白桦ISSR-PCR反应体系的优化  

Optimization of ISSR-PCR Reaction System for Betula platyphylla Suk

作　　者：杨传平[1] 潘华[1] 魏志刚[1] 姜静[1] 

机构地区：[1]东北林业大学,哈尔滨150040

出　　处：《东北林业大学学报》2005年第6期1-3,共3页Journal of Northeast Forestry University

基　　金：国家863课题;东北林业大学校基金课题(2002AA241080);国家自然基金项目(30571513)

摘　　要：以白桦基因组DNA为模板,研究了ISSR的影响因素,建立一套适宜于白桦的ISSR优化反应体系及程序.即20μL反应体系中,Mg2+浓度范围为0.5～3.0 mmol·L-1,dNTPs浓度范围为0.10～0.30 mmol·L-1,Taq聚合酶浓度范围为1.0～2.0 U,模板DNA浓度为30～120 ng,同时含有RNA的模板DNA对ISSR扩增也略有影响,引物浓度范围为0.2～0.4 μmmol·L-1.通过梯度PCR测验,确定了适宜的退火温度为49℃.反应程序为:第一个循环基因组DNA经94℃预变性5 min;30个循环为94℃变性30 s,49℃退火30 s,72℃延伸30 s;最后一个循环完成后,在72℃延伸7min.Using the genomic DNA extracted from Betula as template, the factors influenced ISSR were discussed. A set of ISSR optimized reaction systems and programs were set up. Results showed that for a reaction system with the volume of 20 μL, the optimal parameters were obtainedas Mg^2＋ concentration for ISSR 1.5 ～ 3.0 mmol · L^-1, and the suitable dNTPs concentration 0.10 ～0.25 mmol · L^-1 , proper quantity of Taq polymerase 0.5 ～ 2.0 U, appropriate quantity for template DNA 30 ～ 100 ng, and the concentration of random primer 0.2 ～0.4 μmmol · L^-1. The reaction program was devised for 5 minutes of predenaturalization at 94 ℃, 30 cycles of 30 seconds for denaturalization at 94 ℃, 30 seconds of anneal at 49 ℃, 30 seconds of extension at 72 ℃, and 7 minutes of extension at 72 ℃ in the final cycle.

关 键 词：白桦 ISSR标记 反应体系 

分 类 号：S791.259[农业科学—林木遗传育种]