IDENTIFICATION AND FUNCTIONAL STUDY OF ASTAXANTHIN ACYLTRANSFERASE IN HAEMATOCOCCUS PLUVIALIS
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摘要: 为研究雨生红球藻(Haematococcus pluvialis)的甘油二酯酰基转移酶(Diacylglycerol acyltransferase, DGAT)是否具有催化虾青素酰基化的功能, 首先通过雨生红球藻的cDNA库克隆得到了一个II型DGAT编码区全长序列(DGTT2)。在甘油三酯(Triacylglycerol, TAG)合成缺陷型酵母Saccharomyces cerevisiae H1246中过表达DGTT2基因发现HpDGTT2不能回补H1246的表型, 即不具有典型的DGAT功能。利用分离得到的雨生红球藻的内质网成功地建立了一个体外的虾青素酰基转移酶酶活测定体系, 添加含有重组HpDGTT2的酵母细胞的微粒体后虾青素酯的含量显著高于对照, 初步表明HpDGTT2具有催化雨生红球藻中虾青素酰基化功能。以上结果为进一步探索雨生红球藻中DGTT2的功能及深入理解虾青素合成在代谢水平的调控奠定了基础。Abstract: This study aimed to unravel whether DGATs are involved in the biological processes of astaxanthin acylation in H. pluvialis. A type II DGAT-encoding gene, DGTT2, was identified in H. pluvialis NIES-144 through RNA-seq and cloned from the cDNA library. Overexpression of HpDGTT2 in the TAG-deficient Saccharomyces cerevisiae strain H1246 failed to rescue the TAG-deficient phenotype, indicating that HpDGTT2 cannot utilize diacylglycerol for TAG biosynthesis. Meanwhile, an in vitro enzymatic assay system for measuring astaxanthin:acyl-CoA acyltransferase activity was established successfully by using H. pluvialis endoplasmic reticulum membranes as crude enzymes. When the S. cerevisiae microsomes containing recombinant HpDGTT2 were incorporated into the system, the concentrations of a number of astaxanthin esters increased significantly compared with the control, which indicated that HpDGTT2 possesses astaxanthin acyltransferase activity. This study will provide a basis for further studies on the function and metabolic regulation of HpDGTT2 in astaxanthin biosynthesis.
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图 1 推译的雨生红球藻DGTT2蛋白质氨基酸序列及结构域示意图
其中灰色阴影部分为预测的3个跨膜结构域, 方框内为DGAT2/MGAT功能位点
Figure 1. The putative amino acid sequence and e putative conserved domains of Haematococcus pluvialis DGTT2 protein
The sequences in the shaded boxes and in the red boxes are 3 transmembrane domains and putative conserved domains predicted in Haematococcus pluvialis DGTT2, respectively
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图 2 邻接法构建HpDGTT2氨基酸系统发育进化树(Bootstrap=1000)
Figure 2. Phylogenetic tree constructed from the HpDGTT2 amino acid with the neighbor-joining method (Bootstrap=1000)
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图 3 HpDGTT2和CrDGTT2在S. cerevisiae H1246中的表达及功能回补分析
A. HpDGTT2酵母微粒体蛋白免疫印迹确认; B. HpDGTT2过表达酵母转化子无甘油三酯合成; C. CrDGTT2过表达酵母转化子中检测到甘油三酯
Figure 3. Expression of HpDGTT2 and CrDGTT2 in S. cerevisiae H1246 and functional complementation analysis
A. Western-blotting analysis of S. cerevisiae H1246 harboring pYES2/CT-DGTT2 induction for 8h, 12h, 24h, 48h and 72h, respectively; B. No lipid body observed in HpDGTT2 S. cerevisiae H1246 transformants; C. Lipid body formed in CrDGTT2 S. cerevisiae H1246 transformants
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图 4 虾青素酰基化体外酶活分析体系建立
A. 为雨生红球藻分离的ER的色素组成色谱图。其中箭头和括号在横坐标的垂直投影分别对应游离虾青素和虾青素酯的保留时间; B. 为雨生红球藻高光缺氮6h、9h、12h、15h和24h时分离的内质网的蛋白免疫印迹结果, 其中BIP为细胞器特异性蛋白标记物; C. 为将雨生红球藻内质网作为虾青素酰基化的粗酶时, 反应体系中实验组和对照组的虾青素酯含量的变化
Figure 4. Establishment of astaxanthin acylation in an in vitro enzymatic assay system
A. Chromatogram of separated ER extracted from Haematococcus pluvialis; B. Western-blotting analysis of separated ER in H. pluvialis under high light stress for 6h, 9h, 12h, 15h and 24h, respectively. BIP was used as ER-specific marker; C. Astaxanthin ester (s) content variation between control group and experimental group when seperated ER was used as crude transacylation enzyme in vitro assay
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