中文摘要 水稻(Oryza sativa Tainung, No.67)有九個第一族小分子量熱休克蛋白質基因，分別位於第一對及第三對染色體上。位於第三對染色體上的Oshsp17.3及Oshsp18.0基因啟動子以頭對頭的方式共用一個356 bp的啟動子區域。研究顯示Oshsp17.3啟動子具有一段對Aze有專一性的反應元素(azetidine responsive element；AZRE；GTCCTGGACG)，當AZRE刪除後，對Aze逆境或熱逆境處理敏感性都有降低的趨勢。為改善對照組測定值過高，故改以阿拉伯芥原生質體暫時表現系統對啟動子不同區域片段進行分析。結果顯示，阿拉伯芥原生質體暫時表現系統對熱逆境或鎘離子逆境誘導的敏感性並不明顯。因此改以穩定轉殖(stable transformation)的方式確認AZRE與啟動子AT-rich片段的角色，將p567(－) (Oshsp17.3啟動子全長)、 p567(－)ΔAZRE (Oshsp17.3刪除AZRE片段)、 p567(＋) (Oshsp18.0啟動子全長)，以及P1600 (Oshsp16.9A啟動子全長)和P500 (Oshsp16.9A啟動子刪除AT-rich片段)，利用農桿菌轉殖入阿拉伯芥中，共獲得33個獨立轉殖株品系。以南方墨點分析法選取單套及雙套轉殖基因套數的轉殖品系，進行水稻第一族小分子量熱休克蛋白質基因之啟動子受逆境誘導分析的實驗。 首先針對不同時期的阿拉伯芥轉殖株進行熱逆境與Aze逆境處理觀察組織表現情形。在兩天大的T2轉殖株，熱逆境處理組及Aze逆境處理組與對照組的組織染色結果皆呈現藍色GUS反應，並無差異表現。14天大的T2轉殖株，p567(＋)、p567(－)及p567(－)ΔAZRE的轉殖株，對照組呈微弱或無藍色GUS反應，熱逆境處理組與Aze逆境處理組結果都呈現藍色GUS反應。另方面P1600或P500的熱逆境處理組及Aze逆境處理組與對照組組織染色結果皆呈現明顯藍色GUS反應，無差異表現。一個月大T2轉殖株不同部位逆境處理部份，p567(＋)、p567(－)和p567(－)ΔAZRE熱逆境處理後，組織染色結果皆呈現明顯藍色GUS反應，對照組則沒有藍色GUS反應；在Aze逆境處理部分，p567(＋) 和p567(－)組織染色結果皆呈現藍色GUS反應；但p567(－)ΔAZRE組織染色 結果則與對照組相同無藍色GUS反應出現。分析結果顯示，五組不同啟動子片段的轉殖株並無不同生長時期組織特異性表現的情形。 接著分析轉殖株受逆境處理誘導後GUS活性表現情形。在轉殖株品系P567(＋)及P567(－)，給予熱逆境和Aze逆境處理後，GUS活性呈現受此兩逆境誘導而表現；P567(＋)ΔAZRE給予熱逆境處理後，GUS活性分析顯示轉殖株受熱逆境誘導敏感性不大，給予Aze逆境處理，GUS活性幾乎不受Aze逆境誘導而表現。另方面，在P500的獨立轉殖株品系，GUS活性完全不受熱逆境與Aze逆境誘導而表現。而P1600的獨立轉殖株品系GUS活性受熱逆境誘導而表現，但對Aze逆境處理而誘導的敏感性幾乎沒有反應。本實驗與先前利用基因槍方式對啟動子不同片段區域進行分析結果相近。 最後分別將水稻第一族小分子量熱休克蛋白質基因Oshsp16.9A及大麥(Hordeum vulgare L.)的第三群LEA蛋白質基因HVA1抗逆境基因送入蝴蝶蘭中，期能利用這兩類逆境蛋白質的生理功能增加蝴蝶蘭(Phalaenopsis sp.)對環境逆境的抗性。蝴蝶蘭轉殖工作目前已初步完成，後續兩逆境相關基因Oshsp16.9A及HVA1蛋白質功能性的分析有待進一步的研究來闡明其扮演的角色。 Abstract There are 9 members of the class I small heat shock proteins (sHSP-CI) gene family in rice (Oryza sativa L. cv. Tainong No.67), of which on chromosome 1 and chromosome 3. Interestingly, Oshsp17.3 and Oshsp18.0 on chromosome 3 are linked head-to-head and share a 356-bp putative bi-directional promoter. A possible azetidine (Aze)-responsive element (AZRE;GTCCTGGACG) on Oshsp17.3 promoter was found to be capable of directing expression in response to Aze treatment. The truncation of AZRE decreased the induction of both Aze induction and heat shock (HS) induction. To improve high control value, we constructed a transient expression system using Arabidopsis protoplast to assay Oshsp17.3 promoter. The results showed that there was not significant difference for HS induction and Cd induction. So we use stable transformation to confirm the role of AZRE and AT-rich region. Five constructs ( p567(－): full length of Oshsp17.3 promoter; p567(－)ΔAZRE: AZRE truncation of Oshsp17.3 promoter; p567(＋): full length of Oshsp18.0 promoter; P1600: full length of Oshsp16.9A promoter; P500: AT-rich region truncation of Oshsp16.9A promoter) were generated and introduced into Arabidopsis plants via Agrobacterium. Totally 33 independent lines were constructed for further study. The copy number of transgene was estimated by southern-blot hybridization. One or two copies of transgenic independent line was selected and characterized under stress. Different stage of Arabidopsis transgenic plants were exposed to HS and Aze treatment for histochemical analysis. Two-day-old seedlings and of control and transgenic plants showed constitutive blue GUS staining under stress. Analysis of 14-day-old seedlings revealed that p567(＋), p567(－), and p567(－)ΔAZRE of T2 transgenic plants showed blue GUS staining under HS and Aze stress, but no or less detectable blue GUS staining was found under normal growth conditions. On the other hand, P1600 and P500 transgenic plants showed dominant blue GUS staining under HS and Aze stress. For analysis of one-month-old seedlings of T2 transgenic plants, all tissues in p567(＋) and p567(－) have blue GUS staining under HS and Aze treatment. In contrast, blue GUS staining was detected in HS treated p567(－)ΔAZRE but not in Aze treated p567(－)ΔAZRE. Analysis of GUS activity of the transgenic plants in response to stress induction indicated that GUS activity of p567(＋) and p567(－) increased under HS and Aze treatment. In contrast, p567(－)ΔAZRE showed low expression level of GUS activity under HS stress, and GUS activity was not detected in Aze treated p567(－)ΔAZRE transgenic plants. On the other hands, as we would expect that P1600 showed GUS activity in response to HS but no GUS activity under Aze treatment. Besides, no GUS activity was detected in P500 under HS and Aze stress. Therefore we confirm the role of AZRE on the sHSP-CIs promoter using in vivo system. To improve stress tolerance of Phalaenopsis sp. to environmental stresses, we also try to transform stress genes into this important flower plants in Taiwan. A rice sHSP-CI gene (Oshsp16.9A) and a barley (Hordeum vulgare L.) group 3 LEA protein gene (HVA1) were introduced into Phalaenopsis via Agrobacterium. The works for gene transformation into Phalaenopsis were accomplished. Analysis of stress tolerance conferred by Oshsp16.9A and HVA1 in Phalaenopsis needs further study to characterize in the future.
