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標題: 不同氮源對魚腥藻CH1及CH2穀胺醯胺合成酵素生合成之影響研究
Effect of Different Nitrogen Sources on Glutamine Synthetase Synthesis in Anabaena CH1 and CH2
作者: 張忠凱
Chang, Chung-Kai
關鍵字: heterocyst
vegetative cell
glutamine synthetase
出版社: 植物學系
摘要: 摘要 利用硫酸銨沉澱及電泳部份純化藍綠藻Anabaena CH1之GS蛋白,用來製備 抗CH1 GS血清,藉抗原-抗體作用偵測Anabaena CH1和CH2之GS蛋白生合成 表現量。由CH2對於抗CH1 GS血清的反應可以看出CH1和CH2之GS結構是非 常相似的。 Anabaena CH1和CH2培養在通空氣,培養液中添加NH4Cl或KNO3等不同氮源 條件時,利用異型細胞分離技術,製備異型細胞和營養細胞萃取液,經蛋 白生合成和活性二方面來探討Anabaena CH1和CH2之GS的表現情形。 Anabaena CH1和CH2在異型和營養細胞皆會表現GS。在通空氣時,CH1和 CH2之異型細胞GS蛋白生合成均較營養細胞高約二倍。在GS活性方面,CH1 之異型細胞較營養細胞高約二倍;CH2之異型細胞GS活性則比營養細胞高 約四倍。 對於CH1,當培養液中添加高濃度之NH4Cl(2mM),其異型細胞之GS蛋白 生合成及活性皆受到明顯的抑制;營養細胞之GS蛋白生合成及活性則皆受 到去抑制的作用。當培養液中添加KNO3(30mM),對異型細胞之GS蛋白生 合成有促進作用;營養細胞之GS蛋白生合成則無明顯變化。顯示GS活性之 降低或升高主要是由於GS蛋白生合成受到抑制或去抑制的結果。蛋白生合 成雖是影響GS活性表現的主要因素,但並非唯一因素,GS活性亦會受到其 它因子的複雜調控。由異型細胞和營養細胞之GS蛋白生合成表現應可間接 指出異型細胞和營養細胞GS的基因(glnA)是因應不同的氮源而由二種 proter來調控其表現。對於CH1異型細胞之GS表現而言,ammonium是抑制 因子;nitrate在短期是去抑制因子,長期則會因衍生物質的累積而轉變 為抑制因子。 對於CH2異型細胞之GS蛋白生合成及活性的表現,nitrate扮演了較主要的 抑制角色;對營養細胞之GS表現則有去抑制作用。則需在長期(七十二小 時)的培養才能看出對異型細胞之GS蛋白生合成的抑制作用。由此可說明 對於CH2異型細胞之GS表現的抑制作用可能屬於長期或緩和的效應; nitrate的影響才是抑制CH2之GS表現的主要原因。
Abstract In the filamentous nitrogen-fixing cyanobacteria Anabaena sp. strain CH1 and CH2 a comparative study has been made on the GS protein synthesis and activity between heterocysts and vegetative cells growing in N2, ammonium and nitrate grown conditions. A procedure was developed for the isolation of heterocysts from cyanobacterial filaments and a polyclonal anti- GS serum was prepared from rabbit that immunized with partial purified GS of CH1. The cross-reaction of CH2 GS with anti-CH1 GS serum was strongly. is results implicate that GS of both CH1 and CH2 is highly conserved. The Anabaena CH1 and CH2 both express GS in heterocysts and vegetative cells. In N2 condition, on a quantitative protein basis, the GS content in heterocyst was twofold that of the vegetative cell of CH1 and CH2. The GS specific activity in heterocysts was two- and fourfold higher than that of the vegetative cell of CH1 and CH2. The GS protein synthesis and activity expression of the Anabaena CH1 heterocysts was repressed by NH4Cl(2mM), the GS expression both in protein synthesis and activity of vegetativeell was derepressed in the same condition. These observations indicate that the expression of GS protein synthesis is corresponding to the GS activity, however, GS activity is regulated by additional, more complex mechanism and implicate that the different promoters is availabled by glnA gene of heterocysts and vegetative cells. For GS expression of heterocysts of Anabaena CH1, ammonium is repressor, on the other hand, nitrate is derepressor. In Anabaena CH2, nitrate is repressor of GS expression of heterocysts and derepressor of GS expression of vegetative cell. The repression on GS protein synthesis of ammonium-72hrs grown heterocyst indicated ammonium maybe plays a long-term repressor role.
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