西花蓟马issr-pcr反应体系优化及多态性引物筛选-生物安全学报.doc

西花蓟马ISSR-PCR反应体系的建立与优化( 蒋兴川杨明桂富荣** 云南农业大学 植物保护学院，农业生物多样性与病虫害控制教育部重点实验室，昆明 650201 摘要：西花蓟马是近年来在我国爆发成灾的重要入侵害虫，本文通过改进的法提取西花蓟马的基因组DNA，对西花蓟马ISSR-PCR反应体系中的DNA、引物、Taq聚合酶、buffer缓冲液、dNTPs、Mg和去离子甲酰胺7个影响因素进行了单因素试验，初步筛选出各因素的较优浓度；然后对影响较大的4个因素进行了正交设计试验L16(44)，建立了适合西花蓟马ISSR分子标记的最佳反应体系。即在20μL的反应体系中包含模板DNA 30ng、10×Buffer 2.0 μL、引物1.05μmol/L、Taq聚合酶2.0 U、dNTPs 212.5 μmol/L及MgSO4 2.25 mmol/L。利用采自云南昆明玉溪、、、保山、昭通、丽江、大理普洱9个地方西花蓟马样本所建立的反应体系进行检验，其结果表明优化后的体系适合用于西花蓟马的遗传多样性分析。 关键词：西花蓟马；ISSR-PCR；反应体系；单因素试验；正交设计试验 Optimization of ISSR-PCR Reaction System in Frankliniella occidentalis (Pergande) Xing-chuan JIANG, Ming YANG Fu-rong GUI ** Key Laboratory for Agricultural Biodiversity and Pest Management of Ministry of Education, College of Plant Protection, Yunnan Agricultural University, Kunming 650201, China Abstract: Western flower thrip Frankliniella occidentalis (Pergande), is one of the invasive insect pest occurred in many provinces of China and caused serious economic loss in invaded regions. This paper deals with the optimization of ISSR-PCR reaction system in F. occidentalis. Seven factors including template DNA, primers, Taq DNA Polymerase, Buffer, dNTPs, Mg2+ and formamide deionized were designed with test of single factor and corresponding better concentrations were screened out initially. Then the four more influential factors for ISSR-PCR system were designed employing orthogonal design test with four levels and four factors, and significance test and variance analysis were employed to analyze the PCR amplification data. The results showed that the system, which contains 30ng DNA template, 2.0μL 10×EasyTaq Buffer, 1.05μmol/L primers, 2.0U EasyTaq DNA Polymerase, 212.5μmol/L dNTPs and 2.25mmol/L MgSO4 in 20μL reaction is the optimized ISSR-PCR reaction system. DNA of nine samples of F. occidentalis collected in Kunming, Yuxi, Chuxiong, Honghe, Baoshan, Zhaotong, Lijiang, Dali and Puer in Yunnan province were used to detect suitability of this ISSR-PCR reaction system, and results indic