雁鹅ISSR-PCR反应体系的优化*

本试验以雁鹅基因组DNA为模板，采用正交试验设计L25（55）对PCR反应中5个因素（引物、dNTPs浓度、模板DNA、Mg2+、TapDNA聚合酶）在5个水平上进行优化实验，在初步实验的基础上通过因素内比较进一步优化各因素在ISSR-PCR反应中的浓度，快速准确地建立雁鹅ISSR-PCR的最佳反应体系，为进一步进行雁鹅遗传多样性的研究、遗传图谱构建和基因定位奠定技术基础。在25uL的反应体系中，确定雁鹅ISSR-PCR的最佳反应体系为：ISSR引物的浓度为0.20umol/L，dNTPs浓度为0.28mmol/L，模板DNA的用量为40ng，Mg2+浓度为1.5 mmol/L，Taq DNA聚合酶的用量为1.0U。在正交试验设计的基础上，通过因素内比较进一步优化各因素在ISSR-PCR反应中的浓度，可以更加准确地确立ISSR-PCR的最佳反应体系。

Optimization of ISSR-PCR Reaction System in Yane geese

Using the genomic DNA extracted from Yane geese, the orthogonal design was used to optimize ISSR-PCR amplification system in five levels of five factors(Taq DNA polymerase, Mg2+,DNA template, dNTPs and primer)， respectively. Based on the preliminary experiment, the density within factors of ISSR-PCR was further optimized. The purpose of this experiment was to determine the reaction condition of ISSR-PCR fleetly and accurately, which was basis for the study on genetic diversity, map construction and gene localization of Yane geese. The results showed that a better amplification of ISSR was obtained with the reaction system containing 0.20umol/L primer, 0.28mmol/L dNTPs, 40ng templet DNA of Yane geese, 1.5mmol/L Mg2+,1.0U Taq DNA polymerase in the total volume of 25uL. Based on the orthogonal design experiment, the prime reaction condition of ISSR-PCR can be established through the density within factors of ISSR-PCR further optimized.