DNA甲基化和miRNA介导奥尼罗非鱼DGAT2非加性表达的功能研究

Hybrid Nile tilapia × Blue tilapia is one of the most popular farmed fish in China due to its heterosis. The preliminary work of applicant showed that the DGAT2 was non-additive expression in liver of the hybrid. The present project would focus on key factor of growth and lipid metabolism and investigate the expression pattern and regulatory mechanism of DGAT2 in hybrid Nile tilapia × Blue tilapia. Thus, the project would carry out in following fields: (1) The spatiotemporal expression patterns of DGAT2 in hybrid Nile tilapia × Blue tilapia and its parents would be investigated; (2) We would analyze the changes of non-additive expression pattern of DGAT2 in different nutritional conditions in the hybrid; (3) From the DNA methylation levels, the mechanism of non-additive expression pattern in the hybrid would be illustrated; (4) The functional regulation of microRNA on non-additive expression of DGAT2 in the hybrid would be discussed; (5) And we would analyzed the correlation between non-additive expression pattern of DGAT2 in the hybrid and growth as well as lipid metabolism. From DNA methylation and microRNA levels to gene expression levels and returning to phenotype of hybrid vigor, we would build a bridge between epigenetics and non-additive expression. The project would provide a physiological basis for heterosis and evidences for directional crossbreeding.

奥尼杂交罗非鱼因其杂交优势明显是我国主养的罗非鱼品种之一。申请者前期的研究工作表明，成熟奥尼杂交罗非鱼肝脏中DGAT2的表达呈非加性表达模式。本项目针对奥尼杂交罗非鱼生长和脂代谢关键因子，着力探讨奥尼杂交罗非鱼中DGAT2表达模式及调控机制。项目拟从以下几方面开展研究：（1）分析奥尼罗非鱼与亲本DGAT2的时空表达模式；（2）探讨在不同营养条件下DGAT2在奥尼罗非鱼中的非加性表达模式变化；（3）从DNA甲基化水平阐明DGAT2在奥尼罗非鱼中的非加性表达模式的产生机制；（4）探讨microRNA调控对DGAT2非加性表达模式的作用；（5）并分析DGAT2在奥尼罗非鱼中的非加性表达模式与生长和脂代谢的关联。从甲基化和microRNA水平到基因表达水平，最终回归到杂交优势表型，构建表观遗传学和非加性表达模式间的桥梁。为进一步研究杂交优势奠定重要的生理基础，为定向杂交育种提供依据。

杂交优势被广泛利用于鱼类杂交育种，其产生的关键机制仍是当前研究的重点问题之一。奥尼杂交罗非鱼由于其杂交优势已经成为我国主要养殖的罗非鱼品种之一。本研究在明确了奥尼罗非鱼相对于亲本在血清和肝脏中有着更高水平的甘油三酯累积的基础上，探讨了DGAT2在奥尼罗非鱼中的非加性表达优势。其中，奥尼罗非鱼DGAT2在肝脏和肠道中相对于亲本都有着更高的表达，由于DGAT2是甘油三酯生成途径中的限速酶，这说明高水平表达的DGAT2最终导致了奥尼罗非鱼的甘油三酯水平升高。无论在饲喂、饥饿还是再投喂条件下，奥尼罗非鱼肝脏DGAT2基因相对于亲本均有着较高表达，进一步说明了DGAT2的非加性表达模式属于非条件依赖性，而仅因为其杂交背景所带来的遗传特异性。在此基础上，我们明确了奥尼罗非鱼亚DGAT2等位基因表达特征，证明其双亲亚基因组对非加性表达的贡献基本相似。进一步的研究发现在奥尼罗非鱼肝脏中，DGAT2启动子区域的甲基化整体水平明显低于其亲本的尼罗罗非鱼和奥利亚罗非鱼，各研究个体的DGAT2启动子甲基化水平和DGAT2表达水平呈明显负相关。此外，本研究发现miR-19a-5p可与罗非鱼DGAT2的3’UTR区结合，抑制DGAT2表达，而miR-19a-5p在奥尼罗非鱼中的低表达水平导致了其肝脏DGAT2的表达较高。这些研究结果证明包括甲基化和miRNA在内的表观遗传学水平调控导致了DGAT2的高表达水平，从而影响了奥尼罗非鱼体内甘油三酯水平。以上的研究成果丰富了鱼类杂交优势产生的认识，同时也为精准杂交育种提供了新思路。