KR102008064B1 - Composition and method of controlling virus mediated by small brown planthopper using dsRNA targeting nuclear receptor E75 gene of small brown planthopper - Google Patents

Abstract

The present application discloses a dsRNA that inhibits the expression of apoptotic nucleus receptor E75, and a composition and method for apoptotic control comprising the same. The dsRNA or the composition according to the present invention can effectively inhibit the proliferation of rice stripe leaf blight virus or the like mediated by inhibiting the expression of the apoptotic nucleus receptor E75 protein.

Description

Composition and method of controlling virus mediated by small brown planthopper using dsRNA targeting nuclear receptor E75 gene of small brown planthopper}

The present invention relates to a composition for and control of apoptotic mediated virus using dsRNA specific to the nuclear receptor E75 gene of the crypt.

Anguilla, one of the major pests of rice in Korea, has been affected by the southern regions as the general varieties affected by this have been expanded and grown since the 1980s.

The damage caused by larvae is more damaging by the RSV and the rice black-streaked dwarf virus (RSVV) that they mediate, rather than the direct damage caused by the nectar. The frequency and damage of the virus is increasing year by year. Recently, the overwintering survival rate of annihilate is increasing due to the abnormal high temperature caused by climate change. It was first reported in 2009 in Jeollabuk-do, and it is reported that the amount of anchovy that is flying every year is rapidly increasing, and there is concern about the occurrence of annihilation by the development of southwest wind in mid-May to early June, which is the wheat harvest season in China. Therefore, special countermeasures are needed.

Currently, the control of the virus mediated by the larvae is mainly preventive through the prediction of disease outbreaks using the diagnostic kit, and there is no cure for the virus itself. As a situation which depends entirely on the chemical control used, the control method using RNA interference (RNAi) has recently been developed.

US Patent No. 9,528,123 relates to a dsRNA for insect control, and has disclosed a dsRNA for controlling a CHD3 gene and the like.

US Patent Application Publication No. 2014/0143906 relates to a method of improving resistance to pests of plants, and dsRNAs for pest-derived target genes, such as trypsin progenitors, are disclosed.

Republic of Korea Patent No. 1013092 relates to a rice streaked leaf blight resistant transformed rice and its manufacturing method, dsRNA targeting the RSV-CP-SW gene is disclosed.

However, no dsRNA specific to the neutrophil E75 gene of larvae is disclosed.

The present invention inhibits the proliferation of apoptotic mediator virus through RNA interference induced by using dsRNA specific to the Nuclear Receptor E75 gene of the crypt, thereby controlling the composition and control method To provide.

According to the present application, it is possible to inhibit the expression of the E75 gene of the apoptotic clarifier disclosed herein, thereby inhibiting the proliferation of the apoptotic mediated virus.

In one aspect herein, the present disclosure discloses a gene, polynucleotide or nucleic acid, a vector comprising the gene, and a cell comprising the vector, which encode the apocalyptic E75 protein represented by the sequence of SEQ ID NO: 1 as identified herein.

In another embodiment the present application is a double-stranded RNA comprising two strands of complementary RNA, having a sequence represented by SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5, wherein the dsRNA The expression of the nuclear receptor E75 gene into a protein is suppressed to provide a double-stranded RNA having viral control ability mediated by the apoptotic sphere.

In one embodiment the two strands of the dsRNA are completely complementary, or once the sequence of one strand is determined, the sequence of the other strand can be readily determined by complementarity.

Or may be partially complementary and with a degree of complementarity sufficient to be introduced into the cell to induce RNAi mechanisms, one of ordinary skill in the art will be able to determine appropriate complementarity in view of the description herein, the mechanism of RNAi, and the target sequence according to the invention. .

The dsRNA according to the present disclosure targets the locust nucleus nucleoreceptor E75 gene represented by SEQ ID NO: 1.

In another aspect, the present application provides a vector comprising DNA encoding a double stranded RNA according to the present application, or a plant transformed with the vector. The plant is not limited as long as it is a plant in which the larvae live, i.e., host, but for example, plants and plants including food crops including rice, barley, wheat, crude, corn, and sorghum, barley, dill, poa and vines Including but not limited to.

In another aspect the present disclosure also provides a composition for controlling apoptotic mediated virus comprising a double stranded RNA according to the present disclosure or a vector of the present disclosure.

In one embodiment according to the present invention, the apoptotic mediated virus includes, but is not limited to, Rice stripe virus (RSV) and Rice black-streaked dwarf virus (RBSDV).

In another aspect the present disclosure also relates to a dsRNA according to the present disclosure; A vector expressing the dsRNA; Or a composition comprising the dsRNA or the vector, larvae, larvae of larvae or eggs of larvae; All or part of the larvae host or the seed of the host; Or it provides a method for controlling the locust-mediated virus, comprising the step of contacting with the habitat of the plant or host needing control of the locust-mediated virus.

Apnea bulb mediated viruses that can be controlled by the method according to the present invention include Rice stripe virus (RSV) and Rice black-streaked dwarf virus (RBSDV), wherein the dsRNA herein By suppressing the expression of the E75 gene of the erythrocytes to the protein, the proliferation of the virus mediated by the erythrocytes can be suppressed, and finally, the plant can be rescued from the disease caused by the virus.

The present application uses a dsRNA comprising a sequence complementary to the apoptotic nucleus receptor E75 gene sequence, inhibits the expression of the nuclear receptor E75 protein in a pest body such as larvae, thereby inhibiting the proliferation of apoptotic mediated virus, thereby mediating by a pest such as There is an excellent effect of controlling the apoptotic mediated virus. In addition, the dsRNA of the present application can be treated by plants or habitats such as rice to feed the larvae through the plants to control the larvae-mediated virus, it can prevent the host disease caused by the virus.

1 is an mRNA structure of a gene encoding a nuclear receptor E75 protein expressed in apoptotic cells, according to an embodiment of the present application, the structure of the complementary DNA (cDNA) transcribed from the nuclear receptor E75 gene of the crypts The site where the dsRNA was designed is shown. The apoptotic nucleus receptor E75 gene herein is represented by SEQ ID NO: 1, wherein site 1 is 5'-TATAGTACAAACAAGCCGTGCCGTG-3 'from 2914 to 2939 of SEQ ID NO: 1, and site 2 is 2960 to 2988 of SEQ ID NO: 1 5'-AATCAAAGACTACAAATCGGACTTC-3 ', and site 3 is 5'-TGTAAATGCACTGTTATTCTCCTTC-3' from 3151 through 3176 of SEQ ID NO: 1.
Figure 2 shows the phylogenetic tree of the nuclear receptor E75 sequence. Amino acid sequences inferred from various insects were examined by the neighboring joining method. The number for each node specifies the bootstrap percentage of 1000 copies. The nucleotide sequence accession numbers used herein are as follows: Apis mellifera E75 (GeneBank Accession No. NM_001080110), Bemisia tabaci E75 (GeneBank Accession No. XM_019052056), Blattella germanica E75 (GeneBank Accession No. AM710420), Bombyx mori E75 (GeneBank Accession No. NM_001043577), Choristoneura fumiferana E75 (GeneBank Accession No. U63930), Cimex lectularius E75 (GeneBank Accession No. XM_014384895), Drosophila melanogaster E75A (GeneBank Accessano No. D515 Ega48G) No. × 51549), Galleria mellonella E75 (GeneBank Accession No.U02620), Leptinotarsa decemlineata E75 (GeneBank Accession No. KP340511), Monochamus alternatus E75 (GeneBank Accession No. KX428481), Manduca sexta E75C (GeneBank602190 No. Oncopeltus fasciatus E75A (GeneBank Accession No. EF490808), Tribolium castaneum E75 (GeneBank Accession No. AB894400), Aedes aegypti E78 (GeneBank Accession No. CAO79100), Apis mellifera G78 (GeneBank Accession No. AAA28498). These results indicated that the E75 gene sequence of the larvae derived from the dsRNA of the present application was previously unknown and was registered in GenBank (GeneBank Accession No. KY883671).
Figure 3 shows the transcriptional level of E75 of RSV- bodok (viruliferous) and poison aemyeolgu vivo (L. striatellus). Total RNA was extracted from 10 larvae larvae of RSV- and non-serial larvae and the relative transcription level of E75 was analyzed by qPCR. All analyzes were performed in 3 replicates, indicating that the different characters on the error bars (representing ± standard deviation) were significantly different in Scheffe's post hoc tests (P <0.05).
The E75 gene of apoptotic worms was found to be about 2.2 times more transcribed in RSV-aqueous larvae than non-aqueous larvae. These results suggest that reducing the expression of E75 genes in apoptotic bodies can inhibit RSV proliferation. It is.
Figure 4 is a result of synthesizing dsRNA specific to the nuclear receptor E75 protein gene of larvae and confirmed through agarose gel electrophoresis.
5 is a result confirming the concentration-dependent decrease in the expression of the E75 protein gene in the larvae fed the dsRNA specific to the nuclear receptor E75 protein gene of the larvae. These results indicate that the dsRNA specific for the E75 gene produced herein can be used to effectively reduce the transcription of the E75 gene in apoptotic bodies.
6 is a result confirming the concentration-dependent decrease in the growth of rice stripe leaf blight virus in larvae fed a dsRNA specific to the nuclear receptor E75 protein gene of larvae. These results indicate that by using dsRNA specific to the E75 gene produced herein, by reducing the transcription of E75 gene of the sperm, it can effectively inhibit the growth of rice stripe leaf virus in the constituent body.

The present application is to identify the nuclear receptor E75 protein gene sequence of the larvae, and to suppress the expression of the gene using a specific dsRNA, it is possible to inhibit the proliferation of viruses that are mediated by the larvae causing damage to plants such as rice It is based on the discovery that it can.

Thus, in one embodiment, the present invention, two strands of polyribonucleotides are complementary binding, one of the two strands is represented by SEQ ID NO: 2, the other strand has a sequence complementary to the SEQ ID NO: 2 The present invention relates to double-stranded (ds) RNA capable of inhibiting the expression of the nuclear receptor E75 gene into a protein and inhibiting the proliferation of viruses mediated by apoptotic cells.

The dsRNA according to the invention acts by RNA interference (RNAi) in the cell. RNA interference is a widespread post-transcriptional gene expression regulation mechanism in eukaryotes, including insects and plants (Merkling, SH, van Rij, RP, 2013. Beyond RNAi: antiviral defense strategies in Drosophila and mosquito. J. Insect Physiol. 59 , 159-170). The dsRNA introduced into the cell is processed by Dicer, an RNAse III enzyme, to form a ribonucleo complex called miRNP in the form of siRNA (small interference RNA) to cleave the target gene through complementary binding to the target site, or Inhibit synthesis. As a result, RNA interference induced by dsRNA introduced into cells acts in such a way as to induce the lethality of the mediator by disrupting the physiology of the mediator by inhibiting the expression of the target gene or directly reducing the proliferation of plant viruses in the mediator. Here, in particular, the direct reduction of the propagation of plant viruses in mediators.

The target gene of the dsRNA according to the present application is apoptotic nucleus receptor E75.

Insect nuclear receptor protein E75 is a transcription factor that plays a very important role in a variety of physiological processes, including escape hormone signaling (de Rosny, E., de Groot, A., Jullian-Binard, C., Gaillard, J., Borel, F., Pebay-Peyroula, E., Fontecilla-Camps, JC, Jouve, HM, 2006. Drosophila nuclear receptor E75 is a thiolate hemoprotein.Biochemistry 45, 9727-9734). Stripping hormone signaling is known to regulate ovarian development and follicle maturation processes (Gancz, D., Lengil, T., Gilboa, L., 2011. Coordinated regulation of niche and stem cell precursors by hormonal signaling. PLoS Biol. 9, e1001202). In Drosophila, E75 protein has been reported to be involved in various physiological processes, including follicle survival early in villogenesis (Morris, LX, Spradling, AC, 2012. Steroid signaling within Drosophila ovarian epithelial cells sex-specifically modulates early germ cell development and meiotic entry.PLoS One 7, e46109).

Herein, we identified nuclear receptor E75 specific to larvae, which is represented by SEQ ID NO: 1 and is a gene of unknown new sequence, which shows 66-91% homology with the previously reported homology gene. appear.

In addition, there is no known that it can inhibit the virus proliferation through the regulation of the expression of the nuclear receptor E75 gene, it was first identified here.

Therefore, in one embodiment, the present invention induces the expression of the E75 gene using RNA interference phenomenon, three siRNA binding sites were identified at the 2847 to 3296 site of SEQ ID NO: 1 of the apoptotic E75 gene.

Specifically, siRNA binding sites from which RNA interference may be induced according to the present application are 5'-TATAGTACAAACAAGCCGTGCCGTG-3 'of 2914 to 2939 of SEQ ID NO: 5'-AATCAAAGACTACAAATCGGACTTC- of SEQ ID NO: 1 to 2960 to 2988. 3 ′, 5151-TGTAAATGCACTGTTATTCTCCTTC-3 ′ from 3151 to 3176 of SEQ ID NO: 1 can be targeted.

Such siRNA binding sites can be prepared in the form of dsRNA and used to induce RNA interference.

In this regard, the dsRNA according to the present application may be a dsRNA represented by SEQ ID NO: 2 corresponding to 2847 to 3296th of SEQ ID NO: 1 including all three sites, or three identified at sites 2847 to 3296 of SEQ ID NO: 1. dsRNA of the sequence corresponding to each site of the siRNA target can be used.

Thus, the dsRNA according to the present invention is a sequence of SEQ ID NO: 2, wherein two strands are complementarily joined, or SEQ ID NO: 5'-uauaguacaa acaagccgugccgug-3 'corresponding to each of the three sites; SEQ ID NO: 5'-aaucaaagacuacaaaucggacuuc-3 '; And SEQ ID NO: 5 5'-uguaaaugcacuguuauucuccuuc-3 '.

As used herein, dsRNA or “double stranded RNA” refers to a complex of ribonucleic acid molecules having a duplex structure comprising two nucleic acid strands that are substantially complementary (as defined below) to which sense and antisense are combined. do. Such double-stranded RNA separates into single strands upon introduction into the cell, binding to the target transcriptional RNA in the form of siRNA, thereby preventing the synthesis of proteins from it. Thus, as long as the double strands have complementarity, the two strands of sense and antisense need not be completely complementary, and in this sense, the sequence of the other strand may be appropriately determined with only one strand of sequence.

As used herein, the term “complementary” refers to the ability of two strands of oligonucleotide or polynucleotide to hybridize under certain conditions to form a duplex structure. Various complementarities can be used herein as long as dsRNA is introduced into the cell to achieve the purpose according to the present application. For example, the two strands may be completely complementary or substantially complementary. In one embodiment, it can include up to four, three or two or one mismatched base pair.

The dsRNAs according to the invention can be used in various forms. For example, dsRNA can be synthesized separately to form duplexes. Alternatively, duplexes may be synthesized in one molecule to form duplexes. For example, two strands may be covalently linked, such as by a linker, between the 3'-terminus of one strand and the 5'-terminus of the other strand.

The dsRNAs according to the invention can also be synthesized with DNA encoding them, which can be cloned into appropriate expression vectors or expressed as RNA in vitro. For example, the dsRNA according to the present application can be cloned into a vector for binary transformation expressing the sense and antisense directions at the same time.

In addition, the dsRNAs herein can include substantial modifications at multiple nucleotides and all various types of chemical modifications known in the art.

DsRNA of the Invention Methods widely established in the art, for example, see "Current protocols in nucleic acid chemistry", Beaucage, S.L. et al. (Edrs.), John Wiley & Sons, Inc., New York, NY, USA, can be synthesized and / or modified. Chemical modifications include 2 'modifications, modifications of oligonucleotides at other sites of sugars or bases, introduction of non-natural bases into oligonucleotide chains, covalent attachment to ligands or chemical moieties, and replacement chains of internucleotide phosphate chains. Such as, but not limited to, thiophosphate replacement. One or more of these variations may be used.

The dsRNA herein can be synthesized by standard methods known in the art, for example using automated DNA synthesizers.

The dsRNAs according to the present application can be introduced into plants in a variety of ways, for example see: Kanakala, S., Ghanim, M., 2016. RNA interference in insect vectors for plant viruses. Viruses 8, E329.

The dsRNA according to the present application is not limited to the present theory, but when introduced into a plant cell, RNAi mechanisms present in the cell (Merkling, SH, van Rij, RP, 2013. Beyond RNAi: antiviral defense strategies in Drosophila and mosquito. J. Insect Physiol. 59, 159-170), which are produced as siRNAs (small interference RNAs) by Dicer, a nucleic acid cleavage enzyme, and the corresponding site of the nuclear receptor E75 mRNA, a transcription factor that plays a major role in various physiological functions, including escape hormone signaling. To inhibit its expression into the protein. By suppressing the expression of the larvae by disturbing the physiology of the mediator larvae can induce the death of the larvae, or inhibit the normal development of the larvae by inhibiting the normal escape of the larvae can inhibit the proliferation of the larvae mediated virus. In addition, it affects the hatching rate of larvae eggs, and when treated with eggs, it also affects the development of hatched larvae, which can be effectively used for control.

The dsRNA according to the present application can directly inhibit the growth of plant viruses in the mediator larvae. Thus, various pathogen viruses mediated by crypts can be effectively controlled by the dsRNA according to the present application.

Such viruses include Rice stripe virus (RSV) and Rice black-streaked dwarf virus (RBSDV).

In one embodiment it is used for the control of apoptotic mediated virus. A viral disease, rice streaked leaf blight, is a disease in which rice leaves do not come out at all or the leaves dry out and are called 'rice AIDS'. Mostly, larvae are caused by pathogens.

In another aspect the present application also relates to a transgenic plant or seed thereof comprising a dsRNA according to the present application. The transgenic plant expressing the dsRNA according to the present invention confers resistance to the virus by effectively blocking the infection and proliferation of anthococcus mediated virus, such as rice striation blight virus.

For the production of such transgenic plants, the dsRNA according to the present application is a binary transformation vector (Daisuke Miki and Ko Shimamoto, Plant Cell Physiol. 45 (4): 490-495, 2004), which simultaneously expresses sense and antisense directions. Can be cloned and used by the Gateway system (Invitrogen, USA) method. Such vectors include a variety of promoters, for example, promoters for overexpression, promoters for constant expression, inducible promoters that are expressed in stress conditions, promoters that allow the target genes to be expressed at certain times during plant growth, and certain parts of plant tissues. It may include a hybrid promoter coupled with an enhancer to increase the expression of the promoter or gene of interest only.

Agrobacterium tumefaciens can be transformed using a vector comprising a dsRNA according to the present application, and used for the preparation of a transgenic plant. Such agrobacterium includes a pathogen-removed Ti or Ri plasmid, and an octopin-type strain or a succinamopine-type strain may be used. Subsequently, agrobacterium into which the dsRNA was introduced was co-cultured with rice somatic callus to introduce a target gene into rice somatic cells, and then the target gene was simultaneously expressed in the sense and antisense directions from the rice somatic callus to form a dsRNA. A transgenic rice can be prepared. As marker genes for effectively screening plants into which the target genes have been introduced, antibiotic resistance genes such as NPTII (neomycin phosphotransferase), HPT (hygromycin phosphotransferase), CAT (chloramphenicol acetyltransferase) and gentamicin (gentamicin); herbicide resistance genes such as bar gene; And GUS, GFP (green fluorescent protein), LUX (luciferase) and the like can be used.

Transgenic plants usable by introducing the dsRNA according to the present invention are not particularly limited, as long as they are host plants of the larvae, and include, for example, food crops such as rice, barley, wheat, crude, corn and sorghum, bark, nectar, poa grass, It may include, but is not limited to, plants and plants such as hulling.

In another aspect the present invention also relates to a composition for controlling apoptotic media virus, comprising a vector comprising a dsRNA according to the invention or a DNA encoding the same.

The composition of the present application includes the dsRNA as an active ingredient, but may further include excipients and additives included in other conventional pesticide compositions. For example, it may include, but is not limited to, plasticizers, colorants, brighteners, surfactants, dispersants, emulsifiers, glidants, and the like.

The compositions herein can be formulated in conventional pesticide, or pesticide formulations. For example, the compositions herein can be formulated as emulsions, solutions, hydrating agents, water solubles, powders, granules, fumigations, coatings and the like. Specifically, the dsRNA is included in a titration method to formulate a pesticide preparation. These composition formulations are described in "Catalogue of pesticide formulation types and international coding system", Technical Monograph No. 2, 6th Ed. May 2008, CropLife International. The compositions are described in detail in Mollet and Grubemann, Formulation technology, Wiley VCH, Weinheim, 2001; Or Knowles, New developments in crop protection product formulation, Agrow Reports DS243, T & F Informa, London, 2005.

Specifically, a surfactant, an extender, a disintegrant, a nutrient, and the like may be mixed and pulverized in the composition of the present application to be used as a hydrating agent or dissolved in a liquid formulation. Surfactants include polycarboxylate, sodium lignosulfonate, calcium lignosulfonate sodium dialkyl sulfosuccinate, sodium alkyl sulfonate, polyoxyethylene alkyl phenyl Ethers, sodium tripolyphosphate, sodium alkyl aryl sulfonate, polyoxyethylene alkyl phenyl ether, polyoxyethylene alkyl aryl phosphoric esters, Polyoxyethylene alkyl aryl ether, polyoxyethylene alkyl aryl polymer, polyoxyethylene alkyl aryl polymer special, polyoxyalkylone alkyl phenyl ether, polyoxy Ethylene nonyl phenyl ether ether), sodium sulfonate naphthalene formaldehyde, one or two or more selected from the group consisting of Triton 100 and Tween 80 can be used. Examples of surfactants are listed in McCutcheon's, Vol. 1: Emulsifiers & Detergents, McCutcheon's Directories, Glen Rock, USA, 2008 (International Ed. Or North American Ed.). Bentonite, talc, kaolin, calcium carbonate, and the like may be used as the disintegrant, and one or more of ocher, diatomaceous earth, dextrin, glucose, and starch may be used as the extender.

The compositions herein may comprise an effective amount of dsRNA. The term "effective amount" means an amount of the composition or compound that is sufficient to control the virus on the cultivated plant or does not cause substantial damage to the treated plant. These amounts can vary within wide limits and depend on various factors, the specific species and condition of the cultivated plant treated, the location of habitat, or climatic conditions. When formulated, the timing of use can be used at all times when pests occur.

In another aspect to this, the present application is directed to a dsRNA according to the present application; A vector expressing the dsRNA; Or a composition comprising the dsRNA or the vector, larvae, larvae of larvae or eggs of larvae; All or part of the host or seeds of the host in need of control of the apoptotic mediated virus; Or a method for controlling the locust-mediated virus, comprising contacting the locust or the habitat of the host in need of the locust control.

Anthracnose using the dsRNA or the composition according to the present application includes, but is not limited to, anthracnose or ablative larvae, or eggs or larvae of the pests, which supplement the virus causing the disease, such as rice stripe blight virus, etc. The plants to which the dsRNAs or compositions according to this invention can be applied can be applied to all or part of the plant, for example roots, stems, leaves, and seeds, as well as to cells or tissues of the plant.

Plants to which the dsRNAs or compositions according to the invention can also be applied are those comprising both growing crops and harvested crops.

In addition, the kinds of plants to which the dsRNA or the composition according to the present invention can be applied are the larvae host plants, for example, rice, barley, wheat, rye, millet, crude, corn, oats, and sorghum, as well as blood crops, barley, and ragweed. It may include plants and plants, such as grass, Italian lygras, mustard, sessile, gingko, clam, broadleaf beetle, eggshells, fangs headed, bark and squid.

In addition, the dsRNA or the composition according to the present application can be applied to the habitat where the plant and / or larvae live.

The method herein includes a pest or larvae or eggs thereof, or the habitat of the pest; Or all or part of the plant, or the habitat in which the seed, plant or pest or larvae live, such as soil or water, may be contacted with the composition herein by any method of application known in the art. In this case, "contact" refers to direct contact (dsRNA / composition is applied directly to the pest or plant-typically the leaves, stems or roots of the plant) and indirect contact (dsRNA / composition to the pest or plant growth site). ) Include both.

The methods herein also are utilized by treating plants, plant propagation materials, such as seeds, soil, surfaces, materials or spaces, to be protected with an effective amount of the compositions of the present disclosure. The method may also be carried out both before and after infection of plants, plant propagation materials such as seeds, soil, surfaces, substances or spaces by pests.

The present method can be applied prophylactically to the place where the occurrence of pests is expected.

In another aspect the present invention also relates to a method for inhibiting nucleoreceptor E75 protein expression in a pest comprising treating a dsRNA according to the present application or a composition comprising the same to larvae or larvae or eggs thereof. The dsRNA used in the method, the treatment method may refer to the above-mentioned.

In another aspect the invention also relates to a method of inhibiting the expression of E75 protein in apoptotic cells, thereby inhibiting the proliferation of the mediated virus. The dsRNA used in the method, the treatment method may refer to the above-mentioned.

Hereinafter, examples are provided to help understand the present invention. However, the following examples are provided only to more easily understand the present invention, and the present invention is not limited to the following examples.

Example

Experimental Materials and Methods

Insect Culture Non-viruliferous L. striatellus was collected from pest-free rice paddies (Oriza sativa) at 28 ° C., 80% relative humidity, and 16: 8 (light: dark). It was grown in unvowed rice at the insect breeding in the photoperiod. To obtain RSV-carrier L. striatellus, 80% relative humidity for 5 days in RSV-infected rice of size 5-6 cm in the growth chamber, and unbovine L. under 16: 8 (light: dark) photoperiod conditions. The second instar nymph of the striatellus was raised.

Multiple Sequence Arrangement and Phylogenetic Analysis The CLUSTAL W program (Kumar) was searched for different nuclear receptor E75 sequences by searching the National Center for Biotechnology Information (http: // www.ncbi.nlm.nih.gov/Genbank/index.html). , S., Stecher, G., Tamura, K., 2016. MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets.Mol. Biol. Evol. 33, 18701874.). The phylogeny of LsE75 is inserted into MEGA7 software (Jones, DT, Taylor, WR, Thornton, JM, 1992. The rapid generation of mutation data matrices from protein sequences.Comput. Appl. Biosci. 8, 275-282). Inference was made using the neighborjoining method under the Jones-Taylor-Thornton (JTT) substrate-based model.

To predict dsRNA synthesis candidate siRNA sites, the nucleotide sequence of LsE78 was applied to the BLOCK-iT ™ RNAi Designer (https://rnaidesigner.thermofisher.com/rnaiexpress), and a target gene was predicted to predict at least three putative siRNA sites. DsRNA was designed. A single stranded cDNA of the target gene was synthesized from the total RNA of L. striatellus using the QuantiTect Reverse Transcription Kit (QIAGEN, Germany) according to the manufacturer's manual, and the T7 promoter sequence (5′-TAATACGACTCACTATAG-3) at the 5-end. A target gene comprising a primer set comprising ′) was amplified. The amplified product was used as a template to produce dsRNA for the target gene in Genolution Phamaceuticals (Korea).

To L. striatellus Delivery of dsRNAs Using an assembly feeding chamber according to conventional literature (An et al, 2017. Silencing of rice stripe virus in Laodelphax striatellus using virusderived double-stranded RNAs. J. Asia Pac. Entomol. 20, 695698) DsRNA was ingested into L. striatellus by the mediated feeding RNAi method. A 15 ml conical tube (Fisher Scientific, USA) with a hole for air supply was collected to create a feeding chamber, and a 10 μl tip was placed in the hole to prevent L. striatellus from leaking out of the tube. A Parafilm M film (Bemis, USA) was placed to seal the gap between the feeding chamber component tube cap and the 15 ml tube to make a reservoir for holding the dsRNA solution. 300 microliters of each dsRNA solution (50, 250, and 500 ng / μl in 10% sucrose solution) were dispensed over the reservoir and 3 rice leaves were placed on the dsRNA solution. 15 4 ^th Instar larvae of L. striatellus were infected on 3 dsRNA-transmitted rice leaves. In the feeding chamber, the dsRNA-treated leaves infected with L. striatellus were placed in the growth chamber under 28 ° C., 80% relative humidity, and 16: 8 (light: dark) photoperiod conditions.

Total RNA Extraction and Quantitative PCR ( qPCR ) The fourth instar larvae of RSV-infected L. striatelus grown in dsRNA-treated rice leaves were collected 48 hours after treatment, and their total RNA was collected by QIAzol Lysis Reagent (QIAGEN, Germany). Extracted according to the manufacturer's manual. Complementary DNA was synthesized with oligo-d (T) primers using QuantiTect Reverse Transcription Kit (QIAGEN, Germany). Quantitative CPR was performed using EvaGreen qPCR Master Mix (Applied Biological Materials, Canada) and CFX96 ™ Real-Time System (BIO-RAD, USA) according to the manufacturer's manual. The cycle profile used for qPCR is as follows: preheating step for enzyme activation at 95 ° C. for 10 minutes, followed by 40 cycles of 15 s at 95 ° C., 15 s at 55 ° C. and 15 s at 72 ° C .. ARF ribosylation factor (ARF) was used as a reference gene. Relative transcription levels were calculated using the 2ΔCt method (Pfaffl, MW, 2001. A new mathematical model for relative quantification in real-time RTPCR. Nucleic Acids Res. 29, e45). Primers used for qPCR are listed in Table 1 below.

TABLE 1

Example  1: anguish Nuclear receptors  Sequences of the E75 Protein Gene

The messenger RNA structure of the gene encoding the nucleus receptor E75 protein expressed by the crypts is summarized in FIG. 1. The mRNA was purified from poly-T oligo-attached magnetic beads from total RNA extracted from rice stripe leaf blight virus and non-bovine blight that were distributed from the Southern Crop Division of the National Academy of Crop Science, Rural Development Administration. Cut into smaller pieces. The first strand cDNA was synthesized using reverse transcriptase and random primers as a template, and then second strand cDNA was synthesized. The cDNA fragments thus prepared were amplified by PCR to prepare cDNA library, clusters, and paired-end sequencing using the Illumina system. Raw data obtained through Illumina RNA-sequencing is removed using the NGS QC toolkit v2.3 (National Institute of Plant Genome Research, India) program to remove low quality sequences and adopter sequences, followed by Trinity software (Broad Institute and the Hebrew University of Jerusalem) was used to prepare the EST library through de novo assembly. Each isotig assembled by integrating the 454-pyrosequencing results for the RNAs of the rice stripe blight virus and the non-toxic worms was compared to the protein of Drosophila using BLASTX to complement the DNA base of the neutrophil E75 protein gene of the worms. The sequence was confirmed.

The nucleotide sequence of the gene encoding the nuclear receptor E75 protein expressed by the mutant sphere is shown in SEQ ID NO: 1. Nuclear Receptor E75 protein gene consists of 5,296 nucleotide sequences, including the 5'- and 3'-terminal untranslated regions, of which the open reading frame encodes the Nuclear Receptor E75 protein. frame) consists of 2,454 nucleotide sequences (FIG. 1). The solubilized nuclear receptor E75 protein gene showed 66-91% homology with the previously reported homologous genes (X51548.1, XM022342225.1), but is a novel gene with a different sequence.

Example  2. Annihilator Nuclear receptors  Specific to the E75 Protein Gene dsRNA  making

Search for candidate siRNA sites for the target gene using the BLOCK-It RNAi Designer software (https://rnaidesigner.thermofisher.com/rnaiexpress) provided by Thermofisher, and search for segments containing three or more candidate siRNA sites. It was selected as a target site for the fabrication. The target site of the selected dsRNA is the site corresponding to the 2,847th to 3,296th sequences (SEQ ID NO: 2) in the cDNA of the nuclear receptor E75 protein gene of the larvae (FIG. 1).

Using the QuantiTect Reverse Transcription Kit (QIAGEN, Germany) as a template for the total RNA of the erythrocytes, single-strand cDNA of the nuclear receptor E75 protein gene was synthesized according to the manufacturer's method. The forward primer (T7-LsE75-F, 5'-TAATACGACTCACTATAGTTGCACTCGCTCAAAACTA-3 ') having a single-stranded cDNA of the nuclear receptor E75 protein gene of the annealed chromophore as a template, containing the T7 promoter sequence at the 5'-end. Using a reverse primer (T7-LsE75-R, 5'-TAATACGACTCACTATAGCAAGGTGGTGAAGCTGGAG-3 '), a large amount of the dsRNA target site was synthesized by a gene amplification reaction (Polymerase Chain Reaction, PCR), and commissioned by Genolution Co., Ltd. DsRNA specific for the gene of interest was constructed (FIG. 4).

Example  3. Anchovy Nuclear receptors  Specific to the E75 Protein Gene dsRNA  Regulation of E75 Protein Gene by Treatment

For the successful delivery of dsRNA into the body, the indirect feeding method was performed by absorbing the dsRNA solution through the root of rice and then killing the leaves of rice. 300 ul of dsRNA suspended in 10% sucrose solution was treated with rice at concentrations of 50, 250 and 500 ng / ul, respectively, and 15 larvae IV nymphs were added to each rice treated with dsRNA. Inoculation. As a control, rice treated with dsRNA specific to the same concentration of green fluorescent protein (GFP) gene was used. The rice thus treated was placed in a growth chamber set at a photoperiod of 28 ° C., 80% relative humidity, and 16: 8 (light: dark). After 48 hours of dsRNA treatment, erythrocytes were collected from each rice, and total RNA was extracted using Trizol reagent (Invitrogen, USA) according to the manufacturer's method, and QuantiTect Reverse Transcription Kit (QIAGEN, Germany) was extracted from the total RNA of the extracted larvae. Single stranded complementary DNA was synthesized. Using the single stranded complementary DNA as a template, quantitative Polymerase Chain Reaction (qPCR) was performed using EvaGreen qPCR Mastermix (Applied Biological Materials Inc, Canada) and CFX96TM Real-Time System (BIO-RAD, USA). Was performed. ADP ribosylation factor gene was used as a reference gene to confirm the relative expression level of each gene.For qPCR of the nuclear receptor E75 protein gene, a forward primer (qLsE75-Fw, 5'-CACTCAACGCTGGCCAAGTC-3 ') was used. ) And reverse primer (qLsE75-Re, 5'-CAGCCGGGTACGACTGGTAG-3 '), and forward primer (qARF-Fw, 5'-TTGGACAGTATCAAGACCCATC-3') and reverse for qPCR of the ADP ribosylation factor gene. A reverse primer (qARF-Re, 5'-GCAGCAATGTCATCAATAAGC-3 ') was used.

As shown in the left panel of FIG. 5 (dsGFP), the arkocytes treated with rice treated with dsRNA specific to the green fluorescent protein gene did not change the expression of the nuclear receptor E75 protein gene, whereas the specific expression of the nuclear receptor E75 protein gene. In the larvae treated with rice treated with dsRNA, the expression of the E75 protein gene decreased as the concentration of the treated dsRNA increased (FIG. 5, dsLsE75).

Example  4. Annihilator Nuclear receptors  Specific to the E75 Protein Gene dsRNA  By treatment Rice stripe  Inhibit the growth of the virus

In order to investigate the effect of dsRNA specific to the apoptotic nucleus receptor E75 protein gene on the proliferation of rice stripe leaf blight virus in the apoptotic body, the dsRNA specific to the green fluorescent protein gene or the apoptotic nucleus receptor E75 protein gene was fed in Example 3. Quantitative gene amplification reactions were carried out using EvaGreen qPCR Mastermix (Applied Biological Materials Inc, Canada) and CFX96TM Real-Time System (BIO-RAD, USA), using single-strand complementary DNA synthesized using total RNA extracted from one larvae. Was performed. In order to confirm the relative proliferation rate of the rice stripe leaf blight virus, NS3 gene was used among the genes of the rice stripe leaf blight virus, and the ADP ribosylation factor gene of larvae was used as a reference gene. The NS3 gene of the rice stripe leaf blight virus codes for a protein known as the RNA suppressor of RNAi, which acts against the antiviral defense mechanisms of rice and larvae in the host's body. Is known to play an important role in the proliferation of rice stripe leaf blight virus (Hemmes, H., Kaaij, L., Lohuis, D., Prins, M., Goldbach, R., Schnettler, E., 2009. Binding of small interfering RNA molecules is crucial for RNA interference suppressor activity of rice hoja blanca virus NS3 in plants.Journal of General Virology 90, 1762-1766). For the qPCR of NS3 gene, a forward primer (qNS3-Fw, 5'-GCAGAGCTCTACATTGTGCCA-3 ') and a reverse primer (qNS3-Re, 5'-AACGTGCACTAAGAGGTGGTTT-3') were used.

As shown in FIG. 6, in the larvae fed with dsRNA specific to the green fluorescent protein gene, there was no change in NS3 gene expression of the rice leaf blight virus (FIG. 6, dsGFP), whereas the dsRNA specific to the nuclear receptor E75 protein gene was shown. In feeding larvae, the expression of NS3 gene was significantly decreased as the concentration of the treated dsRNA increased (FIG. 6, dsLsE75). NS3 gene expression of rice stripe leaf virus was reduced by 38.9 ~ 55.5 times in the larvae fed dsRNA specific to the nuclear receptor E75 protein gene.

Although the exemplary embodiments of the present application have been described in detail above, the scope of the present application is not limited thereto, and various modifications and improvements of those skilled in the art using the basic concepts of the present invention defined in the following claims are also provided. It belongs to.

All technical terms used in the present invention, unless defined otherwise, are used in the meaning as commonly understood by those skilled in the art in the related field of the present invention. The contents of all publications described herein by reference are incorporated into the present invention.

<110> Seoul National University R & DB Foundation <120> Composition and method of controlling virus mediated by small          brown planthopper using dsRNA targeting nuclear receptor E75 gene          of small brown planthopper <130> DP201709014P <160> 5 <170> KoPatentIn 3.0 <210> 1 <211> 5296 <212> DNA <213> Laodelphax striatellus <400> 1 ccgcctcaag ctctgactat ggccaggcaa cagcgttttg ttggcttggc ttcacacttc 60 tcgcttcagt cgactcttga acacggtccc agatagttct atgcgtctcc gtgcgttgtt 120 tccattcaaa aattacctag gcgtgaaaat ctactagggt agtgagtgat tgatttttat 180 ctgctccaaa gagaacactt ggatattcac tggtgacaac atttaattcc tagaggggaa 240 atcttctagt gtataggttt actccaccgt tgatatcaac actttgtgtg aaaaactgtg 300 ctcattcaaa ttttgtttgt aaaaactgta ggaacttgtg caattgcaac actgttttgg 360 ggaagttgtt ctcccagtgc tgttacgccc ctcaacccag ttgtagaatt gtgggctatg 420 ggatgtgttt cgacgccgaa taacaccgat aacgatacaa gtatggctcc cacttgtctt 480 accaactcgg cctccgccaa tgttgttatc agcgaggccc tcctaaaagt acaaaacgaa 540 aaggactcta tcatcgaatc ctgctattcg gacatagatg atgattatgt tagtgatgaa 600 aactctgaaa atgttgacgt tgacgactcg tgcaccaatc gtgtttcgtc ttgttgttgc 660 cagggcttct tcagacgcag tatccagcag aagatccagt acaggccatg caccaagaac 720 cagcagtgct ccatactgag gatcaacagg aataggtgcc agtactgtcg gctcaagaag 780 tgcattgctg tcggcatgag tcgagatgct gtgcgattcg gtcgcgtgcc caagcgagaa 840 aaagcgcgca tactggccgc catgcagcag agcaccaact cgcgatctca ggagaaggcg 900 ctcgccgctg agttggaaga cgagcagcga ctgctggcga ctgtcgtcag ggcgcacatt 960 gacacctgcg acttcacgcg cgacaaaatc gaaccgatga tactcagggc gcgacagcag 1020 ccatcctaca ccgcctgtcc acctactctg gcttgcccac tcaaccccaa ccctcagcca 1080 ctgactggtc aacaagaact cctccaggac ttctccaaga ggttttctcc ggccattcgt 1140 ggcgtagtgg agtttgccaa acgcatcccc ggattttctt tcctcgcaca agacgaccag 1200 gtcacattac tcaaggccgg cgtcttcgag gtccttctcg tcagattggc ctgcatgttt 1260 gattctcaga acaacagcat gatttgcctg aacggccaga tcctgaaaag ggagtccatc 1320 cacaacagtt ccaatgccag gttcctgatg gattccatgt ttgactttgc cgaaagactg 1380 aatgccttga gactgtccga tgctgagatt ggtctcttct gctccattgt cgttattgct 1440 gccgatcggc caggcctcag aaataccgac ctcatagaga gaatgcacaa caaactgaaa 1500 acagtgctac aattagtcct caaccagaac catcccgggc agccaaatct ctgccacgaa 1560 ctgatgaaga agattcccga tctgcgcaca ctgaacacgc tgcactcgga aaagctgctc 1620 gcattcaaga tgaccgaaca acagcaactg gcgcagcagc aacagcagat gtggggatcg 1680 atgatggtcg aggacgagtg caacagcaag agccccggcg gcgcctcgtc ctggtcctcc 1740 tcgtccgatg tcaccatgga ggaagtcaag agtccgctcg gctcagtctc cagcaccgag 1800 tccatgtgct cgggcgaagt ggcctcgtta aacgactacc accaaagcca ccatgccagc 1860 agcacgccgc tgctcgctgc caccctcgcc ggcggcgtct gtccgcaccg gcaccgtgcc 1920 aactccggct ctgcctctgg tgatgacatc aagtcacacc acagatcctt cagaaagctc 1980 gactcaccaa gtgactccgg catcgaatca ggcactgaga aactggacaa accaattgga 2040 tcagcaccaa cctcagtctg ttccagtcca cgttcttccc tggaagacaa agatgaccat 2100 catcatcacc cgcaccaccc acccaccaac caccatcatg cccagaatgg ccattcaatg 2160 ccgttccaga gcacacagca ccaaccagct cagcagaatg gcacaacgca ctcagtcgag 2220 gacatgccag ttctgaagcg tgtgctgcag gcgccgcctc tctacgacac aaactctctc 2280 atggatgaag cctacaagcc gcacaaaaag ttcagggcac tgagaaacaa ggactcggcc 2340 gaagccgagc cgatggtgca caactctccg cagctgcacc atcacctcac ctcatcgctg 2400 tccagcactc actcaacgct ggccaagtcg ctgatggaga gtccacgcat gacccccgaa 2460 cagctcaaga gaaccgaaat gatcaacgac ttcatcatgc gtgcaggcaa cgacagtgca 2520 gcatcgtcct accagtcgta cccggctgca cagaggtggc aggccggcac cagtgtcatc 2580 accaccacca gtgcagccaa tgccgccgcc acctctccct ccgcctacat actagtgtcc 2640 accccacccc ccgccgccgc cgcctcgtcg aggatgtacc tctcgccgtc gccctcctca 2700 tccacctctc cccactcgcc ctccgtcatg gagctgcagg tcggcatcgc caactcctcc 2760 tccacatcct cctcacagca gccactcaac ctgtccaaga agacgccgcc tccctcaccc 2820 acccccctct cgcacccgcc tcccaccaag gtggtgaagc tggaggcctg aggtgccgcc 2880 cccacctacc atcatcgccc ccgtgtcatt gtctatagta caaacaagcc gtgccgtgat 2940 gaaccaacca actggtacaa atcaaagact acaaatcgga cttctactga tctctctgag 3000 ttgttgttcg gcgcagcgcg gtacggtgcg cactctatta aatagtcttc attatcaaca 3060 cacaccctgt gctagcaaag agggagagat gaaattctga aggtgccacc ttatgagccc 3120 cccgccgggg acggccaacg tttggtctgt tgtaaatgca ctgttattct ccttcttgat 3180 tttaatgata atctatttgt atgtgtataa tgtatgtatt cgtttagatg tgtgattcga 3240 tatttttaaa aaacaattaa acgtgttgta gttgaaatag ttttgagcga gtgcaaatgt 3300 tgtattttca atgtgaaatt gaagtcatta ggaatattat cattcattca cgttcattta 3360 ttccaaatta ttgattccat tcgttagaaa tcattatctc tttctctttg gaccaaaata 3420 tctgtgtctg tatttgttta taatttacac ttgttaaatt atttttcatc actcacatca 3480 tgtgtaaatt atcacctcat ttgtagaata tctcgttact catttgtata tcttgaagca 3540 tatctcatca tcaatttcac ccgtaataaa ttagagtagg atcatcagtc gtgtttatca 3600 ttaaatgaat caggctgggg aatgacaaat tgtgaatttt aggctcaaag gttaaagttt 3660 gaaagcacca gtcctaatat aaataaatac atgttattat taaaatatat atattattac 3720 gaaaagtgaa gaaatatata ttattcttta attaaataaa tgtgatataa ttgtgtatgt 3780 agggcttgca tttcacatct atgtgtataa atattaagaa aatcagaaac gattacgttt 3840 tagtcttatc ttgtgtaaca taaataatat ttgaaagaat aattaatttg ttaagatgtt 3900 tacttacaaa aaattaaaaa aattgatgga aatgataggt ttaaatgttt gcattctcaa 3960 ctagggctca gaaacgccct tctgtaaata ttatatataa cgactattac tgtcttaaat 4020 aataataatt taagcaaaaa tatgagactg aagggaaggt ctgccatgcc gcctttgccc 4080 ctaaaatatt ccgttgttta tttttcttgt tgtttcttta agttatatta ctaagttgat 4140 ggaggtaggc cagtacctta ttgcgtttta ttattctaat ttaattgaac gaatcaagcc 4200 cacccttttt attattattg taattaatat tgatgataat cgtcctgttg atcgatagta 4260 cataggttgt gttgtgcgtt ttctcgtgtt gattatggta ttggtagatt ctcatgcttg 4320 gtgatttttt tatcagtcca aagccatgtt atttgtatga acagtgtttg tagacaacac 4380 attgcaattt tctggattaa gactgtaaaa ccgatattaa ttcatctcga tcgttttgat 4440 attataatat catatttcgt tgtaattata taatatttaa ttgctgtaga taaatgtgga 4500 atagaacaga aaggcgttag gaatggcaga ggcttcctac aaataattga ataatatata 4560 tattgtcata tatattatat aatcttctat tgtatttgtc tttgtttttt atatatctag 4620 actttttgat ctttagacat cctcaaaatt tttttctgaa gctactctgt tcaattgtaa 4680 tccgtttgaa gttatttacc acttatgcac tatctgaaac agttttcttt gtattgactc 4740 gcaactatta aaagtgatct ttttttaaaa atgaacgaat tgtcaaaaga ttgattaggc 4800 tcaaatgatt tctgtggtgt agttgataat tcatttggca aaggatacaa tacttttgaa 4860 tgtataaatg agcctaagta tgttgtgaat tgtaatctgg gcaatgtttt taatagttca 4920 tttgtatgaa atataatata gatcgatgtt ctctatatac atatttttat gatactttta 4980 aaaacaaaga atatagattt gtataaatca aagaacctac aatatatttt tatttcccta 5040 aaatgaatgg aatcagatta acagtaaaat atcacatatg tttctactac attttagcta 5100 ttgttactta aaacaaacag ttcatcaaat atgtttgtca gcccagtgaa gcagtgatgt 5160 ttagaagtgt atgtttccct ttcaaaaatg gtgtgtgctt gtattgtatg tatcattttt 5220 ttgtaaagct tgggactttt ttgaggtcgt cagattaaac cattgtctgg tgaaaattaa 5280 aaaaaaaaga tcggaa 5296 <210> 2 <211> 450 <212> RNA <213> Artificial Sequence <220> <223> RNA corresponding to 2847 to 3296 <400> 2 caagguggug aagcuggagg ccugaggugc cgcccccacc uaccaucauc gcccccgugu 60 cauugucuau aguacaaaca agccgugccg ugaugaacca accaacuggu acaaaucaaa 120 gacuacaaau cggacuucua cugaucucuc ugaguuguug uucggcgcag cgcgguacgg 180 ugcgcacucu auuaaauagu cuucauuauc aacacacacc cugugcuagc aaagagggag 240 agaugaaauu cugaaggugc caccuuauga gccccccgcc ggggacggcc aacguuuggu 300 cuguuguaaa ugcacuguua uucuccuucu ugauuuuaau gauaaucuau uuguaugugu 360 auaauguaug uauucguuua gaugugugau ucgauauuuu uaaaaaacaa uuaaacgugu 420 uguaguugaa auaguuuuga gcgagugcaa 450 <210> 3 <211> 25 <212> RNA <213> Artificial Sequence <220> <223> RNA corresponding to 2914 to 2939 <400> 3 uauaguacaa acaagccgug ccgug 25 <210> 4 <211> 25 <212> RNA <213> Artificial Sequence <220> <223> RNA corresponding to 2960 to 2988 <400> 4 aaucaaagac uacaaaucgg acuuc 25 <210> 5 <211> 25 <212> RNA <213> Artificial Sequence <220> <223> RNA corresponding to 3151 to 3176 <400> 5 uguaaaugca cuguuauucu ccuuc 25

Claims (13)

A double-stranded RNA (dsRNA) having a sequence represented by SEQ ID NO: 2, wherein the double-stranded RNA inhibits the expression of apoptotic nucleus receptor E75 protein, and has a virus control ability mediated by the apoptotic sphere.
The method of claim 1,
DNA encoding the apoptotic nucleus receptor E75 protein is represented by SEQ ID NO: 1, double stranded RNA.
A vector comprising a DNA encoding a double stranded RNA according to claim 2.
Plant transformed with the vector according to claim 3.
A composition for controlling apoptotic media virus comprising a double stranded RNA according to claim 1 or a vector according to claim 3.
The method of claim 5,
Wherein the virus comprises a rice stripe leaf blight virus or a rice black streaky virus, composition for controlling locust-mediated virus.
A dsRNA having the sequence of SEQ ID NO: 2, a vector expressing the dsRNA, or a composition comprising the dsRNA or the vector comprises: larvae, eggs of larvae or larvae; All or part of the larvae host or the seed of the host; Or contacting with a host's habitat in need of control of said apoptotic mediated virus.
The method of claim 7, wherein
The larvae-mediated virus includes rice streaked blight virus or rice streaked agal virus,
The dsRNA inhibits the expression of the erythrocyte into the protein of the E75 gene, thereby inhibiting the proliferation of the virus mediated by the erythrocyte, a killer mediated virus control method.
The method according to claim 7 or 8,
The apoptotic E75 gene is represented by the sequence of SEQ ID NO: 1, wherein the dsRNA is to bind to the 2847 to 3296 sequence of SEQ ID NO: 1, a method for killing apoptotic virus.
The method according to claim 7 or 8,
Wherein the host is a food crop comprising rice, barley, wheat, crude, corn and sorghum, or a plant and plant including bark, nectar, poa grass, and hulling.
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