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[目的]从烟草cDNA中扩增出1-脱氧木酮糖-5-磷酸合成酶(DXS)基因(去除终止密码子TAA),并对其进行亚细胞定位分析。[方法]利用RT-PCR技术分离、克隆DXS,将烟草DXS基因与绿色荧光蛋白(GFP)基因重组构建亚细胞定位表达载体,转入根癌农杆菌LBA4404后注射侵染本氏烟烟草幼苗进行瞬时表达;并利用激光共聚焦扫描显微镜观察转GFP植株的表达情况。[结果]显微镜观察发现,DXS和GFP融合蛋白仅在本氏烟叶肉细胞(尤其是保卫细胞)的叶绿体中产生绿色荧光,DXS基因在叶绿体中特异表达。[结论]DXS定位在叶绿体中,为接下来对DXS基因功能研究提供了理论依据。
[Objective] The aim of this study was to amplify 1-deoxyxylulose 5-phosphate synthase (DXS) gene (remove the stop codon TAA) from tobacco cDNA and perform subcellular localization analysis. [Method] DXS was isolated and cloned by RT-PCR. The DXS gene and green fluorescent protein (GFP) gene of tobacco were recombined to construct the subcellular localization expression vector. After being transformed into Agrobacterium tumefaciens LBA4404, Transient expression; and the use of laser scanning confocal microscopy GFP transgenic plants expression. [Result] Microscopic examination showed that DXS and GFP fusion protein produced green fluorescence only in the chloroplast of Benbach’s leaf cells (especially guard cells), and DXS gene was specifically expressed in chloroplast. [Conclusion] The mapping of DXS in chloroplast provided the theoretical basis for the study of DXS gene function.