蝴蝶蘭雖原產於熱帶或亞熱帶，比一般植物具有較高的耐熱性，但高溫時間過久，仍會導致休眠，生長遲滯，如再遭遇通風不良，或溼度太高，則容易遭致軟腐病以及其他病害的發生。因此，我們利用PCR篩選扣減式基因庫的技術，選取蝴蝶蘭在高溫逆境下的基因並探討其表現。分析篩選基因的結果，我們找到一些EST，其產物可能是植物面對高溫逆境時所產生的保護蛋白，如熱休克轉錄調節因子 (heat shock transcription factor) 和低分子量的熱休克蛋白 (small heat shock protein) 等。另外我們也找到一些與auxin訊息傳導相關的EST，如AUX1 permease和ChaC-like protein (cation transporter)。而北方墨點分析 (Northern blot) 結果意外發現，這些EST的表現似乎受生理時鐘所調控。由北方墨點分析的結果，啟動子的分析，以及相關的文獻研究，我們推測auxin訊息傳導途徑和蝴蝶蘭高溫逆境的反應可能有關。

Abstract

Phalaenopsis amabilis var. formosa Shimadzu is one of the common tropic and sub-tropic plants that possess some thermotolarance under heat stress. Despite of this capacity, however, the Phalaenopsis would easily be dormant or cease growing, and could be severely infected by pathogen during long-term high temperature. To determine how Phalaenopsis responses to high temperature, we constructed a heat-induced partial cDNA library using PCR-select subtraction. Within the 120 ESTs selected for this study, there are genes encoding the heat shock transcription factor and the small heat shock protein that are involved in known protective mechanisms at higher temperaure. In addition, we obtained some ESTs conding for proteins associated with auxin signal transduction pathway, such as AUX1 permease and Chac-like protein (cation transporter). Interestingly, Northern blot analyses indicated that the transciption levels of those ESTs seem to be regulated by circadian rhythm. Meanwhile, we analyzed the promoter region of these putative auxin-related ESTs to acquirte more information. Our results suggest that the auxin signaling pathway probably interact with heat sock response in Phalaenopsis.

參考文獻

陳文輝 (2002) 科學發展，351期，專題報導。

Bouche N., Fait A., Bouchez D., Moller S. G., and Fromm H. (2003) Mitochondrial succinic-semialdehyde dehydrogenase of the -amino- butyrate shunt is required to restrict levels of reactive oxygen intermediates in plants. Proc. Natl. Acad. Sci. USA 100: 6843-8.

Chang S., Jeff P., and John C. (1993) A simple and efficient method for isolating RNA from pine tree. Plant Mol. Biol. Reporter 11: 113-116.

Caplan A. J. (1999) Hsp90's secrets unfold: new insights from structural and functional studies. Trends Cell Biol. 9: 262-268.

Escobar M. L. G., Salla M., Gunilla H., Jens F., and Carina K. (2001) Heat Stress Response in Pea Involves Interaction of Mitochondrial Nucleoside Diphosphate Kinase with a Novel 86-Kilodalton Protein. Plant Physiol. 126: 69-77.

Ferguson I. B., Susa L., and Judith H. B. (1994) Protein synthesis and beakdown during heat shock of cultured pea (Pyrus communis L.) cells. Plant Physiol. 104: 1429-1437.

Finley D., Ozkaynak E., and Varshavsky A. (1987) The yeast polyubiquitin gene is essential for resistance to high temperature, starvation, and other stresses. Cell 48: 1035-1046.

Glover J. R. and Lindquist S. (1998) Hsp104, Hsp70, and Hsp40: a novel chaperone system that rescues previously aggregated proteins. Cell 94: 73-82.

Harmer S. L., Hogenesch J. B., Straume M., Chang H. S., Han B., Zhu T., Wang X., Kreps J. A., and Kay S.A. (2000) Orchestrated transcription of key pathways in Arabidopsis by the circadian clock. Science 290: 2110-2113.

Harmer S. L., Panda S., and Kay S. A. (2001) Molecular bases of circadian rhythms. Annu. Rev. Cell Dev. Biol. 17: 215-253.

Heckathorn S.A., Downs C.A., Sharkey T.D., Coleman J.S. (1998) The small, methionine-rich chloroplast heat-shock protein protects photosystem II electron transport during heat stress. Plant Physiol. 116: 439-444.
Iba Koh (2002) Acclimative response to temperature stress in higher plants: approaches of gene engineering for temperature tolerance. Annu. Rev. Plant Biol. 53: 225-245.

Kotak Sachin, Markus Port, Arnab Ganguli, Frank Bicker, and Pascal von Koskull-Döring (2004) Characterization of C-terminal domains of Arabidopsis heat stress transcription factors (Hsfs) and identification of a new signature combination of plant class A Hsfs with AHA and NES motifs essential for activator function and intracellular localization. The Plant J. 39: 98-112.

Miller A. J. (2000) Clock proteins: turned over after hours? Curr. Biol. 10: R529-R531.
Moon H., Boyoung L., Giltsu C., Dongjin S., D. Theertha P., Oksun L., Sang-Soo K., Doh H. K., Jaesung N., Jeongdong B., Jong C. H., Sang Y. L., Moo J. C., Chae O. L., and Dae-Jin Y. (2003) NDP kinase 2 interacts with two oxidative stress-activated MAPKs to regulate cellular redox state and enhances multiple stress tolerance in transgenic plants. Proc. Natl. Acad. Sci. USA 100: 358-363.
Oberschmidt O., Grundler F.M.W., and Kleine,M. (2003) Identification of a novel cation transporter gene from sugar beet (Beta vulgaris L.) by DDRT-PCR closely linked to the beet cyst nematode resistance gene Hs1pro-1. Unpublished.

Parsell D. A. and Lindquist S. (1993) The function of heat-shock proteins in stress tolerance: degradation and reactivation of damaged proteins. Annu. Rev. Genet. 27: 437-496.

Parsell D. A., Kowal A. S., Singer M. A., and Lindquist S. (1994) Protein disaggregation mediated by heat-chock protein Hsp104. Nature 372: 475-478.

Reindl A, Fritz S., Jeff S., Csaba K., and László B. (1997) Phosphorylation by a cyclin-dependent kinase modulates DNA binding of the Arabidopsis heat-shock transcription factor HSF1 in vitro. Plant Physiol. 115: 93-100.

Rensing L. and Monnerjahn C. (1996) Heat shock proteins and circadian rhythms. Chronobiol. Int. 13: 239-250.

Riezman H. (2004) Why do cells require heat shock protein to survive heat stress? Cell Cycle 3: 61-63.

Rikin A. (1992) Circadian rhythm of heat resistance in cotton seedlings: synthesis of heat-shock proteins. Eur. J. Cell Biol. 59: 160-5.

Rothman J. E. (1989) Polypeptide chain binding proteins: catalysts of protein folding and related processes in cells. Cell 59: 591-601.

Schaffer R., Landgraf J., Monica A., Simon B., Larson M., and Wisman E. (2001) Microarray analysis of diurnal and circadian-regulated genes in Arabidopsis. The Plant Cell 13: 113-123.

Sun W., Marc V. M., and Nathalie V. (2002) Small heat shock protein and stress tolerance in plants. Biochem. Biophys. Acta 1577: 1-9.

Sweet H.R. (1980) The genus Phalaenopsis. Day Printing Corp., Calif..

Taiz L. and Zeiger Z. (2002) Stress Physiology. In: Plant Physilology 3rd ed, pp 454-457& pp. 602-607. Sinauer Associates, Inc..

Vierling E. (1991) The roles of heat shock proteins in plants. Annu. Rev. Plant Physiol. Plant Mol. Biol. 42: 579-620.

Young M.W. and Kay S. A. (2001) Time zones: a comparative genetics of circadian clocks. Nat. Rev. Genet. 2: 702-715.

Young T. E., Jun L., C. Jane G. L., Robert L. T., Christian C., and Daniel R. G.. (2001) Developmental and Thermal Regulation of the Maize Heat Shock Protein, HSP101. Plant Physiol. 127: 777-791.