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1 CMV CMV CMV Lomonossoff RNA CMV 1989 CMV RNA RNA CMV 1986 CMV CMV 1995 CMV 1993 RNA RNA RNA Roossick 1992 RNA Baulcombe 1986 Gerlach 1987 Harison 1987 Jacquemond 1988 Masuta McGarvey 1994 Pena 1994 Saito 1992 Yie RNA 1989 RNA DNA RNA DN A RNA CMV RNA

2 CMV RNA C MV sat55-1 DDBJ EMBL GenBank accession number AB CMV RNA sat55-1cmv-p II P 1989 CMV-42CM I Y, 1988 CMV-O n2i Y 1986 CMV-Y I Y 1981 CM32 I Y CM I Y CM I Y 1996 CMV Myzus persicae CMV RNA CMV Tochihara V W 0.5 ph6.5 V W 1:1 10,000rpm 10 6W V 6, X100 10mM ph8.0 24W V ,000rpm 90 10mM ph8.0 RNA 1 5 CMV 0.25M ph8.0 8 SDS SDS 1 TAE RNA sat55-1 CM V-P7 Leammli 1970 CMV RNA Peden and Symons, 1973 RNA Gubler and Hoffman 1983 cdna Sma puc119 Pst Sac RNA DNA NPTII pbi121 TOYOBO - GUS CaMV

3 RNA 39 pbi121xbai SacI GUS- sat55-1 puc119psti SacI XbaI PstI35 P35s 35S NOS Hochema1983 LBA4404 YEB MS MS 0.23 mm RNA CTAB Murry and Tompson, 1980 DNA PCR RNAsat55-1 DDBJ EMBL GenBank accession number AB Forward, 5'-TTTT GTTTGTTGGAG - 3 ';Reverse, 5 '- GGGTCCTGTC 1.0mg l TAGAGGA- 3' R 0.1mg l NA mg l NPT mg 5 l 20mg 0.2N NaOH ,000rpm 30

40 1772002 sat55-1 DDBJ EMBL GenBank accession number AB072502 5 l 500 l 0.1M ph8.

4 sat55-1 DDBJ EMBL GenBank accession number AB l 500 l 0.1M ph8.0 PCR PCR Forward, 5'-TCGGCTATGACTGGGCACAACAGA- 3'; Reverse,5'-AAGAAGGCGATAGAAGGCGA TGCG-3' RNA DNA sat55-1 AK1, 5'-GTTTTGTTTGTTGGAGAATTGC G-3';BK2,5'-GGGTCCTGTAGAGGAATGTC-3' PCR TA Invitrogen 310 ABI DNA SIS Ver.7.0 RNA RNA RT-PCR R NA RT-PCR BK PCR RNA 27 DNA PCR RNA T 0 T 0 RNA 27 T 0 T0 2 4 CM V-42CM No No.7 No.27 No

RNA 41 PCR Tsat55-1 174HinfI TOYOBO ;pbi121rna cdna T DNA T CMV 49 37 12

5 RNA 41 PCR Tsat HinfI TOYOBO ;pbi121rna cdna T DNA T CMV CMV-42CM T T No T CMV-42CM No. T T T T No.4 T

6 No.4T CMV-42CM T CMV

7 RNA 43 T T T 100l MS 10 CMV T No T T T T T 100mg l MS

8 PCR NPT 800bp No.4-7 RNA No.4-7 RNA RT-PCR RNA 400bp RNA No.4-7 CMV-42CM RT-PCR T No. - RNA CMV T No.4-7 No.4-7 RNACMV No.4-7 No.4-7 CMV-42CM RNA No nc T No.4 No.4 T No No.4-1 T CMV-42CM nc CMV 3:1 No.4-7 nc CMV RNA RNA nc RT- PCR No T 3 sat55-1 CMV 3 No No. RNA 4-7 No.4-7 RNA CM No.4-7 V No.4-7 No.4-7 RNA No.4-7 CMV No.4-7 RNA CMV-42CM No.4-7 DNA sat AK1 BK g ml PCR 400bp No.4-7 No sat g ml 1 g ml

9 RNA 45 PCR NPTII StyI TOYOBO-38 CMV - CMV MPTII pbi121dna No. - - CMVCMV CMV-42CM 14 -

10 CMV No. - No. 4 P T 3: T 3: CMV-42CM No. - T CMV-42CM CMV No. - nc - N M,n,Mal,St,y N M,n,Mal,St,y M,N M,n,Mal,St,y CMV-42CM Mal, St

11 RNA 47 T CMV 0.1 g 1 g 10 g 100 g CMV-42CM CMV CMV-42CM No.4-38 No.4-7 No CMV CMV CMV-4 2CM No.4-7 No CMV 2 No.4-7 RNA CMV CMV No.4-7 No.4-38 CMV-42CM 10 No.4- No.4-7 No.4-7 RNA CMV CMV CMV 1 RNA RNACMV CM32 14CM 29CM nc No.4-7

12 CMV-42CM 10 RNACMV CMV CMV-CM32 - CMV-14CM - CMV-29CM - nc 11 RNA CMV - CMV-Y nc No No.4-7 RNA CMV 2 RNA CMV-Y1981 RNA CMV-Y nc No

13 RNA 49 12RNA CMV CMV-O n2nc No.4-7 CMV-Y No.4-7 RNA CMV-Y RNA 3 RNA CMV RNA 1986 RNA CMV-O n No.4-7 No.4-7 RNA nc No.4-7 No No.4-7 CM V-O n RNA No.4-7 RNA sat55-1 T27 sat55-1 PCR 6 No C MV T Matzke1998 Chyi 1986; Alberts1989 T 2 CMV CMV T CMV No.4-7 No.4-7

14 No.4-7 No.4 No.4-1 3:1 No.4-7 RNA No.4-7 RNA RNA 6 9 CMV Masuta RNA CMV No.4-7 RNA CMV RNA 1986 RNA CMV RNA 12 RNA CMV RNA RNA No.4-7 RNA RNA RNA CMV Harrison1987 No.4-7 CMV R NA No.4-7 No.4-7 CMV No.4-7 CMV RNA sat55-1 RN A No.4-7 No.4-7 RNA RNA CMV No.4-7 RN A CMV No.4-7 Alberts, B., Bray, D., Lewis, J., Raff, M., Roberts, K. and Watson, J. D : Molecular biology of the cell. 2nd Ed. p , Garl and Publishing Inc, New York. Baulcombe, D., Saunder, G. R., Revan, M. W., Mayo, M. A. and Harrison, B. D : Expression of biologically active viral satellite RNA from the nuclear genome of transformed

15 RNA 51 plants. Nature, Chyi, Y.S., Jorgenson, R.A., Goldstein, D., Tanksley, S.D. and Loaiza-Figueroa, F : Locations and stability of Agrobacteriummediated T-DNA insertions in the Lycopersicon genome. Mol. Gen. Genet., 204, Gerlach, W. L., Llewellyn D. and Haseloff, J : Construction of a plant disease resistance gene from the satellite RNA of tobacco ring spot virus. Nature, Gubler, U. and Hoffman, B. J : A simple and very efficient method for generating cdna libraries. Gene, 25, Harrison, B. D., Mayo, M. A. and Baulcombe, D. C : Virus resistance in transgenic plants that express cucumber mosaic virus satellite RNA. Nature, 328, Hoekema, A., Hirsch, P. R., Hooykaas, P. J. J. and Schilperoort, R. A : A binary plant vector strategy based on separation of vir- and T-region of the Agrobacterium tumegaciens Ti-plasmid. Nature, 303, : : : Jacquemond, M., Ameselem, J. and Tepfer, M : A gene coding for a monomeric form of cucumber mosaic virus satellite RNA confers tolerance to CMV. Mol. Plant-Microbe Interact.,1, Laemmli, U. K : Cleavage of structural proteins during the assembly of the head of bacterophage T4. Nature London, 227, Lomonossoff, G. P : Pathogen-derived resistance to plant viruses. Ann. Rev. Phytopath. 33, Masuta, C., Komari, T. and Takanami, Y : Expression of cucumber mosaic virus satellite RNA from cdna copies in transgenic tobacco plants. Ann. Phytopath. Soc. Japan, 55, Masuta, C., Hayashi, Y., Suzuki, M., Kuwata, S., Takanami, Y. and Koiwai, A : Protective effect of a satellite RNA expressed in transgenic plants on disease incidence after inoculation of cucumber mosaic virus. Ann. Phytopath. Soc. Japan, Matzke, A.J. and Matzke, M.A : Position effects and epigenetic silencing of plant transgenes. Curr Opin Plant Biol., 1 2, McGarvey, P.B., Montasser, M.S. andkaper, J.M : Transgenic tomato plantsexpressing satellite RNA are tolerant to somestrains of cucumber mosaic virus. American Soc. Horticul. Sci., 119, Murray, M. G. and Thompton, W. F : Rapid isolation of high molecular weight plant DNA. Nucleic Acid Res., 8, Peden, K.Q.C. and Symons, R.H : Cucumber mosaic virus contain a functionally divided genome. Virology, 63, Pena, L., Trad, J., Diaz-Ruiz, J.R., McGarvey, P.B. and Kaper, J.M : Cucumber mosaic virus protection in transgenic tobacco plants expressing monomeric, dimeric or partial sequences of a benign satellite RNA. Plant Sci., 100, Roossnick, M.J., Sleat, D. and Palukaitis, P : Satellite RNAs of plant viruses. Microbiol. Rev., 56, Saito, Y., Komari, T., Masuta, C., Hayashi, Y., Kumashiro, T. and Takanami, Y : Cucumber mosaic virus-tolerant transgenic tomato plants expressing a satellite RNA. Theor. Appl. Genet., 83, Tochihara, H Some properties of chrysanthemum mild mottle virus, and comparison of this virus with cucumber mosaic virus. Ann. Phytopathol. Soc. Japan, 36, 1-10.

16 : 131, : 49, Yie, Y., Zhao, F., Zhao, S. Z., Liu, Y. Z., Liu, Y. L. and Tien, P : High resistance to cucumber mosaic virus conferred by satellite RNA and coat protein in transgenic commercial tobacco cultivar G-140. Mol. Plant-Microbe Interact., 5 6, Yie, Y. and Tien, P : Plant virus satellite RNAs and their role in engineering resistance to virus diseases. Seminars in Virology, 4, Yie, Y., Wu, Z. X., Wang, S. Y., Zhao, S. Z., Zhang, T. Q., Yao, G. Y. and Tien, P : Rapid production and field testing of homozygous transgenic tobacco lines with virus resistance conferred by expression of satellite RNA and coat protein of cucumber mosaic virus. Transgenic Research, 4, : RNA CMV.. 40, : cucumovirus R NA.. pp285.

2 カブモザイクウイルス (TuMV) ゲノム由来の遺伝子を導入した Nicotiana benthamiana における TuMV 抵抗性 CP Rp MP CP Rp MP protein-mediated resistance PMR RNA RNA mediated resistancerm

2 カブモザイクウイルス (TuMV) ゲノム由来の遺伝子を導入した Nicotiana benthamiana における TuMV 抵抗性 CP Rp MP CP Rp MP protein-mediated resistance PMR RNA RNA mediated resistancerm 2012 神奈川県農業技術センター研究報告第 155 号 1 2012 155 1) Turnip mosaic virus TuMV resistance of transgenic Nicotiana benthamiana plants containing transgenes derived from TuMV genome Ken NOMURA, Hidetoshi UEKUSA and