EAPSI: Development of a Stable Transformation System in the Red Alga Porphyra Umbilicalis

细菌对包括藻类在内的多细胞生物的正常发育至关重要，细菌可能有助于藻类在环境压力下生存，但这些关联仍然知之甚少。红紫菜经历了包括高温、强光和干燥在内的潮间带压力，但当涨潮时再次被淹没时，紫菜很快就恢复了。紫菜目前是一个广泛的细菌微生物组研究的主题，现在有一个完全测序的基因组，使其成为研究细菌协会如何在分子水平上赋予胁迫耐受性的理想候选者。使这些调查成为可能的一种方法是创建一个系统来操纵紫菜？s基因，即稳定的遗传转化。这项工作是与日本北海道大学的Hiroyuki Mizuta博士和Toshiki Uji博士合作提出的。Uji博士是红藻遗传转化方面的领先专家，他和他的同事最近为紫菜的太平洋表亲海苔（条斑紫菜）开发了一种稳定的转化系统。一个稳定的转化系统将允许对紫菜的应激反应进行研究，包括微生物组的可能影响，并将为基于紫菜基因组的其他科学研究提供宝贵的工具。这项工作的主要目标是建立第一个稳定的转化系统。脐紫菜。将含有热休克因子（HSF）基因和选择标记（潮霉素B抗性）的质粒通过粒子轰击插入脐生假单胞菌和条斑假单胞菌中。将评价转化的原生质体或中性孢子的萌发和发育，并且将对两个物种的转化体进行比较性热休克处理，并分析它们的HSF表达。 这种比较是有用的，因为P. umbilicalis和P. yezoensis是最古老的真核生物群之一（Bangiophyceae）的相关成员，并且它们是经济和文化上重要的藻类。东亚和太平洋夏季研究所计划下的这个奖项支持美国研究生的夏季研究，由NSF和日本科学促进会共同资助。

Bacteria are critical to the normal development of multicellular organisms, including algae, and bacteria may help algae survive environmental stresses, but these associations remain poorly understood. The red alga Porphyra umbilicalis ('laver') experiences intertidal stresses that include high temperature, high light, and desiccation, but the alga recovers quickly when submerged again at high tide. Laver is currently the subject of an extensive bacterial microbiome study, and now has a fully-sequenced genome, making it an ideal candidate for investigations of how bacterial associations confer stress tolerance on a molecular level. One way to enable these investigations is to create a system to manipulate laver?s genes, i.e. stable genetic transformation. This work is proposed in collaboration with Dr. Hiroyuki Mizuta and Dr. Toshiki Uji of Hokkaido University, Japan. Dr. Uji is a leading expert on genetic transformation in red algae, and he and his colleagues recently developed a stable transformation system for nori (Pyropia yezoensis), a Pacific cousin of laver. A stable transformation system will allow research to be conducted on stress responses in laver, including possible effects of the microbiome, and will provide an invaluable tool for other scientific studies based on the Porphyra genome.The primary goal of this work is to establish the first stable transformation system in P. umbilicalis. Plasmids containing a heat shock factor (HSF) gene and a selectable marker (hygromycin B resistance) will be inserted via particle bombardment into both P. umbilicalis and P. yezoensis. Germination and development of transformed protoplasts or neutral spores will be evaluated, and transformants of both species will be subjected to comparative heat shock treatments and their HSF expression analyzed. This comparison is useful because P. umbilicalis and P. yezoensis are related members of one of the oldest eukaryotic groups (Bangiophyceae), and they are both economically and culturally important algae. This award under the East Asia and Pacific Summer Institutes program supports summer research by a U.S. graduate student and is jointly funded by NSF and the Japan Society for the Promotion of Science.