Cloning and Expression Analysis of Ubiquitin-conjugating Enzyme BnaPHO2 Gene in Brassica napus
WANG Ming1, SHAN Qing-Ying1, ZHANG Jing-Jing1, DU Kun2,*
1 College of Biosciences and Biotechnology, Yangzhou University, Yangzhou 225009, China; 2 Basic Experimental Teaching Center of Life Sciences, Yangzhou University, Yangzhou 225009, China
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.
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