环状RNA介导CNS1S1基因影响热应激奶牛乳腺αs1-casein合成及机制研究

热应激与乳蛋白合成密切相关，应激发生时奶牛乳腺αs1-酪蛋白编码基因CSN1S1表达显著下调，但具体机制尚不清楚。前期比较热应激与限饲配对适温奶牛乳腺全转录组数据，发现4个circRNAs竞争性结合miRNA参与CSN1S1上游调控。因此，项目拟通过RNA酶耐受、FISH及核质分离qPCR分析候选circRNA分子特征、亚细胞定位与出核规律；利用荧光素酶报告系统、circRNA pull-down与RIP明确候选circRNA调控网络构成；以乳腺原代细胞为材料，干扰或过表达候选circRNA并设计桥梁miRNA恢复与CSN1S1回补性实验，揭示候选circRNA影响乳腺αs1-酪蛋白合成的分子机制；最后构建候选circRNA腺相关病毒载体，在活体水平上模拟热应激并验证相关ceRNA网络调控乳蛋白合成的作用机理，这对阐明乳蛋白合成机制具有重要意义，可为热应激条件下改善乳品质提供理论依据。

Heat stress is closely related to lactoprotein synthesis in mammary glands of dairy cows, and high ambient temperature can retard the expression of CSN1S1 gene. However, little is known about how heat stress mediates biosynthesis of milk protein in cows. Recently, we performed transcriptome analysis of mammary glands of Holstein cows between heat stress and moderate temperature individuals, and the results showed that 4 circRNA candidates can be as competitive endogenous RNAs to mediate CSN1S1 expression in αs1-casein synthesis. Based on the our previous researches, we will validate the reality, intracellular location and expression of circRNA candidates using RNase R treated analysis, RT-qPCR and Fluorescence in situ hybridization (FISH). In addition, the composition of circRNA-associated network will be further validated by luciferase reporter gene assays, circRNA pull-down and RNA Binding Protein Immunoprecipitation (RIP), respectively. Next, the functionary mechanism of αs1-casein biosynthesis regulated by circRNA candidates will be determined by transfection of over-expression or siRNA recombinant vector, as well as miRNA recovery and CSN1S1 complementation experiments. Finally, with the adeno-associated viral vector of circRNA candidates, we will double-check the circRNA-involved αs1-casein biosynthesis in vitro with heat stress model. In summary, our study will clarify the regulatory mechanisms of milk protein synthesis, and also lead to a new theoretical basis for milk quality improvement under heat stress.