鱼类阿朴脂蛋白14kDa的脂类代谢功能研究

During the embryonic and larval development of oviparous fish, there is a transition from endogenous yolk nourishment to exotrophic period, which is the key stage for the practice of artificial fry rearing. However, little is known about molecular mechanism underlying the two kinds of physiological process. Apo-14 has been cloned from zebrafish, gibel carp and orange-spotted grouper in our previous studies, which is also found in many other fishes by BLAST. Importantly, its transcript is firstly localized in the yolk syncytial layer at a high level during embryogenesis, and then found to be restricted to liver in later embryo and early larvae. Moreover, morpholino knockdown of Apo-14 resulted in severe yolk malabsorption and the disruption of digestive organ. These data indicate that Apo-14 play an important role in yolk lipid metabolism and organogenesis.Recently, a transgenic zebrafish line Tg(Apo14:ORF-GFP) was produced by gene recombination and genetic screening, which can express GFP marked Apo-14 in specific tissue as its endogenous gene expression pattern. In this study physiological assays will be used to verify its function in lipid metabolism. Meanwhile, fluorescent lipid analogs will offer a way to visualize the lipid absorption of YSL in vivo during embryogenesis, and the physiological function will be monitored by the Apo-14 molecular marker in the transgenic line during liver organogenesis. Taken together, this investigation will help to understand the function of Apo-14 in lipid metabolism and provide a new way to highlight the intrinsic mechanism of endo-exotrophic switch as well.

鱼类在胚胎发育过程中，要由最初的卵黄营养供给过渡到外源性营养阶段，这也是鱼苗培育中的关键时期。然而，目前我们对这两种不同的营养方式还缺乏分子水平上的认识。Apo-14基因在鱼类中广泛存在，我们前期研究工作发现其转录本在胚胎的YSL和肝脏中活跃表达，早期敲降Apo-14会造成卵黄吸收障碍，影响消化器官的发育，已有研究结果提示Apo-14在卵黄脂类利用和器官发生中具有重要的作用。本项目构建了组织特异性表达Apo-14的转基因斑马鱼Tg(Apo14:ORF-GFP)，拟利用该实验模型分析成鱼阶段血浆生理指标来证实Apo-14的脂类代谢功能；进一步结合荧光脂类分子探针在体可视地分析胚胎发育过程中YSL的卵黄吸收作用；同时利用Apo-14作为分子标记追踪肝脏原基发生过程中的生理功能。本研究结果不仅有助于揭示Apo-14的脂类代谢功能，而且为阐明鱼类由内源性营养向外源性营养过渡的内在机制提供了新思路。

斑马鱼是理想的脊椎动物模式生物，转基因斑马鱼是研究基因功能的有效工具。近年来建立的Tol2 转座子系统是一种高效的转基因技术，广泛地应用于多种生物。目前人们鱼类阿朴脂蛋白14kDa （apo 14）的功能还不是很清楚。本项目将apo 14 的开放阅读框克隆到Tol2 供体质粒中，构建了组织特异性过表达组成型Apo 14的转基因斑马鱼Tg(Apo14:ORF-GFP)，拟利用该实验模型分析成鱼阶段血浆生理指标来证实Apo-14的脂类代谢功能；进一步结合荧光脂类分子探针在体可视地分析胚胎发育过程中YSL的卵黄吸收作用；同时利用Apo-14作为分子标记追踪肝脏原基发生过程中的生理功能。.本项目构建了转基因斑马鱼Tg(apoP :APO-GFP)，对其表达模式进行了详细追踪，发现在斑马鱼胚胎和幼鱼全身的血液循环中有绿色荧光流动信号，在体证实了APO 14为分泌性蛋白。在转基因斑马鱼Tg(apoP :APO-GFP)的纯系筛选中遇到了F2代性状分离的问题，尝试了Southern Blot分析方法的运用，但是目前没有筛选到表达模式优越的纯合转基因斑马鱼，影响在在生理水平上的定量分析。同时本项目利用干扰鱼类脂类代谢的污染物2, 4-二硝基甲苯(2, 4-DNT)研究了apo 14在脂类代谢中功能，发现2, 4-DNT阻碍胚胎发育过程中YSL在卵黄吸收作用，同时造成肝脏原基发育不良，最终影响鱼体的生长。.