硬叶兜兰叶绿体DNA SSR反应体系的优化及引物筛选  

Optimization of Chloroplast DNA SSR-PCR System and Primer Screening for Paphiopedilum micranthum

作　　者：杨坤梅 杨璧嘉[1] 李宗艳[1] 林萍[1] 

机构地区：[1]西南林业大学园林学院,昆明650224

出　　处：《分子植物育种》2015年第2期443-449,共7页Molecular Plant Breeding

基　　金：园林植物与观赏园艺省级重点学科和重点实验室(500974)资助

摘　　要：本研究以硬叶兜兰为试验材料,通过改良的CTAB法提取总DNA,运用5因素4水平的正交试验设计,综合采用直观量化分析和方差分析两种方法,分析模板DNA用量、Mg2+浓度、d NTPs浓度、引物浓度和Taq酶用量五个因素对cp SSR-PCR扩增的影响,比较不同组合扩增效果的差异,最终确定优化的硬叶兜兰cp SSR-PCR反应体系;并通过梯度PCR确定最佳退火温度。研究结果表明,以DNA模板用量对扩增反应的影响最大,Mg2+浓度的影响最小;较优的反应体系为:模板DNA 45 ng、d NTPs 0.2 mmol/L、引物各0.6μmol/L、Mg2+2.5 mmol/L、Taq DNA聚合酶0.4 U、10×PCR-Buffer,总体积10.0μL。本研究采用该体系对采自滇东南7个居群的12份样本扩增验证其稳定性,并从33对引物中筛选出扩增条带清晰、具明显等位基因、特异性好的12对引物。研究结果说明该优化体系能较好地对硬叶兜兰居群个体遗传差异进行分析。In order to establish an optimized cp SSR-PCR system of Paphiopedilum micranthum,a research on the impacts of five reaction factors（DNA template,Mg2＋,d NTPs,primer concentrations and Taq DNA polymerase） on the reaction system was carried out to compare their amplification results by an orthogonal experimental design L16（45）.Both a visual and quantitative examination method and a variance-analysis method were used to assess their impacts.Total DNA of P.micranthum was extracted from fresh leaves by a modified CTAB method.At the same time,the optimal anneal temperatures of primers were determined by gradient PCR.The results showed that the concentration of DNA template had greatest effects on the reaction system,but the concentration of Mg2＋had a few effects.Based on above research,a suitable amplification system was optimized which contained 45 ng DNA template,0.20 mmol/L d NTPs,0.6 μmol/L primer,2.5 mmol/L Mg2＋and 0.4 U Taq DNA polymerase in the 10 μL PCR volume.It had been used in the research on seven populations of P.micranthum from southeast Yunnan to detect its stability.Finally,12 pairs of primer combination were selected from 33 pairs because the distinct,reproducible,well-resolved bands could be produced and repeated.The optimization reaction system could be suitable and reliable to detect genetic diversity among populations of P.micranthum.

关 键 词：硬叶兜兰 CP SSR 反应体系 正交设计 引物筛选 

分 类 号：S682.31[农业科学—观赏园艺]