VPS28通过泛素化信号通路调控奶牛乳腺上皮细胞中乳蛋白的合成

旨在分析VPS28基因调控乳蛋白合成的分子机制，为奶牛泌乳性状的分子育种奠定理论基础。本研究首先利用RNA干扰（RNA interference，RNAi）技术敲降奶牛原代乳腺上皮细胞（bovine mammary epithelial cells，BMECs）中VPS28基因的表达水平，检测与乳蛋白合成、泛素化-溶酶体和泛素化-蛋白酶体通路相关的11个基因、泛素蛋白的表达水平及蛋白酶体活性；然后抑制BMECs中蛋白酶体和溶酶体的活性，检测酪蛋白相关基因、核糖体蛋白的表达水平；最后利用同位素标记相对和绝对定量（isobaric tags for relative and absolute quantification，iTRAQ）比较蛋白质组学分析敲降前后BMECs的差异表达蛋白。结果表明，敲降VPS28基因后，CSN1S1、CSN2、CSN3、RPS8、UBC、PSMC3、PSMC5基因均显著上调，PSMD12显著下调；抑制蛋白酶体后，CSN1S1、CSN2、CSN3显著上调，RPL13显著下调；抑制溶酶体活性后酪蛋白相关基因表达不显著；iTRAQ结果共筛选出129个差异表达蛋白，下调蛋白主要富集在核糖体、溶酶体、剪切体等相关通路，上调蛋白主要富集在内质网的蛋白质加工、加压素调控的水重吸收过程、RNA转运等通路中。研究表明，VPS28基因可通过泛素化信号通路影响BMECs中乳蛋白的合成。

VPS28 Regulates Milk Protein Synthesis in Bovine Mammary Epithelial Cells through Ubiquitination Signaling Pathway

The aim of this study was to analyze the molecular mechanism of VPS28 gene regulating milk protein synthesis and to lay a theoretical foundation for molecular breeding of lactation traits in dairy cows. In this study, the expression level of VPS28 gene in primary mammary epithelial cells (BMECs) of dairy cows was knocked down by RNAi technology, and the expression level of 11 genes, ubiquitin proteins, and the proteasome activity involved in milk protein synthesis, ubiquitin-lysosome, and ubiquitin-proteasome pathways were detected. Then, the activity of proteasome and lysosome in BMECs were inhibited, and the expression levels of casein-related genes and ribosomal proteins were detected. Finally, iTRAQ comparative proteomics was used to analyze the differentially expressed proteins in BMECs before and after knocking down. After VPS28 knockdown, the CSN1S1, CSN2, CSN3, RPS8, UBC, PSMC3, and PSMC5 genes were significantly up-regulated, while PSMD12 was significantly down-regulated. After proteasome inhibition, the CSN1S1, CSN2, and CSN3 genes were significantly up-regulated, while RPL13 was significantly down-regulated. The expression of casein-related genes was not significant after lysosome inhibition. iTRAQ results showed that there were 129 differentially expressed proteins, the down-regulated proteins were mainly enriched in ribosomes, lysosomes, spliceosomes,and other related pathways, while the up-regulated proteins were mainly enriched in the protein processing in the endoplasmic reticulation, vasopressin-regulated water reabsorption process, RNA transport, and other pathways. The results indicate that VPS28 gene can affect milk protein synthesis through ubiquitination signaling pathways in BMECs.