烟草高亲和硝酸盐转运蛋白基因NtNRT2.5的克隆及表达模式分析

Cloning and expression pattern analysis of tobacco high affinity nitrate transporter gene NtNRT2.5

【背景和目的】高亲和硝酸盐转运蛋白NRT2家族基因在植物硝酸盐吸收转运和再利用过程中发挥重要作用,为探索烟草NRT2家族基因NtNRT2.5对烟草氮素调控的作用。【方法】从烟草烤烟品种K326中克隆得到高亲和硝酸盐转运蛋白基因NtNRT2.5,进行生物信息和表达模式分析,创制NtNRT2.5启动子融合GUS报告基因的K326转基因烟草材料,进行GUS组织化学染色。【结果】NtNRT2.5基因的全长ORF为1509 bp,编码502个氨基酸,属于稳定性疏水蛋白,其蛋白跨膜结构域具有NRT2家族结构特征;烟草NtNRT2.5蛋白与拟南芥AtNRT2.5蛋白亲缘关系最近,功能相似;该基因启动子包含多种激素和胁迫响应元件;NtNRT2.5蛋白定位于细胞质膜;NtNRT2.5基因在烟草各组织都有表达,在下部叶中表达最强,且受低氮诱导表达增强,GUS组织染色结果表明,正常供氮和低氮条件下,在转基因烟草的根(中轴)和下部叶片(叶脉)中都检测到了GUS报告基因,均能显蓝色,且低氮条件下表达更加强烈,蓝色更深。【结论】烟草NtNRT2.5是高亲和硝酸盐转运蛋白的编码基因,该基因受低氮诱导表达增强,可能参与烟草硝酸盐的转运及再利用过程。

[Background and Objective]High-affinity nitrate transporter NRT2 family genes play an important role in nitrate absorption,transport and reuse in plants.This study aims to explore the role of tobacco NRT2 family genes NtNRT2.5 in regulating nitrogen in tobacco.[Methods]The high affinity nitrate transporter gene NtNRT2.5 was cloned from tobacco variety K326,and its bioinformation and expression pattern were analyzed.K326 transgenic tobacco material with GUS reporter gene fusion of NtNRT2.5 promoter was created,and GUS tissue chemical staining was performed.[Results]The full-length ORF of the NtNRT2.5 gene is 1509 bp,encoding 502 amino acids,and is a stable hydrophobic protein.Its transmembrane protein domain has the structural characteristics of the NRT2 family.Tobacco NtNRT2.5 is closely related to Arabidopsis AtNRT2.5 and shares similar function.The gene promoter contains a variety of hormone and stress response elements.The NtNRT2.5 protein is localized to the plasma membrane of the cell.The NtNRT2.5 gene was expressed in all tissues of tobacco,with the strongest expression in the lower leaves,and its expression was enhanced under nitrogen-deficient conditions.GUS tissue staining results showed that,under both regular and low nitrogen conditions,the GUS reporter gene can be detected in the roots(axis)and lower leaves(veins)of transgenic tobacco,displaying a blue hue,with a deeper blue and stronger expression under low nitrogen conditions[Conclusion]Tobacco NtNRT2.5 encodes a high affinity nitrate transporter,and its expression is enhanced under nitrogen-deficient conditions,suggesting its potential involvement in tobacco nitrate transport and reuse.