CAJ | 학술논문

为了快速获得结缕草(Zoysia japonica)SSR反应体系,采用5因素(模板DNA,Mg2+,dNTP, Taq酶和引物)4水平正交设计筛选合适的结缕草SSR体系,并通过随机挑选3对Tm相近的引物对该优化体系进行验证.结果表明:正交设计可应用于SSR-PCR反应体系的优化,20 μl最佳PCR反应体系中包括2 μl 10×buffer、1.0U Taq酶、0.2 mM 引物、100 ng模板DNA、0.2 mM dNTP、2.0 mM Mg+;对结缕草进行梯度退火实验,其最佳退火温度为56～58℃;可行扩增程序是:94℃预变性4 min、进行35个循环的94℃变性30 s、56～58℃退火40 s,72℃延伸1 min;72℃延伸10 min,4℃保存;该最适反应体系的建立,为今后结缕草SSR分析奠定了坚实基础.
위료쾌속획득결루초(Zoysia japonica)SSR반응체계,채용5인소(모판DNA,Mg2+,dNTP, Taq매화인물)4수평정교설계사선합괄적결루초SSR체계,병통과수궤도선3대Tm상근적인물대해우화체계진행험증.결과표명:정교설계가응용우SSR-PCR반응체계적우화,20 μl최가PCR반응체계중포괄2 μl 10×buffer、1.0U Taq매、0.2 mM 인물、100 ng모판DNA、0.2 mM dNTP、2.0 mM Mg+;대결루초진행제도퇴화실험,기최가퇴화온도위56～58℃;가행확증정서시:94℃예변성4 min、진행35개순배적94℃변성30 s、56～58℃퇴화40 s,72℃연신1 min;72℃연신10 min,4℃보존;해최괄반응체계적건립,위금후결루초SSR분석전정료견실기출.
In order to obtain the optimal SSR-PCR system for zoysiagrass (Zoysia japonica), an experimental design of orthogonal diagram L16(45) was employed to evaluate five factors (template DNA, Mg2+, dNTP, Taq DNA polymerase, and primer) at four different levels. Three randomly selected SSR primers (RM84, RM220, RM531) with resembled Tm were used to validate the reaction system. The results indicate that orthogonal design could be used to optimize the SSR-PCR system; the optimal system of terminal volume 20 μl consisted of 2 μl 10×buffer, 1.0U Taq DNA polymerase, 0.2 mM primers, 100ng template DNA, 0.2 mM dNTP, and 2.0 mM Mg+. The optimal annealing temperature was determined to be 56-58℃ by gradient PCR; the suitable thermal cycling conditions with initial melting at 94℃ for 4 min, followed by 35 cycles at 94℃ for 30 s, 56-58℃ for 40 s, 72℃ for 60 s; then keep the reaction mixture at 4℃ after a final extension step of 72℃ for 10 min. The optimized system would be effective as a solid foundation for zoysiagrass SSR analysis.