富营养化湖泊蓝藻水华发生对温度响应生物机制研究

Breakout of Cyanobacterial Bloom in eutrophic waters has been one of the global environmental disasters. Under global warming scenario, to reveal the mechanism of Cyanobacterial Bloom formation and breakout responding to temperature will contribute to controlling Cyanobacterial Bloom, keeping lake-ecosystem healthy and restoring water ecosystem functions. The dominants of Cyanobacterial Bloom are selected as the objects of the research. Field study area in Lake Taihu will be set to determine the critical temperature and effective accumulated temperature during Cyanobacteria dormancy, recruitment and rapid proliferation period, the dominating Cyanobacteria population growth, individual growth, substance metabolism, whole genome sequence will be studied. And microcosm temperature-controlling systems will be built for culturing collected Cyanobacteria communities, to study the critical temperature and effective accumulated temperature during Cyanobacteria dormancy, recruitment and rapid proliferation period. Simultaneously the dominating Cyanobacteria population growth, individual growth, substance metabolism, whole genome sequence under accurate temperature control will be determined. While applying metagenome high throughput sequencing to study the physiological mechanism and molecular regulatory pathway of Cyanobacteria Bloom breakout responding to temperature, to screen temperature induced gene, and explain the action mechanism. We aim at assessing the temperature threshold of the Cyanobacteria rapid multiplication, marking the signal metabolic substance for the Cyanobacteria dormancy starting, recruitment and rapid proliferation period, and interpreting the mechanism of Cyanobacterial Bloom formation and breakout responding to temperature. Finally sound proofs will be provided for predicting the tendency of Cyanobacterial Bloom, and supporting policy decision on managements and protection of lake ecosystem.

富营养化湖泊蓝藻水华暴发是全世界共同面临的环境灾害之一。在全球变暖背景下，研究蓝藻水华发生对温度的响应机理对于防治蓝藻水华、维持湖泊生态系统健康、修复水体生态功能意义重大。项目以水华蓝藻为研究对象，在太湖设置野外观测站点，并在室内构建温度控制培养系统。拟监测水华蓝藻发生过程中的水体温度变化，测定野外蓝藻休眠、复苏、快速生长暴发过程中种群增长、个体生长、物质代谢指标和全基因组序列。在室内模拟蓝藻休眠、复苏、快速生长暴发过程，测定各阶段水体温度阈值和有效积温，测定种群增长、个体生长、物质代谢指标、全基因组序列。应用宏基因组高通量测序，分析蓝藻水华暴发过程中对温度响应的生理生态机制和分子生物学调控途径，筛选温度响应基因，阐明其作用机理。确定水华蓝藻休眠、复苏、快速生长期的标识性代谢产物及其浓度，明确蓝藻种群快速增殖的温度响应阈值，揭示蓝藻水华发生对温度响应机理，为蓝藻水华防控和预警提供理论支持。

综合运用分子生物学手段，检测到太湖水体48细菌门，变形菌门、放线菌门和蓝藻菌门是太湖水体最优势菌门，蓝藻在春季已经达到较高的丰度。水华优势种铜绿微囊藻在8 °C时复苏，16 °C进入指数生长期。在20-25 °C之间藻细胞的比生长速率最大，进入最适生长阶段。铜绿微囊藻代谢活动16 °C显著增强，并在在20 °C时达到峰值，光合系统活性、RuBisCO和FBA酶活性、胞外多糖分泌、藻毒素产量均显著提高。在分子水平上，16 °C时铜绿微囊藻细胞中光合基因rbcL、多糖合成基因capD和藻毒素基因mcyB的表达显著上调，产毒基因拷贝数显著增加。其中光合作用通路变化最显著，差异基因数占差异基因总数 的21.33%。PSII过程中的捕光色素蛋白cp43 (编码基因psbC)、cp47蛋白 (编码基因psbB) 和11 kDa蛋白 (编码基因psb27) 表达上调；ATP酶合成相关的基因、细胞色素编码基因petA表达上调。休眠复苏试验证实暖冬有助于微囊藻提前形成水华，夏季水华的规模更大、持续时间更长。能使藻细胞密度分别增加1.77-2.01倍。MCs可以充当信号分子，使藻细胞适应外界环境变化，气候变暖可能导致蓝藻毒性更高。在最适生长温度（25 °C）下，低浓度（0.5和3 mg-N/L）有机氮源（L-丙氨酸）促进铜绿微囊藻生长；高浓度（12 mg-N/L）铵态氮对铜绿微囊藻会产生毒害作用。6 mg-N/L无机氮源（硝酸盐氮）促进微囊藻毒素产生。氮限制促进ntcA、glnB和mcyB基因表达量上调，促进藻毒素合成，氮饥饿时表达量下降；微囊藻毒素合成基因和氮代谢基因协同调控藻毒素的产生。赖氨酸调控铜绿微囊藻的鸟氨酸-氨循环途径从而抑制其生长。2、4、8 mg/L赖氨酸抑制铜绿微囊藻生长、叶绿素a的合成、光合作用系统和藻毒素的合成。铜绿微囊藻上调了9个参与ABC转运蛋白的基因，而氧化磷酸化和电子传递过程相关的编码基因（NADH脱氢酶与细胞色素c氧化酶）表达丰度显著下降，赖氨酸诱导精氨酸生物合成途径中8个基因的表达下调。铜绿微囊藻对有机磷GP和DNA的利用途径存在差异，有机磷的酶解作用受磷浓度的影响。磷浓度达2.0 mg/L时，GP处理组胞内碱性磷酸酶活性显著增高，GP主要被转运至胞内或间质内进行酶解。分子生物学机制表明16°C水温是蓝藻水华形成的关键温度。监测水温有助于水华预警管理。