摘要
以火炬松(Pinus teada)幼嫩针叶为材料,采用单因素试验设计分析各组分对SSR-PCR扩增结果的影响,并进一步应用正交试验设计,从DNA模板、引物、d NTPs、Mg2+和Taq酶5个因素3个水平对SSR扩增效果进行研究,确定火炬松SSR-PCR的最佳反应体系。结果表明:各因素不同水平浓度对SSR-PCR反应结果均有影响,火炬松SSR-PCR优化后的反应体系为:DNA模板80 ng、引物0.3μmol/L、dNTPs 0.20 mmol/L、Mg^(2+)2.0 mmol/L、Taq酶1.00 U、1×PCR Buffer,总体积25μL。运用该体系从60对引物中筛选出扩增条带清晰、多态性丰富的SSR引物8对,并用不同火炬松家系进行了检验,证明该体系稳定可靠。
Taking fresh needles of Pinus taeda seedlings as material, the SSR - PCR reaction system was conducted by using the single factor experiment, analyzing the impact of each component on SSR -PCR amplification results. Meanwhile, we made orthogonal design for optimizing the SSR - PCR system, which employed three levels and five factors （DNA template, primers, dNTPs, Mg2＋ and Taq polymerase, respectively） on the result of SSR application. The results showed that the different concentration levels of each factor affected the SSR - PCR reaction results, and the optimized SSR - PCR reaction system was ： DNA templates 80 ng, primers 0.3 Mol/L, dNTPs 0. 20 mmol/L, Mg2 ＋ 2.0 mmol/L, Taq polymerase 1. 00 U, 1 × PCR Buffer with total 25 ML reaction volume. It proved that the optimized sys- tem was stable and reliable by testing different families of Pinus taeda, and 8 SSR primers were screened from 60 primers with clear amplified bands and abundant polymorphism.
作者
张蝶
郭小丹
邓亚婷
徐刚标
ZHANG Die GUO Xiao-dan DENG Ya-ting XU Gang-biao(College of Forestry, Central South University of Forestry and Technology, Changsha 410004, China)
出处
《广西林业科学》
2016年第3期253-258,共6页
Guangxi Forestry Science
基金
引进国际先进林业科学技术项目(2013-4-32)
湖南省研究生科研创新项目(CX2016B329)
中南林业科技大学研究生科技创新基金资助项目(CX2016B17)
