U.S. patent application number 14/455368 was filed with the patent office on 2014-11-20 for compositions and methods for the suppression of target polynucleotides from lygus.
The applicant listed for this patent is E I DU PONT DE NEMOURS AND COMPANY, PIONEER HI BRED INTERNATIONAL INC. Invention is credited to RAFAEL HERRMANN, Michael Lassner, Albert Laurence Lu, Mark Edward Nelson, James Kevin Presnail, Janet Ann Rice.
Application Number | 20140343131 14/455368 |
Document ID | / |
Family ID | 40467289 |
Filed Date | 2014-11-20 |
United States Patent
Application |
20140343131 |
Kind Code |
A1 |
HERRMANN; RAFAEL ; et
al. |
November 20, 2014 |
COMPOSITIONS AND METHODS FOR THE SUPPRESSION OF TARGET
POLYNUCLEOTIDES FROM LYGUS
Abstract
Methods and compositions are provided which employ a silencing
element that, when ingested by a pest, such as a pest from the
Lygus genus, they are capable of decreasing the expression of a
target sequence in the pest. In specific embodiments, the decrease
in expression of the target sequence controls the pest and thereby
the methods and compositions are capable of limiting damage to a
plant. The present invention provides various target
polynucleotides from specific polypeptide families as disclosed
herein, and further provides target polynucleotides set forth in
SEQ ID NOS:1-21 or active variants thereof, wherein a decrease in
expression of one or more the sequences in the target pest controls
the pest (i.e., has insecticidal activity). Further provided are
silencing elements which when ingested by the pest decrease the
level of the target polypeptide and thereby control the pest. In
specific embodiment, the pest is Lygus Hesperus. Plants, plant
part, bacteria and other host cells comprising the silencing
elements or an active variant or fragment thereof of the invention
are also provided.
Inventors: |
HERRMANN; RAFAEL;
(Wilmington, DE) ; Lassner; Michael; (Urbandale,
IA) ; Lu; Albert Laurence; (Newark, DE) ;
Nelson; Mark Edward; (Newark, DE) ; Presnail; James
Kevin; (Des Moines, IA) ; Rice; Janet Ann;
(Wilmington, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
PIONEER HI BRED INTERNATIONAL INC
E I DU PONT DE NEMOURS AND COMPANY |
Johnston
Wilmington |
IA
DE |
US
US |
|
|
Family ID: |
40467289 |
Appl. No.: |
14/455368 |
Filed: |
August 8, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12351379 |
Jan 9, 2009 |
8809625 |
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14455368 |
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61021685 |
Jan 17, 2008 |
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61021676 |
Jan 17, 2008 |
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Current U.S.
Class: |
514/44A ;
435/414; 435/415; 435/416; 435/418; 800/302 |
Current CPC
Class: |
Y02A 40/162 20180101;
C12N 15/8218 20130101; C12N 2310/531 20130101; Y02A 40/146
20180101; A01N 57/16 20130101; C12N 15/8286 20130101; C12N 15/113
20130101; C12N 2310/141 20130101 |
Class at
Publication: |
514/44.A ;
435/418; 435/414; 435/416; 435/415; 800/302 |
International
Class: |
C12N 15/82 20060101
C12N015/82; C12N 15/113 20060101 C12N015/113 |
Claims
1. A plant cell having stably incorporated into its genome a
heterologous polynucleotide comprising a silencing element, wherein
said silencing element when ingested by a pest from the Lygus
genus, reduces the level of a target sequence in said pest, and
thereby controls the pest from the Lygus family, and said silencing
element is selected from the group consisting of: a) a
polynucleotide comprising the sense or antisense sequence of the
sequence set forth in SEQ ID NO:283, 289, 292, 304, 316, 319, 322,
325, 328, 331, 334, 337, 340, 343 or 346; b) a polynucleotide
comprising the sense or antisense sequence of a sequence having at
least 95% sequence identity to the sequence set forth in SEQ ID NO:
283, 289, 292, 304, 316, 319, 322, 325, 328, 331, 334, 337, 340,
343 or 346; c) a polynucleotide comprising the sequence set forth
in SEQ ID NO:284, 285, 290, 291, 293, 294, 305, 306, 317, 318, 320,
321, 323, 324, 326, 327, 329, 330, 332, 333, 335, 336, 338, 339,
341, 342, 344, 345, 347 or 348; and d) a polynucleotide comprising
a nucleotide sequence having at least 95% sequence identity to SEQ
ID NO:284, 285, 290, 291, 293, 294, 305, 306, 317, 318, 320, 321,
323, 324, 326, 327, 329, 330, 332, 333, 335, 336, 338, 339, 341,
342, 344, 345, 347 or 348.
2. The plant cell of claim 1, wherein said pest comprises Lygus
hesperus.
3. The plant cell of claim 1, wherein said silencing element
comprises a hairpin RNA.
4. The plant cell of claim 1, wherein said silencing element is
operably linked to a heterologous promoter.
5. The plant cell of claim 1, wherein said plant cell is from a
monocot.
6. The plant cell of claim 5, wherein said monocot is maize,
barley, millet, wheat or rice.
7. The plant cell of claim 1, wherein said plant cell is from a
dicot.
8. The plant cell of claim 7, wherein said plant cell is soybean,
canola, alfalfa, sunflower, safflower, tobacco, Arabidopsis, or
cotton.
9. The plant cell of claim 1, wherein said plant cell has stably
incorporated into its genome a second polynucleotide comprising a
suppressor enhancer element comprising the target pest sequence or
an active variant or fragment thereof, wherein the combined
expression of the silencing element and the suppressor enhancer
element increases the concentration of an inhibitory RNAi specific
for the pest target sequence in said plant cell.
10. A plant or plant part comprising the plant cell of claim 1.
11. The plant or plant part of claim 9, wherein the combined
expression of said silencing element and the suppressor enhancer
element increases the concentration of an inhibitory RNA specific
for the pest target sequence in the phloem of said plant or plant
part.
12. A transgenic seed from the plant of claim 10.
13. A method for controlling Lygus comprising feeding to a Lygus a
composition comprising a silencing element, wherein said silencing
element, when ingested by said Lygus, reduces the level of a target
Lygus sequence and thereby controls the Lygus and said silencing
element comprises: a) a polynucleotide comprising the sense or
antisense sequence of the sequence set forth in SEQ ID NO: 283,
289, 292, 304, 316, 319, 322, 325, 328, 331, 334, 337, 340, 343 or
346; and, b) a polynucleotide comprising the sense or antisense
sequence of a sequence having at least 95% sequence identity to the
sequence set forth in SEQ ID NO: 283, 289, 292, 304, 316, 319, 322,
325, 328, 331, 334, 337, 340, 343 or 346. c) a polynucleotide
comprising the sequence set forth in SEQ ID NO: 284, 285, 290, 291,
293, 294, 305, 306, 317, 318, 320, 321, 323, 324, 326, 327, 329,
330, 332, 333, 335, 336, 338, 339, 341, 342, 344, 345, 347 or 348;
and d) a polynucleotide comprising a nucleotide sequence having at
least 95% sequence identity to SEQ ID NO:284, 285, 290, 291, 293,
294, 305, 306, 317, 318, 320, 321, 323, 324, 326, 327, 329, 330,
332, 333, 335, 336, 338, 339, 341, 342, 344, 345, 347 or 348.
14. The method of claim 13, wherein said composition comprises a
plant or plant part having stably incorporated into its genome a
polynucleotide comprising said silencing element.
15. The method of claim 13, wherein said pest comprises Lygus
hesperus.
16. The method of claim 13, wherein said silencing element
comprises a hairpin RNA.
17. The method of claim 13, wherein said silencing element is
operably linked to a heterologous promoter.
18. The method of claim 13, wherein said plant or plant part has
stably incorporated into its genome a second polynucleotide
comprising a suppressor enhancer element comprising the target pest
sequence or an active variant or fragment thereof, wherein the
combined expression of the silencing element and the suppressor
enhancer element increases the concentration of an inhibitory RNAi
specific for the pest target sequence in said plant.
19. The method claim 18, wherein the combined expression of said
silencing element and the suppressor enhancer element increases the
concentration of an inhibitory RNA specific for the pest target
sequence in the phloem of said plant or plant part.
20. The method of claim 13, wherein said plant is a monocot.
21. The method of claim 20, wherein said monocot is maize, barley,
millet, wheat or rice.
22. The method of claim 13, wherein said plant is a dicot.
23. The method of claim 22, wherein said plant is soybean, canola,
alfalfa, sunflower, safflower, tobacco, Arabidopsis, or cotton.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional
Application No. 61/021,685, filed Jan. 17, 2008 and U.S.
Provisional Application No. 61/021,676; filed Jan. 17, 2008; and is
a continuation of U.S. application Ser. No. 12/351,379 filed Jan.
9, 2009 which now granted as U.S. Pat. No. 8,809,625, which is
incorporated herein by reference in its entirety.
FIELD OF THE INVENTION
[0002] The present invention relates generally to methods of
molecular biology and gene silencing to control pests.
REFERENCE TO A SEQUENCE LISTING SUBMITTED AS A TEXT FILE VIA
EFS-WEB
[0003] The official copy of the sequence listing is submitted
concurrently with the specification as a text file via EFS-Web, in
compliance with the American Standard Code for Information
Interchange (ASCII), with a file name of 366630seqlist.txt, a
creation date of Dec. 23, 2008, and a size of 50 Kb. The sequence
listing filed via EFS-Web is part of the specification and is
hereby incorporated in its entirety by reference herein.
BACKGROUND OF THE INVENTION
[0004] Insect pests are a serious problem in agriculture. They
destroy millions of acres of staple crops such as corn, soybeans,
peas, and cotton. Yearly, these pests cause over $100 billion
dollars in crop damage in the U.S. alone. In an ongoing seasonal
battle, farmers must apply billions of gallons of synthetic
pesticides to combat these pests. Other methods employed in the
past delivered insecticidal activity by microorganisms or genes
derived from microorganisms expressed in transgenic plants. For
example, certain species of microorganisms of the genus Bacillus
are known to possess pesticidal activity against a broad range of
insect pests including Lepidoptera, Diptera, Coleoptera, Hemiptera,
and others. In fact, microbial pesticides, particularly those
obtained from Bacillus strains, have played an important role in
agriculture as alternatives to chemical pest control. Agricultural
scientists have developed crop plants with enhanced insect
resistance by genetically engineering crop plants to produce
insecticidal proteins from Bacillus. For example, corn and cotton
plants genetically engineered to produce Cry toxins (see, e.g.,
Aronson (2002) Cell Mol. Life Sci. 59(3):417-425; Schnepf et al.
(1998) Microbiol. Mol. Biol. Rev. 62(3):775-806) are now widely
used in American agriculture and have provided the farmer with an
alternative to traditional insect-control methods. However, these
Bt insecticidal proteins only protect plants from a relatively
narrow range of pests. Moreover, these modes of insecticidal
activity provided varying levels of specificity and, in some cases,
caused significant environmental consequences. Thus, there is an
immediate need for alternative methods to control pests.
BRIEF SUMMARY OF THE INVENTION
[0005] Methods and compositions are provided which employ a
silencing element that, when ingested by a pest, such as a pest
from the Lygus genus, is capable of decreasing the expression of a
target sequence in the pest. In specific embodiments, the decrease
in expression of the target sequence controls the pest and thereby
the methods and compositions are capable of limiting damage to a
plant. The present invention provides various target
polynucleotides from specific polypeptide families as disclosed
herein, and further provides various target polynucleotides set
forth in SEQ ID NOS:1-21 or active variants or fragments thereof,
wherein a decrease in expression of one or more the sequences in
the target pest controls the pest (i.e., has insecticidal
activity). Further provided are silencing elements, which when
ingested by the pest, decrease the level of expression of one or
more of the target polynucleotides. In specific embodiment, the
pest that is controlled is Lygus Hesperus. Plants, plant parts,
bacteria and other host cells comprising the silencing elements or
an active variant or fragment thereof are also provided.
[0006] In another embodiment, a method for controlling a pest from
the Lygus genus is provided. The method comprises feeding to a pest
from the Lygus genus a composition comprising a silencing element,
wherein said silencing element, when ingested by said Lygus,
reduces the level of a target sequence in the Lygus and thereby
controls the Lygus. Further provided are methods to protect a plant
from Lygus. Such methods comprise introducing into the plant or
plant part a silencing element of the invention. When the plant
expressing the silencing element is ingested by the pest, the level
of the target sequence is decreased and the pest is controlled.
DETAILED DESCRIPTION OF THE INVENTION
[0007] The present inventions now will be described more fully
hereinafter with reference to the accompanying drawings, in which
some, but not all embodiments of the inventions are shown. Indeed,
these inventions may be embodied in many different forms and should
not be construed as limited to the embodiments set forth herein;
rather, these embodiments are provided so that this disclosure will
satisfy applicable legal requirements. Like numbers refer to like
elements throughout.
[0008] Many modifications and other embodiments of the inventions
set forth herein will come to mind to one skilled in the art to
which these inventions pertain having the benefit of the teachings
presented in the foregoing descriptions and the associated
drawings. Therefore, it is to be understood that the inventions are
not to be limited to the specific embodiments disclosed and that
modifications and other embodiments are intended to be included
within the scope of the appended claims. Although specific terms
are employed herein, they are used in a generic and descriptive
sense only and not for purposes of limitation.
I. Overview
[0009] Methods and compositions are provided which employ a
silencing element that, when ingested by a pest, such as a pest
from the Lygus genus, is capable of decreasing the expression of a
target sequence in the pest. In specific embodiments, the decrease
in expression of the target sequence controls the pest and thereby
the methods and compositions are capable of limiting damage to a
plant or plant part. In specific embodiments, the present invention
provides target polynucleotide which encode a potassium channel
polypeptide, a cuticle polypeptide, an endocuticle polypeptide, a
chitin binding polypeptide, a chitinase polypeptide, a hormone
inducible polypeptide, a translation initiation factor, a voltage
dependant channel, an EIF-related polypeptide, a polypeptide having
a coiled coil helix domain, a polypeptide having a zinc finger
domain, a receptor associated finger polypeptide, a lethal timorous
imaginal disc polypeptide, a ribonucleoprotein, a cathepsin
protease polypeptide, a polyprotein deformed destructor, and a
death associated leucine rich polypeptide. The present invention
provides a target polynucleotides set forth in SEQ ID NOS:1-21 or
active variants and fragments thereof. Silencing elements designed
in view of these target polynucleotides are provided which, when
ingested by the pest, decrease the expression of one or more of the
target sequences and thereby controls the pest (i.e., has
insecticidal activity). These results provide the first report of
insecticidal activity of dsRNA against Lygus Hesperus.
[0010] As used herein, by "controlling a pest" or "controls a pest"
is intended any affect on a pest that results in limiting the
damage that the pest causes. Controlling a pest includes, but is
not limited to, killing the pest, inhibiting development of the
pest, altering fertility or growth of the pest in such a manner
that the pest provides less damage to the plant, decreasing the
number of offspring produced, producing less fit pests, producing
pests more susceptible to predator attack, or deterring the pests
from eating the plant.
[0011] By "disease resistance" is intended that the plants avoid
the disease symptoms that are the outcome of plant-pathogen
interactions. That is, pathogens are prevented from causing plant
diseases and the associated disease symptoms, or alternatively, the
disease symptoms caused by the pathogen is minimized or
lessened.
[0012] Reducing the level of expression of the target
polynucleotide or the polypeptide encoded thereby, in the pest
results in the suppression, control, and/or killing the invading
pathogenic organism. Reducing the level of expression of the target
sequence of the pest will reduce the disease symptoms resulting
from pathogen challenge by at least about 2% to at least about 6%,
at least about 5% to about 50%, at least about 10% to about 60%, at
least about 30% to about 70%, at least about 40% to about 80%, or
at least about 50% to about 90% or greater. Hence, the methods of
the invention can be utilized to protect plants from disease,
particularly those diseases that are caused by pests from the Lygus
genus.
[0013] Assays that measure the control of a pest are commonly known
in the art, as are methods to quantitate disease resistance in
plants following pathogen infection. See, for example, U.S. Pat.
No. 5,614,395, herein incorporated by reference. Such techniques
include, measuring over time, the average lesion diameter, the
pathogen biomass, and the overall percentage of decayed plant
tissues. See, for example, Thomma et al. (1998) Plant Biology
95:15107-15111, herein incorporated by reference. See, also the
examples below.
[0014] The invention is drawn to compositions and methods for
protecting plants from a plant pest, such as pests from the
Hemiptera order, or inducing resistance in a plant to a plant pest,
such as pests from the Hemiptera order. The Hemiptera order
comprises four suborders, the Sternorrhyncha (e.g. aphids,
whiteflies), Auchenorrhyncha (e.g. cicadas, leafhoppers),
Coleorrhyncha, and Heteroptera (e.g. true bugs) and about 67,500
species. Accordingly, the compositions and methods are useful in
protecting plants against any member of the Hemiptera order
including those of the family Cicadellidae, Membracidae,
Fulgoridae, Coccidae, Aphididae, Lygaeidae, Pentatomidae, and
Miridae.
[0015] In specific embodiments, the invention is drawn to
compositions and methods for protecting plants from a plant pest,
such as pests from the Lygus genus, or inducing resistance in a
plant to a plant pest, such as pests from the Lygus genus. The
Lygus genus comprises over 40 species of plant feeding insects in
the family Miridae. As used herein, the term "Lygus" or "Lygus Bug"
is used to refer to any member of the Lygus genus. Accordingly, the
compositions and methods are also useful in protecting plants
against any Lygus including, for example, Lygus adspersus, Lygus
alashanensis, Lygus borealis, Lygus elisus, Lygus gemellatus, Lygus
Hesperus, Lygus lineolaris, or Lygus rugulipennis. In particular
embodiment, methods control Lygus Hesperus.
[0016] In other embodiments, the pest is a plant sap-sucking
insect. As used herein, "plant sap-sucking insects" are insects
which feed on plants using their sharp mouth parts which can be
inserted into a plant to take fluid from the plant vascular system.
In one embodiment, these are insects feeding directly on the fluids
in the plant vascular system. In the insertion site, plant cells
can also be damaged which may or may not be used as a food source
by the plant sap-sucking insect. These insects are plant pests
because their feeding reduces the vitality of the crop they feed on
and they can transmit viral disease. Also, such sap-sucking insects
can create a sugar-rich fluid named honeydew that accumulates on
lower plant parts and such parts soon become covered by certain
black or brown fungi known as sooty molds, hence interfering with
photosynthesis.
[0017] Included in such plant sap-sucking insects are aphids or
Homopteran insects of the Aphididae, and plant sap-sucking insects
as used herein include but are not limited to the peach-potato
aphid Myzus persicae, the bean aphid Aphis fabae, the pea aphid
Acyrthosiphumpisun, the cabbage aphid Brevicoryne brassicae, the
grain aphid Sitobion avenae, the rose-grain aphid Metopolophium
dirhodum, the Russian wheat aphid Diuraphis noxia (Mordvilko), the
English grain aphid Macrosiphum avenae, the greenbug aphid
Schizaphis graminum (Rondani), the carrot aphid Cavariella
aegopodii, the potato aphid Macrosiphum euphorbiae, the groundnut
aphid Aphiscraccivora, the cotton aphid Aphis gossypii, the black
citrus aphid Toxoptera aurantii, the brown citrus apid Toxoptera
ciidius, the willow aphid Cavariella spp., the corn leaf aphid
Rhopalosiphum maidis, the aphid Rhopalosiphum padi, the willow leaf
aphids Chaitophorus spp., the black pine aphids Cinara spp., the
Sycamore Aphid Drepanosiphum platanoides, the Spruce aphids
Elatobium spp., Aphis citricola, Lipaphis as Laodelphax striatellus
(small brown planthopper), Nilaparvata lugens (rice brown plant
hopper) and Sogatella furcifera (white-backed rice planthopper),
and Deltocephalidae (or leafhoppers) such as Flexamia DeLong spp.,
Nephotettix cincticeps and Nephotettix virescens, Amrasca
bigutulla, and the potato leafhopper Empoasca filament. Also
included are scales (also named scale insects) such as Aonidiella
aurantii (California red scale), Comstockaspis perniciosa (San Jose
scale), Unaspis citri (citrus snow scale), Pseudaulacaspis
pentagona (white peach scale), Saissetia oleae (brown olive scale),
Lepidosaphes beckii (purple scale), Ceroplastes rubens (red wax
scale) and Icerya purchasi (cottonycushion scale), besides Tingidae
(or lace bugs) and Psyllidae insects, and spittle bugs.
[0018] Further included as plant sap-sucking insects are
Heteropteran insects and Hemipteran insects of the Auchenorrhyncha
that feed from the plants' vascular system, such as sap-sucking
insects of the Cicadoidea (such as Cicadas), Cercopoidea
(spittlebugs or froghoppers), Membracoidea (leafhoppers and
treehoppers), and Fulgoroidea (planthoppers), e.g., the cotton seed
sucker bug Dysdercus peruvianus (Heteroptera, Pyrrhocoridae), the
apple dimpling bug, Campylomma liebknechti (Hemiptera: Miridae) and
the greenmirid, Creontiades dilutus which are cotton sucking insect
pests, and the Lygusbugs (Hemiptera: Miridae, e.g., Lygus
hesperus).
II. Target Sequences
[0019] As used herein, a "target sequence" comprises any sequence
in the pest that one desires to decrease the level of expression.
In specific embodiments, decreasing the level of the target
sequence in the pest controls the pest. For instance the target
sequence can be essential for growth and development. While the
target sequence can be expressed in any tissue of the pest, in
specific embodiments of the invention, the sequences targeted for
suppression in the pest are expressed in cells of the gut tissue of
the pest, cells in the midgut of the pest, and cells lining the gut
lumen or the midgut. Such target sequences can be involved in gut
cell metabolism, growth or differentiation.
[0020] Non-limiting examples of target sequences of the invention
include a polynucleotide set forth in SEQ ID NO:1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21. In
specific embodiments, the silencing element comprises at least or
consists of 15, 20, 22, 25 or greater consecutive nucleotides of
any one of SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, 20, or 21. In further embodiments, the
silencing element comprises or consists of at least one of the
sequences set forth in SEQ ID NO: 22-408. It is recognized that
such silencing elements can further comprise one or more thymine
residues at the 3' end. Such residues can aid in stabilization. The
element can have, for example, 1, 2, 3, 4, 5, 6 or more thymine
residues at its 3' end. In further embodiments, the silencing
element comprises SEQ ID NO: 23 and 24; 26 and 27; 29 and 30; 32
and 33; 35 and 36; 38 and 39; 41 and 42; 44 and 45; 47 and 48; 50
and 51; 53 and 54; 56 and 57; 59 and 60; 62 and 63; 65 and 66; 68
and 69; 71 and 72; 74 and 75; 77 and 78; 80 and 81; 83 and 84; 86
and 87; 89 and 90; 92 and 93; 95 and 96; 98 and 99; 101 and 102;
104 and 105; 107 and 108; 110 and 111; 113 and 114; 116 and 117;
119 and 120; 122 and 123; 125 and 126; 128 and 129; 131 and 132;
134 and 135; 137 and 138; 140 and 141; 143 and 144; 146 and 147;
149 and 150; 152 and 153; 155 and 156; 158 and 159; 161 and 162;
164 and 165; 167 and 168; 170 and 171; 173 and 174; 176 and 177;
179 and 180; 182 and 183; 185 and 186; 188 and 189; 191 and 192;
194 and 195; 197 and 198; 200 and 201; 203 and 204; 206 and 207;
209 and 210; 212 and 213; 215 and 216; 218 and 219; and/or 221 and
222. As exemplified elsewhere herein, decreasing the level of
expression of these target sequence in Lygus controls the pest.
[0021] In specific embodiments, the target sequence comprises SEQ
ID NO:7, 8, 9, 11, 12 or 14. In further embodiments, the silencing
element comprises or consists of at least one of the sequences set
forth in SEQ ID NOS:283, 284, 285, 289, 290, 291, 292, 293, 294,
304, 305, 306, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325,
326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338,
339, 340, 341, 342, 343, 344, 345, 346, 347 or 348. In still
further embodiments, the silencing element comprises or consists of
SEQ ID NOS: 284 and 285, 290 and 291, 293 and 294; 305 and 306; 317
and 318; 320 and 321; 323 and 324; 326 and 327; 329 and 330; 332
and 333; 335 and 336; 338 and 339; 341 and 342; 344 and 345; or 347
and 348. As exemplified elsewhere herein, expression of these
sequences controls lygus.
III. Silencing Elements
[0022] By "silencing element" is intended a polynucleotide which
when ingested by a pest, is capable of reducing or eliminating the
level or expression of a target polynucleotide or the polypeptide
encoded thereby. The silencing element employed can reduce or
eliminate the expression level of the target sequence by
influencing the level of the target RNA transcript or,
alternatively, by influencing translation and thereby affecting the
level of the encoded polypeptide. Methods to assay for functional
silencing elements that are capable of reducing or eliminating the
level of a sequence of interest are disclosed elsewhere herein. A
single polynucleotide employed in the methods of the invention can
comprises one or more silencing elements to the same or different
target polynucleotides.
[0023] In specific embodiments, the target sequence is not a plant
endogenous gene. In other embodiments, while the silencing element
controls pests, preferably the silencing element has no effect on
the normal plant or plant part.
[0024] As discussed in further detail below, silencing elements can
include, but are not limited to, a sense suppression element, an
antisense suppression element, a double stranded RNA, a miRNA, or a
hairpin suppression element. Non-limiting examples of silencing
elements that can employed to decrease expression of these target
Lygus sequences comprise or consists of fragments and variants of
the sense or antisense sequence of the sequence set forth in SEQ ID
NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19, 20, or 21 or SEQ ID NOS: 22-408. or fragments and variants of
the sense or antisense sequence thereof.
[0025] In specific embodiments, the silencing element comprises or
consists of at least one of the sequences set forth in SEQ ID NO:
21-408. It is recognized that such silencing elements can further
comprise one or more thymine residues at the 3' end. Such residues
can aid in stabilization. The element can have, for example, 1, 2,
3, 4, 5, 6 or more thymine residues at its 3' end. In further
embodiments, the silencing element comprises SEQ ID NO: 23 and 24;
26 and 27; 29 and 30; 32 and 33; 35 and 36; 38 and 39; 41 and 42;
44 and 45; 47 and 48; 50 and 51; 53 and 54; 56 and 57; 59 and 60;
62 and 63; 65 and 66; 68 and 69; 71 and 72; 74 and 75; 77 and 78;
80 and 81; 83 and 84; 86 and 87; 89 and 90; 92 and 93; 95 and 96;
98 and 99; 101 and 102; 104 and 105; 107 and 108; 110 and 111; 113
and 114; 116 and 117; 119 and 120; 122 and 123; 125 and 126; 128
and 129; 131 and 132; 134 and 135; 137 and 138; 140 and 141; 143
and 144; 146 and 147; 149 and 150; 152 and 153; 155 and 156; 158
and 159; 161 and 162; 164 and 165; 167 and 168; 170 and 171; 173
and 174; 176 and 177; 179 and 180; 182 and 183; 185 and 186; 188
and 189; 191 and 192; 194 and 195; 197 and 198; 200 and 201; 203
and 204; 206 and 207; 209 and 210; 212 and 213; 215 and 216; 218
and 219; and/or 221 and 222.
[0026] In other embodiments, the silencing element comprises or
consists of at least one of the sequences set forth in SEQ ID
NOS:283, 284, 285, 289, 290, 291, 292, 293, 294, 304, 305, 306,
316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328,
329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341,
342, 343, 344, 345, 346, 347 or 348. In still further embodiments,
the silencing element comprises or consists of SEQ ID NOS: 284 and
285, 290 and 291, 293 and 294; 305 and 306; 317 and 318; 320 and
321; 323 and 324; 326 and 327; 329 and 330; 332 and 333; 335 and
336; 338 and 339; 341 and 342; 344 and 345; or 347 and 348. As
exemplified elsewhere herein, expression of these sequences
controls lygus.
[0027] By "reduces" or "reducing" the expression level of a
polynucleotide or a polypeptide encoded thereby is intended to
mean, the polynucleotide or polypeptide level of the target
sequence is statistically lower than the polynucleotide level or
polypeptide level of the same target sequence in an appropriate
control pest which is not exposed to (i.e., has not ingested) the
silencing element. In particular embodiments of the invention,
reducing the polynucleotide level and/or the polypeptide level of
the target sequence in a pest according to the invention results in
less than 95%, less than 90%, less than 80%, less than 70%, less
than 60%, less than 50%, less than 40%, less than 30%, less than
20%, less than 10%, or less than 5% of the polynucleotide level, or
the level of the polypeptide encoded thereby, of the same target
sequence in an appropriate control pest. Methods to assay for the
level of the RNA transcript, the level of the encoded polypeptide,
or the activity of the polynucleotide or polypeptide are discussed
elsewhere herein.
[0028] i. Sense Silencing Elements
[0029] As used herein, a "sense silencing element" comprises a
polynucleotide designed to express an RNA molecule corresponding to
at least a part of a target messenger RNA in the "sense"
orientation. Expression of the RNA molecule comprising the sense
suppression element reduces or eliminates the level of the target
polynucleotide or the polypeptide encoded thereby. The
polynucleotide comprising the sense suppression element may
correspond to all or part of the sequence of the target
polynucleotide, all or part of the 5' and/or 3' untranslated region
of the target polynucleotide, all or part of the coding sequence of
the target polynucleotide, or all or part of both the coding
sequence and the untranslated regions of the target
polynucleotide.
[0030] Typically, a sense suppression element has substantial
sequence identity to the target polynucleotide, optimally greater
than about 65% sequence identity, more optimally greater than about
85% sequence identity, about 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98% or 99% sequence identity. See, U.S. Pat. Nos. 5,283,184
and 5,034,323; herein incorporated by reference. The sense
suppression element can be any length so long as it does not
interfere with intron splicing and allows for the suppression of
the targeted sequence. The sense suppression element can be, for
example, 15, 20, 22, 25, 30, 50, 100, 150, 200, 250, 300, 350, 400,
450, 500, 600, 700, 900 or longer.
[0031] ii. Antisense Silencing Elements
[0032] As used herein, an "antisense silencing element" comprises a
polynucleotide which is designed to express an RNA molecule
complementary to all or part of a target messenger RNA.
[0033] Expression of the antisense RNA suppression element reduces
or eliminates the level of the target polynucleotide. The
polynucleotide for use in antisense suppression may correspond to
all or part of the complement of the sequence encoding the target
polynucleotide, all or part of the complement of the 5' and/or 3'
untranslated region of the target polynucleotide, all or part of
the complement of the coding sequence of the target polynucleotide,
or all or part of the complement of both the coding sequence and
the untranslated regions of the target polynucleotide. In addition,
the antisense suppression element may be fully complementary (i.e.,
100% identical to the complement of the target sequence) or
partially complementary (i.e., less than 100% identical to the
complement of the target sequence) to the target polynucleotide. In
specific embodiments, the antisense suppression element comprises
at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%
sequence identity to the target polynucleotide. Antisense
suppression may be used to inhibit the expression of multiple
proteins in the same plant. See, for example, U.S. Pat. No.
5,942,657. Furthermore, the antisense suppression element can be
complementary to a portion of the target polynucleotide. Generally,
sequences of at least 25, 50, 100, 200, 300, 400, 450 nucleotides
or greater may be used. Methods for using antisense suppression to
inhibit the expression of endogenous genes in plants are described,
for example, in Liu et at (2002) Plant Physiol. 129:1732-1743 and
U.S. Pat. Nos. 5,759,829 and 5,942,657, each of which is herein
incorporated by reference. In specific embodiments, the antisense
element comprise or consists of the complement of at least 15, 20,
22, 25 or greater contiguous nucleotides of any one of SEQ ID NO:
1-408.
[0034] iii. Double Stranded RNA Silencing Element
[0035] A "double stranded RNA silencing element" or "dsRNA"
comprises at least one transcript that is capable of forming a
dsRNA either before or after ingestion by a pest. Thus, a "dsRNA
silencing element" includes a dsRNA, a transcript or
polyribonucleotide capable of forming a dsRNA or more than one
transcript or polyribonucleotide capable of forming a dsRNA.
"Double stranded RNA" or "dsRNA" refers to a polyribonucleotide
structure formed either by a single self-complementary RNA molecule
or a polyribonucleotide structure formed by the expression of least
two distinct RNA strands. The dsRNA molecule(s) employed in the
methods and compositions of the invention mediate the reduction of
expression of a target sequence, for example, by mediating RNA
interference "RNAi" or gene silencing in a sequence-specific
manner. In the context of the present invention, the dsRNA is
capable of reducing or eliminating the level or expression of a
target polynucleotide or the polypeptide encoded thereby in a
pest.
[0036] The dsRNA can reduce or eliminate the expression level of
the target sequence by influencing the level of the target RNA
transcript, by influencing translation and thereby affecting the
level of the encoded polypeptide, or by influencing expression at
the pre-transcriptional level (i.e., via the modulation of
chromatin structure, methylation pattern, etc., to alter gene
expression). See, for example, Verdel et al. (2004) Science
303:672-676; Pal-Bhadra et al. (2004) Science 303:669-672; Allshire
(2002) Science 297:1818-1819; Volpe et al. (2002) Science
297:1833-1837; Jenuwein (2002) Science 297:2215-2218; and Hall et
al. (2002) Science 297:2232-2237. Methods to assay for functional
iRNA that are capable of reducing or eliminating the level of a
sequence of interest are disclosed elsewhere herein. Accordingly,
as used herein, the term "dsRNA" is meant to encompass other terms
used to describe nucleic acid molecules that are capable of
mediating RNA interference or gene silencing, including, for
example, short-interfering RNA (siRNA), double-stranded RNA
(dsRNA), micro-RNA (miRNA), hairpin RNA, short hairpin RNA (shRNA),
post-transcriptional gene silencing RNA (ptgsRNA), and others.
[0037] In specific embodiments, at least one strand of the duplex
or double-stranded region of the dsRNA shares sufficient sequence
identity or sequence complementarity to the target polynucleotide
to allow for the dsRNA to reduce the level of expression of the
target sequence. As used herein, the strand that is complementary
to the target polynucleotide is the "antisense strand" and the
strand homologous to the target polynucleotide is the "sense
strand."
[0038] In one embodiment, the dsRNA comprises a hairpin RNA. A
hairpin RNA comprises an RNA molecule that is capable of folding
back onto itself to form a double stranded structure. Multiple
structures can be employed as hairpin elements. In specific
embodiments, the dsRNA suppression element comprises a hairpin
element which comprises in the following order, a first segment, a
second segment, and a third segment, where the first and the third
segment share sufficient complementarity to allow the transcribed
RNA to form a double-stranded stem-loop structure.
[0039] The "second segment" of the hairpin comprises a "loop" or a
"loop region." These terms are used synonymously herein and are to
be construed broadly to comprise any nucleotide sequence that
confers enough flexibility to allow self-pairing to occur between
complementary regions of a polynucleotide (i.e., segments 1 and 2
which form the stem of the hairpin). For example, in some
embodiments, the loop region may be substantially single stranded
and act as a spacer between the self-complementary regions of the
hairpin stem-loop. In some embodiments, the loop region can
comprise a random or nonsense nucleotide sequence and thus not
share sequence identity to a target polynucleotide. In other
embodiments, the loop region comprises a sense or an antisense RNA
sequence or fragment thereof that shares identity to a target
polynucleotide. See, for example, International Patent Publication
No. WO 02/00904, herein incorporated by reference. In specific
embodiments, the loop region can be optimized to be as short as
possible while still providing enough intramolecular flexibility to
allow the formation of the base-paired stem region. Accordingly,
the loop sequence is generally less than 1000, 900, 800, 700, 600,
500, 400, 300, 200, 100, 50, 25, 20, 15, 10 nucleotides or
less.
[0040] The "first" and the "third" segment of the hairpin RNA
molecule comprise the base-paired stem of the hairpin structure.
The first and the third segments are inverted repeats of one
another and share sufficient complementarity to allow the formation
of the base-paired stem region. In specific embodiments, the first
and the third segments are fully complementary to one another.
Alternatively, the first and the third segment may be partially
complementary to each other so long as they are capable of
hybridizing to one another to form a base-paired stem region. The
amount of complementarity between the first and the third segment
can be calculated as a percentage of the entire segment. Thus, the
first and the third segment of the hairpin RNA generally share at
least 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99%, up to and including 100% complementarity.
[0041] The first and the third segment are at least about 1000,
500, 400, 300, 200, 100, 50, 40, 30, 25, 22, 20, 15 or 10
nucleotides in length. In specific embodiments, the length of the
first and/or the third segment is about 10-100 nucleotides, about
10 to about 75 nucleotides, about 10 to about 50 nucleotides, about
10 to about 40 nucleotides, about 10 to about 35 nucleotides, about
10 to about 30 nucleotides, about 10 to about 25 nucleotides, about
10 to about 20 nucleotides. In other embodiments, the length of the
first and/or the third segment comprises at least 10-20
nucleotides, 20-35 nucleotides, 30-45 nucleotides, 40-50
nucleotides, 50-100 nucleotides, or 100-300 nucleotides. See, for
example, International Publication No. WO 0200904. In specific
embodiments, the first and the third segment comprises at least 20
nucleotides having at least 85% complementary to the first segment.
In still other embodiments, the first and the third segments which
form the stem-loop structure of the hairpin comprises 3' or 5'
overhang regions having unpaired nucleotide residues.
[0042] In specific embodiments, the sequences used in the first,
the second, and/or the third segments comprise domains that are
designed to have sufficient sequence identity to a target
polynucleotide of interest and thereby have the ability to decrease
the level of expression of the target polynucleotide. The
specificity of the inhibitory RNA transcripts is therefore
generally conferred by these domains of the silencing element.
Thus, in some embodiments of the invention, the first, second
and/or third segment of the silencing element comprise a domain
having at least 10, at least 15, at least 19, at least 20, at least
21, at least 22, at least 23, at least 24, at least 25, at least
30, at least 40, at least 50, at least 100, at least 200, at least
300, at least 500, at least 1000, or more than 1000 nucleotides
that share sufficient sequence identity to the target
polynucleotide to allow for a decrease in expression levels of the
target polynucleotide when expressed in an appropriate cell. In
other embodiments, the domain is between about 15 to 50
nucleotides, about 20-35 nucleotides, about 25-50 nucleotides,
about 20 to 75 nucleotides, about 40-90 nucleotides about 15-100
nucleotides.
[0043] In specific embodiments, the domain of the first, the
second, and/or the third segment has 100% sequence identity to the
target polynucleotide. In other embodiments, the domain of the
first, the second and/or the third segment having homology to the
target polypeptide have at least 50%, 60%, 70%, 80%, 85%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater sequence
identity to a region of the target polynucleotide. The sequence
identity of the domains of the first, the second and/or the third
segments to the target polynucleotide need only be sufficient to
decrease expression of the target polynucleotide of interest. See,
for example, Chuang and Meyerowitz (2000) Proc. Natl. Acad. Sci.
USA 97:4985-4990; Stoutjesdijk et al. (2002) Plant Physiol.
129:1723-1731; Waterhouse and Helliwell (2003) Nat. Rev. Genet.
4:29-38; Pandolfini et al. BMC Biotechnology 3:7, and U.S. Patent
Publication No. 20030175965; each of which is herein incorporated
by reference. A transient assay for the efficiency of hpRNA
constructs to silence gene expression in vivo has been described by
Panstruga et al. (2003) Mol. Biol. Rep. 30:135-140, herein
incorporated by reference.
[0044] The amount of complementarity shared between the first,
second, and/or third segment and the target polynucleotide or the
amount of complementarity shared between the first segment and the
third segment (i.e., the stem of the hairpin structure) may vary
depending on the organism in which gene expression is to be
controlled. Some organisms or cell types may require exact pairing
or 100% identity, while other organisms or cell types may tolerate
some mismatching. In some cells, for example, a single nucleotide
mismatch in the targeting sequence abrogates the ability to
suppress gene expression. In these cells, the suppression cassettes
of the invention can be used to target the suppression of mutant
genes, for example, oncogenes whose transcripts comprise point
mutations and therefore they can be specifically targeted using the
methods and compositions of the invention without altering the
expression of the remaining wild-type allele.
[0045] Any region of the target polynucleotide can be used to
design the domain of the silencing element that shares sufficient
sequence identity to allow expression of the hairpin transcript to
decrease the level of the target polynucleotide. For instance, the
domain can be designed to share sequence identity to the 5'
untranslated region of the target polynucleotide(s), the 3'
untranslated region of the target polynucleotide(s), exonic regions
of the target polynucleotide(s), intronic regions of the target
polynucleotide(s), and any combination thereof. In specific
embodiments a domain of the silencing element shares sufficient
homology to at least about 15 consecutive nucleotides from about
nucleotides 1-50, 50-100, 100-150, 150-200, 200-250, 250-300,
300-350, 350-400, 400-450, 450-500, 550-600, 600-650, 650-700,
750-800, 850-900, 950-1000, 1000-1050, 1050-1100, 1100-1200,
1200-1300, 1300-1400, 1400-1500, 1500-1600, 1600-1700, 1700-1800,
1800-1900, 1900-2000 of the target sequence. In some instances to
optimize the siRNA sequences employed in the hairpin, the synthetic
oligodeoxyribonucleotide/RNAse H method can be used to determine
sites on the target mRNA that are in a conformation that is
susceptible to RNA silencing. See, for example, Vickers et al.
(2003) J. Biol. Chem 278:7108-7118 and Yang et al. (2002) Proc.
Natl. Acad. Sci. USA 99:9442-9447, herein incorporated by
reference. These studies indicate that there is a significant
correlation between the RNase-H-sensitive sites and sites that
promote efficient siRNA-directed mRNA degradation.
[0046] The hairpin silencing element may also be designed such that
the sense sequence or the antisense sequence do not correspond to a
target polynucleotide. In this embodiment, the sense and antisense
sequence flank a loop sequence that comprises a nucleotide sequence
corresponding to all or part of the target polynucleotide. Thus, it
is the loop region that determines the specificity of the RNA
interference. See, for example, WO 02/00904, herein incorporated by
reference.
[0047] In specific embodiments, the silencing element comprising
the hairpin comprises a sequence selected from the group consisting
of a polynucleotide comprising or consist of at least one of the
sequences set forth in SEQ ID NO: 22-408. In further embodiments,
the hairpin comprises a sequence selected from the group consisting
of SEQ ID NO: 23 and 24; 26 and 27; 29 and 30; 32 and 33; 35 and
36; 38 and 39; 41 and 42; 44 and 45; 47 and 48; 50 and 51; 53 and
54; 56 and 57; 59 and 60; 62 and 63; 65 and 66; 68 and 69; 71 and
72; 74 and 75; 77 and 78; 80 and 81; 83 and 84; 86 and 87; 89 and
90; 92 and 93; 95 and 96; 98 and 99; 101 and 102; 104 and 105; 107
and 108; 110 and 111; 113 and 114; 116 and 117; 119 and 120; 122
and 123; 125 and 126; 128 and 129; 131 and 132; 134 and 135; 137
and 138; 140 and 141; 143 and 144; 146 and 147; 149 and 150; 152
and 153; 155 and 156; 158 and 159; 161 and 162; 164 and 165; 167
and 168; 170 and 171; 173 and 174; 176 and 177; 179 and 180; 182
and 183; 185 and 186; 188 and 189; 191 and 192; 194 and 195; 197
and 198; 200 and 201; 203 and 204; 206 and 207; 209 and 210; 212
and 213; 215 and 216; 218 and 219; and/or 221 and 222.
[0048] In other embodiments, the hairpin comprises or consist of a
sequence selected from the group consisting of at least one of the
sequences set forth in SEQ ID NOS: 284 and 285, 290 and 291, 293
and 294; 305 and 306; 317 and 318; 320 and 321; 323 and 324; 326
and 327; 329 and 330; 332 and 333; 335 and 336; 338 and 339; 341
and 342; 344 and 345; or 347 and 348. As exemplified elsewhere
herein, expression of these sequences controls lygus.
[0049] In addition, transcriptional gene silencing (TGS) may be
accomplished through use of a hairpin suppression element where the
inverted repeat of the hairpin shares sequence identity with the
promoter region of a target polynucleotide to be silenced. See, for
example, Aufsatz et al. (2002) PNAS 99 (Suppl. 4):16499-16506 and
Mette et al. (2000) EMBO J 19(19):5194-5201.
[0050] In other embodiments, the dsRNA can comprise a small RNA
(sRNA). sRNAs can comprise both micro RNA (miRNA) and
short-interfering RNA (siRNA) (Meister and Tuschl (2004) Nature
431:343-349 and Bonetta et al. (2004) Nature Methods 1:79-86).
miRNAs are regulatory agents comprising about 19 ribonucleotides
which are highly efficient at inhibiting the expression of target
polynucleotides. See, for example Javier et al. (2003) Nature 425:
257-263, herein incorporated by reference. For miRNA interference,
the silencing element can be designed to express a dsRNA molecule
that forms a hairpin structure containing a 19-nucleotide sequence
that is complementary to the target polynucleotide of interest. The
miRNA can be synthetically made, or transcribed as a longer RNA
which is subsequently cleaved to produce the active miRNA.
Specifically, the miRNA can comprise 19 nucleotides of the sequence
having homology to a target polynucleotide in sense orientation and
19 nucleotides of a corresponding antisense sequence that is
complementary to the sense sequence.
[0051] When expressing an miRNA, it is recognized that various
forms of an miRNA can be transcribed including, for example, the
primary transcript (termed the "pri-miRNA") which is processed
through various nucleolytic steps to a shorter precursor miRNA
(termed the "pre-miRNA"); the pre-miRNA; or the final (mature)
miRNA is present in a duplex, the two strands being referred to as
the miRNA (the strand that will eventually basepair with the
target) and miRNA*. The pre-miRNA is a substrate for a form of
dicer that removes the miRNA/miRNA* duplex from the precursor,
after which, similarly to siRNAs, the duplex can be taken into the
RISC complex. It has been demonstrated that miRNAs can be
transgenically expressed and be effective through expression of a
precursor form, rather than the entire primary form (Parizotto et
al. (2004) Genes & Development 18:2237-2242 and Guo et al.
(2005) Plant Cell 17:1376-1386).
[0052] The methods and compositions of the invention employ
silencing elements that when transcribed "form" a dsRNA molecule.
Accordingly, the heterologous polynucleotide being expressed need
not form the dsRNA by itself, but can interact with other sequences
in the plant cell or in the pest gut after ingestion to allow the
formation of the dsRNA. For example, a chimeric polynucleotide that
can selectively silence the target polynucleotide can be generated
by expressing a chimeric construct comprising the target sequence
for a miRNA or siRNA to a sequence corresponding to all or part of
the gene or genes to be silenced. In this embodiment, the dsRNA is
"formed" when the target for the miRNA or siRNA interacts with the
miRNA present in the cell. The resulting dsRNA can then reduce the
level of expression of the gene or genes to be silenced. See, for
example, U.S. Provisional Application No. 60/691,613, filed Jun.
17, 2005, entitled "Methods and Compositions for Gene Silencing,
herein incorporated by reference. The construct can be designed to
have a target for an endogenous miRNA or alternatively, a target
for a heterologous and/or synthetic miRNA can be employed in the
construct. If a heterologous and/or synthetic miRNA is employed, it
can be introduced into the cell on the same nucleotide construct as
the chimeric polynucleotide or on a separate construct. As
discussed elsewhere herein, any method can be used to introduce the
construct comprising the heterologous miRNA.
IV. Variants and Fragments
[0053] By "fragment" is intended a portion of the polynucleotide or
a portion of the amino acid sequence and hence protein encoded
thereby. Fragments of a polynucleotide may encode protein fragments
that retain the biological activity of the native protein.
Alternatively, fragments of a polynucleotide that are useful as a
silencing element or a suppressor enhancer element do not need to
encode fragment or variant proteins that retain biological
activity. Thus, fragments of a nucleotide sequence may range from
at least about 10, about 15, 20 nucleotides, about 50 nucleotides,
about 75 nucleotides, about 100 nucleotides, 200 nucleotides, 300
nucleotides, 400 nucleotides, 500 nucleotides, 600 nucleotides, 700
nucleotides and up to the full-length polynucleotide employed in
the invention. Methods to assay for the activity of a desired
silencing element are described elsewhere herein.
[0054] "Variants" is intended to mean substantially similar
sequences. For polynucleotides, a variant comprises a deletion
and/or addition of one or more nucleotides at one or more internal
sites within the native polynucleotide and/or a substitution of one
or more nucleotides at one or more sites in the native
polynucleotide. As used herein, a "native" polynucleotide or
polypeptide comprises a naturally occurring nucleotide sequence or
amino acid sequence, respectively. For polynucleotides,
conservative variants include those sequences that, because of the
degeneracy of the genetic code, encode the amino acid sequence of
one of the polypeptides employed in the invention. Variant
polynucleotides also include synthetically derived polynucleotide,
such as those generated, for example, by using site-directed
mutagenesis, but continue to retain the desired activity.
Generally, variants of a particular polynucleotide of the invention
(i.e., a silencing element) will have at least about 40%, 45%, 50%,
55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99% or more sequence identity to that particular
polynucleotide as determined by sequence alignment programs and
parameters described elsewhere herein. A silencing element or
suppressor enhancer element of a variant target sequence need not
encodes a protein, but rather will have the ability to reduce the
level of expression of the target sequence.
[0055] Variants of a particular polynucleotide of the invention
(i.e., the reference polynucleotide) can also be evaluated by
comparison of the percent sequence identity between the polypeptide
encoded by a variant polynucleotide and the polypeptide encoded by
the reference polynucleotide. Percent sequence identity between any
two polypeptides can be calculated using sequence alignment
programs and parameters described elsewhere herein. Where any given
pair of polynucleotides employed in the invention is evaluated by
comparison of the percent sequence identity shared by the two
polypeptides they encode, the percent sequence identity between the
two encoded polypeptides is at least about 40%, 45%, 50%, 55%, 60%,
65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99% or more sequence identity.
[0056] "Variant" protein is intended to mean a protein derived from
the native protein by deletion or addition of one or more amino
acids at one or more internal sites in the native protein and/or
substitution of one or more amino acids at one or more sites in the
native protein. Variant proteins encompassed by the present
invention are biologically active, that is they continue to possess
the desired biological activity of the native protein, as discussed
elsewhere herein. Such variants may result from, for example,
genetic polymorphism or from human manipulation. Biologically
active variants of a native protein will have at least about 40%,
45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the amino
acid sequence for the native protein as determined by sequence
alignment programs and parameters described elsewhere herein. A
biologically active variant of a protein of the invention may
differ from that protein by as few as 1-15 amino acid residues, as
few as 1-10, such as 6-10, as few as 5, as few as 4, 3, 2, or even
1 amino acid residue.
[0057] The following terms are used to describe the sequence
relationships between two or more polynucleotides or polypeptides:
(a) "reference sequence", (b) "comparison window", (c) "sequence
identity", and, (d) "percentage of sequence identity."
[0058] (a) As used herein, "reference sequence" is a defined
sequence used as a basis for sequence comparison. A reference
sequence may be a subset or the entirety of a specified sequence;
for example, as a segment of a full-length cDNA or gene sequence,
or the complete cDNA or gene sequence.
[0059] (b) As used herein, "comparison window" makes reference to a
contiguous and specified segment of a polynucleotide sequence,
wherein the polynucleotide sequence in the comparison window may
comprise additions or deletions (i.e., gaps) compared to the
reference sequence (which does not comprise additions or deletions)
for optimal alignment of the two polynucleotides. Generally, the
comparison window is at least 20 contiguous nucleotides in length,
and optionally can be 30, 40, 50, 100, or longer. Those of skill in
the art understand that to avoid a high similarity to a reference
sequence due to inclusion of gaps in the polynucleotide sequence a
gap penalty is typically introduced and is subtracted from the
number of matches.
[0060] Unless otherwise stated, sequence identity/similarity values
provided herein refer to the value obtained using GAP Version 10
using the following parameters: % identity and % similarity for a
nucleotide sequence using GAP Weight of 50 and Length Weight of 3,
and the nwsgapdna.cmp scoring matrix; % identity and % similarity
for an amino acid sequence using GAP Weight of 8 and Length Weight
of 2, and the BLOSUM62 scoring matrix; or any equivalent program
thereof. By "equivalent program" is intended any sequence
comparison program that, for any two sequences in question,
generates an alignment having identical nucleotide or amino acid
residue matches and an identical percent sequence identity when
compared to the corresponding alignment generated by GAP Version
10.
[0061] (c) As used herein, "sequence identity" or "identity" in the
context of two polynucleotides or polypeptide sequences makes
reference to the residues in the two sequences that are the same
when aligned for maximum correspondence over a specified comparison
window. When percentage of sequence identity is used in reference
to proteins it is recognized that residue positions which are not
identical often differ by conservative amino acid substitutions,
where amino acid residues are substituted for other amino acid
residues with similar chemical properties (e.g., charge or
hydrophobicity) and therefore do not change the functional
properties of the molecule. When sequences differ in conservative
substitutions, the percent sequence identity may be adjusted
upwards to correct for the conservative nature of the substitution.
Sequences that differ by such conservative substitutions are said
to have "sequence similarity" or "similarity". Means for making
this adjustment are well known to those of skill in the art.
Typically this involves scoring a conservative substitution as a
partial rather than a full mismatch, thereby increasing the
percentage sequence identity. Thus, for example, where an identical
amino acid is given a score of 1 and a non-conservative
substitution is given a score of zero, a conservative substitution
is given a score between zero and 1. The scoring of conservative
substitutions is calculated, e.g., as implemented in the program
PC/GENE (Intelligenetics, Mountain View, Calif.).
[0062] (d) As used herein, "percentage of sequence identity" means
the value determined by comparing two optimally aligned sequences
over a comparison window, wherein the portion of the polynucleotide
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 nucleic acid base 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 window of comparison, and
multiplying the result by 100 to yield the percentage of sequence
identity.
V. DNA constructs
[0063] The use of the term "polynucleotide" is not intended to
limit the present invention to polynucleotides comprising DNA.
Those of ordinary skill in the art will recognize that
polynucleotides, can comprise ribonucleotides and combinations of
ribonucleotides and deoxyribonucleotides. Such deoxyribonucleotides
and ribonucleotides include both naturally occurring molecules and
synthetic analogues. The polynucleotides of the invention also
encompass all forms of sequences including, but not limited to,
single-stranded forms, double-stranded forms, hairpins,
stem-and-loop structures, and the like.
[0064] The polynucleotide encoding the silencing element employed
in the methods and compositions of the invention can be provided in
expression cassettes for expression in a plant or organism of
interest. It is recognized that multiple silencing elements
including multiple identical silencing elements, multiple silencing
elements targeting different regions of the target sequence, or
multiple silencing elements from different target sequences can be
used. In this embodiment, it is recognized that each silencing
element can be contained in a single or separate cassette, DNA
construct, or vector. As discussed, any means of providing the
silencing element is contemplated. A plant or plant cell can be
transformed with a single cassette comprising DNA encoding one or
more silencing elements or separate cassettes comprising each
silencing element can be used to transform a plant or plant cell or
host cell. Likewise, a plant transformed with one component can be
subsequently transformed with the second component. One or more
silencing elements can also be brought together by sexual crossing.
That is, a first plant comprising one component is crossed with a
second plant comprising the second component. Progeny plants from
the cross will comprise both components.
[0065] The expression cassette can include 5' and 3' regulatory
sequences operably linked to the polynucleotide of the invention.
"Operably linked" is intended to mean a functional linkage between
two or more elements. For example, an operable linkage between a
polynucleotide of the invention and a regulatory sequence (i.e., a
promoter) is a functional link that allows for expression of the
polynucleotide of the invention. Operably linked elements may be
contiguous or non-contiguous. When used to refer to the joining of
two protein coding regions, by operably linked is intended that the
coding regions are in the same reading frame. The cassette may
additionally contain at least one additional polynucleotide to be
cotransformed into the organism. Alternatively, the additional
polypeptide(s) can be provided on multiple expression cassettes.
Expression cassettes can be provided with a plurality of
restriction sites and/or recombination sites for insertion of the
polynucleotide to be under the transcriptional regulation of the
regulatory regions. The expression cassette may additionally
contain selectable marker genes.
[0066] The expression cassette will include in the 5'-3' direction
of transcription, a transcriptional and translational initiation
region (i.e., a promoter), a polynucleotide comprising the
silencing element employed in the methods and compositions of the
invention, and a transcriptional and translational termination
region (i.e., termination region) functional in plants. The
regulatory regions (i.e., promoters, transcriptional regulatory
regions, and translational termination regions) and/or the
polynucleotides employed in the invention may be native/analogous
to the host cell or to each other. Alternatively, the regulatory
regions and/or the polynucleotide employed in the invention may be
heterologous to the host cell or to each other. As used herein,
"heterologous" in reference to a sequence is a sequence that
originates from a foreign species, or, if from the same species, is
substantially modified from its native form in composition and/or
genomic locus by deliberate human intervention. For example, a
promoter operably linked to a heterologous polynucleotide is from a
species different from the species from which the polynucleotide
was derived, or, if from the same/analogous species, one or both
are substantially modified from their original form and/or genomic
locus, or the promoter is not the native promoter for the operably
linked polynucleotide. As used herein, a chimeric gene comprises a
coding sequence operably linked to a transcription initiation
region that is heterologous to the coding sequence.
[0067] The termination region may be native with the
transcriptional initiation region, may be native with the operably
linked polynucleotide encoding the silencing element, may be native
with the plant host, or may be derived from another source (i.e.,
foreign or heterologous) to the promoter, the polynucleotide
comprising silencing element, the plant host, or any combination
thereof. Convenient termination regions are available from the
Ti-plasmid of A. tumefaciens, such as the octopine synthase and
nopaline synthase termination regions. See also Guerineau et al.
(1991) Mol. Gen. Genet. 262:141-144; Proudfoot (1991) Cell
64:671-674; Sanfacon et al. (1991) Genes Dev. 5:141-149; Mogen et
al. (1990) Plant Cell 2:1261-1272; Munroe et al. (1990) Gene
91:151-158; Ballas et al. (1989) Nucleic Acids Res. 17:7891-7903;
and Joshi et al. (1987) Nucleic Acids Res. 15:9627-9639.
[0068] Additional sequence modifications are known to enhance gene
expression in a cellular host. These include elimination of
sequences encoding spurious polyadenylation signals, exon-intron
splice site signals, transposon-like repeats, and other such
well-characterized sequences that may be deleterious to gene
expression. The G-C content of the sequence may be adjusted to
levels average for a given cellular host, as calculated by
reference to known genes expressed in the host cell. When possible,
the sequence is modified to avoid predicted hairpin secondary mRNA
structures.
[0069] In preparing the expression cassette, the various DNA
fragments may be manipulated, so as to provide for the DNA
sequences in the proper orientation and, as appropriate, in the
proper reading frame. Toward this end, adapters or linkers may be
employed to join the DNA fragments or other manipulations may be
involved to provide for convenient restriction sites, removal of
superfluous DNA, removal of restriction sites, or the like. For
this purpose, in vitro mutagenesis, primer repair, restriction,
annealing, resubstitutions, e.g., transitions and transversions,
may be involved.
[0070] A number of promoters can be used in the practice of the
invention. The polynucleotide encoding the silencing element can be
combined with constitutive, tissue-preferred, or other promoters
for expression in plants.
[0071] Such constitutive promoters include, for example, the core
promoter of the Rsyn7 promoter and other constitutive promoters
disclosed in WO 99/43838 and U.S. Pat. No. 6,072,050; the core CaMV
35S promoter (Odell et al. (1985) Nature 313:810-812); rice actin
(McElroy et al. (1990) Plant Cell 2:163-171); ubiquitin
(Christensen et al. (1989) Plant Mol. Biol. 12:619-632 and
Christensen et al. (1992) Plant Mol. Biol. 18:675-689); pEMU (Last
et al. (1991) Theor. Appl. Genet. 81:581-588); MAS (Velten et al.
(1984) EMBO J. 3:2723-2730); ALS promoter (U.S. Pat. No.
5,659,026), and the like. Other constitutive promoters include, for
example, U.S. Pat. Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597;
5,466,785; 5,399,680; 5,268,463; 5,608,142; and 6,177,611.
[0072] An inducible promoter, for instance, a pathogen-inducible
promoter could also be employed. Such promoters include those from
pathogenesis-related proteins (PR proteins), which are induced
following infection by a pathogen; e.g., PR proteins, SAR proteins,
beta-1,3-glucanase, chitinase, etc. See, for example, Redolfi et
al. (1983) Neth. J. Plant Pathol. 89:245-254; Uknes et al. (1992)
Plant Cell 4:645-656; and Van Loon (1985) Plant Mol. Virol.
4:111-116. See also WO 99/43819, herein incorporated by
reference.
[0073] Of interest are promoters that are expressed locally at or
near the site of pathogen infection. See, for example, Marineau et
al. (1987) Plant Mol. Biol. 9:335-342; Matton et al. (1989)
Molecular Plant-Microbe Interactions 2:325-331; Somsisch et al.
(1986) Proc. Natl. Acad. Sci. USA 83:2427-2430; Somsisch et al.
(1988) Mol. Gen. Genet. 2:93-98; and Yang (1996) Proc. Natl. Acad.
Sci. USA 93:14972-14977. See also, Chen et al. (1996) Plant J.
10:955-966; Zhang et al. (1994) Proc. Natl. Acad. Sci. USA
91:2507-2511; Warner et al. (1993) Plant J. 3:191-201; Siebertz et
al. (1989) Plant Cell 1:961-968; U.S. Pat. No. 5,750,386
(nematode-inducible); and the references cited therein. Of
particular interest is the inducible promoter for the maize PRms
gene, whose expression is induced by the pathogen Fusarium
moniliforme (see, for example, Cordero et al. (1992) Physiol. Mol.
Plant Path. 41:189-200).
[0074] Additionally, as pathogens find entry into plants through
wounds or insect damage, a wound-inducible promoter may be used in
the constructions of the invention. Such wound-inducible promoters
include potato proteinase inhibitor (pin II) gene (Ryan (1990) Ann.
Rev. Phytopath. 28:425-449; Duan et al. (1996) Nature Biotechnology
14:494-498); wun1 and wun2, U.S. Pat. No. 5,428,148; win1 and win2
(Stanford et al. (1989) Mol. Gen. Genet. 215:200-208); systemin
(McGurl et al. (1992) Science 225:1570-1573); WIP1 (Rohmeier et al.
(1993) Plant Mol. Biol. 22:783-792; Eckelkamp et al. (1993) FEBS
Letters 323:73-76); MPI gene (Corderok et al. (1994) Plant J.
6(2):141-150); and the like, herein incorporated by reference.
[0075] Chemical-regulated promoters can be used to modulate the
expression of a gene in a plant through the application of an
exogenous chemical regulator. Depending upon the objective, the
promoter may be a chemical-inducible promoter, where application of
the chemical induces gene expression, or a chemical-repressible
promoter, where application of the chemical represses gene
expression. Chemical-inducible promoters are known in the art and
include, but are not limited to, the maize In2-2 promoter, which is
activated by benzenesulfonamide herbicide safeners, the maize GST
promoter, which is activated by hydrophobic electrophilic compounds
that are used as pre-emergent herbicides, and the tobacco PR-la
promoter, which is activated by salicylic acid. Other
chemical-regulated promoters of interest include steroid-responsive
promoters (see, for example, the glucocorticoid-inducible promoter
in Schena et al. (1991) Proc. Natl. Acad. Sci. USA 88:10421-10425
and McNellis et al. (1998) Plant J. 14(2):247-257) and
tetracycline-inducible and tetracycline-repressible promoters (see,
for example, Gatz et al. (1991) Mol. Gen. Genet. 227:229-237, and
U.S. Pat. Nos. 5,814,618 and 5,789,156), herein incorporated by
reference.
[0076] Tissue-preferred promoters can be utilized to target
enhanced expression within a particular plant tissue.
Tissue-preferred promoters include Yamamoto et al. (1997) Plant J.
12(2):255-265; Kawamata et al. (1997) Plant Cell Physiol.
38(7):792-803; Hansen et al. (1997) Mol. Gen Genet. 254(3):337-343;
Russell et al. (1997) Transgenic Res. 6(2):157-168; Rinehart et al.
(1996) Plant Physiol. 112(3):1331-1341; Van Camp et al. (1996)
Plant Physiol. 112(2):525-535; Canevascini et al. (1996) Plant
Physiol. 112(2):513-524; Yamamoto et al. (1994) Plant Cell Physiol.
35(5):773-778; Lam (1994) Results Probl. Cell Differ. 20:181-196;
Orozco et al. (1993) Plant Mol Biol. 23(6):1129-1138; Matsuoka et
al. (1993) Proc Natl. Acad. Sci. USA 90(20):9586-9590; and
Guevara-Garcia et al. (1993) Plant J. 4(3):495-505. Such promoters
can be modified, if necessary, for weak expression.
[0077] Leaf-preferred promoters are known in the art. See, for
example, Yamamoto et al. (1997) Plant J. 12(2):255-265; Kwon et al.
(1994) Plant Physiol. 105:357-67; Yamamoto et al. (1994) Plant Cell
Physiol. 35(5):773-778; Gotor et al. (1993) Plant J. 3:509-18;
Orozco et al. (1993) Plant Mol. Biol. 23(6):1129-1138; and Matsuoka
et al. (1993) Proc. Natl. Acad. Sci. USA 90(20):9586-9590.
[0078] Root-preferred promoters are known and can be selected from
the many available from the literature or isolated de novo from
various compatible species. See, for example, Hire et al. (1992)
Plant Mol. Biol. 20(2):207-218 (soybean root-specific glutamine
synthetase gene); Keller and Baumgartner (1991) Plant Cell
3(10):1051-1061 (root-specific control element in the GRP 1.8 gene
of French bean); Sanger et al. (1990) Plant Mol. Biol.
14(3):433-443 (root-specific promoter of the mannopine synthase
(MAS) gene of Agrobacterium tumefaciens); and Miao et al. (1991)
Plant Cell 3(1):11-22 (full-length cDNA clone encoding cytosolic
glutamine synthetase (GS), which is expressed in roots and root
nodules of soybean). See also Bogusz et al. (1990) Plant Cell
2(7):633-641, where two root-specific promoters isolated from
hemoglobin genes from the nitrogen-fixing nonlegume Parasponia
andersonii and the related non-nitrogen-fixing nonlegume Trema
tomentosa are described. The promoters of these genes were linked
to a .beta.-glucuronidase reporter gene and introduced into both
the nonlegume Nicotiana tabacum and the legume Lotus corniculatus,
and in both instances root-specific promoter activity was
preserved. Leach and Aoyagi (1991) describe their analysis of the
promoters of the highly expressed rolC and rolD root-inducing genes
of Agrobacterium rhizogenes (see Plant Science (Limerick)
79(1):69-76). They concluded that enhancer and tissue-preferred DNA
determinants are dissociated in those promoters. Teeri et al.
(1989) used gene fusion to lacZ to show that the Agrobacterium
T-DNA gene encoding octopine synthase is especially active in the
epidermis of the root tip and that the TR2' gene is root specific
in the intact plant and stimulated by wounding in leaf tissue, an
especially desirable combination of characteristics for use with an
insecticidal or larvicidal gene (see EMBO J. 8(2):343-350). The
TR1' gene, fused to nptII (neomycin phosphotransferase II) showed
similar characteristics. Additional root-preferred promoters
include the VfENOD-GRP3 gene promoter (Kuster et al. (1995) Plant
Mol. Biol. 29(4):759-772); and rolB promoter (Capana et al. (1994)
Plant Mol. Biol. 25(4):681-691. See also U.S. Pat. Nos. 5,837,876;
5,750,386; 5,633,363; 5,459,252; 5,401,836; 5,110,732; and
5,023,179.
[0079] In one embodiment of this invention the plant-expressed
promoter is a vascular-specific promoter such as a phloem-specific
promoter. A "vascular-specific" promoter, as used herein, is a
promoter which is at least expressed in vascular cells, or a
promoter which is preferentially expressed in vascular cells.
Expression of a vascular-specific promoter need not be exclusively
in vascular cells, expression in other cell types or tissues is
possible. A "phloem-specific promoter" as used herein, is a
plant-expressible promoter which is at least expressed in phloem
cells, or a promoter which is preferentially expressed in phloem
cells.
[0080] Expression of a phloem-specific promoter need not be
exclusively in phloem cells, expression in other cell types or
tissues, e.g., xylem tissue, is possible. In one embodiment of this
invention, a phloem-specific promoter is a plant-expressible
promoter at least expressed in phloem cells, wherein the expression
in non-phloem cells is more limited (or absent) compared to the
expression in phloem cells. Examples of suitable vascular-specific
or phloem-specific promoters in accordance with this invention
include but are not limited to the promoters selected from the
group consisting of: the SCSV3, SCSV4, SCSV5, and SCSV7 promoters
(Schunmann et al. (2003) Plant Functional Biology 30:453-60; the
rolC gene promoter of Agrobacterium rhizogenes (Kiyokawa et al.
(1994) Plant Physiology 104:801-02; Pandolfini et al. (2003) Bio
Med Central (BMC) Biotechnology 3:7,
(www.biomedcentral.com/1472-6750/3/7); Graham et al. (1997) Plant
Mol. Biol. 33:729-35; Guivarc'h et al. (1996); Almon et al. (1997)
Plant Physiol. 115:1599-607; the rolA gene promoter of
Agrobacterium rhizogenes (Dehio et al. (1993) Plant Mol. Biol.
23:1199-210); the promoter of the Agrobacterium tumefaciens T-DNA
gene 5 (Korber et al. (1991) EMBO J. 10:3983-91); the rice sucrose
synthase RSs1 gene promoter (Shi et al. (1994) J. Exp. Bot.
45:623-31); the CoYMV or Commelina yellow mottle badnavirus
promoter (Medberry et al. (1992) Plant Cell 4:185-92; Zhou et al.
(1998) Chin. J. Biotechnol. 14:9-16); the CFDV or coconut foliar
decay virus promoter (Rohde et al. (1994) Plant Mol. Biol.
27:623-28; Hehn and Rhode (1998) J. Gen. Virol. 79:1495-99); the
RTBV or rice tungro bacilliform virus promoter (Yin and Beachy
(1995) Plant J. 7:969-80; Yin et al. (1997) Plant J. 12:1179-80);
the pea glutamin synthase GS3A gene (Edwards et al. (1990) Proc.
Natl. Acad. Sci. USA 87:3459-63; Brears et al. (1991) Plant J.
1:235-44); the inv CD111 and inv CD141 promoters of the potato
invertase genes (Hedley et al. (2000) J. Exp. Botany 51:817-21);
the promoter isolated from Arabidopsis shown to have
phloem-specific expression in tobacco by Kertbundit et al. (1991)
Proc. Natl. Acad. Sci. USA 88:5212-16); the VAHOX1 promoter region
(Tornero et al. (1996) Plant J. 9:639-48); the pea cell wall
invertase gene promoter (Zhang et al. (1996) Plant Physiol.
112:1111-17); the promoter of the endogenous cotton protein related
to chitinase of US published patent application 20030106097, an
acid invertase gene promoter from carrot (Ramloch-Lorenz et al.
(1993) The Plant J. 4:545-54); the promoter of the sulfate
transporter geneSultrl; 3 (Yoshimoto et al. (2003) Plant Physiol.
131:1511-17); a promoter of a sucrose synthase gene (Nolte and Koch
(1993) Plant Physiol. 101:899-905); and the promoter of a tobacco
sucrose transporter gene (Kuhn et al. (1997) Science
275-1298-1300).
[0081] Possible promoters also include the Black Cherry promoter
for Prunasin Hydrolase (PH DL1.4 PRO) (U.S. Pat. No. 6,797,859),
Thioredoxin H promoter from cucumber and rice (Fukuda A et al.
(2005). Plant Cell Physiol. 46(11):1779-86), Rice (RSs1) (Shi, T.
Wang et al. (1994). J. Exp. Bot. 45(274): 623-631) and maize
sucrose synthese -1 promoters (Yang., N-S. et al. (1990) PNAS
87:4144-4148), PP2 promoter from pumpkin Guo, H. et al. (2004)
Transgenic Research 13:559-566), At SUC2 promoter (Truernit, E. et
al. (1995) Planta 196(3):564-70. At SAM-1 (S-adenosylmethionine
synthetase) (Mijnsbrugge K V. et al. (1996) Planr. Cell. Physiol.
37(8): 1108-1115), and the Rice tungro bacilliform virus (RTBV)
promoter (Bhattacharyya-Pakrasi et al. (1993) Plant J.
4(1):71-79).
[0082] The expression cassette can also comprise a selectable
marker gene for the selection of transformed cells. Selectable
marker genes are utilized for the selection of transformed cells or
tissues. Marker genes include genes encoding antibiotic resistance,
such as those encoding neomycin phosphotransferase II (NEO) and
hygromycin phosphotransferase (HPT), as well as genes conferring
resistance to herbicidal compounds, such as glufosinate ammonium,
bromoxynil, imidazolinones, and 2,4-dichlorophenoxyacetate (2,4-D).
Additional selectable markers include phenotypic markers such as
.beta.-galactosidase and fluorescent proteins such as green
fluorescent protein (GFP) (Su et al. (2004) Biotechnol Bioeng
85:610-9 and Fetter et al. (2004) Plant Cell 16:215-28), cyan
florescent protein (CYP) (Bolte et al. (2004) J. Cell Science
117:943-54 and Kato et al. (2002) Plant Physiol 129:913-42), and
yellow florescent protein (PhiYFP.TM. from Evrogen, see, Bolte et
al. (2004) J. Cell Science 117:943-54). For additional selectable
markers, see generally, Yarranton (1992) Curr. Opin. Biotech.
3:506-511; Christopherson et al. (1992) Proc. Natl. Acad. Sci. USA
89:6314-6318; Yao et al. (1992) Cell 71:63-72; Reznikoff (1992)
Mol. Microbiol. 6:2419-2422; Barkley et al. (1980) in The Operon,
pp. 177-220; Hu et al. (1987) Cell 48:555-566; Brown et al. (1987)
Cell 49:603-612; Figge et al. (1988) Cell 52:713-722; Deuschle et
al. (1989) Proc. Natl. Acad. Sci. USA 86:5400-5404; Fuerst et al.
(1989) Proc. Natl. Acad. Sci. USA 86:2549-2553; Deuschle et al.
(1990) Science 248:480-483; Gossen (1993) Ph.D. Thesis, University
of Heidelberg; Reines et al. (1993) Proc. Natl. Acad. Sci. USA
90:1917-1921; Labow et al. (1990) Mol. Cell. Biol. 10:3343-3356;
Zambretti et al. (1992) Proc. Natl. Acad. Sci. USA 89:3952-3956;
Baim et al. (1991) Proc. Natl. Acad. Sci. USA 88:5072-5076;
Wyborski et al. (1991) Nucleic Acids Res. 19:4647-4653;
Hillenand-Wissman (1989) Topics Mol. Struc. Biol. 10:143-162;
Degenkolb et al. (1991) Antimicrob. Agents Chemother. 35:1591-1595;
Kleinschnidt et al. (1988) Biochemistry 27:1094-1104; Bonin (1993)
Ph.D. Thesis, University of Heidelberg; Gossen et al. (1992) Proc.
Natl. Acad. Sci. USA 89:5547-5551; Oliva et al. (1992) Antimicrob.
Agents Chemother. 36:913-919; Hlavka et al. (1985) Handbook of
Experimental Pharmacology, Vol. 78 (Springer-Verlag, Berlin); Gill
et al. (1988) Nature 334:721-724. Such disclosures are herein
incorporated by reference. The above list of selectable marker
genes is not meant to be limiting. Any selectable marker gene can
be used in the present invention.
VI. Compositions Comprising Silencing Elements
[0083] One or more of the polynucleotides comprising the silencing
element can be provided as an external composition such as a spray
or powder to the plant, plant part, seed, a pest, or an area of
cultivation. In another example, a plant is transformed with a DNA
construct or expression cassette for expression of at least one
silencing element. In either compositions, the silencing element,
when ingested by an insect, can reduce the level of a target pest
sequence and thereby control the pest (i.e., any pest from the
Lygus genus, such as, Lygus hesperus). It is recognized that the
composition can comprise a cell (such as plant cell or a bacterial
cell), in which a polynucleotide encoding the silencing element is
stably incorporated into the genome and operably linked to
promoters active in the cell. Compositions comprising a mixture of
cells, some cells expressing at least one silencing element are
also encompassed. In other embodiments, compositions comprising the
silencing elements are not contained in a cell. In such
embodiments, the composition can be applied to an area inhabited by
a pest. In one embodiment, the composition is applied externally to
a plant (i.e., by spraying a field or area of cultivation) to
protect the plant from the pest.
[0084] The composition of the invention can further be formulated
as bait. In this embodiment, the compositions comprise a food
substance or an attractant which enhances the attractiveness of the
composition to the pest.
[0085] The composition comprising the silencing element can be
formulated in an agriculturally suitable and/or environmentally
acceptable carrier. Such carriers can be any material that the
animal, plant or environment to be treated can tolerate.
Furthermore, the carrier must be such that the composition remains
effective at controlling a pest. Examples of such carriers include
water, saline, Ringer's solution, dextrose or other sugar
solutions, Hank's solution, and other aqueous physiologically
balanced salt solutions, phosphate buffer, bicarbonate buffer and
Tris buffer. In addition, the composition may include compounds
that increase the half-life of a composition.
[0086] It is recognized that the polynucleotides comprising
sequences encoding the silencing element can be used to transform
organisms to provide for host organism production of these
components, and subsequent application of the host organism to the
environment of the target pest(s). Such host organisms include
baculoviruses, bacteria, and the like. In this manner, the
combination of polynucleotides encoding the silencing element may
be introduced via a suitable vector into a microbial host, and said
host applied to the environment, or to plants or animals.
[0087] The term "introduced" in the context of inserting a nucleic
acid into a cell, means "transfection" or "transformation" or
"transduction" and includes reference to the incorporation of a
nucleic acid into a eukaryotic or prokaryotic cell where the
nucleic acid may be stably incorporated into the genome of the cell
(e.g., chromosome, plasmid, plastid, or mitochondrial DNA),
converted into an autonomous replicon, or transiently expressed
(e.g., transfected mRNA).
[0088] Microorganism hosts that are known to occupy the
"phytosphere" (phylloplane, phyllosphere, rhizosphere, and/or
rhizoplana) of one or more crops of interest may be selected. These
microorganisms are selected so as to be capable of successfully
competing in the particular environment with the wild-type
microorganisms, provide for stable maintenance and expression of
the sequences encoding the silencing element, and desirably,
provide for improved protection of the components from
environmental degradation and inactivation.
[0089] Such microorganisms include bacteria, algae, and fungi. Of
particular interest are microorganisms such as bacteria, e.g.,
Pseudomonas, Erwinia, Serratia, Klebsiella, Xanthomonas,
Streptomyces, Rhizobium, Rhodopseudomonas, Methylius,
Agrobacterium, Acetobacter, Lactobacillus, Arthrobacter,
Azotobacter, Leuconostoc, and Alcaligenes, fungi, particularly
yeast, e.g., Saccharomyces, Cryptococcus, Kluyveromyces,
Sporobolomyces, Rhodotorula, and Aureobasidium. Of particular
interest are such phytosphere bacterial species as Pseudomonas
syringae, Pseudomonas fluorescens, Serratia marcescens, Acetobacter
xylinum, Agrobacteria, Rhodopseudomonas spheroides, Xanthomonas
campestris, Rhizobium melioti, Alcaligenes entrophus, Clavibacter
xyli and Azotobacter vinlandir, and phytosphere yeast species such
as Rhodotorula rubra, R. glutinis, R. marina, R. aurantiaca,
Cryptococcus albidus, C. diffluens, C. laurentii, Saccharomyces
rosei, S. pretoriensis, S. cerevisiae, Sporobolomyces rosues, S.
odorus, Kluyveromyces veronae, and Aureobasidium pollulans. Of
particular interest are the pigmented microorganisms.
[0090] A number of ways are available for introducing the
polynucleotide comprising the silencing element into the
microorganism host under conditions that allow for stable
maintenance and expression of such nucleotide encoding sequences.
For example, expression cassettes can be constructed which include
the nucleotide constructs of interest operably linked with the
transcriptional and translational regulatory signals for expression
of the nucleotide constructs, and a nucleotide sequence homologous
with a sequence in the host organism, whereby integration will
occur, and/or a replication system that is functional in the host,
whereby integration or stable maintenance will occur.
[0091] Transcriptional and translational regulatory signals
include, but are not limited to, promoters, transcriptional
initiation start sites, operators, activators, enhancers, other
regulatory elements, ribosomal binding sites, an initiation codon,
termination signals, and the like. See, for example, U.S. Pat. Nos.
5,039,523 and 4,853,331; EPO 0480762A2; Sambrook et al. (2000);
Molecular Cloning: A Laboratory Manual (3rd ed.; Cold Spring Harbor
Laboratory Press, Plainview, N.Y.); Davis et al. (1980) Advanced
Bacterial Genetics (Cold Spring Harbor Laboratory, Cold Spring
Harbor, N.Y.; and the references cited therein.
[0092] Suitable host cells include the prokaryotes and the lower
eukaryotes, such as fungi. Illustrative prokaryotes, both
Gram-negative and Gram-positive, include Enterobacteriaceae, such
as Escherichia, Erwinia, Shigella, Salmonella, and Proteus;
Bacillaceae; Rhizobiceae, such as Rhizobium; Spirillaceae, such as
photobacterium, Zymomonas, Serratia, Aeromonas, Vibrio,
Desulfovibrio, Spirillum; Lactobacillaceae; Pseudomonadaceae, such
as Pseudomonas and Acetobacter; Azotobacteraceae and
Nitrobacteraceae. Among eukaryotes are fungi, such as Phycomycetes
and Ascomycetes, which includes yeast, such as Saccharomyces and
Schizosaccharomyces; and Basidiomycetes yeast, such as Rhodotorula,
Aureobasidium, Sporobolomyces, and the like.
[0093] Characteristics of particular interest in selecting a host
cell for purposes of the invention include ease of introducing the
coding sequence into the host, availability of expression systems,
efficiency of expression, stability in the host, and the presence
of auxiliary genetic capabilities. Characteristics of interest for
use as a pesticide microcapsule include protective qualities, such
as thick cell walls, pigmentation, and intracellular packaging or
formation of inclusion bodies; leaf affinity; lack of mammalian
toxicity; attractiveness to pests for ingestion; and the like.
Other considerations include ease of formulation and handling,
economics, storage stability, and the like.
[0094] Host organisms of particular interest include yeast, such as
Rhodotorula spp., Aureobasidium spp., Saccharomyces spp., and
Sporobolomyces spp., phylloplane organisms such as Pseudomonas
spp., Erwinia spp., and Flavobacterium spp., and other such
organisms, including Pseudomonas aeruginosa, Pseudomonas
fluorescens, Saccharomyces cerevisiae, Bacillus thuringiensis,
Escherichia coli, Bacillus subtilis, and the like.
[0095] The sequences encoding the silencing elements encompassed by
the invention can be introduced into microorganisms that multiply
on plants (epiphytes) to deliver these components to potential
target pests. Epiphytes, for example, can be gram-positive or
gram-negative bacteria.
[0096] The silencing element can be fermented in a bacterial host
and the resulting bacteria processed and used as a microbial spray
in the same manner that Bacillus thuringiensis strains have been
used as insecticidal sprays. Any suitable microorganism can be used
for this purpose. Pseudomonas has been used to express Bacillus
thuringiensis endotoxins as encapsulated proteins and the resulting
cells processed and sprayed as an insecticide Gaertner et al.
(1993), in Advanced Engineered Pesticides, ed. L. Kim (Marcel
Decker, Inc.).
[0097] Alternatively, the components of the invention are produced
by introducing heterologous genes into a cellular host. Expression
of the heterologous sequences results, directly or indirectly, in
the intracellular production of the silencing element. These
compositions may then be formulated in accordance with conventional
techniques for application to the environment hosting a target
pest, e.g., soil, water, and foliage of plants. See, for example,
EPA 0192319, and the references cited therein.
[0098] In the present invention, a transformed microorganism can be
formulated with an acceptable carrier into separate or combined
compositions that are, for example, a suspension, a solution, an
emulsion, a dusting powder, a dispersible granule, a wettable
powder, and an emulsifiable concentrate, an aerosol, an impregnated
granule, an adjuvant, a coatable paste, and also encapsulations in,
for example, polymer substances.
[0099] Such compositions disclosed above may be obtained by the
addition of a surface-active agent, an inert carrier, a
preservative, a humectant, a feeding stimulant, an attractant, an
encapsulating agent, a binder, an emulsifier, a dye, a UV
protectant, a buffer, a flow agent or fertilizers, micronutrient
donors, or other preparations that influence plant growth. One or
more agrochemicals including, but not limited to, herbicides,
insecticides, fungicides, bactericides, nematicides, molluscicides,
acaracides, plant growth regulators, harvest aids, and fertilizers,
can be combined with carriers, surfactants or adjuvants customarily
employed in the art of formulation or other components to
facilitate product handling and application for particular target
pests. Suitable carriers and adjuvants can be solid or liquid and
correspond to the substances ordinarily employed in formulation
technology, e.g., natural or regenerated mineral substances,
solvents, dispersants, wetting agents, tackifiers, binders, or
fertilizers. The active ingredients of the present invention (i.e.,
at least one silencing element) are normally applied in the form of
compositions and can be applied to the crop area, plant, or seed to
be treated. For example, the compositions may be applied to grain
in preparation for or during storage in a grain bin or silo, etc.
The compositions may be applied simultaneously or in succession
with other compounds. Methods of applying an active ingredient or a
composition that contains at least one silencing element include,
but are not limited to, foliar application, seed coating, and soil
application. The number of applications and the rate of application
depend on the intensity of infestation by the corresponding
pest.
[0100] Suitable surface-active agents include, but are not limited
to, anionic compounds such as a carboxylate of, for example, a
metal; carboxylate of a long chain fatty acid; an
N-acylsarcosinate; mono- or di-esters of phosphoric acid with fatty
alcohol ethoxylates or salts of such esters; fatty alcohol sulfates
such as sodium dodecyl sulfate, sodium octadecyl sulfate, or sodium
cetyl sulfate; ethoxylated fatty alcohol sulfates; ethoxylated
alkylphenol sulfates; lignin sulfonates; petroleum sulfonates;
alkyl aryl sulfonates such as alkyl-benzene sulfonates or lower
alkylnaphtalene sulfonates, e.g., butyl-naphthalene sulfonate;
salts of sulfonated naphthalene-formaldehyde condensates; salts of
sulfonated phenol-formaldehyde condensates; more complex sulfonates
such as the amide sulfonates, e.g., the sulfonated condensation
product of oleic acid and N-methyl taurine; or the dialkyl
sulfosuccinates, e.g., the sodium sulfonate or dioctyl succinate.
Non-ionic agents include condensation products of fatty acid
esters, fatty alcohols, fatty acid amides or fatty-alkyl- or
alkenyl-substituted phenols with ethylene oxide, fatty esters of
polyhydric alcohol ethers, e.g., sorbitan fatty acid esters,
condensation products of such esters with ethylene oxide, e.g.,
polyoxyethylene sorbitan fatty acid esters, block copolymers of
ethylene oxide and propylene oxide, acetylenic glycols such as
2,4,7,9-tetraethyl-5-decyn-4,7-diol, or ethoxylated acetylenic
glycols. Examples of a cationic surface-active agent include, for
instance, an aliphatic mono-, di-, or polyamine such as an acetate,
naphthenate or oleate; or oxygen-containing amine such as an amine
oxide of polyoxyethylene alkylamine; an amide-linked amine prepared
by the condensation of a carboxylic acid with a di- or polyamine;
or a quaternary ammonium salt.
[0101] Examples of inert materials include, but are not limited to,
inorganic minerals such as kaolin, phyllosilicates, carbonates,
sulfates, phosphates, or botanical materials such as cork, powdered
corncobs, peanut hulls, rice hulls, and walnut shells.
[0102] The compositions comprising the silencing element can be in
a suitable form for direct application or as a concentrate of
primary composition that requires dilution with a suitable quantity
of water or other dilutant before application.
[0103] The compositions (including the transformed microorganisms)
can be applied to the environment of an insect pest (such as a pest
from the Lygus genus) by, for example, spraying, atomizing,
dusting, scattering, coating or pouring, introducing into or on the
soil, introducing into irrigation water, by seed treatment or
general application or dusting at the time when the pest has begun
to appear or before the appearance of pests as a protective
measure. For example, the composition(s) and/or transformed
microorganism(s) may be mixed with grain to protect the grain
during storage. It is generally important to obtain good control of
pests in the early stages of plant growth, as this is the time when
the plant can be most severely damaged. The compositions can
conveniently contain another insecticide if this is thought
necessary. In an embodiment of the invention, the composition(s) is
applied directly to the soil, at a time of planting, in granular
form of a composition of a carrier and dead cells of a Bacillus
strain or transformed microorganism of the invention. Another
embodiment is a granular form of a composition comprising an
agrochemical such as, for example, a herbicide, an insecticide, a
fertilizer, in an inert carrier, and dead cells of a Bacillus
strain or transformed microorganism of the invention.
VII. Plants, Plant Parts, and Methods of Introducing Sequences into
Plants
[0104] In one embodiment, the methods of the invention involve
introducing a polypeptide or polynucleotide into a plant.
"Introducing" is intended to mean presenting to the plant the
polynucleotide or polypeptide in such a manner that the sequence
gains access to the interior of a cell of the plant. The methods of
the invention do not depend on a particular method for introducing
a sequence into a plant, only that the polynucleotide or
polypeptides gains access to the interior of at least one cell of
the plant. Methods for introducing polynucleotide or polypeptides
into plants are known in the art including, but not limited to,
stable transformation methods, transient transformation methods,
and virus-mediated methods.
[0105] "Stable transformation" is intended to mean that the
nucleotide construct introduced into a plant integrates into the
genome of the plant and is capable of being inherited by the
progeny thereof. "Transient transformation" is intended to mean
that a polynucleotide is introduced into the plant and does not
integrate into the genome of the plant or a polypeptide is
introduced into a plant.
[0106] Transformation protocols as well as protocols for
introducing polypeptides or polynucleotide sequences into plants
may vary depending on the type of plant or plant cell, i.e.,
monocot or dicot, targeted for transformation. Suitable methods of
introducing polypeptides and polynucleotides into plant cells
include microinjection (Crossway et al. (1986) Biotechniques
4:320-334), electroporation (Riggs et al. (1986) Proc. Natl. Acad.
Sci. USA 83:5602-5606, Agrobacterium-mediated transformation (U.S.
Pat. No. 5,563,055 and U.S. Pat. No. 5,981,840), direct gene
transfer (Paszkowski et al. (1984) EMBO J. 3:2717-2722), and
ballistic particle acceleration (see, for example, U.S. Pat. No.
4,945,050; U.S. Pat. No. 5,879,918; U.S. Pat. No. 5,886,244; and,
U.S. Pat. No. 5,932,782; Tomes et al. (1995) in Plant Cell, Tissue,
and Organ Culture: Fundamental Methods, ed. Gamborg and Phillips
(Springer-Verlag, Berlin); McCabe et al. (1988) Biotechnology
6:923-926); and Lec1 transformation (WO 00/28058). Also see
Weissinger et al. (1988) Ann. Rev. Genet. 22:421-477; Sanford et
al. (1987) Particulate Science and Technology 5:27-37 (onion);
Christou et al. (1988) Plant Physiol. 87:671-674 (soybean); McCabe
et al. (1988) Bio/Technology 6:923-926 (soybean); Finer and
McMullen (1991) In Vitro Cell Dev. Biol. 27P:175-182 (soybean);
Singh et al. (1998) Theor. Appl. Genet. 96:319-324 (soybean); Datta
et al. (1990) Biotechnology 8:736-740 (rice); Klein et al. (1988)
Proc. Natl. Acad. Sci. USA 85:4305-4309 (maize); Klein et al.
(1988) Biotechnology 6:559-563 (maize); U.S. Pat. Nos. 5,240,855;
5,322,783; and, 5,324,646; Klein et al. (1988) Plant Physiol.
91:440-444 (maize); Fromm et al. (1990) Biotechnology 8:833-839
(maize); Hooykaas-Van Slogteren et al. (1984) Nature (London)
311:763-764; U.S. Pat. No. 5,736,369 (cereals); Bytebier et al.
(1987) Proc. Natl. Acad. Sci. USA 84:5345-5349 (Liliaceae); De Wet
et al. (1985) in The Experimental Manipulation of Ovule Tissues,
ed. Chapman et al. (Longman, New York), pp. 197-209 (pollen);
Kaeppler et al. (1990) Plant Cell Reports 9:415-418 and Kaeppler et
al. (1992) Theor. Appl. Genet. 84:560-566 (whisker-mediated
transformation); D'Halluin et al. (1992) Plant Cell 4:1495-1505
(electroporation); Li et al. (1993) Plant Cell Reports 12:250-255
and Christou and Ford (1995) Annals of Botany 75:407-413 (rice);
Osjoda et al. (1996) Nature Biotechnology 14:745-750 (maize via
Agrobacterium tumefaciens); all of which are herein incorporated by
reference.
[0107] In specific embodiments, the silencing element sequences of
the invention can be provided to a plant using a variety of
transient transformation methods. Such transient transformation
methods include, but are not limited to, the introduction of the
protein or variants and fragments thereof directly into the plant
or the introduction of the transcript into the plant. Such methods
include, for example, microinjection or particle bombardment. See,
for example, Crossway et al. (1986) Mol Gen. Genet. 202:179-185;
Nomura et al. (1986) Plant Sci. 44:53-58; Hepler et al. (1994)
Proc. Natl. Acad. Sci. 91: 2176-2180 and Hush et al. (1994) The
Journal of Cell Science 107:775-784, all of which are herein
incorporated by reference. Alternatively, polynucleotides can be
transiently transformed into the plant using techniques known in
the art. Such techniques include viral vector system and the
precipitation of the polynucleotide in a manner that precludes
subsequent release of the DNA. Thus, the transcription from the
particle-bound DNA can occur, but the frequency with which its
released to become integrated into the genome is greatly reduced.
Such methods include the use particles coated with polyethylimine
(PEI; Sigma #P3143).
[0108] In other embodiments, the polynucleotide of the invention
may be introduced into plants by contacting plants with a virus or
viral nucleic acids. Generally, such methods involve incorporating
a nucleotide construct of the invention within a viral DNA or RNA
molecule. Further, it is recognized that promoters of the invention
also encompass promoters utilized for transcription by viral RNA
polymerases. Methods for introducing polynucleotides into plants
and expressing a protein encoded therein, involving viral DNA or
RNA molecules, are known in the art. See, for example, U.S. Pat.
Nos. 5,889,191, 5,889,190, 5,866,785, 5,589,367, 5,316,931, and
Porta et al. (1996) Molecular Biotechnology 5:209-221; herein
incorporated by reference.
[0109] Methods are known in the art for the targeted insertion of a
polynucleotide at a specific location in the plant genome. In one
embodiment, the insertion of the polynucleotide at a desired
genomic location is achieved using a site-specific recombination
system. See, for example, WO99/25821, WO99/25854, WO99/25840,
WO99/25855, and WO99/25853, all of which are herein incorporated by
reference. Briefly, the polynucleotide of the invention can be
contained in transfer cassette flanked by two non-recombinogenic
recombination sites. The transfer cassette is introduced into a
plant having stably incorporated into its genome a target site
which is flanked by two non-recombinogenic recombination sites that
correspond to the sites of the transfer cassette. An appropriate
recombinase is provided and the transfer cassette is integrated at
the target site. The polynucleotide of interest is thereby
integrated at a specific chromosomal position in the plant
genome.
[0110] The cells that have been transformed may be grown into
plants in accordance with conventional ways. See, for example,
McCormick et al. (1986) Plant Cell Reports 5:81-84. These plants
may then be grown, and either pollinated with the same transformed
strain or different strains, and the resulting progeny having
constitutive expression of the desired phenotypic characteristic
identified. Two or more generations may be grown to ensure that
expression of the desired phenotypic characteristic is stably
maintained and inherited and then seeds harvested to ensure
expression of the desired phenotypic characteristic has been
achieved. In this manner, the present invention provides
transformed seed (also referred to as "transgenic seed") having a
polynucleotide of the invention, for example, an expression
cassette of the invention, stably incorporated into their
genome.
[0111] As used herein, the term plant includes plant cells, plant
protoplasts, plant cell tissue cultures from which plants can be
regenerated, plant calli, plant clumps, and plant cells that are
intact in plants or parts of plants such as embryos, pollen,
ovules, seeds, leaves, flowers, branches, fruit, kernels, ears,
cobs, husks, stalks, roots, root tips, anthers, and the like. Grain
is intended to mean the mature seed produced by commercial growers
for purposes other than growing or reproducing the species.
Progeny, variants, and mutants of the regenerated plants are also
included within the scope of the invention, provided that these
parts comprise the introduced polynucleotides.
[0112] The present invention may be used for transformation of any
plant species, including, but not limited to, monocots and dicots.
Examples of plant species of interest include, but are not limited
to, corn (Zea mays), Brassica sp. (e.g., B. napus, B. rapa, B.
juncea), particularly those Brassica species useful as sources of
seed oil, alfalfa (Medicago sativa), rice (Oryza sativa), rye
(Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare),
millet (e.g., pearl millet (Pennisetum glaucum), proso millet
(Panicum miliaceum), foxtail millet (Setaria italica), finger
millet (Eleusine coracana)), sunflower (Helianthus annuus),
safflower (Carthamus tinctorius), wheat (Triticum aestivum),
soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanum
tuberosum), peanuts (Arachis hypogaea), cotton (Gossypium
barbadense, Gossypium hirsutum), sweet potato (Ipomoea batatus),
cassava (Manihot esculenta), coffee (Coffea spp.), coconut (Cocos
nucifera), pineapple (Ananas comosus), citrus trees (Citrus spp.),
cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musa
spp.), avocado (Persea americana), fig (Ficus casica), guava
(Psidium guajava), mango (Mangifera indica), olive (Olea europaea),
papaya (Carica papaya), cashew (Anacardium occidentale), macadamia
(Macadamia integrifolia), almond (Prunus amygdalus), sugar beets
(Beta vulgaris), sugarcane (Saccharum spp.), oats, barley,
vegetables, ornamentals, and conifers.
[0113] Vegetables include tomatoes (Lycopersicon esculentum),
lettuce (e.g., Lactuca sativa), green beans (Phaseolus vulgaris),
lima beans (Phaseolus limensis), peas (Lathyrus spp.), and members
of the genus Cucumis such as cucumber (C. sativus), cantaloupe (C.
cantalupensis), and musk melon (C. melo). Ornamentals include
azalea (Rhododendron spp.), hydrangea (Macrophylla hydrangea),
hibiscus (Hibiscus rosasanensis), roses (Rosa spp.), tulips (Tulipa
spp.), daffodils (Narcissus spp.), petunias (Petunia hybrida),
carnation (Dianthus caryophyllus), poinsettia (Euphorbia
pulcherrima), and chrysanthemum.
[0114] Conifers that may be employed in practicing the present
invention include, for example, pines such as loblolly pine (Pinus
taeda), slash pine (Pinus elliotii), ponderosa pine (Pinus
ponderosa), lodgepole pine (Pinus contorta), and Monterey pine
(Pinus radiata); Douglas-fir (Pseudotsuga menziesii); Western
hemlock (Tsuga canadensis); Sitka spruce (Picea glauca); redwood
(Sequoia sempervirens); true firs such as silver fir (Abies
amabilis) and balsam fir (Abies balsamea); and cedars such as
Western red cedar (Thuja plicata) and Alaska yellow-cedar
(Chamaecyparis nootkatensis). In specific embodiments, plants of
the present invention are crop plants (for example, corn, alfalfa,
sunflower, Brassica, soybean, cotton, safflower, peanut, sorghum,
wheat, millet, tobacco, etc.). In other embodiments, corn and
soybean plants are optimal, and in yet other embodiments corn
plants are optimal.
[0115] Other plants of interest include grain plants that provide
seeds of interest, oil-seed plants, and leguminous plants. Seeds of
interest include grain seeds, such as corn, wheat, barley, rice,
sorghum, rye, etc. Oil-seed plants include cotton, soybean,
safflower, sunflower, Brassica, maize, alfalfa, palm, coconut, etc.
Leguminous plants include beans and peas. Beans include guar,
locust bean, fenugreek, soybean, garden beans, cowpea, mungbean,
lima bean, fava bean, lentils, chickpea, etc.
VIII. Methods of Use
[0116] The methods of the invention comprise methods for
controlling a pest (i.e., pest from the Lygus genus, such as, Lygus
hesperus) comprising feeding to a pest a composition comprising a
silencing element of the invention, wherein said silencing element,
when ingested by a pest (i.e., pests from the Lygus genus, such as,
Lygus hesperus), reduces the level of a target polynucleotide of
the pest and thereby controls the pest. The pest can be fed the
silencing element in a variety of ways. For example, in one
embodiment, the polynucleotide comprising the silencing element is
introduced into a plant. As the Lygus feeds on the plant or part
thereof expressing these sequences, the silencing element is
delivered to the pest. When the silencing element is delivered to
the plant in this manner, it is recognized that the silencing
element can be expressed constitutively or alternatively, it may be
produced in a stage-specific manner by employing the various
inducible or tissue-preferred or developmentally regulated
promoters that are discussed elsewhere herein. For example, the
silience element could be expressed in aerial plant tissues such
as, the leaves, stem, flower, etc. In other embodiments, the
silencing element is expressed in a root. In these embodiments,
Hemioptera such as grape phylloxera can be targeted.
[0117] In another method, a composition comprising at least one
silencing element of the invention is applied to a plant. In such
embodiments, the silencing element can be formulated in an
agronomically suitable and/or environmentally acceptable carrier,
which are preferably, suitable for dispersal in fields. In
addition, the carrier can also include compounds that increase the
half life of the composition. In specific embodiments, the
composition comprising the silencing element is formulated in such
a manner such that it persists in the environment for a length of
time sufficient to allow it to be delivered to a pest. In such
embodiments, the composition can be applied to an area inhabited by
a pest. In one embodiment, the composition is applied externally to
a plant (i.e., by spraying a field) to protect the plant from
pests.
[0118] In certain embodiments, the constructs of the present
invention can be stacked with any combination of polynucleotide
sequences of interest in order to create plants with a desired
trait. A trait, as used herein, refers to the phenotype derived
from a particular sequence or groups of sequences. For example, the
polynucleotides of the present invention may be stacked with any
other polynucleotides encoding polypeptides having pesticidal
and/or insecticidal activity, such as other Bacillus thuringiensis
toxic proteins (described in U.S. Pat. Nos. 5,366,892; 5,747,450;
5,737,514; 5,723,756; 5,593,881; and Geiser et al. (1986) Gene
48:109), lectins (Van Damme et al. (1994) Plant Mol. Biol. 24:825,
pentin (described in U.S. Pat. No. 5,981,722), and the like. The
combinations generated can also include multiple copies of any one
of the polynucleotides of interest. The polynucleotides of the
present invention can also be stacked with any other gene or
combination of genes to produce plants with a variety of desired
trait combinations including, but not limited to, traits desirable
for animal feed such as high oil genes (e.g., U.S. Pat. No.
6,232,529); balanced amino acids (e.g., hordothionins (U.S. Pat.
Nos. 5,990,389; 5,885,801; 5,885,802; and 5,703,409); barley high
lysine (Williamson et al. (1987) Eur. J. Biochem. 165:99-106; and
WO 98/20122) and high methionine proteins (Pedersen et al. (1986)
J. Biol. Chem. 261:6279; Kirihara et al. (1988) Gene 71:359; and
Musumura et al. (1989) Plant Mol. Biol. 12:123)); increased
digestibility (e.g., modified storage proteins (U.S. application
Ser. No. 10/053,410, filed Nov. 7, 2001); and thioredoxins (U.S.
application Ser. No. 10/005,429, filed Dec. 3, 2001)); the
disclosures of which are herein incorporated by reference.
[0119] The polynucleotides of the present invention can also be
stacked with traits desirable for disease or herbicide resistance
(e.g., fumonisin detoxification genes (U.S. Pat. No. 5,792,931);
avirulence and disease resistance genes (Jones et al. (1994)
Science 266:789; Martin et al. (1993) Science 262:1432; Mindrinos
et al. (1994) Cell 78:1089); acetolactate synthase (ALS) mutants
that lead to herbicide resistance such as the S4 and/or Hra
mutations; inhibitors of glutamine synthase such as
phosphinothricin or basta (e.g., bar gene); and glyphosate
resistance (EPSPS gene)); and traits desirable for processing or
process products such as high oil (e.g., U.S. Pat. No. 6,232,529);
modified oils (e.g., fatty acid desaturase genes (U.S. Pat. No.
5,952,544; WO 94/11516)); modified starches (e.g., ADPG
pyrophosphorylases (AGPase), starch synthases (SS), starch
branching enzymes (SBE), and starch debranching enzymes (SDBE));
and polymers or bioplastics (e.g., U.S. Pat. No. 5,602,321;
beta-ketothiolase, polyhydroxybutyrate synthase, and
acetoacetyl-CoA reductase (Schubert et al. (1988) J. Bacteriol.
170:5837-5847) facilitate expression of polyhydroxyalkanoates
(PHAs)); the disclosures of which are herein incorporated by
reference. One could also combine the polynucleotides of the
present invention with polynucleotides providing agronomic traits
such as male sterility (e.g., see U.S. Pat. No. 5,583,210), stalk
strength, flowering time, or transformation technology traits such
as cell cycle regulation or gene targeting (e.g., WO 99/61619, WO
00/17364, and WO 99/25821); the disclosures of which are herein
incorporated by reference.
[0120] These stacked combinations can be created by any method
including, but not limited to, cross-breeding plants by any
conventional or TopCross methodology, or genetic transformation. If
the sequences are stacked by genetically transforming the plants,
the polynucleotide sequences of interest can be combined at any
time and in any order. For example, a transgenic plant comprising
one or more desired traits can be used as the target to introduce
further traits by subsequent transformation. The traits can be
introduced simultaneously in a co-transformation protocol with the
polynucleotides of interest provided by any combination of
transformation cassettes. For example, if two sequences will be
introduced, the two sequences can be contained in separate
transformation cassettes (trans) or contained on the same
transformation cassette (cis). Expression of the sequences can be
driven by the same promoter or by different promoters. In certain
cases, it may be desirable to introduce a transformation cassette
that will suppress the expression of the polynucleotide of
interest. This may be combined with any combination of other
suppression cassettes or overexpression cassettes to generate the
desired combination of traits in the plant. It is further
recognized that polynucleotide sequences can be stacked at a
desired genomic location using a site-specific recombination
system. See, for example, WO99/25821, WO99/25854, WO99/25840,
WO99/25855, and WO99/25853, all of which are herein incorporated by
reference.
[0121] Methods and compositions are further provided which allow
for an increase in RNAi produced from the silencing element. In
such embodiments, the methods and compositions employ a first
polynucleotide comprising a silencing element for a target pest
sequence operably linked to a promoter active in the plant cell;
and, a second polynucleotide comprising a suppressor enhancer
element comprising the target pest sequence or an active variant or
fragment thereof operably linked to a promoter active in the plant
cell. The combined expression of the silencing element with
suppressor enhancer element leads to an increased amplification of
the inhibitory RNA produced from the silencing element over that
achievable with only the expression of the silencing element alone.
In addition to the increased amplification of the specific RNAi
species itself, the methods and compositions further allow for the
production of a diverse population of RNAi species that can enhance
the effectiveness of disrupting target gene expression. As such,
when the suppressor enhancer element is expressed in a plant cell
in combination with the silencing element, the methods and
composition can allow for the systemic production of RNAi
throughout the plant; the production of greater amounts of RNAi
than would be observed with just the silencing element construct
alone; and, the improved loading of RNAi into the phloem of the
plant, thus providing better control of phloem feeding insects by
an RNAi approach. Thus, the various methods and compositions
provide improved methods for the delivery of inhibitory RNA to the
target organism. See, for example, U.S. Provisional Application No.
61/021,676, entitled "Compositions and Methods for the Suppression
of Target Polynucleotides", filed Jan. 17, 2008 and herein
incorporated by reference in its entirety.
[0122] As used herein, a "suppressor enhancer element" comprises a
polynucleotide comprising the target sequence to be suppressed or
an active fragment or variant thereof. It is recognize that the
suppressor enhancer element need not be identical to the target
sequence, but rather, the suppressor enhancer element can comprise
a variant of the target sequence, so long as the suppressor
enhancer element has sufficient sequence identity to the target
sequence to allow for an increased level of the RNAi produced by
the silencing element over that achievable with only the expression
of the silencing element. Similarly, the suppressor enhancer
element can comprise a fragment of the target sequence, wherein the
fragment is of sufficient length to allow for an increased level of
the RNAi produced by the silencing element over that achievable
with only the expression of the silencing element. Thus, in
specific embodiments, the suppressor enhancer element comprises a
fragment or a variant of a polynucleotide encoding a potassium
channel polypeptide, a cuticle polypeptide, an endocuticle
polypeptide, a chitin binding polypeptide, a chitinase polypeptide,
a hormone inducible polypeptide, a translation initiation factor, a
voltage dependant channel, an EIF-related polypeptide, a
polypeptide having a coiled coil helix domain, a polypeptide having
a zinc finger domain, a receptor associated finger polypeptide, a
lethal timorous imaginal disc polypeptide, a ribonucleoprotein, a
cathepsin protease polypeptide, a polyprotein deformed destructor,
and a death associated leucine rich polypeptide. In still other
embodiments, the suppressor enhancer element comprises a
polynucleotide set forth in SEQ ID NO: 1-42 or an active variant or
fragment thereof.
[0123] It is recognized that multiple suppressor enhancer elements
from the same target sequence or from different target sequences,
or from different regions of the same target sequence can be
employed. For example, the suppressor enhancer elements employed
can comprise fragments of the target sequence derived from
different region of the target sequence (i.e., from the 3'UTR,
coding sequence, intron, and/or 5'UTR). Further, the suppressor
enhancer element can be contained in an expression cassette, as
described elsewhere herein, and in specific embodiments, the
suppressor enhancer element is on the same or on a different DNA
vector or construct as the silencing element. The suppressor
enhancer element can be operably linked to a promoter as disclosed
herein. It is recognized that the suppressor enhancer element can
be expressed constitutively or alternatively, it may be produced in
a stage-specific manner employing the various inducible or
tissue-preferred or developmentally regulated promoters that are
discussed elsewhere herein.
[0124] In specific embodiments, employing both a silencing element
and the suppressor enhancer element allows for the systemic
production of RNAi occurs throughout the entire plant. In further
embodiments, the plant or plant parts of the invention have
improved loading of RNAi into the phloem of the plant over what
would be observed with the expression of the silencing element
construct alone and, thus provide better control of phloem feeding
insects by an RNAi approach. In specific embodiments, the plants,
plant parts, and plant cells of the invention can further be
characterized as allowing for the production of a diversity of RNAi
species that can enhance the effectiveness of disrupting target
gene expression.
[0125] In specific embodiments, the combined expression of the
silencing element and the suppressor enhancer element increases the
concentration of the inhibitory RNA in the plant cell, plant, plant
part, plant tissue or phloem over the level that is achieved when
the silencing element is expressed alone.
[0126] As used herein, an "increased level of inhibitory RNA"
comprises any statistically significant increase in the level of
RNAi produced in a plant having the combined expression when
compared to an appropriate control plant. For example, an increase
in the level of RNAi in the plant, plant part or the plant cell can
comprise at least about a 1%, about a 1%-5%, about a 5% -10%, about
a 10%-20%, about a 20%-30%, about a 30%-40%, about a 40%-50%, about
a 50%-60%, about 60-70%, about 70%-80%, about a 80%-90%, about a
90%-100% or greater increase in the level of RNAi in the plant,
plant part, plant cell, or phloem when compared to an appropriate
control. In other embodiments, the increase in the level of RNAi in
the plant, plant part, plant cell, or phloem can comprise at least
about a 1 fold, about a 1 fold-5 fold, about a 5 fold-10 fold,
about a 10 fold-20 fold, about a 20 fold-30 fold, about a 30
fold-40 fold, about a 40 fold-50 fold, about a 50 fold-60 fold,
about 60 fold-70 fold, about 70 fold-80 fold, about a 80 fold-90
fold, about a 90 fold-100 fold or greater increase in the level of
RNAi in the plant, plant part, plant cell or phloem when compared
to an appropriate control. Methods to assay for an increase in the
level of RNAi are discussed elsewhere herein.
[0127] The following examples are offered by way of illustration
and not by way of limitation.
EXPERIMENTAL
Example 1
Specific Target Genes and Silencing Elements that Cause
Insecticidal Activity Against Lygus Hesperus
[0128] Disruption of insect gene function via RNAi can produce
specific activity against target insects. This specificity is
enhanced by delivery of the dsRNAs via transgenic plants.
Identification of gene function in insects via RNAi has been
largely limited to injection of dsRNAs. In fact, past experiments
have indicated that insects are not capable of systemic RNAi
response based on exposure to dsRNAs.
[0129] As described below, we have demonstrated acute activity of
numerous dsRNA pairs through injection experiments and additionally
have demonstrated insect antagonism through ingestion of dsRNAs.
This evidence identifies several gene/primer pair combinations with
clear insecticidal properties. The use of dsRNAs in transgenic
plants also addresses the potential complication of heterologous
protein expression and the possible risks of allergic reaction,
non-target activity, and environmental- or bioaccumulation. The
data presented below represents the first test of disruption of
these particular genes resulting in insecticidal activity in whole
organisms and the first report of insecticidal activity of dsRNAs
against L. Hesperus.
[0130] The invention describes specific target genes and the dsRNA
sequences causing insecticidal activity against the hemipteran
Lygus hesperus through RNA interference of the target gene's
expression. Disruption of the genes targeted by the dsRNA sequences
may be broadly insecticidal in numerous species. The specific dsRNA
sequences display insecticidal activity upon ingestion indicating
they can be utilized with a transgenic plant mode of delivery.
Table 1 provides the polynucleotide of each target sequence from
Lygus hesperus, a brief description of the function of the protein
encoded by the target sequence, and a SEQ ID NO. Table 2 provides a
summary of the blastx and blastn sequence homology results for each
target sequence from Lygus hesperus. Table 3 provides a summary of
primers used to suppress the target polynucleotides.
TABLE-US-00001 TABLE 1 Target Polynucleotides from Lygus Hesperus.
SEQ ID NO: 1 ilh1c.pk005.o10.f potassium plus
GCGTCACTTGCGACTCGTGTCTGAGCGGCAACTTCAAGGGAAA 151:
GAGGTACAAATGTCTTTCCTGCTACGACTACGATCTGTGCACCAACTGCTACGAGTGTGGCCTCATCAC-
GGGACT 226:
CCACTCAGCAGAGCATCCCATGCAGTGCATCATCACCAGACATGACGTCGACCTGTACTTCGGGGGAGA-
CATGAA 301:
TGGCGACGGAAGTCAGTCCTACACGTGTCCTCATTGTGGTCTAATGGGGTTCAGTTTGTCGCTGTTGAT-
CGAGCA 376:
CGTGAGCGGTGAGCATATCGCGCTGAGCAACGCTGAAGTGATTTGCCCTGTTTGCGCCGCCACGCCAGT-
CAACCG 451:
ACCGAACAACGTTCGCCAGGATTTTTTGGGGCACCTGACGCTGGAGCATCGCTACCCCTCGCGAGAGCT-
GACCGC 526:
CTTCTTCGAAGAGCCCTCGTCCCGACACATGCCGAGTGGCGTCCGCCGGATTCCACCGCCACCAGGGCG-
CAGCGC 601:
TGCCGGGCGTGGACGCCGGGTCGCACGTTCATTTCGGCTCCTCAGGCGCTCTTACTGGACTCACATCCT-
CCAGAG 676:
AAAGTCCGGATCCCATCGCCGAGTTCTTCTCTCAGCTGTCTGgagtcgctcgtcctcaaggtcctgggc-
cgattc SEQ ID NO: 2 ilh1c.pk004.e6.f cuticle protein plus
ACCCCCTGATCGCCGGGATCGTTGCCAATCAGTATCACGCTCAGGATGTG 226:
CTCGGACAGTACACTTACGGCTACTCCGGAnCCCCATCCGCTAAACAAGAGGTTAAGACTGCTGACGGA-
ATAACG 301:
AGAGGATCTTACTCGTACATCGATGGAAACGGGTCTCGTACAGAGCGCTTCTTACGTTGCTGACCCCGT-
CAACGG 376:
GTTCAGAGTCCACGCCACCAACCTGCCCGTGGGACCTGACGGGTCCGTCGCTGCCGCTCCCGTCGCCAG-
GCTCCT 451:
CAGCCCTTTGGCCATCAGCCCTGTGATCAACCTCCACGGCGCTGCTCCCCTCAACCCCGACGGCACCGT-
CGCTGA 526:
CACCCATGAAGTCGCTGTCGCCAAGGCCGCTCACCTCGCTGCCATCAACGAGGCTAGGGCTAGAGGCAA-
GAGGTC 601:
nGCCCCGCTCAACCCCGACGGTACCGTCGCTGACACCCCCGAAGTTGCTGCTGCTAAGTGGGCTCATCT-
CGCTGA 676:
GATCTCCAAAGCTCGTGGAATCCCTCTCGTATACGCTCCCAGATGGTGGGGACCTGGTGCTCCATTGAA-
CGCTGA SEQ ID NO: 3 ilh1c.pk001.e23.f cuticle plus
CTACGCTGGCGGCCCCTCCGCCAAGGAAGAGATCAAGACCGCCGACGG 151:
AATTACCCGCGGAGGATACTCATACATCGACGCCAACGGTATCGTCCAGAGCGCCTCTTACGTGTCGGA-
TCCCGT 226:
CAACGGATTCCGAGTAGCCGCCACTAACCTCCCCGCTGGACCTGCAGTCCCAGCTGGACCTTCAGTGGT-
TGCTGC 301:
TGCTCCAGCTGTCGTTGCTGCTCCTGCTCCAGTTTTGGCTGCTGCCCCTGCTCCAATTGTGGCTTCAGC-
TCCCGT 376:
TTGGGCTGCTCAACCAGCTGTTGTTGCCGCTCCAGCTCCTGTCGCTGTCGCCGAAGGCCCCGCAGTGAC-
CGCCAC 451:
CAACGTCCAGGAAGTTGCTGCCGCTGCTGCTGACGTCCCCGTTGCTGCTGATCTCCCCGAGATCATCGC-
TGCCCG 526:
CTCTCTGCCCACCGTGGTTGCCACCAGGGCCGCCATCGCTCACCCCCTTGCCGCAACCTCCTGGTCCGG-
CATCGT 601:
CCACCACCTGAAGAAGCGTTCCCTTGCCGCCGCTACCGTCGTCACTCCCCTTACTAGTTACCCCGGATC-
TACCGC 676:
TCCCTTGGTTCACGCTTCTCCCGTTATTGCGGCTACACCTGTTATCTCCGCTCACTCGGGTTTGATCGC-
CACTGA 751:
CTCTCTTGTAncctcaccacacctcgttggtgcagtangtnctgtcnagcccctccacaccgccntcct-
canan SEQ ID NO: 4 ilh1c.pk010.f11.f endo cuticle plus
CATTCGGGAGACATTGCAAAACTGGATGCATAGGGGAAGTCTTTA 151:
GGACGGATTTACGGCATACAGTACATGTTACTGTAAGACACGGCGCGTGACTGGACAGCAAGCAGAATG-
GAGGAG 226:
GGACAAGTGTACCAATCATCCGACCAAACCAACTACGTTTACGATGAAGTGTTTCCTGCGCTACCTGAA-
TCAGCC 301:
AACCCGGCACCTCACAACGACATCAAAATTTGCAACAACAAAATGCGTGTTGGCTCATCTGTCATCACT-
CAGGTT 376:
TTCCGAGTGCCAGCAGACGAGCGCCGCTACGATCACAACAACAGCTTTGGGGAAAAGGAATCCGTGAGG-
ACCTGC 451:
TCTGCCATCATGAAGGAAACTGGAGCAGTCATTGAGATCGCCACGAGCAAGGATATGTCTCTGACTTTC-
TTGGTG 526:
ACTGGAAAGACCGACTCAGTGATGGATGCTCGAAGGAAGATACTGAGCAATTTTCAGACCCAAGCTTCA-
TCCAAG 601:
CTCTCTATTCCGAAAGAGCATCACAGGTGGATCCTTGGAAAAGCTGGTGGTCGTCTGAAGGATTTGGAA-
AAATCA 676:
ACAGCCACCAAAATCTCCGTCCCTGGCATAAATGAACACTCGGATGAAATCACTGTGACGGGAACTCGT-
GAAGGG 751: ATCGACAAGGCCATCCATGAAATGCAAGTnatttcggacgaacaatccaagaagn
SEQ ID NO: 5 ilh1c.pk011.m15.f cuticle plus
AACCCCCTGATCGCCGGGATCGTTGCCAATCAGTATCACGCTCAGGATGTG 226:
CTCGGACAGTACACTTACGGCTACTCCGGAnCCCCATCCGCTAAACAAGAGGTTAAGACTGCTGACGGA-
ATAACG 301:
AGAGGATCTTACTCGTACATCGATGGAAACGGGTCTCGTACAGAGCGCTTCTTACGTTGCTGACCCCGT-
CAACGG 376:
GTTCAGAGTCCACGCCACCAACCTGCCCGTGGGACCTGACGGGTCCGTCGCTGCCGCTCCCGTCGCCAG-
GCTCCT 451:
CAGCCCTTTGGCCATCAGCCCTGTGATCAACCTCCACGGCGCTGCTCCCCTCAACCCCGACGGCACCGT-
CGCTGA 526:
CACCCATGAAGTCGCTGTCGCCAAGGCCGCTCACCTCGCTGCCATCAACGAGGCTAGGGCTAGAGGCAA-
GAGGTC 601:
nGCCCCGCTCAACCCCGACGGTACCGTCGCTGACACCCCCGAAGTTGCTGCTGCTAAGTGGGCTCATCT-
CGCTGA 676:
GATCTCCAAAGCTCGTGGAATCCCTCTCGTATACGCTCCCAGATGGTGGGGACCTGGTGCTCCATTGAA-
CGCTGA SEQ ID NO: 6 ilh1c.pk005.d21.f chitin binding plus
CCAACATGTATCTCTCAGTTGTTGGATTGGTGATGGCTTCCGCTG 151:
CTTTCGTCAGCTGCGAGCCATCGAATTTCGCGTGTACTGGCGAGTCGAACTACAAGTATCCTGTGGAGG-
GCTCGT 226:
GCCACAACTACTACCAGTGCGAAAAGGGCTCCACTACGCCTTCAATTCGAGACTGCTCGCTGCCGCTGC-
TTCGAT 301:
TTCGGGATTTCGATCCAGTCAAATTGGAGTGTGACTGGTGCTGGCGGGTAGACTGTTCAGCCAAACCCG-
CACCCC 376:
CACCGACTCCATCGCCGACTCCGGCGCCAACTTCAAGGCCTACTGCTGCGCCGACTACTGGACCAACCT-
CAGCGC 451:
CCACTACTGGACCCACAGCGGCGCCAACCTCAGCGCCCACTGCTGCACCAACCTCAGCTCCCACTGCTG-
CTCCAA 526:
CTCCAGCGCCCACTGCGCCGCCAACTCCAGCGCCCACTGCGGCGCCAACTCCAGCGCCCACTGCAGCAC-
CAACCT 601:
CAGCTCCCACTGCTGCTCCAACTCCAGCGCCTACTGCTGCTCCAACTCCAGCGCCTACTGCTGCGCCAA-
CATCAG 676:
CGCCCTCTACTGGACCCACTGTCGCCCCAACTCGCAGGCCAnctcaagaacccacaatggcaaaaaaat-
cttcga SEQ ID NO: 7 ilh1c.pk011.f4.f chitinase plus
CAAATAAGAAACATGAAGATAGTACCGTTCTTAGTTCTTCTACTT 151:
GTTCAAACGGTTCTTTCCGAAACGACGCCGGCTTCAAACAATCGCCGTATTGTTTGCTACCACACAAGT-
TGGAGT 226:
GCGTATCGTGTCCCAGAGGCAAAATTTACAGCGAAGAACATCAACCCGTACCTTTGCACTCATTTAATA-
TATTCG 301:
TTTGCCAATGTATTGGTAAATGAAGCAACCATCGTTCCTGGTGATGCATGGCAGGATATTGATAACCAT-
CAGTTC 376:
AGAGATTTTGTTGAGTTGAAAACCACATTCAACGAAAACCTGAAAACGTTACTCGCAATAGGAGGCTAC-
AGAGAA 451:
GGGTCGTCGAAGTTCACCCCTATCGCAGCCACCCCCACGAAAAGGGCAGCGTTTGCTCGCAACACGCTC-
AAGTTT 526:
TTGAAAACTTACGGTTTTGACGGGCTCAACATCGATTGGCAGTTCCCTAACGATCAGCATAGAAATGGC-
AGTGTT 601:
GAAGACTATAAGAACTTTGTGTATTTGCTGCAAGATATCGACAAAGTCTTCAGAGAGGAAGCTGCAGCT-
TCCGGG 676:
AAACCTAAAATGATGTTGACCATTTCCGTTCCGGGTAATACGCTGCTAATAGAAAGTGGCTATGATCTA-
CCAAAT 751: CTAGCGAAGTATGTAGAGTTCATGAACGTCCTGAGCTACGATTACCACTTTGCn
SEQ ID NO: 8 ilh1c.pk004.l22.f hormone inducible; JH plus
TCCAGTTCTTTAACGAGGTAACCATGTACAAGACTATCTTAC 151:
CTGAGTTGGGAGCCTTGGATTTGGGTTTGTGCCCGAAGATGTTCCATGGAGAGGCACATAATGGTAAAA-
ATCCTG 226:
AACAAGACATCGTGGTTATTGAAGATTTGTGTCCTCAAGGTTACAAAGTGCCGGAAAAGTTGTTTTTGG-
ACGCTG 301:
ATCACTTAGTGATGGCCATGAAAAAAATAGGGCAACTTCACGGATTATCTTATAAAATGAAAGTATCGT-
CTCCAG 376:
AGAGGTTGTTCGAATTGAGGAATATGCTGATCCCGAAGGTGATTGACGATTCGAAAGGTCTCAATGATG-
CTTGTC 451:
TGGCCAGGGGTTTCAAACCCTTGGTGGAGTCGAGCCCCAGTTACAGTGTAGTGAATAAAGTTTACAAGA-
AACTCG 526:
TCGTAGCGGATGCCGTGGATGTTGCCTATAGTTTACAGAAGCCTGAAGAACCATTCGCCGTTATCACCC-
ACGGTG 601:
ATTTCAATGGTAATAACATATTGTATAAGTATGATGCCAGTGGAAATGTAGTGGATATGAAAATGATAG-
ATTTTG 676:
GTTTCGCTTCTTATTTGGATCCTGCTGTTGACATAGCTTTCTTCCTGTACATGAACTCTTCTCCTGAAA-
CTAGGA 751:
AGCTGCACTGGGATTCATTCCTGAATGCATACTGGGAGGGAGTCATCTCTGTTGCTGGTGATCCn
SEQ ID NO: 9 ilh1c.pk003.d10.f translation initiation factor Plus
CGTGTGCTGATCATTCGATATCCCGGAAACGTGTTTACTTTCCTTTATTG 151:
TGCATAAATACACTTCCGTGGCGGTTCGCGATGTCGAATACAAAAGTCGCGTCTTCCGGTCAACCTAAG-
CTCTCC 226:
TCCCAAGATCTTTCGACTCTTGACGTGACGGCGCTTACCCCATTGTCGCCAGAAGTCATCAGTAGACAA-
GCGACC 301:
ATCAATATCGGTACTATTGGTCACGTGGCACATGGGAAagtcnactgtngnngaaagagtgtctggtgt-
tcnaa SEQ ID NO: 10 ilh1c.pk010.d2.f translation initiation factor
plus TTTTTGGTTTTCATTGAAAATTTCGATAATTTTCCAAAGTTT 151:
TATTATGGTTCAAATTCAAAATGTTTTCTACTGATTTTGATTCTCACAGTATAATTTCGACGTGGAAGT-
GTTAAG 226:
GGCTCAGTGATCGATGGCAGGGAAAGCTTTTTGATAACTATTGGTAAGTCCGAGTTTGTAAGATGACCT-
CTTTTC 301:
AAAAGTGTGTAGGATTTGGTCGGATATTTCCTTTAGGGATTCTCAGAAATCACAATGTTGCATCTCGAA-
GGTGCA 376:
TTCACGGCTCTTCCGTTTTGTACAAAAAGAGGAAAACCAGAGAGGAGAGGAAGTTGCCCAAAGCTATTG-
TTTACT 451:
CTCCCAAATCGAAATGGAAACAAGGCGAGCCCGTTGACGTGTGGAAGCGTATGACAGTGGCGGAGGTAG-
CAAATA 526:
CACTGGGTAAAGATGTAGGACACGTTTTAGAAGTTATGTCGTTCATTGACAACACGGAACAGTACAGAA-
AAGACC 601:
GTGATGTCATCGACAACTTCAAAGTTATAGAAGAAATAGTGAAAAAGTCGGGTCATCGATGTAGGATGG-
CTAGTA 676:
AGCCTACAGAAACTGAAGAAAAAAGTTTTAAAGATGTTGGAcgaagannccttcggattacgttgatcc-
caggcc SEQ ID NO: 11 ilh1c.pk0l1.h12.f voltage dependent channel
Plus TGCCCGAGTGCGTGTTTCGTCAAATAGAAACCCGGGTCTTTTCTGT 151:
AAGAATTAAATCGCAATGGCTCCTCCTTTCTACGCTGATCTAGGTAAGAACGCCCGCGATGTCTTCGGT-
AAAGGG 226:
TACCATTTCGGACTCCTGAAGCTCGACGTCAAGACCAAGACTAACACGGGCGTCGAATTCAGCATCGGC-
GGCGTT 301:
CAAAACCTCGAAACCAAAAACGTAGTCGGCTCTCTCGAGACCAAGTACAAATTCAAGGAGTATGGCGTT-
ACTTTC 376:
ACGGAGAAATGGAACACTGACAACGTACTGGCCACTGAAATCGCCGTTGCTGATTTCTGCGATGGAGCA-
AAAATG 451:
TCCCTTGACACCTCTTTTATCCCTCACAAGGGTGATAAGACCCTGCGATTGAAGGGCGAATTCAAGAAT-
GACACC 526:
TGCGCCATGAACCTTGAAAGCGACTTCAAGTCTGGCGGACCTCTCGTCCGAGGTGGCGCTGTCCTCGGC-
TACGGA 601:
GGCTGGCTATGTGGTTACGCCACGGCCTTCGACGTTAGCAAGAGTAAACTCACCGAAAACAAAGTCACC-
ATGGGA 676:
TTCATCACAAAAGATTTCATCTTGAACACCGTTATCAATGACGGAAGAGTCTTCTCTGGTTCCATCTAC-
CACAAA 751:
GTTAACAGCAAGTTGGAAACTGGAGTCCAGATCTCGTGGGCCTCTGATAACAACAGCACCGACTTCGGC-
ATCGnc SEQ ID NO: 12 ilh1c.pk002.a24.f voltage dependent channel
plus aTTCTGCTTGGGTTTTTTATTTAGTTGAAC 151:
AGTTTTCCGTGGACTCTTATGACGATAACTTCCTCATTCCAAATCTTCTTCGGGACTATCCATCATTTT-
AATCAG 226:
AAGTAGAAGCCGACTATTCTAAAAACCACCTATGGGGCCTCCATTCTTCGCGGATCTCGGTAAAAACTC-
GAGAGA 301:
CATCTTCAATAAAGGTTATAATTTCGGGCTGCTTAAGTTGGACATAAAAACCAGAACAGAAACCGGAGT-
TGAGTT 376:
CGAAATCGGTGGAGTCCAGAACCTTGAGACGAAAAATGTAGCCGGCTCGCTCGAGACTAAGTACAAATT-
CAAGGA 451:
CTTCGGGATCAGCTTTTCGGAGAAATGGAATACGGATAATGTTCTTCAGCTAGAAGTAGCTGCTGCTGA-
TATCTG 526:
CGAAGGAGTCAAAATGTCCTGCATGAGTATCATGACTCCTTCTTCAGATGAGGAGAAAGGTGGCACTGA-
CAAAAT 601:
TTTGAGATTCAAGAGTGAATATAAGAATGCTATCATGGCTGTGAACTTGGAGAGCGATTTCAAAGCTGG-
TGGTCC 676:
GACCTTGGGAGTCTCTGGCGTTTTTGGATTAGGTGGATGGTTGCTCGGAGCTATAGCGGCATTAGATAC-
TGAGAC 751:
TTCGAAAGTGATGACCTTCTCTTTGGGAATGGGAnTTTTAACCAAAGACTTCATACTAAACACCGCTGT-
TATCAA 826: CAAGGGAACAGACTTcann SEQ ID NO: 13 ilh1c.pk009.f20.f
EIF-related factor plus
CGAAAGCAAACAGGACATCATATTTCGCAGGAATAAATTGGAAGA 151:
TCCCATCACACGAGAAAGCAAACCGGACGTCATATTTCGCAGGGTGCGAGGTATCCTCAACAAGCTCAC-
TCCTGA 226:
GAAATTCGATAAGCTAAGCGATGACCTCTTGAAAGAAGAATTTAATTCTGATGTCATTCTCAAAGGCGT-
CATTCT 301:
ATTGGTGTTTGAAAAAGCACTAGATGAGCCGAAGTACAGTGCTATGTATGCTCAGCTCTGCCGGCGACT-
TTGTGA 376:
AGAGATCCGAAGTGCCGACCAGCCTGAACCCTGCCCTTTTCGCCATTTGCTTCTGTCCAGCTGCAAAGC-
TCAGTT 451:
TGAGAGCCGTTCGAAGCACACTAGCAGCAAGCGGAAATCGCTCGGGAACATAAAGTTCATCGGAGAGCT-
TTGCAA 526:
ACTTGGAATCCTTCAGCGCGACATCTTGTACAGGTGTTTGATCCAACTTCTCGAACACAAGACCAAGAC-
GCCTGA 601:
CGAAATGGCCGAAGATCTTGAGTGCGTCTGTCAGATCCTCCGCACTTGCGGCCACATCTTGGACAACGA-
GGAAGC 676:
TCAGAAGCTGATGAATCAACTTTTTGATCGTATGGCGTCCCTCTCCAAGAACGTCAACCTGCCGATCCG-
GATCCG 751: CTTCATGCTCCGTGACATTATCGAGCTCCGGAGGGATAACTGGGTTc SEQ ID
NO: 14 ilh1c.pk002.d9.f coiled coil helix domain ...
CTAACTTTCCTnTTCCGCGTTGTTGCGTTCCTGTGAAATTTCACTAA 151:
AATTGTGATTATTTTATTGTACTCAGAACTATACCTACTTCGTATTTCGATTTGAATACATTCCAAGGG-
CTTTCG 226:
CATGACTCAAACTTTCTTCTAGAAGTGGTTTGTTGCGACGTGTTGAGTTCAATAGTGTGGTATTCACAA-
CCGGTT 301:
TCGCCCATTGGGCCATCAACGAGTATTTTCCAGGTGATGATCTATTGATATGGGGGGCGTAGCCGCTTC-
AAGTCC 376:
TTTGCCAGACGAAGAACCCCGACCTGAAGCTCCAGGCAGCAGGAAGGCAGACGAAGTACCTGCTGAGAG-
TGGTCA 451:
GAACCCCGCAGGACAACCTCACCCAGATGGCGCCAAGGAAGAAGAGAACGGAGATAACGAGGnAAAGGC-
CTGGCC 526:
TTATAAAAGCCGATGGATCTATAAATTGGGATTGCCCATGTTTGGGAGGGATGGCACATGGGCCCTGTG-
GCGATG 601:
AGTTCAGAGCTGCTTTCTCCTGTTTCCACTATTCCACTGCTGAACAAAAAGGCTCCGACTGTTTggnaa-
ccgttc SEQ ID NO: 15 ilh1c.pk003.j7.f zinc finger domain
CACAAAAAGCTCCATACAGGTGAACGCCC 151:
TTTCAAATGCGCACACTGCGTTCGGACTTTCAGCCGGAAAGAGCACTTAGTACGGCATGCCCACTCTCA-
TACAGG 226:
ACAAAAACTCTTCAACTGCGACGTCTGCGGGAAAAGCTTCAGTCGGAAAGACAACGTACGGAAACACCG-
GAAAAC 301:
GCATGAAACGACAGGTCCGTACTCTTGCGAGTTCTGCGGTATGCAGTTCAACGTTCGGCCGTACTATAT-
AATGCA 376:
CAAAAACAAGCACAAAGACGGGTCGTGCGTCCTTGAAGTGAAGAAGGTTGATGTTGAGGAGTCTATCAC-
GTACGA 451:
AGnTCAGGAAGAGTCTCCAGATGTTCATTCGAACGAATCCAATTCCTTCCAACAGGTAACATCTAGCAC-
ATCCAC 526:
TTCAATACTGGAAAAAGCGTTGACGCAAGAAGGCTGAACTTTGGACTTCTTGAATTAACTTTAGGCCAA-
ACTATT 601:
ACAGAGTTGACAAGTATGGAGTGTGCTCAGAGGATTAGTTGGTGGAAGTAACTAGTCCAGAAGCTATTC-
AGAATT 676:
AAGAACTAGAATTGAATGCAACAGCAATCAGTTTGCCCTTTCAGTTTGTGGTTTGTTTTTCTGTTGGAA-
ACTATC 751:
TCTCGGGCATGAATAAGGAGAATGTGTACCAAGTATTTCAGTCTTCCTCTGCTCTGTGATGTAACTCTG-
TGCTTC 826: TTTCCTATACTCGCGTTGGTAATCAA SEQ ID NO: 16
ilh1c.pk003.o10.f receptor associated finger
AAGAATTGTAAATCAATATCAAAATGGAGATGATGAAGTCAGA 151:
TGTCGACTGAAAAGAATGTTTGGATTCCAAGAGATCCAAAAGCTCATTAAATATAACGTAAATACTGTG-
CTGTCC 226:
ATGGACTGGCTTGACCGTAATGCCAAAAACTGTCCGAAATGCAACGnnCCCATTCAGAAAATCAGTGGA-
TGTAAC 301:
CATATGGTATGCTGGAAATGCAAAACATCTTTTTGCTGGCACTGCCTATGCTTTACGTGCATAGGATAG-
TAAGGA 376:
CGTGAGATGATCCTGTAGTTACAGCTCTCTTGCCACTGACTACCTAGAAATATCGCACTAGTCATAGTC-
AGCCAC 451:
CCTCCTCTACCTCGCCATTATCTCATTTGGGCTGTGACAAACACAAACCTCGTGTATATCTCGTATACA-
TTACTA 526:
TGTACCTTGTTTGCGCTGTGACATCTCAGGAACCCCTTGATATGAAAATTTAAGTGGTAAAAAAACTTT-
TTTACG 601:
ATTCCGaaaaaaaaaaaaaaaaaaaacccnactttatgtacaaagttggcattataagaaagcattgat-
atca lethal tumorous imaginal discs SEQ ID NO: 17 ilh1c.pk004.b8.f
GCCTCTCTTCAAGTTTTTGCGGTGTCGGAATGTCAAATTAAATA 151:
ATTGTTCCGATCAAATTAATATGTCTAGCATACGATTTCTTAGAAAATTCAGTGGATTTAGAATCAGTG-
GTTTCC 226:
TAGTAGTGGGGCAGTGTGGTGCGCAGAAAATTTGCAGCTTAGGTCATTTGAAATCTCAAGAGAACTCTA-
GTTTAC 301:
TAAGATTTACCGGTGTTAGTACGAGAAATTTCCATTTGGGAGTGCCATCTCTCGCCAAGAAAGACTACT-
ACGAGA 376:
TCTTGGGCATCTCTAGAAACGCGTCGGTCAAGGAAGTGAAAAAAGCGTACTATCAGCTGGCCAAGAAAT-
ACCATC 451:
CAGACACGAATAAAAnnnnTCCGAACGCCGCCAAGAAGTTTCAAGAAGTATCAGAAGCCTATGAGGTAC-
TGAGTG 526:
ACGACACCAAGAGGAAACAATATGATCAATGGGGTACGACGTCGGAGCAGATGGGCCGAGAAGGTGCTG-
GTACAG 601:
GTCCAGGTAACATGGGCGGCTTCAACTGGCAGTACCGGGCTTCCGTGGACCCTCAGGAGCTCTTCAGGA-
AGATCT 676:
TTGGAGACGCTGCAGGCGGATTTTCCACCGGATTCGACGATTTCGCTGAGTCTAGATTCGGTCACGGTG-
CTGCCG 751:
AAGAAATTCTAATGAAACTCACCTTCTCTCAAGCCGTGCGGGGAGTGAGCAAAGAAATATACGTTAATG-
t SEQ ID NO: 18 ilh1c.pk004.d17.f ribonucleoprotein
GATGATCACnTTTACGGCTAATACTCGTAGATCTCCAGTTT 151:
CGATACAAATTTGAATTCAACGAGATAACCGAATGAAATGACTTGGAAATCAGCTTAAAAGCAGTGAAC-
TCAGCG 226:
GTAGAGGGGAAAATGTCTCACTCCGACTCAAGAACAGGGAAAACCTCCAGAAGTACGAATGAGTCGAAA-
TCAGGA 301:
GCCTCGGGGCGACAGAAAACTTCAAGAACTGAGCCGAAAACCCCGAAAACTGAGTCAAAAACCTCGAAG-
TCAGCG 376:
TCGAAAACCTCGAAGTCAGAAGGGAAGTCTGTGTTGTCTGAAAGTAGAGATAAAAGTAATAAATTTTCA-
AAAAGC 451:
GAATCTGAGTCGTATCGCAAGTCCGATGCGAGGGGACAGCGGGACGAGGCACCAGGACCGTCAGACAGC-
AGAACA 526:
GGAAAAGACGTCACTGGGGATAGGAAAACTAAGAAACAGAAAAGTGAGAAAGGGGTCGACGGATCTACT-
GGAGAA 601:
TCGAAGAAACTCCCAGTGTCGTCCTCAAGAACGTCGGAAGCGCCGCGGAACATCCGAGATCTCTTGAGG-
AGGATC 676:
AACGAGGAGAACGAATCTCAGCCAACACCTTCTTCTCGTCTGAAAGAnCCCAAGCCGGAGAGGACGAAG-
AGTAAA 751:
GTTCCATCCAAAGCACCGCAGGCGAGTATTCCAGATAGGGACGTGGTACGAGCAAAAGCTGCGGAAncg-
gccttg SEQ ID NO: 19 ilh1c.pk004.k13.f cathepsin; protease
GTTCACCAAGGAAGTATCGCACCACATTCTCTGCCAAAC 151:
TACAGCAGGATCCCAAACCACCGGAATGAACACCATTTTGGCTCTCGCAAGTCTGTTGGGCTGCTGTCT-
GGCGGC 226:
GTCCGTTCCGGATTCGAAGTGGGATTCTTTCAAGGCCAAATACGGAAAAACGTACGACGACCCAAAAGT-
CGATAG 301:
TGAGAGACGTAACAACTACGGAAAAACGCTAGAGATGATCAAGGCTCACAACGCACTCTATGGACAGGG-
CCGGGT 376:
GTCCTACTACCTGGCAGAGAACCATCTTGCAGACTTGTCGTCCAGTGAACGAATGAAGTTAAGAGGATT-
CAGAAA 451:
ATCCGAAAGTCAATCGGGCGGCAGAATCCACCAGCACACTGGATTGGGCCGACCCGATTCCGTCGATTG-
GCGAAA 526:
CAAAAGCGTTGTGACCAGCGTCAAAAATCAAGGACAATGTGGTAGCTGCTGGGCTTTCAGTGCGACTGC-
AGCAGT 601:
GGAATCGCAATACGCTATCAAAACCGGGCAATTAGTGGATCTCAGCGAGCAGCAGGTAGTGGACTGTGA-
CCGTAA 676:
TGGTCACGCTTGCAAGTATGGTGACAACCTTGACGCGTTAGGGTATATCGAGGAAGAAGGTCAGGAGCT-
TCTTTC 751:
CTCTTATCCCTACATTGCTGAGCCAGAGACTTGTCAATACGCAGCAGATAAAGTGAAGGTGAAGATTGC-
GAGTTT 826: CCnn SEQ ID NO: 20 ilh1c.pk011.a8.f polyprotein
deformed destructor GTCTTAAGTGATGAAGATGTTGTGTTAGGTTTACCGGG 151:
CGTGCCAGGATACCATGCTATGGAAATGGCAACGTCTGAAGGTTTTCCTTTCACAGCAAGTCGACCACA-
AGGAAG 226:
TTCCAATAAGCGGTGGTTGTTTAACATCAATGAGAATGCCGAGAAGAGATCCTTAATCGCCATGGACCC-
CTTATT 301:
GGTGAAAGTGTTAGAATCGAAGAGGGTTCAGAGAGATCGAGGGTTGATTCCGTGTACCGTTTTTGTAGA-
CTGCTT 376:
GAAGGATTCACGAATAGCGAATGAATCTTACCTCACACCCGGTAAGACTAGGATCTTCTCTATCTCACC-
GGTTGA 451:
CTTTACGATTGAGTTTCGGAAGTATTTCCTTGATATCCTAGCGGCGCAACAACAAAGTCGATTCCACCT-
AGAGCA 526:
TATGGTAGGTATGAATGTTCATTCGCTTGAGTGGACTTTACTAGCCCGCCGTATCCAATCTGTGGGTTC-
TGCAGT 601:
GATCTGTGGTGATTACTCGAACTTTGGTCCTGGTTTGGATAGCGAAGTTGTTGCAGCTGTTGGGGACGT-
TTGGGC 676:
TGATTGGTATGAGTTTTACGAGACCGCTCAGGGCGTCTCGGAAGAGGAGAGAAAGCGACGCCgccnaag-
taagaa SEQ ID NO: 21 ilh1c.pk011.d10.f death associated leucine
rich ... CATGGCGTACGGTGTAACAAGAGTCGTGTTCCGCTGCGAGGAAGCT 151:
CAGGAATCCGGAnAATTGGATCTGTCGGAATGTCAACTCATGCAGGTGCCGGACGCGGTCTACCACTTG-
ATGAGG 226:
CACACGGAACTGAAGGCGTGTAATCTCTCAAGCAACGTCATCACCAAAATTCCCCCGAAATTCGCGGTC-
AACTTT 301:
TCTCTCATTACAGAGCTGAACCTGGCGCACAACCAGATGAGCAAACTCCCGGACGAGCTCGCCG
TABLE-US-00002 TABLE 2 Summary of homology for the target
nucleotides. Lygus target sequences: Homology ilh1c.pk005.o10.f
blastx: melanogaster; drosophila; modulatory; potassium; channel;
isoform; sapiens; homo; rerio; danio; anopheles; musculus; gambiae;
pe . . . blastn: melanogaster; drosophila; isoform; full; insert;
85c-85c; bac; gambiae; str; pest; section; anopheles; length
ilh1c.pk004.e6.f blastx: drosophila; melanogaster; anopheles;
gambiae; pest; cuticle; str; pseudoobscura; a3a; tm-lcp; tm-a3a;
larval; cuticular; tenebri . . . blastn: melanogaster; drosophila;
length; section; full; bac; 92e-92f ilh1c.pk001.e23.f blastx:
drosophila; melanogaster; pseudoobscura; anopheles; gambiae;
buzzatii; pest; str; mellifera; predicted; cuticle; apis; a3a; dbuz
. . . blastn: musculus; mus; bac; library; mouse; r.norvegicus;
roswell; institute; hippocampus; product:hypothetical; female;
containing; can . . . ilh1c.pk010.f11.f blastx: glycoprotein;
endocuticle; structural; drosophila; anopheles; gambiae;
melanogaster; pseudoobscura; pest; str; mori; bombyx; cut . . .
blastn: melanogaster; drosophila; cuticle; larval; lcp-14; m.sexta;
constituent; asap; sexta; gambiae; bombyx; lcp17; subalbatus; mori;
. . . ilh1c.pk011.m15.f blastx: anopheles; gambiae; pest; cuticle;
str; melanogaster; drosophila; lm-acp; isoform; tm-lcp; larval;
a1a; a3a; tm-a3a; tm-a1a; mel . . . blastn: anopheles; gambiae;
melanogaster; drosophila; pest; str; ccp84ab; cuticle; ccp84ad;
ccp84ag; full; insert ilh1c.pk005.d21.f blastx: branchiostoma;
region-containing; floridae; variable; chitin-binding blastn:
cultivar-group; japonica; sativa; oryza; predicted; mucin; insert;
full; collagen; norvegicus; discoideum; alpha; dictyostelium; . . .
ilh1c.pk011.f4.f blastx: danio; rerio; predicted; melanogaster;
anopheles; drosophila; gambiae; chitinase; isoform; acidic; pest;
str; paralichthys; oliv . . . blastn: chitinase; predicted; rerio;
danio; acidic; transcript; taurus; variant; bos; sapiens; b04;
homo; chia; mammalian; chitotriosida . . . ilh1c.pk004.a13.f
blastx: carboxylesterase; gossypii; esterase; aphis;
carboxylic-ester; hydrolase; persicae; myzus; fe4; lygus; juvenile;
polyphemus; ant . . . blastn: carboxylesterase; anisopteromalus;
melanogaster; calandrae; drosophila; esterase; mellifera; apis;
antheraea; est; malathion-sus . . . ilh1c.pk004.i18.f blastx:
esterase; tribolium; castaneum; mellifera; juvenile;
carboxylic-ester; carboxylesterase; hormone; hydrolase; apis;
persicae; myz . . . blastn: carboxylesterase; esterase;
anisopteromalus; m.persicae; calandrae; anopheles; gambiae; pest;
est1; str; mellifera; desago; pred . . . ilh1c.pk004.l22.f blastx:
anopheles; gambiae; drosophila; melanogaster; pest; str;
pseudoobscura; hormone-inducible; juvenile blastn:
ilh1c.pk005.k23.f blastx: esterase; tribolium; castaneum;
melanogaster; anopheles; drosophila; gambiae; carboxylesterase;
pest; athalia; str; juvenile; ro . . . blastn: ilh1c.pk010.g16.f
blastx: melanogaster; drosophila; anopheles; gambiae; pest; str;
jhi-26; juvenile; yakuba; pseudoobscura; hormone-inducible blastn:
ilh1c.pk002.d16.f blastx: vitellogenin; plautia; stali; mellifera;
apis; nipponica; pimpla; clavatus; encarsia; gambiae;
vitellogenin-1; vitellogenin-3; v . . . blastn: vitellogenin;
plautia; aedes; aegypti; stali; vg-b; dictyostelium; discoideum;
vga1; vtg; athalia; periplaneta; formosa; rosae; . . .
ilh1c.pk001.f1.f blastx: vitellogenin; anopheles; gambiae; pest;
str; molitor; encarsia; nigrofuscata; toxorhynchites;
graptopsaltria; quinquefasciatus; . . . blastn: vitellogenin;
aedes; vg-c; aegypti; polynesiensis; vitellogenin-c;
graptopsaltria; nigrofuscata ilh1c.pk005.f15.f blastx:
vitellogenin; plautia; stali; anopheles; vitellogenin-2;
periplaneta; gambiae; mellifera; americana; apis; pest; str;
pimpla; cl . . . blastn: vitellogenin; quinquefasciatus;
dictyostelium; phragmatopoma; californica; discoideum; variant;
pipiens; cement; oreochromis; cu . . . ilh1c.pk003.d10.f blastx:
translation; initiation; eukaryotic; gamma; structural; predicted;
x-linked; musculus; gallus; unnamed; leptinotarsa; decemlinea . . .
blastn: translation; initiation; eukaryotic; structural; musculus;
x-linked; product:eukaryotic; full-length; enriched; mus; library;
in . . . ilh1c.pk010.d2.f blastx: melanogaster; drosophila;
anopheles; gambiae; initiation; pest; str; translation; mellifera;
gallus; apis; predicted; translatio . . . blastn: ilh1c.pk011.g22.f
blastx: elongation; translation; eukaryotic; beta; anopheles;
gambiae; sapiens; synthetic; rerio; danio; pest; homo; str;
construct; gal . . . blastn: anopheles; gambiae; elongation;
translation; eukaryotic; full-length; extremity; mosquito; beta;
malaria; females; african; sing . . . ilh1c.pk011.h12.f blastx:
voltage-dependent; channel; mitochondrial; melanogaster;
drosophila; anopheles; porin; anion-selective; anion; gambiae;
gallus; . . . blastn: anopheles; gambiae; full-length; extremity;
mosquito; malaria; females; african; 5-prime; single; adult; total;
read; made; mito . . . ilh1c.pk001.k13.f blastx:
mannose-6-phosphate; isomerase; phosphomannose; phosphohexomutase;
arabidopsis; schizosaccharomyces; thaliana; cultivar-group; n . . .
blastn: predicted; isomerase; glabrata; candida; bar1; related;
other; japonicum; pmi40; inp51; vid28; yarrowia; neurospora;
mellifera; . . . ilh1c.pk001.k16.f blastx: predicted; cytoplasmic;
poly(a)-binding; polyadenylate; structure; strongylocentrotus;
crystal; isoform; poly(a; x-ray; purpurat . . . blastn: inducible;
cytoplasmic; sapiens; gallus; poly(a; homo; form; finished; pabpc4;
aedes; polya; asap; aegypti; predicted; human; tr . . .
ilh1c.pk001.l22.f blastx: proteasome; non-atpase; drosophila;
musculus; melanogaster; unnamed; isoform; macropain; 26s; mus;
prosome; predicted; danio; re . . . blastn: anopheles; gambiae;
full-length; extremity; proteasome; non-atpase; mosquito; females;
african; malaria; single; macropain; adul . . . ilh1c.pk001.l4.f
blastx: blastn: sativa; oryza; glycine-rich; scapularis;
rna-binding; japonica; ixodes; cultivar-group; nipponbare; trospa;
cultivar; monsanto; . . . ilh1c.pk001.m15.f blastx: glutathione;
s-transferase; anopheles; drosophila; melanogaster; gambiae;
class-sigma; isoform; pest; allergen; str; gst; bla; s . . .
blastn: melanogaster; drosophila; glutathione; isoform; gsts1;
aegypti; insert; aedes; full; extremity; glutathione-s-transferase;
blatt . . . ilh1c.pk001.m17.f blastx: drosophila; melanogaster;
isoform; aegypti; aedes; pseudoobscura; cg118; apis; mellifera;
rna-binding; predicted blastn: isoform; melanogaster; drosophila;
vig; insert; predicted; full; mrnabp; aedes; asap; conserved;
ciona; aegypti; mellifera; i29m . . . ilh1c.pk001.n23.f blastx:
predicted; ankyrin; domain; repeat; sapiens; musculus; unnamed;
isoform; homo; dictyostelium; mus; discoideum; taurus; bos; schi .
. . blastn: domain; strongylocentrotus; repeat; purpuratus;
ankyrin; predicted;; ilh1c.pk001.n6.f blastx: melanogaster;
drosophila; reticulum-type; sarco(endo)plasmic; isoform; atpase;
predicted; calcium; mellifera; anopheles; gambiae . . . blastn:
sarco(endo)plasmic; reticulum-type; calcium; atpase; melanogaster;
mellifera; predicted; drosophila; xenopus; ca-p60a; isoform; . . .
ilh1c.pk001.o17.f blastx: melanogaster; drosophila; dyskerin;
musculus; isoform; predicted; gallus; ribonucleoprotein; mus;
danio; rerio; anopheles; nucle . . . blastn: melanogaster;
drosophila; nop60b; lipolytica; nucleolar; yarrowia; predicted;
dyskerin; isoform; clib99; purpuratus; danio; ribo . . .
ilh1c.pk001.o4.f blastx: drosophila; dehydrogenase; ubiquinone;
melanogaster; subcomplex; nadh; beta; anopheles; musculus; unnamed;
gambiae; predicted; 1 . . . blastn: full-length; nigroviridis;
dehydrogenase; tetraodon; ubiquinone; subcomplex; nadh; beta; asap;
musculus; product:nadh; danio; re . . . ilh1c.pk002.a11.f blastx:
melanogaster; drosophila; anopheles; sapiens; gambiae; interacting;
homo; pest; str; dna-damage-inducible; gadd45gip1; papilloma . . .
blastn: ilh1c.pk002.a24.f blastx: voltage-dependent; channel;
drosophila; melanogaster; mitochondrial; anion; isoform; porin;
sapiens; familiaris; predicted; homo . . . blastn: nigroviridis;
voltage-dependent; full-length; rerio; danio; tetraodon;
mitochondrial; channel; anion; tropicalis; xenopus; porin . . .
ilh1c.pk002.b12.f blastx: drosophila; melanogaster;
aminopeptidase-like; anopheles; pseudoobscura; gambiae; bombyx;
leucyl; pest; mori; str; simulans blastn: tropicalis; xenopus;
aminopeptidase; leucine; gambiae; str; lap3-prov; finished; pest;
anopheles ilh1c.pk003.n7.f blastx: kynurenine; aminotransferase;
drosophila; melanogaster; aegypti; isoform; aedes; transaminase;
aminotrasferase; anopheles; tropi . . . blastn: melanogaster;
drosophila; isoform; anopheles; gambiae; insert; full; single;
fk0aaa5bf12; total; kynurenine; nuclear; ciona; ext . . .
ilh1c.pk003.p4.f blastx: drosophila; melanogaster;
tripeptidyl-peptidase; tripeptidyl; isoform; pseudoobscura;
aminopeptidase; peptidase; anopheles; pred . . . blastn:
tripeptidyl; peptidase; musculus; product:tripeptidyl; sapiens;
full-length; enriched; mus; library; homo; insert; predicted; ri .
. . ilh1c.pk004.c13.f blastx: aminomethyltransferase; cleavage;
glycine; system; drosophila; melanogaster; t-protein; structure;
mitochondrial; crystal; rerio . . . blastn: aminomethyltransferase;
mitochondrial; predicted; norvegicus; cleavage; glycine; rattus;
system; debaryomyces; gcvt; hansenii; c . . . ilh1c.pk004.i5.f
blastx: amino-acid; predicted; synthesis; control; general; 1-like;
musculus; gcn1; sapiens; homo; unnamed; mus; canis; mellifera;
famil . . . blastn: ilh1c.pk001.d8.f blastx: drosophila;
melanogaster; pseudoobscura; isoform; anopheles; gambiae; pest;
str; simulans blastn: melanogaster; drosophila; aminopeptidase;
sapiens; full-length; homo; full; human; length; leucine; fetal;
25-normalized; cs0df0 . . . ilh1c.pk001.e10.f blastx:
e1a-stimulated; cellular; repressor; sapiens; unnamed; musculus;
homo; mus; predicted; anopheles; gambiae; danio; rerio; gallus; . .
. blastn: product:hypothetical; aminoacyl-transfer; musculus;
full-length; synthetases; enriched; containing; library;
e1a-stimulated; ins . . . ilh1c.pk001.i10.f blastx: aminopeptidase;
musculus; melanogaster; drosophila; unnamed; mus; dictyostelium;
membrane-bound; lycopersicon; discoideum; escul . . . blastn:
aminopeptidase; musculus; membrane-bound; full-length; enriched;
mus; library; insert; product:membrane-bound; riken; full; lyco . .
. ilh1c.pk010.p16.f blastx: aminotransferase; phosphoserine;
musculus; unnamed; sapiens; mus; isoform; homo; synthetic;
construct; mellifera; xenopus; rattu . . . blastn:
product:phosphoserine; aminotransferase; full-length; musculus;
yersinia; enriched; library; insert; pestis; riken; blastocyst; . .
. ilh1c.pk003.k14.f blastx: carboxypeptidase; vitellogenic-like;
sapiens; serine; homo; carboxypeptidase-like; predicted; cpvl;
vitellogenic; canis; mellife . . . blastn: full-length;
25-normalized; sapiens; placenta; carboxypeptidase; human; homo;
cot; aegypti; aedes; troglodytes; vitellogenic; se . . .
ilh1c.pk004.a13.f blastx: carboxylesterase; gossypii; esterase;
aphis; carboxylic-ester; hydrolase; persicae; myzus; fe4; lygus;
juvenile; polyphemus; ant . . . blastn: carboxylesterase;
anisopteromalus; melanogaster; calandrae; drosophila; esterase;
mellifera; apis; antheraea; est; malathion-sus . . .
ilh1c.pk001.b20.f blastx: glycine; decarboxylase; decarboxylating;
dehydrogenase; cleavage; melanogaster; system; drosophila; sapiens;
mitochondrial; homo . . . blastn: glycine; decarboxylase;
melanogaster; drosophila; decarboxylating; mitochondrial;
dehydrogenase; cleavage; musculus; product:gly . . .
ilh1c.pk004.i18.f blastx: esterase; tribolium; castaneum;
mellifera; juvenile; carboxylic-ester; carboxylesterase; hormone;
hydrolase; apis; persicae; myz . . . blastn: carboxylesterase;
esterase; anisopteromalus; m.persicae; calandrae; anopheles;
gambiae; pest; est1; str; mellifera; desago; pred . . .
ilh1c.pk004.o2.f blastx: carboxypeptidase; vitellogenic-like;
sapiens;
vitellogenic; serine; homo; carboxypeptidase-like; cpvl; predicted;
canis; pygmaeu . . . blastn: carboxypeptidase; anopheles; gambiae;
vitellogenic; carboxypeptidase- like; full-length;
vitellogenic-like; extremity; mosquito; . . . ilh1c.pk005.m3.f
blastx: proteolipid; vacuolar; drosophila; synthase; melanogaster;
anopheles; isoform; gambiae; kda; atp; pest; mellifera; str;
c-subuni . . . blastn: anopheles; gambiae; full-length; extremity;
mosquito; malaria; african; 5- prime; females; single; total;
adult; made; read; vacu . . . ilh1c.pk002.o3.f blastx:
ubiquitin-specific; ubiquitin; protease; carboxyl-terminal;
thiolesterase; herpesvirus; associated; n-terminal; hydrolase;
usp7h . . . blastn: ubiquitin-specific; protease; ubiquitin;
predicted; specific; herpesvirus; associated; musculus; hausp;
virus-associated; norveg . . . ilh1c.pk003.h22.f blastx:
pyrophosphatase; musculus; dutp; deoxyuridine; unnamed;
triphosphatase; dutpase; human; nucleotidohydrolase; mus;
norvegicus; st . . . blastn: musculus; full-length; enriched; mus;
product:dutpase; triphosphatase; library; insert; riken; full;
deoxyuridine; days; dutpase . . . ilh1c.pk003.i6.f blastx:
inosine-uridine; nucleoside; hydrolase; preferring; cultivar-group;
japonica; sativa; xenopus; oryza; schizosaccharomyces; laevi . . .
blastn: staphylococcus; aureus; subsp; tropicalis; xenopus; mw2;
n315; mu50; finished; strain:mw2; mrsa252; mssa476; col
ilh1c.pk004.a13.f blastx: carboxylesterase; gossypii; esterase;
aphis; carboxylic-ester; hydrolase; persicae; myzus; fe4; lygus;
juvenile; polyphemus; ant . . . blastn: carboxylesterase;
anisopteromalus; melanogaster; calandrae; drosophila; esterase;
mellifera; apis; antheraea; est; malathion-sus . . .
ilh1c.pk003.g17.f blastx: drosophila; melanogaster;
acetyltransferase; choline; anopheles; gambiae; isoform; sapiens;
danio; rerio; predicted; structure; . . . blastn: ilh1c.pk009.k12.f
blastx: drosophila; initiation; melanogaster; translation;
eukaryotic; predicted; anopheles; gambiae; taurus; pest; danio;
rerio; str; b . . . blastn: melanogaster; drosophila; translation;
eukaryotic; initiation; tropicalis; xenopus; 110 kda; rerio; danio;
gambiae; eif3s8-prov; . . . ilh1c.pk009.f20.f blastx:
eif4g-related; danio; rerio; predicted; nat1b; isoform; xenopus;
translation; initiation; eukaryotic; tropicalis; gallus; gamma; . .
. blastn: melanogaster; drosophila; eif4g-related; predicted;
isoform; xenopus; laevis; nat1; translation; mellifera; 49e-49f;
full; eukar . . . ilh1c.pk008.o12.f blastx: translationally;
translationally-controlled; controlled; drosophila; tumor;
melanogaster; mellifera; predicted; anopheles; tctp; . . . blastn:
drosophila; melanogaster; translationally; controlled; anopheles;
gambiae; tumor; tctp; lonomia; bombyx; xylostella; african; in . .
. ilh1c.pk008.k5.f blastx: elongation; rhodopseudomonas; palustris;
translation; bradyrhizobium; melanogaster; gtp-binding; drosophila;
tu:small; domain; m . . . blastn: melanogaster; drosophila;
elongation; insert; rerio; danio; full; brevipalpis; asap; gambiae;
translation; wigglesworthia; marit . . . ilh1c.pk008.c24.f blastx:
translation; eukaryotic; initiation; interacting; sapiens;
melanogaster; drosophila; homo; predicted; familiaris; isoform;
varia . . . blastn: full-length; 25-normalized; sapiens; human;
placenta; homo; cot; interacting; translation; eukaryotic;
initiation; danio; rerio; . . . ilh1c.pk008.b15.f blastx:
melanogaster; musculus; drosophila; predicted; unnamed; anopheles;
translation; mus; gambiae; sapiens; pest; riken; rerio; danio . . .
blastn: sapiens; melanogaster; drosophila; homo; translation;
danio; rerio; et16; fis; length; taurus; bos; spliced;
alternatively; tran . . . ilh1c.pk007.p11.f blastx: translation;
initiation; eukaryotic; drosophila; melanogaster; anopheles;
gambiae; bombyx; eif-5a; pest; spodoptera; danio; reri . . .
blastn: initiation; translation; eukaryotic; arabidopsis; thaliana;
bombyx; eif5a; mori; rerio; danio; eif-5a; 5a-4; gsltsil10za11;
thal . . . ilh1c.pk003.d10.f blastx: translation; initiation;
eukaryotic; gamma; structural; predicted; x-linked; musculus;
gallus; unnamed; leptinotarsa; decemlinea . . . blastn:
translation; initiation; eukaryotic; structural; musculus;
x-linked; product: eukaryotic; full-length; enriched; mus; library;
in . . . blastn: domain; strongylocentrotus; repeat; purpuratus;
ankyrin; predicted; schistosoma; japonicum blastn:
sarco(endo)plasmic; reticulum-type; calcium; atpase; melanogaster;
mellifera; predicted; drosophila; xenopus; ca-p60a; isoform; . . .
ilh1c.pk002.d16.f blastx: vitellogenin; plautia; stali; mellifera;
apis; nipponica; pimpla; clavatus; encarsia; gambiae;
vitellogenin-1; vitellogenin-3; v . . . blastn: vitellogenin;
plautia; aedes; aegypti; stali; vg-b; dictyostelium; discoideum;
vga1; vtg; athalia; periplaneta; formosa; rosae; . . .
ilh1c.pk002.d9.f blastx: coiled-coil-helix-coiled-coil-helix;
containing; predicted; domain; sapiens; musculus; homo; chchd4;
anopheles; norvegicus; gamb . . . blastn:
coiled-coil-helix-coiled-coil-helix; containing; predicted; domain;
gallus; norvegicus; rattus; rerio; danio; chchd4; musculus; . . .
ilh1c.pk002.j6.f blastx: transmembrane; predicted; sapiens;
tropicalis; homo; xenopus; musculus; gallus; norvegicus; mus;
rattus; danio; canis; rerio; te . . . blastn: ilh1c.pk002.k17.f
blastx: proteasome; drosophila; regulatory; melanogaster; isoform;
musculus; non-atpase; 26s; unnamed; predicted; mus; anopheles;
gambia . . . blastn: proteasome; regulatory; melanogaster;
drosophila; predicted; 26s; anopheles; transcript; non-atpase;
gambiae; isoform; variant; . . . ilh1c.pk002.k5.f blastx:
nucleoporin; sapiens; homo; predicted; unnamed; 54 kda; familiaris;
p54; variant; isoform; kda; canis; musculus; macaca; apis; fa . . .
blastn: nucleoporin; product: nucleoporin; full-length; musculus;
enriched; norvegicus; library; insert; riken; rattus; mus; full;
macrop . . . ilh1c.pk002.m21.f blastx: rapamycin-insensitive;
predicted; companion; sapiens; musculus; mtor; homo; insensitive;
pianissimo; rapamycin; mus; rictor; gal . . . blastn:
ilh1c.pk002.n11.f blastx: melanogaster; drosophila;
6-phosphofructo-2-kinase; isoform; 2,6- bisphosphatase;
fructose-2,6-biphosphatase; predicted; fructose . . . blastn:
melanogaster; drosophila; isoform; pfrx; fructose; long; full;
form; insert; 6-phosphofructo; str; 2-kinase; pest; 2,6-bisphosph .
. . ilh1c.pk003.d10.f blastx: translation; initiation; eukaryotic;
gamma; structural; predicted; x-linked; musculus; gallus; unnamed;
leptinotarsa; decemlinea . . . blastn: translation; initiation;
eukaryotic; structural; musculus; x-linked; product: eukaryotic;
full-length; enriched; mus; library; in . . . ilh1c.pk003.d17.f
blastx: mitochondrial; translocase; membrane; inner; predicted;
import; melanogaster; musculus; drosophila; anopheles; isoform;
tim9; ga . . . blastn: nigroviridis; full-length; tetraodon;
mitochondrial; translocase; membrane; gallus; tim9a; inner;
gambiae; purpuratus; danio; fi . . . ilh1c.pk003.h22.f blastx:
pyrophosphatase; musculus; dutp; deoxyuridine; unnamed;
triphosphatase; dutpase; human; nucleotidohydrolase; mus;
norvegicus; st . . . blastn: musculus; full-length; enriched; mus;
product: dutpase; triphosphatase; library; insert; riken; full;
deoxyuridine; days; dutpase . . . ilh1c.pk003.j5.f blastx:
benzodiazepine; receptor; peripheral-type; benzodiazapine;
peripheral; sapiens; musculus; homo; construct; synthetic; unnamed;
m . . . blastn: ilh1c.pk003.j7.f blastx: finger; melanogaster;
zinc; drosophila; musculus; sapiens; predicted; ozf; pygmaeus;
homo; mus; pongo; unnamed; taurus; bos; imp . . . blastn: sapiens;
finger; zinc; homo; predicted; highly; gallus; anopheles; fis;
rerio; danio; gambiae; pest; str; zscan2; 75a; variant; . . .
ilh1c.pk003.l11.f blastx: transmembrane; inducible; musculus;
hormone; growth; gallus; unnamed; mus; norvegicus; anopheles;
pygmaeus; xenopus; gambiae; ra . . . blastn: transmembrane;
product: growth; musculus; inducible; full-length; hormone;
enriched; library; insert; riken; mus; full; growth; o . . .
ilh1c.pk003.l18.f blastx: ubiquitin; polyubiquitin; musculus;
isoform; familiaris; mus; predicted; sapiens; taurus; canis; homo;
bos; caballus; schistosom . . . blastn: polyubiquitin; norvegicus;
rattus; ubiquitin; taeniopygia; variant; guttata; 7-like; pub;
trifallax; ubiquitins; ttu3; sterkiell . . . ilh1c.pk003.o10.f
blastx: receptor-associated; finger; androgen; predicted; triad2;
ring; musculus; caenorhabditis; hfb30; isoform; unnamed; mus;
taurus; . . . blastn: ubiquitin-conjugating; rerio; danio; ariadne;
predicted ilh1c.pk004.b8.f blastx: melanogaster; drosophila;
lethal(2)tumorous; isoform; imaginal; discs; tid56; mitochondrial;
tumorous; l(2)tid; tid58; tid50; br . . . blastn: musculus;
full-length; subfamily; anopheles; product: dnaj; member; gambiae;
mus; hsp40; enriched; adult; library; alternatively; . . .
ilh1c.pk004.d17.f blastx: nucleolar; cerevisiae; assembly;
polymerase; laevis; function; suppressor; ac40; srp40p; purpuratus;
snornps; ribonucleoprotein; . . . blastn: ilh1c.pk004.e16.f blastx:
disrupted; disorder; musculus; drosophila; bipolar; predicted;
melanogaster; unnamed; mus; familiaris; isoform; canis; asparagin .
. . blastn: musculus; disorder; disrupted; bipolar; full-length;
sapiens; enriched; homo; library; mus; predicted; human; insert;
asparagine . . . ilh1c.pk004.g2.f blastx: benzodiazepine; receptor;
peripheral-type; musculus; mitochondrial; unnamed; mus; peripheral;
isoquinoline-binding; pkbs; pbr; t . . . blastn: ilh1c.pk004.k13.f
blastx: cathepsin; proteinase; cysteine; l-like; sapiens; scrofa;
homo; heterodera; glutinosa; globodera; glycines; pallida; sus;
myxine . . . blastn: cysteine; decemlineata; leptinotarsa;
proteinase; intestain; digestive; cathepsin; sapiens; h.americanus;
preproenzyme; homo; vi . . . ilh1c.pk004.m4.f blastx: unc-51-like;
sapiens; kinase; predicted; musculus; homo; isoform; unnamed;
gallus; taurus; mus; ulk3; bos; xenopus; mellifera; l . . . blastn:
melanogaster; drosophila; musculus; predicted; product:
hypothetical; kinase; insert; full; full-length; unc-51-like;
containing; . . . ilh1c.pk005.a7.f blastx: cathepsin;
caenorhabditis; rerio; danio; elegans; procathepsin; tuberaphis;
b-s; rat; olivaceus; fundulus; heteroclitus; paralic . . . blastn:
full-length; sapiens; homo; human; 25-normalized; placenta;
construct; synthetic; cathepsin; cot; ctsb; jurkat; brain; cells;
ce . . . ilh1c.pk005.h9.f blastx: nucleolar; snornp-associated;
interacting; small; predicted; ribonucleoproptein-associated;
55-kda; musculus; sapiens; rerio; da . . . blastn: ilh1c.pk005.i1.f
blastx: melanogaster; drosophila; adenosine; deaminase;
rna-specific; double- stranded; isoform; pre-mrna; editing;
purpuratus; that; pre . . . blastn: ilh1c.pk005.i10.f blastx:
taurus; precystatin; bos; cystatin; hemorrhage; angiopathy; bovine;
unnamed; cerebral; amyloid blastn: ilh1c.pk005.i12.f blastx:
polyphosphate; anopheles; multikinase; drosophila; inositol;
melanogaster; gambiae; pest; musculus; str; predicted; mus; canis;
. . . blastn: melanogaster; drosophila; 21d-21e; insert; section;
full; ipk2; bac ilh1c.pk005.i15.f blastx: cytochrome; melanogaster;
drosophila; periplaneta; fuliginosa; migratoria; dimidiata;
triatoma; locusta; caretta; quadriocellata . . . blastn:
mitochondrial; cytochrome; blackburnia; cytb; isolate; gerris;
insularis; gracilicornis; jk041; jk042; jk045; migratoria; latiab .
. . ilh1c.pk005.i16.f blastx: domain-containing; sapiens; dnaj;
homo; novel; subfamily; anopheles; norvegicus; hsp40; predicted;
gambiae; gallus; rattus; memb . . . blastn: dnaj-1; drosophila;
nigroviridis; full-length; tetraodon; predicted; prokaryotic;
norvegicus; simulans; domain; rerio; danio; sh . . .
ilh1c.pk005.m3.f blastx: proteolipid; vacuolar; drosophila;
synthase; melanogaster; anopheles; isoform; gambiae; kda; atp;
pest; mellifera; str; c-subuni . . . blastn: anopheles; gambiae;
full-length; extremity; mosquito; malaria; african; 5- prime;
females; single; total; adult; made; read; vacu . . .
ilh1c.pk005.o17.f blastx: cathepsin; rerio; danio; virgifera;
b-like;
protease; ventricosus; branchiostoma; olivaceus; paralichthys;
parcxpwnx02; lugens; . . . blastn: product: cathepsin; full-length;
enriched; musculus; library; insert; riken; full; cathepsin; mus;
macrophage; synthetic; constru . . . ilh1c.pk006.c6.f blastx:
vesicle-associated; membrane; vamp-associated; protein-associated;
predicted; tropicalis; xenopus; associated; rerio; danio; iso . . .
blastn: ilh1c.pk006.f2.f blastx: receptor-associated; finger;
predicted; isoform; ring; sapiens; androgen; unnamed; homo;
xenopus; nigroviridis; triad2; rnf14-pr . . . blastn:
ilh1c.pk007.a8.f blastx: membrane-associated; finger; drosophila;
melanogaster; ring; gallus; musculus; c3hc4; predicted; anopheles;
gambiae; taurus; mus . . . blastn: ciona; full; insert;
intestinalis ilh1c.pk007.b21.f blastx: phosphoprotein;
golgi-localized; golgi; predicted; calcium-binding; musculus;
isoform; familiaris; membrane; mus; gallus; anophe . . . blastn:
ilh1c.pk007.g16.f blastx: burkholderia; pseudomallei; membrane;
outer; mallei; hep_hag; family; atcc; s13; exported;
autotransporter; adhesin blastn: musculus; mus; bac; linkage;
group; mouse; zebrafish ilh1c.pk007.j24.f blastx: vitellogenin;
plautia; stali; cynthia; mellifera; samia; apis; clavatus;
japonica; molitor; encarsia; nigrofuscata; saturnia; an . . .
blastn: ilh1c.pk007.o17.f blastx: beta-galactosidase;
galactosidase; familiaris; lysosomal; drosophila; anopheles; canis;
gambiae; melanogaster; beta; pest; fasci . . . blastn:
ilh1c.pk007.p11.f blastx: translation; initiation; eukaryotic;
drosophila; melanogaster; anopheles; gambiae; bombyx; eif-5a; pest;
spodoptera; danio; reri . . . blastn: initiation; translation;
eukaryotic; arabidopsis; thaliana; bombyx; eif5a; mori; rerio;
danio; eif-5a; 5a-4; gsltsil10za11; thal . . . ilh1c.pk007.p13.f
blastx: finger; predicted; sapiens; zinc; homo; musculus; unnamed;
gallus; mus; troglodytes; taurus; human; containing; bos; pan
blastn: ilh1c.pk008.c24.f blastx: translation; eukaryotic;
initiation; interacting; sapiens; melanogaster; drosophila; homo;
predicted; familiaris; isoform; varia . . . blastn: full-length;
25-normalized; sapiens; human; placenta; homo; cot; interacting;
translation; eukaryotic; initiation; danio; rerio; . . .
ilh1c.pk008.f16.f blastx: carboxypeptidase; melanogaster;
drosophila; ochlerotatus; aedes; anopheles; gambiae; aegypti; pest;
str; fluid; polynesiensis; c . . . blastn: carboxypeptidase;
bombyx; fluid; mf-cpa; mori; molting; cpb-i; ochlerotatus;
triseriatus ilh1c.pk008.h14.f blastx: signalosome; cop9; musculus;
predicted; isoform; mus; photomorphogenic; complex; xenopus;
constitutive; arabidopsis; norvegicus; . . . blastn: predicted;
photomorphogenic; constitutive; arabidopsis; norvegicus; thaliana;
rattus; cop9; mellifera; cops8_predicted; apis ilh1c.pk008.j3.f
blastx: receptor; benzodiazepine; benzodiazapine; peripheral;
musculus; peripheral-type; unnamed; synthetic; construct; mus;
sapiens; mi . . . blastn: anopheles; gambiae; full-length;
extremity; mosquito; african; malaria; females; single; total;
adult; read; made; fk0aaa40cb05; . . . ilh1c.pk008.k6.f blastx:
predicted; upstream; element; musculus; fuse; isoform; far;
anopheles; gambiae; unnamed; mus; taurus; rerio; danio; pest; str;
b . . . blastn: musculus; regulatory; splicing; library;
full-length; enriched; mus; product: kh-type; insert; kh-type;
riken; norvegicus; full; . . . ilh1c.pk009.d7.f blastx:
transcriptional; transcription; repressor; potential; isoform;
sapiens; ccr4- not; complex; predicted; not4hp; homo; ccr4-associa
. . . blastn: melanogaster; drosophila; isoform; mellifera;
predicted; apis; homo; not4- n; insert; not4-np; full; sapiens
ilh1c.pk009.f20.f blastx: eif4g-related; danio; rerio; predicted;
nat1b; isoform; xenopus; translation; initiation; eukaryotic;
tropicalis; gallus; gamma; . . . blastn: melanogaster; drosophila;
eif4g-related; predicted; isoform; xenopus; laevis; nat1;
translation; mellifera; 49e-49f; full; eukar . . .
ilh1c.pk010.f11.f blastx: glycoprotein; endocuticle; structural;
drosophila; anopheles; gambiae; melanogaster; pseudoobscura; pest;
str; mori; bombyx; cut . . . blastn: melanogaster; drosophila;
cuticle; larval; lcp-14; m.sexta; constituent; asap; sexta;
gambiae; bombyx; lcp17; subalbatus; mori; . . . ilh1c.pk010.i24.f
blastx: receptor; benzodiazepine; benzodiazapine; peripheral;
peripheral-type; musculus; sapiens; mitochondrial; drosophila;
unnamed; co . . . blastn: ilh1c.pk010.m17.f blastx: ntf2-related;
drosophila; melanogaster; anopheles; export; gambiae; pest;
predicted; musculus; str; nxt1; mus; apis; canis; mell . . .
blastn: ntf2-related; export; sapiens; anopheles; predicted;
gambiae; ntf2-like; homo; pest; strongylocentrotus; str; nxt1;
transcript; . . . ilh1c.pk010.n4.f blastx: receptor; delta; signal;
translocon-associated; sapiens; construct; synthetic; homo; rerio;
danio; anopheles; predicted; gambiae . . . blastn: sapiens;
receptor; synthetic; construct; delta; signal; homo; translocon-
associated; 25-normalized; full-length; ssr4; placenta; . . .
ilh1c.pk011.a8.f blastx: polyprotein; deformed; wing; destructor;
sacbrood; nonstructural; varroa; kakugo; unnamed; venturia;
rna-dependent; homo; sapien . . . blastn: deformed; polyprotein;
wing; isolate; nonstructural; fis; homo; sapiens; kakugo; varroa;
destructor ilh1c.pk011.d10.f blastx: drosophila; melanogaster;
death-associated; leucine-rich; containing; cytoplasmic;
caenorhabditis; anopheles; leucine; repeat; g . . . blastn:
death-associated; leucine-rich; cytoplasmic; small; sclp;
predicted; sexta; gambiae; str; manduca; mellifera; pest; apis;
anophe . . . ilh1c.pk011.e16.f blastx: mediator; transcription;
polymerase; drosophila; predicted; musculus; melanogaster; complex;
sapiens; anopheles; mus; soh1; homo . . . blastn: nigroviridis;
full-length; tetraodon; melanogaster; drosophila; isoform; med31;
full; insert ilh1c.pk009.d7.f blastx: transcriptional;
transcription; repressor; potential; isoform; sapiens; ccr4- not;
complex; predicted; not4hp; homo; ccr4-associa . . . blastn:
melanogaster; drosophila; isoform; mellifera; predicted; apis;
homo; not4- n; insert; not4-np; full; sapiens ilh1c.pk003.d10.f
blastx: translation; initiation; eukaryotic; gamma; structural;
predicted; x-linked; musculus; gallus; unnamed; leptinotarsa;
decemlinea . . . blastn: translation; initiation; eukaryotic;
structural; musculus; x-linked; product: eukaryotic; full-length;
enriched; mus; library; in . . . ilh1c.pk003.f22.f blastx:
transcription; upstream; predicted; nucleolar; danio; rerio;
isoform; ubf-1; autoantigen; nor-90; ubtf; unnamed; human; polymera
. . . blastn: ilh1c.pk004.e16.f blastx: disrupted; disorder;
musculus; drosophila; bipolar; predicted; melanogaster; unnamed;
mus; familiaris; isoform; canis; asparagin . . . blastn: musculus;
disorder; disrupted; bipolar; full-length; sapiens; enriched; homo;
library; mus; predicted; human; insert; asparagine . . .
ilh1c.pk004.m22.f blastx: spinocerebellar; predicted; ataxin;
ataxin-7; ataxia; sapiens; familiaris; gallus; musculus; isoform;
homo; canis; norvegicus; r . . . blastn: ilh1c.pk005.l24.f blastx:
melanogaster; drosophila; isoform; anopheles; gambiae;
caenorhabditis; pest; str; elegans; predicted; f-box; leucine-rich;
lrr-r . . . blastn: melanogaster; drosophila; isoform; full; bac;
clones; asap; conserved; mellifera; apis; aegypti; predicted;
aedes; 99b-99b; sect . . . ilh1c.pk006.c15.f blastx:
caenorhabditis; calpain; anopheles; elegans; gambiae; family;
gallus; calcium-activated; pest; str; isoform; xenopus; neutral; m
. . . blastn: calpain; musculus; ncl-4; sapiens; tract-specific;
product: calpain; homo; mus; transcript; full-length; capn9;
digestive; enrich . . . ilh1c.pk001.f20.f blastx: caenorhabditis;
elegans; predicted; schistosoma; rna-directed; transcriptase;
polymerase; containing; polyprotein; rerio; danio; . . . blastn:
caenorhabditis; zebrafish; linkage; elegans; group; ch211-12p12;
ch211- 260p11; dkey-9p20 ilh1c.pk006.c6.f blastx:
vesicle-associated; membrane; vamp-associated; protein-associated;
predicted; tropicalis; xenopus; associated; rerio; danio; iso . . .
blastn: ilh1c.pk001.h3.f blastx: drosophila; melanogaster;
proteinase; sarcophaga; 26, 29 kda; homologue; predicted;
anopheles; gambiae; gallus; danio; rerio; pest . . . blastn:
anopheles; gambiae; proteinase; sarcophaga; 26, 29 kda;
melanogaster; full- length; drosophila; homologue; extremity;
mosquito; afr . . . ilh1c.pk007.c6.f blastx: predicted; containing;
armadillo; sapiens; repeat; homo; strongylocentrotus; purpuratus;
anopheles; novel; gambiae; pest; str; n . . . blastn: musculus;
complex; cultivar-group; product: adaptor-related; mus;
adaptor-related; japonica; beta; sativa; full-length; oryza; en . .
. ilh1c.pk007.m5.f blastx: transcription; polypeptide; general;
sapiens; musculus; 63 kda; homo; gallus; iiic; iiic-epsilon;
tf3c-epsilon; mus; anopheles; t . . . blastn: ilh1c.pk008.c24.f
blastx: translation; eukaryotic; initiation; interacting; sapiens;
melanogaster; drosophila; homo; predicted; familiaris; isoform;
varia . . . blastn: full-length; 25-normalized; sapiens; human;
placenta; homo; cot; interacting; translation; eukaryotic;
initiation; danio; rerio; . . . ilh1c.pk008.d5.f blastx:
prostaglandin; prostaglandin-endoperoxide; synthase; synthase-2;
cyclooxygenase-2; cox-2; taurus; familiaris; tropicalis; caball . .
. blastn: prostaglandin; synthase; gallus; rerio; predicted;
c.porcellus; danio; cyclooxygenase-2; chicken ilh1c.pk008.f17.f
blastx: cathepsin; proteinase; b-like; e.c.3.4.22.1; schistosoma;
papain-like; virgifera; lysosomal; isotype; mansoni; triatoma;
sm31; d . . . blastn: cathepsin; sapiens; construct; synthetic;
homo; 25-normalized; ctsb; full- length; human; pygmaeus; placenta;
pongo; cot; cs0di02 . . . ilh1c.pk008.g3.f blastx:
pyroglutamyl-peptidase; pyrrolidone-carboxylate; unnamed; musculus;
5- oxoprolyl-peptidase; norvegicus; peptidase; xenopus; sapie . . .
blastn: ilh1c.pk002.o12.f blastx: anopheles; gambiae; trypsin;
pest; str; protease; trypsin-related; chymotrypsin; aegypti; aedes;
chymotrypsin-like; ctenocephali . . . blastn: ilh1c.pk003.d10.f
blastx: translation; initiation; eukaryotic; gamma; structural;
predicted; x-linked; musculus; gallus; unnamed; leptinotarsa;
decemlinea . . . blastn: translation; initiation; eukaryotic;
structural; musculus; x-linked; product: eukaryotic; full-length;
enriched; mus; library; in . . . ilh1c.pk004.o2.f blastx:
carboxypeptidase; vitellogenic-like; sapiens; vitellogenic; serine;
homo; carboxypeptidase-like; cpvl; predicted; canis; pygmaeu . . .
blastn: carboxypeptidase; anopheles; gambiae; vitellogenic;
carboxypeptidase- like; full-length; vitellogenic-like; extremity;
mosquito; . . .
TABLE-US-00003 TABLE 3 Summary of Top Blast Hit sid database
accession# score ilh1c.pk005.o10.f gb AAL28614.1 498
ilh1c.pk004.e6.f gb EAA04403.2 161 ilh1c.pk001.e23.f gb EAA04403.2
160 ilh1c.pk010.f11.f sp Q7M4F2 359 ilh1c.pk011.m15.f sp P83995 300
ilh1c.pk005.d21.f gb AAN62848.1 104 ilh1c.pk011.f4.f gb EAA06494.2
559 ilh1c.pk004.a13.f gb AAT09370.1 634 ilh1c.pk004.i18.f gb
AAT09370.1 648 ilh1c.pk004.l22.f gb EAA03540.3 275
ilh1c.pk005.k23.f gb AAT09370.1 377 ilh1c.pk010.g16.f gb EAA08488.2
148 ilh1c.pk002.d16.f dbj BAA22791.1 567 ilh1c.pk001.f1.f dbj
BAA22791.1 321 ilh1c.pk005.f15.f gb AAB72001.1 245
ilh1c.pk003.d10.f emb CAF92618.1 196 ilh1c.pk010.d2.f gb AAF56796.2
188 ilh1c.pk010.d2.f gb AAM50998.1 188 ilh1c.pk011.g22.f ref
XP_625027.1 437 ilh1c.pk011.h12.f gb AAT01080.1 744
ilh1c.pk001.k13.f ref XP_623842.1 477 ilh1c.pk001.k16.f ref
XP_396057.2 508 ilh1c.pk001.k16.f ref XP_623167.1 508
ilh1c.pk001.l22.f ref XP_392692.2 673 ilh1c.pk001.m15.f gb
AAV31410.1 583 ilh1c.pk001.m17.f ref XP_392925.2 390
ilh1c.pk001.n23.f ref XP_790997.1 235 ilh1c.pk001.n6.f gb
AAD09820.1 384 ilh1c.pk001.o17.f gb AAL90146.1 758
ilh1c.pk001.o17.f gb AAX52682.1 758 ilh1c.pk001.o4.f gb EAA04597.2
302 ilh1c.pk001.o4.f gb EAL32669.1 302 ilh1c.pk002.a11.f gb
EAA05241.2 189 ilh1c.pk002.a24.f gb AAT01080.1 436
ilh1c.pk002.b12.f gb EAA01140.2 269 ilh1c.pk003.n7.f ref
NP_788640.1 760 ilh1c.pk003.p4.f ref XP_395521.2 340
ilh1c.pk004.c13.f gb EAL33114.1 538 ilh1c.pk004.i5.f ref
XP_392968.2 323 ilh1c.pk001.d8.f gb EAA12913.3 565
ilh1c.pk001.e10.f ref NP_001007306.1 255 ilh1c.pk001.i10.f ref
XP_635611.1 284 ilh1c.pk010.p16.f ref XP_396126.2 278
ilh1c.pk003.k14.f ref XP_854245.1 403 ilh1c.pk004.a13.f gb
AAT09370.1 634 ilh1c.pk001.b20.f gb AAF54512.1 741
ilh1c.pk004.i18.f gb AAT09370.1 648 ilh1c.pk004.o2.f gb AAC41580.1
502 ilh1c.pk004.o2.f ref XP_854245.1 502 ilh1c.pk005.m3.f emb
CAA46187.1 706 ilh1c.pk005.m3.f sp P55277 706 ilh1c.pk002.o3.f ref
XP_392848.2 561 ilh1c.pk003.h22.f ref XP_393899.1 331
ilh1c.pk003.i6.f gb AAI08879.1 238 ilh1c.pk004.a13.f gb AAT09370.1
634 ilh1c.pk003.g17.f gb EAA14743.2 158 ilh1c.pk009.k12.f ref
XP_623580.1 583 ilh1c.pk009.f20.f ref XP_394628.2 332
ilh1c.pk008.o12.f dbj BAD52260.1 711 ilh1c.pk008.k5.f gb EAA09467.2
366 ilh1c.pk008.c24.f ref XP_624438.1 804 ilh1c.pk008.b15.f gb
EAA06364.2 466 ilh1c.pk007.p11.f gb ABA54998.1 231
ilh1c.pk003.d10.f emb CAF92618.1 196 ilh1c.pk002.d16.f dbj
BAA22791.1 567 ilh1c.pk002.d9.f gb EAA44856.2 233 ilh1c.pk002.j6.f
gb AAY24048.1 120 ilh1c.pk002.j6.f ref NP_938017.1 120
ilh1c.pk002.k17.f gb EAA01750.2 528 ilh1c.pk002.k5.f dbj BAA91735.1
239 ilh1c.pk002.k5.f dbj BAD97072.1 239 ilh1c.pk002.k5.f dbj
BAE01389.1 239 ilh1c.pk002.k5.f gb AAF67488.1 239 ilh1c.pk002.k5.f
gb AAH12559.1 239 ilh1c.pk002.k5.f ref NP_059122.2 239
ilh1c.pk002.m21.f ref XP_425021.1 404 ilh1c.pk002.n11.f gb
EAA00451.2 968 ilh1c.pk003.d10.f emb CAF92618.1 196
ilh1c.pk003.d17.f gb EAA06794.2 348 ilh1c.pk003.h22.f ref
XP_393899.1 331 ilh1c.pk003.j5.f gb AAK31586.1 153 ilh1c.pk003.j7.f
gb AAY51599.1 268 ilh1c.pk003.j7.f ref NP_650534.1 268
ilh1c.pk003.l11.f emb CAG31191.1 245 ilh1c.pk003.l18.f ref
NP_066289.2 321 ilh1c.pk003.o10.f ref XP_624683.1 196
ilh1c.pk004.b8.f ref XP_394833.1 546 ilh1c.pk004.d17.f gb
AAA35091.1 115 ilh1c.pk004.d17.f gb AAH84355.1 115
ilh1c.pk004.d17.f ref NP_013018.1 115 ilh1c.pk004.e16.f ref
XP_397356.2 317 ilh1c.pk004.g2.f gb EAL33487.1 94 ilh1c.pk004.g2.f
ref XP_531704.2 94 ilh1c.pk004.k13.f gb AAY45870.1 381
ilh1c.pk004.m4.f ref XP_396911.2 421 ilh1c.pk005.a7.f gb AAB65345.1
257 ilh1c.pk005.h9.f ref XP_787944.1 119 ilh1c.pk005.i1.f gb
EAL32477.1 120 ilh1c.pk005.i10.f pir UDBO 105 ilh1c.pk005.i10.f ref
NP_776454.1 105 ilh1c.pk005.i12.f gb EAA03479.2 394
ilh1c.pk005.i15.f gb AAG31619.1 827 ilh1c.pk005.i16.f ref
XP_395584.2 731 ilh1c.pk005.m3.f emb CAA46187.1 706
ilh1c.pk005.m3.f sp P55277 706 ilh1c.pk005.o17.f gb AAQ83887.1 461
ilh1c.pk006.c6.f gb EAA06671.2 201 ilh1c.pk006.f2.f emb CAF96101.1
180 ilh1c.pk007.a8.f gb EAL28755.1 343 ilh1c.pk007.b21.f ref
XP_422806.1 140 ilh1c.pk007.g16.f ref YP_102734.1 129
ilh1c.pk007.j24.f gb AAB72001.1 168 ilh1c.pk007.o17.f ref
NP_001015958.1 118 ilh1c.pk007.o17.f ref NP_001030215.1 118
ilh1c.pk007.p11.f gb ABA54998.1 231 ilh1c.pk007.p13.f dbj
BAA91976.1 337 ilh1c.pk008.c24.f ref XP_624438.1 804
ilh1c.pk008.f16.f sp P04069 273 ilh1c.pk008.h14.f ref XP_391971.1
401 ilh1c.pk008.j3.f ref XP_397432.2 305 ilh1c.pk008.k6.f gb
AAH85379.1 222 ilh1c.pk009.d7.f ref XP_392724.2 1062
ilh1c.pk009.d7.f ref XP_623058.1 1062 ilh1c.pk009.f20.f ref
XP_394628.2 332 ilh1c.pk010.f11.f sp Q7M4F2 359 ilh1c.pk010.i24.f
ref XP_397432.2 190 ilh1c.pk010.m17.f gb EAA12371.3 392
ilh1c.pk010.n4.f ref XP_624607.1 423 ilh1c.pk011.a8.f gb AAP49008.1
433 ilh1c.pk011.a8.f gb AAP49283.1 433 ilh1c.pk011.d10.f ref
XP_623853.1 358 ilh1c.pk011.e16.f ref XP_393244.1 522
ilh1c.pk009.d7.f ref XP_392724.2 1062 ilh1c.pk009.d7.f ref
XP_623058.1 1062 ilh1c.pk003.d10.f emb CAF92618.1 196
ilh1c.pk003.f22.f emb CAF92109.1 360 ilh1c.pk004.e16.f ref
XP_397356.2 317 ilh1c.pk004.m22.f ref XP_541817.2 200
ilh1c.pk004.m22.f ref XP_848286.1 200 ilh1c.pk005.l24.f gb
AAN71350.1 427 ilh1c.pk005.l24.f ref NP_733291.1 427
ilh1c.pk006.c15.f gb EAL38766.1 400 ilh1c.pk001.f20.f gb AAB71256.1
392 ilh1c.pk006.c6.f gb EAA06671.2 201 ilh1c.pk001.h3.f dbj
BAA86911.1 967 ilh1c.pk007.c6.f ref XP_507719.1 126
ilh1c.pk007.m5.f ref XP_342400.2 234 ilh1c.pk008.c24.f ref
XP_624438.1 804 ilh1c.pk008.d5.f ref XP_422297.1 244
ilh1c.pk008.f17.f gb AAT48984.1 386 ilh1c.pk008.g3.f emb CAG00772.1
267 ilh1c.pk008.g3.f ref XP_786904.1 267 ilh1c.pk002.o12.f gb
AAL93243.1 142 ilh1c.pk003.d10.f emb CAF92618.1 196
ilh1c.pk004.o2.f gb AAC41580.1 502 ilh1c.pk004.o2.f ref XP_854245.1
502
TABLE-US-00004 TABLE 4 SEQ ID Target region/ est and Target Region
Antisense sense/ position of gene Sense primer primer % CG
antisense ilh1c.pk5.o1.f 43 AAGAGGTACAAA GAGGUACAAA GGAAAGACAU % CG
= 39.1 22/23/24 TGTCTTTCCTG UGUCUUUCC UUGUACCUC 26 AAGTCAGTCCTA
GUCAGUCCUA GGACACGUGU % CG = 52.2 25/26/27 CACGTGTCCTC CACGUGUCC
AGGACUGAC 353 AACAACGTTCGC CAACGUUCGC AAAAUCCUGG % CG = 39.1
28/29/30 CAGGATTTTTT CAGGAUUUU CGAACGUUG ilh1c.pk4.e6.f 16
AAGACTGCTGAC GACUGCUGAC CGUUAUUCCG % CG = 43.5 31/32/33 GGAATAACGAA
GGAAUAACG UCAGCAGUC 332 AACCCCGACGGC CCCCGACGGC AGCGACGGUG % CG =
69.6 34/35/36 ACCGTCGCTGA ACCGUCGCU CCGUCGGGG 56 AAGATCTCCAAA
GAUCUCCAAA UCCACGAGCU % CG = 43.5 37/38/39 GCTCGTGGAAT GCUCGUGGA
UUGGAGAUC ilh1c.pk1.e23.f 49 AAATTACCCGCG AUUACCCGCG AGUAUCCUCC %
CG = 47.8 40/41/42 GAGGATACTCA GAGGAUACU GCGGGUAAU 256 AATTGTGGCTTC
UUGUGGCUUC ACGGGAGCUG % CG = 47.8 43/44/45 AGCTCCCGTAT AGCUCCCGU
AAGCCACAA 656 AACTCTCTTGTA CUCUCUUGUA UGGUGAGG.U % CG = 43.5
46/47/48 NCCTCACCACA NCCUCACCA ACAAGAGAG ilh1c.pk1.f11.f 37
AAGTCTTTAAGG GUCUUUAAGG UAAAUCCGUC % CG = 39.1 49/50/51 ACGGATTTACG
ACGGAUUUA CUUAAAGAC 24 AATGCGTGTTGG UGCGUGUUGG ACAGAUGAGC % CG =
47.8 52/53/54 CTCATCTGTCA CUCAUCUGU CAACACGCA 543 AAGCTGGTGGTC
GCUGGUGGUC CCUUCAGACG % CG = 52.2 55/56/57 GTCTGAAGGAT GUCUGAAGG
ACCACCAGC 613 AACACTCGGATG CACUCGGAUG CAGUGAUUUC % CG = 43.5
58/59/60 AAATCACTGTG AAAUCACUG AUCCGAGUG ilh1c.pk11.m15.f 28
AATCAGTATCAC UCAGUAUCAC AUCCUGAGCG % CG = 43.5 61/62/63 GCTCAGGATGT
GCUCAGGAU UGAUACUGA 355 AACACCCATGAA CACCCAUGAA GACAGCGACU % CG =
56.5 64/65/66 GTCGCTGTCGC GUCGCUGUC UCAUGGGUG 57 AAGATCTCCAAA
GAUCUCCAAA UCCACGAGCU % CG = 43.5 67/68/69 GCTCGTGGAAT GCUCGUGGA
UUGGAGAUC ilh1c.pk5.d21.f 94 AACTACAAGTAT CUACAAGUAU CUCCACAGGA %
CG = 47.8 70/71/72 CCTGTGGAGGG CCUGUGGAG UACUUGUAG 219 AAATTGGAGTGT
AUUGGAGUGU GCACCAGUCA % CG = 47.8 73/74/75 GACTGGTGCTG GACUGGUGC
CACUCCAAU 57 AACATCAGACGC CAUCAGACGC CAGUAGAGGG % CG = 52.2
76/77/78 CCTCTACTGGA CCUCUACUG CGUCUGAUG ilh1c.pk11.f4.f 51
AAACGGTTCTTT ACGGUUCUUU UCGUUUCGGA % CG = 47.8 79/80/81 CCGAAACGACG
CCGAAACGA AAGAACCGU 225 AACCATCGTTCC CCAUCGUUCC GCAUCACCAG % CG =
47.8 82/83/84 TGGTGATGCAT UGGUGAUGC GAACGAUGG 545 AAAGTCTTCAGA
AGUCUUCAGA AGCUUCCUCU % CG = 47.8 85/86/87 GAGGAAGCTGC GAGGAAGCU
CUGAAGACU ilh1c.pk4.l22.f 79 AAGATGTTCCAT GAUGUUCCAU UGCCUCUCCA %
CG = 47.8 88/89/90 GGAGAGGCACA GGAGAGGCA UGGAACAUC 22 AAATAGGGCAAC
AUAGGGCAAC AUCCGUGAAG % CG = 39.1 91/92/93 TTCACGGATTA UUCACGGAU
UUGCCCUAU 469 AAGAACCATTCG GAACCAUUCG UGAUAACGGC % CG = 47.8
94/95/96 CCGTTATCACC CCGUUAUCA GAAUGGUUC 651 AAGCTGCACTGG
GCUGCACUGG GAAUGAAUCC % CG = 47.8 97/98/99 GATTCATTCCT GAUUCAUUC
CAGUGCAGC ilh1c.pk3.d1.f 88 AATACAAAAGTC UACAAAAGUC GGAAGACGCG % CG
= 47.8 100/101/102 GCGTCTTCCGG GCGUCUUCC ACUUUUGUA 132 AAGATCTTTCGA
GAUCUUUCGA CGUCAAGAGU % CG = 43.5 103/104/105 CTCTTGACGTG CUCUUGACG
CGAAAGAUC 27 AATATCGGTACT UAUCGGUACU GUGACCAAUA % CG = 39.1
106/107/108 ATTGGTCACGT AUUGGUCAC GUACCGAUA ilh1c.pk1.d2.f 96
AATTTCGACGTG UUUCGACGUG UAACACUUCC % CG = 39.1 109/110/111
GAAGTGTTAAG GAAGUGUUA ACGUCGAAA 249 AATGTTGCATCT UGUUGCAUCU
GCACCUUCGA % CG = 43.5 112/113/114 CGAAGGTGCAA CGAAGGUGC GAUGCAACA
422 AACACTGGGTAA CACUGGGUAA CCUACAUCUU % CG = 43.5 115/116/117
AGATGTAGGAC AGAUGUAGG UACCCAGUG ilh1c.pk11.h12.f 29 AACCCGGGTCTT
CCCGGGUCUU UUUACAGAAA % CG = 43.5 118/119/120 TTCTGTAAAGA UUCUGUAAA
AGACCCGGG 22 AAACCTCGAAAC ACCUCGAAAC ACGUUUUUGG % CG = 39.1
121/122/123 CAAAAACGTAG CAAAAACGU UUUCGAGGU 665 AAGTTGGAAACT
GUUGGAAACU CUGGACUCCA % CG = 43.5 124/125/126 GGAGTCCAGAT GGAGUCCAG
GUUUCCAAC ilh1c.pk2.a24.f 72 AAATCTTCTTCG AUCUUCUUCG GGAUAGUCCC %
CG = 39.1 127/128/129 GGACTATCCAT GGACUAUCC GAAGAAGAU 23
AAAACCAGAACA AACCAGAACA UCCGGUUUCU % CG = 43.5 130/131/132
GAAACCGGAGT GAAACCGGA GUUCUGGUU 645 AAGTGATGACCT GUGAUGACCU
CCAAAGAGAA % CG = 43.5 133/134/135 TCTCTTTGGGA UCUCUUUGG GGUCAUCAC
ilh1c.pk9.f2.f 9 AACAGGACATCA CAGGACAUCA UGCGAAAUAU % CG = 43.5
136/137/138 TATTTCGCAGG UAUUUCGCA GAUGUCCUG 21 AAAAAGCACTAG
AAAGCACUAG UCGGCUCAUC % CG = 43.5 139/140/141 ATGAGCCGAAG AUGAGCCGA
UAGUGCUUU 43 AAGTTCATCGGA GUUCAUCGGA GCAAAGCUCU % CG = 43.5
142/143/144 GAGCTTTGCAA GAGCUUUGC CCGAUGAAC 596 AACTTTTTGATC
CUUUUUGAUC ACGCCAUACG % CG = 43.5 145/146/147 GTATGGCGTCC GUAUGGCGU
AUCAAAAAG ilh1c.pk2.d9.f 3 AACTTTCCTNTT CUUUCCUNUU ACAACGCGGA % CG
= 43.5 148/149/150 CCGCGTTGTTG CCGCGUUGU A.AGGAAAG 394 AAGAGAACGGAG
GAGAACGGAG CCUCGUUAUC % CG = 43.5 151/152/153 ATAACGAGGNA AUAACGAGG
UCCGUUCUC 55 AAAAAGGCTCCG AAAGGCUCCG CCAAACAGUC % CG = 43.5
154/155/156 ACTGTTTGGNA ACUGUUUGG GGAGCCUUU ilh1c.pk3.j7.f 4
AAAAAGCTCCAT AAAGCUCCAU UUCACCUGUA % CG = 43.5 157/158/159
ACAGGTGAACG ACAGGUGAA UGGAGCUUU 26 AAAAACAAGCAC AAACAAGCAC
CCCGUCUUUG % CG = 43.5 160/161/162 AAAGACGGGTC AAAGACGGG UGCUUGUUU
669 AAGTATTTCAGT GUAUUUCAGU CAGAGGAAGA % CG = 39.1 163/164/165
CTTCCTCTGCT CUUCCUCUG CUGAAAUAC ilh1c.pk3.o1.f 36 AAGTCAGAATGT
GUCAGAAUGU UUUCAGUCGA % CG = 39.1 166/167/168 CGACTGAAAAG CGACUGAAA
CAUUCUGAC 267 AAGGAACGTGAG GGAACGUGAG CAGGAUCAUC % CG = 43.5
169/170/171 ATGATCCTGTA AUGAUCCUG UCACGUUCC 525 AACCCNACTTTC
CCCNACUUUC UUUGUACAAG % CG = 34.8 172/173/174 TTGTACAAAGT UUGUACAAA
AAAGU.GGG ilh1c.pk4.b8.f 11 AAGTTTTTGCGG GUUUUUGCGG AUUCCGACAC % CG
= 43.5 175/176/177 TGTCGGAATGT UGUCGGAAU CGCAAAAAC 361 AAAANNNNTCCG
AANNNNUCCG GGCGGCGUUC % CG = 43.5 178/179/180 AACGCCGCCAA AACGCCGCC
GGA....UU 668 AAACTCACCTTC ACUCACCUUC GGCUUGAGAG % CG = 47.8
181/182/183 TCTCAAGCCGT UCUCAAGCC AAGGUGAGU ilh1c.pk4.d17.f 56
AATTCAACGAGA UUCAACGAGA AUUCGGUUAU % CG = 34.8 184/185/186
TAACCGAATGA UAACCGAAU CUCGUUGAA 295 AAGTCTGTGTTG GUCUGUGUUG
ACUUUCAGAC % CG = 39.1 187/188/189 TCTGAAAGTAG UCUGAAAGU AACACAGAC
56 AACTCCCAGTGT CUCCCAGUGU UUGAGGACGA % CG = 52.2 190/191/192
CGTCCTCAAGA CGUCCUCAA CACUGGGAG 649 AAGTTCCATCCA GUUCCAUCCA
GCGGUGCUUU % CG = 52.2 193/194/195 AAGCACCGCAG AAGCACCGC GGAUGGAAC
ilh1c.pk4.k13.f 8 AAGGAAGTATCG GGAAGUAUCG AAUGUGGUGC % CG = 43.5
196/197/198 CACCACATTCT CACCACAUU GAUACUUCC 445 AAATCAAGGACA
AUCAAGGACA CUACCACAUU % CG = 39.1 199/200/201 ATGTGGTAGCT AUGUGGUAG
GUCCUUGAU 698 AAAGTGAAGGTG AGUGAAGGUG CGCAAUCUUC % CG = 43.5
202/203/204 AAGATTGCGAG AAGAUUGCG ACCUUCACU ilh1c.pk11.a8.f 14
AAGATGTTGTGT GAUGUUGUGU GUAAACCUAA % CG = 39.1 205/206/207
TAGGTTTACCG UAGGUUUAC CACAACAUC 394 AACAAAGTCGAT CAAAGUCGAU
CUAGGUGGAA % CG = 43.5 208/209/210 TCCACCTAGAG UCCACCUAG UCGACUUUG
515 AACTTTGGTCCT CUUUGGUCCU AUCCAAACCA % CG = 43.5 211/212/213
GGTTTGGATAG GGUUUGGAU GGACCAAAG ilh1c.pk11.d1.f 42 AAGCTACAGGAA
GCUACAGGAA UU.UCCGGAU % CG = 39.1 214/215/216 TCCGGANAATT UCCGGANAA
UCCUGUAGC 136 AAGGCGTGTAAT GGCGUGUAAU GCUUGAGAGA % CG = 43.5
217/218/219 CTCTCAAGCAA CUCUCAAGC UUACACGCC 182 AATTCGCGGTCA
UUCGCGGUCA AGAUAAAGUU % CG = 39.1 220/221/222 ACTTTATCTCT ACUUUAUCU
GACCGCGAA (Note: the sense RNA primer sequence and the antisense
RNA primer sequences shown in table 4 were generated to have 2
thymine residues at the 3' end.)
Example 2
Lygus hesperus Assay Methodology
[0131] Lygus hesperus egg packs are received from the University of
Missouri and placed in Fluon.RTM. AD-1 (AG Fluoropolymers)-treated
containers in a 18.degree. C. incubator, RH.about.40 to 60%, and no
light. Eggs generally hatch after 6 days and then are transferred
to a 25.degree. incubator, RH.about.40 to 60%, 22 hours Day:2 hours
Night. Neonates are fed artificial diet (Bio-Serv F9644B) in
parafilm packets. 1 day old 2.sup.nd instar nymphs are used for
bioassay.
[0132] Liquid samples (20 ul) are dispensed to 96 well microtiter
plates (Falcon Assay Plate 353910) and mixed with 75 ul of
artificial diet (Bio-Serv F9644B). Assays typically include 4 to 5
observations for each sample. The plate is then covered with
slightly stretched parafilm. Several (3 to 8) 1 day old 2.sup.nd
instar nymphs are placed in each well of a 96 well filter plate
(Millipore MABVN1250). The diet/sample plate is flipped on top of
the nymph-infested filter plate so that the wells line up and the
nymphs can feed on the diet/sample mixture through the parafilm.
The filter plate and the diet plate are clamped together using
rubber bands. The plates are placed in a 25.degree. incubator,
RH.about.40 to 60%, and no light, and the assay is scored after 4
days. Each well is scored individually and given a rating of either
0, 1, or 2 based on the following system: [0133] 0=Normal
development--molting to third instar nymphs [0134] 1=Stunted
growth--same size as 1 day old 2.sup.nd instars or slightly larger
[0135] 2=Dead The score is based on the least affected nymph in the
well. For example, if all but one nymph is dead, and that nymph is
stunted, the well would be scored as a 1. If 4 nymphs are stunted
and 2 are normal, the well would be scored as 0. Several
repetitions are run using the same sample and dose. The well values
for all observations are added together and a % effect is
calculated using the following formula:
[0135] Rep 1 ( well value ) + Rep 2 ( well value ) + Rep 3 ( well
value ) + Rep N ( well value ) ( Total number of reps N ) * ( 2 )
.times. 100 = % effect ##EQU00001##
[0136] Where N is the number of observations run for that sample
and dose
Using the % effect numbers and the corresponding doses, an
Effective Concentration (EC) 50 can be calculated using a probit
analysis program (LdP Line).
Results:
[0137] 62 synthetic dsRNA samples (Sigm-Genosys, The Woodlands,
Tex.) were tested at the following concentrations: 35, 17.5, 8.25,
and 4.125 ppm. Of the samples tested, 47 were inactive, while 15
demonstrated varying degrees of activity including stunting and
mortality (highlighted in table 5).
TABLE-US-00005 TABLE 5 Sam- ple id Targeted region sense strand
antisense strand SEQ ID NO A1 ilh1c.pk005.o10.f
AAGAGGTACAAATGTCTTTCCTG GAGGUACAAAUGUCUUUCC GGAAAGACAUUUGUACCUC
223/224/225 B1 ilh1c.pk005.o10.f AAGTCAGTCCTACACGTGTCCTC
GUCAGUCCUACACGUGUCC GGACACGUGUAGGACUGAC 226/227/228 C1
ilh1c.pk005.o10.f AACAACGTTCGCCAGGATTTTTT CAACGUUCGCCAGGAUUUU
AAAAUCCUGGCGAACGUUG 229/230/231 D1 ilh1c.pk004.e6.f
AAGACTGCTGACGGAATAACGAA GACUGCUGACGGAAUAACG CGUUAUUCCGUCAGCAGUC
232/233/234 E1 ilh1c.pk004.e6.f AACCCCGACGGCACCGTCGCTGA
CCCCGACGGCACCGUCGCU AGCGACGGUGCCGUCGGGG 235/236/237 F1
ilh1c.pk004.e6.f AAGATCTCCAAAGCTCGTGGAAT GAUCUCCAAAGCUCGUGGA
UCCACGAGCUUUGGAGAUC 238/239/240 G1 ilh1c.pk001.e23.f
AAATTACCCGCGGAGGATACTCA AUUACCCGCGGAGGAUACU AGUAUCCUCCGCGGGUAAU
241/242/243 H1 ilh1c.pk001.e23.f AATTGTGGCTTCAGCTCCCGTAT
UUGUGGCUUCAGCUCCCGU ACGGGAGCUGAAGCCACAA 244/245/246 A2
ilh1c.pk010.f11.f AAGTCTTTAAGGACGGATTTACG GUCUUUAAGGACGGAUUUA
UAAAUCCGUCCUUAAAGAC 247/248/249 B2 ilh1c.pk010.f11.f
AATGCGTGTTGGCTCATCTGTCA UGCGUGUUGGCUCAUCUGU ACAGAUGAGCCAACACGCA
250/251/252 C2 ilh1c.pk010.f11.f AAGCTGGTGGTCGTCTGAAGGAT
GCUGGUGGUCGUCUGAAGG CCUUCAGACGACCACCAGC 253/254/255 D2
ilh1c.pk010.f11.f AACACTCGGATGAAATCACTGTG CACUCGGAUGAAAUCACUG
CAGUGAUUUCAUCCGAGUG 256/257/258 E2 ilh1c.pk011.m15.f
AATCAGTATCACGCTCAGGATGT UCAGUAUCACGCUCAGGAU AUCCUGAGCGUGAUACUGA
259/260/261 F2 ilh1c.pk011.m15.f AACACCCATGAAGTCGCTGTCGC
CACCCAUGAAGUCGCUGUC GACAGCGACUUCAUGGGUG 262/263/264 G2
ilh1c.pk011.m15.f AAGATCTCCAAAGCTCGTGGAAT GAUCUCCAAAGCUCGUGGA
UCCACGAGCUUUGGAGAUC 265/266/267 H2 ilh1c.pk005.d21.f
AACTACAAGTATCCTGTGGAGGG CUACAAGUAUCCUGUGGAG CUCCACAGGAUACUUGUAG
268/269/270 A3 ilh1c.pk005.d21.f AAATTGGAGTGTGACTGGTGCTG
AUUGGAGUGUGACUGGUGC GCACCAGUCACACUCCAAU 271/272/273 B3
ilh1c.pk005.d21.f AACATCAGACGCCCTCTACTGGA CAUCAGACGCCCUCUACUG
CAGUAGAGGGCGUCUGAUG 274/275/276 C3 ilh1c.pk011.f4.f
AAACGGTTCTTTCCGAAACGACG ACGGUUCUUUCCGAAACGA UCGUUUCGGAAAGAACCGU
277/278/279 D3 ilh1c.pk011.f4.f AACCATCGTTCCTGGTGATGCAT
CCAUCGUUCCUGGUGAUGC GCAUCACCAGGAACGAUGG 280/281/282 ##STR00001##
##STR00002## ##STR00003## ##STR00004## ##STR00005## 283/284/285 F3
ilh1c.pk004.l22.f AAGATGTTCCATGGAGAGGCACA GAUGUUCCAUGGAGAGGCA
UGCCUCUCCAUGGAACAUC 286/287/288 ##STR00006## ##STR00007##
##STR00008## ##STR00009## ##STR00010## 289/290/291 ##STR00011##
##STR00012## ##STR00013## ##STR00014## ##STR00015## 292/293/294 A4
ilh1c.pk004.l22.f AAGCTGCACTGGGATTCATTCCT GCUGCACUGGGAUUCAUUC
GAAUGAAUCCCAGUGCAGC 295/296/297 B4 ilh1c.pk003.d10.f
AATACAAAAGTCGCGTCTTCCGG UACAAAAGUCGCGUCUUCC GGAAGACGCGACUUUUGUA
298/299/300 C4 ilh1c.pk003.d10.f AAGATCTTTCGACTCTTGACGTG
GAUCUUUCGACUCUUGACG CGUCAAGAGUCGAAAGAUC 301/302/303 ##STR00016##
##STR00017## ##STR00018## ##STR00019## ##STR00020## 304/305/306 E4
ilh1c.pk010.d2.f AATTTCGACGTGGAAGTGTTAAG UUUCGACGUGGAAGUGUUA
UAACACUUCCACGUCGAAA 307/308/309 F4 ilh1c.pk010.d2.f
AATGTTGCATCTCGAAGGTGCAA UGUUGCAUCUCGAAGGUGC GCACCUUCGAGAUGCAACA
310/311/312 G4 ilh1c.pk010.d2.f AACACTGGGTAAAGATGTAGGAC
CACUGGGUAAAGAUGUAGG CCUACAUCUUUACCCAGUG 313/314/315 ##STR00021##
##STR00022## ##STR00023## ##STR00024## ##STR00025## 316/317/318
##STR00026## ##STR00027## ##STR00028## ##STR00029## ##STR00030##
319/320/321 ##STR00031## ##STR00032## ##STR00033## ##STR00034##
##STR00035## 322/323/324 ##STR00036## ##STR00037## ##STR00038##
##STR00039## ##STR00040## 325/326/327 ##STR00041## ##STR00042##
##STR00043## ##STR00044## ##STR00045## 328/329/330 ##STR00046##
##STR00047## ##STR00048## ##STR00049## ##STR00050## 331/332/333
##STR00051## ##STR00052## ##STR00053## ##STR00054## ##STR00055##
334/335/336 ##STR00056## ##STR00057## ##STR00058## ##STR00059##
##STR00060## 337/338/339 ##STR00061## ##STR00062## ##STR00063##
##STR00064## ##STR00065## 340/341/342 ##STR00066## ##STR00067##
##STR00068## ##STR00069## ##STR00070## 343/344/345 ##STR00071##
##STR00072## ##STR00073## ##STR00074## ##STR00075## 346/347/348 C6
ilh1c.pk002.d9.f AAAAAGGCTCCGACTGTTTGGNA AAAGGCUCCGACUGUUUGG
CCAAACAGUCGGAGCCUUU 349/350/351 D6 ilh1c.pk003.j7.f
AAAAAGCTCCATACAGGTGAACG AAAGCUCCAUACAGGUGAA UUCACCUGUAUGGAGCUUU
352/353/354 E6 ilh1c.pk003.j7.f AAAAACAAGCACAAAGACGGGTC
AAACAAGCACAAAGACGGG CCCGUCUUUGUGCUUGUUU 355/356/357 F6
ilh1c.pk003.j7.f AAGTATTTCAGTCTTCCTCTGCT GUAUUUCAGUCUUCCUCUG
CAGAGGAAGACUGAAAUAC 358/359/360 G6 ilh1c.pk003.o10.f
AAGTCAGAATGTCGACTGAAAAG GUCAGAAUGUCGACUGAAA UUUCAGUCGACAUUCUGAC
361/362/363 H6 ilh1c.pk003.o10.f AAGGAACGTGAGATGATCCTGTA
GGAACGUGAGAUGAUCCUG CAGGAUCAUCUCACGUUCC 364/365/366 A7
ilh1c.pk004.b8.f AAGTTTTTGCGGTGTCGGAATGT GUUUUUGCGGUGUCGGAAU
AUUCCGACACCGCAAAAACd 367/368/369 B7 ilh1c.pk004.b8.f
AAACTCACCTTCTCTCAAGCCGT ACUCACCUUCUCUCAAGCC GGCUUGAGAGAAGGUGAGU
370/371/372 C7 ilh1c.pk004.d17.f AATTCAACGAGATAACCGAATGA
UUCAACGAGAUAACCGAAU AUUCGGUUAUCUCGUUGAA 373/374/375 D7
ilh1c.pk004.d17.f AAGTCTGTGTTGTCTGAAAGTAG GUCUGUGUUGUCUGAAAGU
ACUUUCAGACAACACAGAC 376/377/378 E7 ilh1c.pk004.d17.f
AACTCCCAGTGTCGTCCTCAAGA CUCCCAGUGUCGUCCUCAA UUGAGGACGACACUGGGAG
379/380/381 F7 ilh1c.pk004.d17.f AAGTTCCATCCAAAGCACCGCAG
GUUCCAUCCAAAGCACCGC GCGGUGCUUUGGAUGGAAC 382/383/384 G7
ilh1c.pk004.k13.f AAGGAAGTATCGCACCACATTCT GGAAGUAUCGCACCACAUU
AAUGUGGUGCGAUACUUCC 385/386/387 H7 ilh1c.pk004.k13.f
AAATCAAGGACAATGTGGTAGCT AUCAAGGACAAUGUGGUAG CUACCACAUUGUCCUUGAU
388/389/390 A8 ilh1c.pk004.k13.f AAAGTGAAGGTGAAGATTGCGAG
AGUGAAGGUGAAGAUUGCG CGCAAUCUUCACCUUCACU 391/392/393 B8
ilh1c.pk011.a8.f AAGATGTTGTGTTAGGTTTACCG GAUGUUGUGUUAGGUUUAC
GUAAACCUAACACAACAUC 394/395/396 C8 ilh1c.pk011.a8.f
AACAAAGTCGATTCCACCTAGAG CAAAGUCGAUUCCACCUAG CUAGGUGGAAUCGACUUUG
397/398/399 D8 ilh1c.pk011.a8.f AACTTTGGTCCTGGTTTGGATAG
CUUUGGUCCUGGUUUGGAU AUCCAAACCAGGACCAAAG 400/401/402 E8
ilh1c.pk011.d10.f AAGGCGTGTAATCTCTCAAGCAA GGCGUGUAAUCUCUCAAGC
GCUUGAGAGAUUACACGCC 403/404/405 F8 ilh1c.pk011.d10.f
AATTCGCGGTCAACTTTATCTCT UUCGCGGUCAACUUUAUCU AGAUAAAGUUGACCGCGAA
406/407/408 (Note: the sense RNA primer sequence and the antisense
RNA primer sequences shown in table 5 were generated to have 2
thymine residues at the 3' end.)
TABLE-US-00006 TABLE 6 % Response (4 reps) Sample 35 ppm 17.5 ppm
8.25 ppm 4.125 ppm A1 37.5 0 0 25 B1 25 0 0 0 C1 0 0 0 0 D1 12.5 0
0 0 E1 0 0 0 0 F1 12.5 0 25 0 G1 0 0 0 0 H1 0 0 0 0 A2 12.5 0 0 25
B2 25 0 0 0 C2 0 0 0 0 D2 0 25 25 0 E2 0 0 25 0 F2 0 0 0 0 G2 0 0 0
0 H2 0 0 0 25 A3 0 0 0 0 B3 0 0 0 0 C3 12.5 0 0 0 D3 0 0 25 25 E3
75 62.5 12.5 0 F3 0 0 0 0 G3 87.5 25 0 0 H3 100 50 12.5 0 A4 25
12.5 0 0 B4 12.5 0 0 0 C4 12.5 0 0 0 D4 62.5 0 0 0 E4 37.5 25 0 0
F4 0 25 0 0 G4 25 0 0 0 H4 100 37.5 0 0 A5 62.5 37.5 0 0 B5 100 75
12.5 0 C5 100 100 50 37.5 D5 100 100 37.5 12.5 E5 100 87.5 50 12.5
F5 100 50 0 0 G5 100 100 50 0 H5 87.5 50 0 25 A6 100 50 25 0 B6 100
37.5 0 0 C6 0 0 0 0 D6 0 0 0 0 E6 0 12.5 0 0 F6 0 0 0 0 G6 0 0 25
25 H6 0 0 0 0 A7 0 0 0 0 B7 0 0 0 0 C7 0 0 0 25 D7 0 0 0 0 E7 0 0 0
0 F7 0 0 0 0 G7 0 0 0 0 H7 0 0 0 0 A8 0 0 0 0 B8 0 0 0 0 C8 12.5 0
0 0 D8 0 0 25 0 E8 0 0 0 0 F8 0 0 0 0
A confirmation test was run with the 14 most active samples. The
activity was confirmed. See, Table 7.
TABLE-US-00007 TABLE 7 % Activity (5 reps) 37.5 ppm 17.5 ppm 8.25
ppm 4.125 ppm 3E 100 50 30 0 3G 90 40 10 0 3H 90 60 10 0 4H 100 40
10 0 5A 90 40 10 0 5B 100 80 50 0 5C 100 90 50 20 5D 100 90 40 10
5E 100 80 40 0 5F 90 40 0 0 5G 100 70 40 10 5H 90 40 10 0 6A 100 40
0 0 6B 90 40 10 0
[0138] New samples were made of 7 of the most active samples. These
were de-salted or were HPLC purified dsRNAs from the same vender
(Sigma-Genosys, the Woodlands, Tex.). The samples tested were 5C,
5D, 5E, 5F, 5G, 5H, and 6A. Only one of the samples, 5C, showed
activity at the doses tested, and only minor stunting was evident.
See, Table 8. Similarly, the desalted primers also had limited
activity.
TABLE-US-00008 TABLE 8 % Effect HPLC De-Salted 35 ppm 50 30 17.5 20
0 8.75 0 10 4.375 0 0 No Mortality, only minor stunting at 35
ppm
The samples were retested at a higher dose (70 ppm) and again,
sample 5C was found to be the only sample active. See, Table 9.
Mortality occurred with the two top doses (35 and 70 ppm).
TABLE-US-00009 TABLE 9 5C % effect HPLC purified De-Salted 70 ppm
100 70 35 ppm 70 30 17.5 ppm 10 0 8.75 ppm 0 0
Dosages were increased again for the HPLC-purified samples and
activity became evident with all the samples tested. Mortality and
stunting occurred at the top three doses. See, Table 10.
TABLE-US-00010 TABLE 10 % Effected ppm 5C 5D 5E 5F 5G 5H 6A 100 100
75 75 75 100 87.5 37.5 50 50 62.5 37.5 25 50 25 25 25 12.5 12.5
12.5 12.5 12.5 12.5 12.5 12.5 0 0 12.5 0 0 0 0
Example 3
Transformation of Maize
[0139] Immature maize embryos from greenhouse donor plants are
bombarded with a plasmid containing the a silencing element of the
invention is operably linked to a maize Ubi1-5UTR-Ubi1 intron and
the selectable marker gene PAT (Wohlleben et al. (1988) Gene
70:25-37), which confers resistance to the herbicide Bialaphos. In
one embodiment, the construct comprises two identical 2 to 300 bp
segments of a target gene in opposite orientations with an intron
segment between them acting as a hairpin loop. In further
embodiments, the construct is driven off of the dMMV promoter.
Alternatively, the selectable marker gene is provided on a separate
plasmid. Transformation is performed as follows. Media recipes
follow below.
Preparation of Target Tissue
[0140] The ears are husked and surface sterilized in 30% Clorox
bleach plus 0.5% Micro detergent for 20 minutes, and rinsed two
times with sterile water. The immature embryos are excised and
placed embryo axis side down (scutellum side up), 25 embryos per
plate, on 560Y medium for 4 hours and then aligned within the 2.5
cm target zone in preparation for bombardment.
[0141] A plasmid vector comprising the silencing element of
interest operably linked to a maize Ubi1-5UTR-Ubi1 intron is made.
This plasmid DNA plus plasmid DNA containing a PAT selectable
marker is precipitated onto 1.1 .mu.m (average diameter) tungsten
pellets using a CaCl.sub.2 precipitation procedure as follows: 100
.mu.l prepared tungsten particles in water; 10 .mu.l (1 .mu.g) DNA
in Tris EDTA buffer (1 .mu.g total DNA); 100 .mu.l 2.5 M
CaCl.sub.2; and, 10 .mu.l 0.1 M spermidine.
[0142] Each reagent is added sequentially to the tungsten particle
suspension, while maintained on the multitube vortexer. The final
mixture is sonicated briefly and allowed to incubate under constant
vortexing for 10 minutes. After the precipitation period, the tubes
are centrifuged briefly, liquid removed, washed with 500 ml 100%
ethanol, and centrifuged for 30 seconds. Again the liquid is
removed, and 105 .mu.l 100% ethanol is added to the final tungsten
particle pellet. For particle gun bombardment, the tungsten/DNA
particles are briefly sonicated and 10 .mu.l spotted onto the
center of each macrocarrier and allowed to dry about 2 minutes
before bombardment.
[0143] The sample plates are bombarded at level #4 in a particle
gun. All samples receive a single shot at 650 PSI, with a total of
ten aliquots taken from each tube of prepared particles/DNA.
[0144] Following bombardment, the embryos are kept on 560Y medium
for 2 days, then transferred to 560R selection medium containing 3
mg/liter Bialaphos, and subcultured every 2 weeks. After
approximately 10 weeks of selection, selection-resistant callus
clones are transferred to 288J medium to initiate plant
regeneration. Following somatic embryo maturation (2-4 weeks),
well-developed somatic embryos are transferred to medium for
germination and transferred to the lighted culture room.
Approximately 7-10 days later, developing plantlets are transferred
to 272V hormone-free medium in tubes for 7-10 days until plantlets
are well established. Plants are then transferred to inserts in
flats (equivalent to 2.5'' pot) containing potting soil and grown
for 1 week in a growth chamber, subsequently grown an additional
1-2 weeks in the greenhouse, then transferred to classic 600 pots
(1.6 gallon) and grown to maturity. Plants are monitored and scored
for the appropriate marker.
[0145] Bombardment medium (560Y) comprises 4.0 g/l N6 basal salts
(SIGMA C-1416), 1.0 ml/l Eriksson's Vitamin Mix (1000.times.
SIGMA-1511), 0.5 mg/l thiamine HCl, 120.0 g/l sucrose, 1.0 mg/l
2,4-D, and 2.88 g/l L-proline (brought to volume with D-I H.sub.2O
following adjustment to pH 5.8 with KOH); 2.0 g/l Gelrite (added
after bringing to volume with D-I H.sub.2O); and 8.5 mg/l silver
nitrate (added after sterilizing the medium and cooling to room
temperature). Selection medium (560R) comprises 4.0 g/l N6 basal
salts (SIGMA C-1416), 1.0 ml/l Eriksson's Vitamin Mix (1000.times.
SIGMA-1511), 0.5 mg/l thiamine HCl, 30.0 g/l sucrose, and 2.0 mg/l
2,4-D (brought to volume with D-I H.sub.2O following adjustment to
pH 5.8 with KOH); 3.0 g/l Gelrite (added after bringing to volume
with D-I H.sub.2O); and 0.85 mg/l silver nitrate and 3.0 mg/l
bialaphos (both added after sterilizing the medium and cooling to
room temperature).
[0146] Plant regeneration medium (288J) comprises 4.3 g/l MS salts
(GIBCO 11117-074), 5.0 ml/l MS vitamins stock solution (0.100 g
nicotinic acid, 0.02 g/l thiamine HCL, 0.10 g/l pyridoxine HCL, and
0.40 g/l glycine brought to volume with polished D-I H.sub.2O)
(Murashige and Skoog (1962) Physiol. Plant. 15:473), 100 mg/l
myo-inositol, 0.5 mg/l zeatin, 60 g/l sucrose, and 1.0 ml/l of 0.1
mM abscisic acid (brought to volume with polished D-I H.sub.2O
after adjusting to pH 5.6); 3.0 g/l Gelrite (added after bringing
to volume with D-I H.sub.2O); and 1.0 mg/l indoleacetic acid and
3.0 mg/l bialaphos (added after sterilizing the medium and cooling
to 60.degree. C.). Hormone-free medium (272V) comprises 4.3 g/l MS
salts (GIBCO 11117-074), 5.0 ml/l MS vitamins stock solution (0.100
g/l nicotinic acid, 0.02 g/l thiamine HCL, 0.10 g/l pyridoxine HCL,
and 0.40 g/l glycine brought to volume with polished D-I H.sub.2O),
0.1 g/l myo-inositol, and 40.0 g/l sucrose (brought to volume with
polished D-I H.sub.2O after adjusting pH to 5.6); and 6 g/l
bacto-agar (added after bringing to volume with polished D-I
H.sub.2O), sterilized and cooled to 60.degree. C.
[0147] To assay for insecticidal activity, a FAW feeding assay can
be performed. Briefly, leaf discs from the transgenic plant are
excised using a 1 cm cork borer or leaf punch. Six leaf discs are
prepared for each plant. The leaves are placed in a 24 well
microtiter plate on top of 500 ul of 0.8% agar. Each leaf disc is
infested with 2 neonate Fall armyworm and the plate is then sealed
with mylar. A small ventilation hole is made for each well and the
plates are then stored in a 28C growth chamber. The assay is scored
for mortality, stunting, and leaf consumption at 96 hours.
Example 4
Agrobacterium-Mediated Transformation of Maize
[0148] For Agrobacterium-mediated transformation of maize with a
silencing element of the invention, the method of Zhao is employed
(U.S. Pat. No. 5,981,840, and PCT patent publication WO98/32326;
the contents of which are hereby incorporated by reference).
Briefly, immature embryos are isolated from maize and the embryos
contacted with a suspension of Agrobacterium, where the bacteria
are capable of transferring the polynucleotide comprising the
silencing element to at least one cell of at least one of the
immature embryos (step 1: the infection step). In this step the
immature embryos are immersed in an Agrobacterium suspension for
the initiation of inoculation. The embryos are co-cultured for a
time with the Agrobacterium (step 2: the co-cultivation step). The
immature embryos are cultured on solid medium following the
infection step. Following this co-cultivation period an optional
"resting" step is contemplated. In this resting step, the embryos
are incubated in the presence of at least one antibiotic known to
inhibit the growth of Agrobacterium without the addition of a
selective agent for plant transformants (step 3: resting step). The
immature embryos are cultured on solid medium with antibiotic, but
without a selecting agent, for elimination of Agrobacterium and for
a resting phase for the infected cells. Next, inoculated embryos
are cultured on medium containing a selective agent and growing
transformed callus is recovered (step 4: the selection step). The
immature embryos are cultured on solid medium with a selective
agent resulting in the selective growth of transformed cells. The
callus is then regenerated into plants (step 5: the regeneration
step), and calli grown on selective medium are cultured on solid
medium to regenerate the plants.
Example 5
Soybean Embryo Transformation
Culture Conditions
[0149] Soybean embryogenic suspension cultures (cv. Jack) are
maintained in 35 ml liquid medium SB196 (see recipes below) on
rotary shaker, 150 rpm, 26.degree. C. with cool white fluorescent
lights on 16:8 hr day/night photoperiod at light intensity of 60-85
.mu.E/m2/s. Cultures are subcultured every 7 days to two weeks by
inoculating approximately 35 mg of tissue into 35 ml of fresh
liquid SB196 (the preferred subculture interval is every 7
days).
[0150] Soybean embryogenic suspension cultures are transformed with
the plasmids and DNA fragments described in the following examples
by the method of particle gun bombardment (Klein et al. (1987)
Nature, 327:70).
Soybean Embryogenic Suspension Culture Initiation
[0151] Soybean cultures are initiated twice each month with 5-7
days between each initiation.
[0152] Pods with immature seeds from available soybean plants 45-55
days after planting are picked, removed from their shells and
placed into a sterilized magenta box. The soybean seeds are
sterilized by shaking them for 15 minutes in a 5% Clorox solution
with 1 drop of ivory soap (95 ml of autoclaved distilled water plus
5 ml Clorox and 1 drop of soap). Mix well. Seeds are rinsed using 2
1-liter bottles of sterile distilled water and those less than 4 mm
are placed on individual microscope slides. The small end of the
seed are cut and the cotyledons pressed out of the seed coat.
Cotyledons are transferred to plates containing SB1 medium (25-30
cotyledons per plate). Plates are wrapped with fiber tape and
stored for 8 weeks. After this time secondary embryos are cut and
placed into SB 196 liquid media for 7 days.
Preparation of DNA for Bombardment
[0153] Either an intact plasmid or a DNA plasmid fragment
containing the silencing element, such as those described in
Example 3, and the selectable marker gene are used for bombardment.
Plasmid DNA for bombardment are routinely prepared and purified
using the method described in the Promega.TM. Protocols and
Applications Guide, Second Edition (page 106). Fragments of the
plasmids carrying the silencing element of interest are obtained by
gel isolation of double digested plasmids. In each case, 100 ug of
plasmid DNA is digested in 0.5 ml of the specific enzyme mix that
is appropriate for the plasmid of interest. The resulting DNA
fragments are separated by gel electrophoresis on 1% SeaPlaque GTG
agarose (BioWhitaker Molecular Applications) and the DNA fragments
containing silencing element of interest are cut from the agarose
gel. DNA is purified from the agarose using the GELase digesting
enzyme following the manufacturer's protocol.
[0154] A 50 .mu.l aliquot of sterile distilled water containing 3
mg of gold particles (3 mg gold) is added to 5 .mu.l of a 1
.mu.g/.mu.l DNA solution (either intact plasmid or DNA fragment
prepared as described above), 50 .mu.l 2.5M CaCl.sub.2 and 20 .mu.l
of 0.1 M spermidine. The mixture is shaken 3 min on level 3 of a
vortex shaker and spun for 10 sec in a bench microfuge. After a
wash with 400 .mu.l 100% ethanol the pellet is suspended by
sonication in 40 .mu.l of 100% ethanol. Five .mu.l of DNA
suspension is dispensed to each flying disk of the Biolistic
PDS1000/HE instrument disk. Each 5 .mu.l aliquot contains
approximately 0.375 mg gold per bombardment (i.e. per disk).
Tissue Preparation and Bombardment with DNA
[0155] Approximately 150-200 mg of 7 day old embryonic suspension
cultures are placed in an empty, sterile 60.times.15 mm petri dish
and the dish covered with plastic mesh. Tissue is bombarded 1 or 2
shots per plate with membrane rupture pressure set at 1100 PSI and
the chamber evacuated to a vacuum of 27-28 inches of mercury.
Tissue is placed approximately 3.5 inches from the
retaining/stopping screen.
Selection of Transformed Embryos
[0156] Transformed embryos were selected either using hygromycin
(when the hygromycin phosphotransferase, HPT, gene was used as the
selectable marker) or chlorsulfuron (when the acetolactate
synthase, ALS, gene was used as the selectable marker).
Hygromycin (HPT) Selection
[0157] Following bombardment, the tissue is placed into fresh SB196
media and cultured as described above. Six days post-bombardment,
the SB196 is exchanged with fresh SB196 containing a selection
agent of 30 mg/L hygromycin. The selection media is refreshed
weekly. Four to six weeks post selection, green, transformed tissue
may be observed growing from untransformed, necrotic embryogenic
clusters. Isolated, green tissue is removed and inoculated into
multiwell plates to generate new, clonally propagated, transformed
embryogenic suspension cultures.
Chlorsulfuron (ALS) Selection
[0158] Following bombardment, the tissue is divided between 2
flasks with fresh SB196 media and cultured as described above. Six
to seven days post-bombardment, the SB196 is exchanged with fresh
SB196 containing selection agent of 100 ng/ml Chlorsulfuron. The
selection media is refreshed weekly. Four to six weeks post
selection, green, transformed tissue may be observed growing from
untransformed, necrotic embryogenic clusters. Isolated, green
tissue is removed and inoculated into multiwell plates containing
SB196 to generate new, clonally propagated, transformed embryogenic
suspension cultures.
Regeneration of Soybean Somatic Embryos into Plants
[0159] In order to obtain whole plants from embryogenic suspension
cultures, the tissue must be regenerated.
Embryo Maturation
[0160] Embryos are cultured for 4-6 weeks at 26.degree. C. in SB196
under cool white fluorescent (Phillips cool white Econowatt
F40/CW/RS/EW) and Agro (Phillips F40 Agro) bulbs (40 watt) on a
16:8 hr photoperiod with light intensity of 90-120 uE/m2s. After
this time embryo clusters are removed to a solid agar media, SB
166, for 1-2 weeks. Clusters are then subcultured to medium SB103
for 3 weeks. During this period, individual embryos can be removed
from the clusters and screened for the appropriate marker or the
ability of the plant, when ingested by Lygus, to control the
Lygus.
Embryo Desiccation and Germination
[0161] Matured individual embryos are desiccated by placing them
into an empty, small petri dish (35.times.10 mm) for approximately
4-7 days. The plates are sealed with fiber tape (creating a small
humidity chamber). Desiccated embryos are planted into SB71-4
medium where they were left to germinate under the same culture
conditions described above. Germinated plantlets are removed from
germination medium and rinsed thoroughly with water and then
planted in Redi-Earth in 24-cell pack tray, covered with clear
plastic dome. After 2 weeks the dome is removed and plants hardened
off for a further week. If plantlets looked hardy they are
transplanted to 10'' pot of Redi-Earth with up to 3 plantlets per
pot. After 10 to 16 weeks, mature seeds are harvested, chipped and
analyzed for proteins
Media Recipes
TABLE-US-00011 [0162] SB 196 - FN Lite liquid proliferation medium
(per liter) - MS FeEDTA - 100x Stock 1 10 ml MS Sulfate - 100x
Stock 2 10 ml FN Lite Halides - 100x Stock 3 10 ml FN Lite P, B, Mo
- 100x Stock 4 10 ml B5 vitamins (1 ml/L) 1.0 ml 2,4-D (10 mg/L
final concentration) 1.0 ml KNO3 2.83 gm (NH4)2SO4 0.463 gm
Asparagine 1.0 gm Sucrose (1%) 10 gm pH 5.8
FN Lite Stock Solutions
TABLE-US-00012 [0163] Stock # 1000 ml 500 ml 1 MS Fe EDTA 100x
Stock Na.sub.2 EDTA* 3.724 g 1.862 g FeSO.sub.4--7H.sub.2O 2.784 g
1.392 g 2 MS Sulfate 100x stock MgSO.sub.4--7H.sub.2O 37.0 g 18.5 g
MnSO.sub.4--H.sub.2O 1.69 g 0.845 g ZnSO.sub.4--7H.sub.2O 0.86 g
0.43 g CuSO.sub.4--5H.sub.2O 0.0025 g 0.00125 g 3 FN Lite Halides
100x Stock CaCl.sub.2--2H.sub.2O 30.0 g 15.0 g KI 0.083 g 0.0715 g
CoCl.sub.2--6H.sub.2O 0.0025 g 0.00125 g 4 FN Lite P, B, Mo 100x
Stock KH.sub.2PO.sub.4 18. g 9.25 g H.sub.3BO.sub.3 0.62 g 0.31 g
Na.sub.2MoO.sub.4--2H.sub.2O 0.025 g 0.0125 g *Add first, dissolve
in dark bottle while stirring
[0164] SB1 solid medium (per liter) comprises: 1 pkg. MS salts
(Gibco/BRL--Cat#11117-066); 1 ml B5 vitamins 1000.times. stock;
31.5 g sucrose; 2 ml 2,4-D (20 mg/L final concentration); pH 5.7;
and, 8 g TC agar.
[0165] SB 166 solid medium (per liter) comprises: 1 pkg. MS salts
(Gibco/BRL--Cat#11117-066); 1 ml B5 vitamins 1000.times. stock; 60
g maltose; 750 mg MgCl2 hexahydrate; 5 g activated charcoal; pH
5.7; and, 2 g gelrite.
[0166] SB 103 solid medium (per liter) comprises: 1 pkg. MS salts
(Gibco/BRL--Cat#11117-066); 1 ml B5 vitamins 1000.times. stock; 60
g maltose; 750 mg MgCl2 hexahydrate; pH 5.7; and, 2 g gelrite.
[0167] SB 71-4 solid medium (per liter) comprises: 1 bottle
Gamborg's B5 salts w/sucrose (Gibco/BRL--Cat#21153-036); pH 5.7;
and, 5 g TC agar.
[0168] 2,4-D stock is obtained premade from Phytotech cat# D
295--concentration is 1 mg/ml.
[0169] B5 Vitamins Stock (per 100 ml) which is stored in aliquots
at -20 C comprises: 10 g myo-inositol; 100 mg nicotinic acid; 100
mg pyridoxine HCl; and, 1 g thiamine. If the solution does not
dissolve quickly enough, apply a low level of heat via the hot stir
plate. Chlorsulfuron Stock comprises 1 mg/ml in 0.01 N Ammonium
Hydroxide
[0170] The article "a" and "an" are used herein to refer to one or
more than one (i.e., to at least one) of the grammatical object of
the article. By way of example, "an element" means one or more
element.
[0171] All publications and patent applications mentioned in the
specification are indicative of the level of those skilled in the
art to which this invention pertains. All publications and patent
applications are herein incorporated by reference to the same
extent as if each individual publication or patent application was
specifically and individually indicated to be incorporated by
reference.
[0172] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it will be obvious that certain changes and
modifications may be practiced within the scope of the appended
claims.
Sequence CWU 1
1
4081643DNALygus hesperus 1gcgtcacttg cgactcgtgt ctgagcggca
acttcaaggg aaagaggtac aaatgtcttt 60cctgctacga ctacgatctg tgcaccaact
gctacgagtg tggcctcatc acgggactcc 120actcagcaga gcatcccatg
cagtgcatca tcaccagaca tgacgtcgac ctgtacttcg 180ggggagacat
gaatggcgac ggaagtcagt cctacacgtg tcctcattgt ggtctaatgg
240ggttcagttt gtcgctgttg atcgagcacg tgagcggtga gcatatcgcg
ctgagcaacg 300ctgaagtgat ttgccctgtt tgcgccgcca cgccagtcaa
ccgaccgaac aacgttcgcc 360aggatttttt ggggcacctg acgctggagc
atcgctaccc ctcgcgagag ctgaccgcct 420tcttcgaaga gccctcgtcc
cgacacatgc cgagtggcgt ccgccggatt ccaccgccac 480cagggcgcag
cgctgccggg cgtggacgcc gggtcgcacg ttcatttcgg ctcctcaggc
540gctcttactg gactcacatc ctccagagaa agtccggatc ccatcgccga
gttcttctct 600cagctgtctg gagtcgctcg tcctcaaggt cctgggccga ttc
6432575DNALygus hesperusmisc_feature81, 426n = A,T,C or G
2accccctgat cgccgggatc gttgccaatc agtatcacgc tcaggatgtg ctcggacagt
60acacttacgg ctactccgga nccccatccg ctaaacaaga ggttaagact gctgacggaa
120taacgagagg atcttactcg tacatcgatg gaaacgggtc tcgtacagag
cgcttcttac 180gttgctgacc ccgtcaacgg gttcagagtc cacgccacca
acctgcccgt gggacctgac 240gggtccgtcg ctgccgctcc cgtcgccagg
ctcctcagcc ctttggccat cagccctgtg 300atcaacctcc acggcgctgc
tcccctcaac cccgacggca ccgtcgctga cacccatgaa 360gtcgctgtcg
ccaaggccgc tcacctcgct gccatcaacg aggctagggc tagaggcaag
420aggtcngccc cgctcaaccc cgacggtacc gtcgctgaca cccccgaagt
tgctgctgct 480aagtgggctc atctcgctga gatctccaaa gctcgtggaa
tccctctcgt atacgctccc 540agatggtggg gacctggtgc tccattgaac gctga
5753722DNALygus hesperusmisc_feature659, 686, 689, 695, 713, 720,
722n = A,T,C or G 3ctacgctggc ggcccctccg ccaaggaaga gatcaagacc
gccgacggaa ttacccgcgg 60aggatactca tacatcgacg ccaacggtat cgtccagagc
gcctcttacg tgtcggatcc 120cgtcaacgga ttccgagtag ccgccactaa
cctccccgct ggacctgcag tcccagctgg 180accttcagtg gttgctgctg
ctccagctgt cgttgctgct cctgctccag ttttggctgc 240tgcccctgct
ccaattgtgg cttcagctcc cgtttgggct gctcaaccag ctgttgttgc
300cgctccagct cctgtcgctg tcgccgaagg ccccgcagtg accgccacca
acgtccagga 360agttgctgcc gctgctgctg acgtccccgt tgctgctgat
ctccccgaga tcatcgctgc 420ccgctctctg cccaccgtgg ttgccaccag
ggccgccatc gctcaccccc ttgccgcaac 480ctcctggtcc ggcatcgtcc
accacctgaa gaagcgttcc cttgccgccg ctaccgtcgt 540cactcccctt
actagttacc ccggatctac cgctcccttg gttcacgctt ctcccgttat
600tgcggctaca cctgttatct ccgctcactc gggtttgatc gccactgact
ctcttgtanc 660ctcaccacac ctcgttggtg cagtangtnc tgtcnagccc
ctccacaccg ccntcctcan 720an 7224700DNALygus
hesperusmisc_feature675, 700n = A,T,C or G 4cattcgggag acattgcaaa
actggatgca taggggaagt ctttaggacg gatttacggc 60atacagtaca tgttactgta
agacacggcg cgtgactgga cagcaagcag aatggaggag 120ggacaagtgt
accaatcatc cgaccaaacc aactacgttt acgatgaagt gtttcctgcg
180ctacctgaat cagccaaccc ggcacctcac aacgacatca aaatttgcaa
caacaaaatg 240cgtgttggct catctgtcat cactcaggtt ttccgagtgc
cagcagacga gcgccgctac 300gatcacaaca acagctttgg ggaaaaggaa
tccgtgagga cctgctctgc catcatgaag 360gaaactggag cagtcattga
gatcgccacg agcaaggata tgtctctgac tttcttggtg 420actggaaaga
ccgactcagt gatggatgct cgaaggaaga tactgagcaa ttttcagacc
480caagcttcat ccaagctctc tattccgaaa gagcatcaca ggtggatcct
tggaaaagct 540ggtggtcgtc tgaaggattt ggaaaaatca acagccacca
aaatctccgt ccctggcata 600aatgaacact cggatgaaat cactgtgacg
ggaactcgtg aagggatcga caaggccatc 660catgaaatgc aagtnatttc
ggacgaacaa tccaagaagn 7005576DNALygus hesperusmisc_feature82, 427n
= A,T,C or G 5aaccccctga tcgccgggat cgttgccaat cagtatcacg
ctcaggatgt gctcggacag 60tacacttacg gctactccgg anccccatcc gctaaacaag
aggttaagac tgctgacgga 120ataacgagag gatcttactc gtacatcgat
ggaaacgggt ctcgtacaga gcgcttctta 180cgttgctgac cccgtcaacg
ggttcagagt ccacgccacc aacctgcccg tgggacctga 240cgggtccgtc
gctgccgctc ccgtcgccag gctcctcagc cctttggcca tcagccctgt
300gatcaacctc cacggcgctg ctcccctcaa ccccgacggc accgtcgctg
acacccatga 360agtcgctgtc gccaaggccg ctcacctcgc tgccatcaac
gaggctaggg ctagaggcaa 420gaggtcngcc ccgctcaacc ccgacggtac
cgtcgctgac acccccgaag ttgctgctgc 480taagtgggct catctcgctg
agatctccaa agctcgtgga atccctctcg tatacgctcc 540cagatggtgg
ggacctggtg ctccattgaa cgctga 5766645DNALygus
hesperusmisc_feature612n = A,T,C or G 6ccaacatgta tctctcagtt
gttggattgg tgatggcttc cgctgctttc gtcagctgcg 60agccatcgaa tttcgcgtgt
actggcgagt cgaactacaa gtatcctgtg gagggctcgt 120gccacaacta
ctaccagtgc gaaaagggct ccactacgcc ttcaattcga gactgctcgc
180tgccgctgct tcgatttcgg gatttcgatc cagtcaaatt ggagtgtgac
tggtgctggc 240gggtagactg ttcagccaaa cccgcacccc caccgactcc
atcgccgact ccggcgccaa 300cttcaaggcc tactgctgcg ccgactactg
gaccaacctc agcgcccact actggaccca 360cagcggcgcc aacctcagcg
cccactgctg caccaacctc agctcccact gctgctccaa 420ctccagcgcc
cactgcgccg ccaactccag cgcccactgc ggcgccaact ccagcgccca
480ctgcagcacc aacctcagct cccactgctg ctccaactcc agcgcctact
gctgctccaa 540ctccagcgcc tactgctgcg ccaacatcag cgccctctac
tggacccact gtcgccccaa 600ctcgcaggcc anctcaagaa cccacaatgg
caaaaaaatc ttcga 6457699DNALygus hesperusmisc_feature699n = A,T,C
or G 7caaataagaa acatgaagat agtaccgttc ttagttcttc tacttgttca
aacggttctt 60tccgaaacga cgccggcttc aaacaatcgc cgtattgttt gctaccacac
aagttggagt 120gcgtatcgtg tcccagaggc aaaatttaca gcgaagaaca
tcaacccgta cctttgcact 180catttaatat attcgtttgc caatgtattg
gtaaatgaag caaccatcgt tcctggtgat 240gcatggcagg atattgataa
ccatcagttc agagattttg ttgagttgaa aaccacattc 300aacgaaaacc
tgaaaacgtt actcgcaata ggaggctaca gagaagggtc gtcgaagttc
360acccctatcg cagccacccc cacgaaaagg gcagcgtttg ctcgcaacac
gctcaagttt 420ttgaaaactt acggttttga cgggctcaac atcgattggc
agttccctaa cgatcagcat 480agaaatggca gtgttgaaga ctataagaac
tttgtgtatt tgctgcaaga tatcgacaaa 540gtcttcagag aggaagctgc
agcttccggg aaacctaaaa tgatgttgac catttccgtt 600ccgggtaata
cgctgctaat agaaagtggc tatgatctac caaatctagc gaagtatgta
660gagttcatga acgtcctgag ctacgattac cactttgcn 6998707DNALygus
hesperusmisc_feature707n = A,T,C or G 8tccagttctt taacgaggta
accatgtaca agactatctt acctgagttg ggagccttgg 60atttgggttt gtgcccgaag
atgttccatg gagaggcaca taatggtaaa aatcctgaac 120aagacatcgt
ggttattgaa gatttgtgtc ctcaaggtta caaagtgccg gaaaagttgt
180ttttggacgc tgatcactta gtgatggcca tgaaaaaaat agggcaactt
cacggattat 240cttataaaat gaaagtatcg tctccagaga ggttgttcga
attgaggaat atgctgatcc 300cgaaggtgat tgacgattcg aaaggtctca
atgatgcttg tctggccagg ggtttcaaac 360ccttggtgga gtcgagcccc
agttacagtg tagtgaataa agtttacaag aaactcgtcg 420tagcggatgc
cgtggatgtt gcctatagtt tacagaagcc tgaagaacca ttcgccgtta
480tcacccacgg tgatttcaat ggtaataaca tattgtataa gtatgatgcc
agtggaaatg 540tagtggatat gaaaatgata gattttggtt tcgcttctta
tttggatcct gctgttgaca 600tagctttctt cctgtacatg aactcttctc
ctgaaactag gaagctgcac tgggattcat 660tcctgaatgc atactgggag
ggagtcatct ctgttgctgg tgatccn 7079275DNALygus
hesperusmisc_feature243, 249, 251, 252, 273n = A,T,C or G
9cgtgtgctga tcattcgata tcccggaaac gtgtttactt tcctttattg tgcataaata
60cacttccgtg gcggttcgcg atgtcgaata caaaagtcgc gtcttccggt caacctaagc
120tctcctccca agatctttcg actcttgacg tgacggcgct taccccattg
tcgccagaag 180tcatcagtag acaagcgacc atcaatatcg gtactattgg
tcacgtggca catgggaaag 240tcnactgtng nngaaagctg tgtctggtgt tcnaa
27510642DNALygus hesperusmisc_feature615, 616n = A,T,C or G
10tttttggttt tcattgaaaa tttcgataat tttccaaagt tttattatgg ttcaaattca
60aaatgttttc tactgatttt gattctcaca gtataatttc gacgtggaag tgttaagggc
120tcagtgatcg atggcaggga aagctttttg ataactattg gtaagtccga
gtttgtaaga 180tgacctcttt tcaaaagtgt gtaggatttg gtcggatatt
tcctttaggg attctcagaa 240atcacaatgt tgcatctcga aggtgcattc
acggctcttc cgttttgtac aaaaagagga 300aaaccagaga ggagaggaag
ttgcccaaag ctattgttta ctctcccaaa tcgaaatgga 360aacaaggcga
gcccgttgac gtgtggaagc gtatgacagt ggcggaggta gcaaatacac
420tgggtaaaga tgtaggacac gttttagaag ttatgtcgtt cattgacaac
acggaacagt 480acagaaaaga ccgtgatgtc atcgacaact tcaaagttat
agaagaaata gtgaaaaagt 540cgggtcatcg atgtaggatg gctagtaagc
ctacagaaac tgaagaaaaa agttttaaag 600atgttggacg aaganncctt
cggattacgt tgatcccagg cc 64211721DNALygus hesperusmisc_feature720n
= A,T,C or G 11tgcccgagtg cgtgtttcgt caaatagaaa cccgggtctt
ttctgtaaga attaaatcgc 60aatggctcct cctttctacg ctgatctagg taagaacgcc
cgcgatgtct tcggtaaagg 120gtaccatttc ggactcctga agctcgacgt
caagaccaag actaacacgg gcgtcgaatt 180cagcatcggc ggcgttcaaa
acctcgaaac caaaaacgta gtcggctctc tcgagaccaa 240gtacaaattc
aaggagtatg gcgttacttt cacggagaaa tggaacactg acaacgtact
300ggccactgaa atcgccgttg ctgatttctg cgatggagca aaaatgtccc
ttgacacctc 360ttttatccct cacaagggtg ataagaccct gcgattgaag
ggcgaattca agaatgacac 420ctgcgccatg aaccttgaaa gcgacttcaa
gtctggcgga cctctcgtcc gaggtggcgc 480tgtcctcggc tacggaggct
ggctatgtgg ttacgccacg gccttcgacg ttagcaagag 540taaactcacc
gaaaacaaag tcaccatggg attcatcaca aaagatttca tcttgaacac
600cgttatcaat gacggaagag tcttctctgg ttccatctac cacaaagtta
acagcaagtt 660ggaaactgga gtccagatct cgtgggcctc tgataacaac
agcaccgact tcggcatcgn 720c 72112724DNALygus
hesperusmisc_feature665, 723, 724n = A,T,C or G 12attctgcttg
ggttttttat ttagttgaac agttttccgt ggactcttat gacgataact 60tcctcattcc
aaatcttctt cgggactatc catcatttta atcagaagta gaagccgact
120attctaaaaa ccacctatgg ggcctccatt cttcgcggat ctcggtaaaa
actcgagaga 180catcttcaat aaaggttata atttcgggct gcttaagttg
gacataaaaa ccagaacaga 240aaccggagtt gagttcgaaa tcggtggagt
ccagaacctt gagacgaaaa atgtagccgg 300ctcgctcgag actaagtaca
aattcaagga cttcgggatc agcttttcgg agaaatggaa 360tacggataat
gttcttcagc tagaagtagc tgctgctgat atctgcgaag gagtcaaaat
420gtcctgcatg agtatcatga ctccttcttc agatgaggag aaaggtggca
ctgacaaaat 480tttgagattc aagagtgaat ataagaatgc tatcatggct
gtgaacttgg agagcgattt 540caaagctggt ggtccgacct tgggagtctc
tggcgttttt ggattaggtg gatggttgct 600cggagctata gcggcattag
atactgagac ttcgaaagtg atgaccttct ctttgggaat 660gggantttta
accaaagact tcatactaaa caccgctgtt atcaacaagg gaacagactt 720cann
72413692DNALygus hesperus 13cgaaagcaaa caggacatca tatttcgcag
gaataaattg gaagatccca tcacacgaga 60aagcaaaccg gacgtcatat ttcgcagggt
gcgaggtatc ctcaacaagc tcactcctga 120gaaattcgat aagctaagcg
atgacctctt gaaagaagaa tttaattctg atgtcattct 180caaaggcgtc
attctattgg tgtttgaaaa agcactagat gagccgaagt acagtgctat
240gtatgctcag ctctgccggc gactttgtga agagatccga agtgccgacc
agcctgaacc 300ctgccctttt cgccatttgc ttctgtccag ctgcaaagct
cagtttgaga gccgttcgaa 360gcacactagc agcaagcgga aatcgctcgg
gaacataaag ttcatcggag agctttgcaa 420acttggaatc cttcagcgcg
acatcttgta caggtgtttg atccaacttc tcgaacacaa 480gaccaagacg
cctgacgaaa tggccgaaga tcttgagtgc gtctgtcaga tcctccgcac
540ttgcggccac atcttggaca acgaggaagc tcagaagctg atgaatcaac
tttttgatcg 600tatggcgtcc ctctccaaga acgtcaacct gccgatccgg
atccgcttca tgctccgtga 660cattatcgag ctccggaggg ataactgggt tc
69214572DNALygus hesperusmisc_feature12, 410, 564n = A,T,C or G
14ctaactttcc tnttccgcgt tgttgcgttc ctgtgaaatt tcactaaaat tgtgattatt
60ttattgtact cagaactata cctacttcgt atttcgattt gaatacattc caagggcttt
120cgcatgactc aaactttctt ctagaagtgg tttgttgcga cgtgttgagt
tcaatagtgt 180ggtattcaca accggtttcg cccattgggc catcaacgag
tattttccag gtgatgatct 240attgatatgg ggggcgtagc cgcttcaagt
cctttgccag acgaagaacc ccgacctgaa 300gctccaggca gcaggaaggc
agacgaagta cctgctgaga gtggtcagaa ccccgcagga 360caacctcacc
cagatggcgc caaggaagaa gagaacggag ataacgaggn aaaggcctgg
420ccttataaaa gccgatggat ctataaattg ggattgccca tgtttgggag
ggatggcaca 480tgggccctgt ggcgatgagt tcagagctgc tttctcctgt
ttccactatt ccactgctga 540acaaaaaggc tccgactgtt tggnaaccgt tc
57215730DNALygus hesperusmisc_feature332n = A,T,C or G 15cacaaaaagc
tccatacagg tgaacgccct ttcaaatgcg cacactgcgt tcggactttc 60agccggaaag
agcacttagt acggcatgcc cactctcata caggacaaaa actcttcaac
120tgcgacgtct gcgggaaaag cttcagtcgg aaagacaacg tacggaaaca
ccggaaaacg 180catgaaacga caggtccgta ctcttgcgag ttctgcggta
tgcagttcaa cgttcggccg 240tactatataa tgcacaaaaa caagcacaaa
gacgggtcgt gcgtccttga agtgaagaag 300gttgatgttg aggagtctat
cacgtacgaa gntcaggaag agtctccaga tgttcattcg 360aacgaatcca
attccttcca acaggtaaca tctagcacat ccacttcaat actggaaaaa
420gcgttgacgc aagaaggctg aactttggac ttcttgaatt aactttaggc
caaactatta 480cagagttgac aagtatggag tgtgctcaga ggattagttg
gtggaagtaa ctagtccaga 540agctattcag aattaagaac tagaattgaa
tgcaacagca atcagtttgc cctttcagtt 600tgtggtttgt ttttctgttg
gaaactatct ctcgggcatg aataaggaga atgtgtacca 660agtatttcag
tcttcctctg ctctgtgatg taactctgtg cttctttcct atactcgcgt
720tggtaatcaa 73016568DNALygus hesperusmisc_feature165, 166, 523n =
A,T,C or G 16aagaattgta aatcaatatc aaaatggaga tgatgaagtc agatgtcgac
tgaaaagaat 60gtttggattc caagagatcc aaaagctcat taaatataac gtaaatactg
tgctgtccat 120ggactggctt gaccgtaatg ccaaaaactg tccgaaatgc
aacgnnccca ttcagaaaat 180cagtggatgt aaccatatgg tatgctggaa
atgcaaaaca tctttttgct ggcactgcct 240atgctttacg tgcataggat
agtaaggacg tgagatgatc ctgtagttac agctctcttg 300ccactgacta
cctagaaata tcgcactagt catagtcagc caccctcctc tacctcgcca
360ttatctcatt tgggctgtga caaacacaaa cctcgtgtat atctcgtata
cattactatg 420taccttgttt gcgctgtgac atctcaggaa ccccttgata
tgaaaattta agtggtaaaa 480aaactttttt acgattccga aaaaaaaaaa
aaaaaaaaac ccnactttct tgtacaaagt 540tggcattata agaaagcatt gcttatca
56817714DNALygus hesperusmisc_feature360, 361, 362, 363n = A,T,C or
G 17gcctctcttc aagtttttgc ggtgtcggaa tgtcaaatta aataattgtt
ccgatcaaat 60taatatgtct agcatacgat ttcttagaaa attcagtgga tttagaatca
gtggtttcct 120agtagtgggg cagtgtggtg cgcagaaaat ttgcagctta
ggtcatttga aatctcaaga 180gaactctagt ttactaagat ttaccggtgt
tagtacgaga aatttccatt tgggagtgcc 240atctctcgcc aagaaagact
actacgagat cttgggcatc tctagaaacg cgtcggtcaa 300ggaagtgaaa
aaagcgtact atcagctggc caagaaatac catccagaca cgaataaaan
360nnntccgaac gccgccaaga agtttcaaga agtatcagaa gcctatgagg
tactgagtga 420cgacaccaag aggaaacaat atgatcaatg gggtacgacg
tcggagcaga tgggccgaga 480aggtgctggt acaggtccag gtaacatggg
cggcttcaac tggcagtacc gggcttccgt 540ggaccctcag gagctcttca
ggaagatctt tggagacgct gcaggcggat tttccaccgg 600attcgacgat
ttcgctgagt ctagattcgg tcacggtgct gccgaagaaa ttctaatgaa
660actcaccttc tctcaagccg tgcggggagt gagcaaagaa atatacgtta atgt
71418716DNALygus hesperusmisc_feature10, 614, 708n = A,T,C or G
18gatgatcacn tttacggcta atactcgtag atctccagtt tcgatacaaa tttgaattca
60acgagataac cgaatgaaat gacttggaaa tcagcttaaa agcagtgaac tcagcggtag
120aggggaaaat gtctcactcc gactcaagaa cagggaaaac ctccagaagt
acgaatgagt 180cgaaatcagg agcctcgggg cgacagaaaa cttcaagaac
tgagccgaaa accccgaaaa 240ctgagtcaaa aacctcgaag tcagcgtcga
aaacctcgaa gtcagaaggg aagtctgtgt 300tgtctgaaag tagagataaa
agtaataaat tttcaaaaag cgaatctgag tcgtatcgca 360agtccgatgc
gaggggacag cgggacgagg caccaggacc gtcagacagc agaacaggaa
420aagacgtcac tggggatagg aaaactaaga aacagaaaag tgagaaaggg
gtcgacggat 480ctactggaga atcgaagaaa ctcccagtgt cgtcctcaag
aacgtcggaa gcgccgcgga 540acatccgaga tctcttgagg aggatcaacg
aggagaacga atctcagcca acaccttctt 600ctcgtctgaa agancccaag
ccggagagga cgaagagtaa agttccatcc aaagcaccgc 660aggcgagtat
tccagatagg gacgtggtac gagcaaaagc tgcggaancg gccttg 71619718DNALygus
hesperusmisc_feature717, 718n = A,T,C or G 19gttcaccaag gaagtatcgc
accacattct ctgccaaact acagcaggat cccaaaccac 60cggaatgaac accattttgg
ctctcgcaag tctgttgggc tgctgtctgg cggcgtccgt 120tccggattcg
aagtgggatt ctttcaaggc caaatacgga aaaacgtacg acgacccaaa
180agtcgatagt gagagacgta acaactacgg aaaaacgcta gagatgatca
aggctcacaa 240cgcactctat ggacagggcc gggtgtccta ctacctggca
gagaaccatc ttgcagactt 300gtcgtccagt gaacgaatga agttaagagg
attcagaaaa tccgaaagtc aatcgggcgg 360cagaatccac cagcacactg
gattgggccg acccgattcc gtcgattggc gaaacaaaag 420cgttgtgacc
agcgtcaaaa atcaaggaca atgtggtagc tgctgggctt tcagtgcgac
480tgcagcagtg gaatcgcaat acgctatcaa aaccgggcaa ttagtggatc
tcagcgagca 540gcaggtagtg gactgtgacc gtaatggtca cgcttgcaag
tatggtgaca accttgacgc 600gttagggtat atcgaggaag aaggtcagga
gcttctttcc tcttatccct acattgctga 660gccagagact tgtcaatacg
cagcagataa agtgaaggtg aagattgcga gtttccnn 71820638DNALygus
hesperusmisc_feature629n = A,T,C or G 20gtcttaagtg atgaagatgt
tgtgttaggt ttaccgggcg tgccaggata ccatgctatg 60gaaatggcaa cgtctgaagg
ttttcctttc acagcaagtc gaccacaagg aagttccaat 120aagcggtggt
tgtttaacat caatgagaat gccgagaaga gatccttaat cgccatggac
180cccttattgg tgaaagtgtt agaatcgaag agggttcaga gagatcgagg
gttgattccg 240tgtaccgttt ttgtagactg cttgaaggat tcacgaatag
cgaatgaatc ttacctcaca 300cccggtaaga ctaggatctt ctctatctca
ccggttgact ttacgattga gtttcggaag 360tatttccttg atatcctagc
ggcgcaacaa caaagtcgat tccacctaga gcatatggta 420ggtatgaatg
ttcattcgct tgagtggact ttactagccc gccgtatcca atctgtgggt
480tctgcagtga tctgtggtga ttactcgaac tttggtcctg gtttggatag
cgaagttgtt 540gcagctgttg gggacgtttg ggctgattgg tatgagtttt
acgagaccgc tcagggcgtc 600tcggaagagg agagaaagcg acgccgccna agtaagaa
63821260DNALygus hesperusmisc_feature59n = A,T,C or G 21catggcgtac
ggtgtaacaa gagtcgtgtt ccgctgcgag gaagctcagg aatccggana 60attggatctg
tcggaatgtc aactcatgca ggtgccggac gcggtctacc acttgatgag
120gcacacggaa ctgaaggcgt gtaatctctc aagcaacgtc atcaccaaaa
ttcccccgaa 180attcgcggtc aacttttctc tcattacaga gctgaacctg
gcgcacaacc agatgagcaa 240actcccggac gagctcgccg 2602223DNALygus
hesperus 22aagaggtaca aatgtctttc ctg 232319RNAArtificial
Sequenceprimer 23gagguacaaa ugucuuucc
192419RNAArtificial Sequenceprimer 24ggaaagacau uuguaccuc
192523DNALygus hesperus 25aagtcagtcc tacacgtgtc ctc
232619RNAArtificial Sequenceprimer 26gucaguccua cacgugucc
192719RNAArtificial Sequenceprimer 27ggacacgugu aggacugac
192822DNALygus hesperus 28aacaacgttc gccaggattt tt
222919RNAArtificial Sequenceprimer 29caacguucgc caggauuuu
193019RNAArtificial Sequenceprimer 30aaaauccugg cgaacguug
193123DNALygus hesperus 31aagactgctg acggaataac gaa
233219RNAArtificial Sequenceprimer 32gacugcugac ggaauaacg
193319RNAArtificial Sequenceprimer 33cguuauuccg ucagcaguc
193423DNALygus hesperus 34aaccccgacg gcaccgtcgc tga
233519RNAArtificial Sequenceprimer 35ccccgacggc accgucgcu
193619RNAArtificial Sequenceprimer 36agcgacggug ccgucgggg
193723DNALygus hesperus 37aagatctcca aagctcgtgg aat
233819RNAArtificial Sequenceprimer 38gaucuccaaa gcucgugga
193919RNAArtificial Sequenceprimer 39uccacgagcu uuggagauc
194023DNALygus hesperus 40aaattacccg cggaggatac tca
234119RNAArtificial Sequenceprimer 41auuacccgcg gaggauacu
194219RNAArtificial Sequenceprimer 42aguauccucc gcggguaau
194323DNALygus hesperus 43aattgtggct tcagctcccg tat
234419RNAArtificial Sequenceprimer 44uuguggcuuc agcucccgu
194519RNAArtificial Sequenceprimer 45acgggagcug aagccacaa
194623DNALygus hesperusmisc_feature13n = A,T,C or G 46aactctcttg
tancctcacc aca 234719RNAArtificial Sequenceprimer 47cucucuugua
nccucacca 194816RNAArtificial Sequenceprimer 48uggugaggca agagag
164923DNALygus hesperus 49aagtctttaa ggacggattt acg
235019RNAArtificial Sequenceprimer 50gucuuuaagg acggauuua
195119RNAArtificial Sequenceprimer 51uaaauccguc cuuaaagac
195223DNALygus hesperus 52aatgcgtgtt ggctcatctg tca
235319RNAArtificial Sequenceprimer 53ugcguguugg cucaucugu
195419RNAArtificial Sequenceprimer 54acagaugagc caacacgca
195523DNALygus hesperus 55aagctggtgg tcgtctgaag gat
235619RNAArtificial Sequenceprimer 56gcuggugguc gucugaagg
195719RNAArtificial Sequenceprimer 57ccuucagacg accaccagc
195823DNALygus hesperus 58aacactcgga tgaaatcact gtg
235919RNAArtificial Sequenceprimer 59cacucggaug aaaucacug
196019RNAArtificial Sequenceprimer 60cagugauuuc auccgagug
196123DNALygus hesperus 61aatcagtatc acgctcagga tgt
236219RNAArtificial Sequenceprimer 62ucaguaucac gcucaggau
196319RNAArtificial Sequenceprimer 63auccugagcg ugauacuga
196423DNALygus hesperus 64aacacccatg aagtcgctgt cgc
236519RNAArtificial Sequenceprimer 65cacccaugaa gucgcuguc
196619RNAArtificial Sequenceprimer 66gacagcgacu ucaugggug
196723DNALygus hesperus 67aagatctcca aagctcgtgg aat
236819RNAArtificial Sequenceprimer 68gaucuccaaa gcucgugga
196919RNAArtificial Sequenceprimer 69uccacgagcu uuggagauc
197023DNALygus hesperus 70aactacaagt atcctgtgga ggg
237119RNAArtificial Sequenceprimer 71cuacaaguau ccuguggag
197219RNAArtificial Sequenceprimer 72cuccacagga uacuuguag
197323DNALygus hesperus 73aaattggagt gtgactggtg ctg
237419RNAArtificial Sequenceprimer 74auuggagugu gacuggugc
197519RNAArtificial Sequenceprimer 75gcaccaguca cacuccaau
197623DNALygus hesperus 76aacatcagac gccctctact gga
237719RNAArtificial Sequenceprimer 77caucagacgc ccucuacug
197819RNAArtificial Sequenceprimer 78caguagaggg cgucugaug
197923DNALygus hesperus 79aaacggttct ttccgaaacg acg
238019RNAArtificial Sequenceprimer 80acgguucuuu ccgaaacga
198119RNAArtificial Sequenceprimer 81ucguuucgga aagaaccgu
198223DNALygus hesperus 82aaccatcgtt cctggtgatg cat
238319RNAArtificial Sequenceprimer 83ccaucguucc uggugaugc
198419RNAArtificial Sequenceprimer 84gcaucaccag gaacgaugg
198523DNALygus hesperus 85aaagtcttca gagaggaagc tgc
238619RNAArtificial Sequenceprimer 86agucuucaga gaggaagcu
198719RNAArtificial Sequenceprimer 87agcuuccucu cugaagacu
198823DNALygus hesperus 88aagatgttcc atggagaggc aca
238919RNAArtificial Sequenceprimer 89gauguuccau ggagaggca
199019RNAArtificial Sequenceprimer 90ugccucucca uggaacauc
199123DNALygus hesperus 91aaatagggca acttcacgga tta
239219RNAArtificial Sequenceprimer 92auagggcaac uucacggau
199319RNAArtificial Sequenceprimer 93auccgugaag uugcccuau
199423DNALygus hesperus 94aagaaccatt cgccgttatc acc
239519RNAArtificial Sequenceprimer 95gaaccauucg ccguuauca
199619RNAArtificial Sequenceprimer 96ugauaacggc gaaugguuc
199723DNALygus hesperus 97aagctgcact gggattcatt cct
239819RNAArtificial Sequenceprimer 98gcugcacugg gauucauuc
199919RNAArtificial Sequenceprimer 99gaaugaaucc cagugcagc
1910023DNALygus hesperus 100aatacaaaag tcgcgtcttc cgg
2310119RNAArtificial Sequenceprimer 101uacaaaaguc gcgucuucc
1910219RNAArtificial Sequenceprimer 102ggaagacgcg acuuuugua
1910323DNALygus hesperus 103aagatctttc gactcttgac gtg
2310419RNAArtificial Sequenceprimer 104gaucuuucga cucuugacg
1910519RNAArtificial Sequenceprimer 105cgucaagagu cgaaagauc
1910623DNALygus hesperus 106aatatcggta ctattggtca cgt
2310719RNAArtificial Sequenceprimer 107uaucgguacu auuggucac
1910819RNAArtificial Sequenceprimer 108gugaccaaua guaccgaua
1910923DNALygus hesperus 109aatttcgacg tggaagtgtt aag
2311019RNAArtificial Sequenceprimer 110uuucgacgug gaaguguua
1911119RNAArtificial Sequenceprimer 111uaacacuucc acgucgaaa
1911223DNALygus hesperus 112aatgttgcat ctcgaaggtg caa
2311319RNAArtificial Sequenceprimer 113uguugcaucu cgaaggugc
1911419RNAArtificial Sequenceprimer 114gcaccuucga gaugcaaca
1911523DNALygus hesperus 115aacactgggt aaagatgtag gac
2311619RNAArtificial Sequenceprimer 116cacuggguaa agauguagg
1911719RNAArtificial Sequenceprimer 117ccuacaucuu uacccagug
1911823DNALygus hesperus 118aacccgggtc ttttctgtaa aga
2311919RNAArtificial Sequenceprimer 119cccgggucuu uucuguaaa
1912019RNAArtificial Sequenceprimer 120uuuacagaaa agacccggg
1912123DNALygus hesperus 121aaacctcgaa accaaaaacg tag
2312219RNAArtificial Sequenceprimer 122accucgaaac caaaaacgu
1912319RNAArtificial Sequenceprimer 123acguuuuugg uuucgaggu
1912423DNALygus hesperus 124aagttggaaa ctggagtcca gat
2312519RNAArtificial Sequenceprimer 125guuggaaacu ggaguccag
1912619RNAArtificial Sequenceprimer 126cuggacucca guuuccaac
1912723DNALygus hesperus 127aaatcttctt cgggactatc cat
2312819RNAArtificial Sequenceprimer 128aucuucuucg ggacuaucc
1912919RNAArtificial Sequenceprimer 129ggauaguccc gaagaagau
1913023DNALygus hesperus 130aaaaccagaa cagaaaccgg agt
2313119RNAArtificial Sequenceprimer 131aaccagaaca gaaaccgga
1913219RNAArtificial Sequenceprimer 132uccgguuucu guucugguu
1913323DNALygus hesperus 133aagtgatgac cttctctttg gga
2313419RNAArtificial Sequenceprimer 134gugaugaccu ucucuuugg
1913519RNAArtificial Sequenceprimer 135ccaaagagaa ggucaucac
1913623DNALygus hesperus 136aacaggacat catatttcgc agg
2313719RNAArtificial Sequenceprimer 137caggacauca uauuucgca
1913819RNAArtificial Sequenceprimer 138ugcgaaauau gauguccug
1913923DNALygus hesperus 139aaaaagcact agatgagccg aag
2314019RNAArtificial Sequenceprimer 140aaagcacuag augagccga
1914119RNAArtificial Sequenceprimer 141ucggcucauc uagugcuuu
1914223DNALygus hesperus 142aagttcatcg gagagctttg caa
2314319RNAArtificial Sequenceprimer 143guucaucgga gagcuuugc
1914419RNAArtificial Sequenceprimer 144gcaaagcucu ccgaugaac
1914523DNALygus hesperus 145aactttttga tcgtatggcg tcc
2314619RNAArtificial Sequenceprimer 146cuuuuugauc guauggcgu
1914719RNAArtificial Sequenceprimer 147acgccauacg aucaaaaag
1914823DNALygus hesperusmisc_feature10n = A,T,C or G 148aactttcctn
ttccgcgttg ttg 2314919RNAArtificial Sequenceprimer 149cuuuccunuu
ccgcguugu 1915016RNAArtificial Sequenceprimer 150acaacgcgga agaaag
1615123DNALygus hesperusmisc_feature22n = A,T,C or G 151aagagaacgg
agataacgag gna 2315219RNAArtificial Sequenceprimer 152gagaacggag
auaacgagg 1915319RNAArtificial Sequenceprimer 153ccucguuauc
uccguucuc 1915423DNALygus hesperusmisc_feature22n = A,T,C or G
154aaaaaggctc cgactgtttg gna 2315519RNAArtificial Sequenceprimer
155aaaggcuccg acuguuugg 1915619RNAArtificial Sequenceprimer
156ccaaacaguc ggagccuuu 1915723DNALygus hesperus 157aaaaagctcc
atacaggtga acg 2315819RNAArtificial Sequenceprimer 158aaagcuccau
acaggugaa 1915919RNAArtificial Sequenceprimer 159uucaccugua
uggagcuuu 1916023DNALygus hesperus 160aaaaacaagc acaaagacgg gtc
2316119RNAArtificial Sequenceprimer 161aaacaagcac aaagacggg
1916219RNAArtificial Sequenceprimer 162cccgucuuug ugcuuguuu
1916323DNALygus hesperus 163aagtatttca gtcttcctct gct
2316419RNAArtificial Sequenceprimer 164guauuucagu cuuccucug
1916519RNAArtificial Sequenceprimer 165cagaggaaga cugaaauac
1916623DNALygus hesperus 166aagtcagaat gtcgactgaa aag
2316719RNAArtificial Sequenceprimer 167gucagaaugu cgacugaaa
1916819RNAArtificial Sequenceprimer 168uuucagucga cauucugac
1916923DNALygus hesperus 169aaggaacgtg agatgatcct gta
2317019RNAArtificial Sequenceprimer 170ggaacgugag augauccug
1917119RNAArtificial Sequenceprimer 171caggaucauc ucacguucc
1917223DNALygus hesperusmisc_feature6n = A,T,C or G 172aacccnactt
tcttgtacaa agt 2317319RNAArtificial Sequenceprimer 173cccnacuuuc
uuguacaaa 1917416RNAArtificial Sequenceprimer 174uuuguacaag aaagug
1617523DNALygus hesperus 175aagtttttgc ggtgtcggaa tgt
2317619RNAArtificial Sequenceprimer 176guuuuugcgg ugucggaau
1917719RNAArtificial Sequenceprimer 177auuccgacac cgcaaaaac
1917823DNALygus hesperusmisc_feature5, 6, 7, 8n = A,T,C or G
178aaaannnntc cgaacgccgc caa 2317919RNAArtificial Sequenceprimer
179aannnnuccg aacgccgcc 1918015RNAArtificial Sequenceprimer
180ggcggcguuc ggauu 1518123DNALygus hesperus 181aaactcacct
tctctcaagc cgt 2318219RNAArtificial Sequenceprimer 182acucaccuuc
ucucaagcc 1918319RNAArtificial Sequenceprimer 183ggcuugagag
aaggugagu 1918423DNALygus hesperus 184aattcaacga gataaccgaa tga
2318519RNAArtificial Sequenceprimer 185uucaacgaga uaaccgaau
1918619RNAArtificial Sequenceprimer 186auucgguuau cucguugaa
1918723DNALygus hesperus 187aagtctgtgt tgtctgaaag tag
2318819RNAArtificial Sequenceprimer 188gucuguguug ucugaaagu
1918919RNAArtificial Sequenceprimer
189acuuucagac aacacagac 1919023DNALygus hesperus 190aactcccagt
gtcgtcctca aga 2319119RNAArtificial Sequenceprimer 191cucccagugu
cguccucaa 1919219RNAArtificial Sequenceprimer 192uugaggacga
cacugggag 1919323DNALygus hesperus 193aagttccatc caaagcaccg cag
2319419RNAArtificial Sequenceprimer 194guuccaucca aagcaccgc
1919519RNAArtificial Sequenceprimer 195gcggugcuuu ggauggaac
1919623DNALygus hesperus 196aaggaagtat cgcaccacat tct
2319719RNAArtificial Sequenceprimer 197ggaaguaucg caccacauu
1919819RNAArtificial Sequenceprimer 198aauguggugc gauacuucc
1919923DNALygus hesperus 199aaatcaagga caatgtggta gct
2320019RNAArtificial Sequenceprimer 200aucaaggaca augugguag
1920119RNAArtificial Sequenceprimer 201cuaccacauu guccuugau
1920223DNALygus hesperus 202aaagtgaagg tgaagattgc gag
2320319RNAArtificial Sequenceprimer 203agugaaggug aagauugcg
1920419RNAArtificial Sequenceprimer 204cgcaaucuuc accuucacu
1920523DNALygus hesperus 205aagatgttgt gttaggttta ccg
2320619RNAArtificial Sequenceprimer 206gauguugugu uagguuuac
1920719RNAArtificial Sequenceprimer 207guaaaccuaa cacaacauc
1920823DNALygus hesperus 208aacaaagtcg attccaccta gag
2320919RNAArtificial Sequenceprimer 209caaagucgau uccaccuag
1921019RNAArtificial Sequenceprimer 210cuagguggaa ucgacuuug
1921123DNALygus hesperus 211aactttggtc ctggtttgga tag
2321219RNAArtificial Sequenceprimer 212cuuugguccu gguuuggau
1921319RNAArtificial Sequenceprimer 213auccaaacca ggaccaaag
1921423DNALygus hesperusmisc_feature19n = A,T,C or G 214aagctacagg
aatccggana att 2321519RNAArtificial Sequenceprimer 215gcuacaggaa
uccgganaa 1921618RNAArtificial Sequenceprimer 216uuuccggauu
ccuguagc 1821723DNALygus hesperus 217aaggcgtgta atctctcaag caa
2321819RNAArtificial Sequenceprimer 218ggcguguaau cucucaagc
1921919RNAArtificial Sequenceprimer 219gcuugagaga uuacacgcc
1922023DNALygus hesperus 220aattcgcggt caactttatc tct
2322119RNAArtificial Sequenceprimer 221uucgcgguca acuuuaucu
1922219RNAArtificial Sequenceprimer 222agauaaaguu gaccgcgaa
1922323DNALygus hesperus 223aagaggtaca aatgtctttc ctg
2322419RNAArtificial Sequenceprimer 224gagguacaaa ugucuuucc
1922519RNAArtificial Sequenceprimer 225ggaaagacau uuguaccuc
1922623DNALygus hesperus 226aagtcagtcc tacacgtgtc ctc
2322719RNAArtificial Sequenceprimer 227gucaguccua cacgugucc
1922819RNAArtificial Sequenceprimer 228ggacacgugu aggacugac
1922923DNALygus hesperus 229aacaacgttc gccaggattt ttt
2323019RNAArtificial Sequenceprimer 230caacguucgc caggauuuu
1923119RNAArtificial Sequenceprimer 231aaaauccugg cgaacguug
1923223DNALygus hesperus 232aagactgctg acggaataac gaa
2323319RNAArtificial Sequenceprimer 233gacugcugac ggaauaacg
1923419RNAArtificial Sequenceprimer 234cguuauuccg ucagcaguc
1923523DNALygus hesperus 235aaccccgacg gcaccgtcgc tga
2323619RNAArtificial Sequenceprimer 236ccccgacggc accgucgcu
1923719RNAArtificial Sequenceprimer 237agcgacggug ccgucgggg
1923823DNALygus hesperus 238aagatctcca aagctcgtgg aat
2323919RNAArtificial Sequenceprimer 239gaucuccaaa gcucgugga
1924019RNAArtificial Sequenceprimer 240uccacgagcu uuggagauc
1924123DNALygus hesperus 241aaattacccg cggaggatac tca
2324219RNAArtificial Sequenceprimer 242auuacccgcg gaggauacu
1924319RNAArtificial Sequenceprimer 243aguauccucc gcggguaau
1924423DNALygus hesperus 244aattgtggct tcagctcccg tat
2324519RNAArtificial Sequenceprimer 245uuguggcuuc agcucccgu
1924619RNAArtificial Sequenceprimer 246acgggagcug aagccacaa
1924723DNALygus hesperus 247aagtctttaa ggacggattt acg
2324819RNAArtificial Sequenceprimer 248gucuuuaagg acggauuua
1924919RNAArtificial Sequenceprimer 249uaaauccguc cuuaaagac
1925023DNALygus hesperus 250aatgcgtgtt ggctcatctg tca
2325119RNAArtificial Sequenceprimer 251ugcguguugg cucaucugu
1925219RNAArtificial Sequenceprimer 252acagaugagc caacacgca
1925323DNALygus hesperus 253aagctggtgg tcgtctgaag gat
2325419RNAArtificial Sequenceprimer 254gcuggugguc gucugaagg
1925519RNAArtificial Sequenceprimer 255ccuucagacg accaccagc
1925623DNALygus hesperus 256aacactcgga tgaaatcact gtg
2325719RNAArtificial Sequenceprimer 257cacucggaug aaaucacug
1925819RNAArtificial Sequenceprimer 258cagugauuuc auccgagug
1925923DNALygus hesperus 259aatcagtatc acgctcagga tgt
2326019RNAArtificial Sequenceprimer 260ucaguaucac gcucaggau
1926119RNAArtificial Sequenceprimer 261auccugagcg ugauacuga
1926223DNALygus hesperus 262aacacccatg aagtcgctgt cgc
2326319RNAArtificial Sequenceprimer 263cacccaugaa gucgcuguc
1926419RNAArtificial Sequenceprimer 264gacagcgacu ucaugggug
1926523DNALygus hesperus 265aagatctcca aagctcgtgg aat
2326619RNAArtificial Sequenceprimer 266gaucuccaaa gcucgugga
1926719RNAArtificial Sequenceprimer 267uccacgagcu uuggagauc
1926823DNALygus hesperus 268aactacaagt atcctgtgga ggg
2326919RNAArtificial Sequenceprimer 269cuacaaguau ccuguggag
1927019RNAArtificial Sequenceprimer 270cuccacagga uacuuguag
1927123DNALygus hesperus 271aaattggagt gtgactggtg ctg
2327219RNAArtificial Sequenceprimer 272auuggagugu gacuggugc
1927319RNAArtificial Sequenceprimer 273gcaccaguca cacuccaau
1927423DNALygus hesperus 274aacatcagac gccctctact gga
2327519RNAArtificial Sequenceprimer 275caucagacgc ccucuacug
1927619RNAArtificial Sequenceprimer 276caguagaggg cgucugaug
1927723DNALygus hesperus 277aaacggttct ttccgaaacg acg
2327819RNAArtificial Sequenceprimer 278acgguucuuu ccgaaacga
1927919RNAArtificial Sequenceprimer 279ucguuucgga aagaaccgu
1928023DNALygus hesperus 280aaccatcgtt cctggtgatg cat
2328119RNAArtificial Sequenceprimer 281ccaucguucc uggugaugc
1928219RNAArtificial Sequenceprimer 282gcaucaccag gaacgaugg
1928323DNALygus hesperus 283aaagtcttca gagaggaagc tgc
2328419RNAArtificial Sequenceprimer 284agucuucaga gaggaagcu
1928519RNAArtificial Sequenceprimer 285agcuuccucu cugaagacu
1928623DNALygus hesperus 286aagatgttcc atggagaggc aca
2328719RNAArtificial Sequenceprimer 287gauguuccau ggagaggca
1928819RNAArtificial Sequenceprimer 288ugccucucca uggaacauc
1928923DNALygus hesperus 289aaatagggca acttcacgga tta
2329019RNAArtificial Sequenceprimer 290auagggcaac uucacggau
1929119RNAArtificial Sequenceprimer 291auccgugaag uugcccuau
1929223DNALygus hesperus 292aagaaccatt cgccgttatc acc
2329319RNAArtificial Sequenceprimer 293gaaccauucg ccguuauca
1929419RNAArtificial Sequenceprimer 294ugauaacggc gaaugguuc
1929523DNALygus hesperus 295aagctgcact gggattcatt cct
2329619RNAArtificial Sequenceprimer 296gcugcacugg gauucauuc
1929719RNAArtificial Sequenceprimer 297gaaugaaucc cagugcagc
1929823DNALygus hesperus 298aatacaaaag tcgcgtcttc cgg
2329919RNAArtificial Sequenceprimer 299uacaaaaguc gcgucuucc
1930019RNAArtificial Sequenceprimer 300ggaagacgcg acuuuugua
1930123DNALygus hesperus 301aagatctttc gactcttgac gtg
2330219RNAArtificial Sequenceprimer 302gaucuuucga cucuugacg
1930319RNAArtificial Sequenceprimer 303cgucaagagu cgaaagauc
1930423DNALygus hesperus 304aatatcggta ctattggtca cgt
2330519RNAArtificial Sequenceprimer 305uaucgguacu auuggucac
1930619RNAArtificial Sequenceprimer 306gugaccaaua guaccgaua
1930723DNALygus hesperus 307aatttcgacg tggaagtgtt aag
2330819RNAArtificial Sequenceprimer 308uuucgacgug gaaguguua
1930919RNAArtificial Sequenceprimer 309uaacacuucc acgucgaaa
1931023DNALygus hesperus 310aatgttgcat ctcgaaggtg caa
2331119RNAArtificial Sequenceprimer 311uguugcaucu cgaaggugc
1931219RNAArtificial Sequenceprimer 312gcaccuucga gaugcaaca
1931323DNALygus hesperus 313aacactgggt aaagatgtag gac
2331419RNAArtificial Sequenceprimer 314cacuggguaa agauguagg
1931519RNAArtificial Sequenceprimer 315ccuacaucuu uacccagug
1931623DNALygus hesperus 316aacccgggtc ttttctgtaa aga
2331719RNAArtificial Sequenceprimer 317cccgggucuu uucuguaaa
1931819RNAArtificial Sequenceprimer 318uuuacagaaa agacccggg
1931923DNALygus hesperus 319aaacctcgaa accaaaaacg tag
2332019RNAArtificial Sequenceprimer 320accucgaaac caaaaacgu
1932119RNAArtificial Sequenceprimer 321acguuuuugg uuucgaggu
1932223DNALygus hesperus 322aagttggaaa ctggagtcca gat
2332319RNAArtificial Sequenceprimer 323guuggaaacu ggaguccag
1932419RNAArtificial Sequenceprimer 324cuggacucca guuuccaac
1932523DNALygus hesperus 325aaatcttctt cgggactatc cat
2332619RNAArtificial Sequenceprimer 326aucuucuucg ggacuaucc
1932719RNAArtificial Sequenceprimer 327ggauaguccc gaagaagau
1932823DNALygus hesperus 328aaaaccagaa cagaaaccgg agt
2332919RNAArtificial Sequenceprimer 329aaccagaaca gaaaccgga
1933019RNAArtificial Sequenceprimer 330uccgguuucu guucugguu
1933123DNALygus hesperus 331aagtgatgac cttctctttg gga
2333219RNAArtificial Sequenceprimer 332gugaugaccu ucucuuugg
1933319RNAArtificial Sequenceprimer 333ccaaagagaa ggucaucac
1933423DNALygus hesperus 334aacaggacat catatttcgc agg
2333519RNAArtificial Sequenceprimer 335caggacauca uauuucgca
1933619RNAArtificial Sequenceprimer 336ugcgaaauau gauguccug
1933723DNALygus hesperus 337aaaaagcact agatgagccg aag
2333819RNAArtificial Sequenceprimer 338aaagcacuag augagccga
1933919RNAArtificial Sequenceprimer 339ucggcucauc uagugcuuu
1934023DNALygus hesperus 340aagttcatcg gagagctttg caa
2334119RNAArtificial Sequenceprimer 341guucaucgga gagcuuugc
1934219RNAArtificial Sequenceprimer 342gcaaagcucu ccgaugaac
1934323DNALygus hesperus 343aactttttga tcgtatggcg tcc
2334419RNAArtificial Sequenceprimer 344cuuuuugauc guauggcgu
1934519RNAArtificial Sequenceprimer 345acgccauacg aucaaaaag
1934623DNALygus hesperusmisc_feature22n = A,T,C or G 346aagagaacgg
agataacgag gna 2334719RNAArtificial Sequenceprimer 347gagaacggag
auaacgagg 1934819RNAArtificial Sequenceprimer 348ccucguuauc
uccguucuc 1934923DNALygus hesperusmisc_feature22n = A,T,C or G
349aaaaaggctc cgactgtttg gna 2335019RNAArtificial Sequenceprimer
350aaaggcuccg acuguuugg 1935119RNAArtificial Sequenceprimer
351ccaaacaguc ggagccuuu 1935223DNALygus hesperus 352aaaaagctcc
atacaggtga acg 2335319RNAArtificial Sequenceprimer 353aaagcuccau
acaggugaa 1935419RNAArtificial Sequenceprimer 354uucaccugua
uggagcuuu 1935523DNALygus hesperus 355aaaaacaagc acaaagacgg gtc
2335619RNAArtificial Sequenceprimer 356aaacaagcac aaagacggg
1935719RNAArtificial Sequenceprimer 357cccgucuuug ugcuuguuu
1935823DNALygus hesperus 358aagtatttca gtcttcctct gct
2335919RNAArtificial Sequenceprimer 359guauuucagu cuuccucug
1936019RNAArtificial Sequenceprimer 360cagaggaaga cugaaauac
1936123DNALygus hesperus 361aagtcagaat gtcgactgaa aag
2336219RNAArtificial Sequenceprimer 362gucagaaugu cgacugaaa
1936319RNAArtificial Sequenceprimer 363uuucagucga cauucugac
1936423DNALygus hesperus 364aaggaacgtg agatgatcct gta
2336519RNAArtificial Sequenceprimer 365ggaacgugag augauccug
1936619RNAArtificial Sequenceprimer 366caggaucauc ucacguucc
1936723DNALygus hesperus 367aagtttttgc ggtgtcggaa tgt
2336819RNAArtificial Sequenceprimer 368guuuuugcgg ugucggaau
1936919RNAArtificial Sequenceprimer 369auuccgacac cgcaaaaac
1937023DNALygus hesperus 370aaactcacct tctctcaagc cgt
2337119RNAArtificial Sequenceprimer 371acucaccuuc ucucaagcc
1937219RNAArtificial Sequenceprimer 372ggcuugagag aaggugagu
1937323DNALygus hesperus 373aattcaacga gataaccgaa tga
2337419RNAArtificial Sequenceprimer 374uucaacgaga uaaccgaau
1937519RNAArtificial Sequenceprimer 375auucgguuau cucguugaa
1937623DNALygus hesperus 376aagtctgtgt tgtctgaaag tag
2337719RNAArtificial Sequenceprimer 377gucuguguug ucugaaagu
1937819RNAArtificial Sequenceprimer 378acuuucagac aacacagac
1937923DNALygus hesperus 379aactcccagt gtcgtcctca aga
2338019RNAArtificial Sequenceprimer 380cucccagugu cguccucaa
1938119RNAArtificial Sequenceprimer 381uugaggacga cacugggag
1938223DNALygus hesperus 382aagttccatc caaagcaccg cag
2338319RNAArtificial Sequenceprimer 383guuccaucca aagcaccgc
1938419RNAArtificial Sequenceprimer 384gcggugcuuu ggauggaac
1938523DNALygus hesperus 385aaggaagtat cgcaccacat tct
2338619RNAArtificial Sequenceprimer 386ggaaguaucg caccacauu
1938719RNAArtificial Sequenceprimer 387aauguggugc gauacuucc
1938823DNALygus hesperus 388aaatcaagga caatgtggta gct
2338919RNAArtificial Sequenceprimer 389aucaaggaca augugguag
1939019RNAArtificial Sequenceprimer 390cuaccacauu guccuugau
1939123DNALygus hesperus 391aaagtgaagg tgaagattgc gag
2339219RNAArtificial Sequenceprimer 392agugaaggug aagauugcg
1939319RNAArtificial Sequenceprimer 393cgcaaucuuc accuucacu
1939423DNALygus hesperus 394aagatgttgt gttaggttta ccg
2339519RNAArtificial Sequenceprimer 395gauguugugu uagguuuac
1939619RNAArtificial Sequenceprimer 396guaaaccuaa cacaacauc
1939723DNALygus hesperus 397aacaaagtcg attccaccta gag
2339819RNAArtificial Sequenceprimer 398caaagucgau uccaccuag
1939919RNAArtificial Sequenceprimer 399cuagguggaa ucgacuuug
1940023DNALygus hesperus 400aactttggtc ctggtttgga tag
2340119RNAArtificial Sequenceprimer 401cuuugguccu gguuuggau
1940219RNAArtificial Sequenceprimer 402auccaaacca ggaccaaag
1940323DNALygus hesperus 403aaggcgtgta atctctcaag caa
2340419RNAArtificial Sequenceprimer 404ggcguguaau cucucaagc
1940519RNAArtificial Sequenceprimer 405gcuugagaga uuacacgcc
1940623DNALygus hesperus 406aattcgcggt caactttatc tct
2340719RNAArtificial Sequenceprimer 407uucgcgguca acuuuaucu
1940819RNAArtificial Sequenceprimer 408agauaaaguu gaccgcgaa 19
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