The gene structure and function of rice in chilling tolerance

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℃；等四項各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；其 中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。再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。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 chillingsensitive 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 mM ABA at 25oC; or (4) 10 mM methyl jasmonate (MeJa) at 25oC. The later two treatments were previously found that could induce chillingresistance 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 Zninduced 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 pretreatment, and their expressions seemed not to be related to endogenous ABA levels