Byun Mi Young, Seo Suyeon, Lee Jungeun, Yoo Yo-Han, Lee Hyoungseok
Division of Life Sciences, Korea Polar Research Institute, Incheon, South Korea.
Polar Science, University of Science and Technology, Incheon, South Korea.
Front Plant Sci. 2021 Jan 8;11:609847. doi: 10.3389/fpls.2020.609847. eCollection 2020.
Mosses number about 13,000 species and are an important resource for the study of the plant evolution that occurred during terrestrial colonization by plants. Recently, the physiological and metabolic characteristics that distinguish mosses from terrestrial plants have received attention. In the Arctic, in particular, mosses developed their own distinct physiological features to adapt to the harsh environment. However, little is known about the molecular mechanisms by which Arctic mosses survive in extreme environments due to the lack of basic knowledge and tools such as genome sequences and genetic transfection methods. In this study, we report the axenic cultivation and transfection of Arctic sp. KMR5045, as a model for bioengineering of Arctic mosses. We also found that the inherent low-temperature tolerance of KMR5045 permitted it to maintain slow growth even at 2°C, while the model moss species failed to grow at all, implying that KMR5045 is suitable for studies of cold-tolerance mechanisms. To achieve genetic transfection of KMR5045, some steps of the existing protocol for were modified. First, protoplasts were isolated using 1% driselase solution. Second, the appropriate antibiotic was identified and its concentration was optimized for the selection of transfectants. Third, the cell regeneration period before transfer to selection medium was extended to 9 days. As a result, KMR5045 transfectants were successfully obtained and confirmed transfection by detection of intracellular Citrine fluorescence derived from expression of a transgene construct. This is the first report regarding the establishment of a genetic transfection method for an Arctic moss species belonging to the Bryaceae. The results of this study will contribute to understanding the function of genes involved in environmental adaptation and to application for production of useful metabolites derived from stress-tolerant mosses.
苔藓约有13000种，是研究植物在陆地定殖过程中发生的植物进化的重要资源。最近，区分苔藓与陆生植物的生理和代谢特征受到了关注。特别是在北极地区，苔藓形成了自身独特的生理特征以适应恶劣环境。然而，由于缺乏诸如基因组序列和基因转染方法等基础知识和工具，关于北极苔藓在极端环境中生存的分子机制知之甚少。在本研究中，我们报告了北极藓属KMR5045的无菌培养和转染，作为北极苔藓生物工程的模型。我们还发现，KMR5045固有的低温耐受性使其即使在2°C时也能保持缓慢生长，而作为模型的苔藓物种根本无法生长，这意味着KMR5045适合用于耐寒机制的研究。为实现KMR5045的基因转染，对现有方案的一些步骤进行了修改。首先，使用1%的溶菌酶溶液分离原生质体。其次，确定合适的抗生素并优化其浓度以用于转染子的筛选。第三，将转移到选择培养基之前的细胞再生期延长至9天。结果，成功获得了KMR5045转染子，并通过检测源自转基因构建体表达的细胞内柠檬黄荧光确认了转染。这是关于建立一种针对属于真藓科的北极苔藓物种的基因转染方法的首次报道。本研究结果将有助于理解参与环境适应的基因功能，并有助于应用于生产源自耐逆苔藓的有用代谢产物。
