Result and Discussion

1. Cloning, Sequencing and Characterization of a Cattleya-ACO A Cattleya-ACO was cloned and sequenced from flower Cattleya orchid Lucky Strike variety using RT-PCR and 3’RACE techniques. Total RNA was purified from Cattleya flower (Figure 7). The presence of genomic DNA was later removed by DNase treatment. The RT-PCR products using ACO degenerate primers were shown in Figure 8. The major band that had expected size of 800 bp. was cloned into pDrive vector (Appendix Figure 2). The recombinant clones were identified by amplifying the recombinant plasmids with M13F and M13R primers (Figure 9). The 3’UTR was amplified using RT-PCR with oligo-dT and 3’end Cattleya-ACO specific primer (Figure 10). The recombinant clones were screened using colony hybridization with DIG labeling Cattleya ACO probe (Figure 11). The plasmid from selected clone was amplified with VFO2 and 3’end Cattleya-ACO specific primer to confirm the authenticity (Figure 12). The Cattleya-ACO cDNA sequence, missing at 5’end, was 1,081 bp. in length (Figure 13) contain an open reading frame which encoded 299 amino acid and 179 nucleotide of 3’ UTR region were. The comparison between Cattleya spp. was done at both nucleotide and amino acid sequences. The nucleotide sequence (ORF and 3’UTR) alignment between Cattleya spp. Lucky Strike variety and Cattleya bicolor indicated that the identity was 89.4% while the identity between Cattleya spp. Lucky Strike variety and Cattleya intermedia was 93.2%. The 3’UTR nucleotide sequence alignment indicated that Cattleya Bicolor and Cattleya spp. Lucky Strike variety was at 88.6% identity. The 3’UTR nucleotide sequence alignment indicated that Cattleya Intermedia and Cattleya spp. Lucky Strike variety was at 85% identity (Table 1.). The significantly difference between Cattleya spp. located at 3’UTR and deletion 8 bp of nucleotide at 3’UTR (TTAATTAG) was observed (Figure 14.). While the amino acid sequence alignment indicated high percent identity between Cattleya spp. at amino acid level were 96.7% (Table 2.). The phylogenetic tree of ACO nucleotide and amino acid sequences from Cattleya spp. Lucky Strike variety between other plants indicated that ACO gene of Cattleya spp. Lucky Strike variety has the closer

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relationship with Cattleya Bicolor and Cattleya Intermedia, respectively. Plant ACO genes were distinguished into two clusters of monocot and dicot plants. (Figure 15. and Figure 16.).

Figure 7. Ethidium bromide stained 1% agarose gel of Cattleya flower total RNA following an electrophoresis at 60 V for 1 hr.

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Figure 8. Ethidium bromide stained 1% agarose gel of open reading frame Cattleya-ACO genes following an electrophoresis at 60 Volt for 1 hr. Lane 1; PCR product size 800 bp.

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Figure 9. Identification the recombinant clones by amplifying the recombinant plasmids with M13F and M13R primers. Products sizes are 1000 bp.

1000 bp 1000 bp

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Figure 10. Ethidium bromide stained 1% agarose gel of 3’untranslated region Cattleya- ACO genes following an electrophoresis at 60 Volt for 1 hr. RT-PCR was carried out using 500 ng of total RNA sample with 3’end Cattleya-ACO specific primer and oligo-dT. Lane 1-2; PCR product size 426 bp.

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Figure 11. Colony hybridization analysis of clone 3’UTR Cattleya-ACOs. Using ORF Cattleya-ACOs as probes. The positive clones were shown in circular. .

Control

Selected colony

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Figure 12. Identification the recombinant clones by amplifying the recombinant plasmids with with 3’ end Cattleya-ACO

specific primer and VFO2. Products size is 229 bp.

250 bp 500 bp

M

229 bp

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1 GTGAATTCGC TTGCGAGAAC TGGGGATTCT TCGAGCTACT GAACCACGGT ATCTCCACAG E F A C E N W G F F E L L N H G I S T 61 AGCTGATGAA CCGAGTCGAA GCCGTGAACA AAGAAAATTA CCGGCGATTC CGCGAGCAGC E L M N R V E A V N K E N Y R R F R E Q 121 GGTTCAAAGA ATTCGCCGCC AAAACCCTTG ATTCCGGCGA GAAGGTCGAC GGCGATAATC R F K E F A A K T L D S G E K V D G D N 181 TGGACTGGGA AAGCACCTTC TTCCTCCGCC ATCTTCCGAA CTCCAACATC TCCCAAATCC L D W E S T F F L R H L P N S N I S Q I 241 CGGATCTGGA CGAGGACTGC CGGAGCACGA TGAAGGAGTT CGCCCGAGAG CTGGAGAAAC P D L D E D C R S T M K E F A R E L E K 301 TGGCGGAGTG GTTGCTGGAT CTGTTGTGCG AGGATTTGGG GCTTGAGAAA GGGTATTTGA L A E W L L D L L C E D L G L E K G Y L 361 AGAGGGTTTT TTGCGGGGGA TCGGATGGAT TGCCGACGTT CGGGACGAAG GTTAGCAATT K R V F C G G S D G L P T F G T K V S N 421 ATCCGCCGTG TCCGAAGCCG GAATTGATAA AGGGGCTGAG AGCTCATACG GATGCGGGAG Y P P C P K P E L I K G L R A H T D A G 481 GGATTATTCT ACTGTTTCAG GACGATAAGG TCAGCGGGCT TCAGCTGCTC AAGGATGGCC G I I L L F Q D D K V S G L Q L L K D G 541 AGTGGATTGA TGTTCCCCCG CTGCGCCATT CCATTGTTGT CAATATCGGA GATCAGCTTG Q W I D V P P L R H S I V V N I G D Q L 601 AGGTGATAAC AAATGGAAAA TACAAGAGTG TGATGCACAG GGTGGTCGCC CAAACCGACG E V I T N G K Y K S V M H R V V A Q T D 661 GCAACCGCAT GTCCATCGCC TCGTTCTACA ACCCCGGCAG CGACGCCGTC ATCTCCCCGG G N R M S I A S F Y N P G S D A V I S P 721 CGCCGGAGCT GGTGGAGAAA GAGGCGGAGG AGGAGAAGAA GGAAACAACT TATCCAAAAT A P E L V E K E A E E E K K E T T Y P K 781 TTGTGTTTCA GGACTACATG AATCTGTATA TTCGCCAGAA ATTTGAGGCT AAGCAGCCGA F V F Q D Y M N L Y I R Q K F E A K Q P 841 GGTTTGAGGC TATGAAGACC ATGGAAACTG TTTCCGGCTC ACAGCTCATT CCTACTGCTT R F E A M K T M E T V S G S Q L I P T A 901 AATTAATTAA TTAATTAGTA GAACATTTTA GTTCATATGG TTTAATTTAT GTGTTGCTGT . 961 TTGTTAGGGT CTAGTGGTTT GATTTGAACA ATTATAATGT GTATTTATCT TGTGTATGTT 1021TGTACCCTAT ATTTATCTAT ATATTATCTT TGGATGGTAA GGTATTAAAAAAAAAAAAAAA

Figure 13. Nucleotide sequence of Cattleya ACC oxidase cDNA and the deduced amino acid sequence. The translation start site is underlined. The asterisk denotes the stop codon.

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Figure 14. The sequence comparison of Cattleya spp. Major difference located at 3’UTR and deletion 8 nucleotide are TTAATTAG (respectived by box).

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(A) open reading frame and 3’untranslated region

Bicolor Intermedia Lucky Bicolor ***** 95.2 89.4

Intermedia ***** ***** 93.2 Lucky ***** ***** *****

(B) 3’untranslated region

Bicolor Intermedia Lucky Bicolor ***** 83.2 88.6

Intermedia ***** ***** 85 Lucky ***** ***** *****

Table1. The nucleotie sequence alignment indicated percent Identity between Cattleya spp. using MegAlign program (Lasergene). (A) In open reading frame and 3’untranslated region part, (B) In 3’untranslated region part.

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Bicolor Intermedia Lucky Bicolor ***** 97.7 96.7

Intermedia ***** ***** 96.7 Lucky ***** ***** *****

Table 2. The amino acid sequence alignment indicated Percent Identity between Cattleya spp. using MegAlign program (Lasergene).

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Figure 15. Phylogenetic tree of ACO nucleotide sequences from Cattleya spp. Lucky Strike variety and other plants. Cluster analysis was done using the MegAlign program (Lasergene). Cattleya intermedia accession : AY598794, Cattleya bicolor accession : AY598793, Laelia anceps accession : AY598795, Phalaenopsis sp. “True Lady” accession : AF004662, Dendrobium crumenatum accession : AF038840, x Doritaenopsis sp. accession : L07912, Oryza sativa accession : AK058296, Phyllostachys edulis accession : AB044747, Carica papaya accession : L76283, Carica papaya ripening-induduced ACO accession : AY077461, Musa acuminate accession : X95599

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Figure 16. Phylogenetic tree of ACO amino acid sequences from Cattleya Lucky Strike variety and other plants. Cluster analysis was done using the MegAlign program (Lasergene). Cattleya intermedia accession:AY598794, Cattleya bicolor accession : AY598793, Laelia anceps accession : AY598795, Phalaenopsis sp. “True Lady” accession : AF004662, Dendrobium crumenatum accession : AF038840, x Doritaenopsis sp. accession : L07912, Oryza sativa accession : AK058296, Phyllostachys edulis accession : AB044747, Carica papaya accession : L76283, Carica papaya ripening-induduced ACO accession : AY077461, Musa acuminate accession : X9559

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2. Identification of Functional Domains in ACO Putative Proteins ACC oxidase is a member of the non-heme iron and ascorbate dependent oxidase family. The functional domains presented in other plant ACC oxidases and members of the non-heme iron and ascorbate dependent oxidase family including ACC binding domain, ascorbate binding domain, ferrous binding domain, bicarbonate binding domain, leucine zipper and basic zipper domains were determined.

ACC is a substrate of all ACOs. Tyrosine, Argenine and Serine which found to be in ACC binding domain through mutagenesis (Dilley et al., 2001) also presented in Cattleya-ACO Lucky Strike variety (Figure 17A). Ascorbate is a cofactor for ACC oxidase activity. Ascorbate binding domain (Arg-X-Ser) is conserved in all ACOs and found in Cattleya-ACO Lucky Strike variety when X is represented by methionine (Figure 17B). Basic zipper (bZIP) domain facilitates oligomerization (Liu et al., 1999). The bZIP domain (consensus sequence, E..L..L..N..L) is found in all ACOs and present in Cattleya-ACO Lucky Strike variety (Figure 18A). Leucine zipper domain is facilitates oligomerization (Liu et al., 1999). The 4 haptad repeats of leucine in Leucine zipper domain are found in all ACOs. The 4 haptad repeats of leucine were also found in Cattleya-ACO Lucky Strike variety (Figure 18B). Ferrus ion is a cofactor for ACC oxidase activity. Fe2+ binding domain is conserved in all ACOs with consensus sequences of HXD/E…H (Rocklin et al., 1999). This conserved region is found at position histidine, threonine, aspatic acid and histidine in Cattleya-ACO Lucky Strike variety when X is represented by threonine (T) and aspartic acid (D) is found in all ACOs (Figure 19.). Bicarbonate is an active form of carbondioxide that activates ACC oxidase activity. Bicarbonate binding domain found through mutagenesis experiment located at argenine 175 (Dilley et al., 2001) is conserved in all ACOs also presented in Cattleya-ACO Lucky Strike variety (Figure 19).

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(A) ACC binding domain

(B) Ascorbate binding domain

Figure 17. Alignment of amino acid sequences in ACOs. (A) Conserved Thr, Arg and Ser in ACC binding domain are respectived by box. (B) Consensus sequence of RXS (R = arginine, X = methionine, S = serine) in ascorbate binding domain are respectived by box.

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(A) Basic zipper (bZIP) domain

(B) Leucine zipper domain

Figure 18. Alignment of amino acid sequence in ACOs. (A) Consensus sequence of Glu…Leu…Leu…Asn…Leu (E…L…L…N…L) in basic zipper (bZIP) domain are respectived by box. (B) The 4 haptad repeats of leucine in leucine zipper domain are respectived by box.

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Figure 19. Alignment of amino acid sequences in ACOs. Consensus sequence of His-X-Asp/Glu…His (HXD/E…H) in Fe2+ binding domains are respectived by box. The X is represented by threonine (T) and aspartic acid (D) is found in all ACOs. The Arg in the bicarbonate binding domain is respectived by box.

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