龙牙百合DNA的提取及ISSR-PCR体系的建立

目的]研究龙牙百合总DNA的提取方法,建立优化的ISSR-PCR反应体系和程序,为种质资源研究奠定基础。方法]利用改良的CTAB法提取龙牙百合基因组DNA,并对影响ISSR扩增反应的各因素进行优化。结果]获得了高质量的龙牙百合基因组DNA;优化的龙牙百合ISSR-PCR反应体系为,25μl体系中含60 ng模板DNA,0.4μmol/L ISSR引物,2.0 mmol/L Mg2+,1.5 UTaq酶,0.2 mmol/LdNTPs;反应程序为,94℃预变性5 min;然后进行40个循环:94℃变性50 s,52℃复性45 s,72℃延伸75 s;循环结束后72℃延伸8 min。结论]建立了适合于龙牙百合的ISSR-PCR反应体系及程序,为种质资源研究奠定了基础。

DNA Extraction from Lilium brownii var. viridulum and the Establishment of ISSR-PCR System

Objective] This research aimed to study the extraction methods of total DNA from Lilium brownii var.viridulum,establish the optimized ISSR-PCR reaction system and procedure and lay the foundation for the germplasm resources study.Method] The genomic DNA was extracted from L.brownii var.viridulum by using improved CTAB method.Different factors that affected ISSR amplification reaction were optimized.Result] High-quality genomic DNA was obtained from L. brownii var.viridulum.The optimized ISSR-PCR reaction system for L.brownii var.viridulum was as follows: 60 ng DNA template,0.4 μmol/L ISSR primer,2.0 mmol/L Mg2+,1.5 U Taq DNA polymerase and 0.2 mmol/L dNTPs in 25 μl system.The reaction procedure was as follows: pre-denaturing at 94 ℃ for 5 min;40 cycles of denaturing at 94 ℃ for 50 s,annealing at 52 ℃ for 45 s and extending at 72 ℃ for 75 s;extending at 72 ℃ for 8 min after the cycles.Conclusion] The optimal ISSR-PCR reaction system and procedure for L.brownii var.viridulum were established and laid the foundation for germplasm resources study.