小麦TILLING分析中CELⅠ酶切及PCR反应体系的优化

Establishment and Optimization of CEL Ⅰ-based TILLING for Wheat

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。

CELⅠ-based TILLING（Targeting Induced Local Lesions In Genomes） platform is very useful for high throughput identification of point mutations within targeted genes and improvement of mutation-detected efficiency in wheat.The space-mutated Xinmai 18（Triticum aestivum L.） SP2 population was used with one SNP of Waxy gene in the population as a positive control.The TILLING protocol for wheat was optimized through improving the method of genomic DNA extraction,the concentration of dNTP,Mg2＋,primers and CELⅠ buffer as well as experimental environment air humidity.It was found that through raising the grinding frequence to 30/s and prolonging the reaction time of KAc to 20min in genomic DNA extraction,the quality and purification of DNA were the best.The concentration of both dNTP and Mg2＋ did not have any influence on PCR products within the set ranges,while primer played an important role in PCR products and the best concentration was 0.4μmol/L.Optimum amount of CELⅠ enzyme was 0.1U in a 20μl of digestion reaction system and ultrapure water substituting for CELⅠ buffer was the best reaction condition.A CELⅠ-based TILLING technique for wheat was established and optimized.