九节茶ISSR反应体系的建立及优化

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.

Establishment and optimization of ISSR reaction system of Sarcandra glabra

Taking genomic DNA extracted from Sarcandra glabra(Thunb.) Nakai leaves as the experimental material,the factors which affected the ISSR-PCR amplification such as suitable concentration for dNTPs,dosage for Taq DNA polymerase,the primers,template DNA,annealing temperature and cycles were selected and optimized.The results showed that the 20 μL ISSR reaction system included: 10×PCR Buffer,0.4 mmol·L-1 dNTPs,2U Taq DNA polymerase,0.6 μmol·μL-1 primer,5 ng template DNA.The optimal PCR amplification process was: 2 minutes at 94 ℃ for predenaturation,then followed by 35 cycles,each with 30 seconds at 94 ℃ for denaturation,30 seconds at 44.8 ℃ for annealing,1 minute at 72 ℃ for extension,finally extension at 72 ℃ for 7 minutes and holding the samples at 4 ℃.The system was applied in the amplification of ten varieties of S.glabra,indicating the suitability and stability of the system.