摘要
【目的】探究吐温-20对转基因棉中Bt蛋白提取效率的影响程度,为转基因植物外源蛋白提取方法标准化,进而精准转Bt基因棉品种审定准入项目检测数据提供理论依据。【方法】配制7种不同吐温-20含量的提取液,提取抗虫棉GK19及非转基因棉泗棉3号苗期叶片、蕾期叶片和小蕾、铃期叶片和小铃中可溶性总蛋白,测定提取液中Bt蛋白和可溶性总蛋白含量,比较不同吐温-20含量提取液蛋白提取效率的差异。【结果】结果表明,吐温-20对抗虫棉中外源Bt蛋白提取效率有显著影响,蛋白提取缓冲液中吐温-20含量为0.55%是最适的。【结论】在蛋白提取缓冲液中加入适量吐温-20可明显提高抗虫棉外源Bt蛋白的提取效率,而不影响标准曲线R平方值(R^2)。过量添加吐温-20,超过一定的限度,检测变异系数加大,检测值可信度降低,检测结果无意义,因此准确控制蛋白提取缓冲液成分含量十分必要。
[ Objective]The effort was focused on to reveal the effect of the content of Tween-20 in the buffer solution on the extracting effi- ciency of Bt protein, as to provide a theoretical basis and references on formulating the standardized methods of protein extraction, and improving the accuracy of the testing data for variety certification admission of transgenic cotton. [ Method] Seven extraction solutions with different amount of Tween-20 were prepared and respectively used for extraction of the total soluble protein in the leaves of cotton seedling, budding stage and cotton boll stage, small buds and small bolls, then the extraction efficieneies of all extraction buffer were compared after the content of foreign Bt protein and total soluble protein were detected. [ Result] Tween-20 could dramatically affect the extraction efficiency of foreign Bt protein, and the most proper concentration of Tween -20 in extraction solution was 0.55 %. [ Conclusion] The extraction efficiency of Bt protein could be improved dramatically when proper amount of Tween-20 was added to the extraction solutions and the R2 value of stand curve remained unaffected. Excessive use of Tween-20 would result in higher variation coefficient and lower reliability thus accuracy control operation is very necessary in the preparation for the protein extraction buffer.
出处
《西南农业学报》
CSCD
北大核心
2017年第8期1777-1783,共7页
Southwest China Journal of Agricultural Sciences
基金
四川省财政基因工程项目-论文基金(2014LWJJ-011
2016LWJJ-010)
四川省财政创新能力提升工程公益深化项目(2016GYSH-032)
关键词
吐温-20
抗虫棉
外源蛋白
提取效率
Tween-20
Insect-resistant cotton
Foreign protein
Extraction efficiency
