华细辛苯丙氨酸解氨酶基因的克隆与生物信息学分析

目的:克隆华细辛Asarum sieboldii苯丙氨酸解氨酶(phenylalanine ammonia-lyase,PAL)基因(AsPAL),并进行序列特征分析。方法:通过同源克隆的原理,利用RT-PCR和c DNA末端快速扩增(RACE)相结合的方法,以华细辛叶片总RNA为模板,获得了该基因的c DNA全长,并通过DNAMAN软件和Ex PASy在线分析等对其进行了生物信息学分析。结果:获得AsPAL全长cDNA,Genbank登录号为KY428932,序列分析表明,所克隆的c DNA含有1个2 157 bp的完整开放阅读框,编码718个氨基酸,预测蛋白质相对分子质量为78 758 Da,理论等电点为6.24,没有跨膜区,含有PAL酶活性中心序列GTITASGDLVPLSYVAG,不含信号肽;氨基酸序列多重比较发现,AsPAL与土肉桂、葡萄、当归和杏的同源性达到80%以上。结论:获得了AsPAL c DNA全长序列,对其进行初步生物信息学分析,为进一步探究AsPAL的功能和华细辛甲基丁香酚生物合成的调控机制奠定了必要的基础。

Cloning and Bioinformatics Analysis of Phenylalanine Ammonia-lyase Gene in Asarum sieboldii

Objective:This study is aimed to clone the key enzyme phenylalanine ammonia-lyase (PAL) from Asarum sieboldii and analyze the sequence characteristics by bioinformatics tools. Method:AsPAL gene was cloned by RT-PCR and RACE strategy by using homologous cloning principle, with the RNA extracted from leaves as the template. The bioinformatics of this gene and its corresponding protein was then analyzed by DNAMAN software and ExPASy online analysis tool. Result:The ORF of AsPAL consisted of 2 157 bp (GenBank accession number:KY428932),encoding 718 amino acid polypeptides. The molecular weight and theoretical isoelectric point of the deduced AsPAL protein were 78 758 Da and 6.24 respectively. Bioinformatics prediction and analysis indicated that AsPAL protein contained the active center sequence GTITASGDLVPLSYIAG, without transmembrane region and signal peptides. Multiple comparisons of amino acid sequences showed that AsPAL had more than 80% homology with PAL of Cinnamomum osmophloeum, Vitis vinifera, Angelica sinensis and Prunus armeniaca. Conclusion:This study cloned and analyzed PAL from A. sieboldii. The preliminary bioinformatic analysis might be used to establish an experimental basis for exploring PAL gene function and further regulation of methyleugenol biosynthesis from A. sieboladii.