鹅掌楸ISSR-PCR反应体系的建立及优化

Establishment and optimization of ISSR-PCR system for Liriodendron

为建立鹅掌楸简单序列重复区间扩增ISSR的PCR优化反应体系,以鹅掌楸叶片基因组DNA为材料,系统地测试了模板DNA、引物、dNTPs、Mg2+浓度、TaqDNA聚合酶用量及退火温度对ISSR-PCR反应体系的影响。结果表明:优化的PCR反应体系为:20μL总体系中,含30 ng模板DNA,0.3μmol.L-1随机引物,0.2 mmol.L-1dNTPs,1.4 mmol.L-1Mg2+,0.8 UTaqDNA聚合酶;最佳退火温度为60℃;PCR反应程序为:94℃预变性5 min;94℃变性1 min,60℃退火45 s,72℃延伸2 min;45个循环;72℃再延伸7 min。

In order to establish the optimal system of ISSR-PCR for Liriodendron,with the genomic DNA of Liriodendron leaves as material,the parameters including template DNA,primers,dNTPs,Mg2＋ concentration,dose of Taq DNA polymerase and annealing temperature to ISSR-PCR were systematically analyzed.The results show that the optimal reaction system of ISSR（20 μL） was： 30 ng template DNA,0.6 μmol·L-1 random primers,0.2 mmol·L-1 dNTPs,1.4 mmol·L-1 Mg2＋,and 0.8 U Taq DNA polymerase;and that the optimized annealing temperature was 60℃;the PCR procedure was： pre-denaturizing at 94℃ for 5 min,denaturizing at 94℃ for 1 min,annealing at 60℃ for 45 s,extension at 72℃ for 2 min,reaction of 45 cycles and re-extension at 72℃ for 7 min.