农杆菌介导的抗菌肽A基因转入沙田柚影响因素研究

Studies on factors influencing ShivaA gene transformation mediated by Agrobacterium in Citrus grandis Osbeck cv.Shatianyu

以沙田柚上胚轴为对象,进行了农杆菌介导的抗菌肽shivaA基因转入沙田柚影响因素研究。研究结果表明转化中抑菌抗菌素以羧苄青霉素为首选,最佳转化条件为菌液稀释至OD6000.5,浸泡时间10min,共培养3d,卡那霉素起始选择浓度50μg/mL。农杆菌再悬浮液与共培养基中加入150μmol/L阿魏酸可提高转化频率。PCR与Southern杂交证明,外源基因已转入沙田柚基因组中。

Transformation and regeneration of Citrus grandis plants were studied by Agrobacterium-mediated. Transgenic plants with ShivaA gene had been obtained. The carbenicillin was better to control Agrobacterium growing after explants were infected. Epicotyls fragment were infecting for 10 min as OD600 of Agrobacterium suspending liquid is 0.5. Epicotyls fragment were co-cultured with Agrobacterium for 3days. The transformation frequency of explants was promoted when 150μmol/L ferulic acid was added in Agrobacterium suspension liquid ＆ co-culture medium. It was useful for anti-Kan positive shoot producing when initial concentration of kanmaycin was 50μg/mL. Foreign gene is proved going to C. grandis by PCR ＆ Southern blot detective.