摘要
以豫杂一号泡桐为材料,通过对影响AFLP技术体系的各主要因素的研究,建立了适于泡桐AFLP分析的技术体系.结果表明最佳酶切体系(20μL)为500 ng模板DNA,3 U的PstI和M seI,在37℃下双酶切3 h;20μL最佳连接体系中为酶切产物15μL,0.25μmol.L-1PstI接头,2.5μmol.L-1M seI接头,1μL 10×T4 Buff-er,2 U T4连接酶,22℃连接18 h;20μL最佳预扩反应体系中5μL稀释10倍的连接产物,100μmol.L-1dNTP,2 U Taq酶,250μmol.L-1PstI和M seI引物(P+AGT/M+AGT),2μL 10×PCR Buffer.20μL最佳选择性扩增反应体系中5μL稀释20倍预扩增产物,100μmol.L-1dNTP,2 U Taq酶,350μmol.L-1PstI和M seI引物(P+AGT/M+AGT),2μL 10×PCR Buffer.最后,筛选出了97对适宜于泡桐AFLP分析的引物.
The establishment of Paulownia AFLP reaction system and its primer selection were investigated through study on the main factors affecting the system quality with Paulownia tomentosa x Paulownia fortune. The results indicated that the optimum conditions for restriction enzyme digestion of the genomic DNA from the Paulownia plant were as follows： 500 ng DNA template, 3 U Pst I and Mse I respectively in 20 μL reaction volume at 37 ℃ for 3 h;The ligation reaction （20 μL） with 15 μL the digestion product, 0.25 μmol·L^-1 Pst I adaper, 2.5 μmol·L^-1 Mse I adapter, 1 μL 10×T4 Buffer,2 U T4 ligation enzyme at 22℃ for 18 h was the optimum condition; the preamplification reaction （20 μL） with 5 μL the ligation fragment which was diluted 10 times, 100 μmol·L^-1 dNTP,2 U Taq enzyme,250 μmol·L^-1 Pst I and Mse I primer （P ＋ AGT/M ＋ AGT） ,2 μL 10 x PCR Buffer was the optimum condition; The selective amplification reaction （20 μL） with 5 μL preamplification product which was diluted 20 times, 100 μmol·L^-1 dNTP,2 U Taq enzyme,350μmol·L^-1 Pst I and Mse I primer （P ＋ AGT/M ＋ AGT） ,2 μL 10× PCR Buffer. Finally,97 pairs of primers were chosen for the AFLP analysis of Paulownia plants.
出处
《河南农业大学学报》
CAS
CSCD
北大核心
2010年第2期145-150,共6页
Journal of Henan Agricultural University
基金
国家自然科学基金项目(30571496)
高等学校博士学科点专项科研基金项目(20050466003)
中国博士后基金项目(20070410874)
