农业生物技术学报2024，Vol.32Issue(11)：2498-2510,13.DOI:10.3969/j.issn.1674-7968.2024.11.005
甘蓝型油菜泛素结合酶BnaPHO2基因克隆及表达分析
Cloning and Expression Analysis of Ubiquitin-conjugating Enzyme BnaPHO2 Gene in Brassica napus
摘要
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 6xHis 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.关键词
甘蓝型油菜/PHO2(PHOSPHATE2)/泛素结合酶/原核表达Key words
Brassica napus/PHOSPHATE2(PHO2)/Ubiquitin-conjugating enzyme/Prokaryotic expression分类
农业科技引用本文复制引用
王明,山清影,张晶晶,杜坤..甘蓝型油菜泛素结合酶BnaPHO2基因克隆及表达分析[J].农业生物技术学报,2024,32(11):2498-2510,13.基金项目
国家自然科学基金(32172019 （）
U20A2028) （）
江苏省高等教育教改研究课题(2021JSJG029) （2021JSJG029）
