Osaki Yasuhide, Kodama Yutaka
Center for Bioscience Research and Education, Utsunomiya University, Utsunomiya, Tochigi, Japan.
PeerJ. 2017 Sep 7;5:e3779. doi: 10.7717/peerj.3779. eCollection 2017.
Particle bombardment is a powerful and relatively easy method for transient expression of genes of interest in plant cells, especially those that are recalcitrant to other transformation methods. This method has facilitated numerous analyses of subcellular localization of fluorescent fusion protein constructs. Particle bombardment delivers genes to the first layer of plant tissue. In leaves of higher plants, epidermal cells are the first cell layer. Many studies have used the epidermal cell layer of onion bulb () as the experimental tissue, because these cells are relatively large. However, onion epidermal cells lack developed plastids (i.e., chloroplasts), thereby precluding subcellular localization analysis of chloroplastic proteins. In this study, we developed a protocol for particle bombardment of the aquatic plant , and showed that it is a useful system for subcellular localization analysis of higher plant proteins. leaflets contain only two cell layers, and cells in the adaxial layer are sufficiently large for observation. The cells in both layers contain well-developed chloroplasts. We fused fluorescent proteins to conventional plant localization signals for the nucleus, cytosol, mitochondria, peroxisome, and chloroplast, and used particle bombardment to transiently express these fusion constructs in leaves. The plant subcellular localization signals functioned normally and displayed the expected distributions in transiently transformed cells, and even chloroplastic structures could be clearly visualized.
粒子轰击是一种在植物细胞中瞬时表达目的基因的强大且相对简便的方法，尤其适用于那些对其他转化方法具有抗性的细胞。该方法促进了对荧光融合蛋白构建体亚细胞定位的大量分析。粒子轰击将基因传递到植物组织的第一层。在高等植物的叶片中，表皮细胞是第一层细胞。许多研究使用洋葱鳞茎的表皮细胞层作为实验组织，因为这些细胞相对较大。然而，洋葱表皮细胞缺乏发育良好的质体（即叶绿体），因此无法进行叶绿体蛋白的亚细胞定位分析。在本研究中，我们开发了一种对水生植物进行粒子轰击的方案，并表明它是用于高等植物蛋白亚细胞定位分析的有用系统。 小叶仅包含两层细胞，近轴层的细胞足够大以便观察。两层细胞均含有发育良好的叶绿体。我们将荧光蛋白与用于细胞核、细胞质、线粒体、过氧化物酶体和叶绿体的传统植物定位信号融合，并使用粒子轰击在 叶片中瞬时表达这些融合构建体。植物亚细胞定位信号正常发挥作用，并在瞬时转化的 细胞中呈现预期的分布，甚至叶绿体结构也能清晰可见。
