南方红豆杉叶绿体非编码序列PCR体系优化及引物筛选

Optimization of PCR Reaction System and Selection of cp DNA Noncoding Primers for Taxus chinensis var. mairei

PCR技术广泛用于分子标记、基因工程等领域,是植物系统发育及种群遗传结构等研究的基础。通过对影响PCR体系扩增的主要因子进行单因素试验,得出南方红豆杉(Taxus chinensis var.mairei)叶绿体DNA(cpDNA)最优体系(30μL)为3μL 10×PCR buffer,1μLDNA模板(60 ng),4.2μL MgCl_2(3.5 m M),8.4μLdNTPs(0.7 m M),0.9μL引物(0.3μM),0.8μL Taq DNA聚合酶(2 U),10.8μL去离子水。利用该体系筛选出叶绿体DNA非编码区通用引物4对,分别为atp I-atp H、psb J-pet A、trn H-psb A和trn L-trn F。

PCR was used widely in fields of molecular markers and gene engineering, which was the basis of research on plant phylogeny and genetic structure of populations. The optimal cpDNA- PCR reaction system of Taxus chinensis var. mairei was established containing 3 μL 10 × PCR buffer, 1 μL template DNA （60 ng）, 4. 2 μL MgC12 （ 3.5 mM）, 8.4 μL dNTPs （ 0. 7 mM）, 0. 9 μL primers （0. 3 μM）, 0. 8 μL Taq DNA polymerase （2 U） and 10. 8 μL ddH20 in a total volume of 30 μL reaction system by single factor test of main factors influencing PCR system amplification. Using this system, 4 pairs of cpDNA noncoding primers which were atpI- atpH, psbJ-petA, trnH-psbA and trnL -trnF were selected.