珍珠黄杨DNA的提取方法比较及ISSR反应体系的优化  

Comparison of Genomic DNA Extraction Methods and Optimization of ISSR-PCR Reaction Systems for Buxus sinica var. parvifolia

作　　者：吕乐燕[1] 季孔庶[1] 黄焱[1] 

机构地区：[1]国家林业局林木遗传与基因工程重点开放实验室南京林业大学,南京210037

出　　处：《分子植物育种》2007年第1期145-149,150,共6页Molecular Plant Breeding

基　　金：国家十五科技攻关项目(2004BA515B0102)资助。

摘　　要：为从珍珠黄杨幼叶中提取高质量的总DNA,比较了1%CTAB、2%CTAB、4%CTAB和1.5%SDS4种DNA提取方法。结果表明:2%CTAB提取的DNAA260/A280值最好,ISSR-PCR扩增效果最佳,是有效提取珍珠黄杨基因组DNA的方法。并在此基础上,以(CT)8G为引物,采用单因素实验法,确立了适合珍珠黄杨的ISSR-PCR反应体系:在总体积20μL的反应体系中,含30ngDNA模板,2.0mmol/LMg2+,0.30μmol/L引物,0.20mmol/LdNTPs,1UTaq酶。In this paper, four different DNA-extracting methods （1% CTAB. 2% CTAB. 4% CTAB and 1.5% SDS） were employed to be compared in order to isolate high-quality genomic DNA from young leaves of Buxus sinic vat. parvifolia. The results showed that the method with 2% CTAB was the best one, which the extracted DNA had an ideal A260/A280 ratio and nice banding pattern amplified by ISSR-PCR. We could recommend the ISSR- PCR reaction system for Buxus sinic var. parvifolia based on optimization with primer （CT）8G by the single factor experiment. The components of 20 μL reaction volume would be as follows： 30 ng genomic DNA as template, 2.0 mmol/L Mg^2＋, 0.30μmol/L primer, 0.20 mmol/L dNTPs, 1 U Taq polymerase and added ddH20 to total volume.

关 键 词：珍珠黄杨(Buxus sinic vat.parvifolia) DNA提取 ISSR-PCR反应体系 

分 类 号：S688.1[农业科学—观赏园艺]