安徽农业科学2009，Vol.37Issue(31)：15160-15162,3.
钩距虾脊兰ISSR-PCR反应体系的建立与优化
Establishment and Optimization of ISSR-PCR Reaction System for Calanthe graciliflora Hayata
摘要
Abstract
[ Objective ] The research aimed to lay the foundation for the germplasm resources researches of Calanthe graciliflora Hayata. [ Method] With C. graciliflora as the test materials,the genomic DNA was extracted from C. graciliflora by using improved CTAB method. Different factors that affected ISSR amplification reaction were optimized. [ Result] High-quality genomic DNA was obtained from C. graciliflora Hayata. And the reaction system and amplified procedure that were suitable for C. graciliflora Hayata were as follows:25μl amplification reactions system contained 2.5μl 10×PCR buffer,2.0 mmol/L MgCl_2,100 ng genomic DNA,240 μmol/L dNTPs,1.75 U Taq DNA polymerase and 0.4 μmol/L ISSR primer. The optimal amplified procedure was as follows: pre-denaturing for 5 min at 94℃ ,40 cycles of denaturing for 30 s at 94℃.annealing for 30 s due to 1 -2℃ lower than denaturing temperature of different primer,extending for 50 s at 72 ℃,finally extending for 7 min at 72℃. and presering at 4 ℃. [ Conclusion ] The optimal system could provide a basis for further study on genetic diversity of C. graciliflora Hayata by ISSR molecular marker.关键词
钩距虾脊兰/DNA提取/ISSR分子标记Key words
Calanthe graciliflora Hayata/DNA extraction/ ISSR分类
农业科技引用本文复制引用
彭德镇,孙小琴,孔令杰,李恩香,杨柏云..钩距虾脊兰ISSR-PCR反应体系的建立与优化[J].安徽农业科学,2009,37(31):15160-15162,3.基金项目
国家环保总局项目(20061A0013). （20061A0013）
