烟草叶片原生质体制备及其在蛋白互作研究中的应用

Preparation of Protoplast in Nicotiana benthamiana Leaf and Application of Protoplasts in Researches on Protein Interaction

[目的]获得高产量的烟草叶片原生质体。[方法]在前人的基础上,在酶浓度和酶解时间上,对简易大量制备本氏烟叶原生质体的条件进行了进一步的探索;并以此为基础,通过双分子荧光互补试验进一步研究了其在蛋白互作研究中的应用。[结果]在纤维素酶浓度为1.3%,离析酶浓度为0.4%,果胶酶浓度为0.3%,酶解4 h,酶解温度28℃,并在简易纯化的条件下制备原生质体,所得到的完整原生质体产量最高,并且细胞碎片和杂质均最少。[结论]该方法为瞬时表达于本氏烟叶中的荧光蛋白研究提供了重要的技术参考。

[Objective] To obtain high yields of Nicotiana benthamiana leaf protoplast.[Method] Different enzyme concentration and digestion time was applied in the experiment on the basis of the predecessors＇experiment,for further exploration of better isolation conditions.And on this basis,a further study on its application in protein interaction was conducted through the bimolecular molecular fluorescence complementation（BiFC） experiments.[Result] The results show that,the Nicotiana benthamiana leaf protoplast were prepared under the condition that the cellulase concentration was 1.3％,the macerozyme concentration was 0.4％,the pectinase concentration was 0.3％,enzymatic hydrolysis time was 4 hours and the reaction temperature was 28 degrees,then in simple purification,we obtained the highest yields of complete protoplasts and the celludar debris and impurities are minimal.[Conclusion] The method provides important technique reference for fluorescent protein research in tobacco leaf.