本文選題：KLF基因 + 雙熒光素酶基因報告載體�。� 參考：《中國畜牧獸醫(yī)》2017年03期

【摘要】：試驗旨在構(gòu)建鋅指蛋白3(KLF3)基因3′-UTR區(qū)雙熒光素酶基因報告載體及其突變載體,初步分析可能調(diào)控KLF3基因表達的miRNAs。首先通過PCR方法擴增KLF3基因的3′-UTR序列,將其克隆到經(jīng)XhoⅠ、NotⅠ雙酶切的雙熒光素酶報告載體中;運用Targetscan軟件預測可能與KLF3基因3′-UTR相互作用的miRNA;使用脂質(zhì)體2000轉(zhuǎn)染試劑將miRNAs mimics與構(gòu)建好的KLF3基因3′-UTR段雙熒光素酶報告載體或突變載體共轉(zhuǎn)染于常規(guī)培養(yǎng)的293T細胞中,檢測熒光素酶活性。結(jié)果表明,KLF3基因3′-UTR可能是miR-21的作用靶位點;雙熒光報告顯示,miR-21 mimics組(0.6900±0.0144)比突變組(1.000±0.0688)和空白對照組(1.000±0.0159)KLF3基因3′-UTR雙熒光素酶基因報告載體和突變載體的活性降低了31%(P0.01)。本試驗成功構(gòu)建了含有KLF3基因3′-UTR段雙熒光素酶基因報告載體與突變載體,初步證實miR-21對KLF3基因有調(diào)控作用。
[Abstract]:The aim of this study was to construct a double luciferase gene reporter vector and its mutant vector in the 3'-UTR region of zinc finger protein 3s KLF3 gene, and to analyze the miRNAs that might regulate the expression of KLF3 gene. Firstly, the 3'-UTR sequence of KLF3 gene was amplified by PCR and cloned into the double luciferase report vector digested by Xho 鈪，

本文編號：1816371