杜氏盐藻LYCB基因克隆及在盐胁迫下的表达分析

LYCBgene cloning and expression analysis in Dunaliella salina under salt stress

[目的]为探究杜氏盐藻(Dunaliella Salina)富集β-胡萝卜素的分子机理。[方法]本文选用从运城盐湖分离的杜氏盐藻株系Ds-YC011为试材,克隆编码番茄红素-β-环化酶(lycopene b-cyclase,LYCB)的基因(DsLYCB)并分析其功能。[结果]DsLYCB基因cDNA全长1 910bp,开放阅读框(ORF)长1 769bp,编码584个氨基酸。生物信息学分析显示,DsLYCB为不稳定亲水性蛋白,无跨膜结构,定位于叶绿体。蛋白二级结构中α-螺旋结构占39.9%,杜氏盐藻DsLYCB蛋白与雨生红球藻亲缘关系最近。qRT-PCR分析表明,在高盐(3mol·L^(-1))胁迫下,DsLYCB基因表达量显著上升,胁迫4h时表达量达峰值,为正常对照(1.5mol·L^(-1))条件下的2倍。高盐(3mol·L^(-1))胁迫下,DsLYCB基因上调表达模式与藻细胞类β-胡萝卜素积累增加相一致,预示着DsLYCB在杜氏盐藻类β-胡萝卜素合成积累中起重要作用。[结论]这为解析微藻类胡萝卜素合成调控机制及遗传修饰提供了科学依据。

[Objective]Present study was proposed in order to understand the molecular mechanism underlying the bio synthesis and accumulation of ~ carotene in Dunaliella salina. [Methods] A D. salina strain Ds YO011 isolated from Yuncheng Salt Lake was selected as genotype to clone lycopene β cyclase （LYCB） gene （DsLYCB） and used as material to analyze its function. [Results]The full length cDNA sequence of DsLYCB was determined as 1 910 bp with 1 769 bp ORF （open reading frame） encoding 584 amino acids. The bioinformatics analysis showed that DsLYCB located in chloroplast was an unstable hydrophilic protein with no transmembrane structure, and a helix secondary structure was predominated （36.3G）. Phylogenetic analysis indicated that DsLYCB was closely related to LYCB protein in Haema tococcus pluvialis. The results of qRT PCR demonstrated that DsLYCB expression was increased significantly under high salt stress （3m01.L-1 NaC1） with its peak value shown at 4 h post treatment, which was two times higher than that of the normal growth condition （1.5 mol-L 1 NaC1）. Under the high salt stress, the upregulated expression pat tern of DsLYCB gene was in accordance with the accumulation pattern of carotenoids in microalgal cells which indicated that DsLYCB may play an important role in the biosynthesis and accumulation of carotenoids in DunaZieHa saZina. [Conclusion~ The present data provided a solid evidence for better understanding of carotenoid biosynthesis regulation mechanism and genetic modification on this pathway.