盐穗木功能未知蛋白 (HcUKPP)的亚细胞定位

藜科的极端盐生植物盐穗木（Halostachys caspica）的高盐胁迫抑制差减文库中有39%的功能未知蛋白（proteins with obscure features, POFs），利用亚细胞定位分析可以初步判断其可能的功能.将盐穗木的1个POF-cDNA序列HcUKPP的编码区构建至pCAMBIA1301-GFP植物表达载体上，冻融法将重组质粒pCAMBIA1301-HcUKPP-GFP转化农杆菌EH105A，利用花序浸染法将基因导入拟南芥，经潮霉素筛选获得T1代阳性幼苗.通过激光扫描共聚焦显微镜观察转基因拟南芥植株的根部细胞. 结果显示,表达GFP蛋白的对照转基因植株中，绿色荧光在细胞核、细胞膜以及细胞质中均能检测到，而表达HcUKPP-GFP融合蛋白的转基因植株中，绿色荧光只在细胞质膜上表达，说明HcUKPP蛋白为细胞质膜相关蛋白.本研究为深入探讨盐穗木未知蛋白的功能奠定了基础.

Subcellular Localization of Function Unknown Protein (HcUKPP) from Halostachys caspica

The suppression subtraction hybridization (SSH) library generated from Halostachys caspica, an extreme halophyte in Chenopodiaceae, following high salt treatment, 39% of the coded proteins with obscure features (POFs) are with unknown function. In this study a POF cDNA from the SSH named Hcukpp was inserted into the plant expression vector pCAMBIA1301-GFP, for testing its predicted subcellular localization. The plasmid was introduced into Agrobacterium tumefaciens strain EH105A using the freeze-thaw method, then transferred into Arabidopsis thaliana by the floral dipping method. The T1 positive transgenic plants were screened with hygromycin. Fluorescence in the selected transgenic seedlings was observed by laser scanning confocal microscopy. The results showed that in the control transgenic Arabidopsis, the GFP fluorescence signal appeared in cell nuclei, cytoplasm and plasma membrane; in the HcUKPP-GFP transgenic Arabidopsis, the fluorescence was only at the plasma membranes. It suggested that HcUKPP is a membrane associated protein.