摘要
[目的]为钩距虾脊兰种质资源研究奠定基础。[方法]以钩距虾脊兰为试验材料,利用改良的CTAB法提取钩距虾脊兰基因组DNA,并对影响ISSR扩增反应的各因素进行优化。[结果]获得了高质量的钩距虾脊兰基因组DNA;同时,建立了最适的钩距虾脊兰ISSR-PCR体系,即25μl PCR反应体积中,2.5μl 10×PCR buffer,2.0 mmol/L MgCl2,100 ng模板DNA,240μmol/L dNTPs,1.75 UTaqDNA聚合酶,0.4μmol/L引物;最佳扩增程序为94℃预变性5 min,然后进行40个循环:94℃变性30 s,复性温度根据各引物的TM值略低1～2℃,30 s,72℃延伸50 s,循环结束后72℃延伸7 min,4℃保存。[结论]这一优化系统的建立为进一步利用ISSR分子标记技术进行钩距虾脊兰遗传多样性研究提供了基础。
[ 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 ：25 μl amplification reactions system contained 2.5 μl10 × PCR buffer,2.0 mmol/L MgC12 , 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 clue to 1 -2 ℃ lower than denaturing temperature of different primer,extending for 50 s at 72 ℃ ,finally extending for 7 rain 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.
出处
《安徽农业科学》
CAS
北大核心
2009年第31期15160-15162,共3页
Journal of Anhui Agricultural Sciences
基金
国家环保总局项目(20061A0013)
