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以九节茶叶片提取的基因组DNA为材料,对影响ISSR-PCR扩增效果的一些因素,诸如dNTPs浓度、TaqDNA聚合酶用量、引物用量、模板DNA用量、退火温度以及循环次数等指标进行筛选和优化.结果表明:20μL ISSR反应体系含10×PCR Buffer、0.4 mmol.L-1dNTPs、2 UTaqDNA聚合酶、0.6μmol.μL-1引物、5 ng模板DNA;PCR扩增程序为:94℃预变性2 min,94℃变性30 s,44.8℃退火30 s,72℃延伸1 min,35个循环,最后于72℃延伸7 min,置4℃保存.应用该ISSR体系对10份九节茶种质进行了扩增,证实了该体系的适用性和稳定性.
The genomic DNA extracted from nine tea leaves was used to screen and analyze the factors influencing the amplification of ISSR-PCR, such as the concentration of dNTPs, the amount of Taq DNA polymerase, the amount of primers, the amount of template DNA, the annealing temperature and the number of cycles The results showed that: 20μL ISSR reaction system containing 10 × PCR Buffer, 0.4 mmol.L-1dNTPs, 2 UTaq DNA polymerase, 0.6μmol.μL-1 primer, 5 ng template DNA; PCR amplification program: 94 ° C pre- 2 min, denaturation at 94 ℃ for 30 s, annealing at 44.8 ℃ for 30 s, extension at 72 ℃ for 1 min for 35 cycles, and finally extension at 72 ℃ for 7 min and storage at 4 ℃ .In this ISSR system, The amplification was carried out to confirm the applicability and stability of the system.