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中国农学通报

中国农学通报 ›› 2010, Vol. 26 ›› Issue (23): 48-52.

所属专题： 生物技术； 园艺

• 生物技术科学 • 上一篇    下一篇

葡萄柚基因组DNA提取方法的比较及ISSR-PCR体系的优化

吴 田 吴田   

  • 收稿日期:2010-09-03 修回日期:2010-09-29 出版日期:2010-12-05 发布日期:2010-12-05
  • 基金资助:

    国家林业局948项目;云南省部级重点学科省高校实验室及校实验室共享平台;西南林业大学大型仪器设备共享基金

Comparison of Genomic DNA Extraction Method and Optimization of ISSR-PCR Amplification for Grapefruit

  • Received:2010-09-03 Revised:2010-09-29 Online:2010-12-05 Published:2010-12-05

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摘要：

对4种基因组DNA提取方法进行比较，得到一种效率较高的、且适用于葡萄柚ISSR-PCR的DNA提取方法。同时，采用正交设计对影响葡萄柚ISSR-PCR的因子进行优化。实验结果表明，改良的SDS法提取的基因组DNA最适宜进行葡萄柚的ISSR-PCR扩增。ISSR-PCR产物在2%琼脂糖凝胶上检测，发现PCR扩增的平均条带数为2.7条。葡萄柚的最优ISSR-PCR反应体系为：20 μL总体积中含有2×buffer(Mg2+)，0.2 mmol/L dNTPs，0.2 μmol/L引物，0.3 U Taq DNA聚合酶，5 ng DNA模板。

关键词: 同源重组, 同源重组, 快速构建, LCHS2基因, RNAi, 表达载体, 酶切连接

Abstract:

An efficient genomic DNA extraction method useful for ISSR-PCR of grapefruit was selected by comparing four DNA extraction methods. Meanwhile, the factors influencing ISSR-PCR for grapefruit were optimized by orthogonal design. The result showed that improved SDS method was suitable for ISSR-PCR amplification. The average amplified bands were 2.7 which were detected in 2% agarose. The optimum amplification condition of ISSR-PCR was including 5 ng DNA template, 0.2 mmol/L dNTPs, 0.2 μmol/L primer, 0.3 U Taq DNA polymerase and 2×buffer (Mg2+) in 20 μL reaction volumes.