CN101370940A

CN101370940A - DsRNA as insect control agent

Info

Publication number: CN101370940A
Application number: CNA200780002294XA
Authority: CN
Inventors: 罗曼·雷马克斯; 劳伦特·库布勒; 格特·普莱廷克; 埃尔丝·万布勒
Original assignee: Devgen NV
Current assignee: Devgen NV
Priority date: 2006-01-12
Filing date: 2007-01-12
Publication date: 2009-02-18

Abstract

The present invention concerns methods for controlling insect infestation via RNAi- mediated gene silencing, whereby the intact insect cell(s) are contacted with a double-stranded RNA from outside the insect cell(s) and whereby the double-stranded RNA is taken up by the intact insect cell(s). In one particular embodiment, the methods of the invention are used to alleviate plants from insect pests. Alternatively, the methods are used for treating and/or preventing insect infestation on a substrate or a subject in need of such treatment and/or prevention. Suitable insect target genes and fragments thereof, dsRNA constructs, recombinant constructs and compositions are disclosed.

Description

DsRNA as insect control agent

Technical field

The present invention relates to field at insect species double center chain RNA (dsRNA) mediated gene silencing.More specifically, the present invention relates to be designed for the genetic constructs of expression corresponding to the dsRNA of new target gene.These constructs are particularly useful in the pest control of RNAi mediation.The invention still further relates to the method that is used for preventing and treating insect, be used to the method that prevents the method for insect pest infestation and use the genetic expression of RNAi downward modulation insect.

Background technology

Insect and other insect can bite or terebra causes damage even dead by it.And many insects are propagated bacterium and other pathogenic agent that causes disease.For example, mosquitoes spread causes the pathogenic agent of malaria, yellow jack, encephalitis and other diseases.Glandular plague (or Bubonic plague) is caused by infected rats and other rodentine bacteriums.The composition that is used to prevent and treat the infringement of microcosmic insect provides with microbiotic, form antiviral and antimycotic composition.The method that is used for pest control (as nematode and insect) infringement adopts the form of chemical composition usually, described chemical composition is applied on the surface of insect stop, perhaps the form with pill, pulvis, tablet, paste or capsule is applied to by the invasion animal.

The insect pest control of important farm crop is very important field, for example destroy the insect pest of plant of Solanaceae, especially the control of coleopteran pest, described plant of Solanaceae refers to potato (Solanumtuberosum) especially, and tomato (Solanum lycopersicum), eggplant (Solanummelongena), capsicum (Solanum capsicum) and nightshade (for example, Solanumaculeastrum, S.bulbocastanum (a kind of wild potato), S.cardiophyllum, S.douglasii, fruit of Biiter Nightshade (S.dulcamara), S.lanceolatum, S.robustum and S.triquetrum).

In the past few decades, be used for preventing and treating the more effective ways of plant insect pest infestation and the exploitation of composition has obtained remarkable break-throughs.Chemical insecticide is very effective in eradicating the insect infringement.

Using seal chinaberry seed extract to carry out biological control has demonstrated the plant coleopteran pest has been had effect.Based on the seal chinaberry commercially available sterilant with nimbin as main activeconstituents.These sterilants are applicable to the insect of wide spectrum.They are as insect growth regulator(IGR); Nimbin stops insect molting by the generation that suppresses a kind of insect hormone (ecdysone).

Use is carried out the alternative that biological control is chemical prevention from the Cry3A albumen and the deutero-insecticidal proteins of bacillus thuringiensis mealworm mutation (Bacillus thuringiensis varietiestenebrionis) and San Diego mutation (varieties san diego).Described Bt (Bacillusthuringiensis) toxin protein is effectively for control colorado potato beetles larva, and this toxin protein or conduct are expressed in preparation that sprays on the plant or the leaf potato.

A kind of alternative biotechnological formulation is dsRNA.In the past few years, become proven technique by the gene (being also referred to as " gene silencing ") that RNA disturbs or " RNAi " reduces in the multicellular organism.

RNA disturbs or " RNAi " is the method (being also referred to as " gene silencing " or " gene silencing of RNA mediation ") of sequence-specific down-regulation of gene expression, it is by sequence and the initial (Fire of double-stranded RNA (dsRNA) that waits to reduce the target gene regional complementarity, A.Trends Genet.Vol.15,358-363,1999; Sharp, P.A.Genes Dev.Vol.15,485-490,2001).

In the past few years, disturb the target gene of (RNAi) downward modulation multicellular organism to become proven technique by RNA.Can be with reference to International Application No. WO 99/32619 (Ka Neiji institute) and WO00/01846 (the applicant).

The dsRNA gene silencing is applied in many different fields, for example the gene silencing of dsRNA mediation in clinical application (WO2004/001013) and plant.In plant, the dsRNA construct that also will be used for gene silencing is designed to be cut and to be processed into siRNA (siRNA).

Although the RNAi technology several years in plant, Caenorhabditis elegans (C.elegans) and the mammalian cell are generally understood in this area, yet genetic expression almost is unknown so far in the RNAi downward modulation insect for using.Since the submission of WO 00/01846 and WO 99/32619 application and announcing, only had to relate to other application of using the RNAi protective plant to avoid insect pest infestation on a small quantity and come forth.These comprise International Application No. WO 01/37654 (DNA Plant Technologies), WO2005/019408 (Bar Ilan university), WO 2005/049841 (CSIRO, BayerCropscience), WO 05/047300 (Univ. Of Utah Research Foundation) and U. S. application 2003/00150017 (Mesa etc.).The invention provides the target gene and the construct of the insect pest control that is used for the RNAi mediation.Correspondingly, the invention provides by suppressing, postpone or reducing in the specific insect target gene expression and come the method and composition of pest control infringement.

Summary of the invention

The invention describes be used to prevent and treat the crop insect pest not based on compound, not based on proteinic novel method.Its activeconstituents is nucleic acid (double-stranded RNA (dsRNA)), and described nucleic acid can be used as the pesticide preparation sprays of leaf (for example, as).The sequence of described dsRNA is part or all of corresponding to the insect indispensable gene, and disturbs (RNAi) to cause the downward modulation of insect target by RNA.Because the downward modulation of mRNA, described dsRNA stops the expression of insect target protein, and therefore causes insect death, cessation of growth cessation or sterile.

But method practical application of the present invention suppresses existence, growth, growth or the breeding of insect or reduces pathogenic or infective any technical field of insect at needs.Method of the present invention need also to be applied to the situation of one or more expression of target gene in the specificity downward modulation insect.Useful especially practical application includes but not limited to: (1) protective plant is avoided the insect pest infringement; (2) medicine in humans and animals or veterinary purpose (insect infection in for example, control, treatment or the prevention humans and animals); (3) the protection article exempt from insect destruction; (4) the perishable article (as food, seed etc.) of protection exempt from insect destruction; And need the control insect usually and/or need prevent that insect from causing any application of destructive.

According to an embodiment, the present invention relates to control cell or the biological method that goes up insect growth, perhaps prevent cell or the biological method of insect pest infestation to the insect infection sensitivity, it comprises insect is contacted with double-stranded RNA, wherein said double-stranded RNA comprises the annealed complementary strand, wherein annealing complementary strand comprises at least a portion complementary nucleotide sequence with insect target gene nucleotide sequence, and this double-stranded RNA is by insect picked-up and therefore control growing or prevent infringement.

Therefore, the invention provides isolating new insect target gene nucleotide sequence, described isolating nucleotide sequence comprises and is selected from following at least a nucleotide sequence:

(i) arbitrary sequence of following expression:

SEQ ID NO

1,3,5,7,9,11,13,15,17,19,21,23,49 to 158,159,160-163,168,173,178,183,188,193,198,203,208,215,220,225,230,240 to 247,249,251,253,255,257,259,275 to 472,473,478,483,488,493,498,503,508 to 513,515,517,519,521,533 to 575,576,581,586,591,596,601,603,605,607,609,621 to 767,768,773,778,783,788,793,795,797,799,801,813 to 862,863,868,873,878,883,888,890,892,894,896,908 to 1040,1041,1046,1051,1056,1061,1066 to 1071,1073,1075,1077,1079,1081,1083,1085,1087,1089,1091,1093,1095,1097,1099,1101,1103,1105,1107,1109,1111,1113,1161 to 1571,1572,1577,1582,1587,1592,1597,1602,1607,1612,1617,1622,1627,1632,1637,1642,1647,1652,1657,1662,1667,1672,1677,1682,1684,1686,1688,1690,1692,1694,1696,1698,1700,1702,1704,1730 to 2039,2040,2045,2050,2055,2060,2065,2070,2075,2080,2085,2090,2095,2100,2102,2104,2106,2108,2120 to 2338,2339,2344,2349,2354,2359,2364,2366,2368,2370,2372,2384 or 2460,2461,2466,2471,2476,2481 or 2486, or its complementary sequence

(ii) the arbitrary sequence with following expression has at least 70%, and preferably at least 75%, 80%, 85%, 90%, more preferably at least 95%, 96%, 97%, 98% or 99% conforming sequence:

SEQ ID NO

1,3,5,7,9,11,13,15,17,19,21,23,49 to 158,159,160-163,168,173,178,183,188,193,198,203,208,215,220,225,230,240 to 247,249,251,253,255,257,259,275 to 472,473,478,483,488,493,498,503,508 to 513,515,517,519,521,533 to 575,576,581,586,591,596,601,603,605,607,609,621 to 767,768,773,778,783,788,793,795,797,799,801,813 to 862,863,868,873,878,883,888,890,892,894,896,908 to 1040,1041,1046,1051,1056,1061,1066 to 1071,1073,1075,1077,1079,1081,1083,1085,1087,1089,1091,1093,1095,1097,1099,1101,1103,1105,1107,1109,1111,1113,1161 to 1571,1572,1577,1582,1587,1592,1597,1602,1607,1612,1617,1622,1627,1632,1637,1642,1647,1652,1657,1662,1667,1672,1677,1682,1684,1686,1688,1690,1692,1694,1696,1698,1700,1702,1704,1730 to 2039,2040,2045,2050,2055,2060,2065,2070,2075,2080,2085,2090,2095,2100,2102,2104,2106,2108,2120 to 2338,2339,2344,2349,2354,2359,2364,2366,2368,2370,2372,2384 to 2460,2461,2466,2471,2476,2481 or 2486, or its complementary sequence, and

The sequence of at least 17 continuous nucleotides that (iii) comprises the arbitrary sequence of following expression:

SEQ ID NO

1,3,5,7,9,11,13,15,17,19,21,23,49 to 158,159,160-163,168,173,178,183,188,193,198,203,208,215,220,225,230,240 to 247,249,251,253,255,257,259,275 to 472,473,478,483,488,493,498,503,508 to 513,515,517,519,521,533 to 575,576,581,586,591,596,601,603,605,607,609,621 to 767,768,773,778,783,788,793,795,797,799,801,813 to 862,863,868,873,878,883,888,890,892,894,896,908 to 1040,1041,1046,1051,1056,1061,1066 to 1071,1073,1075,1077,1079,1081,1083,1085,1087,1089,1091,1093,1095,1097,1099,1101,1103,1105,1107,1109,1111,1113,1161 to 1571,1572,1577,1582,1587,1592,1597,1602,1607,1612,1617,1622,1627,1632,1637,1642,1647,1652,1657,1662,1667,1672,1677,1682,1684,1686,1688,1690,1692,1694,1696,1698,1700,1702,1704,1730 to 2039,2040,2045,2050,2055,2060,2065,2070,2075,2080,2085,2090,2095,2100,2102,2104,2106,2108,2120 to 2338,2339,2344,2349,2354,2359,2364,2366,2368,2370,2372,2384 to 2460,2461,2466,2471,2476,2481 or 2486, or its complementary sequence, perhaps wherein said nucleotide sequence is the lineal homologue of gene of at least 17 continuous nucleotides that comprises the arbitrary sequence of following expression: SEQ ID NO 49 to 158,275 to 472,533 to 575,621 to 767,813 to 862,908 to 1040,1161 to 1571,1730 to 2039,2120 to 2338,2384 to 2460, or its complementary sequence, described nucleotide sequence can be used for preparing the double-stranded RNA that is used to control insect growth of the present invention.

" pest control " that the present invention uses means and kills off the insect pests, or stops insect to grow or growth, or prevents that insect from infecting or infringement.Pest control used herein also comprises offspring's (growth of ovum) of Pest Control.Pest control used herein also comprises existence, growth, growth or the breeding that suppresses insect, or reduces the pathogenic or infectious of insect.It is biological healthy that compound as herein described and/or composition can be used for keeping, and can therapeutic, preventative or general ground makes and is used for pest control or prevention insect growth or growth or infection or infringement.

The specific insect that the present invention relates to is an insect pest.Therefore, " control insect " used herein also comprises the offspring's (for example growth of the ovum of insect pest) who controls insect.Control insect used herein also comprises existence, growth, growth or the breeding that suppresses insect, perhaps reduces the pathogenic or infectious of insect.In the present invention, the control insect can suppress the biological activity in the insect, it causes one or more following features: the insect feed reduces, the insect viability reduces, insect death, suppress differentiation and the growth of insect, following disappearance or ability drop: insect sexual propagation, muscle forms, neotonin forms, the neotonin function is regulated, ion is regulated and transhipment, keeping of cytolemma electromotive force, amino acid whose biosynthesizing, amino acid degradation, sperm forms, pheromone is synthetic, the perception of pheromone, feeler forms, the formation of wing, the formation of foot, grow and differentiation, the formation of ovum, the maturation of larva, the formation of digestive ferment, synthesizing of hemolymph, keeping of hemolymph, neurotransmission, cell fission, energy metabolism, breathe, any component of apoptosis and eukaryotic cytoskeletal structure (as Actin muscle and tubulin).It is biological healthy that compound as herein described and/or composition can be used for keeping, and can therapeutic, preventative or general ground makes and is used for preventing and treating insect, or stop the insect growth or grow or infect or encroach on.Therefore, the present invention can make previous responsive biology that insect pest infestation is produced resistance.

Statement used herein " with ... at least a portion complementation " nucleotides sequence that is meant described nucleotide sequence and target is listed in and surpasses on the length of two Nucleotide, for example surpass at least 15,16,17,18,19,20,21,22,23,24 or the length of more a plurality of continuous nucleotides on complementary fully.

According to another embodiment, the present invention relates to be used for reducing the method for insect expression of target gene, it comprises described insect is contacted with double-stranded RNA, wherein said double-stranded RNA comprises the annealed complementary strand, wherein an annealing complementary strand comprises and at least a portion complementary nucleotide sequence of waiting to reduce insect target gene nucleotide sequence, and wherein said double-stranded RNA is by the insect picked-up and therefore reduce the insect target gene expression.

When countless measure word were modified, " target gene " was meant " at least a " target gene.Similarly, the target biology is meant " at least a " target biology, and RNA molecule or host cell are meant " at least a " RNA molecule or host cell.This also has detailed description in the back.

According to an embodiment, method of the present invention depends on insect and absorbs its outside double-stranded RNA (for example, by feed), and need be at the expressed in insect cells double-stranded RNA.In addition, the present invention also comprises aforesaid method, and wherein said insect contacts with the composition that comprises this double-stranded RNA.

Can express described double-stranded RNA by (such as but not limited to the bacterium) of protokaryon or (such as but not limited to the yeast) host cell or the host living beings of eucaryon.

Described insect can be any insect, means any biology that belongs to animal kingdom, particularly Arthropoda, especially Insecta or Arachnida.Method of the present invention is applicable to all insects to the gene silencing sensitivity of utilizing RNA to disturb to carry out, and can be from its adjacent ambient all insects with the double-stranded RNA internalization.The present invention also is applicable to the insect that is in its any etap.Because insect has abiotic exoskeleton, so they can not be grown but slough off its exoskeleton by periodicity stage by stage with the speed growth of homogeneous.This process is called " casting off a skin ".Stage between casting off a skin is called " age ", and these stages can be used as target of the present invention.Simultaneously, ovum of insect or larva alive also can be used as target of the present invention.All stages in growth cycle (comprising the metamorphosis in the pterygote) all can be used as target of the present invention.Therefore, the single stage (as etap such as larva, pupa, nymphs) all can be used as target.

In one embodiment of the invention, described insect can belong to following order: acarina, Araneida, Anoplura, Coleoptera, Collembola, Dermaptera, Dictyoptera, Diplura, Diptera, Embioptera, Ephemeroptera, Grylloblattodea, Hemiptera, Homoptera, Hymenoptera, Isoptera, lepidopteran, Mallophaga, Mecoptera, Neuroptera, Odonata, Orthoptera, Phasmida,

Wing order, Protura, Corrodentia, Siphonaptera, Anoplura, Thysanura, Strepsiptera, Thysanoptera, Trichoptera and Zoraptera.

In preferred non-limiting embodiments of the present invention and method, described insect is selected from:

(1) Plant insect pests, such as, but not limited to, BPH species (Nilaparvata spp.)) (For example, the brown planthopper (N.lugens)), planthopper species (Laodelphax spp.) (Case For example, brown planthopper (L.striatellus)), leafhopper species (Nephotettix spp.) (Case For example, two points leafhopper (N.virescens), or leafhopper (N.cincticeps), or two Leafhopper (N.nigropictus)), white-backed planthopper species (Sogatella spp.) (For example, Planthopper (S.furcifera)), rod length bug species (Blissus spp.) (For example, the Americas Gu rod length bug (B.leucopterus leucopterus)), black bug species (Scotinophora spp.) (For example, black rice bug (S.vermidulate)), green bug species (Acrosternum spp.) (Example For example, proposed viridula (A.hilare)), species of rice skipper (Parnara spp.) (For example, straight Rice skipper (P.guttata)), Chilo species (Chilo spp.) (For example, stem borer (C. suppressalis), Taiwan's rice borer (C.auricilius) or Chardonnay rice borer (C.polychrysus)), Chilo species (Chilotraea spp.) (For example, rice straw borer (C.polychrysa)), moth Spodoptera species stem (Sesamia spp.) (For example, rice stem borers armyworm (S.inferens)), moth Wo borer species (Tryporyza spp.) (For example, white rice borer (T.innotata) or borer (T. incertulas)), leafroller species (Cnaphalocrocis spp.) (for example, rice longitudinal Leaf borer (C.medinalis)), species leafminer (Agromyza spp.) (For example, Japanese Rice leafminer (A.oryzae) or corn leafminer (A.parvicornis)), tallgrass borer species (Diatraea spp.) (for example, a small sugarcane borer (D.saccharalis) or Southwest corn stalks Grasshopper (D.grandiosella)), Narnaga species (eg, green rice caterpillar (N.aenescens)), yellow moth species (Xanthodes spp.) (E.g., the plow pattern yellow armyworm (X.transversa)), genus Spodoptera Species (Spodoptera spp.) (E.g., Spodoptera frugiperda (S.frugiperda), beet armyworm (S.exigua), Spodoptera littoralis (S.littoralis) or western yellow striped armyworm (S.praefica)), Secret moth species (Mythimna spp.) (Eg, armyworms (Mythmna (Pseudaletia) seperata)), bell moth species (Helicoverpa spp.) (e.g., bollworm (H.zea)), Colaspis species (Colaspis spp.) (For example, Shaw grape leaf beetle (C.brunnea), rice Water weevil species (Lissorhoptrus spp.) (For example, rice water weevil (L.oryzophilus)), Like rice species (Echinocnemus spp.) (For example, as rice (E.squamos)), rice iron A species (Diclodispa spp.) (For example, rice armored (D.armigera)), cereal negative Beetle species (Oulema spp.) (For example, rice Oulema (O.oryzae)), rice weevil genus Species (Sitophilus spp.) (For example, rice weevil (S.oryzae)), Pachydiplosis materiae Species (eg, gall midge (P.oryzae)), hair eyes water fly species (Hydrellia spp.) (Case For example, wheat leaf hair eyes water flies (H.griseola) or Japanese rice hair eyes water flies (H.sasakii)), Chlorops Species (eg, rice straw maggot (C.oryzae)), rootworm species (Diabrotica spp.) (For example, corn rootworm (D.virgifera virgifera), the North American corn rootworm (D.barberi), Fresh cucumber rootworm root subspecies (D.undecimpunctata howardi), Mexican corn root Leaf beetle (D.virgifera zeae), striped cucumber beetle (D.balteata)), pole borer species (Ostrinia spp.) (For example, corn borer (O.nubilalis)), and tiger species (Agrotis spp.) (for example, black cutworm (A.ipsilon)), Elasmopalpus species (e.g., South American corn seedlings moth (E.lignosellus)), comb claw beetle species (Melanotus spp.) (Nematodes), round rhinoceros beetle species (Cyclocephala spp.) (For example, the northern camouflage gold Turtle (C.borealis) or Southern camouflage beetles (C.immaculata)), arc beetle species (Popillia spp.) (For example, Japanese arc beetle (P.japonica)), flea beetle species (Chaetocnema spp.) (For example, copper corn flea beetle (C.pulicaria)), long neck like Species (Sphenophorus spp.) (For example, corn long beak like (S.maidis)), constricted tube Aphid species (Rhopalosiphum spp.) (For example, corn aphid (R.maidis)), round aphid Species (Anuraphis spp.) (For example, corn root aphid (A.maidiradicis)), black locust Species (Melanoplus spp.) (E.g., red foot black locust (M.femurrubrum)), Special Iso black locust (M.differentialis) or migratory black locust (M.sanguinipes)), kind of fly species (Hylemya spp.) (E.g., kind of flies (H.platura)), stay thrips species (Anaphothrips spp.) (for example, yellow stay thrips (A.obscrurus)), fire ant species (Solenopsis spp.) (For example, stolen ants (S.milesta)), mite species (Tetranychus spp.) (E.g., Tetranychus urticae Koch (T.urticae), carmine spider mite (T.cinnabarinus)), bell moth species (For example, Google or Heliothis armigera (H.armigera)), Pectinophora species (cases For example, red bell Gelechiidae (P.gossypiella)), diamond species (Earias spp.) (E.g., Green grain drill armyworm (E.vittella)), Heliothis species (Heliothis spp.) (E.g., tobacco Budworm (H.virescens)), flower elephant species (Anthonomus spp.) (For example, Mexico Brother boll weevil (A.grandis)), Pseudatomoscelis species (eg, cotton pseudo blind spot leg Bug (P.seriatus)), knot winged whitefly species (Trialeurodes spp.) (E.g., junction wings Whiteflies (T.abutiloneus), greenhouse whitefly (T.vaporariorum)), small whitefly species (Bemisia spp.) (E.g., silver leaf whitefly (B.argentifolii)), is a species of aphid (Aphis spp.) (for example, cotton aphid (A.gossypii)), grass species bugs (Lygus spp.) (case For example, the U.S. tarnished plant bug (L.lineolaris) or the Americas tarnished plant bug (L.hesperus)), United States Island species bug (Euschistus spp.) (E.g., spots Americas bug (E.conspersus)), Chlorochroa species (eg, Saybolt bugs (C.sayi)), green stink bug species (Nezara spp.) (For example, green rice bug (N.viridula)), thrips species (Thrips spp.) (E.g., tobacco Thrips (T.tabaci)), flower thrips species (Frankliniella spp.) (For example, tobacco brown thistle Ma (F.fusca) or Western flower thrips (F.occidentalis)), Colorado potato beetle species (Leptinotarsa spp.) (E.g., Colorado potato beetle (L.decemlineata), pseudo potato Leaf beetle (L.juncta) or eggplant Elaeagnus cotyledon leaf beetle (L.texana)), co-claw Oulema species (Lema spp.) (For example, three Oulema (L.trilineata)), eggplant flea beetle species (Epitrix spp.) (for example, the U.S. potato flea beetle (E.cucumeris), tobacco flea beetle (E.hirtipennis) Or tuber flea beetle (E.tuberis)), Epicauta species (Epicauta spp.) (For example, North America Epicauta (E.vittata)), ape leaf beetle species (Phaedon spp.) (For example, horseradish ape Leaf beetle (P.cochleariae)), phytophagous ladybird species (Epilachna spp.) (For example, the ink Mexico bean beetle (E.varivetis)), Acheta species (eg, HOS Ai cricket (A. domesticus)), a small green leafhopper species (Empoasca spp.) (for example, small green beans Cicada (E.fabae)), tumor Aphis (Myzus spp.) (E.g., Myzus persicae (M.persicae)), A psyllids potato species (Paratrioza spp.) (E.g., potato psyllids (P.cockerelli)), Chest wireworm species (Conoderus spp.) (Eg, southern potato nematodes (C.falli) Or tobacco nematodes (C.vespertinus)), Gelechiidae species (Phthorimaea spp.) (E.g., Potato Gelechiidae (P.operculella)), aphid species (Macrosiphum spp.) (Case For example, Euphorbia avenae (M.euphorbiae)), Thyanta species (eg, red shoulder stinkbug (T.pallidovirens)), Gelechiidae species (Phthorimaea spp.) (E.g., potato Gelechiidae (P.operculella)), bell moth species (eg, bollworm), Keiferia genus Species (eg, tomato moth (K.lycopersicella)), wireworm species (Limonius spp.) (Nematodes), Manduca species (eg, tobacco hornworm (M.sexta) or tomato hornworm (M. quinquemaculata)), species leafminer (Liriomyza spp.) (e.g., vegetable spot latent Flies (L.sativae), clover leaf miner (L.trifolli) or South American leafminer (L.huidobrensis)), Drosophila species (Drosophilla spp.) (For example, Drosophila melanogaster (D.melanogaster), D.yakuba, intends dark fruit fly (D.pseudoobscura) or D.simulans (D.simulans)), step A species (Carabus spp.) (For example, grain beetles (C.granulatus)), is a matter chironomid Species (Chironomus spp.) (Eg, midges (C.tentanus)), flea species Ctenocephalides (Ctenocephalides spp.) (For example, Ctenocephalides felis (C.felis)), non-metallic objects like ear beak Kind (Diaprepes spp.) (For example, the root weevil (D.abbreviatus)), tooth beetles materiae Species (Ips spp.) (Eg, dexamethasone tooth beetles (I.pini)), Tribolium species (Tribolium spp.) (for example, Tribolium castaneum (T.castaneum)), Glossina species (Glossina spp.) (For example, thorn Glossina (G.morsitans)), Anopheles species (Anopheles spp.) (Case For example, Gambia Anopheles (A.gambiae)), bell moth genus (eg, cotton bollworm (H.armigera)), Acyrthosiphon species (eg, pea aphid (A.pisum)), bee species (Apis spp.) (Eg, Apis mellifera (A.melifera)), Homalodisca species (eg, glass Leafhopper (H.coagulate)), Aedes species (Aedes spp.) (For example, Egypt markings (Ae. aegypti)), silk moth species (Bombyx spp.) (for example, Bombyx mori (B.mori)), Locust species (Locusta spp.) (For example, locusts (L.migratoria)), cattle ticks materiae Species (Boophilus spp.) (For example, Boophilus microplus (B.microplus)), Acanthoscurria Species (eg, orang chocolate tarantula (A.gomesiana)), A cockroach species (Diploptera spp.) (for example, the Pacific wings folded cockroach (D.punctata)), sleeve butterfly species (Heliconius spp.) (eg, Valsalva sleeve butterfly (H.erato) or Muse Sleeve Butterfly (H.melpomene)), as Insect species (Curculio spp.) (E.g., such as the European oak (C.glandium)), Plutella Species (for example, diamondback moth (P.xylostella)), blunt eye tick species (Amblyomma spp.) (For example, color decorative flower ticks (A.variegatum)), tussah species (Anteraea spp.) (Case For example, Unending (A.yamamai)) and A mosquito species (Armigeres spp.) (Eg, harassment A mosquito (A.subalbatus)); ...

(2) can invade and harass or damage the insect of people and/or animal, such as but not limited to having the Hemiptera of being found in and some Hymenoptera and Diptera (as mosquito, honeybee, wasp, lice, flea and ant) the insect and the arachnid (Arachnidae) (as tick and mite) of piercing-sucking mouthparts: acarina (tick and mite), for example representational section: soft ticks section (Argasidae), Dermanyssidae (Dermanyssidae), hard tick section (Ixodidae), itch mite section (Psoroptidae) or Sarcoptidae (Sarcoptidae), and representational species: Amblyomma species (Amblyomma spp.), Anocentor species (Anocentor spp.), Argas species (Argas spp.), Boophilus species (Boophilusspp.), Cheyletiella species (Cheyletiella spp.), foot mite species (Chorioptes spp.), Demodex species (Demodex spp.), Dermacentor species (Dermacentor spp.), Dermanyssus species (Dermanyssus spp.), Haemaphysalis species (Haemophysalis spp.), Hyalomma species (Hyalomma spp.), hard tick species (Ixodes spp.), the Lynxacarus species, Mesostigmata species (Mesostigmata spp.), back of the body anus mite species (Notoedresspp.), Ornithodoros species (Ornithodoros spp.), Ornithonyssus species (Ornithonyssusspp.), residual beak tick species (Otobius spp.), ear itching mite species (Otodectes spp.), Pneumonyssus species (Pneumonyssus spp.), Psoroptes species (Psoroptes spp.), Rh species (Rhipicephalus spp.), itch mite species (Sarcoptes spp.) or Trombidium species (Trombicula spp.); Anoplura (lice that sucks and bite), for example representative species: ox hair lice species (Bovicola spp.), Haematopinus species (Haematopinus spp.), jaw lice species (Linognathus spp.), chicken lice species (Menopon spp.), Pediculus species (Pediculus spp.), goitre woolly aphid species (Pemphigus spp.), Phylloxera spp species (Phylloxera spp.) or pipe lice species (Solenopotes spp.); Diptera (fly), for example representational species: Aedes species (Aedes spp.), Anopheles species (Anopheles spp.), Calliphora species (Calliphora spp.), Carysomyia species (Chrysomyia spp.), Chrysops species (Chrysops spp.), Callitroga's species (Cochliomyia spp.), Culex species (Culexspp.), Bitting midge species (Culicoides spp.), Cuterebra species (Cuterebra spp.), Hypoderma species (Dermatobia spp.), Gasterophilus species (Gastrophilus spp.), Glossina species (Glossina spp.), Haematobia species (Haematobia spp.), Chrysozona species (Haematopota spp.), Hippobosca species (Hippobosca spp.), Hypoderma species (Hypoderma spp.), Lucilia species (Lucilia spp.), horn fly species (Lyperosiaspp.), Melophagus species (Melophagus spp.), Oestrus species (Oestrus spp.), Lucilia species (Phaenicia spp.), owl midge species (Phlebotomus spp.), Phormia species (Phormia spp.), Sarcophaga species (Sarcophaga spp.), Simulium species (Simulium spp.), Genus Stomoxys species (Stomoxys spp.), Gadfly species (Tabanus spp.), Tannia species or big uranotaenia (Tipula spp.); Mallophaga (lice that bites), for example representational species: Damalina species, cat Linognathus species (Felicola spp.), Heterodoxus species or Trichodectes species (Trichodectes spp.); Siphonaptera (aptery insect), for example Ceratophyllus species (Ceratophyllus spp.), flea species (Pulex spp.) or objective flea species (Xenopsylla spp.); Cimicidae (bedbug), for example representational species: Cimex species (Cimex spp.), Tritominae species, Rhodinius species or Triatoma species (Triatoma spp.) and

(3) cause unfavorable destructive insect, as attacking the insect of food, seed, timber, coating, plastics, clothing etc. to basic unit (substrate) or article.

(4) insect or the arachnid relevant with publilc health and health, comprise housing insect and vermin, such as but not limited to fly, tetranychid, thrips, tick, red poultry mite, ant, cockroach, termite, cricket (comprising the housing cricket), moth, booklice, beetle, earwig, mosquito and flea.Preferred target is cockroach (Blattodea), such as but not limited to Blatella species (Blatella spp.) (for example, Groton bug (Blatella germanica), Periplaneta species (Periplaneta spp.) (for example, periplaneta americana (Periplaneta americana) and Australian cockroach (Periplanetaaustraliasiae), blattaria species (Blatta spp.) (for example, oriental cockroach (Blatta orientalis)) and skin Lian species (Supella spp.) (for example, long palpus blattaria (Supella longipalpa)), ant (Formicoidea), as but (for example be not limited to Solenopsis (Solenopsis spp.), red fire ant (Solenopsis invicta)), MonomoriumMayr species (Monomorium spp.) (for example, MonomoriumMayr (Monomorium pharaonis)), back of a bow ant species (Camponotus spp.) (for example, back of a bow ant species (carpenter ant)), hair ant species (Lasius spp.) (for example, black wool ant (lasius niger)), Pavement Ant species (Tetramorium spp.) (for example, Pavement Ant (Tetramorium caespitum)), Formica rufa species (Myrmica spp.) (for example, little Formica rufa (Myrmica rubra)), ant species (Formica spp.) (carpented ant), (for example lift abdomen ant species (Crematogaster spp.), lift abdomen ant (Crematogaster lineolata)), the smelly ant species of rainbow (Iridomyrmex spp.) (for example, Argentine ant (Iridomyrmexhumilis)), major part ant species (Pheidole spp.) (major part ant) and the Dasymutilla species (for example, Dasymutilla occidentalis (mutillid)), termite (Isoptera and/or Termitidae), such as but not limited to bifurcation Cryptotermes species (Amitermes spp.) (for example black wing reticulitermes flavipe (Amitermes floridensis) in Florida), Reticulitermes species (Reticulitermes spp.) (for example, yellow limb reticulitermes flavipe (Reticulitermes flavips), western reticulitermes flavipe (Reticulitermeshesperus)), formosanes species (Coptotermes spp.) (for example, Taiwan formosanes (Coptotermes formosanus)), principal columns of a hall Cryptotermes species (Incisitermes spp.) (for example, little principal columns of a hall termite (Incisitermes minor)), new Cryptotermes species (Neotermes spp.) (for example, forest tree termite (Neotermes connexus)).

Benefiting from " responsive biological " of the present invention comprises the responsive any biology of insect infringement.Many different biological insects, described biology is animal (as people, domestic animal (as pet (as cat, dog etc.)) and livestock (comprising sheep, milk cow, pig, chicken etc.)) for example.

Thus, of the present invention preferred but in the unrestriced embodiment, described insect or arachnid are selected from:

(1) acarina: the mite class that comprises Ixodides (tick)

(2) Arachnida: Araneida (spider) and Opiliones (daddy longlegs), example comprise latrodectus mactans (Latrodectus mactans) and brown reclusion spider (Loxosceles recluse)

(3) Anoplura: lice, as body louse (Pediculus humanus)

(4) Blattodea: cockroach comprises that the periplaneta americana of Groton bug, Periplaneta and oriental cockroach and the long of skin Lian genus that Australian cockroach, blattaria belong to must blattarias.Most preferred target is a Groton bug.

(5) Coleoptera: beetle, example comprises Bostrichoidea; Large small moth species (Dendroctonusspp.) (Black Turpentine Beetle, dendroctonus frontalis (Southern Pine Beetle), IPS engraver beetle (IPS Engraver Beetle)); Carpet beetle (Carpet Beetles) (Anthrenus species (Anthrenus spp.), the moth-eaten species (Attagenus spp.) of fur); OldHouse Borer (Cerambycidae: house longhorn beetle (Hylotrupes bajulus)); Furniture death watch beetle (Anobiumpunctatum); Tribolium species (flour beetle); Khapra beetle (Trogoderma granarium); Oryzaephilus sarinamensis (bookworms) such as (saw-toothed grain beetle (Toothed Grain Beetle))

(6) Dermaptera: earwig class

(7) Diptera: mosquito (Dulicidae) and fly (Brachycera), example are Anophehnae (as the Anopheles species) and Culicinae (as Aedes fulvus); Tabanidae such as Tabanus punctifer (horse botfly), Glossina morsitans morsitans (tsetse fly), drain flies (Moth files) and Calypteratae (Calyptratae) are as housefly (Musca domestica), flesh fly (Flesh flies) etc.

(8) Heteroptera: bedbug, as bed bug (Cimex lectularius) (bed bug)

(9) Hymenoptera: wasp (Clistogastra) comprises ant (Formicoidea), honeybee (Apoidea): red fire ant, MonomoriumMayr, hunchbacked ant species (carpenter ant), black wool ant, Pavement Ant, little Formica rufa, ant species (carpented ant), act abdomen ant, Argentine ant, major part ant species (major part ant) and Dasymutilla occidentalis (mutillid) etc.

(10) Isoptera: termite, example comprise that the Florida deceives wing reticulitermes flavipe (Amitermesfloridensis), yellow limb reticulitermes flavipe, western reticulitermes flavipe (R.hesperus), Taiwan formosanes, little principal columns of a hall termite, forest tree termite (Neotermes connexus)) and Termitidae

(11) lepidopteran: moth, example comprise rain moth section and knit moth section (as casemaking clothes moth (Tineolabisselliella)) and Pyralidae (as purple plague purpura paddy snout moth's larva (Pyralis farinalis)) etc.

(12) Corrodentia: booklice (Corrodentia)

(13) Siphonaptera: flea, as Pulex irritans (Pulex irritans)

(14) Sternorrhyncha: aphid (Aphidiadae)

(15) silverfish order: moth, example is: special mess silverfish (Thermobia domestica) and silverfish (Lepisma saccharina)

The preferred plant-pathogenic insect of the present invention is plant insect and is selected from: the colorado potato beetles species (for example, colorado potato beetles, pseudo-colorado potato beetles or Thory Elaeagnus Leaf eggplant are chrysomelid), the brown paddy plant hopper species (for example, brown paddy plant hopper), the small brown rice planthopper species (for example, small brown rice planthopper), the rice green leafhopper species (for example, nephotettix bipunctatus, or rice green leafhopper, or two rice green leafhoppers), the white backed planthopper species (for example, white backed planthopper), the straw borer spp species (for example, striped rice borer, goldrimmed moth or rice Dolly snout moth's larva), moth stem Noctua species (for example, Sesamia inferens), moth standing grain snout moth's larva species (for example, white rice borer or yellow rice borer), the chrysomelid species of root firefly (for example, corn root leaf A, the North America corn root leaf A, cucumber 11 asterophyllite first food root subspecies, the zea mexicana root is chrysomelid), the wild snout moth's larva species of bar (for example, Pyrausta nubilalis (Hubern).), slow-witted Thrips species (for example, yellow slow-witted thrips), the Pectinophora species (for example, Pectinophora gossypiella), the Heliothis species (for example, Heliothis virescens), knot wing Aleyrodes species (for example, knot wing aleyrodid, greenhouse whitefly), starch lice species (for example, Bemisia argentifolii), the Aphis species (for example, cotten aphid), the lygus bug species (for example, U.S. tarnished plant bug or America tarnished plant bug), America stinkbug species (for example, spot America stinkbug), the Chlorochroa species (for example, the Sai Shi stinkbug), the Bemisia spp species (for example, Nezara viridula smaragdula Fabricius.), the Thrips species (for example, onion thrips), the flower thrips species (for example, cigarette brown thrip or west flower thrips), the tumor aphid genus species (for example, black peach aphid), the Macrosiphus spp species (for example, root of Beijing euphorbia Macrosiphus spp), bar chinch bug species (for example, America paddy bar chinch bug), the Bemisia spp species (for example, intend green stinkbug), the straw borer spp species (for example, rice bar snout moth's larva (C.polychrysa)), rice water (for example resembles species, rice water weevil), the Rhopalosiphum species (for example, corn leaf aphids) and rounded tail Aphis species (for example, corn root aphid (A.maidiradicis)).

According to a more particular embodiment, method of the present invention is applicable to the colorado potato beetles species.Colorado potato beetles belong to Chrysomelidae or class Diabrotica.Chrysomelid (as flea beetle and corn rootworm) and weevil (as alfalfa weevil) are the insects of particularly important.Flea beetle comprises the chrysomelid worm of multiple small-sized food, and it is a food with multiple grass, cereal and herbal leaf.Flea beetle comprises a plurality of genus (for example, Attica, Apphthona, Argopistes, Disonycha, eggplant phyllotreta, long instep phyllotreta, dive phyllotreta (Prodagricomela), Systena and striped flea beetle).Phyllotreta cruciferae (Phyllotretacruciferae) (also being called rape flea beetle (Rape Flea Beetle)) is the insect of particularly important.Corn rootworm comprises the species (for example, cucumber 11 asterophyllite first (D.undecimpunctataundecimpunctata), cucumber 11 asterophyllite first food root subspecies, D.longicornis, corn root leaf A and striped cucumber are chrysomelid) of chrysomelid genus.Corn rootworm causes extensive destruction to corn and melon (curcubit).Cucumber 11 asterophyllite first (Western Spotted Cucumber Beetle) are the melon insects of Western United States.Alfalfa weevil (also claiming the trifolium weevil) belongs to Phytonomus, and (alfalfa leaf resembles (H.postica), Egyptian Herba Medicaginis leaf and resembles (H.brunneipennis), H.nigrirostris, trifolium leaf and resemble (H.punctata) and H.meles), and is considered to important beans insect.The important clover insect of Egyptian Herba Medicaginis leaf as if Western United States.

The colorado potato beetles species is above 30 kinds.Therefore, the present invention includes the method that is used to prevent and treat the colorado potato beetles species, more specifically to kill insects or stop colorado potato beetles to belong to that insect grows or growth or prevent the method for insect infection or infringement.The specific colorado potato beetles species that the present invention prevented and treated comprises colorado potato beetles (Leptinotarsa decemlineata (Say)) and pseudo-colorado potato beetles (Leptinotarsa juncta (Say)).

CPB (Colorado Potato Beetle, colorado potato beetles) (for example to the potato of our this country, other cultivation and wild tuberosity and the potato species that do not have a stem tuber, S.demissum (Mexico's potato wild species), Fu Liya potato (S.phureja a.o.)) and other nightshade species are (serious) insects, described plant species comprises:

(a) crop species tomato (some tomato species), eggplant, pepper (some Capsicum species), tobacco (some Nicotiana species, it comprises ornamental plant) and gooseberry (Physalis species);

(b) weeds/herbaceous plant species, North America thorn black nightshade (S.carolinense), fruit of Biiter Nightshade (S.dulcamara), belladonna (Atropa species), thorn apple (Datura species), Semen Hyoscyami (poison tobacco species) and chrysanthemum thorn eggplant (S.rostratum).

FPB (False Potato Beetle, pseudo-colorado potato beetles) mainly is found on the thorn black nightshade of North America, also occurs on fruit of Biiter Nightshade, gooseberry and the wintercherry (Physalis species).

Term " insect " comprises the insect of all types and all etap, comprises ovum, larva or nymph, pupa and adult stage.

The present invention extends to method as herein described, and wherein said insect is colorado potato beetles, and described plant is potato, eggplant, tomato, pepper, tobacco, gooseberry or rice, corn or cotton.

The present invention extends to method as herein described, and wherein said insect is that the horseradish ape is chrysomelid, and described plant is leaf mustard, Chinese cabbage, turnip, kale or Plantula Brassicae chinensis.

The present invention extends to method as herein described, wherein said insect is a mexican bean ladybird, and described plant is beans, Kidney bean, string bean, food English Kidney bean, lima bean, mung bean, green soya bean, buphthalmos beans, velvet bean, soybean, cowpea, pigeonpea, trifolium or clover.

The present invention extends to method as herein described, and wherein said insect is that Mexico's cotton boll resembles, and described plant is a cotton.

The present invention extends to method as herein described, wherein said insect is a red flour beetle, described plant is with the form of storage cereal, as flour, cereal, meal, biscuit, beans, seasonings, wheaten food, dessert powder, dried pet food, dried flower, chocolate, nut, seed even dried museum's sample.

The present invention extends to method as herein described, and wherein said insect is a black peach aphid, and described plant is trees such as Prunus, particularly peach, apricot and plum; Solanaceae, Chenopodiaceae, composite family, Cruciferae and vegetable crop cucurbitaceous, it includes but not limited to: choke, asparagus, beans, beet, sprouting broccoli, brussels sprouts, Caulis et Folium Brassicae capitatae, Radix Dauci Sativae, Cauliflower, Hami melon, celery, corn, cucumber, fennel, kale, black salted turnip, turnip, eggplant, lettuce, leaf mustard, gumbo, parsley, parsnip, pea, pepper, potato, radish, spinach, pumpkin, tomato, turnip, Nasturtium officinale and watermelon; Field-crop is such as but not limited to tobacco, sugar beet and Sunflower Receptacle; Flower crop or other ornamental plant.

The present invention extends to method as herein described, and wherein said insect is a brown paddy plant hopper, and described plant is a rice plants.

The present invention extends to method as herein described, and wherein said insect is a striped rice borer, and described plant is rice plants, barley (bareley), Chinese sorghum, corn, wheat or grass.

The present invention extends to method as herein described, wherein said insect is a diamond-back moth, described plant is the Btassica species, such as but not limited to Caulis et Folium Brassicae capitatae, Chinese cabbage, brussels sprouts, kale, Semen Brassicae campestris, sprouting broccoli, Cauliflower, turnip, leaf mustard or radish.

The present invention extends to method as herein described, and wherein said insect is residential house Chinese mugwort Xi, and described plant is any plant as herein described or any organism.

In this article, term " plant " thus comprise the processed any vegetable material that stops or reduce insect growth and/or insect pest infestation of expectation.This comprises whole strain plant, seedling, propagation or reproductive material (as seed, cutting, scion, explant etc.), and vegetable cell and tissue culture etc.Described vegetable material should the expressed rna molecule or is had the ability of expressed rna molecule, described RNA molecule comprises at least a nucleotide sequence, this nucleotide sequence be at least a target gene of insect the sense strand nucleotide sequence at least a portion the RNA complementary sequence or represented its RNA Equivalent, thereby described RNA molecule is absorbed by insect by the interaction of plant and insect, and described RNA molecule can disturb by RNA and suppress target gene or downward modulation target gene expression.

Described target gene can be any target gene as herein described, for example for existence, growth, the growth of insect or breed essential target gene.The present invention relates to any goal gene (this paper can be called " target gene ") that to be reduced in the insect.

Term " down-regulation of gene expression " and " inhibition of gene expression " are used interchangeably, and it refers to that on the protein of target gene and/or mRNA product level measurable or observable genetic expression reduces or eliminates detectable genetic expression fully.Preferably, described downward modulation directly suppresses other expression of gene of insect indistinctively.When with normal genetic expression relatively the time, dsRNA can be calculated as the downward modulation effect of genetic expression and be at least 30%, 40%, 50%, 60%, and is preferred 70%, 80%, and perhaps more preferably 90% or 95%.The character that depends on target gene; can carry out phenotype analytical or use molecular engineering measurement mRNA or protein expression to verify the downward modulation or the inhibition of genetic expression in the insect cell, described molecular engineering such as RNA solution hybridization, PCR, nuclease protection experiment, Northern hybridization, reverse transcription, the little array gene expression of use, antibodies, enzyme-linked immunosorbent assay (ELISA), Western trace, radioimmunoassay (RIA), other immunoassay or fluorescence activated cell analysis (FACS) by pair cell or whole insect.

" target gene " can be pathogenic or infectiously expect repressed almost any gene owing to the growth of its interference insect basically.For example, if method of the present invention is used to stop insect growth and/or infringement, then preferably select existence, growth, growth or breed essential target gene insect, perhaps pathogenic or infective any gene of involved in insect, thus the specificity of target gene is suppressed to cause lethality phenotype or reduction or stops insect pest infestation.

According to a nonrestrictive embodiment, described target gene is when using method downward modulation of the present invention or suppressing its expression, and insect is killed or the breeding of insect or those genes that stunt ends or blocks.Think that this class target gene is that insect existence is necessary, and be called indispensable gene.Therefore, the present invention includes method as herein described, wherein said target gene is an indispensable gene.

According to another nonrestrictive embodiment, described target gene is to work as to use method of the present invention with timing under it, and destruction that infringement of insect or infection, insect are caused and/or insect pest infestation or infection host are biological and/or cause this type of destructive ability to decrease.Usually, term " infringement " and " infection " are used interchangeably all the time.Think the pathogenic or infectious of this class target gene involved in insect.Therefore, the present invention extends to method as herein described, wherein said target gene involved in insect pathogenic or infectious.Select the advantage of back one type target gene to be to have blocked further infection plant of insect or plant part, and suppress its formation offspring.

According to an embodiment, target gene is conservative gene or insect specific gene.

In addition, can use in the method for the invention can guide RNA i or the gene silencing of RNA mediation or any suitable double-stranded RNA fragment of inhibition insect target gene.

In another embodiment, select the substantive gene that participates in insect (as insect) growth, growth and breeding.Exemplary gene includes but not limited to the structure subunit and the β-coatmer gene (as the CHD3 gene) of ribosomal protein.Ribosomal protein such as S4 (RpS4) and S9 (RpS9) participate in the biosynthetic ribosomal structural constituent of protein, and be the component of the little ribosomal subunit of kytoplasm, ribosomal protein such as L9 and L19 participate in the biosynthetic ribosomal structural constituent of protein, and are positioned rrna.β in the Caenorhabditis elegans-coatmer dna encoding the protein, described protein form the subunit of the poly complex body of film bubble coat.In multiple biology, found similar sequence, as Arabidopis thaliana (Arabidopsis thaliana), drosophila melanogaster (Drosophila melanogaster) and yeast saccharomyces cerevisiae (Saccharomyces cerevisiae).Find relevant sequence in multiple biology,, mexican bean ladybird chrysomelid as colorado potato beetles, horseradish ape, Mexico's cotton boll resemble, red flour beetle, black peach aphid, brown paddy plant hopper, striped rice borer, diamond-back moth and residential house Chinese mugwort Xi.

Other target gene that the present invention uses can comprise the gene of for example playing an important role in existence, growth, growth, breeding and infectivity.These target genes for example comprise that lethality knocks out sudden change in housekeeping gene, transcription factor and insect specific gene or nematode or the fruit bat.The target gene that the present invention uses can also be from other biological gene, for example from the gene of insect or arachnid (for example, colorado potato beetles species, phaedon species, epilachna species, flower resemble species, Tribolium species, tumor aphid genus species, brown paddy plant hopper species, straw borer spp species, Plutella species or Acheta species).

Preferred target gene comprises among the table 1A specified gene and from other target biology orthologous gene of (as from other insect).

In the method for the invention, use dsRNA to suppress the pathogenic or infectious of insect growth or interference insect.

Therefore, the present invention relates to comprise the isolating double-stranded RNA of complementary strand of annealing, wherein annealing complementary strand comprises at least a portion complementary nucleotide sequence with the target nucleotide sequences of insect target gene.Described target gene can be any target gene as herein described, perhaps the part of its performance said function.

According to one embodiment of the invention, the isolating double-stranded RNA that comprises the complementary strand of annealing is provided, wherein annealing complementary strand comprises at least a portion complementary nucleotide sequence with insect target gene nucleotide sequence, and wherein said target gene comprises and is selected from following sequence:

(i) arbitrary sequence with following expression has at least 75% conforming sequence: SEQ ID NO1,3,5,7,9,11,13,15,17,19,21,23,49 to 158,159,160-163,168,173,178,183,188,193,198,203,208,215,220,225,230,247,249,251,253,255,257,259,275 to 472,473,478,483,488,493,498,503,513,515,517,519,521,533 to 575,576,581,586,591,596,601,603,605,607,609,621 to 767,768,773,778,783,788,793,795,797,799,801,813 to 862,863,868,873,878,883,888,890,892,894,896,908 to 1040,1041,1046,1051,1056,1061,1071,1073,1075,1077,1079,1081,1083,1085,1087,1089,1091,1093,1095,1097,1099,1101,1103,1105,1107,1109,1111,1113,1161 to 1571,1572,1577,1582,1587,1592,1597,1602,1607,1612,1617,1622,1627,1632,1637,1642,1647,1652,1657,1662,1667,1672,1677,1682,1684,1686,1688,1690,1692,1694,1696,1698,1700,1702,1704,1730 to 2039,2040,2045,2050,2055,2060,2065,2070,2075,2080,2085,2090,2095,2 ¹00,2102,2104,2106,2108,2120 to 2338,2339,2344,2349,2354,2359,2364,2366,2368,2370,2372,2384 to 2460,2461,2466,2471,2476 or 2481, or its complementary sequence, and

The sequence that (ii) comprises at least 17 continuous nucleotides of following arbitrary sequence: SEQ ID NO 1,3,5,7,9,11,13,15,17,19,21,23,49 to 158,159,160-163,168,173,178,183,188,193,198,203,208,215,220,225,230,247,249,251,253,255,257,259,275 to 472,473,478,483,488,493,498,503,513,515,517,519,521,533 to 575,576,581,586,591,596,601,603,605,607,609,621 to 767,768,773,778,783,788,793,795,797,799,801,813 to 862,863,868,873,878,883,888,890,892,894,896,908 to 1040,1041,1046,1051,1056,1061,1071,1073,1075,1077,1079,1081,1083,1085,1087,1089,1091,1093,1095,1097,1099,1101,1103,1105,1107,1109,1111,1113,1161 to 1571,1572,1577,1582,1587,1592,1597,1602,1607,1612,1617,1622,1627,1632,1637,1642,1647,1652,1657,1662,1667,1672,1677,1682,1684,1686,1688,1690,1692,1694,1696,1698,1700,1702,1704,1730 to 2039,2040,2045,2050,2055,2060,2065,2070,2075,2080,2085,2090,2095,2100,2102,2104,2106,2108,2120 to 2338,2339,2344,2349,2354,2359,2364,2366,2368,2370,2372,2384 to 2460,2461,2466,2471,2476 or 2481, or its complementary sequence

Perhaps, wherein said insect target gene is the lineal homologue of insect of gene that comprises at least 17 continuous nucleotides of following arbitrary sequence: SEQ ID NO 49 to 158,275 to 472,533 to 575,621 to 767,813 to 862,908 to 1040,1161 to 1571,1730 to 2039,2120 to 2338,2384 to 2460 or its complementary sequence.

Depend on the mensuration that is used to measure gene silencing, compare with the insect of handling with contrast dsRNA, growth-inhibiting can quantitatively be greater than about 5%, 10%, more preferably from about 20%, 25%, 33%, 50%, 60%, 75%, 80%, most preferably from about 90%, 95% or about 99%.

According to another embodiment of the present invention, isolating double-stranded RNA is provided, wherein at least one described annealing complementary strand comprises the RNA Equivalent of at least a nucleotide sequence of the arbitrary sequence of following expression: SEQ ID NO 1,3,5,7,9,11,13,15,17,19,21,23,49 to 158,159,160-163,168,173,178,183,188,193,198,203,208,215,220,225,230,247,249,251,253,255,257,259,275 to 472,473,478,483,488,493,498,503,513,515,517,519,521,533 to 575,576,581,586,591,596,601,603,605,607,609,621 to 767,768,773,778,783,788,793,795,797,799,801,813 to 862,863,868,873,878,883,888,890,892,894,896,908 to 1040,1041,1046,1051,1056,1061,1071,1073,1075,1077,1079,1081,1083,1085,1087,1089,1091,1093,1095,1097,1099,1101,1103,1105,1107,1109,1111,1113,1161 to 1571,1572,1577,1582,1587,1592,1597,1602,1607,1612,1617,1622,1627,1632,1637,1642,1647,1652,1657,1662,1667,1672,1677,1682,1684,1686,1688,1690,1692,1694,1696,1698,1700,1702,1704,1730 to 2039,2040,2045,2050,2055,2060,2065,2070,2075,2080,2085,2090,2095,2100,2102,2104,2106,2108,2120 to 2338,2339,2344,2349,2354,2359,2364,2366,2368,2370,2372,2384 to 2460,2461,2466,2471,2476 or 2481, perhaps wherein at least one described annealing complementary strand comprises its length and is at least 17 base pairs, preferred its length is at least 18,19,20 or 21, more preferably at least 22, the fragment of the RNA Equivalent of 23 or 24 base pairs.

If method of the present invention is used at host cell or host living beings or host cell or host living beings specificity is controlled growth or the infringement of specific insect, then preferred described double-stranded RNA does not have any significant homology with any host gene, does not perhaps have any significant homology with any indispensable gene of host at least.In this case, the consensus nucleic acid sequence of preferred described double-stranded RNA and any gene of host cell is lower than 30%, more preferably less than 20%, and more preferably less than 10%, even more preferably less than 5%.Sequence identity per-cent should calculate on the total length zone of described double-stranded RNA.If can obtain the genomic sequence data of host living beings, then can use standard biological information science instrument to come the sequence identity of mutual verification and described double-stranded RNA.In one embodiment, there is not the sequence identity that surpasses 21 continuous nucleotides between described dsRNA and the host sequences, promptly in this case, do not have 21 continuous base pairs of described dsRNA in the genome of preferred host living beings.In another embodiment, the sequence identity on 24 continuous nucleotides is lower than about 10% or be lower than about 12.5% between any nucleotide sequence of described dsRNA and host species.

Described double-stranded RNA comprises the annealed complementary strand, and wherein an annealing complementary strand comprises nucleotide sequence corresponding to the target nucleotide sequences of the target gene of waiting to reduce.Another of described double-stranded RNA chain can carry out base pairing with above-mentioned article one chain.

" target region " of described insect target gene or " target nucleotide sequences " can be any suitable zone or the nucleotide sequences of gene.Described target region should comprise at least 17, at least 18 or at least 19 continuous nucleotides of target gene, more preferably at least 20 or at least 21 Nucleotide, and more preferably at least 22,23 or 24 Nucleotide of described target gene.

Preferably the target region of (to small part) described double-stranded RNA and insect target gene has 100% sequence identity.Yet should be understood that having 100% sequence identity on the total length of described double-stranded region is not the prerequisite that functional r NA suppresses.Find that also it is effective that the RNA sequence that has insertion, disappearance and simple point mutation with respect to target sequence suppresses RNA.Term " corresponding to " or " with ... complementation " be used interchangeably at this paper, and when these terms were used in reference to sequence correspondence between the target region of described double-stranded RNA and target gene, should be interpreted as not was the sequence identity of absolute demand 100%.Yet the sequence identity per-cent between described double-stranded RNA and the target region is generally at least 80% or 85% unanimity, preferred at least 90%, 95%, 96% unanimity, perhaps more preferably at least 97%, 98% unanimity, and more preferably at least 99% unanimity.When at least 85% base pairing of two nucleic acid chains, they are " complementary basically ".

Term used herein " complementary " relates to the complementary and DNA-RNA complementarity of DNA-DNA.Similarly, in fact term " RNA Equivalent " refers to that the corresponding base " U " that base in dna sequence dna " T " can be present in the Yeast Nucleic Acid usually substitutes.

Although described dsRNA comprises the sequence corresponding to target region in the target gene, yet whole dsRNA is not the sin qua non corresponding to the sequence of target region.For example, described dsRNA can comprise short non-target region at the flank of target specific sequence, needs only the not RNA rejection of this dsRNA of substantial effect of this sequence.

Thereby described dsRNA can comprise the performance that one or more alternative bases are optimized RNAi.To those skilled in the art, how to change successively each base of described dsRNA and test the activity (for example, in suitable vitro test system) of gained dsRNA thus the performance of optimizing given dsRNA is conspicuous.

The non-natural main chain that described dsRNA can also comprise DNA base, non-natural base or sugar-phosphate backbone connects or modifies, thereby for example improves the stability between preservation term or strengthen resistance to nuclease degradation.

Previous existing report, for efficient gene silencing, the siRNA (siRNA) that forms about 21bp is an ideal.Yet, in applicant's application, show, the minimum length of dsRNA preferably at least about 80-100bp so that effectively absorbed by some insect.There are indications, in invertebrates (as the Caenorhabditis elegans of free living or the Meloidogyne incognita of phytotrophy (Meloidogyne incognita)), these long fragments are more effective in gene silencing, this may since invertebrates absorb these long dsRNA more effective due to.

Also propose recently, compare with 21 aggressiveness (21-mer) siRNA of routine, (short hairpin, sh) the synthetic RNA duplex formed of RNA is that RNA disturbs stronger inductor for synthetic RNA duplex of being made up of 27 aggressiveness flush ends or the bob of stem with 29bp and 2-nt 3 ' overhang folder.Therefore, identified target and be that the molecule of the short hairpin RNA molecule of 27 aggressiveness flush end RNA molecules or stem with 29bp and 2-nt 3 ' overhang is also included within the scope of the present invention based on above-mentioned.

Therefore, in one embodiment, the length of described double-stranded RNA fragment (or zone) itself is preferably 17bp at least, length is preferably 18 or 19bp, more preferably 20bp at least, length is 21bp at least more preferably, or 22bp at least, or 23bp at least, or 24bp, 25bp, 26bp or 27bp at least at least." double-stranded RNA fragment " or " double-stranded RNA zone " is meant the little entity of double-stranded RNA corresponding to (part) target gene.

Usually, described double-stranded RNA is preferably about 17-1500bp, more preferably from about 80-1000bp, most preferably from about 17-27bp or about 80-250bp; Be about 17bp, 18bp, 19bp, 20bp, 21bp, 22bp, 23bp, 24bp, 25bp, 27bp, 50bp, 80bp, 100bp, 150bp, 200bp, 250bp, 300bp, 350bp, 400bp, 450bp, 500bp, 550bp, 600bp, 650bp, 700bp, 900bp, 100bp, 1100bp, 1200bp, 1300bp, 1400bp or 1500bp as the double-stranded RNA zone.

The length upper limit of described double-stranded RNA can be depending on i) insect absorbs the needs of this dsRNA, and ii) this dsRNA is processed into the segmental needs of guide RNA i at cell interior.The influence that selected length also can be subjected to the RNA synthetic method and send the pattern of RNA to cell.Preferably, in the inventive method the length of double-stranded RNA to be used less than 10,000bp, more preferably 1000bp or littler, more preferably 500bp or littler, more preferably 300bp or littler, more preferably 100bp or littler.For any given target gene and insect, the optimum length that described dsRNA effectively suppresses can come to determine by experiment.

Described double-stranded RNA can be double-stranded wholly or in part.Partially double stranded RNA can comprise short strand overhang in the one or both ends of two strands part, as long as described RNA still can be and guided RNAi by the insect picked-up.Described double-stranded RNA also can comprise inner incomplementarity district.

Method of the present invention comprises to the same insect while or two or more different double-stranded RNAs or RNA construct is provided successively, thereby realization is reduced or suppressed multiple target gene or more effectively suppress single kind target gene.

Perhaps, hit a plurality of targets by a kind of double-stranded RNA that hits multiple target sequence is provided, and can more effectively suppress single target more than the segmental existence of the double-stranded RNA corresponding to target gene of a copy.Therefore, in one embodiment of the invention, described double-stranded RNA construct comprises a plurality of dsRNA zone, and at least one chain in each dsRNA zone comprises at least a portion complementary nucleotide sequence with the target nucleotide sequences of insect target gene.According to the present invention, the dsRNA zone in the described RNA construct can with same or different target gene complementations, and/or described dsRNA zone can with the target complementation from identical or different insect species.

Term " hits " at least one chain and target gene or the nucleotide sequence complementation that is meant described dsRNA, and combination with it thus.

In one embodiment, described double-stranded RNA zone comprises a plurality of copies with target gene complementary nucleotide sequence.Perhaps, described dsRNA hit same target gene more than a kind of target sequence.Therefore, the present invention includes isolating double-stranded RNA construct, it comprises at least two copies with at least a portion complementary nucleotide sequence of the nucleotide sequence of insect target.

In this article, term of the present invention " multiple (individual) " means at least 2 kinds (individual), at least 3 kinds (individual), at least 4 kinds (individual), at least 5 kinds (individual), at least 6 kinds (individual) etc.

" another target gene " or " another kind of at least target gene " for example is meant second, third or the 4th kind of target gene etc.

Method of the present invention exploitation and having used is hit more than a kind of dsRNA of above-mentioned target or at the combination of the different dsRNA of above-mentioned different targets.

Therefore, the present invention relates to isolating double-stranded RNA construct, it comprises at least two copies of RNA Equivalent of at least a nucleotide sequence of following expression:

SEQ ID NO 1,3,5,7,9,11,13,15,17,19,21,23,49 to 158,159,160-163,168,173,178,183,188,193,198,203,208,215,220,225,230,247,249,251,253,255,257,259,275 to 472,473,478,483,488,493,498,503,513,515,517,519,521,533 to 575,576,581,586,591,596,601,603,605,607,609,621 to 767,768,773,778,783,788,793,795,797,799,801,813 to 862,863,868,873,878,883,888,890,892,894,896,908 to 1040,1041,1046,1051,1056,1061,1071,1073,1075,1077,1079,1081,1083,1085,1087,1089,1091,1093,1095,1097,1099,1101,1103,1105,1107,1109,1111,1113,1161 to 1571,1572,1577,1582,1587,1592,1597,1602,1607,1612,1617,1622,1627,1632,1637,1642,1647,1652,1657,1662,1667,1672,1677,1682,1684,1686,1688,1690,1692,1694,1696,1698,1700,1702,1704,1730 to 2039,2040,2045,2050,2055,2060,2065,2070,2075,2080,2085,2090,2095,2100,2102,2104,2106,2108,2120 to 2338,2339,2344,2349,2354,2359,2364,2366,2368,2370,2372,2384 to 2460,2461,2466,2471,2476 or 2481, perhaps its length is at least 17 base pairs, preferred its length is at least 18,19,20 or 21 base pairs, more preferably at least 22, at least two copies of the segmental RNA Equivalent of 23 or 24 base pairs.Preferably, described double-stranded RNA comprises the RNA Equivalent of nucleotide sequence SEQ ID NO 159 or 160, perhaps its length be at least 17, the fragment of preferred at least 18,19,20 or 21, more preferably at least 22,23 or 24 base pairs.In another embodiment, the present invention relates to isolating double-stranded RNA construct, it comprises at least two copies of the RNA Equivalent of nucleotide sequence SEQ ID NO 159 or 160.

Therefore, the present invention extends to method as herein described, wherein said dsRNA comprises the annealed complementary strand, and wherein annealing complementary strand comprises complementary and comprise the nucleotide sequence of RNA Equivalent of at least two kinds of nucleotide sequences of selection independently of one another with at least a portion of the target nucleotide sequences of insect target gene.In one embodiment, described dsRNA comprises at least two kinds of the sequence that independently is selected from following expression, the RNA Equivalent of preferred at least three kinds, four kinds or five kinds nucleotide sequences:

SEQ ID NO 1,3,5,7,9,11,13,15,17,19,21,23,49 to 158,159,160-163,168,173,178,183,188,193,198,203,208,215,220,225,230,247,249,251,253,255,257,259,275 to 472,473,478,483,488,493,498,503,513,515,517,519,521,533 to 575,576,581,586,591,596,601,603,605,607,609,621 to 767,768,773,778,783,788,793,795,797,799,801,813 to 862,863,868,873,878,883,888,890,892,894,896,908 to 1040,1041,1046,1051,1056,1061,1071,1073,1075,1077,1079,1081,1083,1085,1087,1089,1091,1093,1095,1097,1099,1101,1103,1105,1107,1109,1111,1113,1161 to 1571,1572,1577,1582,1587,1592,1597,1602,1607,1612,1617,1622,1627,1632,1637,1642,1647,1652,1657,1662,1667,1672,1677,1682,1684,1686,1688,1690,1692,1694,1696,1698,1700,1702,1704,1730 to 2039,2040,2045,2050,2055,2060,2065,2070,2075,2080,2085,2090,2095,2100,2102,2104,2106,2108,2120 to 2338,2339,2344,2349,2354,2359,2364,2366,2368,2370,2372,2384 to 2460,2461,2466,2471,2476 or 2481, perhaps its length is at least 17 base pairs, preferably at least 18,19,20 or 21, more preferably at least 22, the fragment of 23 or 24 base pairs.

Described at least two kinds of nucleotide sequences can derive from target gene as herein described.According to an embodiment preferred, described dsRNA hits insect is survived, grows, grows or breed essential at least a target gene, and hits participation pathogenic or infective at least a gene mentioned above.Perhaps, described dsRNA hits similar several genes, and for example described dsRNA hits at least two kinds of indispensable genes or at least two kinds of genes that participate in same cell function.According to another embodiment, described dsRNA hits at least two kinds of target genes, and described target gene participates in different cell functions.For example, described dsRNA hits two or more genes that participate in protein synthesis (for example ribosomal subunit), intracellular protein transhipment, nuclear mRNA montage or participate in one of function described in the table 1A.

Preferably, the present invention extends to method as herein described, and wherein said insect target gene comprises and is selected from following sequence:

(i) arbitrary sequence with following expression has at least 75% conforming sequence: SEQ ID NO1,3,5,7,9,11,13,15,17,19,21,23,49 to 158,159,160-163,168,173,178,183,188,193,198,203,208,215,220,225,230,247,249,251,253,255,257,259,275 to 472,473,478,483,488,493,498,503,513,515,517,519,521,533 to 575,576,581,586,591,596,601,603,605,607,609,621 to 767,768,773,778,783,788,793,795,797,799,801,813 to 862,863,868,873,878,883,888,890,892,894,896,908 to 1040,1041,1046,1051,1056,1061,1071,1073,1075,1077,1079,1081,1083,1085,1087,1089,1091,1093,1095,1097,1099,1101,1103,1105,1107,1109,1111,1113,1161 to 1571,1572,1577,1582,1587,1592,1597,1602,1607,1612,1617,1622,1627,1632,1637,1642,1647,1652,1657,1662,1667,1672,1677,1682,1684,1686,1688,1690,1692,1694,1696,1698,1700,1702,1704,1730 to 2039,2040,2045,2050,2055,2060,2065,2070,2075,2080,2085,2090,2095,2100,2102,2104,2106,2108,2120 to 2338,2339,2344,2349,2354,2359,2364,2366,2368,2370,2372,2384 to 2460,2461,2466,2471,2476 or 2481, or its complementary sequence, and

Perhaps wherein said insect target gene is the lineal homologue of insect of gene that comprises at least 17 continuous nucleotides of following arbitrary sequence: SEQ ID NO 49 to 158,275 to 472,533 to 575,621 to 767,813 to 862,908 to 1040,1161 to 1571,1730 to 2039,2120 to 2338,2384 to 2460 or its complementary sequence.

DsRNA zone (or fragment) in the described double-stranded RNA can followingly be made up:

A) when a plurality of dsRNA of the single target gene of those targets of combination are regional, in the RNA construct, can make up them with primary order (that is, the order that this zone occurs in target gene),

B) or, can they be become the double-stranded RNA construct with combined in any order randomly or on purpose regardless of described segmental original order,

C) or, a kind of fragment can be in described dsRNA construct repeated several times, for example 1～10 time, for example 1,2,3,4,5,6,7,8,9 or 10 time, perhaps

D) described dsRNA zone (target genes that target is a kind of or different) can be made up with the direction that justice or antisense are arranged.

In addition, target gene described to be made up can be selected from one or more genes of following kind:

E) above-mentioned " essential " gene or " pathogenic gene " comprise critical gene for one or more target insects, when it causes lethal or serious (for example, feed, breeding, growth) phenotype during by silence.Select strong lethality target gene to cause effective RNAi effect.In RNA construct of the present invention, a plurality of dsRNA zone of (very effective) lethal gene that target is identical or different can be made up, and acts on effectiveness, effect or speed in the pest control with further enhancing RNAi.

F) " weak " gene is included in the target gene that has interested especially function in one of cellular pathways as herein described but cause weak phenotype effect when its quilt is reticent separately.In RNA construct of the present invention, thereby the stronger RNAi effect of acquisition can be made up in target a plurality of dsRNA zones a kind of or different weak genes.

G) " insect specificity " gene is included in the gene that does not have remarkable homology counterpart in the non-insect biology, and it can be determined by the bioinformation homology search, for example retrieve by BLAST.Select insect specific target gene to cause the RNAi effect of species specificity, and in non-target biology, do not act on or not significant (unfavorable) effect.

H) " conservative gene " is included in (on amino acid levels) conservative gene between target biology and the non-target biology.In order to reduce possible effect, analyze these effective but conservative genes, and select the target of the target sequence of these conservative gene Variable Areas as the dsRNA zone in the RNA construct to non-target species.At this moment, in nucleotide sequence proficiency assessment conservative property.Therefore, these Variable Areas comprise part (nucleotide sequence level) least conservative in the conservative target gene.

I) " conservative approach " gene comprises the gene that participates in identical biological pathway or cell processes, perhaps is included in the gene that has identical function in the different insects species, and it causes specificity and strong RNAi effect also more effectively to prevent and treat insect;

J) or, RNA construct target of the present invention is from the several genes of different biological approach, it causes cell RNA i effect widely and pest control more effectively.

According to the present invention, all double-stranded RNA zones comprise at least a portion nucleotide sequence complementary at least one chain with any target gene described herein.Yet, if double-stranded RNA zone comprises and the nucleotide sequence of arbitrary target gene described herein part complementary at least one chain, then another double-stranded RNA zone can comprise a part of complementary at least one chain with any other insect target gene (comprising known target gene).

According to another embodiment of the present invention, isolating double-stranded RNA as herein described or RNA construct are provided, it also comprises at least a other sequence and randomly comprises joint.In one embodiment, described another sequence is selected from: the sequence that (i) helps the described dsRNA construct of scale operation; (ii) improve or reduce the sequence of described dsRNA stability; Thereby (iii) allow conjugated protein or other molecule to help the sequence that insect absorbs described RNA construct; (iv) adaptive subsequence, it is in conjunction with acceptor on the insect surfaces or in the tenuigenin or molecule, thus the picked-up, endocytosis and/or the dysuria due to the pressure of the fetus that help insect gulp down; Perhaps (v) other sequence of catalysis dsRNA zone processing.In one embodiment, described joint is the conditionality self-cleaving RNA sequences, preferred pH susceptibility joint or hydrophobic susceptibility joint.In one embodiment, described joint is an intron.

In one embodiment, a plurality of dsRNA zone of described double-stranded RNA construct connects by one or more joints.In another embodiment, described joint is present in and makes the site of described dsRNA zone and another purpose region separation in the described RNA construct.The invention provides the dissimilar joints that are used for described dsRNA construct.

In another embodiment, a plurality of dsRNA zone of described double-stranded RNA construct is not used joint and is connected.

In a specific embodiments of the present invention, described joint can be used for separating a plurality of less dsRNA zones in the insect.Advantageously, described in this case joint sequence can impel the dsRNA with length to be split as less dsRNA zone under given conditions, this causes discharging under these conditions independent dsRNA zone, and these less dsRNA zones cause more effectively gene silencing.The autotomy example of cutover head of suitable conditionality is from the RNA sequence of cutting under high pH condition.The suitable example of these RNA sequences is by (Nucleic Acids Res.2003 May15 such as Borda; 31 (10): 2595-600) describe, the document is incorporated herein by reference.Described sequence source is from the catalytic core of hammerhead ribozyme HH16.

In another aspect of this invention, joint location makes the site that described dsRNA zone and for example another aim sequence are separated in described RNA construct, and described another aim sequence preferably provides certain extra function to described RNA construct.

In a specific embodiments of the present invention, help insect and absorb this dsRNA thereby dsRNA construct of the present invention comprises adaptive son.Design described adaptive son and make it material in conjunction with insect picked-up.Such material can derive from insect or plant.A specific examples of adaptive son is the adaptive son in conjunction with transmembrane protein (for example, the transmembrane protein of insect).Perhaps, described adaptive son can be in conjunction with (plant) metabolite or the nutrition of insect picked-up.

Perhaps, described joint carries out in endosome from cutting.This at construct of the present invention by endocytosis or transcytosis by the insect picked-up and may be favourable during by compartmentation in the endosome insect therefore.Described endosome can have low pH environment, causes described joint to be cut.

When by cell walls transhipment from a cell during to another cell transfer, for example when passing the cell walls of insect pest, above-mentioned in hydrophobic conditions the joint from cutting be particularly useful for dsRNA construct of the present invention.

Intron also can be used as joint." intron " used herein can be any non-coding RNA sequence of messenger RNA(mRNA).The suitable concrete intron sequences that is used for construct of the present invention is rich in U (35-45%) for (1); (2) mean length be 100bp (about 50 and about 500bp between change), can select its base pair or can be at random based on known intron sequences; (3) 5 ' end with-AG:GT-or-CG:GT-is initial, and/or (4) have at its 3 ' end-AG:GC-or-AG:AA.

About 1 base pair to the incomplementarity RNA sequence of about 10,000 base pairs also can be used as joint.

Do not wish to stick to any specific theory or machine-processed, think that insect is from the long double-stranded RNA of its contiguous environment picked-up.Double-stranded RNA is ingested and enters enteron aisle and be transferred to intestinal epithelial cells, is processed into short double-stranded RNA (being called siRNA (siRNA)) in the cell by acting on of endogenous nucleic acid restriction endonuclease then.Then, the siRNA of gained comes mediate rna i by forming multi-component RNA combined enzyme agent (being called RISC or RNA interference silencing complex).

Reduce target gene in the insect cell in order to be implemented in, thereby the double-stranded RNA that adds to the cell walls outside can be can be ingested to enter cell is processed into siRNA mediate rna i then in cell any dsRNA or dsRNA construct, thereby the RNA itself that perhaps adds to outside can be ingested the siRNA that enters cell guide RNA i.

The double-stranded RNA that siRNA is normally short, its length range is 19～25 base pairs, or 20～24 base pairs.In preferred embodiments, can use corresponding to the siRNA that waits to reduce target gene, it has 19,20,21,22,23,24 or 25 base pairs, especially 21 or 22 base pairs.Yet the present invention is not intended to the purposes that is limited to such siRNA.

SiRNA can comprise the strand overhang in the one or both ends of double-stranded part flank.In an especially preferred embodiment, described siRNA can comprise 3 ' outstanding Nucleotide, preferred two 3 ' outstanding thymidine (dTdT) or uridines (UU).If being right after the upstream target-gene sequence of described dsRNA sequence that double-stranded part comprises is AA, then in siRNA, can comprise 3 ' TT or UU overhang.This makes TT or UU overhang and target gene hybridization among the described siRNA.Although the other end of described siRNA also can comprise 3 ' TT or UU overhang, yet it is optional to comprise AA in the downstream target sequence of described siRNA sequence that double-stranded part comprises.In this article, in the siRNA of RNA/DNA mosaic form also is encompassed in.These mosaics comprise: for example, comprise and (for example have 3 ' DNA base overhang, the siRNA of double-stranded RNA dTdT) (as mentioned above), and wherein one or more RNA bases (or ribonucleotide) or even the whole piece chain on all ribonucleotides be replaced by the double-stranded RNA of the polynucleotide form of DNA base (or deoxyribonucleotide).

Described dsRNA can be by two independent (justice and antisense are arranged) RNA chain formation that are annealed to by (non-covalent) base pairing together.Perhaps, described dsRNA can have stem-ring or the hairpin structure that turns back, and two annealing chains of wherein said dsRNA are covalently bound.In this embodiment, the sense strand of described dsRNA and antisense strand are formed by the different zones of the single polynucleotide molecule of the self-complementary of part.If described dsRNA desire is synthesized by expression in vivo (for example in host cell or biology as described below) or by in-vitro transcription, the RNA that then has this structure is easily.Except the ability of the double-stranded part mediate rna i that should not destroy described molecule, the definite character and the sequence of " ring " that connects described two RNA chains is normally unessential for the present invention.The feature of " hair clip " or " stem-ring " RNA that is used for RNAi (referring to, CSIRO WO 99/53050 under one's name for example, its content by with reference to being incorporated herein) normally known in the art.In other embodiments of the present invention, described ring structure can comprise joint sequence or aforesaid other sequence.

Described double-stranded RNA or construct can be prepared by known mode itself.For example, can use chemistry well known in the art or enzymatic RNA synthetic technology at external synthetic double-stranded RNA.In a method, can synthesize two independent RNA chains respectively, thereby annealing forms then double-stranded.In another embodiment, can in host cell or biology, synthesize double-stranded RNA or construct by cell inner expression from suitable expression.Below this method will be discussed in further detail.

The amount of the described double-stranded RNA of insect contact wants to realize described one or more target genes of specificity downward modulation.Described RNA can send the amount of at least one copy to be introduced into each cell delivery.Yet in certain embodiments, the double-stranded RNA of higher dosage (for example, each cell at least 5,10,100,500 or 1000 copies) can more effectively be suppressed.For any given insect genes target, can determine that the best dsRNA that effectively suppresses measures by normal experiment.

Described insect can contact with described double-stranded RNA in any suitable mode that allows insect directly to absorb described double-stranded RNA.For example, described insect can contact the described double-stranded RNA of pure or substantially pure form, for example contains the aqueous solution of described dsRNA.In this embodiment, can only described insect " be soaked " with the aqueous solution that contains described double-stranded RNA.In another embodiment, thus can described insect be contacted with described double-stranded RNA by spraying to insect with the liquid composition that comprises described double-stranded RNA.

Perhaps, described double-stranded RNA can be connected with the food component (as the food component of Mammals pathogenicity bo insect) of described insect, thereby strengthens the picked-up of this insect to described dsRNA.

Described double-stranded RNA can also be mixed in the substratum of described insect growth,, perhaps immerse in the basic unit or article to the insect pest infestation sensitivity perhaps by (or on it) in the article of this insect infestation or the basic unit.

According to another embodiment, described dsRNA is expressed in the cell of bacterium or fungi, and the cell of described bacterium or fungi is absorbed by described insect or ingests.

As the embodiment illustrated, produce any dsRNA of the present invention or dsRNA construct thereby can transform bacterium.These bacteriums can be ingested by insect.In case be ingested, described dsRNA can reply by initial RNAi, and it causes said target mrna degraded and weakening or kills the insect that ingests.

Therefore, in a more particular embodiment, described double-stranded RNA or RNA construct are expressed by (as the bacterium) of protokaryon or (as the yeast) host cell or the host living beings of eucaryon.According to this embodiment, can use any bacterium or the yeast cell that to express dsRNA or dsRNA construct.Described bacterium is selected from gram negative bacterium and gram positive bacterium, such as but not limited to Escherichiaspp (Escherichia spp.) (for example, intestinal bacteria (E.coli), genus bacillus species (Bacillus spp.) (for example, bacillus thuringiensis (B.thuringiensis)), rhizobium species (Rhizobium spp.), lactobacillus species (Lactobacillus spp.), lactococcus species (Lactococcus spp.) etc.Described yeast can be selected from yeast belong species (Saccharomyces spp.) etc.

Some bacterium and host plant have interaction very closely, such as but not limited to symbiotic root nodule bacterium and leguminous plants (Legminosea) (for example, soybean).Such recombinant bacteria can mix (for example, as coating) with seed and be used as soil improvement agent.

Therefore, the present invention also comprises the cell that comprises any nucleotide sequence as herein described or recombinant DNA construction body.The present invention also comprises prokaryotic cell prokaryocyte (such as but not limited to Gram-positive and gram negative bacterium cell) and eukaryotic cell (such as but not limited to yeast cell or vegetable cell).Preferably, described cell is bacterial cell or yeast cell or alga cells.

In other embodiments, described insect can contact with the composition that this paper further describes.Except described dsRNA or DNA, described composition also can comprise vehicle, diluent or carrier.The preferred feature of such composition discusses in more detail following.

Perhaps, bacterium or the yeast cell that produces dsRNA directly can be sprayed onto on the crop.

Therefore, as mentioned above, the invention provides the host cell that comprises RNA construct of the present invention and/or DNA construct and/or expression construct.Preferably, described host cell is bacterium or zymic cell, also can be for example viral.Can use the virus (as baculovirus) of specific infection insect.This has guaranteed the security to Mammals (especially human), because described virus can not infect Mammals, so undesirable RNAi effect can not take place.

Preferably, described bacterial cell or yeast cell be as being inactivated before the biotic pesticide, for example when this reagent is used for environment (as the kitchen) that the mankind or other Mammals may contact.Can realize deactivation by any method, as by thermal treatment, phenol or formaldehyde treated, or pass through mechanical treatment.

In an alternative embodiment, can utilize the virus (as the baculovirus of suitable modification) of deactivation to send the RNAi that dsRNA of the present invention zone is used to mediate insect pest.

Possible application comprises concentrated greenhouse cultivation (for example from the less crop of GMO angle meaning) and field-crop (as soybean) widely.

This method has several advantages, and for example: because there is not the problem of possible plant host cutting (dicing), it allows big dsRNA fragment is sent the intestines inner chamber that enters the insect that ingests; Use bacterium not relate to and produce genetically modified crops, especially be difficult to obtain the crop of transgenosis variant for some as sterilant; Exist extensively and flexibly and use, because can on same farmland, handle different crops and/or the different insect of target simultaneously simultaneously, for example by making up the different bacterium that those produce different dsRNA.

Another aspect of the present invention is the target nucleotide sequences of insect target gene disclosed herein.Described target nucleotide sequences is for design dsRNA construct particularly important of the present invention.The length of these target nucleotide sequences is at least 17, preferably at least 18,19,20 or 21, more preferably at least 22,23 or 24 Nucleotide preferably.The limiting examples of preferred target nucleotide sequences provides in an embodiment.

According to an embodiment, the invention provides the isolating nucleotide sequence of coding double-stranded RNA as herein described or double-stranded RNA construct.

According to a more particular embodiment, the present invention relates to the isolated nucleic acid sequences formed by the arbitrary sequence of following expression: SEQ ID NO 49 to 158,275 to 472,533 to 575,621 to 767,813 to 862,908 to 1040,1161 to 1571,1730 to 2039,2120 to 2338,2384 to 2460, perhaps its at least 17, the fragment of preferred at least 18,19,20 or 21, more preferably at least 22,23 or 24 Nucleotide.

Those of skill in the art will recognize that the homologue that can find these target genes, and these homologues also are used for method of the present invention.

If protein or nucleotide sequence show sequence similarity of " significantly " level or sequence identity more preferably, then they may be homologous.Thereby real homologous sequence is by having dependency from the divergence of common ancestral gene.The sequence homology thing can have two types: (i) when homologue is present in the different plant species, they are called as lineal homologue (orthologue).For example, the α-Zhu Danbai gene of mouse and philtrum is lineal homologue.(ii) the collateral line homologue is the homologous gene a species inside, for example the α in the mouse-and the beta-globin gene be collateral line homologue (paralogue).

Preferred homologue is to comprise with the sequence that is selected from following expression to have at least about 85% or 87.5%, more preferably from about 90%, more preferably at least about 95% and most preferably at least about the gene of 99% conforming sequence: SEQ ID NO 1,3,5,7,9,11,13,15,17,19,21,23,49 to 158,159,160-163,168,173,178,183,188,193,198,203,208,215,220,225,230,247,249,251,253,255,257,259,275 to 472,473,478,483,488,493,498,503,513,515,517,519,521,533 to 575,576,581,586,591,596,601,603,605,607,609,621 to 767,768,773,778,783,788,793,795,797,799,801,813 to 862,863,868,873,878,883,888,890,892,894,896,908 to 1040,1041,1046,1051,1056,1061,1071,1073,1075,1077,1079,1081,1083,1085,1087,1089,1091,1093,1095,1097,1099,1101,1103,1105,1107,1109,1111,1113,1161 to 1571,1572,1577,1582,1587,1592,1597,1602,1607,1612,1617,1622,1627,1632,1637,1642,1647,1652,1657,1662,1667,1672,1677,1682,1684,1686,1688,1690,1692,1694,1696,1698,1700,1702,1704,1730 to 2039,2040,2045,2050,2055,2060,2065,2070,2075,2080,2085,2090,2095,2100,2102,2104,2106,2108,2120 to 2338,2339,2344,2349,2354,2359,2364,2366,2368,2370,2372,2384 to 2460,2461,2466,2471,2476 or 2481, or its complementary sequence.Be used for determining that the method for sequence identity is this area routine, it comprises use Blast software and EMBOSS software (TheEuropean Molecular Biology Open Software Suite (2000), Rice, P.Longden, I.and Bleasby, A.Trends in Genetics 16, (6) pp276-277).Term used herein " consistence " is meant the relation on nucleotide level between the sequence.Compare to determine " consistence per-cent " by the sequence (for example, two or more) to the optimum comparison in comparison window, the sequence part in the wherein said comparison window is compared to comprise with the reference sequences of optimal sequence comparison and is inserted or disappearance.Described reference sequences does not comprise insertion or disappearance.Described reference window is selected from least 10 continuous nucleotides to about 50, about 100 or to about 150 Nucleotide, preferred about 50～150 Nucleotide.Then, by measure between the sequence in the described window consistent Nucleotide number and with this number divided by the few nucleotide in the described window and multiply by 100 and calculate " consistence per-cent ".

Other homologues are the allelotrope that comprises the arbitrary sequence of following expression: SEQ ID NO 1,3,5,7,9,11,13,15,17,19,21,23,49 to 158,159,160-163,168,173,178,183,188,193,198,203,208,215,220,225,230,247,249,251,253,255,257,259,275 to 472,473,478,483,488,493,498,503,513,515,517,519,521,533 to 575,576,581,586,591,596,601,603,605,607,609,621 to 767,768,773,778,783,788,793,795,797,799,801,813 to 862,863,868,873,878,883,888,890,892,894,896,908 to 1040,1041,1046,1051,1056,1061,1071,1073,1075,1077,1079,1081,1083,1085,1087,1089,1091,1093,1095,1097,1099,1101,1103,1105,1107,1109,1111,1113,1161 to 1571,1572,1577,1582,1587,1592,1597,1602,1607,1612,1617,1622,1627,1632,1637,1642,1647,1652,1657,1662,1667,1672,1677,1682,1684,1686,1688,1690,1692,1694,1696,1698,1700,1702,1704,1730 to 2039,2040,2045,2050,2055,2060,2065,2070,2075,2080,2085,2090,2095,2100,2102,2104,2106,2108,2120 to 2338,2339,2344,2349,2354,2359,2364,2366,2368,2370,2372,2384 to 2460,2461,2466,2471,2476 or 2481.Other preferred homologues are to compare the gene that comprises at least a single nucleotide polymorphism (SNP) with the gene of the arbitrary sequence that comprises following expression: SEQ ID NO 1,3,5,7,9,11,13,15,17,19,21,23,49 to 158,159,160-163,168,173,178,183,188,193,198,203,208,215,220,225,230,247,249,251,253,255,257,259,275 to 472,473,478,483,488,493,498,503,513,515,517,519,521,533 to 575,576,581,586,591,596,601,603,605,607,609,621 to 767,768,773,778,783,788,793,795,797,799,801,813 to 862,863,868,873,878,883,888,890,892,894,896,908 to 1040,1041,1046,1051,1056,1061,1071,1073,1075,1077,1079,1081,1083,1085,1087,1089,1091,1093,1095,1097,1099,1101,1103,1105,1107,1109,1111,1113,1161 to 1571,1572,1577,1582,1587,1592,1597,1602,1607,1612,1617,1622,1627,1632,1637,1642,1647,1652,1657,1662,1667,1672,1677,1682,1684,1686,1688,1690,1692,1694,1696,1698,1700,1702,1704,1730 to 2039,2040,2045,2050,2055,2060,2065,2070,2075,2080,2085,2090,2095,2100,2102,2104,2106,2108,2120 to 2338,2339,2344,2349,2354,2359,2364,2366,2368,2370,2372,2384 to 2460,2461,2466,2471,2476 or 2481.

According to another embodiment, the present invention includes the target gene of the lineal homologue of insect of the gene of those any nucleotide sequences that comprise following expression: SEQ ID NO1,3,5,7,9,11,13,15,17,19,21,23,49 to 158,159,160-163,168,173,178,183,188,193,198,203,208,215,220,225,230,247,249,251,253,255,257,259,275 to 472,473,478,483,488,493,498,503,513,515,517,519,521,533 to 575,576,581,586,591,596,601,603,605,607,609,621 to 767,768,773,778,783,788,793,795,797,799,801,813 to 862,863,868,873,878,883,888,890,892,894,896,908 to 1040,1041,1046,1051,1056,1061,1071,1073,1075,1077,1079,1081,1083,1085,1087,1089,1091,1093,1095,1097,1099,1101,1103,1105,1107,1109,1111,1113,1161 to 1571,1572,1577,1582,1587,1592,1597,1602,1607,1612,1617,1622,1627,1632,1637,1642,1647,1652,1657,1662,1667,1672,1677,1682,1684,1686,1688,1690,1692,1694,1696,1698,1700,1702,1704,1730 to 2039,2040,2045,2050,2055,2060,2065,2070,2075,2080,2085,2090,2095,2100,2102,2104,2106,2108,2120 to 2338,2339,2344,2349,2354,2359,2364,2366,2368,2370,2372,2384 to 2460,2461,2466,2471,2476 or 2481.For instance, lineal homologue can comprise any nucleotide sequence of following expression: SEQ ID NO 49 to 123,275 to 434,533 to 562,621 to 738,813 to 852,908 to 1010,1161 to 1437,1730 to 1987,2120 to 2290 and 2384 to 2438, or the fragment of its at least 17,18,19,20,21,22,23,24,25,26 or 27 Nucleotide.Provided the non-limiting tabulation of lineal homologue gene of the segmental insect of 17bp at least that comprises one of sequence of the present invention or Arachnida or sequence in the table 4.

According to another embodiment, the present invention includes the target gene of the lineal homologue of nematode of the gene of those any nucleotide sequences that comprise following expression: 1,3,5,7,9,11,13,15,17,19,21,23,49 to 158,159,160-163,168,173,178,183,188,193,198,203,208,215,220,225,230,247,249,251,253,255,257,259,275 to 472,473,478,483,488,493,498,503,513,515,517,519,521,533 to 575,576,581,586,591,596,601,603,605,607,609,621 to 767,768,773,778,783,788,793,795,797,799,801,813 to 862,863,868,873,878,883,888,890,892,894,896,908 to 1040,1041,1046,1051,1056,1061,1071,1073,1075,1077,1079,1081,1083,1085,1087,1089,1091,1093,1095,1097,1099,1101,1103,1105,1107,1109,1111,1113,1161 to 1571,1572,1577,1582,1587,1592,1597,1602,1607,1612,1617,1622,1627,1632,1637,1642,1647,1652,1657,1662,1667,1672,1677,1682,1684,1686,1688,1690,1692,1694,1696,1698,1700,1702,1704,1730 to 2039,2040,2045,2050,2055,2060,2065,2070,2075,2080,2085,2090,2095,2100,2102,2104,2106,2108,2120 to 2338,2339,2344,2349,2354,2359,2364,2366,2368,2370,2372,2384 to 2460,2461,2466,2471,2476 or 248.For instance, the lineal homologue of nematode can comprise any nucleotide sequence of following expression: SEQ ID NO 124 to 135,435 to 446,563 to 564,739 to 751,853,854,1011 to 1025,1438 to 1473,1988 to 2001,2291 to 2298,2439 or 2440, the perhaps fragment of its at least 17,18,19,20 or 21 Nucleotide.According on the other hand, therefore the present invention comprises any method that nematode grows at biology that is used for controlling as herein described, perhaps be used to prevent any method of nematode infringement to the biology of nematode infections sensitivity, described method comprises elegans cell is contacted with double-stranded RNA, wherein said double-stranded RNA comprises the annealed complementary strand, wherein annealing complementary strand comprises at least a portion complementary nucleotide sequence with the nucleotide sequence of target gene, its comprise following expression arbitrary sequence at least 17,18,19, the fragment of 20 or 21 Nucleotide: SEQ ID NO 124 to 135,435 to 446,563 to 564,739 to 751,853,854,1011 to 1025,1438 to 1473,1988 to 2001,2291 to 2298,2439 or 2440, described double-stranded RNA is by nematode picked-up and therefore control growing or prevent infringement.Provided the non-limiting tabulation of lineal homologue gene of the segmental nematode of the 17bp at least that comprises one of sequence of the present invention or sequence in the table 5.

According to another embodiment, the present invention includes the target gene of the lineal homologue of fungi of the gene of those any nucleotide sequences that comprise following expression: 1,3,5,7,9,11,13,15,17,19,21,23,49 to 158,159,160-163,168,173,178,183,188,193,198,203,208,215,220,225,230,247,249,251,253,255,257,259,275 to 472,473,478,483,488,493,498,503,513,515,517,519,521,533 to 575,576,581,586,591,596,601,603,605,607,609,621 to 767,768,773,778,783,788,793,795,797,799,801,813 to 862,863,868,873,878,883,888,890,892,894,896,908 to 1040,1041,1046,1051,1056,1061,1071,1073,1075,1077,1079,1081,1083,1085,1087,1089,1091,1093,1095,1097,1099,1101,1103,1105,1107,1109,1111,1113,1161 to 1571,1572,1577,1582,1587,1592,1597,1602,1607,1612,1617,1622,1627,1632,1637,1642,1647,1652,1657,1662,1667,1672,1677,1682,1684,1686,1688,1690,1692,1694,1696,1698,1700,1702,1704,1730 to 2039,2040,2045,2050,2055,2060,2065,2070,2075,2080,2085,2090,2095,2100,2102,2104,2106,2108,2120 to 2338,2339,2344,2349,2354,2359,2364,2366,2368,2370,2372,2384 to 2460,2461,2466,2471,2476 or 2481.For instance, the lineal homologue of fungi can comprise any nucleotide sequence of following expression: SEQID NO 136 to 158,447 to 472,565 to 575,752 to 767,855 to 862,1026 to 1040,1475 to 1571,2002 to 2039,2299 to 2338,2441 to 2460, perhaps the fragment of its at least 17,18,19,20,21,22,23,24,25,26 or 27 Nucleotide.According on the other hand, therefore the present invention comprises the growth of fungi on cell or biology that be used to control as herein described, perhaps be used to prevent cell or the biological any method of fungi infringement to the fungi infestation sensitivity, this method comprises the fungal cell is contacted with double-stranded RNA, wherein said double-stranded RNA comprises the annealed complementary strand, wherein annealing complementary strand comprises at least a portion complementary nucleotide sequence with the nucleotide sequence of target gene, its comprise following expression arbitrary sequence at least 17,18,19, the fragment of 20 or 21 Nucleotide: SEQ ID NO 136 to 158,447 to 472,565 to 575,752 to 767,855 to 862,1026 to 1040,1475 to 1571,2002 to 2039,2299 to 2338,2441 to 2460, described double-stranded RNA is by fungi picked-up and therefore control growing or prevent infringement.Provided the non-limiting tabulation of lineal homologue gene of the segmental fungi of the 17bp at least that comprises one of sequence of the present invention or sequence in the table 6.

Term " adjusting sequence " relates to the scope of broad, and it refers to realize the modulability nucleic acid with the expression of its sequence that effectively is connected.

Above-mentioned term comprises promotor and nucleic acid or its synthetic fusion molecule or the derivative that activates or strengthen expression of nucleic acid, is called to activate son or enhanser.Term used herein " effectively connect " is meant functional connection the between " promotor " sequence and purpose nucleic acid molecule, make described " promotor " thus sequence can the initial nucleic acid molecule transcribe and produce suitable dsRNA.

Preferred adjusting sequence is a promotor, and it can be composing type or inducible promoter.Preferred promotor is an inducible promoter, thus the expression of the described RNA molecule of strict control.Preferably can come the inductive promotor by using suitable chemical reagent (as IPTG).Perhaps, the transgenosis with the described RNA molecule of coding places under the control of strong constitutive promoter.Preferably, employed any promotor will instruct described RNA strong expression.The character of the promotor of using can be partly decided by the particular host cell that is used to produce described RNA.In one embodiment, described adjusting sequence comprises phage promoter, as T7, T3, SV40 or SP6 promotor, most preferably T7 promotor.In other embodiments of the present invention, be used for other promotors of expressed rna, it includes but not limited to the promotor from rna plymerase i, rna plymerase ii or rna plymerase iii.Also can use other promotor that derives from yeast or virogene as one sees fit.

In an alternative embodiment, described adjusting sequence comprises promotor, and this promotor is selected from known tac, trc and lac promotor.The inducible promoter that is applicable to host bacterium comprises β-Nei Xiananmei promotor, intestinal bacteria lambda particles phage PL and PR promotor and intestinal bacteria semi-lactosi promotor, pectinose promotor and alkaline phosphatase promoter.Therefore, the present invention also comprises the method that is used to obtain any RNA molecule of the present invention or RNA construct.This method may further comprise the steps: allow described nucleic acid or reorganization (DNA) thus construct transcribe produce downward modulation purpose target gene RNA (when described host cell is ingested by the target biology, perhaps when host cell or from the extract in its source during by the target biological uptake) condition under, isolating nucleic acid of the present invention or reorganization (DNA) construct are introduced in (for example, by conversion, transfection or injection) host cell of the present invention.

Randomly, also one or more transcription termination sequences or " terminator " can be introduced in the recombinant precursor of the present invention.Term " transcription termination sequence " comprises the control sequence of transcriptional units end, and it sends the 3 ' processing of primary transcript and the signal of polyadenylation and Transcription Termination.Described transcription termination sequence is used to prevent that the company that transcribes from reading, thereby described RNA molecule correctly produces in host cell or by host cell.In one embodiment, described terminator comprises T7, T3, SV40 or SP6 terminator, preferred T7 terminator.Also can use other terminator that derives from yeast or virogene as one sees fit.

Can be with in the expression construct as described in other regulatory element (as the enhanser of transcribing or the translating) introducing.

Recombinant precursor of the present invention also can be included in keeps and/or duplicates required replication orgin in the particular cell types.Example is the situation that expression construct need be kept in bacterial cell as the additive type genetic elements in the cell (for example, plasmid or clay molecule).Preferred replication orgin includes but not limited to f1-ori and colE1 ori.

Described recombinant precursor randomly can comprise the selected marker.Term used herein " selected marker " comprises any gene of giving a kind of phenotype of cell, thereby this gene is expressed in described cell and helped identifying and/or select with reorganization of the present invention (expression) construct transfection or cell transformed.The example of suitable selectivity mark comprises the gene of the gene (Ampr) of anti-penbritin, the gene (Tcr) of tetracycline resistance, the gene (Kanr) of anti-kantlex, anti-phosphinothricin and the gene (CAT) of chloramphenicol resistance.Other suitable marker gene provide metabolism proterties, for example manA.Also can use the visible marker gene, it comprises for example beta-Glucuronidase (GUS), luciferase and green fluorescent protein (GFP).

In other embodiments of the present invention, use to can be used for expressing other promotors of dsRNA, it includes but not limited to the promotor from rna plymerase i, rna plymerase ii, rna plymerase iii, T7 RNA polymerase or SP6RNA polysaccharase.These promotors are generally used for external generation dsRNA, described dsRNA are included in the sterilant (antiinsecticidalagent), for example in fluid insecticidal, sprays or powder then.

Therefore, the present invention also comprises the method that is used to produce any double-stranded RNA of the present invention or RNA construct.This method may further comprise the steps:

A. isolating nucleic acid of the present invention or recombinant DNA construction body are contacted with acellular component; Perhaps

B. isolating nucleic acid of the present invention or recombinant DNA construction body are introduced (for example, by conversion, transfection or injection) cell,

Thereby more than carry out allowing described nucleic acid or recombinant DNA construction body to transcribe under the condition that produces described dsRNA or RNA construct.

Randomly, can also introduce one or more transcription termination sequences in the recombinant precursor of the present invention.Term " transcription termination sequence " comprises the control sequence of transcriptional units end, and it sends the 3 ' processing of primary transcript and the signal of polyadenylation and Transcription Termination.Expression construct as described in other regulatory elements (as the enhanser of transcribing or translating) can being introduced.

Recombinant precursor of the present invention also can be included in keeps and/or duplicates required replication orgin in the particular cell types.An example is that expression construct need be kept in bacterial cell as the additive type genetic elements in the cell (for example, plasmid or clay molecule).Preferred replication orgin includes but not limited to f1-ori and colE1 ori.

Described recombinant precursor randomly can comprise the selected marker.Term used herein " selected marker " comprises gives any gene of cell with phenotype, thereby this gene is expressed in described cell and helped identifying and/or select through reorganization of the present invention (expression) construct transfection or cell transformed.The example of suitable selectivity mark comprises the gene of the gene (Ampr) of anti-penbritin, the gene (Tcr) of tetracycline resistance, the gene (Kanr) of anti-kantlex, anti-phosphinothricin and the gene (CAT) of chloramphenicol resistance.Other suitable marker gene provide metabolism proterties, for example manA.Also can use the visible marker gene, it comprises for example beta-Glucuronidase (GUS), luciferase and green fluorescent protein (GFP).

The present invention relates to be used to the method that stops insect on plant, to grow or be used to prevent the insect pest infestation plant.The pending plant of the method according to this invention comprises and is selected from following plant: clover, apple, apricot, choke, asparagus, avocado, banana, barley, beans, beet, blackberry, blueberry, blueberry, sprouting broccoli, brussels sprouts, Caulis et Folium Brassicae capitatae, castor-oil plant, Radix Dauci Sativae, cassava, Cauliflower, cereal, celery, cherry, citrus, the little oranges and tangerines of Ke Laimenshi (clemintine), coffee, corn, cotton, cucumber, eggplant, witloof, eucalyptus, Fructus Fici, grape, natsudaidai, Semen arachidis hypogaeae, gooseberry, Kiwifruit, lettuce, fragrant-flowered garlic, lemon, the lime bitter orange, pine tree, corn, mango, melon, millet, mushroom, oat (nutaot), gumbo, onion, orange, ornamental plant or flower or tree, papaya, parsley, pea, peach, peanut, pea (peat), pepper, persimmon, pineapple, psyllium, plum, pomegranate, potato, pumpkin, witloof, radish, Semen Brassicae campestris, immature fruit of Juteleaf Raspberry, rice, rye, Chinese sorghum, soya bean (soy), soybean (soybean), spinach, strawberry, sugar beet, sugarcane, Sunflower Receptacle, Ipomoea batatas, orange, tea, tobacco, tomato, vine, watermelon, wheat, Chinese yam or summer squash; Preferred potato, eggplant, tomato, pepper, tobacco, gooseberry, rice or vegetable lamb), perhaps seed or stem tuber (for example, clover, apple, apricot, choke, asparagus, avocado, banana, barley, beans, beet, blackberry, blueberry, blueberry, sprouting broccoli, brussels sprouts, Caulis et Folium Brassicae capitatae, castor-oil plant, Radix Dauci Sativae, cassava, Cauliflower, cereal, celery, cherry, citrus, the little oranges and tangerines of Ke Laimenshi, coffee, corn, cotton, cucumber, eggplant, witloof, eucalyptus, Fructus Fici, grape, natsudaidai, Semen arachidis hypogaeae, gooseberry, Kiwifruit, lettuce, fragrant-flowered garlic, lemon, the lime bitter orange, pine tree, corn, mango, melon, millet, mushroom, oat (nut aot), gumbo, orange, ornamental plant or flower or tree, papaya, parsley, pea, peach, peanut, pea (peat), pepper, persimmon, pineapple, psyllium, plum, pomegranate, potato, pumpkin, witloof, radish, Semen Brassicae campestris, immature fruit of Juteleaf Raspberry, rice, rye, Chinese sorghum, soybean, soybean, spinach, strawberry, sugar beet, sugarcane, Sunflower Receptacle, Ipomoea batatas, orange, tea, tobacco, tomato, vine, watermelon, wheat, Chinese yam or summer squash.

The amount of described target biological uptake (preferably ingesting) targeted rna will realize that specificity reduces one or more target genes.Described RNA can send the amount of at least one copy to express in host cell with each cell delivery.Yet in certain embodiments, the double-stranded RNA of higher dosage (for example, at least 5,10,100,500 or 1000 copies of each target biomass cells) can obtain more effective inhibition.For any given target gene and target biology, can determine the effectively optimum quantity of the target RNA molecule of inhibition by normal experiment.

Described target biology can contact with the host cell of expressing described RNA molecule in any suitable manner, thereby allows the target biology to ingest.Preferably, the host cell of expressing described dsRNA can be connected with the food component of described target biology, thereby strengthen this target biology described dsRNA is absorbed.The host cell of expressing described dsRNA can also be introduced in the substratum of described target biological growth,, perhaps be immersed in the biological infringement of the insect responsive basic unit or article perhaps by in biological article of invading and harassing of insect or the basic unit (or on it).

In alternative embodiment, can use the suitable extract that is derived from the host cell of expressing described RNA molecule to realize reducing target gene in the target biology.At this moment, described extract can produce by any appropriate means that the host cell of described RNA molecule is expressed in cracking.For example, can use the technology of handling as supersound process, Fu Shi crushing (French press), freeze thawing and N,O-Diacetylmuramidase (referring to the reference that provides among Sambrook and Russell-Molecular Cloning A laboratory manual-third edition and the table 15-4) to prepare host cell crude extract (lysate).Can be further purified this extract as one sees fit, as long as the ability of the target downward modulation of described extract mediation expression of target gene does not affect adversely.For example can use affinity purification.Add some composition to prevent that the RNA molecular degradation from may also be suitable to described extract.For example, can add the RNA enzyme inhibitors to the extract of the host cell that is derived from expressed rna.In an example, described target biology can contact the host cell of the expressed rna of pure or substantially pure form, for example contains the aqueous solution of described cell extract.In this embodiment, can be simply with the aqueous solution that contains described host cell extract with described target biology (especially insect biology, as insect) " soaking ".In another embodiment, thus can be by making to biological sprinkling of described target that described target is biological to be contacted with the host cell of described expressed rna molecule with the liquid composition that comprises described cell extract.

Come specificity to control growth or the infringement of specific insect if use method of the present invention, then the RNA that expresses in the preferred host cell does not have any significant homology with the gene of non-insect biology, does not especially have any significant homology with any indispensable gene of non-insect biology.Therefore, described non-insect biology is normally to the responsive biology of insect infringement, and the method according to this invention protects described non-insect biology to avoid described insect infringement for this reason.Therefore, for example, non-pest species can comprise plant or mammalian species.Preferably, described mammalian species is human.Described non-target species also can comprise the animal except the people, and it can be exposed to described biology or the basic unit that is protected from infringement.Example comprises birds (it can be a food with protected plant) and livestock and domestic animal (as cat, dog, horse, ox, chicken, pig, sheep etc.).In this case, the consensus nucleic acid sequence of arbitrary gene of preferred described dsRNA and responsive biology or non-target biology is lower than 30%, more preferably less than 20%, and more preferably less than 10%, even more preferably less than 5%.Sequence identity per-cent should calculate on the total length zone of described target RNA.If can obtain the genomic sequence data of the present invention's biology to be protected or arbitrary non-target biology, then can use standard biological information science instrument to come the sequence identity of mutual verification and described target RNA.In one embodiment, there are not 21 sequence identities on the continuous nucleotide between the gene of described RNA molecule and non-insect biology, promptly in this case, do not have 21 continuous nucleotides of described RNA in the genome of preferred described non-insect biology.In another embodiment, the sequence identity on 24 continuous nucleotides is lower than about 10% or be lower than about 12.5% between arbitrary nucleotide sequence of described RNA and non-insect (responsive) species.Especially, can pay special attention to the orthologous gene of non-pest species, because the indispensable gene of described insect biology often can become the target of method of the present invention.Therefore, in one embodiment, described RNA molecule has with the corresponding nucleotide sequence of non-pest species orthologous gene and is lower than 12.5% sequence identity.

In another embodiment, the present invention relates to be used to the composition controlling insect growth and/or prevent or reduce insect pest infestation, said composition comprises at least a double-stranded RNA, wherein said double-stranded RNA comprises the annealed complementary strand, and wherein annealing complementary strand comprises at least a portion complementary nucleotide sequence with the nucleotide sequence of insect target gene.The invention still further relates to the composition that comprises at least a nucleotide sequence as herein described or at least a recombinant DNA construction body.The invention still further relates to the composition that comprises at least a bacterial cell or yeast cell, described bacterial cell or yeast cell to express at least a double-stranded RNA as herein described or double-stranded RNA construct are perhaps expressed at least a nucleotide sequence as herein described or recombinant DNA construction body.Randomly, described composition also comprises at least a appropriate carriers, vehicle or thinner.Described target gene can be arbitrary target gene as herein described.Preferably, described insect target gene is that insect survives, grows, grows or breed necessary.

On the other hand, the present invention relates to aforesaid composition, wherein said insect target gene comprise be selected from following expression arbitrary sequence have at least 75%, preferably at least 80%, 85%, 90%, more preferably at least 95%, 98% or 99% conforming sequence: SEQ ID NO 1,3,5,7,9,11,13,15,17,19,21,23,49 to 158,159,160-163,168,173,178,183,188,193,198,203,208,215,220,225,230,240 to 247,249,251,253,255,257,259,275 to 472,473,478,483,488,493,498,503,508 to 513,515,517,519,521,533 to 575,576,581,586,591,596,601,603,605,607,609,621 to 767,768,773,778,783,788,793,795,797,799,801,813 to 862,863,868,873,878,883,888,890,892,894,896,908 to 1040,1041,1046,1051,1056,1061,1066 to 1071,1073,1075,1077,1079,1081,1083,1085,1087,1089,1091,1093,1095,1097,1099,1101,1103,1105,1107,1109,1111,1113,1161 to 1571,1572,1577,1582,1587,1592,1597,1602,1607,1612,1617,1622,1627,1632,1637,1642,1647,1652,1657,1662,1667,1672,1677,1682,1684,1686,1688,1690,1692,1694,1696,1698,1700,1702,1704,1730 to 2039,2040,2045,2050,2055,2060,2065,2070,2075,2080,2085,2090,2095,2100,2102,2104,2106,2108,2120 to 2338,2339,2344,2349,2354,2359,2364,2366,2368,2370,2372,2384 to 2460,2461,2466,2471,2476,2481 or 2486

Or its complementary sequence, perhaps wherein said insect target gene is the lineal homologue of insect of gene of at least 17 continuous nucleotides that comprises the arbitrary sequence of following expression: SEQ ID NO1,3,5,7,9,11,13,15,17,19,21,23,49 to 158,159,160-163,168,173,178,183,188,193,198,203,208,215,220,225,230,240 to 247,249,251,253,255,257,259,275 to 472,473,478,483,488,493,498,503,508 to 513,515,517,519,521,533 to 575,576,581,586,591,596,601,603,605,607,609,621 to 767,768,773,778,783,788,793,795,797,799,801,813 to 862,863,868,873,878,883,888,890,892,894,896,908 to 1040,1041,1046,1051,1056,1061,1066 to 1071,1073,1075,1077,1079,1081,1083,1085,1087,1089,1091,1093,1095,1097,1099,1101,1103,1105,1107,1109,1111,1113,1161 to 1571,1572,1577,1582,1587,1592,1597,1602,1607,1612,1617,1622,1627,1632,1637,1642,1647,1652,1657,1662,1667,1672,1677,1682,1684,1686,1688,1690,1692,1694,1696,1698,1700,1702,1704,1730 to 2039,2040,2045,2050,2055,2060,2065,2070,2075,2080,2085,2090,2095,2100,2102,2104,2106,2108,2120 to 2338,2339,2344,2349,2354,2359,2364,2366,2368,2370,2372,2384 to 2460,2461,2466,2471,2476,2481 or 2486, or its complementary sequence.

The invention still further relates to composition, its host cell that comprises at least a double-stranded RNA, at least a double-stranded RNA construct, at least a nucleotide sequence, at least a recombinant DNA construction body and/or at least a expression dsRNA of the present invention (for example, bacterial cell or yeast), perhaps the encode virus of dsRNA of the present invention randomly also comprises at least a appropriate carriers, vehicle or thinner.

Described composition can adopt any suitable physical form of using to insect.For example, described composition can adopt solid form (as pulvis, pill or bait formulation), liquid form (as, sprays) or gel form.

According to highly preferred embodiment, described composition adopts the form that insect ingests that is suitable for.

Described composition can comprise other component, and described component acts on to be stablized described dsRNA and/or prevent the dsRNA degraded during the composition standing storage.

Described composition also can comprise enhancing or promote insect to absorb the component of described dsRNA.These can comprise the chemical reagent that for example promotes RNA picked-up to enter cell usually, for example lipofectamin (fat transfection amine) etc.

Described composition also can be included in the component that keeps described host cell viability during the standing storage.

Described composition can take to insect, basic unit, cell (for example, vegetable cell) or by insect infection or to any suitable physical form of the biologic applications of insect pest infestation sensitivity.

In one embodiment, described composition can adopt the form of sprays.Therefore, the user can directly spray insect or basic unit with said composition.

Therefore, the present invention relates to sprays, it comprises the composition that contains at least a bacterial cell or yeast cell, described bacterial cell or yeast cell to express at least a double-stranded RNA as herein described or double-stranded RNA construct, or express at least a nucleotide sequence as herein described or recombinant DNA construction body.More particularly, the present invention relates to sprays as defined above, wherein said bacterial cell comprises at least a sequence of the arbitrary sequence of following expression: SEQ ID NO 1,3,5,7,9,11,13,15,17,19,21,23,49 to 158,159,160-163,168,173,178,183,188,193,198,203,208,215,220,225,230,240 to 247,249,251,253,255,257,259,275 to 472,473,478,483,488,493,498,503,508 to 513,515,517,519,521,533 to 575,576,581,586,591,596,601,603,605,607,609,621 to 767,768,773,778,783,788,793,795,797,799,801,813 to 862,863,868,873,878,883,888,890,892,894,896,908 to 1040,1041,1046,1051,1056,1061,1066 to 1071,1073,1075,1077,1079,1081,1083,1085,1087,1089,1091,1093,1095,1097,1099,1101,1103,1105,1107,1109,1111,1113,1161 to 1571,1572,1577,1582,1587,1592,1597,1602,1607,1612,1617,1622,1627,1632,1637,1642,1647,1652,1657,1662,1667,1672,1677,1682,1684,1686,1688,1690,1692,1694,1696,1698,1700,1702,1704,1730 to 2039,2040,2045,2050,2055,2060,2065,2070,2075,2080,2085,2090,2095,2100,2102,2104,2106,2108,2120 to 2338,2339,2344,2349,2354,2359,2364,2366,2368,2370,2372,2384 to 2460,2461,2466,2471,2476,2481 or 2486, or the fragment of its at least 17 continuous nucleotides.Preferably, described sprays comprises the arbitrary sequence of at least a following expression: SEQ ID NO 1,3,5,7,9,11,13,15,17,19,21,23,49 to 158,159,160-163,168,173,178,183,188,193,198,203,208,215,220,225,230,240 to 247,249,251,253,255,257,259,275 to 472,473,478,483,488,493,498,503,508 to 513,515,517,519,521,533 to 575,576,581,586,591,596,601,603,605,607,609,621 to 767,768,773,778,783,788,793,795,797,799,801,813 to 862,863,868,873,878,883,888,890,892,894,896,908 to 1040,1041,1046,1051,1056,1061,1066 to 1071,1073,1075,1077,1079,1081,1083,1085,1087,1089,1091,1093,1095,1097,1099,1101,1103,1105,1107,1109,1111,1113,1161 to 1571,1572,1577,1582,1587,1592,1597,1602,1607,1612,1617,1622,1627,1632,1637,1642,1647,1652,1657,1662,1667,1672,1677,1682,1684,1686,1688,1690,1692,1694,1696,1698,1700,1702,1704,1730 to 2039,2040,2045,2050,2055,2060,2065,2070,2075,2080,2085,2090,2095,2100,2102,2104,2106,2108,2120 to 2338,2339,2344,2349,2354,2359,2364,2366,2368,2370,2372,2384 to 2460,2461,2466,2471,2476,2481 or 2486, or the fragment of its at least 17 continuous nucleotides.

The invention still further relates to sprays, it comprises at least a composition as herein described or comprises at least a host cell, also comprises at least a adjuvant and randomly at least a tensio-active agent.

The validity of sterilant can be depending on described sprays effectiveness of application.Adjuvant can reduce or eliminate many problems that sprays is used, and described problem is relevant with coverage with stability, solubleness, uncompatibility, suspension, foam formation, drift, evaporation, volatilization, degraded, adhesion, infiltration, the surface tension of sterilant.Adjuvant is designed to realize specific function, comprise wetting, disperse (spread), adhesion, reduce evaporation, reduce volatilization, buffering, emulsification, distribution (disperse), reduce the sprays drift and reduce foam and form.Do not have a kind of adjuvant can realize all these functions, realize multiple function simultaneously yet usually different consistency adjuvants can be made up.These chemical (also being called wetting agent and dispersion agent) are from physically having changed the surface tension of sprays drop.In order to make sterilant correctly bring into play its function, the sprays drop must be able to make leaf wetting and evenly scatter on leaf.Tensio-active agent has enlarged the scope that sterilant covers, thereby has increased the chance of insect contact chemical reagent.When sterilant was put on the leaf of wax or tool hair, tensio-active agent was particularly important.Do not carry out suitable wetting and dispersion, then the sprays drop usually runs off or can not cover these surfaces fully.Yet too much tensio-active agent can cause over-drastic to run off or the precipitation loss, has therefore reduced the effect of sterilant.Insecticidal formulation often comprises tensio-active agent to improve the suspension of this pesticide active ingredient.This is particularly useful for emulsifiable concentrate (EC) prescription.

Term used herein " adjuvant " refers to be added into pesticide product or mixing and any non-biocide materials stable and that use of insecticidal fogging agent composition to improve product in the spray tank.The term " tensio-active agent " that this paper uses in addition refers to that change shows capillary chemical reagent.Tensio-active agent can influence the wetting of liquid and disperse, and can change dispersion, suspension or the precipitation of sterilant in water.There are nonionic surface active agent (neutral), anion surfactant (negative charge) and cats product (positive charge).

In special embodiment, the described host cell that comprises in the described sprays is deactivation, for example realizes by hot deactivation or physical disturbance (going through ground as this paper).

The character of described vehicle and the physical form of described composition can change according to the character of the basic unit of expecting to handle.For example, described composition can be brushed to or be sprayed in pending article or the basic unit or be stamped into pending article or the liquid in the basic unit, or be coating or the powder that is applied to pending article or basic unit.Therefore, in one embodiment, described composition adopts the form of coating on suitable surface, and the insect that its adhesion insect also finally is touched this coating absorbs.

According to an embodiment preferred, described basic unit is intended to the plant or the crop of handling at insect pest infringement.Then, described composition is by the insect internalization or ingest, and said composition can be disturbed by mediate rna therein, thus the control insect.Described sprays is preferably the sprays or the pump formula sprays of pressurization/mist.Described particle can be taked suitable size, thereby it adheres to pending basic unit or adheres to insect, for example adheres to the exoskeleton of insect and/or arachnid, and can be absorbed from here.

In one embodiment, described composition adopts the form of bait formulation.Designing described bait formulation makes it to lure insect to contact with described composition.In case the contact after, described composition by insect internalization (for example by ingesting) thus and mediate rna i kill insects.Described bait formulation can comprise food, as based on proteinic food, for example fish meal.Also can use boric acid as bait formulation.Described bait formulation can be depending on the species as target.Can also use attractive substance.Described attractive substance can be a pheromone, for example male or female pheromone.As an example, the present invention can use " insect pheromone and the application in Pest Management thereof (Insect Pheremones and their use in Pest Management) " (Howse et al, Chapman and Hall, 1998) pheromone of mentioning in.Described attractive substance to described bait formulation, and can maybe can be lured all insects by the specific insect of target with insect attractant.Described bait formulation can adopt any suitable form, as solid, paste, pill or powder form.

Described bait formulation also can be carried go back to colony by insect.Then, this bait formulation can be used as the food source of other member in the colony, thereby to a large amount of insects and may be to the achieve effective control of whole insect pest colony.The bacterium of using double-stranded RNA or expressing dsRNA of the present invention is favourable, because the delayed action to the insect influence of RNAi mediation makes bait formulation be carried go back to colony, thereby has realized at the maximum effect that contacts aspect the insect.

In addition, the composition that contacts with described insect can remain on the stratum corneum of this insect.When cleaning (insect cleaning himself or insect clean mutually), described composition can be ingested, and can therefore mediate its effect in insect.This needs described composition enough stable, thereby even after being exposed to external environmental condition for some time (for example, can be several days), the host cell of described dsRNA or expression dsRNA still remains intact and can mediate rna i.

Described bait formulation can provide with suitable " outer cover " or " trap ".Such outer cover and trap are commercially available, and existing trap can be suitable for comprising composition of the present invention.Scope of the present invention comprises any outer cover or the trap that can attract insect to enter.Described outer cover or trap can for example be box-likes, and can for example provide with the premolding state or can be formed by collapsible cardboard.The suitable material that is used for outer cover or trap comprises plastics and cardboard, especially corrugated cardboard (corrugatedcardboard).The such outer cover or the suitable dimension of trap are loose for for example 7-15cm, and 15-20cm is long and 1-5cm is high.The internal surface of described trap can cover viscous substance, in case make insect enter this trap then be limited to move.Described outer cover or trap can comprise suitable groove, and the inside of described groove can be equipped with described bait formulation in position.It is because insect can not catch after entering easily that trap is different from outer cover, and outer cover conduct " place of feeding ", it provides preferable environment to the insect arachnid, and they can take food and feel the sense of security away from the predator therein.

Therefore, another aspect of the present invention provides at outer cover of insect or trap, and it comprises composition of the present invention, and said composition can merge any feature of composition described herein.

" composition " of the present invention can be " composite reagent box (kit-of-parts) ", and it comprises suitable thinner, vehicle or carrier at the entity that contains RNA (as dsRNA or dsRNA construct, DNA construct, expression construct) in described double-stranded RNA in the container and the independent container; Perhaps comprise one in the container host cell and suitable thinner, vehicle, carrier or the sanitas in independent container at this host cell.The invention still further relates to the double-stranded RNA or the host cell that provide independent, and without any other component.In these embodiments, described dsRNA or host cell can provide with conc forms, as the spissated aqueous solution.It in addition can provide with frozen form or with lyophilized form.The latter may be more stable for long-term preservation, and can be thawed before facing use and/or restore with the suitable dilution agent.

The present invention also is included in the growth of Pest Control biology in the basic unit and/or prevents responsive biological method of being encroached on by the insect biology, and it comprises any composition from significant quantity as herein described to described basic unit and/or the sprays of using.

The present invention also comprises the disease that is used for the treatment of and/or prevents to be caused by the target biology or the method for illness, it comprises to there being this object that treats and/or prevents needs to use composition as herein described or sprays, and wherein the target gene expression downward modulation is effective for treating and/or preventing the disease that is caused by the target biology in the described target biology that is caused by described composition or sprays.Preferred target biology is an insect, especially the insect of this paper detailed description.

The invention still further relates to the medical usage of any double-stranded RNA as herein described, double-stranded RNA construct, nucleotide sequence, recombinant DNA construction body or composition.

Insect and other arthropodss can bite or terebra causes damage even dead by it.In the U.S., annual because honeybee and wasp sting and dead people are than by snakebite and the people of death is more.Many insects can be propagated bacterium and other pathogenic agent that causes disease.During major war between each country, because insect-borne disease and the injured or people that dies are than owing to bullet and bomb and people injured or that die is more.The insect that bites people and domestic animal is to have as Hemiptera and some mostly

The insect of the piercing-sucking mouthparts seen in the homopterous insect.Biting the main discomfort that causes is by due to the enzyme of insect injection object.Tick and trombiculid are different types of mite (Arachnidas), and it is food with the animal blood.Tick can also be propagated virus and other pathogenic agent that cause disease, and described disease comprises Lyme disease and rocky mountain spotted fever.The mite of other kinds can cause the mange of people, dog, cat and other animal.Hemiptera comprises bed bug, kiss worm (kissing bug) and hunts stinkbug (assassin bug) that all these insects have the beak that is used to thrust its host.Biting of pain is to hunt biting of stinkbug in all insects.Sino-U.S. state and South America, the kiss worm participates in causing chagas disease.The caterpillar of some moth can " terebra ".Diptera is the human most important insect order of influence.Bite winged insect (biting flies) and comprise multiple mosquito, buffalo gnat, biting gnats, horsefly etc.These are bitten many persons that are the transmission of disease in the winged insect, as propagating the tsetse fly of nelavan.Fly (as housefly) with licking mouthparts is also propagated bacterium and other pathogenic agent that causes typhoid and other diseases.Maggot is these two kinds larvas of invading the living tissue winged insect of animal.Mosquitoes spread causes the pathogenic agent of malaria, yellow jack, encephalitis and other diseases.Malaria is caused by protozoan parasites, and part life cycle of described protozoan parasites lives in the Anopheles mosquito, and part life cycle is lived in philtrum.The plague also is called glandular plague or Bubonic plague, is caused by infected rats and other rodentine bacteriums.This disease is east ceratophyllus fasciatus (Oriental rat flea) (Siphonaptera) at people's main relay person.Many honeybees, wasp and ant (Hymenoptera) can cause pain even death by its terebra.Dead normally to the allergic result of poisonous substance.Other important stinger comprises hornet (hornets), yellow jacket (yellow jackets) and wasp (paper wasps).Africanized honeybee (Africanized honey bee) or " killer bee " (" killer " bee) are the honeybee strains of human domestication.These two kinds of strains in appearance much at one.Yet Africanized honeybee strain is more aggressive, and attacks with bigger number.

In a specific embodiment, described composition is to be respectively applied for treatment or the prevention mankind or the insect disease of animal or the pharmaceutical composition or the veterinary medicine composition of infection.Such composition comprises at least a double-stranded RNA or RNA construct, at least a carrier, vehicle or the thinner of perhaps encoding the nucleotide sequence of described double-stranded RNA or RNA construct or recombinant DNA construction body (wherein said double-stranded RNA comprises the annealed complementary strand, and wherein an annealing complementary strand comprises and the target nucleotide sequences complementary nucleotide sequence that causes the insect target gene of described disease or infection) and being applicable to pharmaceutical use.

Described composition can be to be suitable for the local composition that uses, as be applied on animal or human's the skin, for example be applied to the liquid composition (drops, gelifying agent, aerosol) of skin, perhaps be used for topical application, perhaps as transdermal patch by brush or sprays, emulsion, ointment etc.

Perhaps, described insect dsRNA is produced by bacterium (for example, lactobacillus) or fungi (for example yeast belong (Sacharomyces) species), and it can be included in the food also as the oral vaccine of resisting insect infection.

Also can be prepared into other conventional pharmaceutical dosage form, comprise tablet, capsule, vaginal suppository, transdermal patch, suppository etc.Selected form depends on the character of target insect and the character of the disease that expectation is treated.

In a specific embodiment, described composition can be coating, paste or pulvis, thereby it can be applied to basic unit and protects described basic unit to avoid the infringement of insect and/or arachnid.In this embodiment, can use any basic unit or the article of the destruction sensitivity that the protection of described composition causes insect pest infestation or insect, for example food and other perishable, and basic unit's (as timber).Termite, powderpost beetles and carpenter ant can be destroyed house and other woodwork.Reticulitermes flavipe (subterraneantermite) and Taiwan formosanes (Formosan termite) are the most serious house insects in southern US and torrid areas.Insect can attack the plant or the animal product of any results.Nearly all other food that is not protected in flour beetle, cereal weevil, paddy snout moth's larva and other stock insect grain, cereal, pet food, chocolate powder and the kitchen galley dresser with storage is food.The clothes that the larva moth erosion of clothes moth is made by animal product (as fur, silk and wool).The larva moth of carpet beetle loses the animal and plant product, comprises grain even museum's sample of leather, fur, cotton, storage.Booklice and moth are the insects in Library.The amylan of these insect moth erosion book bookbindings place.Other insect of invading the house comprises cockroach, and it ingests almost anything.Cockroach is not considered to the particular propagation person of disease, but their contaminated foods and have the smell that makes us unhappy.They are very horrible, and many pest control company is devoted to attempt they are prevented and treated.Modal cockroach in house, provisions shop and restaurant comprises Groton bug, periplaneta americana, oriental cockroach and long palpus blattaria.

The vehicle of described composition and the character of physical form can be depending on the character of the basic unit of expecting processing and change to some extent.For example, pending article or the liquid in the basic unit can be brushed or be sprayed to described composition, perhaps is stamped into pending article or the liquid in the basic unit, or be applied to the pending article or the coating of basic unit.

The present invention comprises also and is used to handle and/or prevent and treat the method for insect to basic unit's infringement that it comprises composition any as herein described from significant quantity to described basic unit or the sprays of using.

The present invention also comprises and being used for the treatment of and/or the method for pre-protection against insect disease or illness, it comprises to there being this object that treats and/or prevents needs to use any composition as herein described or sprays, described composition or sprays comprise at least a double-stranded RNA or double-stranded RNA construct, it comprises the annealed complementary strand, and wherein annealing complementary strand comprises at least a portion complementary nucleotide sequence of insect target gene nucleotide sequence with the insect that causes described insect disease or illness.According to a more particular embodiment, described composition to be administered or sprays comprise and/or express at least a bacterial cell or yeast cell, described bacterial cell or yeast cell to express at least a double-stranded RNA as herein described or double-stranded RNA construct; Perhaps comprise and/or express at least a nucleotide sequence as herein described or recombinant DNA construction body, described RNA or nucleotide sequence and to cause at least a portion nucleotide sequence of insect target gene of the insect of described insect disease or illness be complementary.

In another embodiment of the present invention, described composition is as plant or the propagation of plant or the sterilant of reproductive material (as seed).For instance, described composition can be used as sterilant by spraying to plant tissue or using or spray or mix to soil before and after seedling emerges.

In another embodiment, the invention provides and be used to handle and/or stop the propagation of insect growth and/or insect pest infestation plant or plant or the method for reproductive material, it comprises to plant or composition any as herein described or the sprays of using propagation from significant quantity to plant or reproductive material.

In another embodiment, the present invention relates to any double-stranded RNA or RNA construct or the nucleotide sequence or the recombinant DNA construction body of encode described double-stranded RNA or RNA construct or at least a host cell of expressing dsRNA of the present invention (for example, bacterium or yeast) or the virus of the dsRNA as herein described that encodes or comprise the above-mentioned any composition or the purposes of sprays, it is used to control insect growth, be used to prevent and treat the plant of insect pest infestation, or be used to handle the insect infection of plant the insect infection sensitivity.The insect infection that causes at specific insect and the specified plant handled and are included in the described purposes as previously mentioned.

In a more particular embodiment, the present invention relates to comprise at least a host cell, or at least a host cell of expressing dsRNA of the present invention (for example, bacterium or yeast), or the virus of the dsRNA as herein described that encodes, perhaps relating to the purposes of the sprays that comprises above-mentioned any composition, this sprays is used to control insect growth, be used to prevent and treat the plant of insect pest infestation, or be used for the treatment of the insect infection of plant the insect infection sensitivity.Preferably, described host cell comprises arbitrary sequence at least a of following expression: SEQ ID NO 1,3,5,7,9,11,13,15,17,19,21,23,49 to 158,159,160-163,168,173,178,183,188,193,198,203,208,215,220,225,230,240 to 247,249,251,253,255,257,259,275 to 472,473,478,483,488,493,498,503,508 to 513,515,517,519,521,533 to 575,576,581,586,591,596,601,603,605,607,609,621 to 767,768,773,778,783,788,793,795,797,799,801,813 to 862,863,868,873,878,883,888,890,892,894,896,908 to 1040,1041,1046,1051,1056,1061,1066 to 1071,1073,1075,1077,1079,1081,1083,1085,1087,1089,1091,1093,1095,1097,1099,1101,1103,1105,1107,1109,1111,1113,1161 to 1571,1572,1577,1582,1587,1592,1597,1602,1607,1612,1617,1622,1627,1632,1637,1642,1647,1652,1657,1662,1667,1672,1677,1682,1684,1686,1688,1690,1692,1694,1696,1698,1700,1702,1704,1730 to 2039,2040,2045,2050,2055,2060,2065,2070,2075,2080,2085,2090,2095,2100,2102,2104,2106,2108,2120 to 2338,2339,2344,2349,2354,2359,2364,2366,2368,2370,2372,2384 to 2460,2461,2466,2471,2476,2481 or 2486, or the fragment of its at least 17 continuous nucleotides.

On the other hand, the present invention also provides and has been used to prevent or protective plant is avoided the method combination and the composition of insect infringement.For example, a kind of method provides and has used transgenic method and use double stranded rna molecule and target pest is had the method combination of (organically) compound compositions of toxic Bt insecticidal proteins or chemistry with one or more.Other method provides to be used transgenic method and use the combination of expressing double stranded rna molecule and express the method for described Bt insecticidal proteins in same bacterium or yeast or in different bacteriums or yeast in bacterium or yeast.For example, can use based on method or the technology of RNAi to come target or kill a kind of insect, and use (organically) sterilant of Bt sterilant or chemistry can target or kill another kind of insect according to these methods.

Therefore, the invention still further relates to any composition that is used to handle plant as herein described, sprays or method, wherein said composition comprises the bacterial cell or the yeast of expressing described RNA molecule, and comprise sterilant or comprise and contain or express the bacterial cell of sterilant or yeast cell (can with above mentioned be same bacterium or yeast cell or different bacterium or yeast cell), described sterilant is selected from (organically) sterilant of chemistry, paratin, the bacillus thuringiensis insecticidal proteins, the Xenorhabdus insecticidal proteins, light rod bacterium insecticidal proteins, bacillus laterosporus insecticidal proteins and Bacillus sphaericus insecticidal proteins.Preferably, described bacillus thuringiensis insecticidal proteins is selected from: Cry1, Cry3, TIC851, CryET170, Cry22, binary insecticidal proteins CryET33 and CryET34, binary insecticidal proteins CryET80 and CryET76, binary insecticidal proteins TIC100 and TIC101 and binary insecticidal proteins PS149B1.

Described sprays can be used in the greenhouse or on the farmland.For the water-based spray agent, the biotic pesticide that comprise bacterium (for example, as emulsible suspension) common utility ratio be 25-100 liter/hectare (10-40 liter/acre): per hectare comprises the formulated product (emulsible suspension) that about 2.5-5 rises, and ' bacterial cell ' that wherein said formulated product comprises about 25% (volume/volume) adds ' other composition ' of 75% (volume/volume).The amount of bacterial cell is come in unit, and for example unit definition is among the 1ml 10 ⁹Individual bacterial cell.Depend on the cropping intensity of per hectare and the leaf area of every strain plant, 1 liter formulated product comprises the bacterium of 0.001～10000 unit, preferably at least 0.001,0.003,0.005,0.007,0.01,0.03,0.05,0.07,0.1,0.3,0.5,0.7, more preferably at least 1,3,5,7,10,30,50,70,100,300,500,700, the perhaps more preferably bacterium of at least 1000,3000,5000,7000 or 10000 units.

For example, the common plant density of potato crop plant be about every square metre 4.5 strain plant or per hectare 45,000 strain plants (in a row plantation, distance be 75cm between row and row, between every row's the plant apart from being 30cm).Therefore, the present invention relates to sprays, it comprises at least 0.001,0.003,0.005,0.007,0.01,0.03,0.05,0.07,0.1,0.3,0.5,0.7, more preferably at least 1,3,5,7,10,30,50,70,100,300,500,700, the perhaps more preferably bacterium of at least 1000,3000,5000,7000 or 10000 units, described bacterial expression at least a dsRNA molecule as herein described or dsRNA construct.

The invention still further relates to the test kit that is used for handling plant insect infection, it comprises previous described at least a double-stranded RNA or double-stranded RNA construct or nucleotide sequence or recombinant DNA construction body or host cell or composition or sprays.Described test kit can provide with suitable working instructions.This specification sheets can be printed on the suitable packing that other component is provided, and perhaps can be used as independent entity provides, and for example can adopt the form of page or brochure.This specification sheets can be rolled or folding (for example when storing state), thereby its expansion can be instructed the purposes of described all the other components of test kit then.

Will be further understood that the present invention with reference to following non-limiting example.

Description of drawings

Fig. 1-LD: use the survival of the colorado potato beetles of the artificial diet feeding of handling with dsRNA.Use is with the insect of feed feeding second larval stage of dsRNA (target or the gfp contrast) solution-treated of 50 μ l topical application.After 7 days, feed is replaced with the fresh feed of the dsRNA that comprises topical application.At the 2nd, 5,7,8,9 and 13 day assessment survival insect number.Calculate the per-cent of survival larva with respect to the 0th day (when measuring beginning).Target LD006:(SEQ ID NO 178); Target LD007 (SEQ ID NO 183); Target LD010 (SEQ ID NO 188); Target LD011 (SEQ ID NO 193); Target LD014 (SEQ ID NO 198); Gfp dsRNA (SEQ ID NO 235).

Fig. 2-LD: use the survival of the colorado potato beetles of the artificial diet feeding of handling with dsRNA.Use is with the insect of feed feeding second larval stage of dsRNA (target or the gfp contrast) solution-treated of 50 μ l topical application.After 7 days, it only is fresh feed that feed is replaced to.At the 2nd, 5,6,7,8,9,12 and 14 day assessment survival insect number.Calculate the per-cent of survival larva with respect to the 0th day (when measuring beginning).Target LD001 (SEQ ID NO 163); Target LD002 (SEQID NO 168); Target LD003 (SEQ ID NO 173); Target LD015 (SEQ ID NO215); Target LD016 (SEQ ID NO 220); Gfp dsRNA (SEQ ID NO 235).

Fig. 3-LD: the weight in average of using the colorado potato beetles larva of the leaf of potato dish feeding of handling with dsRNA.Use is with the insect of leaf dish feeding second larval stage of dsRNA (target LD002 or gfp) solution (the 10ng/ μ l) processing of 20 μ l topical application.After 2 days, be transferred to insect on the undressed leaf every day.

Fig. 4-LD: use the survival of the colorado potato beetles of the artificial diet feeding of handling with target LD014 dsRNA and concatermer dsRNA than short-form.Use is with the insect of feed feeding second larval stage of dsRNA (gfp or the target) solution-treated of 50 μ l topical application.At the 3rd, 4,5,6 and 7 day assessment survival insect number.Calculate the per-cent of survival larva with respect to the 0th day (when measuring beginning).

Fig. 5-LD: use is with the survival of the colorado potato beetles larva of the artificial diet feeding of dsRNA (target LD002 (a), target LD007 (b), target LD010 (c), target LD011 (d), target LD014 (e), target LD015 (f), LD016 (g) and target LD027 (the h)) processing of different concns.Use is with the insect of feed feeding second larval stage of the dsRNA solution-treated of 50 μ l topical application.After 7 days, feed is replaced with the fresh feed of the dsRNA that comprises topical application.Assess survival insect number at interval with regular time.Calculate the per-cent of survival larva with respect to the 0th day (when measuring beginning).

Fig. 6-LD. expresses in time the influence of the coli strain of dsRNA (target LD010) to the colorado potato beetles larvae alive.In based on other biological assay of branch of artificial diet, detect two strain bacterial isolateses: (a) AB301-105 (DE3), the data point of pGBNJ003 and pGN29 is represented the average mortality value from 5 different bacterium clones, (b) BL21 (DE3), the data point of pGBNJ003 and pGN29 is represented the average mortality value from 5 different bacterial clones and an independent bacterial clone respectively.Error bar is represented standard deviation.

Fig. 7-LD. express dsRNA (target LD010) coli strain difference clone (a) AB301-105 (DE3) and (b) BL21 (DE3) in the infringement influence to the colorado potato beetles larvae alive in back 12 days.Data point is each clone's of pGN29 and pGBNJ003 an average mortality value.At this time point, AB301-105 (DE3) clone 1 who comprises plasmid pGBNJ003 demonstrates CPB 100% mortality ratio.Error bar is represented standard deviation.

Fig. 8-LD. express dsRNA (target LD010) coli strain difference clone (a) AB301-105 (DE3) and (b) BL21 (DE3) in the influence of back 7 days of infringement to the g and D of the colorado potato beetles larva that survives.Based on the data of table 10, data point is the percentage ratio of each clone's (a pGN29 clone and 5 pGBNJ003 clones) larva weight in average value.Only handle and represent 100% normal larva weight with feed.

The colorado potato beetles larva of Fig. 9-LD. feeding potato plants is in the back 7 days survival of infringement, and described potato plants sprays with the bacterium that produces double-stranded RNA.Calculate survival larva number and be expressed as mortality ratio percentage ratio.Employed bacterial host strains is rna plymerase iii deficient strain AB301-105 (DE3).The insect genes target is LD010.

The colorado potato beetles larva of the survival of Figure 10-LD. feeding potato plants is at the back 11 days growth/development delay of infringement, and described potato plants sprays with the bacterium that produces dsRNA.Employed bacterial host strains is rna plymerase iii deficient strain AB301-105 (DE3).Data plot is represented the per-cent of normal larva weight, only represents 100% normal larva weight with the processing of feed.The insect genes target is LD010.Error bar is represented standard deviation.

Figure 11-LD. back 7 days in infringement, the resistance that the potato that the bacterium of generation double-stranded RNA causes the colorado potato beetles larva damages.Left figure is with bacterium AB301-105 (DE3) the sprinkling plant of 7 units that contain the pGN29 plasmid; Right figure is with bacterium AB301-105 (DE3) the sprinkling plant of 7 units that contain the pGBNJ003 plasmid.Unit definition is in 600nm OD value being the Equivalent of 1 1ml bacterial suspension.The insect genes target is LD010.

Figure 12-LD. uses the survival of the colorado potato beetles adult of the leaf of potato dish feeding of handling with dsRNA.Begin the young adult of leaf dish feeding that two angels handle in order to double-stranded RNA, be placed on then on the undressed leaf of potato.Periodical evaluation survival insect number; Live and the normal insect that seems to move is recorded as movable insect; That live but seem ill and move slowly that insect is recorded as dying insect---in case with its back down, these insects can not stand up.Target LD002 (SEQID NO 168); Target LD010 (SEQ ID NO 188); Target LD014 (SEQ ID NO198); Target LD016 (SEQ ID NO 220); Gfp dsRNA (SEQ ID NO 235).

The bacteriogenic target double-stranded RNA of Figure 13-LD. is to the influence of colorado potato beetles larva.With OD is that 1 50 μ l bacterium AB301-105 (DE3) suspensions through heat treated expression dsRNA (SEQ ID NO 188) are applied topically on the solid artificial diet in each hole of 48 orifice plates.The CPB larva in L2 stage is placed each hole.At the 7th day, to comprising the feed that (a) contains the bacterium of expressing target 10 double-stranded RNAs, (b) contain the feed of the bacterium of empty carrier pGN29, and (c) have only the CPB larva in the flat board of feed to take pictures.

Figure 14-LD. is applied topically to the influence of leaf of potato to CPB larvae alive and growth with colibacillus deactivating AB301-105 (DE3) bacterial strain that contains plasmid pGBNJ003 of difference amount before insect pest infestation.With 10 L1 larvas of treated potato feeding 7 days.The amount that is sprayed at the bacterial suspension on the plant is: the target 10 of 0.25U, 0.08U, 0.025U, 0.008U and the pGN29 (negative control of 0.25U; Also comprise Milli-Q water).A unit (U) is defined as and be the amount of bacteria of 1 1ml culture moderate at the 600nm absorbance.The 1.6ml cumulative volume is sprayed on every strain plant.The insect genes target is LD010.

The resistance of the potato damage that Figure 15-LD back 7 days in infringement, colibacillus deactivating AB301-105 (DE3) bacterial strain that contains plasmid pGBNJ003 cause the CPB larva.(a) water (b) comprises the 0.25U intestinal bacteria AB301-105 (DE3) of pGN29, (c) contains the 0.025U intestinal bacteria AB301-105 (DE3) of pGBNJ003, (d) contains the 0.008U intestinal bacteria AB301-105 (DE3) of pGBNJ003.A unit (U) is defined as and be the amount of bacteria of 1 1ml culture equivalent at the 600nm absorbance.The 1.6ml cumulative volume is sprayed on every strain plant.The insect genes target is LD010.

Fig. 1-PC: feeding target dsRNA is to the survival of the chrysomelid larva of horseradish ape and the influence of growth.Use is with the rape leave dish feeding newborn larvae of 0.1 μ g/ μ l dsRNA (target or the gfp contrast) solution-treated of 25 μ l topical application.After 2 days, described insect is moved to the fresh Ye Panshang through the dsRNA processing.At the 4th day, collect from the larva of a repeated experiments at every kind of processing, and place the culture dish that contains undressed fresh rape leave.Assessed described insect at the 2nd, 4,7,9 and 11 day.(a) use is with the survival of the mexican bean ladybird larva of the rape leave dish feeding of dsRNA processing.Calculate survival larva per-cent with respect to the 0th day (when measuring beginning), (b) use the weight in average of the chrysomelid larva of horseradish ape of the rape leave dish feeding of handling with dsRNA.Weigh together from the weight in average of each multiple insect and definite every larva.Error bar is represented standard deviation.Target 1:SEQ ID 473; Target 3:SEQ ID NO 478; Target 5:SEQ ID NO 483; Target 10:SEQ ID NO 488; Target 14:SEQ ID 493; Target 16:SEQ ID NO 498; Target 27:SEQ ID NO 503; Gfp dsRNA:SEQ ID NO 235.

Fig. 2-PC: use with different concns dsRNA (a) target PC010 and (b) the chrysomelid survival of rape leave dish feeding horseradish ape of target PC027 processing.Nascent larva is placed on the rape leave dish with the dsRNA solution-treated of 25 μ l topical application.At the 2nd day, insect is moved on the treated fresh leaf dish.At the 4th day (at target PC010) and the 5th day (at target PC027), insect is moved to undressed leaf.Survival number the 2nd, 4,7,8,9 and 11 day (at PC010) and the 2nd, 5,8,9 and 12 day (at PC027) assessment insect.Calculate the per-cent of survival larva with respect to the 0th day (when measuring beginning).

Fig. 3-PC: express of in time the influence of intestinal bacteria AB301-105 (DE3) bacterial strain of dsRNA (target PC010) to the chrysomelid larvae alive of horseradish ape.The data point of every kind of processing is represented the average mortality value from 3 different repeated experiments.Error bar is represented standard deviation.Target 10:SEQ IDNO 488.

Fig. 1-EV: use the survival of the mexican bean ladybird of the beans leaf dish feeding of handling with dsRNA.Use is with the nascent larva of beans leaf dish feeding of 1 μ g/ μ l dsRNA (target or the gfp contrast) solution-treated of 25 μ l topical application.After 2 days, described insect is moved on the fresh leaf dish that dsRNA handles.At the 4th day, collect from a kind of larva of processing, and place the plastics casing that contains undressed fresh beans leaf.The the 2nd, 4,6,8 and 10 day assessment insect mortality.Calculate the per-cent of survival larva with respect to the 0th day (when measuring beginning).Target 5:SEQ ID NO 576; Target 10:SEQ ID NO 586; Target 15:SEQ ID NO 591; Target 16:SEQ ID NO 596; GfpdsRNA:SEQ ID NO 235.

Fig. 2-EV: feeding target dsRNA is to the influence of the survival of mexican bean ladybird adult, and to the resistance of the insect pest infestation of snap beans leaf.(a) use is with the survival of the mexican bean ladybird adult of the beans leaf dish feeding of dsRNA processing.Use is with the beans leaf dish feeding adult of 0.1 μ g/ μ l dsRNA (target or the gfp contrast) solution-treated of 75 μ l topical application.After 24 hours, insect is moved on the fresh leaf dish that dsRNA handles.After 24 hours, collect from a kind of adult of processing, and place the plastics casing that comprises undressed fresh potted plant whole strain beans plant.The the 4th, 5,6,7,8 and 11 day assessment insect mortality.Calculate the per-cent of survival larva with respect to the 0th day (when measuring beginning).Target 10:SEQ ID NO 586; Target 15:SEQ ID NO 591; Target 16:SEQ ID NO 596; Gfp dsRNA:SEQ ID NO 235.(b) dsRNA causes the resistance of beans leaf damage to the mexican bean ladybird adult.At the 9th day, visual inspection comprised the leaf damage from the whole strain plant of the insect of a kind of processing (referring to (a)).(i) target 10; (ii) target 15; (iii) target 16; (iv) gfp dsRNA; (v) be untreated.

Fig. 1-TC: use the survival of the red flour beetle of the artificial diet feeding of handling with dsRNA (target 14).Based on the nascent larva of the flour that contains 1mg dsRNA (target 14)/milk mixture feeding.Water in the feed (not containing dsRNA) is contrast.At every kind of processing, carry out repeated experiments 4 times, each repeated experiments is used 10 1 instar larvaes.Assessed the mean number per-cent of the survival of insect at the 6th, 17,31,45 and 60 day.Calculate the per-cent of survival larva with respect to the 0th day (when measuring beginning).Error bar is represented standard deviation.Target TC014:SEQ ID NO 878.

Fig. 1-MP: picked-up dsRNA (target 27) is to the influence of black peach aphid nymph survival.With 1 age insect place and comprise the 50 μ l liquid-state feeds feeding chamber of (containing 2 μ g/ μ l dsRNA (target 27 or gfp dsRNA contrast)).At every kind of processing, set up 5 feeding chambers, wherein each feeding chamber comprises 10 instars.The 8th day assessment survival number after biological assay begins.Error bar is represented standard deviation.Target MP027:SEQ ID NO 1061; Gfp dsRNA:SEQ ID NO 235.

Fig. 1-NL: use the survival of the brown paddy plant hopper of the liquid artificial diet feeding of handling with dsRNA.In dividing other biological assay, use the nymph of feed feeding first to second larval stage of the solution that is supplemented with 2mg/ml dsRNA target: (a) NL002, NL003, NL005, NL010; (b) NL009, NL016; (c) NL014, NL018; (d) NL013, NL015, NL021.Insect needle compares the survival of target and the have only feed and the feed that contains gfp dsRNA contrast of feeding same concentrations.Feed replaced with in per two days the fresh feed that contains dsRNA.Assessment survival every day insect number.

Fig. 2-NL: use is with the survival of the brown paddy plant hopper of the liquid artificial diet feeding of target dsRNA (NL002) processing of different concns.With being supplemented with 1,0.2,0.08 and the nymph of feed feeding first to second larval stage of 0.04mg/ml (final concentration) NL002.Feed replaced with in per two days the fresh feed that contains dsRNA.Assessment survival every day insect number.

Embodiment

Embodiment 1: the target gene in high flux screening in the reticent Caenorhabditis elegans

The library of preparation Caenorhabditis elegans genome range between identical two T7 promotors and the terminator in pGN9A carrier (WO 01/88121), it induces the expression that drives justice and antisense orientation by IPTG under the situation that the T7 polysaccharase is expressed.

With the form of 96 orifice plates, this library is transformed into AB301-105 (DE3) bacterial isolates.In order to screen, with these bacterial cell feeding nuclease defective type Caenorhabditis elegans nuc-1 (e1392) strains in genome range.

Follow following step, in 96 orifice plates with the dsRNA feeding Caenorhabditis elegans nuc-1 (e1392) that produces in described AB301-105 (DE3) bacterial isolates: the nuc-1 ovum is moved in the different plates, thereby and make its 20 ℃ simultaneously the hatching make L1 for synchronization.To comprise the 100 μ L liquid growth mediums of IPTG and the OD that contains Caenorhabditis elegans dsRNA library ₆₀₀1 10 μ L AB301-105 (DE3) bacterial cell cultures add in 96 orifice plates, and each carrier comprises the Caenorhabditis elegans genomic fragment that is used to express described dsRNA in the described library.4 synchronized L1 nematodes are added in each hole, hatched at least 4 to 5 days at 25 ℃.These experiments are carried out in quadruplicate.In described screening, used 6 kinds of contrasts:

-pGN29=negative control, wild-type

-pGZ1=unc-22=ballism phenotype

-pGZ18=chitin synthase=embryonic death

-pGZ25=pos-1=embryonic death

-pGZ59=bli-4D=is acute to cause death

-ACC=acetyl-CoA carboxylase=acute causing death

After 5 days, will compare with the Caenorhabditis elegans nuc-1 (e1392) of the bacterium feeding that produces dsRNA and with empty carrier (pGN29) and other phenotype that contrasts the nematode of feeding.At (embryo) lethality of (acute or larva) lethality of parental generation (P0), first filial generation (F1) or at the retarded growth of P0 the nematode of using described dsRNA feeding is screened, specific as follows: (i) The acute of P0 causes death:L1 aplasia and death, this phenotype does not produce the offspring, and described hole looks quite empty; (ii) (larva) of P0 causes death:P0 is in the stage death more late than L1, and this phenotype does not produce the offspring yet.In the hole, find dead larva or dead adult; (iii) F1 causes death:L1 grows to adult stage and still survival.This phenotype does not produce the offspring.This may be owing to sterile, embryonic death (dead ovum is arranged at the bottom, hole), embryo's stagnation or larva are stagnated (finally causing death); (iv) Cessation of growth cessation and retarded growth/delay:Compare with the hole that comprises normal development and normal back algebraically.

For the target sequence shown in the table 1A, the gained conclusion is: the Caenorhabditis elegans target gene silence that is mediated by dsRNA in nematode (as Caenorhabditis elegans) has material impact to growth and the survival of nematode.

After the reticent experiment of above-mentioned dsRNA, in 24 orifice plates, Caenorhabditis elegans is carried out finer phenotype experiment with high throughput format.According to following steps, with the Caenorhabditis elegans nuc-1 (e1392) in dsRNA library feeding 24 orifice plates that obtain in AB301-105 (DE3) bacterial isolates as mentioned above, specific as follows: as the nuc-1 ovum to be moved in the different plates, thereby and make it make L1 for synchronization 20 ℃ of hatchings simultaneously.Then, 100 synchronized L1 nematodes are soaked in the complete feeding substratum of 500 μ L S (containing 5 μ g/mL cholesterol, 4 μ L/mLPEG and 1mM IPTG) and comprise the OD in Caenorhabditis elegans dsRNA library ₆₀₀Be that each carrier comprises the Caenorhabditis elegans genomic fragment that is used to express described dsRNA in the described library in the mixture of 1 500 μ L AB301-105 (DE3) bacterial cell cultures.The L1 nematode that to be soaked hatched 2 hours 25 ℃ of rollings.

Centrifugal and remove 950 μ L supernatant liquors after, last 5 μ L are moved to (comprising about 10 to 15 nematodes) middle part in 24 each hole of orifice plate through resuspended precipitation, add one deck agar LB liquid nutrient medium.The plate of being inoculated was hatched 2 days at 25 ℃.In the adult stage, choose single adult and hatch and be used to check its offspring in 2 days at 25 ℃.Original position is checked other nematode adult on 24 initial orifice plates.Carry out these experiments in quadruplicate.

Repeat above-mentioned careful phenotypic screen with another batch nematode, unique difference is that second batch of nematode hatched 3 days at 20 ℃.

To comparing with the phenotype of the nematode of Caenorhabditis elegans dsRNA feeding and with the phenotype of the Caenorhabditis elegans nuc-1 (e1392) of described empty carrier feeding.

Based on this experiment, the gained conclusion is: the silence of Caenorhabditis elegans target gene has material impact for growth and the survival of nematode as shown in table 1A, and described target gene is essential for the survival of nematode.Therefore, these genes are desirable target genes that the gene silencing that utilizes dsRNA mediation is prevented and treated (kill or stop its growth) nematode.Therefore, the nematode that the present invention includes above-mentioned Caenorhabditis elegans target gene directly is used to prevent and treat the purposes of nematode infringement (for example nematode is to the infringement of plant) to homologue.

Embodiment 2: the lineal homologue of identifying drosophila melanogaster

As described in example 1 above, identified the deadly sequence of many Caenorhabditis elegans, and described deadly sequence can be used for identifying in other kind directly to homologue.For example, described Caenorhabditis elegans cause death sequence can be used for identifying drosophila melanogaster directly to homologous sequence.That is to say, can use every kind of Caenorhabditis elegans sequence in public database (for example GenBank), retrieve drosophila melanogaster directly to homologue.Selection has (the E value preferably is less than or equal to 1E-30) possible drosophila melanogaster of height sequence homology directly to homologue, described sequence is the best hit results of mutual blast, and the latter refers to the best each other mutually blast hit results of sequence from different biological (for example Caenorhabditis elegans and drosophila melanogaster).For example, use BLAST to compare from the sequence C of Caenorhabditis elegans and the sequence D of drosophila melanogaster.If the drosophila melanogaster sequence D is the best hit results of sequence C, and with all sequences of the Caenorhabditis elegans time series C that compares also be the best hit results of D, D and C are mutual best hit results so.This standard common means the similar sequences that has identity function in the different plant species in the lineal homologue of definition.The drosophila melanogaster sequence of being identified is shown in table 1A.

Embodiment 3: and colorado potato beetles (Colorado potato beetle, CPB)

A. clone portion gene sequence from colorado potato beetles

According to the explanation of manufacturers, use TRIzol reagent (Cat.Nr.15596-026/15596-018, Invitrogen, Rockville, Maryland is USA) from the colorado potato beetles (Colorado beetle of 4 different larval stage; Source: Jeroen van Schaik, Entocare CV Biologische Gewasbescherming, Postbus 162,6700 ADWageningen, the Netherlands) separates high-quality global RNA.According to the explanation of manufacturers, (Cat.Nr.1700 Promega) handles the genomic dna of removing in this RNA prepared product by the DNA enzyme.According to the explanation of manufacturers, use commercial reagent box (SuperScript ^TMThe III reversed transcriptive enzyme, Cat.Nr.18080044, Invitrogen, Rockville, Maryland USA) obtains cDNA.

In order to separate the cDNA sequence that comprises LD001, LD002, LD003, LD006, LD007, LD010, LD011, LD014, LD015, LD016, LC018 and a LD027 gene part, use degenerated primer and use Amplitaq Gold (Cat.Nr.N8080240, Applied Biosystems) to carry out a series of PCR reactions according to the explanation of manufacturers.

Table has provided the degenerated primer sequence of the every kind of gene that is used to increase among the 2-LD, and it has shown the colorado potato beetles target gene that comprises primer sequence and gained cDNA sequence.Use these primers to carry out separately PCR reaction, it uses following condition: 95 ℃ 10 minutes, be 95 ℃ 30 seconds, 55 ℃ of 40 round-robin 1 minute and 72 ℃ 1 minute then, be then 72 ℃ 10 minutes.Analyze gained PCR fragment on sepharose, it is carried out purifying, and (Qiagen), it is cloned into the pCR8/GW/topo carrier, and (Cat.Nr.K2500 20, Invitrogen) also order-checking for QIAquick Gel Extractionkit, Cat.Nr.28706.Table has provided the sequence of the gained PCR product of representing with SEQ ID NO separately among the 2-LD, and it is a partial sequence.Table has provided the corresponding section aminoacid sequence of representing with SEQ ID NO separately among the 3-LD, and wherein the starting point of reading frame indicates in bracket.

B. produce the dsRNA of colorado potato beetles gene

Use commercial reagent box T7 Ribomax ^TMExpress RNAi System (Cat.Nr.P1700, Promega) dsRNA of synthetic milligram level.Obtain initial two kinds of independently single 5 ' t7 rna polymerase promoter templates in two independent PCR reactions, each reaction comprises and the different target sequence of T7 promotor direction.

For every kind of target gene, use specificity T 7 forward primers and specific reverse primers to obtain adopted T7 template.Table has provided the sequence that the every kind of target sequence that is used to increase has each primer of adopted template among the 8-LD.The condition of described PCR reaction is as follows: 95 ℃ 4 minutes, be 95 ℃ 30 seconds, 55 ℃ of 35 round-robin 30 seconds and 72 ℃ 1 minute then, be then 72 ℃ 10 minutes.In the PCR reaction of using condition same as described above, use specificity forward primer and specificity T 7 reverse primers to obtain antisense T7 template.Table has provided the sequence of each primer of the every kind of target gene antisense template that is used to increase among the 8-LD.On sepharose, analyze the PCR product of gained, and by the PCR purification kit (Qiaquick PCR Purification Kit, Cat.Nr.28106, Qiagen) and NaClO ₄Precipitation is carried out purifying.According to the explanation of manufacturers, thereby resulting T7 forward and reverse template mixing are transcribed, gained RNA chain is annealed, carried out DNA enzyme and the processing of RNA enzyme and utilizes sodium-acetate to carry out purifying.Provided the sense strand of every kind of target gene gained dsRNA among the table 8-LD.Table 8-LD has shown dsRNA fragments sequence and the concatermer sequence that is used to prepare the colorado potato beetles target sequence, and it comprises primer sequence.

C. at the activity of colorado potato beetles, use artificial diet that the dsRNA target is screened

Be prepared as follows colorado potato beetles artificial diet (revise from Gelman et al., 2001, J.Ins.Sc., vol.1, no.7,1-10): water and agar are carried out autoclaving, when temperature is reduced to 55 ℃, add remaining composition (as shown in hereinafter Table A).Under this temperature, the described composition of uniform mixing is then with amount five equilibrium to 24 orifice plate (Nunc) of described feed with every hole 1ml.Described artificial diet are at room temperature cooled off and solidify.Feed was preserved for 3 weeks at most at 4 ℃.

Table A: the composition of artificial diet

Composition	The 1L volume
Composition	The 1L volume	Water	768ml
Agar	14g	Water	768ml
Agar	14g	Rolled oats	40g
Torula	60g	Rolled oats	40g
Torula	60g	Lactalbumin hydrolysate	30g
Casein	10g	Lactalbumin hydrolysate	30g
Casein	10g	Fructose	20g
The Wesson salt mixture	4g	Fructose	20g
The Wesson salt mixture	4g	Tomato fruit powder	12.5g
Potato leaf powder	25g	Tomato fruit powder	12.5g
Potato leaf powder	25g	β-Gu Zaichun	1g
Sorbic Acid	0.8g	β-Gu Zaichun	1g
Sorbic Acid	0.8g	Para methyl paraben	0.8g
The Vanderzant vitamine mixture	12g	Para methyl paraben	0.8g
The Vanderzant vitamine mixture	12g	Neomycinsulphate	0.2g
Duomycin	0.130g	Neomycinsulphate	0.2g
Duomycin	0.130g	Rifampin	0.130g
Paraxin	0.130g	Rifampin	0.130g
Paraxin	0.130g	Nystatin	0.050g
Soybean oil	2ml	Nystatin	0.050g
Soybean oil	2ml	Wheatgerm oil	2ml

With concentration is that the 50 μ l dsRNA solution topical application of 1mg/ml are at the most on the solid artificial diet in the orifice bore.Described feed carries out drying in laminar flow cabinet (laminair flow cabin).At every kind of processing, test 24 colorado potato beetles larvas (the 2nd stage), two insects in wherein every hole.Described plate is preserved in the insectarium, and its condition is 25 ± 2 ℃, 60% relative humidity, 16:8 hour illumination: dark photoperiod.Per survival condition of assessing described beetle in 1,2 or 3 day.After 7 days,, replace described feed with the fresh feed of the topical application dsRNA that comprises same concentrations (1mg/ml) for target LD006, LD007, LD010, LD011 and LD014; For LD001, LD002, LD003, LD015 and LD016, only replace described feed with fresh feed.Described dsRNA target and the feed (corresponding to GFP (green fluorescent protein, green fluorescent protein) encoding sequence (SEQ ID NO 235) fragment) that has only feed or comprise topical application dsRNA are compared.

As two independently in the biological assay as shown in, cause larval mortality significantly to increase (Fig. 1 LD-2LD) with the artificial diet feeding colorado potato beetles larva that contains complete naked dsRNA.

After 7 to 14 days, all tested dsRNA finally cause 100% mortality ratio.In whole described biological assay, the feed that contains or do not contain GFP dsRNA makes described insect almost not have or not have death.

Usually, in observed all are measured, with the normal feeding of CPB subordinate phase larva 2 days of the feed feeding that contains or do not contain dsRNA and cast off a skin become the 3rd the stage larva.In this new larval stage, the feed of observing CPB significantly reduces or stops fully, causes mortality ratio to improve.

D. at the activity of colorado potato beetles, use the leaf of potato dish that the dsRNA target is carried out biological assay

Used use the leaf of potato material but not artificial diet as the alternative bioassay method of CPB food source.Use the potato plants leaf in from 2 to 3 ages in week of puncher of suitable dimension to downcut the about 1.1cm of diameter (or 0.95cm ²) the leaf dish.By being applied to paraxial leaf surface, the 20 μ l target LD002 dsRNA of 10ng/ μ l or contrast gfp dsRNA solution prepares treated leaf dish.Make dry 24 holes that also place 24 orifice plates (Nunc) respectively separately of leaf dish.Single only CPB of the 2nd larval stage is placed each hole, and the plastic cover with cotton paper and porous plate covers it then.The plate that will comprise described insect and leaf dish places insectarium, and its condition is 28 ℃ and 16 hours illumination/8 hour dark photoperiods.Make described insect feed leaf dish 2 days, afterwards described insect is moved in the new flat board that comprises treated fresh leaf dish.Afterwards, moved to the flat board that comprise be untreated leaf dish with described insect every day, until the 7th day.Record insect mortality and weight.

Cause larval mortality significantly to increase with the leaf of potato dish feeding colorado potato beetles larva that comprises the complete naked dsRNA (target LD002) that the surface applies (promptly at the 7th day all insect deaths; 100% mortality ratio), contrast the survival not influence of gfp dsRNA to CPB.Target LD002dsRNA had a strong impact on the growth of larva after 2 to 3 days, and with the normal (Fig. 3-LD) of the larvae development of same concentrations gfp dsRNA feeding.

E. at the activity of colorado potato beetles, use artificial diet that the dsRNA than short-form is screened

Present embodiment illustrates: short (60 or 100bp) dsRNA fragment itself or be enough to cause toxicity to colorado potato beetles as the concatermer construct.

Select LD014 to be used for this embodiment, known its is the target of inducing lethality in colorado potato beetles.This genes encoding V-ATP enzyme subunit E (SEQ ID NO 15).

The fragment LD014_F1 (in the 195-294 position of SEQ ID NO 15, SEQ ID NO 159) of 100 base pairs and the fragment LD014_F2 (in the 235-294 position of SEQID NO 15, SEQ ID NO 160) of 60 base pairs have also been selected.Also referring to table 7-LD.

Designed the concatermer of two 300 base pairs, LD014_C1 and LD014_C2 (SEQID NO 161 and SEQ ID NO 162).LD014_C1 comprises 3 repetitions of above-mentioned 100 base pair fragments (SEQ ID NO 159), and LD014_C2 comprises 5 repetitions of above-mentioned 60 base pair fragments (SEQ ID NO 160).Also referring to table 7-LD.

Synthetic fragment LD014_F1 and LD014_F2 are as justice and antisense primer are arranged.Before cloning, thereby these primer annealings are obtained double chain DNA molecule.5 ' and 3 ' end at described primer comprises XbaI and XmaI restriction site respectively, so that clone.

Described concatermer is the synthetic gene of 300 base pairs.Comprise XbaI and XmaI restriction site respectively at this synthetic DNA segmental 5 ' and 3 ' end, so that clone.

With XbaI and 4 kinds of dna moleculars of XmaI digestion, i.e. two kinds of single unit (LD014_F1 and LD014_F2) and two kinds of concatermers (LD014_C1 and LD014_C2), thereby and with its respectively subclone obtain recombinant plasmid p1, p2, p3 and p4 respectively to by among XbaI and the linearizing pBluescriptII SK+ of XmaI digestion.

The generation of double-stranded RNA: use commercial reagent box T7Ribomax ^TMExpress RNAiSystem (Cat.Nr.P1700, Promega) synthetic dsRNA.Independently obtain initial two kinds of independently single 5 ' t7 rna polymerase promoter templates in the PCR reaction at two, each reaction comprises and the different target sequence of T7 promotor direction.For LD014_F1, use specificity T 7 forward primer oGBM159 and specific reverse primers oGBM164 (this paper represents with SEQ ID NO 204 and SEQ ID NO205 respectively) in the PCR reaction, to use following condition generation that adopted T7 template is arranged: 95 ℃ 4 minutes, be 95 ℃ 30 seconds, 55 ℃ of 35 round-robin 30 seconds and 72 ℃ 1 minute then, be then 72 ℃ 10 minutes.Use specificity forward primer oGBM163 and specificity T 7 reverse primer oGBM160 (this paper represents with SEQ ID NO 206 and SEQ ID NO 207 respectively) in the PCR reaction of using condition same as described above, to produce antisense T7 template.On sepharose, analyze gained PCR product, and by the PCR purification kit (Qiaquick PCR Purification Kit, Cat.Nr.28106, Qiagen) and NaClO ₄Precipitation is carried out purifying.According to the explanation of manufacturers, thereby resulting T7 forward and reverse template mixing are transcribed, resulting RNA chain is annealed, carried out DNA enzyme and the processing of RNA enzyme and utilizes sodium-acetate to carry out purifying.The sense strand of gained dsRNA is expressed as SEQ ID NO 203 in this article.

For LD014_F2, use specificity T 7 forward primer oGBM161 and specific reverse primers oGBM166 (this paper represents with SEQ ID NO 209 and SEQ ID NO 210 respectively) in the PCR reaction, to use following condition generation that adopted T7 template is arranged: 95 ℃ 4 minutes, be 95 ℃ 30 seconds, 55 ℃ of 35 round-robin 30 seconds and 72 ℃ 1 minute then, be then 72 ℃ 10 minutes.Use specificity forward primer oGBM165 and specificity T 7 reverse primer oGBM162 (representing with SEQ ID NO 211 and SEQ ID NO 212 respectively) in the PCR reaction of using condition same as described above, to produce antisense T7 template herein.On sepharose, analyze gained PCR product, and by the PCR purification kit (Qiaquick PCR Purification Kit, Cat.Nr.28106, Qiagen) and NaClO ₄Precipitation is carried out purifying.According to the explanation of manufacturers, thereby resulting T7 forward and reverse template mixing are transcribed, resulting RNA chain is annealed, carried out DNA enzyme and the processing of RNA enzyme and utilizes sodium-acetate to carry out purifying.The sense strand of gained dsRNA is expressed as SEQ ID NO 208 in this article.

Similarly, for described concatermer, independently obtain initial two kinds of independently single 5 ' T7 rna polymerase promoter subtemplates in the PCR reaction at two, each reaction comprises and the different target sequence of T7 promotor direction.With XbaI or XmaI described recombinant plasmid p3 and the p4 that comprises LD014_C1 and LD014_C2 carried out linearizing, two kinds of linear fragments to every kind of construct carry out purifying, and being used as the template that in-vitro transcription is measured, described in-vitro transcription is used the T7 promotor of described cloning site flank.Use T7RiboMAX ^TMExpress RNAiSystem (Promega) prepares double-stranded RNA by in-vitro transcription.The sense strand of gained dsRNA (at LD014_C1 and LD014_C2) is expressed as SEQ ID NO213 and 214 in this article respectively.

Shown in Fig. 4-LD, the shorter sequence of target LD014 and concatermer can be induced lethality in colorado potato beetles.

F. at the activity of colorado potato beetles, use artificial diet that the dsRNA of different concns is screened

On the solid artificial diet in 50 μ l dsRNA solution topical application to 24 orifice plate (Nunc) holes, described dsRNA solution adopts a series of ten times of concentration of reducing to 0.01ng/ μ l from 1 μ g/ μ l (for target LD027 from 0.1 μ g/ μ l).Described feed carries out drying in the laminar flow cabinet.At every kind of processing, test 24 colorado potato beetles larvas (the 2nd stage), two insects in wherein every hole.Described plate is preserved in the insectarium, and its condition is 25 ± 2 ℃, 60% relative humidity, 16:8 hour illumination: dark photoperiod.Assess the survival condition of described beetle with regular time interval (maximum 14 days).After 7 days, replace described feed with the fresh feed of the topical application dsRNA that comprises same concentrations.Described dsRNA target is compared when having only feed.

Shown in Fig. 5-LD,, cause high larval mortality with the artificial diet feeding colorado potato beetles larva that contains complete naked dsRNA (concentration is low to moderate 0.1～10ng dsRNA/ μ l) at different targets.

G. the CPB gene fragment clone is advanced in the carrier, described carrier is suitable for bacterium and is created in and has active double-stranded RNA in the insect

Although can use any effective bacterium promotor, into be used in the carrier of host bacterium expression double-stranded RNA (referring to WO 00/01846) yet will clone corresponding to the dna fragmentation of CPB gene target.

Table 8-LD provide the specific primer sequence that is used for the amplified target gene.Employed template is the pCR8/GW/topo carrier that contains any target sequence.Use described primer to carry out PCR reaction, it uses following condition: 98 ℃ 5 minutes, be 98 ℃ 10 seconds, 55 ℃ of 30 round-robin 30 seconds and 72 ℃ 2 minutes then, be then 72 ℃ 10 minutes.On sepharose, analyze gained PCR fragment, it is carried out purifying (QIAquick Gel Extraction kit, Cat.Nr.28706 Qiagen), clones into (with reference to WO00188121A1) and order-checking in the linearizing pGNA49A carrier of SrfI with its flush end.Table has provided among the 8-LD corresponding to the sequence of the gained PCR product of sequence separately.The recombinant vectors that comprises this sequence is called pGBNJ003.

The sequence (forward primer SEQ ID NO 191 and reverse primer SEQ ID NO 190) of the Auele Specific Primer that is used for amplified target gene fragment LD010 is provided among the table 8-LD.Employed template is the pCR8/GW/topo carrier that contains LD010 sequence (SEQ ID NO 11).Use described primer to carry out PCR reaction, it adopts following condition: 98 ℃ 5 minutes, be 98 ℃ 10 seconds, 55 ℃ of 30 round-robin 30 seconds and 72 ℃ 2 minutes then, be then 72 ℃ 10 minutes.On sepharose, analyze gained PCR fragment, it is carried out purifying (QIAquick GelExtraction kit, Cat.Nr.28706 Qiagen), clones into (with reference to WO00188121A1) and order-checking in the linearizing pGNA49A carrier of SrfI with its flush end.Provided sequence among the table 8-LD corresponding to the gained PCR product of SEQ ID NO 188.The recombinant vectors that comprises this sequence is called pGBNJ003.

H. in two coli strains (1) AB301-105 (DE3) and (2) BL21 (DE3), express and produce the double-stranded RNA target

Follow following step, express the double-stranded RNA of the target LD010 of proper level in bacterium, this double-stranded RNA has activity in insect.RNA enzyme III deficient strain (AB301-105 (DE3)) is used for comparing with the wild-type bacterium (BL21 (DE3)) that contains RNA enzyme III.

Transform AB301-105 (DE3) and BL21 (DE3)

Add in ice-cold chemoreception attitude intestinal bacteria AB301-105 (DE3) of 50 μ l or BL21 (DE3) the bacterial strain equal portions 300ng plasmid and soft the mixing.Hatched described cell 20 minutes on ice, afterwards it is carried out 37 ℃ of heat shocks and handled 5 minutes, then described cell is returned on ice and kept again 5 minutes.The SOC substratum of 450 μ l room temperatures is added to described cell, and this suspension was hatched 1 hour on shaking table (250rpm) in 37 ℃.100 μ l bacterial cell suspensions are moved in the 500ml Erlenmeyer flask that contains 150ml LB liquid nutrient medium, and described LB liquid nutrient medium is supplemented with the Pyocianil microbiotic of 100 μ g/ml.Described culture is hatched in 37 ℃ spend the night on Innova 4430 shaking tables (250rpm) (16-18 hours).

Chemical induction double stranded rna expression in AB301-105 (DE3) and BL21 (DE3)

Because there is the hereditary component of all control expression, so in AB301-105 (DE3) or BL21 (DE3) bacterial isolates, can express double-stranded RNA from recombinant vectors pGBNJ003.In the presence of chemical inducer isopropylthiogalactoside (IPTG), the T7 polysaccharase will drive transcribing of described target sequence with antisense and sense orientation, because the flank of described target sequence is rightabout T7 promotor.

Use the absorbancy of suitable spectrophotometer measurement bacterium overnight culture, and its value is adjusted into 1 by adding fresh LB liquid nutrient medium at 600nm.This culture of 50ml is moved in the 50ml Falcon pipe, then at 15 ℃ with the centrifugal described culture of 3000g 10 minutes.Remove supernatant liquor, carry out resuspended with the fresh S perfect medium (the SNC substratum adds 5 μ g/ml cholesterol) that 50ml is supplemented with 100 μ g/ml Pyocianils and 1mMIPTG bacterial precipitation.Induced bacterium 2 to 4 hours in room temperature.

The thermal treatment of bacterium

Thereby make in the test slab the contaminated risk of artificial diet reduce to minimum by the thermal treatment killing bacteria.Yet, to the bacterium of expressing double-stranded RNA heat-treat be not insect induce disturb by RNA due to toxic prerequisite.Institute's inductive bacterial cultures is room temperature with 3000g centrifugal 10 minutes, abandoning supernatant, and will be deposited in 80 ℃ of water-baths and keep 20 minutes.After the thermal treatment, bacterial precipitation is resuspended in the 1.5ml MilliQ water, and suspension is moved in the micro-centrifuge tube.Each renewal is prepared several pipes and is used for biological assay.Described existence-20 of guaranteeing are ℃ standby.

I. at colorado potato beetles, the intestinal bacteria carry out laboratory experiment of expressing dsRNA target LD010 is detected

Use two kinds of bioassay methods to detect the double-stranded RNA that intestinal bacteria produced at the colorado potato beetles larva: (1) is based on the biological assay of artificial diet and (2) biological assay based on plant.

Biological assay based on artificial diet

The artificial diet of preparation colorado potato beetles described in the embodiment 3C of front.Half milliliter of feed is dispensed in each hole of 48 hole porous test panels (Nunc).For every kind of processing, be that (it is equal to about 5 * 10 for 1 50 μ l thermal treatment bacterial suspensions with the OD that expresses dsRNA ⁷Individual bacterial cell) on the solid feed of topical application to the hole, and make this plate dry in the laminar flow case.At every kind of processing, detect the colorado potato beetles larva in 48 the 2nd stages, wherein each contains in the hole of feed and bacterium and comprises a colorado potato beetles larva.Each row (i.e. 8 holes) of plate is as a repetition.Described plate is kept in the insectarium, and its condition is 25 ± 2 ℃, 60 ± 5% relative humidity, 16:8 hour illumination: dark photoperiod.After per 4 days, described beetle is moved to the fresh feed that comprises the topical application bacterium.Per survival condition of assessing described beetle in 1 or 3 day behind the insect pest infestation.For the insect of survival, thereby the weight of the 7th day record larva writes down its g and D after infringement.

For intestinal bacteria AB301-105 (DE3) bacterial strain of RNA enzyme III disappearance, test contains plasmid pGBNJ003 and contains the toxicity of the bacterium of empty plasmid pGN29 (with reference to WO00/188121A1) to CPB in biological assay.The bacterium that comprises the pGBNJ003 plasmid shows that insect mortality in time and obviously increases, yet observes pGN29 and have only the contrast of feed almost not have or do not have death (Fig. 6 a-LD and 7a-LD).Contain behind the artificial diet of the bacterium of expressing dsRNA target LD010 the 7th day at feeding, (table 10-LD, Fig. 8 a-LD, Figure 13-LD) are seriously blocked in the growth of the colorado potato beetles larva of survival.

For e. coli bl21 (DE3) bacterial strain, detect bacterium that comprises plasmid pGBNJ003 and the bacterium that comprises empty carrier pGN29 at the colorado potato beetles larva.Observe similar harmful effect (Fig. 6 b-LD and 7b-LD) in the larva with the feed feeding that contains BL21 (DE3) bacterium with RNA enzyme III deletion mycopremna AB301-105 (DE3).Yet for 5 clones' survival number, BL21 (DE3) will be higher than AB301-105 (DE3); At the 12nd day, the average mortality value of this bacterial strain hanged down 25% approximately than RNA enzyme III deletion mycopremna.In addition, use the weight in average of the survival insect of the feed feeding that contains the BL21 (DE3) that expresses dsRNA (corresponding to target LD010) significantly to reduce (table 10-LD, Fig. 8 b-LD).

G and D with the CPB larva of the feed feeding that comprises one of two bacterial isolateses (containing plasmid pGBNJ003) postpones directly relevant with the feed inhibition, because with the bacterium that comprises empty carrier pGN29 or have only the flat board of feed to compare, from the 4th day dull and stereotyped visible larva ight soil of Kong Zhongwu of changing.This observation is similar to the situation (referring to embodiment 3D) of topical application to the double-stranded RNA feeding CPB of the in-vitro transcription of artificial diet; Here, stopped feed on the 2nd day from handling feed.

Biological assay based on plant

Suspension with the expression dsRNA of chemical induction sprays whole strain potato plants, uses described plant feeding CPB larva then.The potato plants (WageningenUniversity) of " V system " kind grows to the stage that the 8-12 sheet launches leaf from stem tuber in having the plant culturing chamber of following condition: 25 ± 2 ℃, and 60% relative humidity, 16:8 hour illumination: dark photoperiod.Thereby cover described plant on the plant by the 500ml Plastic Bottle is inverted in, bottleneck places earth in the basin securely, cuts an opening in the bottle bottom, and covers and go up fine and closely woven nylon wire in order to ventilate, to reduce inside and condense (condensation) and to prevent that larva from escaping.The colorado potato beetles larva in 15 L1 stages is placed on the treated plant of every strain in the cover.With comprising pGBNJ003 plasmid (clone 1; Fig. 7 a-LD) or the pGN29 plasmid (clone 1; Referring to Fig. 7 a-LD) the suspension of intestinal bacteria AB301-105 (DE3) handle plant.Use the bacteriums of different amounts to plant: 66,22 and 7 units, one of them unit definition is: absorbance is in 1 the 1ml bacterial suspension 10 under the 600nm wavelength ⁹Individual bacterial cell.In each case, with atomizer cumulative volume 1.6ml is sprayed onto on the plant.In this test, handle for every kind and use a strain plant.Counting survival insect number, and write down the weight of every survival insect.

With intestinal bacteria AB301-105 (DE3) the bacterial strain suspension sprinkling plant of expressing, compare it with the pGN29 contrast and cause insect mortality significantly to increase from the target dsRNA of pGBNJ003.When 9 times of the amount of bacterial cell suspension dilutions, the mortality ratio counting (Fig. 9-LD) that remains unchanged.The 11st day survival larva weight in average is than low 10 times approximately of pGN29 (Figure 10-LD) on the plant of spraying with the bacterium that comprises the pGBNJ003 carrier.Compare with the potato plants of using the bacterium sprinkling that comprises empty carrier pGN29, and the remarkable reduction of feed infringement that the CPB larva is caused the potato plants that sprays with the bacterium that comprises the pGBNJ003 plasmid (Figure 11-LD).

These test demonstration, the double-stranded RNA corresponding to the insect genes target sequence that produces in the bacterial expression system of wild-type or RNA enzyme III disappearance has toxicity to insect, and described toxicity shows as the remarkable growth/development delay that increases and survive larva of insect mortality.Find out obviously also that from these experiments provide by using the effective protective plant/crop of spray agent to avoid the example of insect damage, described spray agent is made up of corresponding to the bacterium of the double-stranded RNA of insect genes target expression.

J. at colorado potato beetles, the multiple suspension density of the culture of Escherichia coli of dsRNA target LD010 is expressed in test

The preparation of bacterial cultures and processing are described among the embodiment 3J.Three times of serial dilution things (since 0.25 unit Equivalent) of expressing e. coli rna enzyme III deletion mycopremna AB301-105 (DE3) culture of target LD010 double-stranded RNA are applied to the potato plants mutation " Bintje " that the 8-12 sheet launches the leaf stage.10 colorado potato beetles L1 larvas are placed on the treated plant, handle a strain plant for every kind.The 7th day (after being insect pest infestation 7 days) to insect mortality and retarded growth marking.

Shown in Figure 14-LD, after with treated 1 week of potato plants feeding insect, record high CPB larval mortality (90～100%), described processing is to be that the coli heat inactivated culture of 0.25,0.08 and 0.025 bacterium unit is carried out topical application by fine and closely woven spray concentration, and described intestinal bacteria comprise plasmid pGBNJ003 (being used to express target 10dsRNA).When 0.008 unit, about 1/3rd insect death, still, the survival insect is significantly less than control group (comprise the intestinal bacteria of empty carrier pGN29 and have only water).Compare with the potato plants that sprays with the bacterium that comprises empty carrier pGN29, the CPB larva is to being that the feed infringement that potato plants that the bacterium of 0.025 or 0.008 unit is sprayed causes significantly reduces (Figure 15-LD) with the concentration that comprises the pGBNJ003 plasmid.

K. adult is extremely responsive to the dsRNA corresponding to target gene of oral uptake

Hereinafter the embodiment that is provided has highlighted, and insect imago (being not only the insect of larval stage) is extremely responsive to the dsRNA corresponding to target gene of oral uptake.

Select 4 kinds of targets to be used for this experiment: target 2,10,14 and 16 (being respectively SEQ IDNO 168,188,198 and 220).GFP fragment dsRNA (SEQ ID NO 235) is with comparing.The young adult of random choose (2-3 age in days) does not have preference to the insect sex from the culture of our laboratory rearing.10 adults of every kind of processing selecting.Before handling beginning, described adult is hunger at least 6 hours in advance.At first day that handles, with 4 leaf of potato dish (diameter 1.5cm ²) every adult of feeding, each described leaf of potato dish (synthesizes as described in embodiment 3A by the 25 μ l target dsRNA of topical application 0.1 μ g/ μ l; Topical application is as described in the embodiment 3E) carry out pre-treatment.Every adult is limited in the little culture dish (diameter 3cm) guaranteeing all insects food of equivalent of ingesting, and therefore accepts isodose dsRNA.Second day, as mentioned above once more to 4 treated leaf dishes of every adult feeding.The 3rd day, collect all 10 insects of every kind of processing, and be positioned over together by plastics casing and (in the cover of size 30cm * 20cm * 15cm) form, have fine and closely woven nylon wire in the lid of described plastics casing so that good ventilation to be provided.In described plastics casing, some moist filter paper have been placed in its bottom.Place some (untreated) leaf of potato above the described filter paper, thereby in experimentation, kept adult.From the 5th day, death, survival (movable) and dying insect number are counted thereby carry out periodical evaluation.For dying insect, its back placed down check that can they oneself stand up in several minutes; Only the insect that can not stand up is considered to dying.

In this experiment, show, corresponding to the double-stranded RNA of different targets to the tangible specificity toxic action of colorado potato beetles adult (Figure 12-LD).Demonstrating in the final date of the valuation (the 19th day) corresponding to the segmental double-stranded RNA of gfp does not have toxicity to the CPB adult.This experiment clearly illustrates, when oral delivery touch dsRNA only after several days the survival of CPB adult just significantly reduce.For example, for target 10, at the 5th day, in 10 adults 5 dying (morbid state and move slowly); At the 6th day, 4 death in 10 adults, in the survival insect 3 dying; At the 9th day, it was all dead to observe all adults.

As the conclusion of this experiment, the application extension at the target double-stranded RNA of insect pest can be able to be caused two life stages of the insect pest of crop destruction (being larva and adult) widely to comprising, described insect pest is as the situation of colorado potato beetles.

Embodiment 4: the horseradish ape chrysomelid (Mustard leaf beetle, MLB)

A. clone the partial sequence of the chrysomelid PC001 of horseradish ape, PC003, PC005, PC010, PC014, PC016 and PC027 gene by the PCR of family (family PCR).

According to the explanation of manufacturers, use TRIzol reagent (Cat.Nr.15596-026/15596-018, Invitrogen, Rockville, Maryland, USA) chrysomelid (the horseradish ape is chrysomelid from the horseradish ape; Source: Dr.Caroline Muller, Julius-von-Sachs-Institutefor Biosciences, Chemical Ecology Group, University of Wuerzburg, Julius-von-Sachs-Platz 3, D-97082 Wuerzburg, Germany) the 3rd the stage larva separate high-quality total RNA.According to the explanation of manufacturers, (Cat.Nr.1700, Promega) genomic dna that exists in the RNA prepared product is removed in processing by the DNA enzyme.According to the explanation of manufacturers, use commercial reagent box (Superscript ^TMIII Reverse Transcriptase, Cat.Nr.18080044, Invitrogen, Rockville, Maryland USA) obtains cDNA.

In order to separate the cDNA sequence that comprises PC001, PC003, PC005, PC010, PC014, PC016 and a PC027 gene part, use degenerated primer and use Amplitaq Gold (Cat.Nr.N8080240, Applied Biosystems) to carry out a series of PCR reactions according to the explanation of manufacturers.

Table has provided the degenerated primer sequence of the every kind of gene that is used to increase among the 2-PC.Use these primers to carry out each PCR reaction, it uses following condition: 95 ℃ 10 minutes, be 95 ℃ 30 seconds, 55 ℃ of 40 round-robin 1 minute and 72 ℃ 1 minute then, be then 72 ℃ 10 minutes.Analyze gained PCR fragment on sepharose, it is carried out purifying, and (QIAquick GelExtraction kit, Cat.Nr.28706 Qiagen), are cloned into pCR4/TOPO carrier (Cat.Nr.K4530-20, Invitrogen) also order-checking with it.Table has provided the sequence of representing gained PCR product with SEQID NO separately among the 2-PC, and it is a partial sequence.

Table has provided the corresponding section aminoacid sequence of representing with SEQ ID NO separately among the 3-PC.Table 3-PC provides cDNA clone's aminoacid sequence, and wherein the starting point of reading frame indicates in bracket.

B. produce the dsRNA of the chrysomelid gene of horseradish ape

Use commercial reagent box T7Ribomax ^TMExpress RNAi System (Cat.Nr.P1700, Promega) dsRNA of synthetic milligram level.Independently obtain initial two kinds of independently single 5 ' t7 rna polymerase promoter templates in the PCR reaction at two, each reaction comprises and the different target sequence of T7 promotor direction.

For every kind of target gene, use specificity T 7 forward primers and specific reverse primers to obtain adopted T7 template.Table has provided the sequence that the every kind of target sequence that is used to increase has each primer of adopted template among the 8-PC.Table 8-PC provides the details of the dsRNA fragment of the chrysomelid target sequence of preparation horseradish ape, and it comprises primer sequence.

The condition of PCR reaction is as follows: 95 ℃ 1 minute, be 95 ℃ 30 seconds, 60 ℃ of 20 round-robin 30 seconds and 72 ℃ 1 minute then, be 95 ℃ 30 seconds, 50 ℃ of 15 round-robin 30 seconds and 72 ℃ 1 minute then, be then 72 ℃ 10 minutes.In the PCR reaction of using condition same as described above, use specificity forward primer and specificity T 7 reverse primers to obtain antisense T7 template.Table has provided each primer sequence of the every kind of target gene antisense template that is used to increase among the 8-PC.On sepharose, analyze gained PCR product, and by the PCR purification kit (Qiaquick PCRPurification Kit, Cat.Nr.28106, Qiagen) and NaClO ₄Precipitation is carried out purifying.According to the explanation of manufacturers, thereby resulting T7 forward and reverse template mixing are transcribed, gained RNA chain is annealed, carried out DNA enzyme and the processing of RNA enzyme and utilizes sodium-acetate to carry out purifying.Provide the sense strand of every kind of target gene gained dsRNA among the table 8-PC.

C. the transgenic arabidopsis plant is carried out black peach aphid infringement laboratory test

Obtain transgenic plant

Use flower-dipping method (Clough and Bent (1998) Plant Journal 16 735-743) arabidopsis thaliana transformation plant.The over-ground part of described plant is hatched the several seconds in containing the solution of following material, described solution contains 5% sucrose, from resuspended agrobacterium tumefaciens (Agrobacterium tumefaciens) the bacterial strain C58C1 Rif cell of overnight culture and 0.03% tensio-active agent Silwet L-77.After the inoculation, with transparent plastics cover plant 16 hours to keep humidity.In order to improve transformation efficiency, repeating said steps after the week.Behind seed maturity, stop to water seed that harvests drying and deepfreeze two days.After the sterilization, seed is layered in the growth medium that contains kantlex in order to select to transform plant.

Selected plant is moved to earth in order to produce the T2 seed best.

Biological assay

By being sprouted, isolating T2 seed selects the transgenic arabidopsis plant on the appropriate selection substratum.When the root of these transgenic plant is completed into, they are moved to fresh artificial growth medium or earth, they are grown under top condition.Detect whole strain transgenic plant at the black peach aphid nymph, have remarkable resistance to show the plant damage that cause the nymph that ingests (1), (2) nymph mortality ratio improves, and/or the weight of (3) survival nymph reduces (or any other insect heteroplasia).

D. at the activity of the chrysomelid larva of horseradish ape, use the rape leave dish that dsRNA target carry out laboratory experiment is detected

Embodiment provided below for example understands the dsRNA sensitivity corresponding to self target gene that the chrysomelid larva of horseradish ape is taken in per os.

For the different double-stranded RNA samples of testing needle, adopted use rape (Brassica napus mutation SW Oban to the MLB larva; Source Nick Balaam, Sw Seed Ltd., 49North Road, Abington, Cambridge, CB1 6AS, UK) the leaf material is measured as the leaf dish of food source.Keep described insect culture in insectarium on the rape of identical mutation, its condition is 25 ± 2 ℃, 60 ± 5% relative humidity, 16 hours illumination/8 hour dark photoperiods.Use the rape plant in from 4 to 6 ages in week of puncher of suitable dimension to downcut the about 1.1cm of diameter (or 0.95cm ²) the leaf dish.With the Milli-Q water that contains 0.05% Triton X-100 with the double-stranded RNA diluted sample to 0.1 μ g/ μ l.By being applied to paraxial leaf surface, the diluting soln of target PC001, PC003, PC005, PC010, PC014, PC016, PC027dsRNA and the contrast gfp dsRNA of 25 μ l or 0.05% Triton X-100 prepare treated leaf dish.With dry each hole that also places 24 orifice plates respectively of described leaf dish, 2% agar of 1ml gelation is contained in described hole, and it helps preventing that described leaf dish is withered.Two newborn MLB larvas are placed each hole of described plate, with the sponge plastics lid it is covered then.Described plate (every kind of processing comprises 48 insects) is divided into 4 repetitions, and each repeats to comprise 12 insects (every row).The flat board that will comprise described insect and leaf dish is kept in the insectarium, and its condition is 25 ± 2 ℃, 60 ± 5% relative humidity, 16 hours illumination/8 hour dark photoperiods.With leaf dish feeding insect 2 days, afterwards they are moved to the new flat board that contains the leaf dish of handling recently.Then, began back 4 days, collect from each multiple insect and move to the culture dish that contains unprocessed fresh rape leave in biological assay.Write down larval mortality and weight in average at the 2nd, 4,7,9 and 11 day.

For all tested targets, use the chrysomelid larva of rape leave feeding horseradish ape of handling all to cause larval mortality significantly to increase, shown in Fig. 1 (a) through complete naked target dsRNA.Tested double-stranded RNA at target PC010 caused 100% larva death at the 9th day, and caused 100% larva death at the 11st day at the tested double-stranded RNA of target PC027.Other targets for all, with contrast gfp dsRNA, have only 0.05% Trition X-100 or only have untreated leaf to compare and reached high mortality value: (fiducial interval of mean value percentage ratio ± α 0.05) PC001 (94.4 ± 8.2) at the 11st day, PC003 (86.1 ± 4.1), PC005 (83.3 ± 7.8), PC014 (63.9 ± 20.6), PC016 (75.0 ± 16.8), gfp dsRNA (11.1 ± 8.2), 0.05% Triton X-100 (19.4 ± 10.5) has only leaf (8.3 ± 10.5).

Assess the larva of survival based on their weight in average.For all tested targets, after 4 days, the weight in average of the chrysomelid larva of horseradish ape significantly reduces in described biological assay; Feeding contrasts gfpdsRNA or only has the insect of 0.05% Trition X-100 to grow normally, with the same (Fig. 1 (b)-PC) of larva of the leaf of only ingesting.

E. at the activity of the chrysomelid larva of horseradish ape, use the rape leave dish that different concns dsRNA carry out laboratory experiment is screened

Embodiment 4D is described as mentioned, with 25 μ l from the dsRNA solution topical application of target PC010 or PC027 to the rape leave dish, described dsRNA solution adopts 10 times of series concentration (reducing to 0.1ng/ μ l from 0.1 μ g/ μ l).As negative control, will only there be 0.05% TritonX-100 to be applied to described leaf dish.At every kind of processing, detect 24 chrysomelid newborn larvaes of horseradish ape, two insects in wherein every hole.Preserve described flat board in the insectarium, its condition is 25 ± 2 ℃, 60 ± 5% relative humidity, 16:8 hour illumination: dark photoperiod.At the 2nd day, with described larva move to contain fresh on the new flat board of the leaf dish that dsRNA handles.At the 4th day (at target PC010) and the 5th day (at target PC027), will move to the culture dish that contains the competent leaf material that is untreated from the insect of each repeated experiments.The the 2nd, 4,7,8,9 and 11 day (at the mark PC010) and the 2nd, 5,8,9 and 12 day (at target PC027) assessment described beetle survival condition.

As Fig. 2 (a) with (b), with the chrysomelid high mortality that causes of rape leave dish feeding horseradish ape of the complete naked dsRNA (to be low to moderate the strength of solution of 1ng dsRNA/ μ l) that contains two kinds of different targets (PC010 and PC027).Fiducial interval at average mortality value percentage ratio ± α 0.05 of different concns dsRNA: for target PC010 the 11st day be, 0 μ g/ μ l:8.3 ± 9.4,0.1 μ g/ μ l:100,0.01 μ g/ μ l:79.2 ± 20.6,0.001 μ g/ μ l:58.3 ± 9.4,0.0001 μ g/ μ l:12.5 ± 15.6; At the 12nd day be 0 μ g/ μ l:8.3 ± 9.4,0.1 μ g/ μ l:95.8 ± 8.2,0.01 μ g/ μ l:95.8 ± 8.2,0.001 μ g/ μ l:83.3 ± 13.3,0.0001 μ g/ μ l:12.5 ± 8.2 for PC027.

F. the MLB gene fragment clone is advanced in the carrier, described carrier is suitable for bacterium and is created in and has active double-stranded RNA in the insect

Below be the embodiment that to clone corresponding to the dna fragmentation of MLB gene target in the carrier into, described carrier is used for expressing double-stranded RNA host bacterium, although can use any carrier (with reference to WO0001846) of other any promotor that comprises the T7 promotor or effectively transcribe in bacterium.

Provide the specific primer sequence that is used for amplified target gene fragment PC010 among the table 8-PC.Employed template is the pCR8/GW/topo carrier that contains PC010 sequence (SEQ ID NO 253).Described primer is used for using the PCR that successively decreases (touch-down PCR) reaction of following condition: 95 ℃ 1 minute, be 95 ℃ 30 seconds, 60 ℃ of 20 round-robin 30 seconds (each its temperature that circulates reduces by 0.5 ℃) and 72 ℃ 1 minute then, be 95 ℃ 30 seconds, 50 ℃ of 15 round-robin 30 seconds and 72 ℃ 1 minute then, be then 72 ℃ 10 minutes.On sepharose, analyze gained PCR fragment, it is carried out purifying (QIAquick Gel Extraction kit, Cat.Nr.28706 Qiagen), clones into (with reference to WO00188121A1) and order-checking in the linearizing pGNA49A carrier of SrfI with its flush end.Table 8-PC has provided the sequence corresponding to the gained PCR product of SEQ ID NO 488.The recombinant vectors that comprises this sequence is called pGCDJ001.

G. in a coli strain AB301-105 (DE3), express and produce the double-stranded RNA target

Follow following step, express the double-stranded RNA of the insect target of proper level in bacterium, this double-stranded RNA has activity in insect.In this experiment, use the strains A B301-105 (DE3) of RNA enzyme III disappearance.

Transform AB301-105 (DE3)

Add in ice-cold chemoreception attitude intestinal bacteria AB301-105 (DE3) the bacterial strain equal portions of 50 μ l the 300ng plasmid and soft the mixing.Hatched described cell 20 minutes on ice, afterwards it is carried out 37 ℃ of heat shocks and handled 5 minutes, then described cell is returned on ice and kept again 5 minutes.The SOC substratum of 450 μ l room temperatures is added to described cell, and this suspension was hatched 1 hour on shaking table (250rpm) in 37 ℃.100 μ l bacterial cell suspensions are moved in the 500ml Erlenmeyer flask that contains the 150mlLB liquid nutrient medium, and described LB liquid nutrient medium is supplemented with the Pyocianil microbiotic of 100 μ g/ml.Described culture is hatched in 37 ℃ spend the night on Innova 4430 shaking tables (250rpm) (16-18 hours).

Chemical induction double stranded rna expression in AB301-105 (DE3)

Because there is the hereditary component of all control expression, so in AB301-105 (DE3) bacterial isolates, can express double-stranded RNA from recombinant vectors pGXXX0XX.In the presence of chemical inducer isopropylthiogalactoside (IPTG), the T7 polymkeric substance will drive transcribing of described target sequence with antisense and just direction, because described flank sequence is rightabout T7 promotor.

The thermal treatment of bacterium

Thereby make in the test slab the contaminated risk of artificial diet reduce to minimum by the thermal treatment killing bacteria.Yet, to the bacterium of expressing double-stranded RNA heat-treat be not insect induce disturb by RNA due to toxic prerequisite.Institute's inductive bacterial cultures is room temperature with 3000g centrifugal 10 minutes, abandoning supernatant, and will be deposited in 80 ℃ of water-baths and keep 20 minutes.After the thermal treatment, bacterial precipitation is resuspended in the 0.05% Triton X-100 solution that cumulative volume is 50ml.Describedly guarantee that to have 4 ℃ standby.

H. chrysomelid at the horseradish ape, the intestinal bacteria carry out laboratory experiment of expressing the dsRNA target is detected

The biological assay of leaf dish

At the chrysomelid larva of horseradish ape, use leaf dish bioassay method and detect the target PC010 double-stranded RNA (from plasmid pGCDJ001) that intestinal bacteria produce.As described in embodiment 4, prepare the leaf dish from rape leave.20 μ l bacterial suspensions (absorbance at the 600nm wavelength is 1) are added on each leaf dish.Described leaf dish is placed the hole of 24 orifice plates, and the agar of 1ml gelation is contained in described hole.Two newborn larvaes are added on each leaf dish.For every kind of processing, prepare 3 repeated experiments, wherein each repeated experiments comprises 16 newborn larvaes.Described flat board is kept in the insectarium, and its condition is 25 ± 2 ℃, 60 ± 5% relative humidity, 16:8 hour illumination: dark photoperiod.The 3rd day (being that biological assay began back 3 days), larva is moved to the new flat board that contains fresh treated (same dose) leaf dish.From the 5th day, every other day change the leaf material once.This biological assay is to mortality ratio and weight in average marking.Negative control is with the leaf dish of the bacterial treatment that comprises plasmid pGN29 (empty carrier) and has only leaf.

Behind the chrysomelid larva of rape leave feeding horseradish ape of handling with RNA enzyme III disappearance intestinal bacteria AB301-105 (DE3) bacterial strain that contains plasmid pGCDJ001, described insect mortality significantly increases in time, and in bacterium that comprises plasmid pGN29 that contrasts or only leafed situation, almost do not have or do not have insect death (Fig. 3-PC).

Biological assay based on plant

Bacterial suspension with the expression dsRNA of the chemical induction of heat inactivation is sprayed whole strain plant, uses described plant feeding MLB then.They are grown in the plant culturing chamber.Thereby cover described plant on the plant by the 500ml Plastic Bottle is inverted in, bottleneck places earth in the basin securely, cuts an opening in the bottle bottom, and covers and go up fine and closely woven nylon wire in order to ventilate, to reduce inside and condense and to prevent that larva from escaping.MLB is placed on the treated plant of every strain of cover.Handle plant with intestinal bacteria AB301-105 (DE3) the bacterial strain suspension that comprises pGCDJ001 or pGN29 plasmid.Apply the bacteriums of different amounts to plant: 66,22 and 7 units for example, one of them unit definition is: absorbance is in 1 the 1ml bacterial suspension 10 under the 600nm wavelength ⁹Individual bacterial cell.In each case, with atomizer cumulative volume 1～10ml is sprayed onto on the plant.In this test, handle for every kind and use a strain plant.Counting survival insect number, and write down the weight of every survival insect.

With intestinal bacteria AB301-105 (DE3) the bacterial strain suspension sprinkling plant of expressing, compare it with the pGN29 contrast and cause insect mortality significantly to increase from the target dsRNA of pGCDJ001.These test demonstration, the double-stranded RNA corresponding to the insect genes target sequence that produces in the bacterial expression system of wild-type or RNA enzyme III disappearance is toxic to insect, and described toxicity shows as the remarkable growth/development delay that increases and survive larva of insect mortality.Find out obviously also that from these experiments provide by using the effective protective plant/crop of spray agent to avoid the example of insect damage, described spray agent is made up of corresponding to the bacterium of the double-stranded RNA of insect genes target expression.

Embodiment 5: and mexican bean ladybird (Mexican bean beetle, MBB)

A. clone the portion gene sequence of mexican bean ladybird

According to the explanation of manufacturers, use TRIzol reagent (Cat.Nr.15596-026/15596-018, Invitrogen, Rockville, Maryland is USA) from the mexican bean ladybird (mexican bean ladybird of 4 different larval stage; Source: Thomas Dorsey, Supervising Entomologist, New Jersey Department of Agriculture, Divisionof Plant Industry, Bureau of Biological Pest Control, Phillip AlampiBeneficial Insect Laboratory, PO Box 330, Trenton, New Jersey 08625-0330, USA) the high-quality total RNA of middle separation.According to the explanation of manufacturers, (Cat.Nr.1700 Promega) handles the genomic dna of removing in this RNA prepared product by the DNA enzyme.According to the explanation of manufacturers, use commercial reagent box (SuperScript ^TMThe III reversed transcriptive enzyme, Cat.Nr.18080044, Invitrogen, Rockville, Maryland USA) obtains cDNA.

In order to separate the cDNA sequence that comprises EV005, EV009, EV010, EV015 and an EV016 gene part, use degenerated primer and use Amplitaq Gold (Cat.Nr.N8080240, Applied Biosystems) to carry out a series of PCR reactions according to the explanation of manufacturers.

Table has provided the degenerated primer sequence of the every kind of gene that is used to increase among the 2-EV, and it has shown the mexican bean ladybird target gene that comprises primer sequence and gained cDNA sequence.Use these primers to carry out each PCR reaction, it uses following condition: for EV005 and EV009,95 ℃ 10 minutes, be 95 ℃ 30 seconds, 50 ℃ of 40 round-robin 1 minute and 72 ℃ 1 minute and 30 seconds then, be then 72 ℃ 7 minutes; For EV014,95 ℃ 10 minutes, be 95 ℃ 30 seconds, 53 ℃ of 40 round-robin 1 minute and 72 ℃ 1 minute then, be then 72 ℃ 7 minutes; For EV010 and EV016,95 ℃ 10 minutes, be 95 ℃ 30 seconds, 54 ℃ of 40 round-robin 1 minute and 72 ℃ 1 minute and 40 seconds then, be then 72 ℃ 7 minutes.Analyze gained PCR fragment on sepharose, it is carried out purifying, and (QIAquick Gel Extraction kit, Cat.Nr.28706 Qiagen), are cloned into pCR4/TOPO carrier (Cat.Nr.K4530-20, Invitrogen) also order-checking with it.Table has provided the sequence of the gained PCR product of representing with SEQ ID NO separately among the 2-EV, and it is a partial sequence.Table has provided the corresponding section aminoacid sequence of representing with SEQ IDNO separately among the 3-EV, and wherein the starting point of reading frame indicates in bracket.

B. produce the dsRNA of mexican bean ladybird gene

Use commercial reagent box T7 Ribomax ^TMExpress RNAi System (Cat.Nr.P1700, Promega) dsRNA of synthetic milligram level.Obtain initial two kinds of independently single 5 ' T7 rna polymerase promoter subtemplates in two independent PCR reactions, each reaction comprises and the different target sequence of T7 promotor direction.

For every kind of target gene, use specificity T 7 forward primers and specific reverse primers to obtain adopted T7 template.Table has provided the sequence that the every kind of target sequence that is used to increase has each primer of adopted template among the 8-EV.

The condition of described PCR reaction is as follows: 95 ℃ 1 minute, be 95 ℃ 30 seconds, 60 ℃ of 20 round-robin 30 seconds and 72 ℃ 1 minute then, be 95 ℃ 30 seconds, 50 ℃ of 15 round-robin 30 seconds and 72 ℃ 1 minute then, be then 72 ℃ 10 minutes.In the PCR reaction of using condition same as described above, use specificity forward primer and specificity T 7 reverse primers to obtain antisense T7 template.Table has provided each primer sequence of the every kind of target gene antisense template that is used to increase among the 8-EV.On sepharose, analyze gained PCR product, and by the PCR purification kit (QiaquickPCR Purification Kit, Cat.Nr.28106, Qiagen) and NaClO ₄Precipitation is carried out purifying.According to the explanation of manufacturers, thereby resulting T7 forward and reverse template mixing are transcribed, gained RNA chain is annealed, carried out DNA enzyme and the processing of RNA enzyme and utilizes sodium-acetate to carry out purifying.Provided the sense strand of every kind of target gene gained dsRNA among the table 8-EV.

C. at the activity of mexican bean ladybird larva, use beans leaf dish that dsRNA target carry out laboratory experiment is detected

Embodiment provided below illustrates: mexican bean ladybird (Mexican beanbeetle, MBB) larva the dsRNA sensitivity corresponding to self target gene that per os is taken in.

In order to detect different double-stranded RNA samples, adopted use Kidney bean (Phaseolus vulgaris mutation Montano at the MBB larva; Source Aveve NV, Belgium) the leaf material is measured as the leaf dish of food source.The Kidney bean that uses same mutation in insectarium is to keep the insect culture, and its condition is 25 ± 2 ℃, 60 ± 5% relative humidity, 16 hours illumination/8 hour dark photoperiods.Use the beans plant leaf in from 1 to 2 age in week of puncher of suitable dimension to downcut the about 1.1cm of diameter (or 0.95cm ²) the leaf dish.With the Milli-Q water that contains 0.05% Triton X-100 with the double-stranded RNA diluted sample to 1 μ g/ μ l.By target Ev005 with 25 μ l, Ev010, Ev015, the diluting soln of Ev016 dsRNA and contrast gfp dsRNA or 0.05% Triton X-100 are applied to paraxial leaf surface and prepare treated leaf dish.With dry each hole that also places 24 orifice plates respectively of described leaf dish, 2% agar of 1ml gelation is contained in described hole, and it is withered that it helps anti-uppermost leaf dish.A newborn MBB larva is placed each hole of described plate, with the sponge plastics lid it is covered then.Described plate is divided into 3 repetitions, and each repeats to comprise 8 insects (every row).The flat board that will comprise described insect and leaf dish is kept in the insectarium, and its condition is 25 ± 2 ℃, 60 ± 5% relative humidity, 16 hours illumination/8 hour dark photoperiods.With leaf dish feeding insect 2 days, afterwards they are moved to the new flat board that contains fresh treated leaf dish.Then, began back 4 days in biological assay, moved to the culture dish that contain unprocessed fresh beans leaf with described insect every day, until the 10th day.At the 2nd day and every other day write down larval mortality and weight in average afterwards.

Mexican bean ladybird larva with the Kidney bean leaf feeding of the complete naked target dsRNA that comprises surface applications all causes larval mortality significantly to increase, as shown in Figure 1.After 8 days, the tested double-stranded RNA of target EV010, Ev015 and Ev016 causes 100% mortality ratio, and the dsRNA of target Ev005 is with killing all larvas in 10 days.With comprising contrast gfp dsRNA or only having most of insect of the leaf dish feeding of tensio-active agent Triton X-100 in whole biological assay, to keep survival (Fig. 1-EV).

D. at the activity of mexican bean ladybird adult, use beans leaf dish that dsRNA target carry out laboratory experiment is detected

Embodiment provided below for example understands: the dsRNA sensitivity corresponding to self target gene that the mexican bean ladybird adult is taken in per os.

In being provided with, detecting the MBB adult and resist the double-stranded RNA of topical application to beans leaf dish with the similar biological assay of mexican bean ladybird larva.To be diluted to final concentration from the test dsRNA of every kind of target (Ev010, Ev015 and Ev016) with 0.05% Triton X-100 is 0.1 μ g/ μ l.By 30 μ l test soln parts are applied on each leaf dish beans leaf dish is handled.Make described leaf dish complete drying, be placed on then on the 2% gelation agar thin slice in each hole of 24 orifice plates.Collect three age in days adults from culture hood, 7-8 hour feeding not places an adult each hole (therefore every kind of processing comprises 24 adults) of biological assay plate then.Described plate is kept in the insectarium (preserving 24 hours) under the condition identical with the MBB larva, afterwards described adult is moved to the new plate that comprises the fresh leaf dish of handling through dsRNA.After 24 hours, collect from the adult of every kind of processing, be placed in the plastics casing of 30cm * 15cm * 10cm, described plastics casing comprise two basins undressed 3 the week age beans plant.The assessment insect mortality from the 4th day to the 11st day.

All 3 kinds of target dsRNA (Ev010, Ev015 and Ev016) that ingested by the mexican bean ladybird adult cause mortality ratio significantly to increase since the 4th day (back 4 days of biological assay) beginning, shown in Fig. 2 (a)-EV.From the 5th day, the obvious change of the pattern of ingesting between insect that observes at the beans leaf dish feeding of handling with target dsRNA at first and the insect with the leaf dish feeding that comprises contrast gfp dsRNA or tensio-active agent TritonX-100.Compare with the contrast of having only tensio-active agent with gfp dsRNA, for of the leaf damage reduction (although level difference) of all obviously visible MBB adult of all 3 kinds of targets the beans plant of being untreated; Insect with target 15 feedings damages minimum (Fig. 2 (b)-EV) to the beans leaf.

E. the MBB gene fragment clone is advanced in the carrier, described carrier is suitable for bacterium and is created in and has active double-stranded RNA in the insect

It below is the example of will clone corresponding to the dna fragmentation of MBB gene target in the carrier into, described carrier is used for expressing double-stranded RNA host bacterium, although can use any carrier (with reference to WO0001846) of other any promotor that comprises the T7 promotor or effectively transcribe in bacterium.

Provide the specific primer sequence that is used for the amplified target gene among the table 8-EV.Employed template is the pCR8/GW/topo carrier that comprises any target sequence.Use described primer to carry out PCR reaction, it uses following condition: 98 ℃ 5 minutes, be 98 ℃ 10 seconds, 55 ℃ of 30 round-robin 30 seconds and 72 ℃ 2 minutes then, be then 72 ℃ 10 minutes.On sepharose, analyze gained PCR fragment, it is carried out purifying (QIAquick Gel Extraction kit, Cat.Nr.28706 Qiagen), clones into (with reference to WO00188121A1) and order-checking in the linearizing pGNA49A carrier of SrfI with its flush end.Table has provided among the 8-EV corresponding to the sequence of the gained PCR product of sequence separately.The recombinant vectors that comprises this sequence is called pGXXX0XX.

F. in two coli strains (1) AB301-105 (DE3) and (2) BL21 (DE3), express and produce the double-stranded RNA target

Follow following step, express the double-stranded RNA of the insect target of proper level in bacterium, this double-stranded RNA has activity in insect.The bacterial strain (AB301-105 (DE3)) of RNA enzyme III disappearance is used for comparing with the wild-type bacterium (BL21 (DE3)) that contains RNA enzyme III.

Transform AB301-105 (DE3) and BL21 (DE3)

Because there is the hereditary component of all control expression, so in AB301-105 (DE3) or BL21 (DE3) bacterial isolates, can express double-stranded RNA from recombinant vectors pGXXX0XX.In the presence of chemical inducer isopropylthiogalactoside (IPTG), the T7 polysaccharase will drive transcribing of described target sequence with antisense and sense orientation, because the flank of described target sequence is rightabout T7 promotor.

The thermal treatment of bacterium

G. at mexican bean ladybird, the intestinal bacteria carry out laboratory experiment of expressing the dsRNA target is detected

Biological assay based on plant

Spray whole strain plant with the bacterial suspension of the expression dsRNA of chemical induction, use described plant feeding MBB then.They are grown in the plant culturing chamber.Thereby cover described plant on the plant by the 500ml Plastic Bottle is inverted in, bottleneck places earth in the basin securely, cuts an opening in the bottle bottom, and covers and go up fine and closely woven nylon wire in order to ventilate, to reduce inside and condense and to prevent that larva from escaping.MMB is placed on the plant of the treated mistake of the every strain of cover.Handle plant with intestinal bacteria AB301-105 (DE3) suspension that comprises pGBNJ001 or pGN29 plasmid.Apply the bacteriums of different amounts to plant: 66,22 and 7 units for example, one of them unit definition is: absorbance is in 1 the 1ml bacterial suspension 10 under the 600nm wavelength ⁹Individual bacterial cell.In each case, with atomizer cumulative volume 1～10ml is sprayed onto on the plant.In this test, handle for every kind and use a strain plant.Counting survival insect number, and write down the weight of every survival insect.

With intestinal bacteria AB301-105 (DE3) the bacterial strain suspension sprinkling plant of expressing, compare it with the pGN29 contrast and cause insect mortality significantly to increase from the target dsRNA of pGXXX0XX.These test demonstration, the double-stranded RNA corresponding to the insect genes target sequence that produces in wild-type or RNA enzyme III defective type bacterial expression system is toxic to insect, and described toxicity shows as the remarkable growth/development delay that increases and survive larva of insect mortality.Find out obviously also that from these experiments provide by using the effective protective plant/crop of spray agent to avoid the example of insect damage, described spray agent is made up of corresponding to the bacterium of the double-stranded RNA of insect genes target expression.

Embodiment 6: Mexico's cotton boll resemble (Cotton boll weevil, CBW)

A. clone the portion gene sequence of Mexico cotton boll elephant

According to the explanation of manufacturers, use TRIzol reagent (Cat.Nr.15596-026/15596-018, Invitrogen, Rockville, Maryland, USA) from 3 age Mexico's cotton boll resemble that (Mexico's cotton boll resembles; The source: Dr.Gary Benzon, Benzon Research lnc., 7 Kuhn Drive, Carlisle, Pennsylvania 17013, USA) the high-quality global RNA of middle separation.According to the explanation of manufacturers, (Cat.Nr.1700 Promega) handles the genomic dna of removing in the RNA prepared product by the DNA enzyme.According to the explanation of manufacturers, use commercial reagent box (SuperScript ^TMThe III reversed transcriptive enzyme, Cat.Nr.18080044, Invitrogen, Rockville, Maryland USA) obtains cDNA.

In order to separate the cDNA sequence that comprises AG001, AG005, AG010, AG014 and an AG016 gene part, use degenerated primer and use Amplitaq Gold (Cat.Nr.N8080240, Applied Biosystems) to carry out a series of PCR reactions according to the explanation of manufacturers.

Table has provided the degenerated primer sequence of the every kind of gene that is used to increase among the 2-AG, and it has shown that the Mexico's cotton boll that comprises primer sequence and gained cDNA sequence resembles target gene.Use these primers to carry out separately PCR reaction, it uses following condition: for AG001, AG005 and AG016,95 ℃ 10 minutes, be 95 ℃ 30 seconds, 50 ℃ of 40 round-robin 1 minute and 72 ℃ 1 minute and 30 seconds then, be then 72 ℃ 7 minutes; For AG010,95 ℃ 10 minutes, be 95 ℃ 30 seconds, 54 ℃ of 40 round-robin 1 minute and 72 ℃ 2 minutes and 30 seconds then, be then 72 ℃ 7 minutes; For AG014,95 ℃ 10 minutes, be 95 ℃ 30 seconds, 55 ℃ of 40 round-robin 1 minute and 72 ℃ 1 minute then, be then 72 ℃ 7 minutes.Analyze gained PCR fragment on sepharose, it is carried out purifying, and (QIAquick Gel Extraction kit, Cat.Nr.28706 Qiagen), are cloned into pCR8/GW/TOPO carrier (Cat.Nr.K2500-20, Invitrogen) also order-checking with it.Table has provided the sequence of the gained PCR product of representing with SEQ ID NO separately among the 2-AG, and is called partial sequence.Table has provided the corresponding section aminoacid sequence of representing with SEQ IDNO separately among the 3-AG.

B. produce the dsRNA that Mexico's cotton boll resembles gene

For every kind of target gene, use specificity T 7 forward primers and specific reverse primers to obtain adopted T7 template.Table has provided the sequence that the every kind of target sequence that is used to increase has each primer of adopted template among the 8-AG.The following PCR reaction of successively decreasing: 95 ℃ 1 minute, be 95 ℃ 30 seconds, 60 ℃ of 20 round-robin 30 seconds (each circulation reduces by 0.5 ℃) and 72 ℃ 1 minute then, be 95 ℃ 30 seconds, 50 ℃ of 15 round-robin 30 seconds and 72 ℃ 1 minute then, be then 72 ℃ 10 minutes.In the PCR reaction of using condition same as described above, use specificity forward primer and specificity T 7 reverse primers to obtain antisense T7 template.Table has provided each primer sequence of the every kind of target gene antisense template that is used to increase among the 8-AG.On sepharose, analyze gained PCR product, and by the PCR purification kit (Qiaquick PCR Purification Kit, Cat.Nr.28106, Qiagen) and NaClO ₄Precipitation is carried out purifying.Thereby resulting T7 forward and reverse template mixing are transcribed, gained RNA chain is annealed, carried out DNA enzyme and the processing of RNA enzyme and utilizes sodium-acetate to carry out purifying.Provided the sense strand of every kind of target gene gained dsRNA among the table 8-AG.

C. the CBW gene fragment clone is advanced in the carrier, described carrier is suitable for bacterium and is created in and has active double-stranded RNA in the insect

Below be the embodiment that to clone corresponding to the dna fragmentation of CBW gene target in the carrier into, described carrier is used for expressing double-stranded RNA host bacterium, although can use any carrier (with reference to WO0001846) of other any promotor that comprises the T7 promotor or effectively transcribe in bacterium.

Provided the specific primer sequence that is used for the amplified target gene among the table 8-AG.Employed template is the pCR8/GW/topo carrier that contains any target sequence.Use described primer to carry out PCR reaction, it uses following condition: 98 ℃ 5 minutes, be 98 ℃ 10 seconds, 55 ℃ of 30 round-robin 30 seconds and 72 ℃ 2 minutes then, be then 72 ℃ 10 minutes.On sepharose, analyze gained PCR fragment, it is carried out purifying (QIAquick Gel Extraction kit, Cat.Nr.28706 Qiagen), clones into (with reference to WO00188121A1) and order-checking in the linearizing pGNA49A carrier of SrfI with its flush end.Table has provided among the 8-AG corresponding to the sequence of the gained PCR product of sequence separately.The recombinant vectors that comprises this sequence is called pGXXX0XX.

D. in two coli strains (1) AB301-105 (DE3) and (2) BL21 (DE3), express and produce the double-stranded RNA target

Transform AB301-105 (DE3) and BL21 (DE3)

The thermal treatment of bacterium

E. resemble at Mexico's cotton boll, the intestinal bacteria carry out laboratory experiment of expressing the dsRNA target is detected

Biological assay based on plant

Spray whole strain plant with the bacterial suspension of the expression dsRNA of chemical induction, use described plant feeding CBW then.They are grown in the plant culturing chamber.Thereby cover described plant on the plant by the 500ml Plastic Bottle is inverted in, bottleneck places earth in the basin securely, cuts an opening in the bottle bottom, and covers and go up fine and closely woven nylon wire in order to ventilate, to reduce inside and condense and to prevent that larva from escaping.CBW is placed on the plant of the treated mistake of the every strain of cover.Handle plant with intestinal bacteria AB301-105 (DE3) suspension that comprises pGXXX0XX or pGN29 plasmid.Apply the bacteriums of different amounts to plant: 66,22 and 7 units for example, one of them unit definition is: absorbance is in 1 the 1ml bacterial suspension 10 under the 600nm wavelength ⁹Individual bacterial cell.In each case, with atomizer cumulative volume 1～10ml is sprayed onto on the plant.In this test, handle for every kind and use a strain plant.Counting survival insect number, and write down the weight of every survival insect.

Embodiment 7: and red flour beetle (Red flour beetle, RFB)

A. clone the portion gene sequence of red flour beetle

According to the explanation of manufacturers, use TRIzol reagent (Cat.Nr.15596-026/15596-018, Invitrogen, Rockville, Maryland is USA) from the red flour beetle (red flour beetle of all different stepss; Source: Dr.Lara Senior, InsectInvestigations Ltd., Capital Business Park, Wentloog, Cardiff, CF3 2PX, Wales, UK) the high-quality global RNA of middle separation.According to the explanation of manufacturers, (Cat.Nr.1700 Promega) handles the genomic dna of removing in the RNA prepared product by the DNA enzyme.According to the explanation of manufacturers, use commercial reagent box (SuperScript ^TMThe III reversed transcriptive enzyme, Cat.Nr.18080044, Invitrogen, Rockville, Maryland USA) obtains cDNA.

In order to separate the cDNA sequence that comprises TC001, TC002, TC010, TC014 and a TC015 gene part, use degenerated primer and use Amplitaq Gold (Cat.Nr.N8080240, Applied Biosystems) to carry out a series of PCR reactions according to the explanation of manufacturers.

Table has provided the degenerated primer sequence of the every kind of gene that is used to increase among the 2-TC.Use these primers to carry out each PCR reaction, it uses following condition: 95 ℃ 10 minutes, be 95 ℃ 30 seconds, 50 ℃ of 40 round-robin 1 minute and 72 ℃ 1 minute and 30 seconds then, be 72 ℃ 7 minutes (TC001, TC014, TC015) then; 95 ℃ 10 minutes, be 95 ℃ 30 seconds, 54 ℃ of 40 round-robin 1 minute and 72 ℃ 2 minutes and 30 seconds then, be 72 ℃ 7 minutes (TC010) then; 95 ℃ 10 minutes, be 95 ℃ 30 seconds, 53 ℃ of 40 round-robin 1 minute and 72 ℃ 1 minute then, be 72 ℃ 7 minutes (TC002) then.Analyze gained PCR fragment on sepharose, it is carried out purifying, and (Qiagen), it is cloned into the pCR8/GW/TOPO carrier, and (Cat.Nr.K2500 20, Invitrogen) also order-checking for QIAquick Gel Extraction kit, Cat.Nr.28706.Table has provided the sequence of the gained PCR product of representing with SEQ ID NO separately among the 2-TC, and is called partial sequence.Table has provided the corresponding section aminoacid sequence of representing with SEQ ID NO separately among the 3-TC.

B. produce the dsRNA of red flour beetle gene

Use commercial reagent box T7Ribomax ^TMExpress RNAi System (Cat.Nr.P1700, Promega) dsRNA of synthetic milligram level.Independently obtain initial two kinds of independently single 5 ' T7 rna polymerase promoter subtemplates in the PCR reaction at two, each reaction comprises and the different target sequence of T7 promotor direction.

For every kind of target gene, use specificity T 7 forward primers and specific reverse primers to obtain adopted T7 template.Table has provided the sequence that the every kind of target sequence that is used to increase has each primer of adopted template among the 8-TC.Described PCR reaction conditions is as follows: 95 ℃ 1 minute, be 95 ℃ 30 seconds, 60 ℃ of 20 round-robin 30 seconds (0.5 ℃/circulation) and 72 ℃ 1 minute then, be 95 ℃ 30 seconds, 50 ℃ of 15 round-robin 30 seconds and 72 ℃ 1 minute then, be then 72 ℃ 10 minutes.In the PCR reaction of using condition same as described above, use specificity forward primer and specificity T 7 reverse primers to obtain antisense T7 template.Provided each primer sequence of the every kind of target gene antisense template that is used to increase among the 8-TC.On sepharose, analyze gained PCR product, and by the PCR purification kit (Qiaquick PCR Purification Kit, Cat.Nr.28106, Qiagen) and NaClO ₄Precipitation is carried out purifying.According to the explanation of manufacturers, thereby resulting T7 forward and reverse template mixing are transcribed, gained RNA chain is annealed, carried out DNA enzyme and the processing of RNA enzyme and utilizes sodium-acetate to carry out purifying.Provided the sense strand of every kind of target gene gained dsRNA among the table 8-TC.

C. at the red flour beetle larva, use artificial diet that dsRNA target carry out laboratory experiment is detected

Embodiment provided below for example understands the dsRNA sensitivity corresponding to self target gene that red flour beetle larva (RFB) is taken in per os.

Red flour beetle be maintained at Insect Investigations Ltd. (source: Imperial Collegeof Science, Technology and Medicine, Silwood Park, Berkshire, UK).SOP/251/01 according to company raises insect.In brief, described beetle is placed plastics pot or case.These jars or case have open top in order to ventilate.Thereby net is equipped with at its top and fix with flexible tie and to prevent to escape.Larva raising substratum (flour) is positioned in the container that can raise beetle.Raise the product beetle colony that preserves in temperature-controlling chamber, its condition is 25 ± 3 ℃, 16:8 hour illumination: dark cycle.

To be incorporated in the mixture (whole wheat flour: milk powder ratio is 4:1) of flour and milk powder from the double-stranded RNA (its sequence is corresponding to SEQ ID NO's 799) of target TC014, make it dried overnight.Prepare each repetition respectively: the 100 μ l dsRNA solution (1mgdsRNA) of 10 μ g/ μ l are joined in 0.1g flour/milk mixture.Dry mixture is ground to form fine powder.Insect maintains in the culture dish (55mm diameter), is lined with double-deck filter paper in this culture dish.Treated feed is positioned between the two layers of filter paper.With 10 1 age other larva of Combination be positioned in each ware (repetition).Repeated experiments is carried out in every kind of processing 4 times.Contrast is a Milli-Q water.Assess (survival insect number) with conventional criteria.In process of the test, test condition is 25-33 ℃, 20-25% relative humidity, 12:12 hour illumination: dark photoperiod.

Compare with the feed that has only contrast, use the survival rate of the red flour beetle larva of the artificial diet feeding of handling through target TC014dsRNA significantly to reduce in time, shown in Fig. 1-TC.

D. the RFB gene fragment clone is advanced in the carrier, described carrier is suitable for bacterium and is created in and has active double-stranded RNA in the insect

Below be the embodiment that to clone corresponding to the dna fragmentation of RFB gene target in the carrier into, described carrier is used for expressing double-stranded RNA host bacterium, although can use any carrier (with reference to WO0001846) of other any promotor that comprises the T7 promotor or effectively transcribe in bacterium.

Provide the specific primer sequence that is used for the amplified target gene among the table 8-TC.Employed template is the pCR8/GW/topo carrier that contains any target sequence.Use described primer to carry out PCR reaction, it uses following condition: 98 ℃ 5 minutes, be 98 ℃ 10 seconds, 55 ℃ of 30 round-robin 30 seconds and 72 ℃ 2 minutes then, be then 72 ℃ 10 minutes.On sepharose, analyze gained PCR fragment, it is carried out purifying (QIAquick Gel Extraction kit, Cat.Nr.28706 Qiagen), clones into (with reference to WO00188121A1) and order-checking in the linearizing pGNA49A carrier of SrfI with its flush end.Provided among the 8-TC corresponding to the sequence of the gained PCR product of sequence separately.The recombinant vectors that comprises this sequence is called pGXXX0XX.

E. in two coli strains (1) AB301-105 (DE3) and (2) BL21 (DE3), express and produce the double-stranded RNA target

Follow following step, express the double-stranded RNA of the insect target of proper level in bacterium, this double-stranded RNA has activity in insect.RNA enzyme III deficient strain (AB301-105 (DE3)) is used for comparing with the wild-type bacterium (BL21 (DE3)) that contains RNA enzyme III.

Transform AB301-105 (DE3) and BL21 (DE3)

The thermal treatment of bacterium

F. at red flour beetle, the intestinal bacteria carry out laboratory experiment of expressing the dsRNA target is detected

Biological assay based on plant

Spray whole strain plant with the bacterial suspension of the expression dsRNA of chemical induction, use described plant feeding RFB then.They are grown in the plant culturing chamber.Thereby cover described plant on the plant by the 500ml Plastic Bottle is inverted in, bottleneck places earth in the basin securely, cuts an opening in the bottle bottom, and covers and go up fine and closely woven nylon wire in order to ventilate, to reduce inside and condense and to prevent that larva from escaping.RFB is placed on the plant of the treated mistake of the every strain of cover.Handle plant with intestinal bacteria AB301-105 (DE3) suspension that comprises pGXXX0XX or pGN29 plasmid.Apply the bacteriums of different amounts to plant: 66,22 and 7 units for example, one of them unit definition is: absorbance is in 1 the 1ml bacterial suspension 10 under the 600nm wavelength ⁹Individual bacterial cell.In each case, with atomizer cumulative volume 1～10ml is sprayed onto on the plant.In this test, handle for every kind and use a strain plant.Counting survival insect number, and write down the weight of every survival insect.

Embodiment 8: and black peach aphid (Green peach aphid, GPA)

A. clone the part basic sequence of black peach aphid

According to the explanation of manufacturers, and use TRIzol reagent (Cat.Nr.15596-026/15596-018, Invitrogen, Rockville, Maryland is USA) from black peach aphid nymph (black peach aphid; Source: Dr.Rachel Down, Insect ﹠amp; Pathogen Interactions, CentralScience Laboratory, Sand Hutton, York, YO41 1LZ, UK) the high-quality global RNA of middle separation.According to the explanation of manufacturers, (Cat.Nr.1700 Promega) handles the genomic dna of removing in the RNA prepared product by the DNA enzyme.According to the explanation of manufacturers, use commercial reagent box (SuperScript ^TMThe III reversed transcriptive enzyme, Cat.Nr.18080044, Invitrogen, Rockville, Maryland USA) obtains cDNA.

In order to separate the cDNA sequence that comprises MP001, MP002, MP010, MP016 and a MP027 gene part, use degenerated primer and use Amplitaq Gold (Cat.Nr.N8080240, Applied Biosystems) to carry out a series of PCR reactions according to the explanation of manufacturers.

Table has provided the degenerated primer sequence of the every kind of gene that is used to increase among the 2-MP.Use these primers to carry out separately PCR reaction, it uses following condition: for MP001, MP002 and MP016,95 ℃ 10 minutes, be 95 ℃ 30 seconds, 50 ℃ of 40 round-robin 1 minute and 72 ℃ 1 minute and 30 seconds then, be then 72 ℃ 7 minutes; For MP027, the use PCR program of successively decreasing: 95 ℃ 10 minutes, being 95 ℃ 30 seconds, 60 ℃ of 10 round-robin 40 seconds (every circulation reduces by 1 ℃) and 72 ℃ 1 minute and 10 seconds then, is 95 ℃ 30 seconds, 50 ℃ of 30 round-robin 40 seconds and 72 ℃ 1 minute and 10 seconds then, be then 72 ℃ 7 minutes; For MP010,95 ℃ 10 minutes, be 95 ℃ 30 seconds, 54 ℃ of 40 round-robin 1 minute and 72 ℃ 3 minutes then, be then 72 ℃ 7 minutes.Analyze gained PCR fragment on sepharose, it is carried out purifying, and (QIAquick Gel Extraction kit, Cat.Nr.28706 Qiagen), are cloned into pCR8/GW/TOPO carrier (Cat.Nr.K2500-20, Invitrogen) also order-checking with it.Table has provided the sequence of the gained PCR product of representing with SEQ ID NO separately among the 2-MP, and is called partial sequence.Table has provided the corresponding section aminoacid sequence of representing with SEQ ID NO separately among the 3-MP.

B. produce the dsRNA of black peach aphid gene

Use commercial reagent box T7 Ribomax ^TMExpress RNAi System (Cat.Nr.P1700, Promega) dsRNA of synthetic milligram level.Independently obtain initial two kinds of independently single 5 ' T7 rna polymerase promoter subtemplates in the PCR reaction at two, each reaction comprises and the different target sequence of T7 promotor direction.

For every kind of target gene, use specificity T 7 forward primers and specific reverse primers to obtain adopted T7 template.Table has provided the sequence that the every kind of target sequence that is used to increase has each primer of adopted template among the 8-MP.The following PCR reaction of successively decreasing: 95 ℃ 1 minute, be 20 round-robin 95 ℃ 30 seconds, 55 ℃ 30 seconds (at MP001, MP002, MP016, MP027 and gfp) or 50 ℃ 30 seconds (at MP010) (every circulation reduce by 0.5 ℃) and 72 ℃ 1 minute then, be 95 ℃ 30 seconds, 45 ℃ of 15 round-robin 30 seconds and 72 ℃ 1 minute then, be then 72 ℃ 10 minutes.In the PCR reaction of using condition same as described above, use specificity forward primer and specificity T 7 reverse primers to obtain antisense T7 template.Table has provided each primer sequence of the every kind of target gene antisense template that is used to increase among the 8-MP.On sepharose, analyze gained PCR product, and by the PCR purification kit (Qiaquick PCR Purification Kit, Cat.Nr.28106, Qiagen) and NaClO ₄Precipitation is carried out purifying.According to the explanation of manufacturers, thereby resulting T7 forward and reverse template mixing are transcribed, gained RNA chain is annealed, carried out DNA enzyme and the processing of RNA enzyme and utilizes sodium-acetate to carry out purifying.Provided the sense strand of every kind of target gene gained dsRNA among the table 8-MP.

C. black peach aphid is to the laboratory test of transgenic arabidopsis plant infringement

Obtain transgenic plant

Use flower-dipping method (Clough and Bent (1998) Plant Journal 16 735-743) arabidopsis thaliana transformation plant.The over-ground part of described plant is hatched the several seconds in containing the solution of following material, described solution contains 5% sucrose, from the resuspended agrobacterium tumefaciens bacterial strain C58C1 Rif cell of overnight culture and 0.03% tensio-active agent Silwet L-77.After the inoculation, with transparent plastics cover plant 16 hours to keep humidity.In order to improve transformation efficiency, repeating said steps after the week.Behind seed maturity, stop to water seed that harvests drying and deepfreeze two days.After the sterilization, seed is layered in the growth medium that contains kantlex in order to select to transform plant.

Selected plant is moved to earth in order to produce the T2 seed best.

Biological assay

By being sprouted, isolating T2 seed selects the transgenic arabidopsis plant on the appropriate selection substratum.When the root of these transgenic plant is completed into, they are moved to fresh artificial growth medium or earth, they are grown under top condition.Detect whole strain transgenic plant at the black peach aphid nymph, the plant damage that its demonstration (1) causes the nymph that ingests has remarkable resistance, and (2) nymph mortality ratio improves, and/or the weight of (3) survival nymph reduces (or any other insect heteroplasia).

D. at the activity of black peach aphid, use the liquid artificial diet that dsRNA target carry out laboratory experiment is detected

The liquid artificial diet that are used for black peach aphid based on the feed preparation that is suitable for acyrthosiphum pisim, as described in Febvayet al. (1988) [Influence of the amino acid balance on the improvement of anartificial diet for a biotype of Acyrthosiphon pisum (Homoptera Aphididae) .Can.J.Zool.66:2449-2453], but carried out some modification.Be prepared as follows the amino acid composition of described feed: in mg/100ml, L-Ala 178.71, Beta-alanine 6.22, arginase 12 44.9, l-asparagine 298.55, aspartic acid 88.25, halfcystine 29.59, L-glutamic acid 149.36, glutamine 445.61, glycine 166.56, Histidine 136.02, Isoleucine 164.75, leucine 231.56, lysine hydrochloride 351.09, methionine(Met) 72.35, ornithine (HCl) 9.41, phenylalanine 293, proline(Pro) 129.33, Serine 124.28, Threonine 127.16, tryptophane 42.75, tyrosine 38.63, L-Xie Ansuan 190.85.Except that tyrosine, described amino acid is dissolved in the 30ml Milli-Q water, tyrosine at first was dissolved in before joining aminoacid mixture in the several 1M hydrochloric acid.Form in following 5 * concentrated stoste prepares the vitamine mixture component of described feed: with mg/L, and benzaminic acid 100, xitix 1000, vitamin H 1, calcium pantothenate 50, choline chloride 60 500, folic acid 10, inositol 420, nicotinic acid 100, pyridoxine hydrochloride 25, riboflavin 5, thiamine hydrochloride 25.Riboflavin is dissolved in the 1ml50 ℃ of water, joins then in the vitamine mixture stoste.Described vitamine mixture is divided into every equal portions 20ml, is stored in-20 ℃.One equal portions vitamine mixture is added to described amino acid solution.Respectively with following amount with sucrose and MgSO ₄7H ₂O joins in the described mixture: 20g and 242mg.Be prepared as follows trace metal stoste: in mg/100ml, CuSO ₄5H ₂O4.7, FeCl ₃6H ₂O 44.5, MnCl ₂4H2O 6.5, and NaCl 25.4, ZnCl ₂8.3.With 10ml trace metal solution and 250mgKH ₂PO ₄Add to feed, adding Milli-Q water to liquid feed final volume is 100ml.Is 7 with 1MKOH solution with the pH regulator of described feed.By 0.22 μ m filter (Millipore) with described liquid feed filtration sterilization.

In the controlled environment chamber, have in the aluminium frame cage of 70 μ m mesh with 4 to 6 age in week rape (Brassica napus mutation SW Oban, source Nick Balaam, Sw Seed Ltd., 49North Road, Abington, Cambridge, CB1 6AS UK) raises black peach aphid (black peach aphid; Source: Dr.Rachel Down, Insect ﹠amp; Pathogen Interactions, Central ScienceLaboratory, Sand Hutton, York, YO41 1LZ, UK), the condition of described chamber is as follows: 23 ± 2 ℃, 60 ± 5% relative humidity, 16:8 hour illumination: dark photoperiod.

The day before yesterday that biological assay begins, from rearging cage, collect adult and be placed on the rape leave of plucking recently in the culture dish, and be placed in the insectarium and spend the night.Second day, choose 1 age nymph and move to receptacle.Receptacle comprise place little culture dish (diameter 3cm) 10 1 age nymph, described culture dish is coated with thin individual layer and stretches and seal film (parafilm M), interpolation 50 μ l feeds on to it.Seal the described receptacle of film phonograph seal with another layer, under the condition identical, hatch with raising adult.Every other day change the feed that comprises dsRNA, in the survival rate of the 8th day (being that biological assay began the back the 8th day) assessment insect.At every kind of processing, set up 5 biological assay receptacles (repetition) simultaneously.To test and contrast (gfp) dsRNA solution mix in the feed to final concentration be 2 μ g/ μ l.Described receptacle is kept 23 ± 2 ℃, 60 ± 5% relative humidity, 16:8 hour illumination: dark photoperiod.Utilize GraphPad Prism edition 4 to measure the Mann-Whitney check, whether there were significant differences with intermediate value between definite target 27 (MP027) and the gfp dsRNA.

In described biological assay, use receptacle will be added with the liquid artificial diet feeding black peach aphid nymph of the complete naked dsRNA of target 27 (SEQ ID NO1061), it causes mortality ratio to significantly improve, as shown in Figure 1.Be respectively 2,34 and 82 with target 27, gfp dsRNA and the survival insect average percentage of having only feed to handle.Use Mann-Whitney check relatively target 027 and gfp dsRNA group, obtain single tail P value of 0.004, this shows the intermediate value of target 027 and the big intermediate value of expection of gfpdsRNA, and there were significant differences (P＜0.05).Black peach aphid with the liquid feed feeding that is mixed with target 27dsRNA is significantly less than the black peach aphid (data not shown) that usefulness has only feed or is mixed with the feed feeding of gfp dsRNA contrast.

E. the GPA gene fragment clone is advanced in the carrier, described carrier is suitable for bacterium and is created in and has active double-stranded RNA in the insect

Below be the embodiment that to clone corresponding to the dna fragmentation of GPA gene target in the carrier into, described carrier is used for expressing double-stranded RNA host bacterium, although can use any carrier (with reference to WO0001846) of other any promotor that comprises the T7 promotor or effectively transcribe in bacterium.

Provided the specific primer sequence that is used for the amplified target gene among the table 8-MP.Employed template is the pCR8/GW/topo carrier that contains any target sequence.Use described primer to carry out PCR reaction, it uses following condition: 98 ℃ 5 minutes, be 98 ℃ 10 seconds, 55 ℃ of 30 round-robin 30 seconds and 72 ℃ 2 minutes then, be then 72 ℃ 10 minutes.On sepharose, analyze gained PCR fragment, it is carried out purifying (QIAquick Gel Extraction kit, Cat.Nr.28706 Qiagen), clones into (with reference to WO00188121A1) and order-checking in the linearizing pGNA49A carrier of SrfI with its flush end.Table 8-MP has provided corresponding to the sequence of the gained PCR product of sequence separately.The recombinant vectors that comprises this sequence is called pGXXX0XX.

F. in two intestinal bacteria (1) AB301-105 (DE3) and (2) BL21 (DE3) bacterial strain, express and produce the double-stranded RNA target

Transform AB301-105 (DE3) and BL21 (DE3)

The thermal treatment of bacterium

G. at black peach aphid, the intestinal bacteria carry out laboratory experiment of expressing the dsRNA target is detected

Biological assay based on plant

Spray whole strain plant with the bacterial suspension of the expression dsRNA of chemical induction, use described plant feeding GPA then.They are grown in the plant culturing chamber.Thereby cover described plant on the plant by the 500ml Plastic Bottle is inverted in, bottleneck places earth in the basin securely, cuts an opening in the bottle bottom, and covers and go up fine and closely woven nylon wire in order to ventilate, to reduce inside and condense and to prevent that larva from escaping.GPA is placed on the plant of the treated mistake of the every strain of cover.Handle plant with intestinal bacteria AB301-105 (DE3) suspension that comprises pGXXX0XX or pGN29 plasmid.Apply the bacteriums of different amounts to plant: 66,22 and 7 units for example, one of them unit definition is: absorbance is in 1 the 1ml bacterial suspension 10 under the 600nm wavelength ⁹Individual bacterial cell.In each case, with atomizer cumulative volume 1～10ml is sprayed onto on the plant.In this test, handle for every kind and use a strain plant.Counting survival insect number, and write down the weight of every survival insect.

Embodiment 9: and brown paddy plant hopper (Brown plant hopper, BPH)

A. clone the partial sequence of brown paddy plant hopper

According to the explanation of manufacturers, use commercial reagent box (SuperScript ^TMThe III reversed transcriptive enzyme, Cat.N ° of .18080044, Invitrogen, Rockville, Maryland is USA) from brown paddy plant hopper (source: Dr.J.A.Gatehouse, Dept.Biological Sciences, Durham University, UK) high-quality global RNA obtains cDNA.

In order to separate the cDNA sequence that comprises brown paddy plant hopper NL001, NL002, NL003, NL004, NL005, NL006, NL007, NL008, NL009, NL010, NL011, NL012, NL013, NL014, NL015, NL016, NL018, NL019, NL021, NL022 and a NL027 gene part, use degenerated primer and use Amplitaq Gold (Cat.N ° of .N8080240, Applied Biosystems) to carry out a series of PCR reactions according to the explanation of manufacturers.

Table has provided the degenerated primer sequence of the every kind of gene that is used to increase among the 2-NL.Use these primers to carry out each PCR reaction, it uses following condition: for NL001,95 ℃ 5 minutes, be 95 ℃ 30 seconds, 55 ℃ of 40 round-robin 1 minute and 72 ℃ 1 minute then, be then 72 ℃ 10 minutes; For NL002,95 ℃ 3 minutes, be 95 ℃ 30 seconds, 55 ℃ of 40 round-robin 1 minute and 72 ℃ 1 minute then, be then 72 ℃ 10 minutes; For NL003,95 ℃ 3 minutes, be 95 ℃ 30 seconds, 61 ℃ of 40 round-robin 1 minute and 72 ℃ 1 minute then, be then 72 ℃ 10 minutes; For NL004,95 ℃ 10 minutes, be 95 ℃ 30 seconds, 51 ℃ of 40 round-robin 1 minute and 72 ℃ 1 minute then; For NL005,95 ℃ 10 minutes, be 95 ℃ 30 seconds, 54 ℃ of 40 round-robin 1 minute and 72 ℃ 1 minute then, be then 72 ℃ 10 minutes; For NL006,95 ℃ 10 minutes, be 95 ℃ 30 seconds, 55 ℃ of 40 round-robin 1 minute and 72 ℃ 3 minutes and 30 seconds then, be then 72 ℃ 10 minutes; For NL007,95 ℃ 10 minutes, be 95 ℃ 30 seconds, 54 ℃ of 40 round-robin 1 minute and 72 ℃ 1 minute and 15 seconds then, be then 72 ℃ 10 minutes; For NL008 and NL014,95 ℃ 10 minutes, be 95 ℃ 30 seconds, 53 ℃ of 40 round-robin 1 minute and 72 ℃ 1 minute then, be then 72 ℃ 10 minutes; For NL009, NL011, NL012 and NL019,95 ℃ 10 minutes, be 95 ℃ 30 seconds, 55 ℃ of 40 round-robin 1 minute and 72 ℃ 1 minute then, be then 72 ℃ 10 minutes; For NL010,95 ℃ 10 minutes, be 95 ℃ 30 seconds, 54 ℃ of 40 round-robin 1 minute and 72 ℃ 2 minutes and 30 seconds then, be then 72 ℃ 10 minutes; For NL013,95 ℃ 10 minutes, be 95 ℃ 30 seconds, 54 ℃ of 40 round-robin 1 minute and 72 ℃ 1 minute and 10 seconds then, be then 72 ℃ 10 minutes; For NL015 and NL016,95 ℃ 10 minutes, be 95 ℃ 30 seconds, 54 ℃ of 40 round-robin 1 minute and 72 ℃ 1 minute and 40 seconds then, be then 72 ℃ 10 minutes; For NL018,95 ℃ 10 minutes, be 95 ℃ 30 seconds, 54 ℃ of 40 round-robin 1 minute and 72 ℃ 1 minute and 35 seconds then, be then 72 ℃ 10 minutes; For NL021, NL022 and NL027,95 ℃ 10 minutes, be 95 ℃ 30 seconds, 54 ℃ of 40 round-robin 1 minute and 72 ℃ 1 minute and 45 seconds then, be then 72 ℃ 10 minutes.Analyze gained PCR fragment on sepharose, it is carried out purifying, and (Qiagen), it is cloned into the pCR8/GW/topo carrier, and (Cat.Nr.K2500 20, Invitrogen) also order-checking for QIAquick Gel Extraction kit, Cat.Nr.28706.Table provides the sequence of the gained PCR product of representing with SEQ ID NO separately among the 2-NL, and is called partial sequence.Table has provided the corresponding section aminoacid sequence of representing with SEQ ID NO separately among the 3-NL.

B. clone the partial sequence of brown paddy plant hopper NL023 gene by est sequence

From brown paddy plant hopper cDNA amplification Partial cDNA Sequence NL023, it is corresponding to the brown paddy plant hopper est sequence among the public database GenBank, and its sequence number is CAH65679.2.In order to separate the cDNA sequence that comprises a NL023 gene part, use Auele Specific Primer to carry out a series of PCR reactions according to the explanation of manufacturers based on EST, it uses PerfectShot ^TMExTaq (Cat N ° .RR005A, Takara Bio Inc.).

For NL023, independently use Auele Specific Primer oGBKW002 and oGBKW003 (representing with SEQ ID NO 1157 and SEQID NO 1158 respectively in this article) in the PCR reaction at two, its condition is as follows: 95 ℃ 3 minutes, be 95 ℃ 30 seconds, 56 ℃ of 30 round-robin 30 seconds and 72 ℃ 2 minutes then, be then 72 ℃ 10 minutes.On sepharose, analyze gained PCR fragment, to its carry out purifying (

Gel Extraction kit, Cat.N ° of .28706, Qiagen), and it is cloned into the pCR4-TOPO carrier, and (Cat N ° K4575-40 Invitrogen) also checks order.Represent that with SEQ ID NO 1111 it refers to the partial sequence of NL023 gene to the concensus sequence of two kinds of PCR product order-checking gained herein.Represent corresponding partial amino-acid series with SEQ ID NO1112 herein.

C. produce the dsRNA of brown paddy plant hopper gene

For every kind of target gene, use specificity T 7 forward primers and specific reverse primers to obtain adopted T7 template.Table has provided the sequence that the every kind of target sequence that is used to increase has each primer of adopted template among the 8-NL.The condition of described PCR reaction is as follows: for NL001 and NL002,94 ℃ 4 minutes, be 94 ℃ 30 seconds, 60 ℃ of 35 round-robin 30 seconds and 72 ℃ 1 minute then, be then 72 ℃ 10 minutes; For NL003,94 ℃ 4 minutes, be 94 ℃ 30 seconds, 66 ℃ of 35 round-robin 30 seconds and 72 ℃ 1 minute then, be then 72 ℃ 10 minutes; For NL004, NL006, NL008, NL009, NL010 and NL019,95 ℃ 4 minutes, be 95 ℃ 30 seconds, 54 ℃ of 35 round-robin 30 seconds and 72 ℃ 1 minute then, be then 72 ℃ 10 minutes; For NL005 and NL016,95 ℃ 4 minutes, be 95 ℃ 30 seconds, 57 ℃ of 35 round-robin 30 seconds and 72 ℃ 1 minute then, be then 72 ℃ 10 minutes; For NL007 and NL014,95 ℃ 4 minutes, be 95 ℃ 30 seconds, 51 ℃ of 35 round-robin 30 seconds and 72 ℃ 1 minute then, be then 72 ℃ 10 minutes; For NL011, NL012 and NL022,95 ℃ 4 minutes, be 95 ℃ 30 seconds, 53 ℃ of 35 round-robin 30 seconds and 72 ℃ 1 minute then, be then 72 ℃ 10 minutes; For NL013, NL015, NL018 and NL021,95 ℃ 4 minutes, be 95 ℃ 30 seconds, 55 ℃ of 35 round-robin 30 seconds and 72 ℃ 1 minute then, be then 72 ℃ 10 minutes; For NL023 and NL027,95 ℃ 4 minutes, be 95 ℃ 30 seconds, 52 ℃ of 35 round-robin 30 seconds and 72 ℃ 1 minute then, be then 72 ℃ 10 minutes.In the PCR reaction of using condition same as described above, use specificity forward primer and specificity T 7 reverse primers to obtain antisense T7 template.Table has provided each primer sequence of the every kind of target gene antisense template that is used to increase among the 8-NL.On sepharose, analyze gained PCR product, and (Qiaquick PCRPurification Kit, Cat.Nr.28106 Qiagen) carry out purifying by the PCR purification kit.Thereby resulting T7 forward and reverse template mixing are transcribed, gained RNA chain is annealed, carried out DNA enzyme and the processing of RNA enzyme and utilizes sodium-acetate to carry out purifying.But carrying out following modification: RNA is deposited in 70% ethanol and washes twice.Provide the sense strand of every kind of target gene gained dsRNA among the table 8-NL.

Contrast for described green fluorescent protein (gfp), the template DNA that uses the T7 primer to carry out the PCR reaction is pPD96.12 plasmid (the Fire Lab, http://genome-www.stanford.edu/group/fire/), it contains the wild-type gfp encoding sequence that is separated by 3 synthetic introns.Use commercial reagent box T7Ribomax ^TMExpress RNAiSystem (Cat.N ° of .P1700, Promega) synthetic double-stranded RNA.Independently obtain initial two kinds of independently single 5 ' T7 rna polymerase promoter subtemplates in the PCR reaction at two, each reaction comprises and the different target sequence of T7 promotor direction.For gfp, use specificity T 7FW primer oGAU183 and specificity RV primer oGAU182 (representing with SEQ ID NO 236 and SEQ ID NO 237 respectively) in the PCR reaction, to obtain adopted T7 template herein, its condition is as follows: 95 ℃ 4 minutes, be 95 ℃ 30 seconds, 55 ℃ of 35 round-robin 30 seconds and 72 ℃ 1 minute then, be then 72 ℃ 10 minutes.Use specificity FW primer oGAU181 and specificity T 7 RV primer oGAU184 (representing with SEQ ID NO 238 and SEQ ID NO 239 respectively) in the PCR of condition same as described above reaction, to obtain antisense T7 template herein.On sepharose, analyze gained PCR product, and to its carry out purifying (

PCR Purification Kit, C at.N ° .28106, Qiagen).Explanation according to manufacturers, thereby resulting T7 FW and the mixing of RV template are transcribed, with gained RNA chain annealing, carry out the DNA enzyme and the RNA enzyme is handled and utilize sodium-acetate and Virahol to carry out purifying, be deposited in 70% ethanol and wash twice but carry out following modification: RNA.The sense strand of gained dsRNA is represented with SEQ ID NO 235 herein.

D. at the activity of brown paddy plant hopper, use the liquid artificial diet that dsRNA target carry out laboratory experiment is screened

Be used for the liquid artificial diet (MMD-1) of brown paddy plant hopper as preparation as described in the Koyama (1988) [Artificial rearing and nutritional physiology ofthe planthoppersand leafhoppers (Homoptera:Delphacidae andDeltocephalidae) on a holidic diet JARQ 22 20-27], but changed the final concentration of feed sucrose component: use 14.4% (weight/volume).Feed ingredient is prepared as independently enriched material: 10 * mineral substance stoste (being stored in 4 ℃), 2 * amino acid primary liquid (being stored in-20 ℃) and 10 * VITAMIN stoste (being stored in-20 ℃).Mix described former fluid component to 4/3 * concentration before beginning is faced in biological assay, thereby allow to dilute with test dsRNA solution (4 * concentration), regulate pH to 6.5, filtration sterilization is made the equal portions of about 500 μ l.

In the controlled environment chamber, with 2-3 monthly age paddy rice (coming a paddy rice (Oryza sativacv Taichung Native 1) in the platform) the plant brown paddy plant hopper of feeding, the condition of described chamber is: 27 ± 2 ℃, and 80% relative humidity, 16:8 hour illumination: dark photoperiod.Comprise in the receptacle 10 1 ages or 2 age nymph, it is placed in to be stamped and draws thin individual layer to seal in the little culture dish (diameter 3cm) of film (parafilm M), described sealing is added with 50 μ l feeds on the film.Seal the described chamber of film phonograph seal with another floor, under the condition identical, hatch, but directly do not contact light with raising adult.Every other day change the feed that comprises dsRNA, assess the survival rate of insect every day.At every kind of processing, set up 5 biological assay receptacles (repetition) simultaneously.To test and contrast (gfp) dsRNA solution mix in the feed to final concentration be 2mg/ml.Described receptacle is kept 27 ± 2 ℃, 80% relative humidity, 16:8 hour illumination: dark photoperiod.Use Kaplan-Meier survivorship curve model that the insect survival data is analyzed, use the survival between sequence check (the Prism edition 4 .0) comparative group.

Use receptacle the liquid artificial diet feeding brown paddy plant hopper that external use has added complete naked dsRNA independently as shown in the biological assay as four, cause the nymph mortality ratio significantly to increase (Fig. 1 (a)-(d)-NL; Table 10-NL (a)-(d)) (Durham University).These results show, demonstrate remarkable toxicity to brown paddy plant hopper corresponding to the dsRNA of different BPH indispensable genes.

In these biological assays, gfp dsRNA does not have significant difference to the influence of BPH survival and the survival insect of a feeding feed.

Table 10-NL (a)-(d) shows the survival condition summary with the artificial diet feeding brown paddy plant hopper that is added with the following target of 2mg/ml (final concentration); In table 10-NL (a): NL002, NL003, NL005, NL010; In table 10-NL (b): NL009, NL016; In table 10-NL (c): NL014, NL018; And in table 10-NL (d): NL013, NL015, NL021.In the survival analysis hurdle, the effect of RNAi is following to be indicated: +=compare survival rate significantly descend (α＜0.05) with gfpdsRNA contrast;-=compare no significant difference with gfp dsRNA contrast.Use relatively survivorship curve (, between gfp dsRNA and test dsRNA, and having only between feed and the gfp dsRNA) of sequence check having only feed and having added between the feed of test dsRNA.

E. at the activity of brown paddy plant hopper, use artificial diet that different concns dsRNA carry out laboratory experiment is screened

With 50 μ l added different concns target NL002 dsRNA (be final concentration be 1,0.2,0.08 and 0.04mg/ml) the liquid artificial diet be applied to the brown paddy plant hopper receptacle.The feed that comprises dsRNA is every other day changed once, assesses the survival of insect every day.At every kind of processing, set up 5 biological assay receptacles (repetition) simultaneously.Described receptacle is kept 27 ± 2 ℃, 80% relative humidity, 16:8 hour illumination: dark photoperiod.Use Kaplan-Meier survivorship curve model that the insect survival data is analyzed, use the survival between sequence check (the Prism edition 4 .0) comparative group.

The liquid artificial diet that feeding is added with the complete naked target NL002 dsRNA of different concns cause that the BPH mortality ratio is significantly higher than the only survival rate of feeding feed under the final concentration that is low to moderate 0.04mg dsRNA/ml feed, shown in Fig. 2-NL and table 11-NL.Table 11-NL has summed up with the survival condition in the artificial diet feeding brown paddy plant hopper test of interpolation 1,0.2,0.08 and 0.04mg/ml (final concentration) target NL002.In the survival analysis hurdle, the effect of RNAi is following to be indicated: +=compare survival rate significantly descend (α＜0.05) with gfp dsRNA contrast;-=compare no significant difference with gfp dsRNA contrast.Use relatively survivorship curve of sequence check.

F. the BPH gene fragment clone is entered in the carrier, described carrier is suitable for bacterium and is created in and has active double-stranded RNA in the insect

Below be the embodiment that to clone corresponding to the dna fragmentation of BPH gene target in the carrier into, described carrier is used for expressing double-stranded RNA host bacterium, although can use any carrier (with reference to WO0001846) of other any promotor that comprises the T7 promotor or effectively transcribe in bacterium.

Provide the specific primer sequence that is used for the amplified target gene among the table 8-NL.Employed template is the pCR8/GW/topo carrier that contains any target sequence.Use described primer in PCR reaction, it uses following condition: 98 ℃ 5 minutes, be 98 ℃ 10 seconds, 55 ℃ of 30 round-robin 30 seconds and 72 ℃ 2 minutes then, be then 72 ℃ 10 minutes.On sepharose, analyze gained PCR fragment, it is carried out purifying (QIAquick Gel Extraction kit, Cat.Nr.28706 Qiagen), clones into (with reference to WO00188121A1) and order-checking in the linearizing pGNA49A carrier of SrfI with its flush end.Table 8-NL provides corresponding to the sequence of the gained PCR product of sequence separately.The recombinant vectors that comprises this sequence is called pGXXX0XX.

G. in two coli strains (1) AB301-105 (DE3) and (2) BL21 (DE3), express and produce the double-stranded RNA target

Transform AB301-105 (DE3) and BL21 (DE3)

The thermal treatment of bacterium

H. at brown paddy plant hopper, the intestinal bacteria carry out laboratory experiment of expressing the dsRNA target is detected

Biological assay based on plant

Spray whole strain plant with the bacterial suspension of the expression dsRNA of chemical induction, use described plant feeding BPH then.They are grown in the plant culturing chamber.Thereby cover described plant on the plant by the 500ml Plastic Bottle is inverted in, bottleneck places earth in the basin securely, cuts an opening in the bottle bottom, and covers and go up fine and closely woven nylon wire in order to ventilate, to reduce inside and condense and to prevent that larva from escaping.BPH is placed on the plant of the treated mistake of the every strain of cover.Handle plant with intestinal bacteria AB301-105 (DE3) suspension that comprises pGXXX0XX or pGN29 plasmid.Apply the bacteriums of different amounts to plant: 66,22 and 7 units for example, one of them unit definition is: absorbance is in 1 the 1ml bacterial suspension 10 under the 600nm wavelength ⁹Individual bacterial cell.In each case, with atomizer cumulative volume 1～10ml is sprayed onto on the plant.In this test, handle for every kind and use a strain plant.Counting survival insect number, and write down the weight of every survival insect.

Embodiment 10: and striped rice borer (Rice striped stem borer, SSB)

A. clone the partial sequence of striped rice borer gene by the PCR of family

According to the explanation of manufacturers, and use TRIzol reagent (Cat.Nr.15596-026/15596-018, Invitrogen, Rockville, Maryland, USA) striped rice borer from 4 different larval stage separates high-quality global RNA.According to the explanation of manufacturers, (Cat.Nr.1700 Promega) handles the genomic dna of removing in the RNA prepared product by the DNA enzyme.According to the explanation of manufacturers, use commercial reagent box (SuperScript ^TMThe III reversed transcriptive enzyme, Cat.Nr.18080044, Invitrogen, Rockville, Maryland USA) obtains cDNA.

In order to separate the cDNA sequence that comprises CS001, CS002, CS003, CS006, CS007, CS009, CS011, CS013, CS014, CS015, CS016 and a CS018 gene part, use degenerated primer and use Amplitaq Gold (Cat.Nr.N8080240, Applied Biosystems) to carry out a series of PCR reactions according to the explanation of manufacturers.

Table has provided the degenerated primer sequence of the every kind of gene that is used to increase among the 2-CS.Use these primers to carry out each PCR reaction, it uses following condition: 95 ℃ 10 minutes, be 95 ℃ 30 seconds, 55 ℃ of 40 round-robin 1 minute and 72 ℃ 1 minute then, be then 72 ℃ 10 minutes.Analyze gained PCR fragment on sepharose, it is carried out purifying, and (Qiagen), it is cloned into the pCR4/TOPO carrier, and (Cat.Nr.K2500 20, Invitrogen) also order-checking for QIAquick GelExtraction kit, Cat.Nr.28706.Table provides the sequence of the gained PCR product of representing with SEQ IDNO separately among the 2-CS, and is called partial sequence.Table 3-CS has provided the corresponding section aminoacid sequence of representing with SEQ ID NO separately.

B. produce the dsRNA of striped rice borer gene

For every kind of target gene, use specificity T 7 forward primers and specific reverse primers to obtain adopted T7 template.Table 8-CS has provided the sequence that the every kind of target sequence that is used to increase has each primer of adopted template.Described PCR reaction conditions is as follows: 95 ℃ 4 minutes, be 95 ℃ 30 seconds, 55 ℃ of 35 round-robin 30 seconds and 72 ℃ 1 minute then, be then 72 ℃ 10 minutes.In the PCR reaction of using condition same as described above, use specificity forward primer and specificity T 7 reverse primers to obtain antisense T7 template.Table has provided each primer sequence of the every kind of target gene antisense template that is used to increase among the 8-CS.On sepharose, analyze gained PCR product, and by the PCR purification kit (Qiaquick PCR Purification Kit, Cat.Nr.28106, Qiagen) and NaClO ₄Precipitation is carried out purifying.According to the explanation of manufacturers, thereby resulting T7 forward and reverse template mixing are transcribed, gained RNA chain is annealed, carried out DNA enzyme and the processing of RNA enzyme and utilizes sodium-acetate to carry out purifying.Table 8-CS has provided the sense strand of every kind of target gene gained dsRNA.

C. at the activity of Chilo spp larvae, use artificial diet that dsRNA target carry out laboratory experiment is detected

Striped rice borer (source: Syngenta, Stein, Switzerland) with based on Kamano and Sato, 1985 (Handbook of Insect Rearing Volumes I ﹠amp; II.P Singh andRF Moore, eds., Elsevier Science Publishers, Amsterdam and New York, 1985, pp448) described modified artificial diet are food.In brief, be prepared as follows one liter of feed: join in the 980ml Milli-Q water 20g agar and autoclaving; Make agar-agar soln be cooled to about 55 ℃, add all the other compositions and thoroughly mixing: 40g Semen Maydis powder (Polenta), 20g Mierocrystalline cellulose, 30g sucrose, 30g casein, 20g Fructus Hordei Germinatus (baking), 8g Wesson salt mixture, 12g Vanderzant vitamine mixture, 1.8g xitix, 1.6g Tegosept E (methyl p-hydroxybenzoate), 0.3g duomycin, 0.4g cholesterol and 0.6g L-halfcystine.Feed is cooled to about 45 ℃ to be poured in rearing tray or the cup then.Described feed is placed the horizontal laminar flow cabinet.From raising has the cover of adult moth, shift out and have the rice blade that insect lays eggs, secure it on the solid feed of raising cup or dish.Make the ovum hatching, newborn larvae is used for keeping of biological assay and insect culture.In test and raising, condition is 28 ± 2 ℃, 80 ± 5% relative humidity, 16:8 hour illumination: dark photoperiod.

Use same artificial diet to be used for biological assay, but in this case, described feed is on average poured in 24 orifice plates, the 1ml feed is contained in each hole.In case feed is ready, then the ratio with the 50 μ l target dsRNA of 1 μ g/ μ l is applied to this feed surface (2cm with test formulation ²).Make described dsRNA solution drying, with 21 age moth larvae place each hole.After 7 days, described larva is moved to treated feed fresh in the porous plate.The 14th day (being that biological assay began back 14 days), the number of record survival and dead insects, and check unusual.Every kind of processing is detected 24 larvas altogether.

Carry out an alternative biological assay, wherein used treated rice leaf feeding striped rice borer newborn larvae.To be dipped in the 0.05% Triton X-100 solution that contains 1 μ g/ μ l target dsRNA at the vanelets that comes No. in the rice variety platform, make it dry, and with an every hole that places 24 orifice plates that contain gelation 2% agar.Two newborn larvaes are transferred to the every leaf of handling through dsRNA (every kind of processing comprises 24 larvas) from rearing tray.After 4 and 8 days, described larva is transferred to fresh treated rice blade.At the number of assessment survival in the 4th, 8 and 12 day and dead insects, and write down any unusual.

D. the SSB gene fragment clone is advanced in the carrier, described carrier is suitable for bacterium and is created in and has active double-stranded RNA in the insect

Below be the embodiment that to clone corresponding to the dna fragmentation of SSB gene target in the carrier into, described carrier is used for expressing double-stranded RNA host bacterium, although can use any carrier (with reference to WO0001846) of other any promotor that comprises the T7 promotor or effectively transcribe in bacterium.

Provide the specific primer sequence that is used for the amplified target gene among the table 8-CS.Employed template is the pCR8/GW/topo carrier that contains any target sequence.Use described primer in PCR reaction, it uses following condition: 98 ℃ 5 minutes, be 98 ℃ 10 seconds, 55 ℃ of 30 round-robin 30 seconds and 72 ℃ 2 minutes then, be then 72 ℃ 10 minutes.On sepharose, analyze gained PCR fragment, it is carried out purifying (QIAquick Gel Extraction kit, Cat.Nr.28706 Qiagen), clones into (with reference to WO00188121A1) and order-checking in the linearizing pGNA49A carrier of SrfI with its flush end.Table 8-CS has provided corresponding to the sequence of the gained PCR product of sequence separately.The recombinant vectors that comprises this sequence is called pGXXX0XX.

Transform AB301-105 (DE3) and BL21 (DE3)

The thermal treatment of bacterium

F. at brown paddy plant hopper, the intestinal bacteria carry out laboratory experiment of expressing the dsRNA target is detected

Biological assay based on plant

Spray whole strain plant with the bacterial suspension of the expression dsRNA of chemical induction, use described plant feeding SSB then.They are grown in the plant culturing chamber.Thereby cover described plant on the plant by the 500ml Plastic Bottle is inverted in, bottleneck places earth in the basin securely, cuts an opening in the bottle bottom, and covers and go up fine and closely woven nylon wire in order to ventilate, to reduce inside and condense and to prevent that larva from escaping.SSB is placed on the plant of the treated mistake of the every strain of cover.Handle plant with intestinal bacteria AB301-105 (DE3) suspension that comprises pGXXX0XX or pGN29 plasmid.Apply the bacteriums of different amounts to plant: 66,22 and 7 units for example, one of them unit definition is: absorbance is in 1 the 1ml bacterial suspension 10 under the 600nm wavelength ⁹Individual bacterial cell.In each case, with atomizer cumulative volume 1～10ml is sprayed onto on the plant.In this test, handle for every kind and use a strain plant.Counting survival insect number, and write down the weight of every survival insect.

Embodiment 11: and diamond-back moth (Diamondback moth, DBM)

A. clone the partial sequence of diamond-back moth

According to the explanation of manufacturers, use TRIzol reagent (Cat.Nr.15596-026/15596-018, Invitrogen, Rockville, Maryland is USA) from the diamond-back moth (diamond-back moth of all different larval stage; The source: Dr.Lara Senior, Insect Investigations Ltd., Capital Business Park, Wentloog, Cardiff, CF3 2PX, Wales UK) separates high-quality global RNA.According to the explanation of manufacturers, (Cat.Nr.1700 Promega) handles the genomic dna of removing in the RNA prepared product by the DNA enzyme.According to the explanation of manufacturers, use commercial reagent box (SuperScript ^TMThe III reversed transcriptive enzyme, Cat.Nr.18080044, Invitrogen, Rockville, Maryland USA) obtains cDNA.

In order to separate the cDNA sequence that comprises PX001, PX009, PX010, PX015, a PX016 gene part, use degenerated primer and use Amplitaq Gold (Cat.Nr.N8080240, Applied Biosystems) to carry out a series of PCR reactions according to the explanation of manufacturers.

Table has provided the degenerated primer sequence of the every kind of gene that is used to increase among the 2-PX.Use these primer branches to carry out each PCR reaction, it uses following condition: 95 ℃ 10 minutes, be 95 ℃ 30 seconds, 50 ℃ of 40 round-robin 1 minute and 72 ℃ 1 minute and 30 seconds then, be 72 ℃ 7 minutes (at PX001, PX009, PX015, PX016) then; 95 ℃ 10 minutes, be 95 ℃ 30 seconds, 54 ℃ of 40 round-robin 1 minute and 72 ℃ 2 minutes and 30 seconds then, be 72 ℃ 7 minutes (at PX010) then.Analyze gained PCR fragment on sepharose, it is carried out purifying, and (Qiagen), it is cloned into the pCR8/GW/TOPO carrier, and (Cat.Nr.K2500 20, Invitrogen) also order-checking for QIAquick Gel Extraction kit, Cat.Nr.28706.Table has provided among the 2-PX with SEQ ID NO separately represents the sequence of gained PCR product, and is called partial sequence.Table provides the corresponding section aminoacid sequence of representing with SEQ ID NO separately among the 3-PX.

B. produce the dsRNA of diamond-back moth gene

For every kind of target gene, use specificity T 7 forward primers and specific reverse primers to obtain adopted T7 template.Table provides the sequence that the every kind of target sequence that is used to increase has each primer of adopted template among the 8-PX.Described PCR reaction conditions is as follows: 95 ℃ 1 minute, be 95 ℃ 30 seconds, 60 ℃ of 20 round-robin 30 seconds (0.5 ℃/circulation) and 72 ℃ 1 minute then, be 95 ℃ 30 seconds, 50 ℃ of 15 round-robin 30 seconds and 72 ℃ 1 minute then, be then 72 ℃ 10 minutes.In the PCR reaction of using condition same as described above, use specificity forward primer and specificity T 7 reverse primers to obtain antisense T7 template.Table provides each primer sequence of the every kind of target gene antisense template that is used to increase among the 8-PX.On sepharose, analyze gained PCR product, and by the PCR purification kit (Qiaquick PCR Purification Kit, Cat.Nr.28106, Qiagen) and NaClO ₄Precipitation is carried out purifying.According to the explanation of manufacturers, thereby resulting T7 forward and reverse template mixing are transcribed, gained RNA chain is annealed, carried out DNA enzyme and the processing of RNA enzyme and utilizes sodium-acetate to carry out purifying.Provide the sense strand of every kind of target gene gained dsRNA among the table 8-PX.

C. at the activity of diamond-back moth larva, use artificial diet that dsRNA target carry out laboratory experiment is detected

Diamond-back moth be maintained at Insect Investigations Ltd. (source: NewcastleUniversity, Newcastle-upon-Tyne, UK).With the described insect of Caulis et Folium Brassicae capitatae leaf feeding.Select 1 other larva of Combination in age (about 1 age in days) to be used for this test.Insect is placed Eppendorf pipe (volume 1.5ml).Will (Bio-Serv, NJ USA) place the lid (volume 0.25ml, diameter 8mm) of each pipe according to the commercially available diamond-back moth feed of manufacturers's explanation preparation.When still being liquid, with described feed levelling to remove unnecessary part and to obtain flat surface.

In case described feed is ready, then test formulation is applied to the feed surface, for each repeats the undiluted preparation of 25 μ l (1 μ g/ μ l target dsRNA).Make the test formulation drying, one 1 instar larvae is placed each pipe.Described larva is positioned over the surface of feed in the lid, will manages careful closure then.Described pipe is inverted preservation (covering at it), thereby makes every larva remain on the surface of described feed.Biweekly described larva is transferred to the new Eppendorf pipe that comprises fresh feed.Two a circumferential larva in test provide treated feed, and untreated feed is provided afterwards.

Altogether in 38 days, biweekly assess, all larvas are all dead in the time of the 38th day.When each assessment, assess the survival condition of described insect and check unusual.For every kind of processing, carry out 40 single larvas and repeat.In process of the test, test condition is 23～26 ℃, 50～65% relative humidity, 16:8 hour illumination: dark photoperiod.

D. the DBM gene fragment clone is advanced in the carrier, described carrier is suitable for bacterium and is created in and has active double-stranded RNA in the insect

Below be the embodiment that to clone corresponding to the dna fragmentation of DBM gene target in the carrier into, described carrier is used for expressing double-stranded RNA host bacterium, although can use any carrier (with reference to WO0001846) of other any promotor that comprises the T7 promotor or effectively transcribe in bacterium.

Provided the specific primer sequence that is used for the amplified target gene among the table 8-PX.Employed template is the pCR8/GW/topo carrier that contains any target sequence.Use described primer in PCR reaction, it uses following condition: 98 ℃ 5 minutes, be 98 ℃ 10 seconds, 55 ℃ of 30 round-robin 30 seconds and 72 ℃ 2 minutes then, be then 72 ℃ 10 minutes.On sepharose, analyze gained PCR fragment, it is carried out purifying (QIAquick Gel Extraction kit, Cat.Nr.28706 Qiagen), clones into (with reference to WO00188121A1) and order-checking in the linearizing pGNA49A carrier of SrfI with its flush end.Table 8-PX has provided corresponding to the sequence of the gained PCR product of sequence separately.The recombinant vectors that comprises this sequence is called pGXXX0XX.

Transform AB301-105 (DE3) and BL21 (DE3)

The thermal treatment of bacterium

F. at diamond-back moth, the intestinal bacteria carry out laboratory experiment of expressing the dsRNA target is detected

Biological assay based on plant

Spray whole strain plant with the bacterial suspension of the expression dsRNA of chemical induction, use described plant feeding DBM then.They are grown in the plant culturing chamber.Thereby cover described plant on the plant by the 500ml Plastic Bottle is inverted in, bottleneck places earth in the basin securely, cuts an opening in the bottle bottom, and covers and go up fine and closely woven nylon wire in order to ventilate, to reduce inside and condense and to prevent that larva from escaping.DBM is placed on the plant of the treated mistake of the every strain of cover.Handle plant with intestinal bacteria AB301-105 (DE3) suspension that comprises pGXXX0XX or pGN29 plasmid.Apply the bacteriums of different amounts to plant: 66,22 and 7 units for example, one of them unit definition is: absorbance is in 1 the 1ml bacterial suspension 10 under the 600nm wavelength ⁹Individual bacterial cell.In each case, with atomizer cumulative volume 1～10ml is sprayed onto on the plant.In this test, handle for every kind and use a strain plant.Counting survival insect number, and write down the weight of every survival insect.

Embodiment 12: and residential house Chinese mugwort Xi (House cricket, HC)

A. clone the partial sequence of residential house Chinese mugwort Xi

According to the explanation of manufacturers, use TRIzol reagent (Cat.Nr.15596-026/15596-018, Invitrogen, Rockville, Maryland is USA) from residential house Chinese mugwort Xi (the residential house Chinese mugwort Xi in all different insects stages; Source: Dr.Lara Senior, InsectInvestigations Ltd., Capital Business Park, Wentloog, Cardiff, CF3 2PX, Wales, UK) the high-quality global RNA of middle separation.According to the explanation of manufacturers, (Cat.Nr.1700 Promega) handles the genomic dna of removing in the RNA prepared product by the DNA enzyme.According to the explanation of manufacturers, use commercial reagent box (SuperScript ^TMThe III reversed transcriptive enzyme, Cat.Nr.18080044, Invitrogen, Rockville, Maryland USA) obtains cDNA.

In order to separate the cDNA sequence that comprises AD001, AD002, AD009, AD015 and an AD016 gene part, use degenerated primer and use Amplitaq Gold (Cat.Nr.N8080240, Applied Biosystems) to carry out a series of PCR reactions according to the explanation of manufacturers.

Table provides the degenerated primer sequence of the every kind of gene that is used to increase among the 2-AD.Use these primers to carry out separately PCR reaction, it uses following condition: 95 ℃ 10 minutes, be 95 ℃ 30 seconds, 50 ℃ of 40 round-robin 1 minute and 72 ℃ 1 minute and 30 seconds then, be then 72 ℃ 7 minutes.Analyze gained PCR fragment on sepharose, it is carried out purifying, and (Qiagen), it is cloned into the pCR8/GW/topo carrier, and (Cat.Nr.K2500 20, Invitrogen) also order-checking for QIAquick GelExtraction kit, Cat.Nr.28706.Table provides the sequence of the gained PCR product of representing with SEQ IDNO separately among the 2-AD, and is called partial sequence.Table provides the corresponding section aminoacid sequence of representing with SEQ ID NO separately among the 3-AD.

B. produce the dsRNA of residential house Chinese mugwort Xi gene

Use commercial reagent box T7Ribomax ^TMExpress RNAi System (Cat.Nr.P1700, Promega) dsRNA of synthetic milligram level.Obtain initial two kinds of independently single 5 ' T7 rna polymerase promoter subtemplates in two independent PCR reactions, each reaction comprises and the different target sequence of T7 promotor direction.

For every kind of target gene, use specificity T 7 forward primers and specific reverse primers to obtain adopted T7 template.Table provides the sequence that the every kind of target sequence that is used to increase has each primer of adopted template among the 8-AD.Described PCR reaction conditions is as follows: 95 ℃ 1 minute, be 95 ℃ 30 seconds, 60 ℃ of 20 round-robin 30 seconds (0.5 ℃/circulation) and 72 ℃ 1 minute then, be 95 ℃ 30 seconds, 50 ℃ of 15 round-robin 30 seconds and 72 ℃ 1 minute then, be then 72 ℃ 10 minutes.In the PCR reaction of using condition same as described above, use specificity forward primer and specificity T 7 reverse primers to obtain antisense T7 template.Table provides each primer sequence of the every kind of target gene antisense template that is used to increase among the 8-AD.On sepharose, analyze gained PCR product, and by the PCR purification kit (Qiaquick PCR Purification Kit, Cat.Nr.28106, Qiagen) and NaClO ₄Precipitation is carried out purifying.According to the explanation of manufacturers, thereby resulting T7 forward and reverse template mixing are transcribed, gained RNA chain is annealed, carried out DNA enzyme and the processing of RNA enzyme and utilizes sodium-acetate to carry out purifying.Provided the sense strand of every kind of target gene gained dsRNA among the table 8-AD.

C. at the activity of residential house Chinese mugwort Xi larva, use artificial diet that dsRNA target carry out laboratory experiment is detected

The America cricket be maintained at Insect Investigations Ltd. (source: BladesBiological Ltd., Kent, UK).Described insect is a food with wheat bran particle and Caulis et Folium Brassicae capitatae leaf.Select the mixing sex nymph that is no more than 5 ages in days of same size to be used for this test.With double-stranded RNA with based on particulate state rodent feed (rat and mouse standard feed, the B﹠amp of wheat; KUniversal Ltd., Grimston, Aldbrough, Hull UK) mixes.Described feed BK001P contains following compositions (with the weight descending sort): wheat, soybean, wheat bran, barley, particulate state tackiness agent, rodent 5vit min, mixing fat, Lin Suanergai, mould carb.Fine abrasive grains shape rodent makes any enzyme component inactivation thereby heat-treat in microwave oven, mix then.All rodent are taken from same batch to guarantee consistence.The feed that ground is mixed fully with dsRNA, heavy small-particle such as forms, with it in the ambient temperature overnight drying.

The double-stranded RNA sample of target and gfp contrast is used with the concentration of 10 μ g/ μ l, and its ratio is that the feed that 1g ground adds 1ml dsRNA solution, and obtaining the ratio that applies thus is the 10mgdsRNA/g particle.Change particle weekly.In three week in test, provide treated particle to insect.Untreated particle is provided afterwards.Insect is placed plastic containers with cover (diameter 9cm, dark 4.5cm), and each container comprises 10 insects.Comprise a treated bait particle and a water source (moist cotton balls) in each container, with above-mentioned both place the independently small-sized ship type ware (weigh boat) of weighing.In whole experiment, water is unrestrictedly replenished.

Assess with interval biweekly, be no more than 4 days between twice assessment, until all contrast insect deaths or cast off a skin to adult stage (84 days).In each assessment, survival condition is assessed and checked unusual.From the 46th day, Once you begin cast off a skin to adult, then assessing all insects (survival and death) is nymph or adult.The test the 55th day to the survival insect weigh.Repeated experiments is carried out in every kind of processing 4 times.In process of the test, test condition is 25～33 ℃, 20～25% relative humidity, 12:12 hour illumination: dark photoperiod.D. the HC gene fragment clone is advanced in the carrier, described carrier is suitable for bacterium and is created in and has active double-stranded RNA in the insect

Below be the embodiment that to clone corresponding to the dna fragmentation of HC gene target in the carrier into, described carrier is used for expressing double-stranded RNA host bacterium, although can use any carrier (with reference to WO0001846) of other any promotor that comprises the T7 promotor or effectively transcribe in bacterium.

Provided the specific primer sequence that is used for the amplified target gene among the table 8-AD.Employed template is the pCR8/GW/topo carrier that contains any target sequence.Use described primer in PCR reaction, it uses following condition: 98 ℃ 5 minutes, be 98 ℃ 10 seconds, 55 ℃ of 30 round-robin 30 seconds and 72 ℃ 2 minutes then, be then 72 ℃ 10 minutes.On sepharose, analyze gained PCR fragment, it is carried out purifying (QIAquick Gel Extraction kit, Cat.Nr.28706 Qiagen), clones into (with reference to WO00188121A1) and order-checking in the linearizing pGNA49A carrier of SrfI with its flush end.Table 8-AD has provided corresponding to the sequence of the gained PCR product of sequence separately.The recombinant vectors that comprises this sequence is called pGXXX0XX.

E. in two intestinal bacteria (1) AB301-105 (DE3) and (2) BL21 (DE3) bacterial strain, express and produce the double-stranded RNA target

Transform AB301-105 (DE3) and BL21 (DE3)

The thermal treatment of bacterium

F. at residential house Chinese mugwort Xi, the intestinal bacteria carry out laboratory experiment of expressing the dsRNA target is detected

Biological assay based on plant

Spray whole strain plant with the bacterial suspension of the expression dsRNA of chemical induction, use described plant feeding HC then.They are grown in the plant culturing chamber.Thereby cover described plant on the plant by the 500ml Plastic Bottle is inverted in, bottleneck places earth in the basin securely, cuts an opening in the bottle bottom, and covers and go up fine and closely woven nylon wire in order to ventilate, to reduce inside and condense and to prevent that larva from escaping.HC is placed on the plant of the treated mistake of the every strain of cover.Handle plant with intestinal bacteria AB301-105 (DE3) suspension that comprises pGXXX0XX or pGN29 plasmid.Apply the bacteriums of different amounts to plant: 66,22 and 7 units for example, one of them unit definition is: absorbance is in 1 the 1ml bacterial suspension 10 under the 600nm wavelength ⁹Individual bacterial cell.In each case, with atomizer cumulative volume 1～10ml is sprayed onto on the plant.In this test, handle for every kind and use a strain plant.Counting survival insect number, and write down the weight of every survival insect.

Table 10-NL (a)

¹=use the Kaplan-Meier survivorship curve to analyze analytical data

	x ²	The P value	Sig.Dif. ²
	x ²	The P value	Sig.Dif. ²	Food is right:
NL002	29.06	<0.0001	Be	Food is right:
NL002	29.06	<0.0001	Be	NL003	39.59	<0.0001	Be
NL005	2955	<0.0001	Be	NL003	39.59	<0.0001	Be
NL005	2955	<0.0001	Be	NL010	21.04	<0.0001	Be
Gfp dsRNA is right:				NL010	21.04	<0.0001	Be
Gfp dsRNA is right:				NL002	15.09	0.0001	Be
NL003	22.87	<0.0001	Be	NL002	15.09	0.0001	Be
NL003	22.87	<0.0001	Be	NL005	15.12	<0.0001	Be
NL010	8.838	0.0029	Be	NL005	15.12	<0.0001	Be
NL010	8.838	0.0029	Be	Food is to gfp dsRNA	4.030	0.0447(～0.05)	Not

²α<0.05

Table 10-NL (b)

¹=use the Kaplan-Meier survivorship curve to analyze analytical data

	x ²	The P value	Sig.Dif. ²
	x ²	The P value	Sig.Dif. ²	Food is right:
NL009	11.98	0.0005	Be	Food is right:
NL009	11.98	0.0005	Be	NL016	8.98	0.0027	Be
				NL016	8.98	0.0027	Be
				Gfp dsRNA is right:
NL009	13.69	0.0002	Be	Gfp dsRNA is right:
NL009	13.69	0.0002	Be	NL016	11.37	0.0007	Be
				NL016	11.37	0.0007	Be
				Food is to gfp dsRNA	0.03317	0.8555	Not

²α<0.05

Table 10-NL (c)

¹=use the Kaplan-Meier survivorship curve to analyze analytical data

	x ²	The P value	Sig.Dif. ²
	x ²	The P value	Sig.Dif. ²	Food is right:
NL014	8.088	0.0045	Be	Food is right:
NL014	8.088	0.0045	Be	NL018	10.47	0.0012	Be
				NL018	10.47	0.0012	Be
				Gfp dsRNA is right:
NL014	14.55	0.0001	Be	Gfp dsRNA is right:
NL014	14.55	0.0001	Be	NL018	17.64	<0.0001	Be
				NL018	17.64	<0.0001	Be
				Food is to gfp dsRNA	0.6548	0.4184	Not

²α<0.05

Table 10-NL (d)

¹=use the Kaplan-Meier survivorship curve to analyze analytical data

	x ²	The P value	Sig.Dif. ²
	x ²	The P value	Sig.Dif. ²	Food is right:
NL013	15.73	<0.0001	Be	Food is right:
NL013	15.73	<0.0001	Be	NL015	39.44	<0.0001	Be
NL021	12.75	0,0004	Be	NL015	39.44	<0.0001	Be
NL021	12.75	0,0004	Be
Gfp dsRNA is right:
Gfp dsRNA is right:				NL013	16.42	<0.0001	Be
NL015	39.15	<0.0001	Be	NL013	16.42	<0.0001	Be
NL015	39.15	<0.0001	Be	NL021	14.1	0,0002	Be
				NL021	14.1	0,0002	Be
				Food is to gfpdsRNA	0.1031	0,7481	Not

²α<0.05

Table 11-NL

¹=use the Kaplan-Meier survivorship curve to analyze analytical data

	x ²	The P value	Sig.Dif. ²
	x ²	The P value	Sig.Dif. ²	Food is right:
NL002?1μg/μl	57.53	<0.0001	Be	Food is right:
NL002?1μg/μl	57.53	<0.0001	Be	NL002?0.2μg/μl	74.54	<0.0001	Be
NL002?0.08μg/μl	64	<0.0001	Be	NL002?0.2μg/μl	74.54	<0.0001	Be
NL002?0.08μg/μl	64	<0.0001	Be	NL002?0.04μg/μl	39.49	<0.0001	Be

²α<0.05

Claims

1. isolating nucleotide sequence, it comprises and is selected from following nucleotide sequence:

(i) arbitrary sequence of following expression: SEQ ID NO 1,3,5,7,9,11,13,15,17,19,21,23,49 to 158,159,160-163,168,173,178,183,188,193,198,203,208,215,220,225,230,240 to 247,249,251,253,255,257,259,275 to 472,473,478,483,488,493,498,503,508 to 513,515,517,519,521,533 to 575,576,581,586,591,596,601,603,605,607,609,621 to 767,768,773,778,783,788,793,795,797,799,801,813 to 862,863,868,873,878,883,888,890,892,894,896,908to1040,1041,1046,1051,1056,1061,1066 to 1071,1073,1075,1077,1079,1081,1083,1085,1087,1089,1091,1093,1095,1097,1099,1101,1103,1105,1107,1109,1111,1113,1161 to 1571,1572,1577,1582,1587,1592,1597,1602,1607,1612,1617,1622,1627,1632,1637,1642,1647,1652,1657,1662,1667,1672,1677,1682,1684,1686,1688,1690,1692,1694,1696,1698,1700,1702,1704,1730 to 2039,2040,2045,2050,2055,2060,2065,2070,2075,2080,2085,2090,2095,2100,2102,2104,2106,2108,2120 to 2338,2339,2344,2349,2354,2359,2364,2366,2368,2370,2372,2384 to 2460,2461,2466,2471,2476,2481 or 2486, or its complementary sequence

SEQ ID NO 1,3,5,7,9,11,13,15,17,19,21,23,49 to 158,159,160-163,168,173,178,183,188,193,198,203,208,215,220,225,230,240 to 247,249,251,253,255,257,259,275 to 472,473,478,483,488,493,498,503,508 to 513,515,517,519,521,533 to 575,576,581,586,591,596,601,603,605,607,609,621 to 767,768,773,778,783,78B, 793,795,797,799,801,813 to 862,863,868,873,878,883,888,890,892,894,896,908 to 1040,1041,1046,1051,1056,1061,1066 to 1071,1073,1075,1077,1079,1081,1083,1085,1087,1089,1091,1093,1095,1097,1099,1101,1103,1105,1107,1109,1111,1113,1161 to 1571,1572,1577,1582,1587,1592,1597,1602,1607,1612,1617,1622,1627,1632,1637,1642,1647,1652,1657,1662,1667,1672,1677,1682,1684,1686,1688,1690,1692,1694,1696,1698,1700,1702,1704,1730 to 2039,2040,2045,2050,2055,2060,2065,2070,2075,2080,2085,2090,2095,2100,2102,2104,2106,2108,2120 to 2338,2339,2344,2349,2354,2359,2364,2366,2368,2370,2372,2384 to 2460,2461,2466,2471,2476,2481 or 2486, or its complementary sequence, and

SEQ ID NO 1,3,5,7,9,11,13,15,17,19,21,23,49 to 158,159,160-163,168,173,178,183,188,193,198,203,208,215,220,225,230,240 to 247,249,251,253,255,257,259,275 to 472,473,478,483,488,493,498,503,508 to 513,515,517,519,521,533 to 575,576,581,586,591,596,601,603,605,607,609,621 to 767,768,773,778,783,788,793,795,797,799,801,813 to 862,863,868,873,878,883,888,890,892,894,896,908 to 1040,1041,1046,1051,1056,1061,1066 to 1071,1073,1075,1077,1079,1081,1083,1085,1087,1089,1091,1093,1095,1097,1099,1101,1103,1105,1107,1109,1111,1113,1161 to 1571,1572,1577,1582,1587,1592,1597,1602,1607,1612,1617,1622,1627,1632,1637,1642,1647,1652,1657,1662,1667,1672,1677,1682,1684,1686,1688,1690,1692,1694,1696,1698,1700,1702,1704,1730 to 2039,2040,2045,2050,2055,2060,2065,2070,2075,2080,2085,2090,2095,2100,2102,2104,2106,2108,2120 to 2338,2339,2344,2349,2354,2359,2364,2366,2368,2370,2372,2384 to 2460,2461,2466,2471,2476,2481 or 2486, or its complementary sequence

Perhaps wherein said nucleotide sequence is the lineal homologue of gene of at least 17 continuous nucleotides that comprises the arbitrary sequence of following expression: SEQ ID NO 49 to 158,275 to 472,533 to 575,621 to 767,813 to 862,908 to 1040,1161 to 1571,1730 to 2039,2120 to 2338,2384 to 2460, or its complementary sequence.

2. the polynucleotide sequence of claim 1 is expressed the double-stranded ribonucleoside acid sequence that produces, and wherein the plant insect pest absorbs the growth that described ribonucleoside acid sequence suppresses described insect pest.

3. the ribonucleoside acid sequence of claim 2 wherein absorbs described sequence and suppresses and the expression of complementary nucleotide sequence basically of described sequence.

4. comprise the composition according to the ribonucleoside acid sequence of claim 2 or 3, it also comprises at least a adjuvant and randomly at least a tensio-active agent.

5. the composition that comprises at least a double-stranded RNA, wherein chain comprises and at least a portion complementary nucleotide sequence that is selected from the nucleotide sequence of sequence defined in the claim 1, and it also randomly comprises at least a appropriate carriers, vehicle or thinner.

6. with the polynucleotide cell transformed that comprises nucleotide sequence defined in the claim 1, described nucleotide sequence randomly with regulate sequence and effectively be connected.

7. the cell of claim 6, wherein said cell is a prokaryotic cell prokaryocyte, for example gram-positive cell or gram negative bacterium cell; Perhaps wherein said cell is an eukaryotic cell, for example yeast cell or alga cells.

8. the cell of claim 7, wherein said cell is a bacterial cell.

9. the cell of claim 7, wherein said cell is a yeast cell.

10. the composition that comprises at least a bacterial cell or yeast cell, described bacterial cell or yeast cell comprise at least a polynucleotide defined in the claim 1.

11. the composition of claim 10, wherein said bacterium or yeast cell are inactivated or are killed, for example by thermal treatment or mechanical treatment.

12. comprise at least a bacterium of expressing at least a double-stranded RNA or the composition of yeast cell, a chain of wherein said double-stranded RNA comprises and at least a portion complementary nucleotide sequence that is selected from the nucleotide sequence of sequence defined in the claim 1, also randomly comprises at least a appropriate carriers, vehicle or thinner in the said composition.

13. each composition in claim 5 or 10 to 12, described composition also comprises at least a following sterilant that is selected from: chemical insecticide, paratin, bacillus thuringiensis insecticidal proteins, Xenorhabdus insecticidal proteins, light rod bacterium insecticidal proteins, bacillus laterosporus insecticidal proteins and Bacillus sphaericus insecticidal proteins, described sterilant has the activity of the same plant insect pest defined in antagonism and the claim 2, and perhaps wherein said sterilant has the activity of one or more other plant insect pests of antagonism.

14. each composition in the claim 10 to 12, wherein said at least a bacterium or yeast cell also comprise or also express at least a following sterilant that is selected from: chemical insecticide, paratin, the bacillus thuringiensis insecticidal proteins, the Xenorhabdus insecticidal proteins, light rod bacterium insecticidal proteins, bacillus laterosporus insecticidal proteins and Bacillus sphaericus insecticidal proteins, described sterilant has the activity of the same plant insect pest defined in antagonism and the claim 2, and perhaps wherein said sterilant has the activity of one or more other plant insect pests of antagonism.

15. each composition in claim 5 or 10 to 12, it also comprises at least a other bacterium or yeast cell, described cell comprises or expresses at least a following sterilant that is selected from: chemical insecticide, paratin, the bacillus thuringiensis insecticidal proteins, the Xenorhabdus insecticidal proteins, light rod bacterium insecticidal proteins, bacillus laterosporus insecticidal proteins and Bacillus sphaericus insecticidal proteins, described sterilant has the activity of the same plant insect pest defined in antagonism and the claim 2, and perhaps wherein said sterilant has the activity of one or more specified plant insect pests of antagonism.

16. each composition in the claim 13 to 15, wherein said bacillus thuringiensis insecticidal proteins is selected from: Cry1, Cry3, TIC851, CryET170, Cry22, binary insecticidal proteins CryET33 and CryET34, binary insecticidal proteins CryET80 and CryET76, binary insecticidal proteins TIC100 and TIC101 and binary insecticidal proteins PS149B1.

17. each composition in the claim 13 to 16, it is as killing insect reagent.

18. each composition in the claim 13 to 16, it avoids the medicine of insect pest infestation as prevention or treatment human or animal body.

19. comprise the sprays of each at least a composition in the claim 10 to 18, it also randomly comprises at least a adjuvant and at least a tensio-active agent.

20. comprise the outer cover that is used for insect, trap or the bait of each composition that limits in the claim 10 to 18.

21. the outer cover of the sprays of each composition, claim 19 or claim 20, trap or bait are used for the purposes of kill insects or inhibition insect growth in the claim 10 to 18, described insect is selected from: colorado potato beetles species (Leptinotarsa spp.) (for example, colorado potato beetles (L.decemlineata), pseudo-colorado potato beetles (L.juncta) or Thory Elaeagnus Leaf eggplant chrysomelid (L.texana)), and

Described nucleic acid in the-wherein said composition comprises polynucleotide, or

Bacterium in the-wherein said composition or yeast cell comprise or express polynucleotide,

Described polynucleotide comprise and are selected from following nucleotide sequence:

(i) arbitrary sequence of following expression:

SEQ ID NO 1,3,5,7,9,11,13,15, and 17,19,21,23,49 to 158,159,160 to 163,168,173,178,183,188,193,198,203,208,215,220,225,230,240 to 246, or 2486, or its complementary sequence,

SEQ ID NO 1,3,5,7,9,11,13,15, and 17,19,21,23,49 to 158,159,160 to 163,168,173,178,183,188,193,198,203,208,215,220,225,230,240 to 246, or 2486, or its complementary sequence, and

Perhaps wherein said nucleotide sequence is the lineal homologue of gene of at least 17 continuous nucleotides that comprises the arbitrary sequence of following expression: SEQ ID NO 49 to 158, or its complementary sequence.

22. the outer cover of the sprays of each composition, claim 19 or claim 20, trap or bait are used for the purposes of kill insects or inhibition insect growth in the claim 10 to 18, described insect is selected from: phaedon species (Phaedon spp.) (for example, horseradish ape chrysomelid (P.cochleariae)) and

(i) arbitrary sequence of following expression: SEQ ID NO 247,249,251,253,255,257,259,275 to 472,473, and 478,483,488,493,498,503,508 to 512, or its complementary sequence,

SEQ ID NO 247,249,251,253,255,257,259,275 to 472,473, and 478,483,488,493,498,503,508 to 512, or its complementary sequence, and

SEQ ID NO 247,249,251,253,255,257,259,275 to 472,473, and 478,483,488,493,498,503,508 to 512, or its complementary sequence,

Perhaps wherein said nucleotide sequence is the lineal homologue of gene of at least 17 continuous nucleotides that comprises the arbitrary sequence of following expression: SEQ ID NO 275 to 472, or its complementary sequence.

23. the outer cover of the sprays of each composition, claim 19 or claim 20, trap or bait are used for the purposes of kill insects or inhibition insect growth in the claim 10 to 18, described insect is selected from: epilachna genus (Epilachna spp.) (for example, mexican bean ladybird (E.varivetis)), and

(i) arbitrary sequence of following expression:

SEQ ID NO 513,515,517,519,521,533 to 575,576, and 581,586,591 or 596, or its complementary sequence,

SEQ ID NO 513,515,517,519,521,533 to 575,576, and 581,586,591 or 596, or its complementary sequence, and

Perhaps wherein said nucleotide sequence is the lineal homologue of gene of at least 17 continuous nucleotides that comprises the arbitrary sequence of following expression: SEQ ID NO 533 to 575, or its complementary sequence.

24. the outer cover of the sprays of each composition, claim 19 or claim 20, trap or bait are used for the purposes of kill insects or inhibition insect growth in the claim 10 to 18, described insect is selected from: flower (for example resembles species (Anthonomus spp.), Mexico's cotton boll resembles (A.grandis)), and

(i) arbitrary sequence of following expression:

SEQ ID NO 601,603,605,607,609,621 to 767,768, and 773,778,783 or 788, or its complementary sequence,

SEQ ID NO 601,603,605,607,609,621 to 767,768, and 773,778,783 or 788, or its complementary sequence, and

Perhaps wherein said nucleotide sequence is the lineal homologue of gene of at least 17 continuous nucleotides that comprises the arbitrary sequence of following expression: SEQ ID NO 621 to 767, or its complementary sequence.

25. the outer cover of the sprays of each composition, claim 19 or claim 20, trap or bait are used for the purposes of kill insects or inhibition insect growth in the claim 10 to 18, described insect is selected from: Tribolium species (Tribolium spp.) (for example, red flour beetle (T.castaneum)), and

(i) arbitrary sequence of following expression:

SEQ ID NO 793,795,797,799,801,813 to 862,863, and 868,873,878 or 883, or its complementary sequence,

SEQ ID NO 793,795,797,799,801,813 to 862,863, and 868,873,878 or 883, or its complementary sequence, and

Perhaps wherein said nucleotide sequence is the lineal homologue of gene of at least 17 continuous nucleotides that comprises the arbitrary sequence of following expression: SEQ ID NO 813 to 862, or its complementary sequence.

26. the outer cover of the sprays of each composition, claim 19 or claim 20, trap or bait are used for the purposes of kill insects or inhibition insect growth in the claim 10 to 18, described insect is selected from: tumor aphid genus species (Myzus spp.) (for example, black peach aphid (M.persicae)), and

(i) arbitrary sequence of following expression: SEQ ID NO 888,890,892,894,896,908 to 1040,1041,1046,1051,1056,1061, or 1066 to 1070, or its complementary sequence,

SEQ ID NO 888,890,892,894,896,908 to 1040,1041,1046,1051,1056,1061, or 1066 to 1070, or its complementary sequence, and

SEQ ID NO 888,890,892,894,896,908 to 1040,1041,1046,1051,1056,1061, or 1066 to 1070, or its complementary sequence,

Perhaps wherein said nucleotide sequence is the lineal homologue of gene of at least 17 continuous nucleotides that comprises the arbitrary sequence of following expression: SEQ ID NO 908 to 1040, or its complementary sequence.

27. the outer cover of the sprays of each composition, claim 19 or claim 20, trap or bait are used for the purposes of kill insects or inhibition insect growth in the claim 10 to 18, described insect is selected from: brown paddy plant hopper species (Nilaparvata spp.) (for example, brown paddy plant hopper (N.lugens)), and

(i) arbitrary sequence of following expression:

SEQ ID NO 1071,1073,1075,1077,1079,1081,1083,1085,1087,1089,1091,1093,1095,1097,1099,1101,1103,1105,1107,1109,1111,1113,1161 to 1571,1572,1577,1582,1587,1592,1597,1602,1607,1612,1617,1622,1627,1632,1637,1642,1647,1652,1657,1662,1667,1672 or 1677, or its complementary sequence

SEQ ID NO 1071,1073,1075,1077,1079,1081,1083,1085,1087,1089,1091,1093,1095,1097,1099,1101,1103,1105,1107,1109,1111,1113,1161 to 1571,1572,1577,1582,1587,1592,1597,1602,1607,1612,1617,1622,1627,1632,1637,1642,1647,1652,1657,1662,1667,1672 or 1677, or its complementary sequence, and

Perhaps wherein said nucleotide sequence is the lineal homologue of gene of at least 17 continuous nucleotides that comprises the arbitrary sequence of following expression: SEQ ID NO 1161 to 1571, or its complementary sequence.

28. the outer cover of the sprays of each composition, claim 19 or claim 20, trap or bait are used for the purposes of kill insects or inhibition insect growth in the claim 10 to 18, described insect is selected from: straw borer spp species (Chilo spp.) (for example, striped rice borer (C.suppressalis), goldrimmed moth (C.auricilius) or rice Dolly snout moth's larva (C.polychrysus)), and

(i) arbitrary sequence of following expression: SEQ ID NO 1682,1684,1686,1688,1690,1692,1694,1696,1698,1700,1702,1704,1730 to 2039,2040,2045,2050,2055,2060,2065,2070,2075,2080,2085,2090 or 2095, or its complementary sequence

SEQ ID NO 1682,1684,1686,1688,1690,1692,1694,1696, and 1698,1700,1702,1704,1730 to 2039,2040,2045,2050,2055,2060,2065,2070,2075,2080,2085,2090 or 2095, or its complementary sequence, and

SEQ ID NO 1682,1684,1686,1688,1690,1692,1694,1696, and 1698,1700,1702,1704,1730 to 2039,2040,2045,2050,2055,2060,2065,2070,2075,2080,2085,2090 or 2095, or its complementary sequence,

Perhaps wherein said nucleotide sequence is the lineal homologue of gene of at least 17 continuous nucleotides that comprises the arbitrary sequence of following expression: SEQ ID NO 1730 to 2039, or its complementary sequence.

29. the outer cover of the sprays of each composition, claim 19 or claim 20, trap or bait are used for the purposes of kill insects or inhibition insect growth in the claim 10 to 18, described insect is selected from: the Plutella species (for example, diamond-back moth), and

(i) arbitrary sequence of following expression: SEQ ID NO 2100,2102,2104,2106,2108,2120 to 2338,2339, and 2344,2349,2354 or 2359, or its complementary sequence,

SEQ ID NO 2100,2102,2104,2106,2108,2120 to 2338,2339, and 2344,2349,2354 or 2359, or its complementary sequence, and

SEQ ID NO 2100,2102,2104,2106,2108,2120 to 2338,2339, and 2344,2349,2354 or 2359, or its complementary sequence,

Perhaps wherein said nucleotide sequence is the lineal homologue of gene of at least 17 continuous nucleotides that comprises the arbitrary sequence of following expression: SEQ ID NO 2120 to 2338, or its complementary sequence.

30. the outer cover of the sprays of each composition, claim 19 or claim 20, trap or bait are used for the purposes of kill insects or inhibition insect growth in the claim 10 to 18, described insect is selected from: the Acheta species (for example, residential house Chinese mugwort Xi (A.domesticus)), and

(i) following arbitrary sequence:

SEQ ID NO 2364,2366,2368,2370,2372,2384 to 2460,2461, and 2466,2471,2476 or 2481, or its complementary sequence,

SEQ ID NO 2364,2366,2368,2370,2372,2384 to 2460,2461, and 2466,2471,2476 or 2481, or its complementary sequence, and

Perhaps wherein said nucleotide sequence is the lineal homologue of gene of at least 17 continuous nucleotides that comprises the arbitrary sequence of following expression: SEQ ID NO 2384 to 2460, or its complementary sequence.

31. the outer cover of the sprays of each composition, claim 19 or claim 20, trap or the bait purposes in medicine or animal doctor's application in the claim 10 to 18.

Grow on plant or prevent the method for insect pest infestation plant 32. be used to prevent insect, it comprises each the composition or the sprays of claim 19 in the claim 10 to 18 is applied to described plant.

33. improve the method for output, it comprises each the composition or the sprays of claim 19 in the claim 10 to 18 of significant quantity is applied to plant.

34. the method for claim 32 or 33, wherein said plant is selected from: clover, apple, apricot, choke, asparagus, avocado, banana, barley, beans, beet, blackberry, blueberry, blueberry, sprouting broccoli, brussels sprouts, Caulis et Folium Brassicae capitatae, castor-oil plant, Radix Dauci Sativae, cassava, Cauliflower, cereal, celery, cherry, citrus, the little oranges and tangerines of Ke Laimenshi, coffee, corn, cotton, cucumber, eggplant, witloof, eucalyptus, Fructus Fici, grape, natsudaidai, Semen arachidis hypogaeae, gooseberry, Kiwifruit, lettuce, fragrant-flowered garlic, lemon, the lime bitter orange, pine tree, corn, mango, muskmelon, millet, mushroom, oat, gumbo, onion, orange, ornamental plant or flower or tree, papaya, parsley, pea, peach, peanut, pea, pepper, persimmon, pineapple, psyllium, plum, pomegranate, potato, pumpkin, witloof, radish, rape, immature fruit of Juteleaf Raspberry, rice, rye, Chinese sorghum, soya bean, soybean, spinach, strawberry, sugar beet, sugarcane, Sunflower Receptacle, Ipomoea batatas, orange, tea tree, tobacco, tomato, vine, watermelon, wheat, Chinese yam and summer squash.

35 as claimed in claim any one of 32 to 34, wherein said insect is selected from: MA Potato leaf beetle genus (eg, Colorado potato beetle, Colorado potato beetle or pseudo Elaeagnus eggplant cotyledon leaf beetle), co- Claw Oulema species (Lema spp.) (For example, three Oulema (L.trilineata)), eggplant Flea beetle species (Epitrix spp.) (For example, the U.S. potato flea beetle (E.cucumeris), tobacco Grass flea beetle (E.hirtipennis) or tuber flea beetle (E.tuberis)), Epicauta species (Epicauta spp.) (eg, North America Epicauta (E.vittata)), phytophagous ladybird species (eg, Mexico Colombian bean beetle), ape leaf beetle species (eg, Phaedon A), brown planthopper species (for example, Nilaparvata lugens), small brown planthopper species (Laodelphax spp.) (E.g., brown planthopper (L.striatellus)), Leafhopper species (Nephotettix spp.) (For example, two-point leafhopper (N.virescens), Or leafhopper (N.cincticeps), or two leafhopper (N.nigropictus)), white-backed Planthopper species (Sogatella spp.) (For example, white-backed planthopper (S.furcifera)), Acheta Species (eg, HOS Ai cricket), rod length bug species (Blissus spp.) (For example, the Americas Gu rod length bug (B.leucopterus leucopterus)), black bug species (Scotinophora spp.) (For example, black rice bug (S.vermidulate)), green bug species (Acrosternum spp.) (Example For example, proposed viridula (A.hilare)), species of rice skipper (Parnara spp.) (For example, straight Rice skipper (P.guttata)), Chilo species (eg, stem borer, rice and more Taiwanese rice borer or Korea borer (C.polychrysus)), Chilo species (Chilotraea spp.) (For example, rice straw Borer (C.polychrysa)), stem borers moth species (Sesamia spp.) (For example, rice stem borers Armyworm (S.inferens)), Wo borer moth species (Tryporyza spp.) (For example, white rice borer (T.innotata) or borer (T.incertulas)), leafroller species (Cnaphalocrocis spp.) (For example, rice leafroller (C.medinalis)), maggot Species (Agromyza spp.) (For example, Japanese rice leafminer (A.oryzae) or corn leafminer (A.parvicornis)), tallgrass borer species (Diatraea spp.) (For example, a small sugarcane borer (D. saccharalis) or Southwest corn stalks Grasshopper (D.gtandiosella)), Narnaga species (cases For example, green rice caterpillar (N.aenescens)), yellow moth species (Xanthodes spp.) (Case For example, plow lines yellow armyworm (X.transversa)), Spodoptera species (Spodoptera spp.) (Eg, Spodoptera frugiperda (S.frugiperda), beet armyworm (S.exigua), sea gray wings night Moth (S.littoralis) or western yellow striped armyworm (S.praefica)), Peru moth species (Mythimna spp.) (e.g., armyworm (Mythmna (Pseudaletia) seperata)), bell moth species (Helicoverpa spp.) (E.g., bollworm (H.zea)), Colaspis species (Colaspis spp.) (for example, Shaw grape leaf beetle (C.brunnea)), rice water weevil species (Lissorhoptrus spp.) (for example, rice water weevil (L.oryzophilus)), rice elephant species (Echinocnemus spp.) (for example, as rice (E.squamos)), rice armored species (Diclodispa spp.) (Eg, rice armored (D.armigera)), cereal Oulema species (Oulema spp.) (Eg, rice Oulema (O.oryzae)), rice weevil species (Sitophilus spp.) (Case For example, rice weevil (S.oryzae)), Pachydiplosis species (eg, gall midge (P.oryzae)), Hair eyes water fly species (Hydrellia spp.) (For example, wheat leaf hair eyes water flies (H.griseola) or Day Inage eyes water flies (H.sasakii)), Chlorops species (eg, rice straw maggot (C.oryzae)), Rootworm species (Diabrotica spp.) (For example, corn rootworm (D.virgifera virgifera), the North American corn rootworm (D.barberi), fresh cucumber rootworm root subspecies (D. undecimpunctata howardi), Mexican corn rootworm (D.virgifera zeae), Article Diabrotica pattern (D.balteata)), pole borer species (Ostrinia spp.) (For example, corn Rice borer (O.nubilalis)), and tiger species (Agrotis spp.) (For example, black cutworm (A. ipsilon)), Elasmopalpus species (for example, South America corn seedlings moth (E.lignosellus)), Comb Claw beetle genus (Melanotus spp.) (Nematodes), round rhinoceros beetles genus (Cyclocephala spp.) (For example, the northern camouflage beetles (C.borealis) or Southern camouflage beetles (C.immaculata)), Arc beetle species (Popillia spp.) (E.g., Japanese beetle arc (P.japonica)), Flea beetle species (Chaetocnema spp.) (For example, copper corn flea beetle (C.pulicaria)), Long neck like species (Sphenophorus spp.) (For example, corn long beak like (S.maidis)), Constriction aphid species (Rhopalosiphum spp.) (For example, corn aphid (R.maidis)), round An aphid (Anuraphis spp.) (For example, corn root aphid (A.maidiradicis)), black locust Species (Melanoplus spp.) (E.g., red foot black locust (M.femurrubrum)), Special Iso black locust (M.differentialis) or migratory black locust (M.sanguinipes)), kind of fly species (Hylemyaspp.) (for example, species of flies (H.platura)), stay thrips species (Anophothrips spp.) (for example, yellow stay thrips (A.obscrurus)), fire ant species (Solenopsis spp.) (For example, stolen ants (S.milesta)), mite species (Tetranychus spp.) (E.g., Tetranychus urticae Koch (T.urticae), carmine spider mite (T.cinnabarinus), bell moth species (cases For example, grain or Heliothis armigera (H.armigera)), Pectinophora species (for example, Red bell Gelechiidae (P.gossypiella)), diamond species (Earias spp.) (For example, Tsui pattern Drill armyworm (E.vittella)), Heliothis species (Heliothis spp.) (For example, Heliothis night Moth (H.virescens)), flower elephant species (Anthonomus spp.) (For example, Mexican cotton Bell like (A.grandis)), Pseudatomoscelis species (eg, cotton pseudo-legged bugs spot (P. seriatus)), knot species wing whitefly (Trialeurodes spp.) (e.g., junction wing whitefly (T. abutiloneus), greenhouse whitefly (T.vaporariorum)), small whitefly species (Bemisia spp.) (E.g., silver leaf whitefly (B.argentifolii)), aphid species (Aphis spp.) (E.g., Aphids (A.gossypii)), grass species bugs (Lygus spp.) (For example, the United States tarnished Bugs (L.lineolaris) or the Americas tarnished plant bug (L.hesperus)), the Americas bug species (Euschistus spp.) (e.g., spots Americas bug (E.conspersus)), Chlorochroa species (for example, For example, Saybolt bug (C.sayi)), green bug species (Nezara spp.) (For example, green rice bug (N. viridula)), thrips species (Thrips spp.) (e.g., tobacco thrips (T.tabaci)), Flower thrips species (Frankliniella spp.) (For example, tobacco brown thrips (F.fusca) or Western Flower thrips (F.occidentalis)), small green leafhopper species (Empoasca spp.) (E.g., Bean leafhopper (E.fabae)), phylloxera species (Myzus spp.) (For example, green peach aphid (M. persicae)), a psylla potato species (Paratrioza spp.) (e.g., potato psylla (P. cockerelli)), Chest wireworm species (Conoderus spp.) (eg, southern potato Nematodes (C.falli) or tobacco nematodes (C.vespertinus)), Gelechiidae species (Phthorimaea spp.) (e.g., potato Gelechiidae (P.operculella)), aphid species (Macrosiphum spp.) (e.g., Euphorbia avenae (M.euphorbiae)), Thyanta species (e.g., Red Shoulder stinkbug (T.pallidovirens)), Gelechiidae species (eg, potato Gelechiidae), bell night Moth species (eg, bollworm), Keiferia species (eg, tomato moth), gold Pin worm species (Limonius spp.) (Nematodes), Manduca species (eg, tobacco day Moth (M.sexta) or tomato hornworm (M.quinquemaculata)), leafminer species (Liriomyza spp.) (e.g., vegetable leafminer (L.sativae), clover leafminer (L.trifolli) or South U.S. leafminer (L.huidobrensis)), Drosophila species (Drosophilla spp.) (E.g., Drosophila melanogaster (D.melanogaster), D.yakuba, intends dark fruit flies (D.pseudoobscura) Or D.simulans (D.simulans)), beetles species (Carabus spp.) (For example, grain beetles (C.granulatus)), chironomid species (Chironomus spp.) (Eg, midges (C. tentanus)), Ctenocephalides flea species (Ctenocephalides spp.) (for example, Ctenocephalides felis (C. felis)), non-ear beak like species (Diaprepes spp.) (for example, root weevils (D. abbreviatus)), toothed bark beetle species (Ips spp.) (for example, the United States loose tooth beetles (I.pini)), Tribolium species (Tribolium spp.) (For example, Tribolium castaneum (T.castaneum)), Glossina species (Glossina spp.) (For example, barbed Glossina (G.morsitans)), Anopheles Species (Anopheles spp.) (E.g., Gambia Anopheles (A.gambiae)), is a bell armyworm Species (e.g., cotton bollworm (H.armigera)), Acyrthosiphon species (e.g., pea Bean aphid (A.pisum)), bee species (Apis spp.) (For example, the Italian bees (A. melifera)), Homalodisca species (eg, glass leafhopper (H.coagulate)), Aedes species (Aedes spp.) (For example, Egypt markings (Ae.aegypti)), silk moth is a matter Species (Bombyxspp.) (eg, Bombyx mori (B.mori)), locust species (Locusta spp.) (For example, locusts (L.migratoria)), is a species of ticks (Boophilus spp.) (E.g., Boophilus (B.microplus)), Acanthoscurria species (eg, orang chocolate Tarantulas (A.gomesiana)), A cockroach species (Diploptera spp.) (For example, Pacific Yang wings folded cockroach (D.punctata)), sleeve butterfly species (Heliconius spp.) (Eg, Valsalva Sleeve Butterfly (H.erato) or Muse Sleeve Butterfly (H.melpomene)), weevil species (Curculio spp.) (e.g., such as the European oak (C.glandium)), Plutella species (e.g., diamondback moth (P.xylostella)), flower tick species (Amblyomma spp.) (For example, color decorative flower ticks (A. variegatum)), tussah species (Anteraea spp.) (eg, Cecropia (A.yamamai)) And A mosquito species (Armigeres spp.) (For example, subalbatus (A.subalbatus)). ...

36. be used to the method that prevents that insect from growing in basic unit, it comprises each the composition or the sprays of claim 19 in the claim 10 to 18 is applied to described basic unit.

37. the disease that is used for the treatment of and/or prevents to be caused by the target biology or the method for illness, it comprises each the composition or the sprays of claim 19 in the claim 10 to 18 is applied to and has this to treat and/or prevent the object of needs.