多花黄精ISSR反应体系的建立及正交优化设计

通过对多花黄精ISSR-PCR反应中的Taq酶、buffer(Mg2+)、模板DNA、dNTP以及引物5个关键因素进行了5因素4水平的正交设计试验,确立了适合多花黄精基因组DNA的稳定性强、扩增最多数量谱带的ISSR-PCR最优反应体系,即25μL的反应体系中含有1.0 U Taq DNA聚合酶,3.5 mmol.L-1buffer(Mg2+),80 ng模板DNA,0.08 mmol.L-1dNTP和0.16μmol.L-1引物。采用建立的多花黄精ISSR-PCR最佳反应体系进行退火温度梯度试验,确定了引物UBC841的最适退火温度为54.8℃,且最适退火温度因引物而异。这一优化的ISSR-PCR反应体系的建立为今后利用ISSR技术进行黄精种质资源分类、遗传图谱构建和基因定位奠定了良好的技术基础。

Establishment and optimization of an ISSR reaction system for Polygonatum cyrtonema Hua using orthogonal design

In this paper,the orthogonal design of five factors(Taq DNA polymerase,buffer(Mg2+),DNA tem-plate,dNTP,and primer) in four levels was used to optimize the ISSR-PCR amplification system on Polygonatum sibiricum.Finally,a satisfactory ISSR(Inter Simple Sequence Repeat) reaction system for Polygonatum sibiricum with desirable repeatability and polymorphic bands was established.It contained 1.0 U Taq DNA polymerase,3.5 mmol?L-1 buffer(Mg2+),80 ng DNA template,0.08 mmol?L-1 dNTP and 0.16 μmol?L-1 primer respectively in a 25 μL ISSR reaction system.The optimal annealing temperature of every prime for ISSR-PCR reaction was proposed by gradient PCR with established ISSR reaction,the optimal annealing temperature was 54.8℃ for primer UBC841,and different primers had different annealing temperatures.This optimized ISSR reaction system would provide the basis for the analysis of classification,map construction and gene localization of Polygonatum sibiricum.