- 學位論文
蔗糖濃度與限制氣體交換對花青素生合成和關鍵酵素基因表現之影響–以葡萄懸浮細胞培養生成花青素為例
The effects of sucrose concentration and gas exchange restriction on anthocyanin biosynthesis and key enzyme gene expression in Vitis vinifera cell suspension cultures
摘要
本論文探討葡萄細胞(Vitis vinifera L. Gamay Freaux)於不同蔗糖含量下及不同的植物生長激素下，其對花青素的生合成機制。另外，又以限制氣體交換的方式在不同的蔗糖含量下，探討花青素生合成機制中關鍵酵素基因表現。 本試驗以葡萄癒傷組織作為試驗材料，進行細胞懸浮培養並將培養基隔絕氣體交換二個星期之後再將其恢復；觀察花青素生合成量和花青素生合成關鍵酵素基因表現。另外，也以即時定量聚合酶鏈鎖反應(Real-time Polymerase Chain Reaction)偵測花青素生合成機制中關鍵酵素基因CHS (chalcone synthase)、UFGT(UDP-glucose Flavonoid 3-O-Glucosyl Transferase)…等的基因表現量。 在測定花青素含量結果顯示，培養基中蔗糖含量會影響花青素生合成量；即蔗糖含量愈高則花青素生合成量愈多。其中在產量與細胞生長之間也存在消長(trade-off)的關係，但此關係也會受到培養基環境所影響。限制氣體交換會明顯地抑制花青素生合成量；同時也抑制葡萄細胞生長。在花青素生合成機制中關鍵酵素基因表現方面，與表面白色葡萄細胞比較後得知表面紅色葡萄細胞的CHS2 基因表現上升14.76 倍。 將表面紅色葡萄細胞(FC-01)解除限制氣體交換後，經過三星期培養；其花青素產量在第二星期之後產量達到最高，再以即時定量聚合酶鏈鎖反應(Real-time Polymerase Chain Reaction)偵測花青素生合成基因表現得知，CHS 基因表現量隨培養時間增長而增加；另外，CHi(chalcone isomerase)、F3’H(flavanone 3-hydroxylase)、Dfr(dihydroflavonol 4-reductase)等酵素基因的表現量則隨培養時間增長而降低；而UFGT 則是在培養第二個星期之後，其基因的表現量突然增加；而到了第三個星期則反而降低，由此可知，UFGT 基因的表現量在花青素生合成上扮演著關鍵性的角色。 由以上結果可推論，限制氣體交換會造成CHS 酵素基因活性降低使得花青素初級前趨物無法順利合成而造成UFGT 酵素基因活性必須相對地提高，以形成穩定的花青素。由解除限制氣體交換花青素產量恢復的實驗中，可推論UFGT 在花青素生合成提高時會影響其他基因表現，提高花青素產量。
並列摘要
The aims of this thesis are to investigate the mechanism of anthocyanin biosynthesis under different sucrose level, plant growth regulators or gas restricted from which the expression of gene in anthocyanin biosynthesis were detected. Plant cell suspension was cultured in Gamborg B5 medium. The result showed that, high levels of sucrose enhance the production of anthocyanin. There is a trade-off between growth rate and production of anthocyanin, and it actually influences by cell culture condition. Meanwhile, gas restriction obviously suppresses anthocyanin production and growth simultaneously. On the gene expression of key enzyme in anthocyanin biosynthesis, the comparison bettwen the white surface grape cell, and the red surface grape cell showed that the gene expression of CHS2 increased up to 14.76 fold. Meanwhile, under gas restriction the expression of UFGT in red grape cell increased up to 11.11 fold. Consequently, gas restriction would cause CHS down regulated, implying the activity of UFGT increased to keep up the anthocyanin production. After stop the gas restriction, the production of anthocyanin reached the maximum level at the second week of incubation. On anthocyanin biosynthesis gene expression, the flods of the expression of CHS was proportional to the incubation time. On the other hand, the flods of the expression of Chi (chalcon isomerase)、F3’H ( flavanone 3-hydroxylase)、Dfr (dihydroflavonol 4-reductase) was proportionate inversely. In the second week of incubation, the expression of UFGT suddenly increased, but declined in the third week. The amount of anthocyanin production was parallel with the expression of UFGT. It might conclude that the gene expression of UFGT is critical for anthocyanin biosynthesis.