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標題: 利用小孢子再生之轉殖水稻探討外來基因遺傳之表現及穩定性
Exploring gene expression and stability of foreign gene by microspore embryogenesis in the transgenic rice
作者: 黃詩鈺
Huang, Shih-Yu
關鍵字: haploid
出版社: 生命科學系所
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摘要: 基因的穩定度對性狀的遺傳是相當重要的。本論文利用含有兩個 玉米光合作用基因(phosphoenolpyruvate carboxylase, PEPC, pyruvate,Pi dikinase, PPDK)之轉殖水稻(CK 轉殖水稻)為材料,探討這二個外來基因在小孢子再生雙倍體(dihaploid)之穩定度與表達強度。從小孢子再生的雙倍體選出32 個品系, 針對這兩個轉基因(transgenes) 進行分子、生化、生理分析。西方墨點法顯示,與未轉殖株比較所有的再生雙倍體葉片都有較高的PPDK 蛋白累積量。PCR 也都可以偵測到玉米PPDK 基因的存在。可是只有1/3 的再生雙倍體葉片有PEPC 蛋白的累積,而這些品系也都可以偵測到玉米PEPC 基因的存在。值得注意的是,這些品系的PEPC 酵素活性比CK 轉殖水稻高出30-90%。然而,有三個品系含有玉米PEPC 基因,但 並無PEPC 蛋白的累積與酵素活性。生理上,有一些再生雙倍體品系 的穗重、穀粒重與總乾重值比CK 轉殖水稻有明顯的增加。這與原來 的預測,小孢子再生過程中,經由單倍體自行倍加所產生的同型接合 子(homozygous)後代,會產生大量的變異是相符的。從這兩個玉米 轉基因再生雙倍體的表現來看,基因的穩定度與表達量有很大的變 異,基因會有遺失或缺失的可能。
The stability of genes is very important for genetic traits. In this study,a CK transgenic rice which overexpresses two maize C4 photosynthesis genes encoding phosphoenolpyruvate carboxylase(PEPC) and pyruvate Pi dikinase(PPDK) was used to test the stability and expression level of these two transgenes in dihaploid line derived from microspore embryogenesis. A tatal of thiry-two dihaploid lines were obtained for molecular, biochemical and physiological analysis related to the two genes. Western immuno-blot analysis showed that all lines have high levels of PPDK protein, consistent with the detection of the maize gene in the genome by PCR. However, only eleven lines exhibited high levels of PEPC protein, and the maize gene was also detected in these lines by PCR. Notably, the activities of PEPC in these lines were much higher (30-90%) than that of the parental CK transgenic rice. Interestingly, three dihaploid lines retainrd the maize PEPC gene in their genomes, but did not express PEPC protein and enzyme activity. Physiologically, some dihaploid lines had superior agronomic traits than the parental CK transgenic rice, including panicle weight, tiller number and total biomass while other lines had no significant changes. Taken together, these results III are consistent with the theory that genetic rearrangement will occur during embryogenesis and/or chromosome doubling and generate large variation in phenotype in dihaploid lines derived from microspores.
其他識別: U0005-0102200816434500
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