【作者】 王斌；

【导师】 缪锦来；

【作者基本信息】 国家海洋局第一海洋研究所 ， 海洋生物学， 2007， 硕士

【摘要】 南极是地球上最冷的地区之一,低温是其最基本的特征。海冰是极地物理环境的重要组成部分。南极冰藻就是指生活在南极海冰、海冰边缘或海水中的一大类微藻。海冰中间存在大量网状的盐囊和盐通道,其中海水的盐度可以达到普通海水的3倍,甚至5倍。并且冰藻生活的冰层是一个低光照、寡营养和少气体交换的场所。每年南极的春夏季,海冰的融化时冰藻周围的环境发生巨大的变化,在日积月累海冰的冻溶变化中,冰藻逐渐形成了适应这种变化的生理生化机制。正是冰藻这种特殊的生理特征,使其成为研究低温生物学的良好试验材料;此外,冰藻也是新型活性物质的潜在来源。为了更加合理的开发南极的低温藻类资源,从应用上丰富我国现有的微生物资源,从中开发出有价值的生物活性物质和产品,本文主要以南极衣藻Chlamydomonas sp. L4(南极衣藻L4)为材料,进行叶绿体生理生化特性及其低温环境适应性的研究,以期为南极冰藻抗逆机理的研究和应用开发提供科学依据。对所取得的主要结果概括于下。1利用光学显微镜、扫描和透射电镜对正常培养南极衣藻L4和低温胁迫下的形态和超微结构进行研究。结果表明,南极衣藻L4的主要特点为:细胞壁较厚,无鞭毛端存在着明显的质壁分离现象,细胞内贮存有大量的淀粉颗粒,细胞壁和细胞膜之间充满大量的黑色颗粒物质。南极衣藻L4的叶绿体并不是简单“杯状”,而是以类囊体片层的形式分布在除细胞核区域的整个细胞中,细胞器之间以及淀粉颗粒之间都是类囊体片层结构。海水成冰胁迫后线粒体结构基本没有变化,能够使南极衣藻L4在低温下正常的能量供应,保证其正常代谢。而类囊体片层模糊,扭曲,膨大。采用蔗糖梯度法分离制备的南极衣藻L4叶绿体纯度和得率都很高。2以正常培养的南极衣藻L4作为对照,通过比较低温胁迫过程中类囊体膜色素、类脂、脂肪酸和色素蛋白复合物的变化情况,分析这些成分对南极衣藻L4低温海水成冰环境适应性的作用:(1)富含叶绿素b的集光色素蛋白复合体(LHC)对于南极衣藻L4产生低温适应性发挥着重要作用;叶黄素通过参与低温下植物光合机构中过剩光能的耗散,并且增强膜流动性也对南极衣藻L4低温适应性有着很好的效果。(2)MGDG能够在低温弱光的环境下维持类囊体膜特殊结构的稳定;十八碳-9,12,15-三烯酸不仅维持类囊体膜的流动性,还可以促进重新合成的D1蛋白装配到PSⅡ反应中心的速率;反式十六碳-3-烯酸能够降低“高熔点”PG的含量,促进LHCⅡ解聚。(3)完整PSⅠ复合物、ATP合成酶、细胞色素b6f复合物和RuBP羧化酶在低温环境中能够保持稳定;LHCⅡ在PSⅡ蛋白复合物上结合的紧密程度会影响南极衣藻L4对于低温弱光环境的适应性;LHCⅡ结构的变化即LHCⅡ寡聚体解聚,是低温适应性的重要保障。3通过研究低温胁迫对南极衣藻L4类囊体膜流动性、损伤性和功能的影响,发现:南极衣藻L4通过膜成分的改变来维持其类囊体膜的流动性,以适应低温海水结冰的环境;总体上胁迫期间类囊体膜受到的伤害不大,脂氧合酶活性上升幅度很小,并且很快出现活性下降的趋势,说明南极衣藻L4体内有充分的应激机制,来抵御低温海水成冰胁迫对不饱和脂肪酸的氧化作用,SOD酶是保持类囊体膜不受伤害的关键酶;南极衣藻L4类囊体膜希尔反应活性测定结果表明,净光合速率在胁迫过程中先下降再上升;在海冰环境中,南极衣藻L4类囊体膜H+-ATPase的活性明显增强,从而保障有效的光合磷酸化反应。对南极衣藻L4进行低温胁迫,从藻体形态结构、类囊体膜生化成分和生理功能等方面分析了南极衣藻L4对低温环境适应性的机理。更多还原

【Abstract】 The South Pole is one of the coldest areas on the earth. Cold is characteristic that distinguishes this area from other areas. The sea ice is important constituent for the polar physics environment. Antarctic ice microalgae refers to micro algae which lives in the Antarctic sea ice, the sea ice edge or water. It is within this labyrinth that the sea ice microalgae live with the only liquid being pockets of concentrated brines. At the same time, it is a low sunlight, poor nutrition and less air exchanges in the ice sheet. When sea ice melts in every spring and summer, the environments around the ice microalgae change enormously acclimating the frequent freeze-thaw cycles, Antarctic ice microalgae form gradually a series of physiological and biochemical mechanisms to adapt these extreme changeable environments.Just because of special physiological characteristic, ice microalgae have been used as fine tested materials to cryobiology and potential source of the new-style active substances. For reasonable development of Antarctic ice microalgae, increase of existing microbial resources, discovery of valuable active substances and products, and the new materials for fundamental researches, further study on the adversities acclimation of thylakoid membranes compositon of Antarctic ice microalgae Chlamydmonas sp. L4 is carried out.1 By optical microscope, scan and transmission microscopes, the morphology and ultrastructure of Antarctic ice microalgae Chlamydomonas sp. L4 cultured in normal environment and chilling environment were studied. Comparing with normal temperature green microalgae, the main characteristics of Antarctic ice microalgae Chlamydomonas sp. L4 were as followed: relatively thick cell wall, obvious plasmolysis phenomena at the end; much black particles between cell wall and membrane. In addition, much lipid and starch particles, as the important storage components, played an important role in the survival of Antarctic ice microalgae Chlamydomonas sp. L4 in Antarctic area. The chloroplast of Antarctic ice microalgae Chlamydomonas sp. L4 was irregular and spread all over the cell, and all of these were the adaptation to extreme environment. After chilling stress, the mitochondria was basically steady, which maintained the fundamental metabolism of organisms.But the thylakoid became blurry, contorted and bulgy.The chloroplasts were isolated by centrifugation through sucrose gradient. The grade of purity and the yield of the chloroplasts were appropriate.2 Regarding Antarctic ice microalgae Chlamydomonas sp. L4 normal cultured as control, we compared the changes of pigment, lipoid, fatty acid and pigment protein complexes in thylakoid membranes,and analyzed the functions of each composition: (1) LHC plays an important role in the adversities acclimation of icing ,as it contains abundant chlorophyll b; xanthophylls can asist in the dissipation of photosynthesis under chilling stress, and improve the the mobility of Antarctic ice microalgae Chlamydomonas sp. L4 thylakoid membranes. (2) MGDG can maintain the especial configuration of thylakoid membranes; 18:3ω9 can not only improve the the mobility of the Chlamydomonas sp. L4 thylakoid membranes, but also can accelerate the functiong of D1 protein. 16:1ω3t can reduce the content of the‘high melting point’fatty acid. (3) The content of PSⅠ, H+-ATPase, Cytb6f and RUBPase maintained under chilling stress, possibly it accerlerated the photophosphorylation. LHCⅡis a pivotal component in the adversities acclimation.3 By studying the influence of chilling to the mobility, harm and function of Antarctic ice microalgae Chlamydomonas sp. L4 thylakoid membranes, we got many results: The liquidity of its thylakoid membranes is maintained by adjusting component under chilling stress. As a whole , chilling causes little harm to Chlamydomonas sp. L4 thylakoid membranes, SODase plays an important role in protecting the thylakoid membranes. By analyzing the Hill-reaction activity of Chlamydomonas sp. L4 thylakoid membranes during chilling stress, we found the speed of photosynthesis became lower early and came back later. As a result of the high activity of H+-ATPase in Chlamydomonas sp. L4 thylakoid membranes during chilling stress, the photosynthesis reaction runs natural.We explored a series of physiological and biochemical mechanisms by analystting the the morphology and ultrastructure of Antarctic ice microalgae Chlamydomonas sp. L4, the biochemistry composition of Antarctic ice microalgae Chlamydomonas sp. L4 thylakoid membranes and physiological function of Antarctic ice microalgae Chlamydomonas sp. L4 thylakoid membranes.更多还原