| 结缕草SSR反应体系的正交优化 |
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| 引用本文： | 桂金山,王赟文,FANG Cheng,方程.结缕草SSR反应体系的正交优化[J].草地学报,2008,16(1):89-93,102. |
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| 作者姓名： | 桂金山 王赟文 FANG Cheng 方程 |
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| 作者单位： | 中国农业大学草地研究所,农业部草地植被恢复与重建重点开放实验室,北京,100094;Institute of Grassland Science, China Agricultural University, Ministry of Agriculture Key Laboratory of Grassland Vegetation Restoration and Reconstruction,Beijing 100094,China |
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| 摘 要： | 为了快速获得结缕草(Zoysia japonica)SSR反应体系,采用5因素(模板DNA,Mg2 ,dNTP, Taq酶和引物)4水平正交设计筛选合适的结缕草SSR体系,并通过随机挑选3对Tm相近的引物对该优化体系进行验证.结果表明:正交设计可应用于SSR-PCR反应体系的优化,20 μl最佳PCR反应体系中包括2 μl 10×buffer、1.0U Taq酶、0.2 mM 引物、100 ng模板DNA、0.2 mM dNTP、2.0 mM Mg ;对结缕草进行梯度退火实验,其最佳退火温度为56～58℃;可行扩增程序是:94℃预变性4 min、进行35个循环的94℃变性30 s、56～58℃退火40 s,72℃延伸1 min;72℃延伸10 min,4℃保存;该最适反应体系的建立,为今后结缕草SSR分析奠定了坚实基础.
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| 关 键 词： | 结缕草 SSR标记 反应体系 正交设计 优化 |
| 文章编号： | 1007-0435（2008）01-0089-06 |
| 收稿时间： | 2007-07-26 |
| 修稿时间： | 2007-12-12 |
Optimization of the SSR Reaction System for Zoysiagrass Using Orthogonal Design |
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| FANG Cheng.Optimization of the SSR Reaction System for Zoysiagrass Using Orthogonal Design[J].Acta Agrestia Sinica,2008,16(1):89-93,102. |
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| Authors: | FANG Cheng |
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| Abstract: | In order to obtain the optimal SSR-PCR system for zoysiagrass (Zoysia japonica), an experimental design of orthogonal diagram L16(45) was employed to evaluate five factors (template DNA, Mg2+, dNTP, Taq DNA polymerase, and primer) at four different levels. Three randomly selected SSR primers (RM84, RM220, RM531) with resembled Tm were used to validate the reaction system. The results indicate that orthogonal design could be used to optimize the SSR-PCR system; the optimal system of terminal volume 20 μl consisted of 2 μl 10×buffer, 1.0U Taq DNA polymerase, 0.2 mM primers, 100ng template DNA, 0.2 mM dNTP, and 2.0 mM Mg+. The optimal annealing temperature was determined to be 56-58℃ by gradient PCR; the suitable thermal cycling conditions with initial melting at 94℃ for 4 min, followed by 35 cycles at 94℃ for 30 s, 56-58℃ for 40 s, 72℃ for 60 s; then keep the reaction mixture at 4℃ after a final extension step of 72℃ for 10 min. The optimized system would be effective as a solid foundation for zoysiagrass SSR analysis. |
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| Keywords: | Zoysia iaponica Steud SSR markers Reaction system Orthogonal design Optimization |
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