LncRNA在牙鲆肌肉生长发育中的作用及性别二态性关联分析

The Japanese flounder displays significant sexual dimorphism in growth，with the female grows faster than the male, however, the genetics basis for this phenomenon is not clear. The essence of fish growth is muscle growth. Long chain non-encoding-RNA plays a key role in the process of animal muscle development. It is not still reported that lncRNA affects fish muscle development. On the basis of the previous study on DNA methylation affecting reproduction and growth traits of Japanese flounder, this project is going to study the regulation function of lncRNA, as well as their regulation mechanism of the growth difference between the female and the male. Firstly, the expression profile of lncRNA is identified according to development stage tissues sequenced by RNA-Seq. Then, combined with these results of qRT-PCR, FISH and Western Blot, specific lncRNA and its target genes are screened by bioinformatics analysis. Next to, the regulation relationship between lncRNA and target gene is determined by using the dual luciferase reporter system. Finally, from the aspect of the cellular and individual level, the important lncRNAs function in regulating muscle development are revealed by the use of CRISPRi/Cas9 technology. The results are expected to amplify the molecular regulation and mechanism of differences of muscle growth development and sexual dimorphic in Japanese flounder. And these relevant findings may lay theoretical basis on fish muscle developmental biology and also provide technical support for other fish aquaculture and breeding.

牙鲆雌鱼较雄鱼生长快，但相关遗传基础并不清楚。鱼类的生长，实质上就是肌肉的生长发育，长非编码RNA在动物肌肉发育过程中扮演着关键的角色，而其对鱼类肌肉发育调控的研究鲜见报道。本课题在前期DNA甲基化对牙鲆生殖和生长性状影响研究的基础上，继而拟探讨lncRNA对雌雄牙鲆生长差异的表观遗传调控机制。首先，对发育阶段各组织RNA-Seq分析，确定lncRNA表达谱；然后，结合qRT-PCR、FISH和Western Blot结果，进行生物信息学分析和筛选特异lncRNA及其靶基因；其次，采用双荧光素酶报告系统，确定lncRNA与靶基因的调控关系；最后，利用CRISPRi/Cas9等技术，从细胞和个体水平揭示关键lncRNA在肌肉发育中调控功能。此成果有望诠释牙鲆肌肉生长发育以及性别二态性差异相关的调控分子和作用机理，为鱼类肌肉发育生物学提供理论基础，同时也将为其它鱼类养殖和良种选育提供技术支撑。

长链非编码 RNA 作为ncRNA 的一员，近年来已经成为继 microRNA 后的研究的又一重要的调控性ncRNA。迄今为止，有关鱼类的lncRNA研究甚少，与牙鲆肌肉生长发育相关的lncRNA也未见报道。本研究以牙鲆作为研究对象，基于非编码RNA（lncRNA，miRNA和circRNA）→mRNA（功能基因），在不同生长阶段[JP1阶段：7天的仔鱼，JP2阶段：约90天的幼鱼，JP3阶段（雌鱼）和JP4阶段（雄鱼）：约24个月的成鱼]骨骼肌中筛选出 751个lncRNA；分别鉴定了232、211、194、28、29和14个差异表达的lncRNA以及9549、8673、9181、1821、1080和557个差异表达的mRNA。基于差异共表达分析，鉴定到lncRNA和基因之间存在顺式调控关系的有200, 200, 200, 93, 47, 11对，鉴定到lncRNA和基因之间存在反式调控关系的有29, 19, 24, 38, 8, 47对，鉴定了3805、3720、3627、1779、1780和741个GO 条目，鉴定了79、85、71、31、31和31个KEGG通路。关键的lncRNA，通过原位杂交定位和双荧光素杂交系统分析了共表达基因的调控关系，其中筛选的kirrel基因家族，在斑马鱼上已报道通过CRISPR-cas9技术敲除，发现其对斑马鱼肌细胞融合起重要的调控功能；利用Cytoscape软件，将预测到存在调控关系的lncRNA-mRNA, miRNA-mRNA, lncRNA-miRNA关系对构建成一个相互作用网络。另外，对与肌肉发育相关的功能基因表观遗传分析，并分析急性低氧下与肌肉发育相关基因的调控关系。以上结果阐明牙鲆肌肉生长发育机理，同时也为牙鲆分子育种提供了丰富的分子信息，进一步为牙鲆工厂化养殖提供科学的指导。