Table_2_Genome-Wide Identification and Expression Analysis of Metal Tolerance Protein Gene Family in Medicago truncatula Under a Broad Range of Heavy Metal Stress.DOCX

Metal tolerance proteins (MTPs) encompass plant membrane divalent cation transporters to specifically participate in heavy metal stress resistance and mineral acquisition. However, the molecular behaviors and biological functions of this family in Medicago truncatula are scarcely known. A total of 12 potential MTP candidate genes in the M. truncatula genome were successfully identified and analyzed for a phylogenetic relationship, chromosomal distributions, gene structures, docking analysis, gene ontology, and previous gene expression. M. truncatula MTPs (MtMTPs) were further classified into three major cation diffusion facilitator (CDFs) groups: Mn-CDFs, Zn-CDFs, and Fe/Zn-CDFs. The structural analysis of MtMTPs displayed high gene similarity within the same group where all of them have cation_efflux domain or ZT_dimer. Cis-acting element analysis suggested that various abiotic stresses and phytohormones could induce the most MtMTP gene transcripts. Among all MTPs, PF16916 is the specific domain, whereas GLY, ILE, LEU, MET, ALA, SER, THR, VAL, ASN, and PHE amino acids were predicted to be the binding residues in the ligand-binding site of all these proteins. RNA-seq and gene ontology analysis revealed the significant role of MTP genes in the growth and development of M. truncatula. MtMTP genes displayed differential responses in plant leaves, stems, and roots under five divalent heavy metals (Cd2+, Co2+, Mn2+, Zn2+, and Fe2+). Ten, seven, and nine MtMTPs responded to at least one metal ion treatment in the leaves, stems, and roots, respectively. Additionally, MtMTP1.1, MtMTP1.2, and MtMTP4 exhibited the highest expression responses in most heavy metal treatments. Our results presented a standpoint on the evolution of MTPs in M. truncatula. Overall, our study provides a novel insight into the evolution of the MTP gene family in M. truncatula and paves the way for additional functional characterization of this gene family.

金属耐受蛋白（Metal tolerance proteins, MTPs）是一类定位于植物细胞膜的二价阳离子转运蛋白，可特异性参与重金属胁迫抗性调控与矿质营养获取过程。然而，蒺藜苜蓿（Medicago truncatula）中该基因家族的分子行为与生物学功能仍鲜有研究报道。本研究从蒺藜苜蓿基因组中成功鉴定出12个潜在MTP候选基因，并对其开展系统发育关系、染色体分布、基因结构、分子对接分析、基因本体（Gene Ontology, GO）注释及已有基因表达数据的整合分析。蒺藜苜蓿MTPs（MtMTPs）可进一步划分为3大类阳离子扩散促进因子（Cation Diffusion Facilitator, CDFs）亚家族：锰偏好型CDFs（Mn-CDFs）、锌偏好型CDFs（Zn-CDFs）及铁/锌偏好型CDFs（Fe/Zn-CDFs）。结构分析显示，同亚家族的MtMTPs基因结构相似度较高，且均包含阳离子外排结构域（cation_efflux domain）或ZT二聚体（ZT_dimer）结构域。顺式作用元件分析表明，多种非生物胁迫与植物激素均可诱导多数MtMTP基因的转录本积累。在所有MTP蛋白中，PF16916为其特异性结构域；同时预测发现，GLY、ILE、LEU、MET、ALA、SER、THR、VAL、ASN及PHE等氨基酸残基为上述蛋白配体结合位点的关键结合残基。RNA测序（RNA-seq）与基因本体分析揭示了MTP基因在蒺藜苜蓿生长发育过程中的重要调控作用。在5种二价重金属离子（Cd²+、Co²+、Mn²+、Zn²+及Fe²+）胁迫处理下，MtMTP基因在植株叶片、茎秆与根系中均呈现出差异化表达响应。其中，分别有10个、7个及9个MtMTP基因在叶片、茎秆与根系中至少对一种金属离子处理产生响应。此外，MtMTP1.1、MtMTP1.2与MtMTP4在多数重金属处理组中呈现出最高的表达响应幅度。本研究结果为蒺藜苜蓿MTP家族的进化分析提供了新的视角。总体而言，本研究为蒺藜苜蓿MTP基因家族的进化研究提供了全新见解，也为该家族后续的功能鉴定研究奠定了重要基础。