| 徐欢欢，刘佳佳，李 娜，马正强.小麦叶片瞬时表达体系的优化[J].麦类作物学报,2015,35(12):1617 |
| 小麦叶片瞬时表达体系的优化 |
| Optimization of Transient Expression System in Wheat Leaf |
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| DOI：10.7606/j.issn.1009-1041.2015.12.02 |
| 中文关键词: 小麦 叶片 瞬时表达 体系优化 白粉病 |
| 英文关键词:Wheat Leaf Transient expression System optimization Powdery mildew |
| 基金项目:国家自然科学基金项目（30771165） |
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| 摘要点击次数: 1336 |
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| 中文摘要: |
| 为了解决小麦叶片瞬时表达体系所存在的表达效率低、稳定性差、轰击不均匀等问题，利用GFP和GUS作为报告基因，通过改变叶片处理方式、质粒用量、质粒包裹方法和轰击距离，对小麦叶片瞬时表达体系进行优化，并将优化后的体系应用于小麦抗白粉病相关基因的功能鉴定，验证优化体系的有效性。结果表明，与未优化前的方法相比，叶片处理方式为乙醇，质粒用量为每枪1 μg，质粒包裹方法采用扩大体系法，轰击距离为9 cm时，瞬时表达效率显著提高且轰击更为均匀。应用该体系在感病小麦品种叶片中沉默隐性抗白粉病基因Mlo，显著降低了互作细胞吸器指数，进一步验证了目标基因的功能和优化体系的有效性。 |
| 英文摘要: |
| In order to solve the problems existed in wheat transient expression system, such as low efficiency, poor stability and uneven bombardment, in this study, the transient expression system in wheat leaf has been optimized using GFP and GUS as reporter genes and the effectiveness in functional identification of wheat powdery mildew resistance genes has been verified. Four parameters, including treatment method of wheat leaf, plasmids DNA amount per shot, plasmids coating method and the distance between stopping screen and target cells, have been optimized. The results showed that the efficiency of transient expression was significantly increased by using the parameters combination of leaf treatment with ethanol, 1 μg plasmids DNA concentration per shot, expanded plasmids coating method and 9 cm distance between stopping screen and target cells. Using this optimal system, the recessive resistance gene Mlo was silenced in susceptible wheat leaf, and the haustorium index was decreased, which further confirmed the function of Mlo gene as well as the effectiveness of the optimized system. |
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