大豆孢囊线虫ISSR反应体系的建立与优化  

Establishment and Optimization of ISSR-PCR Reaction System for Heterodera glycines

作　　者：欧师琪 杨飞燕 史树森 OU Shiqi;YANG Feiyan;SHI Shusen(College of Plant Protection, Jilin Agricultural University, Changchun 130118, China)

机构地区：[1]吉林农业大学植物保护学院

出　　处：《吉林农业大学学报》2019年第4期389-393,共5页Journal of Jilin Agricultural University

基　　金：国家自然科学基金项目(31601616)

摘　　要：采用正交设计对大豆孢囊线虫ISSR-PCR反应体系的4因素(Taq酶、模板DNA、d NTPs、引物),在4个用量水平上进行优化试验。结果表明:大豆孢囊线虫ISSR-PCR的最佳反应体系为在25μL反应体系中,含Taq DNA聚合酶(5 U/μL) 0. 34μL、模板DNA 1. 2μL、d NTPs(2. 5 mmol/L) 1. 5μL、引物(10μmol/L)0. 8μL、10×PCR Buffer 2. 5μL。引物PTY26最适退火温度为55℃。用引物PTY188和PTY888对大豆孢囊线虫最佳ISSR-PCR的反应体系进行验证,结果表明该反应体系和程序有较好的重复性和稳定性。The orthogonal design involving four levels of four main factors(d NTPs,primer,Taq DNA polymerase,and DNA template) was used to optimize ISSR-PCR system on Heterodera glycines. The results showed that the optimal 25 μL ISSR-PCR system contained 0. 34 μL Taq DNA polymerase(5 U/μL),1. 2 μL template DNA,1. 5 μL dNTPs(2. 5 mmol/L),0. 8 μL primer(10 μmol/L),and 2. 5 μL 10 × PCR Buffer. And the optimal annealing temperature of primer PTY26 was 55 ℃. The primers PTY188 and PTY888 were used to verify the optimal ISSR-PCR reaction system for H. glycines,and the results showed that the reaction system and procedures had good repeatability and stability.

关 键 词：大豆孢囊线虫 ISSR-PCR 反应体系 反应优化 

分 类 号：S435.651[农业科学—农业昆虫与害虫防治]