2004.3.3

GMO, GMM, GMF and LMO GMO: Genetically Modified Organism GMM: Genetically Modified Microorganism GMF: Genetically Modified Food LMO: Living Modified Organism

Bt

RRS 3. 4. 1. 2.

1. 2. 3. 4. Bt

1. 2. 3. 4. European corn borer Bt corn

(Rootworm) ( ) Above images are from Iowa State University web s

Dow AgroSciences (Novel Bt Protein Developed with Pioneer Hybrid) 15 1999

(Golden rice)

2001 10

Bio (biology) Technology

Bt Technology

Bt Elasped Time: 0 Hours

(Biological vectors) (Physical methods) (Chemical methods)

(Agrobacterium Method)

DNA

(Physical method)

DNA

(Gene Gun)

1 m DNA 430 m/s

(Chemical method)

DNA

1. Bt 3. Bt 2. 4. Bt

1996 2003 ( )

1996 2003 ( ) ( Clive James, ISAAA, 2003)

Source: Clive James, 2003. 67.7 58.7 52.6 44.2 39.9 27.8 11 20.4 (30%) 16 (27%) 13.5 (26%) 10.7 (24%) 7.1 (18%) 4.4 (16%) 3.3 (25%) 47.3 (70%) 42.7 (73%) 39.1 (74%) 33.5 (76%) 32.8 (82%) 23.4 (84%) 9.5 (75%) [mha(percentage)] 2003 2002 2001 2000 1999 1998 1997 1997 2003 ( )

2001 2002 2003 ( ) 2001 % 2002 % 2003 % +/-* %* 39.1 74 42.7 73 47.3 70 +4.6 +11 13.5 26 16.0 27 20.4 30 +4.4 +28 52.6 100 58.7 100 67.7 100 +9.0 +15 * 2002 2003

2003 ( )

1996 2003 ( ) ( Clive James, ISAAA, 2003)

1996 2003 ( ) <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 0.1 <0.1 0.1 0.1 0.2 0.2 0.1 0.1 0.1 <0.1 0.4 0.3 0.2 0.2 0.1 <0.1 2.8 2.1 1.5 0.5 0.3 <0.1 0.0 3.0 4.4 3.5 3.2 3.0 4.0 2.8 1.3 0.1 13.9 13.5 11.8 10.0 6.7 4.3 1.4 0.1 42.8 39.0 35.7 30.3 28.7 20.5 8.1 1.5 (mha) 2003 2002 2001 2000 1999 1998 1997 1996

urce: Clive James, 2003. 67.7 58.7 52.6 44.2 39.9 27.8 11.0 1.7 Total <0.1 ilippines <0.1 rtugal <0.1 raine <0.1 <0.1 <0.1 ance <0.1 <0.1 <0.1 <0.1 rmany <0.1 <0.1 nduras <0.1 <0.1 lombia <0.1 <0.1 <0.1 donesia <0.1 <0.1 <0.1 <0.1 lgaria <0.1 <0.1 <0.1 <0.1 <0.1 0.1 <0.1 <0.1 exico <0.1 <0.1 <0.1 <0.1 uguay (mha) 2003 2002 2001 2000 1999 1998 1997 1996 untry 1996 2003 ( )

2001 2002 2003 ( 2001 % 2002 % 2003 % +/-* %* 35.7 68 11.8 22 3.2 6 1.5 3 0.2 < 1 39.0 66 13.5 23 3.5 6 2.1 4 0.3 1 42.8 63 13.9 21 4.4 6 3.0 4 2.8 4 0.4 1 +3.8 +10 +0.4 +3 +0.9 +25 +0.7 +33 +0.1 +33 52.6 100 58.7 100 67.7 99 +9.0 +15

1996 2003 ( )

1996 2003 ( ) Source: Clive James, 2003. 67.7 58.7 52.6 44.2 39.9 27.8 11 1.7 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 0.0 <0.1 <0.1 <0.1 <0.1 <0.1 0.0 3.6 3.0 2.7 2.8 3.4 2.4 1.2 0.1 7.2 6.8 6.8 5.3 3.7 2.5 1.4 0.8 15.5 12.4 9.8 10.3 11.1 8.3 3.2 0.3 41.4 36.5 33.3 25.8 21.6 14.5 5.1 0.5 (mha) 2003 2002 2001 2000 1999 1998 1997 1996

2001 2002 2003 ( ) 2001 % 2002 % 2003 % +/-* %* 33.3 63 9.8 19 6.8 13 2.7 5 <0.1 <1 <0.1 <1 36.5 62 12.4 21 6.8 12 3.0 5 <0.1 <1 <0.1 <1 41.4 61 15.5 23 7.2 11 3.6 5 <0.1 <1 <0.1 <1 +4.9 +13 +3.1 +25 +0.4 +6 +0.6 +20 () () 52.6 100 58.7 100 67.7 100 +9.0 +15

1996 2003 ( )

1996 2003 ( ) Source: Clive James, 2003. 67.7 58.7 52.6 44.2 39.9 27.8 11 1.7 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 5.8 4.4 4.2 3.2 2.9 0.3 <0.1 12.2 10.1 7.8 8.3 8.9 7.7 4.0 1.1 (Bt) 49.7 44.2 40.6 32.7 28.1 19.8 6.9 0.6 (mha) 2003 2002 2001 2000 1999 1998 1997 1996

1996 2003 ( ) Source: Clive James, 2003. 67.7 58.7 52.6 44.2 39.9 27.8 11 1.7 <0.1 <0.1 <0.1 1.5 2.2 1.8 2.1 1.6 0.4 <0.1 2.6 2.2 1.9 1.7 0.8 <0.1 0.0 3.1 2.4 2.1 1.5 1.3 1 1.1 0.8 3.2 2.5 2.4 2.1 1.5 2 0.2 0.0 3.2 2.2 2.5 1.4 2.1 3.6 3.0 2.7 2.8 3.5 2 1.2 0.1 9.1 7.7 5.9 6.8 7.5 7 3.0 0.3 (Bt) 41.4 36.5 33.3 25.8 21.6 15 5.1 0.5 (mha) 2003 2002 2001 2000 1999 1998 1997 1996

2001 2002 2003 ( ) 2001 % 2002 % 40.6 77 7.8 15 44.2 75 10.1 17 4.2 8 4.4 8 <0.1 <1 <0.1 <1 2003 % 49.7 73 12.2 18 5.8 8 <0.1 +/-* %* +5.5 +12 +2.1 +21 +1.4 +32 52.6 100 58.7 100 67.7 100 +9.0 +15

2003 ( )

2003 ( ) % 76 41.4 55 34 7.2 21 22 3.6 16 140 15.5 11 272 67.7 25 Source: Clive James, 2003.

90 80 70 60 50 40 30 20 10 0 SOYBEANS CORN 1996 1997 1998 1999 2000 2001 2002 2003

Produce in Taiwan Papaya and rice and so on: Just in experimental scale

(PRSV)

: 250 500

(1)

(2)

(3) / / /

(4)

GMO DNA GMO DNA : (crossing) ( )

GMO : (USDA): (Safe to Grow) (EPA): (Safe for the Environment) (FDA): (Safe to Eat)

GMO GMO GMO GMO USDA EPA FDA

~ 10-12 12 } } }

研發階段 之管制 基因改造食品研發階段之管制 a. 國科會 NSC 基因重組 實驗守則 b. 農委會 基因改造植物田間試驗管理規範 COA 基因改造動物田間試驗管理規範

研發階段及上市 之管制 基因改造食品研發及上市階段之管制 c. 環保署 EPA 遺傳工程環境用 藥微生物製劑開 發試驗研究管理 辦法 環境影響評估法 d. 衛生署 DOH 基因改造食品之安全性評估方法 基因改造食品查驗登記辦法 基因改造食品標示辦法

/ / 2~4 GMO 5%

(90.1.1)

- ) (90.1.1

- (90.1.1 )

(GMFAC) 2001 2003

National Laboratories of Foods and Drugs

(1) ( / ) (2)

National Laboratories of Foods and Drugs

(3)

National Laboratories of Foods and Drugs

(4) ( ) ( / )

National Laboratories of Foods and Drugs

(5) (6)

National Laboratories of Foods and Drugs

(7) (8)

92.3.31 Event176 06 92.3.31 Bt11 05 97.04.11 92.04.11 NK603 04 97.07.22 92.07.22 GA21 03 96.10.15 91.10.15 MON810 02 96.07.22 91.07.22 40-3-2 (RRS) 01

07 08 09 10 11 T25 TC1507 DBT418 DLL25 MON863 91.08.16 92.11.17 92.10.16 92.10.20 92.10.16 96.08.16 94.11.17 94.10.16 94.10.20 97. 10.16

Dickson 2/3

: DNA (genomic DNA) : (matrix) DNA (plasmid DNA) :

RRS RRS pepsps Partial E35S, CTP, EPSPS, partial Tnos epsps plec Partial OPF of le1 le1 pmulm2 Event-specific sequences of MON810, NK603 and ivr 1 - (bp) 4994 3870 3432 5.10 (ng) 10-9 3.96 10-9 3.5 10-9

pepspe

plec

pmulm2

DNA :

DNA

酶 (polymerase chain reaction, PCR) (microarray) (Southern blot)

grinding DNA extract DNA isolation Qualitative PCR Analysis TaqMan Chemistry Quantitative PCR Quantitative PCR analysis R Reporter Forward Primer Probe Q 5 R Q Quencher 3 5 5 3 5 Reverse Primer R Q sample 5 3 5 5 3 5

DNA DNA DNA : CTAB (cetyltrimethylammonium bromide)- DNA ( )

PCR DNA PCR

GMO labeling Nonauthorized food PCR Food Sample DNA extraction Checking by other primers GMO screening 35S promotor PCR test Positive Negative PCR tests No GMO labeling for approved GMOs Positive Negative

酶 DNA

DNA CTAB 酶

MON810 Monsanto Co., St. Louis, MO, USA GA21 Monsanto Co., St. Louis, MO, USA 35S Roundup Ready TM

PCR Genomic DNA (Maize: 50 ng) MgCl 2 dntp Primers Taq polymerase 1 PCR buffer Agarose 1X TAE buffer

DNA CTAB cetyltrimethylammonium bromide (CTAB) CTAB extraction buffer: Chloroform/isoamyl alcohol (CI): CTAB precipitation Isopropanol: DNA

DNA Spectrophotometer Model U- 2001 260/280 nm 1.7 260/230 nm 1.7~2.0 DNA 260/280 nm 1.8 260/230 nm 1.8~2.0

DNA Microplate Fluorescence Reader 50 mg CTAB DNA GA21 13.0 ng / L MON810 10.0 ng / L

GA21 G5 GGT GGA AGA GTT CAA TGT ATG G3 CCT CAC TGT TCA GCA GGT TAT Pr-act OTP m-epsps NOS-ter G5 379 bp G3

MON810 M5 GAC GAG TGC TAC CCT ACC TAC M3 CGC CCA ACA ACA AGA TCA AAG E35S pro hsp70 intron cryia zea mays M5 452 bp M3

Zel 1-5 CCT CAG TCG CAC ATA TCT ACT ATA CT Zel 1-3 CTA GAA TGC AGC ACC AAC AAA maize 508 bp G5 GGT GGA AGA GTT CAA TGT ATG G3 CCT CAC TGT TCA GCA GGT TAT GA21 379 bp M5 GAC GAG TGC TAC CCT ACC TAC M3 CGC CCA ACA ACA AGA TCA AAG MON810 452 bp

酶 Qualitative Multiplex PCR Program Ta gradient thermal cycler PCR Express, Thermo Hybaid, UK 55.1 ~70.2 Ta

379 bp 452 bp GA21 MON810 酶 2 L PCR 1-12 55.1, 55.5, 56.3, 57.7, 59.4, 61.4, 63.3, 65.3, 67.6, 69.0, 69.7, 70.2 Mr Bio-100 TM DNA ladder

酶 95 95 95 10 0.5 72 0.5 72 72 65 1 1 7 62 1 1 10 cycles 30 cycles

379 bp 452 bp GA21 MON810 酶 5 L PCR Lanes 1-3 non-gm, GA21 MON810 Lane 4-5 non-gm GM Lane 6 negative control no DNA Mr Bio-100 TM DNA ladder

酶 G5 /G3, M5 /M3 Zel 1-5 /Zel 1-3 GA21, MON810 zein 2 L PCR (508 bp) zein Lane 1-3 non-gm, GA21 MON810 Lane 4 (452 bp) MON810 50% Lane 5-6 non-gm GM (379 bp) GA21 Lane 7 negative control no DNA Mr Bio-100 TM DNA ladder

酶 0.1% 0.1% 379 bp 452 bp GA21 MON810 酶 5 L PCR Lane 1-10 100%, 50%, 25%, 12%, 5%, 1%, 0.5%, 0.1%,0.05% 0.01% GM Lane 11 negative control no DNA Mr Bio-100 TM DNA ladder

(0.2 mol/l) 酶 (508 bp) zein (452 bp) MON810 (379 bp) GA21 G5 /G3, M5 /M3 Zel 1-5 /Zel 1-3 GA21, MON810 zein 2 L PCR Lane 1-10 100%, 50%, 25%, 12%, 5%, 1%, 0.5%, 0.1%, 0.05% 0.01% GM Lane 11 negative control no DNA Mr Bio-100 TM DNA ladder

(Zel 1-5 /Zel 1-3 0.2 mol/l G5 /G3 0.2 mol/l M5 /M3 0.1 mol/l ) 酶 1% (508 bp) zein (452 bp) MON810 (379 bp) GA21 G5 /G3, M5 /M3 Zel 1-5 /Zel 1-3 GA21, MON810 zein 2 L PCR Lane 1-10 100%, 50%, 25%, 12%, 5%, 1%, 0.5%, 0.1%, 0.05% 0.01% GM Lane 11 negative control no DNA Mr Bio- 100TM DNA ladder

CTAB DNA Multiplex PCR 1%

PCR DNA DNA PCR PCR Ct 33 31 29 27 25 23 21 y = -3.2836x + 24.847 R 2 = 0.9564-2 -1.5-1 -0.5 0 0.5 Log (GMO level) Ct 26 24 22 20 18 y = -3.6262x + 23.84 16 R 2 = 0.9555 14-0.5 0 0.5 1 1.5 2 2.5 Log (lectin level)

target DNA standard DNA (linearized plasmid) Amplification + primers +dntps +Taq DNA polymerase Standard PCR product Target PCR product Gel electrophoresis standard DNA band Target DNA band standard DNA target DNA PCR QC-PCR Equivalence point: [target]=[standard]

Polymerization 5 3 5 Strand Displacement Forward Primer R R Probe Probe Q Q 5 3 5 Reverse Primer R: Reporter Q: Quencher Cleavage R Probe Q TaqMan ABI PRISM 7700 Polymerization completed R Probe Q Sequence

PCR

PCR Cycle No. GMO Standard DNA in Real-time Q-PCR Assay 6000 5000 0.10% 0.20% 0.50% 2.00% 4000 FIU 3000 2000 1000 0 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39

DNA (the modified gene) 1 1 2 DNA 3 3 4 4

: Primer Premier 5 (PREMIER Biosoft Int., Palo Alto, CA, USA) : Primer Express (Applied Biosystems, St. Louis, MO, USA)

(5-3 ) bp RR-35S F RR-35S R TCTTGTGCTGATGAAGGTATGTCC TCGGCAGAGGCATCTTCAA Soybean genome/p35s 235 LE103 LE104 GCCCTCTACTCCACCCCCATCC GCCCATCTGCAAGAATTTTTGTG lectin gene 118 EPSPS-B1 NOS-3 TGATGTGATATCTCCACTGACG TTATCCTAGTTTGCGCGCTA P35S/CTP/ epsps/tnos 1977 Re21f Re21r GTGGTTGTATCTCTCTCCCTA ACTTTCTTCCTTCGATCTGTC lectin gene 853 24f CCTCGTGTCGGAAAACCCTGTCAC 24r GCTCATCAGGCAGCCTTCGTATCG epsps 132 24p 5 FAM-CCGGGCTGGGCGCGAAGATCGAACT-TAMRA 3 Sltm1 AACCGGRAGCGTTGCCAG Sltm2 AGCCCATCTGCAAGCCTTT lectin gene 81 Sltmp 5 VIC-TTCGCCGCTTCCTTCAACTTCACCT-TAMRA 3

(5-3 ) bp M810mzpF3 M810mzpR3 M810p TCTTGTGCTGATGAAGGTATGTCC TCGGCAGAGGCATCTTCAA 5 FAM-TTTGCCATTGCCCAGCTATCTGTCAC- TAMRA 3 Maize genome/ P35S 223 Nk603mzpF Nk603mzpR Nk603p CGGCCAGCAAGCCTTGTA TCCCGACTCTCTTCTCAAGCA 5 FAM-CCGCGTTAACAAGCTTACTCGAGGTC ATTC-TAMRA 3 Maize genome/ P-ract 1 113 ivr-f ivr-r ivr-p TGGCGGACGACGACTTGT AAAGTTTGGAGGCTGCCGT 5 VIC-CGAGCAGACCGCCGTGTACTTCTACC- TAMRA 3 Invertase gene Exon 5 79 Zel 1-5 Zel 1-3 CCTCAGTCGCACATATCTACTATACT CTAGAATGCAGCACCAACAAA zein gene 508

PCR 1 TaqMan TM Universal PCR master mix buffer 1 TaqMan buffer A 5 mm MgCl 2 8% glycerol 200 mm dntp (datp, dctp, dgtp) 400 mm dutp 0.125 units Amperaes uraxil-n-glycosylase (UNG) 0.3 units AmpliTaq Gold DNA polymerase 200 nm passive reference dye ROX (6-carboxyrhodamine) UNG 50 2 min 95 95 10 min 15 s 60 1 min

a) (b) (a) ivr 1-F/R;(b) NK603mzpF/R 1310720, 20480, 1280, 80, 20 pmulm2

, C V PCR C V CV (%) n RRS epsps 1.44 0.04 3.00 4 MON810 Host/E35S 0.25 0.09 11.81 4 NK603 Host/P-ract1 0.73 0.01 3.80 4

PCR PCR RRS 24F/24R Sltm1/sltm2 100 100 PCR PCR (%) (nm) 96 97 ON810 M810mzpF3/M810mzpR3 ivr-f/ivr-r 200 200 94 93 NK603 NK603mzpF/NK603mzpR ivr-f/ivr-r 200 200 93 93

PCR Vaïtilingom et al.,1999 Höhne et al., 2002 Holck et al., 2002 Alary et al., 2002 Brodmann et al., 2002 This study PCR 84%~119% 83%~105% 90%~105% 76%~100% 83%~93% 93%~97%

s (%) CV (%) (% ïtilingom et al.,1999 Bt176 Seeds 0.01~100 5~20 - rdal and Holst-Jensen, 01 RRS Seeds - 15~32.7-10~28. hne et al., 2002 E35S Seeds - 19~40-3~41 lck et al., 2002 MON810 - ary et al., 2002 RRS Bt176 Seeds - 3.6~17.23-50~55. odmann et al., 2002 MON810,Event176, Bt11, T25, GA21 Seeds 0.1~100 5~20-10~13 ribara et al., 2002 indo et al., 2002 13 laboratories from pan, Korea, USA) MON810,Event176, Bt11, T25, GA21, and RRS Plasmids 0.5~100 0.5~100 6.2 5.8~32.3-3.2~46. -9.0~38. RRS is study MON810 Plasmids 0.5~100 0.33~17.15-11~22 NK603 V: coefficient of variation.

(enzymelinked immunosorbant assay, ELISA) (Western blot) (Lateral flow strip)

(Lateral flow strip)

(near infrared spectroscopy, NIR) : ( RR ) (chromatography) : GMO (high performance liquid chromatography, HPLC) (chemical ionization mass spectrometry)

PCR ( ) PCR ( ) ELISA RRS Event 176 RRS Event 176 RRS Event 176 RRS Event 176 Bt 11 MON 810 T25

(1%) (1%) (3%) (5%) (5%)

Thank you for your attention Thank you for your attentio

Tel: (02)23630231-3813 Fax: (02)23627044 E-mail: tmpan@ntu.edu.tw Web: http://bst013.bst.ntu.edu.tw/panlab/