高粱原生质体的制备及转化方法研究

Study on the Method of Protoplast Isolation and Transformation of Sorghum bicolor

以高粱幼苗为材料,利用1%的纤维素酶Cellulose R10和0.75%的果胶酶Macerozyme R10处理高粱幼茎,对比不同的酶解时间,发现酶解7h原生质体浓度最高、质量最佳。以经过氯化铯密度梯度离心法获得的高纯度质粒对获得的原生质体进行转化,用激光共聚焦进行荧光观察,结果显示,GFP和YFP在全细胞表达。本研究首次建立了高粱以高粱幼茎为材料的原生质体制备及转化体系,其方法简单、高效,可以用于高粱基因功能研究。

In this study,the seedlings of Sorghum bicolor were used to be incubated with enzyme solution including 1%Cellulose R 10 and 0.75%Macerozyme R 10.Comparing different enzymatic hydrolysis times,we found that the protoplast concentration and quality were the highest within 7 h of enzymatic hydrolysis.The protoplasts were transformed by high-purity plasmids obtained by the density gradient centrifugation method of cesium chloride,and fluorescence observation was carried out by confocal laser microscope.The results showed that GFP and YFP were expressed in the whole cell.In this study,the protoplast isolation and transformation system of Sorghum bicolor by using sorghum young stem as material was established for the first time,the method was simple and efficient,which could be used for the study of Sorghum bicolor gene function.