tctgtcgttg tttgtaggac aggatacgta actgtccaca 60tggaaattga taaatac
773777DNAArtificial SequenceSynthetic oligo2/4 for ITPK5 target
37tagggtattt atcaatttcc atgtggacag ttacgtatcc tgtcctacaa acaacgacag
60atgagctttc ttccatg 773876DNAArtificial SequenceSynthetic oligo3/4
for ITPK5 target 38cctaaattag ggttttgata tgtatatgag gacagtttcg
tatcctggtc aacaatcgac 60ggctacaaat acctaa 763976DNAArtificial
SequenceSynthetic oligo4/4 for ITPK5 target 39agctttaggt atttgtagcc
gtcgattgtt gaccaggata cgaaactgtc ctcatataca 60tatcaaaacc ctaatt
764077DNAArtificial SequenceSynthetic oligo1/4 for mi1ps target
40gatccatgga agaaagctca tctgtcgttg tttgtaggac acaccggcgg taaacgcaca
60tggaaattga taaatac 774177DNAArtificial SequenceSynthetic oligo2/4
for mi1ps target 41tagggtattt atcaatttcc atgtgcgttt accgccggtg
tgtcctacaa acaacgacag 60atgagctttc ttccatg 774276DNAArtificial
SequenceSynthetic oligo3/4 for mi1ps target 42cctaaattag ggttttgata
tgtatatgag cgtttaccac cggtgtgctc aacaatcgac 60ggctacaaat acctaa
764376DNAArtificial SequenceSynthetic oligo4/4 for mi1ps target
43agctttaggt atttgtagcc gtcgattgtt gagcacaccg gtggtaaacg ctcatataca
60tatcaaaacc ctaatt 76444426DNAArtificial SequencePlasmid
44ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
60attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
120gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga
acgtggactc 180caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc
ccactacgtg aaccatcacc 240ctaatcaagt tttttggggt cgaggtgccg
taaagcacta aatcggaacc ctaaagggag 300cccccgattt agagcttgac
ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa 360agcgaaagga
gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
420cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat
tcaggctgcg 480caactgttgg gaagggcgat cggtgcgggc ctcttcgcta
ttacgccagc tggcgaaagg 540gggatgtgct gcaaggcgat taagttgggt
aacgccaggg ttttcccagt cacgacgttg 600taaaacgacg gccagtgagc
gcgcgtaata cgactcacta tagggcgaat tgggtaccgg 660gccctctaga
tgcatgctcg agcggccgcc agtgtgatgg atatctgcag aattcgccct
720tgactactcg agcacctctc actccctttc tctaactagt cttgtgtgca
cccatttatg 780tgtacgtact attatctcat aaataaatat ttttaaaatt
agatgcattt attgatatga 840aaaagttaca agattagttt gttgtgtgtg
agactttgga tcgacagatc gaaaaattaa 900ctaaccggtc agtattgaat
atcaactatt atatgctcca tgcattcgct tatagtttca 960cacaatttgt
tttcttcacg gtctaaaatc agaagattcc atatattttc ttatgacgta
1020aaaggaccac ttataagttg acacgtcagc ccttggattc gtgaggtttt
tctctctact 1080tcacctatct acttttcctc atatcccact gcttttctcc
ttcttgttct tgtttttctc 1140gtttttttct tcttcttctc caagaaaata
gagatcgaaa agattagatc tattttgtgt 1200agcaagaaat tatcattttc
gtttcttcat tcatatattg ttctattatg ttgtacaata 1260atagatactc
gatctcttgt gcgtgcgtaa attttataca agttgtcggc ggatccatgg
1320aagaaagctc atctgtcgtt gtttgtaggc gcagcaccat taagattcac
atggaaattg 1380ataaataccc taaattaggg ttttgatatg tatatgagaa
tcttgatgat gctgcatcaa 1440caatcgacgg ctacaaatac ctaaagcttg
agaaagaaac ttgaagatat tgattgaagt 1500ctggatcgat ctttggtaaa
tctctctctt gattagtttt aagaatcact tttttttttc 1560tgtgtttgaa
catgtttaca tatatcatct atgtctcaat atatatattt tcttaatcta
1620gggtcaatga cggattaggg cgttaattac aatgaatatg gaaaaactat
tttgcctttg 1680atcttgactt gagtgttgat gaacagatgt ataatgttat
gtagtatgta ctgtattttt 1740tctagaatca ttctttagtc tccaactctc
cattaatcaa atgaggtcct tataggtaat 1800gctatgatca agaacaacaa
gatcgtgagc acagatcggc cagttcggtc actttttaaa 1860agagagatgt
tatattgtta atttgttatt atcaggtata ataaatacag aatagttcgt
1920ccagagacca gacattttat agtttcaatt ttatgacagt cttgtaataa
tatttgttta 1980atagtgtgtc accttctatt tctgggttat tacttggtcc
cgaaattttc ttattgttct 2040aattttgtaa tattagaaat ttggttttct
tgccaaatca aatcaaacat tacggtgtgt 2100tgtacattgt accagaactt
ttgttttcaa gtgctcaact tgagaacctc gagtagtcaa 2160gggcgaattc
cagcacactg gcggccgtta ctagttctag agcggccgcc accgcggtgg
2220agctccagct tttgttccct ttagtgaggg ttaattgcgc gcttggcgta
atcatggtca 2280tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc
cacacaacat acgagccgga 2340agcataaagt gtaaagcctg gggtgcctaa
tgagtgagct aactcacatt aattgcgttg 2400cgctcactgc ccgctttcca
gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc 2460caacgcgcgg
ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
2520tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
ggcggtaata 2580cggttatcca cagaatcagg ggataacgca ggaaagaaca
tgtgagcaaa aggccagcaa 2640aaggccagga accgtaaaaa ggccgcgttg
ctggcgtttt tccataggct ccgcccccct 2700gacgagcatc acaaaaatcg
acgctcaagt cagaggtggc gaaacccgac aggactataa 2760agataccagg
cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
2820cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
tcatagctca 2880cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
agctgggctg tgtgcacgaa 2940ccccccgttc agcccgaccg ctgcgcctta
tccggtaact atcgtcttga gtccaacccg 3000gtaagacacg acttatcgcc
actggcagca gccactggta acaggattag cagagcgagg 3060tatgtaggcg
gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg
3120acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
agttggtagc 3180tcttgatccg gcaaacaaac caccgctggt agcggtggtt
tttttgtttg caagcagcag 3240attacgcgca gaaaaaaagg atctcaagaa
gatcctttga tcttttctac ggggtctgac 3300gctcagtgga acgaaaactc
acgttaaggg attttggtca tgagattatc aaaaaggatc 3360ttcacctaga
tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
3420taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
agcgatctgt 3480ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
agataactac gatacgggag 3540ggcttaccat ctggccccag tgctgcaatg
ataccgcgag acccacgctc accggctcca 3600gatttatcag caataaacca
gccagccgga agggccgagc gcagaagtgg tcctgcaact 3660ttatccgcct
ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
3720gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
acgctcgtcg 3780tttggtatgg cttcattcag ctccggttcc caacgatcaa
ggcgagttac atgatccccc 3840atgttgtgca aaaaagcggt tagctccttc
ggtcctccga tcgttgtcag aagtaagttg 3900gccgcagtgt tatcactcat
ggttatggca gcactgcata attctcttac tgtcatgcca 3960tccgtaagat
gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
4020atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
gccacatagc 4080agaactttaa aagtgctcat cattggaaaa cgttcttcgg
ggcgaaaact ctcaaggatc 4140ttaccgctgt tgagatccag ttcgatgtaa
cccactcgtg cacccaactg atcttcagca 4200tcttttactt tcaccagcgt
ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa 4260aagggaataa
gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
4320tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
tatttagaaa 4380aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccac
44264529DNAArtificial SequenceEAT 3' PCR primer 45ctgtgctcac
gatcttgttg ttcttgatc 294629DNAArtificial SequenceEAT 5' PCR primer
46gtcggcggat ccatggaaga aagctcatc 294721RNAArabidopsis thalianaAP2
RNA 47cugcagcauc aucaggauuc u 214821RNAArabidopsis
thalianamisc_feature(0)...(0)EAT miRNA 48agaaucuuga ugaugcugca u
214959DNAArabidopsis thaliana 49accaagtgtt gacaaatgct gcagcatcat
caggattctc tcctcatcat cacaatcag 595059DNAArabidopsis thaliana
50caccgccact gttttcaaat gcagcatcat caggattctc actctcagct acacgccct
595159DNAArabidopsis thaliana 51caccattgtt ctcagttgca gcagcatcat
caggattctc acatttccgg ccacaacct 595259DNAArabidopsis thaliana
52gaaatcgagt ggtgggaatg gcagcatcat caggattctc tcctcaacct tccccttac
595359DNAZea mays 53acgtgccgtt gcaccactct gcagcatcat caggattctc
taccgccgcc ggggccaac 595459DNAZea mays 54acgccagcag cgccgccgct
gcagcatcat caggattccc actgtggcag ctgggtgcg 595535DNAArtificial
SequenceEAT PCR primer 55gactactcga gcacctctca ctccctttct ctaac
355636DNAArtificial SequenceEAT PCR primer 56gactactcga ggttctcaag
ttgagcactt gaaaac 365777DNAArtificial SequenceEAT deletion
oligonucleotide 57gatccatgga agaaagctca tctgtcgttg tttgtaggcg
cagcaccatt aagattcaca 60tggaaattga taaatac 775855DNAArtificial
SequenceEAT deletion oligonucleotide 58cctaaattag ggttttgata
tgtatattca acaatcgacg gctacaaata cctaa 555977DNAArtificial
SequenceEAT deletion oligonucleotide 59tagggtattt atcaatttcc
atgtgaatct taatggtgct gcgcctacaa acaacgacag 60atgagctttc ttccatg
776055DNAArtificial SequenceEAT deletion oligonucleotide
60agctttaggt atttgtagcc gtcgattgtt gaatatacat atcaaaaccc taatt
556130DNAArtificial SequenceS1 probe 61atgcagcatc atcaagattc
tcatatacat 306229DNAArtificial Sequencemir172a-2 PCR primer
62gtcggcggat ccatggaaga aagctcatc
296330DNAArtificial Sequencemir172a-2 PCR primer 63caaagatcga
tccagacttc aatcaatatc 306427DNAArtificial Sequencemir172a-1 PCR
primer 64taatttccgg agccacggtc gttgttg 276528DNAArtificial
Sequencemir172a-1 PCR primer 65aatagtcgtt gattgccgat gcagcatc
286624DNAArtificial SequenceActin PCR primer 66atggcagatg
gtgaagacat tcag 246726DNAArtificial SequenceActin PCR primer
67gaagcacttc ctgtggacta ttgatg 266826DNAArtificial SequenceAP2 PCR
primer 68tttccgggca gcagcaacat tggtag 266929DNAArtificial
SequenceAP2 PCR primer 69gttcgcctaa gttaacaaga ggatttagg
297030DNAArtificial SequenceANT PCR primer 70gatcaacttc aatgactaac
tctggttttc 307130DNAArtificial SequenceANT PCR primer 71gttatagaga
gattcattct gtttcacatg 307221DNAArtificial SequencemiRNA template to
EAT 72agaatcttga tgatgctgca t 2173175DNAArtificial
SequenceSynthetic oligonucleotide 1 for EAT with attB sites
73ttaaacaagt ttgtacaaaa aagcaggctg tcgttgtttg taggcgcagc accattaaga
60ttcacatgga aattgataaa taccctaaat tagggttttg atatgtatat gagaatcttg
120atgatgctgc atcaacaatc gacggcaccc agctttcttg tacaaagtgg tttaa
17574175DNAArtificial SequenceSynthetic oligonucleotide 2 for EAT
with attB sites 74ttaaaccact ttgtacaaga aagctgggtg ccgtcgattg
ttgatgcagc atcatcaaga 60ttctcatata catatcaaaa ccctaattta gggtatttat
caatttccat gtgaatctta 120atggtgctgc gcctacaaac aacgacagcc
tgcttttttg tacaaacttg tttaa 1757521DNAArtificial SequencemiRNA
template for FAD2 75agataagacc aactgtgtca t 2176175DNAArtificial
SequenceSynthetic oligonucleotide 1 for FAD2 76ttaaacaagt
ttgtacaaaa aagcaggctg tcgttgtttg taggcgacac agctggtctt 60atcacatgga
aattgataaa taccctaaat tagggttttg atatgtatat gagataagac
120caactgtgtc atcaacaatc gacggcaccc agctttcttg tacaaagtgg tttaa
17577175DNAArtificial SequenceSynthetic oligonucleotide 2 for FAD2
77ttaaaccact ttgtacaaga aagctgggtg ccgtcgattg ttgatgacac agttggtctt
60atctcatata catatcaaaa ccctaattta gggtatttat caatttccat gtgataagac
120cagctgtgtc gcctacaaac aacgacagcc tgcttttttg tacaaacttg tttaa
1757821DNAArtificial SequencemiRNA template for PDS 78agaaactctt
aaccgtgcca t 2179175DNAArtificial SequenceSynthetic oligonucleotide
1 to target PDS 79ttaaacaagt ttgtacaaaa aagcaggctg tcgttgtttg
taggcggcac ggtcaagagt 60ttcacatgga aattgataaa taccctaaat tagggttttg
atatgtatat gagaaactct 120taaccgtgcc atcaacaatc gacggcaccc
agctttcttg tacaaagtgg tttaa 17580175DNAArtificial SequenceSynthetic
oligonucleotide 2 to target PDS 80ttaaaccact ttgtacaaga aagctgggtg
ccgtcgattg ttgatggcac ggttaagagt 60ttctcatata catatcaaaa ccctaattta
gggtatttat caatttccat gtgaaactct 120tgaccgtgcc gcctacaaac
aacgacagcc tgcttttttg tacaaacttg tttaa 17581907DNAZea mays
81ttaaaaaaat agcgatttgt ttgaagaaag gatcatggcc gagcatcatt caacgtacct
60ctgtagggcg tatgaatcgt tggattagga tcaaagtcgg caacggttaa attcaaggaa
120gaaaacaacg ggcgtggggt cctgtccacg tcatcaggtg accaggcagg
caggcatgcg 180cgccatgcgg cattgcttct gtccccgtgc ccgggcagct
tttggcagcg gatccggacg 240gaacaccacg cgcgcgcgcg cgcggcaggc
acgcaccggc caacttaatc ttgcctccac 300tctgcactag tggggttatt
aacaatttga ttaatccgac actgacgtac tgtgtcaacc 360aatggcaccg
cctatatatt aatcgaacca ttcagctcgt cttaattgcc acccacccac
420ccaccgccat tgccatggtt cacctcattc attctaagct tagacgatgc
agtgatagaa 480attaatactg caaatcagtc agtgtttgcg ggcgtggcat
catcaagatt cacaacccat 540caatccgaac cactgatttg gaatgcatgt
atgagaatct tgatgatgct gcatccgcca 600acaagcgcct acgaacgttt
gtgtgctcat cttcgccatc aatcgagatt ttgtatcttc 660acgtttagct
aaggtgaaag atcgtcatcc catccgccta aagctagctt tgcaaatttt
720tattcgaaac aacgaccatt tctatatatt tcctttctct gttatagtct
ctaattaacg 780cctgtaaact gttgcaccct gcttctgcat cttcttatta
attagttttg tctcttatgg 840atgctaaaca gccatgacgt ttcggacaat
gttcagctcg tacttccttc aatcgggagc 900gccaaaa 907821128DNAZea mays
82tcgtcgatct gatttgcctg ctttatttct tcttcttctt cacaccgagc tagctagcta
60tcttgcttta atttgcctag aacgaataga tccaccgtac tagcttcttg ctcgatctgc
120agcttctcgc ttgtgagcca agagcccggc cagcagtgtc ggccgtgcag
tggcactctc 180tccatcaaca atcaaccctc tctccgtcga catgtggaaa
ggtaggtaga gatagatggt 240gtgtgtaatc cggttccttg gttcttgtgt
ttccgatctc ctctaattaa tcgatctctc 300tacctggcca gctcacttca
cccatgcttg catctagctg ttccaatctg atgcatgata 360tagatgatgc
ttgcggcctc ttcttcttga ttcataggct catcatctat gcctctgtca
420tgcacacact cgtgtctttc ttcttgatgg atacacgtac ggggggttgg
gttgttcaca 480tatatagtag tatagctagt ttattagatg caggtataca
gatcatgagg aagcaagaaa 540ttatgcaaaa cagtcggtgc ttgcaggtgc
agcaccatca agattcacat ccccagctcg 600atctgtgcat gatgagatga
gaatcttgat gatgctgcat cagcaaacac tcacttacat 660cgatctcacc
cctggacaag ctggacagtg aaaccggact gagcaatcga gtactactaa
720aaacttgtcc tcagctcttt atgttttact ttcaattacc ttgcttatat
taattttctt 780tcacttaatt tagttaatta ctgctctctc tctctctctc
tgtctctctc tctctctctc 840tggttttttc atcttgcaaa aaaaatgcag
aaattaatat gtatatgtgt acctcatgat 900tattaaggcc gctgcaccat
gattttatgg tatattatta tcagcttaaa acaggctttc 960ccttttgatt
atatttcaat aattcgttta gcatcattag tttctgcatt tgccgatgat
1020ctcgaggttc tgtttgcaag aagtggctgc actgcagccc tgcagctata
tatacacagg 1080ttcaagttac taattttgtg cttctacaat aatcctatca gtccgcag
112883912DNAZea mays 83cactaatagc tttctatctg atcgattcat catcatccgg
gcatgcatga gcatcatcgt 60ctccagatcg ttgggctctc gcagctacct acattcaaca
ttcaagctcg ctctacatat 120gcatgcaaat ctgcaacact cgctcttggc
agggatacat tcacgccgag agagagagag 180agagagagag agagagagag
agagagagag atgtgtgtgc tgtagtcatc agccagccgg 240tgatttctgg
agtggcatca tcaagattca cacactgcat gccaacataa tgcgcgtgtt
300catgcatcca tcgccgccgc tgcatcatgc atcatatata atatatatat
atgtgtatgt 360gtgggaatct tgatgatgct gcattggata tcaagggcta
tatatatata tggatcaagc 420atatatatat atatatcaga tcaccagtca
tatcgagttc ttccttccag gcttgctagg 480taatttataa cttaaacctt
gttgctgaac taactaattt tacttagcta gctagctact 540actatacttc
attgttagta gtagctagca agaaggaaag taggcatccc ggccggttcg
600taccttcttt ttttttgcac agcaggatct gaccttctgt ataaaatgca
tttttgcctt 660gagttttttt gtttttccac agtaggaggt agctgattct
gatctgctgt ataaaaatgc 720atttttttcc ttttcatttc atggcagaag
gcaatatata ataagaaaag actgaaagga 780aaaggcacca ctgccatgat
ggatcgcatc agtgcatctg ttttgttctt ctaaacgatt 840caggtcatca
ggtgagctag gtgggctaat aagtatatag attaatttct attttgcaca
900tgatttatat gg 912841063DNAZea mays 84catgcatgct gccttacacc
taagctagct agctgttgaa tttgatgcat gacgcatgct 60ttcctcctcc tccgttcgta
gtcgttgtcg ttgtctcagt aatccatcct ctctcttttt 120ttcttgctaa
tacataaaag gggttcagat ggtagctgct agtggttatt cttcttctta
180gacgatgcaa gtatatgtat atggaccacc aaattagctt ctcgtcttgc
cgccggaccg 240ccatcatgca ccttggagaa gcaacagaac gaagctcgct
gctatgctat ctatggatta 300ttgtattgta tatgaatgaa gcagcaagca
aacgtagttc agtacagtcg gtgcttgcag 360gtgcagcacc atcaagattc
acatcgtcca actcatgcat catgcatata tgcatcttca 420atgatgcgtg
cctcgcatgt gtgtgtatat atatatgatg agatgagaat cttgatgatg
480ctgcatcagc agacactcac tagctcatgc atcacctcca agtaataaga
gatgaattga 540attaacgacc atgcagctac tagctctrgt acgtaccact
tcgttctcct ctaatttctt 600tttccattca gtctaccttg tttgctaatc
aacttgttct catataatat atggttccca 660atgcgataag ggttggcctg
caggcttagc tctgcagcag gtagcaccca tgcatggccc 720atgatacata
acatattgat ggatatatac tagcataaaa acatgatgat gcagagcagc
780agcatccatc tcatagctag cataaaaaca tgcatgagct agcagcggca
gttgacgatg 840actcttcgag aggaaggaag gaagcagcag atcgatggac
gcgagacatg agcagtgaca 900gatgcataat gtagcagtac atacagcatt
attgctatta tttgtgccca agcaaattaa 960ggaaggggac caaattgaaa
tatactaatg acattgcaga cggcaccagc agagtccaca 1020gctcgtgaac
ctgtgtaggc tgcctgccga tggtacaatg caa 1063851738DNAZea mays
85ccatcagcaa ctgctcgtag ctccgtcctc atcacttaaa ccttatcatc atcactctct
60cttcctctct cttctggccg gccggtcctt tcacctcact catcttctca gttcattcca
120tggagagcgt cgttcctata tatcatgcat catccaccaa ggccctagct
aagctgctac 180tacctgctag gggttttatt agttgctcaa ccttcgctgg
ccggccttat atatacctag 240ctatagctgt cttgcttgca tagatcatcg
atccatgttg ctagctagct agctccctca 300gttcagttca gttcagttca
gctcagctag ctagctcact cctctcttga gtcgtggtgt 360ccatcacaat
cttctctata tcgatacagg tgaggaggta gctagacaga tcaacaccaa
420tcctctcaac gacatcccct tgttcttgta gagagagttg gtgtaggtcg
aaaggcagat 480agatcatata tagagggaga gatgcatata tggtgtaggg
ttcttcaatt tgtttctatg 540atcgattcat tcgccctgca gccccccctg
cgcatctagt tatgtctcca tccctcctcc 600cttgttcctg atacatatat
atatatgtag gtagtggctc tgtatatacc catgccatct 660ctctcaatct
catctatatc atatataccc atgctttgca tctagctgtt tcatttcttt
720tcactcgtgc tttgaaagat ctggtacagt ccggcctgta ttagtaagaa
cgagttagaa 780aaatacacac gtacgcgcga gaaccatgca tcatcagcta
gctcctctct ttcctctttt 840tttttgttaa tgcatacatt catatatata
ttcccatgaa tgaatgcttt aagcatgagg 900caagcaaaca tcgacagtgg
gtgcttgcag gtgcagcacc accaagattc acatccaact 960ctcacgcatc
ttcagtgatg catgcatgct ctgtgatgtc tcgcagcagc tatatgcata
1020tgtgatgaga tgagaatctt gatgatgctg catcagcaga cactcactca
tcacaccaac 1080gtaccccaac aagggtgaga gacgacgaat cggctgctgg
tatatacata caactgagaa 1140gtcggattac ctttgctgat tattaacttg
tttccattgc tgtgaaatga aactttcaat 1200gcaagggggc tggcctacca
gctggtacta gcaggaatga agagcatata tatatgaaca 1260tgatgatgca
tatatgcaga gcagcaacag cagcatcgtc gtaccatctc atatatatca
1320ttgcaaacat gagcagtagt ggtagttcat gaatcatgat gaaagcaagg
aagaggaagc 1380tagcagtgct ggacgcggat cagatgcaga tcgatggagg
ccggggccgg gggtgtacct 1440acgtagtaca ttgctattat tgtgtccatg
gaagggggac caaagtatgt aatgcgttgc 1500acaccacaca ccagagctgg
ctcagcagct agcagcagcc tgtggtggtg gtggtacaat 1560gcagcgtgta
ctgctgtcgt cccagcagca agttgaaagg taaaagagag aaatatttca
1620gctgacttta ctcatcacgc actctgcctg catgctggct gcaggcctgc
tgtgagtctg 1680tgtgtgtgtg cttgttctct tgctttagtg gtggtgtaga
tcttctattt gctagttt 17388621DNAArabidopsis thaliana 86agaatcttga
tgatgctgca t 218726DNAArtificial SequenceForward PCR primer for
maize miR172a 87ggatcctctg cactagtggg gttatt 268826DNAArtificial
SequenceReverse PCR primer for maize miR172a 88gatatctgca
acagtttaca ggcgtt 268926DNAArtificial SequenceForward PCR primer
for maize miR172b 89ggatcccatg atatagatga tgcttg
269026DNAArtificial SequenceReverse PCR primer for maize miR172b
90gatatcaaga gctgaggaca agtttt 2691170DNAZea mays 91tatacagatc
atgaggaagc aagaaattat gcaaaacagt cggtgcttgc aggtgcagca 60ccatcaagat
tcacatcccc agctcgatct gtgcatgatg agatgagaat cttgatgatg
120ctgcatcagc aaacactcac ttacatcgat ctcacccctg gacaagctgg
1709221DNAZea mays 92agaatcttga tgatgctgca t 219321DNAZea mays
93gtgcagcacc atcaagattc a 2194170DNAArtificial SequencemiRNA
precursor template to target PDS 94tatacagatc atgaggaagc aagaaattat
gcaaaacagt cggtgcttgc agatcctgcc 60tcgcaggttg tcacatcccc agctcgatct
gtgcatgatg agatgagaca acctgcaagg 120caggatcagc aaacactcac
ttacatcgat ctcacccctg gacaagctgg 1709521DNAArtificial SequencemiRNA
template to PDS target 95agacaacctg caaggcagga t
219621DNAArtificial SequencemiRNA template backside to PDS target
96atcctgcctc gcaggttgtc a 2197178DNAArtificial
SequenceOligonucleotide 1 for maize miR172b 97gatctataca gatcatgagg
aagcaagaaa ttatgcaaaa cagtcggtgc ttgcaggtgc 60agcaccatca agattcacat
ccccagctcg atctgtgcat gatgagatga gaatcttgat 120gatgctgcat
cagcaaacac tcacttacat cgatctcacc cctggacaag ctgggtac
17898170DNAArtificial SequenceOligonucleotide 2 for maize miR172b
98ccagcttgtc caggggtgag atcgatgtaa gtgagtgttt gctgatgcag catcatcaag
60attctcatct catcatgcac agatcgagct ggggatgtga atcttgatgg tgctgcacct
120gcaagcaccg actgttttgc ataatttctt gcttcctcat gatctgtata
17099178DNAArtificial SequenceOligonucleotide 1 for maize PDS
target 99gatctataca gatcatgagg aagcaagaaa ttatgcaaaa cagtcggtgc
ttgcagatcc 60tgcctcgcag gttgtcacat ccccagctcg atctgtgcat gatgagatga
gacaacctgc 120aaggcaggat cagcaaacac tcacttacat cgatctcacc
cctggacaag ctgggtac 178100170DNAArtificial SequenceOligonucleotide
2 for maize PDS target 100ccagcttgtc caggggtgag atcgatgtaa
gtgagtgttt gctgatcctg ccttgcaggt 60tgtctcatct catcatgcac agatcgagct
ggggatgtga caacctgcga ggcaggatct 120gcaagcaccg actgttttgc
ataatttctt gcttcctcat gatctgtata 17010126DNAArtificial
Sequenceoligonucleotide 101caccacagtt tgcttatgtc ggatcc
2610230DNAArtificial Sequenceoligonucleotide 102tgacccggga
tgtagagctc ccttcaatcc 3010346DNAArtificial Sequenceoligonucleotide
103atagatcttg atctgacgat ggaagaagag atcctaactt ttcaaa
4610433DNAArtificial Sequenceoligonucleotide 104tgacccggga
tgaagagatc ccatatttcc aaa 3310529DNAArtificial
Sequenceoligonucleotide 105caccaatgat gattacgatg atgagagtc
2910623DNAArtificial Sequenceoligonucleotide 106caaagtttga
tcacgattca tga 2310748DNAArtificial Sequenceoligonucleotide
107gagaatgagg ttgagtttag tctgacttgg ccagtttttt taccaatg
4810846DNAArtificial Sequenceoligonucleotide 108ctgattctgg
tgttggccaa gtcagactaa actctgtttc cttctc 4610948DNAArtificial
Sequenceoligonucleotide 109gagaatgagg ttgatctctt tccagtcttc
agggtttttt taccaatg 4811047DNAArtificial Sequenceoligonucleotide
110gattctggtg tcctgaagac tggaaagaga tctgtttcct tctcttc
4711144DNAArtificial Sequenceoligonucleotide 111tctgacgatg
gaagttcctc gcccgacatt cgaaaatgag ttga 4411245DNAArtificial
Sequenceoligonucleotide 112aaacccggga tgttcctcgc ccggaattcg
aaagaagagt aaaag 45113272DNAArtificial SequencemiR159a precursor
template 113acagtttgct tatgtcggat ccataatata tttgacaaga tactttgttt
ttcgatagat 60cttgatctga cgatggaagt agagctcctt aaagttcaaa catgagttga
gcagggtaaa 120gaaaagctgc taagctatgg atcccataag ccctaatcct
tgtaaagtaa aaaaggattt 180ggttatatgg attgcatatc tcaggagctt
taacttgccc tttaatggct tttactcttc 240tttggattga agggagctct
acatcccggg tc 27211421DNAArtificial SequencemiR159a mature template
114tttggattga agggagctct a 2111521DNAArtificial
SequencemiRPDS(159a) mature template 115tttggaaata tgggatctct t
21116222DNAArtificial SequencemiR169g precursor template
116aatgatgatt acgatgatga gagtctctag ttgtatcaga gggtcttgca
tggaagaata 60gagaatgagg ttgagccaag gatgacttgc cgggtttttt taccaatgaa
tctaattaac 120tgattctggt gtccggcaag ttgaccttgg ctctgtttcc
ttctcttctt ttggatgtca 180gactccaaga tatctatcat catgaatcgt
gatcaaactt tg 22211721DNAArtificial SequencemiR169 mature template
117gagccaagga tgacttgccg g 2111821DNAArtificial
SequencemiRPDSa(169g) mature template 118gagtttagtc tgacttggcc a
2111921DNAArtificial SequencemiR169 mature template 119gagccaagga
tgacttgccg g 2112021DNAArtificial SequencemiRPDSb(169g) mature
template 120gatctctttc cagtcttcag g 211212470DNAArtificial
SequencemiR169 precursor template 121aagctttgat ctttagctct
ttgccaaagc ttcttttgat ttttctattt ctctaatcta 60tccattgacc atttggggtg
atgatattct tcaatttatg ttgttgttta ttgcccatcc 120acagacccac
gtttgatttg tttaatcaaa atatataaac tgacagttgt gccactagtc
180acttgccaat taagcattcc aaagctcctt cctttacatt agtatcaagt
gagactagca 240caagctttta agtccagata aaaagcccca tggaagggaa
gctttcaaga acgagattta 300accgtaaaac ccaatttcga tttccgctaa
taatttggat ccaaaaatct agacaaaatc 360tgataaaatt agacaaagaa
atggataaaa ccccaaaacc cataatcgtc gttgttcttg 420tttgcttcaa
tatcactctt tcccctccaa cgagttagtt agagtgacgt ggcagctgaa
480ctagatttgg agtaacggga tagattaccc ataaagccca ataatgatca
ttacgtgaga 540cataacttgc ttagataacc tcattttatg ggcttagatg
gggtctctag tgttagtcat 600aagctcttaa ataccatttc tagttatata
tcaatcttta gcttggaatt ggatcgttgt 660cctatagtaa aaaaactttt
actattttat gttagcaatc ccacttaaca ttcaatatgt 720ttaaaatgaa
agagtttacc aaaaggaaag aaaaaaaggt tggtaatgaa tttatctaat
780cggatacgat atttcataat ctaatgatgg gatctatcaa taaatagaat
caaagttaac 840tttaacgctt ttgttacctg ttttctttct ttagcaatta
atattaaacg agttttagta 900atataaatat gtttccagtt atataccaaa
ctttatgtaa tattcataag cttgccaaaa 960tttacaagag tttttggaac
gcgcacaaaa ttctcatata tttcttaccc aaaaataaat 1020tttttttttt
tttttacttg tttataatcc tatatgaaca ttgctcatct tccccatttg
1080atggtaattt ttctattcct atatgtaatt aaatcctaac taatgaaatt
gaaaacataa 1140tttgaagata atcaatccta atatctcccg tcttagatct
atttaaatgg tcttatttaa 1200tttcctatat tttggcctaa ttatttattt
gatatagtga atttatggaa gcttcatgtt 1260gatggaataa aaccggctta
tcccaattaa tcgatcggga gctataacac aaatcgaaac 1320tctagtagct
ataaagagtg tgtaatagct ttggatcaca tgtattacta tttatttact
1380agctcgtgca
acaattggct ttgggaaaaa atttatttac tagtactccc ccttcacaat
1440gtgatgagtc tccaaatgat atattctcaa cccaaaggac aatctgaaat
tttcaatata 1500tattccattt tatccgcaac atttgaaatt tgtggcaatg
tttttaaaaa gactatttat 1560aaagaatctt tctaaattgt ttctacgaca
atcgataaca ccttttgttg atcaacccca 1620cacaagacta tgattccaat
cctaagaaac atacgacacg tggattttta tgtcacacta 1680gtacgatgcg
tcgatgcctt cagagtacga atattattca cataaaattc ttattcgaat
1740ttgataatat aaggtcagcc aatcttttaa agtaattata ttcttcaata
tacggttgtg 1800gtcaaaattc cattttattt tgtagcttgc atgcactact
agtttaaaac catgcatgga 1860tttattgcat ataataacat tatatgaatt
ttcaattaaa ttaatccaca catttcccat 1920ttcaatatgc ctataaatac
cttcatcacg agtatgacaa gatcacaaga caagaaaaga 1980aaggtagaga
aaacatgata atgatgatta cgatgatgag agtctctagt tgtatcagag
2040ggtcttgcat ggaagaatag agaatgaggt tgagccaagg atgacttgcc
gggttttttt 2100accaatgaat ctaattaact gattctggtg tccggcaagt
tgaccttggc tctgtttcct 2160tctcttcttt tggatgtcag actccaagat
atctatcatc atgaatcgtg atcaaacttt 2220gtaatttcat tgaaatgtgt
ttttcttgat gcgaattttt tggcttacgg tttttcgatt 2280tgaatgatca
gatttttgtt tttgcactca aactatagtt tcacttaggt tctatttttt
2340tcaggtttat gaatgataaa acaagtaaga ttttatgcta gttttagttc
atttttcgat 2400tcaaattcaa acatcttggt tttggtttag ttaagtttga
tttttcaagt caaatgctat 2460gttttcttgt 2470122268DNANicotiana
benthamiana 122atgcctcaaa ttggacttgt ttctgctgtt aacttgagag
tccaaggtag ttcagcttat 60ctttggagct cgaggtcgtc ttctttggga actgaaagtc
gagatggttg cttgcaaagg 120aattcgttat gttttgctgg tagcgaatca
atgggtcata agttaaagat tcgtactccc 180catgccacga ccagaagatt
ggttaaggac ttggggcctt taaaggtcgt atgcattgat 240tatccaagac
cagagctgga caatacag 268123657DNANicotiana benthamiana 123ggcactcaac
tttataaacc ctgacgagct ttcgatgcag tgcattttga ttgctttgaa 60cagatttctt
caggagaaac atggttcaaa aatggccttt ttagatggta accctcctga
120gagactttgc atgccgattg tggaacatat tgagtcaaaa ggtggccaag
tcagactaaa 180ctcacgaata aaaaagatcg agctgaatga ggatggaagt
gtcaaatgtt ttatactgaa 240taatggcagt acaattaaag gagatgcttt
tgtgtttgcc actccagtgg atatcttgaa 300gcttcttttg cctgaagact
ggaaagagat cccatatttc caaaagttgg agaagctagt 360gggagttcct
gtgataaatg tccatatatg gtttgacaga aaactgaaga acacatctga
420taatctgctc ttcagcagaa gcccgttgct cagtgtgtac gctgacatgt
ctgttacatg 480taaggaatat tacaacccca atcagtctat gttggaattg
gtatttgcac ccgcagaaga 540gtggataaat cgtagtgact cagaaattat
tgatgctaca atgaaggaac tagcgaagct 600tttccctgat gaaatttcgg
cagatcagag caaagcaaaa atattgaagt accatgt 657124781DNANicotiana
benthamiana 124ggagaaagag aaactttctg tcttaagagt aattagcaat
ggcttcctca gttctttcct 60cagcagcagt tgccacccgc agcaatgttg ctcaagctaa
catggttgca cctttcacag 120gtcttaagtc tgctgcctca ttccctgttt
caagaaagca aaaccttgac atcacttcca 180ttgccagcaa cggcggaaga
gtgcaatgca tgcaggtgtg gccaccaatt aacatgaaga 240agtatgagac
tctctcatac cttcccgatt tgagccagga gcaattgctc tccgaaattg
300agtacctttt gaaaaatgga tgggttcctt gcttggaatt cgagactgag
aaaggatttg 360tctaccgtga acaccacaag tcaccaggat actatgatgg
cagatactgg accatgtgga 420agctacctat gttcggatgc actgatgcca
cccaagtgtt ggctgaggtg ggagaggcga 480agaaggaata cccacaggcc
tgggtccgta tcattggatt tgacaacgtg cgtcaagtgc 540agtgcatcag
tttcattgcc tccaagcctg acggctactg agtttcatat taggacaact
600taccctattg tctgtcttta ggggcagttt gtttgaaatg ttacttagct
tctttttttt 660ccttcccata aaaactgttt atgttccttc tttttattcg
gtgtatgttt tggattccta 720ccaagttatg agacctaata attatgattt
tgtgctttgt ttgtaaaaaa aaaaaaaaaa 780a 781125762DNANicotiana
benthamiana 125tctttctgtc ttaagtgtaa ttaacaatgg cttcctcagt
tctttcctca gcagcagttg 60ccacccgcag caatgttgct caagctaaca tggttgcacc
tttcactggt cttaagtcag 120ctgcctcgtt ccctgtttca aggaagcaaa
accttgacat cacttccatt gccagcaacg 180gcggaagagt gcaatgcatg
caggtgtggc caccaattaa caagaagaag tacgagactc 240tctcatacct
tcctgatctg agcgtggagc aattgcttag cgaaattgag tacctcttga
300aaaatggatg ggttccttgc ttggaattcg agactgagcg cggatttgtc
taccgtgaac 360accacaagtc accgggatac tatgacggca gatactggac
catgtggaag ttgcctatgt 420tcggatgcac tgatgccacc caagtgttgg
ccgaggtgga agaggcgaag aaggcatacc 480cacaggcctg gatccgtatt
attggattcg acaacgtgcg tcaagtgcag tgcatcagtt 540tcattgccta
caagccagaa ggctactaag tttcatatta ggacaactta ccctattgtc
600cgactttagg ggcaatttgt ttgaaatgtt acttggcttc tttttttttt
aattttccca 660caaaaactgt ttatgtttcc tactttctat tcggtgtatg
tttttgcatt cctaccaagt 720tatgagacct aataactatg atttggtgct
ttgtttgtaa at 762126683DNANicotiana benthamiana 126tagcaatagc
tttaagctta gaaattattt tcagaaatgg cttcctcagt tatgtcctca 60gcagctgctg
ttgcgaccgg cgccaatgct gctcaagcca acatggttgc acccttcact
120ggcctcaagt ccgcctcctc cttccctgtt accaggaaac aaaaccttga
cattacctcc 180attgctagca atggtggaag agttcaatgc atgcaggtgt
ggccaccaat taacatgaag 240aagtacgaga cactctcata ccttcctgat
ttgagccagg agcaattgct tagtgaagtt 300gagtaccttt tgaaaaatgg
atgggttcct tgcttggaat tcgagactga gcgtggattc 360gtctaccgtg
aacaccacaa ctcaccagga tactacgatg gcagatactg gaccatgtgg
420aagttgccca tgttcgggtg cactgatgcc actcaggtgt tggctgaggt
cgaggaggca 480aagaaggctt acccacaagc ctgggttaga atcattggat
tcgacaacgt ccgtcaagtg 540caatgcatca gttttatcgc ctccaagcca
gaaggctact aaaatctcca tttttaaggc 600aacttatcgt atgtgttccc
cggagaaact gttttggttt tcctgcttcc ttatattatt 660caatgtatgt
ttttgaattc caa 683127700DNANicotiana benthamiana 127aatggcttcc
tcagttatgt cctcagctgc cgctgttgcc accggcgcca atgctgctca 60agccagtatg
gttgcacctt tcactggcct caagtccgca acctccttcc ctgtttccag
120aaaacaaaac cttgacatta cttccattgc tagcaacggc ggaagagttc
aatgcatgca 180ggtgtggcca ccaattaaca agaagaagta cgagacactc
tcataccttc ccgatttgag 240ccaggagcaa ttgcttagtg aagttgagta
cctgttgaaa aatggatggg ttccttgctt 300ggaattcgag actgagcgtg
gattcgtcta ccgtgaacac cacagctcac caggatatta 360tgatggcaga
tactggacca tgtggaagtt gcccatgttc gggtgcactg atgccactca
420ggtgttggct gaggtcgagg aggcaaagaa ggcttaccca caagcctggg
ttagaatcat 480tggattcgac aatgtccgtc aagtgcaatg catcagtttc
atcgcctaca agccagaagg 540ctactagaat ctccattttt aaggcaactt
atcgtatgtg ttccccggag aaactgtttt 600ggtttttcct gcttcattat
attattcaat gtatgttttt gaattccaat caaggttatg 660agaactaata
atgacattta atttgtttct tttctatata 700128698DNANicotiana benthamiana
128taaataatta attgcaacaa tggcttcctc tgtgatttcc tcagctgctg
ccgttgccac 60cggcgctaat gctgctcaag ccagcatggt tgcacccttc actggcctca
aatctgcttc 120ctccttccct gttaccagaa aacaaaacct tgacattaca
tccattgcta gcaatggtgg 180aagagtccaa tgcatgcagg tgtggccacc
aattaacatg aagaagtacg agacactctc 240ataccttcct gatttgagcc
aggagcaatt gcttagtgaa gttgagtatc ttttgaaaaa 300tggatgggtt
ccttgcttgg aattcgagac tgagcgtgga tttgtctacc gtgaacatca
360cagctcacca ggatactacg atggcagata ctggaccatg tggaagttgc
ccatgttcgg 420gtgcactgat gccactcagg tgttggctga ggtcgaggag
gcaaagaagg cttacccaca 480agcctgggtt agaatcattg gattcgacaa
cgtccgtcaa gtgcaatgca tcagttttat 540cgcctccaag ccagaaggct
actaaaatct ccatttttaa ggcaacttat cgtatgtgtt 600ccccggagaa
actgttttgg ttttcctgct tcattatatt attcaatgta tgtttttgaa
660ttccaatcaa ggttatgaga actaataatg acatttaa 698129727DNANicotiana
benthamiana 129gcacgaggct tcctcagtta tgtcctcagc tgccgctgtt
tccaccggcg ccaatgctgt 60tcaagccagc atggtcgcac ccttcactgg cctcaaggcc
gcctcctcct tcccggtttc 120caggaaacaa aaccttgaca ttacttccat
tgctagaaat ggtggaagag tccaatgcat 180gcaggtgtgg ccgccaatta
acaagaagaa gtacgagaca ctctcatacc ttcctgattt 240gagcgtggag
caattgctta gcgaaattga gtaccttttg aaaaatggat gggttccttg
300cttggaattc gagactgagc atggattcgt ctaccgtgaa caccaccact
caccaggata 360ctacgatggc agatactgga cgatgtggaa gttgcccatg
ttcgggtgca ccgatgccac 420tcaggtcttg gctgaggtag aggaggccaa
gaaggcttac ccacaagcct gggtcagaat 480cattggattc gacaacgtcc
gtcaagtgca atgcatcagt ttcatcgcct acaagcccga 540aggctattaa
aatctccatt tttaggacag cttaccctat gtattcaggg gaagtttgtt
600tgaattctcc tggagaaact gttttggttt tcctttgttt taatcttctt
tctattatat 660ttttggattt tactcaagtt tataagaact aataataatc
atttgtttcg ttactaaaaa 720aaaaaaa 72713025DNAArtificial
Sequenceoligonucleotide 130ccactcttct gcaggtgcaa aaacc
2513128DNAArtificial Sequenceoligonucleotide 131acatggtact
tcaatatttt tgctttgc 2813228DNAArtificial Sequenceoligonucleotide
132gatctttgta aaggccgaca gggttcac 2813329DNAArtificial
Sequenceoligonucleotide 133ttcctcagtt ctttcctcag cagcagttg
2913424DNAArtificial Sequenceoligonucleotide 134ctcagttatg
tcctcagcag ctgc 2413524DNAArtificial Sequenceoligonucleotide
135tcctcagtta tgtcctcagc tgcc 2413622DNAArtificial
Sequenceoligonucleotide 136tgtgatttcc tcagctgctg cc
2213723DNAArtificial Sequenceoligonucleotide 137aactcagtag
ccgtcaggct tgg 2313830DNAArtificial Sequenceoligonucleotide
138aatatgaaac ttagtagcct tctggcttgt 3013923DNAArtificial
Sequenceoligonucleotide 139gtttctccgg ggaacacata cga
2314027DNAArtificial Sequenceoligonucleotide 140aaacaaactt
cccctgaata cataggg 2714121RNAArtificial Sequenceoligonucleotide
141uuuggauuga agggagcucu a 2114221RNAArtificial
Sequenceoligonucleotide 142uuuggaaaua ugggaucucu u
2114321RNAArtificial Sequenceoligonucleotide 143aagagauccc
auauuuccaa a 2114421RNAArtificial Sequenceoligonucleotide
144gagccaagga ugacuugccg g 2114521RNAArtificial
Sequenceoligonucleotide 145gaguuuaguc ugacuuggcc a
2114621RNAArtificial Sequenceoligonucleotide 146gaucucuuuc
cagucuucag g 2114721RNAArtificial Sequenceoligonucleotide
147uggccaaguc agacuaaacu c 2114821RNAArtificial
Sequenceoligonucleotide 148ccugaagacu ggaaagagau c
2114921RNAArtificial Sequenceoligonucleotide 149uuucgaauuc
cgggcgagga a 2115021RNANicotiana benthamiana 150uuccuugcuu
ggaauucgag a 21151273DNAArtificialmiRCPC1(159) precursor template
151acagtttgct tatgtcggat ccataatata tttgacaaga tactttgttt
ttcgatagat 60cttgatctga cgatggaaga agaggtgagt aatgttgaaa catgagttga
gcagggtaaa 120gaaaagctgc taagctatgg atcccataag ccctaatcct
tgtaaagtaa aaaaggattt 180ggttatatgg attgcatatc tcaggagctt
taacttgccc tttaatggct tttactcttc 240tttcgatact actcacctct
tcatcccggg tca 27315221DNAArtificialmiRCPC1(159) mature template
152tttcgatact actcacctct t 21153273DNAArtificialmiRCPC3(159)
precursor template 153acagtttgct tatgtcggat ccataatata tttgacaaga
tactttgttt ttcgatagat 60cttgatctga cgatggaagc tcgttggcga caggtgggag
catgagttga gcagggtaaa 120gaaaagctgc taagctatgg atcccataag
ccctaatcct tgtaaagtaa aaaaggattt 180ggttatatgg attgcatatc
tcaggagctt taacttgccc tttaatggct tttactcttc 240ctcccacctg
acgccaacga gcatcccggg tca 27315421DNAArtificialmiRCPC3(159) mature
template 154ctcccacctg acgccaacga g
21155273DNAArtificialmiRP69(159) precursor template 155acagtttgct
tatgtcggat ccataatata tttgacaaga tactttgttt ttcgatagat 60cttgatctga
cgatggaagc cacaagacaa tcgagacttt catgagttga gcagggtaaa
120gaaaagctgc taagctatgg atcccataag ccctaatcct tgtaaagtaa
aaaaggattt 180ggttatatgg attgcatatc tcaggagctt taacttgccc
tttaatggct tttactcttc 240aaagtctcga ttgtcttgtg gcatcccggg tca
27315621DNAArtificialmiRP69(159) mature template 156aaagtctcga
ttgtcttgtg g 21157222DNAArtificialmiRPDS1(169) precursor template
157aatgatgatt acgatgatga gagtctctag ttgtatcaga gggtcttgca
tggaagaata 60gagaatgagg ttgagtttag tctgacttgg ccagtttttt taccaatgaa
tctaattaac 120tgattctggt gttggccaag tcagactaaa ctctgtttcc
ttctcttctt ttggatgtca 180gactccaaga tatctatcat catgaatcgt
gatcaaactt tg 22215821DNAArtificialmiRPDS1(169) mature template
158gagtttagtc tgacttggcc a
21159552DNAArtificialmiRPDS1(169)-CPC3(159) precursor template
159cacctaggaa tgatgattac gatgatgaga gtctctagtt gtatcagagg
gtcttgcatg 60gaagaataga gaatgaggtt gagtttagtc tgacttggcc agttttttta
ccaatgaatc 120taattaactg attctggtgt tggccaagtc agactaaact
ctgtttcctt ctcttctttt 180ggatgtcaga ctccaagata tctatcatca
tgaatcgtga tcaaactttg aagggtgggc 240gactaggaca gtttgcttat
gtcggatcca taatatattt gacaagatac tttgtttttc 300gatagatctt
gatctgacga tggaagctcg ttggcgacag gtgggagcat gagttgagca
360gggtaaagaa aagctgctaa gctatggatc ccataagccc taatccttgt
aaagtaaaaa 420aggatttggt tatatggatt gcatatctca ggagctttaa
cttgcccttt aatggctttt 480actcttcctc ccacctgacg ccaacgagca
tcccgggtca aagggtgggc gactagtcta 540gactcgagta tt
55216021RNAArabidopsis thaliana 160uuuggauuga agggagcucu a
21161184RNAArabidopsis thaliana 161guagagcucc uuaaaguuca aacaugaguu
gagcagggua aagaaaagcu gcuaagcuau 60ggaucccaua agcccuaauc cuuguaaagu
aaaaaaggau uugguuauau ggauugcaua 120ucucaggagc uuuaacuugc
ccuuuaaugg cuuuuacucu ucuuuggauu gaagggagcu 180cuac
18416221RNAArtificialmiRNA 162acuugcucac gcacucgacu g
21163261DNAArtificialmiRNA precursor 163cagtttgctt atgtcggatc
cataatatat ttgacaagat actttgtttt tcgatagatc 60ttgatctgac gatggaagca
gtcgagtgcg tgagcaagtc atgagttgag cagggtaaag 120aaaagctgct
aagctatgga tcccataagc cctaatcctt gtaaagtaaa aaaggatttg
180gttatatgga ttgcatatct caggagcttt aacttgccct ttaatggctt
ttactcttca 240cttgctcacg cactcgactg c 26116421RNAArtificialmiRNA
164aaagucucga uugucuugug g 21165261DNAArtificialmiRNA precursor
165cagtttgctt atgtcggatc cataatatat ttgacaagat actttgtttt
tcgatagatc 60ttgatctgac gatggaagcc acaagacaat cgagactttc atgagttgag
cagggtaaag 120aaaagctgct aagctatgga tcccataagc cctaatcctt
gtaaagtaaa aaaggatttg 180gttatatgga ttgcatatct caggagcttt
aacttgccct ttaatggctt ttactcttca 240aagtctcgat tgtcttgtgg c
261166551DNAArtificialhomo-polymeric pre-miRNA 166cagtttgctt
atgtcggatc cataatatat ttgacaagat actttgtttt tcgatagatc 60ttgatctgac
gatggaagcc acaagacaat cgagactttc atgagttgag cagggtaaag
120aaaagctgct aagctatgga tcccataagc cctaatcctt gtaaagtaaa
aaaggatttg 180gttatatgga ttgcatatct caggagcttt aacttgccct
ttaatggctt ttactcttca 240aagtctcgat tgtcttgtgg catcccgggt
caaagggtgg gcgactagga cagtttgctt 300atgtcggatc cataatatat
ttgacaagat actttgtttt tcgatagatc ttgatctgac 360gatggaagca
gtcgagtgcg tgagcaagtc atgagttgag cagggtaaag aaaagctgct
420aagctatgga tcccataagc cctaatcctt gtaaagtaaa aaaggatttg
gttatatgga 480ttgcatatct caggagcttt aacttgccct ttaatggctt
ttactcttca cttgctcacg 540cactcgactg c 55116723DNATurnip mosaic
virus 167cgatttaggc ggcagataca gcg 2316835DNATurnip mosaic virus
168attctcaatg gtttaatggt ctggtgcatt gagaa 3516926DNATurnip mosaic
virus 169ataaacggaa tgtgggtgat gatgga 2617021DNATurnip mosaic virus
170gatcaggtgg aattcccgat c 2117132DNATurnip mosaic virus
171cacgccaaac ccacatttag gcaaataatg gc 3217268DNATurnip mosaic
virus 172gctgaagcgt acattgaaaa gcgtaaccaa gaccgaccat acatgccacg
atatggtctt 60cagcgcaa 6817380DNATurnip mosaic virus 173gaaatgactt
ctagaactcc aatacgtgcg agagaagcac acatccagat gaaagcagca 60gcactgcgtg
gcgcaaataa 8017426DNATurnip mosaic virus 174acaacggtag agaacacgga
gaggca 26175117RNAArabidopsis thaliana 175gucguuguuu guaggcgcag
caccauuaag auucacaugg aaauugauaa auacccuaaa 60uuaggguuuu gauauguaua
ugagaaucuu gaugaugcug caucaacaau cgacggc 11717660DNAArabidopsis
thaliana 176tagggttttg atatgtatat gagaatcttg atgatgctgc atcaacaatc
gacggctaca 6017760DNAArabidopsis thaliana 177acaaagttct ctatgaaaat
gagaatcttg atgatgctgc atcggcaatc aacgactatt 6017860DNAArabidopsis
thaliana 178gagcctttat tttttggttt gagaatcttg atgatgctgc agcggcaatt
aaatggctta 6017960DNAArabidopsis thaliana 179tagatttttg atgtatgtat
gagaatcttg atgatgctgc agctgcaatc agtggcttac 6018060DNAArabidopsis
thaliana 180aaaagggttc cttatcgagt gggaatcttg atgatgctgc atcagcaaat
acatggctac 6018160DNASolanum lycopersicum 181gctggctatt tgaaactcac
gagaatcttg atgatgctgc atcagcaata aacgactatt 6018260DNAGlycine max
182tccatcggtc tttttgatgt gagaatcttg atgatgctgc atcagccata
aacggcttta 6018360DNASolanum tuberosum 183tgcccaattt ttgaatacat
gagaatcttg atgatgctgc attggcaaat tgatgacttg
6018460DNAOryza sativa 184acgcatgtgt atatatgtgt gggaatcttg
atgatgctgc atcggaaatt aatgactaag 6018560DNAOryza sativa
185gttggctgac tatatgtgat gagaatcttg atgatgctgc atcagcaaac
gctcgactac 6018660DNAOryza sativa 186ttcaagtgta gtcatcgtgc
gtgaatcttg atgatgctgc accagcaaag agccggccgt 6018760DNAOryza sativa
187catatacatc cgatttggct gagaatcttg atgatgctgc atccgcagac
aagcgccttt 601881374DNATurnip mosaic virus 188agtgcggcag gagctaattt
ctggaaaggc ttcgacagat gcttcctcgc ataccgtagt 60gacaatcgcg atcacacatg
ctattcaggg ctagatgtca ctgagtgcgg cgaagtagca 120gcactaatgt
gtttggctat gttcccatgc ggaaagataa cctgtcctga ctgcgtgaca
180gacagtgagc tatcccaagg acaagcaagt ggaccatcta tgaagcacag
gttgacacag 240ctacgcgatg tcatcaagtc aagctaccca cgctttaaac
atgcagtgca gatactagat 300aggtacgagc aatcactgag tagtgcaaac
gagaactacc aagatttcgc agaaatccag 360agcataagcg atggagttga
aaaagctgca ttcccacatg tcaacaagct aaactcaata 420ttgataaagg
gagccacagc gacaggagag gaattctcac aggctacgaa gcatttgctc
480gagatagcac gctacctgaa gaacagaact gagaatatcg agaagggttc
acttaagtct 540tttcgtaaca agatttccca gaaggcgcac atcaacccaa
cactaatgtg cgacaaccaa 600ctcgacagaa atggaaattt catatggggt
gagaggggat accacgcgaa acggttcttt 660agcaactact ttgagataat
cgatccaaag aaaggctaca cccagtacga aacaagagca 720gtgccaaatg
ggtcacggaa acttgcaatc ggcaaactaa tagtcccaac gaacttcgaa
780gttctaaggg aacaaatgaa aggcgaacca gtggaaccat acccagtaac
agccgagtgt 840gtgagcaaat tacagggtga tttcgtccat gcatgttgct
gtgtcacaac agaatcaggc 900gatccaatct tgtctgaaat caaaatgcca
accaaacacc acctagtgat tggtaacagc 960ggcgatccaa agtacataga
tctccctgag atcgaggaga ataaaatgta catagcaaaa 1020gaaggttatt
gctacatcaa tatcttccta gccatgttag tgaatgtcaa ggagtcacag
1080gcaaaggagt tcacgaaagt tgtcagagac aaattagttg gcgaacttgg
caagtggcca 1140actttgctag atgtagcaac cgcttgttat ttcctgaagg
tgttttaccc agacgttgct 1200aacgccgaac tgccacgcat gttagtggac
cacaagacaa agataattca tgttgttgat 1260tcatatgggt cactgtcaac
tggatatcac gtccttaaaa caaacactgt ggaacaactc 1320atcaaattca
cgagatgcaa tctggaatca agtttgaaac actaccgcgt cgga
13741891887DNATurnip yellow mosaic virus 189atgagtaatg gccttccaat
tagcattgga cgcccttgca cccacgactc acagagatcc 60ctctctgcac ccaattctcg
aatccacagt ggattcaatt cgctcctcga tacagaccta 120cccatggtcc
attccgaagg aacttctacc cctactcaac tcctacggca tcccaacatc
180tggtttggga acctcccacc acccccacgc cgcccacaag acaatcgaga
cttttctcct 240ttgcacccac tggtctttcc aggccaccac tcccagctcc
gtcatgttca tgaaacccag 300caagttcaac aaacttgccc aggtaaactc
aaactttcgg gagctgaaga actaccgcct 360gcaccccaac gacagcactc
gttacccctt cacatcacca gaccttcccg ttttccccac 420cattttcatg
cacgacgccc tgatgtatta ccatccgtcc cagatcatgg acctgttctt
480gcggaaacca aacctcgaac gtctgtacgc cagcctcgta gtgccacccg
aggcccatct 540ttccgaccaa tccttctacc caaagttgta cacgtacacg
acgacccgcc acactcttca 600ctacgtccca gagggtcacg aagccggcag
ctacaaccaa ccgtccgacg cccactcttg 660gctccgaatc aattccattc
gcctcggcaa ccaccacctc tcagtgacga tcctggaatc 720ctggggcccc
gtccactcgc tcctaattca acgagggacc cccccccccg acccatcact
780ccaggcccct ccaacactca tggcctcaga cctctttcgg tcttaccaag
agcctcgcct 840cgacgtggtc tccttccgaa tccccgacgc catcgaactt
ccacaggcca cattcctcca 900acaaccactt cgagaccgac tggtcccccg
agccgtctac aacgccctgt tcacctatac 960cagagcagtc cgcacactcc
gaacttcaga cccagcagca ttcgtaagga tgcactcctc 1020caaaccggac
cacgattggg tcacctcgaa tgcctgggac aacctgcaaa ccttcgcact
1080tctgaacgtt cccctccgac caaacgtcgt ctaccacgtt cttcagagcc
caatcgcctc 1140cctaagcctt tacctgaggc aacattggcg ccgtcttacc
gccaccgccg ttcctatcct 1200ttccttccta accctcctgc agcgcttcct
tccattgcct atacctctag cagaggtaaa 1260atccatcaca gccttccgaa
gggagcttta ccgaaagaag gagccccacc accccctcga 1320cgtcttccat
ctccagcacc gcatccgcaa ctaccactcc gcgatctcgg ccgtacgccc
1380ggcttcccca ccccaccaaa aactcccaca cgcactccag aaagccgcat
tactgcttct 1440ccgaccgata tcgcccctct tgacagcgac cccgttcttt
cggtccgaac agaagtccat 1500gctcccgaac gccgaacttt catggaccct
gaagcgcttc gctctgccct ggcaagcctc 1560cctagtcctc ctcgctctgt
cggaatcatc catactgctc cacaaactgt tctccccgcc 1620aaccctccaa
gcccaacacg acacctacca ccgacatctc caccctggat cctacagtct
1680ccagtgggag aggacgccat tgtcgattcc gaggacgaca gcatttcttc
cttttactcc 1740cacgacttcg acagcccctc cggaccgctc cgaagccagt
ctccctcccg ctttcgcctc 1800taccttcgtt ccccgtccac ctccagcggc
atcgagccct ggagcccagc ctcctacgac 1860tacggcagcg cccccgacac cgattga
1887
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