Table_2_Comparative Genomics, Whole-Genome Re-sequencing and Expression Profile Analysis of Nucleobase:Cation Symporter 2 (NCS2) Genes in Maize.DOC

Nucleobase:cation symporter 2 (NCS2) proteins are important for the transport of free nucleobases, participating in diverse plant growth and developmental processes, as well as response to abiotic stress. To date, a comprehensive analysis of the NCS2 gene family has not been performed in maize. In this study, we conducted a comparative genomics analysis of NCS2 genes in 28 plant species, ranging from aquatic algae to land plants, concentrating mainly on maize. Gene duplication events contributed to the expansion of NCS2 genes from lower aquatic plants to higher angiosperms, and whole-genome/segmental and single-gene duplication events were responsible for the expansion of the maize NCS2 gene family. Phylogenetic construction showed three NCS2 subfamilies, I, II, and III. According to homology-based relationships, members of subfamily I are NCS2/AzgA-like genes, whereas those in subfamilies II and III are NCS2/NATs. Moreover, subfamily I exhibited ancient origins. A motif compositional analysis showed that one symbolic motif (motif 4) of the NCS2/NAT genes was absent in subfamily I. In maize, three NCS2/AzgA-like and 21 NCS2/NAT genes were identified, and purifying selection influenced the duplication of maize NCS2 genes. Additionally, a population genetic analysis of NCS2 genes revealed that ZmNCS2–21 showed the greatest diversity between the 78 inbred and 22 wild surveyed maize populations. An expression profile analysis using transcriptome data and quantitative real-time PCR revealed that NCS2 genes in maize are involved in diverse developmental processes and responses to abiotic stresses, including abscisic acid, salt (NaCl), polyethylene glycol, and low (4°C) and high (42°C) temperatures. ZmNCS2 genes with relatively close relationships had similar expression patterns, strongly indicating functional redundancy. Finally, ZmNCS2–16 and ZmNCS2–23 localize in the plasma membrane, which confirmed their predicted membrane structures. These results provide a foundation for future studies regarding the functions of ZmNCS2 proteins, particularly those with potentially important roles in plant responses to abiotic stresses.

碱基阳离子同向转运蛋白2（Nucleobase:cation symporter 2，NCS2）是一类负责游离碱基转运的重要蛋白，广泛参与植物生长发育的各类进程，并响应非生物胁迫。迄今为止，尚未在玉米中开展针对NCS2基因家族的系统性全面分析。本研究针对从水生藻类到陆生植物的28个植物物种中的NCS2基因开展比较基因组学分析，重点聚焦玉米。基因复制事件推动了NCS2基因从低等水生植物到高等被子植物的家族扩张，而玉米NCS2基因家族的扩张则主要由全基因组/片段复制以及单基因复制事件介导。系统发育构建结果显示，NCS2家族可划分为I、II、III三个亚家族。基于同源性关系，I亚家族成员属于NCS2/AzgA类同源基因，而II、III亚家族成员则属于NCS2/NAT型基因。此外，I亚家族具有更为古老的进化起源。基序组成分析结果显示，NCS2/NAT型基因的标志性基序（基序4）在I亚家族中完全缺失。在玉米中，本研究共鉴定得到3个NCS2/AzgA类同源基因与21个NCS2/NAT型基因，且纯化选择作用影响了玉米NCS2基因的复制过程。此外，针对NCS2基因的群体遗传学分析显示，在78份自交玉米种质与22份调研野生玉米种质的群体中，ZmNCS2-21的遗传多样性水平最高。利用转录组数据与实时定量PCR开展的表达谱分析显示，玉米NCS2基因参与多种发育进程，并响应包括脱落酸、盐（NaCl）、聚乙二醇以及低温（4℃）与高温（42℃）在内的各类非生物胁迫。亲缘关系较为接近的ZmNCS2基因具有高度相似的表达模式，这强烈提示其存在功能冗余性。最后，亚细胞定位实验证实ZmNCS2-16与ZmNCS2-23定位于质膜，验证了其预测的膜定位结构特征。本研究结果为后续ZmNCS2蛋白的功能研究，尤其是其在植物响应非生物胁迫过程中的潜在关键作用研究，奠定了坚实的理论基础。