摘要
以黄瓜4d苗龄子叶节和农杆菌GV3101为实验材料,对再生体系和转化体系条件进行优化。再生部分,将培养基中的6-BA和ABA设立浓度梯度,并添加适量的AgNO3,进行再生情况对比,得最佳配方MS+1.5mg/L 6-BA+1.0mg/L ABA+2.0mg/L AgNO3,可直接诱导出芽,最短45d获得完整植株。转化部分,根据敏感性试验确定选择培养卡那霉素(kan)筛选压,并运用gus瞬时表达法对预培养时间、侵染时间、共培养时间和抗氧化剂的添加等影响转化效率的因素进行优化。结果表明,50mg/L kan筛选压即可筛选出阳性株;预培养时间1～2d,侵染时间20～30min,共培养3d时转化效率较高;抗氧化剂的加入可显著提高转化效率。对该转化体系进行验证,PCR结果初步表明外源基因成功整合入黄瓜基因组中。
Cotyledon-nod of 4 days of cucumber cv.gl plant and Agrobacterium tumefaciens GV3101（habored pCAMBIA 2301） were used to optimize the condition for regeneration and transformation system of cucumber.For regeneration,we set grade concentrations of 6-BA and ABA and added proper amount of AgNO3 to get the optimal medium.For genetic transformation,kanamycin sensitivity of cotyledon-nod was tested.The optional parameter of the pre-culture,infection time,co-culture time and the addition of antioxidant were obtained by gus stain.After the optimization,the parameter in transgenic cucumber were investigated by PCR.The results showed that MS ＋ 1.5 mg/L 6-BA ＋ 1.0 mg/L ABA ＋ 2.0 mg/L AgNO3 was the optimal formula,which can induce shooting and a new plantlet can be gotten in 45 days with the formula.The critical concentration of kanamycin was 50 mg/L,and the optimal pre-culture time was 1~2 days,the optimal co-culture time was 3 days after 20~30 min infected by GV3101.The addition of antioxidant enhanced the efficiency distinctly and foreign gene was successful integrated into cucumber genome.
出处
《上海交通大学学报（农业科学版）》
2012年第2期41-47,共7页
Journal of Shanghai Jiaotong University(Agricultural Science)
基金
国家"973"课题(2012CB113902)
国家自然科学基金(31071081)
上海市重点学科建设项目(B209)
关键词
黄瓜
再生
卡那霉素敏感性
抗氧化剂
遗传转化
cucumber
regeneration
kanamycin sensitivity
antioxidant
genetic transformation
