摘要
为探讨高品质实蝇基因组DNA提取,研究模板DNA浓度、引物浓度、TaqDNA聚合酶用量、dNTP浓度、退火温度及时间对ISSR-PCR扩增结果的影响,以3种实蝇为材料,建立通用且稳定的实蝇ISSR-PCR反应体系。结果表明:获得了高品质实蝇基因组DNA;确立了通用且稳定的实蝇ISSR-PCR反应体系:10×PCRBuffer2.5μL,模板DNA50ng,引物0.25μmol/L,TaqDNA聚合酶0.50U,dNTP200μmol/L,最后加ddH2O至25μL;明确了ISSR-PCR扩增程序:94℃预变性5min,94℃变性30s,52.4℃退火45s,72℃延伸90s,循环36次,最后72℃延伸7min,4℃保存。体系的建立弥补了实蝇传统形态检测的不足,为快速准确鉴定、种群异质性及遗传多样性分析奠定了基础。
In order to explore the extraction of high-quality genomic DNA of three kinds of fruit flies, and to study the effect of template DNA concentration, primer concentration, the amount of Taq DNA polymerase, dNTP concentration, annealing temperature and annealing time on ISSR-PCR （inter-simple sequence repeat-polymerase chain reaction） amplified results, the fruit flies ＇ general and stable ISSR-PCR reaction system was established. It was found that the fruit flies ＇ genomic DNA with high quality was obtained, and the fruit flies ＇ general and stable ISSR-PCR reaction system was established：including 2.5 μL 10×PCR Buffer, 50 ng template DNA, 0.25 μmol/L primer, 0.5 U Taq DNA polymerase, 200 μmol/L dNTP and addition of ddH 2 O to 25 μL. The established ISSR-PCR amplification program was as follows： pre-denaturalized at 94℃ for 5 min, denaturalized at 94℃ for 30 s, annealed at 52.4℃ for 45 s, extended at 72℃ for 90 s, 36 cycles, at last extended at 72℃ for 7 min, and then conserved at 4℃. The establishment of optimized system made up for the shortage of fruit flies ＇ traditional morphology observation, and laid a foundation for fast and accurate identification, population heterogeneity and analysis of genetic diversity.
出处
《农学学报》
2011年第3期22-28,共7页
Journal of Agriculture
基金
福建省科技重大专项专题"花卉产业化配套技术研发-盆栽花卉病虫害防治技术研究"(2010NZ0003-2-3)
福建省属公益类科研院所基本科研专项计划项目"漳州出口盆栽花卉病虫害种类调查和防治新技术研究"(2010R1026-4)
福建省科技创新平台建设项目"福建省农作物害虫天敌资源工程技术研究中心"(2008N2002)
