关键词: 马铃薯, 根癌农杆菌, 转基因植株, AtNHX1基因

Abstract:

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.

Key words: Potato, Agrobacterium tumefaciens, Transgenic plant, AtNHX1 gene