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CroE Like Chaperomns在几种藻中的存在、性质及其功能的研究
Thesis Advisor黎尚豪
Degree Grantor中国科学院水生生物研究所
Place of Conferral中国科学院水生生物研究所
Degree Discipline水生生物学
Keyword核酮糖-1,5二磷酸羧化酶/加氧酶亚基结合蛋白 类似groe蛋白的分子伴侣 蓝藻 绿藻 纯化 特性 定位
Abstract豌豆叶片于4 ℃缓冲液中(20 mmol/L Tris-HCl,pH 7.8 5 mmol/L巯基乙醇)提取,提取液在50 ℃水浴中加热10分钟后,12,000 * g离心15分钟,上清液经38%-70%饱和度的(NH_4)_2SO_4分级分离后,沉淀溶于少量缓冲液中,经过Sephadex G-50柱脱盐后,再用DEAE-Sepharose fast flow和Sephacryl S-300柱层析分离,收集RubisCO亚基结合蛋白峰部分。纯化的RubisCO亚基结合蛋白用ND-PAGE及ATP使其解离的特性来鉴定。提纯的RubisCO亚基结合蛋白的得率是20 mg/1.0 kg叶片,比以前报道的高12倍。以纯化的豌豆RubisCO亚基结合蛋白制备出高度专一的多克隆抗体。免疫双扩散结果证明,该抗体仅与RubisCO亚基结合蛋白有一条免疫沉淀线,而与RubisCO无免交叉反应。用此抗体对绿藻Chlamydomonas reinhardtii和多种蓝藻细胞提取液作Western blot分析,结果表明,在绿藻Chlamydomonas reinhardtii和Anabaena siamensis等丝状蓝藻中存在一与豌豆RubisCO结合蛋白具有相似抗原性的蛋白,且亚基分子量相似,均为61KD左右,而在单细胞蓝藻(如Anacystis nidulans)中并不存在这种同源蛋白(groE-like chaperonin)。多细胞蓝及绿藻中这一groE-like chaperonin具有在热致死温度下含量增加,经冰冻处理后不稳定的性质。我们用提纯的豌豆RubisCO亚基结合蛋白进行热稳定性实验,结果发现,该蛋白同样是一热稳定蛋白,并在低温下容易解离。这一结果说明,高等植物的RubisCO亚基结合蛋白藻类的groE-like chaperonin在蛋白的组成结构及性质方面都有一定的同源性。我们用豌豆RubisCO亚基结合蛋白的抗体进行免疫金标记亚显微观测,结果表明,groE- like chaperonin在绿藻Chlamydomonas reinhardtii内主要分布于蛋白核区域及类囊体内;在蓝藻Anabaena siamensis细胞中仅均匀分布于营养细胞,而在成熟的异形胞中并不存在。用水稻RubisCO抗体进行Protein-A免疫金标记,观测表明,RubisCO在在蓝藻Anabaena siamensis细胞中主要分布于营养细胞的羧化体内,而在成熟的异形胞中不存在;RubisCO在绿藻Chlamydomonas reinhardtii中主要分布于蛋白核区域。
Other AbstractPea(Pisum sativum) leaves were extracted with 20 mmol/L Tris-HCl (pH 7.8) at 4 ℃ containing 5 mmol/L mercaptoethanol. The extract was heated at 50 ℃ for 10 min, then centrifuged at 12,000 * g for 15 min. The supernatant was fractionated by 33% to 70% saturation (NH_4)_2SO_4. The precipicate was dissolved in a small volume, desalted with Sephadex G-50 column, and then loaded onto a DEAE-Sepharose fast flow column and then loaded onto a Sephacryl S-300 column. The BP peak fractions were collected. BP was examined by both ND-PAGE and by BP dissociation caused specifically by the addition of ATP. The yields of up t 20 mg/Kg fresh weight of leaves were obtained, which was 12-fold higher than the previous result. A highly specific anti-BP serum was prepared, which gave a single precipitin band with BP, by immunodiffusion tests, but no precipitin band with RubisCO. Western blot using antibodies to BP from pea detected subunits of closely similar size (61 kD or so) in the chlorophyte Chlamydomonas reinhardtii and filamental cyanobacteria (such as Anabaena siamensis), but in unicellular cyanobacteria (such as Anacystis nidulans) these homologous polypeptides couldn't be detected. These polypeptides were increased in amount under heat-shock conditions and decreased at lower temperature. We also found that BP from pea was a heat-stable protein and was easily dissociated at low temperature. This result assumes that homolog exist between BP from pea and groE-like chaperonins from algae both in protein structure and chacterizations. We provide evidence using electron microscopy coupled with antiserum to BP from pea and Protein-A colloidal gold. In filaments of Anabaena siamensis, groE-like chaperonin is absent from mature heterocysts and present well-distributedly in vegetative cells; in Chlamydomonas reinhardtii, groE-like chaperonin is present and it is associated both with pyrenoid and with thylakoid. Using Protein-A immunogold labelling technique, using antibody to RubisCO from rice, we found that in Anabaena siamensis, RubisCO is also absent from mature heterocysts, but present mainly in carboxysome in vegetative cells; in Chlamydomonas reinhardtii, RubisCO is mainly associated with pyrenoid.
Document Type学位论文
Recommended Citation
GB/T 7714
赵若虹. CroE Like Chaperomns在几种藻中的存在、性质及其功能的研究[D]. 中国科学院水生生物研究所. 中国科学院水生生物研究所,1993.
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