| 杨树ISSR反应体系的建立及正交设计优化 |
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| 引用本文： | 汪结明,项艳,吴大强,孙志娟,蔡诚.杨树ISSR反应体系的建立及正交设计优化[J].核农学报,2007,21(5):470-473,513. |
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| 作者姓名： | 汪结明 项艳 吴大强 孙志娟 蔡诚 |
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| 作者单位： | 安徽农业大学林学与园林学院,安徽,合肥,230036 |
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| 摘 要： | 采用正交设计的方法,对杨树ISSR-PCR反应中5个因素(Taq酶、Mg2+、模板DNA、dNTP和引物)在4个水平上进行优化试验。建立了适合杨树的既稳定又能扩出最多数量谱带的ISSR-PCR最佳反应体系,即25μl的反应体系中含有1×buffer,0.12mmol/L dNTP,0.4μmol/L引物,3.5 mmol/L Mg2+,1.5UTaqDNA聚合酶和40ng模板DNA。对杨树ISSR-PCR最佳反应体系的退火温度进行梯度试验,发现引物W95142的最适退火温度为56.1℃,且最适退火温度因引物而异。这一优化的ISSR-PCR反应体系的建立为今后利用ISSR技术进行杨树种质资源分类、遗传图谱构建和基因定位奠定了技术基础。
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| 关 键 词： | 杨树 ISSR 正交设计 反应体系 优化 |
| 文章编号： | 1000-8551（2007）05-470-04 |
| 收稿时间： | 2009-12-31 |
| 修稿时间： | 2006-11-27 |
ESTABLISHMENT OF AN ISSR REACTION SYSTEM FOR POPLAR AND OPTIMIZATION USING ORTHOGONAL DESIGN |
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| WANG Jie-ming,XIANG Yan,WU Da-qiang,SUN Zhi-juan,CAI Cheng.ESTABLISHMENT OF AN ISSR REACTION SYSTEM FOR POPLAR AND OPTIMIZATION USING ORTHOGONAL DESIGN[J].Acta Agriculturae Nucleatae Sinica,2007,21(5):470-473,513. |
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| Authors: | WANG Jie-ming XIANG Yan WU Da-qiang SUN Zhi-juan CAI Cheng |
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| Institution: | College of Forest and Garden, Anhui Agricultural University, Hefei, Anhui 230036 |
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| Abstract: | Orthogonal design was applied for optimizing five factors(Taq DNA polymerase,Mg~(2+),DNA template,dNTP,and primer) in the ISSR-PCR amplification system on poplar respectively.As a result,a satisfactory ISSR reaction system for poplar with desirable repeatability and polymorphic bands was established.In a total volume of 25μl ISSR-PCR system,it contained 1×buffer,0.12mmol/L dNTP,0.4μmol/L primer,3.5 mmol/L Mg~(2+),1.5U Taq DNA polymerase and 40ng template DNA.The optimal annealing temperature of every prime for ISSR-PCR reaction was proposed by gradient PCR.It was found that different primers had different annealing temperature.As far as concerned with primer W95142,the optimal annealing temperature was 56.1℃.This optimized ISSR reaction system would provide the basis for the analysis of diversity,map construction and gene localization of important traits of poplar with ISSR markers. |
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| Keywords: | poplar ISSR orthogonal design reaction system optimization |
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