胡椒ISSR反应体系的建立与优化

Establishment and Optimization of ISSR Reaction System for Piper nigrum L.

通过单因子、双因子实验研究了胡椒ISSR-PCR反应体系中热参数和5个主要成分,即退火温度、循环数、变性时间、退火时间、延伸时间以及Mg2+、dNTPS、引物、模板DNA、TaqDNA聚合酶对扩增结果的影响,建立了适合胡椒ISSR分析的反应体系和扩增程序,即在25μL反应体系中,内含2 mmol/L Mg2+、200μmol/LdNTPS、1×PCR Buffer、2μmol/L引物、100 ng模板、1 U Taq DNA聚合酶。扩增程序为94℃预变性3 min,94℃变性120 s,复性60 s,72℃延伸3 min,循环35个,结束后72℃延伸7 min。这一优化体系的建立为今后利用ISSR标记技术进行胡椒种质鉴定、遗传多样性分析奠定了基础。

Based on the genomic DNA extracted from Piper rtigrum L., this paper presented the effect of the main reaction system elements （Mg2＋, dNTPS, primer, template DNA, Taq DNA polymerase） and hot-cycle parameters （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 Piper nigrum L. were established, that is, 25 μL amplification reaction system containing 1 ×PCR Buffer, 2 mmol/L Mg2＋, 200μmol/L dNTPS, 2 μmol/L primer, 100 ng DNA and 1 U Taq DNA polymerase. The optimal amp：ified procedure was devised for 3 minutes of predenaturalization at 94~C. 35 cycles of 120 seconds for denaturalization at 94℃, 60 seconds of annealing due to denaturing temperature of different primer, 180 seconds of extension at 72℃ and 7 min of extension at 72℃ in the final cycle. This optimal system laid the standardization program of the identification of Piper nigrum L. and the efficient use of its germplasm resource.