摘要
为筛选出黄籽油菜遗传转化中卡拉霉素或卡拉霉素（kanamycin ，Kan or km ）最佳质量浓度，首先将油菜子叶接种在不同质量浓度Kan（0～180 mg／L）的分化培养基上，结果发现Kan质量浓度小于60 mg／L有利于绿苗分化。然后在不同质量浓度Kan的分化培养基上对农杆菌侵染的下胚轴进行筛选，结果表明初代培养Kan质量浓度以20～40 mg／L为宜，同样质量浓度继代培养后可获得抗性愈伤和转基因阳性苗。最后利用Kan抗性对转基因黄籽油菜后代进行研究，苗期到薹花期生长的植株进行叶片涂抹法筛选，Kan最适质量浓度为4 g／L ，时间为4～10 d；T1代抗性分析、GUS染色分析、T1代籽粒的PCR检测说明转基因在T1代、T2代可遗传并出现分离。β葡萄糖苷酸酶基因（GUS）和聚合酶链式反应（PCR）验证叶片涂抹进行Kan抗性筛选的方法准确性高，可达100％。
Cotyledons of yellow seed rape were inoculated onto differentiation media with different concen‐trations of kanamycin （Kan） to screen the optimal Kan concentration in genetic transformation ,and the results indicated that a Kan concentration of less than 60 mg/L was beneficial to green shoot differentia‐tion .Then ,hypocotyls infected by A grabacterium tumefaciens were transferred to differentiation media containing Kan of different concentrations ,and it was found that 20 -40 mg/L Kan concentration in the primary culture was appropriate ,and resistant calli and regenerated positive plantlets were obtained after successive transfer cultures .Finally ,Kan resistance in the transgenic yellow seed rape offspring was stud‐ied .The method of leaf daubing was used in transgenic plant growing from the period of young shoot to flowering ,and the optimum Kan concentration was found to be 4 g/L and the daubing time 4 to 10 days . Kan resistance of T1 generation ,GUS staining analysis ,PCR detection of T1 generation showed that transgenic seeds in T1 and T2 generations was inheritable and segregation occurred .GUS and PCR verifi‐cation in this study demonstrated that the accuracy of the method of leaf daubing to screen resistant plants w as as hig h as 100% .
出处
《西南大学学报（自然科学版）》
CAS
CSCD
北大核心
2015年第11期1-7,共7页
Journal of Southwest University(Natural Science Edition)
基金
国家自然科学基金项目(31171584)
重庆市自然科学基金项目(cstc2011jjA80005)
博士启动项目(104260-20710930)
关键词
卡拉霉素
转基因黄籽油菜
抗性
kanamycin
transgenic yellow-seeded rape
resistance
