Transient Gene Expression in Guard Cell Chloroplasts of Tobacco Using ArF Excimer Laser Microablation
Transient Gene Expression in Guard Cell Chloroplasts of Tobacco Using ArF Excimer Laser Microablation
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DOI：
10.1263/jbb.106.194
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发表时间：
2008-08-01
影响因子：
2.8
通讯作者：
Kobayashi, Akio
中科院分区：
文献类型：
--
作者：
Kajiyama, Shin'ichiro;Joseph, Benesh;Kobayashi, Akio
本文报道了一种利用激光微消融技术将基因导入烟草叶绿体的新方法。将质粒pLD 200-GFP导入烟草（Nicotiana tabacum cv.孵育2-3 d后，在叶绿体中检测到黄原胶保卫细胞和瞬时GFP表达。该技术使用氟化氩（ArF）准分子激光器在质粒包被的金微粒存在下照射细胞表面4 μ m（2）的区域。在激光消融前预处理保卫细胞以促进气孔关闭导致细胞存活率显著增加，并且在试验次数基础上获得了2-3%的瞬时表达率。我们的方法具有独特的优势，如避免了对细胞活力产生不利影响的费力的预处理，以及将转基因特异性递送到复杂叶组织中的所需细胞中。该技术是一种潜在的工具，用于细胞特异性瞬时基因表达研究，以阐明基因调控和表达。
In this paper, we report a novel method for delivering genes into chloroplasts of tobacco cells using laser microablation. The plasmid pLD200-GFP was introduced into chloroplasts of Nicotiana tabacum cv. Xanthi guard cells and transient GFP expression was detected in the chloroplasts after 2-3 d of incubation. The technique uses an argon fluoride (ArF) excimer laser to perforate the cell surface in a 4 mu m(2) area in the presence of plasmid coated gold microparticles. Pretreatment of guard cells to promote stomatal closure prior to laser ablation resulted in a significant increase in the survival rate of cells and a transient expression rate of 2-3% in trial number basis was archived. Our method has unique advantages such as avoiding laborious pretreatments that adversely affect cell viability and specific delivery of transgenes into a desired cell in complex leaf tissue. This technique is a potential tool for cell specific transient gene expression studies for elucidation of gene regulation and expression.