臺灣博碩士論文加值系統

台灣原生的藻種Chlorella sp. DT具有形成蝦紅素的能力。為了進一步探討Chlorella sp. DT最大蝦紅素產量，本研究利用實驗設計法篩選出有助增加藻體中蝦紅素含量的培養因子：硝酸鈉、碳酸鈉及氯化鈣，更進一步以反應曲面法作最適化生產蝦紅素的培養基探討，結果顯示添加2779.08 mg L-1的硝酸鈉、273.97 mg L-1的碳酸鈉以及75.34 mg L-1的氯化鈣於BG-11培養基，經過10天培養與誘導後蝦紅素產量為0.749 mg L-1。並由因子篩選探討的實驗結果，設計兩階段培養Chlorella sp. DT生產蝦紅素的培養基條件，以適合藻體生長的PBD-2培養基累積藻體量，再以Pred-medium培養基進行誘導蝦紅素生成，經過12天的培養與誘導後蝦紅素產量達0.805 mg L-1，實驗結果成功地以一階段及兩階段的培養方式探討Chlorella sp. DT最大的蝦紅素產量。並且為了提升蝦紅素產量，本研究也選殖了Chlorella sp. DT生成蝦紅素的關鍵蛋白質β-carotene ketolase，以農桿菌介導轉殖法將β-carotene ketolase表達至不具蝦紅素形成能力的Nicotiana tabacum及N. benthamiana中，大量表現β-carotene ketolase催化β類胡蘿蔔素代謝形成蝦紅素，使N. tabacum的T0轉殖株葉片組織生成604.6 μg g-1 DW的蝦紅素含量，N. benthamiana的T0轉殖株葉片組織生成385.8 μg g-1 DW的蝦紅素含量，成功以Chlorella sp. DT的β-carotene ketolase基因建立轉基因作物生產蝦紅素的平台。

The Chlorella sp. DT, an endemic species in Taiwan, was found with astaxanthin producing ability. To maximize productivity of astaxanthin in Chlorella sp. DT, NaNO3, Na2CO3 and CaCl2 were selected as inducers by factors screening. Further, response surface methodology was used to optimize the cultural condition for astaxanthin production. The results showed astaxanthin productivity was 0.749 mg L-1 after 10 days of cultivation and induction by used the optimized the cultural condition, BG11 medium with NaNO3 2779.08 mg L-1, Na2CO3 273.97 mg L-1 and CaCl2 75.34 mg L-1. Furthermore, based on factors screening, the culture conditions for two-stage to produce astaxanthin by Chlorella sp. DT were developed. The PBD-2 medium was used to accumulate biomass in green-stage, and the Pred-medium was used for astaxanthin production in induce-stage. Astaxanthin productivity is 0.805 mg L-1 after 12 days of cultivation and induction in two-stage strategy. We successfully maximize the astaxanthin production in Chlorella sp. DT by one and two-stage of cultivation strategies. To increase the production of astaxanthin, we cloned the Chlorella sp. DT β-carotene ketolase (BKT), the key enzyme of astaxanthin synthesis, and overexpressed the BKT gene in Nicotiana tabacum and N. benthamiana by using agrobacterium-mediated transformation. The transgenic N. tabacum and N. benthamiana plants can produce 604.6 and 385.8 μg astaxanthin per g (dry weightof leaves), respectively. This study successfully used β-carotene ketolase gene form Chlorella sp. DT to build a transgenic plant system for astaxanthin production.

摘要................................................................................................................................. i
Abstract .......................................................................................................................... ii
表目錄........................................................................................................................... vi
圖目錄........................................................................................................................ viii
1. 緒論........................................................................................................................... 1
2. 前言........................................................................................................................... 2
2.1 蝦紅素.............................................................................................................. 2
2.2 藻類.................................................................................................................. 4
2.3 藻類生產蝦紅素.............................................................................................. 5
2.4 高等植物生產蝦紅素...................................................................................... 6
2.5 小球藻Chlorella sp. DT ................................................................................. 8
3. 材料方法................................................................................................................. 10
3.1. 實驗材料...................................................................................................... 10
3.1.1. 藻種................................................................................................... 10
3.1.2. 菌種................................................................................................... 10
3.1.3. 質體................................................................................................... 11
3.1.4. 植物................................................................................................... 12
3.1.5. 抗生素............................................................................................... 12
3.1.6. 引子................................................................................................... 12
3.1.7. 酵素................................................................................................... 13
3.1.8. 藥品................................................................................................... 13
3.1.9. 液相層析管柱................................................................................... 14
3.1.10. 有機溶劑......................................................................................... 15
3.1.11. 實驗套組/試劑 ................................................................................ 15
3.1.12. 其他................................................................................................. 15
3.2. 實驗方法...................................................................................................... 15
3.2.1. Chlorella sp. DT 培養 ....................................................................... 15
3.2.2. Chlorella sp. DT 之色素萃取 ........................................................ 16
3.2.3. 高效液相層析儀分析色素萃取物................................................... 17
3.2.4. 葉綠素萃取....................................................................................... 17
3.2.5. 培養基營養因子分析....................................................................... 18
3.2.6. 兩階段培養Chlorella sp. DT 生產蝦紅素條件探討 ..................... 21
3.2.7. Chlorella sp. DT RNA 萃取 ........................................................... 21
3.2.8. cDNA 製備 ..................................................................................... 22
3.2.9. 引子設計......................................................................................... 23
3.2.10. 目標基因增幅與序列分析........................................................... 23
3.2.11. 建立大腸桿菌表現系統 ............................................................... 26
3.2.12. 大腸桿菌表現β-carotene ketolase 與粗萃蛋白質 ..................... 28
3.2.13. SDS-PAGE 凝膠電泳分析 ........................................................... 28
3.2.14. 西方墨點法分析........................................................................... 29
3.2.15. 植物表現系統載體構築............................................................... 30
3.2.16. 農桿菌勝任細胞製備與轉型....................................................... 31
3.2.17. 農桿菌介導轉殖菸草................................................................... 32
3.2.18. 轉殖植株分析............................................................................... 33
4. 結果與討論............................................................................................................. 39
4.1. 反應曲面法最適化Chlorella sp. DT 生產蝦紅素培養基 ........................ 39
4.1.1. 培養基影響因子分析....................................................................... 40
4.1.2. 反應曲面法....................................................................................... 42
4.2. 兩階段培養Chlorella sp. DT 生產蝦紅素產量條件 ................................ 47
4.2.1. 最適Chlorella sp. DT 藻體生長培養基組成 ................................. 48
4.2.2. 最適Chlorella sp. DT 藻體中蝦紅素累積培養基組成 ................. 48
4.3. Chlorella sp. DT 的β-carotene ketolase 基因全長選殖與分析 ................. 50
4.4. 建立大腸桿菌表現重組β-carotene ketolase 系統 .................................. 51
4.4.1. 構築β-carotene ketolase 的大腸桿菌表現載體 ............................. 51
4.4.2. β-carotene ketolase 表現系統建立 .................................................. 52
4.5. 菸草表現重組β-carotene ketolase ............................................................. 53
4.5.1. 表現載體構築................................................................................... 53
4.5.2. 農桿菌介導轉殖菸草....................................................................... 54
4.5.3. 以聚合酶鏈鎖反應篩選菸草轉殖株............................................... 55
4.5.4. 瓶苗時期菸草轉植株外顯型態觀察............................................... 55
4.5.6. 經載體 pBINPLUS-RbcsBKT 轉殖植株南方墨點法分析 .............. 56
4.5.7. 菸草轉植株蝦紅素含量................................................................... 57
4.5.8. 菸草轉植株健化後外顯型態觀察與色素分析............................... 59
5. 結論與展望........................................................................................................... 125
附錄............................................................................................................................ 128
參考文獻.................................................................................................................... 133

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