构树ISSR反应体系的优化与建立

Establishment and Optimization of ISSR Reaction System of Broussonetia papyriferaz

利用正交试验对Taq酶浓度、引物浓度、dNTPs浓度、Mg2+浓度和模板DNA浓度5个因素进行优化,从而建立了一套适宜构树的ISSR-PCR反应体系。在20μL的反应体系中,各反应物的最适宜含量为:0.07 U.μL-1TaqDNA聚合酶、0.35μmol.L-1ISSR引物、0.3 mmol.L-1dNTPs,2.0 mmol.L-1Mg2+、1.2 ng.μL-1模板DNA。PCR扩增程序为:94℃预变性5 min,94℃变性30 s,46℃退火45 s,72℃延伸2 min,40个循环。

The effects of five factors, such as the concentration of Taq DNA polymerase, primers, dNTPs, Mg2~ and DNA template, on ISSR-PCR reaction were optimized by orthogonal tests. Thus a suitable ISSR-PCR reaction system for Broussonetia papyrifera was established. The optimized ISSR-PCR system for B. papyrifera in 20 μL reac- tion mixture contained 0. 07 U·μL^-1 Taq DNA polymerase, 0.35 μmol·L^-1 ISSR primers, 0. 3 mmol·L^-1 dNTPs, 2. 0 mmol·L^-1 Mg^2＋ and 1.2 ng·μL^-1 DNA template. The suitable PCR protocol is preliminary denatur- ation at 94 ℃ for 5 min, 40 cycles of denaturation at 94 ℃ for 30 s, anneal at 46 ℃ for 45 s, extended at 72 ℃ for 2 min. This study could provide some references for the genetic diversity analysis of B. papyrifera.