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研究生: 黃氏妙玲
Hoang Thi Dieu Linh
論文名稱: 兩種台灣蝴蝶蘭雜交種對低溫逆境的生理和分子反應
Physiological and molecular comparisons of two Phalaenopsis amabilis hybrids in response to cold stress
指導教授: 林彩雲
Lin, Tsai-Yun
口試委員:
學位類別: 碩士
Master
系所名稱: 生命科學暨醫學院 - 生物資訊與結構生物研究所
Institute of Bioinformatics and Structural Biology
論文出版年: 2009
畢業學年度: 97
語文別: 英文
論文頁數: 69
中文關鍵詞: 蝴蝶蘭低溫逆境CBF基因表現
外文關鍵詞: Phalaenopsis amabilis, cold stress, CBF, gene expression
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  • 蝴蝶蘭 (Phalaenopsis amabilis) 在台灣花卉產業具有重要的地位。在外銷過程特別是運輸到溫帶地區的國家,低溫逆境常造成植物的損傷而降低其經濟價值。本研究目的是探討兩種蝴蝶蘭雜交種TS97及K51206在低溫逆境下的生理反應。於特定時間間隔收取低溫處理 (4°C) 及對照組 (25°C) 蘭花植株樣品分析其生理改變。由electrolyte leakage的結果發現,在低溫下TS97可維持較好的細胞膜完整性,顯示TS97比K51206有較高的低溫耐受度。測定Fv/Fm數值以比較低溫下這二種蝴蝶蘭的光合作用能力,在4°C下TS97的光合作用較不受低溫所影響。此外選殖PaCBF基因,此基因演繹蛋白質產物為CBF轉錄子,其同源基因參與調控逆境反應基因,以低溫、乾燥、高鹽、滲透壓逆境及離層酸處理並分析PaCBF的mRNA含量變化。結果顯示TS97植株在4□C處理1小時內PaCBF的mRNA含量顯著增加,至10小時達到最高累積量,並且TS97的PaCBF mRNA含量的增加程度高於K51206。另選殖可能受PaCBF所調控的下游基因PaDhn,在低溫下其mRNA含量隨PaCBF的mRNA含量變化增加。我們的結果顯示PaCBF的mRNA含量或許可以做為探討蘭花低溫耐受性的標誌。


    Phalaenopsis amabilis is important in the Taiwan flower industry. During transportation to international markets, especial the temperate area, chilling may damage plants and reduces their values. The goal of this study is to examine the physiological responses to cold of orchids using the TS97 and K51206. Orchids were exposed to 4°C at different time intervals to examine physiological changes. TS97 was found to be more cold tolerant than K51206 as their better maintenance of membrane integrity at 4°C measured with electrolyte leakage. The photosynthetic capacity of TS97 measured as Fv/Fm was also detected to be greater than that of K51206 at 4°C. Moreover, PaCBF coding for a putative CBF transcription was cloned and the mRNA levels were characterized under cold, drought, salinity, osmoticant and ABA treatments. In TS97, the PaCBF mRNA level was significantly induced within 1 hr after exposure to 4°C, and then accumulated to the highest amount at 10 hr of cold stress. The induction of the PaCBF mRNA level was found to be higher in the TS97 than the K51206. The mRNA level of a putative downstream gene of PaCBF, the PaDhn, was found to follow the increase of the PaCBF mRNA level. Our results suggest that PaCBF mRNA level may be used as a marker of cold tolerance in orchid.

    Table of contents Page Acknowledgements 摘要…………………………………………………………………………………i Abstract……………………………………………………………………………..ii Acknowledgements………………………………………………………...………iii Abbreviations………………………………………………………………………iv Tables of contents…………………………………………………………….........vi Lists of tables………………………………………………………………..…......ix List of figures………………………………………………………………….........x Introduction…………………………………………………………………………1 Materials and methods……………………………………………………………...7 1. Plant materials and stress treatments……………………………….........7 2. Total cellular RNA extraction (Hot-phenol method)….…………………9 2.1 Total cellular RNA extraction………………………………….9 2.2 Formaldehyde denatured agarose gel and electrophoresis.........10 3. Full length cDNA synthesis………………………………………….....11 3.1 Reverse transcription…………………………………………..11 3.2 Polymerase chain reaction…………………………………….11 3.3 Ligation………………………………………………………..12 3.4 Competent cell preparation……………………………………13 3.5 Bacterial transformation…………………………………….....13 3.6 Mini-preparation of plasmid DNA………………………….....14 3.7 Restriction enzyme digestion...……………………………......14 3.8 Double-stranded DNA sequencing……………………………14 4. Electrolyte leakage measurement……………………………………....15 5. Photosynthesis measurement…………………………………………...16 6. Relative quantification real-time PCR (qRT-PCR)…………………….17 7. Statistical analysis………………………………………………………18 Results……………………………………………………………………………..19 1. Morphological changes in orchid during cold stress …………………19 2. Low temperature effects on electrolyte leakage of orchids….………..19 3. Effects of low temperature on photosynthesis in orchids......................21 4. Isolation of a full length PaCBF cDNA and a partial PaDhn cDNA....22 5. Expression of PaCBF mRNA level induced by cold treatment……….25 6. Expression of PaDhn mRNA level induced by cold treatment……….27 7. The PaCBF and PaDhn mRNA levels in TS97 flowers under cold stress…………………………………………………………………...28 8. Expression of PaCBF mRNA in different tissues of TS97 under cold stress……………………………………………………………...........28 9. Expression of PaCBF and PaDhn mRNA levels in TS97 and K51206 under drought, salinity, osmoticant and ABA stress...………………...29 Discussion…………………………………………………………………………32 1. TS97 is more cold tolerant than K51206 as indicated by to the better maintenance of membrane integrity at 4°C..…………….. …………...32 2. TS97 maintained a higher photosynthetic capacity under cold stress...34 3. Identification of the CBF in Phalaenopsis amabilis…………………..35 4. Activation of PaCBF and PaDhn mRNA expression may result in a better cold tolerance…………………………………………………...36 5. Conclusion...…………………………………………………………..38 Reference………………………………………………………………………….40 Appendix…………………………………………………………………………..68

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