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，细菌可以帮助藻类在环境压力下生存，但这些联系仍然知之甚少。红藻Porphyra umbilicalis（紫菜）经历了包括高温、强光和干燥在内的潮间带压力，但在涨潮时再次被淹没时，藻类会迅速恢复。紫菜目前是一项广泛的细菌微生物组研究的主题，现在已经有了一个完全测序的基因组，使它成为研究细菌关联如何在分子水平上赋予耐受性的理想候选者。实现这些调查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。宇二博士是研究红藻基因转化的权威专家，他和同事们最近为紫菜的太平洋表亲紫菜（pypia yezoensis）开发了一个稳定的转化系统。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。本研究的主要目的是在脐带草中建立第一个稳定的转化体系。将含有热休克因子（HSF）基因和可选择标记物（潮霉素B抗性）的质粒通过粒子轰击插入脐带假单胞菌和叶斑藻中。将对转化原生质体或中性孢子的萌发和发育进行评估，并对两种转化体进行热休克处理的比较，并分析其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类群之一（Bangiophyceae）的相关成员，它们在经济和文化上都是重要的藻类。该奖项隶属于东亚和太平洋暑期研究所项目，由美国国家科学基金会和日本科学促进会共同资助，支持一名美国研究生进行暑期研究。

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.