转根癌农杆菌介导的AtNHX1基因马铃薯的获得

构建重组表达质粒pBI12135-GZ+AtNHX1（带有CaMV 35S启动子），通过农杆菌将其携带的液泡膜Na⁺/H⁺逆向转运蛋白基因（AtNHX）转化马铃薯栽培品种“甘农薯2号”试管薯薄片和“克新2号”茎段。经根癌农杆菌侵染和共培养后，用50 mg L^-1卡那霉素 +300 mg L^-1头孢霉素筛选抗性芽，试管薯薄片获得30株抗性植株，抗性植株再生率为37%；茎段未获得抗性植株。抗性植株的总DNA用AtNHX1基因的特异性引物进行PCR检测，结果27株为阳性，占90%。Southern杂交结果证实，外源基因多以双拷贝整合到马铃薯的基因组中。Northern杂交结果表明，转基因植株可以进行AtNHX1基因mRNA的正常转录，但植株间存在着转录量的差异。该研究为耐盐马铃薯的培育奠定了良好的基础。

Generation of Transgenic Potato Plants Harboring AtNHX1 Gene Mediated by Agrobacterium tumefaciens

Drought and soil salinization harm are a major challenges that compromise crops growth and lead to soil degeneration and desertification of natural environments. Most of food crops are glycophytes with high susceptibility or low tolerance to soil salinization. Development of new salt-tolerant crop varieties is important to production in large area of saline soils. It is known that the adaptation of plant cells to salt stress requires an improved cellular ion homeostasis that involves organic solute accumulation in cytosol, vacuolar compartmentalization of ions and exclusion of extra Na⁺ from the cells. The latter two functions are implemented by a Na⁺/H⁺ antiporter located on both plasma membrane and tonoplast. In particular, the compartmentalization of Na⁺ can help to absorb and reserve in the ions into the vacuoles，which is necessary to improve their osmotic adjustment and eventually enhance the salt tolerance of the plants. Potatoes are widely cultivated as a dual-purpose crop for food and vegetable consumption in the world. They are also one of the most important economic crops in arid and semi-arid areas in China. However, they are salt sensitive, and difficult to be selected and developed for a new variety with a stronger salt tolerance from limited genetic resources using traditional methodology. In this study, we constructed a pBI12135-GZ+AtNHX1 expression plasmid carrying a constitutive promoter (CaMV 35S), npt II gene (the resistance gene to Kanamycin) and AtNHX1 gene (the tonoplast Na⁺/H⁺ antiporter gene), and then attempted to introduce this constructed expression plasmid into the slices of microtubers of “Gannongsu 2” and the stems of “Kexin 2” potato cultivars mediated by Agrobacterium tumefaciens. After screening the transformed slices and stems on a medium containing 50 mg L^-1 Kanamycin and 300 mg L^-1 Cefotaxime, we obtained 30 transgenic plants from the slices at a regeneration rate of 37%, but none from stems. We further used specific primers to amplify the full-length of the AtNHX1 gene from total genomic DNA of the transgenic plants, of which 27 plants had a positive amplification, indicating 90% of successful transformation. Southern blot showed that two copies of the AtNHX1 gene were inserted into the genome of the transgenic plants and Northern blot hybridization detected a normal expression of the AtNHX1 gene at different levels among the transgenic plants. These results pave the way for the generation of a new potato variety with a stronger salt tolerance.