Abstract：
Hornworts are a deeply diverged lineage of bryophytes and a sister lineage to mosses and liverworts. Hornworts have an array of unique features that can be leveraged to illuminate not only the early evolution of land plants, but also alternative paths for nitrogen and carbon assimilation via cyanobacterial symbiosis and a pyrenoid-based CO2-concentrating mechanism (CCM), respectively. Despite this, hornworts are one of the few plant lineages with limited available genetic tools. Here we report an efficient biolistics method for generating transient expression and stable transgenic lines in the model hornwort, Anthoceros agrestis. An average of 569 (±268) cells showed transient expression per bombardment, with green fluorescent protein expression observed within 48-72 h. A total of 81 stably transformed lines were recovered across three separate experiments, averaging six lines per bombardment. We followed the same method to transiently transform nine additional hornwort species, and obtained stable transformants from one. This method was further used to verify the localization of Rubisco and Rubisco activase in pyrenoids, which are central proteins for CCM function. Together, our biolistics approach offers key advantages over existing methods as it enables rapid transient expression and can be applied to widely diverse hornwort species.
摘要：
刺槐是苔藓和苔藓的姊妹苔藓的苔藓谱系。角草具有一系列独特的功能，不仅可以用来阐明陆地植物的早期进化，而且还有通过蓝细菌共生和基于拟肾素的CO2浓缩机制（CCM）进行氮和碳同化的替代途径，分别。尽管如此,刺耳是为数不多的植物谱系之一，可用的遗传工具有限。在这里，我们报告了一种有效的生物射弹方法，用于在模型中产生瞬时表达和稳定的转基因系，炭疽病。平均569（±268）个细胞显示每次轰击的瞬时表达，在48-72小时内观察到绿色荧光蛋白表达。在三个独立的实验中总共回收了81个稳定转化的品系。平均每次轰炸六行。我们按照同样的方法暂时转化了9种额外的角草，并从中获得稳定的转化体。该方法进一步用于验证Rubisco和Rubisco激活酶在类蛋白中的定位。它们是CCM功能的中心蛋白。一起,与现有方法相比，我们的生物射弹方法具有关键优势，因为它可以实现快速瞬时表达，并且可以应用于广泛多样的龙葵物种。
