Annunziata Rossella, Balestra Cecilia, Marotta Pina, Ruggiero Antonella, Manfellotto Francesco, Benvenuto Giovanna, Biffali Elio, Ferrante Maria Immacolata
Stazione Zoologica Anton Dohrn, 80121, Napoli, Italy.
Sci Rep. 2021 Jan 18;11(1):1681. doi: 10.1038/s41598-021-81238-z.
Due to their abundance in the oceans, their extraordinary biodiversity and the increasing use for biotech applications, the study of diatom biology is receiving more and more attention in the recent years. One of the limitations in developing molecular tools for diatoms lies in the peculiar nature of their cell wall, that is made of silica and organic molecules and that hinders the application of standard methods for cell lysis required, for example, to extract organelles. In this study we present a protocol for intact nuclei isolation from diatoms that was successfully applied to three different species: two pennates, Pseudo-nitzschia multistriata and Phaeodactylum tricornutum, and one centric diatom species, Chaetoceros diadema. Intact nuclei were extracted by treatment with acidified NHF solution combined to low intensity sonication pulses and separated from cell debris via FAC-sorting upon incubation with SYBR Green. Microscopy observations confirmed the integrity of isolated nuclei and high sensitivity DNA electrophoresis showed that genomic DNA extracted from isolated nuclei has low degree of fragmentation. This protocol has proved to be a flexible and versatile method to obtain intact nuclei preparations from different diatom species and it has the potential to speed up applications such as epigenetic explorations as well as single cell ("single nuclei") genomics, transcriptomics and proteomics in different diatom species.
由于硅藻在海洋中数量丰富、具有非凡的生物多样性且在生物技术应用中的使用日益增加，近年来对硅藻生物学的研究受到越来越多的关注。开发硅藻分子工具的局限性之一在于其细胞壁的特殊性质，它由二氧化硅和有机分子组成，阻碍了例如提取细胞器所需的细胞裂解标准方法的应用。在本研究中，我们提出了一种从硅藻中分离完整细胞核的方案，该方案已成功应用于三种不同的物种：两种羽纹硅藻，多列拟菱形藻和三角褐指藻，以及一种中心硅藻，冠盖角毛藻。通过用酸化的NHF溶液处理并结合低强度超声脉冲来提取完整的细胞核，并在与SYBR Green孵育后通过流式细胞分选从细胞碎片中分离出来。显微镜观察证实了分离细胞核的完整性，高灵敏度DNA电泳表明从分离细胞核中提取的基因组DNA片段化程度低。该方案已被证明是一种灵活通用的方法，可从不同的硅藻物种中获得完整的细胞核制剂，并且有潜力加速表观遗传学探索以及不同硅藻物种中的单细胞（“单核”）基因组学、转录组学和蛋白质组学等应用。
