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20μL PCR反应体系含:30 ng模板DNA,0.50μmol.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。
For optimizing ISSR-PCR reaction system of Brassica campestris L. vat. 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 rain 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℃C 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.
出处
《植物研究》
CAS
CSCD
北大核心
2013年第2期238-242,共5页
Bulletin of Botanical Research
基金
国家自然科学基金资助项目(31272170)
安徽省高校自然科学研究重点项目(KJ2012A109)
安徽省年度重点科研项目(11070303026)
关键词
乌塌菜
ISSR-PCR
反应体系
正交设计
Brassica campestris L. var. rosularis
ISSR-PCR
reaction system
orthogonal design
