摘要
[目的]研究提取火棘基因组DNA的最佳方法,并研究适合火棘的稳定的RAPD反应体系,为以后开展火棘的遗传多样性研究、物种资源研究和亲缘关系鉴定等提供重要的参考。[方法]采用改进的CTAB法,成功地提取了火棘基因组DNA,并对RAPD反应体系中的Taq酶、MgC l2、dNTPs和引物4个因素进行4因素4水平的正交试验设计,从中筛选出最佳的优化条件。[结果]电泳结果显示,DNA无降解,杂质少。DNA浓度较高约为500 ng/μl。并以此DNA为模板,成功地建立了RAPD稳定的反应体系:总体积25μl,包括25 ng的DNA,1×buffer,2.3 mmol/LMgC l2,1.0μmol/L引物,0.15 mmol/L dNTPs和2.0 U的TaqDNA聚合酶。RAPD扩增程序为:先在94℃下变性4 m in,然后在94℃下变性40 s,36.8℃下复性50 s,72℃下延伸70 s,反应40个循环,最后72℃延伸4 m in。[结论]改进的CTAB法能成功地提取火棘基因组DNA,利用正交试验设计所建立的RAPD反应体系,可以获得较为稳定、可靠的扩增产物。该体系可应用于火棘遗传多样性、亲缘关系等方面的研究中,为进一步开展火棘分子生物学研究奠定了基础。
[ Objective ] The reference for the research on the diversity and resource of Pyracantha fortuneana and the identification of the relation of its varieties were provided through the study on the optimal method of DNA extraction and stable system of RAPD reaction. [ Method] The genomic DNA of Pyracarathafortuneana was successfully extracted with the improved CTAB method and the optimal condition in the system was selected with the experimental design of 4 factors（ Taq enzyme, MgcL2, dNTPs and primer） at 4 levels. [ Results] The electrophoresis result indicated that the quality and concentration of extracted DNA were very high（500 ng/μl）. After that, the reproducible RAPD reaction system was set up with extracted DNA as template. RAPDs were performed in volumes of 25 μl, containing 25 ng of DNA, 1 × reaction buffer （ TaKaRa Dalian ）, 2.3 mmol/L MgCl2 （ TaKaRa Dalian ）, 1.0 μmol/L primer （ SBS Beijing ）, 0.15 mmol/L mixture of dNTPs （ TaKaRa Dalian） and 2.0 unit Taq DNA Polymerase（ promega Shanghai）. The procedure of RAPD was thermal cycler （HYBAID） was programmed for 1 cycle at 94 ℃ for 4 minutes, followed by 40 cycles at 94 ℃ for 40 seconds, at 36.8 ℃ for 50 seconds and at 72 ℃ for 70 seconds, and finally by 1 cycle at 72℃ for 4 minutes. [ Conclusion] The genomic DNA of Pyracantha fortuneana was extracted with the improved CTAB method and the reaction system was established for the stable and reliable the procedure of RAPD.
出处
《安徽农业科学》
CAS
北大核心
2009年第19期8876-8878,8880,共4页
Journal of Anhui Agricultural Sciences
基金
西南科技大学人才引进基金项目(zk043081)
关键词
火棘
RAPD
反应体系
Pyracantha fortuneana
RAPD
Reaction system
