目的 對連接黑三棱初生代謝及次生代謝途徑的關(guān)鍵酶苯丙氨酸解氨酶（Phenylalanine ammonia lyase，PAL），進行基因全長的克隆和生物學(xué)信息分析。方法 以黑三棱總RNA為模板，采用同源克隆法和RACE技術(shù)克隆黑三棱PAL基因的cDNA全長，并通過DNAMAN軟件和ExPASy在線分析等方法對其生物信息學(xué)進行分析。結(jié)果 獲得黑三棱PAL基因全長cDNA，GenBank注冊號為KF633470，序列分析表明，所克隆的cDNA全長為2 413 bp，包含一個2 151 bp的開放閱讀框架，編碼716個氨基酸的蛋白。預(yù)測該蛋白的相對分子質(zhì)量為1.98×105，等電點為4.84，無信號肽，含有PAL酶活性中心序列GTITASGDLVPLSYIAG。結(jié)論 首次克隆并獲得黑三棱PAL基因全長cDNA，為黑三棱藥效成分生源合成途徑的闡明和改善中藥材品質(zhì)提供科學(xué)依據(jù)。

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 and ExPASy. 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 × 105, 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 cDNA 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.

國家自然科學(xué)基金（81073002）；江蘇省“青藍工程”（2012）；江蘇省中藥學(xué)優(yōu)勢學(xué)科開放課題（2011ZYX1-006）