东方百合ISSR-PCR反应体系的正交优化

Optimizing the ISSR-PCR reaction system for oriental lily based on orthogonal design

以东方百合‘Constanta’和‘Sorbonne’以及9份‘Constanta’בSorbonne’的F1代为试材,采用改良的CTAB法提取百合嫩叶DNA,利用正交设计L16(45)对东方百合ISSR-PCR反应体系的Taq酶、Mg2+、模板DNA、dNTPs和引物5种因素在4个水平上进行优化试验,采用直观分析结合方差分析评价扩增结果。最终获得的最佳反应体系为:在20μL反应体系中,含Taq DNA聚合酶0.5μmol/min、Mg2+1.75 mmol/mL、模板DNA 40～100 ng、dNTPs 0.15 mmol/mL、引物0.6μmol/L、10×PCR Buffer 2μL,不足部分以ddH2O补足。在获得最佳反应体系的基础上采用单因素实验确定了引物最佳退火温度范围为55～45℃,最佳循环次数为30次以及延伸时间为1.5 min。应用该优化的反应体系,对‘Constanta’和‘Sorbonne’以及9份‘Constanta’בSor-bonne’的F1代DNA进行ISSR-PCR扩增,结果显示优化的反应体系具有较高的扩增稳定性。

‘Constanta＇ and ‘Sorbonne＇ and 9 F1 hybrids of ‘Constanta＇ × ‘Sorbonne＇ were used as materials in this experiment,and a modified method of CTAB was applied to extract the genomic DNA from the young leaves.The orthogonal design L16（45）was used to optimize the ISSR-PCR system for oriental lily at four levels and five factors,namely,DNA polymerase,Mg2＋,DNA template,dNTPs and primers.The results of PCR were evaluated by intuitive and variance analysis.Results showed that the best reaction system was 20 μL reaction solution,containing 0.5 μmol/min Taq DNA polymerase,1.75 mmol/mL Mg2＋,40-100 ng template DNA,0.15 mmol/mL dNTPs,0.6 μmol/L primers,2 μL 10×PCR buffer and ddH2O.Using single factor experiment design,we found that 55-45 ℃ was the best touch down PCR annealing temperature,the best cycle times was 30 and the best extend time was 1.5 min.The stabilization of the optimized reaction system was tested by using ‘Constanta＇ and ‘Sorbonne＇ and 9 F1 hybrids of ‘Constanta＇ × ‘Sorbonne＇.