Department of Environmental Life Sciences, Graduate School of Life Sciences, Tohoku University, Sendai, Miyagi, Japan.
Division of Environmental Photobiology, National Institute for Basic Biology, Okazaki, Aichi, Japan.
Sci Rep. 2018 Feb 19;8(1):3237. doi: 10.1038/s41598-018-21499-3.
Coral reef ecosystems rely on stable symbiotic relationship between the dinoflagellate Symbiodinium spp. and host cnidarian animals. The collapse of such symbiosis could cause coral 'bleaching' and subsequent host death. Despite huge interest on Symbiodinium, lack of mutant strains and readily available genetic tools have hampered molecular research. A major issue was the tolerance to marker antibiotics. Here, we isolated Symbiodinium mutants requiring uracil for growth, and hence, useful in transformation screening. We cultured Symbiodinium spp. cells in the presence of 5-fluoroorotic acid (5FOA), which inhibits the growth of cells expressing URA3 encoding orotidine-5'-monophosphate decarboxylase, and isolated cells that require uracil for growth. Sequence analyses and genetic complementation tests using yeast demonstrated that one of the mutant cell lines had a point mutation in URA3, resulting in a splicing error at an unusual exon-intron junction, and consequently, loss of enzyme activity. This mutant could maintain a symbiotic relationship with the model sea anemone Exaiptasia pallida only in sea water containing uracil. Results show that the URA3 mutant will be a useful tool for screening Symbiodinium transformants, both ex and in hospite, as survival in the absence of uracil is possible only upon successful introduction of URA3.
珊瑚礁生态系统依赖于共生甲藻属（Symbiodinium spp.）与宿主刺胞动物之间稳定的共生关系。这种共生关系的崩溃会导致珊瑚“白化”和随后的宿主死亡。尽管人们对共生甲藻属（Symbiodinium）非常感兴趣，但缺乏突变株和现成的遗传工具阻碍了分子研究。一个主要问题是对标记抗生素的耐受性。在这里，我们分离出需要尿嘧啶生长的共生甲藻属（Symbiodinium）突变体，因此可用于转化筛选。我们在 5-氟乳清酸（5FOA）的存在下培养共生甲藻属（Symbiodinium）细胞，5FOA 抑制表达 URA3 编码乳清酸-5'-单磷酸脱羧酶的细胞生长，从而分离出需要尿嘧啶生长的细胞。序列分析和酵母遗传互补测试表明，一个突变细胞系在 URA3 中发生了点突变，导致在一个不寻常的外显子-内含子交界处发生剪接错误，从而导致酶活性丧失。这种突变体只能在含有尿嘧啶的海水中与模式海葵 Exaiptasia pallida 维持共生关系。结果表明，URA3 突变体将是筛选共生甲藻属（Symbiodinium）转化体的有用工具，无论是在宿主内还是在宿主外，因为只有在成功引入 URA3 的情况下，才能在没有尿嘧啶的情况下生存。
