| 摘要: |
| 以香港巨牡蛎(Crassostrea hongkongensis)基因组DNA为模板, 采用正交设计及单因素比较试验,分别对Mg2+、dNTPs、引物、Taq DNA 聚合酶和DNA 模板浓度的PCR 原料进行优化, 并通过温度梯度PCR, 筛选适宜的退火温度。确立了香港巨牡蛎的最适ISSR-PCR 反应体系: 25 μL 反应体系中含PCR1×Buffer, 2.5 mmol/L Mg2+、0.2 mmol/L dNTPs、0.2 μmol/L 引物、1.2 U Taq DNA 聚合酶、20 ng DNA模板。反应程序为: 94℃预变性5 min; 94℃变性1 min, 48～54℃(随引物而确定)退火1 min, 72℃延伸1.5 min, 35 个循环; 72℃延伸10 min。利用所建立的 ISSR-PCR 反应体系对香港巨牡蛎基因组DNA 进行扩增, 获得了清晰、重复性好、多态性高的DNA 谱带, 为进一步利用ISSR 分子标记研究香港巨牡蛎的遗传多样性奠定了基础。 |
| 关键词: 香港巨牡蛎(Crassostrea hongkongensis) ISSR-PCR 体系优化 |
| DOI： |
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| 基金项目:广西科技攻关项目(0992015-1) |
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| Establishment and optimization of an ISSR-PCR reaction system for Crassostrea hongkongensis |
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| Abstract: |
| To establish an ISSR-PCR reaction system in Crassostrea hongkongensis, the factors influencing ISSR-PCR system were explored with the orthogonal design and main parameters comparison test, based on the genomic DNA of C. hongkongensis. The optimized ISSR-PCR system (25 μL) was determined as follows: 1×Buffer, 2.5 mmol/L Mg2+, 0.2 mmol/L dNTPs, 0. 2 μmol/L primer, 1.2 U Taq DNA polymerase, and 20 ng DNA template. The reaction program included an initial denaturation for 5 minutes at 94℃, 35 cycles of denaturation for 1 minutes at 94℃, annealing for 1 minutes at 48～54℃, extension for 1.5 minutes at 72℃, and a final extension of 10 minutes at 72℃. The clear, reproducible, polymorphic products were obtained by the established ISSR-PCR reaction system. The results lay a foundation for the analysis of genetic diversity of C. hongkongensis by ISSR marker. |
| Key words: Crassostrea hongkongensis ISSR-PCR reaction system optimization |
