中国沙棘HrNRAMP1基因克隆及表达分析

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

摘要:

自然抗性相关巨噬细胞蛋白家族(natural resistance-associated macrophage protein, NRAMP)在多种物种二价金属离子转运中发挥着重要作用。为给沙棘低铅积累新品种的分子育种提供理论依据，克隆中国沙棘(Hippophae rhamnoides subsp. sinensis Rousi)的HrNRAMP1基因并探究在重金属铅胁迫下沙棘中该基因的表达模式。参考沙棘基因组数据，采用PCR技术克隆全长HrNRAMP1基因，并对其进行生物信息学分析。利用烟草叶片瞬时表达、实时荧光定量技术分析其亚细胞定位及在不同铅浓度胁迫下的表达模式。结果显示，沙棘HrNRAMP1基因全长1 539 bp，编码512个氨基酸，为疏水性稳定蛋白；生物信息学分析表明，HrNRAMP1蛋白二级结构中α-螺旋占比最大且不含信号肽，预测HrNRAMP1为含11个跨膜区的膜蛋白；通过与13个物种同源基因进行的多序列比对表明HrNRAMP1蛋白具有NRAMP基因家族的典型保守结构域[溶质载体家族5/6 (solute carrier family 5/6, SLC5/6)共有的LeuT折叠区]，系统发育树显示其与茄科亲缘关系最近；亚细胞定位实验表明，HrNRAMP1蛋白定位于质膜；转录组数据(RNA sequencing data, RNA-seq)分析及定量逆转录聚合酶链式反应(real time fluorescence quantitative polymerase chain reaction, qRT-PCR)实验验证结果表明，HrNRAMP1表达量总体上随着环境中铅离子浓度的升高呈现先上升后下调，在铅浓度达2 000 mg/kg时，基因表达量最大，表明HrNRAMP1蛋白可能在调控植物体内铅离子运输及应对重金属铅胁迫中发挥重要作用。本研究为深入解析沙棘响应重金属铅胁迫的分子调控机制及利用基因工程手段培育重金属铅低积累性沙棘新品种提供了研究基础和分子靶标。

Abstract:

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 NRAMP gene 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.