文献类型：期刊文献

中文题名：山苍子AFLP反应体系的建立及其引物筛选

英文题名：Primer Screening and AFLP Amplification Reaction System of Litsea cubeba

作者：田胜平[1] 汪阳东[1] 陈益存[1] 占志勇[1] 斯林林[2]

第一作者：田胜平

机构：[1]中国林业科学研究院亚热带林业研究所;[2]安徽农业大学生命科学学院

年份：2012

卷号：25

期号：2

起止页码：174-181

中文期刊名：林业科学研究

外文期刊名：Forest Research

收录：CSTPCD;;Scopus;北大核心:【北大核心2011】；CSCD:【CSCD2011_2012】；

基金：浙江省科技计划项目(2009C32107);国家林业局重点科研项目(2011-01)

语种：中文

中文关键词：山苍子;基因组DNA;AFLP;多态性引物

外文关键词：Litsea cubeba; genomic DNA; AFLP; the polymorphic primer

分类号：S718.46

摘要：通过对山苍子幼嫩叶片、顶芽、花蕾3种组织的DNA提取效果分析和对影响酶切及选择性扩增效果的4个主要因素(酶切时间、Mg2+浓度、dNTPs浓度、引物浓度)的比较研究,建立了适合于山苍子AFLP分析的技术体系。结果表明,山苍子的顶芽是较好的DNA提取材料;山苍子基因组DNA经5 U EcoR I和5 U Mse I酶切1 h即可完全酶切;最佳的选择性扩增体系为20μL反应体系中含有1.0 U rTaq聚合酶、2.0μL 10×PCR缓冲液、1.8μL 25mmol.L-1MgCl2、1.4μL 2.5 mmol.L-1dNTP、100 ng.μL-1引物各1.0μL。使用该反应体系获得了清晰、稳定的DNA指纹图谱,并筛选出10对多态性较好的AFLP引物组合,为利用AFLP标记技术进一步开展山苍子种群遗传结构、遗传分化等研究奠定了基础。
The effect of DNA extraction was analyzed by comparing the young leaf,terminal bud and flower of Litsea cubeba.The time of DNA digestion and several key factors affecting the PCR selective amplification such as Mg^2＋ concentration,dNTPs concentration and the mount of the selective amplification primer were also trialed.An optimized AFLP reaction system of Litsea cubeba was established.The results showed that the high quality genomic DNA as a PCR template could be isolated from the bud tissue;genomic DNA could be digested in a hour by 5 U EcoR I and 5 U Mse I;The optical selection amplification system was 20 μL reaction mix containing 1.0 U rTaq polymerase,2.0 μL 10×PCR buffer,1.8 μL 25 mmol·L^-1MgCl2,1.4 μL 2.5 mmol·L^-1dNTP,100 ng·μL^-1 primer each 1.0 μL.Clear and stable amplification band patterns can be obtained and 10 pairs of AFLP primers with good genetic diversity were selected according to the optimized reaction system.The results will be an effective protocol for further studying the genetic structure and differentiation of Litsea cubeba population.