國立中山大學學位論文典藏.PDF

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2 (Cattleya bicolor) ACC 酶 (ACC oxidase) RACE (Rapid amplification of cdna ends) ACC oxidase 3 (3 -untranslated region, 3 -UTR) ACC oxidase 3 -UTR cdna (open reading frame, ORF) DNA C. bicolor ACC oxidase (intron) (exon) (C. bicolor) ACC oxidase II

3 Abstract Ethylene, a plant hormone, plays an essential role in many aspects about plant development, growth, ripening, and senescence. In addition, it also regulates several responses when plants suffer stress from drought, flood, herbivore bites, wound, etc. ACC synthase and ACC oxidase belong to two multigene families. In this study, PCR (polymerase chain reaction) and RACE (rapid amplification of cdna ends) methods were used to amplify the ACC oxidase sequences in Cattleya bicolor orchid flower. The results show that there exists differences in the 3 -UTR (untranslated region) of orchid gene sequences. Compare the ACC oxidase sequences, including the cdna ORF (open reading frame) sequences and the amino acid sequences, of several different species, the sequence similarity among the three Laeliinae orchids, namely C.bicolor, C. intermedia, and Laelia anceps, is the highest. The similarity of cdna ORF sequences and amino acid sequences between orchids and the other plants, such as rice, apple and torenia, is comparatively lower. It was proposed that the protein located in cytoplasma (or in mitochondrial matrix space), agrees with the result from analysis of amino acid hydrophilicity prediction. The ultimate goal of this study is to postpone the flower senescence by the way of plant transfection. In the present findings, it only deals with the cloning and analysis of the ACC oxidase genes in C. bicolor. III

4 I..II..III..IV..VI 1 1 ACC 酶 (Escherichia coli) RNA cdna...17 DNA.18 酶 polymerase chain reaction, PCR IV

5 酶 ACC 酶 cdna ACC 酶 DNA V

6 ACC 酶...16 cdna (RACE) 23 pgem T-Easy..27 ReFL5-2/ReFL3-2 cdna..33 C. bicolor cdna..34 ACC 酶 cdna 36 ACC 酶..40 C. bicolor ACC 酶.43 ReFL5-2/ReFL3-2 DNA.46 C. bicolor ACC 酶 DNA cdna.47 VI

7 ACC 酶...21 酶 PCR 25 ACC 酶 cdna. 41 ACC 酶...42 ACC 酶 VII

8 C 2 H 4 Dimitry Nikolayevich Neljubov (Abeles et al., 1992) Gane (1934) (Bleeker, 2001 ) ( ppm) (Burg, 1962) (Hopkins, 1999) 1

9 (Hopkins, 1999) (O Neill et al., 1993; Johnson and Ecker, 1998; Bleeker, 2001; Wang et al., 2002) (Arabidopsis) 酶 (ethylene resistant genes, ETRs) (membrane- spanning domain) (Cu) N- transmembrane domain CTR1 (constitutive triple response 1) RAF like Ser/Thr 酶 (kinase) 酶 cascade) CTR1 EIN2 (ethylene insensitive 2) EIN2 EIN3 (ethylene insensitive 3) (Chang et al., 1993; Kieber et al., 1993; Ecker, 1995; Theologis, 1995; Johnson and Ecker, 1998; Bleeker, 2001; 2

10 Wang et al., 2002) (climacteric) (nonclimacteric) (Biale and Young, 1981) (Liu et al., 1985) (autocatalytic production) (McMurchie et al., 1972; Yang and Hoffman,a1984; Kende, 1993; Woodson, 1994; Oetiker and Yang, 1995) (Stead,1992) 3

11 Wang 2002 Wang et al., 2002 CTR1 Raf-like 酶 N CTR1 EIN2 EIN3 EIN3 (CTR1, constitutive triple response 1; EIN2, ethylene insensitive 2; EIN3, ethylene insensitive 3 4

12 (Lay et al., 1992; Stead, 1992) (post- pollination syndrome) (Whitehead et al., 1984; Borochov and Woodson, 1989; O Neill et al., 1993) (Nichols, 1968; Barden and Hanan, 1972; Liu et al., 1985) (Tang et al., 1994; Jones, 2002) (Stead, 1992; Woodson et al., 1992; O Neill et al., 1993; Tang and Woodson, 1996;aO Neill and Nadeau, 1997; Jones, 2002) (Gilissen, 1977; Jones and Woodson, 1997; Shibuya et al., 2000; Jones, 2002) ACC (Whitehead et al., 1984; Woltering, 1990; Nadeau et al., 1993;aO Neill et 5

13 al., 1993; Woltering et al., 1995; Porat et al., 1995) 1966 Lieberman (methionine, Met) (Lieberman et al., 1966) S-adenosyl-L-methionine (SAM) 酶 (SAM synthetase, EC ) SAM 1-amino- cyclopropane-1-carboxylate (ACC) 酶 (ACC synthase, EC ) SAM 1-aminocyclopropane-1- carboxylate (ACC) 5 -methylthioadenosine (MTA) ACC 酶 (ACC oxidase, EC ) ACC SAM ACC MTA (Murr and Yang, 1975; Adams and Yang, 1977; Adams and Yang, 1979; Yang and Hoffman, 1984) ACC + O 2 C 2 H 4 + CO 2 + HCN + 2H 2 O HCN β-cyanoalanine (Wang et al., 2002) SAM synthetase SAM (Ravanel et al., 1998) ACC synthase SAM ACC MTA 6

14 SAM SAM synthethase ACC synthase SAM ACC (rate-limiting step) ACC (Mayak et al., 1981; Rohwer and Mader, 1981; Vioque et al., 1981; Wang and Yang, 1987) ACC oxidase ACC synthase ACC oxidase ACC oxidase ACC auxin Cd 2+ Li + (Beyer, 1975; Boller and Kende, 1980) (autocatalysis) (autoinhibition) 7

15 Bleecker Kende 2000 (ACC, 1-aminocyclopropane-1-carboxylic acid; AdoMet, S-adenosylmethionine (SAM); KMB, 2-keto-4-methylthiobutyrate; Met, methionine; MTA, 5 -methylthio-adenosine; MTR, 5 -methyl- thioribose; MTR-1-P, 5 -methylthioribose-1-phosphate ) MTR 8

16 (Abeles et al., 1992; Kende, 1993) silver thiosulfate (STS) 2, 5-norbornadiene (NBD) cycloheximide ACC synthase aminooxyacetic acid (AOA) 2-aminoethoxyvinylglycine (AVG) ACC oxidase Fe 2+ phenanthroline PNT Co 2+ Cu 2+ Mn 2+ Mg 2+ (Yang and Hoffman, 1984; Hamilton et al., 1991) ACC 酶 ACC ACC oxidase ACC synthase ACC oxidase ACC oxidase ACC oxidase (Woodson et al., 1992; O Neill et al., 1993; Tang and 9

17 Woodson, 1996; O Neill, 1997; O Neill and Nadeau, 1997) ACC synthase 1990s ACC oxidase in vitro ACC oxidase (reversed biochemistry) cdna (Kende, 1993) Grierson (1985) mrna 35 kda Slater (1985) cdna (differential hybridization) cdna ptom13 ptom13 encode 35 kda Hamilton 1990 ptom13 ptom ACC oxidase ptom13 ACC oxidase Hamilton 1991 Spanu 1991 ptom13 ACC oxidase ACC ACC oxidase 10

18 in vivo ACC oxidase (stereospecificity) CO 2 HCN (Hoffman et al., 1982; Peiser et al., 1984) ptom13 ptom13 flavanone 3-hydroxylase (F3H) 58 ACC oxidase F3H Fe 2+ ascorbate Ververidis John (1991) F3H (Britisch and Grisebach, 1986) ACC oxidase Fe 2+ ascorbate in vitro in vivo Fe 2+ ascorbate ACC oxidase Dong (1992) ACC oxidase 酶 2-oxoglutarate (2-OG) ascorbate ACC oxidase Kao and Yang (1982) CO 2 ACC oxidase CO 2 CO 2 co-factor ACC O 2 ascorbate Fe 2+ CO 2 ACC oxidase ACC oxidase non-heme iron(ii) dependent oxygenase/oxidase ACC oxidase 11

19 isopenicillin N synthase (IPNS) 2-oxoglutarate Fe(II) dependent dioxygenase (aspart- ate, Asp) ACC oxidase (histidine, His) Fe(II) (Zhang et al., 1997) Rocklin (1999) in vitro O 2 ascorbate Fe(II) Fe(II) Fe(II) ACC O 2 CO 2 ACC oxidase (Guy and Kende, 1984) ACC oxidase (Imaseki and Watanabe, 1978; Yu et al., 1980; Yang and Hoffman, 1984) ACC oxidase 肽 (Ververidis and John, 1991; Hamilton et al., 1991) ACC oxidase (Ramassamy et al., 1998) 12

20 ACC 酶 13

21 48hr After Artificial Pollination Total RNA Extracted from Petal Genomic DNA Extracted from Plant Reverse Transcriptase cdna dntp Terminal Deoxynucleotidyl cdna-poly(c) Tail Polymerase Chain Reaction Gel Electrophoresis Gel Extraction Ligase Ligate to pgem T-Easy Vector Sequence and Blast for: 1. cdna Open Reading Frame 2. Genomic DNA Exon/Intron Position 14

22 (Cattleya) NCBI ( (tribe Epidendrea, subtribe Laeliinae) 19 William Cattley (Laelia) Cattleya bicolor (Escherichia coli) JM109 (Yanisch-Perron et al., 1985) JM109 enda1, reca1, gyra96, thi, hsdr17(r - k, m + k ), rela1, supe44, (lac-proab), F, trad36, proab, laql q Z M15 15

23 ACC 酶 C. bicolor C. Y. Hung 16

24 RNA cdna 1. RNA O Neill (1993) 48 ACC oxidase mrna 48 QIAGEN RNeasy Plant Mini Kit RNA 0.1 g 1.5 ml 450 µl RLT buffer 56 QIAshredder spin column 10,000 rpm 1.5 ml 1/ % RNeasy mini spin column 10,000 rpm 15 RNeasy mini spin column RPE buffer 500 µl 10,000 rpm 15 RNA RPE buffer 500 µl 10,000 rpm RNeasy mini spin column 1.5 ml RNase-free water 50 µl 10,000 rpm RNA

25 2. cdna Promega Reverse Transcription System Kit RNase-free 10 µl RNA 1 µl, 0.5 mg/ml oligo (dt) 15 primer mm MgCl 2 4 µl 10 mm dntp 2 µl RNase inhibitor 0.5 µl 15 unit AMV RTase RT 10X buffer 2 µl (100 mm Tris-HCl, 500 mm KCl, 0.1 Triton X-100) RNase-free water 20 µl DNA VIOGENE Plant Genomic DNA Extraction Miniprep System DNA 0.1 g 1.5 ml 400 µl PX1 buffer 4 µl RNase A (100 mg/ml) vortex µl PX2 buffer Shearing tube 10,000 rpm 1.5 ml 18

26 1/2 PX3 buffer % Plant Genomic DNA Mini Column 10,000 rpm 700 µl WS buffer 10,000 rpm DNA WS buffer Plant Genomic DNA Mini Column 1.5 ml 65 TE buffer 150 µl 10,000 rpm -20 酶 polymerase chain reaction, PCR 1. NCBI ACC oxidase mrna ( Doritaenopsis sp., accession no. L07912; Dendrobium hybrid cultivar, accession no. AY506846; Phalaenopsis cv. 'True Lady' accession no. AF004662) BioEdit 5 3 (rapid amplification of cdna ends, RACE) (Cormack and Somssich, 1997) (open reading frame, ORF) 19

27 酶 ORF 2. cdna (rapid amplification of cdna ends, RACE) DNA (transcription) RNA mrna (post-transcription) 5 苷 (methylguanosine) 5 -cap 3 苷 3 -poly(a) tail mrna oligo (dt) 15 酶 cdna cdna 5 -poly(t) head mrna oligo (dt) 15 mrna 3 酶 3 RACE 5 RACE 苷酶 (terminal deoxynucleotidyl transferase, TdTase) mrna cdna 3 苷 (3 -poly(c) tail) cdna poly(g) 酶 mrna 5 酶 10 µl cdna 3 µl ddh 2 O 4 µl 5X buffer 2 µl 20

28 ACC 酶 NCBI ACC oxidase mrna ( Doritaenopsis sp., acc. no. L07912; Dendrobium hybrid, acc. no. AY506846; 丨 Phalaenopsis cv. 'True Lady' acc. no. AF004662) BioEdit (degenerate primers) ACOreIP 5' CTC CGG CTT (CG)GG ACA (CT)GG CGG 3' ACQ450 5' CTC CAA CAT CTC CCA A(AG)T CCC 3' Q1030anti 5' (AC)TT CAT GTA (AG)TC CT(CG) (AG)AA CAC 3' GSP-1 5' CCG CC(AG) TGT CC(CG) AA(AG) CC(AG) GA(CG) 3' 3 RACE BGTT 5' CAC GCG TCG ACT AG (T)15 (ACG) 3 3 RACE RAS51 5' TCC TCG CAC AAC AGA TCC AG 3' 5 RACE RAS52 5' TCC CAG TCC AGA TTA TCG CC 3' 5 RACE NurG 5' GGG GGG GGG GGG GGG (ACT) 3' 5 RACE ReFL5-2 5 AAA CTA GTG GAT CCG AGA ACT AGA GAG AGA TGG 3 ORF ReFL3-2 5 GGT (ATCG)AC CTC GAG ATA TGA ACT AAA TTG TTC C 3 ORF 酶 BamH I: GGATCC; BstE II: GGT(AGTC)ACC; Spe I: ACTAGT; XhoI: CTCGAG 21

29 dctp (10 µm/µl) 1µl TdTase (30 unit/µl) RACE 3. VIOGENE VioTaq TM DNA polymerase 酶 10X PCR buffer 2 mm MgCl 2 5 µl 2.5 mm dntp 4 µl 3 primer 2.5 µl (100 µm/µl) 5 primer (100 µm/µl) 2.5 µl cdna 1 µl Taq polymerase 0.5 µl (5 unit/µl ) ddh 2 O 50 µl 94 pre-denature DNA DNA PCR

30 5 3 mrna cdna RTase 3 poly(a) 5 poly(t) poly(c) 3 cdna ACOreIP TdTase Polymerase 5 poly(t) ACQ450 ACOreIP GSP-1 Q1030anti BGTT nurg ReFL5-2 RAS52 RAS51 5 RACE 3 RACE ReFL3-2 ORF cdna (RACE) RNA (RTase) mrna cdn 苷酶 (TdTase) cdna 3 - 苷 酶 23

31 100 ml 1X TAE buffer (0.04 M Tris-acetate, M EDTA) 1 g AMResco Agarose I TM 50 15~30 1X TAE buffer DNA ladder marker loading dye DNA pipetman 100 V 30 EtBr (ethium bromide, 0.5 µg/ml) (, 1996;, 1997) 2. VIOGENE Gel Extraction System DNA 1.5 ml GEX 500 µl 60 Gel-M column 10,000 rpm 500 µl WF buffer 10,000 rpm 700 µl WS buffer 10,000 rpm WS buffer Gel-M column 1.5 ml 20 µl EB buffer 10,000 rpm DNA 24

32 酶 PCR 酶 40 PCR, 3 RACE 5 RACE (cdna) (genomic DNA) Step 1 Pre-denature 94, 3 min 94, 3 min 94, 3 min 94, 3.5 min 94, 4 min Step 2 Denature 94, 30 sec 94, 30 sec 94, 35 sec 94, 40 sec 94, 50 sec Step 3 Annealing 52, 40 sec 48, 35 sec 50, 35 sec 54, 45 sec 54, 50 sec Step 4 Extension 72, 50 sec 72, 45 sec 72, 45 sec 72, 1.5 min 72, 2 min Step 5 Extension 72, 7 min 72, 6 min 72, 6 min 72, 8 min 72, 10 min 25

33 DNA pgem T-Easy Vector System pgem T-Easy Vector System ampicilin -lactosidase coding region X-gal PCR out-of-frame X-gal 3 (dttp) Taq 酶 3 (datp) 酶 (ligase) pgem T-Easy PCR ampicilin LB LB 26

34 pgem T-Easy 酶 T4 酶 Promega 27

35 pet-32a(+) vector pcambia vector JM109 LB 37 14~16 1 ml 25 ml LB 37 OD ~ ,000 rpm 10 ml, 0.1M CaCl ,500 rpm 1 ml, 0.1M CaCl µl 1.5 ml (, 1997) Hiroaki (1990) 7 µl heat shock µl LB ,000 rpm ampicilin (100 µg/ml) 37 28

36 Viogene Mini-M TM Plasmid DNA Extraction System 1.5 ml LB 10,000 rpm 250 µl MX1 buffer 250 µl MX2 buffer 350 µl MX3 buffer 13,000 rpm Mini-M TM column 10,000 rpm 500 µl WF buffer 10,000 rpm 700 µl WS buffer 10,000 rpm Mini-M TM column Mini-M TM column 50 µl EB buffer 10, 000 rpm pgem T-Easy vector PCR ABI 3100 Genetic Analyzer NCBI pgem T-Easy vector PCR 酶 pet-32a(+) vector 29

37 ( ) 酶 酶 ATP 酶 酶 Sambrook 1989 酶 PCR 酶 2X ligation buffer 10 µl 1 µl Promega T4 ligase 1 µl (4 unit/µl) ddh 2 O 20 µl 16 mrna cdna DNA 酶 酶 vector ( ng ) insert ( kb size ) insert vector ( kb size ) insert ( ng ) vector 30

38 ACC 酶 cdna O Neill (1993) (C. bicolor) 48 RNA RNA 酶 酶 cdna ACOreIP 酶 400 bp BioEdit 3 5 RACE C. bicolor cdna 3 RACE cdna 3 RACE 3 3 (3 -untranslated region, 3 -UTR) 3 ACC oxidase (Barry et al., 1996; Zarembinski and Theologis, 1994) BioEdit 3 RACE 5 RACE 31

39 ReFL5-2 5 (5 -UTR) (open reading frame, ORF) (ATG) ReFL3-2 3 (3 -UTR) 酶 1000 bp NCBI 5 -UTR 3 -UTR ACC oxidase ORF C. bicolor ORF 975 bp NCBI AY aa ORF NCBI ACC oxidase (C. intermedia) (Laelia. anceps) ACC oxidase BioEdit Epidendreae (tribe) Laeliinae (subtribe) C. bicolor, C. intermedia L. anceps Epidendroideae (subfamily) 32

40 Marker Lan Cin Cbi 1000bp- 1000bp ReFL5-2/ReFL3-2 cdna ReFL5-2 ReFL3-2 cdna 酶 cdna 1000 bp (Lan: Laelia anceps, Cin: Cattleya intermedia, Cbi: Cattleya bicolor) 33

41 -43 ctttcctctgtgttctgcttgtgaaactgagaactagagagag 1 atggagagcggaagcttccctgtgatcaacatggagcttcttgag M E S G S F P V I N M E L L E 46 agttctcagcgcccggaagccatggcgcttctccgagaggcctgt S S Q R P E A M A L L R E A C 91 gagaactggggattcttcgagctactgaaccacggtatctccaca E N W G F F E L L N H G I S T 136 gagctgatgaaccgagtcgaaaccgtgaacaaagaaaattaccgg E L M N R V E T V N K E N Y R 181 cgattccgcgagcagcgattcaaagaattcgccgccaaaaccctt R F R E Q R F K E F A A K T L 226 gattccggcgagaaggtcgacggcgataatctggactgggaaagc D S G E K V D G D N L D W E S 271 accttcttcctccgccatcttccgacctccaacatctcccaaatc T F F L R H L P T S N I S Q I 316 ccggatctggacgaggactgccggagcacgatgaaggagttcgcc P D L D E D C R S T M K E F A 361 cgagagctggagaaactggcggagcggttgctggatctgctgtgc R E L E K L A E R L L D L L C 406 gaggatttggggcttgagaaagggtatttgaagagggttttttgc E D L G L E K G Y L K R V F C 451 gggggatcggatggattgccgacgttcgggacgaaggttagcaat G G S D G L P T F G T K V S N C. bicolor (start code) (stop code) 34

42 496 tatccgccgtgtccgaagccggagttgataaaggggctgaaagct Y P P C P K P E L I K G L K A 541 catacggatgcgggagggattattctactgtttcaggacgataag H T D A G G I I L L F Q D D K 586 gtcagcgggcttcagctgctcaaggatggccagtggattgatgtt V S G L Q L L K D G Q W I D V 631 cccccgctgcgccattccattgttgtcaacatcggagatcagctt P P L R H S I V V N I G D Q L 676 gaggtgataacaaatggaaaatacaagagtgtgatgcacagggtg E V I T N G K Y K S V M H R V 721 gtggcccaaaccgacggcaaccgcatgtccatcgcgtcgttctac V A Q T D G N R M S I A S F Y 766 aaccccggcagcgacgccgtgatctccccggcgccggagctggtg N P G S D A V I S P A P E L V 811 gagaaagaggcggaggaggagaagaaggaagcgacttatccaaag E K E A E E E K K E A T Y P K 856 tttgtgtttgaggactacatgaatctgtatattcgcaagaaattt F V F E D Y M N L Y I R K K F 901 gaggccaaggagccgaggtttgaggctatgaagaccatggaaact E A K E P R F E A M K T M E T 946 gtttccggctcccagctcattcctactgcttaa 1192 V S G S Q L I P T A * 991 ttaattaattagtggaacaatttagttcatatggattaatttatg 35

43 Cbi ATGGAGA GCGGAAGCTT CCCTGTGATC AACAT G G A GC T TC T TGAGAG TTCTCAGCGC 57 Cin LAE Dorita A.. T CC.. G.... C Dendro G G Oryza 1 A T GGC. AG T G T T. C C T C G A AC.. G.... C C G AGG.. A. G 60 Apple T G. AG. A A.. T T..... A GC.. CA. TG.. GAGGGAA. A 51 Petunia T G. AG. A A A. T.... G. T.... CA AAG. GA. TG.. GT. G. AA. A Cbi 58 CCGG AAGCCA T G G CGCT TCT CCGAG A G G C C T G T G A G A A C T G G G G A T T C T T C G A G C T AC T G 117 Cin G LAE Dorita C C T C T.. G. A.... T Dendro 28 T. A.. C T G.... T..... T G Oryza 61 GGC. C.CGA.... A.G. CA.... C.. C..... C C A. G Apple 52 AAA. CTA. A.... AAAAGA.. A A... T T.. T.. T..... GG Petunia 52 G. T. CCA. T.... AAA. GA. TAAG.. T.. A..... A C..... T... T. GG Cbi 118 A A C C A C G G T A T C T C C - AC AG A GC T G A T G A A C CGAG T C G A A A CCG T G A A C A A A G A AAA T T A 176 Cin LAE Dorita A......C..G-.... A G.. G.. G..G.. A C Dendro A......C..GA. C.. A A T.. A C Oryza T.. C... G. GC.. G G.. GAG.. G.. G C GG.... G... G. CGC. C Apple 112. GT.. T.. G.. TC. AACTG -.. T. TC.. G.. AC... G.. G. GGC... CA..... GC. C Petunia T.. A... C. ACG TG -. AG. A... G.. AC T.. G.. G. AG A... CA.. G. GTC Cbi 177 C CGG C GAT T C C G C G A G C A G C G A T T C A A A G A A T T C G C C G C C A AAACCCT 224 Cin 111. A LAE Dorita G..... G.. A..... C GT Dendro GAACAT TACCGG T T. 206 Oryza 180. GC C A AC. G... G... G.. AAG..... G.. G..... GC GGCGG. TG Apple 171. AA AG. GTTTG..... AA. G..... G.. GC. G. TG.... GC. AAGG 218 Petunia 171. AA A AG. G. ATG.. A.. AA. G..... G..... G. TT.... GT. AAGC Cbi 225 T G A T T C C G G C G A G A A G G T C G A C G GCGA TAATCTGGAC T G G G A A A G C A C C T T C T T C C T 281 Cin LAE A C Dorita 225 A... A... TG..... C GCCG.. A G Dendro 207 A A.. T C.. T T Oryza 228 G.. GG GCG C..... TGA. GGGC A. C G G. 287 Apple 219 CCT. GAG.. T. TTC.. A... C A.. A. T. A. AG.. A.... T T... CA. T. 275 Petunia 219. C T. GAA.. T. TTC. A.... C T.. G. TTAC.G.. A.... T T... T Cbi 282 C C G C C A T C T T C C G A C C T C C A A C A T C T C C C A A A T C C C G G A T C T G G A C G A G G A C T G C C G G AG 341 Cin LAE C.. T Dorita C.. C C T.. C.. T TC 341 Dendro C.. A G.... C.. C A.. T... G. C Oryza C. GC.. CGT C.. G.. G. CC.... C.. C G. C..... CC... A. A.. CA 347 Apple 276 G TCAA.. G TG.. G. A.. A..... CA. G.. T.. G. A. A... A 335 Petunia 276 GAAA..... C.. C. TT.. T..... T.. TG.. G.... T..... T.. T.. A.. A. A. A.. GA Cbi 342 CACGATGAAG G A G T T C G C CC GAG A G C T G G A G A A A C T G G C G G A G C G G T T G C T G G A T C T G C T 401 Cin T. 335 LAE G T. 260 Dorita 342 A..C A..... G. TG..... A..... C.. C A. AC T. 401 Dendro A.. A..... T. T.. G... C A. A..... C T. 383 Oryza 348 GGT C. A.. T.. GT CG... A. C..... G.. CT..... A.. G C Apple 336 TGT T.. AT TGA. AT.... A... T. A.. A.... A. C CT.. T. 395 Petunia 336 AG T T.... GA.. T.. T.. TA A. AG. T. A..... G..... A... GA. C. TT.... CT ACC 酶 cdna 36

44 Cbi 402 G T G C G A G G A T T T G G G G C T T G A G A A A G G G T A TTTGAAGAGG G TTT T T T G C G G GGGA T C G G A 461 Cin LAE A Dorita A G A. A C Dendro A.... A G A..... A.. C..... C..... T Oryza A.. C.... C.. G..... G.. T.. CC A.. CC.. CGC G... A. 464 Apple T... A.. C. T.. A.. G.. AC CC. T.... A.. CA.... AT A. AA. 452 Petunia T.. AA.. C. T.. C.. G.. A..... T.. CC. T.. A. AT. CC.... AT A Cbi 462 T G G ATTGC C G A C G T T C G G G A C G A A G G T T A G C A A T T A T C C G C C G T G T C C G A A G C C G G A GTT 521 Cin LAE Dorita T G.. T A C. 521 Dendro 444 C T T Oryza 465 C.. C A C.. C..... G.... GC.. C C... C GC.. C.. TC. 521 Apple 453 G A.. T.. T.. C.. C..... G..... C.. C.. T.. A..... C.. C.. A.. CC. 509 Petunia 453 A.. T A. AC.. T..... T.. A.. G..... C..... A.. A.. C.. A.. A.. A.. T Cbi 522 G A T A A A G G G G C T GAAAGC T C A T A C G G A T G C G G G A G G G A T T A T T C T A C T G T T T C A G G A C G A 581 Cin G T LAE G T Dorita A T... G A.. G..... C..... GT T Dendro A T... G C T T Oryza 522 CG. CG. C.. C.. CCGC.. C.. C.. C.. C.. C.. T.. C.. C.. C.. G..... C Apple C..... T.. CCGG.. C.. C.. C..... C.. C.. CC. C.. CT. G.. C.. C..... T Petunia 510 A.. C..... A.. ACG T.. C.. C.. A.. C.. T.. T.. C.. A.. C.. T..... C.. A.. T Cbi 582 T A A G G T C A G C G G G C T T C A G C T G C T C A A G G A T G G CCAG T G G A T T G A T G T T C C C C C GC T G C G 641 Cin LAE Dorita T T C.. GG. A..... C T.. CG Dendro C T.. A T.... T AAG. A T.. CA Oryza 582 CC.... G..... C.. C C.. GG..... G. G.. C.. G.. G.. CA Apple 570 C T.. C.. C..... C C.. AG..... G G.. T.. CA Petunia 570 C.. A.. A.. T.. C.. A.. A.. C..... A.. C..... A..... C T.. CA Cbi 642 C C A T T C C A T T G T T G T C A A C A T C G G A G A T C A G C T T G A G G T G A T A A C A A A T G G A A A A T A C A A 701 Cin 576. T T LAE 501. T T Dorita C..... T.. T G G Dendro T.. T G Oryza CG.... C.. C. C C.. C..... G C.. C.. C.. C. GG Apple C A.... TC. T.. T.. C.. A C.. C.. C..... G Petunia G.. T... C.... T.. C.. A.. C C.. T Cbi 702 G A G T G T G A T G C A C A G G G T G G T G G C C C A A A C C G A C G G C A A C C G C A T G T C C A T C G C G T C G T T 761 Cin T.. A G LAE T.. A G Dorita T.... T T.. A G Dendro G A G.. T.. A.. C Oryza C.. C C. C.. CC. CA. G. GCC C.. C Apple GAA A. T A.. T.... C. A. A..... A.. A.. T.. A Petunia A... A. A A A..... GGC T.. A..... AT. A.. T.. C Cbi 762 C T A C A A C C C C G G C A G C G A C G C C G T G A T C T C C C C G G C G C C G G A G C T G G T G G A G A A A G A G G C 821 Cin A.... C LAE A.... C Dorita T T T C Dendro C.... T A Oryza GC C.... T C. C... C. CC. GCGCC.. C Apple A..... T.. T.. G A... A.. A.. A AC C.. A A Petunia 750 T..... T.. A.. A.. T.. T.. A A T.. A.. A.. A. C T.. T.. T A

45 Cbi 822 G G A G G A G G AG A A G A A GGA A GCGAC T T A T C C A A A G T T T G T G T T TG A G G A C T A C A T 875 Cin A. A A C LAE A. A A C Dorita A AG G AG A. G. AA. T CC.... T Dendro C A C Oryza C.. C GG. G... C..... C Apple 810 A TC G T G.. C.. G.. A. CC..... C.. A Petunia 810 A..... A C.. AC G T C.. C..... A..... A..... T.. T Cbi 876 G A A T C T G T A T ATT C G CAAGA A A T T T G A G G C C A A G G A G C C G A G G T T T G A G G C T A T G A A GAC 935 Cin T LAE T Dorita C..... G G A G G 941 Dendro G G..... C.... G T A Oryza Apple G.. C... GC. G. GGTC.. G.. C A.. A.. A..... A.. C..... AG. 917 Petunia GT. A... GC. G. AC TC.. G... C. A.. T A.. A..... A.. A..... AG Cbi 936 C A T G G A A A C T G TTTCCGGCT CCCAGC TCAT T C C T A C T G C T Cin C LAE C C Dorita 942 T..... G. T.... A T G A.... T.... CG.. A.. C Dendro 915 T..... TG..... A T T A.... T.... CG Oryza Apple 918 AG T. AAGG.. A. T. TTGGTT.GGG N.. AG T. ATA AGTACTGCT 966 Petunia A.GT. AAGA TGG. T. CA...G. C.... T C C. bicolor C. bicolor 苷 NCBI Cbi: Cattleya bicolor (AY598793);Cin: Cattleya intermedia (AY598794); Lae: Laelia anceps (AY598795); Dorita: Doritaenopsis (L07912); Dendro: Dendrobium crumenatum (AF038840); Oryza: Oryza sativa (AF049889); Apple: Malux x Domestica (AB086888); Petunia: Petunia hybrida (M90294) 38

46 Vandeae (tribe) Dendrobeae (tribe) ACC oxidase cdna ORF 60~70 cdna ORF Laeliinae (subtribe) (tribe) cdna ORF ACC oxidase Hamilton (1991) Ververidis John (1991) ACC oxidase 39

47 Cbi 1 -MESGSFPV I NM ELLESSQR PEAMAL L R E A C E N W G F F E L L N H G I STELMN R V E T V N K E N- 58 Cin V Lae dorita QG.... A D L Y H H. 48 dendro G. R. SD... V.. D H. PNE QS HY 49 oryza 1 M AS V A N.. T E E. GAR. E V I. D M..... AH... D E.. R. S. A H. 59 apple ME N..... L.S.NGEG. KAT. EK I K D V S... P.. F L D T.. R LT.. H. 56 petunia ME N.. I. S L DKVNGVE. AAT. E M I K D V.... PR. V. D T.. K MT. G H Cbi Y R RFR E Q R F K E F A A - K T L DSGE K - V DG DN L D W E S T F F L R H L P T S N I S Q I P D L D E DCR 113 Cin Lae E A D dorita S..... T V. N.. EP E D dendro 50 REH N V G 107 oryza ANC.. E K..... R.RM. EA... G A.V KGI V.. R. V.. L A D L.. V. DHY. 115 apple K QCL LV. S. G. EGVQ-. TEV KD M H..... Q.... E V... K D EY. 111 petunia K KCM LV. S. A. EGVQ-. AEV TD M K... I.... E V..... EY Cbi 114 STMKEF A R E L E K L A E R L L D L L C E D L G L E K G Y L K RVF C G - - GSDGLP T F G T K V S N Y P P C P K 171 Cin Lae G I dorita L... N K dendro LG N oryza 116 QV.. Q.. S. I... S.. V N KA. A... S NG S..... R 171 apple 112 NV..... L K Q N.... Q.... KA. Y... T K G N 167 petunia 112 EV. R D.. KR E N NA. Y... S K G - -. N Cbi 172 P E L I K G L K A H T D A G G I I L L F Q D D K V S G L Q L L K D G QW I D V P P L R H S I V V N I G D Q L E V I T N G 231 Cin R Y Lae R Y dorita R E V dendro R T E E M oryza 172. D. VD.. R Q E. V.... M.. A.. A apple 168. D..... R L E. V.... M..... I. L petunia 168. D..... R M L Cbi 232 K Y K S V M H R V V A Q T D G N R M S I A S F Y N P G S D A V I S P A P E L V E K E A E - - E E K - K EATYPKFVF 288 Cin E A T Lae E A T dorita L F... A EK..... KE I dendro N F A KE oryza 232 R L T RP A.... F... A. AG AD. A.. - A A A.. R apple E... I..... T Y... T NQV... S petunia I.. K.. A... L Y... A N KQV Cbi 289 E D Y M N L Y I R K K F E A K E P R F E A M K T M E T V SG SQL I P T A 325 Cin 267 Q Lae 242 Q dorita 281 Q S.. I. MS.. P dendro K.. V. Q DA. IS.. P oryza V. H SAAE. VH AAP.... A apple K.. AGV A V. IKAS FG.GPV. S petunia 283 D... K.. AGL.. Q A...DVK MDP.... A. V 319 ACC 酶 Fe(II) dependent oxigenase/oxidase subfamily Fe(II) Cbi: C. bicolor (AAT02192); Cin: C. intermedia (AAT02193); Lae: L. anceps (AAT02194); Dorita: Doritaenopsis (AAA21611); Dendro: D. crumenatum (AAD02104); Oryza: O. sativa (AAC05507); Apple: Malux x Domestica (BAC53656); Petunia: Petunia hybrida (AAA33708) 40

48 ACC 酶 cdna NCBI Cbi: C. bicolor (AY598793); Cin: C. intermedia (AY598794); Lae: L. anceps (AY598795); Dorita: Doritaenopsis (L07912); Dendro: D. crumenatum (AF038840); Oryza: O. sativa (AF049889); Apple: Malux x Domestica (AB086888); Petunia: Petunia hybrida (M90294) Taxa Cbi Cin Lae Dorita Dendro Oryza Apple Petunia Cbi Cin Lae Dorita Dendro Oryza Apple 75 Petunia 41

49 ACC 酶 NCBI Cbi: C. bicolor (AAT02192); Cin: C. intermedia (AAT02193); Lae: L. anceps (AAT02194); Dorita: Doritaenopsis (AAA21611); Dendro: D. crumenatum (AAD02104); Oryza: O. sativa (AAC05507); Apple: Malux x Domestica (BAC53656); Petunia: Petunia hybrida (AAA33708) Taxa Cbi Cin Lae Dorita Dendro Oryza Apple Petunia Cbi Cin 97 Lae Dorita Dendro Oryza Apple Petunia

50 3 Hopp & Woods Scale Mean Hydrophilicity Profile Scan-window size = Mean Hydrophilicity Position C.. bicolor ACC 酶 Hopp & Woods Scale Mean Hydrophilic Profile Hopp & Woods Scale C. bicolor ACC 酶 43

51 ACC 酶 C. bicolor C. intermedia L. anceps Species Molecular weight (kda) pi value Prediction of Protein Locolization C. bicolor cytoplasm: mitochondrial matrix space: 0.10 C. intermedia cytoplasm: chloroplast (stroma/thylakoid membrane/thylakoid space): 0.20/0.20/0.20 L. anceps cytoplasm mitochondrial (matrix space/inner membrane/intermembrane space): 0.46 / 0.15 /

52 ACC 酶 DNA cdna ORF ReFL5-2 ReFL3-2 C. bicolor DNA 酶 1800 bp C. bicolor cdna ORF (exon) (intron) Tang (1993) Lasserre (1996) Blume (1997) exon intron ReFL5-2 ReFL3-2 C. bicolor intron 669 bp 108 bp DNA 45

53 Marker Cbi 2000 bp bp- 1800bp 1000bp- ReFL5-2/ReFL3-2 DNA C. bicolor 酶 1800 bp 46

54 Cbi_gen 1 T T A A T T T T C T T T G T T C A C G G T T A G T A C C A T C T T T T C T T C G T C G C T C G A T G C G T G G T C G T C 60 Cbi_cDNA Cbi_gen 61 T T T A T C G T T T C A C G G G C T T G T A T C T T G G T C T A T T A C C G A C C A T T C T A A G C A G C A A C T A A A 120 Cbi_cDNA Cbi_gen 121 A C C T T C T C T C A A G A G A G A A C A C A G C A T C T T C T C T C T C T C T C T G G T T T C T T G T C T G C T C T C 180 Cbi_cDNA Cbi_gen 181 T G C T C T C T C T C T C T C T A T C T G C T T T C C T C T G T G T T C T G C T T G T G A A A C T G A G A A C T A G A G 240 Cbi_cDNA Cbi_gen 241 A G A G A T G G A G A G C G G A A G C T T C C C T G T G A T C A A C A T G G A G C T T C T T G A G A G T T C T C A G C G 300 Cbi_cDNA Cbi_gen 301 C C C G G A A G C C A T G G C G C T T C T C C G A G A G G C C T G T G A G A A C T G G G G A T T C T T C G A G GTGAA 360 Cbi_cDNA Cbi_gen 361 GCCCCACTCT CTCTTTAATT CTCTGATTAT CTTTTCCTCG AGCTGACTAA TTGAGAGCTT 420 Cbi_cDNA Cbi_gen 421 TTTTTCTTTT TTGTAGTGAA GTAATGTAAT TTATCTTTCT TATTTTTACT TTTATTTTTT 480 Cbi_cDNA Cbi_gen 481 t AAATGAAAG TTTTGGGAGT GGGTATATGA TTTAGGAACT AAGAGCTTCA CATTGAGTAG 540 Cbi_cDNA Cbi_gen 541 CACTTATGAC TTAATGATTG AGTTTGATAT TTTTAATTAT AAGTTTTTGA GTTCAAAGCC 600 Cbi_cDNA Cbi_gen 601 TATCACATAA GGATACATAT AAATTTAAAT AAATAATTGG AGGACTTTAC AAAAAAAa a a 660 Cbi_cDNA Cbi_gen 661 TTCAAACTCT TTGACTACTA AATTAGCACC CACATAAAAT ATTTCTCCTA AAAAAAGGAG 720 Cbi_cDNA Cbi_gen 721 CTTTTACATA CACAGCACCC GAAATTTATA AATTTACCTA TAACGCACTC AAACTCTAAT 780 Cbi_cDNA Cbi_gen 781 ATTTACAGGG Ga ACGCATAA AACTTATGAC TTCATCTTCT TTTCCCGCCT ATGATGCCCT 840 Cbi_cDNA Cbi_gen 841 GTTGAACATA GGGAGAAAGT ACGAAAAAGA TAAATTTGTA ACTTTTTACG TAATGTTATA 900 Cbi_cDNA Cbi_gen 901 TATATATATA TATGAAAAAT AGAAATAGGA ATAAAATAAA AATTAAGTAT AGCATTCCCC 960 Cbi_cDNA Cbi_gen 961 CTCTTCTTGG ATTTGTTTAT GATTTTCTAA CCAAATATTT TCCa TTATGT ATATAATATG 1020 Cbi_cDNA Cbi_gen 1021 CAGC T A C T G A A C C A C G G T A T C T C C A C A G A G C T G A T G A A C C G A G T C G A A A C C G T G A A C A A A 1080 Cbi_cDNA C. bicolor ACC 酶 DNA cdna intron 47

55 Cbi_gen 1081 G A A A A T T A C C G G C G A T T C C G C G A G C A G C G A T T C A A A G A A T T C G C C G C C A A A A C C C T T G A T 1140 Cbi_cDNA Cbi_gen 1141 T C C G G C G A G A A G G T C G A C G G C G A T A A T C T G G A C T G G G A A A G C A C C T T C T T C C T C C G C C A T 1200 Cbi_cDNA Cbi_gen 1201 C T T C C G A C C T C C A A C A T C T C C C A A A T C C C G G A T C T G G A C G A G G A C T G C C G G A G C A C G A T G 1260 Cbi_cDNA Cbi_gen 1261 A A G G A G T T C G C C C G A G A G C T G G A G A A A C T G G C G G A G C G G T T G C T G G A T C T G C T G T G C G A G 1320 Cbi_cDNA Cbi_gen 1321 G A T T T G G G G C T T G A G A A A G G G T A T T T G A A G A G G G T T T T T T G C G G G G G A T C G G A T G G A T T G 1380 Cbi_cDNA Cbi_gen 1381 C C G A C G T T C G G G A C G A A G G T T A G C A A T T A T C C G C C G T G T C C G A A G C C G G A G T T G A T A A A G 1440 Cbi_cDNA Cbi_gen 1441 G G G C T G A A A G C T C A T A C G G A T G C G G G A G G G A T T A T T C T A C T G T T T C A G G A C G A T A A G G T C 1500 Cbi_cDNA Cbi_gen 1501 A G C G G G C T T C A G C T G C T C A A G G A T G G C C A G T G G A T T G A T G T T C C C C C G C T G C G C C A T T C C 1560 Cbi_cDNA Cbi_gen 1561 A T T G T T G T C A A C A T C G G A G A T C A G C T T G A G GTAATAATTA ATTCATTCAT TCATTCATTC 1620 Cbi_cDNA Cbi_gen 1621 TCTCTCTGAA TCTTCTCATC ATATATATAT TTTTTTTAAA AAAAAATTGA GCTTGAAGTT 1680 Cbi_cDNA Cbi_gen 1681 TGATGAACTG ATCACAGG T G A T A A C A A A T G G A A A A T A C A A G A G T G T G A T G C A C A G G G T G G 1740 Cbi_cDNA Cbi_gen 1741 T G G C C C A A A C C G A C G G C A A C C G C A T G T C C A T C G C G T C G T T C T A C A A C C C C G G C A G C G A C G 1800 Cbi_cDNA Cbi_gen 1801 C C G T G A T C T C C C C G G C G C C G G A G C T G G T G G A G A A A G A G G C G G A G G A G G A G A A G A A G G A A G 1860 Cbi_cDNA Cbi_gen 1861 C G A C T T A T C C A A A G T T T G T G T T T G A G G A C T A C A T G A A T C T G T A T A T T C G C A A G A A A T T T G 1920 Cbi_cDNA Cbi_gen 1921 A G G C C A A G G A G C C G A G G T T T G A G G C T A T G A A G A C C A T G G A A A C T G T T T C C G G C T C C C A G C 1980 Cbi_cDNA Cbi_gen 1981 T C A T T C C T A C T G C T T A A T T A A T T A A T T A G T G G A A C A A T T T A G T T C A T A T G G A T T A A T T T A 2040 Cbi_cDNA Cbi_gen 2041 T G T G T T G C T G T T T G T T A G G G T C T A G T G G T T T G A T T T G A A C A A T T A T A A T G T G T A T T T A T T 2100 Cbi_cDNA Cbi_gen 2101 A A A A A 2105 Cbi_cDNA

56 cdna DNA 酶 ACOreIP bp 3 RACE 3 ACC oxidase ACC oxidase 3 RACE mrna 3 poly (A) tail poly (T) 酶 DNA RACE 酶苷 酶 ACC oxidase DNA 3 NCBI ACC oxidase (Dendrobium) (Phalaenopsis) (Doritaenopsis) 49

57 3 (open reading frame) NCBI Vandeae (tribe) Dendrobeae (tribe) ACC oxidase NCBI ACC oxidase 50

58 Zhang 1997 Fe(II) dependent oxygenase/ oxidase subfamily Fe(II) (aspartate, Asp) (histidine, His) ACC oxidase ACC oxidase Fe(II) ACC oxidase (asparagine, Asn) (tyrosine, Tyr) (Tyr: UAU/UAC) (Asn: AAU/AAC) (His: CAU/CAC) 苷 Fe(II) DNA 酶 ReFL5-2 ReFL3-2 cdna 51

59 5 3 DNA 1800 bp cdna Tang (1993) Lasserre (1996) Blume (1997) ACC oxidase ACC oxidase (antisense) (Aida et al., 1998) 52

60 aminocyclopropane-1-carboxylate 酶 aminocyclopropane-1- carboxylate 酶 Abeles, F. B., Morgen, P. W. and Salveit, M. E. Jr Ethylene in plant Biology, 2 nd Edition. CA: Academic, San Diego, pp 414 Adams, D. O. and Yang, S. F Methionine metabolism in apple tissue: implication of S-adenosylmethionine as an intermediate in the conversion of methionine to ethylene. Plant Physiol. Suppl. 67: No. 274 (Abstr.) Adams, D. O. and Yang, S. F Ethylene biosynthesis: identification of 1-aminocyclopropane-1-carboxylic acid as an intermediate in the conversion of methionine to ethylene. Proc. Natl. Acad. Sci. USA 76: Aida, R., Yoshida, T., Ichimura, K., Goto, R. and Shibata, M Extension of flower longevity in transgenic toreniaplants incorporating ACC oxidase transgene. Plant Science 138: Barden, L. E. and Hanan, J. J Effect of ethylene on carnation keeping life. J. Am. Soc. Hort. Sci. 97:

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