沙棘NRAMP基因家族鉴定及铅胁迫下表达分析

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

摘要/Abstract

摘要：

【目的】NRAMP（natural resistance-associated macrophage protein）基因家族是一类广泛存在于植物中的天然抗性相关巨噬蛋白，对植物中二价金属离子的细胞转运具有关键作用。为挖掘沙棘NRAMP基因家族响应重金属铅胁迫的关键基因，阐明沙棘响应重金属铅胁迫的分子机制。【方法】利用生物信息学技术鉴定沙棘NRAMP家族成员，对编码该家族蛋白的理化性质、系统进化、基因结构、保守基序、顺式作用元件、不同浓度铅胁迫下的NRAMP基因表达谱、种内种间共线性、蛋白互作网络进行综合分析。【结果】NRAMP基因家族的11个成员不均匀分布于7个染色体上，被分为Group1-Group3三大类群。NRAMP基因家族成员的启动子具有丰富的激素应答、分生组织表达和环境应激响应元件。转录组分析表明，沙棘NRAMP基因家族的11个成员在不同浓度的铅离子胁迫下呈现差异表达，其中8个基因在1 000 mg/kg的铅胁迫下显著下调，7个基因在高浓度铅胁迫（5 000mg/kg）下表达上调，并对选定的6个沙棘NRAMP基因进行了RT-qPCR验证。沙棘与单子叶植物小麦NRAMP基因家族的种间共线性比率高于双子叶植物拟南芥。蛋白质互作网络分析表明，沙棘NRAMP基因家族成员HrLNRAMP8可能主要通过乙烯途径调控重金属的吸收转运。【结论】在全基因组范围内从沙棘中系统鉴定得到11个HrLNRAMP家族成员。Group2亚族成员基因结构最复杂且一致性较低；所有的沙棘NRAMP成员都含有NRAMP家族特有的转运基序motif1。不同基因家族成员在铅胁迫条件下的表达具有偏好性，该基因家族通过响应重金属铅离子胁迫来调控基因表达水平，从而降低重金属胁迫带来的伤害。

关键词: 沙棘, 基因家族, 生物信息学分析, 重金属胁迫, 表达分析

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

李嘉欣, 李鸿燕, 刘丽娥, 张恬, 周武. 沙棘NRAMP基因家族鉴定及铅胁迫下表达分析[J]. 生物技术通报, 2024, 40(5): 191-202.

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.

图/表 11

表1 沙棘NRAMP蛋白理化性质与亚细胞定位

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

图1 沙棘NRAMP基因家族染色体定位

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

图2 沙棘、拟南芥、水稻NRAMP蛋白的系统进化分析不同分支颜色代表不同的亚族。红色三角标注的是沙棘NRAMP家族成员、蓝色五角星标注的是拟南芥NRAMP家族成员、黄色圆圈标注的是水稻NRAMP家族成员

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

图3 沙棘NRAMP蛋白的多重比对颜色越深，序列一致性程度越高

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

图4 NRAMP家族外显子-内含子与motif分析 A：沙棘NRAMP家族结构与保守基序；B：沙棘NRAMP家族保守基序组成

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

图5 沙棘HrNRAMP基因启动子顺式作用元件分析

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

图6 沙棘NRAMP基因在不同浓度铅胁迫下的表达分析 CK：对照组，正常生长的中国沙棘幼苗；Pb_500、Pb_1000、Pb_2000、Pb_5000：实验组，铅胁迫浓度依次为500、1 000、2 000 和5 000 mg/kg。下同

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

图7 铅胁迫条件下HrLNRAMP家族部分基因相对表达量数据代表3个生物学重复的平均值，竖线表示标准差，星号表示相应基因在对照组和胁迫组之间差异显著（** P <0.01）

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）

图8 沙棘NRAMP家族种内共线性分析

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

图9 沙棘NRAMP家族种间共线性分析

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

图10 沙棘NRAMP基因的蛋白互作网络线条的粗细代表不同互作强度，线条粗，互作强度大，线条细，互作强度小。其中蓝色代表拟南芥中所有与沙棘存在互作关系的蛋白；红色仅代表存在互作关系的NRAMP蛋白

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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