赤松外生菌根ITS-PCR体系的建立及优化

以CTAB法提取的赤松外生菌根DNA为模板,应用单因子试验及L16（4^5）正交试验,系统的分析了DNA模板、Mg^2＋、dNTPs、引物和Taq酶对ITS-PCR扩增结果的影响,并建立了赤松外生菌根rDNA ITS扩增反应的优化体系,最优反应体系为：20μL体系中,1×PCR buffer 2μL、DNA模板30 ng、Mg^2＋2.0 mmol/L、dNTPs0.2 mmol/L、引物0.2μmol/L、Taq酶0.5 U。

Establishment and Optimization of ITS-PCR Reaction System for Pinus densiflora Ectomycorrihzal

We studied the effects of concentrations of template DNA,Mg^2＋,dNTPs,primer and Taq polymerase on ITS-PCR amplification based on DNA extracted from ectomycorrhizal of Pinus densiflora by the method of CTAB using single factor test and orthogonal design of L16（4^5）.The optimal PCR system was as follows：1×buffer,2.0 mmol/L Mg2 0.2 mmol/L＋,dNTPs,0.2 μmol/L primer,0.5 U Taq DNA polymerase and 30ng genomic DNA templates in 20μl reaction system.The optimal program of amplifying reaction was as follows：95℃ for 5 min followed by 35 cycles of denaturation at 95℃ for 30 s,annealing at 55℃ for 30 s and extension at 72℃ for 1 min.Final extension was at 72℃ for 7 min and then storing at 4℃.Under this reacting system,clear,stable bands to stabilize for the Pinus densiflora ectomycorrihzal molecules study appraisal lays the foundation.