摘要
以柱花草奥克雷品种为材料,采用改良的CTAB法提取基因组DNA,对影响AFLP反应体系的主要因素进行了优化,建立了柱花草的AFLP反应体系。结果表明:20μL为最佳反应体系,酶切体系中DNA模板量为1000ng,用5 U EcoR I 37℃酶切2 h、5 U Mse I 65℃酶切2 h效果最佳;分别取5μL酶切液、1μL T4连接酶(5μL/L)、1μL EcoR I接头、1μL Mse I接头、2μL缓冲液(T4DNA酶自带),于22℃下连接10 min效果最佳;预扩增体系中模板稀释15倍、Mg2+浓度为0.75 mmol/L、Taq酶用量为1 U、dNTPs浓度为0.2 mmol/L、引物浓度为2 ng/μL效果最佳;选择扩增体系中模板稀释20倍、Mg2+浓度为1.25 mmol/L、Taq酶为1 U、dNTPs浓度为0.225 mmol/L、引物浓度为0.4 ng/μL效果最佳。利用热研5号、奥克雷2个品种对8对引物组合进行筛选,筛选出46对引物组合,为利用AFLP标记对柱花草进行分子生物学研究及分子育种奠定基础。
Extract the total DNA of Stylosanthes guianensis using an improved method of CTAB. The Stylosanthes guianensis AFLP system was established and its primers were selected for analysis by optimizing the main factors affecting the AFLP system. The results indicate that the AFLP optimum conditions for restriction enzyme digestion of the Stylosanthes guianensis were as follows： 1 000 ng DNA template, 5 U EcoR I at 3 7℃ for 2 h and 5 U Mse I at 65℃ for 2 h in 20 μL optimum reaction volume; the optimum ligation system （20 μL） was 5 μL the digestion product, 1 μL T4 ligation enzyme（5 μL/L）, 1 μL EcoR I adapter and 1 μL Mse I adapter and 2 μLT4 Buffer reacting at 22℃ for 10 min; The preamplification reaction （20 μL） with 5μ L the ligation fragment which was diluted 15 times, 0.75 mmol/L Mg2＋, 1 U Taq enzyme, 0.2 mmol/L dNTPs, 2ng/μL EcoR I and Mse I primer was the optimum condition; The selective amplification reaction （20 μL） with 5 μL preamplification product which was diluted 20 times, 1.25 mmol/L Mg2＋, 1 U Taq enzyme, 0.225 mmol/L dNTP, 0.4 ng/μL EcoR I and Mse I primer was the optimum condition. Finally, 46 pairs of primers were screened out for the AFLP analysis of Stylosanthes guianensis. Lay a foundation of molecular biology research and molecular breeding of Stylosanthes guianensis by AFLP molecular marker.
出处
《热带农业科学》
2014年第4期46-52,69,共8页
Chinese Journal of Tropical Agriculture
基金
国家自然科学基金(No.30960266
No.31160481)
农业部热带作物种质资源利用重点开放实验室开放基金(No.KFKT:2010 11)
关键词
柱花草
DNA
AFLP
引物筛选
Stylosanthes guianensis
DNA
AFLP
primer selection
