文献类型：期刊文献

中文题名：濒危药用植物厚朴ISSR引物筛选及反应条件的优化

英文题名：Primer screening and optimization of ISSR reaction condition for endangered medicinal plant Magnolia officinalis

作者：于华会[1] 杨志玲[1] 杨旭[1] 谭梓峰[1] 舒枭[2]

第一作者：于华会

机构：[1]中国林业科学研究院亚热带林业研究所;[2]云南农业大学

年份：2009

期号：12

起止页码：2444-2451

中文期刊名：生态学杂志

外文期刊名：Chinese Journal of Ecology

收录：CSTPCD;;Scopus;北大核心:【北大核心2008】；CSCD:【CSCD2011_2012】；

基金：国家科技部公益性林业专项资助项目(200704022)

语种：中文

中文关键词：厚朴;正交设计;引物筛选;ISSR

外文关键词：Magnolia officinalis; orthogonal design; primer screening; ISSR;

分类号：S718.46

摘要：以厚朴DNA为模板,利用正交试验分别对影响厚朴ISSR-PCR反应的Taq酶浓度、dNTP浓度、引物浓度、Mg2+浓度、模板DNA浓度进行了优化,并通过梯度PCR确定不同引物的最佳退火温度和循环次数,最终确定厚朴最佳反应体系及扩增条件为:25μl体系,其中包括1.5mmol.L-1MgCl2,0.3μmol.L-1引物,0.04U.μl-1Taq酶,0.2mmol.L-1dNTP,4ng.μl-1模板DNA,1×Buffer;扩增程序:94℃预变性5min,94℃变性30s,50℃～60℃(退火温度随引物不同而定)退火45s,72℃延伸90s,共40个循环,然后72℃延伸8min,4℃终止反应。此外,还利用优化的反应体系成功筛选出21条ISSR引物,并利用部分引物对厚朴个体进行了遗传多样性分析。
To optimize the inter simple sequence repeat（ISSR）reaction condition for Magnolia officinalis genomic DNA,the concentrations of Mg 2＋,Taq polymerase,primers,deoxyribonucleot-ide triphosphate（dNTP）,and template DNA were studied with an orthogonal experimental design,and the optimal anneal temperature of different primers and cycles were determined through gradient PCR.The optimal PCR system for ISSR analysis was 1.5 mmol MgCl 2·L-1,0.3 μmol primer·L-1 0.04 U Taq polymerase·μl-1,0.2 mmol dNTP·L-1,4 ng DNA template·μl-1,and 1×Buffer in 25 μl reaction solution,and the augmentation procedure was pre-denaturation at 94 ℃ for 5 min,denaturation at 94 ℃ for 30 s,annealing at 50 ℃-60 ℃ for 45 s,extension at 72 ℃ for 90 s,reaction with 40 cycles,and extension at 72 ℃ for 8 min.Twenty-one ISSR primers were screened by using the optimal reaction system,and the genetic diversity was analyzed with some primers.