奶牛STAT5A、PRL、PRLR及GHR基因相关miRNAs的初步研究

发布时间：2018-08-10 07:20

【摘要】：microRNA(miRNA)是一种重要的转录后调控的非编码RNA,很多miRNAs参与了哺乳动物乳腺发育和泌乳等过程。miRNA通过与其靶基因mRNA的3′端非编码区域(3-untranslated region,3′UTR)互补配对,导致沉默复合体降解mRNA或阻碍其翻译。研究表明,STAT5A、PRLR、PRL、GHR基因在调控乳腺泌乳信号通路方面具有重要作用,因此,本研究的目的是探究STAT5A、PRLR、PRL、GHR基因的相关miRNAs,为揭示miRNA调控奶牛的泌乳机理奠定基础。通过targetScan6.0软件预测和分析了STAT5A、PRLR、PRL、GHR基因3’UTR潜在的miRNAs,分别是miRNA2895和miRNA2446(STAT5A)、miR-2393(PRL)、miR-135b(PRLR)、mi R-101(GHR)。通过构建STAT5A、PRLR、PRL、GHR基因的psi-check2双荧光素酶报告载体和pcDNA3.1表达载体,共同转染至HEK293T细胞,并与空白对照组进行对比。40 h后将细胞消化,然后在酶标仪上检测双荧光素酶的活性,结果表明psi-check2-STAT5A3’UTR、psi-check2-PRLR3’UTR、psi-check2-GHR3’UTR的萤火虫荧光素酶活性与海肾荧光素酶活性的比值均有所提高,与对照组相比差异显著(P0.05),表明miR-2895和mi R-2446在载体水平上对STAT5A可能有调控作用;mi R-135b对PRLR、miR-101对GHR基因可能有调控作用;psi-check2-PRL3’UTR的萤火虫荧光素酶活性与海肾荧光素酶的比值变化不显著,差异不显著(P0.05),表明miR-2393对PRL基因的调控作用不显著。
[Abstract]:MicroRNA (miRNA) is an important post-transcriptional non-coding RNA.Many of miRNAs are involved in mammary gland development and lactation in mammals. MiRNAs are paired with the 3-untranslated region 3'noncoding region of its target gene mRNA. The silencing complex leads to the degradation of mRNA or hinders its translation. The results showed that the gene of STAT5A5APRLRGHR plays an important role in the regulation of breast lactation signaling pathway. Therefore, the purpose of this study is to explore the related miRNAs-related genes of STAT5A5A PRLRRGHR gene, so as to lay a foundation for revealing the mechanism of miRNA regulating dairy cows' lactation. The potential miRNAss of the 3'UTR of STAT5Agna PRLRGHR gene are predicted and analyzed by targetScan6.0 software, which are miRNA2895 and miRNA2446 (STAT5A) / miR-2393 (PRL) / miR-135b (PRLR) / mi R-101 (GHR)., respectively. The psi-check2 double luciferase report vector and pcDNA3.1 expression vector of STAT5Agna PRLRGHR gene were constructed and transfected into HEK293T cells. The cells were digested after 40 h compared with the blank control group, and the activity of double luciferase was detected by enzyme labeling instrument. The results showed that the ratio of luciferase activity to luciferase activity in psi-check2-STAT5A3 UTR-psi-check2-PRLR3 UTR-psi-check2-GHR3 UTR was increased, and the ratio of luciferase activity to luciferase activity in sea kidney was increased. Compared with the control group, the difference was significant (P0.05), which indicated that miR-2895 and mi R-2446 might regulate STAT5A at vector level. The ratio of luciferase activity to luciferase of sea kidney luciferase was not significantly different from that of psi-check2-PRL3-UTR, which indicated that mi R-135b could regulate GHR gene. The difference was not significant (P0.05), indicating that the regulation of PRL gene by miR-2393 was not significant.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S823

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