典型藻华甲藻中与维生素B12依赖性营养相关基因的研究

The fact that more than 70% events of marine harmful algal blooms (HABs) are caused by dinoflagellates notifies the vital importance of this group of microalgae in marine ecosystems. Among all well-investigated regulating factors, the trace organic nutrient vitamin B12 has been well documented from both the field surveys and laboratory experiments as a particularly important nutritional factor regulating the dynamics of marine phytoplankton, dinoflagellates in particular, and more than 90% of dinoflagellates investigated are auxotrophs of vitamin B12, a number greater than nearly all others. Therefore, vitamin B12 may play a vital regulating role in the occurrences of HABs. Study from the molecular level on genes pertinent to this trophic mode in dinoflagellates, however, has been almost absent. Based on our work during the past 10 years, particularly those on transcriptomes of the cosmopolitan HAB species Scrippsiella trochoidea and a metatranscriptomic study on a field sample of marine sediment conducted most recently, we hypothesize that "in those dinoflagellates that are auxotrophs for vitamin B12, vitamin B12 is not only involved in the methionine biosynthesis as a cofactor of the gene MetH, but may be also a cofactor required in other key biochemical pathways, which together make most of dinoflagellates auxotrophs of vitamin B12". To test this hypothesis, the proposed project will investigate the clonal cultures of S. trochoidea and other dinoflagellates maintained in the laboratory via an approach combining technologies of physiology, -omics and biochemistry in terms of whether or not they are auxotrophs of vitamin B12, the presence of the genes metH, metE and cobW, the transcriptional responses of these genes to the level and presence of vitamin B12 in the medium, and possible non-methionine biosynthesis biochemical pathways requiring B12 as cofactors. We believe that implementation of this project will greatly elucidate the roles played by vitamin B12 in regulating survival and growth and consequently in regulating bloom dynamics of dinoflagellates. We also anticipate the outputs of this research will provide insights for forecasting, prevention, and control of dinoflagellate HABs.

海洋有害藻华约70%由甲藻引起。在调控藻华的营养因素中，维生素B12已被大量野外和室内研究证明是浮游植物特别是甲藻的重要营养因子，且>90%的甲藻都是B12营养依赖型，因而可以在藻华过程中起关键的调控作用。但对甲藻这一营养方式涉及的相关基因的研究还几乎空白。申请人根据前期工作和近来对锥状斯氏藻转录组和野外样品宏转录组的研究结果，提出维生素B12不仅作为营养依赖型甲藻的MetH辅酶参与甲硫氨酸的合成，还可能是其它重要生化过程的必需因子的科学假设，并拟以锥状斯氏藻和其它甲藻为对象研究它们是否为B12的营养依赖型和这种依赖与metH、metE、cobW等基因的存在性及表达水平的响应关系；是否存在甲硫氨酸合成途径之外必须B12参与的重要生化过程。本研究将从生理、生化和基因表达水平上深度揭示甲藻藻华爆发与一种可能的促发因子的关系，获得对藻华生态学更丰富的机理性认识，最终为藻华预测和防控提供科学依据。

甲藻是引起有害藻华最主要的类群，但长期以来对甲藻营养生态学的研究集中于大量营养盐如氮和磷等，很少关注微量营养如维生素。申请人前期工作证明90%以上的甲藻都是维生素B12的营养依赖型，故探索甲藻对维生素B12依赖性营养的遗传学基础对甲藻生物学及其有害藻华生态学具有重要意义。已有研究证明某些藻类对维生素B12的依赖性营养主要取决于二种甲硫氨酸合成酶基因的有无：以B12为辅酶的metH基因和不以B12为辅酶的metE基因。本研究使用cDNA末端快速扩增技术获得14种甲藻的相关基因全长，并结合实时荧光定量PCR（qRT-PCR）技术比较其中10种甲藻的metH与metE基因在不同维生素B12浓度和添加方式下的表达模式，对其基因功能进行判断。通过整理分析已公开的甲藻转录组和基因组信息，以获得的14种甲藻metH与metE的38个cDNA全长为基础，通过比对数据库共获得61种甲藻的metH与metE基因信息，对其进行系统进化分析。所有结果证明，绝大多数甲藻都由于其metE基因只有C端而无N端结构域从而不能行使甲硫氨酸合成的功能，或者完全缺失metE基因，从而成为B12的营养依赖型。本研究明确地解释了甲藻维生素B12依赖性营养在基因水平的原因。CobW（钴胺素合成酶W）是原核生物维生素B12合成途径中的关键基因，本实验室在甲藻的转录组学研究和宏转录组学研究中，发现了若干含有cobW 结构域的基因（CBWD）。但由于90%以上甲藻都被证明为维生素B12营养依赖型，因此推测甲藻中CBWD蛋白（DinoCBWD）行使其它功能且与维生素B12的合成无关。本研究从四种甲藻的基因组和转录组中找到了90个CBWD基因，并且对有害甲藻剧毒卡尔藻和锥状斯式藻中5个CBWD基因的全长进行了克隆。qRT-PCR结果及系统进化分析表明，DinoCBWD与维生素B12的合成无关。基于系统进化分析结果的提示，通过检测剧毒卡尔藻cobW基因在Zn2+、Fe3+限制和恢复过程中表达规律并结合藻类生长规律，推测DinoCBWD可能参与锌离子内稳态维持和依赖于铁的腈水合酶激活。本项目结果明确解释了甲藻维生素B12依赖性营养的分子机理，并为CBWD蛋白乃至COG0523蛋白家族的功能研究提供了重要基础。