阿拉伯表現細胞質型含銅鋅超氧歧化?之研究

Abstract

超氣歧化?(SOD)是一種含有金屬的酵素，它可以將超氣分子催化成過氣化氫和氣分子。本實驗室之前已成功將水稻細胞質型CuZnSOD轉殖到阿拉伯芥中，並得到數個轉殖系，其中水稻表現量最高的是RCZ5,其total SOD活性為野生型的2~4倍。本論文利用RCZS轉殖株與野生型阿拉伯芥進行巴拉刈及低溫的逆境研究，結果發現RCZS轉殖株對這2種逆境的抗性並沒有提昇。另外，本論文利用農桿菌，以浸泡法將構築之CaMV 35S::AtCSDI（阿拉伯芥細胞質型CuZnSOD cDNA）並含hygromycin為篩選基因之質體轉殖到阿拉伯芥中。經南方氏分析可證實，目前已得到2個Tl轉殖系；經SOD活性測定，T2轉殖株的細胞質型CuZnSOD活性是野生型的1.3至2.5倍，而且植株之簇生葉會從莖頂以外的地方長出、抽苔時的簇生葉片數變異很大、有2~4個初生花芽及果莢變短，種子數減少，畸形種子數增加等的外表型。此結果表示CuZnSOD的增加似乎會影響到植株的正常生長與發育。本論文亦篩選出CuZnSOD反義股(antisense)轉殖株，此序列由CaMV 35S啟動子所啟動。經南方氏分析證實，目前已得到數個含阿拉伯芥CuZnSOD的轉殖系；經SOD活性測定，轉殖株的細胞質型及葉綠體型CuZnSOD活性在T1代中分別為野生型的0.37至0.90倍及0.36至0.73倍，T2植株的細胞質型及葉綠體型CuZnSOD活性似乎會回復到野生型的0.53至1.52倍及0.70至1.79倍。4個品系的轉殖株外表型在T2代第1到6片葉的葉片形狀不規則，葉月上有一些淺色斑點，且生長速度明顯減緩；進入生殖階段後，轉殖株之老化速度較快；此結果表示阿拉伯芥之CuZnSOD活性下降可能會影響植株的生長。

Superoxide dismutase (SOD, EC1.15.1.1) plays an important role in the cellular defense of active oxygen species. To study the resistance of transgenic Arabidopsis with rice cytosolic CuZnSOD overexpression to oxidative stresses, SOD overexpressor and wild-type plants were treated with paraquat, chilling and freezing temperature. The damage caused by these stresses were observed and there were no significant differences between wild-type and transgenic plants. The cDNA coding for Arabidopsis cytosolic CuZnSOD was fused to CaMV 35S promoter and mobilized into Agrobacterium C58 strain. Arabidopsis was transformed by dipping method, and after hygromycin selection, several putative transgenic lines were analyzed for SOD activity by native PAGE. The cytosolic CuZnSOD activities of T2 plants were 1.3 to 2.5 fold higher than the wild-type plants. After Southern analysis, several transgenic plants were demonstrated. The transgenic plants showed abnormal phenotypes, rosette leaf buds formed from several sites of the plants, obvious variance in the rosette leaf numbers when bolting, 2~4 primary inflorescence buds appeared at a time and shorter siliques with some aborted seeds. The seeds from transformed T0 plants were selected for CuZnSOD antisense plants by kanamycin. After Southern analysis, several antisense transgenic plants were demonstrated. The chloroplastic and cytosolic SOD activities of Ti transgenic plants were 27% to 64% and 10% to 63% less than that of wild-type plants. However, decreased activities of both CuZnSOD forms will recover in the T2 antisense plants. The transgenic plants showed the abnormal leaf shapes and colors in the first 6 rosette leaves, decreased growth rate and the aging sever. Accordingly, change in the cytosolic CuZnSOD activitiesin the plants may affect the growth and development of Arabidopsis.