- 學位論文
過量表現 Mitogen-Activated Protein Kinase (OsMAPK3) 水稻轉殖株之耐旱與耐鹽生理機制及基因表現分析
Physiological Mechanism and Gene Expression Profile Analysis in Mitogen-Activated Protein Kinase (OsMAPK3) Over-expression Transgenic Rice (Oryza sativa L.) under Drought and Salt Stresses
摘要
乾旱與高鹽逆境會影響水稻之生長與產量。了解水稻對乾旱及高鹽之耐受機制並提升其耐受性至為重要。先前研究指出大量表現 OsMAPK3 (Mitogen-Activated Protein Kinase) 可提昇轉殖水稻對逆境之耐受性，但其作用機制仍不清楚。本論文針對OsMAPK3轉殖水稻進行分子鑑定、生理、微陣列基因表現及real-time PCR分析等，以期闡明 OsMAPK3 與水稻乾旱與鹽逆境之關係。首先我們確認轉基因之插入位點及OMAPK3轉殖水稻之同型結合株。由活體 H2O2 螢光染色顯示在乾旱與鹽分逆境下轉殖株 H2O2 含量比非轉殖株少；正常生長下，轉殖水稻之光合作用效率、氣孔導度及水分利用效率皆會提高。利用微陣列晶片分析轉植株與非轉殖株間基因表現差異，顯示正常生長下轉殖水稻即能誘導逆境相關基因之表現而提升植株之耐受性。此些基因包括調節滲透物質海藻糖合成基因 TPPA；清除活化氧族相關酵素基因GR1、GR3、CatA 及 CatC；乾旱、鹽分逆境相關的轉錄因子 OsDREB1B、OsDREB2A、OsMyb4、OsZFP252及OsNAC6；離子通道蛋白基因 ClC-a、NHX 及對乾旱反應基因 DHN1等。此外亦發現 OsMAPK3 轉殖水稻種子之發芽率比非轉殖株低，對ABA過度敏感。正常生長下轉殖株之內生 ABA 含量增加。轉殖水稻中受 ABA 誘導的標地基因Rab16A 表現量提高，顯示 OsMAPK3 可能參與 ABA 合成或 ABA 傳遞路徑進而改變植株對 ABA 的敏感性。綜合上述， OsMAPK3 水稻轉殖株之所以有較高的逆境耐受性，可歸因於植株本身較高之抗氧化活性、光合作用與水分利用效率、誘導乾旱與鹽份逆境相關基因之表現，及改變植株對ABA之敏感度所致。
並列摘要
Drought and salt stresses can affect rice growth and productivity. It is important to understand the mechanism and develop drought and salt tolerant rice. Previous study showed that overexpression of OsMAPK3 (Mitogen-Activated Protein Kinase) in rice can increase its stress tolerance. However, the detail action and mechanism remains to be solved. To further reveal the role of OsMAPK3 in rice drought and salt stress tolerance, in this study we conducted molecular characterization, physiology, microarray and real-time PCR analysis in OsMAPK3 overexpression transgenic rice. First, we determined transgene integration site and identified homozygous OsMAPK3 line. Then, in vivo H2O2 fluorescence staining image indicated that the amount of H2O2 in transgenic rice was lower then that of wild type under stress. Under normal growth conditions, the OsMAPK3 overexpressing rice displayed higher photosynthesis rate, stomata conductance and water use efficiency (WUE). By comparison of gene expression profile difference between transgenic and non-transgenic rice with microarray, we were able to confirm that several abiotic-stress related genes that known to increase drought and salt tolerance had already been induced in transgenic rice. These genes included osmolyte-adjustment related gene TPPA, antioxidative enzymes related gene GR1, GR3, CatA and CatC, drought and salt induced transcription factors OsDREB1B, OsDREB2A, OsMyb4, OsZFP252 and OsNAC6, ion channel related protein ClC-a, NHX and downstream drought responsive gene DHN1 etc. In addition, the OsMAPK3 transgenic rice was hypersensitive to ABA with lower seeds germination rate. Under normal growth, OsMAPK3 transgenic rice contains higher content of endogenous ABA and increased gene expression of ABA-responsive gene OsRab16A. This result indicated that OsMAPK3 may participate in ABA biosynthesis or signal transduction pathway then alter the sensitivity of transgenic rice to ABA. Taken together, overexpression of OsMAPK3 in rice could increase its drought and salt stress tolerance because of higher antioxidant activity, photosynthesis and water use efficiency, induction of stress-related gene expression and alteration of ABA sensitivy.