烟草NtNHX2基因的克隆及不同逆境下表达模式分析

Cloning and Expression Patterns Analysis of NtNHX2 Gene in Nicotiana tabacum under Different Stress

为了明确NHX (Na+/H+exchanger)家族成员NHX2在烟草中的功能与对非生物逆境胁迫的响应机制,本研究从烟草栽培品种K326中NtNHX2同源克隆NtNHX2基因,利用qRT-PCR分析该基因在不同非生物逆境下的表达模式,综合生物信息学分析,对其功能和调控机制进行初步预测。该基因开放阅读框为1 605 bp,编码534个氨基酸,预测分子量为59.0 kD,理论等电点pI为8.67。同源性分析结果显示,该基因与美花烟草NHX2 (Nicotiana sylvestris NHX2)、野生烟草NHX2 (Nicotiana attenuate NHX2)等具有较高的氨基酸序列同源性,分别为98.70%、98.14%,组织表达分析结果表明,该基因在根、茎、叶、花中均有表达,且在叶中表达量最高。逆境胁迫处理表明,该基因表达在高盐、低钾、4℃条件下受到抑制,在干旱、ABA处理下受到诱导调控,说明NtNHX2参与高盐、低钾、4℃、干旱、ABA非生物逆境胁迫的响应,为NtNHX2的功能研究、探讨其耐盐机制及基因利用提供了理论依据。

To identify the functions of NHX (Na+/H+ exchanger) family member NHX2 in tobacco and its responding mechanism to abiotic stress, one NtNHX2 gene was cloned from Nicotiana tabacum K326 with homologous cloning strategy. The function of NtNHX2 gene in tobacco was verified by comprehensive bioinformatics analysis and qRT-PCR, followed by the analysis of the expression pattern of NtNHX2 gene in tobacco under different stresses. Comprehensive bioinformatics analysis suggested that the open reading frame of NtNHX2 was 1 605 bp in length, encoding 534 amino acids. NtNHX2 had a predicted molecular weight of 59.0 kDa and the theoretical isoelectric point of 8.67. Homology analysis showed that NtNHX2 had high amino acid sequence homology with protein in Nicotiana sylvestris NHX2, Nicotiana attenuata NHX2, which were 98.70% and 98.14% respectively. Tissue expression analysis showed that the gene was expressed in roots, stems, leaves, and flowers, with the highest expression in leaves. Stress treatment showed that NtNHX2 gene expression was inhibited under high salt, low potassium, and 4℃, and was induced under drought and ABA treatment. So NtNHX2 participated in response to abiotic stresses such as high salt, low potassium, 4℃, drought, and ABA. It has provided a theoretical basis for the functional research of NtNHX2 and the discussion of its salt-tolerance mechanism and gene utilization.