异叶天南星苯丙氨酸解氨酶基因的克隆与蛋白结构分析

Gene cloning and structure characterization of phenylalanine ammonia-lyase from Arisaema heterophyllum

以异叶天南星(Arisaema heterophyllum Blume)为材料,采用逆转录PCR(RT-PCR)法扩增其苯丙氨酸解氨酶(Phenylalanine ammonia-lyase,PAL)基因Ah PAL,获得该基因的开放读码框(ORF),并通过系统的生物信息学软件分析Ah PAL的结构和理化性质。结果显示,Ah PAL的ORF全长为2184 bp,编码727个氨基酸;Ah PAL与郁金香(Tulipa fosteriana W. Irving) PAL的亲缘关系最近,序列相似性达88%。空间结构模型分析结果显示,Ah PAL为同型四聚体,每个单体由3个结构域组成,其中MIO结构域含有PAL酶家族的保守序列和Ala-Ser-Gly三肽活性中心,是Ah PAL酶活性的决定性结构域。利用荧光定量PCR方法检测3个Ah PAL Unigene在根、块茎和叶中的表达情况,发现它们在根中表达量均最高,而在叶和块茎中表达较低。

Phenylalanine ammonia-lyase (PAL) is the first key enzyme in the synthesis of flavonoids and the rate-limiting enzyme of the phenylpropanoid pathway, which is associated with secondary metabolites, growth and development, as well as defense of plants. In this study, the mRNA of Arisaema heterophyllum Blume was used as a template and the full-length open reading frame (ORF) of the phenylalanine ammonia-lyase gene ( AhPAL ) was amplified by real-time polymerase chain reaction (RT-PCR). The structure and physicochemical properties of AhPAL were then analyzed by bioinformatics. Results showed that the ORF of AhPAL was 2,184 bp in length, encoding a protein with 727 amino acids. Furthermore, AhPAL had the closest relationship with PAL of Tulipa fosteriana W. Irving, with a sequence identity of 88%. Structure analysis indicated that AhPAL was a homotypic tetramer, and each monomer consisted of three domains. Among these domains, the MIO domain was highly important for the activity of AhPAL, containing a conserved sequence of the PAL family and Ala-Ser-Gly triad residues. The expression levels of three AhPAL unigenes in the roots, tubers, and leaves were detected by quantitative RT-PCR (qRT-PCR), with higher expression found for all three in the roots compared with that in the leaves and tubers.