摘要
为从分子水平上探讨濒危物种沉水樟种质资源的保护机制,以其基因组DNA为模版,开展ISSR-PCR反应体系的研究,对各反应因子水平、引物退火温度和循环参数进行优化,确定适于沉水樟ISSR-PCR反应的最佳体系：20 μL反应体系中各反应物的最适含量为模板75 ng、引物0.4μmol/L、dNTP 200 μmol/L、Taq酶1.25 U、Mg2＋ 1.5 mmol/L;扩增程序为：94℃预变性7 min; 94℃变性30 s,53.0～58.0℃退火45 s,72℃延伸90 s,45个循环;72℃延伸7 min,4℃保存.
In order to explore the germplasm resource conservation mechanism for the endangered species Cinnamomun micranthum from the molecular level,the most appropriate ISSR-PCR reaction system with its genomic DNA was experimented by optimizing the level of each reacting factors,the primer annealing temperature and cycle parameters.The results showed that the suitable dosage of each reacting factor for C.micranthum ISSR-PCR reaction system was as follows：20 μL reaction system,including 75 ng template,0.4 μmol/L primer,200 μmol/L dNTP,1.25 U Taq enzyme,and 1.5 mmol/L Mg2 ＋.The best amplification program was as：denaturation for 7 min at 94 ℃ in advance,then denaturation at 94 ℃ for 30 s,annealing for 45 s at 53.0-58.0 ℃,extension for 90 s at 72 ℃,after 45 cycles,extension at 72 ℃ for 7 min,preservation at 4 ℃.
出处
《西南林业大学学报（自然科学）》
CAS
2013年第5期40-45,共6页
Journal of Southwest Forestry University：Natural Sciences
基金
国家林业局林业公益性行业科研项目(201304303)资助
高校产学合作科技重大项目(2013N5002)资助
关键词
沉水樟
濒危植物
ISSR
PCR反应体系
Cinnamomun micranthum
endangered plants
ISSR
PCR reaction system
