Ye G N, Daniell H, Sanford J C
Department of Horticultural Sciences, Cornell University, Geneva, NY 14456.
Plant Mol Biol. 1990 Dec;15(6):809-19. doi: 10.1007/BF00039421.
We report here an efficient and highly reproducible delivery system, using an improved biolistic transformation device, that facilitates transient expression of beta-glucuronidase (GUS) in chloroplasts of cultured tobacco suspension cells. Cultured tobacco cells collected on filter papers were bombarded with tungsten particles coated with pUC118 or pBI101.3 (negative controls), pBI505 (positive nuclear control) or a chloroplast expression vector (pHD203-GUS), and were assayed for GUS activity. No GUS activity was detected in cells bombarded with pUC118 or pBI101.3. Cells bombarded with pBI505 showed high levels of expression with blue color being distributed evenly throughout the whole cytosol of the transformants. pHD203-GUS was expressed exclusively in chloroplasts. We base this conclusion on: i) the procaryotic nature of the promoter used in the chloroplast expression vector; ii) delayed GUS staining; iii) localization of blue color within subcellular compartments corresponding to plastids in both shape and size; and iv) confirmation of organelle-specific expression of pHD203-GUS using PEG-mediated protoplast transformation. Chloroplast transformation efficiencies increased dramatically (about 200-fold) using an improved helium-driven biolistic device, as compared to the more commonly used gun powder charge-driven device. Using GUS as a reporter gene and the improved biolistic device, optimal bombardment conditions were established, consistently producing several hundred transient chloroplast transformants per Petri plate. Chloroplast transformation efficiency was found to be increased further (20-fold) with supplemental osmoticum (0.55 M sorbitol and 0.55 M mannitol) in the bombardment and incubation medium. This system provides a highly effective mechanism for introducing and expressing plasmid DNA within higher-plant chloroplasts, and the fact that GUS functions as an effective marker gene now makes many genetic studies possible which were not possible before.
我们在此报告一种高效且高度可重复的递送系统，该系统使用一种改进的生物射弹转化装置，可促进β-葡萄糖醛酸酶（GUS）在培养的烟草悬浮细胞叶绿体中的瞬时表达。将收集在滤纸上的培养烟草细胞用涂有pUC118或pBI101.3（阴性对照）、pBI505（阳性核对照）或叶绿体表达载体（pHD203-GUS）的钨颗粒轰击，并检测GUS活性。用pUC118或pBI101.3轰击的细胞未检测到GUS活性。用pBI505轰击的细胞表现出高水平的表达，蓝色均匀分布在整个转化体的细胞质中。pHD203-GUS仅在叶绿体中表达。我们基于以下几点得出这一结论：i）叶绿体表达载体中使用的启动子的原核性质；ii）GUS染色延迟；iii）蓝色在形状和大小上与质体相对应的亚细胞区室中的定位；iv）使用聚乙二醇介导的原生质体转化确认pHD203-GUS的细胞器特异性表达。与更常用的火药驱动装置相比，使用改进的氦驱动生物射弹装置可使叶绿体转化效率显著提高（约200倍）。以GUS作为报告基因并使用改进的生物射弹装置，建立了最佳轰击条件，每个培养皿始终能产生数百个瞬时叶绿体转化体。发现在轰击和孵育培养基中添加渗透压调节剂（0.55 M山梨醇和0.55 M甘露醇）可进一步提高叶绿体转化效率（20倍）。该系统为在高等植物叶绿体中引入和表达质粒DNA提供了一种高效机制，并且GUS作为有效标记基因这一事实使得许多以前无法进行的遗传研究成为可能。
