皱边石杉内生真菌ISSR-PCR反应条件的优化

以从皱边石杉中分离纯化出的99株内生真菌DNA为材料,优选出10条ISSR引物(UBC842、UBC848、UBC850、UBC856、UBC861、UBC864、UBC868、UBC873、UBC880、UBC887)的最适退火温度,并对其进行ISSR–PCR反应体系单因素试验及正交试验优化,建立皱边石杉内生真菌ISSR–PCR的优化反应体系,即25μL反应体系中,10×PCR Buffer(Mg2+浓度为2.0 mmol/L)2.5μL,dNTPs(10 mmol/L)0.6μL,引物(10 pmol/μL)2.0μL,Taq E(1 U/μL)1.5μL,DNA模板(10 ng/μL)3μL,无菌ddH2O 15.4μL。以UBC873引物对99株内生真菌基因组DNA进行PCR扩增,初步获知皱边石杉内生真菌资源的遗传多样性非常丰富。

Establishment and optimization of ISSR-PCR reaction system for endophytic fungi of Huperzia crispata

DNAs were extracted from 99 endophytic fungi isolated from Huperzia crispata and the best annealing temperatures of 10 primers(UBC842,UBC848,UBC850,UBC856,UBC861,UBC864,UBC868,UBC873,UBC880 and UBC887) were determined for optimization of ISSR-PCR amplification of endophytic fungi from Huperzia crispate through single factor test and orthogonal design.The result showed that the optimal ISSR–PCR reaction volume was 25 μL,containing 2.5 μL of 10×PCR Buffer(2.0 mmol/L Mg2+),0.6 μL of dNTPs(10 mmol/L),2.0 μL of primer(10 pmol/μL),1.5 μL of Taq E(1U/μL),3 μL of template DNA(10 ng/μL) and 15.4 μL of aseptic ddH2O.PCR using primer UBC873 conducted on 99 endophytic fungi showed that the genetic polymorphism for endophytic fungi of Huperzia crispata was abundant.