福建含笑ISSR-PCR反应体系的建立与优化  

Establishment and optimization of ISSR-PCR reaction system of Michelia fujianensis

作　　者：黄宇[1] 陈礼光[1] 夏海涛[1] 荣俊冬[1] 孔玲[1] 廖鹏辉[1] 郑郁善[1] 

机构地区：[1]福建农林大学工业原料林研究所,福建福州350002

出　　处：《福建林学院学报》2009年第2期144-148,共5页Journal of Fujian College of Forestry

基　　金：福建省科技厅重大资助项目(2003I007)

摘　　要：以福建含笑叶片提取的基因组DNA为材料,对影响ISSR-PCR扩增效果的一些因素,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指标进行筛选和优化,确立了可用于福建含笑ISSR-PCR分析最适宜的PCR反应条件:20μL PCR反应体积中含0.4 mmol.L-1dNTPs,2.5 mmol.L-1Mg2+,1.5 UTaqDNA聚合酶,0.6μmol.μL-1引物,30 ng模板DNA。PCR扩增程序:94℃预变性2 min,94℃变性30 s,47.3℃退火30 s,72℃延伸1 min,45个循环,72℃延伸7 min,4℃保存。应用ISSR体系对5份福建含笑种质进行了扩增,证实了该体系的适用性和稳定性。Taking genomic DNA extracted from Michelia fujianensis leaves as the experimental material, the factors which affected the ISSR-PCR amplification such as suitable concentration for dNTPs and Mg^2＋ , then dosage for Taq DNA polymerase, the primer, template DNA and annealing temperature were selected and optimized. The results showed that the suitable PCR conditions for ISSRPCR of M.fujianensis were as follows： In the 20 μL PCR reaction volume, 0.4 mmol · L^-1 dNTPs, 2.5 mmol · L^-1 Mg^2＋ , 1.5 U Taq DNA polymerase, 0.6 μmol · μL^-1 primer, 30 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 47.3 ℃ for annealing, 1 minute at 72℃ for extension, finally extension at 72 ℃ for 7 minutes and holding the samples at 4℃. The ISSR system was applied in the amplification of five varieties of M.fujianensis, indicating the suitability and stability of the system.

关 键 词：福建含笑 ISSR-PCR 体系优化 

分 类 号：S823[农业科学—畜牧学] S791.27[农业科学—畜牧兽医]