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  • 學位論文

潮酶素誘導水稻細胞死亡機制之研究

Studies on the mechanism of cell death caused by hygromycin B in Oryza sativa L.

指導教授 : 洪傳揚

摘要


潮霉素 (Hygromycin B, Hyg) 是水稻轉殖最常用的篩選用抗生素,當轉殖水稻攜帶有 hygromycin phosphotransferase (hpt) 基因即可將 Hyg 磷酸化,避免 Hyg 與核醣體 RNA 結合造成蛋白質不正常轉譯,反之,非轉殖水稻則會受到 Hyg 毒害而死亡,然而 Hyg 導致植物死亡的機制目前仍然未知。為了瞭解 Hyg 造成水稻死亡的機制,本試驗以不同濃度 Hyg處理水稻幼苗,並從生理、生化及分子層次分析水稻的反應。結果顯示水稻經過 Hyg 處理之後,地上部及根部生長皆嚴重受抑制, Evans blue染色顯示 Hyg 會造成植物死亡,同時 malondialdehyde (MDA) 及被羰基化蛋白質含量 (carbonylation) 提高,活性氧族 (reactive oxygen species, ROS) 包括超氧自由基 (O2.-) 、過氧化氫 (H2O2) 及氫氧自由基 (OH.) 在地上部及根部中也隨 Hyg 濃度增加而提高,進一步分析發現 H2O2 主要累積粒線體。以 ascorbate 及 imidazole 處理則可降低 Hyg 造成的傷害,這些結果皆證實 Hyg 會造成水稻產生氧化逆境,進而導致死亡。進一步測定水稻中抗氧化酵素之酵素活性,結果顯示 CuZnSOD 及 MnSOD 酵素活性均受明顯增加,ascorbate peroxide及 glutathione reductase (GR) 活性也分別受到 Hyg 誘導 3 倍和 2.6 倍。基因表現方面,位在葉綠體的 GR3 在 Hyg 處理後6-12 小時間表現量有明顯誘導,而粒線體兩種呼吸作用路徑中關鍵酵素 alternative oxidase I (AOX I) 及 cytochrome c oxidase II (COX II) 在處理 72 小時後蛋白質表現皆增加,基因表現分析結果顯示AOX I 的三種同功酵素中 AOX I a 基因表現量受 Hyg 高度誘導,而 COX II 之基因表現量則無顯著差異。外加 H2O2 及 Menadione 分析水稻基因表現量,結果顯示僅有 H2O2 會誘導 GR3 及 AOXIa 基因表現量,證明Hyg 誘導 GR3及 AOX1a 是因為H2O2累積所導致。綜合以上結果顯示 Hyg 會在葉綠體及粒線體誘導大量 ROS 造成氧化逆境,進而破壞呼吸作用及光合作用,最後導致水稻死亡。

並列摘要


Hygromycin B (Hyg) is the antibiotics most frequenly used in rice transformation. Rice calli is sensitive to Hyg. Transgenic rice carrying hygromycin phosphotransferase (hpt) can phosphorylate Hyg to avoid protein mistranslation, subsequently possesses rice resistant to Hyg. However, mechanism of rice cells sensitive to Hyg remains unclear. To figure out the mechanism, rice seedlings were treated with different concentrations of Hyg and their effects on physiologycal, biochemical and molecular changes were examined. The toxic effect caused by Hyg was observed including the reduction of plant height and root length, protein content, and the increase of malondialdehyde (MDA), carbonylated proteins, as well as Evans blue uptake. Meanwhile, levels of hydrogen peroxide (H2O2), superoxide radical (O2.-), and hydroxyl radical (OH.) were also significantly enhanced by Hyg in shoots. Observation of the localization of H2O2 indicated that H2O2 was mainly accumulated in mitochondria and chloroplast. Conversely, pretreatment of rice with ascorbate or imidazole can attenuate the toxicity, further confirmed that toxic effects caused by Hyg were mediated by oxidative stress. In addition, analysis of the antioxidant enzyme activities showed that CuZnSOD and MnSOD were highly induced. The activities of APX and GR were also induced by 3 folds and 2.6 folds, respectively. Gene expression analysis revealed that the chloroplast-localized GR3 was notably increased after 6-12 hours of Hyg treatment. Studies on the enzymes crucial in the mitochondrial respiration pathway, i.e. alternative oxidase I (AOXI) and cytochrome c oxidase II (COX II), shown that both proteins were significantly induced by Hyg. Reverse-transcription PCR analysis indicated that AOXIa was highly induced by Hyg, but no significant difference was observed in COXII. Futhermore, exogenous application of H2O2 and superoxide generator (menadione) showed that the expression of GR3 and AOXIa were induced by H2O2, instead of superoxide. All the results indicated that Hyg-induced oxidative stress is due to the accumulation of ROS in chloroplast and mitochondria, thus resulted in cell death.

參考文獻


戶刈義次 (1963) 作物學試驗法. 東京農業技術學會印行 p159-176
Agrawal, G. K., Jwa, N. S., Iwahashi, H., and Rakwal, R. (2003). Importance of ascorbate peroxidases OsAPX1 and OsAPX2 in the rice pathogen response pathways and growth and reproduction revealed by their transcriptional profiling. Gene 322, 93-103.
Apel, K., and Hirt, H. (2004). Reactive oxygen species: metabolism, oxidative stress, and signal transduction. Annu Rev Plant Biol 55, 373-99.
Asada, K. (1992). Ascorbate peroxide-a hydrogen peroxide-scavenging enzyme in plant. Physiol Plant 85, 235-241.
Asada, K. (2006). Production and scavenging of reactive oxygen species in chloroplasts and their functions. Plant Physiol 141, 391-6.

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