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拟南芥液泡膜上的Na+/H+逆向转运蛋白是由AtNHX1基因编码的一种重要的植物耐盐性因子。AtNHXS1是利用DNA改组(DNA shuffling)技术对AtNHX1基因进行定向分子进化获得的新基因。利用农杆菌介导的叶盘法将该基因转入烟草中,经过潮霉素和PCR鉴定,得到了10个独立的转基因株系。对其中两个PCR阳性株系进行Southern blot鉴定,确定AtNHXS1以单拷贝的形式成功地插入到烟草的基因组中。荧光定量PCR分析表明,AtNHXS1基因可以利用烟草的转录体系正确转录。在盐处理下,随着盐浓度的提高,植株不同组织部位AtNHXS1基因的表达均有不同程度的提高,其中叶片上调趋势最明显。耐盐性试验结果表明,盐处理下,转基因烟草的长势明显优于野生型。400 mmol/L NaCl处理下,野生型烟草完全死亡,转基因烟草生长受到抑制,但是仍然能够正常生长。研究结果表明,AtNHXS1新基因能够显著提高烟草的耐盐性。
The Na + / H + antiporter on Arabidopsis tonoplast is an important salt tolerance factor encoded by AtNHX1 gene. AtNHXS1 is a novel gene obtained by directed molecular evolution of AtNHX1 gene using DNA shuffling technology. The Agrobacterium tumefaciens-mediated leaf disc method was used to transfer the gene into tobacco. After hygromycin and PCR identification, 10 independent transgenic lines were obtained. Two of the PCR-positive strains were identified by Southern blot, and it was confirmed that AtNHXS1 was successfully inserted into the genome of tobacco in a single copy. Quantitative real-time PCR analysis showed that AtNHXS1 gene could be transcribed correctly using tobacco transcription system. Under salt treatment, the expression of AtNHXS1 gene in different tissues of the plants increased with the increase of salt concentration, and the up-regulation of leaves was the most obvious. Salt tolerance test results show that under salt treatment, the growth of transgenic tobacco was significantly better than the wild type. Under the treatment of 400 mmol / L NaCl, the wild type tobacco completely died and the growth of transgenic tobacco was inhibited, but it still could grow normally. The results show that the AtNHXS1 gene can significantly improve the salt tolerance of tobacco.