文心蘭切花老化相關基因之篩選

Molecular cloning of senescence-ssociated genes in Oncidium cut flower

徐偉恩(Wei-En Hsu)

指導教授 ： 鄭石通

國立臺灣大學/生命科學院/植物科學研究所/碩士(2004年)

https://doi.org/10.6342/NTU.2004.10292

摘要

文心蘭為蘭科文心蘭屬（Oncidium）之作物，原產於亞熱帶及熱帶美洲，因種類多且形態豐富，甚受市場歡迎，現已成為台灣重要的外銷切花之一。在夏秋盛產期，文心蘭較快老化，即使經過繁複的保鮮劑處理，瓶插壽命只能多3-4日。爲了延長其切花壽命，期望能選殖出與老化相關之基因，了解其功能，以期對延
長文心蘭切花壽命有所助益。
本實驗中，將文心蘭從全開到老化分為＋10 (未老化)、－8、－6、－4四個時期，利用mRNA differential display，protein 2-D page assay，Suppression Subtractive Hybridization三種不同的篩選方法，比較＋10與其他各老化時期，選殖出文心蘭因老化所誘導表現的差異性片段。結果共選殖出230個差異性片段（mRNA different display 55個；protein 2-D page assay 2個；Suppression Subtractive hybridization 173個）。而利用文心蘭未老化與老化的RNA對Arabidopsis oligo chip做micro array分析，挑選出14個在文心蘭老化時期中有增加表現量的阿拉伯芥基因，釣取其阿拉伯芥基因片段（計15個）。此外，由台灣大學王淑美實驗室提供一經比對結果為ACC synthase之文心蘭基因片段，一併進行文心蘭cDNA晶片
篩選。
由上述方式，最後選取出246個cDNA片段，經PCR放大後點至玻片上，利用文心蘭未老化與老化的cDNA做探針，對於玻片上的cDNA片段進行偵測，進行cDNA micro array。晶片結果發現，共有48個cDNA片段在文心蘭老化時期表現量為未老化時期表現量兩倍以上。而定序結果發現，在45個序列中，共有30個為已知序列，12個為病毒之序列，另外仍有6個在基因庫中找不到有任何相似之序列，以半定量RT-PCR進行已知序列之篩選，發現有8個基因在文心蘭老化時期中表現量確實有上升的情形，推測其為文心蘭老化相關之基因。而經由阿拉伯芥晶片與文心蘭晶片的雙重篩選後，有6個阿拉伯芥基因在與文心蘭探針雜合下，其基因與文心蘭老化花瓣 cDNA之雜合訊息為未老化花瓣 cDNA之雜合訊息兩倍以上。這些基因參與文心蘭花朵老化過程的位置與機制，仍有待日後進一步分析。

關鍵字

文心蘭 ； 老化

並列摘要

The cut flower of Oncidium Gower Ramsey is one of the most important export flowers in Taiwan. After Oncidium flowers are harvested, the operation of classification and package of flowers always results in the dislodgment of pollinia cap. Consequently, ethylene is produced from flowers within hours, and the senescence of flowers is stimulated. Even the application of preservatives, the vase life of Oncidium cut flowers only increases for another 3-4 days. Therefore, it is important to find senescence-associated genes to increase the vase life of Oncidium cut flowers and delay Oncidium senescence.
In this study, mRNA differential display, suppression subtractive hybridization, and protein 2D analysis were performed to clone senescence-related cDNAs. Also, Arabidopsis oligo chip was also used to isolate up-regulated genes during Oncidium senescence. From above methods, 246 senescence-related cDNAs were further spotted on gene chip. After chip screening, 48 cDNAs were up-regulated during Oncidium senescence. Using NCBI BLAST X, 30 cDNAs were predicted to be known genes, 12 cDNAs were predicted to be Cymbidium mosaic virus, and 6 cDNAs were predicted to be unknown sequences. Eight genes of them were further identified by semi-quantitative RT-PCR, indicating they were up-regulated during Oncidium senescence. Besides, there were 6 Arabidopsis genes were up-regulated through both Arabidopsis oligo chip and Oncidium cDNA chip during Oncidium senescence. Their function in Oncidium senescence will be analysis in the future.

並列關鍵字

senescence ； Oncidium

參考文獻

Adams N. D. and Yang S. F.（1979）Ethylene biosynthesis: Indentification of 1-aminocyclopropane-1-carboxylic acid as an intermediate in the conversion of methionine to ethylene. Proc. Natl. Acad. Sci. USA 76: 70-174

Bartoli, C. G., Simontacchi M., Guiame J. J., Montaldi E., and Puntarulo S.（1995）Antioxidant enzymes and lipid peroxidation during aging of Chrysanthemum

Bekman E. P., Saibo N. J. M., Cataldo A. D., Regalado A. P., Ricardo C. P. and Rodrigues-Pousada C. (2000) Differential expression of four genes encoding 1-aminocyclopropane-1-carboxylate synthase in Lupinus albus during germination, and in response to indole-3-acetic acid and wounding. Planta 211: 663-572.

Bleecker A. B., Kende H.（2000）Ethylene: a gaseous signal molecule in plants. Annu. Rev. Cell Dev. Biol. 16: 1-18.

Bohlmann J., Meyer-Gauen G., Croteau R. (1998) Plant terpenoid synthases: molecular biology and phylogenetic analysis. Proc. Natl. Acad. Sci. USA 95: 4126–4133.