基于Golden Gate技术的齐整小核菌转录单元高效组装系统

A Golden Gate-based efficient assembly system of transcription units for Sclerotium rolfsii

摘要:

【目的】为齐整小核菌代谢工程研究建立高效的转录单元组装系统。【方法】通过应用Golden Gate技术，以mobius assembly为基础，分别设计并构建DNA元件标准化接口改造、单转录单元组装、应用质粒(多转录单元)组装等功能的载体，从而形成一套完整的多转录单元组装系统。【结果】构建了2个用于DNA元件标准化接口改造的Level 0载体，4个用于单转录单元组装的Level 1载体，4个用于应用质粒组装的Level 2载体和13个应用质粒组装的辅助质粒。然后应用此系统为齐整小核菌组装了若干经过标准化接口改造的DNA元件质粒、单转录单元质粒和硬葡聚糖相关基因的功能分析质粒。所构建的最终应用质粒可以同时适用于齐整小核菌的根癌农杆菌介导转化法、电穿孔转化法和原生质体转化法。【结论】此质粒系统具有强大的DNA设计、组装和容纳能力，为未来齐整小核菌代谢工程和功能基因组学研究提供了高效的质粒构建技术平台。

Abstract:

[Objective] To establish an efficient transcription unit assembly system for the metabolic engineering of Sclerotium rolfsii. [Methods] Based on Golden Gate technology and Mobius assembly, the scaffold plasmids for DNA part domestication, single transcription unit assembly, and application plasmid (multi-transcription-unit) assembly, were designed and constructed to provide a complete multi-transcription-unit assembly system. [Results] Two Level 0 vectors for DNA part domestication, four Level 1 vectors for single transcription unit assembly, and four Level 2 vectors and 13 auxiliary plasmids for application plasmid assembly were constructed. Using this system, we assembled tens of plasmids for domesticated DNA parts, single transcription units, and function analysis of scleroglucan-related genes. The constructed application plasmids could be directly applied in Agrobacterium tumefaciens-mediated transformation, electroporation transformation, and protoplast transformation of S. rolfsii. [Conclusion] The plasmid system we built has strong capabilities of DNA design, assembly, and capacity, providing an efficient plasmid construction platform for metabolic engineering and functional genomics research of S. rolfsii in the future.