# Patent

## Title
Lepidopteran moth control using double-stranded RNA constructs

## Abstract

Disclosed is a dsRNA construct that relates to a method to control Lepidopteran moths via double-stranded RNA interference of the PBAN/Pyrokinin gene.

## Background
CROSS-REFERENCE TO RELATED APPLICATION
This present application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Ser. No. 61/422,721, which was filed on Dec. 14, 2010, and is hereby incorporated by reference.


FIELD OF THE INVENTION
This invention relates to a method to control Helicoverpa zea via double-stranded RNA interference of the PBAN/Pyrokinin gene.
BACKGROUND OF INVENTION
Insect pests cost the general public billions of dollars annually in losses. These losses include the expense of controlling insect pests as well as crop loss and property damage caused by the pests. Helicoverpa zea has the capacity to inflict devastating yield losses to agronomically important crops. Depending the type of plant the larva feeds on, the pest is known commonly as the cotton bollworm when it feeds on cotton or corn earworm when it feeds on corn. Helicoverpa zea is typically controlled through the use of pyrethroid and Bacillus thuringiensis (Bt) insecticides.
Chemical pesticides are the primary tools used to combat these insect pests. However, use of traditional chemical pesticides has disadvantages, including non-target effects on neutral or beneficial insects, as well as other animals. Chemical pesticide usage also can lead to chemical residue run-off into streams and seepage into water supplies resulting in ecosystem/environment damage. In addition, animals higher in the food chain are at risk when they consume pesticide contaminated crops or insects. The handling and application of chemical pesticides also presents exposure danger to the public and professionals, and could lead to accidental dispersal into unintended environmentally sensitive areas. In addition, prolonged chemical pesticide application may result in an insect population becoming resistance to a chemical pesticide. In order to control a traditionally chemical resistant-pest, new more potent chemical pesticides must be utilized, which in turn will lead to another resistance cycle. As such, there is a need in the art to control pest populations without the disadvantages of traditional chemical pesticides.
An approach to decrease dependence on chemical pesticides is by causing a specific gene(s) of the target-pest to malfunction by either over expression or silencing gene expression. The silencing approach utilizes RNA interference pathways to knockdown the gene of interest via double-stranded RNA. Double-stranded RNA (dsRNA) induces sequence—specific post-transcriptional gene silencing in many organisms by a process known as RNA interference (RNAi). RNAi is a post-transcriptional, highly conserved process in eukaryotes that leads to specific gene silencing through degradation of the target mRNA. The silencing mechanism is mediated by dsRNA that is homologous in sequence to the gene of interest. The dsRNA is processed into small interfering RNA (siRNA) by an endogenous enzyme called DICER inside the target pest, and the siRNAs are then incorporated into a multi-component RNA-induced silencing complex (RISC), which finds and cleaves the target mRNA. The dsRNA inhibits expression of at least one gene within the target, which exerts a deleterious effect upon the target.
Fire, et al. (U.S. Pat. No. 6,506,559) discloses a process of introducing RNA into a living cell to inhibit gene expression of a target gene in that cell. The RNA has a region with double-stranded structure. Inhibition is sequence-specific in that the nucleotide sequences of the duplex region of the RNA and of a portion of the target gene are identical. Specifically, Fire, et al. (U.S. Pat. No. 6,506,559) discloses a method to inhibit expression of a target gene in a cell, the method comprising introduction of a double-stranded ribonucleic acid into the cell in an amount sufficient to inhibit expression of the target gene, wherein the RNA is a double-stranded molecule with a first ribonucleic acid strand consisting essentially of a ribonucleotide sequence which corresponds to a nucleotide sequence of the target gene and a second ribonucleic acid strand consisting essentially of a ribonucleotide sequence which is complementary to the nucleotide sequence of the target gene. Furthermore, the first and the second ribonucleotide strands are separately complementary strands that hybridize to each other to form the said double-stranded construct, and the double-stranded construct inhibits expression of the target gene.
In using dsRNA in controlling a target insect, one method is to engineer a baculovirus to produce a dsRNA construct in vivo as disclosed in Liu, et al. (U.S. Pat. No. 6,846,482). Salient to Liu is contacting an insect with a recombinant baculovirus wherein a first ribonucleic acid sequence corresponds to at least a portion of at least one gene endogenous to the insect to control the insect. Given the advances made in the field of transfection efficiency and RNA interference, there is a need in the art to utilize RNA interference technology without using a baculovirus as a vector. Such a method would mediate control of a target-pest without depending on variables associated with a baculovirus, such as expression and transfection of dsRNA by the baculovirus.
To utilize RNA interference as a method to regulate gene expression for control, a specific essential gene needs to be targeted. Genes associated with neurohormones represent novel potential targets. One neurohoromone gene family is the pheromone-biosynthesis-activating neuropeptide (PBAN)/pyrokinin gene family. The PBAN/pyrokinin gene produces multiple peptides, each of which are defined by a similar 5-amino-acid C-terminal sequence (FXPRLamide) that is the active core fragment for these peptides as reported in Raina, A. K. and T. G. Kempe (1992). “Structure activity studies of PBAN of Helicoverpa zea (Lepidoptera: Noctuidae).” Insect Biochem Mol Biol 22 (3): 221-225. It was subsequently determined that the five C-terminal amino acids, FXPRLamide, represented the minimal sequence required for activity as reported in Raina, A. K. and T. G. Kempe (1992) id.; Fonagy, A., L. Schoofs, et al. (1992). “Functional cross-reactivities of some locusta myotropins and Bombyx pheromone biosynthesis activating neuropeptide.” J Insect Physiol 38 (9): 651-657; Kuniyoshi, H., H. Nagasawa, et al. (1992). “Cross-activity between pheromone biosynthesis activating neuropeptide (PBAN) and myotropic pyrokinin insect peptides.” Biosci Biotechnol Biochem 56 (1): 167-8; and Raina, A. K. and T. G. Kempe (1990). “A pentapeptide of the C-terminal sequence of PBAN with pheromonotropic activity.” Insect Biochem 20 (8): 849-851.
To date, over 200 PBAN/pyrokinin family peptides including peptides deduced from 37 species PBAN/pyrokinin genes have been identified. While it is one of the largest neuropeptide families in insects, the physiological functions of the PBAN/Pyrokinin peptides are only partially known. Members of the PBAN/pyrokinin family have been shown to have a variety of functions in insects such as: 1) stimulate pheromone biosynthesis in female moths (Raina et al., 1989); 2) induce melanization in moth larvae (Matsumoto et al., 1990; Altstein et al., 1996); 3) induce embryonic diapause and seasonal polyphenism in moths (Suwan et al., 1994; Uehara et al., 2011); 4) stimulate visceral muscle contraction (Nachman et al., 1986; Predel and Nachman, 2001); 5) accelerate puparium formation in several flies (Zdarek et al., 1997; Verleyen et al., 2004); 6) terminate pupal diapause in heliothine moths (Sun et al., 2003; Xu and Denlinger, 2003).
As such there is a need in the art to investigative whether the PBAN/Pyrokinin pathway can be used to interfere with essential developmental and/or reproductive functions of the targeted insect pests and result in abnormal development and/or lack of reproduction.
Furthermore there is a need for novel control methods that would interfere with essential developmental and/or reproductive functions of species that do not have the undesirable characteristics of traditional chemical pesticides. To that end, there is a need to develop dsRNA constructs that are engineered to interfere with essential developmental and/or reproductive functions of specific pest insects that would overcome some of the disadvantages of using traditional pesticides.
BRIEF SUMMARY OF THE INVENTION
Disclosed herewith is a method for controlling Helicoverpa zea, the method comprising: constructing a double-stranded ribonucleic acid construct that is complementary to a gene that encodes an PBAN/Pyrokinin gene sequence, dissolving the double-stranded ribonucleic acid to form a solution, and contacting an effective amount of said solution to Helicoverpa zea, wherein RNA interference is induced and Helicoverpa zea mortality occurs.
In an embodiment of the invention, one strain of the double-stranded ribonucleic acid is complementary to the nucleotide sequence of SEQ. ID. NO. 2. In another embodiment of the invention, one strain of the double-stranded ribonucleic acid is complementary to the nucleotide sequence of SEQ. ID. NO. 3. In an embodiment of the invention, SEQ. ID. NO. 2 and SEQ. ID. NO. 3 form a complementary double-stranded ribonucleic acid construct.
In another embodiment of the invention the double-stranded ribonucleic acid construct is dissolved in water to form a solution. One aspect of the invention is the solution is applied to Helicoverpa zea bait material. In one embodiment, the bait material contains a phagostimulant. Another aspect of the invention is the double-stranded ribonucleic acid construct solution is applied topically to Helicoverpa zea. 
Another embodiment of the invention, disclosed is a double-stranded ribonucleic acid (dsRNA) for inhibiting the expression of a pheromone-biosynthesis-activating neuropeptide/pyrokynin peptide in a cell, wherein said dsRNA comprises a sense strand comprising a first sequence and an antisense strand comprising a second sequence complementary to SEQ ID NO: 2, wherein said first sequence is complementary to said second sequence. One aspect of the invention is the double-stranded ribonucleic acid is expressed in a plant cell.
In another embodiment of the invention, for inhibiting the expression of a pheromone-biosynthesis-activating neuropeptide/pyrokynin peptide in a cell, wherein said dsRNA comprises a sense strand comprising a first sequence and an antisense strand comprising a second sequence complementary to SEQ ID NO: 3, wherein said first sequence is complementary to said second sequence. One aspect of the invention is the double-stranded ribonucleic acid is expressed in a plant cell.



BRIEF DESCRIPTION OF THE DRAWINGS
The present invention together with the disclosed embodiments may best be understood from the following detailed description of the drawings, wherein:
FIG. 1 is a graph depicting pupal mortality of Helicoverpa zea after PBAN dsRNA injection of HezPBAN dsRNA (1 μg/3 μL), water (3 μL), or GFP (1 μg/3 μL) injected into 4-5-day old female pupae (N=13).
FIG. 2 is a photo depicting eclosion failure or problems for the adults leading to mortality from HezPBAN dsRNA injection. Data were analyzed by Kaplan-Meier survival curve comparison (PBAN vs. GFP & water, P=0.011 & 0.002, n≧13).
FIG. 3 is a graph depicting the effect of PBAN RNAi on sex pheromone production in H. zea female adults. HezPBAN dsRNA (1 μg/3 μL), water (3 μL) or GFP (1 μg/3 μL) was injected into 4-5-day old pupae. Sex pheromone was extracted 2-4 hr after light-off from 2-3-day old female adults. Bars with different letters are significantly different (unpaired t-test, two-tailed, PBAN vs. GFP & water, P=0.019 & 0.031, n≧5).



BRIEF DESCRIPTION OF THE SEQUENCES
SEQ. ID. NO. 1: ATGTTCAATCAAACTCAGTTGTTTGTTTTTCTCGCTGTATTCACTACGAGCAGTGTTT TAGGGAATAACAATGATGTTAAGGATGGCGCAGCGAGTGGAGCTCACAGCGACCGA CTAGGTCTTTGGTTCGGTCCCCGGCTAGGCAAGCGCTCGCTCAGAATATCTACCGAA GATAACAGACAAGCATTCTTCAAATTACTCGAAGCCGCCGACGCTTTGAAATACTAT TACGACCAGCTACCTTATGAGATGCAAGCTGATGAACCTGAAACCAGGGTCACCAA GAAGGTGATCTTCACCCCCAAGTTAGGCAGAAGCCTCGCATACGATGACAAAAGCT TTGAGAACGTGGAGTTCACGCCCAGACTCGGCAGGAGACTGTCCGATGATATGCCT GCCACCCCCGCTGACCAGGAAATGTACCGCCAAGACCCTGAACAGATTGACAGCAG GACAAAGTACTTCTCCCCAAGGCTCGGCAGAACCATGAACTTCTCACCACGACTCGG CAGGGAACTGTCTTATGATATGATGCCAAATAAAATCAGGGTAGTCAGGAGTACAA ACAAAACGCGATCAACATAA is the PBAN/pyrokinin gene cDNA of Helicoverpa zea. 
SEQ. ID. NO. 2: is a 5′ to 3′ construct forming one strand of the dsRNA product referred to as HezPBAN dsRNA construct.
SEQ. ID. NO. 2: CAAUGAUGUUAAGGAUGGCGCAGCGAGUGGAGCUCACAGCGACCGACUAGGUCU UUGGUUCGGUCCCCGGCUAGGCAAGCGCUCGCUCAGAAUAUCUACCGAAGAUAAC AGACAAGCAUUCUUCAAAUUACUCGAAGCCGCCGACGCUUUGAAAUACUAUUACG ACCAGCUACCUUAUGAGAUGCAAGCUGAUGAACCUGAAACCAGGGUCACCAAGAA GGUGAUCUUCACCCCCAAGUUAGGCAGAAGCCUCGCAUACGAUGACAAAAGCUUU GAGAACGUGGAGUUCACGCCCAGACUCGGCAGGAGACUGUCCGAUGAUAUGCCUG CCACCCCCGCUGACCAGGAAAUGUACCGCCAAGACCCUGAACAGAUUGACAGCAG GACAAAGUACUUCUCCCCAAGGCUCGGCAGAACCAUGAACUUCUCACCACGACUC GGCAGGGAACUGUCUUAUGAUAUGAUGCCAAAUAAAAUCAGGGUAGUCAGGAGU ACAAACAAAACGCGA is the dsRNA sequence structure of PBAN/pyrokinin gene of Helicoverpa zea. 
SEQ. ID. NO. 3: is a 3′ to 5′ construct forming one strand of the dsRNA product referred to as HezPBAN dsRNA construct.







SEQ. ID. NO. 3:


GUUACUACAAUUCCUACCGCGUCGCUCACCUCGAGUGUCGCUGGCUGAU


 


CCAGAAACCAAGCCAGGGGCCGAUCCGUUCGCGAGCGAGUCUUAUAGAU


 


GGCUUCUAUUGUCUGUUCGUAAGAAGUUUAAUGAGCUUCGGCGGCUGCG


 


AAACUUUAUGAUAAUGCUGGUCGAUGGAAUACUCUACGUUCGACUACUU


 


GGACUUUGGUCCCAGUGGUUCUUCCACUAGAAGUGGGGGUUCAAUCCGU


 


CUUCGGAGCGUAUGCUACUGUUUUCGAAACUCUUGCACCUCAAGUGCGG


 


GUCUGAGCCGUCCUCUGACAGGCUACUAUACGGACGGUGGGGGCGACUG


 


GUCCUUUACAUGGCGGUUCUGGGACUUGUCUAACUGUCGUCCUGUUUCA


 


UGAAGAGGGGUUCCGAGCCGUCUUGGUACUUGAAGAGUGGUGCUGAGCC


 


GUCCCUUGACAGAAUACUAUACUACGGUUUAUUUUAGUCCCAUCAGUCC


 


UCAUGUUUGUUUUGCGCU






DETAILED DESCRIPTION OF THE INVENTION
Disclosed here are specific insect pest dsRNA constructs that target PBAN/Pyrokinin gene expression. Using dsRNA inhibiting expression of the PBAN/Pyrokinin gene as a means of interfering with critical functions of the PBAN/Pyrokinin gene peptide products, a novel method to develop nucleic acid control for pest management is disclosed.
Definitions
As used in the specification and claims, the singular form “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a cell” includes a plurality of cells, including mixtures thereof.
The term “gene” refers to a DNA sequence involved in producing a polypeptide or precursor thereof. The polypeptide can be encoded by a full-length coding sequence or by any portion of the coding sequence, such as exon sequences. In one embodiment of the invention, the gene target is a PBAN/Pyrokinin gene of an insect.
The term “pheromone-biosynthesis-activating neuropeptide/pyrokynin peptide” for Helicoverpa zea, refer to four gene product peptides comprising:









(SEQ. ID. NO. 10)



NDVKDGAASGAHSDRLGLWFGPRL,



 



(SEQ. ID. NO. 11)



VIFTPKL,



 



(SEQ. ID. NO. 12)



SLAYDDKSFENVEFTPRL,



 



(SEQ. ID. NO. 13)



LSDDMPATPADQEMYRQDPEQIDSRTKYFSPRL,



and



 



(SEQ. ID. NO. 14)



TMNFSPRL.






The term “oligonucleotide” refers to a molecule comprising a plurality of deoxyribonucleotides or ribonucleotides. Oligonucleotide may be generated in any manner, including chemical synthesis, DNA replication, reverse transcription, polymerase chain reaction, or a combination thereof. The present invention embodies utilizing the oligonucleotide in the form of dsRNA as means of interfering with a critical developmental or reproductive process that leads to control. Inasmuch as mononucleotides are synthesized to construct oligonucleotides in a manner such that the 5′ phosphate of one mononucleotide pentose ring is attached to the 3′ oxygen of its neighbor in one direction via a phosphodiester linkage, an end of an oligonucleotide is referred to as the “5′ end” if its 5′ phosphate is not linked to the 3′ oxygen of a mononucleotide pentose ring and as the “3′ end” if its 3′ oxygen is not linked to a 5′ phosphate of a subsequent mononucleotide pentose ring. As used herein, a nucleic acid sequence, even if internal to a larger oligonucleotide, also may be said to have 5′ and 3′ ends.
When two different, non-overlapping oligonucleotides anneal to different regions of the same linear complementary nucleic acid sequence, and the 3′ end of one oligonucleotide points towards the 5′ end of the other, the former may be called the “upstream” oligonucleotide and the latter the “downstream” oligonucleotide.
The term “primer” refers to an oligonucleotide, which is capable of acting as a point of initiation of synthesis when placed under conditions in which primer extension is initiated. An oligonucleotide “primer” may occur naturally, as in a purified restriction digest or may be produced synthetically.
A primer is selected to be “substantially complementary” to a strand of specific sequence of the template. A primer must be sufficiently complementary to hybridize with a template strand for primer elongation to occur. A primer sequence need not reflect the exact sequence of the template. For example, a non-complementary nucleotide fragment may be attached to the 5′ end of the primer, with the remainder of the primer sequence being substantially complementary to the strand. Non-complementary bases or longer sequences can be interspersed into the primer, provided that the primer sequence is sufficiently complementary with the sequence of the template to hybridize and thereby form a template primer complex for synthesis of the extension product of the primer.
The term “double-stranded RNA” or “dsRNA” refers to two substantially complementary strands of ribonucleic acid. “Identity,” as used herein, is the relationship between two or more polynucleotide sequences, as determined by comparing the sequences. Identity also means the degree of sequence relatedness between polynucleotide sequences, as determined by the match between strings of such sequences. Identity can be readily calculated (see, e.g., Computation Molecular Biology, Lesk, A. M., eds., Oxford University Press, New York (1998), and Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York (1993), both of which are incorporated by reference herein). While there exist a number of methods to measure identity between two polynucleotide sequences, the term is well known to skilled artisans (see, e.g., Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press (1987); and Sequence Analysis Primer, Gribskov., M. and Devereux, J., eds., M Stockton Press, New York (1991)). Methods commonly employed to determine identity between sequences include, for example, those disclosed in Carillo, H., and Lipman, D., SIAM J. Applied Math. (1988) 48:1073. “Substantially identical” as used herein, means there is a very high degree of homology (preferably 100% sequence identity) between the inhibitory dsRNA and the corresponding part of the target gene. However, dsRNA having greater than 90% or 95% sequence identity may be used in the present invention, and thus sequence variations that might be expected due to genetic mutation, strain polymorphism, or evolutionary divergence can be tolerated. Although 100% identity is preferred, the dsRNA may contain single or multiple base pair random mismatches between the RNA and the target gene, provided that the mismatches occur at a distance of at least three nucleotides from the fusion site.
As used herein, “target gene” refers to a section of a DNA strand of a double-stranded DNA that is complementary to a section of a DNA strand, including all transcribed regions, that serves as a matrix for transcription. The target gene is therefore usually the sense strand.
The term “complementary RNA strand” refers to the strand of the dsRNA, which is complementary to an mRNA transcript that is formed during expression of the target gene, or its processing products. “dsRNA” refers to a ribonucleic acid molecule having a duplex structure comprising two complementary and anti-parallel nucleic acid strands. Not all nucleotides of a dsRNA must exhibit Watson-Crick base pairs. The maximum number of base pairs is the number of nucleotides in the shortest strand of the dsRNA.
As used herein, the term “GFP dsRNA” refers to a control dsRNA construct. The green fluorescent protein (GFP) is commonly used as a reporter gene and was originally isolated from jellyfish and widely used as control in prokaryotic and eukaryotic systems.
“Small interfering RNA” or “siRNA” refers to a short double-strand of ribonucleic acid, approximately 18 to 30 nucleotides in length. The term “RNA interference” or “RNAi” refers to a cellular mechanism for the destruction of targeted ribonucleic acid molecules. Under endogenous conditions, RNAi mechanism operates when dsRNA is cleaved to siRNA via an enzyme, DICER. The siRNA is processed to a single strand of anti-sense ribonucleic acid and coupled with a protein complex named RISC. The antisense RNA then targets a complementary gene construct, such as messenger RNA that is cleaved by ribonuclease. While the examples infra discloses constructing dsRNA constructs via enzymatic techniques with the enzyme RNA polymerase, it is contemplated that siRNA can be constructed via RNA oligonucleotide synthesis such as those disclosed in Scaringe, S., Methods Enzymol., 2000, Vol. 317:3 and incorporated herein by reference.
As used herein, “knock-down” is defined as the act of binding an oligonucleotide with a complementary nucleotide sequence of a gene as such that the expression of the gene or mRNA transcript decreases. In an embodiment, knock-down of a PBAN/pyrokinin gene occurs via injection of a dsRNA that can have multiple negative effects on the target insect, such as incomplete metamorphosis to the adult stage and untimely death of the target insect. Evidence of dsRNA having a negative effect on a targeted insect includes incomplete eclosion.
The term “substantially single-stranded” when used in reference to a nucleic acid product means that the product molecule exists primarily as a single strand of nucleic acid in contrast to a double-stranded product which exists as two strands of nucleic acids which are held together by inter-strand base pairing interactions.
“Oligonucleotide primers matching or complementary to a gene sequence” refers to oligonucleotide primers capable of facilitating the template-dependent synthesis of single or double-stranded nucleic acids. Oligonucleotide primers matching or complementary to a gene sequence may be used in PCRs, RT-PCRs and the like.
The term “corresponds to” as used herein means a polynucleotide sequence homologous to all or a portion of a reference polynucleotide sequence, or a polypeptide sequence that is identical to a reference polypeptide sequence. In contradistinction, the term “complementary to” is used herein to mean that the complementary sequence is homologous to all or a portion of a reference polynucleotide sequence. For example, the nucleotide sequence “TATAC” corresponds to a reference sequence “TATAC” and is complementary to a reference sequence “GTATA”.
An “effective amount” is an amount sufficient to effect desired beneficial or deleterious results. An effective amount can be administered in one or more administrations. In terms of treatment, an “effective amount” is that amount sufficient to make the target pest non-functional by causing an adverse effect on that pest, including (but not limited to) physiological damage to the pest; inhibition or modulation of pest growth; inhibition or modulation of pest reproduction; or death of the pest. In one embodiment of the invention, a dsRNA containing solution is fed to a target insect wherein critical developmental and/or reproductive functions of said insect are disrupted as a result of ingestion.
The term “solvent” includes any liquid that holds another substance in solution. Examples of solvents include but are not limited to water and organic solvents such as acetone, ethanol, dimethyl sulfoxide (DMSO), and dimethylformamide (DMF).
The term “phagostimulant” refers to any substance that will entice the insect to ingest the dsRNA. For insects, suitable phagostimulants include but are not limited to syrups, honey, aqueous solutions of sucrose, artificial sweeteners such as sucralose, saccharin, and other artificial sweeteners, amino acids, and other proteins.
Methods which are well known to those skilled in the art may be used to construct expression vectors containing sequences encoding PBAN/Pyrokinin gene and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Such techniques are described in Sambrook, J. et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y., and Ausubel, F. M. et al. (1989) Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y.
While the examples provided wherein describe dsRNA constructs cloned from GenBank Accession No. U08109 (Helicoverpa zea), it is contemplated that when read in conjunction with the teaching disclosed herein, the construction of other dsRNA constructs targeting PBAN/pyrokinin gene sequences of other insect orders would be feasible to those skilled the in the art. For example, including but not limited to the PBAN/pyrokinin gene/amino acid sequences disclosed in Table 1, it is contemplated that a dsRNA construct targeting the species disclosed in Table 1 of known PBAN/pyrokinin genes would control that respective insect. Additionally it is contemplated that a single dsRNA construct would be effective in controlling a plurality of insect species.









TABLE 1





Insect Order
Species
GenBank Accession #







Lepidoptera
Helicoverpa zea
U08109



Helicoverpa assulta
U96761/AY052417



Helicoverpa armigera
AY043222



Heliothis virescens
AY173075



Agrotis ipsilon
AJ009674



Mamestra brassicae
AF044079



Spodoptera littoralis
AF401480



Spodoptera exigua
AY628764



Pseudaletia separate
P25271 (amino acid




sequence only)



Clostera anastomosis
AY786305



Orgyia thyellina
AB259122



Lymantria dispar
AAB32665 (amino acid




sequence only)



Antheraea pernyi
AY445658



Samia cynthia ricini
AY286544



Manduca sexta
AY172672



Bombyx mori
NM_001043856



Bombyx mandarina
J. Sericultural Science




of Japan, 68: 373-379



Ascotis selenaria cretacea
AB308061



Adoxophyes. sp
AF395670



Plutella xylostella
AY904342


Diptera
Drosophila melanogaster
AY047528



Drosophila mauritiana
EF386943



Drosophila sechellia
EF386942



Drosophila simulans
EF386941



Aedes aegypti
XM_001650839/




XM_001662162



Culex quinquefasciatus
XM_001847366



Anopheles gambiae
XM_307885


Coleoptera
Tribolium castaneum
EFA11568


Hymenoptera
Apis melifera
NM_001110712



Nasonia vitripennis
NM_001167725



Solenopsis invicta
FJ223176



Solenopsis richteri
GQ872200



Solenopsis geminata
GQ872197



Solenopsis pergandei
GQ872198



Solenopsis carolinenesis
GQ872199



Harpegnathos saltator
GL445232



Camponotus floridanus
GL439118



Linepithema humile
Unpublished data


Hemiptera
Acyrthosiphon pisum
Gene ID: ACYPIG135047



(pea aphid)



Rhodnius prolixus
GU230851









Example 1
Constructing dsRNA Construct for Helicoverpa zea 
Cloning and Sequencing of Hez-PBAN/Pyrokinin Gene from Helicoverpa zea 
A mRNA was isolated from the dissected brain-subesophageal ganglion (Br-SGs) of the adult moths (Helicoverpa zea) by Micro Fast mRNA purification kit (Invitrogen), and used to synthesize cDNA with the GeneRacer cDNA synthesis kit (Invitrogen). Cloning the full length Hez-PBAN cDNA was carried out using Generacer kit (Invitrogen) as described by the manufacturer. The primers, 5′-AAGATGTTCAATCAAACTCAGTTG-3′ (SEQ. ID. NO. 4) and 5′-AAATTATGTTGATCGCGTTTTGTTTGT-3′ (SEQ. ID. NO. 5) were designed from the sequence registered on the GenBank (Accession number: U08109). PCR was performed with the following temperature program: 33 cycles at 95° C. for 30 s, 52° C. for 30 s, and 72° C. for 1 min. The PCR product was gel purified and cloned using TOPO TA cloning kit (Invitrogen) and sequenced. The obtained full-length sequence information was aligned and sequences compared with our partial sequence using DNA analysis software.
Construction of Hez-PBAN/Pyrokinin dsRNA Construct and dsGFP Control
To construct Hez-PBAN dsRNA PCR primer set was designed 5′-T7-appended: 5′-TAATACGACTCACTATAGGG GTGTTTGCATTGTGTACCGC-3′ (SEQ. ID. NO. 6), and 5′-TAATACGACTCACTATAGGGTATAGGAAG GGGTTGATGGC-3′ (SEQ. ID. NO. 7), to amplify 508-bp of Hez-PBAN DNA, which serves as the template for dsRNA synthesis using the MEGAscript RNA kit (Ambion). For a negative control, a green fluorescence protein (GFP) dsRNA was purchased from Ambion or was synthesized from a 546-bp GFP DNA template amplified by these primers 5′-TAATACGACTCACTATAGGGACGTAAA CGGCCACA AGTTC-3′ (SEQ. ID. NO. 8) and 5′-TAATACGACTCACTATAGGGTGCTCAGGTAGTGGTTGTCG-3′ (SEQ. ID. NO. 9) using the same kit as above. The length of Hez-PBAN dsRNA (SEQ. ID. NO. 1) was constructed from the full length of Hez-PBAN cDNA, 585-bp (SEQ. ID. NO. 2 and SEQ. ID. NO. 3).
Example 2
Hez-PBAN dsRNA Injected into Pupae and Adult Helicoverpa zea Bioassay
The dsRNA construct comprising the complementary strands of SEQ. ID. NO. 2 and SEQ. ID. NO. 3 was injected into 4 to 5 day old female pupae. Specifically 1 μg of Hez-PBAN dsRNA was injected into 13 female pupae and observed until adult emergence. For a control, 1 μg of GFP dsRNA as disclosed was injected into 13 female pupae. 3 μl of distilled water was also injected into 13 female pupae as an additional control. The pupae were then observed for mortality. As depicted in FIG. 1, it was observed that 53.8% of Helicoverpa zea pupae died as compared to 7.7% mortality and 0.5% mortality of dsRNA GFP and distilled water, respectively. This mortality was recorded as a failure of adult emergence as the pupae developed to adult tissue, but failed to escape from their cocoon as depicted in FIG. 2. Additionally, mortality was recorded as an event if the emerging Helicoverpa zea failed to expand their wings and appendages upon emergence.
Example 3
Inhibition of Pheromone Production via Hez-PBAN dsRNA in Helicoverpa zea 
Pupae Bioassay
The dsRNA construct comprising the complementary strands of SEQ. ID. NO. 2 and SEQ. ID. NO. 3 was injected into Helicoverpa zea pupae. Specifically, 1 μg/3 μL of the dsRNA construct, water (3 μL), or GFP (1 μg/3 μL) was injected into female pupae as detailed in Example 2. Sex pheromone from female adults was then extracted 4-hr after light-off in the 2nd scotophase after adult emergence, then analyzed by via gas chromatography. The Helicoverpa zea pheromone glands were dissected at room temperature and soaked in a hexane solvent containing 100 ng cis-9-tetradecenal (Z9-14:Ald) as an internal standard. A gas chromatography (GC) 6890N (Agilent Technologies) equipped with a 30 m×0.25 mm DB-23 capillary column (J&W) was used to quantify the amount of cis-11-hexadecenal (Z11-16:Ald), a major sex pheromone component for H. zea. The gas chromatography oven temperature was programmed at 80° C. for 1 min, then 10° C./min to 230° C. and held for 8 min. As detailed in FIG. 3, pheromone production was reduced approximately 53% when compared against the water and GFP dsRNA controls.
While the invention has been described with reference to details of the illustrated embodiment, these details are not intended to limit the scope of the invention as defined in the appended claims.

## Claims

1. An isolated double-stranded ribonucleic acid (dsRNA) for inhibiting the expression of a gene that encodes a pheromone-biosynthesis-activating neuropeptide/pyrokynin (PBAN) peptide in a cell consisting of sense strand SEQ ID NO: 2 with the sense strand being complementary to an antisense strand, wherein the dsRNA is capable of inducing RNA interference in Helicoverpa zea.

2. The dsRNA of claim 1, wherein the double-stranded ribonucleic acid is expressed in a transgenic plant cell.

3. A dsRNA for inhibiting the expression of a gene that encodes a pheromone-biosynthesis-activating neuropeptide/pyrokynin peptide in a cell consisting of sense strand SEQ ID NO: 3 with the sense strand being complementary to an antisense strand, wherein the dsRNA is capable of inducing RNA interference in Helicoverpa zea.

4. The double-stranded ribonucleic acid dsRNA of claim 3, wherein the double-stranded ribonucleic acid is expressed in a transgenic plant cell.

5. A method for controlling Helicoverpa zea, the method comprising: constructing a dsRNA that is complementary to a gene that encodes a PBAN/pyrokinin gene sequence wherein the dsRNA consists of SEQ ID NO: 2 or 3, with the sense strand being complementary to an antisense strand, dissolving the dsRNA to form a solution, and contacting at least one μg of dsRNA in said solution to Helicoverpa zea, wherein RNA interference is induced and Helicoverpa zea mortality occurs.

6. The method of claim 5, wherein the dsRNA construct is dissolved in water.

7. The method of claim 5, wherein the solution is incorporated in Helicoverpa zea bait material.

8. The method of claim 7, wherein the bait material contains a phagostimulant.

9. The method of claim 5, wherein the solution is applied topically to Helicoverpa zea.

10. The isolated dsRNA of claim 1, wherein the dsRNA is in solution.

11. The isolated dsRNA of claim 3, wherein the dsRNA is in solution.