采用同源克隆和RACE 法克隆获得喜盐鸢尾(Iris halophila Pall.)Na⁺ / H⁺ 逆转运蛋白基因IhNHX1 的全长序列,该基因序列的全长为1 946 bp,包含1 个长度为1 611 bp 的开放阅读框(ORF),编码537 个氨基酸。序列对比及系统树分析结果表明:IhNHX1 基因编码的氨基酸序列与另外11 种植物NHX1 基因编码的氨基酸序列的一致性高达96. 2%,相同序列占61. 7%,表明该氨基酸序列保守性较高;在系统树上喜盐鸢尾与其他植物的分支长均大于1. 2,表明它们的亲缘关系均较远;IhNHX1 基因编码的氨基酸序列含有2 个保守结构域,即氨氯吡嗪结合位点和CaM 结合结构域,分别是NHX1 蛋白的标志性结合位点和重要调节区域。该蛋白质的二级结构和跨膜结构域分析结果表明:在IhNHX1 基因编码的蛋白质的二级结构中, 琢螺旋占48. 60%、不规则卷曲占32. 03%、延伸链占14. 71%、氢键转角占4. 66%;该蛋白质含有10 个跨膜结构域。此外,对5’RACE 方法中5’端正向引物的优化设计步骤进行了归纳,以提高PCR 扩增的特异性。

Through homology-based cloning and RACE (rapid amplification of cDNA ends) methods, the whole sequence of Na⁺ / H⁺ antiporter gene IhNHX1 of Iris halophila Pall. was cloned. The whole length of this gene is 1 946 bp, which contains an open reading fragment (ORF) with a length of 1 611 bp and encodes 537 amino acids. The results of sequence comparison and phylogenetic tree analysis show that the identity of amino acid sequences encoded by IhNHX1 gene and by NHX1 gene of other eleven species is up to 96. 2%, the same sequence accounts for 61. 7%, meaning that the conservation of this amino acid sequence is higher. The branch length of I. halophila and other species in phylogenetic tree is larger than 1. 2, indicating that their genetic relationship is far away. There are two conserved domains in amino acid sequence encoded by IhNHX1 gene, namely ammonia chloride pyrazine binding site and CaM binding domain, which is the typical binding site and important regulation domain of NHX1 protein, respectively. The analysis results of the second structure and transmembrane domain of this protein show that in the second structure of protein encoded by IhNHX1 gene, αhelix accounts for 48. 60%, random coil does 32. 03%, extended strand does 14. 71% and hydrogen bonding turn does 4. 66%, and there are 10 transmembrane domains in this protein. Moreover, the optimization design steps of 5’end forward primer in 5’RACE method are concluded in order to improve the specificity of PCR amplification.