农杆菌介导马铃薯转化体系的优化及转基因马铃薯的耐盐性研究

Optimization of Agrobacterium tumerfaciens mediated potato transformation system and investigation the salt-tolerance of transgenic potato

通过农杆菌介导的方式将獐茅液泡膜Na^+/H^+逆向转运蛋白基因(AlNHX)表达在马铃薯中,并对影响马铃薯转化的几种因素(抗生素浓度、农杆菌菌液浓度、共培养时间等)进行优化。结果表明:最适农杆菌菌液浓度是OD600=0.6,最适侵染时间是5min,最适共培养时间是2d,马铃薯转化中最适头孢霉素浓度是400mg/L;经PCR检测确认的转基因马铃薯植株在含0.7%NaCl的培养基中可以生长,而野生型马铃薯无法在此培养基中正常生长。在盐胁迫条件下,转基因马铃薯的Na^+、K^+含量及K^+/Na^+的比值均高于野生型马铃薯。研究证实马铃薯植物的的耐盐性可以通过农杆菌转化导入獐茅液泡膜Na^+/H^+逆向转运蛋白基因而得到提高。

Salinity stress is one of the major factors affecting plant growth. The development of high efficiency Agrobacterium mediated transgenic techniques has greatly accelerated the application of transgene in potato. The ORF of vacuolar Na^＋/H^＋ antiporter gene （AINHX） was transferred into potato through Agrobacterium transformation. Factors affecting the transformation frequency （concentration of antibiotics and Agrobacterium ,co-cultivation time） were optimized. The results indicated that： The optimum concentration of Agrobacterium tumefaciens is OD600 = 0.6, the optimum infection time 5 min, the adequate time of co-cultivation is 2 d, the optimum concentration of cefotaxime for potato transformation is 400 mg/L. Transgenic plants confirmed by PCR could survive in the medium containing 0.7% NaCl,while the wild-type plants failed to grow and the contents of Na^＋, K^＋ and K^＋/Na^＋ ratio in transgenic potato were all higher than in wild-type potato under salt stress. These results demonstrated that the salt tolerance of potato can be improved by introducing Na^＋/H^＋ antiporter gene through Agrobacterium transformation.