摘要
通过单因子试验和正交设计,对影响爱玉ISSR-PCR扩增效果的因素,如模板DNA用量、Taq酶用量、dNTPs浓度、引物浓度、延伸时间和循环次数等指标进行优化.实验确立了可用于爱玉ISSR-PCR分析最适宜的反应体系:20μL反应体积中含1ng模板DNA、1.0U Taq酶、0.4μmol/L引物和0.18mmol/L dNTPs;PCR扩增程序为94℃预变性4min;94℃变性45s,53℃退火45s,72℃延伸2.5min,共35个循环;72℃延伸7min,4℃保存.应用该体系对14份爱玉种质进行扩增,证实了该体系的适用性和稳定性.
The factors affecting the ISSR-PCR amplification on Ficus pumila L. vat. awkeotsang such as dosage of template DNA, Taq DNA polymerase, dNTPs, primers and extension time and cycle times were optimized through the combination of single factor and orthogonal design experiment. The results showed that the optimal reaction system for ISSR-PCR of Ficus pumila were as follows：In the 20 μL reaction volume, lng template DNA, 1. 0U Taq DNA polymerase, 0. 4 μmol/L primers, 0. 18 mmol/L dNTPs. The optimal PCR amplification process was：4 minutes at 94 ℃ for predenaturation,then followed by 35 cycles,each with 45 seconds at 94 ℃ for denaturation,45 seconds at 53 ℃ for annealing,2 at 72℃ for extension,finally extension at 72 ℃ for 7 minutes and holding the samples at 4 ℃. The system was applied in the amplification of 14 varieties of indicated the suitability and stability of the system.
出处
《吉首大学学报（自然科学版）》
CAS
2014年第2期73-78,共6页
Journal of Jishou University(Natural Sciences Edition)
基金
宁德师范学院"服务海西建设"资助项目(2010H309)
福建省自然科学基金资助项目(2006JA0399)
福建省大学生创新创业训练计划资助项目(201310398019)
关键词
爱玉
ISSR
反应体系
正交设计
Ficus pumila L. var. awkeotsang
inter-simple sequence repeat （ISSR）
reaction system
orthogonal design
