黄瓜基因组DNA提取及AFLP体系优化研究

Study on Extraction of Genomic DNA and Optimization of AFLP Techniques of Cucumber(Cucumis sativus L.)

[目的]提取黄瓜基因组DNA,并对AFLP体系进行优化。[方法]以黄瓜嫩叶为材料,利用改进的CTAB法提取高质量的黄瓜叶片总DNA,通过优化酶切连接、预扩增、选择性扩增等试验条件建立适合黄瓜的AFLP银染体系,得到清晰的黄瓜AFLP指纹图谱。[结果]DNA模板的质量影响酶切以及后续的连接扩增反应,改良的CTAB提取法可用于黄瓜AFLP分析,形成清晰的AFLP指纹。优化的酶切连接体系为:以37℃酶切连接12 h为宜,酶切连接的基因组DNA用量为200ng,预扩增时Taq酶用量为0.5U/20μl体系,预扩增产物稀释40倍作为选择性扩增的模板。在此优化的体系下引物E41M47扩增出清晰的条带。[结论]为黄瓜品种的分子标记和黄瓜品种间亲缘关系等研究奠定了基础。

[ Objective] The research aimed to study the extraction of genomie DNA and optimization of AFLP techniques of cucumber（ Cucumis satirus L. ）. [ Method ] The improved method of CTAB method was used to extract high quality genomie DNA from tender leaves of cucumber. DNA restriction reaction and ligase reaction, pre-amplification and selective amplification were optimized for establishment of AFLP reaction system and clear DNA fingerprints. [Result] The results indicated that the effects of restriction of DNA and lingation of oligonucleotide adapters and amplification should be influenced by the quality of total DNA. Clear finger map were obtained by the improved CTAB method which was suitable for AFLP analysis on cucumber. The optimized system was as follow： there were 200 ng DNA template, the optimum temperature in the DNA restriction reaction and lingation system was 37 ℃ for 12 h, there were 0.5 U/20 μl Taqase in the system of pre-amplification, the product of pre-amplification were diluted 40 times using for template of amplification. With the optimized system, the AFLP fingerprinting of 2 cucumber materials amplified by primer combination E41 M47 had been obtained. [ Conclusion] The study can establish the basis for the research of the molecular makers of cucumber and the relationship among cucumber varieties.