U.S. patent application number 16/412885 was filed with the patent office on 2019-10-10 for ribosomal protein l40 (rpl40) nucleic acid molecules that confer resistance to coleopteran and hemipteran pests.
The applicant listed for this patent is Dow AgroSciences LLC. Invention is credited to ELANE FISHILEVICH, MEGHAN L. FREY, PREMCHAND GANDRA, WENDY LO, KENNETH NARVA, MURUGESAN RANGASAMY, SARAH E. WORDEN.
Application Number | 20190308702 16/412885 |
Document ID | / |
Family ID | 68096424 |
Filed Date | 2019-10-10 |
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United States Patent
Application |
20190308702 |
Kind Code |
A1 |
NARVA; KENNETH ; et
al. |
October 10, 2019 |
RIBOSOMAL PROTEIN L40 (RPL40) NUCLEIC ACID MOLECULES THAT CONFER
RESISTANCE TO COLEOPTERAN AND HEMIPTERAN PESTS
Abstract
This disclosure concerns nucleic acid molecules and methods of
use thereof for control of coleopteran and/or hemipteran pests
through RNA interference-mediated inhibition of target coding and
transcribed non-coding sequences in coleopteran and/or hemipteran
pests. The disclosure also concerns methods for making transgenic
plants that express nucleic acid molecules useful for the control
of coleopteran and/or hemipteran pests, and the plant cells and
plants obtained thereby.
Inventors: |
NARVA; KENNETH; (ZIONSVILLE,
IN) ; FISHILEVICH; ELANE; (INDIANAPOLIS, IN) ;
RANGASAMY; MURUGESAN; (ZIONSVILLE, IN) ; FREY; MEGHAN
L.; (GREENWOOD, IN) ; LO; WENDY;
(INDIANAPOLIS, IN) ; WORDEN; SARAH E.;
(INDIANAPOLIS, IN) ; GANDRA; PREMCHAND;
(ZIONSVILLE, IN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Dow AgroSciences LLC |
Indianapolis |
IN |
US |
|
|
Family ID: |
68096424 |
Appl. No.: |
16/412885 |
Filed: |
May 15, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15373771 |
Dec 9, 2016 |
10035573 |
|
|
16412885 |
|
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|
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62269382 |
Dec 18, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
B63C 9/20 20130101; C12N
15/8218 20130101; C12N 15/8286 20130101; C07K 14/43563
20130101 |
International
Class: |
B63C 9/20 20060101
B63C009/20 |
Claims
1. An isolated nucleic acid comprising at least one polynucleotide
operably linked to a heterologous promoter, wherein the
polynucleotide is selected from the group consisting of: SEQ ID
NO:1; the complement of SEQ ID NO:1; a fragment of at least 15
contiguous nucleotides of SEQ ID NO:1; the complement of a fragment
of at least 15 contiguous nucleotides of SEQ ID NO:1; a native
coding sequence of a Diabrotica organism comprising any of SEQ ID
NO:9 or SEQ ID NOs:11-15; the complement of a native coding
sequence of a Diabrotica organism comprising any of SEQ ID NO:9 or
SEQ ID NOs:11-15; a fragment of at least 15 contiguous nucleotides
of a native coding sequence of a Diabrotica organism comprising any
of SEQ ID NO:9 or SEQ ID NOs:11-15; the complement of a fragment of
at least 15 contiguous nucleotides of a native coding sequence of a
Diabrotica organism comprising any of SEQ ID NO:9 or SEQ ID
NOs:11-15; SEQ ID NO:3; the complement of SEQ ID NO:3; a fragment
of at least 15 contiguous nucleotides of SEQ ID NO:3; the
complement of a fragment of at least 15 contiguous nucleotides of
SEQ ID NO:3; SEQ ID NO:5; the complement of SEQ ID NO:5; a fragment
of at least 15 contiguous nucleotides of SEQ ID NO:5; the
complement of a fragment of at least 15 contiguous nucleotides of
SEQ ID NO:5; a native coding sequence of a Diabrotica organism
comprising any of SEQ ID NO:10; the complement of a native coding
sequence of a Diabrotica organism comprising any of SEQ ID NO:10; a
fragment of at least 15 contiguous nucleotides of a native coding
sequence of a Diabrotica organism comprising any of SEQ ID NO:10;
the complement of a fragment of at least 15 contiguous nucleotides
of a native coding sequence of a Diabrotica organism comprising any
of SEQ ID NO:10; SEQ ID NO:89; the complement of SEQ ID NO:89; a
fragment of at least 15 contiguous nucleotides of SEQ ID NO:89; the
complement of a fragment of at least 15 contiguous nucleotides of
SEQ ID NO:89; a native coding sequence of a Euschistus organism
comprising SEQ ID NO:91 or SEQ ID NO:92; the complement of a native
coding sequence of a Euschistus organism comprising SEQ ID NO:91 or
SEQ ID NO:92; a fragment of at least 15 contiguous nucleotides of a
native coding sequence of a Euschistus organism comprising SEQ ID
NO:91 or SEQ ID NO:92; the complement of a fragment of at least 15
contiguous nucleotides of a native coding sequence of a Euschistus
organism comprising SEQ ID NO:91 or SEQ ID NO:92.
2. The polynucleotide of claim 1, wherein the polynucleotide is
selected from the group consisting of SEQ ID NO:1; the complement
of SEQ ID NO:1; SEQ ID NO:3; the complement of SEQ ID NO:3; SEQ ID
NO:5; the complement of SEQ ID NO:5; a fragment of at least 15
contiguous nucleotides of SEQ ID NO:1; the complement of a fragment
of at least 15 contiguous nucleotides of SEQ ID NO:1; a fragment of
at least 15 contiguous nucleotides of SEQ ID NO:3; the complement
of a fragment of at least 15 contiguous nucleotides of SEQ ID NO:3;
a fragment of at least 15 contiguous nucleotides of SEQ ID NO:5;
the complement of a fragment of at least 15 contiguous nucleotides
of SEQ ID NO:5; a native coding sequence of a Diabrotica organism
comprising any of SEQ ID NOs:9-15; the complement of a native
coding sequence of a Diabrotica organism comprising any of SEQ ID
NOs:9-15; a fragment of at least 15 contiguous nucleotides of a
native coding sequence of a Diabrotica organism comprising any of
SEQ ID NOs:9-15; and the complement of a fragment of at least 15
contiguous nucleotides of a native coding sequence of a Diabrotica
organism comprising any of SEQ ID NOs:9-15.
3. The polynucleotide of claim 1, wherein the polynucleotide is
selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ
ID NO:5, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ
ID NO:13, SEQ ID NO:14, SEQ ID NO:15, and the complements of any of
the foregoing.
4. The polynucleotide of claim 3, wherein the organism is selected
from the group consisting of D. v. virgifera LeConte; D. barberi
Smith and Lawrence; D. u. howardi; D. v. zeae; D. balteata LeConte;
D. u. tenella; D. speciosa Germar; D. u. undecimpunctata
Mannerheim; Euschistus heros (Fabr.) (Neotropical Brown Stink Bug);
Nezara viridula (L.) (Southern Green Stink Bug); Piezodorus
guildinii (Westwood) (Red-banded Stink Bug); Halyomorpha halys
(stat) (Brown Marmorated Stink Bug); Chinavia hilare (Say) (Green
Stink Bug); Euschistus serous (Say) (Brown Stink Bug); Dichelops
melacanthus (Dallas); Dichelops furcatus (F.); Edessa meditabunda
(F.); Thyanta perditor (F.) (Neotropical Red Shouldered Stink Bug);
Chinavia marginatum (Palisot de Beauvois); Horcias nobilellus
(Berg) (Cotton Bug); Taedia stigmosa (Berg); Dysdercus peruvianus
(Guerin-Meneville); Neomegalotomus parvus (Westwood); Leptoglossus
zonatus (Dallas); Niesthrea sidae (F.); Lygus hesperus (Knight)
(Western Tarnished Plant Bug); and Lygus lineolaris (Palisot de
Beauvois).
5. A plant transformation vector comprising the polynucleotide of
claim 1.
6. A ribonucleic acid (RNA) molecule transcribed from the
polynucleotide of claim 1.
7. A double-stranded ribonucleic acid molecule produced from the
expression of the polynucleotide of claim 1.
8. The double-stranded ribonucleic acid molecule of claim 7,
wherein contacting the polynucleotide sequence with a coleopteran
or hemipteran insect inhibits the expression of an endogenous
nucleotide sequence specifically complementary to the
polynucleotide.
9. The double-stranded ribonucleic acid molecule of claim 8,
wherein contacting said ribonucleotide molecule with a coleopteran
or hemipteran insect kills or inhibits the growth, viability,
and/or feeding of the insect.
10. The double stranded RNA of claim 7, comprising a first, a
second and a third RNA segment, wherein the first RNA segment
comprises the polynucleotide, wherein the third RNA segment is
linked to the first RNA segment by the second polynucleotide
sequence, and wherein the third RNA segment is substantially the
reverse complement of the first RNA segment, such that the first
and the third RNA segments hybridize when transcribed into a
ribonucleic acid to form the double-stranded RNA.
11. The RNA of claim 6, selected from the group consisting of a
double-stranded ribonucleic acid molecule and a single-stranded
ribonucleic acid molecule of between about 15 and about 30
nucleotides in length.
12. A plant transformation vector comprising the polynucleotide of
claim 1, wherein the heterologous promoter is functional in a plant
cell.
13. A cell transformed with the polynucleotide of claim 1.
14. The cell of claim 13, wherein the cell is a prokaryotic
cell.
15. The cell of claim 13, wherein the cell is a eukaryotic
cell.
16. The cell of claim 15, wherein the cell is a plant cell.
17. A plant transformed with the polynucleotide of claim 1.
18. A seed of the plant of claim 17, wherein the seed comprises the
polynucleotide.
19. A commodity product produced from the plant of claim 17,
wherein the commodity product comprises a detectable amount of the
polynucleotide.
20. The plant of claim 17, wherein the at least one polynucleotide
is expressed in the plant as a double-stranded ribonucleic acid
molecule.
21. The cell of claim 16, wherein the cell is a corn cell, soybean
cell, or cotton cell.
22. The plant of claim 17, wherein the plant is a corn plant, a
soybean plant, or a cotton plant.
23. The plant of claim 17, wherein the at least one polynucleotide
is expressed in the plant as a ribonucleic acid molecule, and the
ribonucleic acid molecule inhibits the expression of an endogenous
polynucleotide that is specifically complementary to the at least
one polynucleotide when a coleopteran or hemipteran insect ingests
a part of the plant.
24. The polynucleotide of claim 1, further comprising at least one
additional polynucleotide that encodes an RNA molecule that
inhibits the expression of an endogenous insect gene.
25. A plant transformation vector comprising the polynucleotide of
claim 24, wherein the additional polynucleotide(s) are each
operably linked to a heterologous promoter functional in a plant
cell.
26. A method for controlling a coleopteran or hemipteran pest
population, the method comprising providing an agent comprising a
ribonucleic acid (RNA) molecule that functions upon contact with
the pest to inhibit a biological function within the pest, wherein
the RNA is specifically hybridizable with a polynucleotide selected
from the group consisting of any of SEQ ID NOs:98-110; the
complement of any of SEQ ID NOs:98-110; a fragment of at least 15
contiguous nucleotides of any of SEQ ID NOs:98-110; the complement
of a fragment of at least 15 contiguous nucleotides of any of SEQ
ID NOs:98-110; a transcript of any of SEQ ID NOs:1, 3, 5, 9-15, 89,
and 91-92; the complement of a transcript of any of SEQ ID NOs:1,
3, 5, 9-15, 89, and 91-92; a fragment of at least 15 contiguous
nucleotides of a transcript of any of SEQ ID NOs:1, 3, 5, 9-15, 89,
and 91-92; and the complement of a fragment of at least 15
contiguous nucleotides of a transcript of any of SEQ ID NOs:1, 3,
5, 9-15, 89, and 91-92.
27. The method according to claim 26, wherein the RNA of the agent
is specifically hybridizable with a polynucleotide selected from
the group consisting of SEQ ID NOs:98-100; the complement of SEQ ID
NO:98-100; a fragment of at least 15 contiguous nucleotides of SEQ
ID NO:98-100; the complement of a fragment of at least 15
contiguous nucleotides of SEQ ID NO:98-100; a transcript of SEQ ID
NOs:1, 3, or 5; the complement of a transcript of SEQ ID NOs: 1, 3,
or 5; a fragment of at least 15 contiguous nucleotides of a
transcript of SEQ ID NOs:1, 3, or 5; and the complement of a
fragment of at least 15 contiguous nucleotides of a transcript of
SEQ ID NOs: 1, 3, or 5.
28. The method according to claim 26, wherein the agent is a
double-stranded RNA molecule.
29. A method for controlling a coleopteran pest population, the
method comprising: providing an agent comprising a first and a
second polynucleotide sequence that functions upon contact with the
coleopteran pest to inhibit a biological function within the
coleopteran pest, wherein the first polynucleotide sequence
comprises a region that exhibits from about 90% to about 100%
sequence identity to from about 15 to about 30 contiguous
nucleotides of any of SEQ ID NOs:98-107, and wherein the first
polynucleotide sequence is specifically hybridized to the second
polynucleotide sequence.
30. A method for controlling a hemipteran pest population, the
method comprising: providing an agent comprising a first and a
second polynucleotide sequence that functions upon contact with the
hemipteran pest to inhibit a biological function within the
hemipteran pest, wherein the first polynucleotide sequence
comprises a region that exhibits from about 90% to about 100%
sequence identity to from about 15 to about 30 contiguous
nucleotides of any of SEQ ID NOs:108-110, and wherein the first
polynucleotide sequence is specifically hybridized to the second
polynucleotide sequence.
31. A method for controlling a coleopteran pest population, the
method comprising: providing in a host plant of a coleopteran pest
a transformed plant cell comprising the polynucleotide of claim 2,
wherein the polynucleotide is expressed to produce a ribonucleic
acid molecule that functions upon contact with a coleopteran pest
belonging to the population to inhibit the expression of a target
sequence within the coleopteran pest and results in decreased
growth and/or survival of the coleopteran pest or pest population,
relative to reproduction of the same pest species on a plant of the
same host plant species that does not comprise the
polynucleotide.
32. The method according to claim 31, wherein the ribonucleic acid
molecule is a double-stranded ribonucleic acid molecule.
33. The method according to claim 31, wherein the coleopteran pest
population is reduced relative to a population of the same pest
species infesting a host plant of the same host plant species
lacking the transformed plant cell.
34. The method according to claim 32, wherein the coleopteran pest
population is reduced relative to a coleopteran pest population
infesting a host plant of the same species lacking the transformed
plant cell.
35. A method of controlling coleopteran pest infestation in a
plant, the method comprising providing in the diet of a coleopteran
pest a ribonucleic acid (RNA) that is specifically hybridizable
with a polynucleotide selected from the group consisting of: SEQ ID
NOs:98-107; the complement of any of SEQ ID NOs:98-107; a fragment
of at least 15 contiguous nucleotides of any of SEQ ID NOs:98-107;
the complement of a fragment of at least 15 contiguous nucleotides
of any of SEQ ID NOs:98-107; a transcript of SEQ ID NO:1, SEQ ID
NO:3, or SEQ ID NO:5; the complement of a transcript of SEQ ID
NO:1, SEQ ID NO:3, or SEQ ID NO:5; a fragment of at least 15
contiguous nucleotides of a transcript of SEQ ID NO:1, SEQ ID NO:3,
or SEQ ID NO:5; and the complement of a fragment of at least 15
contiguous nucleotides of a transcript of SEQ ID NO:1, SEQ ID NO:3,
or SEQ ID NO:5.
36. The method according to claim 35, wherein the diet comprises a
plant cell transformed to express the polynucleotide.
37. The method according to claim 35, wherein the specifically
hybridizable RNA is comprised in a double-stranded RNA
molecule.
38. A method of controlling hemipteran pest infestation in a plant,
the method comprising contacting a hemipteran pest with a
ribonucleic acid (RNA) that is specifically hybridizable with a
polynucleotide selected from the group consisting of: SEQ ID
NOs:108-110; the complement of any of SEQ ID NOs:108-110; a
fragment of at least 15 contiguous nucleotides of any of SEQ ID
NOs:108-110; the complement of a fragment of at least 15 contiguous
nucleotides of any of SEQ ID NOs:108-110; a transcript of SEQ ID
NO:89; the complement of a transcript of SEQ ID NO:89; a fragment
of at least 15 contiguous nucleotides of a transcript of SEQ ID
NO:89; and the complement of a fragment of at least 15 contiguous
nucleotides of a transcript of SEQ ID NO:89.
39. The method according to claim 38, wherein contacting the
hemipteran pest with the RNA comprises spraying the plant with a
composition comprising the RNA.
40. The method according to claim 38, wherein the specifically
hybridizable RNA is comprised in a double-stranded RNA
molecule.
41. A method for improving the yield of a crop, the method
comprising: introducing the nucleic acid of claim 1 into a crop
plant to produce a transgenic crop plant; and cultivating the crop
plant to allow the expression of the at least one polynucleotide;
wherein expression of the at least one polynucleotide inhibits
insect pest reproduction or growth and loss of yield due to insect
pest infection, wherein the crop plant is corn, soybean, or
cotton.
42. The method according to claim 41, wherein expression of the at
least one polynucleotide produces an RNA molecule that suppresses
at least a first target gene in an insect pest that has contacted a
portion of the crop plant.
43. The method according to claim 41, wherein the polynucleotide is
selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ
ID NO:5, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ
ID NO:13, SEQ ID NO:14, SEQ ID NO:15, and the complements of any of
the foregoing.
44. The method according to claim 43, wherein expression of the at
least one polynucleotide produces an RNA molecule that suppresses
at least a first target gene in a coleopteran insect pest that has
contacted a portion of the corn plant.
45. A method for producing a transgenic plant cell, the method
comprising: transforming a plant cell with a vector comprising the
nucleic acid of claim 1; culturing the transformed plant cell under
conditions sufficient to allow for development of a plant cell
culture comprising a plurality of transformed plant cells;
selecting for transformed plant cells that have integrated the at
least one polynucleotide into their genomes; screening the
transformed plant cells for expression of a ribonucleic acid (RNA)
molecule encoded by the at least one polynucleotide; and selecting
a plant cell that expresses the RNA.
46. The method according to claim 45, wherein the vector comprises
a polynucleotide selected from the group consisting of: SEQ ID
NO:1; the complement of SEQ ID NO:1; SEQ ID NO:3; the complement of
SEQ ID NO:3; SEQ ID NO:5; the complement of SEQ ID NO:5; a fragment
of at least 15 contiguous nucleotides of SEQ ID NO:1, SEQ ID NO:3,
or SEQ ID NO:5; the complement of a fragment of at least 15
contiguous nucleotides of SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5;
a native coding sequence of a Diabrotica organism comprising any of
SEQ ID NOs:9-15; the complement of a native coding sequence of a
Diabrotica organism comprising any of SEQ ID NOs:9-15; a fragment
of at least 15 contiguous nucleotides of a native coding sequence
of a Diabrotica organism comprising any of SEQ ID NOs:9-15; and the
complement of a fragment of at least 15 contiguous nucleotides of a
native coding sequence of a Diabrotica organism comprising any of
SEQ ID NOs:9-15.
47. The method according to claim 45, wherein the RNA molecule is a
double-stranded RNA molecule.
48. A method for producing a transgenic plant protected against a
coleopteran pest, the method comprising: providing the transgenic
plant cell produced by the method of claim 46; and regenerating a
transgenic plant from the transgenic plant cell, wherein expression
of the ribonucleic acid molecule encoded by the at least one
polynucleotide is sufficient to modulate the expression of a target
gene in a coleopteran pest that contacts the transformed plant.
49. A method for producing a transgenic plant cell, the method
comprising: transforming a plant cell with a vector comprising a
means for providing coleopteran pest protection to a plant;
culturing the transformed plant cell under conditions sufficient to
allow for development of a plant cell culture comprising a
plurality of transformed plant cells; selecting for transformed
plant cells that have integrated the means for providing
coleopteran pest protection to a plant into their genomes;
screening the transformed plant cells for expression of a means for
inhibiting expression of an essential gene in a coleopteran pest;
and selecting a plant cell that expresses the means for inhibiting
expression of an essential gene in a coleopteran pest.
50. A method for producing a transgenic plant protected against a
coleopteran pest, the method comprising: providing the transgenic
plant cell produced by the method of claim 49; and regenerating a
transgenic plant from the transgenic plant cell, wherein expression
of the means for inhibiting expression of an essential gene in a
coleopteran pest is sufficient to modulate the expression of a
target gene in a coleopteran pest that contacts the transformed
plant.
51. A method for producing a transgenic plant cell, the method
comprising: transforming a plant cell with a vector comprising a
means for providing hemipteran pest protection to a plant;
culturing the transformed plant cell under conditions sufficient to
allow for development of a plant cell culture comprising a
plurality of transformed plant cells; selecting for transformed
plant cells that have integrated the means for providing hemipteran
pest protection to a plant into their genomes; screening the
transformed plant cells for expression of a means for inhibiting
expression of an essential gene in a hemipteran pest; and selecting
a plant cell that expresses the means for inhibiting expression of
an essential gene in a hemipteran pest.
52. A method for producing a transgenic plant protected against a
hemipteran pest, the method comprising: providing the transgenic
plant cell produced by the method of claim 51; and regenerating a
transgenic plant from the transgenic plant cell, wherein expression
of the means for inhibiting expression of an essential gene in a
hemipteran pest is sufficient to modulate the expression of a
target gene in a hemipteran pest that contacts the transformed
plant.
53. The nucleic acid of claim 1, further comprising a
polynucleotide encoding a polypeptide from Bacillus thuringiensis,
Alcaligenes spp., or Pseudomonas spp.
54. The nucleic acid of claim 53, wherein the polynucleotide
encodes a polypeptide from B. thuringiensis that is selected from a
group comprising Cry1B, Cry1I, Cry2A, Cry3, Cry7A, Cry8, Cry9D,
Cry14, Cry18, Cry22, Cry23, Cry34, Cry35, Cry36, Cry37, Cry43,
Cry55, Cyt1A, and Cyt2C.
55. The cell of claim 16, wherein the cell comprises a
polynucleotide encoding a polypeptide from Bacillus thuringiensis,
Alcaligenes spp., or Pseudomonas spp.
56. The cell of claim 55, wherein the polynucleotide encodes a
polypeptide from B. thuringiensis that is selected from a group
comprising Cry1B, Cry1I, Cry2A, Cry3, Cry7A, Cry8, Cry9D, Cry14,
Cry18, Cry22, Cry23, Cry34, Cry35, Cry36, Cry37, Cry43, Cry55,
Cyt1A, and Cyt2C.
57. The plant of claim 17, wherein the plant comprises a
polynucleotide encoding a polypeptide from Bacillus thuringiensis,
Alcaligenes spp., or Pseudomonas spp.
58. The plant of claim 57, wherein the polynucleotide encodes a
polypeptide from B. thuringiensis that is selected from a group
comprising Cry1B, Cry1I, Cry2A, Cry3, Cry7A, Cry8, Cry9D, Cry14,
Cry18, Cry22, Cry23, Cry34, Cry35, Cry36, Cry37, Cry43, Cry55,
Cyt1A, and Cyt2C.
59. The method according to claim 45, wherein the transformed plant
cell comprises a polynucleotide encoding a polypeptide from
Bacillus thuringiensis, Alcaligenes spp., or Pseudomonas spp.
60. The method according to claim 59, wherein the polynucleotide
encodes a polypeptide from B. thuringiensis that is selected from a
group comprising Cry1B, Cry1I, Cry2A, Cry3, Cry7A, Cry8, Cry9D,
Cry14, Cry18, Cry22, Cry23, Cry34, Cry35, Cry36, Cry37, Cry43,
Cry55, Cyt1A, and Cyt2C.
Description
PRIORITY CLAIM
[0001] This application claims the benefit of the filing date of
U.S. Provisional Patent Application Ser. No. 62/269,382, filed Dec.
18, 2015, which is incorporated herein in its entirety.
TECHNICAL FIELD
[0002] The present invention relates generally to genetic control
of plant damage caused by coleopteran and/or hemipteran pests. In
particular embodiments, the present disclosure relates to
identification of target coding and non-coding sequences, and the
use of recombinant DNA technologies for post-transcriptionally
repressing or inhibiting expression of target coding and non-coding
sequences in the cells of a coleopteran and/or hemipteran pest to
provide a plant protective effect.
BACKGROUND
[0003] The western corn rootworm (WCR), Diabrotica virgifera
virgifera LeConte, is one of the most devastating corn rootworm
species in North America and is a particular concern in
corn-growing areas of the Midwestern United States. The northern
corn rootworm (NCR), Diabrotica barberi Smith and Lawrence, is a
closely-related species that co-inhabits much of the same range as
WCR. There are several other related subspecies of Diabrotica that
are significant pests in the Americas: the Mexican corn rootworm
(MCR), D. virgifera zeae Krysan and Smith; the southern corn
rootworm (SCR), D. undecimpunctata howardi Barber; D. balteata
LeConte; D. undecimpunctata tenella; D. speciosa Germar; and D. u.
undecimpunctata Mannerheim. The United States Department of
Agriculture estimates that corn rootworms cause $1 billion in lost
revenue each year, including $800 million in yield loss and $200
million in treatment costs.
[0004] Both WCR and NCR eggs are deposited in the soil during the
summer. The insects remain in the egg stage throughout the winter.
The eggs are oblong, white, and less than 0.004 inches (0.010 cm)
in length. The larvae hatch in late May or early June, with the
precise timing of egg hatching varying from year to year due to
temperature differences and location. The newly hatched larvae are
white worms that are less than 0.125 inches (0.3175 cm) in length.
Once hatched, the larvae begin to feed on corn roots. Corn
rootworms go through three larval instars. After feeding for
several weeks, the larvae molt into the pupal stage. They pupate in
the soil, and then they emerge from the soil as adults in July and
August. Adult rootworms are about 0.25 inches (0.635 cm) in
length.
[0005] Corn rootworm larvae complete development on corn and
several other species of grasses. Larvae reared on yellow foxtail
emerge later and have a smaller head capsule size as adults than
larvae reared on corn (Ellsbury et al. (2005) Environ. Entomol.
34:627-634). WCR adults feed on corn silk, pollen, and kernels on
exposed ear tips. If WCR adults emerge before corn reproductive
tissues are present, they may feed on leaf tissue, thereby slowing
plant growth and occasionally killing the host plant. However, the
adults will quickly shift to preferred silks and pollen when they
become available. NCR adults also feed on reproductive tissues of
the corn plant, but in contrast rarely feed on corn leaves.
[0006] Most of the rootworm damage in corn is caused by larval
feeding. Newly hatched rootworms initially feed on fine corn root
hairs and burrow into root tips. As the larvae grow larger, they
feed on and burrow into primary roots. When corn rootworms are
abundant, larval feeding often results in the pruning of roots all
the way to the base of the corn stalk. Severe root injury
interferes with the roots' ability to transport water and nutrients
into the plant, reduces plant growth, and results in reduced grain
production, thereby often drastically reducing overall yield.
Severe root injury also often results in lodging of corn plants,
which makes harvest more difficult and further decreases yield.
Furthermore, feeding by adults on the corn reproductive tissues can
result in pruning of silks at the ear tip. If this "silk clipping"
is severe enough during pollen shed, pollination may be
disrupted.
[0007] Control of corn rootworms may be attempted by crop rotation,
chemical insecticides, biopesticides (e.g., the spore-forming
gram-positive bacterium, Bacillus thuringiensis (Bt)), transgenic
plants that express Bt toxins, or a combination thereof. Crop
rotation suffers from the disadvantage of placing unwanted
restrictions upon the use of farmland. Moreover, oviposition of
some rootworm species may occur in crop fields other than corn or
extended diapause results in egg hatching over multiple years,
thereby mitigating the effectiveness of crop rotation practiced
with corn and soybean.
[0008] Chemical insecticides are the most heavily relied upon
strategy for achieving corn rootworm control. Chemical insecticide
use, though, is an imperfect corn rootworm control strategy; over
$1 billion may be lost in the United States each year due to corn
rootworm when the costs of the chemical insecticides are added to
the costs of the rootworm damage that may occur despite the use of
insecticides. High populations of larvae, heavy rains, and improper
application of the insecticide(s) may all result in inadequate corn
rootworm control. Furthermore, the continual use of insecticides
may select for insecticide-resistant rootworm strains, as well as
raise significant environmental concerns due to the toxicity of
many of them to non-target species.
[0009] Stink bugs and other hemipteran insects (heteroptera) are
another important agricultural pest complex. Worldwide over 50
closely related species of stink bugs are known to cause crop
damage (McPherson & McPherson, R. M. (2000) Stink bugs of
economic importance in America north of Mexico CRC Press). These
insects are present in a large number of important crops including
maize, soybean, fruit, vegetables, and cereals.
[0010] Stink bugs go through multiple nymph stages before reaching
the adult stage. The time to develop from eggs to adults is about
30-40 days. Both nymphs and adults feed on sap from soft tissues
into which they also inject digestive enzymes causing extra-oral
tissue digestion and necrosis. Digested plant material and
nutrients are then ingested. Depletion of water and nutrients from
the plant vascular system results in plant tissue damage. Damage to
developing grain and seeds is the most significant as yield and
germination are significantly reduced. Multiple generations occur
in warm climates resulting in significant insect pressure. Current
management of stink bugs relies on insecticide treatment on an
individual field basis. Therefore, alternative management
strategies are urgently needed to minimize ongoing crop losses.
[0011] RNA interference (RNAi) is a process utilizing endogenous
cellular pathways, whereby an interfering RNA (iRNA) molecule
(e.g., a dsRNA molecule) that is specific for all, or any portion
of adequate size, of a target gene sequence results in the
degradation of the mRNA encoded thereby. In recent years, RNAi has
been used to perform gene "knockdown" in a number of species and
experimental systems; for example, Caenorhabditis elegans, plants,
insect embryos, and cells in tissue culture. See, e.g., Fire et al.
(1998) Nature 391:806-811; Martinez et al. (2002) Cell 110:563-574;
McManus and Sharp (2002) Nature Rev. Genetics 3:737-747.
[0012] RNAi accomplishes degradation of mRNA through an endogenous
pathway including the DICER protein complex. DICER cleaves long
dsRNA molecules into short fragments of approximately 20
nucleotides, termed small interfering RNA (siRNA). The siRNA is
unwound into two single-stranded RNAs: the passenger strand and the
guide strand. The passenger strand is degraded, and the guide
strand is incorporated into the RNA-induced silencing complex
(RISC). Post-transcriptional gene silencing occurs when the guide
strand binds specifically to a complementary sequence of an mRNA
molecule and induces cleavage by Argonaute, the catalytic component
of the RISC complex. This process is known to spread systemically
throughout some eukaryotic organisms despite initially limited
concentrations of siRNA and/or miRNA such as plants, nematodes, and
some insects.
[0013] U.S. Pat. No. 7,612,194 and U.S. Patent Publication Nos.
2007/0050860, 2010/0192265, and 2011/0154545 disclose a library of
9112 expressed sequence tag (EST) sequences isolated from D. v.
virgifera LeConte pupae. It is suggested in U.S. Pat. No. 7,612,194
and U.S. Patent Publication No. 2007/0050860 to operably link to a
promoter a nucleic acid molecule that is complementary to one of
several particular partial sequences of D. v. virgifera
vacuolar-type H.sup.+-ATPase (V-ATPase) disclosed therein for the
expression of anti-sense RNA in plant cells. U.S. Patent
Publication No. 2010/0192265 suggests operably linking a promoter
to a nucleic acid molecule that is complementary to a particular
partial sequence of a D. v. virgifera gene of unknown and
undisclosed function (the partial sequence is stated to be 58%
identical to C56C10.3 gene product in C. elegans) for the
expression of anti-sense RNA in plant cells. U.S. Patent
Publication No. 2011/0154545 suggests operably linking a promoter
to a nucleic acid molecule that is complementary to two particular
partial sequences of D. v. virgifera coatomer beta subunit genes
for the expression of anti-sense RNA in plant cells. Further, U.S.
Pat. No. 7,943,819 discloses a library of 906 expressed sequence
tag (EST) sequences isolated from D. v. virgifera LeConte larvae,
pupae, and dissected midguts, and suggests operably linking a
promoter to a nucleic acid molecule that is complementary to a
particular partial sequence of a D. v. virgifera charged
multivesicular body protein 4b gene for the expression of
double-stranded RNA in plant cells.
[0014] No further suggestion is provided in U.S. Pat. No.
7,612,194, and U.S. Patent Publication Nos. 2007/0050860,
2010/0192265 and 2011/0154545 to use any particular sequence of the
more than nine thousand sequences listed therein for RNA
interference, other than the several particular partial sequences
of V-ATPase and the particular partial sequences of genes of
unknown function. Furthermore, none of U.S. Pat. No. 7,612,194, and
U.S. Patent Publication Nos. 2007/0050860 and 2010/0192265, and
2011/0154545 provides any guidance as to which other of the over
nine thousand sequences provided would be lethal, or even otherwise
useful, in species of corn rootworm when used as dsRNA or siRNA.
U.S. Pat. No. 7,943,819 provides no suggestion to use any
particular sequence of the more than nine hundred sequences listed
therein for RNA interference, other than the particular partial
sequence of a charged multivesicular body protein 4b gene.
Furthermore, U.S. Pat. No. 7,943,819 provides no guidance as to
which other of the over nine hundred sequences provided would be
lethal, or even otherwise useful, in species of corn rootworm when
used as dsRNA or siRNA. U.S. Patent Application Publication No.
U.S. 2013/040173 and PCT Application Publication No. WO 2013/169923
describes the use of a sequence derived from a Diabrotica virgifera
Snf7 gene for RNA interference in maize. (Also disclosed in
Bolognesi et al. (2012) PLoS ONE 7(10): e47534.
doi:10.1371/journal.pone.0047534).
[0015] The overwhelming majority of sequences complementary to corn
rootworm DNAs (such as the foregoing) are not lethal in species of
corn rootworm when used as dsRNA or siRNA. For example, Baum et al.
(2007, Nature Biotechnology 25:1322-1326), describe the effects of
inhibiting several WCR gene targets by RNAi. These authors reported
that 8 of the 26 target genes they tested were not able to provide
experimentally significant coleopteran pest mortality at a very
high iRNA (e.g., dsRNA) concentration of more than 520
ng/cm.sup.2.
[0016] The authors of U.S. Pat. No. 7,612,194 and U.S. Patent
Publication No. 2007/0050860 made the first report of in planta
RNAi in corn plants targeting the western corn rootworm (Baum et
al. (2007) Nat. Biotechnol. 25(11):1322-6). These authors describe
a high-throughput in vivo dietary RNAi system to screen potential
target genes for developing transgenic RNAi maize. Of an initial
gene pool of 290 targets, only 14 exhibited larval control
potential. One of the most effective double-stranded RNAs (dsRNA)
targeted a gene encoding vacuolar ATPase subunit A (V-ATPase),
resulting in a rapid suppression of corresponding endogenous mRNA
and triggering a specific RNAi response with low concentrations of
dsRNA. Thus, these authors documented for the first time the
potential for in planta RNAi as a possible pest management tool,
while simultaneously demonstrating that effective targets could not
be accurately identified a priori, even from a relatively small set
of candidate genes.
SUMMARY
[0017] Disclosed herein are nucleic acid molecules (e.g., target
genes, DNAs, dsRNAs, siRNAs, shRNA, miRNAs, and hpRNAs), and
methods of use thereof, for the control of coleopteran pests,
including, for example, D. v. virgifera LeConte (western corn
rootworm, "WCR"); D. barberi Smith and Lawrence (northern corn
rootworm, "NCR"); D. u. howardi Barber (southern corn rootworm,
"SCR"); D. v. zeae Krysan and Smith (Mexican corn rootworm, "MCR");
D. balteata LeConte; D. u. tenella; D. speciosa Germar; and D. u.
undecimpunctata Mannerheim and hemipteran pests, including, for
example, Euschistus heros (Fabr.) (Neotropical Brown Stink Bug,
"BSB"), Nezara viridula (L.) (Southern Green Stink Bug), Piezodorus
guildinii (Westwood) (Red-banded Stink Bug), Halyomorpha halys
(stat) (Brown Marmorated Stink Bug), Chinavia hilare (Say) (Green
Stink Bug), Euschistus serous (Say) (Brown Stink Bug), Dichelops
melacanthus (Dallas), Dichelops furcatus (F.), Edessa meditabunda
(F.), Thyanta perditor (F.) (Neotropical Red Shouldered Stink Bug),
Chinavia marginatum (Palisot de Beauvois), Horcias nobilellus
(Berg) (Cotton Bug), Taedia stigmosa (Berg), Dysdercus peruvianus
(Guerin-Meneville), Neomegalotomus parvus (Westwood), Leptoglossus
zonatus (Dallas), Niesthrea sidae (F.), Lygus hesperus (Knight)
(Western Tarnished Plant Bug), and Lygus lineolaris (Palisot de
Beauvois). In particular examples, exemplary nucleic acid molecules
are disclosed that may be homologous to at least a portion of one
or more native nucleic acid sequences in a coleopteran and/or
hemipteran pest.
[0018] In these and further examples, the native nucleic acid
sequence may be a target gene, the product of which may be, for
example and without limitation: involved in a metabolic process or
involved in larval/nymph development. In some examples,
post-translational inhibition of the expression of a target gene by
a nucleic acid molecule comprising a sequence homologous thereto
may be lethal in coleopteran and/or hemipteran pests, or result in
reduced growth and/or development. In specific examples, a gene
encoding a fusion protein that is post-translationally cleaved into
ribosomal protein RpL40 and a mono-ubiquitin (referred to herein
collectively as rpL40) may be selected as a target gene for
post-transcriptional silencing. In particular examples, a target
gene useful for post-transcriptional inhibition is the novel gene
referred to herein as rpL40. An isolated nucleic acid molecule
comprising a nucleotide sequence of rpL40 (SEQ ID NOs:1, 3, 5, and
89); the complement of rpL40 (SEQ ID NOs:1, 3, 5, and 89); and
fragments of any of the foregoing is therefore disclosed
herein.
[0019] Also disclosed are nucleic acid molecules comprising a
nucleotide sequence that encodes a polypeptide that is at least 85%
identical to an amino acid sequence within a target gene product
(for example, the product of a gene referred to as RPL40 and/or
mono-ubiquitin, collectively referred to herein as RPL40). For
example, a nucleic acid molecule may comprise a nucleotide sequence
encoding a polypeptide that is at least 85% identical to an amino
acid sequence of SEQ ID NOs:2, 4, 6, 7, 8, and 90 (RPL40 protein).
In particular examples, a nucleic acid molecule comprises a
nucleotide sequence encoding a polypeptide that is at least 85%
identical to an amino acid sequence within a product of RPL40.
Further disclosed are nucleic acid molecules comprising a
nucleotide sequence that is the reverse complement of a nucleotide
sequence that encodes a polypeptide at least 85% identical to an
amino acid sequence within a target gene product.
[0020] Also disclosed are cDNA sequences that may be used for the
production of iRNA (e.g., dsRNA, siRNA, shRNA, miRNA, and hpRNA)
molecules that are complementary to all or part of a coleopteran
and/or hemipteran pest target gene, for example: rpL40. In
particular embodiments, dsRNAs, siRNAs, shRNA, miRNAs, and/or
hpRNAs may be produced in vitro or in vivo by a
genetically-modified organism, such as a plant or bacterium. In
particular examples, cDNA molecules are disclosed that may be used
to produce iRNA molecules that are complementary to all or part of
rpL40 (S SEQ ID NOs:1, 3, 5, and 89).
[0021] Further disclosed are means for inhibiting expression of an
essential gene in a coleopteran and/or hemipteran pest, and means
for protecting a plant from coleopteran and/or hemipteran pests. A
means for inhibiting expression of an essential gene in a
coleopteran and/or hemipteran pest is a single- or double-stranded
RNA molecule consisting of at least one of SEQ ID NO:9 (Diabrotica
rpL40-1 region 1, herein sometimes referred to as rpL40-1 reg1), or
SEQ ID NO:10 (Diabrotica rpL40-3 region 3, herein sometimes
referred to as rpL40-3 reg3), or SEQ ID NO:11 (Diabrotica rpL40-1
version 1, herein sometimes referred to as rpL40-1 v1), or SEQ ID
NO:12 (Diabrotica rpL40-1 version 2, herein sometimes referred to
as rpL40-1 v2), or SEQ ID NO:13 (Diabrotica rpL40-1 version 3,
herein sometimes referred to as rpL40-1 v3), or SEQ ID NO:14
(Diabrotica rpL40-1 version 4, herein sometimes referred to as
rpL40-1 v4), or SEQ ID NO:15 (Diabrotica rpL40-1 version 5, herein
sometimes referred to as rpL40-1 v5), or SEQ ID NO:91 (Euschistus
heros rpL40 region 1, herein sometimes referred to as BSB_rpL40
reg1), or SEQ ID NO:92 (Euschistus heros rpL40 version 1, herein
sometimes referred to as BSB_rpL40 v1), or the complement thereof.
Functional equivalents of means for inhibiting expression of an
essential gene in a coleopteran and/or hemipteran pest include
single- or double-stranded RNA molecules that are substantially
homologous to all or part of a WCR or BSB gene comprising SEQ ID
NOs:1, 3, 5, or 89. A means for protecting a plant from coleopteran
and/or hemipteran pests is a DNA molecule comprising a nucleic acid
sequence encoding a means for inhibiting expression of an essential
gene in a coleopteran and/or hemipteran pest operably linked to a
promoter, wherein the DNA molecule is capable of being integrated
into the genome of a plant.
[0022] Disclosed are methods for controlling a population of an
insect pest (e.g., a coleopteran or hemipteran pest), comprising
providing to an insect pest (e.g., a coleopteran or hemipteran
pest) an iRNA (e.g., dsRNA, siRNA, shRNA, miRNA, and hpRNA)
molecule that functions upon being taken up by the pest to inhibit
a biological function within the pest.
[0023] In some embodiments, methods for controlling a population of
a coleopteran pest comprises providing to the coleopteran pest an
iRNA molecule that comprises all or part of a polynucleotide
selected from the group consisting of: SEQ ID NO:98; the complement
of SEQ ID NO:98; SEQ ID NO:99; the complement of SEQ ID NO:99; SEQ
ID NO:100; the complement of SEQ ID NO:100; SEQ ID NO:101; the
complement of SEQ ID NO:101; SEQ ID NO:102; the complement of SEQ
ID NO:102; SEQ ID NO:103; the complement of SEQ ID NO:103; SEQ ID
NO:104; the complement of SEQ ID NO:104; SEQ ID NO:105; the
complement of SEQ ID NO:105; SEQ ID NO:106; the complement of SEQ
ID NO:106; SEQ ID NO:107; the complement of SEQ ID NO:107; a
polynucleotide that hybridizes to a native rpL40 polynucleotide of
a coleopteran pest (e.g., WCR); the complement of a polynucleotide
that hybridizes to a native rpL40 polynucleotide of a coleopteran
pest; a polynucleotide that hybridizes to a native coding
polynucleotide of a Diabrotica organism (e.g., WCR) comprising all
or part of any of SEQ ID NOs:1, 3, 5 and 9-15; and the complement
of a polynucleotide that hybridizes to a native coding
polynucleotide of a Diabrotica organism comprising all or part of
any of SEQ ID NOs:1, 3, 5, and 9-15.
[0024] In some embodiments, a methods for controlling a population
of a hemipteran pest comprises providing to the hemipteran pest an
iRNA molecule that comprises all or part of a polynucleotide
selected from the group consisting of: SEQ ID NO:108; the
complement of SEQ ID NO:108; SEQ ID NO:109; the complement of SEQ
ID NO:109; SEQ ID NO:110; the complement of SEQ ID NO:110; a
polynucleotide that hybridizes to a native rpL40 polynucleotide of
a hemipteran pest (e.g., BSB); the complement of a polynucleotide
that hybridizes to a native rpL40 polynucleotide of a hemipteran
pest; a polynucleotide that hybridizes to a native coding
polynucleotide of a hemipteran organism (e.g., BSB) comprising all
or part of any of SEQ ID NOs:76, 78, and 80-82; and the complement
of a polynucleotide that hybridizes to a native coding
polynucleotide of a hemipteran organism comprising all or part of
any of SEQ ID NOs:76, 78, and 80-82.
[0025] In particular embodiments, an iRNA that functions upon being
taken up by an insect pest to inhibit a biological function within
the pest is transcribed from a DNA comprising all or part of a
polynucleotide selected from the group consisting of: SEQ ID NOs:1,
3, 5, 9-15, 89, and 91-92; the complement of SEQ ID NOs:1, 3, 5,
9-15, 89, and 91-92; a native coding sequence of a Diabrotica
organism (e.g., WCR) or hemipteran organism (e.g. BSB) comprising
all or part of any of SEQ ID NOs:1, 3, 5, 9-15, 89, and 91-92; the
complement of a native coding sequence of a Diabrotica organism or
hemipteran organism comprising all or part of any of SEQ ID NOs:1,
3, 5, 9-15, 89, and 91-92; a native non-coding sequence of a
Diabrotica organism or hemipteran organism that is transcribed into
a native RNA molecule comprising all or part of any of SEQ ID
NOs:1, 3, 5, 9-15, 89, and 91-92; and the complement of a native
non-coding sequence of a Diabrotica organism or hemipteran organism
that is transcribed into a native RNA molecule comprising all or
part of any of SEQ ID NOs:1, 3, 5, 9-15, 89, and 91-92.
[0026] Also disclosed herein are methods wherein dsRNAs, siRNAs,
shRNAs, miRNAs, and/or hpRNAs may be provided to a coleopteran
and/or hemipteran pest in a diet-based assay, or in
genetically-modified plant cells expressing the dsRNAs, siRNAs,
shRNAs, miRNAs, and/or hpRNAs. In these and further examples, the
dsRNAs, siRNAs, shRNAs, miRNAs, and/or hpRNAs may be ingested by
coleopteran pest larvae and/or hemipteran pest nymphs. Ingestion of
dsRNAs, siRNA, shRNAs, miRNAs, and/or hpRNAs of the invention may
then result in RNAi in the larvae or nymph, which in turn may
result in silencing of a gene essential for viability of the
coleopteran and/or hemipteran pest and leading ultimately to
larval/nymph mortality. Thus, methods are disclosed wherein nucleic
acid molecules comprising exemplary nucleic acid sequence(s) useful
for control of coleopteran and/or hemipteran pests are provided to
a coleopteran and/or hemipteran pest. In particular examples, the
coleopteran and/or hemipteran pest controlled by use of nucleic
acid molecules of the invention may be WCR, NCR, SCR, MCR, D.
balteata, D. u. tenella, D. speciosa, D. u. undecimpunctata,
Euschistus heros, E. serous, Piezodorus guildinii, Halyomorpha
halys, Nezara viridula, Chinavia hilare, C. marginatum, Dichelops
melacanthus, D. furcatus, Edessa meditabunda, Thyanta perditor,
Horcias nobilellus, Taedia stigmosa, Dysdercus peruvianus,
Neomegalotomus parvus, Leptoglossus zonatus, Niesthrea sidae, Lygus
hesperus, and/or Lygus lineolaris.
[0027] The foregoing and other features will become more apparent
from the following Detailed Description of several embodiments,
which proceeds with reference to the accompanying FIGS. 1 and
2.
BRIEF DESCRIPTION OF THE FIGURES
[0028] FIG. 1 includes a depiction of a strategy used to provide
dsRNA from a single transcription template with a single pair of
primers.
[0029] FIG. 2 includes a depiction of a strategy used to provide
dsRNA from two transcription templates.
SEQUENCE LISTING
[0030] The nucleic acid sequences identified in the accompanying
sequence listing are shown using standard letter abbreviations for
nucleotide bases, as defined in 37 C.F.R. .sctn. 1.822. The nucleic
acid and amino acid sequences listed define molecules (i.e.,
polynucleotides and polypeptides, respectively) having the
nucleotide and amino acid monomers arranged in the manner
described. The nucleic acid and amino acid sequences listed also
each define a genus of polynucleotides or polypeptides that
comprise the nucleotide and amino acid monomers arranged in the
manner described. In view of the redundancy of the genetic code, it
will be understood that a nucleotide sequence including a coding
sequence also describes the genus of polynucleotides encoding the
same polypeptide as a polynucleotide consisting of the reference
sequence. It will further be understood that an amino acid sequence
describes the genus of polynucleotide ORFs encoding that
polypeptide.
[0031] Only one strand of each nucleic acid sequence is shown, but
the complementary strand is understood as included by any reference
to the displayed strand. As the complement and reverse complement
of a primary nucleic acid sequence are necessarily disclosed by the
primary sequence, the complementary sequence and reverse
complementary sequence of a nucleic acid sequence are included by
any reference to the nucleic acid sequence, unless it is explicitly
stated to be otherwise (or it is clear to be otherwise from the
context in which the sequence appears). Furthermore, as it is
understood in the art that the nucleotide sequence of a RNA strand
is determined by the sequence of the DNA from which it was
transcribed (but for the substitution of uracil (U) nucleobases for
thymine (T)), a RNA sequence is included by any reference to the
DNA sequence encoding it. In the accompanying sequence listing:
[0032] SEQ ID NO:1 shows a DNA sequence comprising rpL40-1 from
Diabrotica virgifera.
[0033] SEQ ID NO:2 shows an amino acid sequence of a RPL40-1
protein from Diabrotica virgifera.
[0034] SEQ ID NO:3 shows a DNA sequence comprising rpL40-2 from
Diabrotica virgifera.
[0035] SEQ ID NO:4 shows an amino acid sequence of a RPL40-2
protein from Diabrotica virgifera.
[0036] SEQ ID NO:5 shows a DNA sequence comprising rpL40-3 from
Diabrotica virgifera.
[0037] SEQ ID NO:6 shows an amino acid sequence of a RPL40-3
protein from Diabrotica virgifera.
[0038] SEQ ID NO:7 shows an amino acid sequence of a hypothetical
protein protein from Diabrotica virgifera SEQ ID NO:5.
[0039] SEQ ID NO:8 shows an amino acid sequence of a hypothetical
protein protein from Diabrotica virgifera SEQ ID NO:5.
[0040] SEQ ID NO:9 shows a DNA sequence of rpL40-1 reg1 (region 1)
from Diabrotica virgifera that was used for in vitro dsRNA
synthesis (T7 promoter sequences at 5' and 3' ends not shown).
[0041] SEQ ID NO:10 shows a DNA sequence of rpL40-3 reg1 (region 3)
from Diabrotica virgifera that was used for in vitro dsRNA
synthesis (T7 promoter sequences at 5' and 3' ends not shown).
[0042] SEQ ID NO:11 shows a DNA reverse complement sequence of
rpL40-1 v1 (version 1) from Diabrotica virgifera that was used for
in vitro dsRNA synthesis (T7 promoter sequences at 5' and 3' ends
not shown).
[0043] SEQ ID NO:12 shows a DNA sequence of rpL40-1 v2 (version 2)
from Diabrotica virgifera that was used for in vitro dsRNA
synthesis (T7 promoter sequences at 5' and 3' ends not shown).
[0044] SEQ ID NO:13 shows a DNA sequence of rpL40-1 v3 (version 3)
from Diabrotica virgifera that was used for in vitro dsRNA
synthesis (T7 promoter sequences at 5' and 3' ends not shown).
[0045] SEQ ID NO:14 shows a DNA sequence of rpL40-1 v4 (version 4)
from Diabrotica virgifera that was used for in vitro dsRNA
synthesis (T7 promoter sequences at 5' and 3' ends not shown).
[0046] SEQ ID NO:15 shows a DNA sequence of rpL40-1 v5 (version 5)
from Diabrotica virgifera that was used for in vitro dsRNA
synthesis (T7 promoter sequences at 5' and 3' ends not shown).
[0047] SEQ ID NO:16 shows a DNA sequence of a T7 phage
promoter.
[0048] SEQ ID NO:17 shows a DNA sequence of a YFP coding region
segment that was used for in vitro dsRNA synthesis (T7 promoter
sequences at 5' and 3' ends not shown).
[0049] SEQ ID NOs:18 to 31 show primers used to amplify portions of
a rpL40 subunit sequence from Diabrotica virgifera comprising rpL40
reg1 and rpL40 reg3.
[0050] SEQ ID NO:32 shows a YFP protein coding sequence
[0051] SEQ ID NO:33 shows a DNA sequence of Annexin region 1.
[0052] SEQ ID NO:34 shows a DNA sequence of Annexin region 2.
[0053] SEQ ID NO:35 shows a DNA sequence of Beta spectrin 2 region
1.
[0054] SEQ ID NO:36 shows a DNA sequence of Beta spectrin 2 region
2.
[0055] SEQ ID NO:37 shows a DNA sequence of mtRP-L4 region 1.
[0056] SEQ ID NO:38 shows a DNA sequence of mtRP-L4 region 2.
[0057] SEQ ID NOs:29 to 66 show primers used to amplify gene
regions of YFP, Annexin, Beta spectrin 2, and mtRP-L4 for dsRNA
synthesis.
[0058] SEQ ID NO:67 shows a maize DNA sequence encoding a
TIP41-like protein.
[0059] SEQ ID NO:68 shows a DNA sequence of oligonucleotide
T20NV.
[0060] SEQ ID NOs:69 to 73 show sequences of primers and probes
used to measure maize transcript levels.
[0061] SEQ ID NO:74 shows a DNA sequence of a portion of a SpecR
coding region used for binary vector backbone detection.
[0062] SEQ ID NO:75 shows a DNA sequence of a portion of an AAD1
coding region used for genomic copy number analysis.
[0063] SEQ ID NO:76 shows a DNA sequence of a maize invertase
gene.
[0064] SEQ ID NOs:77 to 85 show sequences of primers and probes
used for gene copy number analyses.
[0065] SEQ ID NOs:86 to 88 show sequences of primers and probes
used for maize expression analysis.
[0066] SEQ ID NO:89 shows an exemplary DNA sequence of BSB rpL40
transcript from a Neotropical Brown Stink Bug (Euschistus
heros).
[0067] SEQ ID NO:90 shows an amino acid sequence of a from
Euschistus heros RPL40 protein.
[0068] SEQ ID NO:91 shows a DNA sequence of BSB rpL40 reg1 (region
1) from Euschistus heros that was used for in vitro dsRNA synthesis
(T7 promoter sequences at 5' and 3' ends not shown).
[0069] SEQ ID NO:92 shows a DNA sequence of BSB rpL40 v1 (version
1) from Euschistus heros that was used for in vitro dsRNA synthesis
(T7 promoter sequences at 5' and 3' ends not shown).
[0070] SEQ ID NO:93-94 show primers used to amplify portions of a
from Euschistus heros rpL40 sequence comprising BSB rpL40 regi.
[0071] SEQ ID NO:95 is the sense strand of YFP-targeted dsRNA:
YFPv2
[0072] SEQ ID NO:96-97 show primers used to amplify portions of a
YFP-targeted dsRNA: YFPv2
[0073] SEQ ID NOs:98-110 show exemplary RNAs transcribed from
nucleic acids comprising exemplary rpL40 polynucleotides and
fragments thereof.
[0074] SEQ ID NO:111 shows an IDT Custom Oligo probe rp140 PRB
Setl, labeled with FAM and double quenched with Zen and Iowa Black
quenchers.
[0075] SEQ ID NO:112 shows an exemplary linker polynucleotide,
which forms a "loop" when transcribed in an RNA transcript to form
a hairpin structure:
DETAILED DESCRIPTION
I. Overview of Several Embodiments
[0076] We developed RNA interference (RNAi) as a tool for insect
pest management, using one of the most likely target pest species
for transgenic plants that express dsRNA; the western corn
rootworm. Thus far, most genes proposed as targets for RNAi in
rootworm larvae do not actually achieve their purpose. Herein, we
describe RNAi-mediated knockdown of ribosomal protein L40 (rpL40)
in the exemplary insect pests, western corn rootworm and
Neotropical brown stink bug, which is shown to have a lethal
phenotype when, for example, iRNA molecules are delivered via
ingested or injected rpL40 dsRNA. In embodiments herein, the
ability to deliver rpL40 dsRNA by feeding to insects confers an
RNAi effect that is very useful for insect (e.g., coleopteran and
hemipteran) pest management. By combining rpL40-mediated RNAi with
other useful RNAi targets (e.g., ROP (U.S. patent application
Publication Ser. No. 14/577,811), RNAPII (U.S. patent application
Publication Ser. No. 14/577,854), RNA polymerase II RNAi targets
(U.S. Patent Application No. 62/133,214), RNA polymerase II215 RNAi
targets (U.S. Patent Application No. 62/133,202), RNA polymerase
II33 RNAi targets (U.S. Patent Application No. 62/133,210), ncm
RNAi targets (U.S. Patent Application No. 62/095,487), snap25 RNAi
targets (U.S. Patent Application No. 62/193,502), transcription
elongation factor spt5 RNAi targets (U.S. Patent Application No.
62/168,613), transcription elongation factor spt6 RNAi targets
(U.S. Patent Application No. 62/168,606), COPI alpha (U.S. Patent
Application No. 62/063,199), COPI gamma (U.S. Patent Application
No. 62/063,192), COPI delta (U.S. Patent Application No.
62/063,216), COPI beta (U.S. Patent Application No. 62/063,203),
sec23 (U.S. Patent Application No. 61/989,170), sec24 (U.S. Patent
Application No. 62/061,608), and dre4 (U.S. Patent Application No.
61/989,843)), the potential to affect multiple target sequences,
for example, in larval rootworms, may increase opportunities to
develop sustainable approaches to insect pest management involving
RNAi technologies.
[0077] Disclosed herein are methods and compositions for genetic
control of coleopteran and/or hemipteran pest infestations. Methods
for identifying one or more gene(s) essential to the lifecycle of a
coleopteran and/or hemipteran pest for use as a target gene for
RNAi-mediated control of a coleopteran and/or hemipteran pest
population are also provided. DNA plasmid vectors encoding one or
more dsRNA molecules may be designed to suppress one or more target
gene(s) essential for growth, survival, development, and/or
reproduction. In some embodiments, methods are provided for
post-transcriptional repression of expression or inhibition of a
target gene via nucleic acid molecules that are complementary to a
coding or non-coding sequence of the target gene in a coleopteran
and/or hemipteran pest. In these and further embodiments, a
coleopteran and/or hemipteran pest may ingest one or more dsRNA,
siRNA, shRNA, miRNA, and/or hpRNA molecules transcribed from all or
a portion of a nucleic acid molecule that is complementary to a
coding or non-coding sequence of a target gene, thereby providing a
plant-protective effect.
[0078] Thus, some embodiments involve sequence-specific inhibition
of expression of target gene products, using dsRNA, siRNA, shRNA,
miRNA and/or hpRNA that is complementary to coding and/or
non-coding sequences of the target gene(s) to achieve at least
partial control of a coleopteran and/or hemipteran pest. Disclosed
is a set of isolated and purified nucleic acid molecules comprising
a nucleotide sequence, for example, as set forth in any of SEQ ID
NOs:1, 3, 5, 9-15, 89, 91-92, and fragments thereof. In some
embodiments, a stabilized dsRNA molecule may be expressed from this
sequence, fragments thereof, or a gene comprising one of these
sequences, for the post-transcriptional silencing or inhibition of
a target gene. In certain embodiments, isolated and purified
nucleic acid molecules comprise all or part of SEQ ID NO:1. In
other embodiments, isolated and purified nucleic acid molecules
comprise all or part of SEQ ID NO:3. In still further embodiments,
isolated and purified nucleic acid molecules comprise all or part
of SEQ ID NO:5. In other embodiments, isolated and purified nucleic
acid molecules comprise all or part of SEQ ID NO:9. In yet other
embodiments, isolated and purified nucleic acid molecules comprise
all or part of SEQ ID NOs:10-15, SEQ ID NO:89, or SEQ ID
NOs:91-92.
[0079] Some embodiments involve a recombinant host cell (e.g., a
plant cell) having in its genome at least one recombinant DNA
sequence encoding at least one iRNA (e.g., dsRNA) molecule(s). In
particular embodiments, the dsRNA molecule(s) may be produced when
ingested by a coleopteran and/or hemipteran pest to
post-transcriptionally silence or inhibit the expression of a
target gene in the coleopteran and/or hemipteran pest. The
recombinant DNA sequence may comprise, for example, one or more of
any of SEQ ID NOs:1, 3, 5, 9-15, 89, or 91-92; fragments of any of
SEQ ID NOs:1, 3, 5, 9-15, 89, or 91-92; or a partial sequence of a
gene comprising one or more of SEQ ID NOs:1, 3, 5, 9-15, 89, or
91-92; or complements thereof.
[0080] Some embodiments involve a recombinant host cell having in
its genome a recombinant DNA sequence encoding at least one iRNA
(e.g., dsRNA) molecule(s) comprising all or part of SEQ ID NO:98,
SEQ ID NO:99, SEQ ID NO:100, and/or SEQ ID NO:108 (e.g., at least
one polynucleotide selected from a group comprising SEQ ID
NOs:101-107 and 109-110), or the complement thereof. When ingested
by a coleopteran and/or hemipteran pest, the iRNA molecule(s) may
silence or inhibit the expression of a target gene comprising SEQ
ID NOs:1, 3, 5, 9-15, 89, and/or 91-92, in the coleopteran and/or
hemipteran pest, and thereby result in cessation of growth,
development, reproduction, and/or feeding in the coleopteran and/or
hemipteran pest.
[0081] In some embodiments, a recombinant host cell having in its
genome at least one recombinant DNA sequence encoding at least one
dsRNA molecule may be a transformed plant cell. Some embodiments
involve transgenic plants comprising such a transformed plant cell.
In addition to such transgenic plants, progeny plants of any
transgenic plant generation, transgenic seeds, and transgenic plant
products, are all provided, each of which comprises recombinant DNA
sequence(s). In particular embodiments, a dsRNA molecule of the
invention may be expressed in a transgenic plant cell. Therefore,
in these and other embodiments, a dsRNA molecule of the invention
may be isolated from a transgenic plant cell. In particular
embodiments, the transgenic plant is a plant selected from the
group comprising corn (Zea mays), soybean (Glycine max), and plants
of the family Poaceae.
[0082] Some embodiments involve a method for modulating the
expression of a target gene in a coleopteran and/or hemipteran pest
cell. In these and other embodiments, a nucleic acid molecule may
be provided, wherein the nucleic acid molecule comprises a
nucleotide sequence encoding a dsRNA molecule. In particular
embodiments, a nucleotide sequence encoding a dsRNA molecule may be
operatively linked to a promoter, and may also be operatively
linked to a transcription termination sequence. In particular
embodiments, a method for modulating the expression of a target
gene in a coleopteran and/or hemipteran pest cell may comprise: (a)
transforming a plant cell with a vector comprising a nucleotide
sequence encoding a dsRNA molecule; (b) culturing the transformed
plant cell under conditions sufficient to allow for development of
a plant cell culture comprising a plurality of transformed plant
cells; (c) selecting for a transformed plant cell that has
integrated the vector into its genome; and (d) determining that the
selected transformed plant cell comprises the dsRNA molecule
encoded by the nucleotide sequence of the vector. A plant may be
regenerated from a plant cell that has the vector integrated in its
genome and comprises the dsRNA molecule encoded by the nucleotide
sequence of the vector.
[0083] Thus, also disclosed is a transgenic plant comprising a
vector having a nucleotide sequence encoding a dsRNA molecule
integrated in its genome, wherein the transgenic plant comprises
the dsRNA molecule encoded by the nucleotide sequence of the
vector. In particular embodiments, expression of a dsRNA molecule
in the plant is sufficient to modulate the expression of a target
gene in a cell of a coleopteran and/or hemipteran pest that
contacts the transformed plant or plant cell, for example, by
feeding on the transformed plant, a part of the plant (e.g., root)
or plant cell. Transgenic plants disclosed herein may display
resistance and/or enhanced tolerance to coleopteran and/or
hemipteran pest infestations. Particular transgenic plants may
display resistance and/or enhanced tolerance to one or more
coleopteran and/or hemipteran pests selected from the group
consisting of: WCR, NCR, SCR, MCR, D. balteata, D. u. tenella, D.
speciosa, D. u. undecimpunctata, Euschistus heros, E. serous,
Piezodorus guildinii, Halyomorpha halys, Nezara viridula, Chinavia
hilare, C. marginatum, Dichelops melacanthus, D. furcatus, Edessa
meditabunda, Thyanta perditor, Horcias nobilellus, Taedia stigmosa,
Dysdercus peruvianus, Neomegalotomus parvus, Leptoglossus zonatus,
Niesthrea sidae, Lygus hesperus, and/or Lygus lineolaris.
[0084] Also disclosed herein are methods for delivery of control
agents, such as an iRNA molecule, to a coleopteran and/or
hemipteran pest. Such control agents may cause, directly or
indirectly, impairment in the ability of the coleopteran and/or
hemipteran pest to feed, grow or otherwise cause damage in a host.
In some embodiments, a method is provided comprising delivery of a
stabilized dsRNA molecule to a coleopteran and/or hemipteran pest
to suppress at least one target gene in the coleopteran and/or
hemipteran pest, thereby reducing or eliminating plant damage by a
coleopteran and/or hemipteran pest. In some embodiments, a method
of inhibiting expression of a target gene in a coleopteran and/or
hemipteran pest may result in the cessation of growth, development,
reproduction, and/or feeding in the coleopteran and/or hemipteran
pest. In some embodiments, the method may eventually result in
death of the coleopteran and/or hemipteran pest.
[0085] In some embodiments, compositions (e.g., a topical
composition) are provided that comprise an iRNA (e.g., dsRNA)
molecule of the invention for use in plants, animals, and/or the
environment of a plant or animal to achieve the elimination or
reduction of a coleopteran and/or hemipteran pest infestation. In
particular embodiments, the composition may be a nutritional
composition or food source to be fed to the coleopteran and/or
hemipteran pest. Some embodiments comprise making the nutritional
composition or food source available to the coleopteran and/or
hemipteran pest. Ingestion of a composition comprising iRNA
molecules may result in the uptake of the molecules by one or more
cells of the coleopteran and/or hemipteran pest, which may in turn
result in the inhibition of expression of at least one target gene
in cell(s) of the coleopteran and/or hemipteran pest. Ingestion of
or damage to a plant or plant cell by a coleopteran and/or
hemipteran pest may be limited or eliminated in or on any host
tissue or environment in which the coleopteran and/or hemipteran
pest is present by providing one or more compositions comprising an
iRNA molecule of the invention in the host of the coleopteran
and/or hemipteran pest.
[0086] RNAi baits are formed when the dsRNA is mixed with food or
an attractant or both. When the pests eat the bait, they also
consume the dsRNA. Baits may take the form of granules, gels,
flowable powders, liquids, or solids. In another embodiment, rab5
may be incorporated into a bait formulation such as that described
in U.S. Pat. No. 8,530,440 which is hereby incorporated by
reference. Generally, with baits, the baits are placed in or around
the environment of the insect pest, for example, WCR can come into
contact with, and/or be attracted to, the bait.
[0087] The compositions and methods disclosed herein may be used
together in combinations with other methods and compositions for
controlling damage by coleopteran and/or hemipteran pests. For
example, an iRNA molecule as described herein for protecting plants
from coleopteran and/or hemipteran pests may be used in a method
comprising the additional use of one or more chemical agents
effective against a coleopteran and/or hemipteran pest,
biopesticides effective against a coleopteran and/or hemipteran
pest, crop rotation, or recombinant genetic techniques that exhibit
features different from the features of the RNAi-mediated methods
and RNAi compositions of the invention (e.g., recombinant
production of proteins in plants that are harmful to a coleopteran
and/or hemipteran pest (e.g., Bt toxins)).
II. Abbreviations
[0088] dsRNA double-stranded ribonucleic acid
[0089] GI growth inhibition
[0090] NCBI National Center for Biotechnology Information
[0091] gDNA genomic Deoxyribonucleic Acid
[0092] iRNA inhibitory ribonucleic acid
[0093] ORF open reading frame
[0094] RNAi ribonucleic acid interference
[0095] miRNA micro ribonucleic acid
[0096] shRNA small hairpin ribonucleic acid
[0097] siRNA small inhibitory ribonucleic acid
[0098] hpRNA hairpin ribonucleic acid
[0099] UTR untranslated region
[0100] WCR western corn rootworm (Diabrotica virgifera virgifera
LeConte)
[0101] NCR northern corn rootworm (Diabrotica barberi Smith and
Lawrence)
[0102] MCR Mexican corn rootworm (Diabrotica virgifera zeae Krysan
and Smith)
[0103] PCR Polymerase chain reaction
[0104] RISC RNA-induced Silencing Complex
[0105] SCR southern corn rootworm (Diabrotica undecimpunctata
howardi Barber)
[0106] BSB Neotropical brown stink bug (Euschistus heros
Fabricius)
[0107] YFP yellow fluorescent protein
[0108] SEM standard error of the mean
III. Terms
[0109] In the description and tables which follow, a number of
terms are used. In order to provide a clear and consistent
understanding of the specification and claims, including the scope
to be given such terms, the following definitions are provided:
[0110] Coleopteran pest: As used herein, the term "coleopteran
pest" refers to insects of the genus Diabrotica, which feed upon
corn and other true grasses. In particular examples, a coleopteran
pest is selected from the list comprising D. v. virgifera LeConte
(WCR); D. barberi Smith and Lawrence (NCR); D. u. howardi (SCR); D.
v. zeae (MCR); D. balteata LeConte; D. u. tenella; D. speciosa
Germar; and D. u. undecimpunctata Mannerheim.
[0111] Hemipteran pest: As used herein, the term "hemipteran pest"
refers to insects of the order Hemiptera, including, for example
and without limitation, insects in the families Pentatomidae,
Miridae, Pyrrhocoridae, Coreidae, Alydidae, and Rhopalidae, which
feed on a wide range of host plants and have piercing and sucking
mouth parts. In particular examples, a hemipteran pest is selected
from the list comprising, Euschistus heros (Fabr.) (Neotropical
Brown Stink Bug), Nezara viridula (L.) (Southern Green Stink Bug),
Piezodorus guildinii (Westwood) (Red-banded Stink Bug), Halyomorpha
halys (stat) (Brown Marmorated Stink Bug), Chinavia hilare (Say)
(Green Stink Bug), Euschistus serous (Say) (Brown Stink Bug),
Dichelops melacanthus (Dallas), Dichelops furcatus (F.), Edessa
meditabunda (F.), Thyanta perditor (F.) (Neotropical Red Shouldered
Stink Bug), Chinavia marginatum (Palisot de Beauvois), Horcias
nobilellus (Berg) (Cotton Bug), Taedia stigmosa (Berg), Dysdercus
peruvianus (Guerin-Meneville), Neomegalotomus parvus (Westwood),
Leptoglossus zonatus (Dallas), Niesthrea sidae (F.), Lygus hesperus
(Knight) (Western Tarnished Plant Bug), and Lygus lineolaris
(Palisot de Beauvois).
[0112] Contact (with an organism): As used herein, the term
"contact with" or "uptake by" an organism (e.g., a coleopteran
and/or hemipteran pest), with regard to a nucleic acid molecule,
includes internalization of the nucleic acid molecule into the
organism, for example and without limitation: ingestion of the
molecule by the organism (e.g., by feeding); contacting the
organism with a composition comprising the nucleic acid molecule;
and soaking of organisms with a solution comprising the nucleic
acid molecule.
[0113] Contig: As used herein the term "contig" refers to a DNA
sequence that is reconstructed from a set of overlapping DNA
segments derived from a single genetic source.
[0114] Corn plant: As used herein, the term "corn plant" refers to
a plant of the species, Zea mays (maize).
[0115] Encoding a dsRNA: As used herein, the term "encoding a
dsRNA" includes a gene whose RNA transcription product is capable
of forming an intramolecular dsRNA structure (e.g., a hairpin) or
intermolecular dsRNA structure (e.g., by hybridizing to a target
RNA molecule).
[0116] Expression: As used herein, "expression" of a coding
sequence (for example, a gene or a transgene) refers to the process
by which the coded information of a nucleic acid transcriptional
unit (including, e.g., genomic DNA or cDNA) is converted into an
operational, non-operational, or structural part of a cell, often
including the synthesis of a protein. Gene expression can be
influenced by external signals; for example, exposure of a cell,
tissue, or organism to an agent that increases or decreases gene
expression. Expression of a gene can also be regulated anywhere in
the pathway from DNA to RNA to protein. Regulation of gene
expression occurs, for example, through controls acting on
transcription, translation, RNA transport and processing,
degradation of intermediary molecules such as mRNA, or through
activation, inactivation, compartmentalization, or degradation of
specific protein molecules after they have been made, or by
combinations thereof. Gene expression can be measured at the RNA
level or the protein level by any method known in the art,
including, without limitation, northern (RNA) blot, RT-PCR, western
(immuno-) blot, or in vitro, in situ, or in vivo protein activity
assay(s).
[0117] Genetic material: As used herein, the term "genetic
material" includes all genes and nucleic acid molecules, such as
DNA and RNA.
[0118] Inhibition: As used herein, the term "inhibition", when used
to describe an effect on a coding sequence (for example, a gene),
refers to a measurable decrease in the cellular level of mRNA
transcribed from the coding sequence and/or peptide, polypeptide,
or protein product of the coding sequence. In some examples,
expression of a coding sequence may be inhibited such that
expression is approximately eliminated. "Specific inhibition"
refers to the inhibition of a target coding sequence without
consequently affecting expression of other coding sequences (e.g.,
genes) in the cell wherein the specific inhibition is being
accomplished.
[0119] Isolated: An "isolated" biological component (such as a
nucleic acid or protein) has been substantially separated, produced
apart from, or purified away from other biological components in
the cell of the organism in which the component naturally occurs
(i.e., other chromosomal and extra-chromosomal DNA and RNA, and
proteins). Nucleic acid molecules and proteins that have been
"isolated" include nucleic acid molecules and proteins purified by
standard purification methods. The term also embraces nucleic acids
and proteins prepared by recombinant expression in a host cell, as
well as chemically-synthesized nucleic acid molecules, proteins,
and peptides.
[0120] Nucleic acid molecule: As used herein, the term "nucleic
acid molecule" may refer to a polymeric form of nucleotides, which
may include both sense and anti-sense strands of RNA, cDNA, genomic
DNA, and synthetic forms and mixed polymers of the above. A
nucleotide may refer to a ribonucleotide, deoxyribonucleotide, or a
modified form of either type of nucleotide. A "nucleic acid
molecule" as used herein is synonymous with "nucleic acid" and
"polynucleotide." A nucleic acid molecule is usually at least 10
bases in length, unless otherwise specified. By convention, the
nucleotide sequence of a nucleic acid molecule is read from the 5'
to the 3' end of the molecule. The "complement" of a nucleotide
sequence refers to the sequence, from 5' to 3', of the nucleobases
which form base pairs with the nucleobases of the nucleotide
sequence (i.e., A-T/U, and G-C). The "reverse complement" of a
nucleic acid sequence refers to the sequence, from 3' to 5', of the
nucleobases which form base pairs with the nucleobases of the
nucleotide sequence.
[0121] Some embodiments include nucleic acids comprising a template
DNA that is transcribed into an RNA molecule that is the complement
of an mRNA molecule. In these embodiments, the complement of the
nucleic acid transcribed into the mRNA molecule is present in the
5' to 3' orientation, such that RNA polymerase (which transcribes
DNA in the 5' to 3' direction) will transcribe a nucleic acid from
the complement that can hybridize to the mRNA molecule. Unless
explicitly stated otherwise, or it is clear to be otherwise from
the context, the term "complement" therefore refers to a
polynucleotide having nucleobases, from 5' to 3', that may form
base pairs with the nucleobases of a reference nucleic acid.
Similarly, unless it is explicitly stated to be otherwise (or it is
clear to be otherwise from the context), the "reverse complement"
of a nucleic acid refers to the complement in reverse orientation.
The foregoing is demonstrated in the following illustration:
TABLE-US-00001 ATGATGATG polynucleotide TACTACTAC "complement" of
the polynucleotide CATCATCAT "reverse complement" of the
polynucleo- tide
[0122] Some embodiments of the invention may include hairpin
RNA-forming RNAi molecules. In these RNAi molecules, both the
complement of a nucleic acid to be targeted by RNA interference and
the reverse complement may be found in the same molecule, such that
the single-stranded RNA molecule may "fold over" and hybridize to
itself over region comprising the complementary and reverse
complementary polynucleotides, as demonstrated in the following
illustration:
##STR00001##
[0123] "Nucleic acid molecules" include single- and double-stranded
forms of DNA; single-stranded forms of RNA; and double-stranded
forms of RNA (dsRNA). The term "nucleotide sequence" or "nucleic
acid sequence" refers to both the sense and antisense strands of a
nucleic acid as either individual single strands or in the duplex.
The term "ribonucleic acid" (RNA) is inclusive of iRNA (inhibitory
RNA), dsRNA (double stranded RNA), siRNA (small interfering RNA),
mRNA (messenger RNA), shRNA (small hairpin RNA), miRNA (micro-RNA),
hpRNA (hairpin RNA), tRNA (transfer RNA, whether charged or
discharged with a corresponding acylated amino acid), and cRNA
(complementary RNA). The term "deoxyribonucleic acid" (DNA) is
inclusive of cDNA, genomic DNA, and DNA-RNA hybrids. The terms
"polynucleotide" and "nucleic acid" and "fragments" thereof, or
more generally "segment", will be understood by those in the art as
a functional term that includes both genomic sequences, ribosomal
RNA sequences, transfer RNA sequences, messenger RNA sequences,
operon sequences, and smaller engineered nucleotide sequences that
encode or may be adapted to encode, peptides, polypeptides, or
proteins.
[0124] Oligonucleotide: An oligonucleotide is a short nucleic acid
polymer. Oligonucleotides may be formed by cleavage of longer
nucleic acid segments, or by polymerizing individual nucleotide
precursors. Automated synthesizers allow the synthesis of
oligonucleotides up to several hundred bases in length. Because
oligonucleotides may bind to a complementary nucleotide sequence,
they may be used as probes for detecting DNA or RNA.
Oligonucleotides composed of DNA (oligodeoxyribonucleotides) may be
used in PCR, a technique for the amplification of DNA and RNA
(reverse transcribed into a cDNA) sequences. In PCR, the
oligonucleotide is typically referred to as a "primer", which
allows a DNA polymerase to extend the oligonucleotide and replicate
the complementary strand.
[0125] A nucleic acid molecule may include either or both naturally
occurring and modified nucleotides linked together by naturally
occurring and/or non-naturally occurring nucleotide linkages.
Nucleic acid molecules may be modified chemically or biochemically,
or may contain non-natural or derivatized nucleotide bases, as will
be readily appreciated by those of skill in the art. Such
modifications include, for example, labels, methylation,
substitution of one or more of the naturally occurring nucleotides
with an analog, internucleotide modifications (e.g., uncharged
linkages: for example, methyl phosphonates, phosphotriesters,
phosphoramidates, carbamates, etc.; charged linkages: for example,
phosphorothioates, phosphorodithioates, etc.; pendent moieties: for
example, peptides; intercalators: for example, acridine, psoralen,
etc.; chelators; alkylators; and modified linkages: for example,
alpha anomeric nucleic acids, etc.). The term "nucleic acid
molecule" also includes any topological conformation, including
single-stranded, double-stranded, partially duplexed, triplexed,
hairpinned, circular, and padlocked conformations.
[0126] As used herein with respect to DNA, the term "coding
sequence", "structural nucleotide sequence", or "structural nucleic
acid molecule" refers to a nucleotide sequence that is ultimately
translated into a polypeptide, via transcription and mRNA, when
placed under the control of appropriate regulatory sequences. With
respect to RNA, the term "coding polynucleotide" refers to a
polynucleotide that is translated into a peptide, polypeptide, or
protein. The boundaries of a coding sequence are determined by a
translation start codon at the 5'-terminus and a translation stop
codon at the 3'-terminus. Coding polynucleotides include, but are
not limited to: genomic DNA; cDNA; EST; and recombinant nucleotide
sequences.
[0127] As used herein, "transcribed non-coding polynucleotide"
refers to segments of mRNA molecules such as 5'UTR, 3'UTR and
intron segments that are not translated into a peptide,
polypeptide, or protein. Further, "transcribed non-coding
polynucleotide" refers to a nucleic acid that is transcribed into
an RNA that functions in the cell, for example, structural RNAs
(e.g., ribosomal RNA (rRNA) as exemplified by 5S rRNA, 5.8S rRNA,
16S rRNA, 18S rRNA, 23S rRNA, and 28S rRNA, and the like); transfer
RNA (tRNA); and snRNAs such as U4, U5, U6, and the like.
Transcribed non-coding polynucleotides also include, for example
and without limitation, small RNAs (sRNA), which term is often used
to describe small bacterial non-coding RNAs; small nucleolar RNAs
(snoRNA); microRNAs; small interfering RNAs (siRNA);
Piwi-interacting RNAs (piRNA); and long non-coding RNAs. Further
still, "transcribed non-coding polynucleotide" refers to a
polynucleotide that may natively exist as an intragenic "linker" in
a nucleic acid and which is transcribed into an RNA molecule.
[0128] Genome: As used herein, the term "genome" refers to
chromosomal DNA found within the nucleus of a cell, and also refers
to organelle DNA found within subcellular components of the cell.
In some embodiments of the invention, a DNA molecule may be
introduced into a plant cell such that the DNA molecule is
integrated into the genome of the plant cell. In these and further
embodiments, the DNA molecule may be either integrated into the
nuclear DNA of the plant cell, or integrated into the DNA of the
chloroplast or mitochondrion of the plant cell. The term "genome"
as it applies to bacteria refers to both the chromosome and
plasmids within the bacterial cell. In some embodiments of the
invention, a DNA molecule may be introduced into a bacterium such
that the DNA molecule is integrated into the genome of the
bacterium. In these and further embodiments, the DNA molecule may
be either chromosomally-integrated or located as or in a stable
plasmid.
[0129] Sequence identity: The term "sequence identity" or
"identity", as used herein in the context of two nucleic acid or
polypeptide sequences, refers to the residues in the two sequences
that are the same when aligned for maximum correspondence over a
specified comparison window.
[0130] As used herein, the term "percentage of sequence identity"
may refer to the value determined by comparing two optimally
aligned sequences (e.g., nucleic acid sequences or polypeptide
sequences) over a comparison window, wherein the portion of the
sequence in the comparison window may comprise additions or
deletions (i.e., gaps) as compared to the reference sequence (which
does not comprise additions or deletions) for optimal alignment of
the two sequences. The percentage is calculated by determining the
number of positions at which the identical nucleotide or amino acid
residue occurs in both sequences to yield the number of matched
positions, dividing the number of matched positions by the total
number of positions in the comparison window, and multiplying the
result by 100 to yield the percentage of sequence identity. A
sequence that is identical at every position in comparison to a
reference sequence is said to be 100% identical to the reference
sequence, and vice-versa.
[0131] Methods for aligning sequences for comparison are well-known
in the art. Various programs and alignment algorithms are described
in, for example: Smith and Waterman (1981) Adv. Appl. Math. 2:482;
Needleman and Wunsch (1970) J. Mol. Biol. 48:443; Pearson and
Lipman (1988) Proc. Natl. Acad. Sci. U.S.A. 85:2444; Higgins and
Sharp (1988) Gene 73:237-244; Higgins and Sharp (1989) CABIOS
5:151-153; Corpet et al. (1988) Nucleic Acids Res. 16:10881-10890;
Huang et al. (1992) Comp. Appl. Biosci. 8:155-165; Pearson et al.
(1994) Methods Mol. Biol. 24:307-331; Tatiana et al. (1999) FEMS
Microbiol. Lett. 174:247-250. A detailed consideration of sequence
alignment methods and homology calculations can be found in, e.g.,
Altschul et al. (1990) J. Mol. Biol. 215:403-410.
[0132] The National Center for Biotechnology Information (NCBI)
Basic Local Alignment Search Tool (BLAST.TM.; Altschul et al.
(1990)) is available from several sources, including the National
Center for Biotechnology Information (Bethesda, Md.), and on the
internet, for use in connection with several sequence analysis
programs. A description of how to determine sequence identity using
this program is available on the internet under the "help" section
for BLAST.TM.. For comparisons of nucleic acid sequences, the
"Blast 2 sequences" function of the BLAST.TM. (Blastn) program may
be employed using the default BLOSUM62 matrix set to default
parameters. Nucleic acid sequences with even greater similarity to
the reference sequences will show increasing percentage identity
when assessed by this method.
[0133] Specifically hybridizable/Specifically complementary: As
used herein, the terms "Specifically hybridizable" and
"Specifically complementary" are terms that indicate a sufficient
degree of complementarity such that stable and specific binding
occurs between the nucleic acid molecule and a target nucleic acid
molecule. Hybridization between two nucleic acid molecules involves
the formation of an anti-parallel alignment between the nucleic
acid sequences of the two nucleic acid molecules. The two molecules
are then able to form hydrogen bonds with corresponding bases on
the opposite strand to form a duplex molecule that, if it is
sufficiently stable, is detectable using methods well known in the
art. A nucleic acid molecule need not be 100% complementary to its
target sequence to be specifically hybridizable. However, the
amount of sequence complementarity that must exist for
hybridization to be specific is a function of the hybridization
conditions used.
[0134] Hybridization conditions resulting in particular degrees of
stringency will vary depending upon the nature of the hybridization
method of choice and the composition and length of the hybridizing
nucleic acid sequences. Generally, the temperature of hybridization
and the ionic strength (especially the Na and/or Mg' concentration)
of the hybridization will determine the stringency of
hybridization. The ionic strength of the wash buffer and the wash
temperature also influence stringency. Calculations regarding
hybridization conditions required for attaining particular degrees
of stringency are known to those of ordinary skill in the art, and
are discussed, for example, in Sambrook et al. (ed.) Molecular
Cloning: A Laboratory Manual, 2.sup.nd ed., vol. 1-3, Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, chapters 9
and 11, and updates; and Hames and Higgins (eds.) Nucleic Acid
Hybridization, IRL Press, Oxford, 1985. Further detailed
instruction and guidance with regard to the hybridization of
nucleic acids may be found, for example, in Tijssen, "Overview of
principles of hybridization and the strategy of nucleic acid probe
assays," in Laboratory Techniques in Biochemistry and Molecular
Biology--Hybridization with Nucleic Acid Probes, Part I, Chapter 2,
Elsevier, N Y, 1993; and Ausubel et al., Eds., Current Protocols in
Molecular Biology, Chapter 2, Greene Publishing and
Wiley-Interscience, N Y, 1995, and updates.
[0135] As used herein, "stringent conditions" encompass conditions
under which hybridization will occur only if there is more than 80%
sequence match between the hybridization molecule and a homologous
sequence within the target nucleic acid molecule. "Stringent
conditions" include further particular levels of stringency. Thus,
as used herein, "moderate stringency" conditions are those under
which molecules with more than 80% sequence match (i.e., having
less than 20% mismatch) will hybridize; conditions of "high
stringency" are those under which sequences with more than 90%
match (i.e. having less than 10% mismatch) will hybridize; and
conditions of "very high stringency" are those under which
sequences with more than 95% match (i.e., having less than 5%
mismatch) will hybridize.
[0136] The following are representative, non-limiting hybridization
conditions.
[0137] High Stringency condition (detects sequences that share at
least 90% sequence identity): Hybridization in 5.times.SSC buffer
at 65.degree. C. for 16 hours; wash twice in 2.times.SSC buffer at
room temperature for 15 minutes each; and wash twice in
0.5.times.SSC buffer at 65.degree. C. for 20 minutes each.
[0138] Moderate Stringency condition (detects sequences that share
at least 80% sequence identity): Hybridization in
5.times.-6.times.SSC buffer at 65-70.degree. C. for 16-20 hours;
wash twice in 2.times.SSC buffer at room temperature for 5-20
minutes each; and wash twice in 1.times.SSC buffer at 55-70.degree.
C. for 30 minutes each.
[0139] Non-stringent control condition (sequences that share at
least 50% sequence identity will hybridize): Hybridization in
6.times.SSC buffer at room temperature to 55.degree. C. for 16-20
hours; wash at least twice in 2.times.-3.times.SSC buffer at room
temperature to 55.degree. C. for 20-30 minutes each.
[0140] As used herein, the term "substantially homologous" or
"substantial homology", with regard to a contiguous nucleic acid
sequence, refers to contiguous nucleotide sequences that are borne
by nucleic acid molecules that hybridize under stringent conditions
to a nucleic acid molecule having the reference nucleic acid
sequence. For example, nucleic acid molecules having sequences that
are substantially homologous to a reference nucleic acid sequence
of SEQ ID NO:1 are those nucleic acid molecules that hybridize
under stringent conditions (e.g., the Moderate Stringency
conditions set forth, supra) to nucleic acid molecules having the
reference nucleic acid sequence of SEQ ID NO:1. Substantially
homologous sequences may have at least 80% sequence identity. For
example, substantially homologous sequences may have from about 80%
to 100% sequence identity, such as about 81%; about 82%; about 83%;
about 84%; about 85%; about 86%; about 87%; about 88%; about 89%;
about 90%; about 91%; about 92%; about 93%; about 94% about 95%;
about 96%; about 97%; about 98%; about 98.5%; about 99%; about
99.5%; and about 100%. The property of substantial homology is
closely related to specific hybridization. For example, a nucleic
acid molecule is specifically hybridizable when there is a
sufficient degree of complementarity to avoid non-specific binding
of the nucleic acid to non-target sequences under conditions where
specific binding is desired, for example, under stringent
hybridization conditions.
[0141] As used herein, the term "ortholog" refers to a gene in two
or more species that has evolved from a common ancestral nucleotide
sequence, and may retain the same function in the two or more
species.
[0142] As used herein, two nucleic acid sequence molecules are said
to exhibit "complete complementarity" when every nucleotide of a
sequence read in the 5' to 3' direction is complementary to every
nucleotide of the other sequence when read in the 3' to 5'
direction. A nucleotide sequence that is complementary to a
reference nucleotide sequence will exhibit a sequence identical to
the reverse complement sequence of the reference nucleotide
sequence. These terms and descriptions are well defined in the art
and are easily understood by those of ordinary skill in the
art.
[0143] Operably linked: A first nucleotide sequence is operably
linked with a second nucleic acid sequence when the first nucleic
acid sequence is in a functional relationship with the second
nucleic acid sequence. When recombinantly produced, operably linked
nucleic acid sequences are generally contiguous, and, where
necessary, two protein-coding regions may be joined in the same
reading frame (e.g., in a translationally fused ORF). However,
nucleic acids need not be contiguous to be operably linked.
[0144] The term, "operably linked", when used in reference to a
regulatory sequence and a coding sequence, means that the
regulatory sequence affects the expression of the linked coding
sequence. "Regulatory sequences", or "control elements", refer to
nucleotide sequences that influence the timing and level/amount of
transcription, RNA processing or stability, or translation of the
associated coding sequence. Regulatory sequences may include
promoters; translation leader sequences; introns; enhancers;
stem-loop structures; repressor binding sequences; termination
sequences; polyadenylation recognition sequences; etc. Particular
regulatory sequences may be located upstream and/or downstream of a
coding sequence operably linked thereto. Also, particular
regulatory sequences operably linked to a coding sequence may be
located on the associated complementary strand of a double-stranded
nucleic acid molecule.
[0145] Promoter: As used herein, the term "promoter" refers to a
region of DNA that may be upstream from the start of transcription,
and that may be involved in recognition and binding of RNA
polymerase and other proteins to initiate transcription. A promoter
may be operably linked to a coding sequence for expression in a
cell, or a promoter may be operably linked to a nucleotide sequence
encoding a signal sequence which may be operably linked to a coding
sequence for expression in a cell. A "plant promoter" may be a
promoter capable of initiating transcription in plant cells.
Examples of promoters under developmental control include promoters
that preferentially initiate transcription in certain tissues, such
as leaves, roots, seeds, fibers, xylem vessels, tracheids, or
sclerenchyma. Such promoters are referred to as "tissue-preferred".
Promoters which initiate transcription only in certain tissues are
referred to as "tissue-specific". A "cell type-specific" promoter
primarily drives expression in certain cell types in one or more
organs, for example, vascular cells in roots or leaves. An
"inducible" promoter may be a promoter which may be under
environmental control. Examples of environmental conditions that
may initiate transcription by inducible promoters include anaerobic
conditions and the presence of light. Tissue-specific,
tissue-preferred, cell type specific, and inducible promoters
constitute the class of "non-constitutive" promoters. A
"constitutive" promoter is a promoter which may be active under
most environmental conditions or in most tissue or cell types.
[0146] Any inducible promoter can be used in some embodiments of
the invention. See Ward et al. (1993) Plant Mol. Biol. 22:361-366.
With an inducible promoter, the rate of transcription increases in
response to an inducing agent. Exemplary inducible promoters
include, but are not limited to: Promoters from the ACEI system
that respond to copper; In2 gene from maize that responds to
benzenesulfonamide herbicide safeners; Tet repressor from Tn10; and
the inducible promoter from a steroid hormone gene, the
transcriptional activity of which may be induced by a
glucocorticosteroid hormone (Schena et al. (1991) Proc. Natl. Acad.
Sci. USA 88:10421-10425).
[0147] Exemplary constitutive promoters include, but are not
limited to: Promoters from plant viruses, such as the 35S promoter
from Cauliflower Mosaic Virus (CaMV); promoters from rice actin
genes; ubiquitin promoters; pEMU; MAS; maize H3 histone promoter;
and the ALS promoter, XbaI/NcoI fragment 5' to the Brassica napus
ALS3 structural gene (or a nucleotide sequence similar to said
XbaI/NcoI fragment) (U.S. Pat. No. 5,659,026).
[0148] Additionally, any tissue-specific or tissue-preferred
promoter may be utilized in some embodiments of the invention.
Plants transformed with a nucleic acid molecule comprising a coding
sequence operably linked to a tissue-specific promoter may produce
the product of the coding sequence exclusively, or preferentially,
in a specific tissue. Exemplary tissue-specific or tissue-preferred
promoters include, but are not limited to: A seed-preferred
promoter, such as that from the phaseolin gene; a leaf-specific and
light-induced promoter such as that from cab or rubisco; an
anther-specific promoter such as that from LAT52; a pollen-specific
promoter such as that from Zm13; and a microspore-preferred
promoter such as that from apg.
[0149] Soybean plant: As used herein, the term "soybean plant"
refers to a plant of the species Glycine; for example, Glycine
max.
[0150] Transformation: As used herein, the term "transformation" or
"transduction" refers to the transfer of one or more nucleic acid
molecule(s) into a cell. A cell is "transformed" by a nucleic acid
molecule transduced into the cell when the nucleic acid molecule
becomes stably replicated by the cell, either by incorporation of
the nucleic acid molecule into the cellular genome, or by episomal
replication. As used herein, the term "transformation" encompasses
all techniques by which a nucleic acid molecule can be introduced
into such a cell. Examples include, but are not limited to:
transfection with viral vectors; transformation with plasmid
vectors; electroporation (Fromm et al. (1986) Nature 319:791-793);
lipofection (Felgner et al. (1987) Proc. Natl. Acad. Sci. USA
84:7413-7417); microinjection (Mueller et al. (1978) Cell
15:579-585); Agrobacterium-mediated transfer (Fraley et al. (1983)
Proc. Natl. Acad. Sci. USA 80:4803-4807); direct DNA uptake; and
microprojectile bombardment (Klein et al. (1987) Nature
327:70).
[0151] Transgene: An exogenous nucleic acid sequence. In some
examples, a transgene may be a sequence that encodes one or both
strand(s) of a dsRNA molecule that comprises a nucleotide sequence
that is complementary to a nucleic acid molecule found in a
coleopteran and/or hemipteran pest. In further examples, a
transgene may be an antisense nucleic acid sequence, wherein
expression of the antisense nucleic acid sequence inhibits
expression of a target nucleic acid sequence. In still further
examples, a transgene may be a gene sequence (e.g., a
herbicide-resistance gene), a gene encoding an industrially or
pharmaceutically useful compound, or a gene encoding a desirable
agricultural trait. In these and other examples, a transgene may
contain regulatory sequences operably linked to a coding sequence
of the transgene (e.g., a promoter).
[0152] Vector: A nucleic acid molecule as introduced into a cell,
for example, to produce a transformed cell. A vector may include
nucleic acid sequences that permit it to replicate in the host
cell, such as an origin of replication. Examples of vectors
include, but are not limited to: a plasmid; cosmid; bacteriophage;
or virus that carries exogenous DNA into a cell. A vector may also
be an RNA molecule. A vector may also include one or more genes,
antisense sequences, and/or selectable marker genes and other
genetic elements known in the art. A vector may transduce,
transform, or infect a cell, thereby causing the cell to express
the nucleic acid molecules and/or proteins encoded by the vector. A
vector optionally includes materials to aid in achieving entry of
the nucleic acid molecule into the cell (e.g., a liposome, protein
coating, etc.).
[0153] Yield: A stabilized yield of about 100% or greater relative
to the yield of check varieties in the same growing location
growing at the same time and under the same conditions. In
particular embodiments, "improved yield" or "improving yield" means
a cultivar having a stabilized yield of 105% to 115% or greater
relative to the yield of check varieties in the same growing
location containing significant densities of coleopteran and/or
hemipteran pests that are injurious to that crop growing at the
same time and under the same conditions.
[0154] Unless specifically indicated or implied, the terms "a",
"an", and "the" signify "at least one" as used herein.
[0155] Unless otherwise specifically explained, all technical and
scientific terms used herein have the same meaning as commonly
understood by those of ordinary skill in the art to which this
disclosure belongs. Definitions of common terms in molecular
biology can be found in, for example, Lewin's Genes X, Jones &
Bartlett Publishers, 2009 (ISBN 10 0763766321); Krebs et al.
(eds.), The Encyclopedia of Molecular Biology, Blackwell Science
Ltd., 1994 (ISBN 0-632-02182-9); and Meyers R. A. (ed.), Molecular
Biology and Biotechnology: A Comprehensive Desk Reference, VCH
Publishers, Inc., 1995 (ISBN 1-56081-569-8). All percentages are by
weight and all solvent mixture proportions are by volume unless
otherwise noted. All temperatures are in degrees Celsius.
IV. Nucleic Acid Molecules Comprising a Coleopteran and/or
Hemipteran Pest Sequence
[0156] A. Overview
[0157] Described herein are nucleic acid molecules useful for the
control of coleopteran and/or hemipteran pests. Described nucleic
acid molecules include target sequences (e.g., native genes, and
non-coding sequences), dsRNAs, siRNAs, hpRNAs, shRNA, and miRNAs.
For example, dsRNA, siRNA, shRNA, miRNA and/or hpRNA molecules are
described in some embodiments that may be specifically
complementary to all or part of one or more native nucleic acid
sequences in a coleopteran and/or hemipteran pest. In these and
further embodiments, the native nucleic acid sequence(s) may be one
or more target gene(s), the product of which may be, for example
and without limitation: involved in a metabolic process; involved
in a reproductive process; or involved in larval/nymph development.
Nucleic acid molecules described herein, when introduced into a
cell comprising at least one native nucleic acid sequence(s) to
which the nucleic acid molecules are specifically complementary,
may initiate RNAi in the cell, and consequently reduce or eliminate
expression of the native nucleic acid sequence(s). In some
examples, reduction or elimination of the expression of a target
gene by a nucleic acid molecule comprising a sequence specifically
complementary thereto may be lethal in coleopteran and/or
hemipteran pests, or result in reduced growth and/or
reproduction.
[0158] In some embodiments, at least one target gene in a
coleopteran and/or hemipteran pest may be selected, wherein the
target gene comprises a nucleotide sequence comprising rpL40 (SEQ
ID NOs:1, 3, 5, or 89). In particular examples, a target gene in a
coleopteran and/or hemipteran pest is selected, wherein the target
gene comprises a novel nucleotide sequence comprising rpL40 (SEQ ID
NOs:1, 3, 5, or 89).
[0159] In some embodiments, a target gene may be a nucleic acid
molecule comprising a nucleotide sequence that encodes a
polypeptide comprising a contiguous amino acid sequence that is at
least 85% identical (e.g., about 90%, about 95%, about 96%, about
97%, about 98%, about 99%, about 100%, or 100% identical) to the
amino acid sequence of a protein product of rpL40 (SEQ ID NOs:1, 3,
5, or 89). A target gene may be any nucleic acid sequence in a
coleopteran and/or hemipteran pest, the post-transcriptional
inhibition of which has a deleterious effect on the coleopteran
and/or hemipteran pest, or provides a protective benefit against
the coleopteran and/or hemipteran pest to a plant. In particular
examples, a target gene is a nucleic acid molecule comprising a
nucleotide sequence that encodes a polypeptide comprising a
contiguous amino acid sequence that is at least 85% identical,
about 90% identical, about 95% identical, about 96% identical,
about 97% identical, about 98% identical, about 99% identical,
about 100% identical, or 100% identical to the amino acid sequence
of a protein product of novel nucleotide sequence SEQ ID NOs:1, 3,
5, or 89.
[0160] Provided according to the invention are nucleotide
sequences, the expression of which results in an RNA molecule
comprising a nucleotide sequence that is specifically complementary
to all or part of a native RNA molecule that is encoded by a coding
sequence in a coleopteran and/or hemipteran pest. In some
embodiments, after ingestion of the expressed RNA molecule by a
coleopteran and/or hemipteran pest, down-regulation of the coding
sequence in cells of the coleopteran and/or hemipteran pest may be
obtained. In particular embodiments, down-regulation of the coding
sequence in cells of the coleopteran and/or hemipteran pest may
result in a deleterious effect on the growth, viability,
proliferation, and/or reproduction of the coleopteran and/or
hemipteran pest.
[0161] In some embodiments, target sequences include transcribed
non-coding RNA sequences, such as 5'UTRs; 3'UTRs; spliced leader
sequences; intron sequences; outron sequences (e.g., 5'UTR RNA
subsequently modified in trans splicing); donatron sequences (e.g.,
non-coding RNA required to provide donor sequences for trans
splicing); and other non-coding transcribed RNA of target
coleopteran and/or hemipteran pest genes. Such sequences may be
derived from both mono-cistronic and poly-cistronic genes.
[0162] Thus, also described herein in connection with some
embodiments are iRNA molecules (e.g., dsRNAs, siRNAs, shRNA, miRNAs
and hpRNAs) that comprise at least one nucleotide sequence that is
specifically complementary to all or part of a target sequence in a
coleopteran and/or hemipteran pest. In some embodiments an iRNA
molecule may comprise nucleotide sequence(s) that are complementary
to all or part of a plurality of target sequences; for example, 2,
3, 4, 5, 6, 7, 8, 9, 10, or more target sequences. In particular
embodiments, an iRNA molecule may be produced in vitro, or in vivo
by a genetically-modified organism, such as a plant or bacterium.
Also disclosed are cDNA sequences that may be used for the
production of dsRNA molecules, siRNA molecules, shRNA molecules,
miRNA molecules and/or hpRNA molecules that are specifically
complementary to all or part of a target sequence in a coleopteran
and/or hemipteran pest. Further described are recombinant DNA
constructs for use in achieving stable transformation of particular
host targets. Transformed host targets may express effective levels
of dsRNA, siRNA, shRNA, miRNA and/or hpRNA molecules from the
recombinant DNA constructs. Therefore, also described is a plant
transformation vector comprising at least one nucleotide sequence
operably linked to a heterologous promoter functional in a plant
cell, wherein expression of the nucleotide sequence(s) results in
an RNA molecule comprising a nucleotide sequence that is
specifically complementary to all or part of a target sequence in a
coleopteran and/or hemipteran pest.
[0163] In some embodiments, nucleic acid molecules useful for the
control of coleopteran and/or hemipteran pests may include: all or
part of a native nucleic acid sequence isolated from Diabrotica or
a hemipteran comprising rpL40 (SEQ ID NOs:1, 3, 5, or 89);
nucleotide sequences that when expressed result in an RNA molecule
comprising a nucleotide sequence that is specifically complementary
to all or part of a native RNA molecule that is encoded by rpL40
(SEQ ID NOs:1, 3, 5, or 89); iRNA molecules (e.g., dsRNAs, siRNAs,
shRNA, miRNAs and hpRNAs) that comprise at least one nucleotide
sequence that is specifically complementary to all or part of rpL40
(SEQ ID NOs:1, 3, 5, or 89); cDNA sequences that may be used for
the production of dsRNA molecules, siRNA molecules, shRNA
molecules, miRNA and/or hpRNA molecules that are specifically
complementary to all or part of rpL40 (S SEQ ID NOs:1, 3, 5, or
89); and recombinant DNA constructs for use in achieving stable
transformation of particular host targets, wherein a transformed
host target comprises one or more of the foregoing nucleic acid
molecules.
[0164] B. Nucleic Acid Molecules
[0165] The present invention provides, inter alia, iRNA (e.g.,
dsRNA, siRNA, shRNA, miRNA and hpRNA) molecules that inhibit target
gene expression in a cell, tissue, or organ of a coleopteran and/or
hemipteran pest; and DNA molecules capable of being expressed as an
iRNA molecule in a cell or microorganism to inhibit target gene
expression in a cell, tissue, or organ of a coleopteran and/or
hemipteran pest.
[0166] Some embodiments of the invention provide an isolated
nucleic acid molecule comprising at least one (e.g., one, two,
three, or more) nucleotide sequence(s) selected from the group
consisting of: SEQ ID NO:1; the complement of SEQ ID NO:1; SEQ ID
NO:3; the complement of SEQ ID NO:3; SEQ ID NO:5; the complement of
SEQ ID NO:5; SEQ ID NO:89; the complement of SEQ ID NO:89; a
fragment of at least 15 contiguous nucleotides (e.g., 15, 16, 17,
18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more
contiguous nucleotides) of any of SEQ ID NOs:1, 3, 5, and 89; the
complement of a fragment of at least 15 contiguous nucleotides of
any of SEQ ID NOs:1, 3, 5, and 89; a native coding sequence of a
coleopteran or hemipteran organism (e.g., WCR and BSB) comprising
all or part of any of SEQ ID NOs:1, 3, 5, and 89; the complement of
a native coding sequence of a coleopteran or hemipteran organism
comprising all or part of any of SEQ ID NOs:1, 3, 5, and 89; a
native non-coding sequence of a coleopteran or hemipteran organism
that is transcribed into a native RNA molecule comprising all or
part of any of SEQ ID NOs:1, 3, 5, and 89; the complement of a
native non-coding sequence of a coleopteran or hemipteran organism
that is transcribed into a native RNA molecule comprising all or
part of any of SEQ ID NOs:1, 3, 5, and 89; a fragment of at least
15 contiguous nucleotides of a native non-coding sequence of a
coleopteran or hemipteran organism that is transcribed into a
native RNA molecule comprising all or part of any of SEQ ID NOs:1,
3, 5, and 89; the complement of a fragment of at least 15
contiguous nucleotides of a native non-coding sequence of a
coleopteran or hemipteran organism that is transcribed into a
native RNA molecule comprising all or part of any of SEQ ID NOs:1,
3, 5, and 89; a fragment of at least 15 contiguous nucleotides of a
native coding sequence of a coleopteran or hemipteran organism that
is transcribed into a native RNA molecule comprising SEQ ID NOs:1,
3, 5, and 89; the complement of a fragment of at least 15
contiguous nucleotides of a native coding sequence of a coleopteran
or hemipteran organism that is transcribed into a native RNA
molecule comprising SEQ ID NOs:1, 3, 5, and 89. In particular
embodiments, contact with or uptake by a coleopteran and/or
hemipteran pest of the isolated nucleic acid sequence inhibits the
growth, development, reproduction and/or feeding of the coleopteran
and/or hemipteran pest.
[0167] In some embodiments, a nucleic acid molecule of the
invention may comprise at least one (e.g., one, two, three, or
more) DNA sequence(s) capable of being expressed as an iRNA
molecule in a cell or microorganism to inhibit target gene
expression in a cell, tissue, or organ of a coleopteran and/or
hemipteran pest. Such DNA sequence(s) may be operably linked to a
promoter sequence that functions in a cell comprising the DNA
molecule to initiate or enhance the transcription of the encoded
RNA capable of forming a dsRNA molecule(s). In one embodiment, the
at least one (e.g., one, two, three, or more) DNA sequence(s) may
be derived from a polynucleotide(s) selected from the group
consisting of: SEQ ID NOs:1, 3, 5, and 89. Derivatives of SEQ ID
NOs:1, 3, 5, or 89 include fragments of SEQ ID NOs:1, 3, 5, or 89.
In some embodiments, such a fragment may comprise, for example, at
least about 15 contiguous nucleotides of SEQ ID NOs:1, 3, 5, or 89,
or a complement thereof. Thus, such a fragment may comprise, for
example, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,
29, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160,
170, 180, 190, 200 or more contiguous nucleotides of SEQ ID NOs:1,
3, 5, or 89, or a complement thereof. In these and further
embodiments, such a fragment may comprise, for example, more than
about 15 contiguous nucleotides of SEQ ID NOs:1, 3, 5, or 89, or a
complement thereof. Thus, a fragment of SEQ ID NOs:1, 3, 5, or 89
may comprise, for example, 15, 16, 17, 18, 19, 20, 21, about 25,
(e.g., 22, 23, 24, 25, 26, 27, 28, and 29), about 30, about 40,
(e.g., 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, and 45), about 50,
about 60, about 70, about 80, about 90, about 100, about 110, about
120, about 130, about 140, about 150, about 160, about 170, about
180, about 190, about 200 or more contiguous nucleotides of SEQ ID
NOs:1, 3, 5, or 89, or a complement thereof.
[0168] Some embodiments comprise introducing partial- or
fully-stabilized dsRNA molecules into a coleopteran and/or
hemipteran pest to inhibit expression of a target gene in a cell,
tissue, or organ of the coleopteran and/or hemipteran pest. When
expressed as an iRNA molecule (e.g., dsRNA, siRNA, shRNA, miRNA,
and hpRNA) and taken up by a coleopteran and/or hemipteran pest,
nucleic acid sequences comprising one or more fragments of SEQ ID
NOs:1, 3, 5, or 89 may cause one or more of death, growth
inhibition, change in sex ratio, reduction in brood size, cessation
of infection, and/or cessation of feeding by a coleopteran and/or
hemipteran pest. For example, in some embodiments, a dsRNA molecule
comprising a nucleotide sequence including about 15 to about 300 or
about 19 to about 300 nucleotides that are substantially homologous
to a coleopteran and/or hemipteran pest target gene sequence and
comprising one or more fragments of a nucleotide sequence
comprising SEQ ID NOs:1, 3, 5, or 89 is provided. Expression of
such a dsRNA molecule may, for example, lead to mortality and/or
growth inhibition in a coleopteran and/or hemipteran pest that
takes up the dsRNA molecule.
[0169] In certain embodiments, dsRNA molecules provided by the
invention comprise nucleotide sequences complementary to a target
gene comprising SEQ ID NOs:1, 3, 5, or 89 and/or nucleotide
sequences complementary to a fragment of SEQ ID NOs:1, 3, 5, or 89,
the inhibition of which target gene in a coleopteran and/or
hemipteran pest results in the reduction or removal of a protein or
nucleotide sequence agent that is essential for the coleopteran
and/or hemipteran pest's growth, development, or other biological
function. A selected nucleotide sequence may exhibit from about 80%
to about 100% sequence identity to SEQ ID NOs:1, 3, 5, or 89, a
contiguous fragment of the nucleotide sequence set forth in SEQ ID
NOs:1, 3, 5, or 89, or the complement of either of the foregoing.
For example, a selected nucleotide sequence may exhibit about 81%;
about 82%; about 83%; about 84%; about 85%; about 86%; about 87%;
about 88%; about 89%; about 90%; about 91%; about 92%; about 93%;
about 94% about 95%; about 96%; about 97%; about 98%; about 98.5%;
about 99%; about 99.5%; or about 100% sequence identity to SEQ ID
NOs:1, 3, 5, or 89, a contiguous fragment of the nucleotide
sequence set forth SEQ ID NOs:1, 3, 5, or 89, or the complement of
either of the foregoing.
[0170] In some embodiments, an isolated nucleic acid molecule of
the invention may comprise at least one (e.g., one, two, three, or
more) polynucleotide(s) selected from the group consisting of: SEQ
ID NO:98; the complement of SEQ ID NO:98; SEQ ID NO:99; the
complement of SEQ ID NO:99; SEQ ID NO:100; the complement of SEQ ID
NO:100; a fragment of at least 15 contiguous nucleotides of SEQ ID
NOs:98-100 (e.g., SEQ ID NOs:101-107); the complement of a fragment
of at least 15 contiguous nucleotides of SEQ ID NOs:98-100; a
native coding polynucleotide of a Diabrotica organism comprising
any of SEQ ID NOs:101-107; the complement of a native coding
polynucleotide of a Diabrotica organism comprising any of SEQ ID
NOs:101-107; a fragment of at least 15 contiguous nucleotides of a
native coding polynucleotide of a Diabrotica organism comprising
any of SEQ ID NOs:101-107; and the complement of a fragment of at
least 15 contiguous nucleotides of a native coding polynucleotide
of a Diabrotica organism comprising any of SEQ ID NOs:101-107.
[0171] In other embodiments, an isolated nucleic acid molecule of
the invention may comprise at least one (e.g., one, two, three, or
more) polynucleotide(s) selected from the group consisting of: SEQ
ID NO:108; the complement of SEQ ID NO:108; a fragment of at least
15 contiguous nucleotides of SEQ ID NO:108 (e.g., SEQ ID
NOs:109-110); the complement of a fragment of at least 15
contiguous nucleotides of SEQ ID NO:108; a native coding
polynucleotide of a hemipteran (e.g., BSB) organism comprising any
of SEQ ID NOs:109-110; the complement of a native coding
polynucleotide of a hemipteran organism comprising any of SEQ ID
NOs:109-110; a fragment of at least 15 contiguous nucleotides of a
native coding polynucleotide of a hemipteran organism comprising
any of SEQ ID NOs:109-110; and the complement of a fragment of at
least 15 contiguous nucleotides of a native coding polynucleotide
of a hemipteran organism comprising any of SEQ ID NOs:109-110.
[0172] In some embodiments, a DNA molecule capable of being
expressed as an iRNA molecule in a cell or microorganism to inhibit
target gene expression may comprise a single nucleotide sequence
that is specifically complementary to all or part of a native
nucleic acid sequence found in one or more target coleopteran
and/or hemipteran pest species, or the DNA molecule can be
constructed as a chimera from a plurality of such specifically
complementary sequences.
[0173] In some embodiments, a nucleic acid molecule may comprise a
first and a second nucleotide sequence separated by a "spacer
sequence". A spacer sequence may be a region comprising any
sequence of nucleotides that facilitates secondary structure
formation between the first and second nucleotide sequences, where
this is desired. In one embodiment, the spacer sequence is part of
a sense or antisense coding sequence for mRNA. The spacer sequence
may alternatively comprise any combination of nucleotides or
homologues thereof that are capable of being linked covalently to a
nucleic acid molecule.
[0174] For example, in some embodiments, the DNA molecule may
comprise a nucleotide sequence coding for one or more different RNA
molecules, wherein each of the different RNA molecules comprises a
first nucleotide sequence and a second nucleotide sequence, wherein
the first and second nucleotide sequences are complementary to each
other. The first and second nucleotide sequences may be connected
within an RNA molecule by a spacer sequence. The spacer sequence
may constitute part of the first nucleotide sequence or the second
nucleotide sequence. Expression of an RNA molecule comprising the
first and second nucleotide sequences may lead to the formation of
a dsRNA molecule of the present invention, by specific base-pairing
of the first and second nucleotide sequences. The first nucleotide
sequence or the second nucleotide sequence may be substantially
identical to a nucleic acid sequence native to a coleopteran and/or
hemipteran pest (e.g., a target gene, or transcribed non-coding
sequence), a derivative thereof, or a complementary sequence
thereto.
[0175] dsRNA nucleic acid molecules comprise double strands of
polymerized ribonucleotide sequences, and may include modifications
to either the phosphate-sugar backbone or the nucleoside.
Modifications in RNA structure may be tailored to allow specific
inhibition. In one embodiment, dsRNA molecules may be modified
through a ubiquitous enzymatic process so that siRNA molecules may
be generated. This enzymatic process may utilize an RNAse III
enzyme, such as DICER in eukaryotes, either in vitro or in vivo.
See Elbashir et al. (2001) Nature 411:494-498; and Hamilton and
Baulcombe (1999) Science 286(5441):950-952. DICER or
functionally-equivalent RNAse III enzymes cleave larger dsRNA
strands and/or hpRNA molecules into smaller oligonucleotides (e.g.,
siRNAs), each of which is about 19-25 nucleotides in length. The
siRNA molecules produced by these enzymes have 2 to 3 nucleotide 3'
overhangs, and 5' phosphate and 3' hydroxyl termini. The siRNA
molecules generated by RNAse III enzymes are unwound and separated
into single-stranded RNA in the cell. The siRNA molecules then
specifically hybridize with RNA sequences transcribed from a target
gene, and both RNA molecules are subsequently degraded by an
inherent cellular RNA-degrading mechanism. This process may result
in the effective degradation or removal of the RNA sequence encoded
by the target gene in the target organism. The outcome is the
post-transcriptional silencing of the targeted gene. In some
embodiments, siRNA molecules produced by endogenous RNAse III
enzymes from heterologous nucleic acid molecules may efficiently
mediate the down-regulation of target genes in coleopteran and/or
hemipteran pests.
[0176] In some embodiments, a nucleic acid molecule of the
invention may include at least one non-naturally occurring
nucleotide sequence that can be transcribed into a single-stranded
RNA molecule capable of forming a dsRNA molecule in vivo through
intermolecular hybridization. Such dsRNA sequences typically
self-assemble, and can be provided in the nutrition source of a
coleopteran and/or hemipteran pest to achieve the
post-transcriptional inhibition of a target gene. In these and
further embodiments, a nucleic acid molecule of the invention may
comprise two different non-naturally occurring nucleotide
sequences, each of which is specifically complementary to a
different target gene in a coleopteran and/or hemipteran pest. When
such a nucleic acid molecule is provided as a dsRNA molecule to a
coleopteran and/or hemipteran pest, the dsRNA molecule inhibits the
expression of at least two different target genes in the
coleopteran and/or hemipteran pest.
[0177] C. Obtaining Nucleic Acid Molecules
[0178] A variety of native sequences in coleopteran and/or
hemipteran pests may be used as target sequences for the design of
nucleic acid molecules of the invention, such as iRNAs and DNA
molecules encoding iRNAs. Selection of native sequences is not,
however, a straight-forward process. Only a small number of native
sequences in the coleopteran and/or hemipteran pest will be
effective targets. For example, it cannot be predicted with
certainty whether a particular native sequence can be effectively
down-regulated by nucleic acid molecules of the invention, or
whether down-regulation of a particular native sequence will have a
detrimental effect on the growth, viability, proliferation, and/or
reproduction of the coleopteran and/or hemipteran pest. The vast
majority of native coleopteran and/or hemipteran pest sequences,
such as ESTs isolated therefrom (for example, as listed in U.S.
Pat. Nos. 7,612,194 and 7,943,819), do not have a detrimental
effect on the growth, viability, proliferation, and/or reproduction
of the coleopteran and/or hemipteran pest, such as WCR, NCR, SCR,
BSB, Nezara viridula, Piezodorus guildinii, Halyomorpha halys,
Chinavia hilare, Euschistus serous, Dichelops melacanthus,
Dichelops furcatus, Edessa meditabunda, Thyanta perditor, Chinavia
marginatum, Horcias nobilellus, Taedia stigmosa, Dysdercus
peruvianus, Neomegalotomus parvus, Leptoglossus zonatus, Niesthrea
sidae, Lygus hesperus, and Lygus lineolaris.
[0179] Neither is it predictable which of the native sequences
which may have a detrimental effect on a coleopteran and/or
hemipteran pest are able to be used in recombinant techniques for
expressing nucleic acid molecules complementary to such native
sequences in a host plant and providing the detrimental effect on
the coleopteran and/or hemipteran pest upon feeding without causing
harm to the host plant.
[0180] In some embodiments, nucleic acid molecules of the invention
(e.g., dsRNA molecules to be provided in the host plant of a
coleopteran and/or hemipteran pest) are selected to target cDNA
sequences that encode proteins or parts of proteins essential for
coleopteran and/or hemipteran pest survival, such as amino acid
sequences involved in metabolic or catabolic biochemical pathways,
cell division, reproduction, energy metabolism, digestion, host
plant recognition, and the like. As described herein, ingestion of
compositions by a target organism containing one or more dsRNAs, at
least one segment of which is specifically complementary to at
least a substantially identical segment of RNA produced in the
cells of the target pest organism, can result in the death or other
inhibition of the target. A nucleotide sequence, either DNA or RNA,
derived from a coleopteran and/or hemipteran pest can be used to
construct plant cells resistant to infestation by the coleopteran
and/or hemipteran pests. The host plant of the coleopteran and/or
hemipteran pest (e.g., Z. mays or G. max), for example, can be
transformed to contain one or more of the nucleotide sequences
derived from the coleopteran and/or hemipteran pest as provided
herein. The nucleotide sequence transformed into the host may
encode one or more RNAs that form into a dsRNA sequence in the
cells or biological fluids within the transformed host, thus making
the dsRNA available if/when the coleopteran and/or hemipteran pest
forms a nutritional relationship with the transgenic host. This may
result in the suppression of expression of one or more genes in the
cells of the coleopteran and/or hemipteran pest, and ultimately
death or inhibition of its growth or development.
[0181] Thus, in some embodiments, a gene is targeted that is
essentially involved in the growth, development and reproduction of
a coleopteran and/or hemipteran pest. Other target genes for use in
the present invention may include, for example, those that play
important roles in coleopteran and/or hemipteran pest viability,
movement, migration, growth, development, infectivity,
establishment of feeding sites and reproduction. A target gene may
therefore be a housekeeping gene or a transcription factor.
Additionally, a native coleopteran and/or hemipteran pest
nucleotide sequence for use in the present invention may also be
derived from a homolog (e.g., an ortholog), of a plant, viral,
bacterial or insect gene, the function of which is known to those
of skill in the art, and the nucleotide sequence of which is
specifically hybridizable with a target gene in the genome of the
target coleopteran and/or hemipteran pest. Methods of identifying a
homolog of a gene with a known nucleotide sequence by hybridization
are known to those of skill in the art.
[0182] In some embodiments, the invention provides methods for
obtaining a nucleic acid molecule comprising a nucleotide sequence
for producing an iRNA (e.g., dsRNA, siRNA, shRNA, miRNA, and hpRNA)
molecule. One such embodiment comprises: (a) analyzing one or more
target gene(s) for their expression, function, and phenotype upon
dsRNA-mediated gene suppression in a coleopteran and/or hemipteran
pest; (b) probing a cDNA or gDNA library with a probe comprising
all or a portion of a nucleotide sequence or a homolog thereof from
a targeted coleopteran and/or hemipteran pest that displays an
altered (e.g., reduced) growth or development phenotype in a
dsRNA-mediated suppression analysis; (c) identifying a DNA clone
that specifically hybridizes with the probe; (d) isolating the DNA
clone identified in step (b); (e) sequencing the cDNA or gDNA
fragment that comprises the clone isolated in step (d), wherein the
sequenced nucleic acid molecule comprises all or a substantial
portion of the RNA sequence or a homolog thereof; and (f)
chemically synthesizing all or a substantial portion of a gene
sequence, or a siRNA or miRNA or shRNA or hpRNA or mRNA or
dsRNA.
[0183] In further embodiments, a method for obtaining a nucleic
acid fragment comprising a nucleotide sequence for producing a
substantial portion of an iRNA (e.g., dsRNA, siRNA, shRNA, miRNA,
and hpRNA) molecule includes: (a) synthesizing first and second
oligonucleotide primers specifically complementary to a portion of
a native nucleotide sequence from a targeted coleopteran and/or
hemipteran pest; and (b) amplifying a cDNA or gDNA insert present
in a cloning vector using the first and second oligonucleotide
primers of step (a), wherein the amplified nucleic acid molecule
comprises a substantial portion of a siRNA or shRNA or miRNA or
hpRNA or mRNA or dsRNA molecule.
[0184] Nucleic acids of the invention can be isolated, amplified,
or produced by a number of approaches. For example, an iRNA (e.g.,
dsRNA, siRNA, shRNA, miRNA, and hpRNA) molecule may be obtained by
PCR amplification of a target nucleic acid sequence (e.g., a target
gene or a target transcribed non-coding sequence) derived from a
gDNA or cDNA library, or portions thereof. DNA or RNA may be
extracted from a target organism, and nucleic acid libraries may be
prepared therefrom using methods known to those ordinarily skilled
in the art. gDNA or cDNA libraries generated from a target organism
may be used for PCR amplification and sequencing of target genes. A
confirmed PCR product may be used as a template for in vitro
transcription to generate sense and antisense RNA with minimal
promoters. Alternatively, nucleic acid molecules may be synthesized
by any of a number of techniques (See, e.g., Ozaki et al. (1992)
Nucleic Acids Research, 20: 5205-5214; and Agrawal et al. (1990)
Nucleic Acids Research, 18: 5419-5423), including use of an
automated DNA synthesizer (for example, a P. E. Biosystems, Inc.
(Foster City, Calif.) model 392 or 394 DNA/RNA Synthesizer), using
standard chemistries, such as phosphoramidite chemistry. See, e.g.,
Beaucage et al. (1992) Tetrahedron, 48: 2223-2311; U.S. Pat. Nos.
4,415,732, 4,458,066, 4,725,677, 4,973,679, and 4,980,460.
Alternative chemistries resulting in non-natural backbone groups,
such as phosphorothioate, phosphoramidate, and the like, can also
be employed.
[0185] An RNA, dsRNA, siRNA, miRNA, shRNA, or hpRNA molecule of the
present invention may be produced chemically or enzymatically by
one skilled in the art through manual or automated reactions, or in
vivo in a cell comprising a nucleic acid molecule comprising a
sequence encoding the RNA, dsRNA, siRNA, miRNA, shRNA, or hpRNA
molecule. RNA may also be produced by partial or total organic
synthesis--any modified ribonucleotide can be introduced by in
vitro enzymatic or organic synthesis. An RNA molecule may be
synthesized by a cellular RNA polymerase or a bacteriophage RNA
polymerase (e.g., T3 RNA polymerase, T7 RNA polymerase, and SP6 RNA
polymerase). Expression constructs useful for the cloning and
expression of nucleotide sequences are known in the art. See, e.g.,
U.S. Pat. Nos. 5,593,874, 5,693,512, 5,698,425, 5,712,135,
5,789,214, and 5,804,693. RNA molecules that are synthesized
chemically or by in vitro enzymatic synthesis may be purified prior
to introduction into a cell. For example, RNA molecules can be
purified from a mixture by extraction with a solvent or resin,
precipitation, electrophoresis, chromatography, or a combination
thereof. Alternatively, RNA molecules that are synthesized
chemically or by in vitro enzymatic synthesis may be used with no
or a minimum of purification, for example, to avoid losses due to
sample processing. The RNA molecules may be dried for storage or
dissolved in an aqueous solution. The solution may contain buffers
or salts to promote annealing, and/or stabilization of dsRNA
molecule duplex strands.
[0186] In embodiments, a dsRNA molecule may be formed by a single
self-complementary RNA strand or from two complementary RNA
strands. dsRNA molecules may be synthesized either in vivo or in
vitro. An endogenous RNA polymerase of the cell may mediate
transcription of the one or two RNA strands in vivo, or cloned RNA
polymerase may be used to mediate transcription in vivo or in
vitro. Post-transcriptional inhibition of a target gene in a
coleopteran and/or hemipteran pest may be host-targeted by specific
transcription in an organ, tissue, or cell type of the host (e.g.,
by using a tissue-specific promoter); stimulation of an
environmental condition in the host (e.g., by using an inducible
promoter that is responsive to infection, stress, temperature,
and/or chemical inducers); and/or engineering transcription at a
developmental stage or age of the host (e.g., by using a
developmental stage-specific promoter). RNA strands that form a
dsRNA molecule, whether transcribed in vitro or in vivo, may or may
not be polyadenylated, and may or may not be capable of being
translated into a polypeptide by a cell's translational
apparatus.
[0187] D. Recombinant Vectors and Host Cell Transformation
[0188] In some embodiments, the invention also provides a DNA
molecule for introduction into a cell (e.g., a bacterial cell, a
yeast cell, or a plant cell), wherein the DNA molecule comprises a
nucleotide sequence that, upon expression to RNA and ingestion by a
coleopteran and/or hemipteran pest, achieves suppression of a
target gene in a cell, tissue, or organ of the coleopteran and/or
hemipteran pest. Thus, some embodiments provide a recombinant
nucleic acid molecule comprising a nucleic acid sequence capable of
being expressed as an iRNA (e.g., dsRNA, siRNA, miRNA, shRNA, and
hpRNA) molecule in a plant cell to inhibit target gene expression
in a coleopteran and/or hemipteran pest. In order to initiate or
enhance expression, such recombinant nucleic acid molecules may
comprise one or more regulatory sequences, which regulatory
sequences may be operably linked to the nucleic acid sequence
capable of being expressed as an iRNA. Methods to express a gene
suppression molecule in plants are known, and may be used to
express a nucleotide sequence of the present invention. See, e.g.,
International PCT Publication No. WO06/073727; and U.S. Patent
Publication No. 2006/0200878 A1).
[0189] In specific embodiments, a recombinant DNA molecule of the
invention may comprise a nucleic acid sequence encoding a dsRNA
molecule. Such recombinant DNA molecules may encode dsRNA molecules
capable of inhibiting the expression of endogenous target gene(s)
in a coleopteran and/or hemipteran pest cell upon ingestion. In
many embodiments, a transcribed RNA may form a dsRNA molecule that
may be provided in a stabilized form; e.g., as a hairpin and stem
and loop structure.
[0190] In these and further embodiments, one strand of a dsRNA
molecule may be formed by transcription from a nucleotide sequence
which is substantially homologous to a nucleotide sequence
consisting of SEQ ID NO:1; the complement of SEQ ID NO:1; SEQ ID
NO:3, the complement of SEQ ID NO:3; SEQ ID NO:5; the complement of
SEQ ID NO:5; a fragment of at least 19 contiguous nucleotides of
SEQ ID NOs:1,3, or 5; the complement of a fragment of at least 19
contiguous nucleotides of SEQ ID NOs:1, 3, or 5; a native coding
sequence of a Diabrotica organism (e.g., WCR) comprising SEQ ID
NOs:1, 3, or 5; the complement of a native coding sequence of a
Diabrotica organism comprising SEQ ID NOs:1, 3, or 5; a native
non-coding sequence of a Diabrotica organism that is transcribed
into a native RNA molecule comprising SEQ ID NOs:1, 3, or 5; the
complement of a native non-coding sequence of a Diabrotica organism
that is transcribed into a native RNA molecule comprising SEQ ID
NOs:1, 3, or 5; a fragment of at least 19 contiguous nucleotides of
a native coding sequence of a Diabrotica organism (e.g., WCR)
comprising SEQ ID NOs:1, 3, or 5; the complement of a fragment of
at least 19 contiguous nucleotides of a native coding sequence of a
Diabrotica organism comprising SEQ ID NOs:1, 3, or 5; a fragment of
at least 19 contiguous nucleotides of a native non-coding sequence
of a Diabrotica organism that is transcribed into a native RNA
molecule comprising SEQ ID NOs:1, 3, or 5; and the complement of a
fragment of at least 19 contiguous nucleotides of a native
non-coding sequence of a Diabrotica organism that is transcribed
into a native RNA molecule comprising SEQ ID NOs:1, 3, or 5.
[0191] In other embodiments, one strand of a dsRNA molecule may be
formed by transcription from a nucleotide sequence which is
substantially homologous to a nucleotide sequence consisting of SEQ
ID NO:89; the complement of SEQ ID NO:89; a fragment of at least 15
contiguous nucleotides of SEQ ID NO:89; the complement of a
fragment of at least 15 contiguous nucleotides of SEQ ID NO:89; a
native coding sequence of a hemipteran organism comprising SEQ ID
NO:89; the complement of a native coding sequence of a hemipteran
organism comprising SEQ ID NO:89; a native non-coding sequence of a
hemipteran organism that is transcribed into a native RNA molecule
comprising SEQ ID NO:89; the complement of a native non-coding
sequence of a hemipteran organism that is transcribed into a native
RNA molecule comprising SEQ ID NO:89; a fragment of at least 15
contiguous nucleotides of a native coding sequence of a hemipteran
organism comprising SEQ ID NO:89; the complement of a fragment of
at least 15 contiguous nucleotides of a native coding sequence of a
hemipteran organism comprising SEQ ID NO:89; a fragment of at least
15 contiguous nucleotides of a native non-coding sequence of a
hemipteran organism that is transcribed into a native RNA molecule
comprising SEQ ID NO:89; and the complement of a fragment of at
least 15 contiguous nucleotides of a native non-coding sequence of
a hemipteran organism that is transcribed into a native RNA
molecule comprising SEQ ID NO:89.
[0192] In particular embodiments, a recombinant DNA molecule
encoding a dsRNA molecule may comprise at least two nucleotide
sequence segments within a transcribed sequence, such sequences
arranged such that the transcribed sequence comprises a first
nucleotide sequence segment in a sense orientation, and a second
nucleotide sequence segment (comprising the complement of the first
nucleotide sequence segment) is in an antisense orientation,
relative to at least one promoter, wherein the sense nucleotide
sequence segment and the antisense nucleotide sequence segment are
linked or connected by a spacer sequence segment of from about five
(.about.5) to about one thousand (.about.1000) nucleotides. The
spacer sequence segment may form a loop between the sense and
antisense sequence segments. The sense nucleotide sequence segment
or the antisense nucleotide sequence segment may be substantially
homologous to the nucleotide sequence of a target gene (e.g., a
gene comprising SEQ ID NOs:1, 3, 5, or 89) or fragment thereof. In
some embodiments, however, a recombinant DNA molecule may encode a
dsRNA molecule without a spacer sequence. In embodiments, a sense
coding sequence and an antisense coding sequence may be different
lengths.
[0193] Sequences identified as having a deleterious effect on
coleopteran and/or hemipteran pests or a plant-protective effect
with regard to coleopteran and/or hemipteran pests may be readily
incorporated into expressed dsRNA molecules through the creation of
appropriate expression cassettes in a recombinant nucleic acid
molecule of the invention. For example, such sequences may be
expressed as a hairpin with stem and loop structure by taking a
first segment corresponding to a target gene sequence (e.g., SEQ ID
NOs:1, 3, 5, 89, and fragments thereof); linking this sequence to a
second segment spacer region that is not homologous or
complementary to the first segment; and linking this to a third
segment, wherein at least a portion of the third segment is
substantially complementary to the first segment. Such a construct
forms a stem and loop structure by intramolecular base-pairing of
the first segment with the third segment, wherein the loop
structure forms and comprises the second segment. See, e.g., U.S.
Patent Publication Nos. 2002/0048814 and 2003/0018993; and
International PCT Publication Nos. WO94/01550 and WO98/05770. A
dsRNA molecule may be generated, for example, in the form of a
double-stranded structure such as a stem-loop structure (e.g.,
hairpin), whereby production of siRNA targeted for a native
coleopteran and/or hemipteran pest sequence is enhanced by
co-expression of a fragment of the targeted gene, for instance on
an additional plant expressible cassette, that leads to enhanced
siRNA production, or reduces methylation to prevent transcriptional
gene silencing of the dsRNA hairpin promoter.
[0194] Embodiments of the invention include introduction of a
recombinant nucleic acid molecule of the present invention into a
plant (i.e., transformation) to achieve coleopteran and/or
hemipteran pest-inhibitory levels of expression of one or more iRNA
molecules. A recombinant DNA molecule may, for example, be a
vector, such as a linear or a closed circular plasmid. The vector
system may be a single vector or plasmid, or two or more vectors or
plasmids that together contain the total DNA to be introduced into
the genome of a host. In addition, a vector may be an expression
vector. Nucleic acid sequences of the invention can, for example,
be suitably inserted into a vector under the control of a suitable
promoter that functions in one or more hosts to drive expression of
a linked coding sequence or other DNA sequence. Many vectors are
available for this purpose, and selection of the appropriate vector
will depend mainly on the size of the nucleic acid to be inserted
into the vector and the particular host cell to be transformed with
the vector. Each vector contains various components depending on
its function (e.g., amplification of DNA or expression of DNA) and
the particular host cell with which it is compatible.
[0195] To impart coleopteran and/or hemipteran pest resistance to a
transgenic plant, a recombinant DNA may, for example, be
transcribed into an iRNA molecule (e.g., an RNA molecule that forms
a dsRNA molecule) within the tissues or fluids of the recombinant
plant. An iRNA molecule may comprise a nucleotide sequence that is
substantially homologous and specifically hybridizable to a
corresponding transcribed nucleotide sequence within a coleopteran
and/or hemipteran pest that may cause damage to the host plant
species. The coleopteran and/or hemipteran pest may contact the
iRNA molecule that is transcribed in cells of the transgenic host
plant, for example, by ingesting cells or fluids of the transgenic
host plant that comprise the iRNA molecule. Thus, expression of a
target gene is suppressed by the iRNA molecule within coleopteran
and/or hemipteran pests that infest the transgenic host plant. In
some embodiments, suppression of expression of the target gene in
the target coleopteran and/or hemipteran pest may result in the
plant being resistant to attack by the pest.
[0196] In order to enable delivery of iRNA molecules to a
coleopteran and/or hemipteran pest in a nutritional relationship
with a plant cell that has been transformed with a recombinant
nucleic acid molecule of the invention, expression (i.e.,
transcription) of iRNA molecules in the plant cell is required.
Thus, a recombinant nucleic acid molecule may comprise a nucleotide
sequence of the invention operably linked to one or more regulatory
sequences, such as a heterologous promoter sequence that functions
in a host cell, such as a bacterial cell wherein the nucleic acid
molecule is to be amplified, and a plant cell wherein the nucleic
acid molecule is to be expressed.
[0197] Promoters suitable for use in nucleic acid molecules of the
invention include those that are inducible, viral, synthetic, or
constitutive, all of which are well known in the art. Non-limiting
examples describing such promoters include U.S. Pat. No. 6,437,217
(maize RS81 promoter); U.S. Pat. No. 5,641,876 (rice actin
promoter); U.S. Pat. No. 6,426,446 (maize RS324 promoter); U.S.
Pat. No. 6,429,362 (maize PR-1 promoter); U.S. Pat. No. 6,232,526
(maize A3 promoter); U.S. Pat. No. 6,177,611 (constitutive maize
promoters); U.S. Pat. Nos. 5,322,938, 5,352,605, 5,359,142, and
U.S. Pat. No. 5,530,196 (CaMV 35S promoter); U.S. Pat. No.
6,433,252 (maize L3 oleosin promoter); U.S. Pat. No. 6,429,357
(rice actin 2 promoter, and rice actin 2 intron); U.S. Pat. No.
6,294,714 (light-inducible promoters); U.S. Pat. No. 6,140,078
(salt-inducible promoters); U.S. Pat. No. 6,252,138
(pathogen-inducible promoters); U.S. Pat. No. 6,175,060
(phosphorous deficiency-inducible promoters); U.S. Pat. No.
6,388,170 (bidirectional promoters); U.S. Pat. No. 6,635,806
(gamma-coixin promoter); and U.S. Patent Publication No.
2009/757,089 (maize chloroplast aldolase promoter). Additional
promoters include the nopaline synthase (NOS) promoter (Ebert et
al. (1987) Proc. Natl. Acad. Sci. USA 84(16):5745-5749) and the
octopine synthase (OCS) promoters (which are carried on
tumor-inducing plasmids of Agrobacterium tumefaciens); the
caulimovirus promoters such as the cauliflower mosaic virus (CaMV)
19S promoter (Lawton et al. (1987) Plant Mol. Biol. 9:315-324); the
CaMV 35S promoter (Odell et al. (1985) Nature 313:810-812; the
figwort mosaic virus 35S-promoter (Walker et al. (1987) Proc. Natl.
Acad. Sci. USA 84(19):6624-6628); the sucrose synthase promoter
(Yang and Russell (1990) Proc. Natl. Acad. Sci. USA 87:4144-4148);
the R gene complex promoter (Chandler et al. (1989) Plant Cell
1:1175-1183); the chlorophyll a/b binding protein gene promoter;
CaMV 35S (U.S. Pat. Nos. 5,322,938, 5,352,605, 5,359,142, and
5,530,196); FMV 35S (U.S. Pat. Nos. 5,378,619 and 6,051,753); a
PC1S V promoter (U.S. Pat. No. 5,850,019); the SCP1 promoter (U.S.
Pat. No. 6,677,503); and AGRtu.nos promoters (GenBank.TM. Accession
No. V00087; Depicker et al. (1982) J. Mol. Appl. Genet. 1:561-573;
Bevan et al. (1983) Nature 304:184-187).
[0198] In particular embodiments, nucleic acid molecules of the
invention comprise a tissue-specific promoter, such as a
root-specific promoter. Root-specific promoters drive expression of
operably-linked coding sequences exclusively or preferentially in
root tissue. Examples of root-specific promoters are known in the
art. See, e.g., U.S. Pat. Nos. 5,110,732; 5,459,252 and 5,837,848;
and Opperman et al. (1994) Science 263:221-3; and Hirel et al.
(1992) Plant Mol. Biol. 20:207-18. In some embodiments, a
nucleotide sequence or fragment for coleopteran and/or hemipteran
pest control according to the invention may be cloned between two
root-specific promoters oriented in opposite transcriptional
directions relative to the nucleotide sequence or fragment, and
which are operable in a transgenic plant cell and expressed therein
to produce RNA molecules in the transgenic plant cell that
subsequently may form dsRNA molecules, as described, supra. The
iRNA molecules expressed in plant tissues may be ingested by a
coleopteran and/or hemipteran pest so that suppression of target
gene expression is achieved.
[0199] Additional regulatory sequences that may optionally be
operably linked to a nucleic acid molecule of interest include
5'UTRs that function as a translation leader sequence located
between a promoter sequence and a coding sequence. The translation
leader sequence is present in the fully-processed mRNA, and it may
affect processing of the primary transcript, and/or RNA stability.
Examples of translation leader sequences include maize and petunia
heat shock protein leaders (U.S. Pat. No. 5,362,865), plant virus
coat protein leaders, plant rubisco leaders, and others. See, e.g.,
Turner and Foster (1995) Molecular Biotech. 3(3):225-36.
Non-limiting examples of 5'UTRs include GmHsp (U.S. Pat. No.
5,659,122); PhDnaK (U.S. Pat. No. 5,362,865); AtAnt1; TEV
(Carrington and Freed (1990) J. Virol. 64:1590-7); and AGRtunos
(GenBank.TM. Accession No. V00087; and Bevan et al. (1983) Nature
304:184-7).
[0200] Additional regulatory sequences that may optionally be
operably linked to a nucleic acid molecule of interest also include
3' non-translated sequences, 3' transcription termination regions,
or poly-adenylation regions. These are genetic elements located
downstream of a nucleotide sequence, and include polynucleotides
that provide polyadenylation signal, and/or other regulatory
signals capable of affecting transcription or mRNA processing. The
polyadenylation signal functions in plants to cause the addition of
polyadenylate nucleotides to the 3' end of the mRNA precursor. The
polyadenylation sequence can be derived from a variety of plant
genes, or from T-DNA genes. A non-limiting example of a 3'
transcription termination region is the nopaline synthase 3' region
(nos 3'; Fraley et al. (1983) Proc. Natl. Acad. Sci. USA
80:4803-7). An example of the use of different 3' nontranslated
regions is provided in Ingelbrecht et al., (1989) Plant Cell
1:671-80. Non-limiting examples of polyadenylation signals include
one from a Pisum sativum RbcS2 gene (Ps.RbcS2-E9; Coruzzi et al.
(1984) EMBO J. 3:1671-9) and AGRtu.nos (GenBank.TM. Accession No.
E01312).
[0201] Some embodiments may include a plant transformation vector
that comprises an isolated and purified DNA molecule comprising at
least one of the above-described regulatory sequences operatively
linked to one or more nucleotide sequences of the present
invention. When expressed, the one or more nucleotide sequences
result in one or more RNA molecule(s) comprising a nucleotide
sequence that is specifically complementary to all or part of a
native RNA molecule in a coleopteran and/or hemipteran pest. Thus,
the nucleotide sequence(s) may comprise a segment encoding all or
part of a ribonucleotide sequence present within a targeted
coleopteran and/or hemipteran pest RNA transcript, and may comprise
inverted repeats of all or a part of a targeted coleopteran and/or
hemipteran pest transcript. A plant transformation vector may
contain sequences specifically complementary to more than one
target sequence, thus allowing production of more than one dsRNA
for inhibiting expression of two or more genes in cells of one or
more populations or species of target coleopteran and/or hemipteran
pests. Segments of nucleotide sequence specifically complementary
to nucleotide sequences present in different genes can be combined
into a single composite nucleic acid molecule for expression in a
transgenic plant. Such segments may be contiguous or separated by a
spacer sequence.
[0202] In some embodiments, a plasmid of the present invention
already containing at least one nucleotide sequence(s) of the
invention can be modified by the sequential insertion of additional
nucleotide sequence(s) in the same plasmid, wherein the additional
nucleotide sequence(s) are operably linked to the same regulatory
elements as the original at least one nucleotide sequence(s). In
some embodiments, a nucleic acid molecule may be designed for the
inhibition of multiple target genes. In some embodiments, the
multiple genes to be inhibited can be obtained from the same
coleopteran and/or hemipteran pest species, which may enhance the
effectiveness of the nucleic acid molecule. In other embodiments,
the genes can be derived from different coleopteran and/or
hemipteran pests, which may broaden the range of coleopteran and/or
hemipteran pests against which the agent(s) is/are effective. When
multiple genes are targeted for suppression or a combination of
expression and suppression, a polycistronic DNA element can be
fabricated.
[0203] A recombinant nucleic acid molecule or vector of the present
invention may comprise a selectable marker that confers a
selectable phenotype on a transformed cell, such as a plant cell.
Selectable markers may also be used to select for plants or plant
cells that comprise a recombinant nucleic acid molecule of the
invention. The marker may encode biocide resistance, antibiotic
resistance (e.g., kanamycin, Geneticin (G418), bleomycin,
hygromycin, etc.), or herbicide tolerance (e.g., glyphosate, etc.).
Examples of selectable markers include, but are not limited to: a
neo gene which codes for kanamycin resistance and can be selected
for using kanamycin, G418, etc.; a bar gene which codes for
bialaphos resistance; a mutant EPSP synthase gene which encodes
glyphosate resistance; a nitrilase gene which confers resistance to
bromoxynil; a mutant acetolactate synthase (ALS) gene which confers
imidazolinone or sulfonylurea tolerance; and a methotrexate
resistant DHFR gene. Multiple selectable markers are available that
confer resistance to ampicillin, bleomycin, chloramphenicol,
gentamycin, hygromycin, kanamycin, lincomycin, methotrexate,
phosphinothricin, puromycin, spectinomycin, rifampicin,
streptomycin and tetracycline, and the like. Examples of such
selectable markers are illustrated in, e.g., U.S. Pat. Nos.
5,550,318; 5,633,435; 5,780,708 and 6,118,047.
[0204] A recombinant nucleic acid molecule or vector of the present
invention may also include a screenable marker. Screenable markers
may be used to monitor expression. Exemplary screenable markers
include a .beta.-glucuronidase or uidA gene (GUS) which encodes an
enzyme for which various chromogenic substrates are known
(Jefferson et al. (1987) Plant Mol. Biol. Rep. 5:387-405); an
R-locus gene, which encodes a product that regulates the production
of anthocyanin pigments (red color) in plant tissues (Dellaporta et
al. (1988) "Molecular cloning of the maize R-nj allele by
transposon tagging with Ac." In 18.sup.th Stadler Genetics
Symposium, P. Gustafson and R. Appels, eds. (New York: Plenum), pp.
263-82); a .beta.-lactamase gene (Sutcliffe et al. (1978) Proc.
Natl. Acad. Sci. USA 75:3737-41); a gene which encodes an enzyme
for which various chromogenic substrates are known (e.g., PADAC, a
chromogenic cephalosporin); a luciferase gene (Ow et al. (1986)
Science 234:856-9); an xylE gene that encodes a catechol
dioxygenase that can convert chromogenic catechols (Zukowski et al.
(1983) Gene 46(2-3):247-55); an amylase gene (Ikatu et al. (1990)
Bio/Technol. 8:241-2); a tyrosinase gene which encodes an enzyme
capable of oxidizing tyrosine to DOPA and dopaquinone which in turn
condenses to melanin (Katz et al. (1983) J. Gen. Microbiol.
129:2703-14); and an .alpha.-galactosidase.
[0205] In some embodiments, recombinant nucleic acid molecules, as
described, supra, may be used in methods for the creation of
transgenic plants and expression of heterologous nucleic acids in
plants to prepare transgenic plants that exhibit reduced
susceptibility to coleopteran and/or hemipteran pests. Plant
transformation vectors can be prepared, for example, by inserting
nucleic acid molecules encoding iRNA molecules into plant
transformation vectors and introducing these into plants.
[0206] Suitable methods for transformation of host cells include
any method by which DNA can be introduced into a cell, such as by
transformation of protoplasts (See, e.g., U.S. Pat. No. 5,508,184),
by desiccation/inhibition-mediated DNA uptake (See, e.g., Potrykus
et al. (1985) Mol. Gen. Genet. 199:183-8), by electroporation (See,
e.g., U.S. Pat. No. 5,384,253), by agitation with silicon carbide
fibers (See, e.g., U.S. Pat. Nos. 5,302,523 and 5,464,765), by
Agrobacterium-mediated transformation (See, e.g., U.S. Pat. Nos.
5,563,055; 5,591,616; 5,693,512; 5,824,877; 5,981,840; and
6,384,301) and by acceleration of DNA-coated particles (See, e.g.,
U.S. Pat. Nos. 5,015,580, 5,550,318, 5,538,880, 6,160,208,
6,399,861, and 6,403,865), etc. Techniques that are particularly
useful for transforming corn are described, for example, in U.S.
Pat. Nos. 5,591,616, 7,060,876 and 7,939,3281. Through the
application of techniques such as these, the cells of virtually any
species may be stably transformed. In some embodiments,
transforming DNA is integrated into the genome of the host cell. In
the case of multicellular species, transgenic cells may be
regenerated into a transgenic organism. Any of these techniques may
be used to produce a transgenic plant, for example, comprising one
or more nucleic acid sequences encoding one or more iRNA molecules
in the genome of the transgenic plant.
[0207] The most widely utilized method for introducing an
expression vector into plants is based on the natural
transformation system of various Agrobacterium species. A.
tumefaciens and A. rhizogenes are plant pathogenic soil bacteria
which genetically transform plant cells. The Ti and Ri plasmids of
A. tumefaciens and A. rhizogenes, respectively, carry genes
responsible for genetic transformation of the plant. The Ti
(tumor-inducing)-plasmids contain a large segment, known as T-DNA,
which is transferred to transformed plants. Another segment of the
Ti plasmid, the Vir region, is responsible for T-DNA transfer. The
T-DNA region is bordered by terminal repeats. In modified binary
vectors, the tumor-inducing genes have been deleted, and the
functions of the Vir region are utilized to transfer foreign DNA
bordered by the T-DNA border sequences. The T-region may also
contain a selectable marker for efficient recovery of transgenic
cells and plants, and a multiple cloning site for inserting
sequences for transfer such as a dsRNA encoding nucleic acid.
[0208] Thus, in some embodiments, a plant transformation vector is
derived from a Ti plasmid of A. tumefaciens (See, e.g., U.S. Pat.
Nos. 4,536,475, 4,693,977, 4,886,937, and 5,501,967; and European
Patent No. EP 0 122 791) or a Ri plasmid of A. rhizogenes.
Additional plant transformation vectors include, for example and
without limitation, those described by Herrera-Estrella et al.
(1983) Nature 303:209-13; Bevan et al. (1983) Nature 304:184-7;
Klee et al. (1985) Bio/Technol. 3:637-42; and in European Patent
No. EP 0 120 516, and those derived from any of the foregoing.
Other bacteria such as Sinorhizobium, Rhizobium, and Mesorhizobium
that interact with plants naturally can be modified to mediate gene
transfer to a number of diverse plants. These plant-associated
symbiotic bacteria can be made competent for gene transfer by
acquisition of both a disarmed Ti plasmid and a suitable binary
vector.
[0209] After providing exogenous DNA to recipient cells,
transformed cells are generally identified for further culturing
and plant regeneration. In order to improve the ability to identify
transformed cells, one may desire to employ a selectable or
screenable marker gene, as previously set forth, with the
transformation vector used to generate the transformant. In the
case where a selectable marker is used, transformed cells are
identified within the potentially transformed cell population by
exposing the cells to a selective agent or agents. In the case
where a screenable marker is used, cells may be screened for the
desired marker gene trait.
[0210] Cells that survive the exposure to the selective agent, or
cells that have been scored positive in a screening assay, may be
cultured in media that supports regeneration of plants. In some
embodiments, any suitable plant tissue culture media (e.g., MS and
N6 media) may be modified by including further substances, such as
growth regulators. Tissue may be maintained on a basic medium with
growth regulators until sufficient tissue is available to begin
plant regeneration efforts, or following repeated rounds of manual
selection, until the morphology of the tissue is suitable for
regeneration (e.g., typically about 2 weeks), then transferred to
media conducive to shoot formation. Cultures are transferred
periodically until sufficient shoot formation has occurred. Once
shoots are formed, they are transferred to media conducive to root
formation. Once sufficient roots are formed, plants can be
transferred to soil for further growth and maturation.
[0211] To confirm the presence of a nucleic acid molecule of
interest (for example, a DNA sequence encoding one or more iRNA
molecules that inhibit target gene expression in a coleopteran
and/or hemipteran pest) in the regenerating plants, a variety of
assays may be performed. Such assays include, for example:
molecular biological assays, such as Southern and northern
blotting, PCR, and nucleic acid sequencing; biochemical assays,
such as detecting the presence of a protein product, e.g., by
immunological means (ELISA and/or immuno blots) or by enzymatic
function; plant part assays, such as leaf or root assays; and
analysis of the phenotype of the whole regenerated plant.
[0212] Integration events may be analyzed, for example, by PCR
amplification using, e.g., oligonucleotide primers specific for a
nucleic acid molecule of interest. PCR genotyping is understood to
include, but not be limited to, polymerase-chain reaction (PCR)
amplification of genomic DNA derived from isolated host plant
callus tissue predicted to contain a nucleic acid molecule of
interest integrated into the genome, followed by standard cloning
and sequence analysis of PCR amplification products. Methods of PCR
genotyping have been well described (for example, Rios, G. et al.
(2002) Plant J. 32:243-53) and may be applied to genomic DNA
derived from any plant species (e.g., Z. mays or G. max) or tissue
type, including cell cultures.
[0213] A transgenic plant formed using Agrobacterium-dependent
transformation methods typically contains a single recombinant DNA
sequence inserted into one chromosome. The single recombinant DNA
sequence is referred to as a "transgenic event" or "integration
event". Such transgenic plants are hemizygous for the inserted
exogenous sequence. In some embodiments, a transgenic plant
homozygous with respect to a transgene may be obtained by sexually
mating (selfing) an independent segregant transgenic plant that
contains a single exogenous gene sequence to itself, for example a
T.sub.0 plant, to produce T.sub.1 seed. One fourth of the T.sub.1
seed produced will be homozygous with respect to the transgene.
Germinating T.sub.1 seed results in plants that can be tested for
heterozygosity, typically using an SNP assay or a thermal
amplification assay that allows for the distinction between
heterozygotes and homozygotes (i.e., a zygosity assay).
[0214] In particular embodiments, at least 2, 3, 4, 5, 6, 7, 8, 9
or 10 or more different iRNA molecules that have a coleopteran
and/or hemipteran pest-inhibitory effect are produced in a plant
cell. The iRNA molecules (e.g., dsRNA molecules) may be expressed
from multiple nucleic acid sequences introduced in different
transformation events, or from a single nucleic acid sequence
introduced in a single transformation event. In some embodiments, a
plurality of iRNA molecules are expressed under the control of a
single promoter. In other embodiments, a plurality of iRNA
molecules are expressed under the control of multiple promoters.
Single iRNA molecules may be expressed that comprise multiple
nucleic acid sequences that are each homologous to different loci
within one or more coleopteran and/or hemipteran pests (for
example, the locus defined by SEQ ID NOs:1, 3, 5, or 89), both in
different populations of the same species of coleopteran and/or
hemipteran pest, or in different species of coleopteran and/or
hemipteran pests.
[0215] In addition to direct transformation of a plant with a
recombinant nucleic acid molecule, transgenic plants can be
prepared by crossing a first plant having at least one transgenic
event with a second plant lacking such an event. For example, a
recombinant nucleic acid molecule comprising a nucleotide sequence
that encodes an iRNA molecule may be introduced into a first plant
line that is amenable to transformation to produce a transgenic
plant, which transgenic plant may be crossed with a second plant
line to introgress the nucleotide sequence that encodes the iRNA
molecule into the second plant line.
[0216] The invention also includes commodity products containing
one or more of the sequences of the present invention. Particular
embodiments include commodity products produced from a recombinant
plant or seed containing one or more of the nucleotide sequences of
the present invention. A commodity product containing one or more
of the sequences of the present invention is intended to include,
but not be limited to, meals, oils, crushed or whole grains or
seeds of a plant, or any food or animal feed product comprising any
meal, oil, or crushed or whole grain of a recombinant plant or seed
containing one or more of the sequences of the present invention.
The detection of one or more of the sequences of the present
invention in one or more commodity or commodity products
contemplated herein is de facto evidence that the commodity or
commodity product is produced from a transgenic plant designed to
express one or more of the nucleotides sequences of the present
invention for the purpose of controlling coleopteran and/or
hemipteran plant pests using dsRNA-mediated gene suppression
methods.
[0217] In some aspects, seeds and commodity products produced by
transgenic plants derived from transformed plant cells are
included, wherein the seeds or commodity products comprise a
detectable amount of a nucleic acid sequence of the invention. In
some embodiments, such commodity products may be produced, for
example, by obtaining transgenic plants and preparing food or feed
from them. Commodity products comprising one or more of the nucleic
acid sequences of the invention includes, for example and without
limitation: meals, oils, crushed or whole grains or seeds of a
plant, and any food product comprising any meal, oil, or crushed or
whole grain of a recombinant plant or seed comprising one or more
of the nucleic acid sequences of the invention. The detection of
one or more of the sequences of the invention in one or more
commodity or commodity products is de facto evidence that the
commodity or commodity product is produced from a transgenic plant
designed to express one or more of the iRNA molecules of the
invention for the purpose of controlling coleopteran and/or
hemipteran pests.
[0218] In some embodiments, a transgenic plant or seed comprising a
nucleic acid molecule of the invention also may comprise at least
one other transgenic event in its genome, including without
limitation: a transgenic event from which is transcribed an iRNA
molecule targeting a locus in a coleopteran and/or hemipteran pest
other than the one defined by SEQ ID NOs:1, 3, 5, or 89, such as,
for example, one or more loci selected from the group consisting of
Cafl-180 (U.S. Patent Application Publication No. 2012/0174258),
VatpaseC (U.S. Patent Application Publication No. 2012/0174259),
Rhol (U.S. Patent Application Publication No. 2012/0174260),
VatpaseH (U.S. Patent Application Publication No. 2012/0198586),
PPI-87B (U.S. Patent Application Publication No. 2013/0091600),
RPA70 (U.S. Patent Application Publication No. 2013/0091601), RPS6
(U.S. Patent Application Publication No. 2013/0097730), ROP (U.S.
patent application Publication Ser. No. 14/577,811), RNA polymerase
II (U.S. Patent Application Publication No. 62/133,214), RNA
polymerase II140 (U.S. patent application Publication Ser. No.
14/577,854), RNA polymerase II215 (U.S. Patent Application
Publication No. 62/133,202), RNA polymerase II33 (U.S. Patent
Application Publication No. 62/133,210), ncm (U.S. Patent
Application No. 62/095,487), Dre4 (U.S. patent application Ser. No.
14/705,807), COPI alpha (U.S. Patent Application No. 62/063,199),
COPI beta (U.S. Patent Application No. 62/063,203), COPI gamma
(U.S. Patent Application No. 62/063,192), COPI delta (U.S. Patent
Application No. 62/063,216); a transgenic event from which is
transcribed an iRNA molecule targeting a gene in an organism other
than a coleopteran and/or hemipteran pest (e.g., a plant-parasitic
nematode); a gene encoding an insecticidal protein (e.g., a
Bacillus thuringiensis insecticidal protein, such as, for example,
Cry34Ab1 (U.S. Pat. Nos. 6,127,180, 6,340,593, and 6,624,145),
Cry35Ab1 (U.S. Pat. Nos. 6,083,499, 6,340,593, and 6,548,291), a
"Cry34/35Ab1" combination in a single event (e.g., maize event
DAS-59122-7; U.S. Pat. No. 7,323,556), Cry3A (e.g., U.S. Pat. No.
7,230,167), Cry3B (e.g., U.S. Pat. No. 8,101,826), Cry6A (e.g.,
U.S. Pat. No. 6,831,062), and combinations thereof (e.g., U.S.
Patent Application Nos. 2013/0167268, 2013/0167269, and
2013/0180016); Alcaligenes spp. (e.g., U.S. Patent Application
Publication No. 2014/0033361) or Pseudomonas spp. (e.g., PCT
Application Publication No. WO2015038734) insecticidal protein); a
herbicide tolerance gene (e.g., a gene providing tolerance to
glyphosate, glufosinate, dicamba or 2,4-D (e.g., U.S. Pat. No.
7,838,733)); and a gene contributing to a desirable phenotype in
the transgenic plant, such as increased yield, altered fatty acid
metabolism, or restoration of cytoplasmic male sterility. In
particular embodiments, sequences encoding iRNA molecules of the
invention may be combined with other insect control or with disease
resistance traits in a plant to achieve desired traits for enhanced
control of insect damage and plant disease. Combining insect
control traits that employ distinct modes-of-action may provide
protected transgenic plants with superior durability over plants
harboring a single control trait, for example, because of the
reduced probability that resistance to the trait(s) will develop in
the field.
V. Target Gene Suppression in a Coleopteran and/or Hemipteran
Pest
[0219] A. Overview
[0220] In some embodiments of the invention, at least one nucleic
acid molecule useful for the control of coleopteran and/or
hemipteran pests may be provided to a coleopteran and/or hemipteran
pest, wherein the nucleic acid molecule leads to RNAi-mediated gene
silencing in the coleopteran and/or hemipteran pest. In particular
embodiments, an iRNA molecule (e.g., dsRNA, siRNA, miRNA, shRNA,
and hpRNA) may be provided to the coleopteran and/or hemipteran
pest. In some embodiments, a nucleic acid molecule useful for the
control of coleopteran and/or hemipteran pests may be provided to a
coleopteran and/or hemipteran pest by contacting the nucleic acid
molecule with the coleopteran and/or hemipteran pest. In these and
further embodiments, a nucleic acid molecule useful for the control
of coleopteran and/or hemipteran pests may be provided in a feeding
substrate of the coleopteran and/or hemipteran pest, for example, a
nutritional composition. In these and further embodiments, a
nucleic acid molecule useful for the control of coleopteran and/or
hemipteran pests may be provided through ingestion of plant
material comprising the nucleic acid molecule that is ingested by
the coleopteran and/or hemipteran pest. In certain embodiments, the
nucleic acid molecule is present in plant material through
expression of a recombinant nucleic acid sequence introduced into
the plant material, for example, by transformation of a plant cell
with a vector comprising the recombinant nucleic acid sequence and
regeneration of a plant material or whole plant from the
transformed plant cell.
[0221] b. RNAi-Mediated Target Gene Suppression
[0222] In embodiments, the invention provides iRNA molecules (e.g.,
dsRNA, siRNA, miRNA, shRNA, and hpRNA) that may be designed to
target essential native nucleotide sequences (e.g., essential
genes) in the transcriptome of a coleopteran and/or hemipteran pest
(e.g., WCR, NCR, MCR, BSB, Nezara viridula, Piezodorus guildinii,
Halyomorpha halys, Acrosternum hilare, and Euschistus servus), for
example by designing an iRNA molecule that comprises at least one
strand comprising a nucleotide sequence that is specifically
complementary to the target sequence. The sequence of an iRNA
molecule so designed may be identical to the target sequence, or
may incorporate mismatches that do not prevent specific
hybridization between the iRNA molecule and its target
sequence.
[0223] iRNA molecules of the invention may be used in methods for
gene suppression in a coleopteran and/or hemipteran pest, thereby
reducing the level or incidence of damage caused by the pest on a
plant (for example, a protected transformed plant comprising an
iRNA molecule). As used herein the term "gene suppression" refers
to any of the well-known methods for reducing the levels of protein
produced as a result of gene transcription to mRNA and subsequent
translation of the mRNA, including the reduction of protein
expression from a gene or a coding sequence including
post-transcriptional inhibition of expression and transcriptional
suppression. Post-transcriptional inhibition is mediated by
specific homology between all or a part of an mRNA transcribed from
a gene targeted for suppression and the corresponding iRNA molecule
used for suppression. Additionally, post-transcriptional inhibition
refers to the substantial and measurable reduction of the amount of
mRNA available in the cell for binding by ribosomes.
[0224] In embodiments wherein an iRNA molecule is a dsRNA molecule,
the dsRNA molecule may be cleaved by the enzyme, DICER, into short
siRNA molecules (approximately 20 nucleotides in length). The
double-stranded siRNA molecule generated by DICER activity upon the
dsRNA molecule may be separated into two single-stranded siRNAs;
the "passenger strand" and the "guide strand". The passenger strand
may be degraded, and the guide strand may be incorporated into
RISC. Post-transcriptional inhibition occurs by specific
hybridization of the guide strand with a specifically complementary
sequence of an mRNA molecule, and subsequent cleavage by the
enzyme, Argonaute (catalytic component of the RISC complex).
[0225] In embodiments of the invention, any form of iRNA molecule
may be used. Those of skill in the art will understand that dsRNA
molecules typically are more stable than are single-stranded RNA
molecules, during preparation and during the step of providing the
iRNA molecule to a cell, and are typically also more stable in a
cell. Thus, while siRNA and miRNA molecules, for example, may be
equally effective in some embodiments, a dsRNA molecule may be
chosen due to its stability.
[0226] In particular embodiments, a nucleic acid molecule is
provided that comprises a nucleotide sequence, which nucleotide
sequence may be expressed in vitro to produce an iRNA molecule that
is substantially homologous to a nucleic acid molecule encoded by a
nucleotide sequence within the genome of a coleopteran and/or
hemipteran pest. In certain embodiments, the in vitro transcribed
iRNA molecule may be a stabilized dsRNA molecule that comprises a
stem-loop structure. After a coleopteran and/or hemipteran pest
contacts the in vitro transcribed iRNA molecule,
post-transcriptional inhibition of a target gene in the coleopteran
and/or hemipteran pest (for example, an essential gene) may
occur.
[0227] In some embodiments of the invention, expression of a
nucleic acid molecule comprising at least 15 contiguous nucleotides
of a nucleotide sequence is used in a method for
post-transcriptional inhibition of a target gene in a coleopteran
pest, wherein the nucleotide sequence is selected from the group
consisting of: SEQ ID NO:1; the complement of SEQ ID NO:1; SEQ ID
NO:3; the complement of SEQ ID NO:3; SEQ ID NO:5; the completment
of SEQ ID NO:5; a fragment of at least 15 contiguous nucleotides of
SEQ ID NOs:1, 3, or 5; the complement of a fragment of at least 15
contiguous nucleotides of SEQ ID NOs:1, 3, or 5; a native coding
sequence of a Diabrotica organism (e.g., WCR) comprising SEQ ID
NOs:1, 3, or 5; the complement of a native coding sequence of a
Diabrotica organism comprising SEQ ID NOs:1, 3, or 5; a native
non-coding sequence of a Diabrotica organism that is transcribed
into a native RNA molecule comprising SEQ ID NOs:1, 3, or 5; the
complement of a native non-coding sequence of a Diabrotica organism
that is transcribed into a native RNA molecule comprising SEQ ID
NOs:1, 3, or 5; the complement of a native non-coding sequence of a
Diabrotica organism that is transcribed into a native RNA molecule
comprising SEQ ID NOs:1, 3, or 5; a fragment of at least 15
contiguous nucleotides of a native coding sequence of a Diabrotica
organism (e.g., WCR) comprising SEQ ID NOs:1, 3, or 5; the
complement of a fragment of at least 15 contiguous nucleotides of a
native coding sequence of a Diabrotica organism comprising SEQ ID
NOs:1, 3, or 5; a fragment of at least 15 contiguous nucleotides of
a native non-coding sequence of a Diabrotica organism that is
transcribed into a native RNA molecule comprising SEQ ID NOs:1, 3,
or 5; and the complement of a fragment of at least 15 contiguous
nucleotides of a native non-coding sequence of a Diabrotica
organism that is transcribed into a native RNA molecule comprising
SEQ ID NOs:1, 3, or 5. In certain embodiments, expression of a
nucleic acid molecule that is at least 80% identical (e.g., 80%,
about 81%, about 82%, about 83%, about 84%, about 85%, about 86%,
about 87%, about 88%, about 89%, about 90%, about 91%, about 92%,
about 93%, about 94%, about 95%, about 96%, about 97%, about 98%,
about 99%, about 100%, and 100%) with any of the foregoing may be
used. In these and further embodiments, a nucleic acid molecule may
be expressed that specifically hybridizes to an RNA molecule
present in at least one cell of a coleopteran pest.
[0228] In certain embodiments of the invention, expression of a
nucleic acid molecule comprising at least 15 contiguous nucleotides
of a nucleotide sequence is used in a method for
post-transcriptional inhibition of a target gene in a hemipteran
pest, wherein the nucleotide sequence is selected from the group
consisting of: SEQ ID NO:89; the complement of SEQ ID NO:89; a
fragment of at least 15 contiguous nucleotides of SEQ ID NO:89; the
complement of a fragment of at least 15 contiguous nucleotides of
SEQ ID NO:89; a native coding sequence of a hemipteran organism SEQ
ID NO:89; the complement of a native coding sequence of a
hemipteran organism comprising SEQ ID NO:89; a native non-coding
sequence of a hemipteran organism that is transcribed into a native
RNA molecule comprising SEQ ID NO:89; the complement of a native
non-coding sequence of a hemipteran organism that is transcribed
into a native RNA molecule comprising SEQ ID NO:89; the complement
of a native non-coding sequence of a hemipteran organism that is
transcribed into a native RNA molecule comprising SEQ ID NO:89; a
fragment of at least 15 contiguous nucleotides of a native coding
sequence of a hemipteran organism comprising SEQ ID NO:89; the
complement of a fragment of at least 15 contiguous nucleotides of a
native coding sequence of a hemipteran organism comprising SEQ ID
NO:89; a fragment of at least 15 contiguous nucleotides of a native
non-coding sequence of a hemipteran organism that is transcribed
into a native RNA molecule comprising SEQ ID NO:89; and the
complement of a fragment of at least 15 contiguous nucleotides of a
native non-coding sequence of a hemipteran organism that is
transcribed into a native RNA molecule comprising SEQ ID NO:89. In
certain embodiments, expression of a nucleic acid molecule that is
at least 80% identical (e.g., 80%, about 81%, about 82%, about 83%,
about 84%, about 85%, about 86%, about 87%, about 88%, about 89%,
about 90%, about 91%, about 92%, about 93%, about 94%, about 95%,
about 96%, about 97%, about 98%, about 99%, about 100%, and 100%)
with any of the foregoing may be used. In these and further
embodiments, a nucleic acid molecule may be expressed that
specifically hybridizes to an RNA molecule present in at least one
cell of a hemipteran pest.
[0229] In some embodiments, expression of at least one nucleic acid
molecule comprising at least 15 contiguous nucleotides of a
nucleotide sequence may be used in a method for
post-transcriptional inhibition of a target gene in a coleopteran
pest, wherein the nucleotide sequence is selected from the group
consisting of: SEQ ID NO:1; the complement of SEQ ID NO:1; SEQ ID
NO:3; the complement of SEQ ID NO:3; SEQ ID NO:5; the completment
of SEQ ID NO:5; a fragment of at least 15 contiguous nucleotides of
SEQ ID NOs:1, 3, or 5; the complement of a fragment of at least 15
contiguous nucleotides of SEQ ID NOs:1, 3, or 5; a native coding
sequence of a Diabrotica organism (e.g., WCR) comprising SEQ ID
NOs:1, 3, or 5; the complement of a native coding sequence of a
Diabrotica organism (e.g., WCR) comprising SEQ ID NOs:1, 3, or 5; a
native non-coding sequence of a Diabrotica organism that is
transcribed into a native RNA molecule comprising SEQ ID NOs:1, 3,
or 5; the complement of a native non-coding sequence of a
Diabrotica organism that is transcribed into a native RNA molecule
comprising SEQ ID NOs:1, 3, or 5; a fragment of at least 15
contiguous nucleotides of a native coding sequence of a Diabrotica
organism (e.g., WCR) comprising SEQ ID NOs:1, 3, or 5; the
complement of a fragment of at least 15 contiguous nucleotides of a
native coding sequence of a Diabrotica organism comprising SEQ ID
NOs:1, 3, or 5; a fragment of at least 15 contiguous nucleotides of
a native non-coding sequence of a Diabrotica organism that is
transcribed into a native RNA molecule comprising SEQ ID NOs:1, 3,
or 5; and the complement of a fragment of at least 15 contiguous
nucleotides of a native non-coding sequence of a Diabrotica
organism that is transcribed into a native RNA molecule comprising
SEQ ID NOs:1, 3, or 5. In certain embodiments, expression of a
nucleic acid molecule that is at least 80% identical (e.g., 80%,
about 81%, about 82%, about 83%, about 84%, about 85%, about 86%,
about 87%, about 88%, about 89%, about 90%, about 91%, about 92%,
about 93%, about 94%, about 95%, about 96%, about 97%, about 98%,
about 99%, about 100%, and 100%) with any of the foregoing may be
used. In these and further embodiments, a nucleic acid molecule may
be expressed that specifically hybridizes to an RNA molecule
present in at least one cell of a coleopteran pest. In particular
examples, such a nucleic acid molecule may comprise a nucleotide
sequence comprising SEQ ID NOs:1, 3, or 5.
[0230] In particular embodiments of the invention, expression of a
nucleic acid molecule comprising at least 15 contiguous nucleotides
of a nucleotide sequence is used in a method for
post-transcriptional inhibition of a target gene in a hemipteran
pest, wherein the nucleotide sequence is selected from the group
consisting of: SEQ ID NO:89; the complement of SEQ ID NO:89; a
fragment of at least 15 contiguous nucleotides of SEQ ID NO:89; the
complement of a fragment of at least 15 contiguous nucleotides of
SEQ ID NO:89; a native coding sequence of a hemipteran organism SEQ
ID NO:89; the complement of a native coding sequence of a
hemipteran organism comprising SEQ ID NO:89; a native non-coding
sequence of a hemipteran organism that is transcribed into a native
RNA molecule comprising SEQ ID NO:89; the complement of a native
non-coding sequence of a hemipteran organism that is transcribed
into a native RNA molecule comprising SEQ ID NO:89; the complement
of a native non-coding sequence of a hemipteran organism that is
transcribed into a native RNA molecule comprising SEQ ID NO:89; a
fragment of at least 15 contiguous nucleotides of a native coding
sequence of a hemipteran organism comprising SEQ ID NO:89; the
complement of a fragment of at least 15 contiguous nucleotides of a
native coding sequence of a hemipteran organism comprising SEQ ID
NO:89; a fragment of at least 15 contiguous nucleotides of a native
non-coding sequence of a hemipteran organism that is transcribed
into a native RNA molecule comprising SEQ ID NO:89; and the
complement of a fragment of at least 15 contiguous nucleotides of a
native non-coding sequence of a hemipteran organism that is
transcribed into a native RNA molecule comprising SEQ ID NO:89. In
certain embodiments, expression of a nucleic acid molecule that is
at least 80% identical (e.g., 80%, about 81%, about 82%, about 83%,
about 84%, about 85%, about 86%, about 87%, about 88%, about 89%,
about 90%, about 91%, about 92%, about 93%, about 94%, about 95%,
about 96%, about 97%, about 98%, about 99%, about 100%, and 100%)
with any of the foregoing may be used. In these and further
embodiments, a nucleic acid molecule may be expressed that
specifically hybridizes to an RNA molecule present in at least one
cell of a hemipteran pest. In particular examples, such a nucleic
acid molecule may comprise a nucleotide sequence comprising SEQ ID
NO:89.
[0231] It is an important feature of some embodiments of the
invention that the RNAi post-transcriptional inhibition system is
able to tolerate sequence variations among target genes that might
be expected due to genetic mutation, strain polymorphism, or
evolutionary divergence. The introduced nucleic acid molecule may
not need to be absolutely homologous to either a primary
transcription product or a fully-processed mRNA of a target gene,
so long as the introduced nucleic acid molecule is specifically
hybridizable to either a primary transcription product or a
fully-processed mRNA of the target gene. Moreover, the introduced
nucleic acid molecule may not need to be full-length, relative to
either a primary transcription product or a fully processed mRNA of
the target gene.
[0232] Inhibition of a target gene using the iRNA technology of the
present invention is sequence-specific; i.e., nucleotide sequences
substantially homologous to the iRNA molecule(s) are targeted for
genetic inhibition. In some embodiments, an RNA molecule comprising
a nucleotide sequence identical to a portion of a target gene
sequence may be used for inhibition. In these and further
embodiments, an RNA molecule comprising a nucleotide sequence with
one or more insertion, deletion, and/or point mutations relative to
a target gene sequence may be used. In particular embodiments, an
iRNA molecule and a portion of a target gene may share, for
example, at least from about 80%, at least from about 81%, at least
from about 82%, at least from about 83%, at least from about 84%,
at least from about 85%, at least from about 86%, at least from
about 87%, at least from about 88%, at least from about 89%, at
least from about 90%, at least from about 91%, at least from about
92%, at least from about 93%, at least from about 94%, at least
from about 95%, at least from about 96%, at least from about 97%,
at least from about 98%, at least from about 99%, at least from
about 100%, and 100% sequence identity. Alternatively, the duplex
region of a dsRNA molecule may be specifically hybridizable with a
portion of a target gene transcript. In specifically hybridizable
molecules, a less than full length sequence exhibiting a greater
homology compensates for a longer, less homologous sequence. The
length of the nucleotide sequence of a duplex region of a dsRNA
molecule that is identical to a portion of a target gene transcript
may be at least about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,
26, 27, 28, 29, 30, 35, 40, 45, 25, 50, 100, 200, 300, 400, 500, or
at least about 1000 bases. In some embodiments, a sequence of
greater than 15 to 100 nucleotides may be used. In particular
embodiments, a sequence of greater than about 200 to 300
nucleotides may be used. In particular embodiments, a sequence of
greater than about 500 to 1000 nucleotides may be used, depending
on the size of the target gene.
[0233] In certain embodiments, expression of a target gene in a
coleopteran and/or hemipteran pest may be inhibited by at least
10%; at least 33%; at least 50%; or at least 80% within a cell of
the coleopteran and/or hemipteran pest, such that a significant
inhibition takes place. Significant inhibition refers to inhibition
over a threshold that results in a detectable phenotype (e.g.,
cessation of growth, cessation of feeding, cessation of
development, induced mortality, etc.), or a detectable decrease in
RNA and/or gene product corresponding to the target gene being
inhibited. Although in certain embodiments of the invention
inhibition occurs in substantially all cells of the coleopteran
and/or hemipteran pest, in other embodiments inhibition occurs only
in a subset of cells expressing the target gene.
[0234] In some embodiments, transcriptional suppression in a cell
is mediated by the presence of a dsRNA molecule exhibiting
substantial sequence identity to a promoter DNA sequence or the
complement thereof, to effect what is referred to as "promoter
trans suppression". Gene suppression may be effective against
target genes in a coleopteran and/or hemipteran pest that may
ingest or contact such dsRNA molecules, for example, by ingesting
or contacting plant material containing the dsRNA molecules. dsRNA
molecules for use in promoter trans suppression may be specifically
designed to inhibit or suppress the expression of one or more
homologous or complementary sequences in the cells of the
coleopteran and/or hemipteran pest. Post-transcriptional gene
suppression by antisense or sense oriented RNA to regulate gene
expression in plant cells is disclosed in U.S. Pat. Nos. 5,107,065,
5,231,020, 5,283,184, and 5,759,829.
[0235] C. Expression of iRNA Molecules Provided to a Coleopteran
and/or Hemipteran Pest
[0236] Expression of iRNA molecules for RNAi-mediated gene
inhibition in a coleopteran and/or hemipteran pest may be carried
out in any one of many in vitro or in vivo formats. The iRNA
molecules may then be provided to a coleopteran and/or hemipteran
pest, for example, by contacting the iRNA molecules with the pest,
or by causing the pest to ingest or otherwise internalize the iRNA
molecules. Some embodiments of the invention include transformed
host plants of a coleopteran and/or hemipteran pest, transformed
plant cells, and progeny of transformed plants. The transformed
plant cells and transformed plants may be engineered to express one
or more of the iRNA molecules, for example, under the control of a
heterologous promoter, to provide a pest-protective effect. Thus,
when a transgenic plant or plant cell is consumed by a coleopteran
and/or hemipteran pest during feeding, the pest may ingest iRNA
molecules expressed in the transgenic plants or cells. The
nucleotide sequences of the present invention may also be
introduced into a wide variety of prokaryotic and eukaryotic
microorganism hosts to produce iRNA molecules. The term
"microorganism" includes prokaryotic and eukaryotic species, such
as bacteria and fungi.
[0237] Modulation of gene expression may include partial or
complete suppression of such expression. In another embodiment, a
method for suppression of gene expression in a coleopteran and/or
hemipteran pest comprises providing in the tissue of the host of
the pest a gene-suppressive amount of at least one dsRNA molecule
formed following transcription of a nucleotide sequence as
described herein, at least one segment of which is complementary to
an mRNA sequence within the cells of the coleopteran and/or
hemipteran pest. A dsRNA molecule, including its modified form such
as an siRNA, miRNA, shRNA, or hpRNA molecule, ingested by a
coleopteran and/or hemipteran pest in accordance with the
invention, may be at least from about 80%, about 81%, about 82%,
about 83%, about 84%, about 85%, about 86%, about 87%, about 88%,
about 89%, about 90%, about 91%, about 92%, about 93%, about 94%,
about 95%, about 96%, about 97%, about 98%, about 99%, about 100%,
or 100% identical to an RNA molecule transcribed from a nucleic
acid molecule comprising a nucleotide sequence comprising SEQ ID
NOs:1, 3, 5, or 89. Isolated and substantially purified nucleic
acid molecules including, but not limited to, non-naturally
occurring nucleotide sequences and recombinant DNA constructs for
providing dsRNA molecules of the present invention are therefore
provided, which suppress or inhibit the expression of an endogenous
coding sequence or a target coding sequence in the coleopteran
and/or hemipteran pest when introduced thereto.
[0238] Particular embodiments provide a delivery system for the
delivery of iRNA molecules for the post-transcriptional inhibition
of one or more target gene(s) in a coleopteran and/or hemipteran
plant pest and control of a population of the coleopteran and/or
hemipteran plant pest. In some embodiments, the delivery system
comprises ingestion of a host transgenic plant cell or contents of
the host cell comprising RNA molecules transcribed in the host
cell. In these and further embodiments, a transgenic plant cell or
a transgenic plant is created that contains a recombinant DNA
construct providing a stabilized dsRNA molecule of the invention.
Transgenic plant cells and transgenic plants comprising nucleic
acid sequences encoding a particular iRNA molecule may be produced
by employing recombinant DNA technologies (which basic technologies
are well-known in the art) to construct a plant transformation
vector comprising a nucleotide sequence encoding an iRNA molecule
of the invention (e.g., a stabilized dsRNA molecule); to transform
a plant cell or plant; and to generate the transgenic plant cell or
the transgenic plant that contains the transcribed iRNA
molecule.
[0239] To impart coleopteran and/or hemipteran pest resistance to a
transgenic plant, a recombinant DNA molecule may, for example, be
transcribed into an iRNA molecule, such as a dsRNA molecule, a
siRNA molecule, a miRNA molecule, a shRNA molecule, or an hpRNA
molecule. In some embodiments, an RNA molecule transcribed from a
recombinant DNA molecule may form a dsRNA molecule within the
tissues or fluids of the recombinant plant. Such a dsRNA molecule
may be comprised in part of a nucleotide sequence that is identical
to a corresponding nucleotide sequence transcribed from a DNA
sequence within a coleopteran and/or hemipteran pest of a type that
may infest the host plant. Expression of a target gene within the
coleopteran and/or hemipteran pest is suppressed by the ingested
dsRNA molecule, and the suppression of expression of the target
gene in the coleopteran and/or hemipteran pest results in, for
example, cessation of feeding by the coleopteran and/or hemipteran
pest, with an ultimate result being, for example, that the
transgenic plant is protected from further damage by the
coleopteran and/or hemipteran pest. The modulatory effects of dsRNA
molecules have been shown to be applicable to a variety of genes
expressed in pests, including, for example, endogenous genes
responsible for cellular metabolism or cellular transformation,
including house-keeping genes; transcription factors;
molting-related genes; and other genes which encode polypeptides
involved in cellular metabolism or normal growth and
development.
[0240] For transcription from a transgene in vivo or an expression
construct, a regulatory region (e.g., promoter, enhancer, silencer,
and polyadenylation signal) may be used in some embodiments to
transcribe the RNA strand (or strands). Therefore, in some
embodiments, as set forth, supra, a nucleotide sequence for use in
producing iRNA molecules may be operably linked to one or more
promoter sequences functional in a plant host cell. The promoter
may be an endogenous promoter, normally resident in the host
genome. The nucleotide sequence of the present invention, under the
control of an operably linked promoter sequence, may further be
flanked by additional sequences that advantageously affect its
transcription and/or the stability of a resulting transcript. Such
sequences may be located upstream of the operably linked promoter,
downstream of the 3' end of the expression construct, and may occur
both upstream of the promoter and downstream of the 3' end of the
expression construct.
[0241] Some embodiments provide methods for reducing the damage to
a host plant (e.g., a corn plant) caused by a coleopteran and/or
hemipteran pest that feeds on the plant, wherein the method
comprises providing in the host plant a transformed plant cell
expressing at least one nucleic acid molecule of the invention,
wherein the nucleic acid molecule(s) functions upon being taken up
by the coleopteran and/or hemipteran pest to inhibit the expression
of a target sequence within the coleopteran and/or hemipteran pest,
which inhibition of expression results in mortality, reduced
growth, and/or reduced reproduction of the coleopteran and/or
hemipteran pest, thereby reducing the damage to the host plant
caused by the coleopteran and/or hemipteran pest. In some
embodiments, the nucleic acid molecule(s) comprise dsRNA molecules.
In these and further embodiments, the nucleic acid molecule(s)
comprise dsRNA molecules that each comprise more than one
nucleotide sequence that is specifically hybridizable to a nucleic
acid molecule expressed in a coleopteran and/or hemipteran pest
cell. In some embodiments, the nucleic acid molecule(s) consist of
one nucleotide sequence that is specifically hybridizable to a
nucleic acid molecule expressed in a coleopteran and/or hemipteran
pest cell.
[0242] In other embodiments, a method for increasing the yield of a
corn crop is provided, wherein the method comprises introducing
into a corn plant at least one nucleic acid molecule of the
invention; cultivating the corn plant to allow the expression of an
iRNA molecule comprising the nucleic acid sequence, wherein
expression of an iRNA molecule comprising the nucleic acid sequence
inhibits coleopteran and/or hemipteran pest growth and/or
coleopteran and/or hemipteran pest damage, thereby reducing or
eliminating a loss of yield due to coleopteran and/or hemipteran
pest infestation. In some embodiments, the iRNA molecule is a dsRNA
molecule. In these and further embodiments, the nucleic acid
molecule(s) comprise dsRNA molecules that each comprise more than
one nucleotide sequence that is specifically hybridizable to a
nucleic acid molecule expressed in a coleopteran and/or hemipteran
pest cell. In some embodiments, the nucleic acid molecule(s)
consists of one nucleotide sequence that is specifically
hybridizable to a nucleic acid molecule expressed in a coleopteran
and/or hemipteran pest cell.
[0243] In some embodiments, a method for modulating the expression
of a target gene in a coleopteran and/or hemipteran pest is
provided, the method comprising: transforming a plant cell with a
vector comprising a nucleic acid sequence encoding at least one
nucleic acid molecule of the invention, wherein the nucleotide
sequence is operatively-linked to a promoter and a transcription
termination sequence; culturing the transformed plant cell under
conditions sufficient to allow for development of a plant cell
culture including a plurality of transformed plant cells; selecting
for transformed plant cells that have integrated the nucleic acid
molecule into their genomes; screening the transformed plant cells
for expression of an iRNA molecule encoded by the integrated
nucleic acid molecule; selecting a transgenic plant cell that
expresses the iRNA molecule; and feeding the selected transgenic
plant cell to the coleopteran and/or hemipteran pest. Plants may
also be regenerated from transformed plant cells that express an
iRNA molecule encoded by the integrated nucleic acid molecule. In
some embodiments, the iRNA molecule is a dsRNA molecule. In these
and further embodiments, the nucleic acid molecule(s) comprise
dsRNA molecules that each comprise more than one nucleotide
sequence that is specifically hybridizable to a nucleic acid
molecule expressed in a coleopteran and/or hemipteran pest cell. In
some embodiments, the nucleic acid molecule(s) consists of one
nucleotide sequence that is specifically hybridizable to a nucleic
acid molecule expressed in a coleopteran and/or hemipteran pest
cell.
[0244] iRNA molecules of the invention can be incorporated within
the seeds of a plant species (e.g., corn), either as a product of
expression from a recombinant gene incorporated into a genome of
the plant cells, or as incorporated into a coating or seed
treatment that is applied to the seed before planting. A plant cell
comprising a recombinant gene is considered to be a transgenic
event. Also included in embodiments of the invention are delivery
systems for the delivery of iRNA molecules to coleopteran and/or
hemipteran pests. For example, the iRNA molecules of the invention
may be directly introduced into the cells of a coleopteran and/or
hemipteran pest. Methods for introduction may include direct mixing
of iRNA with plant tissue from a host for the coleopteran and/or
hemipteran pest, as well as application of compositions comprising
iRNA molecules of the invention to host plant tissue. For example,
iRNA molecules may be sprayed onto a plant surface. Alternatively,
an iRNA molecule may be expressed by a microorganism, and the
microorganism may be applied onto the plant surface, or introduced
into a root or stem by a physical means such as an injection. As
discussed, supra, a transgenic plant may also be genetically
engineered to express at least one iRNA molecule in an amount
sufficient to kill the coleopteran and/or hemipteran pests known to
infest the plant. iRNA molecules produced by chemical or enzymatic
synthesis may also be formulated in a manner consistent with common
agricultural practices, and used as spray-on products for
controlling plant damage by a coleopteran and/or hemipteran pest.
The formulations may include the appropriate stickers and wetters
required for efficient foliar coverage, as well as UV protectants
to protect iRNA molecules (e.g., dsRNA molecules) from UV damage.
Such additives are commonly used in the bioinsecticide industry,
and are well known to those skilled in the art. Such applications
may be combined with other spray-on insecticide applications
(biologically based or otherwise) to enhance plant protection from
coleopteran and/or hemipteran pests.
[0245] All references, including publications, patents, and patent
applications, cited herein are hereby incorporated by reference to
the extent they are not inconsistent with the explicit details of
this disclosure, and are so incorporated to the same extent as if
each reference were individually and specifically indicated to be
incorporated by reference and were set forth in its entirety
herein. The references discussed herein are provided solely for
their disclosure prior to the filing date of the present
application. Nothing herein is to be construed as an admission that
the inventors are not entitled to antedate such disclosure by
virtue of prior invention.
[0246] The following EXAMPLES are provided to illustrate certain
particular features and/or aspects. These EXAMPLES should not be
construed to limit the disclosure to the particular features or
aspects described.
EXAMPLES
Example 1
Insect Diet Bioassays
[0247] Sample Preparation and Bioassays
[0248] A number of dsRNA molecules (including those corresponding
to rpL40-1 reg1 (SEQ ID NO:9), rpL40-3 reg1 (SEQ ID NO:10), rpL40-1
ver1 (SEQ ID NO:11), rpL40-1 ver2 (SEQ ID NO:12), rpL40-1 ver3 (SEQ
ID NO:13), rpL40-1 ver4 (SEQ ID NO:14), and rpL40-1 ver5 (SEQ ID
NO:15), were synthesized and purified using a MEGASCRIPT.RTM. RNAi
kit or HiScribe.RTM. T7 In Vitro Transcription Kit. The purified
dsRNA molecules were prepared in TE buffer, and all bioassays
contained a control treatment consisting of this buffer, which
served as a background check for mortality or growth inhibition of
WCR (Diabrotica virgifera virgifera LeConte). The concentrations of
dsRNA molecules in the bioassay buffer were measured using a
NANODROP.TM. 8000 spectrophotometer (THERMO SCIENTIFIC, Wilmington,
Del.).
[0249] Samples were tested for insect activity in bioassays
conducted with neonate insect larvae on artificial insect diet. WCR
eggs were obtained from CROP CHARACTERISTICS, INC. (Farmington,
Minn.).
[0250] The bioassays were conducted in 128-well plastic trays
specifically designed for insect bioassays (C-D INTERNATIONAL,
Pitman, N.J.). Each well contained approximately 1.0 mL of an
artificial diet designed for growth of coleopteran insects. A 60
.mu.L aliquot of dsRNA sample was delivered by pipette onto the
surface of the diet of each well (40 .mu.L/cm.sup.2). dsRNA sample
concentrations were calculated as the amount of dsRNA per square
centimeter (ng/cm.sup.2) of surface area (1.5 cm.sup.2) in the
well. The treated trays were held in a fume hood until the liquid
on the diet surface evaporated or was absorbed into the diet.
[0251] Within a few hours of eclosion, individual larvae were
picked up with a moistened camel hair brush and deposited on the
treated diet (one or two larvae per well). The infested wells of
the 128-well plastic trays were then sealed with adhesive sheets of
clear plastic, and vented to allow gas exchange. Bioassay trays
were held under controlled environmental conditions (28.degree. C.,
.about.40% Relative Humidity, 16:8 (Light: Dark)) for 9 days, after
which time the total number of insects exposed to each sample, the
number of dead insects, and the weight of surviving insects were
recorded. Average percent mortality and average growth inhibition
were calculated for each treatment. Growth inhibition (GI) was
calculated as follows:
GI=[1-(TWIT/TNIT)/(TWIBC/TNIBC)] [0252] where TWIT is the Total
Weight of live Insects in the Treatment; [0253] TNIT is the Total
Number of Insects in the Treatment; [0254] TWIBC is the Total
Weight of live Insects in the Background Check (Buffer control);
and [0255] TNIBC is the Total Number of Insects in the Background
Check (Buffer control).
[0256] Statistical analysis was done using JMP.TM. software (SAS,
Cary, N.C.).
[0257] LC.sub.50 (Lethal Concentration) is defined as the dosage at
which 50% of the test insects are killed. GI.sub.50 (Growth
Inhibition) is defined as the dosage at which the mean growth (e.g.
live weight) of the test insects is 50% of the mean value seen in
Background Check samples.
[0258] Replicated bioassays demonstrated that ingestion of
particular samples resulted in a surprising and unexpected
mortality and growth inhibition of corn rootworm larvae.
Example 2
Identification of Candidate Target Genes
[0259] Multiple stages of WCR (Diabrotica virgifera virgifera
LeConte) development were selected for pooled transcriptome
analysis to provide candidate target gene sequences for control by
RNAi transgenic plant insect resistance technology.
[0260] In one exemplification, total RNA was isolated from about
0.9 gm whole first-instar WCR larvae; (4 to 5 days post-hatch; held
at 16.degree. C.), and purified using the following phenol/TRI
REAGENT-based method (MOLECULAR RESEARCH CENTER, Cincinnati,
Ohio):
[0261] Larvae were homogenized at room temperature in a 15 mL
homogenizer with 10 mL of TRI REAGENT.RTM. until a homogenous
suspension was obtained. Following 5 min. incubation at room
temperature, the homogenate was dispensed into 1.5 mL microfuge
tubes (1 mL per tube), 200 .mu.L of chloroform was added, and the
mixture was vigorously shaken for 15 seconds. After allowing the
extraction to sit at room temperature for 10 min, the phases were
separated by centrifugation at 12,000.times.g at 4.degree. C. The
upper phase (comprising about 0.6 mL) was carefully transferred
into another sterile 1.5 mL tube, and an equal volume of room
temperature isopropanol was added. After incubation at room
temperature for 5 to 10 min, the mixture was centrifuged 8 min at
12,000.times.g (4.degree. C. or 25.degree. C.).
[0262] The supernatant was carefully removed and discarded, and the
RNA pellet was washed twice by vortexing with 75% ethanol, with
recovery by centrifugation for 5 min at 7,500.times.g (4.degree. C.
or 25.degree. C.) after each wash. The ethanol was carefully
removed, the pellet was allowed to air-dry for 3 to 5 min, and then
was dissolved in nuclease-free sterile water. RNA concentration was
determined by measuring the absorbance (A) at 260 nm and 280 nm. A
typical extraction from about 0.9 gm of larvae yielded over 1 mg of
total RNA, with an A.sub.260/A.sub.280 ratio of 1.9. The RNA thus
extracted was stored at -80.degree. C. until further processed.
[0263] RNA quality was determined by running an aliquot through a
1% agarose gel. The agarose gel solution was made using autoclaved
10.times.TAE buffer (Tris-acetate EDTA; 1.times.concentration is
0.04 M Tris-acetate, 1 mM EDTA (ethylenediamine tetra-acetic acid
sodium salt), pH 8.0) diluted with DEPC (diethyl
pyrocarbonate)-treated water in an autoclaved container.
1.times.TAE was used as the running buffer. Before use, the
electrophoresis tank and the well-forming comb were cleaned with
RNAseAway.TM. (INVITROGEN INC., Carlsbad, Calif.). Two .mu.L of RNA
sample were mixed with 8 .mu.L of TE buffer (10 mM Tris HCl pH 7.0;
1 mM EDTA) and 10 .mu.L of RNA sample buffer (NOVAGEN.RTM. Catalog
No 70606; EMD4 Bioscience, Gibbstown, N.J.). The sample was heated
at 70.degree. C. for 3 min, cooled to room temperature, and 5 .mu.L
(containing 1 .mu.g to 2 .mu.g RNA) were loaded per well.
Commercially available RNA molecular weight markers were
simultaneously run in separate wells for molecular size comparison.
The gel was run at 60 volts for 2 hr.
[0264] A normalized cDNA library was prepared from the larval total
RNA by a commercial service provider (EUROFINS MWG Operon,
Huntsville, Ala.), using random priming. The normalized larval cDNA
library was sequenced at 1/2 plate scale by GS FLX 454 Titanium.TM.
series chemistry at EUROFINS MWG Operon, which resulted in over
600,000 reads with an average read length of 348 bp. 350,000 reads
were assembled into over 50,000 contigs. Both the unassembled reads
and the contigs were converted into BLASTable databases using the
publicly available program, FORMATDB (available from NCBI).
[0265] Total RNA and normalized cDNA libraries were similarly
prepared from materials harvested at other WCR developmental
stages. A pooled transcriptome library for target gene screening
was constructed by combining cDNA library members representing the
various developmental stages.
[0266] Candidate genes for RNAi targeting were selected using
information regarding lethal RNAi effects of particular genes in
other insects such as Drosophila and Tribolium. These genes were
hypothesized to be essential for survival and growth in coleopteran
insects. Selected target gene homologs were identified in the
transcriptome sequence database as described below. Full-length or
partial sequences of the target genes were amplified by PCR to
prepare templates for double-stranded RNA (dsRNA) production.
[0267] TBLASTN searches using candidate protein coding sequences
were run against BLASTable databases containing the unassembled
Diabrotica sequence reads or the assembled contigs. Significant
hits to a Diabrotica sequence (defined as better than e.sup.-20 for
contigs homologies and better than e.sup.-10 for unassembled
sequence reads homologies) were confirmed using BLASTX against the
NCBI non-redundant database. The results of this BLASTX search
confirmed that the Diabrotica homolog candidate gene sequences
identified in the TBLASTN search indeed comprised Diabrotica genes,
or were the best hit to the non-Diabrotica candidate gene sequence
present in the Diabrotica sequences. In most cases, Tribolium
candidate genes which were annotated as encoding a protein gave an
unambiguous sequence homology to a sequence or sequences in the
Diabrotica transcriptome sequences. In a few cases, it was clear
that some of the Diabrotica contigs or unassembled sequence reads
selected by homology to a non-Diabrotica candidate gene overlapped,
and that the assembly of the contigs had failed to join these
overlaps. In those cases, Sequencher.TM. v4.9 (GENE CODES
CORPORATION, Ann Arbor, Mich.) was used to assemble the sequences
into longer contigs.
[0268] A candidate target gene encoding Diabrotica rpL40 (SEQ ID
NOs:1, 3, and 5) was identified as a gene that may lead to
coleopteran pest mortality, inhibition of growth, inhibition of
development, or inhibition of reproduction in WCR.
[0269] The sequence of SEQ ID NOs:1, 3, and 5 are novel. The
sequence is not provided in public databases and is not disclosed
in WO/2011/025860; U.S. Patent Application No. 20070124836; U.S.
Patent Application No. 20090306189; U.S. Patent Application No.
US20070050860; U.S. Patent Application No. 20100192265; or U.S.
Pat. No. 7,612,194. The Diabrotica rpL40-1 sequence (SEQ ID NO:1)
is somewhat related to a fragment of a sequence from Aedes aegypti
(GENBANK Accession No. AF418984.1). The closest homolog of the
Diabrotica RPL40-1 amino acid sequence (SEQ ID NO:2) is a
Drosophila melanogaster protein having GENBANK Accession No.
NP_476776.1 (100% similar; 100% identical over the homology
region). The Diabrotica rpL40-2 sequence (SEQ ID NO:3) is somewhat
related to a fragment of a sequence from Dendroctonus ponderosae
(GENBANK Accession No. APGK01027402.1). The closest homolog of the
Diabrotica RPL40-2 amino acid sequence (SEQ ID NO:4) is a
Saccoglossus kowalevskii protein having GENBANK Accession No.
XP_006817704.1 (99% similar; 98% identical over the homology
region). The Diabrotica rpL40-3 sequence (SEQ ID NO:5) is somewhat
related to a short fragment of a sequence from Phyllostachys edulis
(GENBANK Accession No. FP093773.1). The closest homolog of the
Diabrotica RPL40-3 amino acid sequence (SEQ ID NO:6) is a Gregarina
niphandrodes protein having GENBANK Accession No. XP_011128524.1
(96% similar; 88% identical over the homology region). The closest
homolog of the Diabrotica RPL40-3 amino acid sequence (SEQ ID NO:7)
is a Gregarina niphandrodes protein having GENBANK Accession No.
XP_011128522.1 (51% similar; 35% identical over the homology
region). The closest homolog of the Diabrotica RPL40-3 amino acid
sequence (SEQ ID NO:8) is a Gregarina niphandrodes protein having
GENBANK Accession No. XDR24359.1 (62% similar; 44% identical over
the homology region).
[0270] rpL40 dsRNA transgenes can be combined with other dsRNA
molecules to provide redundant RNAi targeting and synergistic RNAi
effects. Transgenic corn events expressing dsRNA that targets rpL40
are useful for preventing root feeding damage by corn rootworm.
rpL40 dsRNA transgenes represent new modes of action for combining
with Bacillus thuringiensis, Alcaligenes spp., or Pseudomonas spp.
insecticidal protein technology in Insect Resistance Management
gene pyramids to mitigate against the development of rootworm
populations resistant to either of these rootworm control
technologies.
[0271] Full-length or partial clones of sequences of a Diabrotica
candidate gene, herein referred to as rpL40, were used to generate
PCR amplicons for dsRNA synthesis.
[0272] SEQ ID NO:1 shows a 574 bp DNA sequence of Diabrotica
rpL40-1.
[0273] SEQ ID NO:3 shows a 652 bp DNA sequence of Diabrotica
rpL40-2.
[0274] SEQ ID NO:5 shows a 4065 bp DNA sequence of Diabrotica
rpL40-3.
[0275] SEQ ID NO:9 shows a 306 bp DNA sequence of rpL40-1 regi.
[0276] SEQ ID NO:10 shows a 361 bp DNA sequence of rpL40-3
regi.
[0277] SEQ ID NO:11 shows a 117 bp DNA sequence of rpL40-1 v1.
[0278] SEQ ID NO:12 shows a 143 bp DNA sequence of rpL40-1 v2.
[0279] SEQ ID NO:13 shows a 141 bp DNA sequence of rpL40-1 v3.
[0280] SEQ ID NO:14 shows a 130 bp DNA sequence of rpL40-1 v4.
[0281] SEQ ID NO:15 shows a 165 bp DNA sequence of rpL40-1 v5.
Example 3
Amplification of Target Genes to Produce dsRNA
[0282] Primers were designed to amplify portions of coding regions
of each target gene by PCR. See Table 1. Where appropriate, a T7
phage promoter sequence (TTAATACGACTCACTATAGGGAGA; SEQ ID NO:16)
was incorporated into the 5' ends of the amplified sense or
antisense strands. See Table 1. Total RNA was extracted from WCR,
and first-strand cDNA was used as template for PCR reactions using
opposing primers positioned to amplify all or part of the native
target gene sequence. dsRNA was also amplified from a DNA clone
comprising the coding region for a yellow fluorescent protein (YFP)
(SEQ ID NO:17; Shagin et al. (2004) Mol. Biol. Evol.
21(5):841-50).
TABLE-US-00002 TABLE 1 Primers and Primer Pairs used to amplify
portions of coding regions of exemplary rpL40 target gene and YFP
negative control gene. SEQ ID Gene ID Primer ID NO: Sequence Pair
rpL40-1 rpL40-1_F 18 TTAATACGACTCACTATAGGGAGA 1 reg1
GAAAATGTCAAGGCGAAAATCCA AG rpL40-1_R 19 TTAATACGACTCACTATAGGGAGA
CTTCTTTGGGCGCAAATTGTTG Pair rpL40-3 rpL40-3_F 20
TTAATACGACTCACTATAGGGAGA 2 reg1 GTCGAGAAGGTCAAGGACTCG rpL40-3_R 21
TTAATACGACTCACTATAGGGAGA AGCTTCACCATCGCGCCGAGGTTC TTC Pair rpL40-1
rpL40- 22 TTAATACGACTCACTATAGGGAGA 3 v1 1_v1_F
CCACCAGATCAGCAACGTCTAATT TTTG rpL40- 23 TTAATACGACTCACTATAGGGAGA
1_v1_R ACCACGTAGACGTAACACCAAATG Pair rpL40-1 rpL40- 24
TTAATACGACTCACTATAGGGAGA 4 v2 1_v2_F TCGAACCATCATTGCGTATTTTGG
rpL40- 25 TTAATACGACTCACTATAGGGAGA 1_v2_R CTTCTTTGGGCGCAAATTGTTGGT
G Pair rpL40-1 rpL40- 26 TTAATACGACTCACTATAGGGAGA 5 v3 1_v3_F
CAAGAGAGGCAAAAAACCAACG rpL40- 27 TTAATACGACTCACTATAGGGAGA 1_v3_R
CTGGTGGAATACCCTCTTTGTC Pair rpL40-1 rpL40- 28
TTAATACGACTCACTATAGGGAGA 6 v4 1_v4_F GCCGTAAGTGCTATGCTCGTTTG rpL40-
29 TTAATACGACTCACTATAGGGAGA 1_v4_R CCAGACATACAGCACAAAAATAC Pair
rpL40-1 rpL40- 30 TTAATACGACTCACTATAGGGAGA 7 v5 1_v5_F
CAGCAACGTCTAATTTTTGCTG rpL40- 31 TTAATACGACTCACTATAGGGAGA 1_v5_R
CATTTTGTCGCAATTGTACTTTTG Pair YFP YFP-F_T7 39
TTAATACGACTCACTATAGGGAGA 8 CACCATGGGCTCCAGCGGCGCCC YFP-R_T7 42
TTAATACGACTCACTATAGGGAGA AGATCTTGAAGGCGCTCTTCAGG
Example 4
RNAi Constructs
[0283] Template preparation by PCR and dsRNA synthesis.
[0284] A strategy used to provide specific templates for rpL40 and
YFP dsRNA production is shown in FIG. 1. Template DNAs intended for
use in rpL40 dsRNA synthesis were prepared by PCR using the primer
pairs in Table 1 and (as PCR template) first-strand cDNA prepared
from total RNA isolated from WCR first-instar larvae. For each
selected rpL40 and YFP target gene region, PCR amplifications
introduced a T7 promoter sequence at the 5' ends of the amplified
sense and antisense strands (the YFP segment was amplified from a
DNA clone of the YFP coding region). The PCR products having a T7
promoter sequence at their 5' ends of both sense and antisense
strands were used as transcription template for dsRNA production.
See FIG. 1. The sequences of the dsRNA templates amplified with the
particular primer pairs were: SEQ ID NO:9 (rpL40-1 reg1), SEQ ID
NO:10 (rpL40-3 reg1), SEQ ID NO:11 (rpL40-1 v1), SEQ ID NO:12
(rpL40-1 v2), SEQ ID NO:13 (rpL40-1 v3), SEQ ID NO:14 (rpL40-1 v4),
SEQ ID NO:15 (rpL40-1 v5), and YFP (SEQ ID NO:17). Double-stranded
RNA for insect bioassay was synthesized and purified using an
AMBION.RTM. MEGASCRIPT.RTM. RNAi kit following the manufacturer's
instructions (INVITROGEN) or HiScribe.TM. T7 High Yield RNA
Synthesis Kit following the manufacturer's instructions (New
England Biolabs). The concentrations of dsRNAs were measured using
a NANODROP.TM. 8000 spectrophotometer (THERMO SCIENTIFIC,
Wilmington, Del.).
[0285] Construction of Plant Transformation Vectors
[0286] Entry vectors harboring a target gene construct for hairpin
formation comprising segments of rpL40 (SEQ ID NOs:1, 3, or 5) are
assembled using a combination of chemically synthesized fragments
(DNA2.0, Menlo Park, Calif.) and standard molecular cloning
methods. Intramolecular hairpin formation by RNA primary
transcripts is facilitated by arranging (within a single
transcription unit) two copies of a segment of a rpL40 target gene
sequence in opposite orientation to one another, the two segments
being separated by a linker polynucleotide (e.g., a loop (such as
SEQ ID NO:112) or an ST-LS1 intron; Vancanneyt et al. (1990) Mol.
Gen. Genet. 220(2):245-50). Thus, the primary mRNA transcript
contains the two rpL40 gene segment sequences as large inverted
repeats of one another, separated by the linker sequence. A copy of
a promoter (e.g. maize ubiquitin 1, U.S. Pat. No. 5,510,474; 35S
from Cauliflower Mosaic Virus (CaMV); Sugarcane bacilliform
badnavirus (ScBV) promoter; promoters from rice actin genes;
ubiquitin promoters; pEMU; MAS; maize H3 histone promoter; ALS
promoter; phaseolin gene promoter; cab; rubisco; LAT52; Zm113;
and/or apg) is used to drive production of the primary mRNA hairpin
transcript, and a fragment comprising a 3' untranslated region
(e.g., a maize peroxidase 5 gene (ZmPer5 3'UTR v2; U.S. Pat. No.
6,699,984), AtUbil0, AtEfl, or StPinII) is used to terminate
transcription of the hairpin-RNA-expressing gene.
[0287] A binary destination vector comprises an herbicide tolerance
gene (aryloxyalknoate dioxygenase; AAD-1 v3) (U.S. Pat. No.
7,838,733(B2), and Wright et al. (2010) Proc. Natl. Acad. Sci.
U.S.A. 107:20240-5) under the regulation of a plant operable
promoter (e.g., sugarcane bacilliform badnavirus (ScBV) promoter
(Schenk et al. (1999) Plant Mol. Biol. 39:1221-30) or ZmUbil (U.S.
Pat. No. 5,510,474)). A 5'UTR and linker are positioned between the
3' end of the promoter segment and the start codon of the AAD-1
coding region. A fragment comprising a 3' untranslated region from
a maize lipase gene (ZmLip 3'UTR; U.S. Pat. No. 7,179,902) is used
to terminate transcription of the AAD-1 mRNA.
[0288] A negative control binary vector, which comprises a gene
that expresses a YFP protein, is constructed by means of standard
GATEWAY.RTM. recombination reactions with a typical binary
destination vector and entry vector. The binary destination vector
comprises a herbicide tolerance gene (aryloxyalknoate dioxygenase;
AAD-1 v3) (as above) under the expression regulation of a maize
ubiquitin 1 promoter (as above) and a fragment comprising a 3'
untranslated region from a maize lipase gene (ZmLip 3'UTR; as
above). An entry vector comprises a YFP coding region under the
expression control of a maize ubiquitin 1 promoter (as above) and a
fragment comprising a 3' untranslated region from a maize
peroxidase 5 gene (as above).
Example 5
Screening of Candidate Target Genes
[0289] Synthetic dsRNA designed to inhibit target gene sequences
identified in EXAMPLE 2 caused mortality and growth inhibition when
administered to WCR in diet-based assays. rpL40 regi, rpL40 v2,
rpL40 v3, and rpL40 v4, were observed to exhibit greatly increased
efficacy in this assay over other dsRNAs screened.
[0290] Replicated bioassays demonstrated that ingestion of dsRNA
preparations derived from rpL40 regi, rpL40 v1, rpL40 v2, rpL40 v3,
rpL40 v4, and rpL40 v5 each resulted in mortality and/or growth
inhibition of western corn rootworm larvae. Table 2 and Table 3
show the results of diet-based feeding bioassays of WCR larvae
following 9-day exposure to these dsRNAs, as well as the results
obtained with a negative control sample of dsRNA prepared from a
yellow fluorescent protein (YFP) coding region (SEQ ID NO:17).
TABLE-US-00003 TABLE 2 Results of rpL40 dsRNA diet feeding assays
obtained with western corn rootworm larvae after 9 days of feeding.
ANOVA analysis found significance differences in Mean % Mortality
and Mean % Growth Inhibition (GI). Means were separated using the
Tukey-Kramer test. Mean % Dose Mortality .+-. Mean GI .+-. Gene
Name (ng/cm.sup.2) No. Rows SEM* SEM rpL40-1 reg1 500 12 91.60 .+-.
1.97 (CD) 0.98 .+-. 0.01 (A) rpL40-1 v1 500 18 11.83 .+-. 2.32
(DEF) 0.44 .+-. 0.04 (B) rpL40-1 v2 500 4 34.56 .+-. 6.62 (A) 0.58
.+-. 0.10 (B) rpL40-1 v3 500 2 36.40 .+-. 1.11 (B) 0.63 .+-. 0.14
(AB) rpL40-1 v4 500 2 61.77 .+-. 14.70 (BC) 0.84 .+-. 0.10 (AB)
rpL40-1 v5 500 4 65.20 .+-. 2.45 (CDE) 0.62 .+-. 0.10 (B) TE** 0 36
18.13 .+-. 1.99 (EF) 0.08 .+-. 0.03 (C) WATER 500 28 13.84 .+-.
2.04 (EF) -0.14 .+-. 0.04 (C) YFP*** 0 28 14.26 .+-. 1.72 (F) 0.05
.+-. 0.04 (D) *SEM = Standard Error of the Mean. Letters in
parentheses designate statistical levels. Levels not connected by
same letter are significantly different (P < 0.05). **TE = Tris
HCl (1 mM) plus EDTA (1 mM) buffer, pH 7.2. ***YFP = Yellow
Fluorescent Protein
TABLE-US-00004 TABLE 3 Summary of oral potency of rpL40 dsRNA on
WCR larvae (ng/cm.sup.2). LC.sub.50 GI.sub.50 Gene Name
(ng/cm.sup.2) Range (ng/cm.sup.2) Range rpL40-1 reg1 12.91
9.88-16.71 4.06 3.28-5.02
[0291] It has previously been suggested that certain genes of
Diabrotica spp. may be exploited for RNAi-mediated insect control.
See U.S. Patent Publication No. 2007/0124836, which discloses 906
sequences, and U.S. Pat. No. 7,612,194, which discloses 9,112
sequences. However, it was determined that many genes suggested to
have utility for RNAi-mediated insect control are not efficacious
in controlling Diabrotica. It was also determined that sequences
rpL40 reg1, rpL40 v1, rpL40 v2, rpL40 v3, rpL40 v4, and rpL40 v5
each provide surprising and unexpected superior control of
Diabrotica, compared to other genes suggested to have utility for
RNAi-mediated insect control.
[0292] For example, Annexin, Beta spectrin 2, and mtRP-L4 were each
suggested in U.S. Pat. No. 7,612,194 to be efficacious in
RNAi-mediated insect control. SEQ ID NO:33 is the DNA sequence of
Annexin region 1 (Reg 1), and SEQ ID NO:34 is the DNA sequence of
Annexin region 2 (Reg 2). SEQ ID NO:35 is the DNA sequence of Beta
spectrin 2 region 1 (Reg 1), and SEQ ID NO:36 is the DNA sequence
of Beta spectrin 2 region 2 (Reg2). SEQ ID NO:37 is the DNA
sequence of mtRP-L4 region 1 (Reg 1), and SEQ ID NO:38 is the DNA
sequence of mtRP-L4 region 2 (Reg 2). A YFP sequence (SEQ ID NO:17)
was also used to produce dsRNA as a negative control.
[0293] Each of the aforementioned sequences was used to produce
dsRNA by the methods of EXAMPLE 3. The strategy used to provide
specific templates for dsRNA production is shown in FIG. 2.
Template DNAs intended for use in dsRNA synthesis were prepared by
PCR using the primer pairs in Table 4 and (as PCR template)
first-strand cDNA prepared from total RNA isolated from WCR
first-instar larvae. (YFP was amplified from a DNA clone.) For each
selected target gene region, two separate PCR amplifications were
performed. The first PCR amplification introduced a T7 promoter
sequence at the 5' end of the amplified sense strands. The second
reaction incorporated the T7 promoter sequence at the 5' ends of
the antisense strands. The two PCR amplified fragments for each
region of the target genes were then mixed in approximately equal
amounts, and the mixture was used as transcription template for
dsRNA production. See FIG. 2. Double-stranded RNA was synthesized
and purified using an AMBION.RTM. MEGAscript.RTM. RNAi kit
following the manufacturer's instructions (INVITROGEN). The
concentrations of dsRNAs were measured using a NANODROP.TM. 8000
spectrophotometer (THERMO SCIENTIFIC, Wilmington, Del.). and the
dsRNAs were each tested by the same diet-based bioassay methods
described above. Table 4 lists the sequences of the primers used to
produce the YFP, Annexin Reg1, Annexin Reg2, Beta spectrin 2 Reg1,
Beta spectrin 2 Reg2, mtRP-L4 Reg1, and mtRP-L4 Reg2 dsRNA
molecules. YFP primer sequences for use in the method depicted in
FIG. 2 are also listed in Table 4. Table 5 presents the results of
diet-based feeding bioassays of WCR larvae following 9-day exposure
to these dsRNA molecules. Replicated bioassays demonstrated that
ingestion of these dsRNAs resulted in no mortality or growth
inhibition of western corn rootworm larvae above that seen with
control samples of TE buffer, water, or YFP protein.
TABLE-US-00005 TABLE 4 Primers and Primer Pairs used to amplify
portions of coding regions of genes. Gene SEQ ID (Region) Primer ID
NO: Sequence Pair 9 YFP YFP-F_T7 39 TTAATACGACTCACTATAGGGAGAC
ACCATGGGCTCCAGCGGCGCCC YFP-R 40 AGATCTTGAAGGCGCTCTTCAGG Pair 10 YFP
YFP-F 41 CACCATGGGCTCCAGCGGCGCCC YFP-R_T7 42
TTAATACGACTCACTATAGGGAGAA GATCTTGAAGGCGCTCTTCAGG Pair 11 Annexin
Ann-F1_T7 43 TTAATACGACTCACTATAGGGAGAG (Reg 1)
CTCCAACAGTGGTTCCTTATC Annexin Ann-R1 44 CTAATAATTCTTTTTTAATGTTCCTG
(Reg 1) AGG Pair 12 Annexin Ann-F1 45 GCTCCAACAGTGGTTCCTTATC (Reg
1) Annexin Ann-R1_T7 46 TTAATACGACTCACTATAGGGAGAC (Reg 1)
TAATAATTCTTTTTTAATGTTCCTGA GG Pair 13 Annexin Ann-F2 T7 47
TTAATACGACTCACTATAGGGAGAT (Reg 2) TGTTACAAGCTGGAGAACTTCTC Annexin
Ann-R2 48 CTTAACCAACAACGGCTAATAAGG (Reg 2) Pair 14 Annexin Ann-F2
49 TTGTTACAAGCTGGAGAACTTCTC (Reg 2) Annexin Ann-R2T7 50
TTAATACGACTCACTATAGGGAGAC (Reg 2) TTAACCAACAACGGCTAATAAGG Pair 15
Beta-spect2 Betasp2-F1_T7 51 TTAATACGACTCACTATAGGGAGAA (Reg 1)
GATGTTGGCTGCATCTAGAGAA Beta-spect2 Betasp2-R1 52
GTCCATTCGTCCATCCACTGCA (Reg 1) Pair 16 Beta-spect2 Betasp2-F1 53
AGATGTTGGCTGCATCTAGAGAA (Reg 1) Beta-spect2 Betasp2-R1_T7 54
TTAATACGACTCACTATAGGGAGAG (Reg 1) TCCATTCGTCCATCCACTGCA Pair 17
Beta-spect2 Betasp2-F2_T7 55 TTAATACGACTCACTATAGGGAGAG (Reg 2)
CAGATGAACACCAGCGAGAAA Beta-spect2 Betasp2-R2 56
CTGGGCAGCTTCTTGTTTCCTC (Reg 2) Pair 18 Beta-spect2 Betasp2-F2 57
GCAGATGAACACCAGCGAGAAA (Reg 2) Beta-spect2 Betasp2-R2_ T7 58
TTAATACGACTCACTATAGGGAGAC (Reg 2) TGGGCAGCTTCTTGTTTCCTC Pair 19
mtRP-L4 L4-F1_T7 59 TTAATACGACTCACTATAGGGAGAA (Reg 1)
GTGAAATGTTAGCAAATATAACATC C mtRP-L4 L4-R1 60
ACCTCTCACTTCAAATCTTGACTTTG (Reg 1) Pair 20 mtRP-L4 L4-F1 61
AGTGAAATGTTAGCAAATATAACAT (Reg 1) CC mtRP-L4 L4-R1_T7 62
TTAATACGACTCACTATAGGGAGAA (Reg 1) CCTCTCACTTCAAATCTTGACTTTG Pair 21
mtRP-L4 L4-F2_ T7 63 TTAATACGACTCACTATAGGGAGAC (Reg 2)
AAAGTCAAGATTTGAAGTGAGAGGT mtRP-L4 L4-R2 64 CTACAAATAAAACAAGAAGGACCC
(Reg 2) C Pair 22 mtRP-L4 L4-F2 65 CAAAGTCAAGATTTGAAGTGAGAGG (Reg
2) T mtRP-L4 L4-R2_T7 66 TTAATACGACTCACTATAGGGAGAC (Reg 2)
TACAAATAAAACAAGAAGGACCCC
TABLE-US-00006 TABLE 5 Results of diet feeding assays obtained with
western corn rootworm larvae after 9 days. Dose Mean Live Mean %
Mean Growth Gene Name (ng/cm.sup.2) Larval Weight (mg) Mortality
Inhibition Annexin-Reg 1 1000 0.545 0 -0.262 Annexin-Reg 2 1000
0.565 0 -0.301 Beta spectrin2 Reg 1 1000 0.340 12 -0.014 Beta
spectrin2 Reg 2 1000 0.465 18 -0.367 mtRP-L4 Reg 1 1000 0.305 4
-0.168 mtRP-L4 Reg 2 1000 0.305 7 -0.180 TE buffer* 0 0.430 13
0.000 Water 0 0.535 12 0.000 YFP** 1000 0.480 9 -0.386 *TE = Tris
HCl (10 mM) plus EDTA (1 mM) buffer, pH 8. **YFP = Yellow
Fluorescent Protein
Example 6
Production of Transgenic Maize Tissues Comprising Insecticidal
dsRNAs
[0294] Agrobacterium-mediated Transformation. Transgenic maize
cells, tissues, and plants that produce one or more insecticidal
dsRNA molecules (for example, at least one dsRNA molecule including
a dsRNA molecule targeting a gene comprising rpL40-1 (SEQ ID NO:1);
rpL40-2 (SEQ ID NO:3); rpL40-3 (SEQ ID NO:5); rpL40-1 reg1 (SEQ ID
NO:9); rpL40-3 reg3 (SEQ ID NO:10); rpL40-1 v1 (SEQ ID NO:11);
rpL40-1 v2 (SEQ ID NO:12); rpL40-1 v3 (SEQ ID NO:13); rpL40-1 v4
(SEQ ID NO:14); rpL40-1 v5 (SEQ ID NO:15); BSB_rpL40 (SEQ ID
NO:89); BSB_rpL40 reg1 (SEQ ID NO:91); or BSB_rpL40 v1 (SEQ ID
NO:92) through expression of a chimeric gene stably-integrated into
the plant genome were produced following Agrobacterium-mediated
transformation. Maize transformation methods employing superbinary
or binary transformation vectors are known in the art, as
described, for example, in U.S. Pat. No. 8,304,604, which is herein
incorporated by reference in its entirety. Transformed tissues were
selected by their ability to grow on Haloxyfop-containing medium
and are screened for dsRNA production, as appropriate. Portions of
such transformed tissue cultures may be presented to neonate corn
rootworm larvae for bioassay, essentially as described in EXAMPLE
1.
[0295] Agrobacterium Culture Initiation.
[0296] Glycerol stocks of Agrobacterium strain DAt13192 cells (WO
2012/016222A2) harboring a binary transformation vector described
above (EXAMPLE 4) were streaked on AB minimal medium plates
(Watson, et al., (1975) J. Bacteriol. 123:255-264) containing
appropriate antibiotics and were grown at 20.degree. C. for 3 days.
The cultures were then streaked onto YEP plates (gm/L: yeast
extract, 10; Peptone, 10; NaCl 5) containing the same antibiotics
and were incubated at 20.degree. C. for 1 day.
[0297] Agrobacterium Culture.
[0298] On the day of an experiment, a stock solution of Inoculation
Medium and acetosyringone was prepared in a volume appropriate to
the number of constructs in the experiment and pipetted into a
sterile, disposable, 250 mL flask. Inoculation Medium (Frame et al.
(2011) Genetic Transformation Using Maize Immature Zygotic Embryos.
IN Plant Embryo Culture Methods and Protocols: Methods in Molecular
Biology. T. A. Thorpe and E. C. Yeung, (Eds), Springer Science and
Business Media, LLC. pp 327-341) contained: 2.2 gm/L MS salts;
1.times.ISU Modified MS Vitamins (Frame et al., ibid.) 68.4 gm/L
sucrose; 36 gm/L glucose; 115 mg/L L-proline; and 100 mg/L
myo-inositol; at pH 5.4.) Acetosyringone was added to the flask
containing Inoculation Medium to a final concentration of 200 .mu.M
from a 1 M stock solution in 100% dimethyl sulfoxide and the
solution was thoroughly mixed.
[0299] For each construct, 1 or 2 inoculating loops-full of
Agrobacterium from the YEP plate were suspended in 15 mL of the
Inoculation Medium/acetosyringone stock solution in a sterile,
disposable, 50 mL centrifuge tube, and the optical density of the
solution at 550 nm (OD.sub.550) was measured in a
spectrophotometer. The suspension was then diluted to OD.sub.550 of
0.3 to 0.4 using additional Inoculation Medium/acetosyringone
mixture. The tube of Agrobacterium suspension was then placed
horizontally on a platform shaker set at about 75 rpm at room
temperature and shaken for 1 to 4 hours while embryo dissection was
performed.
[0300] Ear Sterilization and Embryo Isolation.
[0301] Maize immature embryos were obtained from plants of Zea mays
inbred line B104 (Hallauer et al. (1997) Crop Science 37:1405-1406)
grown in the greenhouse and self- or sib-pollinated to produce
ears. The ears were harvested approximately 10 to 12 days
post-pollination. On the experimental day, de-husked ears were
surface-sterilized by immersion in a 20% solution of commercial
bleach (ULTRA CLOROX.RTM. Germicidal Bleach, 6.15% sodium
hypochlorite; with two drops of TWEEN 20) and shaken for 20 to 30
min, followed by three rinses in sterile deionized water in a
laminar flow hood. Immature zygotic embryos (1.8 to 2.2 mm long)
were aseptically dissected from each ear and randomly distributed
into microcentrifuge tubes containing 2.0 mL of a suspension of
appropriate Agrobacterium cells in liquid Inoculation Medium with
200 .mu.M acetosyringone, into which 2 .mu.L of 10% BREAK-THRU.RTM.
5233 surfactant (EVONIK INDUSTRIES; Essen, Germany) was added. For
a given set of experiments, embryos from pooled ears were used for
each transformation.
[0302] Agrobacterium Co-Cultivation.
[0303] Following isolation, the embryos were placed on a rocker
platform for 5 minutes. The contents of the tube were then poured
onto a plate of Co-cultivation Medium, which contained 4.33 gm/L MS
salts; 1.times.ISU Modified MS Vitamins; 30 gm/L sucrose; 700 mg/L
L-proline; 3.3 mg/L Dicamba in KOH (3,6-dichloro-o-anisic acid or
3,6-dichloro-2-methoxybenzoic acid); 100 mg/L myo-inositol; 100
mg/L Casein Enzymatic Hydrolysate; 15 mg/L AgNO.sub.3; 200 .mu.M
acetosyringone in DMSO; and 3 gm/L GELZAN.TM., at pH 5.8. The
liquid Agrobacterium suspension was removed with a sterile,
disposable, transfer pipette. The embryos were then oriented with
the scutellum facing up using sterile forceps with the aid of a
microscope. The plate was closed, sealed with 3M.TM. MICROPORE.TM.
medical tape, and placed in an incubator at 25.degree. C. with
continuous light at approximately 60 .mu.mol m.sup.-2 s.sup.-1 of
Photosynthetically Active Radiation (PAR).
[0304] Callus Selection and Regeneration of Transgenic Events.
[0305] Following the Co-Cultivation period, embryos were
transferred to Resting Medium, which was composed of 4.33 gm/L MS
salts; 1.times.ISU Modified MS Vitamins; 30 gm/L sucrose; 700 mg/L
L-proline; 3.3 mg/L Dicamba in KOH; 100 mg/L myo-inositol; 100 mg/L
Casein Enzymatic Hydrolysate; 15 mg/L AgNO.sub.3; 0.5 gm/L MES
(2-(N-morpholino)ethanesulfonic acid monohydrate; PHYTOTECHNOLOGIES
LABR.; Lenexa, Kans.); 250 mg/L Carbenicillin; and 2.3 gm/L
GELZAN.TM.; at pH 5.8. No more than 36 embryos were moved to each
plate. The plates were placed in a clear plastic box and incubated
at 27.degree. C. with continuous light at approximately 50 .mu.mol
m.sup.-2 s.sup.-1 PAR for 7 to 10 days. Callused embryos were then
transferred (<18/plate) onto Selection Medium I, which was
comprised of Resting Medium (above) with 100 nM R-Haloxyfop acid
(0.0362 mg/L; for selection of calli harboring the AAD-1 gene). The
plates were returned to clear boxes and incubated at 27.degree. C.
with continuous light at approximately 50 .mu.mol m.sup.-2 s.sup.-1
PAR for 7 days. Callused embryos were then transferred
(<12/plate) to Selection Medium II, which was comprised of
Resting Medium (above) with 500 nM R-Haloxyfop acid (0.181 mg/L).
The plates were returned to clear boxes and incubated at 27.degree.
C. with continuous light at approximately 50 .mu.mol m.sup.-2
s.sup.-1 PAR for 14 days. This selection step allowed transgenic
callus to further proliferate and differentiate.
[0306] Proliferating, embryogenic calli were transferred
(<9/plate) to Pre-Regeneration medium. Pre-Regeneration Medium
contained 4.33 gm/L MS salts; 1.times.ISU Modified MS Vitamins; 45
gm/L sucrose; 350 mg/L L-proline; 100 mg/L myo-inositol; 50 mg/L
Casein Enzymatic Hydrolysate; 1.0 mg/L AgNO.sub.3; 0.25 gm/L MES;
0.5 mg/L naphthaleneacetic acid in NaOH; 2.5 mg/L abscisic acid in
ethanol; 1 mg/L 6-benzylaminopurine; 250 mg/L Carbenicillin; 2.5
gm/L GELZAN.TM.; and 0.181 mg/L Haloxyfop acid; at pH 5.8. The
plates were stored in clear boxes and incubated at 27.degree. C.
with continuous light at approximately 50 .mu.mol m.sup.-2 s.sup.-1
PAR for 7 days. Regenerating calli were then transferred
(<6/plate) to Regeneration Medium in PHYTATRAYS.TM.
(SIGMA-ALDRICH) and incubated at 28.degree. C. with 16 hours
light/8 hours dark per day (at approximately 160 .mu.mol m.sup.-2
s.sup.-1 PAR) for 14 days or until shoots and roots develop.
Regeneration Medium contained 4.33 gm/L MS salts; 1.times.ISU
Modified MS Vitamins; 60 gm/L sucrose; 100 mg/L myo-inositol; 125
mg/L Carbenicillin; 3 gm/L GELLAN.TM. gum; and 0.181 mg/L
R-Haloxyfop acid; at pH 5.8. Small shoots with primary roots were
then isolated and transferred to Elongation Medium without
selection. Elongation Medium contained 4.33 gm/L MS salts;
1.times.ISU Modified MS Vitamins; 30 gm/L sucrose; and 3.5 gm/L
GELRITE.TM.: at pH 5.8.
[0307] Transformed plant shoots selected by their ability to grow
on medium containing Haloxyfop were transplanted from
PHYTATRAYS.TM. to small pots filled with growing medium (PROMIX BX;
PREMIER TECH HORTICULTURE), covered with cups or HUMI-DOMES (ARCO
PLASTICS), and then hardened-off in a CONVIRON growth chamber
(27.degree. C. day/24.degree. C. night, 16-hour photoperiod, 50-70%
RH, 200 .mu.mol m.sup.-2 s.sup.-1 PAR). In some instances, putative
transgenic plantlets were analyzed for transgene relative copy
number by quantitative real-time PCR assays using primers designed
to detect the AAD1 herbicide tolerance gene integrated into the
maize genome. Further, qPCR assays were used to detect the presence
of the linker and/or target sequence in putative transformants.
Selected transformed plantlets were then moved into a greenhouse
for further growth and testing.
[0308] Transfer and Establishment of to Plants in the Greenhouse
for Bioassay and Seed Production.
[0309] When plants reached the V3-V4 stage, they were transplanted
into IE CUSTOM BLEND (PROFILE/METRO MIX 160) soil mixture and grown
to flowering in the greenhouse (Light Exposure Type: Photo or
Assimilation; High Light Limit: 1200 PAR; 16-hour day length;
27.degree. C. day/24.degree. C. night).
[0310] Plants to be used for insect bioassays were transplanted
from small pots to TINUS.TM. 350-4 ROOTRAINTERS.RTM.
(SPENCER-LEMAIRE INDUSTRIES, Acheson, Alberta, Canada) (one plant
per event per ROOTRAINER.RTM.). Approximately four days after
transplanting to ROOTRAINERS.RTM., plants were infested for
bioassay.
[0311] Plants of the T.sub.1 generation were obtained by
pollinating the silks of T.sub.0 transgenic plants with pollen
collected from plants of non-transgenic elite inbred line B104 or
other appropriate pollen donors, and planting the resultant seeds.
Reciprocal crosses were performed when possible.
Example 7
Molecular Analyses of Transgenic Maize Tissues
[0312] Molecular analyses (e.g. RT-qPCR) of maize tissues were
performed on samples from leaves collected from greenhouse grown
plants on the same days that root feeding damage is assessed.
[0313] Results of RT-qPCR assays for the Per5 3'UTR were used to
validate expression of the transgenes. Results of RT-qPCR assays
for linker sequence (which is integral to the formation of dsRNA
hairpin molecules) in expressed RNAs were used to validate the
presence of hairpin transcripts. Transgene RNA expression levels
were measured relative to the RNA levels of an endogenous maize
gene.
[0314] DNA qPCR analyses to detect a portion of the AAD1 coding
region in genomic DNA were used to estimate transgene insertion
copy number. Samples for these analyses were collected from plants
grown in environmental chambers. Results were compared to DNA qPCR
results of assays designed to detect a portion of a single-copy
native gene, and simple events (having one or two copies of rpL40
transgenes) were advanced for further studies in the
greenhouse.
[0315] Additionally, qPCR assays designed to detect a portion of
the spectinomycin-resistance gene (SpecR; harbored on the binary
vector plasmids outside of the T-DNA) were used to determine if the
transgenic plants contained extraneous integrated plasmid backbone
sequences.
[0316] RNA Transcript Expression Level:
[0317] target qPCR. Callus cell events or transgenic plants were
analyzed by real time quantitative PCR (qPCR) of the target
sequence to determine the relative expression level of the full
length hairpin transcript, as compared to the transcript level of
an internal maize gene (SEQ ID NO:67; GENBANK Accession No.
BT069734), which encodes a TIP41-like protein (i.e., a maize
homolog of GENBANK Accession No. AT4G34270; having a tBLASTX score
of 74% identity). RNA was isolated using an Norgen BioTek Total RNA
Isolation Kit (Norgen, Thorold, ON). The total RNA was subjected to
an On Column DNasel treatment according to the kit's suggested
protocol. The RNA was then quantified on a NANODROP 8000
spectrophotometer (THERMO SCIENTIFIC) and concentration was
normalized to 50 ng/.mu.L. First strand cDNA was prepared using a
HIGH CAPACITY cDNA SYNTHESIS KIT (INVITROGEN) in a 10 .mu.L
reaction volume with 5 .mu.L denatured RNA, substantially according
to the manufacturer's recommended protocol. The protocol was
modified slightly to include the addition of 10 .mu.L of 100 .mu.M
T20VN oligonucleotide (IDT) (SEQ ID NO:68; TTTTTTTTTTTTTTTTTTTTVN,
where V is A, C, or G, and N is A, C, G, or T/U) into the 1 mL tube
of random primer stock mix, in order to prepare a working stock of
combined random primers and oligo dT.
[0318] Following cDNA synthesis, samples were diluted 1:3 with
nuclease-free water, and stored at -20.degree. C. until assayed.
Separate real-time PCR assays for the target gene and TIP41-like
transcript were performed on a LIGHTCYCLER.TM. 480 (ROCHE
DIAGNOSTICS, Indianapolis, Ind.) in 10 .mu.L reaction volumes. For
the target gene assay, reactions were run with Primers rp140 (F)
(SEQ ID NO:69) and rp140 (R) (SEQ ID NO:70), and an IDT Custom
Oligo probe rab5 PRB Setl, labeled with FAM and double quenched
with Zen and Iowa Black quenchers (SEQ ID NO:111). For the
TIP41-like reference gene assay, primers TIPmxF (SEQ ID NO:71) and
TIPmxR (SEQ ID NO:72), and Probe HXTIP (SEQ ID NO:73) labeled with
HEX (hexachlorofluorescein) were used.
[0319] All assays included negative controls of no-template (mix
only). For the standard curves, a blank (water in source well) was
also included in the source plate to check for sample
cross-contamination. Primer and probe sequences are set forth in
Table 6. Reaction components recipes for detection of the various
transcripts are disclosed in Table 7, and PCR reactions conditions
are summarized in Table 8. The FAM (6-Carboxy Fluorescein Amidite)
fluorescent moiety was excited at 465 nm and fluorescence was
measured at 510 nm; the corresponding values for the HEX
(hexachlorofluorescein) fluorescent moiety were 533 nm and 580
nm.
TABLE-US-00007 TABLE 6 Oligonucleotide sequences for molecular
analyses of transcript levels in transgenic maize. SEQ Oligo- ID
Target nucleotide NO. Sequence rpl40 rpl40 (F) 58
CACCAGATCAGCAACGTCTAA rpl40 rpl40 (R) 59 CCAAATGGAGTGTCGATTCCT
rpl40 rpl40 111 CGTCCATCTTCCAATTGTTTGCCAGC (FAM- Probe) TIP41
TIPmxF 60 TGAGGGTAATGCCAACTGGTT TIP41 TIPmxR 61
GCAATGTAACCGAGTGTCTCTCAA TIP41 HXTIP 62
TTTTTGGCTTAGAGTTGATGGTGTACTGA (HEX- TGA Probe) *TIP41-like
protein.
TABLE-US-00008 TABLE 7 PCR reaction recipes for transcript
detection. Target Gene TIP-like Gene Component Final Concentration
Roche Buffer 1 X 1X rpl40 (F) 0.4 .mu.M 0 rpl40 (R) 0.4 .mu.M 0
rpl40 (FAM) 0.2 .mu.M 0 HEXtipZM F 0 0.4 .mu.M HEXtipZM R 0 0.4
.mu.M HEXtipZMP (HEX) 0 0.2 .mu.M cDNA (2.0 .mu.L) NA NA Water To
10 .mu.L To 10 .mu.L
TABLE-US-00009 TABLE 8 Thermocycler conditions for RNA qPCR. Target
Gene and TIP41-like Gene Detection Process Temp. Time No. Cycles
Target Activation 95.degree. C. 10 min 1 Denature 95.degree. C. 10
sec 40 Extend 60.degree. C. 40 sec Acquire FAM or HEX 72.degree. C.
1 sec Cool 40.degree. C. 10 sec 1
[0320] Data were analyzed using LIGHTCYCLER.TM. Software v1.5 by
relative quantification using a second derivative max algorithm for
calculation of Cq values according to the supplier's
recommendations. For expression analyses, expression values were
calculated using the .DELTA..DELTA.Ct method (i.e., 2-(Cq TARGET-Cq
REF)), which relies on the comparison of differences of Cq values
between two targets, with the base value of 2 being selected under
the assumption that, for optimized PCR reactions, the product
doubles every cycle.
[0321] Transcript Size and Integrity:
[0322] Northern Blot Assay. In some instances, additional molecular
characterization of the transgenic plants is obtained by the use of
Northern Blot (RNA blot) analysis to determine the molecular size
of the rpL40 hairpin RNA in transgenic plants expressing a rpL40
dsRNA.
[0323] All materials and equipment are treated with RNAZAP
(AMBION/INVITROGEN) before use. Tissue samples (100 mg to 500 mg)
are collected in 2 mL SAFELOCK EPPENDORF tubes, disrupted with a
KLECKO.TM. tissue pulverizer (GARCIA MANUFACTURING, Visalia,
Calif.) with three tungsten beads in 1 mL of TRIZOL (INVITROGEN)
for 5 min, then incubated at room temperature (RT) for 10 min.
Optionally, the samples are centrifuged for 10 min at 4.degree. C.
at 11,000 rpm and the supernatant is transferred into a fresh 2 mL
SAFELOCK EPPENDORF tube. After 200 .mu.L of chloroform are added to
the homogenate, the tube is mixed by inversion for 2 to 5 min,
incubated at RT for 10 minutes, and centrifuged at 12,000.times.g
for 15 min at 4.degree. C. The top phase is transferred into a
sterile 1.5 mL EPPENDORF tube, 600 .mu.L of 100% isopropanol are
added, followed by incubation at RT for 10 min to 2 hr, and then
centrifuged at 12,000.times.g for 10 min at 4.degree. to 25.degree.
C. The supernatant is discarded and the RNA pellet is washed twice
with 1 mL of 70% ethanol, with centrifugation at 7,500.times.g for
10 min at 4.degree. to 25.degree. C. between washes. The ethanol is
discarded and the pellet is briefly air dried for 3 to 5 min before
resuspending in 50 .mu.L of nuclease-free water.
[0324] Total RNA is quantified using the NANODROP8000.RTM.
(THERMO-FISHER) and samples are normalized to 5 .mu.g/10 .mu.L. 10
.mu.L of glyoxal (AMBION/INVITROGEN) are then added to each sample.
Five to 14 ng of DIG RNA standard marker mix (ROCHE APPLIED
SCIENCE, Indianapolis, Ind.) are dispensed and added to an equal
volume of glyoxal. Samples and marker RNAs are denatured at
50.degree. C. for 45 min and stored on ice until loading on a 1.25%
SEAKEM GOLD agarose (LONZA, Allendale, N.J.) gel in NORTHERNMAX
10.times. glyoxal running buffer (AMBION/INVITROGEN). RNAs are
separated by electrophoresis at 65 volts/30 mA for 2 hr and 15
min.
[0325] Following electrophoresis, the gel is rinsed in 2.times.SSC
for 5 min and imaged on a GEL DOC station (BIORAD, Hercules,
Calif.), then the RNA is passively transferred to a nylon membrane
(MILLIPORE) overnight at RT, using 10.times.SSC as the transfer
buffer (20.times.SSC consists of 3 M sodium chloride and 300 mM
trisodium citrate, pH 7.0). Following the transfer, the membrane is
rinsed in 2.times.SSC for 5 minutes, the RNA is UV-crosslinked to
the membrane (AGILENT/STRATAGENE), and the membrane is allowed to
dry at RT for up to 2 days.
[0326] The membrane is prehybridized in ULTRAHYB buffer
(AMBION/INVITROGEN) for 1 to 2 hr. The probe consists of a PCR
amplified product containing the sequence of interest, labeled with
digoxygenin by means of a ROCHE APPLIED SCIENCE DIG procedure.
Hybridization in recommended buffer is overnight at a temperature
of 60.degree. C. in hybridization tubes. Following hybridization,
the blot is subjected to DIG washes, wrapped, exposed to film for 1
to 30 minutes, then the film is developed, all by methods
recommended by the supplier of the DIG kit.
[0327] Transgene copy number determination. Maize leaf pieces
approximately equivalent to 2 leaf punches were collected in
96-well collection plates (QIAGEN). Tissue disruption was performed
with a KLECKO.TM. tissue pulverizer (GARCIA MANUFACTURING, Visalia,
Calif.) in BIOSPRINT96 AP1 lysis buffer (supplied with a
BIOSPRINT96 PLANT KIT; QIAGEN) with one stainless steel bead.
Following tissue maceration, genomic DNA (gDNA) was isolated in
high throughput format using a BIOSPRINT96 PLANT KIT and a
BIOSPRINT96 extraction robot. Genomic DNA was diluted 1:3 DNA:water
prior to setting up the qPCR reaction.
[0328] qPCR analysis. Transgene detection by hydrolysis probe assay
was performed by real-time PCR using a LIGHTCYCLER.RTM.480 system.
Oligonucleotides used in hydrolysis probe assays to detect the
target gene, the linker sequence (e.g., the loop), or to detect a
portion of the SpecR gene (i.e. the spectinomycin resistance gene
borne on the binary vector plasmids; SEQ ID NO:74; SPC1
oligonucleotides in Table 9), were designed using LIGHTCYCLER.RTM.
PROBE DESIGN SOFTWARE 2.0. Further, oligonucleotides used in
hydrolysis probe assays to detect a segment of the AAD-1 herbicide
tolerance gene (SEQ ID NO:75; GAAD1 oligonucleotides in Table 9)
were designed using PRIMER EXPRESS software (APPLIED BIOSYSTEMS).
Table 9 shows the sequences of the primers and probes. Assays were
multiplexed with reagents for an endogenous maize chromosomal gene
(Invertase (SEQ ID NO:76; GENBANK Accession No: U16123; referred to
herein as IVR1), which served as an internal reference sequence to
ensure gDNA was present in each assay. For amplification,
LIGHTCYCLER.RTM.480 PROBES MASTER mix (ROCHE APPLIED SCIENCE) was
prepared at 1.times.final concentration in a 10 .mu.L volume
multiplex reaction containing 0.4 .mu.M of each primer and 0.2
.mu.M of each probe (Table 10). A two step amplification reaction
was performed as outlined in Table 11. Fluorophore activation and
emission for the FAM- and HEX-labeled probes were as described
above; CY5 conjugates were excited maximally at 650 nm and
fluoresce maximally at 670 nm.
[0329] Cp scores (the point at which the fluorescence signal
crosses the background threshold) were determined from the real
time PCR data using the fit points algorithm (LIGHTCYCLER.RTM.
SOFTWARE release 1.5) and the Relative Quant module (based on the
MCt method). Data were handled as described previously (above; RNA
qPCR).
TABLE-US-00010 TABLE 9 Sequences of primers and probes (with
fluorescent conjugate) for gene copy number determination and
binary vector plasmid backbone detection. SEQ ID Name NO: Sequence
Loop - F 86 GGAACGAGCTGCTTGCGTAT Loop- R 87 CACGGTGCAGCTGATTGATG
Loop-P (FAM) 88 TCCCTTCCGTAGTCAGAG GAAD1-F 77 TGTTCGGTTCCCTCTACCAA
GAAD1-R 78 CAACATCCATCACCTTGACTGA GAAD1-P (FAM) 79
CACAGAACCGTCGCTTCAGCAACA IVR1-F 80 TGGCGGACGACGACTTGT IVR1-R 81
AAAGTTTGGAGGCTGCCGT IVR1-P (HEX) 82 CGAGCAGACCGCCGTGTACTTCTACC
SPC1A 83 CTTAGCTGGATAACGCCAC SPC1S 84 GACCGTAAGGCTTGATGAA TQSPEC
(CY5*) 85 CGAGATTCTCCGCGCTGTAGA CY5 = Cyanine-5
TABLE-US-00011 TABLE 10 Reaction components for gene copy number
analyses and plasmid backbone detection. Component Amt. (.mu.L)
Stock Final Conc'n 2x Buffer 5.0 2x 1x Appropriate Forward Primer
0.4 10 .mu.M 0.4 Appropriate Reverse Primer 0.4 10 .mu.M 0.4
Appropriate Probe 0.4 5 .mu.M 0.2 IVR1-Forward Primer 0.4 10 .mu.M
0.4 IVR1-Reverse Primer 0.4 10 .mu.M 0.4 IVR1-Probe 0.4 5 .mu.M 0.2
H.sub.2O 0.6 NA* NA gDNA 2.0 ND** ND Total 10.0 *NA = Not
Applicable **ND = Not Determined
TABLE-US-00012 TABLE 11 Thermocycler conditions for DNA qPCR
Genomic copy number analyses Process Temp. Time No. Cycles Target
Activation 95.degree. C. 10 min 1 Denature 95.degree. C. 10 sec 40
Extend & Acquire 60.degree. C. 40 sec FAM, HEX, or CY5 Cool
40.degree. C. 10 sec 1
Example 8
Bioassay of Transgenic Maize
[0330] In Vitro Insect Bioassays.
[0331] Bioactivity of dsRNA of the subject invention produced in
plant cells is demonstrated by bioassay methods. See, e.g., Baum et
al. (2007) Nat. Biotechnol. 25(11):1322-1326. One is able to
demonstrate efficacy, for example, by feeding various plant tissues
or tissue pieces derived from a plant producing an insecticidal
dsRNA to target insects in a controlled feeding environment.
Alternatively, extracts are prepared from various plant tissues
derived from a plant producing the insecticidal dsRNA and the
extracted nucleic acids are dispensed on top of artificial diets
for bioassays as previously described herein. The results of such
feeding assays are compared to similarly conducted bioassays that
employ appropriate control tissues from host plants that do not
produce an insecticidal dsRNA, or to other control samples.
[0332] Insect Bioassays with Transgenic Maize Events.
[0333] Two western corn rootworm larvae (1 to 3 days old) hatched
from washed eggs are selected and placed into each well of the
bioassay tray. The wells are then covered with a "PULL N' PEEL" tab
cover (BIO-CV-16, BIO-SERV) and placed in a 28.degree. C. incubator
with an 18 hr/6 hr light/dark cycle. Nine days after the initial
infestation, the larvae are assessed for mortality, which is
calculated as the percentage of dead insects out of the total
number of insects in each treatment. The insect samples are frozen
at -20.degree. C. for two days, then the insect larvae from each
treatment are pooled and weighed. The percent of growth inhibition
is calculated as the mean weight of the experimental treatments
divided by the mean of the average weight of two control well
treatments. The data are expressed as a Percent Growth Inhibition
(of the Negative Controls). Mean weights that exceed the control
mean weight are normalized to zero.
[0334] Insect Bioassays in the Greenhouse.
[0335] Western corn rootworm (WCR, Diabrotica virgifera virgifera
LeConte) eggs were received in soil from CROP CHARACTERISTICS
(Farmington, Minn.). WCR eggs were incubated at 28.degree. C. for
10 to 11 days. Eggs were washed from the soil, placed into a 0.15%
agar solution, and the concentration was adjusted to approximately
75 to 100 eggs per 0.25 mL aliquot. A hatch plate was set up in a
Petri dish with an aliquot of egg suspension to monitor hatch
rates.
[0336] The soil around the maize plants growing in ROOTRAINERS.RTM.
was infested with 150 to 200 WCR eggs. The insects were allowed to
feed for 2 weeks, after which time a "Root Rating" was given to
each plant. A Node-Injury Scale was utilized for grading
essentially according to Oleson et al. (2005, J. Econ. Entomol.
98:1-8). Plants which passed this bioassay were transplanted to
5-gallon pots for seed production. Transplants were treated with
insecticide to prevent further rootworm damage and insect release
in the greenhouses. Plants were hand pollinated for seed
production. Seeds produced by these plants were saved for
evaluation at the Ti and subsequent generations of plants.
[0337] Transgenic negative control plants were generated by
transformation with vectors harboring genes designed to produce a
yellow fluorescent protein (YFP). Bioassays were conducted with
negative controls included in each set of plant materials.
TABLE-US-00013 TABLE 12 Root Damage Rating based on greenhouse
bioassay and relative transcript expression level results of rpl40-
expressing maize plants and YFP control plants. Gene of Root Sample
ID Interest ssRNA dsRNA Rating rpl40 Events 126164[1]-002 rpl40
0.291 0.096 1 126164[1]-003 rpl40 0.063 1 126164[1]-005 rpl40 0.178
4.317 1 126164[1]-007 rpl40 0.261 3.945 1 126164[1]-008 rpl40 0.232
3.227 1 126164[1]-010 rpl40 0.136 0.818 1 126164[1]-011 rpl40 0.202
3.387 1 126164[1]-012 rpl40 0.356 4.408 1 126164[1]-013 rpl40 0.346
4.228 1 126164[1]-014 rpl40 0.221 1.945 1 126164[1]-015 rpl40 0.325
2.770 1 126164[1]-018 rpl40 0.31 3.864 1 126164[1]-023 rpl40 0.295
3.204 1 126164[1]-028 rpl40 0.281 4.258 1 126164[1]-030 rpl40 0.295
5.205 1 YFP Events 126944[5]-026 YFPv2 1 126944[6]-031 YFPv2 1
126944[7]-038 YFPv2 126944[7]-051 YFPv2 1 126944[7]-052 YFPv2 1
[0338] The measurement of dsRNA relative to the endogenous
reference gene (TIP-41 like protein) ranges from 0.063 to 5.205.
Root ratings were not significantly different from YFP controls.
The relative expression levels of these constructs could be
insufficient to provide root protection.
Example 9
Transgenic Zea mays Comprising Coleopteran Pest Sequences
[0339] Ten to 20 transgenic T.sub.0 Zea mays plants are generated
as described in EXAMPLE 6. A further 10-20 T.sub.1 Zea mays
independent lines expressing hairpin dsRNA for an RNAi construct
are obtained for corn rootworm challenge. Hairpin dsRNA may be
derived comprising all or part of SEQ ID NOs:1, 3, and 5.
Additional hairpin dsRNAs may be derived, for example, from
coleopteran pest sequences such as, for example, Cafl-180 (U.S.
Patent Application Publication No. 2012/0174258), VatpaseC (U.S.
Patent Application Publication No. 2012/0174259), Rhol (U.S. Patent
Application Publication No. 2012/0174260), VatpaseH (U.S. Patent
Application Publication No. 2012/0198586), PPI-87B (U.S. Patent
Application Publication No. 2013/0091600), RPA70 (U.S. Patent
Application Publication No. 2013/0091601), RPS6 (U.S. Patent
Application Publication No. 2013/0097730), ROP (U.S. patent
application Ser. No. 14/577,811), RNA polymerase 11140 (U.S. patent
application Ser. No. 14/577,854), RNA polymerase II (U.S. Patent
Application No. 62/133,214), RNA polymerase II-215 (U.S. Patent
Application No. 62/133,202), RNA polymerase 33 (U.S. Patent
Application No. 62/133,210), ncm (U.S. Patent Application No.
62/095,487), Dre4 (U.S. patent application Ser. No. 14/705,807),
COPI alpha (U.S. Patent Application No. 62/063,199), COPI beta
(U.S. Patent Application No. 62/063,203), COPI gamma (U.S. Patent
Application No. 62/063,192), or COPI delta (U.S. Patent Application
No. 62/063,216), spt5 (U.S. Patent Application No. 62/168,613), and
spt6 (U.S. Patent Application No. 62/168,606). These are confirmed
through RT-PCR or other molecular analysis methods.
[0340] Total RNA preparations from selected independent T.sub.1
lines are optionally used for RT-PCR with primers designed to bind
in the linker of the hairpin expression cassette in each of the
RNAi constructs. In addition, specific primers for each target gene
in an RNAi construct are optionally used to amplify and confirm the
production of the pre-processed mRNA required for siRNA production
in planta. The amplification of the desired bands for each target
gene confirms the expression of the hairpin RNA in each transgenic
Zea mays plant. Processing of the dsRNA hairpin of the target genes
into siRNA is subsequently optionally confirmed in independent
transgenic lines using RNA blot hybridizations.
[0341] Moreover, RNAi molecules having mismatch sequences with more
than 80% sequence identity to target genes affect corn rootworms in
a way similar to that seen with RNAi molecules having 100% sequence
identity to the target genes. The pairing of mismatch sequence with
native sequences to form a hairpin dsRNA in the same RNAi construct
delivers plant-processed siRNAs capable of affecting the growth,
development and viability of feeding coleopteran pests.
[0342] In planta delivery of dsRNA, siRNA or miRNA corresponding to
target genes and the subsequent uptake by coleopteran pests through
feeding results in down-regulation of the target genes in the
coleopteran pest through RNA-mediated gene silencing. When the
function of a target gene is important at one or more stages of
development, the growth, development, and reproduction of the
coleopteran pest is affected, and in the case of at least one of
WCR, NCR, SCR, MCR, D. balteata LeConte, D. u. tenella, and D. u.
undecimpunctata Mannerheim, leads to failure to successfully
infest, feed, develop, and/or reproduce, or leads to death of the
coleopteran pest. The choice of target genes and the successful
application of RNAi is then used to control coleopteran pests.
[0343] Phenotypic Comparison of Transgenic RNAi Lines and
Nontransformed Zea mays.
[0344] Target coleopteran pest genes or sequences selected for
creating hairpin dsRNA have no similarity to any known plant gene
sequence. Hence it is not expected that the production or the
activation of (systemic) RNAi by constructs targeting these
coleopteran pest genes or sequences will have any deleterious
effect on transgenic plants. However, development and morphological
characteristics of transgenic lines are compared with
nontransformed plants, as well as those of transgenic lines
transformed with an "empty" vector having no hairpin-expressing
gene. Plant root, shoot, foliage and reproduction characteristics
are compared. Plant shoot characteristics such as height, leaf
numbers and sizes, time of flowering, floral size and appearance
are recorded. In general, there are no observable morphological
differences between transgenic lines and those without expression
of target iRNA molecules when cultured in vitro and in soil in the
glasshouse.
Example 10
Transgenic Zea mays Comprising a Coleopteran Pest Sequence and
Additional RNAi Constructs
[0345] A transgenic Zea mays plant comprising a heterologous coding
sequence in its genome that is transcribed into an iRNA molecule
that targets an organism other than a coleopteran pest is
secondarily transformed via Agrobacterium or WHISKERS.TM.
methodologies (see Petolino and Arnold (2009) Methods Mol. Biol.
526:59-67) to produce one or more insecticidal dsRNA molecules (for
example, at least one dsRNA molecule including a dsRNA molecule
targeting a gene comprising SEQ ID NOs:1, 3, or 5). Plant
transformation plasmid vectors prepared essentially as described in
EXAMPLE 4 are delivered via Agrobacterium or WHISKERS.TM.-mediated
transformation methods into maize suspension cells or immature
maize embryos obtained from a transgenic Hi II or B104 Zea mays
plant comprising a heterologous coding sequence in its genome that
is transcribed into an iRNA molecule that targets an organism other
than a coleopteran pest.
Example 11
Transgenic Zea mays Comprising an RNAi Construct and Additional
Coleopteran Pest Control Sequences
[0346] A transgenic Zea mays plant comprising a heterologous coding
sequence in its genome that is transcribed into an iRNA molecule
that targets a coleopteran pest organism (for example, at least one
dsRNA molecule including a dsRNA molecule targeting a gene
comprising SEQ ID NOs:1, 3, or 5) is secondarily transformed via
Agrobacterium or WHISKERS.TM. methodologies (see Petolino and
Arnold (2009) Methods Mol. Biol. 526:59-67) to produce one or more
insecticidal protein molecules, for example, Cry3 or Cry34/Cry35Ab1
insecticidal proteins. Plant transformation plasmid vectors
prepared essentially as described in EXAMPLE 4 are delivered via
Agrobacterium or WHISKERS.TM.-mediated transformation methods into
maize suspension cells or immature maize embryos obtained from a
transgenic B104 Zea mays plant comprising a heterologous coding
sequence in its genome that is transcribed into an iRNA molecule
that targets a coleopteran pest organism. Doubly-transformed plants
are obtained that produce iRNA molecules and insecticidal proteins
for control of coleopteran pests.
Example 12
Mortality of Neotropical Brown Stink Bug (Euschistus heros)
Following rpL40 RNAi Injection
[0347] Neotropical Brown Stink Bug (BSB; Euschistus heros)
Colony.
[0348] BSB were reared in a 27.degree. C. incubator, at 65%
relative humidity, with 16:8 hour light: dark cycle. One gram of
eggs collected over 2-3 days were seeded in 5 L containers with
filter paper discs at the bottom; the containers were covered with
#18 mesh for ventilation. Each rearing container yielded
approximately 300-400 adult BSB. At all stages, the insects were
fed fresh green beans three times per week, a sachet of seed
mixture that contained sunflower seeds, soybeans, and peanuts
(3:1:1 by weight ratio) was replaced once a week. Water was
supplemented in vials with cotton plugs as wicks. After the initial
two weeks, insects were transferred onto new container once a
week.
[0349] BSB Artificial Diet.
[0350] BSB artificial diet prepared as follows (used within two
weeks of preparation). Lyophilized green beans were blended to a
fine powder in a MAGIC BULLET.RTM. blender while raw (organic)
peanuts were blended in a separate MAGIC BULLET.RTM. blender.
Blended dry ingredients were combined (weight percentages: green
beans, 35%; peanuts, 35%; sucrose, 5%; Vitamin complex (e.g.
Vanderzant Vitamin Mixture for insects, SIGMA-ALDRICH, Catalog No.
V1007), 0.9%); in a large MAGIC BULLET.RTM. blender, which was
capped and shaken well to mix the ingredients. The mixed dry
ingredients were then added to a mixing bowl. In a separate
container, water and benomyl anti-fungal agent (50 ppm; 25 .mu.L of
a 20,000 ppm solution/50 mL diet solution) were mixed well and then
added to the dry ingredient mixture. All ingredients were mixed by
hand until the solution was fully blended. The diet was shaped into
desired sizes, wrapped loosely in aluminum foil, heated for 4 hours
at 60.degree. C., then cooled and stored at 4.degree. C.
[0351] BSB Transcriptome Assembly.
[0352] Six stages of BSB development were selected for mRNA library
preparation. Total RNA was extracted from insects frozen at
-70.degree. C. and homogenized in 10 volumes of Lysis/Binding
buffer in Lysing MATRIX A 2 mL tubes (MP BIOMEDICALS, Santa Ana,
Calif.) on a FastPrep.RTM.-24 Instrument (MP BIOMEDICALS). Total
mRNA was extracted using a mirVana.TM. miRNA Isolation Kit (AMBION;
INVITROGEN) according to the manufacturer's protocol. RNA
sequencing using an Illumina.RTM. HiSeg.TM. system (San Diego,
Calif.) provided candidate target gene sequences for use in RNAi
insect control technology. HiSeg.TM. generated a total of about 378
million reads for the six samples. The reads were assembled
individually for each sample using TRINITY assembler software
(Grabherr et al. (2011) Nature Biotech. 29:644-652). The assembled
transcripts were combined to generate a pooled transcriptome. This
BSB pooled transcriptome contains 378,457 sequences.
[0353] BSB rpL40 Ortholog Identification.
[0354] A tBLASTn search of the BSB pooled transcriptome was
performed using as query the Drosophila rpL40 protein isoform A and
B sequences: GENBANK Accession Nos. NP_476776 and NP_001260018. BSB
rpL40 (SEQ ID NO:89) was identified as a Euschistus heros candidate
target gene product with predicted peptide sequence SEQ ID
NO:90.
[0355] Template preparation and dsRNA synthesis. cDNA was prepared
from total BSB RNA extracted from a single young adult insect
(about 90 mg) using TRIzol.RTM. Reagent (LIFE TECHNOLOGIES). The
insect was homogenized at room temperature in a 1.5 mL
microcentrifuge tube with 200 .mu.l, of TRIzol.RTM. using a pellet
pestle (FISHERBRAND Catalog No. 12-141-363) and Pestle Motor Mixer
(COLE-PARMER, Vernon Hills, Ill.). Following homogenization, an
additional 800 .mu.L of TRIzol.RTM. was added, the homogenate was
vortexed, and then incubated at room temperature for five minutes.
Cell debris was removed by centrifugation and the supernatant was
transferred to a new tube. Following manufacturer-recommended
TRIzol.RTM. extraction protocol for 1 mL of TRIzol.RTM., the RNA
pellet was dried at room temperature and resuspended in 200 .mu.l,
of Tris Buffer from a GFX PCR DNA AND GEL EXTRACTION KIT
(Illustra.TM.; GE HEALTHCARE LIFE SCIENCES) using Elution Buffer
Type 4 (i.e. 10 mM Tris-HCl pH8.0). RNA concentration was
determined using a NANODROP.TM. 8000 spectrophotometer (THERMO
SCIENTIFIC, Wilmington, Del.).
[0356] cDNA Amplification.
[0357] cDNA was reverse-transcribed from 5 .mu.g of BSB total RNA
template and oligo dT primer using a SUPERSCRIPT III FIRST-STRAND
SYNTHESIS SYSTEM.TM. for RT-PCR (INVITROGEN), following the
supplier's recommended protocol. The final volume of the
transcription reaction was brought to 100 .mu.L with nuclease-free
water.
[0358] Primers BSB_rpL40-1-For (SEQ ID NO:93) and BSB_rpL40-1-Rev
(SEQ ID NO:94) were used to amplify BSB_rpL40 region 1, also
referred to as BSB_rpL40 reg1 template. The DNA template was
amplified by touch-down PCR (annealing temperature lowered from
60.degree. C. to 50.degree. C. in a 1.degree. C./cycle decrease)
with 1 .mu.l, of cDNA (above) as the template. A fragment
comprising a 410 bp segment of BSB_rpL40 reg1 (SEQ ID NO:91) was
generated during 35 cycles of PCR. The above procedure was also
used to amplify a 301 bp negative control template YFPv2 (SEQ ID
NO:95) using YFPv2-F (SEQ ID NO:96) and YFPv2-R (SEQ ID NO:97)
primers. The BSB_rpL40 and YFPv2 primers contained a T7 phage
promoter sequence (SEQ ID NO:16) at their 5' ends, and thus enabled
the use of YFPv2 and BSB_rpL40 DNA fragments for dsRNA
transcription.
[0359] dsRNA Synthesis.
[0360] dsRNA was synthesized using 2 .mu.L of PCR product (above)
as the template with a MEGAscript.TM. RNAi kit (AMBION) used
according to the manufacturer's instructions. (See FIG. 1). dsRNA
was quantified on a NANODROP.TM. 8000 spectrophotometer and diluted
to 500 ng/.mu.L in nuclease-free 0.1.times.TE buffer (1 mM Tris
HCL, 0.1 mM EDTA, pH7.4).
[0361] Injection of dsRNA into BSB Hemoceol.
[0362] BSB were reared on a green bean and seed diet, as the
colony, in a 27.degree. C. incubator at 65% relative humidity and
16:8 hour light: dark photoperiod. Second instar nymphs (each
weighing 1 to 1.5 mg) were gently handled with a small brush to
prevent injury and were placed in a Petri dish on ice to chill and
immobilize the insects. Each insect was injected with 55.2 nL of a
500 ng/.mu.L dsRNA solution (i.e. 27.6 ng dsRNA; dosage of 18.4 to
27.6 .mu.g/g body weight). Injections were performed using a
NANOJECT.TM. II injector (DRUMMOND SCIENTIFIC, Broomhall, Pa.)
equipped with an injection needle pulled from a Drummond 3.5 inch
#3-000-203-G/X glass capillary. The needle tip was broken and the
capillary was backfilled with light mineral oil, then filled with 2
to 3 .mu.L of dsRNA. dsRNA was injected into the abdomen of the
nymphs (10 insects injected per dsRNA per trial), and the trials
were repeated on three different days. Injected insects (5 per
well) were transferred into 32-well trays (Bio-RT-32 Rearing Tray;
BIO-SERV, Frenchtown, N.J.) containing a pellet of artificial BSB
diet and covered with Pull-N-Peel.TM. tabs (BIO-CV-4; BIO-SERV).
Moisture was supplied by means of 1.25 mL of water in a 1.5 mL
microcentrifuge tube with a cotton wick. The trays were incubated
at 26.5.degree. C., 60% humidity and 16:8 hour light: dark
photoperiod. Viability counts and weights were taken on day 7 after
the injections.
[0363] Injections Identified BSB_rpL40 as a Lethal dsRNA
Target.
[0364] dsRNA that targets segment of YFP coding region, YFPv2 was
used as a negative control in BSB injection experiments. As
summarized in Table 13, 27.6 ng of BSB_rpL40 reg1 dsRNA injected
into the hemoceol of 2.sup.nd instar BSB nymphs produced high
mortality within seven days. The mortality caused by BSB_rpL40 reg1
dsRNA was significantly different from that seen with the same
amount of injected YFPv2 dsRNA (negative control), with p=0.00263
(Student's t-test).
TABLE-US-00014 TABLE 13 Results of BSB_rpL40 reg1 dsRNA injection
into the hemoceol of 2.sup.nd instar Brown Stink Bug nymphs seven
days after injection. Mean % p value Treatment* N Trials Mortality
SEM.dagger. t-test BSB rpL40 reg1 3 100 0 9.89E-05 Not injected 3
10 6 1.58E-01 YFP v2 dsRNA 3 0 0 *Ten insects injected per trial
for each dsRNA. .dagger.SEM- Standard error of the mean
Example 13
Transgenic Zea mays Comprising Hemipteran Pest Sequences
[0365] Ten to 20 transgenic T.sub.0 Zea mays plants harboring
expression vectors for nucleic acids comprising SEQ ID NOs:89, 91,
and/or 92 are generated as described in EXAMPLE 7. A further 10-20
T.sub.1 Zea mays independent lines expressing hairpin dsRNA for an
RNAi construct are obtained for BSB challenge. Hairpin dsRNA may be
derived as set forth in SEQ ID NO:91, SEQ ID NO:92, or otherwise
further comprising SEQ ID NO:89. These are confirmed through RT-PCR
or other molecular analysis methods. Total RNA preparations from
selected independent T.sub.1 lines are optionally used for RT-PCR
with primers designed to bind in the linker of the hairpin
expression cassette in each of the RNAi constructs. In addition,
specific primers for each target gene in an RNAi construct are
optionally used to amplify and confirm the production of the
pre-processed mRNA required for siRNA production in planta. The
amplification of the desired bands for each target gene confirms
the expression of the hairpin RNA in each transgenic Zea mays
plant. Processing of the dsRNA hairpin of the target genes into
siRNA is subsequently optionally confirmed in independent
transgenic lines using RNA blot hybridizations.
[0366] Moreover, RNAi molecules having mismatch sequences with more
than 80% sequence identity to target genes affect corn rootworms in
a way similar to that seen with RNAi molecules having 100% sequence
identity to the target genes. The pairing of mismatch sequence with
native sequences to form a hairpin dsRNA in the same RNAi construct
delivers plant-processed siRNAs capable of affecting the growth,
development and viability of feeding hemipteran pests.
[0367] In planta delivery of dsRNA, siRNA, shRNA, or miRNA
corresponding to target genes and the subsequent uptake by
hemipteran pests through feeding results in down-regulation of the
target genes in the hemipteran pest through RNA-mediated gene
silencing. When the function of a target gene is important at one
or more stages of development, the growth, development, and
reproduction of the hemipteran pest is affected, and in the case of
at least one of Euschistus heros, Piezodorus guildinii, Halyomorpha
halys, Nezara viridula, Acrosternum hilare, and Euschistus serous
leads to failure to successfully infest, feed, develop, and/or
reproduce, or leads to death of the hemipteran pest. The choice of
target genes and the successful application of RNAi is then used to
control hemipteran pests.
[0368] Phenotypic Comparison of Transgenic RNAi Lines and
Nontransformed Zea mays.
[0369] Target hemipteran pest genes or sequences selected for
creating hairpin dsRNA have no similarity to any known plant gene
sequence. Hence it is not expected that the production or the
activation of (systemic) RNAi by constructs targeting these
hemipteran pest genes or sequences will have any deleterious effect
on transgenic plants. However, development and morphological
characteristics of transgenic lines are compared with
nontransformed plants, as well as those of transgenic lines
transformed with an "empty" vector having no hairpin-expressing
gene. Plant root, shoot, foliage and reproduction characteristics
are compared. There is no observable difference in root length and
growth patterns of transgenic and nontransformed plants. Plant
shoot characteristics such as height, leaf numbers and sizes, time
of flowering, floral size and appearance are similar. In general,
there are no observable morphological differences between
transgenic lines and those without expression of target iRNA
molecules when cultured in vitro and in soil in the glasshouse.
Example 14
Transgenic Glycine max Comprising Hemipteran Pest Sequences
[0370] Ten to 20 transgenic T.sub.0 Glycine max plants harboring
expression vectors for nucleic acids comprising SEQ ID NOs:89, 91
and/or 92 are generated as is known in the art, including for
example by Agrobacterium-mediated transformation, as follows.
Mature soybean (Glycine max) seeds are sterilized overnight with
chlorine gas for sixteen hours. Following sterilization with
chlorine gas, the seeds are placed in an open container in a
LAMINAR.TM. flow hood to dispel the chlorine gas. Next, the
sterilized seeds are imbibed with sterile H.sub.2O for sixteen
hours in the dark using a black box at 24.degree. C.
[0371] Preparation of Split-Seed Soybeans.
[0372] The split soybean seed comprising a portion of an embryonic
axis protocol required preparation of soybean seed material which
is cut longitudinally, using a #10 blade affixed to a scalpel,
along the hilum of the seed to separate and remove the seed coat,
and to split the seed into two cotyledon sections. Careful
attention is made to partially remove the embryonic axis, wherein
about 1/2-1/3 of the embryo axis remains attached to the nodal end
of the cotyledon.
[0373] Inoculation.
[0374] The split soybean seeds comprising a partial portion of the
embryonic axis are then immersed for about 30 minutes in a solution
of Agrobacterium tumefaciens (e.g., strain EHA 101 or EHA 105)
containing binary plasmid comprising SEQ ID NOs:89, 91 and/or 92.
The Agrobacterium tumefaciens solution is diluted to a final
concentration of .lamda.=0.6 OD.sub.650 before immersing the
cotyledons comprising the embryo axis.
[0375] Co-Cultivation.
[0376] Following inoculation, the split soybean seed is allowed to
co-cultivate with the Agrobacterium tumefaciens strain for 5 days
on co-cultivation medium (Wang, Kan. Agrobacterium Protocols. 2. 1.
New Jersey: Humana Press, 2006. Print.) in a Petri dish covered
with a piece of filter paper.
[0377] Shoot Induction.
[0378] After 5 days of co-cultivation, the split soybean seeds are
washed in liquid Shoot Induction (SI) media consisting of B5 salts,
B5 vitamins, 28 mg/L Ferrous, 38 mg/L Na.sub.2EDTA, 30 g/L sucrose,
0.6 g/L MES, 1.11 mg/L BAP, 100 mg/L TIMENTIN.TM., 200 mg/L
cefotaxime, and 50 mg/L vancomycin (pH 5.7). The split soybean
seeds are then cultured on Shoot Induction I (SII) medium
consisting of B5 salts, B5 vitamins, 7 g/L Noble agar, 28 mg/L
Ferrous, 38 mg/L Na.sub.2EDTA, 30 g/L sucrose, 0.6 g/L MES, 1.11
mg/L BAP, 50 mg/L TIMENTIN.TM., 200 mg/L cefotaxime, 50 mg/L
vancomycin (pH 5.7), with the flat side of the cotyledon facing up
and the nodal end of the cotyledon imbedded into the medium. After
2 weeks of culture, the explants from the transformed split soybean
seed are transferred to the Shoot Induction II (SI II) medium
containing SII medium supplemented with 6 mg/L glufosinate
(LIBERTY.RTM.).
[0379] Shoot Elongation.
[0380] After 2 weeks of culture on SI II medium, the cotyledons are
removed from the explants and a flush shoot pad containing the
embryonic axis are excised by making a cut at the base of the
cotyledon. The isolated shoot pad from the cotyledon is transferred
to Shoot Elongation (SE) medium. The SE medium consists of MS
salts, 28 mg/L Ferrous, 38 mg/L Na.sub.2EDTA, 30 g/L sucrose and
0.6 g/L MES, 50 mg/L asparagine, 100 mg/L L-pyroglutamic acid, 0.1
mg/L IAA, 0.5 mg/L GA3, 1 mg/L zeatin riboside, 50 mg/L
TIMENTIN.TM., 200 mg/L cefotaxime, 50 mg/L vancomycin, 6 mg/L
glufosinate, 7 g/L Noble agar, (pH 5.7). The cultures are
transferred to fresh SE medium every 2 weeks. The cultures are
grown in a CONVIRON.TM. growth chamber at 24.degree. C. with an 18
h photoperiod at a light intensity of 80-90 mol/m.sup.2 sec.
[0381] Rooting.
[0382] Elongated shoots which developed from the cotyledon shoot
pad are isolated by cutting the elongated shoot at the base of the
cotyledon shoot pad, and dipping the elongated shoot in 1 mg/L IBA
(Indole 3-butyric acid) for 1-3 minutes to promote rooting. Next,
the elongated shoots are transferred to rooting medium (MS salts,
B5 vitamins, 28 mg/L Ferrous, 38 mg/L Na.sub.2EDTA, 20 g/L sucrose
and 0.59 g/L MES, 50 mg/L asparagine, 100 mg/L L-pyroglutamic acid
7 g/L Noble agar, pH 5.6) in phyta trays.
[0383] Cultivation.
[0384] Following culture in a CONVIRON.TM. growth chamber at
24.degree. C., 18 h photoperiod, for 1-2 weeks, the shoots which
have developed roots are transferred to a soil mix in a covered
sundae cup and placed in a CONVIRON.TM. growth chamber (models
CMP4030 and CMP3244, Controlled Environments Limited, Winnipeg,
Manitoba, Canada) under long day conditions (16 hours light/8 hours
dark) at a light intensity of 120-150 mol/m.sup.2 sec under
constant temperature (22.degree. C.) and humidity (40-50%) for
acclimatization of plantlets. The rooted plantlets are acclimated
in sundae cups for several weeks before they are transferred to the
greenhouse for further acclimatization and establishment of robust
transgenic soybean plants.
[0385] A further 10-20 T.sub.1 Glycine max independent lines
expressing hairpin dsRNA for an RNAi construct are obtained for BSB
challenge. Hairpin dsRNA may be derived as set forth in SEQ ID
NOs:91-92, or otherwise further comprising SEQ ID NO:89. These are
confirmed through RT-PCR or other molecular analysis methods. Total
RNA preparations from selected independent T.sub.1 lines are
optionally used for RT-PCR with primers designed to bind in the
linker of the hairpin expression cassette in each of the RNAi
constructs. In addition, specific primers for each target gene in
an RNAi construct are optionally used to amplify and confirm the
production of the pre-processed mRNA required for siRNA production
in planta. The amplification of the desired bands for each target
gene confirms the expression of the hairpin RNA in each transgenic
Glycine max plant. Processing of the dsRNA hairpin of the target
genes into siRNA is subsequently optionally confirmed in
independent transgenic lines using RNA blot hybridizations.
[0386] Moreover, RNAi molecules having mismatch sequences with more
than 80% sequence identity to target genes affect corn rootworms in
a way similar to that seen with RNAi molecules having 100% sequence
identity to the target genes. The pairing of mismatch sequence with
native sequences to form a hairpin dsRNA in the same RNAi construct
delivers plant-processed siRNAs capable of affecting the growth,
development and viability of feeding hemipteran pests.
[0387] In planta delivery of dsRNA, siRNA, shRNA, or miRNA
corresponding to target genes and the subsequent uptake by
hemipteran pests through feeding results in down-regulation of the
target genes in the hemipteran pest through RNA-mediated gene
silencing. When the function of a target gene is important at one
or more stages of development, the growth, development, and
reproduction of the hemipteran pest is affected, and in the case of
at least one of Euschistus heros, Piezodorus guildinii, Halyomorpha
halys, Nezara viridula, Acrosternum hilare, and Euschistus serous
leads to failure to successfully infest, feed, develop, and/or
reproduce, or leads to death of the hemipteran pest. The choice of
target genes and the successful application of RNAi is then used to
control hemipteran pests.
[0388] Phenotypic comparison of transgenic RNAi lines and
nontransformed Glycine max Target hemipteran pest genes or
sequences selected for creating hairpin dsRNA have no similarity to
any known plant gene sequence. Hence it is not expected that the
production or the activation of (systemic) RNAi by constructs
targeting these hemipteran pest genes or sequences will have any
deleterious effect on transgenic plants. However, development and
morphological characteristics of transgenic lines are compared with
nontransformed plants, as well as those of transgenic lines
transformed with an "empty" vector having no hairpin-expressing
gene. Plant root, shoot, foliage and reproduction characteristics
are compared. There is no observable difference in root length and
growth patterns of transgenic and nontransformed plants. Plant
shoot characteristics such as height, leaf numbers and sizes, time
of flowering, floral size and appearance are similar. In general,
there are no observable morphological differences between
transgenic lines and those without expression of target iRNA
molecules when cultured in vitro and in soil in the glasshouse.
Example 15
E. heros Bioassays on Artificial Diet
[0389] In dsRNA feeding assays on artificial diet, 32-well trays
are set up with an .about.18 mg pellet of artificial diet and
water, as for injection experiments (EXAMPLE 12). dsRNA at a
concentration of 200 ng/.mu.l is added to the food pellet and water
sample, 100 .mu.l to each of two wells. Five 2.sup.nd instar E.
heros nymphs are introduced into each well. Water samples and dsRNA
that targets YFP transcript are used as negative controls. The
experiments are repeated on three different days. Surviving insects
are weighed and the mortality rates are determined after 8 days of
treatment.
Example 16
Transgenic Arabidopsis thaliana Comprising Hemipteran Pest
Sequences
[0390] Arabidopsis transformation vectors containing a target gene
construct for hairpin formation comprising segments of rpL40 (SEQ
ID NO:89) are generated using standard molecular methods similar to
EXAMPLE 4. Arabidopsis transformation is performed using standard
Agrobacterium-based procedure. T.sub.1 seeds are selected with
glufosinate tolerance selectable marker. Transgenic T.sub.1
Arabidopsis plants are generated and homozygous simple-copy T.sub.2
transgenic plants are generated for insect studies. Bioassays are
performed on growing Arabidopsis plants with inflorescences. Five
to ten insects are placed on each plant and monitored for survival
within 14 days.
[0391] Construction of Arabidopsis Transformation Vectors.
[0392] Entry clones based on an entry vector harboring a target
gene construct for hairpin formation comprising a segment of rpL40
(SEQ ID NO:89) are assembled using a combination of chemically
synthesized fragments (DNA2.0, Menlo Park, Calif.) and standard
molecular cloning methods. Intramolecular hairpin formation by RNA
primary transcripts is facilitated by arranging (within a single
transcription unit) two copies of a target gene segment in opposite
orientations, the two segments being separated by a linker sequence
(e.g., a loop (such as SEQ ID NO:112) or an ST-LS1 intron
(Vancanneyt et al. (1990) Mol. Gen. Genet. 220(2):245-50)). Thus,
the primary mRNA transcript contains the two rpL40 gene segment
sequences as large inverted repeats of one another, separated by
the linker sequence. A copy of a Arabidopsis thaliana ubiquitin 10
promoter (Callis et al. (1990) J. Biological Chem. 265:12486-12493)
is used to drive production of the primary mRNA hairpin transcript,
and a fragment comprising a 3' untranslated region from Open
Reading Frame 23 of Agrobacterium tumefaciens (AtuORF23 3' UTR v1;
U.S. Pat. No. 5,428,147) is used to terminate transcription of the
hairpin-RNA-expressing gene.
[0393] The hairpin clone within an entry vector described above is
used in standard GATEWAY.RTM. recombination reaction with a typical
binary destination vector to produce hairpin RNA expression
transformation vectors for Agrobacterium-mediated Arabidopsis
transformation.
[0394] The binary destination vector comprises a herbicide
tolerance gene, DSM-2v2 (U.S. Patent App. No. 2011/0107455), under
the regulation of a Cassava vein mosaic virus promoter (CsVMV
Promoter v2, U.S. Pat. No. 7,601,885; Verdaguer et al, (1996) Plant
Molecular Biology, 31:1129-1139). A fragment comprising a 3'
untranslated region from Open Reading Frame 1 of Agrobacterium
tumefaciens (AtuORF1 3' UTR v6; Huang et al, (1990) J. Bacteriol,
172:1814-1822) is used to terminate transcription of the DSM2v2
mRNA.
[0395] A negative control binary construct, which comprises a gene
that expresses a YFP hairpin RNA, is constructed by means of
standard GATEWAY.RTM. recombination reactions with a typical binary
destination vector and entry vector. An entry construct comprises a
YFP hairpin sequence (hpYFP v2-1, SEQ ID NO:89) under the
expression control of an Arabidopsis Ubiquitin 10 promoter (as
above) and a fragment comprising an ORF23 3' untranslated region
from Agrobacterium tumefaciens (as above).
[0396] Production of Transgenic Arabidopsis Comprising Insecticidal
Hairpin RNAs: Agrobacterium-Mediated Transformation.
[0397] Binary plasmids containing hairpin sequences are
electroporated into Agrobacterium strain GV3101 (pMP90RK). The
recombinant Agrobacterium clones are confirmed by restriction
analysis of plasmids preparations of the recombinant Agrobacterium
colonies. A Qiagen Plasmid Max Kit (Qiagen, Cat#12162) is used to
extract plasmids from Agrobacterium cultures following the
manufacture recommended protocol.
[0398] Arabidopsis Transformation and T.sub.1 Selection.
[0399] Twelve to fifteen Arabidopsis plants (c.v. Columbia) are
grown in 4'' pots in the green house with light intensity of 250
mol/m.sup.2, 25.degree. C., and 18:6 hours of light: dark
conditions. Primary flower stems are trimmed one week before
transformation. Agrobacterium inoculums are prepared by incubating
10 .mu.l of recombinant Agrobacterium glycerol stock in 100 ml LB
broth (Sigma L3022)+100 mg/L Spectinomycin+50 mg/L Kanamycin at
28.degree. C. and shaking at 225 rpm for 72 hours. Agrobacterium
cells are harvested and suspended into 5% sucrose+0.04% Silwet-L77
(Lehle Seeds Cat # VIS-02)+10 .mu.g/L benzamino purine (BA)
solution to OD.sub.600 0.8-1.0 before floral dipping. The
above-ground parts of the plant are dipped into the Agrobacterium
solution for 5-10 minutes, with gentle agitation. The plants are
then transferred to the greenhouse for normal growth with regular
watering and fertilizing until seed set.
Example 17
Growth and Bioassays of Transgenic Arabidopsis
[0400] Selection of T.sub.1 Arabidopsis Transformed with Hairpin
RNAi Constructs.
[0401] Up to 200 mg of T.sub.1 seeds from each transformation is
stratified in 0.1% agarose solution. The seeds are planted in
germination trays (10.5''.times.21''.times.1''; T.O. Plastics Inc.,
Clearwater, Minn.) with #5 sunshine media. Transformants are
selected for tolerance to Ignite.RTM. (glufosinate) at 280 g/ha at
6 and 9 days post planting. Selected events are transplanted into
4'' diameter pots. Insertion copy analysis is performed within a
week of transplanting via hydrolysis quantitative Real-Time PCR
(qPCR) using Roche LightCycler480. The PCR primers and hydrolysis
probes are designed against DSM2v2 selectable marker using
LightCycler Probe Design Software 2.0 (Roche). Plants are
maintained at 24.degree. C., with a 16:8 hour light: dark
photoperiod under fluorescent and incandescent lights at intensity
of 100-150 mE/m2.times.s.
[0402] E. heros Plant Feeding Bioassay.
[0403] At least four low copy (1-2 insertions), four medium copy
(2-3 insertions), and four high copy (>4 insertions) events are
selected for each construct. Plants are grown to a flowering stage
(plants containing flowers and siliques). The surface of soil is
covered with .about.50 ml volume of white sand for easy insect
identification. Five to ten 2.sup.nd instar E. heros nymphs are
introduced onto each plant. The plants are covered with plastic
tubes that are 3'' in diameter, 16'' tall, and with wall thickness
of 0.03'' (Item No. 484485, Visipack Fenton Mo.); the tubes are
covered with nylon mesh to isolate the insects. The plants are kept
under normal temperature, light, and watering conditions in a
conviron. In 14 days, the insects are collected and weighed;
percent mortality as well as growth inhibition (1-weight
treatment/weight control) are calculated. YFP hairpin-expressing
plants are used as controls.
[0404] T.sub.2 Arabidopsis seed generation and T.sub.2
bioassays.
[0405] T.sub.2 seed is produced from selected low copy (1-2
insertions) events for each construct. Plants (homozygous and/or
heterozygous) are subjected to E. heros feeding bioassay, as
described above. T.sub.3 seed is harvested from homozygotes and
stored for future analysis.
Example 18
Transformation of Additional Crop Species
[0406] Cotton is transformed with rpL40 (with or without a
chloroplast transit peptide) to provide control of hemipteran
insects by utilizing a method known to those of skill in the art,
for example, substantially the same techniques previously described
in EXAMPLE 14 of U.S. Pat. No. 7,838,733, or Example 12 of PCT
International Patent Publication No. WO 2007/053482.
Example 19
RpL40 dsRNA in Insect Management
[0407] RpL40 dsRNA transgenes are combined with other dsRNA
molecules in transgenic plants to provide redundant RNAi targeting
and synergistic RNAi effects. Transgenic plants including, for
example and without limitation, corn, soybean, and cotton
expressing dsRNA that target rpL40 are useful for preventing
feeding damage by coleopteran and hemipteran insects. RpL40 dsRNA
transgenes are also combined in plants with Bacillus thuringiensis
insecticidal protein technology to represent new modes of action in
Insect Resistance Management gene pyramids. When combined with
other dsRNA molecules that target insect pests, and/or with
Bacillus thuringiensis insecticidal proteins, in a transgenic
plants, a synergistic insecticidal effect is observed that also
mitigates the development of resistant insect populations.
[0408] While the present disclosure may be susceptible to various
modifications and alternative forms, specific embodiments have been
described by way of example in detail herein. However, it should be
understood that the present disclosure is not intended to be
limited to the particular forms disclosed. Rather, the present
disclosure is to cover all modifications, equivalents, and
alternatives falling within the scope of the present disclosure as
defined by the following appended claims and their legal
equivalents.
Sequence CWU 1
1
1121574DNADiabrotica virgifera 1aagttgggta taaaccacgt ttcaagagag
gcaaaaaacc aacggaaaat gcagatattc 60gttaaaactt taaccggtaa gaccatcaca
cttgaggtcg agccaagcga cacaatcgaa 120aatgtcaagg cgaaaatcca
agacaaagag ggtattccac cagatcagca acgtctaatt 180tttgctggca
aacaattgga agatggacgt acattgtccg attacaacat ccaaaaggaa
240tcgacactcc atttggtgtt acgtctacgt ggtggtatca tcgaaccatc
attgcgtatt 300ttggcacaaa agtacaattg cgacaaaatg atttgccgta
agtgctatgc tcgtttgcat 360ccaagagcaa ccaattgccg taagaagaag
tgtggccaca ccaacaattt gcgcccaaag 420aagaagttga agtagacgat
cgtatttttg tgctgtatgt ctggaggaaa acaatgttcg 480gattcgtcca
cagttgttat cattttgaat aatcgaaatt atgtgaatca ttcattgggt
540acagcctgaa taaacttcgt tttctgaaac cggc 5742128PRTDiabrotica
virgifera 2Met Gln Ile Phe Val Lys Thr Leu Thr Gly Lys Thr Ile Thr
Leu Glu1 5 10 15Val Glu Pro Ser Asp Thr Ile Glu Asn Val Lys Ala Lys
Ile Gln Asp 20 25 30Lys Glu Gly Ile Pro Pro Asp Gln Gln Arg Leu Ile
Phe Ala Gly Lys 35 40 45Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr
Asn Ile Gln Lys Glu 50 55 60Ser Thr Leu His Leu Val Leu Arg Leu Arg
Gly Gly Ile Ile Glu Pro65 70 75 80Ser Leu Arg Ile Leu Ala Gln Lys
Tyr Asn Cys Asp Lys Met Ile Cys 85 90 95Arg Lys Cys Tyr Ala Arg Leu
His Pro Arg Ala Thr Asn Cys Arg Lys 100 105 110Lys Lys Cys Gly His
Thr Asn Asn Leu Arg Pro Lys Lys Lys Leu Lys 115 120
1253652DNADiabrotica virgifera 3attaaatatc tgtactttta aatttaccac
gctttttgta acggaaacgt gttcatcaac 60tgtcacttat caaataaact agacttcatt
tttcacttgt ctttttcatt cggaagttag 120cacgttgtgg aataaacact
tccacaatgc aaattttcgt gaaaactctc actggtaaaa 180ccatcaccct
cgaggttgag ccatccgata caatcgaaaa tgttaaagct aagatccaag
240acaaagaagg tatcccccca gaccagcaac gtttgatctt cgctggaaaa
caactcgaag 300atggacgtac cttgtcagac tacaacatcc agaaggaatc
cacccttcac ttggtgctcc 360gattgagagg aggtctcatc gagccttccc
ttcgtatttt agccatgaag tacaactgcg 420agaagatgat ctgccgtaaa
tgttacgcca ggttacaccc aagggccacc aactgcagaa 480agaagaagtg
tggacacacc aacaacttgc gccccaagaa gaagctaaag tagactatta
540cgaggttatt ttttacaatg ttaatatctt acaattccag atgttacttg
aacatttatt 600taactaatta taggaaatga catcaatcct aaaaaaaaaa
aaaaaaaaaa aa 6524128PRTDiabrotica virgifera 4Met Gln Ile Phe Val
Lys Thr Leu Thr Gly Lys Thr Ile Thr Leu Glu1 5 10 15Val Glu Pro Ser
Asp Thr Ile Glu Asn Val Lys Ala Lys Ile Gln Asp 20 25 30Lys Glu Gly
Ile Pro Pro Asp Gln Gln Arg Leu Ile Phe Ala Gly Lys 35 40 45Gln Leu
Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Gln Lys Glu 50 55 60Ser
Thr Leu His Leu Val Leu Arg Leu Arg Gly Gly Leu Ile Glu Pro65 70 75
80Ser Leu Arg Ile Leu Ala Met Lys Tyr Asn Cys Glu Lys Met Ile Cys
85 90 95Arg Lys Cys Tyr Ala Arg Leu His Pro Arg Ala Thr Asn Cys Arg
Lys 100 105 110Lys Lys Cys Gly His Thr Asn Asn Leu Arg Pro Lys Lys
Lys Leu Lys 115 120 12554065DNADiabrotica virgifera 5cagattttcg
tcaagacatt gacggggaag accattaccc tggaggtcga gcccagtgat 60tccatcgaga
ccgtcaaatc caagattcag gataaggaag ggattcctcc tgaccaacag
120cgcttgattt ttgctggcaa gcaactcgag gaaggccgat ctttgtcaga
ctataacatc 180caaaaagaga gtaccatcca tctcgtgctt cgactgagag
gaggtggtat cgagatcgag 240ccaacgttgg cgcaattggc tcgcaagcac
aatattgaga gactcatttg tcgaaaatgt 300tacgccagac tacacatcaa
agctactaat tgccggaaaa aggcctgcgg ccactcatct 360cagctcagac
ccaagaagaa gcctaagagt taataatttt tgaactttct tttttccacg
420aactagctta tgtgtactcg ctcagttgtt tcttctcagc cgccgattct
tctggcccga 480aacccaagtc gtcctctttg tacagaaatg tgtctcgccg
cactatgtcg ctaactaagc 540atgactcgat cgtcgaccac tccagtgggc
gctcacgtgc ttgctgttcg gcgaggtaca 600gcacgcggtt gacgcgattt
ttgaatactg cattgagcat cgacaccacc tcgctcgcct 660cctcccggag
tccgagtttc ctgacggact tgatgccgag cggaccaccg tattcatttg
720acgctcggtc cagtaggcgt tgggtgaacg cccaaccgtc ttctctttga
ggctcttgtg 780ggtggtgcat cccactaccg tagtggtgat tgttagatgc
ggccgttggc ggtgtgtcgt 840cctcagcttc aaagagcatc ctcagactac
cctcagtcga cacaggctgg ttgggtgaga 900gcccccaaaa tgcgctctca
gagaatggcc caagcgtgaa gaacgcccaa tcctccgcgc 960aactggacac
aactggcggc cctgccgaat ttacagtttg ctgagccggc attctccgaa
1020aagctggttc ctgatcaatc aaaccatgat cctctgaatt gtgttccaaa
tgatcagcca 1080acctttcata catacgccga gccgtatctc gtattgctga
atgcgacaaa gcgtccgcgg 1140gtttatagcc ggcacgtgcc tgccgtagca
acgctatgac gaacctcgcc atcgccgtct 1200tcggcactat gatgttcttg
tcgtttagcc aagcgatctg ccgacatgaa tcagaccgcc 1260ccttcatgaa
cgaataatca ggtaccacag ccttcaccgg agcgcgcagg ttattgttca
1320tcatttgtag ctcctgtaga gtcccaggcc gtccaacgag caggggccga
gcggcggagt 1380tcatcaagcc gcgatagggg accagtcggg ggaggccgcg
gagcagttcc ggctgcatat 1440taggtggaga tgagccaacg ccatcgagac
tctgcgggcc gccgaatccc ggcatgtcca 1500tcattccttg ggcactgaga
cttggcgagt tcatcgaatt cggcgtctgg ccttccatca 1560tgctgttcag
agatcccgga ggcgccggct gactggtgaa ggtcggactc tgcccgccag
1620agcccatgtt ttggccaggc ggctgcggca tagcgcctcc agcgtgcatc
atcatttgct 1680gttgctgcat catttgttgc tggagcatgt acggcggctg
ttgtggaggt ggcatcatgc 1740gttgaggcgc cggctggata ttgccagcgt
gcataatcgg cgtcatcgag ttcataccca 1800tcatcgtact ctgcatcata
gggtgatgca ttggcgctga agccatgact tgagggttcg 1860gcgcgccgta
catttgaccc tggggcggcg gctgggctcc gggaggtggc tgccagcgct
1920gttgctgttg ctgagcttgt tgctgtggtg tcggtacata tctattagtt
gggggcctcc 1980atgtattatg catcgtcaga gtggccgtgc gattgagttt
tcgagcataa aaagtgcggc 2040ctcagtgatt gggaatgatc atttcggcag
tcggagtctt gtatggcctg agtatcagag 2100acgtagtcca agagatcttg
ctcgtgctgc gaggcagcaa gggaccgtta atcgtaatga 2160agggctgatg
tttgccattg tccgtgcgtg aagtcaagtc taggactgtg ccgtgacaag
2220ctatggccgt aaactcaatt tccagttcgt agctaactcc aactcgttca
tgtcggaagg 2280tagcctctag actggcaggg atgtagagcg gaactatcgc
aaccagggtc tgcgtaacag 2340tcgtggaggc ttcacacaca gccacggtgt
ccgtcgttat aacaatcagc gttgaatcta 2400cattcgtcaa cgcgtggtag
cttctgaaat ccagccaact caaaccggtc gtggcgttgg 2460gccggtaccc
gaaattttgt ctatgcttgt tccactgaag cgcctcgagc ggagacagtg
2520gcggctgata agacctcgta gtttgtttca atctaacaca cacttccacc
acggccaaag 2580acgaattagt gaattcggtc ttgacataaa ttgtactacc
taggtgcccc tcaagagtgt 2640atgactcatc aacagtggtt ggtggcgatt
tattgccctc gtacactttg gattgcggcg 2700tcgccaaaca aacgtctgct
ggccatatat tgacgacttg gatatcggac agatgggaac 2760cattgatcga
aactctgtag acgggggtag tttgggggcg cacctgtgca tccgcttcaa
2820aagatgagac aaaattgcct gcagttcctt tttgactctt agagctgctc
atgcacctga 2880tcgagccgac aatcttatca atctcaaatc catttccgag
tctttcgatg agattcacct 2940catggacagc gcgctgctga attttctgcc
tcaataactt caagcattct ccaggctctt 3000ctaagaattc gttcaaagct
gcagcagata ctcctgttgc cgtcatatgc ttcgctggcc 3060gagtttcgct
caaagtgcta tgcatatcag cacaggtgta actgagccat ctgagatata
3120agagtccggc atcaaccttt tctttggcga cgagaaaatc attttgtgat
tcttgttcat 3180tgtgtgacca gccagctatc gggagcggaa tgggcagaga
gccagaagtg aaagcggctc 3240cgaaatccca atttaaggcg aggtaggtgg
gggtgaaggg ccgagctggt agcggtggat 3300atataggcgg aggttgtaga
atgatagaag cgagctgcgt gaacttcata aagatgggga 3360tccattcact
taaggggttg atcagaatag gacacgggct aatggttatt gccaagctta
3420tttgcggcgg gattggcggc cctggagcgc cgtttgttgg agtagatatt
tcggactcga 3480tactgtcgag ggtgctggcg cgagctggag ggaggtgcct
cacggatgag ccgccgctcg 3540cgctacgatc attcaaacct accgacccac
tattgttgtg tgaaagtaga gttgctccgc 3600ccgttggccc aatagtcaca
ggcgcgtcat ttctgatagc tagaacgtag tcatagctga 3660atcgtcctcc
tcgtcggatg gacggtggca agctaactgg gatgtagagc tggcatttgt
3720acctgtccac gtcgcctgtg acctcgagag tattgcttag cgcgaacact
cgccctgatt 3780tcttattggt ggtggcggag gtggcttgtt gttgatgtcc
gggggatggg agcgtagtag 3840tggaaacgga ttttccaaac tctctgaggg
ctctagtgac tacctcgtaa ccattttgaa 3900ctttgtcagt agagaataac
gggggggctg atggacagcg atcggtgtcc aatccgttga 3960ccagaccata
cacgtatatt cgatcaccgg cggtgaagtg ggtgtgacaa aaactgatgg
4020actggccacc tctaggcatt catttaatct ggcaagtgtt ccgct
40656130PRTDiabrotica virgifera 6Gln Ile Phe Val Lys Thr Leu Thr
Gly Lys Thr Ile Thr Leu Glu Val1 5 10 15Glu Pro Ser Asp Ser Ile Glu
Thr Val Lys Ser Lys Ile Gln Asp Lys 20 25 30Glu Gly Ile Pro Pro Asp
Gln Gln Arg Leu Ile Phe Ala Gly Lys Gln 35 40 45Leu Glu Glu Gly Arg
Ser Leu Ser Asp Tyr Asn Ile Gln Lys Glu Ser 50 55 60Thr Ile His Leu
Val Leu Arg Leu Arg Gly Gly Gly Ile Glu Ile Glu65 70 75 80Pro Thr
Leu Ala Gln Leu Ala Arg Lys His Asn Ile Glu Arg Leu Ile 85 90 95Cys
Arg Lys Cys Tyr Ala Arg Leu His Ile Lys Ala Thr Asn Cys Arg 100 105
110Lys Lys Ala Cys Gly His Ser Ser Gln Leu Arg Pro Lys Lys Lys Pro
115 120 125Lys Ser 1307665PRTDiabrotica virgifera 7Met Pro Arg Gly
Gly Gln Ser Ile Ser Phe Cys His Thr His Phe Thr1 5 10 15Ala Gly Asp
Arg Ile Tyr Val Tyr Gly Leu Val Asn Gly Leu Asp Thr 20 25 30Asp Arg
Cys Pro Ser Ala Pro Pro Leu Phe Ser Thr Asp Lys Val Gln 35 40 45Asn
Gly Tyr Glu Val Val Thr Arg Ala Leu Arg Glu Phe Gly Lys Ser 50 55
60Val Ser Thr Thr Thr Leu Pro Ser Pro Gly His Gln Gln Gln Ala Thr65
70 75 80Ser Ala Thr Thr Asn Lys Lys Ser Gly Arg Val Phe Ala Leu Ser
Asn 85 90 95Thr Leu Glu Val Thr Gly Asp Val Asp Arg Tyr Lys Cys Gln
Leu Tyr 100 105 110Ile Pro Val Ser Leu Pro Pro Ser Ile Arg Arg Gly
Gly Arg Phe Ser 115 120 125Tyr Asp Tyr Val Leu Ala Ile Arg Asn Asp
Ala Pro Val Thr Ile Gly 130 135 140Pro Thr Gly Gly Ala Thr Leu Leu
Ser His Asn Asn Ser Gly Ser Val145 150 155 160Gly Leu Asn Asp Arg
Ser Ala Ser Gly Gly Ser Ser Val Arg His Leu 165 170 175Pro Pro Ala
Arg Ala Ser Thr Leu Asp Ser Ile Glu Ser Glu Ile Ser 180 185 190Thr
Pro Thr Asn Gly Ala Pro Gly Pro Pro Ile Pro Pro Gln Ile Ser 195 200
205Leu Ala Ile Thr Ile Ser Pro Cys Pro Ile Leu Ile Asn Pro Leu Ser
210 215 220Glu Trp Ile Pro Ile Phe Met Lys Phe Thr Gln Leu Ala Ser
Ile Ile225 230 235 240Leu Gln Pro Pro Pro Ile Tyr Pro Pro Leu Pro
Ala Arg Pro Phe Thr 245 250 255Pro Thr Tyr Leu Ala Leu Asn Trp Asp
Phe Gly Ala Ala Phe Thr Ser 260 265 270Gly Ser Leu Pro Ile Pro Leu
Pro Ile Ala Gly Trp Ser His Asn Glu 275 280 285Gln Glu Ser Gln Asn
Asp Phe Leu Val Ala Lys Glu Lys Val Asp Ala 290 295 300Gly Leu Leu
Tyr Leu Arg Trp Leu Ser Tyr Thr Cys Ala Asp Met His305 310 315
320Ser Thr Leu Ser Glu Thr Arg Pro Ala Lys His Met Thr Ala Thr Gly
325 330 335Val Ser Ala Ala Ala Leu Asn Glu Phe Leu Glu Glu Pro Gly
Glu Cys 340 345 350Leu Lys Leu Leu Arg Gln Lys Ile Gln Gln Arg Ala
Val His Glu Val 355 360 365Asn Leu Ile Glu Arg Leu Gly Asn Gly Phe
Glu Ile Asp Lys Ile Val 370 375 380Gly Ser Ile Arg Cys Met Ser Ser
Ser Lys Ser Gln Lys Gly Thr Ala385 390 395 400Gly Asn Phe Val Ser
Ser Phe Glu Ala Asp Ala Gln Val Arg Pro Gln 405 410 415Thr Thr Pro
Val Tyr Arg Val Ser Ile Asn Gly Ser His Leu Ser Asp 420 425 430Ile
Gln Val Val Asn Ile Trp Pro Ala Asp Val Cys Leu Ala Thr Pro 435 440
445Gln Ser Lys Val Tyr Glu Gly Asn Lys Ser Pro Pro Thr Thr Val Asp
450 455 460Glu Ser Tyr Thr Leu Glu Gly His Leu Gly Ser Thr Ile Tyr
Val Lys465 470 475 480Thr Glu Phe Thr Asn Ser Ser Leu Ala Val Val
Glu Val Cys Val Arg 485 490 495Leu Lys Gln Thr Thr Arg Ser Tyr Gln
Pro Pro Leu Ser Pro Leu Glu 500 505 510Ala Leu Gln Trp Asn Lys His
Arg Gln Asn Phe Gly Tyr Arg Pro Asn 515 520 525Ala Thr Thr Gly Leu
Ser Trp Leu Asp Phe Arg Ser Tyr His Ala Leu 530 535 540Thr Asn Val
Asp Ser Thr Leu Ile Val Ile Thr Thr Asp Thr Val Ala545 550 555
560Val Cys Glu Ala Ser Thr Thr Val Thr Gln Thr Leu Val Ala Ile Val
565 570 575Pro Leu Tyr Ile Pro Ala Ser Leu Glu Ala Thr Phe Arg His
Glu Arg 580 585 590Val Gly Val Ser Tyr Glu Leu Glu Ile Glu Phe Thr
Ala Ile Ala Cys 595 600 605His Gly Thr Val Leu Asp Leu Thr Ser Arg
Thr Asp Asn Gly Lys His 610 615 620Gln Pro Phe Ile Thr Ile Asn Gly
Pro Leu Leu Pro Arg Ser Thr Ser625 630 635 640Lys Ile Ser Trp Thr
Thr Ser Leu Ile Leu Arg Pro Tyr Lys Thr Pro 645 650 655Thr Ala Glu
Met Ile Ile Pro Asn His 660 6658537PRTDiabrotica virgifera 8Gly Arg
Thr Phe Tyr Ala Arg Lys Leu Asn Arg Thr Ala Thr Leu Thr1 5 10 15Met
His Asn Thr Trp Arg Pro Pro Thr Asn Arg Tyr Val Pro Thr Pro 20 25
30Gln Gln Gln Ala Gln Gln Gln Gln Gln Arg Trp Gln Pro Pro Pro Gly
35 40 45Ala Gln Pro Pro Pro Gln Gly Gln Met Tyr Gly Ala Pro Asn Pro
Gln 50 55 60Val Met Ala Ser Ala Pro Met His His Pro Met Met Gln Ser
Thr Met65 70 75 80Met Gly Met Asn Ser Met Thr Pro Ile Met His Ala
Gly Asn Ile Gln 85 90 95Pro Ala Pro Gln Arg Met Met Pro Pro Pro Gln
Gln Pro Pro Tyr Met 100 105 110Leu Gln Gln Gln Met Met Gln Gln Gln
Gln Met Met Met His Ala Gly 115 120 125Gly Ala Met Pro Gln Pro Pro
Gly Gln Asn Met Gly Ser Gly Gly Gln 130 135 140Ser Pro Thr Phe Thr
Ser Gln Pro Ala Pro Pro Gly Ser Leu Asn Ser145 150 155 160Met Met
Glu Gly Gln Thr Pro Asn Ser Met Asn Ser Pro Ser Leu Ser 165 170
175Ala Gln Gly Met Met Asp Met Pro Gly Phe Gly Gly Pro Gln Ser Leu
180 185 190Asp Gly Val Gly Ser Ser Pro Pro Asn Met Gln Pro Glu Leu
Leu Arg 195 200 205Gly Leu Pro Arg Leu Val Pro Tyr Arg Gly Leu Met
Asn Ser Ala Ala 210 215 220Arg Pro Leu Leu Val Gly Arg Pro Gly Thr
Leu Gln Glu Leu Gln Met225 230 235 240Met Asn Asn Asn Leu Arg Ala
Pro Val Lys Ala Val Val Pro Asp Tyr 245 250 255Ser Phe Met Lys Gly
Arg Ser Asp Ser Cys Arg Gln Ile Ala Trp Leu 260 265 270Asn Asp Lys
Asn Ile Ile Val Pro Lys Thr Ala Met Ala Arg Phe Val 275 280 285Ile
Ala Leu Leu Arg Gln Ala Arg Ala Gly Tyr Lys Pro Ala Asp Ala 290 295
300Leu Ser His Ser Ala Ile Arg Asp Thr Ala Arg Arg Met Tyr Glu
Arg305 310 315 320Leu Ala Asp His Leu Glu His Asn Ser Glu Asp His
Gly Leu Ile Asp 325 330 335Gln Glu Pro Ala Phe Arg Arg Met Pro Ala
Gln Gln Thr Val Asn Ser 340 345 350Ala Gly Pro Pro Val Val Ser Ser
Cys Ala Glu Asp Trp Ala Phe Phe 355 360 365Thr Leu Gly Pro Phe Ser
Glu Ser Ala Phe Trp Gly Leu Ser Pro Asn 370 375 380Gln Pro Val Ser
Thr Glu Gly Ser Leu Arg Met Leu Phe Glu Ala Glu385 390 395 400Asp
Asp Thr Pro Pro Thr Ala Ala Ser Asn Asn His His Tyr Gly Ser 405 410
415Gly Met His His Pro Gln Glu Pro Gln Arg Glu Asp Gly Trp Ala Phe
420 425 430Thr Gln Arg Leu Leu Asp Arg Ala Ser Asn Glu Tyr Gly Gly
Pro Leu 435 440 445Gly Ile Lys Ser Val Arg Lys Leu Gly Leu Arg Glu
Glu Ala Ser Glu 450 455 460Val Val Ser Met Leu Asn Ala Val Phe Lys
Asn Arg Val Asn Arg Val465 470 475 480Leu Tyr Leu Ala Glu Gln Gln
Ala Arg Glu Arg Pro Leu Glu Trp Ser 485 490 495Thr Ile Glu Ser Cys
Leu Val Ser Asp Ile Val Arg Arg Asp Thr Phe 500
505 510Leu Tyr Lys Glu Asp Asp Leu Gly Phe Gly Pro Glu Glu Ser Ala
Ala 515 520 525Glu Lys Lys Gln Leu Ser Glu Tyr Thr 530
5359306DNADiabrotica virgifera 9gaaaatgtca aggcgaaaat ccaagacaaa
gagggtattc caccagatca gcaacgtcta 60atttttgctg gcaaacaatt ggaagatgga
cgtacattgt ccgattacaa catccaaaag 120gaatcgacac tccatttggt
gttacgtcta cgtggtggta tcatcgaacc atcattgcgt 180attttggcac
aaaagtacaa ttgcgacaaa atgatttgcc gtaagtgcta tgctcgtttg
240catccaagag caaccaattg ccgtaagaag aagtgtggcc acaccaacaa
tttgcgccca 300aagaag 30610361DNADiabrotica virgifera 10cagattttcg
tcaagacatt gacggggaag accattaccc tggaggtcga gcccagtgat 60tccatcgaga
ccgtcaaatc caagattcag gataaggaag ggattcctcc tgaccaacag
120cgcttgattt ttgctggcaa gcaactcgag gaaggccgat ctttgtcaga
ctataacatc 180caaaaagaga gtaccatcca tctcgtgctt cgactgagag
gaggtggtat cgagatcgag 240ccaacgttgg cgcaattggc tcgcaagcac
aatattgaga gactcatttg tcgaaaatgt 300tacgccagac tacacatcaa
agctactaat tgccggaaaa aggcctgcgg ccactcatct 360c
36111117DNADiabrotica virgifera 11ccaccagatc agcaacgtct aatttttgct
ggcaaacaat tggaagatgg acgtacattg 60tccgattaca acatccaaaa ggaatcgaca
ctccatttgg tgttacgtct acgtggt 11712143DNADiabrotica virgifera
12tcgaaccatc attgcgtatt ttggcacaaa agtacaattg cgacaaaatg atttgccgta
60agtgctatgc tcgtttgcat ccaagagcaa ccaattgccg taagaagaag tgtggccaca
120ccaacaattt gcgcccaaag aag 14313141DNADiabrotica virgifera
13caagagaggc aaaaaaccaa cggaaaatgc agatattcgt taaaacttta accggtaaga
60ccatcacact tgaggtcgag ccaagcgaca caatcgaaaa tgtcaaggcg aaaatccaag
120acaaagaggg tattccacca g 14114130DNADiabrotica virgifera
14gccgtaagtg ctatgctcgt ttgcatccaa gagcaaccaa ttgccgtaag aagaagtgtg
60gccacaccaa caatttgcgc ccaaagaaga agttgaagta gacgatcgta tttttgtgct
120gtatgtctgg 13015165DNADiabrotica virgifera 15cagcaacgtc
taatttttgc tggcaaacaa ttggaagatg gacgtacatt gtccgattac 60aacatccaaa
aggaatcgac actccatttg gtgttacgtc tacgtggtgg tatcatcgaa
120ccatcattgc gtattttggc acaaaagtac aattgcgaca aaatg
1651624DNAArtificial Sequencesynthesized promotor oligonucleotide
16ttaatacgac tcactatagg gaga 2417503DNAArtificial
Sequencesynthesized partial coding region 17caccatgggc tccagcggcg
ccctgctgtt ccacggcaag atcccctacg tggtggagat 60ggagggcaat gtggatggcc
acaccttcag catccgcggc aagggctacg gcgatgccag 120cgtgggcaag
gtggatgccc agttcatctg caccaccggc gatgtgcccg tgccctggag
180caccctggtg accaccctga cctacggcgc ccagtgcttc gccaagtacg
gccccgagct 240gaaggatttc tacaagagct gcatgcccga tggctacgtg
caggagcgca ccatcacctt 300cgagggcgat ggcaatttca agacccgcgc
cgaggtgacc ttcgagaatg gcagcgtgta 360caatcgcgtg aagctgaatg
gccagggctt caagaaggat ggccacgtgc tgggcaagaa 420tctggagttc
aatttcaccc cccactgcct gtacatctgg ggcgatcagg ccaatcacgg
480cctgaagagc gccttcaaga tct 5031849DNAArtificial
Sequencesynthesized primer oligonucleotide 18ttaatacgac tcactatagg
gagagaaaat gtcaaggcga aaatccaag 491946DNAArtificial
Sequencesynthesized primer oligonucleotide 19ttaatacgac tcactatagg
gagacttctt tgggcgcaaa ttgttg 462048DNAArtificial
Sequencesynthesized primer oligonucleotide 20ttaatacgac tcactatagg
gagacagatt ttcgtcaaga cattgacg 482150DNAArtificial
Sequencesynthesized primer oligonucleotide 21ttaatacgac tcactatagg
gagagagatg agtggccgca ggcctttttc 502252DNAArtificial
Sequencesynthesized primer oligonucleotide 22ttaatacgac tcactatagg
gagaccacca gatcagcaac gtctaatttt tg 522348DNAArtificial
Sequencesynthesized primer oligonucleotide 23ttaatacgac tcactatagg
gagaaccacg tagacgtaac accaaatg 482448DNAArtificial
Sequencesynthesized primer oligonucleotide 24ttaatacgac tcactatagg
gagatcgaac catcattgcg tattttgg 482549DNAArtificial
Sequencesynthesized primer oligonucleotide 25ttaatacgac tcactatagg
gagacttctt tgggcgcaaa ttgttggtg 492646DNAArtificial
Sequencesynthesized primer oligonucleotide 26ttaatacgac tcactatagg
gagacaagag aggcaaaaaa ccaacg 462746DNAArtificial
Sequencesynthesized primer oligonucleotide 27ttaatacgac tcactatagg
gagactggtg gaataccctc tttgtc 462847DNAArtificial
Sequencesynthesized primer oligonucleotide 28ttaatacgac tcactatagg
gagagccgta agtgctatgc tcgtttg 472947DNAArtificial
Sequencesynthesized primer oligonucleotide 29ttaatacgac tcactatagg
gagaccagac atacagcaca aaaatac 473046DNAArtificial
Sequencesynthesized primer oligonucleotide 30ttaatacgac tcactatagg
gagacagcaa cgtctaattt ttgctg 463148DNAArtificial
Sequencesynthesized primer oligonucleotide 31ttaatacgac tcactatagg
gagacatttt gtcgcaattg tacttttg 4832705DNAArtificial
Sequencesynthesized protein coding region 32atgtcatctg gagcacttct
ctttcatggg aagattcctt acgttgtgga gatggaaggg 60aatgttgatg gccacacctt
tagcatacgt gggaaaggct acggagatgc ctcagtggga 120aaggttgatg
cacagttcat ctgcacaact ggtgatgttc ctgtgccttg gagcacactt
180gtcaccactc tcacctatgg agcacagtgc tttgccaagt atggtccaga
gttgaaggac 240ttctacaagt cctgtatgcc agatggctat gtgcaagagc
gcacaatcac ctttgaagga 300gatggcaact tcaagactag ggctgaagtc
acctttgaga atgggtctgt ctacaatagg 360gtcaaactca atggtcaagg
cttcaagaaa gatggtcatg tgttgggaaa gaacttggag 420ttcaacttca
ctccccactg cctctacatc tggggtgacc aagccaacca cggtctcaag
480tcagccttca agatctgtca tgagattact ggcagcaaag gcgacttcat
agtggctgac 540cacacccaga tgaacactcc cattggtgga ggtccagttc
atgttccaga gtatcatcac 600atgtcttacc atgtgaaact ttccaaagat
gtgacagacc acagagacaa catgtccttg 660aaagaaactg tcagagctgt
tgactgtcgc aagacctacc tttga 70533218DNADiabrotica virgifera
33tagctctgat gacagagccc atcgagtttc aagccaaaca gttgcataaa gctatcagcg
60gattgggaac tgatgaaagt acaatmgtmg aaattttaag tgtmcacaac aacgatgaga
120ttataagaat ttcccaggcc tatgaaggat tgtaccaacg mtcattggaa
tctgatatca 180aaggagatac ctcaggaaca ttaaaaaaga attattag
21834424DNADiabrotica virgiferamisc_feature(393)..(393)n is a, c,
g, or tmisc_feature(394)..(394)n is a, c, g, or
tmisc_feature(395)..(395)n is a, c, g, or t 34ttgttacaag ctggagaact
tctctttgct ggaaccgaag agtcagtatt taatgctgta 60ttctgtcaaa gaaataaacc
acaattgaat ttgatattcg acaaatatga agaaattgtt 120gggcatccca
ttgaaaaagc cattgaaaac gagttttcag gaaatgctaa acaagccatg
180ttacacctta tccagagcgt aagagatcaa gttgcatatt tggtaaccag
gctgcatgat 240tcaatggcag gcgtcggtac tgacgataga actttaatca
gaattgttgt ttcgagatct 300gaaatcgatc tagaggaaat caaacaatgc
tatgaagaaa tctacagtaa aaccttggct 360gataggatag cggatgacac
atctggcgac tannnaaaag ccttattagc cgttgttggt 420taag
42435397DNADiabrotica virgifera 35agatgttggc tgcatctaga gaattacaca
agttcttcca tgattgcaag gatgtactga 60gcagaatagt ggaaaaacag gtatccatgt
ctgatgaatt gggaagggac gcaggagctg 120tcaatgccct tcaacgcaaa
caccagaact tcctccaaga cctacaaaca ctccaatcga 180acgtccaaca
aatccaagaa gaatcagcta aacttcaagc tagctatgcc ggtgatagag
240ctaaagaaat caccaacagg gagcaggaag tggtagcagc ctgggcagcc
ttgcagatcg 300cttgcgatca gagacacgga aaattgagcg atactggtga
tctattcaaa ttctttaact 360tggtacgaac gttgatgcag tggatggacg aatggac
39736490DNADiabrotica virgifera 36gcagatgaac accagcgaga aaccaagaga
tgttagtggt gttgaattgt tgatgaacaa 60ccatcagaca ctcaaggctg agatcgaagc
cagagaagac aactttacgg cttgtatttc 120tttaggaaag gaattgttga
gccgtaatca ctatgctagt gctgatatta aggataaatt 180ggtcgcgttg
acgaatcaaa ggaatgctgt actacagagg tgggaagaaa gatgggagaa
240cttgcaactc atcctcgagg tataccaatt cgccagagat gcggccgtcg
ccgaagcatg 300gttgatcgca caagaacctt acttgatgag ccaagaacta
ggacacacca ttgacgacgt 360tgaaaacttg ataaagaaac acgaagcgtt
cgaaaaatcg gcagcggcgc aagaagagag 420attcagtgct ttggagagac
tgacgacgtt cgaattgaga gaaataaaga ggaaacaaga 480agctgcccag
49037330DNADiabrotica virgifera 37agtgaaatgt tagcaaatat aacatccaag
tttcgtaatt gtacttgctc agttagaaaa 60tattctgtag tttcactatc ttcaaccgaa
aatagaataa atgtagaacc tcgcgaactt 120gcctttcctc caaaatatca
agaacctcga caagtttggt tggagagttt agatacgata 180gacgacaaaa
aattgggtat tcttgagctg catcctgatg tttttgctac taatccaaga
240atagatatta tacatcaaaa tgttagatgg caaagtttat atagatatgt
aagctatgct 300catacaaagt caagatttga agtgagaggt
33038320DNADiabrotica virgifera 38caaagtcaag atttgaagtg agaggtggag
gtcgaaaacc gtggccgcaa aagggattgg 60gacgtgctcg acatggttca attagaagtc
cactttggag aggtggagga gttgttcatg 120gaccaaaatc tccaacccct
catttttaca tgattccatt ctacacccgt ttgctgggtt 180tgactagcgc
actttcagta aaatttgccc aagatgactt gcacgttgtg gatagtctag
240atctgccaac tgacgaacaa agttatatag aagagctggt caaaagccgc
ttttgggggt 300ccttcttgtt ttatttgtag 3203947DNAArtificial
Sequencesynthesized primer oligonucleotide 39ttaatacgac tcactatagg
gagacaccat gggctccagc ggcgccc 474023DNAArtificial
Sequencesynthesized primer oligonucleotide 40agatcttgaa ggcgctcttc
agg 234123DNAArtificial Sequencesynthesized primer oligonucleotide
41caccatgggc tccagcggcg ccc 234247DNAArtificial Sequencesynthesized
primer oligonucleotide 42ttaatacgac tcactatagg gagaagatct
tgaaggcgct cttcagg 474346DNAArtificial Sequencesynthesized primer
oligonucleotide 43ttaatacgac tcactatagg gagagctcca acagtggttc
cttatc 464429DNAArtificial Sequencesynthesized primer
oligonucleotide 44ctaataattc ttttttaatg ttcctgagg
294522DNAArtificial Sequencesynthesized primer oligonucleotide
45gctccaacag tggttcctta tc 224653DNAArtificial Sequencesynthesized
primer oligonucleotide 46ttaatacgac tcactatagg gagactaata
attctttttt aatgttcctg agg 534748DNAArtificial Sequencesynthesized
primer oligonucleotide 47ttaatacgac tcactatagg gagattgtta
caagctggag aacttctc 484824DNAArtificial Sequencesynthesized primer
oligonucleotide 48cttaaccaac aacggctaat aagg 244924DNAArtificial
Sequencesynthesized primer oligonucleotide 49ttgttacaag ctggagaact
tctc 245048DNAArtificial Sequencesynthesized primer oligonucleotide
50ttaatacgac tcactatagg gagacttaac caacaacggc taataagg
485147DNAArtificial Sequencesynthesized primer oligonucleotide
51ttaatacgac tcactatagg gagaagatgt tggctgcatc tagagaa
475222DNAArtificial Sequencesynthesized primer oligonucleotide
52gtccattcgt ccatccactg ca 225323DNAArtificial Sequencesynthesized
primer oligonucleotide 53agatgttggc tgcatctaga gaa
235446DNAArtificial Sequencesynthesized primer oligonucleotide
54ttaatacgac tcactatagg gagagtccat tcgtccatcc actgca
465546DNAArtificial Sequencesynthesized primer oligonucleotide
55ttaatacgac tcactatagg gagagcagat gaacaccagc gagaaa
465622DNAArtificial Sequencesynthesized primer oligonucleotide
56ctgggcagct tcttgtttcc tc 225722DNAArtificial Sequencesynthesized
primer oligonucleotide 57gcagatgaac accagcgaga aa
225846DNAArtificial Sequencesynthesized primer oligonucleotide
58ttaatacgac tcactatagg gagactgggc agcttcttgt ttcctc
465951DNAArtificial Sequencesynthesized primer oligonucleotide
59ttaatacgac tcactatagg gagaagtgaa atgttagcaa atataacatc c
516026DNAArtificial Sequencesynthesized primer oligonucleotide
60acctctcact tcaaatcttg actttg 266127DNAArtificial
Sequencesynthesized primer oligonucleotide 61agtgaaatgt tagcaaatat
aacatcc 276250DNAArtificial Sequencesynthesized primer
oligonucleotide 62ttaatacgac tcactatagg gagaacctct cacttcaaat
cttgactttg 506350DNAArtificial Sequencesynthesized primer
oligonucleotide 63ttaatacgac tcactatagg gagacaaagt caagatttga
agtgagaggt 506425DNAArtificial Sequencesynthesized primer
oligonucleotide 64ctacaaataa aacaagaagg acccc 256526DNAArtificial
Sequencesynthesized primer oligonucleotide 65caaagtcaag atttgaagtg
agaggt 266649DNAArtificial Sequencesynthesized primer
oligonucleotide 66ttaatacgac tcactatagg gagactacaa ataaaacaag
aaggacccc 49671150DNAZea mays 67caacggggca gcactgcact gcactgcaac
tgcgaatttc cgtcagcttg gagcggtcca 60agcgccctgc gaagcaaact acgccgatgg
cttcggcggc ggcgtgggag ggtccgacgg 120ccgcggagct gaagacagcg
ggggcggagg tgattcccgg cggcgtgcga gtgaaggggt 180gggtcatcca
gtcccacaaa ggccctatcc tcaacgccgc ctctctgcaa cgctttgaag
240atgaacttca aacaacacat ttacctgaga tggtttttgg agagagtttc
ttgtcacttc 300aacatacaca aactggcatc aaatttcatt ttaatgcgct
tgatgcactc aaggcatgga 360agaaagaggc actgccacct gttgaggttc
ctgctgcagc aaaatggaag ttcagaagta 420agccttctga ccaggttata
cttgactacg actatacatt tacgacacca tattgtggga 480gtgatgctgt
ggttgtgaac tctggcactc cacaaacaag tttagatgga tgcggcactt
540tgtgttggga ggatactaat gatcggattg acattgttgc cctttcagca
aaagaaccca 600ttcttttcta cgacgaggtt atcttgtatg aagatgagtt
agctgacaat ggtatctcat 660ttcttactgt gcgagtgagg gtaatgccaa
ctggttggtt tctgcttttg cgtttttggc 720ttagagttga tggtgtactg
atgaggttga gagacactcg gttacattgc ctgtttggaa 780acggcgacgg
agccaagcca gtggtacttc gtgagtgctg ctggagggaa gcaacatttg
840ctactttgtc tgcgaaagga tatccttcgg actctgcagc gtacgcggac
ccgaacctta 900ttgcccataa gcttcctatt gtgacgcaga agacccaaaa
gctgaaaaat cctacctgac 960tgacacaaag gcgccctacc gcgtgtacat
catgactgtc ctgtcctatc gttgcctttt 1020gtgtttgcca catgttgtgg
atgtacgttt ctatgacgaa acaccatagt ccatttcgcc 1080tgggccgaac
agagatagct gattgtcatg tcacgtttga attagaccat tccttagccc
1140tttttccccc 11506822DNAArtificial Sequencesynthesized primer
oligonucleotidemisc_feature(22)..(22)n is a, c, g, or t
68tttttttttt tttttttttt vn 226920DNAArtificial Sequencesynthesized
primer oligonucleotide 69ttgtgatgtt ggtggcgtat 207024DNAArtificial
Sequencesynthesized primer oligonucleotide 70tgttaaataa aaccccaaag
atcg 247121DNAArtificial Sequencesynthesized primer oligonucleotide
71tgagggtaat gccaactggt t 217224DNAArtificial Sequencesynthesized
primer oligonucleotide 72gcaatgtaac cgagtgtctc tcaa
247332DNAArtificial Sequencesynthesized probe oligonucleotide
73tttttggctt agagttgatg gtgtactgat ga 3274151DNAEscherichia coli
74gaccgtaagg cttgatgaaa caacgcggcg agctttgatc aacgaccttt tggaaacttc
60ggcttcccct ggagagagcg agattctccg cgctgtagaa gtcaccattg ttgtgcacga
120cgacatcatt ccgtggcgtt atccagctaa g 1517569DNAArtificial
Sequencesynthesized partial coding region 75tgttcggttc cctctaccaa
gcacagaacc gtcgcttcag caacacctca gtcaaggtga 60tggatgttg
69764233DNAZea mays 76agcctggtgt ttccggagga gacagacatg atccctgccg
ttgctgatcc gacgacgctg 60gacggcgggg gcgcgcgcag gccgttgctc ccggagacgg
accctcgggg gcgtgctgcc 120gccggcgccg agcagaagcg gccgccggct
acgccgaccg ttctcaccgc cgtcgtctcc 180gccgtgctcc tgctcgtcct
cgtggcggtc acagtcctcg cgtcgcagca cgtcgacggg 240caggctgggg
gcgttcccgc gggcgaagat gccgtcgtcg tcgaggtggc cgcctcccgt
300ggcgtggctg agggcgtgtc ggagaagtcc acggccccgc tcctcggctc
cggcgcgctc 360caggacttct cctggaccaa cgcgatgctg gcgtggcagc
gcacggcgtt ccacttccag 420ccccccaaga actggatgaa cggttagttg
gacccgtcgc catcggtgac gacgcgcgga 480tcgttttttt cttttttcct
ctcgttctgg ctctaacttg gttccgcgtt tctgtcacgg 540acgcctcgtg
cacatggcga tacccgatcc gccggccgcg tatatctatc tacctcgacc
600ggcttctcca gatccgaacg gtaagttgtt ggctccgata cgatcgatca
catgtgagct 660cggcatgctg cttttctgcg cgtgcatgcg gctcctagca
ttccacgtcc acgggtcgtg 720acatcaatgc acgatataat cgtatcggta
cagagatatt gtcccatcag ctgctagctt 780tcgcgtattg atgtcgtgac
attttgcacg caggtccgct gtatcacaag ggctggtacc 840acctcttcta
ccagtggaac ccggactccg cggtatgggg caacatcacc tggggccacg
900ccgtctcgcg cgacctcctc cactggctgc acctaccgct ggccatggtg
cccgatcacc 960cgtacgacgc caacggcgtc tggtccgggt cggcgacgcg
cctgcccgac ggccggatcg 1020tcatgctcta cacgggctcc acggcggagt
cgtcggcgca ggtgcagaac ctcgcggagc 1080cggccgacgc gtccgacccg
ctgctgcggg agtgggtcaa gtcggacgcc aacccggtgc 1140tggtgccgcc
gccgggcatc gggccgacgg acttccgcga cccgacgacg gcgtgtcgga
1200cgccggccgg caacgacacg gcgtggcggg tcgccatcgg gtccaaggac
cgggaccacg 1260cggggctggc gctggtgtac cggacggagg acttcgtgcg
gtacgacccg gcgccggcgc 1320tgatgcacgc cgtgccgggc accggcatgt
gggagtgcgt ggacttctac ccggtggccg 1380cgggatcagg cgccgcggcg
ggcagcgggg
acgggctgga gacgtccgcg gcgccgggac 1440ccggggtgaa gcacgtgctc
aaggctagcc tcgacgacga caagcacgac tactacgcga 1500tcggcaccta
cgacccggcg acggacacct ggacccccga cagcgcggag gacgacgtcg
1560ggatcggcct ccggtacgac tatggcaagt actacgcgtc gaagaccttc
tacgaccccg 1620tccttcgccg gcgggtgctc tgggggtggg tcggcgagac
cgacagcgag cgcgcggaca 1680tcctcaaggg ctgggcatcc gtgcaggtac
gtctcagggt ttgaggctag catggcttca 1740atcttgctgg catcgaatca
ttaatgggca gatattataa cttgataatc tgggttggtt 1800gtgtgtggtg
gggatggtga cacacgcgcg gtaataatgt agctaagctg gttaaggatg
1860agtaatgggg ttgcgtataa acgacagctc tgctaccatt acttctgaca
cccgattgaa 1920ggagacaaca gtaggggtag ccggtagggt tcgtcgactt
gccttttctt ttttcctttg 1980ttttgttgtg gatcgtccaa cacaaggaaa
ataggatcat ccaacaaaca tggaagtaat 2040cccgtaaaac atttctcaag
gaaccatcta gctagacgag cgtggcatga tccatgcatg 2100cacaaacact
agataggtct ctgcagctgt gatgttcctt tacatatacc accgtccaaa
2160ctgaatccgg tctgaaaatt gttcaagcag agaggccccg atcctcacac
ctgtacacgt 2220ccctgtacgc gccgtcgtgg tctcccgtga tcctgccccg
tcccctccac gcggccacgc 2280ctgctgcagc gctctgtaca agcgtgcacc
acgtgagaat ttccgtctac tcgagcctag 2340tagttagacg ggaaaacgag
aggaagcgca cggtccaagc acaacacttt gcgcgggccc 2400gtgacttgtc
tccggttggc tgagggcgcg cgacagagat gtatggcgcc gcggcgtgtc
2460ttgtgtcttg tcttgcctat acaccgtagt cagagactgt gtcaaagccg
tccaacgaca 2520atgagctagg aaacgggttg gagagctggg ttcttgcctt
gcctcctgtg atgtctttgc 2580cttgcatagg gggcgcagta tgtagctttg
cgttttactt cacgccaaag gatactgctg 2640atcgtgaatt attattatta
tatatatatc gaatatcgat ttcgtcgctc tcgtggggtt 2700ttattttcca
gactcaaact tttcaaaagg cctgtgtttt agttcttttc ttccaattga
2760gtaggcaagg cgtgtgagtg tgaccaacgc atgcatggat atcgtggtag
actggtagag 2820ctgtcgttac cagcgcgatg cttgtatatg tttgcagtat
tttcaaatga atgtctcagc 2880tagcgtacag ttgaccaagt cgacgtggag
ggcgcacaac agacctctga cattattcac 2940ttttttttta ccatgccgtg
cacgtgcagt caatccccag gacggtcctc ctggacacga 3000agacgggcag
caacctgctc cagtggccgg tggtggaggt ggagaacctc cggatgagcg
3060gcaagagctt cgacggcgtc gcgctggacc gcggatccgt cgtgcccctc
gacgtcggca 3120aggcgacgca ggtgacgccg cacgcagcct gctgcagcga
acgaactcgc gcgttgccgg 3180cccgcggcca gctgacttag tttctctggc
tgatcgaccg tgtgcctgcg tgcgtgcagt 3240tggacatcga ggctgtgttc
gaggtggacg cgtcggacgc ggcgggcgtc acggaggccg 3300acgtgacgtt
caactgcagc accagcgcag gcgcggcggg ccggggcctg ctcggcccgt
3360tcggccttct cgtgctggcg gacgacgact tgtccgagca gaccgccgtg
tacttctacc 3420tgctcaaggg cacggacggc agcctccaaa ctttcttctg
ccaagacgag ctcaggtatg 3480tatgttatga cttatgacca tgcatgcatg
cgcatttctt agctaggctg tgaagcttct 3540tgttgagttg tttcacagat
gcttaccgtc tgctttgttt cgtatttcga ctaggcatcc 3600aaggcgaacg
atctggttaa gagagtatac gggagcttgg tccctgtgct agatggggag
3660aatctctcgg tcagaatact ggtaagtttt tacagcgcca gccatgcatg
tgttggccag 3720ccagctgctg gtactttgga cactcgttct tctcgcactg
ctcattattg cttctgatct 3780ggatgcacta caaattgaag gttgaccact
ccatcgtgga gagctttgct caaggcggga 3840ggacgtgcat cacgtcgcga
gtgtacccca cacgagccat ctacgactcc gcccgcgtct 3900tcctcttcaa
caacgccaca catgctcacg tcaaagcaaa atccgtcaag atctggcagc
3960tcaactccgc ctacatccgg ccatatccgg caacgacgac ttctctatga
ctaaattaag 4020tgacggacag ataggcgata ttgcatactt gcatcatgaa
ctcatttgta caacagtgat 4080tgtttaattt atttgctgcc ttccttatcc
ttcttgtgaa actatatggt acacacatgt 4140atcattaggt ctagtagtgt
tgttgcaaag acacttagac accagaggtt ccaggagtat 4200cagagataag
gtataagagg gagcagggag cag 42337720DNAArtificial Sequencesynthesized
primer oligonucleotide 77tgttcggttc cctctaccaa 207822DNAArtificial
Sequencesynthesized primer oligonucleotide 78caacatccat caccttgact
ga 227924DNAArtificial Sequencesynthesized probe oligonucleotide
79cacagaaccg tcgcttcagc aaca 248018DNAArtificial
Sequencesynthesized primer oligonucleotide 80tggcggacga cgacttgt
188119DNAArtificial Sequencesynthesized primer oligonucleotide
81aaagtttgga ggctgccgt 198226DNAArtificial Sequencesynthesized
probe oligonucleotide 82cgagcagacc gccgtgtact tctacc
268319DNAArtificial Sequencesynthesized primer oligonucleotide
83cttagctgga taacgccac 198419DNAArtificial Sequencesynthesized
primer oligonucleotide 84gaccgtaagg cttgatgaa 198521DNAArtificial
Sequencesynthesized probe oligonucleotide 85cgagattctc cgcgctgtag a
218625DNAArtificial Sequencesynthesized primer oligonucleotide
86gtatgtttct gcttctacct ttgat 258729DNAArtificial
Sequencesynthesized primer oligonucleotide 87ccatgttttg gtcatatatt
agaaaagtt 298834DNAArtificial Sequencesynthesized probe
oligonucleotide 88agtaatatag tatttcaagt atttttttca aaat
3489736DNAEuschistus heros 89gtaagtatag tgcgttaaga ttaattttat
ttggattaag ttcttctttc gtgttttaaa 60tttatagtaa atctaataga taaaaaaact
ctcgtattca gtaatgttta gttattgctc 120tgcttgatga aaccttaatg
aagtagttgg atcagtacaa tgcaaatctt cgtgaaaacc 180ctcacgggta
agaccattac cctagaggtt gaaccttcgg atacaatcga aaatgtaaag
240gctaaaattc aagacaaaga aggaattcct ccagatcagc agaggcttat
ttttgctggc 300aagcagttag aagatggtag aaccttatct gattataaca
ttcaaaaaga gtctaccctt 360cacttggttc ttcgtctgag gggtggtgtc
attgagccaa ctctgaagat ccttgcacag 420aaatacaatt gcgataaaat
gatttgccgc aagtgctatg ctagacttca tccaagagct 480accaattgcc
gcaagaagaa atgtggtcat actaacaaca tccgccccaa gaagaagctg
540aagtagatta tctgcttatt tttcaacctg gatttaaaaa aaaaaaaatt
caagacataa 600aaccatgtta aaataaattt ttaagaaata tatataaaaa
aagtaattgt tttttcttaa 660ttccttttca aatgctatga tttttgtttt
tatgtaatct aatttcaaac tgtacaaaaa 720atatatctgt accaag
73690128PRTEuschistus heros 90Met Gln Ile Phe Val Lys Thr Leu Thr
Gly Lys Thr Ile Thr Leu Glu1 5 10 15Val Glu Pro Ser Asp Thr Ile Glu
Asn Val Lys Ala Lys Ile Gln Asp 20 25 30Lys Glu Gly Ile Pro Pro Asp
Gln Gln Arg Leu Ile Phe Ala Gly Lys 35 40 45Gln Leu Glu Asp Gly Arg
Thr Leu Ser Asp Tyr Asn Ile Gln Lys Glu 50 55 60Ser Thr Leu His Leu
Val Leu Arg Leu Arg Gly Gly Val Ile Glu Pro65 70 75 80Thr Leu Lys
Ile Leu Ala Gln Lys Tyr Asn Cys Asp Lys Met Ile Cys 85 90 95Arg Lys
Cys Tyr Ala Arg Leu His Pro Arg Ala Thr Asn Cys Arg Lys 100 105
110Lys Lys Cys Gly His Thr Asn Asn Ile Arg Pro Lys Lys Lys Leu Lys
115 120 12591410DNAEuschistus heros 91gcaaatcttc gtgaaaaccc
tcacgggtaa gaccattacc ctagaggttg aaccttcgga 60tacaatcgaa aatgtaaagg
ctaaaattca agacaaagaa ggaattcctc cagatcagca 120gaggcttatt
tttgctggca agcagttaga agatggtaga accttatctg attataacat
180tcaaaaagag tctacccttc acttggttct tcgtctgagg ggtggtgtca
ttgagccaac 240tctgaagatc cttgcacaga aatacaattg cgataaaatg
atttgccgca agtgctatgc 300tagacttcat ccaagagcta ccaattgccg
caagaagaaa tgtggtcata ctaacaacat 360ccgccccaag aagaagctga
agtagattat ctgcttattt ttcaacctgg 41092132DNAEuschistus heros
92tgatttgccg caagtgctat gctagacttc atccaagagc taccaattgc cgcaagaaga
60aatgtggtca tactaacaac atccgcccca agaagaagct gaagtagatt atctgcttat
120ttttcaacct gg 1329348DNAArtificial Sequencesynthesized primer
oligonucleotide 93ttaatacgac tcactatagg gagagcaaat cttcgtgaaa
accctcac 489450DNAArtificial Sequencesynthesized primer
oligonucleotide 94ttaatacgac tcactatagg gagaccaggt tgaaaaataa
gcagataatc 5095301DNAArtificial Sequencesynthesized artificial
sequence 95catctggagc acttctcttt catgggaaga ttccttacgt tgtggagatg
gaagggaatg 60ttgatggcca cacctttagc atacgtggga aaggctacgg agatgcctca
gtgggaaagg 120ttgatgcaca gttcatctgc acaactggtg atgttcctgt
gccttggagc acacttgtca 180ccactctcac ctatggagca cagtgctttg
ccaagtatgg tccagagttg aaggacttct 240acaagtcctg tatgccagat
ggctatgtgc aagagcgcac aatcaccttt gaaggagatg 300g
3019647DNAArtificial Sequencesynthesized primer oligonucleotide
96ttaatacgac tcactatagg gagagcatct ggagcacttc tctttca
479746DNAArtificial Sequencesynthesized primer oligonucleotide
97ttaatacgac tcactatagg gagaccatct ccttcaaagg tgattg
4698574RNADiabrotica virgifera 98aaguugggua uaaaccacgu uucaagagag
gcaaaaaacc aacggaaaau gcagauauuc 60guuaaaacuu uaaccgguaa gaccaucaca
cuugaggucg agccaagcga cacaaucgaa 120aaugucaagg cgaaaaucca
agacaaagag gguauuccac cagaucagca acgucuaauu 180uuugcuggca
aacaauugga agauggacgu acauuguccg auuacaacau ccaaaaggaa
240ucgacacucc auuugguguu acgucuacgu ggugguauca ucgaaccauc
auugcguauu 300uuggcacaaa aguacaauug cgacaaaaug auuugccgua
agugcuaugc ucguuugcau 360ccaagagcaa ccaauugccg uaagaagaag
uguggccaca ccaacaauuu gcgcccaaag 420aagaaguuga aguagacgau
cguauuuuug ugcuguaugu cuggaggaaa acaauguucg 480gauucgucca
caguuguuau cauuuugaau aaucgaaauu augugaauca uucauugggu
540acagccugaa uaaacuucgu uuucugaaac cggc 57499652RNADiabrotica
virgifera 99auuaaauauc uguacuuuua aauuuaccac gcuuuuugua acggaaacgu
guucaucaac 60ugucacuuau caaauaaacu agacuucauu uuucacuugu cuuuuucauu
cggaaguuag 120cacguugugg aauaaacacu uccacaaugc aaauuuucgu
gaaaacucuc acugguaaaa 180ccaucacccu cgagguugag ccauccgaua
caaucgaaaa uguuaaagcu aagauccaag 240acaaagaagg uaucccccca
gaccagcaac guuugaucuu cgcuggaaaa caacucgaag 300auggacguac
cuugucagac uacaacaucc agaaggaauc cacccuucac uuggugcucc
360gauugagagg aggucucauc gagccuuccc uucguauuuu agccaugaag
uacaacugcg 420agaagaugau cugccguaaa uguuacgcca gguuacaccc
aagggccacc aacugcagaa 480agaagaagug uggacacacc aacaacuugc
gccccaagaa gaagcuaaag uagacuauua 540cgagguuauu uuuuacaaug
uuaauaucuu acaauuccag auguuacuug aacauuuauu 600uaacuaauua
uaggaaauga caucaauccu aaaaaaaaaa aaaaaaaaaa aa
6521004065RNADiabrotica virgifera 100cagauuuucg ucaagacauu
gacggggaag accauuaccc uggaggucga gcccagugau 60uccaucgaga ccgucaaauc
caagauucag gauaaggaag ggauuccucc ugaccaacag 120cgcuugauuu
uugcuggcaa gcaacucgag gaaggccgau cuuugucaga cuauaacauc
180caaaaagaga guaccaucca ucucgugcuu cgacugagag gaggugguau
cgagaucgag 240ccaacguugg cgcaauuggc ucgcaagcac aauauugaga
gacucauuug ucgaaaaugu 300uacgccagac uacacaucaa agcuacuaau
ugccggaaaa aggccugcgg ccacucaucu 360cagcucagac ccaagaagaa
gccuaagagu uaauaauuuu ugaacuuucu uuuuuccacg 420aacuagcuua
uguguacucg cucaguuguu ucuucucagc cgccgauucu ucuggcccga
480aacccaaguc guccucuuug uacagaaaug ugucucgccg cacuaugucg
cuaacuaagc 540augacucgau cgucgaccac uccagugggc gcucacgugc
uugcuguucg gcgagguaca 600gcacgcgguu gacgcgauuu uugaauacug
cauugagcau cgacaccacc ucgcucgccu 660ccucccggag uccgaguuuc
cugacggacu ugaugccgag cggaccaccg uauucauuug 720acgcucgguc
caguaggcgu ugggugaacg cccaaccguc uucucuuuga ggcucuugug
780gguggugcau cccacuaccg uaguggugau uguuagaugc ggccguuggc
ggugugucgu 840ccucagcuuc aaagagcauc cucagacuac ccucagucga
cacaggcugg uugggugaga 900gcccccaaaa ugcgcucuca gagaauggcc
caagcgugaa gaacgcccaa uccuccgcgc 960aacuggacac aacuggcggc
ccugccgaau uuacaguuug cugagccggc auucuccgaa 1020aagcugguuc
cugaucaauc aaaccaugau ccucugaauu guguuccaaa ugaucagcca
1080accuuucaua cauacgccga gccguaucuc guauugcuga augcgacaaa
gcguccgcgg 1140guuuauagcc ggcacgugcc ugccguagca acgcuaugac
gaaccucgcc aucgccgucu 1200ucggcacuau gauguucuug ucguuuagcc
aagcgaucug ccgacaugaa ucagaccgcc 1260ccuucaugaa cgaauaauca
gguaccacag ccuucaccgg agcgcgcagg uuauuguuca 1320ucauuuguag
cuccuguaga gucccaggcc guccaacgag caggggccga gcggcggagu
1380ucaucaagcc gcgauagggg accagucggg ggaggccgcg gagcaguucc
ggcugcauau 1440uagguggaga ugagccaacg ccaucgagac ucugcgggcc
gccgaauccc ggcaugucca 1500ucauuccuug ggcacugaga cuuggcgagu
ucaucgaauu cggcgucugg ccuuccauca 1560ugcuguucag agaucccgga
ggcgccggcu gacuggugaa ggucggacuc ugcccgccag 1620agcccauguu
uuggccaggc ggcugcggca uagcgccucc agcgugcauc aucauuugcu
1680guugcugcau cauuuguugc uggagcaugu acggcggcug uuguggaggu
ggcaucaugc 1740guugaggcgc cggcuggaua uugccagcgu gcauaaucgg
cgucaucgag uucauaccca 1800ucaucguacu cugcaucaua gggugaugca
uuggcgcuga agccaugacu ugaggguucg 1860gcgcgccgua cauuugaccc
uggggcggcg gcugggcucc gggagguggc ugccagcgcu 1920guugcuguug
cugagcuugu ugcuguggug ucgguacaua ucuauuaguu gggggccucc
1980auguauuaug caucgucaga guggccgugc gauugaguuu ucgagcauaa
aaagugcggc 2040cucagugauu gggaaugauc auuucggcag ucggagucuu
guauggccug aguaucagag 2100acguagucca agagaucuug cucgugcugc
gaggcagcaa gggaccguua aucguaauga 2160agggcugaug uuugccauug
uccgugcgug aagucaaguc uaggacugug ccgugacaag 2220cuauggccgu
aaacucaauu uccaguucgu agcuaacucc aacucguuca ugucggaagg
2280uagccucuag acuggcaggg auguagagcg gaacuaucgc aaccaggguc
ugcguaacag 2340ucguggaggc uucacacaca gccacggugu ccgucguuau
aacaaucagc guugaaucua 2400cauucgucaa cgcgugguag cuucugaaau
ccagccaacu caaaccgguc guggcguugg 2460gccgguaccc gaaauuuugu
cuaugcuugu uccacugaag cgccucgagc ggagacagug 2520gcggcugaua
agaccucgua guuuguuuca aucuaacaca cacuuccacc acggccaaag
2580acgaauuagu gaauucgguc uugacauaaa uuguacuacc uaggugcccc
ucaagagugu 2640augacucauc aacagugguu gguggcgauu uauugcccuc
guacacuuug gauugcggcg 2700ucgccaaaca aacgucugcu ggccauauau
ugacgacuug gauaucggac agaugggaac 2760cauugaucga aacucuguag
acggggguag uuugggggcg caccugugca uccgcuucaa 2820aagaugagac
aaaauugccu gcaguuccuu uuugacucuu agagcugcuc augcaccuga
2880ucgagccgac aaucuuauca aucucaaauc cauuuccgag ucuuucgaug
agauucaccu 2940cauggacagc gcgcugcuga auuuucugcc ucaauaacuu
caagcauucu ccaggcucuu 3000cuaagaauuc guucaaagcu gcagcagaua
cuccuguugc cgucauaugc uucgcuggcc 3060gaguuucgcu caaagugcua
ugcauaucag cacaggugua acugagccau cugagauaua 3120agaguccggc
aucaaccuuu ucuuuggcga cgagaaaauc auuuugugau ucuuguucau
3180ugugugacca gccagcuauc gggagcggaa ugggcagaga gccagaagug
aaagcggcuc 3240cgaaauccca auuuaaggcg agguaggugg gggugaaggg
ccgagcuggu agcgguggau 3300auauaggcgg agguuguaga augauagaag
cgagcugcgu gaacuucaua aagaugggga 3360uccauucacu uaagggguug
aucagaauag gacacgggcu aaugguuauu gccaagcuua 3420uuugcggcgg
gauuggcggc ccuggagcgc cguuuguugg aguagauauu ucggacucga
3480uacugucgag ggugcuggcg cgagcuggag ggaggugccu cacggaugag
ccgccgcucg 3540cgcuacgauc auucaaaccu accgacccac uauuguugug
ugaaaguaga guugcuccgc 3600ccguuggccc aauagucaca ggcgcgucau
uucugauagc uagaacguag ucauagcuga 3660aucguccucc ucgucggaug
gacgguggca agcuaacugg gauguagagc uggcauuugu 3720accuguccac
gucgccugug accucgagag uauugcuuag cgcgaacacu cgcccugauu
3780ucuuauuggu gguggcggag guggcuuguu guugaugucc gggggauggg
agcguaguag 3840uggaaacgga uuuuccaaac ucucugaggg cucuagugac
uaccucguaa ccauuuugaa 3900cuuugucagu agagaauaac gggggggcug
auggacagcg aucggugucc aauccguuga 3960ccagaccaua cacguauauu
cgaucaccgg cggugaagug ggugugacaa aaacugaugg 4020acuggccacc
ucuaggcauu cauuuaaucu ggcaaguguu ccgcu 4065101306RNADiabrotica
virgifera 101gaaaauguca aggcgaaaau ccaagacaaa gaggguauuc caccagauca
gcaacgucua 60auuuuugcug gcaaacaauu ggaagaugga cguacauugu ccgauuacaa
cauccaaaag 120gaaucgacac uccauuuggu guuacgucua cgugguggua
ucaucgaacc aucauugcgu 180auuuuggcac aaaaguacaa uugcgacaaa
augauuugcc guaagugcua ugcucguuug 240cauccaagag caaccaauug
ccguaagaag aaguguggcc acaccaacaa uuugcgccca 300aagaag
306102361RNADiabrotica virgifera 102cagauuuucg ucaagacauu
gacggggaag accauuaccc uggaggucga gcccagugau 60uccaucgaga ccgucaaauc
caagauucag gauaaggaag ggauuccucc ugaccaacag 120cgcuugauuu
uugcuggcaa gcaacucgag gaaggccgau cuuugucaga cuauaacauc
180caaaaagaga guaccaucca ucucgugcuu cgacugagag gaggugguau
cgagaucgag 240ccaacguugg cgcaauuggc ucgcaagcac aauauugaga
gacucauuug ucgaaaaugu 300uacgccagac uacacaucaa agcuacuaau
ugccggaaaa aggccugcgg ccacucaucu 360c 361103117RNADiabrotica
virgifera 103ccaccagauc agcaacgucu aauuuuugcu ggcaaacaau uggaagaugg
acguacauug 60uccgauuaca acauccaaaa ggaaucgaca cuccauuugg uguuacgucu
acguggu 117104143RNADiabrotica virgifera 104ucgaaccauc auugcguauu
uuggcacaaa aguacaauug cgacaaaaug auuugccgua 60agugcuaugc ucguuugcau
ccaagagcaa ccaauugccg uaagaagaag uguggccaca 120ccaacaauuu
gcgcccaaag aag 143105141RNADiabrotica virgifera 105caagagaggc
aaaaaaccaa cggaaaaugc agauauucgu uaaaacuuua accgguaaga 60ccaucacacu
ugaggucgag ccaagcgaca caaucgaaaa ugucaaggcg aaaauccaag
120acaaagaggg uauuccacca g 141106130RNADiabrotica virgifera
106gccguaagug cuaugcucgu uugcauccaa gagcaaccaa uugccguaag
aagaagugug 60gccacaccaa caauuugcgc ccaaagaaga aguugaagua gacgaucgua
uuuuugugcu 120guaugucugg 130107165RNADiabrotica virgifera
107cagcaacguc uaauuuuugc uggcaaacaa uuggaagaug gacguacauu
guccgauuac 60aacauccaaa aggaaucgac acuccauuug guguuacguc uacguggugg
uaucaucgaa 120ccaucauugc guauuuuggc acaaaaguac aauugcgaca aaaug
165108736RNAEuschistus heros 108guaaguauag ugcguuaaga uuaauuuuau
uuggauuaag uucuucuuuc guguuuuaaa 60uuuauaguaa aucuaauaga uaaaaaaacu
cucguauuca guaauguuua guuauugcuc 120ugcuugauga aaccuuaaug
aaguaguugg aucaguacaa ugcaaaucuu cgugaaaacc 180cucacgggua
agaccauuac ccuagagguu gaaccuucgg auacaaucga aaauguaaag
240gcuaaaauuc aagacaaaga aggaauuccu ccagaucagc agaggcuuau
uuuugcuggc 300aagcaguuag aagaugguag aaccuuaucu
gauuauaaca uucaaaaaga gucuacccuu 360cacuugguuc uucgucugag
gggugguguc auugagccaa cucugaagau ccuugcacag 420aaauacaauu
gcgauaaaau gauuugccgc aagugcuaug cuagacuuca uccaagagcu
480accaauugcc gcaagaagaa auguggucau acuaacaaca uccgccccaa
gaagaagcug 540aaguagauua ucugcuuauu uuucaaccug gauuuaaaaa
aaaaaaaauu caagacauaa 600aaccauguua aaauaaauuu uuaagaaaua
uauauaaaaa aaguaauugu uuuuucuuaa 660uuccuuuuca aaugcuauga
uuuuuguuuu uauguaaucu aauuucaaac uguacaaaaa 720auauaucugu accaag
736109410RNAEuschistus heros 109gcaaaucuuc gugaaaaccc ucacggguaa
gaccauuacc cuagagguug aaccuucgga 60uacaaucgaa aauguaaagg cuaaaauuca
agacaaagaa ggaauuccuc cagaucagca 120gaggcuuauu uuugcuggca
agcaguuaga agaugguaga accuuaucug auuauaacau 180ucaaaaagag
ucuacccuuc acuugguucu ucgucugagg ggugguguca uugagccaac
240ucugaagauc cuugcacaga aauacaauug cgauaaaaug auuugccgca
agugcuaugc 300uagacuucau ccaagagcua ccaauugccg caagaagaaa
uguggucaua cuaacaacau 360ccgccccaag aagaagcuga aguagauuau
cugcuuauuu uucaaccugg 410110132RNAEuschistus heros 110ugauuugccg
caagugcuau gcuagacuuc auccaagagc uaccaauugc cgcaagaaga 60aaugugguca
uacuaacaac auccgcccca agaagaagcu gaaguagauu aucugcuuau
120uuuucaaccu gg 132111153DNAArtificial Sequencesynthesized probe
oligonucleotide 111agtcatcacg ctggagcgca catataggcc ctccatcaga
aagtcattgt gtatatctct 60catagggaac gagctgcttg cgtatttccc ttccgtagtc
agagtcatca atcagctgca 120ccgtgtcgta aagcgggacg ttcgcaagct cgt
153112153DNAArtificial Sequencelinker polynucleotide 112agtcatcacg
ctggagcgca catataggcc ctccatcaga aagtcattgt gtatatctct 60catagggaac
gagctgcttg cgtatttccc ttccgtagtc agagtcatca atcagctgca
120ccgtgtcgta aagcgggacg ttcgcaagct cgt 153
* * * * *