【作者】 何薇；

【导师】 王晗；

【作者基本信息】 苏州大学 ， 遗传学， 2015， 硕士

【摘要】 在自然界中所有生物都存在一种近似24小时的昼夜节律,称为生物钟。生物钟在细胞、组织等水平上都具有调节作用。生物钟紊乱会给身体带来极大的危害,例如睡眠失调、代谢失调、衰老提前。生物钟的分子机制主要依赖一个细胞自主调控的转录翻译反馈环路。该环路由一对正向调节因子和一对负向调节因子组成:CLOCK和BMAL形成异二聚体,结合到per和cry启动子区域的E/E’-box来激活它们的转录。随后,PER和CRY蛋白在细胞质结合形成复合体并进入细胞核内,通过作用于CLOCK:BMAL异二聚体,从而反馈抑制其本身转录。斑马鱼中存在clock基因的3个拷贝,它们在不同组织器官中转录振荡。斑马鱼已经演化出不同的clock基因表达机制,并且这三个基因的功能已经出现了分化。但这三个clock基因的具体功能至今还不清楚。本研究利用CRISPR-Cas9技术成功获得了缺失7个碱基的斑马鱼clock1a突变体。荧光素酶报告实验表明突变的clock1a不能激活e4bp4-2b转录活性。实时定量PCR显示,在纯合clock1a突变的斑马鱼幼体中,其反馈环路下游的生物钟基因per1a、per1b、per2、per3、cry1aa、cry1ab、cry1ba、cry1bb表达在正常光暗和持续黑暗条件下都显著改变。clock1a突变体斑马鱼幼鱼在正常光暗条件下的运动振幅增加。而在连续黑暗的情况下,clock1a突变体斑马鱼则完全失去了节律。clock1a敲除斑马鱼幼鱼的高活动量在成鱼中也得到了证实。实验显示在大部分时间点clock1a敲除斑马鱼成鱼的活动量显著高于同龄的野生型成鱼。这些结果表明Clock1a在保持斑马鱼生物钟的正常运转中起到不可或缺的作用。在镜面攻击试验中,clock1a敲除斑马鱼成鱼对于其自身影像更具有攻击性。这些结果表明在斑马鱼中,clock1a敲除斑马鱼成鱼也表现出躁狂症相似的表型。Li+是一种精神稳定药物,常常用来治疗躁狂症。我们使用Li+来处理clock1a敲除斑马鱼幼鱼,来验证其是否能拯救该鱼的异常。实验表明Li+能够有效的使鱼的行为恢复到正常的水平。这也揭示了clock1a在保持生物钟系统的正常运转及正常行为规律上的重要作用。另外,clock1a敲除导致雌鱼所产胚胎量大幅上升,其生殖相关基因lh、lhr、fsh、fshr及卵黄蛋白原基因vtg的表达上调,但体内促黄体素、促卵泡素、雌激素含量并没有受到影响。clock1a敲除对雄鱼生殖没有影响。为了深入研究clock1a的调节功能,我们通过高通量转录组测序检测clock1a-/-突变体胚胎在ZT2(11:00,98hpf)和ZT14(23:00,110hpf)时的基因表达谱。与野生型相比,在ZT2时clock1a-/-突变体有244个基因上调,406个基因下调;而在ZT14时clock1a-/-突变体有184个基因上调,249个基因下调。并且clock1a-/-突变体在不同时间点的差异表达基因也不同,在clock1a-/-突变体中ZT2和ZT14时期有159个相同的差异表达基因。其中凋亡蛋白酶相互作用蛋白Apip的表达水平下降了近7倍,分析该基因发现在其启动子上存在经典的E box元件。apip可能是clock1a的靶基因,这说明clock1a参与细胞凋亡的调节。另外,肌动蛋白结合蛋白anillin的表达也下调,说明clock1a也调节细胞的胞质分裂。本论文构建了clock1a敲除斑马鱼,初步探索了clock1a敲除对其他核心生物钟基因表达以及行为的影响,也揭示了生物钟在行为、生理、生殖方面的作用。为完善现有的生物钟环路做出贡献,也为后续的研究提供了基础。更多还原

【Abstract】 Approximately 24-hour rhythms of activities in all animals from bacteria to human are controlled by the circadian clock. The circadian clock functions at cell and tissue. Circadian misalignment can cause a variety of diseases, such as sleep disorders, metabolic disorders, premature aging and different mental disorders.The mammalian circadian clock controlled by a series of key circadian genes, including Clock and Bmal1 as well as Cryptochromes(Crys) and Period(Pers). CLOCK:BMALl heterodimer bind to E box elements in the promoters of Per and Cry genes and thereby activate their transcriptions. After translation, dimerization and translocation to the nucleus, the PER and CRY proteins physically interact with CLOCK:BMALl complex and thereby inhibit their own transcriptional.In zebrafish, there are three clock genes named clock1 a, clock1 b and clock2. They are rhythmically expressed in different tissues/organs. Zebrafish have evolved different mechanisms controlling the expression of clock genes, and the functions of these three clock genes have diverged. However, how these three clock genes function in zebrafish is still unclear.In this study, we generated a zebrafish clock1 a mutant through CRISPR-Cas9. This clock1 a mutant zebrafish harbors a CRISPR-Cas9 induced 7-bp deletion, which is a frame shift resulting in a truncated Clock1 a with only 101 amino acids. Luciferase reporter assays show that the mutate form of clock1 a lose its activating effect on e4bp4-2b gene. Quantitative Real-Time PCR showed altered expression of key circadian clock genes per1 a, per1 b, per2, per3, cry1 aa, cry1 ab, cry1 ba and cry1 bb in homozygous clock1 a mutant zebrafish larvae under both light/dark(LD) and constant dark(DD) conditions. Behavioral assays show that clock1 a mutant fish display hyperactivity under LD condition and completely lose circadian rhythms under DD condition. Altered expression of key circadian clock genes and disrupted locomotor rhythms in clock1 a mutants suggest that Clock1 a is essential for zebrafish circadian regulation and maintaining zebrafish locomotor rhythms. Hyperactivity phenotype was also observed in clock1 a mutant adult fish, and the mirror-attack test shows that clock1 a mutant adult fish are more aggressive/hyperactive, suggesting zebrafish clock1 a mutant also display mania-like syndrome as well as mice Clock mutant. Lithium is a mood stabilizer that is particularly effective in treating mania. We found Li+ treatment can effectively rescue mania-like behavior.In addition, clock1 a mutants female show increased reproductive capacity. Compared with WT female, clock1 a mutants female produce more embryos. And the expression of lh,lhr,fsh,fshr,vtg are all up-regulated. But clock1 a knockout have no effect on male fertility.We also employed deep sequencing to conduct transcriptome analysis of clock1 a at ZT2(11:00, 98hpf) and ZT14(23:00, 110hpf), and revealed that 244 up-regulated genes and 406 down-regulated genes at ZT2, 184 up-regulated genes and 249 down-regulated genes at ZT14, and 159 differentially expressed genes at both ZT2 and ZT14. Apip is an APAF1 interacting protein, the expression of apip is reduced by 7-fold, which means maybe apip is a target of Clock1 a, and Clock1 a regulates apoptosis.We successfully generated a clock1 a knockout zebrafish. It explored the functions of clock1 a in core circadian clock gene expression, the behavior rhythm and reproduction. It improves the existing feedback loop, also provides a basis for further research.更多还原