| 巨竹ISSR反应体系的建立与优化 |
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| 引用本文： | 李炎梅,何天友,陈凌艳,陈礼光,荣俊冬,郑郁善.巨竹ISSR反应体系的建立与优化[J].甘蔗(福建),2012(1):51-56. |
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| 作者姓名： | 李炎梅 何天友 陈凌艳 陈礼光 荣俊冬 郑郁善 |
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| 作者单位： | 福建农林大学竹类研究所,福建福州350002 |
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| 基金项目： | 福建省科技厅重点项目（2009N0006）;福建省科技重大项目（2011N5002）. |
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| 摘 要： | 以巨竹叶片提取的基因组DNA为材料，用引物UBC810（序列为GAGAGAGAGAGAGAGAT）研究了PCR反应体系的主要成分、退火温度及循环次数对该种植物ISSR扩增结果的影响。结果表明，20μL的反应体系含40ng模板DNA、0．6μmol·L^-1引物，1．0UTaqDNA聚合酶，2．5mmol·L^-1 Mg^2＋，0．25mmol·L^-1dNTPs，1×Buffer。PCR扩增程序为：94℃预变性5min；94℃变性45s，54．5℃复性30S，70℃延伸90S，循环40次；72℃延伸10min，置4c℃保存。
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| 关 键 词： | 巨竹 ISSR-PCR 体系优化 |
Establishment and optimization of ISSR amplification system in Gigantochloa levis |
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| LI Yan-mei,HE Tian-you,CHEN Ling-yan,CHEN Li-guang,RONG Jun-dong,ZHENG Yu-shan.Establishment and optimization of ISSR amplification system in Gigantochloa levis[J].Sugarcane,2012(1):51-56. |
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| Authors: | LI Yan-mei HE Tian-you CHEN Ling-yan CHEN Li-guang RONG Jun-dong ZHENG Yu-shan |
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| Institution: | (Institute of Bamboo Research, Fujian Agriculture and Forestry University, Fuzhou, Fujian 350002, China) |
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| Abstract: | Taking genomie DNA extracted from Gigantochloa levis leaves as the template material, the concentrations of PCR compo- nents, annealing temperature and cycles, whicll affected the ISSR amplification, were optimized with the primer UBC810 （primer sequence ： GAG AGA GAG AGA GAG AT）. The results showed that the 20 μL ISSR reaction system included 40 ng template DNA, 0.6 μmol ·L^-1 primer, 1.0 U Taq DNA polymerase, 2.5 mmol·L^-1 Mg^2＋, 0.25 mmol·L^-1 dNTPs,1×Buffer. The optimal PCR amplification process was 5 minutes at 94 ℃ for predenaturation, followed by 40 cycles ,each with 45 seconds at 94 ℃ for denaturation, 30 seconds at 54.5 ℃ for annealing, 90 seconds at 72℃ for extension,finally extension at 72 ℃ for 10 minutes and holding the samples at 4 ℃. |
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| Keywords: | Gigantochlochloa levis ISSR - PCR system optimization |
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