[Cloning and expression analysis of HrNRAMP1 in Hippophae rhamnoides subsp. sinensis Rousi]

The natural resistance-associated macrophage protein (NRAMP) family plays a crucial role in the transport of divalent metal ions across various species. In this study, we cloned HrNRAMP1 from sea buckthorn (Hippophae rhamnoides subsp. sinensis Rousi) and investigated its expression pattern under lead stress, aiming to provide a theoretical basis for breeding sea buckthorn varieties with reduced lead accumulation. The full-length HrNRAMP1 was cloned via PCR with reference to the genomic data of H. rhamnoides, which was followed by bioinformatics analysis. The subcellular localization and expression patterns of this gene under varying lead stress conditions were examined by transient expression in tobacco leaves and real-time quantitative PCR. The results indicated that the full-length HrNRAMP1 was 1 539 bp, encoding a hydrophobic and stable protein composed of 512 residues. Bioinformatics analysis revealed that the secondary structure of the deduced protein HrNRAMP1 was predominated by α-helices and lacked a signal peptide, which suggested that HrNRAMP1 functioned as a membrane protein with 11 predicted transmembrane domains. Multiple sequence alignment with homologous genes from 13 species demonstrated that HrNRAMP1 contained the solute carrier family 5/6 (SLC5/6) domain, a conserved characteristic domain of the NRAMPgene family. The phylogenetic analysis indicated that HrNRAMP1 was most closely related to members in plants of Solanaceae. The results of tobacco protoplast transformation indicated that HrNRAMP1 was specifically localized to the plasma membrane. RNA sequencing data (RNA-seq) and real time fluorescence quantitative polymerase chain reaction (qRT-PCR) validation showed that HrNRAMP1 expression was initially upregulated and subsequently downregulated with the rise in lead ion concentration, peaking at a lead concentration of 2 000 mg/kg. This study suggests that HrNRAMP1 may play a key role in regulating lead ion transport and responding to heavy metal stress in plants. The research suggests that HrNRAMP1 may play a key role in regulating lead ion transport and responding to heavy metal stress within plants. This finding lays a foundation for further exploration into the molecular regulatory mechanisms by which sea buckthorn responds to heavy metal lead stress, as well as for genetic engineering efforts aimed at developing new sea buckthorn cultivars with reduced accumulation of heavy metal lead.