【作者】 杨礼香；

【导师】 吕应堂；

【作者基本信息】 武汉大学 ， 发育生物学， 2005， 博士

【摘要】 血红蛋白存在于整个生物界。植物中的血红蛋白至少有三类:共生的血红蛋白,非共生的血红蛋白,截短的（truncated）（2-on-2）血红蛋白。共生的血红蛋白主要存在于豆类和非豆类植物的固氮根瘤菌感染的细胞中。非共生的血红蛋白比共生的血红蛋白分布更广泛,不仅存在于含有共生血红蛋白的植物中,而且也存在于其他的植物中。非共生的血红蛋白可以分为两类,其中一类——血红蛋白2（class 2）具有和共生的血红蛋白相似的氧结合特性;另一类——血红蛋白1（class 1）的氧结合特性完全不同于共生的血红蛋白,表达受低氧胁迫和过量氮营养的诱导,因此也称之为压力诱导的血红蛋白（Dordas et al.,2003）。血红蛋白1几乎存在于整个植物界,据推测它可能是共生血红蛋白的进化祖先,因此血红蛋白1在植物的代谢中可能起着非常重要的作用。但迄今为止,血红蛋白1的确切功能还不清楚。 本研究的目的是探索拟南芥血红蛋白1（AtGLB1）的功能。将AtGLB1基因超表达和利用RNAi技术抑制AtGLB1基因的表达,得到不同AtGLB1表达水平的拟南芥株系。通过Northern分析,在纯合的转基因拟南芥中挑选AtGLB1蛋白表达量大的株系（D1）以及表达量小的株系（R11）,进行了后续的研究。研究的主要结果如下: 1.低氧胁迫处理后,三个株系（WT,D1,R11）中的H₂O₂含量和乙醇脱氢酶（ADH）的活性都增加了。但是,三个株系的增加的幅度不一样。其中超表达AtGLB1的株系（D1）中积累的H₂O₂相对较少,对低氧胁迫的抗性增强。而RNAi株系（R11）中积累的H₂O₂相对较多,对低氧胁迫更敏感。在低氧胁迫中拟南芥更多还原

【Abstract】 Hemoglobin (Hb) occurs in all the kingdoms of living organisms. In plants there are at least three distinct types of hemoglobin that have been categorized as symbiotic, nonsymbiotic and truncated hemoglobin (Dordas et al., 2003b). Symbiotic Hbs found in legumes and other nitrogen-fixing plants. The distributing of nonsymbiotic Hbs was more extensive, existing not only in the plants that had symbiotic Hbs, but also in other plants. Nonsymbiotic Hbs could be divided into two species: class 1 and class 2. Class 2 Hbs had similar affinities and dissociation kinetics of oxygen to symbiotic Hbs. Class 1 Hbs had different affinities and dissociation kinetics of oxygen and was called stress-induced hemoglobin, which could be induced by hypoxic stress and excessive nitrogenous nutrition (Dordas et al., 2003) . Class 1 Hbs almost found in whole plant kingdom and maybe the evolutional ancestor of symbiotic Hbs. Therefore, class 1 Hbs might play an important role in plant metabolism. But so far, the function of class 1 Hbs was unclear.The aim of our study is to investigate the function of class 1 Hbs of Arabidopsis (AtGLB1). We gained Arabidopsis lines with different hemoglobin levels using overexpression and RNAi technology. After northern gel blot analysis, we choose D1 (the overexpressing line) and R11 (the underexpressing line) from the transgenic lines for following research. The main results are as follows.After hypoxic stress treatments, the concentration of hydrogen peroxide and the activities of ADH increased in three lines (WT, D1, R11). But the extent of theincrease was different in three lines. The line overexpressing AtGLBl accumulated relatively little H2O2, and has enhanced tolerance to hypoxia. In contrast, the lines in which AtGLBl expression was suppressed by RNAi technique accumulated more H2O2, and were more sensitive to hypoxia. AtGLBl may play certain role in modulation of hydrogen peroxide level during hypoxic stress. Therefore, we detected the tolerance of protoplast and leaves with different hemoglobin 1 levels to exogenous hydrogen peroxide. The result showed that the line with high hemoglobin 1 level were more tolerant, while the line with low hemoglobin 1 level were more sensitive to exogenous hydrogen peroxide. AtGLBl might alleviate the hydrogen peroxide stress.All aerobic organisms have evolved a complex antioxidant system involved in scavenging H2O2, composed of nonenzymatic scavengers (such as ascorbate, glutathione, and hydrophobic molecules) and detoxifying enzymes. The latter include catalases (CAT; EC 1.11.1.6) and ascorbate peroxidases (APX; EC 1.11.1.11). Antioxidant system involved in scavenging H2O2 was also analyzed in our experiment. Before hypoxic stress treatment, the antioxidant system was not different in three lines. After hypoxic stress treatment, the activities of CAT and APX and ascorbate content were, indeed, increased in all three lines examined during the stress treatment, while only Dl can retain these relative high levels up to 48 h of the treatment, suggesting that antioxidant system may play an important role for the low H2O2 level of Arabidopsis over-expressing AtGLBl. However, how this Arabidopsis maintain the high level of these antioxidants for longer time compared with control lines remains unknown currently.The analyses of rapid-scan stopped-flow spectrophotometry showed that recombinant oxygenated AtGLBl could react with H2O2 and lead to the decrease of H2O2 level. The recombinant oxygenated AtGLBl with different concentrations could react with l.OxlO"4 M hydrogen peroxide. The higher the concentration of recombinant oxygenated AtGLBl was, the faster the reaction was. Therefore, AtGLBl might react with H2O2 and scavenge H2O2 directly during hypoxic stress. Alternatively, a recent report indicated that AtGLBl exhibited peroxidase-like activity (Sakamoto et al., 2004). Based on these results, we may also explain our observation for the enhanced tolerance of Arabidopsis over-expressing AtGLBl to hypoxia that hemoglobin 1 may contribute to scavenging H2O2, thus decreasing the H2O2 level and increasing the H2O2 tolerance. This needs to be further tested experimentally.AtGLBl play an important role during hypoxic stress. It is reported that function of a protein is be closely bound up to its location. We studied the location of AtGLB 1 through the instantaneous expression of the recombinant protein of AtGLBl and GFP in onion epidermis. The result showed that AtGLBl located in cytoplasm and nucleolus maily.更多还原