Discovery of Nuclear-Encoded Genes for the Neurotoxin Saxitoxin in Dinoflagellates

Saxitoxin is a potent neurotoxin that occurs in aquatic environments worldwide. Ingestion of vector species can lead to paralytic shellfish poisoning, a severe human illness that may lead to paralysis and death. In freshwaters, the toxin is produced by prokaryotic cyanobacteria; in marine waters, it is associated with eukaryotic dinoflagellates. However, several studies suggest that saxitoxin is not produced by dinoflagellates themselves, but by co-cultured bacteria. Here we show that genes required for saxitoxin synthesis are encoded in the nuclear genomes of dinoflagellates. We sequenced > 1.2 x 106 mRNA transcripts from the two saxitoxin-producing dinoflagellate strains Alexandrium fundyense CCMP1719 and A. minutum CCMP113 using high-throughput sequencing technology. In addition we used in silico transcriptome analyses, RACE, qPCR and conventional PCR coupled with Sanger sequencing. These approaches successfully identified genes required for saxitoxin-synthesis in the two transcriptomes. We focused on sxtA, the unique starting gene of saxitoxin synthesis, and show that the dinoflagellate transcripts of sxtA have the same domain structure as the cyanobacterial sxtA genes. But in contrast to the bacterial homologs, the dinoflagellate transcripts are monocistronic, have a higher GC content, occur in multiple copies, contain typical dinoflagellate spliced-leader sequences and eukaryotic polyA-tails. Further, we investigated 28 saxitoxin-producing and non-producing dinoflagellate strains from six different genera for the presence of genomic sxtA homologs. Our results show very good agreement between the presence of sxtA and saxitoxin-synthesis, except in three strains of A. tamarense, for which we amplified sxtA but did not detect the toxin. Our work opens possibilities to develop molecular tools to detect saxitoxin-producing dinoflagellates in the environment.

石房蛤毒素（Saxitoxin）是一种广泛分布于全球水生环境的强效神经毒素。人类误食携带该毒素的媒介物种后，可引发麻痹性贝类中毒（paralytic shellfish poisoning）——一种可导致麻痹甚至死亡的严重疾病。淡水环境中，该毒素由原核生物蓝细菌（cyanobacteria）合成；海洋环境中，其产生与真核生物甲藻（dinoflagellates）密切相关。

然而此前多项研究认为，石房蛤毒素并非由甲藻自身合成，而是由与其共培养的细菌产生。本研究证实，石房蛤毒素合成所需的功能基因，编码于甲藻的细胞核基因组中。

研究采用高通量测序技术，对两株产毒甲藻——链状亚历山大藻CCMP1719（Alexandrium fundyense CCMP1719）与微小亚历山大藻CCMP113（A. minutum CCMP113）——的超过1.2×10⁶条mRNA转录本进行了测序；同时结合转录组生物信息学分析（in silico transcriptome analyses）、cDNA末端快速扩增技术（RACE）、实时荧光定量PCR（qPCR）以及基于Sanger测序的常规PCR技术开展研究。上述方法成功在两株甲藻的转录组中鉴定出石房蛤毒素合成相关的功能基因。

本研究重点关注石房蛤毒素合成的特异性起始基因sxtA，证实甲藻来源的sxtA转录本与蓝细菌sxtA基因具有完全一致的结构域组成。与细菌同源基因不同的是，甲藻sxtA转录本为单顺反子结构，具有更高的GC含量，以多拷贝形式存在，且包含典型的甲藻剪接前导序列（spliced-leader sequences）与真核生物polyA尾（polyA-tails）。

进一步地，研究团队针对6个不同属的28株产毒与非产毒甲藻菌株，检测其基因组中sxtA同源基因的存在情况。结果显示，除3株塔玛亚历山大藻（A. tamarense）外，sxtA基因的存在与石房蛤毒素合成能力高度吻合：该三株菌株虽成功扩增得到sxtA基因，但未检测到毒素产物。

本研究为开发用于环境中产石房蛤毒素甲藻的分子检测工具提供了可行方向。