U.S. patent application number 14/302286 was filed with the patent office on 2014-09-25 for sugarcane bacilliform viral (scbv) enhancer and its use in plant functional genomics.
This patent application is currently assigned to Dow Agrosciences LLC. The applicant listed for this patent is Dow Agrosciences LLC. Invention is credited to William M. Ainley, John P. Davies, Vaka S. Reddy, Mark A. Thompson.
Application Number | 20140289899 14/302286 |
Document ID | / |
Family ID | 44654474 |
Filed Date | 2014-09-25 |
United States Patent
Application |
20140289899 |
Kind Code |
A1 |
Davies; John P. ; et
al. |
September 25, 2014 |
SUGARCANE BACILLIFORM VIRAL (SCBV) ENHANCER AND ITS USE IN PLANT
FUNCTIONAL GENOMICS
Abstract
Identification of new enhancer sequence has significant utility
in the plant functional genomics. The sugarcane bacilliform
badnavirus (SCBV) transcriptional enhancer has been identified.
This enhancer can be used to increase the rate of transcription
from gene promoters and in activation tagging experiments. A
ten-fold increase in transcription was observed when a 4.times.
array of the SCBV enhancer was placed upstream of a truncated form
of the maize alcohol dehydrogenase minimal promoter. Methods of
using the SCBV transcriptional enhancer are described, as are
chimeric transcription regulatory regions, constructs, cells,
tissues, and organisms that comprise one or more copies of the
enhancer.
Inventors: |
Davies; John P.; (Portland,
OR) ; Reddy; Vaka S.; (Aurora, CO) ; Ainley;
William M.; (Carmel, IN) ; Thompson; Mark A.;
(Zionsville, IN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Dow Agrosciences LLC |
Indianapolis |
IN |
US |
|
|
Assignee: |
Dow Agrosciences LLC
|
Family ID: |
44654474 |
Appl. No.: |
14/302286 |
Filed: |
June 11, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
13220564 |
Aug 29, 2011 |
8785612 |
|
|
14302286 |
|
|
|
|
61402570 |
Aug 30, 2010 |
|
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Current U.S.
Class: |
800/278 ;
435/320.1; 435/412; 435/419; 536/24.1; 800/298; 800/320.1 |
Current CPC
Class: |
A01H 5/10 20130101; C12N
15/8241 20130101; C12N 15/113 20130101; C12N 15/8216 20130101; C12N
15/8261 20130101; Y02A 40/146 20180101 |
Class at
Publication: |
800/278 ;
536/24.1; 435/320.1; 800/298; 800/320.1; 435/419; 435/412 |
International
Class: |
C12N 15/82 20060101
C12N015/82 |
Claims
1. A chimeric transcription regulatory region comprising: one or
more copies of the sugarcane bacilliform viral (SCBV) enhancer
element shown in position 337 to position 618 of SEQ ID NO: 1, or a
homolog thereof; and operably linked thereto, a promoter comprising
an RNA polymerase binding site and a mRNA initiation site, wherein
when a nucleotide sequence of interest is transcribed under
regulatory control of the chimeric transcription regulatory region,
the amount of transcription product is enhanced compared to the
amount of transcription product obtained with the chimeric
transcription regulatory region comprising the promoter and not
comprising the SCBV enhancer sequence(s).
2. The chimeric transcription regulatory region of claim 1, wherein
the promoter is obtained from the upstream region of a plant virus
gene, a bacterial gene, a fungal gene, a plant nuclear gene, a
plant extra-nuclear gene, an invertebrate gene, or a vertebrate
gene.
3. A construct comprising the transcriptional initiation region of
claim 1 operably linked to a transcribable polynucleotide molecule
operably linked to a 3' transcription termination polynucleotide
molecule.
4. The construct of claim 3, wherein said transcribable
polynucleotide molecule confers an agronomic trait to a plant in
which it is expressed.
5. A transgenic plant stably transformed with the construct of
claim 3.
6. The transgenic plant of claim 5, wherein the transcribable
polynucleotide molecule confers an agronomic trait to a plant in
which it is expressed.
7. A seed of the transgenic plant of claim 5, wherein the seed
comprises the construct.
8. The transgenic plant of claim 5, which plant is a maize
plant
9. A transgenic plant cell comprising the chimeric transcription
regulatory region of claim 1.
10. A method of producing a transgenic plant comprising
transforming a plant cell or tissue with the construct of claim
3.
11. The method of claim 10, wherein the transgenic plant is a
dicotyledon.
12. The method of claim 10, wherein the transgenic plant is a
monocotyledon.
13. A plant cell or tissue transformed with the construct of claim
3.
14. The plant cell or tissue of claim 13, wherein the plant cell or
tissue is from a dicotyledon.
15. The plant cell or tissue of claim 13, wherein the plant cell or
tissue is derived from a monocotyledon.
16. A plant cell, fruit, leaf, root, shoot, flower, seed, cutting
and other reproductive material useful in sexual or asexual
propagation, progeny plants inclusive of F1 hybrids, male-sterile
plants and all other plants and plant products derivable from the
transgenic plant of claim 5.
17. A maize plant cell, tissue or plant comprising one or more
copies of the sugarcane bacilliform viral (SCBV) enhancer element
shown in position 337 to position 618 of SEQ ID NO: 1, or a homolog
thereof, in which the one or more copies of the SCBV enhancer
element is inserted into a genome of the maize plant cell, tissue
or plant at a random location.
18. The maize plant cell, tissue or plant of claim 17, wherein the
SCBV enhancer imparts enhanced transcription of a nucleotide
sequence of interest which is under regulatory control of the SCBV
enhancer as compared to transcription of the nucleotide sequence of
interest in the absence of the SCBV enhancer.
Description
PRIORITY CLAIM
[0001] This application is a continuation of co-pending U.S.
application Ser. No. 13/220,564, filed Aug. 29, 2011, which claims
priority to and the benefit of U.S. Provisional Application No.
61/402,570, filed Aug. 30, 2010. The entire disclosure of these
prior applications, as well as the disclosure of International
Application No. PCT/US2011/049532, filed Aug. 29, 2011, and
published as WO 2012/030711, are incorporated herein by reference
in their entirety.
FIELD
[0002] The disclosure relates to the field of plant molecular
biology and genetic engineering, and specifically to polynucleotide
molecules useful for modulating (e.g., enhancing) gene expression
and/or protein production in plants.
PARTIES TO JOINT RESEARCH AGREEMENT
[0003] This application describes and claims certain subject matter
that was developed under a written joint research agreement between
Agrigenetics, Inc., Mycogen Corporation, Exelixis Plant Sciences,
Inc., and Exelixis, Inc., having an effective date of Sep. 4,
2007.
INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED AS AN ASCII TEXT
FILE
[0004] A Sequence Listing is submitted herewith as an ASCII
compliant text file named "Sequence_Listing.txt", created on Jun.
10, 2014, and having a size of .about.1.6 kilobytes, as permitted
under 37 CFR 1.821(c). The material in the aforementioned file is
hereby incorporated by reference in its entirety.
BACKGROUND
[0005] There is an on-going need for genetic regulatory elements
that direct, control or otherwise regulate expression of a
transcribable nucleic acid (e.g., a transgene), for instance for
use in a genetically engineered organism such as a plant. Genetic
regulatory elements typically include 5' untranslated sequences
such a transcription initiation regions that contain transcription
factor and RNA polymerase binding site(s), enhancer/silencer
elements, a TATA box and a CAAT box together with 3'
polyadenylation sequences, transcription stop signals, translation
start and stop signals, splice donor/acceptor sequences and the
like.
[0006] For the purposes of genetic engineering, genetic regulatory
elements are typically included in an expression vector or other
engineered construct, to regulate expression of a transgene
operably linked to the regulatory elements. Well known examples of
promoters used in this fashion are CaMV35S promoter (Nagy et al.
In: Biotechnology in plant science: relevance to agriculture in the
eighties. Eds. Zaitlin et al. Academic Press, Orlando, 1985), maize
ubiquitin promoter (Ubi; Christensen & Quail, Transgenic
Research 5:213, 1996) and the Emu promoter (Last et al., Theor.
Appl. Genet. 81 581, 1991), though many others will be known to
those of ordinary skill. Likewise, enhancers have been isolated
from various sources for use in genetic engineering; these include
the cauliflower mosaic virus (35S CaMV) enhancer, a figwort mosaic
virus (FMV) enhancer, a peanut chlorotic streak caulimovirus
(PC1SV) enhancer, or mirabilis mosaic virus (MMV) enhancer.
[0007] There is an on-going need to identify genetic regulatory
elements, such as enhancer domains, that can be harnessed to
control expression of sequences operably linked thereto, for
instance in heterologous nucleic acid molecules such as vectors and
other engineered constructs.
SUMMARY OF THE DISCLOSURE
[0008] The present disclosure describes novel transcription
regulatory regions comprising an enhancer domain and, under the
enhancing control of the enhancer domain, a transcription
regulatory domain. The enhancer domain comprises a plurality (e.g.,
two to four or more) of copies of a natural but previously
unrecognized SCBV enhancer arranged in tandem. The transcription
regulatory regions (promoters) of the present disclosure provide
enhanced transcription as compared to the promoter in the absence
of the enhancer domain. In one example, a chimeric transcription
regulatory region is disclosed comprising one or more copies of the
SCBV enhancer element shown in position 337 to position 618 of SEQ
ID NO: 1; and operably linked thereto, a promoter comprising an RNA
polymerase binding site and a mRNA initiation site, wherein when a
nucleotide sequence of interest is transcribed under regulatory
control of the chimeric transcription regulatory region, the amount
of transcription product is enhanced compared to the amount of
transcription product obtained with the chimeric transcription
regulatory region comprising the promoter and not comprising the
SCBV enhancer sequence.
[0009] DNA constructs are also provided comprising a described
transcription regulatory region and a DNA sequence to be
transcribed. In one example, a DNA construct comprises a disclosed
transcriptional initiation region operably linked to a
transcribable polynucleotide molecule operably linked to a 3'
transcription termination polynucleotide molecule. The DNA
constructs provide for enhanced transcription of the DNA sequence
to be transcribed. Transgenic plants, plant cells or tissue (such
as a dicotyledon or a monocotyledon plants, plant cells or tissue)
transformed with the disclosed constructs are also disclosed. Also
provided is a plant seed, fruit, leaf, root, shoot, flower, cutting
and other reproductive material useful in sexual or asexual
propagation, progeny plants inclusive of F1 hybrids, male-sterile
plants and all other plants and plant products derivable from the
disclosed transgenic plant. Methods of producing the disclosed
transgenic plants, plant cells or tissue are also provided
herein.
[0010] The foregoing and other features of the disclosure will
become more apparent from the following detailed description, which
proceeds with reference to the accompanying figures.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] FIG. 1 shows the sequence of the SCBV promoter
(corresponding to positions 6758-7596 of GenBank Accession No.
AJ277091.1, "Sugarcane bacilliform IM virus complete genome,
isolate Ireng Maleng" which incorporated by reference herein in its
entirety as it appeared on-line on Apr. 15, 2010); this sequence is
also shown in SEQ ID NO: 1. The enhancer sequences defined in this
study extend from -222 to -503 and are underlined in the Figure
(corresponding to position 337 to position 618 of SEQ ID NO:
1).
[0012] FIGS. 2A and 2B illustrate results of the analysis of the
SCBV promoter. FIG. 2A shows fragments of the SCBV promoter
containing sequences from -839 bp, -576 bp and -333 bp upstream of
the transcription start site and 106 bp downstream of the
transcription start site fused to the luciferase (LUC) reporter
gene. FIG. 2B shows a histogram of the ratio of LUC/GUS activity
from HiII cells co-transformed with the plasmids above and an
UBI::GUS reporter construct. The results show that the promoter
fragment containing sequences from -576 bp upstream of the
transcription start site had 60% of the activity of the promoter
fragment containing 839 bp upstream of the start site. In contrast,
the promoter fragment containing sequences from -333 bp upstream of
the start site had only 10% of the activity of the full-length
promoter (from -839 bp upstream of the transcription start site).
Thus, sequences involved in promoter activity reside upstream of
the -333 bp.
[0013] FIGS. 3A and 3B illustrate that the SCBV enhancer elements
described herein enhance transcription from the maize Adh1
promoter. One, two and four copies of the SCBV promoter sequences
from -503 to -222 were cloned upstream of a truncated maize Adh1
promoter, fused to the firefly luciferase gene (FIG. 3A). For
comparison, 4 copies of the MMV enhancer sequences and 2 copies of
the MMV enhancer and 2 copies of the SCBV promoter were cloned
upstream of the truncated maize Adh1 promoter and fused to the
firefly luciferase gene (FIG. 3A). These constructs were bombarded
into maize Hi-II suspension cells along with the UBI::GUS reporter
construct. As shown in FIG. 3B., constructs containing 1, 2 and 4
copies of the SCBV enhancer had more than 5 times, 6 times and 10
times more activity, respectively, than did cells bombarded with
the truncated Adh1 construct without any enhancers. The 4.times.MMV
construct had 2.5 times the activity as the truncated Adh1
construct and the 2.times.MMV 2.times.SCBV construct had 6 times
the activity as the truncated Adh1 construct.
[0014] FIG. 4 shows accumulation of transcripts close to ("Flanking
gene") the integration site of 4.times.SCBV in transgenic (T)
plants compared non-transgenic (W) control plants, analyzed using
reverse transcription and PCR (RT-PCR). The level of housekeeping
gene GAPDH is shown for comparison. The 4.times.SCBV enhancer
caused increased accumulation of transcripts of genes near where it
integrates; this increase in transcript accumulation probably
results from an increased rate of transcription.
SEQUENCE LISTING
[0015] The nucleic and/or amino acid sequences listed in the
sequence listing below are shown using standard letter
abbreviations for nucleotide bases, and three letter code for amino
acids, as defined in 37 C.F.R. 1.822. Only one strand of each
nucleic acid sequence is shown, but the complementary strand is
understood as included by any reference to the displayed strand.
Nucleic acid sequences (in the Sequence Listing or elsewhere
herein) are presented in the standard 5' to 3' direction, and
protein sequences are presented in the standard amino (N) terminal
to carboxy (C) terminal direction.
[0016] SEQ ID NO: 1 shows the nucleic acid sequence of the SCBV
promoter (corresponding to positions 6758-7596 of GenBank Accession
No. AJ277091.1, "Sugarcane bacilliform IM virus complete genome,
isolate Ireng Maleng" incorporated by reference herein in its
entirety as it appeared on-line on Apr. 15, 2010).
[0017] The enhancer elements described herein are from position 337
to position 618 of SEQ ID NO: 1.
DETAILED DESCRIPTION
I. Abbreviations
[0018] 3' UTR 3'-untranslated region [0019] 5' UTR 5'-untranslated
region [0020] Adh1 alcohol dehydrogenase 1 [0021] asRNA antisense
RNA [0022] cDNA complementary DNA [0023] dsRNA double-stranded RNA
[0024] GAPDH glyceraldehyde 3-phosphate dehydrogenase [0025] KB
kilobytes [0026] kbp kilobase pairs [0027] LUC luciferase [0028]
miRNA microRNA [0029] nt nucleotide [0030] ORF open reading frame
[0031] PCR polymerase chain reaction [0032] RT-PCR reverse
transcription and PCR [0033] SCBV sugarcane bacilliform virus
[0034] siRNA small interfering RNA [0035] ssRNA single stranded RNA
[0036] T.sub.m thermal melting point [0037] UTR untranslated
region
II. Terms
[0038] Unless otherwise noted, technical terms are used according
to conventional usage. Definitions of common terms in molecular
biology may be found in Benjamin Lewin, Genes V, published by
Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al.
(eds.), The Encyclopedia of Molecular Biology, published by
Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A.
Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive
Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN
1-56081-569-8).
[0039] In order to facilitate review of the various embodiments of
the invention, the following explanations of specific terms are
provided:
[0040] 5' and/or 3': Nucleic acid molecules (such as, DNA and RNA)
are said to have "5' ends" and "3' ends" because mononucleotides
are reacted to make polynucleotides in a manner such that the 5'
phosphate of one mononucleotide pentose ring is attached to the 3'
oxygen of its neighbor in one direction via a phosphodiester
linkage. Therefore, one end of a polynucleotide is referred to as
the "5' end" when its 5' phosphate is not linked to the 3' oxygen
of a mononucleotide pentose ring. The other end of a polynucleotide
is referred to as the "3' end" when its 3' oxygen is not linked to
a 5' phosphate of another mononucleotide pentose ring.
Notwithstanding that a 5' phosphate of one mononucleotide pentose
ring is attached to the 3' oxygen of its neighbor, an internal
nucleic acid sequence also may be said to have 5' and 3' ends.
[0041] In either a linear or circular nucleic acid molecule,
discrete internal elements are referred to as being "upstream" or
5' of the "downstream" or 3' elements. With regard to DNA, this
terminology reflects that transcription proceeds in a 5' to 3'
direction along a DNA strand. Promoter and enhancer elements, which
direct transcription of a linked gene, are generally located 5' or
upstream of the coding region. However, enhancer elements can exert
their effect even when located 3' of the promoter element and the
coding region. Transcription termination and polyadenylation
signals are located 3' or downstream of the coding region.
[0042] Agronomic trait: Characteristic of a plant, which
characteristics include, but are not limited to, plant morphology,
physiology, growth and development, yield, nutritional enhancement,
disease or pest resistance, or environmental or chemical tolerance
are agronomic traits. An "enhanced agronomic trait" refers to a
measurable improvement in an agronomic trait including, but not
limited to, yield increase, including increased yield under
non-stress conditions and increased yield under environmental
stress conditions. Stress conditions may include, for example,
drought, shade, fungal disease, viral disease, bacterial disease,
insect infestation, nematode infestation, cold temperature
exposure, heat exposure, osmotic stress, reduced nitrogen nutrient
availability, reduced phosphorus nutrient availability and high
plant density. "Yield" can be affected by many properties including
without limitation, plant height, pod number, pod position on the
plant, number of internodes, incidence of pod shatter, grain size,
efficiency of nodulation and nitrogen fixation, efficiency of
nutrient assimilation, resistance to biotic and abiotic stress,
carbon assimilation, plant architecture, resistance to lodging,
percent seed germination, seedling vigor, and juvenile traits.
Yield can also affected by efficiency of germination (including
germination in stressed conditions), growth rate (including growth
rate in stressed conditions), ear number, seed number per ear, seed
size, composition of seed (starch, oil, protein) and
characteristics of seed fill. Increased yield may result from
improved utilization of key biochemical compounds, such as
nitrogen, phosphorous and carbohydrate, or from improved responses
to environmental stresses, such as cold, heat, drought, salt, and
attack by pests or pathogens. Recombinant DNA used in this
disclosure can also be used to provide plants having improved
growth and development, and ultimately increased yield, as the
result of modified expression of plant growth regulators or
modification of cell cycle or photosynthesis pathways. Additional
examples of agronomic traits, and altering such traits in plants,
are provided herein and/or will be recognized by those of ordinary
skill in the art.
[0043] Alterations: Alterations in a polynucleotide (for example, a
polypeptide encoded by a nucleic acid of the present invention), as
this term is used herein, comprise any deletions, insertions, and
point mutations in the polynucleotide sequence. Included within
this definition are alterations to the genomic DNA sequence that
encodes the polypeptide. Likewise, the term "alteration" may be
used to refer to deletions, insertions, and other mutations in
polypeptide sequences.
[0044] Altering level of production or expression: Changing, either
by increasing or decreasing, the level of production or expression
of a nucleic acid molecule or an amino acid molecule (for example
an siRNA, a miRNA, an mRNA, a gene, a polypeptide, a peptide), as
compared to a control level of production or expression.
[0045] Amplification: When used in reference to a nucleic acid,
this refers to techniques that increase the number of copies of a
nucleic acid molecule in a sample or specimen. An example of
amplification is the polymerase chain reaction, in which a
biological sample collected from a subject is contacted with a pair
of oligonucleotide primers, under conditions that allow for the
hybridization of the primers to nucleic acid template in the
sample. The primers are extended under suitable conditions,
dissociated from the template, and then re-annealed, extended, and
dissociated to amplify the number of copies of the nucleic acid.
The product of in vitro amplification can be characterized by
electrophoresis, restriction endonuclease cleavage patterns,
oligonucleotide hybridization or ligation, and/or nucleic acid
sequencing, using standard techniques. Other examples of in vitro
amplification techniques include strand displacement amplification
(see U.S. Pat. No. 5,744,311); transcription-free isothermal
amplification (see U.S. Pat. No. 6,033,881); repair chain reaction
amplification (see WO 90/01069); ligase chain reaction
amplification (see EP-A-320 308); gap filling ligase chain reaction
amplification (see U.S. Pat. No. 5,427,930); coupled ligase
detection and PCR (see U.S. Pat. No. 6,027,889); and NASBA.TM. RNA
transcription-free amplification (see U.S. Pat. No. 6,025,134).
[0046] Antisense, Sense, and Antigene: DNA has two antiparallel
strands, a 5'.fwdarw.3' strand, referred to as the plus strand, and
a 3'.fwdarw.5' strand, referred to as the minus strand. Because RNA
polymerase adds nucleic acids in a 5'.fwdarw.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).
[0047] 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 (asRNA)
is a molecule of RNA complementary to a sense (encoding) nucleic
acid molecule.
[0048] Antisense inhibition: This term refers to a class of gene
regulation based on cytoplasmic, nuclear, or organelle inhibition
of gene expression (e.g., expression for a host cell genome or the
genome of a pathogen, such as a virus) due to the presence in a
cell of an RNA molecule complementary to at least a portion of the
mRNA being translated.
[0049] cDNA (complementary DNA): A piece of DNA lacking internal,
non-coding segments (introns) and transcriptional regulatory
sequences. cDNA may also contain untranslated regions (UTRs) that
are responsible for translational control in the corresponding RNA
molecule. cDNA is usually synthesized in the laboratory by reverse
transcription from messenger RNA extracted from cells or other
samples.
[0050] Chimeric or Chimera: The product of the fusion of portions
of two or more different polynucleotide or polypeptide molecules.
For instance, the phrases "chimeric sequence" and "chimeric gene"
refer to nucleotide sequences derived from at least two
heterologous parts. Chimeric sequence may comprise DNA or RNA.
[0051] Chimeric transcription regulatory region: An array of
nucleic acid control or regulatory sequences that direct
transcription of a nucleic acid operably linked thereto, which
array is assembled from different polynucleotide sources. For
instance, chimeric transcription regulatory regions as described
herein may be produced through manipulation of known promoters or
other polynucleotide molecules. Chimeric transcription regulatory
regions may combine one or more enhancer domains with one or more
promoters, for example, by fusing a heterologous enhancer domain
from a first native promoter to a second promoter with its own
partial or complete set of regulatory element(s). This disclosure
provides, inter alia, chimeric transcription regulatory regions
that contain at least one SCBV enhancer domain fused (that is,
operably linked) to a promoter active in plant(s).
[0052] Construct: Any recombinant polynucleotide molecule such as a
plasmid, cosmid, virus, autonomously replicating polynucleotide
molecule, phage, or linear or circular single-stranded or
double-stranded DNA or RNA polynucleotide molecule, derived from
any source, capable of genomic integration or autonomous
replication, comprising a polynucleotide molecule where one or more
transcribable polynucleotide molecule has been operably linked.
[0053] Control plant: A plant that does not contain a recombinant
DNA that confers (for instance) an enhanced or altered agronomic
trait in a transgenic plant, is used as a baseline for comparison,
for instance in order to identify an enhanced or altered agronomic
trait in the transgenic plant. A suitable control plant may be a
non-transgenic plant of the parental line used to generate a
transgenic plant, or a plant that at least is non-transgenic for
the particular trait under examination (that is, the control plant
may have been engineered to contain other heterologous sequences or
recombinant DNA molecules). Thus, a control plant may in some cases
be a transgenic plant line that comprises an empty vector or marker
gene, but does not contain the recombinant DNA, or does not contain
all of the recombinant DNAs, in the test plant.
[0054] Cosuppression: The expression of a foreign (heterologous)
gene that has substantial homology to an endogenous gene, resulting
in suppression of expression of both the foreign and the endogenous
gene.
[0055] DNA (deoxyribonucleic acid): DNA is a long chain polymer
which comprises the genetic material of most organisms (some
viruses have genes comprising ribonucleic acid (RNA)). The
repeating units in DNA polymers are four different nucleotides,
each of which comprises one of the four bases, adenine, guanine,
cytosine and thymine bound to a deoxyribose sugar to which a
phosphate group is attached.
[0056] Triplets of nucleotides (referred to as codons) code for
each amino acid in a polypeptide, or for a stop signal. The term
codon is also used for the corresponding (and complementary)
sequences of three nucleotides in the mRNA into which the DNA
sequence is transcribed.
[0057] Unless otherwise specified, any reference to a DNA molecule
includes the reverse complement of that DNA molecule. Except where
single-strandedness is required by the text herein, DNA molecules,
though written to depict only a single strand, encompass both
strands of a double-stranded DNA molecule.
[0058] Encode: A polynucleotide is said to encode a polypeptide if,
in its native state or when manipulated by methods known to those
skilled in the art, the polynucleotide molecule can be transcribed
and/or translated to produce a mRNA for and/or the polypeptide or a
fragment thereof. The anti-sense strand is the complement of such a
nucleic acid, and the encoding sequence can be deduced
therefrom.
[0059] Enhancer domain: A cis-acting transcriptional regulatory
element (a.k.a. cis-element) that confers an aspect of the overall
control of gene expression. An enhancer domain may function to bind
transcription factors, which are trans-acting protein factors that
regulate transcription. Some enhancer domains bind more than one
transcription factor, and transcription factors may interact with
different affinities with more than one enhancer domain. Enhancer
domains can be identified by a number of techniques, including
deletion analysis (deleting one or more nucleotides from the 5' end
or internal to a promoter); DNA binding protein analysis using
DNase I foot printing, methylation interference, electrophoresis
mobility-shift assays, in vivo genomic foot printing by
ligation-mediated PCR, and other conventional assays; or by DNA
sequence comparison with known cis-element motifs using
conventional DNA sequence comparison methods. The fine structure of
an enhancer domain can be further studied by mutagenesis (or
substitution) of one or more nucleotides or by other conventional
methods. Enhancer domains can be obtained by chemical synthesis or
by isolation from promoters that include such elements, and they
can be synthesized with additional flanking nucleotides that
contain useful restriction enzyme sites to facilitate subsequence
manipulation.
[0060] (Gene) Expression: Transcription of a DNA molecule into a
transcribed RNA molecule. More generally, the processes by which a
gene's coded information is converted into the structures present
and operating in the cell. Expressed genes include those that are
transcribed into mRNA and then translated into protein and those
that are transcribed into RNA but not translated into protein (for
example, siRNA, transfer RNA and ribosomal RNA). Thus, expression
of a target sequence, such as a gene or a promoter region of a
gene, can result in the expression of an mRNA, a protein, or both.
The expression of the target sequence can be inhibited or enhanced
(decreased or increased). Gene expression may be described as
related to temporal, spatial, developmental, or morphological
qualities as well as quantitative or qualitative indications.
[0061] Gene regulatory activity: The ability of a polynucleotide to
affect transcription or translation of an operably linked
transcribable or translatable polynucleotide molecule. An isolated
polynucleotide molecule having gene regulatory activity may provide
temporal or spatial expression or modulate levels and rates of
expression of the operably linked transcribable polynucleotide
molecule. An isolated polynucleotide molecule having gene
regulatory activity may include a promoter, intron, leader, or 3'
transcription termination region.
[0062] Gene Silencing: Gene silencing refers to lack of (or
reduction of) gene expression as a result of, though not limited
to, effects at a genomic (DNA) level such as chromatin
re-structuring, or at the post-transcriptional level through
effects on transcript stability or translation. Current evidence
suggests that RNA interference (RNAi) is a major process involved
in transcriptional and posttranscriptional gene silencing.
[0063] Because RNAi exerts its effects at the transcriptional
and/or post-transcriptional level, it is believed that RNAi can be
used to specifically inhibit alternative transcripts from the same
gene.
[0064] Heterologous: A type of sequence that is not normally (e.g.,
in the wild-type sequence) found adjacent to a second sequence. In
one embodiment, the sequence is from a different genetic source,
such as a virus or organism or species, than the second
sequence.
[0065] Hybridization: Oligonucleotides and their analogs hybridize
by hydrogen bonding, which includes Watson-Crick, Hoogsteen or
reversed Hoogsteen hydrogen bonding, between complementary bases.
Generally, nucleic acid consists of nitrogenous bases that are
either pyrimidines (cytosine (C), uracil (U), and thymine (T)) or
purines (adenine (A) and guanine (G)). These nitrogenous bases form
hydrogen bonds between a pyrimidine and a purine, and the bonding
of the pyrimidine to the purine is referred to as base pairing.
More specifically, A will hydrogen bond to T or U, and G will bond
to C. In RNA molecules, G also will bond to U. Complementary refers
to the base pairing that occurs between two distinct nucleic acid
sequences or two distinct regions of the same nucleic acid
sequence.
[0066] 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 concentration) of the
hybridization buffer will determine the stringency of
hybridization. Calculations regarding hybridization conditions
required for attaining particular degrees of stringency are
discussed by Sambrook et al. (ed.), Molecular Cloning: A Laboratory
Manual, 2nd ed., vol. 1-3, Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y., 1989, chapters 9 and 11, herein
incorporated by reference.
[0067] The following is an exemplary set of hybridization
conditions and is not meant to be limiting.
Very High Stringency (Detects Sequences that Share 90% Sequence
Identity)
[0068] Hybridization: 5.times.SSC at 65.degree. C. for 16 hours
[0069] Wash twice: 2.times.SSC at room temperature (RT) for 15
minutes each
[0070] Wash twice: 0.5.times.SSC at 65.degree. C. for 20 minutes
each
High Stringency (Detects Sequences that Share 80% Sequence Identity
or Greater)
[0071] Hybridization: 5.times.-6.times.SSC at 65.degree.
C.-70.degree. C. for 16-20 hours
[0072] Wash twice: 2.times.SSC at RT for 5-20 minutes each
[0073] Wash twice: 1.times.SSC at 55.degree. C.-70.degree. C. for
30 minutes each
Low Stringency (Detects Sequences that Share Greater than 50%
Sequence Identity)
[0074] Hybridization: 6.times.SSC at RT to 55.degree. C. for 16-20
hours
[0075] Wash at least twice: 2.times.-3.times.SSC at RT to
55.degree. C. for 20-30 minutes each.
[0076] In cis: Indicates that two sequences are positioned on the
same piece of RNA or DNA.
[0077] In trans: Indicates that two sequences are positioned on
different pieces of RNA or DNA.
[0078] Industrial crop: Crops grown primarily for consumption by
humans or animals or for use in industrial processes (for example,
as a source of fatty acids for manufacturing or sugars for
producing alcohol). It will be understood that in many instances
either the plant or a product produced from the plant (for example,
sweeteners, oil, flour, or meal) can be consumed; thus, a subset of
industrial crops are food crops. Examples of food crops include,
but are not limited to, corn, soybean, rice, wheat, oilseed rape,
cotton, oats, barley, and potato plants. Other examples of
industrial crops (including food crops) are listed herein.
[0079] Interfering with or inhibiting (expression of a target
sequence): This phrase refers to the ability of a small RNA, such
as a siRNA or a miRNA, or other molecule, to measurably reduce the
expression and/or stability of molecules carrying the target
sequence. A target sequence can include a DNA sequence, such as a
gene or the promoter region of a gene, or an RNA sequence, such as
an mRNA. "Interfering with or inhibiting" expression contemplates
reduction of the end-product of the gene or sequence, e.g., the
expression or function of the encoded protein or a protein, nucleic
acid, other biomolecule, or biological function influenced by the
target sequence, and thus includes reduction in the amount or
longevity of the mRNA transcript or other target sequence. In some
embodiments, the small RNA or other molecule guides chromatin
modifications which inhibit the expression of a target sequence. It
is understood that the phrase is relative, and does not require
absolute inhibition (suppression) of the sequence. Thus, in certain
embodiments, interfering with or inhibiting expression of a target
sequence requires that, following application of the small RNA or
other molecule (such as a vector or other construct encoding one or
more small RNAs), the sequence is expressed at least 5% less than
prior to application, at least 10% less, at least 15% less, at
least 20% less, at least 25% less, or even more reduced. Thus, in
some particular embodiments, application of a small RNA or other
molecule reduces expression of the target sequence by about 30%,
about 40%, about 50%, about 60%, or more. In specific examples,
where the small RNA or other molecule is particularly effective,
expression is reduced by 70%, 80%, 85%, 90%, 95%, or even more.
[0080] Isolated: An "isolated" biological component (such as a
nucleic acid, peptide 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, e.g., other chromosomal and extrachromosomal DNA
and RNA, and proteins. Nucleic acids, peptides and proteins which
have been "isolated" thus include nucleic acids and proteins
purified by standard purification methods. The term also embraces
nucleic acids, peptides and proteins prepared by recombinant
expression in a host cell as well as chemically synthesized nucleic
acids.
[0081] Metabolome: The complement of relatively low molecular
weight molecules (metabolites) that is present in a single
organism, a sample, a tissue, a cell, or whatever other division is
divided. By way of example, metabolomes may include metabolic
intermediates, hormones and other signalling molecules, and
secondary metabolites. Representative metabolomes comprise the
complement of metabolites found within a biological sample, such as
a plant, plant part, or plant sample, or in a suspension or extract
thereof. Examples of such molecules include, but are not limited
to: acids and related compounds; mono-, di-, and tri-carboxylic
acids (saturated, unsaturated, aliphatic and cyclic, aryl,
alkaryl); aldo-acids, keto-acids; lactone forms; gibberellins;
abscisic acid; alcohols, polyols, derivatives, and related
compounds; ethyl alcohol, benzyl alcohol, methanol; propylene
glycol, glycerol, phytol; inositol, furfuryl alcohol, menthol;
aldehydes, ketones, quinones, derivatives, and related compounds;
acetaldehyde, butyraldehyde, benzaldehyde, acrolein, furfural,
glyoxal; acetone, butanone; anthraquinone; carbohydrates; mono-,
di-, tri-saccharides; alkaloids, amines, and other bases; pyridines
(including nicotinic acid, nicotinamide); pyrimidines (including
cytidine, thymine); purines (including guanine, adenine,
xanthines/hypoxanthines, kinetin); pyrroles; quinolines (including
isoquinolines); morphinans, tropanes, cinchonans; nucleotides,
oligonucleotides, derivatives, and related compounds; guano sine,
cytosine, adeno sine, thymidine, inosine; amino acids,
oligopeptides, derivatives, and related compounds; esters; phenols
and related compounds; heterocyclic compounds and derivatives;
pyrroles, tetrapyrroles (corrinoids and porphines/porphyrins, w/w/o
metal-ion); flavonoids; indoles; lipids (including fatty acids and
triglycerides), derivatives, and related compounds; carotenoids,
phytoene; and sterols, isoprenoids including terpenes.
[0082] MicroRNA (miRNA): Small, non-coding RNA gene products of
approximately 21 nucleotides long and found in diverse organisms,
including animals and plants. miRNAs structurally resemble siRNAs
except that they arise from structured, foldback-forming precursor
transcripts derived from miRNA genes. Primary transcripts of miRNA
genes form hairpin structures that are processed by the multidomain
RNaseIII-like nuclease DICER and DROSHA (in animals) or DICER-LIKE1
(DCL1; in plants) to yield miRNA duplexes. The mature miRNA is
incorporated into RISC complexes after duplex unwinding. Plant
miRNAs interact with their RNA targets with perfect or near perfect
complementarity.
[0083] Nucleotide: The term nucleotide includes, but is not limited
to, a monomer that includes a base linked to a sugar, such as a
pyrimidine, purine or synthetic analogs thereof, or a base linked
to an amino acid, as in a peptide nucleic acid (PNA). A nucleotide
is one monomer in an oligonucleotide/polynucleotide. A nucleotide
sequence refers to the sequence of bases in an
oligonucleotide/polynucleotide.
[0084] The major nucleotides of DNA are deoxyadenosine
5'-triphosphate (dATP or A), deoxyguanosine 5'-triphosphate (dGTP
or G), deoxycytidine 5'-triphosphate (dCTP or C) and deoxythymidine
5'-triphosphate (dTTP or T). The major nucleotides of RNA are
adenosine 5'-triphosphate (ATP or A), guanosine 5'-triphosphate
(GTP or G), cytidine 5'-triphosphate (CTP or C) and uridine
5'-triphosphate (UTP or U). Inosine is also a base that can be
integrated into DNA or RNA in a nucleotide (dITP or ITP,
respectively).
[0085] Oil-producing species (of plant): Plant species that produce
and store triacylglycerol in specific organs, primarily in seeds.
Such species include but are not limited to soybean (Glycine
nizax), rapeseed and canola (such as Brassica napus, Brassica rapa
and Brassica campestris), sunflower (Helianthus annus), cotton
(Gossypium hirsutum), corn (Zea mays), cocoa (Theobroina cacao),
safflower (Carthamus tinctorius), oil palm (Elaeis guineensis),
coconut palm (Cocos nucifera), flax (Linum usitatissimuin), castor
(Ricinus commiunis) and peanut (Arachis hypogaea).
[0086] Oligonucleotide: An oligonucleotide is a plurality of
nucleotides joined by phosphodiester bonds, between about 6 and
about 300 nucleotides in length. An oligonucleotide analog refers
to compounds that function similarly to oligonucleotides but have
non-naturally occurring portions. For example, oligonucleotide
analogs can contain non-naturally occurring portions, such as
altered sugar moieties or inter-sugar linkages, such as a
phosphorothioate oligodeoxynucleotide. Functional analogs of
naturally occurring polynucleotides can bind to RNA or DNA
[0087] Operably linked: This term refers to a juxtaposition of
components, particularly nucleotide sequences, such that the normal
function of the components can be performed. Thus, a first nucleic
acid sequence is operably linked with a second nucleic acid
sequence when the first nucleic acid sequence is placed in a
functional relationship with the second nucleic acid sequence. For
instance, a promoter is operably linked to a coding sequence if the
promoter affects the transcription or expression of the coding
sequence. Generally, operably linked DNA sequences are contiguous
and, where necessary to join two protein-coding regions, in the
same reading frame. A coding sequence that is "operably linked" to
regulatory sequence(s) refers to a configuration of nucleotide
sequences wherein the coding sequence can be expressed under the
regulatory control (e.g., transcriptional and/or translational
control) of the regulatory sequences.
[0088] ORF (open reading frame): A series of nucleotide triplets
(codons) coding for amino acids without any termination codons.
These sequences are usually translatable into a peptide.
[0089] Percent sequence identity: The percentage of identical
nucleotides in a linear polynucleotide sequence of a reference
("query") polynucleotide molecule (or its complementary strand) as
compared to a test ("subject") polynucleotide molecule (or its
complementary strand) when the two sequences are optimally aligned
(with appropriate nucleotide insertions, deletions, or gaps
totaling less than 20 percent of the reference sequence over the
window of comparison). Optimal alignment of sequences for aligning
a comparison window are well known to those skilled in the art and
may be conducted using tools such as the local homology algorithm
of Smith and Waterman, the homology alignment algorithm of
Needleman and Wunsch, the search for similarity method of Pearson
and Lipman. Such comparisons are preferably carried out using the
computerized implementations of these algorithms, such as GAP,
BESTFIT, FASTA, and TFASTA available as part of the GCG.RTM.
Wisconsin Package.RTM. (Accelrys Inc., Burlington, Mass.). An
"identity fraction" for aligned segments of a test sequence and a
reference sequence is the number of identical components which are
shared by the two aligned sequences divided by the total number of
components in the reference sequence segment (that is, the entire
reference sequence or a smaller defined part of the reference
sequence). Percent sequence identity is represented as the identity
fraction multiplied by 100. The comparison of one or more
polynucleotide sequences may be to a full-length polynucleotide
sequence or a portion thereof, or to a longer polynucleotide
sequence. Substantial percent sequence identity is at least about
80% sequence identity, at least about 90% sequence identity, or
even greater sequence identity, such as about 98% or about 99%
sequence identity.
[0090] Plant: Any plant and progeny thereof. The term also includes
parts of plants, including seed, cuttings, tubers, fruit, flowers,
etc. In various embodiments, the term plant refers to cultivated
plant species, such as corn, cotton, canola, sunflower, soybeans,
sorghum, alfalfa, wheat, rice, plants producing fruits and
vegetables, and turf and ornamental plant species. The term plant
cell, as used herein, refers to the structural and physiological
unit of plants, consisting of a protoplast and the surrounding cell
wall. The term plant organ, as used herein, refers to a distinct
and visibly differentiated part of a plant, such as root, stem,
leaf or embryo.
[0091] More generally, the term plant tissue refers to any tissue
of a plant in planta or in culture. This term includes a whole
plant, plant cell, plant organ, protoplast, cell culture, or any
group of plant cells organized into a structural and functional
unit.
[0092] Polynucleotide molecule: Single- or double-stranded DNA or
RNA of genomic or synthetic origin; that is, a polymer of
deoxyribonucleotide or ribonucleotide bases, respectively, read
from the 5' (upstream) end to the 3' (downstream) end.
[0093] Polypeptide molecule: A polymer in which the monomers are
amino acid residues which are joined together through amide bonds.
When the amino acids are alpha-amino acids, either the L-optical
isomer or the D-optical isomer can be used, the L-isomers being
preferred. The term polypeptide or protein as used herein
encompasses any amino acid sequence and includes modified sequences
such as glycoproteins. The term polypeptide is specifically
intended to cover naturally occurring proteins, as well as those
that are recombinantly or synthetically produced.
[0094] Post-Transcriptional Gene Silencing (PTGS): A form of gene
silencing in which the inhibitory mechanism occurs after
transcription. This can result in either decreased steady-state
level of a specific RNA target or inhibition of translation
(Tuschl, ChemBiochem, 2: 239-245, 2001). In the literature, the
terms RNA interference (RNAi) and posttranscriptional cosuppression
are often used to indicate posttranscriptional gene silencing.
[0095] Promoter: An array of nucleic acid control sequences which
direct transcription of a nucleic acid, by recognition and binding
of e.g., RNA polymerase II and other proteins (trans-acting
transcription factors) to initiate transcription. A promoter
includes necessary nucleic acid sequences near the start site of
transcription, such as, in the case of a polymerase II type
promoter, a TATA element. Minimally, a promoter typically includes
at least an RNA polymerase binding site together and may also
include one or more transcription factor binding sites which
modulate transcription in response to occupation by transcription
factors. Representative examples of promoters (and elements that
can be assembled to produce a promoter) are described herein.
Promoters may be defined by their temporal, spatial, or
developmental expression pattern.
[0096] A plant promoter is a native or non-native promoter that is
functional in plant cells.
[0097] Protein: A biological molecule, for example a polypeptide,
expressed by a gene and comprised of amino acids.
[0098] Protoplast: An isolated plant cell without a cell wall,
having the potential for being transformed and/or regeneration into
cell culture or a whole plant.
[0099] Purified: The term purified does not require absolute
purity; rather, it is intended as a relative term. Thus, for
example, a purified fusion protein preparation is one in which the
fusion protein is more enriched than the protein is in its
generative environment, for instance within a cell or in a
biochemical reaction chamber. Preferably, a preparation of fusion
protein is purified such that the fusion protein represents at
least 50% of the total protein content of the preparation.
[0100] Recombinant: A recombinant nucleic acid is one that has a
sequence that is not naturally occurring or has a sequence that is
made by an artificial combination of two otherwise separated
segments of sequence. This artificial combination is often
accomplished by chemical synthesis or, more commonly, by the
artificial manipulation of isolated segments of nucleic acids,
e.g., by genetic engineering techniques.
[0101] Similarly, a recombinant protein is one encoded for by a
recombinant nucleic acid molecule.
[0102] Regulatable promoter: A promoter the activity of which is
regulated (directly or indirectly) by an agent, such as a
transcription factor, a chemical compound, an environmental
condition, or a nucleic acid molecule.
[0103] Regulating gene expression: Processes of controlling the
expression of a gene by increasing or decreasing the expression,
production, or activity of an agent that affects gene expression.
The agent can be a protein, such as a transcription factor, or a
nucleic acid molecule, such as a miRNA or an siRNA molecule, which
when in contact with the gene or its upstream regulatory sequences,
or a mRNA encoded by the gene, either increases or decreases gene
expression.
[0104] Regulatory sequences or elements: These terms refer
generally to a class of polynucleotide molecules (such as DNA
molecules, having DNA sequences) that influence or control
transcription or translation of an operably linked transcribable
polynucleotide molecule, and thereby expression of genes. Included
in the term are promoters, enhancers, leaders, introns, locus
control regions, boundary elements/insulators, silencers, Matrix
attachment regions (also referred to as scaffold attachment
regions), repressor, transcriptional terminators (a.k.a.
transcription termination regions), origins of replication,
centromeres, and meiotic recombination hotspots. Promoters are
sequences of DNA near the 5' end of a gene that act as a binding
site for RNA polymerase, and from which transcription is initiated.
Enhancers are control elements that elevate the level of
transcription from a promoter, usually independently of the
enhancer's orientation or distance from the promoter. Locus control
regions (LCRs) confer tissue-specific and temporally regulated
expression to genes to which they are linked. LCRs function
independently of their position in relation to the gene, but are
copy-number dependent. It is believed that they function to open
the nucleosome structure, so other factors can bind to the DNA.
LCRs may also affect replication timing and origin usage.
Insulators (also known as boundary elements) are DNA sequences that
prevent the activation (or inactivation) of transcription of a
gene, by blocking effects of surrounding chromatin. Silencers and
repressors are control elements that suppress gene expression; they
act on a gene independently of their orientation or distance from
the gene. Matrix attachment regions (MARs), also known as scaffold
attachment regions, are sequences within DNA that bind to the
nuclear scaffold. They can affect transcription, possibly by
separating chromosomes into regulatory domains. It is believed that
MARs mediate higher-order, looped structures within chromosomes.
Transcriptional terminators are regions within the gene vicinity
that RNA polymerase is released from the template. Origins of
replication are regions of the genome that, during DNA synthesis or
replication phases of cell division, begin the replication process
of DNA. Meiotic recombination hotspots are regions of the genome
that recombine more frequently than the average during meiosis.
Specific nucleotides within a regulatory region may serve multiple
functions. For example, a specific nucleotide may be part of a
promoter and participate in the binding of a transcriptional
activator protein.
[0105] Isolated regulatory elements that function in cells (for
instance, in plants or plant cells) are useful for modifying plant
phenotypes, for instance through genetic engineering.
[0106] RNA: A typically linear polymer of ribonucleic acid
monomers, linked by phosphodiester bonds. Naturally occurring RNA
molecules fall into three general classes, messenger (mRNA, which
encodes proteins), ribosomal (rRNA, components of ribosomes), and
transfer (tRNA, molecules responsible for transferring amino acid
monomers to the ribosome during protein synthesis). Messenger RNA
includes heteronuclear (hnRNA) and membrane-associated polysomal
RNA (attached to the rough endoplasmic reticulum). Total RNA refers
to a heterogeneous mixture of all types of RNA molecules.
[0107] RNA interference (RNAi): Gene silencing mechanisms that
involve small RNAs (including miRNA and siRNA) are frequently
referred to under the broad term RNAi. Natural functions of RNAi
include protection of the genome against invasion by mobile genetic
elements such as transposons and viruses, and regulation of gene
expression.
[0108] RNA interference results in the inactivation or suppression
of expression of a gene within an organism. RNAi can be triggered
by one of two general routes. First, it can be triggered by direct
cellular delivery of short-interfering RNAs (siRNAs, usually
.about.21 nucleotides in length and delivered in a dsRNA duplex
form with two unpaired nucleotides at each 3' end), which have
sequence complementarity to a RNA that is the target for
suppression. Second, RNAi can be triggered by one of several
methods in which siRNAs are formed in vivo from various types of
designed, expressed genes. These genes typically express RNA
molecules that form intra- or inter-molecular duplexes (dsRNA)
which are processed by natural enzymes (DICER or DCL) to form
siRNAs. In some cases, these genes express "hairpin"-forming RNA
transcripts with perfect or near-perfect base-pairing; some of the
imperfect hairpin-forming transcripts yield a special type of small
RNA, termed microRNA (miRNA). In either general method, it is the
siRNAs (or miRNAs) that function as "guide sequences" to direct an
RNA-degrading enzyme (termed RISC) to cleave or silence the target
RNA. In some cases, it is beneficial to integrate an RNAi-inducing
gene into the genome of a transgenic organism. An example would be
a plant that is modified to suppress a specific gene by an
RNAi-inducing transgene. In most methods that are currently in
practice, RNAi is triggered in transgenic plants by transgenes that
express a dsRNA (either intramolecular or hairpin, or
intermolecular in which two transcripts anneal to form dsRNA).
[0109] RNA silencing: A general term that is used to indicate
RNA-based gene silencing or RNAi.
[0110] Sequence identity: The similarity between two nucleic acid
sequences, or two amino acid sequences is expressed in terms of the
similarity between the sequences, otherwise referred to as sequence
identity. Sequence identity is frequently measured in terms of
percentage identity (or similarity or homology); the higher the
percentage, the more similar the two sequences are. Homologs of the
sequences referenced or disclosed herein, such as homologs of the
SCBV enhancer element, will possess a relatively high degree of
sequence identity when aligned using standard methods.
[0111] Methods of alignment of sequences for comparison are well
known in the art. Various programs and alignment algorithms are
described in: Smith and Waterman (Adv. Appl. Math. 2: 482, 1981);
Needleman and Wunsch (J. Mol. Biol. 48: 443, 1970); Pearson and
Lipman (PNAS USA 85: 2444, 1988); Higgins and Sharp (Gene, 73:
237-244, 1988); Higgins and Sharp (CABIOS 5: 151-153, 1989); Corpet
et al. (Nuc. Acids Res. 16: 10881-90, 1988); Huang et al. (Comp.
Appls Biosci. 8: 155-65, 1992); and Pearson et al. (Methods in
Molecular Biology 24: 307-31, 1994). Altschul et al. (Nature
Genet., 6: 119-29, 1994) presents a detailed consideration of
sequence alignment methods and homology calculations.
[0112] The alignment tools ALIGN (Myers and Miller, CABIOS 4:
11-17, 1989) or LFASTA (Pearson and Lipman, 1988) may be used to
perform sequence comparisons (Internet Program .COPYRGT. 1996, W.
R. Pearson and the University of Virginia, "fasta20u63" version
2.0u63, release date December 1996). ALIGN compares entire
sequences against one another, while LFASTA compares regions of
local similarity. These alignment tools and their respective
tutorials are available on the Internet at
biology.ncsa.uiuc.edu.
[0113] Orthologs or paralogs (more generally, homologs) of the
disclosed sequences are typically characterized by possession of
greater than 75% sequence identity counted over the full-length
alignment with the sequence to which they are compared using ALIGN
set to default parameters. Sequences with even greater similarity
to the reference sequences will show increasing percentage
identities when assessed by this method, such as at least 80%, at
least 85%, at least 90%, at least 92%, at least 95%, or at least
98% sequence identity. In addition, sequence identity can be
compared over the full length of one or both binding domains of the
disclosed fusion proteins. In such an instance, percentage
identities will be essentially similar to those discussed for
full-length sequence identity.
[0114] When significantly less than the entire sequence is being
compared for sequence identity, homologs will typically possess at
least 80% sequence identity over short windows of 10-20 amino
acids, and may possess sequence identities of at least 85%, at
least 90%, at least 95%, or at least 99% depending on their
similarity to the reference sequence. Sequence identity over such
short windows can be determined using LFASTA; methods can be found
at World Wide Web address biology.ncsa.uiuc.edu. One of skill in
the art will appreciate that these sequence identity ranges are
provided for guidance only; it is entirely possible that strongly
significant homologs could be obtained that fall outside of the
ranges provided. The present disclosure provides not only the
peptide homologs that are described above, but also nucleic acid
molecules that encode such homologs.
[0115] An alternative indication that two nucleic acid molecules
are closely related is that the two molecules hybridize to each
other under stringent conditions. Stringent conditions are
sequence-dependent and are different under different environmental
parameters. Generally, stringent conditions are selected to be
about 5.degree. C. to 20.degree. C. lower than the thermal melting
point (T.sub.m) for the specific sequence at a defined ionic
strength and pH. The T.sub.m is the temperature (under defined
ionic strength and pH) at which 50% of the target sequence
hybridizes to a perfectly matched probe. Conditions for nucleic
acid hybridization and calculation of stringencies can be found in
Sambrook et al. (In Molecular Cloning: A Laboratory Manual, Cold
Spring Harbor, N.Y., 1989) and Tijssen (Laboratory Techniques in
Biochemistry and Molecular Biology Part I, Ch. 2, Elsevier, New
York, 1993). Nucleic acid molecules that hybridize under stringent
conditions to the disclosed SCBV enhancer sequences will typically
hybridize to a probe based on either the entire fusion protein
encoding sequence, an entire binding domain, or other selected
portions of the encoding sequence under wash conditions of
0.2.times.SSC, 0.1% SDS at 65.degree. C.
[0116] Nucleic acid sequences that do not show a high degree of
identity may nevertheless encode similar amino acid sequences, due
to the degeneracy of the genetic code. It is understood that
changes in nucleic acid sequence can be made using this degeneracy
to produce multiple nucleic acid sequences that each encode
substantially the same protein.
[0117] 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 are 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.
[0118] Transcribable polynucleotide molecule: Any polynucleotide
molecule capable of being transcribed into a RNA molecule. Methods
are known to those of ordinary skill, for introducing constructs
into a cell in such a manner that the transcribable polynucleotide
molecule is transcribed into a functional mRNA molecule that is
translated and therefore expressed as a protein product. Constructs
may also be constructed to be capable of expressing antisense RNA
molecules, in order to inhibit translation of a specific RNA
molecule of interest. Conventional compositions and methods for
preparing and using constructs and host cells are well known to one
skilled in the art (see for example, Molecular Cloning: A
Laboratory Manual, 3rd edition Volumes 1, 2, and 3. Sambrook et
al., Cold Spring Harbor Laboratory Press, 2000).
[0119] Transcription: The production of an RNA molecule by RNA
polymerase as a complementary copy of a DNA sequence.
[0120] Transcription termination region: Sequences that control
formation of the 3' end of a transcript. Self-cleaving ribozymes
and polyadenylation sequences are examples of transcription
termination sequences.
[0121] Transcriptional gene silencing (TGS): A phenomenon that is
triggered by the formation of dsRNA that is homologous with gene
promoter regions and sometimes coding regions. TGS results in DNA
and histone methylation and chromatin remodeling, thereby causing
transcriptional inhibition rather than RNA degradation. Both TGS
and PTGS depend on dsRNA, which is cleaved into small (21-25
nucleotides) interfering RNAs (Eckhardt, Plant Cell, 14:1433-1436,
2002; Aufsatz et al., Proc. Natl. Acad. Sci. U.S.A.,
99:16499-16506, 2002).
[0122] Transgenic: This term refers to a plant/fungus/cell/other
entity or organism that contains recombinant genetic material not
normally found in entities of this type/species (that is,
heterologous genetic material) and which has been introduced into
the entity in question (or into progenitors of the entity) by human
manipulation. Thus, a plant that is grown from a plant cell into
which recombinant DNA is introduced by transformation (a
transformed plant cell) is a transgenic plant, as are all offspring
of that plant that contain the introduced transgene (whether
produced sexually or asexually).
[0123] Transformation: Process by which exogenous DNA enters and
changes a recipient cell. It may occur under natural conditions, or
artificial conditions using various methods well known in the art.
Transformation may rely on any known method for the insertion of
foreign nucleic acid sequences into a prokaryotic or eukaryotic
host cell. Selection of the method is influenced by the host cell
being transformed and may include, but is not limited to, viral
infection, electroporation, lipofection, and particle
bombardment.
[0124] Transformed: A transformed cell is a cell into which has
been introduced a nucleic acid molecule by molecular biology
techniques. Transformed cells include stably transformed cells in
which the inserted DNA is capable of replication either as an
autonomously replicating plasmid or as part of the host chromosome.
They also include cells that transiently express the inserted DNA
or RNA for limited periods of time. As used herein, the term
transformation encompasses all techniques by which a nucleic acid
molecule might be introduced into such a cell, including
transfection with viral vectors, transformation with plasmid
vectors, and introduction of naked DNA by electroporation,
lipofection, and particle gun acceleration.
[0125] Transposon: A nucleotide sequence such as a DNA or RNA
sequence that is capable of transferring location or moving within
a gene, a chromosome or a genome.
[0126] Transgenic plant: A plant that contains a foreign
(heterologous) nucleotide sequence inserted into either its nuclear
genome or organellar genome.
[0127] Transgene: A nucleic acid sequence that is inserted into a
host cell or host cells by a transformation technique.
[0128] Vector: A nucleic acid molecule as introduced into a host
cell, thereby producing a transformed host cell. A vector may
include nucleic acid sequences that permit it to replicate in the
host cell, such as an origin of replication. A vector may also
include one or more therapeutic genes and/or selectable marker
genes and other genetic elements known in the art. A vector can
transduce, transform or infect a cell, thereby causing the cell to
express nucleic acids and/or proteins other than those native to
the cell. A vector optionally includes materials to aid in
achieving entry of the nucleic acid into the cell, such as a viral
particle, liposome, protein coating or the like.
[0129] Unless otherwise explained, all technical and scientific
terms used herein have the same meaning as commonly understood by
one of ordinary skill in the art to which this invention belongs.
The singular terms "a," "an," and "the" include plural referents
unless context clearly indicates otherwise. Similarly, the word
"or" is intended to include "and" unless the context clearly
indicates otherwise. Hence "comprising A or B" means including A,
or B, or A and B. It is further to be understood that all base
sizes or amino acid sizes, and all molecular weight or molecular
mass values, given for nucleic acids or polypeptides are
approximate, and are provided for description. Although methods and
materials similar or equivalent to those described herein can be
used in the practice or testing of the present invention, suitable
methods and materials are described below. All publications, patent
applications, patents, and other references mentioned herein are
incorporated by reference in their entirety. In case of conflict,
the present specification, including explanations of terms, will
control. In addition, the materials, methods, and examples are
illustrative only and not intended to be limiting.
III. Overview of Several Embodiments
[0130] The present disclosure describes novel transcription
initiation regions comprising an enhancer domain and, under the
enhancing control of the enhancer domain, a transcription
regulatory domain. The enhancer domain comprises a plurality (e.g.,
two to four or more) of copies of a natural but previously
unrecognized SCBV enhancer arranged in tandem. The transcription
regulatory regions (promoters) of the present disclosure provide
enhanced transcription as compared to the promoter in the absence
of the enhancer domain. In one embodiment, a chimeric transcription
regulatory region is disclosed comprising one or more copies of the
SCBV enhancer element shown in position 337 to position 618 of SEQ
ID NO: 1 (or a homolog thereof); and operably linked thereto, a
promoter comprising an RNA polymerase binding site and a mRNA
initiation site, wherein when a nucleotide sequence of interest is
transcribed under regulatory control of the chimeric transcription
regulatory region, the amount of transcription product is enhanced
compared to the amount of transcription product obtained with the
chimeric transcription regulatory region comprising the promoter
and not comprising the SCBV enhancer sequence(s). In some
embodiments, the chimeric transcription regulatory region comprises
a promoter obtained from the upstream region of a plant virus gene,
a bacterial gene, a fungal gene, a plant nuclear gene, a plant
extra-nuclear gene, an invertebrate gene, or a vertebrate gene.
[0131] Also provided are DNA constructs comprising a described
transcription regulatory region and a DNA sequence to be
transcribed. In some embodiments, a DNA construct is disclosed
comprising the transcriptional initiation region operably linked to
a transcribable polynucleotide molecule operably linked to a 3'
transcription termination polynucleotide molecule. In one
embodiment, the transcribable polynucleotide molecule confers an
agronomic trait to a plant in which it is expressed.
[0132] Also provided are transgenic plants. In one embodiment, a
transgenic plant is stably transformed with a disclosed DNA
construct. In some embodiments, the transgenic plant is a
dicotyledon. In other embodiments, the transgenic plant is a
monocotyledon. In one particular embodiment, the transgenic plant
is a maize plant.
[0133] Further provided is a seed of a disclosed transgenic plant.
In one embodiment, the seed comprises the disclosed DNA
construct.
[0134] Even further provided is a transgenic plant cell or tissue.
In one embodiment, a transgenic plant cell or tissue comprises a
disclosed chimeric transcription regulatory region. In some
embodiments, the plant cell or tissue is derived from a
dicotyledon. In other embodiments, the plant cell or tissue is from
a monocotyledon. In one particular embodiment, the plant cell or
tissue is from a maize plant.
[0135] Also provided are methods of producing a disclosed
transgenic plant, plant cell, seed or tissue. In some embodiments,
the method comprises transforming a plant cell or tissue with a
disclosed DNA construct.
[0136] Further provided are a plant cell, fruit, leaf, root, shoot,
flower, seed, cutting and other reproductive material useful in
sexual or asexual propagation, progeny plants inclusive of F1
hybrids, male-sterile plants and all other plants and plant
products derivable from the disclosed transgenic plants.
[0137] Also disclosed is a maize plant cell, tissue or plant
comprising one or more copies of a SCBV enhancer element shown in
position 337 to position 618 of SEQ ID NO: 1. In one embodiment, a
maize plant cell, tissue or plant comprises one or more copies of a
SCBV enhancer element shown in position 337 to position 618 of SEQ
ID NO: 1 in which the one or more copies of the SCBV enhancer
element is inserted into a genome of the maize plant cell, tissue
or plant at a random location. In some embodiments, the SCBV
enhancer imparts enhanced transcription of a nucleotide sequence of
interest which is under regulatory control of the SCBV enhancer as
compared to transcription of the nucleotide sequence of interest in
the absence of the SCBV enhancer.
IV. SCBV Enhancer and its Uses
[0138] The present disclosure provides a previously unrecognized
enhancer region from the Sugarcane Bacilliform badnavirus (SCBV)
genome, which enhancer is useful in enhancing the transcription
efficiency which may result in enhanced transcription of DNA
sequences under control of the enhancer. Of particular interest is
enhanced transcription of gene sequences which may be of the same
genetic origin as the host or of foreign origin, either the
naturally occurring sequences (in both sense and antisense
orientations) or synthetically prepared sequences. The subject
enhancers comprise a plurality of two or more copies of a
previously unrecognized natural SCBV enhancer domain (the sequence
of which is provided in SEQ ID NO: 1, at positions 337 to 618). The
enhancer comprises at least two copies of the enhancer domain
sequence, in some embodiments three or four or more copies,
arranged in tandem.
[0139] Also contemplated are homologous enhancers. Without
intending to be limited in any way, representative homologous
sequences may include those from other SCBV promoters, for instance
from different SCBV isolates such as those described in Braithwaite
et al. (Plant Cell Rep. 23:319-326, 2004; incorporated herein by
reference in its entirety) or in U.S. Pat. No. 5,994,123
(incorporated herein by reference in its entirety).
[0140] A natural enhancer comprises a DNA sequence which in its
native environment is upstream from and within about 600 bp of a
promoter. Taking the initial nucleotide of the mRNA as 0, the
sequence containing an enhancer is from about -50 to about -1,000
bp, usually from about -50 to -950 bp, generally comprising about
-100 to -800 bp. An enhancer domain is cis-acting and desirably is
located within about 10,000 bp, usually about 2,000 bp, more
usually adjacent to or within about 1,000 bp of a transcription
initiation sequence to be enhanced. The enhancer may be in either
orientation with respect to the transcription initiation sequence
and can be located upstream or downstream in relation to the
promoter it enhances, though it is usually upstream.
[0141] The enhancer domain of the present disclosure finds use with
a wide variety of initiation sequences, including promoters that
are naturally found under the control of the enhancer, e.g., in a
cis position (adjacent and homologous) as well as those not
normally associated with the particular enhancer (e.g.,
heterologous). The enhancer domain and transcription initiation
domain may be from the same or different kingdom, family or
species. Species of interest include prokaryotes and eukaryotes,
such as bacteria, plants, insects, mammals, etc. Combinations
include the described SCBV (viral) enhancer domain(s) with a
transcription initiation region of a structural gene of: a host for
SCBV (e.g., from sugarcane), another plant species (e.g., of the
same or a different family), an insect, a vertebrate animal, a
bacterium, a fungus, and so forth.
[0142] The disclosure also contemplates DNA constructs comprising a
subject transcription initiation region and, under the control of
the transcription initiation region, a DNA sequence to be
transcribed. The DNA sequence may comprise a natural open reading
frame including transcribed 5' and 3' flanking sequences.
Alternatively, it may comprise an anti-sense sequence in that it
encodes the complement of an RNA molecule or portion thereof. When
the construct includes an open reading frame (ORF) which encodes a
protein, an enhanced transcription initiation rate is obtained,
usually providing an increased amount of the polypeptide expression
product of the gene. When the construct comprises an anti-sense
sequence, the enhanced transcription of RNA complementary to wild
type suppresses the expression of the wild type mRNA, thereby
decreasing the amount of the polypeptide expression product; it is
contemplated that the wild type mRNA in question may correspond to
a native mRNA of the host cell or a mRNA of a pathogen, such as a
virus or fungus.
[0143] In various embodiments, the DNA sequence to be transcribed
includes: protein encoding sequence(s) of a gene (e.g., from a
plant, animal, bacterium, virus, or fungus), which may include:
natural open reading frame(s) encoding a protein product;
complementary DNA (cDNA) sequences derived from mRNA encoded by a
gene; synthetic DNA giving the desired coding sequence(s); protein
encoding sequence(s) derived from exons of a natural gene, such as
open reading frame(s) produced by exon ligation; and/or
combinations of any two or more thereof. Attached to these
sequences are appropriate transcription termination/polyadenylation
sequences; sequences from a natural gene (e.g., from a plant,
animal, bacterium, virus, or fungus) that encodes a primary RNA
product, that is consisting of exons and introns (e.g., natural
Polymerase II and Polymerase III transcribed genes of eukaryotes);
synthetic DNA sequences that encode a specific RNA or protein
product; sequences of DNA modified from a known coding sequence
(e.g., a natural gene sequence) by mutagenesis (such as site
specific mutagenesis) and/or other genetic engineering technology;
chimeras of any of the above achieved by ligation of DNA fragments,
including chimeras that encode fusion proteins; and/or DNA
sequences encoding the complement of RNA molecules or portions
thereof.
[0144] Enhanced transcription in plants may find use in enhancing
the production of proteins characteristic of the plant
(endogenous--that is, normally found in the wild-type host) or
those proteins from other genetic sources (exogenous--that is, not
normally found in the wild-type host). Examples of types of
sequences to be expressed from the enhancers and chimeric
transcription regulatory regions described herein include:
antisense or small inhibitory RNAs (for gene suppression);
nutritionally important proteins; growth promoting factors;
proteins giving protection to the plant under certain environmental
conditions, e.g., proteins conferring resistance to metal, salt, or
other toxicity; stress related proteins giving tolerance to
extremes of temperature, freezing, etc.; proteins conferring pest
or infection-related protection to the plant, e.g., proteins giving
resistance to bacterial, fungal, or other microbial infection, or
resistance to predation by insects (e.g., B. thuringiensis toxin)
or to other invertebrate or vertebrate animals; compounds of
medical importance outside of the plant, e.g., anti-microbial,
anti-tumor, etc.; proteins or other compounds of specific
commercial value; increased level of proteins, e.g., enzymes of
metabolic pathways (e.g., pathways for production of polyphenolic
compounds or other secondary metabolites); increased levels of
products of structural value to a plant host; and so forth. The
sequences of interest which are transcribed will be of at least
about 8 bp, at least about 12 bp, at least about 20 bp, and may be
one or more kilobase pairs (kbp) in length.
V. Constructs
[0145] Constructs of the present disclosure typically contain a
chimeric transcription regulatory region comprising one or more
copies of the provided SCBV enhancer element operably linked to a
promoter (usually containing at least an RNA polymerase binding
site and a mRNA initiation site), which region is operably linked
to a transcribable polynucleotide molecule operably linked to a 3'
transcription termination polynucleotide molecule. In addition,
constructs may include but are not limited to additional regulatory
polynucleotide molecules from the 3'-untranslated region (3' UTR)
of plant genes (e.g., a 3' UTR to increase mRNA stability of the
mRNA, such as the PI-II termination region of potato or the
octopine or nopaline synthase 3' termination regions). Constructs
may include but are not limited to the 5'-untranslated regions (5'
UTR) of an mRNA polynucleotide molecule which can play an important
role in translation initiation and can also be a genetic component
in a plant expression construct. For example, non-translated 5'
leader polynucleotide molecules derived from heat shock protein
genes have been demonstrated to enhance gene expression in plants
(see for example, U.S. Pat. Nos. 5,659,122 and 5,362,865 each of
which is incorporated by reference in its entirety). Such
additional upstream and downstream regulatory polynucleotide
molecules as are present in the construct may be derived from a
source that is native or heterologous with respect to the other
elements present on the construct.
[0146] Thus, one embodiment is a construct comprising a chimeric
transcription regulatory region itself comprising one or more
copies (e.g., two, three, four or more copies) of the SCBV enhancer
element shown in position 337 to position 618 of SEQ ID NO: 1 (or a
homolog thereof) operably linked to a promoter, operably linked to
a transcribable polynucleotide molecule so as to direct
transcription of said transcribable polynucleotide molecule at a
desired level and/or in a desired tissue or developmental pattern
upon introduction of the construct into a plant cell. The
transcribable polynucleotide molecule in some examples comprises a
protein-coding region of a gene, and the chimeric transcription
regulatory region provides transcription of a functional mRNA
molecule that is translated and expressed as a protein product from
the construct. In another embodiment, the transcribable
polynucleotide molecule comprises an antisense region of a gene,
and the chimeric transcription regulatory region affects
transcription of an antisense RNA molecule or other similar
inhibitory RNA in order to inhibit expression of a specific RNA
molecule of interest in a target host cell.
[0147] Yet more example constructs of the present disclosure
include double Ti plasmid border DNA constructs that have the right
border (RB or AGRtu.RB) and left border (LB or AGRtu.LB) regions of
the Ti plasmid isolated from Agrobacterium tumefaciens comprising a
T-DNA, which along with transfer molecules provided by the
Agrobacterium cells, enable integration of the T-DNA into the
genome of a plant cell. The constructs may also contain plasmid
backbone DNA segments that provide replication function and
antibiotic selection in bacterial cells, for example, an
Escherichia coli origin of replication such as ori322, a broad host
range origin of replication such as oriV or oriRi, and a coding
region for a selectable marker such as Spec/Strp that encodes for
Tn7 aminoglycoside adenyltransferase (aadA) conferring resistance
to spectinomycin or streptomycin, or a gentamicin (Gm, Gent)
selectable marker gene. For plant transformation, representative
host bacterial strains include Agrobacterium tumefaciens ABI, C58,
or LBA4404; however, other strains known to those skilled in the
art of plant transformation can be used.
[0148] Also contemplated are constructs comprising at least one
SCBV enhancer element (optionally in the context of a chimeric
transcription regulatory region), which construct is an activation
tagging construct. Activation tagging is a method by which genes
are randomly and strongly upregulated on a genome-wide scale, after
which specific phenotypes can be screened for and selected.
Components useful in various types of activating tagging constructs
are known; see, for instance: Walden et al., Plant Mol. Biol. 26:
1521-8, 1994 (describing an activation T-DNA tagging construct that
was used to activate genes in tobacco cell culture allowing the
cells to grow in the absence of plant growth hormones);
Miklashevichs et al., Plant J. 12: 489-98, 1997; Harling et al.,
EMBO J. 16: 5855-66, 1997; Walden et al., EMBO J. 13: 4729-36, 1994
(reports of genes isolated from plant genomic sequences flanking
the T-DNA tag and putatively involved in plant growth hormone
responses); Schell et al., Trends Plant Sci. 3: 130, 1998
(discussing investigation of a group of related studies);
Kardailsky et al., Science 286: 1962-1965, 1999 (describing
activation T-DNA tagging and screening of plants for an early
flowering phenotype); Koncz et al., Proc Natl Acad Sci USA
86(21):8467-71, 1989 (describing activation tagging using the
Agrobacterium gene 5 promoter (pg5), which is active only in
proliferating cells and must insert directly adjacent to a plant
gene in order to influence its expression); Wilson et al., Plant
Cell 8: 659-671, 1996 (activation tagging that utilizes a modified
Ds transposon carrying the CaMV 35S promoter and a nos::hpt
selection cassette) and Schaffer et al., Cell 93: 1219-1229, 1998
(illustrating the same system, used to upregulate adjacent plant
genes resulting in dominant gain-of-function mutations 1996); and
Weigel et al., Plant Physiology, 122:1003-1013, 2000 (illustrating
activation tagging vectors that are useful for screening tens of
thousands of transformed plants for morphological phenotypes).
VI. Nucleotide Sequences for Transcription Enhancement
[0149] Exemplary transcribable polynucleotide molecules for
transcription enhancement by incorporation into constructs as
provided herein include, for example, polynucleotide molecules or
genes from a species other than the target species or genes that
originate with or are present in the same species, but are
incorporated into recipient cells by genetic engineering methods
rather than classical reproduction or breeding techniques. The type
of polynucleotide molecule can include but is not limited to a
polynucleotide molecule that is already present in the target plant
cell, a polynucleotide molecule from another plant, a
polynucleotide molecule from a different organism, or a
polynucleotide molecule generated externally, such as a
polynucleotide molecule containing an antisense message of a gene,
or a polynucleotide molecule encoding an artificial, synthetic, or
otherwise modified version of a transgene.
[0150] In one embodiment, a polynucleotide molecule as shown in
positions 337 to 618 of SEQ ID NO: 1 (or two or more copies
thereof) (for instance, in the context of a chimeric transcription
initiation region) is incorporated into a construct such that the
described SCBV enhancer sequence (or series of two or more such
sequences) is operably linked to a transcribable polynucleotide
molecule that is a gene of agronomic interest or other expression
sequence (more generally, a nucleotide sequence of interest). As
used herein, the term "gene of agronomic interest" refers to a
transcribable polynucleotide molecule that includes but is not
limited to a gene that provides a desirable characteristic
associated with plant morphology, physiology, growth and
development, yield, nutritional enhancement, disease or pest
resistance, or environmental or chemical tolerance. The expression
of a gene of agronomic interest is desirable in order to confer an
agronomically important trait, for instance. A gene of agronomic
interest that provides a beneficial agronomic trait to crop plants
may be, for example, one or more sequences conferring to a plant
expressing the gene: herbicide resistance (see, e.g., U.S. Pat.
Nos. 6,803,501; 6,448,476; 6,248,876; 6,225,114; 6,107,549;
5,866,775; 5,804,425; 5,633,435; 5,463,175; and U.S. Publications
US20030135879 and US20030115626), increased yield (see, e.g., U.S.
Pat. RE38,446; U.S. Pat. Nos. 6,716,474; 6,663,906; 6,476,295;
6,441,277; 6,423,828; 6,399,330; 6,372,211; 6,235,971; 6,222,098;
5,716,837), insect control (see, e.g., U.S. Pat. Nos. 6,809,078;
6,713,063; 6,686,452; 6,657,046; 6,645,497; 6,642,030; 6,639,054;
6,620,988; 6,593,293; 6,555,655; 6,538,109; 6,537,756; 6,521,442;
6,501,009; 6,468,523; 6,326,351; 6,313,378; 6,284,949; 6,281,016;
6,248,536; 6,242,241; 6,221,649; 6,177,615; 6,156,573; 6,153,814;
6,110,464; 6,093,695; 6,063,756; 6,063,597; 6,023,013; 5,959,091;
5,942,664; 5,942,658, 5,880,275; 5,763,245; 5,763,241), fungal
disease resistance (see, e.g., U.S. Pat. Nos. 6,653,280; 6,573,361;
6,506,962; 6,316,407; 6,215,048; 5,516,671; 5,773,696; 6,121,436;
6,316,407; 6,506,962), virus resistance (see, e.g., U.S. Pat. Nos.
6,617,496; 6,608,241; 6,015,940; 6,013,864; 5,850,023; 5,304,730),
nematode resistance (see, e.g., U.S. Pat. No. 6,228,992), bacterial
disease resistance (see, e.g., U.S. Pat. No. 5,516,671), plant
growth and development (see, e.g., U.S. Pat. Nos. 6,723,897;
6,518,488), starch production (see, e.g., U.S. Pat. Nos. 6,538,181;
6,538,179; 6,538,178; 5,750,876; 6,476,295), modified oils
production (see, e.g., U.S. Pat. Nos. 6,444,876; 6,426,447;
6,380,462), high oil production (see, e.g., U.S. Pat. Nos.
6,495,739; 5,608,149; 6,483,008; 6,476,295), modified fatty acid
content (see, e.g., U.S. Pat. Nos. 6,828,475; 6,822,141; 6,770,465;
6,706,950; 6,660,849; 6,596,538; 6,589,767; 6,537,750; 6,489,461;
6,459,018), fiber production (see, e.g., U.S. Pat. Nos. 6,576,818;
6,271,443; 5,981,834; 5,869,720), high protein production (see,
e.g., U.S. Pat. No. 6,380,466), fruit ripening (see, e.g., U.S.
Pat. No. 5,512,466), improved digestibility (see, e.g., U.S. Pat.
No. 6,531,648), improved flavor (see, e.g., U.S. Pat. No.
6,011,199), low raffinose (see, e.g., U.S. Pat. No. 6,166,292),
enhanced animal and/or human nutrition (see, e.g., U.S. Pat. Nos.
6,723,837; 6,653,530; 6,541,259; 5,985,605; 6,171,640),
environmental stress resistance (see, e.g., U.S. Pat. No.
6,072,103), desirable peptides (e.g., pharmaceutical or secretable
peptides) (see, e.g., U.S. Pat. Nos. 6,812,379; 6,774,283;
6,140,075; 6,080,560), improved processing traits (see, e.g., U.S.
Pat. No. 6,476,295), industrial enzyme production (see, e.g., U.S.
Pat. No. 5,543,576), nitrogen fixation (see, e.g., U.S. Pat. No.
5,229,114), hybrid seed production (see, e.g., U.S. Pat. No.
5,689,041), biopolymers (see, e.g., U.S. Pat. No. RE37,543; U.S.
Pat. Nos. 6,228,623; 5,958,745 and U.S. Publication No.
US20030028917) and biofuel production (see, e.g., U.S. Pat. No.
5,998,700). The genetic elements, methods, and transgenes described
in the patents and published applications listed above are
incorporated herein by reference.
[0151] Alternatively, a transcribable polynucleotide molecule can
influence an above mentioned (or other) plant characteristic or
phenotypes by encoding an antisense or RNA molecule that causes the
targeted inhibition of expression of an endogenous gene, for
example via antisense, inhibitory RNA (RNAi), or
cosuppression-mediated mechanisms. The RNA could also be a
catalytic RNA molecule (a ribozyme) engineered to cleave a desired
endogenous mRNA product. Thus, any transcribable polynucleotide
molecule that encodes a transcribed RNA molecule that affects a
phenotype, biochemical or morphological change of interest may
benefit from the transcriptional enhancement enabled by the
sequences and constructs provided herein.
[0152] The described SCBV enhancer or chimeric transcription
regulatory region comprising one or more copies thereof can be
incorporated into a construct with one or more marker genes (any
transcribable polynucleotide molecule whose expression can be
screened for or scored in some way) and tested in transient or
stable plant analyses to provide an indication of the regulatory
element's gene expression pattern in stable transgenic plants.
Marker genes for use in the practice of such embodiments include,
but are not limited to transcribable polynucleotide molecules
encoding .beta.-glucuronidase (GUS described in U.S. Pat. No.
5,599,670) and green fluorescent protein (GFP described in U.S.
Pat. Nos. 5,491,084 and 6,146,826), proteins that confer antibiotic
resistance, or proteins that confer herbicide tolerance. Useful
antibiotic resistance markers, including those encoding proteins
conferring resistance to kanamycin (nptII), hygromycin B (aph IV),
streptomycin or spectinomycin (aad, spec/strep) and gentamycin
(aac3 and aacC4) are known in the art. Herbicides for which
transgenic plant tolerance has been demonstrated and the method of
the present invention can be applied, include but are not limited
to: glyphosate, glufosinate, sulfonylureas, imidazolinones,
bromoxynil, delapon, cyclohezanedione, protoporphyrionogen oxidase
inhibitors, and isoxasflutole herbicides. Polynucleotide molecules
encoding proteins involved in herbicide tolerance are known in the
art, and include, but are not limited to a polynucleotide molecule
encoding 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS
described in U.S. Pat. Nos. 5,627,061, 5,633,435, 6,040,497 and in
U.S. Pat. No. 5,094,945 for glyphosate tolerance); polynucleotides
encoding a glyphosate oxidoreductase and a glyphosate-N-acetyl
transferase (GOX described in U.S. Pat. No. 5,463,175 and GAT
described in U.S. publication No. 20030083480); a polynucleotide
molecule encoding bromoxynil nitrilase (Bxn described in U.S. Pat.
No. 4,810,648 for Bromoxynil tolerance); a polynucleotide molecule
encoding phytoene desaturase (crtl) described in Misawa et al.
(Plant J. 4:833-840, 1993) and Misawa et al. (Plant J. 6:481-489,
1994) for norflurazon tolerance; a polynucleotide molecule encoding
acetohydroxyacid synthase (AHAS, aka ALS) described in Sathasiivan
et al. (Nucl. Acids Res. 18:2188-2193, 1990) for tolerance to
sulfonylurea herbicides; a polynucleotide molecule encoding a
dicamba-degrading oxygenase enzyme (described in U.S. Patent
Publications US20030135879 and US20030115626, for dicamba
tolerance); and the bar gene described in DeBlock et al. (EMBO J.
6:2513-2519, 1987) for glufosinate and bialaphos tolerance. The
regulatory elements of the present disclosure can express
transcribable polynucleotide molecules that encode phosphinothricin
acetyltransferase, glyphosate resistant EPSPS, aminoglycoside
phosphotransferase, hydroxyphenyl pyruvate dehydrogenase,
hygromycin phosphotransferase, neomycin phosphotransferase, dalapon
dehalogenase, bromoxynil resistant nitrilase, anthranilate
synthase, glyphosate oxidoreductase and glyphosate-N-acetyl
transferase.
[0153] Constructs containing at least one SCBV enhancer (for
instance, in the context of a chimeric transcription regulatory
region) operably linked to a marker gene or other nucleotide
sequence of interest may be delivered to a tissues (e.g.,
transformed) and the tissues analyzed by the appropriate mechanism,
depending on the marker or sequence that is being transcribed. Such
quantitative or qualitative analyses may be used as tools to
evaluate the potential expression profile of a regulatory element
when operatively linked to a gene of agronomic interest in stable
plants. Marker gene can be used in a transient assay; methods of
testing for marker gene expression in transient assays are known to
those of ordinary skill in the art. Transient expression of marker
genes has been reported using a variety of plants, tissues, and DNA
delivery systems. For example, transient analyses systems include
but are not limited to direct gene delivery via electroporation or
particle bombardment of tissues in any transient plant assay using
any plant species of interest. Such transient systems would include
but are not limited to electroporation of protoplasts from a
variety of tissue sources or particle bombardment of specific
tissues of interest. The present disclosure encompasses use of any
transient expression system to evaluate regulatory elements
operably linked to any transcribable polynucleotide molecule,
including but not limited to marker genes or genes of agronomic
interest. Examples of plant tissues envisioned to test in
transients via an appropriate delivery system would include but are
not limited to leaf base tissues, callus, cotyledons, roots,
endosperm, embryos, floral tissue, pollen, and epidermal
tissue.
VII. Plant Transformation
[0154] A plant transformation construct containing an enhancer
element (or multiple copies thereof) or a chimeric transcription
regulatory region such as is described herein may be introduced
into plants using any plant transformation method. Methods and
materials for transforming plants by introducing a plant expression
construct into a plant genome in the practice of this invention can
include any of the well-known and demonstrated methods including
electroporation (e.g., U.S. Pat. No. 5,384,253), microprojectile
bombardment (e.g., U.S. Pat. Nos. 5,015,580; 5,550,318; 5,538,880;
6,160,208; 6,399,861; and 6,403,865), Agrobacterium-mediated
transformation (e.g., U.S. Pat. Nos. 5,824,877; 5,591,616;
5,981,840; and 6,384,301), and protoplast transformation (e.g.,
U.S. Pat. No. 5,508,184). It will be apparent to those of skill in
the art that a number of transformation methodologies can be used
and modified for production of stable transgenic plants from any
number of target crops of interest.
[0155] Specific methods for transforming dicots are known to those
skilled in the art. By way of example, transformation and plant
regeneration methods have been described for a number of crops
including, but not limited to, cotton (Gossypium hirsutum), soybean
(Glycine max), peanut (Arachis hypogaea), and members of the genus
Brassica.
[0156] Likewise, specific methods for transforming monocots are
also known to those skilled in the art. By way of example
transformation and plant regeneration methods have been described
for a number of crops including, but not limited to, barley
(Hordeum vulgarae); maize (Zea mays); oats (Avena sativa); orchard
grass (Dactylis glomerata); rice (Oryza sativa, including indica
and japonica varieties); sorghum (Sorghum bicolor); sugar cane
(Saccharum sp); tall fescue (Festuca arundinacea); turfgrass
species (e.g. Agrostis stolonifera, Poa pratensis, Stenotaphrum
secundatum); wheat (Triticum aestivum), and alfalfa (Medicago
sativa).
[0157] The transformed plants may be analyzed for the presence of
the gene(s) of interest and the expression level and/or profile
conferred by the chimeric transcription regulatory regions
described herein. Numerous methods are available to those of
ordinary skill in the art for the analysis of transformed plants.
For example, methods for plant analysis include Southern and
northern blot analysis, PCR-based (or other nucleic acid
amplification-based) approaches, biochemical analyses, phenotypic
screening methods, field evaluations, and immunodiagnostic assays
(e.g., for the detection, localization, and/or quantification of
proteins).
[0158] Enhanced expression of genes using the described SCBV
enhancer has been demonstrated in maize, but the enhancer is
expected to function in other plant species, possibly including
dicots as well as monocots. The enhancer element with four copies
of the SCBV upstream region provided the highest level of
expression of the combinations studied herein. Fewer or more copies
of the upstream region, as well as, combinations with enhancer
elements from other sources could also provide advantages for
modulating gene expression. The same activators, constructs and
approaches may be useful for other crop species for which genes may
be identified because genome sequence is available or in progress
(including Sorghum (Sorghum bicolor), Wheat (Triticum aestivum),
Barley (Hordeum vulgare), Foxtail millet (Setaria italica),
Sugarcane (Saccharum officinarum), Miscanthus giganteus or for
which `activated genes` may be identified by future genome
sequencing efforts or perhaps chromosomal synteny (including Oats
(Avena sativa), Rye (Secale cereale), Pearl millet (Pennisetum
glaucum), Finger millet (Eluesine coracana), Proso millet (Panicum
miliaceum), Teff millet (Eragrostis tef)), or for model grass
species for which genomic sequence is available or in progress
(including Purple False Brome (Brachypodium distachyon), Green
bristlegrass (Setaria viridis)).
[0159] The following examples are provided to illustrate certain
particular features and/or embodiments. These examples should not
be construed to limit the invention to the particular features or
embodiments described.
Example 1
Identification of Sequences Comprising Enhancer Element of
Sugarcane Bacilliform Virus (SCBV) Promoter
[0160] This example demonstrates the identification of sequences
including the SCBV promoter enhancer element.
[0161] A promoter fragment derived from the genome of a SCBV
(GenBank Accession No. AJ277091, and described by Geijskes et al.,
Arch. Virol., 147: 2393-2404, 2002) was first examined by transient
expression assays to determine which regions of the promoter
sequence contain enhancer element sequences. In the promoter
analysis study, fragments derived from the SCBV promoter (SEQ ID
NO: 1) containing sequences from -839 to +106 bp (plasmid
pSCBV839), from -576 to +106 bp (plasmid pSCBV576), and from -333
to +106 bp (plasmid pSCBV333) from the transcription start site
(defined as the +1 position) were cloned upstream of a coding
region for a firefly luciferase (LUC) reporter protein.
Transcription was terminated by a copy of the nopaline synthase
(Nos) 3' UTR region (as disclosed in bases 1847 to 2103 of GenBank
Accession No. V00087.1, which is hereby incorporated by reference
in its entirety; and FIG. 1). Transient transcriptional activities
of these constructs were tested by transforming them by particle
bombardment into maize Hi-II suspension cells (described in detail
in Example 2 below) and monitoring activity of the LUC reporter
gene. Luciferase activity was normalized in each experiment by
co-transforming with a equimolar amount of the plasmid DNA
containing an SCBV:LUC construct and DNA of a reference plasmid
harboring a construct consisting of a maize ubiquitin 1 (ubi1) gene
promoter (as disclosed in U.S. Pat. No. 5,510,474 which is hereby
incorporated by reference in its entirety; essentially bases 7 to
1990 of GenBank Accession No. S94464.1, which is hereby
incorporated by reference in its entirety) driving expression of a
GUS (beta-glucuronidase) coding region, and terminated by a maize
PerS 3' UTR terminator (as disclosed in U.S. Pat. No. 6,699,984,
which is hereby incorporated by reference in its entirety; e.g.,
construct ubil:GUS). Two days after bombardment, total protein was
isolated from transformed cells and LUC enzymatic activity
(expressed in Luciferase Units (LU)/mg protein) and GUS enzymatic
activity (expressed in GUS activity units (GU)/.mu.g protein) were
measured by methods found in, for example, (Maliga et al., Methods
in Plant Molecular Biology. A Laboratory Course Manual. Cold Spring
Harbor Laboratory Press, 1995). Relative activities of the test
promoters in the three SCBV:LUC constructs were compared by
normalizing LUC levels to GUS levels as the ratio of LU/mg
protein:GU/.mu.g protein. The transient testing results showed that
LUC activity increased linearly with increasing concentrations of
plasmid DNA bombarded, indicating that LUC activity is correlated
with transcript levels. Further, the SCBV promoter fragment
containing sequences from -576 bp upstream to +106 downstream of
the transcription start site had 66%.+-.2% of the activity of the
full-length promoter fragment (here defined as containing the
sequences from -839 bp upstream to +106 downstream of the start
site). In contrast, the promoter fragment containing sequences from
-333 bp upstream to +106 downstream of the transcription start site
had only 17%.+-.1% of the activity of the full-length promoter.
Thus, sequences for most of the SCBV promoter activity reside
upstream of -333 bp from the transcription start site.
[0162] The portion of the SCBV promoter sequence capable of
enhancing transcription driven by a heterologous minimal promoter
sequence was examined. As defined by these experiments, an enhancer
element is operationally identified as a short (200 to 300 bp)
cis-acting DNA sequence, lacking a TATA-box, that, when placed 5'
proximal to a heterologous minimal promoter sequence, increases the
expression activity of the heterologous minimal promoter in a
reproducible and measurable fashion when tested in either a
transient or stable transformation system. Further, tandem
duplications of the enhancer element provide even higher levels of
expression activity of the heterologous minimal promoter than do
single copies of the enhancer element. The heterologous minimal
promoter element utilized in this Example comprises bases from -100
to +106 of a maize alcohol dehydrogenase 1 (Adh1) gene promoter
(corresponding to bases 997 to 1202 of GenBank Accession No.
X04049, which is hereby incorporated by reference in its
entirety).
[0163] Two fragments derived from the SCBV promoter, comprising
sequences from -503 to -222 bp and from -758 to -222 bp relative to
the transcription start site, were cloned 5' to sequences
comprising a minimal maize Adh1 promoter fused to a coding region
encoding a firefly luciferase (LUC) protein. Transcription of the
chimeric genes was terminated by the Nos 3'UTR as described above.
Maize Hi-II suspension culture cells were transformed by particle
bombardment with DNAs of plasmids harboring LUC and GUS constructs,
and enzymatic activities were measured and compared as above.
Plasmids containing the LUC constructs having the -503 to -222
sequences or the -758 to -222 sequences placed 5' to the minimal
Adh1 promoter showed 6-fold, and 4-fold, respectively, more LUC
activity relative to the minimal Adh1 promoter without the added
SCBV sequences. Thus, sequences within these fragments of the SCBV
promoter enhance transcription activity mediated by a heterologous
maize promoter.
[0164] The abilities of multiple copies of the -503 to -222 bP SCBV
enhancer region to increase expression mediated by the minimal Adh1
promoter was tested by cloning one, two or four copies of the -502
to -222 bp sequences 5' to the minimal maize Adh1 promoter fused to
the LUC coding region (FIG. 3A). Plasmid DNAs harboring the
constructs (as well as plasmid DNA having a reference ubil:GUS
construct) were bombarded into maize Hi-II suspension culture
cells, and LUC and GUS activities were measured and compared as
above. Cells bombarded with constructs containing 1 copy, 2 copies,
or 4 copies of the SCBV enhancer sequence region had more than 5
times, 6 times and 10 times, respectively, more LUC activity than
did cells bombarded with an analogous minimal Adh1 promoter
construct lacking SCBV enhancer sequences (FIG. 3B).
[0165] Nucleic acid bases comprising -502 to -222 bp of the SCBV
promoter, as provided in SEQ ID NO: 1, encode transcriptional
activation activity that can confer superior expression
characteristics to a plant promoter. Further, transcriptional
activation activity is increased by the stacking of multiple tandem
copies of the bases comprising -502 to -222 bp of the SCBV
promoter, as provided in SEQ ID NO: 1. Further still, the methods
and reagents provided herein may be further examined and utilized
to provide even shorter sequences that retain transcriptional
activation activity, or may be combined with other transcriptional
activator elements and plant promoters in new combinations.
Example 2
Transient Expression Testing of SCBV:LUC and ub1:GUS Constructs in
Maize Hi-II Suspension Culture Cells
[0166] This example describes transient expression testing of
SCBV:LUC and ub1:GUS constructs in maize Hi-II suspension culture
cells.
[0167] Maize Hi-II suspension culture cells (Armstrong et al.,
Maize Genet. Coop. Newslett., 65:92-93, 1991) were transformed by
particle bombardment with DNAs of plasmids harboring LUC and GUS
constructs constructed as described above, and enzymatic activities
were measured and compared. Bulk preparations of plasmid DNAs were
prepared using QiAfilter.TM. Plasmid Maxi Kits (Qiagen, Germantown,
Md.) and quantity and quality were analyzed using standard
molecular methods.
[0168] Preparation of Maize Hi-II Suspension Culture Cells for
Bombardment.
[0169] The Hi-II cells were maintained on a shaker at 125 rpm in
H9CP+ medium at 28.degree. in darkness (H9CP medium consists of MS
salts 4.3 gm/L, sucrose 3%, Casamino acids 200 mg/L, myo-inositol
100 mg/L, 2.4-D 2 mg/L, NAA 2 mg/L, 1000.times.MS vitamins 1 mL/L,
L-proline 700 mg/L, and coconut water (Sigma Aldrich, St. Louis,
Mo.) 62.5 mL/L, pH 6.0). Prior to bombardment, the 2-day old Hi-II
cultures were transferred to G-N6 medium (CHU N6 medium 3.98 g/L,
CHU N6 vitamins 1 mL/L (both CHU components from PhytoTechnology
Laboratories.RTM., Lenexa, Kans.), Myo-inositol 100 mg/L, 2,4-D 2
mg/L and Sucrose 3%, pH 6.0) and allowed to grow for 24 hours. On
the day of bombardment, the G-N6 grown cells (2.5 gm of cells) were
transferred to sterile Whatman No. 1 filter disks (55 mm) placed on
G-N6 medium containing 0.5 M D-sorbitol and 0.5 M D-mannitol and
incubated for 4 hours. The osmotically adjusted cells are used for
bombardment.
[0170] Preparation of Gold Particles with Plasmid DNAs and
Bombardment Assay.
[0171] Gold particles (1 .mu.m diameter, BioRad, Hercules, Calif.)
were washed with 70% ethanol for 10 minutes, then three times with
sterile water. The particles were dispensed in 50% glycerol at a
concentration of 120 mg/mL. For a typical experiment, 150 .mu.L (18
mg) of gold particles, approximately 5 .mu.g of plasmid DNA, 150
.mu.L of 2.5 M CaCl.sub.2 and 30 .mu.L 0.2 M spermidine were
combined. The reaction (total volume 375 .mu.L) was incubated at
room temperature for 10 minutes with occasional gentle vortexing.
The DNA coated-gold particles were briefly centrifuged, washed with
420 .mu.L of 70% ethanol and then with 420 .mu.L of 100% ethanol.
The final pellet was resuspended in 110 .mu.L of 100% ethanol and
subjected to a brief sonication (three bursts of 3 seconds each,
with 1 minute between bursts) with a Branson 1450 sonicator.
Aliquots of 12.2 .mu.L of the gold-particles coated with DNA were
spread on each of nine macrocarriers (BioRad, Hercules, Calif.) and
used in bombardment assays using a BioRad PDS1000/He system. The
suspension culture cells were transformed at a target distance of 9
cm using 3510 psi disks and each plate was bombarded 3 times.
Following bombardment, the cells were incubated in the dark at
28.degree. C., first for 12 hours on G-N6 containing D-sorbitol and
D-mannitol medium, then on G-N6 plates for an additional 36 hours.
Cells were collected from the plates, blotted to remove buffer and
extracted with 300 .mu.L of 2.times.CCLT LUC extraction buffer
(Promega Corporation, Madison, Wis.). After centrifugation, about
600 .mu.L of protein extract was collected. Protein concentrations
were estimated using the Bradford assay.
[0172] LUC enzymatic activity (expressed in Luciferase Units
(LU)/mg protein) and GUS enzymatic activity (expressed in GUS
activity units (GU)/.mu.g protein) were measured by methods found
in, for example, Maliga et al. (Methods in Plant Molecular Biology.
A Laboratory Course Manual. Cold Spring Harbor Laboratory Press,
1995). Relative activities of the test promoters in SCBV:LUC
constructs were compared by normalizing LUC levels to GUS levels as
the ratio of LUC/mg protein:GUS/.mu.g protein.
Example 3
Plasmids for Activation Tagging in Maize Plants
[0173] This example describes generation of Agrobacterium
superbinary plasmids.
[0174] The superbinary system is a specialized example of an
Agrobacterium shuttle vector/homologous recombination system
(Komari et al., Meth. Mol. Biol. 343:15-41, 2006, Komari et al.,
Plant Physiol. 114:1155-1160, 2007; see also European Patent No.
EP604662B1 and U.S. Pat. No. 7,060,876 each of which is
incorporated by reference in its entirety). The Agrobacterium
tumefaciens host strain employed with the superbinary system is
LBA4404(pSB1). Strain LBA4404(pSB1) harbors two
independently-replicating plasmids, pAL4404 and pSB1. pAL4404 is a
Ti-plasmid-derived helper plasmid which contains an intact set of
vir genes (from Ti plasmid pTiACH5), but which has no T-DNA region
(and thus no T-DNA left and right border repeat sequences). Plasmid
pSB1 supplies an additional partial set of vir genes derived from
pTiBo542. One example of a shuttle vector used in the superbinary
system is pSB11, which contains a cloning polylinker that serves as
an introduction site for genes destined for plant cell
transformation, flanked by right and left T-DNA border repeat
regions. Shuttle vector pSB11 is not capable of independent
replication in Agrobacterium, but is stably maintained therein as a
co-integrant plasmid when integrated into pSB1 by means of
homologous recombination between common sequences present on pSB1
and pSB11. Thus, the fully modified T-DNA region introduced into
LBA4404(pSB1) on a modified pSB11 vector is productively acted upon
and transferred into plant cells by Vir proteins derived from two
different Agrobacterium Ti plasmid sources (pTiACH5 and pTiBo542).
The superbinary system has proven to be particularly useful in
transformation of monocot plant species (See Hiei et al., Plant J.
6:271-282, 1994, and Ishida et al., Nat. Biotechnol. 14:745-750,
1996).
[0175] A transformation plasmid for production of activation tagged
maize plants can include a cointegrant plasmid formed by homologous
recombination between the superbinary plasmid pSB1 and pEPP1088,
having a pSB11 vector backbone (see European Patent No. EP604662B1
and U.S. Pat. No. 7,060,876 each of which are hereby incorporated
by reference). The cointegrant plasmid is referred to as
pSB1::pEPP1088 or as a ZeaTAG vector. The structure of pEPP1088 was
validated by restriction enzyme analysis and DNA sequence
determination of selected regions of the construct. pEPP1088
contains, positioned between Left (LB) and Right (RB) T-DNA border
sequences provided by the pSB11 plasmid, 4 copies of the -502 to
-222 bp SCBV enhancer sequences described above and a selectable
marker gene comprised of a rice (Oryza sativa) actin gene promoter
with associated intron 1 and 5' UTR (essentially as disclosed as
bases 12 to 1411 of GenBank Accession No. EU155408.1 which is
hereby incorporated by reference in its entirety), a coding
sequence for an AAD-1 herbicide tolerance protein as disclosed in
U.S. Patent Application No. 20090093366, and a 3' UTR terminator
sequence from maize lipase gene essentially as disclosed as bases
921 to 1277 of GenBank Accession No. gbIL35913.11MZELIPASE and in
U.S. Pat. No. 7,179,902 each of which is hereby incorporated by
reference in its entirety.
[0176] The T-DNA of pEPP1088 (and as present in pSB1::pEPP1088)
integrates at random locations in maize chromosomes when introduced
into maize cells by Agrobacterium mediated transformation.
Selection for transformed maize cells is provided by the
constitutively expressed AAD1 selectable marker gene in the T-DNA.
The T-DNA carrying tandem copies of the potent -502 to -222 bp SCBV
transcriptional enhancer activator element causes aberrant
expression of native genes nearby the integration site, thereby, in
some instances, providing new identifiable traits to plants
regenerated from the transformed tissues. Modern molecular biology
methods are available which facilitate the isolation and
identification of the affected genes near the acceptor site, thus
providing the isolated genes for further exploitation.
Example 4
Agrobacterium-Mediated Transformation of Maize
[0177] This example describes generation of Agrobacterium-mediated
transformation of maize
[0178] Immature Embryo Production.
[0179] Seeds from a B104 inbred line were planted into
4-gallon-pots containing Sunshine Custom Blend.RTM. 160 (Sun Gro
Horticulture, Bellevue, Wash.). The plants were grown in a
greenhouse using a combination of high pressure sodium and metal
halide lamps with a 16:8 hour Light:Dark photoperiod. To obtain
immature embryos for transformation, controlled sib-pollinations
were performed. Immature embryos were isolated at 10 to 13 days
post-pollination when embryos were approximately 1.4 to 2.0 mm in
size.
[0180] Infection and Co-Cultivation.
[0181] Maize ears were surface sterilized by immersing in 50%
commercial bleach with Tween 20 (1 or 2 drops per 500 mL) for 10
minutes and triple-rinsed with sterile water. A suspension of
Agrobacterium cells containing a superbinary vector cointegrant
plasmid was prepared by transferring 1 or 2 loops of bacteria grown
on YEP solid medium containing 50 mg/L Spectinomycin, 10 mg/L
Rifampicin, and 50 mg/L Streptomycin at 28.degree. C. for 3 days or
25.degree. C. for 4 days into 5 mL of liquid infection medium (MS
salts, ISU Modified MS Vitamins, 3.3 mg/L Dicamba, 68.4 gm/L
sucrose, 36 gm/L glucose, 700 mg/L L-proline, pH 5.2) containing
100 .mu.M acetosyringone. The solution was gently pipetted up and
down using a sterile 5 mL pipette until a uniform suspension was
achieved, and the concentration was adjusted to an optical density
of 0.3 to 0.5 at 600 nm (OD.sub.600) using an Ultrospec 10 Cell
Density Meter (GE Healthcare/Amersham Biosciences, Piscataway,
N.J.). Immature embryos were isolated directly into a micro
centrifuge tube containing 2 mL of the infection medium. The medium
was removed and replaced twice with 1 to 2 mL of fresh infection
medium, then removed and replaced with 1.5 mL of the Agrobacterium
solution. The Agrobacterium and embryo solution was incubated for 5
minutes at room temperature and then transferred to co-cultivation
medium which contained MS salts, ISU Modified MS Vitamins, 3.3 mg/L
Dicamba, 30 gm/L sucrose, 700 mg/L L-proline, 100 mg/L
myo-inositol, 100 mg/L Casein Enzymatic Hydrolysate, 15 mg/L
AgNO.sub.3, 100 .mu.M acetosyringone, and 2.3 to 3 gm/L Gelzan.TM.
(Sigma-Aldrich, St. Louis, Mo.), at pH 5.8. Co-cultivation
incubation was for 3 to 4 days at 25.degree. C. under either dark
or 24-hour white fluorescent light conditions (approximately 50
.mu.Em.sup.-2s.sup.-1).
[0182] Resting and Selection.
[0183] After co-cultivation, the embryos were transferred to a
non-selection MS-based resting medium containing MS salts, ISU
Modified MS Vitamins, 3.3 mg/L Dicamba, 30 gm/L sucrose, 700 mg/L
L-proline, 100 mg/L myo-inositol, 100 mg/L Casein Enzymatic
Hydrolysate, 15 mg/L AgNO.sub.3, 0.5 gm/L MES
(2-(N-morpholino)ethanesulfonic acid monohydrate; PhytoTechnologies
Labr., Lenexa, Kans.), 250 mg/L Carbenicillin, and 2.3 gm/L
Gelzan.TM., at pH 5.8. Incubation was continued for 7 days at
28.degree. C. under either dark or 24-hour white fluorescent light
conditions (approximately 50 .mu.Em.sup.-2s.sup.-1). Following the
7 day resting period, the embryos were transferred to selective
medium. For selection of maize tissues transformed with a
superbinary plasmid containing a plant expressible AAD1 selectable
marker gene, the MS-based resting medium (above) was used
supplemented with Haloxyfop. The embryos were first transferred to
selection media containing 100 nM Haloxyfop and incubated for 1 to
2 weeks, and then transferred to 500 nM Haloxyfop and incubated for
an additional 2 to 4 weeks. Transformed isolates were obtained over
the course of approximately 5 to 8 weeks at 28.degree. C. under
either dark or 24-hour white fluorescent light conditions
(approximately 50 .mu.Em.sup.-2s.sup.-1). Recovered isolates were
bulked up by transferring to fresh selection medium at 1 to 2 week
intervals for regeneration and further analysis.
[0184] Those skilled in the art of maize transformation will
understand that other methods of selection of transformed plants
are available when other plant expressible selectable marker genes
(e.g., herbicide tolerance genes) are used.
[0185] Pre-Regeneration.
[0186] Following the selection process, cultures exposed to the
24-hour light regime were transferred to an MS-based
pre-regeneration medium containing MS salts, ISU Modified MS
Vitamins, 45 gm/L sucrose, 350 mg/L L-proline, 100 mg/L
myo-inositol, 50 mg/L Casein Enzymatic Hydrolysate, 1 mg/L
AgNO.sub.3, 0.25 gm/L MES, 0.5 mg/L naphthaleneacetic acid, 2.5
mg/L abscisic acid, 1 mg/L 6-benzylaminopurine, 250 mg/L
Carbenicillin, 2.5 gm/L Gelzan.TM., and 500 nM Haloxyfop, at pH
5.8. Incubation was continued for 7 days at 28.degree. under
24-hour white fluorescent light conditions (approximately 50
.mu.Em.sup.-2s.sup.-1).
[0187] Regeneration and Plantlet Isolation.
[0188] For regeneration, the cultures were transferred to an
MS-based primary regeneration medium containing MS salts, ISU
Modified MS Vitamins, 60 gm/L sucrose, 100 mg/L myo-inositol, 125
mg/L Carbenicillin, 2.5 gm/L Gelzan.TM., and 500 nM Haloxyfop, at
pH 5.8. After 2 weeks at 28.degree. under either dark or 24-hour
white fluorescent light conditions (approximately 50
.mu.Em.sup.-2s.sup.-1), tissues were transferred to an MS-based
secondary regeneration medium composed of MS salts, ISU Modified MS
Vitamins, 30 gm/L sucrose, 100 mg/L myo-inositol, 3 gm/L
Gelzan.TM., at pH 5.8, with, or without, 500 nM Haloxyfop.
Regeneration/selection was continued for 2 weeks at 28.degree.
under either 16-hour or 24-hour white fluorescent light conditions
(approximately 50 .mu.Em.sup.-2s.sup.-1). When plantlets reached 3
to 5 cm in length, they were excised and transferred to secondary
regeneration medium (as above, but without Haloxyfop) and incubated
at 25.degree. under 16-hour white fluorescent light conditions
(approximately 50 .mu.Em.sup.-2s.sup.-1) to allow for further
growth and development of the shoot and roots.
[0189] Seed Production.
[0190] Plants were transplanted into Metro-Mix.RTM. 360 soilless
growing medium (Sun Gro Horticulture) and hardened-off in a growth
room. Plants were then transplanted into Sunshine Custom Blend 160
soil mixture and grown to flowering in the greenhouse. Controlled
pollinations for seed production were conducted.
Example 5
SCBV Enhancer Activity in Stably Transformed Maize Cells
[0191] Genomic DNA was isolated (Qiagen DNeasy.RTM. Plant Mini Kit;
Qiagen, Germantown, Md.) from ten To plants regenerated from
transformed B104 immature embryos, and the genomic locations of the
integrated T-DNAs transferred from pSB1::pEPP1088 were determined
by inverse PCR cloning and DNA sequencing of the inverse PCR
amplified products. The identities of genes represented by the
flanking coding regions positioned within 10 kb of the 4.times.SCBV
enhancer were determined by BLAST searches (Altschul et al., J.
Mol. Biol., 215: 403-410, and Karlin et al., Proc. Natl. Acad. Sci.
USA 87: 2264-2268, 1990) using the flanking sequences as query
sequences. Analyses of the BLAST results revealed that the T-DNAs,
and hence the 4.times.SCBV enhancers, were integrated at a
different genomic location in each of the 10 lines, and therefore
the 4.times.SCBV enhancers are flanked by different genes in each
line (Table 1).
[0192] Total RNA was isolated (Qiagen RNeasy.RTM. Plant Mini Kit,
Qiagen, Germantown, Md.) from leaf tissues of the ten T.sub.0
lines. Transcript accumulation of the identified flanking genes was
compared between the appropriate To plants and non-transformed
control plants by reverse transcription and RT-PCR (Real Time PCR),
using primers specific for the relevant genes flanking the
4.times.SCBV enhancers. As a control, transcript accumulation for
the endogenous GAPDH gene was also determined.
[0193] RT-PCR products revealed increased accumulation of
transcripts originating from 3 of the different flanking genes in
these lines. The 4.times.SCBV enhancers are located 2.6 kb and 2.8
kb upstream of the affected flanking genes in 2 of the T.sub.0
lines, and 478 bp downstream of the affected flanking gene in the
third To line. Thus, these results indicate that the 4.times.SCBV
enhancers delivered by T-DNA cause strand-independent increased
accumulation of transcripts of genes nearby the integration site.
Table 1 indicates the flanking genes identified and the results of
analyses of their transcription levels.
TABLE-US-00001 TABLE 1 Effect 4XSCBV enhancer on the RNA
accumulation of the flanking genes in 10 T0 plants. Distance to the
RNA T0 Plant ID 4XSCBV (bp) Flanking Gene Name Accumulation
ZT00031845 1197 P-loop containing NTP hydrolases No change
ZT00032132 5'-UTR A protein that helps vesicular fusion No change
proteins ZT00036435 2644 DEAD-box-like helicase Increased
ZT00034545 1972 High mobility group-like nuclear protein No change
ZT00036729 EST Unknown protein No change ZT00035749 2818 Unknown
protein (GRMZM2G115661) Increased ZT00033904 830 Unknown protein No
change ZT00036426 79 Ribosomal protein L22/L17; T0 plant is No
change tall ZT00036426 2150 Signal peptide No change ZT00035050 478
from the 3'-end Unknown gene (GRMZM2G139336) Increased
[0194] One skilled in the fields of maize genetics and plant
molecular biology will realize that, depending upon the nature of
the affected genes, the increased expression of adjacent genes
induced by 4.times.SCBV enhancers will in some cases confer upon
the transgenic plant new and valuable traits. Collectively, plants
having the 4.times.SCBV enhancers represent a ZeaTAG-marked
population. The traits may be the result of increased accumulation
of the affected gene's encoded protein per se, as for example,
increased accumulation of a nutritionally desirable protein in the
seed, or the result of a downstream effect whereby the gene product
of the immediately affected gene controls the expression of one or
a multitude of other genes (as in the case of, for example,
transcriptional activator/repressor genes). The random nature of
integration location of introduced T-DNAs, coupled with standard
plant breeding methods, may be used to establish large populations
of plants comprising a library of T-DNA bearing plants having
activator elements positioned within an effective distance of all
or most genes within the maize genome, and thus provides the
opportunity for all or most maize genes to be transcriptionally
activated.
[0195] Plant-level screening for phenotypes of economic importance
is possible under growth chamber, greenhouse, or field
environments. As shown here, molecular biology methods such as
inverse PCR enable the isolation of an integrated T-DNA and
substantial lengths of genomic DNA flanking the integrated T-DNA
from plants exhibiting a desirable phenotype. Further, methods such
as genome walking techniques allow the determination of even more
extensive regions of genomic DNA sequence, thus enabling
identification of the genes present in proximity to introduced
activator elements. High throughput methods such as microarray
analysis and more gene specific analytical methods enable
identification and quantification of affected transcript levels.
Candidate genes involved in relevant agronomic traits may thus
identified, isolated, and further characterized and exploited to
provide new and valuable varieties of crops.
[0196] Conversely, the new trait may be the result of disruption of
maize gene function due to the integration of the T-DNA having the
4.times.SCBV enhancers into the coding region or expression
regulatory regions of the maize gene. If such is the case, the
T-DNA having the 4.times.SCBV enhancers and surrounding genomic
regions can be isolated and further characterized.
Example 6
Forward Genetic Screening of the ZeaTAG Population
[0197] This example describes forward genetic screening of the
ZeaTAG population for altered phenotypes.
Drought Stress Screens
[0198] To identify ZeaTAG lines that contain mutations conferring
drought tolerance, plants from individual ZeaTAG events are planted
in a field. Water is withheld to cause drought stress during the
reproductive phase of the growth cycle; roughly 2 weeks prior to
flowering to approximately 2 weeks after flowering. The target is
to achieve 4 weeks of stress period at flowering stage.
Environmental modeling is used to predict accurate corn
evapotranspiration demand based on soil moisture monitoring and
weather data (air temperature, vapor pressure deficit, wind speed,
and net radiation). Plants are monitored for drought symptoms such
as leaf rolling by visual observation, increased leaf temperature
by infrared thermometers, reduced photosynthesis by chlorophyll
fluorescence and reduced yield by measuring grain production.
Plants that show significantly less leaf rolling, lower leaf
temperature, higher rates of photosynthesis or have significantly
more yield under water stress conditions are identified and used in
subsequent screens.
[0199] ZeaTAG events displaying significantly more drought
tolerance are planted in a replicated field trial to confirm the
drought tolerant phenotype. These events are planted in a randomize
split block design with at least 3 replications. One block is
irrigated with water sufficient to prevent water stress. The other
block is grown under water deficient conditions as described above.
Plants are monitored for leaf rolling, increased leaf temperature,
decreased photosynthesis and decreased yield as described above.
Plants with significantly less leaf rolling, lower leaf
temperature, greater photosynthesis or greater yield than
untransformed control plants are considered to have passed the
secondary screen.
Nitrogen Use Efficiency Screens
[0200] To identify ZeaTAG events with greater nitrogen use
efficiency than non-transgenic control plants a primary screen is
performed. Plants containing approximately 40,000 ZeaTAG containing
events are grown in the field under nitrogen deficient conditions.
Plants are grown in fields with less than 35 lbs of N per acre.
Plants are monitored for chlorosis by visual inspection, increased
leaf temperature by infrared thermometers, and decreased yield by
grain harvest. These parameters are compared with non-transgenic
control plants. ZeaTAG lines showing less chlorosis, lower leaf
temperature, higher photosynthetic rates or greater yields than
non-transgenic control lines are evaluated in secondary
screens.
[0201] As a secondary screen, ZeaTAG events displaying
significantly more nitrogen use efficiency are planted in a
replicated field trial to confirm the phenotype. These events are
planted in a randomize split block design with at least 3
replications. One block is irrigated with sufficient nitrogen
fertilizer to prevent nitrogen stress. The other block is grown
under nitrogen deficient conditions as described above. Plants are
monitored for chlorosis by visual inspection, increased leaf
temperature by infrared thermometers, and decreased yield by grain
harvest. Plants with significantly less chlorosis, lower leaf
temperature, greater photosynthesis or greater yield than
untransformed control plants are considered to have passed the
secondary screen.
[0202] Once the phenotype has been confirmed in the secondary
screen, the phenotype is tested for genetic linkage with the ZeaTAG
insertion by screening the progeny of a cross between the
non-transformed parental line and the ZeaTAG line. When plants
containing the ZeaTAG element display the phenotype and plants that
do not contain the ZeaTAG element do not, the phenotype is
considered to be genetically linked with the insert and likely to
be caused by the ZeaTAG element. To identify genes whose expression
may be affected by the ZeaTAG element, the location of the ZeaTAG
element within the genome is determined.
[0203] The genomic location of the ZeaTAG element is determined by
isolating genomic sequences flanking the ZeaTAG element and
comparing these sequences to the genomic sequence of maize.
Sequences flanking the ZeaTAG element can be determined by a number
of molecular biological techniques, including but not limited to,
inverse PCR (iPCR) (Ochman et al., Genetics, 120: 621-6231988),
TAIL (Liu et al., Plant Journal 8: 457-463, 1995) and
ligation-mediated PCR (LMPCR) Prod'hom et al., FEMS Microbiol Lett.
158: 75-81, 1998). These sequences are compared to genomic
sequences by sequence alignment tools such as BLAST to identify the
location of the ZeaTAG element within the genome.
[0204] Genes flaking or interrupted by the ZeaTAG element are
determined by examining the annotated genome. Transcription of
genes flanking the ZeaTAG element may be responsible for the mutant
phenotype. These genes may be over-expressed in wild-type maize to
test whether they can confer a similar phenotype. To test this, the
genes are cloned into transformation vectors driven by strong
promoters or by their own promoter with enhancer sequences flanking
them to enhance transcription. These vectors are introduced into
wild-type maize by transformation and plants resulting from this
transformation are tested for the phenotype.
[0205] Similarly, genes interrupted by the ZeaTAG element may cause
the phenotype. To confirm that a gene interrupted by the element is
responsible for the phenotype, expression of the gene can be
disrupted and plants containing this disruption can be tested for
the phenotype. The disruption of expression of specific genes can
be accomplished by a number of methods know to those skilled in the
art including but not limited to antisense RNA, artificial micro
RNAs and identifying mutations in the gene by TILLING.
Example 7
Reverse Genetic Screening of the ZeaTAG Population
[0206] This example describes reverse genetic screening of the
ZeaTAG population for mutations.
[0207] Reverse genetic screening is looking for mutations affecting
specific genes and subsequently testing the identified line for a
mutant phenotype. The ZeaTAG population can be used in reverse
genetic analyses in several ways including but not limited to
generating a collection Flanking Sequence Tags for the population
(Jeong et al., The Plant Journal 45: 123-132, 2006) and generating
an indexed collection of pooled samples of DNA from the ZeaTAG
population (May et al., Molecular Biotechnology 20: 209-221,
2002).
[0208] A collection of Flanking Sequence Tags is generated by
sampling leaf tissue from the ZeaTAG population, isolating DNA from
each, identification of sequences flanking the insert and storing
the sequences in a searchable database where the sequences are
linked to the events from which they came. Genomic DNA is isolated
using the Qiagen DNAeasy Plant Kit (Qiagen, Germantown, Md.) using
the protocol recommended by the manufacturer. Sequences flanking
the insert are identified using Ligation Mediated PCR (Mueller et
al., Science 246: 780-786, 1989) as modified by Yephremov and
Saedler (Plant Journal 21: 295-305, 2000). Briefly, genomic DNA
from a ZeaTAG line is fragmented restriction enzyme digestion and
denatured. A biotinylated oligonucleotide primer complementary to
the sequence at the end of the ZeaTAG element is hybridized to the
fragmented DNA and extended by DNA polymerase. Streptavidin coated
magnetic beads are added to the mixture to bind DNA fragments
containing DNA fragments extended from this primer. A
double-stranded DNA adaptor of known sequence is ligated to the
unknown end. These fragments are PCR amplified using
oligonucleotides complementary to sequences within the ZeaTAG
element and the DNA adaptor at the other end. The sequence of the
PCR fragment is then determined and mapped to the maize genomic
sequence by BLAST. These sequences locate the site of insertion of
the ZeaTAG element. Genes within a .about.10 kbp may be
up-regulated by the enhancer sequences within the ZeaTAG
element.
[0209] Plants containing insertions in or near genes that are
hypothesized to cause a phenotype can be identified by searching
the database. Plants containing these events can be tested for the
phenotype.
[0210] In view of the many possible embodiments to which the
principles of the disclosed invention may be applied, it should be
recognized that the illustrated embodiments are only preferred
examples of the invention and should not be taken as limiting the
scope of the invention. Rather, the scope of the invention is
defined by the following claims. We therefore claim as our
invention all that comes within the scope and spirit of these
claims.
Sequence CWU 1
1
11839DNASugarcane bacilliform virus 1aagcttattg aatggggaaa
acaaattctt gatccattcc ccaaattcaa gaaggatatg 60tttgaaagaa ctgaacatat
catgatggca acacaagagc ctacgctact atgtggatgc 120aggaagcctg
caatcatgtt aacatcagga acaaggctta atcctcgtag aagattttac
180aagtgtgcca tgaatatctg ccactgctgg tattgggcag atttacttga
agaatacgtg 240caagagagga tcgaagattt catggttgaa aacttcgaca
agaaagcaaa gctggatgaa 300ccaagttcat caaacgttca ccatgatgat
tatgaagaac accgttcgag tgtcatcgac 360aggccaaggc caacagatga
tcatttcaga ccatgggggg atgttacata ctggctgaat 420aaagaagcag
aagagtgcca cacaaggggc gacaacgtcg aaggcgcaga agacgcagtc
480gatctcactg acgtaagcaa tgacgaccag tggaggagat cgtaagcaat
gacgtatgga 540gcgtggagga cccatgaaag cactgagaag gcatctcaac
tttcggtgtg tgagtgcgca 600tcctatgcga tgctttgtac ctttgttagc
tgtgtgtgtc cttttggcat ctgtgccact 660ttacctttgt cggccacgtt
gcctttgctt agcatctacg caagcatagc gctcggctgg 720tgtgtgttcc
ctctgcctat ataaggcatg gttgtatgac tcttacactc atcggtagtt
780caccacatga gtatttgagt caagtttggc ttgaataata agaattacac ctttccgca
839
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