造礁石珊瑚共生虫黄藻离体培养方法的优化

Optimization of in vitro culture method for zooxanthellae associated with reef-building corals

摘要:

【目的】开发一种高效地从造礁石珊瑚中分离、培养共生虫黄藻的技术方法，为珊瑚共生虫黄藻藻种资源储备和生理功能研究积累基础。【方法】首先采用微孔滤网过滤法和密度梯度离心法从造礁石珊瑚组织中直接分离或富集共生虫黄藻细胞，然后用改良的L1培养基在96孔板上对所得细胞进行离体培养，最后进行单细胞分离、培养和(或)平板划线培养获得单克隆虫黄藻细胞系。对所得虫黄藻单克隆藻株进行聚合酶链式反应-限制性内切酶片段长度多态性(polymerase chain reaction-restriction fragment length polymorphism, PCR-RFLP)分析，结合内转录间隔区2 (internal transcribed spacer 2, ITS2)和大亚基(large subunit, LSU)测序进行物种鉴定及系统发育分析。【结果】采用上述方法从涠洲岛的霜鹿角珊瑚(Acropora pruinose)和西沙群岛的丛生盔形珊瑚(Galaxea fascicularis)及柔枝鹿角珊瑚(Acropora tenuis)中分离、培养得到3个虫黄藻株系，编号分别为AP21C1、GF21D1和AT21A113。3株藻的ITS2基因型分别鉴定为C1、D1及A113亚系群，系统发育特征分别与已命名的虫黄藻Cladocopium goreaui、Durusdinium trenchii及Symbiodinium natans基本一致。3株藻细胞在对数生长期均有自旋运动且具有贴壁性，其中AP21C1株系的虫黄藻细胞无法在琼脂平板上生长。【结论】本研究提供了一种高效地对珊瑚共生虫黄藻进行离体培养的方法，将对后续珊瑚共生虫黄藻物种资源的探索、利用、生理功能研究等提供有力的技术理论支持。

Abstract:

[Objective] To develop an efficient method for isolating and cultivating zooxanthellae from reef-building corals and lay a foundation for the research on germplasm banking and physiological functions of zooxanthellae species associated with corals. [Methods] The zooxanthellae were isolated and enriched from reef-building coral tissues by micro-strainer filtration and density gradient centrifugation, and the cells were cultured in 96-well plates with the modified L1 medium. Single clones of zooxanthellae were obtained by single cell isolation, culture, and/or plate streaking. The species and phylogenetic relationship were identified by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis in combination with internal transcribed spacer 2 (ITS2) and large subunit (LSU) sequences. [Results] Three zooxanthellae strains were isolated from Acropora pruinose of Weizhou Island, Galaxea fascicularis and Acropora tenuis of Xisha Islands and numbered AP21C1, GF21D1, and AT21A113, respectively. The three strains showed the ITS2 genotypes of C1, D1, and A113 and the sequences basically consistent with Cladocopium goreaui, Durusdinium trenchii, and Symbiodinium natans, respectively. All the three strains demonstrated self-spinning motion and adherence during the logarithmic growth phase, and strain AP21C1 was unable to grow on agar plates. [Conclusion] This study develops an efficient method for the in vitro culture of zooxanthellae associated with corals and provides a technical and theoretical basis for the research on the germplasm banking and physiological functions of zooxanthellae species associated with corals.