摘要
该文以沙棘木蠢蛾幼虫为材料，用改进的SDS-蛋白酶K法获得了高质量的符合AFLP分析要求的基因组DNA。通过对AFLP试验过程中的酶切-连接、预扩增、选择性扩增等各关键因素的比较研究，建立了一套优化的沙棘木蠹蛾AFEP分子标记体系，获得了清晰的指纹图谱。研究结果表明：利用Eco RⅠ／MseⅠ双酶切系统以酶切-连接2～4h，预扩增产物稀释20倍、Mg^2＋浓度为1．5mol／mL的选择性扩增效果最好。进一步利用该反应体系从80对E＋3，M＋3选择性引物中筛选出10对多态性丰富、分辨率高、谱带清晰的引物组合。该研究结果为利用AFLP标记技术开展沙棘木蠢蛾的种群遗传结构、遗传分化等方面的研究奠定了基础。
In this study, high-quality genomic DNA isolated by improved SDS-proteinase K method from larvae of Holcocerus hippopaecolus was obtained and is approved suitable for AFLP analysis. The AFLP reaction system of H. hippopaecolus was established by optimizing enzyme digestion/ligation reaction, pre-amplification and selective amplification. The optimal reaction time for double enzyme digestion （ EcoR Ⅰ/Mse Ⅰ ） was 2-4 hours. Products of the pre-amplification diluted 20-foM and effects of the selective amplification with 1.5 mol/ mL Mg^2＋ were the best. Ten pairs of selective primer combinations which revealed sufficient amplified bands with high resolution and polymorphism were selected from 80 pairs of E^＋3/M^＋3 primers and clear fingerprints were obtained. The AFLP reaction system can be used to study the population structure and genetic diversity in population of H. hippopaecolus.
出处
《北京林业大学学报》
CAS
CSCD
北大核心
2008年第4期116-120,共5页
Journal of Beijing Forestry University
基金
国家自然科学基金重点项目(30730075)
北京林业大学振兴计划项目(200302002)
长江学者与创新团队发展计划项目(PCSIRT0607)
关键词
沙棘木蠢蛾
AFLP标记
体系建立
引物筛选
Holcocerus hippopaecolus
AFLP marker
system establishment
primer selection
