球等鞭金藻（Isochrysis galbana）岩藻黄素合成途径关键基因挖掘及其对光质的响应机制研究

Fucoxanthin is a major carotenoid that accounts for approximately 10% of total natural carotenoid production. Due to its considerable potential applications in human health, the demand for naturally synthesized fucoxanthin in the global market has increased dramatically. The genes involved in biosynthetic pathway of fucoxanthin are not yet fully understood and this hinders the development of low-cost biotechnological approaches for the production of fucoxanthin. No studies have been done yet to uncover genes involved in biosynthesis pathway of fucoxanthin via integrative genomics approaches. Isochrysis galbana is considered as one of the most promising source of fucoxanthin for commercial production, and its complete genome sequence has been fully assembled by our lab for the first time. Our preliminary studies found that green light-emitting diode light could significantly increase fucoxanthin production in Isochrysis galbana, but the essential genes involved in fucoxanthin biosynthesis remain unclear. In this proposal, the available of the Isochrysis galbana genome, combined with the analyses of comparative genomics and single-cell transcriptome will reveal insights into the regulatory gene networks related to the fucoxanthin biosynthesis pathway in response to light regimes in Isochrysis galbana. We will show genes that are critical for the production of fucoxanthin and identify candidate gene that could be the unknown genes in fucoxanthin biosynthesis pathway. Finally, CRISPR/Cas9-based targeted genome editing will be carried out in model algae Phaeodactylum tricornutum to validate candidate genes. The addition of genomics to the experimental toolkit for Isochrysis galbana will make it an attractive alga not only for fundamental studies of its biology but also enhance prospects for improving commercial production of fucoxanthin.

岩藻黄素是自然界中最丰富的脂溶性类胡萝卜素，在海洋药物、新型功能食品和保健品开发应用上有巨大的商业前景。岩藻黄素合成途径有许多酶基因未知，这限制了利用生物技术手段改良藻种提高其生产效率。目前尚未有报道从多组学水平全面揭示参与岩藻黄素合成途径的关键基因。球等鞭金藻是最具潜力的岩藻黄素生产制备原料藻之一，我们已破译球等鞭金藻全基因组信息。前期我们发现光质会影响球等鞭金藻单细胞岩藻黄素的积累，然而参与岩藻黄素合成途径的关键基因及其对光质的响应机制尚无研究。本项目拟在前期基础上，通过比较基因组手段和单细胞转录组测序技术从基因组结构水平和单细胞转录水平揭示参与岩藻黄素合成途径的关键基因及其响应不同光质的表达调控网络，预测参与岩藻黄素合成途径的未知酶基因。最后基于CRISPR/Cas9技术在三角褐指藻中验证提高岩藻黄素产量的候选基因，为日后构建高产岩藻黄素球等鞭金藻工程藻株奠定实验和理论基础。

岩藻黄素是自然界中最丰富的脂溶性类胡萝卜素，在海洋药物、新型功能食品和保健品开发应用上有巨大的商业前景。等鞭金藻门的球等鞭金藻含有丰富的岩藻黄素和多不饱和脂肪酸，因此近年来备受关注。岩藻黄素合成途径有许多酶基因未知，这限制了利用生物技术手段改良藻种提高其生产效率。目前尚未有报道从多组学水平全面揭示参与岩藻黄素合成途径的关键基因。在本研究中，我们结合二代和三代测序技术首次组装和注释了完整的球等鞭金藻核基因组、叶绿体和线粒体基因组。该球等鞭金藻核质基因组全套数据有助于深入洞悉基因组结构、功能、近缘藻种间物种演化过程。进化分析揭示了等鞭藻门的单系统起源，球等鞭金藻大概在133百万年前与赫氏圆石藻分开。基因家族分析表明，与脂质代谢相关的基因表现出明显的扩张。通过保守结构域预测在球等鞭金藻中鉴定到了39个羟化酶基因，其中一个羟化酶基因（IZ011859）具有催化疏水底物羟基化的功能，与硅甲藻黄素Ddx、岩藻黄素Fx和新叶黄素Nx前人预测的反应过程相似，很可能是Fx合成过程的未知酶，需要进一步的体外实验功能验证。绿光培养7天的球等鞭金藻单细胞岩藻黄素含量显著高于白光培养(p<0.05)。转录组分析表明，PDS, Z-ISO和VDE基因发生了上调，CrtL-b基因发生了下调。基因共表达调控网络分析表明了IgMYB98转录因子（IZ007092）、RPL核糖体蛋白（IZ005089）和ILVD二羟酸脱水酶（IZ008652）是该互作网络的最核心基因，尤其是IgMYB98转录因子。IgMYB98为R2R3MYB转录因子，参与植物次生代谢、类黄酮与类胡萝卜素的生物合成等过程，可能是球等鞭金藻Fx合成的关键调控因子。多组学联合分析结果表明，绿光诱导下能促进β-胡萝卜素、花青素、玉米黄质和岩藻黄素在球等鞭金藻的积累，这可能与IgMYB98、IgZDS、IgPDS、IgLHCX2、IgZEP、IgLCYb和IgNSY的表达显著相关。研究成果解析了球等鞭金藻岩藻黄素生物合成的调控网络，对进一步构建高产岩藻黄素球等鞭金藻藻种、生物饵料开发及医药保健产品的应用将有重要的科学意义。