| 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].亚热带农业研究,2009,5(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. 福建农林大学工业原料林研究所;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，福建省创新平台福建省中药材GAP工程技术研究中心资助项目 |
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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、TaqDNA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0.4 mmol.L-1dNTPs、3.5 mmol.L-1Mg2+、1.5 UTaqDNA聚合酶、0.4μmol.μL-1引物、3 ng模板DNA。PCR扩增程序为:94℃预变性2 min,94℃变性30 s,54.5℃退火30 s,72℃延伸1 min,45个循环,最后72℃延伸7 min,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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| HUANG Yu,HE Tian-you,RONG Jun-dong,LIU Ying-jia,HU Di-ke,ZHENG Yu-shan.Establishment and optimization of ISSR-PCR reaction system of Nelumbo nucifera Gaertn[J].Subtropical Agriculture Research,2009,5(4):284-288. |
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| Authors: | HUANG Yu HE Tian-you RONG Jun-dong LIU Ying-jia HU Di-ke ZHENG Yu-shan |
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| Abstract: | Taking genomic DNA extracted from Nelumbo nucifera Gaertn leaves as the experimental material,the factors which affected 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 μmol·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: | ISSR |
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