盐藻番茄红素β-环化酶全长cDNA的克隆及结构分析

Cloning and Structure Analysis of the Full Length cDNA of Lycopene β-Cyclase from Dunaliella salina

杜氏盐藻是迄今为止工业化最成功的藻类之一,主要特征是能累积大量的β-胡萝卜素,是β-胡萝卜素最好的天然资源。番茄红素β-环化酶是催化番茄红素合成β-胡萝卜素的关键酶。为阐明该酶在盐藻中合成β-胡萝卜素的作用和调控过程,本研究采用RT-PCR结合半巢式PCR方法,首先分离一段包含辅因子黄素腺嘌呤二核苷酸(FAD)结合位点的保守序列,再利用3'RACE、5'RACE方法结合降落PCR,获得盐藻番茄红素β-环化酶(LycB)cDNA的两个末端。完整的cDNA全长2475bp,ORF长1824bp,编码607个氨基酸,其编码蛋白的分子量为67kD,等电点pI为8.45。经分析,盐藻番茄红素β-环化酶在系统进化上介于蓝藻和植物之间,但更靠近高等植物,故将此酶标记为LycB。

The unicellular green alga Dunaliella salina has been successfully used for natural production of β-carotene. Lycopene β-cyclase is the key enzyme for converting lycopene into β-carotene. To disclose the function and regulation process oflycopene β-cyclase in the biosynthesis pathway of β-carotene in D salina, RT-PCR combined with semi-nest PCR methods are firstly employed to isolate a conservative cDNA fragment. Followed by 5＇ RACE, 3＇RACE and Touch-down PCR methods, two ends of the full length cDNA are obtained. Results show that the cDNA is 2475 bp in length including an ORF of 1824 bp, encoding 607 amino acid residues with a molecular weight of 67kD and an isoelectric point of 8.45. Through a variety of analysis, D. salina is deduced phylogeneticly to be closer to higher plant LycB, rather than those of cyanobacteria, and the enzyme was noted as LycB.