Applicability assessment of molecular tools and identification of lines suitable for genetic transformation in the red alga Pyropia yezoensis

Pyropia yezoensis, a red macroalga distributed in the intertidal zone, serves as an important model organism for studying the adaptive mechanisms of intertidal seaweeds in response to abiotic stressors. Additionally, it is a significant economic seaweed due to its high nutrient content and is widely cultivated in China, Japan, and Korea. Despite its importance, existing molecular tools for genetic transformation remain limited and require further development to enhance our understanding of P. yezoensis biology. To address this limitation, based on previous studies, we firstly evaluated the expression of eight fluorescent proteins (FPs) in P. yezoensis. Our findings confirmed that PyZsYellow, PyZsGFP, and PysGFP can be detected successfully their specific fluorescent signals within the cells of P. yezoensis thalli. Notably, PyZsGFP demonstrated effectiveness in sporophytic filaments. 2×SV40 and PyNP may function as nuclear localization signal sequences, effectively directing the FP to localize within the nucleus of cells in P. yezoensis. T2A linker can mediate protein cleavage, to express the multiple genes in one construct, enabling them to function independently in P. yezoensis. Furthermore, we identified that the RZ line exhibited the highest transformation potential, while the middle region of thalli showed the superior transformation efficiency. A significant correlation between cell size and transformation efficiency. These findings will contribute to developing a systematic toolkit for conducting genetic transformations and elucidating gene functions in P. yezoensis.