海南龙血树DNA提取与ISSR反应体系的优化

Isolation Method for Genomic DNA and Optimization of ISSR Reaction Systems for Dracaena cambodiana Pierre ex Gagnep

目的:从海南龙血树叶片中提取出高质量的总DNA,建立与优化海南龙血树ISSR的反应体系。方法:采用4种DNA提取方法,提取海南龙血树叶片中的总DNA,并对DNA进行紫外和电泳检测。采用改良CTAB法提取了基因组DNA模板,对海南龙血树ISSR-PCR反应体系中各个主要影响因子进行了优化和筛选。结果:改良CTAB法提取的DNAA260/A280在1.7～1.9之间,纯度高、杂质少、DNA完整性好。根据PCR产物的琼脂糖凝胶检测结果,由试验得到的最佳反应体系为:60ng模板DNA,1.5mmol/LMg2+,0.25mmol/LdNTPs,1.0μmol/L引物,1UTaq酶,总体积为20μl。结论:改良CTAB法可以从海南龙血树叶片中提取高质量DNA,该反应体系适用于应用ISSR标记开展海南龙血树DNA指纹、遗传多样性等研究。

Objective：To isolate high-quality genomic DNA from leaves of Dracaena cambodiana Pierre ex Gagnep,to establish and optimize ISSR reaction system for Dracaena cambodiana Pierre ex Gagnep.Method：Four isolation methods were used to extract total DNA from young leaves,and the quality of DNA was detected by UV scanning and electrophoresis.DNA was extracted by improved CTAB method,single factor experiment method was used to present the effect of the main reaction system elements on ISSR-PCR.Result：The A260/A280 of total DNA extracted by the Improved CTAB method ranged from 1.7 to 1.9 with high-purity,low levels of impurity and good integrity.Based on the result of PCR products tested on the agarosegel,an optimum reaction system obtained in the test was as follows：60ng DNA template,1.5 mmol/L Mg2＋,0.25mmol/L L dNTPs,1.0μmol/L primer,1 U Taq polymerase,total volume of reaction system being 20μl.Conclusion：The improved CTAB method is suitable for extracting high-quality DNA from young leaves of Dracaena cambodiana Pierre ex Gagnep,The ISSR-PCR system is suitable for the study of the Dracaena cambodiana Pierre ex Gagnep DNA fingerprint,genetic diversity,etc.