| 摘要: |
| 为了利用ISSR分子标记技术对坛紫菜(Porphyra haitanensis)种质资源进行遗传分析及种质鉴定, 获得清晰稳定、重复性好、多态性高的扩增结果, 对影响ISSR-PCR 扩增的多个因素, 包括DNA模板含量、Mg2+浓度、Taq DNA 聚合酶含量、引物浓度、dNT P浓度以及复性温度进行了全面比较和优化, 建立了坛紫菜种质资源ISSR-PCR扩增的最佳反应体系: 25μL 的反应体系中含2.5μL 10×PCR 缓冲液, 5ng模板DNA, 2.5 mmol/ L Mg2+, 1.5 UTaq DNA 聚合酶, 200n mol/L引物, 200μmol/L dNTP。最佳PCR扩增程序: 94℃预变性7min; 每个循环94℃变性1 min, 48℃复性45s, 72℃延伸2min, 共进行35个循环; 循环结束后72℃再延伸7min。 |
| 关键词: 坛紫菜(Porphyra haitanensis) ISSR 实验条件 优化 |
| DOI： |
| 分类号: |
| 基金项目:福建省科技重大专项资助项目(2004NZ03); 厦门市科技局资助项目(3502Z20041051); 厦门市海洋与渔业局资助项目 |
|
| Establishment and optimization of ISSR reaction system for Porphyra haitanensis |
|
|
| Abstract: |
| Porphyra haitanensis is the most economically important seaweeds in south China. The factors which affect the ISSR analysis in the study of the genetic diversity and variety identification of P. haitanensis, such as the amount of template DNA, Taq DNA polymerase, primers concentration, Mg2+ concentration, dNT P concentration and annealing temperature, were studied for optimizing conditions of the ISSR-PCR. The results showed that the optimal conditions being suitable for ISSR- PCR of P. haitanensis were as follows: 25μL reaction system containing 2.5μL 10×PCR Buffer, 5 ng template DNA, 2.5 mmol/ L Mg2+, 1.5 U Taq DNA polymerase (produced by Takara company) , 200n nmo l/ L primers and 200μmol/ L dNT P. With an MJ thermal cycle optimal amplification program was 1 cycle of 7 min at 94℃; 35 cycles of 1 min at 94℃, 45s at 48℃, and 2 min at 72℃; 1 cycle 7 min at 72℃, using block control style. All these results provided a standardizing ISSR-PCR program for the analysis of genetic diversity and variety identification of P. haitanensis. |
| Key words: Porphyra haitanensis inter-simple sequence repeat (ISSR) experimental conditions optimization |
