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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,20μl最佳PCR反应体系中包括2μl 10×buffer、1.0 U Taq酶、0.2 mM引物、100 ng模板DNA、0.2 mMdNTP、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。
In order to obtain the SSR reaction system of Zoysia japonica rapidly, orthogonal design of Zoysia japonica SSR with 5 factors (template DNA, Mg2 +, dNTP, Taq enzyme and primer) was used to screen the SSR system of Zoysia japonica. Tm similar primers to verify the optimization system. The results showed that the orthogonal design could be applied to the optimization of SSR-PCR reaction system. The optimal PCR reaction system consisted of 2 μl 10 × buffer, 1.0 U Taq enzyme, 0.2 mM primer, 100 ng template DNA, 0.2 mM dNTPs, 2.0 mM Mg + The optimal annealing temperature was 56 ~ 58 ℃. The feasible amplification program was as follows: pre-denaturation at 94 ℃ for 4 min, annealing at 94 ℃ for 30 s at 35 cycles, annealing at 56 ~ 58 ℃ 40 s, extension at 72 ℃ for 1 min, extension at 72 ℃ for 10 min, and preservation at 4 ℃. The establishment of this optimal reaction system laid a solid foundation for future SSR analysis of Zoysia japonica.