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。
Protoplasts were isolated from suspension cells of a wheat cultivar“Bodallin”.The protoplasts were transformed by electroporation with uptake of PBC1 plasmid,containing both β glucuronidase(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.
出处
《华北农学报》
CSCD
北大核心
1996年第3期25-30,共6页
Acta Agriculturae Boreali-Sinica
关键词
小麦
原生质体
电激
基因导入
潮霉素
抗性克隆
Wheat protoplasts
Electroporation
Gene transfer
GUS activity
Hygromycin resistant callus
