卡那霉素在转基因芥菜中的应用

Application of kanamycin in transgenic mustard(Brassica juncea Coss.)

为了找出芥菜(Brassica juncea Coss.)遗传转化中最佳的卡那霉素(Kan)筛选浓度,将芥菜的子叶接种于含有不同浓度Kan的分化培养基中,当Kan浓度达到30mg/L时,外植体的分化完全受到抑制。将芥菜种子播种于含有不同浓度Kan的培养基中,当Kan浓度达到200mg/L时,长出的幼苗完全白化;利用叶片涂抹方法,将不同浓度的Kan涂抹于田间生长的植株叶片上,当Kan浓度达到200mg/L时,被处理的叶片完全变白。为了对转基因芥菜后代中外源基因的分离情况进行遗传学分析,分别用200mg/L的Kan处理以npt-Ⅱ基因为选择标记基因的转基因芥菜的种子和转基因芥菜后代植株的叶片,利用χ2测验分析试验结果,4个含有单拷贝外源基因的转基因株系后代,对Kan的抗感分离都符合31的分离规律;而2个含有双拷贝外源基因的转基因株系,其中一个对Kan的抗感分离符合31而不符合151,另一个对Kan的抗感分离既符合31也符合151,双拷贝外源基因在转基因芥菜中的整合方式有待进一步的研究。最后,用PCR分析证实了该方法的准确性,因此,利用Kan对转基因芥菜后代进行筛选是可行的。

In order to find the best screening kanamycin concentration in the genetic transformation of mustard （Brassica juncea Coss.）, the seedling cotyledons of mustard were placed on bud-induced media supplemented with different kanamycin concentrations. The bud differentiation of explants was totally inhibited when the kanamycin concentration was greater than 30 mg/L. The seeds of mustard were placed on germination media supplemented with different concentrations of kanamycin. All the seedlings were white when kanamycin concentration was higher than 200 mg/L. The leaves of mustard in field were smeared with the solutions including different concentrations of kanamycin. The treated leaves showed white when kanamycin concentration was over 200 mg/L. To study the segregation of the alien gene in transgenic mustard offspring, the transgenic mustard seeds and the leaves of the transgenic mustard offspring using npt- Ⅱ gene as assistant selection-marker were treated with 200 mg/L kanamycin, and the results were analyzed by chi-square test. The segregation ratio in the offspring of 4 transgenic lines with single copy of transgene agreed with a ratio of 3 ： 1. The segregation ratio in offspring of the one transgenic line with double copies was agreed with a ratio of 3 ： 1, and the segregation ratio in offspring of the other transgenic line with double copies was agreed with ratios of 3 ： 1 and 15 ： 1 simultaneously. It is indispensable to thoroughly study the insert of the double copies of transgenes in transgenic mustard. PCR technology was used to confirm the above detection methods. It is concluded that applying kanamycin to screen transgenic mustard offspring is feasible.