10种绣线菊DNA的提取及RAPD反应体系的优化  

Studies on DNA Extraction and Establishment of RAPD Reaction System in Spiraea L.

作　　者：房慧旺[1] 刘庆华[1] 王奎玲[1] 刘庆超[1] 唐启和[1] 

机构地区：[1]青岛农业大学环境艺术学院,青岛266109

出　　处：《中国农学通报》2008年第2期67-70,共4页Chinese Agricultural Science Bulletin

基　　金：山东省农业良种工程重大课题(鲁科农字[2005]99号)

摘　　要：旨在筛选出绣线菊属RAPD反应的最佳体系,为绣线菊属植物种质资源遗传多样性和亲缘关系的研究奠定基础。试验以10种绣线菊属植物为材料,采用改良CTAB法进行冬芽和嫩叶的总DNA提取并对影响RAPD扩增效果的因素进行优化。通过单因素优化筛选出最佳的20μl反应体系:10×Taq buffer 2μl,Taq酶1.0U,0.15mmol/L dNTP,DNA 2ng/μl,引物0.2μ mol/L,Mg2+2.0mmol/L;反应程序:94℃预变性4min,然后进行40个循环:94℃变性45s,37℃退火1min,72℃延伸1min,最后72℃延伸5min,4℃终止反应。结果发现,采用改良CTAB法所提取绣线菊冬芽的DNA达到RAPD反应的要求,筛选体系扩增出清晰稳定的多态性条带,可用于对绣线菊属遗传育种方面的研究。In this paper the optimum RAPD reaction system of Spiraea was selected by experiment. It is the basis for preparation of genetic diversity and relatives of Spiraea germplasm resource study. An improved CTAB method was used to extract winter buds and tender leaf of ten Species of Spiraea DNA and factors which affect the RAPD amplification were optimized and selected. A single-factor optimization method used showed that a total volume of 20μl RAPD system of Spiraea consisted of 2μl 10×Taq buffer, 1.0U Taq DNA polymerase, 0.15mmol/L dNTP, 2ng/μl DNA, 0.2txmol/L primer and 2.0mmol/L Mg^2＋. The suitable PCR procedure is one cycle denaturing at 94℃ for 4min, 40 cycles which involves denaturing at 94℃ for 45s, annealing at 37℃ for lmin and extending at 72℃for lmin, one cycle extending at 72℃ for 5min, and then kept at 4℃. The experiment shows that improved CTAB method could extracted winter buds of Spiraea DNA which could meet the requirement of RAPD reaction. The selected RAPD system amplified the polymorphism and its bands are clear.

关 键 词：绣线菊属 DNA提取 RAPD 

分 类 号：Q523[生物学—生物化学]