濒危竹种小蓬竹(Drepanostachyum luodianense)RAPD-PCR反应体系优化

Optimization of RAPD-PCR conditions for a rare and endangered bamboo of Drepanostachyum luodianense

为了建立小蓬竹(Drepanostachyum luodianense)RAPD-PCR反应的最优体系,以小蓬竹基因组DNA为模板,对影响其RAPD扩增的dNTPs浓度、模板DNA浓度、Taq DNA聚合酶量、引物浓度、Mg2+浓度等重要参数进行了单因子和正交试验。试验得出小蓬竹RAPD最优反应体系为:20μL反应体系,10×PCR buffer为1/10体积,dNTPs为100μmol·L-1,模板DNA量为30 ng,Taq DNA聚合酶为1.0 U,引物浓度为0.2μmol·L-1,Mg2+浓度为1.5 mmol·L-1。优化后的RAPD-PCR反应程序为:94℃预变性5 min,然后进入35个循环,即94℃变性1 min,35℃退火30 s,72℃延伸90 s,循环完毕后于72℃延伸7 min,最后在4℃保持。

The Drepanostachyum luodianense RAPD-PCR reaction system was optimized for the analysis of genetic variation and structure of D. luodianense. The genomic DNA of D. luodianense was extracted by improved CTAB method. Then,both single factor test and orthogonal design were applied to study the effects of main factors on the RAPD-PCR system for D. luodianense,in which the main factors included the concentration of dNTPs,primers and Mg2 ＋. The content of template DNA and Taq DNA polymerase and an optimal 20 μL RAPD-PCR reaction system for D. luodianense was established,including 1 /10 volume 10 × PCR buffer,100 μmol·L^-1dNTPs,30 ng template DNA,1. 0 U Taq DNA polymerase,0. 2 μmol·L^-1primers and 1. 50 mmol·L^-1Mg2 ＋. The optimized reaction program was initially at 94℃ for 5 min,followed by 35 cycles at 94℃ for 1 min,at 35℃ for 30 s,at 72℃ for 90 s,and then held at 72℃ for 7 min,and finally kept at 4℃.