【作者】 侯贇；

【导师】 张全斌；

【作者基本信息】 中国科学院研究生院（海洋研究所） ， 海洋化学， 2013， 博士

【摘要】 海藻生活在开放的水体中，易受到病原侵袭和环境改变等因素带来的各种威胁，大规模的病害会给海藻养殖业带来巨大的经济损失。在许多高等植物和海藻免疫反应中，研究者发现寡糖类物质可作为激发子诱导植物的防御反应。本文以条斑紫菜和真江蓠为研究对象，分别考察它们的细胞壁多糖降解产物对其自身的免疫诱导作用。本文首先采用冷水、热水提取和不同浓度碱提取等方法，提取紫菜多糖，再以酸解法降解紫菜多糖，得到紫菜寡糖，分析其对条斑紫菜的免疫诱导活性，发现紫菜寡糖可诱导条斑紫菜产生H2O2。分离纯化活性寡糖，结果表明聚合度1~4的单硫酸化紫菜寡糖可通过激活寡糖氧化酶，引发H2O2产生，抑制由培养密度过高引发的病烂；聚合度为4~13的紫菜寡糖可能激活NADPH氧化酶相关的免疫信号通路，引起氧化爆发。为进一步探索寡糖诱导的海藻防御反应的研究方法，后期研究是在德国基尔霍姆赫兹海洋研究所以真江蓠为研究对象，分析琼胶寡糖对真江蓠的防御诱导作用，对比原产于中国(青岛和荣成)和入侵德国（基尔和Nordstrand）的真江蓠防御反应的异同。研究表明，琼胶寡糖可诱导真江蓠细胞中寡糖氧化酶的表达，从而催化寡糖的氧化脱氢反应，生成醛类和H2O2。真江蓠细胞中寡糖氧化酶的激活与细胞中铁离子结合蛋白和能量的传递相关。琼胶寡糖的诱导不能改变真江蓠表面的细菌数量，但可改变江蓠表面的菌落组成。对比原产地和入侵地的真江蓠，我们发现琼胶寡糖的诱导可两者发生相同的防御反应，即OA氧化酶的表达及H2O2的生成。但不同产地的真江蓠表面细菌数量和菌落组成不同：原产于中国的物种含有更多的细菌，OA可减少原产地（青岛）真江蓠表面的细菌种类，但增加入侵种（Nordstrand）的菌落种类。两种红藻均以其细胞壁多糖的降解产物作为激发子，由此引发的免疫反应具有相似的H2O2产生机理，即寡糖氧化酶的激活，但两者H2O2的产生过程又有很大的差异：真江蓠中H2O2的释放发生在30min内，而条斑紫菜中却持续1d以上。这一现象表明H2O2的产生虽来自同种酶的激活，但海藻内部的信号传导过程却各有不同，H2O2的产生可能并不是单一酶的激活或单一反应的产物。未来的研究可尝试从其它角度分析H2O2的来源，也可从进一步分析寡糖氧化酶的结构、功能及其激发相关的信号通路。更多还原

【Abstract】 Porphyra lives in open ocean where environment is complex, changeful and fullof pathogen. Broad scale diseases in marine agriculture will lead to large economicalloss. Oligosaccharides working as elicitors in both high plants and algae have beenreported by many researches. The present thesis investigated the elicitor activities ofoligo-porphyran and oligoagar in Porphyra yezoensis and Gracilaria vermiculophylla,respectively.Water-extractions and alkaline solution-extractions were employed to obtainnatural polysccharides and oligosaccharides in cell wall of P. yezoensis,Oligosaccharides with different Mw (molecular weight) were obtained by the acidhydrolysis of porphyran from P. yezoensis. The elicitor activities of oligo-porphyranwere investigated. Results showed that oligo-porphyran induced the generation ofH2O2from P. yezoensis. Active oligo-porphyran was seperated by anion-exchangechromatography. Compound of oligo-sulphated galacans with degree ofpolymerization (DP) from1to4induced the expression of cellular oligosaccharidesoxidase, and enabled the P. yezoensis to resist rotting caused by dense incubation.Oligo-sulphated galacans (DP4~13) compound might activate NADPH oxidase,leading to oxidative burst in P. yezoensis.In order to learn new research methods and enforce the communication withother international research groups, the last experimental part of this thesis wascarried out in Kiel, Germany. We investigated the oligoagar (OA)-induced defence responses of G. vermiculophylla and compared the different defence responsesbetween the native (Qingdao and Rongcheng, China) and the invasive (Kiel andNordstrand, Germany) populations. We found that OA induced the generation ofH2O2from G. vermiculophylla which was related to the activation of OA oxidase: OAoxidase catalyzed the oxidation of OA, generating aldehyde and H2O2. After24htreatment of150umol/L OA, G. vermiculophylla had a significant expression of OAoxidase which was related to cellular iron-containing protein and energytransformation. However, OA-treatment couldn’t change the number of G.vermiculophylla surface bacteria, but OA changed the bacterial composition of algalsurface. Both invasive and native populations could response to OA with the similarquantities of OA oxidase expression, but the enzyme activities of native populationswere lower than that of invasive populations because of their less release of aldehydeand H2O2. The surface bacteria in the native and invasive populations are different inquality and quantity. OA reduced the degree of bacterial diversity of native populationfrom Qingdao which had much more bacteria than that in invasive population fromNordstrand, but increased that of invasive population which had fewer bacteria.In the present research, two red seaweeds were chosen and their defenceresponses to the oligosaccharides derived from their cell wall polysaccharides wereinvestigated. For both of the two chosen algae, H2O2was induced by oligosaccharidesby a same mechanism, i.e. the activation of oligosaccharides oxidase. However, thepatterns of H2O2generation were different: the release of H2O2from P. yezoensissustained for more than one day, while that from G. vermiculophylla lasted only forhalf an hour. These results confirmed that defence responses varied in differentspecies, and also indicated that the generation of H2O2in these two red seaweeds maybe not due to the activation of only one enzyme or one signal pathway. Other causesfor H2O2release and the further analysis of oligosaccharide oxidase’s structure,function and relative signal pathways are worthy to be done in the future.更多还原