| 摘要: | 先前研究證實水稻小分子量熱休克蛋白質基因(small heat shock protein ; sHSP) OsHsp16.9A在熱逆境及種子成熟時期可被啟動表現。為研究OsHsp16.9A生理功能，我們建立OsHsp16.9A過量表現與抑制表現的轉殖株，分別是Oshsp16.9A-OE與Oshsp16.9A-RNAi。我們亦建立與OsHsp16.9A氨基酸序列高度相關的OsHsp18.0 (74%相同度及84%相似度)過量表現轉殖株Oshsp18.0-OE作為對照組。型態分析顯示Oshsp16.9A-RNAi植株生長緩慢與延遲抽穗，野生型、Oshsp16.9A-OE、Oshsp18.0-OE植株則正常。Oshsp16.9A-OE米粒中澱粉粒較野生型、Oshsp16.9-RNAi、Oshsp18.0-OE更為緻密且具有較多的不規則多面體。熱耐受性分析顯示高溫處理後，Oshsp16.9A-OE植株種子存活率明顯較野生型、Oshsp16.9A-RNAi、Oshsp18.0-OE植株種子高；高溫處理後，Oshsp16.9A-OE植株幼苗與花粉存活率亦較其它植株高。此外，微陣列和蛋白質體學分析顯示：高溫逆境中，OsHsp16.9A可與Oshsp101直接或間接的共同作用於水稻種子中，亦可正向調控種子中澱粉合成酵素基因表現。在這個三年的計畫中，我們主要目標是研究OsHsp16.9A在種子耐熱機制和降低白堊質上所扮演的角色。首先，我們試圖研究水稻種子中OsHsp16.9A與OsHsp101之間的交互作用。其次，我們計畫釐清OsHsp16.9A過量表現轉殖株中澱粉合成相關酵素的基因表現，測試OsHsp16.9A是否可在這些澱粉合成酵素基因的突變株中執行功能。最後，我們將比較耐熱與熱敏感水稻品系中OsHsp16.9A的累積情形，冀望所得結果能建立Oshsp16.9A表現量為水稻耐熱性的篩選標記。 ;Our previous results have shown that a rice small heat shock protein (sHSP) gene, OsHSP16.9A, was activated under heat stress (HS) and expressed during seed maturation. To further study the physiological function of OsHSP16.9A, we established Oshsp16.9A-overexpression and -underexpression transgenic rice plants, Oshsp16.9A-OE and Oshsp16.9A-RNAi, respectively. In addition, we also developed the transgenic rice plants transformed with Oshsp18.0 (Oshsp18.0-OE), which shares 74% identity (84% similarity) in protein sequence with Oshsp16.9A, as the control. Phenotype analysis revealed that the growth was retarded and heading time was delayed in Oshsp16.9A-RNAi plants compared with the wild-type (WT), Oshsp16.9A-OE, and Oshsp18.0-OE plants. The grains of 16.9A-OEs have more densely packed and irregularly polyhedral starch granules than those of the WT, Oshsp16.9A-RNAis, and Oshsp18.0-OEs. Thermotolerance analysis indicated that 16.9A-OEs showed a significantly increase in seed survivals after heat treatment, whereas WT, Oshsp16.9A-RNAi, and Oshsp18.0-OE seeds were not able to endure HS-induced detrimental effects. In addition, overexpression of Oshsp16.9A can improve the survival rates of seedlings and pollen grains of transgenic rice plants exposed to HS. Furthermore, using microarray and proteomics analysis, we found that Oshsp16.9A directly or indirectly interacted with Oshsp101 to function in seed thermotolerance and upregulated some starch synthesis enzymes in seeds under HS. In this three-year project, our major goals are to further study the role of Oshsp16.9A in seed thermotolerance and reduction of chalkiness. Firstly, we are trying to investigate the interaction between Oshsp16.9A and Oshsp101 in rice seeds. These results will help us to understand the physiological function of Oshsp16.9A and Oshsp101 in rice seeds. Secondly, we plan to characterize the expression patterns of starch synthesis enzymes in Oshsp16.9A-overexpressing rice seeds and test whether Oshsp16.9A can function in the mutants of these starch synthesis enzymes. Finally, we try to compare expression level of Oshsp16.9A in the thermoresistant and thermosensitive rice varieties. Based on these results, we are able to establish Oshsp16.9A as a screening marker for thermotolerant rice. |
