花椰菜的ISSR-PCR反应体系的建立与优化

Establishment and Optimization of ISSR-PCR Reaction System in Cauliflower

以花椰菜基因组为材料,通过单因素试验,对ISSR-PCR反应体系中各种影响因子如dNTPs浓度,DNA模板含量,Taq DNA聚合酶量,引物用量以及最适退火温度等进行了优化和筛选,建立了适合花椰菜的ISSR-PCR反应体系:25μL反应体积:内含10×PCR反应缓冲液(含Mg^(2+))2.5μL、0.5U TaqDNA聚合酶、0.2mmol/L dNTPs、0.5μmol/L引物、60ng模板DNA。确定了适宜的退火温度为48.6℃。扩增程序为94℃预变性5 min;然后94℃变性30s,46.9℃退火45 s,65℃延伸1.5min,35个循环;最后65℃延伸7min,4℃保存。用120条引物对花椰菜基因组进行标记,筛选出引物TI-13可以将这6份花椰菜材料区分开。花椰菜ISSR反应体系的建立为利用ISSR标记技术进行花椰菜品种鉴别、分类、种质资源遗传多样性分析奠定了良好基础。

The influenced factors of ISSR-RCR reaction were optimized and the effect of 5 factors such as annealing temperature,Taq DNA polymerase dosage,DNA templates concentration,primer concentration and dNTPs concentration using single factor experiment based on the cauliflower genomic.Cauliflower reaction system and amplified procedure were established that is 25μL amplification reaction system containing 10×PCR buffer（Mg^（2＋））2.5μL,0.5U Taq DNA polymerase,0.20mmol/L dNTPs,0.5μmol/L primer,60ng template DNA.The optimized annealing temperature is 48.6℃.The optimal amplified procedure was as follows：after a pre-denaturing of 4 min at 94℃,35 cycles were performed with 30s for denaturing at 94℃,annealing of 45s at 48.6℃,extension of 1.5 min at 65℃,7 minutes of extension at 65℃in the final cycle and hold at 4℃.Using 120 primers to marker cauliflower genome,primers TI-13 was selected to separate 6 copies cauliflower material.The extablishment of the ISSR-PCR reaction system could settle favorable foundation for identification of cauliflower cultivars,classification and analysis of the genetic diversity of cauliflower using ISSR molecular marker techniques.