| 杨家强1，王相晶1，郭兆将2，朱勋2，吴青君2*，谢文2，王少丽2，徐宝云2，张友军2.小菜蛾ISSR-PCR反应体系的建立与正交设计优化[J].植物保护,2012,38(6):90-94. |
| 小菜蛾ISSR-PCR反应体系的建立与正交设计优化 |
| Establishment and optimization of ISSR PCR reaction system with orthogonal design for Plutella xylostella |
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| DOI： |
| 中文关键词: 小菜蛾 ISSR 正交设计 反应体系 |
| 英文关键词:Plutella xylostella ISSR orthogonal design reaction system |
| 基金项目: |
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| 摘要点击次数: 1054 |
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| 中文摘要: |
| 以小菜蛾（Plutella xylostella）基因组DNA为模板，采用L16(45)正交试验设计方法，建立小菜蛾的ISSR最佳反应体系。通过梯度退火试验，确定不同引物的最适退火温度。优化得到的反应体系（20 μL)为：Mg2+浓度1.5 mmol/L、Taq DNA聚合酶2.5U、dNTPs浓度0.2 mmol/L、引物浓度1.25 μmol/L、模板DNA量20 ng。反应程序为：94 ℃预变性5.0 min；94 ℃变性45.0 s，40~61 ℃（不同引物退火温度各异）退火1.0 min，72 ℃延伸1.5 min，40个循环；72 ℃延伸10.0 min；10 ℃保存。利用所建立的ISSR PCR反应体系，获得了清晰、重复性好的DNA谱带。 |
| 英文摘要: |
| To establish and optimize ISSR-PCR reaction system for Plutella xylostella, based on the genomic DNA of P. xylostella, the factors influencing ISSR system were explored with the L16(45) orthogonal design. The optimal annealing temperature for ISSR PCR reaction was proposed by gradient PCR. The optimal conditions for ISSR reaction system (20 μL) were determined as follows: 1.5 mmol/L of Mg2+, 2.5U of Taq DNA polymerase, 0.2 mmol/L of dNTPs mixture, 1.25 μmol/L of each primer, and 20ng of template DNA. The reaction program was as followed: initial denaturation for 5.0 min at 94 ℃, 35 cycles of denaturation for 45s at 94 ℃, annealing for 1.0 min at 40-61 ℃, extension for 1.5 min at 72 ℃, with a final extension of 10.0 min at 72 ℃. The clear and reproducible DNA bands were obtained by the established ISSR-PCR reaction system. |
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