摘要
以改良CTAB法提取水葫芦苗基因组DNA为模板,利用正交设计试验对影响ISSR-PCR反应体系的5个因素(TaqDNA聚合酶,dNTP,引物,模板及Mg^(2+))进行了优化筛选。经极差分析和方差分析,最终建立了水葫芦苗ISSR-PCR的最佳反应体系。结果表明,总体积20μL的反应体系中各因素浓度分别为:TaqDNA聚合酶0.025 U/μL、Mg^(2+)1.5 mmol/L、dNTP 1.0 mmol/L、引物0.5μmol/L、模板DNA 1.5 ng/μL。通过筛选得到10条扩增条带清晰的ISSR引物,并通过梯度PCR确定了各引物的最适退火温度。在此基础上对循环次数进行优化,最终确定最佳循环次数为40次。
The genome DNA of Halerpestes cymbalaris was extracted by the improved CTAB method. The orthogonal design method was used to the ISSR-PCR system of H. cymbalarisat four levels of five factors（the concentration of Taq DNA polymerase, d NTPs, primer, template DNA and magnesium ion）. The results were analyzed using the range analysis and variance analysis. A suitable system of the ISSR-PCR reaction of H.cymbalaris was established which contained 0.025 U/μL Taq polymerase, 1.5 mmol/L magnesiumion, 1.0 mmol/L d NTPs, 0.5 μmol/L primer and 1.5 ng/μL template DNA in total 20 μL reaction. Ten ISSR primers with clear bands were selected. And the annealing temperature of each primer was determined by the gradient PCR. On these bases, the optimum cycles were determined. The optimum cycles were 40 times.
出处
《分子植物育种》
CAS
CSCD
北大核心
2017年第8期3145-3152,共8页
Molecular Plant Breeding
基金
青海省科技计划项目(2014-NK-A4-2-1)
国家自然科学基金项目(31300269)共同资助
