桔梗RAPD-PCR体系的正交优化及引物筛选

The optimization of RAPD-PCR system with orthogonal design and primers screening in Platycodon grandiflorum

【目的】建立桔梗RAPD-PCR反应的最佳体系并应用于桔梗遗传图谱的构建。【方法】以2个桔梗亲本GS16和GS106的叶片DNA为模板,对桔梗DNA的提取及对影响桔梗RAPD-PCR扩增的重要参数进行优化试验,并从530个随机引物中筛选出在2个亲本间具多态性的引物。【结果】叶片研磨后用蒸馏水预洗及2次沉淀时加入3 mol/L NaCl(pH 5.2)溶液,提取的基因组DNA纯度较高。优化的最佳反应体系总体积为25μL,内含ddH2O12.4μL、10×buffer 2.5μL、引物0.3μmol/L、dNTPs 200μmol/L、Mg2+2.5 mmol/L、TaqDNA聚合酶0.5 U、DNA模板40 ng;反应程序为:94℃预变性4 min;94℃30 s,35.3℃1 min,72℃2 min,40次循环;72℃10 min。按此优化RAPD条件成功地从530个随机引物中筛选出了115个在两亲本间具多态性的引物。【结论】改良的CTAB法能成功用于桔梗基因组DNA提取,建立的最佳反应体系可用于桔梗遗传图谱的构建。

【Objective】 The study was done to find the optimized reaction system for the construction of genetic link map in Platycodon grandiflorum.【Method】 The method of isolating genomic DNA from the leaves of P.grandiflorum and important factors influencing RAPD-PCR was studied.【Result】 The genomic DNA was successfully extracted from the leaves of P.grandiflorum with distilled water washing after rubbing the leaves,and deposited twice with 3 mol/L NaCl（pH 5.2） solution.The optimal PCR system for RAPD analysis was as follows：ddH2O 12.4 μL,10×buffer 2.5 μL,primer 0.3 μmol/L,dNTPs 200 μmol/L,Mg^2＋ 2.5 mmol/L,Taq DNA polymerase 0.5 U,DNA template 40 ng in 25 μL reaction volume.The reaction program was devised for 94 ℃ 4 minutes,94 ℃ 30 s,35.3 ℃ 1 min,72 ℃ 2 min,followed by 40 cycles,and a final extension at 72 ℃ for 10 minutes.115 primers were screened from 530 10 bp random primers,which could produce stable and polymorphic bands.【Conclusion】 The reformed CTAB method could extract the genomic DNA of P.grandiflorum successfully,and the optimum RAPD reaction system could be used for the genetic link map construction in P.grandiflorum.