芹菜中类胡萝卜素合成相关番茄红素ε–环化酶基因AgLCYE的克隆与表达分析

番茄红素ε–环化酶（lycopene epsilon cyclase，LCYE）是类胡萝卜素合成途径中的关键酶。从‘津南实芹’中克隆获得番茄红素ε–环化酶基因AgLCYE。序列分析结果显示，AgLCYE包含1个1 590 bp的开放阅读框，编码529个氨基酸。氨基酸序列多重比对表明，芹菜和其他物种的LCYE同源性为77.68%，AgLCYE和同属伞形科的胡萝卜LCYE氨基酸序列同源性高达98.07%。AgLCYE蛋白与胡萝卜LCYE蛋白进化关系最近。AgLCYE蛋白属于亲水性蛋白，预测的蛋白质三级结构中有9个α螺旋和18个β折叠。无序化分析结果表明，AgLCYE编码的氨基酸序列有4个无序化区域。用UPLC方法对30、45和60 d龄芹菜叶中叶黄素和α–胡萝卜素的含量进行测定，30 d时叶黄素含量最高，45 d时最低，45和60 d时无显著性差异，叶片中的叶黄素含量均高于叶柄。芹菜叶片和叶柄中均没有检测到α–胡萝卜素的存在。荧光定量表达分析结果显示，随着生长期的增加，叶片中AgLCYE的相对表达量逐渐升高；叶柄中逐渐降低。

Cloning and Expression Profiles Analysis of Carotenoid Biosynthesis Related Gene AgLCYE from Celery

Celery（Apium graveolens L.）is an important vegetable in Apiaceae family，with excellent source of carotenoid. Lycopene epsilon cyclase is a key enzyme in the pathway of carotenoid biosynthesis. Here，the AgLCYE gene encoding lycopene epsilon cyclase was cloned from A. graveolens‘Jinnan Shiqin’. The sequences of AgLCYE gene contained an open reading frame with the length of 1 590 bp，which encoded 529 amino acids. Multiple alignments of the AgLCYE amino acid sequences from celery and other different plants showed a homology of 77.68%. The homology of LCYE was up to 98.07%  between celery and carrot. Phylogenetic tree analysis showed that the LCYE proteins were conserved. The physicochemical property analysis showed that AgLCYE belonged to hydrophilic protein. There were 9 alpha helices and 18 beta folds in the predicted tertiary structure of the protein. The results of disordered analysis showed that the amino acid sequence of AgLCYE protein had four disorder regions. The contents of lutein and α-carotene of celery leaves in different growth stages（30，45，and 60 d）were determined by UPLC. The peak of lutein content was detected at 30 d. The lowest content of lutein was observed in 45 d. There was no significant difference between 45 d and 60 d. The lutein content in leaf blade was higher than that in petiole of celery. The presences of α-carotene were not detected in leaf blade and petiole of celery，respectively. The expression levels of AgLCYE gene in celery were detected by quantitative real-time PCR. In leaf blade，the relative expression levels of AgLCYE gene increased gradually over the process of celery growth and development. On the contrary，the relative expression levels of AgLCYE gene decreased in petiole.