花烟草NaNHX1基因的克隆及其在非生物胁迫下的表达模式

植物NHX家族基因,在植物的生长发育以及生物与非生物胁迫的应答反应中发挥着十分重要的作用。为了探究花烟草Na+/H+逆向转运蛋白的生理功能,为花烟草耐盐分子机制的研究提供参考。采用同源克隆的方法进行基因克隆,对花烟草进行非生物胁迫,并运用qPCR的方法进行基因表达模式分析。结果表明,从花烟草(Nicotiana alata)中克隆了一个属于Na+/H+逆向转运蛋白家族的基因NaNHX1。该基因的开放阅读框全长为1 599 bp,编码了532个氨基酸残基。生物信息学分析结果表明,该基因编码的蛋白分子量为58.4 kD,等电点为5.66;具有Na+/H+逆向转运蛋白家族典型的保守结构域NhaP2;该蛋白属于疏水性蛋白,包含10个跨膜区。NaNHX1基因主要定位于细胞质膜,并含有多个磷酸化位点。同源性分析的结果显示,NaNHX1基因与美花烟草(Nicotiana sylvestris)、茸毛烟草(Nicotiana tomentosiformis)以及番茄(Solanum lycoperisicum)NHX基因的亲缘关系最近,而与拟南芥的NHX基因同源性最低。NaNHX1基因的表达具有组织表达特异性,花中表达量最高,茎中次之,根和叶中表达量较低。在高盐、干旱、低温、ABA、低钾及H2O2等非生物胁迫下,NaNHX1的表达呈现3种不同的表达模式。其中,对高盐及低钾胁迫的响应强烈。本研究的结果表明,NaNHX1基因属于Na+/H+逆向转运蛋白家族,可能参与了花烟草高盐和低钾胁迫,以及其它非生物胁迫响应在内的众多生理过程。

Cloning of Gene NaNHX1 in Nicotiana alata and Its Expression Pattern Under Abiotic Stresses

Plant NHX family genes play very important role in plant growth and development as well as in response to biotic and abiotic stresses.In order to explore the physiological function of Na+/H+ antiporter,and to provide useful references for the study of molecular mechanism of Nicotiana alata in resistance to salt stresses,homologous cloning method was used for gene cloning,abiotic stress was treated to N.alata,and qPCR was conducted to analyze the expression profile of the gene.The results showed that NaNHX1 belonging to Na+/H+ antiporter family was cloned from N.alata.The open reading frame of the gene was 1 599 bp in length and encoded 532 amino acids.Bioinformatics analysis showed that the protein encoded by this gene had a molecular weight of 58.4 kD and an isoelectric point of 5.66.It had a typical conservative domain NhaP2 of Na+/H+ antiporter family.This protein belonged to hydrophobic protein and contained 10 transmembrane regions.NaNHX1 gene was mainly located in the plasma membrane and contained multiple phosphorylation sites.The results of homology analysis revealed that NaNHX1 gene had the closest relationship with the NHX gene of Nicotiana sylvestris,Nicotiana tomentosiformis and Solanum lycoperium,but had the lowest homology with the NHX gene of Arabidopsis thaliana.The expression of NaNHX1 gene was tissue-specific,with the highest expression in flowers,the second in stems,and the lower expression in roots and leaves.Under abiotic stresses such as high salinity,drought,low temperature,ABA,low potassium and hydrogen peroxide,the expression of NaNHX1 presented 3 different patterns,and its response to high salt and low potassium stress was strong.The results of this study suggest that NaNHX1 gene belongs to Na+/H+ antiporter family and may participate in many physiological processes including high salt,low potassium and other abiotic stress responses in N.alata.