U.S. patent application number 15/705312 was filed with the patent office on 2018-04-12 for modulation of transgene expression in plants.
This patent application is currently assigned to Dow AgroSciences LLC. The applicant listed for this patent is Dow AgroSciences LLC. Invention is credited to Andrew J. Bowling, Marcelo Ariel German, Todd P. Glancy, Sandeep Kumar, Heather Pence, Andrew E. Robinson, Shreedharan Sriram, Pohao Wang, Carla N. Yerkes.
Application Number | 20180100162 15/705312 |
Document ID | / |
Family ID | 61830619 |
Filed Date | 2018-04-12 |
United States Patent
Application |
20180100162 |
Kind Code |
A1 |
Kumar; Sandeep ; et
al. |
April 12, 2018 |
Modulation of Transgene Expression in Plants
Abstract
This disclosure concerns the use of endogenous plant RNAi
machinery to preferentially or specifically reduce transgene
expression. In some embodiments, the disclosure concerns specific
reduction of transgene expression in seed tissues of a dicot
plant.
Inventors: |
Kumar; Sandeep; (Carmel,
IN) ; German; Marcelo Ariel; (Lake Oswego, OR)
; Wang; Pohao; (Carmel, IN) ; Glancy; Todd P.;
(Fairmount, IN) ; Sriram; Shreedharan;
(Indianapolis, IN) ; Yerkes; Carla N.;
(Crawfordsville, IN) ; Bowling; Andrew J.;
(Zionsville, IN) ; Pence; Heather; (Indianapolis,
IN) ; Robinson; Andrew E.; (Calvert City,
KY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Dow AgroSciences LLC |
Indianapolis |
IN |
US |
|
|
Assignee: |
Dow AgroSciences LLC
Indianapolis
IN
|
Family ID: |
61830619 |
Appl. No.: |
15/705312 |
Filed: |
September 15, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62406560 |
Oct 11, 2016 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 15/8234 20130101;
C12N 15/8218 20130101; C12N 15/8274 20130101; C12N 15/8223
20130101 |
International
Class: |
C12N 15/82 20060101
C12N015/82 |
Claims
1. A nucleic acid expression cassette comprising a gene of
interest, a plant promoter operably linked to the gene of interest,
and a target site for at least one sRNA molecule present in a dicot
plant.
2. The expression cassette of claim 1, wherein the gene of interest
is an agronomic trait gene, an insect resistance gene, an herbicide
tolerance gene, a non-coding RNA gene, or a selectable marker
gene.
3. The expression cassette of claim 1, wherein the target site is
between about 20 and about 25 nucleotides in length.
4. The expression cassette of claim 1, wherein the dicot plant is
selected from the group consisting of Arabidopsis, soybean,
rapeseed, cotton, and tobacco.
5. A transgenic plant cell comprising the expression cassette of
claim 1, wherein the gene of interest is heterologous to the plant
cell, and wherein the at least one sRNA molecule is an endogenous
sRNA of the plant cell.
6. The transgenic plant cell of claim 5, wherein the endogenous
sRNA is expressed in the plant in a tissue-specific,
tissue-preferred, or cell type-specific manner.
7. A transgenic plant tissue, plant part, or plant, comprising the
transgenic plant cell of claim 5.
8. The transgenic plant tissue, plant part, or plant of claim 7,
wherein the endogenous sRNA is expressed predominantly in seed
tissue.
9. The transgenic plant tissue, plant part, or plant of claim 8,
wherein the sRNA is miR1512 or miR3522.
10. The transgenic plant tissue, plant part, or plant of claim 9,
wherein the target site is selected from the group consisting of
SEQ ID NO:2, SEQ ID NO:4, and combinations thereof.
11. The transgenic plant tissue, plant part, or plant of claim 9,
wherein the gene of interest is an herbicide tolerance gene, and
wherein expression of the herbicide tolerance gene is downregulated
in seed tissue.
12. The transgenic plant tissue, plant part, or plant of claim 7,
wherein the target site is modified for partial or complete
downregulation of the gene of interest.
13. A method for producing a transgenic plant cell, the method
comprising: providing a nucleic acid expression cassette comprising
an herbicide tolerance gene, a plant promoter operably linked to
the gene, and a target site for at least one sRNA molecule; and
transforming a plant cell with the expression cassette, wherein the
plant cell expresses the sRNA.
14. The method of claim 13, wherein the sRNA hybridizes to mRNA
transcribed from the herbicide tolerance gene and thereby reduces
the expression of the herbicide tolerance gene.
15. The method of claim 13, wherein the sRNA is an endogenous sRNA
that is expressed in the plant cell in a tissue-preferred,
tissue-specific, or cell type-specific manner.
16. The method of claim 13, wherein the sRNA is miR1512 or
miR3522.
17. The method of claim 16, wherein the target site is a nucleic
acid having at least 90% sequence identity to a nucleic acid
selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, and
combinations thereof.
18. The method of claim 13, wherein the sRNA is encoded by a
polynucleotide that is operably linked to a non-constitutive
promoter in the genome of the plant cell.
19. The method according to claim 18, wherein the non-constitutive
promoter is a tissue-preferred, tissue-specific, or cell
type-specific.
20. The method according to claim 18, wherein the non-constitutive
promoter is a tissue-specific promoter.
21. The method according to claim 20, wherein the tissue-specific
promoter initiates transcription selectively in vegetative
tissue.
22. A plant tissue comprising a plant cell produced by the method
according to claim 13.
23. A plant comprising the plant tissue of claim 22.
24. A method for producing a transgenic plant cell, the method
comprising: providing a nucleic acid expression cassette comprising
a gene of interest and a target site of a sRNA molecule; providing
a plant cell, wherein the sRNA is expressed predominantly in seed
tissue; and transforming the plant cell with the expression
cassette.
25. The method of claim 24, wherein the sRNA is miR1512.
26. The method of claim 25, wherein the target site is SEQ ID
NO:4.
27. A method for inhibiting expression of a gene of interest in a
plant, comprising growing a plant comprising a nucleic acid
expression cassette comprising a gene of interest, a plant promoter
operably linked to the gene of interest, and a target site for at
least one sRNA molecule present in the plant, wherein expression of
the gene of interest is downregulated only in tissues where the
sRNA molecule is expressed.
28. The method of claim 27, wherein the gene of interest is an
agronomic trait gene, an insect resistance gene, an herbicide
tolerance gene, a non-coding RNA gene, or a selectable marker
gene.
29. The method of claim 27, wherein the plant is a dicot plant
selected from the group consisting of soybean, canola, cotton, and
tobacco.
30. The method of claim 27, wherein the gene of interest is
heterologous to the plant, and wherein the at least one sRNA
molecule is an endogenous sRNA of the plant.
31. The method of claim 30, wherein the endogenous sRNA is
expressed predominantly in seed tissue.
32. The method of claim 31, wherein the sRNA is miR1512.
33. The method of claim 32, wherein the target site is SEQ ID NO:4.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] The present application claims priority to the benefit of
U.S. Provisional Patent Application Ser. No. 62/406,560 filed Oct.
11, 2016 the disclosure of which is hereby incorporated by
reference in its entirety.
FIELD OF THE DISCLOSURE
[0002] The present disclosure relates to polynucleotides,
constructs and methods for the control of transgene expression in
plants. Specific embodiments herein relate to constructs and
methods for utilizing the native RNA interference (RNAi) machinery
of a plant cell to effect tissue-specific expression of a gene of
interest, for example, in seed or root tissues.
BACKGROUND
[0003] RNA interference (RNAi) is a process utilizing endogenous
cellular pathways, whereby an interfering RNA (iRNA) molecule
(e.g., a dsRNA molecule) that is specific for a target gene
sequence results in the reduced expression (e.g., by degradation)
of the mRNA encoded thereby. In recent years, RNAi has been used to
perform gene "knockdown" (i.e., downregulation or inhibition of
expression) in a number of species and experimental systems; for
example, C. elegans, plants, insect embryos, and cells in tissue
culture. See, e.g., Fire et al. (1998) Nature 391:806-11; Martinez
et al. (2002) Cell 110:563-74; McManus and Sharp (2002) Nature Rev.
Genetics 3:737-47.
[0004] RNAi accomplishes degradation of mRNA through an endogenous
pathway including the DICER protein complex. DICER cleaves long
dsRNA molecules into short double-stranded fragments of
approximately 20-24 nucleotides. The inhibitory double-stranded RNA
is unwound into two single-stranded RNAs: the passenger strand and
the guide strand. The passenger strand is degraded, and the guide
strand is incorporated into the RNA-induced silencing complex
(RISC). Post-transcriptional gene silencing (translational
repression) occurs when the guide strand binds specifically to a
complementary sequence of an mRNA molecule and induces cleavage by
Argonaute, the catalytic component of the RISC complex.
[0005] Plant micro-RNAs (miRNAs) are typically produced from
fold-back structures having a partial double-stranded structure
(e.g., "hairpins"), and usually are nearly perfectly complementary
with "target sites," which are found most commonly in
protein-coding regions of the genome. As a result, plant miRNAs
function generally to guide mRNA cleavage. Watson et al. (2005)
FEBS Lett. 579:5982-7. In contrast, animal miRNAs contain
relatively low levels of complementarity to their target sites, and
thus generally do not guide cleavage, but rather function to
repress expression at the translational or co-translational level.
Watson et al. (2005), supra; Tomari and Zamore (2005) Genes Dev.
19(5):517-29. Although miRNA sequences are not conserved between
plants and animals, the RNAi pathways that utilize these genes are
highly similar. Millar and Waterhouse (2005) Funct. Integr.
Genomics 5:129-35. For example, while the biogenesis of miRNAs in
plants is accomplished by a different set of related enzymes than
accomplish the biogenesis of animal miRNAs, the miRNA molecules
themselves have a characteristic structure that is capable of
effecting mRNA cleavage or translational repression, depending on
their degree of sequence complementarity to the target gene.
[0006] In addition to miRNAs, plants also produce endogenous 21-25
nucleotide small inhibitory-RNAs (siRNAs). Most of these differ
from miRNAs, in that they arise from double-stranded RNA (rather
than imperfect fold-back structures), which in some cases are
generated by the activity of RNA-Dependent RNA Polymerases
(RDRs).
[0007] Most plants contain four DICER-LIKE (DCL) proteins, one of
which (DCL1) is necessary for maturation of most miRNA precursors.
Kurihara and Watanabe (2004) Proc. Natl. Acad. Sci. USA
101:12753-8. Animal miRNA precursor processing requires the
sequential nucleolytic activity of DROSHA and DICER. Lee et al.
(2003) Nature 425:415-9. In animals, Exportin-5 (ExpS) regulates
the transport of pre-miRNAs from the nucleus to the cytoplasm.
Bohnsack et al. (2004) RNA 10:185-91.
[0008] Only RNA transcripts complementary to the siRNA and/or miRNA
are cleaved and degraded by RNAi, and thus the knock-down of mRNA
expression is sequence-specific. The gene silencing effect of RNAi
persists for days and, under experimental conditions, can lead to a
decline in abundance of the targeted transcript of 90% or more,
with consequent reduction in levels of the corresponding
protein.
BRIEF SUMMARY OF THE DISCLOSURE
[0009] Described herein are the compositions and methods directed
to a novel plant small RNA (sRNA)-mediated approach for modulation
of transgene expression in plants. The approach utilizes sRNAs
(e.g., endogenous siRNAs and miRNAs) that are expressed in specific
tissues, preferred tissues, or cell types for targeted repression
or knockdown of (trans) gene expression. In some embodiments, a
target site of an sRNA is fused within a transgene expression
cassette sequence that expresses an agronomic trait gene, an insect
resistance gene, an herbicide tolerance gene, a non-coding RNA
gene, or a selectable marker gene. Tissue-specific expression of
sRNA (e.g., endogenous sRNAs) in the tissue results in
downregulation of the transgene, which results in selective
expression of the transgene only in desired tissues. In some
embodiments where the transgene is a herbicide tolerance gene,
downregulation of the transgene confers sensitivity to the
herbicide upon the specific tissue, while other plant tissues where
the sRNA is not expressed remain tolerant.
[0010] In some embodiments, sRNAs are specifically expressed in
pollen, ovule, cotyledon, hypocotyl, anther, pistil, pod, flower,
shoot or stalk tissue of a plant, and in particular embodiments in
leaf, root or seed tissue of a plant, so as to repress/knockdown
the expression of a transgene in these specific tissues. Particular
embodiments include the sRNA-mediated tissue-specific expression of
any transgene in plants. In examples, the transgene is an herbicide
tolerance gene. In some embodiments, sRNAs are specifically or
preferentially expressed in, for example, the root or seed tissue
of a plant, so as to specifically or preferentially reduce
expression of a transgene in the tissue.
[0011] Described herein are nucleic acid expression constructs,
which constructs comprise a gene of interest, a plant promoter
operably linked to the gene of interest, and a target site for at
least one sRNA molecule. In embodiments, RNA may be transcribed
from the gene of interest in vivo when the construct is introduced
into a plant cell, which RNA is then degraded by an RNAi mechanism
under the control of the sRNA molecule(s) only in tissues or at
times where/when the sRNA is also expressed. In particular
embodiments, the gene of interest is an agronomic trait gene (e.g.,
nitrogen use efficiency gene, water use efficiency gene, or
nutritional quality gene (e.g., modified oil profile)), an
herbicide tolerance gene, an insect resistance gene, or a
selectable marker gene. In particular embodiments, the internal
sRNA targeting site is, for example, 20-25 nucleotides in length.
In certain embodiments, the sRNA is miR1512 or miR3522. Further, in
certain embodiments, the target site is SEQ ID NO:2, SEQ ID NO:4,
or combinations thereof.
[0012] Also described herein are transgenic plant cells, plant
tissues, and plants comprising at least one of the foregoing
nucleic acid expression constructs. In particular embodiments, the
sRNA molecule is differentially expressed in different plant cells
and tissues. For example, the gene (e.g., transgene) encoding the
sRNA molecule may be operably linked to a non-constitutive promoter
(e.g., a tissue-specific promoter). In particular embodiments, the
sRNA is an endogenous sRNA of the plant cell. In certain examples,
the sRNA is an endogenous sRNA that is expressed predominantly in
certain tissues (e.g., pollen, ovule, cotyledon, hypocotyl, anther,
pistil, pod, flower, shoot, stalk, root, leaf, or seed tissues),
which sRNA directs degradation specifically in these tissues of RNA
transcribed from the gene of interest, wherein the expression of
RNA transcribed from the gene of interest in other tissues is
essentially unaffected. Also described herein are methods of
producing the transgenic plant cells by transforming plant cells
with the nucleic acid expression cassettes disclosed herein.
[0013] Further described herein are methods for reducing expression
of a gene of interest in a plant by growing a plant comprising the
nucleic acid expression cassettes described herein and comprising a
gene of interest and a sRNA target site. In such methods,
expression of the gene of interest is downregulated only in tissues
or at times where the sRNA molecule is expressed.
[0014] The foregoing and other features will become more apparent
from the following detailed description of several embodiments,
which proceeds with reference to the accompanying figures.
BRIEF DESCRIPTION OF THE FIGURES
[0015] FIG. 1 is a diagram of particular embodiments, wherein
tissue-specific transgene expression modulation is conferred using
a target site of naturally occurring sRNA in a specific tissue.
[0016] FIG. 2 is a schematic of pDAB113181 which contains AAD-12
expression cassette comprising the ATUBI401.8 promoter, AAD-12 v1
coding sequence, and the AtuORF23 3' UTR v1. Further contained on
this plasmid is the pat gene expression cassette, which comprises
the CsVMV promoter v2, pat v6 coding sequence, and AtuORF1 3' UTR
v3 within T-DNA.
[0017] FIG. 3 is a schematic of pDAB113177 which contains AAD-12
expression cassette comprising the ATUBI401.8 promoter, AAD-12 v1
coding sequence, miR3522 target site, and the AtuORF23 3' UTR v1.
Further contained on this plasmid is the pat gene expression
cassette, which comprises the CsVMV promoter v2, pat v6 coding
sequence, and AtuORF1 3' UTR v3 within T-DNA.
[0018] FIG. 4 is a schematic of pDAB113183 which contains AAD-12
expression cassette comprising the ATUBI401.8 promoter, AAD-12 v1
coding sequence, miR1512 target site, and the AtuORF23 3' UTR v1.
Further contained on this plasmid is the pat gene expression
cassette, which comprises the CsVMV promoter v2, pat v6 coding
sequence, and AtuORF1 3' UTR v3 within T-DNA.
[0019] FIG. 5 is a graph of data showing the modulation of
transcript expression of aad12 in leaf using miRNA3522 target
site.
[0020] FIG. 6 is a graph of data showing the modulation of
transcript expression of aad12 in cotyledon using miRNA3522 target
site.
[0021] FIG. 7 is a graph of data showing the modulation of
transcript expression of aad12 in seed coat using miRNA3522 target
site.
[0022] FIG. 8 shows brightfield (left panels) and phase contrast
(right panels) microscopy images showing protein expression of
aad12 in cotyledon and seed coat.
[0023] FIG. 9 is a graph of data showing aad12 protein expression
in leaf using miRNA3522 target site.
[0024] FIG. 10 is a photo showing tolerance of T1 plants to 2,4-D @
2240 g ae/ha 14 day after spray.
SEQUENCE LISTING
[0025] The nucleic acid sequences listed in the accompanying
sequence listing are shown using standard letter abbreviations for
nucleotide bases, as defined in 37 C.F.R. .sctn. 1.822. Only one
strand of each nucleic acid sequence is shown, but the
complementary strand is understood to be included by any reference
to the displayed strand. In the accompanying sequence listing:
[0026] SEQ ID NO:1 shows, in the 5' to 3' direction, an exemplary
miR3522 RNA: UGAGACCAAAUGAGCAGCUGA
[0027] SEQ ID NO:2 shows, in the 5' to 3' direction, an exemplary
miRNA target site HTRNA15975.1 for miRNA3522 (SEQ ID NO:1):
TCAGCTGCTCATTTGGTCTCA
[0028] SEQ ID NO:3 shows, in the 5' to 3' direction, an exemplary
miR1512 RNA: UAACUGAACAUUCUUAGAGCAU
[0029] SEQ ID NO:4 in the 5' to 3' direction, an exemplary miRNA
target site
[0030] HTRNA15976.1 for miRNA1512 (SEQ ID NO:3):
ATGCTCTAAGAATGTTCAGTTA
[0031] SEQ ID NO:5 shows an exemplary expression cassette
comprising an aad12 v1 coding region (underlined), followed by
AtuORF23 3' UTR v3 (italics small case). The uppercase no underline
sequence is a sequence for multiple cloning sites:
TABLE-US-00001 ATGGCTCAGACCACTCTCCAAATCACACCCACTGGTGCCACCTTGGGTGC
CACAGTCACTGGTGTTCACCTTGCCACACTTGACGATGCTGGTTTCGCTG
CCCTCCATGCAGCCTGGCTTCAACATGCACTCTTGATCTTCCCTGGGCAA
CACCTCAGCAATGACCAACAGATTACCTTTGCTAAACGCTTTGGAGCAAT
TGAGAGGATTGGCGGAGGTGACATTGTTGCCATATCCAATGTCAAGGCAG
ATGGCACAGTGCGCCAGCACTCTCCTGCTGAGTGGGATGACATGATGAAG
GTCATTGTGGGCAACATGGCCTGGCACGCCGACTCAACCTACATGCCAGT
CATGGCTCAAGGAGCTGTGTTCAGCGCAGAAGTTGTCCCAGCAGTTGGGG
GCAGAACCTGCTTTGCTGACATGAGGGCAGCCTACGATGCCCTTGATGAG
GCAACCCGTGCTCTTGTTCACCAAAGGTCTGCTCGTCACTCCCTTGTGTA
TTCTCAGAGCAAGTTGGGACATGTCCAACAGGCCGGGTCAGCCTACATAG
GTTATGGCATGGACACCACTGCAACTCCTCTCAGACCATTGGTCAAGGTG
CATCCTGAGACTGGAAGGCCCAGCCTCTTGATCGGCCGCCATGCCCATGC
CATCCCTGGCATGGATGCAGCTGAATCAGAGCGCTTCCTTGAAGGACTTG
TTGACTGGGCCTGCCAGGCTCCCAGAGTCCATGCTCACCAATGGGCTGCT
GGAGATGTGGTTGTGTGGGACAACCGCTGTTTGCTCCACCGTGCTGAGCC
CTGGGATTTCAAGTTGCCACGTGTGATGTGGCACTCCAGACTCGCTGGAC
GCCCAGAAACTGAGGGTGCTGCCTTGGTTTGAGTAGTTAGCTTAATCACC
TAGAGCTCGGTCACCAGCATAATTTTTATTAATGTACTAAATTACTGTTT
TGTTAAATGCAATTTTGCTTTCTCGGGATTTTAAtatcaaaatctattta
gaaatacacaatattttgttgcaggcttgctggagaatcgatctgctatc
ataaaaattacaaaaaaattttatttgcctcaattattttaggattggta
ttaaggacgcttaaattatttgtcgggtcactacgcatcattgtgattga
gaagatcagcgatacgaaatattcgtagtactatcgataatttatttgaa
aattcataagaaaagcaaacgttacatgaattgatgaaacaatacaaaga
cagataaagccacgcacatttaggatattggccgagattactgaatattg
agtaagatcacggaatttctgacaggagcatgtcttcaattcagcccaaa
tggcagttgaaatactcaaaccgccccatatgcaggagcggatcattcat
tgtttgtttggttgcctttgccaacatgggagtccaaggtt
[0032] SEQ ID NO:6 shows an exemplary expression cassette
comprising an aad12 v1 coding region (underlined), followed by
miRNA target site HTRNA15975.1 (bold) for miRNA3522 (SEQ ID NO:1)
and AtuORF23 3' UTR v3 (italics small case). The uppercase no
underline sequence is a sequence for multiple cloning sites:
TABLE-US-00002 ATGGCTCAGACCACTCTCCAAATCACACCCACTGGTGCCACCTTGGGTGC
CACAGTCACTGGTGTTCACCTTGCCACACTTGACGATGCTGGTTTCGCTG
CCCTCCATGCAGCCTGGCTTCAACATGCACTCTTGATCTTCCCTGGGCAA
CACCTCAGCAATGACCAACAGATTACCTTTGCTAAACGCTTTGGAGCAAT
TGAGAGGATTGGCGGAGGTGACATTGTTGCCATATCCAATGTCAAGGCAG
ATGGCACAGTGCGCCAGCACTCTCCTGCTGAGTGGGATGACATGATGAAG
GTCATTGTGGGCAACATGGCCTGGCACGCCGACTCAACCTACATGCCAGT
CATGGCTCAAGGAGCTGTGTTCAGCGCAGAAGTTGTCCCAGCAGTTGGGG
GCAGAACCTGCTTTGCTGACATGAGGGCAGCCTACGATGCCCTTGATGAG
GCAACCCGTGCTCTTGTTCACCAAAGGTCTGCTCGTCACTCCCTTGTGTA
TTCTCAGAGCAAGTTGGGACATGTCCAACAGGCCGGGTCAGCCTACATAG
GTTATGGCATGGACACCACTGCAACTCCTCTCAGACCATTGGTCAAGGTG
CATCCTGAGACTGGAAGGCCCAGCCTCTTGATCGGCCGCCATGCCCATGC
CATCCCTGGCATGGATGCAGCTGAATCAGAGCGCTTCCTTGAAGGACTTG
TTGACTGGGCCTGCCAGGCTCCCAGAGTCCATGCTCACCAATGGGCTGCT
GGAGATGTGGTTGTGTGGGACAACCGCTGTTTGCTCCACCGTGCTGAGCC
CTGGGATTTCAAGTTGCCACGTGTGATGTGGCACTCCAGACTCGCTGGAC
GCCCAGAAACTGAGGGTGCTGCCTTGGTTTGAGTAGTTAGCTTAATCACC
TAGAGCTCGGTCAGCTGCTCATTTGGTCTCATCACCAGCATAATTTTTAT
TAATGTACTAAATTACTGTTTTGTTAAATGCAATTTTGCTTTCTCGGGAT
TTTAAtatcaaaatctatttagaaatacacaatattttgttgcaggcttg
ctggagaatcgatctgctatcataaaaattacaaaaaaattttatttgcc
tcaattattttaggattggtattaaggacgcttaaattatttgtcgggtc
actacgcatcattgtgattgagaagatcagcgatacgaaatattcgtagt
actatcgataatttatttgaaaattcataagaaaagcaaacgttacatga
attgatgaaacaatacaaagacagataaagccacgcacatttaggatatt
ggccgagattactgaatattgagtaagatcacggaatttctgacaggagc
atgtcttcaattcagcccaaatggcagttgaaatactcaaaccgccccat
atgcaggagcggatcattcattgtttgtttggttgcctttgccaacatgg gagtccaaggtt
[0033] SEQ ID NO:7 shows an exemplary expression cassette
comprising an aad12 v1 coding region (underlined), followed by
miRNA target site HTRNA17596.1 (bold) for miRNA1512 (SEQ ID NO:3)
and AtuORF23 3' UTR v3 (italics small case). The uppercase no
underline sequence is a sequence for multiple cloning sites:
TABLE-US-00003 ATGGCTCAGACCACTCTCCAAATCACACCCACTGGTGCCACCTTGGGTGC
CACAGTCACTGGTGTTCACCTTGCCACACTTGACGATGCTGGTTTCGCTG
CCCTCCATGCAGCCTGGCTTCAACATGCACTCTTGATCTTCCCTGGGCAA
CACCTCAGCAATGACCAACAGATTACCTTTGCTAAACGCTTTGGAGCAAT
TGAGAGGATTGGCGGAGGTGACATTGTTGCCATATCCAATGTCAAGGCAG
ATGGCACAGTGCGCCAGCACTCTCCTGCTGAGTGGGATGACATGATGAAG
GTCATTGTGGGCAACATGGCCTGGCACGCCGACTCAACCTACATGCCAGT
CATGGCTCAAGGAGCTGTGTTCAGCGCAGAAGTTGTCCCAGCAGTTGGGG
GCAGAACCTGCTTTGCTGACATGAGGGCAGCCTACGATGCCCTTGATGAG
GCAACCCGTGCTCTTGTTCACCAAAGGTCTGCTCGTCACTCCCTTGTGTA
TTCTCAGAGCAAGTTGGGACATGTCCAACAGGCCGGGTCAGCCTACATAG
GTTATGGCATGGACACCACTGCAACTCCTCTCAGACCATTGGTCAAGGTG
CATCCTGAGACTGGAAGGCCCAGCCTCTTGATCGGCCGCCATGCCCATGC
CATCCCTGGCATGGATGCAGCTGAATCAGAGCGCTTCCTTGAAGGACTTG
TTGACTGGGCCTGCCAGGCTCCCAGAGTCCATGCTCACCAATGGGCTGCT
GGAGATGTGGTTGTGTGGGACAACCGCTGTTTGCTCCACCGTGCTGAGCC
CTGGGATTTCAAGTTGCCACGTGTGATGTGGCACTCCAGACTCGCTGGAC
GCCCAGAAACTGAGGGTGCTGCCTTGGTTTGAGTAGTTAGCTTAATCACC
TAGAGCTCGGATGCTCTAAGAATGTTCAGTTATCACCAGCATAATTTTTA
TTAATGTACTAAATTACTGTTTTGTTAAATGCAATTTTGCTTTCTCGGGA
TTTTAAtatcaaaatctatttagaaatacacaatattttgttgcaggctt
gctggagaatcgatctgctatcataaaaattacaaaaaaattttatttgc
ctcaattattttaggattggtattaaggacgcttaaattatttgtcgggt
cactacgcatcattgtgattgagaagatcagcgatacgaaatattcgtag
tactatcgataatttatttgaaaattcataagaaaagcaaacgttacatg
aattgatgaaacaatacaaagacagataaagccacgcacatttaggatat
tggccgagattactgaatattgagtaagatcacggaatttctgacaggag
catgtcttcaattcagcccaaatggcagttgaaatactcaaaccgcccca
tatgcaggagcggatcattcattgtttgtttggttgcctttgccaacatg
ggagtccaaggtt
[0034] SEQ ID NO:8 shows an ATUBI401.8 promoter, utilized in
certain examples herein:
TABLE-US-00004 GTCGACCTGCAGGTCAACGGATCAGGATATTCTTGTTTAAGATGTTGAAC
TCTATGGAGGTTTGTATGAACTGATGATCTAGGACCGGATAAGTTCCCTT
CTTCATAGCGAACTTATTCAAAGAATGTTTTGTGTATCATTCTTGTTACA
TTGTTATTAATGAAAAAATATTATTGGTCATTGGACTGAACACGAGTGTT
AAATATGGACCAGGCCCCAAATAAGATCCATTGATATATGAATTAAATAA
CAAGAATAAATCGAGTCACCAAACCACTTGCCTTTTTTAACGAGACTTGT
TCACCAACTTGATACAAAAGTCATTATCCTATGCAAATCAATAATCATAC
AAAAATATCCAATAACACTAAAAAATTAAAAGAAATGGATAATTTCACAA
TATGTTATACGATAAAGAAGTTACTTTTCCAAGAAATTCACTGATTTTAT
AAGCCCACTTGCATTAGATAAATGGCAAAAAAAAACAAAAAGGAAAAGAA
ATAAAGCACGAAGAATTCTAGAAAATACGAAATACGCTTCAATGCAGTGG
GACCCACGGTTCAATTATTGCCAATTTTCAGCTCCACCGTATATTTAAAA
AATAAAACGATAATGCTAAAAAAATATAAATCGTAACGATCGTTAAATCT
CAACGGCTGGATCTTATGACGACCGTTAGAAATTGTGGTTGTCGACGAGT
CAGTAATAAACGGCGTCAAAGTGGTTGCAGCCGGCACACACGAGTCGTGT
TTATCAACTCAAAGCACAAATACTTTTCCTCAACCTAAAAATAAGGCAAT
TAGCCAAAAACAACTTTGCGTGTAAACAACGCTCAATACACGTGTCATTT
TATTATTAGCTATTGCTTCACCGCCTTAGCTTTCTCGTGACCTAGTCGTC
CTCGTCTTTTCTTCTTCTTCTTCTATAAAACAATACCCAAAGCTTCTTCT
TCACAATTCAGATTTCAATTTCTCAAAATCTTAAAAACTTTCTCTCAATT
CTCTCTACCGTGATCAAGGTAAATTTCTGTGTTCCTTATTCTCTCAAAAT
CTTCGATTTTGTTTTCGTTCGATCCCAATTTCGTATATGTTCTTTGGTTT
AGATTCTGTTAATCTTAGATCGAAGACGATTTTCTGGGTTTGATCGTTAG
ATATCATCTTAATTCTCGATTAGGGTTTCATAAATATCATCCGATTTGTT
CAAATAATTTGAGTTTTGTCGAATAATTACTCTTCGATTTGTGATTTCTA
TCTAGATCTGGTGTTAGTTTCTAGTTTGTGCGATCGAATTTGTCGATTAA
TCTGAGTTTTTCTGATTAACAG
[0035] SEQ ID NO:9 AAD12 forward nucleotide primer:
TABLE-US-00005 AACCCGTGCTCTTGTTC
[0036] SEQ ID NO:10 AAD12 reverse nucleotide primer:
TABLE-US-00006 GGATGCACCTTGACCAAT
[0037] SEQ ID NO:11 AAD12 fluorescent probe used for transgene copy
number and relative expression detection:
TABLE-US-00007 CAGGCCGGGTCAGCCT
[0038] SEQ ID NO:12 PAT forward nucleotide primer:
TABLE-US-00008 ACAAGAGTGGATTGATGATCTAGAGAGGT
[0039] SEQ ID NO:13 PAT reverse nucleotide primer:
TABLE-US-00009 CTTTGATGCCTATGTGACACGTAAACAGT
[0040] SEQ ID NO:14 PAT fluorescent probe used for transgene copy
number and relative expression detection:
TABLE-US-00010 GGTGTTGTGGCTGGTATTGCTTACGCTGG
[0041] SEQ ID NO:15 Spec forward nucleotide primer:
TABLE-US-00011 CGCCGAAGTATCGACTCAACT
[0042] SEQ ID NO:16 Spec reverse nucleotide primer:
TABLE-US-00012 GCAACGTCGGTTCGAGATG
[0043] SEQ ID NO:17 Spec fluorescent probe used for transgene copy
number and relative expression detection:
TABLE-US-00013 TCAGAGGTAGTTGGCGTCATCGAG
[0044] SEQ ID NO:18 GMS116 forward nucleotide primer:
TABLE-US-00014 GTAATATGGGCTCAGAGGAATGGT
[0045] SEQ ID NO:19 GMS116 reverse nucleotide primer:
TABLE-US-00015 ATGGAGAAGAACATTGGAATTGC
[0046] SEQ ID NO:20 GMS116 fluorescent probe used for transgene
copy number and relative expression detection:
TABLE-US-00016 CCATGGCCCGGTACCATCTGGTC
[0047] SEQ ID NO:21 Soy Actin forward nucleotide primer:
TABLE-US-00017 GTGACTCACACCATCACCAGAGT
[0048] SEQ ID NO:22 Soy Actin reverse nucleotide primer:
TABLE-US-00018 ATCCAGGCTGTCCTCTCCCTATA
[0049] SEQ ID NO:23 Soy Actin fluorescent probe used for transgene
copy number and relative expression detection:
TABLE-US-00019 CAATACCAGTTGTGCGACC
DETAILED DESCRIPTION
[0050] I. Overview of Several Embodiments
[0051] Embodiments herein exploit tissue-specific expression of
sRNAs to repress/knockdown the expression of a transgene in the
specific tissue. sRNAs have been shown to regulate diverse
developmental processes, including organ separation, polarity, and
identity, and to modulate their own biogenesis and function. In
embodiments herein, the precision and specificity of sRNA-mediated
gene regulation is utilized to accomplish the tissue-specific
expression of a transgene. In examples, a target site for a
tissue-specific sRNA (e.g., an endogenous tissue-specific sRNA,
including a miRNA or a siRNA) is engineered into the transgene to
reduce or eliminate expression of the transgene in the tissue.
Embodiments herein may be utilized to target the expression of any
transgene to any particular tissue(s).
[0052] Particular embodiments herein are useful, for example, in
preferentially repressing expression of transgenes in specific
tissues, cell types or developmental stages while continuing to
obtain high expression in other tissues, cells or developmental
stages. The method provides unique means of transgene expression
modulation that overcome limitations of conventional transgene
expression regulated by promoters. Examples include repression of
certain transgene(s): interfering during transformation and tissue
culture stages for transgenic plant production; pollen, egg cell,
zygote or other reproductive stage for seed production; root tissue
for plant health; seed tissue when the transgene trait is not
required in seed; diversified transgene expression modulation when
multiple transgenes are regulated by one promoter.
[0053] II. Abbreviations
[0054] RNA ribonucleic acid
[0055] dsRNA double-stranded ribonucleic acid
[0056] DCL DICER-LIKE proteins
[0057] ELISAs Enzyme Linked Immunosorbant Assays
[0058] RDRs RNA-Dependent RNA Polymerases
[0059] GI growth inhibition
[0060] NCBI National Center for Biological Information
[0061] gDNA genomic DNA
[0062] iRNA inhibitory ribonucleic acid
[0063] ORF open reading frame
[0064] RNAi ribonucleic acid interference
[0065] miRNA micro inhibitory ribonucleic acid
[0066] sRNA small ribonucleic acid
[0067] siRNA small inhibitory ribonucleic acid, or short,
interfering ribonucleic acid
[0068] hpRNA hairpin ribonucleic acid
[0069] RT room temperature
[0070] BSA bovine serum albumin
[0071] UTR untranslated region
[0072] PCR polymerase chain reaction
[0073] RISC RNA-induced Silencing Complex
[0074] III. Terms
[0075] In the description and tables which follow, a number of
terms are used. In order to provide a clear and consistent
understanding of the specification and claims, including the scope
to be given such terms, the following definitions are provided:
[0076] Backcrossing: Backcrossing methods may be used to introduce
a nucleic acid sequence into plants. The backcrossing technique has
been widely used for decades to introduce new traits into plants.
Jensen, N., Ed. Plant Breeding Methodology, John Wiley & Sons,
Inc., 1988. In a typical backcross protocol, the original variety
of interest (recurrent parent) is crossed to a second variety
(non-recurrent parent) that carries a gene of interest to be
transferred. The resulting progeny from this cross are then crossed
again to the recurrent parent, and the process is repeated until a
plant is obtained wherein essentially all of the desired
morphological and physiological characteristics of the recurrent
plant are recovered in the converted plant, in addition to the
transferred gene from the non-recurrent parent.
[0077] Expression: As used herein, "expression" of a nucleic acid
sequence (for example, a gene or a transgene) refers to the process
by which the coded information of a nucleic acid transcriptional
unit (including, e.g., genomic DNA or cDNA) is converted into an
operational, non-operational, or structural part of a cell, often
including the synthesis of a protein, but also including production
of an sRNA. Gene expression can be influenced by external signals;
for example, exposure of a cell, tissue, or organism to an agent
that increases or decreases gene expression. Expression of a gene
can also be regulated anywhere in the pathway from DNA to RNA to
protein. Expression of a gene can also be regulated "predominantly"
in a specific cell, tissue or organ, or at a particular time (e.g.,
during development or differentiation). Expression of a gene is
"predominant" when there is approximately <60% expression in
other non-specific cell, tissue or organ, or stage. Regulation of
gene expression occurs, for example, through controls acting on
transcription, translation, RNA transport and processing,
degradation of intermediary molecules such as mRNA, or through
activation, inactivation, compartmentalization, or degradation of
specific protein molecules after they have been made, or by
combinations thereof. Gene expression can be measured at the RNA
level or the protein level by any method known in the art,
including, without limitation, Northern blot, RT-PCR, Western blot,
or in vitro, in situ, or in vivo protein activity assay(s).
[0078] Genetic material: As used herein, the term "genetic
material" includes all genes, and nucleic acid molecules, such as
DNA and RNA.
[0079] Inhibition/downregulation: As used herein, the terms
"inhibition" and "downregulation," when used to describe an effect
on a coding sequence (for example, a gene), are used
interchangeably and refer to a measurable decrease in the cellular
level of mRNA transcribed from the coding sequence and/or peptide,
polypeptide, or protein product of the coding sequence. In some
examples, expression of a coding sequence may be inhibited such
that expression is essentially eliminated (e.g., expression is
reduced to less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of
its expression in a control cell).
[0080] Isolated: An "isolated" biological component (such as a
nucleic acid or protein) has been substantially separated, produced
apart from, or purified away from other biological components in
the cell of the organism in which the component naturally occurs
(i.e., other chromosomal and extra-chromosomal DNA and RNA, and
proteins), while effecting a chemical or functional change in the
component (e.g., a nucleic acid may be isolated from a chromosome
by breaking chemical bonds connecting the nucleic acid to the
remaining DNA in the chromosome). Nucleic acid molecules and
proteins that have been "isolated" include nucleic acid molecules
and proteins purified by standard purification methods, wherein
there has been a chemical or functional change in the nucleic acid
or protein. The term also embraces nucleic acids and proteins
prepared by recombinant expression in a host cell, as well as
chemically-synthesized nucleic acid molecules, proteins, and
peptides.
[0081] Nucleic acid molecule: As used herein, the term "nucleic
acid molecule" may refer to a polymeric form of nucleotides, which
may include single strands or both sense and anti-sense strands of
RNA, cDNA, genomic DNA, and synthetic forms and mixed polymers of
the above. A nucleotide may refer to a ribonucleotide,
deoxyribonucleotide, or a modified form of either type of
nucleotide. A "nucleic acid molecule" as used herein is synonymous
with "nucleic acid" and "polynucleotide." A nucleic acid molecule
is usually at least 10 bases in length, unless otherwise specified.
The nucleotide sequence of a nucleic acid molecule is read from the
5' to the 3' end of the molecule by convention. The "complement" of
a nucleotide sequence refers to the sequence, from 5' to 3', of the
nucleobases which form base pairs with the nucleobases of the
nucleotide sequence (i.e., A-T/U, and G-C). The "reverse
complement" of a nucleic acid sequence refers to the sequence, from
3' to 5', of the nucleobases which form base pairs with the
nucleobases of the nucleotide sequence.
[0082] "Nucleic acid molecules" include single- and double-stranded
forms of DNA; single-stranded forms of RNA; and double-stranded
forms of RNA (dsRNA). The term "nucleotide sequence" or "nucleic
acid sequence" refers to both the sense and antisense strands of a
nucleic acid as either individual single strands or in the duplex.
The term "ribonucleic acid" (RNA) is inclusive of iRNA (inhibitory
RNA), dsRNA (double-stranded RNA), sRNA (small RNA), siRNA (small
interfering RNA), miRNA (micro-RNA), hpRNA (hairpin RNA), mRNA
(messenger RNA), tRNA (transfer RNA), and cRNA (complementary RNA).
The term "deoxyribonucleic acid" (DNA) is inclusive of cDNA,
genomic DNA, and DNA-RNA hybrids. The term "polynucleotide" will be
understood by those in the art as a structural term that is defined
by its nucleotide sequence, and that includes genomic sequences,
ribosomal RNA sequences, transfer RNA sequences, messenger RNA
sequences, operon sequences, and smaller engineered nucleotide
sequences.
[0083] Small RNA (sRNA): sRNAs are non-coding RNAs that regulate
gene expression by pairing to the message of protein-coding genes
to guide mRNA cleavage or repression of productive translation.
Dugas and Bartel (2004) Cum Opin. Plant Biolo. 7: 512-20. As used
herein, the term "small RNA" (sRNA) includes, for example, microRNA
(miRNA), small interfering RNA (siRNA), trans-acting small
interfering RNA (tasiRNA), and other sRNAs guiding cleavage,
translational repression, and/or gene silencing.
[0084] Small interfering RNA (siRNA): RNA of approximately 21-25
nucleotides that is processed from a dsRNA by a DICER enzyme (in
animals) or a DCL enzyme (in plants). The initial DICER or DCL
products are double-stranded, in which the two strands are
typically 21-25 nucleotides in length and contain two unpaired
bases at each 3' end. The individual strands within the double
stranded siRNA structure are separated, and typically one of the
siRNAs then is associated with a multi-subunit complex, the
RNAi-induced silencing complex (RISC). A typical function of the
siRNA is to guide RISC to the target based on base-pair
complementarity.
[0085] Target nucleic acid (to be inhibited): As used herein, the
term "target" or "target site" in reference to a polynucleotide,
refers to any nucleic acid containing a sequence that interacts
with a miRNA or siRNA, or that has the potential to yield a
sequence that interacts with a miRNA or siRNA (for example, through
transcription of a locus). The target can be a cellular nucleic
acid, such as an mRNA that encodes an essential or nonessential
protein, or a foreign nucleic acid, such as a transgene-derived RNA
molecule comprising a gene of interest linked to a target site for
a specific sRNA. The target can be a DNA sequence corresponding to
a promoter, or a sequence corresponding to any expressed region of
a genome, for instance.
[0086] Oligonucleotide: An oligonucleotide is a short nucleic acid
molecule. Oligonucleotides may be formed by cleavage of longer
nucleic acid segments, or by polymerizing individual nucleotide
precursors. Automated synthesizers allow the synthesis of
oligonucleotides up to several hundred base pairs in length.
Because oligonucleotides may bind to a complementary nucleotide
sequence, they may be used as probes for detecting DNA or RNA.
Oligonucleotides composed of DNA (oligodeoxyribonucleotides) may be
used in PCR, a technique for the amplification of small DNA
sequences. In PCR, the oligonucleotide is typically referred to as
a "primer," which allows a DNA polymerase to extend the
oligonucleotide and replicate the complementary strand.
[0087] A nucleic acid molecule may include either or both naturally
occurring and modified nucleotides linked together by naturally
occurring and/or non-naturally occurring nucleotide linkages.
Nucleic acid molecules may be modified chemically or biochemically,
or may contain non-natural or derivatized nucleotide bases, as will
be readily appreciated by those of skill in the art. Such
modifications include, for example, labels, methylation,
substitution of one or more of the naturally occurring nucleotides
with an analog, inter-nucleotide modifications (e.g., uncharged
linkages: for example, methyl phosphonates, phosphotriesters,
phosphoramidates, carbamates, etc.; charged linkages: for example,
phosphorothioates, phosphorodithioates, etc.; pendent moieties: for
example, peptides; intercalators: for example, acridine, psoralen,
etc.; chelators; alkylators; and modified linkages: for example,
alpha anomeric nucleic acids, etc.). The term "nucleic acid
molecule" also includes any topological conformation, including
single-stranded, double-stranded, partially duplexed, triplexed,
hairpinned, circular, and padlocked conformations.
[0088] As used herein with respect to DNA, the term "coding
sequence" refers to a nucleotide sequence that is transcribed into
RNA when placed under the control of appropriate regulatory
sequences. A "protein coding sequence" is a nucleotide sequence
(DNA or RNA) that is ultimately translated into a polypeptide, via
transcription and mRNA. With respect to RNA, the term "coding
sequence" refers to a nucleotide sequence that is translated into a
peptide, polypeptide, or protein. The boundaries of a coding
sequence are determined by a translation start codon at the
5'-terminus and a translation stop codon at the 3'-terminus. Coding
sequences include, but are not limited to: genomic DNA; cDNA; EST;
and recombinant nucleotide sequences.
[0089] Genome: As used herein, the term "genome" refers to
chromosomal DNA found within the nucleus of a cell, and also refers
to organelle DNA found within subcellular components of the cell.
In some embodiments of the invention, a DNA molecule may be
introduced into a plant cell such that the DNA molecule is
integrated into the genome of the plant cell. In these and further
embodiments, the DNA molecule may be either integrated into the
nuclear DNA of the plant cell, or integrated into the DNA of the
chloroplast or mitochondrion of the plant cell.
[0090] Endogenous: The term "endogenous," as applied to nucleic
acids (e.g., polynucleotides, DNA, RNA (including sRNA), and genes)
herein, refers to one or more nucleic acid(s) that are normally
(e.g., in a wild-type cell of the same type and species) present
within their specific environment or context. For example, an
endogenous gene is one that is normally found in the particular
cell in question and in the same context (e.g., with regard to
regulatory sequences). Endogenous nucleic acids can be
distinguished from exogenous and/or heterologous, for example and
without limitation, by detection in the latter of sequences that
are consequent with recombination from bacterial plasmid;
identification of atypical codon preferences; and amplification of
atypical sequences in a PCR reaction from primers characterized in
a wild-type cell.
[0091] Exogenous: The term "exogenous," as applied to nucleic acids
herein, refers to one or more nucleic acid(s) that are not normally
present within their specific environment or context. For example,
if a host cell is transformed with a nucleic acid that does not
occur in the untransformed host cell in nature, then that nucleic
acid is exogenous to the host cell. The term exogenous, as used
herein, also refers to one or more nucleic acid(s) that are
identical in sequence to a nucleic acid already present in a host
cell, but that are located in a different cellular or genomic
context than the nucleic acid with the same sequence already
present in the host cell. For example, a nucleic acid that is
integrated in the genome of the host cell in a different location
than a nucleic acid with the same sequence is normally integrated
in the genome of the host cell is exogenous to the host cell.
Furthermore, a nucleic acid (e.g., a DNA molecule) that is present
in a plasmid or vector in the host cell is exogenous to the host
cell when a nucleic acid with the same sequence is only normally
present in the genome of the host cell.
[0092] Heterologous: The term "heterologous," as applied to nucleic
acids (e.g., polynucleotides, DNA, RNA, and genes) herein, means of
different origin. For example, if a host cell is transformed with a
nucleic acid that does not occur in the untransformed host cell in
nature, then that nucleic acid is heterologous (and exogenous) to
the host cell. Furthermore, different elements (e.g., promoter,
enhancer, coding sequence, terminator, etc) of a transforming
nucleic acid may be heterologous to one another and/or to the
transformed host. The term heterologous, as used herein, may also
be applied to one or more nucleic acid(s) that are identical in
sequence to a nucleic acid already present in a host cell, but that
are now linked to different additional sequences and/or are present
at a different copy number, etc.
[0093] Sequence identity: The term "sequence identity" or
"identity," as used herein in the context of two nucleic acid or
polypeptide sequences, may refer to the residues in the two
sequences that are the same when aligned for maximum correspondence
over a specified comparison window.
[0094] As used herein, the term "percentage of sequence identity"
may refer to the value determined by comparing two optimally
aligned sequences (e.g., nucleic acid sequences, and amino acid
sequences) over a comparison window, wherein the portion of the
sequence in the comparison window may comprise additions or
deletions (i.e., gaps) as compared to the reference sequence (which
does not comprise additions or deletions) for optimal alignment of
the two sequences. The percentage is calculated by determining the
number of positions at which the identical nucleotide or amino acid
residue occurs in both sequences to yield the number of matched
positions, dividing the number of matched positions by the total
number of positions in the comparison window, and multiplying the
result by 100 to yield the percentage of sequence identity.
[0095] Methods for aligning sequences for comparison are well-known
in the art. Various programs and alignment algorithms are described
in, for example: Smith and Waterman (1981) Adv. Appl. Math. 2: 482;
Needleman and Wunsch (1970) J. Mol. Biol. 48: 443; Pearson and
Lipman (1988) Proc. Natl. Acad. Sci. U.S.A. 85: 2444; Higgins and
Sharp (1988) Gene 73: 237-44; Higgins and Sharp (1989) CABIOS 5:
151-3; Corpet etal. (1988) Nucleic Acids Res. 16: 10881-90; Huang
etal. (1992) Comp. Appl. Biosci. 8: 155-65; Pearson etal. (1994)
Methods Mol. Biol. 24: 307-31; Tatiana et al. (1999) FEMS
Microbiol. Lett. 174: 247-50. A detailed consideration of sequence
alignment methods and homology calculations can be found in, e.g.,
Altschul et al. (1990) J. Mol. Biol. 215: 403-10.
[0096] The National Center for Biotechnology Information (NCBI)
Basic Local Alignment Search Tool (BLAST.TM.; Altschul et al.
(1990)) is available from several sources, including the National
Center for Biotechnology Information (Bethesda, Md.), and on the
internet, for use in connection with several sequence analysis
programs. A description of how to determine sequence identity using
this program is available on the Internet under the "help" section
for BLAST.TM.. For comparisons of nucleic acid sequences, the
"Blast 2 sequences" function of the BLAST.TM. (Blastn) program may
be employed using the default parameters. Nucleic acid sequences
with even greater similarity to the reference sequences will show
increasing percentage identity when assessed by this method.
[0097] Specifically hybridizable/specifically complementary: As
used herein, the terms "specifically hybridizable" and
"specifically complementary" are terms that indicate a sufficient
degree of complementarity such that stable and specific binding
occurs between the nucleic acid molecule and a target nucleic acid
molecule. Hybridization between two nucleic acid molecules involves
the formation of an anti-parallel alignment between the nucleic
acid sequences of the two nucleic acid molecules. The two molecules
are then able to form hydrogen bonds with corresponding bases on
the opposite strand to form a duplex molecule that, if it is
sufficiently stable, is detectable using methods well known in the
art. A nucleic acid molecule need not be 100% complementary to its
target sequence to be specifically hybridizable. However, the
amount of sequence complementarity that must exist for
hybridization to be specific is a function of the hybridization
conditions used.
[0098] Hybridization conditions resulting in particular degrees of
stringency will vary depending upon the nature of the hybridization
method of choice and the composition and length of the hybridizing
nucleic acid sequences. Generally, the temperature of hybridization
and the ionic strength (especially the Na.sup.+ and/or Mg.sup.++
concentration) of the hybridization buffer will determine the
stringency of hybridization, though wash times also influence
stringency. Calculations regarding hybridization conditions
required for attaining particular degrees of stringency are known
to those of ordinary skill in the art, and are discussed, for
example, in Sambrook et al. (ed.) Molecular Cloning: A Laboratory
Manual, 2.sup.nd ed., vol. 1-3, Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y., 1989, chapters 9 and 11; and Hames
and Higgins (eds.) Nucleic Acid Hybridization, IRL Press, Oxford,
1985. Further detailed instruction and guidance with regard to the
hybridization of nucleic acids may be found, for example, in
Tijssen, "Overview of principles of hybridization and the strategy
of nucleic acid probe assays," in Laboratory Techniques in
Biochemistry and Molecular Biology-Hybridization with Nucleic Acid
Probes, Part I, Chapter 2, Elsevier, N.Y., 1993; and Ausubel et
al., Eds., Current Protocols in Molecular Biology, Chapter 2,
Greene Publishing and Wiley-Interscience, NY, 1995.
[0099] As used herein, "stringent conditions" encompass conditions
under which hybridization will only occur if there is less than 20%
mismatch between the hybridization molecule and a homologous
sequence within the target nucleic acid molecule. "Stringent
conditions" include further particular levels of stringency. Thus,
as used herein, "moderate stringency" conditions are those under
which molecules with more than 20% sequence mismatch will not
hybridize; conditions of "high stringency" are those under which
sequences with more than 10% mismatch will not hybridize; and
conditions of "very high stringency" are those under which
sequences with more than 5% mismatch will not hybridize.
[0100] The following are representative, non-limiting hybridization
conditions.
[0101] High Stringency condition (detects sequences that share at
least 90% sequence identity): Hybridization in 5.times.SSC buffer
at 65.degree. C. for 16 hours; wash twice in 2.times.SSC buffer at
room temperature for 15 minutes each; and wash twice in
0.5.times.SSC buffer at 65.degree. C. for 20 minutes each.
[0102] Moderate Stringency condition (detects sequences that share
at least 80% sequence identity): Hybridization in
5.times.-6.times.SSC buffer at 65-70.degree. C. for 16-20 hours;
wash twice in 2.times.SSC buffer at room temperature for 5-20
minutes each; and wash twice in 1.times.SSC buffer at 55-70.degree.
C. for 30 minutes each.
[0103] Non-stringent control condition (sequences that share at
least 50% sequence identity will hybridize): Hybridization in
6.times.SSC buffer at room temperature to 55.degree. C. for 16-20
hours; wash at least twice in 2.times.-3.times.SSC buffer at room
temperature to 55.degree. C. for 20-30 minutes each.
[0104] As used herein, the term "substantially homologous" or
"substantial homology," with regard to a polynucleotide, refers to
polynucleotides that hybridize under stringent conditions to the
reference nucleic acid sequence. For example, polynucleotides that
are substantially homologous to a reference DNA coding sequence are
those polynucleotides that hybridize under stringent conditions
(e.g., the Moderate Stringency conditions set forth, supra) to the
reference DNA coding sequence. Substantially homologous sequences
may have at least 80% sequence identity. For example, substantially
homologous sequences may have from about 80% to 100% sequence
identity, such as about 81%; about 82%; about 83%; about 84%; about
85%; about 86%; about 87%; about 88%; about 89%; about 90%; about
91%; about 92%; about 93%; about 94% about 95%; about 96%; about
97%; about 98%; about 98.5%; about 99%; about 99.5%; and about
100%. The property of substantial homology is closely related to
specific hybridization. For example, a nucleic acid molecule is
specifically hybridizable when there is a sufficient degree of
complementarity to avoid non-specific binding of the nucleic acid
to non-target sequences under conditions where specific binding is
desired, for example, under stringent hybridization conditions.
[0105] As used herein, the term "ortholog" refers to a gene in two
or more species that has evolved from a common ancestral nucleotide
sequence, and may retain the same function in the two or more
species.
[0106] DNA has two antiparallel strands, a 5'- 3' strand, referred
to as the plus strand, and a 3'- 5' strand, referred to as the
minus strand. Because RNA polymerase adds nucleic acids in a 5'- 3'
direction, the minus strand of the DNA serves as the template for
the RNA during transcription. Thus, an RNA transcript will have a
sequence complementary to the minus strand, and identical to the
plus strand (except that U is substituted for T).
[0107] Antisense molecules are molecules that are specifically
hybridizable or specifically complementary to either RNA or the
plus strand of DNA. Sense molecules are molecules that are
specifically hybridizable or specifically complementary to the
minus strand of DNA. Antigene molecules are either antisense or
sense molecules directed to a DNA target. An antisense RNA is a
molecule of RNA complementary to a sense (encoding) nucleic acid
molecule.
[0108] As used herein, two nucleic acid sequence molecules are said
to exhibit "complete complementarity" when every nucleotide of a
sequence read in the 5' to 3' direction is complementary to every
nucleotide of the other sequence when read in the 3' to 5'
direction. A nucleotide sequence that is complementary to a
reference nucleotide sequence will exhibit a sequence identical to
the reverse complement sequence of the reference nucleotide
sequence. These terms and descriptions are well defined in the art
and are easily understood by those of ordinary skill in the
art.
[0109] As used herein, the term "substantially identical" may refer
to nucleotide sequences that are more than 85% identical. For
example, a substantially identical nucleotide sequence may be at
least 85.5%; at least 86%; at least 87%; at least 88%; at least
89%; at least 90%; at least 91%; at least 92%; at least 93%; at
least 94%; at least 95%; at least 96%; at least 97%; at least 98%;
at least 99%; or at least 99.5% identical to the reference
sequence.
[0110] Operably linked: A first nucleotide sequence is operably
linked with a second nucleic acid sequence when the first nucleic
acid sequence is in a functional relationship with the second
nucleic acid sequence. When recombinantly produced, operably linked
nucleic acid sequences are generally contiguous, and, where
necessary to join two protein-coding regions, in the same reading
frame (e.g., in a polycistronic ORF). However, nucleic acids need
not be contiguous to be operably linked.
[0111] The term, "operably linked," when used in reference to a
regulatory sequence and a coding sequence, means that the
regulatory sequence affects the expression of the linked coding
sequence. "Regulatory sequences," or "control elements," refer to
nucleotide sequences that influence the timing and level/amount of
transcription, RNA processing or stability, or translation of the
associated coding sequence. Regulatory sequences may include
promoters; translation leader sequences; introns; enhancers;
stem-loop structures; repressor binding sequences; termination
sequences; polyadenylation recognition sequences; etc. Particular
regulatory sequences may be located upstream and/or downstream of a
coding sequence operably linked thereto. Also, particular
regulatory sequences operably linked to a coding sequence may be
located on the associated complementary strand of a double-stranded
nucleic acid molecule.
[0112] Promoter: As used herein, the term "promoter" refers to a
region of DNA that may be upstream from the start of transcription,
and that may be involved in recognition and binding of RNA
polymerase and other proteins to initiate transcription. A promoter
may be operably linked to a coding sequence for expression in a
cell, or a promoter may be operably linked to a nucleotide sequence
encoding a signal sequence which may be operably linked to a coding
sequence for expression in a cell.
[0113] Some embodiments herein include a "plant promoter." A plant
promoter is a promoter that is capable of initiating transcription
in a plant cell.
[0114] Some embodiments herein include a "tissue-preferred
promoter." A tissue-preferred promoter is a promoter that is
capable of initiating transcription under developmental control,
and include, for example and without limitation: promoters that
preferentially initiate transcription in leaves, pollen, tassels,
roots, seeds, fibers, xylem vessels, tracheids, and sclerenchyma.
Promoters that initiate transcription essentially only in certain
tissues are referred to as "tissue-specific." A "cell
type-specific" promoter primarily drives expression in certain cell
types in one or more organs, for example, vascular cells in roots
or leaves. An "inducible" promoter may be a promoter which may be
under environmental control. Examples of environmental conditions
that may initiate transcription by inducible promoters include
anaerobic conditions and the presence of light. Tissue-specific,
tissue-preferred, cell type specific, and inducible promoters
constitute the class of "non-constitutive" promoters.
[0115] Any inducible promoter may be used in some embodiments of
the invention. See Ward et al. (1993) Plant Mol. Biol. 22: 361-366.
With an inducible promoter, the rate of transcription increases in
response to an inducing agent. Exemplary inducible promoters
include, but are not limited to: Promoters from the ACEI system
that responds to copper; In2 gene from maize that responds to
benzenesulfonamide herbicide safeners; Tet repressor from Tn10; and
the inducible promoter from a steroid hormone gene, the
transcriptional activity of which may be induced by a
glucocorticosteroid hormone (Schena et al. (1991) Proc. Natl. Acad.
Sci. USA 88: 10421-5).
[0116] In contrast to non-constitutive promoters, a "constitutive"
promoter is a promoter that is active under most environmental
conditions. Exemplary constitutive promoters include, but are not
limited to: promoters from plant viruses, such as the 35S promoter
from CaMV; promoters from rice actin genes; ubiquitin promoters;
pEMU; MAS; maize H3 histone promoter; and the ALS promoter,
Xbal/NcoI fragment 5' to the Brassica napus ALS3 structural gene
(or a nucleotide sequence similarity to said Xbal/NcoI fragment)
(PCT International Patent Publication No. WO 96/30530).
[0117] Additionally, any tissue-specific or tissue-preferred
promoter may be utilized in some embodiments of the invention.
Plants transformed with a nucleic acid molecule comprising a coding
sequence operably linked to a tissue-specific promoter may produce
the product of the coding sequence exclusively, or preferentially,
in a specific tissue. Exemplary tissue-specific or tissue-preferred
promoters include, but are not limited to: a root-preferred
promoter, such as that from the phaseolin gene; a leaf-specific and
light-induced promoter such as that from cab or rubisco; an
anther-specific promoter such as that from LAT52; a pollen-specific
promoter such as that from Zm13; and a microspore-preferred
promoter such as that from apg.
[0118] Trait or phenotype: The terms "trait" and "phenotype" are
used interchangeably herein. For the purposes of the present
disclosure, traits of particular interest include agronomically
important traits, as may be expressed, for example, in a crop
plant. In some examples, a trait of particular interest is
herbicide tolerance.
[0119] Transformation: As used herein, the term "transformation"
refers to the transfer of one or more nucleic acid molecule(s) into
a cell. A cell is "transformed" by a nucleic acid molecule
introduced into the cell when the nucleic acid molecule becomes
stably replicated by the cell, either by incorporation of the
nucleic acid molecule into the cellular genome, or by episomal
replication. As used herein, the term "transformation" encompasses
all techniques by which a nucleic acid molecule can be introduced
into such a cell. Examples include, but are not limited to:
transfection with viral vectors; transformation with plasmid
vectors; electroporation (Fromm et al. (1986) Nature 319: 791-3);
lipofection (Felgner et al. (1987) Proc. Natl. Acad. Sci. USA 84:
7413-7); microinjection (Mueller et al. (1978) Cell 15: 579-85);
Agrobacterium-mediated transfer (Fraley et al. (1983) Proc. Natl.
Acad. Sci. USA 80: 4803-7); direct DNA uptake; and microprojectile
bombardment (Klein et al. (1987) Nature 327: 70).
[0120] Transgene: A transgene is an exogenous nucleic acid
sequence. In some examples, a transgene may be a sequence that
encodes one or both strand(s) of a dsRNA molecule that comprises a
nucleotide sequence that is complementary to a target nucleic acid.
In some examples, a transgene may be an antisense nucleic acid
sequence, the expression of which inhibits expression of a target
nucleic acid. In still other examples, a transgene may be a gene
sequence (e.g., a herbicide-tolerance gene), a gene encoding an
industrially or pharmaceutically useful compound, or a gene
encoding a desirable agricultural trait. In these and other
examples, a transgene may contain regulatory sequences operably
linked to the coding sequence of the transgene (e.g., a
promoter).
[0121] Vector: A vector refers to a nucleic acid molecule as
introduced into a cell, for example, to produce a transformed cell.
A vector may include nucleic acid sequences that permit it to
replicate in the host cell, such as an origin of replication.
Examples of vectors include, but are not limited to: a plasmid;
cosmid; bacteriophage; and a virus that carries exogenous DNA into
a cell. A vector may also include one or more expression cassettes
comprising genes, antisense molecules, and/or selectable marker
genes and other genetic elements known in the art. A vector may
transduce, transform, or infect a cell, thereby causing the cell to
express the nucleic acid molecules and/or proteins encoded by the
vector. A vector optionally includes materials to aid in achieving
entry of the nucleic acid molecule into the cell (e.g., a liposome,
protein coating, etc.).
[0122] Unless specifically indicated or implied, the terms "a",
"an", and "the" signify "at least one," as used herein.
[0123] Unless otherwise specifically explained, all technical and
scientific terms used herein have the same meaning as commonly
understood by those of ordinary skill in the art to which this
disclosure belongs. Definitions of common terms in molecular
biology can be found in, for example, Lewin B., Genes V, Oxford
University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.),
The Encyclopedia of Molecular Biology, Blackwell Science Ltd., 1994
(ISBN 0-632-02182-9); and Meyers R. A. (ed.), Molecular Biology and
Biotechnology: A Comprehensive Desk Reference, VCH Publishers,
Inc., 1995 (ISBN 1-56081-569-8). All percentages are by weight and
all solvent mixture proportions are by volume unless otherwise
noted. All temperatures are in degrees Celsius.
[0124] IV. Nucleic Acids and Systems
[0125] Plants and animals use sRNAs to direct the
post-transcriptional and epigenetic regulation of target genes.
Many miRNAs and their corresponding target sequences are highly
conserved. For example, target sequences in plants that are
recognized by related miRNAs in different species often differ only
by several nucleotides. Therefore, the computational prediction of
target sites is possible. Jones-Rhoades and Bartel (2004) Mol. Cell
14: 787-99. Additionally, a functional sRNA target site from one
plant species is likely to be functional in a different plant
species that expresses the targeting sRNA. For example, miRNA
target genes from Arabidopsis heterologously expressed in Nicotiana
are cleaved by endogenous Nicotiana miRNAs. Llave et al. (2002)
Science 297: 2053-6.
[0126] Embodiments herein include nucleic acids comprising a target
site for at least one sRNA molecule. A target site for an sRNA
molecule may be, for example, between about 20-30 nucleotides in
length. For example, such a target site may be between about 20-25
or 20-21 nucleotides in length. In particular examples, the target
site may be 19, about 20, about 21, about 22, about 23, about 24,
about 25, about 26, about 27, about 28, about 29, about 30, or 31
nucleotides in length. Target sites for particular sRNAs may be
identified or engineered computationally, using program parameters
that may be selected within the discretion of those skilled in the
art. Target sites may be identified or engineered to be
specifically targeted by a single sRNA, or to be targeted by a
group of related sRNAs.
[0127] Particular embodiments include nucleic acid expression
cassettes, comprising, for example, a gene or coding region of
interest that comprises an sRNA target site (e.g., an internal
target site that is located within a transcribed portion of the
gene or coding region), such that co-expression in a cell of the
gene or coding region of interest and the targeting sRNA results in
downregulated, including essentially eliminated, expression of the
gene or coding region. Such nucleic acids may be, for example and
without limitation, a heterologous or exogenous nucleic acid in the
genome of a cell (e.g., a plant cell), or a vector.
[0128] In some embodiments, a nucleic acid comprising a gene or
coding region of interest with an sRNA target site further
comprises one or more regulatory sequences that are operably linked
to the gene or coding region of interest, so as to effect the
transcription of the gene or coding region of interest in a cell;
i.e., an expression construct. In examples, the cell is a plant
cell. In particular embodiments, the gene or coding region of
interest is operably linked to a constitutive plant promoter in the
expression construct. By, for example, transforming a plant cell or
tissue with such an expression construct and regenerating a plant
from the plant cell or tissue, a transgenic plant may be produced,
wherein the gene or coding region of interest is transcribed in
every cell or essentially every cell of the plant.
[0129] The nucleic acid sequence of interest may be any nucleic
acid sequence that provides for, or enhances, a beneficial feature
of a resultant transgenic plant. Particularly useful nucleic acid
sequences are those that encode proteins or antisense RNA
transcripts in order to promote increased nutritional values,
higher yields, tolerance to herbicides, insects, or diseases, and
the like. In embodiments, the gene or coding region of interest may
be an agronomic gene or nucleotide sequence encoding a polypeptide
of interest, and may also and/or alternatively include, for example
and without limitation: genes that confer tolerance to an
herbicide, such as an herbicide that inhibits the growing point or
meristem, for example, an imidazolinone or a sulfonylurea
(exemplary genes in this category encode mutant ALS and AHAS
enzymes, as described, for example, by Lee et al. (1988) EMBO J. 7:
1241, and Mild et al. (1990) Theor. Appl. Genet. 80: 449,
respectively); glyphosate tolerance as conferred by, e.g., mutant
5-enolpyruvylshikimate-3-phosphate synthase (EPSPs) genes (via the
introduction of recombinant nucleic acids and/or various forms of
in vivo mutagenesis of native EPSPs genes); aroA genes and
glyphosate acetyl transferase (GAT) genes, respectively); other
phosphono compounds, such as glufosinate phosphinothricin acetyl
transferase (PAT) genes from Streptomyces species, including
Streptomyces hygroscopicus and Streptomyces viridichromogenes); and
pyridinoxy or phenoxy proprionic acids and cyclohexanediones
(ACCase inhibitor-encoding genes). See, e.g., U.S. Pat. Nos.
4,940,835 and 6,248,876 (nucleotide sequences of forms of EPSPs
which can confer glyphosate tolerance to a plant). A DNA molecule
encoding a mutant aroA gene can be obtained under ATCC accession
number 39256. See also U.S. Pat. No. 4,769,061 (nucleotide sequence
of a mutant aroA gene). European patent application No. 0 333 033
and U.S. Pat. No. 4,975,374 disclose nucleotide sequences of
glutamine synthetase genes, which may confer tolerance to
herbicides such as L-phosphinothricin. Nucleotide sequences of
exemplary PAT genes are provided in European application No. 0 242
246, and DeGreef et al. (1989) Bio/Technology 7: 61 (production of
transgenic plants that express chimeric bar genes coding for PAT
activity). Exemplary of genes conferring tolerance to phenoxy
proprionic acids and cyclohexanediones, such as sethoxydim and
haloxyfop, include the Acc1-S1, Acc1-S2 and Acc1-S3 genes described
by Marshall et al. (1992) Theor. Appl. Genet. 83:435. GAT genes
capable of conferring glyphosate tolerance are described, for
example, in WO 2005012515. Genes conferring tolerance to 2,4-D,
phenoxyproprionic acid and/or pyridyloxy auxin herbicides are
described, for example, in WO 2005107437 and WO 2007053482.
[0130] Nucleic acids comprising an agronomic gene or nucleotide
sequence encoding a polypeptide of interest may also include, for
example and without limitation: nitrogen use efficiency gene, water
use efficiency gene, nutritional quality gene (e.g., modified oil
profile), or a gene conferring tolerance to an herbicide that
inhibits photosynthesis, such as a triazine (psbA and gs+genes) or
a benzonitrile (nitrilase gene). See, e.g., Przibila et al. (1991)
Plant Cell 3: 169 (transformation of Chlamydomonas with plasmids
encoding mutant psbA genes). Nucleotide sequences for nitrilase
genes are disclosed in U.S. Pat. No. 4,810,648, and DNA molecules
containing these genes are available under ATCC Accession Nos.
53435; 67441; and 67442. See also Hayes et al. (1992) Biochem. J.
285:173 (cloning and expression of DNA coding for a glutathione
S-transferase).
[0131] Embodiments herein also or alternatively include a set of
nucleic acids, wherein the set includes at least one nucleic acid
comprising a target site for at least one sRNA molecule. In some
embodiments, the set of nucleic acids further includes at least one
nucleic acid encoding at least one sRNA molecule targeting the
nucleic acid comprising the target site therefore. In particular
embodiments, a plant cell may be transformed (either in one step or
in multiple steps) with the nucleic acids of the set. In examples,
when a transgenic plant tissue or plant is regenerated from the
transformed plant cell, the nucleic acid comprising the target site
for the at least one sRNA molecule is expressed essentially in all
cells other than those wherein the sRNA molecule is expressed.
Thus, when the set of nucleic acids includes a nucleic acid
encoding the sRNA molecule under the control of a tissue-specific
promoter, the nucleic acid comprising the target site for the at
least one sRNA molecule is expressed essentially in all cells other
than cells of the tissue wherein the promoter directs expression of
the sRNA molecule.
[0132] In embodiments, the gene or coding region of interest may be
genes that confer resistance to insect pests, such as Cry1A, Cry1C,
Cry1D, Cry1E, Cry 1F, Cry 2A, VIP3, and Cry9. In some embodiments,
the nucleic acid sequences will be useful genes which are
inherently unstable due to their relatively large size (at least
4-5 kb in length), which is known to render the genes more
susceptible to physical, chemical, or enzymatic degradation. Genes
inherently unstable due to their size include insecticidal genes
from Xenorhabdus (see U.S. Pat. No. 6,048,838) and Photorabdus
(e.g., Toxin A).
[0133] In one embodiment, one or more nucleic acids contain one or
more sRNA target sites of the present invention which have been
"stacked" in relation to one another in a particular plant. By use
of the phrase "stacked" or "stacking", it is meant herein that
multiple genes of interest, each gene of interest preferably
conferring a commercially desirable trait, have been transgenically
introduced into a single plant. For example, a corn hybrid with
stacked genes might contain genes for the insect resistance (e.g.,
Cry1F B.t. genes) as well as herbicide resistance genes (e.g.,
glyphosate resistance genes).
[0134] In some embodiments, one or more sRNA target site
sequence(s) of the present invention are functionally linked to a
Toxin A gene from Photorabdus, which is then stacked with one or
more insecticide and/or herbicide resistance genes in a single crop
variety. In some embodiments, the insecticide gene(s) will be from
a Bacillus thuringiensis or Xenorhabdus spp., and the herbicide
gene(s) will be one or more of a glufosinate, glyphosate,
imidazolinone, 2,4-D or sulfonyl urea resistance gene. Of course,
any of the "stacked" insecticide or herbicide genes may be
functionally linked to the miRNA target site of the present
invention.
[0135] The nucleic acid sequence of interest may be derived in
whole or in part from a bacterial genome or episome, eukaryotic
genomic, mitochondrial or plastid DNA, cDNA, viral nucleic acid, or
chemically synthesized nucleic acid. It is contemplated that the
nucleic acid sequence of interest may contain one or more
modifications in either the coding region which could affect the
biological activity or the chemical structure of the expression
product, the rate of expression, or the manner of expression
control. Such modifications include, but are not limited to,
mutations, insertions, deletions, rearrangements and substitutions
of one or more nucleotides. The nucleic acid sequence of interest
may constitute an uninterrupted coding sequence or it may include
one or more introns, bounded by the appropriate plant-functional
splice junctions. The nucleic acid sequence of interest may be a
composite of segments derived from a plurality of sources,
naturally occurring or synthetic. The structural nucleic acid
sequence of interest may also encode a fusion protein, so long as
the experimental manipulations maintain functionality in the
joining of the coding sequences.
[0136] In a further embodiment, one or more miRNA target site
sequence(s) are operably linked to a transgene, wherein the
transgene can be an insecticidal resistance transgene (e.g., B.t.
or dsRNA), an herbicide tolerance transgene, a nitrogen use
efficiency transgene, a water use efficiency transgene, a
nutritional quality transgene, a DNA binding transgene, a
selectable marker transgene, or combinations thereof.
[0137] V. Methods
[0138] In some embodiments herein, a plant cell, plant part, and/or
plant may be genetically modified to comprise at least one nucleic
acid comprising a target site for at least one sRNA molecule by any
of several methods of introducing a heterologous molecule known in
the art, thereby producing a non-natural transgenic plant cell,
plant part, or plant. In particular embodiments herein, a
heterologous molecule is introduced into a plant cell, plant part,
and/or plant by a method selected from, for example and without
limitation: transformation and selective breeding (e.g., backcross
breeding).
[0139] In some embodiments, the nucleic acid is selected such that
the target site is a target of an endogenous sRNA of the plant
wherein the heterologous nucleic acid is introduced. In particular
embodiments, the target site is the target site of an endogenous
sRNA of the plant that is endogenously expressed in the plant in a
tissue-specific, tissue-preferred, or cell type-specific manner.
"Tissue-preferred" sRNA expression means preferential expression in
certain tissues, and include, for example and without limitation:
sRNA that are preferentially expressed in leaves, pollen, tassels,
roots, seeds, fibers, xylem vessels, tracheids, and sclerenchyma.
sRNA expression essentially only in certain tissues is referred to
as "tissue-specific." A "cell type-specific" sRNA is primarily
expressed in certain cell types in one or more organs, for example,
vascular cells in roots or leaves.
[0140] Depending on the particular target gene and the level of
production of the sRNA, embodiments herein may provide partial or
complete downregulation of expression, or function, of the target
gene. The inhibition in target gene of interest expression in
different embodiments is at least a 5%, at least a 10%, at least a
20%, at least a 30%, at least a 50%, at least a 75%, at least an
80%, at least an 85%, at least a 90%, at least a 95%, or a 100%
inhibition in target gene of interest expression. Any plant species
or plant cell may be genetically modified to comprise a
heterologous nucleic acid herein. In some embodiments, the plant
cell that is so genetically modified is capable of regeneration to
produce a plant. In some embodiments, plant cells that are
genetically modified (e.g., host plant cells) include cells from,
for example and without limitation, a higher plant, a
dicotyledonous plant, a monocotyledonous plants, a consumable
plant, a crop plant, and a plant utilized for its oils (e.g., an
oilseed plant). Such plants include, for example and without
limitation: alfalfa; soybean; cotton; rapeseed (canola); linseed;
corn; rice; Brachiaria; wheat; safflower; sorghum; sugarbeet;
sunflower; tobacco; and grasses (e.g., turf grass). In particular
examples, a genetically modified plant cell or plant herein
includes, for example and without limitation: rapeseed (Brassica
napus); Indian mustard (Brassica juncea); Ethiopian mustard
(Brassica carinata); turnip (Brassica rapa); cabbage (Brassica
oleracea); soybean (Glycine max); Linum usitatissimum; corn (Zea
mays); Carthamus tinctorius; Helianthus annuus; tobacco (Nicotiana
tabacum); Arabidopsis thaliana, Brazil nut (Betholettia excelsa);
castor bean (Ricinus communis); coconut (Cocus nucifera); coriander
(Coriandrum sativum); cotton (Gossypium spp.); groundnut (Arachis
hypogaea); jojoba (Simmondsia chinensis); oil palm (Elaeis
guineeis); olive (Olea eurpaea); rice (Oryza sativa); squash
(Cucurbita maxima); barley (Hordeum vulgare); sugarcane (Saccharum
officinarum); Triticum spp. (including Triticum durum and Triticum
aestivum); and duckweed (Lemnaceae sp.). In some embodiments, the
plant may have a particular genetic background, as for elite
cultivars, wild-type cultivars, and commercially distinguishable
varieties.
[0141] According to methods known in the art, nucleic acids can be
introduced into essentially any plant. Embodiments herein may
employ any of the many methods for the transformation of plants
(and production of genetically modified plants) that are known in
the art. Such methods include, for example and without limitation,
biological and physical transformation protocols for dicotyledenous
plants, as well as monocotyledenous plants. See, e.g.,
Goto-Fumiyuki et al. (1999) Nat. Biotechnol. 17:282-6; Mild et al.
(1993) Methods in Plant Molecular Biology and Biotechnology (Glick,
B. R. and Thompson, J. E., Eds.), CRC Press, Inc., Boca Raton,
Fla., pp. 67-88. In addition, vectors and in vitro culture methods
for plant cell and tissue transformation and regeneration of plants
are described, for example, in Gruber and Crosby (1993) Methods in
Plant Molecular Biology and Biotechnology, supra, at pp.
89-119.
[0142] Plant transformation techniques available for introducing a
nucleic acid into a plant host cell include, for example and
without limitation: transformation with disarmed T-DNA using
Agrobacterium tumefaciens or A. rhizogenes as the transformation
agent; calcium phosphate transfection; polybrene transformation;
protoplast fusion; electroporation (D'Halluin et al. (1992) Plant
Cell 4:1495-505); ultrasonic methods (e.g., sonoporation); liposome
transformation; microinjection; contact with naked DNA; contact
with plasmid vectors; contact with viral vectors; biolistics (e.g.,
DNA particle bombardment (see, e.g., Klein et al. (1987) Nature
327:70-3) and microparticle bombardment (Sanford et al. (1987)
Part. Sci. Technol. 5:27; Sanford (1988) Trends Biotech. 6:299,
Sanford (1990) Physiol. Plant 79:206; and Klein et al. (1992)
Biotechnology 10:268); silicon carbide WHISKERS-mediated
transformation (Kaeppler et al. (1990) Plant Cell Rep. 9:415-8);
nanoparticle transformation (see, e.g., U.S. Patent Publication No.
US2009/0104700A1); aerosol beaming; and polyethylene glycol
(PEG)-mediated uptake. In specific examples, a heterologous nucleic
acid may be introduced directly into the genomic DNA of a plant
cell.
[0143] A widely utilized method for introducing an expression
vector into a plant is based on the natural transformation system
of Agrobacterium. Horsch et al. (1985) Science 227:1229. A.
tumefaciens and A. rhizogenes are plant pathogenic soil bacteria
known to be useful to genetically transform plant cells. The Ti and
Ri plasmids of A. tumefaciens and A. rhizogenes, respectively,
carry genes responsible for genetic transformation of the plant.
Kado (1991) Crit. Rev. Plant. Sci. 10:1. Details regarding
Agrobacterium vector systems and methods for Agrobacterium-mediated
gene transfer are also available in, for example, Gruber et al.,
supra, Mild et al., supra, Moloney et al. (1989) Plant Cell Reports
8:238, and U.S. Pat. Nos. 4,940,838 and 5,464,763.
[0144] If Agrobacterium is used for the transformation, the DNA to
be inserted typically is cloned into special plasmids; either into
an intermediate vector or a binary vector. Intermediate vectors
cannot replicate themselves in Agrobacterium. The intermediate
vector may be transferred into A. tumefaciens by means of a helper
plasmid (conjugation). The Japan Tobacco Superbinary system is an
example of such a system (reviewed by Komari et al. (2006) Methods
in Molecular Biology (K. Wang, ed.) No. 343; Agrobacterium
Protocols, 2.sup.nd Edition, Vol. 1, Humana Press Inc., Totowa,
N.J., pp. 15-41). Binary vectors can replicate themselves both in
E. coli and in Agrobacterium. Binary vectors comprise a selection
marker gene and a linker or polylinker which are framed by the
right and left T-DNA border regions. They can be transformed
directly into Agrobacterium (Holsters, 1978). The Agrobacterium
comprises a plasmid carrying a vir region. The Ti or Ri plasmid
also comprises the vir region necessary for the transfer of the
T-DNA. The vir region is necessary for the transfer of the T-DNA
into the plant cell. Additional T-DNA may be contained.
[0145] The virulence functions of the Agrobacterium tumefaciens
host will direct the insertion of a T-strand containing the
construct and adjacent marker into the plant cell DNA when the cell
is infected by the bacteria using a binary T DNA vector (Bevan
(1984) Nuc. Acid Res. 12:8711-21) or the co-cultivation procedure
(Horsch et al. (1985) Science 227:1229-31). Generally, the
Agrobacterium transformation system is used to engineer
dicotyledonous plants. Bevan et al. (1982) Ann. Rev. Genet
16:357-84; Rogers et al. (1986) Methods Enzymol. 118:627-41. The
Agrobacterium transformation system may also be used to transform,
as well as transfer, nucleic acids to monocotyledonous plants and
plant cells. See U.S. Pat. No. 5,591,616; Hernalsteen et al. (1984)
EMBO J 3:3039-41; Hooykass-Van Slogteren et al. (1984) Nature
311:763-4; Grimsley et al. (1987) Nature 325:1677-9; Boulton et al.
(1989) Plant Mol. Biol. 12:31-40; and Gould et al. (1991) Plant
Physiol. 95:426-34.
[0146] The genetic manipulations of a recombinant host herein may
be performed using standard genetic techniques and screening, and
may be carried out in any host cell that is suitable to genetic
manipulation. In some embodiments, a recombinant host cell may be
any organism or microorganism host suitable for genetic
modification and/or recombinant gene expression. In some
embodiments, a recombinant host may be a plant. Standard
recombinant DNA and molecular cloning techniques used here are
well-known in the art and are described in, for example and without
limitation: Sambrook etal. (1989), supra; Silhavy etal. (1984)
Experiments with Gene Fusions, Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y.; and Ausubel et al. (1987) Current
Protocols in Molecular Biology, Greene Publishing Assoc. and
Wiley-Interscience, New York, N.Y.
[0147] Following the introduction of a nucleic acid into a plant
cell, the plant cell may be grown, and upon emergence of
differentiating tissue such as shoots and roots, mature plants can
be generated. In some embodiments, a plurality of plants can be
generated. Methodologies for regenerating plants are known to those
of ordinary skill in the art and can be found, for example, in:
Plant Cell and Tissue Culture (Vasil and Thorpe, Eds.), Kluwer
Academic Publishers, 1994. Genetically modified plants described
herein may be cultured in a fermentation medium or grown in a
suitable medium such as soil. In some embodiments, a suitable
growth medium for higher plants may be any growth medium for
plants, including, for example and without limitation; soil, sand,
any other particulate media that support root growth (e.g.,
vermiculite, perlite, etc.) or hydroponic culture, as well as
suitable light, water and nutritional supplements that facilitate
the growth of the higher plant.
[0148] Transformed plant cells which are produced by any of the
above transformation techniques can be cultured to regenerate a
whole plant that possesses the transformed genotype, and thus the
desired phenotype. Such regeneration techniques rely on
manipulation of certain phytohormones in a tissue culture growth
medium, typically relying on a biocide and/or herbicide marker that
has been introduced together with the desired nucleotide sequences.
Plant regeneration from cultured protoplasts is described in Evans
et al. (1983) "Protoplasts Isolation and Culture," in Handbook of
Plant Cell Culture, Macmillian Publishing Company, New York, pp.
124-176; and Binding (1985) Regeneration of Plants, Plant
Protoplasts, CRC Press, Boca Raton, pp. 21-73. Regeneration can
also be performed from plant callus, explants, organs, pollens,
embryos or parts thereof. Such regeneration techniques are
described generally in Klee et al. (1987) Ann. Rev. Plant Phys.
38:467-86.
[0149] In other embodiments, the plant cells which are transformed
are not capable of regeneration to produce a plant. Such cells may
be employed, for example, in developing a plant cell line having a
relevant phenotype, for example, herbicide tolerance and/or male
sterility.
[0150] A transformed plant cell, callus, tissue or plant may be
identified and isolated by selecting or screening the engineered
plant material for traits encoded by the marker genes present on
the transforming DNA. For instance, selection can be performed by
growing the engineered plant material on media containing an
inhibitory amount of the antibiotic or herbicide to which the
transforming gene construct confers resistance. Further,
transformed plants and plant cells can also be identified by
screening for the activities of any visible marker genes (e.g., the
.beta.-glucuronidase, luciferase, or gfp genes) that may be present
on the recombinant nucleic acid constructs. Such selection and
screening methodologies are well known to those skilled in the
art.
[0151] A transgenic plant containing a heterologous molecule herein
can be produced through selective breeding, for example, by
sexually crossing a first parental plant comprising the molecule,
and a second parental plant, thereby producing a plurality of first
progeny plants. A first progeny plant may then be selected that is
resistant to a selectable marker (e.g., glyphosate, tolerance to
which may be conferred upon the progeny plant by the heterologous
molecule herein). The first progeny plant may then be selfed,
thereby producing a plurality of second progeny plants. Then, a
second progeny plant may be selected that is tolerant to the
selectable marker. These steps can further include the
back-crossing of the first progeny plant or the second progeny
plant to the second parental plant or a third parental plant.
[0152] It is also to be understood that two different transgenic
plants can also be mated to produce offspring that contain two
independently segregating, added, exogenous genes. Selfing of
appropriate progeny can produce plants that are homozygous for both
added, exogenous genes. Back-crossing to a parental plant and
out-crossing with a non-transgenic plant are also contemplated, as
is vegetative propagation. Other breeding methods commonly used for
different traits and crops are known in the art. Backcross breeding
has been used to transfer genes for a simply inherited, highly
heritable trait into a desirable homozygous cultivar or inbred
line, which is the recurrent parent. The resulting plant is
expected to have the attributes of the recurrent parent (e.g.,
cultivar) and the desirable trait transferred from the donor
parent. After the initial cross, individuals possessing the
phenotype of the donor parent are selected and repeatedly crossed
(backcrossed) to the recurrent parent. The resulting parent is
expected to have the attributes of the recurrent parent (e.g.,
cultivar) and the desirable trait transferred from the donor
parent.
[0153] A nucleic acid may also be introduced into a predetermined
area of the plant genome through homologous recombination. Methods
to stably integrate a polynucleotide sequence within a specific
chromosomal site of a plant cell via homologous recombination have
been described within the art. For instance, site specific
integration as described in US Patent Application Publication No.
2009/0111188 A1 involves the use of recombinases or integrases to
mediate the introduction of a donor polynucleotide sequence into a
chromosomal target. In addition, PCT International Patent
Publication No. WO 2008/021207 describes zinc finger
mediated-homologous recombination to stably integrate one or more
donor polynucleotide sequences within specific locations of the
genome. The use of recombinases such as FLP/FRT as described in
U.S. Pat. No. 6,720,475, or CRE/LOX as described in U.S. Pat. No.
5,658,772, can be utilized to stably integrate a polynucleotide
sequence into a specific chromosomal site. Finally, the use of
meganucleases for targeting donor polynucleotides into a specific
chromosomal location is described in Puchta et al. (1996) Proc.
Natl. Acad. Sci. USA 93:5055-60.
[0154] Other various methods for site specific integration within
plant cells are generally known and applicable. Kumar et al. (2001)
Trends Plant Sci. 6(4):155-9. Furthermore, site-specific
recombination systems that have been identified in several
prokaryotic and lower eukaryotic organisms may be applied for use
in plants. Examples of such systems include, but are not limited
to; the R/RS recombinase system from the pSR1 plasmid of the yeast
Zygosaccharomyces rouxii (Araki et al. (1985) J. Mol. Biol.
182:191-203), and the Gin/gix system of phage Mu (Maeser and
Kahlmann (1991) Mol. Gen. Genet. 230:170-6).
[0155] Various assays can be employed in connection with the
nucleic acid molecule of certain embodiments herein. In addition to
phenotypic observations, the following techniques are useful in
detecting the presence of a nucleic acid molecule in a plant cell.
For example, the presence of the molecule can be determined by
using a primer or probe of the sequence, an ELISA assay to detect
an encoded protein, a Western blot to detect the protein, or a
Northern or Southern blot to detect RNA or DNA. Additional
techniques, such as in situ hybridization, enzyme staining, and
immunostaining, also may be used to detect the presence or
expression of a recombinant construct in specific plant organs and
tissues.
[0156] Southern analysis is a commonly used detection method,
wherein DNA is cut with restriction endonucleases and fractionated
on an agarose gel to separate the DNA by molecular weight and then
transferring to nylon membranes. It is then hybridized with the
probe fragment which was radioactively labeled with .sup.32P (or
other probe labels) and washed in an SDS solution.
[0157] Likewise, Northern analysis deploys a similar protocol,
wherein RNA is cut with restriction endonucleases and fractionated
on an agarose gel to separate the RNA by molecular weight and then
transferring to nylon membranes. It is then hybridized with the
probe fragment which was radioactively labeled with .sup.32P (or
other probe labels) and washed in an SDS solution. Analysis of the
RNA (e.g., mRNA) isolated from the tissues of interest can indicate
relative expression levels. Typically, if the mRNA is present or
the amount of mRNA has increased, it can be assumed that the
corresponding transgene is being expressed. Northern analysis, or
other mRNA analytical protocols, can be used to determine
expression levels of an introduced transgene or native gene.
[0158] Nucleic acids herein, or segments thereof, may be used to
design primers for PCR amplification. In performing PCR
amplification, a certain degree of mismatch can be tolerated
between primer and template. Mutations, insertions, and deletions
can be produced in a given primer by methods known to an ordinarily
skilled artisan.
[0159] Hydrolysis probe assay, otherwise known as TAQMAN.RTM. (Life
Technologies, Foster City, Calif.), is another method of detecting
and quantifying the presence of a DNA sequence. Briefly, a FRET
oligonucleotide probe is designed with one oligo within the
transgene and one in the flanking genomic sequence for
event-specific detection. The FRET probe and PCR primers (one
primer in the insert DNA sequence and one in the flanking genomic
sequence) are cycled in the presence of a thermostable polymerase
and dNTPs. Hybridization of the FRET probe results in cleavage and
release of the fluorescent moiety away from the quenching moiety on
the FRET probe. A fluorescent signal indicates the presence of the
flanking/transgene insert sequence due to successful amplification
and hybridization.
[0160] VI. Plants
[0161] Some embodiments herein provide transgenic plants comprising
at least one nucleic acid comprising a target site for at least one
sRNA molecule, such as may be regenerated from stably transformed
plant cells or tissues, or may be produced by introgression of such
a nucleic acid from a donor line. Such plants may be used or
cultivated in any manner, wherein presence of the transforming
polynucleotide(s) of interest is desirable. Accordingly, transgenic
plants may be engineered to, inter alia, have one or more desired
traits, by transformation, and then may be cropped and cultivated
by any method known to those of skill in the art. Particular
embodiments herein provide parts, cells, and/or tissues of such
transgenic plants. Plant parts, without limitation, include seed,
endosperm, ovule and pollen. In some embodiments, the plant part is
seed tissue including seed coat, embryo, cotyledon, endosperm,
radicle, hypocotyl, or epicotyl.
[0162] Representative, non-limiting example plants include
Arabidopsis; field crops (e.g. alfalfa, barley, bean, clover, corn,
cotton, flax, lentils, maize, pea, rape (e.g., canola), rice, rye,
safflower, sorghum, soybean, sunflower, tobacco, and wheat);
vegetable crops (e.g., asparagus, beet, Brassica, broccoli,
Brussels sprouts, cabbage, carrot, cauliflower, celery, cucumber
(cucurbits), eggplant, lettuce, mustard, onion, pepper, potato,
pumpkin, radish, spinach, squash, taro, tomato, and zucchini);
fruit and nut crops (e.g., almond, apple, apricot, banana,
blackberry, blueberry, cacao, cassava, cherry, citrus, coconut,
cranberry, date, hazelnut, grape, grapefruit, guava, kiwi, lemon,
lime, mango, melon, nectarine, orange, papaya, passion fruit,
peach, peanut, pear, pineapple, pistachio, plum, raspberry,
strawberry, tangerine, walnut, and watermelon); tree woods and
ornamentals (e.g., alder, ash, aspen, azalea, birch, boxwood,
camellia, carnation, chrysanthemum, elm, fir, ivy, jasmine,
juniper, oak, palm, poplar, pine, redwood, rhododendron, rose, and
rubber).
[0163] To confirm the presence of a transforming polynucleotide(s)
of interest in a regenerating plant, a variety of assays may be
performed. Such assays include, for example and without limitation:
biochemical assays, such as detecting the presence of a protein
product, e.g., by immunological means (ELISA and/or Western blots)
or by enzymatic function; plant part assays (e.g., leaf or root
assays); and analysis of the phenotype of the plant.
[0164] There are numerous steps in the development of any novel,
desirable plant germplasm, which may begin with the generation of a
transgenic crop plant. In some embodiments, a transgenic plant
comprising at least one nucleic acid comprising a target site for
at least one sRNA molecule (e.g., a male sterile plant) may be used
in a plant breeding and/or germplasm development program.
[0165] Plant breeding begins with the analysis and definition of
problems and weaknesses of the current germplasm, the establishment
of program goals, and the definition of specific breeding
objectives. The next step is selection of germplasm that possess
the traits to meet the program goals. The goal is to combine in a
single variety an improved combination of desirable traits from the
parental germplasm. These important traits may include higher seed
yield, resistance to diseases and insects, better stems and roots,
tolerance to drought and heat, and better agronomic quality.
[0166] The choice of breeding or selection methods depends on the
mode of plant reproduction, the heritability of the trait(s) being
improved, and the type of cultivar used commercially (e.g., F.sub.1
hybrid cultivar, pureline cultivar, etc.). For highly heritable
traits, a choice of superior individual plants evaluated at a
single location will be effective, whereas for traits with low
heritability, selection should be based on mean values obtained
from replicated evaluations of families of related plants. Popular
selection methods include pedigree selection, modified pedigree
selection, mass selection, and recurrent selection.
[0167] The complexity of inheritance influences choice of the
breeding method. Backcross breeding is used to transfer one or a
few favorable genes for a highly heritable trait into a desirable
cultivar. This approach has been used extensively for breeding
disease-resistant cultivars. Various recurrent selection techniques
are used to improve quantitatively inherited traits controlled by
numerous genes. The use of recurrent selection in self-pollinating
crops depends on the ease of pollination, the frequency of
successful hybrids from each pollination, and the number of hybrid
offspring from each successful cross.
[0168] Each breeding program should include a periodic, objective
evaluation of the efficiency of the breeding procedure. Evaluation
criteria vary depending on the goal and objectives, but should
include gain from selection per year based on comparisons to an
appropriate standard, overall value of the advanced breeding lines,
and number of successful cultivars produced per unit of input
(e.g., per year, per dollar expended, etc.).
[0169] Promising advanced breeding lines are thoroughly tested and
compared to appropriate standards in environments representative of
the commercial target area(s) for three or more years. The best
lines are candidates for new commercial cultivars; those still
deficient in a few traits may be used as parents to produce new
populations for further selection.
[0170] These processes, which lead to the final step of marketing
and distribution, usually take from eight to twelve years from the
time the first cross is made. Therefore, development of new
cultivars is a time-consuming process that requires precise forward
planning, efficient use of resources, and a minimum of changes in
direction.
[0171] Pedigree breeding and recurrent selection breeding methods
are used to develop cultivars from breeding populations. Breeding
programs combine desirable traits from two or more cultivars or
various broad-based sources into breeding pools from which
cultivars are developed by selfing and selection of desired
phenotypes. The new cultivars are evaluated to determine which have
commercial potential.
[0172] Pedigree breeding is used commonly for the improvement of
self-pollinating crops. Two parents which possess favorable,
complementary traits are crossed to produce an F.sub.1. An F.sub.2
population is produced by selfing one or several F.sub.1s.
Selection of the best individuals may begin in the F.sub.2
population; then, beginning in the F.sub.3, the best individuals in
the best families are selected. Replicated testing of families can
begin in the F.sub.4 generation to improve the effectiveness of
selection for traits with low heritability. At an advanced stage of
inbreeding (i.e., F.sub.6 and F.sub.7), the best lines or mixtures
of phenotypically similar lines are tested for potential release as
new cultivars.
[0173] Mass and recurrent selections can be used to improve
populations of either self- or cross-pollinating crops. A
genetically variable population of heterozygous individuals is
either identified or created by intercrossing several different
parents. The best plants are selected based on individual
superiority, outstanding progeny, or excellent combining ability.
The selected plants are intercrossed to produce a new population in
which further cycles of selection are continued.
[0174] Backcross breeding has been used to transfer genes for a
simply inherited, highly heritable trait into a desirable
homozygous cultivar or inbred line which is the recurrent parent.
The source of the trait to be transferred is called the donor
parent. The resulting plant is expected to have the attributes of
the recurrent parent (e.g., cultivar) and the desirable trait
transferred from the donor parent. After the initial cross,
individuals possessing the phenotype of the donor parent are
selected and repeatedly crossed (backcrossed) to the recurrent
parent. The resulting plant is expected to have the attributes of
the recurrent parent (e.g., cultivar) and the desirable trait
transferred from the donor parent.
[0175] The single-seed descent procedure in the strict sense refers
to planting a segregating population, harvesting a sample of one
seed per plant, and using the one-seed sample to plant the next
generation. When the population has been advanced from the F.sub.2
to the desired level of inbreeding, the plants from which lines are
derived will each trace to different F.sub.2 individuals. The
number of plants in a population declines each generation due to
failure of some seeds to germinate or some plants to produce at
least one seed. As a result, not all of the F.sub.2 plants
originally sampled in the population will be represented by a
progeny when generation advance is completed.
[0176] In embodiments herein, a transgene comprising a target site
for at least one sRNA molecule may be introduced into a plant
germplasm, for example, to develop novel inbred lines that are
characterized by the tissue-specific expression of the transgene,
under the control of the sRNA molecule. A particular advantage of
such a development program may be that the generality of the RNAi
pathway results in a higher penetrance of the transgenic phenotype
than would otherwise be attainable, for example, by other control
mechanisms.
[0177] The following examples are provided to illustrate certain
particular features and/or embodiments. The examples should not be
construed to limit the disclosure to the particular features or
embodiments exemplified.
EXAMPLES
Example 1
Tissue-Specific sRNAs
[0178] Many plant miRNAs have a distinct developmental and
tissue-specific expression pattern. For example, Arabidopsis
thaliana miR171 (previously known as miR39) accumulates
predominantly in inflorescence tissues. miR171 is produced from an
intergenic region in chromosome III and functionally interacts with
mRNA targets encoding several members of the Scarecrow-like (SCL)
family of putative transcription factors. Llave et al. (2002)
Science 297: 2053-6. The interaction results in tissue-specific
cleavage of target mRNAs within the region of complementarity
between miR171 and a native gene mRNA. Transgene mRNAs carrying the
target site sequence are also recognized and cleaved.
[0179] Small RNA Library Construction and Sequencing: Small RNA
library construction was carried out using Illumina's TruSeq Small
RNA sample preparation kit according to the manufacturer's
recommendations. Briefly, RNA 3' and RNA 5' adapters were
sequentially ligated onto1.mu.g of high-quality purified Small RNA
sample using truncated T4 RNA Ligase 2 and T4 RNA Ligase
respectively. The small RNA fragments were then copied into first
strand cDNA using SuperScript II reverse transcriptase. The cDNA
was converted into double stranded cDNA (ds cDNA) with PCR using
two primers that respectively anneal to the ends of 3' and 5'
adapters. This process selectively enriches those fragments that
have adapter molecules on both ends. The amplified cDNA construct
is then purified on a 6% PAGE gel, normalized to 2nM concentration,
denatured with sodium hydroxide, and diluted in hybridization
buffer for loading onto a Hiseq flow cell. Cluster generation,
primer hybridization and sequencing reactions were carried out
according to Illumina's recommended protocol.
[0180] Data regarding miRNA expression of miR3522 (SEQ ID NO:1) and
miR1512 (SEQ ID NO:3) are shown in Table 1. The data demonstrate
that miR1512 is expressed at high levels in the seed and seed coat,
with medium expression in root, and very low expression in leaves
and other vegetative tissue, whereas miR3522 is expressed at high
or medium levels in all tissues examined except the root tissue.
Given the variation in expression among different soybean tissues,
miR1512 and miR3522 recognition sites were selected to exemplify
tissue-specific transgene expression modulation in a dicot
plant.
[0181] Table 1. miRNA microarray expression profiling of miR1512
and miR3522 in soybean tissues. Numbers represent relative
expression levels.
TABLE-US-00020 TABLE 1 Root 3 mm 5 mm Pods from Seed Embryo miRNA
Construct tip Root Hypocotyl Leaf Stem Flower Pod seed seed 5 mm
seeds Cotyledons coat axis miR1512 113183 3118 0 0 0 15 79 1854
10427 183 23938 70 13532 20 miR3522 113177 649 399 1310 15350 3180
18509 405 8958 30139 1351 26265 2551 3200
Example 2
Plant Transformation Vectors
[0182] In order to investigate whether endogenous miRNA molecules
can be used to confer modulation of transgene expression, the
following nucleic acid constructs were engineered and produced.
[0183] Plant Transformation Constructs. All constructs contain the
pat gene expression cassette, which contains the CsVMV promoter v2
(promoter and 5' untranslated region derived from the Cassava Vein
Mosaic virus; Verdaguer, B.; de Kochko, A.; Beachy, R. N.; Fauquet,
C. (1996), Isolation and expression in transgenic tobacco and rice
plants, of the cassava vein mosaic virus (CVMV) promoter. Plant
Molecular Biology, 31(6): 1129-1139) and the Zea mays Lipase 3'UTR
(labeled as "Zm Lip3 3'UTR"), pat v6 coding sequence (synthetic,
plant-optimized version of phosphinothricin acetyl transferase
(pat) gene, isolated from Streptomyces viridochromogenes, encoding
a protein that confers resistance to inhibitors of glutamine
synthetase comprising phophinothricin, glufosinate, and bialaphos.
Wohlleben, W.; Arnold, W.; Broer, I.; Hillemann, D.; Strauch, E.;
Puehler, A. Nucleotide sequence of the phosphinothricin
N-acetyltransferase gene from Streptomyces viridochromogenes Tu494
and its expression in Nicotiana tabacum. Gene (1988), 70(1):
25-37), and AtuORF1 3' UTR v3 (3' untranslated region (UTR)
comprising the transcriptional terminator and polyadenylation site
of open reading frame 1 (ORF1) of Agrobacterium
tumefacienspTi15955. Barker, R. F.; Idler, K. B.; Thompson, D. V.;
Kemp, J. D. Nucleotide sequence of the T-DNA region from the
Agrobacterium tumefaciens octopine Ti plasmid pTi15955. Plant
Molecular Biology (1983), 2(6): 335-50). pDAB113181 contains AAD-12
expression cassette comprising the ATUBI401.8 promoter (promoter,
5' untranslated region and intron from the Arabidopsis thaliana
polyubiquitin 10 (UBQ10) gene, Norris, S. R.; Meyer, S. E.; Callis,
J. (1993). The intron of Arabidopsis thaliana polyubiquitin genes
is conserved in location and is a quantitative determinant of
chimeric gene expression. Plant Molecular Biology, 21(5): 895-906),
AAD-12 vl (synthetic, plant-optimized version of an
aryloxyalkanoate dioxygenase gene from Delftia acidovorans encoding
an enzyme with alpha ketoglutarate-dependent dioxygenase activity
which results in metabolic inactivation of the herbicide(s) on
which it has enzymatic activity, WO2007/053482), and the AtuORF23
3' UTR v1(3' untranslated region (UTR) comprising the
transcriptional terminator and polyadenylation site of open reading
frame 23 (ORF23) of Agrobacterium tumefaciens pTi15955. Barker, R.
F.; Idler, K. B.; Thompson, D. V.; Kemp, J. D. (1983), Nucleotide
sequence of the T-DNA region from the Agrobacterium tumefaciens
octopine Ti plasmid pTi15955. Plant Molecular Biology, 2(6):
335-50). pDAB113177 contains AAD-12 expression cassette comprising
the ATUBI401.8 promoter (promoter, 5' untranslated region and
intron from the Arabidopsis thaliana polyubiquitin 10 (UBQ10) gene,
Norris, S. R.; Meyer, S. E.; Callis, J. (1993). The intron of
Arabidopsis thaliana polyubiquitin genes is conserved in location
and is a quantitative determinant of chimeric gene expression.
Plant Molecular Biology, 21(5): 895-906), AAD-12 vl (synthetic,
plant-optimized version of an aryloxyalkanoate dioxygenase gene
from Delftia acidovorans encoding an enzyme with an alpha
ketoglutarate-dependent dioxygenase activity which results in
metabolic inactivation of the herbicide(s) on which it has
enzymatic activity, WO2007/053482), and the AtuORF23 3' UTR v 1 (3'
untranslated region (UTR) comprising the transcriptional terminator
and polyadenylation site of open reading frame 23 (ORF23) of
Agrobacterium tumefaciens pTi15955. Barker, R. F.; Idler, K. B.;
Thompson, D. V.; Kemp, J. D. Nucleotide sequence of the T-DNA
region from the Agrobacterium tumefaciens octopine Ti plasmid
pTi15955. Plant Molecular Biology (1983), 2(6): 335-50). The miRNA
target site HTRNA15975.1 (SEQ ID NO:2) for miRNA3522 (SEQ ID NO:1)
was added between AAD-12 vl and AtuORF23 3' UTR v1. pDAB113183
contains AAD-12 expression cassette comprising the ATUBI401.8
promoter (promoter, 5' untranslated region and intron from the
Arabidopsis thaliana polyubiquitin 10 (UBQ10) gene, Norris, S. R.;
Meyer, S. E.; Callis, J. (1993), The intron of Arabidopsis thaliana
polyubiquitin genes is conserved in location and is a quantitative
determinant of chimeric gene expression. Plant Molecular Biology,
21(5): 895-906), AAD-12 vl (synthetic, plant-optimized version of
an aryloxyalkanoate dioxygenase gene from Delftia acidovorans
encoding an enzyme with an alpha ketoglutarate-dependent
dioxygenase activity which results in metabolic inactivation of the
herbicide(s) on which it has enzymatic activity, WO2007/053482),
and the AtuORF23 3' UTR v1(3' untranslated region (UTR) comprising
the transcriptional terminator and polyadenylation site of open
reading frame 23 (ORF23) of Agrobacterium tumefaciens pTi15955.
Barker, R. F., Idler, K. B.; Thompson, D. V.; Kemp, J. D.
Nucleotide sequence of the T-DNA region from the Agrobacterium
tumefaciens octopine Ti plasmid pTi15955. Plant Molecular Biology
(1983), 2(6): 335-50). The miRNA target site HTRNA17596.1 (SEQ ID
NO:2) for miRNA1512 (SEQ ID NO:4) was added between AAD-12 v1 and
AtuORF23 3' UTR v1. AAD-12 expression cassette comprising the
ATUBI401.8 promoter, AAD-12 v1 coding sequence, miR1512 target
site, and the AtuORF23 3' UTR v1.
[0184] miRNA target sites: The miRNA target site sequences for
miR1512 and miR3522 were designed to bind miRNA at the 3' end of
the coding region. Table 2.
TABLE-US-00021 TABLE 2 Exemplary miRNA target sites. SEQ ID NO: 2
TCAGCTGCTCATTTGGTCTCA miRNA3522 SEQ ID NO: 4 ATGCTCTAAGAATGTTCAGTTA
miRNA1512
[0185] Gateway.RTM. (INVITROGEN) entry vectors were constructed by
standard molecular cloning methods, and were used to create plant
transformation vectors pDAB113181, pDAB113177, and pDAB113183.
[0186] The structures of all plasmids were confirmed by restriction
enzyme digests and determinations of the DNA base sequences of
relevant regions were confirmed by standard molecular biological
techniques.
Example 3
Stable Expression in Soybean
[0187] Agrobacterium-mediated transformation was used to stably
integrate a chimeric gene into the plant genome, and thus generate
transgenic soybean cells, tissues, and plants that produce AAD12
mRNAs having miRNA target sites within the mRNA. Control tissues
having no miRNA target sites in the AAD1 mRNA were also
produced.
[0188] Agrobacterium-mediated plant transformation:
Agrobacterium-mediated transformation of soybean (Glycine max cv.,
Maverick) was performed via the split-seed explant with partial
embryo axis soybean transformation protocol described in US
2014/0173774 A1. After transformation, co-cultivation of explants
was carried out for 5 days. The explants were then washed in liquid
Shoot Induction (SI) medium for about 5 to 10 minutes and cultured
onto Shoot Induction-I (SI-I) medium. After 2 weeks of culture at
24.degree. C. with an 18 hour photoperiod, the explants were
transferred to the Shoot Induction-II (SI-II) medium supplemented
with 6 mg/L glufosinate. After 2 weeks on SI-II medium, the
cotyledons were removed from the explants, a flush shoot pad was
excised by making a cut at the base of the cotyledon, and the
isolated shoot was transferred to the Shoot Elongation (SE) medium.
The cultures were transferred to fresh SE medium every two weeks.
Lighting sources were provided with an illumination of 80-90
.mu.moles s.sup.-1 m.sup.-2 for the transformed tissues during
shoot induction and shoot elongation. The elongated shoots were
dipped in 1 mg/L indole 3-butyric acid (IBA) for about 1 to 3
minutes to promote rooting prior to transferring of the isolated
shoots to rooting medium (MS salts, B5 vitamins, 28 mg/L ferrous
sulphate, 38 mg/L Na.sub.2EDTA, 20 g/L sucrose and 0.59 g/L MES, 50
mg/L asparagine, 100 mg/L L-pyroglutamic acid, and 7 g/L NOBLE.TM.
agar, pH 5.6) in phyta trays. Following culturing in the rooting
medium at 24.degree. C., with an 18 hour photoperiod, for 1-2
weeks, the soybean shoots that produced healthy, viable roots were
transferred to soil. The soybean shoots comprising healthy, viable
roots were placed in soil which was contained in an open plastic
sundae cup. The plastic sundae cups containing the transferred
soybean shoots comprising roots were placed in a CONVIRON.TM. for
acclimatization of soybean plantlets. The rooted soybean plantlets
were acclimated in the open sundae cups for several weeks before
the plantlets were transferred to the greenhouse and grown to
maturity for T1 seed production.
[0189] T1 seed generated from self-pollination of single copy T0
events were planted in an artificial soil mix (MetroMix 360.TM.)
contained in 4-inch square pots. The T1 generation is a segregating
population; to distinguish transformed events from null
individuals, the T1 population received a foliar application of 411
g active ingredient (ai)/ha glufosinate ammonium (LIBERTY 280) when
plants reached the 1.sup.st trifoliate leaf stage. Four days after
application (DAA), survivors were sampled for molecular analysis to
determine zygosity and confirm aad12 copy number.
[0190] For each event, 8 homozygous and 8 hemizygous plants were
sampled for transcript and protein analyses, and the following day
(3.sup.rd trifoliate stage), half of the plants received a foliar
application of 2240 g acid equivalent (ae)/ha 2,4-D dimethylamine
(DAM) salt (WEEDAR 64). The remaining plants received no spray
application. Some plants were also sprayed with deionized water as
a sprayer application control. In addition to the transformed
events, the soybean variety, `Maverick`, was included in each
treatment as a non-transformed control.
[0191] Foliar applications were made with a Mandel track sprayer
set to deliver a spray solution at 187 L/ha to a spray area of
0.503 m.sup.2 using an 8002E nozzle and a spray height of 18 inches
above the plant canopy. Plant response to the 2,4-D application was
evaluated at 1, 7, and 14 days after application (DAA). Data were
collected by assessment of visual injury and/or growth reduction as
compared to untreated controls on a scale of 0% to 100% with 0%
corresponding to no injury or growth reduction and 100%
corresponding to complete plant death. Plants were maintained at
28.degree. C./25.degree. C. (day/night) under a 14 h photoperiod
and subirrigated with water or fertilizer as needed.
Example 4
Biochemical and Molecular Analyses of Transgenic Soybean
Tissues
[0192] Protein extraction from soybean leaf: Two 6 mm diameter leaf
samples were stored in a 96 well cluster tube rack at -80.degree.
C. until the day of analysis. Two DAISY.TM. steel BB's and 200
.mu.l of extraction buffer (PBS solution containing 0.05% of Tween
20, 5 .mu.l/ml of Sigma protease inhibitors catalog number 9599,
and 0.75% Ovabumin Sigma catalog number A-5503) were added to each
tube. The samples were milled in a KLECKO.TM. tissue pulverizer for
3 minutes, on the maximum setting. Samples were centrifuged at
3,000.times.g for 5 minutes; 100 .mu.l of the supernatant was
transferred to an empty sample tube. Another 100 .mu.l of
extraction buffer was added to the plant sample and bead milled for
3 additional minutes, centrifuged, and 100 .mu.l of this extract
was combined with the first 100 .mu.l. The combined supernatants
were mixed and analyzed the same day as the extraction.
[0193] ELISA quantification of AAD12 protein: Enzyme Linked
Immunosorbant Assays (ELISAs) were used to measure the production
of AAD12 proteins in soybean leaf. AAD12 protein was quantified
using a ENVIROLOGIX.TM. (catalog number AP018) kit using a Dow
AgroSciences produced AAD12 purified protein as the standard. The
standard curve ranged from 200 ng/ml to 3.1 ng/ml. The ELISAs were
performed using the reagents and instructions essentially as
provided by the suppliers.
[0194] Total RNA extraction for sRNA sequencing Total RNA was
isolated using Plant/Fungi Total RNA Purification Kit (Norgen
#25800). The standard manufacturer's protocol was followed with
.about.30 mg of the ground tissues as starting material including
the on-column DNase treatment step. The concentration and quality
of RNA for each sample was determined by UV spectrometry (Nanodrop
ND-1000 spectrophotometer). Approximately 1 ug of total RNA was
used for sRNA library preparation.
[0195] Hydrolysis Probe qPCR for copy number analysis: Various
types of molecular analyses were employed to screen for low copy,
simple events. Leaf tissue was collected from rooted putative
transgenic plants before transplanting to soil. DNA was extracted
with a QIAGEN MAGATTRACT.TM. kit using THERMO FISHER KINGFISHER.TM.
magnetic particle processors and the supplier's recommended
protocols. Integrated transgene copy number analysis was performed
using specific Hydrolysis Probe assays for the AAD12 and PAT genes.
In addition, contamination by inadvertent integration of the binary
vector plasmid backbone was detected by a Hydrolysis Probe assay
specific for the Spectinomycin resistance gene (Spec) borne on the
binary vector backbone. Hydrolysis Probe assays for endogenous
soybean genes GSM116; (GenBank.TM. Accession No. AK286292.1) and
Actin (GENBANK Accession No. V00450.1) were developed as internal
reference standards. Table 3 lists the oligonucleotide sequences of
the Hydrolysis Probe assay components (synthesized by INTEGRATED
DNA TECHNOLOGIES, Coralville, Iowa). Biplex Hydrolysis Probe PCR
reactions were set up according to Table 4 with about 10 ng DNA,
and assay conditions are presented in Table 5.
TABLE-US-00022 TABLE 3 List of forward and reverse nucleotide
primers and fluorescent probes used for transgene copy number and
relative expression detection. Gene Detected Oligonucleotide
Sequence AAD12 AAD12_F AACCCGTGCTCTTGTTC (SEQ ID NO: 9) AAD12_R
GGATGCACCTTGACCAAT (SEQ ID NO: 10) AAD12_P CAGGCCGGGTCAGCCT (SEQ ID
NO: 11) (FAM* Probe) PAT PAT_F ACAAGAGTGGATTGATGATCTAGAGAGGT (SEQ
ID NO: 12) PAT_R CTTTGATGCCTATGTGACACGTAAACAGT (SEQ ID NO: 13)
PAT_FamP GGTGTTGTGGCTGGTATTGCTTACGCTGG (FAM Probe) (SEQ ID NO: 14)
Spec Spec_F CGCCGAAGTATCGACTCAACT (SEQ ID NO: 15) Spec_R
GCAACGTCGGTTCGAGATG (SEQ ID NO: 16) Spec_P TCAGAGGTAGTTGGCGTCATCGAG
(SEQ ID NO: 17) (FAM Probe) Soybean GMS116_F
GTAATATGGGCTCAGAGGAATGGT (SEQ ID NO: 18) GMS116 GMS116_R
ATGGAGAAGAACATTGGAATTGC (SEQ ID NO: 19) GMS116_P
CCATGGCCCGGTACCATCTGGTC (SEQ ID NO: 20) (HEX* Probe) Soybean Soy
Actin_F GTGACTCACACCATCACCAGAGT (SEQ ID NO: 21) Actin Soy Actin_F
ATCCAGGCTGTCCTCTCCCTATA (SEQ ID NO: 22) Soy Actin_P*
CAATACCAGTTGTGCGACC (SEQ ID NO: 23) (VIC .RTM. Probe) *FAM =
6-Carboxy Fluorescein Amidite; HEX = Hexachlorofluorescein
TABLE-US-00023 TABLE 4 Hydrolysis Probe PCR Mixture for Transgene
DNA Copy Number Analysis. Reaction Component .mu.L Final
Concentration Water 0.5 PVP (10%) 0.1 0.1% ROCHE 2X Master Mix 5 1X
Transgene Forward Primer (10 .mu.M) 0.4 0.4 .mu.M Transgene Reverse
Primer (10 .mu.M) 0.4 0.4 .mu.M Transgene Probe (5 .mu.M) 0.4 0.2
.mu.M GMS116 Forward Primer (10 .mu.M) 0.4 0.4 .mu.M GMS116 Reverse
Primer (10 .mu.M) 0.4 0.4 .mu.M GMS116 Probe (5 .mu.M) 0.4 0.2
.mu.M
TABLE-US-00024 TABLE 5 Thermocycler Conditions for Hydrolysis Probe
PCR Amplification. PCR Steps Temp (.degree. C.) Time No. cycles
Denature/Activation 95 10 min 1 Denature 95 10 sec 40 Anneal/Extend
60 35 sec Acquire 72 1 sec Cool 40 10 sec 1
[0196] For amplification, LIGHTCYCLER.RTM.480 Probes Master mix
(ROCHE APPLIED SCIENCE, Indianapolis, Ind.) was prepared at 1X
final concentration in a 10 .mu.L volume multiplex reaction
containing 0.1% PVP, 0.4 .mu.M of each primer, and 0.2 .mu.M of
each probe. The FAM (6-Carboxy Fluorescein Amidite) fluorescent
moiety was excited at 465 nm, and fluorescence was measured at 510
nm. The corresponding values for the HEX (hexachlorofluorescein)
fluorescent moiety were 533 nm and 580 nm, and for VIC.RTM. the
values were 538 nm and 554 nm. The level of fluorescence generated
for each reaction was analyzed using the ROCHE LIGHTCYCLER.RTM.480
Real-Time PCR system according to the manufacturer's
recommendations. Transgene copy number was determined by comparison
of LIGHTCYCLER.RTM.480 outputs of Target/Reference gene values for
unknown samples to Target/Reference gene values of known copy
number standards (1-Copy representing hemizygous plants, 2-Copy
representing homozygous plants).
[0197] Cp scores; i.e., the point at which the florescence signal
crosses the background threshold using the fit points algorithm
(LIGHTCYCLER.RTM. software release 1.5), and the Relative Quant
module (based on the .DELTA..DELTA.Ct method), were used to perform
the analysis of real time PCR data.
[0198] In the LIGHTCYCLER.RTM. Fit Points Algorithm software, a
graph of the data was made by plotting the logarithm of the input
DNA template concentration against the measured Cp values. The
slope of the curve is a desired comparison parameter; therefore the
initial log input number can be an arbitrary starting point on the
curve, with the caveat that the arbitrary concentration values used
for input DNA template are representative of the actual serial
dilution used. For example, for a 10-fold serial dilution series,
the actual inputs concentrations may be 1000, 100, 10, etc., for
which points the LC480 Fit Points Algorithm software plots 3, 2, 1,
etc. as the logarithms of the inputs. Using a linear regression,
the resulting best fit of this line (input log vs Cp) was then used
to estimate a slope (m) from an equation of the form y=mx+b. There
is an inverse relationship between the starting template amount and
Cp value, and therefore the slope (m) is always negative.
[0199] A perfect (i.e., 100% efficient) PCR reaction doubles the
total template every cycle. PCR efficiency (Eff) is calculated
as:
Eff =10e.sup.(-1/m)
[0200] Thus, the slope (m) of the graph of log input vs Cp will be
-3.3219 for a perfectly efficient reaction (whose efficiency is
defined as 2.00).
[0201] In other words, a 100% efficient PCR reaction is defined
by:
2.0=10 e.sup.(-1/-3.3219)
[0202] The LC480 Fit Points Algorithm software reports the
efficiency value by the first formula. So a 99% efficient reaction
has an Eff value of 1.99, rather than 0.99.
[0203] To express this as a percent efficiency, subtract 1 from
this value and multiply by 100. Or,
% Eff=[(10 e.sup.(-1/m)-1)].times.100
[0204] AAD12 relative transcript analysis: Quantitative Hydrolysis
Probe PCR was also used to detect the relative levels of AAD12
transcript. Leaf tissue was collected at the V4 stage while
cotyledon and seed coat tissue was extracted from immature seed.
Approximately 500 ng total RNA (extracted with a KINGFISHER.TM.
total RNA Kit; THERMO FISHER SCIENTIFIC) was used for cDNA
synthesis using a high capacity cDNA synthesis kit (INVITROGEN) and
random primer T20VN (TTTTTTTTTTTTTTTTTTTTVN; where V is A, C, or G,
and N is A, C, G, or T/U). Typically, a 20 .mu.L reaction contained
2.5 U/.mu.L MULTISCRIBE.TM. reverse transcriptase, 200 nM T20VN
oligonucleotide, and 4 mM dNTPs. The reaction was initiated by
incubation for 10 minutes at 25.degree. C., then synthesis was
performed for 120 minutes at 37.degree. C. and inactivated by 5
minutes at 85.degree. C.
[0205] Newly-synthesized cDNA was used for PCR amplification.
Hydrolysis Probe qPCR set up, running conditions, and signal
capture were the same as given above for DNA copy number analyses.
AAD12 expression data were calculated using 2-.DELTA..DELTA.Ct
relative to the level of Actin.
Example 5
miRNA3522 Target Site-Mediated Optimization of AAD12 mRNA
Expression in Different Tissues Sampled from T1 Transgenic Soybean
Plants
[0206] Transgenic soybean plants were produced wherein the
accumulation of an AAD12 mRNA comprising a miRNA target site was
limited by miRNA3522 cleavage in tissues or cells comprising the
leaf, stem, flower, seed, cotyledons, seed coat, embryo, or other
cells. Micro RNA expression profiling disclosed in Table 1 shows
that miR3522 is not specifically expressed in the root tissue, but
rather is expressed at medium to high levels in a majority of the
above ground tissues of the soybean plant.
[0207] More than 15 plants from each single copy transgenic event
were analyzed for expression analysis. AAD-12 mRNA levels were
calculated relative to the levels of soybean actin mRNA as
described in EXAMPLE 4. The data in FIGS. 5, 6, & 7 show that
there was tissue-specific effect of miRNA3522 miRNA target sites on
AAD12 expression. The accumulation of AAD12 transcript was reduced
in leaf, cotyledon and seed coat tissue samples obtained from
soybean plants transformed with construct pDAB113177 containing
miRNA3522 target site, as compared to the "no miRNA" control plants
transformed with pDAB113181. The AAD12 reduction in these plants is
due to miR3522 that is present in high abundance in the leaf, seed
coat and cotyledon tissue. These results confirm that adding target
sites of naturally occurring miRNA in the transgene can be
exploited to optimize expression of a transgene in a dicot
plant.
Example 6
miRNA1512 Target Site-Mediated Optimization of AAD12 mRNA
Expression in Soybean Seed Tissues
[0208] Transgenic soybean plants were produced wherein the
accumulation of an AAD12 mRNA comprising a miRNA target site was
limited by miRNA1512 cleavage in tissues or cells comprising the
seed, pods, seed coat, and root tip. Micro RNA expression profiling
disclosed in Table 1 shows that miR1512 is not specifically
expressed in the root, hypocotyl, leaf, stem, flower, and cotyledon
but rather is expressed at medium to high levels in seed and seed
coat tissues of the soybean plant.
[0209] Immunolocalization of AAD12 protein was done to demonstrate
the effect of miRNA1512 on expression of AAD12 in seed tissue.
Seeds coats were removed from the cotyledons of mature, dry soybean
seeds. Seeds were oriented with the hilum down and with the median
sagittal plane passing between the left and right cotyledons. The
hilum was removed with a single transverse cut just above the
hilum. The median coronal section, approximately 1 mm thick, was
dissected from the seed, and the coat was removed from the two
cotyledon pieces. This region was chosen because it includes both
thick (lateral) and thin (dorsal) regions of seed coat in the same
piece of tissue. Seed coats and cotyledons of each sample type were
placed in scintillation vials and fixed overnight at 4.degree. C.
in 4% formaldehyde (in 10 mM phosphate buffered saline [PBS] with
1:10,000 Silwet L-77 [Lehle Seeds, Round Rock, Tex., USA]).
Fixative solution was removed the following day, and samples were
rinsed briefly with 10 mM PBS solution. Undiluted acetylated bovine
serum albumin (BSA-c.TM., Aurion, Electron Microscopy Sciences,
Hatfield, Pa., USA) solution was added and allowed to infiltrate
the samples on a rotator at 4.degree. C. for 4 hrs. Samples were
again rinsed briefly with 10 mM PBS to remove excess BSA-c.TM., and
then returned to fresh fixative solution for a second overnight
fixation in 4% formaldehyde. Samples were briefly rinsed with 10 mm
PBS, and dehydrated in a graded ethanol series. Samples were then
infiltrated with LR White resin (Polysciences, Warrington, Pa.) and
polymerized into acrylic blocks; seed coats and corresponding
cotyledons were polymerized together in the same block.
[0210] 500 nm thick sections were generated for all samples using a
DDK histo diamond knife (Delaware Diamond Knives, Inc., Wilmington,
Del., USA) on a Leica EM UC 7 ultramicrotome (Leica Microsystems,
Buffalo Grove, Ill., USA). Tissue sections were stretched and dried
onto Fisherbrand.TM. SuperFrost.TM. Plus slides (Thermo Fisher
Scientific, Pittsburgh, Pa., USA). Two sections (technical
replicates) were generated for each sample type. Sections were
immunolabeled with a custom mouse monoclonal antibody to the AAD12
protein at a concentration of 1:2000 in Da Vinci Green antibody
diluent (Biocare Medical, Concord, Calif., USA), amplified with a
two part probe-polymer HRP secondary antibody system (Biocare
Medical, Concord, Calif., USA), and signal was detected with an
enzymatic deposition of silver (EnzMet.TM. Silver, Nanoprobes,
Inc., Yaphank, N.Y., USA).
[0211] All steps of the immunolocalization were completed in an
opaque humid box (Evergreen Scientific, Vernon, Calif.) at RT.
Tissue sections were first blocked with a PBS-BSA (bovine serum
albumin)-Tween 20 (10 mM PBS with 1% BSA and 0.1% Tween 20, all
reagents from Sigma-Aldrich, St. Louis, Mo., USA) wash solution for
30 min. The wash solution was removed and the primary antibody was
added and incubated for 2.5 hrs with constant agitation. After the
primary antibody incubation, the sections were washed 3 times for 5
min each with the washing solution. The HRP probe solution was
added and incubated for 15 min with agitation, this solution was
removed, another 3 washes for 5 min series was completed, then the
HRP polymer solution was added and incubated for 15 min. Another 3
washes for 5 min each was completed, and the slides were gently
rinsed in deionized water. Slides were allowed to dry to completion
at RT prior to addition of the enzymatic silver solution. Briefly,
the solution was prepared according to manufacturer's instructions
and incubated on the tissue sections for 12 min on the bench at RT
without agitation. Silver solution was removed, and slides were
thoroughly rinsed in deionized water, then blown dry with
compressed air. Slides were then mounted dry with Poly-Mount Xylene
(Polysciences, Inc., Warrington, Pa., USA) and Corning brand cover
glasses (Thermo Fisher Scientific, Pittsburgh, Pa., USA), weighted
on a slide warmer, and cured overnight prior to viewing and
imaging. Slides were imaged on a Leica DM5000 upright microscope
(Leica Microsystems, Buffalo Grove, Ill., USA) with a Leica DFC
7000T digital camera and Leica Application Suite software (version
4.6).
[0212] Brightfield images of the cotyledon and seed coat from the
three sample types are shown on the left panels of the FIG. 8, and
phase contrast images from the same field of view as each
brightfield image are shown on the right. The non-transgenic
`Maverick` cotyledon sections showed a very low level of background
silver deposition, and the seed coat sections show essentially no
background silver. The sections taken from soybean plants
transformed with construct pDAB113183 containing miRNA1512 target
site show levels of staining very similar to `Maverick`, indicating
below-background levels of AAD12 detected by this method. The
sections from "no miRNA" control plants transformed with pDAB113181
showed silver deposition (AAD12 detection) in the cells of the
cotyledon, with heavier staining over the epidermal cells; and
relatively strong staining primarily of the aleurone layer cells in
the seed coat. Scale bar=50 .mu.M.
[0213] The protein expression analysis of leaf tissue showed no
significant change in AAD12 protein in transgenic plants containing
miR1512 target site (pDAB113183), when compared to plants producing
AAD-12 without any target site (pDAB113181). (FIG. 9).
[0214] The transgenic plants containing miR1512 target site
(pDAB113183) demonstrated robust tolerance to 2,4-D application.
Unlike non-transgenic control `Maverick` plants, both pDAB113181
and pDAB113183 did not show any injury after 2,4-D application
(FIG. 10).
[0215] These results confirm that desired and precise regulation of
expression of one or more transgenes can be obtained by adding
selected target sites of native miRNA to the transgene sequence in
a tissue-specific, tissue-preferred manner, or cell type-specific
manner, depending on the target site selected.
Sequence CWU 1
1
23121RNAGlycine max 1ugagaccaaa ugagcagcug a 21221DNAArtificial
Sequencetarget site HTRNA15975.1 2tcagctgctc atttggtctc a
21322RNAGlycine max 3uaacugaaca uucuuagagc au 22422DNAArtificial
Sequencetarget site HTRNA15976.1 4atgctctaag aatgttcagt ta
2251441DNAArtificial Sequenceexpression cassette 5atggctcaga
ccactctcca aatcacaccc actggtgcca ccttgggtgc cacagtcact 60ggtgttcacc
ttgccacact tgacgatgct ggtttcgctg ccctccatgc agcctggctt
120caacatgcac tcttgatctt ccctgggcaa cacctcagca atgaccaaca
gattaccttt 180gctaaacgct ttggagcaat tgagaggatt ggcggaggtg
acattgttgc catatccaat 240gtcaaggcag atggcacagt gcgccagcac
tctcctgctg agtgggatga catgatgaag 300gtcattgtgg gcaacatggc
ctggcacgcc gactcaacct acatgccagt catggctcaa 360ggagctgtgt
tcagcgcaga agttgtccca gcagttgggg gcagaacctg ctttgctgac
420atgagggcag cctacgatgc ccttgatgag gcaacccgtg ctcttgttca
ccaaaggtct 480gctcgtcact cccttgtgta ttctcagagc aagttgggac
atgtccaaca ggccgggtca 540gcctacatag gttatggcat ggacaccact
gcaactcctc tcagaccatt ggtcaaggtg 600catcctgaga ctggaaggcc
cagcctcttg atcggccgcc atgcccatgc catccctggc 660atggatgcag
ctgaatcaga gcgcttcctt gaaggacttg ttgactgggc ctgccaggct
720cccagagtcc atgctcacca atgggctgct ggagatgtgg ttgtgtggga
caaccgctgt 780ttgctccacc gtgctgagcc ctgggatttc aagttgccac
gtgtgatgtg gcactccaga 840ctcgctggac gcccagaaac tgagggtgct
gccttggttt gagtagttag cttaatcacc 900tagagctcgg tcaccagcat
aatttttatt aatgtactaa attactgttt tgttaaatgc 960aattttgctt
tctcgggatt ttaatatcaa aatctattta gaaatacaca atattttgtt
1020gcaggcttgc tggagaatcg atctgctatc ataaaaatta caaaaaaatt
ttatttgcct 1080caattatttt aggattggta ttaaggacgc ttaaattatt
tgtcgggtca ctacgcatca 1140ttgtgattga gaagatcagc gatacgaaat
attcgtagta ctatcgataa tttatttgaa 1200aattcataag aaaagcaaac
gttacatgaa ttgatgaaac aatacaaaga cagataaagc 1260cacgcacatt
taggatattg gccgagatta ctgaatattg agtaagatca cggaatttct
1320gacaggagca tgtcttcaat tcagcccaaa tggcagttga aatactcaaa
ccgccccata 1380tgcaggagcg gatcattcat tgtttgtttg gttgcctttg
ccaacatggg agtccaaggt 1440t 144161462DNAArtificial
Sequenceexpression cassette 6atggctcaga ccactctcca aatcacaccc
actggtgcca ccttgggtgc cacagtcact 60ggtgttcacc ttgccacact tgacgatgct
ggtttcgctg ccctccatgc agcctggctt 120caacatgcac tcttgatctt
ccctgggcaa cacctcagca atgaccaaca gattaccttt 180gctaaacgct
ttggagcaat tgagaggatt ggcggaggtg acattgttgc catatccaat
240gtcaaggcag atggcacagt gcgccagcac tctcctgctg agtgggatga
catgatgaag 300gtcattgtgg gcaacatggc ctggcacgcc gactcaacct
acatgccagt catggctcaa 360ggagctgtgt tcagcgcaga agttgtccca
gcagttgggg gcagaacctg ctttgctgac 420atgagggcag cctacgatgc
ccttgatgag gcaacccgtg ctcttgttca ccaaaggtct 480gctcgtcact
cccttgtgta ttctcagagc aagttgggac atgtccaaca ggccgggtca
540gcctacatag gttatggcat ggacaccact gcaactcctc tcagaccatt
ggtcaaggtg 600catcctgaga ctggaaggcc cagcctcttg atcggccgcc
atgcccatgc catccctggc 660atggatgcag ctgaatcaga gcgcttcctt
gaaggacttg ttgactgggc ctgccaggct 720cccagagtcc atgctcacca
atgggctgct ggagatgtgg ttgtgtggga caaccgctgt 780ttgctccacc
gtgctgagcc ctgggatttc aagttgccac gtgtgatgtg gcactccaga
840ctcgctggac gcccagaaac tgagggtgct gccttggttt gagtagttag
cttaatcacc 900tagagctcgg tcagctgctc atttggtctc atcaccagca
taatttttat taatgtacta 960aattactgtt ttgttaaatg caattttgct
ttctcgggat tttaatatca aaatctattt 1020agaaatacac aatattttgt
tgcaggcttg ctggagaatc gatctgctat cataaaaatt 1080acaaaaaaat
tttatttgcc tcaattattt taggattggt attaaggacg cttaaattat
1140ttgtcgggtc actacgcatc attgtgattg agaagatcag cgatacgaaa
tattcgtagt 1200actatcgata atttatttga aaattcataa gaaaagcaaa
cgttacatga attgatgaaa 1260caatacaaag acagataaag ccacgcacat
ttaggatatt ggccgagatt actgaatatt 1320gagtaagatc acggaatttc
tgacaggagc atgtcttcaa ttcagcccaa atggcagttg 1380aaatactcaa
accgccccat atgcaggagc ggatcattca ttgtttgttt ggttgccttt
1440gccaacatgg gagtccaagg tt 146271463DNAArtificial
Sequenceexpression cassette 7atggctcaga ccactctcca aatcacaccc
actggtgcca ccttgggtgc cacagtcact 60ggtgttcacc ttgccacact tgacgatgct
ggtttcgctg ccctccatgc agcctggctt 120caacatgcac tcttgatctt
ccctgggcaa cacctcagca atgaccaaca gattaccttt 180gctaaacgct
ttggagcaat tgagaggatt ggcggaggtg acattgttgc catatccaat
240gtcaaggcag atggcacagt gcgccagcac tctcctgctg agtgggatga
catgatgaag 300gtcattgtgg gcaacatggc ctggcacgcc gactcaacct
acatgccagt catggctcaa 360ggagctgtgt tcagcgcaga agttgtccca
gcagttgggg gcagaacctg ctttgctgac 420atgagggcag cctacgatgc
ccttgatgag gcaacccgtg ctcttgttca ccaaaggtct 480gctcgtcact
cccttgtgta ttctcagagc aagttgggac atgtccaaca ggccgggtca
540gcctacatag gttatggcat ggacaccact gcaactcctc tcagaccatt
ggtcaaggtg 600catcctgaga ctggaaggcc cagcctcttg atcggccgcc
atgcccatgc catccctggc 660atggatgcag ctgaatcaga gcgcttcctt
gaaggacttg ttgactgggc ctgccaggct 720cccagagtcc atgctcacca
atgggctgct ggagatgtgg ttgtgtggga caaccgctgt 780ttgctccacc
gtgctgagcc ctgggatttc aagttgccac gtgtgatgtg gcactccaga
840ctcgctggac gcccagaaac tgagggtgct gccttggttt gagtagttag
cttaatcacc 900tagagctcgg atgctctaag aatgttcagt tatcaccagc
ataattttta ttaatgtact 960aaattactgt tttgttaaat gcaattttgc
tttctcggga ttttaatatc aaaatctatt 1020tagaaataca caatattttg
ttgcaggctt gctggagaat cgatctgcta tcataaaaat 1080tacaaaaaaa
ttttatttgc ctcaattatt ttaggattgg tattaaggac gcttaaatta
1140tttgtcgggt cactacgcat cattgtgatt gagaagatca gcgatacgaa
atattcgtag 1200tactatcgat aatttatttg aaaattcata agaaaagcaa
acgttacatg aattgatgaa 1260acaatacaaa gacagataaa gccacgcaca
tttaggatat tggccgagat tactgaatat 1320tgagtaagat cacggaattt
ctgacaggag catgtcttca attcagccca aatggcagtt 1380gaaatactca
aaccgcccca tatgcaggag cggatcattc attgtttgtt tggttgcctt
1440tgccaacatg ggagtccaag gtt 146381322DNAArabidopsis thaliana
8gtcgacctgc aggtcaacgg atcaggatat tcttgtttaa gatgttgaac tctatggagg
60tttgtatgaa ctgatgatct aggaccggat aagttccctt cttcatagcg aacttattca
120aagaatgttt tgtgtatcat tcttgttaca ttgttattaa tgaaaaaata
ttattggtca 180ttggactgaa cacgagtgtt aaatatggac caggccccaa
ataagatcca ttgatatatg 240aattaaataa caagaataaa tcgagtcacc
aaaccacttg ccttttttaa cgagacttgt 300tcaccaactt gatacaaaag
tcattatcct atgcaaatca ataatcatac aaaaatatcc 360aataacacta
aaaaattaaa agaaatggat aatttcacaa tatgttatac gataaagaag
420ttacttttcc aagaaattca ctgattttat aagcccactt gcattagata
aatggcaaaa 480aaaaacaaaa aggaaaagaa ataaagcacg aagaattcta
gaaaatacga aatacgcttc 540aatgcagtgg gacccacggt tcaattattg
ccaattttca gctccaccgt atatttaaaa 600aataaaacga taatgctaaa
aaaatataaa tcgtaacgat cgttaaatct caacggctgg 660atcttatgac
gaccgttaga aattgtggtt gtcgacgagt cagtaataaa cggcgtcaaa
720gtggttgcag ccggcacaca cgagtcgtgt ttatcaactc aaagcacaaa
tacttttcct 780caacctaaaa ataaggcaat tagccaaaaa caactttgcg
tgtaaacaac gctcaataca 840cgtgtcattt tattattagc tattgcttca
ccgccttagc tttctcgtga cctagtcgtc 900ctcgtctttt cttcttcttc
ttctataaaa caatacccaa agcttcttct tcacaattca 960gatttcaatt
tctcaaaatc ttaaaaactt tctctcaatt ctctctaccg tgatcaaggt
1020aaatttctgt gttccttatt ctctcaaaat cttcgatttt gttttcgttc
gatcccaatt 1080tcgtatatgt tctttggttt agattctgtt aatcttagat
cgaagacgat tttctgggtt 1140tgatcgttag atatcatctt aattctcgat
tagggtttca taaatatcat ccgatttgtt 1200caaataattt gagttttgtc
gaataattac tcttcgattt gtgatttcta tctagatctg 1260gtgttagttt
ctagtttgtg cgatcgaatt tgtcgattaa tctgagtttt tctgattaac 1320ag
1322917DNAArtificial Sequenceprimer oligonucleotide 9aacccgtgct
cttgttc 171018DNAArtificial Sequenceprimer oligonucleotide
10ggatgcacct tgaccaat 181116DNAArtificial Sequenceprobe
oligonucleotide 11caggccgggt cagcct 161229DNAArtificial
Sequenceprimer oligonucleotide 12acaagagtgg attgatgatc tagagaggt
291329DNAArtificial Sequenceprimer oligonucleotide 13ctttgatgcc
tatgtgacac gtaaacagt 291429DNAArtificial Sequenceprobe
oligonucleotide 14ggtgttgtgg ctggtattgc ttacgctgg
291521DNAArtificial Sequenceprimer oligonucleotide 15cgccgaagta
tcgactcaac t 211619DNAArtificial Sequenceprimer oligonucleotide
16gcaacgtcgg ttcgagatg 191724DNAArtificial Sequenceprobe
oligonucleotide 17tcagaggtag ttggcgtcat cgag 241824DNAArtificial
Sequenceprimer oligonucleotide 18gtaatatggg ctcagaggaa tggt
241923DNAArtificial Sequenceprimer oligonucleotide 19atggagaaga
acattggaat tgc 232023DNAArtificial Sequenceprobe oligonucleotide
20ccatggcccg gtaccatctg gtc 232123DNAArtificial Sequenceprimer
oligonucleotide 21gtgactcaca ccatcaccag agt 232223DNAArtificial
Sequenceprimer oligonucleotide 22atccaggctg tcctctccct ata
232319DNAArtificial Sequenceprobe oligonucleotide 23caataccagt
tgtgcgacc 19
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