乌塌菜ISSR-PCR反应体系的建立及优化

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。

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

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.