水稻OsSAPK6/OSRK1基因之分子鑑定與生理功能分析

Abstract

非生物性逆境對於植物的生長發育與產量品質影響甚鉅，所以探討植物的非生物逆境耐受性之分子機制相當重要。在逆境下植物會伴隨著ABA的生合成，並改變內生基因及代謝物之表現。SnRK2 (Sucrose Non-fermenting 1 Related protein Kinase 2) 是在植物中特有之蛋白磷酸激酶並且會參與在ABA及逆境訊息途徑。在水稻中有10種OsSAPK (Stress Activated Protein Kinase)，本論文針對OsSAPK6在逆境下探討其基因表現，並利用Tos17 OsSAPK6突變株進行生理與分子鑑定分析，以了解OsSAPK6基因於水稻之功能。 首先利用生物資訊的方式分析阿拉伯芥及水稻不同SnRK2之親緣關係，並利用PLACE軟體分析OsSAPK6之1.5kb啟動子順式序列，可確認CRT (C-repeat binding element)、ABRE (ABA-responsive element)、WRKY等DNA結合順式序列，表示OsSAPK6之表現可能會受到逆境誘導或抑制。為瞭解OsSAPK6於逆境下之基因表現，首先利用TNG67處理鹽、乾旱、高溫、低溫等逆境，發現OsSAPK6可受到鹽及乾旱逆境下之誘導。本論文乃針對此兩種逆境進行分析。在不同水稻發育部位與時期，發現OsSAPK6於葉身表現量較高。另外為了比較不同水稻品種(梗/秈)間的差異性，將TCN1(秈稻)以鹽與乾旱處理結果發現OsSAPK6的表現量會較TNG67(梗稻)為低。此外為瞭解OsSAPK6在水稻在逆境下所扮演的角色，本試驗亦比較了日本晴(Nipponbare)及Tos17 OsSAPK6突變株於鹽及乾旱逆境下之外表型及其基因表現。也進一步探討OsSAPK6對於水稻鹽及乾旱耐受性之可能分子機制，偵測Tos17突變株在鹽與乾旱逆境處理下之轉錄因子DREB1A及DREB2A、下游逆境相關基因DHN1及SalT的基因表現。結果顯示四種基因表現相較於WT為低，表示OsSAPK6為一參與水稻鹽及乾旱逆境耐受性之正向調控因子。最後為了解OsSAPK6基因之啟動子活性，將pSAPK6::GUS利用基因槍擊發至水稻胚誘導之癒傷組織上，結果顯示GUS染色有所反應，表示此1.5bk之OsSAPK6啟動子片段為一具功能性之啟動子序列。而為確定OsSAPK6之次細胞表現位置，將OsSAPK6::GFP利用基因鎗將載體擊發至洋蔥表皮細胞上，觀察結果發現OsSAPK6會專一性表現在細胞核中。上述研究結果顯示，OsSAPK6可能參與在水稻鹽及乾旱逆境反應途徑，調控下游轉錄因子，影響逆境反應相關之基因表現，進而影響植株之外表型。

Abiotic stress can greatly affect plant growth and production, so it is important to identify the mechanism of abiotic stress tolerance. When plant encounter abiotic stress, plant accumulate ABA and reprogram gene and metabolites expression. SnRK2 (Sucrose Non-fermenting 1 Related protein Kinase 2) are specific present in plant and regulate ABA and abiotic stress signaling pathway. There are 10 OsSAPKs (Stress Activated Protein Kinase) in Oryza sativa L. In this study, we focus on OsSAPK6 to study the gene expression pattern under various abiotic stresses. We also use Tos17 OsSAPK6 knock-out mutant to dissect possible function of OsSAPK6 gene. At first, we took bioinformatic approach to examine SnRK2 phylogenetic relationship from Arabidopsis and rice then use PLACE to find the putative cis-acting elements in promoter of OsSAPK6 gene. Different cis-acting elements, such as CRT, ABRE and WRKY binding site can be identified in the OsSAPK6 promoter region and indicated that OsSAPK6 expression may be induced under abiotic stress. Using RT-PCR and cDNA from TNG67 treated with salt, drought, high and low temperature, we found that OsSAPK6 expression can be induced by salt and drought stress. Next we monitored the OsSAPK6 expression in different developmental stages and tissues. OsSAPK6 gene was highly expressed in leaf blade. To monitor OsSAPK6 expression in different rice cultivars, we confirmed that OsSAPK6 gene expression was lower in TCN1 than TNG67 under salt and drought stress. Furthermore, to understand function of OsSAPK6 in various stress responses of rice, Tos17 mutants from RGRC were treated by salt and drought stress and the phenotypes and OsSAPK6 gene expression were compared with wild type. Also, to reveal role of OsSAPK6 in the molecular mechanism of stress response, we determined genes expression of two transcription factors (DREB1A & DREB2A) and two down stream stress response genes (DHN1 & SalT) in WT and Tos17 mutants . The expression of DREB1A, DREB2A, DHN1 and SalT was down-regulated in Tos17 mutants as compared with WT. Finally, to analysis the OsSAPK6 promoter activity, we made pSAPK6::GUS plasmid construct and transformed into rice callus. The GUS staining showed blue color on callus and indicated that the 1.5kb DNA fragment of OsSAPK6 promoter is functional. To address the subcellular localization of OsSAPK6 protein, the OsSAPK6::GFP plasmid was particle bombarded to onion epidermis cells and we observed that OsSAPK6 was specifically localized in nucleus. Above all, these results suggested that OsSAPK6 is a positive regulator that involve in the upstream of salt and drought stress tolerance in rice. OsSAPK6 can regulate expression of transcription factors and downstream stress response gene such as DREB1A, DREB2A, DHN1,SalT and affects rice salt and drought stress tolerance.