早熟马铃薯ISSR-PCR反应体系优化及引物筛选  

Optimization of ISSR-PCR reaction system and ISSR primer screening in early potato

作　　者：何海旺[1] 谭冠宁[1] 何虎翼[1] 何新民[1] 王晖[1] 李丽淑[1] 唐洲萍[1] 

机构地区：[1]广西农科院经济作物研究所,广西南宁530007

出　　处：《广东农业科学》2015年第1期133-137,共5页Guangdong Agricultural Sciences

基　　金：广西科技攻关项目(桂科攻1222009-2F);广西自然科学基金(2011GXNSFB018038)

摘　　要：为得出马铃薯ISSR-PCR最佳的反应体系,采用正交试验设计,对r Taq DNA聚合酶、Mg2+、模板DNA、d NTPs和引物进行5因素4水平筛选。结果表明,马铃薯ISSR-PCR的最佳反应体系为:总体积25μL的反应体系中含有r Taq DNA聚合酶0.5 U、Mg2+浓度为2.5 mmol/L、模板DNA用量为75 ng、d NTPs浓度为200μmol/L、引物浓度为0.8μmol/L。这些因素对PCR反应的影响大小顺序为Mg2+浓度>r Taq DNA聚合酶用量>d NTPs浓度=引物浓度>模板DNA用量。应用该最佳反应体系对39条ISSR引物进行筛选,得出15条条带清晰、条带数多、重复性好的引物。并通过退火温度梯度试验得出筛选引物的最佳退火温度。To screen the optimum ISSR-PCR reaction system for potato, the experiment was conducted by using orthogonal design with 4 levels of 5 factors（i.e. r Taq DNA polymerase, Mg2＋, DNA, d NTPs and primers）. Results showed that the optimal reaction system of ISSR-PCR was as follows： 0.5 U r Taq DNA polymerase, 2.5 mmol/L Mg2＋, 75 ng DNA,200 μmol/L d NTPs and 0.8 μmol/L primer in 25 μL reaction volume. And the PCR reaction effects was in the order of Mg2＋ r Taq DNA polymerase d NTPs = primer DNA. Then, 39 ISSR primers were screened by using the optimal reaction system, and 15 primers with clear band, more band number and good repeatability were selected. Finally, the optimized annealing temperatures of the 15 primers were obtained by gradient PCR.

关 键 词：马铃薯 ISSR 反应体系优化 引物筛选 

分 类 号：Q946.2[生物学—植物学] S532[农业科学—作物学]