黄连ISSR反应条件优化的研究

Study on Optimization of ISSR Reaction Conditions for Coptis Chinensis Franch

以黄连(味连,Coptis ChinensisFranch.)基因组DNA为模板,通过单因子、双因子实验研究了ISSR反应体系中主要成分(Mg2+、dNTP、引物、模板、TaqDNA聚合酶)以及热循环参数(退火温度、循环数、变性时间、退火时间、延伸时间)对扩增结果的影响,并找出各自的最适条件,建立了适合黄连ISSR分析的反应体系和扩增程序,即在25μL反应体系中,内含1×PCR buffer、1.5mmol.L-1Mg2+、200μmol.L-1dNTP、0.3μmol.L-1引物、40ng模板、1UTaqDNA聚合酶。扩增程序为94℃预变性5min,然后进行35个循环:94℃变性30s,(据不同引物的退火温度)复性1min,72℃延伸1.5min,循环结束后72℃延伸7min,-4℃保存。这一优化系统的建立为今后利用ISSR标记技术进行黄连鉴定及种质遗传多样性分析提供了一个标准化程序。

Based on the genomic DNA extracted from Coptis Chinensis Franch. , this paper presented the effect of the main reaction system elements （Mg^2＋ ,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, 25 μL amplification reactions system containing 1 × PCRbuffer, 1.5 mmol · L^-1 Mg^2＋, 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 30 s at 94℃, 35 cycles were performed with denaturing of 30 s at 94℃, annealing of 1 min due to denaturing temperature of different primer, extension of 1.5 min 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.