| 摘要: |
| 赫氏颗石藻(Emiliania huxleyi)宿主-病毒(E.huxleyi virus,EhV)的互作过程是影响海洋碳、硫生物地球化学循环及气候变化的重要环节。在共进化过程中,EhV通过基因水平转移从宿主基因组中“劫获”了一组鞘脂从头合成相关酶基因,重构宿主脂代谢网络以支持病毒的特殊需求。目前,对病毒这组相关酶基因的生物学功能尚不十分清楚。以颗石藻病毒EhV-99B1基因组中的甾醇去饱和酶(EhV-SD)和脂肪酸去饱和酶(EhV-FAD)基因为研究对象,构建酿酒酵母重组表达载体pYES2/CT-SD和pYES2/CT-FAD,转化相应的基因缺陷型酵母菌株YMR015C和YGL055W获得重组酵母细胞株,进一步采用UPLC-Q-Exactive-MS非靶向脂质组学技术,比较分析重组酵母和缺陷型酵母细胞脂质的组成和含量变化。结果显示,EhV-SD与EhV-FAD基因在酿酒酵母中成功表达并具有生物学活性。EhV-SD过表达显著改变了重组酵母细胞脂质代谢,含多不饱和酰基链的磷脂酰胆碱(PC,20:4/20:5/20:6)和甘油三酯(TAG,12:3)种类的丰度显著升高;而部分多不饱和脂肪酸(FA,16:2/16:4)和含多不饱和酰基链的磷脂酰甘油(PG,16:2/18:4)丰度则显著降低。EhV-FAD过表达显著增加了重组酵母中单不饱和脂肪酸的生物合成,特别是棕榈油酸(C16:1)和油酸(C18:1)显著积累,而饱和脂肪酸的含量则显著降低,表明EhV-FAD与酵母Δ9FAD具有类似的生物学功能。海洋病毒中脂质代谢相关新基因功能的确定,有助于从代谢角度深入认识海洋病毒-宿主的互作关系,也为相关功能基因在生物技术领域的应用提供科学依据。 |
| 关键词: 海洋颗石藻病毒 甾醇去饱和酶基因SD 脂肪酸去饱和酶基因FAD 酿酒酵母 非靶向脂质组学 |
| DOI：10.11693/hyhz20220400085 |
| 分类号:Q943 |
| 基金项目:国家自然科学基金项目,42076086号,41576166号;福建省自然科学基金项目,2019J01696号。 |
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| FUNCTIONAL ANALYSIS OF STEROL DESATURASE AND FATTY ACID DESATURASE GENES FROM COCCOLITHOPHORES EMILIANIA HUXLEYI VIRUS BASED ON LIPIDOMICS |
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MA Hui, WANG Cai-Feng, LI Gui-Ling, LI Jian, LIU Jing-Wen
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College of Ocean Food and Biological Engineering, Jimei University, Xiamen 361021, China
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| Abstract: |
| The interaction between Emiliania huxleyi (Eh) and its specific lytic virus (EhV) plays a significant role in determining the fate of carbon and sulfur in the ocean and the climate modeling. Viral infection resistance is essentially an arms race of biochemical metabolism. During co-evolution, EhV “hijacked” a series of key enzyme genes involved in de novo biosynthesis pathway of sphingolipids from the host genome through gene horizontal transfer, supporting viral specific needs by rewiring host’ sphingolipid metabolism. At present, the function of these enzymes remains largely unknown. Therefore, the sterol desaturase (EhV-SD) and fatty acid desaturase (EhV-FAD) genes from EhV-99B1 strain were subcloned to construct the yeast recombinant expression vectors pYES2/CT-SD and pYES2/CT-FAD, and were then transformed into those corresponding gene-deficient Saccharomyces cerevisiae strains YMR015C and YGL055W, respectively. Furthermore, UPLC-Q-Exactive-MS untargeted lipidomics technique was used to analyze the changes of total lipids in S. cerevisiae cells. Results show that the EhV-SD and EhV-FAD genes were successfully expressed and biologically active in S. cerevisiae. EhV-SD and EhV-FAD genes overexpression significantly altered the total lipids contents and composition in recombinant S. cerevisiae. The abundance of phosphatidylcholine (PC, 20:4/20:5/20:6) and triglyceride (TAG, 12:3) containing polyunsaturated acyl chains increased significantly, whereas the contents of some polyunsaturated fatty acids (FA, 16:2/16:4) and phosphatidylglycerol (PG, 16:2/18:4) containing polyunsaturated FA decreased significantly. Similarly, EhV-FAD gene overexpression significantly induced the biosynthesis of monounsaturated FA in recombinant S. cerevisiae. In particular, the palmitic oleic acid (C16:1) and oleic acid (C18:1) accumulated remarkably, while the content of saturated fatty acids was declined significantly, suggesting that EhV-FAD and Δ9FAD of S. cerevisiae had similar biological functions. The identification of these new genes from marine virus provided an insight into the interaction between E. huxleyi virus and host, and also expanded their potential application in biotechnology. |
| Key words: Emiliania huxleyi virus sterol desaturase gene SD fatty acid desaturase gene FAD Saccharomyces cerevisiae lipidomics |
