應用綠色螢光蛋白報導基因探討蝴蝶蘭癒合組織基因槍轉殖暫時性表達之最佳物理參數

Optimization of Physical Parameters Affecting Transient Green Fluorescent Protein Expression in Phalaenopsis Callus via Particle Bombardment

源自水母（Aequorea victoria）的綠色螢光蛋白（green fluorescent protein; GFP）為廣泛應用的基因標誌，並在許多生物之細胞中皆可直接活體檢視。就具有較長幼年期與生殖週期的蝴蝶蘭而言，GFP報導系統的非破壞性特色，對現今蝴蝶蘭穩定基因轉殖效率普遍低落的改善，應具相當應用價值。本研究之目的在探討與落實此思維，並採用GFP報導蛋白重構蝴蝶蘭暫時性基因轉殖系統。因此，本試驗以GFP基因作為報導基因，用參試金粒子大小（1.0和1.6μm）、槍擊壓力（650、900、1100和1350psi）和槍擊次數（1和3次）等基因槍轉殖之物理參數，並藉由檢視蝴蝶蘭癒合組織之GFP螢光表達，最適化蝴蝶蘭暫時性基因槍轉殖程序。結果顯示，最佳參試之基因槍轉殖條件為1.6μm金粒子、1100psi槍擊壓力、6cm槍擊距離和一次的轉殖槍擊。此外，文中亦探討後續應用GFP作為篩選標誌，以加速蝴蝶蘭穩定基因轉殖效能提升之可行性。 The green fluorescent protein (GFP) from the jellyfish Aequorea victoria has recently emerged as a genetic marker that can be directly visualized in the living cells of many diverse organisms. The nondestructive assay features of the GFP system should be applicable in phalaenopsis transformation system, as phalaenopsis is well known for its long juvenile period and reproductive cycle, and a tendency of low stable transformation efficiency at present. The purpose of this study was to test the idea and reestablish a transient transformation system of particle bombardment for phalaenopsis using GFP reporter. Physical parameters for DNA delivery into phalaenopsis callus were optimized by monitoring transient GFP gene expression. Optimization of the physical factors was carried out under the following conditions: gold microcarrier size (1.0 and 1.6μm), helium pressure (650, 900, 1100, and 1350 psi), number of bombardments (1 and 3 times) per sample. Optimized bombardment conditions for phalaenopsis callus was bombarding once at 1100 psi, 1.6μm gold particle size, and 6 cm target distance. Besides, the availability of GFP as a selection marker for phalaenopsis stable transformation is discussed.