高粱高亲和硝酸盐转运蛋白NRT2/3基因家族鉴定、表达与DNA变异分析

Identification, expression and DNA variation analysis of high affinity nitrate transporter NRT2/3 gene family in Sorghum bicolor

摘要:

硝态氮是作物吸收无机氮素的主要形态，硝酸盐转运蛋白2（nitrate transporter 2，NRT2）作为高亲和性的转运蛋白，以硝酸盐作为特异性底物，在可利用的硝酸盐受限时，高亲和性转运系统被激活，在硝酸盐吸收、转运过程中发挥着重要作用。大多数NRT2不能单独转运硝酸盐，需在硝酸盐同化相关蛋白2（nitrate assimilation related protein 2，NAR2）的协助下才能完成硝酸盐的吸收或转运。作物氮利用效率受环境条件影响，品种间存在差异，因此培育高氮素利用效率品种有重大意义。高粱（Sorghum bicolor）具有耐贫瘠特性，对土壤中的氮素吸收和利用效率较高。本研究结合高粱基因组数据库对NRT2/3基因家族成员基因结构、染色体定位、理化性质、二级结构与跨膜结构域、信号肽与亚细胞定位、启动子区顺式作用元件、系统进化、单核苷酸多态性（single nucleotide polymorphism，SNP）的识别与注释及选择压力进行了全面分析。通过生物信息学分析，筛选出5个NRT2s（命名为SbNRT2-1a、2-1b、SbNRT2-2–4）基因和2个NAR2s（SbNRT3-1–2）基因，较谷子略少。分布在3条染色体上，分为4个亚家族，同一亚族中基因结构高度相似；高粱NRT2/3亲水性平均值均为正值，表明均为疏水性蛋白；α-螺旋和无规则卷曲占二级结构总量的比例大于70%；亚细胞定位均在质膜上，其中NRT2s蛋白不含信号肽，NRT3s蛋白含信号肽；进一步对其跨膜结构域进行分析，发现NRT2s家族成员跨膜结构域个数均大于10个，而NRT3s家族成员跨膜结构域个数为2个；高粱与玉米（Zea mays）NRT2/3s的共线性较好；蛋白结构域显示存在MFS_1和NAR2蛋白结构域，可执行高亲和力硝酸盐转运；系统进化树分析可知，高粱与玉米和谷子的NRT2/3基因亲缘关系更近；基因启动子顺式作用元件分析发现，SbNRT2/3基因的启动子区均具有数个植物激素和逆境应答元件，可以响应高粱生长和环境变化；基因表达热图显示低氮条件下在根诱导表达的是SbNRT2-1a、SbNRT2-1b和SbNRT3-1，推测可在高粱根部表达并调控对硝酸盐的吸收或转运过程。在SbNRT2-4和SbNRT2-1a等发现多个非同义SNP变异；选择压力分析表明，高粱NRT2/3基因家族在进化过程中受纯化选择作用。SbNRT2/3基因表达及蚜虫侵染影响与基因在不同组织中的表达分析结果一致，SbNRT2-1b和SbNRT3-1在感染蚜虫品系5-27sug根部表达显著，高粱蚜虫侵染叶片显著降低了SbNRT2-3、SbNRT2-4和SbNRT3-2的表达水平。本研究初步对高粱全基因组NRT2/3基因家族进行鉴定、表达与DNA变异分析，为高粱氮高效研究提供了基础。

Abstract:

Nitrate is the main form of inorganic nitrogen that crop absorbs, and nitrate transporter 2 (NRT2) is a high affinity transporter using nitrate as a specific substrate. When the available nitrate is limited, the high affinity transport systems are activated and play an important role in the process of nitrate absorption and transport. Most NRT2 cannot transport nitrates alone and require the assistance of a helper protein belonging to nitrate assimilation related family (NAR2) to complete the absorption or transport of nitrates. Crop nitrogen utilization efficiency is affected by environmental conditions, and there are differences between varieties, so it is of great significance to develop varieties with high nitrogen utilization efficiency. Sorghum bicolor has high stress tolerance and is more efficient in soil nitrogen uptake and utilization. The S. bicolor genome database was scanned to systematically analyze the gene structure, chromosomal localization, physicochemical properties, secondary structure and transmembrane domain, signal peptide and subcellular localization, promoter region cis-acting elements, phylogenetic evolution, single nucleotide polymorphism (SNP) recognition and annotation, and selection pressure of the gene family members. Through bioinformatics analysis, 5 NRT2 gene members (designated as SbNRT2-1a, SbNRT2-1b, SbNRT2-2, SbNRT2-3, and SbNRT2-4) and 2 NAR2 gene members (designated as SbNRT3-1 and SbNRT3-2) were identified, the number of which was less than that of foxtail millet. SbNRT2/3 were distributed on 3 chromosomes, and could be divided into four subfamilies. The genetic structure of the same subfamilies was highly similar. The average value of SbNRT2/3 hydrophilicity was positive, indicating that they were all hydrophobic proteins, whereas α-helix and random coil accounted for more than 70% of the total secondary structure. Subcellular localization occurred on plasma membrane, where SbNRT2 proteins did not contain signal peptides, but SbNRT3 proteins contained signal peptides. Further analysis revealed that the number of transmembrane domains of the SbNRT2s family members was greater than 10, while that of the SbNRT3s were 2. There was a close collinearity between NRT2/3s of S. bicolor and Zea mays. Protein domains analysis showed the presence of MFS_1 and NAR2 protein domains, which supported executing high affinity nitrate transport. Phylogenetic tree analysis showed that SbNRT2/3 were more closely related to those of Z. mays and Setaria italic. Analysis of gene promoter cis-acting elements indicated that the promoter region of SbNRT2/3 had several plant hormones and stress response elements, which might respond to growth and environmental cues. Gene expression heat map showed that SbNRT2-3 and SbNRT3-1 were induced by nitrate in the root and stem, respectively, and SbNRT2-4 and SbNRT2-3 were induced by low nitrogen in the root and stem. Non-synonymous SNP variants were found in SbNRT2-4 and SbNRT2-1a. Selection pressure analysis showed that the SbNRT2/3 were subject to purification and selection during evolution. The expression of SbNRT2/3 gene and the effect of aphid infection were consistent with the expression analysis results of genes in different tissues, and SbNRT2-1b and SbNRT3-1 were significantly expressed in the roots of aphid lines 5-27sug, and the expression levels of SbNRT2-3,SbNRT2-4 and SbNRT3-2 were significantly reduced in sorghum aphid infested leaves. Overall, genome-wide identification, expression and DNA variation analysis of NRT2/3 gene family of Sorghum bicolor provided a basis for elucidating the high efficiency of sorghum in nitrogen utilization.