Ahmed K Z, Omirulleh S, Sági F, Dudits D
Cereal Research Institute, Szeged, Hungary.
Acta Biol Hung. 1997;48(2):209-20.
Direct uptake of reporter gene constructs with the bacterial beta-glucuronidase (GUS) gene fused to various promoters was achieved to embryogenic cell suspension culture-derived protoplasts of GK Ságvári winter wheat (Triticum aestivum L.) with polyethylene glycol (PEG) treatment. Based on GUS specific activity values, it was found that Mg2+ with PEG at 20% final concentration can significantly increase the transient expression in wheat protoplasts in comparison to the Ca(2+)-containing medium. The optimum incubation time in transformation mixture was 5-10 min at 25 degrees C. Transient GUS expression as detected by spectrofluorimetry was positively correlated with the time elapsed after DNA uptake (with maximum activity at 48 h), and the incubation time in GUS reaction mixture. It was also found that the protoplast culture medium plays an important role in the efficiency, and the treated wheat protoplasts cultured in KM medium showed a higher GUS activity than those kept in GM medium. Among the five plasmid constructs 6-16-fold higher promoter activity has been achieved with pKM794 driven by CaMV 35S promoter plus two enhancer elements than with the other constructs tested.
通过聚乙二醇（PEG）处理，将融合了细菌β-葡萄糖醛酸酶（GUS）基因且带有各种启动子的报告基因构建体直接导入了源自GK Ságvári冬小麦（Triticum aestivum L.）胚性细胞悬浮培养物的原生质体中。基于GUS比活性值发现，与含Ca2+的培养基相比，终浓度为20%的PEG与Mg2+可显著提高小麦原生质体中的瞬时表达。转化混合物中的最佳孵育时间为25℃下5 - 10分钟。通过荧光分光光度法检测到的瞬时GUS表达与DNA摄取后经过的时间（48小时时活性最高）以及在GUS反应混合物中的孵育时间呈正相关。还发现原生质体培养基对效率起着重要作用，在KM培养基中培养的经处理的小麦原生质体比在GM培养基中培养的显示出更高的GUS活性。在五个质粒构建体中，由CaMV 35S启动子加两个增强子元件驱动的pKM794的启动子活性比其他测试构建体高6 - 16倍。
