A Toolbox for Constructing a Stable Genetic Transformation Platform Allowing Foreign Fragment Integration in the Genome of Neopyropia yezoensis
A Toolbox for Constructing a Stable Genetic Transformation Platform Allowing Foreign Fragment Integration in the Genome of Neopyropia yezoensis
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DOI：
10.3389/fmars.2022.856790
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发表时间：
2022-05
期刊：
影响因子：
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通讯作者：
Xuexue Cao;Fanna Kong;Bin Sun;J. Yin;Hongzhong Ren;H. Yue;Chenggong Yu;Xianghai Tang;Guo-don Du
中科院分区：
文献类型：
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作者：
Xuexue Cao;Fanna Kong;Bin Sun;J. Yin;Hongzhong Ren;H. Yue;Chenggong Yu;Xianghai Tang;Guo-don Du
条斑新比藻(Neopyropia Yezoensis)，原名约氏新比藻，因其经济价值和典型的潮间带海藻种类而被认为是大型藻类的模式藻类。然而，将外源基因导入其细胞的遗传转化系统效率低下且不稳定。本研究开发了一种更高效的转化工具箱，允许外源基因在条斑夜蛾细胞中稳定表达。我们构建了一个高效的转化平台，其参数为：生物轰击靶距6 cm，金粒子600μg，质粒10μg，氦气1350 psi，真空度30 mm Hg。35d龄的菌体为最适转化条件，转化效率最高。与pPyUBC、pPyDPE2和pPyEF1-a相比，内源启动子pPyACT1能有效调控基因的表达，尤其是外源启动子d35S。最后，外源基因PyGUS和PyHygR在单孢子、配子体和丝状孢子体等不同世代的转化体中稳定表达。Southern blotting分析证实，pYGUS已整合到条斑病菌转化子的基因组中。建立高效的基因表达工具箱为条斑夜蛾功能基因组学研究和分子遗传育种奠定了坚实的基础。
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.