DataSheet_1_Genome-wide identification and characterization of the NPF genes provide new insight into low nitrogen tolerance in Setaria.zip

IntroductionNitrogen (N) is essential for plant growth and yield production and can be taken up from soil in the form of nitrate or peptides. The NITRATE TRANSPORTER 1/PEPTIDE TRANSPORTER family (NPF) genes play important roles in the uptake and transportation of these two forms of N.MethodsBioinformatic analysis was used to identify and characterize the NPF genes in Setaria. RNA-seq was employed to analyze time-series low nitrate stress response of the SiNPF genes. Yeast and Arabidopsis mutant complementation were used to test the nitrate transport ability of SiNRT1.1B1 and SiNRT1.1B2.ResultsWe identified 92 and 88 putative NPF genes from foxtail millet (Setaria italica L.) and its wild ancestor green foxtail (Setaria viridis L.), respectively. These NPF genes were divided into eight groups according to their sequence characteristics and phylogenetic relationship, with similar intron-exon structure and motifs in the same subfamily. Twenty-six tandem duplication and 13 segmental duplication events promoted the expansion of SiNPF gene family. Interestingly, we found that the tandem duplication of the SiNRT1.1B gene might contribute to low nitrogen tolerance of foxtail millet. The gene expression atlas showed that the SiNPFs were divided into two major clusters, which were mainly expressed in root and the above ground tissues, respectively. Time series transcriptomic analysis further revealed the response of these SiNPF genes to short- and long- time low nitrate stress. To provide natural variation of gene information, we carried out a haplotype analysis of these SiNPFs and identified 2,924 SNPs and 400 InDels based on the re-sequence data of 398 foxtail millet accessions. We also predicted the three-dimensional structure of the 92 SiNPFs and found that the conserved proline 492 residues were not in the substrate binding pocket. The interactions of SiNPF proteins with NO3− were analyzed using molecular docking and the pockets were then identified. We found that the SiNPFs- NO3− binding energy ranged from -3.8 to -2.7 kcal/mol.DiscussionTaken together, our study provides a comprehensive understanding of the NPF gene family in Setaria and will contribute to function dissection of these genes for crop breeding aimed at improving high nitrogen use efficiency.

氮素（N）对于植物的生长与产量生产至关重要，植物可从土壤中以硝酸盐或肽的形式吸收氮素。NITRATE TRANSPORTER 1/PEPTIDE TRANSPORTER家族（NPF）基因在两种氮素形式的吸收与运输过程中发挥着至关重要的作用。
方法学上，我们运用生物信息学分析手段，对高粱（Setaria）中的NPF基因进行了鉴定与特征描述。通过RNA测序技术，分析了SiNPF基因在低硝酸盐胁迫时间序列中的应激响应。利用酵母和拟南芥突变体互补实验，测试了SiNRT1.1B和SiNRT1.1B2的硝酸盐运输能力。
结果方面，我们从高粱（Setaria italica L.）及其野生祖先绿色狗尾草（Setaria viridis L.）中分别鉴定出92和88个潜在的NPF基因。这些NPF基因根据其序列特征和系统发育关系被划分为八个组，同一亚家族内具有相似的内含子-外显子结构和基序。二十六次串联重复和十三次片段重复事件促进了SiNPF基因家族的扩张。有趣的是，我们发现SiNRT1.1B基因的串联重复可能对高粱的低氮耐受性有所贡献。基因表达图谱显示，SiNPF基因被分为两大集群，分别主要在根系和地上组织中表达。时间序列转录组分析进一步揭示了这些SiNPF基因对短时和长时间低硝酸盐胁迫的响应。为了提供基因的自然变异信息，我们对这些SiNPF基因进行了单倍型分析，并基于398个高粱品种的重组测序数据，鉴定出2,924个单核苷酸多态性（SNPs）和400个插入/缺失（InDels）。我们还预测了92个SiNPF基因的三维结构，并发现保守的脯氨酸492残基不在底物结合口袋中。通过分子对接技术分析了SiNPF蛋白与NO3−的相互作用，并确定了口袋结构。我们发现SiNPFs-NO3−的结合能介于-3.8至-2.7 kcal/mol之间。
讨论中，本研究对高粱中的NPF基因家族进行了全面探讨，并将有助于这些基因的功能解析，为提高作物氮肥利用效率的育种研究提供理论支持。