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草业学报  2014 

当归苯丙氨酸解氨酶基因片段克隆和组织特异性表达分析

DOI: 10.11686/cyxb20140416, PP. 130-137

Keywords: 当归,苯丙氨酸解氨酶基因,基因克隆,荧光定量PCR,基因表达

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Abstract:

苯丙氨酸解氨酶(PAL)在高等植物苯丙烷类代谢中具有重要作用,与植物体内很多重要的次生代谢产物的合成密切相关。当归主要药效成分阿魏酸是苯丙烷类代谢途径中的产物之一。为了探究阿魏酸生物合成和积累的机理,本研究克隆了当归苯丙氨酸解氨酶基因(PAL)部分序列,运用荧光定量PCR方法分析该基因在当归不同组织部位的表达差异。根据已经克隆的植物PAL保守序列设计一对扩增引物,以当归叶片总RNA为模板,采用反转录PCR(RT-PCR)方法扩增出PAL片段并连接到pGEM-TEasy载体上,转化大肠杆菌DH5α,阳性克隆经PCR检测后测序,得到一段706bp的序列(登录号:KJ000258),内含终止密码子TAA,编码232个氨基酸,与GenBank中注册的伞形科植物珊瑚菜等6个物种的PAL核苷酸序列同源性和氨基酸序列同源性都在80%以上;运用SYBRGreen荧光定量PCR方法,以当归肌动蛋白基因Actin为内参基因,检测PAL在当归不同组织中的表达量,结果显示PAL在叶片中表达量最高,其次是茎和根,叶片和茎中表达量分别是根中表达量的7.5和2.7倍。

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