CAJ | 학술논문

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。
분병안산해안매（PAL）재고등식물분병완류대사중구유중요작용，여식물체내흔다중요적차생대사산물적합성밀절상관。당귀주요약효성분아위산시분병완류대사도경중적산물지일。위료탐구아위산생물합성화적루적궤리，본연구극륭료당귀분병안산해안매기인（PAL）부분서렬，운용형광정량PCR방법분석해기인재당귀불동조직부위적표체차이。근거이경극륭적식물PAL보수서렬설계일대확증인물，이당귀협편총 RNA 위모판，채용반전록PCR（RT-PCR）방법확증출PAL편단병련접도pGEM-T Easy재체상，전화대장간균DH5α，양성극륭경PCR검측후측서，득도일단706 bp 적서렬（등록호：KJ000258），내함종지밀마자 TAA，편마232개안기산，여GenBank중주책적산형과식물산호채등6개물충적PAL핵감산서렬동원성화안기산서렬동원성도재80％이상；운용SYBR Green형광정량PCR방법，이당귀기동단백기인Actin위내삼기인，검측PAL재당귀불동조직중적표체량，결과현시PAL재협편중표체량최고，기차시경화근，협편화경중표체량분별시근중표체량적7．5화2．7배。
Phenylalanine ammonia-lyase (PAL)plays an important role in the phenylpropanoid metabolic path-way in higher plants and is closely related to the synthesis of major secondary metabolites.Ferulic acid,a ma-jor active ingredient in Angelicasinensis,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 ac-cumulation 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 nucleo-tide 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,re-spectively.