| 长柱金丝桃ISSR-PCR反应体系的建立与优化 |
| |
| 引用本文： | 王丽俊,王霞.长柱金丝桃ISSR-PCR反应体系的建立与优化[J].东北林业大学学报,2017,45(10). |
| |
| 作者姓名： | 王丽俊 王霞 |
| |
| 作者单位： | 吉林农业科技学院,吉林省· 吉林市,132101 |
| |
| 基金项目： | 国家级大学生科技创新项目，吉林省大学生科技创新项目(吉农院合字 |
| |
| 摘 要： | 为筛选可用于长柱金丝桃遗传分析的最适ISSR-PCR反应体系,采用正交试验对影响ISSR-PCR反应的5个重要因素(Taq DNA聚合酶、dNTPs、Mg~(2+)、引物和模板DNA浓度)进行4水平正交优化,并对退火温度、循环次数进行筛选。结果表明,20μL ISSR-PCR最佳反应体系包括Taq DNA聚合酶1.0 U、dNTPs 0.2 mmol·L-1、Mg2+3.0 mmol·L~(-1)、引物0.4μmol·L~(-1)、DNA模板40 ng,对ISSR-PCR反应的影响程度由大到小为:Taq DNA聚合酶、引物、Mg2+、dNTPs、DNA模板;引物UBC822的最佳退火温度52.7℃,最佳循环次数41,利用优化的反应体系从61条引物中筛选出12条稳定性好、多态性高的引物,验证试验表明优化的反应体系具有较高的稳定性。
|
| 关 键 词： | 长柱金丝桃 ISSR反应体系 正交试验 引物筛选 |
Establishment and Optimization of ISSR-PCR Reaction System for Hypericum longistylum Oliv |
| |
| Wang Lijun,Wang Xia.Establishment and Optimization of ISSR-PCR Reaction System for Hypericum longistylum Oliv[J].Journal of Northeast Forestry University,2017,45(10). |
| |
| Authors: | Wang Lijun Wang Xia |
| |
| Abstract: | The five important factors ( Taq DNA polymerase , Mg2+, dNTPs, primers and DNA template concentration ) of ISSR-PCR reaction were studied to establish the suitable reaction system by orthogonal experiments , and then the annealing tem-perature and cycles were optimized .The results showed that 20μL ISSR-PCR optimal reaction system including 1.0 U Taq DNA polymerase, 0.2 mmol· L-1 dNTPs, 3.0 mmol· L-1 Mg2+, 0.4μmol· L-1 primers and 40ng DNA template, the de-scending degree of impact on the ISSR-PCR reaction was Taq DNA polymerase , primer, Mg2+, dNTPs, and DNA tem-plate.The best annealing temperature and cycle times of primer UBC 822 were 52.7℃ and 41, and 12 stability, and high polymorphic primers were selected from 61 primers.The optimal reaction system showed a very stable result . |
| |
| Keywords: | Hy pericum longistylum Oliv ISSR Reaction System Orthogonal Experiment Primer Selection |
| 本文献已被 CNKI 万方数据 等数据库收录！ |
|
