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Please use this identifier to cite or link to this item: http://ntour.ntou.edu.tw:8080/ir/handle/987654321/48793

Title: 從三角褐指藻在缺乏磷酸鹽逆境反應的研究到高效率矽藻表現系統的開發
From the Study on Phosphate Starvation Responses to the Development of a High Efficient Expression System in the Marine Diatom Phaeodactylum tricornutum
Authors: Lin, Hung-Yun
林宏運
Contributors: 國立臺灣海洋大學:生命科學暨生物科技學系
Keywords: 矽藻;鹼性磷解酶;缺磷誘導;啟動子;三角褐指藻;矽藻表現系統
diatom;alkaline phosphatase;phosphorus starvation;promoter;Phaeodactylum tricornutum;diatom expression system
Date: 2017
Issue Date: 2018-08-22T06:31:22Z
Abstract: 矽藻不但是海洋生態系中主要的初級生產者,在全球的碳循環中也扮演著相當重要的角色。再者,矽藻具有獨特的矽質外殼並且能夠製造天然色素與不飽和脂肪酸等商業性產物的能力,使得矽藻無論在海洋環境科學研究抑或是生技產業應用方面都相當地受到關注。 矽藻的生長除了需要光照外,還需要吸收營養鹽。其中,磷酸鹽為影響矽藻生長的重要限制因子之一。在缺乏磷酸鹽的條件下,鹼性磷解酶 (alkaline phophatase, APase) 會被三角褐指藻釋放到培養基中並且可以經由活性染色的方式進行快速偵測。藉由質譜分析與資料庫搜尋的方式鑑定出三角褐指藻的鹼性磷解酶 (PtAPase)。即時定量 PCR 的結果顯示 PtAPase 的 mRNA 表現量會隨細胞生長狀態及培養基內磷酸鹽的濃度的不同而出現明顯變化。 經由這項發現,我們進一步鑑定利用三角褐指藻鹼性磷解酶 (PtAPase) 的功能性啟動子來發展新穎的誘導型載體。pPhAP1-EGFP 基因載體包含 PtAPase 的預測啟動子區域 (pPhAP1) 並在下游連結增強型綠色螢光蛋白 (EGFP)。帶有 pPhAP1-EGFP 載體的轉殖矽藻株在高磷酸鹽的環境下不會表現重組蛋白並展現出與野生型矽藻相似的生長曲線。當環境磷酸鹽濃度降低至3.6 μM或更低時,EGFP螢光會受到誘導而表現。這個表現模式與內源性鹼性磷酸酶活性一致,顯示 pPhAP1 的缺磷誘導表現並未受到影響。關於表現載體 EGFP 生產總量,pPhAP1-EGFP轉殖藻株的產量比其他矽藻載體還要高出約 9.3-10.5 倍。這些結果表示 pPhAP1 為優秀的矽藻表現載體,其具有不影響藻類生長、易於調節基因誘導表現與高產量的理想特徵。 綜觀目前的矽藻基因轉殖技術,仍無法克服各轉殖藻株間表現量差異過大的問題。因此,如何快速地篩選出高表現量的轉殖藻株是矽藻重組蛋白表現系統是當今需要克服的主要目標。在此,我們提出一個在轉殖載體額外添加校正型基因卡匣的新設計。良好的校正型卡匣需要由一個誘導型啟動子驅動一個容易被偵測的報導基因,例如 pPhAP1-EGFP 基因卡匣。pPhU6-shSDS1-pPhAP1-EGFP 基因載體除了 pPhAP1-EGFP 之外,還帶有由三角褐指藻 U6 啟動子 (pPhU6) 連結下游抗亞精胺合成酶 shRNA (shSDS1) 的基因卡匣。帶有上述載體的基因轉殖矽藻在一般狀態下亞精胺合成酶 (SDS1) 的抑制程度與在缺磷狀態下的綠色螢光強度呈現正比的關係。未來將會持續評估校正型卡匣發展為啟動子分析工具的可能性。 本論文對於三角褐指藻鹼性磷解酶 (PtAPase) 的研究具有多面向的應用性。監測 PtAPase mRNA的表現量可能有助於監測自然環境和人造生物反應器中矽藻的生理狀況。PtAPase 啟動子 (pPhAP1) 可以利用在重組蛋白的表現平臺。
Based on the ecological status and multiple applications of marine diatoms, it becomes an important research project. Phosphate is one of the key growth-limiting factors of diatoms. Under phosphorus deficient conditions, Phaeodactylum tricornutum releases an alkaline phosphatase (PtAPase) to the medium which is detectable by activity staining. Quantitative RT-PCR results indicated that the induction of APase mRNA transcription is very sensitive to phosphorus availability and population growth.   We constructed a novel transgenic vector (pPhAP1-EGFP) containing the putative promoter region of PtAPase (pPhAP1) with EGFP (enhanced green fluorescent protein) open reading frame attached to downstream side. pPhAP1-EGFP transgenic diatom clones did not express EGFP fluorescence at high phosphate concentrations, but only induced and highly expressed when the ambient phosphate concentration was decreased to 3.6 μM or lower. The expression pattern of endogenous PtAPase and exogenic EGFP in pPhAP1-EGFP transgenic diatom clones were matched, in both the transcriptional and translational levels which indicating that the pPhAP1 has the necessary cis-acting element for phosphate starvation response. In terms of the quantity of EGFP generated by expression vectors, the yield of pPhAP1-EGFP was 9.3 to 10.5 times greater than those of other diatom vectors.   Due to the great amount of individual differences in expression level of the transgene, a rapid screening of clones with high expression transgenes is the main aim in diatom expression system. Here, we propose a novel design of two-cistron system (TCS) containing pPhAP-EGFP cistron. The novel vector, in addition to pPhAP1-EGFP, is also provided with a gene cassette linked by the downstream anti-spermidine synthase shRNA (shSDS1) from the U6 promoter (pPhU6). The inhibition level of spermidine synthase (SDS1) in the general state was positively correlated with the green fluorescence intensity in the phosphorus free condition in the pPhU6-shSDS1-pPhAP1-EGFP transgenic clones. In the future, assessment of the possibility of correction gene cassette as a promoter analysis tool will be confirmed.   The research on PtAPase has a wide range of application potential. Measuring the expression of PtAPase mRNA may help to monitor the physiological condition of diatoms in the natural environment and in the artificial bioreactor. The PtAPase promoter (pPhAP1) may be utilized in the recombinant protein expression platform.
URI: http://ethesys.lib.ntou.edu.tw/cgi-bin/gs32/gsweb.cgi?o=dstdcdr&s=G0010036031.id
http://ntour.ntou.edu.tw:8080/ir/handle/987654321/48793
Appears in Collections:[生命科學暨生物科技學系] 博碩士論文

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