斑马鱼生物钟核心基因cry(cryptochrome) 家族功能研究

As a core circadian gene, it is very important for Cryptochrome (Cry) to maintain the regular biological rhythm and physiological functions of animals. And up to 6 cry genes were found in zebrafish, an important model organism. However, the functions of these zebrafish cry genes remained unclear so far since the related study progress was hindered by the excessive number of genes and their complicated relationship. We will use TALEN and CRISPR/Cas9, the targeted gene knockout technologies which have been rapidly developed in recent years, to construct knockout mutants of all zebrafish cry genes for the first time. These mutants together with followed multiple cry gene knockout mutants could provide the possibility to study the function of zebrafish cry genes. Thus the mechanism how zebrafish cry genes regulated circadian clock can be illustrated based on the locomotor activity assay and RNA-seq results of cry gene knockout mutants. Whether zebrafish Crys are photoreceptors and the influences of zebrafish cry genes on immune system and glucose metabolism pathway also could be studied combined with other biochemical, biophysical and molecular genetics experiments. Meanwhile, the in vivo expression patterns of zebrafish Crys can be learned by the CRISPR/Cas9-based tag knock in mutant. In summary, the above researches will help us clear the cry gene functions in zebrafish, clarify the molecular mechanism of circadian clock.

作为生物钟核心基因，Cry在维持动物体内正常生物节律及生理功能等方面起着关键作用。而斑马鱼这类重要模式生物被发现有多达6个cry基因。可能是其过多的基因数目以及复杂的相互关系阻碍了相关研究，迄今为止人们对斑马鱼cry基因的功能知之甚少。课题组借助近年来发展迅猛的靶向基因敲除技术（TALEN与CRISPR/Cas9），首次获得全部斑马鱼cry基因的敲除突变体，并尝试筛选出其多基因敲除突变体，为研究斑马鱼cry基因功能提供了可能。课题组将分析突变体的行为学规律及转录组高通量测序结果，并结合其他生化，生物物理，分子遗传手段阐明斑马鱼cry基因参与生物钟调控机制，研究Cry蛋白是否具备光受体功能及其对斑马鱼免疫系统与血糖代谢的影响。同时将构建基于CRISPR/Cas9的标签蛋白knock in突变体研究斑马鱼Cry蛋白体内表达模式。本课题将明晰斑马鱼cry基因的功能，阐明斑马鱼生物钟调控分子机理。