《植物生理学报》 2010, 46(10): 1025-1032

荷花液泡膜型Na⁺/H⁺ 逆向转运蛋白基因NnNHX1 的克隆与特性分析

郭会敏, 顾春笋, 刘兆磊*, 陈发棣, 徐迎春

南京农业大学园艺学院, 南京210095

通信作者：刘兆磊；E-mail: lzl@njau.edu.cn；Tel: 025-84396370

摘　要：

采用简并引物和 RACE 等技术从荷花中分离出液泡膜型 Na+/H+ 逆向转运蛋白基因 NHX1 的全长 cDNA 序列, 命名为 NnNHX1。该基因全长 2 237 bp, 预测其编码一个由 538 个氨基酸组成的多肽, 其 N- 端是一个由 11 个跨膜结构组成的疏水 区域, C- 端是一个亲水的尾巴。序列比对表明, NnNHX1 与葡萄 NHX1 等的同源性较高, 并且都具有高度保守的氨氯吡嗪 咪结合位点序列LFFIYLLPPI。系统进化树表明NnNHX1 与液胞膜型Na+/H+ 逆向转运蛋白关系较近, 而与质膜型Na+/H+ 逆 向转运蛋白关系较远。qRT-PCR 结果显示 NnNHX1 基因在植株中属于组成型表达, 但经 NaCl 诱导后, 根中 NnNHX1 的表 达量急剧增高后又迅速下降, 而叶中的表达量却先缓慢增加后又剧烈下降。

关键词：荷花; 液泡膜型Na+/H+逆向转运蛋白基因; 克隆; 荧光定量PCR

收稿：2010-07-01   修定：2010-07-16

资助：上海农业科学技术成果及高新技术产业化项目(沪农科产字[2004]第3号)。

Cloning and Characterization Analysis of a Vacuolar Na⁺ /H⁺ Antiporter Gene NnNHX1 from Nelumbo nucifera

GUO Hui-Min, GU Chun-Sun, LIU Zhao-Lei*, CHEN Fa-Di, XU Ying-Chun

College of Horticulture, Nanjing Agricultural University, Nanjing 210095, China

Corresponding author: LIU Zhao-Lei; E-mail: lzl@njau.edu.cn; Tel: 025-84396370

Abstract:

The cDNA of NHX1 gene which was then designated as NnNHX1 was successfully isolated from Nelumbo nucifera by RACE cloning method. The full-length cDNA of NnNHX1 was 2 237 bp and it was predicted to encode a 538-amino acid protein with 11 hypothetical transmembrane domains in the N-terminal part while a hydrophilic tail in the C-terminal part. The bioinformation analysis of NnNHX1 showed it had high identities with NHX1 from Vitis vinifera and other NHX1s, and all of them had a high degree conserved amiloride binding-site sequence LFFIYLLPPI. The phylogenetic tree analysis showed that NnNHX1 shared the same group with vacuolar Na⁺ /H⁺ antiporter protein, but distinguished from the plasma Na⁺ /H⁺ antiporter protein. The result of fluorescence quantitative real-time PCR showed that NnNHX1 was constitutive expressed, but under the NaCl stress, the expression level of NnNHX1 in roots increased sharply before decreased rapidly; while the expression level of NnNHX1 increased mildly at first, then decreased dramatically in leaves.

Key words: Nelumbo nucifera; vacuolar Na⁺ /H⁺ antiporter gene; cloning; fluorescence quantitative real-time PCR