烟草DXS基因的克隆及其亚细胞定位分析  

Cloning and Subcellular Localization of DXS Gene in Tobacco

作　　者：李尊强 王春军 杨爱国[2] 丁安明[2] 冯全福[2] 徐剑 焦惠鹏 商堃宇 

机构地区：[1]牡丹江烟草科学研究所,黑龙江牡丹江157011 [2]中国农业科学院烟草研究所,烟草行业烟草基因资源利用重点实验室,山东青岛266101 [3]哈尔滨烟叶公司肇东分公司,黑龙江肇东151100

出　　处：《安徽农业科学》2013年第30期11957-11960,共4页Journal of Anhui Agricultural Sciences

基　　金：国家烟草专卖局科技重点项目"优质抗病烤烟新品种选育及育种技术研究"资助(110201002002)

摘　　要：[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 was to amplify the 1-deoxidization xylulose-5-phosphate synthetase gene （DXS） from the tobacco cDNA pool and conduct subcellular localization.[Method] DXS gene was isolated and cloned by RT-PCR method,after the terminator being removed,it was recombined with the green fluorescent protein （GFP） gene and cloned to expression vector for sublocalization.The expression vector was then transformed into Agrobacterium strains GV3101,which was used for injection and infection of the seeding of N.benthaminana for instant expression.The laser confocal scanning microscope was used to survey the expressing of the recombinant gene in transgene plants.[Result] The results showed that the DXS and GFP fusion protein could only located in chloroplast,DXS had a tissue specific expression in chloroplast of the tobacco mesophyll cell （especially in guard cells）.[Conclusion] DXS gene was located in chloroplast,which provides theoretical basis for the future function analysis of the DXS gene.

关 键 词：1-脱氧木酮糖-5-磷酸合成酶(DXS) 绿色荧光蛋白(GFP) 亚细胞定位 叶绿体 

分 类 号：S572[农业科学—烟草工业]