摘要
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.
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.
出处
《福建农林大学学报（自然科学版）》
CSCD
北大核心
2009年第2期139-143,共5页
Journal of Fujian Agriculture and Forestry University:Natural Science Edition
基金
福建省科技厅重大资助项目(2004YZ02-05)
福建省中药材GAP工程技术研究中心资助项目(2008Y2001)
关键词
九节茶
ISSR
反应体系
优化
Sarcandra glabra
ISSR
reaction system
optimization
作者简介
黄宇（1984-），女，硕士研究生．研究方向：森林培育．
通讯作者郑郁善（1960-），男，教授，博士生导师．研究方向：森林培育．Email：zys1960@163．com．
