台湾水青冈ISSR-PCR体系的优化  

Optimization of ISSR-PCR System of Fagus hayatae

作　　者：宋文静[1,2] 金则新[2] 李钧敏[2] 李建辉[2] 

机构地区：[1]杭州师范大学生命与环境科学学院,浙江杭州310036 [2]台州学院生态研究所,浙江临海317000

出　　处：《福建林业科技》2009年第2期18-22,共5页Journal of Fujian Forestry Science and Technology

基　　金：浙江省自然科学基金项目(Y504220)

摘　　要：分析了ISSR-PCR体系中Mg2+、dNTP和BSA浓度,以及模板DNA、引物和TaqDNA聚合酶用量等6个因素对反应结果的影响,建立了适合于台湾水青冈ISSR-PCR扩增的反应体系:10μL PCR反应液中,含10 ng DNA,1.5 mmol.L-1Mg2+,0.125 mmol.L-14×dNTP,1×TaqDNA聚合酶配套缓冲液(10 mmol.L-1Tris-HCl,pH值9.0,50 mmol.L-1KCl,0.1%Triton X-100),0.6 UTaqDNA聚合酶,2 mg.mL-1BSA和3pmol引物。应用优化的ISSR-PCR体系从100个ISSR引物中筛选出12个稳定性好,重复性高的引物,对22株台湾水青冈个体的DNA进行扩增,结果表明优化的ISSR-PCR体系完全适合于台湾水青冈的ISSR分析。This paper analyzed the influence of the 6 factors on the reaction results, the factors included： the concentration of Mg^2＋, dNTP and BSA in ISSR-PCR system, as well as the dosage of the template DNA, primer and Taq DNA polymerase. It established the response system suitable for ISSR-PCR amplification of Fagus hayatae： 10μL of PCR reaction solution, including 10 ng of DNA, 1.5 mmol.L^-1 of Mg^2＋, 0.125 mmol.L^-1 of 4×dNTP, 1×Taq DNA polymerase matching buffer （10 mmol.L^-1 Tris-HCl, pH value of 9.0, 50 mmol.L^-1 of KCl, 0.1% of Triton X-100）, 0.6 U of Taq DNA polymerase, 2 mg.mL^-1 of BSA and 3 pmol primer. With the application of optimized ISSR-PCR system, 12 stability, high repeatability of primers were selected from the 100 ISSR primers. DNA amplification was carried out on 22 F. hayatae individuals, the results showed that the optimization of ISSR-PCR system was completely suitable for F. hayatae ISSR analysis.

关 键 词：台湾水青冈 ISSR—PCR体系 优化 

分 类 号：S718.43[农业科学—林学]