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[目的]筛选经济、稳定性好的水稻PCR反应体系,并检测所选体系在不同的基于PCR反应的分子标记中的通用性。[方法]以CTAB法提取的水稻叶片DNA为模板,应用L16(45)正交设计进行PCR反应体系优化。选用20μl体系的浓度梯度设计,对DNA模板浓度、dNTP、引物浓度、Taq酶、Mg2+浓度5个因素设计4个水平。先以1个SSR标记(MRG6102)对16个不同体系进行筛选。为了验证所选最优体系的稳定性,再选用6个SSR标记(RM213、RM207、RM208、RM155、OSM89、OSM91)对20个水稻品种(系)进行扩增;为检测除SSR标记以外的基于PCR反应标记的通用性,选用2个STS标记(OSR20、OSR32)及2个显性标记(Pibdom,Lys145)进行检测。[结果]16个不同处理组合均扩增出了清晰谱带,但扩增效果及PCR产量有差异。最经济、适用的体系为:20μl反应体系,20ng模板DNA,浓度150μmol/LdNTP,浓度0.2μmol/L引物,1.0UTaqDNA聚合酶,浓度1.5mmol/LMg2+,1×Taqbuffer,剩余体积用超纯水补齐。PCR扩增程序为:95℃预变性3min;94℃变性45s,48～55℃退火45s(不同的引物其最佳退火温度不同),72℃延伸1min(对于其他一些基于PCR反应的标记,如果预期片段较大,可以适当调整延伸时间到1.5min),共36个循环;72℃延伸7min;然后4℃保存。[结论]该研究确定了水稻PCR优化反应体系,该体系也适用于一些其他基于PCR反应的标记。
[Objective] The research aimed to screen economical and stable rice PCR reaction system and to test the versatility of the selected system in different PCR-based molecular markers. [Method] The DNA of rice leaves extracted by CTAB method was used as template and the PCR reaction system was optimized by L16 (45) orthogonal design. The design of the concentration gradient of 20μl system was adopted. Four levels of DNA template concentration, dNTP, primer concentration, Taq enzyme and Mg2 + concentration were designed. Sixteen different systems were screened with one SSR marker (MRG6102). In order to verify the stability of the selected optimal system, 20 SSR markers (RM213, RM207, RM208, RM155, OSM89, OSM91) were selected for amplification. The commonness of PCR reaction markers was detected by two STS markers (OSR20, OSR32) and two dominant markers (Pibdom, Lys145). [Result] The clear bands were amplified in all 16 different combinations, but the amplification effect and PCR yield were different. The most economical and suitable system is: 20μl reaction system, 20ng template DNA, 150μmol / LdNTP, 0.2μmol / L primer, 1.0UTaq DNA polymerase, 1.5mmol / LMg2 +, 1 × Taqbuffer, Qi The PCR procedure was as follows: pre-denaturation at 95 ° C for 3 min, denaturation at 94 ° C for 45 s, annealing at 48-55 ° C for 45 s (optimal annealing temperatures varied for different primers) and extension for 1 min at 72 ° C (for some other PCR-based markers, Expected fragments larger, you can properly adjust the extension time to 1.5min), a total of 36 cycles; 72 ℃ extended 7min; then stored at 4 ℃. [Conclusion] This research confirmed the rice PCR optimization reaction system, and the system was also suitable for some other PCR-based markers.