Plant J|一种在野生型植物Rubisco大亚基基因中引入点突变的方法
光合效率低下限制了作物生产的生产力和可持续性，以及农业对未来社会和环境挑战的适应力。Rubisco是改善的关键目标，因为它在光合作用的碳固定过程中起着核心作用，并且效率很低。将叶绿体编码的Rubisco大亚基rbcL引入突变对于提高酶的催化活性和效率尤为重要。但是，rbcL的操纵受到其在质体组中的位置，许多物种对质体组转化的阻挠以及质体的有效修复系统的阻碍，质体的有效修复系统可防止有效维持由同源重组引入的突变。
本研究，我们介绍了一个系统，其中在模型植物烟草中将许多沉默突变引入rbcL有助于通过其他限制性酶切位点简化筛选。该系统用于成功地从野生型烟草中产生一系列转质体系，其中有40％的转化子在rbcL内具有稳定点突变，从而可以评估这些突变对Rubisco组装和活性的影响。
通过进一步优化，该方法为诱变测试烟草中植物体内的Rubisco功能和修饰其他可进行叶绿体转化的作物中的rbcL提供了可行的方法。该转化策略也可用于在其他叶绿体编码基因中引入点突变。
Photosynthetic inefficiencies limit the productivity and sustainability of crop production, and the resilience of agriculture to future societal and environmental challenges. Rubisco is a key target for improvement as it plays a central role in carbon fixation during photosynthesis and is remarkably inefficient. Introduction of mutations to the chloroplast‐encoded Rubisco large subunit rbcL is of particular interest to improve the catalytic activity and efficiency of the enzyme. However, manipulation of rbcL is hampered by its location in the plastome, with many species recalcitrant to plastome transformation, and by the plastid’s efficient repair system, which can prevent effective maintenance of mutations introduced with homologous recombination. Here we present a system where the introduction of a number of silent mutations into rbcL within the model plant Nicotiana tabacum facilitates simplified screening via additional restriction enzyme sites. This system was used to successfully generate a range of transplastomic lines from wild‐type N. tabacum with stable point mutations within rbcL in 40% of the transformants, allowing assessment of the effect of these mutations on Rubisco assembly and activity. With further optimization, the approach offers a viable way forward for mutagenic testing of Rubisco function in planta within tobacco and modifying rbcL in other crops where chloroplast transformation is feasible. The transformation strategy could also be applied to introduce point mutations in other chloroplast‐encoded genes.
来源：
A procedure to introduce point mutations into the Rubisco large subunit gene in wild‐type plants10.1111/tpj.15196
文章原创，转载请注明来源。
温馨提示：
为方便PaperRSS粉丝们科研、就业等话题交流。我们根据10多个专业方向（植物、医学、药学、人工智能、化学、物理、财经管理、体育等），特建立了30个国内外博士交流群。群成员来源欧美、日韩、新加坡、清华北大、中科院等全球名校。