發(fā)布時間：2019-04-12 07:51

【摘要】：條斑紫菜是我國重要的經(jīng)濟栽培海藻,圍繞其育種育苗以及相關生理生化等方面的研究已經(jīng)展開并積累了一定的研究基礎。性別分化是發(fā)育生物學中重要的科學問題之一,恰當?shù)膶嶒灢牧系倪x擇是促進此研究取得進展的重要因素。條斑紫菜因其葉狀配子體由單層細胞組成,雌雄同株,大部分精子囊細胞分布在其邊緣,從營養(yǎng)細胞向性細胞的分化過程相當于高等植物減數(shù)分裂后子細胞的后續(xù)發(fā)育過程,所以是研究營養(yǎng)細胞向性細胞分化發(fā)育過程這一重要科學問題的理想材料。本實驗中,取新鮮紫菜,放在與其生長條件溫度相同的大量的水體中以維持其良好的生理活性并立即利用刀片分別收集條斑紫菜的營養(yǎng)細胞和精子囊細胞,切下來的細胞迅速在顯微鏡下觀察,營養(yǎng)細胞是獨立的大細胞,每個精子囊細胞分成2-8個不等的部分,兩種材料迅速用液氮冷凍之后分別收集約2 g,于-80℃凍存。綜合比較飽和硫酸銨沉淀法、三氯乙酸／丙酮沉淀法、酚提取法等蛋白質(zhì)提取方法,發(fā)現(xiàn)酚提取方法能夠有效地去除蛋白質(zhì)中的多糖、色素等雜質(zhì),因此確定最適合條斑紫菜蛋白提取的方法為酚提取法。并且酚提取方法中使用的試劑不會對后續(xù)質(zhì)譜鑒定實驗造成影響,利用改良酚法提取兩種材料的可溶性總蛋白之后,雙向電泳檢測提取的蛋白良好,再利用ITRAQ標記技術對兩種材料的蛋白質(zhì)干粉進行差異蛋白的分析,發(fā)現(xiàn)這兩種材料中的差異蛋白共有549種,篩選之后共獲得具有穩(wěn)定差異的蛋白質(zhì)101種,精子囊細胞相對于營養(yǎng)細胞有88種蛋白質(zhì)的含量表現(xiàn)為下調(diào),13種蛋白質(zhì)的含量表現(xiàn)為上調(diào)。這101種蛋白質(zhì)中,與蛋白質(zhì)翻譯有關的蛋白質(zhì)有31種,光合作用有關的蛋白質(zhì)有29種,ATP合成相關的蛋白質(zhì)9種,脂肪酸合成的7種。從這101種差異蛋白中我們篩選與蛋白質(zhì)合成、碳循環(huán)、ATP合成相關的蛋白質(zhì)進行實時熒光定量的實驗,結(jié)果表明,對于營養(yǎng)細胞,肌動蛋白,HSP 90, HSP 70-2,細胞色素f,鈣調(diào)蛋白在精子囊細胞中含量都比較低,微管蛋白和組蛋白H2B含量較高。其中HSP 70-2,鈣調(diào)蛋白與ITRAQ的結(jié)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，

本文編號：2456847