摘要： 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。
关键词:
小麦原生质体,
电激,
基因导入,
GUS活性,
潮霉素抗性克隆
Abstract: Protoplasts were isolated from suspension cells of a wheat cultivar“Bodallin”.The protoplasts were transformed by electroporation with uptake of PBC1 plasmid,containing both βglucuronidase(GUS)and hygromycin B phosphotransferase(HPT) genes.By means of BTX ECM6000 Electroporation System and ASP buffer,maximal GUS activity of the transformed protoplasts was obtained under the condition of 300 V(750V/cm)and 50 ms (app.1000 μF),when an effective concentration (25 μg/ml) of PBC1 plasmid was employed.After gene transfer via electroporation,transient expression of GUS in the protoplasts cultured for 2-3 days was able to reach a high leve1.In addition,GUS activity could be enhanced to certain level,by the aid of calf DNA used as a carrier DNA during electroporation.Protoplasts electroporated with PBC1 DNA were cultured on KMP medium containing 50-100 μg/ml hygromycin for 3-4 months and selection of hygromycin resistant call was conducted.A total of 15 calli from transformed protoplasts survived in selective cultures on 100 μg/m1,hugromycin a concentration that completely inhibited the growth of non transformed wheat callus.
Key words:
Wheat protoplasts,
Electroporation,
Gene transfer,
GUS activity,
Hygromycin resistant callus
中图分类号:
李宏潮, 胡道芬, 尾高志, 町井博明, 平林利郎. 小麦原生质体的电激介导基因转移[J]. 华北农学报, 1996, 11(3): 25-30. doi: 10.3321/j.issn:1000-7091.1996.03.005.
Li Hongchao, Hu Daofen, Toshio Hirabayash I, Hiroaki Machii, Takashi Hagio. Gene Transformation of Wheat Protoplasts by Electroporation[J]. ACTA AGRICULTURAE BOREALI-SINICA, 1996, 11(3): 25-30. doi: 10.3321/j.issn:1000-7091.1996.03.005.