[目的]建立大帽藓科ISSR-PCR实验最佳反应体系.[方法]提取大帽藓科的基因组DNA, 利用5因素4水平的正交设计实验方法对大帽藓科植物ISSR-PCR反应体系进行最佳反应体系的筛选.[结果]大帽藓科植物ISSRPCR的最佳反应体系为(20μL):即20μL体系中, PCR Buffer 2. 0μL、Mg2+2. 25 mmol/L, d NTP 0. 2 mmol/L, 引物0.65μmol/L, Taq DNA聚合酶0. 7 U, 模板DNA 10 ng.5个因素对该反应体系的影响顺序为:Mg2+>引物> d NTP> Taq DNA聚合酶>模板DNA.利用该体系, 在UBC808等13种引物中进行验证, 均能得到有效的扩增条带.[结论]通过对大帽藓科基因组DNA的提取及体系优化, 获得了大帽藓科植物最佳反应体系, 为后续应用ISSR技术鉴定大帽藓科种质资源以及遗传多样性研究奠定了基础.
Optimization of ISSR-PCR system of encalyptaceae
[Objective]Develop the most suitable ISSR-PCR system for Encalyptaceae.[Method]Extract the genomic DNA of the genus, the optimal reaction system of the ISSR-PCR reaction system of the genus Polygonaceae was screened by orthogonal design experiment with 5 factors and 4 levels. [Result] The optimal reaction system for ISSR-PCR of Encalyptaceae is (20 μL): That is, in a 20 μL system, PCR Buffer 2. 0 μL, Mg2+ 2. 25 mmol/L, primer 0. 65 μmol/L, d NTP 0. 2 mmol/L, Taq DNA polymerase 0. 7 U, and template DNA 10 ng. The order of influence of the five factors on the reaction system was Mg2+>primer> d NTP> Taq polymerase> template DNA. Using this system, it was verified in 13 kinds of primers such as UBC808, and an effective amplified band was obtained.[Conclusion]In this experiment, the optimal reaction system of the genus Diptera was obtained by extracting and optimizing the genomic DNA of the genus Polygonaceae, which laid a foundation for the subsequent application of ISSR technology to identify the germplasm resources and genetic diversity of the genus.
NETL
NSTL
万方数据
维普
