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9 years ago
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3 GMO, GMM, GMF and LMO GMO: Genetically Modified Organism GMM: Genetically Modified Microorganism GMF: Genetically Modified Food LMO: Living Modified Organism

4 Bt

11 RRS

12 Bt

13 European corn borer Bt corn

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

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

17 (Golden rice)

23 Bio (biology) Technology

26 Bt Technology

27 Bt Elasped Time: 0 Hours

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

29 (Agrobacterium Method)

30 DNA

31 (Physical method)

32 DNA

33 (Gene Gun)

35 1 m DNA 430 m/s

36 (Chemical method)

40 DNA

42 1. Bt 3. Bt Bt

43 ( )

44 ( ) ( Clive James, ISAAA, 2003)

45 Source: Clive James, (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)] ( )

46 ( ) 2001 % 2002 % 2003 % +/-* %* *

47 2003 ( )

48 ( ) ( Clive James, ISAAA, 2003)

49 ( ) <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 < < < < < (mha)

50 urce: Clive James, 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 exico <0.1 <0.1 <0.1 <0.1 uguay (mha) untry ( )

51 ( 2001 % 2002 % 2003 % +/-* %* <

52 ( )

53 ( ) Source: Clive James, <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 < (mha)

54 ( ) 2001 % 2002 % 2003 % +/-* %* <0.1 <1 <0.1 < <0.1 <1 <0.1 < <0.1 <1 <0.1 < () ()

55 ( )

56 ( ) Source: Clive James, <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 < < (Bt) (mha)

57 ( ) Source: Clive James, <0.1 <0.1 < < < (Bt) (mha)

58 ( ) 2001 % 2002 % <0.1 <1 <0.1 < % <0.1 +/-* %*

59 2003 ( )

60 2003 ( ) % Source: Clive James, 2003.

61 SOYBEANS CORN

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

63 (PRSV)

67 :

70 (1)

71 (2)

72 (3) / / /

73 (4)

74 GMO DNA GMO DNA : (crossing) ( )

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

76 GMO GMO GMO GMO USDA EPA FDA

78 ~ } } }

79 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

80 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

86 / / 2~4 GMO 5%

90 (90.1.1)

91 - ) (90.1.1

92 - ( )

93 (GMFAC)

95 National Laboratories of Foods and Drugs

96 (1) ( / ) (2)

97 National Laboratories of Foods and Drugs

98 (3)

99 National Laboratories of Foods and Drugs

100 (4) ( ) ( / )

101 National Laboratories of Foods and Drugs

102 (5) (6)

103 National Laboratories of Foods and Drugs

104 (7) (8)

105 Event Bt NK GA MON (RRS) 01

106 T25 TC1507 DBT418 DLL25 MON

107

108 Dickson 2/3

109

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

111 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) (ng)

112 pepspe

113 plec

114 pmulm2

115

116 DNA :

117 DNA

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

119 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 Reverse Primer R Q sample

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

121 PCR DNA PCR

122 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

123 酶 DNA

124 DNA CTAB 酶

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

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

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

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

129 DNA Microplate Fluorescence Reader 50 mg CTAB DNA GA ng / L MON ng / L

130 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

131 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

132 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 GA bp M5 GAC GAG TGC TAC CCT ACC TAC M3 CGC CCA ACA ACA AGA TCA AAG MON bp

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

134 379 bp 452 bp GA21 MON810 酶 2 L PCR , 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

135 酶 cycles 30 cycles

136 379 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

137 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

138 酶 0.1% 0.1% 379 bp 452 bp GA21 MON810 酶 5 L PCR Lane %, 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

139 (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 %, 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

140 (Zel 1-5 /Zel 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 %, 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

141 CTAB DNA Multiplex PCR 1%

142 PCR DNA DNA PCR PCR Ct y = x R 2 = Log (GMO level) Ct y = x R 2 = Log (lectin level)

143 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]

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

145 PCR

146 PCR Cycle No. GMO Standard DNA in Real-time Q-PCR Assay % 0.20% 0.50% 2.00% 4000 FIU

147 DNA (the modified gene) DNA

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

149 (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 f CCTCGTGTCGGAAAACCCTGTCAC 24r GCTCATCAGGCAGCCTTCGTATCG epsps p 5 FAM-CCGGGCTGGGCGCGAAGATCGAACT-TAMRA 3 Sltm1 AACCGGRAGCGTTGCCAG Sltm2 AGCCCATCTGCAAGCCTTT lectin gene 81 Sltmp 5 VIC-TTCGCCGCTTCCTTCAACTTCACCT-TAMRA 3

150 (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 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

151 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 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 min 15 s 60 1 min

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

153 , C V PCR C V CV (%) n RRS epsps MON810 Host/E35S NK603 Host/P-ract

154 PCR PCR RRS 24F/24R Sltm1/sltm PCR PCR (%) (nm) ON810 M810mzpF3/M810mzpR3 ivr-f/ivr-r NK603 NK603mzpF/NK603mzpR ivr-f/ivr-r

155 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%

156 s (%) CV (%) (% ïtilingom et al.,1999 Bt176 Seeds 0.01~100 5~20 - rdal and Holst-Jensen, 01 RRS Seeds - 15~ ~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~ ~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., laboratories from pan, Korea, USA) MON810,Event176, Bt11, T25, GA21, and RRS Plasmids 0.5~ ~ ~ ~ ~38. RRS is study MON810 Plasmids 0.5~ ~ ~22 NK603 V: coefficient of variation.

157

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

159 (Lateral flow strip)

160

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

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

163 (1%) (1%) (3%) (5%) (5%)

164 Thank you for your attention Thank you for your attentio

165 Tel: (02) Fax: (02) Web:

SDS 1.3

SDS 1.3 Applied Biosystems 7300 Real-Time PCR System (With RQ Study) SDS 1.3 I. ~ I. 1. : Dell GX280 2.8GHz with Dell 17 Flat monitor 256 MB RAM 40 GB hard drive DVD-RW drive Microsoft Windows XP Operating System