酿酒葡萄品种SSR-PCR体系的优化与建立

Establishment and optimization of SSR-PCR system of Vitis vinifera

以酿酒葡萄品种为原料,研究了聚合酶链式反应(polymerase chain reaction,PCR)体系的主要成分对葡萄简单序列重复(simple sequence repeats,SSR)扩增结果的影响,并确定了各成分的最佳用量。以改良的十六烷基三甲基溴化铵法(hexadecyl trimethyl ammonium bromide,CTAB)从葡萄中提取基因组DNA,通过单因素试验分析SSR-PCR体系中主要成分Mg2+浓度、d NTPs浓度、Taq DNA聚合酶浓度、引物浓度和模板DNA浓度对葡萄SSR-PCR反应体系扩增效果的影响。同时,采用正交优化设计试验确立酿酒葡萄SSR-PCR最佳25μL反应体系为Mg2+浓度为2.5 mmol/L,d NTPs为0.15 mmol/L,Taq DNA聚合酶为1.5 U,引物为0.5μmol/L,DNA含量为40 ng/25μL。通过优化试验的直观分析和方差分析得出PCR体系的主要成分对扩增结果的影响程度依次为:Mg2+>Taq DNA聚合酶>d NTPs>引物>模板DNA。利用此体系对4个酿酒葡萄品种DNA进行SSR-PCR反应体系扩增并进行非变性聚丙烯酰胺凝胶电泳检测,结果扩增条带清晰稳定,说明该体系可用于酿酒葡萄品种SSR标记的研究。

Using Vitis vinifera as raw material,the effect of main components in polymerase chain reaction(PCR)system on the simple sequence repeats(SSR)amplification of vitis vinifera was studied to optimize dosage of each factor.Total DNA of Grape was extracted by improved hexadecyl trimethyl ammonium bromide(CTAB)extraction method.The influences of the concentrations of main components in SSR-PCR system such as Mg 2+,dNTPs,Taq DNA polymerase,primers and the template the vitis vinifera DNA were analyzed through the single factor experiments.Based on orthogonal optimization design,the optimal 25μL reaction system for SSR-PCR of vitis vinifera DNA was established as Mg 2+of 2.5 mmol/L,dNTPs of 0.15 mmol/L,Taq DNA polymerase of 1.5 U,primers of 0.5μmol/L,and DNA template of 40 ng.Based on visual analysis and variance analysis of optimization experiments,the influences of main components in this PCR system were Mg 2+>Taq DNA polymerase>dNTPs>primers>template DNA.4 kinds of vitis vinifera DNA were amplified using this SSR-PCR amplification system and products were detected using non denaturing polyacrylamide gel electrophoresis.There are clear and stable amplification bands in gels,which indicated that this system could be used to study the SSR mark of vitis vinifera.