【作者】 顾文辉；

【导师】 王广策；

【作者基本信息】 中国科学院研究生院（海洋研究所） ， 海洋生物学， 2014， 博士

【摘要】 绿藻在进化地位上介于高等植物和低等光合生物之间，在陆地和海洋环境中都有较广分布。其广泛的分布也使绿藻具有很强的对各种不同环境的适应能力。并且，绿藻生长所经历的多种生境也赋予了其抵抗逆境的生物学特性。尽管不同绿藻适应逆境的机制各不相同，但是很多绿藻在逆境下会合成并且积累类胡萝卜素。而且绿藻中含有的类胡萝卜素种类繁多，并且不同物种在逆境下以合成某一种或某几种类胡萝卜素为主。例如，单细胞淡水绿藻雨生红球藻（Haematococcuspluvialis）在多种逆境下会合成大量酮式类胡萝卜素——虾青素；盐、海水生的单细胞绿藻——杜氏盐藻（Dunaliella salina）在逆境下则合成β-胡萝卜素；而一些大型潮间带海藻如浒苔（Ulva prolifera），虽然也能耐受强光照、干旱等逆境，却并未出现其他两种绿藻具有的大量合成和积累类胡萝卜素的现象。因此，不同种类的类胡萝卜素之于不同的绿藻，在逆境响应中，可能存在不同的协同机制。光合生物中，类胡萝卜素多与类囊体膜蛋白结合；绿藻作为光合生物，执行光合作用的类囊体膜蛋白极为重要，而且很容易受到逆境损伤。本文从蛋白组学的角度，利用不同的比较手段，研究了三种绿藻——雨生红球藻、盐藻和浒苔之间在蛋白组水平上的差异和变化。重点研究了雨生红球藻不同细胞阶段细胞总可溶蛋白组、类囊体膜蛋白组以及三种绿藻在强光照下类囊体膜蛋白组对强光照的响应。盐藻作为与雨生红球藻非常接近的单细胞绿藻，在逆境下积累虾青素合成的前体物质β-胡萝卜素，但是不会积累虾青素。因此，将这两种微藻放在一起比较，为研究虾青素在雨生红球藻光保护中的作用提供比较和参考的样本。另外，能耐受强光的潮间带的大型海藻浒苔，使用相同的强光照处理方式，研究其光合类囊体膜蛋白组的变化，寻找其与单细胞绿藻所不同的光响应形式。本研究论文关于三种绿藻细胞生理和蛋白组研究的主要内容和关键论点包括：（1）研究了雨生红球藻不同阶段细胞的固碳能力和虾青素合成之间的关系。四种细胞中，虽然GV（绿色游动）细胞虾青素的成分最低；GV具有最高的CA酶（carbonic anhydrase,碳酸酐酶）活性，并且同位素示踪分析结果表明GV细胞有最高固定无机碳能力。虽然RC（红色不动孢子）含有最高含量的虾青素，但是其含有最低的CA酶活性。因此，虾青素快速合成的OR细胞具有较快地将无机碳转化为虾青素的能力。（2）雨生红球藻不同细胞阶段的总可溶性蛋白差异荧光双向电泳比较不同生活史阶段细胞蛋白构成差异发现：GV细胞具有细胞快速分裂和生物量积累的蛋白基础；而处于逆境的细胞代谢缓慢。类囊体膜蛋白BN-PAGE和蔗糖密度梯度超速离心-DM增溶SDS聚丙烯酰胺电泳分析差异蛋白显示不同阶段的细胞类囊体膜蛋白保持了主要的光合蛋白。并且鉴定得到一些结合在类囊体膜上的调控蛋白。但是RC细胞可能存在捕光色素蛋白和反应中心蛋白形成的超级蛋白复合体。（3）通过比较雨生红球藻和盐藻强光响应下类囊体膜蛋白组变化，揭示了雨生红球藻在同等光强条件下比盐藻更快地适应了强光。雨生红球藻可能在适应强光的过程中重新调整了碳流向，通过多种机制参与光保护和淀粉与虾青素的合成。在这种机制中，无糖磷酸途径可能参与了抗逆。通过比较研究浒苔强光响应下类囊体膜蛋白组变化，结果表明浒苔的两个捕光色素蛋白系统表现为不同步的变化；并且浒苔的PsbS蛋白在强光照下表现为上调。可能是通过PsbS来启动了NPQ来响应强光。本研究通过对三种绿藻的比较分析，积累类胡萝卜素不同的绿藻对逆境响应的机制不同。先在叶绿体内合成β-胡萝卜素并且随后在细胞质内合成虾青素的雨生红球藻响应强光的机制相对特殊与高效。更多还原

【Abstract】 Green algae evolved at the position between higher plants and lower plants. Andthey are widely distributed both on land and in the sea. The versatile and toughhabitats green algae live in enable them to survive and acclimate. Many green algaespecies synthesize and accumulate carotenoids in the process of stress response.Carotenoids include a wide array of secondary metabolites. For example, the freshwater unicellular microalgae Haematococcus pluvialis are characterized with theability of accumutaing astaxanthin under stress. And Dunaliella salina is able toaccumulate β-carotene intracellularly under stress. However, some macroalgae, Ulvaprolifera for example, show high stress tolerance ability without any carotenoidsaccumulated. Consequently, carotenoids accumulstion and stress acclimation areinterelated, with some synergy effects existed.Thylakoid membrane is where the main process of photosynthesis occurs, makingit important for green algae. In this research, the three green algae, including H.pluvialis, D. salina and U. prolifera were comparatively studied from the thylakoidproteomic perspective. H. pluvialis, which firstly synthesizes β-carotene and convertsit to astaxanthin under stress, was extensively investigated in this research. However,D. salina synthesizes and accumulates β-carotene under stresses. Hence, D. salinacould be used as a suitable comparative object in H. pluvialis photoprotection research.Besides, the macroalga U. prolifera, which shows neither large amount of β-carotenenor astaxanthin accumulation under high light stress, could also cope with high light.The elucidation of high light responsive pattern in U. prolifera might provide someclue revealing the carotenoids role in high light response. Conseqently, the thylakoidmembranes of three green algae were comparatively studied under high light stress.The main research work and conclusions are as follows:(1) The relation of carbon fixation and astaxanthin synthesis in H. pluvialisi wasinvestigated. Among the four types of cells, GV (green vegetative) cells showed the highest CA (carbonic anhydrase) activity and the lowest astaxanthin content level.Radioactive labeling and tracing results also confirmed GV showed highest carbonfixation capability. Comparatively, RC (red cyst) cells were highest in astaxanthincontent while lowest in CA activity. Notably, OR (orange resting) cells showedhighest capability of converting fixed carbon into astaxanthin. Those findings were inaccordance with the fact that OR cells were in the process of rapid astaxanthinsynthesis in H. pluvialis.(2) Comparative analysis using2D DIGE identified some differential expressedproteins among the four types of H. pluvialis cells. The rapid proliferation andbiomass accumulation characteristics GV cells were supported by some up-regulatedproteins. And the other three types of cells, which were under various stress, showedhigher level of proteins involving in low cellular metabolism. Thylakoid membraneproteomics research using BN-PAGE and sucrose gradient ultracentrifugationidentified some regulatory proteins and results also showed that H. pluvialis indifferent stages conserved the photosynthetic proteins. However, RC cells showed thepossibility of LHC-PS super-complex forming as revealed by continuous sucrosegradient ultracentrifugation.(3) By quantitatively monitoring the abundance variations of thylakoid membraneproteins, H. pluvialis showed great high light acclimation capability. Based on thefindings in this study and inspired by results from others, a hyperthetical carbon flowreorientation strategy was proposed, by which the H. pluvialis employed during highlight acclimation. It was theoretically possible that phosphate pentose pathway wasinvolved in stress induced metabolisms including starch and astaxanthin synthesis.Comparatively, U. prolifera showed inbalanced energy distribution pattern betweenthe two photosystems as indicated by the abundance alteration of LHCI and LHCII.Moreover, PsbS protein was monitored to be significantly up-regulated under highlight. Thus it is possible that PsbS activated and enhanced NPQ to funnel excessenergy for photoprotection.更多还原