U.S. patent application number 16/027117 was filed with the patent office on 2019-01-24 for parental rnai suppression of kruppel gene to control hemipteran pests.
The applicant listed for this patent is The Board of Regents of the University of Nebraska, Dow AgroSciences LLC. Invention is credited to Kanika Arora, Elane Fishilevich, Meghan Frey, Ronda L. Hamm, Chitvan Khajuria, Kenneth E. Narva, Blair D. Siegfried, Nicholas P. Storer, Ana Velez, Sarah E. Worden.
Application Number | 20190024112 16/027117 |
Document ID | / |
Family ID | 56127521 |
Filed Date | 2019-01-24 |
United States Patent
Application |
20190024112 |
Kind Code |
A1 |
Siegfried; Blair D. ; et
al. |
January 24, 2019 |
PARENTAL RNAI SUPPRESSION OF KRUPPEL GENE TO CONTROL HEMIPTERAN
PESTS
Abstract
This disclosure concerns nucleic acid molecules and methods of
use thereof for control of hemipteran pests through RNA
interference-mediated inhibition of target coding and transcribed
non-coding sequences in hemipteran pests. The disclosure also
concerns methods for making transgenic plants that express nucleic
acid molecules useful for the control of hemipteran pests, and the
plant cells and plants obtained thereby.
Inventors: |
Siegfried; Blair D.;
(Lincoln, NE) ; Narva; Kenneth E.; (Zionsville,
IN) ; Arora; Kanika; (Indianapolis, IN) ;
Worden; Sarah E.; (Indianapolis, IN) ; Khajuria;
Chitvan; (Chesterfield, MO) ; Fishilevich; Elane;
(Indianapolis, IN) ; Storer; Nicholas P.;
(Kensington, MD) ; Frey; Meghan; (Greenwood,
IN) ; Hamm; Ronda L.; (Carmel, IN) ; Velez;
Ana; (Lincoln, NE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Dow AgroSciences LLC
The Board of Regents of the University of Nebraska |
Zionsville
Lincoln |
IN
NE |
US
US |
|
|
Family ID: |
56127521 |
Appl. No.: |
16/027117 |
Filed: |
July 3, 2018 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
14971550 |
Dec 16, 2015 |
10047374 |
|
|
16027117 |
|
|
|
|
62092784 |
Dec 16, 2014 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
Y02A 40/162 20180101;
C12N 15/113 20130101; C12N 15/8286 20130101; C12N 2310/14 20130101;
Y02A 40/146 20180101; C12N 15/8218 20130101; C12N 15/8261 20130101;
A01N 57/16 20130101 |
International
Class: |
C12N 15/82 20060101
C12N015/82; A01N 57/16 20060101 A01N057/16; C12N 15/113 20100101
C12N015/113 |
Claims
1. A method for controlling a hemipteran pest population, the
method comprising providing an agent comprising a ribonucleic acid
(RNA) molecule that functions upon contact with the hemipteran pest
to inhibit a biological function within the hemipteran pest,
wherein the RNA is specifically hybridizable with a polynucleotide
selected from the group consisting of SEQ ID NOs:15-17; the
complement of any of SEQ ID NOs:15-17; a fragment of at least 15
contiguous nucleotides of any of SEQ ID NOs:15-17; the complement
of a fragment of at least 15 contiguous nucleotides of any of SEQ
ID NOs:15-17; a transcript of SEQ ID NO:1; the complement of a
transcript of SEQ ID NO:1; a fragment of at least 15 contiguous
nucleotides of a transcript of SEQ ID NO:1; and the complement of a
fragment of at least 15 contiguous nucleotides of a transcript of
SEQ ID NO:1.
2. The method according to claim 1, wherein the agent is a
double-stranded RNA molecule.
3. A method for controlling a hemipteran pest population, the
method comprising: introducing into a hemipteran pest, a
ribonucleic acid (RNA) molecule that functions upon contact with
the hemipteran pest to inhibit a biological function within the
hemipteran pest, wherein the RNA is specifically hybridizable with
a polynucleotide selected from the group consisting of any of SEQ
ID NOs:15-17, the complement of any of SEQ ID NOs:15-17, a fragment
of at least 15 contiguous nucleotides of any of SEQ ID NOs:15-17,
the complement of a fragment of at least 15 contiguous nucleotides
of any of SEQ ID NOs:15-17, a transcript of SEQ ID NO:1, the
complement of a transcript of SEQ ID NO:1, a fragment of at least
15 contiguous nucleotides of a transcript of SEQ ID NO:1, and the
complement of a fragment of at least 15 contiguous nucleotides of a
transcript of SEQ ID NO:1, thereby producing a hemipteran pest
having a pRNAi phenotype.
4. The method according to claim 3, wherein the RNA is introduced
into a male hemipteran pest.
5. The method according to claim 3, wherein the RNA is introduced
into a female hemipteran pest, the method further comprising
releasing the female hemipteran pest having the pRNAi phenotype
into the pest population, wherein mating between the female
hemipteran pest having the pRNAi phenotype and male pests of the
population produces fewer viable offspring than mating between
other female pests and male pests of the population.
6. A method for controlling a hemipteran pest population, the
method comprising: providing an agent comprising a first and a
second polynucleotide sequence that functions upon contact with the
hemipteran pest to inhibit a biological function within the
hemipteran pest, wherein the first polynucleotide sequence
comprises a region that exhibits from about 90% to about 100%
sequence identity to from about 19 to about 30 contiguous
nucleotides of SEQ ID NO:15, and wherein the first polynucleotide
sequence is specifically hybridized to the second polynucleotide
sequence.
7. The method according to claim 6, wherein the ribonucleic acid
molecule is a double-stranded ribonucleic acid molecule.
8. The method according to claim 6, wherein the hemipteran pest
population is reduced relative to a population of the same pest
species infesting a host plant of the same host plant species
lacking the transformed plant cell.
9. A method for controlling a hemipteran pest population, the
method comprising: providing in a host plant of a hemipteran pest a
transformed plant cell comprising the polynucleotide of claim 1,
wherein the polynucleotide is expressed to produce a ribonucleic
acid molecule that functions upon contact with a hemipteran pest
belonging to the population to inhibit the expression of a target
sequence within the hemipteran pest and results in decreased
reproduction of the hemipteran pest or pest population, relative to
reproduction of the same pest species on a plant of the same host
plant species that does not comprise the polynucleotide.
10. The method according to claim 9, wherein the ribonucleic acid
molecule is a double-stranded ribonucleic acid molecule.
11. The method according to claim 9, wherein the hemipteran pest
population is reduced relative to a hemipteran pest population
infesting a host plant of the same species lacking the transformed
plant cell.
12. A method of controlling hemipteran pest infestation in a plant,
the method comprising providing in the diet of a hemipteran pest a
ribonucleic acid (RNA) that is specifically hybridizable with a
polynucleotide selected from the group consisting of: SEQ ID
NOs:15-17; the complement of any of SEQ ID NOs:15-17; a fragment of
at least 15 contiguous nucleotides of any of SEQ ID NOs:15-17; the
complement of a fragment of at least 15 contiguous nucleotides of
any of SEQ ID NOs:15-17; a transcript of any of SEQ ID NO:1; the
complement of a transcript of SEQ ID NO:1; a fragment of at least
15 contiguous nucleotides of a transcript of SEQ ID NO:1; and the
complement of a fragment of at least 15 contiguous nucleotides of a
transcript of SEQ ID NO:1.
13. The method according to claim 12, wherein the diet comprises a
plant cell transformed to express the polynucleotide.
14. The method according to claim 12, wherein the specifically
hybridizable RNA is comprised in a double-stranded RNA
molecule.
15. A method for improving the yield of a corn crop, the method
comprising: introducing the nucleic acid of claim 1 into a corn
plant to produce a transgenic corn plant; and cultivating the corn
plant to allow the expression of the at least one polynucleotide;
wherein expression of the at least one polynucleotide inhibits
hemipteran pest reproduction or growth and loss of yield due to
hemipteran pest infection.
16. The method according to claim 15, wherein expression of the at
least one polynucleotide produces an RNA molecule that suppresses
at least a first target gene in a hemipteran pest that has
contacted a portion of the plant.
17. A method for producing a transgenic plant cell, the method
comprising: transforming a plant cell with a vector comprising the
nucleic acid of claim 1; culturing the transformed plant cell under
conditions sufficient to allow for development of a plant cell
culture comprising a plurality of transformed plant cells;
selecting for transformed plant cells that have integrated the at
least one polynucleotide into their genomes; screening the
transformed plant cells for expression of a ribonucleic acid (RNA)
molecule encoded by the at least one polynucleotide; and selecting
a plant cell that expresses the RNA.
18. The method according to claim 17, wherein the RNA molecule is a
double-stranded RNA molecule.
19. A method for producing a hemipteran pest-resistant transgenic
plant, the method comprising: providing the transgenic plant cell
produced by the method of claim 17; and regenerating a transgenic
plant from the transgenic plant cell, wherein expression of the
ribonucleic acid molecule encoded by the at least one
polynucleotide is sufficient to modulate the expression of a target
gene in a hemipteran pest that contacts the transformed plant.
20. The method according to claim 17, wherein the transformed plant
cell comprises a nucleotide sequence encoding a polypeptide from
Bacillus thuringiensis or a PIP-1 polypeptide.
21. The method according to claim 20, wherein the polypeptide from
B. thuringiensis is selected from a group comprising Cry1A, Cry2A,
Cry3A, Cry11A, and Cry51A.
Description
PRIORITY CLAIM
[0001] This application is a divisional of U.S. patent application
Ser. No. 14/971,550, filed on Dec. 16, 2015, which claims the
benefit of the filing date of U.S. Provisional Patent Application
Ser. No. 62/092,784, filed Dec. 16, 2014, for "PARENTAL RNAI
SUPPRESSION OF KRUPPEL GENE TO CONTROL HEMIPTERAN PESTS" which is
incorporated herein in its entirety.
FIELD OF THE DISCLOSURE
[0002] The present invention relates generally to genetic control
of plant damage caused by hemipteran pests. In particular
embodiments, the present disclosure relates to identification of
target coding and non-coding polynucleotides, and the use of
recombinant DNA technologies for post-transcriptionally repressing
or inhibiting expression of target coding and non-coding
polynucleotides in the cells of a hemipteran pest to provide a
plant protective effect.
BACKGROUND
[0003] Stink bugs and other hemipteran insects (heteroptera) are an
important agricultural pest complex. Worldwide, over 50 closely
related species of stink bugs are known to cause crop damage.
McPherson & McPherson (2000) Stink bugs of economic importance
in America north of Mexico, CRC Press. Hemipteran insects are
present in a large number of important crops including maize,
soybean, cotton, fruit, vegetables, and cereals.
[0004] Stink bugs go through multiple nymph stages before reaching
the adult stage. These insects develop from eggs to adults in about
30-40 days. Both nymphs and adults feed on sap from soft tissues
into which they also inject digestive enzymes causing extra-oral
tissue digestion and necrosis. Digested plant material and
nutrients are then ingested. Depletion of water and nutrients from
the plant vascular system results in plant tissue damage. Damage to
developing grain and seeds is the most significant as yield and
germination are significantly reduced. Multiple generations occur
in warm climates resulting in significant insect pressure. Current
management of stink bugs relies on insecticide treatment on an
individual field basis. Therefore, alternative management
strategies are urgently needed to minimize ongoing crop losses.
[0005] RNA interference (RNAi) is a process utilizing endogenous
cellular pathways, whereby an interfering RNA (iRNA) molecule
(e.g., a double stranded RNA (dsRNA) molecule) that is specific for
all, or any portion of adequate size, of a target gene results in
the degradation of the mRNA encoded thereby. In recent years, RNAi
has been used to perform gene "knockdown" in a number of species
and experimental systems; for example, Caenorhabditis elegans,
plants, insect embryos, and cells in tissue culture. See, e.g.,
Fire et al. (1998) Nature 391:806-11; Martinez et al. (2002) Cell
110:563-74; McManus and Sharp (2002) Nature Rev. Genetics
3:737-47.
[0006] RNAi accomplishes degradation of mRNA through an endogenous
pathway including the DICER protein complex. DICER cleaves long
dsRNA molecules into short fragments of approximately 20
nucleotides, termed small interfering RNA (siRNA). The siRNA is
unwound into two single-stranded RNAs: the passenger strand and the
guide strand. The passenger strand is degraded, and the guide
strand is incorporated into the RNA-induced silencing complex
(RISC). Micro inhibitory ribonucleic acids (miRNAs) are
structurally very similar molecules that are cleaved from precursor
molecules containing a polynucleotide "loop" connecting the
hybridized passenger and guide strands, and they may be similarly
incorporated into RISC. Post-transcriptional gene silencing occurs
when the guide strand binds specifically to a complementary mRNA
molecule and induces cleavage by Argonaute, the catalytic component
of the RISC complex. This process is known to spread systemically
throughout some eukaryotic organisms despite initially limited
concentrations of siRNA and/or miRNA such as plants, nematodes, and
some insects.
[0007] Only transcripts complementary to the siRNA and/or miRNA are
cleaved and degraded, and thus the knock-down of mRNA expression is
sequence-specific. In plants, several functional groups of DICER
genes exist. The gene silencing effect of RNAi persists for days
and, under experimental conditions, can lead to a decline in
abundance of the targeted transcript of 90% or more, with
consequent reduction in levels of the corresponding protein. In
insects, there are at least two DICER genes, where DICER1
facilitates miRNA-directed degradation by Argonaute1. Lee et al.
(2004) Cell 117 (1):69-81. DICER2 facilitates siRNA-directed
degradation by Argonaute2. Id.
[0008] The overwhelming majority of sequences complementary to
insect DNAs (such as, for example, the 9,000+ sequences identified
in U.S. Pat. No. 7,612,194 and U.S. Patent Publication Nos.
2007/0050860, 2010/0192265, and 2011/0154545) do not provide a
plant protective effect when used as dsRNA or siRNA. For example,
Baum et al. (2007) Nature Biotechnology 25:1322-1326, describe the
effects of inhibiting several Western corn rootworm (WCR) gene
targets by RNAi. These authors reported that 8 of the 26 target
genes they tested were not able to provide experimentally
significant coleopteran pest mortality at a very high iRNA (e.g.,
dsRNA) concentration of more than 520 ng/cm.sup.2.
[0009] The authors of U.S. Pat. No. 7,612,194 and U.S. Patent
Publication No. 2007/0050860 made the first report of in planta
RNAi in corn plants targeting the western corn rootworm. Baum et
al. (2007) Nat. Biotechnol. 25(11):1322-6. These authors describe a
high-throughput in vivo dietary RNAi system to screen potential
target genes for developing transgenic RNAi maize. Of an initial
gene pool of 290 targets, only 14 exhibited larval control
potential. One of the most effective double-stranded RNAs (dsRNA)
targeted a gene encoding vacuolar ATPase subunit A (V-ATPase),
resulting in a rapid suppression of corresponding endogenous mRNA
and triggering a specific RNAi response with low concentrations of
dsRNA. Thus, these authors documented for the first time the
potential for in planta RNAi as a possible pest management tool,
while simultaneously demonstrating that effective targets could not
be accurately identified a priori, even from a relatively small set
of candidate genes.
[0010] Another potential application of RNAi for insect control
involves parental RNAi (pRNAi). First described in Caenorhabditis
elegans, pRNAi was identified by injection of dsRNA into the body
cavity (or application of dsRNA via ingestion), causing gene
inactivity in offspring embryos. Fire et al. (1998), supra; Timmons
and Fire (1998) Nature 395(6705):854. A similar process was
described in the model coleopteran, Tribolium castaneum, whereby
female pupae injected with dsRNA corresponding to three unique
genes that control segmentation during embryonic development
resulted in knock down of zygotic genes in offspring embryos.
Bucher et al. (2002) Curr. Biol. 12(3):R85-6. Nearly all of the
offspring larvae in this study displayed gene-specific phenotypes
one week after injection. Although injection of dsRNA for
functional genomics studies has been successful in a variety of
insects, uptake of dsRNA from the gut environment through oral
exposure to dsRNA and subsequent down-regulation of essential genes
is required in order for RNAi to be effective as a pest management
tool. Auer and Frederick (2009) Trends Biotechnol.
27(11):644-51.
[0011] Parental RNAi has been used to describe the function of
embryonic genes in a number of insect species, including the
springtail, Orchesella cincta (Konopova and Akam (2014) Evodevo
5(1):2); the brown plant hopper, Nilaparvata lugens; the sawfly,
Athalia rosae (Yoshiyama et al. (2013) J. Insect Physiol.
59(4):400-7); the German cockroach, Blattella germanica (Piulachs
et al. (2010) Insect Biochem. Mol. Biol. 40:468-75); and the pea
aphid, Acyrthosiphon pisum (Mao et al. (2013) Arch Insect Biochem
Physiol 84(4):209-21). The pRNAi response in all these instances
was achieved by injection of dsRNA into the hemocoel of the
parental female.
SUMMARY OF THE DISCLOSURE
[0012] Disclosed herein are nucleic acid molecules (e.g., target
genes, DNAs, dsRNAs, siRNAs, shRNAs, miRNAs, and hpRNAs), and
methods of use thereof, for the control of hemipteran pests,
including, for example, Euschistus heros (Fabr.) (Neotropical Brown
Stink Bug, "BSB"); E. servus (Say) (Brown Stink Bug); Nezara
viridula (L.) (Southern Green Stink Bug); Piezodorus guildinii
(Westwood) (Red-banded Stink Bug); Halyomorpha halys (Stal) (Brown
Marmorated Stink Bug); Chinavia hilare (Say) (Green Stink Bug); C.
marginatum (Palisot de Beauvois); Dichelops melacanthus (Dallas);
D. furcatus (F.); Edessa meditabunda (F.); Thyanta perditor (F.)
(Neotropical Red Shouldered Stink Bug); Horcias nobilellus (Berg)
(Cotton Bug); Taedia stigmosa (Berg); Dysdercus peruvianus
(Guerin-Meneville); Neomegalotomus parvus (Westwood); Leptoglossus
zonatus (Dallas); Niesthrea sidae (F.); Lygus hesperus (Knight)
(Western Tarnished Plant Bug); and L. lineolaris (Palisot de
Beauvois). In particular examples, exemplary nucleic acid molecules
are disclosed that may be homologous to at least a portion of one
or more native nucleic acids in a hemipteran pest. In some
embodiments, hemipteran pests are controlled by reducing the
capacity of an existing generation of the pest to produce a
subsequent generation of the pest. In certain examples, delivery of
the nucleic acid molecules to hemipteran pests does not result in
significant mortality to the pests, but reduces the number of
viable progeny produced therefrom.
[0013] In these and further examples, the native nucleic acid may
be a target gene, the product of which may be, for example and
without limitation: involved in a metabolic process; involved in a
reproductive process; and/or involved in embryonic and/or nymph
development. In some examples, post-transcriptional inhibition of
the expression of a target gene by a nucleic acid molecule
comprising a polynucleotide homologous thereto may result in
reduced growth and/or reproduction of the hemipteran pest. In
specific examples, a kruppel gene is selected as a target gene for
post-transcriptional silencing. In particular examples, a target
gene useful for post-transcriptional inhibition is the novel gene
referred to herein as BSB kruppel (SEQ ID NO:1), referred to herein
in some places as BSB_kr. An isolated nucleic acid molecule
comprising the polynucleotide of SEQ ID NO:1; the complement of SEQ
ID NO:1; and/or fragments of either of the foregoing (e.g., SEQ ID
NO:3 and SEQ ID NO:4) is therefore also disclosed herein.
[0014] Also disclosed are nucleic acid molecules comprising a
polynucleotide that encodes a polypeptide that is at least about
85% identical to an amino acid sequence within a target gene
product (for example, the product of a kruppel gene). For example,
a nucleic acid molecule may comprise a polynucleotide encoding a
polypeptide that is at least 85% identical to SEQ ID NO:2 (BSB
KRUPPEL); and/or an amino acid sequence within a product of BSB_kr.
Further disclosed are nucleic acid molecules comprising a
polynucleotide that is the reverse complement of a polynucleotide
that encodes a polypeptide at least 85% identical to an amino acid
sequence within a target gene product.
[0015] Additionally disclosed are cDNA polynucleotides that may be
used for the production of iRNA (e.g., dsRNA, siRNA, shRNA, miRNA,
and hpRNA) molecules that are complementary to all or part of a
hemipteran pest target gene, for example, a kruppel gene. In
particular embodiments, dsRNAs, siRNAs, shRNAs, miRNAs, and/or
hpRNAs may be produced in vitro, or in vivo by a
genetically-modified organism, such as a plant or bacterium. In
particular examples, cDNA molecules are disclosed that may be used
to produce iRNA molecules that are complementary to all or part of
mRNA transcribed from BSB_kr (SEQ ID NO:1).
[0016] Further disclosed are means for inhibiting expression of an
essential gene in a hemipteran pest, and means for protecting a
plant from a hemipteran pest. A means for inhibiting expression of
an essential gene in a hemipteran pest is a single- or
double-stranded RNA molecule consisting of a polynucleotide
selected from the group consisting of SEQ ID NO:15, SEQ ID NO:16,
and SEQ ID NO:17; and the complements of the foregoing. Functional
equivalents of means for inhibiting expression of an essential gene
in a hemipteran pest include single- or double-stranded RNA
molecules that are substantially homologous to all or part of mRNA
transcribed from a BSB gene comprising SEQ ID NO:1. A means for
protecting a plant from a hemipteran pest is a DNA molecule
comprising a polynucleotide encoding a means for inhibiting
expression of an essential gene in a hemipteran pest operably
linked to a promoter, wherein the DNA molecule is capable of being
integrated into the genome of a maize plant.
[0017] Disclosed are methods for controlling a population of a
hemipteran pest, comprising providing to a hemipteran pest an iRNA
(e.g., dsRNA, siRNA, shRNA, miRNA, and hpRNA) molecule that
functions upon being taken up by the pest to inhibit a biological
function within the pest, wherein the iRNA molecule comprises all
or part of (e.g., at least 15 contiguous nucleotides of) a
polynucleotide selected from the group consisting of: SEQ ID NO:1;
the complement of SEQ ID NO:1; SEQ ID NO:3; the complement of SEQ
ID NO:3; SEQ ID NO:4; the complement of SEQ ID NO:4; a native
coding polynucleotide of a hemipteran organism (e.g., BSB)
comprising all or part of SEQ ID NO:1; the complement of a native
coding polynucleotide of a hemipteran organism comprising all or
part of SEQ ID NO:1; a native non-coding polynucleotide of a
hemipteran organism that is transcribed into a native RNA molecule
comprising all or part of SEQ ID NO:15; and the complement of a
native non-coding polynucleotide of a hemipteran organism that is
transcribed into a native RNA molecule comprising all or part of
SEQ ID NO:15.
[0018] In particular examples, methods are disclosed for
controlling a population of a hemipteran pest, comprising providing
to a hemipteran pest an iRNA (e.g., dsRNA, siRNA, shRNA, miRNA, and
hpRNA) molecule that functions upon being taken up by the pest to
inhibit a biological function within the pest, wherein the iRNA
molecule comprises a polynucleotide selected from the group
consisting of all or part of: SEQ ID NO:15; the complement of SEQ
ID NO:15; SEQ ID NO:16; the complement of SEQ ID NO:16; SEQ ID
NO:17; the complement of SEQ ID NO:17; a polynucleotide that
hybridizes to a native coding polynucleotide of a hemipteran (e.g.,
BSB) organism comprising all or part of SEQ ID NO:1; and the
complement of a polynucleotide that hybridizes to a native coding
polynucleotide of a hemipteran (e.g., BSB) organism comprising all
or part of SEQ ID NO:1.
[0019] Also disclosed herein are methods wherein dsRNAs, siRNAs,
shRNAs, miRNAs, and/or hpRNAs may be provided to a hemipteran pest
in a diet-based assay, or in genetically-modified plant cells
expressing the dsRNAs, siRNAs, shRNAs, miRNAs, and/or hpRNAs. In
these and further examples, the dsRNAs, siRNAs, shRNAs, miRNAs,
and/or hpRNAs may be ingested by a hemipteran pest. Ingestion of
dsRNAs, siRNA, shRNAs, miRNAs, and/or hpRNAs of the invention may
then result in RNAi in the pest, which in turn may result in
silencing of a gene essential for a metabolic process; a
reproductive process; and/or nymph development. Thus, methods are
disclosed wherein nucleic acid molecules comprising exemplary
polynucleotide(s) useful for parental control of hemipteran pests
are provided to a hemipteran pest. In particular examples, the
hemipteran pest controlled by use of nucleic acid molecules of the
invention may be BSB. In some examples, delivery of the nucleic
acid molecules to hemipteran pests does not result in significant
mortality to the pests, but reduces the number of viable progeny
produced therefrom. In some examples, delivery of the nucleic acid
molecules to hemipteran pests results in significant mortality to
the pests, and also reduces the number of viable progeny produced
therefrom.
[0020] The foregoing and other features will become more apparent
from the following Detailed Description of several embodiments,
which proceeds with reference to the accompanying Figures.
BRIEF DESCRIPTION OF THE FIGURES
[0021] FIGS. 1A-1B includes a depiction of the strategy used to
generate dsRNA from a single transcription template with a single
pair of primers (FIG. 1A), and from two transcription templates
(FIG. 1B).
[0022] FIG. 2 includes a depiction of the domain organization of
the Drosophila melanogaster (DME) and Euschistus heros (BSB)
KRUPPEL protein sequences. D. melanogaster and E. heros KRUPPEL
proteins contain four C2H2-type zinc fingers, annotated using SMART
database within InterProScan.
[0023] FIG. 3 includes a summary of modeling data showing the
effect of relative magnitude of a pRNAi effect on female BSB adults
emerging from a "refuge patch" (i.e., that did not express
insecticidal iRNAs or recombinant proteins in a transgenic crop) on
the rate of increase in allele frequencies for resistance to an
insecticidal protein (R) and RNAi (Y) when non-refuge plants
express the insecticidal protein and parental active iRNA.
[0024] FIG. 4 includes a summary of modeling data showing the
effect of relative magnitude of a pRNAi effect on female BSB adults
emerging from a "refuge patch" (i.e., that did not express
insecticidal iRNAs or recombinant proteins in a transgenic crop of
plants comprising BSB nymph-active interfering dsRNA in combination
with the BSB-active insecticidal protein in the transgenic crop) on
the rate of increase in allele frequencies for resistance to an
insecticidal protein (R) and RNAi (Y) when non-refuge plants
express the insecticidal protein and both larval active and
parental active iRNA molecules.
SEQUENCE LISTING
[0025] The nucleic acid sequences listed in the accompanying
sequence listing are shown using standard letter abbreviations for
nucleotide bases, as defined in 37 C.F.R. .sctn. 1.822. The nucleic
acid and amino acid sequences listed define molecules (i.e.,
polynucleotides and polypeptides, respectively) having the
nucleotide and amino acid monomers arranged in the manner
described. The nucleic acid and amino acid sequences listed also
each define a genus of polynucleotides or polypeptides that
comprise the nucleotide and amino acid monomers arranged in the
manner described. In view of the redundancy of the genetic code, it
will be understood that a nucleotide sequence including a coding
sequence also describes the genus of polynucleotides encoding the
same polypeptide as a polynucleotide consisting of the reference
sequence. It will further be understood that an amino acid sequence
describes the genus of polynucleotide ORFs encoding that
polypeptide.
[0026] 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. As the complement and reverse complement
of a primary nucleic acid sequence are necessarily disclosed by the
primary sequence, the complementary sequence and reverse
complementary sequence of a nucleic acid sequence are included by
any reference to the nucleic acid sequence, unless it is explicitly
stated to be otherwise (or it is clear to be otherwise from the
context in which the sequence appears). Furthermore, as it is
understood in the art that the nucleotide sequence of an RNA strand
is determined by the sequence of the DNA from which it was
transcribed (but for the substitution of uracil (U) nucleobases for
thymine (T)), an RNA sequence is included by any reference to the
DNA sequence encoding it. In the accompanying sequence listing:
[0027] SEQ ID NO:1 shows an exemplary E. heros kruppel DNA,
referred to herein in some places as BSB_kr:
TABLE-US-00001 AGGAGCAATAATCTCAATTTCTGAATTTTTTTACTTAAACCACTTTCATT
CTCACCGGCTCAATTATAGTTAAAAATCAGCTGATATATTTTCACTGATA
TAACCCAACCTGATTAACTTAAACACTTAAGTAATAATTACAAGAATTAA
CGGTTAGAGAGTTACTCAAACAAATTTTGTAAAATCTTATCAAACATTGA
TCGAAAAGCCACACAAATATCAACTACTAAATAACATCAACCAAAGTGAT
TGATTATCATAAAATTTTGTTGTTTTATTAAATGGGAAAAAAATAAATTT
TTGAAATCCATAAAAGAGCATAGGGGCATAGAGATGTTAAACAATAAAAA
GCGGAATTTTGCATCTGTCGGTTGGGTAAATTTTACAATATACATTCCAA
TAATAATTATTCCCAGAGATAACCTAATATCAAGTTTCCAGTCACTCTGA
CGTGGAATCACATGTTTTCATTTGAGAAACATACACGCTCAAAAATAGCG
CTTCCATCAATTAAAATATACACAACGAACTTATACTCTAATAATAATAT
ATAATAGAAAACAAAATAAATGAAATGACTATACCGAAACCGGTACATAC
CCGTAGTCGTTTTTACAGTACCTTCAGTAACCCTTACTATATTTCAAAAA
TCATAACTTCAATTATTACATCAATCCAGATCAAGAAATTGTACTCTAAA
ATAAGATATTTTAAATTTATTAATTTATGTACAATAAATCAATTTCGTTT
CGTACCTTATGACGTTAAAGCATTCAAATGGTATAGGGCACTGGCGGTAC
TGTGATAAAAAAATAATAATTTACTAACTAAAGAGGTGAGCCTCCATCCC
ATTCTTCAGAAAGCTCTTCTTCAGATTCAGTCATTGTGAGATTATTATTA
TTGTTGATGTTTTTGTTCTTATGCCTGATAGAAAGATCTTCAGGTTCGGT
CTGCTCAGGCAACGAAAGGTACGCAGGGATAACGTTTACAAGAGCTTGCG
CTTTTCTGTCCCTCGAGTCGCACTTGTGATGCAGGAGGTGGTGTCTCCTT
CTGAATTTGGCACCGCACAATCCGCAGCCGAATGGTTTTTCTCCCTTGTG
AATTAGAGAATGGGCCTTGAGCTGGTTGGAATCAGCGAACCTCGAGGAAC
AGACTTGGCAAGCGTAAGGCCGTTCTCCGGTATGTACCCGTAGGTGTCTC
CTCAAGTTGGCTACTTGCACGAAGTACCTGTCGCAGTGGTCGCAATGGTA
AGGCTTCTCGCCAGTGTGTGTGCGTATGTGGGTGCGGAGATGGTGGTCCC
GTCGAAACCTTCTGTGGCACTCGGGGCACTCGAACGGCCGTTCTCCAGTG
TGGGTTCTTTCGTGATTTTTGAGAACGTGCTTATGCTTGAAGGACTGGTT
ACAAGTGAGGCAGACGAAAGCTCTGTCCCTCCCAGGAGGAGAGTCTTCGT
CCTTCTTCTTCCTCCCTACAGGCTCTGGAGGTGGAGCTGCCCAGAGCAAA
GAGTGCGAGAATAGGGAGCCGCCTCCGATCCCGTGGAGAGCCAAAAGTCC
TGGCATTCCGGCTGCCAGAAGACCGGCCTCTGGACCGTTTTCTTCTCGTT
TGCTTGCTTCCTTCATTGTGCACCTTCCGACGCCTACTACTGAAGTCAAG
CCCTTGGTTGGGTTGTTGGTTGCGTGTATGAGAGACAAGGCCATGGAGGT
GTTATAGAGACGAAGGCATGGCAGGAAC
[0028] SEQ ID NO:2 shows the amino acid sequence of a E. heros
KRUPPEL polypeptide encoded by an exemplary E. heros kruppel
DNA:
TABLE-US-00002 MALSLIHATNNPTKGLTSVVGVGRCTMKEASKREENGPEAGLLAAGMPGL
LALHGIGGGSLFSHSLLWAAPPPEPVGRKKKDEDSPPGRDRAFVCLTCNQ
SFKHKHVLKNHERTHTGERPFECPECHRRFRRDHHLRTHIRTHTGEKPYH
CDHCDRYFVQVANLRRHLRVHTGERPYACQVCSSRFADSNQLKAHSLIHK
GEKPFGCGLCGAKFRRRHHLLHHKCDSRDRKAQALVNVIPAYLSLPEQTE
PEDLSIRHKNKNINNNNNLTMTESEEELSEEWDGGSPL
[0029] SEQ ID NO:3 shows an exemplary E. heros kruppel DNA
(BSB_kr-1), of which the complementary strand is transcribed to
become the sense strand of an exemplary BSB kruppel dsRNA:
TABLE-US-00003 GAGCTTGCGCTTTTCTGTCCCTCGAGTCGCACTTGTGATGCAGGAGGTGG
TGTCTCCTTCTGAATTTGGCACCGCACAATCCGCAGCCGAATGGTTTTTC
CTCCCTTGTGAATTAGAGAATGGGCCTTGAGCTGGTTGGAATCAGCGAAC
CTCGAGGAACAGACTTGGCAAGCGTAAGGCCGTTCTCCGGTATGTACCCG
TAGGTGTCTCCTCAAGTTGGCTACTTGCACGAAGTACCTGTCGCAGTGGT
CGCAATGGTAAGGCTTCTCGCCAGTGTGTGTGCGTATGTGGGTGCGGAGA
TGGTGGTCCCGTCGAAACCTTCTGTGGCACTCGGGGCACTCGAACGGCCG
TTCTCCAGTGTGGGTTCTTTCGTGATTTTTGAGAACGTGCTTATGCTTGA
AGGACTGGTTACAAGTGAGGCAGACGAAAGCTCTG
[0030] SEQ ID NO:4 shows an exemplary E. heros kruppel DNA
(BSB_kr-2), of which the complementary strand is transcribed to
become the sense strand of a further exemplary BSB kruppel
dsRNA:
TABLE-US-00004 AAACATTGATCGAAAAGCCACACAAATATCAACTACTAAATAACATCAAC
CAAAGTGATTGATTATCATAAAATTTTGTTGTTTTATTAAATGGGAAAAA
AATAAATTTTTGAAATCCATAAAAGAGCATAGGGGCATAGAGATGTTAAA
CAATAAAAAGCGGAATTTTGCATCTGTCGGTTGGGTAAATTTTACAATAT
ACATTCCAATAATAATTATTCCCAGAGATAACCTAATATCAAGTTTCCAG
TCACTCTGACGTGGAATCACATGTTTTCATTTGAGAAACATACACGCTCA
AAAATAGCGCTTCCATCAATTAAAATATACACAACGAACTTATACTCTAA
TAATAATATATAATAGAAAACAAAATAAATGAAATGACTATACCGAAACC
GGTACATACCCGTAGTCGTTTTTACAGTACCTTCAGTAACCCTTACTATA
TTTCAAAAATCATAACTTCAATTATTACATCAATCCAGATCAAGAAATTG
TACTCTAAAATAAGATATTTTAAATTTATTAATTTATGTACAATAAATCA
ATTTCGTTTCGTACCTTATGACGTTAAAGCATTCAAATGGTATAGGGCAC TGGCG
[0031] SEQ ID NO:5 shows the nucleotide sequence of a T7 phage
promoter.
[0032] SEQ ID NO:6 presents a YFP hairpin-RNA-forming sequence v2
as found in a plant expression vector. Upper case bases are YFP
sense strand, underlined bases comprise an ST-LS1 intron, lower
case, and non-underlined bases are YFP antisense strand.
TABLE-US-00005 ATGTCATCTGGAGCACTTCTCTTTCATGGGAAGATTCCTTACGTTGTGGA
GATGGAAGGGAATGTTGATGGCCACACCTTTAGCATACGTGGGAAAGGCT
ACGGAGATGCCTCAGTGGGAAAGgactagtaccggttgggaaaggtatgt
ttctgcttctacctttgatatatatataataattatcactaattagtagt
aatatagtatttcaagtatttttttcaaaataaaagaatgtagtatatag
ctattgcttttctgtagtttataagtgtgtatattttaatttataacttt
tctaatatatgaccaaaacatggtgatgtgcaggttgatccgcggttact
ttcccactgaggcatctccgtagcctttcccacgtatgctaaaggtgtgg
ccatcaacattcccttccatctccacaacgtaaggaatcttcccatgaaa
gagaagtgctccagatgacat
[0033] SEQ ID NO:7 shows an exemplary DNA comprising an ST-LS1
intron.
[0034] SEQ ID NOs:8-11 show primers used for PCR amplification of a
BSB_kr-1 and BSB_kr-2 polynucleotide.
[0035] SEQ ID NO:12 shows the DNA template for the sense strand of
an exemplary YFP dsRNA (YFPv2):
TABLE-US-00006 CATCTGGAGCACTTCTCTTTCATGGGAAGATTCCTTACGTTGTGGAGAT
GGAAGGGAATGTTGATGGCCACACCTTTAGCATACGTGGGAAAGGCTAC
GGAGATGCCTCAGTGGGAAAGGTTGATGCACAGTTCATCTGCACAACTG
GTGATGTTCCTGTGCCTTGGAGCACACTTGTCACCACTCTCACCTATGG
AGCACAGTGCTTTGCCAAGTATGGTCCAGAGTTGAAGGACTTCTACAAG
TCCTGTATGCCAGATGGCTATGTGCAAGAGCGCACAATCACCTTTGAAG GAGATGG
[0036] SEQ ID NOs:13 and 14 show primers used for PCR amplification
of a YFP gene.
[0037] SEQ ID NOs:15-17 show exemplary RNAs transcribed from
nucleic acids comprising exemplary kruppel polynucleotides and
fragments thereof.
[0038] SEQ ID NO:18 shows Actin-ORF.
TABLE-US-00007 TACAAAATGTGTGACGAAGAAGTTGCTGCTTTAGTTGTAGACAATGGAT
CTGGTATGTGCAAAGCCGGTTTCGCTGGAGATGATGCACCCCGAGCTGT
ATTCCCATCAATTGTTGGCAGGCCTAGACACCAGGGTGTCATGGTTGGA
ATGGGACAAAAGGACAGTTATGTTGGAGACGAAGCCCAAAGCAAGAGAG
GTATCCTCACCCTGAAATACCCCATTGAACACGGTATCATCACCAACTG
GGACGACATGGAAAAGATCTGGCATCACACCTTCTACAACGAGCTGCGA
GTCGCTCCAGAGGAACACCCCATCCTCCTGACTGAGGCTCCCCTCAACC
CCAAAGCCAACAGGGAGAAGATGACCCAGATCATGTTTGAGACCTTCAA
CACCCCAGCCATGTATGTCGCCATCCAGGCTGTACTCTCCCTCTATGCC
TCCGGTCGTACTACCGGTATTGTACTTGACTCAGGAGATGGTGTCTCCC
ACACCGTACCCATCTATGAAGGTTATGCCCTTCCCCACGCCATCCTCCG
TCTGGATCTTGCTGGACGTGACTTGACTGACTATCTTATGAAGATCCTC
ACCGAGCGTGGTTACAGCTTCACCACCACCGCTGAAAGGGAAATCGTCA
GGGACATCAAGGAAAAACTGTGCTATGTCGCCCTGGACTTTGAGCAGGA
AATGGCCACCGCCGCTGCCTCCACCTCCCTGGAGAAGTCCTATGAACTT
CCCGACGGTCAGGTCATCACCATCGGTAACGAGAGGTTCCGTTGCCCAG
AGGCTCTCTTCCAGCCTTCCTTCTTGGGTATGGAATCTTGCGGTATCCA
TGAGACTGTCTACAACTCCATCATGAAGTGCGACGTTGACATCAGGAAG
GACTTGTACGCCAACACCGTCCTCTCCGGAGGTACCACCATGTACCCAG
GTATTGCTGACAGGATGCAGAAGGAAATCACCGCCCTCGCTCCTTCAAC
CATCAAGATCAAGATCATTGCTCCCCCAGAAAGGAAGTACTCCGTATGG
ATCGGTGGTTCCATCTTGGCTTCCCTGTCCACCTTCCAGCAGATGTGGA
TCTCCAAGCAGGAATACGACGAATCCGGCCCAGGCATCGTCCACCGCAA ATGCTTC
[0039] SEQ ID NOs:19-21 show primers and probes used for actin
qPCR.
DETAILED DESCRIPTION
I. Overview of Several Embodiments
[0040] We developed RNA interference (RNAi) as a tool for insect
pest management, using a target pest species for transgenic plants
that express dsRNA; the Neotropical brown stink bug. Thus far, most
genes proposed as targets for RNAi in particular insects do not
achieve their purpose, and those useful targets that have been
identified involve typically those that cause lethality in the
nymph stage. Herein, we describe RNAi-mediated knockdown of kruppel
(kr) in the Neotropical brown stink bug, which is shown to disrupt
embryonic development when, for example, iRNA molecules are
delivered via kruppel dsRNA provided to adult females. However,
there was almost complete absence of hatching in the eggs collected
from females exposed to kruppel dsRNA. In embodiments herein, the
ability to deliver kruppel dsRNA by injection to adult insects
confers a pRNAi effect that is very useful for insect (e.g.,
hemipteran) pest management. Furthermore, the potential to affect
multiple target sequences in both nymph and adult hemipteran pests
may increase opportunities to develop sustainable approaches to
insect pest management involving RNAi technologies.
[0041] Disclosed herein are methods and compositions for genetic
control of hemipteran pest infestations. Methods for identifying
one or more gene(s) essential to the lifecycle of a hemipteran pest
(e.g., gene(s) essential for normal reproductive capacity and/or
embryonic and/or nymph development) for use as a target gene for
RNAi-mediated control of a hemipteran pest population are also
provided. DNA plasmid vectors encoding an RNA molecule may be
designed to suppress one or more target gene(s) essential for
growth, survival, development, and/or reproduction. In some
embodiments, the RNA molecule may be capable of forming dsRNA
molecules. In some embodiments, methods are provided for
post-transcriptional repression of expression or inhibition of a
target gene via nucleic acid molecules that are complementary to a
coding or non-coding sequence of the target gene in a hemipteran
pest. In these and further embodiments, a hemipteran pest may
ingest one or more dsRNA, siRNA, shRNA, miRNA, and/or hpRNA
molecules transcribed from all or a portion of a nucleic acid
molecule that is complementary to a coding or non-coding sequence
of a target gene, thereby providing a plant-protective effect.
[0042] Other embodiments involve sequence-specific inhibition of
expression of target gene products, using dsRNA, siRNA, shRNA,
miRNA and/or hpRNA that is complementary to coding and/or
non-coding sequences of the target gene(s) to achieve at least
partial control of a hemipteran pest. Disclosed is a set of
isolated and purified nucleic acid molecules comprising a
polynucleotide, for example, as set forth in SEQ ID NO:1, and
fragments thereof. In some embodiments, a stabilized dsRNA molecule
may be expressed from these polynucleotides, fragments thereof, or
a gene comprising one of these polynucleotides, for the
post-transcriptional silencing or inhibition of a target gene. In
certain embodiments, isolated and purified nucleic acid molecules
comprise all or part of any of SEQ ID NOs:1, 3, and 4.
[0043] Some embodiments involve a recombinant host cell (e.g., a
plant cell) having in its genome at least one recombinant DNA
encoding at least one iRNA (e.g., dsRNA) molecule(s). In particular
embodiments, the dsRNA molecule(s) may be produced when ingested by
a hemipteran pest to post-transcriptionally silence or inhibit the
expression of a target gene in the pest or progeny of the pest. The
recombinant DNA may comprise, for example, SEQ ID NO:1, fragments
of SEQ ID NO:1, and a polynucleotide consisting of a partial
sequence of a gene comprising SEQ ID NO:1, and/or complements
thereof.
[0044] Alternative embodiments involve a recombinant host cell
having in its genome a recombinant DNA encoding at least one iRNA
(e.g., dsRNA) molecule(s) comprising all or part of SEQ ID NO:15
(e.g., SEQ ID NOs:15-17). When ingested by a hemipteran pest, the
iRNA molecule(s) may silence or inhibit the expression of a target
kruppel gene (e.g., a DNA comprising all or part of the
polynucleotide of SEQ ID NO:1) in the pest or progeny of the pest,
and thereby result in cessation of reproduction in the pest, and/or
growth, development, and/or feeding in progeny of the pest.
[0045] In some embodiments, a recombinant host cell having in its
genome at least one recombinant DNA encoding at least one RNA
molecule capable of forming a dsRNA molecule may be a transformed
plant cell. Some embodiments involve transgenic plants comprising
such a transformed plant cell. In addition to such transgenic
plants, progeny plants of any transgenic plant generation,
transgenic seeds, and transgenic plant products, are all provided,
each of which comprises recombinant DNA(s). In particular
embodiments, an RNA molecule capable of forming a dsRNA molecule
may be expressed in a transgenic plant cell. Therefore, in these
and other embodiments, a dsRNA molecule may be isolated from a
transgenic plant cell. In particular embodiments, the transgenic
plant is a plant selected from the group comprising corn (Zea
mays), soybean (Glycine max), cotton (Gossypium spp.), and plants
of the family Poaceae.
[0046] Other embodiments involve a method for modulating the
expression of a target gene in a hemipteran pest cell. In these and
other embodiments, a nucleic acid molecule may be provided, wherein
the nucleic acid molecule comprises a polynucleotide encoding an
RNA molecule capable of forming a dsRNA molecule. In particular
embodiments, a polynucleotide encoding an RNA molecule capable of
forming a dsRNA molecule may be operatively linked to a promoter,
and may also be operatively linked to a transcription termination
sequence. In particular embodiments, a method for modulating the
expression of a target gene in a hemipteran pest cell may comprise:
(a) transforming a plant cell with a vector comprising a
polynucleotide encoding an RNA molecule capable of forming a dsRNA
molecule; (b) culturing the transformed plant cell under conditions
sufficient to allow for development of a plant cell culture
comprising a plurality of transformed plant cells; (c) selecting
for a transformed plant cell that has integrated the vector into
its genome; and (d) determining that the selected transformed plant
cell comprises the RNA molecule capable of forming a dsRNA molecule
encoded by the polynucleotide of the vector. A plant may be
regenerated from a plant cell that has the vector integrated in its
genome and comprises the dsRNA molecule encoded by the
polynucleotide of the vector.
[0047] Thus, also disclosed is a transgenic plant comprising a
vector having a polynucleotide encoding an RNA molecule capable of
forming a dsRNA molecule integrated in its genome, wherein the
transgenic plant comprises the dsRNA molecule encoded by the
polynucleotide of the vector. In particular embodiments, expression
of an RNA molecule capable of forming a dsRNA molecule in the plant
is sufficient to modulate the expression of a target gene in a cell
of a hemipteran pest that contacts the transformed plant or plant
cell (for example, by feeding on the transformed plant, a part of
the plant (e.g., leaves or plant cell) or in a cell of a progeny of
the hemipteran pest that contacts the transformed plant or plant
cell (for example, by parental transmission), such that
reproduction of the pest is inhibited. Transgenic plants disclosed
herein may display tolerance and/or protection from hemipteran pest
infestations. Particular transgenic plants may display protection
and/or enhanced protection from one or more pest(s) selected from
the group consisting of: Piezodorus guildinii; Halyomorpha halys;
Nezara viridula; Acrosternum hilare; Euschistus heros; Euschistus
servus, Chinavia hilare; C. marginatum; Dichelops melacanthus; D.
furcatus; Edessa meditabunda; Thyanta perditor; Horcias nobilellus;
Taedia stigmosa; Dysdercus peruvianus; Neomegalotomus parvus;
Leptoglossus zonatus; Niesthrea sidae; Lygus hesperus; and L.
lineolaris.
[0048] Also disclosed herein are methods for delivery of control
agents, such as an iRNA molecule, to a hemipteran pest. Such
control agents may cause, directly or indirectly, an impairment in
the ability of a hemipteran pest population to feed, grow or
otherwise cause damage in a host. In some embodiments, a method is
provided comprising delivery of a stabilized dsRNA molecule to a
hemipteran pest to suppress at least one target gene in the pest or
its progeny, thereby causing parental RNAi and reducing or
eliminating plant damage in a pest host. In some embodiments, a
method of inhibiting expression of a target gene in a hemipteran
pest may result in cessation of reproduction in the pest, and/or
growth, development, and/or feeding in progeny of the pest. In some
embodiments, the method may significantly reduce the size of a
subsequent pest generation in an infestation, without directly
resulting in mortality in the pest(s) that contact the iRNA
molecule. In some embodiments, the method may significantly reduce
the size of a subsequent pest generation in an infestation, while
also resulting in mortality in the pest(s) that contact the iRNA
molecule.
[0049] In some embodiments, compositions (e.g., a topical
composition) are provided that comprise an iRNA (e.g., dsRNA)
molecule for use with plants, animals, and/or the environment of a
plant or animal to achieve the elimination or reduction of a
hemipteran pest infestation. In particular embodiments, the
composition may be a nutritional composition or resource, or food
source to be fed to the hemipteran pest. Some embodiments comprise
making the nutritional composition or food source available to the
pest. Ingestion of a composition comprising iRNA molecules may
result in the uptake of the molecules by one or more cells of the
hemipteran pest, which may in turn result in the inhibition of
expression of at least one target gene in cell(s) of the pest or
its progeny. Ingestion of or damage to a plant or plant cell by a
hemipteran pest infestation may be limited or eliminated in or on
any host tissue or environment in which the pest is present by
providing one or more compositions comprising an iRNA molecule in
the host of the pest.
[0050] The compositions and methods disclosed herein may be used
together in combinations with other methods and compositions for
controlling damage by hemipteran pests. For example, an iRNA
molecule as described herein for protecting plants from hemipteran
pests may be used in a method comprising the additional use of one
or more chemical agents effective against a hemipteran pest,
biopesticides effective against a hemipteran pest, crop rotation,
recombinant genetic techniques that exhibit features different from
the features of RNAi-mediated methods and RNAi compositions (e.g.,
recombinant production of proteins in plants that are harmful to a
hemipteran pest (e.g., Bt toxins)), and/or recombinant expression
of non-parental iRNA molecules (e.g., lethal iRNA molecules that
result in the cessation of growth, development, feeding and/or
cause mortality in the hemipteran pest that ingests the iRNA
molecule).
II. Abbreviations
[0051] BSB Neotropical brown stink bug (Euschistus heros)
[0052] dsRNA double-stranded ribonucleic acid
[0053] GI growth inhibition
[0054] NCBI National Center for Biotechnology Information
[0055] gDNA genomic Deoxyribonucleic Acid
[0056] iRNA inhibitory ribonucleic acid
[0057] ORF open reading frame
[0058] RNAi ribonucleic acid interference
[0059] miRNA micro ribonucleic acid
[0060] siRNA small inhibitory ribonucleic acid
[0061] hpRNA hairpin ribonucleic acid
[0062] shRNA short hairpin ribonucleic acid
[0063] pRNAi parental RNA interference
[0064] UTR untranslated region
[0065] PCR Polymerase chain reaction
[0066] qPCR quantative polymerase chain reaction
[0067] RISC RNA-induced Silencing Complex
[0068] RH relative humidity
[0069] SEM standard error of the mean
[0070] YFP yellow fluorescent protein
III. Terms
[0071] In the description and tables which follow, a number of
terms are used. In order to provide a clear and consistent
understanding of the specification and claims, including the scope
to be given such terms, the following definitions are provided:
[0072] Contact (with an organism): As used herein, the term
"contact with" or "uptake by" an organism (e.g., a hemipteran
pest), with regard to a nucleic acid molecule, includes
internalization of the nucleic acid molecule into the organism, for
example and without limitation: ingestion of the molecule by the
organism (e.g., by feeding); contacting the organism with a
composition comprising the nucleic acid molecule; injection of the
organism with a composition comprising the nucleic acid molecule;
and soaking of organisms with a solution comprising the nucleic
acid molecule.
[0073] Contig: As used herein the term "contig" refers to a DNA
sequence that is reconstructed from a set of overlapping DNA
segments derived from a single genetic source.
[0074] Corn plant: As used herein, the term "corn plant" refers to
a plant of the species, Zea mays (maize).
[0075] Expression: As used herein, "expression" of a coding
polynucleotide (for example, a gene or a transgene) refers to the
process by which the coded information of a nucleic acid
transcriptional unit (including, e.g., gDNA or cDNA) is converted
into an operational, non-operational, or structural part of a cell,
often including the synthesis of a protein. Gene expression can be
influenced by external signals; for example, exposure of a cell,
tissue, or organism to an agent that increases or decreases gene
expression. Expression of a gene can also be regulated anywhere in
the pathway from DNA to RNA to protein. Regulation of gene
expression occurs, for example, through controls acting on
transcription, translation, RNA transport and processing,
degradation of intermediary molecules such as mRNA, or through
activation, inactivation, compartmentalization, or degradation of
specific protein molecules after they have been made, or by
combinations thereof. Gene expression can be measured at the RNA
level or the protein level by any method known in the art,
including, without limitation, northern blot, RT-PCR, western blot,
or in vitro, in situ, or in vivo protein activity assay(s).
[0076] Genetic material: As used herein, the term "genetic
material" includes all genes, and nucleic acid molecules, such as
DNA and RNA.
[0077] Hemipteran pest: As used herein, the term "hemipteran pest"
refers to pest insects of the order Hemiptera, including, for
example and without limitation, insects in the families
Pentatomidae, Miridae, Pyrrhocoridae, Coreidae, Alydidae, and
Rhopalidae, which feed on a wide range of host plants and have
piercing and sucking mouth parts. In particular examples, a
hemipteran pest is selected from the list comprising Euschistus
heros (Fabr.) (Neotropical Brown Stink Bug), Nezara viridula (L.)
(Southern Green Stink Bug), Piezodorus guildinii (Westwood)
(Red-banded Stink Bug), Halyomorpha halys (Stak) (Brown Marmorated
Stink Bug), Chinavia hilare (Say) (Green Stink Bug), Euschistus
servus (Say) (Brown Stink Bug), Dichelops melacanthus (Dallas),
Dichelops furcatus (F.), Edessa meditabunda (F.), Thyanta perditor
(F.) (Neotropical Red Shouldered Stink Bug), Chinavia marginatum
(Palisot de Beauvois), Horcias nobilellus (Berg) (Cotton Bug),
Taedia stigmosa (Berg), Dysdercus peruvianus (Guerin-Meneville),
Neomegalotomus parvus (Westwood), Leptoglossus zonatus (Dallas),
Niesthrea sidae (F.), Lygus hesperus (Knight) (Western Tarnished
Plant Bug), and Lygus lineolaris (Palisot de Beauvois).
[0078] Inhibition: As used herein, the term "inhibition," when used
to describe an effect on a coding polynucleotide (for example, a
gene), refers to a measurable decrease in the cellular level of
mRNA transcribed from the coding polynucleotide and/or peptide,
polypeptide, or protein product of the coding polynucleotide. In
some examples, expression of a coding polynucleotide may be
inhibited such that expression is approximately eliminated.
"Specific inhibition" refers to the inhibition of a target coding
polynucleotide without consequently affecting expression of other
coding polynucleotides (e.g., genes) in the cell wherein the
specific inhibition is being accomplished.
[0079] Isolated: An "isolated" biological component (such as a
nucleic acid or protein) has been substantially separated, produced
apart from, or purified away from other biological components in
the cell of the organism in which the component naturally occurs
(i.e., other chromosomal and extra-chromosomal DNA and RNA, and
proteins), while effecting a chemical or functional change in the
component (e.g., a nucleic acid may be isolated from a chromosome
by breaking chemical bonds connecting the nucleic acid to the
remaining DNA in the chromosome). Nucleic acid molecules and
proteins that have been "isolated" include nucleic acid molecules
and proteins purified by standard purification methods. The term
also embraces nucleic acids and proteins prepared by recombinant
expression in a host cell, as well as chemically-synthesized
nucleic acid molecules, proteins, and peptides.
[0080] Nucleic acid molecule: As used herein, the term "nucleic
acid molecule" may refer to a polymeric form of nucleotides, which
may include both sense and anti-sense strands of RNA, cDNA, gDNA,
and synthetic forms and mixed polymers of the above. A nucleotide
or nucleobase may refer to a ribonucleotide, deoxyribonucleotide,
or a modified form of either type of nucleotide. A "nucleic acid
molecule" as used herein is synonymous with "nucleic acid" and
"polynucleotide." A nucleic acid molecule is usually at least 10
bases in length, unless otherwise specified. By convention, the
nucleotide sequence of a nucleic acid molecule is read from the 5'
to the 3' end of the molecule. The "complement" of a nucleic acid
molecule refers to a polynucleotide having nucleobases that may
form base pairs with the nucleobases of the nucleic acid molecule
(i.e., A-T/U, and G-C).
[0081] Some embodiments include nucleic acids comprising a template
DNA that is transcribed into an RNA molecule that is the complement
of an mRNA molecule. In these embodiments, the complement of the
nucleic acid transcribed into the mRNA molecule is present in the
5' to 3' orientation, such that RNA polymerase (which transcribes
DNA in the 5' to 3' direction) will transcribe a nucleic acid from
the complement that can hybridize to the mRNA molecule. Unless
explicitly stated otherwise, or it is clear to be otherwise from
the context, the term "complement" therefore refers to a
polynucleotide having nucleobases, from 5' to 3', that may form
base pairs with the nucleobases of a reference nucleic acid.
Similarly, unless it is explicitly stated to be otherwise (or it is
clear to be otherwise from the context), the "reverse complement"
of a nucleic acid refers to the complement in reverse orientation.
The foregoing is demonstrated in the following illustration:
TABLE-US-00008 ATGATGATG polynucleotide TACTACTAC "complement" of
the polynucleotide CATCATCAT "reverse complement" of the
polynucleotide
Some embodiments of the invention may include hairpin RNA-forming
RNAi molecules. In these RNAi molecules, both the complement of a
nucleic acid to be targeted by RNA interference and the reverse
complement may be found in the same molecule, such that the
single-stranded RNA molecule may "fold over" and hybridize to
itself over region comprising the complementary and reverse
complementary polynucleotides.
[0082] "Nucleic acid molecules" include all polynucleotides, for
example: single- and double-stranded forms of DNA; single-stranded
forms of RNA; and double-stranded forms of RNA (dsRNA). The term
"nucleotide sequence" or "nucleic acid sequence" refers to both the
sense and antisense strands of a nucleic acid as either individual
single strands or in the duplex. The term "ribonucleic acid" (RNA)
is inclusive of iRNA (inhibitory RNA), dsRNA (double stranded RNA),
siRNA (small interfering RNA), shRNA (small hairpin RNA), mRNA
(messenger RNA), miRNA (micro-RNA), hpRNA (hairpin RNA), tRNA
(transfer RNAs, whether charged or discharged with a corresponding
acylated amino acid), and cRNA (complementary RNA). The term
"deoxyribonucleic acid" (DNA) is inclusive of cDNA, gDNA, and
DNA-RNA hybrids. The terms "polynucleotide" and "nucleic acid," and
"fragments" thereof will be understood by those in the art as a
term that includes both gDNAs, ribosomal RNAs, transfer RNAs,
messenger RNAs, operons, and smaller engineered polynucleotides
that encode or may be adapted to encode, peptides, polypeptides, or
proteins.
[0083] Oligonucleotide: An oligonucleotide is a short nucleic acid
polymer. Oligonucleotides may be formed by cleavage of longer
nucleic acid segments, or by polymerizing individual nucleotide
precursors. Automated synthesizers allow the synthesis of
oligonucleotides up to several hundred bases in length. Because
oligonucleotides may bind to a complementary nucleic acid, they may
be used as probes for detecting DNA or RNA. Oligonucleotides
composed of DNA (oligodeoxyribonucleotides) may be used in PCR, a
technique for the amplification of DNAs. In PCR, the
oligonucleotide is typically referred to as a "primer," which
allows a DNA polymerase to extend the oligonucleotide and replicate
the complementary strand.
[0084] A nucleic acid molecule may include either or both naturally
occurring and modified nucleotides linked together by naturally
occurring and/or non-naturally occurring nucleotide linkages.
Nucleic acid molecules may be modified chemically or biochemically,
or may contain non-natural or derivatized nucleotide bases, as will
be readily appreciated by those of skill in the art. Such
modifications include, for example, labels, methylation,
substitution of one or more of the naturally occurring nucleotides
with an analog, internucleotide modifications (e.g., uncharged
linkages: for example, methyl phosphonates, phosphotriesters,
phosphoramidates, carbamates, etc.; charged linkages: for example,
phosphorothioates, phosphorodithioates, etc.; pendent moieties: for
example, peptides; intercalators: for example, acridine, psoralen,
etc.; chelators; alkylators; and modified linkages: for example,
alpha anomeric nucleic acids, etc.). The term "nucleic acid
molecule" also includes any topological conformation, including
single-stranded, double-stranded, partially duplexed, triplexed,
hairpinned, circular, and padlocked conformations.
[0085] As used herein with respect to DNA, the term "coding
polynucleotide," "structural polynucleotide," or "structural
nucleic acid molecule" refers to a polynucleotide that is
ultimately translated into a polypeptide, via transcription and
mRNA, when placed under the control of appropriate regulatory
elements. With respect to RNA, the term "coding polynucleotide"
refers to a polynucleotide that is translated into a peptide,
polypeptide, or protein. The boundaries of a coding polynucleotide
are determined by a translation start codon at the 5'-terminus and
a translation stop codon at the 3'-terminus. Coding polynucleotides
include, but are not limited to: gDNA; cDNA; EST; and recombinant
polynucleotides.
[0086] As used herein, "transcribed non-coding polynucleotide"
refers to segments of mRNA molecules such as 5'UTR, 3'UTR and
intron segments that are not translated into a peptide,
polypeptide, or protein. Further, "transcribed non-coding
polynucleotide" refers to a nucleic acid that is transcribed into
an RNA that functions in the cell, for example, structural RNAs
(e.g., ribosomal RNA (rRNA) as exemplified by 5S rRNA, 5.8S rRNA,
16S rRNA, 18 S rRNA, 23 S rRNA, and 28S rRNA, and the like);
transfer RNA (tRNA); and snRNAs such as U4, U5, U6, and the like.
Transcribed non-coding polynucleotides also include, for example
and without limitation, small RNAs (sRNA), which term is often used
to describe small bacterial non-coding RNAs; small nucleolar RNAs
(snoRNA); microRNAs; small interfering RNAs (siRNA);
Piwi-interacting RNAs (piRNA); and long non-coding RNAs. Further
still, "transcribed non-coding polynucleotide" refers to a
polynucleotide that may natively exist as an intragenic "spacer" in
a nucleic acid and which is transcribed into an RNA molecule.
[0087] Lethal RNA interference: As used herein, the term "lethal
RNA interference" refers to RNA interference that results in death
or a reduction in viability of the subject individual to which, for
example, a dsRNA, miRNA, siRNA, shRNA, and/or hpRNA is
delivered.
[0088] Parental RNA interference: As used herein, the term
"parental RNA interference" (pRNAi) refers to a RNA interference
phenotype that is observable in progeny of the subject (e.g., a
hemipteran pest) to which, for example, a dsRNA, miRNA, siRNA,
shRNA, and/or hpRNA is delivered. In some embodiments, pRNAi
comprises the delivery of a dsRNA to a hemipteran pest, wherein the
pest is thereby rendered less able to produce viable offspring. A
nucleic acid that initiates pRNAi may or may not increase the
incidence of mortality in a population into which the nucleic acid
is delivered. In certain examples, the nucleic acid that initiates
pRNAi does not increase the incidence of mortality in the
population into which the nucleic acid is delivered. For example, a
population of hemipteran pests may be fed one or more nucleic acids
that initiate pRNAi, wherein the pests survive and mate but produce
eggs that are less able to hatch viable progeny than eggs produced
by pests of the same species that are not fed the nucleic acid(s).
In one mechanism of pRNAi, parental RNAi delivered to a female is
able to knockdown zygotic gene expression in offspring embryos of
the female. Bucher et al. (2002) Curr. Biol. 12(3):R85-6.
[0089] Genome: As used herein, the term "genome" refers to
chromosomal DNA found within the nucleus of a cell, and also refers
to organelle DNA found within subcellular components of the cell.
In some embodiments of the invention, a DNA molecule may be
introduced into a plant cell, such that the DNA molecule is
integrated into the genome of the plant cell. In these and further
embodiments, the DNA molecule may be either integrated into the
nuclear DNA of the plant cell, or integrated into the DNA of the
chloroplast or mitochondrion of the plant cell. The term "genome,"
as it applies to bacteria, refers to both the chromosome and
plasmids within the bacterial cell. In some embodiments of the
invention, a DNA molecule may be introduced into a bacterium such
that the DNA molecule is integrated into the genome of the
bacterium. In these and further embodiments, the DNA molecule may
be either chromosomally-integrated or located as or in a stable
plasmid.
[0090] Sequence identity: The term "sequence identity" or
"identity," as used herein in the context of two polynucleotides or
polypeptides, refers to the residues in the sequences of the two
molecules that are the same when aligned for maximum correspondence
over a specified comparison window.
[0091] As used herein, the term "percentage of sequence identity"
may refer to the value determined by comparing two optimally
aligned sequences (e.g., nucleic acid sequences or polypeptide
sequences) of a molecule over a comparison window, wherein the
portion of the sequence in the comparison window may comprise
additions or deletions (i.e., gaps) as compared to the reference
sequence (which does not comprise additions or deletions) for
optimal alignment of the two sequences. The percentage is
calculated by determining the number of positions at which the
identical nucleotide or amino acid residue occurs in both sequences
to yield the number of matched positions, dividing the number of
matched positions by the total number of positions in the
comparison window, and multiplying the result by 100 to yield the
percentage of sequence identity. A sequence that is identical at
every position in comparison to a reference sequence is said to be
100% identical to the reference sequence, and vice-versa.
[0092] Methods for aligning sequences for comparison are well-known
in the art. Various programs and alignment algorithms are described
in, for example: Smith and Waterman (1981) Adv. Appl. Math. 2:482;
Needleman and Wunsch (1970) J. Mol. Biol. 48:443; Pearson and
Lipman (1988) Proc. Natl. Acad. Sci. U.S.A. 85:2444; Higgins and
Sharp (1988) Gene 73:237-44; Higgins and Sharp (1989) CABIOS
5:151-3; Corpet et al. (1988) Nucleic Acids Res. 16:10881-90; Huang
et al. (1992) Comp. Appl. Biosci. 8:155-65; Pearson et al. (1994)
Methods Mol. Biol. 24:307-31; Tatiana et al. (1999) FEMS Microbiol.
Lett. 174:247-50. A detailed consideration of sequence alignment
methods and homology calculations can be found in, e.g., Altschul
et al. (1990) J. Mol. Biol. 215:403-10.
[0093] The National Center for Biotechnology Information (NCBI)
Basic Local Alignment Search Tool (BLAST.TM.; Altschul et al.
(1990)) is available from several sources, including the National
Center for Biotechnology Information (Bethesda, Md.), and on the
internet, for use in connection with several sequence analysis
programs. A description of how to determine sequence identity using
this program is available on the internet under the "help" section
for BLAST.TM.. For comparisons of nucleic acid sequences, the
"Blast 2 sequences" function of the BLAST.TM. (Blastn) program may
be employed using the default BLOSUM62 matrix set to default
parameters. Nucleic acids with even greater sequence similarity to
the sequences of the reference polynucleotides will show increasing
percentage identity when assessed by this method.
[0094] Specifically hybridizable/Specifically complementary: As
used herein, the terms "Specifically hybridizable" and
"Specifically complementary" are terms that indicate a sufficient
degree of complementarity such that stable and specific binding
occurs between the nucleic acid molecule and a target nucleic acid
molecule. Hybridization between two nucleic acid molecules involves
the formation of an anti-parallel alignment between the nucleobases
of the two nucleic acid molecules. The two molecules are then able
to form hydrogen bonds with corresponding bases on the opposite
strand to form a duplex molecule that, if it is sufficiently
stable, is detectable using methods well known in the art. A
polynucleotide need not be 100% complementary to its target nucleic
acid to be specifically hybridizable. However, the amount of
complementarity that must exist for hybridization to be specific is
a function of the hybridization conditions used.
[0095] 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 acids. Generally, the temperature of hybridization and the
ionic strength (especially the Na.sup.+ and/or Mg.sup.++
concentration) of the hybridization buffer will determine the
stringency of hybridization, though wash times also influence
stringency. Calculations regarding hybridization conditions
required for attaining particular degrees of stringency are known
to those of ordinary skill in the art, and are discussed, for
example, in Sambrook et al. (ed.) Molecular Cloning: A Laboratory
Manual, 2' ed., vol. 1-3, Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, N.Y., 1989, chapters 9 and 11; and Hames and Higgins
(eds.) Nucleic Acid Hybridization, IRL Press, Oxford, 1985. Further
detailed instruction and guidance with regard to the hybridization
of nucleic acids may be found, for example, in Tijssen, "Overview
of principles of hybridization and the strategy of nucleic acid
probe assays," in Laboratory Techniques in Biochemistry and
Molecular Biology-Hybridization with Nucleic Acid Probes, Part I,
Chapter 2, Elsevier, N Y, 1993; and Ausubel et al., Eds., Current
Protocols in Molecular Biology, Chapter 2, Greene Publishing and
Wiley-Interscience, N Y, 1995.
[0096] As used herein, "stringent conditions" encompass conditions
under which hybridization will only occur if there is less than 20%
mismatch between the sequence of the hybridization molecule and a
homologous polynucleotide within the target nucleic acid molecule.
"Stringent conditions" include further particular levels of
stringency. Thus, as used herein, "moderate stringency" conditions
are those under which molecules with more than 20% sequence
mismatch will not hybridize; conditions of "high stringency" are
those under which sequences with more than 10% mismatch will not
hybridize; and conditions of "very high stringency" are those under
which sequences with more than 5% mismatch will not hybridize.
[0097] The following are representative, non-limiting hybridization
conditions.
[0098] High Stringency condition (detects polynucleotides that
share at least 90% sequence identity): Hybridization in 5.times.SSC
buffer at 65.degree. C. for 16 hours; wash twice in 2.times.SSC
buffer at room temperature for 15 minutes each; and wash twice in
0.5.times.SSC buffer at 65.degree. C. for 20 minutes each.
[0099] Moderate Stringency condition (detects polynucleotides that
share at least 80% sequence identity): Hybridization in
5.times.-6.times.SSC buffer at 65-70.degree. C. for 16-20 hours;
wash twice in 2.times.SSC buffer at room temperature for 5-20
minutes each; and wash twice in 1.times.SSC buffer at 55-70.degree.
C. for 30 minutes each.
[0100] Non-stringent control condition (polynucleotides that share
at least 50% sequence identity will hybridize): Hybridization in
6.times.SSC buffer at room temperature to 55.degree. C. for 16-20
hours; wash at least twice in 2.times.-3.times.SSC buffer at room
temperature to 55.degree. C. for 20-30 minutes each.
[0101] As used herein, the term "substantially homologous" or
"substantial homology," with regard to a nucleic acid, refers to a
polynucleotide having contiguous nucleobases that hybridize under
stringent conditions to the reference nucleic acid. For example,
nucleic acids that are substantially homologous to a reference
nucleic acid of any of SEQ ID NOs:1, 3, and 4 are those nucleic
acids that hybridize under stringent conditions (e.g., the Moderate
Stringency conditions set forth, supra) to the reference nucleic
acid of any of SEQ ID NOs:1, 3, and 4. Substantially homologous
polynucleotides may have at least 80% sequence identity. For
example, substantially homologous polynucleotides may have from
about 80% to 100% sequence identity, such as 79%; 80%; about 81%;
about 82%; about 83%; about 84%; about 85%; about 86%; about 87%;
about 88%; about 89%; about 90%; about 91%; about 92%; about 93%;
about 94% about 95%; about 96%; about 97%; about 98%; about 98.5%;
about 99%; about 99.5%; and about 100%. The property of substantial
homology is closely related to specific hybridization. For example,
a nucleic acid molecule is specifically hybridizable when there is
a sufficient degree of complementarity to avoid non-specific
binding of the nucleic acid to non-target polynucleotides under
conditions where specific binding is desired, for example, under
stringent hybridization conditions.
[0102] As used herein, the term "ortholog" refers to a gene in two
or more species that has evolved from a common ancestral nucleic
acid, and may retain the same function in the two or more
species.
[0103] As used herein, two nucleic acid molecules are said to
exhibit "complete complementarity" when every nucleotide of a
polynucleotide read in the 5' to 3' direction is complementary to
every nucleotide of the other polynucleotide when read in the 3' to
5' direction. A polynucleotide that is complementary to a reference
polynucleotide will exhibit a sequence identical to the reverse
complement of the reference polynucleotide. These terms and
descriptions are well defined in the art and are easily understood
by those of ordinary skill in the art.
[0104] Operably linked: A first polynucleotide is operably linked
with a second polynucleotide when the first polynucleotide is in a
functional relationship with the second polynucleotide. When
recombinantly produced, operably linked polynucleotides are
generally contiguous, and, where necessary to join two
protein-coding regions, in the same reading frame (e.g., in a
translationally fused ORF). However, nucleic acids need not be
contiguous to be operably linked.
[0105] The term, "operably linked," when used in reference to a
regulatory genetic element and a coding polynucleotide, means that
the regulatory element affects the expression of the linked coding
polynucleotide. "Regulatory elements," or "control elements," refer
to polynucleotides that influence the timing and level/amount of
transcription, RNA processing or stability, or translation of the
associated coding polynucleotide. Regulatory elements may include
promoters; translation leaders; introns; enhancers; stem-loop
structures; repressor binding polynucleotides; polynucleotides with
a termination sequence; polynucleotides with a polyadenylation
recognition sequence; etc. Particular regulatory elements may be
located upstream and/or downstream of a coding polynucleotide
operably linked thereto. Also, particular regulatory elements
operably linked to a coding polynucleotide may be located on the
associated complementary strand of a double-stranded nucleic acid
molecule.
[0106] Promoter: As used herein, the term "promoter" refers to a
region of DNA that may be upstream from the start of transcription,
and that may be involved in recognition and binding of RNA
polymerase and other proteins to initiate transcription. A promoter
may be operably linked to a coding polynucleotide for expression in
a cell, or a promoter may be operably linked to a polynucleotide
encoding a signal peptide which may be operably linked to a coding
polynucleotide for expression in a cell. A "plant promoter" may be
a promoter capable of initiating transcription in plant cells.
Examples of promoters under developmental control include promoters
that preferentially initiate transcription in certain tissues, such
as leaves, roots, seeds, fibers, xylem vessels, tracheids, or
sclerenchyma. Such promoters are referred to as "tissue-preferred".
Promoters which initiate transcription only in certain tissues are
referred to as "tissue-specific". A "cell type-specific" promoter
primarily drives expression in certain cell types in one or more
organs, for example, vascular cells in roots or leaves. An
"inducible" promoter may be a promoter which may be under
environmental control. Examples of environmental conditions that
may initiate transcription by inducible promoters include anaerobic
conditions and the presence of light. Tissue-specific,
tissue-preferred, cell type specific, and inducible promoters
constitute the class of "non-constitutive" promoters. A
"constitutive" promoter is a promoter which may be active under
most environmental conditions or in most tissue or cell types.
[0107] Any inducible promoter can be used in some embodiments of
the invention. See Ward et al. (1993) Plant Mol. Biol. 22:361-366.
With an inducible promoter, the rate of transcription increases in
response to an inducing agent. Exemplary inducible promoters
include, but are not limited to: Promoters from the ACEI system
that respond to copper; In2 gene from maize that responds to
benzenesulfonamide herbicide safeners; Tet repressor from Tn10; and
the inducible promoter from a steroid hormone gene, the
transcriptional activity of which may be induced by a
glucocorticosteroid hormone (Schena et al. (1991) Proc. Natl. Acad.
Sci. USA 88:0421).
[0108] Exemplary constitutive promoters include, but are not
limited to: Promoters from plant viruses, such as the 35S promoter
from Cauliflower Mosaic Virus (CaMV); promoters from rice actin
genes; ubiquitin promoters; pEMU; MAS; maize H3 histone promoter;
and the ALS promoter, XbaI/NcoI fragment 5' to the Brassica napus
ALS3 structural gene (or a polynucleotide similar to said XbaI/NcoI
fragment) (International PCT Publication No. WO96/30530).
[0109] Additionally, any tissue-specific or tissue-preferred
promoter may be utilized in some embodiments of the invention.
Plants transformed with a nucleic acid molecule comprising a coding
polynucleotide operably linked to a tissue-specific promoter may
produce the product of the coding polynucleotide exclusively, or
preferentially, in a specific tissue. Exemplary tissue-specific or
tissue-preferred promoters include, but are not limited to: A
seed-preferred promoter, such as that from the phaseolin gene; a
leaf-specific and light-induced promoter such as that from cab or
rubisco; an anther-specific promoter such as that from LAT52; a
pollen-specific promoter such as that from Zm13; and a
microspore-preferred promoter such as that from apg.
[0110] Soybean plant: As used herein, the term "soybean plant"
refers to a plant of the species Glycine sp.; for example, G.
max.
[0111] Transformation: As used herein, the term "transformation" or
"transduction" refers to the transfer of one or more nucleic acid
molecule(s) into a cell. A cell is "transformed" by a nucleic acid
molecule transduced into the cell when the nucleic acid molecule
becomes stably replicated by the cell, either by incorporation of
the nucleic acid molecule into the cellular genome, or by episomal
replication. As used herein, the term "transformation" encompasses
all techniques by which a nucleic acid molecule can be introduced
into such a cell. Examples include, but are not limited to:
transfection with viral vectors; transformation with plasmid
vectors; electroporation (Fromm et al. (1986) Nature 319:791-3);
lipofection (Feigner et al. (1987) Proc. Natl. Acad. Sci. USA
84:7413-7); microinjection (Mueller et al. (1978) Cell 15:579-85);
Agrobacterium-mediated transfer (Fraley et al. (1983) Proc. Natl.
Acad. Sci. USA 80:4803-7); direct DNA uptake; and microprojectile
bombardment (Klein et al. (1987) Nature 327:70).
[0112] Transgene: An exogenous nucleic acid. In some examples, a
transgene may be a DNA that encodes one or both strand(s) of a RNA
capable of forming a dsRNA molecule that comprises a polynucleotide
that is complementary to a nucleic acid molecule found in a
hemipteran pest. In further examples, a transgene may be an
antisense polynucleotide, wherein expression of the antisense
polynucleotide inhibits expression of a target nucleic acid,
thereby producing a parental RNAi phenotype. In still further
examples, a transgene may be a gene (e.g., a herbicide-tolerance
gene, a gene encoding an industrially or pharmaceutically useful
compound, or a gene encoding a desirable agricultural trait). In
these and other examples, a transgene may contain regulatory
elements operably linked to a coding polynucleotide of the
transgene (e.g., a promoter).
[0113] Vector: A nucleic acid molecule as introduced into a cell,
for example, to produce a transformed cell. A vector may include
genetic elements that permit it to replicate in the host cell, such
as an origin of replication. Examples of vectors include, but are
not limited to: a plasmid; cosmid; bacteriophage; or virus that
carries exogenous DNA into a cell. A vector may also include one or
more genes, including ones that produce antisense molecules, and/or
selectable marker genes and other genetic elements known in the
art. A vector may transduce, transform, or infect a cell, thereby
causing the cell to express the nucleic acid molecules and/or
proteins encoded by the vector. A vector optionally includes
materials to aid in achieving entry of the nucleic acid molecule
into the cell (e.g., a liposome, protein coating, etc.).
[0114] Yield: A stabilized yield of about 100% or greater relative
to the yield of check varieties in the same growing location
growing at the same time and under the same conditions. In
particular embodiments, "improved yield" or "improving yield" means
a cultivar having a stabilized yield of 105% or greater relative to
the yield of check varieties in the same growing location
containing significant densities of the hemipteran pests that are
injurious to that crop growing at the same time and under the same
conditions, which are targeted by the compositions and methods
herein.
[0115] Unless specifically indicated or implied, the terms "a,"
"an," and "the" signify "at least one," as used herein.
[0116] Unless otherwise specifically explained, all technical and
scientific terms used herein have the same meaning as commonly
understood by those of ordinary skill in the art to which this
disclosure belongs. Definitions of common terms in molecular
biology can be found in, for example, Lewin's Genes X, Jones &
Bartlett Publishers, 2009 (ISBN 10 0763766321); Krebs et al.
(eds.), The Encyclopedia of Molecular Biology, Blackwell Science
Ltd., 1994 (ISBN 0-632-02182-9); and Meyers R. A. (ed.), Molecular
Biology and Biotechnology: A Comprehensive Desk Reference, VCH
Publishers, Inc., 1995 (ISBN 1-56081-569-8). All percentages are by
weight and all solvent mixture proportions are by volume unless
otherwise noted. All temperatures are in degrees Celsius.
IV. Nucleic Acid Molecules Comprising a Hemipteran Pest
Polynucleotide
[0117] A. Overview
[0118] Described herein are nucleic acid molecules useful for the
control of hemipteran pests. Described nucleic acid molecules
include target polynucleotides (e.g., native genes, and non-coding
polynucleotides), dsRNAs, siRNAs, shRNAs, hpRNAs, and miRNAs. For
example, dsRNA, siRNA, miRNA, shRNA, and/or hpRNA molecules are
described in some embodiments that may be specifically
complementary to all or part of one or more native nucleic acids in
a hemipteran pest. In these and further embodiments, the native
nucleic acid(s) may be one or more target gene(s), the product of
which may be, for example and without limitation: involved in a
reproductive process or involved in nymph development. Nucleic acid
molecules described herein, when introduced into a cell (e.g.,
through parental transmission) comprising at least one native
nucleic acid(s) to which the nucleic acid molecules are
specifically complementary, may initiate RNAi in the cell, and
consequently reduce or eliminate expression of the native nucleic
acid(s). In some examples, reduction or elimination of the
expression of a target gene by a nucleic acid molecule specifically
complementary thereto may result in reduction or cessation of
reproduction in the hemipteran pest, and/or growth, development,
and/or feeding in progeny of the pest. These methods may
significantly reduce the size of a subsequent pest generation in an
infestation, for example, without directly resulting in mortality
in the pest(s) that contact the iRNA molecule.
[0119] In some embodiments, at least one target gene in a
hemipteran pest may be selected, wherein the target gene comprises
a kruppel polynucleotide. In particular examples, a target gene in
a hemipteran pest is selected, wherein the target gene comprises a
polynucleotide selected from among any of SEQ ID NOs:1, 3, and
4.
[0120] In other embodiments, a target gene may be a nucleic acid
molecule comprising a polynucleotide that can be reverse translated
in silico to a polypeptide comprising a contiguous amino acid
sequence that is at least about 85% identical (e.g., at least 84%,
85%, about 90%, about 95%, about 96%, about 97%, about 98%, about
99%, about 100%, or 100% identical) to the amino acid sequence of a
protein product of a kruppel polynucleotide (i.e., a KRUPPEL
polypeptide). A target gene may be any nucleic acid in a hemipteran
pest, the post-transcriptional inhibition of which has a
deleterious effect on the capacity of the pest to produce viable
offspring, for example, to provide a protective benefit against the
pest to a plant. In particular examples, a target gene is a nucleic
acid molecule comprising a polynucleotide that can be reverse
translated in silico to a polypeptide comprising a contiguous amino
acid sequence that is at least about 85% identical, about 90%
identical, about 95% identical, about 96% identical, about 97%
identical, about 98% identical, about 99% identical, about 100%
identical, or 100% identical to the amino acid sequence that is the
in silico translation product of SEQ ID NO:2.
[0121] Provided in some embodiments are DNAs, the expression of
which results in an RNA molecule comprising a polynucleotide that
is specifically complementary to all or part of a native RNA
molecule that is encoded by a coding polynucleotide in a hemipteran
pest. In some embodiments, after ingestion of the expressed RNA
molecule by a hemipteran pest, down-regulation of the coding
polynucleotide in cells of the pest, or in cells of progeny of the
pest, may be obtained. In particular embodiments, down-regulation
of the coding polynucleotide in cells of the hemipteran pest may
result in reduction or cessation of reproduction and/or
proliferation in the pest, and/or growth, development, and/or
feeding in progeny of the pest.
[0122] In some embodiments, target polynucleotides include
transcribed non-coding RNAs, such as 5'UTRs; 3'UTRs; spliced
leaders; introns; outrons (e.g., 5'UTR RNA subsequently modified in
trans splicing); donatrons (e.g., non-coding RNA required to
provide donor sequences for trans splicing); and other non-coding
transcribed RNA of target hemipteran pest genes. Such
polynucleotides may be derived from both mono-cistronic and
poly-cistronic genes.
[0123] Thus, also described herein in connection with some
embodiments are iRNA molecules (e.g., dsRNAs, siRNAs, miRNAs,
shRNAs, and hpRNAs) that comprise at least one polynucleotide that
is specifically complementary to all or part of a target nucleic
acid in a hemipteran pest. In some embodiments an iRNA molecule may
comprise polynucleotide(s) that are complementary to all or part of
a plurality of target nucleic acids; for example, 2, 3, 4, 5, 6, 7,
8, 9, 10, or more target nucleic acids. In particular embodiments,
an iRNA molecule may be produced in vitro, or in vivo by a
genetically-modified organism, such as a plant or bacterium. Also
disclosed are cDNAs that may be used for the production of dsRNA
molecules, siRNA molecules, miRNA molecules, shRNA molecules,
and/or hpRNA molecules that are specifically complementary to all
or part of a target nucleic acid in a hemipteran pest. Further
described are recombinant DNA constructs for use in achieving
stable transformation of particular host targets. Transformed host
targets may express effective levels of dsRNA, siRNA, miRNA, shRNA,
and/or hpRNA molecules from the recombinant DNA constructs. Also
described is a plant transformation vector comprising at least one
polynucleotide operably linked to a heterologous promoter
functional in a plant cell, wherein expression of the
polynucleotide(s) results in an RNA molecule comprising a string of
contiguous nucleobases that is specifically complementary to all or
part of a target nucleic acid in a hemipteran pest.
[0124] In particular embodiments, nucleic acid molecules useful for
the control of hemipteran pests may include: all or part of a
native nucleic acid isolated from Euschistus comprising a kruppel
polynucleotide (e.g., SEQ ID NO:1); DNAs that when expressed result
in an RNA molecule comprising a polynucleotide that is specifically
complementary to all or part of a native RNA molecule that is
encoded by kruppel; iRNA molecules (e.g., dsRNAs, siRNAs, miRNAs,
shRNAs, and hpRNAs) that comprise at least one polynucleotide that
is specifically complementary to all or part of a RNA molecule
encoded by kruppel; cDNAs that may be used for the production of
dsRNA molecules, siRNA molecules, miRNA molecules, shRNA molecules,
and/or hpRNA molecules that are specifically complementary to all
or part of an RNA molecule encoded by kruppel; and recombinant DNA
constructs for use in achieving stable transformation of particular
host targets, wherein a transformed host target comprises one or
more of the foregoing nucleic acid molecules.
[0125] B. Nucleic Acid Molecules
[0126] The present invention provides, inter alia, iRNA (e.g.,
dsRNA, siRNA, miRNA, shRNA, and hpRNA) molecules that inhibit
target gene expression in a cell, tissue, or organ of a hemipteran
pest; and DNA molecules capable of being expressed as an iRNA
molecule in a cell or microorganism to inhibit target gene
expression in a cell, tissue, or organ of a hemipteran pest.
[0127] Some embodiments of the invention provide an isolated
nucleic acid molecule comprising at least one (e.g., one, two,
three, or more) polynucleotide(s) selected from the group
consisting of: SEQ ID NO:1; the complement of SEQ ID NO:1; a
fragment of at least 15 contiguous nucleotides (e.g., at least 19
contiguous nucleotides) of SEQ ID NO:1 (e.g., SEQ ID NO:3 and SEQ
ID NO:4); the complement of a fragment of at least 15 contiguous
nucleotides of SEQ ID NO:1; a native coding polynucleotide of a
hemipteran organism (e.g., BSB) comprising SEQ ID NO:1; the
complement of a native coding polynucleotide of a hemipteran
organism comprising SEQ ID NO:1; a fragment of at least 15
contiguous nucleotides of a native coding polynucleotide of a
hemipteran organism comprising SEQ ID NO:1; and the complement of a
fragment of at least 15 contiguous nucleotides of a native coding
polynucleotide of a hemipteran organism comprising SEQ ID NO:1. In
particular embodiments, contact with or uptake by a hemipteran pest
of the isolated polynucleotide inhibits the growth, development,
reproduction and/or feeding of the pest.
[0128] In other embodiments, an isolated nucleic acid molecule of
the invention may comprise at least one (e.g., one, two, three, or
more) polynucleotide(s) selected from the group consisting of: SEQ
ID NO:15; the complement of SEQ ID NO:15; SEQ ID NO:16; the
complement of SEQ ID NO:16; SEQ ID NO:17; the complement of SEQ ID
NO:17; a fragment of at least 15 contiguous nucleotides of any of
SEQ ID NOs:15-17; the complement of a fragment of at least 15
contiguous nucleotides of any of SEQ ID NOs:15-17; a native
polyribonucleotide transcribed in a hemipteran organism from a gene
comprising SEQ ID NO:1; the complement of a native
polyribonucleotide transcribed in a hemipteran organism from a gene
comprising SEQ ID NO:1; a fragment of at least 15 contiguous
nucleotides of a native polyribonucleotide transcribed in a
hemipteran organism from a gene comprising SEQ ID NO:1; and the
complement of a fragment of at least 15 contiguous nucleotides of a
native polyribonucleotide transcribed in a hemipteran organism from
a gene comprising SEQ ID NO:1. In particular embodiments, contact
with or uptake by a hemipteran pest of the isolated polynucleotide
inhibits the growth, development, reproduction and/or feeding of
the pest.
[0129] In alternative embodiments, a nucleic acid molecule of the
invention may comprise at least one (e.g., one, two, three, or
more) DNA(s) capable of being expressed as an iRNA molecule in a
cell or microorganism to inhibit target gene expression in a cell,
tissue, or organ of a hemipteran pest. Such DNA(s) may be operably
linked to a promoter that functions in a cell comprising the DNA
molecule to initiate or enhance the transcription of the encoded
RNA capable of forming a dsRNA molecule(s). In one embodiment, the
at least one (e.g., one, two, three, or more) DNA(s) may be derived
from the polynucleotide of SEQ ID NO:1. Derivatives of SEQ ID NO:1
include fragments of SEQ ID NO:1. In some embodiments, such a
fragment may comprise, for example, at least about 15 contiguous
nucleotides of SEQ ID NO:1 or a complement thereof. Thus, such a
fragment may comprise, for example, 15, 16, 17, 18, 19, 20, 21, 22,
23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 110,
120, 130, 140, 150, 160, 170, 180, 190, 200 or more contiguous
nucleotides of SEQ ID NO:1 or a complement thereof. In some
examples, such a fragment may comprise, for example, at least 19
contiguous nucleotides (e.g., 19, 20, 21, 22, 23, 24, 25, 26, 27,
28, 29, or 30 contiguous nucleotides) of SEQ ID NO:1 or a
complement thereof.
[0130] Some embodiments comprise introducing partially- or
fully-stabilized dsRNA molecules into a hemipteran pest to inhibit
expression of a target gene in a cell, tissue, or organ of the
hemipteran pest. When expressed as an iRNA molecule (e.g., dsRNA,
siRNA, miRNA, shRNA, and hpRNA) and taken up by a hemipteran pest,
polynucleotides comprising one or more fragments of SEQ ID NO:1
(e.g., SEQ ID NO:3 and SEQ ID NO:4), and the complements thereof,
may cause one or more of death, developmental arrest, growth
inhibition, change in sex ratio, reduction in brood size, cessation
of infection, and/or cessation of feeding by a hemipteran pest. In
particular examples, polynucleotides comprising one or more
fragments (e.g., polynucleotides including about 15 to about 300
nucleotides) of SEQ ID NO:1, and the complements thereof cause a
reduction in the capacity of an existing generation of the pest to
produce a subsequent generation of the pest.
[0131] In certain embodiments, dsRNA molecules provided by the
invention comprise polynucleotides complementary to a transcript
from a target gene comprising SEQ ID NO:1, and/or polynucleotides
complementary to a fragment of SEQ ID NO:1, the inhibition of which
target gene in a hemipteran pest results in the reduction or
removal of a polypeptide or polynucleotide agent that is essential
for the pest's or the pest's progeny's growth, development, or
other biological function. A selected polynucleotide may exhibit
from about 80% to about 100% sequence identity to SEQ ID NO:1, a
contiguous fragment of SEQ ID NO:1, or the complement of either of
the foregoing. For example, a selected polynucleotide may exhibit
79%; 80%; about 81%; about 82%; about 83%; about 84%; about 85%;
about 86%; about 87%; about 88%; about 89%; about 90%; about 91%;
about 92%; about 93%; about 94% about 95%; about 96%; about 97%;
about 98%; about 98.5%; about 99%; about 99.5%; or about 100%
sequence identity to SEQ ID NO:1, a contiguous fragment of SEQ ID
NO:1, or the complement of either of the foregoing.
[0132] In some embodiments, a DNA molecule capable of being
expressed as an iRNA molecule in a cell or microorganism to inhibit
target gene expression may comprise a single polynucleotide that is
specifically complementary to all or part of a native
polynucleotide found in one or more target hemipteran pest species,
or the DNA molecule can be constructed as a chimera from a
plurality of such specifically complementary polynucleotides.
[0133] In other embodiments, a nucleic acid molecule may comprise a
first and a second polynucleotide separated by a "linker." A linker
may be a region comprising any sequence of nucleotides that
facilitates secondary structure formation between the first and
second polynucleotides, where this is desired. In one embodiment,
the linker is part of a sense or antisense coding polynucleotide
for mRNA. The linker may alternatively comprise any combination of
nucleotides or homologues thereof that are capable of being linked
covalently to a nucleic acid molecule. In some examples, the linker
may comprise an intron (e.g., as ST-LS1 intron).
[0134] For example, in some embodiments, the DNA molecule may
comprise a polynucleotide coding for one or more different RNA
molecules, wherein each of the different RNA molecules comprises a
first polynucleotide and a second polynucleotide, wherein the first
and second polynucleotides are complementary to each other. The
first and second polynucleotides may be connected within an RNA
molecule by a spacer. The spacer may constitute part of the first
polynucleotide or the second polynucleotide. Expression of an RNA
molecule comprising the first and second nucleotide polynucleotides
may lead to the formation of a dsRNA molecule of the present
invention, by specific intramolecular base-pairing of the first and
second nucleotide polynucleotides. The first polynucleotide or the
second polynucleotide may be substantially identical to a
polynucleotide native to a hemipteran pest (e.g., a target gene, or
transcribed non-coding polynucleotide), a derivative thereof, or a
complementary polynucleotide thereto.
[0135] dsRNA nucleic acid molecules comprise double strands of
polymerized ribonucleotides, and may include modifications to
either the phosphate-sugar backbone or the nucleoside.
Modifications in RNA structure may be tailored to allow specific
inhibition. In one embodiment, dsRNA molecules may be modified
through a ubiquitous enzymatic process so that siRNA molecules may
be generated. This enzymatic process may utilize an RNase III
enzyme, such as DICER in eukaryotes, either in vitro or in vivo.
See Elbashir et al. (2001) Nature 411:494-8; and Hamilton and
Baulcombe (1999) Science 286(5441):950-2. DICER or
functionally-equivalent RNase III enzymes cleave larger dsRNA
strands and/or hpRNA molecules into smaller oligonucleotides (e.g.,
siRNAs), each of which is about 19-25 nucleotides in length. The
siRNA molecules produced by these enzymes have 2 to 3 nucleotide 3'
overhangs, and 5' phosphate and 3' hydroxyl termini. The siRNA
molecules generated by RNase III enzymes are unwound and separated
into single-stranded RNA in the cell. The siRNA molecules then
specifically hybridize with RNAs transcribed from a target gene,
and both RNA molecules are subsequently degraded by an inherent
cellular RNA-degrading mechanism. This process may result in the
effective degradation or removal of the RNA encoded by the target
gene in the target organism. The outcome is the
post-transcriptional silencing of the targeted gene. In some
embodiments, siRNA molecules produced by endogenous RNase III
enzymes from heterologous nucleic acid molecules may efficiently
mediate the down-regulation of target genes in hemipteran
pests.
[0136] In some embodiments, a nucleic acid molecule of the
invention may include at least one non-naturally occurring
polynucleotide that can be transcribed into a single-stranded RNA
molecule capable of forming a dsRNA molecule in vivo through
intermolecular hybridization. Such dsRNAs typically self-assemble,
and can be provided in the nutrition source of a hemipteran pest to
achieve the post-transcriptional inhibition of a target gene. In
these and further embodiments, a nucleic acid molecule of the
invention may comprise two different non-naturally occurring
polynucleotides, each of which is specifically complementary to a
different target gene in a hemipteran pest. When such a nucleic
acid molecule is provided as a dsRNA molecule to a hemipteran pest,
the dsRNA molecule inhibits the expression of at least two
different target genes in the pest.
[0137] C. Obtaining Nucleic Acid Molecules
[0138] A variety of polynucleotides in hemipteran pests may be used
as targets for the design of nucleic acid molecules of the
invention, such as iRNAs and DNA molecules encoding iRNAs.
Selection of native polynucleotides is not, however, a
straight-forward process. Only a small number of native
polynucleotides in the hemipteran pest will be effective targets.
For example, it cannot be predicted with certainty whether a
particular native polynucleotide can be effectively down-regulated
by nucleic acid molecules of the invention, or whether
down-regulation of a particular native polynucleotide will have a
detrimental effect on the growth, viability, proliferation, and/or
reproduction of the hemipteran pest. The vast majority of native
hemipteran pest polynucleotides, such as ESTs isolated therefrom do
not have a detrimental effect on the growth, viability,
proliferation, and/or reproduction of the pest. Neither is it
predictable which of the native polynucleotides that may have a
detrimental effect on a hemipteran pest are able to be used in
recombinant techniques for expressing nucleic acid molecules
complementary to such native polynucleotides in a host plant and
providing the detrimental effect on the pest upon feeding without
causing harm to the host plant.
[0139] In some embodiments, nucleic acid molecules of the invention
(e.g., dsRNA molecules to be provided in the host plant of a
hemipteran pest) are selected to target cDNAs that encode proteins
or parts of proteins essential for hemipteran pest reproduction
and/or development, such as polypeptides involved in metabolic or
catabolic biochemical pathways, cell division, reproduction, energy
metabolism, embryonic development, nymph development,
transcriptional regulation, and the like. As described herein,
contact with compositions by a target organism containing one or
more dsRNAs (e.g., by injection or topical contact), at least one
segment of which is specifically complementary to at least a
substantially identical segment of RNA produced in the cells of the
target pest organism, can result in failure or reduction of the
capacity to mate, oviposit eggs, or produce viable progeny. A
polynucleotide, either DNA or RNA, derived from a hemipteran pest
can be used to construct plant cells resistant to infestation by
the pests. The host plant of the hemipteran pest (e.g., Z. mays, G.
max, and Gossypium sp.), for example, can be transformed to contain
one or more of the polynucleotides derived from the hemipteran pest
as provided herein. The polynucleotide transformed into the host
may encode one or more RNAs that form into a dsRNA structure in the
cells or biological fluids within the transformed host, thus making
the dsRNA available if/when the pest forms a nutritional
relationship with the transgenic host. This may result in the
suppression of expression of one or more genes in the cells of the
pest, and ultimately inhibition of reproduction and/or
development.
[0140] In alternative embodiments, a gene is targeted that is
essentially involved in the growth, development and reproduction of
a hemipteran pest. Other target genes for use in the present
invention may include, for example, those that play important roles
in hemipteran pest viability, movement, migration, growth,
development, infectivity, and establishment of feeding sites. A
target gene may therefore be a housekeeping gene or a transcription
factor. Additionally, a native hemipteran pest polynucleotide for
use in the present invention may also be derived from a homolog
(e.g., an ortholog), of a plant, viral, bacterial or insect gene,
the function of which is known to those of skill in the art, and
the polynucleotide of which is specifically hybridizable with a
target gene in the genome of the target hemipteran pest. Methods of
identifying a homolog of a gene with a known nucleotide sequence by
hybridization are known to those of skill in the art.
[0141] In some embodiments, the invention provides methods for
obtaining a nucleic acid molecule comprising a polynucleotide for
producing an iRNA (e.g., dsRNA, siRNA, miRNA, shRNA, and hpRNA)
molecule. One such embodiment comprises: (a) analyzing one or more
target gene(s) for their expression, function, and phenotype upon
dsRNA-mediated gene suppression in a hemipteran pest; (b) probing a
cDNA or gDNA library with a probe comprising all or a portion of a
polynucleotide or a homolog thereof from a targeted hemipteran pest
that displays an altered (e.g., reduced) reproduction or
development phenotype in a dsRNA-mediated suppression analysis; (c)
identifying a DNA clone that specifically hybridizes with the
probe; (d) isolating the DNA clone identified in step (b); (e)
sequencing the cDNA or gDNA fragment that comprises the clone
isolated in step (d), wherein the sequenced nucleic acid molecule
comprises all or a substantial portion of the RNA or a homolog
thereof; and (f) chemically synthesizing all or a substantial
portion of a gene, or an siRNA, miRNA, hpRNA, mRNA, shRNA, or
dsRNA.
[0142] In further embodiments, a method for obtaining a nucleic
acid fragment comprising a polynucleotide for producing a
substantial portion of an iRNA (e.g., dsRNA, siRNA, miRNA, shRNA,
and hpRNA) molecule includes: (a) synthesizing first and second
oligonucleotide primers specifically complementary to a portion of
a native polynucleotide from a targeted hemipteran pest; and (b)
amplifying a cDNA or gDNA insert present in a cloning vector using
the first and second oligonucleotide primers of step (a), wherein
the amplified nucleic acid molecule comprises a substantial portion
of a siRNA, miRNA, hpRNA, mRNA, shRNA, or dsRNA molecule.
[0143] Nucleic acids of the invention can be isolated, amplified,
or produced by a number of approaches. For example, an iRNA (e.g.,
dsRNA, siRNA, miRNA, shRNA, and hpRNA) molecule may be obtained by
PCR amplification of a target polynucleotide (e.g., a target gene
or a target transcribed non-coding polynucleotide) derived from a
gDNA or cDNA library, or portions thereof. DNA or RNA may be
extracted from a target organism, and nucleic acid libraries may be
prepared therefrom using methods known to those ordinarily skilled
in the art. gDNA or cDNA libraries generated from a target organism
may be used for PCR amplification and sequencing of target genes. A
confirmed PCR product may be used as a template for in vitro
transcription to generate sense and antisense RNA with minimal
promoters. Alternatively, nucleic acid molecules may be synthesized
by any of a number of techniques (See, e.g., Ozaki et al. (1992)
Nucleic Acids Research, 20: 5205-5214; and Agrawal et al. (1990)
Nucleic Acids Research, 18: 5419-5423), including use of an
automated DNA synthesizer (for example, a P.E. Biosystems, Inc.
(Foster City, Calif.) model 392 or 394 DNA/RNA Synthesizer), using
standard chemistries, such as phosphoramidite chemistry. See, e.g.,
Beaucage et al. (1992) Tetrahedron, 48: 2223-2311; U.S. Pat. Nos.
4,980,460, 4,725,677, 4,415,732, 4,458,066, and 4,973,679.
Alternative chemistries resulting in non-natural backbone groups,
such as phosphorothioate, phosphoramidate, and the like, can also
be employed.
[0144] An RNA, dsRNA, siRNA, miRNA, shRNA, or hpRNA molecule of the
present invention may be produced chemically or enzymatically by
one skilled in the art through manual or automated reactions, or in
vivo in a cell comprising a nucleic acid molecule comprising a
polynucleotide encoding the RNA, dsRNA, siRNA, miRNA, shRNA, or
hpRNA molecule. RNA may also be produced by partial or total
organic synthesis--any modified ribonucleotide can be introduced by
in vitro enzymatic or organic synthesis. An RNA molecule may be
synthesized by a cellular RNA polymerase or a bacteriophage RNA
polymerase (e.g., T3 RNA polymerase, T7 RNA polymerase, and SP6 RNA
polymerase). Expression constructs useful for the cloning and
expression of polynucleotides are known in the art. See, e.g.,
International PCT Publication No. WO97/32016; and U.S. Pat. Nos.
5,593,874, 5,698,425, 5,712,135, 5,789,214, and 5,804,693. RNA
molecules that are synthesized chemically or by in vitro enzymatic
synthesis may be purified prior to introduction into a cell. For
example, RNA molecules can be purified from a mixture by extraction
with a solvent or resin, precipitation, electrophoresis,
chromatography, or a combination thereof. Alternatively, RNA
molecules that are synthesized chemically or by in vitro enzymatic
synthesis may be used with no or a minimum of purification, for
example, to avoid losses due to sample processing. The RNA
molecules may be dried for storage or dissolved in an aqueous
solution. The solution may contain buffers or salts to promote
annealing, and/or stabilization of dsRNA molecule duplex
strands.
[0145] In particular embodiments, a dsRNA molecule may be formed by
a single self-complementary RNA strand or from two complementary
RNA strands. dsRNA molecules may be synthesized either in vivo or
in vitro. An endogenous RNA polymerase of the cell may mediate
transcription of the one or two RNA strands in vivo, or cloned RNA
polymerase may be used to mediate transcription in vivo or in
vitro. Post-transcriptional inhibition of a target gene in a
hemipteran pest may be host-targeted by specific transcription in
an organ, tissue, or cell type of the host (e.g., by using a
tissue-specific promoter); stimulation of an environmental
condition in the host (e.g., by using an inducible promoter that is
responsive to infection, stress, temperature, and/or chemical
inducers); and/or engineering transcription at a developmental
stage or age of the host (e.g., by using a developmental
stage-specific promoter). RNA strands that form a dsRNA molecule,
whether transcribed in vitro or in vivo, may or may not be
polyadenylated, and may or may not be capable of being translated
into a polypeptide by a cell's translational apparatus.
[0146] D. Recombinant Vectors and Host Cell Transformation
[0147] In some embodiments, the invention also provides a DNA
molecule for introduction into a cell (e.g., a bacterial cell, a
yeast cell, or a plant cell), wherein the DNA molecule comprises a
polynucleotide that, upon expression to RNA and ingestion by a
hemipteran pest, achieves suppression of a target gene in a cell,
tissue, or organ of the pest. Thus, some embodiments provide a
recombinant nucleic acid molecule comprising a polynucleotide
capable of being expressed as an iRNA (e.g., dsRNA, siRNA, miRNA,
shRNA, and hpRNA) molecule in a plant cell to inhibit target gene
expression in a hemipteran pest. In order to initiate or enhance
expression, such recombinant nucleic acid molecules may comprise
one or more regulatory elements, which regulatory elements may be
operably linked to the polynucleotide capable of being expressed as
an iRNA. Methods to express a gene suppression molecule in plants
are known, and may be used to express a polynucleotide of the
present invention. See, e.g., International PCT Publication No.
WO06/073727; and U.S. Patent Publication No. 2006/0200878 A1)
[0148] In specific embodiments, a recombinant DNA molecule of the
invention may comprise a polynucleotide encoding an RNA that may
form a dsRNA molecule. Such recombinant DNA molecules may encode
RNAs that may form dsRNA molecules capable of inhibiting the
expression of endogenous target gene(s) in a hemipteran pest cell
upon ingestion. In many embodiments, a transcribed RNA may form a
dsRNA molecule that may be provided in a stabilized form; e.g., as
a hairpin and stem and loop structure.
[0149] In other embodiments, one strand of a dsRNA molecule may be
formed by transcription from a polynucleotide that is substantially
homologous to a polynucleotide selected from the group consisting
of SEQ ID NO:1; the complement of SEQ ID NO:1; a fragment of at
least 15 contiguous nucleotides of SEQ ID NO:1; the complement of a
fragment of at least 15 contiguous nucleotides of SEQ ID NO:1; a
native coding polynucleotide of a hemipteran organism (e.g., BSB)
comprising any of SEQ ID NOs:1, 3, and 4; the complement of a
native coding polynucleotide of a hemipteran organism comprising
any of SEQ ID NOs:1, 3, and 4; a fragment of at least 15 contiguous
nucleotides of a native coding polynucleotide of a hemipteran
organism comprising any of SEQ ID NOs:1, 3, and 4; and the
complement of a fragment of at least 15 contiguous nucleotides of a
native coding polynucleotide of a hemipteran organism comprising
any of SEQ ID NOs:1, 3, and 4.
[0150] In particular embodiments, a recombinant DNA molecule
encoding an RNA that may form a dsRNA molecule may comprise a
coding region wherein at least two polynucleotides are arranged
such that one polynucleotide is in a sense orientation, and the
other polynucleotide is in an antisense orientation, relative to at
least one promoter, wherein the sense polynucleotide and the
antisense polynucleotide are linked or connected by a spacer of,
for example, from about five (.about.5) to about one thousand
(.about.1000) nucleotides. The spacer may form a loop between the
sense and antisense polynucleotides. The sense polynucleotide or
the antisense polynucleotide may be substantially homologous to an
RNA encoded by a target gene (e.g., a kruppel gene comprising SEQ
ID NO:1) or fragment thereof. In some embodiments, however, a
recombinant DNA molecule may encode an RNA that may form a dsRNA
molecule without a spacer. In embodiments, a sense coding
polynucleotide and an antisense coding polynucleotide may be
different lengths.
[0151] Polynucleotides identified as having a deleterious effect on
hemipteran pests or a plant-protective effect with regard to
hemipteran pests may be readily incorporated into expressed dsRNA
molecules through the creation of appropriate expression cassettes
in a recombinant nucleic acid molecule of the invention. For
example, such polynucleotides may be expressed as a hairpin with
stem and loop structure by taking a first segment corresponding to
an RNA encoded by a target gene polynucleotide (e.g., a kruppel
gene comprising SEQ ID NO:1, and fragments of either of the
foregoing); linking this polynucleotide to a second segment spacer
region that is not homologous or complementary to the first
segment; and linking this to a third segment, wherein at least a
portion of the third segment is substantially complementary to the
first segment. Such a construct forms a stem and loop structure by
intramolecular base-pairing of the first segment with the third
segment, wherein the loop structure forms comprising the second
segment. See, e.g., U.S. Patent Publication Nos. 2002/0048814 and
2003/0018993; and International PCT Publication Nos. WO94/01550 and
WO98/05770. A dsRNA molecule may be generated, for example, in the
form of a double-stranded structure such as a stem-loop structure
(e.g., hairpin), whereby production of siRNA targeted for a native
hemipteran pest polynucleotide is enhanced by co-expression of a
fragment of the targeted gene, for instance on an additional plant
expressible cassette, that leads to enhanced siRNA production, or
reduces methylation to prevent transcriptional gene silencing of
the dsRNA hairpin promoter.
[0152] Certain embodiments of the invention include introduction of
a recombinant nucleic acid molecule of the present invention into a
plant (i.e., transformation) to achieve hemipteran pest-inhibitory
levels of expression of one or more iRNA molecules. A recombinant
DNA molecule may, for example, be a vector, such as a linear or a
closed circular plasmid. The vector system may be a single vector
or plasmid, or two or more vectors or plasmids that together
contain the total DNA to be introduced into the genome of a host.
In addition, a vector may be an expression vector. Nucleic acids of
the invention can, for example, be suitably inserted into a vector
under the control of a suitable promoter that functions in one or
more hosts to drive expression of a linked coding polynucleotide or
other DNA element. Many vectors are available for this purpose, and
selection of the appropriate vector will depend mainly on the size
of the nucleic acid to be inserted into the vector and the
particular host cell to be transformed with the vector. Each vector
contains various components depending on its function (e.g.,
amplification of DNA or expression of DNA) and the particular host
cell with which it is compatible.
[0153] To impart protection from hemipteran pests to a transgenic
plant, a recombinant DNA may, for example, be transcribed into an
iRNA molecule (e.g., an RNA molecule that forms a dsRNA molecule)
within the tissues or fluids of the recombinant plant. An iRNA
molecule may comprise a polynucleotide that is substantially
homologous and specifically hybridizable to a corresponding
transcribed polynucleotide within a hemipteran pest that may cause
damage to the host plant species. The hemipteran pest may contact
the iRNA molecule that is transcribed in cells of the transgenic
host plant, for example, by ingesting cells or fluids of the
transgenic host plant that comprise the iRNA molecule. Thus,
expression of a target gene is suppressed by the iRNA molecule
within hemipteran pests that infest the transgenic host plant. In
some embodiments, suppression of expression of the target gene in
the target hemipteran pest may result in the plant being tolerant
to attack by the pest.
[0154] In order to enable delivery of iRNA molecules to a
hemipteran pest in a nutritional relationship with a plant cell
that has been transformed with a recombinant nucleic acid molecule
of the invention, expression (i.e., transcription) of iRNA
molecules in the plant cell is required. Thus, a recombinant
nucleic acid molecule may comprise a polynucleotide of the
invention operably linked to one or more regulatory elements, such
as a heterologous promoter element that functions in a host cell,
such as a bacterial cell wherein the nucleic acid molecule is to be
amplified, and a plant cell wherein the nucleic acid molecule is to
be expressed.
[0155] Promoters suitable for use in nucleic acid molecules of the
invention include those that are inducible, viral, synthetic, or
constitutive, all of which are well known in the art. Non-limiting
examples describing such promoters include U.S. Pat. No. 6,437,217
(maize RS81 promoter); U.S. Pat. No. 5,641,876 (rice actin
promoter); U.S. Pat. No. 6,426,446 (maize RS324 promoter); U.S.
Pat. No. 6,429,362 (maize PR-1 promoter); U.S. Pat. No. 6,232,526
(maize A3 promoter); U.S. Pat. No. 6,177,611 (constitutive maize
promoters); U.S. Pat. Nos. 5,322,938, 5,352,605, 5,359,142, and
5,530,196 (CaMV 35S promoter); U.S. Pat. No. 6,433,252 (maize L3
oleosin promoter); U.S. Pat. No. 6,429,357 (rice actin 2 promoter,
and rice actin 2 intron); U.S. Pat. No. 6,294,714 (light-inducible
promoters); U.S. Pat. No. 6,140,078 (salt-inducible promoters);
U.S. Pat. No. 6,252,138 (pathogen-inducible promoters); U.S. Pat.
No. 6,175,060 (phosphorous deficiency-inducible promoters); U.S.
Pat. No. 6,388,170 (bidirectional promoters); U.S. Pat. No.
6,635,806 (gamma-coixin promoter); and U.S. Patent Publication No.
2009/757,089 (maize chloroplast aldolase promoter). Additional
promoters include the nopaline synthase (NOS) promoter (Ebert et
al. (1987) Proc. Natl. Acad. Sci. USA 84(16):5745-9) and the
octopine synthase (OCS) promoters (which are carried on
tumor-inducing plasmids of Agrobacterium tumefaciens); the
caulimovirus promoters such as the cauliflower mosaic virus (CaMV)
19S promoter (Lawton et al. (1987) Plant Mol. Biol. 9:315-24); the
CaMV 35S promoter (Odell et al. (1985) Nature 313:810-2; the
figwort mosaic virus 35S-promoter (Walker et al. (1987) Proc. Natl.
Acad. Sci. USA 84(19):6624-8); the sucrose synthase promoter (Yang
and Russell (1990) Proc. Natl. Acad. Sci. USA 87:4144-8); the R
gene complex promoter (Chandler et al. (1989) Plant Cell
1:1175-83); the chlorophyll a/b binding protein gene promoter; CaMV
35S (U.S. Pat. Nos. 5,322,938, 5,352,605, 5,359,142, and
5,530,196); FMV 35S (U.S. Pat. No. 6,051,753, and 5,378,619); a
PC1SV promoter (U.S. Pat. No. 5,850,019); the SCP1 promoter (U.S.
Pat. No. 6,677,503); and AGRtu.nos promoters (GenBank.TM. Accession
No. V00087; Depicker et al. (1982) J. Mol. Appl. Genet. 1:561-73;
Bevan et al. (1983) Nature 304:184-7).
[0156] In particular embodiments, nucleic acid molecules of the
invention comprise a tissue-specific promoter, such as a
leaf-specific promoter or pollen-specific promoter. In some
embodiments, a polynucleotide or fragment for hemipteran pest
control according to the invention may be cloned between two
tissue-specific promoters oriented in opposite transcriptional
directions relative to the polynucleotide or fragment, and which
are operable in a transgenic plant cell and expressed therein to
produce RNA molecules in the transgenic plant cell that
subsequently may form dsRNA molecules, as described, supra. The
iRNA molecules expressed in plant tissues may be ingested by a
hemipteran pest so that suppression of target gene expression is
achieved.
[0157] Additional regulatory elements that may optionally be
operably linked to a nucleic acid molecule of interest include
5'UTRs located between a promoter element and a coding
polynucleotide that function as a translation leader element. The
translation leader element is present in the fully-processed mRNA,
and it may affect processing of the primary transcript, and/or RNA
stability. Examples of translation leader elements include maize
and petunia heat shock protein leaders (U.S. Pat. No. 5,362,865),
plant virus coat protein leaders, plant rubisco leaders, and
others. See, e.g., Turner and Foster (1995) Molecular Biotech.
3(3):225-36. Non-limiting examples of 5'UTRs include GmHsp (U.S.
Pat. No. 5,659,122); PhDnaK (U.S. Pat. No. 5,362,865); AtAnt1; TEV
(Carrington and Freed (1990) J. Virol. 64:1590-7); and AGRtunos
(GenBank.TM. Accession No. V00087; and Bevan et al. (1983) Nature
304:184-7).
[0158] Additional regulatory elements that may optionally be
operably linked to a nucleic acid molecule of interest also include
3' non-translated elements, 3' transcription termination regions,
or polyadenylation regions. These are genetic elements located
downstream of a polynucleotide, and include polynucleotides that
provide polyadenylation signal, and/or other regulatory signals
capable of affecting transcription or mRNA processing. The
polyadenylation signal functions in plants to cause the addition of
polyadenylate nucleotides to the 3' end of the mRNA precursor. The
polyadenylation element can be derived from a variety of plant
genes, or from T-DNA genes. A non-limiting example of a 3'
transcription termination region is the nopaline synthase 3' region
(nos 3; Fraley et al. (1983) Proc. Natl. Acad. Sci. USA 80:4803-7).
An example of the use of different 3' nontranslated regions is
provided in Ingelbrecht et al., (1989) Plant Cell 1:671-80.
Non-limiting examples of polyadenylation signals include one from a
Pisum sativum RbcS2 gene (Ps.RbcS2-E9; Coruzzi et al. (1984) EMBO
J. 3:1671-9) and AGRtu.nos (GenBank.TM. Accession No. E01312).
[0159] Some embodiments may include a plant transformation vector
that comprises an isolated and purified DNA molecule comprising at
least one of the above-described regulatory elements operatively
linked to one or more polynucleotides of the present invention.
When expressed, the one or more polynucleotides result in one or
more RNA molecule(s) comprising a polynucleotide that is
specifically complementary to all or part of a native RNA molecule
in a hemipteran pest. Thus, the polynucleotide(s) may comprise a
segment encoding all or part of a polyribonucleotide present within
a targeted hemipteran pest RNA transcript, and may comprise
inverted repeats of all or a part of a targeted pest transcript. A
plant transformation vector may contain polynucleotides
specifically complementary to more than one target polynucleotide,
thus allowing production of more than one dsRNA for inhibiting
expression of two or more genes in cells of one or more populations
or species of target hemipteran pests. Segments of polynucleotides
specifically complementary to polynucleotides present in different
genes can be combined into a single composite nucleic acid molecule
for expression in a transgenic plant. Such segments may be
contiguous or separated by a spacer.
[0160] In other embodiments, a plasmid of the present invention
already containing at least one polynucleotide(s) of the invention
can be modified by the sequential insertion of additional
polynucleotide(s) in the same plasmid, wherein the additional
polynucleotide(s) is/are operably linked to the same regulatory
elements as the original at least one polynucleotide(s). In some
embodiments, a nucleic acid molecule may be designed for the
inhibition of multiple target genes. In some embodiments, the
multiple genes to be inhibited can be obtained from the same
hemipteran pest species, which may enhance the effectiveness of the
nucleic acid molecule. In other embodiments, the genes can be
derived from different hemipteran pests, which may broaden the
range of pests against which the agent(s) is/are effective. When
multiple genes are targeted for suppression or a combination of
expression and suppression, a polycistronic DNA element can be
engineered.
[0161] A recombinant nucleic acid molecule or vector of the present
invention may comprise a selectable marker that confers a
selectable phenotype on a transformed cell, such as a plant cell.
Selectable markers may also be used to select for plants or plant
cells that comprise a recombinant nucleic acid molecule of the
invention. The marker may encode biocide resistance, antibiotic
resistance (e.g., kanamycin, Geneticin (G418), bleomycin,
hygromycin, etc.), or herbicide tolerance (e.g., glyphosate, etc.).
Examples of selectable markers include, but are not limited to: a
neo gene which codes for kanamycin resistance and can be selected
for using kanamycin, G418, etc.; a bar gene which codes for
bialaphos resistance; a mutant EPSP synthase gene which encodes
glyphosate tolerance; a nitrilase gene which confers resistance to
bromoxynil; a mutant acetolactate synthase (ALS) gene which confers
imidazolinone or sulfonylurea resistance; and a methotrexate
resistant DHFR gene. Multiple selectable markers are available that
confer resistance to ampicillin, bleomycin, chloramphenicol,
gentamycin, hygromycin, kanamycin, lincomycin, methotrexate,
phosphinothricin, puromycin, spectinomycin, rifampicin,
streptomycin and tetracycline, and the like. Examples of such
selectable markers are illustrated in, e.g., U.S. Pat. Nos.
5,550,318; 5,633,435; 5,780,708 and 6,118,047.
[0162] A recombinant nucleic acid molecule or vector of the present
invention may also include a screenable marker. Screenable markers
may be used to monitor expression. Exemplary screenable markers
include a .beta.-glucuronidase or uidA gene (GUS) which encodes an
enzyme for which various chromogenic substrates are known
(Jefferson et al. (1987) Plant Mol. Biol. Rep. 5:387-405); an
R-locus gene, which encodes a product that regulates the production
of anthocyanin pigments (red color) in plant tissues (Dellaporta et
al. (1988) "Molecular cloning of the maize R-nj allele by
transposon tagging with Ac." In 18.sup.th Stadler Genetics
Symposium, P. Gustafson and R. Appels, eds. (New York: Plenum), pp.
263-82); a .beta.-lactamase gene (Sutcliffe et al. (1978) Proc.
Natl. Acad. Sci. USA 75:3737-41); a gene which encodes an enzyme
for which various chromogenic substrates are known (e.g., PADAC, a
chromogenic cephalosporin); a luciferase gene (Ow et al. (1986)
Science 234:856-9); an xy/E gene that encodes a catechol
dioxygenase that can convert chromogenic catechols (Zukowski et al.
(1983) Gene 46(2-3):247-55); an amylase gene (Ikatu et al. (1990)
Bio/Technol. 8:241-2); a tyrosinase gene which encodes an enzyme
capable of oxidizing tyrosine to DOPA and dopaquinone which in turn
condenses to melanin (Katz et al. (1983) J. Gen. Microbiol.
129:2703-14); and an .alpha.-galactosidase.
[0163] In some embodiments, recombinant nucleic acid molecules, as
described, supra, may be used in methods for the creation of
transgenic plants and expression of heterologous nucleic acids in
plants to prepare transgenic plants that exhibit reduced
susceptibility to hemipteran pests. Plant transformation vectors
can be prepared, for example, by inserting nucleic acid molecules
encoding iRNA molecules into plant transformation vectors and
introducing these into plants.
[0164] Suitable methods for transformation of host cells include
any method by which DNA can be introduced into a cell, such as by
transformation of protoplasts (See, e.g., U.S. Pat. No. 5,508,184),
by desiccation/inhibition-mediated DNA uptake (See, e.g., Potrykus
et al. (1985) Mol. Gen. Genet. 199:183-8), by electroporation (See,
e.g., U.S. Pat. No. 5,384,253), by agitation with silicon carbide
fibers (See, e.g., U.S. Pat. Nos. 5,302,523 and 5,464,765), by
Agrobacterium-mediated transformation (See, e.g., U.S. Pat. Nos.
5,563,055; 5,591,616; 5,693,512; 5,824,877; 5,981,840; and
6,384,301) and by acceleration of DNA-coated particles (See, 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), etc. Techniques that are particularly
useful for transforming corn are described, for example, in U.S.
Pat. Nos. 7,060,876 and 5,591,616; and International PCT
Publication WO95/06722. Through the application of techniques such
as these, the cells of virtually any species may be stably
transformed. In some embodiments, transforming DNA is integrated
into the genome of the host cell. In the case of multicellular
species, transgenic cells may be regenerated into a transgenic
organism. Any of these techniques may be used to produce a
transgenic plant, for example, comprising one or more nucleic acids
encoding one or more iRNA molecules in the genome of the transgenic
plant.
[0165] The most widely utilized method for introducing an
expression vector into plants is based on the natural
transformation system of Agrobacterium. A. tumefaciens and A.
rhizogenes are plant pathogenic soil bacteria which genetically
transform plant cells. The Ti and Ri plasmids of A. tumefaciens and
A. rhizogenes, respectively, carry genes responsible for genetic
transformation of the plant. The Ti (tumor-inducing)-plasmids
contain a large segment, known as T-DNA, which is transferred to
transformed plants. Another segment of the Ti plasmid, the Vir
region, is responsible for T-DNA transfer. The T-DNA region is
bordered by terminal repeats. In modified binary vectors, the
tumor-inducing genes have been deleted, and the functions of the
Vir region are utilized to transfer foreign DNA bordered by the
T-DNA border elements. The T-region may also contain a selectable
marker for efficient recovery of transgenic cells and plants, and a
multiple cloning site for inserting polynucleotides for transfer
such as a dsRNA encoding nucleic acid.
[0166] In some embodiments, a plant transformation vector is
derived from a Ti plasmid of A. tumefaciens (See, e.g., U.S. Pat.
Nos. 4,536,475, 4,693,977, 4,886,937, and 5,501,967; and European
Patent No. EP 0 122 791) or a Ri plasmid of A. rhizogenes.
Additional plant transformation vectors include, for example and
without limitation, those described by Herrera-Estrella et al.
(1983) Nature 303:209-13; Bevan et al. (1983) Nature 304:184-7;
Klee et al. (1985) Bio/Technol. 3:637-42; and in European Patent
No. EP 0 120 516, and those derived from any of the foregoing.
Other bacteria such as Sinorhizobium, Rhizobium, and Mesorhizobium
that interact with plants naturally can be modified to mediate gene
transfer to a number of diverse plants. These plant-associated
symbiotic bacteria can be made competent for gene transfer by
acquisition of both a disarmed Ti plasmid and a suitable binary
vector.
[0167] After providing exogenous DNA to recipient cells,
transformed cells are generally identified for further culturing
and plant regeneration. In order to improve the ability to identify
transformed cells, one may desire to employ a selectable or
screenable marker gene, as previously set forth, with the
transformation vector used to generate the transformant. In the
case where a selectable marker is used, transformed cells are
identified within the potentially transformed cell population by
exposing the cells to a selective agent or agents. In the case
where a screenable marker is used, cells may be screened for the
desired marker gene trait.
[0168] Cells that survive the exposure to the selective agent, or
cells that have been scored positive in a screening assay, may be
cultured in media that supports regeneration of plants. In some
embodiments, any suitable plant tissue culture media (e.g., MS and
N6 media) may be modified by including further substances, such as
growth regulators. Tissue may be maintained on a basic medium with
growth regulators until sufficient tissue is available to begin
plant regeneration efforts, or following repeated rounds of manual
selection, until the morphology of the tissue is suitable for
regeneration (e.g., at least 2 weeks), then transferred to media
conducive to shoot formation. Cultures are transferred periodically
until sufficient shoot formation has occurred. Once shoots are
formed, they are transferred to media conducive to root formation.
Once sufficient roots are formed, plants can be transferred to soil
for further growth and maturation.
[0169] To confirm the presence of a nucleic acid molecule of
interest (for example, a DNA encoding one or more iRNA molecules
that inhibit target gene expression in a hemipteran pest) in the
regenerating plants, a variety of assays may be performed. Such
assays include, for example: molecular biological assays, such as
Southern and northern blotting, PCR, and nucleic acid sequencing;
biochemical assays, such as detecting the presence of a protein
product, e.g., by immunological means (ELISA and/or western blots)
or by enzymatic function; plant part assays, such as leaf or root
assays; and analysis of the phenotype of the whole regenerated
plant.
[0170] Integration events may be analyzed, for example, by PCR
amplification using, e.g., oligonucleotide primers specific for a
nucleic acid molecule of interest. PCR genotyping is understood to
include, but not be limited to, polymerase-chain reaction (PCR)
amplification of gDNA derived from isolated host plant callus
tissue predicted to contain a nucleic acid molecule of interest
integrated into the genome, followed by standard cloning and
sequence analysis of PCR amplification products. Methods of PCR
genotyping have been well described (for example, Rios, G. et al.
(2002) Plant J. 32:243-53) and may be applied to gDNA derived from
any plant species (e.g., Z. mays, G. max, and Gossypium sp.) or
tissue type, including cell cultures.
[0171] A transgenic plant formed using Agrobacterium-dependent
transformation methods typically contains a single recombinant DNA
inserted into one chromosome. The polynucleotide of the single
recombinant DNA is referred to as a "transgenic event" or
"integration event." Such transgenic plants are heterozygous for
the inserted exogenous polynucleotide. In some embodiments, a
transgenic plant homozygous with respect to a transgene may be
obtained by sexually mating (selfing) an independent segregant
transgenic plant that contains a single exogenous gene to itself,
for example a T.sub.0 plant, to produce T.sub.1 seed. One fourth of
the T.sub.1 seed produced will be homozygous with respect to the
transgene. Germinating T.sub.1 seed results in plants that can be
tested for heterozygosity, typically using a SNP assay or a thermal
amplification assay that allows for the distinction between
heterozygotes and homozygotes (i.e., a zygosity assay).
[0172] In particular embodiments, at least 2, 3, 4, 5, 6, 7, 8, 9
or 10 or more different iRNA molecules are produced in a plant cell
that have a hemipteran pest-inhibitory effect. The iRNA molecules
(e.g., dsRNA molecules) may be expressed from multiple nucleic
acids introduced in different transformation events, or from a
single nucleic acid introduced in a single transformation event. In
some embodiments, a plurality of iRNA molecules are expressed under
the control of a single promoter. In other embodiments, a plurality
of iRNA molecules are expressed under the control of multiple
promoters. Single iRNA molecules may be expressed that comprise
multiple polynucleotides that are each homologous to different loci
within one or more hemipteran pests (for example, the loci defined
by SEQ ID NO:1), both in different populations of the same species
of hemipteran pest, or in different species of hemipteran
pests.
[0173] In addition to direct transformation of a plant with a
recombinant nucleic acid molecule, transgenic plants can be
prepared by crossing a first plant having at least one transgenic
event with a second plant lacking such an event. For example, a
recombinant nucleic acid molecule comprising a polynucleotide that
encodes an iRNA molecule may be introduced into a first plant line
that is amenable to transformation to produce a transgenic plant,
which transgenic plant may be crossed with a second plant line to
introgress the polynucleotide that encodes the iRNA molecule into
the second plant line.
[0174] The invention also includes commodity products containing
one or more of the polynucleotides of the present invention.
Particular embodiments include commodity products produced from a
recombinant plant or seed containing one or more of the
polynucleotides of the present invention. A commodity product
containing one or more of the polynucleotides of the present
invention is intended to include, but not be limited to, meals,
oils, crushed or whole grains or seeds of a plant, or any food
product comprising any meal, oil, or crushed or whole grain of a
recombinant plant or seed containing one or more of the
polynucleotides of the present invention. The detection of one or
more of the polynucleotides of the present invention in one or more
commodity or commodity products contemplated herein is de facto
evidence that the commodity or commodity product is produced from a
transgenic plant designed to express one or more of the
polynucleotides of the present invention for the purpose of
controlling plant pests using dsRNA-mediated gene suppression
methods.
[0175] In some aspects, seeds and commodity products produced by
transgenic plants derived from transformed plant cells are
included, wherein the seeds or commodity products comprise a
detectable amount of a nucleic acid of the invention. In some
embodiments, such commodity products may be produced, for example,
by obtaining transgenic plants and preparing food or feed from
them. Commodity products comprising one or more of the
polynucleotides of the invention includes, for example and without
limitation: meals, oils, crushed or whole grains or seeds of a
plant, and any food product comprising any meal, oil, or crushed or
whole grain of a recombinant plant or seed comprising one or more
of the nucleic acids of the invention. The detection of one or more
of the polynucleotides of the invention in one or more commodity or
commodity products is de facto evidence that the commodity or
commodity product is produced from a transgenic plant designed to
express one or more of the iRNA molecules of the invention for the
purpose of controlling hemipteran pests.
[0176] In some embodiments, a transgenic plant or seed comprising a
nucleic acid molecule of the invention also may comprise at least
one other transgenic event in its genome, including without
limitation: a transgenic event from which is transcribed an iRNA
molecule targeting a locus in a hemipteran pest other than the ones
defined by SEQ ID NO:1; a transgenic event from which is
transcribed an iRNA molecule targeting a gene in an organism other
than a hemipteran pest (e.g., a plant-parasitic nematode); a gene
encoding an insecticidal protein (e.g., a Bacillus thuringiensis
insecticidal protein); an herbicide tolerance gene (e.g., a gene
providing tolerance to glyphosate); and a gene contributing to a
desirable phenotype in the transgenic plant, such as increased
yield, altered fatty acid metabolism, or restoration of cytoplasmic
male sterility. In particular embodiments, polynucleotides encoding
iRNA molecules of the invention may be combined with other insect
control and disease traits in a plant to achieve desired traits for
enhanced control of plant disease and insect damage. Combining
insect control traits that employ distinct modes-of-action may
provide protected transgenic plants with superior durability over
plants harboring a single control trait, for example, because of
the reduced probability that resistance to the trait(s) will
develop in the field.
V. Target Gene Suppression in a Hemipteran Pest
[0177] A. Overview
[0178] In some embodiments of the invention, at least one nucleic
acid molecule useful for the control of hemipteran pests may be
provided to a hemipteran pest, wherein the nucleic acid molecule
leads to RNAi-mediated gene silencing in the pest(s). In particular
embodiments, an iRNA molecule (e.g., dsRNA, siRNA, miRNA, shRNA,
and hpRNA) may be provided to the hemipteran host. In some
embodiments, a nucleic acid molecule useful for the control of
hemipteran pests may be provided to a pest by contacting the
nucleic acid molecule with the pest. In these and further
embodiments, a nucleic acid molecule useful for the control of
hemipteran pests may be provided in a feeding substrate of the
pest, for example, a nutritional composition. In these and further
embodiments, a nucleic acid molecule useful for the control of a
hemipteran pest may be provided through ingestion of plant material
comprising the nucleic acid molecule that is ingested by the
pest(s). In certain embodiments, the nucleic acid molecule is
present in plant material through expression of a recombinant
nucleic acid introduced into the plant material, for example, by
transformation of a plant cell with a vector comprising the
recombinant nucleic acid and regeneration of a plant material or
whole plant from the transformed plant cell.
[0179] B. RNAi-Mediated Target Gene Suppression
[0180] In particular embodiments, the invention provides iRNA
molecules (e.g., dsRNA, siRNA, miRNA, shRNA, and hpRNA) that may be
designed to target essential native polynucleotides (e.g.,
essential genes) in the transcriptome of a hemipteran (e.g., BSB)
pest, for example by designing an iRNA molecule that comprises at
least one strand comprising a polynucleotide that is specifically
complementary to the target polynucleotide. The sequence of an iRNA
molecule so designed may be identical to that of the target
polynucleotide, or may incorporate mismatches that do not prevent
specific hybridization between the iRNA molecule and its target
polynucleotide.
[0181] iRNA molecules of the invention may be used in methods for
gene suppression in a hemipteran pest, thereby reducing the level
or incidence of damage caused by the pest on a plant (for example,
a protected transformed plant comprising an iRNA molecule). As used
herein the term "gene suppression" refers to any of the well-known
methods for reducing the levels of protein produced as a result of
gene transcription to mRNA and subsequent translation of the mRNA,
including the reduction of protein expression from a gene or a
coding polynucleotide including post-transcriptional inhibition of
expression and transcriptional suppression. Post-transcriptional
inhibition is mediated by specific homology between all or a part
of an mRNA transcribed from a gene targeted for suppression and the
corresponding iRNA molecule used for suppression. Additionally,
post-transcriptional inhibition refers to the substantial and
measurable reduction of the amount of mRNA available in the cell
for binding by ribosomes.
[0182] In some embodiments wherein an iRNA molecule is a dsRNA
molecule, the dsRNA molecule may be cleaved by the enzyme, DICER,
into short siRNA molecules (approximately 20 nucleotides in
length). The double-stranded siRNA molecule generated by DICER
activity upon the dsRNA molecule may be separated into two
single-stranded siRNAs; the "passenger strand" and the "guide
strand." The passenger strand may be degraded, and the guide strand
may be incorporated into RISC. Post-transcriptional inhibition
occurs by specific hybridization of the guide strand with a
specifically complementary polynucleotide of an mRNA molecule, and
subsequent cleavage by the enzyme, Argonaute (catalytic component
of the RISC complex).
[0183] In other embodiments of the invention, any form of iRNA
molecule may be used. Those of skill in the art will understand
that dsRNA molecules typically are more stable during preparation
and during the step of providing the iRNA molecule to a cell than
are single-stranded RNA molecules, and are typically also more
stable in a cell. Thus, while siRNA and miRNA molecules, for
example, may be equally effective in some embodiments, a dsRNA
molecule may be chosen due to its stability.
[0184] In particular embodiments, a nucleic acid molecule is
provided that comprises a polynucleotide, which polynucleotide may
be expressed in vitro to produce an iRNA molecule that is
substantially homologous to a nucleic acid molecule encoded by a
polynucleotide within the genome of a hemipteran pest. In certain
embodiments, the in vitro transcribed iRNA molecule may be a
stabilized dsRNA molecule that comprises a stem-loop structure.
After a hemipteran pest contacts the in vitro transcribed iRNA
molecule, post-transcriptional inhibition of a target gene in the
pest (for example, an essential gene) may occur.
[0185] In some embodiments of the invention, expression of an iRNA
from a nucleic acid molecule comprising at least 15 contiguous
nucleotides (e.g., at least 19 contiguous nucleotides) of a
polynucleotide are used in a method for post-transcriptional
inhibition of a target gene in a hemipteran pest, wherein the
polynucleotide is selected from the group consisting of: SEQ ID
NO:1; the complement of SEQ ID NO:1; a fragment of at least 15
contiguous nucleotides of SEQ ID NO:1 (e.g., SEQ ID NO:3 and SEQ ID
NO:4); the complement of a fragment of at least 15 contiguous
nucleotides of SEQ ID NO:1; a native coding polynucleotide of a
hemipteran organism comprising any of SEQ ID NOs:1, 3, and 4; the
complement of a native coding polynucleotide of a hemipteran
organism comprising any of SEQ ID NOs:1, 3, and 4; a fragment of at
least 15 contiguous nucleotides of a native coding polynucleotide
of a hemipteran organism comprising any of SEQ ID NOs:1, 3, and 4;
and the complement of a fragment of at least 15 contiguous
nucleotides of a native coding polynucleotide of a hemipteran
organism comprising any of SEQ ID NOs:1, 3, and 4. In certain
embodiments, expression of a nucleic acid molecule that is at least
about 80% identical (e.g., 79%, about 80%, about 81%, about 82%,
about 83%, about 84%, about 85%, about 86%, about 87%, about 88%,
about 89%, about 90%, about 91%, about 92%, about 93%, about 94%,
about 95%, about 96%, about 97%, about 98%, about 99%, about 100%,
and 100%) with any of the foregoing may be used. In these and
further embodiments, a nucleic acid molecule may be expressed that
specifically hybridizes to an RNA molecule present in at least one
cell of a hemipteran pest.
[0186] It is an important feature of some embodiments herein that
the RNAi post-transcriptional inhibition system is able to tolerate
sequence variations among target genes that might be expected due
to genetic mutation, strain polymorphism, or evolutionary
divergence. The introduced nucleic acid molecule may not need to be
absolutely homologous to either a primary transcription product or
a fully-processed mRNA of a target gene, so long as the introduced
nucleic acid molecule is specifically hybridizable to either a
primary transcription product or a fully-processed mRNA of the
target gene. Moreover, the introduced nucleic acid molecule may not
need to be full-length, relative to either a primary transcription
product or a fully processed mRNA of the target gene.
[0187] Inhibition of a target gene using the iRNA technology of the
present invention is sequence-specific; i.e., polynucleotides
substantially homologous to the iRNA molecule(s) are targeted for
genetic inhibition. In some embodiments, an RNA molecule comprising
a polynucleotide with a nucleotide sequence that is identical to
that of a portion of a target gene may be used for inhibition. In
these and further embodiments, an RNA molecule comprising a
polynucleotide with one or more insertion, deletion, and/or point
mutations relative to a target polynucleotide may be used. In
particular embodiments, an iRNA molecule and a portion of a target
gene may share, for example, at least from about 80%, at least from
about 81%, at least from about 82%, at least from about 83%, at
least from about 84%, at least from about 85%, at least from about
86%, at least from about 87%, at least from about 88%, at least
from about 89%, at least from about 90%, at least from about 91%,
at least from about 92%, at least from about 93%, at least from
about 94%, at least from about 95%, at least from about 96%, at
least from about 97%, at least from about 98%, at least from about
99%, at least from about 100%, and 100% sequence identity.
Alternatively, the duplex region of a dsRNA molecule may be
specifically hybridizable with a portion of a target gene
transcript. In specifically hybridizable molecules, a less than
full length polynucleotide exhibiting a greater homology
compensates for a longer, less homologous polynucleotide. The
length of the polynucleotide of a duplex region of a dsRNA molecule
that is identical to a portion of a target gene transcript may be
at least about 25, 50, 100, 200, 300, 400, 500, or at least about
1000 bases. In some embodiments, a polynucleotide of greater than
20-100 nucleotides may be used, for example, a polynucleotide of
100-200 or 300-500 nucleotides may be used. In particular
embodiments, a polynucleotide of greater than about 200-300
nucleotides may be used. In particular embodiments, a
polynucleotide of greater than about 500-1000 nucleotides may be
used, depending on the size of the target gene.
[0188] In certain embodiments, expression of a target gene in a
hemipteran pest may be inhibited by at least 10%; at least 33%; at
least 50%; or at least 80% within a cell of the pest, such that a
significant inhibition takes place. Significant inhibition refers
to inhibition over a threshold that results in a detectable
phenotype (e.g., cessation of reproduction, feeding, development,
etc.), or a detectable decrease in RNA and/or gene product
corresponding to the target gene being inhibited. Although in
certain embodiments of the invention inhibition occurs in
substantially all cells of the pest, in other embodiments
inhibition occurs only in a subset of cells expressing the target
gene.
[0189] In some embodiments, transcriptional suppression is mediated
by the presence in a cell of a dsRNA molecule exhibiting
substantial sequence identity to a promoter DNA or the complement
thereof to effect what is referred to as "promoter trans
suppression." Gene suppression may be effective against target
genes in a hemipteran pest that may ingest or contact such dsRNA
molecules, for example, by ingesting or contacting plant material
containing the dsRNA molecules. dsRNA molecules for use in promoter
trans suppression may be specifically designed to inhibit or
suppress the expression of one or more homologous or complementary
polynucleotides in the cells of the hemipteran pest.
Post-transcriptional gene suppression by antisense or sense
oriented RNA to regulate gene expression in plant cells is
disclosed in U.S. Pat. Nos. 5,107,065; 5,759,829; 5,283,184; and
5,231,020.
[0190] C. Expression of RNAi Molecules Provided to a Hemipteran
Pest
[0191] Expression of iRNA molecules for RNAi-mediated gene
inhibition in a hemipteran pest may be carried out in any one of
many in vitro or in vivo formats. The iRNA molecules may then be
provided to a hemipteran pest, for example, by contacting the iRNA
molecules with the pest, or by causing the pest to ingest or
otherwise internalize the iRNA molecules. Some embodiments of the
invention include transformed host plants of a hemipteran pest,
transformed plant cells, and progeny of transformed plants. The
transformed plant cells and transformed plants may be engineered to
express one or more of the iRNA molecules, for example, under the
control of a heterologous promoter, to provide a pest-protective
effect. Thus, when a transgenic plant or plant cell is consumed by
a hemipteran pest during feeding, the pest may ingest iRNA
molecules expressed in the transgenic plants or cells. The
polynucleotides of the present invention may also be introduced
into a wide variety of prokaryotic and eukaryotic microorganism
hosts to produce iRNA molecules. The term "microorganism" includes
prokaryotic and eukaryotic species, such as bacteria and fungi.
[0192] Modulation of gene expression may include partial or
complete suppression of such expression. In another embodiment, a
method for suppression of gene expression in a hemipteran pest
comprises providing in the tissue of the host of the pest a
gene-suppressive amount of at least one dsRNA molecule formed
following transcription of a polynucleotide as described herein, at
least one segment of which is complementary to an mRNA within the
cells of the hemipteran pest. A dsRNA molecule, including its
modified form such as an siRNA, miRNA, shRNA, or hpRNA molecule,
ingested by a hemipteran pest in accordance with the invention may
be at least from about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or about
100% identical to an RNA molecule transcribed from a kruppel DNA
molecule, for example, comprising a polynucleotide selected from
the group consisting of SEQ ID NOs:1, 3, and 4. Isolated and
substantially purified nucleic acid molecules including, but not
limited to, non-naturally occurring polynucleotides and recombinant
DNA constructs for providing dsRNA molecules of the present
invention are therefore provided, which suppress or inhibit the
expression of an endogenous coding polynucleotide or a target
coding polynucleotide in the hemipteran pest when introduced
thereto.
[0193] Particular embodiments provide a delivery system for the
delivery of iRNA molecules for the post-transcriptional inhibition
of one or more target gene(s) in a hemipteran plant pest and
control of a population of the plant pest. In some embodiments, the
delivery system comprises ingestion of a host transgenic plant cell
or contents of the host cell comprising RNA molecules transcribed
in the host cell. In these and further embodiments, a transgenic
plant cell or a transgenic plant is created that contains a
recombinant DNA construct providing a stabilized dsRNA molecule of
the invention. Transgenic plant cells and transgenic plants
comprising nucleic acids encoding a particular iRNA molecule may be
produced by employing recombinant DNA technologies (which basic
technologies are well-known in the art) to construct a plant
transformation vector comprising a polynucleotide encoding an iRNA
molecule of the invention (e.g., a stabilized dsRNA molecule); to
transform a plant cell or plant; and to generate the transgenic
plant cell or the transgenic plant that contains the transcribed
iRNA molecule.
[0194] To impart protection from hemipteran pests to a transgenic
plant, a recombinant DNA molecule may, for example, be transcribed
into an iRNA molecule, such as a dsRNA molecule, an siRNA molecule,
an miRNA molecule, an shRNA molecule, or an hpRNA molecule. In some
embodiments, an RNA molecule transcribed from a recombinant DNA
molecule may form a dsRNA molecule within the tissues or fluids of
the recombinant plant. Such a dsRNA molecule may be comprised in
part of a polynucleotide that is identical to a corresponding
polynucleotide transcribed from a DNA within a hemipteran pest of a
type that may infest the host plant. Expression of a target gene
within the hemipteran pest is suppressed by the dsRNA molecule, and
the suppression of expression of the target gene in the hemipteran
pest results in the transgenic plant being resistant to the pest.
The modulatory effects of dsRNA molecules have been shown to be
applicable to a variety of genes expressed in pests, including, for
example, endogenous genes responsible for cell division,
chromosomal remodeling, and cellular metabolism or cellular
transformation, including housekeeping genes; transcription
factors; molting-related genes; and other genes which encode
polypeptides involved in cellular metabolism or normal growth and
development.
[0195] For transcription from a transgene in vivo or an expression
construct, a regulatory region (e.g., promoter, enhancer, silencer,
and polyadenylation signal) may be used in some embodiments to
transcribe the RNA strand (or strands). Therefore, in some
embodiments, as set forth, supra, a polynucleotide for use in
producing iRNA molecules may be operably linked to one or more
promoter elements functional in a plant host cell. The promoter may
be an endogenous promoter, normally resident in the host genome.
The polynucleotide of the present invention, under the control of
an operably linked promoter element, may further be flanked by
additional elements that advantageously affect its transcription
and/or the stability of a resulting transcript. Such elements may
be located upstream of the operably linked promoter, downstream of
the 3' end of the expression construct, and may occur both upstream
of the promoter and downstream of the 3' end of the expression
construct.
[0196] In embodiments, suppression of a target gene (e.g., a
kruppel gene) results in a parental RNAi phenotype; a phenotype
that is observable in progeny of the subject (e.g., a hemipteran
pest) contacted with the iRNA molecule. In some embodiments, the
pRNAi phenotype comprises the pest being rendered less able to
produce viable offspring. In particular examples of pRNAi, a
nucleic acid that initiates pRNAi does not increase the incidence
of mortality in a population into which the nucleic acid is
delivered. In other examples of pRNAi, a nucleic acid that
initiates pRNAi also increases the incidence of mortality in a
population into which the nucleic acid is delivered.
[0197] In some embodiments, a population of hemipteran pests is
contacted with an iRNA molecule, thereby resulting in pRNAi,
wherein the pests survive and mate but produce eggs that are less
able to hatch viable progeny than eggs produced by pests of the
same species that are not provided the nucleic acid(s). In some
examples, such pests do not oviposit eggs or oviposit fewer eggs
than what is observable in pests of the same species that are not
contacted with the iRNA molecule. In some examples, the eggs
oviposited by such pests do not hatch or hatch at a rate that is
significantly less than what is observable in pests of the same
species that are not contacted with the iRNA molecule. In some
examples, the nymphs that hatch from eggs oviposited by such pests
are not viable or are less viable than what is observable in pests
of the same species that are not contacted with the iRNA
molecule.
[0198] Transgenic crops that produce substances that provide
protection from insect feeding are vulnerable to adaptation by the
target insect pest population reducing the durability of the
benefits of the insect protection substance(s). Traditionally,
delays in insect pest adaptation to transgenic crops are achieved
by (1) the planting of "refuges" (crops that do not contain the
pesticidal substances, and therefore allow survival of insects that
are susceptible to the pesticidal substance(s)); and/or (2)
combining insecticidal substances with multiple modes of action
against the target pests, so that individuals that are resistant to
one mode of action are killed by a second mode of action.
[0199] In some examples, iRNA molecules (e.g., expressed from a
transgene in a host plant) represent new modes of action for
combining with Bacillus thuringiensis insecticidal protein
technology and/or lethal RNAi technology in Insect Resistance
Management gene pyramids to mitigate against the development of
insect populations resistant to either of these control
technologies.
[0200] Parental RNAi may result in some embodiments in a type of
pest control that is different from the control obtained by lethal
RNAi, and which may be combined with lethal RNAi to result in
synergistic pest control. Thus, in particular embodiments, iRNA
molecules for the post-transcriptional inhibition of one or more
target gene(s) in a hemipteran plant pest can be combined with
other iRNA molecules to provide redundant RNAi targeting and
synergistic RNAi effects.
[0201] Parental RNAi (pRNAi) that causes egg mortality or loss of
egg viability has the potential to bring further durability
benefits to transgenic crops that use RNAi and other mechanisms for
insect protection. pRNAi prevents exposed insects from producing
progeny, and therefore from passing on to the next generation any
alleles they carry that confer resistance to the pesticidal
substance(s). pRNAi is particularly useful in extending the
durability of insect-protected transgenic crops when it is combined
with one or more additional pesticidal substances that provide
protection from the same pest populations. Such additional
pesticidal substances may in some embodiments include, for example,
nymph-active dsRNA; insecticidal proteins (such as those derived
from Bacillus thuringiensis or other organisms); and other
insecticidal substances. This benefit arises because insects that
are resistant to the pesticidal substances occur as a higher
proportion of the population in the transgenic crop than in the
refuge crop. If a ratio of resistance alleles to susceptible
alleles that are passed on to the next generation is lower in the
presence of pRNAi than in the absence of pRNAi, the evolution of
resistance will be delayed.
[0202] For example, pRNAi may not reduce the number of individuals
in a first pest generation that are inflicting damage on a plant
expressing an iRNA molecule. However, the ability of such pests to
sustain an infestation through subsequent generations may be
reduced. Conversely, lethal RNAi may kill pests that already are
infesting the plant. When pRNAi is combined with lethal RNAi, pests
that are contacted with a parental iRNA molecule may breed with
pests from outside the system that have not been contacted with the
iRNA, however, the progeny of such a mating may be non-viable or
less viable, and thus may be unable to infest the plant. At the
same time, pests that are contacted with a lethal iRNA molecule may
be directly affected. The combination of these two effects may be
synergistic; i.e., the combined pRNAi and lethal RNAi effect may be
greater than the sum of the pRNAi and lethal RNAi effects
independently. pRNAi may be combined with lethal RNAi, for example,
by providing a plant that expresses both lethal and parental iRNA
molecules; by providing in the same location a first plant that
expresses lethal iRNA molecules and a second plant that expresses
parental iRNA molecules; and/or by contacting pests with the pRNAi
molecule, and subsequently releasing the contacted pests into the
plant environment, such that they can mate unproductively with the
plant pests.
[0203] Some embodiments provide methods for reducing the damage to
a host plant (e.g., a soybean plant) caused by a hemipteran pest
that feeds on the plant, wherein the method comprises providing in
the host plant a transformed plant cell expressing at least one
nucleic acid molecule of the invention, wherein the nucleic acid
molecule(s) functions upon being taken up by the pest(s) to inhibit
the expression of a target polynucleotide within the pest(s), which
inhibition of expression results in reduced reproduction, for
example, in addition to mortality and/or reduced growth of the
pest(s), thereby reducing the damage to the host plant caused by
the pest(s). In some embodiments, the nucleic acid molecule(s)
comprise dsRNA molecules. In these and further embodiments, the
nucleic acid molecule(s) comprise dsRNA molecules that each
comprise more than one polynucleotide that is specifically
hybridizable to a nucleic acid molecule expressed in a hemipteran
pest cell. In some embodiments, the nucleic acid molecule(s)
consist of one polynucleotide that is specifically hybridizable to
a nucleic acid molecule expressed in a hemipteran pest cell.
[0204] In other embodiments, a method for increasing the yield of a
corn crop is provided, wherein the method comprises introducing
into a corn plant at least one nucleic acid molecule of the
invention; and cultivating the corn plant to allow the expression
of an iRNA molecule comprising the nucleic acid, wherein expression
of an iRNA molecule comprising the nucleic acid inhibits hemipteran
pest damage and/or growth, thereby reducing or eliminating a loss
of yield due to hemipteran pest infestation. In some embodiments,
the iRNA molecule is a dsRNA molecule. In these and further
embodiments, the nucleic acid molecule(s) comprise dsRNA molecules
that each comprise more than one polynucleotide that is
specifically hybridizable to a nucleic acid molecule expressed in a
hemipteran pest cell. In some embodiments, the nucleic acid
molecule(s) consists of one polynucleotide that is specifically
hybridizable to a nucleic acid molecule expressed in a hemipteran
pest cell.
[0205] In some embodiments, a method for increasing the yield of a
plant crop is provided, wherein the method comprises introducing
into a female hemipteran pest (e.g, by injection, by ingestion, by
spraying, and by expression from a DNA) at least one nucleic acid
molecule of the invention; and releasing the female pest into the
crop, wherein mating pairs including the female pest are unable or
less able to produce viable offspring, thereby reducing or
eliminating a loss of yield due to hemipteran pest infestation. In
particular embodiments, such a method provides control of
subsequent generations of the pest. In similar embodiments, the
method comprises introducing the nucleic acid molecule of the
invention into a male hemipteran pest, and releasing the male pest
into the crop (e.g., wherein pRNAi male pests produce less sperm
than untreated controls). In some embodiments, the nucleic acid
molecule is a DNA molecule that is expressed to produce an iRNA
molecule. In some embodiments, the nucleic acid molecule is a dsRNA
molecule. In these and further embodiments, the nucleic acid
molecule(s) comprise dsRNA molecules that each comprise more than
one polynucleotide that is specifically hybridizable to a nucleic
acid molecule expressed in a hemipteran pest cell. In some
embodiments, the nucleic acid molecule(s) consists of one
polynucleotide that is specifically hybridizable to a nucleic acid
molecule expressed in a hemipteran pest cell.
[0206] In some embodiments, a method for modulating the expression
of a target gene in a hemipteran pest is provided, the method
comprising: transforming a plant cell with a vector comprising a
polynucleotide encoding at least one iRNA molecule of the
invention, wherein the polynucleotide is operatively-linked to a
promoter and a transcription termination element; culturing the
transformed plant cell under conditions sufficient to allow for
development of a plant cell culture including a plurality of
transformed plant cells; selecting for transformed plant cells that
have integrated the polynucleotide into their genomes; screening
the transformed plant cells for expression of an iRNA molecule
encoded by the integrated polynucleotide; selecting a transgenic
plant cell that expresses the iRNA molecule; and feeding the
selected transgenic plant cell to the hemipteran pest. Plants may
also be regenerated from transformed plant cells that express an
iRNA molecule encoded by the integrated nucleic acid molecule. In
some embodiments, the iRNA molecule is a dsRNA molecule. In these
and further embodiments, the nucleic acid molecule(s) comprise
dsRNA molecules that each comprise more than one polynucleotide
that is specifically hybridizable to a nucleic acid molecule
expressed in a hemipteran pest cell. In some embodiments, the
nucleic acid molecule(s) consists of one polynucleotide that is
specifically hybridizable to a nucleic acid molecule expressed in a
hemipteran pest cell.
[0207] iRNA molecules of the invention can be incorporated within
the seeds of a plant species (e.g., soybean), either as a product
of expression from a recombinant gene incorporated into a genome of
the plant cells, or as incorporated into a coating or seed
treatment that is applied to the seed before planting. A plant cell
comprising a recombinant gene is considered to be a transgenic
event. Also included in embodiments of the invention are delivery
systems for the delivery of iRNA molecules to hemipteran pests. For
example, the iRNA molecules of the invention may be directly
introduced into the cells of a pest(s). Methods for introduction
may include direct mixing of iRNA into the diet of the hemipteran
pest (e.g., by mixing with plant tissue from a host for the pest),
as well as application of compositions comprising iRNA molecules of
the invention to host plant tissue. For example, iRNA molecules may
be sprayed onto a plant surface. Alternatively, an iRNA molecule
may be expressed by a microorganism, and the microorganism may be
applied onto the plant surface, or introduced into a root or stem
by a physical means such as an injection. As discussed, supra, a
transgenic plant may also be genetically engineered to express at
least one iRNA molecule in an amount sufficient to kill the
hemipteran pests known to infest the plant. iRNA molecules produced
by chemical or enzymatic synthesis may also be formulated in a
manner consistent with common agricultural practices, and used as
spray-on products for controlling plant damage by a hemipteran
pest. The formulations may include the appropriate adjuvants (e.g.,
stickers and wetters) required for efficient foliar coverage, as
well as UV protectants to protect iRNA molecules (e.g., dsRNA
molecules) from UV damage. Such additives are commonly used in the
bioinsecticide industry, and are well known to those skilled in the
art. Such applications may be combined with other spray-on
insecticide applications (biologically based or otherwise) to
enhance plant protection from hemipteran pests.
[0208] All references, including publications, patents, and patent
applications, cited herein are hereby incorporated by reference to
the extent they are not inconsistent with the explicit details of
this disclosure, and are so incorporated to the same extent as if
each reference were individually and specifically indicated to be
incorporated by reference and were set forth in its entirety
herein. The references discussed herein are provided solely for
their disclosure prior to the filing date of the present
application. Nothing herein is to be construed as an admission that
the inventors are not entitled to antedate such disclosure by
virtue of prior invention.
[0209] The following Examples are provided to illustrate certain
particular features and/or aspects. These Examples should not be
construed to limit the disclosure to the particular features or
aspects described.
EXAMPLES
Example 1: RNAi Constructs
Template Preparation by PCR and dsRNA Synthesis
[0210] The strategies used to provide specific templates for
kruppel dsRNA production are shown in FIG. 1A and FIG. 1B. Template
DNAs intended for use in kruppel dsRNA synthesis are prepared by
PCR using specific primers and first-strand cDNA prepared from
total RNA. For kruppel selected target gene regions, two separate
PCR amplifications are performed. FIG. 1. The first PCR
amplification introduces a T7 promoter sequence at the 5' end of
the amplified sense strands. The second reaction incorporates the
T7 promoter sequence at the 5' ends of the antisense strands. The
two PCR amplified fragments for each region of the target genes are
then mixed in approximately equal amounts, and the mixture is used
as transcription template for dsRNA production. FIG. 1.
[0211] For the YFP negative control, a single PCR amplification was
performed. FIG. 1B. The PCR amplification introduced a T7 promoter
sequence at the 5' ends of the amplified sense and antisense
strands. The two PCR amplified fragments for each region of the
target genes were then mixed in approximately equal amounts, and
the mixture was used as transcription template for dsRNA
production. FIG. 1B. dsRNA for the negative control YFP coding
region (SEQ ID NO:6) was produced using specific primers and a DNA
clone of the YFP coding region as template. The PCR product
amplified for kruppel and YFP were used as a template for in vitro
synthesis of dsRNAs using the MEGAscript high-yield transcription
kit (Applied Biosystems Inc., Foster City, Calif.). The synthesized
dsRNAs were purified using the RNeasy Mini kit (Qiagen, Valencia,
Calif.) or an AMBION.RTM. MEGAscript.RTM. RNAi kit, essentially as
prescribed by the manufacturer's instructions. dsRNA preparations
were quantified using a NANODROP.TM. 8000 spectrophotometer (THERMO
SCIENTIFIC, Wilmington, Del.) or equivalent means, and analyzed by
gel electrophoresis to determine purity.
Example 2: Real-Time PCR Analysis
[0212] Quantitative Real-Time PCR: Relative expression levels of
transcripts are analyzed by probe hydrolysis quantitative Real-Time
PCR (qPCR) using Roche LightCycler480. The PCR primers and
hydrolysis probes are designed using LightCycler Probe Design
Software 2.0 (Roche). dsRNA-injected insects or eggs are frozen on
dry ice. Tissue disruption is performed with a Klecko.TM. tissue
pulverizer (Garcia Manufacturing, Visalia, Calif.) in the kit RLT
lysis buffer with one stainless steel bead. Following tissue
maceration, the total RNA is isolated in high throughput format
using the RNeasy96.RTM. kit (Qiagen, #74182) following the kit
protocol with the optional on column DNaseI treatment step at
1/2.times. suggested concentration for 30 minutes and additional
DNaseI digest using Turbo.TM. DNase (AM2238, Life Technologies,
Carlsbad Calif.) for 1 hour at room temperature on the elutant.
[0213] First-strand cDNA synthesis for High-Throughput qPCR assay:
High capacity cDNA RT kit (part#4368813. Life technologies,
Carlsbad Calif.) is used according to manufacturer supplied
protocol with the following modifications. Total RNA is adjusted to
.about.10 ng/.mu.L with nuclease-free water. RNA samples (5 or no
more than 250 ng total RNA) is heated to 70.degree. C. for 10
minutes and cooled to 4.degree. C. Half reactions are initiated by
addition of 5 .mu.L of 2.times. mix. The primer mix, which is
supplied solely as random primers, is first spiked with custom
synthesized T.sub.20VN oligo (Integrated DNA Technologies,
Coralville Iowa) to a final concentration of 2 .mu.M, in order to
improve the sensitivity of 3'UTR based assays. Following first
strand synthesis, the samples are diluted 1:3 with nuclease free
water and stored at -20.degree. C. until ready to be assayed by
high-throughput (HTP) qPCR.
[0214] Non-injected insects and YFP dsRNA-injected insects are used
as controls. The BSB Actin (Act) (SEQ ID NO:18) is used as a
reference gene (Ponton et al. (2011) J Insect Physiol 57
(6):840-50). The primers and probes for the experimental and
reference genes appear in Table 1.
TABLE-US-00009 TABLE 1 Oligonucleotides and probes for BSB probe
hydrolysis qPCR assay. Reference GENE NAME SEQUENCE Actin Actin-F
TCAAGGAAAAACTGTGCTATGT (SEQ ID NO: 19) Actin Actin-R
TACCGATGGTGATGACCTGA (SEQ ID NO: 20) Actin Actin-FAM ACCGCCGCTGCC
(SEQ ID NO: 21)
Example 3: Construction of Plant Transformation Vectors
[0215] An entry vector harboring a target gene construct for dsRNA
hairpin formation comprising segments of kruppel (SEQ ID NO:1) is
assembled using a combination of chemically synthesized fragments
(DNA2.0, Menlo Park, Calif.) and standard molecular cloning
methods. Intramolecular hairpin formation by RNA primary
transcripts is facilitated by arranging (within a single
transcription unit) two copies of a target gene segment in opposite
orientation to one another, the two segments being separated by a
linker sequence (e.g. ST-LS1 intron, SEQ ID NO:7; Vancanneyt et al.
(1990) Mol. Gen. Genet. 220:245-250). Thus, the primary mRNA
transcript contains the two kruppel gene segment sequences as large
inverted repeats of one another, separated by the linker sequence.
A copy of a promoter (e.g. maize ubiquitin 1, U.S. Pat. No.
5,510,474; 35S from Cauliflower Mosaic Virus (CaMV); promoters from
rice actin genes; ubiquitin promoters; pEMU; MAS; maize H3 histone
promoter; ALS promoter; phaseolin gene promoter; cab; rubisco;
LAT52; Zm13; and/or apg) is used to drive production of the primary
mRNA hairpin transcript, and a fragment comprising a 3'
untranslated region, for example and without limitation, a maize
peroxidase 5 gene (ZmPer5 3'UTR v2; U.S. Pat. No. 6,699,984),
AtUbi10, AtEf1, or StPinII is used to terminate transcription of
the hairpin-RNA-expressing gene.
[0216] The entry vector described above is used in standard
GATEWAY.RTM. recombination reactions with a typical binary
destination vector to produce kruppel hairpin RNA expression
transformation vectors for Agrobacterium-mediated plant embryo
transformations.
[0217] A negative control binary vector which comprises a gene that
expresses a YFP hairpin dsRNA is constructed by means of standard
GATEWAY.RTM. recombination reactions with a typical binary
destination vector and entry vector. The entry vector comprises a
YFP hairpin sequence under the expression control of a maize
ubiquitin 1 promoter and a fragment comprising a 3' untranslated
region from a maize peroxidase 5 gene.
[0218] A binary destination vector comprises a herbicide tolerance
gene (aryloxyalknoate dioxygenase; (AAD-1 v3, U.S. Pat. No.
7,838,733, and Wright et al. (2010) Proc. Natl. Acad. Sci. U.S.A.
107:20240-5)) under the regulation of a plant operable promoter
(e.g., sugarcane bacilliform badnavirus (ScBV) promoter (Schenk et
al. (1999) Plant Mol. Biol. 39:1221-30) or ZmUbi1 (U.S. Pat. No.
5,510,474)). 5' UTR and intron from these promoters, are positioned
between the 3' end of the promoter segment and the start codon of
the AAD-1 coding region. A fragment comprising a 3' untranslated
region from a maize lipase gene (ZmLip 3'UTR; U.S. Pat. No.
7,179,902) is used to terminate transcription of the AAD-1
mRNA.
[0219] A further negative control binary vector that comprises a
gene that expresses a YFP protein, is constructed by means of
standard GATEWAY.RTM. recombination reactions with a typical binary
destination vector and entry vector. The binary destination vector
comprises a herbicide tolerance gene (aryloxyalknoate dioxygenase;
AAD-1 v3) (as above) under the expression regulation of a maize
ubiquitin 1 promoter and a fragment comprising a 3' untranslated
region from a maize lipase gene (ZmLip 3'UTR). The entry vector
comprises a YFP coding region under the expression control of a
maize ubiquitin 1 promoter and a fragment comprising a 3'
untranslated region from a maize peroxidase 5 gene.
Example 4: Insect Rearing and Candidate Gene Selection in
Neotropical Brown Stink Bug (Euschistus heros)
[0220] Insect Rearing.
[0221] Neotropical Brown Stink Bugs (BSB; Euschistus heros) were
reared in a 27.degree. C. incubator, at 65% relative humidity, with
16:8 hour light: dark cycle. One gram of eggs collected over 2-3
days was seeded in 5 L containers with filter paper discs at the
bottom; the containers were covered with #18 mesh for ventilation.
Each rearing container yielded approximately 300-400 adult BSB. At
all stages, the insects were fed fresh green beans three times per
week and a sachet of seed mixture containing sunflower seeds,
soybeans, and peanuts (3:1:1 by weight ratio) was replaced once a
week. Water was supplemented in vials with cotton plugs as wicks.
After the initial two weeks, insects were transferred to a new
container once a week.
[0222] RNAi Target Selection.
[0223] Six stages of BSB development were selected for mRNA library
preparation. Total RNA was extracted from insects frozen at
-70.degree. C. and homogenized in 10 volumes of Lysis/Binding
buffer in Lysing MATRIX A 2 mL tubes (MP BIOMEDICALS, Santa Ana,
Calif.) on a FastPrep.RTM.-24 Instrument (MP BIOMEDICALS). Total
mRNA was extracted using a mirVana.TM. miRNA Isolation Kit (AMBION;
INVITROGEN) according to the manufacturer's protocol. RNA
sequencing using an Illumina.RTM. HiSeg.TM. system (San Diego,
Calif.) provided candidate target gene sequences for use in RNAi
insect control technology. HiSeg.TM. generated a total of about 378
million reads for the six samples. Reads were assembled
individually for each sample using TRINITY assembler software
(Grabherr et al. (2011) Nature Biotech. 29:644-652). The assembled
transcripts were combined to generate a pooled transcriptome. This
BSB pooled transcriptome contains 378,457 sequences.
[0224] E. heros Kruppel Ortholog Identification.
[0225] A tBLASTn search of the E. heros pooled transcriptome was
performed using sequences of the Drosophila KRUPPEL (Kr-PA, GENBANK
Accession No. NP_523867) protein. E. heros kruppel (SEQ ID NO:1)
was identified as a Euschistus heros candidate target gene
[0226] The sequence of SEQ ID NO:1 is novel. There was no
significant homologous nucleotide sequence found with a search in
GENBANK. The closest homolog of the Diabrotica KRUPPEL amino acid
sequence (SEQ ID NO:2) is a Oncopeltus fasciatus protein having
GENBANK Accession No. AAT44519.1 (82% similar; 78% identical over
the homology region).
[0227] Template Preparation and dsRNA Synthesis.
[0228] cDNA was prepared from total BSB RNA extracted from a single
young adult insect (about 90 mg) using TRIzol.RTM. Reagent (LIFE
TECHNOLOGIES, Grand Island, N.Y.). The insect was homogenized at
room temperature in a 1.5 mL microcentrifuge tube with 200 .mu.L of
TRIzol.RTM. using a pellet pestle (FISHERBRAND, Grand Island, N.Y.)
and Pestle Motor Mixer (COLE-PARMER, Vernon Hills, Ill.). Following
homogenization, an additional 800 .mu.L of TRIzol.RTM. was added,
the homogenate was vortexed, and then incubated at room temperature
for five minutes. Cell debris was removed by centrifugation and the
supernatant was transferred to a new tube. Following
manufacturer-recommended TRIzol.RTM. extraction protocol for 1 mL
of TRIzol.RTM., the RNA pellet was dried at room temperature and
resuspended in 200 .mu.L Tris Buffer from a GFX PCR DNA AND GEL
EXTRACTION KIT (Illustra.TM.; GE HEALTHCARE LIFE SCIENCES,
Pittsburgh, Pa.) using Elution Buffer Type 4 (i.e. 10 mM Tris-HCl
pH8.0). RNA concentration was determined using a NANODROP.TM. 8000
spectrophotometer (THERMO SCIENTIFIC, Wilmington, Del.).
[0229] cDNA was reverse-transcribed from 5 .mu.g BSB total RNA
template and oligo dT primer using a SUPERSCRIPT III FIRST-STRAND
SYNTHESIS SYSTEM.TM. for RT-PCR (INVITROGEN), following the
supplier's recommended protocol. The final volume of the
transcription reaction was brought to 100 .mu.L with nuclease-free
water.
[0230] Primers for BSB_kr-1, dsRNA BSB_kr-1-F (SEQ ID NO:8) and
BSB_kr-1-R (SEQ ID NO:9), were used to amplify DNA templates for
dsRNA transcription. Primers for BSB_kr-2, dsRNA BSB_kr-2-F (SEQ ID
NO:10) and BSB_kr-2-R (SEQ ID NO:11), were used to amplify DNA
templates for dsRNA transcription. The DNA templates were amplified
using "touch-down" PCR (annealing temperature lowered from
60.degree. C. to 50.degree. C. in a 1.degree. C./cycle decrease)
with 1 .mu.L of cDNA (above) as the template. Fragments comprising
a 434 bp segment of BSB_kr-1 (SEQ ID NO:3) and a 605 bp segment
BSB_kr-2 (SEQ ID NO:4) were generated during 35 cycles of PCR. The
above procedure was used to amplify a 301 bp negative control
template YFPv2 (SEQ ID NO:12) using primers YFPv2_F (SEQ ID NO:13)
and YFPv2_R (SEQ ID NO:14). The BSB-specific and YFPv2 primers
contained a T7 phage promoter sequence (SEQ ID NO:5) at their 5'
ends, enabling the use of the aforementioned BSB DNA fragments for
dsRNA transcription.
[0231] dsRNAs were synthesized using 2 .mu.L of PCR product (above)
as the template with a MEGAscript.TM. RNAi kit (AMBION) or
HiScribe.RTM. T7 In Vitro Transcription Kit, used according to the
manufacturer's instructions. See FIG. 1. dsRNA was quantified on a
NANODROP.TM. 8000 spectrophotometer and diluted to 1 .mu.g/.mu.L in
nuclease-free 0.1.times.TE buffer (1 mM Tris HCL, 0.1 mM EDTA, pH
7.4).
Example 5: dsRNA Injection into 2.sup.nd Instar Neotropical Brown
Stink Bug (Euschistus heros) Nymphs
[0232] BSB Artificial Diet.
[0233] BSB artificial diet was prepared as follows and used within
two weeks of preparation. Lyophilized green beans were blended to a
fine powder in a MAGIC BULLET.RTM. blender while raw (organic)
peanuts were blended in a separate MAGIC BULLET.RTM. blender.
Blended dry ingredients were combined (weight percentages: green
beans, 35%; peanuts, 35%; sucrose, 5%; Vitamin complex (e.g.
Vanderzant Vitamin Mixture for insects, SIGMA-ALDRICH), 0.9%); in a
large MAGIC BULLET.RTM. blender, which was capped and shaken well
to mix the ingredients. The mixed dry ingredients were then added
to a mixing bowl. In a separate container, water and benomyl
anti-fungal agent (50 ppm; 25 .mu.L 20,000 ppm solution/50 mL diet
solution) were mixed well and then added to the dry ingredient
mixture. All ingredients were mixed by hand until the solution was
fully blended. The diet was shaped into desired sizes, wrapped
loosely in aluminum foil, heated for 4 hours at 60.degree. C., then
cooled and stored at 4.degree. C.
[0234] Injection of dsRNA into BSB Hemocoel.
[0235] BSB were reared on a green bean and seed diet, as the colony
described above, in a 27.degree. C. incubator at 65% relative
humidity and 16:8 hour light: dark photoperiod. Second instar
nymphs (each weighing 1 to 1.5 mg) were gently handled with a small
brush to prevent injury and were placed in a Petri dish on ice to
chill and immobilize the insects. Each insect was injected with
55.2 nL of a 500 ng/.mu.L dsRNA solution (i.e., 27.6 ng dsRNA;
dosage of 18.4 to 27.6 .mu.g/g body weight). Injections were
performed using a NANOJECT.TM. II injector (DRUMMOND SCIENTIFIC,
Broomhall, Pa.) equipped with an injection needle pulled from a
Drummond 3.5 inch #3-000-203-G/X glass capillary. The needle tip
was broken and the capillary was backfilled with light mineral oil,
then filled with 2 to 3 .mu.L of dsRNA. dsRNA was injected into the
abdomen of the nymphs (10 insects injected per dsRNA per trial),
and the trials were repeated on three different days. Injected
insects (5 per well) were transferred into 32-well trays (Bio-RT-32
Rearing Tray; BIO-SERV, Frenchtown, N.J.) containing a pellet of
artificial BSB diet and covered with Pull-N-Peel.TM. tabs
(BIO-CV-4; BIO-SERV). Moisture was supplied by means of 1.25 mL
water in a 1.5 mL microcentrifuge tube with a cotton wick. The
trays were incubated at 26.5.degree. C., 60% humidity and 16: 8
hour light: dark photoperiod. Viability counts and weights were
taken on day 7 after the injections.
[0236] Injection of dsRNA that Targets Kruppel mRNA in BSB 2.sup.nd
Instar Nymphs.
[0237] dsRNA homologous to a YFP coding region, YFPv2, was used as
a negative control in BSB injection experiments. As summarized in
Table 2, 27.6 ng BSB_kr-1 dsRNA injected into the hemocoel of
2.sup.nd instar BSB did not lead to increased mortality within
seven days.
TABLE-US-00010 TABLE 2 Results of E. heros kruppel dsRNA injection
into the hemocoel of 2.sup.nd instar E. heros nymphs seven days
after injection. Table shows mean percent mortality, N number of
trials, standard error of the mean (SEM) and a p-value of
two-tailed student t-test. Mean % t-test Treatment mortality SEM N
trials (p) BSB_kr-1 20 5.8 3 2.88E-01 Not injected 13 3.3 3
6.43E-01 YFPv2 dsRNA 10 5.8 3
Example 6: Egg Hatch Following dsRNA Injection in Euschistus
heros
[0238] Injection of dsRNA into Adult BSB Hemocoel.
[0239] BSB were reared as described above for the colony. In the
following exemplification, young adults (zero to three days post
adult molt) were collected and chilled in a secondary container on
ice. The females and males were separated based on structural
dimorphism of the genitalia. Female BSB were handled with
featherweight entomology forceps and injected with dsRNA using a
NANOJECT.TM. II injector (DRUMMOND SCIENTIFIC, Broomhall, Pa.)
equipped with an injection needle pulled from a Drummond 3.5 inch
#3-000-203-G/X glass capillary. The needle tip was broken and the
capillary was backfilled with light mineral oil then filled with 3
.mu.L dsRNA. Ten to twenty females (approximately 90 mg each) per
treatment were injected with dsRNA. Each female was injected into
the abdomen twice consecutively with 69 nL 1 .mu.g/.mu.L dsRNA for
a total of 138 nL (138 ng). Each batch of ten females was moved
into a 1 quart (.about.950 mL) bin with an opening in the lid and
#18 mesh for ventilation. Two adult males were added to each bin of
ten females. The insects were supplied with a vial of water, green
beans, and seeds as described in the rearing procedure. The insects
were kept at 26.5.degree. C., 60% humidity and 16:8 light: dark
photoperiod.
[0240] Surviving female counts, oviposited eggs and egg hatch
numbers were collected on a daily basis starting at nine days after
injection and continued for up to 24 days. Eggs were removed daily
and kept in Petri dishes or multi-well plates on a layer of 1%
agarose in water. Fresh green beans were supplied to adults every
other day. The adult insects were transferred into bins with fresh
water and food every week.
[0241] Females injected with dsRNA that targets a 301 nt sequence
(SEQ ID NO:12) of the YFP coding region were used as a negative
control and compared to un-injected and females injected with
BSB_kr-1 (SEQ ID NO:3) dsRNA.
[0242] Injections of dsRNA targeting kruppel in BSB females has no
significant effect on oviposition. As summarized in Table 3 kruppel
dsRNA-injected females oviposited somewhat lower numbers of eggs
from YFPv2 dsRNA injected and not injected controls. The average #
of eggs oviposited/day/female (Table 3) was not significantly
different from the YFP control. There was a surprising and
unexpected overall hatch rate from females treated with kruppel
dsRNA. The total number of eggs hatched were dramatically reduced
in all replications, ranging from 10% to 31%, as compared to 89%
hatch rate for YFPv2 (Table 4).
TABLE-US-00011 TABLE 3 Number of eggs oviposited per female per day
in three replicates of BSB_kr-1 dsRNA application. Ten to twenty
females were injected with dsRNA targeted against BSB kruppel or
negative control YFPv2. Egg counts encompass 14 days of collection,
starting at day 11 post injection. Two-tailed paired t-tests were
performed in Excel. total # mean # of of eggs eggs/day/ Std. Std.
t-test dsRNA oviposited female Deviation Error (p) Kr-1 rep. 1 662
5.65 1.46 0.390 5.87E-01 Kr-1 rep. 2 742 6.01 1.68 0.450 9.56E-01
Kr-1 rep. 3 385 4.99 2.18 0.583 2.23E-01 YFPv2 904 5.98 1.47
0.393
TABLE-US-00012 TABLE 4 Number of BSB eggs hatched per female per
day based on the eggs oviposited in Table 3. Eggs were collected on
daily basis, with most hatching on days 5, 6, and 7 after
collection. Two-tailed paired t-tests were performed in Excel for
mean # hatched/day/female. total # of % mean # eggs eggs egg
hatched/day/ Std. Std. dsRNA hatched hatch female Deviation Error
t-test (p) Kr-1 65 10% 0.66 0.98 0.261 1.67E-06* rep. 1 Kr-1 133
18% 1.15 0.93 0.249 8.91E-06* rep. 2 Kr-1 120 31% 1.91 1.40 0.373
1.72E-04* rep. 3 YFPv2 805 89% 5.30 1.46 0.391 *indicates
significant difference (p-value < 0.05)
Example 7: Transgenic Zea mays Comprising Hemipteran Pest
Sequences
[0243] Ten to 20 transgenic T.sub.0 Zea mays plants harboring
expression vectors for nucleic acids comprising SEQ ID NO:1, SEQ ID
NO:3, and/or SEQ ID NO:4 are generated as described in EXAMPLE 3. A
further 10-20 T.sub.1 Zea mays independent lines expressing hairpin
dsRNA for an RNAi construct are obtained for BSB challenge. Hairpin
dsRNA may be derived comprising a segment of SEQ ID NO:1. These are
confirmed through RT-PCR or other molecular analysis methods. Total
RNA preparations from selected independent T.sub.1 lines are
optionally used for RT-PCR with primers designed to bind in the
linker of the hairpin expression cassette in each of the RNAi
constructs. In addition, specific primers for each target gene in
an RNAi construct are optionally used to amplify and confirm the
production of the pre-processed mRNA required for siRNA production
in planta. The amplification of the desired bands for each target
gene confirms the expression of the hairpin RNA in each transgenic
Zea mays plant. Processing of the dsRNA hairpin of the target genes
into siRNA is subsequently optionally confirmed in independent
transgenic lines using RNA blot hybridizations.
[0244] Moreover, RNAi molecules having mismatch sequences with more
than 80% sequence identity to target genes affect hemipterans in a
way similar to that seen with RNAi molecules having 100% sequence
identity to the target genes. The pairing of mismatch sequence with
native sequences to form a hairpin dsRNA in the same RNAi construct
delivers plant-processed siRNAs capable of affecting the growth,
development, reproduction, and viability of feeding hemipteran
pests.
[0245] In planta delivery of dsRNA, siRNA, shRNA, hpRNA, or miRNA
corresponding to target genes and the subsequent uptake by
hemipteran pests through feeding results in down-regulation of the
target genes in the hemipteran pest through RNA-mediated gene
silencing. When the function of a target gene is important at one
or more stages of development, the growth, development, and/or
reproduction of the hemipteran pest is affected, and in the case of
at least one of Euschistus heros, Piezodorus guildinii, Halyomorpha
halys, Nezara viridula, Acrosternum hilare, and Euschistus servus
leads to failure to successfully infest, feed, develop, and/or
reproduce, or leads to death of the hemipteran pest. The choice of
target genes and the successful application of RNAi is then used to
control hemipteran pests.
[0246] Phenotypic Comparison of Transgenic RNAi Lines and
Non-Transformed Zea mays.
[0247] Target hemipteran pest genes or sequences selected for
creating hairpin dsRNA have no similarity to any known plant gene
sequence. Hence it is not expected that the production or the
activation of (systemic) RNAi by constructs targeting these
hemipteran pest genes or sequences will have any deleterious effect
on transgenic plants. However, development and morphological
characteristics of transgenic lines are compared with
non-transformed plants, as well as those of transgenic lines
transformed with an "empty" vector having no hairpin-expressing
gene. Plant root, shoot, foliage and reproduction characteristics
are compared. There is no observable difference in root length and
growth patterns of transgenic and non-transformed plants. Plant
shoot characteristics such as height, leaf numbers and sizes, time
of flowering, floral size and appearance are similar. In general,
there are no observable morphological differences between
transgenic lines and those without expression of target iRNA
molecules when cultured in vitro and in soil in the glasshouse.
Example 8: Transgenic Glycine max Comprising Hemipteran Pest
Sequences
[0248] Ten to 20 transgenic T.sub.0 Glycine max plants harboring
expression vectors for nucleic acids comprising SEQ ID NO:1 or a
segment of SEQ ID NO:1 are generated as is known in the art,
including for example by Agrobacterium-mediated transformation, as
follows. Mature soybean (Glycine max) seeds are sterilized
overnight with chlorine gas for sixteen hours. Following
sterilization with chlorine gas, the seeds are placed in an open
container in a LAMINAR.TM. flow hood to dispel the chlorine gas.
Next, the sterilized seeds are imbibed with sterile H.sub.2O for
sixteen hours in the dark using a black box at 24.degree. C.
[0249] Preparation of Split-Seed Soybeans.
[0250] The split soybean seed comprising a portion of an embryonic
axis protocol requires preparation of soybean seed material which
is cut longitudinally, using a #10 blade affixed to a scalpel,
along the hilum of the seed to separate and remove the seed coat,
and to split the seed into two cotyledon sections. Careful
attention is made to partially remove the embryonic axis, wherein
about 1/2-1/3 of the embryo axis remains attached to the nodal end
of the cotyledon.
[0251] Inoculation.
[0252] The split soybean seeds comprising a partial portion of the
embryonic axis are then immersed for about 30 minutes in a solution
of Agrobacterium tumefaciens (e.g., strain EHA 101 or EHA 105)
containing binary plasmid comprising SEQ ID NO:1, SEQ ID NO:3
and/or SEQ ID NO:4. The Agrobacterium tumefaciens solution is
diluted to a final concentration of .lamda.=0.6 OD.sub.650 before
immersing the cotyledons comprising the embryo axis.
[0253] Co-Cultivation.
[0254] Following inoculation, the split soybean seed is allowed to
co-cultivate with the Agrobacterium tumefaciens strain for 5 days
on co-cultivation medium (Agrobacterium Protocols, vol. 2, 2.sup.nd
Ed., Wang, K. (Ed.) Humana Press, New Jersey, 2006) in a Petri dish
covered with a piece of filter paper.
[0255] Shoot Induction.
[0256] After 5 days of co-cultivation, the split soybean seeds are
washed in liquid Shoot Induction (SI) media consisting of B5 salts,
B5 vitamins, 28 mg/L Ferrous, 38 mg/L Na.sub.2EDTA, 30 g/L sucrose,
0.6 g/L MES, 1.11 mg/L BAP, 100 mg/L TIMENTIN.TM., 200 mg/L
cefotaxime, and 50 mg/L vancomycin (pH 5.7). The split soybean
seeds are then cultured on Shoot Induction I (SII) medium
consisting of B5 salts, B5 vitamins, 7 g/L Noble agar, 28 mg/L
Ferrous, 38 mg/L Na.sub.2EDTA, 30 g/L sucrose, 0.6 g/L MES, 1.11
mg/L BAP, 50 mg/L TIMENTIN.TM., 200 mg/L cefotaxime, 50 mg/L
vancomycin (pH 5.7), with the flat side of the cotyledon facing up
and the nodal end of the cotyledon imbedded into the medium. After
2 weeks of culture, the explants from the transformed split soybean
seed are transferred to the Shoot Induction II (SI II) medium
containing SI I medium supplemented with 6 mg/L glufosinate
(LIBERTY.RTM.).
[0257] Shoot Elongation.
[0258] After 2 weeks of culture on SI II medium, the cotyledons are
removed from the explants and a flush shoot pad containing the
embryonic axis are excised by making a cut at the base of the
cotyledon. The isolated shoot pad from the cotyledon is transferred
to Shoot Elongation (SE) medium. The SE medium consists of MS
salts, 28 mg/L Ferrous, 38 mg/L Na.sub.2EDTA, 30 g/L sucrose and
0.6 g/L MES, 50 mg/L asparagine, 100 mg/L L-pyroglutamic acid, 0.1
mg/L IAA, 0.5 mg/L GA3, 1 mg/L zeatin riboside, 50 mg/L
TIMENTIN.TM., 200 mg/L cefotaxime, 50 mg/L vancomycin, 6 mg/L
glufosinate, 7 g/L Noble agar, (pH 5.7). The cultures are
transferred to fresh SE medium every 2 weeks. The cultures are
grown in a CONVIRON.TM. growth chamber at 24.degree. C. with an 18
h photoperiod at a light intensity of 80-90 .mu.mol/m.sup.2
sec.
[0259] Rooting.
[0260] Elongated shoots which developed from the cotyledon shoot
pad are isolated by cutting the elongated shoot at the base of the
cotyledon shoot pad, and dipping the elongated shoot in 1 mg/L IBA
(Indole 3-butyric acid) for 1-3 minutes to promote rooting. Next,
the elongated shoots are transferred to rooting medium (MS salts,
B5 vitamins, 28 mg/L Ferrous, 38 mg/L Na.sub.2EDTA, 20 g/L sucrose
and 0.59 g/L MES, 50 mg/L asparagine, 100 mg/L L-pyroglutamic acid
7 g/L Noble agar, pH 5.6) in phyta trays.
[0261] Cultivation.
[0262] Following culture in a CONVIRON.TM. growth chamber at
24.degree. C., 18 h photoperiod, for 1-2 weeks, the shoots which
have developed roots are transferred to a soil mix in a covered
sundae cup and placed in a CONVIRON.TM. growth chamber (models
CMP4030 and CMP3244, Controlled Environments Limited, Winnipeg,
Manitoba, Canada) under long day conditions (16 hours light/8 hours
dark) at a light intensity of 120-150 .mu.mol/m.sup.2 sec under
constant temperature (22.degree. C.) and humidity (40-50%) for
acclimatization of plantlets. The rooted plantlets are acclimated
in sundae cups for several weeks before they are transferred to the
greenhouse for further acclimatization and establishment of robust
transgenic soybean plants.
[0263] A further 10-20 T.sub.1 Glycine max independent lines
expressing hairpin dsRNA for an RNAi construct are obtained for BSB
challenge. Hairpin dsRNA may be derived as set forth in SEQ ID
NOs:3 and 4, or otherwise further comprising SEQ ID NO:1. These are
confirmed through RT-PCR or other molecular analysis methods, as
known in the art. Total RNA preparations from selected independent
T.sub.1 lines are optionally used for RT-PCR with primers designed
to bind in the linker of the hairpin expression cassette in each of
the RNAi constructs. In addition, specific primers for each target
gene in an RNAi construct are optionally used to amplify and
confirm the production of the pre-processed mRNA required for siRNA
production in planta. The amplification of the desired bands for
each target gene confirms the expression of the hairpin RNA in each
transgenic Glycine max plant. Processing of the dsRNA hairpin of
the target genes into siRNA is subsequently optionally confirmed in
independent transgenic lines using RNA blot hybridizations.
[0264] RNAi molecules having mismatch sequences with more than 80%
sequence identity to target genes affect BSB in a way similar to
that seen with RNAi molecules having 100% sequence identity to the
target genes. The pairing of mismatch sequence with native
sequences to form a hairpin dsRNA in the same RNAi construct
delivers plant-processed siRNAs capable of affecting the growth,
development, reproduction, and viability of feeding hemipteran
pests.
[0265] In planta delivery of dsRNA, siRNA, or miRNA corresponding
to target genes and the subsequent uptake by hemipteran pests
through feeding results in down-regulation of the target genes in
the hemipteran pest through RNA-mediated gene silencing. When the
function of a target gene is important at one or more stages of
development, the growth, development, and/or reproduction of the
hemipteran pest is affected, and in the case of at least one of
Euschistus heros, Piezodorus guildinii, Halyomorpha halys, Nezara
viridula, Acrosternum hilare, and Euschistus servus leads to
failure to successfully infest, feed, develop, and/or reproduce, or
leads to death of the hemipteran pest. The choice of target genes
and the successful application of RNAi is then used to control
hemipteran pests.
[0266] Phenotypic comparison of transgenic RNAi lines and
non-transformed Glycine max. Target hemipteran pest genes or
sequences selected for creating hairpin dsRNA have no similarity to
any known plant gene sequence. Hence it is not expected that the
production or the activation of (systemic) RNAi by constructs
targeting these hemipteran pest genes or sequences will have any
deleterious effect on transgenic plants. However, development and
morphological characteristics of transgenic lines are compared with
non-transformed plants, as well as those of transgenic lines
transformed with an "empty" vector having no hairpin-expressing
gene. Plant root, shoot, foliage and reproduction characteristics
are compared. There is no observable difference in root length and
growth patterns of transgenic and non-transformed plants. Plant
shoot characteristics such as height, leaf numbers and sizes, time
of flowering, floral size and appearance are similar. In general,
there are no observable morphological differences between
transgenic lines and those without expression of target iRNA
molecules when cultured in vitro and in soil in the glasshouse.
Example 9: E. heros Bioassays on Artificial Diet
[0267] In dsRNA feeding assays on artificial diet, 32-well trays
are set up with an .about.18 mg pellet of artificial diet and
water, as for injection experiments. dsRNA at a concentration of
200 ng/.mu.L is added to the food pellet and water sample, 100
.mu.L to each of two wells. Five 2.sup.nd instar E. heros nymphs
are introduced into each well. Water samples and dsRNA that targets
YFP transcript are used as negative controls. The experiments are
repeated on three different days. Surviving insects are weighed and
the mortality rates are determined after 7 days of treatment.
[0268] Feeding bioassays on adult female E. heros are performed as
32-well trays as described above. Young (less than one week of
adulthood) mated females are introduced into bioassay trays with
artificial diet, one per tray. After 7 days of exposure to dsRNA up
to ten adult females are moved to containers with green beans,
water, seeds, and two males. Female viability as well as the
numbers of eggs oviposited and eggs hatched are recorded for the
following two weeks. The data shows that the numbers of eggs
oviposited and/or hatched are significantly reduced.
Example 10: Transgenic Arabidopsis thaliana Comprising Hemipteran
Pest Sequences
[0269] Arabidopsis transformation vectors containing a target gene
construct for hairpin formation comprising segments of kruppel (SEQ
ID NO:1) are generated using standard molecular methods similar to
EXAMPLE 4. Arabidopsis transformation is performed using standard
Agrobacterium-based procedure. T.sub.1 seeds are selected with
glufosinate tolerance selectable marker. Transgenic T.sub.1
Arabidopsis plants are generated and homozygous simple-copy T2
transgenic plants are generated for insect studies. Bioassays are
performed on growing Arabidopsis plants with inflorescences. Five
to ten insects are placed on each plant and monitored for survival
within 14 days.
[0270] Construction of Arabidopsis Transformation Vectors.
[0271] Entry clones based on an entry vector harboring a target
gene construct for hairpin formation comprising a segment of
kruppel (SEQ ID NO:1) are assembled using a combination of
chemically synthesized fragments (DNA2.0, Menlo Park, Calif.) and
standard molecular cloning methods. Intramolecular hairpin
formation by RNA primary transcripts is facilitated by arranging
(within a single transcription unit) two copies of a target gene
segment in opposite orientations, the two segments being separated
by an linker sequence (e.g. ST-LS1 intron; SEQ ID NO:7) (Vancanneyt
et al. (1990) Mol. Gen. Genet. 220(2):245-50). Thus, the primary
mRNA transcript contains the two kruppel gene segment sequences as
large inverted repeats of one another, separated by the linker
sequence. A copy of a promoter (e.g. Arabidopsis thaliana ubiquitin
10 promoter (Callis et al. (1990) J. Biological Chem.
265:12486-12493)) is used to drive production of the primary mRNA
hairpin transcript, and a fragment comprising a 3' untranslated
region from Open Reading Frame 23 of Agrobacterium tumefaciens
(AtuORF23 3' UTR v1; U.S. Pat. No. 5,428,147) is used to terminate
transcription of the hairpin-RNA-expressing gene.
[0272] The hairpin clone within the entry vector described above is
used in standard GATEWAY.RTM. recombination reaction with a typical
binary destination vector to produce hairpin RNA expression
transformation vectors for Agrobacterium-mediated Arabidopsis
transformation.
[0273] The binary destination vector comprises a herbicide
tolerance gene, DSM-2v2 (U.S. Patent App. No. 2011/0107455), under
the regulation of a Cassava vein mosaic virus promoter (CsVMV
Promoter v2, U.S. Pat. No. 7,601,885; Verdaguer et al. (1996) Plant
Mol. Biol. 31:1129-39). A fragment comprising a 3' untranslated
region from Open Reading Frame 1 of Agrobacterium tumefaciens
(AtuORF1 3' UTR v6; Huang et al. (1990) J. Bacteriol. 172:1814-22)
is used to terminate transcription of the DSM2v2 mRNA.
[0274] A negative control binary construct, which comprises a gene
that expresses a YFP hairpin RNA, is constructed by means of
standard GATEWAY.RTM. recombination reactions with a typical binary
destination vector and entry vector. An entry construct comprises a
YFP hairpin sequence (SEQ ID NO:6) under the expression control of
an Arabidopsis Ubiquitin 10 promoter (as above) and a fragment
comprising an ORF23 3' untranslated region from Agrobacterium
tumefaciens (as above).
[0275] Production of Transgenic Arabidopsis Comprising Insecticidal
Hairpin RNAs: Agrobacterium-Mediated Transformation.
[0276] Binary plasmids containing hairpin sequences are
electroporated into Agrobacterium strain GV3101 (pMP90RK). The
recombinant Agrobacterium clones are confirmed by restriction
analysis of plasmids preparations of the recombinant Agrobacterium
colonies. A Qiagen Plasmid Max Kit (Qiagen, Cat#12162) is used to
extract plasmids from Agrobacterium cultures following the
manufacture recommended protocol.
[0277] Arabidopsis Transformation and T.sub.1 Selection.
[0278] Twelve to fifteen Arabidopsis plants (c.v. Columbia) are
grown in 4'' pots in the green house with light intensity of 250
.mu.mol/m.sup.2, 25.degree. C., and 18:6 hours of light: dark
conditions. Primary flower stems are trimmed one week before
transformation. Agrobacterium inoculums are prepared by incubating
10 .mu.L recombinant Agrobacterium glycerol stock in 100 mL LB
broth (Sigma L3022)+100 mg/L Spectinomycin+50 mg/L Kanamycin at
28.degree. C. and shaking at 225 rpm for 72 hours. Agrobacterium
cells are harvested and suspended into 5% sucrose+0.04% Silwet-L77
(Lehle Seeds Cat # VIS-02)+10 .mu.g/L benzamino purine (BA)
solution to OD.sub.600 0.8.about.1.0 before floral dipping. The
above-ground parts of the plant are dipped into the Agrobacterium
solution for 5-10 minutes, with gentle agitation. The plants are
then transferred to the greenhouse for normal growth with regular
watering and fertilizing until seed set.
Example 11: Growth and Bioassays of Transgenic Arabidopsis
[0279] Selection of T.sub.1 Arabidopsis Transformed with Hairpin
RNAi Constructs.
[0280] Up to 200 mg of T.sub.1 seeds from each transformation are
stratified in 0.1% agarose solution. The seeds are planted in
germination trays (10.5''.times.21''.times.1''; T.O. Plastics Inc.,
Clearwater, Minn.) with #5 sunshine media. Transformants are
selected for tolerance to Ignite.RTM. (glufosinate) at 280 g/ha at
6 and 9 days post planting. Selected events are transplanted into
4'' diameter pots. Insertion copy analysis is performed within a
week of transplanting via hydrolysis quantitative Real-Time PCR
(qPCR) using Roche LightCycler480.TM.. The PCR primers and
hydrolysis probes are designed against DSM2v2 selectable marker
using LightCycler.TM. Probe Design Software 2.0 (Roche). Plants are
maintained at 24.degree. C., with a 16:8 hour light: dark
photoperiod under fluorescent and incandescent lights at intensity
of 100-150 mE/m.sup.2s.
[0281] E. heros nymph plant feeding bioassay. At least four low
copy (1-2 insertions), four medium copy (2-3 insertions), and four
high copy (.gtoreq.4 insertions) events are selected for each
construct. Plants are grown to a reproductive stage (plants
containing flowers and siliques). The surface of soil is covered
with .about.50 mL volume of white sand for easy insect
identification. Five to ten 2.sup.nd instar E. heros nymphs are
introduced onto each plant. The plants are covered with plastic
tubes that are 3'' in diameter, 16'' tall, and with wall thickness
of 0.03'' (Item No. 484485, Visipack Fenton Mo.); the tubes are
covered with nylon mesh to isolate the insects. The plants are kept
under normal temperature, light, and watering conditions in a
conviron. In 14 days, the insects are collected and weighed;
percent mortality as well as growth inhibition (1-weight
treatment/weight control) are calculated. YFP hairpin-expressing
plants are used as controls.
[0282] E. heros Adults Plant Feeding Bioassay.
[0283] The pRNAi Arabidopsis T.sub.1 plants are selected and grown
in greenhouse, as described above. One to 5 newly emerged BSB
adults are released on each plant and the entire plant is covered
as described above to prevent adults from escaping. One week after
release, female adults are recovered from each plant and maintained
in the laboratory for egg collection. Depending on the parental
RNAi target and expected phenotype, parameters such as number of
eggs per female, percent egg hatch and nymph mortality are recorded
and compared with control plants.
[0284] T2 Arabidopsis Seed Generation and T2 Bioassays.
[0285] T2 seed is produced from selected low copy (1-2 insertions)
events for each construct. Plants (homozygous and/or heterozygous)
are subjected to E. heros nymph and adult feeding bioassay, as
described above. T3 seed is harvested from homozygotes and stored
for future analysis.
Example 12: Transformation of Additional Crop Species
[0286] Cotton is transformed with kruppel (with or without a
chloroplast transit peptide) to provide control of stink bugs by
utilizing a method known to those of skill in the art, for example,
substantially the same techniques previously described in EXAMPLE
14 of U.S. Pat. No. 7,838,733, or Example 12 of PCT International
Patent Publication No. WO 2007/053482.
Example 13: pRNAi-Mediated Insect Protection
[0287] Parental RNAi that causes egg mortality or loss of egg
viability brings further durability benefits to transgenic crops
that use RNAi and other mechanisms for insect protection. A basic
two-patch model was used to demonstrate this utility.
[0288] One patch contained a transgenic crop expressing
insecticidal ingredients, and the second patch contained a refuge
crop not expressing insecticidal ingredients. Eggs were oviposited
in the two modeled patches according to their relative proportions.
In this example, the transgenic patch represented 95% of the
landscape, and the refuge patch represented 5%. The transgenic crop
expressed an insecticidal protein active against the insect.
[0289] Pest to the insecticidal protein was modeled as monogenic,
with two possible alleles; one (S) conferring susceptibility, and
the other (R) conferring resistance. The insecticidal protein was
modeled to cause 97% mortality of homozygous susceptible (SS)
nymphs that feed on it. There was assumed to be no mortality of
nymphs that are homozygous for the resistance allele (RR).
Resistance to the insecticidal protein was assumed to be
incompletely recessive, whereby the functional dominance is 0.3
(there is 67.9% mortality of nymphs that are heterozygous (RS) for
resistance to the protein that feed on the transgenic crop).
[0290] The transgenic crop also expressed parentally active dsRNA
that, through RNA-interference (pRNAi), causes the eggs of adult
female insects that are exposed to the transgenic crop to be
non-viable. Insect resistance to the pRNAi was also considered to
be monogenic with two possible alleles; one (X) conferring
susceptibility of the adult female to RNAi, and the other (Y)
conferring resistance of the adult female to RNAi. Assuming a high
level of exposure to the dsRNAs, the pRNAi was modeled to cause
99.9% of eggs produced by a homozygous susceptible (XX) female to
be non-viable. The model assumed that pRNAi has no effect on the
viability of eggs produced by homozygous resistant (YY) females.
Resistance to the dsRNA was assumed to be recessive, whereby the
functional dominance is 0.01 (98.9% of eggs produced by a female
that is heterozygous (XY) for resistance to dsRNA are
non-viable).
[0291] In the model, there was random mating among surviving adults
and random oviposition across the two patches in accordance with
their relative proportions. The genotypic frequencies of viable
offspring followed Mendelian genetics for a two-locus genetic
system.
[0292] The effect of pRNAi required the adult females to feed on
plant tissue expressing parental active dsRNA. The interference
with egg development may be lower for adult females emerging from
the refuge crop than from the transgenic crop; adults may feed more
extensively in the patch in which they emerged following nymph
development. Therefore, the relative magnitude of the pRNAi effect
on female adults emerging from the refuge patch was varied, with
the proportion of the pRNAi effect ranging from 0 (no effect of
pRNAi on adult females emerging from the refuge patch) to 1 (same
effect of pRNAi on adult females emerging from the refuge patch as
on adult females emerging from the transgenic patch).
[0293] This model could be easily adjusted to demonstrate the
situation when the effect of pRNAi is also or alternatively
achieved by feeding of adult males on plant tissue expressing
parental active dsRNA.
[0294] Frequencies of the two resistance alleles were calculated
across generations. The initial frequencies of both of the
resistance alleles (R and Y) were assumed to be 0.005. Results were
presented as the number of insect generations for the frequencies
of each of the resistance alleles to reach 0.05. To examine the
resistance delay caused by the pRNAi, simulations that included
pRNAi were compared to simulations that did not include pRNAi, but
were identical in every other way. FIG. 3.
[0295] The model was also modified to include nymph-active
interfering dsRNA in combination with the insecticidal protein in
the transgenic crop. Therein, the nymph RNAi was assigned an effect
of 97% nymph mortality for homozygous RNAi-susceptible nymphs
(genotype XX), and no effect on nymphs that are homozygous
RNAi-resistant (YY). There was 67.9% mortality of nymphs that were
heterozygous for RNAi-resistance (XY). It was assumed that the same
mechanism of resistance applied to both nymph active RNAi and
pRNAi. As before, the pRNAi effect on adult females emerging from
the refuge patch relative to the effect on adult females emerging
from the transgenic patch was varied from 0 to 1. As before, to
examine the resistance delay caused by the pRNAi, simulations that
included pRNAi were compared to simulations that did not include
pRNAi, but were identical in every other way (including nymph
RNAi). FIG. 4.
[0296] A clear resistance management benefit of pRNAi was observed
when the magnitude of the pRNAi effect on egg viability for female
adults emerging from the refuge patch was reduced compared with
magnitude of the effect for adults emerging from the transgenic
patch. The transgenic crops that produced parental active dsRNA in
addition to an insecticidal protein were much more durable compared
with transgenic crops that produced only an insecticidal protein.
Similarly, transgenic crops that produced parental active dsRNA in
addition to both an insecticidal protein and a nymph active dsRNA
were much more durable compared with transgenic crops that produced
only an insecticidal protein and a nymph active dsRNA. In the
latter case, the durability benefit applied to both the
insecticidal protein and the insecticidal interfering dsRNA.
Example 14: Transgenic Plants Comprising a Hemipteran Pest Sequence
and Additional RNAi Constructs
[0297] A transgenic plant comprising a heterologous coding sequence
in its genome that is transcribed into an iRNA molecule that
targets an organism other than a hemipteran pest is secondarily
transformed via Agrobacterium or WHISKERS.TM. methodologies (See
Petolino and Arnold (2009) Methods Mol. Biol. 526:59-67) to produce
one or more insecticidal dsRNA molecules (for example, at least one
dsRNA molecule including a dsRNA molecule targeting a gene
comprising SEQ ID NO:1). Plant transformation plasmid vectors
prepared essentially as described in EXAMPLE 3 are delivered via
Agrobacterium or WHISKERS.TM.-mediated transformation methods into
suspension cells or immature embryos obtained from a transgenic
plant comprising a heterologous coding sequence in its genome that
is transcribed into an iRNA molecule that targets an organism other
than a hemipteran pest.
Example 15: Transgenic Plants Comprising an RNAi Construct and
Additional Hemipteran Pest Control Sequences
[0298] A transgenic plant comprising a heterologous coding sequence
in its genome that is transcribed into an iRNA molecule that
targets a hemipteran pest organism (for example, at least one dsRNA
molecule including a dsRNA molecule targeting a gene comprising SEQ
ID NO:1) is secondarily transformed via Agrobacterium or
WHISKERS.TM. methodologies (see Petolino and Arnold (2009) Methods
Mol. Biol. 526:59-67) to produce one or more insecticidal protein
molecules, for example, Cry3, Cry34 and Cry35 insecticidal
proteins. Plant transformation plasmid vectors prepared essentially
as described in EXAMPLE 3 are delivered via Agrobacterium or
WHISKERS.TM.-mediated transformation methods into suspension cells
or immature embryos obtained from a transgenic plant comprising a
heterologous coding sequence in its genome that is transcribed into
an iRNA molecule that targets a hemipteran pest organism.
Doubly-transformed plants are obtained that produce iRNA molecules
and insecticidal proteins for control of hemipteran pests.
Sequence CWU 1
1
2111728DNAEuschistus heros 1aggagcaata atctcaattt ctgaattttt
ttacttaaac cactttcatt ctcaccggct 60caattatagt taaaaatcag ctgatatatt
ttcactgata taacccaacc tgattaactt 120aaacacttaa gtaataatta
caagaattaa cggttagaga gttactcaaa caaattttgt 180aaaatcttat
caaacattga tcgaaaagcc acacaaatat caactactaa ataacatcaa
240ccaaagtgat tgattatcat aaaattttgt tgttttatta aatgggaaaa
aaataaattt 300ttgaaatcca taaaagagca taggggcata gagatgttaa
acaataaaaa gcggaatttt 360gcatctgtcg gttgggtaaa ttttacaata
tacattccaa taataattat tcccagagat 420aacctaatat caagtttcca
gtcactctga cgtggaatca catgttttca tttgagaaac 480atacacgctc
aaaaatagcg cttccatcaa ttaaaatata cacaacgaac ttatactcta
540ataataatat ataatagaaa acaaaataaa tgaaatgact ataccgaaac
cggtacatac 600ccgtagtcgt ttttacagta ccttcagtaa cccttactat
atttcaaaaa tcataacttc 660aattattaca tcaatccaga tcaagaaatt
gtactctaaa ataagatatt ttaaatttat 720taatttatgt acaataaatc
aatttcgttt cgtaccttat gacgttaaag cattcaaatg 780gtatagggca
ctggcggtac tgtgataaaa aaataataat ttactaacta aagaggtgag
840cctccatccc attcttcaga aagctcttct tcagattcag tcattgtgag
attattatta 900ttgttgatgt ttttgttctt atgcctgata gaaagatctt
caggttcggt ctgctcaggc 960aacgaaaggt acgcagggat aacgtttaca
agagcttgcg cttttctgtc cctcgagtcg 1020cacttgtgat gcaggaggtg
gtgtctcctt ctgaatttgg caccgcacaa tccgcagccg 1080aatggttttt
ctcccttgtg aattagagaa tgggccttga gctggttgga atcagcgaac
1140ctcgaggaac agacttggca agcgtaaggc cgttctccgg tatgtacccg
taggtgtctc 1200ctcaagttgg ctacttgcac gaagtacctg tcgcagtggt
cgcaatggta aggcttctcg 1260ccagtgtgtg tgcgtatgtg ggtgcggaga
tggtggtccc gtcgaaacct tctgtggcac 1320tcggggcact cgaacggccg
ttctccagtg tgggttcttt cgtgattttt gagaacgtgc 1380ttatgcttga
aggactggtt acaagtgagg cagacgaaag ctctgtccct cccaggagga
1440gagtcttcgt ccttcttctt cctccctaca ggctctggag gtggagctgc
ccagagcaaa 1500gagtgcgaga atagggagcc gcctccgatc ccgtggagag
ccaaaagtcc tggcattccg 1560gctgccagaa gaccggcctc tggaccgttt
tcttctcgtt tgcttgcttc cttcattgtg 1620caccttccga cgcctactac
tgaagtcaag cccttggttg ggttgttggt tgcgtgtatg 1680agagacaagg
ccatggaggt gttatagaga cgaaggcatg gcaggaac 17282288PRTEuschistus
heros 2Met Ala Leu Ser Leu Ile His Ala Thr Asn Asn Pro Thr Lys Gly
Leu 1 5 10 15 Thr Ser Val Val Gly Val Gly Arg Cys Thr Met Lys Glu
Ala Ser Lys 20 25 30 Arg Glu Glu Asn Gly Pro Glu Ala Gly Leu Leu
Ala Ala Gly Met Pro 35 40 45 Gly Leu Leu Ala Leu His Gly Ile Gly
Gly Gly Ser Leu Phe Ser His 50 55 60 Ser Leu Leu Trp Ala Ala Pro
Pro Pro Glu Pro Val Gly Arg Lys Lys 65 70 75 80 Lys Asp Glu Asp Ser
Pro Pro Gly Arg Asp Arg Ala Phe Val Cys Leu 85 90 95 Thr Cys Asn
Gln Ser Phe Lys His Lys His Val Leu Lys Asn His Glu 100 105 110 Arg
Thr His Thr Gly Glu Arg Pro Phe Glu Cys Pro Glu Cys His Arg 115 120
125 Arg Phe Arg Arg Asp His His Leu Arg Thr His Ile Arg Thr His Thr
130 135 140 Gly Glu Lys Pro Tyr His Cys Asp His Cys Asp Arg Tyr Phe
Val Gln 145 150 155 160 Val Ala Asn Leu Arg Arg His Leu Arg Val His
Thr Gly Glu Arg Pro 165 170 175 Tyr Ala Cys Gln Val Cys Ser Ser Arg
Phe Ala Asp Ser Asn Gln Leu 180 185 190 Lys Ala His Ser Leu Ile His
Lys Gly Glu Lys Pro Phe Gly Cys Gly 195 200 205 Leu Cys Gly Ala Lys
Phe Arg Arg Arg His His Leu Leu His His Lys 210 215 220 Cys Asp Ser
Arg Asp Arg Lys Ala Gln Ala Leu Val Asn Val Ile Pro 225 230 235 240
Ala Tyr Leu Ser Leu Pro Glu Gln Thr Glu Pro Glu Asp Leu Ser Ile 245
250 255 Arg His Lys Asn Lys Asn Ile Asn Asn Asn Asn Asn Leu Thr Met
Thr 260 265 270 Glu Ser Glu Glu Glu Leu Ser Glu Glu Trp Asp Gly Gly
Ser Pro Leu 275 280 285 3434DNAEuschistus heros 3gagcttgcgc
ttttctgtcc ctcgagtcgc acttgtgatg caggaggtgg tgtctccttc 60tgaatttggc
accgcacaat ccgcagccga atggtttttc tcccttgtga attagagaat
120gggccttgag ctggttggaa tcagcgaacc tcgaggaaca gacttggcaa
gcgtaaggcc 180gttctccggt atgtacccgt aggtgtctcc tcaagttggc
tacttgcacg aagtacctgt 240cgcagtggtc gcaatggtaa ggcttctcgc
cagtgtgtgt gcgtatgtgg gtgcggagat 300ggtggtcccg tcgaaacctt
ctgtggcact cggggcactc gaacggccgt tctccagtgt 360gggttctttc
gtgatttttg agaacgtgct tatgcttgaa ggactggtta caagtgaggc
420agacgaaagc tctg 4344605DNAEuschistus heros 4aaacattgat
cgaaaagcca cacaaatatc aactactaaa taacatcaac caaagtgatt 60gattatcata
aaattttgtt gttttattaa atgggaaaaa aataaatttt tgaaatccat
120aaaagagcat aggggcatag agatgttaaa caataaaaag cggaattttg
catctgtcgg 180ttgggtaaat tttacaatat acattccaat aataattatt
cccagagata acctaatatc 240aagtttccag tcactctgac gtggaatcac
atgttttcat ttgagaaaca tacacgctca 300aaaatagcgc ttccatcaat
taaaatatac acaacgaact tatactctaa taataatata 360taatagaaaa
caaaataaat gaaatgacta taccgaaacc ggtacatacc cgtagtcgtt
420tttacagtac cttcagtaac ccttactata tttcaaaaat cataacttca
attattacat 480caatccagat caagaaattg tactctaaaa taagatattt
taaatttatt aatttatgta 540caataaatca atttcgtttc gtaccttatg
acgttaaagc attcaaatgg tatagggcac 600tggcg 605520DNAArtificial
Sequencepromotor oligonucleotide 5taatacgact cactataggg
206471DNAArtificial SequenceYFP hpRNA-forming sequence 6atgtcatctg
gagcacttct ctttcatggg aagattcctt acgttgtgga gatggaaggg 60aatgttgatg
gccacacctt tagcatacgt gggaaaggct acggagatgc ctcagtggga
120aaggactagt accggttggg aaaggtatgt ttctgcttct acctttgata
tatatataat 180aattatcact aattagtagt aatatagtat ttcaagtatt
tttttcaaaa taaaagaatg 240tagtatatag ctattgcttt tctgtagttt
ataagtgtgt atattttaat ttataacttt 300tctaatatat gaccaaaaca
tggtgatgtg caggttgatc cgcggttact ttcccactga 360ggcatctccg
tagcctttcc cacgtatgct aaaggtgtgg ccatcaacat tcccttccat
420ctccacaacg taaggaatct tcccatgaaa gagaagtgct ccagatgaca t
4717222DNASolanum tuberosum 7gactagtacc ggttgggaaa ggtatgtttc
tgcttctacc tttgatatat atataataat 60tatcactaat tagtagtaat atagtatttc
aagtattttt ttcaaaataa aagaatgtag 120tatatagcta ttgcttttct
gtagtttata agtgtgtata ttttaattta taacttttct 180aatatatgac
caaaacatgg tgatgtgcag gttgatccgc gg 222845DNAArtificial
SequencePrimer BSB_kr-1-F 8ttaatacgac tcactatagg gagacagagc
tttcgtctgc ctcac 45944DNAArtificial SequencePrimer BSB_kr-1-R
9ttaatacgac tcactatagg gagagagctt gcgcttttct gtcc
441047DNAArtificial SequencePrimer BSB_kr-2-F 10ttaatacgac
tcactatagg gagacgccag tgccctatac catttga 471147DNAArtificial
SequencePrimer BSB_kr-2-R 11ttaatacgac tcactatagg gagaaaacat
tgatcgaaaa gccacac 4712301DNAArtificial SequenceSense strand of
YFPv2 dsRNA 12catctggagc acttctcttt catgggaaga ttccttacgt
tgtggagatg gaagggaatg 60ttgatggcca cacctttagc atacgtggga aaggctacgg
agatgcctca gtgggaaagg 120ttgatgcaca gttcatctgc acaactggtg
atgttcctgt gccttggagc acacttgtca 180ccactctcac ctatggagca
cagtgctttg ccaagtatgg tccagagttg aaggacttct 240acaagtcctg
tatgccagat ggctatgtgc aagagcgcac aatcaccttt gaaggagatg 300g
3011347DNAArtificial SequencePrimer YFPv2-F 13ttaatacgac tcactatagg
gagagcatct ggagcacttc tctttca 471446DNAArtificial SequencePrimer
YFPv2-R 14ttaatacgac tcactatagg gagaccatct ccttcaaagg tgattg
46151728RNAEuschistus heros 15aggagcaaua aucucaauuu cugaauuuuu
uuacuuaaac cacuuucauu cucaccggcu 60caauuauagu uaaaaaucag cugauauauu
uucacugaua uaacccaacc ugauuaacuu 120aaacacuuaa guaauaauua
caagaauuaa cgguuagaga guuacucaaa caaauuuugu 180aaaaucuuau
caaacauuga ucgaaaagcc acacaaauau caacuacuaa auaacaucaa
240ccaaagugau ugauuaucau aaaauuuugu uguuuuauua aaugggaaaa
aaauaaauuu 300uugaaaucca uaaaagagca uaggggcaua gagauguuaa
acaauaaaaa gcggaauuuu 360gcaucugucg guuggguaaa uuuuacaaua
uacauuccaa uaauaauuau ucccagagau 420aaccuaauau caaguuucca
gucacucuga cguggaauca cauguuuuca uuugagaaac 480auacacgcuc
aaaaauagcg cuuccaucaa uuaaaauaua cacaacgaac uuauacucua
540auaauaauau auaauagaaa acaaaauaaa ugaaaugacu auaccgaaac
cgguacauac 600ccguagucgu uuuuacagua ccuucaguaa cccuuacuau
auuucaaaaa ucauaacuuc 660aauuauuaca ucaauccaga ucaagaaauu
guacucuaaa auaagauauu uuaaauuuau 720uaauuuaugu acaauaaauc
aauuucguuu cguaccuuau gacguuaaag cauucaaaug 780guauagggca
cuggcgguac ugugauaaaa aaauaauaau uuacuaacua aagaggugag
840ccuccauccc auucuucaga aagcucuucu ucagauucag ucauugugag
auuauuauua 900uuguugaugu uuuuguucuu augccugaua gaaagaucuu
cagguucggu cugcucaggc 960aacgaaaggu acgcagggau aacguuuaca
agagcuugcg cuuuucuguc ccucgagucg 1020cacuugugau gcaggaggug
gugucuccuu cugaauuugg caccgcacaa uccgcagccg 1080aaugguuuuu
cucccuugug aauuagagaa ugggccuuga gcugguugga aucagcgaac
1140cucgaggaac agacuuggca agcguaaggc cguucuccgg uauguacccg
uaggugucuc 1200cucaaguugg cuacuugcac gaaguaccug ucgcaguggu
cgcaauggua aggcuucucg 1260ccagugugug ugcguaugug ggugcggaga
uggugguccc gucgaaaccu ucuguggcac 1320ucggggcacu cgaacggccg
uucuccagug uggguucuuu cgugauuuuu gagaacgugc 1380uuaugcuuga
aggacugguu acaagugagg cagacgaaag cucugucccu cccaggagga
1440gagucuucgu ccuucuucuu ccucccuaca ggcucuggag guggagcugc
ccagagcaaa 1500gagugcgaga auagggagcc gccuccgauc ccguggagag
ccaaaagucc uggcauuccg 1560gcugccagaa gaccggccuc uggaccguuu
ucuucucguu ugcuugcuuc cuucauugug 1620caccuuccga cgccuacuac
ugaagucaag cccuugguug gguuguuggu ugcguguaug 1680agagacaagg
ccauggaggu guuauagaga cgaaggcaug gcaggaac 172816434RNAEuschistus
heros 16gagcuugcgc uuuucugucc cucgagucgc acuugugaug caggaggugg
ugucuccuuc 60ugaauuuggc accgcacaau ccgcagccga augguuuuuc ucccuuguga
auuagagaau 120gggccuugag cugguuggaa ucagcgaacc ucgaggaaca
gacuuggcaa gcguaaggcc 180guucuccggu auguacccgu aggugucucc
ucaaguuggc uacuugcacg aaguaccugu 240cgcagugguc gcaaugguaa
ggcuucucgc cagugugugu gcguaugugg gugcggagau 300gguggucccg
ucgaaaccuu cuguggcacu cggggcacuc gaacggccgu ucuccagugu
360ggguucuuuc gugauuuuug agaacgugcu uaugcuugaa ggacugguua
caagugaggc 420agacgaaagc ucug 43417605RNAEuschistus heros
17aaacauugau cgaaaagcca cacaaauauc aacuacuaaa uaacaucaac caaagugauu
60gauuaucaua aaauuuuguu guuuuauuaa augggaaaaa aauaaauuuu ugaaauccau
120aaaagagcau aggggcauag agauguuaaa caauaaaaag cggaauuuug
caucugucgg 180uuggguaaau uuuacaauau acauuccaau aauaauuauu
cccagagaua accuaauauc 240aaguuuccag ucacucugac guggaaucac
auguuuucau uugagaaaca uacacgcuca 300aaaauagcgc uuccaucaau
uaaaauauac acaacgaacu uauacucuaa uaauaauaua 360uaauagaaaa
caaaauaaau gaaaugacua uaccgaaacc gguacauacc cguagucguu
420uuuacaguac cuucaguaac ccuuacuaua uuucaaaaau cauaacuuca
auuauuacau 480caauccagau caagaaauug uacucuaaaa uaagauauuu
uaaauuuauu aauuuaugua 540caauaaauca auuucguuuc guaccuuaug
acguuaaagc auucaaaugg uauagggcac 600uggcg 605181134DNAEuschistus
heros 18tacaaaatgt gtgacgaaga agttgctgct ttagttgtag acaatggatc
tggtatgtgc 60aaagccggtt tcgctggaga tgatgcaccc cgagctgtat tcccatcaat
tgttggcagg 120cctagacacc agggtgtcat ggttggaatg ggacaaaagg
acagttatgt tggagacgaa 180gcccaaagca agagaggtat cctcaccctg
aaatacccca ttgaacacgg tatcatcacc 240aactgggacg acatggaaaa
gatctggcat cacaccttct acaacgagct gcgagtcgct 300ccagaggaac
accccatcct cctgactgag gctcccctca accccaaagc caacagggag
360aagatgaccc agatcatgtt tgagaccttc aacaccccag ccatgtatgt
cgccatccag 420gctgtactct ccctctatgc ctccggtcgt actaccggta
ttgtacttga ctcaggagat 480ggtgtctccc acaccgtacc catctatgaa
ggttatgccc ttccccacgc catcctccgt 540ctggatcttg ctggacgtga
cttgactgac tatcttatga agatcctcac cgagcgtggt 600tacagcttca
ccaccaccgc tgaaagggaa atcgtcaggg acatcaagga aaaactgtgc
660tatgtcgccc tggactttga gcaggaaatg gccaccgccg ctgcctccac
ctccctggag 720aagtcctatg aacttcccga cggtcaggtc atcaccatcg
gtaacgagag gttccgttgc 780ccagaggctc tcttccagcc ttccttcttg
ggtatggaat cttgcggtat ccatgagact 840gtctacaact ccatcatgaa
gtgcgacgtt gacatcagga aggacttgta cgccaacacc 900gtcctctccg
gaggtaccac catgtaccca ggtattgctg acaggatgca gaaggaaatc
960accgccctcg ctccttcaac catcaagatc aagatcattg ctcccccaga
aaggaagtac 1020tccgtatgga tcggtggttc catcttggct tccctgtcca
ccttccagca gatgtggatc 1080tccaagcagg aatacgacga atccggccca
ggcatcgtcc accgcaaatg cttc 11341922DNAArtificial SequencePrimer
Actin-F 19tcaaggaaaa actgtgctat gt 222020DNAArtificial
SequencePrimer Actin-R 20taccgatggt gatgacctga 202112DNAArtificial
SequenceProbe Actin-FAM 21accgccgctg cc 12
* * * * *