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2 類不兩都理來類利良例說例

3 良 1) 利來不理料省, 率 2) 益連 3) ; 4) 行便 F 1 F 2 異 5) (epistasis) (pleiotropy)

4 Lettuce Linkage Map From: Landry et al Genetics 116:335

5 利來 (1) 異 (protein and isozyme electrophoresis) (2) DNA 切 (Restriction fragment length polymerphisms, RFLPs) (3) 連 (Arbitrarily primed polymerase chain reaction) DNA (Random amplified polymorphic DNA; RAPD)

6 異流異流

9 - 精異

12 切切說 A bb probe A B A B

13 流 DNA DNA DNA extraction cdna library Genomic library 切 Enzyme digestion DNA Gel electrophoresis Probe cloning and preparation Southern blotting DNA Pre-hybridization DNA 識 Probe labeling Hybridization Detection. 切 RFLP 流

and preparation Southern blotting DNA Pre-hybridization

14 切 Soybean DNA Restriction Fragment Length Polymorphisms (RFLPs)

16 連 PCR 流 50mM KCl, 10mM Tris-HCl ph8.3, DNA template DNA 1.5mM MgCl 2, 0.01% gelatine dntp each 200uM(pH 7.0) primer pmole DNA template DNA 1 ug-30 ng DNA polymerase 2.5U/100 ul 100 ul PCR cycles DNA denaturation DNA Primer extention Primer annealing 煉. 連 PCR 流

0) primer 5-100 pmole DNA template DNA 1 ug-30 ng DNA polymerase 2.

17 Random Amplified Polymorphic DNA(RAPD)

19 (FDA)

25 流 (Cell Flow Cytometry) PMT 5 PMT 4 Sample Flow cell Dichroic Filters PMT 2 PMT 3 Scatter Sensor Bandpass Filters PMT 1 Laser

27 Utilization of Genetic Transformation in Broccoli (Brassica olercea var. italica) for Postharvest Yellowing Retardation 利 Long-Fang O. Chen INSTITUTE OF BOTANY, ACADEMIA SINICA, NANKANG, TAIPEI 115, TAIWAN, R.O.C.

28 , (20-25 C ) 易葉綠,

29 不 : 1) 冷冷理 ; 2) ; 3) ; 4) ; 5) 裂類 ; 來老

30 例 : (1) 利冷冷不便 (2) 利例易異

31 利不易異 (4) 利理不良 (5) 利裂 Cytokinins 類易量留不

32 利良狀利 (Zhong 2001, Euphytica 118: )

33 . 來類類糧類,,, 蘿,,,,,,,,,, 類, 梨,, 羅,,, 李,, 理 : 2000

34 . 類糧鈴狀類殺殺殺殺不殺理 : 2000

36 B Fig. 1. Regeneration and transformation of broccoli (Brassica oleracea var. italica). A: Shoots from ipt transformed peduncle explants; B: Close view of shoot regenerated from peduncle explants under Kanamycin selection.

37 Studies on the Transformation of Cytokinins synthatase ( isopentenyl transferase, ipt gene )

38 Plasmid maps (A) psg 529(+) BamHI SpeI NcoI BamHI SacI SpeI RB SAG12 promoter IPT Nos-ter NPTII 1 Kb probe psg529(+) plasmid map LB

39 Fig. 2. Dot assay for neomycin phosphotransferase (NPTII) activity in crude cell extracts of transformants. positive control: pbi121 (1A,1B); psg529(+) (1C); psg766a (1D); negative control: non-transformed leaf (1E); extraction buffer(1f).

40 (A) Primers for ipt were 5 -GATTTGATTAAGCTTTTAACTTGC-3 and 5 -GCCCGCCGTTGGCCTCATGAT-3 for psg529(+) coding for a 1 Kb fragment including segment of SAG12 promoter and ipt gene. (B) Primers for nptii were two 21 mers, 5 -GAGGCTATTCGGCTATGACTG-3 and 5 -ATCGGGAGCGGCGATACCGTA-3 coding for a 0.7 Kb fragment[8]. For psg766a, a primer set 5 -ACCCATGGACCTGCATCTA-3 and 5 -GGAGCTCAGGGCTGGCGTAACC-3 [10] was used. Fig 4. PCR identification of the transgenes(ipt and nptii ) for the (A) psg529(+) and (B) psg766a transformants. Thermal cycles for ipt gene were denatured at 94 C 2 min then 25 cycles of 94 C 30 sec, 60 C 30 sec and 72 C 30 sec, 1 cycle of 72 C 7 min. Thermal cycles for nptii gene were denatured at 94 C 5 min then 35 cycles of 94 C 1 min, 55 C 1 min and 72 C 2 min, C 7 min.

42 Table 1. The effects of seedling ages and explant types on frequencies of shoot regeneration from cotyledon, hypocotyl and peduncle under kanamycin selection after A. tumefaciens infection with ipt genes. Seedling ages or storage time 3 days after in vitro germination 9 days after in vitro germination 2-3 weeks after harvest a Explants Cotyledon Hypocotyl Cotyledon Hypocotyl Peduncle Transformation 4/184 frequency b ( 2.2) 10/175 ( 5.7) 1/ 53 ( 1.9) 1/156 ( 0.6) 18/222 ( 8.1) No. of plantlets recovered c % positive NPTII activity 20/31 (64.5) 34/65 (52.3) 1/ 5 (20.0) 1/ 3 (33.3) 264/372 (71.0)

43 Fig. Southern Hybridization of psg529(a) and (b) psg766 transformants; P: plasmid DNA, a 7 and b 12 are non-transformed control.

44 Fig. Postponing effect on the yellowing of detached leaves of broccoli transformed with ipt gene. A,B,C: transformants (I-126, I-119, P-88), D: nontransformed control, upper row: pictured at the day of detachment, lower row: pictured after detachment and stored at 25 C for 4 days.

45 Figure. Plantlets of transformant at the left (L) and nontransformed control at the right (R) indicate leafy yellowing at the bottom part of the control plant at the stage when the plant starts bolting.

47 Fig. Chromo-meter CR-310, Minolta. 50mm.

48 IT53-12 IT53-14 IT53-22 IT53-32 Hue angle (H o ) Days after harvest IT53 不 ( and : 0.75 kb band; and :1.0 kb band) 類 LDPE 25 Fig 3. Changes of hue angle of the progeny of transformed line IT53 with two types of insert fragments ( and : 0.75 kb band; and :1.0 kb band). Florets was packaged with LDPE-bag and stored at 25 in the dark. Bars are standard errors.

49 Fig. Ipt gene expression detected by RT-PCR on the Day detached (Lanes 1-7) and 4days after stored at 25 C(Lanes 8-14), 6,13 are PCR(-) kanamycin survival plant, 7 and 14 are non-transformed control.

50 (A) (B) ipt 北 (A) psg529(+) ; (B) psg766a Fig. 4. Northern analysis in the progeny of psg529(+) (A) and psg766a (B) transformed. The floret was packaged with a LDPE plastic bag and store at 25 in the dark for 0 day and 5 days. Northern hybridization analysis and the probe was a DIG-labeling 0.75-kb fragment of ipt coding region for the identification of psg766a transformed.

51 Fig. Postponing effect on the yellowing of detached F2 flowers of broccoli transformed with ipt gene. A,B,C,D: transformants (P16-10, P16-11, P16-15, P1 E: non-transformed control, upper row: pictured at the day of detachment, lower row: pictured after detachment and stored at 25 C for 6 days.

56 segregation line Day 5 after detached and stored at 25C in dark

57 Cytokinin synthesizing ipt transformation lines (A) Self-incompatibility line (B) Self-compatibility line

58 Studies on the mers(ethylene response sensor mutant gene) Transformation

59 (A) psmy1h-mersi62f (B) pbi-mersi62f mersi62f (ethylene response sensor, ERS), ERS 列 62 Ile(I) Phe(F). CaMV 35S promoter hygromycin knamycin.

60 Fig. Leaf yellowing comparison in mers transformantsand nontransformed control after continuously applying of 1 ppm ethylene at room temperature.

61 Fig. Northern analysis of mers on transformation lines at the day of Detached (D=0) and 3 days after storing at 25ºC(D=3) in dark.

62 Fig. Leaf yellowing comparison in a mers transformant and non-transformed control after detached and stored at 25 ºC in dark.

63 Fig. Floret yellowing in mers transformation lines in broccoli after detached and stored at 25 ºC in dark for 4 days.

64 Studies on the Anti-chlorophyllase Transformation

65 離不行離不狀不 (Zhong 2001, Euphytica 118: )

66 論利老行不不不狀理切了來

2004级研究生现代免疫学实验技术结业报告

2004级研究生现代免疫学实验技术结业报告 PCR PCR PCR RT-PCR PCR (Polymerase Chain Reaction,PCR) 80 DNA, DNA PCR PCR PCR DNA PCR -- -- DNA DNA 93 DNA PCR DNA DNA ( ) DNA 55 DNA DNA -- TaqDNA dntp DNA -- -- PCR PCR DNA DNA Y (1 X) n Y DNA X (Y)