正交设计优化绿竹ISSR-PCR体系和引物筛选

Optimization of ISSR-PCR Reaction System on Dendrocalamus oldhami by Orthogonal Design,and Selection of Primer

旨在建立绿竹ISSR-PCR最佳反应体系和扩增程序,并筛选适于绿竹ISSR-PCR分析的高多态性引物。以绿竹基因组DNA为ISSR-PCR扩增模板,采用正交试验方法,对d NTPs浓度、Mg2+浓度、Taq DNA聚合酶浓度、引物浓度、模板DNA用量设计5因素4水平试验,采用极差分析法和方差分析法对试验结果进行分析。并对退火温度和循环次数进行筛选,建立绿竹ISSR-PCR最佳反应体系和扩增程序。并利用优化后的体系对100条ISSR引物进行筛选。最终确定的最佳反应体系为:20μL的扩增体系中,d NTPs浓度为0.2 mmol/L,Mg^(2+)浓度为2.0 mmol/L,Taq DNA聚合酶浓度为1.5 U,引物浓度为0.4μmol/L,DNA浓度为60 ng,10×PCR Buffer体积为2μL、剩下用灭菌ddH_2O补全。各因素影响大小依次是:Mg^(2+)>d NTPs>模板DNA>Taq DNA聚合酶>引物。扩增程序为:94℃预变性5 min;94℃变性45 s,(根据引物的退火温度)复性30 s,72℃延伸90 s,循环38次,72℃延伸10 min,4℃保存。以此体系为基础进行引物筛选,在100条ISSR引物中筛选出14条扩增条带清晰、多态性较高、重复性好的引物。

This work is to establish the optimal ISSR-PCR reaction system and amplifying procedure,and to screen high polymorphismprimers for ISSR-PCR analysis of Dendrocalamus oldhami（Munro）. First,the genomic DNA extracted from D. oldhami（Munro）wasused as template for ISSR-PCR amplification,and the orthogonal design（L_（16）（4~5））was to investigate the optimal concentrations of d NTPs,Mg^（2＋）,Taq DNA polymerase,primers and DNA template,and the results were analyzed by range and variance analysis method. Then,theannealing temperature and the number of cycles were screened for establishing the optimal D. oldhami（Munro）ISSR-PCR reaction system andamplifying procedure. Moreover,the optimized system was utilized to screen the 100 ISSR primers. Ultimately,the optimal reaction systemwas determined as follows ：in 20 μL reaction system containing 0.2 mmol/L d NTPs,2.0 mmol/L Mg^（2＋）,1.5U Taq DNA polymerase,0.4 μmol/L primer,60 ng DNA template,2.0 μL 10×Taq Buffer,and dd H_2O completed. The order of the effect by the factors was in Mg^（2＋） d NTPs DNA template Taq DNA polymerase primer. The optimal amplifying procedure was as follows ：pre-denaturing for 5 min at 94℃,38 cycleswere performed with denaturing of 45 s at 94℃,annealing of 30 s due to denaturing temperature of different primer,extension of 90 s at 72℃,a final extension step of 10 min at 72℃ and stored at 4℃. Using this optimized PCR system,14 of 100 primers was selected for their clarity,high polymorphism and repetition.