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1 Res. Plant Dis. 15(3) : (2009) Research in Plant Disease The Krean Sciety f Plant Pathlgy y ù w (Cry1Ac1) w w ³ ûz 1,2 Á y 2 Á 2 Á 2 Á«3 Á½ š 4 Á z 1,2 * 1 w y w œw, 2 w, 3 w, 4 w y w tœw Evaluatin f Disease Resistance f a Leafflder-resistant (Cry1Ac1) Rice Event and Gene Transfer t Plant Pathgens Hy Sng Nam 1,2, Hng-Sik Shim 2, Sang-Mi Yu 2, Se Wn Lee 2, Sn Jng Kwn 3, Myung-KnG Kim 4 and Yng Hn Lee 1,2 * 1 Divisin f Bitechnlgy, Chnbuk Natinal University, Dekjin-dng, Jenju, Jenbuk Krea 2 Agricultural Micrbilgy Divisin, Natinal Academy f Agricultural Science, RDA, Suwn , Krea 3 Bisafety Divisin, Natinal Academy f Agricultural Science, RDA, Suwn , Krea 4 Bi Fd Technlgy, Chnbuk Natinal University, Dekjin-dng, Jenju, Jenbuk , Krea (Received n January 1, 2009; Accepted n Nvember 14, 2009) The genetically mdified leafflder-resistant (Cry1Ac1) rice plant was evaluated fr the changes f resistance by cmparing the ccurrence f majr diseases with a japnica type Krean rice variety, Nakdng which was the mther plant f the transgenic rice event, in greenhuse and field cnditins. There was n difference in the ccurrence f sheath blight and Helminthsprium blight between the tw varieties in the fields. We culdn't find any difference f resistance fr fungal blast and bacterial leaf blight by artificial inculatin in greenhuse. There was als n difference in the susceptibility t sheath blight in artificial inculatin tests cnfirming the results in the fields. The pssibility f gene transfer f Bar and Cry1Ac1 frm the genetically mdified rice plant t naturally infected pathgens such as Fusarium mnilifrme and Pyricularia ryzae in the field cnditins was tested by PCR. And the pssible transfer f thse genes by cntinuus inculatin f Xanthmnas ryzae pv. ryzae and Rhizctnia slani was als tested. Hwever, we culdn't find any pssibility f transfer f the genes in natural and artificial cnditins. Keywrds : Event, Gene transfer, GMO, Leafflder, g, x x ù s š, w x yw ƒ» w ù ù w ö e ƒ,. x t p p ƒ š. w x (GMO; genetically mdified rganisms) p *Crrespnding authr Phne) , Fax) ) ynghnlee@jbnu.ac.kr p w wùwù sƒ w v, y w w w mƒ w. y ù w w» j, m 7 w l» w, s 8 w l vwƒ w ùkù, vw š ú ƒ p. y ù» w w»ƒ z ƒ û š, š, w z ƒ û p w w

2 y ù w (Cry1Ac1) w w ³ 203 ƒ ƒ z š w. y ù w Bt w w {w t s w sƒƒ» w (Ye, 2003). ü w ü m ü w z ü z x ü» x(50% ) g x y. x s x f ³ w w ù w w wš, w x w ü ù w ƒ w ƒ» š (Smalla, 1997; Davis, 1994). ƒ ³ ³(Mazdier, 1989; Trieu-Cut, 1987), ³ z (Heinemann Sprague, 1989), s s s s(curvalin, 1995) s s(hykaas, 1989) w šƒ. w» w, y l ³ ƒ ƒ š (Smith, 1992; Dlittle 1990). w, ƒ ³ x ¾ x (Brer, 1996). ù, w ƒ, r w ƒ w š w (Smith, 1991). x y ù w ù w t w w w w s w 3 w š, w x w f ƒ ³ w.. y ù w w» w ù w» w x (Genetically mdified plants) w s w w s, û û» s y ù w ù w» w w. w. y ù w ù w w w y w» w ³ (Bacterial leaf blight) ³ (Sheath blight) (Fungal blast) w w w. ³(X.. pv. ryzae) PSA(peptne sucrse agar) 28 C 2 z 10 mm MgCl 2 z OD 600 =0.5( 10 8 cfu/ml) w»» ƒ w. ³(Pyricularia grisea) w x ew (KJ101, KJ105, KJ107, KJ201, KJ301, KJ401) x ew (KI1113, KI1117, KI207, KI409) w s k ³ s 5 10 ƒƒ w z 5 1 w. w 2 z k w. wr, ³ PDA (ptat dextrse agar) 10 z x ³ ³w w» w z w. w. ³, ³ j ³ 50 ml w 25 C 7 z w cheese clth m w ³ yw w ƒ ³ w ³ ywš, micrcentrifuge tube ¼ z ƒ tube ³ 1g 1ml 2 CTAB (2% cetyltrimethylammnium brmide, 1.4 M NaCl, 20 mm EDTA, 100 mm Tris-HCL (ph8.0) š 55 C 1 w z 8,000 rpm 4C 15 w. phenl/chlrfrm/isamyl alchl (25:24:1, v/v/v) 2z w z d isprpanl ƒw DNA e k 70% ethanl DNA e š 100 ug RNase Aƒ 0.5 ml TE (1 mm EDTA, 10 mm Tris (ph8.0)) ww z 37 C 1 w RNA ww phenl/chlrfrm/isamyl alchl (25:24:1, v/v/v) 2z š chlrfrm 1z w d ethanl ƒw DNA e k z 70% ethanl DNA e z 20 C w. ³ PSA 2 z ³ ü 2ml micrcentrifuge š NaCl 3z ü z bacterial gdna kit w z w. ³ peptne sucrse agar (1% plypeptne, 1% sucrse and 0.2% L-glutarmate)ù LB 28 C 2 w z ³ z w.

3 204 ûz Á y Á Á Á«Á½ šá z ³ 5M NaCl w z SDS prteinase K ƒƒ 2% 250 ug/ml w w Tris-EDTA (ph 8.0) 600 µl xkw. xk 37 C 1 w z 5M NaCl 100 µl ƒw z. Cetyltrimethylammniumbrmide (CTAB)-NaCl 8µl ƒw z 65 C 10 w z DNA phenl chlrfrm isamyl alchl (25:24:1) w w. p, x y ³ w w» w ³ ³ y ù w ù w z 15 z w. 5~6z w» w ƒƒ w w w z PCR mw Cry1Ac1 Bar w. PCR(Plymerase Chain Reactin). y ù w (Cry1Ac1) f w (Bar) ³ w» w s w ³ w w x w PCR w. Cry1AC1 y w» w Cry1Ac1F-BHI 5' (5'-ga ctg gat CCA TGG ACA ACA ACC CAA AC-3') Cry1Ac-XhI 3' (5'-g act ctc gag TTA AAG ATT GTA CTC AGC-3') v w š, Bar y w» w BarF (5'-ATG AGC CCA GAA CGA CGC CCG-3') BarR (5'-TCA GAT CTC GGT GAC GGG CAG-3') v w. PCR w DNA 1 Taq buffer, 2 mm MgCl 2 ƒƒ dntp 0.25 mm 2 U EX-taq wz (Takara) 25 ulƒ yww z 94 C 1 k z 94 C 45, 55 C 45 w 72 C 45 29z w z 72 C 10 g. w w z 0.8% agarse gel» w ethidium brmide z w. š w. x š w x ƒ y š, 2007 x 23, xpù,, eù,, ã ƒw š. 2007» 75%, g 90% x š, ù x y w ƒ w š. w, x w y w w ƒ w d wš., y ù w w w w y y w» w ù w w GMO s (R. slani), 6CDNG&KUGCUGUQEEWTTGFKPVJGHKGNFUKPCPF Disease Species Surveyed % Occurrence a Level date Plants Leaves Panicles Sheath blight Rhizctnia slani ' " ' Leaf blast Pyricularia grisiae ' <1 <0.1 - " ' <1 <0.1 Panicle blast " ' <1 <0.1 " ' <1 <0.1 Brwn spt Biplaris ryzae ' <0.1 - " ' <0.2 False smut Ustilaginidea virens ' Bakanae disease Fusarium mnilifrme ' < Bacterial leaf blight X. pv. ryzae ' Bacterial grain rt Burkhlderia glumae ' " ' <1 - <0.1 a The ccurrence f the disease was surveyed in the islated fields fr the experiment f genetically mdified plants in Suwn, Gyenggi and Yesan, Chungnam, 2006 and 2007, respectively.

4 (P. grisiae),, Á (Biplaris ryzae), (Ustilaginidea virens), j (Fusarium mnilifrme), (X.. pv. ryzae), ³ (Burkhlderia glumae) š, 2007 û s w,, Á, ³ wƒ (Table 1). wr 2008 w j ù, sƒ w w w» š, w, û s w w w» š w. x s w w t w wš w. ƒ ƒ w 2006 y ù w t ù x ƒ w (Fig. 1), 2007 ù (KI %QORCTKUQP QH UJGCVJ DNKIJV QEEWTTGPEG DGVYGGP NGCHHQNFGTTGUKUVCPVTKEG%T[#ECPF0CMFQPI6JGQEEWTTGPEG QH VJG FKUGCUG YCU UWTXG[GF KP VJG KUQNCVGF HKGNF HQT VJG GZRGTKOGPVQHIGPGVKECNN[OQFKHKGFRNCPVUKP5WYQP y ù w (Cry1Ac1) w w ³ 205 w ù, t (data nt shwn). s Á t w w (Fig. 2). w. ³ w w» w K1, K2 K3»» w w ƒƒ, y ù w t ù j ƒ (Fig. 3). w, ³ w w» w x ew (KJ101, KJ105, KJ107, KJ201, KJ301, KJ401) x ew (KI1113, KI1117, KI207, KI409) yww» w w w t w ƒ (data nt shwn).. x ù wš 1%, g 11% w 3 š wš, ü š 95% wš.» w ù w. w w x y ƒ š, x Ÿ w y w w d w f w ƒ y ƒ. nptii ƒ x y DNA Acinetbacter sp. BD413 x y k ƒ š ƒ ù(gebhard Smalla, 1998), y l ³ (KI %QORCTKUQP QH *GNOKPVJQURQTKWO DNKIJV QEEWTTGPEG DGVYGGP NGCHHQNFGTTGUKUVCPV TKEG %T[#E CPF 0CMFQPI 6JG QEEWTTGPEGQHVJGFKUGCUGYCUUWTXG[GFKP;GUCP (KI4GUKUVCPEGVQVJGTCEGUQHDCEVGTKCNNGCHDNKIJVECWUGFD[: Q RX QT[\CG'CEJ RNCPV YCU KPQEWNCVGF D[ UEKUUQT ENKR KPQEWNCVKQPOGVJQF#RCKTQHUEKUUQTUYCUFKRRGFKPVQDCEVGTKCN UWURGPUKQPQHGCEJTCEGEC EHWONCPFNGCHVKRUYGTGEWV EO DGNQY VJG VKR 6JG KPQEWNCVGF RNCPV YCU UVQTGF KP VJG ITGGPJQWUGCPFVJGRKEVWTGYCUVCMGPYGGMUCHVGTKPQEWNCVKQP

5 206 남효송 심홍식 유상미 이세원 권순종 김명곤 이용훈 을지는 확실하지 않다. 한편 식물체의 DNA는 부식된 식 물잔해에서 토양으로 방출될 수 있는 우려가 있고(Widmer, 1997), 토양입자에 도입된 DNA는 어느 정도 nuclease의 분해로부터 보호되어(Lrenz와 Wackernagel, 1994; Khanna 와 Sttzky, 1992), 농경지에는 지속적으로 형질전환 식물 체로부터 DNA가 농축될 가능성이 있다. 일반적인 토양 조건에서 자연 형질전환의 선결조건은 생태계에 존재하 는 자유 DNA(free DNA)의 이용 가능성, 형질전환 능력 의 획득과 획득한 DNA가 세균 게놈으로 안전적으로 도 입되는 것으로, 이러한 DNA와 형질전환 능력이 있는 세 균을 이용하여 형질전환 식물체의 DNA를 세균에 도입시 키려는 많은 시도가 있었으나 아직까지 성공된 사례는 없 다(Nielsen 등, 1997; Brer 등, 1996). 본 실험에서는 혹명나방저항성벼에서 병원균으로 유전 자가 전이되는지를 알아보기 위하여 포장에서 발병한 도 열병균과 키다리병균을 분리하여 DNA를 분리한 후 PCR 을 통해 내충성유전자(Cry1Ac1) 및 제초제저항성 유전자 (Bar)의 전이여부를 조사하였다. 조사결과 각각의 저항성 유전자는 병원균으로 전이되지 않은 것으로 확인되었다 (Fig. 4). 한편, 키다리병균의 DNA를 이용하여 제초제 저 항성 유전자의 전이여부 조사시 비특이적인 PCR 밴드가 형성되었는데, 이는 저항성 유전자와는 관련이 없었다. 식물의 유전자가 세균으로 전이되었다는 것이 현재까 지 실험적으로 증명된 적은 없을 지라도, 그 가능성을 부 인 할 수는 없다. 본 실험에서는 포장에서 자연 발병한 병원균 이외에 벼의 대표적인 병해인 도열병균과 흰잎마 름병균이 여러세대를 거쳐 혹명나방저항성벼와 계속적으 로 접촉하였을 때 저항성 유전자가 전이되는지의 여부를 Fig. 4. PCR prducts fr the detectin f the transfer f Cry1Ac1 (left panel) and Bar (right panel) gene frm leafflder-resistant rice plant t plant pathgens. The fungus F. mnilifrme (upper rw) and P. grisea (lwer rw) was islated frm naturally infected rice plants in the field. The PCR was perfrmed using the primers specific fr Cry1Ac1 and Bar gene. M; DNA size marker, Cn; psitive cntrl. Fig. 5. PCR prducts fr the detectin f the transfer f Cry1Ac1 (left) and Bar (right) gene frm leafflder-resistant rice plant t plant pathgens. The R. slani (upper rw) and X.. pv. ryzae (lwer rw) was inculated and re-islated cnsecutively at 15 day interval frm each rice plant. M; DNA size marker, Cn; psitive cntrl.

6 y ù w (Cry1Ac1) w w ³ 207 y w» w ³ ³ 15 w. 5~6z w ³ l DNA w ü (Cry1Ac1) w (Bar) ƒ ³ w y (Fig. 5). ³ ³ z w šƒ ù, ü ƒ ³ x ¾ x (Brer, 1996). x w ù w ƒ k w ³ y. ³ w w x y f š. ³ ƒ w w l ³ ƒ w, w w x w ƒ. ¾ l ³ ƒ û x ù, x y(natural transfrmatin), ³ k w (free DNA) DNAƒ ³ wù, x m ³ 40 x y š (Lrenz Wackernagel, 1994). w, k m ü ³ DNAƒ x y ³ ƒ š, m w w ƒ (Nielsen, 1997; Lrenz Wackernagel, 1994; Khanna Sttzky 1992). š w x w w ƒ ù, w v w. x y ù w w w w y s ù w. s Á j ƒ. ³ w w w y w t j š, w w t w s w. x y w (Bar ) y ù w (Cry1Ac1 )ƒ ³ ƒ w» w s w j ³ w z DNA w PCR w ³ y. w, ³ w w ƒ y w» w ³ ³ w z DNA w w w ù y, x k. š x Brer, I., Drge-Laser, W. and Gerke, M Examinatin f the putative hrizntal gene transfer frm transgenic plants t agrbacteria. In: Transgenic Organisms and Bisafety (Schmidt, E.R. and Hankeln, T., Eds.), pp Springer Verlag, Berlin. Curvalin, P Gene transfer frm bacteria t mammalian cells. C. R. Acad. Sci. Ser. III Sci. Vie. 318: Davies, J Inactivatin f antibitics and the disseminatin f resistance genes. Science 264: Dlittle, R. F., Feng, D. F., Andersn, K. L. and Alberr, M. R A naturally ccurring hrizntal gene transfer frm a eukaryte t a prkaryte. J. Ml. Evl. 31: Gebhard, F. and Smalla, K Transfrmatin f Acinetbacter sp. BD413 by transgenic sugar beet DNA. Appl. Envirn. Micrbil. 64: Heinemann, J. A. and Sprague, G. F Bacterial cnjugative plasmids mbilize DNA transfer between bacteria and yeast. Nature 340: Hykaas, P. J. J Transfrmatin f plant cells via Agrbacterium. Plant Ml. Bil. 13: Khanna, M. and Sttzky, G Transfrmatin f Bacillus subtilis by DNA bund n mntmrillnite and effect f DNase n the transfrming ability f bund DNA. Appl. Envirn. Micrbil. 58: Lrenz, M. G. and Wackernagel, W Bacterial gene transfer by natural genetic transfrmatin in the envirnment. Micrbil. Rev. 58: Mazdier, P., Petter, R. and Thmpsn, C Intergeneric cnjugatin between Escherichia cli and Streptmyces species. J. Bacteril. 171: Nielsen, K. M., Gebhard, F., Smalla K., Bnes, A. M. and Van Elsas, J. D Evaluatin f pssible hrizntal gene transfer frm transgenic plants t the sil bacterium Acinetbacter calcaceticus BD413. Ther. Appl. Genet. 95:

7 208 ûz Á y Á Á Á«Á½ šá z Nielsen, K. M., van Weerelt, D. M., Berg, T. N., Bnes, A. M., Hagler, A. N. and van Elsas, J. D Natural transfrmatin and availability f transfrming DNA t Acinetbacter calcaceticus in sil micrcsms. Appl. Envirn. Micrbil. 63: Smalla, K., Wellingtn, E. M. H. and van Elsas, J. D Natural backgrund f bacterial antibitic resistance genes in the envirnment. In: Nrdic Seminar n Antibitic Resistance Marker Genes and Transgenic Plants, pp Nrwegian Bitechnlgy Advisry Bard, Osl. Smith, M. W., Feng, D.-F. and Dlittle, R. F Evlutin by acquisitin: the case fr hrizntal gene transfers. Trends Bichem. Sci. 17: Smith, J. M., Dwsn, C. G. and Spratt, B. G Lcalized sex in bacteria. Nature 349: Trieu-Cut, P., Carlier, C. and Curvalin, P Plasmid transfer by cnjunctin frm Escherichia cli t gram-psitive bacteria. FEMS Micrbil. Lett. 48: Widmer, F., Seidler, R. J., Dnegan, K. K. and Reed, G. L Quantificatin f transgenic plant marker gene persistence in the field. Ml. Ecl. 6: 1-7. Ye, G. Y., Ya, H. W., Shu, Q. Y., Cheng, X., Hu, C., Xia, Y. W., Ga, M. W. and Altsaar, I High levels f stable resistance in transgenic rice with a cry1ab gene frm Bacillus thuringiensis Berliner t rice leafflder, Cnaphalcrcis medinalis (Guenee) under field cnditins. Crp prtectin 22: