CN109588562A - For controlling the composition and method of insect infestations in plant - Google Patents

Abstract

The present invention relates to the controls to pest infestation, this is by inhibiting to realize without one of vertebra harmful organism or a variety of biological functions.The invention discloses the method and compositions for controlling pest infestation, this is to be realized by the way that one or more different recombination double stranded rna molecules are fed to harmful organism by obtaining the reduction of pest infestation to the containment of gene expression.The invention further relates to manufacture expression double stranded rna molecule genetically modified plants method, and for protect plant keep out pest infestation transgenic insecticidal agent specific combination.

Description

For controlling the composition and method of insect infestations in plant

The application be international filing date be on April 8th, 2005 International Application Serial No. PCT/US2005/011816 enter China, Application No. is the invention of 200580019203.4 entitled " for controlling the composition and method of insect infestations in plant " is special The divisional application of benefit application.

Cross-reference to related applications

The present invention claims following U.S. Provisional Applications: being filed in the 60/560,842 of on April 9th, 2004, is filed in 2004 On April 27,60/565,632, be filed on June 11st, 2004 60/579,062, be filed on August 20th, 2004 60/603,421,60/617, the 281 of on October 11st, 2004 are filed in and are filed on April 7th, 2005 60/_________, they every part be all incorporated herein by reference in their entirety.

Technical field

In general, the present invention relates in plant and in animal and on animal to pest infestation carry out Heredity control.More particularly, the present invention relate to modify the endogenous expression of coded sequence in specific pest cell or tissue Method.More specifically, the present invention utilizes recombinant DNA technology, the expression of target coding sequence in pest cell is subject to Contain after transcription or inhibit, this is by being fed with part or all one or more from target coding sequence to harmful organism Thus the double-strand or small interference ribonucleic acid (RNA) molecule of transcription are controlled invasion come what is realized.Therefore, this hair It is bright to be related to inhibiting the sequence-specific that coded sequence is expressed by double-stranded RNA (dsRNA) or siRNA (siRNA), to obtain Obtain the control to the desirable level of harmful organism.

The present invention also provides it is novel, by separation and highly purified nucleic acid molecules comprising but be not limited to use in DsRNA of the present invention or siRNA molecule, non-naturally occurring nucleotide sequence and recombinant dna construct are transcribed, when being introduced into The evil biochron, it can contain or inhibit the expression of endogenous coding sequence or target coding sequence in harmful organism.The present invention also mentions Following genetically modified plants are supplied, (a) described plant contains following nucleotide sequence, the nucleotide sequence coded process separation With highly purified nucleic acid molecules and non-naturally occurring recombinant dna construct, it is used to control to plant pest for transcribing Biological attack dsRNA or siRNA molecule, and (b) show the tolerance of resistance and/or raising to insect infestations. Invention further describes: the composition containing dsRNA nucleotide sequence of the invention is used on plant or on animal or dynamic Topical application in the environment of object, so that pest infestation is eliminated or reduced.

Background technique

The environment of human survival is filled with pest infestation.Harmful organism includes: insect, spider, shellfish, true It is bacterium, bacterium, virus, nematode, flatworm, roundworm, pinworm, hookworm, tapeworm, trypanosoma, blood fluke, horse botfly, flea, flat Lice, mite, lice etc., they be it is in human environment generally existing, applied multiple means and attempted to control these harmful organisms Invasion.Composition for controlling small harmful organism (such as bacterium, fungi and virus) invasion has been provided as antibiotic group Close object, antiviral composition and antifungal composition form.For control larger harmful organism (for example, nematode, flatworm, Roundworm, pinworm, heartworm, tapeworm, trypanosoma, blood fluke etc.) invasion composition typically as the shape of Chemical composition that Formula exists, they can be applied to the known material surface that will appear pest infestation, or with pill, powder, tablet, soft The forms such as cream or capsule are by affected animal consumption.The present invention relates to offers: compared with composition known in the art, for controlling The improved method of pest infestation processed.

Commercial crops are usually the target of attack of insect.In over the past several decades, about exploitation for controlling elder brother in plant The more efficient way and composition of worm invasion, there has been the progress of some substances.Chemical insecticide is harmful for eradicating Biological attack is highly effective.But using chemical insecticide, there are many disadvantages.Chemical insecticide is non-selective.People's meaning Applied chemistry insecticide is intended to control the harmful no vertebra harmful organism for various crop and other plants.But because Lack selectivity, chemical insecticide can also apply its effect for non-targeted animal, also, answer used one in chemical insecticide The section time, it will usually keep field barren.Chemical insecticide is continuously present in environment, is usually metabolized slowly very much, if completely not If being metabolized.They can be accumulated in food chain, in the food chain of especially high carnivore.These chemical insecticides Accumulation leads to development to these reagent resistances and leads to the high-end species of ladder of evolving, the insecticide as mutagens with/ Or carcinogen is had an effect, and irreversible harmful genetic modification is typically resulted in.Therefore people need the side friendly to environment strongly Method is used to controlling or eradicating insect infestations in plant or on plant, that is, selective, environment is inert, non-continuous reservation And it is biodegradable, and can be the method for Pest-resistant management system well.

Composition including Bacillus thuringiensis (B.t.) bacterium can be obtained through commercial channels, It has used as environmentally safe and acceptable insecticide more than 30 years.The insecticidal effect of Bt bacterium is this The protein of a little proprietary generations of bacterium as a result, the protein will not be continuously present in environment, also, for impacted Target species have the selectivity of height, only apply its effect by being absorbed by object noxious livings, they have shown that To the innocuousness of environment and other nontarget organisms (including the mankind).One or more bases containing encoding insecticidal B.t. albumen The genetically modified plants of cause can also obtain in the art, their significant effectives are in the invasion of control harmful insect.Use expression Bt The substance of the recombinant plant of insecticidal protein is applied to environment as a result, in the area using such genetically modified crops to control The amount of the chemical insecticide of fabrication field pest infestation significantly reduces.Chemical insecticide application reduction so that soil more To clean, and the water for flowing to surrounding streams, rivers, pond and lake from soil is also more cleaned.Except these environment are good Except place, the crop field of the anti-insect plant growth of transgenosis, the quantity of advantageous insect is dramatically increased, this is because chemistry kills The reason of the use reduction of worm agent.

Antisense approach and composition are had reported in this field, they are believed can be by single strand RNA molecule (theoretically, Can complementary with height positive-sense strand RNA molecule hybridization in vivo) synthesis apply its effect.Antisense technology is difficult in many systems Middle use, there are three main causes for this.Firstly, the antisense sequences expressed in transformed cells are unstable.Second, table in transformed cells The unstability of the antisense sequences reached is transported to host, cell type or biology far from transgenic cell to by the sequence therewith System causes difficulty.Third, as the difficulty that the transport of unstability and antisense sequences encounters also is made for attempting as follows Made difficulty, the attempt is: target justice nucleosides can effectively be adjusted by providing inside the recombinant cell for encoding the antisense sequences The dosage of acid sequence expression.

Technology for adjusting gene expression dose in cell, tissue or biology rarely has improvement, particularly, lack and be used for Postpone, contain or reduce the technology of the development of specific gene expression by using recombinant DNA technology.In addition, as these hands Section Unpredictability as a result, in eukaryon or prokaryotes adjust specific gene expression, business way Means obtained by diameter.

In the past it has been shown that carrying out the inhibition of double chain RNA mediate to specific gene in a variety of harmful organisms.In drosophila (Tabara et is detected for genetically controlled means to what dsRNA was mediated in Drosophila melanogaster Al., 1998, Science 282:430-431).Tabara et.al. describes a kind of method, turns for transporting to be related to generating DsRNA solution is injected into insect bodies or before embryonic development by the dsRNA of gene insect (it expresses double stranded rna molecule) It is injected into egg capsule.It is shown before researcher, by the way that the solution containing double-strand or siRNA molecule is fed to line Nematode is dipped in the solution by worm, and by injection dsRNA molecule, the gene of double chain RNA mediate can be obtained in nematode Containment.Rajagopal et al. describes the attempt to fail as follows: by being fed with containing the molten of the dsRNA for being specific to target gene Liquid, or by the way that larva to be dipped in the solution, to contain the endogenous gene of harmful insect Spodoptera litura larva, but It is, after dsRNA is injected into the hemolymph of the 5th state larva with micro syringe (microapplicator), Rajagopal et al. is but successfully contained (J.Biol.Chem., 2002,277:46849-46851).Similarly, Mesa et al. (Us 2003/0150017) is predictably described for using the dsRNA for being transported to larva to inhibit lepidoptera The preferred gene seat of larva Helicoverpa atmigera, it is described to be carried through the plant for taking in and being converted into and generating dsRNA Object is realized.It is believed that providing dsRNA molecule in the diet of most of no vertebra pests or will contain It is unpractical that the composition of dsRNA, which is injected into no vertebra harmful organism body,.DsRNA points are provided to no vertebra harmful organism The diet method of son be it is unpractical, because of RNA molecule, even stable double stranded rna molecule, in mild alkalinity or acid It is in environment (for example, found in most of no vertebra harmful organism alimentary canals) and highly unstable, it is easy by ring Nuclease in border is degraded.Accordingly, it is desirable to by containment, delay or reduce base in specific no vertebra harmful organism The improved method that gene expression is adjusted because of expression, the method is in order to control the purpose of pest infestation or for introducing New phenotypic character.

Summary of the invention

The present invention, in one embodiment, the method comprising inhibiting to express without target gene in vertebra harmful organism.It is special Not, the present invention includes to no vertebra harmful organism (particularly, Western corn rootworm (WCR, Diabrotica Virgifera virgifera LeConte)) in one or more target genes the expression method that is adjusted or inhibited, The method can cause feed, growth, development, breeding and propagation to stop, and eventually lead to insect death.The described method includes: Some or all of double-stranded RNA (dsRNA) or its modified forms (for example, siRNA (siRNA) sequence) will be stabilized to draw Enter into no vertebra harmful insect body in cell or extracellular environment (for example, middle intestines), in no vertebra harmful insect body, DsRNA or siRNA are entered in cell, are inhibited to the expression of at least one or more of target gene, and, wherein it is right The inhibition of one or more target gene expression is applied with harmful effect on no vertebra harmful organism.Special consideration should be given to, Method and composition of the invention will be used for: one or more comprising dsRNA molecule by providing in harmful organism diet Composition, any harmful organism host, harmful organism homobium or harmful organism hobby environment in limit or eliminate nothing The invasion of vertebra harmful organism, as long as harmful organism digestive system pH, in the range of about 4.5 to about 9.5, about 5 to big About 9, about 6 to about 7 and about pH 7.0.

The invention discloses illustrative sequence tables, wherein containing Western corn rootworm (WCR, Diabrotica is come from Virgifera nucleotide and amino acid sequence), such as SEQ ID NO:1 to SEQ ID NO:143 and SEQ ID NO:169 To shown in SEQ ID NO:174, also contains and come from other coleopterous insects, including Colorado potato bug (CPB, Leptinotarsa decemlineata) and red flour beetle (RFB, Tribolium castaneum)；From lepidopterid, Including European corn borer (ECB, Ostrinia nubilalis), black cutworm (BCW, Agrotis ipsilon), corn Noctuid (CEW, Helicoverpa zea), armyworm in autumn (FAW, Spodoptera frugiperda), boll weevil (BWV, Anthonomus grandis), silkworm (Bombyx mori) and Manduca sexta, and dipteron is come from, Sequence including Drosophila melanogaster, Anopheles gambiae and Aedes aegypti, such as SEQ Shown in ID NO:144 to SEQ ID NO:159.Sequence table is wrapped in the form of CD-ROM CD with the papery copy of the application It includes.

It is submitting, contain the sequence table information for following sequence: corn corresponding to the computer-reader form of sequence table Rootworm Unigene sequence, est sequence, corn rootworm specific probe sequence, primer sequence, amplification subsequence and coding are double The sequence and v-ATPase of chain RNA sequence and ribosomes L19 from above-mentioned other insects directly to autoploid (SEQ ID NO: 144 to SEQ ID NO:159).

The present invention provides a kind of methods, for containing without in vertebra harmful organism (such as corn rootworm or relative species) Gene expression, described method includes following steps: in the diet of harmful organism provide gene containment amount at least one DsRNA molecule, the dsRNA molecule are by SEQ ID NO:1 to SEQ ID NO:143 and SEQ ID NO in sequence table: 169, to the transcription of nucleotide sequence shown in SEQ ID No:174, at least form in one section of segment and pest cell MRNA sequence it is complementary, and observe the death of harmful organism or feed is suppressed, hinders or stops.

In another aspect of this invention, described method includes following steps: being fed with one warps to harmful organism Cross stable dsRNA molecule or its modified forms, such as siRNA molecule, nucleic acid sequence with selected from by SEQ ID NO:1 extremely The RNA that the nucleotide sequence transcription for the group that SEQ ID NO:143 and SEQ ID NO:169 to SEQ ID NO:174 is constituted comes Molecule at least about 80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99 or about 100% is identical.

Therefore, in another aspect of this invention, SEQ ID NO:1 to the SEQ ID NO:143 that sequence represents is provided And one group of nucleotide sequence by separation and purifying shown in SEQ ID NO:169 to SEQ ID NO:174.It is disclosed herein SEQ ID NO:1 to SEQ ID NO:143 shown in nucleotide sequence be from the complementary DNA prepared by WCR insect larvae (cDNA) library separates and is substantially purified into.SEQ ID NO:169 to SEQ ID NO:174 in sequence table disclosed herein Shown in nucleotide sequence separation and highly purified genomic DNA or nocuousness elder brother from harmful Southern corn rootworm The library mRNA of worm, by the library this class mRNA obtain or based on nucleotide sequence disclosed herein or as is generally known in the art be used as T7 The cDNA nucleotide sequence of phage rna polymerase promoter sequence de novo formation.The present invention provides pass through stable dsRNA Or the expression of siRNA molecule or one or more miRNA, for inhibiting without target in vertebra harmful organism, such as WCR insect The expression of gene.By stable dsRNA, miRNA or siRNA molecule may include relatively at least one promoter with justice or Antisense orientation arrangement at least two coded sequences comprising the nucleotide sequence of positive-sense strand and antisense strand by least about Interval (spacer) sequence of five to about 1,000 nucleotide connects or is coupled, wherein positive-sense strand and antisense chain length are not Together, and wherein, each in two kinds of coded sequences and one of SEQ ID NO:1 to SEQ ID NO:143 in sequence table or Nucleotide sequence shown in one of SEQ ID NO:169 to SEQ ID NO:174 shares at least 90%, at least 95%, at least 98% or even 100% sequence identity.

The present invention also provides non-naturally occurring (NNO) nucleotide sequences, and it is raw without vertebra nocuousness to can be used for targeting Gene in object, for the containment of double chain RNA mediate, to obtain the desired inhibition to target gene.SEQ ID NO:1 is extremely Any one of nucleotide sequence shown in SEQ ID NO:143 or SEQ ID NO:169 to SEQ ID NO:174 can be used for Construct such NNO nucleotide sequence.

The present invention also provides the recombination dNA constructs for the dsRNA molecule that the coding present invention is included, for introducing host Cell.Recombinant dna construct includes the nucleotide sequence that RNA is transcribed by host cell.The RNA of transcription forms at least one DsRNA molecule so that a chain of dsRNA molecule by with selected from by SEQ ID NO:1 to SEQ ID NO:143 and SEQ ID The identical nucleosides of the nucleotide sequence for the group that NO:169 to SEQ ID NO:174 is constituted at least about 80% to about 100% Coded by a part of acid sequence.Recombinant DNA construction physical efficiency generates dsRNA molecule in host cell, can inhibit endogenous gene Or target gene or derivatives thereof or its complementary series absorb transformed host in host cell, or without vertebra harmful organism Expression after cell in pest cell.Nucleotide sequence of the invention is opened in what can be operated in host cell Under promoter sequences control, and it is expressed to generate the RNA sequence for forming dsRNA molecule in host cell.In place Chief cell or without dsRNA molecule is further processed in vertebra harmful organism, forms siRNA molecule.

The present invention also provides the recombinant DNA sequence for Plant Transformation, it is built as containing single stranded RNA can be converted into The non-naturally occurring nucleotide sequence of at least one of molecule.In the case where being provided in the diet of no vertebra harmful organism, Single strand RNA molecule passes through molecule intermolecular hybrid in vivo and forms double stranded rna molecule, inhibits without in vertebra pest cell at least one The expression of kind target gene.Non-naturally occurring nucleotide sequence is operably connected at least one promoter sequence, described Promoter sequence plays a role in transgenic plant cells, and the non-naturally occurring nucleotide sequence being operably connected is turned Record is one or more RNA sequences.RNA sequence itself assembly is double stranded rna molecule, is provided in genetically modified plants In the diet without vertebra harmful organism of upper feed.The supply of dsRNA molecule in the diet of harmful organism, obtains to nocuousness The desired inhibition of one or more target gene expression in biology.

The present invention also provides following recombinant host cells, the host cell has at least one weight in its genome Group DNA sequence dna, the DNA sequence dna can be transcribed in host cell, to generate at least one dsRNA molecule, when by no vertebra When harmful organism digests, the dsRNA molecule plays a role, to inhibit the expression of target gene in harmful organism.DsRNA molecule It is by a part of encoded of following nucleotide sequence, the nucleotide sequence is shown and SEQ ID NO:1 in sequence table To nucleotide sequence shown in SEQ ID NO:143 or SEQ ID:169 to SEQ ID NO:174 at least about 80 to about The 100% phase same sex.It is shown in for constructing targeting WCR gene with the Exemplary nucleotide sequences of the dsRNA reagent inhibited SEQ ID NO:1 to SEQ ID NO:143 and SEQ ID:169 to SEQ ID NO:174 in sequence table.

The present invention also provides the recombinant dna construct for Plant Transformation, the construct is different by least two Non-naturally occurring Sequence composition, when expressing as RNA sequence and provided in the diet of no vertebra harmful organism in vivo When, the sequence can inhibit the expression of at least two different target genes in no vertebra pest cell.The first non-natural Existing sequence is converted into RNA, forms the first at least one dsRNA molecule.A part of quilt of the first dsRNA molecule Coded by a part of the first non-naturally occurring sequence, show and SEQ ID NO:1 to SEQ ID in sequence table Nucleotide sequence shown in NO:143 or SEQ ID:169 to SEQ ID NO:174, the first target gene nucleotide sequence, The phase same sex of at least about 80 to about 100% of its derivative or its complementary series.Second of non-naturally occurring sequence quilt It is converted into RNA, forms second of dsRNA molecule.A part of second of dsRNA molecule is by second of non-naturally occurring sequence Coded by a part of column, show and SEQ ID NO:1 to the SEQ ID NO:143 or SEQ ID:169 in sequence table To the nucleotide sequence of nucleotide sequence shown in SEQ ID NO:174 and second of target gene, its derivative or its The phase same sex of at least about 80 to about 100% of complementary series.Two kinds of non-naturally occurring sequences are operationally placed in Under the control of at least one promoter sequence.Promoter sequence functions, to express first in transgenic plant cells Kind and second of dsRNA molecule.It is in the diet without vertebra harmful organism fed on genetically modified plants, harmful organism is provided The dsRNA molecule of inhibition concentration, harmful organism can obtain to target gene expression in harmful organism the intake of plant cell Desired inhibition.

The present invention also provides the plant cells by conversion, have in its genome mentioned above for plant At least one of recombinant DNA sequence of conversion.Plant cell by conversion generates genetically modified plants, genetically modified plants manufacture Progeny plants, seed and plant product, each of which includes recombinant DNA.

Method and composition of the invention can be applied to any unifacial leaf and dicotyledon, this is depending on wanting without ridge Vertebra harmful organism control, alternatively, it can be by pharmaceutically acceptable formulation application to invertebrate, so as to have without vertebra The reduction of certain level occurs for evil biological attack.Particularly, plant includes but is not limited to: alfalfa, dill (aneth), apple Fruit, apricot, globe artichoke, rocket salad, asparagus, avocado, banana, barley, beans, beet (beet), blackberry, blueberry, blueberry, cabbage, armful son are sweet The small citrus of indigo plant, cabbage, leaf mustard, muskmelon, carrot, cassava, cauliflower, celery, cherry, coriander, citrus, Ke Laimenshi, coffee Coffee, corn, cotton, cucumber, pesudotsuga taxifolia, eggplant, hare's-lettuce, wide leaf lettuce, eucalyptus, fennel, fig, cucurbit, grape, shaddock, "Hami" melon, yam bean, Chinese grooseberry, lettuce, leek, lemon, bitter orange, torch pine, mango, melon, mushroom, nut, oat, okra, Onion, tangerine, decoration with plant, pawpaw, parsley, pea, peach, peanut, pears, pepper, persimmon, pine tree, pineapple, plantain, Lee, pomegranate, White poplar, potato, pumpkin (pumpkin), quince, pine, witloof, radish, raspberry, rice, naked barley, sorghum, Southern Pine, Soybean, spinach, pumpkin (squash), strawberry, beet (sugarbeet), sugarcane, sunflower, sweet potato, storax, mandarin orange, tea, Tobacco, tomato, turf, trailing plant, watermelon, wheat, Chinese yam and small cucumber plant.

The present invention also provides a kind of harmful organisms to control reagent, wherein comprising coming from nucleotide sequence transcription of the invention DsRNA molecule.SEQ ID NO:1 to SEQ ID NO:143 or SEQ ID:169 is extremely in the nucleotide sequence and sequence table Nucleotide sequence shown in SEQ ID NO:174 shares the phase same sex of about 80 to about 100%.In one form, harmful Biocontrol agent includes dsRNA molecule.In another form, harmful organism control reagent includes siRNA molecule.Another In kind form, it includes the recombinant DNA sequence for encoding following mRNA molecules that harmful organism, which controls reagent, and the mRNA molecule can be formed DsRNA or siRNA molecule for introduced plant and microorganism.In still another form, it is micro- life that harmful organism, which controls reagent, Object, contains the recombinant DNA sequence for encoding following mRNA molecules, and the mRNA molecule can form dsRNA or siRNA molecule.Have Evil biocontrol agent is preferably insect or nematode pests biocontrol agent.

People need harmful biocontrol agent to play a role, and to reduce or eliminate the invasion of corn rootworm, but also examine Consider, method and composition shown in this article can be used to obtain the correlated series from other harmful organisms, and utilize this A little derivatives are to control the invasion of other (a variety of) harmful organisms.It is also contemplated that insect pest can be selected from corn rootworm Affiliated same genus, Xiang Tongke or mesh.In addition, the present inventor, which is also contemplated that, can be used the present invention, it is applied to control It makes raw without vertebra from Bugdom and from nematode, fungal pathogens, virus, bacterium and any other pair of plant pest Any species of object.

The present invention also provides the combinations for controlling the method and composition without vertebra pest infestation.A kind of means Provide for protect plant from insect infestation, dsRNA method and composition as described herein and one or more kill Insect agent, the property different from the property that dsRNA method and composition is shown that the insecticide is shown.For example, working as When Bt albumen is provided in the diet of insect pest, the binding mode with method and composition of the invention has just been shown The dramatic different binding mode for being used to control insect pest.It is formulated as the composition of topical application, or use turns base Because of the composition that dsRNA method and composition and Bt method and composition combine that method obtains, collaboration effect can be generated It answers, this is unknown in the past in the field for control insect infestations.Generating one or more dsRNA or siRNA molecule (can inhibit mesh Mark some necessary biological functions in harmful organism) genetically modified plants and one kind or more toxic for object noxious livings Kind B.t. insecticidal protein together provides astonishing synergistic effect.A kind of synergistic effect is (a variety of) dsRNA or (more Kind) expression needed for Bt albumen reduces.When being grouped together, every kind of pest control agent all only needs lower to have Imitate dosage.It is believed that Bt insecticidal protein is manufactured that access aperture, dsRNA or siRNA molecule pass through the access aperture energy The space far from insect gut is more effectively penetrated into, or is more effectively penetrated near wound manufactured by Bt albumen Cell, thus only need lesser amount of Bt or dsRNA that can obtain desired insecticidal as a result, that wants have target The inhibition or containment of evil biological targets biological function.

Multinomial invention is described herein in the present inventor, invades including for controlling without vertebra harmful organism The method attacked, the method are carried out by providing following reagents into the diet of no vertebra harmful organism, the reagent It is constituted included in the ribonucleic acid to play a role when being absorbed by harmful organism, or by the ribonucleic acid, to inhibit harmful raw The expression of the intracellular Target Nucleotide Sequence of object.The ribonucleic acid provided in diet is by being Target Nucleotide Sequence or and mesh The ribonucleotide for marking nucleotide sequence complementation is constituted.Ribonucleotide is transcribed by following continuous DNA sequence dnas Come, the DNA sequence dna there are at least about 19 to about 5000 nucleotide long, selected from by SEQ ID NO:1 to SEQ ID The group that NO:143, SEQ ID:169 to SEQ ID NO:174 and its complementary series are constituted.The method is provided to following cores The building of nucleotide sequence, the nucleotide sequence can be used for expressing RNA molecule, and the RNA molecule is being supplied to harmful organism It is absorbed in diet by harmful organism.Diet can be artificial diet, carried out preparing to it and be protected on such diet with reaching Hold the specific nutrition requirement of harmful organism, the diet can be supplemented with harmful organism control amount, from individual expression system The RNA being purified into system is used for following purposes to the supplement of diet: determining the harmful organism control amount of RNA composition, or really It is fixed --- it, can with one of harmful organism or plurality of target sequence-specific when harmful organism absorbs the diet being added Whether have in conjunction with the specific RNA with partial hybridization, one or more buildings for obtaining some gene containment activity.Diet is also It can be the recombinant cell transformed with DNA sequence dna, the DNA is the expression building for reagent, RNA or gene containment agent 's.When harmful organism absorbs one or more such cells by conversion, it may be observed that desired genotype or phenotypic results, This shows that the reagent has the function of inhibiting Target Nucleotide Sequence expression in pest cell.

No vertebra harmful organism is preferably insect, spider, nematode, flatworm, capsule worm, fungi harmful organism or base Because containing technology other any no vertebra harmful organisms applicatory.It is highly preferred that being in animal or plant without vertebra harmful organism Object invasion aspect no vertebra harmful organism especially under a cloud.More particularly, no vertebra harmful organism is that preference invasion crop is planted Strain, the insect of ornament and/or meadow or nematode or fungi harmful organism.

It selects to express preferably about 19 to about 5000 nucleotide of DNA sequence dna of gene containment agent of the invention Length, at least partly justice or antisense with target sequence present in one or more specific objective pest DNA Chain height in sequence is identical.Phrase " at least partly " is intended to indicate following concepts: selecting the DNA sequence for carrying out expressing gene containment agent Column can be used for expressing following RNA, institute from the single sequence construct obtained from one or more object noxious livings Stating RNA can play a role again in the containment of individual gene or gene family in one or more object noxious livings；Or it can The DNA sequence dna as chimera is constructed from a variety of DNA sequence dnas.A variety of DNA sequence dnas can be with each all from single nocuousness One or more nucleotide sequences in biology obtain, or can be from one or more nucleotide of a variety of different harmful organisms Sequence obtains.Particularly, the sequence selected should can be shown with the nucleotide sequence from pest DNA about The nucleotide sequence identity of 80 to about 100%.The DNA of pest can be identified by following methods: from nocuousness DNA is isolated in biological species, or by being identified the RNA in pest, is oppositely changed RNA sequence For DNA.It is SEQ in shown herein, sequence table that example, which comes out from the sequence of the DNA of corn rootworm pest, ID NO:1 to SEQ ID NO:143, SEQ ID:169 to SEQ ID NO:174 and its complementary series.

Select the DNA sequence dna for carrying out expressing gene containment property RNA molecule that can be included in polynucleotide compositions, described group Object is closed to use in plant cell.Particularly, DNA sequence dna can be included in the carrier for converting plant cell gene group, and And it can be included in expression cassette, the expression cassette contains at least one Plant functional promoters, the promoter and selection DNA sequence dna and other any expression control elements (wanting the expression for obtaining suitable intracellular time or plant space level) It is operably associated.Polynucleotide compositions are introduced into the genome of plant cell and provide transformed cells, if appropriate Selection approach together included then it can be selected, and being regenerated as transgenosis recombinant plant with polynucleotide compositions. Genetically modified plants, event can be provided that in the diet of one or more harmful organisms, to obtain the control to pest infestation System.Genetically modified plants can produce progeny plants, plant cell and seed, each of which contains polynucleotides combination Object.

The present invention provides a kind of methods for protecting plant from insect infestation, and the method is by insect pest There is provided one of following plant cells or it is a variety of realize, the RNA molecules of every kind of the cell all expressing gene containment property, The RNA molecule comes from following DNA sequence dnas, and the DNA sequence dna is selected from the group being made of the sequence illustrated herein.To containing base Because of the intake of the plant cell of containment property RNA, harmful organism or insect-controlling agent, result in a kind of in harmful organism or insect Or the inhibition of multiple biological function.

The present invention provides a kind of compositions, wherein containing two or more different insecticides, wherein every kind all to same The harmful organism of sample or insect species are toxic.As described herein, one of these insecticides can be for raw in nocuousness Contain the RNA molecule of necessary biological function in one or more cells of object.Second of insecticide can be wrapped with the first It includes.Second of insecticide can be can select different from second of gene containment property RNA of the first or second of insecticide The group constituted from following substance: patatin patatin, Bacillus thuringiensis insecticidal egg White, Xenorhabdus insecticidal protein, Photorhabdus insecticidal protein, Bacillus laterosporous insecticidal Albumen, Bacillus sphearicus insecticidal protein and lignin.Bacillus thuringiensis insecticidal protein Can be any type in a large amount of insecticidal proteins, the albumen includes but is not limited to: Cry1, Cry3, TIC851, CryET70, Cry22, binary insecticidal protein CryET33 and CryET34, binary insecticidal protein CryET80 and CryET76, Binary insecticidal protein TIC100 and TIC101, binary insecticidal protein PS149B1, VIP insecticidal protein, TIC900 or related Any kind of insecticidal chimera of albumen, TIC901, TIC1201, TIC407, TIC417 and aforementioned insecticidal protein.

It is targeted or for containment for the function in pest cell or as the physiology of harmful organism or metabolism side The necessary albumen of gene codified that the function (expression for the gene that containment is targeted can be generated) in face is targeted, institute The expectation function for stating albumen is selected from the group being made of following function: muscle is formed, the formation of juvenile hormone, the tune of juvenile hormone Control, ion regulation and transhipment, the synthesis of digestibility enzyme, the holding of cell membrane gesture, amino acid bio synthesis, amino acid degradation, essence Son is formed, the synthesis of pheromones, and pheromones sensing, the formation of day linear protein (antennae), wing is formed, the formation of leg, hair Educate and break up, the formation of ovum, larval maturation, the formation of digestibility enzyme, the synthesis of hemolymph, the holding of hemolymph, neurotransmission, Cell division, energetic supersession, breathing and apoptosis.Preferably, it selects to construct and contain that the DNA sequence dna of construct is from sequence table What nucleotide sequence show, for containing corn rootworm gene obtained.We expect, for controlling without vertebra harmful organism The method of invasion will include: to provide reagent in the diet of no vertebra harmful organism, for example, from the expression of the first DNA sequence dna The first ribonucleotide, functions when being absorbed by harmful organism, to inhibit the biology in the harmful organism Function further contemplates, the first DNA sequence dna is shown with the coded sequence about 85 that obtains from the harmful organism to about 100% nucleotide sequence identity.The first ribonucleotide can hybridize with second of ribonucleotide, described The complementation complementary or substantial with the first ribonucleotide of second of ribonucleotide, second of ribonucleotide sequence Column are expressed by second of DNA sequence dna, and second of DNA sequence dna corresponds to the coded sequence obtained from no vertebra harmful organism, It is selected from the sequence shown in sequence table or its complement herein.Preferably, the first and second of DNA sequence dna include and sequence One of sequence shown in list or a variety of identical continuous sequences, the continuous sequence have about 14 to about 25 or more More continuous nucleotides.

When the insecticide containing pest gene containment amount is (for example, one kind as shown in this paper sequence table or more One or more RNA molecules that the expression of kind of sequence generates) diet when being provided to following no vertebra harmful organisms, the present invention Optimal function is played, the harmful organism shows about 4.5 to about 9.5, about 5.0 to about 9.0, about 5.5 to big About 8.5, about 6.0 to about 8.0, about 6.5 to about 7.0 or about 7.0 digestive system pH.Optionally, described herein Method, nucleic acid, ribonucleic acid, ribonucleotide, composition, plant, plant cell, progeny plants, seed, insect control Preparation, pest control agent, any type in expression cassette are being directed to one or more nocuousness comprising above-mentioned pH in digestive tract Function is provided when providing in the diet of biology.

Diet of the invention can be any enough harmful organism diets comprising but is not limited to, artificial diet or matches Side, plant cell, various plants cell, plant tissue, the root of plant, vegetable seeds and the plant come from vegetable seeds growth Object, wherein RNA molecule that the diet includes pest inhibitory amount, being encoded by following DNA moleculars, the DNA molecular are Or substantially one or more nucleotide sequences shown in the sequence table, or selected from from specific no vertebra harmful organism object Continuous at least about 19 to about 5000 nucleotide of kind of the nucleotide sequence obtained, or be complementary or substantially mutual It mends.

Agriculturally with commercially important product and/or composition (including but not limited to, animal feed, agricultural product and jade Rice product and byproduct, corn product and byproduct are used for the food consumed for the mankind, or for being intended to Composition and agricultural product for mankind's consumption comprising but be not limited to, corn flour, maize flour, corn syrup, corn oil, Cornstarch, puffed rice, corn-dodger, cereal containing corn and corn byproducts etc.) it is intended to fall into the scope of the present invention, if These products and composition contain it is detectable amount, shown in this article, turn base containing any of such nucleotide sequence as diagnosis Because event nucleotide sequence if.These products be it is useful, may be obtained from crop at least because of them, and can give birth to It produces, in the big Tanaka breeding containing less insecticide and organic phosphine, this is that they include into nucleotide of the invention for controlling The result of invasion without vertebra harmful organism in plant.This agricultural products and commodity product are produced from by genetically modified plants Seed produce, wherein the following RNA of Expressed in Transgenic Plant, the RNA is from one or more companies of the invention Continuous nucleotide or the nucleotide and its complement of one or more no vertebra harmful organisms.This agricultural products and commodity product Can also have for control this agricultural products and commodity product without vertebra harmful organism, for example, control plane powder weevil because It there are the RNA that can contain pest gene, the RNA is gene shown in the present invention in agricultural product or commodity product Expressed by sequence.

The present invention also provides computer-readable medium, it is recorded on SEQ ID NO:1 shown in ordered list One into the nucleotide sequence or its complement shown in SEQ ID NO:143 or SEQ ID NO:169 to SEQ ID NO:174 Kind is a variety of, is used for a large amount of computer based applications, including but not limited to, the DNA phase same sex and similarity searching, protein phase The same sex and similarity searching, transcription situation signature analysis, comparison and artificial hybridization analysis between genome.

Specific embodiment

It is hereafter detailed description of the present invention, is provided to that those skilled in the art is assisted to practice the present invention.Ability Domain those of ordinary skill can without departing from present inventive concept or range in the case where improvement is made in embodiment as described herein And variation.

Herein inventors have found that with the introduction of this field on the contrary, double stranded rna molecule (You Wu vertebra object will be contained Kind one or more nucleotide sequences being expressed in the Sequence composition that finds) composition be fed to and therefrom obtain the core Nucleotide sequence without vertebra species, result in the inhibition to one or more biological functions in no vertebra species.Particularly, it invents People's discovery, is fed to corn rootworm for the double stranded rna molecule being made of corn rootworm RNA sequence respectively, and it is such to result in intake The death of the corn rootworm of composition, or development and differentiation are suppressed.

Inventor is subject to the nucleotide sequence of the thousands of kinds of cDNA sequences obtained from every kind without vertebra pest Identification.Be pushed off out by the encoded amino acid sequence of cDNA sequence, and with all known amino acid alignments. The following albumen of coding are much expected to be in cDNA sequence, the albumen has some annotation informations being associated with.With Specific nucleotide sequence and the relevant annotation information of its encoded protein sequence are based on: by cDNA sequence as described herein The publicly available database of the translation amino acid sequence being inferred to and this field in it is same between known amino acid sequence Source property and similitude.For all known amino acid sequences, the amino acid sequence shown in this article being inferred to is carried out BLAST is based on comparison result, assigns possible function to the amino acid sequence that every kind is inferred to.It encodes known in the art Critically important albumen or protein part are (for example, be related to a variety of metabolism or catalysis biochemical route, cell division, numerous for existence Grow, energetic supersession, digestion, nervous function etc. amino acid sequence) cDNA sequence be selected it is raw in no vertebra nocuousness to prepare The double stranded rna molecule provided in the diet of object.As described herein, object noxious livings intake contains one or more dsRNA (its At least one section of segment corresponds to the identical segment of at least one section of height of the RNA generated in object noxious livings cell) combination Object leads to death, hypoevolutism or the other inhibition situations of object noxious livings.These results indicate that raw from no vertebra nocuousness The nucleotide sequence that object obtains, DNA or RNA can be used for building recombination harmful organism host or pest infestation target Homobium.Harmful organism host or homobium can be converted into containing from the nucleotide sequence that no vertebra harmful organism obtains It is one or more.Nucleotide sequence coded one or more RNA of harmful organism host or homobium are transformed into, the RNA exists DsRNA is formed in the intracorporal cell of host or symbiosis or biofluid being converted, so that: if/exist when harmful organism It is fed on transformed host or homobium, dsRNA is can get in the diet of harmful organism, this will lead to harmful organism The containment of one or more gene expressions in cell eventually leads to death, hypoevolutism or the other inhibition situations of harmful organism.

In general, the present invention relates to control the heredity in host organism without vertebra pest infestation.More specifically, The present invention includes the method that pest control agent is transported to no vertebra harmful organism.Such pest control agent directly or Cause indirectly harmful organism keep its own, growth or invasion target host or the ability of homobium it is impaired.The present invention provides Using the method that is stabilized dsRNA in harmful organism diet, this means as target gene in containment harmful organism, Thus to obtain in the host or homobium targeted to harmful organism or the desired control of the pest infestation of surrounding.Use weight Group by stable dsRNA or siRNA molecule, genetically modified plants can be manufactured.

To complete foregoing teachings, the present invention provides a kind of methods, for inhibiting without vertebra harmful organism (particularly, Western corn rootworm (WCR) or other coleopterous insect species) in target gene expression, cause feed, growth, development, Breeding, the stopping propagated, may finally lead to the death of harmful organism.The described method includes: stable double-stranded RNA will be passed through (dsRNA) nucleic acid molecule or its form (such as siRNA (siRNA) molecule) by modification partly or entirely introduce In the alimentation composition relied on to harmful organism as food source, and the alimentation composition feeds harmful organism For be obtainable.To the intake containing double-strand or the composition of siRNA molecule, cause pest cell to the molecule Absorption, to inhibit the expression of at least one of pest cell target gene.It is raw to nocuousness to the inhibition of target gene Object causes harmful influence.DsRNA molecule or siRNA are by SEQ ID NO:1 to SEQ ID NO:143 and SEQ ID NO: 169 into SEQ ID NO:174 nucleotide sequence shown by any sequence constitute, inhibit to lead to life for harmful organism Critically important albumen or nucleotide agent are reduced or remove for long and development or other biological functions.The nucleosides of selection Acid sequence is shown and SEQ ID NO:1 to SEQ ID NO:143 and SEQ ID NO:169 to SEQ ID NO in sequence table: The sequence identity of about 80% at least about 100% of one of nucleotide sequence or its complementary series shown in 174.It is such Inhibition is specific, because the nucleotide sequence from target gene a part is selected from inhibition dsRNA or siRNA, transcription comes Sequence select.The method can be used for inhibiting in harmful organism effective in the expression of at least one target gene of inhibition Many other types of target gene.

The present invention also provides various forms of pest control agents, for obtaining subtracting for desired pest infestation It is few.In one form, pest control agent includes dsRNA molecule.In another form, pest control agent includes SiRNA molecule.In another form, pest control agent includes recombinant dna construct, and the construct can be used for stablizing It is converted into biology and plant, transformed microorganism or plant is enabled to encode dsRNA or siRNA molecule.In another form In, pest control agent is microorganism, wherein the recombinant dna construct containing coding dsRNA or siRNA molecule.

Nucleotide sequence pair by separation and purifying is provided from cDNA library and/or genomic library information.Nucleotide Sequence is used as hot amplimer to being preferably to be obtained from any without vertebra harmful organism, of the invention to generate DsRNA or siRNA molecule.

The present invention provides recombinant dna construct, for obtaining to the specific host of harmful organism targeting or homobium Stable conversion.By conversion, harmful organism targeting host or homobium expression have effective desinsection it is horizontal, from recombination The preferred dsRNA or siRNA molecule of DNA construct, and the molecule is provided in the diet of harmful organism.

As by the convert, host of harmful organism target organism or the example of homobium, the present invention also provides warps Cross the plant cell of conversion and the plant by conversion and their offspring.What plant cell and process by conversion converted Plant expresses one or more dsRNA or siRNA sequence of the invention, and the sequence comes from SEQ ID shown in sequence table One or more DNA sequence dnas shown in NO:1 to SEQ ID NO:143 and SEQ ID NO:169 to SEQ ID NO:174 or Its complementary series.

Vocabulary " gene containment " used herein is at the same time in use, be used to refer to: for reducing the albumen water of generation Flat any known method, albumen generate the result for being genetic transcription into mRNA and subsequent mRNA translation.Gene containment is also used To refer to the reduction of the protein expression from gene or coded sequence, including posttranscriptional gene containment and transcription containment.Base after transcription Because containment be from containment targeting gene or coded sequence transcription come mRNA all or part of with for the corresponding of containment What the homology between double-stranded RNA mediated, refer to the obtainable quality entity for the mRNA in conjunction with ribosomes in cell On or the reduction that can measure.RNA by transcription can be just direction, play so-called total containment effect, or It can be antisense orientation, play so-called antisense containment effect, or generate dsRNA with two kinds of directions, it is dry to play so-called RNA The effect of disturbing (RNAi).Transcription containment is that the presence of dsRNA in cell is mediated, and the dsRNA is gene containment agent, is shown Out with the high sequence identities of promoter DNA sequence or its complementary series, plays and be referred to as that promoter is counter contains.Gene containment Can play a role for natural plant gene relevant to character, for example, provided to plant have drop it is low-level, by natural The albumen of gene coding, or with increasing or decreasing horizontal impacted metabolin.It is following right that gene containment can be also directed to Target gene in the biology of plant pest plays a role, and the harmful organism can absorb or contact the plant containing gene containment agent Object material, the gene containment agent, which is specifically designed, can inhibit or contain one or more homologous sequences in pest cell Or the expression of complementary series.

Disclose in U.S. Patent number 5,107,065,5,759,829,5,283,184 and 5,231,020: antisense or justice are fixed To RNA carry out posttranscriptional gene containment, to regulate and control the gene expression in plant cell.DsRNA is for containing gene in plant Purposes be disclosed WO 99/53050, WO 99/49029, U.S. Patent Application Publication No. 2003/0175965 and 2003/ 0061626, in U.S. Patent Application No. 10/465,800 and U.S. Patent number 6,506,559 and 6,326,193.

In plant carry out posttranscriptional gene containment preferred method using justice orientation and antisense orientation, by stabilization Transcription come RNA, for example, as hair fastener and loop-stem structure.Preferred DNA construct for carrying out posttranscriptional gene containment is It is following such, wherein first segment fragment encoding shows the RNA of antisense orientation, shows and target gene to be contained Perfect homology between segment, the first segment segment are connected with second segment segment, and second segment fragment encoding is shown and the One section of segment has the RNA of height complementarity.Such construct is expected to will form loop-stem structure that (this is by first segment segment Hybridize with second segment segment and to be formed) and from the nucleotide sequence for both connecting section segment ring structure (see WO94/01550, WO98/05770, US 2002/0048814 and US 2003/0018993).

Term " nucleic acid " used herein refers to the DNA or ribonucleic acid base that from 5 ' to 3 ' ends are read Single or double chain polymerization object.Optionally, " nucleic acid " allows also containing base that is non-naturally occurring or being changed by poly- The correct reading of synthase, will not reduce the expression of the polypeptide of the nucleic acid encode.Term " nucleotide sequence " or " nucleic acid sequence " refer to As the individually justice and antisense strand of single-stranded presence or the nucleic acid being present in two-body.Term " ribonucleic acid " (RNA) includes RNAi (inhibitory RNA), dsRNA (double-stranded RNA), siRNA (siRNA), mRNA (mRNA), miRNA (Microrna), TRNA (transfer RNA, quilt or charge is not added by corresponding acylated amino) and cRNA (complementary RNA), term " deoxidation core Ribosomal ribonucleic acid " (DNA) includes cDNA and genomic DNA and DNA RNA hybrid.Vocabulary " nucleic acid fragment ", " nucleic acid sequence piece It is disconnected " or more common " segment " will be understood by those skilled in the art are as follows: it includes genome sequence, ribosomal RNA sequences, turns Transport the nucleotide sequence of RNA sequence, mRNA sequence, operon sequence and smaller engineered mistake, sequence expression or Expression protein, polypeptide or peptide can be transformed into.

Term " harmful organism " used herein refer to insect, spider, shellfish, fungi, bacterium, virus, nematode, Flatworm, roundworm, pinworm, hookworm, tapeworm, trypanosoma, blood fluke, horse botfly, flea, tick, mite, lice etc., they are Nature is generally existing, can take in or contact that one or more (being converted into can table by harmful organism host or homobium Up to double-stranded gene containment agent, or be coated with the containment agent) generate cell, tissue or fluid, or can take in containing The vegetable material of gene containment agent." Pest-resistant " used herein is genetically modified plants, transgenic animals, transgenosis The feature of host or transgenosis homobium, so that the plant, animal, host or homobium have the attack of harmful organism Resistance, the attack typically can be damaged or be lost to the plant, animal, host or homobium.Such harmful organism Resistance is produced from the addition for assigning the recombinant DNA of Pest-resistant there may be from natural mutation, or more typically. There is insect-resistant for render transgenic plant, recombinant DNA can be for example, coding insect lethal or insect inhibitory protein, example Delta endotoxin such as from B.thuringiensis bacterium is (for example, can get in kind in the business of cotton and corn makes ), or it can be transcribed into RNA molecule, dsRNA molecule is formed in the tissue or fluid of recombinant plant.DsRNA molecule portion Subpackage contains: to the identical RNA piece of corresponding RNA segment of DNA sequence encoding in the harmful insect that preference is fed on recombinant plant It is disconnected.The expression of gene is contained by dsRNA in object noxious insect, leads to plant to the containment of gene expression in object noxious insect With insect-resistant.Fire et al. (U.S. Patent number 6,506,599) generally describes the suppression to pest infestation System, only for several nucleotide sequences in nematode kind Caenorhabditis elegans with suppressor function Column provide details.Similarly, Plaetinck et al. (US 2003/0061626) describes dsRNA in a variety of lines The purposes of worm harmful organism kind suppressor function.Mesa et al. (US 2003/0150017) is described: using dsRNA sequence Column remove conversion host cell, with expression and the identical corresponding dsRNA sequence of target sequence height in special pathogen, and especially Describe: building recombinant plant is expressed these dsRNA sequences (for by the biological uptake of a variety of pairs of plant pests), is assisted To the negative tune of gene in pest gene group, and plant is improved to the resistance of pest infestation.

The present invention provides the bases used by stable dsRNA to target genes one or more in object noxious livings The method inhibited by expression.Present invention is particularly useful in eukaryotic gene expression is adjusted, especially present in adjusting insect The expression of gene, the insect shows about 4.5 to about 9.5, more preferably about 5.0 to about 8.0, it is further more excellent The digestive system pH of selection of land about 6.5 to about 7.5.Digestive system with the pH level except these ranges must have plant Harmful biology is not the preferred candidate person of following methods, the method double chain RNA mediate, method for gene containment, It is middle to use the Transfer method for needing to absorb preferred dsRNA molecule.Adjustment effect is suitable for a variety of bases for expressing harmful organism Cause, for example, being responsible for the endogenous gene of endocellular metabolism or cellular transformation, including household's gene, transcription factor and Codocyte The other genes for the polypeptide that intracellular metabolite is related to.

Term " expression " used herein refers to, the transcription of the justice or antisense RNA that obtain from nucleic acid disclosed by the invention It is accumulated with stablizing.Expression also may indicate that translation of the mRNA to polypeptide or protein.Term " justice " RNA used herein refers to Corresponding to following sequences or the rna transcription sheet of segment, when being generated by object noxious livings, the sequence or segment are can pass through Object noxious livings cell translation exists at the form of the mRNA of protein.Term " antisense RNA " used herein refers to and mesh All or part of complementary RNA of the mRNA normally generated in mark pest cell.The complementation of antisense RNA, which can be, to be directed to Specific gene transcript it is any portion of, that is, 5 ' non-coding sequences, 3 ' non-translated sequences, introne or coded sequence.Herein Used in term " rna transcription sheet " refer to, the obtained product of transcription by RNA polymerase catalysis that DNA sequence dna is carried out.When When rna transcription is originally the perfect complementary copy of DNA sequence dna, being referred to as level-one transcript or its can be and transcribe to level-one This carries out the RNA that transcription post-processing obtains, and is referred to as mature rna.

Phrase " inhibition to gene expression " used herein or " inhibit insect cell in target gene expression " refer to, (or observable reduction) is not present in the albumen and/or mRNA product level of target gene.Specificity refers to: inhibiting target gene And without effect and do not have influential ability to the intracellular any gene for generating dsRNA molecule to the other genes of cell.To having The inhibition of the gene expression of target gene may cause the new phenotypic character in harmful insect in evil insect.

In the case where not limiting the scope of the invention, the present invention provides a method in one aspect, is used for: using warp Stable dsRNA strategy is crossed to control the invasion of object noxious livings.The method is related to: manufacture is by stable dsRNA points Son, the gene silencing as a kind of insect-controlling agent, in inducing harmful insect.Insect-controlling agent of the invention can be directly or indirectly Ground induces the posttranscriptional gene silencing event of target gene in insect.The negative tune of target gene expression can be prevented or at least postponed Growth, development, breeding and the propagation to host of insect.Phrase " stable dsRNA molecule is passed through in manufacture " used herein Refer to: using be easily obtained in this field recombinant DNA technology (for example, see Sambrook, et al, In:Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Press, Cold Spring Harbor, New York, 1989), to construct transcription by the method for the DNA nucleotide sequence of stable dsRNA.Of the invention Detailed construction method is disclosed in this specification hereinafter.Term " silencing " used herein refers to, to Target Nucleotide Sequence table What is reached is effective " negative to adjust ", and therefore the sequence ability that generation acts in insect cell disappears.

A part of the invention provides a kind of movement system, and for insect-controlling agent to be transported in insect, this is logical It crosses and they is exposed to the diet containing insect-controlling agent of the present invention to realize.According to one of embodiment, by stable DsRNA or siRNA molecule can be included in insect diet, or can be placed to the top of the diet of insect consumption.

A part of the invention is additionally provided for insect-controlling agent to be transported in insect, this is to pass through: they are sudden and violent Reveal to the microorganism containing insect-controlling agent of the present invention or host, for example, plant, by intake microorganism or host cell or carefully It is intracellular tolerant to realize.According in another embodiment, the present invention relates to: manufacture transgenic plant cells or plant, In containing the recombinant dna construct of the invention by stable dsRNA molecule can be transcribed.Phrase " manufacture used herein Transgenic plant cells or plant " refers to following methods, including using the recombinant DNA technology being easily obtained in this field (for example, seeing Sambrook, et al.), building can transcribe the plant conversion carrier of the invention by stable dsRNA molecule, convert plant Cell or plant, and generate containing by transgenic plant cells or transgenosis transcribing, by stable dsRNA molecule Plant.Particularly, method of the invention may include the recombinant precursor in plant cell, can generate in insect genes groups The dsRNA transcript of nucleotide sequence coded RNA sequence very high homology.Insect genes groups inner nucleotide sequential coding is for elder brother For the survival and propagation of worm in the case where critically important gene, insect existence and infection host will lead to its negative regulator The ability of cell reduces.Therefore, such negative regulator results in the holding viability to insect and propagated adverse effect, because It prevent or reduces insect by the ability of nutrients feed, life from host cell.Utilize above-mentioned insect survival Property and propagated reduction, promote the resistance to insect infection and/or the tolerance of raising in plant cell.In insect Gene can be targeted in the stage of mature (adult), prematurity (larva) or ovum.

In yet another embodiment, the weakening bacterial strain of the avirulence of microorganism is used as the load for insect-controlling agent Body, in this case, the microorganism for carrying such reagent are also referred to as insect-controlling agent.It can be subject to engineering to microorganism to change Make, it made to express the nucleotide sequence of target gene, to generate following RNA molecules, the RNA molecule include in insect cell The RNA sequence complementation of typical case's discovery or homologous RNA sequence.Insect is exposed to the microorganism, so that microorganism is ingested, And the negative tune to target gene expression directly or indirectly mediated by RNA molecule or segment or derivatives thereof.

Alternatively, the present invention provides: insect is exposed to insect-controlling agent of the invention, the controlling agent is included in spray In mist mixer, it is applied to host, for example, the surface of host plant.In a kind of illustrative embodiment, insect is to insect Insect-controlling agent is transported to insect digestive tract by the intake of controlling agent, is then transported in the intracorporal cell of insect.In another kind In embodiment, insect-controlling agent otherwise (such as injection or other physical methods) can also allow that the infection of insect The transport of insect-controlling agent.In yet another embodiment, RNA molecule itself is encapsulated into the matrix of synthesis, such as polymer, And it is applied to host, such as the surface of plant.Insect allows insect-controlling agent to be transported to insect the intake of host cell, Lead to the negative tune to target gene in host.

It is also contemplated that composition of the invention can be included in the seed of plant species, it can be used as and be included in plant The expression product of the recombination of object cellular genome, or it is included in the coating or seed being applied on seed before planting In processing.Plant cell containing recombination is counted as transgenic event herein.

It is believed that insecticidal seed treatment can provide significantly when combining with the transgenic event for providing following protections The advantages of, the invasion protected for keeping out no vertebra harmful organism, the event is for the preferred of object noxious livings Within the scope of validity.In addition, there are following situations it is believed that well known to those skilled in the art, wherein have preferred Validity within the scope of such transgenic event be advantageous.

The invention also includes the seed having more than a kind of transgenic event and plants.Such combination is referred to as " stacking " Transgenic event.The transgenic event of the stacking can be for the event of same object noxious livings or they can To be directed to different object noxious livings.In a preferred embodiment, there is expression 3 albumen of Cry or its insecticidal to become The seed of allogeneic ability also has the ability for expressing at least one other insecticide comprising but be not limited to and 3 egg of Cry The albumen of Bai Butong and/or following RNA molecules, the sequence of the RNA molecule is from the RNA's expressed in object noxious livings Sequence forms duplex-RNA constructs when it is expressed in planting the plant cell that sub or slave seed is grown, wherein object noxious is raw Object results in the inhibition to rna expression in object noxious livings cell to the intake of one or more cells of plant.

In another preferred method, there is the seed for expressing following dsRNA abilities also to have offer herbicide tolerant Property transgenic event, the sequence of the dsRNA comes from object noxious livings.Preferably, provide herbicide tolerant turns base Because event is to provide to glyphosate, the resistance of N- (phosphine carboxymerhyl) glycine (the isopropyl amine salt form including such herbicide) Event.

In the method for the invention, going processing with insecticide includes the seed of transgenic event.It is believed that transgenosis kind Son (shows the bioactivity for object noxious livings, this is insecticidal amount, transgenic seed or comes from the seed growth Plant cell in insecticidal dsRNA production result) with seed is carried out with certain chemical or protein insecticidal agents processing In conjunction with unexpected collaboration can be provided to the seed with such processing, comprising: for object noxious livings to obtaining The damages of genetically modified plants protection, unexpected outstanding effect are provided.Particularly, it is believed that, extremely with about 100gm Certain insecticides (for every 100kg seed) of about 400gm certain will form dsRNA molecule (its sequence be to that can express What the one or more sequences expressed from corn rootworm obtained) the transgenic seed of construct handled, provide to Keep off the unexpected outstanding protection of corn rootworm.In addition, it is believed that such combination is also for the damage for unregistered land tiger The harmful corn plant to protect the state of emergency.Seed of the invention is also believed with the property for reducing insecticide use cost, Because the case where compared with no composition for using invention and method, for the protection for obtaining requirement, less kill can be used Worm agent.In addition, because used less insecticide, and because it is applied before the planting, without individual field Using, it is believed that, method of the invention is therefore all safer for operator and environment, and potentially compared with biography System method is more cheap.

When some effects are mentioned as " collaboration ", this indicate include: transgenic event and insecticide combination pair In the combination acts synergistically of insecticidal activity (or efficiency).But this is not offered as such synergistic effect being limited to insecticidal activity, They should include also following unexpected advantages, for example, increased field of activity, advantageous activity curve (are relevant to damage The reduced type and quantity of evil), reduction, the distribution of insecticide environment of insecticide and application cost are reduced, insecticide is to production exercise Make and the exposure of the personnel of maize planting seed reduces and further advantage well known by persons skilled in the art.

It can be used for the insecticide that combines with method and composition of the invention of the present invention and insecticide (including as seed Processing and coating) and can be found in such as U.S. Patent number 6,551,962 using the method for such composition, full text is logical It crosses and is incorporated herein by reference.

It has been found that the present invention has the agricultural pest for protecting seed and plant to keep out broad range, including elder brother Worm, mite, fungi, yeast, mould, bacterium, nematode, weeds and plastics and saprophyte.

Preferably, seed treatment as described herein and coating are used together with transgenic seed of the invention, particularly, are removed To transgenic seed apply from SEQ ID NO:1 to SEQ ID NO:143 and SEQ ID NO:169 shown in sequence table to Except the dsRNA molecule that sequence shown in SEQ ID NO:174 or its sequence obtain, also to transgenic seed application insecticide. Although it is believed that seed treatment can be to the transgenic seed application for being in any physiological status, it is preferable that seed is in (durable) state of enough lasting stabilities, will not incur damage during treatment process.Typically, seed is from field It is harvesting, removed from genetically modified plants under and separate with any other non-seed vegetable material.Preferably, seed It will also be biologically to be stabilized to following degree, so that processing not will lead to the biology damage to seed.In a kind of reality It applies in mode, for example, processing may be used on following corn seeds, the seed has been harvested, has cleaned and has been dried to moisture content Content is less than about 15% by weight.In another embodiment, seed can be following seeds: it is dried, then It applies (prime) and has gone up water and/or other material, be dried again before then being handled again with insecticide or during processing.Rigid In the limitation just described, it is believed that, in harvest seed and any event between seed can be sowed, processing is applied to seed On.The term as used herein " seed that do not sowed " is for including: to harvest in seed and in ground sowing seed (in order to plant Object germination and growth purpose) between any period.

When mentioning the seed that do not sowed by insecticide " processing ", such processing is not used in including operations described below: Wherein, insecticide is applied to soil, rather than is applied to seed.For example, while being sowed with seed, in soil band (band), such processing is applied in " T " band or ditch dug with a plow, is not considered as included by the present invention.

The combination of insecticide or insecticide can " purely (neat) " application, that is, it is any dilution or there are other Component.But typically, insecticide is applied on seed in the form of insecticidal formulation.The formula can be containing a kind of or more The desired other components of kind comprising but be not limited to, liquid diluent, it is used as the adhesive of pesticide substrate, for coercing The filler of seed is protected under the conditions of compeling and for improving coating elasticity, adhesion and/or the plasticizer of ductility.In addition, right For containing little or no the oiliness insecticidal formulation of filler, it may be necessary to desiccant be added into formula, for example, carbon Sour calcium, kaolin or bentonite, perlite, diatomite or any other adsorbent material.Use of these components in seed treatment Way is known in the art.For example, seeing, U.S. Patent number 5,876,739.Those skilled in the art can be readily selected desired Component, be used for insecticidal formulation, this is depended on processed kind of subtype and the specific insecticide selected.Furthermore, it is possible to Using commercial formulations be easy to get, known insecticides, shown in embodiment as follows.

The component that insecticide of the invention can be used as seed coating is applied on seed.By the way that insecticide of the present invention is added After one of combined embodiment is improved, seed coating method and composition known in the art can be used.For it This kind of coating method and equipment of application are disclosed, for example, U.S. Patent number 5,918,413,5,891,246,5,554, 445, in 5,389,399,5,107,787,5,080,925,4,759,945 and 4,465,017.Seed coating composition is disclosed In, such as U.S. Patent number 5,939,356,5,882,713,5,876,739,5,849,320,5,834,447,5,791,084, 5,661,103、5,622,003、5,580,544、5,328,942、5,300,127、4,735,015、4,634,587、4,383, 391, in 4,372,080,4,339,456,4,272,417 and 4,245,432 etc..

The insecticide used in the coating is those described herein insecticide.The desinsection used in the processing to seed The amount of agent will depend on the type of kind of subtype and active constituent and change,

But the processing will include, and seed is in contact with the insecticide composition of the amount with insecticidal action.Work as insect When being object noxious livings, the amount will be the amount for killing elder brother's worm agent with insecticidal effect.It is used herein have kill The amount of insect effect indicates that the amount of insecticide will kill larva or the harmful organism in pupa stage in growth, or will The lasting amount reduced or postpone the damage that harmful insect generates.

In general, being changed in following ranges in processing to the amount of the insecticide of seed application: to every 100kg seed weight Speech, the pesticide active ingredient of about 100gm to about 2000gm.Preferably, the amount of insecticide is in following ranges: to every The active constituent of about 50gm to about 1000gm for 100kg seed；It is highly preferred that in following ranges: to every 100kg kind The active constituent of about 100gm to about 600gm for son；Even more preferably, in following ranges: to every 100kg kind The active constituent of about 200gm to about 500gm for son.Alternatively, it has been found that being more than big for every 100kg seed The amount of the insecticide of the pesticide active ingredient of about 60gm is preferably, it is highly preferred that being more than about 80gm (every 100kg kind Son).

Necessarily to inhibit germination for the insecticide in handling, but should can be in targeted insect life cycle It can lead to the period to seed or plant injury, effective in protection seed and/or plant.In general, after planting, coating will be effectively big About 0 to 120 day.

Insecticide of the invention can be applied on seed in the form of coating.The use of coating is negative with high insecticide Lotus (can be needed to typically handle fire resisting harmful organism, for example, corn rootworm) when it is especially effective, while prevent by The unacceptable toxicity to plant caused by increased pesticidal loads.

The coating formed with insecticide composition disclosed herein preferably can be by spreading or by matrix movement, by desinsection Agent is discharged at a slow speed in medium around.

Except removing coating, also with one of following compositions or a variety of seed can be handled: other insecticides, including kill true Microbial inoculum and herbicide；Herbicide-safener；Fertilizer and/or biological control agent.These ingredients can be used as individual layer and be added, or It can be added in insecticide coating.

Traditional coating technique and machinery can be used, insecticidal formulation is applied on seed, for example, fluidized bed technology, Roller grinds method, roller electrostatic (rotostatic) seed processing machine and drum coater.Other methods, such as the bed with mouth can also To use.Seed can be classified (presized) in advance before being applied.After coating, typically, seed can be dried, It transfers them in grader and is classified.Such method is known in the art.

" insect-controlling agent " used herein or " gene containment agent " refers to specific RNA molecule, by passing through third section The connected first segment RNA segment of RNA segment and second segment RNA segment are constituted.First segment and second segment RNA segment are in RNA points Within the length of son, they are in height inverted repeat from each other, are connected together by third section RNA segment.First segment and Complementarity between second segment RNA segment causes two sections of segments to hybridize the ability for forming duplex molecule in vivo or in vitro, that is, shape At stem, wherein first segment is connected with every section in second segment segment one end by third section segment, ring is formed, so that total Loop-stem structure is formed, or even more closely hybrid structure can form stem ring knotting (knotted) structure.Second segment and Second segment segment indistinguishably, not respectively corresponds to the justice and antisense sequences of target RNA, and the RNA is object noxious What the target gene transcription of insect came, the gene can be contained by the intake of dsRNA molecule.Insect-controlling agent can also be high The nucleic acid molecules of degree purifying (or separation), more particularly, nucleic acid molecules from genomic DNA (gDNA) or cDNA library or its Nucleic acid fragment molecule.Alternatively, the segment may include smaller oligonucleotides, there are about 15 to about 250 nucleosides Sour residue, it is highly preferred that about 15 to about 30 nucleotide residues." insect-controlling agent " can also refer to DNA construct, described Construct includes by the nucleic acid molecules or its nucleic acid fragment molecule of separation and purifying, and the molecule is from gDNA or cDNA text Library." insect-controlling agent " can also refer to the microorganism comprising this DNA construct, the DNA construct include from gDNA or CDNA library, by separating and the nucleic acid molecules purified or its nucleic acid fragment molecule.Phrase " manufacture insect used herein Controlling agent " refers to following methods, wherein using be easily obtained in this field recombinant DNA technology (for example, see Sambrook, et Al.), the recombinant dna construct for passing through stable dsRNA or siRNA molecule can be transcribed to prepare, can be transcribed with building by steady The carrier of fixed dsRNA or siRNA molecule, and/or conversion or manufacture obtained containing transcription, by stable dsRNA or The cell of siRNA molecule or microorganism.The inventive process provides the production to dsRNA transcript, nucleotide sequence with Target RNA sequence very high homology, the target RNA sequence are that the Target Nucleotide Sequence in object noxious insect genes groups is compiled Code.

Term " genome " used herein is when being used for insect or host cell, not only comprising discovery in core Chromosomal DNA also includes the organelle DNA that finds in the subcellular components of cell.Therefore, DNA of the invention is introduced into Process into plant cell can be carried out by chromosomal integration or by organelle positioning.Term " genome " application It include the chromosome and plasmid in bacterial host cell when to bacterium.Therefore, DNA of the invention is introduced in bacterial host Process in cell can be positioned by chromosomal integration or by plasmid device to carry out.

The inhibition of target gene expression can be quantified, this is produced by measurement endogenous targets RNA or target RNA translation Come what is carried out, the consequence of inhibition can be verified raw albumen by the inspection to cell or biological extrinsic property.For to RNA It is well known to those skilled in the art with the method for protein quantification.It assigns big to ampicillin, bleomycin, chloramphenicol, celebrating Mycin, hygromycin, card receive mycin, lincomycinum, methotrexate (MTX), glufosinate, puromycin, miramycin, rifampin and tetracycline etc. Resistance multiple choices label be obtainable.

In some preferred embodiments, gene expression is inhibited by least 10%, it is preferable that and at least 33%, it is more excellent Selection of land, at least 50%, even more preferably, at least 80%.In particularly preferred embodiment of the invention, insect cell Interior gene expression is inhibited by least 80%, it is highly preferred that at least 90%, it is highly preferred that at least 95%, or at least 99%, so that significant inhibit to occur.It is significant to inhibit to be used to refer to enough inhibition, lead to the phenotype that can be detected RNA corresponding to (for example, larva growth stops, paralysis or death etc.) or repressed target gene and/or protein can The reduction being detected.Although In some embodiments of the present invention, inhibiting to send out in the essentially all of cell of insect It is raw, but in other preferred embodiments, inhibit only to occur in the cell subgroup for expressing the gene.For example, if will Repressed gene plays a significant role in the cell in insect nutrition digestion road, is just enough to the inhibition of these genes within cells Harmful effect is applied to insect.

Advantages of the present invention can include but is not limited to following: being easy to dsRNA introducing insect cell, low concentration can be used DsRNA or siRNA, the validity of the stability and inhibition of dsRNA or siRNA.Using low concentration, by stable The ability of dsRNA avoids many disadvantages of antisense interference.The present invention is not to be limited to use in vitro, or be restricted to it is special Sequence composition, specific target gene group, the specific part of target gene nucleotide sequence, or specific Transgenics or Specific Transfer method, this is runed counter to some in obtainable method known in the art, for example, antisense and holding back altogether System.In addition, genetic manipulation and the biology of non-classical genetic model in be also it is feasible.

In practicing the present invention, following main points are important that the nucleotide sequence come from recombinant precursor transcription In the presence of harmless to the plant cell that wherein they can be expressed according to the present invention, and to the animal food chain especially mankind also without Evil.Because the product of plant may be negative tune that is obtainable, expressing Target Nucleotide Sequence for human consumption It betides in insect.

Therefore, in order to selectively it is desirable that obtaining the inhibition to target gene in the insect species controlled, it is preferable that The sequence identity of corresponding gene should be minuent in target gene and plant or vertebrate.Preferably, sequence identity Degree is less than about 80%.It is highly preferred that sequence identity degree is less than about 70%.Most preferably, sequence identity degree Less than about 60%.

For one kind according to the present invention embodiment there is provided a kind of nucleotide sequence, vivoexpression can lead to following warps The transcription of stable RNA sequence is crossed, the RNA molecule very high homology of target gene in the RNA sequence and insect, the RNA points The encoded RNA sequence of attached bag nucleotide sequence containing insect genes groups.Therefore, it is absorbed in insect and passes through stable RNA sequence After (be contained in diet or be sprayed on plant surface), realize to the corresponding nucleotides sequence of target gene in targeted insect cell The negative tune of column.Holding to insect, survival, proliferation, breeding are led to by the negative nucleotide sequence adjusted in insect and propagated harmful Effect.Therefore, nucleotide sequence of the invention can be used for adjusting or controlling the invasion of the insect within the scope of certain.

Another embodiment according to the present invention provides a kind of nucleotide sequence, the table in microbial cell Up to the transcription for leading to following RNA sequences, the RNA molecule very high homology of target gene, the RNA in the RNA sequence and insect Molecule includes the RNA sequence of insect genes groups nucleotide sequence coding.Therefore, the warp contained in insect intake microbial cell It crosses after stable RNA sequence, the nucleotide sequence that will lead to target gene in insect cell is adjusted by negative.By negative tune in insect Nucleotide sequence leads to holding, survival, proliferation, breeding and the harmful effect of propagation to insect.Therefore, nucleotide of the invention Sequence can be used for adjusting or controlling the invasion of the insect within the scope of certain.

Another embodiment according to the present invention provides a kind of nucleotide sequence, the expression in plant cell Lead to the transcription of following RNA sequences, the RNA molecule very high homology of target gene in the RNA sequence and insect, the RNA points The RNA sequence of attached bag nucleotide sequence containing insect genes groups coding.Therefore, the process contained in insect intake plant cell is steady After fixed RNA sequence, the nucleotide sequence that will lead to target gene in insect cell is adjusted by negative.By the negative nucleosides adjusted in insect Acid sequence leads to holding, survival, proliferation, breeding and the harmful effect of propagation to insect.Therefore, nucleotide sequence of the invention It can be used for adjusting or controlling the invasion of the insect within the scope of certain.

Term " very high homology " used herein or " high homology " are indicated when mentioning with acid sequence tight Under the conditions of careful with any sequence or SEQ ID NO:169 in SEQ ID NO:1 to SEQ ID NO:143 shown in sequence table The nucleotide sequence that coded sequence shown in any sequence into SEQ ID NO:174 or its complementary series can hybridize.Tight Under the conditions of careful with any sequence or SEQ ID NO:169 in SEQ ID NO:1 to SEQ ID NO:143 shown in sequence table The sequence that any sequence or its complementary series into SEQ ID NO:174 can hybridize is that anti-put down occurs between allowing two sequences The sequence that row compares, then two sequences can form hydrogen bond under high stringency conditions with the correspondence base on opposite strand, to form two Body molecule, under high stringency conditions be it is sufficiently stable, can be detected using approach well known.Preferably, such height Any sequence or SEQ ID NO in SEQ ID NO:1 to SEQ ID NO:143 shown in homologous sequence and sequence table: Shown in 169 any sequence into SEQ ID NO:174 control nucleotide sequence or its complementary series have about 65% to About 70% sequence identity, perhaps it is highly preferred that the sequence identity of about 80% to about 85% or most preferably Ground, the sequence identity of about 90% to about 95%, until about 99% sequence identity.

Term " sequence identity " used herein, " sequence similar figures " or " homology " be used to describe two or more Relationship between nucleotide sequence.The percentage of " sequence identity " is measured by following methods between two sequences : compare two sequences for being optimised alignment in comparison window, wherein (do not include addition compared with control sequence or lack), A part of sequence may include addition or missing (that is, notch) in comparison window, for optimizing alignment two sequences.Under Method is stated to calculate percentage: there are identical nucleic acid base or the quantity of the position of amino acid residue in measurement two sequences, with The quantity for generating matching position removes the total number of positions in comparison window with the quantity of matching position, by result multiplied by 100 to generate The percentage of sequence identity.And the identical sequence in each position is considered identical as control sequence when compared with control sequence, Vice versa.If first nucleotide sequence shows the complete complementarity with Article 2 or control sequence, with 5 ' to 3 ' sides When always observing, first nucleotide sequence is considered as the Article 2 or control nucleotide sequence with 3 ' to 5 ' directions to observe Complementary series or be complementary.Herein, with each nucleotide in a sequence of 5 ' to 3 ' directions reading and with 3 ' When each nucleotide for the other sequences read to 5 ' directions is complementary, we say that nucleic acid molecule is shown " completely It is complementary ".The reverse complemental sequence that the nucleotide sequence complementary with control nucleotide sequence will show with compare nucleotide sequence Arrange identical sequence.These terms and description have good detailed description in the art, are that those of ordinary skill in the art are easy In understanding.

" comparison window " used herein refers to, the conceptual segment of at least six continuous position, generally about 50 to about 100, More commonly, about 100 to about 150 continuous positions, wherein two sequences are optimised after alignment, by a sequence Compared with the control sequence with same number continuous position.Compared with control sequence (not including addition or missing), comparison window can To be aligned two sequences for optimizing comprising about 20% or less addition or missing (that is, notch).Those skilled in the art Member will refer to Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575Science Drive Madison, Wis., USA) in for sequence alignment method detailed or refer to Ausubel Being discussed in detail about sequence analysis in et al. (1998).

Target gene of the invention is obtained from insect cell or it is foreign gene, for example, coming from virus, fungi, elder brother The foreign genetic sequence of worm or nematode etc.." acquisition " refers to that sequence is the naturally occurring of the target gene from insect cell gene group Nucleotide sequence all or part；If fruit gene is structural gene, particularly, being coupled with cap, montage one Grade gather polyadenylation mRNA naturally occurring nucleotide sequence a part, the mRNA be found in cell it is naturally occurring DNA sequence dna expression；Or simultaneously all or part of sequence of the RNA of nonstructural gene comprising but it is not limited to tRNA, catalysis RNA, rRNA, Microrna etc..If the sequence obtained is that the nucleotide sequence based on natural RNA manufactures, show With the sequence identity of about 80% to about 100% of native sequences, and can under stringent hybridisation conditions with native sequences Hybridization, then sequence is exactly to obtain from the RNA sequence of these naturally occurring.In one embodiment, target gene includes Any sequence or SEQ ID NO:169 to SEQ ID in SEQ ID NO:1 to SEQ ID NO:143 shown in sequence table Nucleotide sequence shown in any sequence in NO:174 or its complementary series.Depending on specific target gene and dsRNA points The transport dosage of son, this method can make the function of target gene completely or partially lose, or times to fall between What desired containment is horizontal.

It the present invention also provides artificial DNA sequence, can be expressed in cell or microorganism, also, it can inhibit insect The expression of target gene in cell, tissue or organ, wherein artificial DNA sequence, which includes at least, encodes one or more different IPs The dsDNA molecule of nucleotide sequence, wherein each in different nucleotide sequences all includes the just core that intervening sequence is connected Nucleotide sequence and antisense base sequences, the intervening sequence encode dsRNA molecule of the invention.Intervening sequence constitutes justice The part of nucleotide sequence and antisense base sequences will be formed in the dsRNA intramolecular between justice and antisense sequences.Just Nucleotide sequence of sense nucleotide sequence and antisense base sequences and target gene or derivatives thereof or its complementary series height It is identical.DsDNA molecule is operationally placed under the control of promoter sequence, the promoter sequence host cell, It plays a role in tissue or organ, expresses dsDNA, generate dsRNA molecule.In one embodiment, artificial DNA sequence can be from Shown in SEQ ID NO:1 to SEQ ID NO:143 or SEQ ID NO:169 to SEQ ID NO:174 shown in sequence table Nucleotide sequence obtain.

The present invention also provides artificial DNA sequences, for expressing in plant cell, also, as DNA is expressed as RNA And absorbed by object noxious livings, the containment to the expression of target gene in insect pest cell, tissue or organ can be obtained. DsRNA includes at least one or more structural gene sequences, wherein every kind of structural gene sequence all includes that intervening sequence is connected Positive sense nucleotide sequence and antisense base sequences, the interval sequence forms ring in complementary and antisense sequences.Just nucleosides Acid sequence and antisense base sequences and nucleotide sequence of target gene or derivatives thereof or its complementary series height are identical.One Kind or various structures gene order are operationally placed under the control of one or more promoter sequences, wherein at least one It is operable in the cell, tissue or organ of protokaryon or eucaryote, especially insect.In one embodiment, Artificial DNA sequence includes about SEQ ID NO:1 to SEQ ID NO:143 or about SEQ ID NO shown in sequence table: 169 to SEQ ID NO:174 sequence.

Term used herein, for transcribing the " non-naturally occurring of dsRNA or siRNA or its segment of the invention Gene ", " non-naturally occurring coded sequence ", " artificial sequence " or " composite coding sequence " refer to, to be related to any kind of something lost Those of the means preparation of separation and manipulation is passed, the means, which can be prepared, transcribes dsRNA or siRNA or its segment of the invention Coded sequence.This includes separating coded sequence from its naturally occurring state, is manipulated to coded sequence, this is to pass through (1) nucleotides inserted, missing or substitution, (2) segment insertion, deletion or substitution, (3) chemical synthesis, such as deoxyribonucleoside phosphorous Amide chemical method etc., mutation site-specific, to the truncation of coded sequence or any other manipulation or separation method.

This respect according to the present invention can be cloned for the non-naturally occurring gene order of WCR control or its segment To between Liang Zhong operable in transgenic plant cells tissue-specific promoter, such as two kinds of root-specific promoters, The gene order or its segment can be expressed here, and mRNA is generated in transgenic plant cells, to form dsRNA points Son.The dsRNA molecule contained in plant tissue is absorbed by insect, is inhibited to obtain to the expectation that target gene is expressed.

The present invention also provides a kind of methods, for obtaining the nucleic acid comprising following nucleotide sequence, the nucleotides sequence Column can produce dsRNA or siRNA of the invention.In a preferred embodiment, the side for being used to obtain nucleic acid of the invention Method includes: that (a) is gone to detect the library cDNA or gDNA with hybridization probe, and the probe includes the nucleotide sequence from targeted insect Or all or part of its homologue；(b) DNA clone that can hybridize with hybridization probe is identified；(c) it isolates in step (b) The DNA clone of identification；And (d) cDNA the or gDNA segment for being included in separation clone in step (c) is sequenced, In, the nucleic acid molecules being sequenced can transcribe RNA nucleotide sequence or the whole or the overwhelming majority of its homologue.

In another preferred embodiment, of the invention (wherein includes for producing this for obtaining nucleic acid fragment Invention dsRNA or siRNA the overwhelming majority nucleotide sequences) method include the following steps: (a) synthesize first and second Oligonucleolide primers, they are corresponding with the part of one of nucleotide sequence from targeted insect；And (b) use step (a) first and Article 2 Oligonucleolide primers expand cDNA present in cloning vector or gDNA insertion, wherein amplification Obtained nucleic acid molecules can transcribe the overwhelming majority of the overwhelming majority of dsRNA or siRNA of the present invention.

In practicing the present invention, target gene can be obtained from corn rootworm (CRW), such as WCR or SCR, Huo Zhecong Can cause crop plants be compromised and any insect species of the subsequent underproduction obtain.It is presently believed that several standard can It is used to select preferred target gene.The gene is such gene: its protein product has quick circulating rate, makes DsRNA is obtained to inhibit to can lead to the quick reduction of protein level.In some embodiments, expression is selected to drop on a small quantity The gene to the illeffects of insect is caused to be advantageous.If it is desired to targeting broad spectrum insect species, then to select in these objects The highly conserved gene of inter-species.On the contrary, In some embodiments of the present invention, selection is containing each in order to bring specificity The gene in the low region of conservative between kind of insect species or between insect and other biologies.In some embodiments, it selects The gene for not having the known homologue of other biologies is that people want.

Term used herein " from ... obtain " refer to, it can be obtained from specific special source or species special Nucleotide sequence, although need not be directly from the special source or species.

In one embodiment, the gene expressed in insect digestive tract is selected.Target the base expressed in alimentary canal The needs of dsRNA are spread inside insect because avoiding.May include for target gene of the invention, for example, with coding matter Those of the nucleotide sequence very high homology of known alimentary canal expressing gene of the sub- V-ATPase protein component of film quality (Dow et Al., 1997, J.Exp.Biol., 200:237-245；Dow, Bioenerg.Biomemb., 1999,31:75-83).The albumen Complex is the sole energy supplier of epithelial cell example transhipment, the alkalization for middle enteral chamber.V-ATPase is also in geneva (Malpighian) expressed in pipe, the Malpighian tube is that insect hindgut grows body (outgrowth), with mammal kidney The similar mode of organ plays a role in fluid balance and removing toxic substances to xenobiontics.

In another embodiment, gene critically important involved in insect growth, development and breeding is selected.It is exemplary Gene include but is not limited to CHD3 gene and 'beta '-tubulin gene.CHD3 base in Drosophila melanogaster There is the albumen of ATP dependent form DNA helicase activities, the Chromatin assembly/de-assembly being related in core because encoding.Similar sequence Column it has also been found that in it is a variety of biology in, such as Arabidopsis thaliana, Caenorhabditis elegans and Saccharomyces cerevisiae.Beta- microtubule protein gene family encodes is to microcosmic relevant albumen, the albumen The composition part of cytoskeleton.Correlated series it has also been found that in it is a variety of biology in, such as Caenorhabditis elegans and Manduca Sexta。

May include for other target genes of the invention, for example, playing the part of in survival, growth, development, breeding and propagation Drill those of key player.These target genes can be one of the following: household's gene, transcription factor and insect specificity gene Or lethal knockout mutations in Drosophila.The target gene being used in the present invention can also be that from other biologies A bit, for example, coming from nematode (such as C.elegans).In addition, may also come from following plants for nucleotide sequence of the invention The function of object, virus, bacterium or fungal gene, the gene has been established in document, nucleotide sequence and insect genes Target gene shares the similitude of height in group.A method according to the present present invention, for WCR control, target sequence can be with base This is obtained from target WCR insect.Coding and the D.v.virgifera albumen of known albumen homology or its segment, come from WCR Some exemplary goal sequences of cDNA library can be found from sequence table.

The nucleic acid molecules of albumen homology object known to coding from D.virgifera are known (Andersen et Al., US Patent Application Serial No.10/205,189).

Although sequence Primary Reference WCR described in Andersen et aI., in the practice of the invention, it is preferable to use tool There is the DNA segment of following sequences, the sequence is corresponding with gene or coded sequence in object noxious livings to be controlled is needed Sequence has at least about 80% phase same sex, perhaps at least about 90% phase same sex perhaps at least 95% phase same sex or at least The 98% phase same sex, or at least about 100% phase same sex.Sequence with the phase same sex of target sequence less than about 80% does not have more With.Inhibit the one or more genes for being specific to harmful organism, the sequence of the gene corresponds to dsRNA.Independent basis because expression It is unaffected.This species specificity allows to be subject to selectively targeting to pest, thus to the present composition is exposed to Other biologies do not influence.

It is at least about 19 to about 23 nucleotide for DNA segment length of the invention, or about 23 to about 100 nucleotide, but it is less than about 2000 nucleotide.

The present invention is not limited to specific genes as described herein, further include following any genes, the inhibition to the gene Illeffects can be applied to harmful insect.

For many in the insect for the potential target being then controlled through the invention, about most of genes The information of sequence and the phenotype obtained from the mutation to specific gene may be limited.Therefore, the present inventor proposes, from nocuousness Insect selects suitable gene for process of the invention, can by using can to model organism (such as Drosophila), some other insect species, or even nematode species, fungal species, plant species are (in these species Corresponding gene has been analyzed) in the information that obtains of research of corresponding gene carry out.In some cases, it would be possible to by using It comes from, such as the sequence of Drosophila, insect in addition, nematode, fungi or plant (wherein the gene is therefrom cloned) Or gene name, garbled data library, such as GenBank obtain the sequence of targeted insect corresponding gene.Once sequence is obtained, it can The gene segment in the insect suitably selected is expanded to use PCR, for the present invention.

In order to obtain the DNA segment of the corresponding gene in insect species, based on its therefrom cloned in WCR or gene The sequence that finds in its insect designs PCR primer.Primer is designed to: can be expanded the DNA segment of sufficient length, is used for this hair It is bright.DNA (genomic DNA or cDNA) is prepared from insect species, with PCR primer come DNA amplification segment.Amplification condition is selected Are as follows: even if in the case where primer accurately cannot match target sequence, amplification can also be carried out.Alternatively, WCR base can be used Because or other known insect genes be used as probe, from preparing from the gDNA or cDNA library of harmful insect species, clone base Because of (or part of it).Technology for implementing PCR and cloning from library is known.Based in the past from WCR or other elder brothers The gene order cloned in worm species struggles the further details of the method for DNA segment from object noxious insect, is implementing It is provided in example.It will be appreciated by those of ordinary skill in the art that a variety of different technologies can be used for from harmful insect species Isolate gene segment corresponding with the gene separated in the past from other species kinds.

Classified based on feeding methods, may cause the damaged insect of plant and generally fall into three categories, these three types are respectively Belong to Coleoptem, Lepidoptera, Diptera, Orthoptem, Heteroptera, Ctenophalides, Chewing type, suctorial type and the hole drilling type insect of Arachnidiae and Hymenoptera mesh.Chewing type insect eats the tissue of plant, Such as root, leaf, flower, bud and spray, lead to big damage.Example from this big caste includes beetle and its larva. WCR and SCR belongs to chewing type insect.Their larva feeds on the root of plant, particularly, on the root of corn plant, at Worm mainly feeds blade.The gene of any species can be used to open as target from WCR or SCR or in above-mentioned mesh The exhibition present invention.

It has been found that the present invention can be used for protecting seed and plant to keep out extensive agricultural pest, including insect, Mite, fungi, yeast, mould, bacterium, nematode, weeds and parasitism and saprophyte etc..

When insect is object noxious livings of the invention, such harmful organism includes but is not limited to come from Lepidoptera purpose, for example,

Acleris spp.、Adoxophyes spp.、Aegeria spp.、Agrotis spp.、Alabama argillaceae、Amylois spp.、Anticarsia gemmatalis、Archips spp、Argyrotaenia spp.、 Autographa spp.、Busseola fiisca、Cadra cautella、Carposina nipponensis、Chilo spp.、Choristoneura spp.、Clysia ambiguella、Cnaphalocrocis spp.、Cnephasia spp.、 Cochylis spp.、Coleophora spp.、Crocidolomia binotalis、Cryptophlebia leucotreta、Cydia spp.、Diatraea spp.、Diparopsis castanea、Earias spp.、Ephestia spp.、Eucosma spp.、Eupoecilia ambiguella、Euproctis spp.、Euxoa spp.、Grapholita spp.、Hedya nubiferana、Heliothis spp.、Hellula undalis、Hyplumtria cunea、 Keiferia lycopersicella、Leucoptera scitella、Lithocollethis spp.、Lobesia botrana、Lymantria spp.、Lyonetia spp.、Malacosoma spp.、Mamestra brassicae、 Manduca sexta、Operophtera spp.、Ostrinia Nubilalis、Pammene spp.、Pandemis spp.、 Panolis flammea、Pectinophora gossypiella、Phthorimaea operculella、Pieris rapae、Pieris spp.、Plutella xylostella、Prays spp.、Scirpophaga spp.、Sesamia spp.、Sparganothis spp.、Spodoptera spp.、Synanthedon spp.、Thaumetopoea spp.、 Tortrix spp., Trichoplusia ni and Yponomeuta spp.；

From Coleoptera purpose, for example,

Agriotes spp.、Anthonomus spp.、Atomaria linearis、Chaetocnema tibialis、 Cosmopolites spp.、Curculio spp.、Dermestes spp.、Diabrotica spp.、Epilachna spp.、Eremnus spp.、Leptinotarsa decemlineata、Lissorhoptrus spp.、Melolontha spp.、Orycaephilus spp.、Otiorhynchus spp.、Phlyctinus spp.、Popillia spp.、 Psylliodes spp.、Rhizopertha spp.、Scarabeidae、Sitophilus spp.、Sitotroga spp.、 Teuebrio spp., Tribolium spp. and Trogoderma spp.；

From Orthoptera purpose, for example,

Blatta spp.、Blattella spp.、Gryllotalpa spp.、Leucophaea maderae、 Locusta spp., Periplaneta ssp. and Schistocerca spp.；

From Isoptera purpose, for example,

Reticulitemes ssp；

From Psocoptera purpose, for example,

Liposcelis spp.；

From Anoplura purpose, for example,

Haematopinus spp., Linognathus spp., Pediculus spp., Pemphigus spp. and Phylloxera spp.；

From Mallophaga purpose, for example,

Damalineaspp. with Trichodectes spp.；

From Thysanoptera purpose, for example,

Franklinella spp.、Hercinothrips spp.、Taeniothrips spp.、Thrips palmi、 Thrips tabaci and Scirtothrips aurantii；

From Heteroptera purpose, for example,

Cimex spp.、Distantiella theobroma、Dysdercus spp.、Euchistus spp.、 Eurygaster spp.、Leptoeorisa spp.、Nezara spp.、Piesma spp.、Rhodnius spp.、 Sahlbergella singularis, Scotinophara spp., Triatoma spp., Miridae family spp. (such as Lygus hesperus and Lygus lineoloris), Lygaeidae family spp. (such as Blissus leucopterus) With Pentatomidae family spp.；

From Homoptera purpose, such as

Aleurothrixus floccosus、Aleyrodes brassicae、Aonidiella spp.、 Aphididae、Aphis spp.、Aspidiotus spp.、Bemisia tabaci、Ceroplaster spp.、 Chrysomphalus aonidium、Chrysomphalus dictyospermi、Coccus hesperidum、Empoasca spp.、Eriosoma larigerum、Erythroneura spp.、Gascardia spp.、Laodelphax spp.、 Lacanium corrii、Lepidosaphes spp.、Macrosiphus spp.、M.yzus spp.、Nehotettix spp.、Nilaparvata spp.、Paratoria spp.、Pemphigus spp.、Planococcus spp.、 Pseudaulacaspis spp.、Pseudococcus spp.、Psylla ssp.、Pulvinaria aethiopica、 Quadraspidiotus spp.、Rhopallosiphum spp.、Saissetia spp.、Scaphoideus spp.、 Schizaphis spp., Sitobion spp., Trialeurodes vaporariorum, Trioza erytreag and Unaspiscitri；

From Hymenoptera purpose, for example,

Acromynnex、Atta spp.、Cephus spp.、Diprion spp.、Diprionidae、Gilpinia polytoma、Hoplocampa spp.、Lasius sppp.、Monomorium pharaonis、Neodiprion spp、 Solenopsis spp. and Vespa ssp.；

From Diptera purpose, for example,

Aedes spp.、Antherigona soccata、Bibio hortulanus、Calliphora erythrocephala、Ceratitis spp.、Chrysomyia spp.、Culex spp.、Cuterebra spp.、Dacus spp.、Drosophila melanogaster、Fannia spp.、Gastrophilusspp.、Glossina spp.、 Hypoderma spp.、Hyppobosca spp.、Liriomysa spp.、Lucilia spp.、Melanagromyza spp.、Musca ssp.、Oestrus spp.、Orseolia spp.、Oscinella frit、Pegomyia hyoscyami、 Phorbia spp.、Rhagoletis pomonella、Sciara spp.、Stomoxys spp.、Tabanus spp.、 Tannia spp. and Tipula spp.；

From Siphonaptera purpose, for example,

Ceratophyllus spp. and Xenopsylla cheopis, and

From Thysanura purpose, for example,

Lepisma saccharina。

It has been found that when harmful insect is Diabrotica spp., particularly, when harmful organism is Diabrotica Virgifera virgifera (Western corn rootworm, WCR), Diabrotica barberi (Northern corn rootworm, NCR), (Brazil is beautiful by Diabrotica virgifera zeae (Mexican Corn Rootworm, MCR), Diabrotica balteata Rice rootworm, BZR) or Brazilian Com Rootworm complex (BCR, by Diabrotica viridula and Diabrotica Speciosa composition) or Diabrotica undecimpunctata howardii (Southern corn rootworm, SCR) when It waits, the present invention is particularly useful.

The present invention also especially can pierce through plant cell and tissue and the therefrom insect species of sucking fluid effective in controlling, Including but not limited to, the fleahopper (plant bug) in smelly stinkbug (species of Pentatomidae family) and Miridae family, example Such as western fleahopper (western tarnished plant bug, Lygus hesperus species), fleahopper (tarnished Plant bug, Lygus lineolaris species) and white peas or beans fleahopper (pale legume bug, Lygus elisus).

Improvement to method disclosed herein is unexpectedly particularly useful in the crop harmful organism of control Lepidoptera.

The present invention provides stable dsRNA or siRNA molecule is passed through, for controlling insect infestations.DsRNA or siRNA Molecule includes the aggregated ribonucleotide of double-strand, may include the modification to phosphate sugar backbone or nucleosides.In RNA structure Modification can be coupled with tail, to allow specific genetic to inhibit.

In one embodiment, dsRNA molecule can be modified by enzyme method, can produce siRNA molecule. SiRNA can effectively mediate negative tune to act on for some target genes in some insects.This enzyme method can be by eukaryon It is completed in RNAi approach using RNAse III enzyme present in insect, vertebrate, fungi or plant cell or DICER enzyme (Elbashir et al., 2002, Methods, 26 (2): 199-213；Hamilton and Baulcombe, 1999, Science 286:950-952).This method is also using the recombination being introduced into targeted insect cell by recombinant DNA technology DICER or RNAse III is carried out, this is that those skilled in the art are easy to know.It is naturally present in insect or passes through Biggish dsRNA chain can be cut into lesser oligonucleotides by the DICER enzyme and RNAse III of recombinant DNA technology manufacture. DsRNA molecule specifically can be cut into siRNA segment by DICER enzyme, wherein each siRNA segment is about 19-25 core Thuja acid is long, and dsRNA molecule is usually cut into the siRNA of 12-15 base-pair by RNAse III enzyme.Pass through above-mentioned any enzyme The siRNA molecule of generation has 3 ' suspensions and the 5 ' phosphoric acid and 3 ' C-terminals of 2 to 3 nucleotide.It is produced by RNAse III enzyme Raw siRNA, by the identical of Dicer manufacture, therefore, then can be targeted, and after loose in eucaryotic RNA i approach It is degraded by inherent intracellular rna degradation mechanism, is separated into single stranded RNA, and hybridize with the RNA sequence of target gene transcription. This method be can the RNA sequence nucleotide sequence coded to target gene in insect effectively degraded or removed.As a result It is exactly the silencing of the nucleotide sequence especially targeted in insect.To the datail description of enzyme method can Hannon (2002, Nature, 418:244-251) in find.

To inhibit through stabilizing dsrna technology target gene be sequence-specific using of the invention, because, with RNA bis- The corresponding nucleotide sequence of body region is targeted for heredity inhibition.Contain a part of identical nucleotides sequence with target gene The RNA of column is optimized for inhibiting.There is relative target sequence the RNA sequence of insertion, missing and simple point mutation to be also found to have Inhibiting effect.In aspect of performance of the invention, it is preferable that a part of inhibition dsRNA and target gene is shared at least about 80% sequence identity, the perhaps sequence identity higher than about 90% or the sequence identity higher than about 95%, or Person is higher than about 99% sequence identity, or even about 100% sequence identity.Alternatively, two body regions of RNA can By functionally is defined as: the nucleotide sequence that can hybridize with a part of target gene transcript.It shows higher homologous Property the sequence less than overall length can be equivalent to (compensate) it is longer but only have less homology sequence.Identical nucleotides sequence The length of column can be at least about 25,50,100,200,300,400,500 or at least about 1000 bases.In general, answering When the sequence for using more than 20-1100 nucleotide, although being more than the sequence of about 200-300 nucleotide is preferably, to surpass Cross about 500-1000 nucleotide sequence be it is especially preferred, this depend on target gene size.The present invention has such as Lower advantage: it can endure sequence variant, and the variant may be since genetic mutation, strain polymorphism or evolutionary divergence are made At.Level-one transcription product relative to target gene or the mRNA by processing completely, the nucleic acid molecules of introducing may be not required to It is absolutely homologous, it may not be necessary to overall length.Therefore, those skilled in the art it will be appreciated that as disclosed herein, RNA and 100% sequence identity between target gene is for practicing for the present invention not necessarily.

DsRNA molecule can be by synthesizing in vivo or in vitro.DsRNA can be formed by the RNA chain of single self-complementary, or Person can be formed by two complementary RNA chains.The Endogenous RNA polymerase of cell can be with mediate transcription in vivo, or the RNA of clone Polymer can be used for transcribing in vivo or in vitro.It can be by following methods come targeted inhibition: in organ, tissue or cell type In specific transcriptional；The stimulation (for example, infection, stress, temperature, chemical inducer) of environmental condition；And/or in the stage of development Or the engineering transcription that the age carries out.RNA chain may or may not be gather it is polyadenylation；RNA chain can or cannot be by cell Translating equipment is translated as polypeptide.

RNA, dsRNA, siRNA or miRNA of the invention by those skilled in the art by chemical method or enzyme method come Production, this is carried out through manual or automatic reaction or in another organism.It can also be by having partially or completely Machine synthesizes to manufacture RNA；Any ribonucleotide by modification can be introduced by external enzymatic synthesis or organic synthesis.It can lead to Cross intracellular RNA polymerase or phage rna polymerase (for example, T3, T7, SP6) Lai Hecheng RNA.The use of expression construct With manufacture be it is known in the art (see, for example, WO97/32016, U.S. Patent number 5,593,874,5,698,425,5,712, 135,5,789,214 and 5,804,693).If, can be right before introducing cell by chemical synthesis or by external enzymatic synthesis RNA is purified.For example, can be pure from mixture by with solvent or resin extraction, precipitating, electrophoresis, chromatography or combinations thereof Dissolve RNA.Alternatively, RNA can be used, under the case where not purifying or only carrying out minimal purifying to avoid due to sample Loss caused by product processing.RNA can be dried to store, or be dissolved in aqueous solution.Solution can contain buffer and salt, To promote the stabilization and/or annealing of two-body chain.

To be transcribed in vivo from Transgenics or expression construct, it can be used regulatory region (for example, promoter, increasing Hadron, silencer and gather polyadenylation) transcribe (or a plurality of) RNA chain.Therefore, in one embodiment, for manufacturing The nucleotide sequence of RNA molecule can start with having functional one or more in microorganism, fungi or plant host cell Subsequence is operably associated.It is desirable that nucleotide sequence is placed under the control of endogenesis promoter, the endogenous starting Son is generally in host genome.Nucleotide sequence of the present invention under the promoter sequence control being operably connected, Flank can also have additional sequence, and the additional sequence can advantageously influence the steady of the transcript that it is transcribed and/or obtains It is qualitative.The downstream that such sequence is usually located at the upstream for the promoter being operably connected and/or expression construct 3 ' is held, can To exist simultaneously in the downstream of promoter upstream and the end of expression construct 3 ', although having sequence on only such is also to be included 's.

In another embodiment, nucleotide sequence of the invention may include separated by " intervening sequence " it is reversed It repeats.Intervening sequence can be the region comprising following any nucleotide sequences, if necessary, the nucleotide sequence energy Secondary structure between every section of repetition is promoted to be formed.In one embodiment of the invention, intervening sequence be for mRNA just A part of justice or antisense coded sequence.Alternatively, intervening sequence may include can with nucleic acid molecules be covalently attached nucleotide or Any combination of its homologue.Intervening sequence may include the nucleotide sequence that length is at least about 10-100 nucleotide, or Person's length is at least about 100-200 nucleotide, and length is that at least about 200-400 nucleotide or length is at least About 400-500 nucleotide.

For purposes of the invention, dsRNA or siRNA molecule can be obtained from CRW, this is by from corn rootworm The target CRW gene order or part thereof that gDNA or cDNA library obtain carries out polymerase chain (PCR) amplification to realize.It can To prepare WCR larva with method known to persons of ordinary skill in the art, DNA/RNA can be extracted.It can will be different size of Larva for purposes of the present invention from the 1st age to the CRW being fully grown-up to carries out DNA/RNA extraction.Genomic DNA or cDNA text Library can be used for PCR amplification, to produce dsRNA or siRNA.

Then the method being easy to get in this field can be used, PCR amplification and sequencing are carried out to target gene.This field skill Art personnel can change PCR condition, be formed with the PCR product for ensuring optimal.PCR product by verifying be used as Template is used for vivo transcription, to generate the justice and antisense RNA with the minimum limit promoter being included.

Of the invention turns over famous person's proposition, can from any insect species identification in Bugdom and the nucleic acid sequence being purified into For the present invention, to control WCR and other targeted insects.It in one aspect of the invention, can be from the object from coleoptera species Kind is to obtain nucleic acid.Particularly, Diabrotica can be subordinated to and belong to the chrysomelid worm of (coleoptera, Chrysomelidae) to obtain nucleic acid, more Particularly, nucleic acid of the invention can be obtained from virgifera group.Most particularly, nucleic acid of the invention can be from Diabrotica Virgifera virgifera LeConte is obtained, and is commonly known as WCR.It can be used for by isolated nucleic acid, such as identify Target gene, and the following recombinant vectors of building, the carrier can produce of the invention by stable dsRNA or siRNA, use WCR insect infestations are kept out in protection plant.

Therefore, in one embodiment, the present invention includes the nucleosides by separation and purifying from WCR or Lygus Acid sequence is used as insect-controlling agent.It include the SEQ listed in sequence table by the nucleotide sequence for separating and purifying Sequence shown in ID NO:1 to SEQ ID NO:143 or SEQ ID NO:169 to SEQ ID NO:174.

From WCR or other insects, nucleic acid for use in the present invention also may include by separation and highly purified Unigenes and EST nucleic acid molecules or its nucleic acid fragment molecule.EST nucleic acid molecules can encode the marking area of polypeptide, or In fact, the major part of polypeptide.Alternatively, the segment may include smaller oligonucleotides, the oligonucleotides has about 15 to about 250 nucleotide residues, it is highly preferred that about 15 to about 30 nucleotide residues.Alternatively, for of the invention Nucleic acid molecules can come from the cDNA library of the no vertebra pest of WCR, lygus or any other.

Phrase " highly purified nucleic acid " used herein, " artificial sequence ", " by separation and highly purified core Acid " refers to following nucleic acid " by separation and highly purified nucleotide sequence ", and it is adjoint to be no longer accompanied by its native state Substance in some or its structure it is any all different from naturally occurring nucleic acid.The example of highly purified nucleic acid It include: (1) DNA, the partial sequence with naturally occurring genomic DNA molecule, but its flank not in the DNA molecular day So two coded sequences existing for the molecular moiety flank in existing biological genome；(2) carrier or protokaryon are included in Or the nucleic acid in eukaryotic gene group DNA, the means included are so that obtained molecule and any naturally occurring load Body or genomic DNA are all different；(3) independent molecule, for example, cDNA, genomic fragment, pass through polymerase chain reaction (PCR) The segment or restriction fragment of generation；(4) recombinant DNA；And (5) synthetic DNA.Highly purified nucleic acid can also be by one or more CDNA, genomic DNA or synthetic DNA composition.

Nucleic acid molecules and its segment from the no vertebra pest of WCR, Lygus or other can be used for being come From other nucleic acid molecules of other species, dsRNA and siRNA molecule for being wanted in the present invention with generation.Such nucleic acid point Attached bag includes following nucleic acid molecules, the complete coded sequence of the nucleic acid molecule encoding protein and the promoter of such molecule And flanking sequence.In addition, such nucleic acid molecules include: the nucleic acid molecules of encoding gene family member.Use above-mentioned nucleic acid molecules Or its segment goes to screen from D.v.virgifera or the library cDNA or gDNA obtained from Lygus hesperus, can be easy Such molecule must be obtained.Method for such library to be made is well known in the art.

Nucleic acid molecules from WCR or Lygus and its segment can be used for obtaining other nucleic acid molecules, such as nucleic acid is homologous Object, dsRNA and siRNA molecule for being wanted in the present invention with generation.Such homologue includes: wholly or partially to encode The nucleic acid molecules of the protein homology object of other species, plant or other biologies.It goes to sieve using above-mentioned nucleic acid molecules or its segment The library EST, cDNA or gDNA is selected, such molecule can be readily available.Method for such library to be made is this field public affairs Know.The nucleotide sequence of such homologue molecule can be with SEQ ID NO:1 to SEQ shown in sequence table disclosed herein The nucleotide sequence that is found in one of ID NO:143 or SEQ ID NO:169 to SEQ ID NO:174 or a variety of sequences or Its complementary series is different, because not needing complete complementary for hybridization for stablizing.These nucleic acid molecules further include, though So with nucleic acid molecules specific hybrid but it can may lack the molecule of complete complementarity.In a kind of specific implementation, use It can be used for obtaining such sequence (Frohman, M.A.et at, Proc.Natl.Acad.ScL in the method for 3 ' or 5 ' RACE (U.S.A.) (1988) 85:8998-9002；Ohara, O.et at, Proc.Natl.Acad.ScL (U.S.A.) 86:5613- 5611(1989)).In general, any type in above-mentioned nucleic acid molecules or segment all can be used for generating following dsRNA or SiRNA, the RNA are suitable for the invention diet, spraying mixture or recombinant dna construct.

Phrase " coded sequence ", " structural nucleotide sequence " or " structural nucleic acid molecule " used herein refers to, is placed in When under suitable regulating and controlling sequence control, it can be translated into the nucleotide sequence of polypeptide, usually this is carried out by mRNA.Coding The boundary of sequence is determined by the translation initiation codon of 5 ' ends and the translation termination codon of 3 ' ends.Coded sequence It can include but is not limited to, genomic DNA, cDNA, EST and recombinant nucleotide sequence.

Term " recombinant DNA " or " recombinant nucleotide sequence " refer to that the DNA containing genetic engineering modification, the modification is logical The manipulation of mutagenesis, restriction enzyme etc. is crossed to carry out.

The segment of above-mentioned nucleic acid molecules or nucleic acid molecules or other nucleic acid molecules from WCR can be special under certain conditions Property with other making nucleic acid molecular hybridizations.Herein, if two molecules can form antiparallel double-strandednucleic acid structure, two are just said Nucleic acid molecules can mutual specific hybrid.If two nucleic acid molecules show complete complementarity, we just say nucleic acid point Son is the complementary series of another nucleic acid molecules.If two molecules can be with foot under the conditions of at least traditional " low rigor " Enough stability hybridizes mutually, them is allowed to remain the state annealed mutually, we just say that this two molecules are " minimum limits Complementary ".Similarly, if under the conditions of at least traditional " high rigor ", two molecules can be mutual with enough stability Hybridization, allows them to remain the state annealed mutually, we just say that this two molecules are complementary.Traditional high stringency conditions exist Sambrook, et al. and Haymes, et al.In:Nucleic Acid Hybridization, A Practical It is described in Approach, IRL Press, Washington, DC (1985).

Therefore, have with complete complementary away from being allowed, as long as this away from the energy that molecule will not be made to form duplex structure Power completely disappears.Therefore, the segment to make nucleic acid molecules or nucleic acid molecules is used as primer or probe, it is only necessary to sequence It is complementary enough, stable duplex structure can be formed under specific solvent and salinity used.

Can promote the suitable high stringency conditions of DNA hybridization is, for example, being carried out at 6.0x sodium chloride/lemon at about 45 DEG C It in sour sodium (SSC), then goes to wash with 2.0x SSC at 50 DEG C, this is known to the skilled in the art, or can be in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989) are looked in 6.3.1-6.3.6 It arrives.For example, the salinity in cleaning step can be selected from: low rigor is 50 DEG C lower, about 2.0x SSC, 50 under supreme rigor DEG C, about 0.2x SSc.In addition, the temperature in cleaning step can be supreme tight from the increase of (about 22 DEG C) of room temperature under low rigor About 65 DEG C under careful degree.Temperature and salt can all change, alternatively, temperature or salinity can be kept constant, other variables change Become.

For nucleic acid of the invention can with from WCR one or more nucleic acid molecules or its complementary series medium tight Specific hybrid under the conditions of careful, for example, at about 2.0x SSC and about 65 DEG C.It will include: in height for nucleic acid of the invention Under stringency conditions, SEQ ID NO:1 to SEQ ID NO:143 or SEQ the ID NO:169 to SEQ that can be represented with sequence Those of one of nucleic acid molecules or its complementary series disclosed in ID NO:174 or a variety of specific hybrids nucleic acid molecules. Preferably, for nucleic acid molecules of the invention will show SEQ ID NO:1 to the SEQ ID NO:143 represented with sequence or Shown in SEQ ID NO:169 to SEQ ID NO:174 or one or more nucleic acid molecules as disclosed herein have At least about 80%, perhaps at least about 90% perhaps at least about 95% perhaps at least about 98% or even about 100% sequence identity；Alternatively, will be shown and the genomic DNA point from harmful insect for nucleic acid sequence of the invention SEQ ID NO:1 to SEQ ID NO:143 or SEQ ID NO:169 to the SEQ ID NO:174 that separate out, such as sequence represents Shown in one or more nucleic acid molecules have at least about 80%, perhaps at least about 90% or at least about 95%, Perhaps at least about 98% or even about 100% sequence identity.

The whole of nucleic acid from WCR or the overwhelming majority can be used for separating coding from same species or other species CDNA, gDNA and nucleic acid of Diabrotica albumen homology object or its segment.About from the library cDNA and gDNA separate and identify The detailed description of the technology of nucleic acid of the invention, is disclosed in embodiment.

Nucleic acid of the invention can also be to be synthesized completely or partially by methods known in the art, especially when needing When the sequence of favorite plant is provided.Therefore all or part of nucleic acid of the present invention can be using the close of the host's preference selected Numeral synthesizes.The codon of species preference can be by, for example, using in the albumen expressed from specific host species Most frequent codon determines.It can cause mutant that there is the activity slightly changed other modifications of nucleotide sequence.

A part of the present invention provides a kind of delivery system, is used for insect transporting insects controlling agent.Process of the invention Stable dsRNA or siRNA molecule can be introduced directly into the cell of insect, or be introduced into extracellular chamber, void space, leaching Bar system, digestive system, are introduced into the circulatory system of insect, this is can use by orally ingestible or those skilled in the art Other means carry out.For by the oral method introduced include: by the direct mixture of the food of RNA and insect, with And following engineering methods, wherein be used as food species have passed through it is engineered, be able to expression dsRNA or siRNA, so It is fed to be influenced insect afterwards.In one embodiment, for example, dsRNA or siRNA can by microbial expression, And root, stem can be introduced by microbe application to plant surface or by physical means, such as injection.In another embodiment In, can to plant carry out it is genetically engineered, make its expression be enough to kill the insect for having notified infection plant amount dsRNA or siRNA。

Particularly, when practicing the present invention in WCR, it is intracorporal insect will can be introduced by stable dsRNA or siRNA Middle intestines obtain preferably to the inhibition of target gene.As described above, dsRNA or siRNA molecule can be included in diet or cover It is placed on diet, can be absorbed by insect.Under any circumstance, dsRNA of the invention is provided in the meals of object noxious livings In food.Object noxious livings of the invention will show about 4.5 to about 9.5 or about 5 to about 8.5 or about 6 to About 8 or about 6.5 to about 7.7 or about 7.0 pH in digestive tract.The alimentary canal of object noxious livings is determined herein Justice is following positions in harmful organism, and in the position, the food that harmful organism is absorbed is exposed to this hair of energy favorable for absorption The environment of bright dsRNA molecule is extremely arrived without meeting with so that Hydrogen bond cleavage between dsRNA double-strand is to form single-stranded point The pH of son.

In addition, the dsRNA of insect control is transported by spraying application for the purpose of insect infestations in control plant It is sent to plant surface and provides the means of another protection plant.In this case, by engineered to generate and accumulate The bacterium of dsRNA can be fermented, and fermented product is formulated that compatible spray product can be applied with common agricultural.It is described with can To include: suitable sicker and wetting agent needed for high-efficiency blade covering, and the UV for protecting dsRNA to damage from UV Protective agent.Such additives be it is common in bioinsecticidal agent industry, be also well known to those skilled in the art.Similarly, Formula for soil application may include following granular recipes, be used as soil harmful insect (for example, corn rootworm) children The bait of worm.

It is also foreseeable that can be formulated with the consistent mode of conventional agricultural practices by chemistry or enzymatic synthesis production DsRNA is used as spray product, to control insect infestations.The formula may include: conjunction needed for high-efficiency blade covering Suitable sicker and wetting agent, and the UV protective agent for protecting dsRNA to damage from UV.Such additives are that biology kills elder brother It is common in worm agent industry, also it is well known to those skilled in the art.Such application can (be or not with other spraying insecticides It is based on biology) application combination, to improve the protection to plant in terms of keeping out insect feeding damage.

The present inventor proposes that the bacterium bacterial strain for producing insecticidal protein can be used for production and control mesh for insect DsRNA.These bacterial strains show the insect domination property of raising.A variety of different bacterial hosts can be used for producing insect Control dsRNA.Illustrative bacterium may include E.coli, B.thuringiensis, Pseudomonas sp., Photorhabdus sp., Xenorhabdus sp., Serratia entomophila and relevant Serratia sp., B.sphaericus, B.cereus, B.laterosponis, B.popilliae, Clostridium bifermentans and its Its Clostridium species or other gram-positive bacteriums for forming spore.

The invention further relates to the recombinant dna construct for expressing in microorganism.Methods known in the art can be used, The exogenous nucleic acid that target RNA can therefrom be transcribed out is introduced into microbial host cell, for example, bacterial cell or fungal cell.

Nucleotide sequence of the invention can be introduced in the protokaryon and eucaryon host of wide scope, be generated by stable DsRNA or siRNA molecule.Term " microorganism " includes protokaryon and eukaryotic microorganisms species, such as bacterium and fungi.Fungi includes Yeast and there is fungi etc..Illustrative prokaryotes, existing Gram-negative also have it is gram-positive, including Enterobacteriaceae, such as Escherichia, Erwinia, Shigella, Salmonella and Proteus； Bacillaceae；Rhizobiceae, such as Rhizobium；Spirillaceae, for example, photobacteria, Zymomonas, Serratia,Aeromonas,Vibrio,Desulfovibrio,Spirillum；Lactobacillaceae； Pseudomonadaceae, such as Pseudomonas and Acetobacter；Azotobacteraceae, Actinomycetales and Nitrobacteraceae.Eucaryote includes fungi, for example, Phycomycetes and Ascomycetes comprising yeast, for example, Saccharomyces and Schizosaccharomyces；And Basidiomycetes yeast, for example, Rhodotorula, Aureobasidium, Sporobolomyces etc..

For the purpose for insect protection plant, it is known that inhabit the blade face (leaves of plants of a variety of different important crops Son surface) and/or a large amount of microorganisms of rhizosphere (soil of surrounding plant roots) be also possible to for disclosed herein heavy Group construct is manipulated, is bred the desired host cell of propagation, storage, transport and/or mutagenesis.These microorganisms include thin Bacterium, algae and fungi.What it is with special interest is microorganism, for example, bacterium, example belongs to described as follows: Bacillus (including it is following Kind and subspecies: B.thuringiensis kurstaki HD-I, B.thuringiensis kurstaki HD-73, B.thuringiensis sotto、B.thuringiensis berliner、B.thuringiensis thuringiensis、 B.thuringiensis tolworthi、B.thuringiensis dendrolimus、B.thuringiensis alesti、 B.thuringiensis galleriae、B.thuringiensis aizawai、B.thuringiensis subtoxicus、 B.thuringiensis entomocidus, B.thuringiensis tenebrionis and B.thuringiensis san diego)；Pseudomonas, Erwinia, Serratia, Klebsiella, Zanthomonas, Streptomyces, Rhizobium, Rhodopseudomonas, Methylophilius, Agrobacterium, Acetobacter, Lactobacillus, Arthrobacter, Azotobacter, Leuconostoc and Alcaligenes；Fungi, especially ferment Mother, for example, following categories: Saccharomyces, Cryptococcus, Kluyveromyces, Sporobolomyces, Rhodotorula and Aureobasidiuum.What it is with special interest is the bacterial species of such surrounding plants, such as Pseudomonas syringae, Pseudomonas fluorescens, Serratia marcescens, Acetobacter Xylinum, Agrobacterium tumefaciens, Rhodobacter sphaeroides, Xanthomonas Campestris, Rhizobium melioti, Alcaligenes eutrophus and Azotobacter vinlandii；With And the yeast species of surrounding plants, for example, Rhodotorula rubra, R.glutinis, R.marina, R.aurantiaca, Cryptococcus albidus, C.diffluens, C.laurentii, Saccharomyces rosei, S.pretoriensis, S.eerevisiae, Sporobolomyces roseus, S.odorus, Kluyveromyces Veronae and Aureobasidium pollulans.

Bacterium recombinant DNA carrier can be linear or closed cyclic plasmid.Carrier system can be single carrier or Plasmid or two or more carriers or plasmid, they contain the total DNA that will be introduced in bacterial host gene group together.This Outside, bacteria carrier can be expression vector.SEQ ID NO:1 to SEQ ID NO:143 or SEQ ID shown in sequence table NO:169 to SEQ ID NO:1 74 or nucleic acid molecules shown in its segment can be with for example, be adapted for insertion into carrier, the load Body is under the control of suitable promoter, and the promoter plays a role in one or more microbial hosts, with driving The expression of the coded sequence or other DNA sequence dnas that are connected.For this purpose, many carriers be all it is obtainable, to suitable carrier The selection particular host cell that will primarily depend upon the size for the nucleic acid that band is inserted into carrier and will be converted with carrier.Often Kind carrier all contains various ingredients, and the specific host that this depends on its function (DNA amplification or expression DNA) and is compatible with is thin Born of the same parents.Carrier component for bacterium conversion typically includes, but not limited to one of following components or a variety of: signal sequence, duplication Starting point, one or more selectable marker genes and the inducible promoter for allowing exogenous DNA to express.

Expression and cloning vector usually contain selection gene, are also referred to as selected marker.The gene is encoded in selectivity Albumen necessary to the survival of inverted host cell or growth that are grown on culture medium.Typical selection gene encodes following eggs It is white, the albumen energy: (a) assign to antibiotic or other toxin, for example, ampicillin, neomycin, methotrexate (MTX) or Fourth Ring The resistance of element, (b) supplies auxotrophy, or (c) provides the critical nutrients that can not be obtained from complex medium, for example, right For Bacilli, the gene of encoding D-alanine racemase.These can be produced by the cell of heterologous protein or its segment successful conversion The raw albumen assigned to drug resistance, thus, it is possible to survive in selection is handled.

Expression vector for producing mRNA can also be containing following inducible promoters, and the promoter can be by host bacteria Bio-identification, and being operably connected with following nucleic acid, the nucleic acid for example, encoding D .v.virgifera mRNA or its can draw Play the nucleic acid of the segment of interest.Inducible promoter suitable for bacterial host includes beta-lactam enzyme promoters, E.coli λ bacteriophage PL and PR promoter and E.coli galactose promoter, Arabinose promoter, alkaline phosphatase promoter, color Propylhomoserin (trp) promoter and lactose operon promoter and its variant and hybrid promoters, for example, tac promoter.But Other known bacteria-inducible promoter is also suitable.

When mentioning regulating and controlling sequence and structural nucleotide sequence, term " being operably connected " refers to that regulating and controlling sequence leads to phase The expression of structural nucleotide sequence even is adjusted." regulating and controlling sequence " or " control element " refers to positioned at structural nucleotide sequence upstream (5 ' non-coding sequence), intermediate or downstream (3 ' non-translated sequence) nucleotide sequence, can influence dependency structure nucleotide The opportunity and level of transcription, the RNA processing or stability or translation of sequence or quantity.Regulating and controlling sequence may include promoter, turn over It translates boot sequence, introne, enhancer, loop-stem structure, the sub- binding sequence of containment and gathers polyadenylation identification sequence etc..

Alternatively, expression vector can be integrated into bacterial genomes with integration vector.Typically integration vector contains and allows The homologous at least one sequence of the bacterial chromosome of vector integration.Integration looks like homologous in carrier DNA and bacterial chromosome Caused by recombination between DNA.For example, can be integrated into the integration vector of the DNA building from a variety of Bacillus bacterial strains Bacillus chromosome (EP 0,127,328).Integration vector also may include bacteriophage or transposon sequence.Suicide type carries Body is also known in the art.

To containing one or more elements listed above suitable carrier building using the recombinant DNA technology of standard come It carries out.To being cut by isolated plasmid or DNA fragmentation, in addition tail, and it is connected as generating required plasmid again desired Form.The example of obtainable bacterial expression vector includes but is not limited to multi-functional E.coli clone and expression vector, example Such as, Bluescript (Stratagene, La Jolla, CA), wherein for example, D.v.virgifera albumen or its segment energy quilt It connects into carrier, the connection is to meet the reading frame for beta galactosidase amino terminal Met and subsequent seven residues Mode carry out, thus generate hybrid protein；PIN carrier (Van Heeke and Schuster, 1989, J.Biol. Chem.264:5503-5509) etc..

Typically, yeast recombinant precursor includes one of following components or a variety of: promoter sequence, fusion partner sequence Column, boot sequence, translation termination sequence, selected marker.These elements can be combined into expression cassette, and expression cassette can be held in multiple In system, such as extra-chromosomal element (for example, plasmid), it can stablize and be held in host (such as yeast or bacterium).Duplication Son can have two sets of dubbing systems, thus it be allowed to be held in, for example, for expressing in yeast, in prokaryotes host For cloning and expanding.The example of such yeast-bacteria shuttle carrier include YEp24 (Botstein et al., 1979, Gene, 8:17-24), pCI/1 (Brake et al., 1984, Proc.Natl.Acad.Sci USA, 81:4642-4646) and YRp17 (Stinchcomb et al., 1982, J.MoI.Biol., 158:157).In addition, replicon can be high or low copy Several plasmids.High copy number plasmid will be usually with the copy number in about 5 to about 200 ranges, typically, and about 10 to big About 150.Host containing high copy number plasmid will preferably have at least about 10, more preferably at least about 20 copies.

Useful yeast initiating sequence the gene of enzyme can be obtained from encoding metabolic pathway.The example of this genoid includes alcohol Dehydrogenase (ADH) (EP 0 284044), enolase, glucokinase, glucose-6-phosphate isomerase, glyceraldehyde-3-phosphate dehydrogenation Enzyme (GAP or GAPDH), hexokinase, phosphofructokinase, 3-phosphoglycerate mutase and pyruvate kinase (PyK) (EP03215447).The yeast PHO5 gene of encoding acid phosphatase also provides useful promoter sequence (Myanohara Et al., Proc.Natl.Acad.Sci.USA, 80:1,1983).In addition, the synthetic promoter being naturally not present also can be used as Yeast promoter plays a role.The example of such hybrid promoters includes the ADH regulating and controlling sequence being connected with GAP transcriptional activation region (U.S. Patent number 4,876,197 and 4,880,734).Other examples of hybrid promoters include, by ADH2, GAL4, GAL10 or PHO5 gene regulatory sequence group closes the promoter (EP of the transcriptional activation region composition of carbohydrate-splitting enzyme gene (such as GAP or PyK) 0164556).In addition, Yeast promoter may include: in conjunction with yeast RNA polymerase and originate transcription ability, it is non- The naturally occurring promoter of yeast sources.

The example of transcription terminator and other termination sequences (for example, those of coding carbohydrate-splitting enzyme) that can be identified by yeast Son is known to the skilled in the art.

Alternatively, expression construct can be integrated into Yeast genome with integration vector.Typically, integration vector contains and permits Perhaps the homologous at least one sequence of the yeast chromosomal of vector integration, and it is homologous in two kinds of table construct to preferably comprise flank Sequence.Integrate caused by the recombination looked like in carrier and yeast chromosomal between homologous dna (Orr-Weaver et al., 1983, Methods in Enzymol., 101:228-245).Integration vector can be for the particular locus in yeast, this is It include being realized into carrier by selecting suitable homologous sequence.See above the Orr-Weaver et al..It is a kind of or A variety of expression constructs can be integrated, possibly, influence the recombinant protein generated level (Rine et al., 1983, Proc.Natl.Acad.Sci.USA, 80:6750).The chromosome sequence for including in carrier can be used as single segment and be present in In carrier, this will lead to entire carrier integration or its can be used as two segments and exist, two segments and chromosome In adjacent segment it is homologous, expression construct of the flank in carrier will lead to the stable integration of only expression construct.

It is further proposed that nucleotide sequence of the invention is transformed into plant, enable one or more dsRNA molecules With the expression of harmful organism suppression level.Using method obtained by this field, conversion carrier can be easily prepared.Conversion carrier Comprising one or more following nucleotide sequences, the sequence can be transcribed into RNA molecule, and encoded with insect genes groups One or more nucleotide sequence very high homologies and/or complementation, so that: insect absorbs from one or more nucleotide sequence molecules When the RNA that transcription comes, negative tune can be subject to the expression of at least one of nucleotide sequence each in insect genes groups.

Conversion carrier can also indicate dsDNA construct, in addition to this, can also be taken as recombinant molecule, insect-controlling agent, Genetic molecule or chimeric (chimeric) genetic constructs.Chimeric genetic construct of the invention may include, for example, encoding one kind Or a variety of antisense transcript sheets, one or more sense transcript sheets, above-mentioned every kind of one or more nucleotide sequences, wherein The all or part of all or part and following RNA molecules from its transcript is homologous, and the RNA molecule includes insect base Because of a group RNA sequence for nucleotide sequence coding.

In one embodiment, plant conversion carrier is the DNA molecular by separating and purifying, wherein including and this hair The operably associated starting word of bright one or more nucleotide sequences.Nucleotide sequence is selected from as shown in sequence table The group that SEQ ID NO:1 to SEQ ID NO:143 or SEQ ID NO:169 to SEQ ID NO:174 is constituted.Nucleotide sequence Including following segments, all or part of RNA present in encoding target harmful organism rna transcription sheet also has comprising target The evil all or part of inverted repeat of biology RNA.DNA molecular comprising expression vector can also contain functional intron sequences (its upstream for being positioned at coded sequence or even among coded sequence), the molecule can also skim that (5 ') are non-to be turned over containing five Boot sequence (that is, UTR or 5 '-UTR) is translated, is positioned between promoter and translation initiation site.

Plant conversion carrier can contain the sequence from more than one gene, and more than one dsRNA is thus allowed to produce It is raw, for inhibiting the expression of two or more genes in object noxious livings cell.It should be readily apparent to one skilled in the art that having The DNA segment of sequence corresponding to sequence present in different genes is combined into single compound DNA segment, for turning base Because being expressed in plant.Alternatively, can be modified the plasmid of the present invention for having contained at least one DNA segment, this is by by volume Outer DNA segment, which is sequentially inserted between enhancer and promoter and terminator sequence, to carry out.It is being designed to inhibit a variety of In the insect-controlling agent of the present invention of gene, gene to be suppressed can be obtained from same insect species, to enhance insect-controlling agent Efficiency.In some embodiments, gene can be obtained from different insects, to widen effective insect range of reagent.When being directed to It, can be according to Fillatti, number of patent application US 2004- when containing or targeting several genes for the combination of expression and containment 0029283 illustrates and disclosed, to manufacture polycistron DNA element.

In the case where going conversion plant with nucleotide sequence of the invention, selection shows the following startings of following ability Son, the promoter can drive the expression of coded sequence in the specific plant species.There is function in different plant species The promoter of energy is well known in the art.Can be used for expressing the example of the promoter of polypeptide in plant is Odell et al. Induction type, virus, synthesis or constitutive promoter described in (1985, Nature313:810-812), and/or by from the time Regulation, the spatially promoter of regulation and spatiotemporal database.Preferred promoter include enhancing CaMV35S promoter and FMV35S promoter.For purposes of the invention, for example, preferably existing for the optimum control for the species fed in root The highest level expression of these genes is obtained in plant root.A large amount of root enhancing promoter, which has been accredited, to be come out, they are (Lu et al., 2000, J.Plant Phys., 156 (2): 277-283 known in the art；U.S. Patent number 5,837,848 With 6,489,542).Typically, recombinant DNA carrier of the invention or construct will include selected marker, can be in plant cell Upper imparting selects phenotype.Selected marker can also be used to selecting the plant containing the exogenous nucleic acid for encoding polypeptide or albumen of the present invention or Plant cell.Label can encode biocide resistance, antibiotic resistance (for example, card receive mycin, G418 bleomycin, tide it is mould Element etc.) or Herbicid resistant (for example, glyphosate etc.).The example of selected marker includes but is not limited to, and coding card receives chloramphenicol resistance Neo gene, can be selected for mycin, G418 etc. is received using card；Encode the bar gene of bialaphos-resistant；Mutant Epsp synthase gene, coding glyphosate resistance；Nitrilase gene assigns the resistance to Brominal；Mutant acetyl cream Synthase genes (ALS) assign the resistance to imidazolone or sulfonylurea；And the DHFR gene of anti-methopterin.

Recombinant vector or construct of the invention may also include selection markers.Selection markers can be used for monitoring expression.Show The selection markers of example property include beta-glucuronidase or uidA gene (GUS), and coding is known, and there are many enzymes of chromophoric substrate (Jefferson, 1987, Plant Mol.Biol, Rep.5:387-405；Jefferson et al, 1987, EMBO J.6: 3901-3907)；R- locus gene, what coding can regulate and control the generation of anthocyania pigment in plant tissue (red) Product (Dellaporta et al, 1988, Stadler Symposium 11:263-282)；Beta-lactam enzyme gene (Sutcliffe et al., 1978, Proc.Natl.Acad.Sci 75:3737-3741), there are many gene coding is known The enzyme of chromophoric substrate (for example, PADAC, color development cephalosporin)；Luciferase genes (Ow et al., 1986, Science 254:856-859)；XylE gene (Zukowsky et al., 1983, Proc.Natl.Acad.Sci 50:1101-1105), It encodes catechol dioxygenase, and the enzyme can convert the catechol of color development；Alpha-amylase gene (Ikatu et al., 1990, Bio/Technol.8:241-242)；Tyrosinase cdna (Katz et al., 1983, J.Gen.Microbiol.129: 2703-2714), oxidizing tyrosine can be the enzyme of DOPA and DOPA quinone by coding, and DOPA quinone then polymerize (condense) For melanin；Alpha-galactosidase can be catalyzed α-galactose substrate of color development.

Generally, it is preferred to which non-specific location introduces functional recombinant DNA in the plant genome.Under special circumstances, may be used Recombinant dna construct is inserted by site-directed integration.Implant can be made to have functional a variety of fixed point recombination systems there are known, It includes FLP-FRT disclosed in cre-lox disclosed in United States Patent (USP) 4,959,317 and United States Patent (USP) 5,527,695.

In practice, in any single transformation experiment, DNA is only introduced in the target cell of small percentage.Coding choosing The gene for selecting label is used to provide for for identifying that the conversion is by connecing by the efficient system of these cells of stable conversion It receives transgene DNA construct and is integrated into their genome to realize.Preferred marker gene, which provides, to be assigned to choosing The selected marker of the resistance of selecting property reagent (for example, antibiotic or herbicide).Plant of the invention can have to its resistance Any herbicide contributes to the useful reagent of selected marker.The cell that will likely be converted is exposed to selective reagent.It is depositing It will be following cells in the population of living cells, wherein in general, resistance-conferring gene is integrated, and allow cell survival enough Horizontal expression.Cell can further be tested, to verify the stable integration of exogenous DNA.Usually used selectivity mark Remember that gene includes assigning to those of antibiotic resistance, such as receive mycin (nptII), hygromycin B (aph IV) He Qing great to card Mycin (aac3 and aacC4), or to those of Herbicid resistant/tolerance, such as to glufosinate-ammonium (bar or pat), glyphosate (EPSPS) and AMPA (phnO).The example of such selected marker is described in United States Patent (USP) 5,550318,5,633,435,5, In 780,708 and 6,118,047.There is provided the selection markers visually identified transformant can also be used, for example, expression is coloured Or fluorescin (for example, luciferase or green fluorescent protein (GFP)) gene or express beta- glucuronidase base Cause or uidA gene (GUS), it is known that there are many chromophoric substrates for it.

Preferred plant conversion carrier includes the Ti-plasmids from Agrobacterium tumrfaciens (for example, the U.S. 4,536,475,4,693,977,4,886,937,5,501,967 and EP of the patent No. 0 122 is 791).Agrobacterium Rhizogens plasmid (or " Ri ") can also be used, they are known in the art.Other preferred plant conversion carriers include Herrera-Estrella (1983, Nature 303:209-213), Bevan (1983, Nature 304:184-187), Klee (those of disclosed in 1985, Bio/Technol.3:637-642 and EP 0 120516.

Method and composition for converting plant by introducing from recombinant dna construct to Plant Genome includes big Measure any method in means known in the art.A kind of method for constructing the plant by conversion is microinjection blast technique, Such as United States Patent (USP) 5,015,580,5,550,318,5,538,880,6,153,812,6,160,208,6,288,312 and 6,399, Described in 861.Another method for constructing inverted plant is the conversion that Agrobacterium is mediated, such as United States Patent (USP) 5, 159,135,5,824,877,5,591,616 and 6, described in 384,301.Alternatively, other non-Agrobacterium objects can be used Kind, for example, Rhizobium and the other prokaryotic cells for showing following abilities, their energy infection plant cells, by heterologous nucleotide Acid sequence introduces the genome of infected plant cell.

DNA construct of the invention can be introduced into mesh by the way that well known to a person skilled in the art a variety of Conventional reformat technologies Mark plant.With regard to Agrobacterium mediate conversion purpose for, suitable plant conversion carrier include from The carrier that the Ti-plasmids of Agrobacterium tumrfaciens obtains.The plant conversion carrier mediated except Agrobacterium Except, other methods may be alternatively used for for DNA construct of the invention being inserted into plant cell.Such method may include but It is not limited to, for example, electroporation increases the absorption of dissociative DNA using liposome, the fortune for carrying out dissociative DNA is bombarded by microinjection It send and is converted using virus or pollen.

Other something lost can be combined by of the invention by any nucleic acid in isolated nucleic acid by permanently or temporarily means Element, such as promoter, introne, enhancer and untranslated boot sequence etc. is passed to be introduced into plant cell.Coding can be manufactured Coleoptera species RNA, or the RNA from hole drilling type and suctorial type insect species, or preferably D.v.virgifera RNA or Any nucleic acid molecules of Lygus Hesperus RNA, and the means by allowing to generate dsRNA molecule in plant cell will Its introduced plant cell provides one or more specific dsRNA of insecticidal amount in the diet of object noxious insect.

Term " transgenic plant cells " or " genetically modified plants " refer to, plant cell or plant containing exogenous nucleic acid, institute Stating exogenous nucleic acid can obtain from WCR, or obtain from different insect species or any other non-insect species.Genetically modified plants are also used Come include such transgenosis any generation offspring (descendants (decedent), descendant etc.) or all such genetically modified plants Any generation seed comprising the DNA sequence dna of coding RNA, sRNA, dsRNA, siRNA or its segment of the invention The offspring or seed are also importance of the invention.

Typically, contained using the genetically modified plants that Agrobacterium method for transformation is formed and be inserted into item chromosome In single simple recombinant DNA sequence, be referred to as transgenic event.Such genetically modified plants are referred to alternatively as: about insertion Exogenous array, heterozygosis.Homozygous genetically modified plants can be obtained by following methods for Transgenics: will be containing single Independent separate (independent segregant) genetically modified plants of one exogenous gene sequence, for example, F0 plant, certainly with it Body carries out sexual cross (selfing), to generate F1 seed.A quarter in the F1 seed of generation will be that Transgenics are come Say heterozygosis.Make F1 germination, just produce the plant that can be examined by heterozygosity, typically, is increased using SNP test or thermal expansion To carry out, the test allows the difference (that is, zygosity test) between heterozygote and homozygote for test.By heterozygote plant and its Itself or another heterozygote plant hybridize, and only generate heterozygosis offspring.

It, can be by that will have recombinant dna construct other than carrying out directly conversion to plant with recombinant dna construct The first plant and second of plant hybridization for lacking the construct carry out prepare transgenosis plant.For example, for gene containment Recombinant DNA can be introduced in the first plant lines, and the strain can be influenced by converting, and generation can be with second of plant product It is the genetically modified plants of hybridization, the recombinant DNA for being used for gene containment is penetrated into second of plant lines.

Can by the practice present invention come the genetically modified plants that manufacture, including but not limited to, alfalfa, dill, apple, Apricot, globe artichoke, rocket salad, asparagus, avocado, banana, barley, beans, beet (beet), blackberry, blueberry, blueberry, cabbage, brussels sprout, volume The small citrus of heart dish, leaf mustard, muskmelon, carrot, cassava, cauliflower, celery, cherry, coriander, citrus, Ke Laimenshi, coffee, jade Rice, cotton, cucumber, pesudotsuga taxifolia, eggplant, hare's-lettuce, wide leaf lettuce, eucalyptus, fennel, fig, cucurbit, grape, shaddock, Hami Melon, yam bean, Chinese grooseberry, lettuce, leek, lemon, bitter orange, torch pine, mango, melon, mushroom, nut, oat, okra, onion, Tangerine, decoration with plant, pawpaw, parsley, pea, peach, peanut, pears, pepper, persimmon, pine tree, pineapple, plantain, Lee, pomegranate, white poplar, Potato, pumpkin (pumpkin), quince, pine, witloof, radish, raspberry, rice, naked barley, sorghum, Southern Pine, soybean, Spinach, pumpkin (squash), strawberry, beet (sugarbeet), sugarcane, sunflower, sweet potato, storax, mandarin orange, tea, tobacco, Tomato, turf, trailing plant, watermelon, wheat, Chinese yam and small cucumber plant.

In practice, the present invention can control character with other insects in plant and combine, and be used to increase with what acquisition was wanted To the character of the control of insect infestations.Will using different role mode insect control and combination, can provide for insect into The genetically modified plants of row protection have outstanding lasting stability compared with the plant for using single insect to control character, because It is reduced in the probability that field develops resistance.

In over the past several decades, the insecticidal mechanism of B.thuringiensis crystalline protein is conducted extensive research. It has been shown that only after absorbing albumen, albumin crystal albumen just can larval form to insect it is toxic.In Lepidopteran larvae In, alkaline pH and proteolytic enzyme meeting soluble protein in insect midgut, so that the component toxic to insect discharges.These Toxic component destroys midgut epithelial cells, causes insect to stop feed, and final, causes insect death.For this reason, Confirm that B.thuringiensis toxin and is to environment from being effective during handle a variety of harmful insects The insecticide of safety.Coleoptera and hemipteran, and be likely to, diptera, lygus bug and other hole drilling types and suction Suck formula insect and illustrate slightly sour intestinal pH, thus effective in keep out Lepidopteran larvae Bt toxin to above-mentioned harmful organism not It works.The slightly sour intestinal pH of these insects is also believed to be more friendly to composition of the invention, is being not intended to limit In the case where specific theory, it appears that, the alkaline pH of Lepidopteran larvae enteron aisle attempts to show that the attempt of dsRNA effect is lost before being The reason of losing (Fire et al. U.S. Patent number 6,506,559；Mesa et al. patent publication No. US2003/0150017； Rajagopal et al., 2002, J.Biol.Chem.277:46849-46851；Tabara et al., 1998, Science 282:430-431).Therefore it is believed that dsRNA method disclosed herein should be preferentially used for control coleoptera, diptera, half Wing class, lygus bug, hole drilling type and suctorial type insect plant and composition in.Method and composition shown in this article is particularly useful In targeting the gene in following insects for containment, the insect shows about 4.5 to about 9.5, about 5.0 to about 9.0, about 5.5 to about 8.5, about 6.0 to about 8.0, about 6.5 to about 7.7 or about 6.8 to about 7.6 or About 7.0 intestinal pH.But show about 7.5 to about 11.5, or about 8.0 to about 11.0, or about 9.0 to The insect of about 10.0 intestinal pH and other pests, such as lepidopterid larva, also fall into model of the invention In enclosing.The dsRNA for inhibiting Lepidopteran larvae gene is specific to when providing together with one or more Bt albumen in larva diet When, this is especially true, and when with threshold value or higher than the horizontal offer of threshold value, Bt albumen can be according to conventional route to lepidoptera children Worm generates toxicity.Have the presence of virose one or more Bt toxin for insect species of the same race, intestinal pH will be effectively reduced, The stable environment for being directed to double stranded rna molecule is provided, is acted on applying them to the containment of harmful insect target gene.

By one or more generation dsRNA molecules of the invention by stable dsRNA construct in object noxious elder brother It is combined in the diet of worm with one or more insecticidal proteins, so that dsRNA and insecticidal protein have same harmful insect Toxicity will be useful.Insecticidal protein can be obtained from B.thuringiensis, can also can be generated from known in the art Insecticidal protein it is other biology obtain, for example, entomopathogenic nematode bacterial symbiont (for example, Photorhabdus sp., Xenorhabdus sp.), Serratia entomophila and relevant Serratia sp., B.sphaericus, B.cereus, B.laterosporus, B.popilliae, Clostridium bifermentans and insecticidal can be shown The gram-positive bacterium of other formation spores of property.Similarly, it is also proposed that, generate two kinds of dsRNA molecule of the invention Or it is a variety of it is different can be provided in together in single plant by stable dsRNA construct, with ensure insect control phenotype Lasting stability.These dsRNA molecules can target same gene for silencing, alternatively, targeting different genes for sinking It is silent.Two or more differences dsRNA can be combined in same plant, and every kind of dsRNA has toxicity to different harmful insects, It is toxic to same insect species without which kind of dsRNA.

It is expected that certain groups by stable dsRNA construct and one or more insects control protein gene Synergistic effect will be generated by closing, and can enhance the insect control phenotype of genetically modified plants.Using artificial diet or entire plant tissue into Capable insect biologic test, which can be used for determining, uses dsRNA and insect when controlling albumen about dead larvae or growth inhibiting dose Measure response.Those skilled in the art can examine the mixture of dsRNA molecule and insect protein in biologic test, with mirror Make active component, the combination be have for the application (deployment) in insect protection plant concertedness and be people Desired (Tabashnik, 1992).It has been reported that killing the concertedness of harmful insect between different insect control albumen (summary referring to Schnepf et al., 1998).It is expected that between certain dsRNA and certain dsRNA and certain insects There are concertednesses between control albumen.

It is expected that the combination of dsRNA will disclose the unexpected toxicity for being directed to certain harmful insects. DsRNA is fed to lepidoptera nocuousness by Rajagopol et al (2002, J Biol Chem.277:46849-46851) report The larva of biological S.litura, for allow coding in intestines aminopeptidase gene silencing for be do not have it is effective.It is worth mentioning , the alkaline pH environment of intestines is friendly environment for dsRNA in typical lepidoptera, because RNA two-body is in alkaline pH Under denaturation will be expected to cause rapidly to degrade.The B.thuringiensis toxin protein being inserted into midgut epithelium film The hole of formation, resulting in the neutralization of middle intestines pH, (summary is shown in Gill, 1995, Mem.Inst.Osaldo Cruz, Rio de Janeiro, 90:69-74).Therefore, it is only capable of being formed temporary ion channel in lepidoptera midgut epithelium film without will lead to extremely The B.thuringiensis toxin died, which is enough for middle intestines pH to be reduced to be more advantageous to, absorbs dsRNA's by midgut epithelial cell It is horizontal.As an example, it is known that CrylAc albumen is directed to beet armyworm, and Spodoptera exigua is not effective toxin (Chambers et al, 1991, J.Bacteriol.173:3966-3976).But middle intestines pH caused by Cry1Ac albumen It temporarily reduces to have and is used to stablize the dsRNA absorbed altogether, so that them is had the target gene for silencing S.exigua, thus provide The means of unexpected this harmful insect of control.Using any egg of lepidopterid midgut epithelial cells ion regulation can be upset It is white, whether is it can insecticidal, can observe the effect, this to coleoptera, diptera, Hemiptera, lygus bug and its Its hole drilling type and suctorial type harmful insect etc. are also useful.

Some insecticidal proteins from B.thuringiensis, such as Cyt albumen, can lead to sensitive insect larva The temporary opening of midgut epithelium film, this is because the formation of structural porous or the generality class detergent activity due to albumen are made At (Butko, 2003, Appl.Environ.Microbiol.69:2415-2422).Such opening even can be dead to causing DsRNA molecule is assisted to pass through midgut epithelial cell under the protein concentration of suboptimization for dying.It is expected that can cause on insect Any albumen being temporarily open in epithelium promotes regardless of whether energy insecticidal, can assist dsRNA molecule to enter insect cell Cell silencing.

SEQ ID NO:1 to SEQ ID NO:143 or SEQ ID NO:169 to SEQ ID NO shown in sequence table: The nucleotide sequence provided in 174 or its segment or its complementary series, can be by " offer " in a variety of culture mediums, to assist to make With.Its subgroup can also be provided in such culture medium, and the form of the subgroup allows those skilled in the art to check sequence.

Commodity product containing one or more sequences of the present invention, and from contain one or more nucleotide of the present invention Sequence recombinant plant or seed manufacture commodity product as embodiments of the present invention quilt particularly including.Containing a kind of or The commodity product of a variety of sequences of the present invention includes but is not limited to, meat, oil, pulverize or entire grain or vegetable seeds, or Person includes any meat, oil, following pulverizing or entire grain any food product, and the grain is containing one or more The recombinant plant of sequence of the invention or seed.To this in the one or more agricultural product or commodity product for including herein The detection of one or more sequences of invention proves that agricultural product or commodity product are made of genetically modified plants, described Genetically modified plants are configured to express one or more nucleotide sequences of the invention, for what is mediated by using dsRNA Gene contains method to control the purpose of insect infestations.

In a kind of application of present embodiment, nucleotide sequence of the invention can be recorded to computer-readable medium On." computer-readable medium " used herein refers to: conscientiously existing, can be by the expression of computer direct reading and access Medium.Such medium includes but is not limited to: magnetic-based storage media, such as floppy disk, hard disk, storage medium and tape；Optical storage Medium, such as CD-ROM；Electronic storage medium, such as RAM and ROM；The computer document of optical character identification format, Yi Jishang The hybrid of type is stated, for example, magnetism/optical storage medium.It will be readily apparent to those skilled in the art that any at present Known computer-readable medium can be used to manufacture following products, and the product includes to be recorded on computer-readable medium Nucleotide sequence of the invention.

" record " used herein refers to, the method for storage information on a computer-readable medium.Art technology Personnel easily can record information using any method being currently known on a computer-readable medium, to generate comprising this The medium of invention nucleotide sequence information.To those skilled in the art, a variety of different data store organisations are can to obtain , for manufacturing the computer-readable medium for being recorded on nucleotide sequence of the present invention.Selection to data storage medium It will usually be accessed based on selecting by the means of storage information.In addition, a variety of different data processor programs and format are available It stores in by nucleotide sequence of the present invention onto computer-readable medium.Sequence information can be showed in word processing text document, It is formatted with the software that can be obtained through commercial channels, such as WordPerfect and Microsoft Word, or Its form that can be shown as ASCII text document, is stored in database application, such as DB2, Sybase, Oracle etc..Ability Field technique personnel can be easy to use any amount of data processor architecture format (for example, text document or data Library), to obtain the computer-readable medium for being recorded on nucleotide sequence information of the present invention.

It is public for allowing those skilled in the art to access the computer software of the sequence information provided in computer-readable medium Obtained by crowd.BLAST (Altschul et al., J.Mol.Biol.215:403-410 are executed in Sybase system (1990)) and the software of BLAZE (Brutlag, et al, Comp.Chem.17:203-207 (1993)) searching algorithm can by with In identification sequence in open reading frame (ORF), for example, it is provided herein, containing with ORF or protein from other biologies Homology Unigene and EST.Such ORF is the segment of coding protein in sequence of the present invention, can be used for producing commercially Important protein, for example, for Amino acid synthesis, metabolism, transcription, translation, RNA processing, nucleic acid and protein degradation, protein Modification and DNA replication dna, limitation, modification, recombination and the enzyme of reparation.

The present invention also provides the systems for containing sequence information as described herein, particularly, computer based system.This Class system is designed to: can identify the commercially important segment in nucleic acid molecules of the invention.It is used herein " to be based on System for computer " refers to the hardware device, software equipment and data storage device for analyzing nucleotide information of the present invention.This hair The minimum limit hardware device of bright computer based system is set comprising central processor unit (CPU), input equipment, output Standby and data storage device.Those skilled in the art can easily instruct, in currently available computer based system Any one is suitable for the present invention.

The most preferably length of target sequence is about 10 to about 100 amino acid, or about 23 to about 300 nucleosides Sour residue.

" object construction primitive (motif) " or " target primitive " used herein refer to, any sequence for rationally selecting or Combined sequence, wherein the 3-d modelling that is formed selects one or more sequences when being folded based on target primitive.It is known in the art Plurality of target primitive.Protein target primitive includes but is not limited to enzyme active sites and signal sequence.Target set nucleic acid primitive includes But it is not limited to, promoter sequence, cis-acting elements, hairpin structure and inducible expression elements (protein binding sequence).

Embodiment

Inventor is identified herein for controlling the method without vertebra pest infestation, this is by raw in nocuousness Double stranded ribonucleic acid molecule is provided in object diet to carry out.Surprisingly, inventor by harmful organism it is disclosed that take the photograph When taking, double stranded ribonucleic acid molecule can play a role, and inhibit the biological function of harmful organism, cause one of following characteristics or A variety of: harmful organism feed is reduced, and harmful organism survival rate reduces, and harmful organism is dead, the differentiation of harmful organism and development quilt Inhibit, the reduction or disappearance of the sexual propagation ability of harmful organism, muscle are formed, the formation of juvenile hormone, the tune of juvenile hormone Control, ion regulation and transhipment, the holding of cell membrane gesture, amino acid bio synthesis, amino acid degradation, spermiogenesis tail, pheromones Synthesis, pheromones sensing, the formation of day linear protein, wing formed, formation, development and the differentiation of leg, the formation of ovum, larva at Ripe, digestibility enzyme formation, the synthesis of hemolymph, the holding of hemolymph, neurotransmission, cell division, energetic supersession, breathing and Any component of apoptosis and eukaryocyte cytoskeletal structure, for example, Actin and tubulin.Appoint in these characteristics A kind of what or any combination can lead to effective inhibition to pest infestation, in the case where the biology to plant pest, Inhibition to plant invasion.For example, when be used as being supplied to local mode plant, containing being enough to inhibit the amount of harmful organism One or more double stranded ribonucleic acid molecules dietary composition when, be used as seed treatment, the soil as surrounding plants Using, or by plant out of plant cell existing recombinant DNA molecules come biological attack meeting when producing, to plant pest Unexpectedly dramatically reduce.When being applied to single harmful organism, the embodiments shown herein is for illustrating the present invention. But it would be recognized by those skilled in the art that method, formula and idea in embodiment are used with no restriction, can be used for can To consume the institutes of following food sources whether there is or not vertebra pest, to contain related to harmful organism or inside it play Some important features of effect, the food source are that can be formulated for the harmful organism inhibitor containing sufficient amount, by At least one or more of double stranded rna molecule is constituted.

Embodiment 1

Present embodiment describes the identification to following nucleotide sequence, the nucleotide sequence is with the shape of double stranded rna molecule When formula is provided in corn rootworm diet, have for controlling corn rootworm.

Constructed from entire larva and from the middle intestines dissection of incision corn rootworm cDNA library (LIB 149, LIB 150, LD33027, LIB3373), nucleotide sequence information is obtained (see Andersen et al. is filed in 24 purpose July in 2002 U.S. Patent Application Serial Number 10/205,189 is specifically incorporated to herein entirely through reference).In addition, from different hairs are in The entire larva for educating different time in stage and each stage of development, comes construction cDNA library, is come from maximizing The quantity of the different est sequences of Diabrotica species.Respectively by the Western from the first age (1 gram) and third age (2.9 grams) Construct library LIB5444 and LIB5462 in the library mRNA that Corn rootworm larvae obtains.By being inserted into liquid nitrogen, by what is obtained Insect quick freeze.To insect is ground in the mortar and pestle for being held in -20 DEG C, temperature holding is by refrigerating on dry ice And/or liquid nitrogen is added into mortar and is realized until organizing to be grated for fine powder.It usesReagent (Invitrogen), according to manufacturers instruction, RNA is extracted.Using Dynabeads Oligo dT (Dynal Inc., NY), press According to manufacturers instruction, Poly A+RNA is isolated from total serum IgE preparation.Use SuperScript^TM Plasmid System (Invitrogen) come construction cDNA library from Poly A+RNA.Size separation is carried out to cDNA using chromatography.The fourth stage and Pyatyi segment is collected, and is connected into pSPORT1 carrier (Life Technologies Ind., Gaithersburg MD) Sal1 and Not1 restriction enzyme enzyme recognition site between, it is thin into E.coli DH10B Electrocompetent by Electroporation Transformation Born of the same parents.First instar larvae library produces about 420,000 colony forming units.Third instar larvae library produces about 2.78 x 10⁶A colony forming unit.The bacterium colony from LIB149, LIB150 is washed down from plate, by simply shaking uniform mixing, Pour Tris-EDTA buffer into again.The half washed in the substance to get off is loaded into 10% glycerol, dispenses into freezing tubule, storage It is stored in -70 DEG C.The other half be used to generate Plasmid DNA with the micro-purified column of Quiagen or its product of equal value.It will be purified Plasmid DNA is dispensed into micro-centrifuge tube, is stored in -20 DEG C.

In high viscosity culture medium individually amplification from Diabrotica virgifera cDNA library LIB5444 and The bacterium colony of LIB5462.At 37 DEG C, 500 ml LB culture mediums (contain 0.3%SeaPrepWith 50 mg/l carboxylic benzyls Penicillin) in individually placed stirring plate, mixing from LIB5444 about 200,000 colony forming unit and come From the 600 of LIB5462,000 colony forming units, then cooled down 1 hour rapidly in water ice bath, so that bacterial clump is equal Even suspension.Then the library by inoculation is cultivated 42 hours at 30 DEG C.After incubation, 5 are carried out to cell on stirring plate The mixing of minute.It then will be in the centrifugal bottle of media transfer to two 250ml.It is handled 10 minutes in 10,000xg, precipitating is thin Bacterium cell.Culture medium is removed from bottle, cell is resuspended in the LB culture medium of 20ml in total, wherein containing 50mg/l's Carbenicillin.Dimethyl sulfoxide is added to 10%, cell is remained into freezing state.Two kinds of libraries are expanded, until every Milliliter 10⁸The final potency of a colony forming unit.Combine Diabrotica virgiferacDNA library LIB5444 and The sample of LIB5462 is adjusted to the DNA of every microlitre of about 1.25 micrograms in by aseptic distillation and deionized water Concentration is dispensed into 25 freezing tubules, and each freezing tubule contains the DNA of about 8.75 micrograms.Applicant/inventor is in 2004 On June 10, in is by these sample preservations to positioned at 10801University Boulevard, Manassas, Virginia USA The American Type Culture Colleciton (ATCC) of ZIP 20110-2209, is referred to as LIB5444/62. For ATCC to applicant provide preservation receipts, specifying ATCC deposit number is PTA-6072.

Substantially according to described above for the method for generating corn rootworm cDNA library, corn rootworm high molecular weight is prepared CDNA library, i.e. LIB5496 and LIB5498.Respectively from by the first age (1 gram) and third age (1 gram) Western corn rootworm children The library mRNA that worm obtains constructs library LIB5496 and LIB5498.In short, the quick freeze insect in liquid nitrogen, by grinding It mills in alms bowl and pestle, the insect of freezing is become into fine powder.It usesReagent (Invitrogen), according to factory Quotient's specification extracts RNA.Using Dynabeads OligodT (Dynal Inc., NY), according to manufacturers instruction, from total serum IgE PolyA+RNA is isolated in preparation.Use SuperScript^TMPlasmid System (Invitrogen) is from 20 micrograms Poly A+RNA constructs high molecular weight cDNA library.On 1% Ago-Gel in TAE, size separation is carried out to cDNA. The cDNA within the scope of 1 Kb to 10 Kb is collected, is connected between Sal1 the and Not1 restriction site of pSPORT1 carrier, By Electroporation Transformation into E.coli DH10B Electrocompetent cells.LIB5496 produces about 3.5 x 10⁶A bacterium colony is formed Unit.LIB5498 produces about 1.0 x10⁶A colony forming unit.Amplification comes from corn individually in high viscosity culture medium The bacterium colony of rootworm high molecular weight cDNA library LIB5496 and LIB5498.At 37 DEG C, 500ml LB culture medium (contains 0.3% SeaPrep With 50mg/l carbenicillin) in individually placed stirring plate, mixing from LIB5496 and Then about the 600 of LIB5498,000 colony forming unit cool down 1 hour rapidly in water ice bath, so that bacterial clump Even suspension.Then the library by inoculation is cultivated 42 hours at 30 DEG C.After incubation, cell is carried out on stirring plate Mixing in 5 minutes.It then will be in the centrifugal bottle of media transfer to two 250ml.It is handled 10 minutes in 10,000xg, precipitating is thin Bacterium cell.Culture medium is removed from bottle, cell is resuspended in the LB culture medium of 20ml in total, wherein containing 50mg/l's Carbenicillin.Dimethyl sulfoxide is added to 10%, cell is remained into freezing state.Two kinds of libraries are expanded, until every Milliliter 10⁸The final potency of a colony forming unit.The cDNA sequence of insertion is obtained from corn rootworm species specific plasmids library Column information.

The al. corn rootworm library Andersen et and additional sequences from the library LIB5444 and LIB5462 are initial It generates and comes about 18,415 individual est sequences, by about 1.0x10⁷A nucleotide residue is constituted.Est sequence is averaged Length is about 586 nucleotide residues.These est sequences are used for bioinformatics, have obtained contig (contig) assembly of sequence, this is referred to herein as UNIGENE sequence, cannot pass through the overlapping phase same sex and other EST sequences It equips the individual est sequence matched and is referred to herein as singlet (singleton).Then to the library LIB5444 and LIB5462 into The sequencing of row more depth, obtains additional individual est sequence.Will from library, i.e. LIB149, LIB150, LIB3027, The est sequence that LIB3373, LIB5444, LIB5462, LIB5496 and LIB5503 are obtained is showed in sequence table, from SEQ ID NO:1 to SEQ ID NO:143 and SEQ ID NO:169 to SEQ ID NO:174.

If it would be possible, by assembling from the isolated est sequence in the library CRW eDNA as UNIGENE group, these dresses The Unigene sequence matched is showed in sequence table.UNIGENE is the cluster of gene location, is formed from single est sequence and carries sequence Overlapping in column phase same sex region, to form bigger sequence.Every nucleotide sequence shown in sequence table is analyzed, Identify the presence of open reading frame.By the amino acid sequence information being inferred to from open reading frame with can be obtained from public database Known amino acid sequence information compare, to be inferred to amino acid sequence and these known amino acid sequence phase same sexes or similar The degree of property.If any, it with biological function relevant to known amino acid sequence in public database, gives from cDNA library The amino acid sequence that nucleotide sequence information is inferred to adds following annotations.The protein that annotation provides hint property (may be from Provide the specific gene expression of specific cDNA sequence) functional information, but this and indecisive result.Based on hint property Annotation information, certain cDNA sequences are accredited as: coding may relate to the albumen of some biological functions in corn rootworm cell, The function be for life it is critically important, perhaps to ensure cell health and survival for be necessary or may It is related to complete cell, cell holding, fertility etc..

Several eDNA sequences are isolated from the cDNA sequence subgroup that may encode albumen, their inhibition may be led Cause the morbidity or death of CRW or other invertebral living creature Species Cells.Then these sequences are used for dsrna construct molecule, Including into CRW diet.

Thermal expansion is designed based on the cDNA sequence reported in CRW cDNA library increases primer pair.Primer pair is built as A pair of of nucleotide sequence, each member of primer pair show the complete complementarity with justice or antisense sequences.It constructs Primer pair sequence, so that each members show of the centering goes out in its sequence of the 5 ' end containing T7 phage rna polymerase promoter Column, if such as SEQ ID NO:5 nucleotide site 1 is to shown in nucleotide site 23.Preferably, use genomic DNA as Template carries out high-fidelity with the first primer pair for lacking T7 promoter and expands for the first time, generates the first amplicon.Preferably, CDNA or mRNA sequence are used as template, for synthesizing dsRNA molecule used in the present invention, because eukaryon is raw in this field Object genome sequence is considered containing the sequence being not present in mature rna molecule.It then will be from high-fidelity first time amplified reaction The the first amplification subsample generated is used as template in second of hot amplified reaction, with second pair containing T7 promoter sequence Primer pair produces the second amplicon, wherein contains in 5 ' ends of every chain of the second amplicon or in which opens embedded with T7 Mover.The whole nucleotide sequence that the second amplicon is obtained with both direction, by itself and the nucleotide sequence for cDNA report It is compared, if it exists, pointing out the difference between two sequences.In general, genomic DNA is used to prepare as template Sequence with for obtaining the not consistent as further using for dsRNA molecule of level of signifiance containment because have in mRNA or The variation for the genome sequence being not present in cDNA sequence.

Typically, the linearisation DNA profiling that reaction contains about 1 to about 2 microgram is transcribed in vitro, comes from 10X concentrate T7 polymeric enzyme reaction buffer, (wherein concentration is every kind 50 to 100mM) and 1 by ribonucleotide ATP, CTP, GTP and UTP The t7 rna polymerase of a unit.According to manufacturers instruction, in (several minutes to a few hours) progress of about 37 DEG C of incubation a period of times RNA polymerase reaction, this depends on RNA polymerase used.In general, reaction carries out about 2 to about 6 hours, for transcribing Longest is up to the template sequence of about 400 nucleotide, and carries out up to 20 hours, is greater than about 400 for Transcript Length The template sequence of a nucleotide.Reaction is heated to 65 DEG C, 15 minutes, to terminate rna transcription.Rna transcription is precipitated in ethanol Product is washed, and is air-dried, and is resuspended in the water without RNAse, until concentration is every milliliter of about 1 microgram.Using anti- Double-stranded RNA is produced in responsive transcription in vivo to most of transcript of T7 starting substrategy (as outlined above), but It is, by the way that purified RNA is heated to 65 DEG C, to be then slowly cooled to room temperature to ensure justice and antisense RNA segment Correct annealing just obtains the double-stranded RNA of high yield.Then one is carried out to double stranded RNA product at 37 DEG C with DNAse I and RNAse The incubation of hour, to remove any DNA or single stranded RNA present in mixture.According to manufacturers instruction (AMBION MEGASCRIPT RNAi KIT), in purified on columns double stranded RNA product, it is resuspended in 10mM Tris-HCl buffering In liquid (pH 7.5) or water without RNAse, until the concentration of every microlitre 0.1 to 1.0 microgram.

Double-stranded RNA sample is added directly into each hole containing the insect diet being indicated above, or is being added to It is modified before insect diet.RNAse III (AMBION is directed to according to what manufacturer provided to the modification of double-stranded RNA CORPORATION, Austin, Texas) or DICER (STRATAGENE, La Jolla, California) specification come into Row.RNAse III produces the two-body of 21 and 22 nucleotide to the digestion of double-stranded RNA, contains 5 ' phosphorylations End and 3 ' C-terminals with 2-3 base overhang, similar to the two-body short interfering rna of about 21-26 base-pair (siRNA) segment, this is Hamilton et.al. (Science, 1999,286:950-952) and Elbashir et.al. It is (Genes & Development, 2001,15:188-200) identification, dicer enzyme manufacture in eukaryon approach.To this The short interfering rna two-body being collected into is further purified, and is analyzed with polyacrylamide gel electrophoresis sample, with measurement The integrality and efficiency that two-body is formed.Then measured under 250 nanometers of wavelength by spectrophotometry sample purity and Quantity, not used sample is stored in -20 DEG C, for further using.

The sample of siRNA or overall length double-stranded RNA (dsRNA) be used to carry out to be carried out with the object noxious livings of selected quantity Biologic test.According to following processes, the artificial meals of corn rootworm are applied to using the various dose of dsRNA or siRNA as overlapping On food.From Crop Characteristics, Inc., Farmington, Minnesota obtains Diabrotica virgifera The ovum of virgifera (WCR).In the soil, in 24 DEG C, 60% relative humidity, under complete darkness, to the WCR of non-sleep phase Ovum carries out culture in about 13 days.At the 13rd day, the soil containing WCR ovum is placed into the sieve between #30 and #60 mesh, is made Soil is washed away with high pressure garden water.Desinsection (disinfest) is carried out by steeping three minutes surfaces to ovum in LYSOL, is used Sterilizing washing three times, is washed once with 10% formalin solution, then additional three times with sterilizing washing.It will locate in this way The ovum of reason is suspended in sterilizing coffee strainer, at 27 DEG C, 60% relative humidity, and under complete darkness, incubated overnight.

Substantially according to Pleau et al. (Entomologia Experimentalis et Applicata, 2002, It is 105:1-11) described to manufacture insect diet, wherein be made that following change.9.4 grams of SERVA agar is suspended into 540 millis It rises in purified water, stirring is until agar is completely dispersed.Water/agar is mixed and heated to boiling, to be completely dissolved agar, so After be poured into WARING blender.Blender is remained into low speed, at this time by 62.7 grams of BIO-SERV DIET mixtures (F9757), corn root, 1.25 milliliters of non-polluted edible pigment and the 0.6 milliliter of formalin of 3.75 grams of freeze-dryings are added to heat Agar mixture in.Then mixture pH is adjusted to 9.0 by the way that 10% potassium hydroxide reservoir is added.In magnetic stirring heat On plate, with the magnetic stir bar for using sterilizing NALGENE coating, continued with liquid dietary of the high speed to about 600 milliliters of volumes It is mixed, remains at about 48 DEG C to about 60 DEG C, and it is each to be separated into 96 hole round bottom titer plate of FALCOM 200 microlitres of aliquots in hole.The diet in plate is enabled to solidify and be air-dried in the biological air extracting cabinet (biohood) of sterilizing.

Using running fire type micropipettor, the test specimen of 30 (30) microlitre volume is taken, wherein containing different number Reagent or double-stranded RNA are controlled, the insect diet surface in each hole is covered.After application test sample, insect Diet is allowed to be held up in the biological air extracting cabinet of sterilizing one and a half hours (one half hour), so that diffusion of reagents Into diet, and allow diet dry tack free.A WCR newborn larvae is put into each hole with paintbrush.Then close with MYLAR Plate is sealed, is divulged information with insect needle.For every kind of dosage, 12-72 insect larvae is tested, quantity depends on test and sets Meter.Culture in 12-14 days is carried out to biologic test plate under 27 DEG C, 60% relative humidity and complete darkness.At 12-14 days Time point, under every kind of dosage survive larva quantity recorded.Using suitable microbalance, survive for every Larva measures larva quality.It usesStatistical software (SAS Institue, 1995) analyzes data, with Dunnet ' S examines to carry out complete factorial ANOVA, to find the effect (P < 0.05) of processing compared with untreated control.Carry out Tukey- Kramer subsequent (post hoc) is examined, and carrys out all processing of comparison to (P < 0.05).

Following nucleotide sequence is obtained initially as the cDNA sequence identified in intestines cDNA library in corn rootworm (Andersen et a1., ibid), is used for dsrna construct molecule, feeds in harmful organism diet for upchecking Double stranded rna molecule is raised to the inhibitory effect of harmful organism biological function.

Chd3 homologous sequence

Identified in a variety of eucaryotes come CHD gene, corresponding albumen be considered as chromatin reconstruct because Son plays a role.Three kinds of sequence homology structural domains that term CHD has found in CHD albumen: chromo (repair by chromosome organization Decorations person) structural domain, SNF2- relational coiling be mould/ATPase structural domain and DNA binding structural domain, every kind is all believed and can assign Different chromatin related activities.Based on another sequence homology structural domain (PHD Zinc finger domain, typically, with chromosome Related activity is related) whether there is, CHD albumen is divided into two classes.CHD3 GAP-associated protein GAP has PHD Zinc finger domain, still CHD1 GAP-associated protein GAP is then not.Germicidal efficacy imply that CHD3 albumen containment transcription in effect, in some species by It is shown as containing ingredient of the histone as the complex of subunit.The deacetylation of histone is related to transcriptional inactivation, so CHD3 albumen has been implied to be: using as the property of the ingredient of histidine deacetylase complex, can be used as transcription containment agent Play a role (Ogas et al., 1999, PNAS 96:13839-13844).It therefore, can be with to the containment of CHD3 albumen synthesis As useful target, for the inhibition to no vertebra harmful organism with double chain RNA mediate.

SEQ ID NO:4 corresponds to intestines cDNA nucleotide sequence, amino acid sequence translation in CRW and is noted as: with Drosophila melanogaster CHD3 amino acid sequence (GenBank number AF007780) is homologous.SEQ ID NO:5 and SEQ ID NO:40609 is corresponded respectively to: the genome amplification primer (i.e. primer pair) of forward and reverse, and the primer is for producing From birth from crw genomic dna, the amplicon from the library CRW mRNA or from the cDNA generated from this class libraries.Such amplicon Sequence correspond to encoding D .melanogaster CHD3 amino acid sequence homologous body CRW gene a part.SEQ ID NO:5 contains T7 polymerase promoter sequence (nucleotide 1-23) at its 5 ' end, in the promoter sequence and SEQ ID NO:5 CRW Genome Primer sequence (being forcibly appointed as forward primer sequence) shown in 24-45 nucleotide is connected,ItsCorrespond to The the 31st to the 52nd nucleotide shown in SEQ ID NO:4.SEQ ID NO:6 contains T7 polymerase promoter sequence at its 5 ' end Column, as shown in 1-23 nucleotide.T7 promoter sequence is in its 3 ' end and mandatory specified reverse genomic primer sequence phase Even, correspond to 24-44 nucleotide shown in SEQ ID NO:6, the reverse complementary sequence of the sequence is shown in SEQ ID 298-319 nucleotide in NO:4.In using amplified reaction of the crw genomic dna as template, using by SEQ ID NO: The primer pair of 5 and SEQ ID NO:6 composition, generates 335 base-pairs comprising nucleotide sequence shown in SEQ ID NO:7 Amplicon, correspond in CRW genome and encode the parts of following albumen, the protein display goes out and Drosophila The phase same sex of melanogaster CHD3 amino acid sequence about 66%.1-23 nucleosides shown in SEQ ID NO:7 Acid and 314-335 nucleotide reverse complementary sequences correspond to the T7 promoter sequence of any end of amplicon.SEQ ID The genome nucleotide that amplification in NO:7 shown in the 24th nucleotide to the 313rd nucleotide obtains is substantially corresponding to SEQ The cDNA nucleotide sequence being reported in ID NO:4 shown in the 31st nucleotide to the 319th nucleotide, only in addition to following Difference: the 63rd in SEQ ID NO:4,87,117,177,198,213,219-220,246,249 and 261 nucleotide reported Road be T, T, G, G, G, T, T, T, C, C and A respectively, and corresponding position when being compared with SEQ ID NO:7 be the 56th, 80,110,170,191,206,212-213,239,242 and 254 nucleotide contain C, C, A, A, A, C, A, C, G, A and G.This Kind of difference corresponds to the cDNA sequence reported in the past and from nucleotide sequence between the amplification subsequence that genomic DNA template generates About 4% difference in composition, this is consistent with the accuracy that the cDNA sequence reported in the past is likely less than 99% (aforementioned Andersen et al.)。

The amplicons cloned for showing the sequence of corresponding SEQ ID NO:7 is carried into the plasmid that can be replicated in E coli Body recycles the Plasmid DNA of sufficient amount, to allow to restrain (convergent) T7 promoter from the insertion of any end of cloned sequence Carry out external t7 rna polymerase transcription.Double-stranded RNA is generated, biologic test is carried out；One RNA segment is by SEQ ID NO:7 Sequence composition shown in about the 24th nucleotide to as little as the about the 313rd nucleotide is (the difference is that in SEQ ID Each position of thymidine is shown as in NO:7, and there are uracil residues), other RNA segments be in SEQ ID NO:7 about The substantially reverse complementary series of nucleotide sequence shown in 313rd nucleotide to as little as the about the 24th nucleotide, wherein urine Pyrimidine suitably replaces thymidine.The sample of processing double-stranded RNA (dsRNA) is removed, with DICER or RNAse III to generate foot The siRNA (siRNA) enough measured.Sample containing 0.15 part every million siRNA or dsRNA is used for previously described CRW diet biologic test, larva are allowed to feed 13 days.Compared with control, the CRW larva for feeding the diet containing dsRNA is shown Gone out obvious growth inhibition and the death rate, the dsRNA correspond to sequence shown in SEQ ID NO:4 whole or Part.

In biologic test, it is parallel to CHD3 sequence, the other nucleotide sequences obtained from CRW are also examined, Including being noted as that the nucleotide sequence of the CRW equivalent of following albumen may be encoded, the albumen is for example, beta- micro-pipe egg White, 40kDa V-ATPase protein subunit, 26S proteosome subunit p28 albumen, is protected elongation factors albumen EF1 α and EF1 α 48D Young hormone epoxide hydrolase protein, the chloride channel albumen for relying on expansion, Robison ester 1- apodehydrogenase, flesh move egg White 42A albumen, 1 albumen of ADP- ribosylation factor, transcription factor IIB, chitinase protein and ubiquitin combine (conjugating) enzyme.

Beta- tubulin homologous sequence

Tubulin is the important feature component of structure in various kinds of cell in all eukaryocytes, is primarily used to form micro- Pipe.The inhibition formed to micro-pipe in cell will lead to catastrophic effect, including, it is right to the interference that mitosis spindle is formed Fissional obstruction etc..Therefore, the containment formed to tubulin can be the useful target of the inhibition of double chain RNA mediate.

The beta- tubulin correlated series obtained from CRW are identified, for the present invention.SEQ ID NO: 18 correspond to intestines cDNA nucleotide sequence in CRW, and amino acid sequence translation is noted as: with Manduca sexta beta- 1- tubulin amino acid sequence homeologous, and with Drosophila melanogaster beta-1- tubulin ammonia Base acid sequence (GenBank number is respectively AF030547 and M20419) homeologous.SEQ ID NO:19 and SEQ ID NO: 20 correspond respectively to the genome amplification primer (i.e. primer pair) of forward and reverse, and the primer is used for from crw genomic dna, Amplicon is manufactured from the library CRW mRNA or the cDNA generated from thus class libraries.The sequence of such amplicon corresponds to coding The all or part of the CRW gene of beta- tubulin.Each of SEQ ID NO:19 and SEQ ID NO:20 contains 23 cores The T7 promoter sequence of thuja acid, respectively in 1-23 nucleotide.24-44 nucleotide pairs shown in SEQ ID NO:19 It should be in 96-116 nucleotide shown in SEQ ID NO:18.24-44 nucleotide pairs shown in SEQ ID NO:20 Should sequence in SEQ ID:18 shown in 428-448 nucleotide reverse complementary sequence.Use crw genomic dna as mould In the amplified reaction of plate, using the primer pair being made of SEQ ID NO:19 and SEQ ID NO:20, produce comprising SEQ ID The amplicon of 339 base-pairs of nucleotide sequence shown in NO:21 is substantially corresponding to encode following CRW genes Group a part, the protein display gone out with present in Drosophila melanogaster and Manduca sexta The perfect homology of beta- tubulin homologues.Nucleotide sequence shown in SEQ ID NO:21 is substantially corresponding to SEQ Nucleotide sequence in ID NO:18 shown in 96-448 nucleotide.It is corresponding in genome amplification subsequence and cDNA sequence Sequence difference is not observed between sequence.

By the amplicons cloned for showing the sequence for corresponding to SEQ ID NO:21 into plasmid vector, the matter of sufficient amount is recycled Grain DNA is transcribed with allowing the insertion convergence T7 promoter from any end of cloned sequence to carry out external t7 rna polymerase.It produces Raw double-stranded RNA, carries out biologic test for sample；One RNA segment, positive-sense strand, by the about the 24th core in SEQ ID NO:21 Sequence composition shown in thuja acid to as little as the about the 376th nucleotide in SEQ ID NO:21 (the difference is that be shown as There are uracil residues for each position of thymidine), reverse complemental RNA segment or antisense strand are big in SEQ ID NO:21 The substantially reverse complementary series of nucleotide sequence shown in about the 376th nucleotide to as little as the about the 24th nucleotide, wherein Uracil suitably replaces thymidine.The sample of processing double-stranded RNA (dsRNA) is removed, with DICER or RNAse III to generate The siRNA (siRNA) of sufficient amount.Sample containing 0.15 part every million siRNA or dsRNA is used for described previously CRW diet biologic test, larva be allowed to feed 13 days.Compared with control, the CRW larva exhibition of the diet containing dsRNA is fed Obvious growth inhibition and the death rate are shown, the dsRNA corresponds to the complete of sequence shown in SEQ ID NO:18 Portion or part.

40kDa V-ATPase homologous sequence

The intraorganic energetic supersession of eukaryotic systems sub-cellular is very important function.Vacuole atp synthase participates in The ATP of enough levels is kept in vacuole.Therefore, vacuole atp synthase can be the target of the inhibition of double chain RNA mediate.

The nucleotide sequence for encoding following albumen is obtained from CRW, the protein display goes out the phase with 40kDa V-ATPase Like property.The amino acid sequence translation of SEQ ID NO:32 is shown and Manduca sexta 40-kDa V-ATPase subunit ammonia The homology of base acid sequence (GenBank number X98825).SEQ ID NO:33 and SEQ ID NO:34 corresponds respectively to forward direction With reversed genome amplification primer (i.e. primer pair), the primer is used for from crw genomic dna, from the library CRW mRNA or from Thus the cDNA that class libraries generates manufactures amplicon.The sequence of such amplicon should correspond to coding 40kDa V-ATPase's The all or part of CRW gene.But the nucleotide sequence for the amplicon for using crw genomic dna as template and obtaining with The cDNA sequence being reported shown in SEQ ID NO:32 is not consistent.

SEQ ID NO:33 and SEQ ID NO:34 represents the primer of thermal expansion increasing.Every primer is all containing 23 nucleotide T7 promoter sequence, respectively in 1-23 nucleotide.24-40 nucleotide shown in SEQ ID NO:33 correspond to 95-111 nucleotide shown in SEQ ID NO:32.24-43 nucleotide shown in SEQ ID NO:34 correspond to The reverse complementary sequence of sequence in SEQ ID:32 shown in 362-381 nucleotide.Use crw genomic dna as template In amplified reaction, using the primer pair being made of SEQ ID NO:33 and SEQ ID NO:34, produce comprising SEQ ID NO: The amplicon of 291 base-pairs of nucleotide sequence shown in 35.Based on Martinez/Needleman-Wunsch DNA ratio Right, the 24th nucleotide to the 268th nucleotide only illustrates and core shown in SEQ ID NO:32 in SEQ ID NO:35 About 50% homology of nucleotide sequence.Increase the amplification subsequence and SEQ ID NO that primer pair obtains using the thermal expansion of selection: The sequence being reported shown in 32 is not consistent.Preferably, it is made using the library CRW mRNA or the cDNA obtained from this class libraries Make amplicon.

Following amplicons are manufactured, the amplicon is shown corresponding to the about the 95th nucleotide in SEQ ID NO:32 extremely The sequence of about the 381st nucleotide, is cloned the Plasmid DNA that sufficient amount is recycled into plasmid vector, to allow from clone's piece The insertion convergence T7 promoter of the end Duan Renyi carries out external t7 rna polymerase transcription.Double-stranded RNA is generated, sample is carried out Biologic test；One RNA segment, positive-sense strand, by the about the 85th nucleotide in SEQ ID NO:32 to as little as the about the 381st Sequence composition shown in nucleotide is (the difference is that each position for being shown as thymidine in SEQ ID NO:32 is deposited In uracil residues), reverse complemental RNA segment or antisense strand be in SEQ ID NO:32 the about the 381st nucleotide to as little as The substantially reverse complementary series of nucleotide sequence shown in about the 95th nucleotide, wherein uracil suitably replaces thymus gland Pyrimidine.The sample of processing double-stranded RNA (dsRNA) is removed, with DICER or RNAse III to generate the siRNA of sufficient amount (siRNA).Sample containing 0.15 part every million siRNA or dsRNA is used for previously described CRW diet biologic test, Larva is allowed to feed 13 days.Compared with control, the CRW larva for feeding the diet containing dsRNA illustrates obvious life Long inhibition and the death rate, the dsRNA correspond to all or part of sequence shown in SEQ ID NO:32.

EF1 α homologous sequence

Transcription extends and transcription factor is critically important for metabolism, they can be the inhibition for double chain RNA mediate Favo(u)rable target.

At least two CRW cDNA sequences are identified for the present invention, they by with the Zeng Weineng code extension factor 1 The homologue of aIpha (EF1 α).

The amino acid translation of singlet CRW cDNA sequence as shown in SEQ ID NO:36 illustrate with The homology of Drosophila melanogaster EF-1-alpha amino acid sequence (GenBank number X06870).Also from The other sequences for being predicted to be coding EF1 α homologous protein are identified in CRW cDNA in intestines library.These sequences are compared, are produced Raw UNIGENE sequence is predicted to be the EF1 α albumen that can encode referred to herein as 48D as shown in SEQ ID NO:40 Homologue.Several of the sequence for including in the singlet, which are predicted to be, can encode following amino acid sequences, the amino acid sequence Show the homology with a variety of EF1 α homologous protein sequences comprising but be not limited to, Bombyx mori EF1 α (GenBank number D13338), Alternia species EF1 α (GenBank number X03704), Spragueia leo EF1 α (GenBank number U85680), Apis mellifera EF1 α (GenBank number AF015267), Anisakis Simplex EF1 α (GenBank number AJ250539), Papaipema species EF1 α (GenBank number AF151628), Ephedrus persicae EF1 α (GenBank number Z83663), Papilio garamas EF1 α (GenBank number AF044833), Alysia lucicola EF1 α (GenBank number Z83667), Braco species EF1 α (GenBank number Z83669), Histeromerus mystacinus EF1 α (GenBank number Z83666) and Caenorhabditis Elegans EF1 α (GenBank number U41534).

A CRW cDNA sequence for being predicted to be a part of coding EF1 α homologue is referred to herein as B2 sequence Column, are showed in SEQ ID NO:36.SEQ ID NO:37 and SEQ ID NO:38 corresponds respectively to forward and reverse Genome amplification primer (i.e. primer pair, referring to the corresponding or reverse complementary sequence shown in SEQ ID NO:36), the primer For manufacturing amplicon from the library CRW mRNA or the cDNA generated from thus class libraries from crw genomic dna.Such amplicon Sequence correspond to coding EF1 α homologous protein CRW gene all or part.But when use crw genomic dna as The nucleotide sequence of the amplicon obtained when template and the cDNA sequence of report shown in SEQ ID NO:36 are not consistent.

SEQ ID NO:37 and SEQ ID NO:38 represents the sequence for being used for hot amplimer.Each of which contains 23 The T7 promoter sequence of nucleotide, respectively in 1-23 nucleotide.24-44 nucleotide shown in SEQ ID NO:37 Corresponding to 8-29 nucleotide shown in SEQ ID NO:36.24-42 nucleotide pairs shown in SEQ ID NO:38 Should sequence in SEQ ID:36 shown in 310-328 nucleotide reverse complementary sequence.Use crw genomic dna as mould In the amplified reaction of plate, using the primer pair being made of SEQ ID NO:37 and SEQ ID NO:38, produce comprising SEQ ID The amplicon of 933 base-pairs of nucleotide sequence shown in NO:39.Nucleotide sequence shown in SEQ ID NO:39 with 8th nucleotide shown in SEQ ID NO:36 is not consistent to the 328th nucleotide.Preferably, with the library CRW mRNA or The cDNA that is obtained from this class libraries, for example, SEQ ID NO:36 manufactures amplicon.

Using the library CRW mRNA, or the cDNA prepared from this class libraries, to manufacture following amplicons, the amplicon is shown Corresponding to the sequence of the about the 8th nucleotide in SEQ ID NO:36 to the about the 328th nucleotide, is cloned and carried into plasmid Body.The Plasmid DNA of sufficient amount is recycled, external with the insertion convergence T7 promoter progress of permission from any end of cloned sequence T7 rna polymerase transcription.Double-stranded RNA is generated, sample is subjected to biologic test；One RNA segment, positive-sense strand, by SEQ ID Sequence composition shown in about the 8th nucleotide to as little as the about the 328th nucleotide in NO:36 (the difference is that Each position of thymidine is shown as in SEQ ID NO:36, and there are uracil residues), reverse complemental RNA segment or antisense Chain is the reality of nucleotide sequence shown in the about the 328th nucleotide to as little as the about the 8th nucleotide in SEQ ID NO:36 Matter reverse complementary sequence, wherein uracil suitably replaces thymidine.Processing double-stranded RNA is removed with DICER or RNAse III (dsRNA) sample, to generate the siRNA (siRNA) of sufficient amount.Every million siRNA or dsRNA 0.15 will be contained The sample divided is used for previously described CRW diet biologic test, and larva is allowed to feed 13 days.Compared with control, feed contains The CRW larva of the diet of dsRNA illustrates obvious growth inhibition and the death rate, and the dsRNA corresponds to SEQ ID The all or part of sequence shown in NO:36.

The sequence shown in SEQ ID NO:40 goes to the primer pair designed for expanding following crw genomic dna sequences, institute State sequential coding EF1 α 48D homologous protein sequence.SEQ ID NO:41 and SEQ ID NO:42 respectively corresponds forward and reverse Genome amplification primer (that is, primer pair).T7 of each of the SEQ ID NO:41 and SEQ ID NO:42 containing 23 nucleotide Promoter sequence, respectively in 1-23 nucleotide.24-41 nucleotide shown in SEQ ID NO:41 correspond to SEQ 61-79 nucleotide shown in ID NO:40.24-45 nucleotide shown in SEQ ID NO:42 correspond to SEQ The reverse complementary sequence of sequence in ID:40 shown in 562-583 nucleotide.Use crw genomic dna as the amplification of template In reaction, using the primer pair being made of SEQ ID NO:41 and SEQ ID NO:42, produce comprising in SEQ ID NO:43 The amplicon of 569 base-pairs of the nucleotide sequence shown, height correspond to the one of the CRW genome for encoding following albumen Part, the protein display go out in Drosophila melanogaster there is also EF1 α albumen have height it is identical Property.From nucleotide sequence shown in the about the 24th nucleotide to about 546 nucleotide and SEQ ID in SEQ ID NO:43 Nucleotide sequence height shown in about 61-583 nucleotide shown in NO:40 is corresponding.In genome amplification subsequence Sequence difference is not observed between the corresponding sequence in cDNA.

The amplicons cloned of sequence corresponding to SEQ ID NO:43 will be shown into plasmid vector.Recycle the matter of sufficient amount Grain DNA is transcribed with allowing the insertion convergence T7 promoter from any end of cloned sequence to carry out external t7 rna polymerase.It produces Raw double-stranded RNA, carries out biologic test for sample；One RNA segment, positive-sense strand, by the about the 24th core in SEQ ID NO:43 Sequence composition shown in thuja acid to as little as the about the 546th nucleotide in SEQ ID NO:43 (the difference is that be shown as There are uracil residues for each position of thymidine), reverse complemental RNA segment or antisense strand are big in SEQ ID NO:43 The substantially reverse complementary series of nucleotide sequence shown in about the 546th nucleotide to as little as the about the 24th nucleotide, wherein Uracil suitably replaces thymidine.The sample of processing double-stranded RNA (dsRNA) is removed, with DICER or RNAse III to generate The siRNA (siRNA) of sufficient amount.Sample containing 0.15 part every million siRNA or dsRNA is used for described previously CRW diet biologic test, larva be allowed to feed 13 days.Compared with control, the CRW larva exhibition of the diet containing dsRNA is fed Obvious growth inhibition and the death rate are shown, the dsRNA corresponds to the complete of sequence shown in SEQ ID NO:43 Portion or part.

26S proteosome subunit p28 homologous sequence

26S proteosome is big, dependency ATP, multimeric protein enzyme, highly conserved in all eucaryotes.Its There is general function during a variety of short-lived albumen are selectively removed, the albumen is conjugated with ubiquitin connects before this, Then it is degraded by 26S proteasome.Ubiquitin pathway has important role, the control in the control of cell cycle Realized by the selective degradation to a variety of modulins, the albumen include mitotic cell cyclin and to according to The inhibitor for relying the kinases of cyclin, for example, the p27 of mammalian cell.Therefore, 26S proteosome synthesis is held back System and it is formed subunit and degree containment can be double chain RNA mediate inhibition selected objective target (Smith et al., Plant Phys.1997,113:281-291).

The cDNA sequence that intestines library obtains from CRW be accredited as it is homologous with 26S proteosome subunit amino acid Sequence, It is used for the present invention.SEQ ID NO:44 is substantially corresponding to intestines cDNA nucleotide sequence in CRW.The ammonia of SEQ ID NO:44 The translation of base acid sequence illustrates the homology with 26S proteosome subunit p28 albumen (GenBank number AB009619).SEQ ID NO:45 and SEQ ID NO:46 corresponds respectively to the genome amplification primer (i.e. primer pair) of forward and reverse, the primer For manufacturing amplicon from the library CRWmRNA or the cDNA generated from thus class libraries from crw genomic dna.With the generation of this approach Amplicon should show following sequences, all or part of the following CRW genes of sequential coding, the gene coding The homologue of 26S proteosome protein subunit.T7 of each of the SEQ ID NO:45 and SEQ ID NO:46 containing 23 nucleotide Promoter sequence, respectively in 1-23 nucleotide.24-46 nucleotide shown in SEQ ID NO:45 correspond to SEQ 130-152 nucleotide shown in ID NO:34.24-41 nucleotide shown in SEQ ID NO:46 correspond to SEQ The reverse complementary sequence of sequence in ID:44 shown in 423-440 nucleotide.Use crw genomic dna as the amplification of template In reaction, using the primer pair being made of SEQ ID NO:45 and SEQ ID NO:46, produce comprising in SEQ ID NO:47 The amplicon of 1113 base-pairs of the nucleotide sequence shown.Sequence shown in SEQ ID NO:47 and SEQ ID NO:44 institute The sequence shown is simultaneously uncorrelated, therefore not consistent with the cDNA sequence that is reported shown in SEQ ID NO:44.Preferably, it uses The cDNA that CRW mRNA library or thus class libraries obtain manufactures amplicon.

Manufacture shows the amplicon of following sequences, and is cloned into plasmid vector, and the sequence corresponds to SEQ ID The about the 130th nucleotide of NO:44 recycles the Plasmid DNA of sufficient amount, to the about the 440th nucleotide to allow from clone The insertion convergence T7 promoter of any end of segment carries out external t7 rna polymerase transcription.Generate double-stranded RNA, by sample into Row biologic test；One RNA segment, positive-sense strand, by the about the 130th nucleotide in SEQ ID NO:44 to as little as about Sequence composition shown in 440 nucleotide in SEQ ID NO:44 (the difference is that be shown as each position of thymidine Set that there are uracil residues), reverse complemental RNA segment or antisense strand are that the about the 440th nucleotide is extremely in SEQ ID NO:44 The substantially reverse complementary series of nucleotide sequence shown in as little as the about the 110th nucleotide, wherein uracil suitably replaces Thymidine.The sample of processing double-stranded RNA (dsRNA) is removed, with DICER or RNAse III to generate the siRNA of sufficient amount (siRNA).Sample containing 0.15 part every million siRNA or dsRNA is used for previously described CRW diet biologic test, Larva is allowed to feed 13 days.Compared with control, the CRW larva for feeding the diet containing dsRNA illustrates obvious life Long inhibition and the death rate, the dsRNA correspond to all or part of sequence shown in SEQ ID NO:44.

Juvenile hormone epoxide hydrolase protein

Corpus allatum hormone is controlled and regulates and controls, the mistake to necessary biological processes a variety of in insect life cycle Journey includes but is not limited to abnormal, breeding and diapause.When suitable, juvenile hormone (JH) concentration is required in harmful elder brother Reach peak value in worm larva (especially lepidoptera and coleoptera larva) form hemolymph, then it must be degraded, with middle finger Hormone response effect.The enzyme being related to during reducing juvenile hormone concentration is played by two main paths of metabolic degradation to be made With.One approach is related to JH esterase (JHE), can hydrolyze methyl esters, provides corresponding acid.Second of approach is young using protecting Hormone epoxide hydrolase (JHEH) causes glycol to be formed to obtain the hydrolysis to epoxides.JHE is in JH degradation Contribution is better understood, it was found that it is indeclinable between lepidoptera and coleoptera species.Inhibition to JH esterase It is related to serious abnormal variation, including but not limited to, larva entanglement (larval wandering), pupate postponement and deformity The development of intermediate state.On the contrary, people understand less effect of the JHEH in JH metabolism, also, it has shown that in species Between can change, but recent research is directed to following evidences, and the evidence implies that JHEH may be the main way of JH metabolism Diameter (Brandon J.Fetterolf, Doctoral Dissertation, North Carolina State University (Feb.10,2002)Synthesis and Analysis of Mechanism Based Inhibitors of Juvenile Hormone Epoxide Hydrolase from Insect Trichoplusia ni).In at any time, held back with gene Technology processed can be the useful target of the harmful organism inhibition for double chain RNA mediate to the interference of any JH degradation pathway.

The corpus allatum hormone epoxide hydrolase sequence obtained from CRW is identified, the present invention is used for. SEQ ID NO:48 is substantially corresponding to intestines cDNA nucleotide sequence in CRW.The amino acid sequence translation of SEQ ID NO:48 is pre- The homology with juvenile hormone epoxide hydrolase in Manduca sexta (GenBank number U46682) is surveyed.SEQ ID NO:49 and SEQ ID NO:50 corresponds respectively to the amplimer (i.e. primer pair) of forward and reverse, and the primer is used for from CRW Genomic DNA manufactures amplicon from the library CRW mRNA or the cDNA generated from thus class libraries.The sequence of such amplicon should The all or part of CRW gene corresponding to coding JHEH homologous protein.Each of SEQ ID NO:49 and SEQ ID NO:50 T7 promoter sequence containing 23 nucleotide, respectively in 1-23 nucleotide.24-42 shown in SEQ ID NO:49 Position nucleotide corresponds to 7-26 nucleotide shown in SEQ ID NO:48.24-44 shown in SEQ ID NO:50 Nucleotide corresponds to the reverse complementary sequence of the sequence in SEQ ID:48 shown in 360-380 nucleotide.With CRW genome DNA, using the primer pair being made of SEQ ID NO:49 and SEQ ID NO:50, produces packet as in the amplified reaction of template The amplicon of 95 base-pairs of nucleotide sequence shown in the NO:52 of ID containing SEQ.The sequence and SEQ ID NO of amplicon: CDNA sequence shown in 48 does not correspond to.Preferably, use the library CRW mRNA or thus class libraries obtain cDNA as expand Template nucleotide sequence in reaction manufactures amplicon.

By the amplicons cloned for showing the sequence for corresponding to SEQ ID NO:48 into plasmid vector, the matter of sufficient amount is recycled Grain DNA is transcribed with allowing the insertion convergence T7 promoter from any end of cloned sequence to carry out external t7 rna polymerase.It produces Raw double-stranded RNA, carries out biologic test for sample；One RNA segment, positive-sense strand, by the about the 7th nucleosides in SEQ ID NO:48 Sequence composition shown in acid to as little as the about the 380th nucleotide is (the difference is that be shown as chest in SEQ ID NO:48 There are uracil residues for each position of gland pyrimidine), reverse complemental RNA segment or antisense strand be in SEQ ID NO:48 about The substantially reverse complementary series of nucleotide sequence shown in 380th nucleotide to as little as the about the 7th nucleotide, wherein urine Pyrimidine suitably replaces thymidine.The sample of processing double-stranded RNA (dsRNA) is removed, with DICER or RNAse III to generate foot The siRNA (siRNA) enough measured.Sample containing 0.15 part every million siRNA or dsRNA is used for previously described CRW diet biologic test, larva are allowed to feed 13 days.Compared with control, the CRW larva for feeding the diet containing dsRNA is shown Obvious growth inhibition and the death rate are gone out, the dsRNA corresponds to the whole of sequence shown in SEQ ID NO:48 Or part.

Rely on the chloride channel albumen homology sequence of expansion

The chloride channel albumen for relying on expansion is assumed to be: playing the part of angled key in eukaryon zooblast system osmotic adjustment Color.Therefore, show the ability that can express following amino acid sequences nucleotide sequence can be in harmful organism into The useful target that row RNA inhibits, the amino acid sequence are the chloride channel albumen shown with the dependence expansion identified in the past Homology.

It is inferred to rely on the amino acid sequence homologous object of the chloride channel (SDCC) of expansion from CRW cDNA library, is used for The present invention.SEQ ID NO:53 is substantially corresponding to intestines cDNA nucleotide sequence in CRW.The amino acid sequence of SEQ ID NO:53 It is confirmed as: homologous with the SDCC albumen (GenBank number Y08484) in zebra fish Danio rerio.SEQ ID NO:54 The hot amplimer (i.e. primer pair) of forward and reverse is corresponded respectively to SEQ ID NO:55, the primer is used for from CRW base Because of a group DNA, amplicon is manufactured from the library CRW mRNA or the cDNA generated from thus class libraries.The sequence of such amplicon should be right It should be in all or part of the CRW gene of coding SDCC homologous protein.Each of SEQ ID NO:54 and SEQ ID NO:55 contains There is the T7 promoter sequence of 23 nucleotide, respectively in 1-23 nucleotide.24-43 shown in SEQ ID NO:54 Nucleotide corresponds to 78-97 nucleotide shown in SEQ ID NO:53.24-41 cores shown in SEQ ID NO:55 Thuja acid corresponds to the reverse complementary sequence of the sequence in SEQ ID:53 shown in 332-349 nucleotide.With crw genomic dna As in the amplified reaction of template, using the primer pair being made of SEQ ID NO:54 and SEQ ID NO:55, produces and include The amplicon of 318 base-pairs of nucleotide sequence shown in SEQ ID NO:56, with the CRW gene for encoding following albumen A part height of group is corresponding, and the protein display has gone out the perfect homology with SDCC albumen.In SEQ ID NO:56 about Nucleotide sequence shown in 24th nucleotide to the about the 295th nucleotide and 78-349 nucleotide in SEQ ID NO:53 Shown in nucleotide sequence height it is corresponding.

By the amplicons cloned for showing the sequence for corresponding to SEQ ID NO:56 into plasmid vector, the matter of sufficient amount is recycled Grain DNA is transcribed with allowing the insertion convergence T7 promoter from any end of cloned sequence to carry out external t7 rna polymerase.It produces Raw double-stranded RNA, carries out biologic test for sample；One RNA segment, positive-sense strand, by the about the 24th core in SEQ ID NO:56 Sequence composition shown in thuja acid to as little as the about the 295th nucleotide in SEQ ID NO:56 (the difference is that be shown as There are uracil residues for each position of thymidine), reverse complemental RNA segment or antisense strand are big in SEQ ID NO:56 The substantially reverse complementary series of nucleotide sequence shown in about the 295th nucleotide to as little as the about the 24th nucleotide, wherein Uracil suitably replaces thymidine.The sample of processing double-stranded RNA (dsRNA) is removed, with DICER or RNAse III to generate The siRNA (siRNA) of sufficient amount.Sample containing 0.15 part every million siRNA or dsRNA is used for described previously CRW diet biologic test, larva be allowed to feed 13 days.Compared with control, the CRW larva exhibition of the diet containing dsRNA is fed Obvious growth inhibition and the death rate are shown, the dsRNA corresponds to the complete of sequence shown in SEQ ID NO:56 Portion or part.

Robison ester 1- apodehydrogenase homologous sequence

Robison ester 1- apodehydrogenase (G6PD) is catalyzed Robison ester to 6- phosphoglucose acid esters, while adjoint The oxidised form of nicotinamide adenine dinucleotide phosphate (NADP+) is reduced to NADPH.NADPH is as a variety of eukaryons Required confactor in biosynthesis reaction is as known in the art, it is known that it is able to maintain glutathione and goes back original shape for it Formula.Street cleaner of the glutathione being reduced in eucaryote as dangerous oxidation metabolites plays a role, also, in paddy Under the assistance of the sweet peptide peroxidase of Guang, by harmful hydrogen peroxide be changed into water (Beutler et al, 1991, N.Engl.J.Med.324:169-174).Therefore, G6PD can be the suppression in no vertebra harmful organism of double chain RNA mediate The selected objective target of system.

The homologous amino acid sequence of Robison ester 1- apodehydrogenase (G6PD) is inferred to from CRW cDNA library, it will It is used for the present invention.SEQ ID NO:57 is substantially corresponding to intestines cDNA nucleotide sequence in CRW.The amino of SEQ ID NO:57 Acid sequence is confirmed as: showing the homology with G6PD albumen (GenBank number U72484) in fin fish species.SEQ ID NO:58 and SEQ ID NO:59 corresponds respectively to the genome amplification primer (i.e. primer pair) of forward and reverse, and the primer is used In from crw genomic dna, amplicon is manufactured from the library CRWmRNA or the cDNA generated from thus class libraries.The sequence of such amplicon Column should correspond to all or part of the CRW gene of coding G6PD homologous protein.SEQ ID NO:58 and SEQ ID NO:59 Each T7 promoter sequence containing 23 nucleotide, respectively in 1-23 nucleotide.Shown in SEQ ID NO:58 24-46 nucleotide correspond to 113-136 nucleotide shown in SEQ ID NO:57.Shown in SEQ ID NO:59 24-45 nucleotide correspond to the reverse complementary sequence of the sequence in SEQ ID:57 shown in 373-394 nucleotide.With Crw genomic dna uses the primer being made of SEQ ID NO:58 and SEQ ID NO:59 as in the amplified reaction of template It is right, the amplicon of 328 base-pairs comprising nucleotide sequence shown in SEQ ID NO:60 is produced, it is following with encoding A part height of the CRW genome of albumen is corresponding, and the protein display has gone out the homology with G6PD albumen.SEQ ID NO: Nucleotide sequence shown in about the 24th nucleotide to the about the 305th nucleotide and 113- in SEQ ID NO:57 in 60 Nucleotide sequence height shown in 394 nucleotide is corresponding.

By the amplicons cloned for showing the sequence for corresponding to SEQ ID NO:60 into plasmid vector, the matter of sufficient amount is recycled Grain DNA is transcribed with allowing the insertion convergence T7 promoter from any end of cloned sequence to carry out external t7 rna polymerase.It produces Raw double-stranded RNA, carries out biologic test for sample；One RNA segment, positive-sense strand, by the about the 24th core in SEQ ID NO:60 Sequence composition shown in thuja acid to as little as the about the 305th nucleotide in SEQ ID NO:60 (the difference is that be shown as There are uracil residues for each position of thymidine), reverse complemental RNA segment or antisense strand are big in SEQ ID NO:60 The substantially reverse complementary series of nucleotide sequence shown in about the 305th nucleotide to as little as the about the 24th nucleotide, wherein Uracil suitably replaces thymidine.The sample of processing double-stranded RNA (dsRNA) is removed, with DICER or RNAse III to generate The siRNA (siRNA) of sufficient amount.Sample containing 0.15 part every million siRNA or dsRNA is used for described previously CRW diet biologic test, larva be allowed to feed 13 days.Compared with control, the CRW larva exhibition of the diet containing dsRNA is fed Obvious growth inhibition and the death rate are shown, the dsRNA corresponds to the complete of sequence shown in SEQ ID NO:60 Portion or part.

Actin 42A albumen homology sequence

Actin is generally existing, highly conserved eukaryotic protein, is required for cellular activity and movement (Lovato et al., 2001, Insect MoI.Biol.20:333-340).A large amount of CRW cDNA sequence is identified, They are predicted to be possible encoding actin or show the albumen of amino acid sequence structure relevant to actin.Cause This, the gene of encoding actin homologue can be the useful target of the inhibition of double chain RNA mediate in pest cell.

The UNIGENE cluster (cluster 156_1) identified in intestines cDNA library in corn rootworm is by a plurality of singlet EST sequence Column are constituted, wherein every is all predicted to be all or part of energy encoding actin homologous protein.These singlet are aligned to Cluster obtains consensus sequence shown in SEQ ID NO:61, and being predicted to be can encoding actin homologue.In annotation group Homologous actin sequence includes but is not limited to 3 segment of Drosophila melanogaster actin, Helicoverpa Armigera cell fibrinolysin (cytoplasmin) actin A3a (GenBank number X97614), Drosophila Melanogaster actin (GenBank number X06383), hemichordata Saccoglossus kowalevskii flesh are dynamic Albumen mRNA sequence and Strongylocentrotus purpuratus actin (GenBank number X05739).

SEQ ID NO:62 and SEQ ID NO:63 corresponds respectively to genome amplification primer (the i.e. primer of forward and reverse It is right), the primer is used to manufacture amplicon from the library CRWmRNA or the cDNA generated from thus class libraries from crw genomic dna. The sequence of such amplicon should correspond to all or part of the CRW gene of encoding actin homologous protein.SEQ ID T7 promoter sequence of each of the NO:62 and SEQ ID NO:63 containing 23 nucleotide, respectively in 1-23 nucleotide. 24-45 nucleotide shown in SEQ ID NO:62 correspond to 14-35 nucleotide shown in SEQ ID NO:61. 24-45 nucleotide shown in SEQ ID NO:63 correspond to the sequence in SEQ ID:61 shown in 449-470 nucleotide Reverse complementary sequence.In using amplified reaction of the crw genomic dna as template, using by SEQ ID NO:62 and SEQ The primer pair of ID NO:63 composition, produces 503 base-pairs comprising nucleotide sequence shown in SEQ ID NO:64 Amplicon, corresponding with a part height of CRW genome for encoding following albumen, the protein display has gone out and actin Homology.In SEQ ID NO:64 nucleotide sequence shown in the about the 24th nucleotide to the about the 480th nucleotide with Nucleotide sequence height shown in 14-470 nucleotide is corresponding in SEQ ID NO:61.

By the amplicons cloned for showing the sequence for corresponding to SEQ ID NO:64 into plasmid vector, the matter of sufficient amount is recycled Grain DNA is transcribed with allowing the insertion convergence T7 promoter from any end of cloned sequence to carry out external t7 rna polymerase.It produces Raw double-stranded RNA, carries out biologic test for sample；One RNA segment, positive-sense strand, by the about the 24th core in SEQ ID NO:64 Sequence composition shown in thuja acid to as little as the about the 480th nucleotide in SEQ ID NO:64 (the difference is that be shown as There are uracil residues for each position of thymidine), reverse complemental RNA segment or antisense strand are big in SEQ ID NO:64 The substantially reverse complementary series of nucleotide sequence shown in about the 480th nucleotide to as little as the about the 24th nucleotide, wherein Uracil suitably replaces thymidine.The sample of processing double-stranded RNA (dsRNA) is removed, with DICER or RNAse III to generate The siRNA (siRNA) of sufficient amount.Sample containing 0.15 part every million siRNA or dsRNA is used for described previously CRW diet biologic test, larva be allowed to feed 13 days.Compared with control, the CRW larva exhibition of the diet containing dsRNA is fed Obvious growth inhibition and the death rate are shown, the dsRNA corresponds to the complete of sequence shown in SEQ ID NO:64 Portion or part.

1 homologous sequence of ADP- ribosylation factor

ADP ribosylation factor 1 be shown as it is critically important in cell because, they DNA damage reparation, cause Complete effect is embodied during cancer, cell death and Genome stability.Accordingly, it is possible to which double chain RNA mediate can be used Inhibit, the transcription of ADP- ribosylation factor is selectively interfered in no vertebra pest.

Identify a large amount of CRW cDNA sequence, they are predicted to be: coding is shown and ADP- ribosylation factor Homology amino acid sequence.Particularly, a UNIGENE cluster (cluster 88_1) is made of about 30 (30) EST singlet, Wherein every be all predicted to be can encoding actin homologous protein all or part.These singlet are aligned to cluster, are obtained Consensus sequence shown in SEQ ID NO:65.The amino acid sequence translation of singlet CRW cDNA sequence comprising the cluster predicts Following amino acid sequences, the amino acid sequence show the homology with ADP- ribosylation factor homologue.Show with The ADF- ribosylation factor protein sequence packet of the significant homology for the amino acid sequence inferred from the ORF in SEQ ID NO:65 Include but be not limited to Drosophila melanogaster ADP- ribosylation factor (GenBank number Y10618), Drosophilia obscura ADP- ribosylation factor (GenBank number AF025798), Anopheles gambiae ADP- ribosylation factor (GenBank number L11617) and Australian sheep calliphorid (Lycilia cuprina) ADP- core Glycosylated Factor (GenBank number AF218587).

SEQ ID NO:66 and SEQ ID NO:67 corresponds respectively to the amplimer (i.e. primer pair) of forward and reverse, institute Primer is stated for manufacturing amplicon from the library CRW mRNA or the cDNA generated from thus class libraries from crw genomic dna.It is such The sequence of amplicon should correspond to all or part of the CRW gene of coding ADP- ribosylation factor homologous protein.SEQ T7 promoter sequence of each of the ID NO:66 and SEQ ID NO:67 containing 23 nucleotide, respectively in 1-23 nucleosides Acid.24-42 nucleotide shown in SEQ ID NO:66 correspond to 70-88 nucleosides shown in SEQ ID NO:65 Acid.24-40 nucleotide shown in SEQ ID NO:67 correspond in SEQ ID:65 shown in 352-368 nucleotide The reverse complementary sequence of sequence.In using amplified reaction of the crw genomic dna as template, using by SEQ ID NO:66 and The primer pair of SEQ ID NO:67 composition, produces 345 bases comprising nucleotide sequence shown in SEQ ID NO:68 Pair amplicon, corresponding with a part height of CRW genome for encoding following albumen, the protein display has gone out and ADP- The homology of ribosylation factor protein.The about the 24th nucleotide is to the about the 322nd nucleotide institute in SEQ ID NO:68 The nucleotide sequence shown is corresponding with nucleotide sequence height shown in 70-368 nucleotide in SEQ ID NO:65.

By the amplicons cloned for showing the sequence for corresponding to SEQ ID NO:68 into plasmid vector, the matter of sufficient amount is recycled Grain DNA is transcribed with allowing the insertion convergence T7 promoter from any end of cloned sequence to carry out external T7 RNA polymerase.It produces Raw double-stranded RNA, carries out biologic test for sample；One RNA segment, positive-sense strand, by the about the 24th core in SEQ ID NO:68 Sequence composition shown in thuja acid to as little as the about the 322nd nucleotide in SEQ ID NO:68 (the difference is that be shown as There are uracil residues for each position of thymidine), reverse complemental RNA segment or antisense strand are big in SEQ ID NO:68 The substantially reverse complementary series of nucleotide sequence shown in about the 322nd nucleotide to as little as the about the 24th nucleotide, wherein Uracil suitably replaces thymidine.The sample of processing double-stranded RNA (dsRNA) is removed, with DICER or RNAse III to generate The siRNA (siRNA) of sufficient amount.Sample containing 0.15 part every million siRNA or dsRNA is used for described previously CRW diet biologic test, larva be allowed to feed 13 days.Compared with control, the CRW larva exhibition of the diet containing dsRNA is fed Obvious growth inhibition and the death rate are shown, the dsRNA corresponds to the complete of sequence shown in SEQ ID NO:68 Portion or part.

Transcription factor IIB albumen homology sequence

As it was noted above, transcription extends and the translation termination factor is extremely important for metabolism, double-stranded RNA Jie can be The favo(u)rable target for the inhibition led, for controlling or eliminating the invasion of no vertebra harmful organism.

A kind of CRW cDNA sequence is identified, following amino acid sequences can be encoded by being predicted to be, the amino acid sequence Column illustrate the homology with transcription factor IIB albumen.SEQ ID NO:69 is used as the basis of building primer pair, described Primer pair be used to amplify the sequence for encoding following mRNA from CRW genome, and the mRNA is formd for the cDNA sequence The basis of column.

SEQ ID NO:70 and SEQ ID NO:71 corresponds respectively to the hot amplimer (i.e. primer pair) of forward and reverse, The primer is used to manufacture amplicon from the library CRWmRNA or the cDNA generated from thus class libraries from crw genomic dna.It is such The sequence of amplicon should correspond to all or part of the CRW gene of encoding transcription factors IIB homologous protein.SEQ ID NO: T7 promoter sequence of each of the 70 and SEQ ID NO:71 containing 23 nucleotide, respectively in 1-23 nucleotide.SEQ 24-44 nucleotide shown in ID NO:70 correspond to 4-24 nucleotide shown in SEQ ID NO:69.SEQ ID 24-44 shown in NO:71 nucleotide corresponds to the reversed of the sequence in SEQ ID:69 shown in 409-429 nucleotide Complementary series.In using amplified reaction of the crw genomic dna as template, using by SEQ ID NO:70 and SEQ ID NO: The primer pair of 71 compositions, produces the amplicon of 472 base-pairs comprising nucleotide sequence shown in SEQ ID NO:72, It is corresponding with a part height of CRW genome for encoding following albumen, and the protein display has gone out and transcription factor IIB albumen Homology.In SEQ ID NO:72 nucleotide sequence shown in the about the 24th nucleotide to the about the 449th nucleotide with Nucleotide sequence height shown in 4-429 nucleotide is corresponding in SEQ ID NO:69.

By the amplicons cloned for showing the sequence for corresponding to SEQ ID NO:72 into plasmid vector, the matter of sufficient amount is recycled Grain DNA is transcribed with allowing the insertion convergence T7 promoter from any end of cloned sequence to carry out external t7 rna polymerase.It produces Raw double-stranded RNA, carries out biologic test for sample；One RNA segment, positive-sense strand, by the about the 24th core in SEQ ID NO:72 Sequence composition shown in thuja acid to as little as the about the 449th nucleotide in SEQ ID NO:72 (the difference is that be shown as There are uracil residues for each position of thymidine), reverse complemental RNA segment or antisense strand are big in SEQ ID NO:72 The substantially reverse complementary series of nucleotide sequence shown in about the 449th nucleotide to as little as the about the 24th nucleotide, wherein Uracil suitably replaces thymidine.The sample of processing double-stranded RNA (dsRNA) is removed, with DICER or RNAse III to generate The siRNA (siRNA) of sufficient amount.Sample containing 0.15 part every million siRNA or dsRNA is used for described previously CRW diet biologic test, larva be allowed to feed 13 days.Compared with control, the CRW larva exhibition of the diet containing dsRNA is fed Obvious growth inhibition and the death rate are shown, the dsRNA corresponds to the complete of sequence shown in SEQ ID NO:72 Portion or part.

Chitinase protein

Chitin is β (1 → 4) homopolymer of N-acetylglucosamine, is found in insect exoskeleton.Chitin It is formed in the reaction of chitin synthase catalysis from UDP-N- acetylglucosamine.Chitin is structural homopolysaccharide, In the building to this hyperbranched and crosslinking structure, it is related to many enzyme steps.Chitin give insect shape, rigidity and It supports, provides internal, such as muscle is able to the bracket adhered to.Chitin should also be degraded to a certain degree, to regulate and control elder brother The step of involved in worm molting process.Thus, it is believed that double chain RNA mediate, inhibition to albumen in these approach, it will It can be used as means of the control without vertebra pest infestation.

From identifying to showing with chitinase protein homology, intestines cDNA library sequence in corn rootworm translation Amino acid sequence information.From the alignment to two singlet est sequences, a chitinase consensus sequence (UNIGENE is produced Cluster number 716_1；SEQ ID NO:73).From the alignment to four singlet sequences, second of chitinase consensus sequence is produced (UNIGENE cluster number 1238_1；SEQ ID NO:77).The amino acid sequence translation obtained from the ORF of these UNIGENE is annotated Are as follows: chrysomelid (mustard beetle) (Phaedon cochleariae) chitinase amino acid sequence (GenBank of horseradish ape Number Y18011).SEQ ID NO:73 and SEQ ID NO:77 is used as the basis of building primer pair, is used for from CRW gene In group, two sequences are expanded from the library CRW mRNA, or from the cDNA sequence obtained by the library this class mRNA.The nucleosides of such amplicon Acid sequence should correspond to all or part of the gene of chitinase encoding homologous protein.

SEQ ID NO:74 and SEQ ID NO:75 corresponds respectively to the hot amplimer (i.e. primer pair) of forward and reverse, The primer is used to manufacture amplicon from the library CRW mRNA or the cDNA generated from thus class libraries from crw genomic dna.This The sequence of class amplicon should correspond to the complete of the CRW gene of the chitinase encoding homologous protein as shown in SEQ ID NO:73 Portion or part.T7 promoter sequence of each of the SEQ ID NO:74 and SEQ ID NO:75 containing 23 nucleotide, exists respectively 1-23 nucleotide.24-42 nucleotide shown in SEQ ID NO:74 correspond to the shown in SEQ ID NO:73 1-19 nucleotide.24-47 nucleotide shown in SEQ ID NO:75 correspond to 470-493 cores in SEQ ID:73 The reverse complementary sequence of sequence shown in thuja acid.In using amplified reaction of the crw genomic dna as template, using by SEQ The primer pair of ID NO:74 and SEQ ID NO:75 composition, produces comprising nucleotide sequence shown in SEQ ID NO:76 The amplicon of 472 base-pairs, with corresponding, the protein display of a part height for the CRW genome for encoding following albumen The homology with chitinase protein is gone out.The about the 24th nucleotide is to the about the 516th nucleotide in SEQ ID NO:76 Shown in nucleotide sequence it is corresponding with nucleotide sequence height shown in 1-493 nucleotide in SEQ ID NO:76.

By the amplicons cloned for showing the sequence for corresponding to SEQ ID NO:76 into plasmid vector, the matter of sufficient amount is recycled Grain DNA is transcribed with allowing the insertion convergence T7 promoter from any end of cloned sequence to carry out external t7 rna polymerase.It produces Raw double-stranded RNA, carries out biologic test for sample；One RNA segment, positive-sense strand, by the about the 24th core in SEQ ID NO:76 Sequence composition shown in thuja acid to as little as the about the 516th nucleotide in SEQ ID NO:76 (the difference is that be shown as There are uracil residues for each position of thymidine), reverse complemental RNA segment or antisense strand are big in SEQ ID NO:76 The substantially reverse complementary series of nucleotide sequence shown in about the 516th nucleotide to as little as the about the 24th nucleotide, wherein Uracil suitably replaces thymidine.The sample of processing double-stranded RNA (dsRNA) is removed, with DICER or RNAse III to generate The siRNA (siRNA) of sufficient amount.Sample containing 0.15 part every million siRNA or dsRNA is used for described previously CRW diet biologic test, larva be allowed to feed 13 days.Compared with control, the CRW larva exhibition of the diet containing dsRNA is fed Obvious growth inhibition and the death rate are shown, the dsRNA corresponds to the complete of sequence shown in SEQ ID NO:76 Portion or part.

SEQ ID NO:78 and SEQ ID NO:79 corresponds respectively to genome amplification primer (the i.e. primer of forward and reverse It is right), the primer is used to manufacture amplicon from the library CRWmRNA or the cDNA generated from thus class libraries from crw genomic dna. The sequence of such amplicon should correspond to the CRW gene of the chitinase encoding homologous protein as shown in SEQ ID NO:77 It is all or part of.T7 promoter sequence of each of the SEQ ID NO:78 and SEQ ID NO:79 containing 23 nucleotide, respectively In 1-23 nucleotide.24-44 nucleotide shown in SEQ ID NO:78 correspond to shown in SEQ ID NO:77 64-84 nucleotide.24-44 nucleotide shown in SEQ ID NO:79 correspond in SEQ ID:77 779-799 The reverse complementary sequence of sequence shown in nucleotide.In using amplified reaction of the crw genomic dna as template, using by SEQ The primer pair of ID NO:78 and SEQ ID NO:79 composition, produces comprising nucleotide sequence shown in SEQ ID NO:80 The amplicon of 912 base-pairs.The comparison of cDNA sequence shown in SEQ ID NO:77 and amplification subsequence discloses, at two There is substantive difference, only about 32% sequence identity between sequence.Preferably, using primer pair, such as SEQ ID Primer pair shown in NO:78 box 79 and mRNA or cDNA manufacture amplicon as template, to avoid such inconsistent.

By the amplicons cloned for showing the sequence for corresponding to SEQ ID NO:77 into plasmid vector, the matter of sufficient amount is recycled Grain DNA is transcribed with allowing the insertion convergence T7 promoter from any end of cloned sequence to carry out external t7 rna polymerase.It produces Raw double-stranded RNA, carries out biologic test for sample；One RNA segment, positive-sense strand, by the about the 64th core in SEQ ID NO:77 Sequence composition shown in thuja acid to as little as the about the 799th nucleotide in SEQ ID NO:77 (the difference is that be shown as There are uracil residues for each position of thymidine), reverse complemental RNA segment or antisense strand are big in SEQ ID NO:77 The substantially reverse complementary series of nucleotide sequence shown in about the 799th nucleotide to as little as the about the 64th nucleotide, wherein Uracil suitably replaces thymidine.The sample of processing double-stranded RNA (dsRNA) is removed, with DICER or RNAse III to generate The siRNA (siRNA) of sufficient amount.Sample containing 0.15 part every million siRNA or dsRNA is used for described previously CRW diet biologic test, larva be allowed to feed 13 days.Compared with control, the CRW larva exhibition of the diet containing dsRNA is fed Obvious growth inhibition and the death rate are shown, the dsRNA corresponds to the complete of sequence shown in SEQ ID NO:77 Portion or part.

Ubiquitin binding enzyme homologous sequence

Ubiquitin pathway has important role in the control of cell cycle, this is by the spy to a large amount of modulins For specific degradation come what is realized, the albumen includes mitotic cell cyclin, or the kinases to cyclin dependent Inhibitor, for example, the p27 of mammalian cell.Therefore, the gene for encoding ubiquitin and related component can be double-stranded RNA Jie The selected objective target (Smith et al., Plant Phys.1997,113:281-29) for the inhibition led.Rely on the albumen water of ubiquitin Solution approach is one of the main path in eucaryote to intracellular protein selective destruction.The combination of ubiquitin and substrate is by obvious In the enzyme adjustment of diverse array.The targeting of proteolysis can also be dropped with it by multi-subunit 26S protease in the ubiquitination of substrate The step of solution is between peptide is regulated and controled.The complexity of ubiquitin system implys that it for albumen circulation in eukaryocyte regulation Central role, and involve other albumen in approach, including ubiquitin kinase, ubiquitin binding enzyme, ubiquitin-protein ligase And 26S proteosome subunit component.Thus, it is believed that the inhibition to the pathway protein that RNA is mediated will can be used as control nothing The means of vertebra pest infestation use.

Following CRW cDNA library sequences are identified, following amino acid sequences, the amino can be encoded by being predicted to be Acid sequence illustrates the homology with ubiquitin binding enzyme.SEQ ID NO:81 is used as the basis of building primer pair, described Primer pair includes all or part of the ubiquitin binding enzyme from corn rootworm for manufacturing amplicon, the amplicon.

SEQ ID NO:82 and SEQ ID NO:83 corresponds respectively to genome amplification primer (the i.e. primer of forward and reverse It is right), the primer is used to manufacture amplification from the library CRW mRNA or the cDNA generated from thus class libraries from crw genomic dna Son.The sequence of such amplicon should correspond to all or part of the CRW gene of coding ubiquitin binding enzyme homologous protein.SEQ T7 promoter sequence of each of the ID NO:82 and SEQ ID NO:83 containing 23 nucleotide, respectively in 1-23 nucleosides Acid.24-42 nucleotide shown in SEQ ID NO:82 correspond to 16-34 nucleosides shown in SEQ ID NO:81 Acid.24-42 nucleotide shown in SEQ ID NO:83 correspond in SEQ ID:81 shown in 295-313 nucleotide The reverse complementary sequence of sequence.In using amplified reaction of the crw genomic dna as template, using by SEQ ID NO:82 and The primer pair of SEQ ID NO:83 composition, produces 344 bases comprising nucleotide sequence shown in SEQ ID NO:84 Pair amplicon, corresponding with a part height of CRW genome for encoding following albumen, the protein display has gone out and ubiquitin The homology of desmoenzyme.Nucleotide shown in about the 24th nucleotide to the about the 321st nucleotide in SEQ ID NO:84 Sequence is corresponding with nucleotide sequence height shown in 16-313 nucleotide in SEQ ID NO:81.

By the amplicons cloned for showing the sequence for corresponding to SEQ ID NO:84 into plasmid vector, the matter of sufficient amount is recycled Grain DNA is transcribed with allowing the insertion convergence T7 promoter from any end of cloned sequence to carry out external t7 rna polymerase.It produces Raw double-stranded RNA, carries out biologic test for sample；One RNA segment, positive-sense strand, by the about the 24th core in SEQ ID NO:84 Sequence composition shown in thuja acid to as little as the about the 253rd nucleotide in SEQ ID NO:84 (the difference is that be shown as There are uracil residues for each position of thymidine), reverse complemental RNA segment or antisense strand are big in SEQ ID NO:84 The substantially reverse complementary series of nucleotide sequence shown in about the 253rd nucleotide to as little as the about the 24th nucleotide, wherein Uracil suitably replaces thymidine.The sample of processing double-stranded RNA (dsRNA) is removed, with DICER or RNAse III to generate The siRNA (siRNA) of sufficient amount.Sample containing 0.15 part every million siRNA or dsRNA is used for described previously CRW diet biologic test, larva be allowed to feed 13 days.Compared with control, the CRW larva exhibition of the diet containing dsRNA is fed Obvious growth inhibition and the death rate are shown, the dsRNA corresponds to the complete of sequence shown in SEQ ID NO:84 Portion or part.

Glyceraldehyde-3-phosphate dehydrogenase homologous sequence

Sugar decomposition approach is critically important approach in most of biologies, is related to generating metabolism from the degradation of glucose Energy.In the second stage of sugar decomposition approach, an important enzyme is glyceraldehyde-3-phosphate dehydrogenase (G3PDH), is existed In the case where NAD+ and inorganic phosphate, the phase can be catalyzed glyceraldehyde 3-phosphate and be oxidized to glycerol 3-phosphate-phosphoric acid, be subsequently formed NADH.The important component of the reaction is that energy is stored by the formation of NADH.Encode the base of enzyme relevant to sugar decomposition approach Cause, and particularly, coding can become in the gene for possessing the enzyme in the step of being related to can be used to form energy reserve object The particularly useful target of the inhibition of double chain RNA mediate is carried out in no vertebra harmful organism object.

Following CRW eDNA library sequences are identified, following amino acid sequences, the amino can be encoded by being predicted to be Acid sequence illustrates and the homology of glyceraldehyde-3-phosphate dehydrogenase (G3PDH) albumen.From the weight of three singlet est sequences Folded sequence assembly goes out the consensus sequence of cluster shown in SEQ ID NO:85.The amino of ORF in nucleotide sequence SEQ ID NO:85 G3PDH amino acid sequence (the GenBank that acid sequence is illustrated and obtained from Crytococcus curvatus G3PDH gene Number AF126158) and from Drosophila pseudoobscura G3PDH protein amino acid sequence (GenBank compile Number AF025809) homology.Therefore, the amino acid sequence translation of sequence shown in SEQ ID NO:85 is predicted to be CRW A part of G3PDH zymoprotein.Nucleotide sequence shown in SEQ ID NO:85 is used as the base that building thermal expansion increases primer pair Plinth, the primer pair is for manufacturing amplicon, the sequence of the amplicon coding CRW G3PDH enzyme sequence.

SEQ ID NO:86 and SEQ ID NO:87 corresponds respectively to the hot amplimer (i.e. primer pair) of forward and reverse, The primer is used to manufacture amplicon from the library CRW mRNA or the eDNA generated from thus class libraries from crw genomic dna.This The sequence of class amplicon should correspond to all or part of the CRW gene of coding G3PDH homologous protein.SEQ ID NO:86 and T7 promoter sequence of each of the SEQ ID NO:87 containing 23 nucleotide, respectively in 1-23 nucleotide.SEQ ID 24-45 shown in NO:86 nucleotide corresponds to 103-124 nucleotide shown in SEQ ID NO:85.SEQ ID 24-45 shown in NO:87 nucleotide corresponds to the reversed of the sequence in SEQ ID:85 shown in 573-594 nucleotide Complementary series.In using amplified reaction of the crw genomic dna as template, using by SEQ ID NO:86 and SEQ ID NO: The primer pair of 87 compositions, produces the amplicon of 538 base-pairs comprising nucleotide sequence shown in SEQ ID NO:88, It is corresponding with a part height of CRW genome for encoding following albumen, and the protein display has gone out homologous with G3PDH enzyme Property.Nucleotide sequence shown in about the 24th nucleotide to the about the 515th nucleotide and SEQ ID in SEQ ID NO:88 Nucleotide sequence height shown in 103-594 nucleotide is corresponding in NO:85.

By the amplicons cloned for showing the sequence for corresponding to SEQ ID NO:88 into plasmid vector, the matter of sufficient amount is recycled Grain DNA is transcribed with allowing the insertion convergence T7 promoter from any end of cloned sequence to carry out external t7 rna polymerase.It produces Raw double-stranded RNA, carries out biologic test for sample；One RNA segment, positive-sense strand, by the about the 24th core in SEQ ID NO:88 Sequence composition shown in thuja acid to as little as the about the 515th nucleotide in SEQ ID NO:88 (the difference is that be shown as There are uracil residues for each position of thymidine), reverse complemental RNA segment or antisense strand are big in SEQ ID NO:84 The substantially reverse complementary series of nucleotide sequence shown in about the 515th nucleotide to as little as the about the 24th nucleotide, wherein Uracil suitably replaces thymidine.The sample of processing double-stranded RNA (dsRNA) is removed, with DICER or RNAse III to generate The siRNA (siRNA) of sufficient amount.Sample containing 0.15 part every million siRNA or dsRNA is used for described previously CRW diet biologic test, larva be allowed to feed 13 days.Compared with control, the CRW larva exhibition of the diet containing dsRNA is fed Obvious growth inhibition and the death rate are shown, the dsRNA corresponds to the complete of sequence shown in SEQ ID NO:88 Portion or part.

Ubiquitin B homologous sequence

As described above, ubiquitin protein degradation pathway has important role in the control of cell cycle, this is to pass through The selective degradation of a large amount of modulins is realized, the albumen includes mitotic cell cyclin, or to dependence The inhibitor of the kinases of cyclin, for example, the p27 of mammalian cell.Therefore, the base of ubiquitin and related component is encoded Because can be selected objective target (Smith et al., the Plant Phys.1997,113:281- of the inhibition for double chain RNA mediate 291)。

Following CRW cDNA library sequences are identified, following amino acid sequences, the amino can be encoded by being predicted to be Acid sequence illustrates the homology with ubiquitin B albumen.SEQ ID NO is assembled from the overlap of four singlet est sequences: The consensus sequence of UNIGENE cluster shown in 89.The amino acid sequence translation of nucleotide sequence SEQ ID NO:89 illustrate with Polymerization ubiquitin amino acid sequence (GenBank number AF034789) from Amoeba proteus and come from Drosophila The homology of the ubiquitin protein sequence (GenBank number M22428) of melanogaster.Therefore, shown in SEQ ID NO:89 The amino acid sequence translation of sequence, which is believed, can encode ubiquitin B.SEQ ID NO:89 is used as building for hot amplified reaction Primer pair basis, the primer pair be used for amplification coding corn rootworm ubiquitin B amino acid sequence all or part of core Nucleotide sequence.

SEQ ID NO:90 and SEQ ID NO:91 corresponds respectively to the hot amplimer (i.e. primer pair) of forward and reverse, The primer is used to manufacture amplicon from the library CRW mRNA or the cDNA generated from thus class libraries from crw genomic dna.This The sequence of class amplicon should correspond to all or part of the CRW gene of coding ubiquitin B homologous protein.SEQ ID NO:90 and T7 promoter sequence of each of the SEQ ID NO:91 containing 23 nucleotide, respectively in 1-23 nucleotide.SEQ ID 24-40 shown in NO:90 nucleotide corresponds to 62-78 nucleotide shown in SEQ ID NO:89.SEQ ID 24-47 shown in NO:91 nucleotide corresponds to the reversed of the sequence in SEQ ID:89 shown in 399-422 nucleotide Complementary series.In using amplified reaction of the crw genomic dna as template, using by SEQ ID NO:90 and SEQ ID NO: The primer pair of 91 compositions, produces the amplicon of 407 base-pairs comprising nucleotide sequence shown in SEQ ID NO:92, It is corresponding with a part height of CRW genome for encoding following albumen, and the protein display has gone out same with ubiquitin binding enzyme Source property.Nucleotide sequence and SEQ shown in about the 24th nucleotide to the about the 384th nucleotide in SEQ ID NO:92 Nucleotide sequence height shown in 62-422 nucleotide is corresponding in ID NO:89.

By the amplicons cloned for showing the sequence for corresponding to SEQ ID NO:92 into plasmid vector, the matter of sufficient amount is recycled Grain DNA is transcribed with allowing the insertion convergence T7 promoter from any end of cloned sequence to carry out external t7 rna polymerase.It produces Raw double-stranded RNA, carries out biologic test for sample；One RNA segment, positive-sense strand, by the about the 24th core in SEQ ID NO:92 Sequence composition shown in thuja acid to as little as the about the 384th nucleotide in SEQ ID NO:92 (the difference is that be shown as There are uracil residues for each position of thymidine), reverse complemental RNA segment or antisense strand are big in SEQ ID NO:92 The substantially reverse complementary series of nucleotide sequence shown in about the 384th nucleotide to as little as the about the 24th nucleotide, wherein Uracil suitably replaces thymidine.The sample of processing double-stranded RNA (dsRNA) is removed, with DICER or RNAse III to generate The siRNA (siRNA) of sufficient amount.Sample containing 0.15 part every million siRNA or dsRNA is used for described previously CRW diet biologic test, larva be allowed to feed 13 days.Compared with control, the CRW larva exhibition of the diet containing dsRNA is fed Obvious growth inhibition and the death rate are shown, the dsRNA corresponds to the complete of sequence shown in SEQ ID NO:92 Portion or part.

JH esterase homologue

As described above, corpus allatum hormone necessary biological processes a variety of in insect life cycle are controlled and Regulation comprising but be not limited to, abnormal, breeding and diapause.Using gene containment technology to JH synthesis or the interference of degradation pathway It may be the effective target of the harmful organism control of double chain RNA mediate.

The corpus allatum hormone esterase homologous sequence obtained from CRW is identified, the present invention is used for.SEQ ID NO:93 height corresponds to intestines cDNA nucleotide sequence in CRW.The amino acid sequence translation of SEQ ID NO:93 predicts and protects children The homology of hormone esterase (JHE).The amplification that SEQ ID NO:94 and SEQ ID NO:95 corresponds respectively to forward and reverse is drawn Object (i.e. primer pair), the primer are used to make from crw genomic dna from the library CRWmRNA or the cDNA generated from thus class libraries Make amplicon.The sequence of such amplicon should correspond to all or part of the CRW gene of coding JHE homologous protein.SEQ T7 promoter sequence of each of the ID NO:94 and SEQ ID NO:95 containing 23 nucleotide, respectively in 1-23 nucleosides Acid.24-45 nucleotide shown in SEQ ID NO:94 correspond to 58-79 nucleosides shown in SEQ ID NO:93 Acid.24-46 nucleotide shown in SEQ ID NO:95 correspond in SEQ ID:93 shown in 338-360 nucleotide The reverse complementary sequence of sequence.In using amplified reaction of the crw genomic dna as template, using by SEQ ID NO:94 and The primer pair of SEQ ID NO:95 composition, produces 348 bases comprising nucleotide sequence shown in SEQ ID NO:96 Pair amplicon preferably, use the library CRW mRNA or from the cDNA that this class libraries obtains as the template nucleotide in amplified reaction Sequence.

By the amplicons cloned for showing the sequence for corresponding to SEQ ID NO:96 into plasmid vector, the matter of sufficient amount is recycled Grain DNA is transcribed with allowing the insertion convergence T7 promoter from any end of cloned sequence to carry out external t7 rna polymerase.It produces Raw double-stranded RNA, carries out biologic test for sample；One RNA segment, positive-sense strand, by the about the 45th core in SEQ ID NO:96 Sequence composition shown in thuja acid to as little as the about the 302nd nucleotide in SEQ ID NO:96 (the difference is that be shown as There are uracil residues for each position of thymidine), reverse complemental RNA segment or antisense strand are big in SEQ ID NO:96 The substantially reverse complementary series of nucleotide sequence shown in about the 302nd nucleotide to as little as the about the 45th nucleotide, wherein Uracil suitably replaces thymidine.The sample of processing double-stranded RNA (dsRNA) is removed, with DICER or RNAse III to generate The siRNA (siRNA) of sufficient amount.Sample containing 0.15 part every million siRNA or dsRNA is used for described previously CRW diet biologic test, larva be allowed to feed 13 days.Compared with control, the CRW larva exhibition of the diet containing dsRNA is fed Obvious growth inhibition and the death rate are shown, the dsRNA corresponds to the complete of sequence shown in SEQ ID NO:96 Portion or part.

With from the parallel biologic test that the siRNA that double stranded rna molecule generates carries out, to double-strand listed above Ten in RNA molecule are examined.By double stranded RNA sequences sample or the small interference come from double stranded RNA sequences sample preparation (its every kind both corresponds to following amino acid sequences to RNA sample, and it is homologous that the amino acid sequence is noted as selected target gene Object, they include: 40kDa V-ATPase homologue, EF-1-alpha homologue, 26S proteosome subunit p28 homologue, protect children Hormone epoxide hydrolase homologue, beta- tubulin homologues, two kinds of chitinase homologues, turns CHD3 homologue Record factor IIB homologue and JH esterase homologue (correspond respectively to SEQ ID NO:35, SEQ ID NO:39, SEQ ID NO:47, SEQ ID NO:52, SEQ ID NO:7, SEQ ID NO:21, SEQ ID NO:76, SEQ ID NO:80, SEQ ID NO:72 and SEQ ID NO:06)) (to contain quilt for 30 microlitres applied to insect diet every about the concentration of 1,000,010 parts The solution for being adjusted to the double-stranded RNA sample of suitable concentration is added to the microtitre version hole containing 200 microlitres of every hole insect diet In).Every kind of sample uses 18 holes in total.RNA sample diffuses into after diet, and single the first instar larvae only is added to often In a hole.As described above, to biologic test culture about 13 days, morbidity and mortality are monitored daily.By amino acid sequence Variant Cry3Bb1 insecticidal crystalline protein (is named as killing elder brother in English et al. (U.S. Patent number 6,642,030) Worm protein 11 231) it is used as positive control, for observing the insecticidal bioactivity particular for harmful corn rootworm.According to Described in English et al., Cry3Bb is used for diet, the difference is that the concentration of Cry3Bb is adjusted to often in diet Million parts 200-300.Only test is also included in the independent control sample that buffer or water are handled.Double-stranded RNA pair In the same old way product and additional recessive control is used as from the siRNA control sample that double-stranded RNA control sample generates is included in Come (RNAi Kit, AMBION, Austin, Texas).

It is shown using the initial evaluation and test that the double stranded rna molecule obtained from above-mentioned ten sequences carries out, is allowed to feed and contains The larva of the diet of following double-stranded RNAs illustrates the significant death rate compared with control, and the double-stranded RNA corresponds to 40kDa V- ATPase homologue (SEQ ID NO:35), CHD3 homologue (SEQ ID NO:7) and beta- tubulin homologues (SEQ ID NO:31).Based on such as a result, additional biologic test is carried out, to examine whether small interference double-stranded RNA particle compares overall length Double stranded rna molecule is more effective.

Alpha tubulin homologous sequence

Eukaryotic cells usually utilize cytoskeletal structure element, they are not only important to as machinery mount, also heavy It will be in the holding to cell shape.Half flexible microfibril enables cell to move, and helps them in mitosis (cytoplasm point Split) in separation, and, in vertebrate and invertebrate, be used for contraction of muscle.Relatively hard micro-pipe by alpha and Beta tubulin is constituted, they have important role during following: being risen as transporter vesicle and cell a set of high Fast highway plays a role, and for separating chromosome during mitosis (nuclear fission).Flexible intermediate filament provides pair In at least additional intensity of entire cell results.It is also known that cytoskeleton is participated in across cytoplasmic signal process.It considers These functions, it is believed that, any interference to cytoskeleton or even the minor alteration of its integrality all may cause To the disease consequence of cell.

Identify at least one CRW cDNA library sequence, be accredited as to encode show it is same with following albumen The amino acid sequence of source property, the albumen is referred to herein as alpha tubulin, more specifically, referring to sequence herein SEQ ID NO:163 shown in table.The amino acid sequence translation of the sequence as shown in SEQ ID NO:163, which is believed, to be encoded Alpha tubulin or its segment.SEQ ID NO:63 is used as constructing the basis of following sequences, and the sequence is predicted to be: When being expressed in E.coli by T7 or being expressed in plant by Plant functional promoters, double-stranded RNA can be formed.It is used as In such double-stranded RNA coded sequence basis sequence be the 58th nucleotide of SEQ ID NO:97 shown in sequence table extremely 1010th nucleotide.The sequence can be used as RNA molecule expression, is purified, tests in feeding test in vitro, with measurement Inhibition to corn rootworm.

The 58th nucleotide to the 1010th nucleotide that t7 rna polymerase promoter is introduced in SEQ ID NO:97 shows The upstream of nucleotide sequence out generates RNA from the construct (pIC17527).Using three parts of repeat samples, for jade Such RNA is examined in the external feeding test of rice rootworm, it is described to examine for the control of beta tubulin positive (above It is described), 200ppm Cry3Bb and unprocessed control carry out, and measure average mortality.Unprocessed control sample Product illustrate the death rate less than about 3-5%, and all other sample survey illustrates about 20 to about 55% The death rate.Cry3Bb sample display has gone out the death rate of about 20 to about 36%, and pIC17527 sample (15ppm) is then shown The death rate of about 38 to about 45% out.It is similarly D8 (beta tubulin as indicated above) sample of about 15ppm Show the death rate of about 38 to about 52%.Based on these results, alpha tubulin construct is placed on plant function It can be used for maize transformation plant, for the ability for resisting corn rootworm invasion, to from the conversion under the control of property promoter The transformation event of generation is examined.

The nucleotide sequence area shown in SEQ ID NO:97 converts the root from R0 plant.In short, by upper The sequence that dsRNA construct is encoded in SEQ ID NO:97 is stated at 5 ' ends and by being operatively connectable to corn hsp70 introne The sequence that e35S promoter is constituted is connected, and is connected at 3 ' ends with NOS3 ' tanscription termination and poly- polyadenylation sequence.The expression cassette is put It is placed in the downstream of careless glycosides phosphine selection box.Then these connected boxes are placed into Agrobacterium tumefaciens plant Transformation function carrier, new support are named as pMON72829 (alpha tubulin dsRNA construct), are used for maize transformation group It knits, makes it have careless glycosides phosphine tolerance, select event, be transferred into soil.The root of R0 plant is fed to western corn rootworm Larva (WCR, Diabrotica virifera).With the MSOD culture medium containing antibiotic and careless glycosides phosphine by transgenic corns root It is transferred on culture dish, is selected in vitro.Make invasion of the every root by two WCR larvas in each ware with thin head brush.With Parafilm seals these wares, prevents larva from escaping.Test is in 27 DEG C, 60% RH Percival incubator in completely black Dark lower progress.Pollution and larva quality are monitored.After root tissue is fed six days, larva is transferred in 96 orifice plates WCR diet in.Larva is allowed to feed the diet eight days, so that total test is that fortnight is long.To larva quality and survival It is recorded, for analyzing.A kind of analyze is carried out on the basis of larva qualitative data and Dunnett ' s are examined, for seeking Look for the significance,statistical being compared with LH244 (unconverted negative control).WCR larva is feeding two kinds of events, ZM_ After S125922 and ZM_S125938, compared with the growth of the larva of feed negative control plant, hence it is evident that hypoevolutism (α= 0.05) (p < 0.02).The larva of feed negative control plant illustrates about 0.6 to about 0.8mg average larva matter Amount, and the larva for feeding transgenosis root then illustrates about 0.1 to about 0.2mg average larva quality.

After reaching suitable size, by transgenic plant (R0) plantation for using pMON72829 to generate to containing Metromix In 10 inches of bottles of soil.When plant reaches V4 growth phase, make about 1000 Western corn rootworms (WCR, Diabrotica virifera) ovum invades into root area.The non-transgenic corn of same genotype is in similar growth phase It is invaded, to function as negative control.Preincubate is carried out to ovum, makes it possible to hatch in 24 hours of invasion.Larva quilt Allow to unite in root system and feed 3 weeks.Plant is removed from soil, it is cleaned, so that can be subject to for larva feed to root Evaluation and test.Root damage is classified using Node Injury Scale (NIS), wherein 0, expression does not damage, and 1 indicates root Short 1.5 inches of a section within, 2 indicate that two sections are short, and 3 indicate that 3 sections are short.Because after the plant for evaluation and test is conversion Directly from tissue culture, also as transformation event is unique, so only evaluating and testing single plant plant for every kind of event at this time Strain, cannot obtain statistical result.All plant in test all illustrate the sign of larva feed, this shows to obtain success Invasion.Negative control plant root is presented moderate and damages to serious, average out to about 1.9 on Node Injury Scale.It is right Individual plants from eight kinds of different transgenic events are examined.Three plants of root is provided to children in these genetically modified plants The outstanding control of worm feed, Node Injury Scale are average about 0.2 or smaller.Two plants in genetically modified plants Root illustrates moderate feed damage, and three plants of other transgenosis five do not show the control to larva feed.The data Show to be expressed in genetically modified plants and when the plant is provided in corn rootworm diet, coding can form the RNA of dsRNA The Double-stranded nucleotide sequence of sequence is entirely capable of providing the protection for being directed to corn rootworm pest infestation.

About lack the consistent observable death rate or select carry out the sequence of gene containment (including EF1alpha, 26S proteosome subunit and a variety of other cDNA sequences) other effects, a kind of explanation may be, for these For gene, expression is to encode homologue present in the gene group of following albumen, and the albumen has similar function, but Be show sufficiently large sequence difference so that using choosing come contained these sequences when, RNAi approach cannot play work For containing homologue.

Embodiment 2

Present embodiment describes the inhibition of significant harmful organism, this is by containing to the feeding of no vertebra harmful organism from this The diets of the double stranded RNA sequences that harmful organism obtains is realized.

By the sample for the double stranded RNA sequences that application is obtained from six kinds of different corn rootworm cDNA library sequences, to make It prepares enough to raise the artificial diet of Corn rootworm larvae.Corn rootworm larvae by permit feed the diet a few days, compared be only permitted into The corn rootworm for eating control diet, monitors the death rate, disease incidence and hypoevolutism.For the nucleotide sequence in diet It is from SEQ ID NO:35, SEQ ID NO:29, SEQ ID NO:47, SEQ ID NO:52, SEQ ID NO:7 and SEQ ID What sequence shown in NO:31 obtained, they every kind correspond to the nucleotide sequence that obtains from corn rootworm cDNA library, they Infer that amino acid sequence translation corresponds respectively to be noted as 40kDa V-ATPase homologue, EF1 α homologue, 26S albumen Body subunit homologue, juvenile hormone epoxide hydroxylase enzyme homologue, CHD3 homologue and 'beta '-tubulin homologue.

As described above, the double-stranded RNA for corresponding to these sequences is generated.By using RNAse III enzyme to corresponding DsRNA is cut to generate siRNA, it is known that dsRNA can be cut into the dsRNA segment of 12-15bp by the enzyme, and the segment contains There are 3 ' suspensions and the 5 ' phosphoric acid and 3 ' C-terminals of 2-3 nucleotide.The siRNA generated in this way is shown with technical ability Property identical with the siRNA generated with Dicer enzyme (involved in eucaryotic RNA i approach).

As described above, dsRNA and siRNA is loaded in CRW diet with about 0.15ppm.As described above, For every kind of dsRNA or siRNA sample, 12 single Corn rootworm larvaes are individually examined, assessment result after 13 days.

Compared with unprocessed control (UTC), feeds in the larva of the diet containing the following dsRNA sequences of 0.15ppm and see Observed the significant decrease (p < 0.05) of larva quality, the dsRNA sequence be SEQ ID NO:35, SEQ ID NO:52, Shown in SEQ ID NO:7 and SEQ ID NO:31.Corresponding to SEQ ID NO:35, SEQ ID NO:39, SEQ ID NO:47 The significant decrease (p < 0.05) of larva quality is also provided with the siRNA of the sequence of SEQ ID NO:7.But larva sample is big Small to be but not enough to determine foundation: causing larva quality compared with control, utmostly the dsRNA or siRNA of reduction are to become at random actually The result of change? still the specific result of the inhibition to biological functions some in rootworm larvae based on double chain RNA mediate? cause This, is recorded a demerit based on these, with biggish larva sample size, to corresponding to SEQ ID NO:35, SEQ ID NO:39, SEQ ID The RNA sequence of NO:7 and SEQ ID NO:31 is evaluated and tested again.

For each of four kinds of RNA sequences in evaluation and test, dsRNA or siRNA sample is applied in each of 72 holes.It is logical It crosses 30 microlitres of volume applications containing RNA to diet surface and sample is allowed to immerse and allow diet dry tack free, press According to described above, to the dsRNA or siRNA of each hole loading 0.15ppm.Single larva is added in each hole, culture 13 It.Evaluate and test larval mortality and disease incidence, the quality of measurement survival larva.Biological results are shown in table 1.

1. Biological results of table

All siRNA samples are all every hole 0.15ppm

UTC-10mM TrisHCl pH 7.5

STE- standard error

In dsRNA biologic test, 1- phageλ dsRNA, EPICENTER TECHNOLOGIES, Madison, Wisconsin；In siRNA biologic test,RNAi Kit, AMBION, Austin, Texas

2-Cry3Bb variant is 300ppm in dsRNA biologic test, is 200ppm in siRNA biologic test.

Tukey ' s HSD method is used, is mutually compared to all samples, rather than is only compared with any single compare Compared with.It is slow that every kind of examined dsRNA or siRNA observed significant larvae development, this is by compared with survival larva The average quality of unprocessed control reduces to judge.More importantly, double-chain small disturbance RNA sample display has gone out to cause The ability of the death rate and disease incidence (the larva quality based on reduction) of certain level, the level is at least and positive control sample Cry3Bb variant 11231 is equally effective.The above results show what the mRNA sequence present in the corn rootworm cell obtained Any double stranded rna molecule, when being supplied to corn rootworm in the diet of corn rootworm, all effective in inhibiting harmful corn rootworm pair The invasion of plant species.

Embodiment 3

Present embodiment describes the nucleotide sequences for expressing in plant cell, and mention in corn rootworm diet For the effect of such nucleotide sequence.

It goes to construct with the CHD3 coded sequence obtained from corn rootworm cDNA library and encodes the nucleosides for being stabilized double-stranded RNA Acid sequence.Encode cDNA sequence shown in CHD3 amino acid sequence ortholog or homologue a part, SEQ ID NO:171 Column be used to construct primer pair, and the primer pair is used for hot amplified reaction, wherein using corn rootworm genomic templates DNA.SEQ Primer pair energy amplifying doulbe-chain genomic amplicons shown in ID NO:5 and SEQ ID NO:6 a, wherein chain shows SEQ ID Sequence shown in NO:7.Shown in SEQ ID NO:7 nucleotide sequence generates three nucleotide sequence segments.Use SEQ Nucleotide sequence shown in ID NO:7 is as template, in hot amplified reaction, with showing SEQ ID NO:8 and SEQ ID The thermal expansion of sequence shown in NO:9 increases primer pair, produces first nucleotide fragment (SEQ ID NO:174).Use SEQ ID Nucleotide sequence shown in NO:7 is as template, in hot amplified reaction, with showing SEQ ID NO:11 and SEQ ID NO: The thermal expansion of sequence shown in 12 increases primer pair, produces Article 2 nucleotide fragment (SEQ ID NO:13).Use SEQ ID Nucleotide sequence shown in NO:7 is as template, in hot amplified reaction, with showing SEQ ID NO:14 and SEQ ID NO: The thermal expansion of sequence shown in 15 increases primer pair, produces Article 3 nucleotide fragment (SEQ ID NO:16).First silver chain rupture One of 3 ' ends it is complementary with 3 ' ends of one of Article 2 piece chain rupture, thus in the hot amplified reaction containing this two segments, this A little complementary end hybridization, to allow two chains to carry out polymerase-mediated extension from the 3 ' ends of their difference.Article 2 segment 3 ' ends of another chain are complementary with 3 ' ends of one of Article 3 piece chain rupture, thus in the hot amplified reaction containing this two segments In, these complementary end hybridization, to allow two chains to carry out polymerase-mediated extension from the 3 ' ends of their difference.Containing All these segments and their complementary series, i.e., first, in the hot amplified reaction of Article 2 and Article 3 segment, use SEQ Thermal expansion shown in ID NO:8 and SEQ ID NO:15 increases primer sequence, produces the new sequence as shown in SEQ ID NO:17, should When new sequence is positioned under the promoter control to work in plant, it can generate and sequence height shown in SEQ ID NO:17 Identical RNA nucleotide sequence, only in addition to there are uracil residues to replace thymine residue.The RNA nucleotide sequence The reverse complemental that can use Article 3 segment Yu first segment is formed as being stabilized RNA molecule, wherein SEQ ID Part and SEQ ID in NO:17 corresponding to the about the 303rd nucleotide of Article 3 segment to the about the 473rd nucleotide Partial hybridization in NO:17 corresponding to the about the 1st nucleotide of first article of segment to the about the 171st nucleotide, first It is connected with Article 3 segment by Article 2 nucleotide sequence segment, in this embodiment, by corresponding in SEQ ID NO:17 About the 172nd nucleotide of Article 2 segment to the part of the about the 302nd nucleotide indicates.Corresponding to SEQ ID NO:17 Nucleotides sequence be listed in the expression in plant cell and lead to the synthesis for being stabilized RNA molecule.It can will be shown in SEQ ID NO:17 Nucleotide sequence be transcribed into the plant cell of RNA sequence and can be provided that in the diet of corn rootworm.As the homologous egg of CHD3 It is white to synthesize repressed as a result, feeding the corn rootworm stopping feed of such plant cell, it develops and is obstructed for adult beetles, it is numerous It grows and is obstructed, it is dead or influenced by these any or all of.

Building coding is gone to be stabilized double-stranded RNA with the 'beta '-tubulin coded sequence obtained from corn rootworm cDNA library Nucleotide sequence.Coding for beta-tubulin amino acid sequence ortholog or homologue a part, SEQ ID NO:18 Shown in cDNA sequence be used to construct primer pair, the primer pair is used for hot amplified reaction, wherein using corn rootworm gene Group template DNA.Primer pair energy amplifying doulbe-chain genomic amplicons shown in SEQ ID NO:19 and SEQ ID NO:20, wherein one Chain shows sequence shown in SEQ ID NO:21.Shown in SEQ ID NO:21 nucleotide sequence generates three nucleosides Acid sequence segment.Use the nucleotide sequence shown in SEQ ID NO:21 as template, in hot amplified reaction, with showing The thermal expansion of sequence shown in SEQ ID NO:22 and SEQ ID NO:23 increases primer pair, produces first nucleotide fragment (SEQ ID NO:173).Use the nucleotide sequence shown in SEQ ID NO:21 as template, in hot amplified reaction, with showing The thermal expansion of sequence shown in SEQ ID NO:25 and SEQ ID NO:26 increases primer pair, produces Article 2 nucleotide fragment (SEQ ID NO:27).Use the nucleotide sequence shown in SEQ ID NO:21 as template, in hot amplified reaction, with showing SEQ The thermal expansion of sequence shown in ID NO:28 and SEQ ID NO:29 increases primer pair, produces Article 3 nucleotide fragment (SEQ ID NO:36).3 ' ends of one of the first silver chain rupture are complementary with 3 ' ends of one of Article 2 piece chain rupture, thus containing this two silver In disconnected hot amplified reaction, these complementary end hybridization, to allow two chains to carry out polymerase Jie from the 3 ' ends of their difference The extension led.Another chain of Article 2 segment 3 ' end it is complementary with 3 ' ends of one of Article 3 piece chain rupture, thus contain this two In the hot amplified reaction of segment, these complementary ends hybridization, to allow two chains to be polymerize from their 3 ' ends respectively The extension that enzyme mediates.Containing all these segments and their complementary series, i.e., first, Article 2 and Article 3 segment In hot amplified reaction, the thermal expansion shown in SEQ ID NO:22 and SEQ ID NO:29 increases primer sequence, produces such as SEQ ID New sequence shown in NO:31 can generate and SEQ ID when the new sequence is positioned under the promoter control to work in plant The identical RNA nucleotide sequence of the height of sequence shown in NO:31, only in addition to there are uracil residues to replace thymidine residual Base.The RNA nucleotide sequence can use the reverse complemental of Article 3 segment Yu first segment, be formed as being stabilized RNA molecule, wherein corresponding to the about the 338th nucleotide of Article 3 segment to the about the 577th core in SEQ ID NO:31 The part of thuja acid with correspond to the about the 31st nucleotide of first article of segment in SEQ ID NO:31 to the about the 250th nucleosides The partial hybridization of acid, first is connected with Article 3 segment by Article 2 nucleotide sequence segment, in this embodiment, by Part table in SEQ ID NO:31 corresponding to the about the 251st nucleotide of Article 2 segment to the about the 337th nucleotide Show.Nucleotides sequence corresponding to SEQ ID NO:31, which is listed in the expression in plant cell, leads to the synthesis for being stabilized RNA molecule. The plant cell that nucleotide sequence shown in SEQ ID NO:31 is transcribed into RNA sequence can be can be provided that the meals in corn rootworm In food.It synthesizes repressed as 'beta '-tubulin as a result, the corn rootworm for feeding such plant cell stops feed, develops and be Adult beetles are obstructed, and breeding is obstructed, dead or influence by these any or all of.

Embodiment 4

Present embodiment describes one or more combinations with insecticidal action are provided in no vertebra harmful organism diet Object and one or more synergies from the double stranded RNA sequences obtained without vertebra harmful organism, wherein it is described a kind of or Insecticidal effect has been had shown that out when being provided in harmful organism diet before a variety of dsRNA sequences.

As described in Example 3, the double-stranded RNA point obtained from the harmful organism is provided in the diet of no vertebra harmful organism Son leads to the inhibition to biological functions one or more in harmful organism, and thus acts on acquisition insecticidal effect, results in The death of harmful organism or it is some it is other it is being measured to, reduce pest infestation specific environment or host ability Feature.One or more other insecticides are added, and (every kind different mutually, and every kind all by obtaining its desinsection with dsRNA The mode of effect different mode obtains its insecticidal effect), can to control harmful organism it is horizontal improve, and will Further decrease: when inhibition to obtain heap harmful organism is used alone, harmful organism may to one or more insecticides or DsRNA develops a possibility that resistance.

To be examined to this, CRW larva is enabled to feed following diets, including the Cry3Bb rootworm into different content Inhibit the double-stranded RNA of albumen and fixed amount, the RNA is according to preparing described in embodiment 2 or 3, for example, corresponding In the dsRNA of SEQ ID NO:17 or SEQ ID NO:31.It observed the synergy inhibited to harmful organism.Such as embodiment Shown in 2 and 3, the LD50 amount of Cry3Bb be used to obtain the about 50% insect larvae death rate, and survival larva health The companion reduction (being judged by Weight of larvae compared with the reduction of negative control) of degree.Reduce insecticidal protein in diet Amount causes the companion of the death rate to reduce, and the increase of survival larva average weight.Be added correspond to SEQ ID NO:31 or The dsRNA of SEQ ID NO:17, results in the death rate almost in the Cry3Bb of every kind of concentration, and any survival object is put down Equal weight is also greatly reduced.This demonstrate synergies.Concertedness may be by introducing any content Cry3Bb (its by Display are as follows: can in middle goldbeater's skin introduction hole) caused by the interference of intestines in larva is obtained.Hole can permit higher levels of Double-stranded RNA type penetrates in cell, or even penetrates into hemolymph, causes highly efficient to be transported into dsRNA type Thus larva leads to more efficiently reduction in the containment to target mRNA.Hole forms composition and double stranded RNA composition Specific combination leads to increased and concertedness insecticidal action, because dsRNA can more disperse in hemolymph, to far from harmful raw The cell and tissue of object enteron aisle are exerted one's influence.Specific hole forms composition, from B.thuringiensis and The insecticidal toxic protein (no matter whether they can show insecticidal effect to certain specific insect) that relative species obtain, into one Step includes but is not limited to that the hole of this toxoid forms structural domain.This pores, which forms composition, may also include one or more hole shapes At toxin or its structural domain or combinations thereof object, they every kind it is all different, every kind all shows different binding modes, this is Attribute is formed by the channel of every kind of toxin or structural domain to judge comprising, dynamics, the conductance state of ion channel formation Size, total membrane conductance, ion specificity and ion channel gate inhibition's attribute of (conductance state).This pores shape At the combination of composition and the dsRNA molecule for being specific to the containment one or more genes of coleoptera species by particularly including into originally Text.

Embodiment 5

Present embodiment describes the nucleotide sequence fragments of V-ATPase, are provided in CRW species meals in the form of double-stranded RNA When in food, have for controlling harmful insect.

Sequence shown in SEQ ID NO:104 is cDNA clone, represents 2400 nucleotide for encoding following albumen 1870 nucleotide in mRNA, the protein display go out with Drosophila melanogaster vacuole ATPase (68kd, Subunit 2) height sequence identity.Using the primer designed from initial sequence information, to this cDNA grams on two chains It is grand to be sequenced completely.These sequencing primers are listed as SEQ ID NO:105 to SEQ ID NO:120.SEQ ID NO:121 It is primer sequence with SEQ ID NO:122, generates SEQ ID for the cDNA from cloning vector pSPORT (Invitrogen) The copy of NO:104.Every primer contains the T7 promoter sequence of 20 nucleotide, is 1-20 nucleotide.SEQ ID NO: The position 21-45 nucleotide pair in 21-44 position nucleotide and SEQ ID NO:122 in 121 is answered to be inserted into pSPORT carrier The sequence of cDNA flank.These primers allow to containing cDNA segment (its arbitrary end, flank have T7 promoter) DNA profiling into Row amplification, allows to generate double-stranded RNA in vitro with t7 rna polymerase.When by the double-stranded RNA obtained from SEQID NO:104 include into When in CRW diet, about 80% death rate is observed.

By using following amplimer groups, six different zones of SEQ ID NO:104 are examined: SEQ ID NO:123 and SEQ ID NO:124, corresponding to SEQ ID NO:1 1 to 291 nucleotide (referred to as part #1,271 Base-pair)；SEQ ID NO:125 and SEQ ID NO:126, correspond to 292 to 548 nucleotide (referred to as part #2, 260 base-pairs)；SEQ ID NO:127 and SEQ ID NO:128 corresponds to 549 to 830 nucleotide (referred to as portions Divide #3,271 base-pairs)；SEQ ID NO:129 and SEQ ID NO:130 corresponds to 840 to 1345 nucleotide and (is claimed For part #4,505 base-pairs)；SEQ ID NO:131 and SEQ ID NO:132 corresponds to 1360 to 1621 nucleotide (referred to as part #5,261 base-pairs)；SEQ ID NO:133 and SEQ ID NO:136 corresponds to 1540 to 1870 Nucleotide (referred to as part #6,278 base-pairs).Note that part 5 and 6 has been overlapped about 80 base-pairs.When this 6 portions When point separately including into CRW diet, #1, #2, #3 and #4 show the CRW death rate in 94% to 100% range.Part #5 It is not shown with #6 higher than the CRW death rate in unprocessed control more than visible background.The sequence quilt that part #1 is represented Be further divided into three smaller parts, each of these three smaller parts all by the #1 of part at least about 150 to Representated by about 180 contiguous nucleotides, so that first sub-part and second sub-part in the #1 of part in the #1 of part It is overlapped, third sub-part is Chong Die with second sub-part in the #1 of part.To every in these sub-parts in CRW biologic test Kind is tested respectively.Using these three shorter sequences, the death rate between 80% to 90% observed.

Second of the means examined to the bioactivity of the dsRNA molecule obtained from CRW gene are that building itself is mutual The RNA molecule of benefit.Start sub-portfolio by the identical DNA sequence and t7 rna polymerase that will reverse-locate, can synthesize and can itself Complementary single rna molecule.A kind of such RNA molecule be by by 1 in nucleotide sequence shown in SEQ ID NO:104 to What 345 nucleotide and 50 to 325 nucleotide combinations obtained.Obtained sequence is illustrated as SEQ ID NO:137, is named For pIC17527.PIC17527 is cloned into pTOPT2.1 (Invitrogen).Using the T7 promoter in pTOP2.1, generate The dsRNA of about 500 base pair nucleotides, including into CRW diet.The obtained death rate 80% to 100% it Between.

Embodiment 6

Present embodiment describes dsRNA to the oral poison of Colorado potato bug (Leptinotarsa decemlineata) Property.

Using Ambion mirVana kit (catalog number (Cat.No.) 1560) and suggested design (Ambion Inc., Atustin, TX), total serum IgE is isolated from Colorado potato bug (CPB), Leptinotarsa decemlineata.For every kind of preparation, Use the CPB larva for occupying about 200 μ L volumes in micro-centrifuge tube.Use Invitrogen Thermoscript RT-PCR system It unites (catalog number (Cat.No.) 11146) and recommends the scheme (nvitrogen, Carlsbad, CA) for mediating cDNA synthesis for random primer, use 5 microgram total serum IgEs prepare cDNA.The cDNA is used as template, to expand 2 ortholog sequence of V-ATPase A subunit, Wherein using Taq archaeal dna polymerase and oligonucleotide acid primer pr 550 (SEQ ID NO:160) and pr552 (SEQ ID NO: 161).By compare from Manduca sexta (SEQ ID NO:151), Aedes aegypti (SEQ ID NO:152), The nearest V- of Drosophila melanogaster (SEQ ID NO:153) and Diabrotica virgifera (WCR) ATPase A directly to autoploid nucleotide sequence, selects the region of minimum degeneracy, to design above-mentioned primer.Pr550 pairs of primer The position the 230-252 nucleotide in M.sexta gene order is answered, and primer pr552 corresponds to the 1354- in M.sexta gene order 1331 nucleotide.

It is expanded using (touchdown) amplification program is successively decreased, wherein using following loop parameters:

Step 1.94 DEG C, 2 minutes；

Step 2.94 DEG C, 30 seconds；

Step 3.50 DEG C, 2 minutes；

Step 4.72 DEG C, 2 minutes

(step 2-4 carries out 35 circulations, and each circulation is decremented to -0.3 DEG C in step 3)

Step 5.72 DEG C, 10 minutes；And

Step 6.4 DEG C.

The DNA fragmentation of the about 1.2kb expanded from cDNA is cloned into carrier pCR2.1-TOPO (Invitrogen), to generate recombinant plasmid pIC17105.It clones the nucleotide sequence (SEQ ID NO:144) being inserted into and comes from Western corn rootworm, the 2 ortholog sequence of V-ATPase A subunit of Diabrotica virgifera only share 82% Nucleotide sequence identity, still, deduction amino acid sequence about V-ATPase A albumen encoded but shares 97% Sequence identity.

Using primer pr568 (SEQ ID NO:162) and pr569 (SEQ ID NO:163), plasmid pIC17105 is amplified In V-ATPase A ortholog sequence, the primer is designed to " general " primer, for producing from pCR2.1-TOPO The DNA profiling of the raw T7 polymerase promoter sequence with flank.It expands obtained DNA and is used as template, closed for dsRNA At wherein using Ambion MEGAscript^TMKit (catalog number (Cat.No.) 1626) and suggested design (Ambion Inc., Austin, TX).It is purified by being obtained from L.decemlineata V-ATPase A orthologous sequence in insect feeding test DsRNA is fed to the larva of L.decemlineata.

CPB diet by 13.2g/L agar (Serva 11393), 140.3g/L Bio-Serve premix (F9380B), 5ml/L KOH (18.3%w/w) and 1.25ml/L formalin (37%) are constituted.Diet is dispersed into 96 with the aliquot of 200 μ L On orifice plate, briefly dried before application sample.20 μ L sample surveys are applied into each hole, use aqua sterilisa as unprocessed Verification (check) (UTC).Enable plate dry before insect larvae is added.One is added into each hole with fine, soft fur brush newly Raw CPB larva.The plate described in sealable polyester film, is ventilated with insect needle.40 larvas are examined in each processing.27 DEG C, culture in 10-12 days is carried out under 60%RH and complete darkness to biologic test plate.It is slow to these plates record larvae development And the death rate.It uses4 statistical softwares (SAS Institute, Cary, N.C., USA) analyze data.

Oral toxicity of the table 2.dsRNA to CPB larva

It, can be by harmful organism meals based on the oral toxicity biologic test for using CPB specificity V-ATPase dsRNA It is provided in food and expresses one or more following dsRNA sequences to control invasion of the CPB to plant, the dsRNA sequence is special Contain one or more genes in CPB harmful organism.

Embodiment 7

Present embodiment describes what is carried out on the artificial diet using insect specificity dsRNA to a variety of Lepidopteran larvaes The result of biologic test.

Using Ambion mirVana kit (catalog number (Cat.No.) 1560) and suggested design (Ambion Inc., Atustin, TX), from Spodoptera frugiperda, Helicoverpa zea, Agrotis ipsilon and Ostrinia The second age of nubilalis isolates total serum IgE into third instar larvae.It is big in micro-centrifuge tube using occupying for every kind of preparation The larva of about 200 μ L volumes.

Using Invitrogen Thermoscript RT-PCR system (catalog number (Cat.No.) 11146) and recommend to be used for random primer The scheme (nvitrogen, Carlsbad, CA) of cDNA synthesis is mediated, 5 micrograms with every kind in above-mentioned lepidoptera species are total RNA prepares cDNA.The cDNA is used as template, with amplifying specific in the V-ATPase A subunit 2 of every kind of lepidoptera species Ortholog sequence, wherein using Taq archaeal dna polymerase and oligonucleotide acid primer pr 550 (SEQ ID NO:160) and Pr552 (SEQ ID NO:161).

By compare from Manduca sexta, Aedes aegypti, Drosophila melanogaster and The nearest V-ATPase A of Diabrotica virgifera (WCR) selects minimum degeneracy directly to autoploid nucleotide sequence The region of property, to design above-mentioned primer.Primer pr550 corresponds to the position the 230-252 nucleotide in M.sexta gene order, and draws Object pr552 corresponds to the position the 1354-1331 nucleotide in M.sexta gene order.

It is expanded using loop parameter described in PCR scheme and embodiment 6 is successively decreased.The DNA that amplification is obtained is produced Object clones the phase same sex being sequenced into pCR2.1-TOPO to verify them.Recombination matter containing ortholog gene order Grain is listed in Table 3 below.

3. lepidoptera V-ATPase A subunit of table, 2 ortholog sequence

Using primer pr555 (SEQ ID:164) and pr556 (SEQ ID NO:156) amplify plasmid pIC17088, V-ATPase A ortholog sequence in pIC17101, pIC17102, pIC17102, the primer are designed to: being generated Flank reversely has the DNA fragmentation of T7 polymerase promoter, synthesizes for external dsRNA.

Using Ambion MEGAscriptTM kit (catalog number (Cat.No.) 1626) and suggested design (Ambion Inc., Austin, TX), the double-strand for FAW, BCW and CEW ortholog sequence is synthesized from these obtained DNA profilings of amplification It is used for insect biologic test with 10ppm by RNA (dsRNA).

For these tests, artificial lepidoptera diet (165g/L Southland Multiple Species is prepared Diet, 14.48g/L agar), it is dispersed in 128 orifice plates, each 500 μ l of hole.By in sample dispersion to diet, it is placed in In " drying " chamber of 27 DEG C and 35% humidity, wherein excess water evaporation.Once just being gone to invade with single newborn larvae by drying Each hole is attacked, is sealed with the sealable polyester film item of perforation.Culture in six to eight days is carried out to the plate at 27 DEG C.Do not located The insect of reason ran out of whole diets in each hole at six to eight days.50 holes are prepared with every hole 4ml artificial diet All insects that diet is exhausted or almost exhausted before off-test are transferred in new plate by plate.These plates are sealed, it will They are put back into incubator, are evaluated and tested after in total ten to 12 days, then to all biologic tests.

Compared with unprocessed verification, in terms of larval mortality or quality acquisition, lepidopterid is directed to from these The result of the biologic test of species does not show that obvious action (is compared using Tukey-Kramer HSD for all Duis Compared with), using the test, it observed growth (regimen).BT insecticidal egg is being formed using the hole of dsRNA and sub-lethal dose In the biologic test that white combination (known toxic to above-mentioned lepidoptera harmful organism in pervious experiment) carries out, also do not see Observe the influence to larval mortality and quality acquisition.

Embodiment 8

Present embodiment describes for measuring dsRNA to boll weevil, the oral toxicity of Anthonomus grandis Biologic test.

Using Ambion mirVana kit (catalog number (Cat.No.) 1560) and suggested design (Ambion Inc., Atustin, TX), from boll weevil (BWV), the larva of Anthonomus grandis isolates total serum IgE.For every kind of preparation, using accounting for According to the BWV larva of about 200 μ L volumes in micro-centrifuge tube.Use Invitrogen Thermoscript RT-PCR system (mesh Record number is 11146) and recommendation is used for the scheme (nvitrogen, Carlsbad, CA) that random primer mediates cDNA synthesis, with 5 micrograms Total serum IgE prepares cDNA.The cDNA is used as template, to expand 2 ortholog sequence of V-ATPase A subunit, wherein making With Taq archaeal dna polymerase and oligonucleotide acid primer pr 550 (SEQ ID NO:160) and pr552 (SEQ ID NO:161).

By compare from Manduca sexta, Aedes aegypti, Drosophila melanogaster and The nearest V-ATPase A of Diabrotica virgifera (WCR) selects minimum degeneracy directly to autoploid nucleotide sequence The region of property, to design above-mentioned primer.Primer pr550 corresponds to the position the 230-252 nucleotide in M.sexta gene order, and draws Object pr552 corresponds to the position the 1354-1331 nucleotide in M.sexta gene order.

It is expanded using loop parameter described in PCR scheme and embodiment 6 is successively decreased.It will expand to obtain from cDNA The DNA fragmentation of about 1.2kb clone into pCR2.1-TOPO (Invitrogen), insertion is sequenced, to be verified.It uses Primer pr568 (SEQ ID:162) and pr569 (SEQ ID NO:163) amplify V-ATPase A ortholog sequence (SEQ ID NO:149), the primer is designed to " general " primer, for generating from pCR2.1-TOPO there is the T7 of flank to polymerize The DNA profiling of enzyme promoters sequence.

Using Ambion MEGAscriptTM kit (catalog number (Cat.No.) 1626) and suggested design (Ambion Inc., Austin, TX), the DNA profiling obtained from the amplification synthesizes double-stranded RNA (dsRNA).

To carry out boll weevil, the biologic test of Anthonomtus grandis Boheman illustrates according to manufacturer Book uses artificial insect's diet (Bioserv-F9247B based on agar；Gast and Davich, 1966).By about 200 μ The thawing diet of l is dispersed into 96-well microtiter plate, enables its cooling and solidifying.Then it will contain about the sample (20 of 10ppm dsRNA μ l) cover on diet, enable its drying, the dsRNA corresponding to V-ATPase A identical sources object sequence (SEQ ID NO: 149).Then the insect ovum (0-14) in 25 μ l0.1% agar is distributed on diet.Then aperture seal item (Zymark# is used 72281) sealing titer plate is gone.Culture in ten to 12 days is carried out to test at 27 DEG C, by the accumulation of measurement larva excrement come Assessment activity.Be not observed to larval mortality or quality acquisition influence, but this may be boll weevil it is specific into Eat the result of physiology.The dosage of the dsRNA of intake may be significantly reduced by digging a hole in diet, therefore significantly reduce use Feed any effect that can be observed when physiology in surface.It include in an uniform way that will likely be obtained significantly into mountain city by dsRNA The death rate and reduction quality obtain.

Other target gene sequences from boll weevil can be cloned, and be used as template, for synthesizing in vitro Then dsRNA can examine dsRNA in insect biologic test, to evaluate their effect.For example, ribosomal protein L19 (rpl19) gene is used as the template to synthesize for dsRNA.For design degeneracy oligonucleotide primer purpose, to come From Bombyx mori (SEQ ID NO:154), Drosophila melanogaster (SEQ ID NO:155), Anopholes Gambiae's (SEQ ID NO:156) and Diabrotica virgifera (SEQ ID NO:157) is directed to rpl19 directly to same The nucleotide sequence of source object is compared, and identifies the shared region of minimum degeneracy.Primer pr574 (SEQ ID NO:166) With pr577 (SEQ ID NO:168) or primer pr575 (SEQ ID NO:167) and pr577 (SEQ ID NO:168) can by with In amplifying possible rpl19 ortholog sequence from a variety of different insects species.

It is expanded using amplification scheme and loop parameter as described in Example 6 is successively decreased.It will be from boll weevil The DNA fragmentation for the about 0.4kb that cDNA is expanded is cloned into carrier pCR2.1-POTO (Invitrogen), and insertion is sequenced, To be verified.The straight Xiang Tongyuan of rpl79 is amplified using primer pr568 (SEQ ID:162) and pr569 (SEQ ID NO:163) Object sequence (SEQ ID NO:149), the primer is designed to " general " primer, has for generating from pCR2.1-TOPO The DNA profiling of the T7 polymerase promoter sequence of flank.

Embodiment 9

Present embodiment describes for measuring dsRNA to red flour beetle, the oral poison of Tribolium castaneum larva The biologic test of property.

Some harmful insects be commercially it is especially important because they can invade the agricultural product system from specific crop production Product and the material by processing.A kind of special such harmful organism is red flour beetle.In the agricultural production from specific crop production Exist in product product and material by processing be specific to inhibit in such harmful organism one kind of one or more genes or A variety of dsRNA types will be used to control such pest infestation.

Using Ambion mir Vana kit (catalog number (Cat.No.) 1560) and suggested design (Ambion Inc., Atustin, TX), from red flour beetle (RFB), the larva of Tribolium castaneum isolates total serum IgE.For every kind of preparation, using accounting for According to the RFB larva of about 200 μ L volumes in micro-centrifuge tube.Use Invitrogen Thermoscript RT-PCR system (mesh Record number is 11146) and recommendation is used for the scheme (nvitrogen, Carlsbad, CA) that random primer mediates cDNA synthesis, with 5 micrograms Total serum IgE prepares cDNA.The cDNA is used as template, to expand 2 ortholog sequence of V-ATPase A subunit, wherein making With Taq archaeal dna polymerase and oligonucleotide acid primer pr 550 (SEQ ID NO:160) and pr552 (SEQ ID NO:161).

By compare from Manduca sexta, Aedes aegypti, Drosophila melanogaster and The nearest V-ATPase A of Diabrotica virgifera (WCR) selects minimum degeneracy directly to autoploid nucleotide sequence The region of property, to design above-mentioned primer.Primer pr550 corresponds to the position the 230-252 nucleotide in M.sexta gene order, and draws Object pr552 corresponds to the position the 1354-1331 nucleotide in M.sexta gene order.

It is expanded using loop parameter described in PCR scheme and embodiment 6 is successively decreased.It will expand to obtain from cDNA The DNA fragmentation of about 1.2kb clone into pCR2.1-TOPO (Invitrogen), insertion is sequenced, to be verified.It uses Primer pr568 (SEQ ID:162) and pr569 (SEQ ID NO:163) amplify V-ATPase A ortholog sequence (SEQ ID NO:150), the primer is designed to " general " primer, for generating from pCR2.1-TOPO there is the T7 of flank to polymerize The DNA profiling of enzyme promoters sequence.

Using Ambion MEGAscriptTM kit (catalog number (Cat.No.) 1626) and suggested design (Ambion Inc., Austin, TX), the DNA profiling obtained from the amplification synthesizes double-stranded RNA (dsRNA), is used for insect biologic test.It will Wheat flour and water and the dsRNA homogeneous mixture for corresponding to V-ATPase A ortholog sequence (SEQ ID NO:150), Its drying is enabled, composition is used as the substrate of red flour beetle biologic test.After a couple of days culture, by from flour/dsRNA Weevil larva is extracted in mixture to observe insecticidal effect.

Other target gene sequences from red flour beetle can be cloned, and be used as template, for synthesizing in vitro Then dsRNA can examine dsRNA in insect biologic test, to evaluate their effect.For example, ribosomal protein L19 (rpl19) gene is used as the template to synthesize for dsRNA.For design degeneracy oligonucleotide primer purpose, to come From Bombyx mori, Drosophila melanogaster, Anopholes gambiae and Diabrotica virgifera The nucleotide sequence for rpl19 ortholog be compared, identify the shared region of minimum degeneracy.Primer Pr574 (SEQ ID NO:166) and pr577 (SEQ ID NO:168) or primer pr575 (SEQ ID NO:167) and pr577 (SEQ ID NO:168) can be used for amplifying possible rpl19 ortholog sequence from a variety of different insects species.

It is expanded using amplification scheme and loop parameter as described in Example 6 is successively decreased.It will be from red flour beetle The DNA fragmentation for the about 0.4kb that cDNA is expanded is cloned into carrier pCR2.1-POTO (Invitrogen), and insertion is sequenced, To be verified.The straight Xiang Tongyuan of rpl19 is amplified using primer pr568 (SEQ ID:162) and pr569 (SEQ ID NO:163) Object sequence (SEQ ID NO:159), the primer is designed to " general " primer, has for generating from pCR2.1-TOPO The DNA profiling of the T7 polymerase promoter sequence of flank.

Embodiment 10

Present embodiment describes the mouths for measuring dsRNA dialogue grub (white grub) and iron wire worm (wireworm) The biologic test of taking poison property.

Using Ambion mir Vana kit (catalog number (Cat.No.) 1560) and suggested design (Ambion Inc., Atustin, TX), total serum IgE is isolated from the larva of white grub or iron wire worm.For every kind of preparation, using occupying about 200 μ in micro-centrifuge tube The larva of L volume.Using Invitrogen Thermoscript RT-PCR system (catalog number (Cat.No.) 11146) and recommend for random Primer mediates the scheme (nvitrogen, Carlsbad, CA) of cDNA synthesis, prepares cDNA with 5 microgram total serum IgEs.The cDNA quilt It is used as template, to expand 2 ortholog sequence of V-ATPase A subunit, wherein using Taq archaeal dna polymerase and core of living alone as a widow Thuja acid primer pr 550 (SEQ ID NO:160) and pr552 (SEQ ID NO:161).

By compare from Manduca sexta, Aedes aegypti, Drosophila melanogaster and The nearest V-ATPase A of Diabrotica virgifera (WCR) selects minimum degeneracy directly to autoploid nucleotide sequence The region of property, to design above-mentioned primer.Primer pr550 corresponds to the position the 230-252 nucleotide in M.sexta gene order, and draws Object pr552 corresponds to the position the 1354-1331 nucleotide in M.sexta gene order.

It is expanded using loop parameter described in PCR scheme and embodiment 7 is successively decreased.It will expand to obtain from cDNA The DNA fragmentation of about 1.2kb clone into pCR2.1-TOPO (Invitrogen), insertion is sequenced, to be verified.It uses Primer pr568 (SEQ ID:162) and pr569 (SEQ ID NO:163) amplify V-ATPase A ortholog sequence (SEQ ID NO:150), the primer is designed to " general " primer, for generating from pCR2.1-TOPO there is the T7 of flank to polymerize The DNA profiling of enzyme promoters sequence.

Using Ambion MEGAscriptTM kit (catalog number (Cat.No.) 1626) and suggested design (Ambion Inc., Austin, TX), the DNA profiling obtained from the amplification synthesizes double-stranded RNA (dsRNA), is used for insect biologic test.Number After its biologic test, the effect of oral toxicity is observed.

It can be cloned come other target gene sequences of make clear one's meaning and position grub or iron wire worm, and be used as template, for external DsRNA is synthesized, then dsRNA can be examined in insect biologic test, to evaluate their effect.For example, ribosomes Protein L 19 (rpl19) gene is used as the template to synthesize for dsRNA.For design degeneracy oligonucleotide primer purpose, To from Bombyx mori, Drosophila melanogaster, Anopholes gambiae and Diabrotica The nucleotide sequence for rpl19 ortholog of virgifera is compared, and identifies the consensus of minimum degeneracy Domain.Primer pr574 and pr577 or primer pr575 and pr577 can be used for amplifying possibility from a variety of different insects species Rpl19 ortholog sequence.

It is expanded using amplification scheme and loop parameter as described in Example 7 is successively decreased.It will be from red flour beetle The DNA fragmentation for the about 0.4kb that cDNA is expanded is cloned into carrier pCR2.1-POTO (Invitrogen), and insertion is sequenced, To be verified.The straight Xiang Tongyuan of rpl19 is amplified using primer pr568 (SEQ ID:162) and pr569 (SEQ ID NO:163) Object sequence, the primer are designed to " general " primer, for generating from pCR2.1-TOPO there is the T7 polymerase of flank to open The DNA profiling of promoter sequences.

Embodiment 11

Present embodiment describes for measuring dsRNA to mosquito, the biologic test of the oral toxicity of Aedes aegypti.

Using Ambion mir Vana kit (catalog number (Cat.No.) 1560) and suggested design (Ambion Inc., Atustin, TX), total serum IgE is isolated from the larva of Aedes aegypti.For every kind of preparation, using occupying about 200 μ L in micro-centrifuge tube The Aedes aegypti larva of volume.Using Invitrogen Thermoscript RT-PCR system (catalog number (Cat.No.) 11146) and Recommend the scheme (nvitrogen, Carlsbad, CA) for mediating cDNA synthesis for random primer, is prepared with 5 microgram total serum IgEs cDNA.The cDNA is used as template, to expand 2 ortholog sequence of V-ATPaseA subunit, wherein poly- using Taq DNA Synthase and oligonucleotide acid primer pr 550 (SEQ ID NO:160) and pr552 (SEQ ID NO:161).

By compare from Manduca sexta, Aedes aegypti, Drosophila melanogaster and The nearest V-ATPase A of Diabrotica virgifera (WCR) selects minimum degeneracy directly to autoploid nucleotide sequence The region of property, to design above-mentioned primer.Primer pr550 corresponds to the position the 230-252 nucleotide in M.sexta gene order, and draws Object pr552 corresponds to the position the 1354-1331 nucleotide in M.sexta gene order.

It is expanded using loop parameter described in PCR scheme and embodiment 7 is successively decreased.It will expand to obtain from cDNA The DNA fragmentation of about 1.2kb clone into pCR2.1-TOPO (Invitrogen), insertion is sequenced, to be verified.It uses Primer pr568 (SEQ ID:162) and pr569 (SEQ ID NO:163) amplify V-ATPase A ortholog sequence (SEQ ID NO:150), the primer is designed to " general " primer, for generating from pCR2.1-TOPO there is the T7 of flank to polymerize The DNA profiling of enzyme promoters sequence.

Using Ambion MEGAscriptTM kit (catalog number (Cat.No.) 1626) and suggested design (Ambion Inc., Austin, TX), the DNA profiling obtained from the amplification synthesizes double-stranded RNA (dsRNA), is used for insect biologic test.Number After its biologic test, the effect to insect larvae is observed.

Other target gene sequences from mosquito can be cloned, and be used as template, for synthesizing dsRNA in vitro, so After dsRNA can be examined in insect biologic test, to evaluate their effect.For example, ribosomal protein L 19 (rpl19) template that gene is used as to synthesize for dsRNA.For the purpose for designing degeneracy oligonucleotide primer, to coming from Bombyx mori, Drosophila melanogaster, Anopholes gambiae and Diabrotica virgifera It is compared for the nucleotide sequence of rpl19 ortholog, identifies the shared region of minimum degeneracy.Primer pr574 With pr577 or primer pr575 and pr577 can be used for amplifying from a variety of different insects species possible rpl19 directly to Homologue sequence.

It is expanded using amplification scheme and loop parameter as described in Example 7 is successively decreased.It will be expanded from cDNA To the DNA fragmentation of about 0.4kb clone into carrier pCR2.1-POTO (Invitrogen), insertion is sequenced, to be tested Card.Rpl19 ortholog sequence is amplified using primer pr568 (SEQ ID:162) and pr569 (SEQ ID NO:163), The primer is designed to " general " primer, for generating the T7 polymerase promoter sequence with flank from pCR2.1-TOPO The DNA profiling of column.

Using Ambion MEGAscriptTM kit (catalog number (Cat.No.) 1626) and suggested design (Ambion Inc., Austin, TX), the DNA profiling obtained from the amplification synthesizes double-stranded RNA (dsRNA), is used for insect biologic test.

Other mosquito class species can be included in the scope of the present invention.Suitable Oligonucleolide primers can be used, to coming from The suitable targets gene order of Aedes, Culex and Anopholes species is expanded, and is cloned into carrier pCR2.1- POTO (Invitrogen) is sequenced insertion, to be verified.Use primer pr568 (SEQ ID:162) and pr569 (SEQ ID NO:163) clone's target sequence is amplified, the primer is designed to " general " primer, for producing from pCR2.1-TOPO The DNA profiling of the raw T7 polymerase promoter sequence with flank.

Using Ambion MEGAscriptTM kit (catalog number (Cat.No.) 1626) and suggested design (Ambion Inc., Austin, TX), double-stranded RNA (dsRNA) is synthesized from these obtained DNA profilings of amplification, is used for insect biologic test.

Embodiment 12

Present embodiment describes: dsRNA made from the 3 ' UTR regions from V-ATPase is the tune how shown to target Control.

The segment (dsRNA of ca.300bp) of 3 ' UTR of WCR V-ATPase has been used for carrying out WCR biologic test, still But fail to show hypoevolutism and the death rate in 12 days biologic tests.Sizable in V-ATPase coding region It is disconnected that significant hypoevolutism and the death rate are but illustrated within the scope of a certain concentration.To from feeding 3 ' UTR of WCR V-ATPase The total serum IgE that segment 4 days WCR larvas are extracted carries out the hybridization of the Northern marking (and going to be detected with coding region probe) Display: compared with unprocessed larva, V-ATPase target RNA significantly reduces (being summarised as NBP#7497215).But it leaves The signal that can be detected, this shows with 3 ' UTR dsRNA segments (compared with coding region segment is used) to the knockout of target It is more invalid, and/or the contribution from possible second of V-ATPase gene, second of gene has and the first V- Visibly different 3 ' the UTR of ATPase gene of MP.Exist with genome more than one to the Southern marking hybridization data that WCR is carried out Kind of hybrid gene sequence it is identical of views, but inspection to EST and there is the family PCR of limitation not show possible the but Two kinds of genes are transcribed.

Want herein it is important to note that, although it is crucial for determining that larvae development of sening as an envoy to is slow and kills the potential target of larva , but hybridized by the Northern marking or quantitative PCR carries out easy detection to the expression of target gene can also find transport With the target of RNAi strategy.The above results add be conceived to V-ATPase target other Northern experiment it has been shown that In insect, in a few hours existing for dsRNA, influence of the RNA for Transcript abundance be can be seen that.

Embodiment 13

Present embodiment describes a kind of methods, and silencing methods for being mediated using ta-siRNA are executed to harmful insect The containment of gene.

Used the method for gene silencing in the biology to plant pest it has recently found that trans-acting siRNA (ta-siRNA) class group (Dalmay et al., Cell 101:543-553,2000；Mourrain et al., Cell 101: 533-542,2000；Peragine et al, Genes and Development, 18:2368-2379,2004；Vazquez Et al, MoI Cell 16 (l): 69-79,2004；Yu et al., Mol Plant Microbe Interact 16:206- 216,2003).Ta-siRNA is that the single stranded RNA transcript targeted from naturally occurring miRNA obtains.Using Microrna with It induces method of the ta-siRNA for carrying out gene silencing in plant and is described in U.S. Provisional Patent Application Serial No. 60/ In 643,136 (Carrington et al.2004), it is incorporated herein by reference in their entirety.At least one is identified in jade The harmful organism specificity miRNA expressed in rice rootworm larvae intestinal epithelial cell.Then with harmful organism specificity miRNA Go to identify: the miRNA expressed in this sequence of at least one target rna transcription, the sequence and cell is complementary.Corresponding target Sequence is no more than the short sequence of 21 contiguous nucleotides, in the cell type with functional r NAi approach, rna transcription When this part of corresponding miRNA is contacted, the short sequence leads to the cutting to the transcript of silencer mediation. Once identifying miRNA target sequence, at least one miRNA target sequence is merged with Article 2 sequence, the Article 2 Sequence corresponds to a part for the pest gene that this method will be used to carry out silencing.For example, by miRNA target sequence and jade The sequence fusion of rice rootworm vacuole ATPase (V-ATPase) gene.MiRNA target sequence can be placed on V-ATPase gene 5 ' end, 3 ' end or be embedded among the gene.Using a variety of miRNA target sequences for corresponding to a variety of miR-96 genes, or Same miRNA target sequence use is used for multiple times in miRNA target sequence and the chimera of V-ATPase sequence may be Preferably.V-ATPase sequence can be random length, but minimum 21bp.

Using any substance in a large amount of suitable promoters or other transcriptional regulatory elements, by miRNA target sequence and The chimera of v-ATPase is expressed in plant cell, at least will be provided in harmful organism diet cell type (for example, For controlling the corn root of corn rootworm) in can transcribe.

This method can have additional advantage: longer RNA molecule can be transported to object noxious livings.Typically, exist The dsRNA generated in plant can be short rna by Dicer rapid processing, when being fed to some harmful organisms in a manner of external source, this It may not use.In the method, single-stranded transcript is generated in plant cell, is absorbed by harmful organism, in harmful organism DsRNA is converted into cell, in cell, then being processed into can be to one of one or more harmful organisms or a variety of base The ta-siRNA of silencing after being transcribed because caused by.

Embodiment 14

Present embodiment describes compared with the sequence in other sources from non-CRW and right to CRW cDNA sequence (1) identification of the sequence as the sequence in other sources and the exclusive sequence of (2) CRW.It is guarded in two kinds of biologies CDNA sequence is potential RNAi candidate sequence, can be used for the expression and function of genes for targeting both biologies.Alternatively, choosing Following sequences for the containment of CRW gene may be what people needed out, in (a) other harmful organisms, (b) nontarget organism with And (c) it is chosen to be contained in Sequence Transformed Plant Genome with CRW exist without the known homologous sequence of the sequence.

Six CRW cDNA sequences are selected, are compared for the sequence in other sources.Specific sequence includes coding Alpha- tubulin, beta- tubulin, CHD3, vacuolar proton pump E subunit, V-ATPase A subunit and silk-fibroin The sequence of (thread protein).Nucleotide sequence is shown in SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO: 100, SEQ ID NO:101, SEQ ID NO:102, in SEQ ID NO:103.Use NCBI megablast program (Altschul et al., J.Mol.Biol.215:403-410,1990), by CRW cDNA sequence and from the more of GenBank All open cDNA of kind biology are compared, and search parameter is as follows:

-W21-b50-v50

This needs the perfect matching of at least 21 bodies (21-mer), only retains best 50 matchings and alignment.To result into Row filtering only includes belonging to InsectaMeshBiology, and honeybee (Apis mellifera) is left out.Although only existing It is analyzed in six CRW cDNA sequences, but same method can be used for cDNA all in CRW or other target organisms Or unigene sequence, without having excessive burden and experiment.

Using six CRW cDNA sequences, 145 kinds of matchings in total are identified in the insect biology different from 20 kinds.They Including several pests, for example, acyrthosiphum pisim (Acyrthosiphon pisum), Asia diaphorina citri (Diaphorina ) and pediculus humanus capitis (Pediculus humanus) citri.

These results are showed in the following table 4, which show search sequence with hit matching between sequence, match percentage Than the insect species that the phase same sex and acquisition hit sequence.For example, identifying, from the in SEQ ID NO:98 the 844th to 1528 In the segment of position nucleotide and the GenBank sequence number GI:47521748 from acyrthosiphum pisim (Acyrthosiphon pisum) The segment height of 812nd to 128 nucleotide is identical.The phase same sex of this two sequences shared about 85%.

Embodiment 15

Present embodiment describes: using the matching for sharing model with known array and existing structural domain, to public herein The protein functional structural domain and gene family of the nucleotide sequence opened translated to identify prediction.

Protein sequence is generated with " translater " program first, Unigene is translated as titanium sequence by described program, this is to pass through What following step carried out: the homology with known albumen；The prediction of gene structure from the beginning started based on model；And it is longest Opening code-reading frame (ORF).Correction is as being sequenced caused by mistake position of frameing shift.Then for Pfam database, (covering is many common The big collection of Hidden Markov model (HMM) and a variety of sequence alignments of protein family) search for protein sequence (The Pfam Protein Families Database, Bateman et al., Nucleic Acids Research 32:D138-D141, 2004).Using default rigor, with program HMMPAM (Durbin et al., Biological Sequence Analysis: Probabilistic Models of Proteins and Nucleic Acids, Cambridge University Press, 1998) albumen HMM model is searched for.Further filtered, only retain desired value be 0.1 or smaller those With as significant matching.In 20303 corn rootworm peptide sequences, identified with 1317 kinds of different protein structure domains and family 4199 (21%) items.

Analysis result is showed in region feature (feature) of sequence table file, with following characteristics: Pfam, Pfam description and the quantity copied with structural domain in the matching level, desired value (E- value) and peptide sequence of HMMPFAM score.

Embodiment 16

Present embodiment describes a kind of methods, for providing the DNA sequence dna for being used for the gene silencing that dsRNA is mediated.More Body, present embodiment describes the selections to the improved DNA in the gene silencing that can be used for dsRNA mediation, this is under It states step to realize: (a) selecting initial DNA sequence dna from target gene comprising the contiguous nucleotide more than 21；(b) Identify at least one shorter DNA sequence dna, the region from initial dna sequence, by be predicted to be do not generate it is undesired The region of polypeptide is constituted；And (c) select the DNA sequence dna of the gene silencing for dsRNA mediation comprising at least one is shorter DNA sequence dna.Undesired polypeptide includes but is not limited to, with the polypeptide of sensitization homologous peptide and with known polypeptide toxin Homologous polypeptide.

WCR V-ATPase has shown that out: can play a role in corn rootworm feeding test, to examine dsRNA to mediate Silencing (means as control larva growth).From Western corn rootworm (WCR) (Diabrotica cirgifera Virgifera LeConte) the cDNA sequence of vacuole ATPase gene of MP (V-ATPase) be selected, be used as initial DNA Sequence (SEQ ID NO:104).It is screened for following regions DNA sequence dna initial to this, in this region, each Connected segments comprising at least 21 nucleotide only in 21 contiguous nucleotides in known vertebrate sequence less than 21 Matching.It identifies three and is more than the segment of about 100 contiguous nucleotides, wherein being hit without above-mentioned 21/21；First sequence The disconnected position the 739-839 nucleotide corresponding in SEQ ID NO:104 of column-slice, Article 2 sequence fragment correspond to SEQ ID NO: The position 849-987 nucleotide in 104, Article 3 sequence fragment correspond to the position the 998-1166 nucleotide in SEQ ID NO:104. This three sequences are combined, are constructed gomphosis DNA array (SEQ ID NO:1), for mutually deserved CRW V-ATPase coded sequence Carry out the gene silencing of dsRNA mediation.Novel gomphosis DNA array is examined in CRW biologic test described above.

All disclosures, patent and the disclosed patent application mentioned in this specification all pass through reference and are incorporated into this Text, this with particularly, individually statement it is individually open herein or patent is incorporated herein by reference by every kind.

Sequence table

<110>Monsanto Technology LLC

J. A. Bao nurse

L. A. gilbert is gloomy

D. K. Kovalick

T. J. La Luosa

M. Lu

T. R. I. Meng Yikewa

J. K. Luo Baici

W. Wu

B. it opens

<120>for controlling the composition and method of insect infestations in plant

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<160> 174

<210> 1

<211> 409

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 1

ggacaagaaa cttgcccaac gtaagcactt cccttcagta gactggcttg gatcatattc 60

caaatattta agagcattgg acgactttta tgacaaaaac tttattcctc ttagaaccaa 120

agttaaggaa attcttcagg aagaagatga tctagccgaa attgtgcagc tggtaggtaa 180

agcatctctg gcagaaacgg acaaaatcac cttggaaatt gccaggcttc ttaaagaaga 240

caaaactcat actcttctta tgacagattc tgtccattct ataaaactgt cggtatgttg 300

agaaacatga tcggtttgta cgacatggcg agacacgctg tagaatcaac cgcacaatca 360

gaaaataaga tcacttggaa cgtaataaga gattcaatga gtggaattt 409

<210> 2

<211> 157

<212> DNA

<213> Diabrotica virgifera

<400> 2

atgtttcagg tgggctcaat aagcaccaac tttcaatttt atttttcatt tttgtattta 60

tttacagtaa ctcctcagtt tgctaacaat attacattgt taacgcattc atatgttgtt 120

taatataata gttttggaat ataattacaa gtttgtc 157

<210> 3

<211> 338

<212> DNA

<213> Diabrotica virgifera

<400> 3

atttttattc tgttaatagt ttttcacatt tcatgtttca cacatactta gatctagtca 60

agattgttag agttttggca aagaaattaa ataaaaattc ttttcataaa aatcatttct 120

ttaatattac attagagaaa aattatattt ttatactgag tacaaatttg aacaagttat 180

taattttaag ttacaaaata cgcttttata ggttaacaat tatcaaagcg cttaaatcta 240

atagatacta cacaacatta aggactgcaa accatatctt tcacgaagta atccctacta 300

gtgaccaatt gctcgctagg agcagatgca aattacac 338

<210> 4

<211> 458

<212> DNA

<213> Diabrotica virgifera

<400> 4

aaaagagtga ggaaacaggt taattataat gacggaggaa tgacaactga cacacgagaa 60

gatacgacat ggcaagaaaa tctctctgat taccattctg acttttctgc gggatcggat 120

gaggataagg aagacgatga tttcgatgag aagaacgacg ccgatttaag cagaaggagt 180

cgaagaaaga tggaaaggaa agacgagaag gatcgtcctt taccaccgtt actagccaga 240

gttggcggca atattgaagt actcggtttt aatgccaggc agcgtaaagc gttccttaat 300

gctattatgc gctacggaat gccaccacaa gacgctttca attcacagtg gctggtgaga 360

gatcttcgag gaaaatctga gaagatattc aaggcttacg tgtctctctt tatgaggcat 420

ctttgcgaac ctggtgcaga taatgctgat acgtttgc 458

<210> 5

<211> 45

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 5

taatacgact cactataggg agagacggag gaatgacaac tgaca 45

<210> 6

<211> 44

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 6

taatacgact cactataggg agattccgta gcgcataata gcat 44

<210> 7

<211> 335

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 7

taatacgact cactataggg agagacggag gaatgacaac tgacacacga gaagacacga 60

catggcaaga aaatctctcc gattaccatt ctgacttttc tgcgggatca gatgaggata 120

aggaagacga tgatttcgat gagaagaacg acgccgattt aagcagaaga agtcgaagaa 180

agatggaaag aaaagacgag aaggaccgtc cactaccacc gttactagcc agagttgggg 240

gaaatattga agtgctcggt tttaatgcca ggcagcgtaa agcgttcctt aatgctatta 300

tgcgctacgg aatctcccta tagtgagtcg tatta 335

<210> 8

<211> 29

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 8

tctgaattct ccgtagcgca taatagcat 29

<210> 9

<211> 21

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 9

gacgccgatt taagcagaag a 21

<210> 10

<211> 171

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 10

tctgaattct ccgtagcgca taatagcatt aaggaacgct ttacgctgcc tggcattaaa 60

accgagcact tcaatatttc ccccaactct ggctagtaac ggtggtagtg gacggtcctt 120

ctcgtctttt ctttccatct ttcttcgact tcttctgctt aaatcggcgt c 171

<210> 11

<211> 45

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 11

tcttctgctt aaatcggcgt cgaagacacg acatggcaag aaaat 45

<210> 12

<211> 32

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 12

gatcccgcag aaaagtcaga atggtaatcg ga 32

<210> 13

<211> 112

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 13

tcttctgctt aaatcggcgt cgaagacacg acatggcaag aaaatctctc cgattaccat 60

tctgactttt ctgcgggatc tccgattacc attctgactt ttctgcggga tc 112

<210> 14

<211> 24

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 14

ctccgattac cattctgact tttc 24

<210> 15

<211> 30

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 15

tctggatcct tccgtagcgc ataatagcat 30

<210> 16

<211> 245

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 16

ctccgattac cattctgact tttctgcggg atcagatgag gataaggaag acgatgattt 60

cgatgagaag aacgacgccg atttaagcag aagaagtcga agaaagatgg aaagaaaaga 120

cgagaaggac cgtccactac caccgttact agccagagtt gggggaaata ttgaagtgct 180

cggttttaat gccaggcagc gtaaagcgtt ccttaatgct attatgcgct acggaaggat 240

ccaga 245

<210> 17

<211> 473

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 17

tctgaattct ccgtagcgca taatagcatt aaggaacgct ttacgctgcc tggcattaaa 60

accgagcact tcaatatttc ccccaactct ggctagtaac ggtggtagtg gacggtcctt 120

ctcgtctttt ctttccatct ttcttcgact tcttctgctt aaatcggcgt cgaagacacg 180

acatggcaag aaaatctctc cgattaccat tctgactttt ctgcgggatc tccgattacc 240

attctgactt ttctgcggga tcagatgagg ataaggaaga cgatgatttc gatgagaaga 300

acgacgccga tttaagcaga agaagtcgaa gaaagatgga aagaaaagac gagaaggacc 360

gtccactacc accgttacta gccagagttg ggggaaatat tgaagtgctc gttttaatgc 420

caggcagcgt aaagcgttcc ttaatgctat tatgcgctac ggaaggatcc aga 473

<210> 18

<211> 536

<212> DNA

<213> Diabrotica virgifera

<400> 18

accgccatca tgttattggc atcacacatg tgctgtgtga gctctggaac tttcaacggt 60

ctgtattgtt ggctgcctct tgaggtgagt ggagcgaatc cgggcatgaa gaagtggaga 120

cgggggaagg gaaccatgtt gacagccaat tttctaagat cagcattcaa ctgacctggg 180

aacctaagac aggtggttac accggacatt gtgagggata ccaaatggtt taagtctcca 240

tatgtgggtg ttgtgagttt caaagttctg aagcaaatgt catagagagc ttcattatca 300

atacagtatg tttcatctgt gttttctacc aattgatgta ctgaaagtgt ggcattgtat 360

ggttctacta cggtatctga tactttgggt gaggggacta ctgagtatgt gttcataatt 420

ctgtctgggt attcttcacg gatttttgag atagggaggg tacccatacc tgatccagta 480

ccacctccaa gtgagtgtgt gagttggaat ccttgtaaac aatcacatga tcagct 536

<210> 19

<211> 44

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 19

taatacgact cactataggg agagaatccg ggcatgaaga agtg 44

<210> 20

<211> 44

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 20

taatacgact cactataggg agacaaaaat ccgtgaagaa tacc 44

<210> 21

<211> 399

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 21

taatacgact cactataggg agagaatccg ggcatgaaga agtggagacg ggggaaggga 60

accatgttga cagccaattt tctaagatca gcattcaact gacctgggaa cctaagacag 120

gtggttacac cggacattgt gagggatacc aaatggttta agtctccata tgtgggtgtt 180

gtgagtttca aagttctgaa gcaaatgtca tagagagctt cattatcaat acagtatgtt 240

tcatctgtgt tttctaccaa ttgatgtact gaaagtgtgg cattgtatgg ttctactacg 300

gtatctgata ctttgggtga ggggactact gagtatgtgt tcataattct gtctgggtat 360

tcttcacgga tttttgtctc cctatagtga gtcgtatta 399

<210> 22

<211> 26

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 22

actgaattct ccgggcatga agaagt 26

<210> 23

<211> 19

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 23

tattcttcac ggatttgac 19

<210> 24

<211> 267

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 24

actgaattct ccgggcatga agaagtggag acgggggaag ggaaccatgt tgacagccaa 60

ttttctaaga tcagcattca actgacctgg gaacctaagg caggtggtta caccggacat 120

tgtgagggat accaaatggt ttaagtctcc gtatgtgggt gttgtgagtt tcaaagttct 180

gaagcaaatg tcatagagag cttcattatc aatacagtat gtttcatctg tgttttctac 240

caattgatgt caaatccgtg aagaata 267

<210> 25

<211> 17

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 25

caaatccgtg aagaata 17

<210> 26

<211> 19

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 26

ctgaaagtgt ggcgttgta 19

<210> 27

<211> 106

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 27

caaatccgtg aagaataccc agatagaatt atgaacacat actcagtagt cccctctccc 60

aaagtatcag ataccgtagt agaaccatac aacgccacac tttcag 106

<210> 28

<211> 22

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 28

tagaaccata caacgccaca ct 22

<210> 29

<211> 25

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 29

tctggatcca cgggggaagg gaacc 25

<210> 30

<211> 257

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 30

tagaaccata caacgccaca ctttcagtac atcaattggt agaaaacaca gatgaaacat 60

actgtattga taatgaagct ctctatgaca tttgcttcag aactttgaaa ctcacaacac 120

ccacatacgg agacttaaac catttggtat ccctcacaat gtccggtgta accacctgcc 180

ttaggttccc aggtcagttg aatgctgatc ttagaaaatt ggctgtcaac atggttccct 240

tcccccgtgg atccaga 257

<210> 31

<211> 586

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 31

actgaattct ccgggcatga agaagtggag acgggggaag ggaaccatgt tgacagccaa 60

ttttctaaga tcagcattca actgacctgg gaacctaagg caggtggtta caccggacat 120

tgtgagggat accaaatggt ttaagtctcc gtatgtgggt gttgtgagtt tcaaagttct 180

gaagcaaatg tcatagagag cttcattatc aatacagtat gtttcatctg tgttttctac 240

caattgatgt caaatccgtg aagaataccc agatagaatt atgaacacat actcagtagt 300

cccctctccc aaagtatcag ataccgtagt agaaccatac aacgccacac tttcagtaca 360

tcaattggta gaaaacacag atgaaacata ctgtattgat aatgaagctc tctatgacat 420

ttgcttcaga actttgaaac tcacaacacc cacatacgga gacttaaacc atttggtatc 480

cctcacaatg tccggtgtaa ccacctgcct taggttccca ggtcagttga atgctgatct 540

tagaaaattg gctgtcaaca tggttccctt cccccgtgga tccaga 586

<210> 32

<211> 399

<212> DNA

<213> Diabrotica virgifera

<400> 32

acgcgtccag ttaatatccc gtgagatatt tttgcagtcc ttttaataag attcttcata 60

attcaccatg aagggctgcg ttttcaacat cgacaacggt tatttggaag gcctgtgtcg 120

tggctttaaa tgtgggatcc tgaaacacgc cgattatttg aatttggtcc agtgtgaaac 180

tcttgaagat ttaaaactgc acttgcaagg cactgactat ggaacttttt tggccaatga 240

accttcacct ttgtcagtat ccgtcatcga ttcaagactt cgacaaaaac tcctgattga 300

gttccagcac atgcgtaacc aagcagtaga gcctctctcg acatttatgg gcttcattac 360

ctacagttac atgatcgaca acataatttt gcttattac 399

<210> 33

<211> 40

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 33

taatacgact cactataggg agaaacggtt atttggaagg 40

<210> 34

<211> 43

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 34

taatacgact cactataggg agattgtcga tcatgtaact gta 43

<210> 35

<211> 291

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 35

taatacgact cactataggg agaaacggtt atttgcaagg tttgactgta tattattatt 60

tttatgtatc gacatygatg aattgattta tttatcgtaa agaaattcaa atacatttaa 120

agcttcaaat attaataata atgaacaagc tttgaagggt tacaaacaac caatcgatct 180

attaatatag tctattgact ctgaagttgc aaacggtaat agggccaatg caatggtttg 240

attctcccta cagttacatg atcgacaact ccctatagtg agtcgtatta a 291

<210> 36

<211> 451

<212> DNA

<213> Diabrotica virgifera

<400> 36

ccacatacca cgctatgaaa cccccttata tggggccgat ctaacaggag tgtggaactc 60

tatatccatt atatctaaaa tggttaacag aaaagaattc ataattaaca ttcaattgcc 120

attgtacacg tatattctgg taaatctact actactggac atttaattta caaatgtagt 180

ggtatcgaca aacttaccat cgaaaagttc caaaaagaat cccaacaaat gggtaaaggc 240

taattcaaat atgcctgggt actctacata cttacagccc atagagaacg tggtattacc 300

attgatattg ctgtgcggaa attcgaaaca gctaaatact attgaaccat cattgatgcc 360

cctggcacag atatttcatt aataacatta tcactggtac attacaatct gactgtgctg 420

tactcattga tgcaactggt acttggtaat t 451

<210> 37

<211> 44

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 37

taatacgact cactataggg agacacgcta tgaaaccccc ttat 44

<210> 38

<211> 42

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 38

taatacgact cactataggg agatttcgaa tttccgcaca gc 42

<210> 39

<211> 933

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 39

taatacgact cactataggg agatttcgaa tttccgcaca gcaagtgtgt aacttaaatt 60

tcaaaaaact tttcgttgtc ccaatttttt ttcataatct tagcggttac gattcgcata 120

tgatgattag agatcttgcg aaaaatggta gtatcagctt actaccaata aataaggaaa 180

agtatatttc atttacaata tacgattctg aggtcagtat taggttgagg ttcgttgatt 240

cactgagatt tttaaattca tcattggaca agttggctgc cacattgcaa cctgaggatt 300

taagatattt agctagcgaa tttccaaata ccactaccga acaaatggaa ttattgaaac 360

gaaaaggcat attcccatac gaatatattg agtctttcaa taaattgaat gaaacgcaac 420

taccatcaat tgataaattt tacagctcat tatcgggtga aaacatctcc aaaaatatgt 480

atcatcatgc tcagaatgtt tggcagtcat tcggtattaa aaatattttg gaatatagta 540

tgttgtacat gaaaactgat attatgttac tgacttgcat ttttgaaaat tttcgacaaa 600

aatgtcgaag tacatacagt cttgatcctg catggtacta taccatgcct ggattttctt 660

gggatgcaat gcttaaatat actggatgta aacttgaact gctgaatgat atcgataaaa 720

tcatgtttat tgagaaagct atccgaggtg gtataagtca agtaagtaat cggtattctg 780

aggcaaataa caaatacatg cataattatg atccatcaaa gcctagtaaa tatgtgctat 840

atttagatgt caacaatttg tatggttggg caatgtctca attattacca taagggggtt 900

tcatagcgtg tctccctata gtgagtcgta tta 933

<210> 40

<211> 918

<212> DNA

<213> Diabrotica virgifera

<400> 40

cccaagcgtc cgcccacgcg tccgcccacg cggccccccc cgccgcccgc acggtgtgga 60

cctcgcgcct ggtgttacat cccaagtagt gttcctttta ttctaagttt aatttcgaac 120

agttgcattt actttatttc caaacaatca aaatgggtaa agaaaagatt catattaaca 180

tcgttgtcat tggacacgta gattctggta aatctactac tactggacat ttaatttaca 240

aatgtggtgg tatcgacaaa cgtaccatcg aaaagttcga aaaagaagcc caagaaatgg 300

gtaaaggttc attcaaatat gcctgggtac tcgacaaact taaggccgag agagaacgtg 360

gtattaccat tgatattgct ttgtggaaat tcgaaacagc taaatactat gtaaccatca 420

ttgatgcccc tggacacaga gatttcatta agaacatgat cactggtaca tcacaagctg 480

actgtgctgt actcattgtt gcagctggta ctggtgaatt tgaagcaggt atttcaaaga 540

atggacaaac acgtgaacat gctcttcttg ctttcaccct tggtgtaaaa caacttattg 600

ttggtgtcaa caaaatggac tcgactgaac cagcatacag tgaatcacgt ttcgaggaaa 660

tcaagaagga agtatcctca tacatcaaga aaattggtta caacccagct gccgttgctt 720

tcgtaccaat ttcaggatgg cacggagaca acatgttaga aggatctgac aagatgccat 780

ggttcaaggg atggcaaatc gaacgtaaag aaggaaaagc tgaaggaaag tgcttgattg 840

aggctttgga tgctatcctt cccccacctc gtccaactga gaaacccctc cgtcttccac 900

tccaggatgt ctacaaaa 918

<210> 41

<211> 41

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 41

taatacgact cactataggg agacctcgcg cctggtgtta c 41

<210> 42

<211> 45

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 42

taatacgact cactataggg agaccaaggg tgaaagcaag aagag 45

<210> 43

<211> 569

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 43

taatacgact cactataggg agacctcgcg cctggtgtta catcccaagt agtgttcctt 60

ttattctaag tttaatttcg aacagttgca tttactttat ttccaaacaa tcaaaatggg 120

taaagaaaag attcatatta acatcgttgt cattggacac gtagattctg gtaaatctac 180

tactactgga catttaattt acaaatgtgg tggtatcgac aaacgtacca tcgaaaagtt 240

cgaaaaagaa gcccaagaaa tgggtaaagg ttcattcaaa tatgcctggg tactcgacaa 300

acttaaggcc gagagagaac gtggtattac cattgatatt gctttgtgga aattcgaaac 360

agctaaatac tatgtaacca tcattgatgc ccctggacac agagatttca ttaagaacat 420

gatcactggt acatcacaag ctgactgtgc tgtactcatt gttgcagctg gtactggtga 480

atttgaagca ggtatttcaa agaatggaca aacacgtgaa catgctcttc ttgctttcac 540

ccttggtctc cctatagtga gtcgtatta 569

<210> 44

<211> 440

<212> DNA

<213> Diabrotica virgifera

<400> 44

tcgcgggccg acacacgcct ccatattaag tcttgaaagt catttttaaa aacattttaa 60

tttaaaagta gtatttttaa gatttttcat tttcacacca gttcataatg gcatctggtt 120

caatatacga cgctgcacat aagggagatt ttgaatatgt ttcccaaaag attgaagagg 180

atccactaat tataaaagca ccagactcta gtaaaaggct tctaattcat tgggcagttc 240

tcagcggaaa tgtaaagctt gttactcatt tactggaact tggatcttct gtgaacccct 300

cggatgatac agatatgaca ccattaatat tagcttcatc ggctggccat accgaagttg 360

tcaaattgtt attaaaaaaa tgtgatgatg tcaatcataa aaatgcacag ggtcattcat 420

cacttcagta tgcagcctcc 440

<210> 45

<211> 46

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 45

taatacgact cactataggg agaacgctgc acataaggga gatttt 46

<210> 46

<211> 41

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 46

taatacgact cactataggg agaggaggct gcatactgaa g 41

<210> 47

<211> 1113

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 47

taatacgact cactataggg agaacgctgc acataaggga gattttgact atgtttcaga 60

aaagattgaa gagtttccaa taattttaga agcaccagac tctgtaagtt gtaattattt 120

gtatattatt atctaacgtt aatctctaga cgaaccttta ttatatccaa tctctaccgt 180

gcaatactaa aatgtaccac gtacatttgt agttcctttg atttttttaa tttcattttt 240

ttaacaggtt ttgcaaatgt atacattttt attttggtta catagtcagg ttatacagtc 300

cgtataattt caataatttc tctatctagt atagaaccca tgtcacctta tcaacctatt 360

atcctgcata tttaaatgca gtaaaaccac atttaaaaac atattttctg gtatctcata 420

gccatttgta tcctctaatg gatgtgcata ggcttcttct aaagttttct agtttctatg 480

aagttacaaa gtagtttcct gttattctct tgagaacatt tgttcatatg ataggtggtt 540

catattatct gacagtttca gcttacaagt gaagcagtag catctccaga agatgccaac 600

ccctagtgtt ggtgaaacgt cgagaactac ttgacagtct aagagcccca acaaacagtt 660

taacaagttg gtgtgcattt agttgataga attctgtcag gttcttggat actccattgt 720

attggtttat tttatttaac taatttcctc tctcttggtt ctctttacta ttccaaacct 780

aaaaattttt tattgtatag attcattttg ttgttgagct tatatattgt gctattgacc 840

aataatcaaa tactttttag agtaagaggc ttgtaattca ttgggcagtt ctcagcggaa 900

atgtaaagct tgttacctat ttactgaaac ttggatctcc tgtgaactcc tcagatgata 960

cagatatgac accattaata ttagcttcat cagctggcca taccgaagtt gtcaaattgt 1020

tattaaaaaa atgtgatgat gtcaatcata aaaatgcaca gggccattca tcacttcagt 1080

atgcagcctc cctccctata gtgagtcgta tta 1113

<210> 48

<211> 425

<212> DNA

<213> Diabrotica virgifera

<400> 48

aggattttct gaagctgccg aagtaactgg actcaatcca gcccaaatat ccgtcattat 60

gaagaacctg atggctcgat tgggattcca gaagtactac cttcagggag gtgattgggg 120

ttccgcaata gtagccaact tagcatcatt attcccagaa aaagtgctgg gagtccattc 180

caatatgtgt atggtcaata gtatgctttc taatctaaaa ttagcattgg gtagttttat 240

gccatccttg attgttgatg ctgacaagca acatctcctt tatcccagaa tgaaacattt 300

tggattcctt atattggaaa gtggttatat gcatcttcag ggtagtaaac cagataccgt 360

tggtgtcgct ctacgtgata gccctgtagg tcttgcagct tacatcatag agaagtttca 420

cacat 425

<210> 49

<211> 42

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 49

taatacgact cactataggg agatctgaag ctgccgaagt aa 42

<210> 50

<211> 44

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 50

taatacgact cactataggg agatatcacg tagagcgaca ccaa 44

<210> 51

<211> 47

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 51

taatacgact cactataggg agactatgat gtaagctgca agaccta 47

<210> 52

<211> 95

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 52

taatacgact cactataggg agatcgcgat gagtagaccc aacacctctg gtaagggctc 60

tcatgcattg tttctcccta tagtgagtcg tatta 95

<210> 53

<211> 427

<212> DNA

<213> Diabrotica virgifera

<400> 53

gacgcgcggg tcgatgcaag actctagata gagtcgtaat attgtcaact ttttcgtttc 60

ggtaaaattt atactaacta gccgtcagaa aagttactaa ttctccagtt atttaattga 120

gaatttgact ttattcgtca ctagcgcaat aactcagtat ggtgattatt aattcattta 180

aacaccccga gtctcctatt aggtatcaac aggacgatgt tcaagtctac ttagacaaga 240

aagatttggg cctgggaact ttatttgtta gtgaaagcac attatgctgg caacaagaag 300

agaacaatgg ttttgctatt gaatattcaa gtatttcctt gcatgccata tctaaagatt 360

taaacattca ttctacagaa tgtgtatacc tcgtgacaga tggacatatt actatgccag 420

gtgacag 427

<210> 54

<211> 43

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 54

taatacgact cactataggg agactagccg tcagaaaagt tac 43

<210> 55

<211> 41

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 55

taatacgact cactataggg agaatggcat gcaaggaaat a 41

<210> 56

<211> 318

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 56

taatacgact cactataggg agactagccg tcagaaaagt tactaattct ccagttattt 60

aattgagaat ttgactttat tcgtcactag cgcaataact cagtatggtg attattaatt 120

catttaaaca ccccgagtct cctattaggt atcaacagga cgatgttcaa gtctacttag 180

acaagaaaga tttgggcctg ggaactttat ttgttagtga aagcacatta tgctggcaac 240

aagaagagaa caatggtttt gctattgaat attcaagtat ttccttgcat gccattctcc 300

ctatagtgag tcgtatta 318

<210> 57

<211> 431

<212> DNA

<213> Diabrotica virgifera

<400> 57

caattattca cagaacaaca attatacaga atagaccact atttgggtaa ggaaatggta 60

cagaatttaa tgacacttcg atttggtaac agaatcttta accccacatg gaacagtgac 120

catatagctt ccatccaaat aaattgtaag gaacccttcg gaactgaagg cagaggaggg 180

tattttgacg aattcggcat tattagggat gtaatgcaga atcatatttt acaaattcta 240

gctctagtag ctatggaaaa accagcttca gttcaaccag acgatataag aaatgaaaag 300

gtaaaggtat taaaaagtat agctccaata aagctcaagg acgttgtatt gggtcagtac 360

gttggaaatc ctgatggaca aggtaatgcg aaattgggat acttagatga tccgagtgtt 420

cctaaagatt c 431

<210> 58

<211> 47

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 58

taatacgact cactataggg agaacagtga ccatatagct tccatcc 47

<210> 59

<211> 45

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 59

taatacgact cactataggg agaatttcgc attaccttgt ccatc 45

<210> 60

<211> 328

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 60

taatacgact cactataggg agaacagtga ccatatagct tccatccaaa taaattgtaa 60

ggaacccttc ggaactgaag gcagaggagg gtattttgac gaattcggca ttattaggga 120

tgtaatgcag aatcatattt tacaaattct agctctagta gctatggaaa aaccagcttc 180

agttcaacca gacgatataa gaaatgaaaa ggtaaaggta ttaaaaagta tagctccaat 240

aaagctcaag gacgttgtat tgggtcagta cgttggaaat cctgatggac aaggtaatgc 300

gaaattctcc ctatagtgag tcgtatta 328

<210> 61

<211> 483

<212> DNA

<213> Diabrotica virgifera

<400> 61

acccacgcct accccgcccc gtgatattta gtgcttactt ggtacagcag tttcagtgct 60

gtgctttaga ataatttatt ttttaacatt tatatagaaa tcaaatacta accaatcaac 120

atgtgtgaag aagaagttgc cgctttagtc gtagacaatg gatccggtat gtgcaaagct 180

ggttttgctg gggatgatgc acctcgtgct gtattccctt caattgttgg acgcccaaga 240

catcagggtg tgatggtagg aatgggacaa aaagattcct atgtaggtga tgaagctcaa 300

agtaaaagag gtatccttac cttaaaatac cccatcgagc acggaatagt cacaaactgg 360

gatgatatgg agaaaatttg gcatcataca ttctacaatg aactcagagt agccccagaa 420

gaacaccctg ttctgttgac agaagctcct ctcaacccca aggccaacag ggaaaagatg 480

aca 483

<210> 62

<211> 45

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 62

taatacgact cactataggg agaccgcccc gtgatattta gtgct 45

<210> 63

<211> 45

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 63

taatacgact cactataggg agactgttgg ccttggggtt gagag 45

<210> 64

<211> 503

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 64

taatacgact cactataggg agaccgcccc gtgatattta gtgcttactt ggtacagcag 60

tttcagtgct gtgctttaga ataatttatt ttttaacatt tatatagaaa tcaaatacta 120

accaatcaac atgtgtgaag aagaagttgc cgctttagtc gtagacaatg gatccggtat 180

gtgcaaagct ggttttgctg gggatgatgc acctcgtgct gtattccctt caattgttgg 240

acgcccaaga catcagggtg tgatggtagg aatgggacaa aaagattcct atgtaggtga 300

tgaagctcaa agtaaaagag gtatccttac cttaaaatac cccatcgagc acggaatagt 360

cacaaactgg gatgatatgg agaaaatttg gcatcataca ttctacaatg aactcagagt 420

agccccagaa gaacaccctg ttctgttgac agaagctcct ctcaacccca aggccaacag 480

tctccctata gtgagtcgta tta 503

<210> 65

<211> 407

<212> DNA

<213> Diabrotica virgifera

<400> 65

aggtgaatgt tatatcgttt ttcaaagtgt aaggtgttta ttttcaaaaa gtttataaaa 60

taagcaatca ctatgggtaa tgtgtttgca aatttattca aaggcctctt tggcaaaaag 120

gaaatgagga tattgatggt acgactcgat gcagctggta aaaccacaat tttatataaa 180

cttaaattag gagaaattgt aacaactatt ccaacaattg gatttaatgt ggagactgta 240

gaatataaga acattagttt tacagtatgg gatgtaggtg gtcaagataa aattaggcca 300

ttgtggagac actatttcca aaacacacaa cgcctaattt tcgtagtaga cagtaaccac 360

acggaaacta acactgaggc taaagattaa ttaatgcgtt agttggg 407

<210> 66

<211> 42

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 66

taatacgact cactataggg agaactatgg gtaatgtgtt tg 42

<210> 67

<211> 40

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 67

taatacgact cactataggg agagtttccg tgtggttact 40

<210> 68

<211> 345

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 68

taatacgact cactataggg agaactatgg gtaatgtgtt tgcaaattta ttcaaaggcc 60

tctttggcaa aaaggaaatg aggatattga tggtacgact cgatgcagct ggtaaaacca 120

caattttata taaacttaaa ttaggagaaa ttgtaacaac tattccaaca attggattta 180

atgtggagac tgtagaatat aagaacatta gttttacagt atgggatgta ggtggtcaag 240

ataaaattag gccattgtgg agacactatt tccaaaacac acaacgccta attttcgtag 300

tagacagtaa ccacacggaa actctcccta tagtgagtcg tatta 345

<210> 69

<211> 456

<212> DNA

<213> Diabrotica virgifera

<400> 69

tcgcgggtcg atacaagcgt ctaaacacac gttctgatga catcaatttc taaaaatgtt 60

cgcaaattcc taccaaagcg gcttcatttc aatattctac agcgtaggaa gtaatccact 120

agcattatgg gacaagcagg taaagaacgg acatatcaga cggattatgg acgatgatgt 180

gaaatcatta gttttggaaa tatctggaac taatgtagct actacttata taacgtgccc 240

catcaaacca cgagcttcac ttggaatcag attacctttt ctgattatga ttataaagaa 300

tatgaagaag tactttacat ttgaaattca aatattagat gataaagata tgcgtagaag 360

gtttagaata tcaaatttcc aatcatccac caaagtgaga ccgttctgta caacgatgcc 420

aatgggactc agcagtggct ggaatcaagt tcaatt 456

<210> 70

<211> 44

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 70

taatacgact cactataggg agacgggtcg atacaagcgt ctaa 44

<210> 71

<211> 44

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 71

taatacgact cactataggg agaagtccca ttggcatcgt tgta 44

<210> 72

<211> 472

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 72

taatacgact cactataggg agacgggtcg atacaagcgt ctaaacacac gttctgatga 60

catcaatttc taaaaatgtt cgcaaattcc taccaaagcg gcttcatttc aatattctac 120

agcgtaggaa gtaatccact agcattatgg gacaagcagg taaagaacgg acatatcaga 180

cggattatgg acgatgatgt gaaatcatta gttttggaaa tatctggaac taatgtagct 240

actacttata taacgtgccc catcaaacca cgagcttcac ttggaatcag attacctttt 300

ctgattatga ttataaagaa tatgaagaag tactttacat ttgaaattca aatattagat 360

gataaagata tgcgtagaag gtttagaata tcaaatttcc aatcatccac caaagtgaga 420

ccgttctgta caacgatgcc aatgggactt ctccctatag tgagtcgtat ta 472

<210> 73

<211> 503

<212> DNA

<213> Diabrotica virgifera

<400> 73

cacgcgtcca aaatcaatcc ttgaaaaaag gcaacttcac ggaactttta caaaaactta 60

gtaaggttct aaaaccccag ggatacttat taagtgcagc agctccggga gcacgtgata 120

aaattgatga accttacgac attccagcga tttcaaagct actagacttg gtcaatgtta 180

tggttttcga tttccacggc gcttttgaca actatgtagg acatatctca ccgctttttc 240

ccgctaaagt tgactacgat tactataata ataaaacata caatgtggat acaggaattc 300

aatattggtt gaatggtggt gcagatcctg caaaattaaa cttgggtgtt gtcgcttatg 360

gaagaacttt tactttggct gataaaaata ataccgctct atatgctcct gtcaaaggtg 420

gaggtacagt tggaccttat tcacaacaat ctggatattt gggatataat gagatttgca 480

gatactatac cgactcaact tac 503

<210> 74

<211> 42

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 74

taatacgact cactataggg agacacgcgt ccaaaatcaa tc 42

<210> 75

<211> 47

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 75

taatacgact cactataggg agatcggtat agtatctgca aatctca 47

<210> 76

<211> 417

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 76

taatacgact cactataggg agacaaacat caggtgcgga aaaaacacga gaggctaata 60

ccaagtgatg ccgacgttgc caaatggaga tgtgttggtc ttctgtcagt cacaaccgca 120

aggcagtgtg aaatgtgaag ttatgtgatt tactttgaaa aaaacagata aggattacgt 180

aagatgagca attcatgtac tagtacaatt aaagttattg aaaataacac aattcttgta 240

gaatggcaaa aacatcatta tggtcatatt tttgattcgc aacatattaa tttacaaaag 300

aaagataata acataatagg ttcaaagcta atatcggagt cccatagcaa aggtaaaaaa 360

attgtttttc ttttttttct tacttaaaaa attctctccc tatagtgagt cgtatta 417

<210> 77

<211> 927

<212> DNA

<213> Diabrotica virgifera

<400> 77

aggtaaatgc tcaacatgaa ggtgctagtg ttactctcgg tactatctgc atttcttgtt 60

tgccaaacat caggtgcgga aaaacgggtc gtttgttatt tcgccagttg gaccatttat 120

agagcaagaa aaggtgcttt cgatgtcagt aatatagatc catcgctgtg tacacacatt 180

aattttgctt tccttggtct taatgaagat ggttctattc acattttgga ttcctgggag 240

tcaagtgatg ctggtggtca tgagggtttt aaacatctcg tagagcttaa aaagaccaat 300

cctgacctta aggtatgtgt aagtatgggc ggttggaacg aaggttccaa gcagtattca 360

gcagtagcat cagatccagc aaaaagagta aaacttgcag atgaggtttt agcttttatc 420

gaaaattggg gcttcgatgg ttttgatttg gattgggaat atccaggatt acgaggagga 480

aacgaaacta ttgataaaga gaattatgtc gaacttttga aagctcttag tgacgttctt 540

gagcccaaag gatacttact cagtgtagcc actgcaggcg ccgttgaaaa aatcgacgtt 600

ggatttgacg tctcagttat aaatgagttg gtggatatga ttaacgttat ggtttttgat 660

tttcatggag catttgagaa ctttgtagga cacgtttcac cattgttccc agctcaagtt 720

gattacgaat atgaagctaa tagtacatac aatgtagaca caggaatcca acactggata 780

ttgagtggtg cagatcccgc aaaaataaac ctcggcattg tcacctatgg aagaacctat 840

accttagctg ataaaaccaa tacttctctt tatgcaaatg ttaccggtgg tggtaataca 900

gggccatatt ctgcacaatc tggatat 927

<210> 78

<211> 44

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 78

taatacgact cactataggg agacaaacat caggtgcgga aaaa 44

<210> 79

<211> 44

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 79

taatacgact cactataggg agacgggatc tgcaccactc aata 44

<210> 80

<211> 912

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 80

taatacgact cactataggg agacgggatc tgcaccactc aataatatta aaactactat 60

aaaaatatga ctagttattc agaagattta atcataatct aaaaaagtgc aatacatttt 120

taataaacta tgatttattt atcccgcggt aaacactaaa aacactatat attatacata 180

aagataaatt aatacagtca aatactatta atttattctc tgaagtacgg gccattactt 240

tgtttacatg tttgtatact aacctgtaga acgttattcc tgaagatttt ttattaacat 300

tgcttctgct actgcaacta cgttgagaac aagaagccat tattatgcac tatcacaata 360

tattattaca gtttctataa aagtattaaa aaactaaaaa tattcgaaag acaacaaacg 420

taaacaaaca tatacgatct gtcaaaagtg tcacaacaat cttaacgata tggccgaagt 480

gaggtcgttt ttagtcacgt gatgccttct ccatagattc taactcgatg gtgtagacgc 540

aaatagcgac atctgataat aaaatcgtga actaattttc gaaaccaaat tcagaatttc 600

gctttaatct gtgccttcta agaattgcaa ggcaagacag acgttgataa agatgttaga 660

tataagtttg atataagtag atataagttt gattattact tacaataggg acagcatcta 720

attattttta gcacactcac ttgctgccaa caatactggc cgcaaaacta ggtaatagag 780

aaatagtgta tattaaggaa tgaactgact ggtcgcaagc tcttgcttgt cggacctttc 840

cttacgaagt tgcttgacga ctgtattatt tttccgcacc tgatgtttgt ctccctatag 900

tgagtcgtat ta 912

<210> 81

<211> 342

<212> DNA

<213> Diabrotica virgifera

<400> 81

ggagcgaagg catctctctc catcccgacc tctcgtggcc gccgcgaaga aaaggagctt 60

atcatggctt caaaacgtat cctgaaggaa ctgaaggact tgcagaaaga tcctccgaga 120

tcatgcagtg caggtccttc tggcgaggat atgttccatt ggcaggcaac aattatgggt 180

cctcctgata gtccctatgc tggaggtgtt ttcttagtga atatccattt ccccccggac 240

taccccttca agcctccgaa ggtatcgttc aagacaaagg tcttccatcc gaacatcaat 300

agcaatggag gcatatgcct cgacattctg aaggagcaat gg 342

<210> 82

<211> 42

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 82

taatacgact cactataggg agactctcca tcccgacctc tc 42

<210> 83

<211> 42

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 83

taatacgact cactataggg agatgcctcc attgctattg at 42

<210> 84

<211> 344

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 84

taatacgact cactataggg agactctcca tcccgacctc tcgtggccgc cgcgaagaaa 60

aggagcttat catggcttca aaacgtatcc tgaaggaact gaaggacttg cagaaagatc 120

ctccgagatc atgcagtgca ggtccttctg gcgaggatat gttccattgg caggcaacaa 180

ttatgggtcc tcctgatagt ccctatgctg gaggtgtttt cttagtgaat atccatttcc 240

ccccggacta ccccttcaag cctccgaagg tatcgttcaa gacaaaggtc ttccatccga 300

acatcaatag caatggaggc atctccctat agtgagtcgt atta 344

<210> 85

<211> 674

<212> DNA

<213> Diabrotica virgifera

<400> 85

tcggcggccg gtaaggaact ttaaaccgga atggtcaaaa aacaaaatcc tggcataatg 60

gggaaaattg gaattaacgg ttttggccga attggccgcc tggtaccccg tgcagctctt 120

gaaaaaggag ttgaagtagt agctgtcaac gatcccttcc ttgatgtcga ctacatggta 180

tacttgttca aatttgactc tacccacggt cgctacaagg gatgtgtcaa cagtgatggc 240

aaaaacttag ttgttgatgg caaagtcatt tccgtacacc aagaaagaga cccagctgct 300

attccatggg gcaaagctgg tgcagattat gtagtagaat ctaccggagt gttcaccaca 360

attgaaaagg ccaagaaaca tcttgacggt ggtgctaaga aagtcatcat ctcagctcca 420

tctgctgatg ctccaatgta tgtatgtggt gttaacttgg atgcctacaa tccagctgat 480

cccgtaatct ctaacgcttc ttgcactacc aactgccttg ctccactcgc caaagtcatc 540

cacgacaact tcgaaatcgt tgaaggtttg atgaccaccg tacatgccac aaccgccaca 600

caaaaaactg tcgacggacc ctctggaaaa ttgtggcgtg acggtcgtgg tgccggacaa 660

aacatcatcc cagc 674

<210> 86

<211> 45

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 86

taatacgact cactataggg agagtacccc gtgcagctct tgaaa 45

<210> 87

<211> 45

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 87

taatacgact cactataggg agaggttgtg gcatgtacgg tggtc 45

<210> 88

<211> 538

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 88

taatacgact cactataggg agagtacccc gtgcagctct tgaaaaagga gttgaagtag 60

tagctgtcaa cgatcccttc cttgatgtcg actacatggt atacttgttc aaatttgact 120

ctacccacgg tcgctacaag ggatgtgtca acagtgatgg caaaaactta gttgttgatg 180

gcaaagtcat ttccgtacac caagaaagag acccagctgc tattccatgg ggcaaagctg 240

gtgcagatta tgtagtagaa tctaccggag tgttcaccac aattgaaaag gccaagaaac 300

atcttgacgg tggtgctaag aaagtcatca tctcagctcc atctgctgat gctccaatgt 360

atgtatgtgg tgttaacttg gatgcctaca atccagctga tcccgtaatc tctaacgctt 420

cttgcactac caactgcctt gctccactcg ccaaagtcat ccacgacaac ttcgaaatcg 480

ttgaaggttt gatgaccacc gtacatgcca caacctctcc ctatagtgag tcgtatta 538

<210> 89

<211> 551

<212> DNA

<213> Diabrotica virgifera

<400> 89

atagaagttg aaccatctga tactattgag aatgtgaaag ctaagatcca agataaggaa 60

ggtatcccac cagaccagca aagattgatc tttgcaggta aacagctgga agatggtaga 120

accttgtctg actataacat ccagaaagag tccactcttc acttggtact gagattgaga 180

ggaggtatgc agatcttcgt caagacacta actggaaaga ccatcacttt ggaagttgaa 240

ccatctgata ccattgagaa tgtcaaagct aagatccaag ataaggaagg tatcccacca 300

gatcagcaaa gattgatctt tgcaggtaaa cagctagaag atggtagaac tttgtctgat 360

tataacatcc agaaagagtc cactcttcac ttggtactta gattgagagg aggtatgcac 420

attttcgtca agacattgac tggtaatacc atcacattag aagttgaacc atctgatact 480

attgagaatg tgaaagctaa gattcaagat aaggaaggta tcccaccaga tcagcaaaga 540

ttgatctttg c 551

<210> 90

<211> 40

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 90

taatacgact cactataggg agagtatccc accagaccag 40

<210> 91

<211> 47

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 91

taatacgact cactataggg agaatgtgca tacctcctct caatcta 47

<210> 92

<211> 407

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 92

taatacgact cactataggg agagtatccc accagaccag caaagattga tctttgcagg 60

taaacagctg gaagatggta gaaccttgtc tgactataac atccagaaag agtccactct 120

tcacttggta ctgagattga gaggaggtat gcagatcttc gtcaagacac taactggaaa 180

gaccatcact ttggaagttg aaccatctga taccattgag aatgtcaaag ctaagatcca 240

agataaggaa ggtatcccac cagatcagca aagattgatc tttgcaggta aacagctaga 300

agatggtaga actttgtctg attataacat ccagaaagag tccactcttc acttggtact 360

tagattgaga ggaggtatgc acattctccc tatactgagt cgtatta 407

<210> 93

<211> 401

<212> DNA

<213> Diabrotica virgifera

<220>

<221> misc_feature

<222> (369)..(369)

<223>n is a, c, g, or t

<400> 93

gtaatgttca tgttttgtgt gtagaaaaac gctaaaactg tgtgcaggca catcctttcg 60

cgatgagtag acccaacaca aactgttttc aagtcttacc gaacaatagc agatggctat 120

cgacacaaga ttctggaatt tttcccaaac gtcacactga ctactatgta tttaatatgg 180

gaagacagga agtgttagtg gaaggatggt ggggaacaaa actgggatgg actggggttt 240

tggatggagt gaacctggcg cctggcaatg gttacagaat tgtagtcagt gataaaccat 300

attttgtaac agctgtgaaa ataacaaata aaacaactgt aagggctctc atgcattgtt 360

ctgagatana cggttatcct ctgcggagtc aaggaactga c 401

<210> 94

<211> 45

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 94

taatacgact cactataggg agatcgcgat gagtagaccc aacac 45

<210> 95

<211> 46

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 95

taatacgact cactataggg agaaacaatg catgagagcc cttaca 46

<210> 96

<211> 348

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 96

taatacgact cactataggg agacgcgatg agtagaccca acacaaactg ttttcaagtc 60

ttaccgaaca atagcagatg gctatcgaca caagattctg gaatttttcc caaacgtcac 120

actgactact atgtatttaa tatgggaaga caggaagtgt tagtggaagg atggtgggga 180

acaaaactgg gatggactgg ggttttggat ggagtgaacc tggcgcctgg caatggttac 240

agaattgtag tcagtgataa accatatttt gtaacagctg tgaaaataac aaataaaaca 300

actgtaaggg ctctcatgca ttgtttctcc ctatactgag tcgtatta 348

<210> 97

<211> 1168

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 97

acgctgacaa gctgactcta gcagatcacc gtcttcgata ccaagcggcc tgaattcgcg 60

tgaatcgtat ctcagtccat cgttggccaa gccggagtcc aaataggtaa tgcctgctcg 120

ggagttgtac tcgcctggaa cagggcatcc aacctgacgg tcagatgcca tcagacaaga 180

ctgttggagg aggagatgac agtttcaaca cattcttcag tgaaactggt gccggcaaac 240

atgtacctag agcagtattt gtagatttgg aaccaacagt agtagatgaa gtacgtaccg 300

gcacataccg tcaattgttc cacccagaac aactcatcac tggcaaagaa gatgccgcca 360

ataactacta gaggtcacta tacaattggt aaagaaatag ttgacttggt attggacaga 420

atccgtaaat tggctgatca atgccatagt caacagatag acgttccatc aacaaagaag 480

tgaaaccaga tccagtacca ccaccgaagg agtggaagat caagaaacct tgaagtccag 540

tacattgatc agccaattta cggattctgt ccaataccaa gtcaactatt tctttaccaa 600

ttgtatagtg acctctagta gttattggcg gcatcttctt tgccagtgat gagttgttct 660

gggtggaaca attgacggta tgtgccggta cgtacttcat ctactactgt tggttccaaa 720

tctacaaata ctgctctagg tacatgtttg ccggcaccag tttcactgaa gaatgtgttg 780

aaactgtcat ctcctcctcc aacagtcttg tctgatggca tctgaccgtc aggttggatg 840

ccctgttcca ggcgagtaca actcccgagc aggcattacc tatttggact ccggcttggc 900

caacgatgga ctgagatacg attcacgcat tttggttgat gagtttttaa cttttacacc 960

acaaatgaaa caaaattacg acagacgttc agattagcta gtaatgcagc ggatccgatc 1020

gttcaaacat ttggcaataa agtttcttaa gattgaatcc tgttgccggt cttgcgatga 1080

ttatcatata atttctgttg aattacgtta agcatgtaat aattaacatg taatgcatga 1140

cgttatttat gagatgggtt tttatgat 1168

<210> 98

<211> 2131

<212> DNA

<213> Diabrotica virgifera

<400> 98

tcgcggcgac acacacccct ctaaacacgc tatcattggt cccacgcgcc gctagctagc 60

gatcgcgagc gagcgcccgc ccccccgccc gggaagctgc attactagct aatctgaacg 120

tctgtcgtaa ttttgtttca tttgtggtgt aaaagttaaa actcatcaac caaaatgcgt 180

gaatgtatct cagtccatgt tggccaagcc ggagtccaaa tcggtaatgc ctgctgggag 240

ttgtactgcc tggaacatgg catccaacct gacggtcaga tgccatcaga caagactgtt 300

ggaggaggag atgacagttt caacacattc ttcagtgaaa ctggtgccgg caaacatgta 360

cctagagcag tatttgtaga tttggaacca acagtagtag atgaagtacg taccggcaca 420

taccgtcaat tgttccaccc agaacaactc atcactggca aagaagatgc cgccaataac 480

tatgctagag gtcactatac aattggtaaa gaaatagttg acttggtatt ggacagaatc 540

cgtaaattgg ctgatcaatg tactggactt caaggtttct tgattttcca ctccttcggt 600

ggtggtactg gatctggttt cacttctttg ttgatggaac gtctatctgt tgactatggt 660

aaaaaatcaa aactggaatt cgccatctac ccagctcctc aagtatctac tgctgtagta 720

gaaccataca actccatctt gaccacccac accactcttg aacactcaga ctgtgccttt 780

atggtagata atgaagccat ctatgacatc tgcagacgta atctagacat cgagcgccca 840

acctacacca acttgaacag acttattggc caaatcgtat cctcaatcac agcttctcta 900

agattcgatg gtgctctaaa tgttgacttg acagaattcc aaactaactt ggttccttac 960

cctcgtattc acttccctct tgtcacctat gccccagtaa tttccgctga aaaggcttac 1020

catgaacaac tttccgtagc tgaaatcacc aatgcctgtt tcgaacctgc caaccagatg 1080

gtaaaatgtg atcccagaca tggtaaatac atggcttgct gtatgttgta cagaggggat 1140

gttgtaccaa aggatgtaaa tgctgctatt gcaaccatta agaccaaacg taccatccaa 1200

ttcgtagact ggtgtccaac tggtttcaaa gtaggtatca actaccaacc accaactgtt 1260

gtacctggag gtgatttggc taaagtacaa cgtgccgtat gcatgttgtc caacactaca 1320

gctattgctg aagcctgggc aagattggac cacaaattcg atcttatgta tgccaagaga 1380

gctttcgtcc actggtatgt aggagagggt atggaagaag gtgaattctc tgaagctcgt 1440

gaagatttgg ctgctttgga gaaagattat gaagaagttg gtatggactc cggagaaggt 1500

gagggtgaag gagctgaaga atattaaatt tgattccaaa catgacaaat cacttgtttt 1560

taagacaaaa aattcctttc aattttttta cactttttca ttacttttct gtgaaacgat 1620

tatttaaagt ctgatttaat ttaatacaga attttttacg agcaaaaaaa aaaaagggcg 1680

gcccccgatt gcgcatatag ctacattaaa gatcgtggcc tctgtctaga gactgactac 1740

aagtatccat gattaaacgg agactgcaaa cagtgtaatc tcggttatca ataaccatcc 1800

aatgaactag tgcatgctgt agtatcataa cacgaagtaa gcatcttcac ttgaggaatg 1860

tattatactg tgtgagccaa tatcagtatg tgacaacact aaatgagact ggccatagat 1920

aaaacctaca gcgcctttag gacgacttcc tatactagaa tccggtggaa aaccagttcc 1980

tcaaagcact gctatctgca ggcatctgtc taacgtatgc aaatcttggt ggtaaagacg 2040

caaaggtaaa tcttgttata tatattgctg atcaagcgta tgatgatatg aagaaaccta 2100

tgggcgaata catgagatac agggatgaaa g 2131

<210> 99

<211> 1720

<212> DNA

<213> Diabrotica virgifera

<400> 99

gacacgggcc cgaatatccc cggccgctct ctacgatcaa cgcatcgaag agagcgttct 60

gtgttttcta gtaatagtta tttaatacat tttataaatc aaaatgaggg aaatcgttca 120

catccaagct ggacaatgcg gtaaccaaat tggagccaaa ttctgggaaa tcatctctga 180

tgaacacgga atcgacccca ccggagccta ccatggagac tctgacctcc aacttgaaag 240

aatcaatgtc tactacaacg aggcctccgg cggaaaatac gtaccccgcg ccatcctcgt 300

cgacttggaa cccggtacca tggattcagt aaggtcgggt cccttcggac aaatcttcag 360

accagacaac ttcgtgtttg gacagtctgg agctggaaac aactgggcca agggacatta 420

cacagaaggt gctgaattag ttgattcagt attagatgtt gtaaggaaag aagctgaatc 480

atgtgattgt ttacaaggat tccaactcac acactcactt ggaggtggta ctggatcagg 540

tatgggtacc ctccttatct caaaaatccg tgaagaatac ccagacagaa ttatgaacac 600

atactcagta gtcccctcac ccaaagtatc agataccgta gtagaaccat acaatgccac 660

actttcagta catcaattgg tagaaaacac agatgaaaca tactgtattg ataatgaagc 720

tctctatgac atttgcttca gaactttgaa actcacaaca cccacatatg gagacttaaa 780

ccatttggta tccctcacaa tgtccggtgt aaccacctgt cttaggttcc caggtcagtt 840

gaatgctgat cttagaaaat tggctgtcaa catggttccc ttcccccgtc tccacttctt 900

catgcccgga ttcgctccac tcacctcaag aggcagccaa caatacagag cgttgacagt 960

tccagagctc acacagcaaa tgtttgatgc caagaacatg atggcggctt gtgatcccag 1020

acacggaagg taccttacag tagctgcagt attcagaggt aggatgtcaa tgaaagaagt 1080

tgacgaacag atgctcaaca tccagaacaa gaacagcagc tacttcgtcg aatggatccc 1140

caacaacgtt aaaacagccg tttgtgatat cccaccaaga ggtctcaaga tgtctgccac 1200

tttcatcggc aactcaaccg ccatccaaga attgttcaaa cgtatctccg aacaatttac 1260

agctatgttc aggaggaaag ctttcttgca ttggtacacc ggagaaggta tggatgaaat 1320

ggaattcacg gaagcagaat ccaacatgaa cgacttggta tcagaatacc aacagtacca 1380

agaagccaca gctgacgaag atgccgaatt cgacgaagac caggaagccg aagtcgacga 1440

gaactaaatt tcatacgtta attttggatc tgaaatcaaa gctttataac ttttatattt 1500

gtctcctctc cttttatttt ttatttaagc atgttttttg tacagtctct acattcccgt 1560

ttgtaaattt cgaatacact acttaaatta ttccaagact gactttttgt tgcttgtgtt 1620

tctggaattt caggaagtgt ttagatattt aacatgtttt gcgaactgtt tttttatgaa 1680

taggcattaa aactgctgcc attacttata ctcagaggca 1720

<210> 100

<211> 1175

<212> DNA

<213> Diabrotica virgifera

<400> 100

tgacaactga cacacgagaa gatacgacat ggcaagaaaa tctctctgat taccattctg 60

acttttctgc gggatcggat gaggataagg aagacgatga tttcgatgag aagaacgacg 120

ccgatttaag cagaaggagt cgaagaaaga tggaaaggaa agacgagaag gatcgtcctt 180

taccaccgtt actagccaga gttggcggca atattgaagt actcggtttt aatgccaggc 240

agcgtaaagc gttccttaat gctattatgc gctacggaat gccaccacaa gacgctttca 300

attcacagtg gctggtgaga gatcttcgag gaaaatctga gaagatattc aaggcttacg 360

tgtctctctt tatgaggcat ctttgcgaac ctggtgcaga taatgctgat acatttgcgg 420

acggtgtgcc gagggaagga ctgagtaggc aacatgtttt gacaaggatt ggtgtgatgt 480

cacttataag aaagaaggtt caggagttcg aacacatcaa cggcgagtat agcatgccgg 540

aagtaatcaa aaagagcatt atggatcaaa ataaaatcaa tgccgccggc accgccacca 600

caagcgaagc agaaacgcct aaaagtgcta ctaccagtac tagtgctacg ccagctacaa 660

gtgctgctcc cagtcccgct cccacacaag gagaagataa agataaggat aaagattccg 720

ttcagagtga cgaaaataaa gataaagaag tggttaataa aacggaaacc gaagatgaag 780

agaagaaaac gggagaatct tcaacagaaa agccgaaaac tgaaccggaa gaagtgaaag 840

aagcttctcc gaaaaccgaa attcccgaag ctagttccga agctgataaa tctgagatca 900

aatccgaagt cgatacctcg tctgtaacca gcgaggaaaa gaaagaagag aaagaggaag 960

aggccaaaaa ggaagaaccc gaagagacca aaatggaaat acaggaggag gaacttgtta 1020

aagaggagaa aaaagaagaa gaggatgata agaagaagga ggaaattaag aaagaggtgg 1080

aaaagaagga agaggatgac gttatggtta ttgatgatga taaagataag aaggacaaaa 1140

aggaaatcga tctcgaagcc aagaagcgtt tcatg 1175

<210> 101

<211> 1176

<212> DNA

<213> Diabrotica virgifera

<400> 101

cccatgcggc cgcccatttt tattgagcaa attgttcaga aagttgctgg gcgtagtcgg 60

gaaaaacatt gtttaaatcc ctttaatttc ctctaagtcg aaagaaaaag gctcaaaatg 120

gctctcagcg acgcagatgt acaaaagcag atcaagcaca tgatggcttt cattgagcaa 180

gaagccaatg aaaaggccga ggaaattgat gcaaaggctg aagaagaatt caacatcgaa 240

aagggccgtc tggtccaaca acagaggctc aagattatgg agtactacga gaaaaaagag 300

aagcaagtag aactccagaa aaaaatccaa tcatcaaaca tgttgaacca ggcaagattg 360

aaggtattga aagtaaggga agaccatgta cgtgccgttt tggaagatgc tcgcaaacgt 420

cttggtgagg taaccagaga ttcaggcaaa tatacacaaa tcctggaaag tctcatcctc 480

caagggctct atcagctctt cgaaaaggac atcaccatta gagtacgccc tcaggacaga 540

gaattggtaa aatctatcat gcctaacgtc tcccaaaagt acaaggacat aaccggtaaa 600

gacgtaaatc taaaaatcga cgacgagagc cacctttctc aagaaaccac cggaggaatc 660

gaactgttgg ccttgagaaa caagatcaaa atcaacaata ctctggaagc ccgtcttgag 720

ctcatctcac aacaattgat tccccagatc cgtaatgctc tgttcggacg caacgtcaac 780

agaaaattca ctgattaagt attttttgga tactgtgtat tgcctgtatt ttatatagta 840

ttgtaaaaca ttgttggttg cttagacaga tcttcaaaaa ccttttaaac tactatgtat 900

atacgatata tataataaac cattcctttt tttgaagtat tttaaacagt taagtttgtt 960

gttaccctaa ttgtatcctt gtcaagcaga tattttttaa aatccttaga aaattattag 1020

gtttcagtta tactacctta ttttttttct caaatatatt catattttat gtttatatgt 1080

atataaaaaa attatttttt tcttgtgaga aaatcatcgc aataaaattt attgttagtc 1140

caacaaaaaa aaaatggtgg ccgctttgtt ttttat 1176

<210> 102

<211> 2410

<212> DNA

<213> Diabrotica virgifera

<400> 102

cggacgcgtg ggggagaaac ataacatcca tccacaaata tgtcgaaagt aaggatcgga 60

gatgaagaga aggaagggca gtatggttat gtccatgctg tctcaggtcc agtcgttact 120

gctgagaaaa tgtctggttc tgctatgtac gaactggtac gtgtcggata ctatgagctg 180

gtaggagaaa tcattagatt ggaaggtgac atggctacta ttcaggtata cgaagaaaca 240

tcaggtgtaa ctgttggtga tccagtatta agaactggta aaccactttc agtagaactt 300

ggacctggta ttatgggttc catttttgat ggtatccaac gtccattgaa agacatttgt 360

gacgctactg atagtattta catccccaag ggtattaacg taccttcttt atcgagaaca 420

gcaaaatggg acttcaaccc aatcaacatc aagttgggat ctcacttaac tggaggtgat 480

atatatggtc tagttcatga aaacaccctt gtcaaacaca aaatgattct gcctcctaga 540

gctaagggta ctgtaaccta cattgcagaa ccaggaaact acactgttga tgaagtagta 600

ttggaaactg aatttgatgg tgatcgtacc aaatatacta tgttgcaagt atggcctgta 660

cgtcaagcaa ggccagtcag tgaaaaatta cctgccaacc atcctctgct tacaggacag 720

cgtgtacttg atgctctttt cccatgtgta cagggtggta ctactgccat tcccggagct 780

ttcggttgtg gaaaaactgt aatttcacaa tctctttcca aatattccaa ctctgatgtc 840

attatctacg tcggttgcgg agaaagaggt aacgaaatgt ctgaagtatt gagagatttc 900

cctgaattga ctgttgaaat tgacgggcac actgaatcta ttatgaaacg taccgcattg 960

gtcgccaaca catctaacat gcctgtagct gctcgtgaag cttctatcta tactggtatt 1020

actctttctg aatacttccg tgatatgggt tacaacgtat ctatgatggc tgactcgaca 1080

tcacgttggg ccgaagcttt gagagaaatt tcaggtcgtt tggctgaaat gcctgccgat 1140

tccggttatc cggcttactt aggtgcccgt ttggcttcct tctacgaacg tgctggtcgc 1200

gttaaatgtt taggtaatcc agacagagaa ggatccgttt caattgtagg agccgtatca 1260

cctcctggtg gtgatttctc agatcctgtt accactgcta ctcttggtat tgtacaggtg 1320

ttctggggtt tggacaagaa acttgcccaa cgtaagcact tcccttcagt agactggctt 1380

ggatcatatt ccaaatattt aagagcattg gacgactttt atgacaaaaa cttccaagag 1440

tttattcctc ttagaaccaa agttaaggaa attcttcagg aagaagatga tctagccgaa 1500

attgtgcagc tggtaggtaa agcatctctg gcagaaacgg acaaaatcac cttggaaatt 1560

gccaggcttc ttaaagaaga tttcttgcaa caaaactcat actcttctta tgacagattc 1620

tgtccattct ataaaactgt cggtatgttg agaaacatga tcggtttgta cgacatggcg 1680

agacacgctg tagaatcaac cgcacaatca gaaaataaga tcacttggaa cgtaataaga 1740

gattcaatga gtggaatttt atatcaactt agcagtatga aatttaagga tcccgtaaaa 1800

gatggtgaag ctaaaatcaa ggcagatttt gatcaattat atgaagatat tcagcaggcc 1860

ttcagaaact tagaagatta aatcttttta aggaaatttt cctattttgt tcatcagtgt 1920

aagtttaaaa atatagcgat atttatcaaa aagaataata aggcctctat ccctcacttc 1980

tgtgaatatt aatatggccg tactaaagat agtaactaaa gataggtttt ctcttttttg 2040

atattatcct gtacaaaata aattatgtaa attgttgaat atgtgtatag tttttttggg 2100

tgagggtaca gtgcttatta aatacttttt aaacattttt cccgccattc caattactat 2160

taagtttttt cgttttaata cttttttaaa tatacaggtg cttaatatcg tttatatttt 2220

cagtattact tggttttctt catgtaaatt gttttaaatt tttcttttac ccttttaatc 2280

ttgtatatta cattacccaa ttaaagttaa ttgtacagat taagataaac gagtatctta 2340

taacatctat tagattgtta gaatcaataa atgtagtgta attgttctgt tttgaacaaa 2400

taaatgcatc 2410

<210> 103

<211> 1575

<212> DNA

<213> Diabrotica virgifera

<400> 103

atctgacagt ttctacagta tagttgcagt gttcagtgga aaatattcaa ttaagatatt 60

cctagcgttc agacgtgtgc tctgatttca tggtactaaa atggcagtag ttcaatcaaa 120

ttacattcaa aatatacctt cttttggatg tgtagaccaa cctgacaacg gctccaaaac 180

aacaagagaa tcattagtag aagtgtcttc atcacgtcca cgccaagaag actactcagt 240

atatgagaac agactggcat ctttcactaa ctggcccaac acccaagtgt caagagaatc 300

attagctcga gctggtttta tatatacagg tcaagatgac atcgttatct gccctatttg 360

taagatagag ggataccatt gggtatcagg agacaatcca atggatgatc atcgtgtttg 420

gaatcccaac tgcccctttc ttaatagaag agataacatc gagcacgatc actctgtagg 480

ttctagagac acttgtggac tttttggcat agaattgtta ccaaattcag ttcctgaaga 540

taatacaagt aatttacaaa aattagggat ccaacctgga acaggtccac aaaatcaaga 600

caaaattacg ttagaaagcc ggttagcaac attccagggt tggccaaaga gcattaaaca 660

gaggccttct gagttagctg aggcgggatt ttattacaca ggagctgggg accaaactgt 720

gtgcttttat tgtggtgggg gattaaaaga ctgggatgaa ggagatgatc cttgggagca 780

acatgccctt tggtttagca aatgtgtgtt tctcaatttg aaaaagggca aagaattcat 840

cgatcaagta aagaggaagg ctgatccaca attttcaatt cctggaccta gcggtactca 900

agccaaagag gaaccgactg ctactgaatc ttcaagtgat aaacaaagtg aaacagtgaa 960

aacaaaatca gatagggaaa gtttcgcaac tgacacaact ttgtgcaaaa tttgctttaa 1020

aaacgaactt ggtgttgttt tcttgccttg tggacatatt gttgcttgtg tagattgtgc 1080

tgctgcacta aaaacatgtg ctgtatgccg aaaaccttta gaggccacag tcagagcgtt 1140

cctatcataa atttttattc tgttaatagt ttttcacatt tcatgtttca cacatactta 1200

gatctagtca agattgttag agttttggca aagaaattaa ataaaaattc ttttcataaa 1260

aatcatttct ttaatattac attagagaaa aattatattt ttatactgag tacaaatttg 1320

aacaagttat taattttaag ttacaaaata cgcttttata ggttaacaat tatcaaagcg 1380

cttaaatcta atagatacta cacaacatta aggactgcaa accatatctt tcacgaagta 1440

atccctacta gtgaccaatt gctcgctagg agcagatgca aattacacaa atttactata 1500

aatctgacat taaaacttag gtgtatgttt gtgtgtatgt tatgtattga tcataataat 1560

atagtaattt ataat 1575

<210> 104

<211> 1870

<212> DNA

<213> Diabrotica virgifera

<400> 104

gtcgacccac gcgtccgaat ttgatggtga tcgtaccaaa tatactatgt tgcaagtatg 60

gcctgtacgt caagcaaggc cagtcagtga aaaattacct gccaaccatc ctctgcttac 120

aggacagcgt gtacttgatg ctcttttccc atgtgtacag ggtggtacta ctgccattcc 180

cggagctttc ggttgtggaa aaactgtaat ttcacaatct ctttccaaat attccaactc 240

tgatgtcatt atctacgtcg gttgcggaga aagaggtaac gaaatgtctg aagtattgag 300

agatttccct gaattgactg ttgaaattga cgggcacact gaatctatta tgaaacgtac 360

cgcattggtc gccaacacat ctaacatgcc tgtagctgct cgtgaagctt ctatctatac 420

tggtattact ctttctgaat acttccgtga tatgggttac aacgtatcta tgatggctga 480

ctcgacatca cgttgggccg aagctttgag agaaatttca ggtcgtttgg ctgaaatgcc 540

tgccgattcc ggttatccgg cttacttagg tgcccgtttg gcttccttct acgaacgtgc 600

tggtcgcgtt aaatgtttag gtaatccaga cagagaagga tccgtttcaa ttgtaggagc 660

cgtatcacct cctggtggtg atttctcaga tcctgttacc actgctactc ttggtattgt 720

acaggtgttc tggggtttgg acaagaaact tgcccaacgt aagcacttcc cttcagtaga 780

ctggcttgga tcatattcca aatatttaag agcattggac gacttttatg acaaaaactt 840

ccaagagttt attcctctta gaaccaaagt taaggaaatt cttcaggaag aagatgatct 900

agccgaaatt gtgcagctgg taggtaaagc atctctggca gaaacggaca aaatcacctt 960

ggaaattgcc aggcttctta aagaagattt cttgcaacaa aactcatact cttcttatga 1020

cagattctgt ccattctata aaactgtcgg tatgttgaga aacatgatcg gtttgtacga 1080

catggcgaga cacgctgtag aatcaaccgc acaatcagaa aataagatca cttggaacgt 1140

aataagagat tcaatgagtg gaattttata tcaacttagc agtatgaaat ttaaggatcc 1200

cgtaaaagat ggtgaagcta aaatcaaggc agattttgat caattatatg aagatattca 1260

gcaggccttc agaaacttag aagattaaat ctttttaagg aaattttcct attttgttca 1320

tcagtgtaag tttaaaaata tagcgatatt tatcaaaaag aataataagg cctctatccc 1380

tcacttctgt gaatattaat atggccgtac taaagatagt aactaaagat aggttttctc 1440

ttttttgata ttatcctgta caaaataaat tatgtaaatt gttgaatatg tgtatagttt 1500

ttttgggtga gggtacagtg cttattaaat actttttaaa catttttccc gccattccaa 1560

ttactattaa gttttttcgt tttaatactt ttttaaatat acaggtgctt aatatcgttt 1620

atattttcag tattacttgg ttttcttcat gtaaattgtt ttaaattttt cttttaccct 1680

tttaatcttg tatattacat tacccaatta aagttaattg tacagattaa gataaacgag 1740

tatcttataa catctattag attgttagaa tcaataaatg tagtgtaatt gttctgtttt 1800

gaacaaataa atgcatcaaa aaaaaaaaaa aaaaaaaaaa aaaggaaaaa aaaaaaaaaa 1860

gggcggccgc 1870

<210> 105

<211> 24

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 105

ctaatagatg ttataagata ctcg 24

<210> 106

<211> 24

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 106

cgagtatctt ataacatcta ttag 24

<210> 107

<211> 23

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 107

gtaatactga aaatataaac gat 23

<210> 108

<211> 23

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 108

atcgtttata ttttcagtat tac 23

<210> 109

<211> 20

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 109

agcactgtac cctcacccaa 20

<210> 110

<211> 20

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 110

ttgggtgagg gtacagtgct 20

<210> 111

<211> 21

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 111

gtgagggata gaggccttat t 21

<210> 112

<211> 21

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 112

aataaggcct ctatccctca c 21

<210> 113

<211> 19

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 113

aacttacact gatgaacaa 19

<210> 114

<211> 19

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 114

ttgttcatca gtgtaagtt 19

<210> 115

<211> 20

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 115

aggcctgctg aatatcttca 20

<210> 116

<211> 20

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 116

tgaagatatt cagcaggcct 20

<210> 117

<211> 21

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 117

ctgccttgat tttagcttca c 21

<210> 118

<211> 21

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 118

gtgaagctaa aatcaaggca g 21

<210> 119

<211> 20

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 119

gattgtgcgg ttgattctac 20

<210> 120

<211> 19

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 120

gtagaatcaa ccgcacaat 19

<210> 121

<211> 43

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 121

taatacgact cactataggg tacgtaagct tggatcctct aga 43

<210> 122

<211> 44

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 122

taatacgact cactataggg tgcaggtacc ggtccggaat tccc 44

<210> 123

<211> 41

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 123

taatacgact cactataggg cgcgtccgaa tttgatggtg a 41

<210> 124

<211> 41

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 124

taatacgact cactataggg gttacctctt tctccgcaac c 41

<210> 125

<211> 41

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 125

taatacgact cactataggg gaagtattga gagatttccc t 41

<210> 126

<211> 41

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 126

taatacgact cactataggg ggaatcggca ggcatttcag c 41

<210> 127

<211> 41

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 127

taatacgact cactataggg gcttacttag gtgcccgttt g 41

<210> 128

<211> 40

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 128

taatacgact cactataggg ataaaagtcg tccaatgctc 40

<210> 129

<211> 40

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 129

taatacgact cactataggg ccaagagttt attcctctta 40

<210> 130

<211> 41

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 130

taatacgact cactataggg gctatatttt taaacttaca c 41

<210> 131

<211> 40

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 131

taatacgact cactataggg gaataataag gcctctatcc 40

<210> 132

<211> 40

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 132

taatacgact cactataggg taaacgatat taagcacctg 40

<210> 133

<211> 40

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 133

taatacgact cactataggg acatttttcc cgccattcca 40

<210> 134

<211> 39

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 134

taatacgact cactataggg gatgcattta tttgttcaa 39

<210> 135

<211> 36

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 135

gcgtagaatt cgttcaaaac agaacaatta cactac 36

<210> 136

<211> 56

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 136

taatacgact cactataggg gcgtagaatt cgttcaaaac agaacaatta cactac 56

<210> 137

<211> 53

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 137

ggccttaagc tagcgcaatt ggatcccatt tattgattct aacaatctaa tag 53

<210> 138

<211> 26

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 138

ggccttaagc tagcgcaatt ggatcc 26

<210> 139

<211> 25

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 139

gatggtgaag ctaaaatcaa ggcag 25

<210> 140

<211> 53

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 140

ctgccttgat tttagcttca ccatcggaaa ttttcctatt ttgttcatca gtg 53

<210> 141

<211> 608

<212> DNA

<213> Diabrotica virgifera

<400> 141

gcgtagaatt cgttcaaaac agaacaatta cactacattt attgattcta acaatctaat 60

agatgttata agatactcgt ttatcttaat ctgtacaatt aactttaatt gggtaatgta 120

atatacaaga ttaaaagggt aaaagaaaaa tttaaaacaa tttacatgaa gaaaaccaag 180

taatactgaa aatataaacg atattaagca cctgtatatt taaaaaagta ttaaaacgaa 240

aaaacttaat agtaattgga atggcgggaa aaatgtttaa aaagtattta ataagcactg 300

taccctcacc caaaaaaact atacacatat tcaacaattt acataattta ttttgtacag 360

gataatatca aaaaagagaa aacctatctt tagttactat ctttagtacg gccatattaa 420

tattcacaga agtgagggat agaggcctta ttattctttt tgataaatat cgctatattt 480

ttaaacttac actgatgaac aaaataggaa aatttcctta aaaagattta atcttctaag 540

tttctgaagg cctgctgaat atcttcatat aattgatcaa aatctgcctt gattttagct 600

tcaccatc 608

<210> 142

<211> 533

<212> DNA

<213> Diabrotica virgifera

<400> 142

ggccttaagc tagcgcaatt ggatcccatt tattgattct aacaatctaa tagatgttat 60

aagatactcg tttatcttaa tctgtacaat taactttaat tgggtaatgt aatatacaag 120

attaaaaggg taaaagaaaa atttaaaaca atttacatga agaaaaccaa gtaatactga 180

aaatataaac gatattaagc acctgtatat ttaaaaaagt attaaaacga aaaaacttaa 240

tagtaattgg aatggcggga aaaatgttta aaaagtattt aataagcact gtaccctcac 300

ccaaaaaaac tatacacata ttcaacaatt tacataattt attttgtaca ggataatatc 360

aaaaaagaga aaacctatct ttagttacta tctttagtac ggccatatta atattcacag 420

aagtgaggga tagaggcctt attattcttt ttgataaata tcgctatatt tttaaactta 480

cactgatgaa caaaatagga aaatttccga tggtgaagct aaaatcaagg cag 533

<210> 143

<211> 1114

<212> DNA

<213> Diabrotica virgifera

<400> 143

cgtagaattc gttcaaaaca gaacaattac actacattta ttgattctaa caatctaata 60

gatgttataa gatactcgtt tatcttaatc tgtacaatta actttaattg ggtaatgtaa 120

tatacaagat taaaagggta aaagaaaaat ttaaaacaat ttacatgaag aaaaccaagt 180

aatactgaaa atataaacga tattaagcac ctgtatattt aaaaaagtat taaaacgaaa 240

aaacttaata gtaattggaa tggcgggaaa aatgtttaaa aagtatttaa taagcactgt 300

accctcaccc aaaaaaacta tacacatatt caacaattta cataatttat tttgtacagg 360

ataatatcaa aaaagagaaa acctatcttt agttactatc tttagtacgg ccatattaat 420

attcacagaa gtgagggata gaggccttat tattcttttt gataaatatc gctatatttt 480

taaacttaca ctgatgaaca aaataggaaa atttccttaa aaagatttaa tcttctaagt 540

ttctgaaggc ctgctgaata tcttcatata attgatcaaa atctgccttg attttagctt 600

caccatcgaa attttcctat tttgttcatc agtgtaagtt taaaaatata gcgatattta 660

tcaaaaagaa taataaggcc tctatccctc acttctgtga atattaatat ggccgtacta 720

aagatagtaa ctaaagatag gttttctctt ttttgatatt atcctgtaca aaataaatta 780

tgtaaattgt tgaatatgtg tatagttttt ttgggtgagg gtacagtgct tattaaatac 840

tttttaaaca tttttcccgc cattccaatt actattaagt tttttcgttt taatactttt 900

ttaaatatac aggtgcttaa tatcgtttat attttcagta ttacttggtt ttcttcatgt 960

aaattgtttt aaatttttct tttacccttt taatcttgta tattacatta cccaattaaa 1020

gttaattgta cagattaaga taaacgagta tcttataaca tctattagat tgttagaatc 1080

aataaatggg atccaattgc gctagcttaa ggcc 1114

<210> 144

<211> 1125

<212> DNA

<213> Leptinotarsa decemlineata

<400> 144

ggtgacatgg ccaccatcca ggtatatgaa gaaacttctg gagtaacggt gggagatcct 60

gtgttgcgta ccggtaaacc tctatctgtg gaacttgggc caggtattat gggttccatc 120

tttgatggta tccaacgtcc gctgaaagac atctgcgaca tgacggaaag tatctacatt 180

cccaagggtg tgaacgtgcc ttcactctcc agaactatca aatgggaatt caacccaatc 240

aacatcaagt tgggatccca cttgacaggt ggagatattt atggtatggt ccacgaaaac 300

acccttgtta agcacaaaat gatcctccca ccaaaatcta agggaacagt tacatacgtg 360

gcagaaccag gaaactatac cgttgatgaa gttgtattgg aaactgaatt tgatggagaa 420

aggtcaaaat acactatgtt acaagtctgg ccagttcgac aggcaagacc tgttagtgaa 480

aaactcccag ccaatcaccc gcttctcaca ggacagcgtg tattggactc tcttttccca 540

tgtgtgcaag gaggaaccac tgctattccc ggtgctttcg gttgtggtaa aactgtaatt 600

tcccagtcac tttccaagta ttccaactct gatgtcattg tgtatgtagg ttgtggagag 660

agaggtaatg agatgtctga agtattgaga gatttccctg aactgactgt ggaaattggt 720

ggtgagaccg aatctatcat gaaacgtacc gccttggttg caaacacctc caacatgcct 780

gtcgctgccc gtgaggcttc tatttatact ggtattaccc tgtctgaata tttccgtgat 840

atgggttaca acgtttctat gatggctgac tctacatcac gttgggctga agctttgaga 900

gaaatttcag gacgtttggc tgaaatgcct gctgattccg gttacccagc ctatttgggt 960

gctcgtcttg cctctttcta tgaacgtgct ggtcgcgtca aatgtttggg taaccctgac 1020

agagaaggat cggtttctat tgtaggagca gtatctccac ccggtggtga cttttcagat 1080

cccgttactt cagcaacttt aggtatcgta caggtgttct ggggt 1125

<210> 145

<211> 1125

<212> DNA

<213> Spodoptera frugiperda

<400> 145

ggtgacatgg ccaccatcca ggtatacgaa gaaacatcag gcgtaactgt aggtgacccc 60

gtgctgcgta ccggcaagcc cctgtccgta gagctcggac ctggtatcct cggctccatc 120

tttgacggta tccagcggcc actgaaggac atcaacgagc tcacacagtc catctacatc 180

cccaagggtg tcaacgtacc ctgccttgga cgtgatgtct cctgggaatt caaccccttg 240

aatgttaagg tcggctccca catcaccgga ggagacttgt acggtatcgt acacgagaac 300

acattggtta agcacaagat gttgatccca cccaaggcca agggtaccgt cacctacgtc 360

gcgccctccg gcaactacaa agtcactgac gtagtgttgg agacggagtt cgacggcgag 420

aaggagaagt acacgatgtt gcaagtatgg ccggtgcgcc agccccgccc cgtcactgag 480

aagctgcccg ccaaccaccc cctgctcacc ggacagagag tgctcgactc tctcttccct 540

tgtgtccagg gtggtaccac ggccatcccc ggcgccttcg gttgtggcaa gactgtcgtc 600

tcacaggctc tgtccaagta ctccaactct gacgtcatca tctacgtcgg atgcggtgaa 660

cgtggtaacg agatgtctga ggtactgcgt gacttccccg agctgacggt ggagatcgag 720

ggcatgaccg agtccatcat gaagcgtacc gcgctcgtcg ccaacacctc caacatgcct 780

gtagccgccc gagaggcttc catctacacc ggtatcaccc tctctgagta cttccgtgac 840

atgggttaca acgtgtccat gatggctgac tccacctctc gttgggccga ggctcttcgt 900

gagatctcag gtcgtctggc tgagatgcct gccgactccg gttaccccgc ctacctggga 960

gcccgtctgg cctcgttcta cgagcgtgcc ggacgcgtga agtgcctggg taaccccgac 1020

agggagggct ccgtgtccat cgtgggcgcc gtgtcgccgc ccggaggtga cttctccgac 1080

cccgtgacgg ccgccacgct gggtatcgtg caggtgttct ggggt 1125

<210> 146

<211> 1126

<212> DNA

<213> Agrotis ipsilon

<400> 146

ggtgacatgg ccaccatcca ggtatacgaa gaaacatcag gtgtaacagt gggcgacccc 60

gtactgcgta ctggcaagcc tctgtccgtg gaactgggtc ctggtatcct gggctccatc 120

tttgacggta tccagcgtcc tctgaaggac attaacgagc tcacacagtc catctacatc 180

cccaagggtg tgaacgtgcc cagtctatcc agggatatcg cctgggaatt tgagcccatg 240

aacctgaaga tcgggtccca catcactggc ggagacctgt acgccatcgt ccgcgagaac 300

accctggtga agcacaagat gttgatcccg cccaaggcca agggtaccgt cacatacatc 360

gcgcccgctg gcaactacca cgtcactgac gtggttctgg agacagagtt cgacggtgag 420

aaggagaagt acagcatgtt acaagtgtgg cccgtgaggc agccgcggcc ggtcgctgag 480

aagctccccg ccaaccatcc gctgctcacc gggcagaggg tactcgactc gctgttcccc 540

tgtgtgcagg gtggtacgac ggccatcccc ggagccttcg gttgcgggaa gactgtcatc 600

tcacaggcgt tgtccaagta ctccaactcc gatgtcatcg tctacgtcgg ttgcggagag 660

cgtggtaacg agatgtctga agtactgcgg gacttcccgg agctgaccgt agagatcggc 720

ggcgtcaccg agtccatcat gaagagaacc gcgctggtcg ccaacacatc caacatgcct 780

gtcgccgccc gagaggcttc catctatacc ggtatcactc tgtcggagta cttccgtgac 840

atgggctaca acgtgtccat gatggccgac tccacgtctc gttgggcgga ggccctccgt 900

gagatctctg gtcgtctggc cgagatgccg gcggactccg ggtacccggc ctacctggga 960

gcacgactgg cctccttcta cgagcgagcc ggacgagtca agtgtctggg taaccccgac 1020

agggaaggtt ccgtatccat cgtgggcgcc gtgtctcctc ccggcggaga cttctccgac 1080

cctgtgacgg ccgcgaccct gggtatcgtg caggtgttct ggggta 1126

<210> 147

<211> 1126

<212> DNA

<213> Helicoverpa zea

<400> 147

ggtgacacgg ccaccatcca ggtatacgag gaaacctcag gtgtaaccgt gggtgacccc 60

gtactccgta ccggcaagcc cctgtccgtg gagttgggcc ccggtatcct gggctccatc 120

tttgacggta tccagcgtcc cctgaaagac attaacgagc tcacacagtc catctacatc 180

cccaagggtg tgaacgtacc ctctctggct agggatgtca gctgggaatt cgttcccatg 240

aacgttaaga cgggctccca catcaccgga ggagacctgt acggtctggt gcacgagaac 300

acgctggtga agcaccgcat gctgatcccg cccaaggcca agggtaccgt cacatacatc 360

gcgcccgctg gcaactacaa agtcactgac gtagtgctgg agacggagtt cgacggcgag 420

agggagaagt acacgatgtt gcaggtgtgg ccggtgcgcc agccgcggcc cgtcaccgag 480

aagctccccg ccaaccatcc gctgctcacc ggacagaggg tgctcgactc actcttccct 540

tgcgtacagg gtggtacaac tgccatcccc ggagctttcg gttgcggcaa gactgtcatc 600

tcgcaggcgc tgtccaagta ctccaactcc gatgtcattg tgtacgtcgg gtgcggagag 660

cgtggtaacg agatgtccga agtactgcgt gacttccccg agctgacggt ggagatcgag 720

ggcgtgacgg agtccatcat gaagcgaact gccctcgtcg ccaacacctc caacatgcct 780

gtcgccgccc gagaggcttc catctacact ggtatcactc tatccgagta cttccgtgac 840

atgggttaca acgtgtccat gatggctgac tccacgtccc gttgggccga agccctgcgt 900

gagatctcgg gtcgcctggc ggagatgccg gccgactccg gctaccccgc atacctgggc 960

gctaggttag cttccttcta cgagagagcc ggacgcgtca agtgtctggg taaccccgac 1020

agggaaggtt ccgtatccat cgtgggtgcc gtatctcccc ccggaggtga cttctctgac 1080

cctgtaactg cggccacgct gggtattgtg caggtgttct ggggta 1126

<210> 148

<211> 1126

<212> DNA

<213> Ostrinia nubilalis

<400> 148

ggtgacacgg ccaccatcca ggtatacgaa gagacctcag gtgtgaccgt cggtgatccc 60

gtgctccgaa ccggcaagcc tctgtccgtc gagctgggtc cgggtatcct gggttccata 120

ttcgacggca tccagcgccc gctgaaggac atcaacgaac tgacgcagtc catctacatc 180

cccaagggag tcaacgtgcc ctgcctggcc aggaaccacg actgggagtt caacccgctt 240

aacgttaagg tcggctccca catcaccggc ggagacttgt acggtatcgt gcacgaaaat 300

accctggtga agcacaaaat gctgatgccg cccaaggcta aaggcaccat cacctacatc 360

gcgcctgccg gcaactacaa cgtcactgat gtggtgctgg agacagagtt tgacggcgaa 420

aagaactcct acaccatgtt gcaagtgtgg cccgtgcgcc agcccagacc ctgcactgag 480

aagctgcccg ccaaccaccc gctgctaact gggcagcgtg tgctggactc actcttcccc 540

tgtgtccagg gcggcaccac cgccatcccc ggcgccttcg gttgcggcaa gactgtcatc 600

tcgcaagcgc tgtccaagta ctccaactct gacgtcatcg tctacgtcgg ctgcggagag 660

cgtggtaacg agatgtctga ggtactgcga gacttccctg agctgagcgt ggagatcgac 720

ggcgtgacgg aatccatcat gaagcgcaca gcgctcgtgg ccaacacctc caacatgcct 780

gtggctgccc gtgaggcctc catctatact ggtatcaccc tatccgagta cttccgcgac 840

atgggttaca acgtgtcaat gatggcggat tccacatcgc gttgggcgga ggcgctgcgc 900

gagatctcgg gccgtctggc cgagatgccg gcggattccg gctacccggc ctacctgggc 960

gcccggctgg cctccttcta cgagcgagcg ggacgcgtga agtgtctcgg aaaccccgac 1020

agggaaggtt ccgtatccat cgtgggcgcc gtgtcgccac ccggaggaga cttctcggac 1080

ccggtgacgg cggcgaccct gggtatcgtg caggtgttct ggggta 1126

<210> 149

<211> 1125

<212> DNA

<213> Anthonomus grandis

<400> 149

ggtgacatgg ccaccatcca ggtatatgaa gaaacctcag gtgtaacagt aggcgaccct 60

gtcctaagaa cgggcaaacc tctgtcagta gaactgggac ctggtatcat gggttccatt 120

tttgatggta tccaacgtcc cttaaaagac attaacgact tgacccagtc catttacatc 180

cccaagggtg taaatgtgcc atgtctgtcc aggacagccc agtgggaatt caatcccgtc 240

cacatcaaga tgggttctca tttgaccgga ggcgacatct atggtatggt ccatgaaaac 300

actttggtga aacacaaaat gattttgcct ccaaaggcaa agggtactgt gacatatatc 360

gccgaggcag gcaactatac tgtggacgat gtggtacttg agaccgaatt cgacggagaa 420

cgcaccaaat acaccatgtt gcaagtgtgg cccgtacgtc aaccgagacc tgtgagcgaa 480

aaattgccgg ccaaccaccc actgctcacc ggacaacgtg tactcgattc acttttcccc 540

tgtgtgcaag gaggtaccac cgccatcccc ggcgctttcg gttgcggtaa aaccgtaatt 600

tcacaggcct tgtccaaata ttccaactcc gatgtcatca tttacgtcgg ttgcggtgaa 660

agaggtaacg aaatgtctga agtactacgt gacttcccgg agttaacggt cgaaatcgac 720

ggtgccaccg aatccatcat gaaacgtacc gctttggtgg cgaacacctc caacatgccc 780

gtggccgccc gtgaggcctc catttatacc ggaatcactt tgtccgagta tttccgtgat 840

atgggttaca acgtttcgat gatggccgac tccacctcac gttgggccga agccttaaga 900

gaaatttcag gtcgtttggc tgaaatgccc gccgattccg gttatcccgc ttacttggga 960

gcacgtttgg cctcgttcta cgaacgtgcc ggtcgcgtta agtgtttagg taatccggac 1020

agagagggct ccgtgtccat cgtaggcgca gtatcgccac ctggtggtga cttctcagat 1080

cccgtcactt ccgccacttt gggtatcgta caggtgttct ggggt 1125

<210> 150

<211> 1125

<212> DNA

<213> Tribolium castaneum

<400> 150

ggtgacatgg ccaccatcca ggtatacgaa gaaacttcag gtgttacggt gggtgatcca 60

gtcttacgaa ctggtaaacc cttgtcggtg gagctgggcc caggtattat gggttcgatt 120

tttgacggta tccagagacc gctgaaggac atcaacgagc tcacgcaaag tatttacatt 180

cctaagggtg ttaatgtgcc atcgttgtcg cgtacgacta agtgggagtt tgccccattg 240

aatatcaagt tggggtcaca tctgacaggc ggtgatattt acgggatcgt ccatgaaaac 300

actctcgtca agcataaaat gctgctgccg cccaaagcca aggggactgt cacatacgtc 360

gccgatcccg gaaattacac agtcgatgaa gtcgtcttgg agacggaatt cgacggcgag 420

aggaccaaat acaccatgtt gcaagtgtgg cctgtgcgtc agccccgccc tgtcagcgag 480

aaattgccag ccaatcaccc cctattaact ggtcaacgcg tactcgactc acttttcccg 540

tgcgtccaag ggggtaccac cgccattccc ggagctttcg gttgtggtaa gaccgtaatc 600

tcgcaatctc tctccaaata ttccaactct gacgttatca tttgcgtcgg ttgcggggag 660

cgtggtaacg aaatgtctga agtattgcgg gacttccccg aactgacagt cgaaatcgaa 720

ggccaaacag agtctatcat gaaacgtacc gctcttgtcg ccaacacctc taacatgcct 780

gtagccgccc gtgaggcttc aatttacacc ggtattacac tgtctgagta tttccgtgat 840

atgggttaca acgtgtcgat gatggccgat tccacctcgc gttgggccga agctttgaga 900

gaaatttccg gtcgtttagc tgaaatgccc gccgattctg ggtaccccgc gtatttgggg 960

gcccgtttgg cttcgtttta cgagcgtgca gggcgtgtta aatgcttggg taaccctgat 1020

cgtgaaggtt ccgtttctat tgtcggggcc gtatcgcccc ctggtggtga tttctctgat 1080

cccgtcacct cagctacctt gggtatcgta caggtgttct ggggt 1125

<210> 151

<211> 2860

<212> DNA

<213> Manduca sexta

<400> 151

ggttcgtctc atccatcttt ctcgtctcaa caggacacac agatagtaca aaatggcgag 60

caaaggcggt ttgaagacga tcgccaatga ggagaatgag gagaggttcg gatacgtgtt 120

cgccgtgtcc ggtcctgtcg taacagcgga gaagatgtcc ggatccgcta tgtacgagct 180

ggtgcgcgtc ggttacaacg agctggtggg agaaatcatc cgtcttgagg gtgacatggc 240

caccatccag gtatacgagg agacctcagg cgtcacagtc ggtgaccctg tgctgcgtac 300

cggcaagccc ttgtccgtgg aactcggccc cggtatcctg ggctccatct ttgacggtat 360

ccagcgtcca ctgaaggaca tcaacgagct cacacaatcc atctacatcc ccaagggtgt 420

gaacgtgccc tcgctcgcca gggaggttga ctgggaattc aaccccctca atgttaaggt 480

cggctcccac atcaccggcg gagacctgta cggtatcgtg cacgagaaca cgctcgtgaa 540

gcacaagatg ttgatgccgc cgcgcgccaa gggtaccgtc acctacatcg cgcccgccgg 600

caactacaaa gtcactgatg tagtgttgga gacagagttc gacggcgaga aggcgcagta 660

cacgatgttg caggtgtggc ccgtgcgtca gccccgtccc gtcaccgaga agctccccgc 720

caaccacccg ctgctcactg gacagagagt actcgactcc ctcttcccct gtgtccaggg 780

cggtaccact gccatccccg gagccttcgg ttgcggcaaa actgtcatct cacaggcgct 840

gtccaagtac tccaactctg acgtcatcat ctacgtcggt tgcggagagc gtggtaacga 900

gatgtctgag gtactgcgtg acttccctga gctgacggtg gagatcgagg gtgtgacgga 960

gtccatcatg aagcgtaccg ccctcgtcgc caacacatcc aacatgcctg tcgctgcccg 1020

tgaggcttcc atctacacag gaatcaccct ttccgagtac ttccgtgaca tgggttacaa 1080

tgtgtccatg atggctgact cgacctcccg ttgggccgag gctcttcgtg agatctcagg 1140

tcgtctagct gagatgcctg ccgattccgg ttaccctgcg tacctgggag cccgtctggc 1200

ctccttctac gagcgtgccg gtagagtcaa gtgtctcgga aaccctgaca gggaaggttc 1260

ggtgtccatc gtgggtgccg tgtcgccgcc cggaggtgac ttctcggacc ccgtgacggc 1320

ggccacgctg ggtatcgtgc aggtgttctg gggtctcgac aagaaactcg cgcagaggaa 1380

gcacttcccc tccatcaact ggcttatctc ttacagcaag tacatgcgtg ctttggatga 1440

cttttatgag aagaactacc ccgaattcgt gccccttagg actaaggtca aggagatcct 1500

gcaggaggaa gaggacctgt cagaaatcgt gcagttggtc ggtaaagcct cgctcgccga 1560

gactgacaag atcaccctcg aggtcgccaa actgcttaaa gacgacttct tgcaacagaa 1620

cagctactcg tcatacgatc gattctgtcc gttctacaag accgtgggca tgcttaagaa 1680

catcatctcg ttctacgaca tgtcgcggca cgcggtggag tccacggccc agtccgacaa 1740

caaggtcacg tggaacgtga tccgcgacgc catgggcaac gtactctacc aactctcctc 1800

catgaagttc aaggacccag tgaaagacgg cgaggccaag atcaaggcag atttcgacca 1860

gctgttggag gatatgtccg ccgccttccg taacctcgag gactaagcac agccgtacta 1920

cagtacagta cagtagggag cgcccacgag ccgcgccgcg acatcctccg cagccgagag 1980

gacatcttta tcgacttgtt ttcatgttgt catttttatt ataatttatt gattaatatg 2040

aggatatatt ttttcgtatt ctattcacgt ccggagcgtt ttgagacagt tttttcgagt 2100

ctggagtgtt ttgcatttta tcgatattat cgagtgtcgg gcgtcgttaa ggcggtgctg 2160

ttagcgaggt atgcgttatg acacacgcat atatcgtaat aacagcgttg tttaaacggg 2220

tctgtgcgca ggcgcagttc gtgggcggtc gtgttgttat agtaattatg tagtgttaaa 2280

tatattacaa catcgattcc agaggatggt gtcgcgggct agaactccga cagcgcgaaa 2340

gcctacaaag ggcgtggctt gtaaacggca caataaggcc gactaacaat tctccgttat 2400

ttgaaatagc agttcaaaca cagtcgtcac agtggcggta gtccgaatgt ttggacctgg 2460

gttggtgttt ataaagttcg cccaatctat tgtaaatata taacaggttc gctgttctag 2520

cccgcgggcc gttacggcgt ttagttgttt tatgaaatct atttatgtac tatatcgatc 2580

ggtaaaccgt gatttataat caaatatcct cttgcattcc acgttgttgt tagaaatata 2640

gaattcaaaa cgtttgttgt tttcgagagc tttttacgct taatatggat gttcattcag 2700

tattaatata atgcgtacga gtacgcagta acaatagtca gcactaatat gtctactcgc 2760

tgtttgaaat tctgtgacgt tacgtgttga gaaattatta taaataataa taaaatattg 2820

taaacaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2860

<210> 152

<211> 3097

<212> DNA

<213> Aedes aegypti

<400> 152

caggttggcc agtctttcag tcagtcagtc ttgtgatacc attttgcttc gctcggtgtg 60

tggagtttgc atttttccca tcccatctct ctcgacaact gcagcaccta agagcagaag 120

gaagcagagc aggaggaacg gatcgtaaca atgtccaccc tgaagaagat ctccgatgag 180

gaccgcgagt ccaaattcgg atatgtgttc gccgtatccg gtcctgtcgt cacggccgag 240

cggatgtccg gttcggctat gtacgagttg gtccgcgtcg gttactacga gctggtcggt 300

gagatcatcc gtttggaagg tgacatggcc accatccagg tatacgagga aacctccggt 360

gtcaccgtcg gcgatcccgt gctgcgtacc ggcaagcccc tctccgtcga actcggtcca 420

ggtattatgg gtagcatctt tgacggtatc cagcgtccac tgaaggacat taacgaactg 480

accagctcga tctacatccc cgaagggtgt gaacattccc tgcttgtccc gtacaggagg 540

ctggggattc aaccccttga acgtaaaggg ttgggctctc acatcaccgg aagagatctg 600

tacggtttgg tgcacgagaa taccctggtc aagcacaagc tgttggtccc gccacgcgcc 660

aagggtacag ttcgttacat tgctccaccc ggaaattaca ccgtcgacga catcattctg 720

gagacggaat tcgacggtga gatcaacaag tggtctatgt tgcaggtgtg gcccgtgcgt 780

cagccacgtc cagtgactga gaagttgccc gccaatcatc ctctgctgac tggtcagcgt 840

gtgttggatt cgctgttccc ttgtgtccag ggtggtacca ctgccatccc cggagctttc 900

ggttgcggta agactgtcat ctcgcaggcc ctgtccaagt actccaactc cgatgtcatt 960

atctacgtcg gttgcggaga acgtggtaac gaaatgtctg aagtattgcg tgatttccct 1020

gagctgtcgg ttgagattga cggtgttacg gagtccatca tgaagcgtac cgcgctggtt 1080

gccaacacct ccaacatgcc tgtcgctgct cgtgaagctt ccatctacac cggtattacc 1140

ttgtccgagt acttccgtga tatgggttac aacgtatcca tgatggctga ctcgacctct 1200

cgttgggccg aagctcttcg agaaatttcc ggtcgtctgg ctgagatgcc tgccgattcc 1260

ggttatcctg cctacctggg tgcacgtttg gcctccttct acgagcgtgc cggtcgtgtc 1320

aagtgtctcg gtaaccctga acgtgaaggt tcggtgtcca tcgtcggtgc cgtatcgccc 1380

cctggtggtg atttctccga tcccgtcaca tccgccaccc tcggtatcgt acaggtgttc 1440

tggggtctgg acaagaaact ggcccagcgt aagcatttcc cctcgatcaa ctggttgatc 1500

tcctacagca agtacatgcg cgcccttgat gacttctacg ataagaactt ccaggagttt 1560

gtacccactg cgtacaaggt taaggagatc ctgcaggagg aagaagattt gtccgaaatt 1620

gtgcagctgg tcggtaaggc atcgctggca gaaaccgata agatcaccct tgaggtagcc 1680

aagctgctca aggatgattt cctgcagcag aactcgtact cggcgtacga tcgattctgt 1740

ccgttctaca agacggtcgg tcgtatgctg cgaaacatga tcggattcta cgatatggct 1800

cgccacgccg tcgaaaccac cgcccagtcg gagaacaaga tcacctggaa cgtgatccgt 1860

gactcgatgg gcaacatcct gtaccagctg tcgtcgatga agttcaagga cccgggaagg 1920

atggcgaaga agatcaaggc cgatttcgac caactgtacg aagacctgca gcaggcgttc 1980

cgcaacctgg aagattaaat tctcccgcac attcgtggtc tcttcaatgc gaaattcttg 2040

aacagtttat tgtttcagta acatagcaaa gaaatgttcg tagcatagtg caaacaaaac 2100

atcaaaatga gaaacacgaa acacagcaaa agtgtagggc cctccttggc atcatgataa 2160

accaacaaca tccattaagt aaaatgcttc taggtcacca ttttacaggc gtatttaggt 2220

ttaaacattt atttacacaa attattgcaa gaaaaagatt aagagaacaa atctataaag 2280

cgagtgtaac atatacattt agaaacggcg aaacactaca acaactacag aaccacacgg 2340

cagaacagaa acaaatttta gtaggtaagt gatattgcaa gtgttgtccg acggcgtagg 2400

aaaaggttag cgaacggaat aacgttcaat cggaaattgt cttcgaaagt ttccgcttgc 2460

atgcgtgtct caaatgcgaa taaaacgtat aaacaatcgt ggtgaaactt aacatcagtg 2520

atgatataat caaaggggat taaaatgaaa cacgtggaca aaagatctat aaagaaaaac 2580

tctcagctag aatagttcaa gacgtggcga agcgtatcat aaatagaata atatgtaaac 2640

cacggttaat gggaaaataa gaagaaactt tcgattgagt atgttataga aacttatcca 2700

tgtatgatgt ataaatcgct aattaatcgt ataagaaata acagaacaag ttttattata 2760

ggtgtaagcc aatcaagttg ttatatcagt ttaaatatta tttagtgaat atagttttac 2820

ttttaatttt gtagtgtcgt ttttccatcg gtaggatcgg aaacgagaat cgatgattga 2880

ttgactgttg acaaatgaaa tgaaagttaa atttattatg cttttttgtt tgtgtgaaca 2940

gaattgaaga gccgccgcgt cgtttcggtc aatgcaagcg accgacggct cgtatctgtc 3000

ctgtacattt ttgtcgatga gcagaaaata tatgagaata aaaccctcta aaaaattgca 3060

ttccgcgtaa aaaaaaaaaa aaaaaaaaaa aaaaaaa 3097

<210> 153

<211> 2533

<212> DNA

<213> Drosophila melanogaster

<400> 153

cgaaaacacg cacacagact gcaagtgtgt tagataataa gtgcagcaca agtccacact 60

tgagtaaaat aatccctaaa aaagccgaat atcaattagt tttccaagga gcttgaaaaa 120

gtgcgtcgaa aaaacagaat aaagcaaaat gtccaacctt aagcgtttcg atgatgagga 180

gcgtgagtcc aaatatggac gtgtcttcgc tgtctccggt cctgtcgtca ccgccgaggc 240

catgtctgga tcagctatgt acgagttggt ccgcgtcggc tactacgagc tggtgggcga 300

gatcatccgt ctggagggtg acatggccac catccaggtg tacgaggaga cctctggcgt 360

aactgtcgga gatccggtgc tgcgtaccgg caagcctctt tccgtggagc tgggacccgg 420

tatcatgggc agcatctttg acggtatcca gcgtcccctg aaggacatta acgagctgac 480

cgaatccatc tacatcccca agggtgtgaa cgtgcccagt ttgtcccgcg tggccagctg 540

ggagttcaac cccctgaacg tcaaggtcgg ctcccacatc accggaggtg acctgtacgg 600

tctggtgcat gagaacactc tggtcaagca caagatgatt gtgaaccccc gcgccaaggg 660

aacagtgcgc tacatcgccc cctccggcaa ctacaaggtc gacgatgtcg tcctggagac 720

cgagttcgat ggagagatca ccaagcacac catgttgcag gtgtggccag tgcgtcagcc 780

acgtcccgtg accgagaagc tgcccgccaa ccaccccctg ctcaccggac agcgtgtgct 840

cgactcgctc ttcccctgtg tccagggcgg taccaccgcc attcccggag ctttcggttg 900

cggcaagact gtgatctcgc aggctctgtc caagtactcc aactccgatg tcatcatcta 960

cgtcggttgc ggtgagcgtg gtaacgagat gtctgaggta ctgcgtgact tccccgagct 1020

gtccgtggag atcgacggtg tcaccgagtc catcatgaag cgtaccgccc ttgtggccaa 1080

cacctccaac atgcctgtgg ctgctcgtga ggcctccatc tacactggta tcaccttgtc 1140

cgaatacttc cgtgatatgg gttacaacgt gtccatgatg gctgattcca cctcccgttg 1200

ggctgaggct cttcgtgaaa tttctggtcg tctcgctgag atgcctgccg attccggcta 1260

cccagcctac ttgggagccc gtctggcctc cttctacgag cgtgccggtc gcgttaagtg 1320

cttgggtaac cccgagcgcg agggatccgt gtccattgtc ggagctgtgt ctcctcctgg 1380

tggtgacttc tccgatcccg tgacctccgc cactctgggt atcgtgcagg tgttctgggg 1440

tctcgacaag aagttggccc agcgcaagca cttcccctcg atcaactggc tcatctccta 1500

ctcgaagtac atgcgtgctc tggatgactt ctatgacaag aacttccccg aattcgtgcc 1560

gctgcgtacc aaggtcaagg agatcctgca ggaggaggag gatctgtctg agatcgtgca 1620

actggtcggc aaggcctctc tggccgaaac cgacaagatc acgctggagg tggccaagct 1680

gctgaaggac gatttcctgc agcagaactc ctactcctcg tacgatcgct tctgcccctt 1740

ctacaagacc gtgggcatgt tgaggaacat catcgacttc tacgacatgg cccgtcactc 1800

cgtggagtct acggctcagt ctgagaacaa gatcacctgg aacgtgattc gtgaggcaat 1860

gggcaacatt atgtaccagc tgtcatccat gaagttcaag gaccccgtta aggatggtga 1920

ggccaagatc aaggctgact tcgagcagct gcacgaggac ctgcagcagg ccttcagaaa 1980

tctggaggac tagagaccgc gctggcccta cttttacact ctaatcttat atttgttata 2040

tagttaacgt ttaaaaatga aagcagtcaa aaaccatccg aaaaagccta atcaaacacc 2100

aacaattccg tgctgcattc gatgaaaaac aaaagtccaa caaataccac aacttcttgg 2160

tgcctgcgag agatgtaaac attccggcct gcggttaata ctttccccta accacgcccc 2220

ctccgcccct tgaagggcaa ctctaggcaa cagcaactac aacgtcctgc tatgtacttc 2280

catttacaac aacaacacca acatacactt gaataaaagt acacggacac tggcgcacac 2340

acaacacata cataaaagac acaaatacaa atgcatgcat aaatagtatt attgtttaat 2400

gaatggaaat tcttgtttat ttgtgaaaaa agtcatgttt tctccctgtt tgtttgttaa 2460

atttatgtaa atatttaaag tatgaaatat taaatgtacg aataaagtgc aacaacaaat 2520

acatttaatg taa 2533

<210> 154

<211> 603

<212> DNA

<213> Bombyx mori

<400> 154

atgagttccc tcaagctgca gaagaggctt gcagcctctg ttatgcgatg tggtaaaaag 60

aaggtgtggt tggatccaaa tgaaatcaat gagatcgcaa acaccaactc cagacagaac 120

atccgtaaga tgatcaagga tggtctcgtc atcaagaaac ctgtagcagt acactcccgc 180

gctcgtgtcc gcaaaaacac agaagcacgt agaaagggtc gtcactgtgg ctttggtaag 240

agaagaggta cagccaatgc gcgtatgcca cagaaggaac tatgggtaca aagacaaagg 300

gttttaagaa aattgctcct gaagtacaga actgccaaga agattgacag gcatctatac 360

cactcactct acatgaaggc gaagggtaat gtgttcaaga acaagcgtgt gctcatggag 420

tacatccaca ggaagaaggc tgagaaggcc aggacgaaga tgcttagcga ccaggctgag 480

gcccgccgca ataaagtgaa ggaggcacgc aagcgccgcg aggaacgtat tgccgccaag 540

aaggaggaac tgctgcagac cttcgctaga gaagacgaag ccgcgcttac cgctaagaag 600

taa 603

<210> 155

<211> 612

<212> DNA

<213> Drosophila melanogaster

<400> 155

atgagttctc taaagctcca gaagaggctc gcagcctccg tgctgcgatg cggcaagaag 60

aaggtctggt tggatcccaa tgaaatcaac gagatcgcta acacaaactc gcgtcagaac 120

attcgcaagc ttatcaagga tggtctgatc atcaagaagc ccgtcgtggt ccactcccgt 180

taccgtgtgc gcaaaaacac cgaggcccgc cgcaaggacc gtcactgcgg attcggaaag 240

cgtaagggta ctgcgaacgc ccgcatgcct accaagctgc tgtggatgca gcgccagccg 300

ttctgccgcc gcctgttgaa gaagtaccgc gacagcaaga agattgacag gcacctgtac 360

cacgacctgt acatgaagtg caagggtaac gtgttcaaga acaagcgcgt cctcatggag 420

tacatccaca agaagaaggc tgagaagcag cgcagcaaga tgctggctga tcaggccgag 480

gctcgccgac agaaggtgcg tgaggcccgc aagcgccgcg aggagcgtat tgccaccaag 540

aagcaggagc tcatcgccct gcatgctaag gaggacgaga tcgctgccaa ggccgccacc 600

gcgggtcact aa 612

<210> 156

<211> 567

<212> DNA

<213> Anopheles gambiae

<400> 156

atgcgatgcg gcaagaagaa ggtgtggttg gatcctaatg aaatcaacga gattggaaac 60

accaactcgc gacaaaacat tcgcaaactg atcaaggatg gtctgatcat caagaagccg 120

gtggtggtcc actcgcgtta ccgtgtgcgc aaaaacacga tcgctcgccg caagggtcgc 180

cactgcggtt atggtaagcg aaagggtacg gccaatgccc gtatgcccca gaagctgctc 240

tggatgaacc gtatgcgtgt gctgcgtcgt ctgctgaaga agtaccgtga ggcgaagaaa 300

atcgaccgtc acctgtacca cgacctgtac atgcgtgcga agggtaacgt gttcaagaac 360

aagcgtatcc tgatcgagca catccacaag aggaaggcgg agaaggcccg ctccaagatg 420

ctgagcgatc aggccgaagc caagcgtacc aaggttcgtg aggcccgtcg tcgtcgcgag 480

gaacgtattg ccaccaagcg ccaggagctt ctgcagacga tcgctaagga agaggagacc 540

gcgcagcatg ttgccgctac tggaaag 567

<210> 157

<211> 652

<212> DNA

<213> Diabrotica virgifera

<400> 157

cacgttgaga ggtgcatttg cacgatgagt tccttaaaac ttcagaagag gctagcagcc 60

tctgttatgc gatgtggtaa aaagaaagta tggttggacc ctaatgaaat caacgaaatt 120

gccaacacta actcaagaca gaacatccgt aagttgataa aggatggtct tattattaag 180

aagcccgtag ctgtacattc ccgtgcccgt gttcgcaaaa acactgaagc ccgcaggaaa 240

ggaaggcact gcggttttgg taaaaggaag ggtactgcta atgcccgtac cccgcaaaag 300

gaattatgga ttcaacgcat gagagttttg cgtcgtctcc ttaaaaaata cagggaagct 360

aaaaaaattg acagacatct ataccactca ctctacatga aggccaaggg taacgtattc 420

aagaacaagc gtgtccttat ggaatacatc cacaagaaga aggcagagaa agcccgtgcc 480

aagatgttgg cagaccaggc caatgccaga aggatgaagg taaaacaggc tagagaaaga 540

cgtgaggaac gtatcgccac aaagaaacaa gaagttttgc agaactacat gagggaggat 600

gaagctgcgg ccactaagaa ataagttaat tgttttataa gatgactata tt 652

<210> 158

<211> 402

<212> DNA

<213> Anthonomus grandis

<400> 158

tgagatgtgg taagaagaag gtatggttgg accctaatga aattaacgag attgccaaca 60

ccaactcgag gcaaaacatc cgtaaattga tcaaggatgg tttgatcatt aagaaaccgg 120

tggcagtgca ctctagggct cgtgtccgta aaaacacaga agctcgcagg aagggaaggc 180

actgcggttt cggtaagagg aaaggtacag cgaacgctcg tatgcctcaa aaggaactat 240

ggatccaaag gatgcgtgtc ttgaggcgtc tcctgaaaaa atacagggaa gccaaaaaga 300

tcgacaggca tctgtaccac gccctgtaca tgaaggccaa gggtaacgtg ttcaagaaca 360

agagagtgtt gatggaatac atccacaaga agaaggctga ga 402

<210> 159

<211> 403

<212> DNA

<213> Tribolium castaneum

<400> 159

tgagatgcgg taagaagaag gtatggttag atccgaacga aatcaacgag atcgccaaca 60

cgaattcacg ccagaacatc cgcaaattga tcaaagatgg tctcatcatc aaaaagcccg 120

tcgctgtgca ctccagagcc cgcgtccgca agaacacgga ggcccgcagg aagggacgcc 180

attgcggctt cggcaagagg aaaggtacag ccaatgcgcg tatgccccag aaggagctct 240

ggatacagag gatgcgggtc ttgaggaggc tcctcaagaa gtatcgcgag gccaaaaaga 300

tcgacagaca tctttaccat tcgctgtata tgaaggccaa gggcaacgtc ttcaagaaca 360

agagggtcct tatggagtac atccacaaga ggaaggccga gaa 403

<210> 160

<211> 23

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 160

ggtgacatgg ccaccatcca ggt 23

<210> 161

<211> 24

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 161

accccagaac acctgyacra tacc 24

<210> 162

<211> 44

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 162

ttaatacgac tcactatagg gagaccagtg tgctggaatt cgcc 44

<210> 163

<211> 44

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 163

ttaatacgac tcactatagg gagaggatat ctgcagaatt cgcc 44

<210> 164

<211> 45

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 164

ttaatacgac tcactatagg gagacctgtc cgtagagctc ggacc 45

<210> 165

<211> 44

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 165

ttaatacgac tcactatagg gagaggcacg ctcgtagaac gagg 44

<210> 166

<211> 22

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<400> 166

tgmgatgygg yaaraagaar gt 22

<210> 167

<211> 26

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<220>

<221> misc_feature

<222> (23)..(23)

<223>n is a, c, g, or t

<400> 167

tgmgatgygg yaaraagaar gtntgg 26

<210> 168

<211> 25

<212> DNA

<213>artificial sequence

<220>

<223>it synthesizes

<220>

<221> misc_feature

<222> (6)..(6)

<223>n is a, c, g, or t

<400> 168

ttctcngcct tcytcytgtg gatgt 25

<210> 169

<211> 2713

<212> DNA

<213> Diabrotica virgifera

<400> 169

cggacgcgtg agcggacgcg tgggcggacg cgtgggcgga cgcgtgggcg gacgcgtggg 60

cggacgcgtg ggcggacgcg tgggtggcaa cccacgcgtc cgctagttag tgctcgccgg 120

cgagcgcccg cgcccccgcc ccgaaagctg cattactagc taatctgaac gtctgtcgta 180

attttgtttc atttgtggtg taaaagttaa aactcatcaa ccaaaatgcg tgaatgtatc 240

tcagtccatg ttggccaagc cggagtccaa atcggtaatg cctgctggga gttgtactgc 300

ctggaacatg gcatccaacc tgacggtcag atgccatcag acaagactgt tggaggagga 360

gatgacagtt tcaacacatt cttcagtgaa actggtgccg gcaaacatgt acctagagca 420

gtatttgtag atttggaacc aacagtagta gatgaagtac gtaccggcac ataccgtcaa 480

ttgttccacc cagaacaact catcactggc aaagaagatg ccgccaataa ctatgctaga 540

ggtcactata caattggtaa agaaatagtt gacttggtat tggacagaat ccgtaaattg 600

gctgatcaat gtactggact tcaaggtttc ttgattttcc actccttcgg tggtggtact 660

ggatctggtt tcacttcttt gttgatggaa cgtctatctg ttgactatgg taaaaaatca 720

aaactggaat tcgccatcta cccagctcct caagtatcta ctgctgtagt agaaccatac 780

aactccatct tgaccaccca caccactctt gaacactcag actgtgcctt tatggtagat 840

aatgaagcca tctatgacat ctgcagacgt aatctagaca tcgagcgccc aacctacacc 900

aacttgaaca gacttattgg ccaaatcgta tcctcaatca cagcttctct aagattcgat 960

ggtgctctaa atgttgactt gacagaattc caaactaact tggttcctta ccctcgtatt 1020

cacttccctc ttgtcaccta tgccccagta atttccgctg aaaaggctta ccatgaacaa 1080

ctttccgtag ctgaaatcac caatgcctgt ttcgaacctg ccaaccagat ggtaaaatgt 1140

gatcccagac atggtaaata catggcttgc tgtatgttgt acagagggga tgttgtacca 1200

aaggatgtaa atgctgctat tgcaaccatt aagaccaaac gtaccatcca attcgtagac 1260

tggtgtccaa ctggtttcaa agtaggtatc aactaccaac caccaactgt tgtacctgga 1320

ggtgatttgg ctaaagtaca acgtgccgta tgcatgttgt ccaacactac agctattgct 1380

gaagcctggg caagattgga ccacaaattc gatcttatgt atgccaagag agctttcgtc 1440

cactggtatg taggagaggg tatggaagaa ggtgaattct ctgaagctcg tgaagatttg 1500

gctgcttttc ttatcatctc tatttttttt acgatcctta accgcataac accgtatcta 1560

tcattgtgaa attaggtgtg aaaggtgttt aaaaatgagg ttccttattc tacttgccgt 1620

attggctgta gctgtgaatg ctacatcaat ccaccaacaa tgggctacat ttaaggtaaa 1680

ccattccaag aagtacggac atcttaaaga agagcaagtt cgcttccaag ttttctctca 1740

aaatctccgc aaaattgaag aacacaatgc aagataccag aatggtgaag tgtccttcta 1800

cttgggggtt aatcagttcg cagatatgac ttcagaggaa ttcaaggcta tgcttgactc 1860

ccaactcatt cacaagccta agcgaaacat tacatcccgc tttgtagctg atcctcaatt 1920

gactgttcca gaatcaattg actggagaga aaagggggca gttgctccca taagggacca 1980

agggcaatgc ggatcatgtt gggcatttag tgcagctggt gctcttgaag gacaaagatt 2040

tttaaagcag aacgtactag aagtactgag tacccaacag ttagtagatt gttccggtga 2100

ttacgacaat gaaggctgca atggtggttg gccccattgg gcatataact acattaaaga 2160

tcatggcctc tgtctagagt ctgattacaa gtatcaagga ttagacggtg actgcaaaca 2220

gtgtaatccg gttatcaaaa ccatcaatgg ctatgcatct gtagatcaaa ctgaagaagc 2280

acttaaggag gctgtaggta ctgctggccc aatatcagta tgtgtcaacg ctaattggga 2340

ctggcaactg tacagcgggg gtatccttga tagccaaagt tgtccaggcg gcattttaaa 2400

ccatgcagtt ttagctgttg gatatggttc agaaaatggt aaagactttt ggcttatcaa 2460

gaattcatgg gacacttatt ggggagaagc aggttatttg agattagtac gtggtacaaa 2520

ccagtgcggt atcaatgaag tggccgatta tcctctccta taattttaaa aattgtcatg 2580

ccttacagtt tatataatga aacatgaata aaaatattat aactttaaaa aaaaaaaaaa 2640

agggcggccg acttttttta aaaaaaaaaa aaaaaaaaca aaaaaaaata aaaaaaaaaa 2700

agggggggcc ccc 2713

<210> 170

<211> 1363

<212> DNA

<213> Diabrotica virgifera

<400> 170

cccacccgtc ccgtggtcga gaaaagtact aatagtgata tctacgtttt tcgtttgttt 60

aaaatgtagt gacatttctg ttaaagtctt caaaaacgga gacgtagtta atttaaatat 120

taactcaatt tctgagttaa agcaaaatgt agtccacaga ggtgaactag acgacattga 180

aattgaggac caaaatgttc cagtagtacc aaacaatttg ctgaatggaa tcactgccac 240

ggaacttcac attattagat cccaagtaag agatgttgaa cctggtgcat tcgatggagc 300

ttctatggtt aacctaatgt tgtatgaaaa ccaaattgca agaataagaa aaggtatatt 360

taacaaaaac tcatttaata tacttgcttt gcagaataac gttatatcta atatagaaga 420

tgaagccttt gacggtacaa ccatcgcgat actggacttt ggttttaaca agatggaaaa 480

attgacttca aaaatgttcg ctggttcaaa tattacaaat cttaacttac aatcgaacct 540

aataagtaac atagaagatg gtacctttca gaaaatcgat aatttgaata aattagactt 600

aagcggtaac caattggaag ttattggaca cgtctttaga aacctgacaa acctaaatga 660

attgcacttg gatggaaacc gaatcaaaac acttgaacct ggatgctttg gtggttctgg 720

gatctactgg ctttattttg caggtaacca actgactcat attgtaaagg gagtgtttta 780

taaagtacca gtatccttat tggatttcac taataacaaa atttcaaaaa ttgataaagg 840

agccttagct ggtctttcaa cgctaacatt tgttcagtta tctaataaca atataggaga 900

tttgaagctg tccactcttg gcgatctcaa tactgcttta aatggtctat ctttgagtga 960

taacggcatt tcaaatatcg atattggagt gttcaaaaat actaaaatcg atatgttgga 1020

cttaagcaaa aaccatataa aatcaattaa aaaaggactg ttccagaatg ttaaaatgta 1080

cactattaat ttgagtgaaa atgaaattac tgaaatagag gaagatgctt ttggtgatat 1140

cgaggattta agtcacatag atgtgagctt gaacaaactt acagaagtta agaagagaat 1200

gttcagtcta ccattggatg aagttaattg gaagataatg taataactaa aatcgataat 1260

gatgcccctc gtgtccttcc gctgtcacgt cttcagataa aaataatcct attggctgca 1320

agaacaaagc ttaaaataat ggtgtattta ataataatgg aat 1363

<210> 171

<211> 1215

<212> DNA

<213> Diabrotica virgifera

<400> 171

aaaagagtga ggaaacaggt taattataat gacggaggaa tgacaactga cacacgagaa 60

gatacgacat ggcaagaaaa tctctctgat taccattctg acttttctgc gggatcggat 120

gaggataagg aagacgatga tttcgatgag aagaacgacg ccgatttaag cagaaggagt 180

cgaagaaaga tggaaaggaa agacgagaag gatcgtcctt taccaccgtt actagccaga 240

gttggcggca atattgaagt actcggtttt aatgccaggc agcgtaaagc gttccttaat 300

gctattatgc gctacggaat gccaccacaa gacgctttca attcacagtg gctggtgaga 360

gatcttcgag gaaaatctga gaagatattc aaggcttacg tgtctctctt tatgaggcat 420

ctttgcgaac ctggtgcaga taatgctgat acatttgcgg acggtgtgcc gagggaagga 480

ctgagtaggc aacatgtttt gacaaggatt ggtgtgatgt cacttataag aaagaaggtt 540

caggagttcg aacacatcaa cggcgagtat agcatgccgg aagtaatcaa aaagagcatt 600

atggatcaaa ataaaatcaa tgccgccggc accgccacca caagcgaagc agaaacgcct 660

aaaagtgcta ctaccagtac tagtgctacg ccagctacaa gtgctgctcc cagtcccgct 720

cccacacaag gagaagataa agataaggat aaagattccg ttcagagtga cgaaaataaa 780

gataaagaag tggttaataa aacggaaacc gaagatgaag agaagaaaac gggagaatct 840

tcaacagaaa agccgaaaac tgaaccggaa gaagtgaaag aagcttctcc gaaaaccgaa 900

attcccgaag ctagttccga agctgataaa tctgagatca aatccgaagt cgatacctcg 960

tctgtaacca gcgaggaaaa gaaagaagag aaagaggaag aggccaaaaa ggaagaaccc 1020

gaagagacca aaatggaaat acaggaggag gaacttgtta aagaggagaa aaaagaagaa 1080

gaggatgata agaagaagga ggaaattaag aaagaggtgg aaaagaagga agaggatgac 1140

gttatggtta ttgatgatga taaagataag aaggacaaaa aggaaatcga tctcgaagcc 1200

aagaagcgtt tcatg 1215

<210> 172

<211> 3363

<212> DNA

<213> Diabrotica virgifera

<400> 172

accacgcatc cgcccacgcg tccgcccacg cgtccgccca cgcgtccgat tgaattactc 60

taatattttt tttttatttt cattttttat ttatttaata atttaaacta ttttaacttt 120

aattataaac caaaatattt taaaactaaa aaaactaatt taaaattcaa ttgaaaatga 180

taataaattt attttcttct ttcgacccta catctaattt taatttacca ataaactgat 240

taagaacagt attaggtcta ttaattattc catctagatt ttgattaatc ccctctcgtt 300

ataattattt atgaataaag attattataa cattacataa agaatttaaa gttttaattg 360

gaaattataa atcccaagga agaacattaa tttttatctc actatttaga ttaattttat 420

ttaataattt tcttggatta ttcccgtata tttttactag aacaagacat ataactttaa 480

cattaagatt agctttacca ttatgattga gatttataat ttatggatga ataaataata 540

ctattcatat attagctcat ttagttcctc aaggaactcc tccgatttta ataccattta 600

tagtttgtat tgaaacaatt agaaatgtaa ttcgacctgg aacattagca gtacgtttaa 660

ctgctaatat aatcgcagga cacttattaa taactctttt aggaaatact ggaccaataa 720

tatcaatcta tatattaaat attttaatta ttgtccaact tttactatta attttagaaa 780

cagcagtatc tataattcaa tcttatgtat ttgctgtttt aagaacacta tattctagag 840

aagtaaatta atgtcaaatc ataaaaatca tccttatcat ttagtagata ttagaccatg 900

acctttatta ggagctttta gagcaatatt aacaatatta ggaataatta aatgatttca 960

tttatataat aataatttac taataattgg attattaatt acaagattaa ttatatatca 1020

atgatgacga gatattgtac gagaaggaac ttatcaaggc cttcatacct ttagtagtta 1080

ctaaaggttt acgttgagga ataattttat ttattacttc agaagtatta ttttttatat 1140

catttttttg aggatttttt catagatcat tagcaccaac tattgaatta ggaatacttt 1200

gacctcctaa aggaattcaa gcctttaacc cattagaaat ccctttatta aatactttaa 1260

ttcttttaac ttcgggatta actgtaactt gagcccatca tagcctaata gaaaaataat 1320

ttttctcaag gacttcaagg attaattttt acagtaacat taggaattta ttttactatt 1380

ttacaaggat atgaatatat tgaatcacct tttgcaattt ctgattcaat ttatggatct 1440

tcatttttta tagcaacagg ttttcatgga ttacatgtaa ttattggaac aaccttctta 1500

ttaatttgtt taattcgcca ttatttaaat catttttcat cgacacatca ctttggtttt 1560

gaagcagcag cttgatactg acattttgta gatgtagtat gattattctt atatatttca 1620

atttactgat gaggtagatt gagtaaatac gtctaccgtt ctcctttaaa tgacgctatt 1680

tgtgctcctg aaagagagca aaagtgccca tggaatccga aggctgacag atctacctca 1740

attcatacaa cggtcaattg gcaagctcca cgtccaaaaa tactgccaaa tgccttgcac 1800

gcaattggta atactccatt gatcaagctt aacagaatac ctcagcaaga aggtttggaa 1860

tgtgatatat atgtaaaatg tgagttcttt aatcctggtg gatcagtaaa agatcgcatg 1920

gcaaacagaa tactgacaga tgccgagaat gaaggtatct taaaaccagg atgtaccatt 1980

atagagccgt cttcaggaaa tactggcatt ggtttggcta tggcagctgc tattaaagga 2040

tataggtgta taatcgtaat gtcagaaaaa atatccaaag agaaagaata cgtaatgaga 2100

gctttgggag ctgaagttat tagatgtcct gtcacagcta attcgttttc tccatatgga 2160

atgtttggta ctgtccatcg tttatcaaaa gaaattccca acagtattat ttttgatcag 2220

ttctctaatc ccggaaatcc actgactcac tacgatacta cagcagaaga aatttatgat 2280

caatgcgaca aaaaagtaga tatgataata atgggagctg gaacaggtgg taccgttacg 2340

ggtataggaa gaaaatttaa agagatttct cccaatacgg aaatcgtttg tgcagatcca 2400

attggatcat cttttgcttt accagaaatt ataaataaaa ctgacgttac tttctgggag 2460

atagaaggta tgggctacga tttcattccc tcaaccttag accgcaaagt cattgacact 2520

tggattaaag taggtgatga gaatgccctg ccaatggcaa gaaggttgat taaggatgaa 2580

ggccttttga ttggggctag cagtggagct atgatgtggg cggctattca agcagcgaaa 2640

gctaaaaatt atggccctgg taaaagggtt gtagttatgt taccagatag tattaggaac 2700

tacttaacaa agttcgtatg tgaccaatgg atggaagagc gaaatcttca gccttgtgta 2760

aatacaaaca accacccgtg gtggaattta aatgtctccc aattaaatct tcctgtacca 2820

caaactgtac cgataaattc ttccattgaa cagactttga atctaatgaa gaaacttgga 2880

cttaaccaga tacctgcatt ggatgatcaa gggggtgttg ttggagtact ttcaatgcag 2940

ctaattatta acaaacttac atctggtaat gctacactca atgacccaat agcagatgct 3000

atagaccgac tttatcccag agttgagaaa tctgctaata ttggactcgt ctcaagagta 3060

ttggaacgtg agccttattt ggtaattttg gatacacaag gtaaaggacc ttccaagata 3120

aataagcctg caggcgttgt aactccttta gattttctac agtttatcca gaagcagcat 3180

taaatataga ggagactaat atttccacca tttaacaaaa gtaatcacca taaagtgata 3240

aaataaataa tacctaatat aatagaaata ttagaaataa tagaaattat agattataat 3300

aaataaataa gtattataat caaaaaaaaa aaaaaaaggg gcggcgcccc tttttttttt 3360

ttt 3363

<210> 173

<211> 843

<212> DNA

<213> Diabrotica virgifera

<400> 173

ctcataatat tatgccaaaa atttaaaata gtatttcgag gagaaattct ttataaaaaa 60

aattgtatct ttctttatat ttctggtgat atttatgaaa aacacaccag caatatgttt 120

gctatatcga ggagatcaat agctgcttta acacaaatca gatcaaagac agacaaggcc 180

gttttggacg aaattattcg agtagatcat gctggagaat tgggagcaga tcgtatttat 240

gcaggccaga tgttcattct aggcagcact tcaaaagcac ctttgataag acatatgtgg 300

gaacaagaaa aacatcacaa agctacattc gaagatctaa ttagaaaaaa acgtgttaga 360

cctacagtaa tgactcctat ttggaatgtt gcaggcttcg ccttaggagc aggatcagca 420

ttgcttggag acaaagcagc tatggcgtgt actgtggctg tcgaaacagt aattgtagat 480

cattataatg accaactgag aactctgttg gaagatccag agtgtgataa agagcttgta 540

gaaactatta agaagtttag agacgaggaa caagaacatc atgaccatgg cattgatcag 600

ggagcaaagc agactccttt ttatgaagcg tttactaatg tcattaaagc tggatgcaaa 660

gcagctatag caatatcgaa agtagtttaa cttgtgttta tgtacatatt atgtagttga 720

ttgtgaaata tatgttgtta aatttgtaaa gtattgacag tattatatat ttttggatat 780

aaagttagtc ccactatgtg tacagaaaaa tctaataaaa taaaatcaat ttaaatacag 840

att 843

<210> 174

<211> 704

<212> DNA

<213> Diabrotica virgifera

<400> 174

aaagaccttg aagatcttct accatggcat gagaagcctg tgaagataca ggtcttagtt 60

ctagacatgt agagggacat aatctgtgtt atattttaga tcacaaaaga gtgcaattaa 120

ggctgttgtg tgatgagtac tagacaggaa gaaagagcag actcccagga tatctcgtgg 180

ttgagtctta tgatcaacaa tatacatatt ttgcatcaga atcttgatag atcaggctat 240

cttctaatta ttcttctatt ttttgttttt ttctcgagtt agctcagttt tttcctattt 300

tttttttggt acttttgcta gatatatttt acacatactc atttttatga gtcttaagtg 360

caatacgttg gtaacggaat actggttatt tgtcattcct tccttgtcgt acctaggttg 420

tttctcttta cttcaatagt tacaatgact atttgatttt tgattgtgtc aagctataca 480

agaaataaga gagtaatcag gagagagaaa gagagaaaag attgagtaat ctgtaagaca 540

tcaaaagatg aaaagaccta gaacatcttc tatcatagtt gtaagaggat gatgaaaggc 600

acaggtatta gttcaatcca gataaaaaat gaagtgttaa aagacataga agaaaaactt 660

ttgtgtacag tcgtacagta gacataggaa tacagcgaag atgc 704

Claims (45)

1. a kind of method for controlling without vertebra pest infestation comprising: it is mentioned in the diet of no vertebra harmful organism For reagent, the reagent includes that can play a role to inhibit target gene in the harmful organism when being absorbed by the harmful organism The ribonucleic acid of expression, wherein the ribonucleic acid by be the target sequence ribonucleotide or with the target The ribonucleotide of sequence complementation is constituted, wherein the ribonucleotide be from selected from by SEQ ID NO:1 to The DNA sequence dna transcription for the group that SEQ ID NO:143, SEQ ID NO:169 to SEQ ID NO:174 and its complementary series are constituted comes 's.

2. the method for claim 1, wherein the no vertebra harmful organism is selected from by the biological and right of plant pest Animal pest biology constitute group, wherein the biology to plant pest be selected from by harmful insect, mite class harmful organism and The group that nematode pests are constituted.

3. can play a role when in the diet for being provided in no vertebra harmful organism by isolated ribonucleic acid and control nothing Vertebra pest infestation, the effect is realized by inhibiting the expression of target sequence in the harmful organism, described Ribonucleic acid includes the ribonucleotide come from the transcription of following DNA sequence dnas, the DNA sequence dna with selected from by SEQ ID NO: 1 to SEQ ID NO:143, the nucleotide sequence for the group that SEQ ID NO:169 to SEQ ID NO:174 and its complementary series are constituted About 80 to about 100% is identical.

4. a method of for inhibiting the expression of Target Nucleotide Sequence in the biology to plant pest, which comprises Reagent is provided in the diet of harmful organism, inhibits the harmful organism nucleotide sequence when it is absorbed by the harmful organism The expression of column gene, the reagent includes: the ribonucleic acid of following DNA sequence dna expression, the DNA sequence dna and the harmful organism Present in nucleotide coding sequence about 80 to about 100% it is identical, the nucleotide coding sequence be selected from by SEQ ID NO: 1 to SEQ ID NO:143, the group that SEQ ID NO:169 to SEQ ID NO:174 and its complementary series are constituted.

5. a kind of polynucleotide compositions in plant cell, wherein including expression cassette, the expression cassette includes: with nucleosides The Plant functional promoters that sequence elements are operably connected, the element include about 50 to about 5000 continuous Nucleotide, the continuous nucleotide show and are selected from by SEQ ID NO:1 to SEQ ID NO:143, SEQ ID NO:169 The phase same sex of the nucleotide coding sequence about 80 to about 100% of the group constituted to SEQ ID NO:174 and its complementary series, Wherein, critically important in the harmful organism is inhibited to the intake of the polynucleotide compositions to the invertebral living creature of plant pest Biological function.

6. the plant cell comprising polynucleotide compositions described in claim 5, from the regenerated plant of the plant cell, from The progeny plants and the seed generated from the progeny plants that the regenerated plant generates, wherein the progeny plants packet Containing the polynucleotide compositions, and wherein, the seed includes the polynucleotide compositions.

7. a kind of insect control agent, wherein comprising the ribonucleotide generated from the expression of following DNA sequence dnas, it is described DNA sequence dna is selected from by SEQ ID NO:1 to SEQ ID NO:143, SEQ ID NO:169 to SEQ ID NO:174 and its complementation The group of Sequence composition, wherein when being absorbed by no vertebra harmful organism, the ribonucleotide plays a role inhibition and institute State the expression of the basic complementary nucleotide sequence of DNA sequence dna.

8. a kind of method for protecting plant from insect infestation, which comprises provide institute in the insect diet One or more cells of plant are stated, the cell expresses RNA molecule from following DNA sequence dnas, and the DNA sequence dna is selected from by SEQ The group that ID NO:1 to SEQ ID NO:143, SEQ ID NO:169 to SEQ ID NO:174 and its complementary series are constituted, wherein The insect leads to the inhibition to biological function in the insect to the intake of one or more cells.

9. method according to claim 8, wherein the plant cell for expressing the RNA includes selected from by potato ball Stem storage protein patatin,Bacillus thuringiensisInsecticidal protein,XenorhabdusInsecticidal protein,PhotorhabdusInsecticidal protein,Bacillus laterosporousInsecticidal protein andBacillus sphearicus The insecticide for the group that insecticidal protein is constituted.

10. method as claimed in claim 9, wherein describedBacillus thuringiensisInsecticidal protein be selected from by Cry1, Cry3, TIC851, CryET70, Cry22, binary insecticidal protein CryET33 and CryET34, binary insecticidal protein The group that CryET80 and CryET76, binary insecticidal protein TIC100 and TIC101, binary insecticidal protein PS149B1 are constituted.

11. a kind of method, the expression of desired gene product, the method in one or more cells for inhibiting harmful insect It include: to provide the RNA molecule of gene containment amount to one or more cells, wherein described in the diet of the insect RNA molecule with gene containment effect is generated from following DNA sequence dnas, and the DNA sequence dna is selected from by SEQ ID NO:1 extremely The group that SEQ ID NO:143, SEQ ID NO:169 to SEQ ID NO:174 and its complementary series are constituted, wherein to the tool The intake for the RNA for having gene containment to act on leads to the drop of the level of desired gene product described in the insect pest cell It is low.

12. method as claimed in claim 11, wherein the DNA sequence dna be selected from by SEQ ID NO:4, SEQ ID NO:17, SEQ ID NO: 18、SEQ ID NO:31、SEQ ID NO:32、SEQ ID NO:35、SEQ ID NO:36、SEQ ID NO: 39、SEQ ID NO:40、SEQ ID NO:43、SEQ ID NO:44、SEQ ID NO:47、SEQ ID NO:48、SEQ ED NO:52、SEQ ID NO:53、SEQ ID NO:56、SEQ TD NO:57、SEQ ID NO:60、SEQ ID NO:60、SEQ ID NO:61、SEQ ID NO:64、SEQ ID NO:65、SEQ ID NO:68、SEQ ID NO:69、SEQ ID NO:72、SEQ ID NO:73、SEQ ID NO:76、SEQ ID NO:77、SEQ ID NO:80、SEQ ID NO:81、SEQ ID NO:84、SEQ ID NO:85, SEQ ID NO:88, EQ ID NO:89, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:169 to SEQ The group that ID NO:174 and its complementary series are constituted.

13. method as claimed in claim 12, wherein the DNA sequence dna is selected from by SEQ ID NO:17 and SEQ ID NO: The group that 19-21 contiguous nucleotide is constituted shown in 31.

14. a kind of expression cassette, wherein including the DNA sequence dna as shown in claim 13.

15. method as claimed in claim 11, wherein the target gene encodes following albumen, the forecast function of the albumen Selected from being formed by muscle, the formation of juvenile hormone, the regulation of juvenile hormone, ion regulation and transhipment, the synthesis of digestibility enzyme, carefully The holding of after birth gesture, amino acid bio synthesis, amino acid degradation, spermiogenesis tail, the synthesis of pheromones, pheromones sensing, antenna The formation of albumen, wing are formed, formation, development and the differentiation of leg, formation, larval maturation, the formation of digestibility enzyme, blood strangury of ovum Bar synthesis, hemolymph the group that is constituted of holding, neurotransmission, cell division, energetic supersession, breathing and apoptosis.

16. method as claimed in claim 11, wherein the insect be selected from byDiabrotica virgifera、Diabrotica barberiWithDiabrotica undecimpunctataThe group of compositionDiabrotica spp.。

17. a kind of method, for controlling the invasion without vertebra harmful organism comprising: in the diet of no vertebra harmful organism There is provided a kind of reagent, the reagent includes: the first RNA sequence can play inhibition when being absorbed by the harmful organism The effect of biological function in the harmful organism, wherein the ribonucleotide shows and comes from the harmful organism Coded sequence about 85 to about 100% nucleotide sequence identity, and can be miscellaneous with second of ribonucleotide It hands over, second of ribonucleotide is complementary with the first described ribonucleotide, and wherein, from the nocuousness The coded sequence that biology obtains is selected from by SEQ ID NO:1 to SEQ ID NO:143, SEQ ID NO:169 to SEQ ID The group that NO:174 and its complementary series are constituted.

18. a kind of method, for controlling the invasion without vertebra harmful organism comprising: in the diet of no vertebra harmful organism There is provided a kind of reagent, the reagent includes: the first RNA sequence can play inhibition when being absorbed by the harmful organism The effect of biological function in the harmful organism, wherein the ribonucleotide is shown to be obtained with from the harmful organism The nucleotide sequence of about 95 to about 100% of the coded sequence obtained at least about 14 to about 25 contiguous nucleotides The phase same sex, and capable of hybridizing with second of ribonucleotide, second of ribonucleotide and it is described the first Ribonucleotide is complementary, and wherein, and the coded sequence obtained from the harmful organism is selected from by SEQ ID NO:1 The group constituted to SEQ ID NO:143, SEQ ID NO:169 to SEQ ID NO:174 and its complementary series.

19. a kind of nucleotide sequence, following RNA molecules are encoded, when being provided in harmful organism diet with pest inhibitory amount When middle, the RNA molecule can inhibit the harmful organism fed in the diet containing the RNA molecule, wherein the core Nucleotide sequence is selected from by SEQ ID NO:1 to SEQ ID NO:143, SEQ ID NO:169 to SEQ ID NO:174 and its complementation The group of Sequence composition.

20. nucleotide sequence as claimed in claim 19, wherein the diet is selected from by artificial diet, plant cell, a variety of Plant cell, plant tissue, the root of plant, vegetable seeds and the group constituted from the plant that vegetable seeds growth comes, wherein The diet includes the RNA molecule of pest inhibitory amount.

21. nucleotide sequence as claimed in claim 19, wherein the sequence includes at least big shown in SEQ ID NO:97 About 19 contiguous nucleotides.

22. nucleotide sequence as claimed in claim 22, it is further characterized by about 19 contiguous nucleotides choosings From the 58th nucleotide shown in SEQ ID NO:97 to about 1010 nucleotide.

23. a kind of method, for protecting plant from insect infestation, which comprises

A) plant cell for expressing following RNA is provided in the diet of the insect, the RNA can contain the base of the insect Cause, and function critically important for insect survival is executed, the function is selected from the group being made of following function: You Haisheng The reduction of object feed, the reduction of harmful organism survival rate, the apoptosis of pest cell, the suppression that harmful organism is broken up and is developed System, the disappearance or reduction of the ability or demand of harmful organism sexual propagation, muscle are formed, muscle cramp, contraction of muscle, are protected children and are swashed The formation of element, the regulation of juvenile hormone, ion regulation and transhipment, the holding of cell membrane gesture, amino acid bio synthesis, amino acid drop Solution, spermiogenesis tail, the synthesis of pheromones, pheromones sensing, the formation of day linear protein, wing are formed, the formation of leg, the shape of ovum At larval maturation, the formation of digestibility enzyme, the synthesis of hemolymph, the holding of hemolymph, neurotransmission, larval stage is abnormal, changes Any component of pupa, the emergence pupated, cell division, energetic supersession, breathing and eukaryocyte cytoskeletal structure；

B) culture includes the plant of the plant cell.

24. a kind of method, for improving the yield of the crop produced from the crop plant that experience harmful insect invades, the method Include the following steps:

A) crop plant is cultivated using the seed comprising one or more following RNA molecules, the RNA molecule can be contained One or more target genes in following harmful insects, the harmful insect are that the crop absorbed in its diet is planted A part of strain, wherein it is critically important that one or more target genes execute at least one of insect physiology and metabolism Function, the critically important function are selected from the group being made of following function: the feed of harmful organism, the survival of harmful organism The differentiation and development of rate, the apoptosis of pest cell, harmful organism or any pest cell, harmful organism it is sexual numerous It grows, muscle is formed, muscle cramp, contraction of muscle, the formation and/or reduction of juvenile hormone, the regulation of juvenile hormone, ion regulation And transhipment, the holding of cell membrane gesture, amino acid bio synthesis, amino acid degradation, spermiogenesis tail, the synthesis of pheromones, pheromones Sensing, the formation of day linear protein, wing are formed, the formation of leg, the formation of ovum, larval maturation, the formation of digestibility enzyme, hemolymph Synthesis, the holding of hemolymph, neurotransmission, larval stage is abnormal, pupate, the emergence pupated, cell division, energetic supersession, Breathing, the synthesis and holding of cytoskeletal structure, nucleotide metabolism, nitrogen metabolism, the use of water, the holding and sensibility of water Perception；And

B) raising of the percentage yield of the crop plant is observed.

25. agricultural product or commodity product that the seed generated from genetically modified plants produces, wherein the genetically modified plants are from one Kind or a variety of continuous nucleotide sequences express RNA, and the nucleotide sequence is selected from by SEQ ID NO:1 to SEQ ID NO: 143, the group that SEQ ID NO:169 to SEQ ID NO:174 and its complementary series are constituted, and wherein, it is described one or more It is observable that nucleotides sequence, which is listed in the agricultural product or commodity product,.

26. length is the associated nucleotide sequence of at least about 21 nucleotide, selected from the SEQ by expressing as RNA sequence The group that ID NO:1-143 and 169-174 are constituted, and be provided in the diet of harmful insect, wherein harmful insect is pressed down The intake of the RNA of amount processed inhibits the harmful organism further to feed the diet.

27. a kind of plant cell, the associated nucleotide sequence by length by least about 21 nucleotide is converted, the core Nucleotide sequence is selected from the group being made of SEQ ID NO:1-143 and 169-174, wherein the nucleotides sequence is listed in the plant As the RNA segment expression being provided in harmful insect diet in object cell, wherein (a) described harmful insect is to the RNA's Intake leads to the containment to following genes, and the gene encodes the albumen from the gene expression, and (b) presses down to harmful insect The intake of the RNA of amount processed inhibits the harmful organism further to feed the diet.

28. a kind of method is used for selected nucleotide sequence, the sequence is for expressing RNA, and the RNA is for inhibiting harmful elder brother The expression of worm cytogene, described method includes following steps:

(a) from the bio-separation mRNA to plant pest；

(b) RNA is generated from all or part of of the cDNA sequence obtained by the mRNA；

(c) RNA is provided in the diet of the harmful insect；

(d) RNA for inhibiting the gene expression in the harmful insect is selected；And

(e) select the nucleotide sequence that can hybridize with the RNA, the RNA from SEQ ID NO:1-143 and 169-174 or The group that its complementary series is constituted.

29. a kind of method, for controlling insect pest, which comprises in the diet of the insect pest Two or more are provided and has virose insecticidal reagent for same insect species, wherein the examination of the first insecticidal Agent includes the RNA molecule expressed from DNA sequence dna, wherein when the RNA molecule is absorbed by the harmful organism, can inhibit described in Biological function in harmful organism, wherein by a part of exhibition for the DNA sequence dna that at least about 21 contiguous nucleotides are constituted Nucleotide sequence identity with the coded sequence about 85% to about 100% obtained from the harmful organism, Yi Jiqi are shown In, as the first described insecticidal reagent provides second of insecticidal reagent in the diet, wherein kill for described second Insect reagent from described the first is different.

30. method as claimed in claim 29 obtains wherein the harmful organism is corn rootworm from the harmful organism The coded sequence is selected from the group being made of SEQ ID NO:1-143 and 169-174.

31. a kind of method, for controlling coleopteran pest infestation, which comprises in the meals of coleopteran pest A kind of reagent is provided in food, the reagent includes following ribonucleic acid, and the ribonucleic acid by the harmful organism when being absorbed The effect for inhibiting target sequence expression in the harmful organism can be played, wherein the ribonucleic acid is by being the target sequence Ribonucleotide or the ribonucleotide complementary with the target sequence constitute, wherein the nucleotide sequence It is from selected from by SEQ ID NO:1 to SEQ ID NO:143, SEQ ID NO:169 to SEQ ID NO:174 and its complementary series What the DNA sequence dna transcription of the group of composition came.

32. method as claimed in claim 31, wherein the coleopteran pest is selected from by Leptinotarsa spp Harmful organism, Tribolium spp harmful organism, Anthonomous spp harmful organism, Cyclocephala spp nocuousness are raw The group that object and Polyphaga spp harmful organism are constituted.

33. method as claimed in claim 32, wherein the Leptinotarsa spp harmful organism be selected from byDiabrotica virgifera virgifera(Western corn rootworm, WCR),Diabrotica barberi (Northern corn rootworm, NCR),Diabrotica virgifera zeae(Mexican Corn Rootworm, MCR),Diabrotica balteata (Brazilian Com Rootworm, BZR),Diabrotica viridula (Brazilian Com Rootworm, BZR),Diabrotica speciosi (Brazilian Com Rootworm, BZR) andDiabrotica undecimpunctata howardiiThe group that (Southern corn rootworm, SCR) is constituted.

34. a kind of method, for inhibiting expression of the Target Nucleotide Sequence in the coleopteran pest to plant pest, institute The method of stating includes: that following reagents are provided in the diet of the harmful organism, and the reagent by the harmful organism when being absorbed It can inhibit expression of the following nucleotide sequence in the harmful organism, the reagent includes the core expressed from following DNA sequence dnas Ribosomal ribonucleic acid, the DNA sequence dna plant about 100% identical, institute with nucleotide coding sequence present in the harmful organism about 80 State nucleotide sequence be selected from by SEQ ID NO:1 to SEQ ID NO:143, SEQ ID NO:169 to SEQ ID NO:174 and its The group that complementary series is constituted.

35. a kind of polynucleotide compositions in plant cell, wherein including expression cassette, the expression cassette includes: with core The Plant functional promoters that nucleotide sequence element is operably connected, the element include about 50 to about 5000 continuous Nucleotide, the continuous nucleotide show with selected from by SEQ ID NO:1 to SEQ ID NO:143, SEQ ID NO: 169 to SEQ ID NO:174 and its complementary series constitute group nucleotide coding sequence about 80 to about 100% The phase same sex, wherein can inhibit described to the intake of the polynucleotide compositions to the coleopteran pest of plant pest has Critically important biological function in evil biology.

36. the plant cell comprising polynucleotide compositions described in claim 35, from the regenerated plant of plant cell, from again The progeny plants and the seed generated from the progeny plants that raw plant generates, wherein the progeny plants include described Polynucleotide compositions, and wherein, the seed includes the polynucleotide compositions.

37. method as claimed in claim 31, wherein DNA sequence dna is selected from by SEQ ID NO:4, SEQ ID NO:17, SEQ ID NO: 18、SEQ ID NO:31、SEQ ID NO:32、SEQ ID NO:35、SEQ ID NO:36、SEQ ID NO:39、 SEQ ID NO:40、SEQ ID NO:43、SEQ ID NO:44、SEQ ID NO:47、SEQ ID NO:48、SEQ ED NO: 52、SEQ ID NO:53、SEQ ID NO:56、SEQ TD NO:57、SEQ ID NO:60、SEQ ID NO:60、SEQ ID NO:61、SEQ ID NO:64、SEQ ID NO:65、SEQ ID NO:68、SEQ ID NO:69、SEQ ID NO:72、SEQ ID NO:73、SEQ ID NO:76、SEQ ID NO:77、SEQ ID NO:80、SEQ ID NO:81、SEQ ID NO:84、SEQ ID NO:85, SEQ ID NO:88, EQ ID NO:89, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:169 to SEQ The group that ID NO:174 and its complementary series are constituted.

38. a kind of method, the expression of desired gene product, institute in one or more cells for inhibiting coleopteran insect pest The method of stating includes: to provide the RNA molecule of gene containment amount to one or more cells in the diet of the insect, In, the RNA molecule with gene containment effect is generated from following DNA sequence dnas, and the DNA sequence dna is selected from by SEQ ID The group that NO:1 to SEQ ID NO:143, SEQ ID NO:169 to SEQ ID NO:174 and its complementary series are constituted, wherein right The intake of the RNA with gene containment effect causes desired gene product described in the insect pest cell horizontal Reduction.

39. method as claimed in claim 38, wherein the coleopteran pest is selected from by Leptinotarsa spp Harmful organism, Tribolium spp harmful organism, Anthonomous spp harmful organism, Cyclocephala spp nocuousness are raw The group that object and Polyphaga spp harmful organism are constituted.

40. method as claimed in claim 39, the Leptinotarsa spp harmful organism be selected from byDiabrotica virgifera virgifera(Western corn rootworm, WCR),Diabrotica barberi(Northern corn rootworm, NCR),Diabrotica virgifera zeae(Mexican Corn Rootworm, MCR),Diabrotica balteata (Brazil Corn rootworm, BZR),Diabrotica viridula (Brazilian Com Rootworm, BZR),Diabrotica speciosi (bar Western corn rootworm, BZR) andDiabrotica undecimpunctata howardii(Southern corn rootworm, SCR) structure At group.

41. method as claimed in claim 38, wherein the DNA sequence dna be selected from by SEQ ID NO:4, SEQ ID NO:17, SEQ ID NO: 18、SEQ ID NO:31、SEQ ID NO:32、SEQ ID NO:35、SEQ ID NO:36、SEQ ID NO: 39、SEQ ID NO:40、SEQ ID NO:43、SEQ ID NO:44、SEQ ID NO:47、SEQ ID NO:48、SEQ ED NO:52、SEQ ID NO:53、SEQ ID NO:56、SEQ TD NO:57、SEQ ID NO:60、SEQ ID NO:60、SEQ ID NO:61、SEQ ID NO:64、SEQ ID NO:65、SEQ ID NO:68、SEQ ID NO:69、SEQ ID NO:72、SEQ ID NO:73、SEQ ID NO:76、SEQ ID NO:77、SEQ ID NO:80、SEQ ID NO:81、SEQ ID NO:84、SEQ ID NO:85, SEQ ID NO:88, EQ ID NO:89, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:169 to SEQ The group that ID NO:174 and its complementary series are constituted.

42. a kind of method, for controlling the invasion of coleopteran pest comprising: in the diet of coleopteran pest There is provided a kind of reagent, the reagent includes: the first RNA sequence can play inhibition when being absorbed by the harmful organism The effect of biological function in the harmful organism, wherein the ribonucleotide is shown to be obtained with from the harmful organism The nucleotide sequence of about 95 to about 100% of the coded sequence obtained at least about 14 to about 25 contiguous nucleotides The phase same sex, and capable of hybridizing with second of ribonucleotide, second of ribonucleotide and it is described the first Ribonucleotide is complementary, and wherein, and the coded sequence obtained from the harmful organism is selected from by SEQ ID NO:1 The group constituted to SEQ ID NO:143, SEQ ID NO:169 to SEQ ID NO:174 and its complementary series.

43. method as claimed in claim 42, the diet is selected from by artificial diet, plant cell, various plants cell, plants Object tissue, the root of plant, vegetable seeds and the group constituted from the plant that vegetable seeds growth comes, wherein the diet packet The RNA molecule containing pest inhibitory amount.

44. method as claimed in claim 42, wherein the sequence includes at least about 19 shown in SEQ ID NO:97 Contiguous nucleotide.

45. method as claimed in claim 44, it is further characterized by about 19 contiguous nucleotides are selected from SEQ 58th nucleotide shown in ID NO:97 is to about 1010 nucleotide.