JP2014505480A - Virus-resistant transgenic silkworm - Google Patents

Abstract

  Viral resistant transgenic silkworms and methods for their production are provided herein. The transgenic silkworms disclosed herein exhibit simultaneous and constitutive expression of double stranded RNA of at least two viral genes. The transgenic silkworm of the present invention is produced by RNA interference (RNAi) mediated suppression of baculovirus viral genes.

Description

  The present invention relates to the use of RNA interference (RNAi) for the suppression of baculovirus infection in transgenic insects, domesticated silkworms, Bombyx mori L .. With the PiggyBac transposon-based vector, we have two essential viral genes: two for early early-1 (ie-1), late effector factors (lef1 and 3) and p74. Eighteen homozygous lines of transgenic silkworms that constitutively produce strand RNA were obtained.

The domesticated silkworm, Bombix mori, is a major lepidopteran insect of significant economic importance and is also regarded as an ideal lepidopteran model system for genome analysis (Nagaraju J and Goldsmith MR (2002) Silkworm genomics-progress and prospects.Current Science 83: 415-425; Nagaraju J, Klimenko V and Couble P (2000) The silkworm Bombyx mori, a model genetic system.In Encyclopedia of Genetics, ed.Reeves E. (Fitzroy Dearborn, London), pp. 219-239). In addition to its usefulness as an economic insect and genetic tool, the industrial application of silkworms has been augmented by successful germline gene transfer using PiggyBac transposon-based vectors (Tamura T, Thibert C, Royer C, Kanda T, Abraham E, Kamba M, Komoto N, Thomas JL, Mauchamp B, Chavancy G, Shirk P, Fraser M, Prudhomme JC, Couble P, Toshiki T, Chantal T, Corinne R, Toshio K, Eappen A, Mari K, Natuo K, Jean-Luc T, Bernard M, Gerard C, Paul S, Malcolm F, Jean-Claude P and Pierre C (2000) Germline transformation of the silkworm Bombyx mori L. using a piggyBac transposon- derived vector. Nature Biotechnology 18: 81-84). The Lepidoptera provide livelihoods for millions of farmers in India, but farmers' incomes are impacted by severe baculovirus disease caused by the virus Bombyx Mori nuclear polyhedrosis virus (BmNPV) Is done. Almost 50% of crop losses are accounted for by baculovirus. The robust nature of the polyhedral viral body produced at the end of the baculovirus infection cycle facilitates widespread viral infections, especially in the summer and hot and humid seasons. Therefore, the development of BmNPV resistant silkworm strains is the primary priority for stabilizing silk cocoon yields and improving silk production and consequently improving the sericulture economy.

  BmNPV is a member of the Baculoviridae family and is one of 14 major families of invertebrate viruses that infect arthropods, primarily insects (van Regenmortel MH, Fauquet CM, Bishop DHL, Carstens EB, Estes MK, Lemon SM, Maniloff J, Mayo MA, McGeoch DJ, Pringle CR and Wickner RB (2000) Seventh Report of the International Committee on Taxonomy of Viruses (Academic Press, San Diego, Wien New York .; Murphy FA, Fauquet CM Bishop DHL, Ghabrial SA, Jarvis AW, Martelli GP, Mayo MA and Summers MD (1995) Sixth Report of the International Committee on Taxonomy of Virus. (Springer Verlag, Wien, New York). BmNPV is host-specific and highly infectious to Bombix Mori N (BmN) cells, but does not replicate in Spodoptera frugiperda 21 (Sf21) cells (Kondo A and Maeda S (1991) Host range expansion by recombination of the baculovir Journal of Virology 65: 3625-3632) Bombyx mori nuclear polyhedrosis virus and Autographa californica nuclear polyhedrosis virus.Journal of Virology 65: 3625-3632) Life cycle of baculovirus is polyhedral inclusion body (PIB, inclusion body-derived virus (occlusion derived virus). (also known as `` virus '') occurs when you eat virus particles that are resistant to the environment left on the leaves, and when you enter the alkaline environment of the midgut, through the receptor into the midgut cells Release free virions that gain invasion. In the cell, the viral gene is transcribed from the early promoter by host RNA polymerase II from the early promoter-1 (ie-1), ie-2, and begins transcription in a transient manner (Huh). NE and Weaver RF (1990a) Categorizing some early and late transcripts directed by the Autographa californica nuclear polyhedrosis virus. Journal of General Virology 71: 2195-2200). This is followed by the expression of other late effector factors (lef) while Amanitin-resistant viral RNA polymerase initiates transcription of the very late gene (Huh NE and Weaver RF (1990b) Identifying the RNA polymerases that synthesize specific transcripts of the Autographa californica nuclear polyhedrosis virus. Journal of General Virology 71: 195-201). Finally, the viral particles encapsulated in the polyhedron are released into the environment by cell lysis and complete degradation of the infected larvae. In contrast to PIB, free virus (known as Budded Virus-BV), which forms in cells without a multifaceted cover, causes intra-individual infection. Start. The larval tracheal system provides the main channel for baculoviruses to pass through the basal layer and spread the infection (Engelhard E, Kam-Morgan L, Washburn J and Volkman L (1994) The insect tracheal system: A conduit for the systemic spread of Autographa californica multiple nuclear polyhedrosis virus. Proceedings National Academy of Sciences USA 91: 3224-3227).

  By using plasmid DNA replication, lef genes such as ie-1, lef-1, lef-2 and lef-3 have been shown to be essential for replication (Lu A and Miller L (1995) The roles of eighteen baculovirus late expression factor genes in transcription and DNA replication. Journal of Virology 69: 975-982). The baculovirus immediate early-1 (ie-1) gene is one of five essential genes required for viral replication (Lu A and Miller L (1995) The roles of eighteen baculovirus late expression factor genes in transcription and DNA Journal of Virology 69: 975-982; Kool M, Ahrens C, Goldbach R, Rohrmann G and Vlak J (1994) Identification of genes involved in DNA replication of the Autographa californica baculovirus.Proceedings National Academy of Sciences USA 91: 11212 -11216). IE-1 is suggested to activate the promoters of early genes including ie-2, 39K and p35 (Carson D, Summers M and Guarino L (1991) Molecular analysis of a baculovirus regulatory gene.Virology 182: 279-286; Guarino LA and Summers MD (1986) Functional mapping of a trans-activating gene required for expression of a baculovirus delayed-early gene.Journal of Virology 57: 563-571; Nissen MS and Friesen PD (1989) Molecular analysis of the transcriptional regulatory region of an early baculovirus gene.Journal of Virology 63: 493-503), directly or indirectly involved in the expression of late genes (Passarelli A and Miller L (1993) Three baculovirus genes involved in late and very late gene expression: ie-1, ie-n, and lef-2. Journal of Virology 67: 2149-2158). One of the late expression factors important for DNA replication is lef-1, a gene involved in late and extreme late gene expression (Kool M, Ahrens C, Goldbach R, Rohrmann G and Vlak J (1994) Identification of genes involved. in DNA replication of the Autographa californica baculovirus. Proceedings National Academy of Sciences USA 91: 11212-11216). LEF-1 is a DNA primase essential for initiation of DNA replication (Isobe R, Kojima K, Matsuyama T, Quan GX, Kanda T, Tamura T, Sahara K, Asano SI and Bando H (2004) Use of RNAi technology to confer enhanced resistance to BmNPV on transgenic silkworms. Archives Virology 149: 1931-1940). However, RNAi-mediated knockdown of lef-1 alone did not show complete suppression of baculovirus growth (Isobe R, Kojima K, Matsuyama T, Quan GX, Kanda T, Tamura T, Sahara K, Asano SI and Bando H (2004) Use of RNAi technology to confer enhanced resistance to BmNPV on transgenic silkworms. Archives Virology 149: 1931-1940).

  The Lef-3 gene is another essential early class of genes encoded by baculovirus that is required for DNA replication (Lu A and Miller L (1995) The roles of eighteen baculovirus late expression factor genes in transcription and DNA. Journal of Virology 69: 975-982; Kool M, Ahrens C, Goldbach R, Rohrmann G and Vlak J (1994) Identification of genes involved in DNA replication of the Autographa californica baculovirus.Proceedings National Academy of Sciences USA 91: 11212 -11216; Li Y, AL Passarelli AL and Miller LK (1993) Identification, sequence, and transcriptional mapping of lef-3, a baculovirus gene involved in late and very late gene expression. Journal of Virology 67: 5260-5268). LEF-3 mediates nuclear localization of P143, a putative DNA helicase (Wu Y and Carstens EB (1998) A baculovirus single-stranded DNA binding protein, LEF-3, mediates the nuclear localization of the putative helicase P143. Virology 247: 32-40, Chen Z and Carstens EB (2005) Identification of domains in Autographa californica multiple nucleopolyhedrovirus late expression factor 3 required for nuclear transport of P143. Journal of Virology 79: 10915-10922) A single-stranded DNA binding protein that forms (Wu Y and Carstens EB (1998) A baculovirus single-stranded DNA binding protein, LEF-3, mediates the nuclear localization of the putative helicase P143. Virology 247: 32-40) .

  P74 is essential for virulence (Kuzio J, Jaques R and Faulkner P (1989) Identification of p74, a gene essential for virulence of baculovirus occlusion bodies.Virology 173: 759-763) but is associated with DNA replication. Vulology 173: 759-763; Faulkner (Kuzio J, Jaques R and Faulkner P (1989) Identification of p74, a gene essential for virulence of baculovirus occlusion bodies. P, Kuzio J, Williams GV and Wilson JA (1997) Analysis of P74, a PDV envelope protein of Autographa californica nucleopolyhedrovirus required for occlusion body infectivity in vivo. Journal of General Virology 78: 3091-310). It is transcribed late in the life cycle of baculovirus and is thought to help adhere viral particles to the midgut during oral inoculation (Haas-Stapleton EJ, Washburn JO and Volkman LE (2004) P74 mediates specific binding of Autographa californica multiple nucleopolyhedrovirus occlusion-derived virus to primary cellular targets in the midgut epithelia of Heliothis virescens larvae. Journal of Virology 78: 6786-6791).

  RNA interference (RNAi) is a phenomenon of selective mRNA destruction leading to gene knockdown (Fire A, Xu S, Montgomery M, Kostas S, Driver S and Mello C (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391: 806-811). RNAi is currently considered an ancient antiviral strategy (Lindenbach B and Rice C (2002) RNAi targeting an animal virus: news from the front.Molecular Cell 9: 925-927; Downward J (2004) RNA interference. British Medical Journal 328: 1245-1248), part of which is also used by cellular machinery for gene regulation (Denli AM and Hannon GJ (2003) RNAi: an ever-growing puzzle. Trends in Biochemical Sciences 28: 196-201; Agrawal N, Dasaradhi PV, Mohmmed A, Malhotra P, Bhatnagar RK and Mukherjee SK (2003) RNA interference: biology, mechanism, and applications. Microbiology and Molecular Biology Review 67: 657-685). RNAi has been shown to work in many arthropod species, including silkworm B. mori (Riu HL, Li WX and Ding SW (2004) RNA-based immunity in insects. In SGM symposium 63: Microbe-vector interactions in vector-borne diseases, ed.SH Gillespie, GLSAO (Cambridge University Press) pp. 63-74), demonstrated to suppress AcNPV proliferation in Spodoptera frugiperda (Valdes VJ, Sampieri A, Sepulveda J and Vaca L (2003) Using double-stranded RNA to prevent in vitro and in vivo viral infections by recombinant baculovirus.Journal of Biological Chemistry 278: 19317-19324) On the other hand, RNAi targeting lef-1 alone and ie-1 alone is B It was not effective to efficiently suppress BmNPV proliferation in mori (Isobe R, Kojima K, Matsuyama T, Quan GX, Kanda T, Tamura T, Sahara K, Asano SI and Bando H (2004) Use of RNAi technology to confer enhanced resistance to BmNPV on transgenic silkworms. Archives Virology 149: 1931-1 940; Kanginakudru S, Royer C, Edupalli SV, Jalabert A, Mauchamp B, Chandrashekaraiah, Prasad SV, Chavancy G, Couble P and Nagaraju J (2007) Targeting ie-1 gene by RNAi induces baculoviral resistance in lepidopteran cell lines and in the transgenic silkworms, Insect Molecular Biology 16: 635-644).

Nagaraju J and Goldsmith MR (2002) Silkworm genomics-progress and prospects.Current Science 83: 415-425 Nagaraju J, Klimenko V and Couble P (2000) The silkworm Bombyx mori, a model genetic system.In Encyclopedia of Genetics, ed.Reeves E. (Fitzroy Dearborn, London), pp. 219-239 Tamura T, Thibert C, Royer C, Kanda T, Abraham E, Kamba M, Komoto N, Thomas JL, Mauchamp B, Chavancy G, Shirk P, Fraser M, Prudhomme JC, Couble P, Toshiki T, Chantal T, Corinne R , Toshio K, Eappen A, Mari K, Natuo K, Jean-Luc T, Bernard M, Gerard C, Paul S, Malcolm F, Jean-Claude P and Pierre C (2000) Germline transformation of the silkworm Bombyx mori L. using a piggyBac transposon-derived vector. Nature Biotechnology 18: 81-84 van Regenmortel MH, Fauquet CM, Bishop DHL, Carstens EB, Estes MK, Lemon SM, Maniloff J, Mayo MA, McGeoch DJ, Pringle CR and Wickner RB (2000) Seventh Report of the International Committee on Taxonomy of Viruses (Academic Press, San Diego, Wien New York. Murphy FA, Fauquet CM, Bishop DHL, Ghabrial SA, Jarvis AW, Martelli GP, Mayo MA and Summers MD (1995) Sixth Report of the International Committee on Taxonomy of Virus. (Springer Verlag, Wien, New York) Kondo A and Maeda S (1991) Host range expansion by recombination of the baculoviruses Bombyx mori nuclear polyhedrosis virus and Autographa californica nuclear polyhedrosis virus. Journal of Virology 65: 3625-3632 Huh NE and Weaver RF (1990a) Categorizing some early and late transcripts directed by the Autographa californica nuclear polyhedrosis virus.Journal of General Virology 71: 2195-2200 Huh NE and Weaver RF (1990b) Identifying the RNA polymerases that synthesize specific transcripts of the Autographa californica nuclear polyhedrosis virus.Journal of General Virology 71: 195-201 Engelhard E, Kam-Morgan L, Washburn J and Volkman L (1994) The insect tracheal system: A conduit for the systemic spread of Autographa californica multiple nuclear polyhedrosis virus.Proceedings National Academy of Sciences USA 91: 3224-3227 Lu A and Miller L (1995) The roles of eighteen baculovirus late expression factor genes in transcription and DNA replication.Journal of Virology 69: 975-982 Kool M, Ahrens C, Goldbach R, Rohrmann G and Vlak J (1994) Identification of genes involved in DNA replication of the Autographa californica baculovirus.Proceedings National Academy of Sciences USA 91: 11212-11216 Carson D, Summers M and Guarino L (1991) Molecular analysis of a baculovirus regulatory gene.Virology 182: 279-286 Guarino LA and Summers MD (1986) Functional mapping of a trans-activating gene required for expression of a baculovirus delayed-early gene.Journal of Virology 57: 563-571 Nissen MS and Friesen PD (1989) Molecular analysis of the transcriptional regulatory region of an early baculovirus gene.Journal of Virology 63: 493-503 Passarelli A and Miller L (1993) Three baculovirus genes involved in late and very late gene expression: ie-1, ie-n, and lef-2.Journal of Virology 67: 2149-2158 Isobe R, Kojima K, Matsuyama T, Quan GX, Kanda T, Tamura T, Sahara K, Asano SI and Bando H (2004) Use of RNAi technology to confer enhanced resistance to BmNPV on transgenic silkworms.Archives Virology 149: 1931-1940 Li Y, A L Passarelli AL and Miller LK (1993) Identification, sequence, and transcriptional mapping of lef-3, a baculovirus gene involved in late and very late gene expression.Journal of Virology 67: 5260-5268 Wu Y and Carstens EB (1998) A baculovirus single-stranded DNA binding protein, LEF-3, mediates the nuclear localization of the putative helicase P143. Virology 247: 32-40 Chen Z and Carstens EB (2005) Identification of domains in Autographa californica multiple nucleopolyhedrovirus late expression factor 3 required for nuclear transport of P143. Journal of Virology 79: 10915-10922 Kuzio J, Jaques R and Faulkner P (1989) Identification of p74, a gene essential for virulence of baculovirus occlusion bodies. Virology 173: 759-763 Faulkner P, Kuzio J, Williams GV and Wilson JA (1997) Analysis of P74, a PDV envelope protein of Autographa californica nucleopolyhedrovirus required for occlusion body infectivity in vivo. Journal of General Virology 78: 3091-310 Haas-Stapleton EJ, Washburn JO and Volkman LE (2004) P74 mediates specific binding of Autographa californica multiple nucleopolyhedrovirus occlusion-derived virus to primary cellular targets in the midgut epithelia of Heliothis virescens larvae. Journal of Virology 78: 6786-6791 Fire A, Xu S, Montgomery M, Kostas S, Driver S and Mello C (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans.Nature 391: 806-811 Lindenbach B and Rice C (2002) RNAi targeting an animal virus: news from the front.Molecular Cell 9: 925-927 Downward J (2004) RNA interference. British Medical Journal 328: 1245-1248 Denli AM and Hannon GJ (2003) RNAi: an ever-growing puzzle.Trends in Biochemical Sciences 28: 196-201 Agrawal N, Dasaradhi PV, Mohmmed A, Malhotra P, Bhatnagar RK and Mukherjee SK (2003) RNA interference: biology, mechanism, and applications.Microbiology and Molecular Biology Review 67: 657-685 Riu HL, Li W-X and Ding S-W (2004) RNA-based immunity in insects. In SGM symposium 63: Microbe-vector interactions in vector-borne diseases, ed. S. H. Gillespie, G. L. S. A. O. (Cambridge University Press) pp. 63-74 Valdes VJ, Sampieri A, Sepulveda J and Vaca L (2003) Using double-stranded RNA to prevent in vitro and in vivo viral infections by recombinant baculovirus. Journal of Biological Chemistry 278: 19317-19324 Kanginakudru S, Royer C, Edupalli SV, Jalabert A, Mauchamp B, Chandrashekaraiah, Prasad SV, Chavancy G, Couble P and Nagaraju J (2007) Targeting ie-1 gene by RNAi induces baculoviral resistance in lepidopteran cell lines and in the transgenic silkworms Insect Molecular Biology 16: 635-644 Thomas JL, Da Rocha M, Besse A, Mauchamp B and Chavancy G (2002) 3xP3-EGFP marker facilitates screening for transgenic silkworm Bombyx mori L. from the embryonic stage onwards.Insect Biochemistry Molecular Biology 32: 247-253 Van der Linden AM and Plasterk RH (2004) Shotgun cloning of transposon insertions in the genome of Caenorhabditis elegans.Computational and Functional Genomics 5: 225-229 Nagaraja GM and Nagaraju J (1995) Genome fingerprinting of the silkworm, Bombyx mori, using random arbitrary primers. Electrophoresis 16: 1633-1638 http://sgp.dna.affrc.go.jp/KAIKO/ Sambrook J and Russel DW (2001) In Molecular Cloning: A Laboratory Manual. Cold Springs Harbor Laboratory Press, Cold Springs Harbor, New York

  One aspect of the invention is a small hairpin RNA (shRNA) for RNAi-mediated suppression of baculovirus infection in insects, the group consisting of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14 The present invention relates to a short hairpin RNA (shRNA) comprising an RNA sense strand comprising a fragment of two or more nucleotide sequences selected from: a spacer sequence, and an antisense RNA strand complementary to the sense RNA strand.

Another aspect of the invention is a recombinant DNA construct comprising:
A polynucleotide having the nucleotide sequence set forth in SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14 in the antisense orientation and producing a dsRNA molecule; or SEQ ID NO: 11, A sense strand of a polynucleotide comprising a fragment of two or more nucleotide sequences selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14 and its complementary polynucleotide; or SEQ ID NO: 11, SEQ ID NO: A sense strand of a polynucleotide comprising a fragment of two or more nucleotide sequences selected from the group consisting of 12, SEQ ID NO: 13 and SEQ ID NO: 14, its complementary polynucleotide and spacer polynucleotide sequences; or A set comprising polynucleotides encoding the shRNA disclosed in the present invention that are operably linked For example relates to a DNA construct.

  Yet another aspect of the present invention is a method of producing a transgenic insect resistant to baculovirus comprising transforming an insect with a recombinant construct disclosed in the present invention, and resistance to baculovirus. Selecting a transgenic insect exhibiting

  Yet another aspect of the present invention relates to a baculovirus resistant transgenic insect that expresses a small hairpin RNA (shRNA) disclosed in the present invention or a recombinant DNA construct disclosed in the present invention.

  A further aspect of the invention relates to transgenic baculovirus resistant insect progeny expressing the small hairpin RNA (shRNA) disclosed in the present invention or the recombinant DNA construct disclosed in the present invention.

Figure 2 shows the PiggyBac-based RNAi vectors pPiggy-Sense-3XP3-GFP, pPiggy-Antisense-3XP3-DsRed2 and pPiggy-Multigene flip-flop-3XP3-DsRed2 used to obtain transgenic silkworms. Left ITR and Right ITR indicate the left and right terminal inverted repeats of the piggyBac transposable element. Each construct consists of four viral genes: ie-1 (310bp), lef-1 (326bp), lef-3 (316bp), p74 (310bp), marker gene gfp (800bp) and 200bp SV40 polyadenylation site segment including. A) The pPiggy-Sense-3XP3-GFP construct (9677 bp) consists of four viral target gene segments that are transcribed as sense strands under the widely expressed silkworm cytoplasmic actin (A3) promoter. The gene encoding green fluorescent protein (GFP) is under the 3XP3 promoter, but 3XP3 is expressed in the eye and other neural tissues to identify transgenic silkworms during the late embryonic and moth stages It is used as a marker gene. B) The pPiggy-Antisense-3XP3-DsRed2 construct (10097 bp) consists of four viral target gene segments transcribed as antisense strands under the widely expressed silkworm cytoplasmic actin (A3) promoter. The gene encoding red fluorescent protein (DsRed2) is placed under the 3XP3 promoter, but 3XP3 is expressed in the individual eye and other neural tissues, and transforms transgenic silkworms during the late embryonic and moth stages. Used as a marker gene for identification. C) pPiggy-Multigene flip- with each part of the essential baculovirus gene in sense and antisense orientation, separated by a spacer (323 bp), driven by a widely expressed silkworm cytoplasmic actin (A3) promoter flop-3XP3-DsRed2 construct (12422bp). The gene encoding red fluorescent protein (DsRed2) is under the 3XP3 promoter, but 3XP3 is expressed in the eye and other neural tissues to identify transgenic silkworms during the late embryonic and moth stages It is used as a marker gene. D) Essential baculovirus gene (gfp segment in the sense and antisense orientation), separated by spacer (323 bp), separated by spacer (323 bp), driven by a widely expressed silkworm cytoplasmic actin (A3) promoter PPiggy-Multigene flip-flop-3XP3-DsRed2 construct with each part of (not). The gene encoding red fluorescent protein (DsRed2) is under the 3XP3 promoter, but 3XP3 is expressed in the eye and other neural tissues to identify transgenic silkworms during the late embryonic and moth stages It is used as a marker gene. E) Three essential baculovirus genes in the sense and antisense orientation (no p74 and gfp segments) separated by a spacer (323 bp), driven by a widely expressed silkworm cytoplasmic actin (A3) promoter ) PPiggy-Multigene flip-flop-3XP3-DsRed2 construct with each part. A gene encoding red fluorescent protein (DsRed2), which is under the 3XP3 promoter, is expressed in the eye and other neural tissues and is a marker gene for identifying transgenic silkworms during the late embryonic and moth stages It is used as It is a figure which shows the molecular characteristic analysis of a silkworm transgenic body. Transposable Element Display (TED) method. It is a figure which shows the molecular characteristic analysis of a silkworm transgenic body. The copy number and site of transgene insertion in the transgenic lines was characterized using the TED assay. Seven Nistari transgenic lines (170A, 170B, 164C, 164B, 154C, 154D and 118A) showed single copy insertion. It is a figure which shows the molecular characteristic analysis of a silkworm transgenic body. The presence of a signature sequence TTAA that is characteristic of piggyBac-mediated integration of the transgene with respect to the left arm of piggyBac. TTAA sequences that are characteristic of piggyBac-mediated transfer are shown in red. It is a figure which shows the molecular characteristic analysis of a silkworm transgenic body. Mapping of transgene to chromosomal location in various transgenic lines expressing dsRNA for essential baculovirus genes. It is a figure which shows BmNPV resistance in the transgenic and non-transgenic Nistari (NM) strain | stump | stock of a silkworm. Fourth-instar larvae were given BmNPVP10-GFP at a dose of 12000 PIB / larvae. Mortality was calculated based on the number of moths that appeared. Bars indicate standard deviation. Transgenic line expressing TAFib6-non-target dsRNA; transgenic Nistari line with IE-1 FF-single viral gene target; transgenic with four essential baculovirus target genes expressed in MFF-sense and antisense orientation Strain; Nistari transgenic strain with four viral target gene segments transcribed in Sense (ABS) -sense direction; Antisense (AS)-Nistari transgenic with four viral gene segments transcribed in antisense direction Lines; and transgenic lines obtained by crossing Hybrid-Sense × AS-Sense and AS lines. FIG. 6 shows BmNPV-induced mortality in pure lines or hybrids of transgenic and non-transgenic (CSR2) lines. Mortality was reduced in lines with the transgene. Bars indicate standard deviation. Transgenic lines expressing TAFib6-non-target dsRNA; IE-1 FF (E) -transgenic Nistari line with single viral gene target; Multiple gene FF (MFF) -four essential expressed in sense and antisense orientation Transgenic line with baculovirus target gene; Nistari transgenic line with four viral target gene segments transcribed in the Sense (ABS) -sense direction; Antivirus (AS)-four viruses transcribed in the anti-sense direction Nistari transgenic lines with gene segments; transgenic lines obtained by crossing Hybrid-Sense × AS-Sense and AS lines. It is a figure which shows the polyhedral inclusion body (PIB) titer in various transgenic worms. The number of polygonal inclusion bodies formation is much lower in transgenic lines compared to non-transgenic control lines. CSR2-control CSR2 line, NM-control Nistari line; IE-1 FF (E) -transgenic Nistari line with single viral gene target; Sense (ABS) -segment of 4 viral target genes transcribed in sense direction Nistari transgenic lines with Antisense (AS) -Nistari transgenic lines with four viral gene segments transcribed in the antisense direction; obtained by crossing Hybrid (ABS x AS) -sense and antisense lines Transgenic lines; and Multiple gene FF (MFF) -transgenic Nistari lines with four essential baculovirus target genes expressed in the sense and antisense orientation. FIG. 4 shows four baculovirus genes ie-1, lef-1, lef-3 and p74 initially cloned in pCRII TOPO vector (Invitrogen). The resulting plasmids were named Topo-ie-1, Topo-lef-1, Topo-lef-3, and Topo-p74. These vectors with the correct insert were confirmed by restriction digestion and DNA sequencing for later use in the cloning step. FIG. 5 shows various steps involved in the construction of pPiggysense-3XP3-GFP, pPiggyantisense-3XP3-DsRed2 and pPiggyMultigene flip-flop-3XP3-DsRed2. Provide step-by-step instructions in writing. See Figure 7a. See Figure 7a. See Figure 7a. See Figure 7a. See Figure 7a. See Figure 7a. See Figure 7a.

Objects of the present invention The main object of the present invention is to produce transgenic silkworms that show improved resistance to viral infection.

  One of the objects of the present invention is the careful selection of appropriate viral genes that are essential for early baculovirus propagation.

  Another object of the present invention is the identification of DNA segments of such viral genes.

  Another object of the present invention is the identification of essential viral genes in both sense and antisense orientation, under a widely active promoter, to ensure transgene expression in all tissues This is the construction of a composite vector having segments.

  Yet another object of the present invention is the inclusion of a suitable selectable marker gene under the control of a promoter that is expressed in the early or late embryonic stage as well as the adult stage to allow easy screening of transgenic littermates.

  Yet another object of the present invention is the simultaneous RNAi-mediated suppression of multiple essential viral genes and evaluation of their effects on baculovirus propagation in transgenic silkworms.

A further object of the invention is the feasibility study of transferring transgenic features into F ₁ hybrids of transgenic silkworm strains × non-transgenic silkworm strains.

Detailed Description of the InventionDefinitions As used herein, the term "antisense" has a homology of greater than 50%, preferably greater than 80%, with respect to the target gene as defined herein. Represents a gene or nucleotide sequence derived therefrom linked to a promoter in the reverse 5 'to 3' direction.

  As used herein, the term “RNAi” (RNA interference) corresponds to the whole or part of a target gene, generates double-stranded RNA, and introduces sequences that provide target gene-specific gene silencing (repression) and Represents suppression of target gene expression by expression. In general, such RNAi sequences comprise a linear arrangement of partial sense and antisense DNA sequences whose expression forms RNA interference.

  A “polynucleotide” is a nucleic acid molecule comprising a plurality of polymerized nucleotides, eg, at least about 15 consecutive polymerized nucleotides. The polynucleotide can be a nucleic acid, an oligonucleotide, a nucleotide, or any fragment thereof. In many cases, the polynucleotide comprises a nucleotide sequence that encodes the polypeptide (or protein) or a domain or fragment thereof. In addition, the polynucleotide can include a promoter, intron, enhancer region, polyadenylation site, translation initiation site, 5 ′ or 3 ′ untranslated region, reporter gene, selectable marker, and the like. The polynucleotide can be single-stranded or double-stranded DNA or RNA. A polynucleotide optionally includes a modified base or a modified backbone. The polynucleotide can be, for example, genomic DNA or RNA, transcript (such as mRNA), cDNA, PCR product, cloned DNA, synthetic DNA or RNA, and the like. Polynucleotides can be combined with carbohydrates, lipids, proteins, or other materials to perform specific activities such as transformation or to form useful compositions such as peptide nucleic acids (PNA). A polynucleotide can comprise a sequence that is in the sense or antisense orientation. “Oligonucleotide” is substantially equivalent to the terms ampere primer, primer, oligomer, element, target, and probe, and is preferably single stranded.

  “Gene” or “gene sequence” refers to a partial or complete coding sequence of a gene, its complement, and its 5 ′ or 3 ′ untranslated region. A gene is also a functional unit of inheritance, and in a physical perspective, a specific segment or sequence of nucleotides along the molecule of DNA (or RNA in the case of RNA viruses) that is involved in the production of a polypeptide chain. The latter can be subjected to subsequent processing such as chemical modification or folding to obtain a functional protein or polypeptide. A gene can be isolated, partially isolated, or found with the genome of an organism. As an example, a transcription factor gene encodes a transcription factor polypeptide that is functional or may require processing to function as an initiator of transcription.

  In operation, a gene can be defined by a cis-trans assay. A cis-trans test is a genetic test that can be used to determine whether two mutations occur in the same gene and to determine the limits of a genetically active unit. A gene generally includes a region preceding the coding region (“leader”; upstream) and a region following the coding region (“trailer”; downstream). A gene can also include intervening, non-coding sequences termed “introns” located between individual coding segments termed “exons”. Most genes have a regulatory sequence in the associated promoter region, 5 'of the transcription start codon (there are some genes that do not have an identifiable promoter). Gene function can also be regulated by enhancers, operators, and other regulatory elements.

  Construct: Unless otherwise stated, the term “construct” refers to a recombinant genetic molecule comprising one or more isolated polynucleotide sequences of the present invention.

  The genetic construct used for transgene expression in the host organism includes a gene promoter sequence operably linked to the open reading frame and optionally a gene termination sequence 3 'downstream of the open reading frame. The open reading frame can be oriented in the sense or antisense direction, depending on the intended use of the gene sequence. The construct can also include a selectable marker gene and other regulatory elements for gene expression.

  Vector: A DNA molecule that is capable of replication in a host cell and / or can be operably linked to effect replication of a segment with another DNA segment. Plasmids, phagemids, cosmids, phages, viruses, YACs, and BACs are all typical vectors.

  The term “vector” refers to a nucleic acid molecule that is used to introduce a polynucleotide sequence into a host cell, thereby creating a transformed host cell. In addition to the genetic constructs described above, a “vector” refers to genetic material, such as an origin of replication, a selectable marker gene, and other genetic elements known in the art (eg, One or more nucleic acid sequences that allow it to replicate in one or more host cells, such as sequences for integration into the genome.

  The present invention relates to RNA of baculovirus propagation in transgenic silkworms by simultaneously targeting more than 1, preferably more than 2, more preferably more than 3, most preferably more than 4 viral genes. Provides interference (RNAi) mediated suppression. The present invention is a method for the production of a transgenic silkworm that is resistant to baculovirus, wherein the transgenic silkworm comprises at least two viral genes, preferably three viral genes, more preferably four viral genes. Also provided are methods that demonstrate the co-expression of double-stranded RNA. Furthermore, the present invention provides a method for the transfer of virus resistance characteristics into hybrid silkworms.

  The present invention relates to RNA of baculovirus propagation in transgenic silkworms by simultaneously targeting more than 1, preferably more than 2, more preferably more than 3, most preferably more than 4 viral genes. Interference (RNAi) mediated suppression, wherein the viral gene is selected from the group consisting of early-1 gene (ie-1), late effector factors lef-1, lef-3 and p74 genes Provides suppression specifically.

  The present invention is a method for the production of a transgenic silkworm that is resistant to baculovirus, wherein the transgenic silkworm comprises at least two viral genes, preferably three viral genes, more preferably four viral genes. Also provided is a method that exhibits co-expression of double stranded RNA and the viral gene is selected from the group consisting of early-1 gene (ie-1), late effector factors lef-1, lef-3 and p74 genes.

  The present invention relates to transgenic silkworms and their progeny that constitutively and simultaneously express double-stranded RNA of at least two viral genes, preferably three viral genes, more preferably four viral genes, wherein the viral genes are Further provided is a transgenic silkworm selected from the group consisting of an early-1 gene (ie-1), a late effector factor lef-1, lef-3 and p74 gene and progeny thereof.

  Furthermore, the present invention provides a method for the transfer of virus resistance characteristics into hybrid silkworms.

  The present invention includes an early-1 gene (ie-1) having the nucleotide sequence set forth in SEQ ID NO: 11, a late effector factor gene (lef-1) having the nucleotide sequence set forth in SEQ ID NO: 12, and the sequence set forth in SEQ ID NO: 13. Also provided are polynucleotides of the lef-3 gene having a nucleotide sequence and the p74 gene having the nucleotide sequence set forth in SEQ ID NO: 14.

  The present invention further provides a spacer polynucleotide, wherein the spacer nucleotide is set forth in SEQ ID NO: 15.

In this study, we chose multiple essential viral gene segments, ie-1, lef-1, lef-3 and p74 genes, to produce double-stranded RNA (dsRNA) and generate a composite vector. We have introduced transgenic vectors to construct transgenic silkworms that express dsRNA in all tissues of the transgenic silkworm. We expressed in transgenic constructs and larval and adult eye cells (single eyes) so that dsRNA for the selected gene is expressed in all tissues under a broadly active promoter, A marker gene was created that allows easy screening of individuals. We also succeeded in transferring the transgenic properties to the F ₁ hybrid.

  In accordance with the present invention, in one embodiment of the present invention, in the sense-1 orientation of the early-1 gene (ie-1), the late effector factors lef-1, lef-3 and the p74 gene in the polynucleotide and antisense orientation A recombinant vector comprising polynucleotides of early-1 gene (ie-1), late effector factors lef-1, lef-3 and p74 gene, wherein the polynucleotides in the sense and antisense directions are separated by spacer DNA An isolated recombinant vector is provided.

  In one embodiment of the invention, at least two polys selected from the group consisting of early-1 gene (ie-1), late effector factors lef-1, lef-3 and p74 genes in sense and antisense orientation A recombinant vector comprising nucleotides is provided wherein the polynucleotides in sense and antisense orientation are separated by spacer DNA.

  In one embodiment of the invention, at least three polys selected from the group consisting of an early-1 gene (ie-1), late effector factors lef-1, lef-3 and p74 genes in sense and antisense orientation A recombinant vector comprising nucleotides is provided wherein the polynucleotides in sense and antisense orientation are separated by spacer DNA.

  In another embodiment of the invention, polynucleotides of the early-1 gene in the sense direction (ie-1), late effector factors lef-1 and lef-3 genes and the early-1 gene in the antisense direction (ie -1), a recombinant vector comprising polynucleotides of the late effector factor lef-1 and lef-3 gene, wherein the polynucleotides in sense direction and antisense direction are separated by spacer DNA Is provided.

  In another embodiment of the invention, polynucleotides of early-1 gene (ie-1) and late effector factor lef-1 gene in sense direction and early-1 gene (ie-1) in antisense direction, and A recombinant vector comprising a late effector factor lef-1 gene polynucleotide, wherein the polynucleotides in sense and antisense orientation are separated by spacer DNA, is provided.

  In another embodiment of the invention, the early -1 gene in the sense direction (ie-1), the late effector factors lef-1 and p74 gene polynucleotides and the early -1 gene in the antisense direction (ie-1) A recombinant vector comprising a late effector factor lef-1 and a polynucleotide of the p74 gene, wherein the polynucleotide in sense and antisense orientation is separated by spacer DNA is provided.

  In another embodiment of the invention, polynucleotides of the early-1 gene (ie-1), lef-3 and p74 genes in the sense direction and the early-1 gene (ie-1) in the antisense direction, lef- A recombinant vector comprising a polynucleotide of 3 and p74 genes, wherein the polynucleotide in sense and antisense orientation is separated by spacer DNA is provided.

  In another embodiment of the invention, polynucleotides of early-1 gene (ie-1) and p74 gene in the sense direction and polynucleotides of early-1 gene (ie-1) and p74 gene in the antisense direction A recombinant vector comprising a polynucleotide in which the polynucleotides in sense and antisense orientation are separated by spacer DNA is provided.

  In a further embodiment of the invention, a method for the production of a transgenic silkworm resistant to baculovirus, wherein said method comprises an early-1 gene (ie-1) in a sense direction and an antisense direction, a late effector Introducing a recombinant vector comprising at least two polynucleotides selected from the group consisting of the factors lef-1, lef-3 and p74 genes, wherein the polynucleotides in sense and antisense orientation are mediated by spacer DNA An isolated method is provided.

  In a preferred embodiment of the present invention, transgenic silkworms and their progeny that constitutively and simultaneously express double-stranded RNA of at least two viral genes, wherein the viral gene is an early-1 gene (ie-1), Provided are transgenic silkworms and their progeny selected from the group consisting of late effector factors lef-1, lef-3 and p74 genes.

  In another preferred embodiment of the invention, a transgenic silkworm and its progeny that constitutively and simultaneously expresses double-stranded RNA of at least three viral genes, wherein the viral gene is an early-1 gene (ie-1 ), A transgenic silkworm selected from the group consisting of late effector factors lef-1, lef-3 and p74 genes and progeny thereof.

  In another preferred embodiment of the present invention, transgenic silkworms and their progeny that express constitutively and simultaneously double-stranded RNA of four viral genes, wherein the viral gene is an early-1 gene (ie-1) A transgenic silkworm selected from the group consisting of late effector factors lef-1, lef-3 and p74 genes and their progeny is provided.

  The transgenic silkworm disclosed in the present invention exhibits a high level of RNA interference mediated resistance to baculovirus infection.

  One embodiment of the present invention is a short hairpin RNA (shRNA) for RNAi-mediated suppression of baculovirus infection in insects, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14. Provided is a small hairpin RNA (shRNA) comprising an RNA sense strand comprising a fragment of two or more nucleotide sequences selected from the group consisting of, a spacer sequence, and an antisense RNA strand complementary to the sense RNA strand .

  Another embodiment of the present invention is a small hairpin RNA (shRNA) for RNAi-mediated suppression of baculovirus infection in insects from SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14. Small hairpin RNA (shRNA) comprising an RNA sense strand comprising at least 15 contiguous nucleotides of two or more nucleotide sequences selected from the group consisting of, a spacer sequence, and an antisense RNA strand complementary to the sense RNA strand )I will provide a.

  Another embodiment of the present invention is a short hairpin RNA (shRNA) for RNAi-mediated suppression of baculovirus infection in insects, a fragment of the nucleotide sequence set forth in SEQ ID NO: 11, the sequence set forth in SEQ ID NO: 12. An RNA sense strand comprising a fragment of a nucleotide sequence, a fragment of a nucleotide sequence set forth in SEQ ID NO: 13 and a fragment of a nucleotide sequence set forth in SEQ ID NO: 14, a spacer sequence, and an antisense RNA strand complementary to the sense RNA strand, Small hairpin RNA (shRNA) is provided.

  Another embodiment of the present invention is a short hairpin RNA (shRNA) for RNAi-mediated suppression of baculovirus infection in insects, wherein the sense RNA strand is a fragment of the nucleotide sequence set forth in SEQ ID NO: 11, Provided is a short hairpin RNA (shRNA) comprising a fragment of the nucleotide sequence set forth in No. 12 and a fragment of the nucleotide sequence set forth in SEQ ID No. 13, a spacer sequence, and an antisense RNA strand complementary to the sense RNA strand .

  Another embodiment of the present invention is a short hairpin RNA (shRNA) for RNAi-mediated suppression of baculovirus infection in insects, wherein the sense RNA strand is a fragment of the nucleotide sequence set forth in SEQ ID NO: 11, Provided is a short hairpin RNA (shRNA) comprising a fragment of the nucleotide sequence set forth in No. 12 and a fragment of the nucleotide sequence set forth in SEQ ID No. 14, a spacer sequence, and an antisense RNA strand complementary to the sense RNA strand .

  Yet another embodiment of the present invention is a short hairpin RNA (shRNA) for RNAi-mediated suppression of baculovirus infection in insects, wherein the sense RNA strand is a fragment of the nucleotide sequence set forth in SEQ ID NO: 11, Provided short hairpin RNA (shRNA) comprising a fragment of the nucleotide sequence set forth in SEQ ID NO: 13 and a fragment of the nucleotide sequence set forth in SEQ ID NO: 14, a spacer sequence, and an antisense RNA strand complementary to the sense RNA strand To do.

  Yet another embodiment of the present invention is a short hairpin RNA (shRNA) for RNAi-mediated suppression of baculovirus infection in insects, wherein the sense RNA strand is a fragment of the nucleotide sequence set forth in SEQ ID NO: 12, Provided short hairpin RNA (shRNA) comprising a fragment of the nucleotide sequence set forth in SEQ ID NO: 13 and a fragment of the nucleotide sequence set forth in SEQ ID NO: 14, a spacer sequence, and an antisense RNA strand complementary to the sense RNA strand To do.

  A further embodiment of the present invention provides an shRNA of the present invention comprising the spacer sequence set forth in SEQ ID NO: 15.

Another embodiment of the invention is a recombinant DNA construct comprising:
A polynucleotide having the nucleotide sequence set forth in SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14 in the antisense orientation and producing a dsRNA molecule; or SEQ ID NO: 11, A sense strand of a polynucleotide comprising a fragment of two or more nucleotide sequences selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14 and its complementary polynucleotide; or SEQ ID NO: 11, SEQ ID NO: A sense strand of a polynucleotide comprising a fragment of two or more nucleotide sequences selected from the group consisting of 12, SEQ ID NO: 13 and SEQ ID NO: 14, its complementary polynucleotide and spacer polynucleotide sequences; or A set comprising polynucleotides encoding the shRNA disclosed in the present invention that are operably linked To provide the example DNA constructs.

  Yet another embodiment of the present invention is a recombinant DNA construct disclosed in the present invention, wherein the promoter is a constitutive promoter such as a silkworm cytoplasmic actin (Bmactin) promoter or a tissue specific such as a Pax (3XP3) promoter. A recombinant DNA construct that is a promoter is provided.

  The recombinant DNA construct disclosed in the present invention includes two promoters, one of which includes a constitutive promoter that is widely expressed in a gene encoding silkworm cytoplasmic actin protein (Bmactin), and the other is a Pax protein. It contains two promoters, including a tissue specific promoter for the gene encoding (3XP3).

  One embodiment of the present invention provides a host cell that is the E. coli strain one shot INV110 (Invitrogen), which is a chemically competent strain.

Yet another embodiment of the present invention is a recombinant DNA construct comprising a polynucleotide encoding the shRNA of the present invention, wherein the polynucleotide encoding the shRNA comprises:
a sense strand comprising two or more nucleotide sequences selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19,
b. a spacer sequence, and
c. providing a recombinant DNA construct comprising an antisense strand complementary to the sense strand;

  The recombinant DNA construct disclosed in the present invention further comprises a reporter gene.

  A further embodiment of the invention is a recombinant vector comprising a polynucleotide encoding a small hairpin RNA (shRNA), wherein the shRNA is from SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14. Recombinant vectors comprising an RNA sense strand comprising a fragment of two or more nucleotide sequences selected from the group consisting of, a spacer sequence, and an antisense RNA strand complementary to the sense RNA strand are provided.

A further embodiment of the invention is a recombinant vector comprising a recombinant construct, wherein the recombinant construct comprises
A polynucleotide having the nucleotide sequence set forth in SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14 in the antisense orientation and producing a dsRNA molecule; or SEQ ID NO: 11, A sense strand of a polynucleotide comprising a fragment of two or more nucleotide sequences selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14 and its complementary polynucleotide; or SEQ ID NO: 11, SEQ ID NO: A sense strand of a polynucleotide comprising a fragment of two or more nucleotide sequences selected from the group consisting of 12, SEQ ID NO: 13 and SEQ ID NO: 14, its complementary polynucleotide and spacer polynucleotide sequences; or A set comprising polynucleotides encoding the shRNA disclosed in the present invention that are operably linked To provide the e vector.

  Another embodiment of the present invention provides a host cell comprising a polynucleotide encoding a small hairpin RNA (shRNA) disclosed in the present invention, or a recombinant construct of the present invention or a recombinant vector of the present invention.

  Another embodiment of the invention provides a host cell such as E. coli or an insect cell such as a silkworm.

  Yet another embodiment of the present invention is a method of producing a transgenic insect resistant to baculovirus comprising transforming an insect with the recombinant construct of the present invention, and resistance to baculovirus. Selecting a transgenic insect to be displayed.

  Yet another embodiment of the present invention is a method of producing a transgenic silkworm that is resistant to baculovirus, comprising transforming a silkworm with the recombinant construct of the present invention, and resistance to baculovirus. Selecting a transgenic silkworm to be displayed.

  Yet another embodiment of the present invention is a method for the production of a transgenic bombix mori resistant to baculovirus comprising transforming bombix mori with the recombinant construct of the present invention; Selecting a transgenic insect exhibiting resistance to baculovirus.

  Yet another embodiment of the present invention is a method for the production of a transgenic bombix mori resistant to baculovirus comprising transforming bombix mori with the recombinant construct of the present invention; Selecting a transgenic insect exhibiting resistance to baculovirus, and providing a method wherein the silkworm is a Bombyx mori Nistari strain.

  A further embodiment of the invention provides a baculovirus resistant transgenic insect that expresses the short hairpin RNA (shRNA) of the invention.

  A further embodiment of the invention provides a baculovirus resistant transgenic insect that expresses a recombinant DNA construct of the invention.

  A further embodiment of the present invention provides a baculovirus resistant transgenic silkworm that expresses the short hairpin RNA (shRNA) of the present invention.

  A further embodiment of the present invention provides a baculovirus resistant transgenic silkworm that expresses the recombinant DNA construct of the present invention.

  A further embodiment of the present invention provides baculovirus resistant transgenic insect progeny expressing the short hairpin RNA (shRNA) of the present invention.

  A further embodiment of the present invention provides baculovirus resistant transgenic insect progeny expressing the recombinant DNA construct of the present invention.

  A further embodiment of the present invention provides baculovirus resistant transgenic silkworm progeny expressing the short hairpin RNA (shRNA) of the present invention.

  A further embodiment of the present invention provides baculovirus resistant transgenic silkworm progeny expressing the recombinant DNA construct of the present invention.

The invention specifically provides for the production of domesticated silkworm strains (Bombix mori) with high levels of baculovirus resistance produced by RNAi and gene transfer. By constructing a piggyBac transposon-based vector, we have double-stranded RNA for four baculovirus genes: the immediate early-1 gene (ie-1), the late effector factors lef-1, lef-3 and p74 genes Several homozygous lines of transgenic silkworms that constitutively produce were obtained. These homozygous lines showed suppression of B. mori nuclear polyhedrosis virus (BmNPV) infection as shown by the low mortality in the transgenic worms when compared to non-transgenic controls. A decrease in virus growth was also confirmed by Western blot analysis and other conventional methods. This antiviral property was successfully transferred to F ₁ hybrids of transgenic silkworm strains crossed with non-transgenic strains of B. mori. These results demonstrated that we succeeded in constructing silkworm strains with high levels of resistance by linking to silkworm gene transfer and simultaneously targeting multiple essential baculovirus genes.

  The RNAi mechanism has been used with contrasting results regarding suppressing baculovirus infection in Lepidoptera. There was the first report suggesting successful almost complete suppression of AcNPV proliferation in Sf9 cell lines and Spodoptera frugiperda by transient RNAi-mediated targeting of IE-1 (Kanginakudru S, Royer C, Edupalli SV, Jalabert A, Mauchamp B, Chandrashekaraiah, Prasad SV, Chavancy G, Couble P and Nagaraju J (2007) Targeting ie-1 gene by RNAi induces baculoviral resistance in lepidopteran cell lines and in the transgenic silkworms.Insect Molecular Biology 16: 635-644) . Isobe et al. (Li Y, AL Passarelli AL and Miller LK (1993) Identification, sequence, and transcriptional mapping of lef-3, a baculovirus gene involved in late and very late gene expression.Journal of Virology 67: 5260-5268) We reported a similar observation of moderate viral suppression but not complete protection in transgenic silkworm B. mori by targeting the lef-1 gene. These contrasting results may be due to differences in the target gene since it is known that the target gene / region will determine the success of RNAi-mediated gene silencing. In view of obtaining heritable, RNAi-induced baculovirus silencing, we have targeted baculoviruses in transgenic silkworms by targeting the essential baculovirus genes ie-1, lef-1, lef-3 and p74 A study was started to observe the effectiveness of deterrence.

  To obtain transgenic silkworms, we have piggyBac transposon with selectable markers 3XP3-GFP and 3XP3-DsRed, four viral gene sense, antisense and DNA to express double-gene flip-flop double-stranded RNA A base vector was constructed (Figure 1). Since the 3XP3-GFP selectable gene marker is expressed in the embryo, it allowed easy screening of transgenic silkworms at the early stages of development. Furthermore, GFP expression from 3XP3-GFP is restricted to certain tissues. We exploited this property of the 3XP3-GFP construct to facilitate easy screening of both the transgenic body and the progression of baculovirus infection. We obtained several lines of transgenic silkworms by co-injecting freshly born eggs of the Nistari strain with plasmids and helper plasmids to produce dsRNA. By controlled breeding, we obtained 18 homozygous transgenic lines, which we tested for their antiviral activity using recombinant GFP-expressing BmNPV, BmNPV-P10GFP. We tested oral infectivity by administering virus and calculating mortality.

  Our results show that regardless of the route of infection, transgenic silkworms were much more resistant than non-transgenic silkworm lines. Western blot analysis and microscopic observation confirm that virus growth was less in the transgenic body than in the non-transgenic body.

  In conclusion, we now note that RNAi-mediated baculovirus resistance is possible in Lepidoptera, with particular emphasis on domesticated silkworm B. mori, and a transgene for RNAi-mediated virus suppression of infection. It has been shown that a transgenic strain of silkworms with a piglet could be successfully produced by piggyBac-mediated germline gene transfer. It will be interesting to understand the effects of multigene targeting, where various essential baculovirus genes are targeted. The use of better universal promoters, especially those activated by baculovirus, needs to be investigated as a double-stranded driver of the target RNA. We suggest that further research in this direction will teach us to use RNAi as a tool for baculovirus suppression of infection.

  The invention is described in detail with the help of the following examples. However, the examples should not be construed as limiting the scope of the invention.

(Example)
The following examples described herein are for illustrative purposes only, and various changes or modifications to be considered will be suggested to one skilled in the art, and the spirit and scope of this application as well as the appended claims. It should be understood that it should be included within the scope of

(Example 1)
vector
pPIG3XP3GFP-FF
The vector pPIGA3GFP-FF expresses green fluorescent protein (GFP) under the silkworm cytoplasmic actin promoter, an almost universal promoter. The gfp driven by this promoter is widely expressed and therefore, even if not integrated, G0 embryos or cells containing the vector fluoresce, making it difficult to screen transgenics . To overcome this problem, we have previously introduced a piggyBac element inverted repeat for integration and a GFP gene under the control of a synthetic eye-specific promoter (3XP3) as a selectable marker for gene transfer. 3XP3-based vectors (Kanginakudru S, Royer C, Edupalli SV, Jalabert A, Mauchamp B, Chandrashekaraiah, Prasad SV, Chavancy G, Couble P and Nagaraju J (2007) Targeting ie-1 gene by RNAi induces baculoviral resistance in lepidopteran cell lines and in the transgenic silkworms. Insect Molecular Biology 16: 635-644). This synthetic promoter is recognized in vivo by the Pax protein, and downstream genes are expressed in a tissue-specific manner. The plasmid pPIG3XP3GFP-FF is an A3GFP cassette of pPIGA3GFP-FF, which was developed by Thomas et al. (Thomas JL, Da Rocha M, Besse A, Mauchamp B and Chavancy G (2002) 3xP3-EGFP marker facilitates screening for transgenic silkworm Bombyx mori L. from the It was constructed by replacing with 3XP3GFP cassette of embryonic stage onwards. Insect Biochemistry Molecular Biology 32: 247-253). For this, the 3XP3-GFP fragment was amplified by PCR and cloned into a TA vector from which it was restriction digested and mobilized to pPIGA3GFP-FF between the NgoMIV and SfiI sites. The construct pPIG3XP3GFP-FF was confirmed again by selective restriction digestion and sequencing.

Sense, Antisense, and Multigene Flip-Flop Vectors In order to obtain transgenic silkworm strains that are resistant to viruses, we have begun work targeting multiple essential viral genes. The vector was constructed using the piggyBac vector backbone. These constructs are pPiggysense-3XP3-GFP, pPiggyantisense-3XP3-DsRed2, and pPiggymultigene flip-flop-3XP3-DsRed2. A diagrammatic representation of the vector used is shown in FIG. The vectors, ie-1, lef-, encoding the immediate early 1 gene, late expression factor 1, 3 and the coat protein of B. mori nuclear polygonal virus, respectively, using primers with specific restriction enzyme sites. 1, constructed by amplifying the regions of the lef-3 and p74 genes and the gfp gene of pPIGA3GFP (Tamura T, Thibert C, Royer C, Kanda T, Abraham E, Kamba M, Komoto N, Thomas JL, Mauchamp B, Chavancy G, Shirk P, Fraser M, Prudhomme JC, Couble P, Toshiki T, Chantal T, Corinne R, Toshio K, Eappen A, Mari K, Natuo K, Jean-Luc T, Bernard M, Gerard C, Paul S, Malcolm F, Jean-Claude P and Pierre C (2000) Germline transformation of the silkworm Bombyx mori L. using a piggyBac transposon-derived vector. Nature Biotechnology 18: 81-84). The amplicon was cloned into the T-overhang of the pCR II Topo vector (Invitrogen) (Figure 6). The resulting plasmids were named Topo-gfp, Topo-ie-1, Topo-lef-1, Topo-lef-3, and Topo-p74. These vectors with the correct insert were confirmed by restriction digestion and DNA sequencing. These genes produce virus-specific proteins and do not share any homology with known human or insect genes. The target region of ie-1, lef-1, lef-3, p74 is nucleotide positions 141 to 450 of the nucleotide sequence set forth in SEQ ID NO: 11 with respect to the ie-1 gene and set forth in SEQ ID NO: 12 with respect to the lef-1 gene From nucleotide position 80 to 405 of the nucleotide sequence of, nucleotide position 391 to 706 of the nucleotide sequence set forth in SEQ ID NO: 13 for the lef-3 gene, and from nucleotide position 1243 of the nucleotide sequence set forth in SEQ ID NO: 14 for the p74 gene 1552.

  BmNPV ie-1, lef-1, lef-3 and p74 and gfp DNA were amplified by PCR using the primers set forth in SEQ ID NOs: 1-10.

A typical PCR reaction consisted of a 50 μl final volume with 25 pmoles of forward and reverse primers each. PCR amplification, for each reaction, 10mM Tris-HCl, (50mM KCl, 1.5mM MgCl 2, containing 0.01% gelatin and 0.01% Triton X-100 pH8.3) , 1mM dNTP and 5U of Taq DNA polymerase (NEB , US). Temperature cycling was performed on a thermal cycler (PE9700, Applied Biosystems) using the following conditions: initial denaturation at 95 ° C for 5 minutes; 95 ° C for 30 seconds; 50 ° C for 30 seconds and 72 ° C for 2 seconds 35 cycles per minute, and 10 min final extension at 72 ° C. PCR products were quantified on a 2.0% agarose gel and purified using a Qiagen PCR purification column according to the manufacturer's protocol. The purified product was cloned into the above Topo vector.

  The pTopo clone was transformed into one shot INV110 chemically competent E. coli (Invitrogen) (since it contains dam and dcm deletions that allow restriction digestion with dam- and dcm-sensitive restriction enzymes. dam and dcm The deficiency allows the production of DNA that is not methylated at these sites). Blue / white screening was performed on LB agar plates containing 40 μl X-Gal (40 mg / ml) and 40 μl isopropyl β-D-thiogalactoside (IPTG, 100 mM). White colonies were picked for insert analysis through digestion. Colonies that gave the correct insert of the expected size were picked.

  For DNA sequencing, 250 ng of plasmid was used in a sequencing reaction containing 8 μl of Ready reaction mix (BigDye terminator, BDTv 3.0, Applied Biosystems, Foster City, Calif.) And 5 pmoles of M13 sequencing primer. The cycling conditions used were: 25 cycles of 96 ° C for 10 seconds, 50 ° C for 5 seconds, 60 ° C for 4 minutes. Samples were precipitated with ethanol, washed with 70% ethanol, and resuspended in Hi-Di ™ formamide (Applied Biosystems). Sequencing was performed on ABI Prism 3100 Genetic Analyzer (Applied Biosystems) to confirm the integrity of each gene fragment.

The constructs pPiggysense-3XP3-GFP, pPiggyantisense-3XP3-DsRed2 and pPiggyMultigene flip-flop-3XP3-DsRed2 were obtained in the following series of cloning steps (Figure 7):
1.The 316 bp fragment of lef-3 (SEQ ID NO: 18) was cloned into the pA3DS-FSG vector backbone (7624 bp) using the SalI-NheI site to obtain a single gene construct, pA3ΔS-lef3 (6057 bp) Was generated. Transformants were verified by XmnI (internal site of lef-3) restriction digest and XmnI / ScaI double digestion. XmnI digestion was linearized into a 6.05 kb fragment of a single gene construct, and double digestion with ScaI (site in the main chain) followed a 323 bp product or insert size and 5.73 kb according to the DNA strider map pattern. Fragments were released.
2. The 310 bp fragment of ie-1 (SEQ ID NO: 16) was cloned into the pA3ΔS-lef3 vector using the BspEI-SalI site to generate pA3ΔS-ie1lef3SG, two gene constructs (5929 bp). Transformants were examined by continuous digestion using the XmnI / HpaI restriction system. Xmn I linearization of the two gene constructs yielded a 5929 bp fragment, and XmnI / Hpa I sequential digestion yielded the expected three bands of 339 bp, 1071 bp and 4519 bp fragments.
3. The 800 bp fragment of gfp was cloned into the pA3ΔS-ie1lef3 vector backbone using the PvuI-BamHI site to generate pA3ΔS-gfpie1lef3, the three gene construct (6665 bp). Transformants were examined by PvuI / EcoRI restriction analysis that generated an 834 bp insert and a 5834 bp backbone.
4. In addition to the pA3ΔS-gfpie1lef3 vector backbone, pA3ΔS-gfplef1ie1lef3 (6650 bp), four gene products were generated using BspEI / BamHI, a 326 bp fragment of the lef-1 clone (SEQ ID NO: 17). Transformants were confirmed through HpaI and NdeI restriction digests that yielded three fragments of expected lengths 188 bp, 1410 bp, 5040 bp and two fragments of expected lengths 1361 bp and 5289 bp, respectively.
5. The 310 bp fragment of p74 (SEQ ID NO: 19) was cloned into the pA3ΔS-gfplef1ie1lef3 vector backbone using the AgeI-XbaI site to generate five gene constructs, pA3ΔS-gfple1ie1lef3p74 (6160 bp). Transformants were confirmed through SalI restriction digestion. It gave a 520 bp insert and a 5640 bp backbone.
A construct directed to 5 gene senses, pA3ΔS-gfplef1ie1lef3p74, was digested with an internal NheI-XbaI site to generate a pool directed to sense and antisense of the 5 gene fragments. Transformants were screened for plasmids directed to antisense. This was confirmed by HindIII / XmnI digestion, where the antisense colony yielded a 1.3 kb fragment while the colony directed to the sense yielded a 2.2 kb fragment.
Sense construct-A3: A promoter fused with gfp with four genes and a poly A tail was cloned into pPiggyP25il2- (3XP3-GFP) TQ using BglII-NheI and BglII-XbaI sites to create pPiggy-Sense A −3XP3-GFP vector (FIG. 1A) was generated.
Antisense construct—The antisense fragment obtained above was cloned into pPiggyP25il2- (3XP3-DsRed2) TQ to generate the pPiggy-Antisense-3XP3.DsRed2 vector (FIG. 1B).
6.Multigene Flip-Flop construct-A3 above: A promoter in which gfp is fused with the four genes is cloned into the pP25 (sp *) DsRed vector backbone using the BglII-AgeI site and the spacer region (sequence A sense fragment having the number 15), an intermediate vector type pP25 (sp *) DsRed-A3ΔS Sense.Spacer was generated.
7.5 Antisense fragments of the genes were cloned into the middle form of the sense-spacer vector backbone using the Sse8337I-AflII site and the multigene construct flip-flop, pP25 (sp *) DsRed-A3ΔS Sense .Spacer.Antisense.Poly A region was generated.
8. The intermediate shuttle vector, pP25 (sp *) Sense-Spacer-Antisense vector, was digested at the BglII and SfiI sites to generate a flip-flop version of the insert. This insert was cloned into PiggyP25IL2- (3XP3-DsRed2) TQ using the BglII / SfiI site to generate pPiggyA3gfplef1ie1lef3p74.spacer.p74lef3ie1lef1gfp.3XP3-DsRed2 vector (FIG. 1C) with DsRed2 as a selectable marker gene. Generated.

  Therefore, a recombinant vector comprising a polynucleotide encoding the shRNA of the present invention, wherein the polynucleotide encoding the shRNA comprises a fragment of two or more nucleotides arranged in a different order, Can be constructed according to requirements and functionality.

(Example 2)
Germline gene transfer in B. mori Germline gene transfer was essentially performed by Tamura et al. (Tamura T, Thibert C, Royer C, Kanda T, Abraham E, Kamba M, Komoto N, Thomas JL, Mauchamp B, Chavancy G , Shirk P, Fraser M, Prudhomme JC, Couble P, Toshiki T, Chantal T, Corinne R, Toshio K, Eappen A, Mari K, Natuo K, Jean-Luc T, Bernard M, Gerard C, Paul S, Malcolm F Jean-Claude P and Pierre C (2000) Germline transformation of the silkworm Bombyx mori L. using a piggyBac transposon-derived vector. Nature Biotechnology 18: 81-84).

  About 1000 silkworm eggs were microinjected with 1 μg / μl of pPiggy-Sense-3XP3-GFP and an equimolar ratio of helper plasmid. In the same way, other constructs, pPiggy-Antisense-3XP3-DsRed2 and pPiggy-Multigene flip-flop-3XP3-DsRed2, were also injected with helper plasmids to isolate the eggs and generate various lines of transgenics . The resulting eggs were incubated and analyzed for integration of each construct by expression of fluorescence in the eye. By controlled breeding, 18 homozygous lines of transgenic silkworms were obtained. Transgenic lines obtained by using pPiggy-Sense-3XP3-GFP, pPiggy-Antisense-3XP3-DsRed2 and pPiggy-Multigene flip-flop-3XP3-DsRed2 were expressed as Sense (S) and Antisense (AS), respectively. And called the Multigene flip-flop (MFF) line. The resulting transgenic line contains 3 Senses, 2 Antisense, 5 Sense × Antisense hybrids and 8 MFF.

(Example 3)
Molecular characterization of transgenic bodies
MFF transgenic lines carrying transgenes that produce double-stranded RNA for the four essential baculovirus genes, with respect to transgene insert sites, their copy number and their physical location in the silkworm genome as follows: Characterized:
3.1 Transposable factor display (TED) method The transposable factor display (TED) method is applied to van der Linden and Plasterk 2004 (Van der Linden AM and Plasterk RH (2004) Shotgun cloning of transposon insertions in the genome of Caenorhabditis elegans.Computational and Functional Genomics 5: 225-229) was used to identify the site of insertion and the copy number of the insertion according to the modified protocol (Figure 2A). Briefly, the genomic DNA from the transgenic line was analyzed according to Nagaraja and Nagaraju 1995 (Nagaraja GM and Nagaraju J (1995) Genome fingerprinting of the silkworm, Bombyx mori, using random arbitrary primers.Electrophoresis 16: 1633-1638). Extracted and digested with Sau 3A. The resulting digested DNA was ligated into a vector cassette with Sau 3A overhangs (Oligo 503 and 504). Two rounds of PCR were designed into a piggyBac transgene near the left inverted repeat (LIR) transposon-specific LIR outer (SEQ ID NO: 20-5'-CTCACGCGGTCGTTATAGTT) and LIR inner (SEQ ID NO: 21 -5'-GGCGACTGAGATGTCCTAAA) primer and van der Linden and Plasterk adapter specific 505 and 337NEW primers. The first round of PCR was performed using the LIR outer and 505 primers, and the second round of PCR was performed using the LIR inner and 337NEW primers using MBI taq. After the second round PCR, the expected size of the amplicon exceeded 120 bp of the transgene portion and 120 bp with unknown length of genomic DNA. The PCR product from the second amplification step was used for sequencing. After the second PCR reaction, the formation of a single PCR amplification product indicated the presence of a single copy insertion of the transgene in the transgenic line (FIG. 2B). TED was used to target the left arm of the piggyBac transposon.
3.2 Location of the transgene on different chromosomes of transgenic lines To confirm the location of the insertion, the PCR products were sequenced and blasted against the entire B. mori genome sequence. In all transgenic lines, it was discovered that the genomic integration of the transgene was mediated by piggyBac, as evidenced by the presence of the signature sequence TTAA characteristic of piggyBac-mediated integration shown in FIG. 2C. These sequences are blasted against the silkworm physical map assembly of the sequence (http://sgp.dna.affrc.go.jp/KAIKO/), and the chromosomal location and the relative position of the transgene on the chromosome are translated. Determined for each of the transgenic lines (Table 1 and FIG. 2D).

(Example 4)
Hybrid of transgenic female and non-transgenic male
Non-dormant varieties of Nistari are relatively resistant to baculovirus infection (polygonal inclusion bodies (PIB) with LD50-4000 per larva), and dormant varieties of CSR2 are more susceptible to viral infection (LD50-250PIB / larva). To clearly demonstrate suppression of viral infection, we crossed transgenic Nistari females with silkworm non-transgenic BmNPV sensitive CSR2 variants. Therefore, we obtained 13 hybrid lines, including 7 S-As x CSR2 lines, 4 MFF x CSR2 lines and one experimental control line TAFib6 x CSR2 and NM x CSR2. Hybrids were tested for virus resistance orally as follows.

(Example 5)
Viral inoculation and mortality analysis oral inoculation in transgenic silkworms We performed oral BmNPV infection of transgenic lines and control non-transgenic NM silkworms at 12000 PIB per 4th instar larvae. 100 healthy larvae of each strain were given PIB of BmNPV-P10GFP, retaining only the larvae that consumed all the viral particles, and on the fresh mulberry leaves until the surviving larvae reached the moth stage Raised.

Recombinant BmNPV virus (BmNPV-P10GFP) was used to generate PIB. Baculovirus was amplified in BmN cells and free virus in cell culture medium was used for experimental purposes. To obtain BmNPV-P10GFP PIB, free virus in the cell culture medium was injected into the body cavity of CSR2 strain larvae, and hemolymph was collected from the infected larvae 5 to 6 days after infection. Fluorescent PIB was purified by frequent washes and counted using a hemocytometer. Wild-type BmNPV was collected from haemolymph of infected silkworms recovered from sericulture farmers. Hemolymph was used to obtain free virus. Free virus titer was calculated and diluted by plaque assay to give a final concentration of 1 × 10 ⁷ BV / ml. PIB was used in experiments involving oral inoculation of silkworms.

(Example 6)
Western blot by using antibody against GP64 A commercially available antibody against the GP64 coat protein of baculovirus was obtained from Novagen. These antibodies were cross-reacted with BmNPV GP64. Infected sputum from transgenic and control strains were purified from lysis buffer (50 mM Tris-Cl, pH 8.0, 120 mM NaCl, 0.5% (v / v) NP-40, 0.2 containing protease inhibitor cocktail (Sigma). (Sodium ortho orthovanadate, 100 mM NaF). (Sambrook J and Russel DW (2001) In Molecular Cloning: A Laboratory Manual. Cold Springs Harbor Laboratory Press, Cold Springs Harbor, New York). Equal amounts of protein were denatured in SDS-Loading dye (tube as loading control). (Using phosphorus), separated by 10% SDS-PAGE and blotted onto Hybond P ⁺ membranes. Western blots were performed with mouse anti-GP64 antibody and primary antibody was detected using an appropriate secondary antibody conjugated to horseradish peroxidase. Protein bands were visualized with chemiluminescent detection reagents (ECL detection kit, Amersham) according to the manufacturer's protocol. The developed bands were quantified densitometrically by using QuantityOne software from the BioRad gel documentation system.

(Example 7)
Silkworm Successful Germline Gene Transfer with RNAi Transgenes Silkworm germline gene transfer was performed using all three constructs: pPiggy-Sense-3XP3-GFP, pPiggy-Antisense-3XP3-DsRed2, and pPiggy-Multigene flip- Achieved using flop-3XP3-DsRed2. Several lines were selected as shown in Table 2 based on the intensity of GFP / RFP fluorescence in the eye driven by the 3XP3 promoter. They were mated with siblings to obtain several homozygous lines of transgenic silkworms described in Table 2.

  A hybrid between the transgenic lines of pPiggySense-3XP3-GFP, pPiggyAntisense-3XP3-DsRed2, termed hybrid Sense × AS, was also obtained and used in the viral assay.

(Example 8)
BmNPV diet experiments show reduced mortality in transgenics We performed oral BmNPV infection of transgenic lines and control non-transgenic (NM) silkworms at 12000 PIB per 4th instar larvae. This virus dose, because it exceeds a known LD ₅₀ (LD ₅₀ about 4000PIB / larvae in the third instar) on Nistari stock, we are, a study was initiated at this dose. TAFib6, a transgenic line expressing non-targeted dsRNA was also used as a control. The polyhedron was spread on a small piece of 1 cm diameter fresh leaf. 100 larvae of each line were given PIB and only the larvae that consumed all the leaf pieces were retained and raised on fresh mulberry leaves until the end of larval life. Silkworm mortality was estimated by counting the number of moths that appeared. Virus-induced mortality was confirmed by analyzing larvae / pupae or moths that succumbed to infection. The mortality rate for each strain is shown in FIG.

  A prominent feature of the oral assay is that two multigene flip-flops were observed while 100% mortality was observed in the non-transgenic Nistari (NM) and CSR2 control lines in the group given the 4-year-old PIB. The lines (MFF154C and 170B) show only 20% mortality. The results suggest that mortality was less than 25% in MFF lines, followed by between 25-35% in sense and antisense hybrids. These results also indicate that multigene targeting is particularly effective at high viral infection loads and that RNAi knockdown is beneficial for inhibiting viral growth. Kanginakudru et al. (Kanginakudru S, Royer C, Edupalli SV, Jalabert A, Mauchamp B, Chandrashekaraiah, Prasad SV, Chavancy G, Couble P and Nagaraju J (2007) Targeting ie-1 gene by RNAi induces baculoviral resistance in lepidopteran cell lines and In the transgenic silkworms. Insect Molecular Biology 16: 635-644), this multi-viral gene-targeted transgenic has at least a 2-fold increased survival after oral virus inoculation. Indicated.

(Example 9)
Transgenic female and non-transgenic male hybrids are also resistant to viruses. To test whether the protective effect of transgenic lines is observed in hybrids of transgenic and non-transgenic lines, we A hybrid of Nistari strain and non-transgenic CSR2 was obtained and inoculated into the hybrid as described above. Trends in virus-induced mortality and the ability of the transgene to suppress mortality were similar to the parent strain in the hybrid (FIG. 4). Viability is higher in CSR2 hybrids of MFF lines (≤75%) than in hybrids with CSR2 sense-antisense hybrids (40-70%), and most in non-transgenic hybrids of NM and CSR2 Less (<20%). These data support the hypothesis that RNAi-mediated suppression of baculovirus replication will benefit hybrids of transgenic and non-transgenic silkworms.

Baculovirus resistance in the transgenic body was measured as determined by the number of PIBs per microliter of hemolymph and the number of PIBs was determined as shown in FIG. It was found to be much lower in multiviral gene targeted transgenic lines compared to non-transgenic control lines.

SEQ ID NO: 11 shows the nucleotide sequence of the ie-1 gene (1755 bp, NCBI reference number: AY048770.1, Bombyx Mori nuclear polyhedrosis virus K1 immediate early protein (ie-1) gene, all coding region)

SEQ ID NO: 12 shows the nucleotide sequence of the entire lef-1 gene sequence (813 bp; NCBI reference number: NC_001962.1; Bombix Mori NPV, whole genome)

SEQ ID NO: 13 shows the nucleotide sequence of the entire lef-3 gene sequence (1158 bp; NCBI reference number: NC_001962.1; Bombix Mori NPV, whole genome)

SEQ ID NO: 14 shows the nucleotide sequence of the entire p74 gene sequence (1938 bp; NCBI reference number: NC_001962.1; Bombix Mori NPV, whole genome)

SEQ ID NO: 15 shows the nucleotide sequence (323 bp) of the spacer consisting of the dsRed (red fluorescent protein) part

SEQ ID NO: 16: nucleotide sequence of the target region of the ie-1 gene used in the shRNA construct, the entire ie1 of BmNPV is 1755 bp and the target region is 141-450 bp (310 bp) ie1 (Accession number AY048770)

SEQ ID NO: 17: nucleotide sequence of the target region of the lef 1 gene used in the shRNA construct, the entire lef1 of BmNPV is 813 bp, and the target region is 80-405 bp (326 bp) lef1 (Accession number L33180)

SEQ ID NO: 18: nucleotide sequence of the target region of the lef 3 gene used in the shRNA construct, the entire lef3 of BmNPV is 1158 bp and the target region is 391-706 bp (316 bp) lef3 (accession number L33180)

SEQ ID NO: 19: Nucleotide sequence of the target region of the p74 gene used in the shRNA construct, the entire p74 of BmNPV is 1938 bp and the target region is 1243-1552 bp (310 bp) p74 (Accession number L33180)

Claims (24)

A small hairpin RNA (shRNA) for RNAi-mediated suppression of baculovirus infection in insects,
an RNA sense strand comprising a fragment of two or more nucleotide sequences selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14,
b. a spacer sequence, and
c. shRNA comprising an antisense RNA strand complementary to the sense RNA strand.   2. The shRNA of claim 1 wherein the fragment comprises at least 15 contiguous nucleotides.   The sense RNA strand includes a fragment of the nucleotide sequence set forth in SEQ ID NO: 11, a fragment of the nucleotide sequence set forth in SEQ ID NO: 12, a fragment of the nucleotide sequence set forth in SEQ ID NO: 13, and a fragment of the nucleotide sequence set forth in SEQ ID NO: 14. The shRNA according to claim 1.   2. The shRNA of claim 1, wherein the sense RNA strand comprises a fragment of the nucleotide sequence set forth in SEQ ID NO: 11, a fragment of the nucleotide sequence set forth in SEQ ID NO: 12, and a fragment of the nucleotide sequence set forth in SEQ ID NO: 13.   2. The shRNA of claim 1, wherein the sense RNA strand comprises a fragment of the nucleotide sequence set forth in SEQ ID NO: 11, a fragment of the nucleotide sequence set forth in SEQ ID NO: 12, and a fragment of the nucleotide sequence set forth in SEQ ID NO: 14.   2. The shRNA of claim 1, wherein the sense RNA strand comprises a fragment of the nucleotide sequence set forth in SEQ ID NO: 11, a fragment of the nucleotide sequence set forth in SEQ ID NO: 13, and a fragment of the nucleotide sequence set forth in SEQ ID NO: 14.   2. The shRNA of claim 1, wherein the sense RNA strand comprises a fragment of the nucleotide sequence set forth in SEQ ID NO: 12, a fragment of the nucleotide sequence set forth in SEQ ID NO: 13, and a fragment of the nucleotide sequence set forth in SEQ ID NO: 14.   2. The shRNA of claim 1, wherein the spacer sequence comprises SEQ ID NO: 15. a polynucleotide having the nucleotide sequence set forth in SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14, wherein the polynucleotide is in the antisense orientation and produces a dsRNA molecule; or
b. a sense strand of a polynucleotide comprising a fragment of two or more nucleotide sequences selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14 and complementary polynucleotides thereof; or
c. a sense strand of a polynucleotide comprising a fragment of two or more nucleotide sequences selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14, its complementary polynucleotide and spacer poly Nucleotide sequence; or
d. A recombinant DNA construct comprising a polynucleotide encoding an shRNA according to claim 1 operably linked to a promoter.   The recombinant DNA construct according to claim 9, wherein the promoter is a constitutive promoter or a tissue-specific promoter.   11. The recombinant DNA construct according to claim 10, wherein the constitutive promoter is a silkworm cytoplasmic actin (Bmactin) promoter.   11. The recombinant DNA construct according to claim 10, wherein the tissue-specific promoter is Pax (3XP3) promoter. A polynucleotide encoding shRNA
a sense strand comprising two or more nucleotide sequences selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19,
b. a spacer sequence, and
c. The recombinant DNA construct of claim 9, comprising an antisense strand complementary to the sense strand.   The recombinant DNA construct of claim 9, further comprising a reporter gene.   A recombinant vector comprising the polynucleotide encoding the short hairpin RNA (shRNA) according to claim 1 or the recombinant construct according to claim 9.   A host cell comprising the polynucleotide encoding the short hairpin RNA (shRNA) according to claim 1 or the recombinant construct according to claim 9 or the recombinant vector according to claim 15.   17. A host cell according to claim 16, which is an E. coli or insect cell.   The host cell according to claim 17, wherein the insect is a silkworm.   A method for producing a transgenic insect resistant to baculovirus, comprising transforming an insect with the recombinant construct according to claim 9, and selecting a transgenic insect exhibiting resistance to baculovirus. And a method comprising.   The method according to claim 19, wherein the insect is a silkworm.   21. The method according to claim 20, wherein the silkworm is Bombix Mori.   10. A baculovirus resistant transgenic insect that expresses the short hairpin RNA (shRNA) according to claim 1 or the recombinant DNA construct according to claim 9.   10. A baculovirus resistant transgenic insect progeny expressing the short hairpin RNA (shRNA) of claim 1 or the recombinant DNA construct of claim 9.   24. A baculovirus resistant transgenic insect according to claim 22 or a baculovirus resistant transgenic insect progeny according to claim 23, wherein the insect is a silkworm.