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白花丹参鲨烯合酶SQS2的克隆与原核表达分析

Keywords: 白花丹参,SQS2,克隆,原核表达

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Abstract:

该研究根据丹参的转录组数据提供的基因片段设计引物,采用逆转录聚合酶链式反应方法,从白花丹参中克隆鲨烯合酶SQS2的全长cDNA序列,命名为SmSQS2,GenBank注册号KM244731。序列长度为1597bp,包含1245bp的开放阅读框,推测编码414个氨基酸,含有115bp的5'UTR和237bp的3'UTR。利用生物信息学软件对获得的序列进行同源性分析,得出白花丹参SQS2的与紫花丹参SQS2的序列无明显差异。原核表达分析结果表明SmSQS2在大肠杆菌中能表达出与预测蛋白大小相当的目标蛋白,对影响蛋白表达的4个因素,即诱导温度、诱导时间、IPTG浓度和诱导时宿主菌的吸光度(A600)进行了优化,得出SmSQS2蛋白表达的最佳条件为:IPTG终浓度0.2mmol·L-1,宿主菌的吸光度(A600)为0.6,温度30℃,诱导时间20h。这为进一步研究白花丹参鲨烯合酶SQS2在丹参萜类和甾醇类生物合成途径中的作用提供了理论依据。

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