青稞硝酸盐转运蛋白基因HvnNPF4.5的克隆和亚细胞定位

Cloning and Subcellular Localization Analysis of Nitrate Transporter Gene HvnNPF4.5 from Hulless Barley

植物硝酸盐转运蛋白家族NPFs(Nitrate Transpoter1/Peptide Transporter Family)属于一类低亲和硝酸盐转运蛋白家族,在植物氮素吸收和转运过程中发挥着重要作用,NPFs基因家族是目前植物氮素吸收、转运和同化相关基因家族研究的热点。该研究从“昆仑14”青稞中克隆了HvnNPF4.5基因和其启动子区域序列,并对HvnNPF4.5基因和蛋白结构、系统进化和亚细胞定位进行了研究。结果表明:HvnNPF4.5基因序列含有2个内含子,启动子区域有与干旱以及脱落酸、赤霉素、生长素相关的顺式作用元件;HvnNPF4.5蛋白由614个氨基酸组成,分子量为66875.31 Da,总原子数为9465个,亲水系数为0.317,理论等电点为8.85,不稳定指数36.98,脂溶性指数为100.43,有8个磷酸化位点和12个跨膜结构,没有信号肽;HvnNPF4.5蛋白二级结构中α螺旋、延长链、β转角、无规则卷曲分别占48.83%、11.83%、3.5%、35.83%;HvnNPF4.5和其他物种的同源蛋白都有MFS结构域,将HvnNPF4.5与其他植物的氨基酸序列进行比较并构建系统进化树,发现青稞HvnNPF4.5与大麦HvNPF4.5、节节麦AtsNPF4.5的亲缘关系最近;亚细胞定位结果显示HvNPF4.5定位在细胞膜上。

NPFs(Nitrate Transpoter1/Peptide Transporter Family)belongs to a low-affinity nitrate transporter family,which plays an important role in plant nitrogen uptake and transport.The NPFs is currently a hot spot for research related to nitrogen uptake,transport and assimilation in plants.In this study,the HvnNPF4.5 gene and promoter region sequence were cloned from'Kunlun 14'hulless barley,and the HvnNPF4.5 gene and protein structure and phylogenetic tree were analyzed by bioinformatics software.In addition,the subcellular localization of HvnNPF4.5 was also performed.The results have shown that HvnNPF4.5 contains 2 introns and the promoter region contains a large number of cis-acting elements related to drought,abscisic acid,gibberellins and auxin.HvnNPF4.5 contains 614 amino acids,of which the molecular weight is 66875.31 Da,total number of atoms is 9465,GRAVY is 0.317,theoretical pI is 8.85,instability index is 36.98,and aliphatic index is 100.43.HvnNPF4.5 protein contains 8 phosphorylation sites,12 transmembrane structures and no signal peptide.The proportions ofα-helix,extended strand,β-turn and random coil of HvnNPF4.5 are 48.83%,11.83%,3.5%and 35.83%,respectively.Both HvnNPF4.5 and other homologous proteins of other species contains MFS domain.By comparing HvnNPF4.5 amino acid sequences with those of other plants and constructing phylogenetic tree,it is found that HvnNPF4.5 is closely related to HvNPF4.5 and AtsNPF4.5.Subcellular localization results have showed that HvnNPF4.5 is localized in the cell membrane.