| Summary: | This study aimed to explore genes associated with milk protein content in dairy cows and their relationships with <span style="font-variant: small-caps;">l</span>-leucine. Ten primiparous Holstein cows (93.8 ± 11.56 milking days) fed the same diet were divided into two groups depending on their milk protein contents (group High, 3.34 ± 0.10%; and group Low, 2.86 ± 0.05%). Milk epithelial cells (MECs) were isolated from the collected morning milk and differentially expressed proteins in MECs were explored by two-dimensional gel electrophoresis (2-DE). Then, the mRNA expression of these proteins was detected by real time PCR in MAC-T cells incubated with three different media named positive control (PC), negative control (NC), and <span style="font-variant: small-caps;">l</span>-leucine depletion (NO-leu). Results showed that ten proteins were differentially expressed in MECs from cows in group High. They included seven down-regulated ones (heat shock protein beta-1 (HSPB1), 78 kDa glucose-regulated protein (GRP-78), <span style="font-variant: small-caps;">l</span>-lactate dehydrogenase B chain (LDH-B), malate dehydrogenase, cytoplasmic (MDH1), annexin I (ANXA1), cytokeratin-7 (CK-7), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH)), and three up-regulated ones (prohibitin (PHB), beta casein (CSN2), and alpha S1 casein (CSN1S1)). When <span style="font-variant: small-caps;">l</span>-leucine was depleted from the medium, not only proteins content was lowered (<i>p</i> < 0.05), but also the LDH-B mRNA expression was decreased in MAC-T cells (<i>p</i> < 0.05). In conclusion, LDH-B is negatively associated with the milk protein content of dairy cows and has a positive association with <span style="font-variant: small-caps;">l</span>-leucine.
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