臺灣博碩士論文加值系統

　　水稻(Oryza sativa L.)在台灣及其他亞熱帶地區早春的幼苗時期經常遭受冷害，此冷害可能導致作物生長受阻甚至降低產量。為了進一步探究調控冷害的分子機制，本研究以差異表現法(differential display)並配合銀染的技術來選殖與水稻幼苗低溫耐性表現相關的cDNAs。以低溫敏感的印度型水稻台中在來1號(TN 1)及低溫抗性的日本型台農67號(TNG 67)幼苗，經過(1)5℃；(2)25℃(對照組)；(3) 10 mM ABA，25℃；或(4)10 mM methyl jasmonate (MeJa)，25℃；各24小時處理後，取其根部之total RNA供進行差異表現法試驗。結果由600個差異表現片段中，成功再擴增出362個差異表現之cDNA片段，經基因庫比對結果，發現有153個(42.3 %) cDNA與已知基因有極高的相似性；61個cDNA分別與rab16D、heat shock protein、SalT、Zn-induced protein……等29個已知功能基因有極高的相似性。
另外以RNA slot blot分析結果發現，有58個cDNA clones在低溫或ABA處理下有極高的表現。利用北方墨點分析法(northern-blot analysis)自58個差異表現的片段中，選出28個在低溫、ABA及其它逆境處理下的表現與對照組有明顯差異的cDNA clones。其中有部分cDNA分別在低溫、ABA、MeJa、缺水、鹽害及熱休克(heat shock)處理下的水稻根部或地上部具專一性表現。以11個分別具有不同低溫耐性及ABA內生量的水稻品種，來測試這28個cDNA clones的表現，發現有6個基因在根部或地上部的表現量與電解質滲漏量呈顯著負相關，顯示這6個基因與低溫耐性相關。
低溫逆境下ABA前處理可導致部分低溫耐性基因在低溫敏感的水稻台中在來1號幼苗中大量表現，顯示這些基因可能參與水稻幼苗受ABA調節所產生的低溫耐性。另外，部分基因在未經ABA前處理的水稻幼苗中大量表現，顯然它們的表現不受ABA的影響。本試驗的結果也顯示，差異表現法可有效的選殖水稻幼苗低溫相關基因。

　　Rice (Oryza sativa L.) seedlings frequently suffer from chilling injury during early spring in Taiwan as well as in other subtropical areas. The chilling injury may cause severe growth retardation and yield reduction. To gain more insights of molecular mechanisms of chilling tolerance, cDNAs associated with chilling tolerance in rice seedlings were isolated using differential display of mRNA. Seedlings of a chilling-sensitive indica type cultivar (cv. Taichung Native 1, TN 1), and a chilling-tolerant japonica type cultivar (cv. Tainung 67, TNG 67) were treated for 24 h with either: (1) 5oC; (2) 25oC (control); (3) 10-5 mM ABA at 25oC; or (4) 10-5 M methyl jasmonate (MeJa) at 25oC. The later two treatments were previously found that could induce chilling-resistance in TN1 seedlings. Total RNA were isolated from the roots of seedlings and used for differential display. Clones of 362 cDNA were re-amplified and isolated from 600 differentially expressed cDNA fragments. Sequences of 153 cDNA clones had significant similarities to the sequences known in the GenBank. Sixty-one of these clones, which could be separated to 29 groups, showed high homology to the sequences with known functions, i.e., rab16D, heat shock protein, SalT, and Zn-induced protein.
Slot-blot analysis revealed that 58 cDNA clones could be regulated by ABA, MeJA or chilling treatments. Among these 58 differentially expressed cDNA clones, 28 cDNA clones could be enhanced by ABA or chilling using northern blot analysis. In addition, 6 clones of the 28 clones showed differential expressions under stresses including chilling, osmotic, salt, heat shock, and ABA or MeJa treatments. By testing their expressions in 11 cultivars, which have different chilling tolerance and different chilling-induced ABA levels, six cDNA clones showed positively correlated with chilling tolerance. Some of these cDNA clones showed much higher transcript levels after exposure to chilling stress in TN 1 seedlings that pre-treated with ABA. Therefore, these clones might be involved in the ABA-mediated acclimation of chilling resistance in TN 1 seedlings. In contrast, transcript levels of other cDNA clones were up-regulated in seedlings without ABA pre-treatment, and their expressions seemed not to be related to endogenous ABA levels. In the current study, the technique of mRNA differential display was successfully used to identify genes associated with chilling tolerance in rice seedlings.

封面
目
表目錄
圖目錄
中文摘要
英文摘要
縮寫字
第一章 緒言
第二章前人研究
第三章利用差異表現法選殖水稻幼苗低溫耐性基因及低溫耐性相關基因之表現
第一節 前言
第二節 材料與方法
第三節 結果
第四節 討論
第四章 水稻低溫耐性相關基因與低溫耐性表現之關係
第一節 前言
第二節 材料與方法
第三節 結果
第四節 討論
第五章
參考文獻
附錄

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