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1 Res. Plant Dis. 15(3) : (2009) Research in Plant Disease The Korean Society of Plant Pathology GM s ü š w Cucumber mosaic virus v y 1 Á y Á»x 1 Á * w w, 1 w y w Comparative Analysis of Coat Protein Gene of Isolates of Cucumber mosaic virus Isolated from Pepper Plants in Two GMO Environmental Risk Assessment Fields Jin Sung Hong 1, Ho Seop Park, Ki Hyun Ryu 1 and Jang Kyung Choi* Department of Applied Biology, Kangwon National University, Chuncheon , Korea 1 Division of Environment & Life Sciences, Seoul Women's University, Seoul , Korea (Received on November 2, 2009; Accepted on December 2, 2009) Twelve Cucumber mosaic virus (CMV) isolates were isolated from genetically modified (GM) and non-gm Capsicum annuum in two GM fields, Namyangju and Anseong, and their properties were investigated in this study. Coat protein (CP) gene of the CMV isolates were synthesized by RT-PCR using genus-specific primers which designed to amplify a DNA fragment of 950 bp. Purified cdna fragments were cloned into the pgem- T easy vector for sequence determination. Nucleotide sequences (internal 657 bp) of CMV isolates were compared with Fny-CMV CP sequences and there were no significant collection site specific sequence similarities found. When predicted amino acid sequences (219 amino acids) were compared with Fny-CMV CP amino acids sequences, there were 96.8% to 97.3% similarities found from Namyangju collections and 95.9% to 96.8% similarities from Anseong collections. The phylogenetic analysis with nucleotide sequences showed definite differences in CMVs which have been isolated from the two regions. Keywords : Coat protein gene, Cucumber mosaic virus, Genetically modified pepper, Nucleotide sequence, Regional difference š w 60 š (Kim Choi, 2002), ü Cucumber mosaic virus(cmv) 10 š (Kim, 1990; w wz, 2004; Choi, 2005). CMV, Pepper mild mottle virus(pmmov), Pepper mottle virus(pepmov), Broad bean wilt virus (BBWV) w vw š. CMV Bromoviridae Cucumovirus genus w t (Murphy, 1995). 30 nm x, 3 ƒ RNA ƒ š (Peden Symons, 1973). ƒ» ƒ *Corresponding author Phone) , Fax) ) jkchoi@kangwon.ac.kr š CMV ƒ 1000 š š (Douine, 1979; Peter Garcia, 2003), š ƒ swš (Choi, 2005; Cho, 2006).» w w š t y w š, ü Agrobacterium x y w w Fny- CMV CP CMV ü x š (Lee, 2009) û GM s x wš. GM s š l CMV x j y wš GM Non-GM š xw CMV p, w CMV,» Fny-CMV GM š CMV w» w w. œ.» û GM

166 홍진성 박호섭 류기현 최장경 Fig. 1. RT-PCR products from Pepper CMV isolates using CMV specific primer. M, 100 bp DNA marker; Lane 1~12, PCR product of 12 CMV field isolates.

2 166 홍진성 박호섭 류기현 최장경 Fig. 1. RT-PCR products from Pepper CMV isolates using CMV specific primer. M, 100 bp DNA marker; Lane 1~12, PCR product of 12 CMV field isolates. 포장에서 시험 재배되고 있는 CMV 내병성 유전자 변형 고추에 대하여 모자이크 병징을 나타내는 GM 고추와 Non-GM 고추로부터 2회에 걸쳐 바이러스 병징이 뚜렷 한 8개체로부터 12점의 잎을 선발 채집하였다 M 인 산완충액을 이용한 즙액접종을 Nicotiana benthamiana에 실시하였다. 바이러스가 증식된 N. benthamiana의 이병엽 을 Chenopodium amaranticolor의 잎에 접종한 후 단일병 반분리를 3회 실시하였으며, 분리된 바이러스를 다시 N. benthamiana에 증식시켜 그 이병엽을 공시재료로 이용하 였다. Fig. 2. Nucleotide sequence differences from Fny-CMV coat protein gene. Different nucleotide sequences of field collected pepper CMV isolates (1-12) were indicated by bold letters.

3 GM s ü š w Cucumber mosaic virus v 167 CMV RT-PCR j. GM š Non- GMš w CMV,» CMV š w ƒ w y w (Table 1). CMV k N. benthamiana l Total RNA w z CMV specific primer (KI2TGFKEVGFCOKPQCEKFUGSWGPEGFKHHGTGPEGUHTQO(P[%/8EQCVRTQVGKP&KHHGTGPVCOKPQCEKFUGSWGPEGUQHHKGNFEQNNGEVGFRGRRGT %/8KUQNCVGUQHVJGVYQTGIKQPUYGTGOCTMGFCUDQZGU

4 168 y Á y Á»xÁ w v sww CMV RNA3 w RT-PCR w, 12 CMV 950 bp ƒ y (Fig. 1). ƒ cdna r w pgem-t easy l j w. CMV v sww cdna r j wz EcoR I w 950 bp j» j w» w.». Fny-CMV v» š CMV w» w v sw cdna w» w. 941 bp 942 bp» y w» CMV v 3' y. v 657»,» l 218» w. û s š CMV v w»» š Fny-CMV» w (Fig. 2, 3). ƒ s š w CMV» Fny-CMV 92.9% 93.8%¾ š. ù û š s CMVƒ 96.8% 97.3%, š s CMV 95.9% 96.8% 6CDNG %QORCTKUQP QH PWENGQVKFG CPF COKPQ CEKF UGSWGPEG JQOQNQIKGUCOQPI(P[%/8CPF2GRRGT%/8KUQNCVGU 2GTEGPVCIGQHEQCVRTQVGKPKFGPVKV[ %/8KUQNCVGU C 0WENGQVKFG #OKPQCEKF (P[%/8 a CMV isolates were collected from Ansung GMO environmental risk assessement field (1-3 and 5-6; GM isolates, 4; Non-GM isolate) and Deokso GMO environmental risk assessement field (7-8 and 10-12; GM isolates, 9; Non-GM isolate). (KI 2J[NQIGPGVKE CPCN[UKU DCUGF QP PWENGQVKFG UGSWGPEG QH EQCVRTQVGKPQH(P[%/8CPF2GRRGT%/8KUQNCVGU CMV ùk ü (Table 1). p, Fig. 3, û CMV Fny-CMV 88» Lysine(K) sww s CMV Arginine(R) sww, 91» Leucine(L) sww z Valine(V) sww y. v» w m w û ƒƒ w CMV x w w y w (Fig. 4)., û GM Non-GM š 8 w ƒƒ, GM Non-GM š» ƒ q. û GM š s j w l w p w. CMV RNA3 v s ww 3' w p v w RT-PCR w, 950 bp ƒ y. PCR w j w v» m» Fny-CMV v» w. û ƒ Fny- CMV» p w» w m CMVƒ y ùkû. wr, ƒ CMV v» mw y Fny-CMV v w, û CMVƒ 96.8%

5 GM s ü š w Cucumber mosaic virus v %, š s CMV 95.9% 96.8%. p û CMV Fny-CMV 88» Lysine(K) sww CMV Arginine(R) sww, 91» Leucine(L) sww z Valine(V) sww y. ww, û š ƒƒ CMV p ùkü y. w» (KRF C00560) 21 ( y : ) w tw tw. š x Cho, J. D., Lee, S. H., Choi, G. S., Lim, T. J., Chung, B. N., Ryu, K. H. and Kim, J. S Viruses and their mixed infections on red peppers at Jeolla-do Province in 2005.(Abstr.) Plant Pathol. J. 21: Choi, G. S., Kim, J. H., Lee, D. H., Kim, J. S. and Ryu, K. H Occurrence and distribution of viruses infecting pepper in Korea. Plant Pathol. J. 21: Choi, J. K., Kim, H. J., Hong, J. S., Kim, D. W. and Lee, S. Y Identification and differentiation of Cucumber mosaic virus isolates in Korea. Korean J. Plant Pathol. 14: Choi, S. K., Choi, J. K., Park, W. M. and Ryu, K. H RT- PCR detection and identification of three species of cucumoviruses with a genus-specific single pair of primers. J. Virol. Methods 83: Douine, L., Auiot, J. B., Marchoux, G. and Archange, P Recensement d' especes vegetales sensibles au virus de la mosaique de concombre (CMV) etude bibliographique. Ann. Phytopathol. Soc. Japan. 11: w wz w. 4q. w wz 779 pp. Palukaitis, P. and Garcia-Arenal, F Cucumoviruses. Adv. Virus Res. 62: Kim, J. S. and Choi, G. S Viruses infecting pepper and their characteristics. J. Kor. Capsicum Res. Coop. 7: Kim, J. S., Kim, S. K., Choi, G. S. and Lee, M. W Virus disease incidence and symptom appearance in red pepper. Korean J. Plant Pathol. 6: Lee, Y. H., Jung, M., Shin, S. H., Lee, J. H., Choi, S. H., Her, N. H., Lee, J. H., Ryu, K. H., Peak, K. Y. and Harn, C. H Transgenic peppers that are highly tolerant to a new CMV pathotype. Plant Cell Rep. 28: Murphy, F. A Bromoviridae. In: Virus Taxonomy, ed. by F.A. Murphy, pp Springer-Verlag, Wien., Austria. Peden, K. W. C. and Symons, R. H Cucumber mosaic virus contains a functionally divided genome. Virology 53:

16-15(3)-07(최장경).fm

16-15(3)-07(최장경).fm Res. Plant Dis. 15(3) : 254-258 (2009) Research in Plant Disease The Korean Society of Plant Pathology t (Lagenaria leucantha var. gourda) w IB m Cucumber mosaic virus Áy 1 Á»x 1 Á t 2 Á * w w, 1 w y w,