大麦EcoTILLING技术体系的优化

Optimization of EcoTILLING Technique in Barley

【目的】建立适合于大麦的EcoTILLING技术体系,用于鉴定大麦目的基因或特定区域的自然突变。【方法】利用M13通用接头作荧光标记,采用巢式PCR对大麦目的基因进行扩增,从PCR扩增的退火温度及模板量、CELⅠ的用量和酶切处理时间等方面对大麦EcoTILLING技术体系进行了优化。【结果】用于第一次PCR扩增(10μL反应体系)的大麦基因组DNA模板最佳用量为20 ng,第一次PCR扩增产物稀释3倍后作为第二次PCR扩增的模板,2次PCR扩增的适宜退火温度均为58℃;在20μL CELⅠ酶切体系中,最适酶量为0.4μL(3 U.μL-1),45℃条件下最佳酶切处理时间为17.5 min。【结论】优化的EcoTILLING技术体系能有效地发现大麦自然群体中特定区域的DNA多态性,降低了试验成本,为在大麦及其它作物上的广泛应用提供借鉴。

【Objective】The goal of this work is to establish an optimized EcoTILLING protocol for identification of natural variations within target genes and specified regions of genomes in barley.【Method】The universal adapter M13 was labeled with fluorescent dyes,the nested PCR with specific primer combination and M13 adapter was performed for the targeted region.The EcoTILLING protocol for barley was optimized,including the template amount and annealing temperature in PCR amplification,CELⅠamount and reaction time in cleavage of mispaired heteroduplex DNA.【Result】The results showed that the optimal amount of genomic DNA in a 10 μL of reaction system was 20 ng as template for the first round of PCR,and the first PCR product was diluted 3-fold and used as a template for the second round.The proper annealing temperature for both first-and second-round PCRs was 58℃.Optimum amount of CELⅠ enzyme was 0.4 μL（3 U·μL-1） in a 20 μL of digestion reaction system,and 17.5 min at 45℃ was the optimal time for the cleavage of heteroduplex DNAs.【Conclusion】The experiment indicates that the optimized EcoTILLING protocol can effectively detect DNA polymorphisms for natural population of barley with lower cost,and it could provide a foundation for larger scale efforts in reverse genetics and characterization of natural nucleotide variation in barley.