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花生AhRRF基因的克隆与原核表达

DOI: 10.11869/j.issn.100-8551.2015.10.1867, PP. 1867-1875

Keywords: 花生,核糖体循环因子,克隆,序列分析,原核表达

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Abstract:

核糖体循环因子在蛋白质合成体系中发挥着重要作用,利用电子克隆和RT-PCR技术,从花生中克隆到1个核糖体循环因子基因,命名为AhRRF(Genbank登录号KF621303).序列分析表明,该基因开放阅读框为837bp,编码278个氨基酸,推测编码蛋白质的分子量为30.94kD,等电点为9.63,无信号肽序列,AhRRF编码蛋白包含4个α-螺旋、6个β-折叠和一些无规则卷曲结构.系统发生分析结果表明,AhRRF编码蛋白与蒺藜苜蓿、鹰嘴豆、大豆和菜豆的核糖体循环因子具有较高的同源性.实时荧光定量PCR结果显示,AhRRF基因在花生的叶、花、根和幼果中均有表达,但叶中表达量最高,花中最低;UV-B辐射处理1h时叶中的表达量显著上升,为对照的3.43倍,之后开始下降,24h时显著低于对照.将构建的重组质粒pET28a-AhRRF转化大肠杆菌BL21(DE3),以IPTG诱导重组蛋白RRF的表达,SDS-PAGE检测表明,重组蛋白的分子量大小约为30kD,其大小与推测的大小一致.本研究为进一步探索UV-B辐射对花生分子机理的影响奠定基础.

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