- 期刊
新型綠螢光基因AcGFP及ZsGFP在水稻之表現、評估與應用
Expression, Evaluation and Application of Novel Green Fluorescent Protein Genes, ＂AcGFP＂ and ＂ZsGFP＂, in Rice
摘要
高效率、無害的基因表現篩選系統為所有作物基因轉殖必須克服的問題，爰此本研究建構新型＂AcGFP＂及＂ZsGFP＂報導基因表現系統， 並於水稻進行基因功能評估。首先完成pCAMBIA35SAcGFP及pCAMBIA35SZsGFP表現載體之構築，利用農桿菌媒介轉形法，將螢光基因導入水稻基因組。結果發現，兩轉殖系經感染癒傷組織，培養14 d後，即可觀察到綠色螢光。而T1種子於無菌播種14 d後，＂35S::AcGFP＂轉殖系可在幼苗的胚盤及根部等組織觀察到綠色螢光；＂35S::ZsGFP＂轉殖系也可在幼苗的胚盤及胚乳表面糊粉層等組織觀察到綠色螢光。然而，兩轉殖系幼苗的芽鞘及幼葉可能因葉綠素的干擾，無法觀察到綠色螢光。此外，＂35S::AcGFP＂轉殖系各組織的螢光表現皆高於＂35S::ZsGFP＂轉殖系，可能肇因於＂35S::ZsGFP＂系統的表現效率低所致。以＂AcGFP＂分析玉米＂Ubi-1＂及水稻＂GluB5＂與＂GluC＂啟動子活性，結果發現＂Ubi-1::AcGFP＂轉殖系在感染癒傷組織及發芽芽體，均可觀察到螢光表現；而＂GluB5::AcGFP＂及＂GluC::AcGFP＂轉殖系亦可在感染癒傷組織及發芽芽體觀察到螢光。這些結果顯示，玉米＂Ubi-1＂、水稻＂GluB5＂與＂GluC啟動子皆可在水稻細胞中驅動＂AcGFP＂表現，生產具有功能的螢光蛋白質。本研究所建構的這些綠螢光基因表現載體系統，應可作為植物基因轉殖的早期篩選標誌，應用於協助較具挑戰性之植物轉殖平台的建立，亦可作為其它重要基因與啟動子的功能分析。
並列摘要
The aim of this study is to construct novel expression system driving reporter genes, ＂AcGFP＂ and ＂ZsGFP＂, for functional evaluation of rice genes. Two constructs, pCAMBIA35S＂AcGFP＂ and pCAMBIA35S＂ZsGFP＂, were completed and then introduced into rice genomes by ＂Agrobacterium＂-mediated transformation. The results displayed that the transformed calli of two transgenic lines present green fluorescence after culture for 14 days. The radicles and roots of ＂35S::ZsGFP＂ lines presented green fluorescence in the seed lings at 14 days after germination. At the same time, the radicles and endosperm aleurone layer of ＂35S::ZsGFP＂ lines also displayed green fluorescence. However, the coleoptiles and spires of two transgenic lines did not be observed green fluorescence since the interference of chlorophyll fluorescence. The ＂35S::ZsGFP＂ lines showed stronger fluorescence than those of ＂35S::ZsGFP＂ since the poor expression efficiency of ＂35S::ZsGFP＂ system. Finally, ＂AcGFP＂ was applied to analyze the activities of maize ＂Ubi-1＂, rice ＂GluB5＂ and ＂GluC＂ promoters. The results displayed that transformed calli and shoots of ＂Ubi-1::AcGFP＂ lines could express green fluorescence. The transformed calli and shoots of ＂GluB5::AcGFP＂ and ＂GluC::AcGFP＂ lines also expressed green fluorescence. These results suggest that maize ＂Ubi-1＂, rice ＂GluB5＂ and ＂GluC＂ promoters could drive ＂AcGFP＂ and produce functional fluorescent proteins. Therefore, the expression systems established in this study can provide as selectable markers in the initial stages of plant transformation and are applicable in the development of transgenic platforms and functional analysis of plant genes.