论文部分内容阅读
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。
OBJECTIVE: To clone asarum sieboldii phenylalanine ammonia-lyase (PAL) gene (AsPAL) and analyze its sequence characteristics. Methods: By using the principle of homologous cloning, the total length of c DNA of the gene was obtained by RT-PCR and rapid amplification of cDNA ends (RACE) DNAMAN software and Ex PASy online analysis of its bioinformatics analysis. Results: The full-length AsPAL cDNA was obtained with GenBank accession number KY428932. Sequence analysis showed that the cloned cDNA contained a 2 157 bp complete open reading frame (ORF) encoding 718 amino acids. The predicted protein molecular weight was 78,758 Da. The theoretical isoelectric point was 6.24, there was no transmembrane region, and the nucleotide sequence of GERTASGDLVPLSYVAG containing PAL enzyme contained no signal peptide. Multiple amino acid sequence comparison revealed that the homology of AsPAL with Cinnamon, Grape, Angelica and Apricot reached more than 80%. CONCLUSION: The full-length AsPAL c DNA sequence was obtained and preliminary bioinformatics analysis was carried out, which laid the necessary foundation for further exploring the function of AsPAL and the regulatory mechanism of asarone.