摘要
[目的]建立栝楼的RAPD-PCR体系并对该体系进行优化。[方法]以栝楼叶片为材料,采用CTAB法提取栝楼叶片基因组DNA,利用正交设计对RAPD-PCR体系进行优化。[结果]各因素水平变化对PCR反应影响大小依次为:Mg2+、TaqDNA聚合酶、引物和dNTPs。通过试验分析,优化的栝楼RAPD反应体系为:在25μl反应体系中,含10×buffer2.5μl,Mg2+2.0mmol/L,Taq酶1U,引物0.8μmol/L,dNTPs0.1mmol/L。反应程序为94℃预变性2mim;94℃变性30s,37℃退火温度40s,72℃延伸1.5mim,36次循环;72℃延伸10mim,4℃保存。[结论]该研究建立的栝楼RAPD反应体系稳定可靠,为栝楼性别鉴定、遗传多样性分析、亲缘关系分析等方面的研究提供了有效的方法。
[Objective] The aim was to establish and optimize the RAPD-PCR system of Trichosanthes kirilowii Maxim. [Method] With the leaves of T. kirilowii as materials, the genomic DNA of T. kirilowii leaves were extracted by CTAB method and the orthogonal design was used to optimize the RAPD-PCR system of T. kirilowii. [Result] The effects of level changes of each factor on PCR reaction in the following order was Mg2＋, Taq DNA polymerase, primer and dNTPs. Through experimental analysis, the optimized RAPD-PCR system of T. kirilowii was established, namely, 25 μl reaction system containing 2.5 μl 10×buffer, 2.0 mmol/L Mg2＋, 1 U Taq polymerase, 0.8 μmol/L primer and 0.1 mmol/L dNTPs. The reaction program was pre-denaturation at 94 ℃ for 2 min, followed by 36 cycles of denaturation at 94 ℃ for 30 s, anneal at 37 ℃ for 40 s, extension at 72 ℃ for 1.5 min, and a final extension at 72 ℃ for 10 min, kept at 4 ℃. [Conclusion]The established RAPD-PCR system of T. kirilowii by the research was stable and reliable, which provided the effective method for studies such as sex identification, genetic diversity analysis and phylogenetic analysis of T. kirilowii.
出处
《安徽农业科学》
CAS
北大核心
2010年第13期7150-7152,共3页
Journal of Anhui Agricultural Sciences
关键词
栝楼
RAPD
分子标记
优化
Trichosanthes kirilowii Maxim
RAPD
Molecular markers
Optimization
