矮化蓖麻RAPD-PCR反应体系及扩增程序的优化

Optimization of RAPD-PCR System and Amplification Program of dwarf Castor

目的优化矮化蓖麻RAPD-PCR反应体系及扩增程序。方法采用正交试验设计,优化RAPD-PCR反应体系(引物、dNTP、Taq酶和Mg2+浓度)及扩增程序(退火温度、退火时间、变性时间、延伸时间和循环次数)。结果25μl反应体系中最佳浓度配比为:引物0.36μmol/L,dNTP0.24mmol/L,Taq酶0.05U/μl,Mg2+1.20mmol/L;最佳扩增程序为:94℃预变性2min;94℃变性45s,36℃退火45s,72℃延伸80s,共35个循环;最后72℃再延伸5min。结论已优化了矮化蓖麻RAPD-PCR的反应体系及扩增程序,为应用RAPD-PCR技术对蓖麻株高性状进行研究奠定了基础。

Objective To optimize the RAPD-PCR system and amplification program of dwarf castor. Methods The RAPDPCR system including primers, dNTP, Taq enzyme and magnesium ion concentration as well as amplification program including temperature and time for annealing, times for denaturalization and extension and the number of cycle were optimized by orthogonal test. Results An optimal 25 μl reaction system consisted of 0. 36 μmol / L primers, 0. 24 mmol / L dNTP, 0. 05 U / μl Taq enzyme and 1.2 mmol/L magnesium ion. The amplification program was optimized as follows： denaturalization at 94℃ for 45 s, annealing at 36℃ for 45 s, extension at 72℃ for 80 s; 35 cycles, then extension at 72℃ for 5 min. Conclusion The RAPD-PCR system and amplification program of dwarf castor was optimized.