小菜蛾ISSR-PCR反应体系的建立与正交设计优化  

Establishment and optimization of ISSR-PCR reaction system with orthogonal design for Plutella xylostella

作　　者：杨家强[1] 王相晶[1] 郭兆将[2] 朱勋[2] 吴青君[2] 谢文[2] 王少丽[2] 徐宝云[2] 张友军[2] 

机构地区：[1]东北农业大学生命科学学院,哈尔滨150030 [2]中国农业科学院蔬菜花卉研究所,北京100081

出　　处：《植物保护》2012年第6期90-94,共5页Plant Protection

基　　金：公益性行业(农业)科研专项(201103021);国家科技支撑计划(2012BAD19B06);国家自然科学基金(31071709)

摘　　要：以小菜蛾(Plutella xylostella)基因组DNA为模板,采用L16(45)正交试验设计方法,建立小菜蛾的ISSR最佳反应体系。通过梯度退火试验,确定不同引物的最适退火温度。优化得到的反应体系(20μL)为:Mg2+浓度1.5mmol/L、Taq DNA聚合酶2.5U、dNTPs浓度0.2mmol/L、引物浓度1.25μmol/L、模板DNA量20ng。反应程序为:94℃预变性5.0min;94℃变性45.0s,40～61℃(不同引物退火温度各异)退火1.0min,72℃延伸1.5min,40个循环;72℃延伸10.0min;10℃保存。利用所建立的ISSR-PCR反应体系,获得了清晰、重复性好的DNA谱带。To establish and optimize ISSR-PCR reaction system for Plutella xylostella, based on the genomic DNA of P. xylostella, the factors influencing ISSR system were explored with the L16 （45 ） orthogonal design. The optimal annealing temperature for ISSR-PCR reaction was proposed by gradient PCR. The optimal conditions for ISSR reaction system （20 μL） were determined as follows： 1.5 mmol/L of Mg2＋ , 2.5U of Taq DNA polymerase, 0.2 mmol/L of dNTPs mixture, 1.25 μmol/L of each primer, and 20ng of template DNA. The reaction program was as followed： initial denaturation for 5.0 min at 94 ℃, 35 cycles of denaturation for 45s at 94 ℃, annealing for 1.0 min at 40--61 ℃, extension for 1.5 min at 72 ℃, with a final extension of 10.0 min at 72 ℃. The clear and reproducible DNA bands were obtained by the established ISSR-PCR reaction system.

关 键 词：小菜蛾 ISSR 正交设计 反应体系 

分 类 号：S433.4[农业科学—农业昆虫与害虫防治]