拟南芥DUF647家族成员RUS4植物表达载体的构建及亚细胞定位

ROOT UV-B SENSITIVE4 (RUS4)是拟南芥DUF647蛋白家族的一个功能未知的成员。RUS1和RUS2曾经报道和根UV-B-感应途径相关联,并在拟南芥早期幼苗形态发生和发育中发挥重要作用。为了探究RUS4的分子功能,本研究对RUS4蛋白进行了亚细胞定位分析。首先,我们通过Gateway TOPO载体系统,构建了融合蛋白表达载体pMDC83-RUS4;然后,通过农杆菌介导的花苞转化法,获得了转化pMDC83-RUS4的转基因拟南芥株系;对转基因植物叶肉原生质体的荧光显微镜观察显示,RUS4-GFP信号与叶绿体的自发荧光共定位,说明RUS4蛋白定位叶绿体中。该结果为进一步研究RUS4的分子功能奠定了良好的基础。

Construction of the Plant Expression Vector and Subcellular Localization of DUF647 Family Member RUS4 in Arabidopsis thaliana

ROOT UV-B SENSITIVE4(RUS4) is a member of the Domain of Unknown Function 647(DUF647)protein family that is conserved in diverse eukaryotic organisms, but has no known biological function. The DUF6-47-containing proteins RUS1 and RUS2 have previously been associated with root UV-B-sensing pathway that plays a major role in Arabidopsis early seedling morphogenesis and development. Here, we assessed its subcellular localization using a translational fusion to the Aequorea victoria green fluorescent protein GFP(RUS4-GFP). We first created the entry clone using pENTR directional TOPO cloning kit;subsequently, the RUS4 fragment in the entry clone was transferred to pMDC83 destination vector by using the Gateway LR Clonase enzyme mix. Then,we introduced the translational fusion 35 Spro:RUS4-GFP into Arabidopsis(Col-0) plants. The distribution of RUS4-GFP was examined in mesophyll protoplasts. We found that the RUS4-GFP signal is highly localized to the chloroplasts, confirming that the protein is chloroplast localized. This study will facilitate analysis of the function and molecular mechanisms by which RUS4 activity regulates Arabidopsis growth and development.