小麦TILLING突变体检测中基因组DNA提取方法的优化

Optimization of Genomic DNA Extraction Methods for Mutant Screen in a Wheat TILLING Population

为了优化小麦高通量TILLING突变体扫描所需基因组DNA提取的方法,对利用SDS法、改良SDS法、CTAB法、改良CTAB法、AxyPrep DNA提取试剂盒、Qiagen DNeasy Plant Mini Kit试剂盒以及再生Qiagen硅胶柱方法提取的小麦基因组DNA进行了系统比较。结果表明,尽管7种方法所提取DNA的纯度及琼脂糖凝胶电泳检测结果均良好,但再生Qiagen硅胶柱方法、CTAB法和改良SDS法的DNA浓度显著高于其他方法。根据小麦细胞分裂素氧化酶基因1(TaCKX1)的DNA序列设计引物,以新鲜DNA样品用于PCR扩增大于1 000bp的片段时,后4种方法扩增效果优于前3种方法;DNA在-20℃下保存100d后重新进行PCR扩增时,则只有Qiagen试剂盒和再生Qiagen硅胶柱2种方法提取的DNA能扩增出大于1 000bp的片段。采用本实验室与新西兰坎特伯雷大学合作建立的再生硅胶柱与自配提取液提取小麦基因组DNA,既保证了DNA的高质量,又降低了使用试剂盒的成本,为小麦TILLING库的构建及其他相关试验提供了一种经济有效的DNA提取方法。

In order to optimize the genomic DNA extraction methods for efficient mutant screening in wheat TILLING population, seven different DNA extraction methods, such as SDS method, modified SDS method, CTAB method,modified CTAB method, AxyPrep DNA column method, Qiagen DNeasy Plant Mini Kit method and regenerated Qiagen column method, were compared for their effectiveness on genomic DNA extraction. The results showed that the DNA concentration varied among different extraction methods,and the DNA concentration of regenerated Qiagen column method, CTAB method, and modified SDS method were higher than that of the other method. The quality of DNA extracted by all the seven methods in terms of purity and integrity were satisfied when detected with agarose gel e-lectrophoresis. However, when these DNA samples were used as PCR template to amplify products more than 1000 bp in length, the amplification efficiency of the modified CTAB method, AxyPrep DNA column method,Qiagen DNeasy Plant Mini Kit method and regenerated Qiagen column method was much better than that of the other three methods. After storing the DNA samples in -20℃ for 100 days,the target gene sequences longer than 1 000 bp could only be amplified from the DNA sam-ples extracted using Qiagen DNeasy Plant Mini Kit method and regenerated Qiagen column method. The newly developed method combining the regenerated Qiagen column with home-made solutions al-lowed us to extract high quality genomic DNA from a wheat TILLING population, with much higher DNA concentration and lower cost than the Qiagen DNeasy Plant Mini Kit. This will provide an effi-cient and economical genomic DNA extraction method for TILLING procedure in wheat.