香蕉果實後熟相關基因MhDnaJ之啟動子活性與基因功能分析

Abstract

香蕉 (Musa spp.) 為大型單子葉草本植物，是臺灣重要的經濟作物。本研究針對香蕉果實後熟相關MhDnaJ基因，進行過量表達 (overexpression)、基因默化 (RNA interference, RNAi)、蛋白質定位 (protein localization) 及啟動子活性分析 (promoter activity analysis)，以瞭解香蕉果實後熟相關蛋白質MhDnaJ之功能及其啟動子之表現特性。蛋白質定位質體以2 X CaMV 35S啟動子驅動MhDnaJ融合綠色螢光蛋白 (green fluorescent protein, GFP) ，分別以阿拉伯芥原生質體轉殖與基因槍法轉殖至洋蔥表皮細胞，進行暫時性表現分析，以共軛焦顯微鏡觀察，MhDnaJ::GFP融合蛋白於細胞核內表達。MhDnaJ啟動子引導報導基因GUS之菸草轉殖植株，經南方氏雜交分析確認均為轉殖株，於T1轉殖株播種後5-30天啟動子皆無表現，進一步分析菸草R1轉殖株花序與果實不同生長發育階段之啟動子活性，顯示MhDnaJ啟動子於柱頭發育早期 (stages 2-4) 與後期 (stage 8)、雄蕊發育後期 (stages 8-9) 與果實發育早期 (stages 10-11) 表現，顯示啟動子活性於花器表現。不同非生物性逆境與生長調節劑誘導處理，皆無法誘導啟動子表現。

Banana (Musa spp.), the largest herbaceous monocot flowering plant, is an important crop in Taiwan. This research aims to understand gene function of banana ripening-associated gene MhDnaJ by employing gene overexpression studies and gene silencing through RNA interference strategy. Furthermore, the localization of MhDnaJ protein and promoter activity were studied. MhDnaJ cDNA was fused with green fluorescence protein gene driven by 2 X CaMV 35S promoter, transformed to Arabidopsis protoplasts and onion epidermal cells, then analyzed by confocal fluorescence microscopy for protein localization. The MhDnaJ protein was found to be localized in the nucleus as evidenced by green fluorescence. Twelve putative transgenic tobacco plants containing promoter construct were confirmed by Southern blot analysis. No expression of MhDnaJ promoter was detected in 5 to 30 day-old plants, but was found to be expressed in flower (pistil: stages 2-4, 8; stamen: stages 8-9) and fruit (stages 10-11) as detected by GUS histochemical staining. None of the abiotic stresses or any plant growth regulators could induce MhDnaJ promoter activity.