基于转录组和microRNA信息的牙鲆白化发生机制研究

Japanese flounder (Paralichthys olivaceus)is a marine fish which is ecomomically important as a food resource, and has been widely cultured in the north of China. The albinism in Japanese flounder aquaculture has become a common phenomenon and influenced its large-scale farming and market value. So far, the study on albinism of Japanese flounder was only focused on bait nutrients, environment, physiology and so on. So, it is necessary to investigate the mechanism of albinism in Japanese flounder from molecular genetics. In this project, the albinism and normal Japanese flounder are used for the transcriptome sequencing and miRNA sequencing to screen albinism related genes and miRNA. In parallel, the GO and KEGG pathway analysis for miRNA targeted genes are conducted. The regulation mechanism of miRNA in albinism is studied by analyzing the expression pattern of miRNA, the targeted mRNA verification and miRNA function. Then, the structure, function and regulation network of the albinism related genes are analyzed with time and space expression pattern, over-expression, loss of function and Co-IP. The results of this project will not only be helpful to fully understand the molecular mechanism of Japanese flounder albinism, but also provide important theoretical guidance to control the albinism fish.

牙鲆是我国北方重要的海水养殖鱼类，苗种生产中普遍存在体色白化现象，严重影响了苗种品种和市场价值。目前针对牙鲆白化的研究仅停留于营养、环境及生理等方面，亟需开展分子调控机制方面研究。本项目将以牙鲆白化和正常样品为材料，在获得转录组数据基础上，进行microRNA深度测序，拟从转录和转录后水平上揭示牙鲆白化发生机制。通过mRNA与miRNA联合分析，筛选并鉴定牙鲆白化关键miRNA及关键基因，并对关键基因进行GO和KEGG富集分析。通过对牙鲆白化miRNA的表达模式、功能和靶基因验证分析，揭示miRNA在牙鲆白化发生过程中的调控机制。利用定量PCR、原位杂交、过表达和抑制表达、Co-IP等方法分析牙鲆白化关键基因的时空表达规律、功能及其调控网络，确定牙鲆白化关键基因的作用机制；本项目完成后，不仅有助于深入揭示牙鲆白化发生的分子机制，而且将为利用分子育种手段防治牙鲆白化现象提供重要理论指导。

牙鲆等鲆鲽鱼类中普遍存在体色白化问题，严重制约了养殖产业健康可持续发展。本项目针对牙鲆白化现象，开展了正常和白化个体有眼侧皮肤的转录组及miRNA测序分析，鉴定出tyr、tyrp1、oca2、slc45a2、mitf等差异表达基因235个及mmu-miR-143-5p_R+2、pol-miR-199a-5p_R+1等差异表达miRNA33个，GO和KEGG富集分析揭示黑色素合成、酪氨酸代谢、卵磷脂代谢、光传导通路等通路在牙鲆白化发生的重要作用。利用双荧光素酶报告基因系统验证了mmu-miR-143-5p_R+2和tyrp1a的负调控关系，并阐明了其在不同体色及组织中的表达模式。利用甲状腺激素T3、T4及甲状腺激素抑制剂TU进行了牙鲆鱼苗的处理实验，发现甲状腺激素处理能显著提高鱼苗的白化比例，选取对照组和T3处理组42天鱼苗进行了RNA-seq和miRNA-seq的测序分析，鉴定出具有显著差异表达趋势的146个基因和12个miRNA，GO和KEGG分析显著富集到糖代谢途径、酪氨酸代谢等通路；采用视黄酸注射、不同波长光照实验等揭示了视蛋白可以通过促进视黄酸合成来调控比目鱼类体色不对称发育。利用半滑舌鳎高比例白化家系进行QTL分析，定位到与眼睛移动异常和白化相关QTL位点q-10M和rps6kb2基因，同时还利用构建的白化相关连锁图谱鉴定出3个白化显著相关位点，包含tyrp2、gpr143等2407个基因，并富集到酪氨酸激酶活性、AMPK信号通路等，为揭示半滑舌鳎等鲆鲽鱼类白化现象分子机制奠定重要基础。建立了牙鲆正常和白化皮肤细胞系，并开展了病毒感染及基因转染分析，为牙鲆白化相关基因或miRNA的功能研究提供了细胞平台。本项目相关结果获2018年度中国水产科学院大渔创新奖1项，发表研究论文7篇，其中SCI文章6篇，申请国家发明专利1项。本项目的实施揭示了牙鲆等鲆鲽鱼类体色发育及白化现象的分子机制，为鲆鲽鱼类变态异常及白化问题的防治提供了重要理论指导。