摘要
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。
IL-4 gene transformation mediated by pollen-tube pathway method of Chinese cabbage for molecular detection of descendants using PPT resistance screening, PCR amplification, PCR-Southern hybrid, ELISA, Western hybrid methods, combined with statistical analysis study IL-4 inheritance of genes in transgenic Chinese cabbage in future generations. Results indicated that： （1） PPT resistance in transgenic Chinese cabbage, PPT resistance positive rates of T4 generation was 33.43%, the positive rate of PCR amplification of IL-4 gene was 1.39%. IL-4 separation plant does not meet Mendel＇ s law of segregation. （2） ELISA test on 14 ones of PCR-positive strains of 5 strains in the T4 generation, 7 samples of which are positive, IL4 expression was about 326. 87 - 1233. 13 ng/g FW, the ones of IL4 expression within the same strains significance of difference. （3） Western confirned IL-4 in transgenic Chinese cabbage hybrid offspring in the regular expression. It indicated that IL-4 gene has been incorporated into the Chinese cabbage in the genome with PCR-southern hybrid, and that gene still exists in transgenic plants＇ future generations.
出处
《中国生物工程杂志》
CAS
CSCD
北大核心
2013年第6期131-137,共7页
China Biotechnology
基金
黑龙江省自然科学基金资助项目(C0014)
关键词
花粉管通道法
大白菜
白细胞介素4非孟德尔遗传
Pollen-tube pathway method Chinese cabbage Interleukin-4 （IL-4） Non-MendelianInheritance
