泌乳期差异表达microRNA对奶牛乳腺κ-酪蛋白的调控作用研究

项目名称： 泌乳期差异表达microRNA对奶牛乳腺κ-酪蛋白的调控作用研究

项目编号： No.31501978

项目类型： 青年科学基金项目

立项/批准年度： 2016

项目学科： 畜牧学与草地科学

项目作者： 李文清

作者单位： 河南农业大学

项目金额： 22万元

中文摘要： microRNA可以调节乳成分，κ-酪蛋白是牛奶中的一种重要蛋白质，但对κ-酪蛋白起调控作用的microRNA尚不清楚。在奶牛不同泌乳阶段的乳腺组织中，κ-酪蛋白表达显著不同，推测泌乳期差异表达的microRNA对κ-酪蛋白的表达起一定的调控作用。本项目筛选不同泌乳阶段奶牛乳腺组织差异表达的microRNA，在牛乳腺上皮细胞中分别过表达筛选出的microRNA，检测κ-酪蛋白的蛋白表达变化。将引起κ-酪蛋白显著变化的microRNA进行双荧光报告基因分析，检测出与κ-酪蛋白3’-UTR直接结合的microRNA，其它均为间接起作用的microRNA。将间接起作用的单个或成簇microRNA导入牛乳腺上皮细胞，检测信号调节通路JAK-STAT和mTOR中关键基因的mRNA和蛋白表达变化。综合上述各方面结果，解析对κ-酪蛋白表达起直接和间接调控作用的microRNA，及其主要的调节路径。

中文关键词： κ-酪蛋白；microRNA；奶牛；泌乳期；；牛乳腺上皮细胞

英文摘要： MicroRNA can regulate milk composition. κ-casein(CSN3) is a kind of important proteins in milk. But it is not clear that which microRNAs can regulate kappa casein expression. In bovine mammary tissue of different lactating stages, expression of κ-casein is markedly different. It was speculated that differentially expressed microRNAs in lactating period played a certain role in regulating κ-casein expression. The differentially expressed microRNAs at different stages of lactation were screened in this project. Selected microRNAs were over expressed in bovine mammary epithelial cells(BMEC), respectively. Then, the change of κ-casein protein was detected in BMEC. In order to make the analysis of dual fluorescence reporter gene, microRNAs that significantly affected κ-casein expression were transfected into tool cells. Through this step, microRNAs that directly combined with CSN3 3’-UTR were detected and others were indirect acting microRNAs. In order to detect the expression change of mRNA and protein of key genes in JAK-STAT signal pathway and mTOR signal pathway, indirect acting microRNA singly or in cluster was transfected into BMEC. To synthesize the above-mentioned results, microRNAs which direct and indirect regulated κ-casein expression and its mainly regulatory pathways were revealed.

英文关键词： κ-casein;microRNA;cow;lactating period;bovine mammary epithelial cells