Department of Biology, Northeastern University, Boston, MA, USA.
Department of Bioengineering, Northeastern University, Boston, MA, USA.
Methods Mol Biol. 2022;2505:249-262. doi: 10.1007/978-1-0716-2349-7_18.
Catharanthus roseus produces medicinal terpenoid indole alkaloids, including the critical anti-cancer compounds vinblastine and vincristine in its leaves. Recently, we developed a highly efficient transient expression method relying on Agrobacterium-mediated transformation of seedlings to facilitate rapid and high-throughput studies on the regulation of terpenoid indole alkaloid biosynthesis in C. roseus . We detail our optimized protocol known as efficient Agrobacterium-mediated seedling infiltration method (EASI), including the development of constructs used in EASI and an example experimental design that includes appropriate controls. We applied our EASI method to rapidly screen and evaluate transcriptional activators and repressors and promoter activity. Our EASI method can be used for promoter transactivation studies or transgene overexpression paired with downstream analyses like quantitative PCR or metabolite analysis. Our protocol takes about 16 days from sowing seeds to obtaining the results of the experiment.
长春花产生药用萜类吲哚生物碱，包括叶子中的关键抗癌化合物长春碱和长春新碱。最近，我们开发了一种高效的瞬时表达方法，依赖于农杆菌介导的幼苗转化，以促进长春花萜类吲哚生物碱生物合成的快速和高通量研究。我们详细介绍了我们优化的协议，称为高效农杆菌介导的幼苗浸润法（EASI），包括在 EASI 中使用的构建体的开发和一个包含适当对照的示例实验设计。我们应用我们的 EASI 方法来快速筛选和评估转录激活子和抑制剂以及启动子活性。我们的 EASI 方法可用于启动子反式激活研究或转基因过表达与下游分析（如定量 PCR 或代谢物分析）相结合。我们的方案从播种种子到获得实验结果大约需要 16 天。
