Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/36205
標題: 利用農桿菌方式轉殖單細胞綠藻
Genetic transformation of green algae- Chlorella sp. strain DT by Agrobacterium tumefaciens
作者: 呂權蓁
Lu, Chung-Chan
關鍵字: Chlorella;單細胞綠藻;Agrobacterium;BaMV;農桿菌;竹嵌紋病毒
出版社: 生物科技學研究所
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摘要: 
綠藻(Chlorella sp.)為小球型單細胞綠藻,具有低成本易大量增殖培養之特性,所以具備生產基因工程蛋白質之潛力。目前運用於綠藻基因轉殖的技術包含了 PEG 融合、基因槍與電穿孔法等方式,但是轉殖效率皆不高。本實驗為建構綠藻之農桿菌轉殖系統,將綠螢光蛋白(Green florescent protein,GFP)基因構築於竹嵌紋病毒(Bamboo mosaic virus, BaMV)載體系統(pCAMBG),並轉型於農桿菌進行綠藻的基因轉殖。 綠藻細胞與農桿菌於黑暗中共同培養 48 小時同時加入 100 μM Acetosyringone(AS)轉殖效率可達 92.5 ± 6.4/ 108 cells。轉殖成功的綠藻,利用序列專一性的引子對,經由聚合酵素鏈鎖反應可增幅出大小約 1 kb 的轉殖基因片段,而南方漬染法分析可於轉殖株中偵測轉殖基因片段的存在;於反轉錄聚合酵素反應亦可利用序列專一性引子對增幅出轉殖基因 Hygromycin、BaMV-CP 及 GFP 的 RNA 表達;以西方漬染法也可偵測到 BaMV 鞘蛋白及與綠螢光蛋白之累積。另外,於共軛焦顯微鏡觀察中,亦證實轉殖綠藻具螢光蛋白表現,此結果首度證明農桿菌轉殖系統可成功應用於綠藻中。為了證實 BaMV 病毒載體是否可在轉殖綠藻中複製並表現外源蛋白,因此構築一個病毒突變載體(pCAMBGdGDD),剔除 BaMV RNA 聚合酵素(RNA polymerase)活性區(GDD motif),使RNA聚合酵素失去複製活性作為對照組 ,與只有單一花椰菜嵌紋病毒(Cauliflower mosaic virus, CaMV)35S 啟動子 的 pCAMBG 轉型綠藻,進行鞘蛋白累積量與 GFP 表現量的比較,發現 pCAMBGdGDD 轉殖株不會有 CP 及 GFP 的蛋白累積;同時為了增加外源蛋白的表現量, 構築具有兩組 35S 啟動子的病毒載體(pCAM2pBG),與只有一組 35S 啟動子的 pCAMBG 病毒載體接種煙草進行鞘蛋白與綠螢光蛋白之累積的比較,可以測得 pCAM2pBG 接種葉鞘蛋白與綠螢光蛋白累積較 pCAMBG 接種葉多。 本研究期望建立一個以植物病毒載體於綠藻中表現蛋白的生產平台,同時為首次證實植物病毒可於綠藻細胞複製及表現基因。

Chlorella sp. is an unicellular green algae with spherical morphology, which can be cultured inexpensively on a large scale. These characteristics provide rationale for using Chlorella as a new system for foreign protein expression. Different methods have been developed for the nuclear transformation of Chlorella sp., such as polyethylene glycol-mediated transformation, particle bombardment and electroporation. However the transformation frequencies of these methods are very low. In this study we established a nuclear transformation system of Chlorella sp. by Agrobacterium tumefaciens C58C1. Bamboo mosaic virus (BaMV) viral vector
carrying the green florescent protein (GFP) gene constructed in a plant binary vector (pCAMBG) was used as a reporter system. The highest nuclear transformation frequency, 92.5 ± 6.4 cells/108 cells, was achieved when Chlorella cells were co- cultivated with A. tumefaciens in dark for 48 hours, in the presence of 100 μM Acetosyringone (AS). Polymerase chain reaction (PCR) results showed a precise amplification of a 1 kb gene fragment from Agrobacterium transformed Chlorella. Southern blot analysis confirmed the integration of transgene fragment in transformed Chlorella. In the transformed cells, hygromycin resistance gene, BaMV CP and GFP
can be detected by RT-PCR. BaMV coat protein and GFP accumulation in
transformed Chlorella sp. was also confirmed by western blot analysis. In addition, the transformed Chlorella showed the expression of GFP as detected by confocal laser scanning microscope. To confirm the replication of BaMV viral vector in Chlorella sp. cells. Mutant pCAMBGdGDD was constructed with a deletion of GDD motif to disrupt the replicase activity as a negative control. To compare with pCAMBG for its BaMV CP accumulation and GFP expression, we found pCAMBGdGDD transgenic lines lack of BaMV CP accumulation and GFP expression. Moreover, transformation of Chlorella cells with the plasmid
pCAM2pBG, which carries duplicate 35S promoter of Cauliflower mosaic virus, as compared to pCAMBG for efficiency in expressing BaMV CP and GFP. Here, we report the first evidence of the successful transformation of Chlorella sp. by A. tumefaciens harboring the pCAMBG plasmid. We had established a system that can be applied for foreign protein expression with plant viral vector in green algae. This work is a first demonstration that a higher plant virus replicates in green algae.
URI: http://hdl.handle.net/11455/36205
其他識別: U0005-1008200923073800
Appears in Collections:生物科技學研究所

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