甜瓜半粒干种子DNA提取方法的优化

Optimization of DNA extraction method for half melon dry seeds

【目的】优化甜瓜半粒干种子DNA提取方法,为甜瓜相关分子研究提供技术支持。【方法】以甜瓜半粒干种子为材料,比较改良CTAB法、SDS法、高盐低pH法和尿素法4种提取方法的DNA提取效果,并对改良CTAB法中的提取液CTAB浓度、裂解时间、苯酚抽提次数等进行优化,以琼脂糖凝胶电泳和紫外分光光度计分别检测DNA的质量和纯度。最后通过SSR分析验证优化后改良CTAB法提取DNA的效果。【结果】4种提取方法中,改良CTAB法提取的DNA提取率最高,且DNA质量和纯度均高于其他提取方法。优化得到改良CTAB法的最佳提取条件为:提取液CTAB浓度2%,裂解时间20 min,苯酚抽提1次[先用苯酚/氯仿/异戊醇(25∶24∶1)抽提1次,再用氯仿/异戊醇(24∶1)抽提1次]。在此条件下,提取的甜瓜半粒干种子DNA质量和纯度较好,以此为模板进行SSR分子标记分析时,电泳条带稳定、清晰,基本无杂带,可清楚区分品种间的差异。【结论】优化后的改良CTAB法提取甜瓜半粒干种子DNA效果较好,可满足甜瓜种子分子标记和遗传多样性分析等分子实验需求。

【Objective】The aim of the study was to optimize the extraction method of half melon dry seed DNA,laying a foundation for the molecular analysis of melon seed.【Method】Using half melon dry seeds as material,the DNA extraction effects of four extraction methods including modified CTAB method,SDS method,low pH medium with high salt method( LPHS)and urea method were compared,and the CTAB concentration,cracking time and times of phenol extraction in the modified CTAB method were optimized,the quality and purity of the extracted DNA were detected by agarose gel electrophoresis and spectrophotometer. Finally,the effects of DNA extracted by the modified CTAB method after optimization was verified by SSR analysis.【Result】Among the four extraction methods,the modified CTAB method had the highest extraction rate of DNA,and the quality and purity of DNA were higher than other extraction methods. The optimal extraction conditions of the improved CTAB method after optimization were as follows:CTAB concentration of extraction solution was 2%,cracking time was 20 min,and phenol was extracted once[phenol/chloroform/isoamyl alcohol (25∶24∶1)extracted the first time,chloroform/isoamyl alcohol(24∶1)extracted the second time]. Under this condition, DNA quality and purity of the extracted half dry seeds of melon were fine. When SSR molecular marker analysis was carried out based on the extracted seeds,the electrophoresis bands were stable,clear and basically free of heterogenous bands,which could clearly distinguish the differences between individual varieties.【Conclusion】The optimized CTAB method can extract the melon seed DNAeffectively,which can meet the needs of melon seed molecular markers and genetic diversity analysis.