摘要
[目的]研究东北红豆杉RAPD反应体系的优化条件。[方法]以东北红豆杉叶片为材料,用改进CTAB法提取DNA,分别从模板DNA浓度(10、20、30、40、50 ng)、引物浓度(5、10、15、20、25 pmol/μl)、dNTP浓度(0.1、0.15、0.2、0.25、0.3 mmol/L),TaqDNA聚合酶量(0.5、1.0、1.5、2.0、2.5 U)及镁离子浓度(0.5、1.0、1.5、2.0、2.5 mmol/L)5个方面对东北红豆杉RAPD扩增效果的影响进行分析。[结果]用改良的CTAB法提取红豆杉基因组DNA,所得DNA质量较好,无杂质存在,条带整齐一致,而且能够取得比较理想的RAPD扩增效果。通过各因子的比较分析,建立了东北红豆杉RAPD优化体系:20μl PCR反应总体积中含模板DNA10 ng,Mg2 +2.0 mmol/L,引物15pmol/μl,dNTP0.2 mmol/L,TaqDNA聚合酶1.0 U,10×buffer缓冲液2μl,以双蒸水补充至20μl。[结论]为进一步探索与东北红豆杉性别鉴定相关的研究提供技术支持。
[Objective] The research aimed to study the optimization conditions of RAPD reaction system of Taxus cuspidata.[Method] Using the leaves of Taxus cuspidata as materials,DNA was extracted by using modified CTAB method.The influences of template DNA concentration,primer concentration,dNTP concentration,Taq DNA polymerase amount and magnesium ion concentration on the amplification effects of RAPD of Taxus cuspidate were analyzed.[Result] Through analysis and comparison of various factors,the optimized reaction system was established as follows：in a total volume of 20 μl,containing 10 ng DNA,2.0 mmol/L Mg2＋,0.2 mmol/L dNTPs,15 pmol/μl random primer,1.0 U Taq polymerase,2 μl 10×buffer,and the supplementary ddH2O.[Conclusion] The research provided technical support for further discussion on the related studies with the sexual identification of Taxus cuspidate.
