摘要
以金银花叶片基因组DNA为模板,通过单因素试验,研究了退火温度、Taq DNA聚合酶的用量及模板DNA、引物、dNTPs、Mg2+浓度等6种因素对ISSR-PCR扩增的影响,建立了适合于金银花IS-SR-PCR反应体系和扩增程序,即在20μl反应体系中,内含1×PCR反应缓冲液(Mg2+free)、1.5UTaq DNA聚合酶、0.15mmol/L dNTPs、0.4μmol/L引物、1.5mmol/LMgCl2、60ng模板DNA。确定了适宜的退火温度为49.9℃。扩增程序为94℃预变性5min;35个循环为94℃变性30s,49.9℃退火30s,72℃延伸1.5min;最后72℃延伸7min,4℃保存。利用优化反应体系,从100条ISSR引物中筛选出10条稳定性和重复性高的引物;以这10条引物对22个金银花品种基因组DNA扩增,共扩增出108条带,其中多态性条带96条,多态性条带比率为88.9%。金银花ISSR反应体系的建立为利用ISSR标记技术进行金银花品种鉴别、分类、种质资源遗传多样性分析奠定了良好基础。
Based on the genomic DNA extracted from honeysuckle, the factors influenced ISSR were optimized and the effect of 6 factors such as annealing temperature, Taq DNA polymerase dosage, DNA templates concentration,primer concentration, dNTPs concentration tested using single factor experiment. A reaction system and Mg^2＋ concentration on ISSR amplification were and amplified procedure suitable for honeysuckle were established, that is 201μl amplification reactions system containing 1×PCR buffer （Mg^2＋free）, 1.5U Taq DNA polymerase, 0.15mmol/L dNTPs, 0.4μmol/L primer, 1.5mmol/L MgC12, 60ng template DNA. The optimized annealing temperature is 49.9%. The optimal amplified procedure was as follows： after a pre-denaturing of 5 min at 94℃, 35 cycles were performed with 30 s for denaturing at 94℃, annealing of 30 s at 49.9℃, extension of 1.5 min at 72℃, 7 minutes of extension at 72% in the final cycle and hold at 4℃. Using these optimal amplification conditions, 10 stable and repeatable ISSR primers were selected from total 100 primers. The genome DNA of 22 honeysuckle cultivars were amplified with 10 ISSR primers and 108 bands were amplified totally, including 96 polymorphism bands with polymorphism rate of 88.9%. The establishment of the ISSR-PCR reaction system could settle favorable foundation for identification of honeysuckle cultivars, classification and analysis of the genetic diversity of honeysuckle using ISSR molecular marker techniques.
出处
《中国农学通报》
CSCD
2008年第11期73-77,共5页
Chinese Agricultural Science Bulletin
基金
湖南省农业科技成果转化资金项目"优质高产花蕾型金银花新品种及繁育技术中试与示范"(06NK4034)。
关键词
金银花
ISSR
反应体系
优化
Honeysuckle, ISSR, reaction system, optimization
