植物研究2013，Vol.33Issue(2)：238-242,5.DOI:10.7525/j.issn.1673-5102.2013.02.020
乌塌菜ISSR-PCR反应体系的建立及优化
Establishment and Optimization of ISSR-PCR Reaction System for Brassica campestris L.var.rosularis
摘要
Abstract
For optimizing ISSR-PCR reaction system of Brassica campestris L.var.rosularis,single factor gradient and orthogonal design experiments were conducted.The main factors affecting ISSR-PCR amplification i.e.suitable concentration of primer,dNTP,Mg2+ and Taq DNA polymerase were studied.Furthermore,the annealing temperature and cycling numbers were optimized on the base of the above tests.An ideally ISSR-PCR reaction system was established,namely 20 μL reaction system containing template DNA 30 ng,0.50 μmol · L-1 primer,0.25 mmol · L-1 dNTP,1 mmol · L-1 Mg2+ and Taq DNA polymerase 1.0 U.The optimal PCR amplification program was; 3 min at 941 for predenaturation,followed by 35 cycles of 30 sec at 94℃ for denaturation,1 min at 50℃ for annealing,90 sec at 72 ℃ for extension,finally extension at 72℃ for 7 min and holding the samples at 4℃.This optimized ISSR-PCR reaction system would provide the basis for the analysis of germplasm classification and identification in B.campestris.关键词
乌塌菜/ISSR-PCR/反应体系/正交设计Key words
Brassica campestris L. var. rosularis/ISSR-PCR/reaction system/orthogonal design分类
农业科技引用本文复制引用
宋江华,赵颖,汪承刚,张灵凤,张慧..乌塌菜ISSR-PCR反应体系的建立及优化[J].植物研究,2013,33(2):238-242,5.基金项目
国家自然科学基金资助项目(31272170) （31272170）
安徽省高校自然科学研究重点项目(KJ2012A109) （KJ2012A109）
安徽省年度重点科研项目(11070303026) （11070303026）
