- 學位論文
不同培養條件對痲瘋樹懸浮細胞生成脂肪酸及蛋白質的影響
Effect of the different culture conditions on formation of fatty acids and proteins in Jatropha curcas suspension cells
摘要
綠色能源是全球趨勢，不僅可以解決能源短缺的問題，還可以克服溫室氣體大量增加的問題，痲瘋樹 ( Jtropha curcas L.)為大戟科植物，種子有很高的含油量約40%，但其具有毒性不可食用故適合做為生質柴油。本實驗目的是利用無菌實生苗誘導癒傷組織並建立植株再生系統，以量化繁殖痲瘋樹種苗與利用懸浮細胞進行脂肪酸及蛋白質的分析，結果顯示在MS培養基 (Murashige and Skoog, 1962)添加1.5 mg/L BA和0.5 mg/L IBA多芽誘導比例與芽體個數最高；另一方面將下胚軸誘導的癒傷組織放入液態培養基分別以光照和黑暗進行懸浮培養，顯示懸浮培養細胞在黑暗中生長速度較光照快，但光照處理的細胞含脂質小胞器顆粒數較高，因植物會將光合作用產物轉換成脂質的形式儲存；萃取脂肪酸經由薄層色層分析，發現懸浮培養細胞會因型態差異不同而累積不同的脂肪酸與其衍生物。懸浮培養細胞以細胞活性、脂肪酸種類特性選擇體外培養條件，為明白不同條件脂質的代謝機制以比較蛋白質體學來分析蛋白質的變化，差異表現的蛋白質透過液相質譜儀分析資料庫比對後，在黑暗處理中比對到Peroxidase 68 OS、Hypothetical protein CHLREDRAFT_150509、glutathione S-transferase omega、translationally controlled tumor protein、annexin-like protein、Glyceraldehyde-3-phosphate dehydrogenase、major allergen Pru ar，其中與植物致病相關的蛋白major allergen Pru ar在光照及黑暗皆有發現，且黑暗處理表現較多。本研究對於植物脂質生合成有很大的幫助，未來希望懸浮培養細胞能以生物反應器的模式培養並可用來生產生質柴油，可以降低生質柴油在生產過程中的成本，提高生質柴油的特性，使消費者更能接受，也可以增進對高等植物脂肪酸生合成的了解。
並列摘要
A gradual substitution of fossil energy to renewable energy is a global trend, not only it resolves and increases the critical energy shortage, but also crisis of greenhouse effect. Jatropha curcas is an inedible crop, the seeds containing about 40% lipids can be used as biodiesel. The aim of the current research project was to apply a nonconventional bioprocess for in vitro biomass and oil production of Jatropha cells. The seeds of Jatropha curcas were used for aseptic seeding. The methods of callus induction, suspension cell culture and embryogenesis of Jatropha were established. The best inducing rate of buds was achieved by adding 1.5 mg/L BA and 0.5mg/L IBA in MS. The suspension cells were cultured in light and dark conditions. Once friable callus was obtained, and cell suspension cultures were established. The cell viability, fatty acid content, and characteristics were used to select the most promising cell line according to its fatty acid profile and ability to grow and develop under in vitro conditions. Nile red staining showed that suspension cells in light accumulate more lipid than in dark. To characterize the metabolic mechanism of lipid accumulation in Jatropha suspension culture cells, comparative proteomic technique was employed to profile protein changes during different growth condition. The fatty acids were extracted and run through thin layer chromatography (TLC), and the results showed that suspension cells in light and dark accumulated different fatty acids and their derivatives. Differences growth conditions of Jatropha were related to their fatty acid profiles, oil content. We used comparative proteomics to understand the metabolic mechanism of lipid. The protein were identified by LC/MS. Peroxidase 68 OS、Hypothetical protein CHLREDRAFT_150509、glutathione S-transferase omega、translationally controlled tumor protein、annexin-like protein、Glyceraldehyde-3-phosphate dehydrogenase、major allergen Pru ar were found in dark. Major allergen Pru ar was found more in dark than light, and it was associated with plant pathogenic. It is helpful in plant lipid synthesis and may make the Jatropha suspension cultured cells can be as bioreactor to produce biodiesel. It can reduce cost in the production process and improve the properties of biodiesel. Also improving the understanding of fatty acids biosynthesis in higher plants.