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当前位置: 首页 > 文章 > 毛梾ISSR-PCR反应体系的建立 北方园艺 2013 (22) 114-119
Position: Home > Articles > Establishment of ISSR-PCR Reaction System of Cornus walteri Northern Horticulture 2013 (22) 114-119

毛梾ISSR-PCR反应体系的建立

作  者：
李娜;张存旭;刘伟;宋利伟;王豪;杨玲
单  位：
西北农林科技大学林学院
关键词：
毛梾;ISSR;单因素2次循环试验;体系优化;引物筛选
摘  要：
以毛梾叶片为试材,通过单因素2次循环试验对毛梾ISSR反应体系进行优化,建立了毛梾ISSR-PCR的最佳反应体系和扩增程序。结果表明:PCR反应体系总体积为25μL,其中10×Buffer 2.5μL,模板DNA用量50ng,Taq DNA聚合酶用量1.5U,Mg2+浓度2.0mmol/L,引物浓度0.6μmol/L,dNTPs浓度0.15mmol/L;扩增程序:94℃预变性5min;94℃变性50s,48.8~59.1℃退火60s,72℃延伸1.5min,38个循环;最后72℃延伸10min。在此基础上,从100条ISSR通用引物中筛选出17条多态性好、稳定性高的引物,并优化了它们的退火温度。这一优化体系的建立,为开展毛梾种质资源鉴定、遗传多样性及分子育种方面的研究提供了技术依据和参考。
译  名：
Establishment of ISSR-PCR Reaction System of Cornus walteri
作  者：
LI Na;ZHANG Cun-xu;LIU Wei;SONG Li-wei;WANG Hao;YANG Ling;College of Forestry,Northwest Agricultural and Forestry University;
关键词：
Cornus walteri;;ISSR;;single factor with two circular tests;;system optimization;;primer screening
摘  要：
Using leaves of Cornus walteri as material,ISSR reaction conditions of Cornus walteri were optimized systematically via single factor with two circular tests,and the optimal ISSR reaction system and thermal cycling conditions of Cornus walteri were established.The results showed that the optimal ISSR reaction system included a total volume 25μL containing 2.5μL 10×Buffer,50ng template DNA,1.5UTaq DNA polymerase,2.0mmol/L MgCl2, 0.6μmol/L primer,0.15mmol/L dNTPs.Thermal cycling conditions were as follows:denaturation 5min at 94℃; 38cycles of 50sat 94℃,60sat 48.8~59.1℃,1.5min at 72℃;and a final extension of 10min at 72℃at the end of the amplification.Based on the optimized reaction system,17primers with superior stability and polymorphism were screened out from 100ISSR primer candidates.Subsequently,annealing temperatures of selected primers were subjected to further optimization.As a consequence,the optimized system in this study may provide technological base and reference for identification of germplasm resources,genetic diversity and molecular breeding of Cornus walter.

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