伯乐树cpDNA-PCR反应体系的优化与引物筛选  

Optimization of cpDNA-PCR system for Bretschneidera sinensi and primers screening

作　　者：胡尚力[1] 徐刚标[1] 梁艳[1] 刘雄盛[1] 肖玉菲[1] 郝博搏[1] 

机构地区：[1]中南林业科技大学生命科学与技术学院,湖南长沙410004

出　　处：《中南林业科技大学学报》2013年第7期67-71,共5页Journal of Central South University of Forestry & Technology

基　　金：林业公益性行业科研专项经费项目(201104033);湖南省研究生科研创新项目(CX2012B326)

摘　　要：伯乐树为国家一级保护植物,在研究被子植物的系统发育和古地理、古气候等方面具有重要的科学价值。cpDNA-PCR反应是在分子水平上确定伯乐树系统分类的基础。建立了伯乐树cpDNA-PCR反应体系,对影响扩增反应的因素(Mg2+浓度、dNTP浓度、引物浓度以及模板DNA浓度)进行了优化,得到了伯乐树cpDNA-PCR扩增反应的最佳体系,并筛选出了适合伯乐树分子谱系地理研究的非编码区序列和引物。伯乐树cpDNA-PCR最佳反应体系为:30μL反应体系含有10×PCR buffer、2.9 mmol Mg2+、120 mmol dNTP、上下游引物各11μmol、DNA模版30 ng以及3个单位的Taq酶。适合于伯乐树分子谱系地理学研究的3对引物及其退火温度为PipetB1411F-PipetD738R(52℃)、psbA-trnHGUG(59℃)、trnL-trnF(56℃)。Bretschneidera sinensis is a kind of national first-grade protected plants and has important scientific value in studying angiosperm’s phylogeny,paleogeography and paleoclimate.The cpDNA-PCR reaction is the basis of determinting the phylogenetic classification of B.sinensis at the molecular level.The concentrations of Mg2＋,dNTPs,primers and template DNA affecting the cpDNAPCR that will affect the amplified reaction were optimized to establish the cpDNA-PCR for B.sinensis,and the suitable non-coding sequences and primers for B.sinensis on molecular phylogeography were screened.The optimum 30 μL reaction system contained 10×PCR buffer,2.9 mmol Mg2＋,120 mmol dNTPs,11 μmol primers,30 ng template DNA and 3 units Taq polymerase.The 3 pairs of primers that are appropriate for the geographical study of molecular genealogy of B.sinensis and their corresponding annealing temperatures were screened out as followings： PipetB1411F-PipetD738R（52℃）,psbA-trnHGUG（59℃）,trnL-trnF（56℃）.

关 键 词：伯乐树 CPDNA PCR体系优化 引物筛选 

分 类 号：S794.9[农业科学—林木遗传育种] Q78[农业科学—林学]