【作者】 李娜；

【导师】 侯林；

【作者基本信息】 辽宁师范大学 ， 细胞生物学， 2018， 硕士

【摘要】 中国卤虫（Artemia sinica）是一类生活在高盐水体中的小型甲壳动物,也是重要的饵料生物,具有极强抗逆能力,也是分子生物学和发育生物学研究的重要实验动物,在胚胎学和分子生物学等研究方面发挥越来越重要的作用。膜联蛋白（Annexin）家族是一个功能多的可溶性钙离子依赖的膜结合蛋白超家族,它们是细胞内Ca²⁺信号通路的一个必不可少的部分,遍布于脊椎动物、无脊椎动物、植物等真核生物（酵母除外）和原核生物中。膜联蛋白（Annexin）家族有12个成员,有研究表明,利用RNA干扰抑制Annexin A3的表达对DNA的合成有抑制作用。还有很多生理过程都有Annexin 3的介入,包括肿瘤发生、耐药性和转移。但Annexin 3在中国卤虫滞育胚胎重启的过程中的作用还不明确。此次实验利用RACE技术首次克隆了中国卤虫As-anx 3基因的cDNA全长,对As-anx 3做生物信息学及分子进化方面的分析。As-anx 3全长1024bp,包含948bp的开放阅读框,编码315个氨基酸,有四个经典的ANX结构域。采用免疫荧光技术对As-ANX3进行亚细胞定位分析,实验结果显示,As-ANX 3定位在卤虫细胞的细胞膜上。利用Real-time PCR与Western blotting手段,分析了Annexin 3的表达模式,As-anx 3在0h时,表达量最高,随着卤虫的发育,滞育时积攒的膜联蛋白被一点点的消耗,As-anx 3表达量减少,说明膜联蛋白在胚胎重启中发挥了重要的作用。在盐度刺激与温度胁迫实验中,As-ANX 3在温度15℃,盐度28‰时,表达量最高,之后随着温度升高或降低,盐度升高,表达量均下调。可以得出结论As-ANX 3可受温度和盐度的调控,盐度过高或温度过低时,As-ANX 3表达量下降。如遭遇恶劣环境,如低温或高盐,卤虫的生长发育或繁殖短暂的停止,形成滞育卵,当环境适宜卤虫生长时,滞育卵恢复发育,滞育胚胎重启动与温度盐度有关,而As-ANX 3也受温度和盐度的调控,说明As-ANX 3是胚胎发育过程中一种重要的蛋白。本文分析了中国卤虫滞育胚胎重启过程中Annexin 3的作用,为甲壳动物和其他无脊椎动物滞育胚胎重启动的分子机制的研究提供科学依据。更多还原

【Abstract】 Artemia sinica is a small crustacean that lives in a high salt water body,It’s also an important bait,extremely resilient,It is also an important laboratory animal in molecular biology and developmental biology,It plays an increasingly important role in embryology and molecular biology.The Annexin family is a multifunctional soluble calcium ion dependent membrane bound protein superfamily,they are essential for intracellular Ca²⁺signaling pathways,It is found in vertebrates,invertebrates,plants and other eukaryotes（except yeasts）and prokaryotes.The animal cell Annexin is regulated by the cytoplasmic free Ca²⁺ratio on the outside of the cell.All in all,a lot of physiological and biochemical processes involve the involvement of animal Annexin,such as regulation of membrane dynamics,participation in Ca²⁺signaling pathway,participation in Ca²⁺concentration regulation and adjustment of cytoskeleton,formation of membrane ion channels,etc.Many physiological processes involve Annexin 3,these include tumor occurrence,drug resistance and metastasis.Therefore,Annexin 3 has a very important role in the process of restarting the embryo of Artemia sinica in China,but its regulatory mechanism is unclear.In this experiment,RACE technology was used for the first time to clone the full length of cDNA of the Chinese Artemia sinica As-anx 3 gene,In present study,the full-length of As-anx 3 cDNA was 1024 bp,encoding a 315 amino acid protein,open reading box containing 948bp,there are four classical ANX domains.The subcellular localization analysis of As-anx was analyzed by immunofluorescence technique,experimental results show that,As-ANX 3 is located on the cell membrane of the Artemia sinica cell.Using real-time PCR and Western blotting,the expression pattern of Annexin is analyzed,As-anx has the highest expression at 0h,with the development of Artemia sinica,the membrane protein that accumulates during diapause is consumed by a little bit,As-anx expression decreased,It is indicated that membrane protein plays an important role in embryo restart.In the experiment of salinity stimulus and temperature stress,As-ANX 3 at temperature 15,when the salinity is28‰,the expression quantity is the highest,and then as the temperature goes up or down,the salinity goes up,the quantity of expression was down-regulated.It can be concluded that As-ANX 3 can be controlled by temperature and salinity,when salinity is too high or too low,As-ANX 3 expression decreased,As-ANX 3 is an important protein in embryo development.This paper analyzes the role of Annexin 3 in the restarting process of Chinese Artemia sinica diapause embryos,the study provides scientific basis for the study of the molecular mechanism of embryo reactivation of crustaceans and other invertebrate animals.更多还原