浒苔叶绿体NAD(P)H脱氢酶的组成、分布及其介导的环式电子传递对失水胁迫的响应

Ulva prolifera (Chlorophyta) is a representative species of intertidal macro-algae and is also one of the main causative species of green tides. U. prolifera is highly tolerant against many stresses such as dehydration and osmotic stress. Generally, under stress conditions, chloroplast NAD(P)H dehydrogenase (NDH)-dependent cyclic electron flow plays a vital role. Recently, we have found that NDH-dependent cyclic electron flow exist in macro-algae. Based on these results, in this program U. prolifera are chosen to investigate deeply the relationship between NDH-dependent cyclic electron flow and dehydration. We will analyze the activity of NDH during dehydration and re-hydration, separate and purify the thylakoid membranes and the NDH which will be compared with that of higher plants and Chiamydomonas reinhardtii. Moreover, we will determine the distribution of the NDH in thylakoid membrane and the responses of the composition and distribution of NDH to dehydration and re-hydration, and further clarify the link between the composition and function of the NDH during dehydration. The completion of this program will help us to understand not only the biology of U. prolifera, but also the responses of photosynthetic electron flow especially NDH-dependent cyclic electron flow to stresses. Furthermore, the completion of this program has an important significance for clarifying the cause and vicissitude of green tides.

浒苔属于绿藻门, 是潮间带大型海藻的代表物种，也是形成“绿潮”的主要原因种。浒苔具有极强的抗逆性如耐受极端失水和渗透胁迫等。一般认为,胁迫条件下光合生物叶绿体NAD(P)H脱氢酶（NDH）介导的环式电子传递起到十分重要的作用。我们近期的研究结果表明，大型海藻中存在NDH介导的环式电子传递。为此，本项目将以浒苔为研究对象，探究浒苔NDH介导的环式电子传递与失水胁迫的协同关系;测定失水胁迫以及复水过程NDH的活性;分离正常状态的NDH，与高等植物和莱茵衣藻的NDH进行比较,解析浒苔NDH的组成及其在类囊体膜上的分布特征;进一步分离失水和复水状态的NDH，比较分析其与正常状态下NDH的组成、分布以及功能上的异同。本项目的完成不仅可以加深我们对浒苔生物学特性的认知，而且可以丰富我们对光合电子传递尤其是NDH介导的环式电子传递对逆境响应的认识,尤其对阐明“绿潮”的成因和兴衰过程具有重要的意义。

浒苔是潮间带大型绿藻的代表物种，也是形成近年来全球性的生态灾害—“绿潮”的主要原因种。浒苔具有极强的抗逆性如耐受失水和高盐胁迫等。该项目的主要研究内容：比较分析不同胁迫状态(高盐胁迫盐度为7%，9%和11%)不同处理时间的浒苔NDH的活性以及NDH介导的环式电子传递的运转状态；分析了浒苔转录组中NDH的序列，设计了浒苔NDH的多肽并制备了浒苔NDH蛋白多克隆抗体，免疫印迹分析了不同盐度胁迫（盐度为7%，9%和11%）状态下浒苔NDH含量的变化趋势；研究了浒苔类囊体的结构特征，探究了浒苔类囊体膜蛋白对高盐胁迫的响应特征及机理。该项目建立了浒苔NDH的活性检测方法，在活体状态下检测了不同盐度胁迫状态下浒苔NDH的活性，发现在盐胁迫下浒苔NDH活性显著升高；利用自己制备的NDH的多克隆抗体，发现了在高盐状态下，浒苔NDH表达显著升高；通过透射电镜，确定浒苔的类囊体的结构特征，筛选了增溶浒苔类囊体膜蛋白的增溶剂（Digitonin，β-DM和α-DM）并确定了增溶浓度，发现了盐度胁迫条件下形成的PSII-LHCII-PSI的特异性三元复合体，其具有生理的形成可以通过PSI消耗过多能量从而保护光合膜结构。本项目的完成，为后续浒苔NDH的结构解析奠定了基础。