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標題: 汞離子還原酶在小球藻Chlorella sp. DT的表現之研究
Functional expression of mercuric reductase in microalga Chlorella sp. DT
作者: 陳孟偉
Chen, Meng-Wei
關鍵字: Chlorella
mercuric ion reductase
出版社: 生命科學系
摘要: 近來由於人類的工業活動,導致大量的汞離子被排放到環境水域中造成污染。汞離子對生物具有高度的毒性,汞離子進入生物體內會增加活性氧(reactive oxygen species, ROS)的生成並導致氧化逆境造成細胞傷害甚至死亡。merA基因為葛蘭氏陽性菌中抗汞操縱子(mercury resistance operon, mer operon)的一員,其基因產物汞離子還原酶(mercuric reductase, MerA),能催化汞離子Hg2+的還原使二價汞還原成毒性較弱的元素汞Hg0進而揮發到大氣中。在這個研究中,我們希望藉由Chlorella來表現MerA,並以此為媒介來加速排除含汞汙水裡的汞離子。我們分別利用兩種已構築merA基因的載體pHm3A及pHm3A/35S來進行Chlorella的基因轉殖。pHm3A構築了一段稻米actin1啟動子來驅動merA基因的表現,而pHm3A/35S則是利用花椰菜鑲嵌病毒35S (CaMV35S)啟動子來驅動merA基因。再者,利用聚合酶鏈鎖反應(PCR)及南方墨點法(Southern blotting)來確認轉基因已成功插入Chlorella的染色體基因組中。此外,在具有汞離子逆境條件下,轉殖株(transformants)相較於野生株(wild type),表現出較高的光合作用活性及較強的汞離子排除能力。在超氧歧化酶(superoxide dismutase, SOD)的活性測試中,轉殖株則表現出較少的超氧歧化酶活性;這個結果顯示在遭受汞離子逆境時,轉殖株細胞相較於野生株,遭受較輕微的氧化壓力。 綜合以上的結果,我們發現轉殖株對汞離子的抗性及排除能力有著顯著的提升, merA基因不只成功的插入Chlorella的基因組中而且具有功能性的表現。 小球藻Chlorella是一種單細胞綠藻。因為其具有真核生物的生理特性及低廉的培養成本,有潛力成為一種新型式的生物反應器(bioreactor)來表現異源蛋白。
Chlorella is an attractive organism for heterogeneous protein expression because of its eukaryotic characteristics and low cost for large-scale culture. Mercury enters the water table as a result of industrial activities such as textile and paper, and as a product of the mining for gold. Mercury usually occurs in nature as a cation, Hg2+. Mercury ions are toxic to plants and animals due to its tendency to cause the production of active oxygen species. Mercuric reductase (MerA), the gene product of merA from Bacillus sp. RC607, performs the enzymatic reduction of Hg2+ to volatile uncharged Hg0 which lacks significant affinity for any functional group and less toxic to organisms. The exploitation of MerA-mediated functions by transgenic Chlorella for the removal of Hg2+ from industrial wastewater was purposed in this study. Protoplasts of Chlorella sp. DT were transformed with two vectors containing merA gene. Vector pHm3A was constructed with an actin1 promoter to drive the merA gene and with a hpt gene as a selection marker conferring resistance to antibiotic hygromycin B. Vector pHm3A/35 was modified from vector pHm3A by replacing the actin1 promoter with CaMV35S promoter. The presence of introduced DNA was determined by PCR amplification of the merA gene from genomic DNA isolated from transformants. The stable integration of introduced DNA was confirmed by Southern blotting analysis. In this study, the results of molecular analysis showed that merA was successfully integrated into the genome of Chlorella, and those of bioassay showed that the tolerance of the transformants to Hg2+ was promoted. It was suggested that merA gene was expressed functionally in Chlorella.
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