Table_1_A Toolbox for Constructing a Stable Genetic Transformation Platform Allowing Foreign Fragment Integration in the Genome of Neopyropia yezoensis.docx

Neopyropia yezoensis, previously known as Pyropia yezoensis, is regarded as a model macroalgae because of its economic value and typical intertidal seaweed species. However, the genetic transformation system to introduce foreign genes into its cells is inefficient and not stable. This study developed a more efficient transformation toolbox that allows the stable expression of foreign genes in N. yezoensis cells. We constructed an efficient transformation platform with the parameters of biolistic bombardment of 6 cm target distance, 600 μg gold particles/shot, 10 μg plasmid DNA/shot, 1,350 psi of helium, and 30 mmHg vacuum pressure. Thalli at 35 days of age were the most suitable transformation conditions, in which the highest transformation efficiency was generated. The endogenous promoter pPyACT1 could control gene expression efficiently compared to pPyUBC, pPyDPE2, and pPyEF1-a, especially the exogenous promoter d35S. Finally, the foreign genes PyGUS and PyHygR were stably expressed in different generations of transformants, including monospores, gametophytes, and filamentous sporophytes. Southern blotting analysis confirmed that PyGUS was integrated into the genome of N. yezoensis transformants. Establishing an efficient gene expression toolbox provides a strong foundation for functional genomics research and molecular genetic breeding on N. yezoensis.

条斑紫菜（Neopyropia yezoensis，曾用名Pyropia yezoensis）兼具经济价值与典型潮间带海藻特征，被视为大型藻类的模式生物。然而，当前将外源基因导入其细胞的遗传转化系统存在效率低下、稳定性不足的缺陷。本研究开发了一套更为高效的转化工具盒，可实现外源基因在条斑紫菜细胞中的稳定表达。本研究构建了一套高效转化平台，其基因枪轰击参数为：靶距6 cm、每枪使用600 μg金颗粒、10 μg质粒DNA、氦气压力1350 psi、真空度30 mmHg。培养35天的叶状体为最适转化材料，此时可获得最高的转化效率。与pPyUBC、pPyDPE2及pPyEF1-α相比，内源性启动子pPyACT1可更高效地调控基因表达，其中外源启动子d35S的调控效果尤为显著。最终，外源基因PyGUS与PyHygR可在转化子的不同世代中稳定表达，涵盖单孢子、配子体及丝状孢子体。Southern印迹分析证实，PyGUS已整合至条斑紫菜转化子的基因组中。这套高效基因表达工具盒的建立，为条斑紫菜的功能基因组学研究与分子遗传育种奠定了坚实基础。