WO2010140675A1 - 害虫防除方法 - Google Patents
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- WO2010140675A1 WO2010140675A1 PCT/JP2010/059499 JP2010059499W WO2010140675A1 WO 2010140675 A1 WO2010140675 A1 WO 2010140675A1 JP 2010059499 W JP2010059499 W JP 2010059499W WO 2010140675 A1 WO2010140675 A1 WO 2010140675A1
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- C—CHEMISTRY; METALLURGY
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
- A01N43/90—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having two or more relevant hetero rings, condensed among themselves or with a common carbocyclic ring system
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N61/00—Biocides, pest repellants or attractants, or plant growth regulators containing substances of unknown or undetermined composition, e.g. substances characterised only by the mode of action
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/50—Isolated enzymes; Isolated proteins
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/60—Isolated nucleic acids
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- C—CHEMISTRY; METALLURGY
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
- C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
- C12N15/8286—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for insect resistance
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/14—Type of nucleic acid interfering N.A.
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
- Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants
Definitions
- the present invention relates to a pest control means. Specifically, the present invention relates to a pest control method, a pest control agent used in the method, and a transgenic plant.
- This application claims priority based on Japanese Patent Application No. 2009-136701 filed on June 5, 2009, the entire contents of which are incorporated by reference.
- Chemical pesticides containing organic compounds or inorganic compounds as active ingredients have been used for pest control.
- Chemical pesticides are generally toxic and have low action specificity, so they are highly toxic to humans and livestock and have a large impact on the environment. There is also a problem that a resistive system is likely to appear.
- biological pesticides have been developed as a technique for replacing or supplementing chemical pesticides.
- Biological pesticides have many advantageous properties such as low persistence in the environment, low toxicity to the human body, high action specificity, and resistance to resistance. ing.
- insects themselves, insect extracts, microorganisms themselves, microorganism extracts, plant extracts, and the like are used for the biological pesticide.
- RNAi RNA interference
- JP 2002-159230 A Japanese Patent No. 3033959 Japanese Patent No. 3532943
- IAP apoptosis inhibitory factor
- IAP inhibitor of apoptosis
- dsRNA double-stranded RNA
- the above strategy targeting IAP can be applied to any pest at various stages of development. In other words, it has excellent versatility and a wide range of applications.
- a highly specific control effect can be obtained by targeting IAP.
- the type (range) of pests to be controlled can be adjusted by selecting an appropriate IAP (one or more) according to the purpose. That is, it is possible to exhibit an effect that is not possible with conventional biological pesticides, that the control target range can be arbitrarily set.
- RNA that can specifically inhibit IAP expression is adopted as an IAP inhibitor (typically when RNAi is used)
- only specific pests are specifically selected depending on the level of specificity. It can be controlled. That is, it is possible to exhibit very high specificity.
- RNAi when RNAi is used, it is considered that the possibility of emergence of a resistant strain is extremely low due to its property, in addition to being easy to obtain a quick control effect.
- genetic modification operations are not performed on plants, there is no genetic influence. Thus, many advantages can be obtained when RNA is used as an IAP inhibitor in the above strategy.
- a method for controlling pests which comprises incorporating an inhibitor against an inhibitor of apoptosis (IAP) into the body of a target pest.
- IAP apoptosis
- the target pest is an agricultural pest, a sanitary pest or an unpleasant pest.
- the pest control method according to [1] wherein the target pest is an insect of the order Coleoptera, Grasshopper, Lepidoptera or Cockroach.
- [4] The method for controlling pests according to [1], wherein the target pest is an insect of the order of Coleoptera: Ladybugidae, Grasshopper: Locustaceae, Lepidoptera: Noctuidae, or Cockroachidae: [5]
- [6] The method for controlling pests according to any one of [1] to [5], wherein the inhibitor is a compound selected from the group consisting of the following (a) to (d): (a) siRNA targeting a gene encoding an apoptosis inhibitor of the target pest; (b) a nucleic acid construct for generating siRNA targeting a gene encoding an apoptosis inhibitor of the target pest in a cell; (c) an antisense nucleic acid targeting a transcript of a gene encoding an apoptosis inhibitor of the target pest; (d) A ribozyme that targets a transcription product of a gene encoding an apoptosis inhibitor of the target pest.
- the gene includes the sequence shown in SEQ ID NO: 1, the sequence shown in SEQ ID NO: 3, the sequence shown in SEQ ID NO: 5, the sequence shown in SEQ ID NO: 16, the sequence shown in SEQ ID NO: 17, or the sequence shown in SEQ ID NO: 19.
- [8] A plant that is harmed by the target pest is preliminarily retained by applying, spraying, or spraying a drug containing the inhibitor, and the inhibitor is incorporated into the target pest by ingesting the plant.
- the pest control method according to any one of [1] to [7], which is characterized in that it is characterized.
- a feed containing the inhibitor is placed at a place where the target pest is generated or invaded, and the inhibitor is taken into the body of the target pest by feeding the feed.
- the pest control method according to any one of [7] to [7].
- Results of experiments using Nijuya Hoshi Tento as test insects The time-dependent change of the larva which ingested the potato leaf spotted with RNA (Hv-iap dsRNA) which targeted the IAP gene is shown. In the control group, potato leaves spotted with H 2 O were used. Results of experiments using Nijuya Hoshi Tento as test insects. The state of the potato leaf 64 hours after the start of feeding is shown. The upper row is the test group (group fed with potato leaves spotted with Hv-iap dsRNA), and the lower row is the control group. Results of experiments using Nijuya Hoshi Tento as test insects. The time required from the start of feeding to death was compared between the test group and the control group. An average of 5 animals was shown.
- the result of an experiment using the giant tobacco as a test insect Indicates the time taken from the start of feeding to death.
- dsRNA) which targeted the IAP gene is shown.
- an artificial diet supplemented with GFP dsRNA was fed.
- Results of experiments using a cockroach as a test insect The time-dependent change of the larva fed with Bl-iapiadsRNA aqueous solution is shown. Movement (behavior) and survival status were examined.
- DsRed dsRNA aqueous solution was fed. Results of experiments using Nijuya Hoshi Tento as test insects.
- Hv-iap The time-dependent change of the Aedes nigras larvae which ingested the RNA extracted from the leaf of Nicotiana * benthamiana which expressed iap (Hv-iap) is shown.
- a control group a group fed with Hv-iap synthesized dsRNA (positive control) and a group fed with total RNA extracted from N. benthamiana leaf tissue expressing only virus (negative control) were provided.
- the first aspect of the present invention relates to a method for controlling pests.
- the “pest” in the present invention is not particularly limited. In general, pests are roughly classified into agricultural pests, sanitary pests, and unpleasant pests.
- Agricultural pests refer to pests that harm agricultural crops (including horticultural crops and also preserved agricultural crops). Pests that damage stored crops are sometimes referred to as “stored grain pests”.
- Hagiene pest refers to a pest that harms the human hygienic environment.
- an “unpleasant pest” refers to a pest that harms a person's mood by appearance or movement.
- the present invention can also be applied to pests that damage human property (termites, stains, etc.) and pests that damage livestock (mosquitoes, parasites, etc.).
- Examples of the pest classification include butterflies (Coleoptera, Noctuidae, Pteridomyceae, Tortoidaceae, Anophelesaceae, Singingidae, Diptera, Totsugaidae, Higgariidae, Dokugaidae, etc.) , Pheasantaceae, aphids, whiteflies, scales, bugs, grubs, stink bugs, bark beetles, etc., coleoptera (carabidae, beetles, ladybirds, beetles) , Fly flies (fly flies, flies flies, flies flies, flies flies, flies flies, flies flies, etc.), locusts (eg, grasshoppers, locusts, ombattas), thrips (Thripsidae, zebraidae) Thripid
- Lepidoptera examples include Nikameiga, Knotomeiga, Ichimonse Seri, Yonetotou, Awayotou, Futaobikogaaga, Lotus japonicus, Shirochimomodarameiga, Himeimomodarameiga, soybeans Sayamushiga, Mameshinagaiga, Kabura They are: Tobacco moths, mushrooms, white moths, white butterflies, white butterflies, scallops, and tanaginaginawa.
- Examples of the bugs of the order of the stink bugs are the brown planthopper, white-spotted plant, brown-tailed planthopper, black-spotted leafhopper, white-spotted leafhopper, red-footed beetle, red-bellied beetle, spider helicopter, blue-headed beetle, southern blue-headed beetle, white-winged beetle It is a spruce beetle, a white stink bug, a white-headed stink bug, a hornbill beetle, a beetle bug, a beetle stink bug, a spotted stink bug, a gear aphid, a corn aphid, a corn aphid.
- Examples of the order of the order of the order of the order of the order of the order of the order of the order of the order Coleoptera are rice weevil, rice squirrel beetle, rice weevil, fruit beetle, marc beetle moth, doganebuibui, ganbububui, bean gannet, red-billed beetle, beetle weevil, beetle It is a ladybug, a ladybug, a scallop, a swordbug, a cucumber beetle, and a hornbill beetle.
- Examples of pests of the order of the fly are rice flies, rice-spotted leaf flies, mugi-catered flies, seed fly, soybean flies, soybean-branched flies, legumes, tomato leaf-flies, eggplant leaf flies and eggplant leaf flies.
- grasshopper pests are Cobainago and Hananagainago.
- thrips-like pests are the rice thrips and the southern thrips.
- Pyrethrum pests are root-knot nematodes, root-knot nematodes, and cyst nematodes.
- pests are Goats's beetles and Pinus sorghum.
- mite pests are wheat mites, urticae mites, kanzawa spider mites, kenagakonadani mites, and mites.
- arachnoid pests are snails and slugs.
- An example of the insect of the order Coleoptera is roundworm.
- An example of a moth of the posterior moth flukes is Yokogawa flukes.
- An example of the pest of the walled flukes is Schistosoma japonicum.
- Examples of cockroach pests are German cockroaches, black cockroaches, American cockroaches, and cockroaches. Examples of spotted pests are Yamato and Ami.
- the present invention is preferably applied to insects of the order Coleoptera, Grasshopper, Lepidoptera or Cockroach, more preferably Coleoptera ladybird It is applied to insects of the family, grasshoppers, locusts, butterflies, cockroaches, cockroaches.
- Coleoptera ladybird insects here are Nigiri Ayame and Nami Tento, and a specific example of the grasshopper Locust family is Kobaneiago.
- a specific example of an insect of the Lepidoptera Noctuidae is a giant tobacco moth
- a specific example of a cockroach is an Lepidoptera.
- control means a state in which there is no harm or less harm by pests. Insect pests (disinfect), suppress pest growth, suppress pest growth, keep pests away (repellency), and detoxify or reduce pests (for example, Impairing the feeding ability of agricultural pests) is all included in the concept of “control”.
- an inhibitory substance (IAP inhibitory substance) against apoptosis inhibiting factor (hereinafter referred to as “IAP”) is incorporated into the body of the target pest.
- IAP inhibitor is used as a general term for substances that inhibit the action of IAP.
- the type of IAP inhibitor is not limited as long as it has an activity to inhibit IAP.
- Target pests are pests to be controlled. In other words, it is a pest controlled by the present invention. Two or more types of pests may be used as target pests.
- an IAP inhibitor corresponding to the target pest is used. Specifically, a substance that can inhibit IAP expressed by the target pest in the body is adopted as an “IAP inhibitor”. If an IAP inhibitor corresponding to two or more kinds of pests is used, or if two or more kinds of IAP inhibitors are used in combination, two or more kinds of pests can be controlled.
- IAP inhibitory substances include nucleic acids that inhibit IAP gene expression and substances that specifically bind to IAP (antibodies, low molecular weight compounds, etc.). The former will be described in detail below.
- a substance that specifically binds to IAP can be obtained or prepared using a binding assay that targets IAP.
- antibodies that specifically bind to IAP can be prepared using immunological techniques, phage display methods, ribosome display methods, and the like.
- a compound selected from the group consisting of the following (a) to (d) is used as the IAP inhibitor.
- siRNA targeting a gene encoding an apoptosis inhibitor of the target pest a nucleic acid construct for generating siRNA targeting a gene encoding an apoptosis inhibitor of the target pest in a cell;
- RNAi RNA interference
- the expression of IAP is inhibited by RNAi, and a control effect is obtained.
- target pests can be specifically controlled.
- due to its nature it is considered that the possibility that a resistant system appears will be extremely low.
- genetic modification operations are not performed on plants, there is no genetic influence.
- RNAi refers to a phenomenon in which expression of a target gene can be suppressed by introducing RNA having a sequence homologous to the target gene (particularly, homologous to mRNA corresponding to the target gene) into the target cell.
- dsRNA double-stranded RNA
- Two or more types of dsRNA may be used for one target gene.
- RNAi targeting a mammalian cell gene a short dsRNA (siRNA) of about 21 to 23 nucleotides is used.
- siRNA short dsRNA
- a long dsRNA of several hundred nucleotides or more is rather preferable. Is preferred as a more effective one.
- the length of dsRNA used for RNAi is, for example, 30 nucleotides or more, preferably 200 nucleotides or more (for example, 200 to 500 nucleotides in length).
- dsRNA is preferable for causing effective expression suppression, it does not prevent the use of single-stranded RNA.
- the dsRNA to be used is not necessarily divided into two molecules of a sense strand and an antisense strand.
- the dsRNA may have a structure in which the sense strand and the antisense strand constituting the dsRNA are linked by a hairpin loop.
- You may decide to use dsRNA which consists of modified RNA. Examples of modifications herein include phosphorothioation and the use of modified bases (eg, fluorescently labeled bases).
- nucleic acid construct (b) is used for such means.
- DsRNA used in RNAi method can be prepared by chemical synthesis or in vitro or in vivo using an appropriate expression vector.
- the expression vector method is particularly effective for preparing relatively long dsRNA.
- a sequence unique to the target nucleic acid continuous sequence
- programs and algorithms for selecting an appropriate target sequence have been developed.
- Non-Patent Documents 1 to 4 (Baum, JA et al. (2007) Control of coleopteran insect pests through RNA interference. Nat. Biotechnol. 25, 1322-1326; 326Mao, YB et al. (2007) Silencing a cotton bollworm P450; monooxygenase gene by plant-mediated RNAi impairs larval tolerance of gossypol. Nat. Biotechnol. 25, 1307-131; Price, DRG and Gatehouse JA (2008) RNAi-mediated crop protect .26 No.7, 393-400; Whangbo, JS and Hunter CP (2008) Environmental RNA interference. Trends in Genetics Vol. 24 No. 6, 297-305) reports on pest control using RNAi. Yes, it will be helpful in carrying out the present invention. Therefore, all the contents of these documents are incorporated by reference.
- (C) is a compound used for expression inhibition by the antisense method.
- an antisense construct that generates RNA complementary to a unique portion of mRNA corresponding to a target gene when it is transcribed is usually used.
- Such an antisense construct (also referred to as an antisense nucleic acid) is introduced into a target cell in the form of an expression plasmid, for example.
- an antisense construct an oligo that, when introduced into a target cell, hybridizes with the DNA sequence of the target gene or its corresponding mRNA sequence (collectively referred to as “target nucleic acid”) to inhibit its expression.
- Nucleotide probes can also be employed.
- oligonucleotide probe one that is resistant to endogenous nucleases such as exonuclease and / or endonuclease is preferably used.
- endogenous nucleases such as exonuclease and / or endonuclease
- oligodeoxyribonucleotide derived from a region containing a translation initiation site (for example, a region of ⁇ 10 to +10) of mRNA corresponding to the target gene is preferable.
- the complementarity between the antisense nucleic acid and the target nucleic acid is strict, but some mismatch may exist.
- the ability of an antisense nucleic acid to hybridize to a target nucleic acid generally depends on both the degree of complementarity and the length of both nucleic acids. Usually, the longer the antisense nucleic acid used, the more stable duplexes (or triplexes) can be formed with the target nucleic acid, even if the number of mismatches is large.
- One skilled in the art can ascertain the degree of acceptable mismatch using standard techniques.
- the antisense nucleic acid may be DNA, RNA, a chimeric mixture thereof, or a derivative or modified type thereof. Moreover, it may be single-stranded or double-stranded. By modifying the base moiety, sugar moiety, or phosphate skeleton moiety, the stability, hybridization ability, etc. of the antisense nucleic acid can be improved.
- the antisense nucleic acid can be synthesized by a conventional method, for example, using a commercially available automatic DNA synthesizer (for example, Applied Biosystems). For example, Stein et al. (1988), Nucl. Acids Res. 16: 3209 and Sarin et al., (1988), Proc. Natl. Acad. Sci. USA 85: 7448- 7451 etc. can be referred to.
- a promoter for example, actin promoter or ie1 promoter
- actin promoter or ie1 promoter that acts strongly in the target cell. That is, when a construct containing an antisense nucleic acid arranged under the control of such a promoter is introduced into a target cell, a sufficient amount of the antisense nucleic acid can be transcribed by the action of the promoter.
- inhibition of expression by a ribozyme is used (in the case of the compound (d) above).
- a ribozyme that cleaves mRNA with a site-specific recognition sequence can be used to destroy the mRNA corresponding to the target gene, but a hammerhead ribozyme is preferably used.
- a hammerhead ribozyme for example, Haseloff and Gerlach, 1988, Nature, 334: 585-591 can be referred to.
- ribozymes may be constructed using modified oligonucleotides, for example, for the purpose of improving stability and targeting ability.
- a nucleic acid construct in which DNA encoding the ribozyme is placed under the control of a promoter (eg, actin promoter or ie1 promoter) that acts strongly in insect cells is used. It is preferable to do.
- SEQ ID NO: 1 is the sequence of the IAP gene (partial cDNA) of Nijuya Hoshi Tento.
- SEQ ID NO: 16 is the full-length cDNA sequence of the same gene.
- the amino acid sequence encoded by the sequence is shown in SEQ ID NO: 2.
- SEQ ID NO: 3 is the sequence of the Nami Tento IAP gene (partial cDNA).
- SEQ ID NO: 4 is the sequence of the IAP gene (partial cDNA) of Cobainago.
- SEQ ID NO: 6 The amino acid sequence encoded by the sequence is shown in SEQ ID NO: 6.
- SEQ ID NO: 17 is the sequence of the IAP gene (partial cDNA) of tobacco tobacco.
- the amino acid sequence encoded by this sequence is shown in SEQ ID NO: 18.
- SEQ ID NO: 19 is the sequence of the IAP gene (partial cDNA) of the cockroach.
- the amino acid sequence encoded by the sequence is shown in SEQ ID NO: 20.
- the method for incorporating the IAP inhibitor is not particularly limited, and an appropriate one may be selected for each target pest.
- the target pest for example, a chemical (agricultural chemical) containing an IAP inhibitor is applied, sprayed, or sprayed to the plant that is damaged by the target pest.
- the target pest feeds the plant, the IAP inhibitor is taken into the target pest.
- the food containing the IAP inhibitor food
- the target pest feeds on the food, which causes the IAP inhibitor to enter the target pest. Is taken in.
- a gene encoding an IAP inhibitor is introduced into a plant to be damaged and modified, the IAP inhibitor is taken into the target pest when the transgenic plant is ingested.
- a transgenic plant used in such a method (A) siRNA targeting the gene encoding IAP of the target pest, (B) antisense nucleic acid targeting the transcript of the gene encoding IAP of the target pest Alternatively, or (C) a plant that has been genetically modified to express a ribozyme that targets a transcript of a gene encoding the target pest IAP can be used.
- the pest control agent of the present invention is provided, for example, in the form of an agrochemical or a bait.
- the pest control agent of the present invention contains an inhibitory substance (IAP inhibitory substance) against the IAP of the target pest.
- IAP inhibitor examples of the IAP inhibitor here are the following (a) to (d). Details of each of these compounds are as described above.
- siRNA targeting a gene encoding the target pest IAP (a) siRNA targeting a gene encoding the target pest IAP; (b) a nucleic acid construct that generates an siRNA targeting a gene encoding an IAP of a target pest in a cell; (c) an antisense nucleic acid targeting a transcript of a gene encoding the target pest IAP; (d) A ribozyme targeting a transcript of a gene encoding the target pest IAP.
- the form of the agrochemical of the present invention is not particularly limited. For example, it is prepared in a solid, liquid or gel form.
- Specific examples of dosage forms include powders, granules, wettable powders, water solvents, emulsions, liquids, oils, aerosols, microcapsules, pastes, smoke agents, fumigants, and coating agents.
- the content of the active ingredient in the agricultural chemical of the present invention varies depending on the form of the agricultural chemical, but is, for example, 0.1% by weight to 50% by weight.
- the agrochemical of the present invention contains an IAP inhibitor as an active ingredient (active ingredient), but may contain one or more other active ingredients in combination.
- active ingredients include insecticides (organophosphates, carbamates, synthetic pyrethroids, nereistoxins, neonicotinoids, GABA inhibitors, GABA analogues, respiratory inhibitors, IGR agents (insect growth inhibitors) Agent), BT agent), repellent and attractant.
- components (additives) that can be contained in the agricultural chemical of the present invention in addition to the active components include surfactants (anionic surfactants, cationic surfactants, amphoteric surfactants, nonionic surfactants).
- Thickener Thickener, flow aid, binder, spreading agent, spreading agent, wetting agent, bulking agent, carrier, stabilizer, pH adjuster, anti-caking agent, antiseptic, antifreeze agent, anti Oxidizing agents, UV absorbers, UV scattering agents, dispersants, colorants, and fragrances.
- transgenic plant A further aspect of the present invention (third aspect) relates to a transgenic plant.
- a gene encoding an inhibitor for IAP of the target pest is introduced.
- the transgenic plant of the present invention comprises (A) siRNA targeting a gene encoding IAP of the target pest, (B) antisense targeting a transcript of the gene encoding IAP of the target pest. It has been genetically modified to express a nucleic acid or (C) a ribozyme targeted to a transcript of a gene encoding the target pest IAP.
- the transgenic plant of the present invention can be produced by introducing a gene construct (such as a recombinant vector) containing a target gene (the gene encoding the above siRNA, antisense nucleic acid or ribozyme) into a plant body or plant cell. it can.
- a recombinant vector as a gene construct can be constructed by inserting a target gene into an appropriate vector.
- the vectors here pBI, pPZP, and pSMA vectors are preferably used. When these vectors are used, gene introduction via Agrobacterium is possible.
- pBI binary vectors or intermediate vector systems are suitable.
- a binary vector is a shuttle vector that can replicate in E. coli and Agrobacterium.
- Another example of a vector that can be used is a pUC vector.
- a target gene can be directly introduced into a plant.
- Examples of pUC vectors are pUC18, pUC19, and pUC9.
- Plant virus vectors (cauliflower mosaic virus, kidney bean mosaic virus, tobacco mosaic virus, etc.) can also be used.
- purified DNA including the target gene
- sequences promoter, enhancer, terminator, etc.
- Other sequences useful for expressing the target gene at a high level for example, an intron sequence of a specific gene, a sequence of a 5 ′ non-translated region, etc. may be linked.
- a constitutive promoter or a promoter that functions under specific conditions can be used, and may be selected as necessary. Further, the origin of the promoter is not particularly limited. Specific examples of the promoter include ubiquitin promoter, cauliflower mosaic virus 35S promoter, actin promoter, and nopaline synthase gene promoter.
- Enhancers are used to increase the expression efficiency of target genes.
- An example of an enhancer is an enhancer region comprising an upstream sequence within the CaMV35S promoter.
- a terminator is a sequence that terminates transcription of a gene.
- the terminator of CaMV 35S RNA gene, the terminator of nopaline synthase gene, the terminator of octopine synthase gene, etc. can be used.
- selection marker genes are ampicillin resistance gene, neomycin resistance gene, hygromycin resistance gene, ⁇ -glucuronidase gene, and luciferase gene.
- the method for introducing the target gene into the plant is not particularly limited.
- an Agrobacterium method, an electroporation method, a particle Kurgan method, a polyethylene glycol (PEG) method, or the like can be employed.
- the Agrobacterium method is particularly preferable.
- the target gene is introduced by a method of co-culturing with Agrobacterium having Ti plasmid or a method of fusing with Agrobacterium spheroplasted (Spheroplast method).
- the target gene When using a tissue piece, the target gene is introduced by a method of infecting a sterile cultured leaf piece (leaf disc) of a target plant or a method of infecting a callus.
- the target gene When a plant body is used (in planta method), the target gene is introduced by direct treatment of Agrobacterium to water-absorbing seeds, seedlings (seedlings), potted plants, and the like.
- the particle gun method is also a preferable introduction method.
- a sample plant body, plant organ, plant tissue, protoplast, etc.
- a gene introduction device eg, BIOLISTIC POS 1000 / He; BioRad.
- the treatment conditions vary depending on the sample, but usually the pressure is about 1000-1100 psi and the distance is about 5-10 cm.
- a recombinant vector containing the target gene and a recombinant vector containing a selectable marker gene may be mixed, and simultaneously shot these two recombinant vectors into the sample (co-transformation).
- Whether or not the target gene has been incorporated into the plant body can be confirmed by PCR, Southern hybridization, Northern hybridization, Western blotting, or the like.
- the target gene was incorporated using a reporter gene (beta glucuronidase (GUS), luciferase (LUC), Green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), beta galactosidase (LacZ), etc.) May be confirmed.
- GUS beta glucuronidase
- LOC luciferase
- GFP Green fluorescent protein
- CAT chloramphenicol acetyltransferase
- LacZ beta galactosidase
- the kind of plant to be transformed is not particularly limited, and examples thereof include useful crops such as cereals, vegetables and fruit trees, and ornamental plants such as foliage plants. Moreover, either a monocotyledonous plant or a dicotyledonous plant may be sufficient. Examples of monocotyledonous plants include: Gramineae (rice, barley, wheat, corn, sugarcane, buckwheat, sorghum, millet, millet, etc.), liliaceae (asparagus, lily, onion, leek, Japanese chestnut, etc.), ginger (ginger) , Myoga, turmeric, etc.).
- dicotyledonous plants for example, Brassicaceae (Arabidopsis, cabbage, rapeseed, cauliflower, broccoli, radish, etc.), solanaceae (tomato, eggplant, potato, tobacco, etc.), legumes (soybean, peas, green beans, alfalfa, etc.) ), Cucurbitaceae (cucumber, melon, pumpkin, etc.), crustaceae (carrot, celery, honeybee, etc.), asteraceae (lettuce, etc.), mallow (cotton, okra, etc.), red crustaceae (sugar beet, spinach, etc.), Mention may be made of plants belonging to the genus Euphoridae (eucalyptus, clove etc.) and willow genus (poplar etc.).
- ⁇ plant '' includes plant bodies, plant organs (e.g. leaves, petals, stems, roots, rhizome seeds, etc.), plant tissues (e.g. epidermis, phloem, parenchyma, xylem, vascular bundles, etc.) and plant cells.
- plant cell also includes seed suspension cultures, embryos, meristem sites, callus tissue, leaves and root-derived cells, and gametophytes (embryo, pollen) and precursor cells thereof.
- organs or individuals can be regenerated from the obtained transformed cells by a known tissue culture method. Such operations can be easily performed by those skilled in the art. An example is shown below.
- the transformed plant cells are cultured in a sterile callus-forming medium (containing carbon sources, sugars, vitamins, minerals, plant hormones such as auxin and cytokinin), and proliferated in an indefinite form.
- Differentiated callus is formed (callus induction).
- the formed callus is transferred to a new medium containing a plant growth regulator such as auxin and further grown (subcultured). If callus induction is performed in a solid medium such as agar and subculture is performed in liquid culture, each culture can be performed efficiently.
- callus grown by subculture is cultured under appropriate conditions to induce organ redifferentiation (redifferentiation induction) to regenerate the plant body.
- Induction of regeneration can be performed by appropriately setting the type and amount of various components such as plant growth regulators such as auxin and cytokinin, carbon sources, and the like, light, temperature, etc. in the medium.
- plant growth regulators such as auxin and cytokinin, carbon sources, and the like
- somatic embryos, adventitious roots, adventitious shoots, adventitious foliage, etc. are formed and grown into complete plants.
- Storage or the like may be performed in a state before becoming a complete plant body (for example, encapsulated artificial seeds, dried embryos, freeze-dried cells and tissues).
- transgenic plant of the present invention produced as described above produces an inhibitor to the target pest IAP.
- an IAP inhibitor is incorporated into the target pest body, resulting in a control effect.
- the term “transgenic plant” in the present invention refers to “T1 generation” produced by transformation treatment, “T2 generation” which is a progeny obtained from the seed of the plant, and further progeny plants thereafter (T3 Generation, T4 generation, etc.).
- Non-Patent Documents 1 and 2 (Baum, JA et al. (2007) Control of coleopteran insect pests through RNA interference. Nat. Biotechnol. 25, 1322-1326; Mao, YB et al. (2007) Silencing a cotton bollworm P450 monooxygenase gene by plant-mediated RNAi impairs larval tolerance of gossypol. Nat. Biotechnol. 25, 1307-131) reports an example of a recombinant plant in which dsRNA against a specific target is forcibly expressed. As these reports demonstrate, it is a promising pest control measure to express dsRNA against a target in a plant and cause an inhibitory effect by RNAi.
- dsRNA against IAP of the target pest is forcibly expressed.
- dsRNA against the target pest IAP is forcibly expressed in the plant as a result of genetic manipulation.
- RNA from TRIZOL is obtained from the larvae primordial larvae of the larvae of the larvae, the embryos of Namitentou (Ha), and the larvae of the white larvae (Oy). Extracted by the guanidine hydrochloride method using TECHNOLOGIES. Using this RNA as a template, based on SMARTTM RACE cDNA Amplification Kit (CLONTECH), Superscript II reverse transcriptase (GIBCO BRL) is used to separate 5'Rapid amplification of cDNA ends (RACE) and 3'RACE. Strand cDNA was synthesized.
- PCR Polymerase chain reaction
- Hv-iap-03 5′-GATCCGTGGGAACAGCACGCGCTTT-3 ′ (25mer)
- Hv-iap-04 5′-GAAACAACGCAGAGGAAAGCTCGAC-3 ′ (25mer) (SEQ ID NO: 13)
- the first PCR conditions for RACE were: denaturation (94 ° C, 5 seconds); extension (72 ° C, 3 minutes), 5 cycles, denaturation (94 ° C, 5 seconds); annealing (70 ° C, 10 seconds); After 5 cycles (72 ° C., 3 minutes), denaturation (94 ° C., 5 seconds); annealing (68 ° C., 10 seconds); extension (72 ° C., 3 minutes) were performed 25 cycles.
- the second PCR conditions were denaturation (94 ° C., 5 seconds); extension (72 ° C., 3 minutes) for 5 cycles, denaturation (94 ° C., 5 seconds); annealing (70 ° C., 10 seconds); extension (72 ° C.
- PCR product After PCR, add agarose (Agarose II [DOJINDO]) to TBE (89 mM Tris, 89 mM boric acid, 2 mM EDTA), and add ethidium bromide to 0.5 mg / ml. PCR products were separated by electrophoresis on 1% Agarose II agarose gel. A band containing the desired PCR product was cut out, and the PCR product was recovered by Mag Extractor (Toyobo) and used as an insert for subcloning.
- Mag Extractor Toyobo
- PCR was performed under the conditions of 25 cycles of denaturation (95 ° C., 30 seconds); annealing (55 ° C., 30 seconds); extension (72 ° C., 30 seconds).
- the selected clones were cultured with shaking overnight in LB medium at 37 ° C., and plasmid DNA was prepared by FlexiPrep Kit (Amerciam Pharmacia Biotech) according to the protocol of the kit.
- the base sequence was determined according to the protocol using BigDye Terminator v3.1 Cycle Sequencing kit. The obtained base sequence was analyzed by DNASIS.
- RNAi method Amplification of template for RNA synthesis
- the iap of Nijuya Hoshi Tento uses the sub-cloned RT-PCR product amplified with iap1 and iap3 primers on the EcoRV site of pBluescript KS + vector (Stratagene) It was.
- the Nami Tento iap and the Kobaneiago iap were obtained by subcloning RT-PCR products amplified with iap2 and iap3 primers.
- T7 RNA polymerase promoter sequence is added to the vector sequence so that it can be used in common with any gene cloned in the above vector.
- the following PCR primers were used.
- T7-KS primer 5'-TAATACGACTCACTATAGGGAGACCACTCGAGGTCGACGGTATC-3 '(SEQ ID NO: 14)
- T7-SK primer 5'-TAATACGACTCACTATAGGGAGACCACCGCTCTAGAACTAGTGGATC-3 '(SEQ ID NO: 15)
- PCR was performed under the following conditions, and 4-8 reaction tubes were prepared to obtain a sufficient amount of PCR product.
- the PCR product obtained by the above reaction was concentrated by ethanol precipitation, then subjected to agarose electrophoresis, and purified from the gel using Mag Extractor (Toyobo).
- RNA was synthesized according to MEGAscrtipt T7 Kit (Ambion) and dissolved in an appropriate amount of nuclease-free ultrapure water.
- RNA solution was incubated at 65 ° C. for 30 minutes using a heat block, and then returned to room temperature over 1-2 hours. A small amount of this double-stranded RNA was used, and concentration measurement and confirmation by agarose electrophoresis were performed. Double-stranded RNA that was confirmed to be synthesized was adjusted to a concentration of 2 ⁇ g / ⁇ l, and divided into small portions and stored at ⁇ 80 ° C.
- FIG. 1 shows the change over time of larvae (test group) that ingested potato leaves spotted with Hv-iap dsRNA. Compared with the control, the amount of food intake and exercise decreased from an early stage after the start of food intake. In addition, when potato leaves were observed 64 hours after the start of feeding (FIG. 2), a marked difference was observed in the amount of food consumed between the test group (upper) and the control group (lower). In addition, all the larvae in the test group died within 4 days, and all the larvae in the control group survived after 4 days (FIG. 3). As mentioned above, by incorporating Hv-iap dsRNA into the body and inhibiting IAP, eating disorders occurred rapidly, and survival and growth were inhibited. That is, it was shown that inhibition of IAP exerts a rapid and excellent control effect.
- Nami Tento (Coleoptera, Ladybirdidae) Ha-iap dsRNA and gfp dsRNA were each given to 2 male adults. All individuals fed Ha-iap dsRNA had a marked reduction in food intake within 24 hours. Food intake for 4.5 days was less than 10 mg for all individuals receiving Ha-iap dsRNA, compared to 20 mg for all individuals receiving gfp dsRNA. On the other hand, individuals given Ha-iap dsRNA died after 7.5 and 9 days. In contrast, all individuals given gfp dsRNA survived even after 33 days. As described above, it was shown that the inhibition of IAP exerts an excellent control effect for Nami Tento.
- Cobaineago (Battaridae, Locustidae) Oy-iap dsRNA and gfp dsRNA were each given to 2 adults. As a result, individuals given Oy-iap dsRNA died after 9 and 12 days. On the other hand, all individuals given gfp dsRNA survived even after 25 days. Thus, it was shown that the inhibition of IAP also exerts an excellent control effect for Cobainago. Note that the amount of rice leaf intake after dsRNA feeding was not examined because it was difficult to measure experimentally.
- RNA was extracted from the testis by the guanidine hydrochloride method using TRIZOL (LIFE TECHNOLOGIES). Using this RNA as a template, based on SMART TM RACE cDNA Amplification Kit (CLONTECH), Superscript II reverse transcriptase (GIBCO BRL) is used to separate 5'Rapid amplification of cDNA ends (RACE) and 3'RACE 1 Single-stranded cDNA was synthesized.
- TRIZOL LIFE TECHNOLOGIES
- PCR Polymerase chain reaction
- PCR was performed under the conditions of 25 cycles of denaturation, 95 ° C./30 seconds; annealing, 55 ° C./30 seconds; extension, 72 ° C./30 seconds.
- the selected clones were cultured with shaking in LB at 37 ° C. overnight, and plasmid DNA was prepared by Axy Prep Plasmid Miniprep Kit (Axygen Scientific) according to the protocol of the kit.
- RNAi method Amplification of template for RNA synthesis
- the RT-PCR product amplified with the primers of iap1 and iap3 was subcloned into the TOPO vector (pCR4-TOPO).
- pCR4-TOPO TOPO vector
- a T7 RNA polymerase promoter sequence is added to the vector sequence so that it can be used in common with any gene cloned in the above vector.
- the following PCR primers were used.
- T7-PCR4R primer 5′-TAATACGACTCACTATAGGGAGACCACCGAATTGAATTTAGCGGC-3 ′
- T7-PCR4L primer 5′-TAATACGACTCACTATAGGGAGACCACGTCCTGCAGGTTTAAACG-3 ′
- PCR was performed under the following conditions, and 4-8 reaction tubes were prepared to obtain a sufficient amount of PCR product.
- the PCR product obtained by the above reaction was concentrated by ethanol precipitation, then subjected to agarose electrophoresis, and purified from the gel using Mag Extractor (Toyobo).
- RNA was synthesized according to MEGAscrtipt T7 Kit (Ambion) and dissolved in an appropriate amount of nuclease-free ultrapure water. In order to anneal double-stranded RNA, the obtained RNA solution was incubated at 65 ° C. for 30 minutes using a heat block, and then returned to room temperature over 1-2 hours. A small amount of this double-stranded RNA was used, and concentration measurement and confirmation by agarose electrophoresis were performed.
- the double-stranded RNA that has been confirmed to be synthesized is adjusted to a concentration of 10 ⁇ g / ⁇ l for the tobacco iap (Har-iap) and 5 ⁇ g / ⁇ l for the cockroach iap (Bl-iap), and subdivided into batches- Stored at 80 ° C.
- Test plant Nicotiana benthamiana was sold by the Japan Tobacco Inc. Leaf Tobacco Research Institute. Seeds were sown in a polyethylene pot containing Kureha Soil (Kureha Co., Ltd.) and Soil Mix (Sakata Seed Co., Ltd.), and the plants on the 10th day after sowing were transplanted to the same polyethylene pot and placed in a constant temperature room at 25 ° C. For 24 hours under light conditions. The treated plant body was transferred to a constant temperature room at 22 ° C. and allowed to stand under a light condition of 16 hours and a dark condition of 8 hours.
- the upper layer was transferred to a new 1.5 ml Eppendorf tube and collected, and 500 ⁇ l of isopropanol was added to the solution, stirred, allowed to stand for 10 minutes, and then centrifuged (12,000 ⁇ g, 10 minutes, 4 ° C.). The resulting precipitate was washed with 70% ethanol, air-dried for 10 minutes, and dissolved in 40 ⁇ l of DEPC-treated water to obtain total RNA.
- Spectrophotometer ND-1000 Spectrophotometer, NanoDrop
- PVX vector pGR107 binary vector containing PVX potato virus X
- Tobacco rattle virus as a vector for A 389 bp Hv-iap cDNA fragment to be inserted into Plant J. 25, 237-245.
- the following PCR primers to which restriction enzyme sequences were added were used.
- PCR was performed under the following conditions, and the following two reaction tubes were prepared to obtain a sufficient amount of PCR product.
- the PCR product obtained by the above reaction was concentrated by ethanol precipitation, then subjected to agarose electrophoresis, and purified from the gel using Mag Extractor (Toyobo).
- Mag Extractor Toyobo
- the obtained cDNA fragment was digested with NotI and incorporated into pGR107 (Ratctliff et al. 2001), and E. coli was transformed and cultured using this to purify plasmid DNA.
- Transformation of A. tumefaciens by electroporation Transformation of Agrobacterium tumefaciens was performed by the method of Hellens et al. (Hellens, RP, Edwards, EA, Leyland, NR, Bean, S., and Mullineaux, PM (2000).
- pGreen A versatile and flexible binary Ti vector for Agrobacterium-mediated plant transformation. Plant Mol. Biol. 42, 819-832.
- the helper plasmid pSoup (10 ng) was added and 40 ⁇ l of A. tumefaciens GV3101 electrocell dissolved on ice was allowed to stand on ice for 30 minutes.
- the solution was transferred to a 14 ml polypropylene round tube (BECTON DICKINSON) and allowed to stand on ice for 10 minutes, then 960 ⁇ l SOC medium [2% tryptone peptone, 0.5% yeast extract, 0.05% NaCl, 10 mM MgCl 2 , 10 mM MgSO 4 , 20 mM glucose, pH 7.0], and shaken at 28 ° C. for 1 hour.
- kanamycin 50 ⁇ g / ml
- rifampicin 50 ⁇ g / ml
- YEB medium 0.1% yeast extract, 0.5% beef extract, 0.5% tryptone peptone, 0.5% sucrose, 2 mM MgSO 4 , 2% agar, pH 7.2
- 20 ⁇ l was plated on the medium and cultured at 28 ° C. for 30 hours.
- RT-PCR RT-PCR was performed as follows using ReverTra Ace-Plus- (registered trademark) (TOYOBO). 0.5 ⁇ g of total RNA and 25 pmol of Oligo (dT) 20 primer were mixed in 6 ⁇ l of solution, treated at 65 ° C. for 5 minutes, and immediately cooled on ice. Subsequently, the remaining RT-PCR reaction solution [1 x RT reaction solution, 1 mM dNTPs, 1 unit / ⁇ l RNase inhibitor, 5 unit / ⁇ l ReverTra Ace (registered trademark)] was added, and 30 ° C. in 10 ⁇ l reaction solution The reverse transcription reaction was carried out for 10 minutes at 42 ° C. for 1 hour at 85 ° C.
- the PCR reaction was performed using ExTaq (Takara) in 10 ⁇ l of reaction solution per 1 ⁇ l of the reverse transcription reaction solution at an annealing temperature of 53 ° C. for 20 seconds and an extension reaction at 74 ° C. for 30 seconds under 27 cycles. .
- the base sequences of primers used for RT-PCR are shown below.
- Hv-iap dsRNA contained in the total RNA extracted from N. benthamiana leaf tissue is expected to be a trace amount
- Hv-iap synthesized dsRNA was used as a positive control. In this case, after feeding 20 ng, potato leaves were fed to the larvae. Thereafter, larvae were observed over time.
- an IAP inhibitor is incorporated into a pest body to inhibit IAP and obtain a control effect.
- the present invention is highly versatile and can be applied to various pests. Further, it can be applied to pests at various stages of development.
- SEQ ID NO: 7 Description of artificial sequence: iap sense primer (iap-01)
- SEQ ID NO: 8 Description of artificial sequence: iap sense primer (iap-02)
- SEQ ID NO: 9 Description of artificial sequence: iap antisense primer (iap-03)
- SEQ ID NO: 10 Description of artificial sequence: 5′RACE primer (Hv-iap-01)
- SEQ ID NO: 11 Description of artificial sequence: 5′RACE primer (Hv-iap-02)
- SEQ ID NO: 12 Description of artificial sequence: 3′RACE primer (Hv-iap-03)
- SEQ ID NO: 13 Description of artificial sequence: 3′RACE primer (Hv-iap-04)
- SEQ ID NO: 14 Description of artificial sequence: PCR primer (T7-KS)
- SEQ ID NO: 21 Description of artificial sequence: iap antisense primer (i
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Abstract
Description
そこで本発明は、従来の生物農薬が抱える上記問題の多くを解決し、優れた防除効果を発揮する害虫防除手段を提供することを課題とする。
[1]アポトーシス阻害因子(Inhibitor of apoptosis;IAP)に対する阻害物質を標的害虫の体内に取り込ませること、を特徴とする害虫防除方法。
[2]前記標的害虫が農業害虫、衛生害虫又は不快害虫である、[1]に記載の害虫防除方法。
[3]前記標的害虫がコウチュウ目、バッタ目、チョウ目又はゴキブリ目の昆虫である、[1]に記載の害虫防除方法。
[4]前記標的害虫がコウチュウ目テントウムシ科、バッタ目イナゴ科、チョウ目ヤガ科又はゴキブリ目ゴキブリ科の昆虫である、[1]に記載の害虫防除方法。
[5]前記標的害虫がニジュウヤホシテントウ、ナミテントウ、コバネイナゴ、オオタバコガ又はチュウトウゴキブリである、[1]に記載の害虫防除方法。
[6]前記阻害物質が、以下の(a)~(d)からなる群より選択される化合物である、[1]~の[5]いずれか一項に記載の害虫防除方法:
(a)前記標的害虫のアポトーシス阻害因子をコードする遺伝子を標的としたsiRNA;
(b)前記標的害虫のアポトーシス阻害因子をコードする遺伝子を標的としたsiRNAを細胞内で生成する核酸コンストラクト;
(c)前記標的害虫のアポトーシス阻害因子をコードする遺伝子の転写産物を標的としたアンチセンス核酸;
(d)前記標的害虫のアポトーシス阻害因子をコードする遺伝子の転写産物を標的としたリボザイム。
[7]前記遺伝子が配列番号1に示す配列、配列番号3に示す配列、配列番号5に示す配列、配列番号16に示す配列、配列番号17に示す配列、又は配列番号19に示す配列を含む、[6]に記載の害虫防除方法。
[8]前記標的害虫が害する植物に、前記阻害物質を含有する薬剤を塗布、散布又は噴霧によって予め保持させておき、該植物の摂取によって前記阻害物質を前記標的害虫の体内に取り込ませることを特徴とする、[1]~[7]のいずれか一項に記載の害虫防除方法。
[9]前記標的害虫の発生箇所又は侵入経路に、前記阻害物質を含有する餌を置き、該餌の摂食によって前記阻害物質を前記標的害虫の体内に取り込ませることを特徴とする、[1]~[7]のいずれか一項に記載の害虫防除方法。
[10]前記阻害物質をコードする遺伝子が導入されたトランスジェニック植物の摂取によって前記阻害物質を前記標的害虫の体内に取り込ませることを特徴とする、[1]~[7]のいずれか一項に記載の害虫防除方法。
[11]前記阻害物質が、以下の(A)~(C)からなる群より選択される化合物である、[10]に記載の害虫防除方法:
(A)前記標的害虫のアポトーシス阻害因子をコードする遺伝子を標的としたsiRNA;
(B)前記標的害虫のアポトーシス阻害因子をコードする遺伝子の転写産物を標的としたアンチセンス核酸;
(C)前記標的害虫のアポトーシス阻害因子をコードする遺伝子の転写産物を標的としたリボザイム。
[12]標的害虫のアポトーシス阻害因子に対する阻害物質を含有することを特徴とする、害虫防除剤。
[13]前記阻害物質が、以下の(a)~(d)からなる群より選択される化合物である、[12]に記載の害虫防除剤:
(a)前記標的害虫のアポトーシス阻害因子をコードする遺伝子を標的としたsiRNA;
(b)前記標的害虫のアポトーシス阻害因子をコードする遺伝子を標的としたsiRNAを細胞内で生成する核酸コンストラクト;
(c)前記標的害虫のアポトーシス阻害因子をコードする遺伝子の転写産物を標的としたアンチセンス核酸;
(d)前記標的害虫のアポトーシス阻害因子をコードする遺伝子の転写産物を標的としたリボザイム。
[14]標的害虫のアポトーシス阻害因子に対する阻害物質をコードする遺伝子が導入されたトランスジェニック植物。
本発明の第1の局面は害虫防除方法に関する。本発明における「害虫」は特に限定されない。一般に、害虫は農業害虫、衛生害虫、及び不快害虫に大別される。「農業害虫」とは、農作物(園芸作物を含む。また、保存中の農作物も含む)を害する害虫をいう。保存中の農作物を害する害虫については「貯穀害虫」と呼ぶことがある。「衛生害虫」とは、ヒトの衛生環境を害する害虫をいう。同様に、「不快害虫」とは、外見や動きなどによってヒトの気分を害する害虫をいう。ヒトの財産を害する害虫(シロアリ、シミなど)や家畜を害する害虫(蚊、寄生虫など)に対しても本発明を適用可能である。
(a)前記標的害虫のアポトーシス阻害因子をコードする遺伝子を標的としたsiRNA;
(b)前記標的害虫のアポトーシス阻害因子をコードする遺伝子を標的としたsiRNAを細胞内で生成する核酸コンストラクト;
(c)前記標的害虫のアポトーシス阻害因子をコードする遺伝子の転写産物を標的としたアンチセンス核酸;
(d)前記標的害虫のアポトーシス阻害因子をコードする遺伝子の転写産物を標的としたリボザイム。
IAP阻害物質の取り込ませ方は特に限定されず、標的害虫毎に適当なものを選択すればよい。植物を食害する害虫が標的害虫の場合には、例えば、標的害虫が害する植物に、IAP阻害物質を含有する薬剤(農薬)を塗布、散布又は噴霧によって予め保持させておく。このようにすれば、標的害虫が植物を摂食した際、IAP阻害物質が標的害虫の体内に取り込まれる。一方、IAP阻害物質を含有する餌(餌剤)を標的害虫の発生箇所又は侵入経路に置いておくことにすれば、標的害虫が当該餌を摂食し、これによってIAP阻害物質が標的害虫の体内に取り込まれる。また、食害される植物にIAP阻害物質をコードする遺伝子を導入して改変しておけば、当該トランスジェニック植物を摂食した際、IAP阻害物質が標的害虫の体内に取り込まれる。このような方法に用いられるトランスジェニック植物として、(A)標的害虫のIAPをコードする遺伝子を標的としたsiRNA、(B)標的害虫のIAPをコードする遺伝子の転写産物を標的としたアンチセンス核酸、又は(C)標的害虫のIAPをコードする遺伝子の転写産物を標的としたリボザイム、が発現するように遺伝子改変された植物を用いることができる。
本発明の第2の局面は、上記防除方法に利用可能な害虫防除剤に関する。本発明の害虫防除剤は例えば農薬の形態又は餌の形態で提供される。本発明の害虫防除剤は、標的害虫のIAPに対する阻害物質(IAP阻害物質)を含有する。ここでのIAP阻害物質の例は、以下の(a)~(d)である。これらの各化合物の詳細は上で述べた通りである。
(a)標的害虫のIAPをコードする遺伝子を標的としたsiRNA;
(b)標的害虫のIAPをコードする遺伝子を標的としたsiRNAを細胞内で生成する核酸コンストラクト;
(c)標的害虫のIAPをコードする遺伝子の転写産物を標的としたアンチセンス核酸;
(d)標的害虫のIAPをコードする遺伝子の転写産物を標的としたリボザイム。
本発明の更なる局面は(第3の局面)はトランスジェニック植物に関する。本発明のトランスジェニック植物には、標的害虫のIAPに対する阻害物質をコードする遺伝子が導入されている。典型的には、本発明のトランスジェニック植物は、(A)標的害虫のIAPをコードする遺伝子を標的としたsiRNA、(B)標的害虫のIAPをコードする遺伝子の転写産物を標的としたアンチセンス核酸、又は(C)標的害虫のIAPをコードする遺伝子の転写産物を標的としたリボザイム、が発現するように遺伝子改変されている。
アポトーシス阻害因子(IAP)に注目し、IAPの発現を特異的に阻害可能な二本鎖RNA(dsRNA)による防除効果を検討した。
<材料と方法>
1.アポトーシス阻害因子ホモログcDNAのクローニングおよび塩基配列の決定
(1)供試昆虫
ニジュウヤホシテントウ(Henosepilachna vigintioctopunctata)については名古屋大学 大学院生命農学研究科圃場のジャガイモ葉上より採集して継代飼育した個体、ナミテントウ(Harmonia axyridis)については当研究室で継代飼育した個体、コバネイナゴ(Oxya yezoensis)については名古屋大学 大学院生命農学研究科圃場の水田より採集した幼虫を用いた。
ニジュウヤホシテントウ(Hv)については前蛹期の幼虫の翅原基、ナミテントウ(Ha)については胚、コバネイナゴ(Oy)については幼虫の後肢より、全RNAをTRIZOL(LIFE TECHNOLOGIES)を用いた塩酸グアニジン法により抽出した。このRNAを鋳型として、SMARTTM RACE cDNA Amplification Kit(CLONTECH)に基づき、Superscript II reverse transcriptase (GIBCO BRL)を用いて5′Rapid amplification of cDNA ends(RACE)用と3′RACE用とに分けて1本鎖cDNAを合成した。
上記の通り調製した1本鎖cDNAを鋳型として、ポリメラーゼチェーン反応(PCR)を行った。PCRのTaq DNAポリメラーゼにはAmpli Taq Gold(PERKIN ELMER)を用いた。PCRに用いたプライマーを以下に示す。
(iapセンスプライマー)
iap-01: 5′-GCIGAIGCIGGITTYTWYTA-3′(20mer)(配列番号7)
iap-02: 5′-GAYKIICCITGGGARSARCAYG-3′(22mer)(配列番号8)
(iapアンチセンスプライマー)
iap-03: 5′-CAIGYIRYIAIRTGICCRCAIGG-3′(23mer)(配列番号9)
尚、WはA+T、SはC+G、YはC+T、RはA+G、KはT+G、Iはイノシンを表す。
上記のように調製した1本鎖cDNAを鋳型として用い、SMARTTM RACE cDNA Amplification Kit(CLONTECH)のプロトコルに基づいてRACEを行った。用いたプライマーは、ニジュウヤホシテントウiapホモログcDNAの部分配列をもとに、5′RACE、3′RACE用に各2本ずつ設計した。設計したプライマーは以下の通りである。
(5′RACE用)
Hv-iap-01: 5′-CTTCGACCCAATCTTTCAGACCGCC-3′(25mer)(配列番号10)
Hv-iap-02: 5′-AAAGCGCGTGCTGTTCCCACGGATC-3′(25mer)(配列番号11)
Hv-iap-03: 5′-GATCCGTGGGAACAGCACGCGCTTT-3′(25mer)(配列番号12)
Hv-iap-04: 5′-GAAACAACGCAGAGGAAAGCTCGAC-3′(25mer)(配列番号13)
PCR後、TBE(89 mM Tris、89 mM ホウ酸、2 mM EDTA)にアガロース(Agarose II [DOJINDO])を添加し、0.5 mg/mlとなるようにエチジウムブロマイドを加えた1% Agarose IIアガロースゲルによる電気泳動により、PCR産物を分離した。目的とするPCR産物を含んだバンドを切り出し、Mag Extractor(Toyobo)によりPCR産物を回収してサブクローニング用のインサートとして用いた。
上記のように調製したインサートをpBluescriptTMKS(+)(pBS)のEco RV認識部位に挿入した。ライゲーション反応は、DNA Ligation Kit Ver. 2(TaKaRa)を用いて行った。このライゲーション反応液を、大腸菌(XL1-Blue)への形質転換に用いた。この形質転換した大腸菌培養液をLBプレートに引き、37℃で一晩培養した。ホワイトコロニーを選出し、PCR法を用いて目的のPCR断片が挿入されたクローンを選択した。このときのPCRのTaq DNAポリメラーゼにはAmpli Taq Gold (PERKIN ELMER)を用い、プライマーはSKプライマーおよびKSプライマーを用いた。PCRは25サイクルを変性(95℃、30秒間);アニーリング(55℃、30秒間);伸長(72℃、30秒間)の条件で行った。選択したクローンをLB培地中で37℃で一晩振とう培養し、FlexiPrep Kit(Amerciam Pharmacia Biotech)によりキットのプロトコルに従ってプラスミドDNAを調製した。
BigDye Terminator v3.1 Cycle Sequencing kitを用いて、プロトコルに従って塩基配列を決定した。得られた塩基配列はDNASISで解析した。
(1)RNA合成用の鋳型の増幅
ニジュウヤホシテントウのiapは、pBluescript KS+ベクター(Stratagene)のEcoRVサイトにiap1とiap3のプライマーで増幅されたRT-PCR産物がサブクローニングされたものを用いた。ナミテントウのiapおよびコバネイナゴのiapは、同様にiap2とiap3のプライマーで増幅されたRT-PCR産物がサブクローニングされものを用いた。
(T7-KSプライマー)
5'-TAATACGACTCACTATAGGGAGACCACTCGAGGTCGACGGTATC-3'(配列番号14)
(T7-SKプライマー)
5'-TAATACGACTCACTATAGGGAGACCACCGCTCTAGAACTAGTGGATC-3'(配列番号15)
テンプレートDNA(20~50 ng)+ H2O 37.75μl
10×バッファー 5μl
2 mM dNTP 5μl
10 pmol/μl T7-KSプライマー 1μl
10 pmol/μl T7-SKプライマー 1μl
AmpliTaq Gold (Perkin Elmer) 0.25μl
合計 50μl
[95℃で9分]→[94℃で60秒→55℃で30秒→72℃で30秒]×40サイクル→72℃で7分→12℃で∞
上記鋳型DNAを1μg用い、MEGAscrtipt T7 Kit(Ambion)に従ってRNAを合成し、適量のヌクレアーゼ・フリーの超純水に溶解した。
この二本鎖RNAを少量用い、濃度測定およびアガロース電気泳動による確認を行った。
合成が確認された二本鎖RNAは、2μg/μlの濃度に調整し、一回分ずつに小分けして-80℃で保存した。
ニジュウヤホシテントウの場合、3齢幼虫を実験に用いた。5μlのHv-iap dsRNAをジャガイモ葉に10カ所程度スポットした。このジャガイモ葉を幼虫に与え、RNA溶液をスポットした領域をすべて摂食させた。対照区には、水を用いた。その後、経時的に幼虫を観察した。
1.ニジュウヤホシテントウ(コウチュウ目、テントウムシ科)
Hv-iap dsRNAをスポットしたジャガイモ葉を摂食した幼虫(試験区)の経時的変化を図1に示す。対照区と比較して、摂食開始後の早い段階から摂食量及び運動量が減少した。また、摂食開始から64時間後のジャガイモの葉を観察したところ(図2)、試験区(上段)と対照区(下段)の間で摂食量に顕著な差を認めた。また、試験区の幼虫は全て4日以内に死亡し、対照区の幼虫は4日を経過してもすべて生存した(図3)。以上のように、Hv-iap dsRNAを体内に取り込ませてIAPを阻害したことにより、速やかに摂食障害が生じ、生存・成長が阻害された。即ち、IAPの阻害が迅速且つ優れた防除効果を発揮することが示された。
Ha-iap dsRNAおよびgfp dsRNAをそれぞれ雄成虫2頭に与えた。Ha-iap dsRNAを与えた個体はすべて、24時間以内に摂食量が著しく減少した。4.5日間の摂食量は、Ha-iap dsRNAを与えた個体はすべて10 mg以下であったのに対し、gfp dsRNAを与えた個体はすべて20 mgであった。一方、Ha-iap dsRNAを与えた個体は、7.5日後と9日後に死亡した。これに対して、gfp dsRNAを与えた個体は33日経過してもすべて生存した。以上のように、ナミテントウについても、IAPの阻害が優れた防除効果を発揮することが示された。
Oy-iap dsRNAおよびgfp dsRNAをそれぞれ成虫2頭に与えた。結果、Oy-iap dsRNAを与えた個体は9日後と12日後に死亡した。一方、gfp dsRNAを与えた個体は25日経過してもすべて生存した。このように、コバネイナゴについても、IAPの阻害が優れた防除効果を発揮することが示された。尚、dsRNA摂食後の、イネ葉の摂食量は、実験上測定困難であったため検討しなかった。
別の昆虫を用い、IAPを標的とした防除戦略の有効性を確認した。
<材料と方法>
1.IPAホモログcDNAのクローニングおよび塩基配列の決定
(1)供試昆虫
オオタバコガ(Helicoverpa armigera)、チュウトウゴキブリ(Blatta lateralis、レッドローチあるいはトルキスタンローチとも呼ばれる)、及びニジュウヤホシテントウ(Henosepilachna vigintioctopunctata)を用いた。
オオタバコガ(Har)については蛹化後6日の蛹の前翅原基、ゴキブリ(Bl)については終齢幼虫の後翅原基、ニジュウヤホシテントウ(Hv)については成虫の卵巣および精巣より、全RNAをTRIZOL(LIFE TECHNOLOGIES)を用いた塩酸グアニジン法により抽出した。このRNAを鋳型として、SMARTTM RACE cDNA Amplification Kit(CLONTECH)に基づき、Superscript II reverse transcriptase (GIBCO BRL)を用いて5′Rapid amplification of cDNA ends(RACE)用と3′RACE用とに分けて1本鎖cDNAを合成した。
上記の通り調製した1本鎖cDNAを鋳型として、Polymerase chain reaction(PCR)を行った。PCRのTaq DNAポリメラーゼにはAmpli Taq Gold (PERKIN ELMER)を用いた。PCRに用いたプライマーを以下に示す。
(iapセンスプライマー)
iap-01: 5′-GCIGAIGCIGGITTYTWYTA-3′(20mer)(配列番号7)
iap-02: 5′-GAYKIICCITGGGARSARCAYG-3′(22mer)(配列番号8)
(iapアンチセンスプライマー)
iap-03: 5′-CAIGYIRYIAIRTGICCRCAIGG-3′(23mer)(配列番号9)
iap-08: 5′-GCRCAYTTISCRCAIGCIACIAC-3′(23mer)(配列番号21)
尚、WはA+T、SはC+G、YはC+T、RはA+G、KはT+G、Iはイノシンを表す。
それぞれの種に対応したセンスプライマーおよびアンチセンスプライマーの組み合わせ、および反応条件は下記の通りである。
iap cDNA
A.<材料と方法>1.(4)と同様の方法で行った。ただしサンプルは前回と異なり、上記のように成虫の卵巣および精巣を用いた。
A.<材料と方法>1.(5)と同様の方法で行った。
上記のように調製したインサートをTOPO TA Cloning Kit(Invitrogen)を用いTOPO vector (pCR4-TOPO)に挿入した。この反応液を、大腸菌(DHαTM-T1R)への形質転換に用いた。この形質転換した大腸菌培養液をLBプレートに引き、37℃で一晩培養した。生じたコロニーを選出し、PCR法を用いて目的のPCR断片が挿入されたクローンを選択した。このときのPCRのTaq DNAポリメラーゼにはAmpli Taq Gold (PERKIN ELMER)を用い、プライマーはT3プライマーおよびT7プライマーを用いた。PCRは25サイクルを変性、95℃/ 30秒間;アニーリング、55℃/ 30秒間;伸長、72℃/ 30秒間の条件で行った。選択したクローンをLB中で37℃で一晩振とう培養し、Axy Prep Plasmid Miniprep Kit(Axygen Scientific)によりキットのプロトコルに従ってプラスミドDNAを調製した。
A.<材料と方法>1.(7)と同様の方法で行った。
(1)RNA合成用の鋳型の増幅
オオタバコガおよびゴキブリのiapは、TOPO vector (pCR4-TOPO)にiap1とiap3のプライマーで増幅されたRT-PCR産物がサブクローニングされたものを用いた。二本鎖RNAを合成するための鋳型として使用するPCR産物を得るため、上記のベクターにクローニングしたどの遺伝子にも共通して使用可能となるように、ベクター配列にT7 RNAポリメラーゼのプロモーター配列を付加した下記のPCRプライマーを使用した。
(T7-PCR4Rプライマー)
5'- TAATACGACTCACTATAGGGAGACCACCGAATTGAATTTAGCGGC -3'(配列番号22)
(T7-PCR4Lプライマー)
5'- TAATACGACTCACTATAGGGAGACCACGTCCTGCAGGTTTAAACG -3'(配列番号23)
テンプレートDNA(20~50 ng)+ H2O 37.75μl
10×バッファー 5μl
2 mM dNTP 5μl
10 pmol/μl T7-PCR4Rプライマー 1μl
10 pmol/μl T7-PCR4Lプライマー 1μl
AmpliTaq Gold (Perkin Elmer) 0.25μl
合計 50μl
[95℃で9分]→[94℃で60秒→55℃で30秒→72℃で30秒]×40サイクル→72℃で7分→12℃で∞
上記鋳型DNAを1μg用い、MEGAscrtipt T7 Kit(Ambion)に従ってRNAを合成し、適量のヌクレアーゼ・フリーの超純水に溶解した。二本鎖RNAのアニーリングを行うため、得られたRNA溶液はヒートブロックを用い65°Cで30分間インキュベートした後、1~2時間かけて室温に戻した。この二本鎖RNAを少量用い、濃度測定およびアガロース電気泳動による確認を行った。合成が確認された二本鎖RNAは、オオタバコガiap(Har-iap)の場合10μg/μl、ゴキブリiap(Bl-iap)の場合5μg/μlの濃度に調整し、一回分ずつに小分けして-80℃で保存した。
オオタバコガの場合、2齢幼虫を実験に用いた。1μl(10μg)のHar-iap dsRNAを少量の人工飼料(インセクタLFS、日本農産工)に添加した。この飼料を幼虫に摂食させた。GFP dsRNAを同様に用いた。その後、経時的に幼虫を観察した。
1.ニジュウヤホシテントウ
IAPをコードする全長cDNA配列(配列番号16)が確認された。
Har-iap dsRNAを添加した人工飼料を摂食させた幼虫(試験区)を経時的に観察した。図4に示す通り、試験区の幼虫は全て4日以内に死亡した。対照区の幼虫は4日を経過しても生存した。このように、IPAの阻害によって迅速且つ顕著な防除効果を発揮できることが示された。
Bl-iap dsRNA水溶液を摂食させた幼虫(試験区)を経時的に観察した。試験区の幼虫は極めて早い段階から行動障害を示した(図5)。また、試験区の幼虫はほとんどが1日以内に死亡した。これに対して対照区の幼虫には行動障害や生存・成長阻害は認められなかった。このように、IAPの阻害によって迅速且つ極めて強い防除効果を発揮できることが示された。
本法が遺伝子導入植物(由来のRNA)を用いた場合でも機能することを確かめるために、ウイルスベクターによりニジュウヤホシテントウのiap(Hv-iap)を発現させたベンサミアナタバコ(Nicotiana benthamiana)葉から抽出したRNAを用いてニジュウヤホシテントウへの摂食試験を行った。
(1)供試植物
ベンサミアナタバコ(Nicotiana benthamiana)は、日本たばこ産業株式会社葉たばこ研究所より分譲を受けた。種子をクレハソイル(株式会社クレハ)とソイルミックス(株式会社サカタのタネ)を入れたポリエチレンポットに播種し、播種後10日目の植物体を同様のポリエチレンポットに移植し、25℃の恒温室にて24時間の明条件下で生育させた。処理を行った植物体は22℃の恒温室に移し、16時間の明条件および8時間の暗条件下で静置した。
ニジュウヤホシテントウ(Henosepilachna vigintioctopunctata)
全RNAの抽出はTRIZOL Reagent(invitrogen)を用いて、以下の方法で行った。ジルコニアビーズ2粒が入った2 mlのサンプリングチューブ(株式会社アシスト)にN. benthamiana葉組織およそ100 mgを入れ、液体窒素で凍結し、Shake Master(bio medical science)を用いて破砕した。そこへ1 mlのTRIZOL Reagentを加えてよく懸濁した。懸濁液を遠心分離して(12,000 x g、10分間、4℃)上層を新しい1.5 ml容エッペンドルフチューブに移し回収し、この溶液に200μlのクロロホルムを加えて撹拌した後、遠心分離した(12,000 x g、15分間、4℃)。上層を新しい1.5 ml容エッペンドルフチューブに移し回収し、この溶液に500μlのイソプロパノールを加えて撹拌して10分間静置した後、遠心分離した (12,000 x g、10分間、4℃)。得られた沈澱を70%エタノールで洗浄した後10分間風乾し、40μlのDEPC処理水に溶解して全RNAとした。分光光度計(ND-1000 Spectrophotometer、NanoDrop)を用いてA260を測定することによりRNA量を定量し、実験に用いた。
PVX(potato virus X)を含むpGR107バイナリーベクター(Ratcliff, F., Martin-Hernandez, A.M., and Baulcombe, D.C. (2001). Tobacco rattle virus as a vector for analysis of gene function by silencing. Plant J. 25, 237-245.)に挿入する389 bpのHv-iap cDNA断片をPCRにより調製した。制限酵素配列を付加した下記のPCRプライマーを使用した。
(Hv-iap-NotI-センスプライマー)
5’-ATAAGAATGCGGCCGCGCGGAGGCGGGGTTTTATTAC-3’(配列番号24)
(Hv-iap-NotI-アンチセンスプライマー)
5’-ATAGTTTAGCGGCCGCCAGGCGATGAGATGGCCACA-3’(配列番号25)
テンプレートDNA(20~50 ng)+ H2O 37.75 ml
10×バッファー 5 ml
2 mM dNTP 5 ml
10 pmol/μl Hv-iap-NotI-センスプライマー 1 ml
10 pmol/μl Hv-iap-NotI-アンチセンスプライマー 1 ml
AmpliTaq Gold (Perkin Elmer) 0.25 ml
合計 50 ml
[95℃で9分]→[94℃で60秒→55℃で30秒→72℃で30秒]×25サイクル→72℃で7分→12℃で∞
Agrobacterium tumefaciensの形質転換は、Hellensらの方法(Hellens, R.P., Edwards, E.A., Leyland, N.R., Bean, S., and Mullineaux, P.M. (2000). pGreen: A versatile and flexible binary Ti vector for Agrobacterium-mediated plant transformation. Plant Mol. Biol. 42, 819-832.)に準じて行った。導入するベクター(10 ng)と共に、ヘルパープラスミドpSoup(10 ng)を加えて氷上で溶解したA. tumefaciens GV3101のエレクトロセル40μlを氷上で30分間静置した。この溶液をあらかじめ氷冷したキュベットに移し、遺伝子導入装置Micro PulserTM (BIO-RAD)を用いてエレクトロポレート(V=1.44 kV、T=2.5 kV/resistance、C=all out、R=R5 129)して形質転換した。溶液を14 mlポリプロピレンラウンド・チューブ(BECTON DICKINSON)に移し氷上に10分間静置した後、960μl SOC培地[2 % トリプトン ペプトン、0.5 % 酵母エキス、0.05% NaCl、10 mM MgCl2、10 mM MgSO4、20 mMグルコース、pH 7.0]を加え、28℃で1時間振とうした。YEB培地[0.1 % 酵母エキス、0.5 % ビーフエキス、0.5 % トリプトン ペプトン、0.5%シュークロース、2 mM MgSO4、2 % 寒天、pH 7.2]にカナマイシン(50μg/ml)およびリファンピシン(50μg/ml)を加えた培地に20μlプレーティングし、28℃で30時間培養した。
389 bpのHv-iap cDNA断片を含むpGR107ベクターを保持するアグロバクテリウムを、50μg/mlのカナマイシン、50μg/mlのリファンピシンおよび5μg/mlのテトラサイクリンを含むLB液体培地中で28℃で2日間培養した。培養液をLB液体培地で5~10倍希釈し、OD600=0.5-1.0になるまで28℃でさらに培養した。アグロバクテリウムを遠心分離(1,700 x g、15分)により回収し、5 mlの導入緩衝液に懸濁後、再び遠心分離により集菌した。沈澱したアグロバクテリウムをOD600=0.5になるように150μMアセトシリンゴンを含む導入緩衝液に懸濁し、室温で2時間静置した後、注射筒を用いて播種後約3週間のN. benthamiana葉の細胞間隙に注入した。アグロバクテリウム注入後3~4週間の植物の上位葉を実験に用いた。
RT-PCRは、ReverTra Ace-Plus-(登録商標)(TOYOBO)を用いて以下のように行った。6μlの溶液中に0.5μgの全RNAと25 pmolのOligo(dT)20プライマーを混合し、65℃で5分間処理した後、直ちに氷冷した。続いて残りのRT-PCR反応液[1 x RT反応液、1 mM dNTPs、1 unit/μl RNase inhibitor、5 unit/μl ReverTra Ace(登録商標)]を加え、10μlの反応液中で、30℃で10分間、42℃で1時間、85℃で5分間の条件で逆転写反応を行った。PCR反応はExTaq(Takara)を用い、逆転写反応液1μlに対して10μlの反応液中で、アニーリング温度を53℃、20秒間、伸長反応を74℃、30秒間で27サイクルの条件で行った。RT-PCRに用いたプライマーの塩基配列を以下に示す。
(Hv-iap-NotI-センスプライマー)
5’-ATAAGAATGCGGCCGCGCGGAGGCGGGGTTTTATTAC-3’(配列番号24)
(Hv-iap-NotI-アンチセンスプライマー)
5’-ATAGTTTAGCGGCCGCCAGGCGATGAGATGGCCACA-3’(配列番号25)
(EF-1αセンスプライマー)
5’-TGTGGAAGTTTGAGACCACC-3’(配列番号26)
(EF-1αアンチセンスプライマー)
5’-GCAAGCAATGCGTGCTCAC-3’(配列番号27)
ニジュウヤホシテントウのiap(Hv-iap)は、A.<材料と方法>2.(1)(2)と同じものを使用した。
ニジュウヤホシテントウの3齢幼虫を実験に用いた。ニジュウヤホシテントウは食草ではないN. benthamiana葉を摂食しないため、植物で発現させたRNAに含まれると思われるHv-iap dsRNAおよびsiRNAの効果を検討した。Hv-iap遺伝子断片を導入したウイルスを発現させたN. benthamiana葉より全RNAを抽出し、2μgを摂食させた。この実験の対照区には、ウイルスのみを発現させたN. benthamiana葉組織より抽出した全RNA 2μgを用いた。摂食させた後、ジャガイモ葉を幼虫に与えた。また、N. benthamiana葉組織より抽出した全RNAに含まれるHv-iap dsRNAは微量と予想されるため、ポジティブコントロールとして、Hv-iap 合成dsRNAを用いた。この場合、20 ngを摂食させた後、ジャガイモ葉を幼虫に与えた。その後、経時的に幼虫を観察した。
Hv-iap dsRNA導入ウイルスを発現させたN. benthamiana葉より抽出した全RNAを摂食させた幼虫(試験区)は、一匹を除き、43時間後には殆ど摂食を停止し、67時間後には完全に摂食を停止した(図6)。この結果は、RNAを植物内で合成させた場合であってもIAP阻害効果が十分に得られることを示す。また、標的害虫のIAPの発現を特異的に阻害するRNAが合成されるように植物体を組み換えること(即ち、トランスジェニック植物の作出)が、防除戦略として有効であることを強く示唆する。尚、20 ngという極微量のdsRNAを摂食させた場合(ポジティブコントロール)において十分な摂食停止効果が得られたことも注目に値する。
本明細書の中で明示した論文、公開特許公報、及び特許公報などの内容は、その全ての内容を援用によって引用することとする。
配列番号8:人工配列の説明:iapセンスプライマー(iap-02)
配列番号9:人工配列の説明:iapアンチセンスプライマー(iap-03)
配列番号10:人工配列の説明:5′RACE用プライマー(Hv-iap-01)
配列番号11:人工配列の説明:5′RACE用プライマー(Hv-iap-02)
配列番号12:人工配列の説明:3′RACE用プライマー(Hv-iap-03)
配列番号13:人工配列の説明:3′RACE用プライマー(Hv-iap-04)
配列番号14:人工配列の説明:PCR用プライマー(T7-KS)
配列番号15:人工配列の説明:PCR用プライマー(T7-SK)
配列番号21:人工配列の説明:iapアンチセンスプライマー(iap-08)
配列番号22:人工配列の説明:PCR用プライマー(T7-PCR4R)
配列番号23:人工配列の説明:PCR用プライマー(T7-PCR4L)
配列番号24:人工配列の説明:Hv-iap-NotI-センスプライマー
配列番号25:人工配列の説明:Hv-iap-NotI-アンチセンスプライマー
配列番号26:人工配列の説明:EF-1αセンスプライマー
配列番号27:人工配列の説明:EF-1αアンチセンスプライマー
Claims (14)
- アポトーシス阻害因子(Inhibitor of apoptosis;IAP)に対する阻害物質を標的害虫の体内に取り込ませること、を特徴とする害虫防除方法。
- 前記標的害虫が農業害虫、衛生害虫又は不快害虫である、請求項1に記載の害虫防除方法。
- 前記標的害虫がコウチュウ目、バッタ目、チョウ目又はゴキブリ目の昆虫である、請求項1に記載の害虫防除方法。
- 前記標的害虫がコウチュウ目テントウムシ科、バッタ目イナゴ科、チョウ目ヤガ科又はゴキブリ目ゴキブリ科の昆虫である、請求項1に記載の害虫防除方法。
- 前記標的害虫がニジュウヤホシテントウ、ナミテントウ、コバネイナゴ、オオタバコガ又はチュウトウゴキブリである、請求項1に記載の害虫防除方法。
- 前記阻害物質が、以下の(a)~(d)からなる群より選択される化合物である、請求項1~5のいずれか一項に記載の害虫防除方法:
(a)前記標的害虫のアポトーシス阻害因子をコードする遺伝子を標的としたsiRNA;
(b)前記標的害虫のアポトーシス阻害因子をコードする遺伝子を標的としたsiRNAを細胞内で生成する核酸コンストラクト;
(c)前記標的害虫のアポトーシス阻害因子をコードする遺伝子の転写産物を標的としたアンチセンス核酸;
(d)前記標的害虫のアポトーシス阻害因子をコードする遺伝子の転写産物を標的としたリボザイム。 - 前記遺伝子が配列番号1に示す配列、配列番号3に示す配列、配列番号5に示す配列、配列番号16に示す配列、配列番号17に示す配列、又は配列番号19に示す配列を含む、請求項6に記載の害虫防除方法。
- 前記標的害虫が害する植物に、前記阻害物質を含有する薬剤を塗布、散布又は噴霧によって予め保持させておき、該植物の摂取によって前記阻害物質を前記標的害虫の体内に取り込ませることを特徴とする、請求項1~7のいずれか一項に記載の害虫防除方法。
- 前記標的害虫の発生箇所又は侵入経路に、前記阻害物質を含有する餌を置き、該餌の摂食によって前記阻害物質を前記標的害虫の体内に取り込ませることを特徴とする、請求項1~7のいずれか一項に記載の害虫防除方法。
- 前記阻害物質をコードする遺伝子が導入されたトランスジェニック植物の摂取によって前記阻害物質を前記標的害虫の体内に取り込ませることを特徴とする、請求項1~7のいずれか一項に記載の害虫防除方法。
- 前記阻害物質が、以下の(A)~(C)からなる群より選択される化合物である、請求項10に記載の害虫防除方法:
(A)前記標的害虫のアポトーシス阻害因子をコードする遺伝子を標的としたsiRNA;
(B)前記標的害虫のアポトーシス阻害因子をコードする遺伝子の転写産物を標的としたアンチセンス核酸;
(C)前記標的害虫のアポトーシス阻害因子をコードする遺伝子の転写産物を標的としたリボザイム。 - 標的害虫のアポトーシス阻害因子に対する阻害物質を含有することを特徴とする、害虫防除剤。
- 前記阻害物質が、以下の(a)~(d)からなる群より選択される化合物である、請求項12に記載の害虫防除剤:
(a)前記標的害虫のアポトーシス阻害因子をコードする遺伝子を標的としたsiRNA;
(b)前記標的害虫のアポトーシス阻害因子をコードする遺伝子を標的としたsiRNAを細胞内で生成する核酸コンストラクト;
(c)前記標的害虫のアポトーシス阻害因子をコードする遺伝子の転写産物を標的としたアンチセンス核酸;
(d)前記標的害虫のアポトーシス阻害因子をコードする遺伝子の転写産物を標的としたリボザイム。 - 標的害虫のアポトーシス阻害因子に対する阻害物質をコードする遺伝子が導入されたトランスジェニック植物。
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Also Published As
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US20120151631A1 (en) | 2012-06-14 |
JP5305489B2 (ja) | 2013-10-02 |
EP2438813A1 (en) | 2012-04-11 |
JPWO2010140675A1 (ja) | 2012-11-22 |
EP2438813A4 (en) | 2013-10-02 |
US9051569B2 (en) | 2015-06-09 |
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