EAGER Collaborative Research: Developing Transformation Technologies for Porphyra

联合基因组研究所正在对海洋红藻的基因组进行测序。序列信息将帮助科学家解决许多基本问题，因为紫菜属一种古老的真核生物谱系，是重要的人类食物（“紫菜”），具有复杂的生活史，甚至与其他潮间带生物相比，具有不同寻常的耐压代谢。基于计算机的新基因组数据分析将足以解决一些研究问题，但大多数研究（例如，紫菜对极端干燥或强光的耐受性的基础）将需要基于生物信息学分析的实验方法。本项目将开发紫菜稳定遗传转化的关键技术，使此类实验工作成为可能。研究人员将把重点放在转化中性芽孢上，因为这些大量产生的芽孢缺乏细胞壁。这将使通过电穿孔等方法转化细胞成为可能。根据JGI测序项目提供的信息，使用卟啉基因的原生卟啉启动子，合成密码子优化的报告基因。孢子将用选择性抗生素处理以恢复转化的孢子，并将在孢子成熟到成虫时评估转化的稳定性。两位pi和一名博士后将在两个参与的实验室合作开发转化技术。该博士后将在转化技术发展过程中研究生殖途径或应激生理学。由于全面的博士后培训和参与卟啉NSF研究协调网络，他/她将为卟啉模型系统的个人和合作进步做好充分的准备。该项目将允许科学界在紫菜的实验研究中使用全基因组测序项目的数据，从而在代谢、进化和发育生物学领域取得根本性的进展。

The genome of the marine red alga Porphyra umbilicalis is being sequenced by the Joint Genome Institute. The sequence information will help scientists address many fundamental questions, because Porphyra spp. belong to an ancient eukaryotic lineage, are important human foods ("nori"), have complex life histories, and---even compared to other intertidal organisms--- possess an unusually stress-tolerant metabolism. Computer-based analyses of the new genomic data will be sufficient to address some research questions, but most studies (e.g., the basis of Porphyra's tolerance to extreme drying or high light) will require experimental approaches based upon bioinformatics analyses. This project will develop the essential technology of stable genetic transformation in Porphyra to make such experimental work possible. The investigators will focus on transforming neutral spores of P. umbilicalis because these abundantly-produced spores lack a cell wall. This should make it possible to transform the cell by electroporation, among other approaches. Native Porphyra promoters of Porphyra genes will be used, based on information provided by the JGI sequencing project, and codon-optimized reporter genes will be synthesized. Spores will be treated with selective antibiotics to recover transformed sporelings, and the stability of the transformation will be assessed as sporelings mature to adults. The PIs and a postdoctoral associate will work across both participating laboratories to develop transformation technologies. The postdoctoral associate will investigate reproductive pathways or stress physiology during the development of transformation techniques. S/he will be well-prepared to make individual and collaborative advances with the Porphyra model system due to the comprehensive postdoctoral training and participation in the Porphyra NSF Research Coordination Network. This project will allow the scientific community to use the data from the whole genome sequencing project in experimental research on Porphyra, leading to fundamental advances in the areas of metabolism, evolution, and developmental biology.