根癌农杆菌介导D32基因转化烟草的条件研究

Study on the Conditions of Transferring D32 Gene into Tobacco Mediated by Agrobacterium tumefaciens

以烟草品种中烟99的无菌苗叶片为转化受体材料,通过根癌农杆菌C58C1介导对大豆中克隆的抗逆性基因D32进行转化,获得了抗卡那霉素的再生植株,并对转化植株进行了PCR检测.结果表明,烟草叶片分化和再生的卡那霉素选择压力为150mg/L;外植体预培养对转化率有影响;优化的烟草转化方法是:经预培养2d的外植体用OD600值为0.7的菌液侵染5min,共培养2d后用无菌水冲洗5～6次,羧苄青霉素(Cb)和头孢霉素(Cef)浓度为400mg/L的脱菌液浸泡120min,超净工作台上吹风60min,于筛选分化培养基生长50d,可获得26.7%卡那抗性苗.对抗性植株经PCR检测证明,外源D32基因已初步整合到烟草基因组中.

By mediation of Agrobacterium tumefaciens C58 C1 , D32 gene derived from soybean was introduced into sterile leaves of tobacco＇ Zhongyan 99＇. The key factors affecting the transformation were assessed and the transgenic plants were generated and further tested using PCR. The results showed that the optimal kanamycin concentration for tobacco leaves differentiation and regeneration was 150 mg/L and preculture had an impact on transformation efficiency. The optimized transformation conditions and procedures were as follows：explants were firstly immersed into liquid bacterium at OD600 of 0.7 for 5 min prior to preculture,followed by a two-day long co-culture before being transferred to and cultured in a differentiation medium for 50 days. A proportion 26.7% of the transgenic plants were kanamycin resistant. The PCR eventually detected that the D32 gene has preliminarily inserted into the tobacco genome.