亮氨酸感受器LARS和Sestrin2协同调节奶牛乳蛋白合成的机制研究

乳蛋白合成调控一直是奶牛育种和营养学领域关注的重要科学问题。申请人前期研究发现LARS和SESN2可能作为细胞内亮氨酸感受器参与奶牛乳蛋白合成的调控，但详细机制未知。本研究拟首先通过活体和离体细胞试验揭示奶牛乳腺LARS和SESN2的亮氨酸识别功能、识别阈值；进一步分别对其氨基酸识别结构进行定点基因编辑获得突变乳腺上皮细胞株，检测mTORC1下游信号分子活性、亮氨酸转运蛋白LAT1的表达、亚细胞分布及其氨基酸转运活性，确定这两个基因通过mTORC1调控乳蛋白合成；研究基因突变对LARS和SESN2相互之间表达与活性的影响，初步确定二者的关系；通过比较分析细胞内LARS和SESN2的不同表达与活性组合对Rag GTP酶活性状态的影响及其与mTORC1活性的关系，进而全面揭示LARS和SESN2协同调控乳蛋白表达的机制。研究成果可为奶牛多基因聚合育种以及乳蛋白生产的精准营养调控提供理论指导。

The mechanism of milk protein synthesis has always been a scientific question to dairy cattle breeding and nutrition scientists. The applicant's previous studies showed that LARS and SESN2 may be involved in the regulation of cow milk protein synthesis as intracellular leucine receptors, but the detailed mechanism is unknown. In this study, leucine recognition thresholds of LARS and SESN2 in dairy cows will be revealed through in vivo and in vitro. Furthermore, the mutant mammary epithelial cell lines will be made by site-specific editing of the amino acid recognition domain of LARS and SESN2. The effects of LARS and SESN2 mutation on mTORC1 downstream pathway, expression of leucine transporter LAT1, subcellular distribution and amino acid transporter activity will be analyzed, so as to confirm that LARS and SESN2 regulate milk protein synthesis through mTORC1 pathway. The effects of the mutation on the expression and activity of LARS and SESN2 each other will also be investigated to analyze their relationship. Furthermore, the interaction of LARS and SESN2 with Rag GTP and their effects on the activity of Rag GTPase , thereby on mTORC1 activity will also be analyzed. The ultimate goal is to reveal the coordinately control of cow milk protein synthesis by LARS and SESN2. The research results can provide theoretical guidance for polygene pyramid breeding and precise nutrition regulation on milk protein production of dairy cows.