江蓠受精过程中的细胞识别及性别特异凝集素的研究

The study of sexual reproduction of algae will not only help to understand both the process of reproductive evolution and its regulation mechanisms, but also help to guide the algal cultivation method and even the industrial upgrading. Cell-cell recognition system usually depends on complementary binding between carbohydrate moieties on the surface of one gamete with specific sugar-binding lectins on another gamete. Lectin belongs to the protein or glycoprotein with non-immune origin. The sex specific lectin in algae has now been purified and realized gradually. Due to the non-motility and small size of spermatia, red algal fertilization has been regarded as inherently inefficient. However, the facts that the carpogonium has long trichogyne extending to algal surface and the high efficient recognition and adhesion between spermatia and trichogyne may enhance the efficiency of fertilization. This project are going to use Gracilaria lemaneiformis, the important agarophyte, as the main experimental material to observe the fertilization process and to isolate and purify the sex specific lectin of Gracilaria. Then the encoding gene will be cloned from the cDNA library according to the acquired amino acid sequence of the lectin. Also, a female specific expressed protein GMF01 achieved earlier will be analysed for its expression localization and other characters. Through these efforts, we are trying to make it clear the cellular process of fertilization as well as its recognition mechanisms in Gracilaria lemaneiformis.

对藻类有性生殖的研究既有助于了解生殖进化的过程和调控机理，也将有助于对藻类的养殖方式进行指导和产业升级。受精时细胞间的识别系统一般是依赖于一个配子细胞表面的糖基片段与另一个配子细胞上的糖基结合蛋白（即凝集素）的特异性互补结合。凝集素是非免疫起源的蛋白质或糖蛋白，藻类中性别特异凝集素已被逐步分离和了解。人们一般认为红藻的精子个体较小且没鞭毛不能运动，这会造成其受精效率低下。但是红藻的果胞具有延伸到藻体表面的受精丝以及精子和受精丝之间高效的识别粘连机制，这或许可以帮助其实现较高的受精效率。本项目将以产琼胶红藻龙须菜为主要研究对象，对江蓠属物种的受精过程进行显微观察以及性别特异凝集素的分离和纯化，并将根据获得的凝集素序列从cDNA文库中克隆相应的编码基因。同时还将对之前获得的龙须菜雌配子体特异表达蛋白GMF01进行定位和相关性质的分析。通过这些研究以期搞清龙须菜精卵受精的细胞学过程及其识别机制。

以江蓠属物种真江蓠为主要研究目标，首次对江蓠的精子进行了荧光显微观察和受精过程的扫描电镜观察，进而分析了受精过程的影响因素，揭示了较高的光强，中温，低氮浓度和短光时更有利于真江蓠的受精并证实无论雄配子体存在与否精子都可以与果胞进行受精；还通过抑制实验对受精中精卵识别所涉及的凝集素进行了筛查，发现了真江蓠精卵识别的主要凝集素是位于雌配子体受精丝上的ConA类的凝集素；同时还对hsp70和gmf01这些与真江蓠性别或世代相关的基因进行了转录分析，首次在真江蓠中克隆了3个hsp70基因和1个hsp90基因，并分析了它们的细胞定位和在不同世代藻体中的转录水平，发现hsp70-3在雌配子体中的转录最显著提高，可能与它作为分子伴侣的功能相关。

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