陆地棉硝酸盐转运体NRT基因家族鉴定及表达分析

Identification and expression analysis of nitrate transporter NRT gene family in upland cotton(Gossypium hirsutum L.)

硝酸盐转运体(nitrate transporters,NRTs)在植物氮素吸收、利用和存储等过程中发挥着重要作用。本研究利用HMM软件和Blastp方法,在陆地棉中,鉴定出了106个GhNRT1/PTR(NPF)(Nitrate transporter 1(NRT1)/Peptide Transporter(PTR)family(NPF))和14个GhNRT2(Nitrate transporter 2 family)成员,对它们的保守结构域、系统发育关系、理化性质、亚细胞定位、保守基序、基因结构、启动子区顺式作用元件和表达模式进行了分析。结果表明,GhNPF均具有典型的PTR2(Peptide Transporter 2 family,肽转运蛋白)结构域,个别蛋白(GhNPF2.6bD、GhNPF4.1cA和GhNPF2.14aD)出现了2个PTR2和/或其他结构域,表明棉花NPF进化保守性较低;GhNRT2具有典型的MFS_1(Major Facilitator Superfamily,主要促进子超家族)单域。多数GhNRTs定位于细胞质膜上,为疏水性蛋白。系统发育分析显示GhNRTs可分为10个类群,相同类群具有相似的基因结构及基序分布。顺式作用元件组成表明,大部分GhNRTs的表达可能与植物激素、非生物胁迫和光反应等相关。此外,GhNPF不同亚类间的表达模式存在一定差异,但同一亚类内不同成员的表达模式相对保守;GhNRT2基因则主要在根中高表达。盐胁迫处理后的转录组数据分析发现,近1/5的GhNRTs基因表达量发生了显著上调或下调,表明其可能参与棉花盐胁迫应答。选择6个GhNRTs基因检测其在根系、幼叶、功能叶和老叶中的表达对不同NO_(3)^(-)供应水平的响应发现,GhNPF6.3dA和GhNPF7.3aA可能具有双亲和吸收NO_(3)^(-)的能力,GhNPF6.2bD则可能编码高亲和NO_(3)^(-)转运蛋白;三者在功能叶和老叶中可能参与NO_(3)^(-)卸载。这些结果与拟南芥等植物中的报道不同。上述研究结果为进一步揭示棉花硝酸盐转运蛋白的功能提供了参考,为解析棉花氮素吸收和利用机制提供了初步依据。

Nitrate transporters(NRTs)play an important role in plant nitrogen absorption,utilization,and storage.In this study,106 GhNRT1/PTR(NPF)(Nitrate transporter 1(NRT1)/Peptide Transporter(PTR)family(NPF))and 14 GhNRT2(Nitrate transporter 2 family)were identified from Gossypium hirsutum L.(TM-1)by HMM software and Blastp method.The conserved domains,phylogenetic relationships,physicochemical properties,subcellular localization,conserved motifs,gene structure,promoter cis-acting elements,and expression patterns of these GhNRTs were analyzed.The results showed that GhNPF had a typical PTR2(Peptide Transporter 2 family)domain.Two PTR2 and/or other domains were found in individual proteins(GhNPF2.6bD,GhNPF4.1cA,and GhNPF2.14aD),indicating that GhNPF was less evolutionarily conserved.The GhNRT2 had a typical MFS_1(Major Facilitator Superfamily)domain.Most proteins were located on the cytoplasmic membrane with hydrophobic properties.Phylogenetic analysis showed that these GhNRTs could be divided into 10 groups,and the same group had similar gene structure and motif distribution.The composition of cis-acting elements indicated that the relative expression levels of most GhNRTs could be related to plant hormones,abiotic stress,and light response.In addition,the relative expression patterns of GhNPF were different among the diverse subgroups,but the relative expression patterns of different members in the same subgroup were mostly conserved.GhNRT2 genes were mainly expressed in roots.Moreover,the transcriptome data with salt stress treatment revealed that the relative levels of nearly 1/5 GhNRTs were significantly up-regulated or down-regulated,indicating that they probably function in response to salt stress.Six GhNRTs were selected to detect the response of their expression in roots,young leaves,functional leaves,and old leaves to different NO_(3)^(-)supply levels.The results showed that GhNPF6.3dA and GhNPF7.3aA may have the ability to absorb NO_(3)^(-)with dual affinity,while GhNPF6.2bD may encode high-affinity NO_(3)^(-)transporter.The three may be involved in NO_(3)^(-)unloading in functional leaves and old leaves.These results were different from those reported in plants such as Arabidopsis.In conclusion,the results provide a reference for further functional characterization of nitrate transporters and provide a preliminary basis for the mechanism analysis of nitrogen absorption and utilization in cotton.