中间偃麦草Na^+/H^+逆向转运蛋白的分子克隆及生物信息学分析

Molecular cloning and bioinformatics analysis of a vacuolar Na^+/H^+ antiporter gene in Elytrigia intermedia

根据小麦液泡膜Na+/H+逆转运蛋白基因TaNHX1的全长序列设计引物,通过RT-PCR直接扩增的方法从中间偃麦草(Elytrigia intermedia)中克隆到了TaNHX1的同源基因,命名为TiNHX1(Acession Numeber:EF409418)。TiNHX1最大开放阅读框为1 641 bp,编码含有546个氨基酸残基、分子量为59.8 kDa的蛋白,预测等电点8.0。TiNHX1含有38个碱性氨基酸,36个酸性氨基酸,256个疏水氨基酸及129个极性氨基酸。二级结构预测表明该蛋白含约44%的α-螺旋、21%的β-折叠、4%的β-转角和29%的不规则卷曲。亲疏水性分析显示,TiNHX1含有12个连续的疏水片断,其中10个可能构成穿膜螺旋。序列分析显示,TiNHX1与小麦(Triticum aestivum)、长穗偃麦草(Elytrigia elongate)、水稻(Oryza sativa)、小盐芥(Thellungiella halophila)、拟南芥(Arabidopsis thaliana)等植物的液泡膜Na+/H+逆向转运蛋白高度同源,序列相似性分别为97%、96%、85%、68%、67%。序列比对结果以及进化树分析均表明TiNHX1应为定位于中间偃麦草液胞膜上的Na+/H+逆向转运蛋白。

TiNHX1, homologous with the TaNHX1 gene encoding a vacuole Na^＋/H^＋ antiporter was cloned from Elytrigia intermedia by RT-PCR, with primers designed according to the sequence of TaNHX1. The largest open reading frame of TiNVHX1 gene has 1 641bp in length and encoded a protein of 546 amino acid residues. The estimated molecular weight and isoelectric points of the putative protein was 59.8 kDa and 8.0, respectively. Components of amino acids encoded by TiNHX1 contained 38 basic amino acids, 36 acidic amino acid, 256 hydrophobic amino acids and 129 polar amino acids. The predicted secondary structure composition for the protein has about 44% alpha helixes, 21% extended strand, 4% beta turn and 29% random coil. Hydrophobic analysis indicated that the TiNHX1 contained 10 potential transmembrane segments. Blast result and the phylogenetic analysis showed that TiNHX1, AtNHX1, OsNHX1, GmNHX1, TaNHX1 share a cluster.