马齿苋ISSR-PCR反应体系与反应条件的优化

目的]优化马齿苋ISSR-PCR反应体系与反应条件。方法]以马齿苋为试验材料,采用改良的CTAB法提取总DNA,对ISSRPCR引物进行筛选,同时进行ISSR-PCR反应体系和反应条件的优化,电泳检测ISSR-PCR产物。结果]8个ISSR引物适合马齿苋的ISSR-PCR分析;最适反应体系为总体系25.0μL,其中2×Taq Platinum PCR Master Mix 12.5μL,10μmol/L引物2.0μL,40 mg/L DNA模板1.0μL,dd H_2O 9.5μL;最适反应条件为94℃预变性360 s;94℃变性60 s,54℃退火60 s,72℃延伸90 s,30次循环;72℃再延伸300 s。结论]建立了马齿苋ISSR-PCR扩增的最佳反应体系和扩增程序,为进一步研究马齿苋提供基础资料。

Optimization of ISSR-PCR Reaction System and Reaction Condition of Portulaca oleracea L.

Objective] To optimize ISSR-PCR reaction system and reaction condition of Portulaca oleracea L.Method] Taking P.oleracea L.as the research object,using improved CTAB method to extract total DNA of P.oleracea L.,the ISSR-PCR primers were screened,at the same time ISSR-PCR reaction system and reaction condition were optimized to test ISSR-PCR products by electrophoresis.Result] Eight primers were suitable for ISSR-PCRof P.oleracea L..The optimal reaction system was that total reaction system 25.0 μL,ineluding 12.5 μL 2 × Taq Platinum PCR Master Mix,2.0 μL 10 μmol/L primers,1.0 μL 40 mg/L DNA templates,9.5 μL ddH2O.The optimal reaction condition was that pre-degeneration at 94 ℃ for 360 s,going to 30 cycles:degeneration at 94 ℃ for 60 s,annealing at 54 ℃ for 60 s,extension at 72 ℃ for 90 s,then extension at 72 ℃ for 300 s after 30 cycles.Conclusion]The optimizing ISSR-PCR reaction system and reaction condition of P.oleracea L.were established to provide basis for further study P.oleracea L..