摘要
【背景】西花蓟马是近年来在我国局部地区暴发成灾的重要入侵害虫。【方法】本文通过改进的STE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、Mg2+和去离子甲酰胺等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。【结果】在20μL的反应体系中包含模板DNA30ng、10×Buffer2.0μL、引物1.05μmol·L-1、Taq聚合酶2.0U、dNTPs212.5μmol·L-1及MgSO42.25mmol·L-1。【结论与意义】利用采自云南昆明、玉溪、楚雄、红河、保山、昭通、丽江、大理和普洱9个地方的西花蓟马样本对所建立的反应体系进行检验,结果表明优化后的体系适用于西花蓟马的遗传多样性分析,本研究可为进一步研究西花蓟马的种群结构奠定基础。
[ Background ] Western flower thrips, Frankliniella occidentalis （Pergande）, is one of the invasive insect pests occurring in many provinces of China and causing serious economic loss in invaded regions. [ Method] In the present study, inter-simple se- quence repeat （ISSR-PCR） system in F. occidentalis was optimized. Seven factors including template DNA, primers, Taq DNA pol- ymerase, buffer, dNTPs, Mg2~ and deionized formamide were designed with a single factor test, and then the corresponding better concentrations were screened out initially. After that four more influential factors for ISSR-PCR system were designed employing or- thogonal design test with four levels and four factors, and significance test and variance analysis were employed to analyze the PCR amplification data. [Result]The system, containing 30 ng DNA template, 2.00L 10 xEasyTaq buffer, 1.05 ttmol.L-1 primers, 2.0 U EasyTaq DNA polymerase, 212.5 ttmol＂ L-l dNTPs and 2.25 retool. L-I MgSO4 in 20 I^L reaction, gave the best results in ISSR- PCR system. [ Conclusion and significance] DNA of nine samples of F. occidentalis collected in Kunming, Yuxi, Chuxiong, Hong- he, Baoshan, Zhaotong, Lijiang, Dali and Puer in Yunnan Province were used to detect suitability of this ISSR-PCR system. The results indicated that the system is suitable for genetic diversity detection of F. occidentalis.
出处
《生物安全学报》
2011年第3期198-206,共9页
Journal of biosafety
基金
国家自然科学基金项目(30860069)
973计划项目(2011CB100404)
云南省科技创新团队高层次科技人才培引工程(2011CI128)
关键词
西花蓟马
ISSR-PCR
反应体系
单因素试验
正交设计试验
Frankliniella occidentaliz
ISSR-PCR
reaction system
single factor test
design of orthogonal testing
