农杆菌介导的抗菌肽A基因转入沙田柚影响因素研究

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。

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

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 OD_ 600 of Agrobacterium suspending liquid is 0.5. Epicotyls fragment were co-cultured with Agrobacterium for 3days. The transformation frequency of explants was promoted when 150 μmol/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 50 μg/mL. Foreign gene is proved going to C.grandis by PCR & Southern blot detective.