蘭科是被子植物中最大家族，也是生態與演化最重要的代表性植物。瞭解其起源與多樣化有助於植物的研究。儘管擁有獨特的發育生殖生物學，以及專一的授粉和生態策略，但相對於其他植物，具有豐富物種蘭科，其在分子生物學研究上，仍然相對缺乏。迄今，蘭科植物中，只有少數在樹蘭亞科的幾個物種被利用來進行分子生物學之研究。基因資料庫不僅幫助基因預測，也助益未來基因體的註解。為此，本文建構高效能網路表現序列標籤資料庫，提供基因序列搜尋、序列比對、EST序列片段自動化處理流程及Gene Ontology註解資訊、Pathway分析與基因網路預測等數種工具。基於蝴蝶蘭之表現序列標籤資料庫資訊，實際建立了44K寡核酸微矩陣列，並應用於蝴蝶蘭花轉錄體分析，這些分子生物學工具將有效率的協助植物學家了解隱藏在蘭花演化及生殖發育之秘密。

Orchidaceae is one of largest family of the angiosperms, and represents one of the most ecologically and evolutionarily significant plants. An understanding of orchid origin and diversification will help the plant research. Despite their unique developmental reproductive biology, as well as specialized pollination and ecological strategies, orchids remain under-represented in molecular studies relative to other species-rich plant families. Nowadays, among the subfamilies of Orchidaceae, only a few species are studied. Genetic databases would be useful not only for gene discovery but also for future genomic annotation. For this purpose, this dissertation proposed and implemented an efficient, web-accessible database for the orchid ESTs database. It provides several tools to search ESTs sequences, sequence similarity search, automatic pre-processing of EST reads, annotated information with Gene Ontology, KEGG pathways as well as gene network prediction. Based on the Phalaenopsis ESTs information in the database, an 44K oligomicroarray was developed and applied to transcriptomic analysis of floral organs. The molecular and biochemical tools can efficiently assist biologists in understanding the evolution and reproductive development of orchids.

Aarts, M.G., Keijzer, C.J., Stiekema, W.J., Pereira, A., 1995. Molecular characterization of the CER1 gene of Arabidopsis involved in epicuticular wax biosynthesis and pollen fertility. Plant Cell 7, 2115–2127.

Abbruscato P, Nepusz T, Mizzi L, Del Corvo M, Morandini P, Fumasoni I, Michel C, Paccanaro A, Guiderdoni E, Schaffrath U, Morel JB, Piffanelli P, Faivre-Rampant O., 2012. OsWRKY22, a monocotWRKY gene, plays a role in the resistance response to blast. Mol. Plant Pathol. 13, 828–841.

Alagna, F., D’Agostino, N., Torchia, L., Servili, M., Rao, R., Pietrella, M.et al. (2009) Comparative 454 pyrosequencing of transcripts from two olive genotypes during fruit development. BMC Genomics 10, 399.

Albert, V.A., Soltis, D.E., Carlson, J.E., Farmerie, W.G., Wall, P.K., Ilut, D.C. et al. (2005) Floral gene resources from basal angiosperms for comparative genomics research. BMC Plant Biol. 5, 5.

Ashburner, M., Ball, C.A., Blake, J.A., Botstein, D., Butler, H., Cherry, J.M.et al. (2000) Gene ontology: tool for the unification of biology. Nat. Genet. 25, 25–29.

Atwood, J.T. (1986) The size of Orchidaceae and the systematic distribution of epiphytic orchids. Selbyana 9, 16.

Bai, Y., Meng, Y., Huang, D., Qi, Y., Chen, M., 2011. Origin and evolutionary analysis of the plant-specific TIFY transcription factor family. Genomics 98, 128–136.

Bennett, S.T., Barnes, C., Cox, A., Davies, L. and Brown, C. (2005) Toward the 1,000 dollars human genome. Pharmacogenomics 6,373–382.

Branda˜o, M.M., Dantas, L.L. and Silva-Filho, M.C. (2009) AtPIN:Arabidopsis thaliana protein interaction network. BMC Bioinformatics 10, 454.

Cozzolino, S. and Widmer, A. (2005) Orchid diversity: an evolutionary consequence of deception?. Trends Ecol. Evol. 20, 487–494.

Yu-Yun Chang, Nai-Hsuan Kao, Jen-Ying Li, Wei-Han Hsu, Yu-Ling Liang, Jia-Wei Wu and Chang-Hsien Yang. (2010) Characterization of the possible roles for B class MADS box genes in regulation of perianth formation in orchid. Plant Physiol. 152, 837–853.
Chang, Y.Y., Chu, Y.W., Chen, C.W., Leu, W.M. and Yang, C.H. (2011) Characterization of Oncidium ‘Gower Ramsey’ transcriptomes using 454 GS-FLX pyrosequencing and their application to the identification of genes associated with flowering time. Plant Cell Physiol.52, 1532–1545.

Chase, M.W., Williams, N.H., Donisete de Faria, A., Neubig, K.M., Amaral, M.C.E., Whitten,W.M., 2009. Floral convergence in Oncidiinae (Cymbidieae; Orchidaceae): an expanded concept of Gomesa and a new genus Nohawilliamsia. Ann. Bot. 104, 387–402.

Chen, Y.Y., Lee, P.F., Hsiao, Y.Y., Wu, W.L., Pan, Z.J., Lee, Y.I. et al. (2012) C- and D-class MADS-box genes from Phalaenopsis equestris (Orchidaceae) display functions in gynostemium and ovule development. Plant Cell Physiol. 53, 1053–1067.

Cozzolino, S., Widmer, A., 2005. Orchid diversity: an evolutionary consequence of deception?Trends Ecol. Evol. 20, 487–494.

Delseny, M., Han, B. and Hsing, Y.I. (2010) High throughput DNA sequencing: the new sequencing revolution. Plant Sci. 179,407–422.

Laurent G Deluc, Jérôme Grimplet, Matthew D Wheatley, Richard L Tillett, David R Quilici, Craig Osborne, David A Schooley, Karen A Schlauch, John C Cushman, and Grant R Cramer., 2007. Transcriptomic and metabolite analyses of Cabernet Sauvignon grape berry development. BMC Genomics 8, 429.

Dressler, R.L., 2005. How many orchid species? Selbyana 26, 155–158.

Duan, H., Schuler, M.A., 2005. Differential expression and evolution of the Arabidopsis CYP86A subfamily. Plant Physiol. 137, 1067–1081.

Endress, P.K. (2001) Evolution of floral symmetry. Curr. Opin. Plant Biol. 4: 86–91.

F. Azuaje, "A computational neural approach to support the discovery of gene function and classes of cancer," IEEE Transactions on Biomedical Engineering, vol. 48, pp. 332-339, 2001.

Fu, C.H., Chen, Y.W., Hsiao, Y.Y., Pan, Z.J., Liu, Z.J., Huang, Y.M. et al.(2011) OrchidBase: a collection of sequences of the transcriptome derived from orchids. Plant Cell Physiol. 52: 238–243.

Godard, K.-A., White, R., Bohlmann, J., 2008. Monoterpene-induced molecular responses in Arabidopsis thaliana. Phytochemistry 69, 1838–1849.

Golz, J.F., Hudson, A., 1999. Plant development: YABBYs claw to the fore. Curr. Biol. 9, R861–R863.

Go´rniaka, M., Paunb, O. and Chase, M.W. (2010) Phylogenetic relationships within Orchidaceae based on a low-copy nuclear coding gene, Xdh: congruence with organellar and nuclear ribosomal DNA results. Mol. Phylogenet. Evol. 56: 784–795.

Guilfoyle, T.J., Hagen, G., 2007. Auxin response factors. Curr. Opin. Plant Biol. 10, 453–460.

Yu-Yun Hsiao, Wen-Chieh Tsai, Chang-Sheng Kuoh, Tian-Hsiang Huang, Hei-Chia Wang, Tian-Shung Wu, Yann-Lii Leu, Wen-Huei Chen and Hong-Hwa Chen., 2006. Comparison of transcripts in Phalaenopsis bellina and Phalaenopsis equestris (Orchidaceae) flowers to deduce the monoterpene biosynthesis pathway. BMC Plant Biol. 6, 14.

Hsiao, Y.Y., Chen, Y.W., Huang, S.C., Pan, Z.J., Fu, C.H., Chen, W.H. et al.(2011a) Gene discovery using next-generation pyrosequencing to develop ESTs for Phalaenopsis orchids. BMC Genomics 12, 360.

Hsiao, Y.Y., Huang, T.H., Fu, C.H., Huang, S.C., Chen, Y.J., Huang, Y.M.et al. (2013) Transcriptomic analysis of floral organs from Phalaenopsis orchid by using oligonucleotide microarray. Gene (in press).

Hsiao, Y.Y., Jeng, M.F., Tsai, W.C., Chuang, Y.C., Li, C.Y., Wu, T.S. et al.(2008) A novel homodimeric geranyl diphosphate synthase from the orchid Phalaenopsis bellina lacking a DD(X)2–4D motif. Plant J. 55, 719–733.

Hsiao, Y.Y., Pan, Z.J., Hsu, C.C., Yang, Y.P., Hsu, Y.C., Chuang, Y.C. et al.(2011b) Research on orchid biology and biotechnology. Plant Cell Physiol. 52, 1467–1486.

Chia-Chi Hsu, Yu-Lin Chung, Tien-Chih Chen, Yu-Ling Lee, Yi-Tzu Kuo, Wen-Chieh Tsai, Yu-Yun Hsiao, Yun-Wen Chen, Wen-Luan Wu, Hong-Hwa Chen, 2011. An overview of the Phalaenopsis orchid genome through BAC end sequence analysis. BMC Plant Biol. 11, 3.

Hu, W., Ma, H., 2006. Characterization of a novel putative zinc finger gene MIF1: involvement in multiple hormonal regulation of Arabidopsis development. Plant J. 45, 399–422.

Huang P, Ju HW, Min JH, Zhang X, Chung JS, Cheong HS, Kim CS., 2012. Molecular and physiological characterization of the Arabidopsis thaliana Oxidation-related Zinc Finger 2, a plasma membrane protein involved in ABA and salt stress response through the ABI2-mediated signaling pathway. Plant Cell Physiol. 53, 193–203.

Jakoby M, Weisshaar B, Dröge-Laser W, Vicente-Carbajosa J, Tiedemann J, Kroj T, Parcy F; bZIP Research Group., 2002. bZIP transcription factors in Arabidopsis. Trends Plant Sci. 7, 106–111.

Joubès J, Raffaele S, Bourdenx B, Garcia C, Laroche-Traineau J, Moreau P, Domergue F, Lessire R., 2008. The VLCFA elongase gene family in Arabidopsis thaliana: phylogenetic analysis, 3D modelling and expression profiling. Plant Mol. Biol. 67, 547–566.

So-Young Kim, Pil-Yong Yun, Tatsuya Fukuda, Toshinori Ochiai, Jun Yokoyama, Toshiaki Kameya, Akira Kanno., 2007. Expression of a DEFICIENS-like gene correlates with the differentiation between sepal and petal in the orchid, Habenaria radiata (Orchidaceae). Plant Sci. 172, 319–326.

Kocyan, A. and Endress, P.K. (2001) Floral structure and development of Apostasia and Neuwiedia (Apostasioideae) and their relationships to other Orchidaceae. Int. J. Plant Sci. 162,847–867.

Kocyan, A., Qiu, Y.-L., Endress, P.K. and Conti, E. (2004) A phylogenetic analysis of Apostasioideae (Orchidaceae) based on ITS, trnL-F and matK sequences. Plant Syst. Evol. 247: 203–213.

Kanehisa, M., Goto, S., Kawashima, S., Okuno, Y. and Hattori, M. (2004) The KEGG resource for deciphering the genome. Nucleic Acids Res.32, D277–D280.

Labarga, A., Valentin, F., Anderson, M. and Lopez, R. (2007) Web services at the European Bioinformatics Institute. Nucleic Acids Res. 35,W6–W11.

Haifeng Li, Wanqi Liang, Ruidong Jia, Changsong Yin, Jie Zong, Hongzhi Kong and Dabing Zhang, 2010. The AGL6-like gene OsMADS6 regulates floral organ and meristem identities in rice. Cell Res. 20, 299–313.

Li, Y., Pi, L., Huang, H., Xu, L., 2012. ATH1 and KNAT2 proteins act together in regulation of plant inflorescence architecture. J. Exp. Bot. 63, 1423–1433.

Li, R., Zhu, H., Ruan, J., Qian, W., Fang, X., Shi, Z. et al. (2010) De novo assembly of human genomes with massively parallel short read sequencing. Genome Res. 20, 265–272.

Zhong-Jian Liu, Li-Jun Chen, Sing-Chi Chen, Jing Cai, Wen-Chieh Tsai, Yu-Yun Hsiao, Wen-Hui Rao, Xue-Yong Ma, Guo-Qiang Zhang., 2011. Paraholcoglossum and Tsiorchis, two new orchid genera established by molecular andmorphological analyses of the Holcoglossum alliance. PLoS One 6, e24864.

Lynch, D.V., Dunn, T.M., 2004. An introduction to plant sphingolipids and a review of recent advances in understanding their metabolism and function. New Phytol. 161, 677–702.

Margulies, M., Egholm, M., Altman, W.E., Attiya, S., Bader, J.S.,Bemben, L.A. et al. (2005) Genome sequencing in microfabricated high-density picolitre reactors. Nature 437, 376–380.

Meyers, B.C., Galbraith, D.W., Nelson, T., Agrawal, V., 2004. Methods for transcriptional profiling in plants. Be fruitful and replicate. Plant Physiol. 135, 637–652.

Mondragon-Palomino,M., Theissen,G., 2008.MADS about the evolution of orchid flowers. Trends Plant Sci. 13, 51–59.

Nakashima K, Tran LS, Van Nguyen D, Fujita M, Maruyama K, Todaka D, Ito Y, Hayashi N, Shinozaki K, Yamaguchi-Shinozaki K., 2007. Functional analysis of a NAC-type transcription factor OsNAC6 involved in abiotic and biotic stress-responsive gene expression in rice. Plant J. 51, 617–630.

Otero, J.T. and Flanagan, N.S. (2006) Orchid diversity—beyond deception. Trends Ecol. Evol. 21: 64–65.

Pan, Z.J., Cheng, C.C., Tsai, W.C., Chung, M.C., Chen, W.H., Hu, J.M.et al. (2011) The duplicated B-class MADS-box genes display dualistic characters in orchid floral organ identity and growth. Plant Cell Physiol. 52: 1515–1531.

Peakall, R. (2007) Speciation in the Orchidaceae: confronting the challenges. Mol. Ecol. 16: 2834–2837.

Rhee, S.Y., Beavis, W., Berardini, T.Z., Chen, G., Dixon, D., Doyle, A. et al.(2003) The Arabidopsis Information Resource (TAIR): a model organism database providing a centralized, curated gateway to Arabidopsis biology, research materials and community. Nucleic Acids Res. 1, 224–228.

Rowland, O., Zheng, H., Hepworth, S.R., Lam, P., Jetter, R., Kunst, L., 2006. CER4 encodes an alcohol-forming fatty acyl-coenzyme A reductase involved in cuticular wax production in Arabidopsis. Plant Physiol. 142, 866–877.

Rushton DL, Tripathi P, Rabara RC, Lin J, Ringler P, Boken AK, Langum TJ, Smidt L, Boomsma DD, Emme NJ, Chen X, Finer JJ, Shen QJ, Rushton PJ., 2012. WRKY transcription factors: key components in abscisic acid signalling. Plant Biotechnol. J. 10, 2–11.

Silvera, K., Santiago, L.S., Cushman, J.C., Winter, K., 2009. Crassulacean acid metabolism and epiphytism linked to adaptive radiations in the Orchidaceae. Plant Physiol. 149, 1838–1847.

Skipper, M., Johansen, L.B., Pedersen, K.B., Frederiksen, S., Johansen, B.B., 2006. Cloning and transcription analysis of an AGAMOUS- and SEEDSTICK ortholog in the orchid Dendrobium thyrsiflorum (Reichb. f.). Gene 366, 266–274.

Song IJ, Nakamura T, Fukuda T, Yokoyama J, Ito T, Ichikawa H, Horikawa Y, Kameya T, Kanno A., 2006. Spatiotemporal expression of duplicate AGAMOUS orthologues during floral development in Phalaenopsis. Dev. Genes Evol. 216, 301–313.

Su, C.L., Chao, Y.T., Alex Chang, Y.C., Chen, W.C., Chen, C.Y., Lee, A.Y.et al. (2011) De novo assembly of expressed transcripts and global analysis of the Phalaenopsis aphrodite transcriptome. Plant Cell Physiol. 52, 1501–1514.

Swarbreck D, Wilks C, Lamesch P, Berardini TZ, Garcia-Hernandez M, Foerster H, Li D, Meyer T, Muller R, Ploetz L, Radenbaugh A, Singh S, Swing V, Tissier C, Zhang P, Huala E., et al., 2007. The Arabidopsis Information Resource (TAIR): gene structure and function annotation. Nucleic Acids Res. 36, D1009–D1014.

Tan, J., Wang, H.L. and Yeh, K.W. (2005) Analysis of organ-specific, expressed genes in Oncidium orchid by subtractive expressed sequence tags library. Biotechnol. Lett. 27: 1517–1528.

Teh, S.L., Chan, W.S., Abdullah, J.O. and Namasivayam, P. (2010) Development of expressed sequence tag resources for Vanda Mimi Palmer and data mining for EST-SSR. Mol. Biol. Rep. 38: 3903–3909.

Tholl, D., Chen, F., Petri, J., Gershenzon, J., Pichersky, E., 2005. Two sesquiterpene synthases are responsible for the complex mixture of sesquiterpenes emitted from Arabidopsis flowers. Plant J. 42, 757–771.

Tremblay, R.L., Ackerman, J.D., Zimmerman, J.K. and Calvo, R.N. (2005) Variation in sexual reproduction in orchids and its evolutionary consequences: a spasmodic journey to diversification. Biol. J. Linn. Soc. 84: 1–54.

Tsai,W.C., Chuang,M.H., Kuoh, C.S., Chen,W.H., Chen, H.H., 2004. Four DEF-likeMADS box genes displayed distinct floral morphogenetic roles in Phalaenopsis orchid. Plant Cell Physiol. 45, 831–844.

Tsai WC, Lee PF, Chen HI, Hsiao YY, Wei WJ, Pan ZJ, Chuang MH, Kuoh CS, Chen WH, Chen HH., 2005. PeMADS6, a GLOBOSA/PISTILLATA-like gene in Phalaenopsis equestris involved in petaloid formation, and correlated with flower longevity and ovary development. Plant Cell Physiol. 46, 1125–1139.

Wen-Chieh Tsai, Yu-Yun Hsiao, Shu-Hua Lee, Chun-Wei Tung, Dan-Ping Wang, Hei-Chia Wang, Wen-Huei Chen, Hong-Hwa Chen., 2006. Expression analysis of the ESTs derived from the flower buds of Phalaenopsis equestris. Plant Sci. 170, 426–432.

Tsai, W.C. and Chen, H.H. (2006) The orchid MADS-box genes controlling floral morphogenesis. ScientificWorldJournal 6,1933–1944.

Tsai, W.C., Hsiao, Y.Y., Lee, S.H., Tung, C.W., Wang, D.P., Wang, H.C.et al. (2006) Expression analysis of the ESTs derived from the flower buds of Phalaenopsis equestris. Plant Sci. 170, 426–432.

Tsai, W.C., Kuoh, C.S., Chuang, M.H., Chen, W.H. and Chen, H.H. (2004) Four DEF-like MADS box genes displayed distinct floral morphogenetic roles in Phalaenopsis orchid. Plant Cell Physiol. 45, 831–844.

Tsai, W.C., Lee, P.F., Chen, H.I., Hsiao, Y.Y., Wei, W.J., Pan, Z.J. et al. (2005) PeMADS6, a GLOBOSA/PISTILLATA-like gene in Phalaenopsis equestris involved in petaloid formation, and correlated with flower longevity and ovary development. Plant Cell Physiol. 46, 1125–1139.

Wen-Chieh Tsai, Yu-Yun Hsiao, Zhao-Jun Pan, Chia-Chi Hsu, Ya-Ping Yang, Wen-Huei Chen, Hong-Hwa Chen., 2008a.Molecular biology of orchid flowers: with emphasis on Phalaenopsis. Adv. Bot. Res. 47, 99–145.

Tsai, W.C., Pan, Z.J., Hsiao, Y.Y., Jeng, M.F., Wu, T.F., Chen, W.H. et al.(2008) Interactions of B-class complex proteins involved in tepal development in Phalaenopsis orchid. Plant Cell Physiol. 49, 814–824.

Voelckel, C., Borevitz, J.O., Kramer, E.M., Hodges, S.A., 2010. Within and between whorls: comparative transcriptional profiling of Aquilegia and Arabidopsis. PLoS One 5, e9735.

V. Roth and T. Lange, "Bayesian class discovery in microarray datasets," IEEE Transactions on Biomedical Engineering, vol. 51, pp. 707-718, 2004.

Wang, C.Y., Chiou, C.Y., Wang, H.L., Krishnamurthy, R., Venkatagiri, S., Tan, J. et al. (2008) Carbohydrate mobilization and gene regulatory profile in the pseudobulb of Oncidium orchid during the flowering process. Planta 227, 1063–1077.

Wang, S.Y., Lee, P.F., Lee, Y.I., Hsiao, Y.Y., Chen, Y.Y., Pan, Z.J. et al.(2011) Duplicated C-class MADS-box genes reveal distinct roles in gynostemium development in Cymbidium ensifolium (Orchidaceae). Plant Cell Physiol. 52, 563–577.

Wang, W., Wang, Y., Zhang, Q., Qi, Y. and Guo, D. (2009) Global characterization of Artemisia annua glandular trichome transcriptome using 454 pyrosequencing. BMC Genomics 10, 465.

W Patrick Wechter, Amnon Lev, Karen R Harris, Angela R Davis, Zhangjun Fei, Nurit Katzir, James J Giovannoni, Ayelet Salman-Minkov, Alvaro Hernandez, Jyothi Thimmapuram, Yaakov Tadmor, Vitaly Portnoy and Tova Trebitsh., 2008. Gene expression in developing watermelon fruit. BMC Genomics 9, 275.

Weigel, D., Meyerowitz, E.M., 1994. The ABCs of floral homeotic genes. Cell 78, 203–209.

Wellmer, F., Riechmann, J.L., Alves-Ferreira, M., Meyerowitz, 2004. Genome-wide analysis of spatial gene expression in Arabidopsis flowers. Plant Cell 16, 1314–1326.

Wu, T., Qin, Z., Zhou, X., Feng, Z. and Du, Y. (2010) Transcriptome profile analysis of floral sex determination in cucumber. J. Plant Physiol. 167, 905–913.

Xu, Y.H., Wang, J.W., Wang, S., Wang, J.Y., Chen, X.Y., 2004. Characterization of GaWRKY1, a cotton transcription factor that regulates the sesquiterpene synthase gene (1)-δ-cadinene synthase-A. Plant Physiol. 135, 507–515.

Xu, Y., Teo, L.L., Zhou, J., Kumar, P.P., Yu, H., 2006. Floral organ identity genes in the orchid Dendrobium crumenatum. Plant J. 46, 54–68.

Yu, H. and Goh, C.J. (2001) Molecular genetics of reproductive biology in orchids. Plant Physiol. 127, 1390–1393.

Zheng, H., Rowland, O., Kunst, L., 2005. Disruptions of the Arabidopsis Enoyl-CoA reductase gene reveal an essential role for very-long-chain fatty acid synthesis in cell expansion during plant morphogenesis. Plant Cell 17, 1467–1481.