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以厚朴DNA为模板,利用正交试验分别对影响厚朴ISSR-PCR反应的Taq酶浓度、dNTP浓度、引物浓度、Mg2+浓度、模板DNA浓度进行了优化,并通过梯度PCR确定不同引物的最佳退火温度和循环次数,最终确定厚朴最佳反应体系及扩增条件为:25μl体系,其中包括1.5mmol.L-1MgCl2,0.3μmol.L-1引物,0.04U.μl-1Taq酶,0.2mmol.L-1dNTP,4ng.μl-1模板DNA,1×Buffer;扩增程序:94℃预变性5min,94℃变性30s,50℃～60℃(退火温度随引物不同而定)退火45s,72℃延伸90s,共40个循环,然后72℃延伸8min,4℃终止反应。此外,还利用优化的反应体系成功筛选出21条ISSR引物,并利用部分引物对厚朴个体进行了遗传多样性分析。
Using Magnolia officinalis DNA as template, the orthogonal design was used to optimize Taq polymerase, dNTP, primer, Mg2 + and template DNA, respectively, to determine the ISSR-PCR reaction of Magnolia officinalis. Optimal reaction system and amplification conditions were determined as follows: 25μl system, including 1.5mmol.L-1MgCl2, 0.3μmol.L-1 primer, 0.04U.μl-1Taq enzyme, 0.2 1 × Buffer; Amplification program: denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 30s, annealing at 50 ℃ ~ 60 ℃ (the annealing temperature varies with the primers) for 45s, Extension at 72 ° C for 90 s for 40 cycles, then extension at 72 ° C for 8 min, stopping the reaction at 4 ° C. In addition, 21 ISSR primers were successfully screened by using optimized reaction system, and some primers were used to analyze the genetic diversity of Magnolia officinalis individuals.