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小麦原生质体的电激介导基因转移

DOI: 10.3321/j.issn:1000-7091.1996.03.005, PP. 25-30

Keywords: 小麦原生质体,电激,基因导入,GUS活性,潮霉素抗性克隆

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Abstract:

从小麦品种”Bodalin“胚性悬浮细胞分离出原生质体,通过电激将质粒PBC1DNA(携带β-葡萄糖苷酸酶(GUS)标记基因和潮霉素抗性基因hph)导入原生质体。采用BTX电激系统和ASP电激缓冲液,最佳电激条件为300V(750V/cm)和50ms(约1000μF),转化的原生质体内GUS的活性最高;质粒DNA的有效使用浓度为25μg/ml.电激处理后,原生质体培养2~3天,GUS基因表达最强,宜于检测其瞬时表达;牛胸腺DNA可协助提高GUS基因的导入效果。质粒PBC1DNA处理的原生质体培养于添加潮霉素的KMP培养基。经4个月抗性筛选,选择获得15个潮霉素抗性克隆。

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