摘要
应用改良CTAB法提取了无患子基因组DNA,该方法具有操作过程简单、耗时少、能有效降低无患子基因组DNA提取过程中酚类化合物、多糖等杂质的污染。正交试验研究了dNTP浓度、引物浓度、Taq DNA聚合酶这3个因素对ISSR-PCR反应的影响,建立并优化了适合于无患子ISSR-PCR的反应体系。优化的反应总体系20μl含1×PCR Buffer,0.4 mmol.L-1dNTP,0.8μmol.L-1引物,0.75 U Taq DNA聚合酶,20 ng DNA模板。使用此ISSR扩增反应体系,获得了部分不同种质无患子DNA的清晰条带,验证了该体系的稳定性。优化的反应体系为后续无患子遗传多样性的研究奠定了基础。
The genomic D NA was extracted from the leaves of Sapindus mukorossi by using the modified CTAB method. This method is of easy manipulation and can effectively reduce contamination of phenolic compounds and polysaccharide in the process of DNA extraction. The orthogonal test was used to optimize ISSR reaction system for Sapindus mukorossi, including the dosage of dNTP, primer and Taq DNA polymerase, which were thought to influence the efficiency of ISSR reaction. As a result, an optimal ISSR-PCR reaction system （20 μl） including 1×PCR Buffer,0.4 mmol·L^-1 dNTP, 1.0 μmol·L-1 primer, 1.0 U Taq DNA polymerase and 20 ng DNA template was obtained. With this optimal ISSR amplification system, various clear and polymorphic bands obtained with DNA samples from some germplasm of Sapindus mukorossi, which indicated were the stability of the current system. The present study will set a molecular foundation for the study of genetic diversity of Sapindus mukorossi.
出处
《东南园艺》
2013年第1期19-23,共5页
Southeast Horticulture
基金
福建省农科院导师制青年科技创新基金项目(2012DQB-26)
福建省农业科学院科技下乡"双百"行动项目(sbmx1301)
作者简介
姜翠翠（1983-），女，博士，助理研究员，从事果树遗传育种与生物技术
通讯作者
