狼尾蕨基因组DNA提取及SSR反应体系的建立  

Extraction of genomic DNA and establishment of SSR reaction system for Davallia bullata

作　　者：贾新平[1] 叶晓青[1] 梁丽建[1] 邓衍明[1] 佘建明[1] 孙晓波[1] 

机构地区：[1]江苏省农业科学院农业生物技术研究所,江苏省农业生物学重点实验室,江苏南京210014

出　　处：《江苏农业学报》2013年第6期1456-1462,共7页Jiangsu Journal of Agricultural Sciences

基　　金：江苏省农业科技自主创新基金项目[CX(13)5057]

摘　　要：为了研究蕨类植物基因组DNA的提取方法及建立适合狼尾蕨的SSR-PCR反应体系,以狼尾蕨为试验材料,利用改良的CTAB方法提取狼尾蕨叶片基因组DNA,分析SSR-PCR反应体系的主要成分(DNA模板浓度、Mg2+浓度、dNTPs浓度、引物浓度和Taq DNA聚合酶)对扩增结果的影响,并对引物的适宜退火温度进行优化。结果表明:引物DB-23的最佳退火温度为54℃;20μl PCR反应体系中,DNA模板浓度为30 ng/μl,引物浓度为1.5μmol/L,Mg2+浓度为2.0 mmol/L,dNTPs浓度为0.20 mmol/L,Taq DNA聚合酶加入量为1.5 U。利用建成的SSRPCR反应体系对8个蕨类品种进行扩增,扩增谱带清晰且有较高的多态性,说明该体系稳定可靠。The extraction method of fern genomic DNA and establishment of SSR-PCR reaction system for Davallia bullata were studied. The genomic DNA of Davallia bullata was isolated using improved CTAB method. Five factors including DNA template concentration, Mg2＋ concentration, dNTPs concentration and primer concentration and Taq DNA polymerase on the amplification were analyzed, and the annealing temperature was optimized. The results revealed that the optimal annealing temperature of primer DB-23 was 54 ℃, and the 20 μl reaction system was comprised of： 30 ng/μl template DNA, 1.5 Ixmol/L primer, 2. 00 mmol/L Mg2＋, 0. 20 mmol/L dNTPs and 1.5 U Taq DNA polymerase. With the optimized reaction sys- tem, SSR fragments of eight pteridophyte cuhivars showed clear band, and good polymorphism and stability.

关 键 词：狼尾蕨 DNA提取 SSR 反应体系 

分 类 号：S682.35[农业科学—观赏园艺]