| 荷花ISSR—PCR反应体系的建立及优化 |
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| 引用本文： | 黄宇,;何天友,;荣俊冬,;刘颖嘉,;胡迪科,;郑郁善.荷花ISSR—PCR反应体系的建立及优化[J].甘蔗(福建),2009(4):284-288. |
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| 作者姓名： | 黄宇 ;何天友 ;荣俊冬 ;刘颖嘉 ;胡迪科 ;郑郁善 |
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| 作者单位： | [1]福建农林大学工业原料林研究所; [2]福建农林大学园林学院,福建福州350002 |
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| 基金项目： | 福建省科技重大专项资助项目（2004YZ02-05）;福建省创新平台福建省中药材CAP工程技术研究中心资助项目（2008Y2001）. |
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| 摘 要： | 以荷花叶片提取的基因组DNA为材料，通过对影响ISSR—PCR扩增效果的一些因素，如dNTPs浓度、Mg2＋浓度、TαDNA聚合酶用量、引物用量、模板DNA用量以及退火温度等进行筛选和优化，确立了可用于荷花ISSR—PCR分析的最适宜的PCR反应体系：20μL PCR反应体积含O．4mmol·L-1 dNTPs、3．5mmol·L-1Mg2＋、1．5U TαqDNA聚合酶、0．4μmol·μL-1引物、3ng模板DNA。PCR扩增程序为：94℃预变性2min，94oC变性30s，54．5℃退火30s，72℃延伸1min，45个循环，最后72℃延伸7min，置4℃保存。应用该ISSR体系对6份荷花种质进行了扩增，证实了该体系的适用性和稳定性。
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| 关 键 词： | 荷花 ISSR 反应体系 优化 |
Establishment and optimization of ISSR - PCR reaction system of Nelumbo nucifera Gaertn |
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| Institution: | HUANG Yu , HE Tian-you, RONG Jun-dong , LIU Ying-jia , HU Di-ke , ZHENG Yu-shan ( 1. Institute of Industrial Forest ; 2. College of Landscape, Fujian Agriculture and Forestry University, Fuzhou, Fujian 350002, China) |
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| Abstract: | Taking genomic DNA extracted from Nelumbo nucifera Gaertn leaves as the experimental material, the factors which af- fected the ISSR - PCR amplification such as suitable concentration for dNTPs and Mg2 ＋ , then dosage for Taq DNA polymerase, the primer, template DNA and annealing temperature were selected and optimized. The results showed that the suitable PCR system for ISSR -PCR of N. nucifera Gaertn were as follows： the 20 μL PCR reaction volume including, 0.4 mmol · L-1 dNTPs, 3.5 mmol · L-1Mg2＋ , 1.5 U Taq DNA polymerase, 0.4 p.mol· μL-1 primer, 3 ng template DNA. The optimal PCR amplification process was： 2 minutes at 94 ℃ for predenaturation, then followed by 45 cycles, each with 30 seconds at 94 ℃ for denaturation, 30 seconds at 54.5 ℃ for annealing, 1 minute at 72 ℃ for extension, finally extension at 72 ℃ for 7 minutes and holding the samples at 4 ℃. The system was applied in the amplification of six varieties of N. nucifera Gaertn indicating the suitability and stability of the system. |
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| Keywords: | Nelumbo nucifera Gaertn ISSR reaction system optimization |
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