Identification and Expression Analysis of the NRAMP Family in Seabuckthorn Under Lead Stress

doi:10.13560/j.cnki.biotech.bull.1985.2023-1161

Abstract

Abstract:

【Objective】The NRAMP（natural resistance-associated macrophage protein）gene family plays a pivotal role in the transport of divalent metal ions at the cellular level. To identify the key NRAMP family genes in seabuckthorn (Hippophae rhamnoides) that confer resistance to heavy metal lead stress and to elucidate the associated molecular mechanisms.【Method】Using bioinformatics techniques, we identified seabuckthorn's NRAMP gene family members and comprehensively analyzed their physicochemical properties, phylogenetic relationships, gene structures, conserved motifs, cis-acting elements, expression profiles under varying Pb stress concentrations, intraspecific and interspecific variability, and protein interaction networks. 【Result】The 11 NRAMP gene family members were distributed across seven chromosomes and categorized into Groups 1-Group 3. Promoters of the NRAMP genes contained numerous hormonal, meristematic, and environmental stress response elements. Transcriptome analysis indicated differential expressions of NRAMP genes in seabuckthorn under varying lead ion concentrations, with eight genes significantly down-regulated at 1 000 mg/kg and seven up-regulated at 5 000 mg/kg lead levels. RT-qPCR validated the expression patterns of six chosen NRAMP genes in seabuckthorn. The seabuckthorn NRAMP genes showed greater interspecific co-linkage with monocotyledonous wheat than with the dicot Arabidopsis thaliana. Protein network analysis suggested that HrLNRAMP8 potentially regulates heavy metal uptake and translocation primarily via the ethylene pathway. 【Conclusion】A systematic identification of 11 HrLNRAMP family members from seabuckthorn was achieved at the whole-genome level. Members of the Group2 subfamily have the most complex gene structures and lower consistency. All seabuckthorn NRAMP members contain the unique transport domain motif1 of the NRAMP family. The study demonstrates preferential expression of different gene family members under lead stress conditions. This gene family regulates gene expression levels in response to heavy metal lead ion stress, thereby mitigating damage caused by heavy metal stress.

Key words: seabuckthorn（Hippophae rhamnoides）, NRAMP gene family, bioinformatics analysis, heavy metal stress, expression analysis

LI Jia-xin, LI Hong-yan, LIU Li-e, ZHANG Tian, ZHOU Wu. Identification and Expression Analysis of the NRAMP Family in Seabuckthorn Under Lead Stress[J]. Biotechnology Bulletin, 2024, 40(5): 191-202.

Figures/Tables 11

Table 1 Physicochemical properties and subcellular localization of seabuckthorn NRAMP protein

基因名称 Gene name	基因ID Gene ID	蛋白序列长度 Protein sequence length/aa	分子量 Molecular weight/kD	理论等电点 Isoelectric point	亚细胞定位 Subcellular localization
HrLNRAMP1	Hiprha1gene15634	512	56.21	4.89	质膜Plas
HrLNRAMP2	Hiprha1gene30069	513	55.52	9.53	质膜Plas
HrLNRAMP3	Hiprha1gene21177	626	69.13	8.50	质膜Plas
HrLNRAMP4	Hiprha1gene09270	1 291	141.46	5.79	质膜Plas
HrLNRAMP5	Hiprha1gene12503	530	58.39	4.93	质膜Plas
HrLNRAMP6	Hiprha1gene10061	532	57.72	9.33	质膜Plas
HrLNRAMP7	Hiprha1gene12853	538	58.68	5.94	质膜Plas
HrLNRAMP8	Hiprha1gene11195	1 295	141.63	6.08	质膜Plas
HrLNRAMP9	Hiprha1gene27351	513	56.33	4.73	质膜Plas
HrLNRAMP10	Hiprha1gene30205	502	54.94	8.50	质膜Plas
HrLNRAMP11	Hiprha1gene16210	327	36.24	6.40	液泡膜Vacu

Fig. 1 Chromosome mapping of the NRAMP gene family in Seabuckthorn

Fig. 2 Phylogenetic analysis of NRAMP proteins in H. rhamnoides, A. thaliana and O. sativa Different branch colors indicate different subfamilies. Red triangles are labeled with H. rhamnoides NRAMP family members, blue pentagrams are labeled with Arabidopsis thaliana NRAMP family members, and yellow circles are labeled with Oryza sativa NRAMP family members

Fig. 3 Multiple alignment of H. rhamnoides NRAMP family proteins The darker the color, the more consistent the sequence

Fig. 4 NRAMP family exon-intron and motif analysis A: Structure and conserved motifs of the NRAMP family in seabuckthorn. B: Conserved motif composition of the NRAMP family in seabuckthorn

Fig. 5 Analysis of cis-acting elements in the promoter of the HrNRAMP gene in seabuckthorn

Fig. 6 Expression analysis of NRAMP gene in H. rhamnoi-des under different concentrations of lead stress CK: Control group, normal-growing seabuckthorn seedlings. Pb_500, Pb_1000, Pb_2000, Pb_5000: Experimental groups, with Pb stress concentrations of 500, 1 000, 2 000, and 5 000 mg/kg. The same below

Fig. 7 Relative expressions of partial genes of HrLNRAMP gene family under lead stress conditions Data are means（±SD）of three biological replicates. Vertical bars indicate standard deviations. Asterisks indicate corresponding genes significantly upregulated or downregulated between the treatment and control（** P < 0.01）

Fig. 8 Intraspecific collinearity analysis of NRAMP gene family of H. rhamnoides

Fig. 9 Interspecific collinearity analysis of NRAMP gene family of H. rhamnoides

Fig. 10 Interaction protein network of HrLNRAMP geness in seabuckthorn Line thickness indicates the interaction strength; thicker lines denote stronger interactions, while thinner lines signify weaker ones. Blue denotes Arabidopsis proteins interacting with seabuckthorn, while red is specific to NRAMP proteins engaging in such interactions

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