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在小麦中建立稳定的基于CELⅠ酶切的目的基因突变位点检测技术,有助于高通量鉴定目的基因片段的点突变及提高突变检测效率。本研究以冬小麦品种新麦18空间诱变SP2群体为材料,以小麦糯质基因Waxy为目标片段,通过优化基因组DNA提取方法、调整PCR反应体系中dNTP、Mg2+及引物浓度、改变目标片段CELⅠ酶切缓冲液成分,以及调整纯化过程中的空气相对湿度等方式,优化了小麦TILLING技术体系。在利用PVP-40法提取DNA过程中,研磨器振动频率提高到30/s,KAc溶液的反应时间延伸为20min时,基因组DNA质量和纯度最佳;在设定的浓度范围内dNTP和Mg2+浓度对产物影响差异不明显,均能高效扩增出目的条带。引物浓度对产物影响差异显著,最佳引物浓度为0.4μmol/L。20μl酶切体系中,最佳CEL I酶浓度为0.1U且利用超纯水代替CELⅠ缓冲液。最终在小麦中建立起了基于CELⅠ酶切的高通量TILLING筛选技术体系。
The establishment of a stable CEL-I-based target gene mutation detection technology in wheat helps to identify high-throughput point mutations in the target gene fragment and improve the mutation detection efficiency. In this study, the wheat variety Waxy Waxy was used as the target material to optimize the genomic DNA extraction method. The concentration of dNTP, Mg2 + and primer in the PCR reaction system was adjusted to change the target fragment CELⅠ enzyme Cut the buffer composition, and adjust the purification process of air relative humidity and other ways to optimize the wheat TILLING technology system. In the process of extraction of DNA by PVP-40, the quality and purity of genomic DNA were the best when the vibration frequency of grinder was increased to 30 / s and the reaction time of KAc solution was extended to 20min. The concentration of dNTP and Mg2 + The product of the difference is not obvious, can efficiently amplify the purpose of the band. The effect of primer concentration on the product was significant, the best primer concentration was 0.4μmol / L. In a 20 μl digestion system, the optimal CEL I enzyme concentration was 0.1 U and ultrapure water was used instead of CEL I buffer. Finally, a high-throughput TILLING screening system based on CEL I was established in wheat.