条斑紫菜叶状配子体营养细胞和生殖细胞的蛋白质组分析比较
发布时间：2019-04-12 07:51
【摘要】：条斑紫菜是我国重要的经济栽培海藻,围绕其育种育苗以及相关生理生化等方面的研究已经展开并积累了一定的研究基础。性别分化是发育生物学中重要的科学问题之一,恰当的实验材料的选择是促进此研究取得进展的重要因素。条斑紫菜因其叶状配子体由单层细胞组成,雌雄同株,大部分精子囊细胞分布在其边缘,从营养细胞向性细胞的分化过程相当于高等植物减数分裂后子细胞的后续发育过程,所以是研究营养细胞向性细胞分化发育过程这一重要科学问题的理想材料。本实验中,取新鲜紫菜,放在与其生长条件温度相同的大量的水体中以维持其良好的生理活性并立即利用刀片分别收集条斑紫菜的营养细胞和精子囊细胞,切下来的细胞迅速在显微镜下观察,营养细胞是独立的大细胞,每个精子囊细胞分成2-8个不等的部分,两种材料迅速用液氮冷冻之后分别收集约2 g,于-80℃冻存。综合比较饱和硫酸铵沉淀法、三氯乙酸／丙酮沉淀法、酚提取法等蛋白质提取方法,发现酚提取方法能够有效地去除蛋白质中的多糖、色素等杂质,因此确定最适合条斑紫菜蛋白提取的方法为酚提取法。并且酚提取方法中使用的试剂不会对后续质谱鉴定实验造成影响,利用改良酚法提取两种材料的可溶性总蛋白之后,双向电泳检测提取的蛋白良好,再利用ITRAQ标记技术对两种材料的蛋白质干粉进行差异蛋白的分析,发现这两种材料中的差异蛋白共有549种,筛选之后共获得具有稳定差异的蛋白质101种,精子囊细胞相对于营养细胞有88种蛋白质的含量表现为下调,13种蛋白质的含量表现为上调。这101种蛋白质中,与蛋白质翻译有关的蛋白质有31种,光合作用有关的蛋白质有29种,ATP合成相关的蛋白质9种,脂肪酸合成的7种。从这101种差异蛋白中我们筛选与蛋白质合成、碳循环、ATP合成相关的蛋白质进行实时荧光定量的实验,结果表明,对于营养细胞,肌动蛋白,HSP 90, HSP 70-2,细胞色素f,钙调蛋白在精子囊细胞中含量都比较低,微管蛋白和组蛋白H2B含量较高。其中HSP 70-2,钙调蛋白与ITRAQ的结果相反,其他的都一致。
[Abstract]:Porphyra yezoensis is an important economic cultivated seaweed in China. The research on its breeding and breeding, physiology and biochemistry has been carried out and accumulated a certain basis. Sex differentiation is one of the important scientific problems in developmental biology. The selection of appropriate experimental materials is an important factor to promote the progress of this research. Because of its leaflike gametophyte is composed of monolayer cells, androgynous, most of the sperm vesicle cells are distributed at its edge. The differentiation process of vegetative cells is equivalent to the subsequent development of the progeny cells after meiotic division of higher plants. Therefore, it is an ideal material to study the process of vegetative cell differentiation and development. In this experiment, fresh porphyra was taken and placed in a large amount of water at the same temperature as its growth condition to maintain its good physiological activity and immediately collect vegetative cells and spermatozoa cells from porphyra yezoensis using blades. The cells were quickly observed under microscope. The vegetative cells were independent large cells. Each sperm vesicle cell was divided into 8 different parts. The two materials were rapidly frozen with liquid nitrogen and then collected about 2 g respectively and frozen at-80 鈩，
本文编号：2456847
[Abstract]:Porphyra yezoensis is an important economic cultivated seaweed in China. The research on its breeding and breeding, physiology and biochemistry has been carried out and accumulated a certain basis. Sex differentiation is one of the important scientific problems in developmental biology. The selection of appropriate experimental materials is an important factor to promote the progress of this research. Because of its leaflike gametophyte is composed of monolayer cells, androgynous, most of the sperm vesicle cells are distributed at its edge. The differentiation process of vegetative cells is equivalent to the subsequent development of the progeny cells after meiotic division of higher plants. Therefore, it is an ideal material to study the process of vegetative cell differentiation and development. In this experiment, fresh porphyra was taken and placed in a large amount of water at the same temperature as its growth condition to maintain its good physiological activity and immediately collect vegetative cells and spermatozoa cells from porphyra yezoensis using blades. The cells were quickly observed under microscope. The vegetative cells were independent large cells. Each sperm vesicle cell was divided into 8 different parts. The two materials were rapidly frozen with liquid nitrogen and then collected about 2 g respectively and frozen at-80 鈩，
本文编号：2456847
本文链接：https://www.wllwen.com/shoufeilunwen/zaizhiyanjiusheng/2456847.html
