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植物研究 ›› 2013, Vol. 33 ›› Issue (2): 238-242.doi: 10.7525/j.issn.1673-5102.2013.02.020

• 论文 • 上一篇    下一篇

乌塌菜ISSR-PCR反应体系的建立及优化

宋江华;赵颖;汪承刚*;张灵凤;张慧   

  1. 安徽农业大学园艺学院,合肥 230036
  • 收稿日期:1900-01-01 修回日期:1900-01-01 出版日期:2013-03-20 发布日期:2013-03-20
  • 通讯作者: 汪承刚
  • 基金资助:
     

Establishment and Optimization of ISSR-PCR Reaction System for Brassica campestris L. var. rosularis

SONG Jiang-Hua;ZHAO Ying;WANG Cheng-Gang*;ZHANG Ling-Feng;ZHANG Hui   

  1. College of Horticulture,Anhui Agricultural University,Hefei 230036
  • Received:1900-01-01 Revised:1900-01-01 Online:2013-03-20 Published:2013-03-20
  • Contact: WANG Cheng-Gang
  • Supported by:
     

摘要: 为获得乌塌菜ISSR-PCR的最佳反应体系,采用单因素浓度梯度试验和正交优化设计相结合的方法,研究了引物浓度、dNTP浓度、Mg2+浓度、Taq DNA聚合酶用量对PCR反应的影响。并在此基础上对退火温度和循环数进一步优化,最终确立了适合乌塌菜ISSRPCR反应的最佳体系和程序。即20 μL PCR反应体系含:30 ng模板DNA,0.50 μmol·L-1引物,0.25 mmol·L-1 dNTP,1 mmol·L-1 Mg2+,1.0 U Taq DNA聚合酶。PCR扩增程序为:94℃预变性3 min;94℃变性30 s,50℃退火1 min,72℃延伸90 s,35个循环;72℃延伸7 min,4℃保存。这一优化的ISSR-PCR反应体系的建立为今后利用ISSR技术对乌塌菜进行种质资源的分类鉴定奠定了基础。

关键词: 乌塌菜, ISSR-PCR, 反应体系, 正交设计

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 94℃ 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.

Key words: Brassica campestris L. var. rosularis, ISSR-PCR, reaction system, orthogonal design

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