【摘要】 PHO2 (PHOSPHATE2)作为磷转运蛋白的负调控因子，通过行使其泛素结合酶活性在植物体内磷的代谢平衡中发挥作用。本研究以拟南芥(Arabidopsis thaliana) PHO2为基础，探究甘蓝型油菜(Brassica napus)中与之同源性最高的BnaPHO2蛋白特性及其功能。以生物信息学数据为参考，对BnaPHO2基因表达模式、亚细胞定位以及酵母自激活活性进行分析，并利用原核表达系统获得BnaPHO2融合蛋白。结果显示，BnaPHO2蛋白由865个氨基酸组成，分子量为95.7 kD，蛋白理化特性稳定；保守结构区域含有与泛素蛋白和泛素连接酶共价结合的位点；在系统发育上与同科的白菜(Brassica rapa)和甘蓝(Brassica oleracea)的亲缘关系较近。qRT-PCR结果显示，BnaPHO2基因在油菜的根、叶等营养器官中的相对表达量较高，且积极响应高磷胁迫。烟草(Nicotiana tabacum)瞬时转化结果显示，BnaPHO2定位于细胞膜和细胞核。酵母自激活检测表明，BnaPHO2全长无自激活活性。在28℃下经1 mmol/L IPTG诱导16 h后，分别获得N端融合6×His标签和C端融合谷胱甘肽巯基转移酶(glutathione S-transferase, GST)标签的BnaPHO2纯化蛋白。BnaPHO2蛋白特性的阐明及异源蛋白的表达为后续开展体外互作、泛素化等研究提供理论参考和基础资料。更多还原

【Abstract】 PHO2(PHOSPHATE2) functions as a negative regulator of phosphorus transport proteins and plays a role in the metabolic homeostasis of phosphorus in plants by exercising its ubiquitin-binding enzyme activity. In this study, Arabidopsis thaliana PHO2 was used as the basis to investigate properties and functions of BnaPHO2 protein, which has the highest homology with it in Brassica napus. With biological information data as a reference, the BnaPHO2 gene expression mode, subcellular localization, and yeast self-activation activity were analyzed, and the BnaPHO2 fusion protein was obtained using the prokaryotic expression system. The results showed that the BnaPHO2 protein consisted of 865 amino acids with a molecular weight of 95.7 kD, and the physicochemical properties of the protein were stable. The conserved structural region contained sites covalently bound to ubiquitin proteins and ubiquitin ligases, and phylogenetically it was closely related to cabbage(Brassica rapa) and kale(Brassica oleracea) of the same family. The results of qRT-PCR showed that the relative expression of BnaPHO2 gene was high in nutrient organs such as roots and leaves, and it positively responded to high phosphorus stress. Tobacco(Nicotiana tabacum) transient transformation results showed that BnaPHO2 was localized in the cell membrane and nucleus. Yeast self-activation assay showed that BnaPHO2 had no self-activating activity in its full length. BnaPHO2 was induced by 1 mmol/L IPTG at 28 ℃ for 16 h, and the purified proteins were obtained with N-terminal fusion of 6×His tag or C-terminal fusion of glutathione S-transferase(GST) tag. The elucidation of BnaPHO2 protein characterization and heterologous protein expression provides theoretical reference and basic information for subsequent studies of in vitro interactions and ubiquitination.更多还原