过量表达AtNHXS1新基因提高烟草耐盐性的研究

Overexpression of A New Evolved Na^+/H^+ Antiporter Gene AtNHXS1 Improved Salt Tolerance of Tobacco(Nicotiana tabacum L.)

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。

AtNHXS1,a new vacuolar Na^＋/H^＋ antiporter gene which was obtained from Arabidopsis thaliana AtNHX1 gene by DNA shuffling technology,was transformed into tobacco by Agrobacterium-mediated leaf disc.After hygromycin screened and PCR test,ten transgenic T2 lines were identified,and two lines of which were single gene insertion were used for further salt tolerance assays.Real-time PCR analyses showed that the expression levels of AtNHXS1 mRNA were significantly higher after salt treatment among different tissues,especially in leaves in transgenic lines.After salt stress,the growth status of transgenic lines were dominant better than the wild type lines.Under the treatment of 400 mmol/L NaCl,the transgenic plants grew well but the wild type plants almost nearly died.The result suggested that the transgenic AtNHXS1 tobacco could significantly improve the ability of plant salt tolerance.