气候变化对全球农业构成重大威胁,需要创新的解决方案。植物合成生物学,特别是叶绿体工程,有希望成为应对这一挑战的可行方法。叶绿体为基因工程提供了多种有利的性状,但是,这些细胞器中遗传工具和遗传部分表征的发展受到产生转体生物所需的漫长时间尺度的阻碍。为了应对这些挑战,我们已经建立了一个通用的协议,用于产生高活性的基于叶绿体的无细胞基因表达(CFE)系统,该系统来自各种植物物种,包括小麦(单子叶),菠菜,和白杨树(双子叶)。我们表明,这些系统与常规使用的T7RNA聚合酶以及内源性叶绿体聚合酶一起工作,允许在转录和翻译水平上对调控序列进行详细的表征和原型化。为了证明高等植物叶绿体基因表达中启动子和5'和3'非翻译区(UTR)的表征平台,我们分析了235个UTR的集合,103'UTR,和6个叶绿体启动子,评估了它们在菠菜和小麦提取物中的表达,并发现表达模式的一致性,表明跨物种相容性。展望未来,我们的叶绿体CFE系统为植物合成生物学开辟了新的途径,提供原型工具,用于理解基因表达和开发工程植物,这可以帮助满足不断变化的全球气候的需求。
Climate change poses a significant threat to global agriculture, necessitating innovative solutions. Plant synthetic biology, particularly chloroplast engineering, holds promise as a viable approach to this challenge. Chloroplasts present a variety of advantageous traits for genetic engineering, but the development of genetic tools and genetic part characterization in these organelles is hindered by the lengthy time scales required to generate transplastomic organisms. To address these challenges, we have established a versatile protocol for generating highly active chloroplast-based cell-free gene expression (CFE) systems derived from a diverse range of plant species, including wheat (monocot), spinach, and poplar trees (dicots). We show that these systems work with conventionally used T7 RNA polymerase as well as the endogenous chloroplast polymerases, allowing for detailed characterization and
prototyping of regulatory sequences at both transcription and translation levels. To demonstrate the platform for characterization of promoters and 5\' and 3\' untranslated regions (UTRs) in higher plant chloroplast gene expression, we analyze a collection of 23 5\'UTRs, 10 3\'UTRs, and 6 chloroplast promoters, assessed their expression in spinach and wheat extracts, and found consistency in expression patterns, suggesting cross-species compatibility. Looking forward, our chloroplast CFE systems open new avenues for plant synthetic biology, offering
prototyping tools for both understanding gene expression and developing engineered plants, which could help meet the demands of a changing global climate.