珙桐cpDNA非编码序列引物反应条件优化与筛选

Optimization of reaction condition and selection of primers for Davidia involucrate cpDNA noncoding

实验采用改良CTAB方法提取总DNA,以珙桐及其近缘种基因组DNA为材料,优化cpDNA非编码序列PCR扩增条件,优据优化的PCR扩增条件,首次筛选出适合于珙桐分子谱系地理学研究的cpDNA非编码序列和引物。结果表明:25μL PCR反应体系中含有50 ng DNA,10×Buffer,2.5 mmol MgCl2,0.4 mmol dNTPs,0.2μmolpri mer1,0.2μmol pri mer2,1.25 U Pfu DNA Polymerase。适宜的PCR反应程序为94℃5 min,94℃1 min,退火时间45 s,退火温度和72℃延伸时间依不同的引物和产物而不同,35个循环,72℃总延伸7 min。稳定性好的、扩增条带清晰且单一的trnSGCU和trnGUUC、F71和R1516、rps16—F和rps16—R、atpB—49和rbcL—188四对引物可作为进一步珙桐分子谱系地理学研究的引物。

Genomic DNA was extracted from Davidia involucrate and its related species with the improved CTAB method.PCR amplification conditions fit for cpDNA noncoding sequences were determined,and cpDNA noncoding sequences and primers that were applicable to study on molecular phylogeography in D.involucrate were screened out.Results show：（1） The favorable reaction system of PCR was composed of 25μL in volume,containing 50ng DNA,10×Buffer,2.5mmol MgCl2,0.4mmol dNTPs,0.2μmol primer 1,0.2μmol primer 2,and 1.25U Pfu DNA polymerase.（2） The PCR procedure was： pre-denaturizing at 94℃ for 5minutes;denaturizing at 94℃ for 1minute;annealing for 45seconds;annealing temperature and extension time at 72℃ varied with primers and the products;reaction of 35 cycles and re-extension at 72℃ for 7minutes.（3） Four pairs of primers were selected according to stability,unicity and clarity in PCR reaction,i.e.trn^SGCU and trn^GUUC,F71 and R1516,rps16-F and rps16-R,atpB-49 and rbcL-188.The primers could be used for molecular research on phylogeography in D.involucrate.