萝卜ISSR-PCR反应体系的正交设计优化

本研究利用正交设计L16(45)对萝卜ISSR-PCR反应体系的5因素(Taq酶、Mg2+、模板DNA、dNTP、引物)在4个水平上进行优化试验。研究结果表明,获得了最佳的反应体系,在10μL反应体系中,含TaqDNA聚合酶0.6U﹑Mg2+2.0mmo/L﹑模板DNA 40ng﹑dNTP 250μmol/L﹑引物0.25μmol/L﹑10×PCRBuffer,不足部分以ddH2O补足。确立了以UBC876为引物的最优反应条件,退火温度为48℃,总循环数为40次。新优化的萝卜ISSR-PCR的反应体系和程序有很好的重复性和稳定性好。此研究为今后利用ISSR技术对萝卜进行种质资源分类﹑遗传图谱构建和基因定位奠定了基础。

The Orthogonal Optimization of ISSR-PCR System in Radish

In this research,the orthogonal design was used to optimize ISSR-PCR system on Radish in for levels of five factors(dNTP,primer,Mg~(2+),Taq DNA polymerase,and DNA template)respectively.The results showed that the orthogonal experiment design was a practicable method and in a total volume of 10μL SRAP-PCR system,it contains 0.6 U Taq DNA polymerase,2.0 mmol/L Mg~(2+) 40 ng template DNA,250 μmol/L dNTP,0.25 μmol/L primer,10×PCR buffer and ddH_2O.The suitable PCR procedure was denaturing temperature at 48℃for the forty cycles with UBC876 primer.The new optimized ISSR-PCR system and procedure of Radish had good repeatability and stability.This work would provide basis for the analysis of diversity,genetic map construction and gene localization by using ISSR technique of radish in the future.