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植物原生质体广泛应用于植物基因功能研究中,包括瞬时基因表达、亚细胞定位、蛋白互作和蛋白活性分析等。当前,小麦基因的亚细胞定位和功能分析,大多利用模式植物拟南芥等异源的原生质体,易于造成研究结果的不准确。为避免这种情况,小麦原生质体制备及高效转化体系的建立与应用是必需的。在PEG介导的小麦原生质体转化过程中,原生质体分泌的核酸酶大量降解质粒DNA,转化效率的提高因此受到阻碍。为了建立小麦原生质体的高效转化体系,本文测试了抑制胞外核酸酶活性的因素和提高质粒DNA浓度等多个条件对转化效率的影响。结果表明,转化过程中加入双倍用量的质粒DNA进行转化,且始终保持低温环境(1℃)用以抑制核酸酶酶活性,可以使小麦原生质体的转化效率提高至85%。本文还将该系统成功地应用于2个小麦抗病相关蛋白的亚细胞定位研究,证明了该系统的高效性和实用性。该研究对未来相关研究有一定参考价值。
Plant protoplasts are widely used in plant gene function research, including transient gene expression, subcellular localization, protein interaction and protein activity analysis. At present, the subcellular location and function analysis of wheat genes are mostly heterologous protoplasts, such as the model plant Arabidopsis thaliana, which easily lead to inaccurate findings. To avoid this situation, the establishment and application of wheat protoplast preparation and efficient transformation system are necessary. During PEG-mediated transformation of wheat protoplasts, protoplast-secreted nucleases degrade plasmid DNA in large quantities and thus the increase in transformation efficiency is hindered. In order to establish an efficient transformation system of wheat protoplasts, we tested the effect of multiple factors such as the factors inhibiting the activity of extracellular nuclease and increasing the concentration of plasmid DNA on transformation efficiency. The results showed that the conversion efficiency of wheat protoplasts could be improved up to 85% by adding double amount of plasmid DNA into the transformation process and always maintaining the low temperature environment (1 ℃) to inhibit the nuclease activity. The system was also successfully applied to the subcellular localization of two wheat disease-related proteins, which proved the system’s efficiency and practicability. This research has certain reference value to the related research in the future.