泡桐属植物ISSR-PCR反应体系的优化

摘要

为建立和优化泡桐属植物高效、稳定的ISSR-PCR反应体系,本试验采用了5因素4水平的正交试验以及单因素梯度优化相结合的方法,筛选出了泡桐属植物最适的ISSR-PCR反应体系,即20μL体系中含buffer 2.5μL,Taq酶2.0U,Mg2+ 3.75 mmol·L-1,0.4 mmol·L-1dNTPs,引物0.35 mmol·L-1,模板40 ng.扩增程序为:94℃预变性5 min,然后94℃变性30 s,55℃复性45 s,72℃延伸90 s,40个循环,72℃延伸7 min,最后4℃保温.%To establish and optimize the efficient and stable ISSR-PCR reaction system for the genus Paulownia plants,this study adopted the five-factor and four-level orthogonal design and single-factor gradient optimization method to establish and screen the plants of the genus Paulownia optimal ISSRPCR system,in 20 μL reaction solution,containing 2.5 μL buffer,2.0 U Taq-polymerase,3.75 mmol ·L-1 of ig2+,0.4 mmol · L-1 of dNTPs,0.35 mmol · L-1 of primers,and 40 ng of template DNA.Am-plification program was devised:initial denaturating at 94 ℃ for 5 min,denaturing at 94 ℃ for 30 s,annealing at 55 ℃ for 45 s,and extension at 72 ℃ for 90 s,40 cycles ; ending with a final extension at72 ℃ for 7 min,then stored at 4 ℃.