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采用PPT抗性筛选、PCR扩增、PCR-Southern杂交、ELISA、Western杂交等方法,对花粉管通道法介导IL-4基因转化大白菜的后代进行了分子生物学检测,同时结合统计分析手段研究了IL-4基因在转基因大白菜后代中的遗传规律。结果表明:(1)PPT抗性的转基因大白菜,T4代植株PPT抗性的阳性率为33.43%,IL-4基因PCR扩增的阳性率为1.39%。IL-4的分离情况不符合孟德尔分离定律。(2)对T4代5个株系的14个PCR阳性株进行ELISA检测,其中有7份样品为阳性,IL-4表达量约为326.87～1233.13 ng/g FW,同一株系内各株间IL-4的表达量差异显著。(3)Western杂交进一步证实IL-4在转基因大白菜后代中能正常表达。PCR-southern杂交表明IL-4基因已整合到大白菜基因组中,且在转基因后代植株中仍然存在。
PPO resistance screening, PCR amplification, PCR-Southern hybridization, ELISA and Western blotting were used to detect the molecular biology of the progeny of Chinese cabbage transformed by IL-4 gene mediated by pollen tube pathway. At the same time, with statistical analysis The genetic rules of IL-4 gene in transgenic Chinese cabbage were studied. The results showed that: (1) The positive rate of PPT resistance in transgenic Chinese cabbage and T4 plants with PPT resistance was 33.43%, and the positive rate of IL-4 gene PCR amplification was 1.39%. IL-4 separation does not meet the Mendelian law of separation. (2) 14 PCR positive strains of 5 strains of T4 were tested by ELISA. Among them, 7 samples were positive and the expression of IL-4 was about 326.87 ~ 1233.13 ng / g FW. Among the strains in the same strain IL-4 expression was significantly different. (3) Western blot further confirmed that IL-4 was normally expressed in transgenic Chinese cabbage. PCR-southern hybridization indicates that the IL-4 gene has been integrated into the Chinese cabbage genome and remains in transgenic progeny plants.