烟草硝酸盐转运蛋白NtNRT1.5基因的克隆及功能分析

【背景和目的】低亲和硝酸盐转运蛋白NRT1家族基因在植物吸收和转运硝态氮过程中发挥重要作用，为研究烟草NRT1家族基因在烟草氮素利用中的作用。【方法】从栽培烟草品种K326中克隆获得NtNRT1.5的全长cDNA序列，利用生物信息分析、qRT-PCR技术和转基因技术系统分析NtNRT1.5基因的序列特征、表达模式及在提高烟草氮素利用效率方面的功能。【结果】NtNRT1.5全长序列包含1800bp开放阅读框、编码599个氨基酸，编码的蛋白质具有NRT基因家族的共有结构特征，与拟南芥AtNRT1.5相似性达到59.28%。亚细胞定位结果表明NtNRT1.5蛋白定位在细胞膜上，其启动子中包含多个与硝酸盐（A(C/G)TCA）、胁迫（MYB）、根系特异表达（ROOT MOTIF BOX）相关的响应元件。表达模式分析结果表明NtNRT1.5主要在根部表达，其次是衰老叶片和茎，新叶基本不表达；低氮抑制其在根系中的表达，说明NtNRT1.5主要负责正常条件下的硝酸盐的吸收；低氮条件下，NtNRT1.5过表达显著提高了转基因烟株的氮素利用率。【结论】NtNRT1.5基因在烟草氮素的吸收和转运上行使重要...

Cloning and functional analysis of nitrate transporter NtNRT1.5 gene in Nicotiana tabacum

  Background  Low-affinity nitrate transporter NRT1 family genes play an important role in the absorption and transport of nitrate nitrogen in plants.  Methods  NtNRT1.5 was cloned from cultivated tobacco variety K326. The bioinformation analysis, qRT-PCR and transgenic technology were used to systematically analyze the sequence characteristics, expression pattern of NtNRT1.5 and its function in improving nitrogen use efficiency of tobacco.  Results  NtNRT1.5 full-length sequence contains 1800bp open reading frame and encodes 599 amino acids. The encoded protein has the common structural characteristics of NRT gene family, which is 59.28% similar to that of arabidopsis AtNRT1.5. Subcellular localization showed that NtNRT1.5 protein is located on the cell membrane, and its promoter contains multiple response elements related to nitrate(A(C/G)TCA), stress (MYB) and root motif box. Expression pattern analysis showed that NtNRT1.5 was mainly expressed in roots, followed by senescent leaves and stems, but not in new leaves. Low nitrogen inhibited its expression in roots, indicating that NtNRT1.5 is mainly responsible for nitrate absorption under normal conditions. Under low nitrogen condition, the overexpression of NtNRT1.5 significantly increased the nitrogen use efficiency of transgenic tobacco plants.  Conclusion  NtNRT1.5 gene plays an important role in the absorption and transport of nitrogen in tobacco. It can increase the plant biomass by improving the nitrogen utilization rate of overexpressed tobacco. This study provides a theoretical basis for cultivating new varieties of tobacco with high nitrogen utilization in the later stage.