南亚热带水库拟柱孢藻的产毒能力对其生态优势的影响与机制研究

Cylindrospermopsis raciborskii originated from tropical regions was currently widely distributed and even formed blooms throughout most of reservoirs in southern subtropical China. The species has become a predominant toxic cyanobacteria in this region which jeopardize drinking water safety, but the mechanisms for bloom formation is still obscure. Previous studies have shown that toxin production might improve the competitive edge of C. raciborskii and play an important role in its dispersal around the world. C. raciborskii from southern China is able to produce cylindrospermopsin (CYN), does the toxic potential contribute to its bloom occurrences? In this study, in order to evaluate the influence of toxic potential on C. raciborskii’s dominance, quantitative PCR ( toxic cell abundance) and enzyme linked immunosorbent assay (CYN concentration) will be performed to measure toxic potential of C. raciborskii, in the meantime the seasonal dynamics of phytoplankton and zooplankton will be examined with microscopic observation. By competitive experiments and comparative genomics, we will compare the competitive differences of toxic and non-toxic Cylindrospermopsis strains and try to understand the geneticfeatures of competitive edge of toxic Cylindrospermopsis. To clarify the underlying role of CYN in defense against planktivores, we will compare the fitness and growth of Daphnia fed on toxic or non-toxic C. raciborskii, and concurrently analyze extracellular CYN concentration and expression level of CYN biosynthetic genes. After adding CYN to sympatric phytoplankton cultures, the allelopathic role of CYN will be domonstrated by the activity of alkaline phosphatase, which will test whether CYN is benefitial for the Pi availability of Cylindrospermopsis. Our work will contribute to clarifying the presence of C.raciborskii in the reservoirs of southern subtropical China,and also to interpreting CYN synthesis and its ecological role.

起源于热带的拟柱孢藻已在我国南亚热带广泛发生并形成水华，成为危害该地区供水安全的首要产毒蓝藻，亟需阐明其优势形成机制。产毒被认为能提高拟柱孢藻竞争力，是驱动其在全球成功拓展的重要因子。南亚热带水库拟柱孢藻可产拟柱孢藻毒素（CYN），这样的产毒能力是否促进其种群优势的形成？本项目用qPCR（产毒细胞数）和ELISA（CYN浓度）测定水库拟柱孢藻的产毒能力，分析浮游植物组成变化，评估产毒能力高低对拟柱孢藻水华生消的影响；室内开展竞争实验和全基因组比较，了解产毒/非产毒拟柱孢藻竞争力差异，解析产毒藻株竞争优势的遗传基础；喂养实验中比较产毒/非产毒藻对Daphnia的毒性，结合CYN浓度和合成酶基因的转录，认识产毒在捕食防御中的作用；观测CYN产生对共存浮游植物碱性磷酸酶的诱导，确认CYN有利于拟柱孢藻获得无机磷的真实性。本研究有助于了解南亚热带拟柱孢藻的发生规律，阐明CYN在其中的作用和生态功能

拟柱孢藻已在南亚热带水库广泛发生并占据优势，产毒是否在其中起作用还有待阐明。本项目主要从CYN的化感作用和诱导防御2方面证实产毒在该藻优势形成中的作用，并通过比较基因组揭示拟柱孢藻灵活的磷利用潜能，有利该藻在磷波动下获得优势。具体结论如下：1..对广东省20座水库的历史资料和本次调查的115座水库数据分析确认拟柱孢藻在90%以上水库发生，而产毒基因型在种群中不占优势，竞争实验表明营养充足条件可使混合种群中产毒株的优势度和CYN产量显著增加，因此营养的可获得性在调节产毒和非产毒拟柱孢藻的相对优势中发挥重要作用， CYN可能对产毒种群的生态优势形成具重要意义。对野外数据的进一步分析发现磷不足水库中CYN的存在更有利于CYN产生者获得优势，室内实验表明CYN在磷不足下通过激活PIK3/Akt-cGMP/PKG信号通路，显著诱导胞内氮分配和蛋白输出系统，从而促进微囊藻、小球藻和栅藻等浮游植物分泌更多的碱性磷酸酶(ALP)，由此同域拟柱孢藻可获得ALP释放的无机磷来支持生长。2..与非产毒株相比，产毒拟柱孢藻不但能显著抑制大型溞的生长，还使其停止产卵和繁殖，因此可更显著降低浮游动物摄食，有利于拟柱孢藻种群生长；更为重要是，拟柱孢藻对浮游动物牧食具诱导防御策略，当捕食压力达到一定强度时，拟柱孢藻显著增长藻丝长度，迅速增加CYN产量，从而增强对大型溞摄食的机械干扰和毒性作用，显著减少浮游动物的摄食，维持自身种群优势。3..完成中国产毒藻株QDH7和非产毒藻株N8全基因组测序基础上，结合其他23株不同产地和产毒特性拟柱孢藻进行泛基因组和系统进化分析，发现两藻株拥有不同数量的特异基因， N8与亚洲藻株亲缘关系近，产毒藻株QDH7则与非洲藻株关系近。比较基因组分析表明不同产毒能力的藻株均拥有完整的磷吸收代谢通路基因，具有灵活利用环境中不同形态磷源的潜能，从而适应磷波动的环境条件并形成优势。