摘要
以荷花叶片提取的基因组DNA为材料,通过对影响ISSR-PCR扩增效果的一些因素,如dNTPs浓度、Mg2+浓度、TaqDNA聚合酶用量、引物用量、模板DNA用量以及退火温度等进行筛选和优化,确立了可用于荷花ISSR-PCR分析的最适宜的PCR反应体系:20μL PCR反应体积含0.4 mmol.L-1dNTPs、3.5 mmol.L-1Mg2+、1.5 UTaqDNA聚合酶、0.4μmol.μL-1引物、3 ng模板DNA。PCR扩增程序为:94℃预变性2 min,94℃变性30 s,54.5℃退火30 s,72℃延伸1 min,45个循环,最后72℃延伸7 min,置4℃保存。应用该ISSR体系对6份荷花种质进行了扩增,证实了该体系的适用性和稳定性。
Taking genomic DNA extracted from Nelumbo nucifera Gaertn leaves as the experimental material, the factors which af- fected the ISSR - PCR amplification such as suitable concentration for dNTPs and Mg2 ＋ , then dosage for Taq DNA polymerase, the primer, template DNA and annealing temperature were selected and optimized. The results showed that the suitable PCR system for ISSR -PCR of N. nucifera Gaertn were as follows： the 20 μL PCR reaction volume including, 0.4 mmol · L-1 dNTPs, 3.5 mmol · L-1Mg2＋ , 1.5 U Taq DNA polymerase, 0.4 p.mol· μL-1 primer, 3 ng template DNA. The optimal PCR amplification process was： 2 minutes at 94 ℃ for predenaturation, then followed by 45 cycles, each with 30 seconds at 94 ℃ for denaturation, 30 seconds at 54.5 ℃ 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 six varieties of N. nucifera Gaertn indicating the suitability and stability of the system.
出处
《亚热带农业研究》
2009年第4期284-288,共5页
Subtropical Agriculture Research
基金
福建省科技重大专项资助项目(2004YZ02-05)
福建省创新平台福建省中药材GAP工程技术研究中心资助项目(2008Y2001)
关键词
荷花
ISSR
反应体系
优化
Nelumbo nucifera Gaertn
ISSR
reaction system
optimization
