摘要
目的对连接黑三棱初生代谢及次生代谢途径的关键酶苯丙氨酸解氨酶(phenylalanine ammonia lyase,PAL)进行基因全长的克隆和生物学信息分析。方法以黑三棱总RNA为模板,采用同源克隆法和RACE技术克隆黑三棱PAL基因的cDNA全长,并通过DNAMAN软件和ExPASy在线分析等方法对其生物信息学进行分析。结果获得黑三棱PAL基因全长cDNA,GenBank注册号为KF633470,序列分析表明,所克隆的cDNA全长为2 413 bp,包含一个2 151 bp的开放阅读框架,编码716个氨基酸的蛋白。预测该蛋白的相对分子质量为1.98×105,等电点为4.84,无信号肽,含有PAL酶活性中心序列GTITASGDLVPLSYIAG。结论首次克隆并获得黑三棱PAL基因全长cDNA,为黑三棱药效成分生源合成途径的阐明和改善中药材品质提供科学依据。
Objective To clone the full-length cDNA encoding phenylalanine ammonia lyase （PAL）, which is the key enzyme that links primary metabolism to secondary metabolism in Sparganium stoloniferum and to perform bioinformatic analysis. Methods With the total RNA as template, the full length cDNA of PAL in S. stoloniferum was cloned through homology-based cloning approach and rapid amplification of cDNA ends （RACE） technique. The bioinformatics of the cloning PAL gene was analyzed by DNAMAN software and ExPASy on line. Results The full-length cDNA （2 413 bp） of PAL gene was obtained （GenBank accession number KF633470）, with an open reading frame （ORF） of 2 151 bp and encoding 716 amino acid polypeptides. The relative molecular mass of PAL calculated was 1.98 × 10^5, the isoelectric point was 4.84, and there was no signal peptide in PAL. The protein sequence contained the active center sequence GTITASGDLVPLSYIAG Conclusion The eDNA encoding PAL from S. stoloniferum is cloned and reported for the first time. This work provides a scientific basis for exploring the biosynthetic pathway of the medicinal ingredient and improving its quality in S. stoloniferum.
出处
《中草药》
CAS
CSCD
北大核心
2014年第3期403-409,共7页
Chinese Traditional and Herbal Drugs
基金
国家自然科学基金资助项目(81073002)
江苏省"青蓝工程"(2012)
江苏省中药学优势学科开放课题(2011ZYX1-006)
关键词
黑三棱
苯丙氨酸解氨酶
基因克隆
信号肽
生物信息学分析
Sparganium stoloniferum （Graebn.） Buch.-Ham.ex Juz.
phenylalanine ammonia lyase
gene cloning
signal peptide
bioinformatics analysis
