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

摘要/Abstract

摘要： 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-T Easy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706 bp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SYBR Green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。

Abstract: Phenylalanine ammonia-lyase (PAL) plays an important role in the phenylpropanoid metabolic pathway in higher plants and is closely related to the synthesis of major secondary metabolites. Ferulic acid,a major active ingredient in Angelica sinensis,is one of the intermediate products in phenylpropanoid pathway. In this paper,the cDNA fragment of phenylalanine ammonia-lyase gene (PAL) was cloned and its tissue-specific expression by fluorescence quantitative PCR was analyzed for exploring the mechanism of biosynthesis and accumulation of ferulic acid in Angelica sinensis. A pair of amplification primers was designed according to the conservative sequences of the cloned PAL. Extracting the total RNA from the leaf tissue of A. sinensis as a template,PAL fragments were obtained by reverse transcription polymerase chain reaction (RT-PCR) and connected to pGEM-T Easy vector then transformed into Escherichia coli DH5α. The positive clone identified by PCR was sequenced and 706 bp sequence (accession number: KJ000258) including termination codon TAA was obtained,which encoding 232 amino acids. The sequence identity analysis suggested that both the nucleotide sequence and its corresponding amino acid sequence shared over 80% of homology with GenBank PALs from Glehnia littoralis and five other higher plant species. Taking actin of A. sinensis as a reference gene,SYBR green fluorescence quantitative RT-PCR was used to detect the relative expression levels of PAL in leaf,stem and root of A. sinensis. The findings were PAL expressed at the highest level in leaf,followed by stem and root,and the relative expression levels in leaf and stem was 7.5 and 2.7 times relative to root,respectively.

中图分类号:

S567.23⁺9

雒军，王引权，温随超，李静，张金林，夏琦. 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[J]. 草业学报, 2014, 23(4): 130-137.

LUO Jun,WANG Yin-quan,WEN Sui-chao,LI Jing,ZHANG Jin-lin,XIA Qi. Cloning and tissue-specific expression analysis of phenylalanine ammonia-lyase gene fragment in Angelica sinensis[J]. Acta Prataculturae Sinica, 2014, 23(4): 130-137.

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