唐古特白刺液泡膜Na^+/H^+逆向运输蛋白基因NtNHX1的克隆与表达分析

Isolation and Expression Analysis of a Vacuolar Membrane Na^+/H^+ Antiporter Gene NtNHX1 from Nitraria tangutorum

植物液泡膜Na+/H+逆向转运蛋白具有将胞质中过多的Na+区隔化在液泡内,从而减轻过量Na+对细胞质的伤害,同时维持细胞的渗透势,利于植物抵御盐胁迫。以2年生唐古特白刺嫩叶为试材,采用RT-PCR和RACE方法从叶片中获得1个NHX基因cDNA全长,命名为NtNHX1,GenBank登录号为KF751928。序列分析结果表明:NtNHX1全长2 134 bp,包含281 bp的5’非编码区、218 bp的3’非编码区和29 bp的poly(A)尾巴。该cDNA编码1个544个氨基酸的多肽,等电点为8.14,推测分子质量为59.9 kDa。通过与其他物种的NHX1氨基酸序列比对,NtNHX1基因与柑橘同源性最高达81%。NtNHX1基因存在12个跨膜结构域,其中TM3跨膜结构域上具有高度保守的氨氯吡嗪脒结合位点(LFFIYLLPPI)。该位点与Na+有竞争作用。相对荧光定量PCR分析表明,NtNHX1在根、茎、叶中均有表达,叶中的表达量明显高于茎和根;在高浓度盐(200 mmol·L-1NaCl)处理下,NtNHX1在叶中的转录水平显著增强,在处理12 h后表达量达到最大值。由此推断,NtNHX1基因的表达与盐胁迫的诱导和调节有关。

The plant Na＋/H＋ antiporter （NHX） can regionalize the excess Na＋ of cytoplasm in the vacuole. This process can avoid the noxious effects of Na ＋ in the cytosol and maintain osmotic balance by using Na ＋ amassed in the vacuole to drive water into the cell, thus facilitating the plants to resist salt stress. In this work, a full-length cDNA sequence of NtNHXI gene was obtained from leaves of Nitraria tangutorum using RT-PCR and RACE, named NtNHX1 （GenBank accession number KF751928）. Sequence analysis indicated that NtNHX,I is 2 184 bp in full length, containing a 5＇-untranslated region （5＇-UTR） of 281 bp, a 3＇-UTR of 218 bp and 29 bp of the ploy（A） tail. The NtNHX1 cDNA encodes a protein of 544 amino acids with a pI of 8.14 and a deduced molecular mass of 59.9 kDa. Amino acid sequence alignment with other species showed that the N. tangutorum of NtNHX1 displayed the highest similarity（81% ） to citrus. NtNHX1 has twelve putative transmembrane （TM） domain structures, of which the TM3 transmembrane structure includes the conserved amilorude-binding sites （LFFIYLLPPI）. The amilorude-binding site was found to be responsible for playing a competitive role. Relative Real-Time PCR analysis indicated that NtNHX1 expressed in root, stems and leaves. The expression in leaves was highest. The transcript level of NtNHX1 in the leaves was obviously increased by 200 mmol· L-1 NaCl treatment, and the expression of NtNHX1 reached to the highest in 12 h of treatment. The study indicated that the expression of NtNHX1 gene was related to the induction and regulation of salt stress.