U.S. patent application number 16/027066 was filed with the patent office on 2018-10-25 for parental rnai suppression of chromatin remodeling genes to control coleopteran 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 | 20180305713 16/027066 |
Document ID | / |
Family ID | 56127536 |
Filed Date | 2018-10-25 |
United States Patent
Application |
20180305713 |
Kind Code |
A1 |
Siegfried; Blair D. ; et
al. |
October 25, 2018 |
PARENTAL RNAI SUPPRESSION OF CHROMATIN REMODELING GENES TO CONTROL
COLEOPTERAN 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: |
56127536 |
Appl. No.: |
16/027066 |
Filed: |
July 3, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14971515 |
Dec 16, 2015 |
10053706 |
|
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16027066 |
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62092747 |
Dec 16, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
Y02A 40/146 20180101;
A01N 57/16 20130101; C07K 2319/21 20130101; A61P 7/04 20180101;
A01N 63/10 20200101; Y02A 40/162 20180101; C12N 15/8218 20130101;
C12N 15/113 20130101; C12N 2310/14 20130101; C12N 15/8286
20130101 |
International
Class: |
C12N 15/82 20060101
C12N015/82; A01N 57/16 20060101 A01N057/16; A01N 63/02 20060101
A01N063/02; C12N 15/113 20100101 C12N015/113 |
Claims
1. An isolated nucleic acid molecule comprising a polynucleotide
operably linked to a heterologous promoter, wherein the
polynucleotide comprises: SEQ ID NO:5; the complement of SEQ ID
NO:5; a fragment of at least 15 contiguous nucleotides of SEQ ID
NO:5; the complement of a fragment of at least 15 contiguous
nucleotides of SEQ ID NO:5; a native coding sequence of a
hemipteran insect comprising SEQ ID NO:5; the complement of a
native coding sequence of a hemipteran insect comprising SEQ ID
NO:5; SEQ ID NO:7; the complement of SEQ ID NO:7; a fragment of at
least 15 contiguous nucleotides of SEQ ID NO:7; the complement of a
fragment of at least 15 contiguous nucleotides of SEQ ID NO:7; a
native coding sequence of a hemipteran insect comprising SEQ ID
NO:7; the complement of a native coding sequence of a hemipteran
insect comprising SEQ ID NO:7; SEQ ID NO:10; the complement of SEQ
ID NO:10; a fragment of at least 15 contiguous nucleotides of SEQ
ID NO:10; the complement of a fragment of at least 15 contiguous
nucleotides of SEQ ID NO:10; a native coding sequence of a
hemipteran insect comprising SEQ ID NO:10; the complement of a
native coding sequence of a hemipteran insect comprising SEQ ID
NO:10; SEQ ID NO:8; SEQ ID NO:64; the complement of SEQ ID NO:8;
the complement of SEQ ID NO:64; a fragment of at least 15
contiguous nucleotides of SEQ ID NO:8; the complement of a fragment
of at least 15 contiguous nucleotides of SEQ ID NO:8; a fragment of
at least 15 contiguous nucleotides of SEQ ID NO:64; the complement
of a fragment of at least 15 contiguous nucleotides of SEQ ID
NO:64; a native coding sequence of a hemipteran insect comprising
SEQ ID NO:8; the complement of a native coding sequence of a
hemipteran insect comprising SEQ ID NO:8; a native coding sequence
of a hemipteran insect comprising SEQ ID NO:64; the complement of a
native coding sequence of a hemipteran insect comprising SEQ ID
NO:64; a native coding sequence of a hemipteran insect that is
transcribed into a native RNA molecule comprising SEQ ID NO:69; the
complement of a native coding sequence of a hemipteran insect that
is transcribed into a native RNA molecule comprising SEQ ID NO:69;
a fragment of at least 15 contiguous nucleotides of a native coding
sequence of a hemipteran insect comprising SEQ ID NO:16; the
complement of a fragment of at least 15 contiguous nucleotides of a
native coding sequence of a hemipteran insect comprising SEQ ID
NO:16; SEQ ID NO:10; SEQ ID NO:65; the complement of SEQ ID NO:10;
the complement of SEQ ID NO:65; a fragment of at least 15
contiguous nucleotides of SEQ ID NO:10; the complement of a
fragment of at least 15 contiguous nucleotides of SEQ ID NO:10; a
fragment of at least 15 contiguous nucleotides of SEQ ID NO:65; the
complement of a fragment of at least 15 contiguous nucleotides of
SEQ ID NO:65; a native coding sequence of a hemipteran insect
comprising SEQ ID NO:10; the complement of a native coding sequence
of a hemipteran insect comprising SEQ ID NO:10; a native coding
sequence of a hemipteran insect comprising SEQ ID NO:65; the
complement of a native coding sequence of a hemipteran insect
comprising SEQ ID NO:65; a native coding sequence of a hemipteran
insect that is transcribed into a native RNA molecule comprising
SEQ ID NO:70; the complement of a native coding sequence of a
hemipteran insect that is transcribed into a native RNA molecule
comprising SEQ ID NO:70; a fragment of at least 15 contiguous
nucleotides of a native coding sequence of a hemipteran insect
comprising SEQ ID NO:17; the complement of a fragment of at least
15 contiguous nucleotides of a native coding sequence of a
hemipteran insect comprising SEQ ID NO:17; SEQ ID NO:12; SEQ ID
NO:66; the complement of SEQ ID NO:12; the complement of SEQ ID
NO:66; a fragment of at least 15 contiguous nucleotides of SEQ ID
NO:12; the complement of a fragment of at least 15 contiguous
nucleotides of SEQ ID NO:12; a fragment of at least 15 contiguous
nucleotides of SEQ ID NO:66; the complement of a fragment of at
least 15 contiguous nucleotides of SEQ ID NO:66; a native coding
sequence of a hemipteran insect comprising SEQ ID NO:12; the
complement of a native coding sequence of a hemipteran insect
comprising SEQ ID NO:12; a native coding sequence of a hemipteran
insect comprising SEQ ID NO:66; the complement of a native coding
sequence of a hemipteran insect comprising SEQ ID NO:66; a native
coding sequence of a hemipteran insect that is transcribed into a
native RNA molecule comprising SEQ ID NO:71; the complement of a
native coding sequence of a hemipteran insect that is transcribed
into a native RNA molecule comprising SEQ ID NO:71; a fragment of
at least 15 contiguous nucleotides of a native coding sequence of a
hemipteran insect comprising SEQ ID NO:18; the complement of a
fragment of at least 15 contiguous nucleotides of a native coding
sequence of a hemipteran insect comprising SEQ ID NO:18; SEQ ID
NO:14; SEQ ID NO:67; the complement of SEQ ID NO:14; the complement
of SEQ ID NO:67; a fragment of at least 15 contiguous nucleotides
of SEQ ID NO:14; the complement of a fragment of at least 15
contiguous nucleotides of SEQ ID NO:14; a fragment of at least 15
contiguous nucleotides of SEQ ID NO:67; the complement of a
fragment of at least 15 contiguous nucleotides of SEQ ID NO:67; a
native coding sequence of a hemipteran insect comprising SEQ ID
NO:14; the complement of a native coding sequence of a hemipteran
insect comprising SEQ ID NO:14; a native coding sequence of a
hemipteran insect comprising SEQ ID NO:67; the complement of a
native coding sequence of a hemipteran insect comprising SEQ ID
NO:67; a native coding sequence of a hemipteran insect that is
transcribed into a native RNA molecule comprising SEQ ID NO:72; the
complement of a native coding sequence of a hemipteran insect that
is transcribed into a native RNA molecule comprising SEQ ID NO:72;
a fragment of at least 15 contiguous nucleotides of a native coding
sequence of a hemipteran insect comprising SEQ ID NO:19; the
complement of a fragment of at least 15 contiguous nucleotides of a
native coding sequence of a hemipteran insect comprising SEQ ID
NO:19; SEQ ID NO:30; the complement of SEQ ID NO:30; a fragment of
at least 15 contiguous nucleotides of SEQ ID NO:30; the complement
of a fragment of at least 15 contiguous nucleotides of SEQ ID
NO:30; a native coding sequence of a hemipteran insect comprising
SEQ ID NO:30; the complement of a native coding sequence of a
hemipteran insect comprising SEQ ID NO:30; a native coding sequence
of a hemipteran insect that is transcribed into a native RNA
molecule translated into the polypeptide of SEQ ID NO:31; the
complement of a native coding sequence of a hemipteran insect that
is transcribed into a native RNA molecule translated into the
polypeptide of SEQ ID NO:31; a fragment of at least 15 contiguous
nucleotides of a native coding sequence of a hemipteran insect
comprising SEQ ID NO:30; the complement of a fragment of at least
15 contiguous nucleotides of a native coding sequence of a
hemipteran insect comprising SEQ ID NO:30; SEQ ID NO:32; the
complement of SEQ ID NO:32; a fragment of at least 15 contiguous
nucleotides of SEQ ID NO:32; the complement of a fragment of at
least 15 contiguous nucleotides of SEQ ID NO:32; a native coding
sequence of a hemipteran insect comprising SEQ ID NO:32; the
complement of a native coding sequence of a hemipteran insect
comprising SEQ ID NO:32; a native coding sequence of a hemipteran
insect that is transcribed into a native RNA molecule translated
into the polypeptide of SEQ ID NO:33; the complement of a native
coding sequence of a hemipteran insect that is transcribed into a
native RNA molecule translated into the polypeptide of SEQ ID
NO:33; a fragment of at least 15 contiguous nucleotides of a native
coding sequence of a hemipteran insect comprising SEQ ID NO:32; and
the complement of a fragment of at least 15 contiguous nucleotides
of a native coding sequence of a hemipteran insect comprising SEQ
ID NO:32.
2. The nucleic acid molecule of claim 1, wherein the polynucleotide
encodes a double-stranded ribonucleic acid (dsRNA) molecule, the
polynucleotide further comprising a second nucleotide sequence and
a third nucleotide sequence, wherein the third nucleotide sequence
is operably linked to the first nucleotide sequence by the second
nucleotide sequence, and wherein the third nucleotide sequence is
substantially the reverse complement of the first nucleotide
sequence.
3. A double-stranded ribonucleic acid (dsRNA) molecule comprising a
polyribonucleotide encoded by the polynucleotide of the nucleic
acid molecule of claim 1.
4. The double-stranded ribonucleic acid molecule of claim 3,
wherein contacting said ribonucleotide molecule with a hemipteran
pest kills or inhibits the growth, reproduction, and/or feeding of
the pest.
5. A cell transformed with the polynucleotide of claim 1.
6. A plant transformation vector comprising the polynucleotide of
claim 1, wherein the heterologous promoter is functional in a plant
cell.
7. The plant of claim 6, wherein the at least one polynucleotide is
expressed in the plant as a double-stranded ribonucleic acid
molecule.
8. The plant of claim 6, wherein the at least one polynucleotide is
expressed in the plant as a ribonucleic acid molecule, and the
ribonucleic acid molecule inhibits the expression of an endogenous
polynucleotide that is specifically complementary to the at least
one polynucleotide when a hemipteran pest ingests a part of the
plant.
9. The polynucleotide of claim 1, further comprising at least one
additional polynucleotide that encodes an RNA molecule that
inhibits the expression of an endogenous pest gene.
10. The polynucleotide of claim 9, wherein the additional
polynucleotide encodes an iRNA molecule that results in a parental
RNAi phenotype.
11. The polynucleotide of claim 9, wherein the additional
polynucleotide encodes an iRNA molecule that inhibits the
expression of a hunchback or kruppel gene.
12. The polynucleotide of claim 9, wherein the additional
polynucleotide encodes an iRNA molecule that results in decreased
growth and/or development and/or mortality in a hemipteran pest
that contacts the iRNA molecule (lethal RNAi).
13. A plant transformation vector comprising the polynucleotide of
claim 9, wherein the additional polynucleotide(s) are each operably
linked to a heterologous promoter functional in a plant cell.
14. 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:43-62 and 68-72, the complement of any of SEQ ID NOs:43-62
and 68-72, a fragment of at least 15 contiguous nucleotides of any
of SEQ ID NOs:43-62 and 68-72, the complement of a fragment of at
least 15 contiguous nucleotides of any of SEQ ID NOs:43-62 and
68-72, a transcript of any of SEQ ID NO:1, SEQ ID NO:8, SEQ ID
NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:30, SEQ ID NO:32, SEQ
ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, and SEQ ID
NO:67, the complement of a transcript of any of SEQ ID NO:1, SEQ ID
NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:30, and
SEQ ID NO:32, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID
NO:66, and SEQ ID NO:67, a fragment of at least 15 contiguous
nucleotides of a transcript of any of SEQ ID NO:1, SEQ ID NO:8, SEQ
ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:30, and SEQ ID
NO:32, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, and
SEQ ID NO:67, and the complement of a fragment of at least 15
contiguous nucleotides of a transcript of any of SEQ ID NO:1, SEQ
ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:30,
and SEQ ID NO:32, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID
NO:66, and SEQ ID NO:67 thereby producing a hemipteran pest having
a pRNAi phenotype.
15. 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:43, SEQ ID NO:45, SEQ ID NO:46, SEQ ID
NO:47, SEQ ID NO:48, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:68, SEQ
ID NO:69, SEQ ID NO:70, SEQ ID NO:71, and SEQ ID NO:72, and wherein
the first polynucleotide sequence is specifically hybridized to the
second polynucleotide sequence.
16. The method according to claim 15, wherein the agent is a
double-stranded RNA molecule.
17. The method according to claim 15, 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.
18. The method according to claim 17, wherein the ribonucleic acid
molecule is a double-stranded ribonucleic acid molecule.
19. The method according to claim 17, 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.
20. 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:43-62 and 68-72, the complement of any of SEQ ID NOs:43-62 and
68-72, a fragment of at least 15 contiguous nucleotides of any of
SEQ ID NOs:43-62 and 68-72, the complement of a fragment of at
least 15 contiguous nucleotides of any of SEQ ID NOs:43-62 and
68-72, a transcript of any of SEQ ID NO:1, SEQ ID NO:8, SEQ ID
NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:30, SEQ ID NO:32, SEQ
ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, and SEQ ID
NO:67, the complement of a transcript of any of SEQ ID NO:1, SEQ ID
NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:30, SEQ
ID NO:32, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66,
and SEQ ID NO:67, a fragment of at least 15 contiguous nucleotides
of a transcript of any of SEQ ID NO:1, SEQ ID NO:8, SEQ ID NO:10,
SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:30, SEQ ID NO:32, SEQ ID
NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, and SEQ ID NO:67,
and the complement of a fragment of at least 15 contiguous
nucleotides of a transcript of any of SEQ ID NO:1, SEQ ID NO:8, SEQ
ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:30, SEQ ID NO:32,
SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, and SEQ ID
NO:67.
21. The method according to claim 20, wherein the diet comprises a
plant cell transformed to express the polynucleotide.
22. The method according to claim 20, wherein the specifically
hybridizable RNA is comprised in a double-stranded RNA
molecule.
23. The method according to claim 20, wherein the RNA molecule is a
double-stranded RNA molecule.
24. A method for producing a transgenic plant cell, the method
comprising: transforming a plant cell with a vector comprising a
means for protecting a plant from a hemipteran pest; 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 means for protecting a plant from a hemipteran
pest into their genomes; screening the transformed plant cells for
expression of a means for inhibiting expression of an essential
gene in a hemipteran pest; and selecting a plant cell that
expresses the means for inhibiting expression of an essential gene
in a hemipteran pest.
25. A method for producing a hemipteran pest-resistant transgenic
plant, the method comprising: providing the transgenic plant cell
produced by the method of claim 24; and regenerating a transgenic
plant from the transgenic plant cell, wherein expression of the
means for inhibiting expression of an essential gene in a
hemipteran pest is sufficient to modulate the expression of a
target gene in a hemipteran pest that contacts the transformed
plant.
Description
PRIORITY CLAIM
[0001] This application is a divisional of U.S. patent application
Ser. No. 14/971,515 filed, on Dec. 16, 2015, which claims the
benefit of the filing date of U.S. Provisional Patent Application
Ser. No. 62/092,747, filed Dec. 16, 2014, the contents of which are
incorporated herein in its entirety by this reference.
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, 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.
[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 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 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 chromatin remodeling 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 chromatin remodeling gene referred to herein as
BSB_Brahma (SEQ ID NO:1 and SEQ ID NO:63). In particular examples,
a target gene useful for post-transcriptional inhibition is the
novel chromatin remodeling gene referred to herein as BSB_mi-2 (SEQ
ID NO:8 and SEQ ID NO:64). In particular examples, a target gene
useful for post-transcriptional inhibition is the novel chromatin
remodeling gene referred to herein as BSB_iswi-1 (SEQ ID NO:10 and
SEQ ID NO:65). In particular examples, a target gene useful for
post-transcriptional inhibition is the novel chromatin remodeling
gene referred to herein as BSB_chd1 (SEQ ID NO:14 and SEQ ID
NO:67). In particular examples, a target gene useful for
post-transcriptional inhibition is the novel chromatin remodeling
gene referred to herein as BSB_iswi-2 (SEQ ID NO:12 and SEQ ID
NO:66). In particular examples, a target gene useful for
post-transcriptional inhibition is the novel chromatin remodeling
gene referred to herein as BSB_ino80 (SEQ ID NO:30). In particular
examples, a target gene useful for post-transcriptional inhibition
is the novel chromatin remodeling gene referred to herein as
BSB_domino (SEQ ID NO:32).
[0014] An isolated nucleic acid molecule comprising the
polynucleotide of SEQ ID NO:1; the complement of SEQ ID NO:1; SEQ
ID NO:8; the complement of SEQ ID NO:8; SEQ ID NO:10; the
complement of SEQ ID NO:10; SEQ ID NO:12; the complement of SEQ ID
NO:12; SEQ ID NO:14; the complement of SEQ ID NO:14; SEQ ID NO:30;
the complement of SEQ ID NO:30; SEQ ID NO:32; the complement of SEQ
ID NO:32; SEQ ID NO:63; the complement of SEQ ID NO:63; SEQ ID
NO:64; the complement of SEQ ID NO:64; SEQ ID NO:65; the complement
of SEQ ID NO:65; SEQ ID NO:66; the complement of SEQ ID NO:66; SEQ
ID NO:67; the complement of SEQ ID NO:67; and/or fragments of any
of the foregoing (e.g., SEQ ID NO:3, SEQ ID NO:16, SEQ ID NO:17,
SEQ ID NO:18, and SEQ ID NO:19) is therefore disclosed herein.
[0015] 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 chromatin
remodeling gene product (for example, the product of a brahma,
mi-2, iswi-1, chd1, iswi-2, ino80, or domino gene). For example, a
nucleic acid molecule may comprise a polynucleotide encoding a
polypeptide that is at least 85% identical to a polypeptide
selected from the group consisting of SEQ ID NO:2 (BSB BRAHMA); an
amino acid sequence within a product of BSB brahma; SEQ ID NO:9
(BSB MI-2); an amino acid sequence within a product of BSB mi-2;
SEQ ID NO:11 (BSB ISWI-1); an amino acid sequence within a product
of BSB iswi-1; SEQ ID NO:15 (BSB CHD1); an amino acid sequence
within a product of BSB chd1; SEQ ID NO:13 (BSB ISWI-2); an amino
acid sequence within a product of BSB iswi-2; SEQ ID NO:31 (BSB
INO80); an amino acid sequence within a product of BSB ino80; SEQ
ID NO:33 (BSB DOMINO); and an amino acid sequence within a product
of BSB domino. 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 chromatin remodeling gene
product.
[0016] Also 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 chromatin remodeling
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_brahma (SEQ ID NO:1 and SEQ ID NO:63),
BSB_mi-2 (SEQ ID NO:8 and SEQ ID NO:64), BSB_iswi-1 (SEQ ID NO:10
and SEQ ID NO:65), BSB_chd1 (SEQ ID NO:14 and SEQ ID NO:67),
BSB_iswi-2 (SEQ ID NO:12 and SEQ ID NO:66), BSB_ino80 (SEQ ID
NO:30), and BSB_domino (SEQ ID NO:32).
[0017] 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:44; SEQ ID NO:49;
SEQ ID NO:50; SEQ ID NO:51; SEQ ID NO:52; and the complements
thereof. 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 encoding a
ATP-dependent remodeling enzyme, such as mRNAs comprising SEQ ID
NO:43; SEQ ID NO:45; SEQ ID NO:46; SEQ ID NO:47; SEQ ID NO:48; SEQ
ID NO:53; or SEQ ID NO:54. 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
soybean plant.
[0018] 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:43;
the complement of SEQ ID NO:43; SEQ ID NO:44; the complement of SEQ
ID NO:44; SEQ ID NO:45; the complement of SEQ ID NO:45; SEQ ID
NO:46; the complement of SEQ ID NO:46; SEQ ID NO:47; the complement
of SEQ ID NO:47; SEQ ID NO:48; the complement of SEQ ID NO:48; SEQ
ID NO:49; the complement of SEQ ID NO:49; SEQ ID NO:50; the
complement of SEQ ID NO:50; SEQ ID NO:51; the complement of SEQ ID
NO:51; SEQ ID NO:52; the complement of SEQ ID NO:52; SEQ ID NO:53;
the complement of SEQ ID NO:53; SEQ ID NO:54; the complement of SEQ
ID NO:54; SEQ ID NO:55; the complement of SEQ ID NO:55; SEQ ID
NO:56; the complement of SEQ ID NO:56; SEQ ID NO:57; the complement
of SEQ ID NO:57; SEQ ID NO:58; the complement of SEQ ID NO:58; SEQ
ID NO:59; the complement of SEQ ID NO:59; SEQ ID NO:60; the
complement of SEQ ID NO:60; SEQ ID NO:61; the complement of SEQ ID
NO:61; SEQ ID NO:62; the complement of SEQ ID NO:62; SEQ ID NO:68;
the complement of SEQ ID NO:68; SEQ ID NO:69; the complement of SEQ
ID NO:69; SEQ ID NO:70; the complement of SEQ ID NO:70; SEQ ID
NO:71; the complement of SEQ ID NO:71; SEQ ID NO:72; the complement
of SEQ ID NO:72; a polynucleotide that hybridizes to a coding
polynucleotide of a hemipteran organism (e.g., BSB) comprising all
or part of any of SEQ ID NO:1, SEQ ID NO:8, SEQ ID NO:10, SEQ ID
NO:12, SEQ ID NO:14, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:63, SEQ
ID NO:64, SEQ ID NO:65, SEQ ID NO:66, and SEQ ID NO:67; and the
complement of a polynucleotide that hybridizes to a coding
polynucleotide of a hemipteran organism comprising all or part of
any of SEQ ID NO:1, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID
NO:14, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:63, SEQ ID NO:64, SEQ
ID NO:65, SEQ ID NO:66, and SEQ ID NO:67.
[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] FIG. 1A includes a depiction of the strategy used to
generate dsRNA from a single transcription template with a single
pair of primers, and from two transcription templates (FIG.
1B).
[0022] FIG. 2 includes a phylogenetic tree representation of the
sequence alignment of ATP-dependent remodelers from D. v. virgifera
(WCR), E. heros, and Drosophila melanogaster. For comparison, the
tree also contains human BRAHMA, Saccharomyces cerevisiae SNF2, and
Iswi homologs from the brown marmorated stink bug, Halyomorpha
halys. The alignment was performed using MUSCLE (100 iterations) in
MEGA 6.06. Bootstrap values (MEGA) support the topology of the
ATP-dependent remodeler family branches on the maximum likelihood
phylogeny tree.
[0023] FIG. 3A-3E includes a representation of the domain
architecture of ATP-dependent chromatin remodeling enzymes of
Diabrotica virgifera virgifera (WCR), Euschistus heros (BSB) and
Drosophila melanogaster (Dme). The graphical representation is of
Pfam output, with domains shaded and labeled. The proteins are
organized by families and aligned with respect to SNF2 domain.
"Squiggly" lines represent truncation/discontinuity for
representation purposes.
[0024] FIGS. 4A-4C includes data regarding E. heros adult female
survival, oviposition, and egg hatch rates following dsRNA
injections that target chromatin remodeling ATPases. Females were
injected with dsRNA at 0 to 2 days post adult molt. FIG. 4A shows
the effects on female survival: twenty females were injected with
each dsRNA and survival rate was monitored for 23 days. FIG. 4B
shows the effects on oviposition: eggs collected from
dsRNA-injected females starting at 9 days post-injection. The
oviposition rates plotted are per day per female, based on each
week of collection. FIG. 4C shows the effects on egg hatching: eggs
hatched based on the numbers of eggs laid in FIG. 4B. Means
comparisons were performed with YFP as control using Dunnett's
test, .dagger. p<0.001, **p<0.05.
[0025] FIG. 5 includes data showing the percent knockdown of
chromatin remodeling ATPases in E. heros ovaries. Relative
expression is represented by 2.sup.-.DELTA..DELTA.Ct. E. heros
muscle actin transcript was used as a reference gene and ovaries
from non-injected females as negative controls. Four sets of
ovaries were used in each qRT-PCR experiment. Means comparisons
were performed using Student's t-test; .dagger.p<0.001.
[0026] FIGS. 6A-6B includes data showing the development and hatch
rates of eggs oviposited by Brahma dsRNA-injected E. heros females.
Ovipositing females were injected with dsRNA at 14 to 16 days post
adult molt. FIG. 6A shows the effects on oviposition: eggs
collected from dsRNA-injected females starting at 1 day
post-injection. The number of eggs plotted are per day per female,
binned into three-day intervals. FIG. 6B shows the effects on egg
hatching: eggs hatched based on the numbers in FIG. 6A. Means
comparisons were performed with Dunnett's test using non-injected
insects as controls, *indicates significance at p<0.05.
**indicates significance at p<0.001
[0027] FIGS. 7A-7H includes data showing the effects on ovaries of
E. heros females injected with brm or mi-2 dsRNA. FIGS. 7(A-B) show
ovaries of non-injected E. heros females at zero and four days
after adult molt, provided for developmental comparison. FIGS.
7(C-D) show ovaries of females injected with YFP dsRNA, and FIGS.
7(E-F) show Brahma dsRNA ovaries at 9 and 14 days post injection.
FIG. 7(E) shows lack of ovariole elongation and lack oocyte
development, and FIG. 7(F) shows decaying oocytes. FIGS. 7(G-H)
show mi-2 dsRNA at 9 and 14 days post injection. FIG. 7(H) shows
lack of ovariole elongation, and FIG. 7(G) shows somewhat elongated
ovaries with no mature oocytes.
[0028] FIG. 8 includes a summary of modeling data showing the
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). FIG. 8
illustrates the effect 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.
[0029] FIG. 9 includes a summary of modeling data showing the
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). FIG. 9
illustrates the effect 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
[0030] 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.
[0031] 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:
[0032] SEQ ID NO:1 shows an exemplary Euschistus heros chromatin
remodeling gene DNA, referred to herein in some places as
Brahma.
[0033] SEQ ID NO:2 shows the amino acid sequence of a E. heros
BRAHMA polypeptide encoded by an exemplary E. heros chromatin
remodeling gene DNA.
[0034] SEQ ID NO:3 shows an exemplary E. heros chromatin remodeling
gene DNA, referred to herein in some places as BSB_brm-1, which is
used in some examples for the production of a dsRNA.
[0035] SEQ ID NO:4 shows the nucleotide sequence of a T7 phage
promoter.
[0036] SEQ ID NO:5 shows a segment of an exemplary YFPv2 gene,
which is used in some examples for the production of a dsRNA.
[0037] SEQ ID NOs:6 and 7 show primers used for PCR amplification
of a YFPv2 sequence, used in some examples for dsRNA
production.
[0038] SEQ ID NO:8 shows a further exemplary E. heros chromatin
remodeling gene DNA, referred to herein in some places as
BSB_mi-2.
[0039] SEQ ID NO:9 shows the amino acid sequence of a E. heros MI-2
polypeptide encoded by an exemplary E. heros chromatin remodeling
gene DNA.
[0040] SEQ ID NO:10 shows a further exemplary E. heros chromatin
remodeling gene DNA, referred to herein in some places as
BSB_iswi-1.
[0041] SEQ ID NO:11 shows the amino acid sequence of a E. heros
ISWI-1 polypeptide encoded by an exemplary E. heros chromatin
remodeling gene DNA.
[0042] SEQ ID NO:12 shows a further exemplary E. heros chromatin
remodeling gene DNA, referred to herein in some places as
BSB_iswi-2.
[0043] SEQ ID NO:13 shows the amino acid sequence of a E. heros
ISWI-2 polypeptide encoded by an exemplary E. heros chromatin
remodeling gene DNA.
[0044] SEQ ID NO:14 shows a further exemplary E. heros chromatin
remodeling gene DNA, referred to herein in some places as
BSB_chd1.
[0045] SEQ ID NO:15 shows the amino acid sequence of a E. heros
CHD1 polypeptide encoded by an exemplary E. heros chromatin
remodeling gene DNA.
[0046] SEQ ID NO:16 shows an exemplary E. heros chromatin
remodeling gene DNA, referred to herein in some places as
BSB_mi-2-1, which is used in some examples for the production of a
dsRNA.
[0047] SEQ ID NO:17 shows an exemplary E. heros chromatin
remodeling gene DNA, referred to herein in some places as
BSB_iswi-1-1, which is used in some examples for the production of
a dsRNA.
[0048] SEQ ID NO:18 shows a further exemplary E. heros chromatin
remodeling gene DNA, referred to herein in some places as
BSB_iswi-2-1, which is used in some examples for the production of
a dsRNA.
[0049] SEQ ID NO:19 shows a further exemplary E. heros chromatin
remodeling gene DNA, referred to herein in some places as
BSB_chd1-1, which is used in some examples for the production of a
dsRNA.
[0050] SEQ ID NOs:20-29 show primers used to amplify gene regions
of chromatin remodeling genes.
[0051] SEQ ID NO:30 shows a further exemplary E. heros chromatin
remodeling gene DNA, referred to herein in some places as
BSB_ino80.
[0052] SEQ ID NO:31 shows the amino acid sequence of a E. heros
INO80 polypeptide encoded by an exemplary E. heros chromatin
remodeling gene DNA.
[0053] SEQ ID NO:32 shows a further exemplary E. heros chromatin
remodeling gene DNA, referred to herein in some places as
BSB_domino.
[0054] SEQ ID NO:33 shows the amino acid sequence of a E. heros
DOMINO polypeptide encoded by an exemplary E. heros chromatin
remodeling gene DNA.
[0055] SEQ ID NOs:34-37 show exemplary DNAs encoding dsRNA
sequences for targeting SNF2-Helicase regions of insect (e.g.,
Euschistus heros, Diabrotica, Tribolium, and Drosophila
melanogaster) chromatin remodeling gene DNA.
[0056] SEQ ID NOs:38-41 show exemplary DNAs encoding dsRNA
sequences for targeting chromatin remodeling domains (Chromodomain,
Bromodomain, or HAND-SLIDE regions) of insect (e.g., Euschistus
heros, Diabrotica, Tribolium, and Drosophila melanogaster)
chromatin remodeling gene DNA.
[0057] SEQ ID NO:42 shows an exemplary DNA encoding a YFP v2
hairpin-forming RNA; containing sense polynucleotides, a loop
polynucleotide (underlined) including an intron, and antisense
polynucleotide (bold font):
TABLE-US-00001 ATGTCATCTGGAGCACTTCTCTTTCATGGGAAGATTCCTTACGTTGTGGA
GATGGAAGGGAATGTTGATGGCCACACCTTTAGCATACGTGGGAAAGGCT
ACGGAGATGCCTCAGTGGGAAAGGACTAGTACCGGTTGGGAAAGGTATGT
TTCTGCTTCTACCTTTGATATATATATAATAATTATCACTAATTAGTAGT
AATATAGTATTTCAAGTATTTTTTTCAAAATAAAAGAATGTAGTATATAG
CTATTGCTTTTCTGTAGTTTATAAGTGTGTATATTTTAATTTATAACTTT
TCTAATATATGACCAAAACATGGTGATGTGCAGGTTGATCCGCGGTTACT
TTCCCACTGAGGCATCTCCGTAGCCTTTCCCACGTATGCTAAAGGTGTGG
CCATCAACATTCCCTTCCATCTCCACAACGTAAGGAATCTTCCCATGAAA
GAGAAGTGCTCCAGATGACAT
[0058] SEQ ID NOs:43-62 show exemplary RNAs transcribed from
nucleic acids comprising exemplary chromatin remodeling gene
polynucleotides and fragments thereof.
[0059] SEQ ID NO:63 shows the open reading frame of an exemplary E.
heros Brahma DNA.
[0060] SEQ ID NO:64 shows the open reading frame of an exemplary E.
heros mi-2 DNA.
[0061] SEQ ID NO:65 shows the open reading frame of an exemplary E.
heros iswi-1 DNA.
[0062] SEQ ID NO:66 shows the open reading frame of an exemplary E.
heros DNA.
[0063] SEQ ID NO:67 shows the open reading frame of an exemplary E.
heros chd1 DNA.
[0064] SEQ ID NOs:68-72 show further exemplary RNAs transcribed
from nucleic acids comprising exemplary chromatin remodeling gene
polynucleotides and fragments thereof.
[0065] SEQ ID NO:73 shows the open reading frame of an exemplary
muscle actin gene.
[0066] SEQ ID NOs:74-91 show oligonucleotides and probes used for
BSB probe hydrolysis qPCR assay.
DETAILED DESCRIPTION
I. Overview of Several Embodiments
[0067] 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
chromatin remodeling genes (e.g., brahma, mi-2, iswi-1, chd1,
iswi-2, ino80, and domino) in the Neotropical brown stink bug,
which is shown to disrupt embryonic development when, for example,
iRNA are molecules are delivered via chromatin remodeling
gene-targeting dsRNA fed to adult females. There was almost
complete absence of hatching in the eggs collected from females
exposed to chromatin remodeling gene-targeting dsRNA. In
embodiments herein, the ability to deliver chromatin remodeling
gene-targeting dsRNA by feeding 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.
[0068] 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.
[0069] Some 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; SEQ ID
NO:8; SEQ ID NO:10; SEQ ID NO:12; SEQ ID NO:14; SEQ ID NO:30; SEQ
ID NO:32; SEQ ID NO:63; SEQ ID NO:64; SEQ ID NO:65; SEQ ID NO:66;
SEQ ID NO:67; 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 NO:1; SEQ ID NO:3; SEQ ID NO:8; SEQ ID NO:10; SEQ ID NO:12; SEQ
ID NO:14; SEQ ID NO:16; SEQ ID NO:17; SEQ ID NO:18; SEQ ID NO:19;
SEQ ID NO:30; SEQ ID NO:32; SEQ ID NO:63; SEQ ID NO:64; SEQ ID
NO:65; SEQ ID NO:66; and SEQ ID NO:67.
[0070] 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, any of SEQ ID NO:1; SEQ
ID NO:8; SEQ ID NO:10; SEQ ID NO:12; SEQ ID NO:14; SEQ ID NO:30;
SEQ ID NO:32; SEQ ID NO:63; SEQ ID NO:64; SEQ ID NO:65; SEQ ID
NO:66; SEQ ID NO:67; fragments of any of SEQ ID NO:1; SEQ ID NO:8;
SEQ ID NO:10; SEQ ID NO:12; SEQ ID NO:14; SEQ ID NO:30; SEQ ID
NO:32; SEQ ID NO:63; SEQ ID NO:64; SEQ ID NO:65; SEQ ID NO:66; SEQ
ID NO:67 (e.g., SEQ ID NO:3, SEQ ID NO:16, SEQ ID NO:17, SEQ ID
NO:18, and SEQ ID NO:19); and a polynucleotide consisting of a
partial sequence of a gene comprising one of SEQ ID NO:1; SEQ ID
NO:8; SEQ ID NO:10; SEQ ID NO:12; SEQ ID NO:14; SEQ ID NO:30; SEQ
ID NO:32; SEQ ID NO:63; SEQ ID NO:64; SEQ ID NO:65; SEQ ID NO:66;
SEQ ID NO:67; fragments of any of SEQ ID NO:1; SEQ ID NO:8; SEQ ID
NO:10; SEQ ID NO:12; SEQ ID NO:14; SEQ ID NO:30; SEQ ID NO:32; SEQ
ID NO:63; SEQ ID NO:64; SEQ ID NO:65; SEQ ID NO:66; SEQ ID NO:67;
and/or complements thereof.
[0071] Some 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:43 (e.g.,
SEQ ID NO:44); all or part of SEQ ID NO:45 (e.g., SEQ ID NO:49);
all or part of SEQ ID NO:46 (e.g., SEQ ID NO:50); all or part of
SEQ ID NO:47 (e.g., SEQ ID NO:51); all or part of SEQ ID NO:48
(e.g., SEQ ID NO:52); all or part of SEQ ID NO:53; and all or part
of SEQ ID NO:54. When ingested by a hemipteran pest, the iRNA
molecule(s) may silence or inhibit the expression of a target
chromatin remodeling gene (e.g., a DNA comprising all or part of a
polynucleotide selected from the group consisting of SEQ ID NO:1;
SEQ ID NO:8; SEQ ID NO:10; SEQ ID NO:12; SEQ ID NO:14; SEQ ID
NO:30; SEQ ID NO:32; SEQ ID NO:63; SEQ ID NO:64; SEQ ID NO:65; SEQ
ID NO:66; and SEQ ID NO:67) 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.
[0072] 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 sp.), and plants of
the family Poaceae.
[0073] Some 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.
[0074] 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.
[0075] 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.
[0076] 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.
[0077] 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, and/or feeding in
the hemipteran pest that contacts the iRNA molecule).
II. Abbreviations
[0078] BSB Neotropical brown stink bug (Euschistus heros)
[0079] dsRNA double-stranded ribonucleic acid
[0080] GI growth inhibition
[0081] NCBI National Center for Biotechnology Information
[0082] gDNA genomic Deoxyribonucleic Acid
[0083] iRNA inhibitory ribonucleic acid
[0084] ISWI Imitation SWI/imitation switch
[0085] ORF open reading frame
[0086] RNAi ribonucleic acid interference
[0087] miRNA micro ribonucleic acid
[0088] siRNA small inhibitory ribonucleic acid
[0089] hpRNA hairpin ribonucleic acid
[0090] shRNA short hairpin ribonucleic acid
[0091] pRNAi parental RNA interference
[0092] UTR untranslated region
[0093] PCR Polymerase chain reaction
[0094] qPCR quantitative polymerase chain reaction
[0095] RISC RNA-induced Silencing Complex
[0096] RH relative humidity
[0097] SEM standard error of the mean
[0098] YFP yellow fluorescent protein
III. Terms
[0099] 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:
[0100] 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; and soaking of
organisms with a solution comprising the nucleic acid molecule.
[0101] 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.
[0102] Corn plant: As used herein, the term "corn plant" refers to
a plant of the species, Zea mays (maize). The terms "corn plant"
and "maize" are used interchangeably herein.
[0103] Cotton plant: As used herein, the term "cotton plant" refers
to a plant of the species Gossypium sp.; for example, G.
hirsutum.
[0104] 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).
[0105] Genetic material: As used herein, the term "genetic
material" includes all genes, and nucleic acid molecules, such as
DNA and RNA.
[0106] 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 (Stal) (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).
[0107] 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.
[0108] 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.
[0109] 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).
[0110] 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-00002 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.
[0111] "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.
[0112] 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.
[0113] 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.
[0114] 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.
[0115] 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 "linker" in
a nucleic acid and which is transcribed into an RNA molecule.
[0116] 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.
[0117] 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.
[0118] 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.
[0119] 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.
[0120] 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.
[0121] 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.
[0122] 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.
[0123] 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.
[0124] 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.sup.nd ed., vol. 1-3, Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y., 1989, chapters 9 and 11; and Hames
and Higgins (eds.) Nucleic Acid Hybridization, IRL Press, Oxford,
1985. Further detailed instruction and guidance with regard to the
hybridization of nucleic acids may be found, for example, in
Tijssen, "Overview of principles of hybridization and the strategy
of nucleic acid probe assays," in Laboratory Techniques in
Biochemistry and Molecular Biology-Hybridization with Nucleic Acid
Probes, Part I, Chapter 2, Elsevier, N Y, 1993; and Ausubel et al.,
Eds., Current Protocols in Molecular Biology, Chapter 2, Greene
Publishing and Wiley-Interscience, N Y, 1995.
[0125] 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.
[0126] The following are representative, non-limiting hybridization
conditions.
[0127] 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.
[0128] 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.
[0129] 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.
[0130] 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 NO:1, SEQ ID NO:3, SEQ ID NO:8, SEQ
ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:17,
SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:30, SEQ ID NO:32, SEQ ID
NO:63; SEQ ID NO:64; SEQ ID NO:65; SEQ ID NO:66; and SEQ ID NO:67
are those nucleic acids that hybridize under stringent conditions
(e.g., the Moderate Stringency conditions set forth, supra) to the
reference nucleic acid of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:8,
SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID
NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:30, SEQ ID NO:32, SEQ
ID NO:63; SEQ ID NO:64; SEQ ID NO:65; SEQ ID NO:66; and SEQ ID
NO:67. 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.
[0131] 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.
[0132] 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.
[0133] 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.
[0134] 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.
[0135] 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.
[0136] 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).
[0137] 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).
[0138] 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.
[0139] Soybean plant: As used herein, the term "soybean plant"
refers to a plant of the species Glycine sp.; for example, G.
max.
[0140] 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).
[0141] Transgene: An exogenous nucleic acid. In some examples, a
transgene may be a DNA that encodes one or both strand(s) of an 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).
[0142] 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.).
[0143] 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.
[0144] Unless specifically indicated or implied, the terms "a,"
"an," and "the" signify "at least one," as used herein.
[0145] 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
[0146] A. Overview
[0147] 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 nucleic acids in a
hemipteran pest. In these and further embodiments, the 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 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.
[0148] In some embodiments, at least one target gene in a
hemipteran pest may be selected, wherein the target gene comprises
a chromatin remodeling polynucleotide (e.g., a gene). In particular
examples, such a chromatin remodeling gene in a hemipteran pest is
selected, wherein the target gene comprises a polynucleotide
selected from among BSB_Brahma (SEQ ID NO:1 and SEQ ID NO:63);
BSB_mi-2 (SEQ ID NO:8 and SEQ ID NO:64); BSB_iswi-1 (SEQ ID NO:10
and SEQ ID NO:65); BSB_chd1 (SEQ ID NO:14 and SEQ ID NO:67);
BSB_iswi-2 (SEQ ID NO:12 and SEQ ID NO:66); BSB_ino80 (SEQ ID
NO:30); and BSB_domino (SEQ ID NO:32). For example, a target gene
in certain embodiments comprises a chromatin remodeling
polynucleotide selected from among SEQ ID NO:1, SEQ ID NO: 8, SEQ
ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:30, SEQ ID NO:32,
SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID
NO:67; and fragments of any of the foregoing (e.g., SEQ ID NO:3,
SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:19).
[0149] In some embodiments, a chromatin remodeling polynucleotide
encodes a member of the group of "ATP-dependent remodeling
enzymes," a class of ATPases that contain a SNF2 domain (sucrose
non-fermenting, originally identified in Saccharomyces cerevisiae).
ATP-dependent remodeling enzymes include, for example and without
limitation, BRAHMA and its orthologs; MI-2 and its orthologs; ISWI,
its paralogs, and its orthologs (e.g., ISWI-1 and ISWI-2); CHD1 and
its orthologs; INO80 and its orthologs; and DOMINO and its
orthologs. Chromatin remodelers (e.g., ATP-dependent remodeling
enzymes) exert lasting epigenetic effects by mobilizing nucleosomes
and thus changing the access of the transcriptional machinery to
DNA.
[0150] ATP-dependent remodeling enzymes share the same functional
domains and sequence-level conservation. In Pfam
(pfam.sanger.ac.uk) searches, ATP-dependent remodeling enzymes can
be identified by a combination of SNF2 family N-terminal and
Helicase conserved C-terminal (SNF2-Helicase) domains. Thus, RNAi
target sites can be designed within the conserved SNF2 family
N-terminal and Helicase C-terminal domains (here referred to as
SNF2-Helicase) that are common to all chromatin remodelers, as well
as chromatin binding or other functional domains that are conserved
within each family, which include but are not limited to
bromodomain, chromodomain, and HAND-SLIDE domains.
[0151] In some 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 chromatin remodeling gene. 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 a Brahma, mi-2, iswi-1, chd1, iswi-2, ino80,
or domino gene. Examples of such translation products include, for
example and without limitation: SEQ ID NO:2; SEQ ID NO:9; SEQ ID
NO:11; SEQ ID NO:13; SEQ ID NO:15; SEQ ID NO:31; and SEQ ID
NO:33.
[0152] 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 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.
[0153] 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.
[0154] 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.
Therefore, 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 are specifically
complementary to all or part of a target nucleic acid in a
hemipteran pest.
[0155] In particular examples, nucleic acid molecules useful for
the control of hemipteran pests may include: all or part of a
nucleic acid isolated from a hemipteran insect (e.g., BSB)
comprising a chromatin remodeling gene polynucleotide (e.g., any of
SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12,
SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID
NO:19, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:63, SEQ ID NO:64, SEQ
ID NO:65, SEQ ID NO:66, and SEQ ID NO:67); DNAs that when expressed
result in an RNA molecule comprising a polynucleotide that is
specifically complementary to all or part of a RNA molecule that is
encoded by chromatin remodeling gene; 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
an RNA molecule encoded by a chromatin remodeling gene; 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 a chromatin remodeling gene; 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.
[0156] B. Nucleic Acid Molecules
[0157] 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.
[0158] 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; SEQ ID NO:63; the complement of SEQ ID
NO:1; the complement of SEQ ID NO:63; a fragment of at least 15
contiguous nucleotides (e.g., at least 19 contiguous nucleotides)
of SEQ ID NO:1 or SEQ ID NO:63 (e.g., SEQ ID NO:3); the complement
of a fragment of at least 15 contiguous nucleotides of SEQ ID NO:1
or SEQ ID NO:63; a coding polynucleotide of a hemipteran insect
(e.g., BSB) comprising SEQ ID NO:1 or SEQ ID NO:63; the complement
of a coding polynucleotide of a hemipteran insect comprising SEQ ID
NO:1 or SEQ ID NO:63; a fragment of at least 15 contiguous
nucleotides of a coding polynucleotide of a hemipteran insect
comprising SEQ ID NO:1 or SEQ ID NO:63; and the complement of a
fragment of at least 15 contiguous nucleotides of a coding
polynucleotide of a hemipteran insect comprising SEQ ID NO:1 or SEQ
ID NO:63. 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.
[0159] Alternative 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:8; SEQ ID NO:64; the complement of SEQ ID
NO:8; the complement of SEQ ID NO:64; a fragment of at least 15
contiguous nucleotides (e.g., at least 19 contiguous nucleotides)
of SEQ ID NO:8 or SEQ ID NO:64 (e.g., SEQ ID NO:16); the complement
of a fragment of at least 15 contiguous nucleotides of SEQ ID NO:8
or SEQ ID NO:64; a coding polynucleotide of a hemipteran insect
comprising SEQ ID NO:8 or SEQ ID NO:64; the complement of a coding
polynucleotide of a hemipteran insect comprising SEQ ID NO:8 or SEQ
ID NO:64; a fragment of at least 15 contiguous nucleotides of a
coding polynucleotide of a hemipteran insect comprising SEQ ID NO:8
or SEQ ID NO:64; and the complement of a fragment of at least 15
contiguous nucleotides of a coding polynucleotide of a hemipteran
insect comprising SEQ ID NO:8 or SEQ ID NO:64. 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.
[0160] Particular 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:10; SEQ ID NO:65; the complement of SEQ ID
NO:10; the complement of SEQ ID NO:65; a fragment of at least 15
contiguous nucleotides (e.g., at least 19 contiguous nucleotides)
of SEQ ID NO:10 or SEQ ID NO:65 (e.g., SEQ ID NO:17); the
complement of a fragment of at least 15 contiguous nucleotides of
SEQ ID NO:10 or SEQ ID NO:65; a coding polynucleotide of a
hemipteran insect comprising SEQ ID NO:10 or SEQ ID NO:65; the
complement of a coding polynucleotide of a hemipteran insect
comprising SEQ ID NO:10 or SEQ ID NO:65; a fragment of at least 15
contiguous nucleotides of a coding polynucleotide of a hemipteran
insect comprising SEQ ID NO:10 or SEQ ID NO:65; and the complement
of a fragment of at least 15 contiguous nucleotides of a coding
polynucleotide of a hemipteran insect comprising SEQ ID NO:10 or
SEQ ID NO:65. 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.
[0161] 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:12; SEQ ID NO:66; the complement of SEQ ID
NO:12; the complement of SEQ ID NO:66; a fragment of at least 15
contiguous nucleotides (e.g., at least 19 contiguous nucleotides)
of SEQ ID NO:12 or SEQ ID NO:66 (e.g., SEQ ID NO:18); the
complement of a fragment of at least 15 contiguous nucleotides of
SEQ ID NO:12 or SEQ ID NO:66; a coding polynucleotide of a
hemipteran insect comprising SEQ ID NO:12 or SEQ ID NO:66; the
complement of a coding polynucleotide of a hemipteran insect
comprising SEQ ID NO:12 or SEQ ID NO:66; a fragment of at least 15
contiguous nucleotides of a coding polynucleotide of a hemipteran
insect comprising SEQ ID NO:12 or SEQ ID NO:66; and the complement
of a fragment of at least 15 contiguous nucleotides of a coding
polynucleotide of a hemipteran insect comprising SEQ ID NO:12 or
SEQ ID NO:66. 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.
[0162] Other 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:14; SEQ ID NO:67; the complement of SEQ ID
NO:14; the complement of SEQ ID NO:67; a fragment of at least 15
contiguous nucleotides (e.g., at least 19 contiguous nucleotides)
of SEQ ID NO:14 or SEQ ID NO:67 (e.g., SEQ ID NO:19); the
complement of a fragment of at least 15 contiguous nucleotides of
SEQ ID NO:14 or SEQ ID NO:67; a coding polynucleotide of a
hemipteran insect comprising SEQ ID NO:14 or SEQ ID NO:67; the
complement of a coding polynucleotide of a hemipteran insect
comprising SEQ ID NO:14 or SEQ ID NO:67; a fragment of at least 15
contiguous nucleotides of a coding polynucleotide of a hemipteran
insect comprising SEQ ID NO:14 or SEQ ID NO:67; and the complement
of a fragment of at least 15 contiguous nucleotides of a coding
polynucleotide of a hemipteran insect comprising SEQ ID NO:14 or
SEQ ID NO:67. 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.
[0163] 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:30; the complement of SEQ ID NO:30; a
fragment of at least 15 contiguous nucleotides (e.g., at least 19
contiguous nucleotides) of SEQ ID NO:30; the complement of a
fragment of at least 15 contiguous nucleotides of SEQ ID NO:30; a
coding polynucleotide of a hemipteran insect comprising SEQ ID
NO:30; the complement of a coding polynucleotide of a hemipteran
insect comprising SEQ ID NO:30; a fragment of at least 15
contiguous nucleotides of a coding polynucleotide of a hemipteran
insect comprising SEQ ID NO:30; and the complement of a fragment of
at least 15 contiguous nucleotides of a coding polynucleotide of a
hemipteran insect comprising SEQ ID NO:30. 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.
[0164] Other 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:32; the complement of SEQ ID NO:32; a
fragment of at least 15 contiguous nucleotides (e.g., at least 19
contiguous nucleotides) of SEQ ID NO:32; the complement of a
fragment of at least 15 contiguous nucleotides of SEQ ID NO:32; a
coding polynucleotide of a hemipteran insect comprising SEQ ID
NO:32; the complement of a coding polynucleotide of a hemipteran
insect comprising SEQ ID NO:32; a fragment of at least 15
contiguous nucleotides of a coding polynucleotide of a hemipteran
insect comprising SEQ ID NO:32; and the complement of a fragment of
at least 15 contiguous nucleotides of a coding polynucleotide of a
hemipteran insect comprising SEQ ID NO:32. 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.
[0165] In some 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:43; the complement of SEQ ID NO:43; SEQ ID NO:44; the
complement of SEQ ID NO:44; SEQ ID NO:45; the complement of SEQ ID
NO:45; SEQ ID NO:46; the complement of SEQ ID NO:46; SEQ ID NO:47;
the complement of SEQ ID NO:47; SEQ ID NO:48; the complement of SEQ
ID NO:48; SEQ ID NO:49; the complement of SEQ ID NO:49; SEQ ID
NO:50; the complement of SEQ ID NO:50; SEQ ID NO:51; the complement
of SEQ ID NO:51; SEQ ID NO:52; the complement of SEQ ID NO:52; SEQ
ID NO:53; the complement of SEQ ID NO:53; SEQ ID NO:54; the
complement of SEQ ID NO:54; SEQ ID NO:55; the complement of SEQ ID
NO:55; SEQ ID NO:56; the complement of SEQ ID NO:56; SEQ ID NO:57;
the complement of SEQ ID NO:57; SEQ ID NO:58; the complement of SEQ
ID NO:58; SEQ ID NO:59; the complement of SEQ ID NO:59; SEQ ID
NO:60; the complement of SEQ ID NO:60; SEQ ID NO:61; the complement
of SEQ ID NO:61; SEQ ID NO:62; the complement of SEQ ID NO:62; SEQ
ID NO:68; the complement of SEQ ID NO:68; SEQ ID NO:69; the
complement of SEQ ID NO:69; SEQ ID NO:70; the complement of SEQ ID
NO:70; SEQ ID NO:71; the complement of SEQ ID NO:71; SEQ ID NO:72;
the complement of SEQ ID NO:72; a polyribonucleotide transcribed in
a hemipteran insect from a gene comprising SEQ ID NO:1, SEQ ID
NO:3, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID
NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:30, SEQ
ID NO:32, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66,
or SEQ ID NO:67; the complement of a polyribonucleotide transcribed
in a hemipteran insect from a gene comprising SEQ ID NO:1, SEQ ID
NO:3, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID
NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:30, SEQ
ID NO:32, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66,
or SEQ ID NO:67; a fragment of at least 15 contiguous nucleotides
of a polyribonucleotide transcribed in a hemipteran insect from a
gene comprising SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:8, SEQ ID
NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:17, SEQ
ID NO:18, SEQ ID NO:19, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:63,
SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, or SEQ ID NO:67; and the
complement of a fragment of at least 15 contiguous nucleotides of a
polyribonucleotide transcribed in a hemipteran insect from a gene
comprising SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:10, SEQ
ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18,
SEQ ID NO:19, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:63, SEQ ID
NO:64, SEQ ID NO:65, SEQ ID NO:66, or SEQ ID NO:67. 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. In some embodiments,
contact with or uptake by the insect occurs via feeding on plant
material or bait comprising the iRNA. In some embodiments, contact
with or uptake by the insect occurs via spraying of a plant
comprising the insect with a composition comprising the iRNA.
[0166] In some 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, SEQ ID NO:8, SEQ ID NO:10,
SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:30, SEQ ID NO:32, SEQ ID
NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, and SEQ ID NO:67.
Derivatives of SEQ ID NO:1, SEQ ID NO:8, SEQ ID NO:10, SEQ ID
NO:12, SEQ ID NO:14, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:63, SEQ
ID NO:64, SEQ ID NO:65, SEQ ID NO:66, and SEQ ID NO:67 includes
fragments of these polynucleotides. In some embodiments, such a
fragment may comprise, for example, at least about 15 contiguous
nucleotides of SEQ ID NO:1, SEQ ID NO:8, SEQ ID NO:10, SEQ ID
NO:12, SEQ ID NO:14, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:63, SEQ
ID NO:64, SEQ ID NO:65, SEQ ID NO:66, or SEQ ID NO:67, 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,
SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID
NO:30, SEQ ID NO:32, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ
ID NO:66, or SEQ ID NO:67, 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, SEQ ID NO:8,
SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:30, SEQ ID
NO:32, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, or
SEQ ID NO:67, or a complement thereof.
[0167] 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 any of SEQ ID
NO:1, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID
NO:30, SEQ ID NO:32, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ
ID NO:66, and SEQ ID NO:67; 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 any of S SEQ ID NO:1, SEQ ID NO:8, SEQ ID NO:10,
SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:30, SEQ ID NO:32, SEQ ID
NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, and SEQ ID NO:67;
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.
[0168] In certain embodiments, dsRNA molecules provided by the
invention comprise polynucleotides complementary to a transcript
from a target gene comprising SEQ ID NO:1, SEQ ID NO:8, SEQ ID
NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:30, SEQ ID NO:32, SEQ
ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, and SEQ ID
NO:67, and/or polynucleotides complementary to a fragment of SEQ ID
NO:1, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID
NO:30, SEQ ID NO:32, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ
ID NO:66, and SEQ ID NO:67, 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, SEQ ID
NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:30, SEQ
ID NO:32, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66,
or SEQ ID NO:67, a contiguous fragment of SEQ ID NO:1, SEQ ID NO:8,
SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:30, SEQ ID
NO:32, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, or
SEQ ID NO:67, 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, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID
NO:30, SEQ ID NO:32, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ
ID NO:66, or SEQ ID NO:67, a contiguous fragment of SEQ ID NO:1,
SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID
NO:30, SEQ ID NO:32, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ
ID NO:66, or SEQ ID NO:67, or the complement of any of the
foregoing.
[0169] 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.
[0170] In some 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).
[0171] 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 linker. The linker 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.
[0172] 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.
[0173] 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.
[0174] C. Obtaining Nucleic Acid Molecules
[0175] 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 polynucleotides is not, however, a straight-forward
process. Only a small number of polynucleotides in the hemipteran
pest will be effective targets. For example, it cannot be predicted
with certainty whether a particular polynucleotide can be
effectively down-regulated by nucleic acid molecules of the
invention, or whether down-regulation of a particular
polynucleotide will have a detrimental effect on the growth,
viability, proliferation, and/or reproduction of the hemipteran
pest. The vast majority of pest polynucleotides, such as ESTs
isolated therefrom (e.g., the coleopteran pest polynucleotides
listed in U.S. Pat. No. 7,612,194), do not have a detrimental
effect on the growth, viability, proliferation, and/or reproduction
of the pest. Neither is it predictable which of the 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 polynucleotides in a host plant and
providing the detrimental effect on the pest upon feeding without
causing harm to the host plant.
[0176] 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 of compositions by a target organism containing one or more
dsRNAs, 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, lay 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 or G. max), 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.
[0177] Thus, in some 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 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.
[0178] 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.
[0179] 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 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.
[0180] 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.
[0181] 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.
[0182] In 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.
[0183] D. Recombinant Vectors and Host Cell Transformation
[0184] 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)
[0185] 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.
[0186] In alternative embodiments, one strand of a dsRNA molecule
may be formed by transcription from a polynucleotide which is
substantially homologous to the RNA encoded by a polynucleotide
selected from the group consisting of SEQ ID NO:1, SEQ ID NO:8, SEQ
ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:30, SEQ ID NO:32,
SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, and SEQ ID
NO:67; the complement of SEQ ID NO:1, SEQ ID NO:8, SEQ ID NO:10,
SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:30, SEQ ID NO:32, SEQ ID
NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, and SEQ ID NO:67;
a fragment of at least 15 contiguous nucleotides of SEQ ID NO:1,
SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID
NO:30, SEQ ID NO:32, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ
ID NO:66, and SEQ ID NO:67; the complement of a fragment of at
least 15 contiguous nucleotides of SEQ ID NO:1, SEQ ID NO:8, SEQ ID
NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:30, SEQ ID NO:32, SEQ
ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, and SEQ ID
NO:67; a coding polynucleotide of a hemipteran insect (e.g., BSB)
comprising SEQ ID NO:1, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12,
SEQ ID NO:14, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:63, SEQ ID
NO:64, SEQ ID NO:65, SEQ ID NO:66, and SEQ ID NO:67; the complement
of a coding polynucleotide of a hemipteran insect comprising SEQ ID
NO:1, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID
NO:30, SEQ ID NO:32, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ
ID NO:66, and SEQ ID NO:67; a fragment of at least 15 contiguous
nucleotides of a coding polynucleotide of a hemipteran insect
comprising SEQ ID NO:1, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12,
SEQ ID NO:14, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:63, SEQ ID
NO:64, SEQ ID NO:65, SEQ ID NO:66, and SEQ ID NO:67; and the
complement of a fragment of at least 15 contiguous nucleotides of a
coding polynucleotide of a hemipteran insect comprising SEQ ID
NO:1, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID
NO:30, SEQ ID NO:32, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ
ID NO:66, and SEQ ID NO:67.
[0187] 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 linker of,
for example, from about five (.about.5) to about one thousand
(.about.1000) nucleotides. The linker may form a loop between the
sense and anti sense polynucleotides. The sense polynucleotide or
the antisense polynucleotide may be substantially homologous to an
RNA encoded by a target gene (e.g., a chromatin remodeling gene
comprising SEQ ID NO:1, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12,
SEQ ID NO:14, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:63, SEQ ID
NO:64, SEQ ID NO:65, SEQ ID NO:66, or SEQ ID NO:67) or fragment
thereof. In some embodiments, however, a recombinant DNA molecule
may encode an RNA that may form a dsRNA molecule without a linker.
In embodiments, a sense coding polynucleotide and an antisense
coding polynucleotide may be different lengths.
[0188] 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 chromatin
remodeling gene comprising SEQ ID NO:1, SEQ ID NO:8, SEQ ID NO:10,
SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:30, SEQ ID NO:32, SEQ ID
NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, or SEQ ID NO:67,
and fragments thereof); linking this polynucleotide to a second
segment linker 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.
[0189] 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-protective
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.
[0190] To impart protection from a hemipteran pest 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.
[0191] 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.
[0192] 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
U.S. Pat. No. 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. Nos. 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).
[0193] 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.
[0194] 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); AtAntl; 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).
[0195] 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).
[0196] 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 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 linker.
[0197] In some 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) 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 a different insect (e.g., 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.
[0198] 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.
[0199] 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 xylE 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.
[0200] 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.
[0201] 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.
[0202] 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.
[0203] Thus, 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.
[0204] 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.
[0205] 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.
[0206] 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.
[0207] 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 or G. max) or tissue type,
including cell cultures.
[0208] 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 an SNP assay or a
thermal amplification assay that allows for the distinction between
heterozygotes and homozygotes (i.e., a zygosity assay).
[0209] 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-protective 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, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID
NO:14, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:63, SEQ ID NO:64, SEQ
ID NO:65, SEQ ID NO:66, and SEQ ID NO:67), both in different
populations of the same species of hemipteran pest, or in different
species of hemipteran pests.
[0210] 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.
[0211] 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.
[0212] 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.
[0213] 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, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12,
SEQ ID NO:14, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:63, SEQ ID
NO:64, SEQ ID NO:65, SEQ ID NO:66, and SEQ ID NO:67; 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
[0214] A. Overview
[0215] 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.
[0216] B. RNAi-mediated Target Gene Suppression
[0217] In embodiments, the invention provides iRNA molecules (e.g.,
dsRNA, siRNA, miRNA, shRNA, and hpRNA) that may be designed to
target essential 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.
[0218] 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.
[0219] In 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).
[0220] In 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.
[0221] 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.
[0222] 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; SEQ ID NO:8; the complement of
SEQ ID NO:8; SEQ ID NO:10; the complement of SEQ ID NO:10; SEQ ID
NO:12; the complement of SEQ ID NO:12; SEQ ID NO:14; the complement
of SEQ ID NO:14; SEQ ID NO:30; the complement of SEQ ID NO:30; SEQ
ID NO:32; the complement of SEQ ID NO:32; SEQ ID NO:63; the
complement of SEQ ID NO:63; SEQ ID NO:64; the complement of SEQ ID
NO:64; SEQ ID NO:65; the complement of SEQ ID NO:65; SEQ ID NO:66;
the complement of SEQ ID NO:66; SEQ ID NO:67; the complement of SEQ
ID NO:67; 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 fragment of at least 15 contiguous
nucleotides of SEQ ID NO:8; the complement of a fragment of at
least 15 contiguous nucleotides of SEQ ID NO:8; a fragment of at
least 15 contiguous nucleotides of SEQ ID NO:10; the complement of
a fragment of at least 15 contiguous nucleotides of SEQ ID NO:10; a
fragment of at least 15 contiguous nucleotides of SEQ ID NO:12; the
complement of a fragment of at least 15 contiguous nucleotides of
SEQ ID NO:12; a fragment of at least 15 contiguous nucleotides of
SEQ ID NO:14; the complement of a fragment of at least 15
contiguous nucleotides of SEQ ID NO:14; a fragment of at least 15
contiguous nucleotides of SEQ ID NO:30; the complement of a
fragment of at least 15 contiguous nucleotides of SEQ ID NO:30; a
fragment of at least 15 contiguous nucleotides of SEQ ID NO:32; the
complement of a fragment of at least 15 contiguous nucleotides of
SEQ ID NO:32; a fragment of at least 15 contiguous nucleotides of
SEQ ID NO:63; the complement of a fragment of at least 15
contiguous nucleotides of SEQ ID NO:63; a fragment of at least 15
contiguous nucleotides of SEQ ID NO:64; the complement of a
fragment of at least 15 contiguous nucleotides of SEQ ID NO:64; a
fragment of at least 15 contiguous nucleotides of SEQ ID NO:65; the
complement of a fragment of at least 15 contiguous nucleotides of
SEQ ID NO:65; a fragment of at least 15 contiguous nucleotides of
SEQ ID NO:66; the complement of a fragment of at least 15
contiguous nucleotides of SEQ ID NO:66; a fragment of at least 15
contiguous nucleotides of SEQ ID NO:67; the complement of a
fragment of at least 15 contiguous nucleotides of SEQ ID NO:67; a
coding polynucleotide of a hemipteran insect comprising SEQ ID
NO:1; the complement of a coding polynucleotide of a hemipteran
insect comprising SEQ ID NO:1; a coding polynucleotide of a
hemipteran insect comprising SEQ ID NO:8; the complement of a
coding polynucleotide of a hemipteran insect comprising SEQ ID
NO:8; a coding polynucleotide of a hemipteran insect comprising SEQ
ID NO:10; the complement of a coding polynucleotide of a hemipteran
insect comprising SEQ ID NO:10; a coding polynucleotide of a
hemipteran insect comprising SEQ ID NO:12; the complement of a
coding polynucleotide of a hemipteran insect comprising SEQ ID
NO:12; a coding polynucleotide of a hemipteran insect comprising
SEQ ID NO:14; the complement of a coding polynucleotide of a
hemipteran insect comprising SEQ ID NO:14; a coding polynucleotide
of a hemipteran insect comprising SEQ ID NO:30; the complement of a
coding polynucleotide of a hemipteran insect comprising SEQ ID
NO:30; a coding polynucleotide of a hemipteran insect comprising
SEQ ID NO:32; the complement of a coding polynucleotide of a
hemipteran insect comprising SEQ ID NO:32; a coding polynucleotide
of a hemipteran insect comprising SEQ ID NO:63; the complement of a
coding polynucleotide of a hemipteran insect comprising SEQ ID
NO:63; a coding polynucleotide of a hemipteran insect comprising
SEQ ID NO:64; the complement of a coding polynucleotide of a
hemipteran insect comprising SEQ ID NO:64; a coding polynucleotide
of a hemipteran insect comprising SEQ ID NO:65; the complement of a
coding polynucleotide of a hemipteran insect comprising SEQ ID
NO:65; a coding polynucleotide of a hemipteran insect comprising
SEQ ID NO:66; the complement of a coding polynucleotide of a
hemipteran insect comprising SEQ ID NO:66; a coding polynucleotide
of a hemipteran insect comprising SEQ ID NO:67; the complement of a
coding polynucleotide of a hemipteran insect comprising SEQ ID
NO:67; a fragment of at least 15 contiguous nucleotides of a coding
polynucleotide of a hemipteran insect comprising SEQ ID NO:1; the
complement of a fragment of at least 15 contiguous nucleotides of a
coding polynucleotide of a hemipteran insect comprising SEQ ID
NO:1; a fragment of at least 15 contiguous nucleotides of a coding
polynucleotide of a hemipteran insect comprising SEQ ID NO:8; the
complement of a fragment of at least 15 contiguous nucleotides of a
coding polynucleotide of a hemipteran insect comprising SEQ ID
NO:8; a fragment of at least 15 contiguous nucleotides of a coding
polynucleotide of a hemipteran insect comprising SEQ ID NO:10; the
complement of a fragment of at least 15 contiguous nucleotides of a
coding polynucleotide of a hemipteran insect comprising SEQ ID
NO:10; a fragment of at least 15 contiguous nucleotides of a coding
polynucleotide of a hemipteran insect comprising SEQ ID NO:12; the
complement of a fragment of at least 15 contiguous nucleotides of a
coding polynucleotide of a hemipteran insect comprising SEQ ID
NO:12; a fragment of at least 15 contiguous nucleotides of a coding
polynucleotide of a hemipteran insect comprising SEQ ID NO:14; the
complement of a fragment of at least 15 contiguous nucleotides of a
coding polynucleotide of a hemipteran insect comprising SEQ ID
NO:14; a fragment of at least 15 contiguous nucleotides of a coding
polynucleotide of a hemipteran insect comprising SEQ ID NO:30; the
complement of a fragment of at least 15 contiguous nucleotides of a
coding polynucleotide of a hemipteran insect comprising SEQ ID
NO:30; a fragment of at least 15 contiguous nucleotides of a coding
polynucleotide of a hemipteran insect comprising SEQ ID NO:32; and
the complement of a fragment of at least 15 contiguous nucleotides
of a coding polynucleotide of a hemipteran insect comprising SEQ ID
NO:32; a fragment of at least 15 contiguous nucleotides of a coding
polynucleotide of a hemipteran insect comprising SEQ ID NO:63; and
the complement of a fragment of at least 15 contiguous nucleotides
of a coding polynucleotide of a hemipteran insect comprising SEQ ID
NO:63; a fragment of at least 15 contiguous nucleotides of a coding
polynucleotide of a hemipteran insect comprising SEQ ID NO:64; and
the complement of a fragment of at least 15 contiguous nucleotides
of a coding polynucleotide of a hemipteran insect comprising SEQ ID
NO:64; a fragment of at least 15 contiguous nucleotides of a coding
polynucleotide of a hemipteran insect comprising SEQ ID NO:65; and
the complement of a fragment of at least 15 contiguous nucleotides
of a coding polynucleotide of a hemipteran insect comprising SEQ ID
NO:65; a fragment of at least 15 contiguous nucleotides of a coding
polynucleotide of a hemipteran insect comprising SEQ ID NO:66; and
the complement of a fragment of at least 15 contiguous nucleotides
of a coding polynucleotide of a hemipteran insect comprising SEQ ID
NO:66; a fragment of at least 15 contiguous nucleotides of a coding
polynucleotide of a hemipteran insect comprising SEQ ID NO:67; and
the complement of a fragment of at least 15 contiguous nucleotides
of a coding polynucleotide of a hemipteran insect comprising SEQ ID
NO:67. 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.
[0223] 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.
[0224] 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.
[0225] 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.
[0226] 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.
[0227] C. Expression of IRNA Molecules Provided to a Hemipteran
Pest
[0228] 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.
[0229] 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 chromatin
remodeling gene DNA molecule, for example, comprising a
polynucleotide selected from the group consisting of SEQ ID NO:1,
SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID
NO:30, SEQ ID NO:32, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ
ID NO:66, and SEQ ID NO:67. 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.
[0230] 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.
[0231] 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,
a miRNA molecule, a shRNA molecule, or a 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.
[0232] 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.
[0233] In embodiments, suppression of a target gene (e.g., a
chromatin remodeling 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.
[0234] 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 lay eggs or lay 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.
[0235] 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.
[0236] 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 (e.g., Cry1A, Cry2A, Cry3A, Cry11A, and Cry51A) 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.
[0237] 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.
[0238] 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, Alcaligenes spp., Pseudomonas spp., 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.
[0239] 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 female and/or male
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.
[0240] 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. 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.
[0241] In some 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.
[0242] 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.
[0243] 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.
[0244] 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 or bait 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.
[0245] 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.
[0246] 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: Identification of Candidate Target Genes
[0247] RNAi Target Selection.
[0248] In one example, six stages of BSB development were selected
for mRNA library preparation. Additional samples were prepared
using BSB midgut and salivary glands. Brown stink bug midguts and
salivary glands were dissected from 10 and 25 mixed sex adults
respectively under a dissecting microscope on a chilled clean glass
slide and immediately frozen on dry ice. 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. Hi
Seq.TM. system (San Diego, Calif.) provided candidate target gene
sequences for use in RNAi insect control technology. HiSeq.TM.
generated a total of about 378 million reads for the six samples.
The 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.
[0249] BSB_Brahma, Mi-2, Iswi-1, Iswi-2, Chd1, Ino80, and Domino
Ortholog Identification.
[0250] tBLASTn searches of the BSB pooled transcriptome were
performed using sequences of the Drosophila BRAHMA (brm-PA, GENBANK
Accession No. NP_536745 and NP_536746), MI-2 (Mi-2-PA, GENBANK
Accession No. NP_001014591.1, NP_001163476.1, NP_001262078.1,
NP_649154.2, and NP_001014591.1), ISWI (Iswi-PA, GENBANK Accession
No. NP_523719, NP_725203, and NP_725204), and CHD1 (Chd1-PA,
GENBANK Accession No. NP_477197 and NP_001245851) proteins as
queries. BSB_brahma (SEQ ID NO:1; SEQ ID NO:63), mi-2 (SEQ ID NO:8;
SEQ ID NO:64), iswi-1 (SEQ ID NO:10; SEQ ID NO:65), iswi-2 (SEQ ID
NO:12; SEQ ID NO:66), chd1 (SEQ ID NO:14; SEQ ID NO:67), ino80 (SEQ
ID NO:30), and domino (SEQ ID NO:32) transcripts were identified as
BSB candidate target genes.
[0251] BSB homology info. The BSB_brahma (SEQ ID NO:1) is somewhat
(72% identity) related to a fragment of a sequence from Ciona
intestinalis (GENBANK Accession No. AK116913.1). The closest
homolog of the BSB BRAHMA amino acid sequence (SEQ ID NO:2) is a
Camponotus floridanus protein having GENBANK Accession No.
EFN67856.1 (79% similar; 70% identical over the homology region).
The BSB_mi-2 (SEQ ID NO:8) is somewhat (76% identity) related to a
fragment of a sequence from Acyrthosiphon pisum (GENBANK Accession
No. XM_008186702.1). The closest homolog of the BSB MI-2 amino acid
sequence (SEQ ID NO:9) is a Bombus impatiens protein having GENBANK
Accession No. XP 003493868.1 (79% similar; 71% identical over the
homology region). The BSB iswi-1 (SEQ ID NO:10) is somewhat (75%
identity) related to a fragment of a sequence from Bobmus impatiens
(GENBANK Accession No. XM_003486758.1). The closest homologs of the
BSB ISWI-1 amino acid sequence (SEQ ID NO:11) are a Megachile
rotundata and Apis dorsata proteins having GENBANK Accession Nos.
XP_003708682.1 and XP_006615660.1 respectively (91% similar; 84%
identical over the homology region). The BSB iswi-2 (SEQ ID NO:12)
is somewhat (76% identity) related to a fragment of a sequence from
Latimeria chalumnaw (GENBANK Accession No. XM_005994941.1). The
closest homolog of the BSB ISWI-2 amino acid sequence (SEQ ID
NO:13) is a Cerapachys biroi protein having GENBANK Accession No.
EZA60706.1 (93% similar; 84% identical over the homology region).
The BSB chd1 (SEQ ID NO:14) is somewhat (77% identity) related to a
fragment of a sequence from Apis mellifera (GENBANK Accession No.
XM_006565933.1). The closest homolog of the BSB CHD1 amino acid
sequence (SEQ ID NO:15) is a Riptortus pedestris protein having
GENBANK Accession No. BAN20905.1 (94% similar; 88% identical over
the homology region). The BSB_ino80 (SEQ ID NO:30) is somewhat (79%
identity) related to a fragment of a sequence from Hydra
magnipapillata (GENBANK Accession No. XM_002164516.2). The closest
homolog of the BSB INO80 amino acid sequence (SEQ ID NO:31) is a
Zootermopsis nevadensis protein having GENBANK Accession No.
KDR11347.1 (74% similar; 64% identical over the homology region).
The BSB_domino (SEQ ID NO:32) is somewhat (80% identity) related to
a fragment of a sequence from Apis florea (GENBANK Accession No.
XR_143356.1). The closest homolog of the BSB DOMINO amino acid
sequence (SEQ ID NO:33) is a Nasonia vitripennis protein having
GENBANK Accession No. XP_008210745.1 (71% similar; 58% identical
over the homology region).
[0252] These genes encode SNF2-type chromatin remodeler proteins,
which correspond to a subunit of the chromatin remodeler complexes
that play global roles in mobilizing nucleosomes. See, for example,
Brizuela et al. (supra); Kal et al. (2000) Genes Devel. 14:1058-71;
and Tamkun et al. (1992) Cell 68:561-72. Although they share a
SNF2-Helicase domain, most chromatin remodelers within each species
have non-redundant functions that are conferred by the additional
domains they comprise. These characteristics present chromatin
remodeling ATPases as attractive targets for
multi-generational/parental RNAi.
[0253] The SWI2/SNF2 (mating type switch/sucrose non-fermenting)
family of the ATP-dependent remodeling enzymes contains a
bromodomain, which binds acetylated histones. While yeasts and
vertebrates contain several SWI2/SNF2 proteins, only one SWI2/SNF2
protein, BRAHMA, has been identified in Drosophila. BRAHMA is
well-conserved, and yet distinct, from other insect SNF2-containing
proteins, with the putative orthologs clustering closely on a
phylogenetic tree. FIG. 2. The human BRAHMA (BRM) as well as the
Saccharomyces cerevisiae SNF2 protein cluster together with insect
BRAHMAs. Furthermore, the orthologs of the Drosophila BRAHMA
maintain overall protein domain conservation including the SNF2
ATPase/helicase, the bromodomain as well as additional domains:
conserved Gln, Leu, Gln motif domain (QLQ), DNA-binding HSA domain,
and BRK (brahma and kismet) domain. FIG. 3A.
[0254] BRAHMA is known to incorporate into BAP (Brahma Associated
Proteins) and PBAP (Polybromo-associated BAP) chromatin remodeling
complexes. The loss of Drosophila brahma impairs overall
transcription by RNA polymerase II (Pol II), suggesting a broad
function for the BRAHMA complexes. In Drosophila, the maternal
contribution of brahma is needed for early embryogenesis, while the
zygotic brahma expression is necessary for late embryonic
development. In addition to embryogenesis, Drosophila brahma is
involved in gametogenesis. Brahma RNAi-treated female BSB produce
no viable eggs. Table 5. Further, BSB females whose brahma was
depleted via RNAi lay no eggs altogether. Tables 3 and 4.
[0255] The ISWI (Imitation SWI/imitation switch) family is defined
by histone-biding domain that comprises the HAND, SANT, and SLIDE
domains in a HAND-SANT-SLIDE architecture (also annotated as
HAND-SLIDE). In Drosophila, the ISWI family of ATP-dependent
remodeling enzymes has only one member, ISWI. The Drosophila ISWI
can confer multiple functions by integrating into various complexes
that include ATP-dependent chromatin assembly and remodeling factor
(ACF), nucleosome remodeling factor (NURF), and chromatin
accessibility complex (CHRAC). Loss of ISWI in Drosophila results
in dramatic chromosome condensation defects.
[0256] BSB express at least two iswi homologs (SEQ ID NO:10 and SEQ
ID NO:12 (with SEQ ID NO:12 being partial sequence). The complete
BSB ISWI protein contains the SNF2 ATPase/helicase, HAND-SANT-SLIDE
(identified as HAND and SLIDE by Pfam) and DNA-binding domain
(DBINO). FIG. 3B. The identified ISWI-2 protein from BSB comprises
only HAND-SANT-SLIDE domains. FIG. 3B. The contig that comprises
iswi-2 (SEQ ID NO:12) is 1316 nucleotides long; based on the
alignment with known Drosophila ISWI protein this contig does not
contain the first half of the ISWI protein sequence. Therefore, it
is reasonable to assume that the current BSB transcriptome assembly
contains an incomplete sequence of iswi-2 transcript.
[0257] The parental RNAi applications of both BSB_Iswi-1 and
BSB_Iswi-2 result in both egg laying and egg hatch defects. Tables
4 and 5.
[0258] Proteins of the CHD (chromodomain helicase DNA-binding)
family of ATP-dependent remodeling enzymes contain two
amino-terminal chromodomains [chromatin organization modifier].
FIG. 3C. The Drosophila CHD proteins include CHD1, MI-2, CHD3, and
KISMET. The CHD family is further subdivided into three
subfamilies, herein referred to as subfamilies I, II, and III. The
Drosophila CHD1 belongs to CHD subfamily I, which has a C-terminal
DNA-binding domain. FIG. 1C (DUF4208). In Drosophila, CHD1 protein
shows similar distribution patterns to BRAHMA, yet chd1 mutant
flies are viable. Interestingly, the Drosophila chd1 is needed for
gametogenesis. BSB females subjected to chd1 RNAi show a
significant decrease in both egg production and hatch rates. Tables
4 and 5.
[0259] MI-2 and CHD3 belong to subfamily II. Enzymes of the CHD
subfamily II have no DNA-binding domain, but have Zn-finger-like
domains called PHD (plant homeodomain) fingers. The BSB ortholog of
MI-2 mirrors the Drosophila domain arrangement, and includes the
SNF2 ATPase/helicase domain, the double chromodomain, PHD fingers,
and CHDNT domain that is associated with PHD finger-containing
chromodomain helicases, as well as other conserved domains of
unknown functions, DUF1087 and DUF1086. FIG. 3D. The Drosophila
MI-2 is known to associate with the NuRD (Nucleosome Remodeling
Deacetylase) and dMec (Drosophila MEP-1 containing complex)
complexes. Maternal expression of mi-2 is necessary for
gametogenesis. BSB females whose mi-2 was depleted via RNAi lay
very few eggs. Table 4.
[0260] The third subfamily of CHD proteins is represented by KISMET
in Drosophila; in humans this subfamily comprises CHD5-9. Like
other CHD proteins, KISMET contains an SNF2 domain and a
chromodomain. FIG. 3E. Unlike other CHD subfamilies, KISMET has
characteristics of both CHD and SWI2/SNF2 proteins, in that it has
a BRK domain that is common to both BRAHMA and KISMET. Although BRK
is a well-established feature of Drosophila KISMET, a standard Pfam
analysis did not identify this domain in BSB. FIG. 3E. Loss of
either maternal or zygotic function of kismet causes defects during
Drosophila embryogenesis and the insects die during early larval
stages, while oogenesis is unaffected.
Example 2: Degenerate Sequences Comprising Chromatin Remodelers
[0261] Brahma and its homologs, as well as mi-2 and other chromatin
remodelers and their orthologs, share the same functional domains
and sequence-level conservation. RNAi target sites were designed
within the conserved SNF2 family N-terminal and Helicase C-terminal
domains (here referred to as SNF2-Helicase) that are common to all
chromatin remodelers, as well as chromatin binding and other
functional domains that are conserved within each family (including
bromodomain, chromodomain, and HAND-SLIDE domains). RNAi target
sequences that are common to Diabrotica virgifera virgifera,
Euschistus heros, Tribolium castaneum, and Drosophila melanogaster
were designed. The DNA nucleotides and RNAi nucleotides are listed
according to the standard IUPAC code:
[0262] A=Adenine
[0263] C=Cytosine
[0264] G=Guanine
[0265] T=Thymine
[0266] R=A or G
[0267] Y=C or T
[0268] S=G or C
[0269] W=A or T
[0270] K=G or T
[0271] M=A or C
[0272] B=C or G or T
[0273] D=A or G or T
[0274] H=A or C or T
[0275] V=A or C or G
[0276] N=A or C or G or T
[0277] dsRNA encoding sequences targeting SNF2-Helicase regions
(SEQ ID NOs:34-37) and chromatin remodeling domains (SEQ ID
NOs:38-41) were designed by aligning the amino acid sequences for
each target protein from four species, Diabrotica v. virgifera, E.
heros, Tribolium castaneum, and Drosophila melanogaster, using
Vector NTI Align X (Invitrogen, Grand Island, N.Y.). Highly
homologous regions of the amino acid sequence containing at least 8
amino acids within the SNF2 domain or chromatin remodeling domain
specific to each target protein were selected. The corresponding
nucleotide sequence for each species from each target was then
aligned also using the Align X program. Where there was a
misalignment across the four species the nucleotides were replaced
with nucleotides as shown above. Finally, the sequence was aligned
against the nucleotide sequence from Apis mellifera to determine if
the sequence would also target that species. If the sequence could
also target the protein from A. mellifera either new regions were
chosen or the sequence was shortened to at least 21 bases which did
not target A. mellifera proteins.
Example 3: Preparation of RNAi Molecules
[0278] Template Preparation and dsRNA Synthesis.
[0279] 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.).
[0280] 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.
[0281] Primers were used to amplify DNA templates for dsRNA
transcription. Table 1. 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
cDNA (above) as the template. Fragments comprising a 499 bp segment
of Brahma (i.e., BSB_brm-1; SEQ ID NO:3), a 496 bp segment of mi-2
(i.e., BSB_mi-2-1; SEQ ID NO:16), a 481 bp segment of iswi-1 (i.e.,
BSB_iswi-1-1; SEQ ID NO:17), a 490 bp segment of iswi-2 (i.e.,
BSB_iswi-2-1; SEQ ID NO:18), and a 496 bp segment of chd1 (i.e.,
BSB_chd1-1; SEQ ID NO:19) were generated during 35 cycles of PCR. A
301 pb template for dsRNA termed YFPv2 (SEQ ID NO:5) was
synthesized using primers YFPv2_F. (SEQ ID NO:6) and YFPv2_R (SEQ
ID NO:7). The BSB-specific and YFPv2 primers contained a T7 phage
promoter sequence (SEQ ID NO:4) at their 5' ends, enabling the use
of the aforementioned BSB DNA fragments for dsRNA
transcription.
TABLE-US-00003 TABLE 1 Primer pairs used to amplify DNA templates
for dsRNA transcription. Gene (Re- Primer gion) ID Sequence Pair
brahma BSB_ TTAATACGACTCACTATAGGGAGAG 1 brm-1-F
ATGATGAAGAAGATGCAAGTAC (SEQ ID NO: 20) BSB_
TTAATACGACTCACTATAGGGAGAC brm-1-R TCCACTCCCTCGGGTC (SEQ ID NO: 21)
Pair mi-2 BSB_ TTAATACGACTCACTATAGGGAGAG 2 Mi-2-1-F
ACTACCTCGAGGGTGAAGG (SEQ ID NO: 22) BSB_ TTAATACGACTCACTATAGGGAGAG
Mi-2-1-R TAATTCTTCAACAGCTTTATCGTC (SEQ ID NO: 23) Pair iswi-1 BSB_
TTAATACGACTCACTATAGGGAGAC 3 Iswi-1-1-F AAAAATTGAAACTGACCGTTCTAG
(SEQ ID NO: 24) BSB_ TTAATACGACTCACTATAGGGAGAG Iswi-1-1-R
CTAATGTTGATTTTGGTACGATG (SEQ ID NO: 25) Pair iswi-2 BSB_
TTAATACGACTCACTATAGGGAGAG 4 Iswi-2-1-F TTCAAGATTTCCAATTTTTCCCAC
(SEQ ID NO: 26) BSB_ TTAATACGACTCACTATAGGGAGAG Iswi-2-1-R
AAACGGTGCTCTATATCGACTC (SEQ ID NO: 27) Pair chd1 BSB_
TTAATACGACTCACTATAGGGAGAC 5 Chd1-1-F AGCTGGAACCATATATTCTACGAC (SEQ
ID NO: 28) BSB_ TTAATACGACTCACTATAGGGAGAG Chd1-1-R
TGAATTTTCAGCATTGAAATGATCG (SEQ ID NO: 29) Pair YFPv2 YFPv2_F
TTAATACGACTCACTATAGGGAGAG 6 CATCTGGAGCACTTCTCTTTCA (SEQ ID NO: 6)
YFPv2_R TTAATACGACTCACTATAGGGAGAC CATCTCCTTCAAAGGTGATTG (SEQ ID NO:
7)
[0282] 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. 1B. 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 4: Brahma dsRNA Injection of 2nd Instar Euschistus heros
Nymphs
[0283] Insect Rearing.
[0284] 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.
[0285] BSB Artificial Diet.
[0286] 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.
[0287] Injection of dsRNA into BSB Hemocoel.
[0288] 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 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.
[0289] Injection of dsRNA that Targets Brahma mRNA in BSB 2.sup.nd
Instar Nymphs.
[0290] dsRNA that targets segment of YFP coding region, YFPv2, was
used as a negative control in BSB injection experiments. As
summarized in Table 2, at least ten 2.sup.nd instar BSB nymphs
(1-1.5 mg each) were injected into the hemoceol with 55.2 nL
BSB_brm-1 (500 ng/.mu.L) for an approximate final concentration of
18.4-27.6 .mu.g dsRNA/g insect. Percent mortality was scored seven
days after dsRNA injection. The mortality determined for BSB_brm-1
dsRNA was not significantly different from that seen with the same
amount of injected YFPv2 dsRNA (negative control), with p=0.279
(Student's t-test). There was also no significant difference
between the YFPv2 dsRNA injected and not injected treatments.
TABLE-US-00004 TABLE 2 Results of BSB_brm-1 dsRNA injection into
the hemoceol of 2.sup.nd instar E. heros nymphs seven days after
injection. Table shows mean percent mortality, N number of trials,
and standard error of the mean (SEM). Means comparisons were
performed with YFP dsRNA as control, using a Student's t-test with
Dunnett's adjustment in JMP .RTM. Pro 11; p-value shown. Treatment
Mean % mortality SEM N trials t-test (p) BSB_brm-1 27 12.0 3 0.3039
not injected 13 3.3 3 0.9384 YFPv2 dsRNA 10 5.8 3 *Ten insects
injected per trial for each dsRNA.
Example 5: Parental RNAi Effects Following dsRNA Injection in
Euschistus heros
[0291] Injection of dsRNA into BSB Hemocoel.
[0292] BSB were reared as described above for the colony. In the
following exemplification, young adults (up to one week 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.
[0293] Surviving female counts, oviposition, and egg hatch numbers
were collected on a daily basis starting seven to nine days after
injection and continued for up to 16 days. Eggs were removed daily
and kept in Petri dishes or multi-well plates on a layer of 1%
agarose in water. The adult insects were transferred into bins with
fresh water and food every week.
[0294] Injections of dsRNA that target brahma, iswi-1, iswi-2,
mi-2, and/or chd1 in BSB females decreased egg laying. Females
injected with dsRNA that targets a 301 nt sequence of the YFP
coding region were used as a negative controls, and compared to
un-injected and females injected with BSB_brm-1 dsRNA (SEQ ID
NO:3). As summarized in Table 3, un-injected females did not lay
statistically different numbers of eggs from YFPv2 controls. On the
other hand, BSB_brm-1 dsRNA-injected females oviposited no
eggs.
[0295] Injection of 138 ng chromatin-remodeling ATPase dsRNA had no
effect on viability or no immediate effect on viability of the
adult female BSB. FIG. 4A. Injection of BSB_brahma dsRNA (BSB_brm-1
(SEQ ID NO:3)) and of dsRNAs that target BSB_mi-2-1 (SEQ ID NO:16),
BSB_iswi-1-1 (SEQ ID NO:17), BSB_iswi-2-1 (SEQ ID NO:18), and
BSB_chd1-1 (SEQ ID NO:19) of BSB greatly decreased oviposition or
eliminated oviposition altogether, as compared to negative YFPv2
dsRNA controls (SEQ ID NO:5) Table 4 and FIG. 4B. Oviposition by
BSB females injected with dsRNAs BSB_brm-1 (SEQ ID NO:3),
BSB_mi-2-1 (SEQ ID NO:16), BSB_iswi-1-1 (SEQ ID NO:17),
BSB_iswi-2-1 (SEQ ID NO:18), and BSB_chd1-1 (SEQ ID NO:19) were
significantly different from that observed with the same amount of
injected YFPv2 dsRNA (SEQ ID NO:5), with p<0.05 (Table 4 and
FIG. 4B). No eggs were produced by BSB_brm-1 and very few or none
by BSB_mi-2-1 injected females. BSB_brm-1 (SEQ ID NO:3), BSB_mi-2-1
(SEQ ID NO:16), BSB_iswi-1-1 (SEQ ID NO:17), and BSB_chd1-1 (SEQ ID
NO:19) dsRNA caused significant knockdown of transcript levels in
the BSB ovary. FIG. 5. The transcript of BSB_iswi-2-1 (SEQ ID
NO:18) was not readily detected by probe hydrolysis PCR.
[0296] The numbers of eggs hatched in the experiment below shows
that the number of offspring produced from females injected with
dsRNAs for BSB_Brahma, mi-2, iswi-1, iswi-2, and chd1 were
significantly lower than the control. Table 5 and FIG. 4C. Egg
hatch rates of BSB females injected with dsRNAs BSB_brm-1 (SEQ ID
NO:3), BSB_mi-2-1 (SEQ ID NO:16), BSB_iswi-1-1 (SEQ ID NO:17),
BSB_iswi-2-1 (SEQ ID NO:18), and BSB_chd1-1 (SEQ ID NO:19) were
significantly different from those observed with the same amount of
injected negative control YFPv2 dsRNA (SEQ ID NO:5), with p<0.05
(Student's t-test).
TABLE-US-00005 TABLE 3 Brahma pRNAi: number of eggs oviposited per
female per day. Ten females were injected with each dsRNA targeted
against BSB brahma and negative control, YFPv2. Counts of
oviposited eggs were collected starting on day 7 post injection,
for up to 15 consecutive days. The N number of days during which
eggs were collected varies between treatments due to female
mortality impact of some dsRNAs. Means comparisons were performed
with YFPv2 dsRNA as control, using a Student's t-test with
Dunnett's adjustment in JMP .RTM. Pro 11. total # of mean # eggs of
eggs/ oviposited day/ Std. Std. N dsRNA in 15 days female Deviation
Error days T-test (p) YFPv2 1280 8.66 1.84 0.48 15 not inj. 1429
7.32 2.66 0.69 15 0.6697 BSB_brm-1 0 0 0 0 13 <0.0001* *p-values
<0.05.
TABLE-US-00006 TABLE 4 Oviposition by E. heros females injected
with chromatin remodelers dsRNA. Total numbers of eggs oviposited
in 15 days and average numbers of eggs per female injected with
negative control YFPv2 dsRNA or chromatin remodeling ATPase dsRNAs.
Twenty females were injected with each dsRNA. Egg counts started on
day 9 post-injection and continued for 15 consecutive days. The N
number of days during which eggs were collected varied between
treatments due to female mortality in brm and mi-2 treatments.
Means comparisons were performed on average numbers of eggs
oviposited by females, using daily oviposition values. YFPv2 dsRNA
was used as control for Student t-test with Dunnett's adjustment in
JMP. N = number of days; SEM = standard error of the mean. Total #
Average # of eggs of eggs/ N dsRNA in 15 days day/female SEM Days
p-Value YFPv2 1629 6.75 0.357 15 BSB_brm-1 0 0.00 0.000 10
<0.0001* BSB_chd1-1 496 2.65 0.338 15 <0.0001* BSB_iswi-1-1
209 0.84 0.142 15 <0.0001* BSB_iswi-2-1 1097 5.54 0.433 15
0.0171* BSB_mi-2-1 42 0.22 0.085 13 <0.0001* *significantly
different from YFPv2 dsRNA p < 0.05.
TABLE-US-00007 TABLE 5 Total and average numbers of eggs hatched
from E. heros females injected with chromatin remodelers dsRNA.
Total numbers of eggs hatched in 15-day collection and average
number of eggs hatched per female per day of oviposition, from
females injected with negative control YFPv2 dsRNA or chromatin
remodeling dsRNAs. Twenty females were injected with each dsRNA.
Nymph emergence was evaluated from eggs oviposited on day 9
post-injection for 15 consecutive days. Means comparisons were
performed on numbers of eggs hatched each day per female, using
daily values. YFPv2 dsRNA was used as the control for Student-t
test with Dunnett's adjustment in JMP. SEM = standard error of the
mean. Total # of Average # eggs hatched of eggs from 15-day
hatched/ dsRNA collection female/day SEM p-Value YFPv2 1321 5.47
0.257 BSB brm-1 0 0.00 0.000 <0.0001* BSB chd1-1 51 0.28 0.054
<0.0001* BSB_iswi-1-1 93 0.39 0.062 <0.0001* BSB_iswi-2-1 312
1.63 0.253 <0.0001* BSB_mi-2-1 34 0.17 0.067 <0.0001*
*significantly different from YFPv2 dsRNA p < 0.05.
[0297] To determine the onset of pRNAi response, oviposting
females, 14 to 16 days post adult molt, were injected with
BSB_brm-1 (SEQ ID NO:3) dsRNA. FIG. 6 shows that egg hatch was
inhibited by day 4 post-injection (FIG. 6B) and oviposition halted
by day 7 (FIG. 6A).
[0298] Based on the complete lack of oviposition in E. heros in
response to Brahma dsRNA and severe inhibition of oviposition in
response to mi-2 dsRNA, we investigated the state of oocyte and
ovary development in parent females. The females were examined 9
and 14 days post injection. By day nine after injection, control
females began oviposition. Since brm dsRNA injections led to
lethality within about two weeks, day 14 was chosen to capture
phenotypes from the last surviving females. FIG. 4A. E. heros
ovaries were dissected in 1.times.PBS under stereo microscope, and
then fixed in 4% paraformaldehyde/1.times.PBS solution for 2 hours
on ice. Trachea surrounding the ovaries was removed with #5 biology
forceps. Images of three to four sets of ovaries for each treatment
were captured with a Leica M205 FA stereo microscope (WETZLAR,
Germany). Mature eggs and developing oocytes were observed in YFP
dsRNA-injected females. FIGS. 7C and D. Brahma and mi-2
dsRNA-injected females showed lack of ovary development and
ovariole elongation. FIG. 7. These insects showed no maturing
oocytes or mature eggs (FIGS. 7E, G, and H), or oocytes that were
in a state of decay (FIG. 7F).
[0299] Contact with dsRNA molecules encoding sequences targeting
SNF2-Helicase regions (SEQ ID NOs:34-37) and chromatin remodeling
domains (SEQ ID NOs:38-41) by adult BSB females is demonstrates to
a have surprising, dramatic and reproducible effect on egg
viability. The mated females exposed to dsRNA produce a lower
number of eggs than females exposed to untreated diet or diet
treated with YFPv2 dsRNA.
[0300] The above results clearly document the systemic nature of
RNAi in BSB adults, and the potential to achieve a parental effect
where genes associated with embryonic development are knocked down
in the eggs of females that are exposed to dsRNA. These
observations confirm that the dsRNA can be taken up translocated to
tissues (e.g., developing ovarioles) other than the point of
contact (e.g., midgut or hemocoel).
[0301] The ability to knock down the expression of genes involved
with embryonic development such that the eggs do not hatch, offers
a unique opportunity to achieve and improve control of BSB. Because
adults readily feed on above-ground reproductive tissues, adult BSB
can be exposed to iRNA control agents by transgenic expression of
dsRNA to achieve plant protection in the subsequent generation by
preventing eggs from hatching. Delivery of the dsRNA through
transgenic expression of dsRNA in plants, or by contact with
surface-applied iRNAs, provides an important stacking partner for
other transgenic approaches that target nymphs directly and enhance
the overall durability of pest management strategies.
Example 6: Quantitive Real-Time PCR Analysis
[0302] E. heros tissues for qRT-PCR were collected from zero to
three day-old females injected with dsRNA. After seven days, female
ovaries were dissected under a stereo microscope in nuclease-free
1.times.PBS (pH 7.4) and frozen individually on dry ice in
collection microtubes. Tissue disruption was performed with the RL
lysis buffer and the Klecko.TM. tissue pulverizer (GARCIA
MANUFACTURING, Visalia, Calif.). Following tissue maceration, the
total RNA was isolated in high throughput format using the
Norgen.RTM. Total RNA Purification 96-well kit (NoRGEN BIOTEK
CORP., Ontario, Canada) following the manufacturer's protocol using
Turbo.TM. DNase (LIFE TECHNOLOGIES, Carlsbad, Calif.) for 1 hour at
37.degree. C. on the elutant. cDNA synthesis was performed using
the high capacity cDNA RT kit (LIFE TECHNOLOGIES, Carlsbad, Calif.)
according to the manufacturer's protocol with the following
modifications. Total RNA was adjusted to 50 ng/.mu.L with
nuclease-free water. RNA samples were heated to 70.degree. C. for
10 minutes and cooled to 4.degree. C. Half reactions were initiated
by addition of 5 .mu.L 2.times.mix. The primer mix, which is
supplied solely as random primers, was 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 were diluted 1:3 with nuclease-free
water.
[0303] E. heros qRT-PCR primers and hydrolysis probes were designed
using LightCycler.RTM. Probe Design Software 2.0 (ROCHE, Basel,
Switzerland) for the reference gene and Primer Express.RTM.
Software Version 3.0 (APPLIED BIOSYSTEMS, Grand Island, N.Y.) for
the target genes. Table 6. Non-injected insects were used as
controls. E. heros muscle actin (SEQ ID NO:73) was used as the
reference gene. Probes were labeled with FAM (6-Carboxy Fluorescein
Amidite). The final primer concentration was 0.4 and the final
probe concentration was 0.2 .mu.M (in 10 reaction volumes).
Relative transcript levels were analyzed by probe hydrolysis
qRT-PCR using LightCycler.RTM. 480. All assays included negative
controls of no-template (mix only). For the standard curves, a
blank was included in the source plate to check for sample
cross-contamination. PCR cycling conditions included a 10 minute
target activation incubation at 95.degree. C., followed by 40
cycles of denaturation at 95.degree. C. for 10 seconds,
anneal/extend at 60.degree. C. for 40 seconds, and FAM acquisition
at 72.degree. C. for 1 second. The reaction was followed by a 10
second cool-down at 40.degree. C. E. heros iswi-2 was not detected
reliably both in the negative controls and dsRNA exposed females,
therefore iswi-2 data was omitted from the final results. The data
was analyzed using LightCycler.RTM. Software v1.5 and relative
changes in expression were calculated using 2.sup.-.DELTA..DELTA.Ct
method (Livak and Schmittgen (2001) Methods 25:402-8).
TABLE-US-00008 TABLE 6 Oligonucleotides and probes for BSB probe
hydrolysis qPCR assay and primer efficacy results. MGB = Minor
Groove Binder probes from Applied Biosystems. Pro- Primer duct
Effic- Length iency NAME SEQUENCE (bp) Slope (%) Refer- ence GENE
Actin, Act42A- TCAAGGAAAAACTG 120 -3.77 92 muscle F TGCTATGT (SEQ
ID NO: 74) Actin, Act42A- TACCGATGGTGATG muscle R ACCTGA (SEQ ID
NO: 75) Actin, Act42A- ACCGCCGCTGCC muscle FAM (SEQ ID NO: 76)
Target GENE brahma brm-F TCATCAAGGACAAG 205 -3.54 93.5 GCAGT (SEQ
ID NO: 77) brahma brm-R GACGGGAGGAGAAA GTTTAGA (SEQ ID NO: 78)
brahma brm- CGACGAGGGACACA FAM GGATG (SEQ ID NO: 79) mi-2 mi-2-
GATGAGGGCTTGCT 149 -3.55 95.5 F GTT (SEQ ID NO: 80) mi-2 mi-2-
GAGGCGGGAAGTAT R TGAC (SEQ ID NO: 81) mi-2 mi-2- ATGAGGAAGGAAGC FAM
AGAAGTGC (SEQ ID NO: 82) iswi-1 iswi- GAGTTCAACGAAGA 155 -3.67 94.5
1-F AGACAGTAA (SEQ ID NO: 83) iswi-1 iswi- CGATGAGCACGATC R CATAG
(SEQ ID NO: 84) iswi-1 iswi- TTAGCCACCGCAGA 1-FAM TGTAGTCA (SEQ ID
NO: 85) iswi-2 iswi- ACGTAAGGGAGATG 65 -3.96 89 2- GATCTATTTCA
F_MGB (SEQ ID NO: 86) iswi-2 iswi- CAGGGCTGCTTTTA 2- TCACTCTGT
R_MGB (SEQ ID NO: 87) iswi-2 iswi- CTCCACCTGTCTCT 2- G FAM_MGB (SEQ
ID NO: 88) chd1 chd1-F CAACAGTGGCTGGT 68 -3.71 93 CCTTCA (SEQ ID
NO: 89) chd1 chd1-R ACCAACTTGTGACA TTGACGAAA (SEQ ID NO: 90) chd1
chd1- TCTGGTTTCAGCTC FAM TT (SEQ ID NO: 91)
Example 7: Construction of Plant Transformation Vectors
[0304] Entry vectors harboring a target gene construct for dsRNA
hairpin formation comprising segments of one of various chromatin
remodeling genes (SEQ ID NO:1 or SEQ ID NO:63 (brahma); SEQ ID NO:8
or SEQ ID NO:64 (BSB_mi-2); SEQ ID NO:10 or SEQ ID NO:65
(BSB_iswi-1); SEQ ID NO:12 or SEQ ID NO:66 (BSB_iswi-2); SEQ ID
NO:14 or SEQ ID NO:67 (BSB_chd1); SEQ ID NO:30 (BSB_ino80); and SEQ
ID NO:32 (BSB_domino)) 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 orientation to one another, the two segments
being separated by a linker sequence (e.g. ST-LS1 intron;
Vancanneyt et al. (1990) Mol. Gen. Genet. 220:245-250). Thus, the
primary mRNA transcript contains the two brahma or ortholog 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.
[0305] The entry vector described above is used in standard
GATEWAY.RTM. recombination reactions with a typical binary
destination vector to produce hairpin RNA expression transformation
vectors for Agrobacterium-mediated plant embryo
transformations.
[0306] 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.
[0307] 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 ZmUbil (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.
[0308] 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 8: Transgenic Zea mays Comprising Hemipteran Pest
Sequences
[0309] Ten to 20 transgenic T.sub.0 Zea mays plants harboring
expression vectors for nucleic acids comprising a segment of SEQ ID
NO:1, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID
NO:30, SEQ ID NO:32, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ
ID NO:66, or SEQ ID NO:67 are generated as described in EXAMPLE 5.
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,
SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID
NO:30, SEQ ID NO:32, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ
ID NO:66, or SEQ ID NO:67. 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.
[0310] 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.
[0311] 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, Chinavia hilare, Euschistus serous,
Dichelops melacanthus, Dichelops furcatus, Edessa meditabunda,
Thyanta perditor, Chinavia marginatum, Horcias nobilellus, Taedia
stigmosa, Dysdercus peruvianus, Neomegalotomus parvus, Leptoglossus
zonatus, Niesthrea sidae, or Lygus lineolaris 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.
[0312] Phenotypic Comparison of Transgenic RNAi Lines and
Non-Transformed Zea mays.
[0313] 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: Transgenic Glycine max Comprising Hemipteran Pest
Sequences
[0314] Ten to 20 transgenic T.sub.0 Glycine max plants harboring
expression vectors for nucleic acids comprising a segment of SEQ ID
NO:1, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID
NO:30, SEQ ID NO:32, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ
ID NO:66, or SEQ ID NO:67 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.
[0315] Preparation of Split-Seed Soybeans.
[0316] 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.
[0317] Inoculation.
[0318] 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 a segment of SEQ ID NO:1, SEQ
ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:30,
SEQ ID NO:32, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID
NO:66, or SEQ ID NO:67. 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.
[0319] Co-Cultivation.
[0320] 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.
[0321] Shoot Induction.
[0322] 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.).
[0323] Shoot Elongation.
[0324] 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 Na2EDTA, 30 g/L sucrose and 0.6 g/L
IVIES, 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.
[0325] Rooting.
[0326] 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 Na2EDTA, 20 g/L sucrose and
0.59 g/L IVIES, 50 mg/L asparagine, 100 mg/L L-pyroglutamic acid 7
g/L Noble agar, pH 5.6) in phyta trays.
[0327] Cultivation.
[0328] 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.
[0329] 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 comprising a segment of SEQ
ID NO:1, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ
ID NO:30, SEQ ID NO:32, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65,
SEQ ID NO:66, or SEQ ID NO:67. 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.
[0330] 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.
[0331] 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, Chinavia hilare, Euschistus servus, Dichelops
melacanthus, Dichelops furcatus, Edessa meditabunda, Thyanta
perditor, Chinavia marginatum, Horcias nobilellus, Taedia stigmosa,
Dysdercus peruvianus, Neomegalotomus parvus, Leptoglossus zonatus,
Niesthrea sidae, or Lygus lineolaris 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.
[0332] 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 10: E. heros Bioassays on Artificial Diet
[0333] 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.
[0334] 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 11: Transgenic Arabidopsis thaliana Comprising Hemipteran
Pest Sequences
[0335] Arabidopsis transformation vectors containing a target gene
construct for hairpin formation comprising segments of BSB_brahma
(SEQ ID NO:1 or SEQ ID NO:63), BSB_mi-2 (SEQ ID NO:8 or SEQ ID
NO:64), BSB_iswi-1 (SEQ ID NO:10 or SEQ ID NO:65), BSB_iswi-2 (SEQ
ID NO:12 or SEQ ID NO:66), BSB_chd1 (SEQ ID NO:14 or SEQ ID NO:67),
BSB_ino80 (SEQ ID NO:30), and/or BSB_domino (SEQ ID NO:32) are
generated using standard molecular methods similar to EXAMPLE 5.
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.
[0336] Construction of Arabidopsis transformation vectors.
[0337] Entry clones based on an entry vector harboring a target
gene construct for hairpin formation comprising a segment of
BSB_brahma (SEQ ID NO:1 or SEQ ID NO:63), BSB_mi-2 (SEQ ID NO:8 or
SEQ ID NO:64), BSB_iswi-1 (SEQ ID NO:10 or SEQ ID NO:65),
BSB_iswi-2 (SEQ ID NO:12 or SEQ ID NO:66), BSB_chd1 (SEQ ID NO:14
or SEQ ID NO:67), BSB_ino80 (SEQ ID NO:30), and/or BSB_domino (SEQ
ID NO:32) 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) (Vancanneyt et al. (1990) Mol. Gen.
Genet. 220(2):245-50). Thus, the primary mRNA transcript contains
the two chromatin remodeling 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 ofAgrobacterium tumefaciens
(AtuORF23 3' UTR v1; U.S. Pat. No. 5,428,147) is used to terminate
transcription of the hairpin-RNA-expressing gene.
[0338] 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.
[0339] 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.
[0340] 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 (hpYFP v2, SEQ ID NO:42) 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).
[0341] Production of Transgenic Arabidopsis Comprising Insecticidal
Hairpin RNAs: Agrobacterium-Mediated Transformation.
[0342] Binary plasmids containing hairpin sequences are
electroporated into an Agrobacterium strain. 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.
[0343] Arabidopsis transformation and T.sub.1 Selection.
[0344] 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 12: Growth and Bioassays of transgenic Arabidopsis
[0345] Selection of T.sub.1 Arabidopsis Transformed with Hairpin
RNAi Constructs.
[0346] 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.
[0347] E. heros Nymph Plant Feeding Bioassay.
[0348] At least four low copy (1-2 insertions), four medium copy
(2-3 insertions), and four high copy (>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.
[0349] 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.
[0350] T.sub.2 Arabidopsis Seed Generation and T2 Bioassays.
[0351] 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 13: Transformation of Additional Crop Species
[0352] Cotton is transformed with Brahma, mi-2, iswi-1, iswi-2,
chd1, ino80, and/or domino (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 14: pRNAi-Mediated Insect Protection
[0353] 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.
[0354] 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.
[0355] Pest resistance 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).
[0356] 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).
[0357] 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.
[0358] 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 are expected
to 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).
[0359] 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.
[0360] 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. 8.
[0361] The model was also modified to include nymph-active
interfering dsRNA in combination with the BSB-active 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. 9.
[0362] 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 larval active dsRNA. In the
latter case, the durability benefit applied to both the
insecticidal protein and the insecticidal interfering dsRNA.
Sequence CWU 1
1
9114958DNAEuchistus heros 1cttggctagt actcttccgt gacgtcacgt
tcgccatatt gttagagttt gtcttgcctc 60tggatagtta tgttgattct ttttaagtga
ttttgaagat ttcctgacca ttttatcacg 120aaaaactatt ttaaacagcg
ctattgctcc ttataatacg tgtgattcaa caacgatgga 180cggagacagc
ggtggtatgg cgagcccttc gccacagcct cagtcgtcac caatgccccc
240tccacaagct ccatcaccta tgggcccgcc gcagggcgcc ccatcgccaa
tgcccccttc 300taaccaacag gcggcctcac caatgggtcc accgcaccac
ccccacagcc cgacaggtta 360ccaaggaggg atgccacaca tgaatggacc
aaatggtgtt cctcctggta tgcagcaggc 420tactcaaaca tttcagcctc
atcagcaatt gccaccccac cagcaaccac caatgcagac 480tgctcctggt
gggcctgcta gtggtggagg acaagaaaat cttagcgctc tccagcgtgc
540aatagattct atggaagaga aagggcttca ggaagatcca cgttactcgc
agctgcttgc 600gttgagggca aggcatgcca acatggaacc tccggttagg
cctccatctc agcttgttgg 660gggtgggttc agcggtgagg gtggtgcccc
tcctcctgct aaacacagct tcagcgcgaa 720ccaactgcaa caacttcgag
tgcagatcat ggcgtatcgc ctacttgcta ggaaccaacc 780tctttcccag
cagctagctt tggctgtgca aggcaaacgc ctcgacagcc ctggcgagtc
840caactaccag catcctccta gtgaaggagc aggaggtgtt ggtggagaag
gaagtggaga 900cgggggatcg tcgaacggcc tgatgacgca gccgatgcgt
gccccatgcc cccctggtgg 960ccagccccca acggcctcac cgatgacagg
ccagatggca cctcctactg ggccagctcc 1020tgtaaggcca cctcctcccg
gtgtgtctcc tacacctccg cgccctcctc agcaggttcc 1080tggtgctccg
ggggccccac aaccaaagca aaatagggtt accaccatgc caagaccgca
1140tggtttagat cccattctta ttctccagga aagagagaat agagtagccg
ctaggattgt 1200acataggatg gaagaattat caaatttacc agctacgatg
cctgaagacc ttcgaataaa 1260agcgcagata gaacttaggg ccttgagggt
acttaacttc caaaggcaat taagagcaga 1320ggtgatagct tgtactagac
gcgatacaac attagaaaca gctgtaaatg tgaaagctta 1380taaacgaacg
aagaggcaag gcttacggga agccagagct acggaaaagc ttgaaaaaca
1440acagaaactt gagacagaaa ggaagaagag acaaaaacac caggaatatc
tgagcactat 1500attgcaacat tgcaaagact tcaaagaatt ccatagaaat
aatgttgcta aagttggtag 1560attaaataag gctgtgatga attaccatgc
gaatgccgag cgtgaacaga agaaagagca 1620agaaaggata gaaaaagaac
gtatgagaag gcttatggct gaggatgaag agggttacag 1680gaaactgatt
gatcagaaaa aagataagag attggcattc cttctttcac aaactgatga
1740atatattgcc aatcttactg aaatggtgaa gcagcataaa atggaacaac
agcgtaagca 1800ggaacaagaa gagcaacaaa aacggaagag gaaaaagaaa
aagaagaata gggaaggaga 1860tccagatgat gaaagctctc agatgtcaga
tttacatgtt agcgttatag aagcagcaac 1920tggtcggcag ctgacggggg
aggatgctcc attggccagc cagcttggga gctggttgga 1980ggcacacccg
ggctgggagc ctttggaaga tagcgaagat gaagatgatg aagaggacag
2040cgacgaggaa ggtgatgata acagtagatc aaaaggtggt ttttcaatga
taggaaaaga 2100tgaagctgat agcaagttat ctgttgaaga cgaagctcga
gaaatgataa agaaagcgaa 2160gattgaagat gatgaataca agaacacgac
cgaagaacat acatactaca gcatcgctca 2220caccgtgcat gaaattgtca
ccgaacaagc ttcaatcatg attaacggta aattgaaaga 2280atatcaaatt
aaaggtcttg aatggttggt ttctttatac aacaacaact tgaatggaat
2340cctcgccgac gagatgggcc ttggcaagac aattcaaaca ataggtctca
ttacttattt 2400gatggagaag aagaaagtaa atggtcctta cctcattatt
gttcctctgt caacattatc 2460caattgggtt ttggaattcg agaaatgggc
tccttcagtg tttgtggtag cttataaagg 2520ttctcctgca atgaggagaa
ctttacaatc acagatgcgc tcgacgaagt tcaatgtcct 2580gctcacgacc
tacgagtatg tcatcaagga caaggcagta cttgcaaagt tgcattggaa
2640gtacatgata atcgacgagg gacacaggat gaaaaaccac cattgtaagc
tgacgcaggt 2700gctgaacacc cattatttgg cacctcaccg cctccttctc
acgggcacac ctctccagaa 2760caaactacct gagctctggg ctcttctaaa
ctttctcctc ccgtccatct tcaagtcgtg 2820ttctacgttt gagcaatggt
tcaatgcacc atttgctacc actggagaaa aggttgagtt 2880gaatgaggaa
gaaacaattt tgattatcag gcgtttacat aaggtccttc gacctttcct
2940ccttcgtcga ctgaaaaagg aagtcgaaag tcagttgcca gagaaaattg
aatacatcgt 3000caagtgtgat atgtctggtc tccaacgtgt actttatagg
cacatgcaga gtaaaggagt 3060cctgcttacc gatggttctg agaagggcaa
gcagggtaaa ggaggagcta aagcgctaat 3120gaacacgatc gtccaattga
ggaagctttg caatcatcct ttcatgttcc atcatattga 3180agaaaaatat
tgtgatcacg ttggccagaa caacgttgtc acagggcctg atctgttccg
3240agtttctggt aaatttgaat tcctcgatcg tatattgcca aaactgaagg
ccacgagcca 3300tagggtactt cttttctgtc aaatgactca gctgatgacc
atcatggagg attatttgtc 3360ttggagaggg ttctcctacc ttcgtcttga
tggtacgacc aaatctgaag accgaggaga 3420tcttctgaaa aaattcaaca
atccagaaag tgaatatttt attttcttgc tctcaaccag 3480agctggaggt
ctcggattga acttacaggc tgcagatact gtcattatat ttgattcaga
3540ttggaaccct catcaggatt tacaagctca agacagagct cataggattg
gacagcaaaa 3600cgaagttcgt gttttgcggc taatgacagt aaattctgtt
gaggagcgta ttcttgcagc 3660tgctcggtac aagctgaata tggatgagaa
agtcattcag gctggtatgt ttgaccagaa 3720atctacagga accgagaggc
agaaatttct gcaaaacatc cttcatcaag atgatgcaga 3780tgatgaggaa
aatgaagttc cagatgatga aatggttaat cgtatgattg cgcgaacaga
3840agatgaattc aacctcttcc agaaaatcga tttagaaagg aggagggaag
aggctaaact 3900tggacctaac aggaagtcaa ggcttgtaga agaggcggaa
ttacctgact ggcttgtaaa 3960gaatgacgat gagattgaga agtggactta
tgaagaaacc gaggtccaaa tgggaagagg 4020taataggcag aggaaggaag
tagattatac agatagtttg actgaaaaag aatggttaaa 4080ggccattgat
gacaatgtag atgattttga tgacgatgaa gaggaagagg taaaaacaaa
4140gaaaagaggc aagagaagaa gaaggggaga ggatgatgaa gaagatgcaa
gtacttcaaa 4200gagaaggaaa tattctccat ctgaaaacaa actgaggagg
cgtatgcgta acctcatgaa 4260cattgttgtt aagtatactg acagtgactc
gagagtactc agtgaaccat tcatgaaact 4320tccctctcgc cataagtacc
cagactacta tgagttgatc aagaaaccta tagacatcaa 4380gaggatattg
gccaaagtag aagagtgtaa atatgctgac atggatgaat tagaaaagga
4440ttttatgcaa ctttgtaaaa atgctcagac atacaatgag gaggcctcat
tgatctatga 4500agattcgata gtattagaaa gtgttttctc taatgctcgt
caaaaagtag agcaggataa 4560tgattcagat gatgatgaaa gtaaaggtga
ccaagaagat gctgcatcag acacttcatc 4620cgtcaaaatg aaattgaaac
taaagcctgg gaggacccga gggagtggag ctggtggtaa 4680aaggaggaga
agaaaatata tctctgaaga tgaagacgaa gaccatagcg aagtttcctt
4740aatgtaatgc ctcttcactg tcctttgtaa ttattagttt tcatcggtgt
tcggtacctg 4800tcagtcaagg gagaagctaa gctttttagt tgactattga
agaatttagg actgagttct 4860gtttttgttt tttttgtttg tttttttttg
gataaatgta tttaatagat aaaatgtttc 4920gcttatatat atatttttta
ctggttttgt aattggcc 495821523PRTEuchistus heros 2Met Asp Gly Asp
Ser Gly Gly Met Ala Ser Pro Ser Pro Gln Pro Gln 1 5 10 15 Ser Ser
Pro Met Pro Pro Pro Gln Ala Pro Ser Pro Met Gly Pro Pro 20 25 30
Gln Gly Ala Pro Ser Pro Met Pro Pro Ser Asn Gln Gln Ala Ala Ser 35
40 45 Pro Met Gly Pro Pro His His Pro His Ser Pro Thr Gly Tyr Gln
Gly 50 55 60 Gly Met Pro His Met Asn Gly Pro Asn Gly Val Pro Pro
Gly Met Gln 65 70 75 80 Gln Ala Thr Gln Thr Phe Gln Pro His Gln Gln
Leu Pro Pro His Gln 85 90 95 Gln Pro Pro Met Gln Thr Ala Pro Gly
Gly Pro Ala Ser Gly Gly Gly 100 105 110 Gln Glu Asn Leu Ser Ala Leu
Gln Arg Ala Ile Asp Ser Met Glu Glu 115 120 125 Lys Gly Leu Gln Glu
Asp Pro Arg Tyr Ser Gln Leu Leu Ala Leu Arg 130 135 140 Ala Arg His
Ala Asn Met Glu Pro Pro Val Arg Pro Pro Ser Gln Leu 145 150 155 160
Val Gly Gly Gly Phe Ser Gly Glu Gly Gly Ala Pro Pro Pro Ala Lys 165
170 175 His Ser Phe Ser Ala Asn Gln Leu Gln Gln Leu Arg Val Gln Ile
Met 180 185 190 Ala Tyr Arg Leu Leu Ala Arg Asn Gln Pro Leu Ser Gln
Gln Leu Ala 195 200 205 Leu Ala Val Gln Gly Lys Arg Leu Asp Ser Pro
Gly Glu Ser Asn Tyr 210 215 220 Gln His Pro Pro Ser Glu Gly Ala Gly
Gly Val Gly Gly Glu Gly Ser 225 230 235 240 Gly Asp Gly Gly Ser Ser
Asn Gly Leu Met Thr Gln Pro Met Arg Ala 245 250 255 Pro Cys Pro Pro
Gly Gly Gln Pro Pro Thr Ala Ser Pro Met Thr Gly 260 265 270 Gln Met
Ala Pro Pro Thr Gly Pro Ala Pro Val Arg Pro Pro Pro Pro 275 280 285
Gly Val Ser Pro Thr Pro Pro Arg Pro Pro Gln Gln Val Pro Gly Ala 290
295 300 Pro Gly Ala Pro Gln Pro Lys Gln Asn Arg Val Thr Thr Met Pro
Arg 305 310 315 320 Pro His Gly Leu Asp Pro Ile Leu Ile Leu Gln Glu
Arg Glu Asn Arg 325 330 335 Val Ala Ala Arg Ile Val His Arg Met Glu
Glu Leu Ser Asn Leu Pro 340 345 350 Ala Thr Met Pro Glu Asp Leu Arg
Ile Lys Ala Gln Ile Glu Leu Arg 355 360 365 Ala Leu Arg Val Leu Asn
Phe Gln Arg Gln Leu Arg Ala Glu Val Ile 370 375 380 Ala Cys Thr Arg
Arg Asp Thr Thr Leu Glu Thr Ala Val Asn Val Lys 385 390 395 400 Ala
Tyr Lys Arg Thr Lys Arg Gln Gly Leu Arg Glu Ala Arg Ala Thr 405 410
415 Glu Lys Leu Glu Lys Gln Gln Lys Leu Glu Thr Glu Arg Lys Lys Arg
420 425 430 Gln Lys His Gln Glu Tyr Leu Ser Thr Ile Leu Gln His Cys
Lys Asp 435 440 445 Phe Lys Glu Phe His Arg Asn Asn Val Ala Lys Val
Gly Arg Leu Asn 450 455 460 Lys Ala Val Met Asn Tyr His Ala Asn Ala
Glu Arg Glu Gln Lys Lys 465 470 475 480 Glu Gln Glu Arg Ile Glu Lys
Glu Arg Met Arg Arg Leu Met Ala Glu 485 490 495 Asp Glu Glu Gly Tyr
Arg Lys Leu Ile Asp Gln Lys Lys Asp Lys Arg 500 505 510 Leu Ala Phe
Leu Leu Ser Gln Thr Asp Glu Tyr Ile Ala Asn Leu Thr 515 520 525 Glu
Met Val Lys Gln His Lys Met Glu Gln Gln Arg Lys Gln Glu Gln 530 535
540 Glu Glu Gln Gln Lys Arg Lys Arg Lys Lys Lys Lys Lys Asn Arg Glu
545 550 555 560 Gly Asp Pro Asp Asp Glu Ser Ser Gln Met Ser Asp Leu
His Val Ser 565 570 575 Val Ile Glu Ala Ala Thr Gly Arg Gln Leu Thr
Gly Glu Asp Ala Pro 580 585 590 Leu Ala Ser Gln Leu Gly Ser Trp Leu
Glu Ala His Pro Gly Trp Glu 595 600 605 Pro Leu Glu Asp Ser Glu Asp
Glu Asp Asp Glu Glu Asp Ser Asp Glu 610 615 620 Glu Gly Asp Asp Asn
Ser Arg Ser Lys Gly Gly Phe Ser Met Ile Gly 625 630 635 640 Lys Asp
Glu Ala Asp Ser Lys Leu Ser Val Glu Asp Glu Ala Arg Glu 645 650 655
Met Ile Lys Lys Ala Lys Ile Glu Asp Asp Glu Tyr Lys Asn Thr Thr 660
665 670 Glu Glu His Thr Tyr Tyr Ser Ile Ala His Thr Val His Glu Ile
Val 675 680 685 Thr Glu Gln Ala Ser Ile Met Ile Asn Gly Lys Leu Lys
Glu Tyr Gln 690 695 700 Ile Lys Gly Leu Glu Trp Leu Val Ser Leu Tyr
Asn Asn Asn Leu Asn 705 710 715 720 Gly Ile Leu Ala Asp Glu Met Gly
Leu Gly Lys Thr Ile Gln Thr Ile 725 730 735 Gly Leu Ile Thr Tyr Leu
Met Glu Lys Lys Lys Val Asn Gly Pro Tyr 740 745 750 Leu Ile Ile Val
Pro Leu Ser Thr Leu Ser Asn Trp Val Leu Glu Phe 755 760 765 Glu Lys
Trp Ala Pro Ser Val Phe Val Val Ala Tyr Lys Gly Ser Pro 770 775 780
Ala Met Arg Arg Thr Leu Gln Ser Gln Met Arg Ser Thr Lys Phe Asn 785
790 795 800 Val Leu Leu Thr Thr Tyr Glu Tyr Val Ile Lys Asp Lys Ala
Val Leu 805 810 815 Ala Lys Leu His Trp Lys Tyr Met Ile Ile Asp Glu
Gly His Arg Met 820 825 830 Lys Asn His His Cys Lys Leu Thr Gln Val
Leu Asn Thr His Tyr Leu 835 840 845 Ala Pro His Arg Leu Leu Leu Thr
Gly Thr Pro Leu Gln Asn Lys Leu 850 855 860 Pro Glu Leu Trp Ala Leu
Leu Asn Phe Leu Leu Pro Ser Ile Phe Lys 865 870 875 880 Ser Cys Ser
Thr Phe Glu Gln Trp Phe Asn Ala Pro Phe Ala Thr Thr 885 890 895 Gly
Glu Lys Val Glu Leu Asn Glu Glu Glu Thr Ile Leu Ile Ile Arg 900 905
910 Arg Leu His Lys Val Leu Arg Pro Phe Leu Leu Arg Arg Leu Lys Lys
915 920 925 Glu Val Glu Ser Gln Leu Pro Glu Lys Ile Glu Tyr Ile Val
Lys Cys 930 935 940 Asp Met Ser Gly Leu Gln Arg Val Leu Tyr Arg His
Met Gln Ser Lys 945 950 955 960 Gly Val Leu Leu Thr Asp Gly Ser Glu
Lys Gly Lys Gln Gly Lys Gly 965 970 975 Gly Ala Lys Ala Leu Met Asn
Thr Ile Val Gln Leu Arg Lys Leu Cys 980 985 990 Asn His Pro Phe Met
Phe His His Ile Glu Glu Lys Tyr Cys Asp His 995 1000 1005 Val Gly
Gln Asn Asn Val Val Thr Gly Pro Asp Leu Phe Arg Val 1010 1015 1020
Ser Gly Lys Phe Glu Phe Leu Asp Arg Ile Leu Pro Lys Leu Lys 1025
1030 1035 Ala Thr Ser His Arg Val Leu Leu Phe Cys Gln Met Thr Gln
Leu 1040 1045 1050 Met Thr Ile Met Glu Asp Tyr Leu Ser Trp Arg Gly
Phe Ser Tyr 1055 1060 1065 Leu Arg Leu Asp Gly Thr Thr Lys Ser Glu
Asp Arg Gly Asp Leu 1070 1075 1080 Leu Lys Lys Phe Asn Asn Pro Glu
Ser Glu Tyr Phe Ile Phe Leu 1085 1090 1095 Leu Ser Thr Arg Ala Gly
Gly Leu Gly Leu Asn Leu Gln Ala Ala 1100 1105 1110 Asp Thr Val Ile
Ile Phe Asp Ser Asp Trp Asn Pro His Gln Asp 1115 1120 1125 Leu Gln
Ala Gln Asp Arg Ala His Arg Ile Gly Gln Gln Asn Glu 1130 1135 1140
Val Arg Val Leu Arg Leu Met Thr Val Asn Ser Val Glu Glu Arg 1145
1150 1155 Ile Leu Ala Ala Ala Arg Tyr Lys Leu Asn Met Asp Glu Lys
Val 1160 1165 1170 Ile Gln Ala Gly Met Phe Asp Gln Lys Ser Thr Gly
Thr Glu Arg 1175 1180 1185 Gln Lys Phe Leu Gln Asn Ile Leu His Gln
Asp Asp Ala Asp Asp 1190 1195 1200 Glu Glu Asn Glu Val Pro Asp Asp
Glu Met Val Asn Arg Met Ile 1205 1210 1215 Ala Arg Thr Glu Asp Glu
Phe Asn Leu Phe Gln Lys Ile Asp Leu 1220 1225 1230 Glu Arg Arg Arg
Glu Glu Ala Lys Leu Gly Pro Asn Arg Lys Ser 1235 1240 1245 Arg Leu
Val Glu Glu Ala Glu Leu Pro Asp Trp Leu Val Lys Asn 1250 1255 1260
Asp Asp Glu Ile Glu Lys Trp Thr Tyr Glu Glu Thr Glu Val Gln 1265
1270 1275 Met Gly Arg Gly Asn Arg Gln Arg Lys Glu Val Asp Tyr Thr
Asp 1280 1285 1290 Ser Leu Thr Glu Lys Glu Trp Leu Lys Ala Ile Asp
Asp Asn Val 1295 1300 1305 Asp Asp Phe Asp Asp Asp Glu Glu Glu Glu
Val Lys Thr Lys Lys 1310 1315 1320 Arg Gly Lys Arg Arg Arg Arg Gly
Glu Asp Asp Glu Glu Asp Ala 1325 1330 1335 Ser Thr Ser Lys Arg Arg
Lys Tyr Ser Pro Ser Glu Asn Lys Leu 1340 1345 1350 Arg Arg Arg Met
Arg Asn Leu Met Asn Ile Val Val Lys Tyr Thr 1355 1360 1365 Asp Ser
Asp Ser Arg Val Leu Ser Glu Pro Phe Met Lys Leu Pro 1370 1375 1380
Ser Arg His Lys Tyr Pro Asp Tyr Tyr Glu Leu Ile Lys Lys Pro 1385
1390 1395 Ile Asp Ile Lys Arg Ile Leu Ala Lys Val Glu Glu Cys Lys
Tyr 1400 1405 1410 Ala Asp Met Asp Glu Leu Glu Lys Asp Phe Met Gln
Leu Cys Lys 1415 1420 1425 Asn Ala Gln Thr Tyr Asn Glu Glu Ala Ser
Leu Ile Tyr Glu Asp 1430 1435 1440 Ser Ile Val Leu Glu Ser Val Phe
Ser Asn Ala Arg Gln Lys Val 1445 1450 1455 Glu Gln Asp Asn Asp Ser
Asp Asp Asp Glu Ser Lys Gly Asp Gln 1460 1465 1470 Glu Asp Ala Ala
Ser Asp Thr Ser Ser Val Lys Met Lys Leu Lys 1475 1480 1485 Leu Lys
Pro Gly Arg Thr Arg Gly Ser Gly Ala Gly Gly Lys Arg 1490 1495 1500
Arg Arg Arg Lys Tyr Ile Ser Glu Asp Glu Asp Glu Asp His Ser
1505
1510 1515 Glu Val Ser Leu Met 1520 3499DNAEuchistus heros
3gatgatgaag aagatgcaag tacttcaaag agaaggaaat attctccatc tgaaaacaaa
60ctgaggaggc gtatgcgtaa cctcatgaac attgttgtta agtatactga cagtgactcg
120agagtactca gtgaaccatt catgaaactt ccctctcgcc ataagtaccc
agactactat 180gagttgatca agaaacctat agacatcaag aggatattgg
ccaaagtaga agagtgtaaa 240tatgctgaca tggatgaatt agaaaaggat
tttatgcaac tttgtaaaaa tgctcagaca 300tacaatgagg aggcctcatt
gatctatgaa gattcgatag tattagaaag tgttttctct 360aatgctcgtc
aaaaagtaga gcaggataat gattcagatg atgatgaaag taaaggtgac
420caagaagatg ctgcatcaga cacttcatcc gtcaaaatga aattgaaact
aaagcctggg 480aggacccgag ggagtggag 499420DNAArtificial SequenceT7
phage promoter 4taatacgact cactataggg 205301DNAArtificial
SequenceYFPv2 dsRNA sense strand encoding sequence 5catctggagc
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 301647DNAArtificial
SequencePrimer YFPv2-F 6ttaatacgac tcactatagg gagagcatct ggagcacttc
tctttca 47746DNAArtificial SequencePrimer YFPv2-R 7ttaatacgac
tcactatagg gagaccatct ccttcaaagg tgattg 4686346DNAEuchistus heros
8atctcggtgc tgtggatcgt ccttagtgat tgttttctaa tatagtttgt aattatatag
60tgttttatgc gttgatatcg gtgatattag tgaataatag tgaagtgttg atgttttatt
120tctaatggcg tctgaagaag aagttgacga gtgtttacca gttgacgatg
aagttgacac 180tagtgttgtt caacaagaag gcactgaaga aaattcacct
gacagtgatg aaagaagtag 240gatagaggaa gaagatgacg agtatgaccc
tgaggatgcg aggaaaaaaa agaaaggtaa 300aaagagaaaa gccaaagggg
aaagcaaaaa agaaaagaaa cgtaaaaaaa ggaagaagaa 360tgatagtgct
gaagaaagtg agggaggcgg ggaagaagaa ggcgattccg attatggaag
420aaaatctaag aagtctaaag gaacttcaca accaaaacca gtgcagcaag
attcttctgg 480aggtgtacct tcagtagaag aagtttgcag cctttttgga
cttacagatg tacagattga 540ctataccgaa gatgattacc aaaatctgac
tacgtataaa ctttttcaac aacatgttcg 600tcctattctt gccaaggaca
accagaaggt tcccatcgga aaaatgatga tgctcgtggc 660tgcaaaatgg
agagattttt gcaattccaa tccaaacgct caacaggaac cagatccaga
720agcttcagaa gaacaggaat attctaaacc taccaggaca cgaccttcac
gagtttcaac 780tacacaaaat gatgatgaag aagacgacga tgctgacgaa
cgagggagga aaaagagaag 840tggacgaagt aaaaagtcat caggaaagaa
gtccgctcct ccggccacaa ccaaggtccc 900taccctcaag atcaagatag
gaaaaagaaa acagaattcc gatgaagaag atgaaggttc 960agttggtgcc
gtttctgaaa gggactcaga tgctgaattc gagcaaatgc tcgcagaagc
1020tgaagaagtt aataaacctg aaggtgttgt agaagaagaa gaaggtgcag
aggtggctcc 1080tgtacctaag aaaaaggcca aaacgaaaat tggtaataaa
aagaaaagga aaaagacacg 1140gactactaac aagtttccag acagtgaagc
tggttatgaa acagatcatc aggactattg 1200tgaagtttgt caacaaggag
gtgaaataat attatgtgat acgtgccctc gagcttatca 1260tttggtctgt
ttggatcccg aattggaaga tacgccagaa ggcaaatggt catgccctca
1320ttgtgaaggt gaaggtgtac aggaaaaaga agatgatgtc catcaagaat
tttgcagagt 1380ttgtaaagat ggtggagaac ttttatgctg tgattcttgc
ccttctgcat accacacatt 1440ctgtttgaac cctccattga cagatattcc
agatggtgac tggaagtgcc cacgttgttc 1500ggcgaagcct ttgagaggta
aagtgtcaaa gattcttact tggaggtggt tggaatctcc 1560cagtagtaaa
gatgaagaag acaatactaa aaaacgaaac aggcagaggc aaagagaata
1620tttcgtcaag tgggcagata tgtcttattg gcactgtagt tgggtgtctg
aacttcagat 1680ggatgttttt catactcaaa tgatcaggag ttatattcgt
aaatatgata tggacgaacc 1740tcccaaacta gaagaaccct tggatgaagc
agacaataga atgaagagga tacgagaggc 1800aaatatcaat gagcaagaat
tagaagagaa atattacaag tatggtatca aaccagagtg 1860gcttattgtg
cagagggtaa ttaaccatcg cactataagg gatggaagca atctgtacct
1920cgtcaaatgg agggacctcc cttatgacca ggcgacttgg gaggaagaag
tcaccgatat 1980ccctggcttg aagaaagcta ttgaatatta caatgagatg
agggcttgct gtttaggtga 2040atctaaaaaa ctaaaaaaag gtaaaggtaa
aagatcaaag agagatcaag atgatgagga 2100aggaagcaga agtgcaggaa
tgatgggcgt cggtggacca gctactggtc aatacttccc 2160gcctcctgaa
aagcctgtca cagatttgaa aaagaaatac gataaacagc cggactatct
2220cgacgtctcc ggtatgtgcc ttcatcctta ccaattagaa ggtttaaatt
ggttgaggta 2280ttcctggggg caaggaacag acactattct tgccgatgag
atgggtcttg gaaaaaccat 2340tcagacaatt actttcctct attctcttta
caaagagggt cattgtaaag gccccttcct 2400tgtgagtgta cccttatcta
caattatcaa ttgggaaaga gagttcgaaa cttgggcgcc 2460agacttctac
gttgtcacat atgtcggaga caaagattct cgtgctgtaa tacgtgaaaa
2520tgaattttca ttcgatgata atgctgttag aggaggaaga ggtgtttcta
aagttcgctc 2580ttctgcaata aagtttcatg tactgctaac atcttatgaa
cttatctcta tcgatgtcac 2640ttgccttgga tcgatcgagt gggcagtgct
tgtagtagat gaagcacaca ggctgaaaag 2700taatcagagc aagttcttta
ggcttcttgc ttcataccac attgcttata aacttctgct 2760gacaggaact
ccgttgcaaa acaatctaga agaattgttt catttactta atttccttac
2820gccggaaaaa ttcaacgacc ttgcgacatt tcaaaacgaa ttcgctgata
tttcaaaaga 2880agaacaagtc aaaagacttc atgagttact cgggccgcat
atgttgagga gattaaaagc 2940tgatgtactc aagaatatgc ctacaaaatc
tgagttcatt gttagagttg aactctcccc 3000gatgcagaag aagtactaca
aatatattct cacaaggaat ttcgaagctt taaatccaaa 3060aggaggcggt
caacaagtat ctcttttgaa cattatgatg gatcttaaaa aatgctgtaa
3120tcatccatac ctgtttcctg ctgcttctca ggaagctcct ttaggaccaa
gcggatctta 3180cgatcttcaa gggttaatca aagcatctgg aaaattgata
cttctgtcga aaatgctgag 3240acggctcaaa gaagagggtc acagagtact
gattttctct caaatgacaa aaatgttgga 3300cttattagaa gactacctcg
agggtgaagg ttataaatat gaacgtattg acggtacgat 3360caccggtagc
ttaagacaag aagctatcga tcggtttaac gcccctggag ctcaacaatt
3420tgtttttctt ttgtccactc gtgcgggagg tcttggtatt aatctcgcta
ctgcagatac 3480agttattatt tatgactctg actggaatcc tcataacgat
attcaggcct tttcgagagc 3540acacaggata gggcaagcaa acaaggttat
gatttatcga tttgtgacac gagcgtctgt 3600tgaagaaaga gtaacgcaag
tggctaagag aaaaatgatg ttaacccatc ttgtcgtacg 3660accaggtatg
ggtggcaagc aagcaaattt cactaagcaa gaacttgatg atattttaag
3720gtttggaaca gaagaacttt tcaaagaaga gcagggtaaa gaagatgaag
ccattcatta 3780tgacgataaa gctgttgaag aattacttga ccggtcgaag
atgggtattg aacagaaaga 3840aaactggtct aatgaatatc tttcttcttt
caaagtggca agttatgtta ctaaagaaga 3900agacgaagat gaggaaatag
gaacagaggt aataaaacag gaagcagaaa atacagaccc 3960agcttattgg
gtcaaactgt tgaggcacca ttatgagcaa caacaagagg atatttctcg
4020aactctcggt aaaggaaaaa ggattcgaaa acaggtgaat tacatcgacg
gtggagtgat 4080ggactcaaga gagaacgccg attcgacgtg gcaagacaac
ctctctgact ataattcaga 4140cttctctgct ccttctgatg atgacaagga
agacgatgac tttgatgaga aaaatgatga 4200tggaacgaga aagaagcgta
ggccagaaag gagggaggac aaagataggc ctctacctcc 4260tcttcttgcc
cgagtcggtg gaaacattga ggtcctggga ttcaacgcca gacagcgtaa
4320agcattcttg aatgctatta tgaggtatgg aatgccacct caagatgcat
tcaactcgca 4380gtggcttgtt cgagacctga ggggtaaatc tgagaagcat
ttcaaggcat acgtatccct 4440ctttatgagg catttgtgtg agcctggcgc
ggacaatgcc gaaacattcg cggatggtgt 4500tccaagggaa ggtcttagtc
ggcagcatgt tctcacaagg ataggtgtga tgtcactcat 4560taggaaaaag
gttcaagaat ttgagcaaat taatggatat tactcgatgc ctgaaatgtt
4620gaagaaacca cttgttgatg ccggattgca taaaacaagt gctagcagta
taggtgaagg 4680tgctagtagt tccggtacac ctgcaacatc agctgctcca
agtccagctc ctactctttt 4740ggataagaca caaattgaag atttgagtga
aaaagaagat ccgtcaaaga ctgaagataa 4800aaccaccgat gattccaaac
cctcagaaga ggctaaagct gcagatgatg caaataagcc 4860tcaggctgaa
ggagaaaagg cagaaggatc ttctaatgca aaccaaactt ctgaagctga
4920aggaagcgat gagaaaaaac ccaaagaaga accgatggat gtagatggtg
aaggagaggc 4980taaagatagt gataagacag aaaaacaaga aggtactgac
gaaaaagatg tagccctaaa 5040agaggaagaa aaggatgaag aggtcaacaa
agagaaggga gaggaaacag aggaaaagaa 5100ggttatcgat tttgaagaag
acaaatctaa aaggaaattt atgttcaata tcgctgatgg 5160aggatttact
gagctccata ccttatggca aaatgaagag aaagctgcag tacctggtag
5220ggagtacgag atctggcata ggaggcatga ctattggctg ttgggtggaa
tcgttaccca 5280tggctatggt cggtggcaag atattcaaaa tgatattaga
tttgctatta tcaacgaacc 5340atttaagatg gatgttggaa aaggaaattt
cttagaaatt aaaaataaat ttcttgccag 5400gaggtttaag cttcttgagc
aagctctggt gattgaagaa cagttaagac gtgcagctta 5460tttaaatctg
acgcaagatc caaatcaccc agcaatgtca ctgaatgcaa gatttgcaga
5520ggttgaatgt ctagccgaat ctcaccaaca cctctcgaag gaaagtcttg
ctggcaacaa 5580acctgcaaat gcagtgttac ataaagtatt gaaccaatta
gaggagcttc tgtcggatat 5640gaaatctgac gtatctcgac taccagccac
tctagccaga attccacctg tagcccagag 5700gctacagatg tctgaacggt
caatactttc taggttggct gcaactactt ctcctgcgac 5760gcccaccacg
tcccatcaaa ctggtatgat aagcagtcag ttccctgctg gatttcaatc
5820agggcagttg actggaacgt ttccgaatgc cagttttacc aacttcaggc
cccagtattc 5880agttcctggg caaactgcag cccagggttt tcccggtaat
tgataattga aagctggacg 5940gtaattgtct gcgagtgaat tctccatgag
taaataatag gttttttttt ttttttaaga 6000aagaaataaa agaagcgttt
tgtttagttt tgttgatagt tctctttatt tctttcaatt 6060ttgttttagc
ggaaaaaaaa atgttcatta taagtaactt ataaattgga catgctaatt
6120aaatttccta ttagattatt ttgttatttg taagtttttc ggtattgtaa
gaatgtctat 6180atgtgtaaga ggttgtacaa gattgcctaa ataccttgta
ttatttattt ttactattga 6240ataaaaaaaa aaaataatta acttcgatct
taggttaagg gtaataaaaa aaaatgttac 6300tggaaaaaaa aatagaaaaa
ataaaaaaga tagcctttcc ccttac 634691938PRTEuchistus heros 9Met Ala
Ser Glu Glu Glu Val Asp Glu Cys Leu Pro Val Asp Asp Glu 1 5 10 15
Val Asp Thr Ser Val Val Gln Gln Glu Gly Thr Glu Glu Asn Ser Pro 20
25 30 Asp Ser Asp Glu Arg Ser Arg Ile Glu Glu Glu Asp Asp Glu Tyr
Asp 35 40 45 Pro Glu Asp Ala Arg Lys Lys Lys Lys Gly Lys Lys Arg
Lys Ala Lys 50 55 60 Gly Glu Ser Lys Lys Glu Lys Lys Arg Lys Lys
Arg Lys Lys Asn Asp 65 70 75 80 Ser Ala Glu Glu Ser Glu Gly Gly Gly
Glu Glu Glu Gly Asp Ser Asp 85 90 95 Tyr Gly Arg Lys Ser Lys Lys
Ser Lys Gly Thr Ser Gln Pro Lys Pro 100 105 110 Val Gln Gln Asp Ser
Ser Gly Gly Val Pro Ser Val Glu Glu Val Cys 115 120 125 Ser Leu Phe
Gly Leu Thr Asp Val Gln Ile Asp Tyr Thr Glu Asp Asp 130 135 140 Tyr
Gln Asn Leu Thr Thr Tyr Lys Leu Phe Gln Gln His Val Arg Pro 145 150
155 160 Ile Leu Ala Lys Asp Asn Gln Lys Val Pro Ile Gly Lys Met Met
Met 165 170 175 Leu Val Ala Ala Lys Trp Arg Asp Phe Cys Asn Ser Asn
Pro Asn Ala 180 185 190 Gln Gln Glu Pro Asp Pro Glu Ala Ser Glu Glu
Gln Glu Tyr Ser Lys 195 200 205 Pro Thr Arg Thr Arg Pro Ser Arg Val
Ser Thr Thr Gln Asn Asp Asp 210 215 220 Glu Glu Asp Asp Asp Ala Asp
Glu Arg Gly Arg Lys Lys Arg Ser Gly 225 230 235 240 Arg Ser Lys Lys
Ser Ser Gly Lys Lys Ser Ala Pro Pro Ala Thr Thr 245 250 255 Lys Val
Pro Thr Leu Lys Ile Lys Ile Gly Lys Arg Lys Gln Asn Ser 260 265 270
Asp Glu Glu Asp Glu Gly Ser Val Gly Ala Val Ser Glu Arg Asp Ser 275
280 285 Asp Ala Glu Phe Glu Gln Met Leu Ala Glu Ala Glu Glu Val Asn
Lys 290 295 300 Pro Glu Gly Val Val Glu Glu Glu Glu Gly Ala Glu Val
Ala Pro Val 305 310 315 320 Pro Lys Lys Lys Ala Lys Thr Lys Ile Gly
Asn Lys Lys Lys Arg Lys 325 330 335 Lys Thr Arg Thr Thr Asn Lys Phe
Pro Asp Ser Glu Ala Gly Tyr Glu 340 345 350 Thr Asp His Gln Asp Tyr
Cys Glu Val Cys Gln Gln Gly Gly Glu Ile 355 360 365 Ile Leu Cys Asp
Thr Cys Pro Arg Ala Tyr His Leu Val Cys Leu Asp 370 375 380 Pro Glu
Leu Glu Asp Thr Pro Glu Gly Lys Trp Ser Cys Pro His Cys 385 390 395
400 Glu Gly Glu Gly Val Gln Glu Lys Glu Asp Asp Val His Gln Glu Phe
405 410 415 Cys Arg Val Cys Lys Asp Gly Gly Glu Leu Leu Cys Cys Asp
Ser Cys 420 425 430 Pro Ser Ala Tyr His Thr Phe Cys Leu Asn Pro Pro
Leu Thr Asp Ile 435 440 445 Pro Asp Gly Asp Trp Lys Cys Pro Arg Cys
Ser Ala Lys Pro Leu Arg 450 455 460 Gly Lys Val Ser Lys Ile Leu Thr
Trp Arg Trp Leu Glu Ser Pro Ser 465 470 475 480 Ser Lys Asp Glu Glu
Asp Asn Thr Lys Lys Arg Asn Arg Gln Arg Gln 485 490 495 Arg Glu Tyr
Phe Val Lys Trp Ala Asp Met Ser Tyr Trp His Cys Ser 500 505 510 Trp
Val Ser Glu Leu Gln Met Asp Val Phe His Thr Gln Met Ile Arg 515 520
525 Ser Tyr Ile Arg Lys Tyr Asp Met Asp Glu Pro Pro Lys Leu Glu Glu
530 535 540 Pro Leu Asp Glu Ala Asp Asn Arg Met Lys Arg Ile Arg Glu
Ala Asn 545 550 555 560 Ile Asn Glu Gln Glu Leu Glu Glu Lys Tyr Tyr
Lys Tyr Gly Ile Lys 565 570 575 Pro Glu Trp Leu Ile Val Gln Arg Val
Ile Asn His Arg Thr Ile Arg 580 585 590 Asp Gly Ser Asn Leu Tyr Leu
Val Lys Trp Arg Asp Leu Pro Tyr Asp 595 600 605 Gln Ala Thr Trp Glu
Glu Glu Val Thr Asp Ile Pro Gly Leu Lys Lys 610 615 620 Ala Ile Glu
Tyr Tyr Asn Glu Met Arg Ala Cys Cys Leu Gly Glu Ser 625 630 635 640
Lys Lys Leu Lys Lys Gly Lys Gly Lys Arg Ser Lys Arg Asp Gln Asp 645
650 655 Asp Glu Glu Gly Ser Arg Ser Ala Gly Met Met Gly Val Gly Gly
Pro 660 665 670 Ala Thr Gly Gln Tyr Phe Pro Pro Pro Glu Lys Pro Val
Thr Asp Leu 675 680 685 Lys Lys Lys Tyr Asp Lys Gln Pro Asp Tyr Leu
Asp Val Ser Gly Met 690 695 700 Cys Leu His Pro Tyr Gln Leu Glu Gly
Leu Asn Trp Leu Arg Tyr Ser 705 710 715 720 Trp Gly Gln Gly Thr Asp
Thr Ile Leu Ala Asp Glu Met Gly Leu Gly 725 730 735 Lys Thr Ile Gln
Thr Ile Thr Phe Leu Tyr Ser Leu Tyr Lys Glu Gly 740 745 750 His Cys
Lys Gly Pro Phe Leu Val Ser Val Pro Leu Ser Thr Ile Ile 755 760 765
Asn Trp Glu Arg Glu Phe Glu Thr Trp Ala Pro Asp Phe Tyr Val Val 770
775 780 Thr Tyr Val Gly Asp Lys Asp Ser Arg Ala Val Ile Arg Glu Asn
Glu 785 790 795 800 Phe Ser Phe Asp Asp Asn Ala Val Arg Gly Gly Arg
Gly Val Ser Lys 805 810 815 Val Arg Ser Ser Ala Ile Lys Phe His Val
Leu Leu Thr Ser Tyr Glu 820 825 830 Leu Ile Ser Ile Asp Val Thr Cys
Leu Gly Ser Ile Glu Trp Ala Val 835 840 845 Leu Val Val Asp Glu Ala
His Arg Leu Lys Ser Asn Gln Ser Lys Phe 850 855 860 Phe Arg Leu Leu
Ala Ser Tyr His Ile Ala Tyr Lys Leu Leu Leu Thr 865 870 875 880 Gly
Thr Pro Leu Gln Asn Asn Leu Glu Glu Leu Phe His Leu Leu Asn 885 890
895 Phe Leu Thr Pro Glu Lys Phe Asn Asp Leu Ala Thr Phe Gln Asn Glu
900 905 910 Phe Ala Asp Ile Ser Lys Glu Glu Gln Val Lys Arg Leu His
Glu Leu 915 920 925 Leu Gly Pro His Met Leu Arg Arg Leu Lys Ala Asp
Val Leu Lys Asn 930 935 940 Met Pro Thr Lys Ser Glu Phe Ile Val Arg
Val Glu Leu Ser Pro Met 945 950 955 960 Gln Lys Lys Tyr Tyr Lys Tyr
Ile Leu Thr Arg Asn Phe Glu Ala Leu 965 970 975 Asn Pro Lys Gly Gly
Gly Gln Gln Val Ser Leu Leu Asn Ile Met Met 980 985 990 Asp Leu Lys
Lys Cys Cys Asn His Pro Tyr Leu Phe Pro Ala Ala Ser 995 1000 1005
Gln Glu Ala Pro Leu Gly Pro Ser Gly Ser Tyr Asp Leu Gln Gly 1010
1015 1020 Leu Ile Lys Ala Ser Gly Lys Leu Ile Leu Leu Ser Lys Met
Leu 1025 1030 1035 Arg Arg Leu Lys Glu Glu Gly His Arg Val Leu Ile
Phe Ser Gln 1040 1045 1050 Met Thr Lys Met Leu Asp Leu Leu Glu Asp
Tyr Leu Glu Gly Glu 1055 1060 1065 Gly Tyr Lys Tyr Glu Arg Ile Asp
Gly Thr Ile Thr Gly Ser Leu 1070 1075 1080 Arg Gln Glu Ala Ile Asp
Arg Phe Asn Ala Pro Gly Ala Gln Gln 1085 1090 1095 Phe Val Phe Leu
Leu Ser Thr Arg Ala Gly Gly Leu Gly Ile Asn 1100 1105 1110
Leu Ala Thr Ala Asp Thr Val Ile Ile Tyr Asp Ser Asp Trp Asn 1115
1120 1125 Pro His Asn Asp Ile Gln Ala Phe Ser Arg Ala His Arg Ile
Gly 1130 1135 1140 Gln Ala Asn Lys Val Met Ile Tyr Arg Phe Val Thr
Arg Ala Ser 1145 1150 1155 Val Glu Glu Arg Val Thr Gln Val Ala Lys
Arg Lys Met Met Leu 1160 1165 1170 Thr His Leu Val Val Arg Pro Gly
Met Gly Gly Lys Gln Ala Asn 1175 1180 1185 Phe Thr Lys Gln Glu Leu
Asp Asp Ile Leu Arg Phe Gly Thr Glu 1190 1195 1200 Glu Leu Phe Lys
Glu Glu Gln Gly Lys Glu Asp Glu Ala Ile His 1205 1210 1215 Tyr Asp
Asp Lys Ala Val Glu Glu Leu Leu Asp Arg Ser Lys Met 1220 1225 1230
Gly Ile Glu Gln Lys Glu Asn Trp Ser Asn Glu Tyr Leu Ser Ser 1235
1240 1245 Phe Lys Val Ala Ser Tyr Val Thr Lys Glu Glu Asp Glu Asp
Glu 1250 1255 1260 Glu Ile Gly Thr Glu Val Ile Lys Gln Glu Ala Glu
Asn Thr Asp 1265 1270 1275 Pro Ala Tyr Trp Val Lys Leu Leu Arg His
His Tyr Glu Gln Gln 1280 1285 1290 Gln Glu Asp Ile Ser Arg Thr Leu
Gly Lys Gly Lys Arg Ile Arg 1295 1300 1305 Lys Gln Leu Tyr Lys Val
Asn Tyr Ile Asp Gly Gly Val Met Asp 1310 1315 1320 Ser Arg Glu Asn
Ala Asp Ser Thr Trp Gln Asp Asn Leu Ser Asp 1325 1330 1335 Tyr Asn
Ser Asp Phe Ser Ala Pro Ser Asp Asp Asp Lys Glu Asp 1340 1345 1350
Asp Asp Phe Asp Glu Lys Asn Asp Asp Gly Thr Arg Lys Lys Arg 1355
1360 1365 Arg Pro Glu Arg Arg Glu Asp Lys Asp Arg Pro Leu Pro Pro
Leu 1370 1375 1380 Leu Ala Arg Val Gly Gly Asn Ile Glu Val Leu Gly
Phe Asn Ala 1385 1390 1395 Arg Gln Arg Lys Ala Phe Leu Asn Ala Ile
Met Arg Tyr Gly Met 1400 1405 1410 Pro Pro Gln Asp Ala Phe Asn Ser
Gln Trp Cys Ser Arg Leu Val 1415 1420 1425 Arg Asp Leu Arg Gly Lys
Ser Glu Lys His Phe Lys Ala Tyr Val 1430 1435 1440 Ser Leu Phe Met
Arg His Leu Cys Glu Pro Gly Ala Asp Asn Ala 1445 1450 1455 Glu Thr
Phe Ala Asp Gly Val Pro Arg Glu Gly Leu Ser Arg Gln 1460 1465 1470
His Val Leu Thr Arg Ile Gly Val Met Ser Leu Ile Arg Lys Lys 1475
1480 1485 Val Gln Glu Phe Glu Gln Ile Asn Gly Tyr Tyr Ser Met Pro
Glu 1490 1495 1500 Met Leu Lys Lys Pro Leu Val Asp Ala Gly Leu His
Lys Thr Ser 1505 1510 1515 Ala Ser Ser Ile Gly Glu Gly Ala Ser Ser
Ser Gly Thr Pro Ala 1520 1525 1530 Thr Ser Ala Ala Pro Ser Pro Ala
Pro Thr Leu Leu Asp Lys Thr 1535 1540 1545 Gln Ile Glu Asp Leu Ser
Glu Lys Glu Asp Pro Ser Lys Thr Glu 1550 1555 1560 Asp Lys Thr Thr
Asp Asp Ser Lys Pro Ser Glu Glu Ala Lys Ala 1565 1570 1575 Ala Asp
Asp Ala Asn Lys Pro Gln Ala Glu Gly Glu Lys Ala Glu 1580 1585 1590
Gly Ser Ser Asn Ala Asn Gln Thr Ser Glu Ala Glu Gly Ser Asp 1595
1600 1605 Glu Lys Lys Pro Lys Glu Glu Pro Met Asp Val Asp Gly Glu
Gly 1610 1615 1620 Glu Ala Lys Asp Ser Asp Lys Thr Glu Lys Gln Glu
Gly Thr Asp 1625 1630 1635 Glu Lys Asp Val Ala Leu Lys Glu Glu Glu
Lys Asp Glu Glu Val 1640 1645 1650 Asn Lys Glu Lys Gly Glu Glu Thr
Glu Glu Lys Lys Val Ile Asp 1655 1660 1665 Phe Glu Glu Asp Lys Ser
Lys Arg Lys Phe Met Phe Asn Ile Ala 1670 1675 1680 Asp Gly Gly Phe
Thr Glu Leu His Thr Leu Trp Gln Asn Glu Glu 1685 1690 1695 Lys Ala
Ala Val Pro Gly Arg Glu Tyr Glu Ile Trp His Arg Arg 1700 1705 1710
His Asp Tyr Trp Leu Leu Gly Gly Ile Val Thr His Gly Tyr Gly 1715
1720 1725 Arg Trp Gln Asp Ile Gln Asn Asp Ile Arg Phe Ala Ile Ile
Asn 1730 1735 1740 Glu Pro Phe Lys Met Asp Val Gly Lys Gly Asn Phe
Leu Glu Ile 1745 1750 1755 Lys Asn Lys Phe Leu Ala Arg Arg Phe Lys
Leu Leu Glu Gln Ala 1760 1765 1770 Leu Val Ile Glu Glu Gln Leu Arg
Arg Ala Ala Tyr Leu Asn Leu 1775 1780 1785 Thr Gln Asp Pro Asn His
Pro Ala Met Ser Leu Asn Ala Arg Phe 1790 1795 1800 Ala Glu Val Glu
Cys Leu Ala Glu Ser His Gln His Leu Ser Lys 1805 1810 1815 Glu Ser
Leu Ala Gly Asn Lys Pro Ala Asn Ala Val Leu His Lys 1820 1825 1830
Val Leu Asn Gln Leu Glu Glu Leu Leu Ser Asp Met Lys Ser Asp 1835
1840 1845 Val Ser Arg Leu Pro Ala Thr Leu Ala Arg Ile Pro Pro Val
Ala 1850 1855 1860 Gln Arg Leu Gln Met Ser Glu Arg Ser Ile Leu Ser
Arg Leu Ala 1865 1870 1875 Ala Thr Thr Ser Pro Ala Thr Pro Thr Thr
Ser His Gln Thr Gly 1880 1885 1890 Met Ile Ser Ser Gln Phe Pro Ala
Gly Phe Gln Ser Gly Gln Leu 1895 1900 1905 Thr Gly Thr Phe Pro Asn
Ala Ser Phe Thr Asn Phe Arg Pro Gln 1910 1915 1920 Tyr Ser Val Pro
Gly Gln Thr Ala Ala Gln Gly Phe Pro Gly Asn 1925 1930 1935
103391DNAEuchistus heros 10agagggggta ggcgcacagc tttcctcaca
tcgaacaata tcttagtgaa tgaatggctt 60tattggccgg ttcaaaatct tgttaaatgt
tggtttgata tatatttata ctaacgttat 120ttaacgcagc tcacaccaat
aaaaatgtcg aagccaaatg aagttagttt ggatacaaca 180gatactgttg
aaatttctaa tgaatcttcg ggagacacag agtcgtccaa gggtaaaaat
240gaagattttg aaacaaaaat tgaaactgac cgttctagaa gatttgagtt
tctgttgaag 300cagacagaaa ttttttcaca ttttatgaca aatcaaggaa
agtcgaacag ccctgcaaag 360cctaaagtcg gccgtcctag aaaggaaact
aataaattgg caccagccgg tggtgatggt 420tctgccgacc atcggcatcg
tatgaccgag caggaagaag atgaagaact gcttgctgaa 480agtaatactt
cttcaaaatc cttagcaagg tttgacgctt ctccttttta tattaaaagc
540ggagagttga gggattacca gatacgtggt ttgaattgga tgatatccct
ctacgaacac 600ggtataaatg gtatacttgc tgatgagatg ggtttaggta
aaactctcca aactatttct 660ctccttggtt acatgaagca ttatagaaat
ataccagggc cacatatggt catcgtacca 720aaatcaacat tagctaattg
gatgaatgaa tttaaaaagt ggtgcccaac cctgcgtgct 780gtctgtttaa
tcggagatca ggaaacgagg aatgcgttca tcagagacac tcttatgccg
840ggtgaatggg atgtctgcgt tacatcttat gaaatgatca tacgagaaaa
gagcgttttc 900aagaagttca actggaggta tatggtcatt gacgaagccc
acaggatcaa gaatgaaaaa 960tccaaactct ccgagattgt gagagagttc
aaaacgacga atcgattact cctgaccggt 1020actcctttac aaaataacct
ccacgaattg tggtctcttc ttaacttcct cttaccagat 1080gttttcaatt
catcagatga ttttgattca tggtttaata ccaatacctt ccttggcgat
1140aattctcttg tcgagagatt acatgctgta ctgagacctt tcctcctaag
aagattgaaa 1200tctgaggtag agaaaaaact caaaccgaag aaagaagtca
aaatctacgt tggattgagt 1260aaaatgcaga gagaatggta tactaaagtt
ctaatgaaag atatagacat tgtaaacggt 1320gctggccgag tcgaaaaaat
gcgcctccaa aacatcctca tgcagttgag gaagtgcagt 1380aatcaccctt
atctcttcga cggagctgaa ccaggtccac cttactcaac tgatgagcat
1440ctggtatata acagtggaaa aatggtaata ttagacaagc ttcttcctaa
attgcaagaa 1500caaggatcac gagttctggt tttcagccaa atgacaagga
tgattgatat tctcgaagat 1560tactgttatt ggagaggata taattactgt
cgtcttgatg gtaatacacc tcatgaggat 1620aggcagagac agattaatga
gttcaacgaa gaagacagta agaaattcat tttcatgttg 1680tcgactcgtg
cgggtggttt gggtatcaat ttagccaccg cagatgtagt cattttgtac
1740gattcggatt ggaaccctca aatggatctc caggctatgg atcgtgctca
tcgtattggt 1800caaaagaaac aagtcaaagt gttcaggatg ataactgaaa
acacagttga agagaaaatt 1860gttgagagag ctgaaataaa actccgcctc
gataagttgg tcatccaaca aggcaggctg 1920gtagacaata aaacggcact
caacaaagat gaaatgttga atatgatccg tcacggtgcc 1980aatcatgtat
ttgccagtaa agattctgaa atcaccgatg aagacattga cactattttg
2040gaaaaaggcg aagcaaggac ggaagaaatg aataaaaaac ttgaacaact
cggtgattct 2100aatttgaaag acttcatgat ggaaaccccg actgagtcag
tttaccaatt cgaaggagag 2160gattacaggg aaaagcagaa agttttagga
ataggaagtt ggatagaacc tccaaaaaga 2220gaacgtaaag ctaattacgc
tgtcgatgcc tattttaggg aagcattgag agtatcagaa 2280cctaaagctc
ccaaggcacc gaggcctcct aaacagccta tagttcaaga tttccaattc
2340tttcctcctc gtctctttga gctattggac caggagatct attacttcag
gaaaactgtg 2400ggctacaaag ttcctaaaaa tcctgaatta ggttctgatg
catcacgtgt ccaaaaggaa 2460gaacaaagaa agatagatga ggcagaacct
ttatcagaag aagaactcgc tgaaaaggaa 2520aaacttctta cgcagggttt
taccaattgg actaaaagag atttcaacca gtttattaaa 2580gctaatgaaa
aatatggtcg tgatgatatt gacaatattt caaaagaagt agaaggaaaa
2640actccagaag aagtaagagc ttattcagaa gtgttctggg aacgatgtaa
cgaattgcag 2700gacatagatc gtatcatggg gcagatcgac aggggagagg
ctaaaattca aaggagagca 2760agtattaaga aagctctcga tacaaagatg
agccggtaca gagccccatt tcatcaactt 2820cgcatctcct acggtacgaa
taagggtaag aactataccg aggaagaaga tagattcctt 2880gtctgtatgt
tgcataagct tggttttgac aaggaaaatg tgtacgaaga acttagagcg
2940atggtcaggt gtgcgcctca gttcagattc gactggttca tcaaatcgag
aacagccatg 3000gaattgcaga ggcgttgtaa tactctaatt actctcatcg
aaagagaaaa tcaggaactt 3060gaggagaggg aaagagccga gaagaggaaa
ggaagaggaa gtgggcgtgg tcctggttcc 3120ggtaaaagga aaggagacgg
ttccatttca tctccccctc ctgtccctgg ccaaggggat 3180aagaacagcc
ccgccagaaa aaagaaaaaa atgtagtttc acctcctcat gaaaggaact
3240cattttaaga tatctttttc tagatattta ttttgtgaaa actgtgatgt
attttatatc 3300cgttccgaaa agctctactg ttttgacagt tttattaatt
agtggggtgg ggaggaaata 3360tagccccctc accccccaat aattcataaa t
3391111023PRTEuchistus heros 11Met Ser Lys Pro Asn Glu Val Ser Leu
Asp Thr Thr Asp Thr Val Glu 1 5 10 15 Ile Ser Asn Glu Ser Ser Gly
Asp Thr Glu Ser Ser Lys Gly Lys Asn 20 25 30 Glu Asp Phe Glu Thr
Lys Ile Glu Thr Asp Arg Ser Arg Arg Phe Glu 35 40 45 Phe Leu Leu
Lys Gln Thr Glu Ile Phe Ser His Phe Met Thr Asn Gln 50 55 60 Gly
Lys Ser Asn Ser Pro Ala Lys Pro Lys Val Gly Arg Pro Arg Lys 65 70
75 80 Glu Thr Asn Lys Leu Ala Pro Ala Gly Gly Asp Gly Ser Ala Asp
His 85 90 95 Arg His Arg Met Thr Glu Gln Glu Glu Asp Glu Glu Leu
Leu Ala Glu 100 105 110 Ser Asn Thr Ser Ser Lys Ser Leu Ala Arg Phe
Asp Ala Ser Pro Phe 115 120 125 Tyr Ile Lys Ser Gly Glu Leu Arg Asp
Tyr Gln Ile Arg Gly Leu Asn 130 135 140 Trp Met Ile Ser Leu Tyr Glu
His Gly Ile Asn Gly Ile Leu Ala Asp 145 150 155 160 Glu Met Gly Leu
Gly Lys Thr Leu Gln Thr Ile Ser Leu Leu Gly Tyr 165 170 175 Met Lys
His Tyr Arg Asn Ile Pro Gly Pro His Met Val Ile Val Pro 180 185 190
Lys Ser Thr Leu Ala Asn Trp Met Asn Glu Phe Lys Lys Trp Cys Pro 195
200 205 Thr Leu Arg Ala Val Cys Leu Ile Gly Asp Gln Glu Thr Arg Asn
Ala 210 215 220 Phe Ile Arg Asp Thr Leu Met Pro Gly Glu Trp Asp Val
Cys Val Thr 225 230 235 240 Ser Tyr Glu Met Ile Ile Arg Glu Lys Ser
Val Phe Lys Lys Phe Asn 245 250 255 Trp Arg Tyr Met Val Ile Asp Glu
Ala His Arg Ile Lys Asn Glu Lys 260 265 270 Ser Lys Leu Ser Glu Ile
Val Arg Glu Phe Lys Thr Thr Asn Arg Leu 275 280 285 Leu Leu Thr Gly
Thr Pro Leu Gln Asn Asn Leu His Glu Leu Trp Ser 290 295 300 Leu Leu
Asn Phe Leu Leu Pro Asp Val Phe Asn Ser Ser Asp Asp Phe 305 310 315
320 Asp Ser Trp Phe Asn Thr Asn Thr Phe Leu Gly Asp Asn Ser Leu Val
325 330 335 Glu Arg Leu His Ala Val Leu Arg Pro Phe Leu Leu Arg Arg
Leu Lys 340 345 350 Ser Glu Val Glu Lys Lys Leu Lys Pro Lys Lys Glu
Val Lys Ile Tyr 355 360 365 Val Gly Leu Ser Lys Met Gln Arg Glu Trp
Tyr Thr Lys Val Leu Met 370 375 380 Lys Asp Ile Asp Ile Val Asn Gly
Ala Gly Arg Val Glu Lys Met Arg 385 390 395 400 Leu Gln Asn Ile Leu
Met Gln Leu Arg Lys Cys Ser Asn His Pro Tyr 405 410 415 Leu Phe Asp
Gly Ala Glu Pro Gly Pro Pro Tyr Ser Thr Asp Glu His 420 425 430 Leu
Val Tyr Asn Ser Gly Lys Met Val Ile Leu Asp Lys Leu Leu Pro 435 440
445 Lys Leu Gln Glu Gln Gly Ser Arg Val Leu Val Phe Ser Gln Met Thr
450 455 460 Arg Met Ile Asp Ile Leu Glu Asp Tyr Cys Tyr Trp Arg Gly
Tyr Asn 465 470 475 480 Tyr Cys Arg Leu Asp Gly Asn Thr Pro His Glu
Asp Arg Gln Arg Gln 485 490 495 Ile Asn Glu Phe Asn Glu Glu Asp Ser
Lys Lys Phe Ile Phe Met Leu 500 505 510 Ser Thr Arg Ala Gly Gly Leu
Gly Ile Asn Leu Ala Thr Ala Asp Val 515 520 525 Val Ile Leu Tyr Asp
Ser Asp Trp Asn Pro Gln Met Asp Leu Gln Ala 530 535 540 Met Asp Arg
Ala His Arg Ile Gly Gln Lys Lys Gln Val Lys Val Phe 545 550 555 560
Arg Met Ile Thr Glu Asn Thr Val Glu Glu Lys Ile Val Glu Arg Ala 565
570 575 Glu Ile Lys Leu Arg Leu Asp Lys Leu Val Ile Gln Gln Gly Arg
Leu 580 585 590 Val Asp Asn Lys Thr Ala Leu Asn Lys Asp Glu Met Leu
Asn Met Ile 595 600 605 Arg His Gly Ala Asn His Val Phe Ala Ser Lys
Asp Ser Glu Ile Thr 610 615 620 Asp Glu Asp Ile Asp Thr Ile Leu Glu
Lys Gly Glu Ala Arg Thr Glu 625 630 635 640 Glu Met Asn Lys Lys Leu
Glu Gln Leu Gly Asp Ser Asn Leu Lys Asp 645 650 655 Phe Met Met Glu
Thr Pro Thr Glu Ser Val Tyr Gln Phe Glu Gly Glu 660 665 670 Asp Tyr
Arg Glu Lys Gln Lys Val Leu Gly Ile Gly Ser Trp Ile Glu 675 680 685
Pro Pro Lys Arg Glu Arg Lys Ala Asn Tyr Ala Val Asp Ala Tyr Phe 690
695 700 Arg Glu Ala Leu Arg Val Ser Glu Pro Lys Ala Pro Lys Ala Pro
Arg 705 710 715 720 Pro Pro Lys Gln Pro Ile Val Gln Asp Phe Gln Phe
Phe Pro Pro Arg 725 730 735 Leu Phe Glu Leu Leu Asp Gln Glu Ile Tyr
Tyr Phe Arg Lys Thr Val 740 745 750 Gly Tyr Lys Val Pro Lys Asn Pro
Glu Leu Gly Ser Asp Ala Ser Arg 755 760 765 Val Gln Lys Glu Glu Gln
Arg Lys Ile Asp Glu Ala Glu Pro Leu Ser 770 775 780 Glu Glu Glu Leu
Ala Glu Lys Glu Lys Leu Leu Thr Gln Gly Phe Thr 785 790 795 800 Asn
Trp Thr Lys Arg Asp Phe Asn Gln Phe Ile Lys Ala Asn Glu Lys 805 810
815 Tyr Gly Arg Asp Asp Ile Asp Asn Ile Ser Lys Glu Val Glu Gly Lys
820 825 830 Thr Pro Glu Glu Val Arg Ala Tyr Ser Glu Val Phe Trp Glu
Arg Cys 835 840 845 Asn Glu Leu Gln Asp Ile Asp Arg Ile Met Gly Gln
Ile Asp Arg Gly 850 855 860 Glu Ala Lys Ile Gln Arg Arg Ala Ser Ile
Lys Lys Ala Leu Asp Thr 865 870 875 880 Lys Met Ser Arg Tyr Arg Ala
Pro Phe His Gln Leu Arg Ile Ser Tyr 885 890 895 Gly Thr Asn Lys Gly
Lys Asn Tyr Thr Glu Glu Glu Asp Arg Phe Leu 900
905 910 Val Cys Met Leu His Lys Leu Gly Phe Asp Lys Glu Asn Val Tyr
Glu 915 920 925 Glu Leu Arg Ala Met Val Arg Cys Ala Pro Gln Phe Arg
Phe Asp Trp 930 935 940 Phe Ile Lys Ser Arg Thr Ala Met Glu Leu Gln
Arg Arg Cys Asn Thr 945 950 955 960 Leu Ile Thr Leu Ile Glu Arg Glu
Asn Gln Glu Leu Glu Glu Arg Glu 965 970 975 Arg Ala Glu Lys Arg Lys
Gly Arg Gly Ser Gly Arg Gly Pro Gly Ser 980 985 990 Gly Lys Arg Lys
Gly Asp Gly Ser Ile Ser Ser Pro Pro Pro Val Pro 995 1000 1005 Gly
Gln Gly Asp Lys Asn Ser Pro Ala Arg Lys Lys Lys Lys Met 1010 1015
1020 121316DNAEuchistus heros 12aatgaataaa aaacttgaac aacttggtgt
tgattcatca ttaaaagatt tcatgatgga 60ggctcccact gagtctgtct atcagtttga
aggcgaagat tatagagaaa agcaaaaagt 120ttttggaatt ggaaattgga
ttgaaccacc aaaacgagaa cgtaaagcaa attatgcagt 180agatgcctat
tttagagaag cactgagagt ttcagaacct aaagctccaa aggcccctag
240gccaccaaag caacccatag ttcaagattt ccaatttttc ccacctcgtc
tgtttgagct 300gttagatcaa gaaatatact attttcgaaa aactgtttgc
tacaaggttc ctaaaaatcc 360ggagttagga tcagatgctt ctcgtataca
aagggaagag caaagaaaaa ttgatgaagc 420tgagccgttg actgaggaag
agctagctga gaaagaaaac ttattgaccc agggttttac 480taattggact
aaaagagatt ttaaccagtt cataaaagct aatgaaaaat atggacgtga
540tgatattgat aatatctcaa aagatgttga agggaagact ccagaagaag
tacgagcata 600ctctgaagta ttttgggaaa ggtgcaatga actacaggcc
atagatcgta tcatggggca 660gattgataga ggtgaagcga aaattcaaag
aagagccagt attaaaaaag ctttagatac 720aaagatgagt cgatatagag
caccgtttca tcaactacga attgcttatg gtacgaacaa 780ggggaaaaat
tacacagaag aagaagacag attccttgtg tgcatgctac ataagcttgg
840ctttgataaa gaaaatgtgt atgaggaact tagggcgatg gtgaggtgtg
ctcctcagtt 900taggtttgat tggttcatca agtctcgaac agctttggaa
ttgcaaagac gttgtaatac 960tctaatcacg ttaattgaaa gggaaaacca
agaattagaa gaaagggaaa aagtagaaaa 1020aaggaaaagt cgaggcagta
atgggcgtgg tcccagttct ggtaaacgta agggagatgg 1080atctatttca
tctccacctg tctctgtaca gagtgataaa agcagccctg ctcggaaaaa
1140gaaaaagtat atctctgttg agtaaattta tcttaaaact gggagtagat
acccaattct 1200cattatcggg tgatcaagga atcaatctca tataggagcc
taaaacttca ttagtttgta 1260attgaatatt taatttacat ctctagtttc
caaatattgt ttcttttaca tctgta 131613387PRTEuchistus heros 13Met Asn
Lys Lys Leu Glu Gln Leu Gly Val Asp Ser Ser Leu Lys Asp 1 5 10 15
Phe Met Met Glu Ala Pro Thr Glu Ser Val Tyr Gln Phe Glu Gly Glu 20
25 30 Asp Tyr Arg Glu Lys Gln Lys Val Phe Gly Ile Gly Asn Trp Ile
Glu 35 40 45 Pro Pro Lys Arg Glu Arg Lys Ala Asn Tyr Ala Val Asp
Ala Tyr Phe 50 55 60 Arg Glu Ala Leu Arg Val Ser Glu Pro Lys Ala
Pro Lys Ala Pro Arg 65 70 75 80 Pro Pro Lys Gln Pro Ile Val Gln Asp
Phe Gln Phe Phe Pro Pro Arg 85 90 95 Leu Phe Glu Leu Leu Asp Gln
Glu Ile Tyr Tyr Phe Arg Lys Thr Val 100 105 110 Cys Tyr Lys Val Pro
Lys Asn Pro Glu Leu Gly Ser Asp Ala Ser Arg 115 120 125 Ile Gln Arg
Glu Glu Gln Arg Lys Ile Asp Glu Ala Glu Pro Leu Thr 130 135 140 Glu
Glu Glu Leu Ala Glu Lys Glu Asn Leu Leu Thr Gln Gly Phe Thr 145 150
155 160 Asn Trp Thr Lys Arg Asp Phe Asn Gln Phe Ile Lys Ala Asn Glu
Lys 165 170 175 Tyr Gly Arg Asp Asp Ile Asp Asn Ile Ser Lys Asp Val
Glu Gly Lys 180 185 190 Thr Pro Glu Glu Val Arg Ala Tyr Ser Glu Val
Phe Trp Glu Arg Cys 195 200 205 Asn Glu Leu Gln Ala Ile Asp Arg Ile
Met Gly Gln Ile Asp Arg Gly 210 215 220 Glu Ala Lys Ile Gln Arg Arg
Ala Ser Ile Lys Lys Ala Leu Asp Thr 225 230 235 240 Lys Met Ser Arg
Tyr Arg Ala Pro Phe His Gln Leu Arg Ile Ala Tyr 245 250 255 Gly Thr
Asn Lys Gly Lys Asn Tyr Thr Glu Glu Glu Asp Arg Phe Leu 260 265 270
Val Cys Met Leu His Lys Leu Gly Phe Asp Lys Glu Asn Val Tyr Glu 275
280 285 Glu Leu Arg Ala Met Val Arg Cys Ala Pro Gln Phe Arg Phe Asp
Trp 290 295 300 Phe Ile Lys Ser Arg Thr Ala Leu Glu Leu Gln Arg Arg
Cys Asn Thr 305 310 315 320 Leu Ile Thr Leu Ile Glu Arg Glu Asn Gln
Glu Leu Glu Glu Arg Glu 325 330 335 Lys Val Glu Lys Arg Lys Ser Arg
Gly Ser Asn Gly Arg Gly Pro Ser 340 345 350 Ser Gly Lys Arg Lys Gly
Asp Gly Ser Ile Ser Ser Pro Pro Val Ser 355 360 365 Val Gln Ser Asp
Lys Ser Ser Pro Ala Arg Lys Lys Lys Lys Tyr Ile 370 375 380 Ser Val
Glu 385 141827DNAEuchistus heros 14gataaatatg aataagaaaa ttttaaattt
atttgtttca ttaaaaaatt atcttatggg 60tttattgatt ataaattggt tcaatcataa
aatacgagat acataagatt gtattatcat 120aacaaaccca atctctagta
tcgtcatcct gctgttctgg ttcactctga gtttctttat 180cttcatcaaa
agcaaaactt gcaactttaa aagcagaaag taattcatca ccaacagtgg
240ctggtccttc atctctggtt tcagctcttc tcaaaatttc gtcaatgtca
caagttggtt 300cttcatcacc atcttcttca tctttaaata attcttcagc
cccaaatttt aaaatagcag 360taagttcttc tttgttaaaa ggcgcactgg
atgaagaatt ttttttatcc aggacagttc 420tacctgtagt atccattctt
tgtataacta aatgatctaa gaccattttt tgtttggccc 480gctcgacaat
attttcctca acagaacttt tagtaacaag tctgtatatg ttcacctgat
540ttttctgacc gattctatga gctctagctt gtgcttgcaa atcattttgt
ggattccaat 600cagagtcaaa tataatgaca gtatcagctg ttgctaaatt
aatgcccaaa ccaccagcac 660gagttgataa taagaaacag aaatctggtg
aattttcagc attgaaatga tcgagggctt 720gctttctcaa ttcaccttta
attgaaccgt ctaaacgttg gaaagggaaa tgtctcattt 780gaagatactc
agccagtata tccaacattc gtaccatttg agaaaatata agtactctat
840gcccagtttc tttaaggcga acaagcaact tgtccaacag aagtaatttc
cctgagcctt 900ttaacaattg ctgtaagtag tcttcagttt ttgcttcatt
ttctaatggt tttattagat 960gtgcatgatt acagcatttt tttaattcaa
taacaatatt tataaatgta ctaggagaac 1020ctttgactcc ttttcgaaga
gcagaataat ttttggacaa aatccacctg taatactgct 1080tctgtacaga
tgtcatttca acacgtaata tttgttccac tttagctggt aaagatttct
1140caacatcctt cttaactcgt cgtagaatat atggttccag ctgtctgtgc
aacttagtat 1200agcctttatt agcagagttg tcatgttctt tttcaaattc
ttcccagtta ttaaatctgt 1260tgggcataat aaagtgaagc aacgcccaaa
gctctttaag actattttgc aaaggagtgc 1320ctgttataag aagcctatgg
ttggtatcaa actctttcaa tgttttgtat aataatgaat 1380catcattttt
caatctgtgt gcttcatcaa ccataaggat agcccagctt atactaccca
1440aaaatgcttt gtctttaaga acaatttcat atgtagtaag aatggcattg
aattttaacc 1500ttttcgaacc tgaatagcac cattcataat tacgtataac
atcacgggag tttatatcac 1560caatataagt tacaacattc atttctggag
cccataatga aaactccctc tgccatgaag 1620tcatcgtaga taaagggaca
acaattaaaa atggtccata caactggtga gtatgaaata 1680aataatacaa
actgcagata gtctgaatag ttttaccaag acccatttca tcagccaaaa
1740taatagaatt ttctttacac cacgaatgaa ccaaccaatt caaaccactg
atttgataat 1800ctctcaaaac caatacctgg tcaccac 1827151454PRTEuchistus
heros 15Met Pro Gln Lys Asp Gly Ser Glu Asp Ser Ala Ser Glu Ser Asp
Lys 1 5 10 15 Asp Gln Gly Asn Gln Glu Glu Ser Asp Asn Ser Ser Ser
Glu Ser Gly 20 25 30 Ser Gly Ser Glu Ser Asp Ser Ala Ser Ser Ala
Ser Ser Ser Ser Lys 35 40 45 Ser Ser Asp Ser Gly Ser Asp Tyr Lys
Ser Lys Thr Ser Asn Ser Ser 50 55 60 Arg Gly Lys Asn Asp Ile Lys
Gln Tyr Trp Glu Glu Asn Pro Asp Val 65 70 75 80 Tyr Gly Ile Arg Arg
Ser Asn Arg Gln Arg Lys Glu Pro Ser Arg Leu 85 90 95 Asn Thr Gly
Asp Ser Asp Ser Ser Glu Lys Thr Lys Arg Ser Val Lys 100 105 110 Arg
Ser Ser Pro Lys Ser Trp Asn Ser Asp Thr Ser Tyr Asp Ser Glu 115 120
125 Thr Asp Lys Glu Ser Lys Arg Pro Pro Pro Ser Lys Pro Pro Gly Gly
130 135 140 Arg Arg Arg Pro Ala Lys Thr Thr Arg Lys Pro Lys Ser Arg
Ile Arg 145 150 155 160 Asn Arg Ala Tyr Ser Asp Ser Ser Glu Ser Ser
Tyr Glu Ser Glu Asp 165 170 175 Asp Asn Asn Arg Arg Thr Lys Ser Arg
Arg Gly Val Thr Ser Val Ser 180 185 190 Tyr Lys Glu Ala Ser Asp Glu
Lys Thr Asp Ser Asp Glu Leu Leu Glu 195 200 205 Pro Asp Pro Glu Pro
Val Glu Pro Ala Pro Pro Asp Thr Ser Glu Thr 210 215 220 Ile Glu Lys
Val Leu Ala Gln Arg Ile Gly Lys Lys Gly Val Val Gly 225 230 235 240
Asn Gln Thr Thr Val Tyr Ala Val Glu Glu Asn Gly Asp Pro Asn Ser 245
250 255 Asn Tyr Glu Ser Leu Asp Lys Asp Glu Thr Glu Val Gln Tyr Leu
Ile 260 265 270 Lys Trp Lys Gly Trp Ser His Ile His Asn Thr Trp Glu
Ser Glu Leu 275 280 285 Ser Leu Lys Glu Gln Lys Val Lys Gly Val Lys
Lys Leu Glu Asn Phe 290 295 300 Val Lys Arg Glu Glu Asp Ile Arg Phe
Trp Lys Glu His Thr Thr Pro 305 310 315 320 Glu Asp Ile Glu Tyr Tyr
Glu Cys Gln Leu Glu Leu Gln Gln Glu Leu 325 330 335 Leu Lys Ser Tyr
Asn Arg Val Glu Arg Ile Ile Ala Val Ser Lys Thr 340 345 350 Asp Gly
Gln Val Glu Tyr Tyr Val Lys Trp Glu Ser Leu Pro Tyr Ser 355 360 365
Glu Ala Thr Trp Glu Asp Ser Gly Leu Ile Glu Lys Lys Trp Pro Lys 370
375 380 Lys Ile Lys Glu Phe Lys Glu Arg Glu Asp Ser Lys Arg Thr Pro
Ser 385 390 395 400 Lys Leu Cys Arg Val Leu Lys Ala Arg Pro Lys Phe
Ile Lys Ile Glu 405 410 415 Asp Gln Pro Glu Tyr Met Gly Gly Asp Gln
Val Leu Val Leu Arg Asp 420 425 430 Tyr Gln Ile Ser Gly Leu Asn Trp
Leu Val His Ser Trp Cys Lys Glu 435 440 445 Asn Ser Ile Ile Leu Ala
Asp Glu Met Gly Leu Gly Lys Thr Ile Gln 450 455 460 Thr Ile Cys Ser
Leu Tyr Tyr Leu Phe His Thr His Gln Leu Tyr Gly 465 470 475 480 Pro
Phe Leu Ile Val Val Pro Leu Ser Thr Met Thr Ser Trp Gln Arg 485 490
495 Glu Phe Ser Leu Trp Ala Pro Glu Met Asn Val Val Thr Tyr Ile Gly
500 505 510 Asp Ile Asn Ser Arg Asp Val Ile Arg Asn Tyr Glu Trp Cys
Tyr Ser 515 520 525 Gly Ser Lys Arg Leu Lys Phe Asn Ala Ile Leu Thr
Thr Tyr Glu Ile 530 535 540 Val Leu Lys Asp Lys Ala Phe Leu Gly Ser
Ile Ser Trp Ala Ile Leu 545 550 555 560 Met Val Asp Glu Ala His Arg
Leu Lys Asn Asp Asp Ser Leu Leu Tyr 565 570 575 Lys Thr Leu Lys Glu
Phe Asp Thr Asn His Arg Leu Leu Ile Thr Gly 580 585 590 Thr Pro Leu
Gln Asn Ser Leu Lys Glu Leu Trp Ala Leu Leu His Phe 595 600 605 Ile
Met Pro Asn Arg Phe Asn Asn Trp Glu Glu Phe Glu Lys Glu His 610 615
620 Asp Asn Ser Ala Asn Lys Gly Tyr Thr Lys Leu His Arg Gln Leu Glu
625 630 635 640 Pro Tyr Ile Leu Arg Arg Val Lys Lys Asp Val Glu Lys
Ser Leu Pro 645 650 655 Ala Lys Val Glu Gln Ile Leu Arg Val Glu Met
Thr Ser Val Gln Lys 660 665 670 Gln Tyr Tyr Arg Trp Ile Leu Ser Lys
Asn Tyr Ser Ala Leu Arg Lys 675 680 685 Gly Val Lys Gly Ser Pro Ser
Thr Phe Ile Asn Ile Val Ile Glu Leu 690 695 700 Lys Lys Cys Cys Asn
His Ala His Leu Ile Lys Pro Leu Glu Asn Glu 705 710 715 720 Ala Lys
Thr Glu Asp Tyr Leu Gln Gln Leu Leu Lys Gly Ser Gly Lys 725 730 735
Leu Leu Leu Leu Asp Lys Leu Leu Val Arg Leu Lys Glu Thr Gly His 740
745 750 Arg Val Leu Ile Phe Ser Gln Met Val Arg Met Leu Asp Ile Leu
Ala 755 760 765 Glu Tyr Leu Gln Met Arg His Phe Pro Phe Gln Arg Leu
Asp Gly Ser 770 775 780 Ile Lys Gly Glu Leu Arg Lys Gln Ala Leu Asp
His Phe Asn Ala Glu 785 790 795 800 Asn Ser Pro Asp Phe Cys Phe Leu
Leu Ser Thr Arg Ala Gly Gly Leu 805 810 815 Gly Ile Asn Leu Ala Thr
Ala Asp Thr Val Ile Ile Phe Asp Ser Asp 820 825 830 Trp Asn Pro Gln
Asn Asp Leu Gln Ala Gln Ala Arg Ala His Arg Ile 835 840 845 Gly Gln
Lys Asn Gln Val Asn Ile Tyr Arg Leu Val Thr Lys Ser Ser 850 855 860
Val Glu Glu Asn Ile Val Glu Arg Ala Lys Gln Lys Met Val Leu Asp 865
870 875 880 His Leu Val Ile Gln Arg Met Asp Thr Thr Gly Arg Thr Val
Leu Asp 885 890 895 Lys Lys Asn Ser Ser Ser Ser Ala Pro Phe Asn Lys
Glu Glu Leu Thr 900 905 910 Ala Ile Leu Lys Phe Gly Ala Glu Glu Leu
Phe Lys Asp Glu Glu Asp 915 920 925 Gly Asp Glu Glu Pro Thr Cys Asp
Ile Asp Glu Ile Leu Arg Arg Ala 930 935 940 Glu Thr Arg Asp Glu Gly
Pro Ala Thr Val Gly Asp Glu Leu Leu Ser 945 950 955 960 Ala Phe Lys
Val Ala Ser Phe Ala Phe Asp Glu Asp Lys Glu Thr Gln 965 970 975 Ser
Glu Pro Glu Gln Gln Asp Asp Asp Thr Arg Asp Trp Asp Glu Ile 980 985
990 Ile Pro Glu Thr Tyr Arg Gln Lys Val Glu Glu Glu Glu Arg Ala Lys
995 1000 1005 Glu Met Glu Asp Leu Tyr Leu Pro Pro Arg Ser Arg Lys
Thr Leu 1010 1015 1020 Gln Gln Ile Asn His Ser Glu Ser Asp Ala Asp
Gly Lys Ala Asn 1025 1030 1035 Lys Lys Lys Arg Lys Lys Gly Glu Glu
Asn Glu Thr Thr Glu Glu 1040 1045 1050 Gly Ser Asp Glu Glu Lys Pro
Arg Lys Arg Gly Arg Pro Arg Gly 1055 1060 1065 Asn Lys Gly Ser Ser
Lys Glu Val Ile Lys Gly Phe Asn Asp Ala 1070 1075 1080 Glu Ile Arg
Arg Phe Ile Arg Ser Phe Lys Lys Phe Pro Ala Pro 1085 1090 1095 Leu
Lys Arg Leu Asp Ala Ile Ala Cys Asp Ala Glu Leu Gln Glu 1100 1105
1110 Lys Pro Leu Ala Glu Leu Arg Lys Leu Gly Asp Met Leu Lys Gln
1115 1120 1125 Arg Cys Lys Ala Cys Leu Gly Asp Gln Thr Lys Glu Asn
Leu Thr 1130 1135 1140 Asp Ala Asn Glu Glu Asn Thr Gly Thr Ser Gly
Arg Lys Arg Gly 1145 1150 1155 Arg Gly Pro Ser Ala Lys Leu Gly Gly
Val Ser Val Asn Ala Lys 1160 1165 1170 Ser Leu Leu Ala Cys Glu Lys
Glu Leu Glu Pro Leu Asp Ile Glu 1175 1180 1185 Ile Pro Leu Asp Pro
Asn Glu Arg Asn Lys Trp Val Leu Asp Val 1190 1195 1200 Arg Val Lys
Pro Ala Asn Phe Asp Cys Asp Trp Asp Val Asn Asp 1205 1210 1215 Asp
Ser Ala Leu Leu Arg Gly Val Tyr Gln Tyr Gly Met Gly Ser 1220 1225
1230 Trp Glu Ala Ile Lys Met Asp Pro Ser Ile Gly Ile Ser Asp Lys
1235 1240 1245 Ile Leu Ser Asn Asn Gly Ser Lys Pro Gln Thr Lys His
Leu Ala 1250 1255 1260 Ser Arg Ala Glu Tyr Leu Leu Lys Val Leu Lys
Lys Ser Ile Asp 1265 1270 1275 Gln Arg Gln Gly Ser Thr Val Lys Thr
Lys Arg Gln Arg Lys Arg 1280
1285 1290 Asp Asn Lys Ala Thr Ser Arg Glu Ile Ile Glu Asp Lys Asp
Asp 1295 1300 1305 Ser Ser Gly Gly Glu Leu Pro Ala Glu Ser Val Ser
Thr Pro Ser 1310 1315 1320 Gln Asp Ser Phe Asn His Lys Asp Ile Lys
Leu Glu Glu Asn Glu 1325 1330 1335 Glu Asp Lys Lys Lys Gly Lys Lys
Lys Glu Thr Gln Lys Lys Lys 1340 1345 1350 Lys Lys Asn Glu Ser Gly
Pro Met His Phe Thr Ala Asn Ser Glu 1355 1360 1365 Pro Arg Ala Leu
Asp Val Leu Gly Asp Leu Glu Pro Ser Ile Phe 1370 1375 1380 Asn Glu
Cys Lys Glu Lys Met Arg Pro Val Lys Lys Ala Leu Lys 1385 1390 1395
Ala Leu Asp Asn Pro Asp Gln Ser Leu Gly Pro Gln Glu Gln Val 1400
1405 1410 Asn His Thr Arg Gln Cys Leu Val Gln Ile Gly Asp Gln Ile
Asn 1415 1420 1425 Lys Cys Leu Met Glu Tyr Lys Glu Ser Asp Ile Ile
Lys Gln Trp 1430 1435 1440 Arg Arg Cys Val Ser Ser Asn Phe Val Ile
Val 1445 1450 16496DNAEuchistus heros 16gactacctcg agggtgaagg
ttataaatat gaacgtattg acggtacgat caccggtagc 60ttaagacaag aagctatcga
tcggtttaac gcccctggag ctcaacaatt tgtttttctt 120ttgtccactc
gtgcgggagg tcttggtatt aatctcgcta ctgcagatac agttattatt
180tatgactctg actggaatcc tcataacgat attcaggcct tttcgagagc
acacaggata 240gggcaagcaa acaaggttat gatttatcga tttgtgacac
gagcgtctgt tgaagaaaga 300gtaacgcaag tggctaagag aaaaatgatg
ttaacccatc ttgtcgtacg accaggtatg 360ggtggcaagc aagcaaattt
cactaagcaa gaacttgatg atattttaag gtttggaaca 420gaagaacttt
tcaaagaaga gcagggtaaa gaagatgaag ccattcatta tgacgataaa
480gctgttgaag aattac 49617481DNAEuchistus heros 17caaaaattga
aactgaccgt tctagaagat ttgagtttct gttgaagcag acagaaattt 60tttcacattt
tatgacaaat caaggaaagt cgaacagccc tgcaaagcct aaagtcggcc
120gtcctagaaa ggaaactaat aaattggcac cagccggtgg tgatggttct
gccgaccatc 180ggcatcgtat gaccgagcag gaagaagatg aagaactgct
tgctgaaagt aatacttctt 240caaaatcctt agcaaggttt gacgcttctc
ctttttatat taaaagcgga gagttgaggg 300attaccagat acgtggtttg
aattggatga tatccctcta cgaacacggt ataaatggta 360tacttgctga
tgagatgggt ttaggtaaaa ctctccaaac tatttctctc cttggttaca
420tgaagcatta tagaaatata ccagggccac atatggtcat cgtaccaaaa
tcaacattag 480c 48118490DNAEuchistus heros 18gttcaagatt tccaattttt
cccacctcgt ctgtttgagc tgttagatca agaaatatac 60tattttcgaa aaactgtttg
ctacaaggtt cctaaaaatc cggagttagg atcagatgct 120tctcgtatac
aaagggaaga gcaaagaaaa attgatgaag ctgagccgtt gactgaggaa
180gagctagctg agaaagaaaa cttattgacc cagggtttta ctaattggac
taaaagagat 240tttaaccagt tcataaaagc taatgaaaaa tatggacgtg
atgatattga taatatctca 300aaagatgttg aagggaagac tccagaagaa
gtacgagcat actctgaagt attttgggaa 360aggtgcaatg aactacaggc
catagatcgt atcatggggc agattgatag aggtgaagcg 420aaaattcaaa
gaagagccag tattaaaaaa gctttagata caaagatgag tcgatataga
480gcaccgtttc 49019496DNAEuchistus heros 19cagctggaac catatattct
acgacgagtt aagaaggatg ttgagaaatc tttaccagct 60aaagtggaac aaatattacg
tgttgaaatg acatctgtac agaagcagta ttacaggtgg 120attttgtcca
aaaattattc tgctcttcga aaaggagtca aaggttctcc tagtacattt
180ataaatattg ttattgaatt aaaaaaatgc tgtaatcatg cacatctaat
aaaaccatta 240gaaaatgaag caaaaactga agactactta cagcaattgt
taaaaggctc agggaaatta 300cttctgttgg acaagttgct tgttcgcctt
aaagaaactg ggcatagagt acttatattt 360tctcaaatgg tacgaatgtt
ggatatactg gctgagtatc ttcaaatgag acatttccct 420ttccaacgtt
tagacggttc aattaaaggt gaattgagaa agcaagccct cgatcatttc
480aatgctgaaa attcac 4962038DNAArtificial SequencePrimer Mi2.T7.F
20taatacgact cactataggg aagaaggcat agaacaga 382139DNAArtificial
SequencePrimer Mi2.T7.R 21taatacgact cactataggg tcagaatggt
aatcagaga 392238DNAArtificial SequencePrimer ISWI30.T7.F
22taatacgact cactataggg tgaatcagtc taccaatt 382338DNAArtificial
SequencePrimer ISWI30.T7.R 23taatacgact cactataggg ggttctgact
catctatt 382439DNAArtificial SequencePrimer ISWI2.T7.F 24taatacgact
cactataggg ttgctcaatc ctacataca 392538DNAArtificial SequencePrimer
ISWI2.T7.R 25taatacgact cactataggg gaataccaac aggctact
382638DNAArtificial SequencePrimer KSMT.T7.F 26taatacgact
cactataggg gatcaaattc aagcaact 382738DNAArtificial SequencePrimer
KSMT.T7.R 27taatacgact cactataggg ttcttcctaa accatgtt
382838DNAArtificial SequencePrimer CHD1.T7.F 28taatacgact
cactataggg tttgcttcct tctttcaa 382939DNAArtificial SequencePrimer
CHD1.T7.R 29taatacgact cactataggg cttctttgtt aaacggatt
39304493DNAEuchistus heros 30gcatatacag aaacaatcaa aagatcaaat
ctaactcgtt caaacagcag gatgaagaaa 60actaggaaga attctcgacc tttatttgtg
gctggtggtt ttgcagcggc agctgagaag 120atgcctgagt aggcctgatt
gagagtgcag gtacggatgg ctttcttccc ggtcctctca 180gtcctctggg
gccacgtgag ccaatatgac cagggccgac gggcctcaac cgtggcctcc
240ctggacctcg acgattgctt cggttgctta aagcaggcat gaaaggcctc
gaaatttttg 300gtggtgtctg gctgataggt tcctcctggt cggtgttgtt
atcattggag tcttggctcg 360atttcataac gtccgtgccc ttcggactct
cagctggtga tttcacacct tggtaaactt 420caacgacccc atcttcagtc
ttcatatgtt tcaccgtgtc cttgtaattt cctagagcat 480tttttctttt
tctttctctt tccctgtaga tatcttgcct tgattcttca agttgtctcc
540tctctgcttg cttctgccga tatttctgtt ctatttctgc atcatcaagc
aatagagaaa 600caacctcttt cggtttaaga gtgtctggtt tgaaattacc
cccactgatg acaacccttt 660ggatttcact tttttcttga gctctctgca
agatcctttc ttcgatggtc cctttacaaa 720tgagccgata gactgtgact
tgtttcgttt gtccaagacg atgggcacgg tccatcgctt 780gttggtcaac
agtagggttc caatcactgt catagaatat cacagtatca gctgcggtaa
840ggttgattcc aagacctcca gctcgcgtac tcaacaggaa tacaaatatg
tcctctcttg 900tttgaaagtc agcaaccata tcccttctgt ctgatatctt
tgaagaacca tctaacctca 960tatatgtgtg ctttctgtac cacatatatt
cctctaacaa gtcgatcatc cttgtcatct 1020gggagtaaat tagagctcga
tgcccttgtt ccttcagcct ggtgagaagc ccatccaaga 1080cgtacagctt
tccagcatca gttaccagtg tctgtttgtc aggtatgact atactcgacc
1140agccagtgat tggtcgaaga ctgagtaggc caagaggagg tgggcactgg
aacccaggtt 1200cttgtttatc cccttcccaa agaacctgcc acagtctccc
gtcctccccc cagaggtgag 1260aagagatcca gctccacctt cggttcgagc
tgtacagccg tctgcttttc tttatctcca 1320ccttgacagt aggagtgaag
aggaatggag gtaaatagac tggctgacat gagagaatct 1380gaggcttcct
cacaatatgt ggcaatgctg gtaataagtt agcactatct tctccaccgc
1440actctccctt agccttcttc gacctgacta tgcgatgttc tatcgtttca
ggcatacttt 1500ctatcctcac ggtggaatgg gaaaacactt ggttggaata
agtggtgaac acaagattgt 1560ttgggtagct cctgtcttca atgattccga
cgagtttcct tctcactcct tgcctccttg 1620ctataatatt gtgccacctg
aaataaatcc caaaaaacat tagcttatac agttctccta 1680cagataacct
caacagtcga gtgaaagaaa atgaactatc cctctcggat gagtcatcac
1740tgaataaaga tctatgagaa tgaaaaggat taaaaggcga taagtagttc
atgagaagat 1800gaagtttatc cccagggaac acagcatctg tgatgagagc
aggcacaatg taatcttccg 1860tagccatgga aaaaggggat cgaggttccc
ttcgctcgaa tagttcaggg tgattacaga 1920ccttacgaaa ttgcatcacg
aggttcatca aatttgaagt gatactctga gctgattggt 1980aagaagatcc
agaagagtgt agcaaatctt caattcgaat cttctttttc acagctgaat
2040ataacatctt ctgcctcgtc gtcagaggac agtacaccat gatttctatt
ttatctgaca 2100gttcattctc cacatctgtt tttactctcc gcaacatgaa
tggttttagg atcatatgta 2160aacgggacaa atgcttttca tcaatactgg
ttttatgctc tgcatgactt tctatatctt 2220ttgaaaacca ttcattgaac
tcatcgtgtg aatcaaacat tgagggcatt atgaaatgaa 2280gaagagccca
aagttcagcc attgagtttt gaataggtgt tccactcaga agtaatctgt
2340tgcggcaatt gaatccaaga agcaatttcc aacgcatgct tgtagtgctt
ttgatagcct 2400gagcttcgtc tagaattaaa tactgccatt ttatcctatt
gaagtatttt atatcagtaa 2460ttacaagctg atagcttgtg atcacaacat
ggaaactggc atctttagta tgtaaacctt 2520tttgatccca aaattgacgt
aatattttcc tttcctgctg atttccccaa taaggcacaa 2580ctttgaaatc
aggtacaaaa cgctgcattt cttgctgcca attatgtaat gtagaagcgg
2640gcgatattat gagaaatgga ccccaaacag agtatttttc agcaatatgg
caaagaaagg 2700ctatcgattg gactgtcttt cccaatccca tttcatctgc
caagattcca ttaatacctt 2760ggtcatataa attcacaagc catgtcattc
cctttatttg atatcccttg agagtaccac 2820ggaatatctg cggttggggt
ttatcttcac caacatctcc atcctcttcc attttctggc 2880ttactccaaa
ttctcttgct cgtgcttctt ccagaaaaaa caccttttca actttctttc
2940gtactttttc tttctctgcc tcgcaatcgt agtcatccaa aggtaggagt
ctaggattag 3000cttcctcttc aagttggctc aggattcgaa gttgatcttc
agtcgttccg ccaccgagct 3060tacgggacat aaagtgagca tagagttctg
tttgagttat gaggaaattt aattttcttt 3120gctgcctctt agcctccatc
agttctacat ccaacttcct ttgttcctct gcttctttct 3180ccattcttct
tcttgtttcc ctttccaccc tctcaaatct tttccagtat acttgcattt
3240cccgtgtcaa tctttttgct ctccaaataa cctctttcat attcttttgc
gattgcattg 3300cacgttgtcg acagtgcctc atacagttag tagcagctcg
cctgcaagct gttagaattt 3360ctttatggtt acttatccta tagcgctgaa
cctttccaat ttcttttttc gccatgttgg 3420cccaaatttt acgcctgcga
tgtgccatga tttcggcagc tttgttttgg gctgattttt 3480ttctaaggct
catttccttt ttagaccttg acattatttg taggtctggt tcttctttga
3540tttttctttt ctttttttct acactaaaac tttcatgtgg ttgatcttgt
tttttcaact 3600tttctttttt ttgaaaaaca aatttctttt tcttaacaaa
accagaactt gaactctgat 3660gctcaggata cttatcgaaa ttggagagga
gtcctgtgcc atagtacata tactcttggt 3720ttttagaatt gtggtagaat
ttttttttat atttgtttct aagtacatgt tcacggagca 3780tgtcttgtaa
gtcctcttca gttatttctt catcatcaga ggaatctgat gtgtcagtca
3840atagaacatc aactaaccac tgcctatcta agcttacatt actcaagttg
taaagcctct 3900tcttgtcagc tatcctatct tctttagttg ctgttatacc
attccatacc gtttctccgg 3960tataggcatc aacaccagct aattccgtgt
cagaatcact ggaggcttct ccatcctcac 4020ctaatggttg ttttaaaaag
tcttcaacat atgttaggaa aggagctatg tccaagcttt 4080tttctagctt
ttgataataa agaggtttgg caatttctgt tttcactacc atgtttgttt
4140tatcatcact catactgcaa aaatcaatga catcaaaaga tatctctgca
tcccagttag 4200aaaatattac taaactgaaa tgtaaaactt atatagaatc
atatttaaaa tgagttgaac 4260aaactattac gcttgtcaca tttttagtaa
accacaccca aattaatatc tacttttata 4320cataaaccta atcagaatat
cagtcagtcc atactagacg attgtaaaaa tgtgctaggg 4380gtcaaataaa
aaaggaaagt gaaattaggt tagtatatat tgaaagacgc atctcctttt
4440cagagattca gtgaaatatt tcagccagct gggttagcct gacagaattc aag
4493311363PRTEuchistus heros 31Met Ser Asp Asp Lys Thr Asn Met Val
Val Lys Thr Glu Ile Ala Lys 1 5 10 15 Pro Leu Tyr Tyr Gln Lys Leu
Glu Lys Ser Leu Asp Ile Ala Pro Phe 20 25 30 Leu Thr Tyr Val Glu
Asp Phe Leu Lys Gln Pro Leu Gly Glu Asp Gly 35 40 45 Glu Ala Ser
Ser Asp Ser Asp Thr Glu Leu Ala Gly Val Asp Ala Tyr 50 55 60 Thr
Gly Glu Thr Val Trp Asn Gly Ile Thr Ala Thr Lys Glu Asp Arg 65 70
75 80 Ile Ala Asp Lys Lys Arg Leu Tyr Asn Leu Ser Asn Val Ser Leu
Asp 85 90 95 Arg Gln Trp Leu Val Asp Val Leu Leu Thr Asp Thr Ser
Asp Ser Ser 100 105 110 Asp Asp Glu Glu Ile Thr Glu Glu Asp Leu Gln
Asp Met Leu Arg Glu 115 120 125 His Val Leu Arg Asn Lys Tyr Lys Lys
Lys Phe Tyr His Asn Ser Lys 130 135 140 Asn Gln Glu Tyr Met Tyr Tyr
Gly Thr Gly Leu Leu Ser Asn Phe Asp 145 150 155 160 Lys Tyr Pro Glu
His Gln Ser Ser Ser Ser Gly Phe Val Lys Lys Lys 165 170 175 Lys Phe
Val Phe Gln Lys Lys Glu Lys Leu Lys Lys Gln Asp Gln Pro 180 185 190
His Glu Ser Phe Ser Val Glu Lys Lys Lys Arg Lys Ile Lys Glu Glu 195
200 205 Pro Asp Leu Gln Ile Met Ser Arg Ser Lys Lys Glu Met Ser Leu
Arg 210 215 220 Lys Lys Ser Ala Gln Asn Lys Ala Ala Glu Ile Met Ala
His Arg Arg 225 230 235 240 Arg Lys Ile Trp Ala Asn Met Ala Lys Lys
Glu Ile Gly Lys Val Gln 245 250 255 Arg Tyr Arg Ile Ser Asn His Lys
Glu Ile Leu Thr Ala Cys Arg Arg 260 265 270 Ala Ala Thr Asn Cys Met
Arg His Cys Arg Gln Arg Ala Met Gln Ser 275 280 285 Gln Lys Asn Met
Lys Glu Val Ile Trp Arg Ala Lys Arg Leu Thr Arg 290 295 300 Glu Met
Gln Val Tyr Trp Lys Arg Phe Glu Arg Val Glu Arg Glu Thr 305 310 315
320 Arg Arg Arg Met Glu Lys Glu Ala Glu Glu Gln Arg Lys Leu Asp Val
325 330 335 Glu Leu Met Glu Ala Lys Arg Gln Gln Arg Lys Leu Asn Phe
Leu Ile 340 345 350 Thr Gln Thr Glu Leu Tyr Ala His Phe Met Ser Arg
Lys Leu Gly Gly 355 360 365 Gly Thr Thr Glu Asp Gln Leu Arg Ile Leu
Ser Gln Leu Glu Glu Glu 370 375 380 Ala Asn Pro Arg Leu Leu Pro Leu
Asp Asp Tyr Asp Cys Glu Ala Glu 385 390 395 400 Lys Glu Lys Val Arg
Lys Lys Val Glu Lys Val Phe Phe Leu Glu Glu 405 410 415 Ala Arg Ala
Arg Glu Phe Gly Val Ser Gln Lys Met Glu Glu Asp Gly 420 425 430 Asp
Val Gly Glu Asp Lys Pro Gln Pro Gln Ile Phe Arg Gly Thr Leu 435 440
445 Lys Gly Tyr Gln Ile Lys Gly Met Thr Trp Leu Val Asn Leu Tyr Asp
450 455 460 Gln Gly Ile Asn Gly Ile Leu Ala Asp Glu Met Gly Leu Gly
Lys Thr 465 470 475 480 Val Gln Ser Ile Ala Phe Leu Cys His Ile Ala
Glu Lys Tyr Ser Val 485 490 495 Trp Gly Pro Phe Leu Ile Ile Ser Pro
Ala Ser Thr Leu His Asn Trp 500 505 510 Gln Gln Glu Met Gln Arg Phe
Val Pro Asp Phe Lys Val Val Pro Tyr 515 520 525 Trp Gly Asn Gln Gln
Glu Arg Lys Ile Leu Arg Gln Phe Trp Asp Gln 530 535 540 Lys Gly Leu
His Thr Lys Asp Ala Ser Phe His Val Val Ile Thr Ser 545 550 555 560
Tyr Gln Leu Val Ile Thr Asp Ile Lys Tyr Phe Asn Arg Ile Lys Trp 565
570 575 Gln Tyr Leu Ile Leu Asp Glu Ala Gln Ala Ile Lys Ser Thr Thr
Ser 580 585 590 Met Arg Trp Lys Leu Leu Leu Gly Phe Asn Cys Arg Asn
Arg Leu Leu 595 600 605 Leu Ser Gly Thr Pro Ile Gln Asn Ser Met Ala
Glu Leu Trp Ala Leu 610 615 620 Leu His Phe Ile Met Pro Ser Met Phe
Asp Ser His Asp Glu Phe Asn 625 630 635 640 Glu Trp Phe Ser Lys Asp
Ile Glu Ser His Ala Glu His Lys Thr Ser 645 650 655 Ile Asp Glu Lys
His Leu Ser Arg Leu His Met Ile Leu Lys Pro Phe 660 665 670 Met Leu
Arg Arg Val Lys Thr Asp Val Glu Asn Glu Leu Ser Asp Lys 675 680 685
Ile Glu Ile Met Val Tyr Cys Pro Leu Thr Thr Arg Gln Lys Met Leu 690
695 700 Tyr Ser Ala Val Lys Lys Lys Ile Arg Ile Glu Asp Leu Leu His
Ser 705 710 715 720 Ser Gly Ser Ser Tyr Gln Ser Ala Gln Ser Ile Thr
Ser Asn Leu Met 725 730 735 Asn Leu Val Met Gln Phe Arg Lys Val Cys
Asn His Pro Glu Leu Phe 740 745 750 Glu Arg Arg Glu Pro Arg Ser Pro
Phe Ser Met Ala Thr Glu Asp Tyr 755 760 765 Ile Val Pro Ala Leu Ile
Thr Asp Ala Val Phe Pro Gly Asp Lys Leu 770 775 780 His Leu Leu Met
Asn Tyr Leu Ser Pro Phe Asn Pro Phe His Ser His 785 790 795 800 Arg
Ser Leu Phe Ser Asp Asp Ser Ser Glu Arg Asp Ser Ser Phe Ser 805 810
815 Phe Thr Arg Leu Leu Arg Leu Ser Val Gly Glu Leu Tyr Lys Leu Met
820 825 830 Phe Phe Gly Ile Tyr Phe Arg Trp His Asn Ile Ile Ala Arg
Arg Gln 835 840 845 Gly Val Arg Arg Lys Leu Val Gly Ile Ile Glu Asp
Arg Ser Tyr Pro 850 855 860 Asn Asn Leu Val Phe Thr Thr Tyr Ser Asn
Gln Val Phe Ser His Ser 865 870 875 880 Thr Val Arg Ile Glu Ser Met
Pro Glu Thr Ile Glu His Arg Ile Val 885 890 895 Arg Ser Lys Lys Ala
Lys Gly Glu Cys Gly Gly Glu Asp Ser Ala Asn 900 905 910 Leu Leu Pro
Ala Leu Pro His Ile Val Arg Lys Pro Gln Ile Leu Ser 915 920 925 Cys
Gln Pro Val Tyr Leu Pro Pro Phe Leu Phe Thr Pro Thr Val Lys 930
935 940 Val Glu Ile Lys Lys Ser Arg Arg Leu Tyr Ser Ser Asn Arg Arg
Trp 945 950 955 960 Ser Trp Ile Ser Ser His Leu Trp Gly Glu Asp Gly
Arg Leu Trp Gln 965 970 975 Val Leu Trp Glu Gly Asp Lys Gln Glu Pro
Gly Phe Gln Cys Pro Pro 980 985 990 Pro Leu Gly Leu Leu Ser Leu Arg
Pro Ile Thr Gly Trp Ser Ser Ile 995 1000 1005 Val Ile Pro Asp Lys
Gln Thr Leu Val Thr Asp Ala Gly Lys Leu 1010 1015 1020 Tyr Val Leu
Asp Gly Leu Leu Thr Arg Leu Lys Glu Gln Gly His 1025 1030 1035 Arg
Ala Leu Ile Tyr Ser Gln Met Thr Arg Met Ile Asp Leu Leu 1040 1045
1050 Glu Glu Tyr Met Trp Tyr Arg Lys His Thr Tyr Met Arg Leu Asp
1055 1060 1065 Gly Ser Ser Lys Ile Ser Asp Arg Arg Asp Met Val Ala
Asp Phe 1070 1075 1080 Gln Thr Arg Glu Asp Ile Phe Val Phe Leu Leu
Ser Thr Arg Ala 1085 1090 1095 Gly Gly Leu Gly Ile Asn Leu Thr Ala
Ala Asp Thr Val Ile Phe 1100 1105 1110 Tyr Asp Ser Asp Trp Asn Pro
Thr Val Asp Gln Gln Ala Met Asp 1115 1120 1125 Arg Ala His Arg Leu
Gly Gln Thr Lys Gln Val Thr Val Tyr Arg 1130 1135 1140 Leu Ile Cys
Lys Gly Thr Ile Glu Glu Arg Ile Leu Gln Arg Ala 1145 1150 1155 Gln
Glu Lys Ser Glu Ile Gln Arg Val Val Ile Ser Gly Gly Asn 1160 1165
1170 Phe Lys Pro Asp Thr Leu Lys Pro Lys Glu Val Val Ser Leu Leu
1175 1180 1185 Leu Asp Asp Ala Glu Ile Glu Gln Lys Tyr Arg Gln Lys
Gln Ala 1190 1195 1200 Glu Arg Arg Gln Leu Glu Glu Ser Arg Gln Asp
Ile Tyr Arg Glu 1205 1210 1215 Arg Glu Arg Lys Arg Lys Asn Ala Leu
Gly Asn Tyr Lys Asp Thr 1220 1225 1230 Val Lys His Met Lys Thr Glu
Asp Gly Val Val Glu Val Tyr Gln 1235 1240 1245 Gly Val Lys Ser Pro
Ala Glu Ser Pro Lys Gly Thr Asp Val Met 1250 1255 1260 Lys Ser Ser
Gln Asp Ser Asn Asp Asn Asn Thr Asp Gln Glu Glu 1265 1270 1275 Pro
Ile Ser Gln Thr Pro Pro Lys Ile Ser Arg Pro Phe Met Pro 1280 1285
1290 Ala Leu Ser Asn Arg Ser Asn Arg Arg Gly Pro Gly Arg Pro Arg
1295 1300 1305 Leu Arg Pro Val Gly Pro Gly His Ile Gly Ser Arg Gly
Pro Arg 1310 1315 1320 Gly Leu Arg Gly Pro Gly Arg Lys Pro Ser Val
Pro Ala Leu Ser 1325 1330 1335 Ile Arg Pro Thr Gln Ala Ser Ser Gln
Leu Pro Leu Gln Asn His 1340 1345 1350 Gln Pro Gln Ile Lys Val Glu
Asn Ser Ser 1355 1360 326108DNAEuchistus heros 32acaggcttat
atcatctaac aattatgaat gtcatgagat caaaaatatt atccgaacta 60aattaaaaac
acaaaaacga atgacagaaa gatcggcctg aatcagttta aattagtcct
120ttgattgtga aaaattttaa agctttgaat ccttaattat aataataaac
tattaaccgg 180cagtgtatca tgaagctaaa cttgtactta tagtcaatgg
aacaacatct gatggacgct 240ttgttttact taaccgacca actttcttgc
tgaccttact aatctcatta cttgaggcag 300cagaagctct tctagtacga
attacaaggt tagggttcga ataaggtggt gggggaggat 360taggaggagg
gggaggtgta ctacgccgtt taccactctt aaattttcta ctttgccctg
420atgagggttt ccccccattt tgaggactgt catctaaagt ccacagatca
attgaaacac 480gaccatgaga tctagtcctt ggagtgccct cctcgatact
acttccacta tgacaatttc 540ttcccccgcc cccactagct cttgttttat
tgacctgatt tcgcgaatca acgccagagt 600aagtgagaac agcttccgac
tcagcttccc cagaaggtga ttttctatct ccttgcagag 660cagccagtct
accagcctcc cactcttttt tttgttgttc gatttctgct tcagctgcag
720ccaactgctc ttttgaccaa gcagcatcat tttcttccat gaatttcatg
gcatacctct 780caacagcaga aagctgttgc ataagattgt gaagttctaa
ttctgccttg ctcatttctt 840gcccaactcc ttcatgggta tcaataggta
tattttcatc gaactctgcc aactcagcag 900cagcttctgc tttagcaacc
ttggctgccg caacatcgga ttcatcctca gcttgggcca 960ttgcactctc
gagcgcacca attgctactt tctcatcaga gttcgcaacc atctgtgtgt
1020cttcaggatt ctgagcagtt ttatcactat tatgaagaac ttctgccatc
cttctggaag 1080catcattctc ggaggtgtca acattaaaca gatcttgaat
tgttgaactc ttaaagtaag 1140ctgtagtaaa gtttcctcct tcaatggcta
catctcccag cattctcttt tggttcgctt 1200ttttaagtat attttcctca
acagtttttt cgctgatcaa tctataaata tgtacatctc 1260tcgtttggcc
gattctgtgg catcgatctt gagcttgggc atccatggta ggattccaat
1320cactatcata aaatataaca gtgtctgcac cagttaaatt aataccaact
cctccagatc 1380ttgtggatag aataaagcaa aatattctct tgtcagcatt
aaagcgttcc attaagagct 1440gtctctgatc tactttcgtt gttccatcaa
gacgaagata tatgtgcccg tgaaagttaa 1500gaaatgcttc cagtacgtct
aacattctag tcatttgtgt aaatattaat atccgatgat 1560ggtcagcttt
caatcttcga agaagcttgt ctaatgattg aagctttcca cagtcatact
1620gtattagtct tctatcaggg aactgcgtac tcattgctga tgatatgcta
tgaagtaatc 1680gaagtttagg tcttagccaa gtatcaacta atgatagcct
cttctcttct tggaacatct 1740ttgaaggtgg cggatgtggc acatgtaaac
ggacaggttg gctggaaaca gccggtacgt 1800acaaaacaaa cctagagaat
atatcagaca actccgcaac tcggtcttcg atggaatgaa 1860tagctgctgt
gagagcatga gtctgattcc aaaataacaa ggggtttgaa gataaagcat
1920tcttacagtg aactgttcca atgcagtctt tagtttcatc aggagcatca
tccaatgtta 1980aagatgatag aaggtcagaa ccataaattg gaagggcctg
acacctttgt tcattgattc 2040taacaatcag ttccaacttc tcctttcgcc
gtttttttct aagattttct aggtattcat 2100ctctcaaatc ttcattcgga
ttagattctt ctttgttatt tttatttgag ttcaaagtac 2160tttgtcttgt
tactttttta agagaaggta atgaattgac gttcgatcca tttccttgag
2220ctggctctga cgatatgctc acaggtgaat tgtttacagt agtcaaactg
ttttgaacaa 2280cagtagctgc agaagtgatg tttaatggag gtaccctcat
gataggccga tttgaattta 2340tttgggtcac agaagtaggg ttgactagtt
tagctacatt gcttccctgt ttggatacca 2400cagttaatct ttgcccagta
gtagtcatta cagtagtagc acctccttga ggagtaataa 2460ccggttgtgg
agagagtacc agctgcctac cggtaggggt attaactaat tgtgcaaatt
2520gagggaccat acgaccagca ttgccttcat ttttaaccac gcctccacta
gtagttgtta 2580atgaagccac tgagattgct ttcatggcac caggttgtaa
taactgaagg taattaggaa 2640tctgaccaga agtaggattc gccagtctta
aagttacact ctggctagca ttttgtccaa 2700gctttatcaa tggtgatgtt
ccaacttttg ttgaaaaaga agaaatccga tgattattac 2760taacttgtac
aattggccta acagggctag aaacctgagg tggttggggc aatatccgtt
2820tcttgacatt aattttaacc cttcctttag ggcaaggtgg tagatcagga
ggtgcagaat 2880caattgtgcg aatgaagtca ggattaactt tgtatttacg
agctctgtga gcaacaaatg 2940ctaataacca tagttcaaat tcaattatcc
gcaaatttag gaaaactaga tcgacatgtt 3000taaacggatc ataatctaaa
gcactccaaa ctatagaagg aacatggtat tcaagagaat 3060ccatttgaaa
cggagacacg gtagggcgaa cttcgaacag gttaggatga ttacacactt
3120tcctcagctg catcagtaca ttaatgacac tcaataaact accagaagca
agtgtttctt 3180ttgttttagc tctagacatg aaatcatcat ataaatatcg
ttgcctgttg gataaccgac 3240acattactat atgttcatat ttctttggca
tttgcgtttc tacttcacac tttaatcttc 3300ttaacagaaa cggacgcaac
actttatgaa gtcttttaat aatagtgtca ttgtactcag 3360aattcccttc
aatcatgcct gttactggat tagaaaacca ttctttaaat tcacgatgcg
3420attcaaaaac attaggcata agaaaatgca ttaatgacca gagttccata
agattatttt 3480gtaatggagt accagtgagt agtaaccgcc tttgagtttg
aaaattcaat aagagttgcc 3540aacgttgtga tttaaaattt ttgatatttt
gagcttcatc taaaattaaa tatttccatt 3600tttttctacg aaaactctga
tgatcctgta taactaactt ataagaggta atgcagatat 3660ggaatgcatt
aggttttgtc caccctgatc gtttcaattt ccgttctttt tgagttccat
3720aataagttaa tattttgaat gctggacacc attttttaaa ctccatttcc
cagtttaaca 3780tgacagacgt agggacaatg attaaatgag gtccccaatt
acctttttca caagcaagat 3840gagctattaa tgctatggtt tgaattgtct
ttccgagacc catttcgtca gcaagtatac 3900catttagttt cctatcatac
atagtaacta accagtctaa cccaatgtgc tgatattctc 3960ttaagggatg
tttaagaaga aaaggaactt tcgtaacaac acttgtagat gatagagtgt
4020ttcctttagg ctgaagactt tcagctatag cagcaacatc atttatttcc
ttgtctttgt 4080cttggttccc ttcctctaca tgatgcgagt catttatgag
agccttcaaa gtcgaatctt 4140ccttgtcact tgaagaactg tcttcttctt
cttcttcttc actaacttca tcttcaaaat 4200cttcttcttc tgaggcttca
tcagattctt cattcaggat ttctttcctt tttcttgatc 4260tttgggaact
gggatctccg ttcgaaggta aaggttcgga cgggagctcg ataccatatt
4320ttgctctaag ttgttctatg ctcatatttc cttcctcctt taaatcatct
atttcttgtt 4380tataatccac tgttccttca gtcttttctt gttccagaat
tgtttcttca tcatctgaag 4440aatcaccaga atcttgatat tccatgtcat
catcctcaac atcttcatcc atgctgacag 4500gagttgatat agaatccctt
tgagcaaggt agttggaaag tagatcttca agtggaagct 4560cgctctcttt
tttcagaaga tccacttcat cttgattatc acatttatca tcattagcag
4620aaagtgcttc ttccttagca atcgtctctt catcgtcgtc agacggatct
tctggctcaa 4680attcatcgtc agagtgatgc ctgggagaag ttggtcttga
agtgttcatg ctttcagcaa 4740caagacttga atacttttca gtttgatcaa
caataaaact taagtgttga tcaagtgctt 4800tttttctctt ttcttctaac
ctagtttgtt gtttgaattc aactagcttt tcaacatttg 4860accagaactg
tttgatttct ttcgcaataa aagaagctat gcgttttaac tgcatttctt
4920gagctttgat agctttctgt actagagctt ctttttcttg aaaatgtttt
tgaaccattc 4980ttgcacactt ttttgcagct gctttcttcc atttcctctc
ctgggcaaaa tctgcagcca 5040gccaagccat ttcttctagc agataatccc
aatgagcttt agcccttggc aattcatgga 5100ctttaggtaa tcttttctct
ggccataatc catccttttg taactctcct accctttgca 5160ttacatacgc
ctcttgttta gctctttcaa caatttgttc ctggctattg actccatcca
5220ccggagaagc attttttaca gctgatttca ctgtagtcag tttttggaat
ttggaagata 5280attctgcttt ggtaggacta gcagatgatg gaacccgtac
atcagtattg cttcctggcc 5340tgttcccagt aagagcagat tgtagaagag
agtgattttg tgattgcaga accttataca 5400gatcctctcc gtcattacca
ggatctaact tatttgtttt tagaaatgtc tgtaaaggaa 5460ctacaggcaa
acgagacctc cacggactga agtctgagag gtttccattt gcctgtagat
5520aacagagaag agcacatctc tctgcaaact ttgtctgaaa tcttttattt
ttattatcgt 5580ttaaatcttt tattcttttt ctaaaggaat tatattcatc
aagcggcagc ttgcgttttc 5640ttgttgaaga tgacgacaaa cttaatgaaa
gtgatggttg agggttacct tggctaggag 5700gttggagatt ggtcatcata
ggagaatcgg acaggataga agtcggggag gagcggactt 5760ggagtcgccc
cccttggctc gagtttgcca accttgtcat tgatggtgaa ccctgttgca
5820gggttggcaa gagaggagcc aaggggagca cttgagatcc agcacaaggt
ttttcaagaa 5880ctggcttttc aaagccaagt tgaagattgt tcatatcaaa
ttcattgtgt tatcacaatt 5940ttcttcgtcc atcaacaata aaatcaagaa
aatgatcttc ggtaacgaac tttaggaaag 6000aactcatttt actaatttat
tagccaatta attctgattt tattcaaatt ccgtggagaa 6060atattcctat
gccacattct cttcaatgca acatggcgtt aggttcag 6108331790PRTEuchistus
heros 33Met Asn Asn Leu Gln Leu Gly Phe Glu Lys Pro Val Leu Glu Lys
Pro 1 5 10 15 Cys Ala Gly Ser Gln Val Leu Pro Leu Ala Pro Leu Leu
Pro Thr Leu 20 25 30 Gln Gln Gly Ser Pro Ser Met Thr Arg Leu Ala
Asn Ser Ser Gln Gly 35 40 45 Gly Arg Leu Gln Val Arg Ser Ser Pro
Thr Ser Ile Leu Ser Asp Ser 50 55 60 Pro Met Met Thr Asn Leu Gln
Pro Pro Ser Gln Gly Ser Asn Thr Asp 65 70 75 80 Val Arg Val Pro Ser
Ser Ala Ser Pro Thr Lys Ala Glu Leu Ser Ser 85 90 95 Lys Phe Gln
Lys Leu Thr Thr Val Lys Ser Ala Val Lys Asn Ala Ser 100 105 110 Pro
Val Asp Gly Val Asn Ser Gln Glu Gln Ile Val Glu Arg Ala Lys 115 120
125 Gln Glu Ala Tyr Val Met Gln Arg Val Gly Glu Leu Gln Lys Asp Gly
130 135 140 Leu Trp Pro Glu Lys Arg Leu Pro Lys Val His Glu Leu Pro
Arg Ala 145 150 155 160 Lys Ala His Trp Asp Tyr Leu Leu Glu Glu Met
Ala Trp Leu Ala Ala 165 170 175 Asp Phe Ala Gln Glu Arg Lys Trp Lys
Lys Ala Ala Ala Lys Lys Cys 180 185 190 Ala Arg Met Val Gln Lys His
Phe Gln Glu Lys Glu Ala Leu Val Gln 195 200 205 Lys Ala Ile Lys Ala
Gln Glu Met Gln Leu Lys Arg Ile Ala Ser Phe 210 215 220 Ile Ala Lys
Glu Ile Lys Gln Phe Trp Ser Asn Val Glu Lys Leu Val 225 230 235 240
Glu Phe Lys Gln Gln Thr Arg Leu Glu Glu Lys Arg Lys Lys Ala Leu 245
250 255 Asp Gln His Leu Ser Phe Ile Val Asp Gln Thr Glu Lys Tyr Ser
Ser 260 265 270 Leu Val Ala Glu Ser Met Asn Thr Ser Arg Pro Thr Ser
Pro Arg His 275 280 285 His Ser Asp Asp Glu Phe Glu Pro Glu Asp Pro
Ser Asp Asp Asp Glu 290 295 300 Glu Thr Ile Ala Lys Glu Glu Ala Leu
Ser Ala Asn Asp Asp Lys Cys 305 310 315 320 Asp Asn Gln Asp Glu Val
Asp Leu Leu Lys Lys Glu Ser Glu Leu Pro 325 330 335 Leu Glu Asp Leu
Leu Ser Asn Tyr Leu Ala Gln Arg Asp Ser Ile Ser 340 345 350 Thr Pro
Val Ser Met Asp Glu Asp Val Glu Asp Asp Asp Met Glu Tyr 355 360 365
Gln Asp Ser Gly Asp Ser Ser Asp Asp Glu Glu Thr Ile Leu Glu Gln 370
375 380 Glu Lys Thr Glu Gly Thr Val Asp Tyr Lys Gln Glu Ile Asp Asp
Leu 385 390 395 400 Lys Glu Glu Gly Asn Met Ser Ile Glu Gln Leu Arg
Ala Lys Tyr Gly 405 410 415 Ile Glu Leu Pro Ser Glu Pro Leu Pro Ser
Asn Gly Asp Pro Ser Ser 420 425 430 Gln Arg Ser Arg Lys Arg Lys Glu
Ile Leu Asn Glu Glu Ser Asp Glu 435 440 445 Ala Ser Glu Glu Glu Asp
Phe Glu Asp Glu Val Ser Glu Glu Glu Glu 450 455 460 Glu Glu Asp Ser
Ser Ser Ser Asp Lys Glu Asp Ser Thr Leu Lys Ala 465 470 475 480 Leu
Ile Asn Asp Ser His His Val Glu Glu Gly Asn Gln Asp Lys Asp 485 490
495 Lys Glu Ile Asn Asp Val Ala Ala Ile Ala Glu Ser Leu Gln Pro Lys
500 505 510 Gly Asn Thr Leu Ser Ser Thr Ser Val Val Thr Lys Val Pro
Phe Leu 515 520 525 Leu Lys His Pro Leu Arg Glu Tyr Gln His Ile Gly
Leu Asp Trp Leu 530 535 540 Val Thr Met Tyr Asp Arg Lys Leu Asn Gly
Ile Leu Ala Asp Glu Met 545 550 555 560 Gly Leu Gly Lys Thr Ile Gln
Thr Ile Ala Leu Ile Ala His Leu Ala 565 570 575 Cys Glu Lys Gly Asn
Trp Gly Pro His Leu Ile Ile Val Pro Thr Ser 580 585 590 Val Met Leu
Asn Trp Glu Met Glu Phe Lys Lys Trp Cys Pro Ala Phe 595 600 605 Lys
Ile Leu Thr Tyr Tyr Gly Thr Gln Lys Glu Arg Lys Leu Lys Arg 610 615
620 Ser Gly Trp Thr Lys Pro Asn Ala Phe His Ile Cys Ile Thr Ser Tyr
625 630 635 640 Lys Leu Val Ile Gln Asp His Gln Ser Phe Arg Arg Lys
Lys Trp Lys 645 650 655 Tyr Leu Ile Leu Asp Glu Ala Gln Asn Ile Lys
Asn Phe Lys Ser Gln 660 665 670 Arg Trp Gln Leu Leu Leu Asn Phe Gln
Thr Gln Arg Arg Leu Leu Leu 675 680 685 Thr Gly Thr Pro Leu Gln Asn
Asn Leu Met Glu Leu Trp Ser Leu Met 690 695 700 His Phe Leu Met Pro
Asn Val Phe Glu Ser His Arg Glu Phe Lys Glu 705 710 715 720 Trp Phe
Ser Asn Pro Val Thr Gly Met Ile Glu Gly Asn Ser Glu Tyr 725 730 735
Asn Asp Thr Ile Ile Lys Arg Leu His Lys Val Leu Arg Pro Phe Leu 740
745 750 Leu Arg Arg Leu Lys Cys Glu Val Glu Thr Gln Met Pro Lys Lys
Tyr 755 760 765 Glu His Ile Val Met Cys Arg Leu Ser Asn Arg Gln Arg
Tyr Leu Tyr 770 775 780 Asp Asp Phe Met Ser Arg Ala Lys Thr Lys Glu
Thr Leu Ala Ser Gly 785 790 795 800 Ser Leu Leu Ser Val Ile Asn Val
Leu Met Gln Leu Arg Lys Val Cys 805 810 815 Asn His Pro Asn Leu Phe
Glu Val Arg Pro Thr Val Ser Pro Phe Gln 820 825 830 Met Asp Ser Leu
Glu Tyr His Val Pro Ser Ile Val Trp Ser Ala Leu 835 840 845 Asp Tyr
Asp Pro Phe Lys His Val Asp Leu Val Phe Leu Asn Leu Arg 850 855 860
Ile Ile Glu Phe Glu Leu Trp Leu Leu Ala Phe Val Ala His Arg Ala 865
870 875 880 Arg Lys Tyr Lys Val Asn Pro Asp Phe Ile Arg Thr Ile Asp
Ser Ala 885 890 895 Pro Pro Asp Leu Pro Pro Cys Pro Lys Gly Arg Val
Lys Ile Asn Val 900 905 910 Lys Lys Arg Ile Leu Pro Gln
Pro Pro Gln Val Ser Ser Pro Val Arg 915 920 925 Pro Ile Val Gln Val
Ser Asn Asn His Arg Ile Ser Ser Phe Ser Thr 930 935 940 Lys Val Gly
Thr Ser Pro Leu Ile Lys Leu Gly Gln Asn Ala Ser Gln 945 950 955 960
Ser Val Thr Leu Arg Leu Ala Asn Pro Thr Ser Gly Gln Ile Pro Asn 965
970 975 Tyr Leu Gln Leu Leu Gln Pro Gly Ala Met Lys Ala Ile Ser Val
Ala 980 985 990 Ser Leu Thr Thr Thr Ser Gly Gly Val Val Lys Asn Glu
Gly Asn Ala 995 1000 1005 Gly Arg Met Val Pro Gln Phe Ala Gln Leu
Val Asn Thr Pro Thr 1010 1015 1020 Gly Arg Gln Leu Val Leu Ser Pro
Gln Pro Val Ile Thr Pro Gln 1025 1030 1035 Gly Gly Ala Thr Thr Val
Met Thr Thr Thr Gly Gln Arg Leu Thr 1040 1045 1050 Val Val Ser Lys
Gln Gly Ser Asn Val Ala Lys Leu Val Asn Pro 1055 1060 1065 Thr Ser
Val Thr Gln Ile Asn Ser Asn Arg Pro Ile Met Arg Val 1070 1075 1080
Pro Pro Leu Asn Ile Thr Ser Ala Ala Thr Val Val Gln Asn Ser 1085
1090 1095 Leu Thr Thr Val Asn Asn Ser Pro Val Ser Ile Ser Ser Glu
Pro 1100 1105 1110 Ala Gln Gly Asn Gly Ser Asn Val Asn Ser Leu Pro
Ser Leu Lys 1115 1120 1125 Lys Val Thr Arg Gln Ser Thr Leu Asn Ser
Asn Lys Asn Asn Lys 1130 1135 1140 Glu Glu Ser Asn Pro Asn Glu Asp
Leu Arg Asp Glu Tyr Leu Glu 1145 1150 1155 Asn Leu Arg Lys Lys Arg
Arg Lys Glu Lys Leu Glu Leu Ile Val 1160 1165 1170 Arg Ile Asn Glu
Gln Arg Cys Gln Ala Leu Pro Ile Tyr Gly Ser 1175 1180 1185 Asp Leu
Leu Ser Ser Leu Thr Leu Asp Asp Ala Pro Asp Glu Thr 1190 1195 1200
Lys Asp Cys Ile Gly Thr Val His Cys Lys Asn Ala Leu Ser Ser 1205
1210 1215 Asn Pro Leu Leu Phe Trp Asn Gln Thr His Ala Leu Thr Ala
Ala 1220 1225 1230 Ile His Ser Ile Glu Asp Arg Val Ala Glu Leu Ser
Asp Ile Phe 1235 1240 1245 Ser Arg Phe Val Leu Tyr Val Pro Ala Val
Ser Ser Gln Pro Val 1250 1255 1260 Arg Leu His Val Pro His Pro Pro
Pro Ser Lys Met Phe Gln Glu 1265 1270 1275 Glu Lys Arg Leu Ser Leu
Val Asp Thr Trp Leu Arg Pro Lys Leu 1280 1285 1290 Arg Leu Leu His
Ser Ile Ser Ser Ala Met Ser Thr Gln Phe Pro 1295 1300 1305 Asp Arg
Arg Leu Ile Gln Tyr Asp Cys Gly Lys Leu Gln Ser Leu 1310 1315 1320
Asp Lys Leu Leu Arg Arg Leu Lys Ala Asp His His Arg Ile Leu 1325
1330 1335 Ile Phe Thr Gln Met Thr Arg Met Leu Asp Val Leu Glu Ala
Phe 1340 1345 1350 Leu Asn Phe His Gly His Ile Tyr Leu Arg Leu Asp
Gly Thr Thr 1355 1360 1365 Lys Val Asp Gln Arg Gln Leu Leu Met Glu
Arg Phe Asn Ala Asp 1370 1375 1380 Lys Arg Ile Phe Cys Phe Ile Leu
Ser Thr Arg Ser Gly Gly Val 1385 1390 1395 Gly Ile Asn Leu Thr Gly
Ala Asp Thr Val Ile Phe Tyr Asp Ser 1400 1405 1410 Asp Trp Asn Pro
Thr Met Asp Ala Gln Ala Gln Asp Arg Cys His 1415 1420 1425 Arg Ile
Gly Gln Thr Arg Asp Val His Ile Tyr Arg Leu Ile Ser 1430 1435 1440
Glu Lys Thr Val Glu Glu Asn Ile Leu Lys Lys Ala Asn Gln Lys 1445
1450 1455 Arg Met Leu Gly Asp Val Ala Ile Glu Gly Gly Asn Phe Thr
Thr 1460 1465 1470 Ala Tyr Phe Lys Ser Ser Thr Ile Gln Asp Leu Phe
Asn Val Asp 1475 1480 1485 Thr Ser Glu Asn Asp Ala Ser Arg Arg Met
Ala Glu Val Leu His 1490 1495 1500 Asn Ser Asp Lys Thr Ala Gln Asn
Pro Glu Asp Thr Gln Met Val 1505 1510 1515 Ala Asn Ser Asp Glu Lys
Val Ala Ile Gly Ala Leu Glu Ser Ala 1520 1525 1530 Met Ala Gln Ala
Glu Asp Glu Ser Asp Val Ala Ala Ala Lys Val 1535 1540 1545 Ala Lys
Ala Glu Ala Ala Ala Glu Leu Ala Glu Phe Asp Glu Asn 1550 1555 1560
Ile Pro Ile Asp Thr His Glu Gly Val Gly Gln Glu Met Ser Lys 1565
1570 1575 Ala Glu Leu Glu Leu His Asn Leu Met Gln Gln Leu Ser Ala
Val 1580 1585 1590 Glu Arg Tyr Ala Met Lys Phe Met Glu Glu Asn Asp
Ala Ala Trp 1595 1600 1605 Ser Lys Glu Gln Leu Ala Ala Ala Glu Ala
Glu Ile Glu Gln Gln 1610 1615 1620 Lys Lys Glu Trp Glu Ala Gly Arg
Leu Ala Ala Leu Gln Gly Asp 1625 1630 1635 Arg Lys Ser Pro Ser Gly
Glu Ala Glu Ser Glu Ala Val Leu Thr 1640 1645 1650 Tyr Ser Gly Val
Asp Ser Arg Asn Gln Val Asn Lys Thr Arg Ala 1655 1660 1665 Ser Gly
Gly Gly Gly Arg Asn Cys His Ser Gly Ser Ser Ile Glu 1670 1675 1680
Glu Gly Thr Pro Arg Thr Arg Ser His Gly Arg Val Ser Ile Asp 1685
1690 1695 Leu Trp Thr Leu Asp Asp Ser Pro Gln Asn Gly Gly Lys Pro
Ser 1700 1705 1710 Ser Gly Gln Ser Arg Lys Phe Lys Ser Gly Lys Arg
Arg Ser Thr 1715 1720 1725 Pro Pro Pro Pro Pro Asn Pro Pro Pro Pro
Pro Tyr Ser Asn Pro 1730 1735 1740 Asn Leu Val Ile Arg Thr Arg Arg
Ala Ser Ala Ala Ser Ser Asn 1745 1750 1755 Glu Ile Ser Lys Val Ser
Lys Lys Val Gly Arg Leu Ser Lys Thr 1760 1765 1770 Lys Arg Pro Ser
Asp Val Val Pro Leu Thr Ile Ser Thr Ser Leu 1775 1780 1785 Ala Ser
1790 34240DNAArtificial SequenceSNF2-helicase degenerate dsRNA
sequencemisc_feature(216)..(216)n is a, c, g, or
tmisc_feature(222)..(222)n is a, c, g, or t 34cgsythctyy tmacsggyac
hcctctvcar aayaarctwc chgarytstg ggcbytdcth 60aayttyytvc tbccstcbat
yttyaarwsb tgytcbacdt tygarcartg gttcaaygcv 120cchttygcha
cmacbggmga raargtygar ytdaaygarg argaracvat yytkatyaty
180mgdcgtytdc ayaargtyyt kcgwccktty ytvytnmgdc gnytvaaaaa
rgargtmgar 2403527DNAArtificial SequenceSNF2-helicase degenerate
dsRNA sequencemisc_feature(21)..(21)n is a, c, g, or t 35mghgcygtbt
gyythatygg ngaycar 273660DNAArtificial SequenceSNF2-helicase
degenerate dsRNA sequence 36tayaarctyc tvytsacmgg machccgytb
caraacaayc tmgargaryt rttycatytr 603761DNAArtificial
SequenceSNF2-helicase degenerate dsRNA sequence 37garttygaya
cbaaycaymg rctkcthath acwggbacyc ckytvcaraa ywskytdaar 60g
613823DNAArtificial SequenceBromodomain degenerate dsRNA sequence
38ytswsygaac crttyatgaa ryt 233965DNAArtificial SequenceHAND-SLIDE
degenerate dsRNA sequence 39gchgtvgatg cytayttymg vgargcwytv
mgdgtytchg arccyaargc dccdaargch 60cchmg 654036DNAArtificial
SequenceChromodomain degenerate dsRNA
sequencemisc_feature(33)..(33)n is a, c, g, or t 40mghaartrbg
ayatggavga rvvdccbaar ytngar 364156DNAArtificial
SequenceChromodomain degenerate dsRNA
sequencemisc_feature(29)..(29)n is a, c, g, or t 41bhggdaarad
dggrkkbryb ggmaaymwna chacdrtsta ykmhrtagar gaaaay
5642471DNAArtificial SequenceYFPv2 hpRNA encoding sequence
42atgtcatctg 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
471434958RNAEuchistus heros 43cuuggcuagu acucuuccgu gacgucacgu
ucgccauauu guuagaguuu gucuugccuc 60uggauaguua uguugauucu uuuuaaguga
uuuugaagau uuccugacca uuuuaucacg 120aaaaacuauu uuaaacagcg
cuauugcucc uuauaauacg ugugauucaa caacgaugga 180cggagacagc
ggugguaugg cgagcccuuc gccacagccu cagucgucac caaugccccc
240uccacaagcu ccaucaccua ugggcccgcc gcagggcgcc ccaucgccaa
ugcccccuuc 300uaaccaacag gcggccucac caaugggucc accgcaccac
ccccacagcc cgacagguua 360ccaaggaggg augccacaca ugaauggacc
aaaugguguu ccuccuggua ugcagcaggc 420uacucaaaca uuucagccuc
aucagcaauu gccaccccac cagcaaccac caaugcagac 480ugcuccuggu
gggccugcua gugguggagg acaagaaaau cuuagcgcuc uccagcgugc
540aauagauucu auggaagaga aagggcuuca ggaagaucca cguuacucgc
agcugcuugc 600guugagggca aggcaugcca acauggaacc uccgguuagg
ccuccaucuc agcuuguugg 660ggguggguuc agcggugagg guggugcccc
uccuccugcu aaacacagcu ucagcgcgaa 720ccaacugcaa caacuucgag
ugcagaucau ggcguaucgc cuacuugcua ggaaccaacc 780ucuuucccag
cagcuagcuu uggcugugca aggcaaacgc cucgacagcc cuggcgaguc
840caacuaccag cauccuccua gugaaggagc aggagguguu gguggagaag
gaaguggaga 900cgggggaucg ucgaacggcc ugaugacgca gccgaugcgu
gccccaugcc ccccuggugg 960ccagccccca acggccucac cgaugacagg
ccagauggca ccuccuacug ggccagcucc 1020uguaaggcca ccuccucccg
gugugucucc uacaccuccg cgcccuccuc agcagguucc 1080uggugcuccg
ggggccccac aaccaaagca aaauaggguu accaccaugc caagaccgca
1140ugguuuagau cccauucuua uucuccagga aagagagaau agaguagccg
cuaggauugu 1200acauaggaug gaagaauuau caaauuuacc agcuacgaug
ccugaagacc uucgaauaaa 1260agcgcagaua gaacuuaggg ccuugagggu
acuuaacuuc caaaggcaau uaagagcaga 1320ggugauagcu uguacuagac
gcgauacaac auuagaaaca gcuguaaaug ugaaagcuua 1380uaaacgaacg
aagaggcaag gcuuacggga agccagagcu acggaaaagc uugaaaaaca
1440acagaaacuu gagacagaaa ggaagaagag acaaaaacac caggaauauc
ugagcacuau 1500auugcaacau ugcaaagacu ucaaagaauu ccauagaaau
aauguugcua aaguugguag 1560auuaaauaag gcugugauga auuaccaugc
gaaugccgag cgugaacaga agaaagagca 1620agaaaggaua gaaaaagaac
guaugagaag gcuuauggcu gaggaugaag aggguuacag 1680gaaacugauu
gaucagaaaa aagauaagag auuggcauuc cuucuuucac aaacugauga
1740auauauugcc aaucuuacug aaauggugaa gcagcauaaa auggaacaac
agcguaagca 1800ggaacaagaa gagcaacaaa aacggaagag gaaaaagaaa
aagaagaaua gggaaggaga 1860uccagaugau gaaagcucuc agaugucaga
uuuacauguu agcguuauag aagcagcaac 1920uggucggcag cugacggggg
aggaugcucc auuggccagc cagcuuggga gcugguugga 1980ggcacacccg
ggcugggagc cuuuggaaga uagcgaagau gaagaugaug aagaggacag
2040cgacgaggaa ggugaugaua acaguagauc aaaagguggu uuuucaauga
uaggaaaaga 2100ugaagcugau agcaaguuau cuguugaaga cgaagcucga
gaaaugauaa agaaagcgaa 2160gauugaagau gaugaauaca agaacacgac
cgaagaacau acauacuaca gcaucgcuca 2220caccgugcau gaaauuguca
ccgaacaagc uucaaucaug auuaacggua aauugaaaga 2280auaucaaauu
aaaggucuug aaugguuggu uucuuuauac aacaacaacu ugaauggaau
2340ccucgccgac gagaugggcc uuggcaagac aauucaaaca auaggucuca
uuacuuauuu 2400gauggagaag aagaaaguaa augguccuua ccucauuauu
guuccucugu caacauuauc 2460caauuggguu uuggaauucg agaaaugggc
uccuucagug uuugugguag cuuauaaagg 2520uucuccugca augaggagaa
cuuuacaauc acagaugcgc ucgacgaagu ucaauguccu 2580gcucacgacc
uacgaguaug ucaucaagga caaggcagua cuugcaaagu ugcauuggaa
2640guacaugaua aucgacgagg gacacaggau gaaaaaccac cauuguaagc
ugacgcaggu 2700gcugaacacc cauuauuugg caccucaccg ccuccuucuc
acgggcacac cucuccagaa 2760caaacuaccu gagcucuggg cucuucuaaa
cuuucuccuc ccguccaucu ucaagucgug 2820uucuacguuu gagcaauggu
ucaaugcacc auuugcuacc acuggagaaa agguugaguu 2880gaaugaggaa
gaaacaauuu ugauuaucag gcguuuacau aagguccuuc gaccuuuccu
2940ccuucgucga cugaaaaagg aagucgaaag ucaguugcca gagaaaauug
aauacaucgu 3000caagugugau augucugguc uccaacgugu acuuuauagg
cacaugcaga guaaaggagu 3060ccugcuuacc gaugguucug agaagggcaa
gcaggguaaa ggaggagcua aagcgcuaau 3120gaacacgauc guccaauuga
ggaagcuuug caaucauccu uucauguucc aucauauuga 3180agaaaaauau
ugugaucacg uuggccagaa caacguuguc acagggccug aucuguuccg
3240aguuucuggu aaauuugaau uccucgaucg uauauugcca aaacugaagg
ccacgagcca 3300uaggguacuu cuuuucuguc aaaugacuca gcugaugacc
aucauggagg auuauuuguc 3360uuggagaggg uucuccuacc uucgucuuga
ugguacgacc aaaucugaag accgaggaga 3420ucuucugaaa aaauucaaca
auccagaaag ugaauauuuu auuuucuugc ucucaaccag 3480agcuggaggu
cucggauuga acuuacaggc ugcagauacu gucauuauau uugauucaga
3540uuggaacccu caucaggauu uacaagcuca agacagagcu cauaggauug
gacagcaaaa 3600cgaaguucgu guuuugcggc uaaugacagu aaauucuguu
gaggagcgua uucuugcagc 3660ugcucgguac aagcugaaua uggaugagaa
agucauucag gcugguaugu uugaccagaa 3720aucuacagga accgagaggc
agaaauuucu gcaaaacauc cuucaucaag augaugcaga 3780ugaugaggaa
aaugaaguuc cagaugauga aaugguuaau cguaugauug cgcgaacaga
3840agaugaauuc aaccucuucc agaaaaucga uuuagaaagg aggagggaag
aggcuaaacu 3900uggaccuaac aggaagucaa ggcuuguaga agaggcggaa
uuaccugacu ggcuuguaaa 3960gaaugacgau gagauugaga aguggacuua
ugaagaaacc gagguccaaa ugggaagagg 4020uaauaggcag aggaaggaag
uagauuauac agauaguuug acugaaaaag aaugguuaaa 4080ggccauugau
gacaauguag augauuuuga ugacgaugaa gaggaagagg uaaaaacaaa
4140gaaaagaggc aagagaagaa gaaggggaga ggaugaugaa gaagaugcaa
guacuucaaa 4200gagaaggaaa uauucuccau cugaaaacaa acugaggagg
cguaugcgua accucaugaa 4260cauuguuguu aaguauacug acagugacuc
gagaguacuc agugaaccau ucaugaaacu 4320ucccucucgc cauaaguacc
cagacuacua ugaguugauc aagaaaccua uagacaucaa 4380gaggauauug
gccaaaguag aagaguguaa auaugcugac auggaugaau uagaaaagga
4440uuuuaugcaa cuuuguaaaa augcucagac auacaaugag gaggccucau
ugaucuauga 4500agauucgaua guauuagaaa guguuuucuc uaaugcucgu
caaaaaguag agcaggauaa 4560ugauucagau gaugaugaaa guaaagguga
ccaagaagau gcugcaucag acacuucauc 4620cgucaaaaug aaauugaaac
uaaagccugg gaggacccga gggaguggag cuggugguaa 4680aaggaggaga
agaaaauaua ucucugaaga ugaagacgaa gaccauagcg aaguuuccuu
4740aauguaaugc cucuucacug uccuuuguaa uuauuaguuu ucaucggugu
ucgguaccug 4800ucagucaagg gagaagcuaa gcuuuuuagu ugacuauuga
agaauuuagg acugaguucu 4860guuuuuguuu uuuuuguuug uuuuuuuuug
gauaaaugua uuuaauagau aaaauguuuc 4920gcuuauauau auauuuuuua
cugguuuugu aauuggcc 495844499RNAEuchistus heros 44gaugaugaag
aagaugcaag uacuucaaag agaaggaaau auucuccauc ugaaaacaaa 60cugaggaggc
guaugcguaa ccucaugaac auuguuguua aguauacuga cagugacucg
120agaguacuca gugaaccauu caugaaacuu cccucucgcc auaaguaccc
agacuacuau 180gaguugauca agaaaccuau agacaucaag aggauauugg
ccaaaguaga agaguguaaa 240uaugcugaca uggaugaauu agaaaaggau
uuuaugcaac uuuguaaaaa ugcucagaca 300uacaaugagg aggccucauu
gaucuaugaa gauucgauag uauuagaaag uguuuucucu 360aaugcucguc
aaaaaguaga gcaggauaau gauucagaug augaugaaag uaaaggugac
420caagaagaug cugcaucaga cacuucaucc gucaaaauga aauugaaacu
aaagccuggg 480aggacccgag ggaguggag 499456346RNAEuchistus heros
45aucucggugc uguggaucgu ccuuagugau uguuuucuaa uauaguuugu aauuauauag
60uguuuuaugc guugauaucg gugauauuag ugaauaauag ugaaguguug auguuuuauu
120ucuaauggcg ucugaagaag aaguugacga guguuuacca guugacgaug
aaguugacac 180uaguguuguu caacaagaag gcacugaaga aaauucaccu
gacagugaug aaagaaguag 240gauagaggaa gaagaugacg aguaugaccc
ugaggaugcg aggaaaaaaa agaaagguaa 300aaagagaaaa gccaaagggg
aaagcaaaaa agaaaagaaa cguaaaaaaa ggaagaagaa 360ugauagugcu
gaagaaagug agggaggcgg ggaagaagaa ggcgauuccg auuauggaag
420aaaaucuaag aagucuaaag gaacuucaca accaaaacca gugcagcaag
auucuucugg 480agguguaccu ucaguagaag aaguuugcag ccuuuuugga
cuuacagaug uacagauuga 540cuauaccgaa gaugauuacc aaaaucugac
uacguauaaa cuuuuucaac aacauguucg 600uccuauucuu gccaaggaca
accagaaggu ucccaucgga aaaaugauga ugcucguggc 660ugcaaaaugg
agagauuuuu gcaauuccaa uccaaacgcu caacaggaac cagauccaga
720agcuucagaa gaacaggaau auucuaaacc uaccaggaca cgaccuucac
gaguuucaac 780uacacaaaau gaugaugaag aagacgacga ugcugacgaa
cgagggagga aaaagagaag 840uggacgaagu aaaaagucau caggaaagaa
guccgcuccu ccggccacaa ccaagguccc 900uacccucaag aucaagauag
gaaaaagaaa acagaauucc gaugaagaag augaagguuc 960aguuggugcc
guuucugaaa gggacucaga ugcugaauuc gagcaaaugc ucgcagaagc
1020ugaagaaguu aauaaaccug aagguguugu agaagaagaa gaaggugcag
agguggcucc 1080uguaccuaag aaaaaggcca aaacgaaaau ugguaauaaa
aagaaaagga aaaagacacg 1140gacuacuaac aaguuuccag acagugaagc
ugguuaugaa acagaucauc aggacuauug 1200ugaaguuugu caacaaggag
gugaaauaau auuaugugau acgugcccuc gagcuuauca 1260uuuggucugu
uuggaucccg aauuggaaga uacgccagaa ggcaaauggu caugcccuca
1320uugugaaggu gaagguguac aggaaaaaga agaugauguc caucaagaau
uuugcagagu 1380uuguaaagau gguggagaac uuuuaugcug ugauucuugc
ccuucugcau accacacauu 1440cuguuugaac ccuccauuga cagauauucc
agauggugac uggaagugcc cacguuguuc 1500ggcgaagccu
uugagaggua aagugucaaa gauucuuacu uggagguggu uggaaucucc
1560caguaguaaa gaugaagaag acaauacuaa aaaacgaaac aggcagaggc
aaagagaaua 1620uuucgucaag ugggcagaua ugucuuauug gcacuguagu
ugggugucug aacuucagau 1680ggauguuuuu cauacucaaa ugaucaggag
uuauauucgu aaauaugaua uggacgaacc 1740ucccaaacua gaagaacccu
uggaugaagc agacaauaga augaagagga uacgagaggc 1800aaauaucaau
gagcaagaau uagaagagaa auauuacaag uaugguauca aaccagagug
1860gcuuauugug cagaggguaa uuaaccaucg cacuauaagg gauggaagca
aucuguaccu 1920cgucaaaugg agggaccucc cuuaugacca ggcgacuugg
gaggaagaag ucaccgauau 1980cccuggcuug aagaaagcua uugaauauua
caaugagaug agggcuugcu guuuagguga 2040aucuaaaaaa cuaaaaaaag
guaaagguaa aagaucaaag agagaucaag augaugagga 2100aggaagcaga
agugcaggaa ugaugggcgu cgguggacca gcuacugguc aauacuuccc
2160gccuccugaa aagccuguca cagauuugaa aaagaaauac gauaaacagc
cggacuaucu 2220cgacgucucc gguaugugcc uucauccuua ccaauuagaa
gguuuaaauu gguugaggua 2280uuccuggggg caaggaacag acacuauucu
ugccgaugag augggucuug gaaaaaccau 2340ucagacaauu acuuuccucu
auucucuuua caaagagggu cauuguaaag gccccuuccu 2400ugugagugua
cccuuaucua caauuaucaa uugggaaaga gaguucgaaa cuugggcgcc
2460agacuucuac guugucacau augucggaga caaagauucu cgugcuguaa
uacgugaaaa 2520ugaauuuuca uucgaugaua augcuguuag aggaggaaga
gguguuucua aaguucgcuc 2580uucugcaaua aaguuucaug uacugcuaac
aucuuaugaa cuuaucucua ucgaugucac 2640uugccuugga ucgaucgagu
gggcagugcu uguaguagau gaagcacaca ggcugaaaag 2700uaaucagagc
aaguucuuua ggcuucuugc uucauaccac auugcuuaua aacuucugcu
2760gacaggaacu ccguugcaaa acaaucuaga agaauuguuu cauuuacuua
auuuccuuac 2820gccggaaaaa uucaacgacc uugcgacauu ucaaaacgaa
uucgcugaua uuucaaaaga 2880agaacaaguc aaaagacuuc augaguuacu
cgggccgcau auguugagga gauuaaaagc 2940ugauguacuc aagaauaugc
cuacaaaauc ugaguucauu guuagaguug aacucucccc 3000gaugcagaag
aaguacuaca aauauauucu cacaaggaau uucgaagcuu uaaauccaaa
3060aggaggcggu caacaaguau cucuuuugaa cauuaugaug gaucuuaaaa
aaugcuguaa 3120ucauccauac cuguuuccug cugcuucuca ggaagcuccu
uuaggaccaa gcggaucuua 3180cgaucuucaa ggguuaauca aagcaucugg
aaaauugaua cuucugucga aaaugcugag 3240acggcucaaa gaagaggguc
acagaguacu gauuuucucu caaaugacaa aaauguugga 3300cuuauuagaa
gacuaccucg agggugaagg uuauaaauau gaacguauug acgguacgau
3360caccgguagc uuaagacaag aagcuaucga ucgguuuaac gccccuggag
cucaacaauu 3420uguuuuucuu uuguccacuc gugcgggagg ucuugguauu
aaucucgcua cugcagauac 3480aguuauuauu uaugacucug acuggaaucc
ucauaacgau auucaggccu uuucgagagc 3540acacaggaua gggcaagcaa
acaagguuau gauuuaucga uuugugacac gagcgucugu 3600ugaagaaaga
guaacgcaag uggcuaagag aaaaaugaug uuaacccauc uugucguacg
3660accagguaug gguggcaagc aagcaaauuu cacuaagcaa gaacuugaug
auauuuuaag 3720guuuggaaca gaagaacuuu ucaaagaaga gcaggguaaa
gaagaugaag ccauucauua 3780ugacgauaaa gcuguugaag aauuacuuga
ccggucgaag auggguauug aacagaaaga 3840aaacuggucu aaugaauauc
uuucuucuuu caaaguggca aguuauguua cuaaagaaga 3900agacgaagau
gaggaaauag gaacagaggu aauaaaacag gaagcagaaa auacagaccc
3960agcuuauugg gucaaacugu ugaggcacca uuaugagcaa caacaagagg
auauuucucg 4020aacucucggu aaaggaaaaa ggauucgaaa acaggugaau
uacaucgacg guggagugau 4080ggacucaaga gagaacgccg auucgacgug
gcaagacaac cucucugacu auaauucaga 4140cuucucugcu ccuucugaug
augacaagga agacgaugac uuugaugaga aaaaugauga 4200uggaacgaga
aagaagcgua ggccagaaag gagggaggac aaagauaggc cucuaccucc
4260ucuucuugcc cgagucggug gaaacauuga gguccuggga uucaacgcca
gacagcguaa 4320agcauucuug aaugcuauua ugagguaugg aaugccaccu
caagaugcau ucaacucgca 4380guggcuuguu cgagaccuga gggguaaauc
ugagaagcau uucaaggcau acguaucccu 4440cuuuaugagg cauuugugug
agccuggcgc ggacaaugcc gaaacauucg cggauggugu 4500uccaagggaa
ggucuuaguc ggcagcaugu ucucacaagg auagguguga ugucacucau
4560uaggaaaaag guucaagaau uugagcaaau uaauggauau uacucgaugc
cugaaauguu 4620gaagaaacca cuuguugaug ccggauugca uaaaacaagu
gcuagcagua uaggugaagg 4680ugcuaguagu uccgguacac cugcaacauc
agcugcucca aguccagcuc cuacucuuuu 4740ggauaagaca caaauugaag
auuugaguga aaaagaagau ccgucaaaga cugaagauaa 4800aaccaccgau
gauuccaaac ccucagaaga ggcuaaagcu gcagaugaug caaauaagcc
4860ucaggcugaa ggagaaaagg cagaaggauc uucuaaugca aaccaaacuu
cugaagcuga 4920aggaagcgau gagaaaaaac ccaaagaaga accgauggau
guagauggug aaggagaggc 4980uaaagauagu gauaagacag aaaaacaaga
agguacugac gaaaaagaug uagcccuaaa 5040agaggaagaa aaggaugaag
aggucaacaa agagaaggga gaggaaacag aggaaaagaa 5100gguuaucgau
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5160aggauuuacu gagcuccaua ccuuauggca aaaugaagag aaagcugcag
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uuggguggaa ucguuaccca 5280uggcuauggu cgguggcaag auauucaaaa
ugauauuaga uuugcuauua ucaacgaacc 5340auuuaagaug gauguuggaa
aaggaaauuu cuuagaaauu aaaaauaaau uucuugccag 5400gagguuuaag
cuucuugagc aagcucuggu gauugaagaa caguuaagac gugcagcuua
5460uuuaaaucug acgcaagauc caaaucaccc agcaauguca cugaaugcaa
gauuugcaga 5520gguugaaugu cuagccgaau cucaccaaca ccucucgaag
gaaagucuug cuggcaacaa 5580accugcaaau gcaguguuac auaaaguauu
gaaccaauua gaggagcuuc ugucggauau 5640gaaaucugac guaucucgac
uaccagccac ucuagccaga auuccaccug uagcccagag 5700gcuacagaug
ucugaacggu caauacuuuc uagguuggcu gcaacuacuu cuccugcgac
5760gcccaccacg ucccaucaaa cugguaugau aagcagucag uucccugcug
gauuucaauc 5820agggcaguug acuggaacgu uuccgaaugc caguuuuacc
aacuucaggc cccaguauuc 5880aguuccuggg caaacugcag cccaggguuu
ucccgguaau ugauaauuga aagcuggacg 5940guaauugucu gcgagugaau
ucuccaugag uaaauaauag guuuuuuuuu uuuuuuaaga 6000aagaaauaaa
agaagcguuu uguuuaguuu uguugauagu ucucuuuauu ucuuucaauu
6060uuguuuuagc ggaaaaaaaa auguucauua uaaguaacuu auaaauugga
caugcuaauu 6120aaauuuccua uuagauuauu uuguuauuug uaaguuuuuc
gguauuguaa gaaugucuau 6180auguguaaga gguuguacaa gauugccuaa
auaccuugua uuauuuauuu uuacuauuga 6240auaaaaaaaa aaaauaauua
acuucgaucu uagguuaagg guaauaaaaa aaaauguuac 6300uggaaaaaaa
aauagaaaaa auaaaaaaga uagccuuucc ccuuac 6346463391RNAEuchistus
heros 46agagggggua ggcgcacagc uuuccucaca ucgaacaaua ucuuagugaa
ugaauggcuu 60uauuggccgg uucaaaaucu uguuaaaugu ugguuugaua uauauuuaua
cuaacguuau 120uuaacgcagc ucacaccaau aaaaaugucg aagccaaaug
aaguuaguuu ggauacaaca 180gauacuguug aaauuucuaa ugaaucuucg
ggagacacag agucguccaa ggguaaaaau 240gaagauuuug aaacaaaaau
ugaaacugac cguucuagaa gauuugaguu ucuguugaag 300cagacagaaa
uuuuuucaca uuuuaugaca aaucaaggaa agucgaacag cccugcaaag
360ccuaaagucg gccguccuag aaaggaaacu aauaaauugg caccagccgg
uggugauggu 420ucugccgacc aucggcaucg uaugaccgag caggaagaag
augaagaacu gcuugcugaa 480aguaauacuu cuucaaaauc cuuagcaagg
uuugacgcuu cuccuuuuua uauuaaaagc 540ggagaguuga gggauuacca
gauacguggu uugaauugga ugauaucccu cuacgaacac 600gguauaaaug
guauacuugc ugaugagaug gguuuaggua aaacucucca aacuauuucu
660cuccuugguu acaugaagca uuauagaaau auaccagggc cacauauggu
caucguacca 720aaaucaacau uagcuaauug gaugaaugaa uuuaaaaagu
ggugcccaac ccugcgugcu 780gucuguuuaa ucggagauca ggaaacgagg
aaugcguuca ucagagacac ucuuaugccg 840ggugaauggg augucugcgu
uacaucuuau gaaaugauca uacgagaaaa gagcguuuuc 900aagaaguuca
acuggaggua uauggucauu gacgaagccc acaggaucaa gaaugaaaaa
960uccaaacucu ccgagauugu gagagaguuc aaaacgacga aucgauuacu
ccugaccggu 1020acuccuuuac aaaauaaccu ccacgaauug uggucucuuc
uuaacuuccu cuuaccagau 1080guuuucaauu caucagauga uuuugauuca
ugguuuaaua ccaauaccuu ccuuggcgau 1140aauucucuug ucgagagauu
acaugcugua cugagaccuu uccuccuaag aagauugaaa 1200ucugagguag
agaaaaaacu caaaccgaag aaagaaguca aaaucuacgu uggauugagu
1260aaaaugcaga gagaauggua uacuaaaguu cuaaugaaag auauagacau
uguaaacggu 1320gcuggccgag ucgaaaaaau gcgccuccaa aacauccuca
ugcaguugag gaagugcagu 1380aaucacccuu aucucuucga cggagcugaa
ccagguccac cuuacucaac ugaugagcau 1440cugguauaua acaguggaaa
aaugguaaua uuagacaagc uucuuccuaa auugcaagaa 1500caaggaucac
gaguucuggu uuucagccaa augacaagga ugauugauau ucucgaagau
1560uacuguuauu ggagaggaua uaauuacugu cgucuugaug guaauacacc
ucaugaggau 1620aggcagagac agauuaauga guucaacgaa gaagacagua
agaaauucau uuucauguug 1680ucgacucgug cgggugguuu ggguaucaau
uuagccaccg cagauguagu cauuuuguac 1740gauucggauu ggaacccuca
aauggaucuc caggcuaugg aucgugcuca ucguauuggu 1800caaaagaaac
aagucaaagu guucaggaug auaacugaaa acacaguuga agagaaaauu
1860guugagagag cugaaauaaa acuccgccuc gauaaguugg ucauccaaca
aggcaggcug 1920guagacaaua aaacggcacu caacaaagau gaaauguuga
auaugauccg ucacggugcc 1980aaucauguau uugccaguaa agauucugaa
aucaccgaug aagacauuga cacuauuuug 2040gaaaaaggcg aagcaaggac
ggaagaaaug aauaaaaaac uugaacaacu cggugauucu 2100aauuugaaag
acuucaugau ggaaaccccg acugagucag uuuaccaauu cgaaggagag
2160gauuacaggg aaaagcagaa aguuuuagga auaggaaguu ggauagaacc
uccaaaaaga 2220gaacguaaag cuaauuacgc ugucgaugcc uauuuuaggg
aagcauugag aguaucagaa 2280ccuaaagcuc ccaaggcacc gaggccuccu
aaacagccua uaguucaaga uuuccaauuc 2340uuuccuccuc gucucuuuga
gcuauuggac caggagaucu auuacuucag gaaaacugug 2400ggcuacaaag
uuccuaaaaa uccugaauua gguucugaug caucacgugu ccaaaaggaa
2460gaacaaagaa agauagauga ggcagaaccu uuaucagaag aagaacucgc
ugaaaaggaa 2520aaacuucuua cgcaggguuu uaccaauugg acuaaaagag
auuucaacca guuuauuaaa 2580gcuaaugaaa aauauggucg ugaugauauu
gacaauauuu caaaagaagu agaaggaaaa 2640acuccagaag aaguaagagc
uuauucagaa guguucuggg aacgauguaa cgaauugcag 2700gacauagauc
guaucauggg gcagaucgac aggggagagg cuaaaauuca aaggagagca
2760aguauuaaga aagcucucga uacaaagaug agccgguaca gagccccauu
ucaucaacuu 2820cgcaucuccu acgguacgaa uaaggguaag aacuauaccg
aggaagaaga uagauuccuu 2880gucuguaugu ugcauaagcu ugguuuugac
aaggaaaaug uguacgaaga acuuagagcg 2940auggucaggu gugcgccuca
guucagauuc gacugguuca ucaaaucgag aacagccaug 3000gaauugcaga
ggcguuguaa uacucuaauu acucucaucg aaagagaaaa ucaggaacuu
3060gaggagaggg aaagagccga gaagaggaaa ggaagaggaa gugggcgugg
uccugguucc 3120gguaaaagga aaggagacgg uuccauuuca ucucccccuc
cugucccugg ccaaggggau 3180aagaacagcc ccgccagaaa aaagaaaaaa
auguaguuuc accuccucau gaaaggaacu 3240cauuuuaaga uaucuuuuuc
uagauauuua uuuugugaaa acugugaugu auuuuauauc 3300cguuccgaaa
agcucuacug uuuugacagu uuuauuaauu aguggggugg ggaggaaaua
3360uagcccccuc accccccaau aauucauaaa u 3391471316RNAEuchistus heros
47aaugaauaaa aaacuugaac aacuuggugu ugauucauca uuaaaagauu ucaugaugga
60ggcucccacu gagucugucu aucaguuuga aggcgaagau uauagagaaa agcaaaaagu
120uuuuggaauu ggaaauugga uugaaccacc aaaacgagaa cguaaagcaa
auuaugcagu 180agaugccuau uuuagagaag cacugagagu uucagaaccu
aaagcuccaa aggccccuag 240gccaccaaag caacccauag uucaagauuu
ccaauuuuuc ccaccucguc uguuugagcu 300guuagaucaa gaaauauacu
auuuucgaaa aacuguuugc uacaagguuc cuaaaaaucc 360ggaguuagga
ucagaugcuu cucguauaca aagggaagag caaagaaaaa uugaugaagc
420ugagccguug acugaggaag agcuagcuga gaaagaaaac uuauugaccc
aggguuuuac 480uaauuggacu aaaagagauu uuaaccaguu cauaaaagcu
aaugaaaaau auggacguga 540ugauauugau aauaucucaa aagauguuga
agggaagacu ccagaagaag uacgagcaua 600cucugaagua uuuugggaaa
ggugcaauga acuacaggcc auagaucgua ucauggggca 660gauugauaga
ggugaagcga aaauucaaag aagagccagu auuaaaaaag cuuuagauac
720aaagaugagu cgauauagag caccguuuca ucaacuacga auugcuuaug
guacgaacaa 780ggggaaaaau uacacagaag aagaagacag auuccuugug
ugcaugcuac auaagcuugg 840cuuugauaaa gaaaaugugu augaggaacu
uagggcgaug gugaggugug cuccucaguu 900uagguuugau ugguucauca
agucucgaac agcuuuggaa uugcaaagac guuguaauac 960ucuaaucacg
uuaauugaaa gggaaaacca agaauuagaa gaaagggaaa aaguagaaaa
1020aaggaaaagu cgaggcagua augggcgugg ucccaguucu gguaaacgua
agggagaugg 1080aucuauuuca ucuccaccug ucucuguaca gagugauaaa
agcagcccug cucggaaaaa 1140gaaaaaguau aucucuguug aguaaauuua
ucuuaaaacu gggaguagau acccaauucu 1200cauuaucggg ugaucaagga
aucaaucuca uauaggagcc uaaaacuuca uuaguuugua 1260auugaauauu
uaauuuacau cucuaguuuc caaauauugu uucuuuuaca ucugua
1316481827RNAEuchistus heros 48gauaaauaug aauaagaaaa uuuuaaauuu
auuuguuuca uuaaaaaauu aucuuauggg 60uuuauugauu auaaauuggu ucaaucauaa
aauacgagau acauaagauu guauuaucau 120aacaaaccca aucucuagua
ucgucauccu gcuguucugg uucacucuga guuucuuuau 180cuucaucaaa
agcaaaacuu gcaacuuuaa aagcagaaag uaauucauca ccaacagugg
240cugguccuuc aucucugguu ucagcucuuc ucaaaauuuc gucaauguca
caaguugguu 300cuucaucacc aucuucuuca ucuuuaaaua auucuucagc
cccaaauuuu aaaauagcag 360uaaguucuuc uuuguuaaaa ggcgcacugg
augaagaauu uuuuuuaucc aggacaguuc 420uaccuguagu auccauucuu
uguauaacua aaugaucuaa gaccauuuuu uguuuggccc 480gcucgacaau
auuuuccuca acagaacuuu uaguaacaag ucuguauaug uucaccugau
540uuuucugacc gauucuauga gcucuagcuu gugcuugcaa aucauuuugu
ggauuccaau 600cagagucaaa uauaaugaca guaucagcug uugcuaaauu
aaugcccaaa ccaccagcac 660gaguugauaa uaagaaacag aaaucuggug
aauuuucagc auugaaauga ucgagggcuu 720gcuuucucaa uucaccuuua
auugaaccgu cuaaacguug gaaagggaaa ugucucauuu 780gaagauacuc
agccaguaua uccaacauuc guaccauuug agaaaauaua aguacucuau
840gcccaguuuc uuuaaggcga acaagcaacu uguccaacag aaguaauuuc
ccugagccuu 900uuaacaauug cuguaaguag ucuucaguuu uugcuucauu
uucuaauggu uuuauuagau 960gugcaugauu acagcauuuu uuuaauucaa
uaacaauauu uauaaaugua cuaggagaac 1020cuuugacucc uuuucgaaga
gcagaauaau uuuuggacaa aauccaccug uaauacugcu 1080ucuguacaga
ugucauuuca acacguaaua uuuguuccac uuuagcuggu aaagauuucu
1140caacauccuu cuuaacucgu cguagaauau augguuccag cugucugugc
aacuuaguau 1200agccuuuauu agcagaguug ucauguucuu uuucaaauuc
uucccaguua uuaaaucugu 1260ugggcauaau aaagugaagc aacgcccaaa
gcucuuuaag acuauuuugc aaaggagugc 1320cuguuauaag aagccuaugg
uugguaucaa acucuuucaa uguuuuguau aauaaugaau 1380caucauuuuu
caaucugugu gcuucaucaa ccauaaggau agcccagcuu auacuaccca
1440aaaaugcuuu gucuuuaaga acaauuucau auguaguaag aauggcauug
aauuuuaacc 1500uuuucgaacc ugaauagcac cauucauaau uacguauaac
aucacgggag uuuauaucac 1560caauauaagu uacaacauuc auuucuggag
cccauaauga aaacucccuc ugccaugaag 1620ucaucguaga uaaagggaca
acaauuaaaa augguccaua caacugguga guaugaaaua 1680aauaauacaa
acugcagaua gucugaauag uuuuaccaag acccauuuca ucagccaaaa
1740uaauagaauu uucuuuacac cacgaaugaa ccaaccaauu caaaccacug
auuugauaau 1800cucucaaaac caauaccugg ucaccac 182749496RNAEuchistus
heros 49gacuaccucg agggugaagg uuauaaauau gaacguauug acgguacgau
caccgguagc 60uuaagacaag aagcuaucga ucgguuuaac gccccuggag cucaacaauu
uguuuuucuu 120uuguccacuc gugcgggagg ucuugguauu aaucucgcua
cugcagauac aguuauuauu 180uaugacucug acuggaaucc ucauaacgau
auucaggccu uuucgagagc acacaggaua 240gggcaagcaa acaagguuau
gauuuaucga uuugugacac gagcgucugu ugaagaaaga 300guaacgcaag
uggcuaagag aaaaaugaug uuaacccauc uugucguacg accagguaug
360gguggcaagc aagcaaauuu cacuaagcaa gaacuugaug auauuuuaag
guuuggaaca 420gaagaacuuu ucaaagaaga gcaggguaaa gaagaugaag
ccauucauua ugacgauaaa 480gcuguugaag aauuac 49650481RNAEuchistus
heros 50caaaaauuga aacugaccgu ucuagaagau uugaguuucu guugaagcag
acagaaauuu 60uuucacauuu uaugacaaau caaggaaagu cgaacagccc ugcaaagccu
aaagucggcc 120guccuagaaa ggaaacuaau aaauuggcac cagccggugg
ugaugguucu gccgaccauc 180ggcaucguau gaccgagcag gaagaagaug
aagaacugcu ugcugaaagu aauacuucuu 240caaaauccuu agcaagguuu
gacgcuucuc cuuuuuauau uaaaagcgga gaguugaggg 300auuaccagau
acgugguuug aauuggauga uaucccucua cgaacacggu auaaauggua
360uacuugcuga ugagaugggu uuagguaaaa cucuccaaac uauuucucuc
cuugguuaca 420ugaagcauua uagaaauaua ccagggccac auauggucau
cguaccaaaa ucaacauuag 480c 48151490RNAEuchistus heros 51guucaagauu
uccaauuuuu cccaccucgu cuguuugagc uguuagauca agaaauauac 60uauuuucgaa
aaacuguuug cuacaagguu ccuaaaaauc cggaguuagg aucagaugcu
120ucucguauac aaagggaaga gcaaagaaaa auugaugaag cugagccguu
gacugaggaa 180gagcuagcug agaaagaaaa cuuauugacc caggguuuua
cuaauuggac uaaaagagau 240uuuaaccagu ucauaaaagc uaaugaaaaa
uauggacgug augauauuga uaauaucuca 300aaagauguug aagggaagac
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aacuacaggc cauagaucgu aucauggggc agauugauag aggugaagcg
420aaaauucaaa gaagagccag uauuaaaaaa gcuuuagaua caaagaugag
ucgauauaga 480gcaccguuuc 49052496RNAEuchistus heros 52cagcuggaac
cauauauucu acgacgaguu aagaaggaug uugagaaauc uuuaccagcu 60aaaguggaac
aaauauuacg uguugaaaug acaucuguac agaagcagua uuacaggugg
120auuuugucca aaaauuauuc ugcucuucga aaaggaguca aagguucucc
uaguacauuu 180auaaauauug uuauugaauu aaaaaaaugc uguaaucaug
cacaucuaau aaaaccauua 240gaaaaugaag caaaaacuga agacuacuua
cagcaauugu uaaaaggcuc agggaaauua 300cuucuguugg acaaguugcu
uguucgccuu aaagaaacug ggcauagagu acuuauauuu 360ucucaaaugg
uacgaauguu ggauauacug gcugaguauc uucaaaugag acauuucccu
420uuccaacguu uagacgguuc aauuaaaggu gaauugagaa agcaagcccu
cgaucauuuc 480aaugcugaaa auucac 496534493RNAEuchistus heros
53gcauauacag aaacaaucaa aagaucaaau cuaacucguu caaacagcag gaugaagaaa
60acuaggaaga auucucgacc uuuauuugug gcuggugguu uugcagcggc agcugagaag
120augccugagu aggccugauu gagagugcag guacggaugg cuuucuuccc
gguccucuca 180guccucuggg gccacgugag ccaauaugac cagggccgac
gggccucaac cguggccucc 240cuggaccucg acgauugcuu cgguugcuua
aagcaggcau gaaaggccuc gaaauuuuug 300guggugucug gcugauaggu
uccuccuggu cgguguuguu aucauuggag ucuuggcucg 360auuucauaac
guccgugccc uucggacucu cagcugguga uuucacaccu ugguaaacuu
420caacgacccc aucuucaguc uucauauguu ucaccguguc cuuguaauuu
ccuagagcau 480uuuuucuuuu ucuuucucuu ucccuguaga uaucuugccu
ugauucuuca aguugucucc 540ucucugcuug cuucugccga uauuucuguu
cuauuucugc aucaucaagc aauagagaaa 600caaccucuuu cgguuuaaga
gugucugguu ugaaauuacc cccacugaug acaacccuuu 660ggauuucacu
uuuuucuuga gcucucugca agauccuuuc uucgaugguc ccuuuacaaa
720ugagccgaua gacugugacu uguuucguuu guccaagacg augggcacgg
uccaucgcuu 780guuggucaac aguaggguuc caaucacugu cauagaauau
cacaguauca gcugcgguaa 840gguugauucc aagaccucca gcucgcguac
ucaacaggaa uacaaauaug uccucucuug 900uuugaaaguc agcaaccaua
ucccuucugu cugauaucuu ugaagaacca ucuaaccuca 960uauaugugug
cuuucuguac cacauauauu ccucuaacaa gucgaucauc cuugucaucu
1020gggaguaaau uagagcucga ugcccuuguu ccuucagccu ggugagaagc
ccauccaaga 1080cguacagcuu uccagcauca guuaccagug ucuguuuguc
agguaugacu auacucgacc 1140agccagugau uggucgaaga cugaguaggc
caagaggagg ugggcacugg aacccagguu 1200cuuguuuauc cccuucccaa
agaaccugcc acagucuccc
guccuccccc cagaggugag 1260aagagaucca gcuccaccuu cgguucgagc
uguacagccg ucugcuuuuc uuuaucucca 1320ccuugacagu aggagugaag
aggaauggag guaaauagac uggcugacau gagagaaucu 1380gaggcuuccu
cacaauaugu ggcaaugcug guaauaaguu agcacuaucu ucuccaccgc
1440acucucccuu agccuucuuc gaccugacua ugcgauguuc uaucguuuca
ggcauacuuu 1500cuauccucac gguggaaugg gaaaacacuu gguuggaaua
aguggugaac acaagauugu 1560uuggguagcu ccugucuuca augauuccga
cgaguuuccu ucucacuccu ugccuccuug 1620cuauaauauu gugccaccug
aaauaaaucc caaaaaacau uagcuuauac aguucuccua 1680cagauaaccu
caacagucga gugaaagaaa augaacuauc ccucucggau gagucaucac
1740ugaauaaaga ucuaugagaa ugaaaaggau uaaaaggcga uaaguaguuc
augagaagau 1800gaaguuuauc cccagggaac acagcaucug ugaugagagc
aggcacaaug uaaucuuccg 1860uagccaugga aaaaggggau cgagguuccc
uucgcucgaa uaguucaggg ugauuacaga 1920ccuuacgaaa uugcaucacg
agguucauca aauuugaagu gauacucuga gcugauuggu 1980aagaagaucc
agaagagugu agcaaaucuu caauucgaau cuucuuuuuc acagcugaau
2040auaacaucuu cugccucguc gucagaggac aguacaccau gauuucuauu
uuaucugaca 2100guucauucuc cacaucuguu uuuacucucc gcaacaugaa
ugguuuuagg aucauaugua 2160aacgggacaa augcuuuuca ucaauacugg
uuuuaugcuc ugcaugacuu ucuauaucuu 2220uugaaaacca uucauugaac
ucaucgugug aaucaaacau ugagggcauu augaaaugaa 2280gaagagccca
aaguucagcc auugaguuuu gaauaggugu uccacucaga aguaaucugu
2340ugcggcaauu gaauccaaga agcaauuucc aacgcaugcu uguagugcuu
uugauagccu 2400gagcuucguc uagaauuaaa uacugccauu uuauccuauu
gaaguauuuu auaucaguaa 2460uuacaagcug auagcuugug aucacaacau
ggaaacuggc aucuuuagua uguaaaccuu 2520uuugauccca aaauugacgu
aauauuuucc uuuccugcug auuuccccaa uaaggcacaa 2580cuuugaaauc
agguacaaaa cgcugcauuu cuugcugcca auuauguaau guagaagcgg
2640gcgauauuau gagaaaugga ccccaaacag aguauuuuuc agcaauaugg
caaagaaagg 2700cuaucgauug gacugucuuu cccaauccca uuucaucugc
caagauucca uuaauaccuu 2760ggucauauaa auucacaagc caugucauuc
ccuuuauuug auaucccuug agaguaccac 2820ggaauaucug cgguuggggu
uuaucuucac caacaucucc auccucuucc auuuucuggc 2880uuacuccaaa
uucucuugcu cgugcuucuu ccagaaaaaa caccuuuuca acuuucuuuc
2940guacuuuuuc uuucucugcc ucgcaaucgu agucauccaa agguaggagu
cuaggauuag 3000cuuccucuuc aaguuggcuc aggauucgaa guugaucuuc
agucguuccg ccaccgagcu 3060uacgggacau aaagugagca uagaguucug
uuugaguuau gaggaaauuu aauuuucuuu 3120gcugccucuu agccuccauc
aguucuacau ccaacuuccu uuguuccucu gcuucuuucu 3180ccauucuucu
ucuuguuucc cuuuccaccc ucucaaaucu uuuccaguau acuugcauuu
3240cccgugucaa ucuuuuugcu cuccaaauaa ccucuuucau auucuuuugc
gauugcauug 3300cacguugucg acagugccuc auacaguuag uagcagcucg
ccugcaagcu guuagaauuu 3360cuuuaugguu acuuauccua uagcgcugaa
ccuuuccaau uucuuuuuuc gccauguugg 3420cccaaauuuu acgccugcga
ugugccauga uuucggcagc uuuguuuugg gcugauuuuu 3480uucuaaggcu
cauuuccuuu uuagaccuug acauuauuug uaggucuggu ucuucuuuga
3540uuuuucuuuu cuuuuuuucu acacuaaaac uuucaugugg uugaucuugu
uuuuucaacu 3600uuucuuuuuu uugaaaaaca aauuucuuuu ucuuaacaaa
accagaacuu gaacucugau 3660gcucaggaua cuuaucgaaa uuggagagga
guccugugcc auaguacaua uacucuuggu 3720uuuuagaauu gugguagaau
uuuuuuuuau auuuguuucu aaguacaugu ucacggagca 3780ugucuuguaa
guccucuuca guuauuucuu caucaucaga ggaaucugau gugucaguca
3840auagaacauc aacuaaccac ugccuaucua agcuuacauu acucaaguug
uaaagccucu 3900ucuugucagc uauccuaucu ucuuuaguug cuguuauacc
auuccauacc guuucuccgg 3960uauaggcauc aacaccagcu aauuccgugu
cagaaucacu ggaggcuucu ccauccucac 4020cuaaugguug uuuuaaaaag
ucuucaacau auguuaggaa aggagcuaug uccaagcuuu 4080uuucuagcuu
uugauaauaa agagguuugg caauuucugu uuucacuacc auguuuguuu
4140uaucaucacu cauacugcaa aaaucaauga caucaaaaga uaucucugca
ucccaguuag 4200aaaauauuac uaaacugaaa uguaaaacuu auauagaauc
auauuuaaaa ugaguugaac 4260aaacuauuac gcuugucaca uuuuuaguaa
accacaccca aauuaauauc uacuuuuaua 4320cauaaaccua aucagaauau
cagucagucc auacuagacg auuguaaaaa ugugcuaggg 4380gucaaauaaa
aaaggaaagu gaaauuaggu uaguauauau ugaaagacgc aucuccuuuu
4440cagagauuca gugaaauauu ucagccagcu ggguuagccu gacagaauuc aag
4493546108RNAEuchistus heros 54acaggcuuau aucaucuaac aauuaugaau
gucaugagau caaaaauauu auccgaacua 60aauuaaaaac acaaaaacga augacagaaa
gaucggccug aaucaguuua aauuaguccu 120uugauuguga aaaauuuuaa
agcuuugaau ccuuaauuau aauaauaaac uauuaaccgg 180caguguauca
ugaagcuaaa cuuguacuua uagucaaugg aacaacaucu gauggacgcu
240uuguuuuacu uaaccgacca acuuucuugc ugaccuuacu aaucucauua
cuugaggcag 300cagaagcucu ucuaguacga auuacaaggu uaggguucga
auaagguggu gggggaggau 360uaggaggagg gggaggugua cuacgccguu
uaccacucuu aaauuuucua cuuugcccug 420augaggguuu ccccccauuu
ugaggacugu caucuaaagu ccacagauca auugaaacac 480gaccaugaga
ucuaguccuu ggagugcccu ccucgauacu acuuccacua ugacaauuuc
540uucccccgcc cccacuagcu cuuguuuuau ugaccugauu ucgcgaauca
acgccagagu 600aagugagaac agcuuccgac ucagcuuccc cagaagguga
uuuucuaucu ccuugcagag 660cagccagucu accagccucc cacucuuuuu
uuuguuguuc gauuucugcu ucagcugcag 720ccaacugcuc uuuugaccaa
gcagcaucau uuucuuccau gaauuucaug gcauaccucu 780caacagcaga
aagcuguugc auaagauugu gaaguucuaa uucugccuug cucauuucuu
840gcccaacucc uucaugggua ucaauaggua uauuuucauc gaacucugcc
aacucagcag 900cagcuucugc uuuagcaacc uuggcugccg caacaucgga
uucauccuca gcuugggcca 960uugcacucuc gagcgcacca auugcuacuu
ucucaucaga guucgcaacc aucugugugu 1020cuucaggauu cugagcaguu
uuaucacuau uaugaagaac uucugccauc cuucuggaag 1080caucauucuc
ggagguguca acauuaaaca gaucuugaau uguugaacuc uuaaaguaag
1140cuguaguaaa guuuccuccu ucaauggcua caucucccag cauucucuuu
ugguucgcuu 1200uuuuaaguau auuuuccuca acaguuuuuu cgcugaucaa
ucuauaaaua uguacaucuc 1260ucguuuggcc gauucugugg caucgaucuu
gagcuugggc auccauggua ggauuccaau 1320cacuaucaua aaauauaaca
gugucugcac caguuaaauu aauaccaacu ccuccagauc 1380uuguggauag
aauaaagcaa aauauucucu ugucagcauu aaagcguucc auuaagagcu
1440gucucugauc uacuuucguu guuccaucaa gacgaagaua uaugugcccg
ugaaaguuaa 1500gaaaugcuuc caguacgucu aacauucuag ucauuugugu
aaauauuaau auccgaugau 1560ggucagcuuu caaucuucga agaagcuugu
cuaaugauug aagcuuucca cagucauacu 1620guauuagucu ucuaucaggg
aacugcguac ucauugcuga ugauaugcua ugaaguaauc 1680gaaguuuagg
ucuuagccaa guaucaacua augauagccu cuucucuucu uggaacaucu
1740uugaaggugg cggauguggc acauguaaac ggacagguug gcuggaaaca
gccgguacgu 1800acaaaacaaa ccuagagaau auaucagaca acuccgcaac
ucggucuucg auggaaugaa 1860uagcugcugu gagagcauga gucugauucc
aaaauaacaa gggguuugaa gauaaagcau 1920ucuuacagug aacuguucca
augcagucuu uaguuucauc aggagcauca uccaauguua 1980aagaugauag
aaggucagaa ccauaaauug gaagggccug acaccuuugu ucauugauuc
2040uaacaaucag uuccaacuuc uccuuucgcc guuuuuuucu aagauuuucu
agguauucau 2100cucucaaauc uucauucgga uuagauucuu cuuuguuauu
uuuauuugag uucaaaguac 2160uuugucuugu uacuuuuuua agagaaggua
augaauugac guucgaucca uuuccuugag 2220cuggcucuga cgauaugcuc
acaggugaau uguuuacagu agucaaacug uuuugaacaa 2280caguagcugc
agaagugaug uuuaauggag guacccucau gauaggccga uuugaauuua
2340uuugggucac agaaguaggg uugacuaguu uagcuacauu gcuucccugu
uuggauacca 2400caguuaaucu uugcccagua guagucauua caguaguagc
accuccuuga ggaguaauaa 2460ccgguugugg agagaguacc agcugccuac
cgguaggggu auuaacuaau ugugcaaauu 2520gagggaccau acgaccagca
uugccuucau uuuuaaccac gccuccacua guaguuguua 2580augaagccac
ugagauugcu uucauggcac cagguuguaa uaacugaagg uaauuaggaa
2640ucugaccaga aguaggauuc gccagucuua aaguuacacu cuggcuagca
uuuuguccaa 2700gcuuuaucaa uggugauguu ccaacuuuug uugaaaaaga
agaaauccga ugauuauuac 2760uaacuuguac aauuggccua acagggcuag
aaaccugagg ugguuggggc aauauccguu 2820ucuugacauu aauuuuaacc
cuuccuuuag ggcaaggugg uagaucagga ggugcagaau 2880caauugugcg
aaugaaguca ggauuaacuu uguauuuacg agcucuguga gcaacaaaug
2940cuaauaacca uaguucaaau ucaauuaucc gcaaauuuag gaaaacuaga
ucgacauguu 3000uaaacggauc auaaucuaaa gcacuccaaa cuauagaagg
aacaugguau ucaagagaau 3060ccauuugaaa cggagacacg guagggcgaa
cuucgaacag guuaggauga uuacacacuu 3120uccucagcug caucaguaca
uuaaugacac ucaauaaacu accagaagca aguguuucuu 3180uuguuuuagc
ucuagacaug aaaucaucau auaaauaucg uugccuguug gauaaccgac
3240acauuacuau auguucauau uucuuuggca uuugcguuuc uacuucacac
uuuaaucuuc 3300uuaacagaaa cggacgcaac acuuuaugaa gucuuuuaau
aauaguguca uuguacucag 3360aauucccuuc aaucaugccu guuacuggau
uagaaaacca uucuuuaaau ucacgaugcg 3420auucaaaaac auuaggcaua
agaaaaugca uuaaugacca gaguuccaua agauuauuuu 3480guaauggagu
accagugagu aguaaccgcc uuugaguuug aaaauucaau aagaguugcc
3540aacguuguga uuuaaaauuu uugauauuuu gagcuucauc uaaaauuaaa
uauuuccauu 3600uuuuucuacg aaaacucuga ugauccugua uaacuaacuu
auaagaggua augcagauau 3660ggaaugcauu agguuuuguc cacccugauc
guuucaauuu ccguucuuuu ugaguuccau 3720aauaaguuaa uauuuugaau
gcuggacacc auuuuuuaaa cuccauuucc caguuuaaca 3780ugacagacgu
agggacaaug auuaaaugag guccccaauu accuuuuuca caagcaagau
3840gagcuauuaa ugcuaugguu ugaauugucu uuccgagacc cauuucguca
gcaaguauac 3900cauuuaguuu ccuaucauac auaguaacua accagucuaa
cccaaugugc ugauauucuc 3960uuaagggaug uuuaagaaga aaaggaacuu
ucguaacaac acuuguagau gauagagugu 4020uuccuuuagg cugaagacuu
ucagcuauag cagcaacauc auuuauuucc uugucuuugu 4080cuugguuccc
uuccucuaca ugaugcgagu cauuuaugag agccuucaaa gucgaaucuu
4140ccuugucacu ugaagaacug ucuucuucuu cuucuucuuc acuaacuuca
ucuucaaaau 4200cuucuucuuc ugaggcuuca ucagauucuu cauucaggau
uucuuuccuu uuucuugauc 4260uuugggaacu gggaucuccg uucgaaggua
aagguucgga cgggagcucg auaccauauu 4320uugcucuaag uuguucuaug
cucauauuuc cuuccuccuu uaaaucaucu auuucuuguu 4380uauaauccac
uguuccuuca gucuuuucuu guuccagaau uguuucuuca ucaucugaag
4440aaucaccaga aucuugauau uccaugucau cauccucaac aucuucaucc
augcugacag 4500gaguugauau agaaucccuu ugagcaaggu aguuggaaag
uagaucuuca aguggaagcu 4560cgcucucuuu uuucagaaga uccacuucau
cuugauuauc acauuuauca ucauuagcag 4620aaagugcuuc uuccuuagca
aucgucucuu caucgucguc agacggaucu ucuggcucaa 4680auucaucguc
agagugaugc cugggagaag uuggucuuga aguguucaug cuuucagcaa
4740caagacuuga auacuuuuca guuugaucaa caauaaaacu uaaguguuga
ucaagugcuu 4800uuuuucucuu uucuucuaac cuaguuuguu guuugaauuc
aacuagcuuu ucaacauuug 4860accagaacug uuugauuucu uucgcaauaa
aagaagcuau gcguuuuaac ugcauuucuu 4920gagcuuugau agcuuucugu
acuagagcuu cuuuuucuug aaaauguuuu ugaaccauuc 4980uugcacacuu
uuuugcagcu gcuuucuucc auuuccucuc cugggcaaaa ucugcagcca
5040gccaagccau uucuucuagc agauaauccc aaugagcuuu agcccuuggc
aauucaugga 5100cuuuagguaa ucuuuucucu ggccauaauc cauccuuuug
uaacucuccu acccuuugca 5160uuacauacgc cucuuguuua gcucuuucaa
caauuuguuc cuggcuauug acuccaucca 5220ccggagaagc auuuuuuaca
gcugauuuca cuguagucag uuuuuggaau uuggaagaua 5280auucugcuuu
gguaggacua gcagaugaug gaacccguac aucaguauug cuuccuggcc
5340uguucccagu aagagcagau uguagaagag agugauuuug ugauugcaga
accuuauaca 5400gauccucucc gucauuacca ggaucuaacu uauuuguuuu
uagaaauguc uguaaaggaa 5460cuacaggcaa acgagaccuc cacggacuga
agucugagag guuuccauuu gccuguagau 5520aacagagaag agcacaucuc
ucugcaaacu uugucugaaa ucuuuuauuu uuauuaucgu 5580uuaaaucuuu
uauucuuuuu cuaaaggaau uauauucauc aagcggcagc uugcguuuuc
5640uuguugaaga ugacgacaaa cuuaaugaaa gugaugguug aggguuaccu
uggcuaggag 5700guuggagauu ggucaucaua ggagaaucgg acaggauaga
agucggggag gagcggacuu 5760ggagucgccc cccuuggcuc gaguuugcca
accuugucau ugauggugaa cccuguugca 5820ggguuggcaa gagaggagcc
aaggggagca cuugagaucc agcacaaggu uuuucaagaa 5880cuggcuuuuc
aaagccaagu ugaagauugu ucauaucaaa uucauugugu uaucacaauu
5940uucuucgucc aucaacaaua aaaucaagaa aaugaucuuc gguaacgaac
uuuaggaaag 6000aacucauuuu acuaauuuau uagccaauua auucugauuu
uauucaaauu ccguggagaa 6060auauuccuau gccacauucu cuucaaugca
acauggcguu agguucag 610855240RNAArtificial SequenceSNF2/Helicase
degenerate dsRNA sequencemisc_feature(216)..(216)n is a, c, g, or
umisc_feature(222)..(222)n is a, c, g, or u 55cgsyuhcuyy umacsggyac
hccucuvcar aayaarcuwc chgaryusug ggcbyudcuh 60aayuuyyuvc ubccsucbau
yuuyaarwsb ugyucbacdu uygarcarug guucaaygcv 120cchuuygcha
cmacbggmga raarguygar yudaaygarg argaracvau yyukauyauy
180mgdcguyudc ayaarguyyu kcgwcckuuy yuvyunmgdc gnyuvaaaaa
rgargumgar 2405627RNAArtificial SequenceSNF2/Helicase degenerate
dsRNA sequencemisc_feature(21)..(21)n is a, c, g, or u 56mghgcygubu
gyyuhauygg ngaycar 275760RNAArtificial SequenceSNF2/Helicase
degenerate dsRNA sequence 57uayaarcuyc uvyusacmgg machccgyub
caraacaayc umgargaryu ruuycauyur 605861RNAArtificial
SequenceSNF2/Helicase degenerate dsRNA sequence 58garuuygaya
cbaaycaymg rcukcuhauh acwggbacyc ckyuvcaraa ywskyudaar 60g
615923RNAArtificial SequenceBromodomain degenerate dsRNA sequence
59yuswsygaac cruuyaugaa ryu 236065RNAArtificial SequenceHAND-SLIDE
degenerate dsRNA sequence 60gchguvgaug cyuayuuymg vgargcwyuv
mgdguyuchg arccyaargc dccdaargch 60cchmg 656136RNAArtificial
SequenceChromodomain degenerate dsRNA
sequencemisc_feature(33)..(33)n is a, c, g, or u 61mghaarurbg
ayauggavga rvvdccbaar yungar 366256RNAArtificial
SequenceChromodomain degenerate dsRNA
sequencemisc_feature(29)..(29)n is a, c, g, or u 62bhggdaarad
dggrkkbryb ggmaaymwna chacdrusua ykmhruagar gaaaay
56634569DNAEuchistus heros 63atggacggag acagcggtgg tatggcgagc
ccttcgccac agcctcagtc gtcaccaatg 60ccccctccac aagctccatc acctatgggc
ccgccgcagg gcgccccatc gccaatgccc 120ccttctaacc aacaggcggc
ctcaccaatg ggtccaccgc accaccccca cagcccgaca 180ggttaccaag
gagggatgcc acacatgaat ggaccaaatg gtgttcctcc tggtatgcag
240caggctactc aaacatttca gcctcatcag caattgccac cccaccagca
accaccaatg 300cagactgctc ctggtgggcc tgctagtggt ggaggacaag
aaaatcttag cgctctccag 360cgtgcaatag attctatgga agagaaaggg
cttcaggaag atccacgtta ctcgcagctg 420cttgcgttga gggcaaggca
tgccaacatg gaacctccgg ttaggcctcc atctcagctt 480gttgggggtg
ggttcagcgg tgagggtggt gcccctcctc ctgctaaaca cagcttcagc
540gcgaaccaac tgcaacaact tcgagtgcag atcatggcgt atcgcctact
tgctaggaac 600caacctcttt cccagcagct agctttggct gtgcaaggca
aacgcctcga cagccctggc 660gagtccaact accagcatcc tcctagtgaa
ggagcaggag gtgttggtgg agaaggaagt 720ggagacgggg gatcgtcgaa
cggcctgatg acgcagccga tgcgtgcccc atgcccccct 780ggtggccagc
ccccaacggc ctcaccgatg acaggccaga tggcacctcc tactgggcca
840gctcctgtaa ggccacctcc tcccggtgtg tctcctacac ctccgcgccc
tcctcagcag 900gttcctggtg ctccgggggc cccacaacca aagcaaaata
gggttaccac catgccaaga 960ccgcatggtt tagatcccat tcttattctc
caggaaagag agaatagagt agccgctagg 1020attgtacata ggatggaaga
attatcaaat ttaccagcta cgatgcctga agaccttcga 1080ataaaagcgc
agatagaact tagggccttg agggtactta acttccaaag gcaattaaga
1140gcagaggtga tagcttgtac tagacgcgat acaacattag aaacagctgt
aaatgtgaaa 1200gcttataaac gaacgaagag gcaaggctta cgggaagcca
gagctacgga aaagcttgaa 1260aaacaacaga aacttgagac agaaaggaag
aagagacaaa aacaccagga atatctgagc 1320actatattgc aacattgcaa
agacttcaaa gaattccata gaaataatgt tgctaaagtt 1380ggtagattaa
ataaggctgt gatgaattac catgcgaatg ccgagcgtga acagaagaaa
1440gagcaagaaa ggatagaaaa agaacgtatg agaaggctta tggctgagga
tgaagagggt 1500tacaggaaac tgattgatca gaaaaaagat aagagattgg
cattccttct ttcacaaact 1560gatgaatata ttgccaatct tactgaaatg
gtgaagcagc ataaaatgga acaacagcgt 1620aagcaggaac aagaagagca
acaaaaacgg aagaggaaaa agaaaaagaa gaatagggaa 1680ggagatccag
atgatgaaag ctctcagatg tcagatttac atgttagcgt tatagaagca
1740gcaactggtc ggcagctgac gggggaggat gctccattgg ccagccagct
tgggagctgg 1800ttggaggcac acccgggctg ggagcctttg gaagatagcg
aagatgaaga tgatgaagag 1860gacagcgacg aggaaggtga tgataacagt
agatcaaaag gtggtttttc aatgatagga 1920aaagatgaag ctgatagcaa
gttatctgtt gaagacgaag ctcgagaaat gataaagaaa 1980gcgaagattg
aagatgatga atacaagaac acgaccgaag aacatacata ctacagcatc
2040gctcacaccg tgcatgaaat tgtcaccgaa caagcttcaa tcatgattaa
cggtaaattg 2100aaagaatatc aaattaaagg tcttgaatgg ttggtttctt
tatacaacaa caacttgaat 2160ggaatcctcg ccgacgagat gggccttggc
aagacaattc aaacaatagg tctcattact 2220tatttgatgg agaagaagaa
agtaaatggt ccttacctca ttattgttcc tctgtcaaca 2280ttatccaatt
gggttttgga attcgagaaa tgggctcctt cagtgtttgt ggtagcttat
2340aaaggttctc ctgcaatgag gagaacttta caatcacaga tgcgctcgac
gaagttcaat 2400gtcctgctca cgacctacga gtatgtcatc aaggacaagg
cagtacttgc aaagttgcat 2460tggaagtaca tgataatcga cgagggacac
aggatgaaaa accaccattg taagctgacg 2520caggtgctga acacccatta
tttggcacct caccgcctcc ttctcacggg cacacctctc 2580cagaacaaac
tacctgagct ctgggctctt ctaaactttc tcctcccgtc catcttcaag
2640tcgtgttcta cgtttgagca atggttcaat gcaccatttg ctaccactgg
agaaaaggtt 2700gagttgaatg aggaagaaac aattttgatt atcaggcgtt
tacataaggt ccttcgacct 2760ttcctccttc gtcgactgaa aaaggaagtc
gaaagtcagt tgccagagaa aattgaatac 2820atcgtcaagt gtgatatgtc
tggtctccaa cgtgtacttt ataggcacat gcagagtaaa 2880ggagtcctgc
ttaccgatgg ttctgagaag ggcaagcagg gtaaaggagg agctaaagcg
2940ctaatgaaca cgatcgtcca attgaggaag ctttgcaatc atcctttcat
gttccatcat 3000attgaagaaa aatattgtga tcacgttggc cagaacaacg
ttgtcacagg gcctgatctg 3060ttccgagttt ctggtaaatt tgaattcctc
gatcgtatat tgccaaaact gaaggccacg 3120agccataggg tacttctttt
ctgtcaaatg actcagctga tgaccatcat ggaggattat 3180ttgtcttgga
gagggttctc ctaccttcgt cttgatggta cgaccaaatc tgaagaccga
3240ggagatcttc tgaaaaaatt caacaatcca gaaagtgaat attttatttt
cttgctctca 3300accagagctg gaggtctcgg attgaactta caggctgcag
atactgtcat tatatttgat 3360tcagattgga accctcatca ggatttacaa
gctcaagaca gagctcatag gattggacag 3420caaaacgaag ttcgtgtttt
gcggctaatg acagtaaatt ctgttgagga gcgtattctt 3480gcagctgctc
ggtacaagct gaatatggat gagaaagtca ttcaggctgg tatgtttgac
3540cagaaatcta caggaaccga gaggcagaaa tttctgcaaa acatccttca
tcaagatgat 3600gcagatgatg aggaaaatga agttccagat gatgaaatgg
ttaatcgtat gattgcgcga 3660acagaagatg aattcaacct cttccagaaa
atcgatttag aaaggaggag ggaagaggct 3720aaacttggac ctaacaggaa
gtcaaggctt gtagaagagg cggaattacc tgactggctt 3780gtaaagaatg
acgatgagat tgagaagtgg acttatgaag aaaccgaggt ccaaatggga
3840agaggtaata ggcagaggaa ggaagtagat tatacagata gtttgactga
aaaagaatgg 3900ttaaaggcca ttgatgacaa tgtagatgat tttgatgacg
atgaagagga agaggtaaaa 3960acaaagaaaa gaggcaagag aagaagaagg
ggagaggatg atgaagaaga tgcaagtact 4020tcaaagagaa ggaaatattc
tccatctgaa aacaaactga ggaggcgtat gcgtaacctc 4080atgaacattg
ttgttaagta tactgacagt gactcgagag tactcagtga accattcatg
4140aaacttccct ctcgccataa
gtacccagac tactatgagt tgatcaagaa acctatagac 4200atcaagagga
tattggccaa agtagaagag tgtaaatatg ctgacatgga tgaattagaa
4260aaggatttta tgcaactttg taaaaatgct cagacataca atgaggaggc
ctcattgatc 4320tatgaagatt cgatagtatt agaaagtgtt ttctctaatg
ctcgtcaaaa agtagagcag 4380gataatgatt cagatgatga tgaaagtaaa
ggtgaccaag aagatgctgc atcagacact 4440tcatccgtca aaatgaaatt
gaaactaaag cctgggagga cccgagggag tggagctggt 4500ggtaaaagga
ggagaagaaa atatatctct gaagatgaag acgaagacca tagcgaagtt
4560tccttaatg 4569646222DNAEuchistus heros 64atggcgtctg aagaagaagt
tgacgagtgt ttaccagttg acgatgaagt tgacactagt 60gttgttcaac aagaaggcac
tgaagaaaat tcacctgaca gtgatgaaag aagtaggata 120gaggaagaag
atgacgagta tgaccctgag gatgcgagga aaaaaaagaa aggtaaaaag
180agaaaagcca aaggggaaag caaaaaagaa aagaaacgta aaaaaaggaa
gaagaatgat 240agtgctgaag aaagtgaggg aggcggggaa gaagaaggcg
attccgatta tggaagaaaa 300tctaagaagt ctaaaggaac ttcacaacca
aaaccagtgc agcaagattc ttctggaggt 360gtaccttcag tagaagaagt
ttgcagcctt tttggactta cagatgtaca gattgactat 420accgaagatg
attaccaaaa tctgactacg tataaacttt ttcaacaaca tgttcgtcct
480attcttgcca aggacaacca gaaggttccc atcggaaaaa tgatgatgct
cgtggctgca 540aaatggagag atttttgcaa ttccaatcca aacgctcaac
aggaaccaga tccagaagct 600tcagaagaac aggaatattc taaacctacc
aggacacgac cttcacgagt ttcaactaca 660caaaatgatg atgaagaaga
cgacgatgct gacgaacgag ggaggaaaaa gagaagtgga 720cgaagtaaaa
agtcatcagg aaagaagtcc gctcctccgg ccacaaccaa ggtccctacc
780ctcaagatca agataggaaa aagaaaacag aattccgatg aagaagatga
aggttcagtt 840ggtgccgttt ctgaaaggga ctcagatgct gaattcgagc
aaatgctcgc agaagctgaa 900gaagttaata aacctgaagg tgttgtagaa
gaagaagaag gtgcagaggt ggctcctgta 960cctaagaaaa aggccaaaac
gaaaattggt aataaaaaga aaaggaaaaa gacacggact 1020actaacaagt
ttccagacag tgaagctggt tatgaaacag atcatcagga ctattgtgaa
1080gtttgtcaac aaggaggtga aataatatta tgtgatacgt gccctcgagc
ttatcatttg 1140gtctgtttgg atcccgaatt ggaagatacg ccagaaggca
aatggtcatg ccctcattgt 1200gaaggtgaag gtgtacagga aaaagaagat
gatgtccatc aagaattttg cagagtttgt 1260aaagatggtg gagaactttt
atgctgtgat tcttgccctt ctgcatacca cacattctgt 1320ttgaaccctc
cattgacaga tattccagat ggtgactgga agtgcccacg ttgttcggcg
1380aagcctttga gaggtaaagt gtcaaagatt cttacttgga ggtggttgga
atctcccagt 1440agtaaagatg aagaagacaa tactaaaaaa cgaaacaggc
agaggcaaag agaatatttc 1500gtcaagtggg cagatatgtc ttattggcac
tgtagttggg tgtctgaact tcagatggat 1560gtttttcata ctcaaatgat
caggagttat attcgtaaat atgatatgga cgaacctccc 1620aaactagaag
aacccttgga tgaagcagac aatagaatga agaggatacg agaggcaaat
1680atcaatgagc aagaattaga agagaaatat tacaagtatg gtatcaaacc
agagtggctt 1740attgtgcaga gggtaattaa ccatcgcact ataagggatg
gaagcaatct gtacctcgtc 1800aaatggaggg acctccctta tgaccaggcg
acttgggagg aagaagtcac cgatatccct 1860ggcttgaaga aagctattga
atattacaat gagatgaggg cttgctgttt aggtgaatct 1920aaaaaactaa
aaaaaggtaa aggtaaaaga tcaaagagag atcaagatga tgaggaagga
1980agcagaagtg caggaatgat gggcgtcggt ggaccagcta ctggtcaata
cttcccgcct 2040cctgaaaagc ctgtcacaga tttgaaaaag aaatacgata
aacagccgga ctatctcgac 2100gtctccggta tgtgccttca tccttaccaa
ttagaaggtt taaattggtt gaggtattcc 2160tgggggcaag gaacagacac
tattcttgcc gatgagatgg gtcttggaaa aaccattcag 2220acaattactt
tcctctattc tctttacaaa gagggtcatt gtaaaggccc cttccttgtg
2280agtgtaccct tatctacaat tatcaattgg gaaagagagt tcgaaacttg
ggcgccagac 2340ttctacgttg tcacatatgt cggagacaaa gattctcgtg
ctgtaatacg tgaaaatgaa 2400ttttcattcg atgataatgc tgttagagga
ggaagaggtg tttctaaagt tcgctcttct 2460gcaataaagt ttcatgtact
gctaacatct tatgaactta tctctatcga tgtcacttgc 2520cttggatcga
tcgagtgggc agtgcttgta gtagatgaag cacacaggct gaaaagtaat
2580cagagcaagt tctttaggct tcttgcttca taccacattg cttataaact
tctgctgaca 2640ggaactccgt tgcaaaacaa tctagaagaa ttgtttcatt
tacttaattt ccttacgccg 2700gaaaaattca acgaccttgc gacatttcaa
aacgaattcg ctgatatttc aaaagaagaa 2760caagtcaaaa gacttcatga
gttactcggg ccgcatatgt tgaggagatt aaaagctgat 2820gtactcaaga
atatgcctac aaaatctgag ttcattgtta gagttgaact ctccccgatg
2880cagaagaagt actacaaata tattctcaca aggaatttcg aagctttaaa
tccaaaagga 2940ggcggtcaac aagtatctct tttgaacatt atgatggatc
ttaaaaaatg ctgtaatcat 3000ccatacctgt ttcctgctgc ttctcaggaa
gctcctttag gaccaagcgg atcttacgat 3060cttcaagggt taatcaaagc
atctggaaaa ttgatacttc tgtcgaaaat gctgagacgg 3120ctcaaagaag
agggtcacag agtactgatt ttctctcaaa tgacaaaaat gttggactta
3180ttagaagact acctcgaggg tgaaggttat aaatatgaac gtattgacgg
tacgatcacc 3240ggtagcttaa gacaagaagc tatcgatcgg tttaacgccc
ctggagctca acaatttgtt 3300tttcttttgt ccactcgtgc gggaggtctt
ggtattaatc tcgctactgc agatacagtt 3360attatttatg actctgactg
gaatcctcat aacgatattc aggccttttc gagagcacac 3420aggatagggc
aagcaaacaa ggttatgatt tatcgatttg tgacacgagc gtctgttgaa
3480gaaagagtaa cgcaagtggc taagagaaaa atgatgttaa cccatcttgt
cgtacgacca 3540ggtatgggtg gcaagcaagc aaatttcact aagcaagaac
ttgatgatat tttaaggttt 3600ggaacagaag aacttttcaa agaagagcag
ggtaaagaag atgaagccat tcattatgac 3660gataaagctg ttgaagaatt
acttgaccgg tcgaagatgg gtattgaaca gaaagaaaac 3720tggtctaatg
aatatctttc ttctttcaaa gtggcaagtt atgttactaa agaagaagac
3780gaagatgagg aaataggaac agaggtaata aaacaggaag cagaaaatac
agacccagct 3840tattgggtca aactgttgag gcaccattat gagcaacaac
aagaggatat ttctcgaact 3900ctcggtaaag gaaaaaggat tcgaaaacag
gtgaattaca tcgacggtgg agtgatggac 3960tcaagagaga acgccgattc
gacgtggcaa gacaacctct ctgactataa ttcagacttc 4020tctgctcctt
ctgatgatga caaggaagac gatgactttg atgagaaaaa tgatgatgga
4080acgagaaaga agcgtaggcc agaaaggagg gaggacaaag ataggcctct
acctcctctt 4140cttgcccgag tcggtggaaa cattgaggtc ctgggattca
acgccagaca gcgtaaagca 4200ttcttgaatg ctattatgag gtatggaatg
ccacctcaag atgcattcaa ctcgcagtgg 4260cttgttcgag acctgagggg
taaatctgag aagcatttca aggcatacgt atccctcttt 4320atgaggcatt
tgtgtgagcc tggcgcggac aatgccgaaa cattcgcgga tggtgttcca
4380agggaaggtc ttagtcggca gcatgttctc acaaggatag gtgtgatgtc
actcattagg 4440aaaaaggttc aagaatttga gcaaattaat ggatattact
cgatgcctga aatgttgaag 4500aaaccacttg ttgatgccgg attgcataaa
acaagtgcta gcagtatagg tgaaggtgct 4560agtagttccg gtacacctgc
aacatcagct gctccaagtc cagctcctac tcttttggat 4620aagacacaaa
ttgaagattt gagtgaaaaa gaagatccgt caaagactga agataaaacc
4680accgatgatt ccaaaccctc agaagaggct aaagctgcag atgatgcaaa
taagcctcag 4740gctgaaggag aaaaggcaga aggatcttct aatgcaaacc
aaacttctga agctgaagga 4800agcgatgaga aaaaacccaa agaagaaccg
atggatgtag atggtgaagg agaggctaaa 4860gatagtgata agacagaaaa
acaagaaggt actgacgaaa aagatgtagc cctaaaagag 4920gaagaaaagg
atgaagaggt caacaaagag aagggagagg aaacagagga aaagaaggtt
4980atcgattttg aagaagacaa atctaaaagg aaatttatgt tcaatatcgc
tgatggagga 5040tttactgagc tccatacctt atggcaaaat gaagagaaag
ctgcagtacc tggtagggag 5100tacgagatct ggcataggag gcatgactat
tggctgttgg gtggaatcgt tacccatggc 5160tatggtcggt ggcaagatat
tcaaaatgat attagatttg ctattatcaa cgaaccattt 5220aagatggatg
ttggaaaagg aaatttctta gaaattaaaa ataaatttct tgccaggagg
5280tttaagcttc ttgagcaagc tctggtgatt gaagaacagt taagacgtgc
agcttattta 5340aatctgacgc aagatccaaa tcacccagca atgtcactga
atgcaagatt tgcagaggtt 5400gaatgtctag ccgaatctca ccaacacctc
tcgaaggaaa gtcttgctgg caacaaacct 5460gcaaatgcag tgttacataa
agtattgaac caattagagg agcttctgtc ggatatgaaa 5520tctgacgtat
ctcgactacc agccactcta gccagaattc cacctgtagc ccagaggcta
5580cagatgtctg aacggtcaat actttctagg ttggctgcaa ctacttctcc
tgcgacgccc 5640accacgtccc atcaaactgg tatgataagc agtcagttcc
ctgctggatt tcaatcaggg 5700cagttgactg gaacgtttcc gaatgccagt
tttaccaact tcaggcccca gtattcagtt 5760cctgggcaaa ctgcagccca
gggttttccc ggtaattgat aattgaaagc tggacggtaa 5820ttgtctgcga
gtgaattctc catgagtaaa taataggttt tttttttttt ttaagaaaga
5880aataaaagaa gcgttttgtt tagttttgtt gatagttctc tttatttctt
tcaattttgt 5940tttagcggaa aaaaaaatgt tcattataag taacttataa
attggacatg ctaattaaat 6000ttcctattag attattttgt tatttgtaag
tttttcggta ttgtaagaat gtctatatgt 6060gtaagaggtt gtacaagatt
gcctaaatac cttgtattat ttatttttac tattgaataa 6120aaaaaaaaaa
taattaactt cgatcttagg ttaagggtaa taaaaaaaaa tgttactgga
6180aaaaaaaata gaaaaaataa aaaagatagc ctttcccctt ac
6222653072DNAEuchistus heros 65atgtcgaagc caaatgaagt tagtttggat
acaacagata ctgttgaaat ttctaatgaa 60tcttcgggag acacagagtc gtccaagggt
aaaaatgaag attttgaaac aaaaattgaa 120actgaccgtt ctagaagatt
tgagtttctg ttgaagcaga cagaaatttt ttcacatttt 180atgacaaatc
aaggaaagtc gaacagccct gcaaagccta aagtcggccg tcctagaaag
240gaaactaata aattggcacc agccggtggt gatggttctg ccgaccatcg
gcatcgtatg 300accgagcagg aagaagatga agaactgctt gctgaaagta
atacttcttc aaaatcctta 360gcaaggtttg acgcttctcc tttttatatt
aaaagcggag agttgaggga ttaccagata 420cgtggtttga attggatgat
atccctctac gaacacggta taaatggtat acttgctgat 480gagatgggtt
taggtaaaac tctccaaact atttctctcc ttggttacat gaagcattat
540agaaatatac cagggccaca tatggtcatc gtaccaaaat caacattagc
taattggatg 600aatgaattta aaaagtggtg cccaaccctg cgtgctgtct
gtttaatcgg agatcaggaa 660acgaggaatg cgttcatcag agacactctt
atgccgggtg aatgggatgt ctgcgttaca 720tcttatgaaa tgatcatacg
agaaaagagc gttttcaaga agttcaactg gaggtatatg 780gtcattgacg
aagcccacag gatcaagaat gaaaaatcca aactctccga gattgtgaga
840gagttcaaaa cgacgaatcg attactcctg accggtactc ctttacaaaa
taacctccac 900gaattgtggt ctcttcttaa cttcctctta ccagatgttt
tcaattcatc agatgatttt 960gattcatggt ttaataccaa taccttcctt
ggcgataatt ctcttgtcga gagattacat 1020gctgtactga gacctttcct
cctaagaaga ttgaaatctg aggtagagaa aaaactcaaa 1080ccgaagaaag
aagtcaaaat ctacgttgga ttgagtaaaa tgcagagaga atggtatact
1140aaagttctaa tgaaagatat agacattgta aacggtgctg gccgagtcga
aaaaatgcgc 1200ctccaaaaca tcctcatgca gttgaggaag tgcagtaatc
acccttatct cttcgacgga 1260gctgaaccag gtccacctta ctcaactgat
gagcatctgg tatataacag tggaaaaatg 1320gtaatattag acaagcttct
tcctaaattg caagaacaag gatcacgagt tctggttttc 1380agccaaatga
caaggatgat tgatattctc gaagattact gttattggag aggatataat
1440tactgtcgtc ttgatggtaa tacacctcat gaggataggc agagacagat
taatgagttc 1500aacgaagaag acagtaagaa attcattttc atgttgtcga
ctcgtgcggg tggtttgggt 1560atcaatttag ccaccgcaga tgtagtcatt
ttgtacgatt cggattggaa ccctcaaatg 1620gatctccagg ctatggatcg
tgctcatcgt attggtcaaa agaaacaagt caaagtgttc 1680aggatgataa
ctgaaaacac agttgaagag aaaattgttg agagagctga aataaaactc
1740cgcctcgata agttggtcat ccaacaaggc aggctggtag acaataaaac
ggcactcaac 1800aaagatgaaa tgttgaatat gatccgtcac ggtgccaatc
atgtatttgc cagtaaagat 1860tctgaaatca ccgatgaaga cattgacact
attttggaaa aaggcgaagc aaggacggaa 1920gaaatgaata aaaaacttga
acaactcggt gattctaatt tgaaagactt catgatggaa 1980accccgactg
agtcagttta ccaattcgaa ggagaggatt acagggaaaa gcagaaagtt
2040ttaggaatag gaagttggat agaacctcca aaaagagaac gtaaagctaa
ttacgctgtc 2100gatgcctatt ttagggaagc attgagagta tcagaaccta
aagctcccaa ggcaccgagg 2160cctcctaaac agcctatagt tcaagatttc
caattctttc ctcctcgtct ctttgagcta 2220ttggaccagg agatctatta
cttcaggaaa actgtgggct acaaagttcc taaaaatcct 2280gaattaggtt
ctgatgcatc acgtgtccaa aaggaagaac aaagaaagat agatgaggca
2340gaacctttat cagaagaaga actcgctgaa aaggaaaaac ttcttacgca
gggttttacc 2400aattggacta aaagagattt caaccagttt attaaagcta
atgaaaaata tggtcgtgat 2460gatattgaca atatttcaaa agaagtagaa
ggaaaaactc cagaagaagt aagagcttat 2520tcagaagtgt tctgggaacg
atgtaacgaa ttgcaggaca tagatcgtat catggggcag 2580atcgacaggg
gagaggctaa aattcaaagg agagcaagta ttaagaaagc tctcgataca
2640aagatgagcc ggtacagagc cccatttcat caacttcgca tctcctacgg
tacgaataag 2700ggtaagaact ataccgagga agaagataga ttccttgtct
gtatgttgca taagcttggt 2760tttgacaagg aaaatgtgta cgaagaactt
agagcgatgg tcaggtgtgc gcctcagttc 2820agattcgact ggttcatcaa
atcgagaaca gccatggaat tgcagaggcg ttgtaatact 2880ctaattactc
tcatcgaaag agaaaatcag gaacttgagg agagggaaag agccgagaag
2940aggaaaggaa gaggaagtgg gcgtggtcct ggttccggta aaaggaaagg
agacggttcc 3000atttcatctc cccctcctgt ccctggccaa ggggataaga
acagccccgc cagaaaaaag 3060aaaaaaatgt ag 3072661164DNAEuchistus
heros 66atgaataaaa aacttgaaca acttggtgtt gattcatcat taaaagattt
catgatggag 60gctcccactg agtctgtcta tcagtttgaa ggcgaagatt atagagaaaa
gcaaaaagtt 120tttggaattg gaaattggat tgaaccacca aaacgagaac
gtaaagcaaa ttatgcagta 180gatgcctatt ttagagaagc actgagagtt
tcagaaccta aagctccaaa ggcccctagg 240ccaccaaagc aacccatagt
tcaagatttc caatttttcc cacctcgtct gtttgagctg 300ttagatcaag
aaatatacta ttttcgaaaa actgtttgct acaaggttcc taaaaatccg
360gagttaggat cagatgcttc tcgtatacaa agggaagagc aaagaaaaat
tgatgaagct 420gagccgttga ctgaggaaga gctagctgag aaagaaaact
tattgaccca gggttttact 480aattggacta aaagagattt taaccagttc
ataaaagcta atgaaaaata tggacgtgat 540gatattgata atatctcaaa
agatgttgaa gggaagactc cagaagaagt acgagcatac 600tctgaagtat
tttgggaaag gtgcaatgaa ctacaggcca tagatcgtat catggggcag
660attgatagag gtgaagcgaa aattcaaaga agagccagta ttaaaaaagc
tttagataca 720aagatgagtc gatatagagc accgtttcat caactacgaa
ttgcttatgg tacgaacaag 780gggaaaaatt acacagaaga agaagacaga
ttccttgtgt gcatgctaca taagcttggc 840tttgataaag aaaatgtgta
tgaggaactt agggcgatgg tgaggtgtgc tcctcagttt 900aggtttgatt
ggttcatcaa gtctcgaaca gctttggaat tgcaaagacg ttgtaatact
960ctaatcacgt taattgaaag ggaaaaccaa gaattagaag aaagggaaaa
agtagaaaaa 1020aggaaaagtc gaggcagtaa tgggcgtggt cccagttctg
gtaaacgtaa gggagatgga 1080tctatttcat ctccacctgt ctctgtacag
agtgataaaa gcagccctgc tcggaaaaag 1140aaaaagtata tctctgttga gtaa
1164671665DNAEuchistus heros 67atgggtcttg gtaaaactat tcagactatc
tgcagtttgt attatttatt tcatactcac 60cagttgtatg gaccattttt aattgttgtc
cctttatcta cgatgacttc atggcagagg 120gagttttcat tatgggctcc
agaaatgaat gttgtaactt atattggtga tataaactcc 180cgtgatgtta
tacgtaatta tgaatggtgc tattcaggtt cgaaaaggtt aaaattcaat
240gccattctta ctacatatga aattgttctt aaagacaaag catttttggg
tagtataagc 300tgggctatcc ttatggttga tgaagcacac agattgaaaa
atgatgattc attattatac 360aaaacattga aagagtttga taccaaccat
aggcttctta taacaggcac tcctttgcaa 420aatagtctta aagagctttg
ggcgttgctt cactttatta tgcccaacag atttaataac 480tgggaagaat
ttgaaaaaga acatgacaac tctgctaata aaggctatac taagttgcac
540agacagctgg aaccatatat tctacgacga gttaagaagg atgttgagaa
atctttacca 600gctaaagtgg aacaaatatt acgtgttgaa atgacatctg
tacagaagca gtattacagg 660tggattttgt ccaaaaatta ttctgctctt
cgaaaaggag tcaaaggttc tcctagtaca 720tttataaata ttgttattga
attaaaaaaa tgctgtaatc atgcacatct aataaaacca 780ttagaaaatg
aagcaaaaac tgaagactac ttacagcaat tgttaaaagg ctcagggaaa
840ttacttctgt tggacaagtt gcttgttcgc cttaaagaaa ctgggcatag
agtacttata 900ttttctcaaa tggtacgaat gttggatata ctggctgagt
atcttcaaat gagacatttc 960cctttccaac gtttagacgg ttcaattaaa
ggtgaattga gaaagcaagc cctcgatcat 1020ttcaatgctg aaaattcacc
agatttctgt ttcttattat caactcgtgc tggtggtttg 1080ggcattaatt
tagcaacagc tgatactgtc attatatttg actctgattg gaatccacaa
1140aatgatttgc aagcacaagc tagagctcat agaatcggtc agaaaaatca
ggtgaacata 1200tacagacttg ttactaaaag ttctgttgag gaaaatattg
tcgagcgggc caaacaaaaa 1260atggtcttag atcatttagt tatacaaaga
atggatacta caggtagaac tgtcctggat 1320aaaaaaaatt cttcatccag
tgcgcctttt aacaaagaag aacttactgc tattttaaaa 1380tttggggctg
aagaattatt taaagatgaa gaagatggtg atgaagaacc aacttgtgac
1440attgacgaaa ttttgagaag agctgaaacc agagatgaag gaccagccac
tgttggtgat 1500gaattacttt ctgcttttaa agttgcaagt tttgcttttg
atgaagataa agaaactcag 1560agtgaaccag aacagcagga tgacgatact
agagattggg tttgttatga taatacaatc 1620ttatgtatct cgtattttat
gattgaacca atttataatc aataa 1665684569RNAEuchistus heros
68auggacggag acagcggugg uauggcgagc ccuucgccac agccucaguc gucaccaaug
60cccccuccac aagcuccauc accuaugggc ccgccgcagg gcgccccauc gccaaugccc
120ccuucuaacc aacaggcggc cucaccaaug gguccaccgc accaccccca
cagcccgaca 180gguuaccaag gagggaugcc acacaugaau ggaccaaaug
guguuccucc ugguaugcag 240caggcuacuc aaacauuuca gccucaucag
caauugccac cccaccagca accaccaaug 300cagacugcuc cuggugggcc
ugcuaguggu ggaggacaag aaaaucuuag cgcucuccag 360cgugcaauag
auucuaugga agagaaaggg cuucaggaag auccacguua cucgcagcug
420cuugcguuga gggcaaggca ugccaacaug gaaccuccgg uuaggccucc
aucucagcuu 480guugggggug gguucagcgg ugaggguggu gccccuccuc
cugcuaaaca cagcuucagc 540gcgaaccaac ugcaacaacu ucgagugcag
aucauggcgu aucgccuacu ugcuaggaac 600caaccucuuu cccagcagcu
agcuuuggcu gugcaaggca aacgccucga cagcccuggc 660gaguccaacu
accagcaucc uccuagugaa ggagcaggag guguuggugg agaaggaagu
720ggagacgggg gaucgucgaa cggccugaug acgcagccga ugcgugcccc
augccccccu 780gguggccagc ccccaacggc cucaccgaug acaggccaga
uggcaccucc uacugggcca 840gcuccuguaa ggccaccucc ucccggugug
ucuccuacac cuccgcgccc uccucagcag 900guuccuggug cuccgggggc
cccacaacca aagcaaaaua ggguuaccac caugccaaga 960ccgcaugguu
uagaucccau ucuuauucuc caggaaagag agaauagagu agccgcuagg
1020auuguacaua ggauggaaga auuaucaaau uuaccagcua cgaugccuga
agaccuucga 1080auaaaagcgc agauagaacu uagggccuug aggguacuua
acuuccaaag gcaauuaaga 1140gcagagguga uagcuuguac uagacgcgau
acaacauuag aaacagcugu aaaugugaaa 1200gcuuauaaac gaacgaagag
gcaaggcuua cgggaagcca gagcuacgga aaagcuugaa 1260aaacaacaga
aacuugagac agaaaggaag aagagacaaa aacaccagga auaucugagc
1320acuauauugc aacauugcaa agacuucaaa gaauuccaua gaaauaaugu
ugcuaaaguu 1380gguagauuaa auaaggcugu gaugaauuac caugcgaaug
ccgagcguga acagaagaaa 1440gagcaagaaa ggauagaaaa agaacguaug
agaaggcuua uggcugagga ugaagagggu 1500uacaggaaac ugauugauca
gaaaaaagau aagagauugg cauuccuucu uucacaaacu 1560gaugaauaua
uugccaaucu uacugaaaug gugaagcagc auaaaaugga acaacagcgu
1620aagcaggaac aagaagagca acaaaaacgg aagaggaaaa agaaaaagaa
gaauagggaa 1680ggagauccag augaugaaag cucucagaug ucagauuuac
auguuagcgu uauagaagca 1740gcaacugguc ggcagcugac gggggaggau
gcuccauugg ccagccagcu ugggagcugg 1800uuggaggcac acccgggcug
ggagccuuug gaagauagcg aagaugaaga ugaugaagag 1860gacagcgacg
aggaagguga ugauaacagu agaucaaaag gugguuuuuc aaugauagga
1920aaagaugaag cugauagcaa guuaucuguu gaagacgaag cucgagaaau
gauaaagaaa 1980gcgaagauug aagaugauga auacaagaac acgaccgaag
aacauacaua cuacagcauc 2040gcucacaccg ugcaugaaau ugucaccgaa
caagcuucaa ucaugauuaa cgguaaauug 2100aaagaauauc aaauuaaagg
ucuugaaugg uugguuucuu uauacaacaa caacuugaau 2160ggaauccucg
ccgacgagau gggccuuggc aagacaauuc aaacaauagg ucucauuacu
2220uauuugaugg agaagaagaa aguaaauggu ccuuaccuca uuauuguucc
ucugucaaca 2280uuauccaauu ggguuuugga auucgagaaa ugggcuccuu
caguguuugu gguagcuuau 2340aaagguucuc cugcaaugag gagaacuuua
caaucacaga ugcgcucgac gaaguucaau 2400guccugcuca cgaccuacga
guaugucauc aaggacaagg caguacuugc aaaguugcau 2460uggaaguaca
ugauaaucga cgagggacac aggaugaaaa accaccauug uaagcugacg
2520caggugcuga acacccauua uuuggcaccu caccgccucc uucucacggg
cacaccucuc 2580cagaacaaac uaccugagcu cugggcucuu cuaaacuuuc
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aguugcaagu uuugcuuuug augaagauaa agaaacucag 1560agugaaccag
aacagcagga ugacgauacu agagauuggg uuuguuauga uaauacaauc
1620uuauguaucu cguauuuuau gauugaacca auuuauaauc aauaa
1665731134DNAEuchistus heros 73tacaaaatgt 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 11347422DNAArtificial SequencePrimer Actin42A-F
74tcaaggaaaa actgtgctat gt 227520DNAArtificial SequencePrimer
Actin42A-R 75taccgatggt gatgacctga 207612DNAArtificial
SequenceProbe Actin42A-FAM 76accgccgctg cc 127719DNAArtificial
SequenceProbe brm-F 77tcatcaagga caaggcagt 197821DNAArtificial
SequencePrimer brm-R 78gacgggagga gaaagtttag a 217919DNAArtificial
SequenceProbe brm-FAM 79cgacgaggga cacaggatg 198017DNAArtificial
SequencePrimer mi-2-F 80gatgagggct tgctgtt 178118DNAArtificial
SequencePrimer mi-2-R 81gaggcgggaa gtattgac 188222DNAArtificial
SequenceProbe mi-2-FAM 82atgaggaagg aagcagaagt gc
228323DNAArtificial SequencePrimer iswi-1-F 83gagttcaacg aagaagacag
taa 238419DNAArtificial SequencePrimer iswi-R 84cgatgagcac
gatccatag 198522DNAArtificial SequenceProbe iswi-1-FAM 85ttagccaccg
cagatgtagt ca 228625DNAArtificial SequencePrimer iswi-2-F_MGB
86acgtaaggga gatggatcta tttca 258723DNAArtificial SequencePrimer
iswi-2-R_MGB 87cagggctgct tttatcactc tgt 238815DNAArtificial
SequenceProbe iswi-2-FAM_MGB 88ctccacctgt ctctg 158920DNAArtificial
SequencePrimer chd1-F 89caacagtggc tggtccttca 209023DNAArtificial
SequencePrimer chd1-R 90accaacttgt gacattgacg aaa
239116DNAArtificial SequenceProbe chd1-FAM 91tctggtttca gctctt
16
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