中国樱桃S-RNase基因PCR扩增技术体系的建立

Establishment of PCR reaction system of S-RNase gene in Chinese cherry cultivars(Cerasus pseudocerasus)

通过对TaqDNA聚合酶、Mg2+、dNTP、通用引物、模板DNA浓度等反应参数的系统研究,建立了中国樱桃S-RNase基因特异PCR扩增体系。该体系反应的总体积25μL,其中Taq酶1.25U,MgCl22.5mmol/L,dNTP0.15mmol/L,通用引物0.25μmol/L,模板DNA 50 ng。反应程序为:94℃预变性3 min;33个循环的94℃变性1 min,56℃退火45 s,72℃延伸1.5 min;最后72℃延伸10 min。利用优化的扩增体系在中国樱桃的不同品种中获得有效扩增,进一步证明了该体系具有良好的稳定性和重复性。

A special PCR reaction system has been established for S-RNase gene in Chinese cherry through the optimization of the concentration of Taq DNA polymerase, Mg2＋, dNTP, universal primers and template DNA. The reaction volume was 25 μL, containing 1.25 U Tat/DNA polymerase, 2.5 mmol/L MgCl2, 0.15 mmol/L dNTP, 0.25 μmol/L universal primers and 50 ng DNA. The PCR cycle was designed as following, pre-denaturing at 94℃ for 3 rain, denaturing at 94 ℃ for 1 min, annealing at 56℃ for 45 s, extension at 72 ℃ for 1.5 rain, total 33 cycles, then extension at 72 ℃ for 10 min. The stability and repeatability of the system had been validated by the effective amplification of S-RNase genes in Chinese cherry cultivars.