2014-01-012024-05-14https://scholars.lib.ntu.edu.tw/handle/123456789/657274摘要：植物對應非生物逆境如鹽分或滲透壓，藉著調節不同的訊號機制來反應。植物的蔗醣中性轉化酵素CINV是細胞質催化蔗糖水解的酵素，早先研究中，AtCINV1被發現存在於細胞核、細胞質和細胞膜之中，AtCINV1基因並參與滲透壓逆境反應。在阿拉伯芥乾旱誘導蛋白Di19有七個基因，AtDi19-2在高濃度鹽分逆境處理下，於轉錄階段增加表現，AtDi19-2被發現存在於細胞核和細胞質中，但是它的蛋白表現和功能仍然未知。14-3-3是個鷹架蛋白，它調節許多訊號傳導機制。在我們初期資料顯示，藉著串聯親合純化或pull-down，14-3-3被發現分別與CINV1和Di19-2分別有交互作用，然而，交互作用機制未明。我們假設阿拉伯芥的CINV1和Di19-2在鹽分或滲透壓逆境反應扮演重要角色，並且CINV1也參與離層酸訊息傳遞機制。這個計劃的目標擬在探究AtCINV1和AtDi19-2的在鹽分或滲透壓逆境反應，和AtCINV1於離層酸訊號傳導中的功能。對於AtCINV1的研究，AtCINV1和AtDi19-2的過度表現植株及T-DNA插入突變株，在鹽分、滲透壓逆境反應或離層酸反應作用下的外表型。我們將使用微矩陣及蛋白質體分析，以揭示在鹽分、滲透壓逆境反應或離層酸處理之下的knockout突變體和過分表現植株之下游受調控基因或蛋白。而且，14-3-3影響CINV1和Di19-2細胞內地域性的機制及重要性也將被調查。我們希望這項研究最終可應用轉植作物以改進作物的逆境抗性。<br> Abstract: Plants respond to salt stress by adjusting various signaling transduction pathways i.e. ABA-dependent signaling pathway. Neutral invertase CINV is a cytosolic enzyme which catalyzes sucrose hydrolysis in cytosol in plant cells. In a previous study, Arabidopsis CINV1 gene is involved in osmotic stress response. CINV1 was found to be sub-cellular localized in the nucleus, cytosol, and membrane. Drought-inducible protein 19 (Di19) is encoded by seven genes with unknown functions. Previous studies showed that AtDi19-2 transcript level increased in response to high-salt stress. CINV1 was found to be subcellular localized in the nucleus and cytosol, but its protein expression pattern and functions remain unknown. 14-3-3 is a scaffold protein which regulates many signaling pathways. In our preliminary data, 14-3-3 was found to be interacting with neutral invertase CINV1 and Di19-2 by use of tandem affinity purification or pull-down respectively. We have tested and confirmed the interaction between 14-3-3omega and neutral invertase CINV1 by yeast two-hybrid screen. T-DNA insertion mutant cinv1 appeared to be more sensitive to salt stress in the root phenotype. In addition, AtDi19-2 was found to be present in the nucleus and cytoplasm by transient expression analysis in Arabidopsis protoplasts. We hypothesize that CINV1 and Di19-2 play important roles in salt or osmotic stress response, and CINV1 is involve in abscisic acid (ABA) signaling pathway in Arabidopsis thaliana. The ultimate goal of this project is to elucidate the function of neutral invertase AtCINV1 and AtDi19-2 in salt or osmotic stress response, and AtCINV1 in ABA signal transduction pathway. For the study of AtCINV1 and AtDi19-2, over-expression line and T-DNA insertion knockout line of CINV1 and AtDi19-2 will be generated and screened out, and subjected to phenotyping to observe salt, osmotic stress, or ABA treatment. In particular, we will observe root phenotype. We will use microarray analysis and 2D-gel based and gel-free proteomic analysis to reveal the up-regulated and down-regulated genes or proteins using knockout mutant and over-expression line under salt, osmotic stress, or ABA treatment. Moreover, the functional importance of 14-3-3 binding on the invertase activity or subcellular localization change will be investigated. We hope that this study will lead to a better understanding of the function of Arabidopsis sucrose hydrolysis enzyme CINV1, Di19-2, and 14-3-3 genes in the osmotic or salt stress response, and hopefully can eventually be applied to engineer transgenic crop to improve abiotic stress resistance.蔗醣中性轉化酵素乾旱誘導蛋白19鹽分細胞核14-3-3neutral invertaseDi19saltnucleus14-3-3