RESULTS AND DISCUSSIONS

1. Cloning and Sequencing The papaya genomic DNA was used as material for amplification with degenerate primers ETR1 amd ETR2. There are various of PCR product but the size that was estimated at 835 bp in ETR1 and 379 bp in ETR2 were selected to cloning into pDrive and transformation into E. coli DH5-α . The recombinant DNA was detected with PCR technique by ETR1 and ETR2 primers (Figure 4).

M 1 2 3 4 5 6

Figure 2 Ethidium bromide stained 1% agarose gel of PCR product with ETR1 primer following the electrophoresis at 60 volt for 1 hour. Molecular weight marker is shown on the left. Lane 1 papaya DNA amplified with ETR1F1 (forward) R1 (reverse), lane 2 amplified

with ETR1F1R2, lane 3 amplified with ETR1F1R3, lane 4 amplified with ETR1F2R1, lane 5 amplified with ETR1F2R2, lane 6 amplified with ETR1F2R3.

1000 bp 750 bp 500 bp 250 bp

~ 800 bp

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M 1 2 3 4 5 6 7 8 9

Figure 3 Ethidium bromide stained 1% agarose gel of PCR product with ETR2 primer following the electrophoresis at 60 volt for 1 hour. Molecular weight marker is on shown on the

left. Lane 1 papaya DNA amplified with ETR2F1R1, lane 2 amplified with ETR2F1R2, lane

3 amplified with ETR2F1R3, lane 4 amplified with ETR2F2R1, lane 5 amplified with ETR2F2R2, lane 6 amplified with ETR2F2R3.

1000 bp 750 bp 500 bp

250 bp ~ 400 bp

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M C 1 2 3 4 5 6 7 8 9 10

Figure 4 Ethidium bromide stained 1% agarose gel of PCR product of recombinant Plasmid.

Molecular weight marker is on shown on the left. Lane 1-15 recombinant plasmid

DNA clone#3 amplified with ETR1 size 835 base pairs, lane 16-17 clone #4

amplified with ETR2 size 376 base pairs.

M 11 12 13 14 15 16 17

835 bp

835 bp 379 bp

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2. Sequence comparison ETR1 sequence (835 bp) from papaya was compared to ethylene receptor genes of other plant including passion fruit (Passiflora edulis, AB015496, 21.8% identity), apple (Malus domestica, AF032448, 21.7% identity), geranium (Pelagonium x hortorum, AF141928, 23.4% identity), mango (Mangifera indica, AF227742, 23.0% identity), grape (Vitis vinifera, AF243474, 22.5% identity), papaya (Carica papaya, AF311942, 20.8% identity), rose (Rosa hybrida, AF380127, 22.0% identity), pear (Pyrus communis, AF38652, 22.45% identity), peach (Prunus persica, AF376830, 24.2% identity) and european beach (Fagus sylvalica, FS4420143, 22.8% identity). The results of sequence distance were shown in Table 5. Sequence distance revealed low similarity between CP-ETR1 and other plants. ETR2 sequence (376 bp) from papaya was compared to ethylene receptor genes of other plants including cucumber (Cucumis sativas, AB026500, 76.3% identity), rose (Rosa hybrida, AF154119, 76.6% identity), snapdragon (Antirrhinum majus, AY159363, 48.7% identity), tomato (Lycopersicon esculentum, AY600438, 70.6% percent identity) and strawberry (Fragaria x ananassa, FRX297513, 77.1% identity). The results of sequence distance were shown in Table 6. Sequence distance revealed high similarity. These results were used to design specific primers to CP-ETR1 and CP-ETR2, according to the conservative regions. In this study, partial sequences of two ethylene receptor gene, CP-ETR1 and CP-ETR2 were compared to other ethylene receptors. The sequence comparison between CP-ETR1 to other plants was shown low similarity while sequence distance between CP-ETR2 with other was shown high similarity. Although, the sequence of CP-ETR1 was divergence to other ethylene receptors but this is the partial sequence of CP-ETR1. The distance between CP-ETR1 and other plant could be change when complete cDNA sequences are used. Nevertheless, the partial sequences were sufficient for designing specific primers of CP-ETR1 and CP-ETR2.

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CP-ETR 1 Sequence comparison Partial sequence of papaya ETR1 (CP-ETR1) was cloned and sequenced. Percentage of

identity between plant ETR1 sequences was showed in Table 5. Sequence comparison between CP-ETR1 and other plant ETR1 genes was shown in Figure 8. Figure 5 ClustalW comparison between CP-ETR1 and other plant ETR1 genes. F227742Magifera CCAGCTGATGAATTGTTGATGAAGTATCAATATATTTCGGATTTCTTCATTGCACTTGCT 297 CPETR1F1R3 ------------------------------------------------------------ FSY420193Fagus ------------------------------------------------------------ AF243474Vitis TATTTTTCCATCCCTCTAGAGCTGATCTACTTTGTTAAGAAATCTGCAGTGTTTCCCTAT 332 AF227742Magifera TATTTTTCGATTCCTCTAGAGCTGATCTACTTTGTGAAGAAATCTGCTGTGTTTCCATAT 357 CPETR1F1R3 ------------------------------------------------------------ FSY420193Fagus ------------------------------------------------------------ AF243474Vitis AGATGGGTGCTTGTGCAGTTTGGTGCTTTTATTGTCCTATGTGGAGCAACGCATCTTATT 392 AF227742Magifera AGATGGGTCCTTGTGCAGTTTGGTGCTTTCATAGTTTTGTGTGGGGCAACACATCTTATT 417 CPETR1F1R3 ------------------------------------------------------------ FSY420193Fagus ------------------------------------------------------------ AF243474Vitis AACTTGTGGACTTTTGGAATGCATTCGAGAAATGTTGAAGTAGTTATGACCACTGCAAAG 452 AF227742Magifera AACTTGTGGACTTTTAGTATGCATTCAAGAACTGTGGCAATAGTAATGACCACTGCCAAG 477 CPETR1F1R3 ------------------------------------------------------------ FSY420193Fagus ------------------------------------------------------------

AF243474Vitis GTTTTGACTGCTGTGGTGTCTTGTGTAACTGCCCTAATGCTGGTTCACATTATTCCTGAT 512 AF227742Magifera GTTTTCACTGCTGCTGTGTCTTGTGCGACGGCCCTTATGCTCGTGCATATAATTCCTGAT 537 CPETR1F1R3 ------------------------------GCATCAAAACCA--TCAGAGCATACC-GTT 27 FSY420193Fagus ------------------------------GCATCAAAACCA--TCAGAGCATACC-GTT 27 ** * * ** * ** ** * * AF243474Vitis CTACTGAGTGTGAAGACTAGAGAACTATTTTTGAAAAACAAAGCTGCTGAGCTTGATCGA 572 AF227742Magifera CTGTTAAGTGTTAAAACAAGAGAACTATTTTTGAAAAACAAGGCTGCAGAACTTGATAGA 597 CPETR1F1R3 TTGTCGAAAGTTCAGGCCAATCATTAATTTGGAAATTGGAGAGATGCAGAAGCGGAACAC 87 FSY420193Fagus TTGTCGAAAGTTCAGGCCAATCATTAATTTGGAAATTGGAGAGATGCAGAAGCGGAACAC 87 * * ** * * * * **** ** * * *** ** ** AF243474Vitis GAAATGGGTCTGA-TTCGTACGCAGGAAGAAACAGGGAGGCATGTTAGAATGCTGACTCA 631 AF227742Magifera GAAATGGGCCTGA-TCCGCACACACGAAGAAACCGGTCGGCATGTTAGGATGTTGACGCA 656 CPETR1F1R3 GAACCGCAACAACCTCCCCAGGCATGTAGTTTCCTGCAAG-AGGCCGTAATCTTGCCACA 146 FSY420193Fagus GAACCGCAACAACCTCCCCAGGCATGTAGTTTCCTGCAAG-AGGCCGTAATCTTGCCACA 146 *** * * * * * ** * ** * * * * * ** ** * ** AF243474Vitis TGAGA--TCAGAAGCACTCTTGATAGGCATACTATACTAAAGACCACGCTTATAGAGTTA 689 AF227742Magifera TGAGA--TCAGAAGCACTTTGGATAGACATACCATACTAAAGACCACTCTTGTCGAATTG 714 CPETR1F1R3 GGACAATTTGGTGATATTTTTACTGCACGACTACTACTGAAGACTTGATTGATCA----- 201 FSY420193Fagus GGACAATTTGGTGATATTTTTACTGCACGACTACTACTGAAGACTTGATTGATCA----- 201 ** * * * * * * * * **** ***** * *

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AF243474Vitis GGTAGAACTTTGTCTCTGGAAGAGTGTGCATTGTGGA----TGCCAACACGTACTGGCTT 745 AF227742Magifera GGAAGAACTTTGGCACTGGAAGAGTGTGCCTTATGGA----TGCCGACGCAAACTGGTTT 770 CPETR1F1R3 --CAGGATGTTGA-ATAGGTACCGTATACCCAATGGGGTTCTGCTGACGGAGAGTGTAAG 258 FSY420193Fagus --CAGGATGTTGA-ATAGGTACCGTATACCCAATGGGGTTCTGCTGACGGAGAGTGTAAG 258 ** * *** ** * ** * * *** *** ** * ** AF243474Vitis AGAGCTTCAACTTTCCTACACTCTGCGTCAACAGAATCCTGTTGGCTTTACCATACCCAT 805 AF227742Magifera AGAGCTTCAACTCTCATACACACTTCATCAACAGAATCCTGTTGGGTATACTGTGCCCAT 830 CPETR1F1R3 AAAGTTGTAAGTCTAACCCAATACGTGTTGGCA----TCCACAAGGCACACTCTTCCAGT 314 FSY420193Fagus AAAGTTGTAAGTCTAACCCAATACGTGTTGGCA----TCCACAAGGCACACTCTTCCAGT 314 * ** * ** * * ** * ** * * ** * ** * AF243474Vitis -TCAGCATCCTGTTATTAATCAAGTCTTCAGTAGCAATCGTGCAGTTAAGATATCACCAA 864 AF227742Magifera -CCAACTTCCTGTGATCAGTCAAGTATTCACCAGCAATCATGCGGTAAAGATATCACCAA 889 CPETR1F1R3 GCCAATGTTCTAC--CTAGTTCAACTAGAGTGGTCTTTAAAATAGTATGTCTATCAAGAG 372 FSY420193Fagus GCCAATGTTCTAC--CTAGTTCAACTAGAGTGGTCTTTAAAATAGTATGTCTATCAAGAG 372 ** * ** * * * * * ** ***** * AF243474Vitis ATTGCCCAGTGGCAAGATTGCGGCCTCTTGCAGGAAAATACATGCCTGGGGAGGTTGTTG 924 AF227742Magifera ATTGTCCAGTGGCTAGGTTAAGGCCTCTTGCAGGAAAATATATGCCTGGCGAGGTGGTTG 949 CPETR1F1R3 TGCTTCTAATCTCATGAGTCAACATCCTAACATGCCGGCCTGTTTCTTCCTGTGTGCGGA 432 FSY420193Fagus TGCTTCTAATCTCATGAGTCAACATCCTAACATGCCGGCCTGTTTCTTCCTGTGTGCGGA 432 * * * * * * ** ** * * ** ** AF243474Vitis CTGTGCGTGTACCACTTCTACATCTCTCTAATTTCCAAATCAATGACTGGCCTGAGCTTT 984 AF227742Magifera CTGTTCGTGTTCCACTCCTACATCTATTAAATTTTCAAATTAATGATTGGCCAGAACTTT 1009 CPETR1F1R3 TTAAACCCATTTCTCTGTCAAGCTCCGCAGCTTTGTTCTTCAGAAATAGCTCTCTAGTTT 492 FSY420193Fagus TTAAACCCATTTCTCTGTCAAGCTCCGCAGCTTTGTTCTTCAGAAATAGCTCTCTAGTTT 492 * * * * ** * *** * * * * * *** AF243474Vitis CAACAAAGCGCTATGCTTTGATGGTTTTGATGCTTCCCTCAGACAGTGCAAGGCAATGGC 1044 AF227742Magifera CTACAAAACGCTATGCTTTGATGGTTTTGATGCTTCCCTCAGATAGTGCAAGGCAGTGGC 1069 CPETR1F1R3 TCACACTCAACAAATCAGGAATAATATGGACA---AGCATAAGTGCAGTTGCACAA-GAC 548 FSY420193Fagus TCACACTCAACAAATCAGGAATAATATGGACA---AGCATAAGTGCAGTTGCACAA-GAC 548 *** * * * ** * * ** * * * ** * * AF243474Vitis ATGTTCATGAACTGGAACTTGTTGAAGTTGTTGCTGACCAGGTGGCAGTGGCTCTTTCAC 1104 AF227742Magifera ATGTCCATGAGTTGGAACTTGTTGAAGTGGTTGCTGATCAGGTGGCTGTTGCTCTTTCAC 1129 CPETR1F1R3 ACAACCGC-AGTCAAAACCT-TTGCAGTGGTCATC-ACCA--CTGCCACGGTTCTAGTGT 603 FSY420193Fagus ACAACCGC-AGTCAAAACCT-TTGCAGTGGTCATC-ACCA--CTGCCACGGTTCTAGTGT 603 * * * *** * *** *** ** * ** ** * *** AF243474Vitis ATGCTGCAATCTTAGAAGAGTCAATGAGAGCAAGGGATCTTCTTATGGAGCAGAATGTT- 1163 AF227742Magifera ATGCTGCCATCTTAGAGGAGTCAATGAGAGCAAGGGATCTTCTTGTGGTGCAGAATATC- 1188 CPETR1F1R3 GCATGGTAAACGTCCATAAATTAATAAGAT----GAGTTGCCCCACAAAGAACAATAAAA 659 FSY420193Fagus GCATGGTAAACGTCCATAAATTAATAAGAT----GAGTTGCCCCACAAAGAACAATAAAA 659 * * * * * * * *** *** * * * * * *** AF243474Vitis GCACTTGACCTAGCCAGGAG------AGAAGCAGAAACAGCAGTTCGTGCTCGCAATGAT 1217 AF227742Magifera GCGCTTGACCGTGCCAGAAG------AGAAGCAGAAACAGCCATTTGTGCTCGTAATGAT 1242 CPETR1F1R3 GCACCAAACTGCACAAGGACCCATCTATATGGAAACACAGCAGATT-TCTTCACAAAG-T 717 FSY420193Fagus GCACCAAACTGCACAAGGACCCATCTATATGGAAACACAGCAGATT-TCTTCACAAAG-T 717 ** * ** * ** * * * * * * ***** * * ** ** * * AF243474Vitis TTCTTGGCTGTTATGAACCATGAGATGAGAACACCTATGCATGCCATCATTGCACTCTCA 1277 AF227742Magifera TTCCTGTCCGTTATGAACCATGAAATGAGAACTCCAATGCATGCGATCATTGCCCTTTCT 1302 CPETR1F1R3 AGATTAGC--TCAAGA-----GGGATGGAAAAATAAGCAAGTGCAATGAA-GAAATCTGA 769 FSY420193Fagus AGATTAGC--TCAAGA-----GGGATGGAAAAATAAGCAAGTGCAATGAA-GAAATCTGA 769 * * * * ** * *** ** *** ** * * * *

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AF243474Vitis TCCTTGCTGCAAGAAACTGAGCTGACTCCTGAGCAGCGCCTGATGGTTGAAGCCATCCTA 1337 AF227742Magifera TCTTTACTGCAAGAAACTGAGTTGACCCCTGAGCAGCGACTGATGGTTGAAACAATTCTT 1362 CPETR1F1R3 AATATACTGATATTTCATCAACTTTTCATAAGATGGCCAGTGCGGCTCAATACAAT---- 825 FSY420193Fagus AATATACTGATATTTCATCAACTTTTCATAAGATGGCCAGTGCGGCTCAATACAAT---- 825 * *** * * * * ** ** * * * * ** AF243474Vitis AAGAGCAGTAGTCTTTTGGCTACACTCATAAATGATGTACTAGATCTTTCAAGGCTTGAA 1397 AF227742Magifera AAGAGCGGTAATCTCTTAGCTACTCTAATAAATGATGTATTAGATCTTTCAAGGCTTGAA 1422 CPETR1F1R3 ------------------------------------------------------------ FSY420193Fagus ------------------------------------------------------------ AF243474Vitis GATGGTAGCCTTCAACTTGAAAATGGAACTTTTAACCTTCATGCGTTATTCAGGGAGGTC 1457 AF227742Magifera GATGGTAGCCTACAACTTCAACTTGGAACTTTTAACCTTCATGCTGTATTTAAAGAGGTG 1482 CPETR1F1R3 ------------------------------------------------------------ FSY420193Fagus ------------------------------------------------------------ AF243474Vitis CTTTACTTGATCAAGCCTATTGCGTCTGTCAAAAAGTTGTTTGTTTCATCAAGTTTGGCT 1517 AF227742Magifera CTTAATCTGATCAAACCTATTGCATCAGTCAAGAAGTTGCTTGTTGCATTGAATTTGGCT 1542 CPETR1F1R3 ------------------------------------------------------------ FSY420193Fagus ------------------------------------------------------------ AF243474Vitis CCAGACTTGCCTGAATTTGCTGTTGGTGATGAAAAACGCCTCATGCAAATTATGTTAAAT 1577 AF227742Magifera CCTGATCTTCCTGAATATGCTGTTGGCGATGAAAAACGCTTTATGCAAACTCTCTTAAAT 1602 CPETR1F1R3 ------------------------------------------------------------ FSY420193Fagus ------------------------------------------------------------ AF243474Vitis GTTGTTGGTAATGCTGTGAAATTCTCGAATGAGGGAAACATCTCAATTTTTGCTTTTATT 1637 AF227742Magifera GTTGTTGGTAATGCTGTGAAATTCACAAAAGAGGGCAACATCTTAATCACTGCTTTTGTT 1662 CPETR1F1R3 ------------------------------------------------------------ FSY420193Fagus ------------------------------------------------------------ AF243474Vitis GCAAAATCAGAATCTTTAAGAGATTCTCGCGCGCCAGATTTTTTCCCAGTGCACAGTGAT 1697 AF227742Magifera GCAAAATCAGAATCTTTTCTAGATTCTCGAACCCCAGAGTTCTTCCCAGTGCCAAGTGAT 1722 CPETR1F1R3 ------------------------------------------------------------ FSY420193Fagus ------------------------------------------------------------ AF243474Vitis AATCACTTTTATTTGCGTGTACAGGTTAAGGATTCAGGAGCAGGAATTGACCCCCAAGAT 1757 AF227742Magifera AATCATTTTTATCTGCGTGTACAGGTTAAAGATTCAGGATCAGGCATTAGCCCCCAAGAT 1782 CPETR1F1R3 ------------------------------------------------------------ FSY420193Fagus ------------------------------------------------------------ AF243474Vitis ATTTCTAAGCTGTTCACCAAATTTGCACAATGTCAATCACTAGCAGCTAGAAATTCTGGT 1817 AF227742Magifera ATTCCCAAGTTATTTACAAAGTTTGCTCAAAATCAATCAATAGCAACCAGAAATTCCCAT 1842 CPETR1F1R3 ------------------------------------------------------------ FSY420193Fagus ------------------------------------------------------------

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AF243474Vitis GGCAGTGGACTTGGCCTTGCAATTTGTAAGAGGTTTGTAAATCTTATGGGAGGACATATT 1877 AF227742Magifera GGCAGTGGACTTGGCCTTGCTATTTGTAAGAGGTTTGTTAATCTTATGGAGGGACATATT 1902 CPETR1F1R3 ------------------------------------------------------------ FSY420193Fagus ------------------------------------------------------------ AF243474Vitis TGGATAGAAAGTGAAGGGCTTGGAAAGGGAAGCACTGCTGTCTTCATTGTGAAACTTGGG 1937 AF227742Magifera TGGATTGAAAGTGAAGGCCTCGGCAAGGGATGCACTGCTGTCTTTGTTGTGAAACTTGGG 1962 CPETR1F1R3 ------------------------------------------------------------ FSY420193Fagus ------------------------------------------------------------ AF243474Vitis ATTCCAGAGTACTCAAATGAATCTAAGCTCCCTTTTTTTCCTAAAGTGTCAGCAAACCAT 1997 AF227742Magifera ATCCCAGAGAGCTCGAACGAATCTAAGCTCTCTTTCATGCCGAAAATGTCAG--GACCA- 2019 CPETR1F1R3 ------------------------------------------------------------ FSY420193Fagus ------------------------------------------------------------ AF243474Vitis TGTCAGACAAATTTTCCAGGACTCAAAGTTCTTGTTATGGATGACAATGGGGTTAGCAGA 2057 AF227742Magifera ---CAGACAAATTTTACAGGTCTCAAAGTTCTTGTCCTGGATGATAACGGAGTTAGCAGG 2076 CPETR1F1R3 ------------------------------------------------------------ FSY420193Fagus ------------------------------------------------------------ AF243474Vitis TCAGTGACAAAGGGTCTGCTTGTGCACCTGGGATGTGATGTAACAACTGTAAGCTCAGCT 2117 AF227742Magifera TCAGTGACGAAAGGGCTTTTGGTGCACCCTGGATGTGATGTGATGACGGTTGGCTCGAGC 2136 CPETR1F1R3 ------------------------------------------------------------ FSY420193Fagus ------------------------------------------------------------ AF243474Vitis GATGACTGCTTGCGGGTTGTTTCTCAGGAACACAATGTGGTGTTCTTAGATGTGTGTATG 2177 AF227742Magifera GAGGAGTGCCTACATGTTGTATCTCAGGAACACAAGGTGGTCTTCATGGATGTCGGCATA 2196 CPETR1F1R3 ------------------------------------------------------------ FSY420193Fagus ------------------------------------------------------------ AF243474Vitis CCTGGTGTTGATGGTTATGAAGTTGCTATTCGTATACATGAAAAATTTACAACATGTCGT 2237 AF227742Magifera CCTGGTATAGATGGTTATGAAGTTGCTGTCCACATACACCAAAAGTTTACAAGGCGTCAT 2256 CPETR1F1R3 ------------------------------------------------------------ FSY420193Fagus ------------------------------------------------------------ AF243474Vitis CAAGGGCCATTGCTGGTAGCACTTATGGGAAACACGGACAGAGTGACAAAGGAGAACTAC 2297 AF227742Magifera GAAAGGCCATTAATAGTAGCGCTGACTGGAAATACTGACAAAATAACCAAGGAGAATTGC 2316 CPETR1F1R3 ------------------------------------------------------------ FSY420193Fagus ------------------------------------------------------------ AF243474Vitis TTGAGGGTGGGTATAAATGGAGTTGTACTGAAACCTGTTTCAGTTGATAAGATGAGGTGT 2357 AF227742Magifera ATGAGTGTTGGAATGGATGGGGTTATATTGAAACCCATTTCGCTAGAAAAAATGAGGAGC 2376 CPETR1F1R3 ------------------------------------------------------------ FSY420193Fagus ------------------------------------------------------------

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AF243474Vitis GTATTATCAGAGGTTCTAGAGCACCGTTTTCTGTTTGAGACCTGTTGAGTTGTAGGGAGT 2417 AF227742Magifera GTTTTATTAGGCCTTTTGGAGCACCGGGTTCTATTCGAGGCCATATAAGCGCAGAGGAGC 2436 CPETR1F1R3 ------------------------------------------------------------ FSY420193Fagus ------------------------------------------------------------ AF243474Vitis GCTTGCATGAGAAGCCAAAAAAGGGTGAGTGCTTCTTCAAGCCAAAATTAGAGTCTGTAC 2477 AF227742Magifera A--------AGCTGCCGGATGCCATTGAGC----CTGGAAAAAAATAGTACAGGTAATGC 2484 CPETR1F1R3 ------------------------------------------------------------ FSY420193Fagus ------------------------------------------------------------ AF243474Vitis ATAACTTATACGGCAATGCAGAAATATTGTGGAAGATGTAGTGATGGTGGTAATAGTGTA 2537 AF227742Magifera ATGTTTTTCTC-------TAGAGATATTTAGTTTTGTGTACATACCCAG---ATGGCGTT 2534 CPETR1F1R3 ------------------------------------------------------------ FSY420193Fagus ------------------------------------------------------------ AF243474Vitis ACATAATATGCTACAAGCAGGTTTGTTTATTCTTCTTGTTCAACTTGTAAAATGAAAACT 2597 AF227742Magifera TTAGACCATTGGAGTGATTTGAGTGATGACAAATCACAAGAAATTCGAAAAGC--AACTT 2592 CPETR1F1R3 ------------------------------------------------------------ FSY420193Fagus ------------------------------------------------------------ AF243474Vitis CAAATAAATGGTAAATTAACATATTGTTTAAAAAAAAAAAAAAAAAAAAAAAAAAAA 2654 AF227742Magifera TAAGCTCGTGCCGAATTC--------------------------------------- 2610 CPETR1F1R3 --------------------------------------------------------- FSY420193Fagus ---------------------------------------------------------

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Table 7 Sequence distances of CP-ETR1 sequences from papaya and other plant ETR1 genes. The analysis was done using the MegAlign program (DNASTAR).

1. Passion fruit (Passiflora edulis) : AB 015496 2. Apple (Malus domestica) : AF 032448 3. Geranium (Pelargonium x hortorum) : AF 141928 4. Mango(Mangifera indica) : AF227742 5. Grape (Vitis vinifera) : AF 243474 6. Papaya (Carica papaya) : AF 311942 7. Rose (Rosa hybrida) : AF 380127 8.Pear (Pyrus communis) : AF 386520 9. Peach (Prunus persica) : AF 396830 10. European beach (Fagus sylvalica) : FSY 420193 11. CP-ETR1

Percent Identity 1 2 3 4 5 6 7 8 9 10 11

1.Passifon fruit *** 66.2 66.6 68.9 68.6 57.8 77.7 53.4 56.4 83.0 21.8 2.Apple *** *** 68.4 70.3 69.8 57.5 87.6 72.2 64.3 85.3 21.7 3.Geranium *** *** *** 68.3 69.5 58.3 82.9 59.1 60.0 84.2 23.4 4.Mango *** *** *** *** 71.0 56.9 83.6 60.5 61.8 83.9 23.0 5.Grape *** *** *** *** *** 59.8 82.3 59.0 61.7 84.3 22.5 6.Papaya *** *** *** *** *** *** 67.0 53.4 52.7 69.1 20.8 7.Rose *** *** *** *** *** *** *** 87.6 89.2 85.3 22.0 8.Pear *** *** *** *** *** *** *** *** 45.2 85.2 22.4 9.Peach *** *** *** *** *** *** *** *** *** 87.2 24.2 10.Beech *** *** *** *** *** *** *** *** *** *** 22.8 11.CP-ETR1 *** *** *** *** *** *** *** *** *** *** ***

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CP-ETR 2 Sequence comparison Partial sequence of papaya ETR2 (CP-ETR2) was cloned and sequenced. Percentage of identity between plant ETR2 sequences was showed in Table 6. Sequence comparison between CP-ETR2 and other plant ETR2 genes was shown in Figure 9. Figure 6 ClustalW comparison between CP-ETR2 and other plant ETR2 genes. FRX297513 AGACTGTCTAGGCCAGCGCTTTACACGGCGCGAGGTGGCCGGCGGTTTAGTGCCCTCAGT 60 AF154119 ------------------------------------------------------------ AB026500 ------------------------------------------------------------ CPETR2 ------------------------------------------------------------ AY600438 ------------------------------------------------------------ FRX297513 TTGGTTTTTAATTTTAAACAATATTCACTCCTTTTCTCCTTCTGTACTGTGTAATTATTT 120 AF154119 ------------------------------------------------------------ AB026500 ------------------------------------------------------------ CPETR2 ------------------------------------------------------------ AY600438 ------------------------------------------------------------ FRX297513 TTTTCTTTATATATCGGGATTTGTTTACTTACTTGGTTTTGGTTTCGAATTTTGGAGAGA 180 AF154119 ------------------------------------------------------------ AB026500 ------------------------------AGCTCCACGCGGTGGCGGCCGCTCTAGAAT 30 CPETR2 ------------------------------------------------------------ AY600438 ------------------------------------------------------------ FRX297513 AGGGTTGGGTGCCTAGATTGTGGTTTTAGTATTTATATGAGAGTGGTGGTTGTGATGGGT 240 AF154119 ------------------------------------------------------------ AB026500 AG---TGGATCCCCCG-----GGCTGCAGGAATTCGGCACGAGGATTAATGGATGAGGGA 82 CPETR2 ------------------------------------------------------------ AY600438 ------------------------------------------------------------ FRX297513 CATTGTTGATGGCTGCAGAGGAGTTTGAGATTGTTGCATGTAAGATCTGATGATGATGAA 300 AF154119 ------------------------------------------------------------ AB026500 GATGG--GATTGC----GAATCGTTTGAGGTTTGTGCATGT--GCTGGAATGCTAA-GAT 133 CPETR2 ------------------------------------------------------------ AY600438 ------------------------------------------------------------ FRX297513 CAAGAACATATCGATGGTGGTTGAGTTGGAATCTGGGTTGAAACGTCTCTAGTGTGCTTT 360 AF154119 ------------------------------------------------------------ AB026500 CTGAAGC---TGGATGTGAATGGAATCTGA---TGTATCGGCGACTGGTTGGTTTTCCTT 187 CPETR2 ------------------------------------------------------------ AY600438 ------------------------------------------------------------ FRX297513 TGTCAGGAAATGGGAATTAGAGGTTGTCAATTTGTGATTGTAGTCCATGGATGTTTTAGC 420 AF154119 ------------------------------------------------------------ AB026500 TGTCAGGAAATGGGAATTGGAGATGCGTGTACTGTTGTTATAGTTAATGCCAAGTGCGAC 247 CPETR2 ------------------------------------------------------------ AY600438 ------------------------------------------------------------ FRX297513 TCAAGTTTTAGTCTGTAGTGGGGGTTTGAACGTTCGGCGTTGTTTTTACTGATTGATGGA 480 AF154119 ------------------------------------------------------------ AB026500 TCCGTTATCGGCAAGTTGTTGGGGCTTCAATCTCTCG----AATTTTGGGCGTTAATGGA 303 CPETR2 ------------------------------------------------------------ AY600438 ------------------------------------------------------------

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FRX297513 TTGAAGAAGC--AGTCTTTGGATGCAATGTTAAAAGCATTAGCATCTGGGCTATCGATTT 538 AF154119 ------------------------------------------------------------ AB026500 TTGAAGAATCTTGGGTTTTGGATGCAATGTTAAAGGCATTGCCATCTGGGTTTCTGATTT 363 CPETR2 ------------------------------------------------------------ AY600438 --------------------------ATGTTGAGGACGTTAGCATCAGCTTTGTTGGTTT 34 FRX297513 CTTTGCTACTGGTGTGTGTTTCGGCGTCTGATGGTGGATTTCCGCGGTGCAATTGTGACG 598 AF154119 ------------------------------------------------------------ AB026500 TGTTATTACTGGCATCTGTTTCTGCTGCCGACAACGGATTTCCGAGATGTAATTGTGATG 423 CPETR2 ------------------------------------------------------------ AY600438 TGTCCTTCTTTGTTTCCTTATCGGCTGCTGATAATGGTTTCCCGCGATGTAACTGTGATG 94 FRX297513 ACGAGGGAAGCTTCTGGAGCATTGATAGCATACTAGAATGTCAGCGAGTGAGCGATTTCT 658 AF154119 ------------------------------------------------------------ AB026500 ATGAGGGTAGTTTATGGAGCATCGACAGCATTTTGGAGTGTCAGCGTGTGAGTGATTTTT 483 CPETR2 ------------------------------------------------------------ AY600438 ATGAGGGA---TTTTGGAGCATTGAGAGTATCTTAGAGTGCCAAAAGATTAGTGATCTCT 151 FRX297513 TGATCGCGGTGGCCTACTTTTCGATCCCCATTGAGCTGCTTTACTTTGTCAGCTGCTCGA 718 AF154119 ------------------------------------------------------------ AB026500 TGATTGCTGTGGCCTACTTCTCTATTCCTATTGAACTTCTCTATTTCGTTAGTTGCTCCA 543 CPETR2 ------------------------------------------------------------ AY600438 TTATTGCGATTGCGTATTTTTCCATCCCAATTGAGCTCCTTTACTTTGTCAGTTGTTCTA 211 FRX297513 ATGTACCTTTCAAATGGGTTCTCTTTGAGTTTATTGCCTTCATTGTGCTATGTGGAATGA 778 AF154119 -----------------------------TTTATCGCGTTTATCGTGCAATGTGGAATGA 31 AB026500 ATGTACCCTTCAAATGGGTTCTATTTCAGTTTATTGCCTTCATTGTTCTATGTGGGTTGA 603 CPETR2 ---------------------------AGTTTGTTGCCTTCATTGTGCTATGTGGACTCA 33 AY600438 ACTTTCCATTCAAATGGGTGCTCTTCCAATTTATTGCATTCATTGTTCTGTGTGGGATGA 271 *** * ** ** ** ** * ***** * * FRX297513 CACATTTGCTGAATGGCTGGACTTA---TGGTCCTCAC------CCGTTTCAGTTGATGC 829 AF154119 CACATTTGCTGAATGGCTGGACTTA---TGGTCCTCAC------CCTTTTCAGCTAATGC 82 AB026500 CCCATTTGCTCAATGGCTGGACTTA---TGGCCCCCAC------TCATTCCAATTGATGC 654 CPETR2 CCCATTTGCTCAACGGATGGACCTA---TGGCCCCCATACCCATCCATTTCAGCTCATGC 90 AY600438 CTCATTTGCTCAATTTCTGGACTTACTATGGCCAACAC------CCGTTTCAGCTTATGC 325 * ******** ** ***** ** *** * ** * ** ** * **** FRX297513 TCGCTCTCACAGTTTTCAAAATTCTCACTGCTCTTGTATCATGTGCCACAGCTATAACGC 889 AF154119 TTGCTCTCACAGTTTTCAAAATTCTCACTGCTCTGGTATCATGTGCCACTGCTATAACGC 142 AB026500 TAGCTCTCACTGTCTTCAAAATTCTTACCGCCCTGGTTTCCTGTGCTACTGCTATAACCC 714 CPETR2 TAGCACTCACTGTCTTCAAGATCCTCACTGCTTTGGTCTCATGTGCCACTGCTATTACTC 150 AY600438 TTGCTTTAACCATTTTTAAAGTCCTCACTGCACTGGTATCCTTCGCCACGGCTATAACCC 385 * ** * ** * ** ** * ** ** ** * ** ** * ** ** ***** ** * FRX297513 TCATCACTCTTATTCCTTTGCTTCTCAAAGTGAAAGTGAGAGAATTCATGTTGAAGAAGA 949 AF154119 TCATCACTCTCATTCCTTTGCTTCTCAAGGTCAAAGTGAGAGAATTTATGTTGAAGAAGA 202 AB026500 TCATCACACTCATTCCTTTGCTTCTCAAGGTGAAAGTGAGAGAGTTTATGTTGAAGGAGA 774 CPETR2 TCATTACTCTTATTCCTTTGCTTCTTAAAGTCAAGGTAAGGGAGTTCATGTTGAAGAAAA 210 AY600438 TTATTACCCTCTTTCCTATGCTGCTTAAAGTCAAGGTGAGGGAATTTATGCTGAAAAAGA 445 * ** ** ** ***** **** ** ** ** ** ** ** ** ** *** **** * * FRX297513 AAACTTGGGACCTTGGAAGAGAGGTTGGAATTATAATGAGACAGAAAGAAGCTGGAATGC 1009 AF154119 AGACTTGGGACCTTGGAAGAGAGGTTGGGATTATAATGAGACAGAAAGAAGCTGGAATGC 262 AB026500 AGACATGGGATCTAGGAAGAGAAGTTGGTATGATACTGAAGCAGAAAGAAGCTGGCTTAC 834 CPETR2 AGGCCTGGGATCTGGGGCGAGAAGTTGGGATCATAATGAAACAGAAAGAGACAGGATTGC 270 AY600438 AGACTTGGGATCTTGGTAGAGAAGTTGGATTAATAAAGATGCAAAAAGAAGCTGGATGGC 505 * * ***** ** ** **** ***** * *** ** ** ***** * ** * FRX297513 ATGTCCGAATGCTTACCCAAGAGATTCGCAAGTCCCTTGATAGACATACAATATTGTCCA 1069 AF154119 ATGTTCGAATGCTTACCCAAGAGATTCGCAAGTCTCTTGATAGACATACAATATTGTCCA 322 AB026500 ACGTTCGGATGCTTACTCAAGAGATCCGCAAGTCTCTTGATCGACATACTATACTTTACA 894 CPETR2 ATGTTCGCATGCTTACTCAAGAGATCCGTAAGTCACTAGATAGGCATACTATTTTATACA 330 AY600438 ATGTTCGGATGCTTACACAGGAGATTCGAAAGTCACTTGACCGTCATACAATACTCTACA 565 * ** ** ******** ** ***** ** ***** ** ** * ***** ** * * ** FRX297513 CAACCCTTTTTGAGCTATCTGAGACATTGGGTTTGCAGTACTGTGCAGTTTGGATGCCTA 1129 AF154119 CAACCCTCTTTGAGCTATCTGAGACATTGGGTTTGCAGTACTGTGCAGTTTGGATGCCTA 382 AB026500 CGACCATGTTTGAGCTGTCTGAAACTTTGGGTTTGCACTACTGTGCAGTTTGGATGCCTA 954

32

CPETR2 CAACTTTGGTTGAGCTCTCCAAGACATTGGGTTTGCACTACTGCGCAGTTTGGA------ 384 AY600438 CAACTCTGGTGGAGCTATCAAAGACGCTGGATTTGCATAACTGTGCTGTTTGGAAGCCCA 625 * ** * * ***** ** * ** *** ****** **** ** ******* FRX297513 ATGAAATTAAAACAGAGATGATCCTGACCCATGAGTTGAAAGGGAAGAACTATTCTAATA 1189 AF154119 ATGAAATTAAAACGGAGATGATCCTGACCCATGAGTTGAAAGGGAGGAATTATTCTAATA 442 AB026500 ATGAGAGCAAGACACTGATGAATTTGACTCATGAGTTGAAAGATCGCAGCTTCTCAAATG 1014 CPETR2 ------------------------------------------------------------ AY600438 ATGAGAATAAAACTGAGATGAACCTAATTCATGAGCTGAGAGACAGTAGCTTTAATAGCG 685 FRX297513 TGTACAACTTTTCTATACCAATTGGCGATCCAGATGTTGTACTTATTAAAGGGAGTGATG 1249 AF154119 TGTACAACTTTTCTATACCAATAGGTGATCCAGATGTTGCACTTATCAAAGGGAGTGATG 502 AB026500 GATACAATGTCTTCATACCAATAAGTGATTCTGATGTCATTAAAATCAAGGGCAGCGATG 1074 CPETR2 ------------------------------------------------------------ AY600438 CGTATAATTTACCTATCCCGAGAAGTGATCCAGATGTTATACAGGTTAAGGAGAGTGACG 745 FRX297513 GGGTCAACATTCTTCGGCCAGATTCAGCACTTGTATGTGGAAGCAGTGGTGATTCTGGTG 1309 AF154119 GGGTCAACATCCTTGGGCCAGATTCAATACTCGTGTCCGGAAGCAGTGGTGATTTTGGTG 562 AB026500 GTGTTAACGTTCTTGGGCCTAATTCGGCACTTGTTGTCGCTAACTGTGGCGAGTCTGATG 1134 CPETR2 ------------------------------------------------------------ AY600438 GAGTAAAGATACTTGATGCCGACTCCCCACTTGCTGTTGCGAGTAGTGGAGGGAGTAGGG 805 FRX297513 AGCCGGGACCAGTAGCTGCAATTCGGATGCCAATGCTTCGGGTTTCCAATTTTAAAGGGG 1369 AF154119 AGCCGGGACCAGTAGCTGCAATACGGATGCCAATGCTTCGGGTTTCCAATTTCAAAGGGG 622 AB026500 AACGTGGTCCTGCAGCAGCGATTAGGATGCCAATGCTACGTGTCTCCAACTTCAAGGGAG 1194 CPETR2 ------------------------------------------------------------ AY600438 AACCAGGAGCTGTGGCTGCAATTAGGATGCCGATGCTTAAAGTGTCGAACTTCAAAGGTG 865 FRX297513 GGACTCCTGAGTTGATCCAGACTTGTTATGCGATATTGGTTCTGGTTCTACCTGGTGGAG 1429 AF154119 GGACCCCTGAGTTCATCCAGACTTGTTATGCGATTTTGGTTCTGGTTCTCGCTGGTGGAC 682 AB026500 GAACTCCTGAGATAGTTCCAACTTACTATGCGATTTTGGTTTTAGTTCTCCCTGGTGGAC 1254 CPETR2 ------------------------------------------------------------ AY600438 GAACTCCTGAACTTGTCCCAGAATGTTATGCCATACTGGTTTTGGTTCTACCTAGTGAAC 925 FRX297513 AGCCTAGATCTTGGAGCAGTCAGGAACTTGAGATAATTAAGGTGGTTGCTGACCAGGTGG 1489 AF154119 AGCCTAGATCTTGGAGCAGCCAGGAACTTGAGATAATTAAGGTGGTTGCCGATCAGGTGG 742 AB026500 AACCTAGATCTTGGAATAATCAGGAACTTGAGATAATAAAGGTGGTTGCTGACCAGGTGG 1314 CPETR2 ------------------------------------------------------------ AY600438 AAGGTAGATCATGGTGCAGCCAGGAAATTGAGATAGTCAGGGTTGTGGCTGATCAGGTTG 985 FRX297513 CTGTGGCCTTATCCCATGCTGCAATCCTTGAAGAGTCCCAACTTATGCGGGAACAATTGG 1549 AF154119 CTGTGGCTTTATCCCATGCTGCAATCCTTGAAGAGTCCCAACTAATGCGCGAGAAA---- 798 AB026500 CTGTTGCTCTCTCCCATGCTGCTCTTCTGGAGGAGTCCCAGCTCATGAGAGATAAACTGG 1374 CPETR2 ------------------------------------------------------------ AY600438 CTGTGGCTCTGTCCCATGCTGCAATTCTTGAAGAGTCTCAGCATATGAGAGAAACATTGG 1045 FRX297513 CTGAGCAAAACCGGGCCTTGCAACAGGCGAAAATGAATGCCATGATGGCAAGCCATGCAA 1609 AF154119 ------------------------------------------------------------ AB026500 CTGAGCAGAATCGAGATCTGCAACAGGCCAAGGAGAATGCTCTGATGGCAAGCCAAGCTA 1434 CPETR2 ------------------------------------------------------------ AY600438 AAGAGCAAAACCGAGCTCTGGAACAAGCAAAGCAGGATGCACTTAGGGCGAGTCAAGCAA 1105 FRX297513 GAAACTCATTCCAAAAGGTGATGAGTGATGGGATGAGAAGGCCAATGCATTCAGTATTGG 1669 AF154119 ------------------------------------------------------------ AB026500 GAAACTCCTTCCAGAAGGTGATGAGTGATGGGATGAGGAGACCTATGCATTCCATCATGG 1494 CPETR2 ------------------------------------------------------------ AY600438 GGAATGCATTTCAGATGGTTATGAGCCATGGTCTGAGAAGACCCATGCACTCAATATTGG 1165 FRX297513 GGCTGCTTTCCATGATGCAGGATGAGAGTTTGAATAATGATCAGCGAGTTATTGTTGATG 1729 AF154119 ------------------------------------------------------------ AB026500 GTTTGCTTTCAATGTTGCAGAATGAGAATATGAATGATGACCAACGAATTATACTCGATG 1554 CPETR2 ------------------------------------------------------------ AY600438 GTCTGCTCTCCTTGTTGCAAGATGAGAAATTGGGTAATGAGCAGCGGCTTCTTGTGGATT 1225

33

FRX297513 CAATGGTAAGGACAAGCAATGTCTTATCGACGTTGATAAATGATGCTATGGATAATCCAG 1789 AF154119 ------------------------------------------------------------ AB026500 CCATGGTGAGGACTGGCAATGTTGTCTCTACACAGATAGATGATGTTATGGAACATCCAA 1614 CPETR2 ------------------------------------------------------------ AY600438 CAATGGTTAAAACCAGTAATGTCGTGTCAACCCTAATAGATGATGTGATGGATACTTCAA 1285 FRX297513 CCAAGGATAGTGGAAGATTTCCTTTGGAG------ATGAGGCCTTTCCGGTTACAACCAA 1843 AF154119 ------------------------------------------------------------ AB026500 TTAAGGATAGTGCAAGATTTCCTTTGGAGTTGGAGATGAGATCTTTTAGGTTACATTCTA 1674 CPETR2 ------------------------------------------------------------ AY600438 CAAAGGACAACGGTAGATTCCCTTTGGAG------ATGAGATATTTTCAGCTACATTCCA 1339 FRX297513 TGATAAAAGAAGCAGCTTGCCTTGCCAAATGCTTGTGTGTGTATAGGGGGTTTGGTTTTG 1903 AF154119 ------------------------------------------------------------ AB026500 TGATAAAGGAGGCAGCTTGTCTTGCCAAGTGCTTGTGTGCATATAAGGGCTTTGGTTTTG 1734 CPETR2 ------------------------------------------------------------ AY600438 TGATAAAAGAAGCTGCTTGTCTTGCCAAGTGTTTGTGTGCTTATAGGGGTTATAATATTT 1399 FRX297513 CAATTGAGGTTGACAAGTCCATAGCTGATCATGTAATTGGAGATGAAAGAAGGGTTTTTC 1963 AF154119 ------------------------------------------------------------ AB026500 CCTTTGAAGTTCAGAGGTCTCTGCCTGATCACGTCATGGGCGATGAAAGAAGGGTTTTTC 1794 CPETR2 ------------------------------------------------------------ AY600438 CCATTGAGGTTGACAAATCTTTGCCAAATCATGTTCTCGGTGATGAAAGAAGAGTTTTTC 1459 FRX297513 AGGTGATTTTGCATATGGTTGGTAGCCTGTTGAATGGAA---ACCAGGGCGGAGGGTTGG 2020 AF154119 ------------------------------------------------------------ AB026500 AGGTGCTTTTGCATATGGTGGGGAGCCTATTAAATGACATCAACCAGGGAGGAGGATATG 1854 CPETR2 ------------------------------------------------------------ AY600438 AAGTTATTCTTCATATGGTTGGAAATCTTTTGAAGG------ACCCCAATGGAGGTCTTC 1513 FRX297513 TCGTATTTCGGGTTTCTTCAGAGAATGGAAGTCAGGGGAGGAATGATCAAAGATGGGCTG 2080 AF154119 ------------------------------------------------------------ AB026500 CTTTGTTTCGGGTTGTGGCTGAGAGTGGAAGTCAGGGACGGAATGACCAAAGGTGGGGTA 1914 CPETR2 ------------------------------------------------------------ AY600438 TCACATTTAGGGTTCTCCCAGAAAGTGTAAGTAGGGAAGGCATTGGTGGAGCATGGAGAA 1573 FRX297513 CTTGGAGACAGAACTCATCTGATAGTGGTGATGTATATATTAGATTTGAGATTGGGATAA 2140 AF154119 ------------------------------------------------------------ AB026500 ATTGGAGACAAAACTCTTCTGAT---GGGGATGCCTTTATCAGATTTGAGGTTGGGATAA 1971 CPETR2 ------------------------------------------------------------ AY600438 CAAGGAGGTCAAACTCATCTCGT---GATAACGCCTATATCAGGTTTGAAGTTGGAACAA 1630 FRX297513 GCAATGGTGGTTCTCAATCAGATATGACAAGTCCAATTATGCAGCTTGTTGGTAGCAGAT 2200 AF154119 ------------------------------------------------------------ AB026500 ATAAGAGCAATTCTCAATCAGAGGGCTCTATTCCAAATATGGTATCTGGTGATCGAAGAT 2031 CPETR2 ------------------------------------------------------------ AY600438 GCAATAATCATTCTCAGCCAGAGGGGACCATGT------TGCCACATTACAGGCCAAAAC 1684 FRX297513 ACAACAGTGAAGGAGTTGAGGAGAACTTGAGCTTCAACATTTGCAAAAGGCTAGTGCAGT 2260 AF154119 ------------------------------------------------------------ AB026500 ACGCCAGTGATGGAGCTGAGGAACGCTTGAGTTTTACCATCTGCAAGAAGCTTGTTAAGT 2091 CPETR2 ------------------------------------------------------------ AY600438 GCTGCAGTAAGGAGATGGATGAAGGCTTGAGTTTCACTGTGTGCAGAAAGCTGGTTCAGG 1744 FRX297513 TGATGCAAGGA-AACAT--ATGGCCAATTCCTAACCCTCAAGGATTTCCTCAAAGCATGG 2317 AF154119 ------------------------------------------------------------ AB026500 TGATGCAAGGG-AATAT--ATGGGTAATCCCTAATCCTCAAGGATTTACACGAAGCATGG 2148 CPETR2 ------------------------------------------------------------ AY600438 TATTCTATTGCTAATACCAGCATCTGAGTATGTATATTCTGGAGTTTAT--AAACCAAAA 1802 FRX297513 CACTTGTTC-TCCGGTTTCAAACTCGACCATCCATTGCAATAGCCATCTCTGAACCTGG- 2375 AF154119 ------------------------------------------------------------

34

AB026500 CACTTGTTC-TTCGTTTTCAACTTCGACCCTCCATAGCAGTGGCCATGCCTGAACCTGG- 2206 CPETR2 ------------------------------------------------------------ AY600438 AACTGTTTCATTTGGTTCTATTCCCTTCTTTCTGTGGTTTATAGTACACTCAACTTTGAT 1862 FRX297513 AGGATCATCAGAGCACTCGCATTCCAATTCTATATTCAGAGGCTTGCAAG-------TTC 2428 AF154119 ------------------------------------------------------------ AB026500 AGAATCATCTGAACATCCACACTCCAACTCCATCTTCAGAGGATTGCAAG-------TTA 2259 CPETR2 ------------------------------------------------------------ AY600438 AAAATCATTCTGTTATAGGT-TAAAAGAGAAAAAATGATAGTATTACAAAAGAAAAATGA 1921 FRX297513 TGTTAACTGATGAAGATGATGTGAATAGG-CTTGTGACTCGGAAGCTGCTTGAGAAACTA 2487 AF154119 ------------------------------------------------------------ AB026500 TTTTAGCCGATGCCGATGACATGAACAGA-GCTGTGACACGAAAAATGCTCGAGAAGTTG 2318 CPETR2 ------------------------------------------------------------ AY600438 TATTTAAGCCTGACTAGTTTTTAAATTTCTACTGCAATTGGATGAGACCTTTTAAATTGT 1981 FRX297513 GGTTGCAATGTAACCGCTGTTTCCTCAGGATTCG----AATGCCTTTCTGCCATTGGTCC 2543 AF154119 ------------------------------------------------------------ AB026500 GGCTGCAACGTGACTGCTGTTTCTTCTGGATTCG----AATGTCTCACAGTCATGGCACC 2374 CPETR2 ------------------------------------------------------------ AY600438 GATTTCTGGATGGCGTTAACTACTACAATTTCCATGTCAAAGAAGATAAAGCAATTCATG 2041 FRX297513 CTCTGGAGCTTCTGT--CCAAGTTGTCTTCCTAGATCTCCAAATGACGGAGCTGGATGGT 2601 AF154119 ------------------------------------------------------------ AB026500 AGCTGGTTCTTCAAT--TCAAGTGGTGCTCTTGGATCTTCACATGCCAGAATTAGATGGT 2432 CPETR2 ------------------------------------------------------------ AY600438 ACCTTGATTGCCTGTCATGTAATTAAATATGTCGTTTTCCCTGTGAATGAGAAATATGAT 2101 FRX297513 CTTGAAGTTGC----AATGAGAATTAGAAAATTCCGAAGCCGCACTT---GGCCATTGAT 2654 AF154119 ------------------------------------------------------------ AB026500 TTTGAAGTTAC----AACAAGGATCAGAAAGTTTAGAAGCCAGAATTATAGGCCAGTGAT 2488 CPETR2 ------------------------------------------------------------ AY600438 ACTAAAGTTGCTTAAGCTGTTTGTTGCGGAACTTTTAATCCCCATTT---GTTTGGGAAT 2158 FRX297513 CATTGCCGTAACTGCCAGTGCAGATGATGATATTTGGGATAGATGCATGCAGATTGGGAT 2714 AF154119 ------------------------------------------------------------ AB026500 CATTGCATTAACTGCAAGTGCAGGTGAAGAT---TGGGAGAGATGCGTGCAGATTGGGAT 2545 CPETR2 ------------------------------------------------------------ AY600438 GAAATCTGAAACCACTCATACAAGTTGGTAGTACCGTAGTACTTGTTCTCTTTTTTCTCC 2218 FRX297513 CAACGGAGTCATCCGAAAACCAGTATTGTTGCAA---GGCATTGCCAGTGAGCTTAGACG 2771 AF154119 ------------------------------------------------------------ AB026500 GAACGGTGTCATACGAAAACCAGTTCAGTTACAG---GGAATCGCCCATGAGCTTCGCCG 2602 CPETR2 ------------------------------------------------------------ AY600438 CTTCGATTATAATTTAAGTGCA-TATAGTTGTGGTTTGGGGTAGGCACTAACATCTTGCT 2277 FRX297513 AGTCCTGGTGCAGGCAAACAAAATTGTGTGATGAG-------ACCGTTTGCTCAATGGAT 2824 AF154119 ------------------------------------------------------------ AB026500 TGCTCTGCTGCAAGCAAGCAAGGTTGTGTGAGGAGGAGAAGGATAGAGAGCCTGGCAGTT 2662 CPETR2 ------------------------------------------------------------ AY600438 GGTATGCAAATATGATGAACAGTTGATGCAAGGAG---ACATCTGGGTAATCCCAAATCC 2334 FRX297513 GAA-----GTAGCTAATGTTTCTTTGATCTTTCTTT-----------GGCTCCAAAATCA 2868 AF154119 ------------------------------------------------------------ AB026500 AAATGCTTATGTGCAAAAATCTGGTTATCTTGATAGA--------GAGGTAGCAAGCAAA 2714 CPETR2 ------------------------------------------------------------ AY600438 AGAAGGTTTTGATCAAAGCATGGCTGTCGTTCTTGGGCTTCAACTGCGGCCATCAATTGC 2394 FRX297513 TGATTCCTTCCCCGAACATCAAAAACATAGAGAGATGTTACACCAGCATTCA--CAGTTA 2926 AF154119 ------------------------------------------------------------ AB026500 TATAATTAGTCTCTGACCTCAAAAGTCT---GAGTTTCCCCATCAAATCTCTT-CCGCCA 2770 CPETR2 ------------------------------------------------------------ AY600438 CATAGGCATTCCTGAATATGGGGAATCTTCTGATCATTCGCATCCACACTCACTCCTCCA 2454

35

FRX297513 GAATGGTA------T---ACAGGATTAAATTAGCTCCTTTTTCTTTC------------- 2964 AF154119 ------------------------------------------------------------ AB026500 GAGAGGAA------TGTTACGAACTTGAAAAAATGTTCAACAATTTCACAGTGAAAAATG 2824 CPETR2 ------------------------------------------------------------ AY600438 AGGTGTTAAAGTTCTGTTAGCAGATTATGATGACGTGAATAGAGCGGTAACAAGTAAGCT 2514 FRX297513 ------------------------------------------------------------ AF154119 ------------------------------------------------------------ AB026500 GTATACAATAGTTGTCCTTACTCTCTTTTCAAAAAAAAAAAAAAAAA------------- 2871 CPETR2 ------------------------------------------------------------ AY600438 ACTTGAAAAATTGGGATGCAGTGTTTCTGCAGTTTCATCTGGACGTGACTGTATTGGTGT 2574 FRX297513 ------------------------------------------------------------ AF154119 ------------------------------------------------------------ AB026500 ------------------------------------------------------------ CPETR2 ------------------------------------------------------------ AY600438 TCTTAGCCCTGCTGTATCCTCATTCCAAATCGTCCTTTTGGATCTTCACCTGCCTGATTT 2634 FRX297513 ------------------------------------------------------------ AF154119 ------------------------------------------------------------ AB026500 ------------------------------------------------------------ CPETR2 ------------------------------------------------------------ AY600438 GGATGGCTTCGAAGTAACCATGAGAATTCGGAAGTTTGGTAGCCACAACTGGCCATTGAT 2694 FRX297513 ------------------------------------------------------------ AF154119 ------------------------------------------------------------ AB026500 ------------------------------------------------------------ CPETR2 ------------------------------------------------------------ AY600438 AGTTGGTTTAACTGCGACTGCTGATGAAAATGTTACTGGAAGATGCCTGCAGATTGGAAT 2754 FRX297513 ------------------------------------------------------------ AF154119 ------------------------------------------------------------ AB026500 ------------------------------------------------------------ CPETR2 ------------------------------------------------------------ AY600438 GAATGGACTTATTCGTAAACCAGTGCTATTGCCAGGTATCGCTGATGAGCTTCAAAGGGT 2814 FRX297513 ---------------------------- AF154119 ---------------------------- AB026500 ---------------------------- CPETR2 ---------------------------- AY600438 TCTGCTACGGGGAAGTAGAATGATGTAA 2842

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Table 8 Sequence distances of CP-ETR2 sequences from papaya and other plant ETR2 genes. The analysis was done using the MegAlign program (DNASTAR).

Percent Identity 1 2 3 4 5 6

1.Cucumber *** 74.9 39.5 48.9 64.1 76.3

2.Rose *** *** 37.3 69.7 93.2 76.6 3.Snapdragon *** *** *** 38.5 39.3 48.7 4.Tomato *** *** *** *** 51.7 70.6 5.Strawberry *** *** *** *** *** 77.1

6.CP-ETR2 *** *** *** *** *** ***

1. Cucumber (Cucumis sativas) : AB 026500 2. Rose (Rosa hybrida) : AF 154119 3. Snapdragon (Antirrhinum majus) : AY 159363 4. Tomato (Lycopersicon esculentum) : AY 600438 5. Strewberry (Fragaria x ananassa) : FRX 297513 6. CP-ETR2

37

3. Expression of CP-ETR1 and CP-ETR2 gene Total RNA from various-times were extracted and measured by agarose gel electrophoresis and spectrophotometer (Figure 9 and Figure 10), the result showed a good quality of total RNA. The expression levels of CP-ETR1 and CP-ETR2 at various times after PRSV inoculation were determined using RT-PCR techniques. The results were shown the differential expression of CP-ETR1. In control plant (PRSV free), CP-ETR1 was detected from 0 min after inoculation and the expression level was decreased at 30 min and 2 hr. CP-ETR1 had the highest level at 6 hr and decreased until day 3 (Figure 11). Wounding is known to induce plant responses (Johnson and Ecker, 1998). Therefore, the use of celite might unintentionally induced CP-ETR1 expression. In PRSV inoculated plants, CP-ETR1 expression started at 30 min and reached its peak at 6 hr. The expression continued at lower level until 3 days after inoculation (Figure 12). CP-ETR2 expression, however, was not detected either in PRSV free of PRSV inoculated plants (Figure 13 and 14). The possibility of primer faulty of PCR reaction was eliminated by the amplification of CP-ETR2 plasmid DNA and the RT-PCR experiment control of CP-ACO2. CP-ACO2 was previously shown expressing in papaya leaves (Kumdee, 2003).

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Figure 7 Ten week-old plant of papaya KhaekNuan variety

39

Figure 8 Papaya Ring Spot Virus inoculation (a) Instrument of Virus inoculation (b) and (c) The viral

suspension was applied on the leap surface (d) The excess was washed out.

(a) (b)

(d) (c)

40

M 1 2 3 4

Figure 9 Ethidium bromide stained 1% agarose gel total RNA of papaya leaf following the

electrophoresis at 60 volt for 45 min. Molecular weight marker is shown on the left.

M 1 2 3 4 5 6 7 8 9 10 11 12 M

Figure 10 Ethidium bromide stained 1% agarose gel total RNA of papaya leaf after DNase I

treatment following the electrophoresis at 60 volt for 45 min. Molecular weight marker are shown on the left and the right.

DNA

RNA

41

M 1 2 3 4 5 6 7 8 9

Figure 11 Ethidium bromide stained 1% agarose gel of RT-PCR product of the PRSV free inoculated papaya collecting at the various times following the electrophoresis at 60 volt for 1 hr. RT-PCR was carried out using 500 µg of total RNA sample with CP- ETR1 primer. Lane1 at 0 min, lane2 at 30 min, lane3 at 2 hr, lane4 at 6 hr, lane5 at 1 hr, lane6 at 24 hr, lane7 at 3 d, lane8 at 7 d and lane 9 control (non-inoculated papaya).

M 1 2 3 4 5 6 7 8 9

Figure 12 Ethidium bromide stained 1% agarose gel of RT-PCR product of PRSV inoculated papaya collecting at various times following the electrophoresis at 60 volt for 1 hr. RT-PCR was carried out using 500 µg of total RNA sample with CP-ETR1 primer. Lane1 at 0 min, lane2 at 30 min, lane3 at 2 hours, lane4 at 6 hours, lane5 at 12 hours, lane6 at 24 hours, lane7 at 3 days, lane8 at 7days and lane9 control (non-inoculated papaya).

835 bp

42

835 bp

M 1 2 3 4 5 6 7 8 9 C Figure 13 Ethidium bromide stained 1% agarose gel of RT-PCR product of PRSV free inoculated

papaya collecting at various times following the electrophoresis at 60 volt for 1 hr. RT-PCR was carried out using 500 µg of total RNA sample with CP-ETR2 primer. Lane1 at 0 min, lane2 at 30 min, lane3 at 2 hours, lane4 at 6 hours, lane5 at 12 hours, lane6 at 24 hours, lane7 at 3 days, lane8 at 7days and lane9 control (non-inoculated papaya). And RT-PCR control of CP-ACO2.

M 1 2 3 4 5 6 7 8 C Figure 14 Ethidium bromide stained 1% agarose gel of RT-PCR product of PRSV inoculated Papaya collecting at various times following the electrophoresis at 60 volt for 1 hr. RT-PCR was carried out using 500 µg of total RNA sample with CP-ETR1 primer. Lane1 at 0 min, lane2 at 30 min, lane3 at 2 hours, lane4 at 6 hours, lane5 at 12 hours, lane6 at 24 hours, lane7 at 3 days, lane8 at 7days. And RT-PCR control of CP-ETR2.

800 bp

379 bp

43