关键词: 黄连, ISSR, 反应体系, 热循环参数

Abstract: Based on the genomic DNA extracted from Coptis Chinensis Franch., this paper presented the effect of the main reaction system elements(Mg²⁺、dNTP、primer、template DNA、Taq DNA polymerase) and hot-cycle parameter(annealing temperature、cycles、denaturing time、annealing time and extension time) on ISSR-PCR which were tested by single or dual factor experiment, respectively to determine its optimal levels. A reaction system and amplified procedure suitable for Coptis Chinensis Franch were established,that is, 25μL amplification reactions system containing 1×PCRbuffer、1.5mmol·L^-1 Mg²⁺、200μmol·L^-1 dNTP、0.3μmol·L^-1 primer、40 ng template DNA、1 U Taq DNA polymerase.The optimal amplified procedure was as follows: after a pre-denaturing of 30s at 94℃, 35 cycles were performed with denaturing of 30s at 94℃, annealing of 1min due to denaturing temperature of different primer, extension of 1.5min at 72℃, a final extension step of 7 min at 72℃ and hold at -4℃. This optimal system laid the standardization program of the identification of Coptis Chinensis Franch. and the efficient use of its germplasm resource.

Key words: Coptis Chinensis Franch., ISSR, reaction system, hot-cycle parameter