女贞ISSR-PCR反应体系的建立与优化

Establishment and Optimization of ISSR-PCR Reaction System of Ligustrum lucidum Ait.

利用单因子试验,测试了女贞ISSR-PCR反应体系中的主要影响因子。经过优化实验,建立了一套适合于女贞的稳定的ISSR-PCR反应体系:10×buffer2.5μL,2.0mmol·L-1的MgCl2,200μmol·L-1的dNTPs,1.5U的Taq酶,0.4的μmol·L-1的引物,10ng的DNA模板。PCR扩增程序为:94℃预变性4min,94℃变性40s,52～62℃退火45s,72℃延伸120s,进行35个循环,72℃延伸8min,4℃保存。应用该优化的ISSR-PCR实验体系对12份女贞种质材料进行了扩增,均能扩增出丰富稳定的条带。

Single factor experiment was used to test the effect factors of ISSR-PCR reaction system. After the optimization, a suitable ISSR-PCR reaction system of Ligustrum lucidum Ait. was established as follows： 10 × buffer 2.5 μL, 2.0 mmol · L^-1 MgC12,200 μmol · L^-1 dNTPs, 1.5 U Taq polymerase, 0.4μmol · L^-1 primers, 10 ng DNA template, which were contained in 25 μL reaction solution. The optimized amplification program was that predenaturing at 94℃ for 4 min, denaturing at 94℃ for 40 s, annealing at 52 ℃-60 ℃ for 45 s, extension at 72℃ for 120 s, for 35 cycles, extension at 72 ℃ for 8 min at last. The products were stored at 4℃. The optimized ISSR-PCR experiment system was applied in the amplification of 12 varieties of germplasm materials of Ligustrum lucidum Ait and the results indicated that the rich and stable bands can be amplified.